CN109529040A

CN109529040A - LGR4 and R-spondin binding inhibitors and its purposes in oncotherapy

Info

Publication number: CN109529040A
Application number: CN201710861974.2A
Authority: CN
Inventors: 杜冰; 谭炳合; 李伟
Original assignee: Shanghai Bioray Biotechnology Co Ltd; East China Normal University
Current assignee: Shanghai Bioray Biotechnology Co Ltd; East China Normal University
Priority date: 2017-09-21
Filing date: 2017-09-21
Publication date: 2019-03-29
Anticipated expiration: 2037-09-21
Also published as: CN109529040B; WO2019057120A1

Abstract

The present invention provides a kind of LGR4 and R-spondin binding inhibitors and its purposes in oncotherapy.Specifically, present invention discover that inhibiting the combination of LGR4 and R-spondin, it has been able to suppress the M2 type polarization of tumor-associated macrophage, while having improved the M1 type macrophage and CD8 for having anti-tumor function in tumor tissues⁺The ratio of T lymphocyte can be used for treating the tumor type rich in the macrophages infiltration R-spondin albumen of height expression simultaneously.

Description

LGR4 and R-spondin binding inhibitors and its purposes in oncotherapy

Technical field

The present invention relates to immunotherapy of tumors field, relate more specifically to LGR4 and R-spondin binding inhibitors and its Purposes in oncotherapy.

Background technique

Lung cancer is one of global incidence and the highest malignant tumour of lethality, since diet, environment, smoking etc. are a variety of The effect of factor, disease incidence of the lung cancer in the whole world can not have always been high any more, every to have millions of people to be diagnosed as lung cancer every year, wherein Most survival rates are no more than 5 years, and lung cancer has become the big chronic illness for seriously threatening human health and life.Serious bone Transfer and frequent recurrence rate are the main reason for causing lung cancer patient dead, due to lung special structure construction and cell It constitutes, while lung carcinoma cell often has lower immunogenicity, causes the therapeutic effect of a variety of targeted therapies and immunotherapy It is extremely limited.Lung cancer often forms special tumor microenvironment when occurring in lung, apparent feature first is that there are numerous The infiltration of immunocyte especially macrophage, this prognosis poor with lung cancer patient and lower survival rate are closely related.

Tumor-associated macrophage (Tumor Associated Macrophages, TAM) is that one kind is found largely to soak Moisten in the macrophage of kinds of tumors happening part, clinical data and zoopery all prove, the macrophage in tumor tissues It is closely related with the survival rate and prognosis situation of cancer patient or lotus knurl experimental animal.It is thin currently based on tumour correlation macrophage The tumor therapeuticing method of born of the same parents has become the very attractive new direction in tumour immunotherapy field, and target tumor correlation macrophage is thin The great clinical value of the immunotherapy of born of the same parents.

Summary of the invention

The purpose of the present invention is to provide a kind of LGR4 and R-spondin binding inhibitors and its in oncotherapy Purposes.

Specifically, the purpose of the present invention is to provide a kind of closes or blocks tumor tissues using Lgr4 Extracellular domain protein The combination of middle R-spondin albumen and endogenous Lgr4 receptor, and then modulate tumor associated macrophages function polarization conversion, And then inhibit the application of the occurrence and development of tumour.More specifically, the invention mainly relates to Lgr4 Extracellular domain proteins in oncotherapy The application in field.

In the first aspect of the present invention, a kind of purposes of inhibitor that inhibition LGR4 and R-spondin is combined is provided, It is used to prepare a preparation or composition, the preparation or composition are for regulating and controlling the function Phenotypic change of macrophage.

In another preferred example, the function Phenotypic change of the regulation macrophage, which refers to, inhibits macrophage to M2 Phenotype polarization, and/or macrophage is promoted to polarize to M1 phenotype.

In another preferred example, the function Phenotypic change of the regulation macrophage, which refers to, promotes macrophage by M2 Phenotypic change is M1 phenotype.

In another preferred example, the macrophage is tumor-associated macrophage.

In another preferred example, the preparation or composition are also used to one or more purposes selected from the group below:

(i) ratio of M1 phenotype macrophage is improved；

(ii) CD8 is improved⁺The ratio of T cell；

(iii) tumour is treated；

(iv) tumor tissues are adjusted and microenvironment is immunized.

In another preferred example, the tumour is rich in macrophages infiltration and/or high expression R-spondin albumen Tumour.

In another preferred example, the inhibitor is selected from the group:

(a) specificity inhibits LGR4 expression and/or active antagonist；

(b) specificity inhibits R-spondin expression and/or active antagonist；

(c) analogue of LGR4；

(d) analogue of R-spondin；

(e) any combination of above-mentioned items.

In another preferred example, the antagonist include MicroRNA, siRNA, shRNA, or combinations thereof.

In another preferred example, the antagonist includes antibody, preferably monoclonal antibody.

In another preferred example, the inhibitor is LGR4 Extracellular domain protein.

In another preferred example, the LGR4 Extracellular domain protein includes Extracellular domain protein full-length proteins, Extracellular domain protein Segment.

In another preferred example, the inhibitor is the fusion protein comprising LGR4 Extracellular domain protein.

In another preferred example, the inhibitor is the CAR-T cell for integrating LGR4 Extracellular domain protein encoding gene.

In another preferred example, the LGR4 and R-spondin derives from people or non-human mammal.

In another preferred example, the amino acid sequence of the LGR4 Extracellular domain protein is as shown in SEQ ID NO.:1.

In another preferred example, the nucleotide sequence such as SEQ ID NO.:2 institute of the LGR4 Extracellular domain protein is encoded Show.

In another preferred example, the composition is pharmaceutical composition.

In another preferred example, described pharmaceutical composition includes the inhibitor that (a) inhibits LGR4 and R-spondin to combine； (b) pharmaceutically acceptable carrier.

In another preferred example, the dosage form of described pharmaceutical composition is injection type or external drug dosage forms.

In another preferred example, described pharmaceutical composition can pass through the side of subcutaneous injection, intravenous injection, intramuscular injection Formula administration.

In the second aspect of the present invention, providing a kind of screening to promote macrophage by M2 Phenotypic change is M1 phenotype The method of drug candidate, the method includes the steps:

(a) untested compound is provided, and is detecting suppression of the untested compound to LGR4 and R-spondin combination Situation processed, so that the untested compound for being combined with inhibiting effect to LGR4 and R-spondin is selected, as the chemical combination through primary dcreening operation Object；And

(b) effect of the compounds towards macrophages Phenotypic change through primary dcreening operation is tested, so that selecting has promotion huge Phagocyte is the compound of M1 phenotype by M2 Phenotypic change, as drug candidate.

In the third aspect of the present invention, promoting macrophage with providing a kind of non-therapeutic by M2 Phenotypic change is M1 The method of phenotype, comprising steps of

(a) in the presence of the inhibitor for inhibiting LGR4 and R-spondin to combine, macrophage is cultivated, to promote macrophage Cell is M1 phenotype by M2 Phenotypic change.

In another preferred example, the macrophage is tumor-associated macrophage.

In the fourth aspect of the present invention, a kind of method of the function Phenotypic change of regulation macrophage, including step are provided It is rapid:

(i) to need object application inhibit LGR4 and R-spondin in conjunction with inhibitor or include the inhibitor Composition.

In another preferred example, the object includes people and non-human mammal.

It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, Not repeated them here.

Detailed description of the invention

Fig. 1 shows that the missing of Lgr4 causes the polarized decrease of M2 type macrophage.

Figure 1A shows the differential expression of Lgr4 in LLC tumor-bearing mice Macrophage in Spleen and tumor-associated macrophage.

Figure 1B shows that Lgr4 ligand Rspo albumen promotes wild type macrophage F4/80+CD206+M2 type macrophage Polarization, and this kind of facilitation of Lgr4 knockout type macrophage is weakened.

Fig. 1 C, Fig. 1 D and Fig. 1 E respectively illustrate Lgr4 knock out macrophage in M2 marker protein Agr1, Ym-1, CD206mRNA expression reduces.

Fig. 1 F shows that Lgr4 knocks out M2 marker protein Agr1, CD206 protein expression level in macrophage and reduces.

Fig. 2 shows differential expression of the LGR4 in different tumor tissues.

Fig. 3 shows the building of Lgr4 extracellular fragment prokaryotic expression carrier.

Fig. 3 A shows LGR4 extracellular fragment structural domain.

Fig. 3 B shows p-ET8a prokaryotic expression carrier and LGR4 Extracellular domain protein expression cassette insertion position.

Fig. 4 shows that LGR4 Extracellular domain protein significantly improves tumor microenvironment, inhibits the occurrence and development of LLC tumour.

Fig. 4 A shows LGR4 Extracellular domain protein dose-dependent inhibition LLC tumor development.

Fig. 4 B shows that Rspo/Lgr4 access blocks the growth for significantly inhibiting LLC tumour.

Fig. 4 C, which shows that Rspo/Lgr4 access blocks, reduces M2 type macrophage (F4/80+ in LLC tumor microenvironment CD206+) ratio, while improving the ratio of cd8 t cell (CD3+CD8+) and M1 type macrophage (F4/80+CD16/32+).

Fig. 5 shows after conditioned medium Fiber differentiation 7 days of the colony stimulating factor containing M-CSF, F4/80⁺At Ripe macrophage purity can be arrived to 98% above, meet subsequent experimental requirement.

Fig. 6 shows the electrophoresis result of the LGR4-ECD molecular weight of albumen of expression.

Fig. 7 shows the structure of typical shRNA a kind of.

Specific embodiment

The present inventor is surprised to find that LGR4 gene being capable of modulate tumor tissue by depth studying extensively for the first time The function Phenotypic change of middle tumor-associated macrophage.Specifically, after LGR4 gene and its ligand R-spondin protein binding Tumor-associated macrophage can be promoted to polarize towards M2 phenotype.The LGR4 Extracellular domain protein of prokaryotic expression, amino acid sequence As shown in SEQ ID NO:1, by subcutaneous administrations mode, which can be in conjunction with endogenous LGR4 Receptor Competition R-spondin albumen so that the M2 type of tumor-associated macrophage be inhibited to polarize, while improving in tumor tissues and having The M1 type macrophage and CD8 of anti-tumor function⁺The ratio of T lymphocyte can be used for treating rich in macrophages infiltration simultaneously The tumor type of height expression R-spondin albumen.The present invention relates to Lgr4 can be used as adjust tumor tissues be immunized microenvironment into And inhibit the application of the drug target and Lgr4 Extracellular domain protein of tumor development on clinical therapy of tumor, it wraps It includes LGR4 extracellular fragment gene integration into CART to promote effect of the CART on treatment solid tumor.Complete this on this basis Invention.

Tumor-associated macrophage

Tumor-associated macrophage (Tumor Associated Macrophages, TAM) is that one kind is found largely to soak Moisten in the macrophage of kinds of tumors happening part, clinical data and zoopery all prove, the macrophage in tumor tissues It is closely related with the survival rate and prognosis situation of cancer patient or lotus knurl experimental animal.Macrophage is according to its phenotype and function Energy can be roughly divided into two major class, i.e., proinflammatory antitumor M1 type and the scorching M2 phenotype for promoting tumour of suppression, and stimulate according to receiving The difference of signal can mutually convert between amphitypy macrophage, be presented as the heterogeneity and plasticity of macrophage height. TAM possesses a large amount of features similar with the anti-inflammatory rush M2 type macrophage of tumour, is able to suppress antitumor immune response, promotees It is monitored into neoplastic cells escape immunity of organism；Meanwhile by secrete the cell factors such as a large amount of IL-10, TGF-β, VEGF and Growth factor promotes angiogenesis, fibrosis, Epithelial and stromal conversion etc. inside tumor tissues；TAM can also be with surrounding The even other immunocyte interactions of tumour cell, stroma cell and communication, are the proliferation of tumour cell, survive, invade It attacks, shift and provide good local microenvironment.It is had become currently based on the tumor therapeuticing method of tumor-associated macrophage swollen The very attractive new direction in tumor immunotherapy field, the great clinical application of the immunotherapy of target tumor associated macrophages Value.

LGR4

Lgr4 full name is (the leucine-rich repeat- of g protein coupled receptor 4 rich in leucine motive Containing G protein-coupled receptor 4), also it is (the G protein of g protein coupled receptor 48 Couple receptor 48), belong to Lgr class g protein coupled receptor family II, Gene ID:107515.

Lgr4 receptor can be with R-spondin protein family (R-spondin 1-4) high specific and high-affinity knot It closes, IC50 is only 2-230nM, thus the secretory protein family is considered as the endogenic ligand of Lgr4.Lgr4 and R- Classical wnt/ β-catenin signal path can greatly be enhanced after spondin protein binding, in adult stem cell and hair follicle Development of stem cells, bone cells balance, genital tract development, eyelid development are equal even in the mode identification procedure of macrophage Play important adjustment effect.However, the combination of Lgr4 and R-spondin albumen can not activate classical G-protein signal Access.It has been confirmed that LGR4 and its ligand R-spondin the albumen unconventionality expression in a variety of human malignancies, most In the case of its expression be above normal tissue, including lung cancer, gastric cancer, colorectal cancer, breast cancer, prostate cancer etc., LGR4/ R- Spondin promotes the proliferation growth of tumour cell by wnt wnt associated signal paths.

It has been found that Lgr4 is thin in mice lung cancer tissue tumor correlation macrophage compared to the macrophage of normal tissue Also up-regulated expression is presented in born of the same parents, and confirms that Lgr4 is just regulating and controlling the function of macrophage M2 phenotype by internal experiment in vitro Can polarization, in conjunction with R-spondin in cancerous lung tissue and Lgr4 in lung cancer tumor associated macrophages height expression, Tumour cell sheet can directly acted on by the interaction of Lgr4/R-spondin in blocking cancerous lung tissue by implying The microenvironment depended on for existence while body by the function phenotype of modulate tumor associated macrophages to modulate tumor cell, And then inhibit the occurrence and development of tumour, there is great application value and meaning for the immunization therapy of tumour.

LGR4 and R-spondin binding inhibitors

As used herein, " LGR4 and R-spondin binding inhibitors ", " suppression for inhibiting LGR4 and R-spondin to combine Preparation " is used interchangeably, and is the preparation or composition for referring to specificity and LGR4 and R-spondin being inhibited to combine.

In another preferred example, LGR4 the and R-spondin binding inhibitors are LGR4 inhibitor, R-spondin Inhibitor, LGR4 analogue, and/or R-spondin analogue.

In another preferred example, LGR4 the and R-spondin binding inhibitors are shown in SEQ ID NO.:1 LGR4 Extracellular domain protein.

In another preferred example, LGR4 the and R-spondin binding inhibitors be anti-LGR4 monoclonal antibody and/ Or anti-LGR4 monoclonal antibody.

RNA interferes (RNAi)

In the present invention, a kind of effective LGR4 and R-spondin binding inhibitors are RNA interferings.

As used herein, term " RNA interference (RNA interference, RNAi) " refers to: some small double-stranded RNAs The expression that can efficiently, specifically block internal specific gene, promotes mRNA to degrade, and lures that cells show goes out specific gene and lacks into The phenotype of mistake is also referred to as RNA intervention or RNA interference.RNA interference is that the gene in mRNA level in-site of high special is heavy Silent mechanism.

As used herein, term " siRNA (small interfering RNA, siRNA) " refers to a kind of short-movie Section double stranded rna molecule, can be using the mRNA of homologous complementary sequence as the target specific mRNA of degradation, this process is exactly RNA Interference channel (RNA interference pathway).

In the present invention, RNA interfering includes siRNA, shRNA and corresponding construction.In the present invention, described RNA interfering can specifically be directed to the RNA interfering of R-spondin for the RNA interfering of LGR4, and/or specificity.It is preferred that Ground, the RNA interfering can specifically be directed to the bond area of LGR4 and R-spondin.

In the present invention, a kind of typical shRNA is as shown in the Formula II in Fig. 7,

In formula,

Seq’_{It is positive}For Seq_{It is positive}The corresponding RNA sequence of sequence or sequence fragment；

Seq’_ReverselyFor with Seq '_{It is positive}The sequence being substantially complementary；

X ' is nothing；Or for positioned at Seq '_{It is positive}And Seq '_ReverselyBetween intervening sequence, and the intervening sequence and Seq '_{It is positive} And Seq '_ReverselyIt is complementary,

| | it indicates in Seq_{It is positive}And Seq_ReverselyBetween the hydrogen bond that is formed.

The regulation of the function Phenotypic change of macrophage

The present invention provides a kind of purposes of inhibitor that inhibition LGR4 and R-spondin is combined, and are used to prepare a preparation Or composition, the preparation or composition are for regulating and controlling the function Phenotypic change of macrophage.

The present invention also provides a kind of methods of the function Phenotypic change of regulation macrophage, comprising steps of (i) to needs Object application inhibit LGR4 and R-spondin in conjunction with inhibitor or composition comprising the inhibitor.

The present invention relates to the medicine groups containing LGR4 and R-spondin binding inhibitors and pharmaceutically acceptable carrier Close object.

Pharmaceutically acceptable carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, Pulvis, and combinations thereof.Pharmaceutical preparation should match with administration mode.Pharmaceutical composition of the invention can be made into injection shape Formula, such as the aqueous solution with physiological saline or containing glucose and other adjuvants are prepared by conventional method.Such as tablet With the pharmaceutical composition of capsule etc, can be prepared by conventional method.Pharmaceutical composition such as injection, solution, tablet and glue Capsule preferably aseptically manufactures.Pharmaceutical composition of the invention can also be made into pulvis for Neulized inhalation.It is a kind of preferred Dosage form is oral preparation.In addition, pharmaceutical composition of the present invention can be also used together with other therapeutic agents.

It, can be with one or more pharmaceutically acceptable carriers when pharmaceutical composition of the present invention is used for such use Or excipient mixing, such as solvent, diluent, and can be administered orally with following form: tablet, capsule, can divide pill Scattered powder, particle or suspension (containing such as from about 0.05-5% suspending agent), syrup (containing such as from about 10-50% sugar) and elixir (containing about 20-50% ethyl alcohol), or about 0.05- (is contained in isotonic medium with sterile injectable solution or suspension form 5% suspending agent) carry out parenteral routes.For example, these pharmaceutical preparations contain the about 0.01-99% mixed with carrier, more preferably Ground is about the active constituent of 0.1%-90% (weight).

Pharmaceutical composition of the invention can be administered by conventional route, including (but being not limited to): flesh It is interior, peritonaeum is interior, in intravenous, subcutaneous, intradermal, oral, tumor or local administration.Preferred administration route includes oral administration, flesh Interior administration or intravenous administration smear administration.

In addition, pharmaceutical composition of the invention can also be with the drug combination of other treatment tumour.

Main advantages of the present invention include:

1. the present invention filters out Lgr4 gene and discloses the pole that Lgr4/R-spondin participates in macrophage M2 phenotypic function Change process；Based on this fact, using Lgr4 Extracellular domain protein as the competitive binding albumen of R-spondin1 in tumor tissues, Inhibit tumor-associated macrophage in tumor tissues to polarize towards the M2 type for promoting tumour, while improving M1 type macrophage and CD8⁺The ratio of T cell, to inhibit the occurrence and development of tumour.

2. the present invention is using Lgr4/R-spondin1 as molecular target or drug target, to treat the high table of the two The tumor type reached.

3. the present invention provides a kind of Lgr4 Extracellular domain protein isolated and purified, the Lgr4 Extracellular domain protein have as The amino acid sequence of SEQ ID NO:1, the sequence are located at 28-528 of Lgr4 protein amino acid sequence.

4. Lgr4 Extracellular domain protein of the present invention can be specifically bound with R-spondin1, the albumen can pass through Perhaps mammalian cell expression acquisition can be with monomer or fusion protein form expression and in conjunction with R- for prokaryotic expression Spondin1 albumen.

5. the present invention constructs the prokaryotic expression carrier of Lgr4 Extracellular domain protein, which is PET28a expression vector, structure The expression vector for building completion can be described as PET28a-Lgr4ECD expression vector, express through host e. coli BL21 bacterial strain, wherein ECD means extracellular binding structural domain.Lgr4 extracellular domain has been carried out crystal parsing at present, in conjunction with R-spondin albumen Region is apparent.But in view of protein folding three-dimensional structure after bond strength or affinity and expression, the present invention is still adopted Expression Lgr4 extracellular fragment full length sequence strategy is taken, the amino acid sequence of the extracellular fragment overall length is as shown in SEQ ID NO:1.

6. Lgr4 Extracellular domain protein of the present invention uses subcutaneous administration mode, experiment mice is had no obviously not after injection Good reaction.Above-mentioned albumen can also take intravenous injection, intramuscular injection or drug administration by injection mode.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate this hair It is bright rather than limit the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to routine Condition, or according to the normal condition proposed by manufacturer.Unless otherwise stated, otherwise percentage and number are calculated by weight.

Embodiment 1

Lgr4 expresses water in the building and tumor-associated macrophage of mouse LLC lung carcinoma cell subcutaneous transplantation knurl model Flat detection

Mouse lewis lung cancer cell system LLC cell is purchased from American type culture collection (ATCC), by its explanation It is required that be incubated in dulbecco ' s modified eagle medium (DMEM) complete medium (10% fetal calf serum, 100 μ g/ml streptomysins, 100U/ml penicillin, nonessential amino acid are diluted purchased from GIBCO company and by operation instructions).It will LLC cell culture is in 6cm culture dish, when to its length to 80%-90% ware floor space, discards upper layer culture medium, and 37 DEG C Preheating PBS buffer solution washes twice, and 500 μ l, 0.25% trypsase+0.02%EDTA is added in ware and digests in 37 DEG C 30s is then added isometric DMEM complete medium and terminates digestion, centrifugation, and cell is resuspended with PBS buffer solution, adjusts cell Concentration is to 1 × 10⁷/ml。

Take C57BL/6 mouse (East China Normal University's experiment of above-mentioned 8 week old of cell suspension subcutaneous injection or so size Animal center, SPF grades), every mouse injects 200 μ l.

After two weeks, cervical dislocation puts to death tumor-bearing mice, and operation removes tumor tissues and spleen and by it in 5ml It smashs digestion 5 minutes in 0.25% tryptic digestive juice to pieces, isometric DMEM complete medium is added and terminates digestion, is centrifuged, with Cell is resuspended with PBS afterwards, and adjusts cell density to 1 × 10⁸/ml.

Respectively by the above-mentioned cell suspension of 2ml as in flow cytometer showed pipe, the big of 3 μ l APC fluorophors label is added in every pipe Mouse anti-mouse F4/80 monoclonal antibody (Biolegend company), be sufficiently mixed uniformly be placed on 4 DEG C be protected from light condition be incubated for 40 points Clock, then the PBS buffer solution washing with 2ml containing 2% fetal calf serum three times, 1ml PBS buffer solution is resuspended for the last time above-mentioned Cell.

Flow cytometer (BD company) separation and collection F4/80⁺Tumor-associated cell and Macrophage in Spleen, wait collect Cell number reaches 2 × 10⁵When stop collect.

By the cell being collected into 1000rpm/min centrifugation 3 minutes, supernatant is abandoned, 1ml Trizol lysate is added in each pipe (TAKARA company) cracks 2 minutes, and subsequent 4 DEG C of condition 12000rpm/min are centrifuged 10 minutes, takes upper strata aqueous phase, and 200 μ are added L chloroform (the raw work in Shanghai), stands 2 minutes, 4 DEG C of condition 12000rpm/min are centrifuged 5 minutes, take upper strata aqueous phase, are added isometric Isopropanol (the raw work in Shanghai) is stood after five minutes, and 4 DEG C of condition 12000rpm/min are centrifuged 5 minutes, and precipitating is with 800 μ l with DEPC water Washing is resuspended in 75% ethyl alcohol prepared, and supernatant is abandoned in 4 DEG C of condition 8000rpm/min centrifugation after five minutes, after RNA precipitate dries with 50 μ l RNAase Free water or the dissolution of DEPC water, test total serum IgE extracting concentration and purity.

The total serum IgE of above-mentioned extracting is subjected to reverse transcription and obtains cDNA, used kit is the reagent of TAKARA company production Box, operating procedure and program follow production specification, and the RNA total amount of reverse transcription is 1000ng, response procedures are as follows: 37 DEG C 30 points Clock, 85 DEG C 5 seconds, 12 DEG C preservation.

The cDNA that above-mentioned reverse transcription is obtained is as template, using β-actin as reference gene, real-time quantitative PCR detection Lgr4 expression, used kit are the SYBR kit of TAKARA company production, reaction system, operating procedure and program Follow production specification.

As a result as shown in Figure 1A, expression of the Lgr4 in lung cancer tumor associated macrophages is apparently higher than normal spleen Dirty tissue macrophages.

Embodiment 2

Bone marrow derived macrophage (Bone Marrow derived Macrophages, BMM) external evoked training It supports

The method of the external evoked culture of BMM is as follows:

1,6-8 weeks C57BL/6 mouse is taken, cervical dislocation is put to death, the disinfection of 75% alcohol body surface.

2, abdominal cut and periphery skin expose groin, two hind legs of mouse are cut along groin, are careful not to Cut femur.

3, hindlimb muscle is removed with the scissors and tweezers crossed by 75% ethanol postincubation, isolates shin bone and femur (thigh Bone and focile).

4, in an aseptic environment (such as in superclean bench), shin bone and femur both ends are successively cut off, exposes ossis, is used Syringe is drawn 5ml or so BMM CMC model and is blown and beaten repeatedly, until ossis is whitened, since shin bone is compared with femur Carefully, it is proposed that purged using 1ml gauge hypodermic device.

5, blown and beaten repeatedly with pipettor marrow purging liquid, until it is invisible compared with large crumb (piping and druming can not exert oneself Suddenly, it avoids causing cell serious shearing force to damage).It is prepared as bone marrow cell suspension at this time.

6, above-mentioned cell suspension is passed through into 45 μm of aperture screen filtrations, counted, fishplate bar.Generally 1 is inoculated in 6cm culture dish ×10⁷A total cell is advisable.

7, cell is placed in 37 DEG C, 5%CO₂And it is cultivated 5-7 days in the incubator of suitable humidity, the culture of replacement in every 2 days Liquid.

8, at the 5th or the 7th day, cell is digested 10 points with trypsase (0.25% trypsase+0.02%EDTA) Clock terminates, and is resuspended, and adjustment cell density is 1 × 10⁷/ ml, for fishplate bar in six well culture plates, every hole cell number is 1 × 10⁶, culture Base is changed to common DMEM complete medium, then respectively with 10ng/mlIL-4,500ng/ml recombinant murine R-spondin1 (R& D), 500ng/ml recombinant murine R-spondin3 handles cell, according to subsequent different detection levels, impose respectively different time or Time point processing.

Wherein, the CMC model based component of BMM is induced are as follows: DMEM basal medium, 10%FBS, 100U/ml penicillin, 100 μ g/ml streptomysins, 15%L929 cells and supernatant.

As a result as shown in figure 5, after the conditioned medium Fiber differentiation 7 days, F4/80⁺Full-brown macrophage purity 98% can be reached above, show that the Fiber differentiation mode has feasibility.

Embodiment 3

The fluidic cell of BMM dyes

The Lgr4 that embodiment 2 is prepared^+/+And Lgr4^-/-BMM is incubated at six well culture plates, is changed to common DMEM It is handled cell 24 hours with 500ng/ml R-spondin1,500ng/mlR-spondin3 respectively after complete medium, digestion Cell is resuspended, and adjustment cell density is 1 × 10⁶/ ml takes 200 μ l cell suspensions to be placed in flow cytometer showed pipe and is dyed.It keeps away Under the conditions of light, successively marked with the rat anti-mouse F4/80 monoclonal antibody (APC-anti-F4/80) and FITC of APC label 4 DEG C of the rat anti-mouse CD206 monoclonal antibody (FITC-anti-CD206) of note is protected from light incubation 40 minutes, and antibody dosage is 0.1μg/10⁵Cell.After dyeing, PBS buffer solution washing cell of each effective 1ml containing 0.2% fetal calf serum is three times (800rpm/min, 3 minutes) is finally resuspended cell with 200 μ lPBS buffers, carries out flow cytometry analysis.It is set when dyeing Isotype control group, single dye group and experimental group are set, wherein fluorescent marker Isotype control IgG antibody is added in Isotype control group, single to contaminate Group is added separately to APC-anti-F4/80 antibody or FITC-anti-CD206 antibody, and then two kinds of antibody are simultaneously for experimental group It is added.

As a result as shown in Figure 1B, wherein F4/80⁺CD206⁺Cell is M2 type macrophage, it is seen that R-spondin1 and R- Spondin3 can promote Lgr4^+/+Rather than Lgr4^-/-Macrophage is towards M2 direction polarization.

Embodiment 4

The expression of real-time quantitative PCR detection Ym1, Arg1 and CD206

The Lgr4 that embodiment 2 is prepared^+/+And Lgr4^-/-BMM is incubated at six well culture plates, is changed to common DMEM With 10ng/ml recombinant murine IL-4 processing 4 hours after complete medium, culture solution is abandoned, PBS buffer solution washes twice, Trizol (TAKARA) method extracted total RNA, reverse transcription obtain cDNA, and real-time quantitative PCR method detects each group Ym1, Arg1 and CD206 Expression.Used kit is the SYBR kit of TAKARA company production, and reaction system, operating procedure and program follow Produce specification.

As a result such as Fig. 1 C, shown in Fig. 1 D, Fig. 1 E, the missing of Lgr4 inhibits the function in the direction macrophage M2 to polarize.

Embodiment 5

Protein immunoblot Westernblot detects Arg1, the expression of CD206

The Lgr4 that embodiment 2 is prepared^+/+And Lgr4^-/-BMM is incubated at six well culture plates, is changed to common DMEM It with 10ng/ml recombinant murine after complete medium, handles 24 hours, abandons culture solution, PBS buffer solution washes twice, and every hole is with 200 μ L contains the strong RIPA lysate of protease inhibitors of phosphatases cocktail (Selleckchem company, the U.S., 100 × liquid storage) (500mMTris pH7.0,150mM NaCl, 0.1% Triton-X-100,1% NaTDC, 0.1%SDS) is split on ice Solution 30 minutes, cell lysate is collected into clean EP pipe, and 12000rpm/min is centrifuged 2 minutes, supernatant is transferred to another 40 μ 5 × Loading of l Buffer (formula sees below) are added in clean EP pipe, every pipe, mix 100 DEG C of water-baths of postposition and boil 10 minutes, then the sample by this after being sufficiently denaturalized carried out PAGE gel electrophoresis, wherein 5% polyacrylamide is concentrated Glue, 10% resolving polyacrylamide gel.Every 15 μ l, 60V low-voltage electrophoresis of hole applied sample amount after twenty minutes, replaces 100V high voltage Electrophoresis 120 minutes；Electrophoresis is terminated, the gel with albumen is transferred to 0.2 μm of nitrocellulose filter (NC film, U.S. Millipore company) progress semidry method transferring film operation, transferring film voltage 100V, the time 90 minutes；After transferring film, it will succeed Transfer has the NC film of albumen to be placed in the PBS buffer solution containing 5% bovine serum albumin(BSA) (BSA), and jog closes 120 points on shaking table Clock；After PBS washs NC film twice, is indicated to cut NC film according to Marker size, the NC film with destination protein band is distinguished With rabbit-anti mouse Arg1 antibody, 6 antibody of rabbit-anti MuCD20 and the anti-mouse β-actin antibody of mouse are incubated overnight in 4 DEG C of conditions, PBST It washs (10 minutes, 3 times), the mountain sheep anti mouse and green fluorescence group being coupled respectively with green fluorescence group under the conditions of being protected from light The goat antirabbit secondary antibody of coupling is incubated for, incubation time 120 minutes, PBST wash (10 minutes, 3 times), then with Odyssey fluorescence is swept film instrument and is exposed, and wherein β-actin is loading internal reference.

It is as follows to test related reagent formula used:

PBS buffer solution: sodium chloride (NaCl), 8g；Potassium chloride (KCl), 0.2g；Disodium hydrogen phosphate (Na2HPO4), 1.44g；Potassium dihydrogen phosphate (KH2PO4), 0.24g；PH 7.2 is adjusted, 1L is settled to.

1 × electrophoretic buffer: 25mMTris, 250mM glycine, 0.1%SDS.

1 × transferring film buffer: 48mMTris, 39mM glycine, 0.037%SDS, 20% methanol.

5 × Loading Buffer:0.25M Tris-HCl, pH6.8,25% beta -mercaptoethanol, 10%SDS, 0.5% Bromophenol blue, 50% glycerol.Cell pyrolysis liquid is added in used time, is allowed to be diluted to 1 × working solution.

PBST: the PBS buffer solution containing 0.1%Tween20.

Institute is purchased from BioRad company using electrophoresis tank, transferring film slot number its necessary accessories.

As shown in fig. 1F, the missing of Lgr4 makes M2 correlating markings Gene A rg1, CD206 table in macrophage to experimental result Up to horizontal down-regulation.

Embodiment 6

The retrieval of tumour database

TCGA (https: //tcga-data.nci.nih.gov/tcga/tcgaHome2.jsp) and oncomine The retrieval of (https: //www.oncomine.org/resource/login.html) tumour database is grasped according to site description Make.Wherein TCGA database uses cbioportal (http://www.cbioportal.org/index.do) gopher It is retrieved, retrieval adenocarcinoma of lung patients database selects " Lung Adenocarcinoma (TCGA, Provisional) " data Group；Oncomine retrieval uses " RSOP1 ", " cancer vs normal analysis " and " TCGA Lung 2 " data Group.

Result is analyzed as shown in Fig. 2, LGR4 and its ligand RSPO1 is expressed in adenocarcinoma of lung patient's tumor specimen in high.

Embodiment 7

The building of Lgr4 prokaryotic expression carrier and Lgr4 Extracellular domain protein expression and purification

Lgr4 prokaryotic expression carrier is to construct applicant's early period, and expression carrier used thereof skeleton is PET28a (+), passes through base Because the polynucleotide sequence of LGR4 gene is connected into its multiple cloning sites by Engineering operation technology, after expression, destination protein Lgr4 born of the same parents Outer segment albumen n end amalgamation and expression 6 × his label protein.Recombinant plasmid transformed BL21 Escherichia coli apply band kalamycin resistance LB agarose culture plate, after being incubated overnight, picking monoclonal positive bacterium colony, be placed in containing kalamycin resistance LB liquid training It supports base shaking table culture to stay overnight, overnight bacterium solution is all transferred in fresh LB liquid medium of the 1L containing kalamycin resistance, 37 DEG C, 220rpm/min shaking table shakes bacterium.Bacterium solution OD value is monitored with spectrophotometer at any time, when bacterium solution OD value reaches 0.6-0.8, The IPTG that 1ml 0.8M is added carries out inducing expression.It takes 1ml bacterium solution 2000rpm/min to be centrifuged before induction two minutes, abandons supernatant, 100 μ l 1 × SDS loading Buffer are added into thallus, 100 DEG C of water-baths boil sample 10 minutes, as not inducing pair According to group, the IPTG inducing expression time is 4 hours.

Microorganism collection and cracking: after induction, by bacterium solution in batches with 11000rpm/min centrifugation 15 minutes, in abandoning Clearly, 100 μ l 1 × SDS loading Buffer are added in a small amount of thallus of picking, and 100 DEG C of water-baths boil sample 10 minutes, as luring Lead control group.The lysis buffer (associated formula sees below, and reagent used below is same) of 20ml, whirlpool vibration is added in remaining thallus It swings device sufficiently to shake up, with -80 DEG C after refrigerator multigelation 3 times, bacterium solution is placed in and carries out ultrasonication, broken condition on ice are as follows: 400W, each ultrasonic time 5 seconds, interval 5 seconds, number are 300 times, before ultrasonication, and 1ml albumen is added into bacterium solution Enzyme inhibitor cocktai l (Sel leckchem company, the U.S., 100 × liquid storage).

Collecting protein and purifying: the bacterium solution 11000rpm/min after ultrasonication is centrifuged 10 minutes, careful to be sucked out Clearly, a small amount of precipitating and supernatant are taken respectively, 100 μ l 1 × SDS loading Buffer are added, and 100 DEG C of water-baths boil sample 10 and divide Clock gives over to electrophoresis loading, and secreting, expressing albumen is primarily present in supernatant, and albumen is then primarily present in precipitating when inclusion body is expressed In.

Due to destination protein amalgamation and expression 6 × his label protein, therefore destination protein can be purified with affinity chromatography method, Nickel column used is that the Ni-NTA Agarose of QIAGEN company production purifies beads.Before protein purification:

Clean nickel column: Ni-NTA Agarose used is saved with 20% alcohol solution dipping in 4 DEG C of conditions.Using going to 4mlNi-NTA Agarose beads is perfused in plastic column, slightly settles, then fills it up with double dehydrated column cleanings 2 into pillar It is secondary, efflux is abandoned, is carried out under the conditions of operating in 4 DEG C above.

Balance nickel column: equilibration buffer is filled it up with into the nickel column after cleaning, efflux is abandoned, is operated under the conditions of 4 DEG C above It carries out.

Albumen sample crosses column purification: sample liquid carefully filled into pillar, is perfused several times, column was stood, abandons efflux, with On operate in 4 DEG C under the conditions of carry out.

Washing nickel column: after sample crosses column, washing buffer is filled it up with into nickel column, stood column, is repeated twice, and is abandoned Efflux carries out under the conditions of operating in 4 DEG C above.

Elute albumen: into the nickel column after washing plus 2ml elution buffer, collection efflux carry out under the conditions of 4 DEG C.

Protein concentration and preservation: the ultrafiltration concentration pipe (being purchased from U.S. mil lipore company) of selection 7OKD specification uses It is preceding to be cleaned twice with double dehydrations, cleaning process: 4 DEG C of centrifugations, 6000rpm/min, 5 minutes.

Protein sample is added, 6000rpm/min is centrifuged after twenty minutes, outwells collecting pipe bottom waste stream, mend into super filter tube Add sterile PBS buffer solution, until filling it up with, continues to be centrifuged, so be repeated 2 times, PBS buffer is finally made to replace eluent In imidazoles.

After concentration, raffinate is the destination protein being concentrated in super filter tube upper layer, and BCA method measures total protein concentration Afterwards, packing is stored in -80 degrees Celsius of ultralow point of refrigerators, and when use avoids multigelation.

Related reagent used in this example and its formula are as follows:

Lysis buffer: 50mM Tris, 300mMNaCl, pH8.0

Washing buffer: 50mM NaH₂PO₄, 300mMNaCl, pH8.0

Elution buffer: 50mM NaH₂PO₄, 300mMNaCl, 250mM imidazoles, pH8.0

Equilibration buffer: 50mM NaH₂PO₄, 300mMNaCl, 20mM imidazoles, pH8.0

As a result as shown in fig. 6, the LGR4-ECD molecular weight of albumen size given expression to is 72KD or so, purity is high, without miscellaneous Band.

Embodiment 8

Lgr4 Extracellular domain protein treatment concentration relies on experiment

It chooses 8 week old or so male C57BL/6 mouse and is divided into three groups, every group of 8 mouse.Yellow Jackets anesthesia, with scraping Hair device removes back hair, and subsequent every mouse is subcutaneously in left side dorsal injection 5 × 10⁵LLC cell.It is thin from injection tumour Start within second day after born of the same parents, be administered respectively to three groups of right side of mice dorsal scs, are as follows: PBS blank control group, 10 μ g dosage groups, 20 μ g dosage group.Successive administration 5 days, starts within the tenth day after injecting tumour cell to use vernier caliper measurement mouse tumor volume, adopt With gross tumor volume=length × wide², 2 calculation formula.

As a result as shown in Figure 4 A, it is shown that the variation figure of gross tumor volume at any time, it is seen that as Lgr4 Extracellular domain protein is given The rising of concentration, gross tumor volume are obviously suppressed.

Embodiment 9

Lgr4 Extracellular domain protein Experiment on therapy

It chooses 8 week old or so male C57BL/6 mouse and is divided into six groups, every group of 8 mouse.Yellow Jackets anesthesia, with scraping Hair device removes back hair, and subsequent every mouse is subcutaneously in left side dorsal injection 5 × 10⁵LLC cell.It is thin from injection tumour Start within second day after born of the same parents, be administered respectively to six groups of right side of mice dorsal scs, are as follows: PBS blank control group, 20 μ gLgr4 are extracellular Section albumen dosage group, irrelevant antibody IgG control group, anti-mouse R-spondin1 monoclonal antibody is (according to the neutralization meter of specification Amount calculates, and 14 μ g are administered in every mouse), DMSO blank control group, BLZ945 (CSF-1R micromolecular inhibitor, it is possible to reduce swollen M2 type macrophage in tumor microenvironment) 200mg/kg weight group (group can be used as positive controls).Successive administration 5 days, in Start within the tenth day after injection tumour cell with vernier caliper measurement mouse tumor volume, using gross tumor volume=length × wide²,2 Calculation formula,

As a result as shown in Figure 4 B, it is shown that the variation figure of gross tumor volume at any time, it is seen that with Lgr4 Extracellular domain protein agent The increase of amount, anti-mouse R-spondin1 monoclonal antibody and BLZ945 all have obvious inhibition effect to mouse tumor volume Fruit.

Embodiment 10

Tumor tissues macrophage and T cell analysis after the administration of tumor formation mouse

Operation clip as above states the tumor tissues of six groups of mouse, smashs to pieces and disappears in 0.25% tryptic digestive juice of 5ml Change 5 minutes, isometric DMEM complete medium is added and terminates digestion, then cell is resuspended with PBS in centrifugation, and it is close to adjust cell It spends to 1 × 10⁸/ml.

Respectively by the 200 above-mentioned cell suspensions of μ l as in flow cytometer showed pipe, point good Isotype control pipe, single dye is managed and double dye pipes, The rat anti-mouse monoclonal antibody of 0.5 μ l fluorophor label is added in every pipe, is sufficiently mixed uniformly that being placed on 4 DEG C is protected from light item Part is incubated for 40 minutes, and then the PBS buffer solution washing with 1ml containing 2% fetal calf serum three times, for the last time delays 200 μ lPBS Above-mentioned cell is resuspended in fliud flushing.Flow cytometer (BD company) analyzes F4/80 in each tissue⁺CD206⁺M2 type macrophage, F4/ 80⁺CD16/32⁺M1 type macrophage and CD3⁺CD8⁺T cell ratio.

As a result as shown in Figure 4 C, Lgr4 Extracellular domain protein and anti-mouse R-spondin1 monoclonal antibody can obviously subtract Few M2 type tumor-associated macrophage enhances CD8 simultaneously⁺The infiltration of T cell.

All references mentioned in the present invention is incorporated herein by reference, just as each document coverlet It is solely incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.

Sequence table

<110>East China Normal University

Shanghai Bang Yao Biotechnology Co., Ltd

<120>LGR4 and R-spondin binding inhibitors and its purposes in oncotherapy

<130> P2017-1528

<160> 2

<170> PatentIn version 3.5

<210> 1

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<213>homo sapiens (Homo sapiens)

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Ser Gly Lys Gly Leu Thr Ala Val Pro Glu Gly Leu Ser Ala Phe Thr

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Gln Ala Leu Asp Ile Ser Met Asn Asn Ile Thr Gln Leu Pro Glu Asp

35 40 45

Ala Phe Lys Asn Phe Pro Phe Leu Glu Glu Leu Gln Leu Ala Gly Asn

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Asp Leu Ser Phe Ile His Pro Lys Ala Leu Ser Gly Leu Lys Glu Leu

65 70 75 80

Lys Val Leu Thr Leu Gln Asn Asn Gln Leu Lys Thr Val Pro Ser Glu

85 90 95

Ala Ile Arg Gly Leu Ser Ala Leu Gln Ser Leu Arg Leu Asp Ala Asn

100 105 110

His Ile Thr Ser Val Pro Glu Asp Ser Phe Glu Gly Leu Val Gln Leu

115 120 125

Arg His Leu Trp Leu Asp Asp Asn Ile Leu Thr Glu Val Pro Val Arg

130 135 140

Pro Leu Ser Asn Leu Pro Thr Leu Gln Ala Leu Thr Leu Ala Leu Asn

145 150 155 160

Asn Ile Ser Ser Ile Pro Asp Phe Ala Phe Thr Asn Leu Ser Ser Leu

165 170 175

Val Val Leu His Leu His Asn Asn Lys Ile Lys Ser Leu Ser Gln His

180 185 190

Cys Phe Asp Gly Leu Asp Asn Leu Glu Thr Leu Asp Leu Asn Tyr Asn

195 200 205

Asn Leu Asp Glu Phe Pro Gln Ala Ile Lys Ala Leu Pro Ser Leu Lys

210 215 220

Glu Leu Gly Phe His Ser Asn Ser Ile Ser Val Ile Pro Asp Gly Ala

225 230 235 240

Phe Ala Gly Asn Pro Leu Leu Arg Thr Ile His Leu Tyr Asp Asn Pro

245 250 255

Leu Ser Phe Val Gly Asn Ser Ala Phe His Asn Leu Ser Asp Leu His

260 265 270

Ser Leu Val Ile Arg Gly Ala Ser Leu Val Gln Trp Phe Pro Asn Leu

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Ala Gly Thr Val His Leu Glu Ser Leu Thr Leu Thr Gly Thr Lys Ile

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Ser Ser Ile Pro Asp Asp Leu Cys Gln Asn Gln Lys Met Leu Arg Thr

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Leu Asp Leu Ser Tyr Asn Asp Ile Arg Asp Leu Pro Ser Phe Asn Gly

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Leu Ser Arg Asn Leu Ile Arg Glu Ile His Ser Gly Ala Phe Ala Lys

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gggacaaaaa taagcagcat acctgatgat ctgtgccaaa accaaaagat gctgcggact 960

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gaagaaattt cattgcagcg taatcaaatc tccctgataa aggaaactac ttttcaaggc 1080

ctaacatccc taaggattct agatctgagt agaaacctga ttcgtgaaat tcacagtgga 1140

gcttttgcga agcttgggac aattactaac ctggatgtga gtttcaatga attaacctca 1200

tttcctacgg aaggcctgaa tgggctcaat caacttaaac ttgtgggtaa cttccagctg 1260

aaagatgcct tggcagccag agactttgcc aatctcaggt ctctatcagt accatatgct 1320

tatcagtgtt gtgcattttg ggggtgtgac tcttatgcaa atttaaacac agaagataac 1380

agcccccaag accacagtgt gacaaaagag aaaggtgcta cagatgcagc aaatgccacc 1440

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gct 1503

Claims

1. a kind of purposes for the inhibitor for inhibiting LGR4 and R-spondin to combine, which is characterized in that be used to prepare a preparation or group Object, the preparation or composition are closed for regulating and controlling the function Phenotypic change of macrophage.

2. purposes as described in claim 1, which is characterized in that the function Phenotypic change of the regulation macrophage refers to suppression Macrophage processed polarizes to M2 phenotype, and/or macrophage is promoted to polarize to M1 phenotype.

3. purposes as described in claim 1, which is characterized in that the macrophage is tumor-associated macrophage.

4. purposes as described in claim 1, which is characterized in that the preparation or composition is also used to one selected from the group below Or multiple purposes:

(i) ratio of M1 phenotype macrophage is improved；

(ii) CD8 is improved⁺The ratio of T cell；

(iii) tumour is treated；

(iv) tumor tissues are adjusted and microenvironment is immunized.

5. purposes as described in claim 1, which is characterized in that the inhibitor is selected from the group:

(a) specificity inhibits LGR4 expression and/or active antagonist；

(b) specificity inhibits R-spondin expression and/or active antagonist；

(c) analogue of LGR4；

(d) analogue of R-spondin；

(e) any combination of above-mentioned items.

6. purposes as described in claim 1, which is characterized in that the inhibitor is LGR4 Extracellular domain protein.

7. purposes as described in claim 1, which is characterized in that the inhibitor is to integrate LGR4 Extracellular domain protein to encode base The CAR-T cell of cause.

8. purposes as claimed in claims 6 or 7, which is characterized in that the amino acid sequence of the LGR4 Extracellular domain protein is such as Shown in SEQ ID NO.:1.

9. a kind of screening promotes macrophage by the method for the drug candidate that M2 Phenotypic change is M1 phenotype, which is characterized in that institute State method comprising steps of

(a) untested compound is provided, and is detecting the untested compound to the inhibition feelings of LGR4 and R-spondin combination Condition, so that the untested compound for being combined with inhibiting effect to LGR4 and R-spondin is selected, as the compound through primary dcreening operation；With And

(b) effect for testing the compounds towards macrophages Phenotypic change through primary dcreening operation promotes macrophage thin to select and have Born of the same parents are the compound of M1 phenotype by M2 Phenotypic change, as drug candidate.

10. promoting to a kind of non-therapeutic method of the macrophage by M2 Phenotypic change for M1 phenotype, which is characterized in that including step It is rapid:

(a) in the presence of the inhibitor for inhibiting LGR4 and R-spondin to combine, macrophage is cultivated, to promote macrophage It is M1 phenotype by M2 Phenotypic change.