Application of anti-CD150 antibodies and immune checkpoint inhibitor antibodies in drug preparation

Abstract

This application proposes the use of anti-CD150 antibodies and immune checkpoint inhibitor antibodies in drug preparation, belonging to the field of biomedical technology. This application proposes the combined use of anti-CD150 antibodies and immune checkpoint inhibitor antibodies to prepare drugs for treating drug-resistant tumors. By combining anti-CD150 antibodies with immune checkpoint inhibitor antibodies, the drug resistance problem of immune checkpoint inhibitor antibodies is overcome, and it was unexpectedly discovered that the combination of the two can produce a synergistic effect, effectively killing tumors and prolonging the survival of target subjects.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, specifically, it relates to the use of anti-CD150 antibodies and immune checkpoint inhibitor antibodies in the preparation of drugs and drug combinations. Background Technology

[0002] In recent years, immunotherapy strategies based on immune checkpoint inhibitors have become an important treatment for various malignant tumors. This type of therapy, by relieving the immunosuppressive microenvironment and restoring the body's own immune system's ability to kill tumor cells, has significantly prolonged the survival of patients with various tumors such as melanoma, lung cancer, and liver cancer.

[0003] However, in clinical practice, only a portion of patients benefit from this therapy in the long term, with the majority exhibiting primary or secondary resistance. Drug resistance not only reduces the clinical efficacy of immune checkpoint inhibitors but also poses a significant challenge to cancer treatment. Therefore, overcoming drug resistance and expanding the beneficiary population of immunotherapy has become a core issue in the field of cancer immunotherapy. Summary of the Invention

[0004] This application aims to at least partially address one of the technical problems in the related art. To this end, this application proposes the use of anti-CD150 antibodies and immune checkpoint inhibitor antibodies in the preparation of drugs. By combining anti-CD150 antibodies with immune checkpoint inhibitor antibodies, the drug resistance problem of immune checkpoint inhibitor antibodies is overcome, and it is unexpectedly discovered that the combination of the two can produce a synergistic effect, effectively killing tumors and prolonging the survival of target subjects.

[0005] Specifically, the technical solution of this application is as follows: In one aspect, this application proposes the use of anti-CD150 antibodies and immune checkpoint inhibitor antibodies in the preparation of a drug for the treatment of drug-resistant tumors.

[0006] By combining anti-CD150 antibodies with immune checkpoint inhibitor antibodies, the resistance of tumors to immune checkpoint inhibitor antibodies (such as anti-PD-1 and anti-PD-L1 antibodies) can be effectively overcome. Anti-CD150 antibodies target SLAMF1 (CD150)-positive B cells, relieving their suppression of T cells. Simultaneously, immune checkpoint inhibitor antibodies block inhibitory signal transduction, synergistically activating T cells and promoting cytokine release, effectively enhancing the killing ability against tumor cells.

[0007] The combined treatment regimen provided in this embodiment overcomes the resistance of immune checkpoint inhibitor antibodies and also has a broad-spectrum anti-tumor effect. It is particularly suitable for tumor types that have developed resistance to PD-1 / PD-L1 immune checkpoint inhibitors, thus broadening the scope of immunotherapy and improving the survival and treatment efficacy of the target subjects.

[0008] The combination therapy regimens of this application are applicable to various types of tumors, such as liver cancer, colon cancer, melanoma, and lung cancer. These tumor types often exhibit varying degrees of immune resistance; for example, the efficacy of using anti-PD-1 / PD-L1 antibodies alone is often limited. However, by combining anti-CD150 antibodies with immune checkpoint inhibitor antibodies, the therapeutic effect can be effectively improved, overcoming immune resistance, especially in patients who have developed resistance to PD-1 / PD-L1 inhibitors, significantly improving survival and efficacy. Through the combination therapy strategy of this embodiment, the anti-tumor immune response can be re-stimulated, achieving a breakthrough from "ineffective" to "effective," with higher tumor growth inhibition rates, increased proportion of complete tumor regression, and significantly prolonged survival of the target subjects.

[0009] In an exemplary embodiment, the immune checkpoint inhibitor antibody includes at least one of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, and anti-TIGIT antibody. In practical applications, suitable immune checkpoint inhibitor antibodies can be selected for combination therapy based on the specific tumor type, immune escape mechanism, and the patient's drug resistance.

[0010] For example, anti-PD-1 or anti-PD-L1 antibodies, as widely used immune checkpoint inhibitors in clinical practice, have been proven to be effective in treating various malignant tumors. However, some patients develop drug resistance during treatment with these antibodies, leading to a significant decline in treatment efficacy. By combining anti-CD150 antibodies with anti-PD-1 or anti-PD-L1 antibodies, the immune response of T cells can be effectively enhanced, overcoming drug resistance to immune checkpoint inhibitor antibodies and thus restoring their effective tumor-killing ability.

[0011] In an exemplary preferred embodiment, the immune checkpoint inhibitor antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

[0012] Secondly, this application proposes a drug combination. According to embodiments of this application, the drug combination includes: an anti-CD150 antibody and an immune checkpoint inhibitor antibody.

[0013] By combining anti-CD150 antibodies with immune checkpoint inhibitor antibodies, the drug resistance problem of immune checkpoint inhibitor antibodies can be overcome, effectively killing tumors and achieving a synergistic anti-tumor effect.

[0014] In an exemplary embodiment, the immune checkpoint inhibitor antibody includes at least one of anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, and anti-TIGIT antibody. In practical applications, suitable immune checkpoint inhibitor antibodies can be selected for combination therapy based on the specific tumor type, immune escape mechanism, and the patient's drug resistance.

[0015] In an exemplary preferred embodiment, the immune checkpoint inhibitor antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody. Immune checkpoint inhibitor antibodies restore the tumor-killing effect of immune cells by blocking tumor immune escape mechanisms. Due to the problem of tumor resistance to immune checkpoint inhibitor antibodies, this embodiment introduces the combined use of an anti-CD150 antibody and an immune checkpoint inhibitor antibody, effectively enhancing the efficacy of immunotherapy. This combined treatment strategy can be personalized according to different tumor types and drug resistance mechanisms, further improving treatment efficacy.

[0016] It is understood that the drug combination therapy described in this embodiment is applicable to the treatment of various types of tumors, including but not limited to liver cancer, colon cancer, melanoma, and lung cancer. By combining anti-CD150 antibodies with immune checkpoint inhibitor antibodies, this embodiment can address the problem of immune resistance in different tumor types. It is particularly suitable for clinically common tumor types resistant to immune checkpoint inhibitors; this combination therapy strategy can significantly improve treatment efficacy and increase the breadth and depth of treatment.

[0017] In an exemplary preferred embodiment, the effective dose of the anti-CD150 antibody is preferably from 1 mg / kg to 10 mg / kg, measured by body weight. The dose can be adjusted according to the patient's specific condition to achieve the best therapeutic effect. Meanwhile, the effective dose of the anti-PD-1 antibody or anti-PD-L1 antibody is preferably from 2 mg / kg to 4 mg / kg, measured by body weight, or a fixed dose of 200 mg / dose to 400 mg / dose. The dose selection in this embodiment ensures safety and effectiveness in clinical use. Through reasonable dose adjustment, not only can the therapeutic effect be improved, but adverse reactions can also be minimized, and patient tolerability can be enhanced.

[0018] In an exemplary embodiment, the drug combination includes not only anti-CD150 antibodies and immune checkpoint inhibitor antibodies, but also cytokines. Cytokines, as immunomodulatory factors, can regulate the body's immune response, enhance the activity and function of immune cells, and thus improve the efficacy of immunotherapy. By incorporating cytokines into the drug combination, the immune system can be further activated, and the anti-tumor immune response can be strengthened. In particular, under the synergistic effect of immune checkpoint inhibitor antibodies and anti-CD150 antibodies, cytokines can accelerate the remodeling of the tumor microenvironment, enhance the recognition and clearance of tumors by immune cells, thereby significantly improving therapeutic efficacy and overcoming immune escape mechanisms.

[0019] In an exemplary embodiment, the drug includes pharmaceutically acceptable excipients.

[0020] In this application, "pharmaceutical acceptable" means that a substance or composition must be chemically and / or toxicologically compatible with other components of the formulation and / or the mammals to which it is treated. Preferably, "pharmaceutical acceptable" as used herein means approved by federal regulatory agencies or national governments or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, particularly in humans.

[0021] In this application, "pharmaceuticalally acceptable excipients" may include sugars, including monosaccharides or polysaccharides such as lactose, sucrose, mannitol, and sorbitol; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and methyl cellulose; calcium phosphates, such as dicalcium phosphate and tricalcium phosphate; sodium sulfate; calcium sulfate; polyvinylpyrrolidone; polyvinyl alcohol; stearic acid; alkaline earth metal stearates, such as magnesium stearate and calcium stearate; vegetable oils, such as peanut oil, cottonseed oil, sesame oil, olive oil, and corn oil; nonionic, cationic, and anionic surfactants; ethylene glycol polymers; fatty alcohols; and hydrolyzed cereal solids, as well as other nontoxic and compatible fillers, binders, disintegrants, buffers, preservatives, antioxidants, lubricants, colorants, and other excipients commonly used in pharmaceutical formulations.

[0022] In an exemplary embodiment, the excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

[0023] In this application, "carrier" includes any solvent, pharmaceutical stabilizer, or combination thereof, all of which are known to those skilled in the art. Except in cases where any conventional carrier is incompatible with the active ingredient, its use in therapeutic or pharmaceutical compositions is covered.

[0024] In an exemplary preferred embodiment, the drug combination is preferably an injectable preparation, such as a lyophilized powder or solution for injection. Injectable preparations, as a commonly used method of drug delivery, have high bioavailability and direct action on the bloodstream, ensuring that the drug exerts its therapeutic effect rapidly and effectively. By using injectable form, the drug can quickly reach therapeutic concentrations, thereby producing clinical effects more quickly, especially for diseases such as tumors that urgently require rapid control, where injectable forms can provide more immediate efficacy. Furthermore, the use of injectable preparations allows for precise control of drug dosage and administration method, ensuring the safety and standardization of treatment.

[0025] Thirdly, this application proposes the application of anti-CD150 antibodies and immune checkpoint inhibitor antibodies in the treatment of drug-resistant tumors.

[0026] According to embodiments of this application, the combined application of anti-CD150 antibodies and immune checkpoint inhibitor antibodies can effectively overcome the problem of tumor resistance to immune checkpoint inhibitor antibodies (such as anti-PD-1 or anti-PD-L1 antibodies). Anti-CD150 antibodies, through synergistic action with immune checkpoint inhibitor antibodies, restore the function of the immune system and enhance the immune response of T cells against tumor cells. During treatment, anti-CD150 antibodies can help reverse immune escape mechanisms in the tumor microenvironment and enhance the killing ability of immune cells, thereby effectively improving the anti-tumor effect. This combined therapy not only overcomes resistance to immunosuppressant antibodies but also enhances the persistent immune response of the immune system, making tumor treatment more efficient.

[0027] The combined use of anti-CD150 antibodies and immune checkpoint inhibitor antibodies in this application is particularly suitable for tumor types that have developed resistance to immune checkpoint inhibitors. In these patients, using immune checkpoint inhibitor antibodies alone often fails to produce sufficient therapeutic effects. By introducing anti-CD150 antibodies as a combination therapy component, the tumor-killing ability can be improved while overcoming drug-resistant tumors. This embodiment provides a more effective treatment option for patients resistant to immune checkpoint inhibitors in clinical practice.

[0028] Fourthly, embodiments of this application provide a treatment method for drug-resistant tumors. According to embodiments of this application, the method includes: administering an effective dose of a drug combination to a subject; wherein the drug combination is as shown in any example of the second aspect.

[0029] In an exemplary preferred embodiment, the drug combination may be an anti-CD150 antibody with an anti-PD-1 antibody or an anti-PD-L1 antibody. This treatment method is particularly suitable for tumor patients who have developed resistance to immune checkpoint inhibitor antibodies (such as anti-PD-1 or anti-PD-L1 antibodies). By using anti-CD150 antibodies in combination with immune checkpoint inhibitor antibodies, the immune escape mechanisms of drug-resistant tumors can be overcome, thereby effectively enhancing the efficacy of tumor treatment.

[0030] In exemplary embodiments, the dosage should be flexibly adjusted based on factors such as the patient's weight, tumor type, and immune escape mechanisms to achieve optimal therapeutic effects. Preferably, the effective dose of the anti-CD150 antibody can be selected from 1 mg / kg to 10 mg / kg, measured by body weight. The effective dose of the immune checkpoint inhibitor antibody (such as anti-PD-1 antibody or anti-PD-L1 antibody) should also be adjusted according to the patient's weight, preferably from 2 mg / kg to 4 mg / kg; or a fixed dose can be used, such as 200 mg / dose to 400 mg / dose. This dosage range has been validated through clinical trials to ensure the full efficacy of the drug while reducing the risk of adverse reactions.

[0031] To ensure the efficacy of the combined drug regimen, the choice of dosing frequency is crucial. Preferably, the combined drug regimen is administered once every 3 to 4 days. This dosing frequency ensures an effective drug concentration in the body and guarantees the sustained anti-tumor effect. By appropriately adjusting the dosing frequency, tumor progression can be effectively controlled, drug-related adverse reactions can be reduced, and patient treatment tolerance can be improved.

[0032] During treatment, drug resistance can be determined based on the proportion of CD150-positive B cells in the tumor. A proportion of CD150-positive B cells greater than a predetermined threshold indicates that the tumor is resistant to immune checkpoint inhibitor antibodies, requiring combined treatment with anti-CD150 antibodies. In an exemplary embodiment, this predetermined threshold is selected from 35%-45%.

[0033] Monotherapy with relevant immune checkpoint inhibitors has limited efficacy in treating drug-resistant or refractory tumors. This embodiment introduces the combined use of anti-CD150 antibodies and immune checkpoint inhibitor antibodies, effectively overcoming the immune escape mechanisms of drug-resistant tumors and enhancing the intensity and persistence of the immune response. Particularly in patients with a high proportion of CD150-positive B cells in their tumors, combination therapy significantly improves treatment efficacy, restores the immune system's ability to clear tumors, and thus effectively prolongs patient survival. Attached Figure Description

[0034] To more clearly illustrate the technical solutions in the embodiments of this application, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0035] Figure 1 A schematic diagram illustrating the combined treatment principle provided in the embodiments of this application; Figure 2 This is a schematic diagram of the mouse model processing flow provided in the embodiments of this application; Figure 3 A schematic diagram of tumor growth curves for the HEPA1-6 liver cancer model provided in this application embodiment; Figure 4 A schematic diagram of the tumor growth curve of the MC38 colorectal cancer model provided in the embodiments of this application; Figure 5 A schematic diagram of the tumor growth curve of the B16-F10 melanoma model provided in the embodiments of this application; Figure 6 Schematic diagram of flow cytometry detection results for different treatment groups provided in the embodiments of this application; Figure 7 CD8 for different processing groups provided in the embodiments of this application + IFN-γ + Statistical results diagram. Detailed Implementation

[0036] 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.

[0037] It should be noted that the terms "first," "second," etc., in the specification, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover non-exclusive inclusion; for example, a process, method, system, product, or server that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or devices.

[0038] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0039] In the embodiments of this application, antibodies include monoclonal antibodies, polyclonal antibodies, nanobodies, and antigen-binding fragments.

[0040] In the embodiments of this application, anti-CD150 antibody refers to an antibody that specifically recognizes and binds to the CD150 molecule. CD150, also known as SLAMF1, is a cell surface protein that is widely expressed on activated B cells, T cells, and dendritic cells.

[0041] In this application, immune checkpoint inhibitor antibodies are a class of antibodies that restore the body's anti-tumor immune response by targeting immune checkpoint molecules (such as PD-1, PD-L1, CTLA-4, LAG-3, TIGIT, etc.). Immune checkpoint molecules typically play an inhibitory role in the immune response, limiting the activity of immune cells. During tumor immune escape, tumor cells express immune checkpoint molecules that bind to receptors on the surface of immune cells, inhibiting the killing function of immune cells. Immune checkpoint inhibitor antibodies restore the tumor-killing effect by blocking these inhibitory signals, relieving the inhibitory state of immune cells.

[0042] In this embodiment, the anti-PD-1 antibody is an antibody that targets the PD-1 (programmed death receptor-1) molecule. PD-1 is an immune checkpoint receptor located on the surface of T cells. When it binds to PD-L1 on the surface of tumor cells, it can inhibit the activity of T cells, leading to immune tolerance or immune escape. The anti-PD-1 antibody enhances the body's anti-tumor immune response by binding to and blocking the PD-1 receptor, thereby restoring the function of T cells.

[0043] In this embodiment, the anti-PD-L1 antibody is an antibody that targets the PD-L1 (programmed death-ligand-1) molecule. PD-L1 is one of the common immune checkpoint molecules on the surface of tumor cells, capable of binding to the PD-1 receptor and suppressing the immune function of T cells. By binding to and blocking the PD-L1 molecule, the anti-PD-L1 antibody prevents its interaction with PD-1, thereby relieving the immunosuppressive effect of T cells and restoring the immune system's ability to recognize and eliminate tumors.

[0044] In this embodiment, the anti-CTLA-4 antibody is an antibody that targets the CTLA-4 (cytotoxic T-lymphocyte antigen-4) molecule. CTLA-4 is an inhibitory receptor on the surface of immune cells, involved in regulating T cell activity, especially in the early stages of immunity. By binding to its ligands (such as B7-1 and B7-2), CTLA-4 can inhibit T cell activation and proliferation. The anti-CTLA-4 antibody enhances T cell activity and promotes the body's anti-tumor immune response by blocking the function of the CTLA-4 receptor.

[0045] In this embodiment, the anti-LAG-3 antibody is an antibody targeting the LAG-3 (lymphocyte activation gene-3) molecule. LAG-3 is an immune checkpoint molecule mainly expressed on activated T cells, capable of binding to its ligand and inhibiting the T cell immune response. The anti-LAG-3 antibody relieves immunosuppression and enhances the anti-tumor effect of T cells by blocking the binding of LAG-3 to its ligand.

[0046] In this embodiment, the anti-TIGIT antibody is an antibody that targets the TIGIT (T cell immunoglobulin and ITIM domain) molecule. TIGIT is an immune checkpoint molecule expressed on T cells and NK cells, and participates in suppressing immune responses. The anti-TIGIT antibody relieves the immunosuppression of T cells and restores their tumor-killing effect by blocking the binding of TIGIT to its ligand.

[0047] In this application, drug-resistant tumors refer to a type of tumor that, after initial treatment, gradually loses its response to immune checkpoint inhibitors, chemotherapeutic drugs, or targeted drugs due to various immune escape mechanisms or treatment-induced drug resistance mechanisms. The formation of drug-resistant tumors is usually related to changes in tumor cells and their microenvironment, such as increased expression of immune checkpoint molecules and decline in immune cell function.

[0048] The embodiments of this application will now be described in more detail, examples of which are illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain this application, and should not be construed as limiting this application. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0049] Example 1: In vivo experimental validation of the combined treatment of drug-resistant tumors with anti-CD150 antibody and anti-PD-1 antibody. This embodiment uses a mouse subcutaneous xenograft tumor model to verify the anti-tumor effect and mechanism of action of the combined treatment of drug-resistant tumors with anti-SLAMF1 antibody and immune checkpoint inhibitor antibody. The mechanism of action of the combined treatment strategy is illustrated in the figure below. Figure 1 As shown.

[0050] In the PD-1 resistant tumor microenvironment, a high proportion of SLAMF1 exists within the tumor tissue. + B cells, which possess immunosuppressive functions, can limit the efficacy of anti-PD-1 monotherapy by weakening the effector activity of cytotoxic T cells. To address this resistance mechanism, this embodiment employs a combination therapy strategy using an anti-SLAMF1 antibody to target the inhibitory SLAMF1... + B cells are targeted for elimination or functional regulation, while anti-PD-1 antibodies block inhibitory immune checkpoint signals on the surface of T cells, thereby forming a synergistic immune activation effect in the tumor microenvironment, significantly reversing the immunosuppressive state, and promoting CD8 activation. + T cell reactivation promotes IFN-γ secretion and enhances tumor-killing ability.

[0051] To verify the in vivo antitumor efficacy of the above combined treatment strategy, subcutaneous xenograft tumor models of HEPA1-6 hepatocellular carcinoma, MC38 colorectal cancer, and B16-F10 melanoma were established in mice. The treatment process is as follows: Figure 2 As shown, the specific steps are as follows: Tumor cell preparation. HEPA1-6, MC38, and B16-F10 tumor cells were cultured routinely in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C in a 5% CO2 incubator. When the cells reached 70%–80% confluence, they were digested with trypsin and collected by centrifugation. The resulting cell pellet was washed twice with sterile PBS or basal RPMI-1640 medium to thoroughly remove residual serum and medium components, then resuspended and viable cell counted. Finally, the cell suspension concentration was adjusted to 1×10⁻⁶ cells / mL using PBS or RPMI-1640 medium. 7 One cell / mL for use.

[0052] Anesthetized the experimental animals. C57BL / 6 mice were placed in an induction box and anesthesia was induced using an isoflurane gas anesthesia machine with an induction concentration set at 3%. After the mice were anesthetized, they were transferred to the surgical operating board, and a mixture of gas containing 1%-1.5% isoflurane was continuously supplied through the nose to maintain a stable depth of surgical anesthesia.

[0053] Tumor cells were subcutaneously inoculated under anesthesia. Using a sterile insulin syringe or a 1 mL syringe equipped with a 25-27G needle, the prepared tumor cell suspension was drawn up and inoculated, with 5 × 10⁶ HEPA1-6 cells in each syringe. 5 50 μL per animal, 1 × 102 MC38 cells 6 Each individual, with 5 × 10 B16-F10 cells. 5Injection site: Right abdomen or upper groin area of ​​the right hind limb of the mouse. Gently lift the skin and insert the needle horizontally into the subcutaneous tissue. Slowly inject to form a distinct wheal. After removing the needle, gently press the injection site with a sterile cotton swab to prevent leakage.

[0054] After inoculation, turn off the anesthesia vaporizer, move the mice to a warm, clean bedding, and continuously supply pure oxygen for 1–2 minutes to promote isoflurane clearance. Once the mice have fully recovered their spontaneous activity, return them to their cages for routine feeding and observation.

[0055] Starting from day 5-7 after tumor inoculation, the long diameter (L) and short diameter (W) of the tumor were measured every 2-3 days using calipers, and the result was calculated using the formula V = (L × W). 2 The tumor volume was calculated as () / 2. Before the first administration (usually 6-7 days after inoculation), mice were randomly and evenly divided into 4 groups of 5-8 mice each, based on tumor volume, ensuring no significant difference in initial tumor volume among the groups. The groups were as follows: isotype control group, anti-PD-1 monotherapy group, anti-SLAMF1 monotherapy group, and anti-PD-1 and anti-SLAMF1 combination therapy group.

[0056] The antibody therapy phase began on day 7 post-vaccination, with administration administered on days 7, 10, 13, 16, and 19. In the monotherapy group, each antibody was diluted to 1 mg / mL with sterile PBS and filtered through a 0.22 μm filter for sterilization. In the combination therapy group, the stock solutions of anti-PD-1 monoclonal antibody and anti-SLAMF1 monoclonal antibody were diluted separately with PBS and mixed in equal volumes to ensure a final concentration of 1 mg / mL for both antibodies, guaranteeing consistent dosage of both antibodies in each injection. All antibody solutions were briefly stored at 4°C and prepared and used immediately.

[0057] The administration method was intraperitoneal injection. Mice were restrained with their heads slightly lower than their tails. The needle was inserted at approximately a 45° angle into the lower abdomen, avoiding the midline and bladder region, and 200 μL of antibody solution was slowly injected. The monotherapy group received a dose of 200 μg antibody per mouse, while the combination therapy group received 200 μL of a mixture containing 200 μg anti-PD-1 antibody and 200 μg anti-SLAMF1 antibody.

[0058] Tumor volume and mouse weight changes were continuously monitored during treatment. If a single tumor exceeded 1500 mm², [further action was taken]. 3If a mouse loses more than 20% of its initial body weight, develops ulcers or infections in its tumor, or exhibits obvious signs of distress, it should be euthanized immediately in accordance with animal ethics requirements. The experimental endpoint is typically set as 28 days after the last administration of medication, or when the tumor in the control group reaches the ethical endpoint. At the endpoint, all mice are sacrificed, and tumor tissue, spleen, and tumor-draining lymph nodes are collected, weighed, photographed, and preserved in liquid nitrogen or fixative as needed for subsequent flow cytometry, immunohistochemistry, and transcriptome analysis.

[0059] Experimental results are as follows Figures 3-5 As shown. In HEPA 1-6 liver cancer ( Figure 3 ) and MC38 colorectal cancer ( Figure 4 In models where monotherapy for immunotherapy is prone to resistance, both anti-PD-1 monotherapy and anti-SLAMF1 monotherapy showed a certain degree of tumor growth inhibition. However, the combination therapy of anti-PD-1 and anti-SLAMF1 significantly enhanced the tumor inhibition effect, with a tumor inhibition rate significantly higher than either monotherapy group (p<0.05), effectively delaying tumor progression.

[0060] In rapidly growing and highly aggressive B16-F10 melanoma ( Figure 5 In the model, anti-SLAMF1 monotherapy had limited efficacy, and although anti-PD-1 monotherapy had some inhibitory effect, the combination therapy group showed more significant and durable tumor growth control ability, and its anti-tumor effect was significantly better than that of the monotherapy group (p<0.01).

[0061] In summary, the combined treatment of anti-PD-1 and anti-SLAMF1 antibodies demonstrated a clear synergistic anti-tumor effect in various tumor models with different biological characteristics, showing significant advantages, especially in drug-resistant and highly invasive tumor models. The results of this embodiment indicate that this combined immunotherapy strategy can provide a new and feasible technical solution for overcoming tumor immunotherapy resistance by reshaping the tumor microenvironment, relieving immunosuppression, and enhancing effector T cell function.

[0062] Example 2: Combined therapy enhances T-cell immune response in the tumor microenvironment In this embodiment, tumor-infiltrating lymphocytes (TILs) were isolated from tumor tissues of tumor-bearing mice, and the effector function of cytotoxic T cells was detected by flow cytometry to evaluate the effect of combined treatment with anti-PD-1 antibody and anti-SLAMF1 antibody on T cell immune response in the tumor microenvironment.

[0063] At the treatment endpoint, tumor-bearing mice that had completed grouped treatment were euthanized. These mice included the isotype IgG control group, the anti-PD-1 monotherapy group, the anti-SLAMF1 monotherapy group, and the anti-PD-1 and anti-SLAMF1 combination therapy group. Subcutaneous tumor tissue was completely dissected under aseptic conditions, and after removing surrounding non-tumor tissue, it was accurately weighed and immediately placed in pre-cooled RPMI 1640 basal medium containing 1% penicillin-streptomycin antibiotics for temporary storage on ice.

[0064] Subsequently, tumor tissue digestion and single-cell suspension preparation were performed. The tumor tissue was transferred to a sterile culture dish and thoroughly minced to approximately 1 mm using sterile surgical scissors. 3 Small, fragmented tissue pieces were prepared. The minced tissue was transferred to 15 mL sterile centrifuge tubes, and 3-5 mL of preheated (37°C) tumor digestion working solution was added, depending on the volume of the tumor tissue. This digestion working solution used RPMI 1640 basal medium as the solvent and contained 1 mg / mL collagenase IV, 0.1 mg / mL DNase I, and 2% fetal bovine serum. The centrifuge tubes were placed in a 37°C shaker and digested at 200 rpm for 30-45 minutes, with vigorous vortexing every 10 minutes to promote complete tissue dissociation. After digestion, an equal volume of complete culture medium containing 10% fetal bovine serum was immediately added to terminate the enzymatic reaction. The resulting cell suspension was filtered through a 70 μm cell sieve, the filtrate was collected, washed with pre-cooled PBS, and centrifuged at 300 × g for 5 minutes. The supernatant was discarded, and the cell pellet was obtained.

[0065] To enrich tumor-infiltrating lymphocytes, discontinuous Percoll density gradient centrifugation was used for separation. First, 70% and 40% Percoll solutions were prepared with PBS. In a 15 mL centrifuge tube, 3 mL of 70% Percoll solution was added as the bottom layer, followed by resuspending the cell pellet in 3 mL of 40% Percoll solution. This 40% Percoll solution was carefully stacked on top of the 70% Percoll solution layer to avoid interfacial mixing. Centrifugation was performed at 25°C for 30 minutes at 750 × g, acceleration 1, and deceleration 0. After centrifugation, lymphocytes were mainly enriched in the interfacial layer between the 70% and 40% Percoll solutions. This interfacial layer of cells was carefully aspirated and transferred to a new centrifuge tube. The cells were washed thoroughly with excess PBS and centrifuged at 500 × g for 5 minutes. The supernatant was discarded, yielding highly purified tumor-infiltrating lymphocytes, which were then counted.

[0066] The isolated TILs were then analyzed by flow cytometry staining. First, cell stimulation was performed by culturing cells with 50 ng / mL PMA and 1 μg / mL iomycin for 3.5 hours. Monensin was then added to a final concentration of 2 μM and cultured for another 30 minutes to promote intracellular cytokine accumulation. After stimulation, the cells were washed with PBS. Next, viable cell staining was performed by resuspending cells in PBS, adding Fixed Viability Stain 700 (FVS700) stain, and incubating at 4°C in the dark for 15 minutes. After incubation, the cells were washed twice with PBS containing 2% fetal bovine serum.

[0067] Cell surface antigen staining was then performed. Cells were resuspended in PBS, and a pre-titrated mixture of cell surface antibodies, including anti-mouse CD45, CD3, CD4, and CD8 antibodies, was added. The mixture was incubated at 4°C in the dark for 30 minutes, followed by washing twice with PBS. After surface staining, intracellular cytokine staining was performed using a commercially available intracellular fixation and permeabilization kit. Following the manufacturer's instructions, cells were first fixed and permeabilized with a fixative / permeabilization agent, incubated at 4°C in the dark for 30-60 minutes, washed after permeabilization, and anti-mouse IFN-γ antibody or its isotype control antibody was added. The mixture was incubated at 4°C in the dark for 60 minutes, followed by washing twice.

[0068] Finally, the stained cells were resuspended in 100-200 μL of PBS, filtered through a 35 μm filter, transferred to flow cytometry tubes, and immediately placed on a flow cytometer for analysis. During flow cytometry data acquisition, at least 10,000 events were acquired within the live-cell gate (FVS700 negative). For data analysis, the lymphocyte population was first delineated in the FSC-A / SSC-A scatter plot, and further screening of FVS700 cells was performed within this population. - Live cells, and then CD45 is sorted from the live cells. + White blood cell population. In CD45 + Further screening of CD3 in cells + T lymphocytes, and in CD3 + T cell population analysis CD4 - CD8 + Cell subsets, ultimately in CD8 + IFN-γ levels are counted within T cell gates. + Cell percentage. All data were analyzed using FlowJo software and compared with isotype control groups to exclude non-specific signal interference.

[0069] Test results as follows Figure 6 and Figure 7As shown. The results indicated that, compared with the isotype control group and the monotherapy group, the anti-PD-1 and anti-SLAMF1 combination therapy group had a higher number of effector cytotoxic T cells (CD8+) in tumor tissue. + IFN-γ + The significantly increased proportion suggests that this combined treatment strategy can synergistically enhance the functional activity of effector T cells in the tumor microenvironment.

[0070] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0071] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0072] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. The use of anti-CD150 antibodies and immune checkpoint inhibitor antibodies in the preparation of a drug for the treatment of drug-resistant tumors.

2. The application according to claim 1, characterized in that, The immune checkpoint inhibitor antibodies include at least one of the following: anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, anti-LAG-3 antibody, and anti-TIGIT antibody.

3. The application according to claim 2, characterized in that, The immune checkpoint inhibitor antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody.

4. The application according to any one of claims 1-3, characterized in that, The tumors include: liver cancer, colon cancer, melanoma, and lung cancer.

5. A drug combination, characterized in that, include: Anti-CD150 antibodies and immune checkpoint inhibitor antibodies.

6. The drug combination according to claim 5, characterized in that, The immune checkpoint inhibitor antibodies include at least one of the following: anti-PD-1 antibody, anti-PD-L1 antibody, anti-CTLA-4 antibody, and anti-LAG-3 antibody; Preferably, the immune checkpoint inhibitor antibody is an anti-PD-1 antibody or an anti-PD-L1 antibody; Preferably, the drug combination comprises an anti-CD150 antibody and an anti-PD-1 antibody; Preferably, the drug combination comprises an anti-CD150 antibody and an anti-PD-L1 antibody; Preferably, the effective dose of the anti-CD150 antibody, based on body weight, is selected from 1 mg / kg to 10 mg / kg; Preferably, the effective dose of the anti-PD-1 antibody or the anti-PD-L1 antibody, on a body weight basis, is selected from 2 mg / kg to 4 mg / kg; Preferably, the effective dose of the anti-PD-1 antibody or anti-PD-L1 antibody is selected from 200 mg / dose to 400 mg / dose on a fixed dosage.

7. The drug combination according to claim 6, characterized in that, The drug combination includes: cytokines; Optionally, the pharmaceutical composition further comprises: pharmaceutically acceptable excipients; Optionally, the excipients include one or more pharmaceutically acceptable excipients, diluents, stabilizers, or carriers.

8. The drug combination according to any one of claims 5-7, characterized in that, The drugs are combined in the form of an injectable formulation.

9. Application of anti-CD150 antibodies and immune checkpoint inhibitor antibodies in the treatment of drug-resistant tumors.

10. A treatment method for drug-resistant tumors, characterized in that, include: Administering an effective dose of a drug combination to a subject; wherein the drug combination is as described in any one of claims 5-8; Preferably, the drug combination comprises an anti-CD150 antibody and an anti-PD-1 antibody; Preferably, the drug combination comprises an anti-CD150 antibody and an anti-PD-L1 antibody; Preferably, the effective dose of the anti-CD150 antibody, based on body weight, is selected from 1 mg / kg to 10 mg / kg; Preferably, the effective dose of the anti-PD-1 antibody or the anti-PD-L1 antibody, on a body weight basis, is selected from 2 mg / kg to 4 mg / kg; Preferably, the effective dose of the anti-PD-1 antibody or anti-PD-L1 antibody, measured in fixed doses, is selected from 200 mg / dose to 400 mg / dose; Preferably, the combined administration frequency of the drugs is once every 3-4 days; Preferably, the proportion of CD150-positive B cells in the tumor is greater than a predetermined threshold; Preferably, the predetermined threshold is selected from 35%-45%.

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