A sialylglycan-sialic acid binding immunoglobulin-like lectin signaling pathway blocker and uses thereof

Abstract

This invention discloses a sialyl polysaccharide-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker, containing one or more N-acetylneuraminic acid derivatives, used to manufacture immunotherapy synergistic drugs or immunomodulatory drugs. The immunotherapy synergistic drugs are used in combination with immunotherapy drugs, and the immunomodulatory drugs are used to enhance the regulation of immunity.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a sialic acid-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker and its uses. Background Technology

[0002] With an aging population and changing lifestyles, the risk of developing cancer is increasing, and the incidence and mortality rates of malignant tumors are rising year by year. Currently, cancer has become one of the leading causes of death, seriously threatening human health. Tumor cells are genetically unstable, prone to mutation and variation, leading to rapid cell proliferation and suppressed apoptosis, forming an abnormal tumor microenvironment (TME). The TME is composed of different cell types, extracellular matrix components, and soluble factors supporting tumor growth and progression. It typically has a high immunosuppressive effect, preventing immune cells from clearing tumor cells, thus negatively regulating tumor immunotherapy and affecting its efficacy. Therefore, overcoming the immunosuppressive TME is crucial for developing effective tumor immunotherapies.

[0003] Tumor cells typically express abnormal amounts of sialic acid on their cell surface. Sialic acid is a family of negatively charged sugar molecules that end in glycan chains covering glycoproteins and glycolipids. Sialoglycans on tumor cells participate in tumor cell-extracellular matrix interactions and tumor cell-immune cell interactions, protecting tumor cells from immune recognition and killing. Sialoglycans achieve immunosuppressive regulation through specific interactions with sialic acid-binding immunoglobulin-like lectins (Siglec) on the surface of various immune cells. Siglec is an immunoreceptor containing an immunoreceptor tyrosine inhibitory motif (ITIM), independent of the major histocompatibility complex (MHC), which regulates the body's immune response through interactions with sialoglycans on the surface of target cells. TME can enhance the abnormally high expression of sialic acid in tumor cells and also stimulate Siglec expression on tumor-infiltrating immune cells. Dysregulation of the sialoglycan-Siglec axis in tumor tissue contributes to the formation of immunosuppressive TMEs, severely affecting tumor immunotherapy.

[0004] The Siglec receptor family is a major target for many immune functions of tumor sialic acid glycans in regulating tumor-infiltrating immune cells, and in human effector CD4 receptors. + T cells, CD8 + Only inhibitory Siglec cells, primarily Siglec-7 and Siglec-9, were identified on T cells and NK cells, but no activating Siglec cells were found. Tumor cells utilize Siglec cells to suppress the function of effector immune cells, suggesting that Siglec cells on lymphocytes function as a negative "checkpoint" for glucose immunity, similar to activated CD8+. +PD-1 checkpoints on T cells.

[0005] NK cells express Siglec-7 and Siglec-9. Siglec-7 is expressed on almost all NK cells and binds to α2-8 sialic acid. Siglec-9 is selectively expressed on CD56 cells. dim Siglec-9 binds to α2-3 sialic acid on a subset of NK cells (the cytotoxic subset of NK cells). This binding of Siglec-9 to α2-3 sialic acid on target cells induces an inhibitory signaling cascade by recruiting tyrosine phosphatase SHP-1, counteracting phosphorylation-mediated immune activation by other activating signaling molecules and inhibiting the tumor-killing ability of NK cells. Degrading sialyl glycans on the surface of tumor cells with sialylase, or blocking the sialyl glycan-Siglec-7 / 9 interaction with Siglec-blocking antibodies, can enhance the in vitro tumor-killing effect of human NK cells.

[0006] CD8 isolates from patients with non-small cell lung cancer (NSCLC), colorectal cancer, ovarian cancer, and melanoma + Siglec is highly expressed on T cells, with Siglec-9 being the most highly expressed. Similar to human NK cells, the interaction between sialyl glycan and Siglec-9 can lead to the cloning of human CD8 cells in vitro. + T cell ITIM phosphorylation and SHP-1 recruitment inhibit T cell receptor (TCR) signaling and cytotoxic function.

[0007] Tumor sialyl-Siglec interactions downregulate antitumor immunity by modulating dendritic (DC) cell function. DCs express multiple inhibitory Siglec family members, including Siglec-3, -5, -7, and -9 (human DCs) and Siglec-E, -G, and -H (mouse DCs), which affect DC maturation, antigen presentation, and activation of tumor-specific CD8+. + The capacity of T cells. Certain tumors can utilize sialyl-Siglec interactions to modulate dendritic (DC) function at multiple levels, thereby affecting CD4+. + and CD8 + The induction of T cell responses has negative effects.

[0008] Myeloid cells in the TME are mainly composed of immunosuppressive macrophages, neutrophils, and myeloid-derived suppressor cells (MDSCs), which can limit effective anti-tumor immune responses. Siglec is widely expressed in human bone marrow cell subsets.

[0009] Tumor cells suppress immune cell function by upregulating sialic acid on the cell surface. Viral-infected cells can also regulate immune cell function through similar pathways. For example, in HIV infection, the HIV virus prefers to infect cells with high levels of fucose and sialic acid on their cell surface. Sialic acid is a key component in HIV infection of CD4 cells. + Essential for T cells. After HIV infects cells, the infected cells further increase the amount of sialic acid on their surface to send immunosuppressive signals to other immune cells, evading immune surveillance and allowing the HIV virus to quickly control and spread to more parts of the body. The sialic acid on the surface of HIV-infected cells pairs with Siglec on the surface of NK cells, sending an inhibitory signal and stopping the attack on HIV-infected cells.

[0010] In summary, the MHC independence of Siglec molecules on the surface of immune cells makes them a target for tumor cells or virus-infected cells to evade host immune surveillance. Therefore, it is necessary to develop novel, low-cost drugs that block the sialic acid-sialic acid binding immunoglobulin-like lectin signaling pathway as synergists or immunomodulators in immunotherapy. Summary of the Invention

[0011] In view of this, the technical problem to be solved by the present invention is to provide an N-acetylneuraminic acid derivative for use as a signaling pathway blocker, which can be incorporated into the sialic acid bio-metabolic pathway, replace the N-acetylneuraminic acid at the terminal end of sialoglycan molecules on the surface of abnormally expressed sialylated cells such as tumor cells and virus-infected cells, change the molecular structure and charge characteristics at the terminal end of sialoglycan molecules, reduce its affinity for sialic acid-binding immunoglobulin-like lectin (Siglec), block the signaling pathway of sialoglycan-sialic acid-binding immunoglobulin-like lectin interaction, relieve the immunosuppressive signal on immune cells, thereby enabling immune cells to exert an immune killing effect on tumor cells or virus-infected cells.

[0012] As a first aspect of the present invention, a sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker is provided, comprising one or more N-acetylneuraminic acid derivatives.

[0013] Preferably, the N-acetylneuraminic acid derivative is selected from N-acetylneuraminic acid esters.

[0014] Preferably, the N-acetylneuraminic acid ester is selected from 2,4,7,8,9-penta-O-acetyl-N-acetylneuraminic acid deuterated methyl ester, 4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid deuterated methyl ester, N-acetylneuraminic acid methyl ester, N-acetylneuraminic acid ethyl ester, 2,4,7,8,9-penta-O-acetyl-N-acetylneuraminic acid methyl ester, 4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester, and N-acetylneuraminic acid deuterated methyl ester. The following are included in the list of N-acetylneuraminic acid deuterated ethyl ester, N-acetylneuraminic acid propyl ester, N-acetylneuraminic acid isopropyl ester, N-acetylneuraminic acid n-butyl ester, N-acetylneuraminic acid sec-butyl ester, N-acetylneuraminic acid tert-butyl ester, N-acetylneuraminic acid isobutyl ester, N-acetylneuraminic acid methyl ester cyclodextrin inclusion complex, N-acetylneuraminic acid ethyl ester cyclodextrin inclusion complex, N-acetylneuraminic acid deuterated methyl ester cyclodextrin inclusion complex, and N-acetylneuraminic acid deuterated ethyl ester cyclodextrin inclusion complex.

[0015] Preferably, the N-acetylneuraminic acid derivative is selected from the group with the following general structural formula: N-acetylneuraminic acid esters, wherein R1 is H or OH, and R2 is a benzene ring substituent, a biotin substituent, a folic acid substituent, or an RGD substituent; and / or

[0016] R2 is OH, and R1 is a benzene ring substituent, a biotin substituent, a folic acid substituent, or an RGD substituent; and / or

[0017] R1 is a benzene ring substituent, a biotin substituent, a folic acid substituent, or an RGD substituent; R2 is a benzene ring substituent, a biotin substituent, a folic acid substituent, or an RGD substituent.

[0018] Wherein, the benzene ring substituent is ; and / or

[0019] The biotinylate substituent is And / or the folic acid substituents are selected from...

[0020] and / or And / or the RGD-type substituents are

[0021]

[0022] Preferably, the N-acetylneuraminic acid derivative is selected from the group with the following general structural formula: The compound wherein R is an alkyl, cycloalkyl, substituted alkyl, substituted cycloalkyl, thioester, thioether, ether group, disulfide ester, dithiomethyl, S-CH3, S-CH2-CH3, aryl, substituted aryl, methionine, methionine-zinc, sodium, phenol or phenol derivative.

[0023] Preferably, the N-acetylneuraminic acid derivative is an N-acetylneuraminic acid derivative cyclodextrin inclusion complex.

[0024] Preferably, the cyclodextrin is selected from any one of β-cyclodextrin or its derivatives, γ-cyclodextrin or its derivatives, α-cyclodextrin or its derivatives, wherein the cyclodextrin is hydroxypropyl-β-cyclodextrin, or hydroxypropyl-γ-cyclodextrin, or dimethyl-β-cyclodextrin, or dimethyl-γ-cyclodextrin, or sulfobutyl-β-cyclodextrin.

[0025] In a second aspect of the invention, the use of the signaling pathway blocker of the first aspect of the invention to alter the molecular structure and charge characteristics of the sialyte terminals on the surface of tumor cells or virus-infected cells is disclosed; wherein the tumor cells or virus-infected cells are tumor cells or virus-infected cells with abnormal sialylation.

[0026] The signaling pathway blocker is used to block the immunosuppressive signaling pathway between sialylated glycans on the surface of tumor cells or virus-infected cells and sialic acid-binding immunoglobulin-like lectins on the surface of immune cells.

[0027] In a third aspect of the invention, the use of the signaling pathway blocker described in the second aspect of the invention in the manufacture of synergistic immunotherapy drugs is disclosed.

[0028] Preferably, the immunotherapy synergist is used in combination with other immunotherapy drugs;

[0029] The immunotherapy drugs include immune cell preparations, immune checkpoint inhibitors, tumor vaccine preparations, T-cell connective drugs, NK-cell connective drugs, antibody drugs with ADCC activity, and immunomodulators.

[0030] As a synergistic agent for immunotherapy, it is used to enhance the cytotoxic activity of immune cell preparations against tumor cells, enhance the cytotoxic activity of immune cell preparations against virus-infected cells, enhance the therapeutic effect of tumor treatment by synergizing with immune checkpoint inhibitors, enhance the therapeutic effect of tumor treatment by synergizing with tumor vaccine preparations, enhance the immune clearance effect of tumor cells by synergizing with T-cell connective drugs, enhance the immune clearance effect of tumor cells by synergizing with NK-cell connective drugs, enhance the immune clearance effect of tumor cells by synergizing with antibody drugs with ADCC activity, enhance the immune clearance effect of tumor cells by synergizing with immunomodulatory drugs, and enhance the immune clearance effect of virus-infected cells by synergizing with immunomodulatory drugs.

[0031] Preferably, the immune cell preparations include NK cell preparations, CAR-NK cell preparations, αβT cell preparations, γδT cell preparations, TIL cell preparations, NKT cell preparations, CAR-T cell preparations, TCR-T cell preparations, CIK cell preparations, DC-CIK cell preparations, macrophage (Mφ) preparations, CAR-Mφ cell preparations, neutrophil preparations, dendritic cell (DC) preparations, B cell preparations, myeloid-derived suppressor cell (MDSC) preparations, and Treg cell preparations.

[0032] The immune checkpoint inhibitors include PD-1 antibody preparations, PD-L1 antibody preparations, CTLA-4 antibody preparations, LAG-3 antibody preparations, TIM-3 antibody preparations, TIGIT antibody preparations, and VISTA antibody preparations.

[0033] The tumor vaccine formulations include whole-cell tumor vaccines, tumor peptide vaccines, genetically engineered mRNA tumor vaccines, genetically engineered DNA tumor vaccines, tumor subunit vaccines, heat shock protein tumor vaccines, and antibody tumor vaccines.

[0034] Preferably, the immunotherapy synergist is used in combination with the immunotherapy drug for the treatment of tumors and the prevention and treatment of potential malignant diseases and carcinogenesis;

[0035] The tumors include solid tumors and hematologic malignancies;

[0036] The solid tumors include lung cancer, breast cancer, esophageal cancer, gastric cancer, cardia cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, thyroid cancer, parathyroid cancer, kidney cancer, bladder cancer, penile cancer, malignant melanoma, and glioma.

[0037] The hematologic malignancies include leukemia, multiple myeloma, and malignant lymphoma;

[0038] The potential malignant diseases include leukoplakia, atrophic gastritis, cirrhosis, pulmonary nodules, adenomatous polyps, Barrett's esophagus, Crohn's disease, ulcerative colitis, borderline ovarian tumors, familial adenomatous polyposis (FAP), Lynch syndrome, BRCA1 / 2 gene mutation diseases, myelodysplastic syndrome (MDS), monoclonal gammopathy (MGUS), thyroid nodules, melanocytic nevi, endometrial hyperplasia, cervical intraepithelial neoplasia (CIN), intraductal carcinoma in situ (DCIS) of the breast, and lobular carcinoma in situ (LCIS) of the breast.

[0039] Preferably, the immunotherapy synergist is used in combination with the immunotherapy drug to treat viral infections;

[0040] The viral infections mentioned include human immunodeficiency virus (HIV) infection, influenza virus infection, coronavirus infection, rhinovirus infection, mumps virus infection, respiratory syncytial virus (RSV) infection, cytomegalovirus infection, hepatitis B virus infection, hepatitis C virus infection, human papillomavirus (HPV) infection, Ebola virus infection, herpes simplex virus infection, adenovirus infection, rubella virus infection, norovirus infection, Zika virus, parainfluenza virus infection, measles virus infection, avian influenza virus infection, varicella-zoster virus infection, Newcastle disease virus infection, Sendai virus, and rotavirus infection.

[0041] Preferably, the signaling pathway blocker is used to manufacture an immunomodulatory drug, the immunomodulatory drug is used to enhance the anti-tumor immunity of cancer patients, and the immunomodulatory drug is used to treat tumors and prevent potential malignant diseases from becoming cancerous;

[0042] The tumors include solid tumors and hematologic malignancies;

[0043] The solid tumors include lung cancer, breast cancer, esophageal cancer, gastric cancer, cardia cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, thyroid cancer, parathyroid cancer, kidney cancer, bladder cancer, penile cancer, malignant melanoma, and glioma.

[0044] The hematologic malignancies include leukemia, multiple myeloma, and malignant lymphoma;

[0045] The potential malignant diseases include leukoplakia, atrophic gastritis, cirrhosis, pulmonary nodules, adenomatous polyps, Barrett's esophagus, Crohn's disease, ulcerative colitis, borderline ovarian tumors, familial adenomatous polyposis (FAP), Lynch syndrome, BRCA1 / 2 gene mutation diseases, myelodysplastic syndrome (MDS), monoclonal gammopathy (MGUS), thyroid nodules, melanocytic nevi, endometrial hyperplasia, cervical intraepithelial neoplasia (CIN), intraductal carcinoma in situ (DCIS) of the breast, and lobular carcinoma in situ (LCIS) of the breast.

[0046] Preferably, the signaling pathway blocker is used to manufacture an immunomodulatory drug, which is used to enhance the antiviral immunity of virus-infected individuals.

[0047] The immunomodulatory drug is used to treat viral infections;

[0048] The viral infections mentioned include human immunodeficiency virus (HIV) infection, influenza virus infection, coronavirus infection, rhinovirus infection, mumps virus infection, respiratory syncytial virus (RSV) infection, cytomegalovirus infection, hepatitis B virus infection, hepatitis C virus infection, human papillomavirus (HPV) infection, Ebola virus infection, herpes simplex virus infection, adenovirus infection, rubella virus infection, norovirus infection, Zika virus, parainfluenza virus infection, measles virus infection, avian influenza virus infection, varicella-zoster virus infection, Newcastle disease virus infection, Sendai virus, and rotavirus infection.

[0049] In a fourth aspect of the invention, a medicament and a pharmaceutical composition are provided as an inhibitor of the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway, comprising an inhibitor of the signaling pathway described in the first aspect of the invention, and a pharmaceutically acceptable dosage form.

[0050] The dosage forms of the drug and drug composition are tablets, capsules, injections, granules, suppositories, nasal sprays, oral sprays, nebulized inhalers, powders, powder inhalers, ointments, or gels.

[0051] In a fifth aspect of the invention, a method for blocking the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway is provided, characterized in that it includes blocking the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway using an effective amount of the signaling pathway blocker described in the first aspect of the invention.

[0052] Tumor cells express abnormal amounts of sialic acid on their surface. Sialic acid is a family of negatively charged sugar molecules that end in glycan chains covering glycoproteins and glycolipids. Sialoglycans on tumor cells participate in tumor cell-extracellular matrix interactions and tumor cell-immune cell interactions, forming a protective region that shields tumor cells from immune recognition and killing. Sialoglycans play a role in tumor immune evasion as immunomodulatory sugars. Sialoglycans achieve immunosuppressive regulation through specific interactions with sialic acid-binding immunoglobulin-like lectins (Siglec). Tumor cells with abnormal sialic acid expression can interact with the Siglec family to regulate immune cell function in the tumor microenvironment. The tumor microenvironment can enhance abnormal sialic acid secretion from tumor cells and also stimulate Siglec expression in infiltrating immune cells.

[0053] Dysregulation of the sialic acid-Siglec axis in tumor tissue contributes to the formation of an immunosuppressive tumor microenvironment, hindering immunotherapy for tumors. Viral infection can also regulate immune cell function through similar pathways. For example, in HIV infection, the HIV virus prefers to infect cells with high levels of fucose and sialic acid on their surface. Sialic acid is essential for HIV infection of CD4+ T cells. After HIV infection, infected cells further increase their surface sialic acid levels to send immunosuppressive signals to other immune cells, evading immune surveillance and allowing the HIV virus to rapidly spread and control the virus to more parts of the body. The sialic acid on the surface of HIV-infected cells seeks out NK cells that can eliminate them and pairs with their corresponding receptor, Siglec. Once bound, Siglec sends inhibitory signals to NK cells, causing them to stop attacking other cells, allowing HIV to replicate unchecked.

[0054] The method of the present invention can block the signaling pathway of sialic acid-sialic acid binding to immunoglobulin-like lectin (Siglec), thereby relieving the immunosuppressive state of immune cells. In this way, immune cells can recognize the aforementioned tumor cells or virus-infected cells, thereby exerting the effect of immunotherapy.

[0055] It is a common phenomenon for tumor cells to express abnormal amounts of sialic acid on their cell surface. Sialic acid carries a negative charge and is a glycan chain that coats the ends of glycoproteins and glycolipids. High expression of sialic acid in tumor cells results in a greater negative charge on their surface. This excessive negative charge is a characteristic of tumor cells, leading to increased repulsion between them and making them more prone to detachment and entry into the bloodstream or lymphatic system. The negative charge on the surface of tumor cells may be related to the dynamic balance of tumor dissemination, activation, and dormancy within the body. For example, tumor cells may interact with positively charged molecules in their surrounding environment through their negative surface charge, thereby altering their adhesion, migration, and invasive abilities, promoting metastasis and colonization within the body.

[0056] Sialoglycosides on tumor cells participate in tumor cell-extracellular matrix interactions and tumor cell-immune cell interactions, forming a protective region that shields tumor cells from immune recognition and killing. Sialoglycosides function as immunomodulatory sugars in tumor immune evasion. Their immunomodulatory function is achieved through a specific interaction with sialic acid-binding immunoglobulin-like lectin (Siglec). In human effector CD4... + T cells, CD8 + Only inhibitory Siglecs, primarily Siglec-7 and Siglec-9, were identified on T cells and NK cells, but no activating Siglecs were found. Siglecs on lymphocytes act as negative "checkpoints" for glycoimmune dysfunction, and tumor cells utilize the Siglec signaling pathway to suppress the cytotoxic effects of effector immune cells.

[0057] Siglec-9 is a major histocompatibility complex (MHC)-independent inhibitory receptor expressed on a subset of natural killer (NK) cells. Siglec-9 inhibits NK cell cytotoxicity by binding to sialyl polysaccharides on target cells.

[0058] Tumor cells with aberrant sialic acid expression can interact with the Siglec family to regulate immune cell function in the tumor microenvironment. The tumor microenvironment can enhance abnormal sialic acid secretion from tumor cells and stimulate Siglec expression in infiltrating immune cells. Dysregulation of the sialic acid-Siglec axis in tumor tissue contributes to the formation of an immunosuppressive tumor microenvironment, hindering immunotherapy. A close relationship exists between the sialylation characteristics and negative charge characteristics of tumor cell surfaces, which can promote tumor metastasis and affect the efficacy of immunotherapy.

[0059] Many malignant tumor cells express tumor-derived antibodies at high levels, which can promote malignant behavior of tumor cells, mediate tumor immune escape, induce inflammation, and activate platelet aggregation. Tumors expressing these antibodies are mainly various types of epithelial cell tumors, such as breast cancer, colon cancer, cervical cancer, lung cancer, laryngeal cancer, nasopharyngeal cancer, pancreatic cancer, liver cancer, prostate cancer, oral cancer, thyroid cancer, parathyroid cancer, esophageal cancer, gastric cancer, kidney cancer, and bladder cancer.

[0060] Tumor-derived antibodies possess a unique glycosylation modification, occurring at Asn162 rather than Asn297. Through this specific sialylation modification, tumor-derived antibodies exert immunosuppressive effects. The function of tumor-derived antibodies can significantly shift from promoting immune responses to suppressing them, with their immunosuppressive function heavily dependent on sialylation at the Ans162 ​​site. These tumor-derived sialylated IgG molecules can inhibit dendritic cells (DCs) and CD4+ by binding to the sialic acid receptor DC-SIGN on DCs. + The function of T cells.

[0061] Siglec is present on most immune cells and shares a common N-terminal domain that recognizes sialic acid-containing polysaccharides. Siglec is highly expressed in tumor-infiltrating immune cells and acts as an immune checkpoint regulating anti-cancer immunity. Tumor-derived sialylated IgG molecules also interact with the surface of NK and T immune cells, activating inhibitory signals and suppressing the function of NK and T immune cells.

[0062] Latent malignant diseases (also known as precancerous lesions or precursor lesions) may not have yet developed into cancer, but they carry the risk of transforming into aggressive cancers. The most direct danger of latent malignant diseases lies in their potential to evolve into cancer over time. This transformation can take years or even decades, but once it occurs, the cancer can progress rapidly and have a severe impact on health. While latent malignant diseases are not immediately fatal, their health hazards should not be ignored. Early detection, proactive management, and appropriate interventions are crucial for mitigating these harms. Strengthening the body's immunity, particularly through modern immunotherapies such as NK cell preparations, tumor vaccines, and immune checkpoint inhibitors, can enhance the body's immune system's ability to recognize and eliminate mutations, variants, and carcinogenesis, and can effectively prevent the transformation of latent malignant diseases into cancer to a certain extent.

[0063] In the early stages of carcinogenesis, during the process from molecular mutation to cellular variation, the immune system can more effectively deal with a small number of abnormal cells. At this time, the lesion has not yet formed obvious tumor tissue, and the immune response is more likely to control these initial changes. Therefore, strengthening immunity as an early intervention can significantly improve the effectiveness of preventing tumor development.

[0064] Regular health checkups and biomarker testing, combined with individualized immune status assessments, can provide a better understanding of a patient's immune function, allowing for adjustments to immune enhancement strategies. For example, targeted immune enhancement in advance can reduce the risk of cancer in individuals with a higher genetic risk or exposure to specific carcinogens.

[0065] The key to the efficacy of immunotherapy drugs lies in enhancing the function of the body's immune cells (especially NK cells, T cells, and DC cells). The immunotherapy synergistic drug provided by this invention, when used in combination with immunotherapy drugs, can create a synergistic effect, more effectively enhance the body's cellular immune function, and can greatly help prevent potential malignant diseases from developing into cancer.

[0066] After a virus infects a cell, NK cells recognize the infected cell through a series of complex mechanisms. Many viral infections lead to reduced or absent expression of major histocompatibility complex class I (MHC-I) molecules on the surface of host cells. Normally, MHC-I molecules bind to inhibitory receptors on NK cells (such as KIR and CD94 / NKG2A), transmitting a "self" signal that prevents NK cells from attacking healthy cells. When MHC-I expression is reduced, this inhibitory signal weakens or disappears, making NK cells more susceptible to activation and attacking infected cells lacking MHC-I expression. Viral infection or other forms of cellular stress can cause the expression of specific stress-inducible ligands on the surface of host cells, such as MIC-A / B and ULBP1-6. These ligands can bind to activation receptors on NK cells (such as NKG2D), providing activation signals. Activation of the NKG2D receptor enhances the cytotoxic activity of NK cells and promotes their attack on target cells. If virus-infected cells have been labeled with specific antibodies, NK cells can recognize and bind to these antibodies via the FcγRIIIa (CD16) receptor on their surface. Once bound, NK cells release effector molecules such as perforin and granzymes, directly killing the antibody-labeled target cells. Cytokines produced during viral infection, such as interferon-α / β (IFN-α / β), interleukin-12 (IL-12), IL-15, and IL-18, can enhance NK cell activity. These cytokines not only directly stimulate NK cells but also promote the function of other immune cells, creating a more potent antiviral response environment. NK cells can also recognize unique markers on the surface of certain virus-infected cells through direct contact. For example, some viruses may alter the glycosylation pattern on the host cell membrane, which could become a new target for NK cell recognition.

[0067] Viruses infect cells via sialic acid receptors, and the resulting abnormal expression of sialylation on the cell surface is a common phenomenon in many viral infections. The following describes the relationship between several specific viruses and sialylic acid receptors on the cell membrane surface during host cell infection, and the impact of viral infection on sialylation of the host cell surface:

[0068] 1. Influenza Virus: The hemagglutinin (HA) protein of the influenza virus specifically recognizes and binds to α2,3- or α2,6-linked sialic acid on the surface of host cells. This specificity determines the virus's host range and tissue tropism. The influenza virus also encodes sialylase (NA), an enzyme that cleaves sialic acid on the surface of host cells, helping to release newly generated viral particles from the cell surface and preventing viral particles from reattaching to the same cell. Studies have shown that influenza virus infection can lead to an increase in the level of sialylation on the surface of host cells, which may contribute to further viral spread.

[0069] 2. Ebola Virus: The Ebola virus glycoprotein (GP) mediates viral adsorption and endocytosis by recognizing sialic acid on the cell surface. The presence of sialic acid enhances viral infectivity. The level of sialylation on the host cell surface may change after Ebola virus infection, but research in this area is relatively limited.

[0070] 3. Human Immunodeficiency Virus (HIV): The HIV envelope glycoprotein gp120 is highly glycosylated, containing multiple sialylation sites. These sialylation modifications help the virus evade recognition by the host's immune system. Although HIV itself does not encode sialylase, HIV-infected cells may exhibit altered sialylation levels, which may affect viral infectivity and transmission.

[0071] 4. Coronaviruses: Some coronaviruses, such as MERS-CoV and SARS-CoV, can enhance their infectivity by recognizing sialic acid on the cell surface. However, SARS-CoV-2 primarily infects by binding its spike protein (S protein) to the ACE2 receptor on the surface of host cells. Although SARS-CoV-2 mainly relies on the ACE2 receptor, the level of sialylation on the cell surface may change after infection, which could affect the infection of other viruses or the host immune response.

[0072] 5. Cytomegalovirus (CMV): CMV's glycoproteins gB and gH / gL complexes can mediate viral adsorption and endocytosis by recognizing sialic acid on the cell surface. After CMV infection, the level of sialylation on the host cell surface may increase, which facilitates further viral spread and evasion of the immune system.

[0073] 6. Herpes Simplex Virus (HSV): HSV's glycoprotein gD can mediate viral adsorption and endocytosis by recognizing sialic acid on the cell surface. After HSV infection, the level of sialylation on the host cell surface may change, which may affect viral infectivity and transmission.

[0074] 7. Respiratory Syncytial Virus (RSV): The RSV F protein (fusion protein) mediates viral adsorption and fusion by recognizing sialic acid on the cell surface. After RSV infection, the level of sialylation on the host cell surface may change, which may affect further viral transmission and infection efficiency.

[0075] 8. Newcastle Disease Virus (NDV): The NDV HN protein (hemagglutinin-neuraminidase protein) mediates viral adsorption and fusion by recognizing sialic acid on the cell surface. NDV also encodes sialylase, which can cleave sialic acid on the host cell surface, helping newly generated viral particles to be released from the cell surface.

[0076] 9. Adenovirus: Adenovirus's fibrin protein mediates viral adsorption by recognizing sialic acid on the cell surface. After adenovirus infection, the level of sialylation on the host cell surface may change, potentially affecting further viral transmission and infection efficiency.

[0077] 10. Rubella Virus: The E1 and E2 glycoproteins of rubella virus can mediate viral adsorption and endocytosis by recognizing sialic acid on the cell surface. After infection with rubella virus, the level of sialylation on the surface of host cells may change, which may affect the further spread of the virus and the efficiency of infection.

[0078] 11. Hepatitis C Virus (HCV): The E2 glycoprotein of HCV mediates viral adsorption by recognizing sialic acid on the cell surface. After HCV infection, the level of sialylation on the host cell surface may change, which may affect further viral transmission and infection efficiency.

[0079] 12. Norovirus: The VP1 protein of norovirus mediates viral adsorption by recognizing sialic acid on the cell surface. After norovirus infection, the level of sialylation on the host cell surface may change, which may affect further viral transmission and infection efficiency.

[0080] 13. Parainfluenza Virus: The hemagglutinin-neuraminidase protein (HN) of parainfluenza virus can mediate viral adsorption and fusion by recognizing sialic acid on the cell surface. Parainfluenza virus also encodes sialylase, which can cleave sialic acid on the host cell surface, helping newly generated viral particles to be released from the cell surface.

[0081] 14. Measles Virus: The hemagglutinin protein (H) of measles virus can mediate viral adsorption by recognizing sialic acid on the cell surface. After infection with measles virus, the level of sialylation on the host cell surface may change, which may affect further viral transmission and infection efficiency.

[0082] 15. Avian Influenza Virus: The hemagglutinin (HA) protein of avian influenza virus specifically recognizes and binds to α2,3-linked sialic acid on the surface of host cells. This specificity determines the virus's host range and tissue tropism. Avian influenza virus also encodes sialylase (NA), an enzyme that cleaves sialic acid on the surface of host cells, facilitating the release of newly generated viral particles from the cell surface.

[0083] 16. Rotavirus: The VP4 protein of rotavirus mediates viral adsorption by recognizing sialic acid on the cell surface. After rotavirus infection, the level of sialylation on the host cell surface may change, which may affect further viral transmission and infection efficiency.

[0084] 17. Human Papillomavirus (HPV): The L1 protein of HPV mediates viral adsorption by recognizing sialic acid on the cell surface. After HPV infection, the level of sialylation on the surface of host cells may change, which may affect the further spread of the virus and the efficiency of infection.

[0085] These viruses infect cells using sialic acid receptors on the cell surface through different mechanisms, potentially altering the level of sialylation on the host cell surface. These changes may facilitate viral adsorption, endocytosis, release, spread, and evasion of the immune system. Specific mechanisms and effects vary by virus type, but sialic acid plays a crucial role in viral infection, mediating complex interactions between virus-infected cells and immune cells. It negatively regulates immune cell function through the sialyl-sialic acid-immunoglobulin-like lectin signaling pathway, thereby enabling viruses to evade the body's immune system's recognition, killing, and clearance of virus-infected cells.

[0086] Specifically, the present invention has at least the following beneficial effects:

[0087] The sialic acid-sialic acid binding immunoglobulin-like lectin signaling pathway blocker of the present invention can modify the sialic acid-expressing sialic acid on the surface of tumor cells or virus-infected cells, thereby converting the electronegative N-acetylneuraminic acid carboxyl group at the end of glycolipids or glycoproteins into an electronegative chemical group. This alters the local molecular structure and charge characteristics of the sialic acid-expressing sialic acid on the surface of tumor cells or virus-infected cells, thus reshaping the local immune microenvironment of sialic acid-expressing sialic acid on the surface of these cells.

[0088] Specifically, based on this principle, substances such as N-acetylneuraminic acid esters or analogues can block the sialic acid-sialic acid binding immunoglobulin-like lectin signaling pathway. They can modify the sialic acid glycans on the surface of tumor cells or virus-infected cells with abnormal sialic acid expression, thereby converting the electronegative carboxyl groups of N-acetylneuraminic acid at the ends of glycolipids or glycoproteins into electronegative ester groups (such as methyl ester, ethyl ester, propyl ester, etc.). This alters the local molecular structure and charge characteristics of the sialic acid glycans at the ends of glycolipids or glycoproteins, reshaping the local immune microenvironment of cells with abnormal sialic acid expression, such as tumor cells or virus-infected cells.

[0089] The sialic acid-glycan-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker provided by this invention can be used as an immunotherapy synergist in combination with immunotherapy drugs to assist in the treatment of neoplastic diseases or viral infectious diseases with abnormally high sialic acid expression.

[0090] 1. Tumor cells with abnormal sialic acid expression are modified by N-acetylneuraminic acid esters or analogues, which alters the surface charge, reduces the negative charge, decreases intercellular repulsion, and reduces tumor cell shedding and metastasis.

[0091] 2. Tumor cells with abnormal sialic acid expression undergo modification with N-acetylneuraminic acid esters or analogues. This alters the local molecular structure and charge characteristics of the sialic acid glycans on their surface, leading to a decrease in the affinity of these modified sialic acid receptors for Siglec family members, which are immunosuppressive receptors on the surface of immune cells. This blocks the sialic acid-sialic acid binding immunoglobulin-like lectin signaling pathway, relieving its immunosuppressive effect on immune cells and thus promoting the killing of tumor cells by immune cells, thereby enhancing the N-acetylneuraminic acid... Esters have a synergistic effect on immunotherapy, and this synergistic effect is universal for various tumors. That is, regardless of the type of tumor, as long as the tumor cells of that type of tumor highly express sialic acid, N-acetylneuraminic acid esters or analogues, as blockers of the sialic acid-sialic acid-immunoglobulin-like lectin signaling pathway, can relieve the negative regulation of immune cells by blocking the sialic acid-sialic acid-immunoglobulin-like lectin signaling pathway on the surface of immune cells, thereby better exerting the functions of immune recognition and immune killing of tumor cells. Figure 1 ).

[0092] 3. N-acetylneuraminic acid esters or analogues are incorporated into the glycosylation process of abnormally sialylated IgG derived from tumors, modifying the N-acetylneuraminic acid of Asn162 into N-acetylneuraminic acid esters or analogues. This reduces the affinity of IgG for sialic acid-binding immunoglobulin-like lectin (Siglec) molecules on the surface of DCs, NK cells, and T cells, preventing the activation of Siglec inhibitory signals, relieving immunosuppression of these immune cells, and allowing for better immune recognition and killing effects. Figure 1 ).

[0093] 4. In viral infections with abnormal sialic acid expression, the local molecular structure of sialyl glucomannan on the cell surface is altered. This leads to a decrease in the affinity between these modified sialyl receptors and members of the Siglec family of immunosuppressive receptors on the surface of immune cells. This blocks the sialyl glucomannan-sialyl-immunoglobulin-like lectin signaling pathway, relieving its immunosuppressive effect on immune cells. Consequently, it promotes the killing of virus-infected cells by immune cells, exerting a synergistic effect of N-acetylneuraminic acid esters or analogues on immunotherapy. This synergistic effect is universal for various viral infections; that is, regardless of the type of viral infection, as long as the surface of the virus-infected cells express sialyl, N-acetylneuraminic acid esters or analogues, acting as blockers of the sialyl glucomannan-sialyl-immunoglobulin-like lectin signaling pathway on the surface of immune cells, can relieve the negative regulation of immune cells and better exert the functions of immune recognition and immune killing of virus-infected cells.

[0094] 5. It possesses universal synergistic effect in immunotherapy. Various immunotherapeutic drugs ultimately exert their effects by acting on endogenous or exogenous immune cells, enhancing their immune recognition and killing functions. These immune cells all express members of the Siglec family of immunosuppressive receptors on their surface. Therefore, the sialyl polysaccharide-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker provided by this invention has universal synergistic effect in combination with other immunotherapeutic drugs. It is suitable for use as a synergistic agent in various ways: synergistically increasing the cytotoxic activity of immune cell preparations against tumor cells; synergistically increasing the cytotoxic activity of immune cell preparations against virus-infected cells; synergistically enhancing the therapeutic effect of immune checkpoint inhibitors; synergistically enhancing the therapeutic effect of tumor vaccines; synergistically increasing the immune clearance efficacy of T-cell connector drugs against tumor cells; synergistically increasing the immune clearance efficacy of NK-cell connector drugs against tumor cells; synergistically increasing the immune clearance efficacy of antibody drugs with ADCC activity against tumor cells; synergistically increasing the immune clearance efficacy of immunomodulatory drugs against tumor cells; and synergistically increasing the immune clearance efficacy of immunomodulatory drugs against virus-infected cells.

[0095] 6. The sialic acid-glycan-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker of the present invention is used as a synergistic agent in immunotherapy, in combination with immunotherapeutic drugs, to enhance the body's immunity and better identify and kill mutated and variant cells. By working synergistically with immunotherapeutic drugs for potentially malignant diseases, it can eliminate tumors in precancerous lesions or early cancer stages, thereby reducing the incidence of malignant tumors.

[0096] 7. The sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker of the present invention can be used as an immunomodulatory drug for neoplastic diseases. In other words, as long as a certain tumor has the characteristic of abnormally high expression of sialic acid on the cell membrane surface, the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker provided by the present invention can be used as an immunomodulatory drug to modify the sialyl-sialic acid on the surface of the tumor cells, change the local molecular structure and charge characteristics, thereby reducing the affinity of sialyl-binding immunoglobulin-like lectin on the surface of the patient's own immune cells (such as T cells, NK cells, etc.), blocking the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway, relieving the inhibition of immune cell function by this signaling pathway, and promoting the recognition and killing of tumor cells. Attached Figure Description

[0097] Figure 1This is a schematic diagram illustrating the principle of relieving the immunosuppression of immune cells by Siglec on the surface of tumor cells through TJ101 (N-acetylneuraminic acid methyl ester) blocking the sialic acid-sialic acid binding immunoglobulin-like lectin signaling pathway.

[0098] Figure 2 This refers to the weight statistics of male mice in Example 1;

[0099] Figure 3 This is a statistical analysis of the weight of female mice in Example 1;

[0100] Figure 4 This is a statistical analysis of the organ coefficients of the experimental mice in Example 1;

[0101] Figure 5 The results are statistically significant from Example 2. Among them, (A) is a photograph of the tumor after the experiment was completed, (B) is a statistical analysis of the weight of the tumor after the experiment was completed, (C) is a statistical analysis of the tumor volume during the experiment, and (D) is a statistical analysis of the weight of the mice during the experiment.

[0102] Figure 6 The results of the killing activity of NK cells combined with N-acetylneuraminic acid methyl ester (TJ101) against human lung adenocarcinoma H1975 cells in Example 3 are shown in Figure A. (Figure A: Killing activity curve of NK cells combined with N-acetylneuraminic acid methyl ester (TJ101) against human lung adenocarcinoma H1975 cells; Figure B: Killing activity of NK cells combined with N-acetylneuraminic acid methyl ester (TJ101) against human lung adenocarcinoma H1975 cells after 12 hours; Figure C: Killing activity of NK cells combined with N-acetylneuraminic acid methyl ester (TJ101) against human lung adenocarcinoma H1975 cells after 24 hours.)

[0103] Figure 7 This is the result of the killing activity of NK cells combined with N-acetylneuraminic acid methyl ester (TJ101) against human non-small cell lung cancer Hcc827 cells in Example 4; (Figure A, killing activity curve of NK cells combined with N-acetylneuraminic acid methyl ester (TJ101) against human non-small cell lung cancer Hcc827 cells; Figure B, killing activity of NK cells combined with N-acetylneuraminic acid methyl ester (TJ101) against human non-small cell lung cancer Hcc827 cells after 12 hours; Figure C, killing activity of NK cells combined with N-acetylneuraminic acid methyl ester (TJ101) against human non-small cell lung cancer Hcc827 cells after 24 hours.)

[0104] Figure 8The results of Example 5 show the cytotoxic activity of NK cells combined with 9-biotinylated-N-acetylneuraminic acid methyl ester (SJ301) against human non-small cell lung cancer Hcc827 cells; (Figure A, cytotoxic activity curve of NK cells combined with 9-biotinylated-N-acetylneuraminic acid methyl ester (SJ301) against human non-small cell lung cancer Hcc827 cells; Figure B, cytotoxic activity of NK cells combined with 9-biotinylated-N-acetylneuraminic acid methyl ester (SJ301) against human non-small cell lung cancer Hcc827 cells after 6 hours; Figure C, cytotoxic activity of NK cells combined with 9-biotinylated-N-acetylneuraminic acid methyl ester (SJ301) against human non-small cell lung cancer Hcc827 cells after 12 hours.)

[0105] Figure 9 This is the complete liver function and blood lipid test report of patient He Moumou from Example 7, dated February 19, 2024;

[0106] Figure 10 This is the complete liver function and blood lipid test report of patient He Moumou from Example 7, dated March 5, 2024;

[0107] Figure 11 These are the thyroid ultrasound examination results of patient Feng Moumou from Example 9, dated July 11, 2023 and December 8, 2023;

[0108] Figure 12 These are the CT scan, tumor marker, and liver and kidney function test results of patient Yang from Example 10, dated November 15, 2024 and December 18, 2024.

[0109] Figure 13 This is the blood routine test report of patient Chen from Example 14, dated April 13, 2024;

[0110] Figure 14 This is the blood routine test report of patient Chen Moumou from Example 14, dated April 23, 2024. Detailed Implementation

[0111] In existing anticancer or antiviral drugs, there is no precedent for blocking the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway by modifying sialyl glucan to change its local molecular structure and charge characteristics. Through dedicated research, the inventors of this invention have obtained a broader-spectrum drug and its application for blocking the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway. Specifically, as a first aspect of this invention, a sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker is provided, which contains one or more N-acetylneuraminic acid derivatives.

[0112] In a preferred embodiment, the N-acetylneuraminic acid derivative is selected from N-acetylneuraminic acid esters.

[0113] In a preferred embodiment, the N-acetylneuraminic acid ester is selected from 2,4,7,8,9-penta-O-acetyl-N-acetylneuraminic acid deuterated methyl ester, 4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid deuterated methyl ester, N-acetylneuraminic acid methyl ester, N-acetylneuraminic acid ethyl ester, 2,4,7,8,9-penta-O-acetyl-N-acetylneuraminic acid methyl ester, 4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester, and N-acetylneuraminic acid... Any one or more of the following: deuterated methyl ester, deuterated ethyl ester of N-acetylneuraminic acid, propyl ester of N-acetylneuraminic acid, isopropyl ester of N-acetylneuraminic acid, n-butyl ester of N-acetylneuraminic acid, sec-butyl ester of N-acetylneuraminic acid, tert-butyl ester of N-acetylneuraminic acid, isobutyl ester of N-acetylneuraminic acid, methyl ester cyclodextrin inclusion complex of N-acetylneuraminic acid, ethyl ester cyclodextrin inclusion complex of N-acetylneuraminic acid, deuterated methyl ester cyclodextrin inclusion complex of N-acetylneuraminic acid, and deuterated ethyl ester cyclodextrin inclusion complex of N-acetylneuraminic acid.

[0114] In a preferred embodiment, the N-acetylneuraminic acid derivative is selected from the group with the following general structural formula: N-acetylneuraminic acid esters, wherein R1 is H or OH, and R2 is a benzene ring substituent, a biotin substituent, a folic acid substituent, or an RGD substituent; and / or

[0115] R2 is OH, and R1 is a benzene ring substituent, a biotin substituent, a folic acid substituent, or an RGD substituent; and / or

[0116] R1 is a benzene ring substituent, a biotin substituent, a folic acid substituent, or an RGD substituent; R2 is a benzene ring substituent, a biotin substituent, a folic acid substituent, or an RGD substituent.

[0117] Wherein, the benzene ring substituent is ; and / or

[0118] The biotinylate substituent is and / or

[0119] The folic acid substituents are selected from...

[0120] and / or

[0121] and / or

[0122] The RGD-type substituent is

[0123]

[0124] In a preferred embodiment, the N-acetylneuraminic acid derivative is selected from the group with the following general structural formula: The compound wherein R is an alkyl, cycloalkyl, substituted alkyl, substituted cycloalkyl, thioester, thioether, ether group, disulfide ester, dithiomethyl, S-CH3, S-CH2-CH3, aryl, substituted aryl, methionine, methionine-zinc, sodium, phenol or phenol derivative.

[0125] In a preferred embodiment, the N-acetylneuraminic acid derivative is selected from N-acetylneuraminic acid derivative cyclodextrin inclusion complexes.

[0126] In a preferred embodiment, the cyclodextrin is selected from any one of β-cyclodextrin or its derivatives, γ-cyclodextrin or its derivatives, α-cyclodextrin or its derivatives, wherein the cyclodextrin is hydroxypropyl-β-cyclodextrin, or hydroxypropyl-γ-cyclodextrin, or dimethyl-β-cyclodextrin, or dimethyl-γ-cyclodextrin, or sulfobutyl-β-cyclodextrin.

[0127] In a second aspect of the invention, the signaling pathway blocker is used to alter the molecular structure and charge characteristics of the sialyte terminals on the surface of tumor cells or virus-infected cells; wherein the tumor cells or virus-infected cells are tumor cells or virus-infected cells with abnormal sialylation.

[0128] The signaling pathway blocker is used to block the immunosuppressive signaling pathway between sialylated glycans on the surface of tumor cells or virus-infected cells and sialic acid-binding immunoglobulin-like lectins on the surface of immune cells.

[0129] In a third aspect of the invention, the use of the signaling pathway blocker described in the second aspect of the invention in manufacturing an immunotherapy synergistic drug is disclosed.

[0130] In a preferred embodiment, the immunotherapy synergist is used in combination with other immunotherapy drugs;

[0131] The immunotherapy drugs include immune cell preparations, immune checkpoint inhibitors, tumor vaccine preparations, T cell connector drugs, NK cell connector drugs, antibody drugs with ADCC activity, and immunomodulators.

[0132] As a preferred embodiment, as an immunotherapy synergist (immunotherapy synergist drug), it is used to synergistically increase the cytotoxic activity against tumor cells with immune cell preparations, to synergistically increase the cytotoxic activity against virus-infected cells with immune cell preparations, to synergistically enhance the tumor treatment effect with immune checkpoint inhibitors, to synergistically enhance the tumor treatment effect with tumor vaccine preparations, to synergistically increase the immune clearance effect against tumor cells with T cell connector drugs, to synergistically increase the immune clearance effect against tumor cells with NK cell connector drugs, to synergistically increase the immune clearance effect against tumor cells with antibody drugs with ADCC activity, to synergistically increase the immune clearance effect against tumor cells with immunomodulatory drugs, and to synergistically increase the immune clearance effect against virus-infected cells with immunomodulatory drugs.

[0133] In a preferred embodiment, the immune cell preparation includes NK cell preparations, CAR-NK cell preparations, αβT cell preparations, γδT cell preparations, TIL cell preparations, NKT cell preparations, CAR-T cell preparations, TCR-T cell preparations, CIK cell preparations, DC-CIK cell preparations, and macrophages. preparation, Cellular preparations, neutrophil preparations, dendritic cell (DC) preparations, B cell preparations, myeloid-derived suppressor cell (MDSC) preparations, Treg cell preparations;

[0134] In a preferred embodiment, the immune checkpoint inhibitors include PD-1 antibody preparations, PD-L1 antibody preparations, CTLA-4 antibody preparations, LAG-3 antibody preparations, TIM-3 antibody preparations, TIGIT antibody preparations, and VISTA antibody preparations.

[0135] In a preferred embodiment, the tumor vaccine formulation includes whole-cell tumor vaccines, tumor peptide vaccines, genetically engineered mRNA tumor vaccines, genetically engineered DNA tumor vaccines, tumor subunit vaccines, heat shock protein tumor vaccines, and antibody tumor vaccines.

[0136] In a preferred embodiment, the immunotherapy synergist is used in combination with the immunotherapy drug to treat tumors and prevent potential malignant diseases from becoming cancerous.

[0137] The tumors include solid tumors and hematologic malignancies;

[0138] The solid tumors include lung cancer, breast cancer, esophageal cancer, gastric cancer, cardia cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, thyroid cancer, parathyroid cancer, kidney cancer, bladder cancer, penile cancer, malignant melanoma, and glioma.

[0139] The hematologic malignancies include leukemia, multiple myeloma, and malignant lymphoma;

[0140] The potential malignant diseases include leukoplakia, atrophic gastritis, cirrhosis, pulmonary nodules, adenomatous polyps, Barrett's esophagus, Crohn's disease, ulcerative colitis, borderline ovarian tumors, familial adenomatous polyposis (FAP), Lynch syndrome, BRCA1 / 2 gene mutation diseases, myelodysplastic syndrome (MDS), monoclonal gammopathy (MGUS), thyroid nodules, melanocytic nevi, endometrial hyperplasia, cervical intraepithelial neoplasia (CIN), intraductal carcinoma in situ (DCIS) of the breast, and lobular carcinoma in situ (LCIS) of the breast.

[0141] In a preferred embodiment, the immunotherapy synergist is used in combination with the immunotherapy drug to treat viral infections;

[0142] The viral infections mentioned include human immunodeficiency virus (HIV) infection, influenza virus infection, coronavirus infection, rhinovirus infection, mumps virus infection, respiratory syncytial virus (RSV) infection, cytomegalovirus infection, hepatitis B virus infection, hepatitis C virus infection, human papillomavirus (HPV) infection, Ebola virus infection, herpes simplex virus infection, adenovirus infection, rubella virus infection, norovirus infection, Zika virus, parainfluenza virus infection, measles virus infection, avian influenza virus infection, varicella-zoster virus infection, Newcastle disease virus infection, Sendai virus, and rotavirus infection.

[0143] In a preferred embodiment, the signaling pathway blocker is used to manufacture an immunomodulatory drug, which is used to enhance the anti-tumor immunity of cancer patients and to treat tumors and prevent potential malignant diseases from becoming cancerous.

[0144] The tumors include solid tumors and hematologic malignancies;

[0145] The solid tumors include lung cancer, breast cancer, esophageal cancer, gastric cancer, cardia cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, thyroid cancer, parathyroid cancer, kidney cancer, bladder cancer, penile cancer, malignant melanoma, and glioma.

[0146] The hematologic malignancies include leukemia, multiple myeloma, and malignant lymphoma;

[0147] The potential malignant diseases include leukoplakia, atrophic gastritis, cirrhosis, pulmonary nodules, adenomatous polyps, Barrett's esophagus, Crohn's disease, ulcerative colitis, borderline ovarian tumors, familial adenomatous polyposis (FAP), Lynch syndrome, BRCA1 / 2 gene mutation diseases, myelodysplastic syndrome (MDS), monoclonal gammopathy (MGUS), thyroid nodules, melanocytic nevi, endometrial hyperplasia, cervical intraepithelial neoplasia (CIN), intraductal carcinoma in situ (DCIS) of the breast, and lobular carcinoma in situ (LCIS) of the breast.

[0148] In a preferred embodiment, the signaling pathway blocker is used to manufacture an immunomodulatory drug, the immunomodulatory drug is used to enhance the antiviral immunity of virus-infected individuals, and the immunomodulatory drug is used to treat viral infections;

[0149] The viral infections mentioned include human immunodeficiency virus (HIV) infection, influenza virus infection, coronavirus infection, rhinovirus infection, mumps virus infection, respiratory syncytial virus (RSV) infection, cytomegalovirus infection, hepatitis B virus infection, hepatitis C virus infection, human papillomavirus (HPV) infection, Ebola virus infection, herpes simplex virus infection, adenovirus infection, rubella virus infection, norovirus infection, Zika virus, parainfluenza virus infection, measles virus infection, avian influenza virus infection, varicella-zoster virus infection, Newcastle disease virus infection, Sendai virus, and rotavirus infection.

[0150] Fourthly, the present invention provides a medicament and a pharmaceutical composition for use as an inhibitor of the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway, comprising the signaling pathway inhibitor described in the first aspect of the present invention, and a pharmaceutically acceptable dosage form.

[0151] The dosage forms of the drug and drug composition are tablets, capsules, injections, granules, suppositories, nasal sprays, oral sprays, nebulized inhalers, powders, powder inhalers, ointments, or gels.

[0152] Fifthly, the present invention provides a method for blocking the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway, characterized in that it includes blocking the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway using an effective amount of the signaling pathway blocker described in the first aspect of the present invention.

[0153] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0154] I. Animal Experiments

[0155] Example 1: Acute toxicity test of N-acetylneuraminic acid methyl ester

[0156] In the preliminary acute toxicity test, when the dosage of N-acetylneuraminic acid methyl ester reached 5.1 g / kg / d, no mice died after three days of observation. Therefore, N-acetylneuraminic acid methyl ester was considered to be a low-toxicity compound. According to the "Technical Guidelines for Acute Toxicity Testing of Chemical Drugs" (Guideline No.: [H]GPT1-1), the maximum dose method was used in this embodiment.

[0157] SPF-grade ICR mice were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. SPF (Specific Pathogen Free) animals are laboratory animals that, apart from pathogens that should be excluded from clean animals, do not carry major potential infectious agents, opportunistic pathogens, or pathogens that significantly interfere with scientific experiments. ICR mice are named after the Institute of Cancer Research in the United States; ICR stands for the first three letters of the Institute of Cancer Research. They have since been introduced worldwide and have become the internationally recognized closed-colony mouse species.

[0158] SPF-grade ICR mice were grouped by sex, with five mice of the same sex placed in each cage. The ambient temperature was 20-25℃, and the relative humidity was around 60%. The mice were given free access to water and food and were allowed to acclimatize for 5 days.

[0159] Mice were randomly divided into groups of 20 mice each, with half male and half female, according to the different drugs used. The groups were as follows:

[0160] 1) Saline control group (control group);

[0161] 2) SA (sialic acid) group (5g / kg);

[0162] 3) TJ101 (N-acetylneuraminic acid methyl ester) group (5g / kg);

[0163] 4) TD102 (N-acetylneuraminic acid deuterated methyl ester) group (5g / kg);

[0164] 5) TB201 (N-acetylneuraminic acid methyl ester-hydroxypropyl-β-cyclodextrin inclusion complex) group (5g / kg);

[0165] 6) LT301 (polylysine-sialic acid binary polymer) group (5g / kg).

[0166] SA, TJ101, TD102, TB201, and LT301 solutions were all administered via gavage, once on the first day of the experiment. Mice were fasted for 12 hours before administration but allowed free access to water. During the administration period, mouse weight was measured every two days, and acute toxicity indicators were observed for 14 consecutive days. After the observation period, mice were euthanized by cervical dislocation, and the heart, liver, spleen, kidneys, testes, and lungs were dissected. Organ coefficients were calculated. Subsequently, one group was fixed in 4% paraformaldehyde, and the other group was placed in a -80°C freezer.

[0167] In this embodiment, acute toxicity tests were conducted on five compounds, namely SA (sialic acid), TJ101 (N-acetylneuraminic acid methyl ester), TD102, TB201, and LT301, using the maximum dose method. The effects on skin, mucous membranes, coat color, eyes, respiration, circulation, spontaneous activity and central nervous system behavior, and body weight were observed for 14 days. The animals were then sacrificed, and their organ coefficients were measured.

[0168] Organization Coefficient = Organ weight / Body weight.

[0169] LT301 male mice exhibited toxicity characteristics including spontaneous activity, exploration, grooming, reduced movement, drowsiness, genital redness and ulceration, and spleen enlargement. No significant differences were found between the other groups (SA, TJ101, TD102, TB201) and the normal control group. The results of this example indicate that TJ101 (N-acetylneuraminic acid methyl ester) had no effect on the body weight of male mice in the acute toxicity test. Figure 2 It had no effect on the body weight of female mice. Figure 3 It had no effect on the organ coefficients of the heart, liver, spleen, kidney, testis, and lungs in the experimental mice. Figure 4 Therefore, it has good security.

[0170] Example 2: Effect of N-acetylneuraminic acid methyl ester on H22 subcutaneous xenograft tumors

[0171] 1) Preparation of a mouse liver cancer model: Ascites fluid cells from mouse H22 liver cancer cells passaged three times were collected and diluted 1:1 with 0.9% sodium chloride injection. The cells were then mixed using a 1ml pipette. After disinfecting the skin around the injection site with an alcohol swab, 0.2 ml of H22 cell suspension was drawn into a disposable syringe and slowly injected subcutaneously into the axilla of the mouse's right forelimb. After injection, the area was gently pressed with a sterile cotton ball to promote absorption of the suspension. The entire procedure was completed within 60 minutes. The mouse was observed for 10 minutes after injection. If the mouse's general condition and activity level were normal, it was returned to its cage.

[0172] On days 2-3, a subcutaneous mass the size of a soybean (approximately 3mm × 5mm measured with calipers) was observed at the inoculation site of the mice, indicating that the H22 hepatocellular carcinoma-bearing mouse model was successfully established.

[0173] 2) Administration method: After successful modeling, all model mice were immediately randomly divided into five groups:

[0174] ① Control group (n=7); ② Cisplatin group (n=7) (cisplatin, a platinum-containing anticancer drug, administered at a concentration of 25 mg / kg) via intraperitoneal injection twice a week; ③ TJ101 low-dose group (n=75 mg / kg); ④ TJ101 medium-dose group (n=750 mg / kg); ⑤ TJ101 high-dose group (n=700 mg / kg).

[0175] On the day the model was successfully established, the medication was administered via gavage once a day.

[0176] 3) General condition observation: During the drug administration period, the general condition of mice in each group was observed and recorded every 2 days, including eating, drinking, hair changes, mental state, and spontaneous activity; the body weight of mice was monitored using an electronic balance. The maximum length and maximum width of the tumor in the axillary region of the mice were measured using calipers, and the tumor volume was calculated as (the length of the major axis multiplied by the square of the minor axis divided by 2).

[0177] 4) Calculation of tumor weight inhibition rate: On the second day after the last administration, weigh all mice and measure the tumor volume. Then, euthanize them by cervical dislocation and dissect the subcutaneous tumor tissue. Weigh the tissue and calculate the tumor weight inhibition rate.

[0178] Tumor weight suppression rate (%) = (CT) / C × 100%

[0179] (T: average tumor weight in the treatment group; C: average tumor weight in the saline group).

[0180] 5) Experimental Results: Through the establishment of the H22 cell mouse subcutaneous xenograft model, the results are as follows: Figure 5 As shown in the figure. Compared with the control group, both the cisplatin group and the TJ101 group showed significant growth inhibition on H22 cell xenografts. Based on the tumor inhibition rate calculation, the tumor inhibition rate of the positive control cisplatin group was 67.59%, while the tumor inhibition rates of the low-dose TJ101 group (75 mg / kg), medium-dose TJ101 group (150 mg / kg), and high-dose TJ101 group (300 mg / kg) were 44.23%, 57.05%, and 54.37%, respectively. Furthermore, monitoring of body weight changes in mice during the treatment period revealed no significant changes in body weight in the TJ101 (N-acetylneuraminic acid methyl ester) groups, demonstrating the safety of TJ101 (N-acetylneuraminic acid methyl ester) treatment.

[0181] II. Cell-level experiments

[0182] Example 3: In vitro killing activity test of N-acetylneuraminic acid methyl ester (TJ101) combined with NK cells against human lung adenocarcinoma H1975 cells

[0183] 1) Preparation of NK cells (natural killer cells): Under aseptic conditions in a biosafety cabinet, pipette 20 μl of NK cells (prepared from human peripheral blood PBMCs; NK cell in vitro expansion kit purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd.), mix with trypan blue at a 1:1 ratio, and measure cell density. As needed, transfer an appropriate amount of NK cells to a new sterile T75 flask, add an appropriate amount of culture medium to adjust the cell density to 102. 6 / ml, place in a 5% CO2 incubator and continue incubation for 24 hours for later use.

[0184] 2) Preparation of adherent tumor cell suspension: Under aseptic conditions, take human lung adenocarcinoma H1975 cells cultured in T75 flasks, remove the culture medium under aseptic conditions, wash twice with 3 ml of PBS (phosphate-buffered saline), and add 1.5 ml of trypsin solution containing EDTA. Incubate at 37°C, observing the cell digestion under an inverted microscope. If cytoplasmic retraction and cell intervals increase, add fresh culture medium to stop digestion. Transfer the digested H1975 cells to a 15 ml centrifuge tube, centrifuge at 800 rpm for 5 min, remove the supernatant, resuspend in 3 ml of culture medium, count a small amount, and adjust the cell density to 8 × 10⁶ cells / mL. 4 / ml.

[0185] 3) Preparation of 96-well E-Plate: Add 50 μl of serum-free culture medium to each well of the 96-well E-Plate. Place the 96-well E-Plate on a Real-Time Label-Free Cell Analyzer (RTCA Station). The RTCA system will automatically scan the plate ("Scan Plate") to check for good contact (the "Message" page will display "Connection OK"). Click the "Layout" page and set the cell names and numbers for each well. Click the start icon to begin baseline detection and confirm that the selected wells are in good contact. Remove the 96-well E-Plate and add 100 μl of well-mixed tumor cell suspension to each well, ensuring a cell count of 8 × 10⁶ cells per well. 3 Cells / 100μl. Divided into 5 groups: control group, NK group, combination drug group 1 (NK+TJ101 low concentration), combination drug group 2 (NK+TJ101 medium concentration), and combination drug group 3 (NK+TJ101 high concentration).

[0186] In combination therapy group 1 (NK+TJ101 low concentration), a low concentration of N-acetylneuraminic acid methyl ester was added, with a final concentration of 50 μM; in combination therapy group 2 (NK+TJ101 medium concentration), a medium concentration of N-acetylneuraminic acid methyl ester was added, with a final concentration of 100 μM; and in combination therapy group 3 (NK+TJ101 high concentration), a high concentration of N-acetylneuraminic acid methyl ester was added, with a final concentration of 200 μM.

[0187] Table 1 Experimental setup for Example 3

[0188]

[0189] Subsequently, the 96-well E-Plate was placed in a clean bench at room temperature for 30 minutes. The 96-well E-Plate was then placed on the RTCA Station in the incubator. After the system automatically scanned the "Scan Plate," step 2 began to dynamically monitor cell proliferation.

[0190] 4) RTCA program setup: After baseline measurements, cell index (CI) was measured every 15 minutes to record cell growth. After adding effector cells, the killing activity against tumor cells in each group was measured every 15 minutes to observe the activity.

[0191] Table 2 Parameter Settings for the RTCA Program

[0192]

[0193] 5) Addition of NK cells: On the second day, terminate step 2 and remove the 96-well E-Plate from the RTCAStation. Add 50 μl of NK cells to each well, with a cell density of 3.2 × 10⁻⁶ cells per group. 5 / ml, target-effect ratio 2:1. Reinsert the E-Plate96 into the RTCA Station. The RTCA system will automatically perform a scan (“Scan Plate”) -> check for good contact (the “Message” page will display “Connection OK”). Proceed to step 3.

[0194] 6) Test Results: The results of this embodiment are as follows Figure 5 As shown. From Figure 6 It can be seen that after adding N-acetylneuraminic acid methyl ester to the culture medium of human lung adenocarcinoma H1975 cells, the killing activity of NK cells against human lung adenocarcinoma H1975 cells increased with the increase of the concentration of N-acetylneuraminic acid methyl ester. Figure 6A). At 12 h, the cell inhibition rate was 0.00% in the control group, 29.43% in the NK group, 46.86% in the combined drug group 1 (low concentration of NK+TJ101), 61.73% in the combined drug group 2 (medium concentration of NK+TJ101), and 69.51% in the combined drug group 3 (high concentration of NK+TJ101). At 24 h, the cell inhibition rate was 0.00% in the control group, 37.44% in the NK group, 54.55% in the combined drug group 1 (low concentration of NK+TJ101), 75.01% in the combined drug group 2 (medium concentration of NK+TJ101), and 79.21% in the combined drug group 3 (high concentration of NK+TJ101). Figure 6 B). Figure 6 The cell inhibition rate of B cells was as follows: combined drug group 3 (NK + high concentration of TJ101) > combined drug group 2 (NK + medium concentration of TJ101) > combined drug group 1 (NK + low concentration of TJ101) > NK group. This indicates that TJ101 has a synergistic effect on the tumor killing activity of NK cells, and within a certain range, this enhancement of killing activity is dose-dependent on N-acetylneuraminic acid methyl ester.

[0195] The above experimental results indicate that the addition of N-acetylneuraminic acid methyl ester to the culture medium of human lung adenocarcinoma H1975 cells can ultimately be presented to the surface of H1975 tumor cells via the intracellular sialic acid metabolic pathway. This causes the carboxyl group of sialic acid at the terminal end of glycolipids or glycoproteins on the surface of H1975 tumor cells to be converted into a methyl ester group, thereby altering the local molecular structure and charge characteristics of sialic acid glycans at the terminal end of glycolipids or glycoproteins. This leads to a decrease in the affinity between these modified sialic acid receptors and members of the Siglec family of immunosuppressive receptors on the surface of NK cells, blocking the sialic acid-sialic acid binding immunoglobulin-like lectin signaling pathway, relieving its immunosuppressive effect on NK cells, and thus promoting NK cells to kill H1975 tumor cells, exerting a synergistic effect of N-acetylneuraminic acid methyl ester on tumor immunotherapy.

[0196] Example 4: In vitro killing activity test of N-acetylneuraminic acid methyl ester (TJ101) combined with NK cells against human non-small cell lung cancer Hcc827 cells

[0197] 1) Preparation of NK cells: Under aseptic conditions in a biosafety cabinet, pipette 20 μl of NK cells (prepared from human peripheral blood PBMCs; NK cell in vitro expansion kit purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd.), mix with trypan blue at a 1:1 ratio, and measure cell density. As needed, transfer an appropriate amount of NK cells to a new sterile T75 flask, add an appropriate amount of culture medium to adjust the cell density to 102. 6 / ml, place in a 5% CO2 incubator and continue incubation for 24 hours for later use.

[0198] 2) Preparation of adherent tumor cell suspension: Under aseptic conditions, take human non-small cell lung cancer (NSCLC) Hcc827 cells cultured in T75 flasks, remove the culture medium under aseptic conditions, wash twice with 3 ml of PBS, and add 1.5 ml of trypsin solution containing EDTA. Incubate at 37°C, observing the digestion of cells under an inverted microscope. If cytoplasmic retraction and cell intervals increase, add fresh culture medium to stop digestion. Transfer the digested human NSCLC Hcc827 cells to a 15 ml centrifuge tube, centrifuge at 800 rpm for 5 min, remove the supernatant, resuspend in 3 ml of culture medium, count a small amount, and adjust the cell density to 8 × 10⁻⁶ cells. 4 / ml.

[0199] 3) Preparation of 96-well E-Plate: Add 50 μl of serum-free culture medium to each well of the 96-well E-Plate. Place the 96-well E-Plate on a Real-Time Label-Free Cell Analyzer (RTCA Station). The RTCA system will automatically scan the plate ("Scan Plate") to check for good contact (the "Message" page will display "Connection OK"). Click the "Layout" page and set the cell names and numbers for each well. Click the start icon to begin baseline detection and confirm that the selected wells are in good contact. Remove the 96-well E-Plate and add 100 μl of well-mixed tumor cell suspension to each well, ensuring a cell count of 8 × 10⁶ cells per well. 3 Cells / 100μl. Divided into 5 groups: control group, NK group, combination drug group 1 (NK+TJ101 low concentration), combination drug group 2 (NK+TJ101 medium concentration), and combination drug group 3 (NK+TJ101 high concentration).

[0200] In combination drug group 1 (NK+TJ101 low concentration), a low concentration of N-acetylneuraminic acid methyl ester was added to the final concentration of 50 μM. In combination drug group 2 (NK+TJ101 medium concentration), a medium concentration of N-acetylneuraminic acid methyl ester was added to the final concentration of 100 μM. In combination drug group 3 (NK+TJ101 high concentration), a high concentration of N-acetylneuraminic acid methyl ester was added to the final concentration of 200 μM.

[0201] Table 3 Experimental setup for Example 4

[0202]

[0203] Subsequently, the 96-well E-Plate was placed in a clean bench at room temperature for 30 minutes. The 96-well E-Plate was then placed on the RTCA Station in the incubator. After the system automatically scanned the "Scan Plate," step 2 began to dynamically monitor cell proliferation.

[0204] 4) RTCA program settings

[0205] After baseline measurements, the cell index (CI) was measured every 15 minutes to record cell growth. After the addition of effector cells, the killing activity against tumor cells in each group was measured every 15 minutes to observe the activity.

[0206] Table 4. Parameter settings for the RTCA program

[0207]

[0208]

[0209] 5) Addition of NK cells: On the second day, terminate step 2 and remove the 96-well E-Plate plate from the RTCAStation. Add 50 μl of NK cells to each well, with a cell density of 3.2 × 10⁻⁶ cells per group. 5 / ml, target-effect ratio 2:1. Reinsert the E-Plate96 into the RTCA Station. The RTCA system will automatically perform a scan (“Scan Plate”) -> check for good contact (“Connection OK” will be displayed on the “Message” page). Proceed to step 3.

[0210] 6) Experimental Results: The results of Example 4 are as follows Figure 6 As shown. From Figure 7 It can be seen that after adding N-acetylneuraminic acid methyl ester to the culture medium of human non-small cell lung cancer (NSCLC) Hcc827 cells, the killing activity of NK cells against Hcc827 cells in each combination drug group was significantly increased compared with that of the NK group alone. Among them, the killing activity of low-concentration combination drug group 1 and medium-concentration combination drug group 2 against Hcc827 cells was similar, while the high-concentration combination drug group 3 showed the best killing activity against Hcc827 cells. Figure 7A). At 12 h, the cell inhibition rate was 0.00% in the control group, 18.34% in the NK group, 28.62% in the combined drug group 1 (low concentration of NK+TJ101), 36.61% in the combined drug group 2 (medium concentration of NK+TJ101), and 49.79% in the combined drug group 3 (high concentration of NK+TJ101). At 24 h, the cell inhibition rate was 0.00% in the control group, 18.28% in the NK group, 35.55% in the combined drug group 1 (low concentration of NK+TJ101), 39.39% in the combined drug group 2 (medium concentration of NK+TJ101), and 55.58% in the combined drug group 3 (high concentration of NK+TJ101). Figure 7 B). Figure 7 The cell inhibition rate of B cells was as follows: combined drug group 3 (NK + high concentration of TJ101) > combined drug group 2 (NK + medium concentration of TJ101) ≈ combined drug group 1 (NK + low concentration of TJ101) > NK group. This indicates that TJ101 has a synergistic effect on the tumor killing activity of NK cells, and within a certain range, this enhancement of killing activity is dose-dependent on N-acetylneuraminic acid methyl ester.

[0211] The above results indicate that the addition of N-acetylneuraminic acid methyl ester to the culture medium of human non-small cell lung cancer Hcc827 cells can ultimately be presented to the surface of Hcc827 tumor cells via the intracellular sialic acid metabolic pathway. This causes the carboxyl group of sialic acid at the terminal end of glycolipids or glycoproteins on the surface of Hcc827 tumor cells to be converted into a methyl ester group, thereby altering the local molecular structure and charge characteristics of sialic acid glycans at the terminal end of glycolipids or glycoproteins. This leads to a decrease in the affinity between these modified sialic acid receptors and members of the Siglec family of immunosuppressive receptors on the surface of NK cells, blocking the sialic acid-sialic acid binding immunoglobulin-like lectin signaling pathway, relieving its immunosuppressive effect on NK cells, and thus promoting NK cells to kill Hcc827 tumor cells, exerting a synergistic effect of N-acetylneuraminic acid methyl ester on tumor immunotherapy.

[0212] Example 5: In vitro killing activity assay of 9-biotinyl-N-acetylneuraminic acid methyl ester (SJ301) combined with NK cells against human non-small cell lung cancer Hcc827 cells.

[0213] 1) Preparation of NK cells and SJ301: Under aseptic conditions in a biosafety cabinet, pipette 20 μL of NK cells (prepared from human peripheral blood PBMCs; NK cell in vitro expansion kit purchased from Beijing Tongli Haiyuan Biotechnology Co., Ltd.), mix with trypan blue at a 1:1 ratio, and measure cell density. As needed, transfer an appropriate amount of NK cells to a new sterile T75 flask, add an appropriate amount of culture medium to adjust the cell density to 102.6 The sample was incubated at 5% CO2 for 24 hours. 9-Biotinyl-N-acetylneuraminic acid methyl ester (SJ301) was prepared according to the method described in Example 1 of CN 110128490A, wherein compound (4) is 9-Biotinyl-N-acetylneuraminic acid methyl ester (SJ301).

[0214] 2) Preparation of adherent tumor cell suspension: Under aseptic conditions, take human non-small cell lung cancer Hcc827 cells cultured in T75 flasks, aspirate the culture medium under aseptic conditions, wash twice with 3 ml of PBS, and add 1.5 ml of trypsin solution containing EDTA. Incubate at 37°C. During incubation, observe the digestion of cells under an inverted microscope. If the cytoplasm shrinks and the cell intervals increase, add fresh culture medium to stop the digestion.

[0215] Digested human non-small cell lung cancer Hcc827 cells were transferred to 15ml centrifuge tubes, centrifuged at 800rpm for 5min, the supernatant was discarded, and the cells were resuspended in 3ml of culture medium. A small number of cells were counted, and the cell density was adjusted to 8×10⁻⁶. 4 / ml.

[0216] 3) Preparation of 96-well E-Plate: Add 50 μl of serum-free culture medium to each well of the 96-well E-Plate. Place the 96-well E-Plate on the Real-Time Label-Free Cell Analyzer (RTCA Station). The RTCA system will automatically scan (“ScanPlate”) to check for good contact (the “Message” page will display “Connection OK”). Click the “Layout” page to set the cell names and numbers for the test wells. Click the start icon to begin baseline detection and confirm that the selected wells are in good contact.

[0217] Remove the 96-well E-Plate plate and add 100 μl of well-mixed tumor cell suspension to each well, ensuring a cell count of 8 × 10⁶ cells per well. 3Cells / 100μl. Divided into 5 groups: control group, NK group, combination therapy group 1 (NK + SJ301 low concentration), combination therapy group 2 (NK + SJ301 medium concentration), and combination therapy group 3 (NK + SJ301 high concentration). Combination therapy group 1 (NK + TJ101 low concentration) was supplemented with a low concentration of 9-biotinylated-N-acetylneuraminic acid methyl ester (SJ301) to a final concentration of 50μM; combination therapy group 2 (NK + TJ101 medium concentration) was supplemented with a medium concentration of 9-biotinylated-N-acetylneuraminic acid methyl ester (SJ301) to a final concentration of 100μM; and combination therapy group 3 (NK + TJ101 high concentration) was supplemented with a high concentration of 9-biotinylated-N-acetylneuraminic acid methyl ester (SJ301) to a final concentration of 200μM.

[0218] Table 5. Experimental setup for Example 5

[0219]

[0220] Place the 96-well E-Plate in a clean bench at room temperature for 30 minutes. Then place the 96-well E-Plate on the RTCA Station in the incubator. After the system automatically scans the "Scan Plate," begin step 2 to dynamically monitor cell proliferation.

[0221] 4) RTCA program setup: After baseline measurements, cell index (CI) was measured every 15 minutes to record cell growth. After adding effector cells, the killing activity against tumor cells in each group was measured every 15 minutes to observe the activity.

[0222] Table 6. Parameter settings for the RTCA program

[0223]

[0224] 5) Addition of NK cells: On the second day, terminate step 2 and remove the 96-well E-Plate from the RTCAStation. Add 50 μl of NK cells to each well, with a cell density of 3.2 × 10⁻⁶ cells per group. 5 / ml, target-effect ratio 2:1. Reinsert the E-Plate96 into the RTCA Station. The RTCA system will automatically perform a scan (“Scan Plate”) -> check for good contact (“Connection OK” will be displayed on the “Message” page). Proceed to step 3.

[0225] 6) Experimental Results: The results of Example 5 are as follows Figure 7 As shown. From Figure 8It can be seen that after adding 9-biotinyl-N-acetylneuraminic acid methyl ester (SJ301) to the culture medium of human non-small cell lung cancer Hcc827 cells, the killing activity of NK cells against human non-small cell lung cancer Hcc827 cells was basically the same under low, medium and high dose concentrations, suggesting that 9-biotinyl-N-acetylneuraminic acid methyl ester (SJ301) can exert a synergistic effect on tumor killing of NK cells at relatively low dose concentrations. Figure 8 A). At 6 h, the cell inhibition rate was 0.00% in the control group, 20.15% in the NK group, 48.65% in the combined drug group 1 (low concentration of NK+SJ301), 48.60% in the combined drug group 2 (medium concentration of NK+SJ301), and 47.28% in the combined drug group 3 (high concentration of NK+SJ301). At 12 h, the cell inhibition rate was 0.00% in the control group, 33.27% in the NK group, 58.47% in the combined drug group 1 (low concentration of NK+SJ301), 59.25% in the combined drug group 2 (medium concentration of NK+SJ301), and 55.72% in the combined drug group 3 (high concentration of NK+SJ301). Figure 8 B). Figure 8 The cell inhibition rate of B cells was approximately equal to that of the combined drug group 3 (NK + high concentration of SJ301) ≈ combined drug group 2 (NK + medium concentration of SJ301) ≈ combined drug group 1 (NK + low concentration of SJ301) > NK group. This indicates that SJ301 has a synergistic effect on the tumor killing activity of NK cells. Furthermore, after adding 9-biotinyl-N-acetylneuraminic acid methyl ester (SJ301) to the culture medium of human non-small cell lung cancer Hcc827 cells, the tumor killing activity of NK cells against human non-small cell lung cancer Hcc827 cells was basically the same under low, medium and high dose concentrations. This suggests that 9-biotinyl-N-acetylneuraminic acid methyl ester (SJ301) can exert a synergistic effect on the tumor killing activity of NK cells at relatively low dose concentrations.

[0226] The above results demonstrate that the addition of 9-biotinyl-N-acetylneuraminic acid methyl ester (SJ301) to the culture medium of human non-small cell lung cancer (Hcc827) cells allows it to be ultimately presented to the surface of Hcc827 tumor cells via the intracellular sialic acid metabolic pathway. This causes the carboxyl group of sialic acid at the terminal end of glycolipids or glycoproteins on the surface of Hcc827 tumor cells to be converted into a methyl ester group, thereby altering the local molecular structure and charge characteristics of the sialic acid glycans at the terminal end of glycolipids or glycoproteins. This leads to a decrease in the affinity between these modified sialic acid receptors and members of the Siglec family of immunosuppressive receptors on the surface of NK cells, blocking the sialic acid-sialic acid binding immunoglobulin-like lectin signaling pathway, relieving its immunosuppressive effect on NK cells, and thus promoting NK cell killing of Hcc827 tumor cells. This demonstrates the synergistic effect of 9-biotinyl-N-acetylneuraminic acid methyl ester (SJ301) on tumor immunotherapy.

[0227] III. Clinical Trials

[0228] Example 6: Preparation of N-acetylneuraminic acid methyl ester enteric-coated tablets

[0229] 1) Using N-acetylneuraminic acid methyl ester as the core raw material, N-acetylneuraminic acid methyl ester enteric-coated tablets are prepared, each tablet containing 80 mg of N-acetylneuraminic acid methyl ester. The material composition and proportions (mass fraction) are as follows:

[0230] N-acetylneuraminic acid methyl ester, 26.67%;

[0231] Anhydrous lactose, 36.17%;

[0232] Microcrystalline cellulose, 36.16%;

[0233] Magnesium stearate, 1.00%.

[0234] 2) Tableting method: Select a tablet press with a 9mm punch; the tablet weight test is 300mg±5%, and the hardness is 60~80N.

[0235] 3) Coating method: Select orange organic enteric coating; prepare the coating powder with organic solvent (80-85% ethanol-water mixture) to a solid content of 10% and a weight gain of 8-12%; slowly add the coating powder to the stirring organic solvent and stir the solution for 45 minutes; set the product temperature to 33℃, set the coating speed to 5-10 revolutions before the weight gain is 5%, and set the coating speed to 15-20 revolutions after the weight gain is 5%; harvest the finished product after coating is completed.

[0236] 4) Disintegration time: Six tablets of the coated product were placed in a solution with a pH of 1 and did not disintegrate within 12 hours; six tablets of the coated product were placed in a solution with a pH of 10 and completely disintegrated within 20 minutes.

[0237] Example 7: Effects of N-acetylneuraminic acid methyl ester on human liver function and lipid metabolism

[0238] Volunteer He, male, 47 years old, took 160mg of N-acetylneuraminic acid methyl ester enteric-coated tablets orally once daily on an empty stomach every morning from February 19, 2024 to March 5, 2024. Liver function and blood lipid tests were performed on February 19, 2024 and March 5, 2024, respectively. Figure 9 , Figure 10 The results showed that N-acetylneuraminic acid methyl ester had no effect on human liver function and lipid metabolism.

[0239] Example 8: N-acetylneuraminic acid methyl ester combined with NK cell preparations as adjuvant therapy for a patient with multiple tumors (who subsequently suffered from breast cancer and colon cancer).

[0240] Patient Zhu, female, 56 years old, was diagnosed with breast cancer in April 2014 and underwent tumor resection followed by targeted therapy. In July 2018, she was diagnosed with colon cancer and underwent tumor resection and adjuvant chemotherapy. In April 2023, she received combined treatment with allogeneic NK cell therapy and N-acetylneuraminic acid methyl ester enteric-coated tablets. The specific regimen was as follows: allogeneic NK cell therapy 5 billion units / infusion, once a month for 3 consecutive months; N-acetylneuraminic acid methyl ester enteric-coated tablets 160 mg orally on an empty stomach 2 hours before the NK cell therapy infusion, and then 160 mg orally once daily on an empty stomach every morning for 5 consecutive days. After receiving the combined treatment of NK cell therapy and N-acetylneuraminic acid methyl ester enteric-coated tablets, the patient reported significant improvement in her mental state and physical strength. As of the date of this invention application, there were no signs of tumor recurrence.

[0241] Example 9: N-acetylneuraminic acid methyl ester combined with NK cell preparations as adjuvant therapy for a patient with multiple tumors (who successively suffered from rectal cancer, endometrial cancer, and cervical adenocarcinoma).

[0242] Patient Chen, female, 62 years old, was diagnosed with rectal cancer in August 2012 and underwent tumor resection surgery and adjuvant chemotherapy. In April 2023, during a follow-up visit, she was diagnosed with endometrial cancer and cervical adenocarcinoma (pathological examination could not confirm that the cancer was caused by rectal cancer metastasis). She underwent tumor resection surgery and adjuvant chemotherapy. In March 2024, she received combined treatment with allogeneic NK cell therapy and N-acetylneuraminic acid methyl ester enteric-coated tablets. The specific regimen was: 5 billion allogeneic NK cell agents per dose, once a month for 3 consecutive months; 160 mg of N-acetylneuraminic acid methyl ester enteric-coated tablets orally on an empty stomach 2 hours before the NK cell infusion, followed by 160 mg of N-acetylneuraminic acid methyl ester enteric-coated tablets orally once daily on an empty stomach for 5 consecutive days. After receiving the combined treatment with NK cell therapy and N-acetylneuraminic acid methyl ester enteric-coated tablets, the patient reported significant improvement in sleep and fatigue, and weight gain. As of the date of this invention application, there were no signs of tumor recurrence.

[0243] Examples 8-9 illustrate that the sialic acid-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker provided by this invention, when used in combination with immunotherapeutic drugs, can synergistically enhance the treatment of tumors with abnormally high sialic acid expression. In other words, as long as a certain tumor has the characteristic of abnormally high expression of sialic acid on its cell membrane surface, the sialic acid-sialic acid-binding immunoglobulin-like lectin signaling pathway provided by this invention can be used to modify the sialic acid-sialic acid-binding immunoglobulin-like lectin signaling pathway on the surface of the tumor cells, thereby changing the local molecular structure and charge characteristics, thereby reducing the affinity of sialic acid for binding immunoglobulin-like lectins on the surface of immune cells (such as T cells, NK cells, etc.), blocking the sialic acid-sialic acid-binding immunoglobulin-like lectin signaling pathway, relieving the inhibition of immune cell function by this signaling pathway, and promoting the recognition and killing of tumor cells.

[0244] Various immunotherapeutic drugs, such as immune cell preparations (directly supplementing exogenous immune cells, such as NK cell preparations, CAR-NK cell preparations, αβT cell preparations, γδT cell preparations, TIL cell preparations, NKT cell preparations, CAR-T cell preparations, TCR-T cell preparations, CIK cell preparations, DC-CIK cell preparations, macrophage (Mφ) preparations, CAR-Mφ cell preparations, neutrophil preparations, dendritic cell (DC) preparations, B cell preparations, myeloid-derived suppressor cell (MDSC) preparations, Treg cell preparations, etc.), immune checkpoint inhibitors (enhancing the body's anti-tumor immune response by regulating the activity of T cells and NK cells), and tumor vaccine preparations (activating the patient's own immune system). These drugs, including those that induce cellular and humoral immune responses to control or eliminate tumors, T-cell connective drugs (engineered antibody drugs that guide T cells to kill tumors), NK-cell connective drugs (engineered antibody drugs that guide NK cells to kill tumors), antibody drugs with ADCC activity (engineered antibody drugs that guide NK cells and macrophages to kill tumors), and immunomodulators (which enhance the tumor-killing function of immune cells), all ultimately exert their therapeutic effects by promoting the killing of tumor cells by immune cells. These immune cells structurally express sialic acid-binding immunoglobulin-like lectins (Siglec) on their surface. Therefore, the sialic acid-sialic acid binding immunoglobulin-like lectin signaling pathway blocker provided by this invention is universally applicable as a synergistic agent for immunotherapy, and can be used in combination with immunotherapy drugs to treat tumors (including solid tumors and hematological malignancies) with abnormally high sialic acid expression. Suitable tumors include, but are not limited to, lung cancer, breast cancer, esophageal cancer, gastric cancer, cardia cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, thyroid cancer, parathyroid cancer, kidney cancer, bladder cancer, penile cancer, malignant melanoma, glioma, leukemia, multiple myeloma, and malignant lymphoma.

[0245] References:

[0246] [1] Filip Filipsky, Heinz Regulation of sialic acid metabolismincancer.Carbohydr Res.2024 May:539:109123.

[0247] [2]Yixian Wang,Zhan Xu,Kuan-Lin Wu,et al.Siglec-15 / sialic acid axisas a central glyco-immune checkpoint in breast cancer bone metastasis.ProcNatl Acad Sci U S A.2024Jan 30;121(5):e2312929121.

[0248] [3]Roland Schauer,Johannis P Kamerling.Exploration of the Sialic AcidWorld.Adv Carbohydr Chem Biochem.2018:75:1-213.

[0249] [4]Olivia Joan Adams,Michal A Stanczak,Stephan von Gunten,etal.Targeting sialic acid-Siglec interactions to reverse immune suppression incancer.Glycobiology.2018Sep 1;28(9):640-647.

[0250] [5]Eline J H van Houtum,Christian Büll,Lenneke A M Cornelissen,etal.Siglec Signaling in the Tumor Microenvironment.Front Immunol.2021Dec 13:12:790317.

[0251] [6]Heinz Kunio Kawanishi,Cijo George Vazhappilly,et al.Tools tostudy and target the Siglec-sialic acid axis in cancer.FEBS J.2021Nov;288(21):6206-6225.

[0252] [7]Isabella Fraschilla,Shiv Pillai.Viewing Siglecs through the lensof tumor immunology.Immunol Rev.2017Mar;276(1):178-191.

[0253] [8]John Daly,Mattias Carlsten,Michael O'Dwyer.Sugar Free:NovelImmunotherapeutic Approaches Targeting Siglecs and Sialic Acids to EnhanceNatural Killer Cell Cytotoxicity Against Cancer.Front Immunol.2019May 9:10:1047.

[0254] [9]Yuzuru Sakamoto,Sachiyo Yoshio,Hiroyoshi Doi,et al.IncreasedFrequency of Dysfunctional Siglec-7-CD57+PD-1+Natural Killer Cells inPatients With Non-alcoholic Fatty Liver Disease.Front Immunol.2021Feb 22:12:603133.

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[10] Zhuo Yang,Yingqin Hou,Geramie Grande,et al.Targeted desialylationand cytolysis of tumour cells by fusing a sialidase to a bispecific T-cellengager.Nat Biomed Eng.2024May;8(5):499-512.

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[11] Meheli Ghosh, Priyodarshini Hazarika, SJ Dhanya, et al. Exploration of sialic acid receptors as a potential target for cancer treatment: Acomprehensive review. Int J Biol Macromol. 2024 Feb; 257 (Pt1): 128415.

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[12] Alessandro Natoni,Raghvendra Bohara,Abhay Pandit,et al.TargetedApproaches to Inhibit Sialylation of Multiple Myeloma in the Bone MarrowMicroenvironment.Front Bioeng Biotechnol.2019Oct 4:7:252.

[0258]

[13] Osamu Suzuki,Masafumi Abe.Recent progress and new perspectives inlymphoma glycobiology.Fukushima J Med Sci.2013;59(1):1-14.

[0259] Example 10: N-acetylneuraminic acid methyl ester combined with NK cell therapy downgraded the TI-RADS grade of suspected malignant thyroid nodules, reducing the risk of malignancy.

[0260] Patient Feng, female, 50 years old, underwent thyroid and cervical lymph node ultrasound examination on July 11, 2023. Ultrasound description: The thyroid gland was normal in size and shape, with a smooth surface and intact capsule. The left lobe and isthmus showed homogeneous internal echoes. A hypoechoic lesion measuring approximately 6.5mm × 4.2mm was visible in the right lobe, with indistinct borders and an irregular shape. Color Doppler flow imaging (CDFI) showed linear blood flow signals within the lesion. No enlarged lymph nodes were observed in the neck. Figure 11A) Ultrasound examination results suggested suspected malignancy, TI-RADS 4C (suspicious malignant nodule, malignancy risk 50-90%). On July 11, 2023, the patient was given a combination of allogeneic NK cell therapy and N-acetylneuraminic acid methyl ester enteric-coated tablets. The specific regimen was as follows: allogeneic NK cell therapy, dose of 5 billion / bag; 2 hours before NK cell therapy infusion, 160 mg of N-acetylneuraminic acid methyl ester enteric-coated tablets were taken orally on an empty stomach, followed by 160 mg of N-acetylneuraminic acid methyl ester enteric-coated tablets once daily on an empty stomach for 5 consecutive days. On December 8, 2023, the patient underwent a repeat ultrasound examination of the thyroid gland and cervical lymph nodes. Ultrasound description: The thyroid gland appeared normal in size and shape, with a smooth surface and intact capsule. The left lobe and isthmus showed homogeneous echoes. Two hypoechoic lesions were visible in the right lobe, with relatively clear borders and regular shapes. Color Doppler flow imaging (CDFI) showed no obvious abnormal blood flow signals, with the larger lesion measuring approximately 3.8 mm × 2.1 mm. No enlarged lymph nodes were observed in the neck. Figure 11 B). Two ultrasound examinations showed that after intervention with NK cells combined with N-acetylneuraminic acid methyl ester, the thyroid nodule in the right lobe of the thyroid gland shrank from 6.5mm×4.2mm to 3.8mm×2.1mm, the boundary changed from unclear to clear, the shape changed from irregular to regular, and the abnormal blood flow signal inside disappeared. The TI-RADS category was 3 (possibly benign, with a malignancy risk of less than 2%). The comprehensive assessment showed that the probability of malignancy of the patient's thyroid nodule was significantly reduced.

[0261] Example 10 illustrates that the sialic acid-glycan-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker of the present invention, when used in combination with immunotherapy drugs, can enhance the body's immunity, better identify and kill mutated and variant cells, eliminate tumors in their early stages, and prevent tumor development. The sialic acid-glycan-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker of the present invention, as an immunotherapy synergistic agent, when applied in conjunction with immunotherapy drugs to potentially malignant diseases, can eliminate tumors in precancerous lesions or early-stage cancer, thereby reducing the incidence of malignant tumors. Precancerous lesions refer to certain lesions that have the potential to become cancerous if they continue to develop.

[0262] Potential malignant diseases (precancerous lesions) include, but are not limited to:

[0263] (1) Leukoplakia: White patches appear on the mucous membranes of the mouth, esophagus or other parts of the body that do not disappear and cannot be scraped off. Although most are benign, a small number of cases may develop into squamous cell carcinoma.

[0264] (2) Atrophic gastritis: This is a chronic gastritis in which the gastric mucosa thins and glands decrease or disappear. Long-term atrophic gastritis increases the risk of gastric cancer, especially when accompanied by intestinal metaplasia.

[0265] (3) Cirrhosis: Whether caused by viral hepatitis (such as hepatitis B and C), alcohol abuse or other reasons, cirrhosis is an important risk factor for hepatocellular carcinoma.

[0266] (4) Pulmonary nodules: Small round shadows found on chest X-rays or CT scans. Most are benign, but some pulmonary nodules with certain characteristics may indicate the presence or risk of lung cancer.

[0267] (5) Adenomatous polyps: especially adenomas in the colorectal region, which may progress to colon cancer over time.

[0268] (6) Barrett's esophagus: The replacement of normal cells in the lower esophagus with abnormal cells due to gastric acid reflux increases the risk of esophageal adenocarcinoma.

[0269] (7) Chronic inflammatory bowel diseases: such as Crohn's disease and ulcerative colitis, which are associated with an increased risk of colon cancer.

[0270] (8) Borderline ovarian tumors are not clearly malignant tumors, but they have the potential to become malignant.

[0271] (9) Genetic syndromes: Familial adenomatous polyposis (FAP), Lynch syndrome, and BRCA1 / 2 gene mutations all significantly increase the risk of developing certain types of cancer.

[0272] (10) Pre-leukemic state: such as myelodysplastic syndrome (MDS), which is a group of diseases that affect the blood and bone marrow and may develop into acute myeloid leukemia.

[0273] (11) Lymphoproliferative disorders: including monoclonal gammopathy (MGUS), which has the potential to progress to multiple myeloma or other B-cell lymphomas.

[0274] (12) Thyroid nodules: Most are benign, but about 5-10% may be malignant or have the risk of malignant transformation.

[0275] (13) Dysplastic nevi: Atypical or large pigmented nevi may transform into melanoma.

[0276] (14) Endometrial hyperplasia: especially complex hyperplasia and cases with atypical cellular changes, increases the risk of endometrial cancer.

[0277] (15) Cervical intraepithelial neoplasia (CIN): This refers to abnormal changes in cervical cells, which are divided into different grades. High-grade lesions may develop into cervical cancer.

[0278] (16) Atypical breast hyperplasia: This type of lesion includes ductal carcinoma in situ (DCIS) and lobular carcinoma in situ (LCIS), which are precursor lesions of breast cancer.

[0279] Example 11: N-acetylneuraminic acid methyl ester treatment for pancreatic cancer patients

[0280] Patient Yang, male, 82 years old, was diagnosed with late-stage pancreatic cancer on September 2, 2024. The patient refused hospitalization for treatment. A plain CT scan on November 15, 2024, revealed a nodular shadow (approximately 1.5cm × 1.2cm) in the subpleural region of the left upper lobe of the lung, and a mass-like soft tissue density shadow (approximately 4.2cm × 3.6cm) in the right lower lobe of the lung, with lobulated margins, located in the mediastinum, and without significantly enlarged lymph nodes. No significant pleural effusion was observed bilaterally. The head of the pancreas appeared full and of uneven density. No effusion was observed in the abdominal or pelvic cavities. Enlarged lymph nodes were observed in the retroperitoneum, some fused, with indistinct borders, the largest measuring approximately 2.7cm in diameter. Figure 12 A). Tumor marker test on November 15, 2024: Carbohydrate antigen 199 was 64.17 U / ml (reference range 0-30 U / ml). Figure 12 C). Liver and kidney function tests conducted on November 15, 2024 showed the following results: total protein 61.0 g / L (reference range 60-83 g / L), albumin 32.6 g / L (reference range 35-55 g / L), albumin-to-globulin ratio 1.15 (reference range 1.25-2.5), creatinine 56 μml / L (reference range 64-104 μml / L). Total bilirubin, direct bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, gamma-glutamyl transferase (GGT), lactate dehydrogenase, blood urea nitrogen, and uric acid were all within the normal range and no abnormalities were observed. Figure 12 E). Starting November 15, 2024, the patient began taking N-acetylneuraminic acid methyl ester enteric-coated tablets orally on an empty stomach, 80 mg three times daily, continuously. A follow-up CT scan on December 18, 2024, showed a nodular shadow in the subpleural region of the left upper lobe, approximately 1.5 cm × 1.2 cm; a mass-like soft tissue density shadow in the right lower lobe, approximately 4.4 cm × 3.5 cm, with lobulated margins, located in the mediastinum, without significantly enlarged lymph nodes; no significant pleural effusion was observed bilaterally. The head of the pancreas was full with uneven density, while the tail of the pancreas was atrophied. A small amount of pelvic effusion was present, and enlarged lymph nodes were seen in the retroperitoneum, some fused, with indistinct borders, the largest approximately 3.7 cm in diameter. Figure 12 B). Tumor marker test on December 18, 2024: Carbohydrate antigen 199 decreased to 44.91 U / ml (reference range 0-30 U / ml). Figure 12D). Liver and kidney function tests conducted on December 18, 2024, showed that total protein increased to 64.8 g / L (reference range 60-83 g / L), albumin increased to 35.6 g / L (reference range 35-55 g / L), albumin-to-globulin ratio increased to 1.22 (reference range 1.25-2.5), and creatinine increased to 61 μml / L (reference range 64-104 μml / L). Total bilirubin, direct bilirubin, alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase, gamma-glutamyl transferase (GGT), lactate dehydrogenase (LDH), blood urea nitrogen, and uric acid were all within the normal range and no abnormalities were observed. Figure 12 F). Based on comprehensive assessment, the patient's condition was SD (Stable Disease, meaning the total increase in the longest diameter of a single or several tumors is less than 30% or the decrease is less than 20%), and no side effects such as abnormal liver or kidney function were observed.

[0281] Example 11 illustrates that the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker of the present invention can be applied as an immunomodulatory drug to neoplastic diseases. In other words, as long as a certain tumor has the characteristic of abnormally high expression of sialic acid on the cell membrane surface, the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker provided by the present invention can be used as an immunomodulatory drug to modify the sialyl-sialic acid on the surface of the tumor cells, change the local molecular structure and charge characteristics, thereby reducing the affinity of sialyl-binding immunoglobulin-like lectin on the surface of the patient's own immune cells (such as T cells, NK cells, etc.), blocking the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway, relieving the inhibition of immune cell function by this signaling pathway, and promoting the recognition and killing of tumor cells. Since the overexpression of abnormal amounts of sialic acid on the surface of tumor cells is a common feature, the sialic acid-glycan-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker provided by this invention has universal applicability as an immunomodulatory drug for the treatment of tumors with abnormally high sialic acid expression (including solid tumors and hematological malignancies). Suitable tumors include, but are not limited to, lung cancer, breast cancer, esophageal cancer, gastric cancer, cardia cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, thyroid cancer, parathyroid cancer, kidney cancer, bladder cancer, penile cancer, malignant melanoma, glioma, leukemia, multiple myeloma, and malignant lymphoma.

[0282] References:

[0283] [1] Filip Filipsky, Heinz Regulation of sialic acid metabolismincancer.Carbohydr Res.2024May:539:109123.

[0284] [2]Yixian Wang,Zhan Xu,Kuan-Lin Wu,et al.Siglec-15 / sialic acid axisas a central glyco-immune checkpoint in breast cancer bone metastasis.ProcNatl Acad Sci U S A.2024Jan 30;121(5):e2312929121.

[0285] [3]Roland Schauer,Johannis P Kamerling.Exploration of the Sialic AcidWorld.Adv Carbohydr Chem Biochem.2018:75:1-213.

[0286] [4]Olivia Joan Adams,Michal A Stanczak,Stephan von Gunten,etal.Targeting sialic acid-Siglec interactions to reverse immune suppression incancer.Glycobiology.2018Sep 1;28(9):640-647.

[0287] [5]Eline J H van Houtum,Christian Büll,Lenneke A M Cornelissen,etal.Siglec Signaling in the Tumor Microenvironment.Front Immunol.2021Dec 13:12:790317.

[0288] [6]Heinz Kunio Kawanishi,Cijo George Vazhappilly,et al.Tools tostudy and target the Siglec-sialic acid axis in cancer.FEBS J.2021Nov;288(21):6206-6225.

[0289] [7]Isabella Fraschilla,Shiv Pillai.Viewing Siglecs through the lensof tumor immunology.Immunol Rev.2017Mar;276(1):178-191.

[0290] [8]John Daly,Mattias Carlsten,Michael O'Dwyer.Sugar Free:NovelImmunotherapeutic Approaches Targeting Siglecs and Sialic Acids to EnhanceNatural Killer Cell Cytotoxicity Against Cancer.Front Immunol.2019May 9:10:1047.

[0291] [9]Yuzuru Sakamoto,Sachiyo Yoshio,Hiroyoshi Doi,et al.IncreasedFrequency of Dysfunctional Siglec-7-CD57+PD-1+Natural Killer Cells inPatients With Non-alcoholic Fatty Liver Disease.Front Immunol.2021Feb 22:12:603133.

[0292]

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[0293]

[11] Alessandro Natoni,Raghvendra Bohara,Abhay Pandit,et al.TargetedApproaches to Inhibit Sialylation of Multiple Myeloma in the Bone MarrowMicroenvironment.Front Bioeng Biotechnol.2019Oct 4:7:252.

[0294]

[12] Osamu Suzuki,Masafumi Abe.Recent progress and new perspectives inlymphoma glycobiology.Fukushima J Med Sci.2013;59(1):1-14.

[0295] Example 12: N-acetylneuraminic acid methyl ester as an adjunct therapy for influenza virus infection;

[0296] Ms. Mao, female, 66 years old, developed a fever of 37.9℃ on March 22, 2024, accompanied by chills, cough, runny nose, fatigue, and general muscle discomfort. She was treated with a combination of Sanjiu Cold Relief Granules, acetaminophen, and N-acetylneuraminic Acid Methyl Ester Enteric-coated Tablets. The dosage of N-acetylneuraminic Acid Methyl Ester Enteric-coated Tablets was 2 tablets (160mg) orally on an empty stomach, followed by 160mg of N-acetylneuraminic Acid Methyl Ester Enteric-coated Tablets once daily on an empty stomach every morning. The patient felt a significant reduction in all symptoms that evening, and all symptoms disappeared after three days of continuous medication.

[0297] Example 13: NK+N-acetylneuraminic acid methyl ester treatment for COVID-19 infection;

[0298] Ms. Yang, 76 years old, developed a fever of 38.6℃ on May 14, 2024, accompanied by sore throat, cough, fatigue, and muscle aches in both lower limbs. She was diagnosed with COVID-19 (second infection) after a COVID-19 antigen test. After self-medicating with amoxicillin and acetaminophen, her fever and muscle aches improved, but she still felt fatigued, and her cough and sore throat worsened. On May 16, she received combined treatment with allogeneic NK cell therapy and N-acetylneuraminic acid methyl ester enteric-coated tablets. The regimen was: 3 billion allogeneic NK cells, followed by 160mg of N-acetylneuraminic acid methyl ester enteric-coated tablets orally on an empty stomach 2 hours before infusion, and then 160mg of N-acetylneuraminic acid methyl ester enteric-coated tablets orally once daily on an empty stomach in the morning. On May 17, her temperature returned to normal, her mild cough and sore throat significantly improved. She continued taking N-acetylneuraminic acid methyl ester enteric-coated tablets until May 19, when all symptoms disappeared.

[0299] Example 14: N-acetylneuraminic acid methyl ester treatment for varicella-zoster virus infection, relieving pain and enhancing patient immunity.

[0300] Patient Chen, male, 53 years old, presented on April 13, 2024, with right-sided facial pain accompanied by herpes zoster for 3 days and dizziness for 2 days. He was diagnosed with herpes zoster and treated with N-acetylneuraminic acid methyl ester enteric-coated tablets orally on an empty stomach, 160 mg once daily for 7 consecutive days. On April 23, a follow-up examination showed significant pain relief, and blood tests showed enhanced immune function, with the white blood cell count decreasing from 8.53 × 10⁻⁶. 9 / L increased to 12.59×10 9 / L, neutrophils increased from 7.09 × 10 9 / L increased to 9.88×10 9 / L, lymphocytes increased from 0.7×10 9 / L increased to 1.78×10 9 / L( Figure 13 , Figure 14 ).

[0301] Examples 12-14 illustrate that the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker of the present invention can be used to manufacture immunotherapeutic synergistic drugs or immunomodulatory drugs. By modifying the sialyl-sialyl glycan on the surface of virus-infected cells, it alters the local molecular structure and charge characteristics, thereby reducing the affinity of sialyl-binding immunoglobulin-like lectin on the surface of immune cells (autologous immune cells such as T cells, NK cells, DC cells, Mg² cells, etc., or allogeneic immune cells such as NK cells, γδT cells, etc.), blocking the sialyl-sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway, relieving the inhibition of immune cell function by this signaling pathway, promoting the recognition and killing of virus-infected cells by immune cells, and thus clearing the virus. Viruses infect cells by binding to sialic acid receptors. After infection, the virus causes abnormal expression of sialyl on the surface of infected cells, NK cells, Immune cells, such as cellular cells, can recognize and kill virus-infected cells; these characteristics are common in many viral infections. Therefore, the immunomodulatory drug manufactured based on the sialic acid-glycan-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker of this invention has universal applicability for treating viral infectious diseases. As long as the surface of the virus-infected cells expresses sialic acid, and the immune cells (such as NK cells, etc.) in the infected person are present... Cells (such as immune cells) can recognize and kill these virus-infected cells. Immunomodulatory drugs manufactured based on the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker of this invention are suitable for treating such viral infections. By blocking the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway between virus-infected cells and immune cells, the negative regulation of immune cells is relieved, enhancing the recognition and killing function of immune cells against virus-infected cells, thereby clearing the virus. Suitable viruses include, but are not limited to, human immunodeficiency virus infection, influenza virus infection, coronavirus infection, rhinovirus infection, mumps virus infection, respiratory syncytial virus infection, cytomegalovirus infection, hepatitis B virus infection, hepatitis C virus infection, human papillomavirus infection, Ebola virus infection, herpes simplex virus infection, adenovirus infection, rubella virus infection, norovirus infection, Zika virus, parainfluenza virus infection, measles virus infection, avian influenza virus infection, varicella-zoster virus infection, Newcastle disease virus infection, Sendai virus, and rotavirus infection.

[0302] References:

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[0304] [2]Pratima Saini, Opeyemi S Adeniji, Devivasha Bordoloi, et al.Siglec-9Restrains Antibody-Dependent Natural Killer Cell Cytotoxicity against SARS-CoV-2.mBio.2023Feb 28;14(1):e0339322.

[0305] [3]Renee R Anderko, Robbie B Mailliard. Mapping the interplay between NK cells and HIV: therapeutic implications. J Leukoc Biol. 2023 Feb 1; 113(2):109-138.

[0306] [4]Wei Zuo,Xiangyu Zhao.Natural killer cells play an important rolein virus infection control:Antiviral mechanism,subset expansion and clinicalapplication.Clin Immunol.2021Jun:227:108727.

[0307] [5]Kyle W Kroll,Spandan V Shah,Olivier A Lucar,et al.Mucosal-homingnatural killer cells are associated with aging in persons living withHIV.Cell Rep Med.2022Oct 18;3(10):100773.

[0308] [6]Jennifer E Stencel-Baerenwald,Kerstin Reiss,Dirk M Reiter,etal.Thesweet spot:defining virus-sialic acid interactions.Nat Rev Microbiol.2014Nov;12(11):739-49.

[0309] [7]Brian R Wasik,Karen N Barnard,Colin R Parrish.Effects of SialicAcid Modifications on Virus Binding and Infection.Trends Microbiol.2016Dec;24(12):991-1001.

[0310] [8]Keijo Fukushima,Tadanobu Takahashi,Takashi Suzuki.Characterizationof Human Parainfluenza Virus Receptor Using Terminal Sialic Acid Linkage-Modified Cells.Methods Mol Biol.2022:2556:169-178.

[0311] [9]Chee Wah Tan,Catherine Hong Huan Hor,Swee Sen Kwek,et al.Cellsurfaceα2,3-linked sialic acid facilitates Zika virus internalization.EmergMicrobes Infect.2019;8(1):426-437.

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[10] Weiqi Hong,Jingyun Yang,Jun Zou,et al.Histones released byNETosis enhance the infectivity of SARS-CoV-2by bridging the spike proteinsubunit 2and sialic acid on host cells.Cell Mol Immunol.2022May;19(5):577-587.

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[11] Dàlia Patricia Resa-Infante, Gallemí,et al.Role ofSiglecs in viral infections:A double-edged sword interaction.Mol AspectsMed.2023Apr:90:101113.

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[12] Tadahiro Suenaga,Yasuko Mori,Tatsuo Suzutani,et al.Regulation ofSiglec-7-mediated varicella-zoster virus infection of primary monocytes bycis-ligands.Biochem Biophys Res Commun.2022Jul 12:613:41-46.

[0315]

[13] Mohammad Khavani,Aliyeh Mehranfar,Mohammad R K Mofrad.On thepotentials of sialic acid derivatives as inhibitors for the mumps virus:Amolecular dynamics and quantum chemistry investigation.Virus Res.2023Mar:326:199050.

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[14] Yue Liu,Marchel G Hill,Thomas Klose,et al.Atomic structure of arhinovirus C,a virus species linked to severe childhood asthma.Proc Natl AcadSci U S A.2016Aug 9;113(32):8997-9002.

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[15] Che C Colpitts,Luis M Schang.A small molecule inhibits virionattachment to heparan sulfate-or sialic acid-containing glycans.JVirol.2014Jul;88(14):7806-17.

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[16] R M Ravindranath,M C Graves.Attenuated murine cytomegalovirusbinds to N-acetylglucosamine,and shift to virulence may involve recognitionof sialic acids.J Virol.1990Nov;64(11):5430-40.

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[17] You Nie,Dandan Liu,Wen Yang,et al.Increased expression of TIGITand KLRG1 correlates with impaired CD56bright NK cell immunity in HPV16-related cervical intraepithelial neoplasia.Virol J.2022Apr 12;19(1):68.

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[18] Jeremy R Teuton,Curtis R Brandt.Sialic acid on herpes simplexvirus type 1envelope glycoproteins is required for efficient infection ofcells.J Virol.2007Apr;81(8):3731-9.

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[19] Alexander T Baker,Rosie M Mundy,James A Davies,et al.Humanadenovirus type 26uses sialic acid-bearing glycans as a primary cell entryreceptor.Sci Adv.2019Sep 4;5(9):eaax3567.

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[20] Moeko Yamasaki,Yuta Kanai,Yoshiki Wakamura,et al.Characterizationof Sialic Acid-Independent Simian Rotavirus Mutants in Viral Infection andPathogenesis.J Virol.2023Jan31;97(1):e0139722.

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[21] Aparna Talekar,Anne Moscona,Matteo Porotto.Measles virus fusionmachinery activated by sialic acid binding globular domain.J Virol.2013Dec;87(24):13619-27.

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[22] Amy Lam,Orville O Kirkland,Papa Freduah Anderson,et al.Single-virus assay reveals membrane determinants and mechanistic features of Sendaivirus binding.Biophys J.2022Mar15;121(6):956-965.

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[23] Keijo Fukushima,Tadanobu Takahashi,TakashiSuzuki.Characterization of Human Parainfluenza Virus Receptor Using TerminalSialic Acid Linkage-Modified Cells.Methods Mol Biol.2022:2556:169-178.

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[25] Celeste C Goh, Krystal M Roggerson, Hai-Chon Lee, et al. Hepatitis CVirus-Induced Myeloid-Derived Suppressor Cells Suppress NK Cell IFN-γProduction by Altering Cellular Metabolism via Arginase-1. J Immunol. 2016Mar1;196(5):2283-92.

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[26] Niklas K Benedikt Strunz, Hans-Gustaf Ljunggren. Naturalkiller cells in antiviral immunity. Nat Rev Immunol. 2022Feb; 22(2):112-123.

[0329] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A sialyl polysaccharide-sialic acid-binding immunoglobulin-like lectin signaling pathway blocker, characterized in that, It contains one or more N-acetylneuraminic acid derivatives.

2. The signal pathway blocking agent according to claim 1, characterized in that, The N-acetylneuraminic acid derivative is selected from N-acetylneuraminic acid esters. The N-acetylneuraminic acid esters are selected from 2,4,7,8,9-penta-O-acetyl-N-acetylneuraminic acid deuterated methyl ester, 4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid deuterated methyl ester, N-acetylneuraminic acid methyl ester, N-acetylneuraminic acid ethyl ester, 2,4,7,8,9-penta-O-acetyl-N-acetylneuraminic acid methyl ester, 4,7,8,9-tetra-O-acetyl-N-acetylneuraminic acid methyl ester, N-acetylneuraminic acid deuterated methyl ester, N... - Any one or more of the following: N-acetylneuraminic acid deuterated ethyl ester, N-acetylneuraminic acid propyl ester, N-acetylneuraminic acid isopropyl ester, N-acetylneuraminic acid n-butyl ester, N-acetylneuraminic acid sec-butyl ester, N-acetylneuraminic acid tert-butyl ester, N-acetylneuraminic acid isobutyl ester, N-acetylneuraminic acid methyl ester cyclodextrin inclusion complex, N-acetylneuraminic acid ethyl ester cyclodextrin inclusion complex, N-acetylneuraminic acid deuterated methyl ester cyclodextrin inclusion complex, and N-acetylneuraminic acid deuterated ethyl ester cyclodextrin inclusion complex.

3. The signal pathway blocking agent according to claim 1, characterized in that, The N-acetylneuraminic acid derivative is selected from those with the following general structural formula characteristics. N-acetylneuraminic acid esters, among which, R1 is H or OH, and R2 is a benzene ring substituent, a biotin substituent, a folic acid substituent, or an RGD substituent; and / or R2 is OH, and R1 is a benzene ring substituent, a biotin substituent, a folic acid substituent, or an RGD substituent; and / or R1 is a benzene ring substituent, a biotinylated substituent, a folic acid substituent, or an RGD substituent; R2 is a benzene ring substituent, a biotinylated substituent, a folic acid substituent, or an RGD substituent; wherein, the benzene ring substituent is... and ; and / or The biotinylate substituent is and / or The folic acid substituents are selected from... and / or and / or The RGD-type substituent is 4. The signal pathway blocking agent according to claim 1, characterized in that, The N-acetylneuraminic acid derivative is selected from those with the following general structural formula characteristics. The compound wherein R is an alkyl, cycloalkyl, substituted alkyl, substituted cycloalkyl, thioester, thioether, ether group, disulfide ester, dithiomethyl, S-CH3, S-CH2-CH3, aryl, substituted aryl, methionine, methionine-zinc, sodium, phenol or phenol derivative.

5. The signal pathway blocking agent according to claim 1, characterized in that, The N-acetylneuraminic acid derivative is an N-acetylneuraminic acid derivative cyclodextrin inclusion complex.

6. The signal pathway blocking agent according to claim 5, characterized in that, The cyclodextrin is selected from any one of β-cyclodextrin or its derivatives, γ-cyclodextrin or its derivatives, α-cyclodextrin or its derivatives, wherein the cyclodextrin is hydroxypropyl-β-cyclodextrin, or hydroxypropyl-γ-cyclodextrin, or dimethyl-β-cyclodextrin, or dimethyl-γ-cyclodextrin, or sulfobutyl-β-cyclodextrin.

7. The signal pathway blocking agent according to any one of claims 1-6, characterized in that, The signaling pathway blocker is used to alter the molecular structure and charge characteristics of the sialyte terminals on the surface of tumor cells or virus-infected cells; the tumor cells or virus-infected cells are tumor cells or virus-infected cells with abnormal sialylation. The signaling pathway blocker is used to block the immunosuppressive signaling pathway between sialylated glycans on the surface of tumor cells or virus-infected cells and sialic acid-binding immunoglobulin-like lectins on the surface of immune cells.

8. The use of the signaling pathway blocker according to any one of claims 1-5 in the preparation of synergistic immunotherapy drugs.

9. The application according to claim 8, characterized in that, The immunotherapy synergist is used in combination with other immunotherapy drugs; The immunotherapy drugs include immune cell preparations, immune checkpoint inhibitors, tumor vaccine preparations, T-cell connective drugs, NK-cell connective drugs, antibody drugs with ADCC activity, and immunomodulators.

10. The application according to claim 9, characterized in that, The immune cell preparations include NK cell preparations, CAR-NK cell preparations, αβT cell preparations, γδT cell preparations, TIL cell preparations, NKT cell preparations, CAR-T cell preparations, TCR-T cell preparations, CIK cell preparations, DC-CIK cell preparations, macrophage (Mφ) preparations, CAR-Mφ cell preparations, neutrophil preparations, dendritic cell (DC) preparations, B cell preparations, myeloid-derived suppressor cell (MDSC) preparations, and Treg cell preparations.

11. The application according to claim 9, characterized in that, The immune checkpoint inhibitors include PD-1 antibody preparations, PD-L1 antibody preparations, CTLA-4 antibody preparations, LAG-3 antibody preparations, TIM-3 antibody preparations, TIGIT antibody preparations, and VISTA antibody preparations.

12. The application according to claim 9, characterized in that, The tumor vaccine formulations include whole-cell tumor vaccines, tumor peptide vaccines, genetically engineered mRNA tumor vaccines, genetically engineered DNA tumor vaccines, tumor subunit vaccines, heat shock protein tumor vaccines, and antibody tumor vaccines.

13. The application according to claim 9, characterized in that, The immunotherapy synergist is used in combination with the immunotherapy drug to treat tumors and prevent the transformation of potential malignant diseases into cancer. The tumors include solid tumors and hematologic malignancies; The solid tumors include lung cancer, breast cancer, esophageal cancer, gastric cancer, cardia cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, thyroid cancer, parathyroid cancer, kidney cancer, bladder cancer, penile cancer, malignant melanoma, and glioma. The hematologic malignancies include leukemia, multiple myeloma, and malignant lymphoma; The potential malignant diseases include leukoplakia, atrophic gastritis, cirrhosis, pulmonary nodules, adenomatous polyps, Barrett's esophagus, Crohn's disease, ulcerative colitis, borderline ovarian tumors, familial adenomatous polyposis, Lynch syndrome, BRCA1 / 2 gene mutation diseases, myelodysplastic syndrome, monoclonal gammopathy, thyroid nodules, melanocytic nevi, endometrial hyperplasia, cervical intraepithelial neoplasia, intraductal carcinoma in situ of the breast, and lobular carcinoma in situ of the breast.

14. The application according to claim 9, characterized in that, The immunotherapy synergist is used in combination with the immunotherapy drug to treat viral infections; The viral infections mentioned include human immunodeficiency virus infection, influenza virus infection, coronavirus infection, rhinovirus infection, mumps virus infection, respiratory syncytial virus infection, cytomegalovirus infection, hepatitis B virus infection, hepatitis C virus infection, human papillomavirus infection, Ebola virus infection, herpes simplex virus infection, adenovirus infection, rubella virus infection, norovirus infection, Zika virus, parainfluenza virus infection, measles virus infection, avian influenza virus infection, varicella-zoster virus infection, Newcastle disease virus infection, Sendai virus, and rotavirus infection.

15. The use of the signaling pathway blocker according to any one of claims 1-5 in the preparation of an immunomodulatory drug, wherein the immunomodulatory drug is used to enhance the anti-tumor immunity of cancer patients, or to treat cancer and prevent potential malignant diseases from becoming cancerous; The tumors include solid tumors and hematologic malignancies; The solid tumors include lung cancer, breast cancer, esophageal cancer, gastric cancer, cardia cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, thyroid cancer, parathyroid cancer, kidney cancer, bladder cancer, penile cancer, malignant melanoma, and glioma. The hematologic malignancies include leukemia, multiple myeloma, and malignant lymphoma; The potential malignant diseases include leukoplakia, atrophic gastritis, cirrhosis, pulmonary nodules, adenomatous polyps, Barrett's esophagus, Crohn's disease, ulcerative colitis, borderline ovarian tumors, familial adenomatous polyposis, Lynch syndrome, BRCA1 / 2 gene mutation diseases, myelodysplastic syndrome, monoclonal gammopathy, thyroid nodules, melanocytic nevi, endometrial hyperplasia, cervical intraepithelial neoplasia, intraductal carcinoma in situ of the breast, and lobular carcinoma in situ of the breast.

16. The use of the signaling pathway blocker according to any one of claims 1-5 in the preparation of an immunomodulatory drug, wherein the immunomodulatory drug is used to enhance the antiviral immunity of a virus-infected person or to treat a virus infection; The viral infections mentioned include human immunodeficiency virus infection, influenza virus infection, coronavirus infection, rhinovirus infection, mumps virus infection, respiratory syncytial virus infection, cytomegalovirus infection, hepatitis B virus infection, hepatitis C virus infection, human papillomavirus infection, Ebola virus infection, herpes simplex virus infection, adenovirus infection, rubella virus infection, norovirus infection, Zika virus, parainfluenza virus infection, measles virus infection, avian influenza virus infection, varicella-zoster virus infection, Newcastle disease virus infection, Sendai virus, and rotavirus infection.

17. A medicament and pharmaceutical composition for use as an inhibitor of the sialyl-sialic acid-binding immunoglobulin-like lectin signaling pathway, characterized in that, Includes a signaling pathway blocker according to any one of claims 1-5, and a pharmaceutically acceptable dosage form. The dosage forms of the drug and drug composition are tablets, capsules, injections, granules, suppositories, nasal sprays, oral sprays, nebulized inhalers, powders, powder inhalers, ointments, or gels.

18. A method for blocking the sialic acid-sialic acid binding immunoglobulin-like lectin signaling pathway, characterized in that, A drug or pharmaceutical composition comprising a signaling pathway blocker according to any one of claims 1-5 that blocks the sialic acid-sialic acid-binding immunoglobulin-like lectin signaling pathway.

19. A drug for treating tumors, characterized in that... It contains N-acetylneuraminic acid derivatives.

20. The medicament for treating tumors according to claim 19, characterized in that, The tumors mentioned are characterized by high expression of sialic acid on the cell membrane surface, including but not limited to lung cancer, breast cancer, esophageal cancer, gastric cancer, cardia cancer, colon cancer, rectal cancer, anal cancer, liver cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer, prostate cancer, oral cancer, laryngeal cancer, nasopharyngeal cancer, thyroid cancer, parathyroid cancer, kidney cancer, bladder cancer, penile cancer, malignant melanoma, glioma, leukemia, multiple myeloma, and malignant lymphoma.

21. The medicament for treating tumors according to claim 19 or 20, characterized in that, The N-acetylneuraminic acid derivative is selected from N-acetylneuraminic acid esters.

22. The medicament for treating tumors according to claim 21, characterized in that, The N-acetylneuraminic acid ester is N-acetylneuraminic acid methyl ester.

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