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Preparation method and application of bispecific antibody targeting mouse T lymphocyte CD3 and human tumor antigen HER2

A bispecific antibody and target cell technology, which is applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, anti-tumor drug, etc., can solve the problem of limited curative effect of solid tumors, and achieve increased immunity Therapy, efficacy enhancement effect

Active Publication Date: 2015-04-29
WUHAN YZY BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, immunotherapy can only remove a small amount of scattered tumor cells, and has limited efficacy for advanced solid tumors

Method used

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  • Preparation method and application of bispecific antibody targeting mouse T lymphocyte CD3 and human tumor antigen HER2
  • Preparation method and application of bispecific antibody targeting mouse T lymphocyte CD3 and human tumor antigen HER2
  • Preparation method and application of bispecific antibody targeting mouse T lymphocyte CD3 and human tumor antigen HER2

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Example 1: Preparation of bispecific antibody (HER2×mCD3, M806)

[0068] 1. Bispecific antibody sequence design

[0069] The bispecific antibody targeting HER2 and mCD3 (clone number: 2C11) was named M806, as image 3 As shown, the anti-HER2 is in the form of IgG, including anti-HER2 heavy chain and light chain, and the anti-mCD3 is in the form of ScFv-Fc, including anti-mCD3 VH, VL, and Fc domains.

[0070] Anti-HER2 heavy chain (amino acid sequence SEQ ID NO.1)

[0071] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0072] Anti-HER2 light chain (amino acid sequence SE...

Embodiment 2

[0091] Example 2: Preparation of mouse tumor cell lines expressing human tumor antigen HER2

[0092] 1. Murine Tumor Cell Preparation

[0093] B16 mouse melanoma cells and CT26.WT mouse colon cancer cells were purchased from the Cell Bank of the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences.

[0094] 2. Acquisition of antigen gene and construction of expression vector

[0095] The cultured SK-BR-3 cells (ATCC, HTB-30) expressing human HER2 were collected, centrifuged at 400×g for 5 min, and the supernatant was discarded. Add the same volume of PBS as the culture medium before centrifugation, resuspend the pellet, centrifuge at 400×g for 2-3 minutes, and discard the supernatant. Using Invitrogen's The reagent kit (15596-026) was operated according to the instructions to extract the total RNA of the cells.

[0096] Firstly, random primers in the 5'RACE FULL kit (purchased from TAKARA, product number D315) of Takara Company ...

Embodiment 3

[0108] Example 3: In Vitro Cell Killing Detection

[0109] 1. Mouse Splenocyte Preparation

[0110] Bluntly dissect the mouse spleen, inject PBS repeatedly into the spleen with a syringe, collect the outflowing spleen cell suspension, centrifuge at 400×g for 10 minutes, resuspend the pellet in PBS, and act with 5 times the volume of red blood cell lysate (beyotime, C3702) for 5 minutes Centrifuge at 400×g for 5 minutes, wash once with PBS and resuspend the cells in 10% FBS-1640 medium to 1×10 6 cells / ml, spare.

[0111] 2. Preparation of murine tumor cells expressing human tumor antigens

[0112] B16-HER2 / CT26-HER2 cells were cultured in RPMI-1640 (GIBCO, C11875) + 10% FBS (BI, 04-001-1A). After culture, the cells were digested with 0.25% trypsin and detected by Vi-Cell cell counter Cell number and viability, collect the cells and centrifuge to remove the supernatant, resuspend in 1% FBS-PBS to make a single cell suspension, stain with CFSE at a final concentration of 5 μ...

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Abstract

The invention provides a bispecific antibody as well as a preparation method and an application thereof. Meanwhile, the invention also provides a mouse tumor cell line expressing human-derived HER2 protein and an application of the cell line. The bispecific antibody provided by the invention can set up a bridge between the target cell and the functional molecule (cell) to activate an oriented immunoreaction, and has a broad application prospect in tumor immunotherapy. A tumor cell in-vivo efficacy experiment of the bispecific antibody provided by the invention is developed in an animal model with a complete immune system, wherein the tumor is killed by the cells of the immune system, and the efficacy of the bispecific antibody mediating the immune cells to kill the tumor cells can be effectively reflected, and thus a perfect efficacy evaluation method is provided for developing a bispecific antibody drug targeting immune cells and tumor cells.

Description

technical field [0001] The present invention relates to the technical field of immunology. Specifically, it involves the construction and preparation of bispecific antibodies, the design and establishment of mouse tumor models, and the research on the in vitro and in vivo drug efficacy and mechanism of bispecific antibodies. Provide powerful methods and models for the research of drug mechanism in the process of double antibody drug development, especially the study of drug efficacy in preclinical animals. Background technique [0002] Bispecific antibody (bispecific antibody, BiAb) is an artificial antibody containing two specific antigen-binding sites, which can build a bridge between target cells and functional molecules (cells) to produce oriented effector functions. BiAb has broad application prospects in biomedicine, especially in tumor immunotherapy. Killing tumor cells through BiAb-mediated cytotoxicity is a hot topic in current immunotherapy application research. ...

Claims

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Application Information

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IPC IPC(8): C07K16/46C07K16/30C12N15/85A61K39/395A61P35/00
Inventor 王涛胡柳罗振戴晴刘敬松范克索周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
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