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Quick construction method of CAR-T (chimeric antigen receptor T-cells) toxicity indictor cells

A technology for constructing methods and cells, applied in chemical instruments and methods, reverse transcription RNA viruses, botanical equipment and methods, etc., can solve the problems of large workload, low success rate, and long time-consuming indicator cells, so as to reduce costs, The effect of omitting preparation steps and saving preparation time

Active Publication Date: 2019-08-16
山东昂科诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] However, no matter which method is used, it must first go through a series of tedious steps such as preparing lentivirus, infecting cells, and screening stable cell lines; secondly, for different CAR-T cells, it is necessary to prepare a series of corresponding indicator cells. The entire construction process of indicator cells has problems such as long time-consuming, huge workload, and low success rate; finally, the detection of multi-target CAR-T cytotoxicity requires indicator cells to express multiple tumor antigens at the same time, which also means that indicator cells Build difficulty increases

Method used

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  • Quick construction method of CAR-T (chimeric antigen receptor T-cells) toxicity indictor cells
  • Quick construction method of CAR-T (chimeric antigen receptor T-cells) toxicity indictor cells
  • Quick construction method of CAR-T (chimeric antigen receptor T-cells) toxicity indictor cells

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Experimental program
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Effect test

Embodiment 1

[0051] 1. Construction of lentiviral plasmid

[0052] 1. Whole gene synthesis figure 1 The sequence of the gene fragment shown is SED NO: 1, which contains IgK leader, avidin, CD28 transmembrane region, T2A sequence and Luciferase derived from Firefly, and the upstream and downstream restriction sites are designed to be XhoI and BamHI respectively;

[0053] 2. Digest the above gene fragment and pLVX-IRES-Puro plasmid with XhoI and BamHI; cut the gel and recover, and connect with conventional molecular biology methods;

[0054] 3. Transform the ligation product into Stbl3 competent cells, and spread the LB medium plate (ampicillin resistance)

[0055] 4. Pick a single clone and culture it in LB medium for 8-12 hours;

[0056] 5. Small plasmid extraction, double enzyme digestion with XhoI and BamHI, combined with gel electrophoresis to verify whether the band size is correct, and further plasmid sequencing to verify the correctness with universal primers; the universal primers...

Embodiment 2

[0084] Construction of K562 cells carrying CD19 antigen

[0085] 1. Cultivate the K562-avidin-Luc cells constructed in step 3 in a 12-well plate so that they are in the logarithmic growth phase;

[0086] 2. See SEQ NO:2 for the amino acid sequence of the ectodomain of the CD19 antigen protein. This sequence can be entrusted to the company for exogenous expression. For easy purification, a histidine tag is added at the end of the sequence. After protein purification, entrust the company to carry out biotinylation treatment. Alternatively, the biotinylated CD19 ectodomain protein is commercially available and can be purchased, such as from ACROBiosystems.

[0087] 3. Take 1×10 6 To each K562-avidin-Luc cell, add the biotinylated CD19 antigen ectodomain protein obtained above to a final concentration of 10 μg / ml, and incubate at 4°C for 30 minutes;

[0088] 4. Centrifuge to remove the supernatant, resuspend with IMDM complete medium (containing 10% FBS), repeat this step once,...

Embodiment 3

[0090] Example 3 Construction of K562 cells carrying CD19 and CD30 antigens

[0091] 1. Cultivate the K562-avidin-Luc cells constructed in step 3 in a 12-well plate so that they are in the logarithmic growth phase;

[0092] 2. Prepare biotinylated CD19 and CD30 extracellular domain proteins;

[0093] 3. Take 1×10 6 K562-avidin-Luc cells, at the same time, add the biotinylated CD19 and CD30 ectodomain proteins to a final concentration of 10 μg / ml, and incubate at 4°C for 60 minutes;

[0094] 4. Centrifuge to remove the supernatant, resuspend with IMDM complete medium (containing 10% FBS), repeat this step once, and obtain K562 cells carrying CD19 and CD30 antigens.

[0095] The obtained K562 cells carrying CD19 and CD30 antigens stably express Luciferase and combine the extracellular domains of CD19 and CD30 antigens, which can be used as indicator cells for detecting the toxicity of CAR-T cells targeting both CD19 and CD30.

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Abstract

The invention discloses a quick construction method of CAR-T (chimeric antigen receptor T-cells) toxicity indictor cells, which comprises the steps of first, constructing a lentiviral plasmid having asequence shown as SEQ NO: 1; second, performing large-scale extraction on the lentiviral plasmid, and performing virus packaging to prepare lentivirus; third, infecting the lentivirus with common cells X; fourth, adding one or more biotin-processed proteins A into universal cells U, and tightly combining with avidin of membranes of the universal cells U to obtain CAR-T toxicity indicator cells. The quick construction method of CAR-T toxicity indictor cells has the advantages that by adding the different biotin-processed proteins A or their combination, the cells U with Luciferase fragments can be converted quickly into various indicator cells suitable for CAR-T toxicity detection, the complex preparation steps are omitted greatly, preparation time is saved, and the cost is reduced.

Description

technical field [0001] The invention relates to the technical field of cellular immunotherapy, in particular to a method for rapidly constructing CAR-T toxicity indicator cells. Background technique [0002] Chimeric antigen receptor T cell therapy CAR-T has great application prospects and value in the field of tumor treatment. CAR-T has become a promising candidate in the field of tumor treatment due to its high treatment accuracy, definite clinical efficacy and good safety. research direction. [0003] In medical research and clinical application, the determination of CAR-T cytotoxicity is an unavoidable and important issue, and CAR-T cytotoxicity is also one of the important criteria for measuring the quality of CAR-T products. The currently commonly used method for in vitro detection of cytotoxicity is to mix CAR-T cells with target cells, and after a period of culture, use flow cytometry antibody labeling and then use flow cytometry to measure. This method faces probl...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/62
CPCC07K14/70503C07K2319/00C07K2319/02C07K2319/03C07K2319/60C12N15/86C12N2740/15043C12N2800/107
Inventor 唐东起张文
Owner 山东昂科诺生物科技有限公司
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