Application for directionally killing bladder cancer cells T24 through CD3*B7H3 dual-specificity antibody

A bispecific antibody, bladder cancer cell technology, applied in the direction of antibodies, anti-tumor drugs, drug combinations, etc., can solve the problems of unsatisfactory survival rate after bladder cancer surgery, unable to curb tumor recurrence and drug resistance, etc.

Inactive Publication Date: 2019-06-28
张曼
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Despite the existence of various treatment methods such as surgery, radiotherapy, and chemotherapy, the postoperative survival rate of bladder cancer is still very poor
These treatments can limit tumor development and growth, but they cannot prevent tumor recurrence and drug resistance

Method used

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  • Application for directionally killing bladder cancer cells T24 through CD3*B7H3 dual-specificity antibody
  • Application for directionally killing bladder cancer cells T24 through CD3*B7H3 dual-specificity antibody
  • Application for directionally killing bladder cancer cells T24 through CD3*B7H3 dual-specificity antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 cell culture

[0021] Human bladder cancer T24 cell line was purchased from ATCC cell bank. Cell lines were cultured in RPMI 1640 medium with 15% fetal bovine serum. Cells were cultured at 37°C in an incubator containing 5% carbon dioxide.

Embodiment 2

[0022] Example 2 Isolation of peripheral blood mononuclear cells and preparation of activated T lymphocytes (ATC) for cryopreservation

[0023] Peripheral blood mononuclear cells (PBMCs) were obtained by separating peripheral blood from healthy donors provided by Beijing Blood Bank by Ficoll density gradient centrifugation. 1 × 10 in RPMI-1640 medium supplemented with 10% FBS and 5 μg / ml anti-CD3 mAb (eBioscience, San Diego, CA, USA) and 100 IU / ml recombinant human IL-2 6 / ml cultured PBMC. The fresh medium required for cell culture contains 100IU / ml recombinant human IL-2. On day 13, ATC with expanded average expressing CD3+ (CD4+ and %CD8+) was cryopreserved for further use.

Embodiment 3

[0024] Example 3 Anti-CD3×anti-B7-H3 bispecific antibody (B7-H3Bi-Ab) was synthesized and conjugated to activated T cells

[0025] Anti-B7-H3 monoclonal antibody (R&D System, Minneapolis, MN, USA) was reacted with sulfo-SMCC, and anti-CD3 (OKT3, eBioscience) was reacted with Traut's reagent. Thaw cryopreserved ATC and mix with B7-H3Bi-Ab at 50ng / 10 6 Cells were concentrated for 30 min at room temperature and cells were subsequently washed to eliminate unbound antibody. Grayscale analysis of western blot bands was performed using ImagJ to calculate the conjugation rate.

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Abstract

The invention provides application for directionally killing human bladder cancer cells T24 through a coupling CD3*B7H3 dual-specificity antibody, particularly, the coupling CD3*B7H3 dual-specificityantibody can enhance the T24 bladder cancer cell killing effect of the activated T cell (ATC), and an immunity treatment target spot is provided for bladder cancer targeting treatment. Researches prove that B7H3 can achieve high expression in the T24 bladder cancer cell; compared with the ATC of the uncoupled CD3*B7H3 dual-specificity antibody, the capability of the ATC combined with the CD3*B7H3dual-specificity antibody for directionally killing the T24 bladder cancer cells is enhanced, and the remarkable cell toxin activity is achieved for the T24 bladder cancer cells. When co-culture is performed on the condition that the effective target ratio of the ATC combined with the CD3*B7H3 dual-specificity antibody to the tumor cells is 10:1, the killing effect is remarkably increased. Meanwhile, the level of the ATC combined with the CD3*B7H3 dual-specificity antibody for secreting a gamma-interferon (IFN-gamma) and an alpha-tumor necrosis factor (TNF-alpha) is increased.

Description

technical field [0001] The purpose of the present invention is to provide an application of conjugated CD3×B7H3 bispecific antibody for targeted killing of human bladder cancer cell T24, specifically, the conjugated CD3×B7H3 bispecific antibody can enhance the effect of activated T cells (ATC) on T24 bladder cancer The killing effect of cells provides a new immunotherapy target for the targeted therapy of bladder cancer. Background technique [0002] Bladder cancer is the fourth most common cancer in men and the 11th most common cancer in women. In 2017, there were an estimated 79,030 new bladder cancer cases and 19,870 deaths in the United States, and the incidence and mortality rates of men were four times higher than those of women. Initially, about 85% of bladder cancer patients are diagnosed as superficial bladder cancer, more than 50% of superficial bladder cancer will recur, and only 46% of stage III patients and 15% of stage 4 patients can reach stage five. years o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/395A61P35/00
Inventor 张曼马婉茹雷婷
Owner 张曼
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