Novel chimeric antigen receptor and application thereof

A chimeric antigen receptor, antigen technology, applied in the field of biomedical transformation

Active Publication Date: 2017-01-04
北京领柯生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although CAR-T targeting tumor cells is currently one of the most promising cancer treatment options, it has targeting non-tumor "off-target effects" (Morgan RA, Yang JC, Kitano M, et al. Case report of asrious adverse event following the administration of T cells transduced with a chimeric antigen receptor recognizing ERBB2[J].Mol Ther, 2010, 18(4): 843-851) and "cytokine storm" (Wrzesinski C, Paulos CM, Kaiser A, et al .Increased intensity lymphodepletion enhances tumor treatment efficacy of adoptively transferred tumor- specific T cells[J].J Immunother, 2010, 33( 1): 1- 7) Toxic effects

Method used

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  • Novel chimeric antigen receptor and application thereof
  • Novel chimeric antigen receptor and application thereof
  • Novel chimeric antigen receptor and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0072] Example 1, Design of Recombinant Chimeric Antigen Receptors with Light-Inducible Elements

[0073] The present invention selects the CD19-CAR gene with better clinical effect, and the constituent elements are as follows: CD19 scFv, CD8hinge, CD8 transmembrane, 4-1BB intracellular, CD3ζ;

[0074] LOV2 used in the present invention (aa 404-546 of Avena sativa Phototropin 1, NPH1-1, GenBank: AAC05083.1) derived from oat ( Avena sativa ), according to people ( Homo spaice ) codon preference for codon optimization, the amino acid sequence is shown in SEQ ID No: 1, and the nucleotide sequence is shown in SEQ ID No: 13.

[0075] Insert LOV2 into the CD19-CAR antigen binding region, transmembrane structure region, co-stimulatory signal transduction region and T cell signal transduction region before and after or between the functional domains, as follows:

[0076] liCAR5: CD19 scFv-connecting peptide 1-LOV2-connecting peptide 2-CD8 hinge region-CD8 transmembrane domain-4...

Embodiment 2

[0082] Example 2, Expression of Recombinant Chimeric Antigen Receptors with Light-Inducible Elements

[0083] 1. Construction of co-expression vectors Lenti-liCAR5, Lenti-liCAR6, Lenti-liCAR7, Lenti-liCAR8 and Lenti-liCAR9

[0084] Lenti-EF1α-CD19CAR (CD3ZetaQQ) was purchased from iCartab Biomedical Technology (Suzhou) Co., Ltd. (http: / / www.icartab.com.cn). Plasmid pUCK-AsLOV2 was purchased from the company.

[0085] Using the classic CD19-CAR molecule (SEQ ID No: 45) and the LOV2 molecule (SEQ ID No: 13) optimized according to human codon preference as templates, the primers were designed as follows:

[0086] liCAR5:

[0087] CALOV (EcoRI): 5'-TTCGAATTCGCCGCCACCATG-3' SEQ ID No: 29;

[0088] CALOV5aR: 5'-GCTCCCACTCCCGCTTCCGCTGGACACGGTGACCAGA-3' SEQ ID No: 33;

[0089] GSLOVF: 5'-GGAAGCGGGAGTGGGAGCCTTGCAACCACCTTGGAAAG-3' SEQ ID No: 31;

[0090] EALOVR: 5'-CCGCTGCTTCTTTGGCCGCTGCTTCCAGCTCTTTGGCGGCCTCATC-3' SEQ ID No: 32;

[0091] EALOV5cF: 5'-AGCGGCCAAAGAAGCAGCGGCCAAAACCACTA...

Embodiment 3

[0131] Example 3. Study on the Killing Activity of Recombinant Chimeric Antigen Receptor T Cells with Light-Inducible Elements

[0132] Inoculate 100 μl 1×10 4 Target cells (leukemia cells Raji) per well were added to 96-well cell culture plates, and Jurkat-CAR5, Jurkat-CAR6, Jurkat-CAR7, Jurkat-CAR8, Jurkat-CAR9 cells (expressing liCAR5 -9 Jurkat cells), make up the culture solution to 200μl, respectively, under light conditions (LED light, 465nm, 30μmolm -2 the s -1 , exposure for 4h) and 37°C, 5% CO under dark conditions 2 The incubator co-cultured for 48h.

[0133] (1) Experiment 1

[0134] After 48 hours of co-cultivation, the cell culture suspensions were collected and centrifuged to collect the supernatant, and the IL-2 content was detected using the human IL-2 ELISA kit. For specific steps, refer to the instruction manual of the human IL-2 ELISA kit. Specific results such as figure 2 shown. figure 2 It shows that the IL-2 activity of Jurkat-CAR5, Jurkat-CAR6,...

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Abstract

The invention discloses a recombined chimeric antigen receptor with a photoinduction element. The chimeric antigen receptor comprises an antigen combining area, a transmembrane structure area, a co-stimulatory signal transduction area and T cell signal transduction area functional structure domains, wherein the photoinduction element is inserted at the N terminal or C terminal of the recombined chimeric antigen receptor or between the functional structure domains. According to the chimeric antigen receptor, photoinduction element LOV2 genes are cloned into plasmids Lenti-EF1alpha-CD19CAR with a molecular cloning method to establish a co-expression vector of the recombined chimeric antigen receptor with the photoinduction element, 293T cells are used for packaging to obtain a lentiviral vector with photoinduction CAR molecules. After the recombined chimeric antigen receptor is expressed in T cells through the lentiviral vector, the damage toxicity of T cells is reversely controlled by LOV2, and an importable approach is provided for improving the safety of CAR-T in treating cancer clinically.

Description

technical field [0001] The invention relates to the field of biomedical transformation, in particular to a recombinant chimeric antigen receptor with a light-inducible element, its coding gene and its application. Background technique [0002] The immune system is the body's defense system. On the one hand, it plays the role of eliminating bacteria, viruses, and foreign substances. On the other hand, it eliminates aging cells and mutated cells in the body. Some mutated cells will become cancer cells. Cancer immunotherapy is to mobilize the body's own immune system to kill cancer cells and inhibit their proliferation through artificial intervention. [0003] 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 important methods for the treatment of malignant tumors. It has achieved good curative effect in the clinical treatment of vari...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10
Inventor 应述欢
Owner 北京领柯生物科技有限公司
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