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A preparing method of bispecific antibodies targeting a mouse T lymphocyte CD3 and a human tumor antigen EpCAM, and applications of the bispecific antibodies

A bispecific antibody, target cell technology, applied in the direction of anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, antibody, antitumor drug, etc. Therapeutic, efficacy-enhancing effect

Active Publication Date: 2015-07-22
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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  • A preparing method of bispecific antibodies targeting a mouse T lymphocyte CD3 and a human tumor antigen EpCAM, and applications of the bispecific antibodies
  • A preparing method of bispecific antibodies targeting a mouse T lymphocyte CD3 and a human tumor antigen EpCAM, and applications of the bispecific antibodies
  • A preparing method of bispecific antibodies targeting a mouse T lymphocyte CD3 and a human tumor antigen EpCAM, and applications of the bispecific antibodies

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1: Preparation of bispecific antibody (EpCAM×mCD3, M706)

[0061] 1. Bispecific antibody sequence design

[0062] The bispecific antibody targeting EpCAM and mCD3 was named M706, such as image 3 , the anti-EpCAM here is in the form of IgG, including anti-EpCAM heavy chain and light chain, and the anti-mCD3 is in the form of ScFv-Fc, including anti-mCD3 VH, VL, and Fc domains.

[0063] Anti-EpCAM heavy chain (amino acid sequence SEQ ID NO.1)

[0064] EVQLLEQSGAELVRPGTSVKISCKASGYAFTNYWLGWVKQRPGHGLEWIGDIFPGSGNIHYNEKFKGKATLTADKSSSTAYMQLSSLTFEDSAVYFCARLRNWDEPMDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0065] Anti-EpCAM light chain (amino acid sequence SEQ ID NO.2) ...

Embodiment 2

[0084] Example 2: Preparation of mouse tumor cell lines expressing human tumor antigen EpCAM

[0085] 1. Murine Tumor Cell Preparation

[0086] 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.

[0087] 2. Acquisition of antigen gene and construction of expression vector

[0088] The cultured HCT116 cells expressing human EpCAM 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 400g 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.

[0089] Firstly, random primers in the 5'RACE FULL kit (purchased from TAKARA, product number D315) of Takara Company were used to re...

Embodiment 3

[0101] Example 3: In Vitro Cell Killing Detection

[0102] 1 Mouse splenocyte preparation:

[0103] Bluntly separate the mouse spleen, inject PBS into the spleen repeatedly with a syringe, collect the outflowing spleen cell suspension, centrifuge at 400g for 10min, resuspend the pellet in PBS, act with 5 times the volume of red blood cell lysate (beyotime, C3702) for 5min, and then centrifuge at 400g 5min, after washing once with PBS, resuspend the cells in 10% FBS-1640 medium to 1×10 6 cells / ml, spare.

[0104] 2 Preparation of mouse tumor cells expressing human tumor antigens

[0105] B16-EpCAM / CT26-EpCAM cells were cultured in RPMI-1640 (GIBCO, C11875) + 10% FBS (BI, 04-001-1A). After culture, the cells were digested with 0.25% trypsin for 20 seconds, collected and centrifuged. Supernatant, resuspended to 1% FBS-PBS to make a single cell suspension, Vi-Cell cell counter to detect cell number and viability, stained with CFSE at a final concentration of 5uM, 5% CO 2 , i...

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Abstract

The invention provides bispecific antibodies, a preparing method thereof and uses of the bispecific antibodies, and provides murine tumor cell lines expressing human-derived EpCAM protein and uses of the cell lines. The bispecific antibodies can set up a bridge between a target cell and a functional molecule (cell), stimulate directional immune responses, and have a wide application prospect in immunotherapy of tumor. A tumor cell in-vivo drug efficacy experiment is performed in an animal model with a complete immune system, kills tumor by utilization of immune system cells, effectively reflects drug efficacy of the bispecific antibodies mediating the immune cells to kill tumor cells, and provides a good drug efficacy assessment method for development of bispecific antibody drugs 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 张敬胡伶俐王瑞周祥范克索周鹏飞
Owner WUHAN YZY BIOPHARMA CO LTD
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