A bispecific antibody targeting robo1 and its preparation and application

A bispecific antibody and specific technology, applied in the field of bispecific antibody and its preparation and application, can solve the problems of time-consuming and laborious yield, production and purification of bispecific antibody, etc.

Active Publication Date: 2020-11-17
四川阿思科力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] In the above two methods, although the sources of bispecific antibodies produced by the hybridoma method are reliable, due to the random pairing and assembly of light chains and heavy chains, various antibody molecular forms can be produced, making the production and purification of bispecific antibodies very difficult. difficulty
The chemical cross-linking method produces bispecific antibodies with relatively uniform components, but it is time-consuming and labor-intensive and the yield is very low.

Method used

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  • A bispecific antibody targeting robo1 and its preparation and application
  • A bispecific antibody targeting robo1 and its preparation and application
  • A bispecific antibody targeting robo1 and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0107] Example 1 Preparation of bispecific antibody ZD037

[0108] Material:

[0109] Cell line ExpiCHO-S cells (Gibco Catalog No.A29127);

[0110] Transfection kit ExpiFectamine CHO Transfection Kit (Gibco CatalogNo.A29129);

[0111] OptiPRO SFM (Gibco Catalog No. 12309-050);

[0112] Medium ExpiCHO Expression Medium (Gibco Catalog No.A29100-01);

[0113] Culture conditions: 37°C, 8% CO2 incubator, shaker culture;

[0114] pCDNA3.4 plasmid vector;

[0115] 1.1 ZD037 protein expression

[0116] ZD037 molecular design form such as figure 1 shown. In this example, the ZD037 molecule containing two heavy chains (heavy chain A and heavy chain B) and one light chain is taken as an example for illustration. Wherein, the amino acid sequence of heavy chain A is shown in SEQ ID NO: 8, and its gene sequence is shown in SEQ ID NO: 11; the amino acid sequence of heavy chain B is shown in SEQ ID NO: 10, and its gene sequence is shown in SEQ ID NO : shown in 13; the amino acid sequ...

Embodiment 2

[0135] Example 2 Tumor cell ROBO1 expression analysis and identification

[0136] In this example, the liver cancer cell MHCC97-H cells highly expressing the ROBO1 antigen were selected as the target cells. In order to confirm that MHCC97-H cells are highly expressed ROBO1 molecules, flow staining was performed, and the specific method is as follows:

[0137] Take 5*10 5 MHCC97-H cells were used for staining. Incubate MHCC97-H cells with the primary antibody for 45min, take 50ul of the co-incubation and place it on ice, and wash it once with the flow staining buffer; incubate the MHCC97-H cells with the secondary antibody for 30min; then wash it once with the flow staining buffer , then resuspended in 120ul FACS reagent, and analyzed by flow cytometry. The experimental results are as Figure 3a and Figure 3b shown.

[0138] Depend on Figure 3a and Figure 3b It can be seen that MHCC97-H cells highly express ROBO1 molecules.

Embodiment 3

[0139] Example 3 Tumor killing assay detects drug activity

[0140] Use the IncuCyte Zoom long-term dynamic cell imaging analysis system to observe the state of cells in real time. Add tumor cell MHCC97-H and immune cell PBMC to the well plate and observe it under the analysis system to record the whole killing process of immune cells. The killing effect was detected by NucView488Caspase-3.

[0141] NucView488Caspase-3 is a dead cell dye. When tumor cells are killed, the dye can bind to DNA, and it turns green after laser excitation. The green fluorescent signal can be captured and recorded by IncuCyte Zoom equipment. By analyzing the green fluorescence intensity signals of different experimental wells, the killing effect of each experimental group can be judged. The stronger the fluorescence intensity, the better the killing effect.

[0142] Reagents and materials:

[0143] Cells: MHCC97-H, PBMC;

[0144] Drug: ZD037;

[0145] Medium: DMEM (10% FBS, 5% double antibody) (Gi...

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Abstract

The invention discloses a BsAb (bispecific monoclonal antibody). The BsAb contains an antigen binding domain (R structural domain for short) which specifically binds to a tumor cell surface antigen molecule and an antigen binding domain (I structural domain for short) which specifically binds to immune cells such as T cells, NKT cells and / or CIK cells. The I structural domain and the R structuraldomain are connected directly or are connected through connecting peptide. The provided BsAb taking ROBO1 as a target can binds CD3 molecules on the surfaces of the T cells and ROBO1 molecules on thesurfaces of tumor cells simultaneously, accordingly, the distance between the tumor cells and the T cells is shortened, the static T cells can be rapidly activated, the tumor cells can be killed rapidly and effectively, and a novel approach for development of anti-tumor (with high expression of ROBO1molecules) drugs is provided.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a bispecific antibody targeting ROBO1 and its preparation and application. Background technique [0002] Bispecific antibody (bispecific monoclonal antibody, BsAb) can double recognize tumor target cells and immune effector cells, so it has both antibody specificity and mediate cytotoxicity of effector cells, and properly designed bispecific antibodies can bind and aggregate Effector cells at the tumor site, activate the activity of effector cells, and induce the lysis of tumor cells. It does not exist in the natural state and can only be prepared artificially. [0003] Bispecific antibodies can be obtained in the following ways: [0004] ① Hybridoma method: Hybridoma cells are immortalized cell lines formed by the fusion of B cells and myeloma cells that can produce specific monoclonal antibodies. The hybridoma cells formed by further hybridization of two hybridoma cells can prod...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/46C12N15/13C12N15/63A61K39/395A61P35/00
CPCA61K2039/505C07K16/2803C07K16/2809C07K16/30C07K2317/31C07K2317/734
Inventor 李华顺任宝永刘鹏
Owner 四川阿思科力生物科技有限公司
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