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Genetically engineered lymphocyte targeting Human EGFR (Epidermal Growth Factor Receptor), preparation method and application of genetically engineered lymphocyte

A targeting and host cell technology, applied in the field of lymphocytes, can solve the problem of not using lymphocytes to treat tumors, etc.

Active Publication Date: 2015-04-22
WEST VAC BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] So far, there is no report on the use of EGFR-targeted chimeric antigen receptor technology to transform lymphocytes to treat tumors

Method used

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  • Genetically engineered lymphocyte targeting Human EGFR (Epidermal Growth Factor Receptor), preparation method and application of genetically engineered lymphocyte
  • Genetically engineered lymphocyte targeting Human EGFR (Epidermal Growth Factor Receptor), preparation method and application of genetically engineered lymphocyte
  • Genetically engineered lymphocyte targeting Human EGFR (Epidermal Growth Factor Receptor), preparation method and application of genetically engineered lymphocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1 Acquisition of EGFR-targeted CAR full-length gene and construction of expression plasmid vector

[0033] 1 experimental route

[0034] The CAR gene is composed of SP hIL-2, scFv that recognizes EGFR, IgG2-Fc, and CD28TM-CD3ζ. The SP hIL-2 sequence is included in the upstream amplification primer by primer synthesis; the EGFR targeting scFv sequence is obtained from the amplification of anti-EGFR hybridoma cells, that is, after optimization of ribosome display technology. The specific steps are as follows: prepare anti-EGFR hybrid The total RNA was extracted from tumor cells, and the anti-EGFR heavy and light chain genes were respectively amplified by RT-PCR, and sent to Invitrogen for sequencing. The sequencing results were analyzed by Jellyfish software, and the sequences that could be translated correctly were compared by VBASE2 and GeneBank, and the specificity and affinity were evolved by ribosome display technology, and the anti-EGFR single-chain antibod...

Embodiment 2

[0084] Example 2 Transfection and expression detection of lymphocytes

[0085] 1 experimental route

[0086] 1) Culture of lymphocytes

[0087] (1) Lymphocytes were isolated from the peripheral blood of 5 voluntary blood donors by density gradient centrifugation: inactivated human plasma and rhIL-2 ( Shandong Quangang Pharmaceutical, Quanqi, 2 million IU / cartridge) to a final concentration of 3% and 300IU / mL, as a conventional medium for lymphocytes.

[0088] (2) Activation and expansion of lymphocytes: Add mouse anti-human CD3 and CD28 antibodies (BD Bioscience, CD3 antibody product number: 555337, CD28 antibody product number: 55725) to the lymphocyte culture medium to a final concentration of 1 μg / mL. The isolated lymphocytes were diluted with this medium to a cell density of 3×10 6 . 37°C, 5% CO 2 nourish. After 72 hours, the culture medium was replaced with the regular culture medium for lymphocytes.

[0089] 2) Transfection of lymphocytes

[0090] (1) Collect the...

Embodiment 3

[0105] Example 3 In vitro killing activity detection of CAR gene-modified lymphocytes

[0106] In this experiment, a cytotoxicity detection kit (CytoTox from Promega Corporation) was used. Non-Radioactive Cytotoxicity Assay Kit, Cat. No. G1780) was used to detect the killing activity of CAR gene-modified lymphocytes in vitro.

[0107] 1 experimental route

[0108] 1) Lymphocytes, human epidermal squamous cell line A431 and human ovarian cancer cell line A2780 were collected 16 hours after transfection, and the cells were counted;

[0109] 2) Add 4,000 tumor cells in a 96-well plate as target cells; add effector cells in the target cell wells according to the ratio of 10, 20 and 40 effector cells: target cells. Make 3 auxiliary wells for each group; set aside 3 wells for target cells and effector cells in each group to detect the release of spontaneous lactate dehydrogenase (Lactate Dehydrogenase, LDH); centrifuge at 250G for 4min; 37°C, 5% CO 2 Incubation in the incubator ...

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Abstract

The invention relates to the field of genetic engineering, and in particular relates to an anti-EGFR ((Epidermal Growth Factor Receptor) chimeric antigen receptor, and a lymphocyte for expressing the antigen receptor. The technical solution of the invention provides a new effective selection for the anti-tumor technical field. The technical scheme is that a single-chain antibody targeting the EGFR is provided. The single-chain antibody is further designed to be constructed into the chimeric antigen receptor by means of a genetic engineering technology, and the structure of the gained chimeric antigen receptor is anti-EGFR (scFv)-IgG2 (Fc)-CD28-CD3 Zeta. According to the genetically engineered lymphocyte targeting the human EGFR, the chimeric antigen receptor is transfected into the lymphocyte by the nucleofaction technology, and thus the nonspecific lymphocyte can be endowed with the capacity of specifically distinguishing the EGFR tumor antigen and targeting and killing the EGFR over-expression tumor cells. According to the technical scheme, the adoptive cell therapy and the genetic therapy are organically combined, so that remarkable effect of killing the tumor is gained, and a novel effective selection is provided for the field.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to an anti-EGFR chimeric antigen receptor and lymphocytes capable of expressing the antigen receptor. Background technique [0002] Epidermal growth factor receptor (EGFR), a membrane surface receptor with tyrosine kinase activity, plays an important regulatory role in many physiological processes of cells [1,2]. EGFR is structurally composed of three parts: the extracellular region, the transmembrane region and the intracellular region, in which the extracellular region has two cysteine-rich regions, and the intracellular region has a typical ATP binding site and tyrosine kinase Area. EGFR is highly expressed on the surface of many malignant tumor cells [3], including epidermal squamous cell carcinoma, breast cancer, ovarian cancer, non-small cell lung cancer, kidney cancer and head and neck cancer. Normally, EGFR tyrosine kinase exists in the form of a monomer. When the specif...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K16/28C07K19/00C12N15/11C12N15/63C12N5/10A61K48/00A61P35/00
Inventor 魏于全李炯周西坤
Owner WEST VAC BIOPHARMA CO LTD
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