WO2019057120A1

WO2019057120A1 - Lgr4 and r-spondin binding inhibitor and use thereof in tumor therapy

Info

Publication number: WO2019057120A1
Application number: PCT/CN2018/106739
Authority: WO
Inventors: 杜冰; 谭炳合; 李伟; 刘明耀
Original assignee: 上海邦耀生物科技有限公司; 华东师范大学
Priority date: 2017-09-21
Filing date: 2018-09-20
Publication date: 2019-03-28
Also published as: CN109529040A; CN109529040B

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

LGR4 and R-spondin binding inhibitors and their use in tumor therapy

Technical field

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.

Background technique

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 (TAM) 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.

Summary of the invention

It is an object of the present invention to provide an LGR4 and R-spondin binding inhibitor and its use in the treatment of tumors.

Specifically, 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.

In a first aspect of the invention, there is provided 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.

In another preferred embodiment, 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.

In another preferred embodiment, the functional phenotypic transformation of the regulated macrophage means promoting the conversion of the macrophage from the M2 phenotype to the M1 phenotype.

In another preferred embodiment, the macrophage is a tumor-associated macrophage.

In another preferred embodiment, the formulation or composition is also for one or more uses selected from the group consisting of:

(i) increasing the proportion of M1 phenotype macrophages;

(ii) increasing the proportion of CD8 ⁺ T cells;

(iii) treating the tumor;

(iv) Regulating the immune microenvironment of tumor tissue.

In another preferred embodiment, the tumor is a tumor that is enriched in macrophage infiltration and/or that highly expresses R-spondin protein.

In another preferred embodiment, the inhibitor is selected from the group consisting of:

(a) an antagonist that specifically inhibits LGR4 expression and/or activity;

(b) an antagonist that specifically inhibits the expression and/or activity of R-spondin;

(c) structural analogs of LGR4;

(d) a structural analogue of R-spondin;

(e) any combination of the above.

In another preferred embodiment, the antagonist comprises a microRNA, an siRNA, a shRNA, or a combination thereof.

In another preferred embodiment, the antagonist comprises an antibody, preferably a monoclonal antibody.

In another preferred embodiment, the inhibitor is an LGR4 extracellular segment protein.

In another preferred embodiment, the LGR4 extracellular segment protein comprises an extracellular segment protein full length protein, an extracellular segment protein fragment.

In another preferred embodiment, the inhibitor is a fusion protein comprising an LGR4 extracellular domain protein.

In another preferred embodiment, the inhibitor is a CAR-T cell that integrates an LGR4 extracellular segment protein encoding gene.

In another preferred embodiment, the LGR4 and R-spondin are derived from a human or a non-human mammal.

In another preferred embodiment, the amino acid sequence of the LGR4 extracellular segment protein is set forth in SEQ ID NO.: 1.

In another preferred embodiment, the nucleotide sequence encoding the LGR4 extracellular segment protein is set forth in SEQ ID NO.: 2.

In another preferred embodiment, the composition is a pharmaceutical composition.

In another preferred embodiment, the pharmaceutical composition comprises (a) an inhibitor that inhibits binding of LGR4 and R-spondin; and (b) a pharmaceutically acceptable carrier.

In another preferred embodiment, the pharmaceutical composition is in the form of an injectable form or a topical pharmaceutical form.

In another preferred embodiment, the pharmaceutical composition can be administered by subcutaneous injection, intravenous injection, intramuscular injection.

In a second aspect of the invention, there is provided a method of screening for a drug candidate that promotes the conversion of a macrophage from an M2 phenotype to an M1 phenotype, the method comprising the steps of:

(a) providing a test compound, and detecting the inhibition of binding of the test compound to LGR4 and R-spondin, thereby selecting a test compound which inhibits the binding of LGR4 and R-spondin as a preliminary sieve Compound;

(b) testing the effect of the sifted compound on macrophage phenotype switching, thereby selecting a compound having a phenotype that promotes the conversion of the macrophage from the M2 phenotype to the M1 phenotype as a drug candidate.

In a third aspect of the invention, there is provided a method of non-therapeutically promoting the conversion of a macrophage from a M2 phenotype to an M1 phenotype, comprising the steps of:

(a) Culture macrophages in the presence of inhibitors that inhibit the binding of LGR4 and R-spondin, thereby promoting the conversion of macrophages from the M2 phenotype to the M1 phenotype.

In a fourth aspect of the invention, a method of regulating a functional phenotype conversion 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.

In another preferred embodiment, the subject comprises a human and a non-human mammal.

It is to be understood that within the scope of the present invention, the various technical features of the present invention and the various technical features specifically described hereinafter (as in the embodiments) may be combined with each other to form a new or preferred technical solution. Due to space limitations, we will not repeat them here.

DRAWINGS

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.

Figure 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.

Figure 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.

Figure 4A shows that LGR4 extracellular protein dose-dependently inhibits the development of LLC tumorigenesis.

Figure 4B shows that Rspo/Lgr4 pathway blockade significantly inhibits the growth of LLC tumors.

Figure 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.

Detailed ways

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. M2-type polarization of related macrophages, and increase the ratio of M1 macrophages and CD8 ⁺ T lymphocytes with anti-tumor function in tumor tissues, which can be used to treat macrophage-infiltrating and high-expression R-spondin The type of tumor of the protein. 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 macrophage

Tumor Associated Macrophages (TAM) 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. At the same time, by secreting a large amount of IL-10, TGF-β, VEGF, etc. 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. 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.

LGR4

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/β-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. It has been confirmed that 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.

Applicants have found that 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. At the same time, 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.

In another preferred embodiment, the LGR4 and R-spondin binding inhibitors are LGR4 inhibitors, R-spondin inhibitors, LGR4 structural analogs, and/or R-spondin structural analogs.

In another preferred embodiment, the LGR4 and R-spondin binding inhibitor is the LGR4 extracellular protein represented by SEQ ID NO.: 1.

In another preferred embodiment, the LGR4 and R-spondin binding inhibitor is an anti-LGR4 monoclonal antibody and/or an anti-LGR4 monoclonal antibody.

RNA interference (RNAi)

In the present invention, a class of potent LGR4 and R-spondin binding inhibitors are interfering RNAs.

As used herein, the term "RNA interference (RNAi)" 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.

As used herein, the term "small interfering RNA (siRNA)" 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).

In the present invention, interfering RNA includes siRNA, shRNA, and corresponding constructs. In the present invention, the interfering RNA may specifically target an interfering RNA of LGR4, and/or an interfering RNA specific for R-spondin. Preferably, the interfering RNA can specifically target a binding region of LGR4 to R-spondin.

In the present invention, a typical shRNA is shown in Formula II in Figure 7,

In the formula,

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;

X 'is absent; or located Seq' _forward and Seq 'spacer sequence between the _reverse, and the spacer sequence Seq' _forward and Seq 'no _reverse complementary,

|| represents a hydrogen bond formed between the Seq _forward and the Seq _reverse .

Regulation of functional phenotypic transformation of macrophages

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 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. Furthermore, the pharmaceutical compositions of the invention may also be used with other therapeutic agents.

When the pharmaceutical composition of the present invention is 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). For example, 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.

The pharmaceutical 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. Preferred routes of administration include oral administration, intramuscular administration or intravenous administration, and administration by smearing.

In addition, the pharmaceutical compositions of the invention may also be combined with other agents for treating tumors.

The main advantages of the invention include:

1. 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.

2. The present invention uses Lgr4/R-spondin1 as a molecular target or a drug target to treat tumor types with high expression.

3. 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.

4. 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. .

5. 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. At present, the Lgr4 extracellular domain has been crystallized, and its binding to the R-spondin protein region has been clarified. However, in view of the binding strength or affinity and the three-dimensional structure of the folded protein after expression, 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.

6. 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 invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are not intended to limit the scope of the invention. The experimental methods in the following examples which do not specify the specific conditions are usually in accordance with conventional conditions or according to the conditions recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated.

Example 1

Construction of a subcutaneous xenograft model of mouse LLC lung cancer cells and detection of Lgr4 expression in tumor-associated macrophages

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 μg/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 μl 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 μl.

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.

2 ml of each of the above cell suspensions was placed in a flow analysis tube, and 3 μl of APC fluorophore-labeled rat anti-mouse F4/80 monoclonal antibody (Biolegend) was added to each tube, thoroughly mixed and placed at 4 ° C in the dark. Incubate for 40 minutes, then wash three times with 2 ml of 2% fetal bovine serum in PBS buffer, and resuspend the above cells in 1 ml of PBS buffer for the last time.

Flow cytometry (BD) sorted and collected F4/80 ⁺ tumor-associated cells and spleen macrophages, and the collection was stopped when the number of collected cells reached 2×10 ⁵ .

The collected cells were centrifuged at 1000 rpm/min for 3 minutes, the supernatant was discarded, and 1 ml of Trizol lysate (TAKARA) was added to each tube, and lysed for 2 minutes, followed by centrifugation at 12,000 rpm/min for 10 minutes at 4 ° C, and the upper aqueous phase was taken and added. 200 μl of chloroform (Shanghai Shenggong), allowed to stand for 2 minutes, centrifuged at 12000 rpm/min for 5 minutes at 4 ° C, took the upper aqueous phase, and added an equal volume of isopropyl alcohol (Shanghai Shenggong) for 5 minutes, 4 ° C conditions 12000 rpm / After centrifugation for 5 minutes at min, the pellet was resuspended in 800 μl of 75% ethanol in DEPC water, centrifuged at 8000 rpm/min for 5 minutes at 4 ° C, and the supernatant was discarded. After RNA precipitation was dried, it was dissolved in 50 μl 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, β-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.

Results As shown in Figure 1A, the expression level of Lgr4 in lung cancer tumor-associated macrophages was significantly higher than that in normal spleen tissue macrophages.

Example 2

In vitro induction culture of mouse bone marrow-derived macrophages (BMM)

The method of inducing BMM in vitro is as follows:

1. Take C57BL/6 mice for 6-8 weeks, the cervical vertebrae are dislocated and killed, and 75% alcohol body surface disinfection.

2, cut the abdomen and peripheral skin, expose the groin, cut the two hind limbs of the mouse along the groin, be careful not to cut the femur.

3. Peel the hind limb muscles with scissors and tweezers treated with 75% alcohol to separate the tibia and femur (thigh bone and calf bone).

4, in a sterile environment (such as in the ultra-clean workbench), cut the ends of the tibia and femur in turn, expose the marrow cavity, use a syringe to absorb about 5ml BMM conditional culture and repeatedly blow until the bone marrow cavity is white, because the tibia The femur is fine and it is recommended to use a 1ml syringe for purging.

5. Repeat the pipetting of the bone marrow washing solution with a pipette until the large mass is not visible to the naked eye (the blowing force is too strong to avoid serious shear damage to the cells). At this time, a bone marrow cell suspension was prepared.

6. The above cell suspension was filtered through a 45 μm pore size sieve, counted, and plated. It is preferred to inoculate 1 x 10 ⁷ total cells in a 6 cm culture dish.

7. 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.

8. On the fifth or seventh day, 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.

Among them, the conditioned medium components for inducing BMM were: DMEM basal medium, 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, and 15% L929 cell culture supernatant.

The results are shown in Fig. 5. After 7 days of culture induction by the conditioned medium, the purity of mature macrophages of F4/80 ⁺ can reach 98% or more, indicating that the induced culture method is feasible.

Example 3

Flow cytometry of BMM

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 μl of the cell suspension in a flow analysis tube for staining. 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) were protected from light in the dark. Incubate at 4 ° C for 40 minutes in the dark, and the amount of antibody is 0.1 μg/10 ⁵ cells. After the end of the staining, the tubes were washed three times with 800 ml of PBS buffer containing 0.2% fetal bovine serum (800 rpm/min, 3 minutes), and finally the cells were resuspended in 200 μl of PBS buffer for flow cytometry analysis. When staining, the same type of control group, single staining group and experimental group were set, in which the same type control group was added with fluorescent labeled isotype control IgG antibody, and the single staining group was separately added with APC-anti-F4/80 antibody or FITC-anti-CD206 antibody. Then both antibodies are added at the same time.

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.

Example 4

Real-time quantitative PCR for detection of Ym1, Arg1 and CD206 expression

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.

As a result, as shown in FIG. 1C, FIG. 1D, and FIG. 1E, the deletion of Lgr4 inhibited the functional polarization of the macrophage M2 direction.

Example 5

Western blot analysis of Arg1, CD206 expression

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 μl 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. 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 μl 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 μl 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 0.2μm nitrocellulose membrane (NC membrane, Millipore, USA). Semi-dry transfer operation, transfer voltage 100V, time 90 minutes; after transfer film, the successfully transferred protein NC membrane was placed in PBS buffer containing 5% bovine serum albumin (BSA), shaker After gently shaking for 120 minutes; after washing the NC membrane twice with PBS, the NC membrane was cut according to the Marker size indication, and the NC membrane with the target protein band was rabbit anti-mouse Arg1 antibody, rabbit anti-mouse CD206 antibody and mouse anti-mouse β. -actin antibody was incubated overnight at 4 °C, washed with PBST (10 min, 3 times), and protected with green fluorescent group-conjugated goat anti-mouse and green fluorescent-conjugated goat anti-rabbit secondary antibody in the dark. Incubation, incubation time 120 min, PBST wash (10 min, 3 times), followed by exposure with an Odyssey fluorescence sweeper with β-actin as the loading internal reference.

The relevant reagents used in the experiment were formulated as follows:

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.

5 x Loading Buffer: 0.25 M Tris-HCl, pH 6.8, 25% β-mercaptoethanol, 10% SDS, 0.5% bromophenol blue, 50% glycerol. The cell lysate was added during use and diluted to 1 x working solution.

PBST: PBS buffer containing 0.1% Tween20.

The electrophoresis tank and the transfer tank extremely supporting equipment were purchased from BioRad.

As shown in Fig. 1F, the deletion of Lgr4 down-regulated the expression levels of M2-related marker genes Arg1 and CD206 in macrophages.

Example 6

Tumor database search

The TCGA (https://tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp) and 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.

The results of the analysis are shown in Figure 2. LGR4 and its ligand RSPO1 are highly expressed in tumor specimens of lung adenocarcinoma patients.

Example 7

Construction of prokaryotic expression vector of Lgr4 and purification of extracellular domain of Lgr4

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. Overnight, 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 μl 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.

Collection and lysis of the cells: After the end of the induction, the bacteria were centrifuged at 11,000 rpm/min for 15 minutes, the supernatant was discarded, and a small amount of cells were picked and added to 100 μl of 1×SDS loading Buffer, and the sample was boiled in a 100 ° C water bath for 10 minutes. As an induction control group. The remaining cells were added with 20 ml of lysis buffer (the relevant formula is shown below, the reagents used below are the same), the vortex shaker was shaken well, and after repeated freezing and thawing with the -80 °C refrigerator for 3 times, the bacterial solution was placed on ice for sonication. 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.

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 μl 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.

Since 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:

Cleaning the nickel column: The 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.

Balance the nickel column: Fill the cleaned nickel column with the equilibration buffer and discard the effluent. The above operation is 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.

After adding the protein sample and centrifuging at 6000 rpm/min for 20 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.

After the concentration is completed, the remaining liquid in the upper layer of the ultrafiltration tube is the concentrated protein of interest. After 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.

The relevant reagents used in this example and their formulations are as follows:

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

As a result, as shown in Fig. 6, the molecular weight of the expressed LGR4-ECD protein was about 72 KD, and the purity was high, and there was no impurity band.

Example 8

Lgr4 extracellular protein therapy concentration-dependent experiment

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. From the second day after the injection of tumor cells, the mice in each group were subcutaneously administered to the right side of the PBS blank control group, 10 μg dose group, and 20 μg dose group. After 5 days of continuous administration, the tumor volume of the mice was measured with a vernier caliper on the tenth day after the injection of the tumor cells, and the calculation formula of the tumor volume = length × width ² , 2 was used.

The results are shown in Fig. 4A, showing 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.

Example 9

Lgr4 extracellular protein therapy experiment

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. From the second day after the injection of tumor cells, the mice were administered subcutaneously to the right side of the six groups of mice: PBS blank control group, 20 μg Lgr4 extracellular protein dose group, irrelevant antibody IgG control group, anti-mouse R-spondin1 Monoclonal antibody (14 μg 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). After 5 days of continuous administration, the tumor volume of the mice was measured with a vernier caliper on the tenth day after the injection of the tumor cells, and the calculation formula of the tumor volume = length × width ² , 2 was used.

The results are shown in 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. .

Example 10

Analysis of tumor tissue macrophages and T cells after administration of tumor-bearing 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.

200 μl of each of the above cell suspensions into a flow analysis tube, separate the isotype control tubes, single stained tubes and double stained tubes, and add 0.5 μl of fluorophore-labeled rat anti-mouse monoclonal antibody to each tube, and mix well. After incubation for 40 minutes at 4 ° C in the dark, it was washed three times with 1 ml of 2% fetal bovine serum in PBS buffer, and the cells were resuspended in 200 μl of PBS buffer for the last time. Flow cytometry (BD) analyzed the ratio of F4/80 ⁺ CD206 ⁺ M2 macrophages, F4/80 ⁺ CD16/32 ⁺ M1 macrophages, and CD3 ⁺ CD8 ⁺ T cells in each tissue.

As shown in Fig. 4C, 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.

All documents mentioned in the present application are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the the In addition, it should be understood that various modifications and changes may be made by those skilled in the art in the form of the present invention.

Claims

Use of an inhibitor for inhibiting the binding of LGR4 and R-spondin, characterized in that it is used to prepare a preparation or composition for regulating the functional phenotypic conversion of macrophages.
The use according to claim 1, wherein the functional phenotypic transformation of the regulated macrophage means inhibiting macrophage polarization to the M2 phenotype and/or promoting macrophage to the M1 phenotype pole. Chemical.
The use according to claim 1, wherein said macrophage is a tumor-associated macrophage.
The use according to claim 1 wherein said formulation or composition is further for one or more uses selected from the group consisting of:

(i) increasing the proportion of M1 phenotype macrophages;

(ii) increasing the proportion of CD8 + T cells;

(iii) treating the tumor;

(iv) Regulating the immune microenvironment of tumor tissue.
The use according to claim 1 wherein said inhibitor is selected from the group consisting of:

(a) an antagonist that specifically inhibits LGR4 expression and/or activity;

(b) an antagonist that specifically inhibits the expression and/or activity of R-spondin;

(c) structural analogs of LGR4;

(d) a structural analogue of R-spondin;

(e) any combination of the above.
The use according to claim 1, wherein the inhibitor is an LGR4 extracellular segment protein.
The use according to claim 1, wherein the inhibitor is a CAR-T cell which integrates an LGR4 extracellular segment protein-encoding gene.
The use according to claim 6 or 7, wherein the amino acid sequence of the LGR4 extracellular segment protein is as shown in SEQ ID NO.: 1.
A method of screening for a drug candidate that promotes conversion of a macrophage from an M2 phenotype to an M1 phenotype, the method comprising the steps of:

(a) providing a test compound, and detecting the inhibition of binding of the test compound to LGR4 and R-spondin, thereby selecting a test compound which inhibits the binding of LGR4 and R-spondin as a preliminary sieve Compound;

(b) testing the effect of the sifted compound on macrophage phenotype switching, thereby selecting a compound having a phenotype that promotes conversion of the macrophage from the M2 phenotype to the M1 phenotype as a drug candidate.
A method for non-therapeuticly promoting the conversion of a macrophage from a M2 phenotype to an M1 phenotype, comprising the steps of:

(a) Culture macrophages in the presence of inhibitors that inhibit the binding of LGR4 and R-spondin, thereby promoting the conversion of macrophages from the M2 phenotype to the M1 phenotype.