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BCMA-based (B cell maturation antigen-based) chimeric antigen receptor and preparation method and application thereof

A chimeric antigen receptor and antibody technology, which is applied in the fields of biochemical equipment and methods, antibody medical components, chemical instruments and methods, etc., can solve the problem that the killing effect of tumor cells is not long-lasting, the killing power cannot be exerted, and the tumor cells cannot be fully exerted. Killing effect and other issues, to achieve the effect of good tumor targeting, strong killing ability, and good therapeutic effect

Inactive Publication Date: 2016-08-10
李斯文 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the lack of co-stimulatory signals for T cell activation, the first-generation CAR T cells have basically no killing effect on tumor cells; while the second-generation and third-generation CAR T cells have obvious killing effects on tumor cells, but due to the CD3 activation signal It causes the apoptosis of T cells, which makes T cells apoptotic in a short time and cannot exert lasting lethality. The killing effect of the second-generation and third-generation CAR T cells on tumor cells is not durable. On the other hand, due to the first The combination of the second and third generation CAR T cells with the antigen and the signal transmitted after the combination are relatively weak, and the second and third generation CAR T cells cannot fully exert their killing effect on tumor cells

Method used

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  • BCMA-based (B cell maturation antigen-based) chimeric antigen receptor and preparation method and application thereof
  • BCMA-based (B cell maturation antigen-based) chimeric antigen receptor and preparation method and application thereof
  • BCMA-based (B cell maturation antigen-based) chimeric antigen receptor and preparation method and application thereof

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preparation example Construction

[0051] Specifically, in an embodiment of the present invention, the method for preparing the DNA sequence of the BCMA-based chimeric antigen receptor described in the present invention includes the following steps: extracting mRNA in PBMC, and obtaining cDNA through Vazyme reverse transcription kit; and Using the obtained cDNA as a template, primers were designed, and the guide sequence encoding CD8a (BamHI restriction site was introduced upstream), the hinge region and transmembrane region of CD8a (NheI restriction site was introduced upstream), and the intracellular signal of CD137 were respectively obtained by PCR. domain, the OX40 intracellular signaling domain, the DNA fragment of the CD3ζ intracellular signaling domain (the SalI restriction site is introduced downstream), the intracellular domain of CD3ζmut, and these fragments are combined by overlappingPCR to obtain CD8-CD28- CD3ζ, CD8-CD28-CD137-CD3, CD8-CD28-OX40-CD3ζ, the restriction site NheI / SalI was introduced int...

Embodiment 1

[0070] Example 1 Construction of recombinant vector pELPS-BCMAscFv-CD8-CD28-CD3ζ

[0071] Based on the vector pELPS-19-BB-ζ derived from the patent US_2015_0093822_A1, the restriction endonuclease BamHI / SalI was used for double digestion, and the promega gel recovery kit was used to recover large fragments. The specific method was carried out according to the instructions.

[0072] The Fab sequence of the BCMA antibody was derived from PDB: 4ZFO, using pUC-4ZFO-Fab-H and pUC-4ZFO-Fab-L (sequence synthesized by Genscript) as templates, designed primers, and obtained VH and VL by PCR. A linker was introduced to obtain BCMAscFv by overlappingPCR. The downstream of the BCMAscFv sequence was introduced with restriction site NheI.

[0073] The mRNA in PBMC was extracted, cDNA was obtained by Vazyme reverse transcription kit, and primers were designed using the obtained cDNA as a template, and the CD8a guide sequence (introduced upstream BamHI restriction site), CD8a transmembrane r...

Embodiment 2

[0077] Example 2 Construction of recombinant vector pELPS-BCMAscFv-CD8-CD28-CD137-CD3ζ

[0078] Based on the vector pELPS-19-BB-ζ derived from the patent US_2015_0093822_A1, the restriction endonuclease BamHI / SalI was used for double digestion, and the promega gel recovery kit was used to recover large fragments. The specific method was carried out according to the instructions.

[0079] The Fab sequence of the BCMA antibody was derived from PDB: 4ZFO, using pUC-4ZFO-Fab-H and pUC-4ZFO-Fab-L (sequence synthesized by Genscript) as templates, designed primers, and obtained VH and VL by PCR. A linker was introduced to obtain BCMAscFv by overlappingPCR. The downstream of the BCMAscFv sequence was introduced with restriction site NheI.

[0080] The mRNA in PBMC was extracted, cDNA was obtained by Vazyme reverse transcription kit, and primers were designed using the obtained cDNA as a template, and the CD8a guide sequence (introduced upstream BamHI restriction site), CD8a transmemb...

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Abstract

The invention provides a BCMA-based (B cell maturation antigen-based) chimeric antigen receptor, comprising following cis-form cascade domains: CD8a leader region, scFv fragment BCMA scFv of anti-BCMA antibody, CD8a hinge region and transmembrane region, CD28 intracellular signal domain and CD3 Zeta intracellular signal domain; the CD3 Zeta intracellular signal domain is wild CD3 Zetaintracellular signal domain or mutant CD3 Zeta mut intracellular signal domain; the BCMA-based chimeric antigen receptor can enhance anti-apoptotic capability of T-cells and enhance CAR T-cell and antigen bonding and signal conduction; T-cells with the BCMA-based chimeric antigen receptor show good tumor targeting performance in in-vivo experiments, tumors diminish significantly after two weeks of dosage, and the T-cells have excellent therapeutic effect in vivo.

Description

technical field [0001] The invention relates to the field of biological products, in particular to a BCMA-based antigen chimeric receptor and its preparation method and application. Background technique [0002] The main pathology of multiple myeloma (MM) is the unlimited expansion and enrichment of plasma cells in the bone marrow, leading to osteonecrosis. The current treatment for MM is mainly through antibodies targeting MM cell surface-associated antigens, and adoptive immunotherapy based on these antibodies. For example, although immunotherapy based on CD19 CART cells has achieved good curative effect in acute lymphoblastic leukemia, CD19 is also expressed on the surface of normal B lymphocytes, but rarely expressed in MM cells, so the effect on MM is very limited Satisfactory. [0003] BCMA (B-cell maturation antigen) is a B cell maturation antigen, a type III transmembrane protein composed of 185 amino acid residues, belonging to the TNF receptor superfamily, and it...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N5/10A61K35/17A61P35/00
CPCC07K14/70517A61K35/17A61K2039/5156A61K2039/5158C07K14/7051C07K14/70521C07K16/2878C07K16/3053C07K2317/51C07K2317/515C07K2317/622C07K2319/02C07K2319/03C07K2319/33C07K2319/74C12N5/0636C12N2510/00
Inventor 李斯文赵磊
Owner 李斯文
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