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Cell strain for detecting activity of antibody-immune agonist coupled drug and detection method

A technology for drug activity and antibody detection, applied in botany equipment and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc., can solve the problem of large difference in PBMC activity, high cost, PBMC and antigen-positive cells Co-cultivation system is not sensitive enough to achieve the effect of simple operation and low cost

Active Publication Date: 2022-04-26
GENEQUANTUM HEALTHCARE SUZHOU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Cell strain for detecting activity of antibody-immune agonist coupled drug and detection method
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  • Cell strain for detecting activity of antibody-immune agonist coupled drug and detection method

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preparation example Construction

[0102] The preparation method of "trastuzumab" in the present invention mainly includes: transferring the nucleic acid sequences of the heavy chain and the light chain into expression cells, culturing and purifying the obtained.

[0103] 2. 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: HER2, TLR7 and NF-κB-alkaline phosphatase are expressed in HEK293 cells, which are used to detect the activity of TLR7 immune agonists targeting HER2 antibodies; another example: PD-L1, TLR8 and NF- κB-luciferase, used to detect the activity of an antibody-TLR8 immune agonist targeting PD-L1.

[0105] Cell line selection method:

[0106] Method 1: TLR7 agonist or TLR8 agonist reporter gene cell lines for detection of small molecule agonists can be purchased (such as HEK-hTLR7 (HEK-Bl...

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, and can also be 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 cell confluence of 70%~80% and good growth state, incubate with PBS for 2~3min, beat the cells and blow them with a pipette, and trypan blue staining and counting.

[0136] 2. Resuspend the cells with HEK medium (for detecting the activity of alkaline phosphatase, HEK-Blue medium from InvivoGen (Product No. hb-det2)) and adjust the cell density to 4×10 5 cells / ml, plated at 100 µL / well, and the experimental group was added with immune stimulants (see Table 1 for the immune stimulants used in this example). ...

Embodiment 2

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

[0147] 1. HER2-GFP plasmid construction, lentiviral packaging concentration and titer detection are provided by Jinweizhi Biotechnology Co., Ltd. HER2-GFP lentiviral expression plasmid map as shown in 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 status, digest, count, and inoculate about 1.5×10 6 Cells were cultured in T25 flasks and cultured overnight at 37°C in a cell culture incubator.

[0149] 3. Prepare a mixture of DMEM complete medium and polybrene, the final concentration of polybrene is 10µg / mL; before infection, take it out from the refrigerator and put it on ice to melt the virus stock solution, suck off the original cell culture medium, and add DMEM The mixture of complete medium and polybrene, then add the virus stock solution into the cells, the MOI (Multiplicity of inf...

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Abstract

The invention provides a cell strain for detecting the activity of an antibody-immune agonist coupled drug and a detection method. The cell strain expresses an antigen or a fragment thereof capable of being combined with an antibody in the antibody-immune agonist coupled drug, a receptor capable of being combined with an immune agonist in the antibody-immune agonist coupled drug, and an immune signal path reporter gene. The cell strain provided by the invention can be used for activity detection of immune agonist coupled drugs with different target antigens and different activity levels. The detection method disclosed by the invention can overcome the defects of a PBMC and antigen positive cell co-culture mode, and has the advantages of simplicity in operation, low cost, higher sensitivity, larger detection dynamic range and the like; and the activity result of the antibody-immune agonist coupled drug obtained by the detection method is closer to the verification result of an in-vivo pharmacological experiment.

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 method, and it 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. The currently widely used clinical tumor immunotherapy uses checkpoint inhibitors (checkpoint inhibitors, CPIs) represented by monoclonal antibodies targeting PD-1 and PD-L1, which have shown good results in multiple tumor types. However, 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 availabili...

Claims

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Application Information

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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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