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A chimeric antigen receptor targeting epcam and its application

A chimeric antigen receptor and targeting technology, which can be used to target specific cell fusion, polypeptides containing positioning/targeting motifs, applications, etc., can solve rare problems and achieve long retention time and safety Good, low immunogenicity effect

Active Publication Date: 2021-01-05
上海科棋药业科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Targeting EpCAM CAR-T therapy has better efficacy and broader application prospects in the field of malignant solid tumors, but reports on CAR molecules and CAR-T cells targeting EpCAM are rare.

Method used

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  • A chimeric antigen receptor targeting epcam and its application
  • A chimeric antigen receptor targeting epcam and its application
  • A chimeric antigen receptor targeting epcam and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] This embodiment is the construction of a chimeric antigen receptor molecule targeting EpCAM, which includes the following steps:

[0046] Firstly, based on the monoclonal antibody MOC31 targeting EpCAM, humanization was carried out. Humanized transformation is carried out by conventional CDR region transplantation methods in the field, see literature (Jones PT, Dear PH, Foote J, et al. Replacing the complementarity-determining regions in a human antibody with those from amouse. Nature. 1986May 29-Jun 4; 321(6069):522-5.; Sandhu JS. A rapid procedure for the humanization of monoclonal antibodies. Gene. 1994 Dec 15; 150(2):409-10.). By grafting the CDR region of MOC31 into the framework region of the highly homologous human variable region, the humanized antibody was named MOCF. And its sequence was transformed into a single-chain variable region sequence MOCF scFv; at the same time, the EPCAM single-chain variable region sequence MOCB scFv derived from the paper was use...

Embodiment 2

[0048] This embodiment is the construction of MOCF / MOCB-ICOSBBZ CAR-T cells, which includes the following steps:

[0049] Packaging of CAR lentivirus: Firstly, the constructed lentiviral plasmids pHAGE-EF1a-MOCF-ICOSBBZ, pHAGE-EF1a-MOCF-ICOSBBZ and lentiviral system auxiliary packaging plasmids pMD2.G and pSPAX2. Spread 1.8×10E7 293T into T175 culture flasks one day before transfection. 1 hour before transfection, the medium of 293T cells was replaced with 30ml of serum-free medium. The plasmid was co-transfected into 293T cells using the calcium phosphate precipitation method, and the cell culture medium was replaced with 60ml of complete medium DMEM+10%FBS 24 hours after transfection. The cell supernatant was harvested 48 hours after transfection, and 60ml of fresh complete medium was added. After 72 hours, the cell supernatant was collected again, and the cells were discarded. The collected cell supernatant was centrifuged at 5000g for 3min to remove impurities, then fi...

Embodiment 3

[0052] This example is the identification of EpCAM target cells and the determination of the activity of EpCAM CAR-T cells.

[0053] Identification of EpCAM target cells: In order to detect whether EpCAM CAR-T can effectively kill tumor cells, the expression of EpCAM on the surface of different tumor cells was first identified. The results showed that the lung cancer cell line H1650 highly expressed EpCAM, while the lung cancer cell line A549 did not express EpCAM , the result is as image 3 shown.

[0054] IFN-γ and IL2 secretion experiment: MOCF-ICOSBBZ, MOCB-ICOSBBZ two kinds of CAR-T cells and control T cells three kinds of effector cells and target cells H1650 and control target cells A549 were used to target at a ratio of 5:1 in a 96-well plate Than mixed. After culturing at 37°C for 24 hours, the expression of IFN-γ and IL2 in the supernatant was detected by standard ELISA method. The result is as Figure 4 As shown, when MOCF-ICOSBBZ and MOCB-ICOSBBZ two effector c...

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Abstract

The present invention relates to a chimeric antigen receptor targeting EpCAM, which comprises a recognition region, a hinge region, a transmembrane region, and an intracellular signal region, an extracellular recognition region sequence is a humanized single-chain variable region antibody MOCF that recognizes EpCAM, and its sequence is shown as SEQ ID NO. 3; and specifically, the chimeric antigenreceptor comprises a sequence shown as SEQ ID NO. 13. The invention also relates to an application of the above-described chimeric antigen receptor and a preparation method of the chimeric antigen receptor. The present invention employs the humanized antibody which has lower immunogenicity than a conventional murine scFv, the CAR-T cells prepared on the basis are less likely to produce a HAMA effect in vivo, and may have a longer retention time in vivo; the CAR-T cell MOCF-ICOSBBZ prepared by the above humanized antibody has lower background level cytokines release, and better safety in clinical applications; by targeting EpCAM protein on the surface of tumor cells and activating a signaling pathway downstream of T cells, T cells are empowered to kill EpCAM-targeted tumor cells, and EpCAM-positive tumors can be treated efficiently and specifically.

Description

technical field [0001] The present invention relates to the technical field of cellular immunotherapy, in particular to a chimeric antigen receptor targeting EpCAM and its application. Background technique [0002] Epithelial cell adhesion molecule (EpCAM), also known as CD326, is a 40kD transmembrane glycoprotein. It is specifically expressed in a variety of tumor tissues, whereas in normal tissues its expression is restricted to the basolateral side of epithelial cells, making their apical side inaccessible. EpCAM is highly expressed in a variety of epithelial cell-derived tumors, including a variety of adenocarcinomas, such as colon, stomach, pancreas, lung, ovary, breast, etc. As a signaling molecule, EpCAM downstream activates the Wnt signaling pathway, thereby regulating the cleavage of proteins in the membrane, which in turn leads to tumorigenesis. In tumor tissues, EpCAM expression changes from the basal layer to uniform expression on the cell membrane surface, mak...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K35/17A61P35/00
CPCA61K35/17A61P35/00C07K14/70517C07K16/30C07K2317/24C07K2317/622C07K2317/73C07K2319/02C07K2319/03C07K2319/33C12N5/0636C12N15/86C12N2740/15043
Inventor 郝瑞栋李彦涛易桥勇红丽张大挺刘根桃吴国祥
Owner 上海科棋药业科技有限公司