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Transgenic lymphocyte co-expressing anti-mesothelin (MSLN) chimeric antigen receptor and immune checkpoint inhibitory molecule and application thereof

A chimeric antigen receptor, lymphocyte technology, applied to receptor/cell surface antigen/cell surface determinant, anti-receptor/cell surface antigen/cell surface determinant immunoglobulin, anti-animal/human immunoglobulin protein and other directions, which can solve problems such as dyspnea in patients

Active 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 immune checkpoint inhibitory molecule and application thereof
  • Transgenic lymphocyte co-expressing anti-mesothelin (MSLN) chimeric antigen receptor and immune checkpoint inhibitory molecule and application thereof
  • Transgenic lymphocyte co-expressing anti-mesothelin (MSLN) chimeric antigen receptor and immune checkpoint inhibitory molecule and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0231] Cell lines and basic experimental techniques used in the embodiments of the present invention are as follows:

[0232] Generation of lentivirus and transduction of human T lymphocytes

[0233] 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 (at a concentration of 1 μg / μl). The supernatant was collected at 24 hours and 48 hours, and centrifuged for 2 hours using an ultracentrifuge at 28,000 rpm...

Embodiment 2

[0241] Example 2 Construction of vectors that co-express shRNA for silencing cellular immune checkpoints and anti-MSLN chimeric antigen receptors

[0242] 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 sequences containing the U6 promoter and human PD1 shRNA (iPD1) or CBL-B shRNA (iCBL-B) or CTLA4 shRNA (iCTLA4) were cloned into the LV-MSLN CAR vector plasmid to construct LV-MSLN CAR / iPD1 or LV-MSLN CAR / iPD1 or LV- MSLN CAR / iCBL or LV-MSLNCAR / iCTLA4. figure 1 is a schematic representation of a lentiviral vector containing the sequence encod...

Embodiment 3

[0243] Example 3 T lymphocytes co-expressing PD1 shRNA and anti-MSLN chimeric antigen receptor have the characteristics of higher cell proliferation ability

[0244] In this example, peripheral blood lymphocytes were obtained from anonymous blood donors. Peripheral blood lymphocytes were separated by gradient centrifugation using Ficoll-Hypaque. In the presence of T lymphocyte activating factor magnetic beads CD3 / CD28 (purchased from Invitrogen, Carlsbad, CA), activated T lymphocytes were transduced with lentiviral vectors, expanded and cultured in vitro, and the method was as described in Example 1. 2-7 days after lentiviral vector transduction, the transduced T cells (the number of cells is 1×10 6 / hole) with MSLN + After 4 days of MSTO-211H co-culture, cell numbers are currently detected by flow cytometry. Experimental results such as figure 2 shown. figure 2 The results showed that the number of T lymphocytes transduced with LV-MSLN CAR / iPD1 was significantly highe...

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Abstract

The invention provides a transgenic lymphocyte, a construct, and a therapeutic composition for treating cancer. The cellular immune checkpoint of the transgenic lymphocyte is silenced, and the transgenic lymphocyte expresses 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 connected with the transmembrane region and comprises the intracellular segment of CD28 and a CD3 zeta chain. The transgenic lymphocyte has the characteristic of resisting tumor cell-mediated immunosuppression, is substantially strengthened in the ability of 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 method of cell activity. 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 called megak...

Claims

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Application Information

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