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Anti-BCMA antigen chimeric antigen receptor and application thereof

A chimeric antigen receptor and antigen recognition technology, applied in the field of genetic engineering, can solve the problems of tumor cells off-target, unable to solve the survival crisis of MM patients, and unable to cure them

Active Publication Date: 2018-12-18
CHONGQING PRECISION BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although initial treatment of MM is sensitive to a variety of cytotoxic drugs, it is currently incurable due to the high probability of relapse in highly heterogeneous and drug-resistant MM
In 2016, there were 30,330 newborn MM patients in the United States, and 12,650 patients died of MM. Although there is no systematic statistics on the incidence of MM and the survival of patients in China, it can be seen from the data of the United States that the existing drugs are far from being able to solve MM patients. existential crisis
[0005] The commonly used antigen recognition region is single-chain antibody (scFV). However, not all scFV derived from anti-BCMA antibodies can be used as the antigen recognition region in the CAR structure. The specificity, affinity, stability and connection with different hinge regions of different scFV Both may lead to the inability of the constructed CAR-transduced CAR-T cells to effectively target tumor cells or even off-target

Method used

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  • Anti-BCMA antigen chimeric antigen receptor and application thereof
  • Anti-BCMA antigen chimeric antigen receptor and application thereof
  • Anti-BCMA antigen chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1 Construction of chimeric antigen receptor virus containing BCMA scFv

[0064] In order to construct a more effective chimeric antigen receptor against BCMA, we used 8 antibody strains to construct its scFV and designed BCMA(1), BCMA(2), BCMA(3), BCMA(4), BCMA(5), BCMA( 6), BCMA(7), BCMA(8), BCMA(9) and BCMA(10) have a total of ten groups of CARs, including 3 structures such as figure 1 shown.

[0065] 1 Synthesis of gene sequences of chimeric antigen receptors targeting BCMA containing different scFvs

[0066] Synthesize different single-chain antibody ScFv containing anti-human BCMA antigen, IgG4 or human CD8 hinge region (also known as hinge region), CD28 transmembrane region (abbreviated as TM), CD28 and / or CD137 and CD3 co-stimulatory signal combination The resulting chimeric antigen receptor has a structure such as figure 1 As shown, from left to right are marker, original plasmid and restriction plasmid.

[0067] 2 Construction of lentiviral vectors ...

Embodiment 2

[0082] Example 2 Expression of BCMA in different tumor cells

[0083] The tumor cells Raji and NCI-H929 cultured for 10 days were collected into 10ml centrifuge tubes respectively, and the expression of BCMA was detected by flow cytometry using APC-BCMA antibody (BD Company).

[0084] The result is as image 3 As shown, NCI-H929 is a high-expression BCMA cell, and Raji is a BCMA-negative cell.

Embodiment 3

[0085] Example 3 Detection of CAR Transfection T Cell Ability and CAR-T Cell Killing Ability

[0086] 1. Detection of the ability of BCMA-CAR to transfect T lymphocytes

[0087] 1 Isolation of human peripheral blood mononuclear cells

[0088] Human peripheral blood mononuclear cells were separated by gradient centrifugation to obtain PBMC cells, which were cultured by adding RPMI1640 complete medium containing 10% FBS.

[0089] 2 Lentiviral vector infection of T lymphocytes

[0090] Mononuclear cell PBMCs obtained in step 1 were activated by adding anti-CD3 monoclonal antibody on the first day; lentivirus infection was carried out in the first 3 days; lentiviral vector corresponding to 5MOI was added, and uninfected T lymphocytes were used as blank control; after 24 hours, culture The base was replaced with RPMI 1640 complete medium containing 500 IU / ml recombinant human IL-2, and the culture was continued for 10-20 days.

[0091] During the culture process, the virus-infec...

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Abstract

The invention belongs to the field of genetic engineering, and concretely relates to a BCMA-targeting chimeric antigen receptor and an application thereof. The chimeric antigen receptor comprises an antigen recognition region, a hinge region, a transmembrane region and an intracellular signal domain; and the amino acid sequence of an extracellular recognition region is represented by SEQ ID NO.1 or SEQ ID NO.2 or SEQ ID NO.3. The chimeric antigen receptor can effectively remove BCMA-expressing tumor target cells after being expressed in immune cells, has no toxic or side effects on antigen-negative cells, and can be used for the targeted therapy of tumors.

Description

technical field [0001] The invention belongs to the field of genetic engineering, relates to a BCMA-targeted chimeric antigen receptor and its application, and also relates to a virus vector containing the BCMA-targeted chimeric antigen receptor and its application. Background technique [0002] Multiple myeloma is also known as multiple myeloma, abbreviated as MM. According to statistics, the high incidence age of MM is 65-74 years old. Complications include hypercalcemia, renal insufficiency, anemia and infection. Although initial treatment of MM is sensitive to a variety of cytotoxic drugs, due to high heterogeneity and high recurrence probability of drug-resistant MM, it is currently incurable. In 2016, there were 30,330 newborn MM patients in the United States, and 12,650 patients died of MM. Although there is no systematic statistics on the incidence of MM and the survival of patients in China, it can be seen from the data of the United States that the existing drugs a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K35/17A61P35/00
CPCA61P35/00A61K35/17C07K14/7051C07K16/2878C07K2317/622C07K2319/33C12N2510/00
Inventor 张巍徐艳敏陈军黄霞单娟娟赵文旭赵永春张茜真
Owner CHONGQING PRECISION BIOTECH CO LTD
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