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A novel chimeric antigen receptor (CAR) targeting CD19 and its application

A chimeric antigen receptor and targeting technology, applied in the direction of DNA / RNA fragments, application, recombinant DNA technology, etc., can solve the problems of nucleic acid construction, nucleic acid introduction, foreign gene expression difficulties, etc., achieve good application prospects, improve the transformation Effect of improving dyeing efficiency and reducing difficulty of operation

Active Publication Date: 2020-06-23
浙江玉安康瑞生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The antigen-binding domain is used to recognize target tumor cells, usually using the scFv region of the antibody targeting the tumor antigen, which includes the light chain variable region of the antibody (light chain variable, V L ) and heavy chain variable region (heavy chain variable, V H ), and a Linker is used between the two. Although the molecular weight of the scFv region is significantly smaller than that of the complete antibody structure, which is beneficial to the recombination construction of the CAR gene to a certain extent, its nucleotide length is still relatively large in molecular biology. It brings certain difficulties to subsequent nucleic acid construction, nucleic acid introduction, and exogenous gene expression, so it is still necessary to further simplify this region in order to reduce the difficulty of molecular biology operations, improve the success rate of transfection and reduce production costs

Method used

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  • A novel chimeric antigen receptor (CAR) targeting CD19 and its application
  • A novel chimeric antigen receptor (CAR) targeting CD19 and its application
  • A novel chimeric antigen receptor (CAR) targeting CD19 and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0039] Example 1 Determination and Synthesis of Chimeric Antigen Receptor Gene Sequence

[0040] 1.1 Identification of the nanobody gene targeting CD19.

[0041] Human CD19 protein was used to immunize alpaca, and the anti-human CD19 nanobody was obtained through screening by phage display technology. Specifically: select healthy adult dromedary camels, separate and purify human CD19 extracellular domain protein expressed by yeast to obtain human CD19 extracellular domain protein. Mix 15 μg / Kg of human CD19 ectodomain protein with Freund's adjuvant at a ratio of 1:1, and immunize by multi-point subcutaneous injection on the back for a total of 6-8 times with an interval of 4 weeks. Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for the remaining immunizations. After the immunization, 100 mL of peripheral blood from the dromedary camel neck was collected to construct a phage display library. Positive clones were scre...

Embodiment 2

[0048] Example 2 Preparation of plasmid vector carrying chimeric antigen receptor gene

[0049] 2.1 Digestion of the target product and linearization of the plasmid vector

[0050] The CAR01 and CAR02 gene sequences were amplified and obtained by PCR, and Xba I and Not I restriction sites were added at both ends of the sequences. The target gene fragment and the lentiviral vector plasmid pCDH-CMV-MCS-EF1-GFP-T2A-Puro (purchased from Addgene) were subjected to Xba I and Not I double enzyme digestion reaction. The enzyme digestion reaction conditions were: 37°C, enzyme digestion 30min. The 50μL enzyme digestion system is as follows:

[0051] 10×buffer: 5 μL;

[0052] DNA5μg;

[0053] Xba I enzyme: 2 μL;

[0054] Not I enzyme: 2 μL;

[0055] Make up volume with deionized water.

[0056] 2.2 Recovery of digested products

[0057] The CAR01, CAR02 DNA fragments and pCDH-CMV-MCS-EF1-GFP-T2A-Puro DNA fragments were electrophoresed on 1.5% agarose gel respectively, and the agaro...

Embodiment 3

[0084] Packaging, concentration and titer determination of embodiment 3 lentivirus

[0085] 3.1 Extraction of lentiviral vector plasmid

[0086] 1) Inoculate 50mL of Amp-resistant liquid LB culture medium with the original bacterial solution with correct sequencing, and cultivate overnight at 37°C with a shaker at 200rpm.

[0087] 2) Centrifuge at 4000rpm for 2min, discard the supernatant, and collect the bacteria.

[0088] 3) The plasmid was extracted using Takara MiniBEST Plasmid Purification KitVer 4.0 (purchased from Takara Company) plasmid extraction kit, and the specific method was carried out according to the instructions.

[0089] 3.2 Packaging of lentiviral vector

[0090] 1) 293T cells were treated with 1×10 6 The amount of Cells / well was seeded in a 6-well cell culture plate at 37°C, 5% CO 2 Culture under the conditions, and proceed to the next step when the confluence of the cells reaches 80%.

[0091] 2) Gently suck off the cell culture supernatant in the 6-w...

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Abstract

The invention belongs to the technical field of biopharming, and specifically relates to a novel chimeric antigen receptor (CAR) targeting CD19 and applications thereof. The chimeric antigen receptorincludes signal peptide, nanoantibodies targeting CD19, a transmembrane region, costimulatory factors and an intracellular signal domain; and the intracellular signal domain is the mutant CD3[zeta] intracellular signal domain. Through the using of the nanoantibodies targeting CD19 and the mutant CD3[zeta] intracellular signal domain, the molecular weight of the chimeric antigen receptor can be reduced, convenient nucleic acid transfection and molecular biology operation can be achieved, antineoplastic activity can be enhanced, stronger tumor killing effects can be achieved, and therefore, novel approaches can be provided for the development of a novel tumor immunotherapy; and the chimeric antigen receptor can be used in CAR-T therapy, and shows good application prospects.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a novel chimeric antigen receptor (CAR) targeting CD19 and its application. Background technique [0002] Malignant tumors are one of the diseases that seriously threaten human health. According to incomplete statistics, the annual incidence of malignant tumors in China has reached more than 4 million cases, among which the incidence of lymphoma, leukemia, multiple myeloma and other hematological tumors remains high. , and studies in recent years have shown that the incidence of hematological malignancies continues to increase, and the age of onset has also advanced. Increased pollution, unhealthy living habits and other factors are related. However, there is still a lack of effective treatment methods for hematological malignancies at present, and traditional chemotherapy, radiotherapy and surgery are difficult to produce satisfactory therapeutic effects. Major de...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00A61P35/02
Inventor 王振李静
Owner 浙江玉安康瑞生物科技有限公司
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