Wnt4 neutralizing antibodies, methods of making and using the same

Abstract

This invention relates to a WNT4 neutralizing antibody and its preparation method. The WNT4 neutralizing antibody is prepared by immunization with the antigen shown in SEQ ID NO:1. The WNT4 neutralizing antibody can be used for inflammatory bowel disease, as well as colorectal cancer and intestinal fibrosis associated with inflammatory bowel disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to WNT4 neutralizing antibodies, their preparation methods, and applications. Background Technology

[0002] Inflammatory bowel disease (IBD) is a chronic, relapsing inflammatory disease of the gastrointestinal tract, primarily including Crohn's disease (CD) and ulcerative colitis (UC). Its pathogenesis involves the interaction of multiple mechanisms, including genetic susceptibility, gut microbiota dysbiosis, environmental factors, and immune abnormalities, leading to persistent and chronic intestinal inflammation. Data from the Global Burden of Disease Study (GBD) 2021 shows that in 2021, there were approximately 3.83 million people with IBD globally, with about 375,000 new cases and 42,400 deaths. The overall absolute number of cases and the economic burden are on the rise, with a particularly pronounced upward trend in newly industrialized countries.

[0003] Chronic inflammation in intestinal dysplasia (IBD) can affect the entire digestive tract and multiple systems throughout the body, and its complications are increasingly becoming a pressing clinical problem. Besides simple intestinal inflammation, common complications include fibrotic strictures, fistulas, abscesses, perforation, toxic megacolon, colitis-associated colorectal cancer, and extraintestinal manifestations. These complications significantly increase the probability of hospitalization and surgery, while also reducing patients' quality of life. Among these, intestinal fibrosis and colitis-associated colorectal cancer are the two most common complications.

[0004] Intestinal fibrosis is one of the most common complications of Crohn's disease (CD), with approximately 70% of CD patients developing fibrostenotic stricture within 10 years of diagnosis. Its core pathological mechanism involves abnormal activation of myofibroblasts, excessive deposition of extracellular matrix (ECM), and epithelial-mesenchymal transition (EMT) driven by chronic inflammation, involving multiple signaling pathways (such as TGF-β / Smad, TL1A / IL-13, YAP / TAZ, and NOD2-related inflammation-fibrosis crosstalk) and gut microbiota dysbiosis (such as AIEC and Bacteroides fragilis). Clinically, it manifests as recurrent intestinal obstruction, abdominal pain, and malnutrition, leading to significantly increased hospitalization rates, hormone dependence, and surgical rates. Anti-TNF biologics can delay the need for surgery in some patients, but there are currently no specific anti-fibrotic drugs; endoscopic balloon dilation (EBD) and surgical procedures (resection or stricture repair) are the main interventions. However, the postoperative recurrence rate remains as high as 50%. Intestinal fibrosis has become the leading cause of disability and surgery in CD patients, and research on novel anti-fibrotic targets is urgently needed.

[0005] Colitis-associated colorectal cancer (CAC) is one of the most serious complications of acute colitis-related colorectal cancer (IBD). IBD patients have a 1.5–2.4 times higher risk of developing colorectal cancer than the general population, and this risk increases significantly with disease progression and lesion size. While CAC accounts for approximately 1–2% of all colorectal cancers, it is the leading cause of death in 10–15% of IBD patients. Compared to sporadic colorectal cancer, CAC has a earlier age of onset, is more often located proximally, is multifocal, has a higher tumor stage, and infiltrates more surrounding structures, making surgery more challenging. Early screening and strict follow-up are currently the main prevention and control strategies. However, unlike the common adenoma-adenocarcinoma sequence in sporadic colorectal cancer, the flattened morphology of precancerous dysplastic lesions in CAC makes them difficult to identify endoscopically, resulting in a generally poor prognosis.

[0006] Intestinal fibroblasts, a highly heterogeneous group of cells, play a crucial role in intestinal development, homeostasis, and disease. Under physiological conditions, intestinal fibroblasts, as supporting cells, maintain intestinal epithelial homeostasis by secreting growth factors. For example, telocytes provide a suitable microenvironment for intestinal stem cells by paracrine secretion of WNT4, WNT5a, and BMPs. Trophoblasts at the base of crypts secrete WNT2a, R-spondins, and Gremlin1, participating in crypt homeostasis maintenance and damage repair.

[0007] Against the backdrop of chronic inflammation in inflammatory bowel disease, intestinal fibroblasts undergo abnormal activation, becoming core effector cells in two key complications: intestinal fibrosis and colitis-associated colorectal cancer. In the course of intestinal fibrosis, intestinal fibroblasts drive fibrotic stenosis through excessive deposition of extracellular matrix. In the development and progression of colitis-associated colorectal cancer, intestinal fibroblasts transform into cancer-associated fibroblasts (CAFs), promoting tumorigenesis, progression, and immune escape by remodeling the tumor microenvironment (TME).

[0008] In the pathological process of intestinal fibrosis, intestinal fibroblasts are activated into myofibroblasts, which, in conjunction with myofibroblasts derived from epithelial-mesenchymal transition (EMT), endothelial-mesenchymal transition (EMT), and smooth muscle cell transdifferentiation, secrete extracellular matrix that deposits in the intestinal wall, leading to intestinal wall thickening and ultimately intestinal stenosis and obstruction. Regarding the development of colorectal cancer, studies have found that a high-fat diet promotes colon tumorigenesis by increasing the levels of primary and secondary bile acids in the mouse intestine and stimulating WNT2b expression in tumor-associated fibroblast-like mesenchymal stem cells (MAMSCs) via FXR. In the progression of colorectal cancer, tumor-associated fibroblasts (CAFs) activate their own FZD8-mediated WNT signaling pathway by autosecreting WNT2, promoting extracellular matrix remodeling and angiogenic factor expression, thereby promoting tumor invasion. Regarding the treatment and drug resistance of colorectal cancer, CAFs can secrete long non-coding RNA (lncRNA) H19 and colon cancer-associated lncRNA (CCAL) into tumor stem cells via exosomes, promoting chemotherapy resistance.

[0009] Large-scale epidemiological surveys have found a positive correlation between the incidence of inflammatory bowel disease (IBD) and the level of economic development, suggesting that lifestyle changes caused by economic development may be related to an increase in IBD incidence. Recent research has found a close relationship between dietary factors and the occurrence and development of IBD. Previous studies have shown that a high-sugar diet and its subsequent effects can promote the occurrence and development of IBD. Maniyar's team found that high intake of added sugar and sugary drinks significantly increased the risk of IBD. Ahsan's team found that high sugary drink intake was significantly associated with increased hospitalization and emergency room visits in IBD patients, as well as disease severity and elevated inflammatory markers. Khan's team found that a high-sugar diet disrupts the intestinal mucus barrier by remodeling the gut microbiome and increasing the proportion of mucus-degrading bacteria, promoting bacterial invasion and the development of colitis. Fecal transplantation experiments confirmed the microbiome-mediated pathogenicity. Burr's team found that a high-sugar diet significantly inhibits the proliferation of colonic epithelial stem cells and impairs the epithelial barrier's regenerative capacity through metabolic reprogramming (locking stem cells to glucose metabolism and reducing ATP production), thereby aggravating intestinal damage in IBD models. Ma's team used animal experiments to demonstrate that high fructose intake can significantly aggravate DSS-induced and T-cell transfer-induced colitis, promote Th1 / Th17 cell differentiation by inducing ROS production and the glutamine metabolism-mTORC1 pathway, independently of risk factors such as obesity and abnormal blood sugar. However, some mechanisms still need further clarification.

[0010] Although there are no direct literature reports on the effects of high-sugar diets on IBD complications (such as intestinal fibrosis / stenosis), multiple experiments have shown that high-sugar diets can significantly exacerbate IBD-like colitis, damage the epithelial barrier and regenerative capacity, and activate fibrosis-related pathways such as epithelial-mesenchymal transition, which may indirectly promote the progression of chronic inflammation to fibrosis / stenosis.

[0011] The human WNT4 gene is located on chromosome 1p36.12, and in mice on chromosome 4. The WNT4 protein contains conserved serine / cysteine ​​residues, and its post-translational modifications include palmitoylation (crucial for secretion and receptor binding) and N-glycosylation (affecting protein stability and transport). Its domains contain approximately 350-400 amino acids common to the WNT family, exhibiting a specific disulfide bond pattern formed by multiple cysteine ​​residues, stabilizing its three-dimensional folding. As a secretory glycoprotein, WNT4 belongs to the WNT family (Wingless-type MMTV integration site family, WNT family) of signaling molecules. This family plays a crucial role in regulating cell proliferation, differentiation, and migration, and is essential for embryonic development and adult tissue homeostasis.

[0012] In the context of IBD, the WNT / β-catenin signaling pathway is highly activated, interacting with inflammatory pathways such as NF-κB and STAT3 to jointly influence epithelial regeneration and intestinal homeostasis. WNT4 also plays a crucial role in the process of intestinal fibrosis secondary to inflammatory bowel disease. Studies by Colleen McGregor et al. found that fistula-associated stromal fibroblasts in CD patients significantly induced the expression of WNT2, WNT4, and WNT5A, accompanied by upregulation of their receptor Frizzled (FZD). WNT4 drives abnormal activation of morphogenetic signaling and the PCP pathway, promoting fibroblast proliferation, migration, ECM remodeling, and fistula dilation. This shifts repair towards a pathological invasive-fibrotic state, leading to chronic tissue rigidity and persistent fistulas. In colorectal cancer, WNT4 primarily activates the classical WNT / β-catenin signaling pathway. Mutations in upstream regulators (such as APC) lead to persistent activation of the WNT / β-catenin pathway, thereby promoting cell survival, epithelial-mesenchymal transition (EMT), angiogenesis, and metastasis. Hypoxic colorectal cancer cells can secrete WNT4 into normoxic cells via exosomes, activating the WNT / β-catenin pathway and promoting tumor progression. CXCL14+INHBA+ cancer cells (CAFs) promote tumor stroma activation and tumor cell proliferation by secreting WNT4.

[0013] Inflammatory bowel disease (IBD) and its secondary complications, intestinal fibrosis and colorectal cancer, threaten human health with their increasing incidence and mortality, while current treatment options remain limited. Therefore, evaluating the efficacy of WNT4 neutralizing antibodies against IBD and its secondary complications, intestinal fibrosis and colorectal cancer, may provide new insights into the systemic treatment of IBD. Summary of the Invention

[0014] Based on this, the purpose of this invention is to provide a WNT4 neutralizing antibody and its preparation method, as well as the application of the WNT4 neutralizing antibody in the treatment of colon tumors and colitis.

[0015] In a first aspect, the present invention provides a WNT4 neutralizing antibody, which is prepared by immunization with the antigen shown in SEQ ID NO:1.

[0016] In one embodiment, the antibody is a polyclonal antibody.

[0017] Secondly, the present invention provides a method for preparing the WNT4 neutralizing antibody described in the first aspect, comprising immunizing with the antigen shown in SEQ ID NO:1, collecting antiserum, and purifying it to obtain the WNT4 neutralizing antibody.

[0018] In one embodiment, the method includes immunizing animals with the antigen shown in SEQ ID NO:1 and collecting the serum of the immunized animals for affinity purification.

[0019] In one embodiment, the immunized animal is a rabbit or a mouse, preferably a rabbit.

[0020] In one embodiment, the immunization route is intradermal injection, preferably at multiple points on the back.

[0021] Thirdly, the present invention provides the use of the WNT4 neutralizing antibody described in the first aspect in the preparation of a medicament for treating inflammatory bowel disease.

[0022] The application of the WNT4 neutralizing antibody in the preparation of a drug for treating intestinal fibrosis caused by inflammatory bowel disease.

[0023] The application of the WNT4 neutralizing antibody in the preparation of drugs for treating colorectal cancer.

[0024] In one embodiment, the colorectal cancer is colitis-associated colorectal cancer.

[0025] Fourthly, the present invention provides a drug whose active ingredient includes the WNT4 neutralizing antibody described in the first aspect.

[0026] In some embodiments, the dosage form of the drug is an injection.

[0027] The present invention has the following beneficial effects: The present invention obtains a WNT4 neutralizing antibody, which can be used for inflammatory bowel disease, as well as the complications of inflammatory bowel disease, colitis-associated colorectal cancer and intestinal fibrosis. Attached Figure Description

[0028] Figure 1 This diagram illustrates the changes in the mouse intestine, including chronic inflammation and tumor formation, following treatment with an AOM / DSS-induced colitis-associated colorectal cancer model. A shows the trend of mouse body weight change; B shows a photograph of changes in mouse colon length; and C shows the degree of colon shortening in mice.

[0029] Figure 2 This diagram illustrates the effect of intravenous injection of WNT4 neutralizing antibody on colon tumor growth in mice. In the diagram, A shows a longitudinally dissected photograph of the entire mouse colon; B shows the tumor count results within the mouse colon.

[0030] Figure 3 This diagram illustrates the effect of intravenous injection of WNT4 neutralizing antibody on colon tumor volume and tumor burden in mice. In the diagram, A represents the calculated colon tumor volume in mice; B represents the calculated number of colon tumors in mice.

[0031] Figure 4 This diagram illustrates the effect of intravenous injection of WNT4 neutralizing antibody on the pathological results of mouse colon tissue. In the diagram, A shows the immunohistochemical staining results observed under a light microscope; B shows the pathological scoring results.

[0032] Figure 5 This diagram illustrates the effect of intravenous injection of WNT4 neutralizing antibody on the proliferation of colon tumors in mice. In the diagram, A shows the immunohistochemical staining results of Ki-67 under a light microscope; B shows the data analysis results of Ki-67; C shows the immunohistochemical staining results of PCNA under a light microscope; and D shows the data analysis results of PCNA.

[0033] Figure 6 This is a schematic diagram of the development process for WNT4 neutralizing antibodies.

[0034] Figure 7 The results of the WB experiment for the WNT4 neutralizing antibody are shown.

[0035] Figure 8 The results of the cell immunofluorescence assay for WNT4 neutralizing antibody are shown.

[0036] Figure 9 Masson staining results of colon in chronic TNBS mice; the blue area indicates collagen fiber deposition.

[0037] Figure 10 The percentage of collagen fiber-positive areas in the colon stained with Masson staining in chronic TNBS mice.

[0038] Figure 11 Masson staining of the colon in chronic DSS mice.

[0039] Figure 12 The percentage of collagen fiber-positive areas in the colon stained with Masson staining in chronic DSS mice.

[0040] Figure 13 To detect intestinal barrier permeability in mice using the FITC-Dextran method.

[0041] Figure 14 To construct a plasmid map expressing WNT4. Detailed Implementation

[0042] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0043] Unless otherwise specified, experimental methods in the following examples were performed under standard conditions, such as those described in the fourth edition of *Molecular Cloning: A Laboratory Manual*, edited by Green and Sambrook, published in 2013, or according to the manufacturer's recommendations. All commonly used chemical reagents used in the examples are commercially available products.

[0044] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0045] Furthermore, as used herein, the term "or" is an inclusive "or" sign and is equivalent to the term "and / or," unless the context clearly specifies otherwise. The term "based on" is not exclusive and allows for basing on other factors not described, unless the context clearly specifies otherwise.

[0046] To facilitate understanding of this technology, some terms and phrases are defined below.

[0047] WNT, Wingless-type MMTV integration site.

[0048] WNT4, Wingless-type MMTV integration site family, member 4.

[0049] WNT4 neutralizing antibody, also known as neutralizing anti-human WNT4 antibody.

[0050] Main instruments and equipment

[0051] Table 1:

[0052]

[0053] Main reagents:

[0054] Table 2:

[0055]

[0056] Experimental animals were immunized with recombinantly expressed exogenous proteins to induce antibody production against the antigen. The specific antiserum produced by the experimental animals was collected and subjected to ELISA titer testing. The antibodies were then purified using the protein antigen through affinity assay, and the purified antibodies were further tested for ELISA titer. A schematic diagram of the polyclonal antibody development process described in this invention is shown below. Figure 6 As shown.

[0057] The antigen can be obtained by synthesis or by purifying a plasmid containing the human WNT4 coding sequence shown in SEQ ID NO:2 after cell transfection and culture.

[0058] The present invention will be further described in detail below with reference to specific embodiments.

[0059] Example 1: Preparation of WNT4 neutralizing antibody

[0060] I. Antigen Preparation

[0061] A recombinant protein expression system was used to produce 3.5 mg of WNT4 recombinant protein for immunization and subsequent screening.

[0062] 1. Immunogen protein (SEQ ID NO:1):

[0063] SNWLYLAKLSSVGSISEEETCEKLKGLIQRQVQMCKRNLEVMDSVRRGAQLAIEECQYQFRNRRWNCSTLDSLPVFGKVVTQGTREAAFVYAISSAGVAFAVTRACSSGELEKCGCDRTVHGVSPQGFQWSGCSDNIAYGVAFSQSFVDVRERSKGASSSRALM NLHNNEAGRKAILTHMRVECKCHGVSGSCEVKTCWRAVPPFRQVGHALKEKFDGATEVEPRRVGSSRALVPRNAQFKPHTDEDLVYLEPSPDFCEQDMRSGVLGTRGRTCNKTSKAIDGCELLCCGRGFHTAQVELAERCSCKFHWCCFVKCRQCQRLVELHTCR

[0064] The aforementioned immunogenic peptides were entered into the IEDB database for comparison, yielding seven linear B-cell WNT4 epitopes. This suggests that the resulting antibody targets multiple WNT4 epitopes, i.e., a polyclonal antibody.

[0065] 2. Expression and purification

[0066] I. Large-scale extraction of recombinant plasmids

[0067] 1) Construct the recombinant plasmid using conventional methods. First, design the human WNT4 coding region sequence (SEQ ID NO: 1).NO:2):AGCAACTGGCTGTACCTGGCCAAGCTGTCCTCCGTGGGAAGCATCAGCGAGGAGGAGACATGTGAGAAGCTGAAGGGCCTGATCCAGAGACAGGTGCAGATGTGTAAGAGGAACCTGGAGGTGATGGATAGCGTGAGGAGGGGCGCTCAGCTGGCTATTGAGGAGTGTCAGTACCAGTTTAGGAATAGGAGATGGAATTGTAGCACCCTGGATAGCCTGCCTGTGTTTGGCAAGGTGGTGACCCAGGGCACCAGAGAAGCCGCTTTCGTGTACGCCATCAGCTCCGCTGGCGTGGCTTTCGCTGTGACAAGAGCCTGTAGCAGCGGCGAGCTGGAGAAGTGTGGCTGTGATAGGACCGTGCACGGCGTGTCTCCCCAAGGATTTCAGTGGTCCGGCTGCTCCGATAACATCGCCTACGGCGTGGCCTTTAGCCAGTCCTTTGTGGACGTGAGAGAGAGGAGCAAGGGCGCTAGCAGCAGCAGAGCTCTGATGAACCTGCACAACAACGAGGCCGGCAGGAAGGCTATCCTGACCCATATGAGGGTGGAGTGTAAGTGCCACGGCGTGTCCGGAAGCTGTGAGGTTAAGACATGTTGGAGAGCCGTGCCTCCTTTTAGGCAGGTGGGCCATGCCCTGAAGGAGAAGTTCGATGGCGCCACAGAGGTGGAGCCTAGAAGAGTGGGCTCCTCCAGGGCTCTGGTGCCTAGGAATGCCCAGTTTAAGCCTCACACCGATGAGGACCTGGTGTACCTGGAGCCCAGCCCTGATTTCTGTGAGCAGGACATGAGGAGCGGCGTGCTGGGAACAAGGGGAAGAACATGTAATAAGACATCCAAGGCCATCGACGGCTGCGAGCTGCTGTGTTGTGGCAGAGGATTCCACACCGCCCAGGTGGAACTGGCTGAGAGATGTAGCTGTAAGTTCCACTGGTGTTGCTTCGTGAAGTGCAGACAGTGTCAGAGGCTGGTGGAGCTGCACACCTGCAGA。

[0068] The plasmid was constructed by Jiangsu Saisofe Biotechnology Co., Ltd. Primers were designed and synthesized according to the sequence, and amplification was performed. Simultaneously, the empty vector was double-digested with the corresponding restriction endonuclease to linearize it. The PCR product recovered from the gel was then mixed with the linearized vector in a specific ratio, and recombinase was added. The mixture was incubated at 52°C for 40 min to complete homologous recombination ligation. The ligation product was transformed into competent cells, and after ice bath, heat shock, and recovery, the cells were plated on antibiotic-containing plates and cultured overnight. Single colonies were picked and positive clones were screened by colony PCR. The plasmid was then extracted and full-length sequencing was performed using forward, reverse, and walking primers, ultimately obtaining a 100% accurate human WNT4 recombinant plasmid. The constructed recombinant plasmid was transformed into TOP10 strains and plated on LB plates (containing antibiotics) and cultured overnight at 37°C.

[0069] 2) Select single colonies containing recombinant plasmids and add them to 3 mL of LB liquid medium. Incubate overnight at 37°C.

[0070] 3) Take 700 μl of bacterial culture to preserve the culture, and inoculate the remaining bacterial culture into 300 mL of LB liquid medium (containing resistance) and incubate overnight at 37°C.

[0071] 4) Collect bacterial cells and perform large-scale plasmid extraction.

[0072] Please see the plasmid map. Figure 14 .

[0073] II. Transfection Culture

[0074] 1) Follow the standard passage procedure to passage HEK293 cells.

[0075] 2) Once the cell density reaches 0.5 × 10⁶ cells / mL, seed 80 mL of the system cells into a shake flask and incubate for 24 h in a shaker incubator at 37°C, 120 rpm, and 5% CO₂ concentration, until the cell density reaches 1 × 10⁶ cells / mL. 6 cells / mL.

[0076] 3) Take 80 μg of plasmid (filtered and sterilized) and add it to 8 mL of PBS. Then vortex mix for 3 seconds to ensure thorough mixing.

[0077] 4) Add 0.32 mL of filtered sterilized PEI solution (0.5 mg / mL) to the PBS / plasmid mixture.

[0078] 5) Let the PEI-plasmid mixture stand at room temperature for 20 minutes.

[0079] 6) Add the plasmid / PEI mixture into the cells (cell density 1.5-2.0 × 10⁶).6 (cells / mL).

[0080] 7) After transfection, incubate for 6 days in a shaker incubator at 37℃, 120rpm, and 5% carbon dioxide concentration.

[0081] 8) Centrifuge at 3000g for 5 min at 4℃ to collect the supernatant of the culture medium, and store at -80℃ or use it for subsequent purification.

[0082] III. Purification and Detection

[0083] 1) The culture medium supernatant was filtered using a 0.22 μm filter.

[0084] 2) Prepare a Protein A / G resin gravity column and load the supernatant protein solution at a flow rate of 1 mL / min.

[0085] 3) Wash the column with PBS buffer (pH 7.4) until the effluent is free of protein (G250 detection solution does not change color).

[0086] 4) Elute with PBS buffer (pH 3.0) and collect the eluent in fractions until the G250 detection solution does not change color.

[0087] 5) Take 10 μl of each stage flow-through buffer for SDS-PAGE electrophoresis detection, and neutralize the collected eluent to pH 7.4, then concentrate it and perform final concentration SDS-PAGE electrophoresis detection. Obtain the immunogenic antigen (hereinafter referred to as WNT4 recombinant protein) used for antibody preparation.

[0088] II. Antibody Preparation

[0089] 1. Animal immunization

[0090] Animals: Healthy female New Zealand White rabbits, 4 months old, 2.1 kg.

[0091] Adjuvants: Freund's complete adjuvant was used for the first major injection, and Freund's incomplete adjuvant was used for subsequent booster injections. Both adjuvants were thoroughly mixed with an equal volume of antigen before injection.

[0092] Immunogen: The recombinant WNT4 protein prepared above.

[0093] immunity:

[0094] Multiple injections on the back:

[0095] First immunization: On day 1, the antigen used for immunization is Freund's complete adjuvant + WNT4 recombinant protein.

[0096] Second immunization: On day 14, the antigen used for immunization was Freund's incomplete adjuvant + WNT4 recombinant protein.

[0097] Third immunization: On day 28, the antigen used for immunization is Freund's incomplete adjuvant + WNT4 recombinant protein.

[0098] Fourth immunization: On day 42, the antigen used for immunization is Freund's incomplete adjuvant + WNT4 recombinant protein.

[0099] Blood was collected after the fourth immunization: On day 49, 1 mL of blood was collected from the ear vein and the antiserum titer was detected by ELISA.

[0100] Fifth immunization: On day 56, the antigen used for immunization is Freund's incomplete adjuvant + WNT4 recombinant protein.

[0101] Final bloodletting: On the 63rd day, whole blood was collected from the carotid artery, left to stand overnight at 4°C, and then the serum was collected and frozen.

[0102] 4. Antiserum purification

[0103] The serum obtained above was subjected to affinity purification:

[0104] 1) Take an appropriate amount of CNBr-activated Bestarose 4B into the purification column, wash three times with 10mM HCl (about 30mL), then wash with binding buffer to remove residual hydrochloric acid, add the antigen dissolved in PBS to the beads, add buffer and incubate overnight at 4°C, then wash three times with acid, water and alkali for column connection, and finally wash with PBS for later use.

[0105] 2) Incubate the antiserum with the affinity purification column overnight.

[0106] 3) Wash with pH 5.0 HCl to remove impurities and antibodies.

[0107] 4) Elute with 0.15M glycine buffer at pH 2.5, then quickly neutralize with 10×PBS buffer to prepare affinity-purified antibodies.

[0108] 5) Dialyze the PBS buffer solution to change the buffer.

[0109] 6) The Bradford method was used to detect the concentration of purified antibodies.

[0110] The antibody concentration was determined to be 1.187 mg / mL using the Bradford method.

[0111] 5. Indirect ELISA detection of antiserum and affinity-purified antibody titers.

[0112] Indirect ELISA detection

[0113] 1) Coating antigen: The WNT4 recombinant protein prepared above was diluted with 0.05 mol / L carbonate (pH 9.6) to 6 μg / mL, 100 μL / well, and incubated overnight at 4°C.

[0114] 2) Washing: Remove the plate and wash it three times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0115] 3) Blocking: Add 150 μL of 5% skim milk powder (PBST) blocking solution to each well and block at 37°C for 60 min.

[0116] 4) Washing: Remove the plate and wash it three times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0117] 5) Add primary antibody: Dilute the antiserum at a ratio of 1:1000, then serially dilute and incubate at 37°C for 1 min.

[0118] 6) Washing: Remove the plate and wash it three times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0119] 7) Add secondary antibody: horseradish enzyme-labeled goat anti-rabbit IgG (H+L), diluted 1:8000, and incubated at 37℃ for 45 min.

[0120] 8) Washing: Remove the plate and wash it five times with 0.05% Tween-20 (PBST), 3 minutes each time.

[0121] 9) Color development: Add 100 μL of substrate solution (TMB) per well, react for 5-10 min, and finally add 100 μL of 2 mol / L sulfuric acid to terminate the reaction.

[0122] 10) OD value measurement: OD value is measured at a wavelength of 450nm using an enzyme-linked immunosorbent assay (ELISA) reader.

[0123] result:

[0124] 1. Serum titer testing

[0125]

[0126] 2. Purified polyclonal antibody titer detection

[0127]

[0128] Note: The table shows the titer data measured by an ELISA reader. The purified antibody titer is ≥1024K.

[0129] Using the method described above, WNT4 neutralizing antibodies, i.e. neutralizing anti-human WNT4 antibodies, can be obtained stably.

[0130] A WNT4 neutralizing antibody of 1.187 mg / mL was obtained and used in the following experiments.

[0131] Example 2

[0132] To verify that the WNT4 neutralizing antibody prepared in Example 1 can specifically target the recombinant WNT4 protein, we first used Western Blot method for verification.

[0133] Prepare a 13.8% PAGE gel. Electrophore the WNT4 recombinant protein prepared in Example 1 at U=80 V for 30 min. When the sample enters the separating gel, adjust the V to U=120 V and electrophoresis for 60 min. After electrophoresis, remove the SDS-PAGE gel. Add 500 mL of transfer buffer to the container to prepare the transfer "sandwich": Open the electrotransfer clamp, place a dedicated sponge pad soaked in transfer buffer on each side, and then place four sheets of filter paper soaked in transfer buffer on each side. Place the gel flat on the cathode (black) side filter paper, and finally place the NC membrane flat on the gel. Remove air bubbles, clamp the electrotransfer clamp, and ensure there are no air bubbles between the NC membrane and the gel, the blot membrane and the transfer pad, and the transfer pad and the gel. Fill the transfer tank with transfer buffer, insert the electrotransfer clamp, place the transfer tank in an ice bath, connect the electrodes, and turn on the current. The NC membrane in the transfer clamp should be aligned with the positive electrode of the transfer tank. Transfer at a constant current of 280 mA for 60 min. After transfer, any unbound protein sites on the membrane need to be blocked by treating with skim milk powder at room temperature for 60 min to prevent non-specific binding by primary or secondary antibodies. Take 5 μL of the WNT4 neutralizing antibody prepared in Example 1 and rabbit anti-human IgG (negative control) (Maisi Biotechnology, GS60005), dilute them 1:1000 with universal antibody diluent to prepare primary antibody, immerse the NC membrane, and incubate overnight at 4°C on a shaker. After incubation, wash the membrane 3 times for 3 min each with TBST. Select HRP-labeled goat anti-rabbit secondary antibody, dilute it to 1:5000 with TBST, incubate at room temperature for 60 min, and wash the membrane 3 times for 3 min each with TBST after incubation. Perform chemiluminescence imaging according to the instructions of the chemiluminescence kit (Yisheng Biotechnology, 36208ES76).

[0134] The results showed that, compared with rabbit anti-human IgG (negative control), a distinct protein band was observed in the 55kDa-72kDa range when the WNT4 neutralizing antibody was used as the primary antibody. No distinct bands were observed in the remaining regions. Since the recombinant WNT4 protein used has an fc tag, its molecular weight is 25kDa larger than the theoretical molecular weight, meaning the final predicted observed molecular weight was 64kDa-70kDa, consistent with the results.

[0135] The results were then validated using cell immunofluorescence with a cell line expressing WNT4. PANC-1 cells were selected as the positive expression cell line. Cells with 80%–90% confluence were washed with PBS, then digested with trypsin at 37°C for 3 min. Digestion was stopped when cells became rounded and intercellular spaces increased. Cells were centrifuged and resuspended. Sterile cell slides were placed at the bottom of 24-well plates, cell suspension was added, and culture medium was replenished. Cells were cultured until 40%–60% confluence was reached, at which point the culture medium was removed, and the cells were washed with PBS. 4% paraformaldehyde was added for fixation at room temperature for 10 min, followed by PBS washing for 5 min × 3 times. PBS permeabilization buffer containing 0.1% Triton X-100 was added, and the cells were incubated at room temperature for 30 min, followed by PBS washing for 5 min × 3 times. PBS was aspirated from the slides, and immunoblocking buffer was added and incubated at room temperature for 30 min. Primary antibody working solution was prepared by diluting WNT4 neutralizing antibody and immunofluorescence primary antibody at a volume ratio of 1:1000, and added until the cell layer was completely covered. An equal volume and concentration of rabbit anti-human IgG (negative control) were added to the negative control wells, and the cells were incubated overnight at 4°C. The primary antibody was removed, and the cells were washed with PBS for 5 minutes. Repeat the process three times a day, diluting the anti-rabbit Alexa Fluor 488 fluorescent secondary antibody at a ratio of 1:400 with immunofluorescence secondary antibody dilution buffer under light-protected conditions. After adding the antibody, incubate at room temperature for 60 minutes. Remove the secondary antibody in the dark, wash with PBS for 5 minutes three times a day, add DAPI-containing immunofluorescence mounting medium to a glass slide, remove the slide with tweezers, and invert the cell side onto the mounting medium to seal the slide. Finally, observe and photograph the slide under a fluorescence microscope using an appropriate filter.

[0136] The results showed that in the group using WNT4 neutralizing antibody as primary antibody, the cytoplasm showed green fluorescence, indicating that it could target the corresponding protein, and the nucleus showed blue DAPI fluorescence; in the group using rabbit anti-human IgG (negative control) as primary antibody, no green fluorescence was observed, only nuclear DAPI fluorescence.

[0137] That is, from Figure 7 As can be seen, using WNT4 neutralizing antibody as the primary antibody, a band at 72-100 kDa can be recognized, while no band was observed using rabbit anti-human IgG (negative control), indicating that the constructed WNT4 neutralizing antibody can target the WNT4 recombinant protein in vitro.

[0138] from Figure 8 As can be seen, PANC-1 cells constitutively express WNT4 protein. When using WNT4 neutralizing antibody as the primary antibody and anti-rabbit Alexa Fluor 488 as the secondary antibody, green fluorescence was observed under a fluorescence microscope, indicating that the WNT4 neutralizing antibody bound the WNT4 protein in PANC-1 cells. No green fluorescence was observed in the group using rabbit anti-human IgG (negative control) as the primary antibody. The cell nucleus was represented by the blue fluorescent dye DAPI.

[0139] Example 3

[0140] Intravenous injection of WNT4 neutralizing antibody has a therapeutic effect on AOM / DSS-induced colitis-associated colorectal cancer in mice under a high-sugar diet.

[0141] NCD Normal Diet

[0142] HSD (High-Sugar Diet)

[0143] NCD+CAC: A mouse model of colorectal cancer based on a normal diet.

[0144] HSD+CAC: A mouse model of colorectal cancer on a high-sugar diet.

[0145] HSD+CAC+CAb: High-sugar diet colorectal cancer model mice + rabbit anti-human IgG (negative control); HSD+CAC+PAb: High-sugar diet colorectal cancer model mice + PD-1 antibody.

[0146] HSD+CAC+WAb: High-sugar diet colorectal cancer model mice + WNT4 neutralizing antibody.

[0147] 1. Seven-week-old male C57BL / 6J mice were randomly divided into seven groups of six to eight mice each. Two groups received no disease modeling but only dietary intervention: one group received no treatment as a negative control, and the other group received a high-sugar diet control with 10% sucrose solution instead of drinking water. Of the remaining five groups, one group developed an AOM / DSS-induced colitis-associated colorectal cancer (CAC) model under normal dietary conditions without any special treatment; the other four groups developed CAC models under a high-sugar diet. The CAC model was established as follows: After acclimatization, mice were intraperitoneally injected with AOM (MedChemExpress, HY-111375, 10 mg / kg); after a one-week rest, DSS was administered in the drinking water in cycles. Each cycle included 7 days of continuous drinking of 2% DSS (MP Biomedicals, 9011-18-1, hereinafter the same) solution, followed by 14 days of normal drinking water, for a total of three cycles. All mice on a high-sugar diet (including the high-sugar diet control group) were switched to drinking plain water during the period of drinking DSS. Of the four groups modeled under a high-sugar diet, one group was designated as the high-sugar diet model group and received no special treatment; the other three groups were injected weekly via tail vein with the following drugs during a two-week interval before drinking DSS: WNT4 neutralizing antibody (250 μg / mouse) prepared in Example 1, PD-1 antibody (MedChemExpress, HY-P99144, 12.5 mg / kg), and rabbit anti-human IgG (negative control) (Maisi Biotechnology, GS60005, 250 μg / mouse), for a total of six injections. After modeling, serum and colon tissue were collected.

[0148] The experimental results are as follows:

[0149] 1. Intravenous injection of WNT4 neutralizing antibody significantly improved the general condition and colonic lesions in mice with colorectal cancer following a high-sugar diet.

[0150] With treatment in the CAC model, the mouse intestines underwent changes such as chronic inflammation and tumor formation. Due to multiple factors including inflammation, edema, and tumors, the colon in the model group mice showed a trend of shortening. Therefore, the degree of colon shortening in mice is an important indicator for assessing the severity of intestinal inflammation and tumors in mice. Compared with colorectal cancer model mice injected with rabbit anti-human IgG (negative control), injection of WNT4 neutralizing antibody into colorectal cancer model mice significantly reduced the length of colon shortening, and the reduction trend was more pronounced compared to PD-1 antibodies currently used in colorectal cancer immunotherapy. Furthermore, the mice generally showed better condition than the model group, and their body weight did not decrease significantly compared to the model group. Figure 1 As shown.

[0151] 2. Intravenous injection of WNT4 neutralizing antibody significantly reduced the number of colon tumors in mice with a high-sugar diet for colorectal cancer.

[0152] Tumors formed within the colon were counted by longitudinally dissecting the entire colon of mice. Mice injected with WNT4 neutralizing antibodies showed a significantly lower number of colonic tumors than mice with a colorectal cancer model injected with rabbit anti-human IgG (negative control). Figure 2 As shown.

[0153] 3. Intravenous injection of WNT4 neutralizing antibody significantly reduced colon tumor volume and tumor burden in a high-sugar diet-fed colorectal cancer model mouse.

[0154] Images of the mouse colon were acquired longitudinally. The long and short diameters of the tumor were measured using ImageJ software. The formula (tumor volume = long diameter * short diameter) was used. 2 / 2) Calculate tumor volume. Statistical analysis showed that the colon tumor volume in mice injected with WNT4 neutralizing antibodies was significantly lower than that in colorectal cancer model mice injected with rabbit anti-human IgG (negative control).

[0155] Tumor burden is an indicator of the amount of tumor load in an individual, calculated as tumor volume divided by the number of tumors. Analysis showed that mice injected with WNT4 neutralizing antibodies had a significantly lower colonic tumor burden than mice with a colorectal cancer model injected with rabbit anti-human IgG (negative control). Figure 3 As shown.

[0156] 4. Intravenous injection of WNT4 neutralizing antibody significantly reduced the histopathological score of colon tissue in mice with a high-sugar diet for colorectal cancer.

[0157] Mouse colon tissue was paraffin-embedded, sectioned, and stained with hematoxylin and eosin (HE). Pathological scoring was performed under a light microscope using the following indicators: ... Figure 4 As shown in Figure A.

[0158] Intravenous injection of WNT4 neutralizing antibodies significantly reduced the colonic tissue pathological score in a high-sugar diet-fed colorectal cancer model mouse. Figure 4 As shown in B.

[0159] 5. Intravenous injection of WNT4 neutralizing antibody significantly reduced the proliferative capacity of colon tumors in a high-sugar diet-fed colorectal cancer model mouse.

[0160] 6. Ki-67 and PCNA, indicators of proliferative capacity, were selected. Whole colon tissue from mice was paraffin-embedded, sectioned pathologically, and immunohistochemically stained. ImageJ software (IHC Profiler) was used for analysis. Statistical analysis of the obtained data revealed that intravenous injection of WNT4 neutralizing antibody significantly reduced the proliferative capacity of colon tumors in a high-sugar diet colorectal cancer model mouse. Figure 5 As shown.

[0161] The above results indicate that: First, compared to the rabbit anti-human IgG (negative control) group, mice in the WNT4 neutralizing antibody group generally had better health, as evidenced by a less pronounced trend in weight loss. Second, compared to NCD, HSD+AOM / DSS significantly shortened the colon length and increased the number of tumors in mice; the WNT4 neutralizing antibody injection group alleviated the colon length shortening effect and resulted in fewer colon tumors. (Volume = (Length × Width)) 2 The value was calculated as 1 / 2, indicating that compared to HSD, HSD+AOM / DSS significantly increased the number and size of tumors, while the use of WNT4 neutralizing antibody significantly inhibited this increase. H&E staining and tumor malignancy were quantitatively analyzed using pathological scoring according to the criteria shown in the table below:

[0162]

[0163] Compared to NCD, HSD+AOM / DSS significantly increased tumor malignancy, while the use of WNT4 neutralizing antibody significantly inhibited this increase. IHC staining showed Ki-67 and PCNA proliferation markers; compared to NCD, HSD significantly increased the proliferative capacity of colon tumors, while WNT4 neutralizing antibody could inhibit this increased effect. These findings suggest that HSD promotes the development and progression of CAC, while specifically targeting and neutralizing WNT4 can inhibit this process.

[0164] Example 4: WNT4 neutralizing antibody inhibits the development and progression of chronic TNBS / DSS-induced intestinal fibrosis in mice.

[0165] WNT4 neutralizing antibodies inhibited the development of chronic TNBS-induced intestinal fibrosis in mice. The model groups involved (N=6-8) are as follows:

[0166] NCD Normal Diet

[0167] HSD (High-Sugar Diet)

[0168] NCD+TNBS normal diet + chronic TNBS intestinal fibrosis model mice

[0169] HSD+TNBS high-sugar diet + chronic TNBS intestinal fibrosis model mice

[0170] HSD+TNBS+CAb high-sugar diet + chronic TNBS intestinal fibrosis model mice + rabbit anti-human IgG (negative control)

[0171] HSD+TNBS+WAb high-sugar diet + chronic TNBS intestinal fibrosis model mice + WNT4 neutralizing antibody.

[0172] First, a TNBS model was constructed. Seven-week-old male C57BL / 6J mice were randomly divided into six groups of 6-8 mice each. One group was trained to develop a chronic TNBS intestinal fibrosis model under a normal diet, while the other three groups were trained to develop a chronic TNBS intestinal fibrosis model under a high-sugar (HSD) diet. Based on previous literature, a 5% (wt / vol) TNBS solution (Sigma, P2297) was diluted to a 1% (wt / vol) TNBS pre-sensitization solution using a 4:1 mixture of acetone and olive oil. After hair removal on the back of the mice, 150 μL of this solution was added to the skin for pre-sensitization. One week after pre-sensitization, TNBS enemas were administered weekly for a total of six weeks, with concentrations of 0.75%, 1.5%, and 2.5% (the last four enemas were all at 2.5%). Before enema, mice needed to be induced to defecate sufficiently to ensure that the colonic administration tube could reach 4 cm from the anus (Wirtz S, et al., Nat Protoc. 2017;12(7):1295-1309). Simultaneously with the enema procedure, three groups of mice with a chronic TNBS intestinal fibrosis model established under HSD were treated as follows: one group received a tail vein injection of WNT4 neutralizing antibody (250 μg / mouse), and another group received a rabbit anti-human IgG injection (negative control) (Maisi Biotechnology, GS60005, 250 μg / mouse), both injected once a week for a total of 6 times; the remaining group received no special treatment (no drugs) and served as a positive control. After modeling, the intestinal barrier function of the mice was assessed using the FITC-Dextran method. Specific methods: After fasting mice for 4 hours, FITC-dextran was dissolved in PBS at a dose of 0.6 mg / g body weight and administered orally in 0.1 ml volume. Three hours after gavage, blood was collected and serum was separated (to avoid hemolysis). A standard curve was prepared by serially diluting known concentrations of FITC-dextran using normal mouse serum. The test serum and standard were added to 96-well plates (70 μL per well), and fluorescence intensity was detected using a microplate reader at an excitation wavelength of 495 nm and an emission wavelength of 520 nm. The concentration of FITC-dextran in serum was quantified using the standard curve to assess intestinal permeability (elevated concentration indicates barrier damage). Subsequently, mouse serum and colon tissue were collected for further analysis.

[0173] The results showed (see Figures 9-10 Masson staining revealed that HSD significantly increased the degree of intestinal fibrosis in TNBS-induced mice compared to NCD. Injection of WNT4 neutralizing antibody alleviated the degree of intestinal fibrosis, while injection of rabbit anti-human IgG (negative control) had no such effect.

[0174] Meanwhile, the FITC-D results showed that injection of the WNT4 neutralizing antibody described in Example 1 could improve chronic TNBS-induced colonic barrier function damage in mice and restore colonic barrier integrity. Figure 13 ).

[0175] WNT4 neutralizing antibodies inhibited the development of intestinal fibrosis induced by chronic DSS in mice. The model groups involved (N=6-8) are as follows:

[0176] NCD: Normal diet;

[0177] HSD (High-Sugar Diet)

[0178] NCD+DSS normal diet + chronic DSS intestinal fibrosis model mice;

[0179] HSD+DSS high-sugar diet + chronic DSS intestinal fibrosis model mice;

[0180] HSD+DSS+CAb high-sugar diet + chronic DSS intestinal fibrosis model mice + rabbit anti-human IgG (negative control);

[0181] HSD+DSS+WAb high-sugar diet + chronic DSS intestinal fibrosis model mice + WNT4 neutralizing antibody.

[0182] Seven-week-old male C57BL / 6J mice were randomly divided into six groups of 6-8 mice each. Two groups received no disease modeling but only dietary intervention: one group received plain water as a normal diet control; the other group received 10% sucrose solution as a high-sugar diet control. The remaining four groups were used to establish a chronic DSS intestinal fibrosis model. One group was modeled under a normal diet without special treatment. The other three groups were modeled under a high-sugar diet: one group served as the high-sugar diet model group without special treatment; one group received a tail vein injection of rabbit anti-human IgG (negative control) (Maisi Biotechnology, GS60005, 250 μg / mouse); and one group received a WNT4 neutralizing antibody injection of 250 μg / mouse. All mice on the high-sugar diet (including the high-sugar diet control group) were switched to plain water during the DSS period. The chronic DSS model was established using a cyclic induction method: each cycle consisted of 7 consecutive days of drinking 2% DSS solution, followed by 14 days of normal water intake, repeated for a total of 3 cycles. For the two groups requiring antibody injection, the mice were injected once a week during each 14-day normal drinking water period, for a total of 6 injections. After the modeling was completed, serum and colon tissue were collected from each group of mice.

[0183] The previous experiment showed that the proportion of collagen fibers positive in the colon stained with Masson staining in mice with chronic TNBS increased. This suggests that chronic TNBS modeling can lead to an increase in the proportion of collagen fibers in the mouse colon, and HSD can enhance this effect; WNT4 neutralizing antibody can significantly alleviate intestinal fibrosis in mice induced by chronic TNBS. Figure 10 ), and chronic TNBS modeling significantly damaged the intestinal barrier in mice, and HSD could exacerbate barrier damage; WNT4 neutralizing antibody injection significantly improved the intestinal barrier in mice ( Figure 13 ).

[0184] The results of this experiment found (see...) Figures 11-12 Masson staining showed that, compared with a normal diet, a high-sugar diet significantly aggravated intestinal fibrosis in mice with chronic DSS; injection of WNT4 neutralizing antibody could alleviate this degree of fibrosis, while injection of rabbit anti-human IgG (negative control) had no such effect.

[0185] from Figure 12 It can be seen that chronic DSS modeling can lead to an increase in the proportion of collagen fibers in the mouse colon, and HSD can enhance this effect; WNT4 neutralizing antibody can significantly alleviate chronic DSS-induced intestinal fibrosis in mice.

[0186] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. WNT4 neutralizing antibody, characterized by, The WNT4 neutralizing antibody is prepared by immunization with the antigen shown in SEQ ID NO:

1.

2. The method for preparing the WNT4 neutralizing antibody according to claim 1, characterized in that, The preparation method includes immunizing animals with the antigen shown in SEQ ID NO:1, collecting the serum of the immunized animals for affinity purification, and obtaining the WNT4 neutralizing antibody.

3. The preparation method according to claim 2, characterized in that, The immunized animal is a rabbit or a mouse, preferably a rabbit.

4. The preparation method according to claim 2 or 3, characterized in that, The immunization route is intradermal injection, preferably at multiple points on the back.

5. The use of the WNT4 neutralizing antibody of claim 1 in the preparation of a medicament for treating inflammatory bowel disease.

6. The use of the WNT4 neutralizing antibody of claim 1 in the preparation of a medicament for treating intestinal fibrosis caused by inflammatory bowel disease.

7. The use of the WNT4 neutralizing antibody according to claim 1 in the preparation of a medicament for treating colorectal cancer.

8. The application according to claim 7, wherein the colorectal cancer is colitis-associated colorectal cancer.

9. A drug characterized by, The active ingredient of the drug includes the WNT4 neutralizing antibody as described in claim 1.

10. The medicament according to claim 9, characterized in that, The drug is in the form of an injection.

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