Application of CD3xB7H3 bispecific antibody in directional killing of human bladder cancer cells

A bispecific antibody and bladder cancer cell technology, applied in tumor/cancer cells, animal cells, vertebrate cells, etc., can solve the problems of unsatisfactory survival rate after bladder cancer surgery, failure to curb tumor recurrence and drug resistance

Inactive Publication Date: 2019-07-05
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 of CD3xB7H3 bispecific antibody in directional killing of human bladder cancer cells
  • Application of CD3xB7H3 bispecific antibody in directional killing of human bladder cancer cells
  • Application of CD3xB7H3 bispecific antibody in directional killing of human bladder cancer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 cell culture

[0020] Human bladder cancer cells were cultured with 15% fetal bovine serum. Cells were cultured at 37°C in an incubator containing 5% carbon dioxide.

Embodiment 2

[0021] Example 2 Isolation of peripheral blood mononuclear cells and preparation of activated T lymphocytes (ATC) for cryopreservation

[0022] 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

[0023] Example 3 Anti-CD3×anti-B7-H3 bispecific antibody (B7-H3Bi-Ab) was synthesized and conjugated to activated T cells

[0024] 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 aims to provide an application of coupling CD3xB7H3 bispecific antibody in directional killing of human bladder cancer cells, and particularly, the coupling CD3xB7H3 bispecific antibodycan enhance the killing effect of activated T cells (ATC) on human bladder cancer cells and provides a new immunotherapy target for targeted therapy of drug resistance of bladder cancer. The researchof the invention proves that B7H3 is highly expressed in human bladder cancer cells; and compared with ATC which is not coupled with the CD3xB7H3 bispecific antibody, the ATC which is combined with the CD3xB7H3 bispecific antibody has enhanced capability of directionally killing the human bladder cancer cells, and has obvious cytotoxic activity on the human bladder cancer cells. When ATC combinedwith the CD3xB7H3 bispecific antibody and tumor cells are co-cultured at the multiplicity of infection of 10:1, the killing effect is significantly increased. Meanwhile, the ATC combined with the CD3xB7H3 bispecific antibody has increased level of secreting interferon-gamma (IFN-gamma) and tumor necrosis factor-alpha (TNF-alpha).

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 cells, specifically, the coupling of CD3×B7H3 bispecific antibody can enhance the effect of activated T cells (ATC) on human bladder cancer cells. It provides a new immunotherapy target for the targeted therapy of bladder cancer drug resistance. 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 of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12N5/0781A61P35/00
CPCA61K35/17A61K47/42C12N5/0635C12N5/0693
Inventor 张曼马婉茹雷婷
Owner 张曼
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