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A kind of cell line and detection method for detecting the activity of antibody-immune agonist conjugated drug

A technology for detecting antibody and drug activity, applied in the detection of programmed cell death, animal cells, vertebrate cells, etc., can solve the problems of high cost, large difference in PBMC activity, cumbersome detection methods, etc., and achieve low cost and simple operation Effect

Active Publication Date: 2022-07-01
GENEQUANTUM HEALTHCARE SUZHOU
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0007] 1. The detection method of the PBMC and antigen-positive cell co-culture system is relatively cumbersome, involving multiple steps such as blood collection, PBMC separation, cell culture, plating and ELISA, and the cost of materials and labor is high;
[0008] 2. The activity of PBMCs from different blood donors varies greatly, which has a significant impact on the experimental results;
[0009] 3. For some agonists with relatively weak activity, the co-culture system of PBMC and antigen-positive cells is not sensitive enough, and the changes of cytokines are often unable to be detected;
[0010] 4. For different targets and agonists, different inflammatory cytokines need to be screened to select appropriate detection indicators, which cannot be used universally among multiple targets and agonists

Method used

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  • A kind of cell line and detection method for detecting the activity of antibody-immune agonist conjugated drug
  • A kind of cell line and detection method for detecting the activity of antibody-immune agonist conjugated drug
  • A kind of cell line and detection method for detecting the activity of antibody-immune agonist conjugated drug

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preparation example Construction

[0102] The preparation method of "trastuzumab" in the present invention is mainly as follows: the nucleic acid sequences of the heavy chain and the light chain are transferred into the expression cells, which are obtained after culturing and purifying.

[0103] Second, the construction of cell lines:

[0104] The genome of the reporter gene cell line includes the target gene (the nucleic acid sequence encoding the tumor antigen or its fragment), the nucleic acid sequence encoding TLR7 or TLR8 and the reporter gene. For example: HEK293 cells express HER2, TLR7 and NF-κB-alkaline phosphatase to detect the activity of antibodies targeting HER2-TLR7 immune agonists; another example: HEK293 cells express PD-L1, TLR8 and NF- κB-luciferase to detect the activity of PD-L1-targeting antibody-TLR8 immune agonists.

[0105] How to choose a cell line:

[0106] Method 1: TLR7 agonist or TLR8 agonist reporter gene cell lines for small molecule agonist detection can be purchased (such as H...

Embodiment 1

[0133] Activity detection of TLR7 / 8 immune agonists in HEK-hTLR7 and HEK-hTLR8 cell lines

[0134] The reporter gene cell line of TLR7 or TLR8 agonist used for small molecule detection can be purchased commercially or constructed according to the experimental method mentioned above. In this example, the reporter gene cell line of TLR7 or TLR8 agonist (HEK - hTLR7 and HEK-hTLR8) were purchased from InvivoGen.

[0135] 1. Take HEK-hTLR7 and HEK-hTLR8 cells with a cell confluency of 70%-80% and a good growth state respectively, incubate with PBS for 2-3 minutes, scatter the cells and disperse them with a pipette, and count them by trypan blue staining.

[0136] 2. Resuspend the cells in HEK medium (for detecting the activity of alkaline phosphatase, InvivoGen's HEK-Blue medium, the product number is hb-det2), adjust the cell density to 4 × 10 5 Cells / ml, plated at 100µL / well, and immune agonists were added to the experimental group (the immune agonists used in this example are s...

Embodiment 2

[0146] Construction of HEK-hTLR7-HER2-GFP and HEK-hTLR8-HER2-GFP stably transfected cell lines

[0147] 1. The HER2-GFP construction plasmid, lentiviral packaging concentration and titer detection are provided by Golden Vision Biotechnology Co., Ltd. HER2-GFP lentiviral expression plasmid map such as Figure 4 shown.

[0148] 2. Inoculate cells: take HEK-hTLR7 cell lines and HEK-hTLR8 cell lines with a confluence of 70%~80% and good growth conditions, digest, count, and inoculate about 1.5×10 cells. 6 Cells were cultured in T25 flasks and placed in a cell incubator at 37°C overnight.

[0149] 3. Prepare a mixture of DMEM complete medium and polybrene, and the final concentration of polybrene is 10 µg / mL; before infection, remove the virus stock solution from the refrigerator and place it on ice to thaw the virus stock solution, aspirate the original cell culture medium, and add DMEM A mixture of complete medium and polybrene, and then the virus stock solution was added to t...

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Abstract

The present invention provides a cell strain and a detection method for detecting the activity of an antibody-immune agonist conjugated drug. ‑Immune agonist receptors and immune signaling pathway reporter genes in immune agonist drug conjugates. The cell line of the present invention can be used for the activity detection of different target antigens and immune agonist conjugated drugs with different activity levels. The detection method of the present invention can overcome the shortcomings of the co-culture method of PBMC and antigen-positive cells, and has the advantages of simple operation, low cost, higher sensitivity, larger detection dynamic range, and the like, and the antibody obtained by the detection method of the present invention has the advantages of ‑The activity results of the immune agonist-conjugated drug are closer to the results of in vivo efficacy experiments.

Description

technical field [0001] The invention relates to a method for detecting the activity and efficacy of an antibody-immune agonist conjugated drug, in particular to a cell line and a detection method for detecting the activity of an antibody-immune agonist conjugated drug. Background technique [0002] Tumor immunotherapy is a very promising cancer treatment and is also a hot direction in drug development. The principle of tumor immunotherapy is to eliminate tumors and prevent tumor recurrence by enhancing host immune function. Currently widely used in clinical tumor immunotherapy is the use of checkpoint inhibitors (checkpoint inhibitors, CPI) represented by monoclonal antibodies targeting PD-1 and PD-L1, which have shown good results in multiple tumor types efficacy, but many cancer patients remain unresponsive to checkpoint inhibitors or develop new disease progression after treatment. One reason is that many solid tumors are internally immunosuppressed, with low neoantigen...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/10C12N15/867C12N15/65C12N15/12C12Q1/02
CPCC12N5/0686C12N15/86C12N15/65C07K14/70596C07K14/82C07K14/71G01N33/5041G01N33/5008C12N2510/00C12N2740/15043C12N2800/107C12N2503/02G01N2500/10
Inventor 徐涵文马赛秦刚
Owner GENEQUANTUM HEALTHCARE SUZHOU
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