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WO2019057120A1 - Inhibiteur de la liaison entre le lgr4 et la r-spondine et son utilisation en thérapie antitumorale - Google Patents

Inhibiteur de la liaison entre le lgr4 et la r-spondine et son utilisation en thérapie antitumorale Download PDF

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Publication number
WO2019057120A1
WO2019057120A1 PCT/CN2018/106739 CN2018106739W WO2019057120A1 WO 2019057120 A1 WO2019057120 A1 WO 2019057120A1 CN 2018106739 W CN2018106739 W CN 2018106739W WO 2019057120 A1 WO2019057120 A1 WO 2019057120A1
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lgr4
phenotype
tumor
spondin
macrophage
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Chinese (zh)
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杜冰
谭炳合
李伟
刘明耀
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East China Normal University
Bioray Laboratories Inc
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East China Normal University
Bioray Laboratories Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A61K49/0008—Screening agents using (non-human) animal models or transgenic animal models or chimeric hosts, e.g. Alzheimer disease animal model, transgenic model for heart failure
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to the field of tumor immunotherapy, and more particularly to LGR4 and R-spondin binding inhibitors and their use in the treatment of tumors.
  • Lung cancer is one of the most common malignant tumors with global morbidity and mortality. Due to various factors such as diet, environment and smoking, the global incidence of lung cancer has been high. Millions of people have been diagnosed with lung cancer every year. Most of them have a survival rate of no more than five years. Lung cancer has become a serious problem that threatens human health and life. Severe bone metastasis and frequent recurrence rates are the leading causes of death in lung cancer patients. Due to the special structural structure and cellular composition of the lungs, lung cancer cells tend to have lower immunogenicity, resulting in multiple targeted therapies and immunotherapy. The therapeutic effect is extremely limited. Lung cancer often forms a special tumor microenvironment in the lungs. One of its obvious features is the infiltration of many immune cells, especially macrophages, which is closely related to the poor prognosis and lower survival rate of lung cancer patients.
  • Tumor Associated Macrophages are a class of macrophages found to be infiltrated in a variety of tumorigenic sites. Clinical data and animal experiments have demonstrated that macrophages and cancer patients in tumor tissues or The survival rate and prognosis of tumor experimental animals are closely related. At present, tumor-based macrophage-based tumor therapy has become an attractive new direction in the field of tumor immunotherapy, and immunotherapy targeting tumor-associated macrophages has great clinical value.
  • the object of the present invention is to provide a method for blocking or blocking the binding of R-spondin protein to endogenous Lgr4 receptor in tumor tissues by using Lgr4 extracellular segment protein, thereby regulating the function-dependent macrophage functional polarization conversion. Further, the application of inhibiting the occurrence and development of tumors. More specifically, the invention relates primarily to the use of Lgr4 extracellular protein in the field of tumor therapy.
  • a use of an inhibitor for inhibiting the binding of LGR4 and R-spondin for the preparation of a formulation or composition for regulating the function of macrophages Type conversion.
  • the functional phenotypic transformation of the regulated macrophage means inhibiting macrophage polarization to the M2 phenotype and/or promoting macrophage polarization to the M1 phenotype.
  • the functional phenotypic transformation of the regulated macrophage means promoting the conversion of the macrophage from the M2 phenotype to the M1 phenotype.
  • the macrophage is a tumor-associated macrophage.
  • formulation or composition is also for one or more uses selected from the group consisting of:
  • the tumor is a tumor that is enriched in macrophage infiltration and/or that highly expresses R-spondin protein.
  • the inhibitor is selected from the group consisting of:
  • the antagonist comprises a microRNA, an siRNA, a shRNA, or a combination thereof.
  • the antagonist comprises an antibody, preferably a monoclonal antibody.
  • the inhibitor is an LGR4 extracellular segment protein.
  • the LGR4 extracellular segment protein comprises an extracellular segment protein full length protein, an extracellular segment protein fragment.
  • the inhibitor is a fusion protein comprising an LGR4 extracellular domain protein.
  • the inhibitor is a CAR-T cell that integrates an LGR4 extracellular segment protein encoding gene.
  • the LGR4 and R-spondin are derived from a human or a non-human mammal.
  • amino acid sequence of the LGR4 extracellular segment protein is set forth in SEQ ID NO.: 1.
  • nucleotide sequence encoding the LGR4 extracellular segment protein is set forth in SEQ ID NO.: 2.
  • the composition is a pharmaceutical composition.
  • the pharmaceutical composition comprises (a) an inhibitor that inhibits binding of LGR4 and R-spondin; and (b) a pharmaceutically acceptable carrier.
  • the pharmaceutical composition is in the form of an injectable form or a topical pharmaceutical form.
  • the pharmaceutical composition can be administered by subcutaneous injection, intravenous injection, intramuscular injection.
  • a method of screening for a drug candidate that promotes the conversion of a macrophage from an M2 phenotype to an M1 phenotype comprising the steps of:
  • a method of non-therapeutically promoting the conversion of a macrophage from a M2 phenotype to an M1 phenotype comprising the steps of:
  • the macrophage is a tumor-associated macrophage.
  • the inhibitor is an LGR4 extracellular segment protein.
  • a method of regulating a functional phenotype conversion of a macrophage comprising the steps of:
  • the subject comprises a human and a non-human mammal.
  • Figure 1 shows that deletion of Lgr4 causes a decrease in polarization of M2 type macrophages.
  • Figure 1A shows the differential expression of Lgr4 in spleen macrophages and tumor-associated macrophages of LLC tumor-bearing mice.
  • Figure 1B shows that the Lgr4 ligand Rspo protein promotes the polarization of wild-type macrophage F4/80+CD206+M2 type macrophages, whereas the promotion of Lgr4 knockout macrophages is attenuated.
  • Figure 1C, Figure 1D and Figure 1E show the decrease in the expression levels of the M2 marker proteins Agr1, Ym-1, CD206 mRNA in Lgr4 knockout macrophages, respectively.
  • FIG. 1F shows the decreased expression levels of the M2 marker proteins Agr1, CD206 in Lgr4 knockout macrophages.
  • Figure 2 shows the difference in expression of LGR4 in different tumor tissues.
  • Figure 3 shows the construction of a prokaryotic expression vector for the extracellular domain of Lgr4.
  • FIG. 3A shows the LGR4 extracellular domain.
  • Figure 3B shows the p-ET8a prokaryotic expression vector and the LGR4 extracellular protein expression cassette insertion site.
  • Figure 4 shows that the extracellular domain of LGR4 significantly improves the tumor microenvironment and inhibits the development of LLC tumors.
  • FIG. 4A shows that LGR4 extracellular protein dose-dependently inhibits the development of LLC tumorigenesis.
  • FIG. 4B shows that Rspo/Lgr4 pathway blockade significantly inhibits the growth of LLC tumors.
  • FIG 4C shows that Rspo/Lgr4 pathway blockade reduces the proportion of M2 macrophages (F4/80+CD206+) in the LLC tumor microenvironment, while increasing CD8T cells (CD3+CD8+) and M1 macrophages (F4/80+) The ratio of CD16/32+).
  • Figure 5 shows that the purity of mature macrophages of F4/80 + can reach 98% or more after induction of culture with conditioned medium containing M-CSF colony-stimulating factor for 7 days, which meets the requirements of subsequent experiments.
  • Figure 6 shows the results of electrophoresis of the molecular weight of the expressed LGR4-ECD protein.
  • Figure 7 shows the structure of a typical shRNA.
  • the present inventors have extensively and intensively studied, and for the first time, unexpectedly found that the LGR4 gene can regulate the functional phenotypic transformation of tumor-associated macrophages in tumor tissues. Specifically, binding of the LGR4 gene to its ligand R-spondin protein promotes the polarization of tumor-associated macrophages toward the M2 phenotype.
  • the prokaryotic expression of the LGR4 extracellular segment protein has the amino acid sequence shown in SEQ ID NO: 1, and the protein can compete with the endogenous LGR4 receptor for binding to the R-spondin protein by subcutaneous injection, thereby inhibiting the tumor.
  • the invention relates to the application of Lgr4 as a drug target for regulating tumor tissue immune microenvironment and inhibiting tumor development and the application of Lgr4 extracellular protein in clinical treatment of tumor, including integrating LGR4 extracellular gene into CART to promote CART in Treatment of solid tumors.
  • the present invention has been completed on this basis.
  • Tumor Associated Macrophages are a class of macrophages found to be infiltrated in a variety of tumorigenic sites. Clinical data and animal experiments have demonstrated that macrophages and cancer patients in tumor tissues or The survival rate and prognosis of tumor experimental animals are closely related. Macrophages can be broadly classified into two broad categories according to their phenotype and function, namely the pro-inflammatory and anti-tumor M1 type and the anti-inflammatory and tumor-promoting M2 phenotype, and the two types of macrophages are different depending on the received stimulation signals. They can be transformed into each other, reflecting the high heterogeneity and plasticity of macrophages.
  • TAM has a large number of features similar to anti-inflammatory and tumor-promoting M2 macrophages, which can inhibit the anti-tumor immune response and promote tumor cell immune surveillance.
  • IL-10 IL-10
  • TGF- ⁇ vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • Cytokines and growth factors promote angiogenesis, fibrosis, and epithelial-mesenchymal transition in tumor tissues; TAM can also interact and communicate with surrounding tumor cells, stromal cells, and even other immune cells to promote tumor cell proliferation. Survival, invasion, and metastasis provide a good local microenvironment.
  • tumor-based macrophage-based tumor therapy has become an attractive new direction in the field of tumor immunotherapy, and immunotherapy targeting tumor-associated macrophages has great clinical value.
  • Lgr4 is called leucine-rich repeat-containing G protein-coupled receptor 4, also called G protein couple receptor 48. It belongs to the Lgr class G protein coupled receptor family II, Gene ID: 107515.
  • the Lgr4 receptor binds to the R-spondin protein family (R-spondin 1-4) with high specificity and high affinity, and its IC50 is only 2-230 nM, so the secreted protein family is considered to be an endogenous ligand for Lgr4.
  • Lgr4 binds to R-spondin protein and greatly enhances the classical wnt/ ⁇ -catenin signaling pathway in adult stem cell and hair follicle stem cell development, skeletal cell balance, reproductive tract development, eyelid development, and even pattern recognition in macrophages. Both play an important regulatory role. However, the binding of Lgr4 to the R-spondin protein does not activate the classical G protein signaling pathway.
  • LGR4 and its ligand R-spondin protein are abnormally expressed in various human malignant tumors, and in most cases, their expression is higher than normal tissues, including lung cancer, gastric cancer, colorectal cancer, breast cancer, prostate cancer, etc.
  • LGR4/R-spondin promotes the proliferation of tumor cells through wnt or wnt-related signaling pathways.
  • Lgr4 is also up-regulated in tumor-associated macrophages in mouse lung cancer tissues compared to normal tissue macrophages, and it has been demonstrated in vivo and in vitro that Lgr4 positively regulates the function of the M2 phenotype of macrophages.
  • High expression of R-spondin in lung cancer tissues and Lgr4 in lung cancer-associated macrophages suggests that it can directly act on tumor cells by blocking the interaction of Lgr4/R-spondin in lung cancer tissues.
  • by regulating the functional phenotype of tumor-associated macrophages to regulate the microenvironment on which tumor cells depend, and thus inhibiting the occurrence and development of tumors it has great application value and significance for tumor immunotherapy.
  • LGR4 and R-spondin binding inhibitors As used herein, “LGR4 and R-spondin binding inhibitors”, “inhibitors that inhibit LGR4 and R-spondin binding” are used interchangeably and refer to a formulation or composition that is capable of specifically inhibiting the binding of LGR4 and R-spondin.
  • the LGR4 and R-spondin binding inhibitors are LGR4 inhibitors, R-spondin inhibitors, LGR4 structural analogs, and/or R-spondin structural analogs.
  • the LGR4 and R-spondin binding inhibitor is the LGR4 extracellular protein represented by SEQ ID NO.: 1.
  • the LGR4 and R-spondin binding inhibitor is an anti-LGR4 monoclonal antibody and/or an anti-LGR4 monoclonal antibody.
  • RNA interference RNA interference
  • a class of potent LGR4 and R-spondin binding inhibitors are interfering RNAs.
  • RNA interference means that some small double-stranded RNA can efficiently and specifically block the expression of specific genes in the body, promote mRNA degradation, and induce cells to exhibit specific gene deletions. Phenotype, which is also known as RNA intervention or RNA interference. RNA interference is a highly specific mechanism of gene silencing at the mRNA level.
  • small interfering RNA refers to a short-segment double-stranded RNA molecule that is capable of degrading specific mRNAs with mRNAs of homologous complementary sequences. This process is the RNA interference pathway (RNA). Interference pathway).
  • interfering RNA includes siRNA, shRNA, and corresponding constructs.
  • the interfering RNA may specifically target an interfering RNA of LGR4, and/or an interfering RNA specific for R-spondin.
  • the interfering RNA can specifically target a binding region of LGR4 to R-spondin.
  • Seq 'Forward Forward sequence corresponds to Seq RNA sequences or fragments of sequences
  • Seq' reverse is a sequence that is substantially complementary to the Seq' forward ;
  • the present invention provides the use of an inhibitor that inhibits the binding of LGR4 and R-spondin for the preparation of a formulation or composition for regulating the functional phenotypic transformation of macrophages.
  • the invention also provides a method of modulating a functional phenotype switch of a macrophage comprising the steps of: (i) administering to a subject in need thereof an inhibitor that inhibits binding of LGR4 and R-spondin or a composition comprising the inhibitor.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an LGR4 and R-spondin binding inhibitor and a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, powders, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration.
  • the pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • Pharmaceutical compositions such as tablets and capsules can be prepared by conventional methods.
  • Pharmaceutical compositions such as injections, solutions, tablets and capsules are preferably manufactured under sterile conditions.
  • the pharmaceutical combination of the invention may also be formulated as a powder for nebulization.
  • a preferred dosage form is an oral formulation.
  • the pharmaceutical compositions of the invention may also be used with other therapeutic agents.
  • the pharmaceutical composition of the present invention When used in the above-mentioned use, it may be mixed with one or more pharmaceutically acceptable carriers or excipients such as a solvent, a diluent, etc., and may be orally administered in the form of a tablet: , pills, capsules, dispersible powders, granules or suspensions (containing, for example, about 0.05-5% suspension), syrup (containing, for example, about 10-50% sugar), and elixirs (containing about 20-50% ethanol), or Parenteral administration is carried out as a sterile injectable solution or suspension (containing about 0.05-5% suspending agent in isotonic medium).
  • these pharmaceutical preparations may contain from about 0.01% to about 99%, more preferably from about 0.1% to about 90%, by weight of the active ingredient in admixture with the carrier.
  • compositions of this invention may be administered by conventional routes including, but not limited to, intramuscular, intraperitoneal, intravenous, subcutaneous, intradermal, oral, intratumoral or topical administration.
  • routes of administration include oral administration, intramuscular administration or intravenous administration, and administration by smearing.
  • compositions of the invention may also be combined with other agents for treating tumors.
  • the present invention screens out the Lgr4 gene and reveals that Lgr4/R-spondin is involved in the polarization process of M2 phenotypic function of macrophages; based on this fact, Lgr4 extracellular domain protein is used as a competitive binding protein of R-spondin1 in tumor tissues. Inhibition of tumor-associated macrophages in tumor tissues toward M2-type polarization of tumor-promoting, and at the same time increasing the proportion of M1-type macrophages and CD8 + T cells, thereby inhibiting the occurrence and development of tumors.
  • the present invention uses Lgr4/R-spondin1 as a molecular target or a drug target to treat tumor types with high expression.
  • the present invention provides an isolated and purified Lgr4 extracellular segment protein having the amino acid sequence of SEQ ID NO: 1, which is located at positions 28-528 of the amino acid sequence of the Lgr4 protein.
  • the Lgr4 extracellular segment protein of the present invention can specifically bind to R-spondin1, which can be obtained by prokaryotic expression or expression of mammalian cells, and can be expressed as a monomer or a fusion protein and binds to R-spondin1 protein. .
  • the present invention constructs a prokaryotic expression vector of Lgr4 extracellular segment protein, which is a PET28a expression vector, and the constructed expression vector can be called PET28a-Lgr4ECD expression vector, expressed by host E. coli BL21 strain, wherein ECD means cell Outer binding domain.
  • ECD means cell Outer binding domain.
  • the Lgr4 extracellular domain has been crystallized, and its binding to the R-spondin protein region has been clarified.
  • the present invention still adopts a strategy for expressing the full-length sequence of the extracellular segment of Lgr4, and the full-length amino acid sequence of the extracellular segment is shown in SEQ ID NO: 1.
  • the Lgr4 extracellular segment protein of the present invention is administered subcutaneously, and the experimental mice have no obvious adverse reactions after the injection.
  • the above proteins may also be administered intravenously, intramuscularly or by injection.
  • the 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 as required (10% fetal bovine serum, 100 ⁇ g/ml streptomycin). , 100 U / ml penicillin, non-essential amino acids were purchased from GIBCO and diluted according to the instructions for use).
  • the LLC cells were cultured in a 6 cm culture dish. When they were as long as 80%-90% of the bottom area, the upper medium was discarded, and the PBS buffer was washed twice at 37 ° C, and 500 ⁇ l of 0.25% pancreas was added to the dish.
  • the protease + 0.02% EDTA was digested at 37 ° C for 30 s, then the digestion was terminated by adding an equal volume of DMEM complete medium, centrifuged, and the cells were resuspended in PBS buffer, and the cell concentration was adjusted to 1 ⁇ 10 7 /ml.
  • the above cell suspension was subcutaneously injected into C57BL/6 mice (East China Normal University Experimental Animal Center, SPF grade) of about 8 weeks old, and each mouse was injected with 200 ⁇ l.
  • mice Two weeks later, the tumor-bearing mice were sacrificed by cervical dislocation.
  • the tumor tissue and spleen were surgically removed and lysed in 5 ml of 0.25% trypsin digest for 5 minutes. Digestion was carried out by adding an equal volume of DMEM complete medium, and centrifugation was performed. The cells were then resuspended in PBS and the cell density was adjusted to 1 ⁇ 10 8 /ml.
  • BD Flow cytometry
  • Trizol lysate TAKARA
  • RNA precipitation was dried, it was dissolved in 50 ⁇ l of RNAase Free water or DEPC water. Total RNA extraction concentration and purity were tested.
  • the extracted total RNA was subjected to reverse transcription to obtain cDNA.
  • the kit used was a kit manufactured by TAKARA. The procedure and procedure were in accordance with the manufacturer's instructions.
  • the total amount of reverse-transcribed RNA was 1000 ng, and the reaction procedure was: 37 ° C for 30 minutes. Store at 85 ° C for 5 seconds at 12 ° C.
  • the cDNA obtained by the above reverse transcription was used as a template, ⁇ -actin was used as an internal reference gene, and the expression level of Lgr4 was detected by real-time quantitative PCR.
  • the kit used was a SYBR kit produced by TAKARA, and the reaction system, operating procedures and procedures were in accordance with the production specifications.
  • the method of inducing BMM in vitro is as follows:
  • the above cell suspension was filtered through a 45 ⁇ m pore size sieve, counted, and plated. It is preferred to inoculate 1 x 10 7 total cells in a 6 cm culture dish.
  • the cells were cultured in an incubator at 37 ° C, 5% CO 2 and a suitable humidity for 5-7 days, and the culture solution was changed every 2 days.
  • the cells were digested with trypsin (0.25% trypsin + 0.02% EDTA) for 10 minutes, terminated, resuspended, adjusted to a cell density of 1 ⁇ 10 7 /ml, and plated at six wells.
  • the culture plate was 1 ⁇ 10 6 per well, and the medium was changed to normal DMEM complete medium, followed by 10 ng/ml IL-4, 500 ng/ml recombinant mouse R-spondin1 (R&D), 500 ng/ml recombinant mouse R.
  • the cells were treated with -spondin3 and treated at different times or time points according to different subsequent detection levels.
  • the conditioned medium components for inducing BMM were: DMEM basal medium, 10% FBS, 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 15% L929 cell culture supernatant.
  • the Lgr4 +/+ and Lgr4 -/- BMM prepared in Example 2 were cultured in a six-well culture plate, and replaced with normal DMEM complete medium, and then treated with 500 ng/ml R-spondin 1,500 ng/ml R-spondin 3 for 24 hours. Digest the cells, resuspend, adjust the cell density to 1 ⁇ 10 6 /ml, and take 200 ⁇ l of the cell suspension in a flow analysis tube for staining.
  • APC-labeled rat anti-mouse F4/80 monoclonal antibody APC-anti-F4/80 monoclonal antibody
  • FITC-labeled rat anti-mouse CD206 monoclonal antibody FITC-anti-CD206
  • Fig. 1B The results are shown in Fig. 1B, in which the F4/80 + CD206 + cells are M2 type macrophages, and it can be seen that both R-spondin1 and R-spondin3 can promote Lgr4 +/+ instead of Lgr4 -/- macrophages toward the M2 direction. Chemical.
  • the Lgr4 +/+ and Lgr4 -/- BMM prepared in Example 2 were cultured in a six-well culture plate, replaced with normal DMEM complete medium, and treated with 10 ng/ml recombinant mouse IL-4 for 4 hours, and the culture medium was discarded.
  • the buffer was washed twice, total RNA was extracted by Trizol (TAKARA) method, cDNA was obtained by reverse transcription, and the expression levels of Ym1, Arg1 and CD206 in each group were detected by real-time quantitative PCR.
  • the kit used was a SYBR kit manufactured by TAKARA, and the reaction system, procedures and procedures were followed by the manufacturer's instructions.
  • the Lgr4 +/+ and Lgr4 -/- BMM prepared in Example 2 were cultured in a six-well culture plate, replaced with normal DMEM complete medium, and then treated with 10 ng/ml recombinant mice for 24 hours, and the culture medium and PBS buffer were discarded. Wash twice, each well with 200 ⁇ l of strong RIPA lysate containing protease phosphatase inhibitor cocktail (Selleckchem, USA 100 ⁇ stock solution) (500 mM Tris pH 7.0, 150 mM NaCl, 0.1% Triton-X-100, 1% deoxygenation Sodium cholate, 0.1% SDS) was lysed on ice for 30 minutes.
  • Selleckchem strong RIPA lysate containing protease phosphatase inhibitor cocktail
  • Cell lysates were collected into clean EP tubes, centrifuged at 12000 rpm/min for 2 minutes, and the supernatant was transferred to another clean EP tube, 40 ⁇ l of each tube was added.
  • Loading Buffer (formulation see below), mix and then cook in a 100 ° C water bath for 10 minutes, then carry out the fully denatured sample for SDS-PAGE gel electrophoresis, 5% polyacrylamide concentrate, 10% polyacrylamide Separate the glue. After loading 15 ⁇ l per well and electrophoresis at 60 V for 20 minutes, replace 100V high voltage electrophoresis for 120 minutes. Stop the electrophoresis and transfer the gel with protein to 0.2 ⁇ m nitrocellulose membrane (NC membrane, Millipore, USA).
  • PBS buffer sodium chloride (NaCl), 8g; potassium chloride (KCl), 0.2g; disodium hydrogen phosphate (Na2HPO4), 1.44g; potassium dihydrogen phosphate (KH2PO4), 0.24g; pH 7.2, set Tolerance to 1L.
  • 1 x running buffer 25 mM Tris, 250 mM glycine, 0.1% SDS.
  • 1 x transfer buffer 48 mM Tris, 39 mM glycine, 0.037% SDS, 20% methanol.
  • PBST PBS buffer containing 0.1% Tween20.
  • the electrophoresis tank and the transfer tank extremely supporting equipment were purchased from BioRad.
  • the TCGA https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp
  • oncomine https://www.oncomine.org/resource/login.html
  • tumor database searches are performed according to the site instructions.
  • the TCGA database was searched using the cbioportal (http://www.cbioportal.org/index.do) search tool.
  • the Lung Adenocarcinoma (TCGA, Provisional) data set was searched for the lung adenocarcinoma patient database; the oncomine search was performed using the RSOP1 , "cancer vs normal analysis" and "TCGA Lung 2" data sets.
  • LGR4 and its ligand RSPO1 are highly expressed in tumor specimens of lung adenocarcinoma patients.
  • the prokaryotic expression vector of Lgr4 was constructed by the applicant in the early stage.
  • the expression vector backbone was PET28a(+), and the polynucleotide sequence of LGR4 gene was ligated into its multiple cloning site by genetic engineering technique. After expression, the extracellular domain of the target protein Lgr4 was expressed. The N-terminal fusion of the protein expresses a 6 ⁇ his tagged protein.
  • the recombinant plasmid was transformed into BL21 Escherichia coli and coated with kanamycin-resistant LB agarose culture plate. After overnight culture, the monoclonal positive colonies were picked and placed in a LB liquid medium shaker culture containing kanamycin resistance.
  • the overnight bacterial solution was transferred to 1 L of fresh LB liquid medium containing kanamycin resistance, and shaken at 37 ° C, 220 rpm / min shaker.
  • the OD value of the bacterial liquid was monitored by a spectrophotometer at any time. When the OD value of the bacterial liquid reached 0.6-0.8, 1 ml of 0.8 M IPTG was added for induction expression. Before the induction, 1 ml of the bacterial solution was centrifuged at 2000 rpm/min for two minutes, the supernatant was discarded, 100 ⁇ l of 1 ⁇ SDS loading Buffer was added to the cells, and the sample was boiled in a 100 ° C water bath for 10 minutes. As an uninducible control group, the expression time of IPTG induction was 4 hour.
  • the crushing condition was: 400 W, each ultrasonic time was 5 seconds, the interval was 5 seconds, and the number was 300 times. Before the ultrasonication, 1 ml of protease inhibitor cocktail (Selleckchem, 100 ⁇ stock solution) was added to the bacterial solution.
  • protease inhibitor cocktail Selleckchem, 100 ⁇ stock solution
  • Protein collection and purification Centrifuge the sonicated liquid at 11000 rpm/min for 10 minutes, carefully aspirate the supernatant, take a small amount of precipitate and supernatant, add 100 ⁇ l of 1 ⁇ SDS loading Buffer, and boil for 10 minutes in a 100 °C water bath. It is left for electrophoresis loading, and the secreted expressed protein is mainly present in the supernatant, and the protein is mainly present in the precipitate when the inclusion body is expressed.
  • the target protein is fused to express the 6 ⁇ his tagged protein
  • the target protein can be purified by nickel column affinity chromatography, and the nickel column used is Ni-NTA Agarose purified beads produced by QIAGEN. Before protein purification:
  • Ni-NTA Agarose used was stored in a 20% ethanol solution and stored at 4 °C. 4 ml of Ni-NTA Agarose beads were poured into a plastic column, and the mixture was slightly sedimented. The column was filled with double dehydration column and washed twice, and the effluent was discarded. The above operation was carried out at 4 °C.
  • Protein-like column purification The sample solution is carefully filled into the column, sub-perfused, and the column is allowed to stand, and the effluent is discarded. The above operation is carried out at 4 °C.
  • washing the nickel column After the sample is over the column, fill the nickel column with the washing buffer, let stand the column, repeat twice, and discard the effluent. The above operation is carried out at 4 °C.
  • Elution of protein 2 ml of elution buffer was added to the washed nickel column, and the effluent was collected and subjected to 4 °C.
  • Protein Concentration and Storage Select a concentrated ultrafiltration tube (purchased from Millipore, USA) of 7OKD size, and wash it twice with double dehydration before use. The cleaning process: centrifugation at 4 ° C, 6000 rpm / min, 5 minutes.
  • the waste liquid at the bottom of the collection tube was drained, and the sterile PBS buffer was added to the ultrafiltration tube. After the filling was completed, the centrifugation was continued, and this was repeated twice, and finally the PBS was buffered. The liquid replaces the imidazole in the eluent.
  • the remaining liquid in the upper layer of the ultrafiltration tube is the concentrated protein of interest.
  • the total protein concentration is determined by the BCA method, it is stored in an ultra-low-range refrigerator at -80 ° C, and avoids repeated freezing and thawing during use.
  • Lysis buffer 50 mM Tris, 300 mM NaCl, pH 8.0
  • Wash buffer 50 mM NaH 2 PO 4 , 300 mM NaCl, pH 8.0
  • Elution buffer 50 mM NaH 2 PO 4 , 300 mM NaCl, 250 mM imidazole, pH 8.0
  • Equilibration buffer 50 mM NaH 2 PO 4 , 300 mM NaCl, 20 mM imidazole, pH 8.0
  • the molecular weight of the expressed LGR4-ECD protein was about 72 KD, and the purity was high, and there was no impurity band.
  • mice of 8 weeks old Male C57BL/6 mice of 8 weeks old were divided into three groups of 8 mice each.
  • the pentobarbital sodium was anesthetized, and the back hair was removed with a stripper, and then each mouse was subcutaneously injected with 5 ⁇ 10 5 LLC cells in the left back.
  • the mice in each group were subcutaneously administered to the right side of the PBS blank control group, 10 ⁇ g dose group, and 20 ⁇ g dose group.
  • Fig. 4A shows changes in tumor volume over time. It can be seen that as the concentration of Lgr4 extracellular protein is increased, the tumor volume is significantly inhibited.
  • mice of about 8 weeks old Male C57BL/6 mice of about 8 weeks old were divided into six groups of 8 mice each.
  • the pentobarbital sodium was anesthetized, and the back hair was removed with a stripper, and then each mouse was subcutaneously injected with 5 ⁇ 10 5 LLC cells in the left back.
  • mice were administered subcutaneously to the right side of the six groups of mice: PBS blank control group, 20 ⁇ g Lgr4 extracellular protein dose group, irrelevant antibody IgG control group, anti-mouse R-spondin1 Monoclonal antibody (14 ⁇ g per mouse according to the neutralization of the instructions), DMSO blank control group, BLZ945 (CSF-1R small molecule inhibitor, can reduce M2 macrophage in tumor microenvironment) 200mg /kg body recombination (this group can be used as a positive control group).
  • Fig. 4B showing the change of tumor volume with time. It can be seen that with the increase of Lgr4 extracellular protein dose, anti-mouse R-spondin1 monoclonal antibody and BLZ945 have obvious inhibitory effects on tumor volume in mice. .
  • mice The tumor tissues of the above six groups of mice were surgically excised and minced in 5 ml of 0.25% trypsin digest for 5 minutes. The digestion was terminated by adding an equal volume of DMEM complete medium, and then centrifuged, then resuspended in PBS, and the cells were adjusted. Density to 1 ⁇ 10 8 /ml.
  • both the Lgr4 extracellular protein and the anti-mouse R-spondin1 monoclonal antibody significantly reduced M2 tumor-associated macrophages and enhanced CD8 + T cell infiltration.

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Abstract

L'invention concerne un inhibiteur de la liaison entre le LGR4 et la R-spondine et son utilisation en thérapie antitumorale. En particulier, grâce à l'inhibition de la liaison entre le LGR4 et la R-spondine, la polarisation des macrophages de type M2 associés à une tumeur peut être inhibée, tandis que le rapport entre les macrophages M1 à effet antitumoral et les lymphocytes T CD8+ dans le tissu tumoral est accru, si bien que la présente invention peut être utilisée pour traiter des types de tumeurs dans lesquelles l'infiltration des macrophages est importante et qui présentent une forte expression de protéines R-spondine.
PCT/CN2018/106739 2017-09-21 2018-09-20 Inhibiteur de la liaison entre le lgr4 et la r-spondine et son utilisation en thérapie antitumorale Ceased WO2019057120A1 (fr)

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