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

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

Active Publication Date: 2013-05-22
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: Obtaining the full-length gene of EGFR targeting CAR 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 through primer synthesis; the EGFR targeting scFv sequence is amplified from anti-EGFR hybridoma cells, that is, optimized by ribosome display technology. The specific steps are as follows: Preparation of anti-EGFR hybrid Tumor cells, extract total RNA, and then use RT-PCR to amplify the heavy and light chain genes against EGFR, and send them to Invitrogen for sequencing. The sequencing results were analyzed by Jellyfish software, and the sequences that could be correctly translated were compared through VBASE2 and GeneBank, and their specificity and affinity were evolved by ribosome display technology. The resulting anti-EGFR single-chain antibody with high affinity ...

Embodiment 2

[0084] Example 2 Transfection and expression detection of lymphocytes

[0085] 1 experimental route

[0086] 1) Culture of lymphocytes

[0087] (1) Lymphocytes were separated from the peripheral blood of five voluntary blood donors by density gradient centrifugation: Inactivated human plasma and rhIL-2 were added to X-VIVO15 medium (Lonza, Item No. 04-744Q) ( Shandong Quangang Pharmaceutical, Quanqi, 2 million IU / branch) to a final concentration of 3% and 300 IU / mL, as a conventional medium for lymphocytes.

[0088] (2) Activation and expansion of lymphocytes: Add mouse anti-human CD3 and CD28 antibodies (BDBioscience, CD3 antibody product number: 555337, CD28 antibody product number: 55725) to the lymphocyte culture medium to a final concentration of 1μg / mL. Dilute the separated lymphocytes with this medium to make the cell density 3×10 6 . 37℃,5%CO 2 to cultivate. After 72h, change the medium to a regular medium for lymphocytes.

[0089] 2) Transfection of lymphocytes

[0090] (1) ...

Embodiment 3

[0105] Example 3 Detection of in vitro killing activity of CAR gene modified lymphocytes

[0106] This experiment uses a cytotoxicity detection kit (Promega CytoTox Non-Radioactive Cytotoxicity Assay Kit, catalog number G1780) for the detection of the in vitro killing activity of CAR genetically modified lymphocytes.

[0107] 1 experimental route

[0108] 1) Collect lymphocytes and human epidermal squamous cell line A431 and human ovarian cancer cell line A2780 after 16 hours of transfection, and count the cells;

[0109] 2) Add 4000 tumor cells in a 96-well plate as target cells; add effector cells in the target cell wells at the ratio of effector cells: target cells of 10, 20 and 40. Make 3 auxiliary holes for each group; set aside 3 holes for target cells and effector cells in each group to detect the release of spontaneous lactate dehydrogenase (LDH); centrifuge at 250G, 4min; 37℃, 5% CO 2 Incubate for 6h in the incubator;

[0110] 3) Add Lysis Solution to the target cell wells of...

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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 present 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) is a membrane surface receptor with tyrosine kinase activity, which plays an important regulatory role in many physiological processes of cells [1,2]. EGFR is structured by three parts: extracellular region, transmembrane region and intracellular region. The extracellular region has two cysteine-rich regions, and the intracellular region has typical ATP binding sites and tyrosine kinases. 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 malignancies. Under normal circumstances, EGFR tyrosine kinase exists in the form of a monomer. When the s...

Claims

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Application Information

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Patent Type & Authority Applications(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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