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CD19-based chimeric antigen receptor and application thereof

A technology of chimeric antigen receptors and antigens, applied in the fields of application, antibody medical components, antibody mimics/stents, etc., can solve the problems of large side effects, high toxicity of immune factor storms, etc., and achieve high expression levels and enhanced immune effects Effect

Pending Publication Date: 2018-08-10
BEIJING MEIKANG JIMIAN BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the chimeric antigen receptor immune factor storm based on CD19 in the prior art has relatively high toxicity and side effects

Method used

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  • CD19-based chimeric antigen receptor and application thereof
  • CD19-based chimeric antigen receptor and application thereof
  • CD19-based chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of Chimeric Antigen Receptor

[0062] (1) Synthesize Secretory signal peptide, CD19 antigen binding domain, CD8α and / or CD28 transmembrane domain, CD28 signaling domain, CD27 signaling domain, CD3ζ signaling domain, 2A sequence and cysteine ​​through whole gene synthesis Protease 9 domains, such as figure 1 Shown, namely Secretory-CD19-CD28-CD27-CD3ζ-2A-iCasp9;

Embodiment 2

[0063] Example 2 lentiviral packaging

[0064] (1) Use 293T cells and culture for 17-18 hours;

[0065] (2) Add fresh DMEM containing 10% FBS;

[0066] (3) Add the following reagents to a sterile centrifuge tube: Take DMEM from each well, add helper DNA mix (pNHP, pHEF-VSV-G) and pTYF DNA vector, and vortex;

[0067] (4) Add Superfect or any transgenic material to the centrifuge tube, and let stand at room temperature for 7-10 minutes;

[0068] (5) Add the DNA-Superfect mixture in the centrifuge tube to each cultured cell drop by drop, and vortex to mix well;

[0069] (6) 37°C 3% CO 2 Cultivate in the incubator for 4-5 hours;

[0070] (7) Suck away the culture fluid of the culture medium, wash the culture medium with 293 cell culture fluid, and add the culture fluid to continue culturing;

[0071] (8) Return the culture medium to 3% CO 2 Culture overnight in an incubator, and observe the transfection efficiency with a fluorescence microscope the next morning.

Embodiment 3

[0072] Example 3 Purification and Concentration of Lentivirus

[0073] 1) Virus purification

[0074] Remove cell debris by centrifugation at 1000g for 5 minutes to obtain virus supernatant, filter the virus supernatant with a 0.45 micron low protein binding filter, divide the virus into aliquots, and store at -80°C;

[0075] Typically, transfected cells can produce 10 6 to 10 7 Lentiviral vectors for titration of transducing units.

[0076] 2) Concentrate lentiviral vectors with Centricon filters

[0077] (1) Add virus supernatant to Centricon filter tube, then centrifuge at 2500g for 30 minutes;

[0078] (2) Shake the filter tube, then centrifuge at 400g for 2 minutes, and collect the concentrated virus into the collection cup. Finally, pool the virus in all tubes into one centrifuge tube.

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Abstract

The invention relates to a CD19-based chimeric antigen receptor and application thereof, in particular to a lentivirus vector material built by a chimeric antigen receptor T (CAR-T) cell technology using a tumor specific target point CD19 as the basis, a method, and application thereof to anti-tumor treatment. The chimeric antigen receptor is formed by serially connecting an antigen combination structure domain, a membrane spaning structure domain, a costimulatory signal conduction region, a CD3 zeta signal conduction structure domain and an inducible suicide fusion structure domain, wherein the antigen combination structure domain is combined with the tumor surface antigen; the tumor surface antigen is CD19. The chimeric antigen receptor is subjected to specific gene transformation on theT cell stimulation signals. Compared with other chimeric antigen receptors, the chimeric antigen receptor provided by the invention has a better reaction effect and higher safety, so that the CAR-T cells have higher immune effects and low side effects; the treatment effect and safety of the CAR-T cells are enhanced.

Description

technical field [0001] The present invention relates to the field of tumor cell immunotherapy, in particular to a CD19-based chimeric antigen receptor and its application, specifically the chimeric antigen receptor T (CAR-T) cell technology based on tumor-specific target CD19 The construction method and its application in anti-tumor therapy. Background technique [0002] B lymphocyte malignancies, including acute lymphocytic leukemia (B cell acute lymphocyticleukemia, B-ALL), B cell lymphoma (B cell lymphoma) and chronic lymphocytic leukemia (chronic lymphocytic leukemia, CLL)), regardless of adult Or children, are a large number of blood diseases. One of the traditional approaches to treating these patients has been the use of chemotherapy, radiotherapy, stem cell transplantation, small molecule drugs, and antibody drugs. Although these therapies can cure some patients, many people still die due to the malignant reaction to chemotherapy and radiotherapy, drug resistance, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/867C12N5/10A61K35/17A61P35/00A61P35/02
CPCA61P35/00A61P35/02A61K35/17C12N5/0636C07K14/7051C07K14/70578C07K16/2803C07K2319/03C07K2319/02A61K2039/5156C07K2317/622C07K2317/73C07K2319/33C07K14/70521C07K2319/036C12N9/6472A61K39/001112A61K2039/5158A61K2039/804C12N2510/00C12N2501/515C12N2501/599A61K38/00C07K16/1045C07K16/3061
Inventor 章睿
Owner BEIJING MEIKANG JIMIAN BIOTECH CO LTD
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