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Isolated binding proteins, nucleic acids, vectors, CAR-T cells and applications of b7-h3 antigen binding domains

A technology of B7-H3 and binding domain, which is applied in the direction of anti-animal/human immunoglobulin, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, and cells modified by introducing foreign genetic material, etc. It can solve the problems of slow research progress and no target

Inactive Publication Date: 2020-12-11
北京善科生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CAR-T has achieved very good clinical therapeutic effects in the treatment of lymphoma, leukemia and other blood system tumors, the research progress of CAR-T in solid tumors is relatively slow. One of the important reasons is that there is no suitable target, because usually Antigens highly expressed in solid tumors are also expressed in some normal tissues

Method used

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  • Isolated binding proteins, nucleic acids, vectors, CAR-T cells and applications of b7-h3 antigen binding domains
  • Isolated binding proteins, nucleic acids, vectors, CAR-T cells and applications of b7-h3 antigen binding domains
  • Isolated binding proteins, nucleic acids, vectors, CAR-T cells and applications of b7-h3 antigen binding domains

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Embodiment 1

[0068] 1. B7-H3 antibody screening

[0069] BALB / c mice were immunized by subcutaneous injection with purified B7-H3-His protein mixed with Freund's adjuvant, once every two weeks, and immunized three times in total. On the 35th day, blood was collected and the anti-B7-H3 antibody in the serum was detected by ELISA. When the OD value after 4000-fold dilution was greater than 1.0, it indicated that the serum contained a high concentration of anti-B7-H3 antibody. On the 39th day, the mice were immunized again, and the mouse spleen cells were collected on the 42nd day, and a single cell suspension was prepared, and fused with myeloma cells F0 by PEG. After 7 days of culture, the supernatants of the fused hybridoma clones were screened by ELISA to detect their specificity and sensitivity. After re-screening and subcloning, monoclonal 2D6 was obtained which could stably secrete anti-B7-H3 antibodies.

[0070] 2. Plasmid construction

[0071] In order to construct a CAR targeting ...

Embodiment 2

[0093] 1. Co-culture killing experiment

[0094] Non-small cell lung cancer cells were divided into 5×10 5 Each well was added to a 24-well cell culture plate, and after 16 hours, 5×10 5 NT, B7-H3-CAR-28 or B7-H3-CAR-BB CAR-T cells, the ratio of T cells to cancer cells is 1:5. After 5 days of co-culture, the suspended T cells and the adherent cancer cells were collected, and the T cells and cancer cells were labeled with CD3 and B7-H3 antibodies respectively, and then the proportion of residual cancer cells was detected by flow cytometry. Zombie Aqua Dye (Biolegend) removes dead cells.

[0095] 2. Using the H522 lung cancer metastasis model to evaluate the effect of B7-H3-CAR-T on tumor killing in mice

[0096] The H522 lung cancer cells overexpressing luciferase were injected into NSG mice through the tail vein, and each mouse was injected with 2×10 6 H522 cells were used to construct a lung cancer metastasis model in mice. After 14 days, tumor-bearing mice were treated ...

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Abstract

The invention relates to the field of CAR-T cells, in particular to isolated binding proteins, nucleic acid, a vector, a CAR-T cell in a B7-H3 antigen binding domain and an application thereof. According to an scFV fragment obtained by the B7-H3 antigen, a second generation of B7-H3-CAR using CD28 or 4-1BB as a costimulatory molecule is designed and constructed, and the capability of killing cancer cells is verified in B7-H3 positive non-small cell lung cancer, and a result shows that when the ratio of T cells to cancer cells is 1: 5, the B7-H3-CAR-T can still kill B7-H3 positive non-small cell lung cancer cells very efficiently, and a large amount of type I interferon, such as IFN gamma and TNF alpha, is released.

Description

technical field [0001] The present invention relates to the field of CAR-T cells, in particular, to isolated binding proteins, nucleic acids, vectors, CAR-T cells and applications of B7-H3 antigen-binding domains. Background technique [0002] Lung cancer is the tumor with the highest cancer mortality rate in the world, with an increase of about 1.3 million new cases every year, and about 80-85% of newly diagnosed lung cancers are non-small cell lung cancers, such as adenocarcinoma, squamous cell carcinoma, and large cell carcinoma. In 2017, there were about 800,000 new lung cancer cases and 700,000 deaths in China. The incidence and mortality rate of lung cancer accounted for about 40% of the world's lung cancer, far higher than other countries. In China, lung cancer is also the cancer with the highest morbidity and mortality rate. With the increasing air pollution in recent years, it is expected that the number of lung cancer patients will further increase in the future, s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C12N15/13C12N5/10C12N15/867A61K35/17A61P35/00
Inventor 陈模江王刚刘金超
Owner 北京善科生物科技有限公司