Application of combination of B7-H3 inhibitor and HSPA8 inhibitor in preparation of medicine for treating breast cancer

Abstract

The invention discloses application of combination of a B7-H3 inhibitor and an HSPA8 inhibitor in preparation of a medicine for treating breast cancer, and belongs to the technical field of biological medicines. The invention proves that the antitumor effect of the B7-H3 targeted therapy can be enhanced by targeted inhibition of HSPA8 for the first time. Growth of breast cancer cells can be remarkably inhibited by inhibiting expression of HSPA8 and B7-H3 at the same time, compared with independent inhibition and combined inhibition of HSPA8 and B7-H3, an obvious synergistic effect is shown, an anti-tumor immune microenvironment can be activated, CD8 + T cell infiltration / activation can be enhanced, organism immunity can be enhanced, and improvement of prognosis of breast cancer patients is facilitated. The invention provides a'B7-H3 blocking + HSPA8 inhibition 'combined medication strategy, and provides a new treatment strategy with transformation prospect for treating refractory breast cancer, especially triple negative breast cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of B7-H3 inhibitors in combination with HSPA8 inhibitors in the preparation of drugs for treating breast cancer. Background Technology

[0002] Among the various subtypes of breast cancer, triple-negative breast cancer (TNBC) accounts for approximately 15%-20% of all breast cancer cases. It is characterized by its high invasiveness and recurrence rate, and lacks expression of estrogen / progesterone receptors and HER2 receptors. Therefore, TNBC patients exhibit a significantly worse prognosis. Due to its insensitivity to traditional treatments and the lack of clear biomarkers, treatment options for TNBC are extremely limited. An effective treatment strategy is urgently needed to overcome the treatment challenges of TNBC, especially addressing its core bottlenecks of high invasiveness and insensitivity to existing treatments.

[0003] Immune checkpoint B7-H3, an emerging immunomodulatory molecule, is abnormally overexpressed in various solid tumors but minimally expressed in normal tissues. This unique and selective expression pattern promotes the formation of an immune escape phenotype, thereby promoting cancer cell proliferation, invasion, and drug resistance. Notably, T lymphocytes, a core component of anti-tumor immunity, are significantly suppressed in the presence of high B7-H3 expression. Given the widespread overexpression of B7-H3 in tumors and its association with the immunosuppressive microenvironment, various B7-H3-targeted therapies, including monoclonal antibodies (mAbs), antibody-drug conjugates, and CAR-T cell therapy, are undergoing clinical evaluation and have shown encouraging preliminary efficacy. However, the specific role and molecular mechanisms of B7-H3 in the progression of TNBC remain not fully elucidated.

[0004] The advent of immunotherapy has ushered in a new era in cancer treatment and is also a highly promising treatment option for breast cancer. In recent years, with the expanding application of immune checkpoint inhibitors (ICIs), some patients have developed primary or acquired resistance, making it difficult for monotherapy to restore functional immune circulation. These patients typically benefit little from ICI monotherapy. Therefore, combining ICIs with other therapies to enhance anti-tumor effects has become a hot research topic. Summary of the Invention

[0005] The purpose of this invention is to provide the application of B7-H3 inhibitors combined with HSPA8 inhibitors in the preparation of drugs for treating breast cancer, thereby addressing the problems existing in the prior art. This invention addresses the lack of effective targeted drugs for breast cancer, especially triple-negative breast cancer, as well as the low response rate to immunotherapy and the prevalence of primary / acquired drug resistance. It proposes a novel combined intervention strategy that simultaneously inhibits B7-H3 and HSPA8, synergistically disrupting tumor cell protein homeostasis and survival networks, activating the anti-tumor immune microenvironment, and thus enhancing anti-tumor efficacy.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides the use of a B7-H3 inhibitor in combination with an HSPA8 inhibitor in the preparation of a pharmaceutical composition for treating breast cancer.

[0007] Optionally, the B7-H3 inhibitor includes, but is not limited to, nucleic acid intervention molecules targeting B7-H3, B7-H3 antibodies, B7-H3 antibody-drug conjugates, and CAR-T-related components.

[0008] Preferably, the B7-H3 inhibitor is a nucleic acid intervention molecule targeting B7-H3 or a B7-H3 antibody.

[0009] More preferably, the nucleic acid intervention molecules targeting B7-H3 include siRNA, shRNA, and ASO.

[0010] Further preferably, the sequence of the siRNA is shown in SEQ ID NO.2.

[0011] Optionally, the HSPA8 inhibitor includes, but is not limited to, nucleic acid intervention molecules that target HSPA8, compounds that inhibit HSPA8, HSPA8 antibodies, HSPA8 antibody-drug conjugates, and CAR-T-related components.

[0012] Preferably, the HSPA8 inhibitor is a nucleic acid intervention molecule that targets HSPA8 or a compound that inhibits HSPA8.

[0013] More preferably, the nucleic acid intervention molecules targeting HSPA8 include siRNA, shRNA, and ASO.

[0014] Further preferably, the sequence of the siRNA is shown in SEQ ID NO.3.

[0015] More preferably, the compound that inhibits HSPA8 is VER155008.

[0016] Optionally, the breast cancer includes triple-negative breast cancer.

[0017] The present invention also provides a pharmaceutical composition for treating breast cancer, the pharmaceutical composition comprising a B7-H3 inhibitor and an HSPA8 inhibitor.

[0018] Optionally, the B7-H3 inhibitor includes, but is not limited to, nucleic acid intervention molecules targeting B7-H3, B7-H3 antibodies, B7-H3 antibody-drug conjugates, and CAR-T-related components.

[0019] Preferably, the B7-H3 inhibitor is a nucleic acid intervention molecule targeting B7-H3 or a B7-H3 antibody.

[0020] More preferably, the nucleic acid intervention molecules targeting B7-H3 include siRNA, shRNA, and ASO.

[0021] Further preferably, the sequence of the siRNA is shown in SEQ ID NO.2.

[0022] Optionally, the HSPA8 inhibitor includes, but is not limited to, nucleic acid intervention molecules targeting HSPA8, HSPA8 antibodies, HSPA8 antibody-drug conjugates, and CAR-T-related components.

[0023] Preferably, the HSPA8 inhibitor is a nucleic acid intervention molecule that targets HSPA8 or a compound that inhibits HSPA8.

[0024] More preferably, the nucleic acid intervention molecules targeting HSPA8 include siRNA, shRNA, and ASO.

[0025] Further preferably, the sequence of the siRNA is shown in SEQ ID NO.3.

[0026] More preferably, the compound that inhibits HSPA8 is VER155008.

[0027] The present invention also provides the use of the above-described pharmaceutical composition in the preparation of a medicament for treating breast cancer.

[0028] The present invention also provides a medicament for treating breast cancer, the medicament comprising the above-described pharmaceutical composition.

[0029] Optionally, the drug may also contain a pharmaceutically acceptable carrier or excipient.

[0030] The present invention discloses the following technical effects: This invention is the first to demonstrate that targeted inhibition of HSPA8 can enhance the anti-tumor effect of B7-H3 targeted therapy. In vitro and in vitro experiments confirmed that simultaneous inhibition of HSPA8 and B7-H3 expression significantly inhibited the growth of breast cancer cells. Compared to the individual inhibition of HSPA8 and B7-H3, the combined inhibition showed a significant synergistic effect and could activate the anti-tumor immune microenvironment and enhance CD8 expression. + T-cell infiltration / activation enhances the body's immunity and helps improve the prognosis of breast cancer patients.

[0031] This invention proposes a combined treatment strategy of "B7-H3 blockade + HSPA8 inhibition" to address the current challenges in breast cancer treatment. This strategy offers a promising new approach for overcoming refractory breast cancer, especially triple-negative breast cancer. HSPA8 inhibition therapy can form a "platform-based" combination with existing anti-B7-H3 therapies (mAb / ADC / CAR-T, etc.), lowering the development threshold and facilitating translational research. Furthermore, it allows for the prediction and analysis of patient treatment outcomes based on B7-H3 / HSPA8 expression levels, improving the accuracy of clinical applications. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 This is a schematic diagram illustrating the principle of the combined strategy of "B7-H3 blockade + HSPA8 inhibition"; Figure 2 Figure 1 shows the results of HSPA8 expression analysis in TNBC tissues; where A represents the HSPA8 expression level in normal breast tissue and TNBC tissues; B represents the Kaplan-Meier curve of TNBC patient prognosis and HSPA8 expression level; C represents the immunofluorescence staining image of TNBC tumors and adjacent tumor samples; and D represents the statistical results of immunohistochemical scores and the proportion of positive cells. Figure 3 The figure shows the results of the B7-H3 expression analysis in TNBC tissue; where A represents the B7-H3 expression level in normal breast tissue and TNBC tissue; and B represents the Kaplan-Meier curve of TNBC patient prognosis and B7-H3 expression level. Figure 4The image shows the results of B7-H3 expression analysis in TNBC cells and normal cells. A represents the immunofluorescence staining of TNBC tumor and adjacent tumor samples; B represents the statistical results of immunohistochemical scores and the proportion of positive cells; C represents the detection results of B7-H3 gene levels in TNBC cell lines and normal breast epithelial cell lines; D represents the detection results of B7-H3 protein levels in TNBC cell lines and normal breast epithelial cell lines; and E represents the statistical results of B7-H3 protein expression levels in TNBC cell lines and normal breast epithelial cell lines. Figure 5 Figure 1 shows the validation results of the combined knockdown of B7-H3 and HSPA8; where A represents the gene expression level detection results after combined knockdown of B7-H3 and HSPA8 in TNBC cells; B represents the Western blot detection results after combined knockdown of B7-H3 and HSPA8 in TNBC cells; and C represents the statistical results of protein expression level after combined knockdown of B7-H3 and HSPA8 in TNBC cells. Figure 6 Figure (A) and statistical results (B) of cell scratch assay to inhibit TNBC cell growth by combined knockdown of B7-H3 and HSPA8. Figure 7 Figure (A) and statistical results (B) of the transwell experiment for inhibiting TNBC cell growth by jointly knocking down B7-H3 and HSPA8; Figure 8 Figure (A) and statistical results (B) of the colony formation experiment to inhibit TNBC cell growth by jointly knocking down B7-H3 and HSPA8. Figure 9 Flow cytometry (A) and statistical results of apoptosis rate (B) for the combined knockdown of B7-H3 and HSPA8 to inhibit the growth of TNBC cells. Figure 10 The cell cycle assay results (A) and cell cycle statistics (B) show the combined effect of knocking down B7-H3 and HSPA8 to inhibit TNBC cell growth. Figure 11Figure 1 shows the in vivo experimental results of the combined anti-B7-H3 and anti-HSPA8 therapy inhibiting tumor growth. A is the flowchart of the in vivo experimental procedure; B is the tumor growth curve showing the change in tumor volume over time for each group; C is the curve showing the increase in mouse body weight over time for each group; D is the statistical result of tumor weight for each group at the end of the experiment; E is a representative image of the tumors for each group at the end of the experiment; F is the detection image of the expression levels of B7-H3, HSPA8, CD8, CD4, FOXP3, PCNA, KI-67, TUNEL, and Caspase9 in the tumor tissues of each group of mice; G is the statistical result of the expression levels of B7-H3, HSPA8, CD8, CD4, FoxP3, PCNA, Ki-67, TUNEL, and Caspase9 in the tumor tissues of each group of mice. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] The technical concept of this invention is as follows: Previous studies have found that B7-H3 can maintain the correct folding and functional stability of key oncogenic proteins in tumor cells by regulating the tumor protein homeostasis network, providing core support for their malignant proliferation, invasion, and immune escape, suggesting that it may be a key target for combination therapy of TNBC. Due to genomic instability and high metabolic rate, tumor cells are highly dependent on members of the Hsp70 family. HSPA8 is a constitutively expressed chaperone protein that not only participates in the folding of nascent proteins but is also a key executor of molecular chaperone-mediated autophagy (CMA). Previous studies have shown that HSPA8 can degrade key tumor suppressor factors or signaling molecules such as p53 and IKKβ through the CMA pathway, thereby promoting tumor survival.

[0040] Previous research by the inventors' team revealed that B7-H3 activates the p38MAPK signaling pathway, thereby upregulating the transcription factor SP1, which in turn drives HSPA8 expression to drive the malignant phenotype of TNBC (see...). Figure 1 These findings suggest that combined inhibition of B7-H3 and HSPA8 may be a promising strategy. To verify this hypothesis, this invention used a "B7-H3 blockade + HSPA8 inhibition" combination therapy in in vivo and in vitro experiments, observing a significant improvement in anti-tumor efficacy when both genes were simultaneously inhibited. This invention elucidates for the first time the molecular mechanism by which B7-H3 maintains TNBC cell protein homeostasis and invasiveness by activating the p38MAPK / SP1 signaling axis and upregulating HSPA8 transcriptionally. The "B7-H3 blockade + HSPA8 inhibition" combination strategy demonstrates therapeutic potential by synergistically inhibiting tumor cell survival pathways and activating the anti-tumor immune microenvironment.

[0041] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the biological materials or reagents involved in the following embodiments can be purchased through conventional channels; the experimental methods involved, unless otherwise specified, are conventional technical methods in the art.

[0042] Example 1 1. Experimental Methods 1.1 Bioinformatics Analysis B7-H3 expression in normal breast tissue and triple-negative breast cancer tissue was investigated using the UALCAN database (https: / / ualcan.path.uab.edu / ) in the The Cancer Genome Atlas (TCGA) database, and differential expression was analyzed using the Wilcoxon assay. The association between B7-H3, HSPA8, and patient survival was explored using the KM plotter database (https: / / kmplot.com / analysis / ), and log-rank test was used for analysis. Enrichment analysis was performed using "clusterProfiler" and "org.", presented using the R packages "Hs.eg.db", "enrichplot", and "ggplot2". Correlation analysis was performed using Spearman correlation analysis and presented using the R packages "ggplot2", "ggpubr", and "ggExtra".

[0043] Human mammary epithelial cell line MCF-10A (KGG3103-1) and TNBC cell lines MDA-MB-231 (KGG3220-1) and BT-549 (KGG3221-1) were all derived from KeyGEN Biotechnology in Nanjing, China. All human cell lines were identified by short tandem repeat (STR) analysis to confirm their identity. Gene expression differences between normal cells and TNBC cells were analyzed.

[0044] Twenty-one paraffin-embedded TNBC tumors and 19 adjacent tumor samples from Wuxi Maternal and Child Health Hospital were used as an intra-tissue cohort to verify the association between B7-H3 and HSPA8 expression and TNBC. This study received ethical approval from the Ethics Committee of Wuxi Maternal and Child Health Hospital (approval number 2021-01-0927-28).

[0045] 1.2 In vitro experiments 1.2.1 Cell Culture Human mammary epithelial cell line MCF-10A (KGG3103-1) and TNBC cell lines MDA-MB-231 (KGG3220-1) and BT-549 (KGG3221-1) were all derived from KeyGEN Biotechnology in Nanjing, China. All human cell lines were identified by short tandem repeat (STR) analysis to confirm their identity. Cells were cultured in their respective recommended media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% carbon dioxide.

[0046] Various cell lines were divided into three groups: control group (si-NC), B7H3 knockdown group (si-B7H3), and combined knockdown group (si-B7H3+si-HSPA8). The control group was transfected with si-NC, the B7H3 knockdown group was transfected with si-B7H3, and the combined knockdown group was transfected with both si-B7H3 and si-HSPA8. The siRNA was designed and synthesized by KeyGEN Biotech, and transient transfection was performed using Lipofectamine 3000 (ThermoFisher, L3000015) at a final concentration of 100 nM.

[0047] The siRNA sequences involved are as follows: si-NC: UUCUCCGAACGUGUCACGUdTdT, SEQ ID NO.1; si-B7H3: CAAAGAAGAUGAUGGACAAGAdTdT, SEQ ID NO.2; si-HSPA8:GCUGGUCUCAAUGUACUUATT, SEQ ID NO. 3.

[0048] 1.2.2 Cell Scratch Assay Cells were seeded in 10% FBS medium and cultured until cell confluence reached 90%-100%. After changing to FBS-free medium, and culturing for 12 hours, a vertical scratch was made in the center of the cell monolayer using a sterile pipette tip. Images of the same location were taken under a microscope at 0 h, 24 h, and 48 h, and the scratch width was measured and the scratch healing rate was calculated.

[0049] 1.2.3 Transwell Experiment Transwell assays consist of two parts: migration assay (without Matrigel (Corning) added to the chamber) and invasion assay (with Matrigel added to the chamber). Cells were seeded into the upper chamber in FBS-free medium, while the lower chamber was treated with 10% FBS medium. After 48 hours of culture, cells that migrated or invaded the membrane bottom surface were fixed with paraformaldehyde and stained with crystal violet dye. The stained cells were then visualized and quantified.

[0050] 1.2.4 Cloning Experiment Cells were seeded into 10% FBS medium and cultured for 12 days until visible monoclonal colonies formed. The medium was discarded, and the cells were fixed with paraformaldehyde and stained with crystal violet dye. The number of clones with ≥50 cells was counted, and the colony formation rate was calculated.

[0051] 1.2.5 Flow cytometry Cells were treated with 0.25% trypsin (EDTA-free) and double-stained using the Annexin V-APC / 7-AAD apoptosis detection kit (KeyGEN, KGA1106-50). Apoptosis was assessed using CytoFLEX flow cytometry.

[0052] 1.2.6 Immunofluorescence analysis Cells receiving each treatment were washed twice with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature. After fixation, cells were infiltrated with 0.1% Triton X-100 (Solarbio, T8200). The reaction was terminated by treatment with 10% goat serum for 30 minutes, followed by overnight co-incubation with primary antibodies at 4°C. Antibodies used included SP1 (1:200 dilution, Proteintech, 21962-1-AP), HSPA8 (1:200 dilution, abcam, 3180), and B7-H3 (1:200 dilution, CST, 14058). The following day, cells were incubated with fluorescently labeled secondary antibodies at room temperature. Cell nuclei were contrast stained with DAPI (1:1000 dilution, Beyotime, C1002), and cells were visualized using a confocal microscope (Nikon A1+, Japan).

[0053] 1.2.7 RNA Extraction and Real-Time Quantitative PCR Total RNA was extracted from cells using an RNA extraction reagent (Vazyme, R701-01). cDNA was synthesized using a HiScript IIQ RT SuperMix (Vazyme, R222-01). Real-time quantitative PCR was then performed using ChamQ SYBR qPCR master mix (Vazyme, Q321-02), with β-actin as a normalization control.

[0054] The primer sequences used are as follows: B7-H3-F: 5'- GGGCTGTGTGTGTCTCAT-3', SEQ ID NO.4; B7-H3-R: 5'-TTTCTCCAGCACACGAAGC-3', SEQ ID NO.5; HSPA8-F: 5'-ACCTACTCTTGTGTGGGTGTT-3', SEQ ID NO.6; HSPA8-R: 5'-GACATAGCTTGGAGTGGTTCG-3', SEQ ID NO.7; HSPA2-F: 5'-GATGTGTCGGTTCTCTCCATTG-3', SEQ ID NO.8; HSPA2-R:5′-CTTCCATGAAGTGGTTCACGA-3′,SEQ ID NO.9; HSPA6-F:5′-GGTCCCGGCCTATTTCAACG-3′,SEQ ID NO. HSPA6-R:5′-GGACACGTCGAAAGTGCCA-3′,SEQ ID NO. β-actin-F:5′-GCAGAAGGAGATCACTGCCCT-3′,SEQ ID NO.12; β-actin-R:5′-GCTGATCCACATCTGCTGGAA-3′,SEQ ID NO.13.

[0055] 1.2.8 Western blot Cells were harvested on ice and homogenized using RIPA buffer (Solarbio, R0020), supplemented with PMSF (Solarbio, P0100) and a phosphatase inhibitor (Abcam, ab201113). Protein concentration in the supernatant was determined using the BCA assay (Vazyme, E112-01). Equal volumes of protein were separated on 8%–12% SDS-PAGE gels (Vazyme, E302-01, E303-01, E304-01) and subsequently transferred to polyvinyl chloride (PVDF) membranes (Millipore, IPVH00010). Cells were blocked with NcmBlot blocking buffer (NCM). Biotech (P30500) blocking membranes were incubated overnight with primary antibodies: HSPA8 (1:1000 dilution, Abcam, ab51052), SP1 (1:1000 dilution, Proteintech, 21962-1-AP), B7-H3 (1:1000 dilution, Proteintech, 83152-5-RR), p-P38MAPK (1:1000 dilution, Proteintech, 28796-1-AP). P38MAPK (1:1000 dilution, Proteintech, 14064-1-AP), HSPA6 (1:1000 dilution, Proteintech, 13616-1-AP), HSPA2 (1:1000 dilution, Proteintech, 12797-1-AP), β-actin (1:1000 dilution, BOSTER, P60709). The following day, the membrane was incubated with HRP-bound secondary antibody. Specific protein bands were observed using the SuperPico ECL chemiluminescence kit (Vazyme, E422-01) and imaged using the ChemiDoc XRS+ system (Biorad, USA). Protein expression levels were normalized using β-actin as a control.

[0056] 1.3 In vivo experiments (animal experiments) Female BALB / c mice aged 5-6 weeks (purchased from the Shanghai Laboratory Animal Center) were selected as experimental subjects. Figure 11 The experimental procedure shown in A involves preparing a suspension of 4T1 breast cancer cells (concentration 1×10⁻⁶). 7 The tumor (cells / mL) was subcutaneously injected into the right axilla of mice at a volume of 0.1 mL / mouse. The tumor diameter was measured using calipers; when the tumor volume reached approximately 100 mm... 3Mice were randomly divided into four groups: control group, anti-B7-H3 group, anti-HSPA8 group, and anti-B7-H3 + anti-HSPA8 group, with five mice in each group. Treatment began at this time, with each group receiving the corresponding drug. The anti-B7-H3 group received B7-H3 monoclonal antibody (BioXCell, BE0124) twice a week, 160 μg / mouse, intraperitoneally; the anti-HSPA8 group received an HSPA8 inhibitor (VER155008) three times a week, 3 mg / kg, intraperitoneally; the anti-B7-H3 + anti-HSPA8 group received both B7-H3 monoclonal antibody and HSPA8 inhibitor at the same dosages as the anti-B7-H3 and anti-HSPA8 groups; the control group received an equal volume of PBS. The experiment lasted for two weeks to comprehensively evaluate the treatment effect. During the experiment, tumor size was measured periodically to dynamically observe the anti-tumor effect. At the end of the experiment, mice were euthanized using carbon dioxide (Euthanex Chamber). The tumor was then removed from the euthanized animal, and its weight was recorded. The excised tumor tissue was subjected to tumor-infiltrating immunocellular analysis and immunofluorescence staining. ImageJ software was used for quantitative analysis of fluorescence.

[0057] 1.4 Statistical Analysis Statistical analyses and graphs were presented using R language V.4.3 and GraphPad Prism V.9.5. Group differences were assessed using Student's t-test or Wilcoxon test for two groups, and one-way ANOVA was performed using Tukey's multiple comparison test or Kruskal-Wallis test for multiple groups. Prognostic values ​​for categorical variables were assessed using the log-rank test. All analyses were considered statistically significant with p < 0.05.

[0058] 2. Experimental Results 2.1 Bioinformatics Analysis Results This invention analyzed the gene expression of normal breast tissue and triple-negative breast cancer tissue in the TCGA database, and the results are as follows: Figure 2 As shown, HSPA8 expression was significantly increased in TNBC tissue compared to normal breast tissue. Figure 2 (A). Kaplan-Meier curves show that in TNBC patients, higher HSPA8 expression is associated with worse overall survival (OS) and recurrence-free survival (RFS). Figure 2 B). Subsequently, an internal cohort was used to validate HSPA8 expression in TNBC and the association between B7-H3 and HSPA8 expression, and the results showed that HSPA8 levels were significantly elevated in TNBC tumors (B). Figure 2 (CD).

[0059] To further evaluate the role of B7-H3 in the pathological progression of TNBC, in-depth analysis of the TCGA database was performed. Results are as follows: Figure 3 As shown, compared with adjacent normal tissue and other subtypes, B7-H3 mRNA showed a significant upregulation trend in TNBC tissue. Figure 3 Survival analysis further revealed that patients with high B7-H3 expression had significantly shorter overall survival (OS) and disease-free survival (DFS), suggesting that it is a potent biomarker for predicting TNBC prognosis. Figure 3 The finding was subsequently validated using tissue microarray and multiplex immunofluorescence techniques. The B7-H3 protein showed a diffuse, strongly positive distribution in tumor nests, while its expression was weak in adjacent tumor samples (including stroma and normal glands). Figure 4 Furthermore, Western blot and qPCR results showed that the expression level of B7-H3 in multiple TNBC cell lines was significantly higher than that in normal mammary epithelial cells (AB). Figure 4 (CE). These clinical and cytological evidences together establish the pathological basis for B7-H3 as a key oncogenic factor in TNBC.

[0060] 2.2 Combined knockdown of B7-H3 and HSPA8 enhanced the inhibitory effect of B7-H3 knockdown on cancer cells in vitro. This invention investigates whether combined knockdown of B7-H3 and HSPA8 can enhance the anti-tumor effect on TNBC cells. First, this invention uses siRNA technology to knock down B7-H3 and HSPA8 in cells, and the results are as follows... Figure 5 As shown in the AC, qPCR and Western blot analyses confirmed that B7-H3 and HSPA8 could be double knocked down.

[0061] Furthermore, in vitro cell scratch assays and transwell assays confirmed that the combined knockdown of HSPA8 and B7-H3 had a stronger inhibitory effect on the migration and invasion of TNBC cells than the knockdown of B7-H3 alone. Figure 6 and Figure 7 Clonogenic assays showed that, compared to B7-H3 knockdown alone, the combined knockdown of HSPA8 and B7-H3 had a stronger inhibitory effect on TNBC cell proliferation. Figure 8 Apoptosis and cell cycle assays showed that combined knockdown of HSPA8 and B7-H3 led to more pronounced TNBC cell apoptosis and more significant cell cycle arrest. Figure 9 and Figure 10 ).

[0062] The results show that the combined knockdown of B7-H3 and HSPA8 resulted in the strongest therapeutic effect, indicating that the combined knockdown of B7-H3 and HSPA8 has a synergistic anti-tumor effect.

[0063] 2.4 Combination therapy strategies inhibit tumor growth and reshape the immune microenvironment in vivo. This invention established a TNBC mouse model using the 4T1 breast cancer cell line and investigated the synergistic antitumor effects of B7-H3 and HSPA8 in vivo by administering B7-H3 inhibitors and HSPA8 inhibitors. The results are as follows: Figure 11 As shown. Figure 11 As shown in the BE (Body-Earnings Intervention) curves, tumor volume growth curves and tumor weight analysis revealed that the tumor growth rate was slower in the anti-B7-H3 + anti-HSPA8 group compared to the control group and the treatment-alone group. Figure 11 As shown in Figure D, compared to the control group, the tumor inhibition rate of the anti-B7-H3 + anti-HSPA8 group was 85.3%, the tumor inhibition rate of the anti-B7-H3 group was 36.3%, and the tumor inhibition rate of the anti-HSPA8 group was 43.6%. This indicates that the tumor inhibition rate of the anti-B7-H3 + anti-HSPA8 group was higher than that of the anti-B7-H3 group and the anti-HSPA8 group alone, and even higher than the sum of the tumor inhibition rates of the anti-B7-H3 group and the anti-HSPA8 group, suggesting a synergistic effect of combined anti-B7-H3 and anti-HSPA8 treatment. Furthermore, no significant weight loss was observed in the treatment group compared to the untreated group, highlighting the tolerability of the treatment. Immunohistochemical staining for B7-H3 and HSPA8 also corroborated the results of the in vitro experiments, such as... Figure 11 As shown in FG, immunofluorescence analysis of CD8 and CD4 showed that CD8 in the anti-B7-H3+ anti-HSPA8 group was significantly higher. + T cells and CD4 + T levels are higher. CD8 is one of the most crucial immune cells in tumor immunity. + T cell recruitment and activation are crucial for inducing an effective anti-tumor immune response. Furthermore, immunofluorescence results confirmed that tumor tissues in the anti-B7-H3 + anti-HSPA8 group exhibited low expression of FOXP3, KI-67, and PCNA, and high expression of Caspase9. Consistent with this, immunofluorescence TUNEL staining and quantitative analysis showed that the combination therapy significantly increased tumor cell apoptosis.

[0064] The aforementioned in vitro and in vivo experiments consistently demonstrate that targeting HSPA8 enhances the tumor-suppressive effect of anti-B7-H3 therapy, providing a novel treatment option for TNBC patients eligible for anti-B7-H3 therapy. This combination therapy also significantly improves the infiltration and activation of immune cells, enhancing patient immunity and effectively improving prognosis. These results confirm the potential of this method in enhancing the efficacy of TNBC immunotherapy.

[0065] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of B7-H3 inhibitors in combination with HSPA8 inhibitors in the preparation of pharmaceutical compositions for the treatment of breast cancer.

2. Use according to claim 1, characterized in that, The B7-H3 inhibitors include nucleic acid intervention molecules that target B7-H3, B7-H3 antibodies, B7-H3 antibody-drug conjugates, and CAR-T-related components.

3. Use according to claim 1, characterized in that, The HSPA8 inhibitors include nucleic acid intervention molecules that target HSPA8, compounds that inhibit HSPA8, HSPA8 antibodies, HSPA8 antibody-drug conjugates, and CAR-T-related components.

4. Use according to claim 1, characterized in that, The breast cancer mentioned includes triple-negative breast cancer.

5. A pharmaceutical composition for treating breast cancer, characterized by, The pharmaceutical composition contains a B7-H3 inhibitor and an HSPA8 inhibitor.

6. The pharmaceutical composition of claim 5, wherein, The B7-H3 inhibitors include nucleic acid intervention molecules that target B7-H3, B7-H3 antibodies, B7-H3 antibody-drug conjugates, and CAR-T-related components.

7. The pharmaceutical composition of claim 5, wherein, The HSPA8 inhibitors include nucleic acid intervention molecules that target HSPA8, HSPA8 antibodies, HSPA8 antibody-drug conjugates, and CAR-T-related components.

8. Use of the pharmaceutical composition according to any one of claims 5-7 in the preparation of a medicament for treating breast cancer.

9. A medicament for treating breast cancer, characterized by comprising a compound of the formula (I) or a pharmaceutically acceptable salt thereof. The drug comprises the pharmaceutical composition according to any one of claims 5-7.

10. The medicament according to claim 9, characterized in that, The drug also contains pharmaceutically acceptable carriers or excipients.

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