Use of amphotericin B or its combination agent in the preparation of an antitumor drug

Abstract

The present application relates to the use of amphotericin B or its combination in the preparation of anti-tumor drugs. The present application first confirms that amphotericin B directly binds to Toll-like receptor 2 and activates the downstream signaling pathway as a Toll-like receptor 2 agonist, thereby enhancing the phagocytic ability of macrophages to tumor cells; it is first confirmed that amphotericin B combined with therapeutic antibodies can produce a synergistic anti-tumor effect, and the above synergistic effect is mainly due to the regulatory effect of amphotericin B on macrophage function, including enhancing Fc gamma receptor expression and polarizing macrophages to M1 direction, which provides a solid foundation for the clinical transformation of amphotericin B and is expected to provide new treatment options for cancer patients.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to a novel pharmaceutical use of amphotericin B, specifically the use of amphotericin B or its combination in the preparation of antitumor drugs. Background Technology

[0002] Amphotericin B (AmB) is a polyene antifungal drug that has been widely used to treat severe systemic fungal infections since its discovery in the 1950s. AmB works by binding to ergosterol in the fungal cell membrane, creating membrane pores, leading to leakage of cell contents and cell death. In traditional applications, AmB's primary mechanism of action is against the membrane integrity of fungal cells, while research on its potential value in the field of anticancer treatment is relatively limited.

[0003] In recent years, studies have shown that AmB exhibits certain anti-cancer potential, and it has been discovered to have the ability to activate immune cells. A study published in Nature Neuroscience showed that AmB can inhibit the spread of brain tumor initiating cells (BTIC) by activating macrophages and microglia. In animal experiments, treatment of mice containing human BTIC with AmB nearly doubled their survival time and significantly reduced brain tumor growth rate (PMID: 24316889). These findings suggest that AmB may exert its anti-tumor effect by modulating the immune system, rather than directly killing tumor cells. However, the current technology regarding the anti-cancer mechanism and immunomodulatory mechanism of AmB is unclear, and related research is still insufficient. Although existing studies have shown that AmB may regulate immune cell function by affecting the Toll-like receptor (TLR) signaling pathway, they have not clearly explained how AmB regulates immune cell function or whether it works through specific signaling pathways. In particular, there is a lack of direct evidence regarding the interaction between AmB and immune cell surface receptors (such as Toll-like receptor 2, TLR2) (e.g., whether AmB can bind to TLR2 and how it activates downstream signaling pathways to regulate macrophage function). The lack of research on these mechanisms has prevented the full utilization of AmB's immunomodulatory properties and has failed to provide a theoretical basis for targeted immunomodulatory therapy, thus limiting the design and development of drugs based on the AmB-TLR2 interaction.

[0004] Regarding drug combination therapy, existing technologies have included research on the combination of AmB with other drugs. For example, Chinese patent CN106474164A discloses an anticancer pharmaceutical composition of Ganoderma lucidum extract synergistically with amphotericin B. This composition enhances anticancer efficacy by administering Ganoderma lucidum extract first, followed by AmB, through a fractional administration method. Chinese patent CN110801433B discloses a targeted drug composition co-loaded with amphotericin B and doxorubicin. This composition utilizes a β-cyclodextrin derivative as a carrier to achieve synergistic delivery of the two drugs, demonstrating good targeting ability and therapeutic efficiency in cell models. These studies suggest that the combination of AmB with other anticancer drugs may produce a synergistic effect and improve therapeutic efficacy. However, the aforementioned combination therapy with AmB mainly focuses on the effect of directly killing tumor cells, rather than through mechanisms such as immune regulation. Furthermore, although it is known that AmB can activate macrophages, its enhancing effect on macrophage phagocytosis and its value in combination with therapeutic antibodies have not been systematically studied. For example, whether AmB can enhance antibody-dependent phagocytosis (ADCP), whether AmB can enhance the anti-cancer effect of therapeutic antibodies, and how AmB promotes macrophage phagocytosis of tumor cells in vitro and in vivo, etc., current technologies have not provided sufficient experimental data.

[0005] The toxicity of AmB also limits its application in cancer treatment. As an antifungal drug, AmB can cause serious side effects such as fever and multiple organ failure when administered in excessive intravenous doses. Although there are some strategies to reduce the toxicity of AmB in the existing technology, such as developing liposomal formulations or structural derivatives, these methods are mainly aimed at antifungal treatment. For cancer treatment, which may require long-term or high-dose administration, their safety and efficacy still face challenges.

[0006] Current technologies lack assessments of the in vivo anticancer effects of AmB in immunocompetent animal models. Most studies focus on the direct antitumor activity of AmB, while insufficient research has been conducted on the relationship between its immunomodulatory effects and anticancer efficacy. In particular, key parameters such as the synergistic effect of AmB combined with antibodies in vivo, optimal dosing regimens, and pharmacokinetic characteristics have not yet been established, failing to provide sufficient evidence for clinical translation.

[0007] In summary, although existing technologies have revealed some potential applications of AmB in the field of cancer treatment, its specific mechanisms, optimal application methods, and combination therapy strategies still require further in-depth research. These various limitations severely restrict the expansion of AmB's application in cancer treatment; therefore, a systematic solution is urgently needed to clarify AmB's immune regulatory mechanisms and combination therapy strategies to fully realize its anti-cancer potential. Summary of the Invention

[0008] To overcome at least one deficiency in the prior art, this invention provides the application of amphotericin B (AmB) or its combination agents in the preparation of antitumor drugs. Specifically, it develops a series of new cancer treatment regimens based on AmB, covering multiple aspects from mechanism exploration to application development. These regimens systematically address the deficiencies in the prior art (such as the unclear anticancer mechanism of AmB, the lack of systematic research on the combined application of AmB and therapeutic antibodies, the toxicity of AmB, the lack of research on AmB as a Toll-like receptor 2 (TLR2) agonist, and the lack of in vivo evaluation of the anticancer effect of AmB), thus fully leveraging the potential of AmB in the field of anticancer therapy.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: The first aspect of the invention is to provide the use of amphotericin B (AmB) in the preparation of formulations that bind to Toll-like receptor 2 (TLR2) or in the preparation of direct agonists of Toll-like receptor 2.

[0010] A second aspect of the invention is the use of amphotericin B (AmB) in the preparation of formulations that enhance macrophage phagocytosis or in the preparation of formulations that enhance the antitumor effect of antibodies.

[0011] Furthermore, in the above applications, AmB directly binds to TLR2, and the affinity KD value between AmB and TLR2 is 1.29 × 10⁻⁶. -6 M.

[0012] Furthermore, in the aforementioned applications, TLR2 is a key target for AmB, and the immune activation effect of AmB depends on TLR2 expression. AmB is a direct agonist of TLR2 and activates downstream signaling, inducing macrophages to enhance their phagocytic capacity and antigen-presenting function, thus exhibiting immunomodulatory effects.

[0013] Furthermore, in the above applications, AmB combined with antibodies has a synergistic anti-tumor effect.

[0014] Furthermore, in the above applications, AmB activates Toll-like receptor 2 (TLR2) on the surface of macrophages, initiating downstream signaling pathways (including NF-κB).

[0015] Furthermore, in the above applications, AmB upregulates the expression of Fcγ receptors on the surface of macrophages, enhancing their binding ability to the antibody Fc fragment.

[0016] Furthermore, in the above applications, AmB upregulates MHC2 and downregulates CD206 on the surface of macrophages, causing macrophages to polarize towards M1.

[0017] Furthermore, the applications described in the first aspect of the present invention or the applications described in the second aspect of the present invention include: the use of amphotericin B or combinations thereof in the preparation of antitumor drugs or drugs for treating autoimmune diseases.

[0018] Furthermore, in the antitumor drug or the drug for treating autoimmune diseases, the amphotericin B has the ability to promote antibody-dependent macrophage phagocytosis as a Toll-like receptor 2 agonist.

[0019] Furthermore, the tumors include gastric cancer, liver cancer, colorectal cancer, colorectal adenocarcinoma, esophageal cancer, pancreatic cancer, gallbladder cancer, lung cancer, nasopharyngeal carcinoma, laryngeal cancer, breast cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, kidney cancer, bladder cancer, ureteral cancer, leukemia, lymphoma, multiple myeloma, thyroid cancer, osteosarcoma, melanoma, and brain tumors. Specifically, the tumors include glioblastoma, lymphoma, breast cancer, colorectal adenocarcinoma, and leukemia.

[0020] Furthermore, the autoimmune diseases include immune diseases caused by abnormal B cells, such as the therapeutic antibody rituximab used below, which can be used to treat autoimmune diseases associated with B cell abnormalities.

[0021] Furthermore, the combined treatment also includes: therapeutic antibodies, immunomodulators, and immune checkpoint inhibitors. Specifically, when AmB is used in combination with therapeutic antibodies, it can significantly enhance the phagocytic efficiency of macrophages against tumor cells.

[0022] Furthermore, the therapeutic antibodies include anti-CD20 antibodies, anti-EGFR antibodies, and anti-HER2 antibodies.

[0023] Further, the anti-CD20 antibody includes rituximab, ofamumab, ofamumab, thiamozolomide, and tosimozolomide; preferably, the anti-CD20 antibody is rituximab.

[0024] Further, the anti-EGFR antibody includes cetuximab, panitumumab, nimotuzumab, and nexituzumab; preferably, the anti-EGFR antibody is cetuximab.

[0025] Further, the anti-HER2 antibody includes trastuzumab, pertuzumab, magnusumab, and magnusumab; preferably, the anti-HER2 antibody is trastuzumab.

[0026] Furthermore, the immune checkpoint inhibitors include anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-CTLA-4 antibodies, anti-CD47 antibodies or fusion proteins, and anti-Sirpα antibodies or fusion proteins.

[0027] Further, the anti-PD-L1 antibody includes atezolizumab, durvalumab, avelumab, sugemalimab, envorimab, adebelimab, and bemosubaimab; preferably, the anti-PD-L1 antibody is atezolizumab.

[0028] Further, the anti-PD-1 antibody includes pembrolizumab, nivolumab, semipril, dostialil, retivalimab, sintilimab, camrelizumab, tislelizumab, dolilizumab, cerilimab, and bamphetimab; preferably, the anti-PD-1 antibody is pembrolizumab.

[0029] Further, the anti-CTLA-4 antibody includes ipilimumab and texilimumab; preferably, the anti-CTLA4 antibody is ipilimumab.

[0030] Furthermore, the immunomodulatory agent includes an interleukin-2 (IL-2) inhibitor. Based on the content of prior art CN1703236A, it can be reasonably concluded that combining AmB with an IL-2 inhibitor can reduce AmB treatment-related toxicities; specifically, AmB combined with an IL-2 inhibitor significantly reduces nephrotoxicity and systemic inflammatory response without affecting its anticancer efficacy.

[0031] Furthermore, the IL-2 inhibitor includes dalizumab.

[0032] Furthermore, the forms of amphotericin B include: amphotericin B powder, liposomal amphotericin B, amphotericin B prodrug, etc.

[0033] Furthermore, when using amphotericin B powder, it can be pre-prepared into a corresponding injection solution. Specifically, it is prepared using PBS containing 5% glucose, and the concentration can be adjusted according to the actual application.

[0034] Furthermore, the liposomal amphotericin B is a liposomal AmB that targets tumor tissue, which can increase the local drug concentration in the tumor and reduce systemic toxicity.

[0035] Furthermore, the amphotericin B prodrug is an AmB prodrug activated in the tumor microenvironment (such as under specific pH or enzyme conditions), which can enhance therapeutic specificity.

[0036] Furthermore, in the application of preparing antitumor drugs, the amphotericin B is used to prepare at least one of the following drugs: a drug for activating the downstream signaling pathway of Toll-like receptor 2, a drug for enhancing the expression of Fcγ receptors on the surface of macrophages, a drug for polarizing macrophages toward M1, and a drug for inhibiting tumor growth.

[0037] Furthermore, the downstream signaling pathway of the Toll-like receptor 2 includes NF-κB.

[0038] Furthermore, the method of co-administering AmB with therapeutic antibodies in tumor cells includes: first, pretreating macrophages with AmB and co-culturing them for a certain period of time; then adding tumor cells and co-incubating with therapeutic antibodies to allow the antibodies to fully bind to tumor cell surface antigens; wherein, the concentration of AmB is 1 nM~10 μM, and AmB can increase antibody-dependent macrophage phagocytosis by 3-5 times.

[0039] Furthermore, AmB and the therapeutic antibody were administered sequentially to mice twice a week at the following dosages: AmB 0.8 mg / kg and rituximab 10 mg / kg.

[0040] A third aspect of the invention provides a combination formulation as described above, comprising amphotericin B and a therapeutic antibody, an immunomodulator, and an immune checkpoint inhibitor.

[0041] Compared with the prior art, the present invention, by adopting the above technical solution, has the following beneficial effects: This invention fully explores the application potential of amphotericin B in the field of cancer treatment, and systematically addresses many shortcomings of existing technologies through the following multiple dimensions: (1) Clarifying the immunomodulatory mechanism of AmB: Existing technologies have failed to elucidate the specific mechanism by which AmB enhances the anticancer effect of antibodies. This invention is the first to demonstrate that AmB directly binds to the TLR2 protein and acts as a TLR2 agonist to activate downstream signaling pathways, thereby enhancing the phagocytic capacity of macrophages against tumor cells. The elucidation of this mechanism will provide a theoretical basis for the immunomodulatory effect of AmB and a scientific basis for its combined application with therapeutic antibodies.

[0042] (2) Establishing a treatment strategy combining AmB with antibodies: Existing technologies lack a systematic approach to combining AmB with therapeutic antibodies. This invention develops a method for synergistic treatment with AmB and antibodies, verifying its ability to promote macrophage phagocytosis of tumor cells through in vitro and in vivo experiments, and optimizing the order of administration, dosage ratio, and timing of treatment (see the experimental verifications in Examples 3, 4, and 5 below) to maximize the therapeutic effect. This solves the problem of unclear combined treatment strategies in existing technologies.

[0043] (3) Verify the in vivo anticancer effect of AmB in multiple cancer models: Existing in vivo anticancer studies of AmB mainly focus on glioblastoma. This invention expands the application scope of AmB by evaluating its in vivo effect in combination with antibodies in multiple tumor models (including lymphoma, colon adenocarcinoma, leukemia, etc.) and examining its impact on tumor growth and animal survival, providing a basis for clinical application in more cancer types.

[0044] (4) Development of AmB-based anticancer drug formulations: This invention will explore various applications of AmB, including: combination with traditional antibodies and combination with other immunomodulators. These drug formulations will fully utilize the immunomodulatory activity and cytotoxicity of AmB, expanding its application scenarios in cancer treatment.

[0045] In summary, this invention establishes a complete AmB anti-cancer application system, from mechanism research to treatment strategies, from in vitro validation to in vivo evaluation, providing comprehensive support for the clinical translation of AmB in the field of cancer treatment. Attached Figure Description

[0046] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this invention, illustrate exemplary embodiments of the invention and are for illustrative purposes only, and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic diagram showing the results of high-throughput screening in one embodiment of the present invention, which revealed that amphotericin B can promote the monoclonal antibody-mediated immune killing effect. Figure 2 This is a schematic diagram illustrating the results of in vitro verification of the effect of combining AmB with antibodies to promote macrophage phagocytosis of tumor cells (human B lymphoma cell line Raji and human colorectal adenocarcinoma epithelial cell line HT29) in one embodiment of the present invention. Figure 3 This is a schematic diagram showing the results of verifying the interaction mechanism between AmB and TLR2 in one embodiment of the present invention; wherein, part A: the binding assay of AmB to the full-length TLR2 protein (FULL), extracellular domain (ECD), intracellular domain (ICD), deletion mutant (DEL); part B: AmB can directly act on human TLR2 protein; part C: the phagocytic effect of AmB depends on TLR2.

[0047] Figure 4 This is a schematic diagram illustrating the results of an embodiment of the present invention verifying the mechanism by which AmB activates the NF-κB pathway, promotes the expression of Fcγ receptor, upregulates MHC2 on the surface of macrophages, and downregulates CD206. Part A shows the subcellular localization of p65 (NF-κB pathway) detected by immunofluorescence; Part B shows the expression of Fcγ receptor (FcγR) detected by flow cytometry; and Part C shows the expression of MHC2 and CD206 detected by flow cytometry. Figure 5 This is a schematic diagram illustrating the results of in vivo verification of the antitumor effect of AmB-enhanced antibody drugs in a Raji cell subcutaneous tumor model according to one embodiment of the present invention. Figure 6This is a schematic diagram of the results of an in vivo experiment verifying the anticancer effect of AmB combined with an antibody in a systemic chronic B-cell leukemia model according to one embodiment of the present invention. Detailed Implementation

[0048] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental materials in the following embodiments that do not specify their source are all commercially available raw materials. The equipment used in each step of the following embodiments is conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or conditions recommended by the manufacturer. Unless otherwise stated, all parts are parts by weight, and all percentages are percentages by mass. Unless otherwise defined or stated, all professional and scientific terms used in the present invention have the same meaning as those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the methods of the present invention.

[0049] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but this is not intended to limit the scope of the invention.

[0050] The technical solution of the present invention will be illustrated by way of example in the following embodiments.

[0051] Example 1 - High-throughput screening revealed that AmB can promote monoclonal antibody-mediated immune killing effects. This embodiment uses high-throughput screening to discover that amphotericin B (AmB) can promote monoclonal antibody-mediated immune killing effects, specifically including the following steps: High-throughput drug screening using tumor cell-macrophage co-culture: In the high-throughput screening experiment, FDA-approved small molecule compounds were co-incubated with primary macrophages (i.e., mouse bone marrow-derived macrophages, BMDM) at 0.05 M / 50 μL for 24 h. Subsequently, Raji cells expressing GFP-luciferase (ATCC catalog number: Bio-69201) were cultured at 0.05 × 10⁻⁶ cells. 650 μL of rituximab (anti-CD20 antibody, HY-P9913) was added to the wells, and the cells were cultured in 96-well plates for 24 hours with or without 2 ng / μL. Luciferin substrate was then added to the wells, and the luminescence signal was measured using Cytation 3. Wells containing only Raji cells and with the added drug were used as controls for calculating phagocytosis (phagocytosis = 0%). The normalized phagocytosis index was calculated as the difference in phagocytosis between the drug-treated group and the dimethyl sulfoxide (DMSO) control group.

[0052] The above high-throughput screening results are as follows: Figure 1 As shown, the antifungal drug amphotericin B was found to promote antibody-mediated macrophage-dependent tumor cell clearance. Further testing of the dosage of amphotericin B revealed that, under conditions of 1 nM to 10 μM, amphotericin B exhibited a dose-dependent effect in promoting macrophage phagocytosis and clearance of tumor cells.

[0053] Example 2 - AmB has the ability to promote antibody-dependent phagocytosis (ADCP). This embodiment verifies that AmB has the ability to promote antibody-dependent phagocytosis (ADCP), specifically verifying the enhancing effect of AmB combined with therapeutic antibodies on the phagocytosis of tumor cells by macrophages, including the following steps: (1) Materials and methods; Cell lines: human B lymphoma cell line Raji (CD20 positive) (ATCC catalog number: Bio-69201), mouse bone marrow-derived macrophages BMDM (differentiated into macrophages by M-CSF), human colorectal adenocarcinoma epithelial cell line HT29 (EGFR positive) (ATCC catalog number: Bio-69131), and human renal epithelial cell line 293T (ATCC catalog number: Bio-72947).

[0054] Reagents: AmB (Selleck-S1636), rituximab (anti-CD20 antibody, HY-P9913), cetuximab (anti-EGFR antibody, HY-P9905).

[0055] (2) Experimental steps; Lentiviral expression vectors containing GFP-Luc (Addgene catalog number: #193674) were used. Lentiviral viruses were packaged and collected in 293T cells (ATCC catalog number: Bio-72947) and subsequently used to transduce Raji and HT29 cells. Raji and HT29 cells were seeded during logarithmic growth phase and transduced using standard lentiviral infection methods. After transduction, cells were sorted by flow cytometry to obtain stable GFP-positive expression lines. Expression was finally confirmed by fluorescence microscopy and luciferase activity assay. Bone marrow-derived macrophages (BMDM) did not require labeling.

[0056] Co-culture phagocytosis assay procedure: Primary macrophages (i.e., mouse bone marrow-derived macrophages BMDM) were pretreated with AmB (1 nM-10 μM) for 24 h. Then, tumor cells were added at a macrophage-to-tumor cell ratio of 1:2 (Raji cells: group 1 without rituximab, group 2 with 0.5 μg / mL rituximab; HT29 cells: group 1 without cetuximab, group 2 with 0.5 μg / mL cetuximab). A dimethyl sulfoxide (DMSO) control group was set up for all cases. After co-culturing for 24 hours, the phagocytic efficiency of macrophages (remaining tumor cells / control tumor cells) was quantified by the chemiluminescence value of tumor cells.

[0057] (3) Experimental results: The results are as follows Figure 2 As shown, the macrophage phagocytic rate in the combined treatment group was significantly higher than that in each single-drug group and the control group. AmB at a concentration of 1 nM could promote antibody-mediated macrophage phagocytosis of tumor cells, increasing the macrophage phagocytic rate of tumor cells from approximately 20% to nearly 100%. The promoting effect of AmB was concentration-dependent; significant phagocytic enhancement was observed within the range of 1 nM to 10 μM, and the phagocytic rate gradually increased with increasing AmB concentration. These 1 nM-10 μM concentrations are far lower than the antifungal treatment concentration of AmB, suggesting that its immunomodulatory effect and direct cytotoxicity are achieved through different mechanisms.

[0058] The combination of the aforementioned therapeutic antibody and AmB showed significant killing effects on B lymphoma cells and colon cancer cells in in vitro experiments, providing a theoretical basis for the clinical application of AmB in combination with therapeutic antibodies.

[0059] Example 3 - Specific Mechanism of AmB Promoting Antibody-Dependent Phagocytosis Based on the verification in Example 2 that AmB has the ability to promote antibody-dependent phagocytosis, its mechanism of action was further verified, specifically including the following steps: (1) Verify the direct binding of AmB to TLR2 and its downstream signal activation using biophysical and molecular biological methods, specifically including the following experimental steps: ① Materials and methods; Cells: HEK293T cells (derived from ATCC), HEK293 cell line overexpressing human TLR2 fused with red fluorescent protein RFP (TLR2-RFP), wild-type and TLR2 knockout mouse-derived macrophages RAW264.7 cells; Protein: Lysis buffer of protein obtained from HEK293 cells co-expressed with human TLR2 fused with red fluorescent protein RFP; ② Experimental methods; 1) Micro-thermal surge (MST): HEK293T cells expressing TLR2-RFP protein and HEK293T cells expressing RFP protein were lysed. The TLR2-RFP and RFP protein lysates were co-incubated with AmB small molecule compounds of varying concentrations. The binding constant (KD) was then measured using the Binding mode of the micro-thermal surge instrument.

[0060] 2) Gene knockout technology: The TLR2 protein in RAW264.7 cells was knocked out using CRISPR-Cas9 technology to obtain TLR2 knockout RAW264.7 cells. Wild-type and TLR2 knockout RAW264.7 macrophages were pretreated with AmB, respectively, and co-cultured for 24 h. Then, MEC1 tumor cells were added and co-incubated with rituximab (0.5 μg / mL) antibody for 24 h. The control group was treated with DMSO. The phagocytic efficiency was then evaluated using a tumor cell chemiluminescence quantitative analysis system.

[0061] ③ Experimental results: such as Figure 3 As shown in Part A, AmB can bind to the full-length (FULL) and extracellular domain (ECD) of the recombinant TLR2 protein; as Figure 3 As shown in Part B, the MST results indicate that AmB binds to TLR2 with a high affinity, and the equilibrium dissociation constant (KD) is 1.29 × 10⁻⁶. -6 M; such as Figure 3 As shown in section C, the AmB-induced increase in phagocytosis was significantly attenuated in TLR knockout macrophages, confirming that TLR2 is a key target of AmB action.

[0062] The experimental results above show that AmB can directly bind to recombinant Toll-like receptor 2 (TLR2) protein. The immune activation effect of AmB depends on TLR2 expression. This is the first time that AmB has been confirmed at the molecular level as a direct agonist of TLR2. Its direct binding to TLR2 and activation of downstream signals explains the molecular mechanism of its immunomodulatory effect and provides a basis for developing immunotherapeutic strategies based on AmB-TLR2 interaction.

[0063] (2) Immunofluorescence was used to locate p65 (NF-κB pathway) in subcellular regions. The experimental procedure was as follows: 0.05 M / 50 μL of macrophages (i.e. mouse bone marrow-derived macrophages BMDM) were co-incubated with 0.01-10 μM AmB (Selleck-S1636) for 24 hours. After AmB treatment, the cells were washed with PBS, fixed and permeabilized with 4% paraformaldehyde and Triton X-100, respectively, followed by blocking with 5% BSA. Then, the cells were incubated with anti-NF-κB antibody. After washing off the antibody, fluorescent secondary antibody was added for incubation. Finally, the cell nuclei were stained with DAPI and the fluorescence signal was detected.

[0064] The results are as follows Figure 4 As shown in Part A, when the AmB concentration is 0, p65 is mainly distributed in the cytoplasm. As the AmB concentration increases, p65 gradually translocates to the nucleus, indicating that AmB can activate the NF-κB pathway. These results suggest that AmB activates Toll-like receptor 2 (TLR2) on the surface of macrophages, initiating the downstream signaling pathway NF-κB.

[0065] (3) The expression changes of FcγR were detected by flow cytometry. The experimental procedure was as follows: 0.05 M / 50 μL of macrophages (i.e. mouse bone marrow-derived macrophages BMDM) were co-incubated with 5 μM AmB (Selleck-S1636) for 24 hours. The macrophages treated with AmB were digested and made into single-cell suspensions. The single-cell suspensions were then stained with anti-FcγR flow cytometry antibody (9E9). After washing away the excess antibody, flow cytometry was performed.

[0066] The results are as follows Figure 4 As shown in Part B, the average fluorescence intensity of Fcγ receptor (FcγR) in the AmB-treated group was significantly higher than that in the control group. The signal distribution in the FcγR dimension of the AmB-treated group cells shifted upward overall, indicating that AmB promoted the expression of FcγR. That is, AmB upregulated the expression of Fcγ receptor on the surface of macrophages and enhanced its binding ability to the antibody Fc fragment.

[0067] (4) Flow cytometry was used to detect the expression of MHC2 and CD206. The experimental procedure was as follows: 50 μL of 0.05 M macrophages (i.e., mouse bone marrow-derived macrophages BMDM) were co-incubated with 5 μM AmB (Selleck-S1636) for 24 hours. The macrophages treated with AmB were digested and made into single-cell suspensions. First, the suspensions were stained with effervescent antibody APC-anti-MHC2 (catalog number: 107613). Then, the single-cell suspensions were permeabilized and fixed using the BD Cytofix / Cytoperm™ Plus Kit (catalog number: 555028). Finally, the suspensions were stained with FITC-anti-CD206 (C068C2) flow cytometry antibody. Excess flow cytometry antibody was washed away, and the samples were then analyzed by flow cytometry.

[0068] The results are as follows Figure 4 As shown in section C, the expression of MHC2 in the AmB-treated group was significantly higher than that in the control group, while the expression of CD206 was significantly lower than that in the control group. This indicates that AmB upregulates MHC2 and downregulates CD206 on the surface of macrophages, inhibiting macrophages from developing towards M2 and polarizing macrophages towards M1.

[0069] The experimental results above show that AmB works synergistically with therapeutic antibodies through the aforementioned mechanism, thus achieving better anti-tumor effects.

[0070] Example 4 - Anticancer effect of AmB combined with antibody in subcutaneous tumors in vivo This embodiment evaluates the in vivo anticancer effect of AmB combined with an antibody in a mouse model of subcutaneous tumor, and specifically includes the following experimental steps: (1) Materials and methods; Animals: Severely immunodeficient BALB / c NCG mice (lacking T, B, and NK cells, containing only macrophages; NCG (NOD / ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22 / Gpt); Strain NO. T001475), female, 6-8 weeks old; Model: Subcutaneous xenograft model of human B-cell lymphoma Raji (CD20 positive; derived from ATCC); Raji cells in the logarithmic growth phase were collected by centrifugation, resuspended in PBS, and injected at 0.5 × 10⁻⁶ cells per site. 6 Cells were seeded on the backs of mice. Three weeks later, the mice were randomly divided into groups for drug treatment.

[0071] (2) Experimental steps; Experimental grouping and treatment: Control group: intravenous injection of normal saline, twice a week; AmB monotherapy group: intratumoral injection of AmB 1 mg / kg, twice a week; Rituximab monotherapy group: intravenous injection of rituximab 10 mg / kg, twice a week; combination group: sequential administration of AmB (1 mg / kg) and rituximab (10 mg / kg), twice a week.

[0072] Monitoring indicators: Tumor volume, mouse weight and survival time were monitored regularly using vernier calipers (measured every 2 days); after the experiment, tumor tissue was collected for tumor size and weight analysis. Tumor size analysis mainly analyzed macrophage infiltration and phagocytosis rate of macrophages within the tumor.

[0073] (3) Experimental results: such as Figure 5 As shown, in the Raji cell subcutaneous tumor model, the combined treatment group exhibited the strongest tumor growth inhibition effect: after 28 days of treatment, the average tumor volume in the combined group was 75±15 mm. 3 The tumor growth inhibition rate was significantly lower than that of the AmB monotherapy group (710±75 mm³), the rituximab monotherapy group (500±50 mm³), and the control group (920±65 mm³). The combination therapy achieved a tumor growth inhibition rate of 85%, which was significantly higher than that of the monotherapy group (AmB monotherapy: 5%; antibody monotherapy: 50%).

[0074] The experimental results above demonstrate that the combination of AmB and rituximab produces a synergistic anti-tumor effect in subcutaneous tumors and significantly inhibits tumor growth.

[0075] Example 5 - Anticancer effect of AmB combined with antibody in vivo in systemic chronic B-cell leukemia This embodiment evaluates the in vivo anticancer effect of AmB combined with an antibody in systemic chronic B-cell leukemia, specifically including the experimental steps: (1) Materials and methods; Animals: Severely immunodeficient BALB / c NCG mice (lacking T, B, and NK cells, containing only macrophages; NCG (NOD / ShiLtJGpt-Prkdcem26Cd52Il2rgem26Cd22 / Gpt); Strain NO. T001475), female, 6-8 weeks old; Model: A systemic disseminated tumor model of human chronic B-cell leukemia MEC1 (CD20 positive; derived from ATCC); MEC1 cells in the logarithmic growth phase were collected by centrifugation, resuspended in PBS, and injected at 0.5 × 10⁻⁶ cells per site. 6 The cells were slowly injected into the body via the tail vein. Four days later, the mice were randomly divided into groups for drug treatment.

[0076] (2) Experimental steps; Experimental grouping and treatment: Control group: intravenous injection of normal saline, twice a week; AmB monotherapy group: AmB 1 mg / kg tail vein injection, twice a week; Rituximab monotherapy group: rituximab 10 mg / kg intravenous injection, twice a week; Combination group: AmB (1 mg / kg) and rituximab (10 mg / kg) were administered sequentially, twice a week.

[0077] Monitoring indicators: Tumor growth, mouse weight, and survival were monitored using in vivo imaging of mice (measured every 3 days).

[0078] (3) Experimental results: such as Figure 6 As shown, in the MEC1 disseminated B-cell leukemia model, the combination therapy group exhibited the strongest tumor growth inhibition effect: after 22 days of treatment, compared with other groups, the combination therapy group showed a significantly more significant tumor inhibition effect; and increased the lifespan of mice from about 20 days to as high as 40 days. Furthermore, the combination therapy group could also inhibit the weight loss caused by tumor progression.

[0079] The experimental results above demonstrate that the combination of AmB and rituximab produces a synergistic anti-tumor effect in systemic tumors and significantly inhibits tumor growth.

[0080] As can be seen from the above embodiments, the present invention verifies that: (1) AmB has a significant enhancement effect on the anti-cancer effect of antibodies: It is the first time that the combination of AmB and therapeutic antibodies (such as rituximab and trastuzumab) can produce a synergistic anti-tumor effect; in vitro experiments show that AmB can increase the phagocytic activity of antibody-dependent macrophages by 3-5 times; in vivo experiments show that the combination of AmB and rituximab can produce a synergistic anti-tumor effect in vivo and significantly inhibit tumor growth. The above synergistic effect is mainly due to the regulatory effect of AmB on macrophage function, including enhancing Fcγ receptor expression and polarizing macrophages towards M1. (2) AmB has in vivo anti-cancer activity: In various mouse tumor models, the combination of AmB and antibodies shows significant in vivo anti-cancer activity. In subcutaneous tumor and systemic tumor models, the combined treatment group showed the strongest tumor growth inhibition effect. The above effect can be achieved at a low dose of AmB (1 mg / kg), which is far lower than the conventional dose of its antifungal treatment (3-5 mg / kg), indicating that its anticancer effect is not related to direct cytotoxicity, but is achieved through an immune regulation mechanism. (3) This invention has discovered a novel mechanism of action: AmB has TLR2 agonist activity. For the first time, it has been confirmed at the molecular level that AmB is a direct agonist of TLR2. The affinity KD value of AmB to TLR2 was measured by MST technology to be 1.29 × 10. -6M, a high-affinity binding receptor, induces macrophages to enhance their phagocytic capacity and antigen-presenting function by activating the TLR2 signaling pathway (which initiates the downstream NF-κB signaling pathway). This discovery not only explains the immunomodulatory role of AmB but also provides new insights for developing TLR2 agonist-based immunotherapies.

[0081] In summary, this invention not only discovers a new application of AmB in cancer treatment but also elucidates its mechanism of action and develops various optimization strategies to improve its efficacy and safety. The experimental validations provided by the above findings lay a solid foundation for the clinical translation of AmB and hold promise for offering new treatment options for cancer patients.

[0082] The specific embodiments of the present invention have been described in detail above, but they are only examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to the present invention are also within the scope of the present invention. Therefore, all equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. The use of amphotericin B in the preparation of formulations that bind to Toll-like receptor 2 or in the preparation of direct agonists of Toll-like receptor 2.

2. Application of amphotericin B in the preparation of formulations that enhance macrophage phagocytosis or in the preparation of formulations that enhance the antitumor effect of antibodies.

3. A combination preparation containing amphotericin B, characterized in that, The combination therapy also includes at least one of therapeutic antibodies, immunomodulators, and immune checkpoint inhibitors.

4. The combined formulation according to claim 3, characterized in that, The therapeutic antibodies include anti-CD20 antibodies, anti-EGFR antibodies, and anti-HER2 antibodies; the immunomodulators include IL-2 inhibitors; and the immune checkpoint inhibitors include anti-PD-L1 antibodies, anti-PD-1 antibodies, anti-CTLA-4 antibodies, anti-CD47 antibodies or fusion proteins, and anti-Sirpα antibodies or fusion proteins.

5. The combination formulation according to claim 3, characterized in that, The forms of amphotericin B include: amphotericin B powder, liposomal amphotericin B, and amphotericin B prodrug.

6. The use of amphotericin B or its combination in the preparation of antitumor drugs or drugs for treating autoimmune diseases, characterized in that, The combined formulation is as described in any one of claims 3 to 5.

7. The application according to claim 6, characterized in that, In the antitumor drug or the drug for treating autoimmune diseases, the amphotericin B has the ability to promote antibody-dependent macrophage phagocytosis and activate the tumor immune microenvironment as a Toll-like receptor 2 agonist.

8. The application according to claim 6, characterized in that, The tumors include gastric cancer, liver cancer, colorectal cancer, colorectal adenocarcinoma, esophageal cancer, pancreatic cancer, gallbladder cancer, lung cancer, nasopharyngeal carcinoma, laryngeal cancer, breast cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, kidney cancer, bladder cancer, ureteral cancer, leukemia, lymphoma, multiple myeloma, thyroid cancer, osteosarcoma, melanoma, and brain tumors; the autoimmune diseases include immune diseases caused by abnormal B cells.

9. The application according to claim 6, characterized in that, In the application described, the amphotericin B is used to prepare at least one of the following drugs: a drug for activating the downstream signaling pathway of Toll-like receptor 2, a drug for enhancing the expression of Fcγ receptors on the surface of macrophages, a drug for polarizing macrophages toward M1, and a drug for inhibiting tumor growth.

10. The application according to claim 9, characterized in that, The downstream signaling pathway of the Toll-like receptor 2 includes NF-κB.

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