A pharmaceutical composition for treating breast cancer and use thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a pharmaceutical composition for treating breast cancer and application thereof. The pharmaceutical composition provided by the application comprises a B7-H4 antibody drug conjugate and a HER2 targeted preparation; the B7-H4 antibody drug conjugate is covalently coupled by an anti-B7-H4 monoclonal antibody, a protease-cleavable linker and a small molecule toxin topoisomerase I inhibitor; and the HER2 targeted preparation is selected from any one or more of trastuzumab, lapatinib, pertuzumab, trastuzumab emtansine, trastuzumab deruxtecan and neratinib. The application utilizes the combination of the B7-H4 antibody drug conjugate and the HER2 targeted treatment preparation. This strategy can not only enhance the treatment effect, but also overcome the immune escape phenomenon by simultaneously targeting HER2 and the immune checkpoint B7-H4 related to HER2 drug resistance.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a pharmaceutical composition for treating breast cancer and its application. Background Technology

[0002] HER2-positive breast cancer is a malignant tumor caused by the overexpression of human epidermal growth factor receptor 2 (HER2), accounting for approximately 15-20% of breast cancer cases. Cancers with HER2 overexpression, such as breast cancer, gastric cancer, and colon cancer, are typically treated with HER2-targeted therapies, including small molecule inhibitors, monoclonal antibodies, and antibody-drug conjugates (ADCs). These therapies have achieved significant results, but drug resistance remains a major obstacle in the treatment process. Drug resistance may stem from HER2 receptor mutations or alterations, activation of compensatory pathways, and the heterogeneity of molecular subtypes of HER2-positive tumors.

[0003] In the field of immunotherapy, although immune checkpoint inhibitors (ICBs) based on PD-1 / PD-L1 inhibition have shown clinical benefits in various HER2-negative cancers, the objective response rate of these drugs in HER2-positive cancer patients is significantly lower than that in HER2-negative patients, making monotherapy with immunotherapy insufficient to meet clinical needs. Therefore, attempts have been made to combine ICBs with anti-HER2 targeted agents, but these combination products only target a single PD-1 / PD-L1 immune checkpoint, resulting in limited improvement in objective response rates. Summary of the Invention

[0004] To address the limitation in existing technologies where ICB combined with anti-HER2 targeted agents only targets a single immune checkpoint, PD-1 / PD-L1, resulting in limited improvement in objective response rates, this invention provides a pharmaceutical composition for treating breast cancer and its application. To achieve the above objective, this invention employs the following technical solution.

[0005] The present invention provides a pharmaceutical composition for treating breast cancer, comprising a B7-H4 antibody-drug conjugate (B7-H4 ADC) and a HER2-targeting agent, wherein the molar ratio of the B7-H4 antibody-drug conjugate to the HER2-targeting agent is 1:0.2 to 1:5.

[0006] The B7-H4 antibody-drug conjugate is covalently coupled with an anti-B7-H4 monoclonal antibody, a protease-cleavable linker, and a small molecule toxin topoisomerase I inhibitor; wherein the molar ratio of the anti-B7-H4 monoclonal antibody, the protease-cleavable linker, and the small molecule toxin topoisomerase I inhibitor is 1:5~7:5~7.

[0007] The HER2-targeting agent is selected from any one or more of trastuzumab, lapatinib, pertuzumab, trastuzumab emtansine (T-DM1), trastuzumab dextromethorphan (T-DXd / DS-8201), and neratinib.

[0008] The pharmaceutical composition is used to overcome resistance to HER2-targeted therapy and enhance therapeutic efficacy by reshaping the tumor immune microenvironment.

[0009] Existing combination products of ICBs and anti-HER2 targeted agents only target a single immune checkpoint, PD-1 / PD-L1, and do not contain a B7-H4 targeting component, resulting in limited improvement in objective response rates. This invention, by revealing the activation mechanism of the ATF3 / B7-H4 axis after HER2-targeted therapy, proposes a pharmaceutical composition with a different component design than existing combination products. This composition comprises a B7-H4 antibody-drug conjugate and a HER2-targeting agent, used in a synergistic and effective ratio. This composition uses a B7-H4 ADC to target and eliminate B7-H4-positive cells while simultaneously blocking the HER2 signaling pathway with the HER2-targeting agent, achieving synergistic intervention on both the HER2 pathway and the B7-H4-mediated immune escape pathway. Since B7-H4 expression is upregulated after HER2-targeted therapy and is closely related to drug resistance, this invention's composition, by simultaneously targeting HER2 and B7-H4, can effectively overcome the drug resistance-related pathways that existing combination products cannot intervene in due to the absence of a B7-H4 targeting component, significantly improving therapeutic efficacy. The innovation of this technical solution lies in using B7-H4 as a target. By combining anti-B7-H4 ADC with HER2-targeting drugs, it overcomes the drug resistance problem of existing HER2-targeted therapies and further enhances the immunotherapy effect by reshaping the tumor immune microenvironment. It has shown superior efficacy in multiple preclinical models.

[0010] Preferably, the anti-B7-H4 monoclonal antibody is a humanized monoclonal antibody or a fully human monoclonal antibody.

[0011] Preferably, the anti-B7-H4 monoclonal antibody (B7-H4 ADC) comprises a heavy chain variable region and a light chain variable region.

[0012] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.2.

[0013] Preferably, the chemical structure of is shown in formula (I): Formula (I).

[0014] The protease-cleavable linker has a maleimide-valine-citrulline-p-aminobenzyloxycarbonyl (MC-Val-Cit-PABC) backbone structure, wherein valine-citrulline (Val-Cit) is a dipeptide cleavage unit; the maleimide terminus of the linker is covalently linked to the anti-B7-H4 monoclonal antibody, and the p-aminobenzyloxycarbonyl terminus of the linker is covalently linked to the topoisomerase I inhibitor.

[0015] Specifically, the protease-cleavable linker is a dedicated cleavable linker for antibody-drug conjugates, with a core comprising a valine-citrulline (Val-Cit) dipeptide cleavage unit. The linker adopts the classic MC-Val-Cit-PABC backbone structure, wherein one end of the valine-citrulline dipeptide is covalently linked to the B7-H4 targeting antibody via a maleimide group (MC) as the antibody linker, and the other end is covalently linked to the cytotoxic drug molecule via a p-aminobenzyloxycarbonyl (PABC) group as the drug linker.

[0016] Preferably, the small molecule toxin topoisomerase I inhibitor is SHR-9265, whose chemical structure is shown in formula (II): Formula (II).

[0017] The present invention also provides the use of the pharmaceutical composition in the preparation of medicaments for the prevention and / or treatment of breast cancer.

[0018] Preferably, the breast cancer is HER2-positive breast cancer.

[0019] Preferably, the drug further comprises pharmaceutically acceptable excipients.

[0020] Preferably, the pharmaceutically acceptable excipients include any one or more of buffers, lyophilization protectants, and surfactants.

[0021] The buffer is selected from one or more of histidine, phosphate and acetate; the freeze-drying protectant is selected from one or more of sucrose, trehalose, glucose, mannitol and lactose; and the surfactant is selected from one or more of polysorbate 80 (Tween 80), polysorbate 20 (Tween 20) and poloxamer 188.

[0022] Preferably, the drug treatment targets HER2 and B7-H4 simultaneously to synergistically overcome immune escape and reverse drug resistance.

[0023] Compared with the prior art, the present invention has the following beneficial effects: 1. This invention provides a pharmaceutical composition for treating breast cancer, comprising a B7-H4 antibody-drug conjugate (B7-H4 ADC) and a HER2-targeting agent, used in a synergistic and effective combination. The B7-H4 ADC is formed by covalently coupling an anti-B7-H4 monoclonal antibody to a topoisomerase I inhibitor via a protease-cleavable linker; the HER2-targeting agent is selected from any one or more of trastuzumab, pertuzumab, trastuzumab emtansine, trastuzumab dexamethasone, lapatinib, or neratinib. Existing technologies combining ICBs with anti-HER2-targeting agents only target a single PD-1 / PD-L1 immune checkpoint, and their product components do not contain a B7-H4 targeting component, resulting in limited improvement in objective response rates. Based on in-depth research into the immune escape mechanism in the treatment of HER2-positive breast cancer, this invention proposes an innovative treatment strategy: a novel breast cancer treatment strategy based on the combination of B7-H4 antibody-drug conjugate (B7-H4 ADC) and HER2-targeting therapy. This strategy combines the synergistic effect of HER2-targeted therapy with B7-H4 immune checkpoint-targeting drugs, aiming to overcome the resistance problem of HER2-targeted therapy and improve the treatment effect of HER2-positive breast cancer by improving the immune microenvironment and enhancing the immune response.

[0024] 2. The novel breast cancer treatment strategy proposed in this invention, based on B7-H4 antibody-drug conjugate (B7-H4 ADC) combined with HER2-targeted therapy, demonstrates significant technical effects in several aspects, including: (1) Overcoming resistance to HER2-targeted therapy: The combined treatment regimen of this invention effectively overcomes drug resistance in HER2-targeted therapy by targeting the B7-H4 immune checkpoint and working synergistically with the HER2 receptor. Studies have shown that B7-H4 expression is significantly upregulated in HER2-positive breast cancer after HER2-targeted therapy, and this upregulation is closely related to immune escape and resistance to anti-HER2 drugs. By combining a B7-H4-specific ADC (B7-H4 ADC) with HER2-targeted therapy, this immune escape pathway can be blocked, significantly improving the efficacy of HER2-targeted therapy. Experimental data indicate that high B7-H4 expression is associated with tumor immunosuppression, and inhibiting B7-H4 can restore the tumor immune response, enhance anti-tumor immune effects, thereby overcoming drug resistance and achieving a more durable therapeutic effect.

[0025] (2) Enhance the efficacy of immunotherapy: This invention elucidates the role of the ATF3 / B7-H4 axis in HER2-targeted therapy and proposes a combined approach using an antibody-drug conjugate targeting B7-H4 (B7-H4 ADC) with HER2-targeted therapy. B7-H4 inhibits the infiltration of CD8+ T cells, thereby suppressing the immune system's recognition and clearance of tumor cells. By combining B7-H4 targeting, T cell activity can be effectively restored, anti-tumor immune responses enhanced, and treatment efficacy improved.

[0026] Preclinical studies (such as cancer cell lines, organoids, and patient-derived xenograft models) have validated the effectiveness of this combination therapy in HER2-positive breast cancer, showing that the combination therapy group has significant anti-tumor effects, especially in patients with anti-HER2 resistant breast cancer.

[0027] (3) Clinical potential of novel combination therapy strategies: The technical solution of this invention has received strong support in preclinical models, indicating that the combination therapy can effectively control the tumor growth of HER2-positive breast cancer, especially in breast cancer patients resistant to HER2-targeted therapy, showing a stronger anti-tumor effect.

[0028] This technical approach has also been preliminarily validated in clinical trials. The anti-B7-H4 ADC has shown remarkable efficacy in HER2-positive breast cancer patients, especially in advanced breast cancer patients resistant to anti-HER2 therapy. The combination therapy can effectively control disease progression and shows broad clinical application prospects.

[0029] (4) Economic benefits: The technical solution of this invention has significant economic benefits, specifically reflected in the following aspects: 1) Improve the treatment efficacy of HER2-positive breast cancer and reduce the treatment cycle for patients: The combined use of B7-H4 antibody-drug conjugates (B7-H4 ADCs) and HER2-targeted drugs can significantly improve the treatment outcomes for HER2-positive breast cancer patients, reduce drug resistance, and prolong progression-free survival (PFS). This has positive implications for reducing repeated treatments and lowering medical costs, significantly improving the cost-effectiveness of treatment.

[0030] Clinically, patients resistant to HER2-targeted therapy typically require second or third-line treatment, which is costly. By employing the combination therapy strategy of this invention, not only can the effective treatment time be extended, but the occurrence of ineffective treatment can also be reduced, thereby lowering the overall treatment cost.

[0031] 2) Promote the commercialization of novel immunotherapies: The B7-H4 antibody-drug conjugate (B7-H4 ADC) used in this invention, as a novel immunotherapy drug, has demonstrated high clinical efficacy in the treatment of HER2-positive breast cancer. The realization of this technology can not only accelerate the development of new drugs but also promote the commercialization of ADC-type immunotherapy drugs, generating substantial market revenue for pharmaceutical companies.

[0032] Based on existing clinical trial data, B7-H4 ADC has shown some efficacy in other types of cancer (such as triple-negative breast cancer and ovarian cancer), which provides the possibility for further expanding its indications and increasing its market share. Attached Figure Description

[0033] Figure 1 This is a schematic diagram illustrating the mechanism of action of B7-H4 ADC combined with HER2-targeting agents in the present invention.

[0034] Figure 2 The figures show a Western blotting image and a statistical graph of B7-H4 protein expression levels in HER2-positive breast cancer cells after treatment with the HER2-targeting drug (trastuzumab). A is a Western Blot diagram showing the expression level of B7-H4 protein in HER2-positive breast cancer cells after treatment with HER2-targeting drugs. B is a statistical graph showing the mRNA expression level of HER2-positive breast cancer cells after treatment with HER2-targeted drugs.

[0035] Figure 3 The images show Western blotting and ChIP-qPCR validation results of ATF3-regulated B7-H4 expression in HER2-positive breast cancer cells in this invention; wherein: A is a Western blotting image of ATF3 regulating B7-H4 expression in HER2-positive breast cancer cells. B represents the ChIP-qPCR validation result of ATF3 regulating B7-H4 expression in HER2-positive breast cancer cells.

[0036] Figure 4 The results of CCK-8 assays and colony formation experiments on HER2-positive breast cancer cells treated with single or combined drugs in this invention are shown below; wherein: A represents HER2-positive breast cancer cells, specifically CCK-8 cells, treated with different concentrations of B7-H4 ADC in combination with a HER2-targeting agent. B represents the clonogenic assay results of HER2-positive breast cancer cells after treatment with single or combined drugs. C is a statistical graph showing the results of clone formation experiments on HER2-positive breast cancer cells after treatment with single or combined drugs.

[0037] Figure 5 The images shown are light micrographs and statistical graphs of organoid models derived from HER2-positive patients in this invention after treatment with single or combined drugs; wherein: A shows light micrographs of organoid models derived from HER2-positive patients after treatment with single or combined drugs. B is a statistical graph showing the maximum diameter of organoid models derived from HER2-positive patients after treatment with single or combined drugs.

[0038] Figure 6 These are the experimental results of xenograft models derived from HER2-positive patients in this invention, after treatment with single or combined drugs; wherein: A shows tumor images of xenograft models from HER2-positive patients after treatment with single or combination drugs. B is a statistical chart of tumor weight in Figure A; C represents the change in tumor volume over time as shown in Figure A. Detailed Implementation

[0039] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0040] The mechanism of action of B7-H4 ADC combined with HER2-targeting agents in this invention is as follows: Figure 1 As shown.

[0041] Example 1: Validation of the regulatory effect of HER2-targeting agents on B7-H4 expression at the cellular level.

[0042] HER2-positive breast cancer cells HCC1954 (Wuhan Pronosai, catalog number: CL-0756) were treated with HER2-targeting agents (trastuzumab, MedChemExpress, catalog number: HY-P9907) at concentrations of 0 μg / mL, 10 μg / mL, and 20 μg / mL, with the 0 μg / mL trastuzumab treatment group serving as the control group. After 24 h of drug treatment, the culture medium was discarded, cells were washed with pre-cooled PBS, and total protein was extracted from each group. Protein concentration was quantified using the BCA method, and the loading volume was standardized. After routine SDS-PAGE electrophoresis, membrane transfer, blocking with 5% (w / v) skim milk at room temperature, overnight incubation with primary antibody at 4°C, and incubation with secondary antibody at room temperature, ECL chemiluminescence solutions A and B were mixed at a 1:1 (v / v) ratio and evenly covered the membrane surface. Chemiluminescence images were acquired using a chemiluminescence imaging system. Parallel cell samples were collected simultaneously, and total RNA was extracted from the cells using the RNAfast200 Total RNA Rapid Extraction Kit (Shanghai Feijie, catalog number: 220010). After the RNA purity was verified by Nanodrop, cDNA was synthesized using a reverse transcription kit (Aikerui, catalog number: AG11728). Using the cDNA as a template, qPCR was performed using TSINGKE® Master qPCR Mix (SYBR Green I, catalog number: TSE201) to amplify the RNA. B7-H4 Genes and internal reference β-Actin gene; the amplification program included pre-denaturation, 40 rounds of amplification, and melting curve analysis, using 2 -ΔΔCt Method calculation B7-H4 relative mRNA expression level. "Overnight" refers to a time ≥12 hours.

[0043] like Figure 2 Western blot and qPCR results showed that, compared with the control group, the protein and mRNA expression levels of B7-H4 in HCC1954 cells treated with 10 μg / mL and 20 μg / mL trastuzumab were significantly upregulated in a dose-dependent manner. This demonstrates that the HER2-targeting agent has a regulatory effect on B7-H4 expression.

[0044] Example 2: Validation of the role of the ATF3 / B7-H4 axis in HER2-targeted therapy at the cellular level.

[0045] HEK293T cells were co-transfected with the pLKO.1-puro backbone shATF3 plasmid (ordered from Biopharmaceuticals) and the negative control shNC plasmid, along with packaging plasmids psPAX2 and pMD2.G, to package lentivirus. Viral supernatant was collected, filtered, and used to infect HCC1954 cells. Stable cell lines were obtained by continuous selection with puromycin at a final concentration of 1 μg / mL for 14 days. The ATF3 knockdown efficiency was verified by qPCR and Western blotting, successfully constructing the HCC1954-shATF3 stable knockdown cell line and the control HCC1954-shNC cell line. The HCC1954 control and knockdown cell lines were treated with PBS and trastuzumab at 10 μg / mL, respectively. Cell pellets were collected after 24 hours of continuous treatment in each group. Total protein was extracted from each group of cells using the same steps as in Example 1. Protein quantification was performed using BCA, followed by SDS-PAGE electrophoresis, transfer to a membrane, blocking, and incubation with primary and secondary antibodies before Western blotting detection. Figure 3 Western blot results in cell line A showed that in the HCC1954 control group, trastuzumab treatment increased the expression level of B7-H4 protein, but ATF3 protein level did not change significantly. However, in the ATF3 knockdown cell line, the expression level of B7-H4 was significantly reduced and no longer affected by trastuzumab.

[0046] HCC1954 wild-type cells from the control group and the trastuzumab-treated group were used for ChIP-qPCR verification. Cells were cross-linked with 1% (v / v) formaldehyde at room temperature for 10 min, and the cross-linking was terminated with glycine. Cells were lysed to extract chromatin, which was then sonicated to fragments of 200bp-1000bp. DNA was enriched by incubation overnight at 4°C using an ATF3-specific antibody (Abcam, catalog number ab254268) and an IgG negative antibody. The protein-DNA complex was purified by protein G magnetic bead capture, washing, decross-linking, and proteinase K digestion. Using the enriched DNA as a template, targeted... B7-H4 Gene promoter region-specific primers were used for qPCR amplification, and the ChIP-qPCR results are as follows: Figure 3 As shown in B, trastuzumab treatment significantly increased the enrichment level of ATF3 protein in the promoter region of the B7-H4 gene, suggesting that under trastuzumab stimulation, ATF3 regulates the transcription of the B7-H4 gene by directly binding to the B7-H4 promoter.

[0047] Overnight stays refer to stays of 12 hours or more.

[0048] Targeted B7-H4 Information on gene promoter region-specific primers is as follows: upstream primer of the target gene: 5'-GGAGGAAAGTCCTGCTGAATG-3' (SEQ ID NO.3).

[0049] Downstream primer for the target gene: 5'-AGTCTTGACTTTGGGAGGGTG-3' (SEQ ID NO.4).

[0050] The reaction system for qPCR amplification is as follows: Real-time quantitative PCR was performed using TSINGKE® Master qPCR Mix (SYBR Green I, catalog number: TSE201) from TSINGKE Biotechnology. The composition of the 20 μL reaction system was as follows: cDNA template: 8 μL.

[0051] upstream primer of the target gene (10 μmol / L): 1 μL.

[0052] Downstream primer for the target gene (10 μmol / L): 1 μL.

[0053] 2×TSINGKE® Master qPCR Mix (SYBR Green I): 10μL.

[0054] Total volume: 20 μL.

[0055] Three technical replicates were set up for each sample group.

[0056] The reaction procedure for qPCR amplification is as follows: Pre-denaturation stage: 95℃, 3 min.

[0057] Amplification cycle (40 cycles in total): 95℃, 10s (denaturation); 60℃, 30s (annealing and extension, with simultaneous acquisition of fluorescence signals).

[0058] Melting curve analysis: 95℃, 15s; 60℃, 60s; continuously heated to 95℃ at a rate of 0.3℃ / s, and fluorescence was continuously collected to determine the specificity of the amplified product.

[0059] Example 3: In vitro verification of the synergistic therapeutic effect of combined medication.

[0060] Using HER2-positive breast cancer cells HCC1954 (HCC1954 wild-type cells) as a model, trastuzumab (0-80 μg / mL) and B7-H4 ADC (0-300 μg / mL) were administered in combination. After 72 hours of continuous incubation, CCK-8 solution was added, and after 2 hours of incubation, the absorbance at 450 nm was measured using a microplate reader. The cell proliferation inhibition rate was calculated, and plotted as shown below. Figure 4The combined drug therapy thermogram is shown in Figure A. Subsequently, a plate colony formation experiment was conducted, with control, trastuzumab, B7-H4 ADC, and combined drug therapy groups. Cells in each group were treated sequentially with PBS, trastuzumab, B7-H4 ADC, and a combination of two drugs. After 24 hours of treatment with the corresponding drugs, the cells were digested and resuspended, seeded at the same cell density in culture dishes, and cultured for 14 days, maintaining drug intervention throughout. After culture, cells were fixed with 4% (w / v) paraformaldehyde and stained with crystal violet. Figure 4 B in the figure represents the representative result, and the number of clones is counted as follows: Figure 4 As shown in C, the cell proliferation inhibition rate of the combined group was significantly higher than that of the single-drug group.

[0061] Among them, B7-H4 ADC, or B7-H4 antibody-drug conjugate, is covalently coupled with an anti-B7-H4 monoclonal antibody, a protease-cleavable linker, and a small molecule toxin topoisomerase I inhibitor; wherein the molar ratio of the anti-B7-H4 monoclonal antibody, the protease-cleavable linker, and the small molecule toxin topoisomerase I inhibitor is 1:6:6.

[0062] The anti-B7-H4 monoclonal antibody contains a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1 (see the hu2F7 heavy chain variable region corresponding to SEQ ID NO:17 in Chinese patent application with publication number CN112351797B): EVQLVESGGGLVQPGGSLRLSCAASGFTFSNYYMSWVRQAPGKGLEWVAYVSSGGGSTYYSDSVKGRFTISRDNAKNTLYLQMSSLRAEDTAVYYCTRESYSQGNYFDYWGQGTTVTSS.

[0063] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.2 (see the hu2F7 light chain variable region corresponding to SEQ ID NO:17 in Chinese patent application with publication number CN112351797B): EIVMTQSPATLSLSPGERATLSCRASQSISDYLHWYQQKPGQSPRLLIKFASQSISGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQNGHSFSLTFQGGTKLEIK.

[0064] The chemical structure of the protease-cleavable linker is shown in formula (I): Formula (I).

[0065] The small molecule toxin topoisomerase I inhibitor is SHR-9265, whose chemical structure is shown in formula (II): Formula (II).

[0066] Trastuzumab and B7-H4 ADC combination therapy is a drug composition for the treatment of breast cancer obtained by combining trastuzumab and B7-H4 ADC (B7-H4 antibody-drug conjugate).

[0067] Using organoids derived from HER2-positive breast cancer patients as a model, we set up a control group, a trastuzumab group, a B7-H4 ADC group, and a combination drug group. Each group was given PBS, 20 μg / mL of trastuzumab, 20 μg / mL of B7-H4 ADC, and the combination of the two drugs for continuous intervention and culture, respectively. The growth of organoids was observed and statistically analyzed regularly. Figure 5 The experimental results still showed that the inhibitory effect of the combination group was significantly higher than that of the single-drug group. This indicates that trastuzumab and B7H4 ADC have a synergistic anti-tumor proliferation effect.

[0068] Example 4: Animal model to verify the in vivo efficacy of combined drug therapy.

[0069] A patient-derived xenograft model was constructed using BALB / c nude mice. The experimental animals were purchased from the Experimental Animal Center of Xi'an Jiaotong University. Fresh tumor tissue from HER2-positive breast cancer patients was collected, transported in pre-cooled sterile culture medium, and necrotic and connective tissues were removed. The tumor tissue was minced, resuspended, and subcutaneously inoculated into the backs of BALB / c nude mice to construct the patient-derived xenograft model. After the primary xenograft tumors stabilized, they were passaged and expanded to obtain tumor-bearing mice for experimentation. The xenograft model was established when the tumor volume reached 100 mm². 3 ~120mm 3 Mice were randomly divided into a control group, a trastuzumab group, a B7-H4 ADC group, and a combination therapy group. They were given saline, trastuzumab, B7-H4 ADC, and B7-H4 ADC trastuzumab, respectively. The drugs in each group were injected into mice via the tail vein at a solution concentration of 0.5 mg / kg.

[0070] like Figure 6 As shown, after the dosing cycle, the tumor volume and weight in the combination therapy group were significantly lower than those in the single therapy groups (trastuzumab group and B7-H4 ADC group).

[0071] In summary, this invention overcomes the drug resistance problem in existing treatments for HER2-positive breast cancer by combining B7-H4 antibody-drug conjugates (B7-H4 ADCs) with HER2-targeted therapy, and improves the immune microenvironment, thus significantly enhancing treatment efficacy. This technology not only provides clinicians with a new treatment option but also brings considerable economic benefits to pharmaceutical companies. Although some side effects and individual variability issues may exist, the advantages and market potential of this invention make it a significant innovation in the field of breast cancer treatment.

[0072] It should be noted that when numerical ranges are involved in this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, this invention describes preferred embodiments.

[0073] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments, all of which fall within the scope of the invention.

Claims

1. A pharmaceutical composition for treating breast cancer, characterized in that, It comprises a B7-H4 antibody-drug conjugate and a HER2-targeting formulation, wherein the molar ratio of the B7-H4 antibody-drug conjugate to the HER2-targeting formulation is 1:0.2~5; The B7-H4 antibody-drug conjugate is covalently coupled with an anti-B7-H4 monoclonal antibody, a protease-cleavable linker, and a small molecule toxin topoisomerase I inhibitor; wherein the molar ratio of the anti-B7-H4 monoclonal antibody, the protease-cleavable linker, and the small molecule toxin topoisomerase I inhibitor is 1:5~7:5~7. The HER2-targeting agent is selected from any one or more of trastuzumab, lapatinib, pertuzumab, trastuzumab emtansine, detrastuzumab, and neratinib.

2. The pharmaceutical composition according to claim 1, characterized in that, The anti-B7-H4 monoclonal antibody is a humanized monoclonal antibody or a fully human monoclonal antibody.

3. The pharmaceutical composition according to claim 2, characterized in that, The anti-B7-H4 monoclonal antibody contains a heavy chain variable region and a light chain variable region; The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.1; the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

2.

4. The pharmaceutical composition according to claim 1, characterized in that, The chemical structure of the protease-cleavable linker is shown in formula (I): Equation (I).

5. The pharmaceutical composition according to claim 1, characterized in that, The small molecule toxin topoisomerase I inhibitor is SHR-9265, and its chemical structure is shown in formula (II): Formula (II).

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for the prevention and / or treatment of breast cancer.

7. The application according to claim 6, characterized in that, The breast cancer mentioned is HER2-positive breast cancer.

8. The application according to claim 7, characterized in that, The drug also contains pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The pharmaceutically acceptable excipients include any one or more of buffers, lyophilization protectants, and surfactants.

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