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Technology of detecting CART (chimeric antigen receptor-T) cell expression in vivo and application of technology

An in vivo detection and cell technology, applied in recombinant DNA technology, DNA/RNA fragments, microbial determination/inspection, etc., to achieve the effect of increasing accuracy and improving accuracy

Inactive Publication Date: 2018-07-17
HRAIN BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the copy number of CAR in the genome is related to carrier characteristics, virus titer, cell state, etc., which will further lead to errors in estimating CAR cells by detecting the CAR content in the peripheral blood genome

Method used

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  • Technology of detecting CART (chimeric antigen receptor-T) cell expression in vivo and application of technology
  • Technology of detecting CART (chimeric antigen receptor-T) cell expression in vivo and application of technology
  • Technology of detecting CART (chimeric antigen receptor-T) cell expression in vivo and application of technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Extraction of genomic DNA from peripheral blood mononuclear cells (PBMC)

[0024] 1. Handling materials

[0025] Cells should be processed into cell suspension first, then centrifuged at 10,000rpm (~11,200×g) for 1min, the supernatant is decanted, 200μl buffer GA is added, and shaken to complete suspension;

[0026] 2. Add 20μl Proteinase K (Tiangen) solution and mix well.

[0027] 3. Add 200μl buffer GB (Tiangen), fully invert and mix well, place at 70°C for 10min, the solution should be clear, centrifuge briefly to remove the water droplets on the inner wall of the tube cap.

[0028] 4. Add 200μl of absolute ethanol (Sinopharm Group), shake and mix well for 15sec. At this time, flocculent precipitation may appear. Centrifuge briefly to remove water droplets on the inner wall of the cap.

[0029] 5. Put the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3 (the adsorption column is placed in the collection tube) (Tiangen), c...

Embodiment 2

[0036] Example 2 Preparation of Plasmids and Cell Standards

[0037] 1. Extract the CD19CAR-CD28 plasmid, and use a spectrophotometer to determine the concentration and purity of the extracted plasmid DNA (the ratio of OD260 / OD280 is about 1.8).

[0038] 2. According to the formula of plasmid copy number calculation: (6.02×10 23 )×(ng / ul×10 -9 ) / (DNA length×660)=copies / ul. Calculate the number of copies in the plasmid DNA of the corresponding quality.

[0039] Standard 1: CD19CAR plasmid from 1X10 6 Dilute 6 points to 320copies and add them to 40ng PBMC cell genomic DNA. QPCR detects CD19CAR.

[0040] Group

1

2

3

4

5

6

Plasmid

1.00E+06

2.00E+05

4.00E+04

8.00E+03

1.60E+03

3.20E+02

DNA

40ng

40ng

40ng

40ng

40ng

40ng

[0041] Standard 2: CD19CART cells whose CAR expression rate has been measured by flow cytometry, diluted 1:4 into untransfected PBMC cells, extract genomic DNA, and take 40ng DNA QPCR to detect CD19CAR( figure 1 ).

[0042] Standard 3: Dilute the genome of PBMC ...

Embodiment 3

[0044] Example 3 QPCR detection

[0045] 1. Primer design: Design CD19CAR and internal reference calibration gene CDKN1A QPCR primers as follows:

[0046] Types of

Name

Sequence

Upstream primer sequence

CD19CAR

GGGTTCCAAGCCGATTCAGT

Downstream primer sequence

CD19CAR

CAAGTTGCTAATGGTCAGGGAATA

Upstream primer sequence

CDKN1A

GAAAGCTGACTGCCCCTATTTG

Downstream primer sequence

CDKN1A

GAGAGGAAGTGCTGGGAACAAT

[0047] 2. Prepare the PCR reaction solution according to the following components (please prepare the reaction solution on ice):

[0048] Reagent

Usage amount

PCR upstream primer (10μM)

0.75μl

PCR downstream primer (10μM)

0.75μl

ROX Reference Dye II(50×)

0.25μl

DNA template

2.0μl

dH2O (sterilized distilled water)

21.175μl

Total

25.0μl

[0049] 3. Computer program:

[0050] 7500 quantitative PCR instrument absolute quantification (ABI) experimental amplification conditions are as follows

[0051]

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Abstract

The invention discloses a detection method of in-vivo expression efficiency of a CART (chimeric antigen receptor-T) cell. The detection method comprises the following steps of: adding plasmid expressing CD19CAR into an untransfected PBMC (peripheral blood mononuclear cell) to extract genome as a standard I, adding the CART cell with the known expression efficiency into the untransfected PBMC to extract genome as a standard II, using gradient dilution PBMC genome to measure internal reference CDKN1A as a standard III, applying the three standards in combination to create a standard curve, and detecting a virus copy number of the CART cell integrated into full genome DNA (deoxyribonucleic acid) and a percentage content of the CART cell in the PBMC by using a fluorescent quantitative PCR (polymerase chain reaction) technology after a linear relationship is shown. According to the technology, an in-vivo expression situation of the CART cell can be calculated; a calculation method is simpleand convenient; and the technology is high in precision and convenient in practical operation.

Description

Technical field [0001] The invention belongs to the field of chimeric antigen receptors, and specifically uses fluorescent quantitative PCR and flow cytometry methods to detect the expression of CART cells in vivo. Background technique [0002] Chimeric Antigen Receptor-T cell (CART) T cells are T cells that have been genetically modified to recognize specific target antigens in an MHC non-restrictive manner and continue to activate expansion. In the 2012 International Cell Therapy Association Annual Meeting, it was pointed out that biological immune cell therapy has become the fourth method of cancer treatment besides surgery, radiotherapy, and chemotherapy, and will become a necessary method for cancer treatment in the future. CART cell reinfusion therapy is the most clear and effective form of immunotherapy in current tumor treatment. A large number of studies have shown that CART cells can effectively recognize tumor antigens, cause specific anti-tumor immune responses, and ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q2545/114C12Q2537/16C12Q2561/113
Inventor 黄飞金涛王海鹰何凤史子啸
Owner HRAIN BIOTECHNOLOGY CO LTD