Anti-BCMA CAR as well as expression vector and application thereof

A technology of expressing vectors and vectors, which is applied in the field of genetic engineering, can solve problems such as safety problems, limited efficacy, and the loss of antigen binding activity of antibodies, and achieve the effects of high safety, reduced immunogenicity, and good ability to remove tumor cells

Active Publication Date: 2020-11-17
CHONGQING PRECISION BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the modification of antibodies usually leads to the loss of the original antigen-binding activity of the antibody. Therefore, it is necessary to modify the antibody, and then use a large number of antigen-antibody binding specificity and affinity tests to screen for active antibody sequences.
At present, clinical trials of targeted drugs targeting BCMA have limited efficacy and safety issues

Method used

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  • Anti-BCMA CAR as well as expression vector and application thereof
  • Anti-BCMA CAR as well as expression vector and application thereof
  • Anti-BCMA CAR as well as expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Preparation of Lentivirus Expressing Chimeric Antigen Receptor Targeting Human BCMA Antigen

[0058] (1) Preparation of chimeric antigen receptor targeting human BCMA antigen

[0059] Synthesize chimeric antigen receptor sequences containing single-chain antibody ScFv against human BCMA antigen, hFc hinge region, transmembrane region and intracellular signal segment. Its structure is as figure 1 shown. Wherein the humanized single-chain antibody nucleotide sequence of the anti-human BCMA antigen is shown in SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22 or SEQ ID NO: 23 or SEQ ID NO: 24; hinge region The nucleotide sequence is the DNA sequence encoding amino acid SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 19; the transmembrane region is derived from CD8 or CD28 sequence; the intracellular signal segment source In CD28 or CD137 and CD3 sequence.

[0060] (2) Construction of lentiviral vectors expressing chimeric antigen receptors

[006...

Embodiment 2B

[0067] Example 2 Preparation of Chimeric Antigen Receptor Modified T Cells for BCMA Antigen

[0068] (1) Lentivirus infection of T cells

[0069] 1) Isolation of human peripheral blood mononuclear cells

[0070] Lymphocytes were separated by gradient centrifugation; after centrifugation, the second white lymphocyte layer was taken, washed with physiological saline, and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human peripheral blood mononuclear cells.

[0071] 2) Lentiviral vector infects T lymphocytes

[0072] After the obtained PBMC cells were activated by anti-CD3 and CD28 monoclonal antibodies, they were infected with lentivirus; after infection, they were cultured for about 10 days to verify the biological characteristics of CAR-T cells in vitro and in vivo. The chimeric antigen receptor T cells obtained are named after the corresponding lentiviral vector.

[0073] 3) Expression detection of chimeric antigen receptor (CAR) targeting human BCMA ...

Embodiment 3

[0076] Example 3. Verification of the anti-tumor effect of T lymphocytes expressing chimeric antigen receptors targeting BCMA

[0077] BCMA-positive H929 cells stably expressing firefly luciferase (referred to as H929-luc), negative cells K562-Luc, and K562-BCMA-Luc cells with high BCMA expression constructed in vitro by K562 were used as target cells. The expression of BCMA in target cells was detected as follows: figure 2 and image 3 As shown, H929 was detected as BCMA high expression positive cells by flow cytometry and RT method, and K562 was BCMA negative cells.

[0078] Because there are too many CARs, the effectiveness verification is divided into 3 groups; the method in Example 2 is used to prepare CAR-T cells by using 3 batches of buffy coat to prepare CAR-T cells. The buffy coat is a source of peripheral blood mononuclear cells from healthy donors. The first batch of CAR-T cells was plated with effector cells according to the effector-target ratio of 16:1 / 8:1 / 4:1 / ...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to an anti-BCMA antigen CAR and an expression vector, a CAR cell and application thereof. The CAR comprises an antigen recognition region capable of recognizing a BCMA antigen, a hinge region, a transmembrane region and an intracellular signal domain. The antigen recognition region for recognizing the BCMA antigen is an anti-BCMA single-chain antibody, wherein the heavy-chain amino acid sequence of the antigen recognition region is shown as any one of SEQ ID NO: 1-5, and the light-chain amino acid sequence of the antigen recognition region is shown as any one of SEQ ID NO: 6-10. The CAR can be stably expressed in patient-derived T lymphocytes, has better capability of removing tumor cells, and can beused for adoptive cell therapy aiming at hematological malignancies in preparation of drugs for treating hematological malignancies. The anti-BCMA antigen CAR cell not only can effectively remove tumor target cells expressing the BCMA antigen, but also has no toxic effect on negative antigen, namely tumor cells not expressing the BCMA, so that the safety is very high.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to an anti-BCMA antigen CAR and its expression vector, CAR cells and applications. Background technique [0002] The full name of BCMA is tumor necrosis factor receptor superfamily member 17, also known as B-cell maturation antigen (BCMA) or CD269 or TNFRSF13A. It is mainly expressed on the surface of mature B lymphocytes and plasma cells, and is highly expressed in MM tissues and cells. There are many suitable targets for blood diseases, and myeloma (MM) is one of the malignant blood diseases. [0003] Multiple myeloma, also known as multiple myeloma, abbreviated as MM, is highly heterogeneous and drug-resistant. MM has a high probability of recurrence. Currently, it cannot be cured. Existing drugs are far from being able to solve the survival crisis of MM patients. However, CAR-T therapy targeting CD19 has a better effect than traditional drugs in hematolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00A61P35/02
CPCC07K16/2878C07K14/7051C12N15/86A61K39/001117A61P35/00A61P35/02C07K2319/03C07K2319/33C12N2740/15043A61K2039/804
Inventor 单娟娟张巍赵文旭徐艳敏陈军赵永春黄霞张茜真
Owner CHONGQING PRECISION BIOTECH CO LTD
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