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T cell capable of specifically recognizing NY-ESO-1, as well as application of combination thereof with cell factor

A specific, cellular technology, applied in the fields of immunology and medicine, can solve problems such as difficult to effectively reach tumor tissue

Inactive Publication Date: 2018-01-09
SHENZHEN BEIKE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing studies have shown that mesenchymal stem cells can specifically chemotaxis to tumor tissue, while traditional drug delivery methods are often difficult to effectively reach the interior of tumor tissue due to the disorder of tumor tissue

Method used

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  • T cell capable of specifically recognizing NY-ESO-1, as well as application of combination thereof with cell factor
  • T cell capable of specifically recognizing NY-ESO-1, as well as application of combination thereof with cell factor
  • T cell capable of specifically recognizing NY-ESO-1, as well as application of combination thereof with cell factor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1 Construction of recombinant expression vector pGreen puro-TCR (NY-ESO-1) and pGreen puro-TCR (NY-ESO-1)-IL2

[0032] The recombinant expression vectors pGreen puro-TCR(NY-ESO-1) and pGreen puro-TCR(NY-ESO-1)-IL2 were constructed.

[0033] The structure of the insert fragment of the vector is as follows figure 1 shown.

[0034] 1. pGreen puro-TCR (NY-ESO-1)

[0035] The insert of pGreen puro-TCR (NY-ESO-1) includes the first nucleic acid fragment encoding the alpha chain of TCR and the second nucleic acid fragment encoding the beta chain of TCR; the first nucleic acid fragment and the second nucleic acid fragment are connected by T2A sequence .

[0036] The build process includes:

[0037] A nucleic acid fragment encoding NY-ESO-1TCRα-T2A-NY-ESO-1TCRβ (referred to as the TCR (NY-ESO-1) sequence) was synthesized consisting of a first nucleic acid comprising the α chain encoding NY-ESO-1-TCR Fragment (base sequence as shown in SEQ ID No.1), including the ...

Embodiment 2

[0044] Example 2 Construction of recombinant expression vector pGreen puro-LIGHT

[0045] The insert of pGreen puro-LIGHT includes a fourth nucleic acid segment encoding LIGHT.

[0046] The build process includes:

[0047] A nucleic acid fragment encoding LIGHT (referred to as a LIGHT sequence) is synthesized. The base sequence of the LIGHT sequence is shown in SEQID No.5.

[0048] EcoR I and BamH I restriction endonuclease site sequences were added at both ends of the nucleic acid fragment of the LIGHT sequence. The nucleic acid fragment was double-digested with the lentiviral core vector pGreen puro with restriction endonucleases EcoR I and BamH I, and the digested products were gel-recovered and ligated with T4 ligase overnight at 16°C. After ligation, transform Escherichia coli HB2151 competent, smear the plate, pick out the monoclonal double enzyme digestion and sequence identification the next day, and obtain the recombinant expression vector pGreen puro-LIGHT.

Embodiment 3

[0049] The packaging of embodiment 3 lentiviruses

[0050] The resulting recombinant expression vectors pGreen puro-TCR (NY-ESO-1) or pGreen puro-TCR (NY-ESO-1)-IL2 or pGreen puro-LIGHT were combined with lentiviral backbone plasmids pMDLg PRRE, pRSV-Rev and pMD2 .Equal amount of G (Addgene) was transfected into Hek293T cells (ATCC, product number CRL-3216) through Lipofectamine 2000 liposomes (Life, Invitrogen, product number 11668-019) for lentivirus packaging.

[0051]Follow the instructions of the supplier of lentiviral transformation reagents. The specific steps include: culture 293T cells with 1640 medium containing 10% FBS; 5 / cm 2 Transfer the density to a culture dish with a diameter of 15cm and cultivate for 20h to ensure that the cell confluence is 80-90% during transfection; replace the medium with 1640 medium without serum and set aside; take two EP tubes and put them in the EP tubes respectively Add 1ml 1640, the lentiviral vector pGreen puro-TCR (NY-ESO-1), p...

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Abstract

The invention provides a T cell. The T cell comprises nucleic acid for encoding a TCR (T cell receptor) capable of specifically recognizing NY-ESO-1, expresses the TCR capable of specifically recognizing the NY-ESO-1, and meanwhile, can also express a T cell of IL-2 (interleukin 2). The invention also provides the T cell cultured together with a mesenchymal stem cell for expressing LIGHT. The invention further provides a medicinal composition containing the T cell. The invention further provides use of the T cell in preparation of a medicament for preventing or treating cancers.

Description

technical field [0001] The present invention relates to the fields of immunology and medicine. Specifically, the present invention provides T cells expressing a T cell receptor (TCR) that specifically recognizes NY-ESO-1. The present invention also provides the use of the combination of T cells and cytokines in treating cancer or preparing medicines for treating cancer. Background technique [0002] In recent years, tumor immunotherapy has developed rapidly, but the overall effect is still not good. The reason may be related to tumor immune escape through various mechanisms. For example, tumor cells express the same antigens as normal tissue cells to evade the recognition of the body's immune system; secrete immunosuppressive factors such as TGF-β and IL-10 to inhibit the function of immune active cells; down-regulate TNF-α receptors, Fas Anti-tumor effects of cytokines and cytotoxic T lymphocytes (cytotoxic lymphocytes, CTLs), and down-regulation of HLA through loss of t...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867A61K35/17A61K35/28A61P35/00
Inventor 刘沐芸胡祥刘未斌李琼书刘曲波
Owner SHENZHEN BEIKE BIOTECH