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Photo-induced dimer type chimeric antigen receptor

A chimeric antigen receptor, light-induced technology, applied in the field of biomedical transformation, can solve problems such as the publication of research results, and achieve the effect of improving safety

Active Publication Date: 2017-09-08
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

It is a relatively safe and convenient method to control the activity of CAR-T cells through light-induced dimerization, but there are relatively few such studies at present, and no successful research results have yet been published.

Method used

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  • Photo-induced dimer type chimeric antigen receptor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] Example 1, Design of Light-Induced Dimer Type Recombination Chimeric Antigen Receptor

[0052] In the present invention, the CD19-CAR molecule, which is currently the most widely studied and used molecule, is selected as an example to construct the first monomer and the second monomer of light-induced dimer recombination and antigen receptor respectively. A total of two groups of light-induced dimer-type chimeric antigen receptors were designed, named combination 1 and combination 2 respectively (for the sequence structure, see figure 1 ).

[0053] Combination 1 first monomer components are as follows: CD8α signal peptide (amino acid sequence shown in SEQ ID No: 2, nucleotide sequence shown in SEQ ID No: 1), anti-CD19 scFv (amino acid sequence shown in SEQ ID No: 4, the nucleotide sequence is shown in SEQ ID No: 3), CD8 hinge (the amino acid sequence is shown in SEQ ID No: 6, the nucleotide sequence is shown in SEQ ID No: 5), CD8 transmembrane (amino acid The sequence...

Embodiment 2

[0065] Example 2, Light Induced Expression of Dimeric Chimeric Antigen Receptor

[0066] 1. Construction of co-expression vector

[0067] Lenti-EF1α plasmid vector was purchased from iCartab Biomedical Technology (Suzhou) Co., Ltd. (http: / / www.icartab.com.cn).

[0068] According to the sequences encoded by SEQ ID NO: 35 and SEQ ID NO: 40, Beijing Aoke Biotechnology Co., Ltd. was entrusted to carry out the whole gene synthesis, and then inserted into the EcoRI and BamHI restriction sites of the Lenti-EF1α plasmid. Transformed into E.coli (TOP10, purchased from Quanshijin Company), after correct sequencing, the plasmid was extracted and purified using Qiagen’s plasmid purification kit, and the endotoxin-free plasmid of the two combined recombinant expression vectors was obtained, that is, Lenti- LID-CD19CAR-1 and Lenti-LID-CD19CAR-2.

[0069] 2. Packaging of lentivirus vector Lentivirus

[0070] Inoculate 5 × 10 in gelatin-precoated 15-cm dishes 6 HEK 293T cells were cultur...

Embodiment 3

[0076] Example 3, Analysis of cell killing activity of LID CD19-CAR Jurkat T cells

[0077] (1) Detection of IL-2 secretion

[0078] The degree of activation of immune cells was evaluated by the level of IL-2 secreted by cells. Combine K562 and K562-CD19 (K562 cells obtained by transfection with stable CD19 phenotype) at 1×10 5 The concentration per well was plated in a 96-well plate, and 100 μl of target cells were added to each well. The LID CD19-CAR Jurkat T cells (LID CD19-CAR-1 Jurkat T cells and LIDCD19-CAR-2 Jurkat T cells) according to the ratio of target cells: effector cells = 1:1, 1:3, 1:10, respectively co-culture with target cells. Set both illuminated and non-illuminated groups. When co-cultivating in the light group, blue LED light (1.2mW / cm 2 at450nm) was irradiated for 6h; the non-illumination group wrapped the cell culture plate with tinfoil. At 37°C, 5% CO 2 After 18-20 hours of co-cultivation in the incubator, the cell culture suspensions were colle...

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Abstract

The invention discloses a photo-induced dimer type chimeric antigen receptor. The photo-induced dimer type chimeric antigen receptor is composed of two monomers, wherein a first monomer is sequentially composed of a signal peptide, an extracellular antigen binding area, an extracellular hinge area, a transmembrane area, a costimulatory signal transduction area and an optically-controlled element and polymerization element 1 functional structure domain; a second monomer is sequentially composed of a signal peptide, an extracellular affine area, a transmembrane area, a costimulatory signal transduction area and a polymerization element 2 and T cell signal transduction area functional structure domain. The photo-induced dimer type chimeric antigen receptor is applied to mediating immune response of T cells; either monomer has no activity when not in a coupled mode, so that polymerization and depolymerization of the photo-induced dimer type chimeric antigen receptor can be controlled through blue light irradiation.

Description

technical field [0001] The invention relates to the field of biomedical transformation, in particular to a light-induced dimer type chimeric antigen receptor and its encoding gene and application. Background technique [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 currently one of the most promising methods for the treatment of malignant tumors. It has achieved good curative effect in the clinical treatment of various solid tumors and blood tumors. Among them, chimeric antigen receptor (chimeric antigen receptor, CAR) T...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62
CPCC07K14/7051C07K14/70517C07K14/70578C07K16/2803C07K2317/622C07K2319/02C07K2319/03C07K2319/33
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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