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Application of CD3*B7H3 bispecific antibody in directionally killing doxorubicin-resistant bladder cancer cell PUMC-91/ADM

A technology of PUMC-91 and bladder cancer cells, which is used in medical preparations, anti-tumor drugs, and drug combinations with inactive ingredients, etc. question

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 of CD3*B7H3 bispecific antibody in directionally killing doxorubicin-resistant bladder cancer cell PUMC-91/ADM
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  • Application of CD3*B7H3 bispecific antibody in directionally killing doxorubicin-resistant bladder cancer cell PUMC-91/ADM

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Example 1 cell culture

[0021] The doxorubicin-resistant human transitional cell bladder cancer cell line PUMC-91 / ADM was established by gradually increasing the dose of doxorubicin in its parent cell line PUMC-91, and the final concentration of doxorubicin was 1.0 μg / ml. Cultured 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 aims at providing application of a coupled CD3*B7H3 bispecific antibody in directionally killing a doxorubicin-resistant human transitional cell bladder cancer cell PUMC-91 / ADM, specially relates to the coupled CD3*B7H3 bispecific antibody which can enhance the killing effect of an activated T cell (ATC) on the doxorubicin-resistant bladder cancer cell PUMC-91 / ADM, and provides a novel immunotherapy target for targeted therapy of the drug resistance of the bladder cancer. The high expression of B7H3 in the doxorubicin-resistant bladder cancer cell PUMC-91 / ADM is verified throughthe research; compared with the ATC of an uncoupled CD3*B7H3 bispecific antibody, the ability of ATC of the CD3*B7H3 bispecific antibody to directionally kill the doxorubicin-resistant bladder cancercell PUMC-91 / ADM is enhanced, and the doxorubicin-resistant bladder cancer cell PUMC-91 / ADM has significant cytotoxic activity. When the ATC combined with the CD3*B7H3 bispecific antibody is co-cultured with a tumor cell under the condition that the multiplicity of infection is 10:1, the killing effect is significantly increased. The level of the ATC combined with the CD3*B7H3 bispecific antibodysecreting 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 a CD3×B7H3 bispecific antibody for directional killing of doxorubicin-resistant human transitional cell bladder cancer PUMC-91 / ADM. Specifically, the CD3×B7H3 bispecific antibody can enhance The killing effect of activated T cells (ATC) on PUMC-91 / ADM doxorubicin-resistant human transitional cell bladder cancer cells 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...

Claims

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Application Information

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IPC IPC(8): A61K35/17A61K47/68A61P35/00
Inventor 张曼马婉茹张敏雷婷
Owner 张曼
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