Use of ormdl3 degradation agent in her2-positive disease

Abstract

ORMDL3 has an interactive relationship with HER2-positive diseases, and an ORMDL3 degradation agent is used in HER2-positive diseases, particularly in HER2-refractory diseases.

Description

Use of ORMDL3 degraders in HER2-positive diseases Technical Field

[0001] This disclosure relates to the pharmaceutical field, specifically to the relationship between ORMDL3 and HER2-positive diseases and the use of ORMDL3 degraders in HER2-positive diseases, especially HER2-refractory diseases. Background Technology

[0002] Breast cancer is a heterogeneous disease composed of different molecular and histological subtypes. Common breast cancer classifications include human epidermal growth factor receptor 2 positive (HER2+ / HER2-positive) breast cancer, Luminal A breast cancer, Luminal B breast cancer, and triple-negative breast cancer (TNBC).

[0003] HER2-positive breast cancer is characterized by ERBB2 / neu oncogene amplification and loss of estrogen receptor (ER) and progesterone receptor (PR) expression, exhibiting a HER2+ / ER- / PR- profile, accounting for 20%–25% of all breast cancers. Luminal A breast cancer is characterized by HER2- / ER+ / PR+, some Luminal B breast cancers are characterized by HER2+ / ER+ / PR+, and triple-negative breast cancers are characterized by HER2- / ER- / PR-. HER2-positive breast cancer is clinically associated with increased malignancy, metastasis rate, and mortality, and is characterized by high invasiveness and poor prognosis. Currently, first-line targeted therapies, such as trastuzumab, pertuzumab, lapatinib, and neratinib, have achieved some therapeutic effects against the HER2 target. However, some patients do not respond to these drugs, experience breast cancer recurrence, metastasis of the primary breast cancer, or develop drug resistance after a period of treatment. Therefore, finding a new target and / or therapeutic agent is of great significance. Summary of the Invention

[0004] ORMDL3 is described in the prior art as an asthma susceptibility gene expressed in the respiratory epithelium. This disclosure provides ORMDL3 as a novel target for HER2-positive disease, particularly HER2-positive subtype breast cancer. This disclosure also reveals that ORMDL3 is a novel and preferential interactor with HER2 in metastatic breast cancer, directly binding to HER2 in the mammary ductal epithelium, and that the interaction between ORMDL3 and HER2 is specific for HER2-positive subtype breast cancer. Loss of ORMDL3 significantly attenuates tumorigenesis and metastasis in HER2-positive subtype breast cancer, but has no effect on other subtypes (Luminal A, Luminal B, and triple-negative subtypes).

[0005] In a first aspect, this disclosure provides methods for treating and / or preventing HER2-positive disease, including administering an effective amount of an ORMDL3 degrading agent to a subject in need. In some embodiments, the ORMDL3 degrading agent provided herein is selected from FTY720.

[0006] In a second aspect, this disclosure provides the use of an ORMDL3 degrading agent in the preparation of a medicament for the treatment and / or prevention of HER2-positive diseases. In some embodiments, the ORMDL3 degrading agent provided herein is selected from FTY720.

[0007] In a third aspect, this disclosure provides ORMDL3 degrading agents for the treatment and / or prevention of HER2-positive diseases. In some embodiments, the ORMDL3 degrading agents provided herein are selected from FTY720.

[0008] In a fourth aspect, this disclosure provides a method for inhibiting the proliferation and / or metastasis of HER2-positive samples in vitro for non-therapeutic purposes, comprising administering an ORMDL3 degrading agent to the sample. In some embodiments, the ORMDL3 degrading agent provided herein is selected from FTY720.

[0009] In a fifth aspect, this disclosure provides the correlation between ORMDL3 and HER2. ORMDL3 can serve as a novel target for HER2-positive disease, particularly HER2-positive subtype breast cancer. In metastatic breast cancer, ORMDL3 is a novel and preferential interactor with HER2, directly binding to HER2 in the mammary ductal epithelium, and the interaction between ORMDL3 and HER2 is specific for HER2-positive subtype breast cancer. The absence of ORMDL3 significantly attenuates tumorigenesis and metastasis in HER2-positive subtype breast cancer, but has no effect on other subtypes (Luminal A, Luminal B, and triple-negative subtypes). In a sixth aspect, this disclosure provides the use of ORMDL3 for the detection of HER2-positive disease.

[0010] In a seventh aspect, this disclosure provides (1) a detection reagent comprising a substance for detecting ORMDL3 and / or (2) a substance for detecting ORMDL3, wherein the reagent or the substance for detecting ORMDL3 is used for the diagnosis or auxiliary diagnosis of HER2-positive diseases. In some embodiments, the substance for detecting ORMDL3 provided herein is selected from anti-ORMDL3 antibodies or antigen-binding fragments thereof, ORMDL3 gene-specific primers, and ORMDL3 gene-specific probes.

[0011] In an eighth aspect, this disclosure provides the use of substances for detecting ORMDL3 in the preparation of reagents or kits for diagnosing or assisting in the diagnosis of HER2-positive diseases. In some embodiments, the substances for detecting ORMDL3 provided herein are selected from anti-ORMDL3 antibodies or antigen-binding fragments thereof, ORMDL3 gene-specific primers, and ORMDL3 gene-specific probes.

[0012] In a ninth aspect, this disclosure provides a diagnostic reagent or kit for diagnosing or assisting in the diagnosis of HER2-positive diseases, comprising the substance provided herein for detecting ORMDL3. Attached Figure Description

[0013] Figure 1 is a scatter plot showing the correlation between HER2 and ORMDL3 expression.

[0014] Figure 2 shows the mRNA expression levels of ORMDL3 in the HER2-negative (HER2-) and HER2-positive (HER2+) groups.

[0015] Figure 3 shows the survival time of breast cancer patients of different subtypes based on the expression of ORMDL3.

[0016] Figures 4A-C show the immunoprecipitation results of the interaction between ORMDL3 and HER2 in metastatic lesions of HER2-positive breast cancer patients, spontaneous tumorigenesis in HER2-positive breast cancer mice, or HER2-positive breast cancer cells.

[0017] Figures 5A-B show the staining results of ORMDL3 and HER2 co-localization in metastatic lesions and HER2-positive breast cancer cells of patients with HER2-positive breast cancer.

[0018] Figures 6A-D show the evaluation results of the effect of ORMDL3 on the metastatic ability of breast cancer cells of different subtypes. The left side of each figure shows the results of staining and photographing metastatic cells (which have migrated from inside the ventricle to the outside), the upper right shows the results of Western blot analysis of ORMDL3 knockdown efficiency, and the lower right shows a bar chart of quantitative statistical analysis of cancer cell metastatic ability. ***P<0.001.

[0019] Figures 7A-D show the evaluation results of the in vivo metastatic effect of ORMDL3 on different subtypes of breast cancer. The left side of each figure shows the fluorescence intensity in mice, and the right side shows the Western blot results of gene knockout efficiency and the fluorescence quantitative statistical bar charts of tumor cell metastasis ability in mice. *** P<0.001.

[0020] Figures 8A-B show whole lung images, H&E staining, and lung metastasis counts in HER2-positive (MMTV-neu) and triple-negative (MMTV-PyVT) breast cancer mice and gene knockout hybrid mice. The left side of each figure shows a photograph of the mouse's whole lung tissue, the middle shows the H&E staining results of lung metastases after sectioning, and the right side is a statistical graph of the number of lung metastases. *** P<0.001.

[0021] Figure 9 shows the immunoblotting results of ORMDL3 protein levels after drug treatment.

[0022] Figure 10A shows the evaluation results of the metastatic ability of different subtypes of breast cancer cell lines after drug treatment.

[0023] Figure 10B is a quantitative statistical bar chart showing the metastatic ability of different breast cancer cell lines after drug treatment. *** P<0.001.

[0024] Figures 11A-D show the evaluation results of the metastatic ability of tumor cells in mice with different subtypes of breast cancer after treatment with solvents or drugs. The left side of each figure shows the fluorescence intensity photographs in mice, and the right side shows the fluorescence quantitative statistical bar charts of the metastatic ability of tumor cells in mice in the solvent group and the drug treatment group. *** P<0.001.

[0025] Figures 12A-B show the whole lung images, H&E staining, and number of lung metastases in HER2-positive (MMTV-neu) and triple-negative (MMTV-PyVT) breast cancer mice after treatment with solvents or drugs. The left side of each figure shows a photograph of the mouse's whole lung tissue, the middle shows the H&E staining results of lung metastases after sectioning, and the right side is a statistical graph of the number of lung metastases. *** P<0.001.

[0026] Figure 13 shows the effects of different drugs on the viability of HER2-positive breast cancer cells, with the horizontal axis representing the logarithmic concentration of the drug.

[0027] Figure 14 shows the effects of different drugs on the metastatic ability of HER2-positive breast cancer. The left side of the figure shows the results of stained photographs of metastatic cells (that have migrated from inside the ventricle to the outside), while the right side shows a bar chart of quantitative statistics on the metastatic ability of tumor cells after drug treatment. *** P<0.001.

[0028] Figures 15A-C show the effects of FTY720 on the cell viability of drug-resistant cell lines, with the horizontal axis representing the logarithmic concentration of the drug.

[0029] Figures 16A-B show the metastasis results of cancer cells after treatment with FTY720 on drug-resistant cell lines. The left side of each figure shows stained images of metastatic cells (thrash cells from inside the ventricle to the outside), and the right side is a bar chart quantitatively analyzing the metastatic ability of tumor cells after drug treatment. *** P<0.001.

[0030] Figures 17A-B are line graphs showing the change in tumor volume over time in mice with HER2-positive (MMTV-neu) and triple-negative (MMTV-PyVT) breast cancer, respectively, after treatment with FTY720. 17A corresponds to HER2-positive (MMTV-neu) breast cancer, and 17B corresponds to triple-negative (MMTV-PyVT) breast cancer. Detailed Implementation

[0031] The following specific embodiments illustrate the implementation of this application. Those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this specification.

[0032] Unless otherwise stated, all figures used in this specification and claims to represent content, concentration, proportion, mass, volume, time, temperature, thickness, technical effect, etc., should in any instance be understood to be modified by the terms “about” or “approximately”. Therefore, unless indicated to the contrary, the numerical parameters listed in the following specification and appended claims are approximate values. They can vary for those skilled in the art depending on the desired properties and effects sought through this disclosure, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or in a manner understood by those skilled in the art.

[0033] Although the numerical ranges and parameters described in this disclosure are approximate, the values ​​presented in the specific embodiments are provided as precisely as possible. However, any numerical value will inherently contain some errors, which are necessarily caused by the standard deviation found in its corresponding test measurements. Each numerical range given in this specification will include every narrower numerical range falling within that wider range, as if these narrower numerical ranges were explicitly stated herein.

[0034] Unless otherwise stated, the terminology used herein has the common meaning understood by one of ordinary skill in the art. It may vary for those skilled in the art depending on the desired properties and effects sought through this application, and each numerical parameter should be interpreted according to the number of significant figures and conventional rounding methods or as understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the experimental procedures in organic chemistry, medicinal chemistry, and biology described herein are well-known and commonly used in the art. Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. Where multiple definitions exist for terms used herein, the definitions in this section shall prevail unless otherwise stated.

[0035] definition

[0036] When used in this document, the expression “A and / or B” includes three cases: (1) A; (2) B; and (3) A and B. The expression “A, B and / or C” includes seven cases: (1) A; (2) B; (3) C; (4) A and B; (5) A and C; (6) B and C; and (7) A, B and C. The meanings of similar expressions can be deduced by analogy.

[0037] When used in this article, FTY720 is an immunomodulatory drug and a modulator of the sphingosine 1-phosphate (S1P) receptor, also known as fingolimod hydrochloride, CAS number 162359-56-0, and has the molecular formula [missing information].

[0038] In this article, "HER2" stands for Human Epidermal Growth Factor Receptor 2. "HER2 positive" refers to an amplification of the human epidermal growth factor receptor 2 (HER2) gene or overexpression of the protein. For example, HER2-positive diseases are those caused by amplification of the HER2 gene or overexpression of the HER2 protein. In some clinical cases, the diagnostic criteria for HER2-positive diseases are an immunohistochemical result showing HER2 with three plus signs (IHC 3+) or two plus signs (IHC 2+); and a positive FISH result.

[0039] When used in this article, “ORMDL3” stands for serum mucin-like protein 3, which is one of the three members of the ORMDL family.

[0040] When used in this article, "HER2-positive disease cell metastasis or migration," for example, in vivo metastasis or migration of HER2-positive cancer cells refers to the process by which HER2-positive cancer cells detach from the primary tumor, spread to other parts of the body via the bloodstream or lymphatic system, and form new tumors in distant organs or tissues. Cancer cell metastasis is a leading cause of cancer death because it makes cancer more difficult to treat. In vitro metastasis or migration of HER2-positive cancer cells refers to the spread of HER2-positive cancer cells from their originating site to other sites; this in vitro metastasis or migration can be detected by conventional methods, such as the Transwell assay.

[0041] In this article, "Herceptin" is marketed as "Herceptin," also known as Trastuzumab. Herceptin is an anti-HER2 monoclonal antibody that blocks the attachment of human epidermal growth factor to HER2 by attaching itself to HER2, thereby inhibiting cancer cell growth. Clinically, it can be used for breast cancer and gastric cancer with HER2 overexpression. "T-DXd" is marketed as "Enhertu," also known as DS-8201. T-DXd is an antibody-drug conjugate composed of an anti-HER2 antibody and a cytotoxic topoisomerase I inhibitor. This drug exhibits durable antitumor activity. "Lapatinib" is a small molecule tyrosine kinase inhibitor that inhibits the EGFR and HER2 signaling pathways and can be used to treat HER2-positive breast cancer.

[0042] When used in this article, "refractory disease" includes intractable disease, disease that has metastasized from its original location, recurrent disease, and disease that is resistant or intolerant to initial treatment or develops resistance or tolerance during treatment.

[0043] Methods and uses

[0044] This disclosure provides the use of ORMDL3 degrading agents in the preparation of medicaments for the treatment and / or prevention of HER2-positive diseases.

[0045] In some embodiments, the medicaments provided herein for treating and / or preventing HER2-positive disease may achieve one or more of the following (a)-(g):

[0046] (a) Preventing the occurrence of HER2-positive diseases;

[0047] (b) Prevent the development of HER2-positive disease;

[0048] (c) Inhibits the metastasis of HER2-positive disease cells;

[0049] (d) Inhibits the proliferation of HER2-positive disease cells;

[0050] (e) Treatment of drug-resistant HER2-positive diseases;

[0051] (f) Inhibits the proliferation of HER2-positive, drug-resistant disease cells;

[0052] (g) Inhibits the transfer of HER2-positive disease-resistant cells.

[0053] This disclosure provides an ORMDL3 degrading agent for the treatment and / or prevention of HER2-positive diseases.

[0054] In some embodiments, the ORMDL3 degrading agent provided herein can achieve any one or more of the following (a)-(g):

[0055] (a) Preventing the occurrence of HER2-positive diseases;

[0056] (b) Prevent the development of HER2-positive disease;

[0057] (c) Inhibits the metastasis of HER2-positive disease cells;

[0058] (d) Inhibits the proliferation of HER2-positive disease cells;

[0059] (e) Treatment of drug-resistant HER2-positive diseases;

[0060] (f) Inhibits the proliferation of HER2-positive, drug-resistant disease cells;

[0061] (g) Inhibits the transfer of HER2-positive disease-resistant cells.

[0062] This disclosure provides methods for treating and / or preventing HER2-positive disease, including administering an effective amount of an ORMDL3 degrader to a subject in need.

[0063] In some implementations, the methods provided herein for treating and / or preventing HER2-positive disease can achieve one or more of the following (a)-(g):

[0064] (a) Preventing the occurrence of HER2-positive diseases;

[0065] (b) Prevent the development of HER2-positive disease;

[0066] (c) Inhibits the metastasis of HER2-positive disease cells;

[0067] (d) Inhibits the proliferation of HER2-positive disease cells;

[0068] (e) Treatment of drug-resistant HER2-positive diseases;

[0069] (f) Inhibits the proliferation of HER2-positive, drug-resistant disease cells;

[0070] (g) Inhibits the transfer of HER2-positive disease-resistant cells.

[0071] In some embodiments, the HER2-positive disease or HER2-positive disease cell transfer described herein includes in vivo transfer and / or in vitro transfer.

[0072] In some embodiments, the drug resistance described herein includes resistance to drugs used to treat HER2-positive diseases. In some embodiments, the drug resistance described herein includes resistance to any one or more of Herceptin, T-DXd, and Lapatinib.

[0073] In some embodiments, the ORMDL3 degrading agent provided herein is selected from FTY720.

[0074] In some embodiments, the effective amount provided herein does not exceed 5 mg / kg, for example, not exceeding 4.5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg. In some preferred embodiments, the effective amount provided herein does not exceed 1 mg / kg, for example, 0.2 mg / kg, 0.1 mg / kg, or 0.05 mg / kg.

[0075] In some embodiments, the HER2-positive diseases described herein include, but are not limited to, HER2-positive breast cancer, ovarian cancer, gastric cancer, non-small cell lung cancer, colorectal cancer, endometrial cancer, cervical cancer, bile duct cancer, gastric and esophageal cancer, and salivary gland cancer. In some preferred embodiments, the HER2-positive disease described herein is HER2-positive breast cancer.

[0076] In some embodiments, the ORMDL3 degrading agents or drugs provided herein are administered by one or more of the following methods: subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal, nasal, pulmonary, ocular, and topical application.

[0077] In some embodiments, the medicaments provided in this disclosure for treating and / or preventing HER2-positive diseases comprise the ORMDL3 degrading agents provided herein and one or more pharmaceutically acceptable carriers or excipients.

[0078] The medicaments disclosed herein can be formulated in any manner known in the art, including but not limited to dosage forms such as tablets, capsules, small capsules, suspensions, powders, lyophilized preparations, suppositories, eye drops, skin patches, oral soluble preparations, sprays, aerosols, and other solid, semi-solid, or liquid systems.

[0079] The pharmaceutical products disclosed herein can be immediate-release and / or modified-release formulations, including delayed-release, sustained-release, pulsatile-release, controlled-release, targeted-release, and programmed-release formulations.

[0080] The term "pharmaceutically acceptable carrier" includes pharmaceutically acceptable materials, compositions, or carriers, such as liquid or solid fillers, diluents, excipients, solvents, or encapsulating materials, relating to carrying or delivering the peptides of this disclosure within or to a subject, enabling them to perform their intended function. Each salt or carrier must be "acceptable" in the sense of compatibility with other components of the formulation and not harmful to the subject. Some examples of materials that can be used as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth gum; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate. Esters and ethyl lauryl ester; agar; buffers, such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free raw water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solution; diluents; granulators; lubricants; binders; disintegrants; wetting agents; emulsifiers; colorants; release agents; coating agents; sweeteners; flavoring agents; preservatives; antioxidants; plasticizers; gelling agents; thickeners; hardeners; setting agents; suspending agents; surfactants; humectants; carriers; stabilizers; and other non-toxic, compatible substances used in pharmaceutical preparations, or any combination thereof.

[0081] When used in this document, the term “subject” includes animals such as vertebrates, preferably mammals such as dogs, cats, pigs, cattle, sheep, horses, rodents (e.g., mice, rats, or guinea pigs) or primates (e.g., gorillas, chimpanzees, and humans).

[0082] When used in this document, the term “treatment” means to alleviate or improve a disease or disorder (i.e., to slow or stop the development of the disease or at least one clinical symptom); or to alleviate or improve at least one physical parameter or biomarker associated with the disease or disorder.

[0083] In this document, "effective amount" means an amount administered by any of the methods described above or by any other means known in the art that is sufficient to elicit a desired therapeutic, preventative, or inhibitory effect, resulting in benefit or achieving a certain effect compared to a corresponding subject who did not receive that amount. This amount is sufficiently low within the range of reasonable medical judgment to avoid serious side effects. The effective amount of the polypeptide derivative, pharmaceutical composition, or composition described herein will vary depending on factors such as the choice of polypeptide derivative, pharmaceutical composition, or composition; the route of administration; the severity of the disease being treated; the age, body type, weight, and physical condition of the patient being treated; the patient's medical history; the duration of treatment; the nature of concurrent treatments; and the desired therapeutic effect, but can still be determined by those skilled in the art in a conventional manner.

[0084] This disclosure provides a method for inhibiting the proliferation and / or metastasis of HER2-positive samples in vitro for non-therapeutic purposes, including administering an ORMDL3 degrading agent to the sample.

[0085] In some embodiments, the ORMDL3 degrading agent provided herein is selected from FTY720.

[0086] In some embodiments, the HER2-positive samples provided herein include, but are not limited to, HER2-positive cells or tissues. In some preferred embodiments, the HER2-positive samples provided herein are selected from HER2-positive subtype breast cancer cells and HER2-positive subtype drug-resistant breast cancer cells. In some preferred embodiments, the drug resistance provided herein includes resistance to drugs used to treat HER2-positive diseases. In some preferred embodiments, the drug resistance provided herein includes resistance to any one or more of Herceptin, T-DXd, and Lapatinib.

[0087] This disclosure provides the use of ORMDL3 for the detection of HER2-positive diseases.

[0088] Detection substances, detection reagents, and kits

[0089] This disclosure provides (1) a detection reagent comprising a substance for detecting ORMDL3 and / or (2) a substance for detecting ORMDL3, wherein the detection reagent or the substance for detecting ORMDL3 is used for the diagnosis or auxiliary diagnosis of HER2-positive diseases. In some embodiments, the substance for detecting ORMDL3 is selected from anti-ORMDL3 antibodies or their antigen-binding fragments, ORMDL3 gene-specific primers, and ORMDL3 gene-specific probes. In some embodiments, the detection reagents provided herein, in addition to comprising the substance for detecting ORMDL3 provided herein, further comprise other conventional detection reagents.

[0090] This disclosure provides the use of substances for detecting ORMDL3 in the preparation of reagents or kits for diagnosing or assisting in the diagnosis of HER2-positive diseases. In some embodiments, the substances for detecting ORMDL3 provided herein are selected from anti-ORMDL3 antibodies or antigen-binding fragments thereof, ORMDL3 gene-specific primers, and ORMDL3 gene-specific probes.

[0091] This disclosure provides diagnostic reagents or kits for diagnosing or assisting in the diagnosis of HER2-positive diseases, comprising substances for detecting ORMDL3. In some embodiments, the substances provided herein for detecting ORMDL3 are selected from anti-ORMDL3 antibodies or their antigen-binding fragments, ORMDL3 gene-specific primers, and ORMDL3 gene-specific probes. In some embodiments, the diagnostic kits provided herein comprise the substances provided herein for detecting ORMDL3 and instructions for use.

[0092] In some implementations, “auxiliary diagnosis” as described herein means that, in addition to detecting biomarkers (such as ORMDL3), other physiological parameters (such as sample biopsy data, changes in body temperature, changes in weight, pain index, blood test data such as white blood cell count, and other biomarker detections) can be used in combination for diagnosis.

[0093] The various embodiments and preferences disclosed above can be combined with each other (as long as they are not inherently contradictory), and all embodiments formed by such combinations are considered as part of the disclosure of this application.

[0094] Example

[0095] The following description, in conjunction with the accompanying drawings, illustrates exemplary embodiments of this application, including various details of these embodiments to aid understanding. It should be understood that these are merely exemplary and are in no way intended to limit the scope of protection of this application. The scope of protection of this application is defined only by the claims. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

[0096] Example 1. The relationship between ORMDL3 and HER2 and the specificity of ORMDL3 for HER2-positive subtype breast cancer.

[0097] (1) Bioinformatics database analysis

[0098] a. Correlation between HER2 and ORMDL3 mRNA levels

[0099] Expression data of HER2 and ORMDL3 in breast cancer tumor samples (n=1075) were analyzed in the Cancer Genome Atlas (TCGA) database. Statistical analysis was performed on the fragments per thousand bases of transcription per million mapped reads (FPKM) provided by the TCGA database, resulting in a scatter plot (Figure 1) of HER2 and ORMDL3 expression levels. As shown in Figure 1, it can be seen that the mRNA level of ORMDL3 is positively correlated with that of HER2 in breast cancer tumor samples.

[0100] b. mRNA levels of ORMDL3 in HER2- and HER2+ breast cancers, respectively

[0101] Patient samples from the Cancer Genome Atlas Project-RNA (TCGA-RNA), TCGA, and Metabrilliant Breast Cancer Gene Expression Profiles (METABRIC) databases were analyzed using the Breast Cancer Integrated Platform website (http: / / www.omicsnet.org / bcancer / database). All breast cancer samples were divided into HER2-negative (HER2-) and HER2-positive (HER2+) groups based on HER2 expression. The mRNA expression of ORMDL3 in both groups was statistically analyzed, and Figure 2 was created based on the expression levels. As shown in Figure 2, ORMDL3 is highly expressed in the HER2-positive (HER2+) group in the TCGA-RNA, TCGA, and METABRIC databases, further demonstrating the correlation between ORMDL3 and HER2 expression.

[0102] c. The relationship between ORMDL3 and the prognosis of different subtypes of breast cancer

[0103] Patient samples from the TCGA database were analyzed using the website http: / / www.omicsnet.org / bcancer / database. All breast cancer samples were divided into four groups according to clinical classification criteria (molecular subtypes): HER2-positive, Luminal A, Luminal B, and triple-negative. Survival times for each subtype of breast cancer were calculated based on ORMDL3 expression, and Figure 3 shows the results. As shown in Figure 3, ORMDL3 specifically affects the survival of patients with HER2-positive breast cancer, but has no effect on the other three subtypes. Furthermore, among HER2-positive breast cancer patients, those in the high ORMDL3 expression group had a lower survival rate compared to those in the low ORMDL3 expression group.

[0104] (2) Immunoprecipitation detection of the intrinsic interaction between ORMDL3 and HER2

[0105] a. Total protein extraction. Weigh the appropriate mass of lung metastasis samples from HER2-positive breast cancer patients (Shanghai Xinchao Biotechnology Co., Ltd.) or metastasis samples from MMTV-neu spontaneous tumorigenetic mice (Jackson Laboratory, #002376) (HER2-positive subtyped breast cancer samples), and add radioimmunoprecipitation lysis buffer (RIPA) (Beyotime Biotechnology Co., Ltd., #P0013B) at a ratio of 10 μL lysis buffer per milligram of tissue for homogenization; lyse human breast cancer SK-BR-3 cells (HER2-positive breast cancer cell line, ATCC cell bank, #HTB-30), approximately 1 × 10 7 Add 500 μL of RIPA lysis buffer to each cell. After centrifugation, collect the supernatant, determine the protein concentration, and set aside for later use; b. Take 10% of the protein lysis buffer as the total protein sample (WCL group), and the remaining 90% as the immunoprecipitation (IP) sample. Bind the IP sample to the antibody. IgG antibody (CST, #2729) was added to the control group, and HER2 antibody (CST, #2165) was added to the experimental group, and incubated overnight at 4°C; c. Balance protein A / G magnetic beads (Thermo, #88802), add 200 μL of lysis buffer, and incubate at 4°C for 10 minutes each time, discarding the supernatant, repeating three times. Add 20 μL of magnetic beads to each sample and continue to vortex at 4°C for 4-6 hours; d. Centrifuge the above samples at 4°C for 90 seconds at 500 g, then transfer the supernatant to a new Eppendorf tube (for IP efficiency testing) and store at -30°C for later use; resuspend the remaining magnetic beads with RIPA lysis buffer, vortex at room temperature for 10 minutes, then centrifuge at 4°C for 90 seconds at 500 g, discard the supernatant, and repeat 3 times; e. Add 50 μL of loading buffer (Dingguo Changsheng Biotechnology Co., Ltd., #WB-0081) to each sample, vortex at room temperature for 10 minutes, centrifuge at 1000 g for 90 seconds, then transfer the supernatant to a new Eppendorf tube and store at -30°C for later use; f. Detect the binding of HER2 and ORMDL3 by Western blot.

[0106] The Western blot results are shown in Figures 4A-C. As shown in Figures 4A-C, the imaging results show that the HER2 antibody group can detect the protein band of ORMDL3 in human breast cancer metastatic lesion samples (Figure 4A), spontaneous tumorigenic mouse metastatic lesion samples (Figure 4B), and SK-BR-3 cells (Figure 4C), proving that HER2 can have an endogenous interaction with ORMDL3.

[0107] (3) Immunofluorescence detection of the relationship between ORMDL3 and HER2 localization

[0108] a. Dehydrate and embed adjacent breast tissue from HER2-positive breast cancer patients (Shanghai Xinchao Biotechnology Co., Ltd.) to obtain frozen sections; seed SK-BR-3 cells onto slides in 24-well plates for subsequent slide climbing; b. Preheat 4% paraformaldehyde to 37°C, add 200 μL of paraformaldehyde to each well, fix for 15 minutes, and wash 3 times with phosphate-buffered saline (PBS); c. Block at room temperature for 1 hour (blocking solution is 3% bovine serum albumin (BSA), prepared using 1×PBS); d. Prepare antibody solutions by diluting HER2 antibody (CST Biotechnology Co., Ltd., #2165) and ORMDL3 antibody (abcam, #ab211522) at a ratio of 1:100 using 3% sheep serum, and mix 1:1 after dilution. Remove the slides from the 24-well plate and add 40 μL of diluted antibody solution to each slide's tissue or cell tissue. Place the slides in a humidified chamber and incubate overnight at 4°C. Allow the humidified chamber to stand at room temperature for 30 minutes and wash three times with PBS. e. Prepare a 1:100 solution of fluorescent secondary antibody (Thermo, #A11029) using blocking buffer, add it to the slides, place them in a humidified chamber, and incubate at room temperature for 1 hour. Wash three times with PBS. Mount the slides using mounting medium containing DAPI dye.

[0109] The staining results are shown in Figures 5A-B. The staining results show that HER2 and ORMDL3 are co-localized on the cell membrane in both patient tissues and cells.

[0110] Example 2. Knockout of ORMDL3 weakens in vitro migration of HER2-positive breast cancer subtypes.

[0111] (1) HER2-positive breast cancer cells SK-BR-3 (#HTB-30), HCC1954 (#CRL-2338), 1028 (cell number #301, primary cell line), Luminal A breast cancer cells MCF-7 (#HTB-22), T-47D (#HTB-133), ZR-75-1 (#CRL-1500), Luminal B breast cancer cells BT-474 (#HTB-20), and triple-negative breast cancer cells MDA-MB-231 (#HTB-26) and BT-549 (#HTB-122) were digested and counted at 1.5 × 10⁻⁶ cells per well. 5 The cells were seeded in 12-well plates and allowed to adhere to the plate (all cells were purchased from ATCC Cell Bank (American Type Culture Collection)).

[0112] (2) Gene interference (interference sequence purchased from Guangzhou Ruibo Biotechnology Co., Ltd.). The control group (siNC, using 5'-UUCUCCGAACGUGUCACGUAA-3' (SEQ ID NO:1)) and the interference group (each interference group used a different siRNA sequence, named 1# (5'-GGUUGGAGUAAGUGUUGUA-3' (SEQ ID NO:2)), 2# (5'-CAGGCCUAAAAACCUUAGU-3' (SEQ ID NO:3)), and 3# (5'-AGUGGUCGAAGAUGUGAUU-3' (SEQ ID NO:4)), for a total of three groups) were set up. The interference system was prepared using opti-mem (serum-reduced medium): a. Discard the original culture medium of the cells in culture step (1) and add 0.4 mL of complete medium containing 10% serum; b. Add 2 μL of iMAX (Thermo, #13778150) to 50 μL opti-mem medium, mix well to obtain solution A, and let stand for 5 minutes; c. Add 50 μL of iMAX (Thermo, #13778150) to 50 μL opti-mem medium, mix well to obtain solution A, and let stand for 5 minutes; Add 60 ng siRNA (siRNA selected from control group, 1#, 2#, or 3#) to opti-mem medium, mix well to obtain solution B, and let stand for 5 minutes; d. Mix solution A with solution B to obtain solution C, and let stand for 15 minutes; e. Slowly add solution C to the cells in step (1), and after 6 hours, replace the culture medium containing the interference system with complete culture medium containing 10% serum;

[0113] (3) A second intervention is performed 12 hours later (repeating the previous step);

[0114] (4) After 72 hours, collect and count the cells, then resuspend them in serum-free medium to obtain a cell suspension (2.5 × 10⁻⁶). 5 (cells / mL);

[0115] (5) Take out the 24-well plate with the permeable cell culture chamber (Transwell chamber), remove the chamber, add 0.6 mL of complete culture medium containing 10% serum to the well plate; place the chamber into the well plate, add 0.4 mL of cell suspension to the chamber, and incubate for 24 hours (the chamber is placed in the culture plate, the chamber is called the upper chamber, and the culture plate is called the lower chamber. The upper and lower culture media are separated by a polycarbonate membrane. Serum-free culture medium is added to the upper chamber, and serum-containing culture medium is added to the lower chamber. The cells to be studied are seeded in the upper chamber. Because the polycarbonate membrane is permeable, the nutrients in the lower culture medium can affect cell movement in the upper chamber, etc.).

[0116] (6) Remove the chamber, use a vacuum pump to remove the liquid in the well plate and chamber, and gently wipe away the cells that have not penetrated the filter membrane in the chamber with a cotton swab; fix in 100% methanol for 5 minutes, and wash with distilled water; stain with 0.1% crystal violet for 15 minutes, wash with distilled water, and observe the degree of staining; wipe off excess liquid with a cotton swab, air dry, and take a picture;

[0117] (7) Count the cells and obtain the cell migration inhibition rate using the following formula:

[0118] The results are shown in Figures 6A-D. As can be seen from Figures 6A-D, knocking down ORMDL3 significantly inhibited the in vitro migration of HER2-positive breast cancer, while having no effect on the migration ability of other breast cancer subtypes, indicating that ORMDL3 can regulate the metastasis of HER2-positive breast cancer with subtype specificity.

[0119] Example 3. ORMDL3 specifically regulates in vivo spread of HER2-positive subtype breast cancer.

[0120] (1) Cell lines of different subtypes were selected (HER2-positive breast cancer cells MDA-MB-453, Luminal A breast cancer cells T-47D, Luminal B breast cancer cells BT-474, or triple-negative breast cancer cells MDA-MB-231); sgRNA sequences were designed using CRISPR / Cas9 technology, and ORMDL3 was knocked out in each subtype cell line (the knockout sequence is 5'-GUACAGCACGAUGGGUGUGA-3' (SEQ ID NO: 5')). NO:5)) Construct stable gene knockout breast cancer cell lines; Construct different subtypes of breast cancer cell lines carrying luciferase reporter genes (using calcium transfer kit (Zhongke Maichen Company, #CTK001) and packaging plasmids VSV-G (Addgene, #12259) and PAX2 (Addgene, #12260) for lentiviral packaging, and infecting the above subtypes of cell lines (including wild-type and gene knockout cells) with the virus respectively, and then screening the virus-infected cells for resistance, leaving cells containing luciferase expression);

[0121] (2) In the mouse experiments of each subtype of breast cancer, immunodeficient NOG mice (Vital River Animal Company, #408) were randomly divided into two groups (n=3), which were set as control group and gene knockout group respectively;

[0122] (3) The constructed breast cancer cells of various subtypes expressing Luciferase were digested and counted, and resuspended in physiological saline to a concentration of 1×10⁻⁶ cells / mL. 7Each mouse was injected with 200 μL of cell suspension via the tail vein (wild-type breast cancer cells were injected via the tail vein in the control group, and gene knockout breast cancer cells were injected via the tail vein in the gene knockout group).

[0123] (4) Prepare luciferase substrate solution with physiological saline (Perkin Elmer, #122799) to a final concentration of 30 mg / mL. Inject 100 μL of substrate solution into each mouse intraperitoneally 5 minutes before the shooting.

[0124] (5) Half an hour after the tail vein injection of cells, in vivo imaging was performed to record the initial enrichment of tumor cells in mice.

[0125] (6) Subsequently, in vivo imaging was performed weekly to observe fluorescence luminescence, and the fluorescence intensity of the mouse metastatic lesions was statistically analyzed and plotted. The results are shown in Figures 7A-D.

[0126] In vivo fluorescence results showed that tumor cells spread to different locations in all control mice. Fluorescence values ​​indicated that, compared with the control group, knockout of ORMDL3 significantly inhibited the metastasis of HER2-positive breast cancer cells in mice, while having no effect on the metastasis of the other three subtypes (Luminal A, Luminal B, and triple-negative breast cancer cells). This suggests that ORMDL3 affects the metastasis of HER2-positive breast cancer cells, and knockout of ORMDL3 significantly inhibited the spread of HER2-positive breast cancer cells in mice, demonstrating subtype specificity.

[0127] Example 4. ORMDL3 specifically regulates in vivo metastasis of HER2-positive subtype breast cancer.

[0128] Hematoxylin and eosin (H&E) staining is used to stain the cell nucleus blue-purple with hematoxylin and the cytoplasm and extracellular matrix red or pink with eosin to visualize cell morphology or tissue structure. In this example, MMTV-neu (HER2-positive breast cancer spontaneous tumorigenesis mouse model) and MMTV-PyVT (triple-negative breast cancer spontaneous tumorigenesis mouse model) were selected to observe tumor metastases in mouse lung tissue using H&E staining. The control group mice used in this experiment were non-ORMDL3 gene knockout MMTV-neu or MMTV-PyVT mice.

[0129] Specific steps (1) Animal model construction: Use CRISPR / Cas9 technology to construct ORMDL3 gene knockout MMTV-neu or MMTV-PyVT mice, cross them with spontaneous tumor-forming mice and identify the genotype. Tissue samples are taken 6 months after obtaining the model mice.

[0130] (2) Tissue embedding and sectioning: After photographing the lung tissue of the control group or gene knockout group mice, the tissue was immersed in 4% paraformaldehyde and placed at room temperature for 24 hours. Then, it was dehydrated using a tissue dehydrator. After dehydration, it was placed in a pathological tissue embedding machine for embedding. Note that the cut surface to be obtained should be placed at the bottom of the embedding box. After filling with paraffin, it was placed on a cold stage for shaping. Paraffin sections were made using a microtome with a thickness of 3μm. The sections were placed on glass slides and dried.

[0131] (3) Baking the slides: Place the glass slides in an oven at 65°C for 1-3 hours;

[0132] (4) Dewaxing: Quickly immerse the baked glass slide in xylene I solution for 15 minutes, followed by immersion in xylene II solution for 15 minutes;

[0133] (5) Dexylene removal: The slides treated in step (4) are treated with 100% ethanol for 2 minutes, 100% ethanol for 2 minutes, 95% ethanol for 2 minutes, 95% ethanol for 2 minutes, 90% ethanol for 2 minutes, and 80% ethanol for 2 minutes; then washed twice with distilled water for 2 minutes each time.

[0134] (6) Hematoxylin staining: After hematoxylin staining, let stand at room temperature for 5 minutes, rinse 3 times with tap water, differentiate with 1% hydrochloric acid-80% ethanol solution, rinse 3 times with warm water to return to blue.

[0135] (7) Eosin staining: After eosin staining, let stand at room temperature for 1-3 minutes;

[0136] (8) Dehydration: 80% ethanol for 2 minutes, 90% ethanol for 2 minutes, 95% ethanol for 2 minutes, 95% ethanol for 2 minutes, 100% ethanol for 2 minutes, 100% ethanol for 2 minutes;

[0137] (9) Transparency: Immerse the glass slide processed in step (8) in xylene I solution for 2 minutes and xylene II solution for 2 minutes;

[0138] (10) Seal the slide. After drying the slide processed in step (9), scan or photograph it.

[0139] The results are shown in Figures 8A-B. The photographs of mouse lung tissue show that the MMTV-neu control group (non-gene knockout group) had more severe lung metastases. Knocking out ORMDL3 significantly improved lung metastasis, while in the MMTV-PyVT model, knocking out ORMDL3 showed almost no change in lung metastasis. H&E staining further indicated that in the MMTV-neu mouse model, the control group mice had more tumor metastases in their lungs, and knocking out ORMDL3 significantly reduced the number and area of ​​metastatic lesions. However, in the triple-negative MMTV-PyVT mouse model, compared to the control group, the number of lung metastases in the ORMDL3 knockout group was not significantly improved. This suggests that ORMDL3 affects the metastasis of HER2-positive breast cancer in mice, and knocking out ORMDL3 significantly inhibits the metastasis of HER2-positive breast cancer, exhibiting subtype specificity.

[0140] Example 5. FTY720 degradation of ORMDL3 protein levels

[0141] (1) HER2-positive breast cancer cells SK-BR-3 were digested with trypsin and counted, at a ratio of 2.5 × 10⁶ cells per well. 5 One cell was seeded in a 12-well plate and allowed to adhere to the plate.

[0142] (2) Weigh FTY720 and dissolve it in DMSO to prepare a 100 mM stock solution. Before the experiment, dilute it 1000 times with serum-free medium to obtain a 100 μM working solution (prepare fresh for use). Set up a blank control group (solvent group, serum-free medium containing 0.01‰ DMSO, FTY720 concentration of 0) and a drug treatment group (FTY720 concentrations of 0.1 μM, 0.5 μM and 1 μM), with 1 mL per well; treat the above HER2 positive breast cancer cells SK-BR-3 with serum-free medium containing 0.01‰ DMSO or different concentrations of FTY720;

[0143] (3) After 24 hours, discard the supernatant culture medium, wash three times with physiological saline, digest and count the cells, and dilute the cells with serum-free culture medium to obtain a cell suspension (2.5 × 10⁻⁶). 5 (cells / mL);

[0144] (4) Collect 0.6 mL of cell suspension, centrifuge at 4°C, resuspend the precipitate with physiological saline, and centrifuge again to obtain cell precipitate;

[0145] (5) Add 45 μL of RIPA cell lysis buffer to the cell pellet, vortex at 4°C for 30 minutes, and use Bradford (Coomassie Brilliant Blue staining method) to prepare a standard curve and determine the protein concentration.

[0146] (6) Add loading buffer (Dingguo Changsheng Biotechnology Co., Ltd., #WB-0091) according to the final supernatant volume, mix well, heat in a 100℃ metal bath for 20 minutes, take it out and centrifuge at 12000rpm for 10 seconds in a room temperature centrifuge, and set aside for use.

[0147] (7) Western Blot: Prepare a 10% SDS-PAGE protein gel, with a constant voltage of 80V for the upper stacking gel. After the markers are separated, adjust the voltage to 120V. Prepare a "sandwich" transfer apparatus in the following order: black side of plate - sponge - filter paper - gel - activated PVDF membrane - filter paper - sponge - white side of plate. Transfer conditions: constant current 250mA, 2 hours; 5% milk at room temperature for 1 hour or at 4℃ overnight.

[0148] (8) Antibody incubation:

[0149] Primary antibody labeling: Wash the blocked milk with TBST, add the prepared ORMDL3 antibody (diluted at 1:1000) or tubulin antibody (CST, #2128; diluted at 1:5000) and incubate overnight at 4°C on a shaker;

[0150] Secondary antibody labeling: Recover the primary antibody, wash the PVDF membrane with TBST, dilute the corresponding species of HRP (horseradish peroxidase) labeling secondary antibody (CST, #7074) at a ratio of 1:5000, and incubate on a shaker at room temperature for 1 hour;

[0151] (9) Development: Mix the developing solution A and solution B (ECL chemiluminescence detection kit, Thermo, #34577) in a 1:1 ratio and then develop.

[0152] As shown in Figure 9, FTY720 significantly downregulated the protein level of ORMDL3 in a concentration-dependent manner.

[0153] Example 6. Specificity of ORMDL3 degrading agent on the migration of HER2-positive breast cancer cells

[0154] (1) HER2-positive breast cancer cells SK-BR-3, Luminal A-type breast cancer cells MCF-7, Luminal B-type breast cancer cells BT-474, and triple-negative breast cancer cells MDA-MB-231 were digested with trypsin and counted, at a ratio of 2.5 × 10⁻⁶ cells per well. 5 One cell was seeded in a 12-well plate and allowed to adhere to the plate.

[0155] (2) Weigh FTY720 and dissolve it in DMSO to prepare a 100 mM stock solution. Before the experiment, dilute it 1000 times with serum-free medium to obtain a 100 μM working solution (prepare fresh for use). Set up a blank control group (solvent group) (serum-free medium containing 0.01‰ DMSO) and a drug treatment group (FTY720 drug concentration of 1 μM), with 1 mL per well; treat the above-mentioned breast cancer cells of different subtypes with serum-free medium containing 0.01‰ DMSO or FTY720 solution;

[0156] (3) After 24 hours, discard the supernatant culture medium, wash three times with physiological saline, digest and count the cells, and dilute the different subtypes of breast cancer cells with serum-free culture medium to obtain a cell suspension (2.5 × 10⁻⁶). 5 (cells / mL);

[0157] (4) Take out the 24-well plate with the Transwell chamber, remove the chamber, and add 0.6 mL of complete culture medium containing 10% serum into the well plate;

[0158] (5) Place the chamber into the well plate, slowly add 0.4 mL of cell suspension into the chamber, and incubate in an incubator at 37°C with 5% CO2 for 24 hours;

[0159] (6) Remove the chamber, use a vacuum pump to remove the liquid in the well plate and chamber, and gently wipe away the cells that have not penetrated the filter membrane in the chamber with a cotton swab; fix in 100% methanol for 5 minutes, and wash with distilled water; stain with 0.1% crystal violet for 15 minutes, wash with distilled water, and observe the degree of staining; wipe off excess liquid with a cotton swab, air dry, and take a picture;

[0160] (7) Count the cells and obtain the cell migration inhibition rate using the following formula:

[0161] The results, shown in Figures 10A and 10B, indicate that FTY720 significantly inhibited the in vitro metastasis of HER2-positive breast cancer cells, and the relative migration rate of HER2-positive breast cancer cells in the FTY720 group was lower than that in the solvent group. FTY720 had no significant effect on the metastasis of other breast cancer cell types, and the relative migration rates of other breast cancer cells in the FTY720 group were the same as those in the solvent group, maintaining a relatively high level. This demonstrates that FTY720 significantly inhibited the metastasis of HER2-positive breast cancer cells but not the metastasis of other breast cancer cell types, indicating that FTY720 exhibits type specificity.

[0162] Example 7. ORMDL3 degrader specifically affects the in vivo spread of HER2-positive subtype breast cancer.

[0163] (1) Construct breast cancer cell lines MDA-MB-453, Luminal A breast cancer cells MCF-7, Luminal B breast cancer cells BT-474, and triple-negative breast cancer MDA-MB-231 carrying the Luciferase reporter gene (using a calcium transfer kit (Zhongke Maichen Company, #CTK001) and packaging plasmids VSV-G (Addgene, #12259) and PAX2 (Addgene, #12260) for lentiviral packaging, and then screening the virus-infected cells for resistance, leaving cells containing Luciferase expression);

[0164] (2) Immunodeficient NOG mice were randomly divided into two groups (n=3), namely a control group (0.9% sodium chloride solution, solvent group) and an FTY720 administration group (dose of 0.05mg / kg);

[0165] (3) The constructed cells were digested and counted, and resuspended in physiological saline to a concentration of 1 × 10⁻⁶ cells per milliliter. 7 200 μL of cell suspension was injected into the tail vein of mice.

[0166] (4) Prepare FTY720 stock solution (concentration of 500mM), weigh the mice, and administer FTY720 or the same volume of physiological saline orally once a day at a dose of 0.05mg / kg. Due to the different metastatic abilities of breast cancer cells of different subtypes, the tumors in the control group mice were metastatic and stably enriched after treatment (HER2 positive mice were treated for 45 days, Luminal A mice for 35 days, Luminal B mice for 21 days, and triple negative mice for 28 days).

[0167] (5) Prepare luciferase substrate solution with physiological saline (Perkin Elmer, #122799) to a final concentration of 30 mg / mL. Inject 100 μL of substrate solution into each mouse intraperitoneally 5 minutes before each shooting.

[0168] (6) Half an hour after the tail vein injection of cells, in vivo imaging was performed to record the initial enrichment of tumor cells in mice; in vivo imaging was performed once a week during the drug treatment period to observe fluorescence luminescence, and the fluorescence intensity of mouse metastatic lesions was statistically analyzed and plotted in Figures 11A-D.

[0169] The in vivo fluorescence results are shown in Figures 11A-D. Tumor cell proliferation occurred at different locations in all control mice. Fluorescence values ​​indicated that, compared to the control group, FTY720 treatment significantly improved the metastasis of HER2-positive breast cancer cells in mice, with significantly lower fluorescence values. However, the metastasis of the other three types of breast cancer cells remained unchanged, with fluorescence values ​​similar to the solvent group, and all maintaining high fluorescence values. This suggests that FTY720 significantly inhibited the proliferation of HER2-positive breast cancer cells in mice but could not significantly inhibit the proliferation of other types of breast cancer cells. This inhibitory effect of FTY720 is cancer type-specific.

[0170] Example 8. ORMDL3 degrader specifically inhibits in vivo metastasis of HER2-positive subtype breast cancer.

[0171] (1) Animal model construction: Using a spontaneous tumorigenesis mouse model of breast cancer, MMTV-neu (HER2-positive breast cancer spontaneous tumorigenesis mouse model) and MMTV-PyVT (triple-negative breast cancer spontaneous tumorigenesis mouse model) were randomly divided into two groups: a solvent control group (0.9% sodium chloride solution) and a drug treatment group (FTY720 0.2 mg / kg), with 8 mice in each group; when the mouse tumors grew to 0.4 cm 3 Mice were weighed, and FTY720 stock solution (500mM concentration) or an equal volume of physiological saline was administered orally once daily, or orally with physiological saline once daily, until the tumor volume in the control group was approximately 1.5 cm. 3 draw materials;

[0172] (2) Tissue embedding and sectioning: After photographing the mouse lung tissue, it was immersed in 4% paraformaldehyde and left at room temperature for 24 hours. Then it was dehydrated using a tissue dehydrator. After dehydration, it was placed in a pathological tissue embedding machine for embedding. The cut surface to be obtained was placed at the bottom of the embedding box, filled with paraffin, and then placed on a cold stage for shaping. Paraffin sections were made using a microtome with a thickness of 3μm, placed on glass slides, and dried.

[0173] (3) Baking the slides: Place the glass slides in a 65℃ oven for 1-3 hours;

[0174] (4) Dewaxing: Quickly immerse the baked glass slide in xylene I solution for 15 minutes, followed by immersion in xylene II solution for 15 minutes;

[0175] (5) Dexylene removal: Place the glass slide after step (4) in 100% ethanol for 2 minutes, 100% ethanol for 2 minutes, 95% ethanol for 2 minutes, 95% ethanol for 2 minutes, 90% ethanol for 2 minutes, and 80% ethanol for 2 minutes; wash twice with distilled water for 2 minutes each time.

[0176] (6) Hematoxylin staining: After staining, let stand at room temperature for 5 minutes, rinse 3 times with tap water, differentiate with 1% hydrochloric acid-80% ethanol solution, rinse 3 times with warm water to return to blue.

[0177] (7) Eosin staining: After staining, let stand at room temperature for 1-3 minutes;

[0178] (8) Dehydration: 80% ethanol for 2 minutes, 90% ethanol for 2 minutes, 95% ethanol for 2 minutes, 95% ethanol for 2 minutes, 100% ethanol for 2 minutes, 100% ethanol for 2 minutes;

[0179] (9) Transparency: Immerse the glass slide processed in step (8) in xylene I solution for 2 minutes and xylene II solution for 2 minutes;

[0180] (10) Seal the slide. After drying the slide processed in step (9), scan or photograph it.

[0181] The results are shown in Figures 12A-B. The photographs of mouse lung tissue reveal that lung metastases were more severe in the MMTV-neu control group, and FTY720 treatment significantly inhibited lung metastasis, while no significant improvement was observed in the MMTV-PyVT group. H&E staining further indicated that in the MMTV-neu mouse model, the control group mice developed more tumor metastases in their lungs, and treatment significantly reduced the number and area of ​​metastatic lesions in a dose-dependent manner. However, in the triple-negative MMTV-PyVT mouse model, the number of lung metastases in the treatment group was not significantly improved compared to the control group. This demonstrates that FTY720 significantly inhibits the metastasis of HER2-positive breast cancer and exhibits subtype specificity.

[0182] Example 9. Effects of first- and second-line clinical medications on the proliferation of HER2-positive subtype breast cancer.

[0183] (1) HER2-positive breast cancer HCC1954 cells were digested and counted, and 5 × 10⁶ cells were collected per well. 3 One cell was seeded in a 96-well plate, with three replicates per group, and allowed to adhere to the plate wall.

[0184] (2) Weigh different drugs (FTY720, Herceptin, T-DXd, Lapatinib) and dissolve them in DMSO to prepare a stock solution with a concentration of 100mM. A drug concentration gradient was set up, and the drug stock solutions were serially diluted using serum-free medium: FTY720 drug final concentrations were 0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, and 100 μM, with a volume of 100 μL per well; Herceptin drug final concentrations were 0, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, and 20 μM, with a volume of 100 μL per well; T-DXd drug final concentrations were 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2.5, and 5 μM, with a volume of 100 μL per well; Lapatinib drug final concentrations were 0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, and 100 μM, with a volume of 100 μL per well.

[0185] (3) After 24 hours of drug intervention on breast cancer cells, 20 μL of MTS solution (Promega, #G3582) was added directly to each well and incubated in a 5% CO2, 37°C incubator for 2 hours.

[0186] (4) Measure the absorbance at 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the relative cell viability and half-maximal inhibitory concentration (IC50). 50 .

[0187] The results are shown in Figure 13. The IC50 of FTY720 after 24 hours of intervention in HCC1954 cells was measured. 50 The value was 9.89 μM. The IC50 values ​​were compared with those of first- and second-line clinical medications, and the values ​​of Herceptin, T-DXd, and Lapatinib were determined. 50 The values ​​were 6.59 μM, 0.99 μM, and 13.10 μM, respectively. In summary, the effect of FTY720 on the proliferation of HER2-positive breast cancer cells is almost comparable to that of first- and second-line clinical treatments.

[0188] Example 10. The effect of first- and second-line clinical medications on the metastasis of HER2-positive subtype breast cancer.

[0189] (1) HER2-positive breast cancer cells HCC1954 (#CRL-2338) were digested and counted, at a density of 2.5 × 10⁶ cells per well. 5 One cell was seeded in a 12-well plate and allowed to adhere to the plate.

[0190] (2) Weigh different drugs (FTY720, Herceptin, T-DXd, Lapatinib) and dissolve them in DMSO to prepare a 100 mM stock solution. Before the experiment, dilute the stock solution 1000 times with serum-free medium to obtain a 100 μM working solution. Set up a blank control group (serum-free medium containing 0.01‰ DMSO, drug concentration of 0) and a drug administration group (final drug concentration of 1 μM), with 1 mL per well.

[0191] (3) After drug intervention, breast cancer cells were treated for 24 hours. The supernatant culture medium was discarded, and the cells were washed three times with physiological saline. The cells were digested and counted. The cells were then diluted with serum-free culture medium to obtain a cell suspension (2.5 × 10⁻⁶). 5 (cells / mL);

[0192] (4) Take out the 24-well plate with the Transwell chamber, remove the chamber, and add 0.6 mL of complete culture medium containing 10% serum into the well plate;

[0193] (5) Place the chamber into the well plate, slowly add 0.4 mL of cell suspension into the chamber, and incubate in a 5% CO2, 37°C incubator for 24 hours;

[0194] (6) Remove the chamber, use a vacuum pump to remove the liquid in the well plate and chamber, and gently wipe away the cells that have not penetrated the filter membrane in the chamber with a cotton swab; fix in 100% methanol for 5 minutes, and wash with distilled water; stain with 0.1% crystal violet for 15 minutes, wash with distilled water, and observe the degree of staining; wipe off excess liquid with a cotton swab, air dry, and take a picture;

[0195] (7) Count the cells and calculate the cell migration inhibition rate according to the formula in Example 6.

[0196] As shown in Figure 14, 1 μM FTY720 significantly inhibited the metastasis of HER2-positive breast cancer cells. Furthermore, compared with first- and second-line clinical treatments, FTY720 showed significantly better therapeutic effects on HER2-positive breast cancer metastases than Herceptin and Lapatinib, and was comparable to T-DXd in treating HER2-positive breast cancer metastases.

[0197] Example 11. ORMDL3 degrading agents affect the proliferation of drug-resistant HER2-positive subtype breast cancer.

[0198] (1) Culture of HER2-positive drug-resistant breast cancer cell lines:

[0199] a. Herceptin-resistant cell line: 100 nM Herceptin was used as the drug concentration for screening resistant cell lines, and HCC1954 cells were cultured in basal medium containing 10% serum for 2 months; b. T-DXd-resistant cell line: 100 nM T-DXd was used as the drug concentration for screening resistant cell lines, and HCC1954 cells were cultured in basal medium containing 10% serum for 2 months; c. Lapatinib-resistant cell line: 1 μM Lpatinib was used as the drug concentration for screening resistant cell lines, and HCC1954 cells were cultured in basal medium containing 10% serum for 6 months.

[0200] (2) Different drug-resistant cell lines were digested and counted separately, at a density of 5 × 10⁶ cells per well. 3 One cell was seeded in a 96-well plate, with three replicates per group, and allowed to adhere to the plate wall.

[0201] (3) Weigh different drugs (FTY720, Herceptin, T-DXd, Lapatinib) and dissolve them in DMSO to prepare stock solutions with a concentration of 100 mM. Solvent groups and drug administration groups were set up, and the drug stock solutions were serially diluted with serum-free medium: Herceptin drug final concentrations were 0, 0.01, 0.05, 0.1, 0.5, 1, 2, 5, 10, 20 μM, with a volume of 100 μL per well; T-DXd drug final concentrations were 0, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5, 1, 2.5, 5 μM, with a volume of 100 μL per well; Lapatinib drug final concentrations were 0, 0.1, 0.5, 1, 2, 5, 10, 20, 50, 100 μM, with a volume of 100 μL per well; FTY720 was set with the same drug concentration as the control drug according to the corresponding group.

[0202] (4) After drug intervention for 24 hours, 20 μL of MTS solution (Promega, #G3582) was added directly to each well and incubated in a 5% CO2, 37°C incubator for 2 hours.

[0203] (5) Measure the absorbance at 405 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and calculate the relative cell viability and half-maximal inhibitory concentration (IC50). 50 .

[0204] As shown in Figures 15A-C, FTY720 significantly affected the cell viability of different drug-resistant cell lines, demonstrating that FTY720 can solve the drug resistance problems caused by Herceptin, T-DXd, and Lapatinib.

[0205] Example 12. ORMDL3 degrading agents affect metastasis in drug-resistant HER2-positive subtype breast cancer.

[0206] (1) The Herceptin and T-DXd resistant cell lines described in Example 11 above were digested and counted separately, at a density of 2.5 × 10⁶ cells per well. 5 One cell was seeded in a 12-well plate and allowed to adhere to the plate.

[0207] (2) Weigh different drugs (FTY720, Herceptin, T-DXd) and dissolve them in DMSO to prepare a 100 mM stock solution. Before the experiment, dilute the stock solution 1000 times with serum-free medium to obtain a 100 μM working solution. Set up a blank control group (serum-free medium containing 0.01‰ DMSO, drug concentration of 0) and drug administration groups (final drug concentrations of 0.5, 1, and 2 μM), with 1 mL of system per well;

[0208] (3) After drug intervention, the supernatant culture medium was discarded 24 hours later. The cells were washed three times with physiological saline, digested, and counted. The cells were then diluted with serum-free culture medium to obtain a cell suspension (2.5 × 10⁻⁶). 5 (cells / mL);

[0209] (4) Take out the 24-well plate with the Transwell chamber, remove the chamber, and add 0.6 mL of complete culture medium containing 10% serum into the well plate;

[0210] (5) Place the chamber into the well plate, slowly add 0.4 mL of cell suspension into the chamber, and incubate in a 5% CO2, 37°C incubator for 24 hours;

[0211] (6) Remove the chamber, use a vacuum pump to remove the liquid in the well plate and chamber, and gently wipe away the cells that have not penetrated the filter membrane in the chamber with a cotton swab; fix in 100% methanol for 5 minutes, and wash with distilled water; stain with 0.1% crystal violet for 15 minutes, wash with distilled water, and observe the degree of staining; wipe off excess liquid with a cotton swab, air dry, and take a picture;

[0212] (7) Count the cells and calculate the cell migration inhibition rate according to the formula in Example 6.

[0213] As shown in Figures 16A-B, FTY720 effectively addressed the resistance issues arising from first-line clinical drug Herceptin and second-line clinical drug T-DXd. In vitro treatment with different concentrations of FTY720 significantly inhibited the migration ability of drug-resistant HER2-positive breast cancer cells in a concentration-dependent manner.

[0214] Example 13. Effect of ORMDL3 degrading agent on mouse breast cancer cell proliferation

[0215] (1) Animal model construction: Using a mouse model of spontaneous tumor formation of breast cancer, HER2-positive (MMTV-neu) and triple-negative (MMTV-PyVT) mice were divided into 4 groups (solvent group: 0.9% sodium chloride solution; drug administration group: 0.05 mg / kg, 0.1 mg / kg, 0.2 mg / kg FTY720, n=10) and 2 groups (solvent group: 0.9% sodium chloride solution; drug administration group: 0.2 mg / kg FTY720, n=8) according to the drug administration dosage.

[0216] (2) When the mouse tumor grows to 0.4cm 3 The tumor was administered orally once daily or with the same volume of sodium chloride solution once daily, and the tumor volume was measured and recorded every 3 days until the tumor volume in the control group was approximately 1.5 cm. 3 .

[0217] The results are shown in Figures 17A-B: Low-dose FTY720 (0.2 mg / kg) effectively inhibited the proliferation of HER2-positive breast cancer tumors, while the same dose had no effect on the proliferation of triple-negative breast cancer cells. Furthermore, according to Example 8, 0.2 mg / kg of FTY720 significantly inhibited the metastasis of HER2-positive breast cancer, exhibiting subtype specificity.

Claims

1. Methods for treating and / or preventing HER2-positive disease, including administering an effective amount of an ORMDL3 degrader to subjects in need.

2. The method according to claim 1, wherein the method can implement any one or more of the following items (a)-(g): (a) Preventing the occurrence of HER2-positive diseases; (b) Prevent the development of HER2-positive disease; (c) Inhibits the metastasis of HER2-positive disease cells; (d) Inhibits the proliferation of HER2-positive disease cells; (e) Treatment of drug-resistant HER2-positive diseases; (f) Inhibits the proliferation of HER2-positive, drug-resistant disease cells; (g) Inhibits the transfer of HER2-positive disease-resistant cells.

3. The method according to claim 2, wherein the drug resistance includes resistance to any one or more of Herceptin, T-DXd, and Lapatinib.

4. The method according to claim 1 or 2, wherein the ORMDL3 degrading agent is selected from FTY720.

5. The method according to claim 3, wherein the effective amount does not exceed 5 mg / kg, for example, not exceeding 4.5 mg / kg, 4 mg / kg, 3 mg / kg, 2 mg / kg, or 1 mg / kg, preferably, the effective amount is 0.2 mg / kg, 0.1 mg / kg, or 0.05 mg / kg.

6. The method according to claim 1 or 2, wherein the HER2-positive disease is selected from HER2-positive breast cancer, ovarian cancer, gastric cancer, non-small cell lung cancer, colorectal cancer, endometrial cancer, cervical cancer, bile duct cancer, gastric and esophageal cancer, and salivary gland cancer, preferably HER2-positive breast cancer.

7. The method according to claim 1 or 2, wherein the ORMDL3 degrading agent is administered by one or more of the following methods: subcutaneous injection, intramuscular injection, intravenous injection, intraperitoneal injection, intrathecal injection, oral administration, transdermal, nasal, pulmonary, ocular, and topical application.

8. A method for inhibiting the proliferation and / or metastasis of HER2-positive samples in vitro for non-therapeutic purposes, comprising administering an ORMDL3 degrading agent to the sample.

9. The method according to claim 8, wherein the ORMDL3 degrading agent is selected from FTY720.

10. The method according to claim 8, wherein the HER2-positive sample comprises HER2-positive cells or tissues, preferably HER2-positive subtype breast cancer cells or HER2-positive subtype drug-resistant breast cancer cells, and preferably the drug resistance comprises resistance to any one or more of Herceptin, T-DXd, and Lapatinib.

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