EpCAM-specific chimeric antigen receptor and encoding gene and application thereof

[0029] Example 1: Construction of EpCAM-specific chimeric antigen receptor retroviral vector

[0030] A kind of EpCAM-specific chimeric antigen receptor proposed by the present invention is composed of human anti-EpCAM single-chain antibody, CH2CH3 of human antibody IgG1, transmembrane region and intracellular signal structure of CD28, intracellular signal structure of CD137, and CD3ζ. The intracellular signal structure is formed in series, and its amino acid sequence is shown in SEQ ID NO.1.

[0031] 1. Using the human anti-human EpCAM monoclonal antibody sequence published in 2001, the VH and VL sequences were selected for the design of the scFv sequence in the CAR vector, and the codons were optimized using OptimumGeneTM gene design software from GenScript Biotechnology Company. For further optimization, NcoI and BAMHI restriction sites were added to both ends of the optimized sequence. The optimized sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd., and th

[0035] Example 2: Preparation of EpCAM-specific chimeric antigen receptor modified T lymphocytes

[0036] 1. Packaging of retroviruses containing EpCAM-specific chimeric antigen receptors

[0037] Extract the retroviral packaging vector pRD114 and the EpCAM-scFv-CH2CH3-CD28-CD137-CD3ζ retroviral vector with the operating instructions in the endotoxin-free plasmid extraction kit (Tiangen Bio) and co-transfect them into 293T cells. The cell supernatant was collected 48 hours after transfection, centrifuged at 4000rpm for 10min, and the cells and cell debris in the supernatant were removed. Filter with a 0.45 μm membrane filter, aliquot and freeze for later use.

[0038] 2. Preparation of T lymphocytes

[0039] Take 20mL of fresh anticoagulated blood from healthy volunteers, and separate peripheral blood mononuclear cells (PBMC) with lymphatic separation fluid (GE Company). The isolated cells were stimulated with CD3 and CD28 plates for 48 hours, and induced with T lymphocyte med

[0044] Example 3: The killing effect of chimeric antigen receptor EpCAM-specific CAR-T cells on EpCAM-related tumors

[0045] 1. Detection of tumor lethality of CAR-T cells

[0046] The EpCAM-positive colon cancer cell HT-29 was adjusted to 5×10 6 / mL, 50 μL per well, according to the effector cell and target cell ratio of 4:1, add tumor cells 2.5×10 5 After the cells were completely adhered to the wall, T cells and CAR-T cells were collected, and the cell concentration was adjusted to 1.25×10 6 / mL, 50 μL per well, and then perform flow cytometry (cultured for 24h) and MTT detection (cultured for 4h) respectively.

[0047] First, cells were collected for CD3 staining, and flow cytometric analysis of tumor cells (CD3 - ) and T cells (CD3 + ), the result is as Figure 5 shown. Depend on Figure 5 It can be seen that the chimeric antigen receptor EpCAM-specific CAR-T cells obtained in the present invention can effectively kill colon cancer cells.

[0048] Secondly, in t