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Transgenic lymphocyte co-expressing anti-mesothelin (MSLN) chimeric antigen receptor and non-function EGFR and application thereof

A technology of chimeric antigen receptors and lymphocytes, applied in the field of biomedicine, can solve problems such as breathing difficulties of patients, and achieve high safety effects

Inactive Publication Date: 2018-07-31
BEIJING MARINO BIOTECH PTY LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Rapidly growing pleural effusion often leads to severe dyspnea in patients. Palliative surgery can only temporarily improve the quality of life of these advanced patients, but cannot cure them

Method used

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  • Transgenic lymphocyte co-expressing anti-mesothelin (MSLN) chimeric antigen receptor and non-function EGFR and application thereof
  • Transgenic lymphocyte co-expressing anti-mesothelin (MSLN) chimeric antigen receptor and non-function EGFR and application thereof
  • Transgenic lymphocyte co-expressing anti-mesothelin (MSLN) chimeric antigen receptor and non-function EGFR and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Cell lines and basic experimental techniques used in the embodiments of the present invention are as follows:

[0087] Generation of lentivirus and transduction of human T lymphocytes

[0088] Replication-defective lentiviral vectors were generated and collected by centrifugation for transduction of human T lymphocytes. The following is a brief introduction to the production and collection of lentiviral vectors: 293T cells were placed on a cell culture dish with a bottom area of ​​150-cm2, and according to the instructions, Express-In (purchased from Open Biosystems / ThermoScientific, Waltham, MA) Viral transduction of 293T cells. Add 15 μg of lentiviral transgenic plasmid, 5 μg of pVSV-G (VSV glycoprotein expression plasmid), 10 μg of pCMVR8.74 plasmid (Gag / Pol / Tat / Rev expression plasmid) and 174 μl of Express- In (concentration is 1 μg / μl). The supernatants were collected at 24 hours and 48 hours, and centrifuged for 2 hours using an ultracentrifuge at 28,000 rpm (B...

Embodiment 2

[0095] Example 2 Construction of vectors co-expressing non-functional EGFR and anti-MSLN chimeric antigen receptors

[0096] In this example, the inventors cloned the sequence encoding the single-chain antibody against human MSLN, the ζ-chain sequence of the 4-1BB intracellular segment and the T cell receptor combination into a lentiviral vector containing the EF-1 promoter ( lentiviral vector), during the cloning process, the selected restriction enzymes were XbaI and NotI double enzyme digestion, and NotI and XhoI double enzyme digestion. Lentiviral plasmid with antigen receptor complex (LV-MSLN CAR). The sequence containing synthetic IRES and expressing non-functional EGFR was cloned into LV-MSLN CAR vector plasmid to construct LV-MSLN CAR / tEGFR. figure 1 is a schematic diagram of a lentiviral vector, including sequences encoding an anti-MSLN chimeric antigen receptor, an IRES, and a sequence encoding a non-functional EGFR. The sequence of the anti-MSLN chimeric antigen r...

Embodiment 3

[0097] Example 3 Anti-EGFR antibody can effectively kill and eliminate T lymphocytes co-expressing non-functional EGFR and anti-MSLN chimeric antigen receptor

[0098] In this example, peripheral blood lymphocytes were obtained from anonymous blood donors. Peripheral blood lymphocytes were separated by gradient centrifugation using Ficoll-Hypaque. T lymphocytes and T cell activator magnetic beads CD3 / CD28 (purchased from Invitrogen, Carlsbad, CA) in 5% CO 2 , Incubated at 37 degrees Celsius for 72 hours, the medium was added with 2mmol / L glutamine, 10% high temperature inactivated fetal calf serum (FCS) (purchased from Sigma-Aldrich Co.) and 100U / ml of penicillin / chain RPMI medium 1640 (purchased from Invitrogen Gibco Cat. no. 12633-012) with antimycin double antibody. After activating and culturing for 72 hours, the cells were rinsed with washing solution to wash away the magnetic beads. The T cells were planted on cell culture dishes covered with recombinant fibronectin f...

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Abstract

The invention provides a transgenic lymphocyte, a construct, and a therapeutic composition for treating cancer. The transgenic lymphocyte expresses a non-function EGFR and a chimeric antigen receptor.The chimeric antigen receptor comprises an extracellular region, which comprises the heavy-chain variable region and the light-chain variable region of a single-chain antibody that specifically recognizes the antigen MSLN; a transmembrane region, which is connected with the extracellular region and embedded into the cell membrane of a T lymphocyte; and an intracellular region, which is connectedwith the transmembrane region and comprises the intracellular segment of CD28 or 4-1BB and a CD3 zeta chain. The transgenic lymphocyte has the ability of directionally killing tumor cells, and particularly has remarkable directional killing effect on tumors with high expression of MSLN.

Description

technical field [0001] The present invention relates to the field of biomedicine, in particular, the present invention relates to a T lymphocyte, a lentivirus, a transgenic lymphocyte, a construct, a therapeutic composition for treating cancer and a lymphatic Approaches to cell therapy safety. Background technique [0002] Mesothelin (MSLN) is a differentiation antigen whose expression in normal human tissues is limited to mesothelial cells lining the pleura, pericardium and peritoneum. However, interstitin is highly expressed in a variety of human cancer tissues, including almost all mesothelioma and pancreatic cancer and about 70% of ovarian cancer and about 50% of lung adenocarcinoma and other cancers, such as cholangiocarcinoma, gastric cancer, intestinal cancer , Esophageal cancer, Breast cancer. The mesenchymal gene encodes a 71KDa precursor protein, which is then processed into a 31KDa shedding fragment and a 40KDa protein fragment. The 31KDa shedding fragment is ca...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N7/01C12N15/867C12N15/864A61K35/17A61P35/00
CPCC12N7/00C12N15/86C07K14/7051C07K14/70521C07K14/70578C07K14/70596C07K16/30C07K2319/03C07K2317/56C12N2740/15021C12N2740/15043C12N2750/14143A61K2039/505A61K2239/25A61K39/4611A61K39/464468A61K39/4631A61P35/00C12N5/10C12N15/864C12N15/867
Inventor 严勇朝朱益林陈思毅
Owner BEIJING MARINO BIOTECH PTY LTD
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