Application of Rab001 combined PD-1 monoclonal antibody in preparation of anti-breast cancer tumor drugs

Abstract

The invention relates to the technical field of anti-breast cancer tumor drugs, in particular to application of Rab001 combined with a PD-1 monoclonal antibody in preparation of anti-breast cancer tumor drugs. The invention discloses an application of Rab001 combined with a PD-1 monoclonal antibody in preparation of an anti-breast cancer tumor drug, the Rab001 promotes antigen presentation, antibody generation, formation of a tertiary lymphatic structure (TLS) and regulation of a tumor microenvironment by increasing the number and activity of peripheral circulating B lymphocytes and M1 type macrophages, M1 / M2, CD8 + T and B cells in tumors, so that the treatment effect of the PD-1 monoclonal antibody is enhanced.

Description

Technical Field

[0001] This application relates to the technical field of anti-breast cancer drugs, and in particular to the application of Rab001 in combination with PD-1 monoclonal antibody in the preparation of anti-breast cancer drugs. Background Technology

[0002] With the continuous advancement of cancer treatment technologies, immunotherapy, especially immune checkpoint inhibitors (ICIs) such as PD-1 monoclonal antibodies, is increasingly widely used in tumor treatment. However, although PD-1 monoclonal antibodies have shown good efficacy in some "hot" tumors, their therapeutic effect in solid tumors such as breast cancer remains limited. The response rate to ICI treatment in most types of tumors is only 20%–40%, mainly due to a variety of factors, including intratumoral cytotoxicity of CD8. + The depletion or functional defects of T lymphocytes, and the complex immunosuppressive mechanisms in the tumor microenvironment.

[0003] In recent years, research on immunotherapy has deepened, aiming to improve the response rate of tumors to ICI treatment. Studies have found that, in addition to T lymphocytes, B lymphocytes also play a crucial role in tumor immunotherapy. B cells can promote anti-tumor immune responses through various mechanisms, including enhancing antigen presentation, producing anti-tumor antibodies, and inducing tertiary lymphoid structures (TLS). These findings provide a theoretical basis for developing novel combination therapy regimens.

[0004] While the potential of B cells in tumor immunotherapy is widely recognized, effectively activating and utilizing their anti-tumor activity remains a significant technical challenge. Furthermore, PD-1 monoclonal antibodies alone have limited efficacy in "cold" tumors or drug-resistant solid tumors, prompting researchers to explore combination therapy strategies to improve treatment outcomes. However, no mature technological approach currently exists that can fully integrate the synergistic effect of B cell activation and PD-1 monoclonal antibody therapy to significantly improve the treatment efficacy of solid tumors such as breast cancer.

[0005] Moreover, existing combination therapy strategies primarily focus on enhancing the anti-tumor activity of T cells through radiotherapy, chemotherapy, or targeted drugs, while paying less attention to the role of B cells. Although these strategies have improved the response rate of ICI treatment to some extent, problems such as unstable efficacy and significant side effects still exist. In addition, the development of specific drugs targeting B cell activation is relatively lagging, and there is a lack of effective mechanisms for synergistic effects with PD-1 monoclonal antibodies.

[0006] Therefore, current combination therapy strategies have significant limitations in activating and utilizing the anti-tumor activity of B cells, failing to fully realize the potential of B cells in tumor immunotherapy. To overcome these limitations, it is necessary to develop new combination therapy strategies that activate B cells and combine them with PD-1 monoclonal antibody therapy, aiming to achieve higher tumor treatment response rates and lower side effects. Summary of the Invention

[0007] The purpose of this application is to provide the application of Rab001 in combination with PD-1 monoclonal antibody in the preparation of anti-breast cancer drugs. Rab001 enhances the therapeutic effect of PD-1 monoclonal antibody by increasing the number and activity of peripheral circulating B lymphocytes and tumor-bearing M1 macrophages, M1 / M2, CD8+ T and B cells, thereby promoting antigen presentation, antibody production, formation of tertiary lymphoid structures (TLS) and regulation of the tumor microenvironment.

[0008] To solve the above-mentioned technical problems, this application is implemented as follows:

[0009] The primary objective of this application is to provide the use of Rab001 in combination with PD-1 monoclonal antibodies in the preparation of anti-breast cancer drugs.

[0010] The second objective of this application is to provide the application of Rab001 in combination with PD-1 monoclonal antibody in the preparation of anti-breast cancer tumor inhibitors.

[0011] In some specific embodiments, the anti-breast cancer tumor drug or anti-breast cancer tumor inhibitor can increase the content of M1 macrophages and tumor-killing macrophages in tumor tissue.

[0012] In some specific embodiments, the anti-breast cancer tumor drug or anti-breast cancer tumor inhibitor can reduce the size of breast cancer tumors.

[0013] In some specific embodiments, the anti-breast cancer tumor drug or anti-breast cancer tumor inhibitor includes an effective dose of Rab001 and an effective dose of PD-1 monoclonal antibody, with the ratio of the effective dose of Rab001 to the effective dose of PD-1 monoclonal antibody being (1-9):10. In specific mouse studies, the effective dose of Rab001 in the anti-breast cancer tumor drug or anti-breast cancer tumor inhibitor is 1 mg / kg to 9.0 mg / kg, and the effective dose of the PD-1 monoclonal antibody is 10 mg / kg. Based on body surface area conversion, the effective adult dose range of Rab001 as an anti-breast cancer tumor drug or anti-breast cancer tumor inhibitor is approximately 0.08 mg / kg to 0.72 mg / kg, and the effective adult dose of the PD-1 monoclonal antibody is approximately 0.8 mg / kg. The actual clinical dose needs to be determined through clinical trials.

[0014] In some specific embodiments, the anti-breast cancer tumor drug or anti-breast cancer tumor inhibitor also includes a pharmaceutically acceptable carrier or excipient.

[0015] In some specific embodiments, the administration methods of the anti-breast cancer tumor drug or anti-breast cancer tumor inhibitor are subcutaneous, local perfusion of tumor tissue, oral, intraperitoneal or intravenous administration.

[0016] A third objective of this application is a pharmaceutical composition comprising Rab001 or a pharmaceutically acceptable salt or solvate thereof.

[0017] PD-1 or its pharmaceutically acceptable salts or solvates.

[0018] In some specific embodiments, the weight ratio of Rab001 to PD-1 in the pharmaceutical composition is (1-9):10.

[0019] The beneficial effects of this application are as follows:

[0020] The application of Rab001 in combination with PD-1 monoclonal antibody in the preparation of anti-breast cancer drugs described in this application involves Rab001 increasing peripheral circulating B lymphocytes and intratumoral M1 macrophages, M1 / M2 ratio, and CD8+. + The number and activity of T and B cells promote antigen presentation, antibody production, and the formation of tertiary lymphoid structures (TLS), reshaping the tumor microenvironment to make it more conducive to the infiltration and activation of immune cells, thereby enhancing the therapeutic effect of PD-1 monoclonal antibody; the combination of Rab001 and PD-1 monoclonal antibody significantly reduced the size of breast cancer tumors and improved the tumor growth inhibition rate.

[0021] The application of Rab001 combined with PD-1 monoclonal antibody in the preparation of anti-breast cancer drugs described in this application is a novel combination therapy strategy. By targeting immune regulation and enhancing immune cell function, it improves the efficacy of tumor immunotherapy and provides a new treatment approach for breast cancer. Attached Figure Description

[0022] Figure 1 This is a graph showing the effect of the combination of Rab001 and PD-1 antibodies on mouse body weight in Example 1.

[0023] Figure 2 This is a graph showing the effect of the combined use of Rab001 and PD-1 antibodies on the growth of mouse breast cancer tumors in Example 1.

[0024] Where: *: P<0.05; **: P<0.01; ***: P<0.001 vs. 6D or 13D; a: P<0.05; b: P<0.01; C: P<0.001 vs. solvent group;

[0025] Figure 3 This is a graph showing the inhibition rate of Rab001 and PD-1 antibodies in combination against mouse breast cancer tumor growth in Example 1.

[0026] Figure 4 This is a bar graph showing the effect of the combination of Rab001 and PD-1 antibodies on tumor weight in mouse breast cancer in Example 1.

[0027] Where: *: P<0.05; **: P<0.01; ***: P<0.001 vs. 6D or 13D; a: P<0.05; b: P<0.01; C: P<0.001 vs. solvent group;

[0028] Figure 5 This is a graph showing the effect of the combined use of Rab001 and PD-1 antibodies on the number of total peripheral lymphocytes in mice, as described in Example 1.

[0029] Where: *: P<0.05; **: P<0.01; ***: P<0.001; a: P<0.05; b: P<0.01; C: P<0.001 vs. baseline;

[0030] Figures 6-9 The combined use of Rab001 and PD-1 antibodies in Example 1 was used to target M1, M1 / M2, and CD8 in mouse breast cancer tumor tissue. + A bar chart showing the effect of the T and B cell ratio;

[0031] Where: Note: *: P<0.05; **: P<0.01; ***: P<0.001 vs. 6D or 13D; a: P<0.05; b: P<0.01; C: P<0.001 vs. solvent group;

[0032] Figure 10 This is a graph showing the effect of different doses of Rab001 and PD-1 antibody combined on mouse body weight in Example 2;

[0033] Figure 11 This is a graph showing the effect of different doses of Rab001 and PD-1 antibody combined on the growth of mouse breast cancer tumors in Example 2.

[0034] Where: Note: *: P<0.05; **: P<0.01; ***: P<0.001 vs. 6D or 13D; a: P<0.05; b: P<0.01; C: P<0.001 vs. solvent group;

[0035] Figure 12 This is a graph showing the inhibition rate of Rab001 and PD-1 antibodies in combination against mouse breast cancer tumor growth in Example 1.

[0036] Figure 13 This is a bar graph showing the effect of different doses of Rab001 and PD-1 antibody combined on tumor weight in mouse breast cancer in Example 2.

[0037] Where: Note: *: P<0.05; **: P<0.01; ***: P<0.001 vs. 6D or 13D; a: P<0.05; b: P<0.01; C: P<0.001 vs. solvent group;

[0038] Figure 14 This is a graph showing the effect of different doses of Rab001 and PD-1 antibody combined on the number of peripheral total lymphocytes in mice in Example 2.

[0039] Where: *: P<0.05; **: P<0.01; ***: P<0.001; a: P<0.05; b: P<0.01; C: P<0.001 vs. baseline;

[0040] Figures 15-18 The effects of different doses of Rab001 and PD-1 antibody combined on M1, M1 / M2, and CD8 in mouse breast cancer tumor tissue, as shown in Example 2. + A bar chart showing the effect of the T and B cell ratio;

[0041] Where: *: P<0.05; **: P<0.01; ***: P<0.001 vs. 6D or 13D; a: P<0.05; b: P<0.01; C: P<0.001 vs. solvent group.

[0042] Figure 19 The image shows the results of detecting mouse body weight using the combination of Rab001 and PD-1 antibodies in Example 1.

[0043] Figure 20 The figure shows the results of detecting the effect of the combination of Rab001 and PD-1 antibodies on the growth and size of mouse breast cancer tumors in Example 1. Detailed Implementation

[0044] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings.

[0045] Terminology Explanation:

[0046] Rab001 is a polypeptide compound composed of two ligands covalently linked by a chemical chain. One ligand is a highly derived synthetic tripeptide, LLP2A, which exhibits high affinity and specificity for α4β1 integrin; the other ligand is a bone-targeting bisphosphate (alendronate). Because α4β1 integrin is highly expressed in stem cells (MSCs), this drug utilizes its dual-targeting action to draw stem cells to bone tissue, thereby treating various bone diseases. This compound was originally developed as a clinical candidate for the treatment of bone diseases such as osteonecrosis, osteoporosis, and fractures. The specific chemical structure of the compound is shown below:

[0047]

[0048] α4β1 integrin is a cell surface receptor belonging to the integrin family; it is expressed in various cell types, including stem cells and lymphocytes (such as B lymphocytes). α4β1 integrin plays an important role in cell adhesion, migration, proliferation, and differentiation.

[0049] PD-1 monoclonal antibody, or programmed death-1 (PD-1) monoclonal antibody, is an immune checkpoint receptor expressed on T cells that negatively regulates T cell activity and function. PD-1 monoclonal antibody restores T cell activity and enhances the immune system's ability to kill tumors by blocking the binding of PD-1 to its ligand PD-L1.

[0050] M1 macrophages are a polarized state of macrophages with pro-inflammatory and anti-tumor properties. M1 macrophages can secrete a variety of cytokines (such as IL-12 and TNF-alpha), activate cytotoxic T cells, and promote the killing of tumor cells.

[0051] Tumor-killing macrophages refer to macrophages with the ability to directly kill tumor cells, typically including M1 type macrophages. They can kill tumor cells through phagocytosis, secretion of cytokines, or induction of immunogenic cell death.

[0052] B cells, or B lymphocytes, are an important cell type in the immune system. B cells are primarily responsible for producing antibodies, which kill tumor cells or mark them for destruction by other immune cells through antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC).

[0053] Cytotoxic CD8 + T lymphocytes, a subset of T cells, also known as cytotoxic T cells (CTLs), are capable of recognizing and killing infected or tumor cells. CD8 +T cells play a crucial role in tumor immunotherapy, and their quantity and activity are closely related to the tumor's response rate to immunotherapy. Studies have shown that cytotoxic CD8 cells... + T lymphocytes play a leading role in the response to ICI immunotherapy for cancer; therefore, cytotoxic CD8 cells... + The number and activity of T lymphocytes within tumor tissue are closely correlated with the tumor's response rate to ICI immunotherapy. Immunotherapy is highly effective for hematologic malignancies but has limited efficacy against solid tumors. Most types of tumors respond to ICI therapy with only about 20%-40%. Possible reasons include tumor infiltration of CD8+ cells. + The limited number or functional defects of T lymphocytes necessitate the clinical combination of various effective treatments, including radiotherapy, chemotherapy / targeted therapies, etc., to improve the tumor's response rate to ICI immunotherapy. Since cancer remains a leading cause of death, this unmet clinical need necessitates the development of more new and effective therapeutic drugs.

[0054] ICI (immune checkpoint inhibitors) are drugs that inhibit immune checkpoint signaling pathways (such as PD-1 / PD-L1, CTLA-4, etc.). ICI restores the immune system's anti-tumor ability by relieving the suppression of tumor cells by the immune system.

[0055] "Hot" tumors refer to tumors infiltrated with a large number of immune cells (such as T cells and B cells). These tumors usually respond well to immunotherapy. However, in some cases, the immune cells in "hot" tumors may become depleted or dysfunctional due to prolonged exposure to tumor antigens and chronic antigen stimulation.

[0056] ADCC (antibody-dependent cytotoxicity) refers to the killing effect of antibodies on tumor cells by binding to antigens on the surface of tumor cells, which in turn bind to immune cells with Fc receptors (such as NK cells, macrophages, etc.).

[0057] CDC (complement-dependent cytotoxicity) refers to the process by which antibodies bind to antigens on the surface of tumor cells, activating the complement system, causing complement components to deposit on the surface of tumor cells and form membrane attack complexes, thereby triggering tumor cell lysis and death.

[0058] Tertiary lymphoid structures (TLS) are structures that form within the tumor microenvironment, resembling secondary lymphoid organs such as lymph nodes and the spleen. TLS can serve as centers for immune activation and T-cell initiation, playing a crucial role in anti-tumor immune responses.

[0059] Antigen presentation refers to the process by which immune cells (such as B cells and dendritic cells) take up, process, and present antigens (such as tumor antigens) to T cells. Antigen presentation is one of the key steps in initiating a specific immune response.

[0060] Cross-presentation refers to the process by which antigen-presenting cells (such as dendritic cells) process exogenous antigens (such as tumor antigens) into peptides, which then bind to MHC class I molecules and present them to CD8. + The process of T cells. Cross-presentation is CD8. + One of the important ways in which T cells recognize and kill infected or tumor cells.

[0061] Current findings indicate that PD-1 monoclonal antibodies have limited efficacy in "hot" tumors, partly due to pre-existing T cell depletion and immunosuppressive signals in the tumor microenvironment limiting immune cell activity, resulting in insufficient numbers or dysfunction of immune cells in some tumor patients. Therefore, this application provides the use of Rab001 in combination with PD-1 monoclonal antibodies in the preparation of anti-breast cancer drugs.

[0062] The present application will be further described through the following embodiments and other content.

[0063] Example 1

[0064] 1. Main experimental materials and instruments

[0065] Materials: 32 BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0066] Reagents: Flow cytometry antibodies (BD, Biolegend, USA);

[0067] Rab001 was synthesized by Chengdu Shengnuo Biopharmaceutical Co., Ltd., which signed an agreement with RabPharma.

[0068] The PD-1 monoclonal antibody is an anti-mPD-1 antibody, purchased from Bioxcell in the United States;

[0069] Instruments: Flow cytometer (BD LSR Fortessa, USA); Centrifuge (Eppendorf 5920R, Germany); Cell counter (Nexcelom Bioscience Cellometer Auto T4, USA); Cell dissociator (Miltenyi Biotec cgentleMACS, Germany) TM Dissociators).

[0070] 2. Experimental Methods

[0071] A murine EMT6 subcutaneous transplantation tumor model of breast cancer was established by subcutaneously inoculating BALB / c mice with EMT6 tumor cells. The experiment was divided into four groups: a solvent control group, an anti-mPD-1 10 mg / kg monotherapy group, a Rab001 1.6 mg / kg monotherapy group, and a combination therapy group of anti-mPD-1 10 mg / kg and Rab001 1.6 mg / kg, with eight mice in each group. Efficacy was evaluated based on tumor inhibition index (TGI), and safety was evaluated based on changes in animal body weight and mortality.

[0072] The experimental mice were subcutaneously inoculated with 5×10⁶ mice on their right back. 5 EMT6 cells were resuspended in PBS (0.1 ml / cell), and tumor growth was observed regularly.

[0073] Before administration, weigh all animals and measure tumor volume using calipers. Continue administration until the tumor reaches an average volume of 93 mm. 3 Mice were randomly assigned to groups based on tumor size. Since tumor size affects treatment effectiveness, a randomized controlled trial was used, grouping mice according to their tumor size to ensure similar tumor sizes across groups. StudyDirector was used. TM (Version 3.1.399.19, vendor Studylog System, Inc., S. San Francisco, CA, USA) Selected the Matched distribution method for grouping. The grouping day is defined as day 0.

[0074] Following tumor cell inoculation, routine monitoring included monitoring tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the animal's activity level, food and water intake, weight gain or loss, and any abnormalities observed in the eyes, coat, or other areas. Clinical symptoms observed during the experiment were recorded in the raw data. After drug administration began, tumor size and mouse weight were measured two or three times per week.

[0075] Formula for calculating tumor volume: Tumor volume (mm) 3 )=1 / 2×(a×b 2 (Where a represents the major axis and b represents the minor axis). The experiment uses StudyDirector. TM(Version 3.1.399.19, supplier: Studylog System, Inc.) The software collects data, including measurements of tumor length and short diameter and animal weight. Raw data, measured using a balance and calipers, is directly imported into the software, and any changes are recorded. All procedures, including drug administration, tumor measurement, and weight measurement, are performed in a biosafety cabinet or laminar flow hood. Cells are resuspended in PBS (0.1 ml / mouse), and tumor growth is observed regularly. Mouse weight is measured.

[0076] Peripheral blood lymphocyte counts were performed using routine blood tests; flow cytometry was used to analyze the percentage of lymphocyte subsets in breast cancer tumor tissue.

[0077] 3. Results:

[0078] (1) Effect of Rab001 combined with PD-1 antibody on mouse body weight

[0079] See results Figure 19 and Figure 1 The curves showing the effect of the combination of Rab001 and PD-1 antibody on mouse body weight revealed that repeated administration of Rab001 combined with PD-1 antibody had no significant effect on mouse body weight.

[0080] (2) Effects of Rab001 combined with PD-1 antibody on the growth of breast cancer tumors in mice

[0081] See results Figure 20 and Figure 2 , Figure 3 , Figure 4 It is evident that Rab001 combined with PD-1 antibody significantly inhibits the growth of mouse breast cancer tumors. Specifically, on Day 20, after administration of Rab001 combined with PD-1 antibody, the growth of mouse breast cancer tumors was significantly smaller than that of PD-1 antibody alone. The inhibition rate of Rab001 combined with PD-1 antibody on the growth of mouse breast cancer tumors reached 54.16%, while the inhibition rate of PD-1 antibody alone was 24.98%.

[0082] (3) Effect of Rab001 combined with PD-1 antibody on the number of peripheral blood lymphocytes in mice

[0083] The results are shown in Table 1-1 and Figure 5 The results showed that Rab001 combined with PD-1 antibody significantly increased the number of peripheral blood lymphocytes in mice; specifically, one hour after the third administration of Rab001 combined with PD-1 antibody, the number of peripheral blood lymphocytes in mice was significantly higher than that 24 hours before administration.

[0084] Table 1-1 shows the effect of Rab001 on the number of peripheral blood lymphocytes in mice (10). 9 / L)

[0085]

[0086] (4) Rab001 combined with PD-1 antibody showed that it inhibited M1 macrophages, M1 / M2, and CD8 in mouse breast cancer tumor tissue. + The effect of T and B cell ratio

[0087] The results are shown in Tables 1-2 and 2-3. Figure 6 , Figure 7 , Figure 8 and Figure 9 The results showed that after administration of Rab001 in combination with PD-1 antibody, M1 macrophages, M1 / M2, and CD8 were reduced in mouse breast cancer tumor tissue. + The ratio of T cells to B cells increases.

[0088] Table 1-2 shows the effects of Rab001 combined with PD-1 antibody on B cells, M1 macrophages, M1 / M2, and CD8+ in mouse breast cancer tumor tissue. + The effect of T cell proportion (%)

[0089]

[0090] Example 2

[0091] 1. Main experimental materials and instruments

[0092] Materials: 48 BALB / c mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0093] Reagents: Flow cytometry antibodies (BD, Biolegend, USA);

[0094] Instruments: Flow cytometer (BD LSR Fortessa, USA); Centrifuge (Eppendorf 5920R, Germany); Cell counter (Nexcelom Bioscience Cellometer Auto T4, USA); Cell dissociator (Miltenyi Biotec cgentleMACS, Germany) TM Dissociators).

[0095] 2. Experimental Methods

[0096] The experimental mice were subcutaneously inoculated with 5×10⁶ mice on their right back. 5 EMT6 cells were resuspended in PBS (0.1 ml / cell), and tumor growth was observed regularly until the tumor reached an average volume of 100 mm². 3 When the tumor is in its left or right position, the medication is administered to patients in random groups based on tumor size.

[0097] The trial was divided into four groups: a solvent control group, an anti-mPD-1 10 mg / kg monotherapy group, an anti-mPD-1 10 mg / kg and Rab001 1.0 mg / kg combination therapy group (low-dose group), an anti-mPD-1 10 mg / kg and Rab001 9.0 mg / kg combination therapy group (low-dose group), and a Rab001 9.0 mg / kg monotherapy group. Eight mice were treated in each group. Efficacy was evaluated based on tumor inhibition rate (TGI), and safety was evaluated based on changes in animal body weight and mortality.

[0098] Before administration, all animals were weighed and tumor volume was measured using calipers. Given that tumor volume affects treatment effectiveness, a randomized controlled trial was conducted, grouping mice according to their tumor volume to ensure similarity in tumor volume between groups. Grouping was performed using StudyDirector™ (version 3.1.399.19, vendor StudylogSystem, Inc., S. San Francisco, CA, USA). The "Matched distribution" randomization method was selected, which minimizes inter-group differences in tumor volume. This algorithm matches individual measurements of all selected animals to the mean of all selected animals. When grouping animals for the experiment, the mean weight of all selected animals was first calculated. Animals with similar mean weights were then paired. These paired animals were then assigned to different experimental groups, ensuring that the mean weight of each group matched, or was as close as possible to, the overall mean weight of all selected animals. This method ensures balanced starting conditions between experimental groups, reducing bias in experimental results. The day the groups are finalized is defined as Day 1 of the experiment, and medication is administered according to the experimental design on Day 1. All subsequent experimental procedures and observations will begin from this day.

[0099] Following tumor cell inoculation, routine monitoring included tumor growth and the impact of treatment on normal animal behavior. Specific monitoring included the experimental animals' activity levels, food and water intake, weight gain or loss, and any abnormalities observed in the eyes, coat, or other areas. Clinical symptoms observed during the experiment were recorded in the raw data. After drug administration began, mouse body weight and tumor size were measured two or three times per week. The tumor volume was calculated using the formula: Tumor volume (mm³) = 1 / 2 × (a × b) 2(Where a represents the major axis and b represents the minor axis). Data was collected using StudyDirector™ software (version 3.1.399.19, vendor: StudylogSystem, Inc.), including measurements of the tumor's major and minor axes and animal weight. Raw data, measured using a balance and vernier calipers, was directly imported into the software, and any changes were recorded. All procedures, including drug administration, tumor measurement, and weight measurement, were performed in a biosafety cabinet or clean bench.

[0100] 3. Results:

[0101] (1) Effects of different doses of Rab001 combined with PD-1 antibody on mouse body weight

[0102] The results are shown in Table 2-1 and Figure 10 The results showed that multiple administrations of different doses of Rab001 combined with PD-1 antibody had no significant effect on mouse body weight.

[0103] Table 2-1 shows the effect of different doses of Rab001 combined with PD-1 antibody on mouse body weight (g).

[0104]

[0105] (2) Effects of different doses of Rab001 combined with PD-1 antibody on the growth of breast cancer tumors in mice

[0106] The results are shown in Table 2-2 and Figure 11 , Figure 12 and Figure 13 The results showed that high-dose Rab001, in combination with PD-1 antibody, had a greater inhibitory effect on the growth of breast cancer tumors in mice compared with low-dose Rab001.

[0107] Table 2-2 shows the effects of different doses of Rab001 combined with PD-1 antibody on mouse breast cancer tumors.

[0108]

[0109] (3) Effects of different doses of Rab001 combined with PD-1 antibody on the number of peripheral blood lymphocytes in mice

[0110] The results are shown in Tables 2-3 and 3-3. Figure 14 The results showed that, before and after the third administration, the increase in the number of peripheral blood lymphocytes in mice was more significant after administration of high-dose Rab001 and low-dose Rab001 combined with PD-1 antibody.

[0111] Table 2-3 shows the effect of different doses of Rab001 combined with PD-1 antibody on the number of peripheral blood lymphocytes in mice (10). 9 / L)

[0112]

[0113] (4) Different doses of Rab001 combined with PD-1 antibody showed effects on M1 macrophages, M1 / M2, and CD8 in mouse breast cancer tumor tissue. + The effect of T and B cell ratio

[0114] The results are shown in Tables 2-4 and 4. Figure 15 , 16 The results showed that high-dose Rab001 combined with low-dose PD-1 antibody administration resulted in lower M1 / M2 ratios and CD8+ levels in mouse breast cancer tumor tissue. + The number of T and B cells increased significantly.

[0115] Table 2-4 shows the effects of different doses of Rab001 combined with PD-1 antibody on M1 macrophages, M1 / M2, and CD8+ in mouse breast cancer tumor tissue. + The effect of T and B cell ratio

[0116]

[0117] In summary, Rab001 exhibits high affinity and specificity for integrin α4β1. Since α4β1 integrin is also expressed in lymphocytes, including B lymphocytes, completed in vivo animal and clinical trials have observed a transient increase in the number of circulating lymphocytes, particularly B lymphocytes, after Rab001 administration. This increase in peripheral blood lymphocyte count may, in turn, increase the number of lymphocytes, including B lymphocytes, within tumor tissues through blood circulation. Utilizing this effect of Rab001, it can be combined with PD-1 monoclonal antibodies to enhance the efficacy of PD-1 monoclonal antibody therapy for breast cancer through synergistic effects, expanding the application of Rab001 in the field of tumor immuno-checkpoint therapy.

[0118] The embodiments of this application have been described above with reference to the accompanying drawings. However, this application is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of this application without departing from the spirit and scope of the claims, and all of these forms are within the protection scope of this application.

Claims

1. Application of Rab001 in combination with PD-1 monoclonal antibody in the preparation of anti-breast cancer drugs.

2. The application according to claim 1, characterized in that, The aforementioned anti-breast cancer tumor drugs include anti-breast cancer tumor inhibitors.

3. The application according to claim 1, characterized in that, The aforementioned anti-breast cancer tumor drugs or anti-breast cancer tumor inhibitors can increase the content of M1 macrophages and tumor-killing macrophages in tumor tissue.

4. The application according to claim 1, characterized in that, The aforementioned anti-breast cancer tumor drugs or anti-breast cancer tumor inhibitors can reduce the size of breast cancer tumors.

5. The application according to claim 1, characterized in that, The anti-breast cancer tumor drug or anti-breast cancer tumor inhibitor includes an effective dose of Rab001 and an effective dose of PD-1 monoclonal antibody, with the ratio of the effective dose of Rab001 to the effective dose of PD-1 monoclonal antibody being (1-9):

10.

6. The application according to claim 1, characterized in that, The anti-breast cancer tumor drugs or anti-breast cancer tumor inhibitors also include pharmaceutically acceptable carriers or excipients.

7. The application according to claim 1, characterized in that, The administration methods for the anti-breast cancer tumor drugs or anti-breast cancer tumor inhibitors are subcutaneous, local perfusion of tumor tissue, oral, intraperitoneal or intravenous administration.

8. A pharmaceutical composition, characterized in that, This includes Rab001 or its pharmaceutically acceptable salts or solvates; PD-1 or its pharmaceutically acceptable salts or solvates.

9. The pharmaceutical composition according to claim 8, characterized in that, In the pharmaceutical composition, the weight ratio of Rab001 to PD-1 is (1-9):10.

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