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Regulation and control method of expression in chimeric antigen receptor

A chimeric antigen receptor and expression system technology, applied in the field of biomedical transformation, can solve problems such as patient death and cytokine storm

Active Publication Date: 2017-08-29
胜武(北京)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] Although CAR-T targeting tumor cells is currently one of the most promising cancer treatment options, when too many signal transductions reach the threshold, the strong immune response in the body may cause severe diseases such as "cytokine storm" , and even lead to patient death

Method used

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  • Regulation and control method of expression in chimeric antigen receptor
  • Regulation and control method of expression in chimeric antigen receptor
  • Regulation and control method of expression in chimeric antigen receptor

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Example 1. Design of Expression Vectors for Unnatural Amino Acid-Dependent Recombinant Chimeric Antigen Receptors

[0052] The present invention selects the currently researched and most widely used CD19-CAR molecule as an example, uses the plasmid pUltra as the backbone, and constructs the lentiviral transfection plasmid of the non-natural amino acid-dependent recombinant chimeric antigen receptor as follows ( figure 1 ):

[0053] Lentiviral Transfer Plasmids: Lenti-RS / tRNA / CAR

[0054] 5'LTR(truncated)-ψ-RRE-UbC promoter-PylRS-2A-CD19CAR-(U6-tRNA-H1-tRNA)*2-3'LTR [△U3 (U6-tRNA-H1-tRNA)-R- U5]

[0055] Among them, the CAR molecule UAG—Signal peptide-scFv-hinge-transmembrane domain-4-1BB-CD3 zeta.

[0056] The plasmid construction was synthesized by Beijing Aoke Biotechnology Co., Ltd. and sequenced to confirm the correct sequence.

Embodiment 2

[0057] Embodiment 2, the packaging of lentiviral vector

[0058] Inoculate 5 × 10 in gelatin-precoated 15-cm dishes 6 293T cells at 37 °C, CO 2 Incubate overnight in an incubator. When the confluency reaches 70%, prepare the plasmid for transfection. Two hours before transfection, replace with fresh serum-free medium for transfection.

[0059] Take out the transfection reagent 100 μM PEI (Polyethyleneimine) from the refrigerator, and heat it in a water bath at 60°C for 15 minutes until it is completely dissolved. Take out the plasmids Lenti-RS / tRNA / CAR, pMDL g / pRRE, pRSV-Rev, pMD2.G from the refrigerator and dissolve at room temperature.

[0060] Preparation of PEI / DNA complex: take 2ml PBS, add 10μg Lenti-RS / tRNA / CAR, 5μg pMDL g / pRRE, 2.5μg pRSV-Rev, 2.5μg pMD2.G, after blowing and mixing well, add 18μl 100μM PEI, Immediately inhale and mix well, and let stand at room temperature for 10 minutes. Add the PEI / DNA complex obtained after standing still to the cell culture ...

Embodiment 3

[0062] Example 3, lentivirus-mediated cell transfection

[0063] Add Polybrene to an appropriate volume of serum-free RPMI 1640 to a final concentration of 8 μg / ml, use the culture medium to resuspend the Jurkat cells collected by centrifugation, and dilute 5×10 5 The density per well was seeded in a 6-well plate. According to the virus titer and cell number, add the lentivirus concentrate with MOI=10, shake the culture plate gently to mix. Placed at 37°C, 5% CO 2 Culture in an incubator, and change to RPMI 1640 medium containing 10% FBS after 16-24 hours to continue the culture.

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Abstract

The invention discloses a regulation and control method of expression in a chimeric antigen receptor. An expression system enables coexpression of the chimeric antigen receptor, unnatural amino acid is coded to the corresponding UAG orthogonal unnatural amino acid arginyl tRNA and tRNA pair, the nonsense codon UAG is introduced into an amino acid sequence of the chimeric antigen receptor for coding the unnatural amino acid, the expression system is lack of the unnatural amino acid, the chimeric antigen receptor cannot be expressed, and when the expression system adds the unnatural amino acid, the chimeric antigen receptor can be completely expressed. The recombinant chimeric antigen receptor having unnatural amino acid-dependence keeps targeting and killing efficiency on CAR-T, and increases the controlling performance on two specific characters, a control switch is selected as a micromolecular compound, and the method provides an important approach for increasing the safety for cancer clinical treatment for CAR-T.

Description

technical field [0001] The invention relates to the field of biomedical transformation, in particular to a method for regulating the expression of chimeric antigen receptors dependent on unnatural amino acids. technical background [0002] As a public health problem, cancer's morbidity and mortality are increasing year by year, and cancer has become one of the leading causes of death. Adoptive immunotherapy is an important treatment plan to improve the body's immune effect on tumors in addition to traditional surgical treatment, radiotherapy and chemotherapy. Adoptive cellular immunotherapy (ACI) refers to the infusion of autologous or allogeneic immune cells activated in vitro into patients to kill tumor cells in patients. It is one of the important means of treating malignant tumors and has been used in many Good curative effect has been achieved in the clinical treatment of solid tumors and hematological tumors. Among them, chimeric antigen receptor (chimeric antigen re...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/867
CPCC12N15/635C12N15/86C12N2740/15043C12N2840/002
Inventor 应述欢高鹏李玉霞
Owner 胜武(北京)生物科技有限公司
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