Preparation method of Mesothelin chimeric antigen receptor modified T cell with targeted high-intensity tumor killing activity

A chimeric antigen receptor, high-strength technology, applied in the field of biomedicine, can solve problems such as adverse reactions, immaturity, and failure to achieve therapeutic effects, and achieve high proliferative activity and good safety.

Pending Publication Date: 2022-02-08
YANAN HOSPITAL OF KUNMING CITY
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Problems solved by technology

However, the research of this technology in solid tumors is not yet mature enough to achieve the desired therapeutic effect, and still faces many challenges, including: the special pathological characte...

Method used

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  • Preparation method of Mesothelin chimeric antigen receptor modified T cell with targeted high-intensity tumor killing activity
  • Preparation method of Mesothelin chimeric antigen receptor modified T cell with targeted high-intensity tumor killing activity
  • Preparation method of Mesothelin chimeric antigen receptor modified T cell with targeted high-intensity tumor killing activity

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Embodiment Construction

[0039] The present invention will be further described in detail below in conjunction with the examples.

[0040] Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be considered as limiting the scope of the present invention. If no specific technique or condition is indicated in the examples, it shall be carried out according to the technique or condition described in the literature in this field or according to the product specification. The materials or equipment used are not indicated by the manufacturer, and they are all conventional products that can be obtained through purchase.

[0041] Ampicillin stock solution, Solarbio, Cat. No. A1170.

[0042] Puromycin, Thermo Fisher Scientific, Cat. No. A1113803.

[0043] The MSLN-CAR-puro plasmid was constructed by ShanghaiTech University. (It is inconvenient for us to count this sequence, so we will not write it.)

[0044] Preparation of LB sol...

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Abstract

The invention relates to a preparation method of Mesothelin chimeric antigen receptor modified T cells with targeted high-intensity tumor killing activity, which comprises the following steps: transforming MSLN-CAR-puro plasmids into escherichia coli competent cells, conducting coating, screening and culturing on a solid culture medium, and extracting plasmids after propagating a grown single colony; preparing human peripheral blood mononuclear lymphocytes (PBMCs); and extracting plasmids, mixing the extracted plasmids, Super PiggyBac Transspoase plasmids and an electrotransfection buffer solution, transfecting PBMC (peripheral blood mononuclear cells), conducting culturing by using an X-VIVO culture medium, activating and amplifying CD3 positive T cells by using CD3/CD28 magnetic beads, and screening and amplifying the transfected T cells. The mesothelin-targeted MSLN-CAR-T cell is constructed through a PiggyBac transposon system, so that the mesothelin-targeted MSLN-CAR-T cell has targeted killing performance on solid tumors and is good in safety, and a research basis is provided for related clinical transformation.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a preparation method of T cells modified by Mesothelin chimeric antigen receptors with targeted high-intensity tumor-killing activity. Background technique [0002] As one of the diseases that seriously threaten human health, cancer not only harms patients physically and mentally, but also brings huge economic burdens to families, society and the country. At present, common clinical treatment methods for malignant tumors include surgery, radiotherapy and chemotherapy, and targeted therapy, but such treatments cannot effectively control the progression and recurrence of some malignant tumors, nor can they effectively prolong the survival of such patients and improve their quality of life. Therefore, there is an urgent need to find new treatments. [0003] In recent years, immunotherapy has become an important means of tumor treatment, bringing hope to overcome ma...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C12N15/62A61K39/00A61P35/00
CPCC12N15/85C12N5/0636C07K16/30C07K16/28C07K14/7051A61K39/001111A61P35/00C12N2510/00C12N2800/107C12N2501/2302C12N2501/2321C07K2319/33A61K2039/5156A61K2039/86A61K2039/828
Inventor 谭晶李汝红王文举黄文文段德仁林杼颖侯宗柳孟明耀李琳
Owner YANAN HOSPITAL OF KUNMING CITY
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