WO2021135350A1 - 阻断ccl28趋化通路的胃癌治疗方法 - Google Patents

阻断ccl28趋化通路的胃癌治疗方法 Download PDF

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WO2021135350A1
WO2021135350A1 PCT/CN2020/113566 CN2020113566W WO2021135350A1 WO 2021135350 A1 WO2021135350 A1 WO 2021135350A1 CN 2020113566 W CN2020113566 W CN 2020113566W WO 2021135350 A1 WO2021135350 A1 WO 2021135350A1
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ccl28
gastric cancer
cells
catenin
expression
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高维强
马斌
冀露
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上海交通大学
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    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57446—Specifically defined cancers of stomach or intestine
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6872—Intracellular protein regulatory factors and their receptors, e.g. including ion channels

Definitions

  • the present invention belongs to the field of biomedicine. Specifically, the present invention relates to a method for treating gastric cancer by blocking the CCL28 chemotactic pathway.
  • the fast-developing immunotherapy methods have brought hope to countless cancer patients.
  • the molecular characteristics of gastric cancer indicate that EBV-related subtypes of gastric cancer show elevated PD-L1 expression, suggesting that anti-PD immunotherapy may be effective for these patients.
  • the development of these immunotherapy methods in gastric cancer encounters many practical problems.
  • the immune escape mechanism of tumors is also a major obstacle to the immunotherapy of tumors.
  • Regulatory T cells are an important type of immunosuppressive cell involved in the immune evasion of tumors.
  • the purpose of the present invention is to provide an effective treatment plan for refractory gastric cancer while reducing side effects.
  • the first aspect of the present invention provides a use of CCL28 gene or CCL28 protein or its detection reagent for preparing reagents or kits for detecting gastric cancer, said gastric cancer is Wnt/ ⁇ -catenin signaling pathway and chemokine
  • the expression of CCL28-related molecules is also abnormally up-regulated in gastric cancer.
  • the pathological manifestations of gastric cancer have one or more pathological manifestations selected from the following group:
  • the pathological manifestations of gastric cancer also include low body immune response.
  • the gastric cancer includes gastric cancer in situ, intestinal gastric cancer, and diffuse gastric cancer.
  • the gastric cancer includes Helicobacter pylori-negative gastric cancer.
  • the gastric tissue includes gastric cancer tumor tissue, gastric adjacent tissue, or a combination thereof.
  • the related expression of the Wnt/ ⁇ -catenin signaling pathway is selected from the group consisting of: the expression level of transcription factor TCF1, the expression level of transcription factor TCF4, the content of ⁇ -catenin, and the nuclear ectopic ⁇ -catenin Level, transcriptional activity of ⁇ -catenin/TCF4 complex, phosphorylation level of GSK3 ⁇ , or a combination thereof.
  • the chemokine CCL28-related molecules include: CCL28 gene (including the nucleotide sequence of the genome, cDNA sequence, and/or mRNA), and the gene of the CCL28 receptor includes the nucleotide sequence of the genome , CDNA sequence, and/or mRNA), CCL28 protein, CCL28 receptor protein, or a combination thereof.
  • the CCL28 receptor includes CCR3 and/or CCR10.
  • the kit includes: reagents for quantitatively detecting the protein or mRNA of CCL28-related molecules and corresponding labels or instructions.
  • the reagents include specific primers, specific antibodies, probes and/or chips for CCL28 related molecules.
  • the above-mentioned reagent includes a detection chip, such as a nucleic acid chip and a protein chip.
  • the nucleic acid chip includes a substrate and a cancer-related gene-specific oligonucleotide probe spotted on the substrate, and the cancer-related gene-specific oligonucleotide probe Including probes that specifically bind to CCL28-related genes or mRNA.
  • the protein chip includes a substrate and a specific antibody of a cancer-related protein spotted on the substrate, and the specific antibody of the cancer-related protein includes a specific antibody of an anti-CCL28-related protein.
  • the CCL28-related protein includes fusion protein and non-fusion protein.
  • the reagent or kit includes: CCL28 gene (or nucleic acid molecule) or CCL28 protein as a standard.
  • the CCL28 gene (or nucleic acid molecule) or CCL28 protein includes wild type and/or mutant type.
  • the reagent or kit further includes: a detection reagent for detecting CCL28 gene or CCL28 protein.
  • the second aspect of the present invention provides a kit for detecting gastric cancer.
  • the kit contains a container containing a detection reagent for detecting CCL28-related protein or mRNA; and a label or instruction manual.
  • the label or instructions indicate that the kit is used to detect gastric cancer.
  • the gastric cancer is gastric cancer in which the Wnt/ ⁇ -catenin signaling pathway and the expression of chemokine CCL28-related molecules are abnormally up-regulated at the same time.
  • the content indicated in the label or manual is selected from the following group:
  • the detection reagents include: specific primers, specific antibodies, probes and/or chips.
  • the kit is used for detecting isolated human tumor tissue samples or blood samples.
  • the tumor tissue sample is a gastric cancer sample.
  • the third aspect of the present invention provides a use of a CCL28 inhibitor for preparing a pharmaceutical composition for inhibiting the growth or proliferation of cancer cells, or for preparing a pharmaceutical composition for treating gastric cancer;
  • the gastric cancer is Wnt/ ⁇ -catenin signaling pathway and the expression of chemokine CCL28-related molecules are abnormally up-regulated at the same time in gastric cancer.
  • the inhibitor is selected from the following group: an antibody or small molecule inhibitor targeting CCL28 and/or its receptor protein; a targeting nucleic acid molecule targeting CCL28 and/or its receptor gene or Gene editor; or a combination thereof.
  • the CCL28 receptor includes CCR3 and/or CCR10.
  • the antibody is selected from the group consisting of polyclonal antibodies, monoclonal antibodies, chimeric antibodies, bispecific antibodies, antibody conjugates, small molecule antibodies, antibody fusion proteins, and combinations thereof.
  • the small molecule antibody is selected from the group consisting of single-chain antibody ScFv, Fab antibody, Fv fragment, and combinations thereof.
  • the ScFv antibody includes a secreted single-chain antibody expressed (including overexpression) in the therapeutic cell.
  • the therapeutic cells include mesenchymal stem cells and CAR-T cells.
  • the vector includes: bacterial plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.
  • the inhibitor is selected from the group consisting of plant extract inhibitors, small molecule compound inhibitors, nucleic acid inhibitors, peptide inhibitors, polysaccharide inhibitors, viral vector inhibitors, lipids Plastid carrier inhibitor, or nanoparticle carrier inhibitor.
  • the pharmaceutical composition further includes other Wnt/ ⁇ -catenin signaling pathway inhibitors.
  • the pharmaceutical composition can synergistically inhibit the activation of abnormal Wnt/ ⁇ -catenin signaling pathway in tumor tissues.
  • the pharmaceutical composition can inhibit the infiltration of tumor tissue by regulatory T (Treg) cells, and at the same time improve the activity of peripheral immunity.
  • Treg regulatory T
  • the fourth aspect of the present invention provides a non-therapeutic in vitro method for inhibiting the growth or proliferation of cancer cells, including the step of culturing cancer cells in the presence of a CCL28 inhibitor, thereby inhibiting the growth or proliferation of cancer cells.
  • the method includes adding CCL28-related molecular inhibitors to the culture system of cancer cells, thereby inhibiting the growth or proliferation of cancer cells.
  • the cancer cells are gastric cancer cells.
  • the gastric cancer cells are selected from: AGS cell line, SC7901 cell line, AZ-521 cell line, primary gastric cancer cell, or a combination thereof.
  • the fifth aspect of the present invention provides a method for screening candidate compounds for the treatment of cancer, including the steps:
  • test group In the test group, add a test compound to the cell culture system, and observe the expression and/or activity of CCL28-related molecules in the cells of the test group; in the control group, the cell culture system does not Adding a test compound, and observing the expression and/or activity of CCL28-related molecules in the cells of the control group;
  • the test compound is a candidate compound for the treatment of cancer that has an inhibitory effect on the expression and/or activity of CCL28-related molecules.
  • the cells include: cancer cells or normal cells;
  • the cells are gastric cancer cells or gastric cells.
  • the method further includes the steps:
  • step (b) The candidate compound obtained in step (a) is further tested for its inhibitory effect on the growth or proliferation of cancer cells.
  • the step (b) includes the step: in the test group, the cancer cell culture system is added with a test compound, and the number and/or growth of the cancer cells are observed; in the control group, the cancer cells No test compound is added to the cell culture system, and the number and/or growth of cancer cells are observed; among them, if the number or growth rate of cancer cells in the test group is lower than that of the control group, it indicates that the test compound is the growth of cancer cells Or a candidate compound for the treatment of cancer with an inhibitory effect on proliferation.
  • the sixth aspect of the present invention provides a method for inhibiting or treating gastric cancer, comprising the steps of: administering a safe and effective amount of a CCL28-related molecular inhibitor to a subject (mammal) in need of treatment; the gastric cancer is Wnt/ ⁇ - The catenin signaling pathway and the expression of chemokine CCL28-related molecules are abnormally up-regulated at the same time.
  • Figure 1 shows that the ⁇ -catenin signaling pathway induces CCL28 expression in gastric cancer.
  • A is the qPCR detection of gastric cancer cell line SGC7901 overexpressing ⁇ -catenin.
  • Figure B shows the detection of ⁇ -catenin and CCL28 protein levels in gastric cancer cell lines SGC7901 and AGS that overexpress or knock down ⁇ -catenin.
  • C and D are the immunohistochemical detection of ⁇ -catenin and CCL28 in the tissue chip of gastric cancer patients, and the correlation analysis result of the positive rate.
  • FIG. 2 shows that CCL28 is a direct transcriptional regulatory target gene of the ⁇ -catenin signaling pathway.
  • Figure A is a schematic diagram of the binding site of ⁇ -catenin/TCF transcription factor complex in the promoter of CCL28 gene.
  • B the chromatin immunoprecipitation experiment proved that ⁇ -catenin binds to the predicted sites 1 and 3 in the CCL28 gene promoter.
  • C is that overexpression of ⁇ -catenin up-regulates the activity of the CCL28 promoter, and mutation of the core sequence of binding sites 1 and 3 (CCL28.mut) makes the regulation of ⁇ -catenin lose.
  • D is that overexpression of ⁇ -catenin up-regulates the activity of the Wnt pathway reporter gene TOFlash and CCL28 promoter.
  • Wnt inhibitor iCRT14 treatment reduced the activity of the two reporter genes.
  • E is that compared with the control shRNA sequence shScr, the shRNA targeting the Wnt pathway transcription factors TCF/LEF family members TCF1 (shTCF1) and TCF4 (shTCF4) knocked down their expression by using RNA interference, and the CCL28 promoter activity was reduced , While knocking down the expression of LEF1 (shLEF1) has no effect on the CCL28 promoter activity.
  • Figure F shows that overexpression of the transcription factors TCF1 and TCF4 up-regulated the activity of the CCL28 promoter, and overexpression of LEF1 had no effect on the activity of the CCL28 promoter. It shows that the transcription factors TCF and TCF4 cooperate with ⁇ -catenin to regulate the expression of CCL28.
  • Figure 3 shows that the Wnt pathway inhibitor iCRT14 reduces the expression of CCL28 in mouse gastric cancer.
  • the expression of CCL28 protein in the stomach of mice treated with Wnt pathway inhibitor iCRT14 was found to be significantly reduced by Western blotting (A) and ELISA (B).
  • vehicle represents a drug solvent (medium).
  • Figure 4 shows that the expression of CCL28 is associated with the development of gastric cancer.
  • the Oncomine database analysis found that the expression of CCL28 mRNA in intestinal (Intestinal Type) and diffuse type (Diffuse Type) gastric cancers is higher than that of normal tissues.
  • the immunohistochemical analysis of gastric cancer patient tissues shows that the protein expression level of CCL28 is higher than that in grade II and III gastric cancer, which has a higher degree of deterioration.
  • Figure 5 shows that gastric cancer cells activated by the ⁇ -catenin signaling pathway induce the expression of CCL28 to regulate the migration of T cells.
  • A is the Western blot analysis of ⁇ -catenin and CCL28 under the conditions of SGC7901 cell line overexpressing ⁇ -catenin (Vector) and knocking down CCL28 (shScr).
  • B is a flow chart of regulatory T cells (Tregs), CD4 + T cells, and CD8 + T cells.
  • C and D are the statistical results of the proportion and absolute number of regulatory T cells (Tregs), CD4 + T cells, and CD8 + T cells. *P ⁇ 0.05;**P ⁇ 0.01.
  • Figure 6 shows that CCL28 antibody treatment inhibits H.felis/MNU-induced gastric cancer progression in situ in mice.
  • Figure A is the CCL28 antibody treatment model diagram.
  • Figure B is the anatomical diagram of the stomach treated with CCL28 antibody and the statistics of the tumor area. *P ⁇ 0.05.
  • C is the HE staining with CCL28 antibody treatment, Alcianlan staining and H+K+ATPase immunohistochemical staining.
  • D is the pathological statistical results of the gastric body (Corpus) and gastric antrum (Antrum). *P ⁇ 0.05; **P ⁇ 0.01; ***P ⁇ 0.001.
  • Figure 7 shows that CCL28 antibody treatment alleviates the suppression of the immune microenvironment.
  • A is the flow cytometry diagram and statistical diagram of regulatory T cells (Tregs).
  • B is the flow cytometry diagram and statistical diagram of IFN ⁇ + CD4 + T cells.
  • C is the flow cytometry diagram and statistical diagram of IFN ⁇ + CD8 + T cells. *P ⁇ 0.05;***P ⁇ 0.001.
  • Figure 8 shows that CCL28 antibody blockade has no significant therapeutic effect in melanoma and breast cancer xenograft models.
  • A is the growth curve of melanoma B16 subcutaneously transplanted tumor.
  • Figure B is the growth curve of breast cancer 4T1 subcutaneously transplanted tumor.
  • Figure C is the survival rate of melanoma B16 tumor-bearing mice.
  • D is the survival rate of breast cancer 4T1 tumor-bearing mice.
  • Figure 9 shows the expression level of CCL28 protein in various human tissues.
  • the Human Protein Atlas database it was found that CCL28 protein is highly expressed in the brain, gastrointestinal tract, and pancreas.
  • the term “about” means that the value can vary from the recited value by no more than 1%.
  • the expression “about 100” includes all values between 99 and 101 (eg, 99.1, 99.2, 99.3, 99.4, etc.).
  • the term "containing” or “including (including)” can be open, semi-closed, and closed. In other words, the term also includes “substantially consisting of” or “consisting of”.
  • Chemokines are a class of cytokines that regulate cell migration and play a vital role in the formation of tumor micro-immune environment and the occurrence and development of tumors.
  • C-C motif chemokine ligand 28 (C-C motif chemokine ligand 28, CCL28), located at 5p12, encodes 8 exons.
  • CCL28 is a chemokine that mediates the migration of cells expressing its receptor CCR3 or CCR10.
  • CCL28 is expressed in the epithelial cells of the digestive tract, lung, breast and other tissues (as shown in Figure 9).
  • the present invention proves for the first time that the expression of Wnt/ ⁇ -catenin-CCL28 pathway is up-regulated in the pathological process of gastric cancer, and for the first time the therapeutic effect of CCL28 antibody blocking in a gastric cancer model is officially confirmed.
  • the abnormally up-regulated expression of chemokine CCL28-related molecules refers to the mRNA expression of CCL28-related molecules in a certain tissue (such as gastric cancer tissue) C1 and the mRNA expression of CCL28-related molecules in normal tissues (such as adjacent tissues)
  • the level of ⁇ -catenin in the cytoplasm is strictly controlled by the multi-protein destruction complex composed of APC, Axin and glycogen synthase kinase-3 ⁇ (GSK-3 ⁇ ).
  • the multi-protein destruction complex can phosphorylate ⁇ -catenin and further induce its ubiquitination and proteasome-mediated degradation.
  • Wnts pathway stimulation will inhibit the activity of the destruction complex, break the stability of cytoplasmic ⁇ -catenin, and translocate cytoplasmic ⁇ -catenin to the nucleus to activate transcription together with the LEF/TCF family.
  • the activation program of this ⁇ -catenin transcription target is Catenin responsive transcription (CRT) is a key aspect of cell responsiveness to specific Wnt stimuli.
  • Wnt-related carcinogenesis is caused by CRT misregulation, so targeting the ⁇ -catenin-TCF/LEF complex has become an ideal therapeutic target.
  • the Wnt/ ⁇ -catenin pathway is one of the most common oncogene pathways in colorectal cancer, gastric cancer and other types of cancer. It plays an important role in the occurrence and development of cancer and is also one of the targets of concern in cancer treatment.
  • CTNNB1 and APC are common and significantly mutated genes. Taking gastric cancer as an example, about 70% of gastric cancer patients are accompanied by mutations in the Wnt/ ⁇ -catenin signaling pathway.
  • Wnt signaling pathway components may be achieved by up-regulating positive regulators or down-regulating negative regulators, and ultimately lead to the activation of the canonical Wnt pathway.
  • Wnt/ ⁇ -catenin signaling pathway abnormalities are found in most cancer types, and it is a very important oncogene pathway in tumorigenesis and development.
  • the increased expression of ⁇ -catenin is negatively correlated with the infiltration of lymphocytes in the tumor, suggesting that Wnt/ ⁇ -catenin can also lead to immune evasion of tumors and resistance to immunotherapy.
  • the present invention proves for the first time that in the pathological process of gastric cancer, ⁇ -catenin and the transcription factor TCF/LEF complex can up-regulate the expression of CCL28 in gastric cancer by binding to the CCL28 promoter region.
  • This Wnt/ ⁇ -catenin-CCL28 pathway is involved in gastric cancer.
  • the up-regulation of expression in the pathological process is unique.
  • CCL28 antibody can significantly alleviate the progression of gastric cancer, but has no similar effect on other solid tumors such as melanoma and breast tumor.
  • the abnormally up-regulated expression of Wnt/ ⁇ -catenin signaling pathway related molecules means that the mRNA expression B1 of Wnt/ ⁇ -catenin signaling pathway related molecules in a certain tissue (such as gastric cancer tissue) is compared with that in normal tissues (para-cancerous tissues).
  • the ratio of mRNA expression B0 (B1/B0) of Wnt/ ⁇ -catenin signaling pathway related molecules is ⁇ 1.2, preferably ⁇ 1.5, more preferably, ⁇ 2.0 or ⁇ 2.5.
  • iCRT14 is a catenin response transcription inhibitor.
  • RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway.Gonsalves et al.Proc.Natl.Acad.Sci.USA,2011;108:595)
  • the screening identified 34 molecules with statistically significant inhibitory effects on the activity of the dTF12-luciferase reporter gene (total hit rate is about 0.3%), and these compounds are called CRT inhibitors (iCRT).
  • iCRT14 belongs to the ⁇ -catenin reactive transcription inhibitor of the thiazolidinedione class, and is a Wnt/ ⁇ -catenin pathway inhibitor.
  • Empirical formula (Hill notation) C 21 H 17 N 3 O 2 S. It is an effective inhibitor of ⁇ -catenin and transcription factor TCF/LEF complex. It can disrupt the ⁇ -catenin-TCF/LEF interaction in a dose-dependent manner, and cause G0/G1 arrest in colon tumor cell lines, resulting in a continuous decrease in cell proliferation and tumor growth in colon cancer cells.
  • the present invention finds for the first time that in gastric cancer, the regulatory factors TCF1 and TCF4 non-LEF1 cooperate with ⁇ -catenin to regulate the transcription of the CCL28 gene.
  • the S33Y. ⁇ -catenin plasmid is transferred into gastric cancer cells to inhibit ⁇ -catenin phosphorylation, so as to achieve the purpose of overexpressing ⁇ -catenin.
  • the said DNA plasmid of S33Y. ⁇ -catenin refers to a DNA plasmid carrying a fragment in which serine (S33) at position 33 of the amino acid sequence of ⁇ -catenin is mutated to tyrosine (Y).
  • S33 is the phosphorylation site of GSK3 ⁇ , and ⁇ -catenin enters the degradation pathway after phosphorylation at this site.
  • the mutant S33Y. ⁇ -catenin can escape the phosphorylation and degradation pathways, and then be more efficiently enriched in the cell.
  • This mutation has also been detected in samples from some patients with gastrointestinal tumors.
  • the invention is used to study the effect of abnormal activation of Wnt/ ⁇ -catenin pathway in tumor cells.
  • Helicobacter pylori is considered to be one of the main causes of chronic gastritis. Therefore, the establishment of a mouse model of gastric cancer with Helicobacter pylori is of great significance to the study of the occurrence and development of human gastric cancer.
  • C57BL/6 mice have significant resistance to colonization of various Helicobacter pylori strains in the stomach. Therefore, it is very important to use H.felis (a close relative of Helicobacter pylori) to establish a mouse model of gastric cancer. This strain is isolated from the stomach of cats and is easily colonized in the stomach of mice. It can induce severe gastritis and atrophy in mice.
  • MNU N-methyl-N-nitrosourea
  • Surgical removal of the anterior stomach before use of MNU helps promote the occurrence of well-differentiated adenocarcinoma of the glandular stomach, and the incidence rate is 100% after 40 weeks of use. Therefore, the gastric glands are very sensitive to the carcinogenic effects of MNU.
  • 240ppm MNU was dissolved in drinking water and administered for 5 consecutive weeks (every other week). The results of experiments on 6 strains of mice showed that this method can induce gastric cancer. Therefore, this program uses a combination of H. felis infection and MNU administration to establish a mouse gastric cancer model.
  • Immune evasion has been recognized as a new sign of cancer. Understanding the tumor immune microenvironment is essential for discovering new therapeutic targets and predicting and immunotherapy response. The normal immune system will curb the occurrence and development of tumors. As the cancer progresses, the tumor will produce many immune evasion mechanisms, leading to the enhancement of tumor growth.
  • Regulatory T (Treg) cells are a type of immunosuppressive cells that have been found to participate in the process of accelerating tumor progression in a variety of cancer types. Regulatory T cells (Treg) are an important type of immunosuppressive cell involved in the immune evasion of tumors. Its increase will lead to a decrease in the function and number of anti-tumor effector immune cells, as well as the weakening or even loss of the efficacy of tumor immunotherapy or other therapies.
  • the use of CCL28 antibody can effectively inhibit the migration process of targeted regulatory T cells to tumors without affecting the apoptosis of regulatory T cells.
  • it only targets the Treg cells in the gastric cancer tumor tissue and the spleen, without reducing the ratio of Treg cells in the blood, and can also improve the overall immune response.
  • the present invention provides a gastric cancer that inhibits CCL28 to treat Wnt/ ⁇ -catenin signaling pathway-CCL28 abnormally elevated gastric cancer.
  • Application of the CCL28 antibody treatment provided by the present invention can significantly reduce the tumor area, obvious changes in dysplasia and intestinal transformation, and obviously alleviate the phenomenon of normal cell loss.
  • the present invention finds that ⁇ -catenin/CCL28 is positively correlated with the occurrence and progression of gastric cancer, and thus provides a detection method for the diagnosis, screening and prognosis of gastric cancer, which can screen the incidence of gastric cancer in the population Rate and accurately detect the metastasis of gastric cancer.
  • the inhibitory effect of the present invention on ⁇ -catenin/CCL28 only targets Treg cells in gastric cancer tumor tissue and spleen, without reducing the ratio of Treg cells in the blood, and can improve the overall immune response.
  • the method of the present invention to block the CCL28 chemotactic pathway to treat malignant gastric cancer targets and regulates the migration process of T cells to tumors without affecting the apoptosis of regulatory T cells, which may provide more targeted regulatory T cell immunotherapy.
  • the present invention provides a therapeutic idea of inhibiting the Wnt/ ⁇ -catenin signaling pathway in tumor cells by inhibiting CCL28.
  • gastric cancer with Wnt/ ⁇ -catenin signaling pathway and CCL28 abnormally elevated at the same time is a disease phenotype that is significantly different from other tumors, even solid tumors, and is mainly reflected in the effectiveness of blocking the CCL28 chemotaxis pathway Inhibiting the development of malignant gastric cancer, but the effect on other tumor models is not clear, especially for melanoma cell B16 and breast cancer cell 4T1 models.
  • Human gastric cancer cell lines AGS and SC7901 were cultured in RPMI 1640 (Life Technologies) medium containing 10% serum (Gbico) and 1% penicillin/streptomycin dual antibody (Life Technologies).
  • RNA in the cell line and mouse gastric tissue was extracted using RNeasy Mini Kit (Qiagen), and then used PrimeScript RT Reagent Kit (Takara) for reverse transcription to synthesize cDNA.
  • QPCR is performed using SYBR Green PCR Master Mix kit (Takara) and ABI 7900HT Fast Real-Time PCR System (Applied Biosystems). The method of ⁇ Ct is used for quantitative calculation.
  • the protein in gastric cancer cell lines and mouse gastric tissues was lysed with RIPA lysate (Thermo Scientific) with protease inhibitor (Roche), and the protein concentration was detected with BCA Protein Assay Reagent (Thermo Scientific).
  • the detected proteins are as follows: ⁇ -catenin (Abcam), CCL28 (R&D Systems), active ⁇ -catenin (Merck), GAPDH (Abcam) and ⁇ -tubulin (Abcam).
  • Wnt/ ⁇ -catenin reporter plasmid M50 Super 8X TOPFlash (Addgene plasmid #12456) and mutant control M51 Super 8X FOPFlash (Addgene plasmid #12457) are gifts from Randall Moon.
  • the human CCL28 gene promoter (2.8kb, relative to the transcription start site is -2576/+205) was cloned into the firefly luciferase reporter gene construct pGL4.
  • the Hieff MutTM multi-site directed mutagenesis kit (Yeasen, Shanghai) was used to introduce mutations in the potential TCF/LEF binding site on the CCL28 promoter to generate a mutant CCL28 promoter reporter construct (pGL4-CCL28.mut).
  • SGC7901 cells were fixed with 1% formaldehyde solution for 15 minutes, and then 0.125M glycine solution was added to stop the reaction. The reaction solution was washed away with PBS, and then Farnham lysis solution was added to collect the cells. Centrifugation to collect the cell clusters, resuspend the cells in RIPA lysis buffer, and use an sonicator to break the DNA in the cells. The DNA fragments were incubated with the previously coupled antibody and Dynabeads (purchased from ThermoFisher) complex overnight at 4 degrees rotation. The antibodies are anti- ⁇ -catenin and control IgG (Abcam).
  • the DNA-protein-antibody-Dynabeads complex is precipitated using a magnet, and the DNA fragments are finally eluted with a buffer.
  • Primers were designed for the three different potential binding sites of ⁇ -catenin on the CCL28 promoter to perform quantitative PCR to check the content of DNA fragments at the three sites in the sample.
  • the calculation method of relative enrichment is the relative binding of ⁇ -catenin to the potential DNA binding site of the control IgG.
  • Fresh peripheral blood was separated by Ficoll-Paque Plus (purchased from GE Healthcare) gradient centrifugation, and then resuspended into single cells in PBS and counted in a hemocytometer.
  • Ficoll-Paque Plus purchased from GE Healthcare
  • SGC7901 gastric cancer cell line culture medium supernatant was spread on the lower layer of 8- ⁇ m-pore transwell chambers (purchased from Corning), and 2 million peripheral blood mononuclear cells resuspended in PBS containing 1% serum were spread in the chamber. Place in a 37°C incubator for 6 hours, collect the PBMC that migrated to the lower layer and use flow cytometry to detect regulatory T cells, CD4 + and CD8 + T cells.
  • CD3[SK7] purchased from eBioscience
  • CD4[RPA -T4] purchased from eBioscience
  • CD8a[OKT8] purchased from eBioscience
  • CD25[BC96] purchasedd from BioLegend
  • FOXP3[206D] purchasedd from BioLegend
  • H.felis Helicobacter felis
  • MNU N-Methyl-N-nitrosourea
  • Antibody treatment plan intraperitoneal injection of 50mg/kg CCL28 monoclonal antibody (purchased from R&D Systems), the control group uses the isotype control IgG antibody of this antibody. Refer to Figure 4A for the specific time.
  • the spleen and stomach use a tissue homogenizer gentleMACS Octo dissociator (purchased from Miltenyi Biotec) for cell separation.
  • Stomach tissue is decomposed with 1mg/ml collagenase IV (purchased from Thermo Fisher) and 50 ⁇ g/ml (20U/ml) DNase I (stock: 5mg/ml) (purchased from Sigma-Aldrich). Red blood cells are used as red blood cells in the tissue The lysate (purchased from eBioscience) was removed.
  • the antibodies used in flow cytometry were purchased from BioLegend or eBioscience.
  • a cell activator purchased from BioLegend
  • Cyto-FastTM Fix/Perm Buffer Set purchased from BioLegend
  • the nucleoprotein was processed using Foxp3/Transcription Factor Staining Buffer Set (purchased from BD Biosciences).
  • Flow cytometry data were processed by FACS Aria II cytometer (purchased from BD Biosciences) and FlowJo software.
  • Paraffin sections of tissues of patients with gastric cancer were purchased from Alenabio.
  • the mouse stomach tissue was embedded in paraffin and cut into 5 micron tissue sections, deparaffinized, antigen retrieval, and blocked with the following antibodies ⁇ -catenin (purchased from Abcam), H+K+ATPase ⁇ (ATP4B) (purchased from Abcam) ), FOXP3 (purchased from Abcam), CCL28 (purchased from R&D Systems) the primary antibody overnight, incubate the second day with the secondary antibody, and use DAB Peroxidase Substrate Kit (purchased from Gene Tech, Shanghai) to develop color, and finally stain with hematoxylin Check and rehydrate and seal the film.
  • the quantification of ⁇ -catenin and CCL28 uses ImageJ software for grayscale analysis and statistics.
  • Example 1 ⁇ -catenin signaling pathway induces CCL28 expression in gastric cancer
  • the effect of the Wnt/ ⁇ -catenin signaling pathway in gastric cancer cell lines on the expression of chemokines was detected.
  • the method is as follows: 1) Overexpress ⁇ -catenin in gastric cancer cell SGC7901, and use plasmid vector to express the mutant ⁇ -catenin (ie S33Y. ⁇ -catenin) with mutation of GSK3 ⁇ phosphorylation site, so as to realize the continuous ⁇ -catenin Activate, and then use qPCR to detect changes in chemokine expression; 2) In human gastric cancer cell lines AGS and SGC7901, by knocking down or overexpressing ⁇ -catenin, using WB to detect changes in CCL28 expression at the protein level; 3 ) After immunohistochemical staining in clinical gastric cancer tissue samples, statistical analysis of the positive rates of ⁇ -catenin and CCL28 verifies the correlation between ⁇ -catenin and CCL28.
  • Example 2 is the direct target gene of ⁇ -catenin signaling pathway in human gastric cancer cells
  • the structural correlation between CCL28 and ⁇ -catenin signaling pathway is analyzed in combination with bioinformatics, and then the relationship between the two is verified in a cell model.
  • the method is as follows: Use bioinformatics to find three potential binding sites of ⁇ -catenin/TCF transcription complex in the promoter region of CCL28, and use chromatin immunoprecipitation to analyze whether ⁇ -catenin is enriched at the potential binding sites , Clone the CCL28 promoter and the CCL28 promoter sequence with the potential binding site sequence mutated into the luciferase reporter vector to check the transcription activity of the CCL28 promoter.
  • ⁇ -catenin/TCF shRNA knocked down TCF/LEF transcription factor family members or overexpressed TCF/LEF members to detect the transcriptional activity of CCL28 promoter.
  • Wnt signaling pathway inhibitor iCRT14 also reduced the upregulation of ⁇ -catenin on CCL28 promoter activity ( Figure 2D).
  • the Wnt signaling pathway inhibitor iCRT14 prevents the binding of ⁇ -catenin and TCF and the binding of ⁇ -catenin/TCF transcription complex on the promoter DNA, thereby inhibiting the Wnt signaling pathway.
  • CCL28 is used as the target gene of the Wnt signaling pathway. , Its transcriptional activity has also been suppressed. After shRNA knockdown or gene overexpression, the CCL28 promoter activity was tested, and it was found that the transcription factors TCF1 and TCF4 cooperated with ⁇ -catenin to regulate the transcription of CCL28 gene, but not LEF1.
  • the in situ mouse gastric cancer was induced by Helicobacter H. felis infection and the carcinogen MNU, and then the Wnt signaling pathway inhibitor iCRT14 was injected intraperitoneally to detect the expression level of CCL28 protein in the stomach.
  • Example 4 CCL28 is correlated with the degree of development of gastric cancer
  • the immunohistochemical analysis further analyzed the correlation between CCL28 and the expression level of ⁇ -catenin.
  • the results showed that in different degrees of gastric cancer samples, there was a significant positive correlation between the expression level of ⁇ -catenin and the expression level of CCL28 ( Figure 1C and 1D).
  • CCL28 is the target gene of the Wnt/ ⁇ -catenin signaling pathway in gastric cancer cells, which suggests that CCL28 is a potential pathogenic factor in gastric cancer.
  • the positive rate of CCL28 is higher.
  • Example 5 Gastric cancer cells activated by the ⁇ -catenin signaling pathway induce the expression of CCL28 to regulate the migration of T cells
  • in vitro migration experiments were used to test whether Wnt/ ⁇ -catenin-activated tumor cells can recruit human Treg cells through CCL28.
  • the method is as follows: construct a gastric cancer cell line that overexpresses ⁇ -catenin in the gastric cancer cell line SGC7901, or overexpresses ⁇ -catenin while using shRNA to knock down CCL28, collect the cell culture supernatant, spread it in the lower layer of the transwell, and spread 1X10 in the upper chamber After 6 peripheral blood mononuclear cells were cultured in a 37°C incubator for 4 hours, the cells that migrated to the lower layer were collected, and the proportion and number of immune cells were analyzed by flow cytometry.
  • peripheral blood mononuclear cells that migrated to the lower layer were analyzed by flow cytometry for changes in immune cells (Figure 5B).
  • Example 6 CCL28 antibody treatment inhibits H.felis/MNU-induced gastric cancer progression
  • H. felis/MNU gastric cancer model animals were used. At the 29th week after the start of MNU treatment (the figure indicates the end of 28 weeks and the beginning of 29 weeks), the model mice were given CCL28 monoclonal antibody once a week. To block CCL28 activity, the experimental scheme is shown in Figure 6A.
  • Example 8 CCL28 antibody has no obvious therapeutic effect on melanoma and breast cancer
  • the method is as follows: use mouse melanoma cells B16 and breast cancer cells 4T1 to establish subcutaneous transplantation tumors: take the cells in the logarithmic growth phase, trypsinize them, and resuspend them in PBS. C57BL/6 mice were anesthetized for 6-8 weeks, the forelimb armpits of the mice were treated with a hair shaver, and the bare skin was disinfected with 75% alcohol. The cell suspension with adjusted cell density was inoculated into the armpits of the forelimbs of mice, and the tumor growth was observed and recorded every day.
  • B16 xenograft mice and 4T1 xenograft mice were treated with CCL28 antibody on day 3-7 after cell inoculation, and the treatment method and dosage were the same as in Example 6.
  • Wnt/ ⁇ -catenin one of the common oncogene pathways in cancer, Wnt/ ⁇ -catenin, has a synergistic effect with the expression of CCL28, and this effect then increases the number of regulatory T cells in the tumor.
  • CCL28 monoclonal blocking antibody can effectively reduce the infiltration of regulatory T cells in the tumor and inhibit the development of the tumor.
  • the present invention proved through comparative experiments that the combined effect of Wnt/ ⁇ -catenin and CCL28 is uniquely present in a special pathological situation—gastric cancer, but in other tumors, and even other solid tumors such as melanoma Cell B16 and breast cancer cells are not present in the 4T1 model.
  • the present invention establishes a method to block the CCL28 chemotactic pathway, and then targets and regulates the migration process of T cells to tumors, and does not affect the apoptosis of regulatory T cells, and may provide better immunotherapy for targeted regulatory T cells. It is a novel and effective means. Moreover, for long-term patients suffering from gastric cancer and other patients undergoing immunotherapy, low autoimmunity and its derivative diseases are an important cause of death. Therefore, the method provided by the present invention can not only effectively inhibit gastric cancer metastasis, without reducing the patient's own immunity, but also improve the overall immunity.
  • the present invention also confirms the correlation between CCL28 and its receptor levels and the occurrence and severity of gastric cancer through a large amount of clinical data for the first time, so that a detection system can be developed to accurately insight into the occurrence, metastasis and prognosis of gastric cancer.
  • CCL28 blockade therapy has great potential for clinical application in cancer treatment, and may improve the efficacy of other immunotherapies.

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Abstract

本发明提供了一种通过阻断CCL28趋化通路的胃癌治疗方法。具体地，提供了一种CCL28基因或CCL28蛋白的用途，用于制备检测胃癌的试剂或试剂盒，所述的胃癌是Wnt/β-catenin信号通路与趋化因子CCL28相关分子表达同时异常上调型的胃癌。同时本发明提供了一种检测试剂盒、一种药物组合物的用途、和一种体内外抑制癌细胞生长的方法。

Description

阻断CCL28趋化通路的胃癌治疗方法 技术领域
本发明属于生物医药领域，具体地，本发明涉及通过阻断CCL28趋化通路的胃癌治疗方法。
背景技术
快速发展的免疫治疗方法为无数肿瘤病人带来了希望。胃癌的分子特征表明，EBV相关亚型的胃癌显示PD-L1表达升高，提示抗PD的免疫治疗对这些患者可能有效。然而，相较于黑素瘤和肺癌，由于胃癌免疫微环境和分子机制的复杂性，这些免疫治疗方法在胃癌中的发展遇到许多实用性难题。肿瘤的免疫逃逸机制也是肿瘤的免疫治疗的一大障碍。调节T细胞(Treg)是一类参与到肿瘤的免疫逃避中的重要免疫抑制型细胞。它的增多会导致抗肿瘤的效应免疫细胞的功能和数量降低，同时也会导致肿瘤的免疫治疗或其他疗法的功效的减弱甚至丧失。长期罹患胃癌和其他经受免疫治疗的患者而言，自身免疫力低下及其衍生疾病是致死的重要原因。
因此，本领域急需一种有效的针对难治性胃癌同时减少副作用的治疗方案。
发明内容
本发明的目的是提供一种有效的针对难治性胃癌同时减少副作用的治疗方案。
本发明的第一方面，提供了一种CCL28基因或CCL28蛋白或其检测试剂的用途，用于制备检测胃癌的试剂或试剂盒，所述的胃癌是Wnt/β-catenin信号通路与趋化因子CCL28相关分子表达同时异常上调型的胃癌。
在另一优选例中，所述的胃癌的病理表现具有选自下组的一个或多个病理表现：
(1)胃部组织中Wnt/β-catenin信号通路相关表达上调；
(2)胃部组织中趋化因子CCL28相关表达上调；
(3)胃部肿瘤；
(4)胃壁细胞丢失；及
(5)胃部组织调节T(Treg)细胞增多。
在另一优选例中，所述的胃癌的病理表现还包括机体免疫反应低下。
在另一优选例中，所述胃癌包括原位胃癌、肠型胃癌以及弥散性胃癌。
在另一优选例中，所述胃癌包括幽门螺杆菌阴性的胃癌。
在另一优选例中，所述的胃部组织包括胃癌肿瘤组织、胃部癌旁组织、或其组合。
在另一优选例中，所述的Wnt/β-catenin信号通路相关表达选自下组：转录因子TCF1的表达水平、转录因子TCF4的表达水平、β-catenin的含量、β-catenin核异位水平、β-catenin/TCF4复合物的转录活性、GSK3β磷酸化水平或其组合。
在另一优选例中，所述的趋化因子CCL28相关分子包括：CCL28基因(包括基因组的核苷酸序列、cDNA序列、和/或mRNA)、CCL28受体的基因包括基因组的核苷酸序列、cDNA序列、和/或mRNA)、CCL28蛋白、CCL28受体蛋白、或其组合。
在另一优选例中，所述的CCL28的受体包括CCR3、和/或CCR10。
在另一优选例中，所述的试剂盒包括：对CCL28相关分子的蛋白或mRNA进行定量检测的试剂以及相应的标签或说明书。
在另一优选例中，所述的试剂包括CCL28相关分子的特异性引物、特异性抗体、探针和/或芯片。
在另一优选例中，上述的试剂包括：检测用芯片，例如核酸芯片和蛋白质芯片。
在另一优选例中，所述的核酸芯片包括基片和点样在基片上的癌症相关基因的特异性寡核苷酸探针，所述的癌症相关基因的特异性寡核苷酸探针包括与CCL28相关基因或mRNA特异性结合的探针。
在另一优选例中，所述的蛋白质芯片包括基片和点样在基片上的癌症相关蛋白的特异性抗体，所述的癌症相关蛋白的特异性抗体包括抗CCL28相关蛋白的特异性抗体。
在另一优选例中，所述的CCL28相关蛋白包括融合蛋白和非融合蛋白。
在另一优选例中，在所述试剂或试剂盒中，包括：作为标准品的CCL28基因(或核酸分子)或CCL28蛋白。
在另一优选例中，所述的CCL28基因(或核酸分子)或CCL28蛋白包括野生型和/或突变型。
在另一优选例中，在所述试剂或试剂盒中，还包括：用于检测CCL28基因或CCL28蛋白的检测试剂。
本发明的第二方面，提供了一种用于检测胃癌的试剂盒，所述的试剂盒含有一容器，所述容器中含有检测CCL28相关蛋白或mRNA的检测试剂；以及标签或说明书，所述标签或说明书注明所述试剂盒用于检测胃癌。
在另一优选例中，所述的胃癌是Wnt/β-catenin信号通路与趋化因子CCL28相关分子表达同时异常上调型的胃癌。
在另一优选例中，所述的标签或说明书中注明内容，选自下组：
a)当检测对象的CCL28相关蛋白的mRNA表达量A1与癌旁组织的CCL28相关蛋白的mRNA表达量A0之比(A1/A0)≥2，则提示该检测对象患胃癌的几率高于普通人群；
b)当检测对象的CCL28相关蛋白的mRNA表达量与癌旁组织的CCL28相关蛋白的mRNA表达量之比A1/A0≥2，如果该A1/A0比值越高，则提示所述检测对象患胃癌的恶性程度更高；和
c)当检测对象的CCL28相关蛋白的mRNA表达量与癌旁组织的CCL28相关蛋白的mRNA表达量之比A1/A0≥2，如果该A1/A0比值越高，则提示所述检测对象患胃癌的预后更差、转移率更高。
在另一优选例中，所述的检测试剂包括：特异性引物、特异性抗体、探针和/或芯片。
在另一优选例中，所述的试剂盒用于检测离体的人肿瘤组织样品或血液样品。
在另一优选例中，所述的肿瘤组织样品为胃癌样品。
本发明的第三方面，提供了一种CCL28抑制剂的用途，用于制备抑制癌细胞生长或增殖的药物组合物，或用于制备治疗胃癌的药物组合物；所述的胃癌是Wnt/β-catenin信号通路与趋化因子CCL28相关分子表达同时异常上调型的胃癌。
在另一优选例中，所述抑制剂选自下组：靶向CCL28和/或其受体蛋白的抗体或小分子抑制剂；靶向CCL28和/或其受体基因的靶向核酸分子或基因编辑器；或其组合。
在另一优选例中，所述的CCL28的受体包括CCR3、和/或CCR10。
在另一优选例中，所述抗体选自下组：多克隆抗体、单克隆抗体、嵌合抗体、双特异性抗体、抗体偶联物、小分子抗体、抗体融合蛋白，及其组合。
在另一优选例中，所述小分子抗体选自下组：单链抗体ScFv，Fab抗体， Fv片段，及其组合。
在另一优选例中，所述ScFv抗体包括在治疗细胞中表达(包括过表达)的分泌型单链抗体。
在另一优选例中，所述治疗细胞包括间充质干细胞、CAR-T细胞。
在另一优选例中，所述载体包括：细菌质粒、噬菌体、酵母质粒、植物细胞病毒、哺乳动物细胞病毒如腺病毒、逆转录病毒、或其他载体。
在另一优选例中，所述抑制剂选自下组：植物提取物抑制剂、小分子化合物抑制剂、核酸类抑制剂、肽类抑制剂、多糖类抑制剂、病毒载体抑制剂、脂质体载体抑制剂、或纳米颗粒载体抑制剂。
在另一优选例中，所述药物组合物还包括其他Wnt/β-catenin信号通路抑制剂。
在另一优选例中，所述药物组合物可以协同抑制肿瘤组织中的异常的Wnt/β-catenin信号通路激活。
在另一优选例中，所述药物组合物能够抑制调节T(Treg)细胞浸润肿瘤组织，同时提高外周免疫的活性。
本发明的第四方面，提供了一种体外非治疗性的抑制癌细胞生长或增殖的方法，包括步骤：在CCL28抑制剂存在下，培养癌细胞，从而抑制癌细胞生长或增殖。
在另一优选例中，所述的方法包括向癌细胞的培养体系中添加CCL28相关分子抑制剂，从而抑制癌细胞生长或增殖。
在另一优选例中，所述的癌细胞是胃癌细胞。
在另一优选例中，所述的胃癌细胞选自：AGS细胞系、SC7901细胞系、AZ-521细胞系、原代胃癌细胞、或其组合。
本发明的第五方面，提供了一种筛选治疗癌症的候选化合物的方法，包括步骤：
(a)测试组中，在细胞的培养体系中添加测试化合物，并观察所述测试组的细胞中CCL28相关分子的表达量和/或活性；在对照组中，在相同细胞的培养体系中不添加测试化合物，并观察对照组的所述细胞中CCL28相关分子的表达量和/或活性；
其中，如果测试组中细胞的CCL28相关分子的表达量和/或活性小于对照组，就表明该测试化合物是对CCL28相关分子的表达和/或活性有抑制作用的 治疗癌症的候选化合物。
在另一优选例中，所述的细胞包括：癌细胞或正常细胞；
在另一优选例中，所述的细胞为胃癌细胞或胃细胞。
在另一优选例中，所述方法还包括步骤：
(b)对于步骤(a)中获得的候选化合物，进一步测试其对癌细胞生长或增殖的抑制作用。
在另一优选例中，所述步骤(b)中包括步骤：测试组中，癌细胞的培养体系中添加测试化合物，并观察癌细胞的数量和/或生长情况；在对照组中，在癌细胞的培养体系中不添加测试化合物，并观察癌细胞的数量和/或生长情况；其中，如果测试组中癌细胞的数量或生长速度小于对照组，就表明该测试化合物是对癌细胞的生长或增殖有抑制作用的治疗癌症的候选化合物。
本发明的第六方面，提供了一种抑制或治疗胃癌的方法，包括步骤：给需要治疗的对象(哺乳动物)施用安全有效量的CCL28相关分子抑制剂；所述的胃癌是Wnt/β-catenin信号通路与趋化因子CCL28相关分子表达同时异常上调型的胃癌。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了β-catenin信号通路在胃癌中诱导CCL28表达。图中A为，过表达β-catenin的胃癌细胞系SGC7901趋化因子qPCR检测。图中B为，过表达或敲低β-catenin的胃癌细胞系SGC7901和AGS中β-catenin，CCL28蛋白水平检测。图中C,D为，胃癌病人组织芯片中β-catenin，CCL28免疫组化检测，并阳性率相关性分析结果。
图2显示了CCL28是β-catenin信号通路直接转录调控靶基因。图中A为，CCL28基因启动子中β-catenin/TCF转录因子复合体结合位点示意图。图中B为，染色质免疫沉淀实验证明了β-catenin结合在CCL28基因启动子中的预测位点1和3上。图中C为，过表达β-catenin上调了CCL28启动子活性，将结合位点1和3的核心序列突变(CCL28.mut)使β-catenin的调控作用丧失。图中D为，过表达β-catenin上调了Wnt通路报告基因TOPflash以及CCL28启动子活性。Wnt抑制剂 iCRT14处理使两个报告基因的活性降低。图中E为，利用RNA干扰，与对照shRNA序列shScr相比，靶向Wnt通路转录因子TCF/LEF家族成员TCF1(shTCF1)和TCF4(shTCF4)的shRNA敲低其表达之后，CCL28启动子活性降低，而敲低LEF1的表达(shLEF1)对CCL28启动子活性无影响。图F为，过表达转录因子TCF1和TCF4上调CCL28启动子活性，过表达LEF1对CCL28启动子活性无影响。说明转录因子TCF和TCF4协同β-catenin调控CCL28的表达。
图3显示了Wnt通路抑制剂iCRT14降低小鼠胃癌中CCL28的表达。在H.felis/MNU诱导的小鼠原位胃癌模型中，Wnt通路抑制剂iCRT14处理的小鼠胃部CCL28蛋白的表达通过免疫印迹法(A)和ELISA(B)检测发现有显著降低。图中，vehicle表示药物溶剂(介质)。
图4显示了CCL28的表达与胃癌的发展相关。图中A为，Oncomine数据库分析发现肠型(Intestinal Type)和弥散型(Diffuse Type)胃癌中CCL28 mRNA的表达均高于正常组织。图中B为，胃癌病人组织的免疫组化分析表明在恶化程度更高的II和III级胃癌中CCL28的蛋白表达水平比I级更高。*P<0.05；***P<0.001。
图5显示了β-catenin信号通路激活的胃癌细胞诱导CCL28表达来实现调节T细胞的迁移。图中A为，SGC7901细胞系同时过表达β-catenin(Vector)和敲低CCL28(shScr)条件下β-catenin和CCL28的免疫蛋白印迹分析。图中B为，调节T细胞(Tregs)，CD4 +T细胞，CD8 +T细胞流式图。图中C,D为，调节T细胞(Tregs)，CD4 +T细胞，CD8 +T细胞比例及绝对数量统计结果。*P<0.05；**P<0.01。
图6显示了CCL28抗体治疗抑制H.felis/MNU诱导的小鼠原位胃癌进展。图中A为，CCL28抗体治疗模式图。图中B为，CCL28抗体治疗胃解剖图及肿瘤面积统计。*P<0.05。图中C为，CCL28抗体治疗HE染色，阿尔辛兰染色及H+K+ATPase免疫组化染色。图中D为，胃体(Corpus)和胃窦(Antrum)部位病理统计结果。*P<0.05；**P<0.01；***P<0.001。
图7显示了CCL28抗体治疗缓解免疫微环境的抑制性。图中A为，调节T细胞(Tregs)流式分析图和统计图。图中B为，IFNγ +CD4 +T细胞流式分析图和统计图。图中C为，IFNγ +CD8 +T细胞流式分析图和统计图。*P<0.05；***P<0.001。
图8显示了CCL28抗体阻断在黑色素瘤与乳腺癌移植瘤模型中无显著的治疗效果。图中A为，黑色素瘤B16皮下移植瘤的生长曲线。图中B为，乳腺 癌4T1皮下移植瘤的生长曲线。图中C为，黑色素瘤B16荷瘤小鼠的生存率。图中D为，乳腺癌4T1荷瘤小鼠的生存率。
图9显示了CCL28蛋白在人类各组织中的表达水平。在The Human Protein Atlas数据库中发现CCL28蛋白在脑部、胃肠道、和胰腺有较高表达。
具体实施方式
本发明人经过广泛而深入的研究，首次意外地发现Wnt/β-catenin在胃癌细胞中上调CCL28的表达继而使肿瘤中的调节T细胞增多。利用阻断CCL28趋化通路的方法，可非常有效地减少了肿瘤中调节T细胞的浸润并抑制了肿瘤的发展。进一步研究发现，这种治疗效果仅针对胃癌模型，对于其他实体瘤如黑色素瘤和乳腺癌并无明显效果。
在此基础上，发明人完成了本发明。
术语说明
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
如本文所用，在提到具体列举的数值中使用时，术语“约”意指该值可以从列举的值变动不多于1％。例如，如本文所用，表述“约100”包括99和101和之间的全部值(例如，99.1、99.2、99.3、99.4等)。
如本文所用，术语“含有”或“包括(包含)”可以是开放式、半封闭式和封闭式的。换言之，所述术语也包括“基本上由…构成”、或“由…构成”。
趋化因子与CCL28
趋化因子是一类调节细胞迁移的细胞因子，在肿瘤微免疫环境的构成以及肿瘤的发生发展中起到了至关重要的作用。
C-C基序趋化因子配体28(C-C motif chemokine ligand 28，CCL28)，位于5p12，编码8个外显子。CCL28是一种趋化因子，介导了表达其受体CCR3或CCR10的细胞的迁移。CCL28在消化道、肺、乳腺等组织上皮细胞中表达(如图9所示)。
本发明首次证明了Wnt/β-catenin-CCL28通路在胃癌病理进程中表达上调，也在胃癌模型中首次正式了CCL28抗体阻断的治疗效果。
本发明中，趋化因子CCL28相关分子表达异常上调是指某一组织(如胃癌组织)的CCL28相关分子的mRNA表达量C1与正常组织(如癌旁组织)中的 CCL28相关分子的mRNA表达量C0之比(C1/C0)≥1.2，较佳地，≥1.5，更佳地，≥2.0或≥2.5。
Wnt/β-catenin通路
在细胞中，胞浆中的β-catenin水平受APC、Axin和糖原合成酶激酶-3β(GSK-3β)组成的多蛋白破坏复合物严格控制。多蛋白破坏复合物能磷酸化β-catenin，进一步诱导其泛素化和蛋白酶体介导的降解。Wnts通路刺激则会抑制破坏复合物的活性，打破胞浆β-catenin稳定，胞浆β-catenin易位至细胞核与LEF/TCF家族一起激活转录，这种β-catenin转录靶点的激活程序就是连环蛋白反应转录效应(catenin responsive transcription，CRT)，是细胞对特定Wnt刺激反应性的一个关键方面。
大部分(如果不是全部)Wnt相关的致癌作用是由CRT错误调控导致的，因此靶向β-catenin-TCF/LEF复合物成为理想的治疗靶点。Wnt/β-catenin通路是肠癌、胃癌等类型癌症中最常见的致癌基因通路之一，在癌症的发生发展中起着重要的作用，也是癌症治疗中备受关注的靶点之一。在经典Wnt信号通路中，CTNNB1和APC是其中常见的显著突变的基因。以胃癌为例，约70％的胃癌病人都伴有Wnt/β-catenin信号通路的突变。除了基因突变，许多Wnt信号通路组分的改变可能通过上调正调节因子或下调负调节因子来实现，并最终导致经典Wnt通路的激活。有研究表明幽门螺旋杆菌感染可以导致胃上皮细胞中Wnt/β-catenin信号通路的激活。总之，这些发现表明了Wnt/β-catenin信号通路在癌症发病机制中的关键作用。
Wnt/β-catenin信号通路异常在大多数癌症类型中都有发现，是肿瘤发生发展中一个非常重要的癌基因通路。通过对多种肿瘤样品的分析，β-catenin的表达增加与肿瘤中淋巴细胞的浸润具有负相关性，提示Wnt/β-catenin也会导致肿瘤的免疫逃避已经对免疫治疗的耐受性。
本发明首次证明了在胃癌病理进程中，β-catenin和转录因子TCF/LEF复合体通过与CCL28启动子区域结合，从而上调胃癌中CCL28的表达，这种Wnt/β-catenin-CCL28通路在胃癌病理进程中表达上调是独特的。在本发明的一个实施例中，使用相同的处理方式和剂量，CCL28抗体能显著缓解胃癌的进展，但对于其他实体瘤如黑色素瘤和乳腺瘤并无类似效果。
本发明中，Wnt/β-catenin信号通路相关分子表达异常上调是指某一组织(如胃癌组织)的Wnt/β-catenin信号通路相关分子的mRNA表达量B1与正常组织(癌旁组织)中的Wnt/β-catenin信号通路相关分子的mRNA表达量B0之比 (B1/B0)≥1.2，较佳地，≥1.5，更佳地，≥2.0或≥2.5。
Wnt/β-catenin信号通路抑制剂iCRT14
在本文中，使用的Wnt/β-catenin信号通路抑制剂为iCRT14。iCRT14是一种连环蛋白反应转录抑制剂。
在最初的研究中(An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway.Gonsalves et al.Proc.Natl.Acad.Sci.USA,2011；108:595),实验初筛鉴定出34个对dTF12-荧光素酶报告基因活性有统计学显著抑制作用的分子(总命中率约0.3％)，称这些化合物称为CRT抑制剂(iCRT)。
iCRT14属于噻唑烷二酮类的β-连环蛋白反应性转录抑制剂，是一种Wnt/β-catenin通路抑制剂。经验分子式(希尔表示法)C 21H 17N 3O 2S。它是连环蛋白β-catenin和转录因子TCF/LEF复合体的有效抑制剂。它可以剂量依赖性方式破坏β-连环蛋白-TCF/LEF的相互作用，并在结肠肿瘤细胞系中引起G0/G1的停滞，从而导致结肠癌细胞中细胞增殖和肿瘤生长减少的持续减弱。
而本发明首次发现，在胃癌中，调控因子TCF1和TCF4非LEF1协同β-catenin调控CCL28基因的转录。
S33Y.β-catenin
本发明中，使用向胃癌细胞中转入S33Y.β-catenin质粒，以抑制β-catenin磷酸化，从而实现过表达β-catenin的目的。
所述的S33Y.β-catenin的DNA质粒是指携带将β-catenin氨基酸序列第33位的丝氨酸(S33)突变为酪氨酸(Y)的片段的DNA质粒。S33是GSK3β的磷酸化位点，此位点磷酸化之后β-catenin进入降解途径。将S33突变为酪氨酸(Y)之后的突变体S33Y.β-catenin能逃脱磷酸化和降解途径，继而在细胞内更高效地富集。此突变也在一些胃肠道肿瘤病人的样品中检测到。本发明用以研究Wnt/β-catenin通路异常激活在肿瘤细胞中的作用。
H.felis/MNU诱导胃癌模型
幽门螺杆菌被认为是慢性胃炎的主要病因之一。因此用幽门螺旋杆菌建立胃癌小鼠模型对研究人类胃癌发生发展有十分重要的意义。C57BL/6小鼠对各种幽门螺杆菌菌株在胃部的定植具有显著的抵抗性。因此，使用H.felis(幽门螺杆菌的近亲)来建立胃癌小鼠模型就十分重要了。这种菌株是从猫的胃中分离 出来的，并且很容易在小鼠的胃中定植。其可诱发小鼠严重的胃炎和萎缩。felis感染的小鼠表现为胃的SPEM、异型增生和浸润性肿瘤，且观察期较长。该模型可在胃体沿胃小弯的鳞柱状交界处(SCJ)观察到广泛的异型增生病变，并发生大的息肉状胃窦肿瘤。
通过对螺杆菌感染的小鼠模型的研究，研究人员确定了胃癌发生中其他辅助因子的影响，如性别、饮食和合并感染。幽门螺杆菌感染前使用N-甲基-N-亚硝基脲(MNU)，可诱发更严重的癌前病变并增加胃癌发病率。MNU在小鼠模型中诱导胃癌发生的作用十分重要，通过每周两次胃内给药(0.5mg MNU)可导致大多数Balb/c小鼠因前胃鳞癌而死亡。MNU使用前手术切除前胃有助于促进腺胃高分化腺癌的发生，使用40周后发生率为100％。因此，胃腺体对MNU的致癌作用十分敏感。240ppm MNU溶解于饮用水中，连续5周(每隔一周)给药，在6个品系的小鼠实验中的结果显示，该方法可诱发胃癌。因此，本方案使用H.felis感染和MNU给药结合的方法建立小鼠胃癌模型。
免疫逃避
免疫逃避已被公认为癌症的新标志。了解肿瘤免疫微环境对于发现新的治疗靶点以及预测和免疫治疗反应至关重要。正常的免疫系统会扼制肿瘤的发生发展。随着癌症的进程，肿瘤会产生许多免疫逃避机制，导致肿瘤生长的加强。
小鼠模型和肿瘤病人样本的数据表明，髓样抑制细胞(MDSCs)和M2型巨噬细胞参与胃癌的免疫抑制。调节性T(Treg)细胞是一类免疫抑制型细胞，其在多种癌症类型中被发现参与加速肿瘤进展的过程。调节T细胞(Treg)是一类参与到肿瘤的免疫逃避中的重要免疫抑制型细胞。它的增多会导致抗肿瘤的效应免疫细胞的功能和数量降低，同时也会导致肿瘤的免疫治疗或其他疗法的功效的减弱甚至丧失。
本发明的一个优选实施例中，使用CCL28抗体能有效抑制靶向调节T细胞向肿瘤的迁移过程，而不影响调节T细胞的凋亡。同时，仅靶向胃癌肿瘤组织和脾脏中的Treg细胞，而不会降低血液中的Treg细胞的比率，还可以提高整体的免疫反应。
本发明的技术方案具有如下优点：
1.本发明提供了一种通过抑制CCL28，治疗Wnt/β-catenin信号通路-CCL28同时异常升高型的胃癌。应用本发明提供的CCL28抗体治疗能使肿瘤面积显著减少、在发育异常和肠型转化方面的明显变化、明显被缓解正常细胞丢失的现 象。
2.本发明发现β-catenin/CCL28与胃癌的发生和恶化具有正相关性，因而提供了一种用于胃癌的诊断、筛查与预后的判断的检测方法，能够筛查群体中的胃癌发病率和精准察觉胃癌的转移情况。
3.本发明抑制β-catenin/CCL28其效果仅靶向胃癌肿瘤组织和脾脏中的Treg细胞，而不会降低血液中的Treg细胞的比率，可以提高整体的免疫反应。
4.本发明阻断CCL28趋化通路治疗恶性胃癌的方法，靶向调节T细胞向肿瘤的迁移过程，而不影响调节T细胞的凋亡，可能为靶向调节T细胞的免疫疗法提供更为新颖有效的手段。
5.本发明提供了一种通过抑制CCL28，从而抑制肿瘤细胞中Wnt/β-catenin信号通路的治疗思路。
6.本发明发现Wnt/β-catenin信号通路-CCL28同时异常升高型的胃癌是一种与其他肿瘤，甚至实体瘤发展显著不同的疾病表型，主要体现在阻断CCL28趋化通路能有效抑制恶性胃癌发展，但对于其他肿瘤模型的效果并不明确，尤其是对于黑色素瘤细胞B16和乳腺癌细胞4T1模型无效。
下面结合具体实施，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
通用实验方法
1.细胞培养
人类胃癌细胞系AGS和SC7901培养在含10％血清(Gbico)和1％青霉素/链霉素双抗(Life Technologies)的RPMI 1640(Life Technologies)培养基中。
2.RNA纯化和定量PCR
细胞系和小鼠胃组织中的总RNA使用RNeasy Mini Kit(Qiagen)抽提，随后用PrimeScript RT Reagent Kit(Takara)进行反转录合成cDNA。QPCR使用SYBR Green PCR Master Mix kit(Takara)和ABI 7900HT Fast Real-Time PCR System(Applied Biosystems)来进行。定量计算使用ΔΔCt的方法。
3.免疫蛋白印记
胃癌细胞系和小鼠胃组织中的蛋白用加有蛋白酶抑制剂(Roche)的RIPA裂 解液(Thermo Scientific)裂解，蛋白浓度用BCA Protein Assay Reagent(Thermo Scientific)检测。检测蛋白如下：β-catenin(Abcam),CCL28(R&D Systems),activeβ-catenin(Merck),GAPDH(Abcam)andβ-tubulin(Abcam)。
4.荧光色酶报告基因活性检测
Wnt/β-catenin报告质粒M50 Super 8X TOPFlash(Addgene质粒#12456)和突变体对照M51 Super 8X FOPFlash(Addgene质粒#12457)是来自Randall Moon的馈赠。将人类CCL28基因的启动子(2.8kb,相对于转录起始位点为-2576/+205)克隆到萤火虫荧光素酶报告基因构建体pGL4中。使用Hieff Mut TM多位点定向诱变试剂盒(Yeasen，Shanghai)引入CCL28启动子上潜在TCF/LEF结合位点的突变以产生突变CCL28启动子报道构建体(pGL4-CCL28.mut)。使用jetPRIME转染试剂(Polybus Transfection)转染质粒。通过与含有海肾荧光素酶基因的pRL-CMV报告基因共转染来标准化转染效率。使用Dual-Glo荧光素酶测定系统(Promega)测量萤火虫和海肾荧光素酶活性。
5.染色质免疫沉淀ChIP
SGC7901细胞利用1％的甲醛溶液固定15分钟，之后加入0.125M的甘氨酸溶液终止反应。利用PBS洗去反应液，然后加入Farnham裂解液收集细胞。离心收集细胞团，将细胞重悬浮到RIPA裂解液中，利用超声仪使细胞中的DNA断裂。将DNA片段与事先耦连好的抗体和Dynabeads(购自ThermoFisher)复合物在4度旋转孵育过夜。抗体为抗β-catenin和对照IgG(Abcam)。利用磁石将DNA-蛋白-抗体-Dynabeads复合物沉淀下来，最终用缓冲液将DNA片段洗脱下来。针对CCL28启动子上面3个不同的β-catenin潜在结合位点设计了引物进行定量PCR，来检查样品中三处位点DNA片段的含量。相对富集的计算方法为β-catenin相对于对照IgG在潜在DNA结合位点上的相对结合。
6.人外周血单核细胞分离和迁移实验
使用Ficoll-Paque Plus(购自GE Healthcare)梯度离心分离新鲜外周血，随后用PBS重悬成单个细胞并使用血球计数皿计数。
SGC7901胃癌细胞系培养液上清铺在8-μm-pore transwell chambers(购自Corning)的下层，小室中铺2百万个重悬在含1％血清的PBS中的外周血单核细胞。37℃培养箱中放置6小时，收集迁移到下层的PBMC并用流式检测调节T细胞，CD4 +和CD8 +T细胞，使用到的抗体如下：CD3[SK7](购自eBioscience),CD4[RPA-T4](购自eBioscience),CD8a[OKT8](购自eBioscience),CD25[BC96](购自BioLegend)和FOXP3[206D](购自BioLegend)。
7.原位胃癌小鼠模型和治疗方案
为了建立Helicobacter felis(H.felis)和N-Methyl-N-nitrosourea(MNU)诱导的胃癌小鼠模型，8周龄大的野生型小鼠通过灌胃的方式给H.felis菌种(ATCC49179)，一周三次，随后隔周给含有240ppm MNU的食用水，共计12周。
抗体治疗方案：腹腔注射50mg/kg CCL28单克隆抗体(购自R&D Systems)，对照组使用此抗体的同型对照IgG抗体，具体时间参照图4A。
8.流式分析
为了检测外周血、脾脏和胃组织中的免疫细胞，脾脏和胃使用组织匀浆器gentleMACS Octo dissociator(购自Miltenyi Biotec)来进行细胞分离。胃组织用1mg/ml胶原酶IV(购自Thermo Fisher)and 50μg/ml(20U/ml)DNA酶I(stock:5mg/ml)(购自Sigma-Aldrich)进行分解，组织中的红细胞使用红细胞裂解液(购自eBioscience)去除。流式检测中使用的抗体购自BioLegend或eBioscience公司。对于胞内抗原检测，需用细胞激活剂(购自BioLegend)刺激6小时。胞间抗原染色使用Cyto-Fast TM Fix/Perm Buffer Set(购自BioLegend)。核蛋白使用Foxp3/Transcription Factor Staining Buffer Set(购自BD Biosciences)处理。流式数据由FACS Aria II cytometer(购自BD Biosciences)和FlowJo软件处理。
9.免疫组化
胃癌病人的组织石蜡切片购自Alenabio公司。小鼠胃组织包埋于石蜡中并切成5微米的组织切片，脱蜡，抗原修复，封闭之后用如下抗体β-catenin(购自Abcam),H+K+ATPaseβ(ATP4B)(购自Abcam),FOXP3(购自Abcam),CCL28(购自R&D Systems)一抗四度过夜，第二天二抗孵育，并使用DAB Peroxidase Substrate Kit(购自Gene Tech,上海)显色，最后用苏木素染核并复水封片。β-catenin和CCL28的定量使用ImageJ软件进行灰度分析统计。
10.B16黑色素瘤和4T1乳腺癌皮下移植瘤模型
取对数生长期的B16或4T1细胞，PBS调整细胞浓度为3X10 6个/毫升。取6周龄大的WT雌鼠，眼科剪剪去小鼠右后肢外侧鼠毛，并用75％酒精消毒，每只小鼠皮下注射0.1毫升细胞悬液。随后观察到肿瘤出现5mm直径的肿瘤时，记为时间起点，游标卡尺测量并计算肿瘤大小。
11.统计分析
数据表示为平均值±SD。组间的统计学显著性通过双尾非配对Student t检验(GraphPad Prism)计算。P<0.05被认为具有统计学意义。
实施例1.β-catenin信号通路在胃癌中诱导CCL28表达
在本实施例中，检测胃癌细胞系中的Wnt/β-catenin信号通路对趋化因子表达的影响。方法如下：1)在胃癌细胞SGC7901中过表达β-catenin，并使用质粒载体表达GSK3β磷酸化位点突变的突变型β-catenin(即S33Y.β-catenin)，从而实现了β-catenin的持续激活，随后用qPCR检测趋化因子表达的变化；2)分别在人类胃癌细胞系AGS、SGC7901中，通过敲低或者过表达β-catenin，用WB在蛋白水平来检测CCL28表达水平的变化；3)在临床胃癌组织样本中通过免疫组化染色后，β-catenin和CCL28阳性率的统计分析，验证β-catenin与CCL28的相关性。
结果:
qPCR结果表明，相较于对照质粒，过表达β-catenin后，变化最为明显的趋化因子是CCL28(图1A)。
在SGC7901和AGS人类胃癌细胞系(源自中国科学院细胞库)中，野生型(WT)或抵御降解的突变体S33Y.β-catenin的过表达也增加了CCL28蛋白水平(图1B)。相反，这两种细胞系中β-catenin的敲低则导致CCL28蛋白水平降低(图1B)。
实施例2.在人胃癌细胞中CCL28是β-catenin信号通路的直接靶基因
在本实施例中，结合生物信息学分析CCL28与β-catenin信号通路结构相关性，然后在细胞模型中验证两者的关系。方法如下：利用生物信息学在CCL28的启动子区域找到3个β-catenin/TCF转录复合体的潜在结合位点，利用染色质免疫沉淀技术分析β-catenin是否在潜在的结合位点上富集，将CCL28启动子和突变了潜在结合位点序列的CCL28启动子序列克隆到荧光素酶报告载体中来检查CCL28启动子的转录活性。利用β-catenin/TCF，shRNA敲低TCF/LEF转录因子家族成员或过表达TCF/LEF成员之后检测CCL28启动子的转录活性。
结果：
生物信息学分析发现了在CCL28基因的启动子区域有三个潜在的β-catenin/TCF转录复合体的结合区域(图2A)。
在人的胃癌细胞SGC7901中，利用染色质免疫沉淀分析发现β-catenin结合在启动子位点1和3上(图2B)。
对着两个结合位点序列进行突变之后，β-catenin对CCL28启动子活性的上调作用消失(图2C)，这说明：β-catenin是通过在CCL28的启动子上结合位点来直接调控CCL28基因的转录。
利用Wnt信号通路抑制剂iCRT14同样降低了β-catenin对CCL28启动子活性的上调作用(图2D)。Wnt信号通路抑制剂iCRT14处理阻碍细胞中了β-catenin和TCF的结合以及β-catenin/TCF转录复合体在启动子DNA上的结合，从而抑制了 Wnt信号通路，CCL28作为Wnt信号通路的靶基因，其转录活性也被抑制掉了。通过shRNA敲低或者基因过表达之后检测CCL28启动子活性，发现了转录因子TCF1和TCF4协同β-catenin调控CCL28基因的转录，而非LEF1。
实施例3.Wnt信号通路抑制剂iCRT14抑制小鼠原位胃癌中CCL28的表达
在本实施例中，利用螺旋杆菌H.felis感染和致癌剂MNU诱导原位发生的小鼠胃癌，再通过腹腔注射Wnt信号通路抑制剂iCRT14，检测胃部的CCL28蛋白表达水平。
结果如图3所示。蛋白免疫印迹法(图3A)和ELISA(图3B)分析均表明，iCRT14使胃部的CCL28蛋白表达显著降低，说明在体内CCL28表达也受β-catenin通路的调控。
实施例4.CCL28与胃癌的发展程度相关
在本实施例中，免疫组织化学分析进一步分析CCL28与β-catenin表达水平的相关性。结果表明，在不同程度的胃癌样品中，β-catenin表达水平与CCL28表达水平存在着显著正相关性(图1C和1D)。
基于Oncomine数据库，进一步对CCL28与胃癌发展程度的进行分析。结果表明，在肠型以及弥散型胃癌中CCL28的mRNA水平都比正常组织高(图4A)。胃癌病人组织样品的免疫组化染色也表明，在恶性等级更高中的肿瘤中CCL28的蛋白水平也更高(图4B)。
总之，这些结果表明CCL28是胃癌细胞中Wnt/β-catenin信号通路的靶基因，这提示CCL28是胃癌中的潜在致病因子。此外，随着胃癌发展或恶性等级增加，CCL28的阳性率更高。
实施例5.β-catenin信号通路激活的胃癌细胞诱导CCL28表达来实现调节T细胞的迁移
在本实施例中，通过体外迁移实验检测了Wnt/β-catenin激活的肿瘤细胞是否能够通过CCL28募集人Treg细胞。方法如下：构建在胃癌细胞系SGC7901中过表达β-catenin，或过表达β-catenin同时利用shRNA敲低CCL28的胃癌细胞系，收集细胞培养液上清，铺在transwell下层，上层小室中铺1X10 6个外周血单核细胞，37℃培养箱中培养4小时后，收集迁移至下层的细胞，并通过流式细胞术分析其免疫细胞的比例和数量。
结果：
在SGC7901细胞中通过shRNA敲低S33Y.β-catenin诱导的CCL28(图5A)。
在迁移实验中，迁移到下层的外周血单核细胞用流式的方法分析其中免疫细胞变化(图5B)。
在胃癌细胞系SGC7901过表达β-catenin的条件下，调节T细胞无论在CD4 +T细胞中的比例还是绝对数量都明显增加，敲低β-catenin则减少(图5C,D)。这些结果提示β-catenin激活的胃肿瘤细胞在体外通过CCL28募集Treg细胞。
流式结果未显示总CD4 +或CD8 +T细胞募集的任何差异(图3C,D)，提示了β-catenin/CCL28对Treg的独特作用。
实施例6.CCL28抗体治疗抑制H.felis/MNU诱导的胃癌进展
在本实施例中，采用H.felis/MNU胃癌模型动物，在MNU处理开始后第29周(图中指示为28周结束，29周开始)，每周一次给予模型小鼠CCL28单克隆抗体以阻断CCL28活性，实验方案如图6A所示。
结果：在用抗体处理8周后，CCL28抗体治疗组小鼠模型的胃中的肿瘤面积显著减少(图6B)。组织学分析显示CCL28抗体疗法在发育异常和肠型转化方面的明显变化(图6C)。在H+K+ATPase的免疫组化分析中看到，使用CCL28抗体治疗后，壁细胞丢失的现象在胃窦部位明显被缓解(图6D)。这提示，抑制CCL28可有效缓解胃癌的进展。
实施例7.CCL28抗体治疗缓解免疫微环境的抑制性
在本实施例中，对于实施例6中的用CCL28抗体进行治疗的模型动物，在第36周结束时，对小鼠胃肿瘤样本进行FACS分析。
结果如图7所示。FACS分析证实CCL28抗体处理的小鼠胃中调节T(Treg)细胞显著减少(图7A)。有趣的是，抗CCL28治疗也降低了脾脏中Treg细胞的比率，但没有降低血液中的Treg细胞的比率，这表明在脾脏和胃中存在依赖于趋化因子CCL28的Treg募集机制。除了对于胃部的直接效用，CCL28抗体治疗在脾脏中的作用也可以提高整体的免疫反应，因为有效的免疫疗法需要外周的免疫激活和协调。总之，这些数据表明抗CCL28疗法通过抑制Treg的募集，有效地抑制了H.felis/MNU诱导的胃癌的进展。
值得一提的是，通过检测IFNγ+的CD4 +/CD8 +T细胞(图7B,C)，结果表明，这些效应T细胞在脾脏或外周血中有一定程度的提高，但在胃部并没有明显变化，说明CCL28抗体治疗可以提高外周免疫的活性，但对于效应T细胞浸润到实体瘤的过程则收效甚微。
实施例8.CCL28抗体对黑色素瘤和乳腺癌无明显治疗功效
在本实施例中，验证CCL28抗体治疗对于其他癌症的治疗作用。方法如下：利用小鼠黑色素瘤细胞B16和乳腺癌细胞4T1建立皮下移植瘤：取对数生长期的细胞，胰酶消化后PBS重悬配用。将6-8周C57BL/6小鼠麻醉，剃毛器处理小鼠前肢腋下，并用75％酒精消毒裸露皮肤。取已调整好细胞密度的细胞悬液接种于小鼠前肢腋下，每天观察记录肿瘤生长情况。其中B16细胞5X10 5个/只,4T1细胞1X10 6个/只。B16移植瘤小鼠和4T1移植瘤小鼠在细胞接种后第3-7天用CCL28抗体处理，处理方式和剂量如实施例6相同。
结果表明，用CCL28抗体或对照IgG抗体治疗之后，CCL28抗体在小鼠移植瘤的生长(图8A和B)以及生存率(图8C和D)上均没有展现出明显的治疗功效。
总结与讨论
本发明人发现了在胃癌细胞中，癌症中常见的致癌基因通路之一Wnt/β-catenin与CCL28的表达的协同作用，而这种作用继而使肿瘤中的调节T细胞增多。通过后续大量创造性的研究，本发明人意外地发现，在胃癌中，利用CCL28的单克隆阻断抗体能够有效地减少肿瘤中调节T细胞的浸润并抑制了肿瘤的发展。这些创造性的研究成果提示Wnt/β-catenin除了直接调控细胞增殖和存活之外的免疫调节机制，并提供新的免疫治疗靶点CCL28。
更出乎意料的是，本发明通过对比实验证明，Wnt/β-catenin与CCL28的联动作用，是在特殊的病理情景—胃癌中独特地存在，而在其他肿瘤、甚至其他实体肿瘤如黑色素瘤细胞B16和乳腺癌细胞4T1模型中不存在的。
另一方面，最近的研究提示调节T细胞的凋亡可能导致更高的免疫抑制性，直接靶向调节T细胞使其凋亡的免疫治疗方法可能无法实现最佳的治疗效果。但是，本发明通过建立起阻断CCL28趋化通路的方法，继而靶向调节T细胞向肿瘤的迁移过程，并不影响调节T细胞的凋亡，可能为靶向调节T细胞的免疫疗法提供更为新颖有效的手段。而且对于长期罹患胃癌和其他经受免疫治疗的患者而言，自身免疫力低下及其衍生疾病是致死的重要原因。因此，本发明提供的方法不但能有效抑制胃癌转移，同时不降低病人的自身免疫力，还会提高整体的免疫力。
除此之外，本发明还首次通过大量的临床数据证实CCL28及其受体水平与胃癌的发生和严重程度的相关性，由此可以开发出精准洞察胃癌发生、转移和预后的检测体系。
总的来看，CCL28阻断疗法在癌症治疗中具有极大的临床应用潜力，而且 可能会提高其他免疫疗法的功效。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (15)

  1. 一种CCL28基因或CCL28蛋白或其检测试剂的用途，其特征在于，用于制备检测胃癌的试剂或试剂盒，所述的胃癌是Wnt/β-catenin信号通路与趋化因子CCL28相关分子表达同时异常上调型的胃癌。
  2. 如权利要求1所述的用途，其特征在于，所述的胃癌的病理表现具有选自下组的一个或多个病理表现：
    (1)胃部组织中Wnt/β-catenin信号通路相关表达上调；
    (2)胃部组织中趋化因子CCL28相关表达上调；
    (3)胃部肿瘤；
    (4)胃壁细胞丢失；及
    (5)胃部组织调节T细胞增多。
  3. 如权利要求1所述的用途，其特征在于，所述胃癌包括原位胃癌、肠型胃癌以及弥散性胃癌。
  4. 如权利要求1所述的用途，其特征在于，所述的Wnt/β-catenin信号通路相关表达，包括转录因子TCF1和TCF4的表达水平、β-catenin的含量、β-catenin核异位水平、β-catenin/TCF4复合物的转录活性、GSK3β磷酸化水平或其组合。
  5. 如权利要求1所述的用途，其特征在于，所述的趋化因子CCL28相关分子，包括CCL28及其受体的基因、蛋白、或其组合。
  6. 如权利要求1所述的用途，其特征在于，所述的试剂包括CCL28相关分子的特异性引物、特异性抗体、探针和/或芯片。
  7. 一种用于检测胃癌的试剂盒，所述的试剂盒含有一容器，所述容器中含有检测CCL28相关蛋白或mRNA的检测试剂；以及标签或说明书，所述标签或说明书注明所述试剂盒用于检测胃癌。
  8. 如权利要求7所述的试剂盒，其特征在于，所述的胃癌是Wnt/β-catenin信号通路趋化因子CCL28相关分子表达同时异常上调型的胃癌。
  9. 如权利要求7所述的试剂盒，其特征在于，所述的检测试剂包括：特异性引物、特异性抗体、探针和/或芯片。
  10. 如权利要求7所述的试剂盒，其特征在于，所述的标签或说明书中注明内容，选自下组：
    a)当检测对象的CCL28相关蛋白的mRNA表达量A1与癌旁组织的CCL28相关蛋白的mRNA表达量A0之比(A1/A0)≥2，则提示该检测对象患胃癌的几率高于普通人群；
    b)当检测对象的CCL28相关蛋白的mRNA表达量与癌旁组织的CCL28相关蛋白的mRNA表达量之比A1/A0≥2，如果该A1/A0比值越高，则提示所述检测对象患胃癌的恶性程度越高；和
    c)当检测对象的CCL28相关蛋白的mRNA表达量与癌旁组织的CCL28相关蛋白的mRNA表达量之比A1/A0≥2，如果该A1/A0比值越高，则提示所述检测对象患胃癌的预后越差、转移率越高。
  11. 一种CCL28抑制剂的用途，其特征在于，用于制备抑制癌细胞生长或增殖的药物组合物，或用于制备治疗胃癌的药物组合物；所述的胃癌是Wnt/β-catenin信号通路与趋化因子CCL28相关分子表达同时异常上调型的胃癌。
  12. 如权利要求11所述的用途，其特征在于，所述CCL28抑制剂选自下组：
    靶向CCL28和/或其受体蛋白的抗体或小分子抑制剂；靶向CCL28和/或其受体基因的靶向核酸分子或基因编辑器；或其组合。
  13. 一种体外非治疗性的抑制癌细胞生长或增殖的方法，其特征在于，包括步骤：在CCL28抑制剂存在下，培养癌细胞，从而抑制癌细胞生长或增殖。
  14. 一种筛选治疗癌症的候选化合物的方法，包括步骤：
    (a)测试组中，在细胞的培养体系中添加测试化合物，并观察所述测试组的细胞中CCL28相关分子的表达量和/或活性；在对照组中，在相同细胞的培养体系中不添加测试化合物，并观察对照组的所述细胞中CCL28相关分子的表达量和/或活性；
    其中，如果测试组中细胞的CCL28相关分子的表达量和/或活性小于对照组，就表明该测试化合物是对CCL28相关分子的表达和/或活性有抑制作用的治疗癌症的候选化合物。
  15. 一种抑制或治疗胃癌的方法，包括步骤：给需要治疗的对象施用安全有效量的CCL28相关分子抑制剂；所述的胃癌是Wnt/β-catenin信号通路趋化因子CCL28相关分子表达同时异常上调型的胃癌。
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