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PD-L1 antibody secretion anti-mesothelin CAR-T cell tumor immunotherapy

A PD-L1, lymphocyte technology, applied in the field of biomedicine, can solve problems such as breathing difficulties of patients, and achieve the effects of enhanced proliferation and survival ability, high safety, and enhanced killing ability

Inactive Publication Date: 2020-05-01
ILIFESEQ LTD CORP
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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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  • PD-L1 antibody secretion anti-mesothelin CAR-T cell tumor immunotherapy
  • PD-L1 antibody secretion anti-mesothelin CAR-T cell tumor immunotherapy
  • PD-L1 antibody secretion anti-mesothelin CAR-T cell tumor immunotherapy

Examples

Experimental program
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Effect test

Embodiment 1

[0107] Cell lines and basic experimental techniques used in the embodiments of the present invention are as follows:

[0108] Generation of lentivirus and transduction of human T lymphocytes

[0109] 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 supernatants were collected at 24 hours and 48 hours, and centrifuged for 2 hours using an ultracentrifuge at 28,000 r...

Embodiment 2

[0114] Example 2 Construction of vectors that co-express anti-PD-L1 single-chain antibody and IgG Fc fusion protein containing amino acid mutations and anti-MSLN chimeric antigen receptor

[0115] 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 of the anti-PD-L1 single-chain antibody (LS1clone) and the IgG Fc fusion protein containing amino acid mutations were cloned into the LV-MSLNCAR vector plasmid to construct a lentiviral vector αLS1-QL / M-CAR. figure 1 It is a schematic diagram of the lentiviral vector, including the sequence e...

Embodiment 3

[0116] Example 3 Vector αLS1-QL / M-CAR transduces T lymphocytes to secrete anti-PD-L1 single-chain antibody and IgG Fc fusion protein containing amino acid mutations to inhibit the effect of PD-1:PD-L1

[0117] 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. 3-7 days after lentiviral vector transduction, harvest the transduced T cells (the number of cells is 2×10 6 / well) culture supernatant for PD-1:PD-L1 inhibition experiment. 100 ng / well of PD-L1 protein (Aerobiosystems, Boston, MA) was used to coat 96-well ELISA plates. 10ng of biotin-labeled PD1 (Aerobiosystems, Boston, MA) was mixed...

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Abstract

The invention proposes PD-L1 antibody secretion anti-mesothelin CAR-T cell tumor immunotherapy. CAR-T cells co-express an anti-PD-L1 fusion antibody, non-functional EGFR and a chimeric antigen receptor, wherein the anti-PD-L1 fusion antibody is formed by linking an anti-PD-L1 single-chain antibody to an IgG1Fc fragment containing amino acid mutation. The transgenic lymphocyte secretes the anti-PD-L1 single-chain antibody and an IgG Fc fusion antibody, and has the characteristics of resisting tumor cell-mediated immunosuppression, significantly enhanced killing ability to tumor cells, especially a significant targeted killing effect on tumors highly expressing mesothelin and PD-L1 molecules, and high safety.

Description

technical field [0001] The present invention relates to the field of biomedicine, specifically, the present invention relates to T lymphocytes, lentiviruses, transgenic lymphocytes, constructs, therapeutic compositions for treating cancer, and methods for improving lymphocyte activity and treatment safety. Background technique [0002] Mesothelin (MSLN) is a differentiation antigen, and its expression in normal human tissues is limited to mesothelial cells lining the pleura, pericardium and peritoneum. However, mesothelin 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 mesothelin gene encodes a 71KDa precursor protein, which is then processed into a 31KDa shedding fragment and a 40KDa protein fragment. The 31KDa shedding f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10C07K19/00C07K16/46C12N15/62C12N15/13C12N15/867A61K35/17A61P35/00
CPCC12N5/0636C07K16/30C07K14/7051C07K14/70517C07K14/70578C07K16/2827C12N15/86A61P35/00C12N2510/00C07K2317/622C07K2319/00C07K2319/03C07K2319/33C07K2319/74C12N2800/107C12N2740/15043A61K39/464411A61K2239/54A61K2239/38A61K39/4631A61K39/4611A61K2239/31A61K39/464468C07K16/46C07K19/00C12N5/10C12N15/62C12N15/867C12N15/864
Inventor 黄雪芬陈思毅
Owner ILIFESEQ LTD CORP
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