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Protein complex and application thereof

A complex, protein technology, applied in the field of immunology and molecular biology, can solve problems such as immunosuppression

Active Publication Date: 2021-09-17
QINGDAO SINO-CELL BIOMEDICINE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a protein complex, an immunosuppressive-co-stimulatory signal conversion molecule, a polynucleotide, an expression vector, a cell, an immune effector cell, a pharmaceutical composition and Its use in the preparation of drugs for the prevention or treatment of tumors aims to solve the technical problem of immunosuppression of reinfused cells by the tumor microenvironment in the existing ACT therapy

Method used

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  • Protein complex and application thereof
  • Protein complex and application thereof
  • Protein complex and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] 1.1SWITCH lentivirus packaging and harvesting

[0105] first day:

[0106] 1) Replace the cultured 293FT (purchased from ATCC) with 9 mL of antibiotic-free DMEM+10% FBS before transfection;

[0107] 2) Set up 2 sets of EP tubes;

[0108] 3) Three EP tubes A: OPTi-MEM 1.5ml+main plasmid 20ug+pMDLg.PRRE 10ug+PRSV-Rev 5ug+PMD2.G 5ug. The total plasmid ratio is 40ug (4:2:1:1); and the corresponding three EP tubes B: OPti-MEM 1.5ml+lipo3000 41μL. The plasmids in this step are all purchased from Addgene, and the main plasmid of the first EP tube A is empty plasmid, the main plasmid of the second EP tube A is the main plasmid of the multi-cloning site inserted into the TIGIT-switch DNA fragment, and the third The main plasmid of tube A of EP is the main plasmid in which the TIGIT-switch-link DNA fragment is inserted into the multiple cloning site.

[0109] 4) The three EP tubes B were mixed separately and left at room temperature for 5 minutes;

[0110] 5) Slowly drop the...

Embodiment 2

[0132] Example 2 TIL specimen (LA20201222-2-LC) switch TIL cell effect verification test

[0133] 1) Centrifuge the TIL cells obtained from the initial culture, resuspend with AIM-V+10% AB serum+P / S complete medium and adjust the cell density to 1×10 6 / mL, spread 1mL cell suspension in each well of a 24-well plate; add CD3 monoclonal antibody 100ng / mL, IL-23000U / mL;

[0134] 2) When the expansion doubles twice and is in the logarithmic growth phase, use AIM-V+10% AB serum without P / S to adjust the cell density to 2×10 6 / mL, seed a 24-well plate, 0.5mL per well;

[0135] 3) Take the three lentivirus concentrates obtained according to the scheme of Example 1 out of the -80°C refrigerator, and put them on ice to melt slowly;

[0136] 4) Lentivirus infects T cells at a ratio of MOI=20, that is, each cell corresponds to 20 lentiviruses, and each well needs 2×10 lentiviruses 7 indivual. Calculate the volume required for each virus according to the virus titer, add it to T cell...

Embodiment 3

[0143] Example 3 TIL cell SWITCH and SWITCH-link effect verification experiment of TIL sample (LA2021011901)

[0144] 1) Separate the tumor tissue block of lung cancer patient (LA2021011901) by enzymatic hydrolysis, and use IL-2 for primary culture amplification amplification, and the TIL cells obtained after the primary culture are counted by centrifugation, and AIM-V+10%AB serum+P / Resuspend in S complete medium and adjust the cell density to 1×10 6 / mL, spread 1mL cell suspension in each well of a 24-well plate; add CD3 monoclonal antibody 100ng / mL, IL-23000U / mL;

[0145] 2) When the expansion doubles twice and is in the logarithmic growth phase, adjust the cell density to 1×10 6 / mL, seed a 24-well plate, 1 mL per well;

[0146] 3) Take the three lentivirus concentrates obtained according to the scheme of Example 1 out of the -80°C refrigerator, and put them on ice to melt slowly;

[0147] 4) Lentivirus infects TIL cells at a ratio of MOI=10, that is, each cell correspo...

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Abstract

The invention belongs to the technical field of immunology and molecular biology, and particularly relates to a protein complex and application thereof. The protein complex comprises an extracellular domain of TIGIT, a transmembrane domain of TIGIT, an intracellular domain of CD28 and an intracellular domain of 4-1BB, the structural design of the protein complex not only can relieve the original inhibition of a tumor microenvironment on immune effector cells, but also a costimulatory signal molecule can further enhance the immune function of the immune effector cells in turn. In addition, the protein complex can also improve the multiplication capacity of the immune effector cells and realize mass amplification of the immune effector cells.

Description

technical field [0001] The present invention relates to the fields of immunology and molecular biology, in particular to a protein complex, an immunosuppressive-co-stimulatory signal conversion molecule, a polynucleotide, an expression vector, a cell, and an immune effector cell , and a pharmaceutical composition and its use in the preparation of drugs for preventing or treating tumors. Background technique [0002] Adoptive Cell Therapy (ACT) is a method to reinfuse autologous or allogeneic immune cells that have been treated in vitro to patients to enhance the patient's immune function and achieve therapeutic purposes. Currently, tumor ACT is progressing rapidly, and has achieved very good results in the clinical treatment of various types of malignant tumors. [0003] Among various ACTs, the use of tumor infiltrating lymphocytes (Tumor Infiltrating Lymphocyte, TIL) has been proven to be a very successful method in tumor treatment. The main reason for the success of ACT ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/867C12N5/10A61K39/00A61P35/00
CPCC07K14/7051C07K14/70503C12N5/0636C12N15/86A61K39/001111A61P35/00C07K2319/02C07K2319/03C07K2319/33C12N2510/00C12N2740/15043
Inventor 龚拯李亚腾李斌李文静高青刁树青王泽瑶
Owner QINGDAO SINO-CELL BIOMEDICINE CO LTD
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