Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

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

A chimeric antigen receptor, specific technology, applied in the field of cellular immunity

Inactive Publication Date: 2016-09-21
SINOBIOWAY CELL THERAPY CO LTD
View PDF3 Cites 7 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

EpCAM's antibody is currently approved by the FDA in Europe for the treatment of cancerous ascites, but there is no EpCAM's CART in clinical application

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • EpCAM-specific chimeric antigen receptor and encoding gene and application thereof
  • EpCAM-specific chimeric antigen receptor and encoding gene and application thereof
  • EpCAM-specific chimeric antigen receptor and encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[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...

Embodiment 2

[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 ...

Embodiment 3

[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 th...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses an EpCAM (Epithelial cell adhesion molecule)-specific chimeric antigen receptor. The EpCAM-specific chimeric antigen receptor is obtained by serially connecting a single-chain antibody of a human anti-EpCAM, CH2CH3 of a human antibody IgG1, a transmembrane region and an intracellular signal structure of CD28, an intracellular signal structure of CD137 and an intracellular signal structure of CD3[zeta]. The invention further discloses an amino acid sequence and a nucleic acid sequence of the chimeric antigen receptor. The invention further discloses application of the chimeric antigen receptor to preparation of medicines for treating EpCAM-related tumors, namely modification of T lymphocytes, and the modified lymphocytes can be applied to treatment of the EpCAM-related tumors.

Description

technical field [0001] The invention relates to the technical field of cellular immunity, in particular to an EpCAM-specific chimeric antigen receptor and its coding gene and application. Background technique [0002] EpCAM (Epithelial cell adhesion molecule) epithelial cell adhesion molecule belongs to the family of adhesion molecules, also known as 17-A, ESA, EGP40, Trop-1, KSA, CD326, TACSTDI, C017-1A, GA733-2, etc. EpCAM is a single transmembrane protein with a molecular weight of 30-40 kDa encoded by the tumor-associated calcium signal transducer I (TACSTDI) gene, involved in regulating cell adhesion, mediating signal transduction, cell migration, Functions such as proliferation and differentiation (Kurtz JE, DufourP. Adecatumumab: an ant1-EpCAM monoclonal antibody, from the bench to the bedside [J]. Expert Opin BioITher, 2010, 10(6): 951-958; Trzpis M, McLaughlin PM, de LeijLM, et al. Epithelial cell adhesion molecule. More than a carcinoma marker and adhesion molecul...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
IPC IPC(8): C07K19/00C12N15/62A61K35/17A61P35/00
CPCC07K16/2821A61K35/17C07K14/7051C07K14/70521C07K14/70578C07K2317/622C07K2319/02C07K2319/33
Inventor 宋晓彤许国贞刘振云
Owner SINOBIOWAY CELL THERAPY CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com