lgr4 and r-spondin binding inhibitors and their use in tumor therapy

A technique for inhibitors and uses, which is applied in the field of LGR4 and R-spondin binding inhibitors and its application in tumor therapy, and can solve problems such as limited therapeutic effect and low immunogenicity

Active Publication Date: 2020-11-13
EAST CHINA NORMAL UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Severe bone metastasis and frequent recurrence rate are the main causes of death of lung cancer patients. Due to the special structural structure and cell composition of the lung, lung cancer cells often have low immunogenicity, resulting in a variety of targeted therapies and immunotherapy. Therapeutic effect is extremely limited

Method used

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  • lgr4 and r-spondin binding inhibitors and their use in tumor therapy
  • lgr4 and r-spondin binding inhibitors and their use in tumor therapy
  • lgr4 and r-spondin binding inhibitors and their use in tumor therapy

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Construction of mouse LLC lung cancer cell subcutaneous xenograft model and detection of Lgr4 expression level in tumor-associated macrophages

[0103] Mouse Lewis lung cancer cell line LLC cells were purchased from the American Type Culture Collection (ATCC) and cultured in dulbecco's modified eagle medium (DMEM) complete medium (10% fetal bovine serum, 100 μg / ml streptomycin) according to its instructions. , 100U / ml penicillin, non-essential amino acids were purchased from GIBCO and diluted according to the instruction manual). Cultivate LLC cells in a 6cm culture dish. When it grows to 80%-90% of the bottom area of ​​the dish, discard the upper medium, wash twice with preheated PBS buffer at 37°C, and add 500μl 0.25% trypsin to the dish. Protease + 0.02% EDTA was digested at 37°C for 30s, then an equal volume of DMEM complete medium was added to stop the digestion, centrifuged, and the cells were resuspended with PBS buffer, and the cell concentration was adjusted to...

Embodiment 2

[0113] Induction and culture of mouse bone marrow derived macrophages (BMM) in vitro

[0114] The method of in vitro induction and culture of BMM is as follows:

[0115] 1. Take 6-8 week old C57BL / 6 mice, kill them by cervical dislocation, and disinfect the body surface with 75% alcohol.

[0116] 2. Cut open the abdomen and peripheral skin to expose the groin, and cut off the two hind limbs of the mouse along the groin, taking care not to cut off the femur.

[0117] 3. Use scissors and tweezers treated with 75% alcohol to peel off the muscles of the hindlimb, and separate the tibia and femur (femur and shank).

[0118] 4. In a sterile environment (such as in an ultra-clean workbench), cut open the two ends of the tibia and femur in sequence to expose the bone marrow cavity. Use a syringe to absorb about 5ml of BMM conditional culture and repeat blowing until the bone marrow cavity turns white. The femur is thin, and it is recommended to use a 1ml syringe for flushing.

[01...

Embodiment 3

[0126] Flow cytometric staining of BMM

[0127] The Lgr4 that embodiment 2 prepares + / + and Lgr4 - / - BMM was cultured in a six-well culture plate, replaced with ordinary DMEM complete medium, and treated cells with 500ng / ml R-spondin1 and 500ng / ml R-spondin3 for 24 hours, digested cells, resuspended, and adjusted the cell density to 1×10 6 / ml, take 200 μl of cell suspension and place it in a flow analysis tube for staining. Under dark conditions, use APC-labeled rat anti-mouse F4 / 80 monoclonal antibody (APC-anti-F4 / 80) and FITC-labeled rat anti-mouse CD206 monoclonal antibody (FITC-anti-CD206) in sequence Incubate at 4°C in the dark for 40 minutes, the amount of antibody used is 0.1 μg / 10 5 cell. After staining, each tube was washed with 1ml of PBS buffer containing 0.2% fetal bovine serum three times (800rpm / min, 3 minutes), and finally the cells were resuspended in 200μl of PBS buffer for flow cytometry analysis. Set the isotype control group, single-staining group and...

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Abstract

A LGR4 and R-spondin binding inhibitor and a use thereof in tumor therapy; specifically, by means of inhibiting the binding of LGR4 and R-spondin, M2-type polarization of tumor-associated macrophages may be inhibited, while the ratio of M1 macrophages having an anti-tumor function to CD8+ T lymphocytes in tumor tissue is increased, and thus the present invention may be used to treat types of tumors that are rich in macrophage infiltration and that have a high expression of R-spondin proteins.

Description

technical field [0001] The present invention relates to the field of tumor immunotherapy, and more specifically relates to LGR4 and R-spondin binding inhibitors and their application in tumor treatment. Background technique [0002] Lung cancer is one of the malignant tumors with the highest morbidity and mortality in the world. Due to the effects of diet, environment, smoking and other factors, the incidence of lung cancer in the world has always been high, and millions of people are diagnosed with lung cancer every year. , most of which have a survival rate of no more than five years. Lung cancer has become a chronic disease that seriously threatens human health and life. Severe bone metastasis and frequent recurrence rate are the main causes of death of lung cancer patients. Due to the special structural structure and cell composition of the lung, lung cancer cells often have low immunogenicity, resulting in a variety of targeted therapies and immunotherapy. Therapeutic ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K45/00A61K38/17A61K49/00A61P35/00
CPCA61K38/1709A61K45/00A61K49/0004A61K49/0008A61K38/17A61K49/00A61P35/00
Inventor 杜冰谭炳合李伟
Owner EAST CHINA NORMAL UNIV
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