Application of ICAM1 or coding gene thereof in preparation of inflammatory breast cancer treatment or diagnosis product

Abstract

The invention relates to the field of biological pharmacy and biological medicine, in particular to application of ICAM1 or a coding gene thereof in preparation of a product for treating or diagnosing inflammatory breast cancer. The invention provides application of an ICAM1 antibody-drug conjugate in preparation of a product. The product at least has one of the following effects: 1) treating inflammatory breast cancer; 2) inhibiting lymphatic vessel generation; and 3) the curative effect of the immune checkpoint inhibitor on the inflammatory breast cancer is improved. According to the application, it is determined that ICAM1 can be used as a potential therapeutic target of inflammatory breast cancer, and two ADCs, namely ICAM1-MMAE and ICAM1-DXd, related to ICAM1 are reasonably designed and developed. Experimental data confirm that ICAM1-DXd can be used as an optimal ADC for targeted therapy of inflammatory breast cancer, inhibits the generation of lymphatic vessels, and also shows a synergistic therapeutic effect after being combined with an immune checkpoint inhibitor PD1 for treatment. Finally, the application provides important basis and viewpoint for developing ADC drugs for treating inflammatory breast cancer.

Description

Technical Field

[0001] This application relates to the fields of biopharmaceuticals and biomedicine, and in particular to the use of ICAM1 or its encoding gene in the preparation of products for the treatment or diagnosis of inflammatory breast cancer. Background Technology

[0002] Inflammatory breast cancer (IBC) is a rare and highly metastatic subtype of breast cancer, accounting for 2% of all breast cancers. Its course is rapid, with a metastasis rate as high as 40%. Due to the lack of effective treatment options, IBC patients have a poor clinical prognosis, with a mortality rate of approximately 7%-10%. About 40% of patients have distant metastases at diagnosis. Studies have shown that IBC can induce new lymphangiogenesis, and its lymphangiogenic capacity is stronger than that of non-IBC patients. Currently, treatment options for inflammatory breast cancer are limited. Most patients have metastases at diagnosis, making surgical resection and radiotherapy / chemotherapy less effective and less efficient. Clinical studies have shown that initial chemotherapy with anthracyclines followed by adjuvant therapy with taxanes can improve clinical outcomes. Regarding targeted therapy, HER2 overexpression rates in IBC are 36%-60%, and experiments have shown that adjuvant therapy with trastuzumab can significantly improve the complete response rate.

[0003] Antibody-drug conjugates (ADCs) are a novel type of targeted therapy composed of monoclonal antibodies, linkers, and cytotoxic molecules. ADCs work by using monoclonal antibodies to highly specifically bind to tumor cell surface antigens, delivering cytotoxic molecules into tumor cells. Intracellular proteases then hydrolyze the linker, releasing the cytotoxic molecules and leading to tumor cell death. ADCs exhibit high specificity, a long plasma half-life, improved in vivo stability and efficacy, and can overcome multidrug resistance, thus enhancing therapeutic effects. However, currently, there is still a lack of effective ADC drug targets for treating intracranial thrombocytopenic purpura (IBC).

[0004] ICAM1 (intercellular adhesion molecule 1), a transmembrane glycoprotein, is widely expressed on the surface of immune and endothelial cells and belongs to the immunoglobulin superfamily. It promotes leukocyte adhesion and migration by binding to LFA-1 (lymphocyte function-associated antigen 1), participating in immune responses and inflammatory reactions. ICAM1 also affects intercellular signaling, cell proliferation, apoptosis, and polarity. Its overexpression enhances the adhesion of tumor cells to surrounding tissues, promoting invasion and metastasis, and accelerating tumor growth and metastasis. Summary of the Invention

[0005] To address the technical problems in the prior art, the purpose of this application is to provide the use of ICAM1 or its encoding gene in the preparation of products for the treatment or diagnosis of inflammatory breast cancer.

[0006] To achieve the above and other related objectives, this application provides, on the one hand, the use of ICAM1 antibody-drug conjugates in the preparation of products, including said products having at least one of the following effects:

[0007] 1) Treatment of inflammatory breast cancer;

[0008] 2) Inhibits lymphangiogenesis;

[0009] 3) Improve the efficacy of immune checkpoint inhibitor therapy for inflammatory breast cancer.

[0010] A second aspect of this application provides an antibody-drug conjugate for treating inflammatory breast cancer, comprising a drug with cytotoxic effects, said drug being conjugated to an ICAM1 antibody via a linker to form the antibody-drug conjugate.

[0011] A third aspect of this application provides a pharmaceutical composition comprising an effective amount of an immune checkpoint inhibitor and the ICAM1 antibody-drug conjugate used in the aforementioned purposes.

[0012] The fourth aspect of this application provides the use of the aforementioned pharmaceutical composition in the preparation of a product for treating inflammatory breast cancer.

[0013] Compared with the prior art, the beneficial effects of this application are as follows:

[0014] This application identifies ICAM1 as a potential therapeutic target for inflammatory breast cancer and rationally designs and develops two ICAM1-related ADCs: ICAM1-MMAE and ICAM1-DXd. Experimental data confirm that ICAM1-DXd can serve as the optimal ADC for targeted therapy of inflammatory breast cancer, inhibiting lymphangiogenesis, and also demonstrating synergistic therapeutic effects when combined with the immune checkpoint inhibitor PD-1. Finally, this application provides important evidence and perspectives for the development of ADC drugs for the treatment of inflammatory breast cancer. Attached Figure Description

[0015] Figure 1 To enable unbiased and quantitative analysis of cell membrane protein expression in two inflammatory breast cancer cell lines (SUM149PT and KPL4) and normal epithelial cells (HEK-293) using flow cytometry; and to visualize the expression of ICAM1, TROP2, and HER2 using single-photon confocal microscopy.

[0016] Figure 2 Immunohistochemistry was used to stain tumor tissue from patients with inflammatory breast cancer; ICAM1 was identified as a potential target for IBC using public datasets.

[0017] Figure 3Human lymphatic endothelial cells were co-cultured with inflammatory breast cancer cells; ICAM1 expression was detected by flow cytometry and single-photon confocal microscopy; the expression of ICAM1 and NF-κb in HLECs before and after induction was detected by immunoblotting, and the expression of the target gene / internal reference gene was quantified. *p<0.05.

[0018] Figure 4 To quantify the efficiency of ICAM1 internalization in inflammatory breast cancer cell lines by flow cytometry; and to visualize the ICAM1 internalization process using single-photon confocal microscopy.

[0019] Figure 5 This is a schematic diagram of the in vivo distribution of PBS, IgG-Cy3, and ICAM1-Cy3. IVIS was used to photograph isolated organs and tumors of nude mice. The data were analyzed using GraphPad, and the results are shown in the figure (*p<0.05; **p<0.01; ***p<0.001; no significant difference ns).

[0020] Figure 6 To quantify the efficiency of ICAM1 internalization in human lymphatic endothelial cells by flow cytometry; to visualize the internalization process of ICAM1 by single-photon confocal microscopy; and to demonstrate the co-localization of ICAM1 (red) and LYVE-1 (green) in tumor tissue by multiplex immunofluorescence.

[0021] Figure 7 This is a schematic diagram of the ICAM1 DXd molecular structure.

[0022] Figure 8 To detect and compare the cell line killing effects of DXd and first-line chemotherapy drugs using the CCK-8 assay, the IC50 was calculated. 50 .

[0023] Figure 9 This diagram illustrates the process of establishing and treating inflammatory breast cancer in nude mice. The figure shows photographs of the ex vivo tumor and illustrates the changes in body weight and tumor volume of each group of nude mice at different time points. Significant differences were observed between the ICAM1-DXd and ICAM1-MMAE treatment groups and the PBS group. No significant changes in body weight were observed in any of the nude mice during treatment (*p<0.05; **p<0.01; ***p<0.001; no significant difference in ns).

[0024] Figure 10 This diagram illustrates the modeling and treatment process for inflammatory breast cancer in immunocompetent mice. The figure shows photographs of the isolated tumor and illustrates the changes in body weight and tumor volume of each group of nude mice at different time points. Significant differences were observed between the ICAM1-DXd and ICAM1-MMAE treatment groups and the PBS group. No significant changes in body weight were observed in any of the nude mice during treatment (*p<0.05; **p<0.01; ***p<0.001; no significant difference in ns).

[0025] Figure 11 To label tumor lymphatic vessels with immunohistochemistry, the positive rate of lymphatic vessel staining was calculated. The data were analyzed using GraphPad and the results are shown in the figure (*p<0.05; no significant difference in ns).

[0026] Figure 12 Differential expression heatmaps were generated among the PBS, ICAM1-MMAE, and ICAM1-DXd treatment groups; GO enrichment analysis was performed; and flow cytometry was used to analyze immune cells in tumors.

[0027] Figure 13 This is a schematic diagram of the process of establishing an inflammatory breast cancer model in immune-healthy mice and combining immunotherapy. The changes in body weight and tumor volume of each group of nude mice were recorded at time points. The combination therapy group had a better tumor-suppressing effect than any single drug group. There were no significant changes in body weight of nude mice in any group during the treatment process (*p<0.05; **p<0.01; ***p<0.001; no significant difference ns). Detailed Implementation

[0028] To make the inventive objectives, technical solutions, and beneficial effects of this application clearer, the following description, in conjunction with embodiments, further illustrates this application. It should be understood that the embodiments described are for illustrative purposes only and are not intended to limit the scope of the application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and those skilled in the art can easily understand other advantages and effects of this application from the content disclosed in this description.

[0029] The inventors of this application, through extensive research and exploration, discovered the use of ICAM1 or its encoding gene in the preparation of products for the treatment or diagnosis of inflammatory breast cancer, and completed this application based on this discovery.

[0030] This application provides, in one aspect, the use of ICAM1 antibody-drug conjugates in the preparation of products, including said products having at least one of the following effects:

[0031] 1) Treatment of inflammatory breast cancer;

[0032] 2) Inhibits lymphangiogenesis;

[0033] 3) Improve the efficacy of immune checkpoint inhibitor therapy for inflammatory breast cancer.

[0034] In some implementations, the drug is conjugated to an ICAM1 antibody via a linker to form an ICAM1 antibody-drug conjugate.

[0035] In some embodiments, the drug in the ICAM1 antibody-drug conjugate is selected from monomethylaurestatin E, monomethylaurestatin F, compuratin A, cytarabine, gemcitabine, maytansine, DM1, DM4, chachiin and its derivatives, doxorubicin, pyrrolobenzodiazepines and their derivatives, SN-38, camptothecin analogs, α-ammantin, and tubulin lysin analogs. In a specific embodiment of this application, the drug is selected from MMAE or DXd.

[0036] In some implementations, the ICAM1 antibody is conjugated to four or eight drugs.

[0037] In some implementations, the linker is Mc-VC-PAB and / or Mc-GGFG.

[0038] In some embodiments, the ICAM1 antibody-drug conjugate is: ICAM1 conjugated to MMAE, with a drug-antibody ratio of 4, and the linker is Mc-VC-PAB.

[0039] In some embodiments, the ICAM1 antibody-drug conjugate is: ICAM1 conjugated with Dxd, the drug-antibody ratio is 8, and the linker is Mc-GGFG.

[0040] Drug-antibody ratio (DAR) refers to the average number of payload molecules attached to a single monoclonal antibody.

[0041] The specific structure of the ICAM1 antibody-drug conjugate in this application is as follows: Figure 7 As shown.

[0042] In some embodiments, the immune checkpoint inhibitor is selected from one or more combinations of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors. In a specific embodiment of this application, the immune checkpoint inhibitor is selected from PD-1 inhibitors.

[0043] A second aspect of this application provides an antibody-drug conjugate for treating inflammatory breast cancer, comprising a drug with cytotoxic effects, said drug being conjugated to an ICAM1 antibody via a linker to form the antibody-drug conjugate.

[0044] In some embodiments, the drug is selected from monomethylaurestatin E, monomethylaurestatin F, compuratin A, cytarabine, gemcitabine, maytansine, DM1, DM4, chachiin and its derivatives, doxorubicin, pyrrolobenzodiazepines and their derivatives, SN-38, camptothecin analogs, α-ammantin, and tubulin lysin analogs; preferably, the drug is selected from DXd. The linker is Mc-GGFG. In a specific embodiment of this application, the antibody conjugate is ICAM1-DXd conjugated with DXd, the drug-antibody ratio is 8, and the linker is Mc-GGFG. Experimental data of this application confirm that ICAM1-DXd has the highest tumor inhibition rate in all test groups, approximately 81.5%, significantly higher than ICAM1-MMAE (58.7%) and doxorubicin (46.1%). The results indicate that ICAM1-DXd is the optimal targeted therapy for IBC.

[0045] A third aspect of this application provides a pharmaceutical composition comprising an effective amount of an immune checkpoint inhibitor and the ICAM1 antibody-drug conjugate used in the aforementioned purposes.

[0046] In some embodiments, the immune checkpoint inhibitor is selected from one or more combinations of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors. In a specific embodiment of this application, the immune checkpoint inhibitor is selected from PD-1 inhibitors.

[0047] In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient.

[0048] "Pharmaceutical acceptable" means that when the molecular basis and the composition are properly administered to animals or humans, they do not produce adverse, allergic, or other adverse reactions.

[0049] "Pharmaceutically acceptable carriers or excipients" should be compatible with immune checkpoint inhibitors and ICAM1 antibody-drug conjugates, meaning they can be mixed with them without significantly reducing the drug's efficacy under normal circumstances. Specific examples of substances that can serve as pharmaceutically acceptable carriers or excipients include sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents, stabilizers, antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0050] The pharmaceutical compositions provided in this application are adaptable to any form of administration, including oral or parenteral administration. For example, they can be administered via pulmonary, nasal, rectal, and / or intravenous injection; more specifically, they can be administered intradermally, subcutaneously, intramuscularly, intra-articularly, intraperitoneally, pulmonaryly, or orally, via the mouth, sublingually, nasally, percutaneously, vaginally, or orally, or via parenteral administration. Those skilled in the art can select appropriate formulations based on the administration method. For example, formulations suitable for oral administration may include, but are not limited to, pills, tablets, chewable tablets, capsules, granules, drops, or syrups. Similarly, formulations suitable for parenteral administration may include, but are not limited to, solutions, suspensions, rehydrated dry formulations, or sprays. Furthermore, suppositories are typically suitable for rectal administration.

[0051] The fourth aspect of this application provides the use of the aforementioned pharmaceutical composition in the preparation of a product for treating inflammatory breast cancer. Experimental data from this application confirm that ICAM1-DXd can serve as an optimal ADC for targeted therapy of inflammatory breast cancer, inhibiting lymphangiogenesis, and also demonstrating a synergistic therapeutic effect when combined with the immune checkpoint inhibitor PD-1. Finally, the research in this application provides important evidence and perspectives for the development of ADC drugs for treating inflammatory breast cancer.

[0052] The present application will be further illustrated by the following examples, but these examples do not limit the scope of the present application.

[0053] Example 1

[0054] Experimental methods

[0055] 1. Cell Culture

[0056] SUM149 cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin; KPL4 cells were cultured in L-15 medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin; HEK-293T cells were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin; all cells were cultured in an incubator containing 5% CO2 at 37°C.

[0057] 2. Target validation

[0058] 2.1 Quantitative analysis of protein expression on cell membranes

[0059] Flow cytometry was used to assess the expression of surface proteins in IBC cells and normal cells. Cells in the logarithmic growth phase were collected by trypsin digestion and the cell concentration was adjusted to 1 × 10⁻⁶. 6 Cells were blocked at 1% BSA in PBS on ice for 30 min. After blocking, 1 μg of PE-labeled antibody was added, mixed well, and stained at room temperature in the dark for 45 min. Cells were then washed three times with PBS, centrifuged at 1200 rpm for 3 min, and analyzed by flow cytometry.

[0060] 2.2 Visualization of ICAM1 localization in cells

[0061] The location of ICAM1 in cells was determined using confocal microscopy. Cells in the logarithmic growth phase were collected by trypsin digestion and the cell concentration was adjusted to 1 × 10⁻⁶. 6 Cells were seeded at a density of 1 / mL in 35 mm confocal dishes and incubated overnight in an incubator. The confocal dishes were then removed from the incubator and blocked with PBS containing 1% BSA for 30 min. After blocking, 1 μg of PE-labeled antibody was added and stained at room temperature in the dark for 45 min. The samples were then washed three times with PBS, fixed with 4% paraformaldehyde for 15 min, stained with Hoechst 33342 for the nuclei, rinsed with PBS 1-2 times, and detected using a confocal microscope (Nikon) with an A1R HD25 microscope.

[0062] 2.3 Analysis of ICAM1 antibody internalization efficiency

[0063] 2.3.1 Quantitative analysis by flow cytometry

[0064] Cells in the logarithmic growth phase were collected by trypsin digestion, and the cell concentration was adjusted to 1×10⁻⁶. 6Cells were blocked with PBS containing 1% BSA for 30 min. After blocking, 1 μg of PE-labeled antibody was added, mixed, and stained at room temperature in the dark for 45 min. Cells were then washed three times with PBS and incubated at 37°C at time points of 240 min, 120 min, 60 min, 30 min, 15 min, and 0 min. After incubation, 0.5 μg of PE-labeled anti-mouse IgG1 was added and incubated for 45 min. After rinsing with PBS 1-2 times, the MFI of each cell was measured using CytoFLEX LX (Beckman).

[0065] 2.3.2 Internalization of Single-Photon Confocal Visualization ICAM1

[0066] Cells in the logarithmic growth phase were collected by trypsin digestion, and the cell concentration was adjusted to 1×10⁻⁶. 6 Cells were seeded at a density of 1 / mL in 35 mm confocal dishes and incubated overnight in an incubator. The confocal dishes were then removed from the incubator and blocked with PBS containing 1% BSA for 30 min. After blocking, 1 μg of PE-labeled antibody was added, and the dishes were stained at room temperature in the dark for 45 min. The staining was then repeated three times with PBS. The dishes were incubated at 37°C at time points of 240 min, 120 min, 60 min, 30 min, 15 min, and 0 min. The samples were fixed with 4% paraformaldehyde for 15 min, and the nuclei were stained with Hoechst 33342. After rinsing three times with PBS, the samples were examined using a Nikon A1R HD25 microscope via confocal microscopy.

[0067] 2.4 Construction of ICAM1 antibody-drug conjugates

[0068] ICAM1 antibodies were constructed by covalently coupling with two different linkers and payloads (MC-VC-PAB-MMAE and MC-GGFG-DXd). The DAR value of the ICAM1 ADCs was determined using hydrophobic interaction chromatography.

[0069] 2.5 In vitro cytotoxicity assay

[0070] Human inflammatory breast cancer cells (SUM149PT and KPL4) in the logarithmic growth phase were seeded in 96-well plates at a cell density of 5 × 10⁻⁶ cells / well. 3 The cells were incubated overnight in a cell culture incubator. ICAM1 ADC, ICAM1 monoclonal antibody, and the chemotherapeutic drug doxorubicin were serially diluted 10-fold from 0 to 10 μg / mL and cultured in a cell culture incubator for 72 h. 10 μL of LCK8 reagent was added to each well and incubated for 1-2 h. The absorbance was detected using a microplate reader at 450 nm.

[0071] 2.6 Preparation of ICAM1 monoclonal antibody conjugated with Cy3

[0072] ICAM1 monoclonal antibody and Cy3 were added to a 1.5 mL EP tube at a molar ratio of 1:10. The pH was adjusted to 8.5 by adding 10 M NaOH, and the reaction was carried out overnight at room temperature with shaking, protected from light. Unbound Cy3 was removed by filtration using a Cytiva G-25 desalting column to obtain the ICAM1-Cy3 compound.

[0073] 2.7 Establishment of tumor-bearing mouse model and organ imaging

[0074] 2.7.1 Establish a SUM149 tumor xenograft model to evaluate the specific targeting and biodistribution of ICAM1 tumors in vivo to major organs.

[0075] 5×10 6 One SUM149 cell was seeded into the fat pad of the third pair of mammary glands on the right side of mice. When the tumor grew to a volume of 100-200 mm, 3 Mice were randomly divided into three groups of five each. PBS, IgG-Cy3, and ICAM1-Cy3 were injected via tail vein distribution at a concentration of 5 mg / kg. After 48 hours, mice in each group were sacrificed by cervical dislocation, and their organs (brain, heart, liver, lungs, kidneys, and spleen) were removed. The organs were then imaged and photographed using an IVIS Lumina III imaging system.

[0076] 2.7.2 Establish a SUM149 tumor xenograft model to evaluate the antitumor activity of ICAM1 antibody-drug conjugate.

[0077] 5×10 6 SUM149 cells were inoculated into the fat pads of the third pair of mammary glands on the right side of mice. When the tumors grew to a volume of 100-200 mm³, the mice were randomly divided into five groups of five mice each. PBS, IgG-MMAE, IgG-DXd, ICAM1-MMAE, and ICAM1-DXd were injected via tail vein at a concentration of 5 mg / kg, once weekly. Mouse weight was recorded, and tumor volume changes were measured using calipers. Tumor volume = 1 / 2 tumor length (mm) × [tumor width (mm)] 2 The experiment was conducted 3-4 times per week. After the experiment, the mice in each group were sacrificed by cervical dislocation, and the tumors were removed and weighed.

[0078] 2.8 Establish a 4T1-ICAM1 tumor immune model to evaluate the antitumor activity of ICAM1 antibody-drug conjugates.

[0079] First, 4T1 cell lines were infected with ICAM1 lentivirus to obtain monoclonal lines through dilution and then expanded. 2×10⁶ cells were then cultured. 6Four T1-ICAM1 cells were seeded into the fat pads of the third pair of mammary glands on the right side of mice. When the tumors grew to a volume of 100-200 mm³, the mice were randomly divided into five groups of five mice each. PBS, doxorubicin, ICAM1, ICAM1-MMAE, and ICAM1-DXd were injected via tail vein at a concentration of 5 mg / kg, once weekly. Mouse weight was recorded, and tumor volume changes were measured using calipers. Tumor volume = 1 / 2 tumor length (mm) × [tumor width (mm)] 2 Mice were euthanized 3-4 times per week at the end of the experiment by cervical dislocation, and tumors were removed and weighed. Tumor samples were then subjected to single-cell dissociation for fluorescence-activated cell sorting (FACS) analysis and RNA-seq, or collected, rapidly frozen, and embedded in paraffin blocks.

[0080] 2.9 Establish a 4T1-ICAM1 tumor immune model to evaluate the antitumor activity of ICAM1 antibody-drug conjugate combined with PD1 antibody.

[0081] 2×10 6 Four T1-ICAM1 cells were seeded into the third pair of fat pads on the right side of mice. When the tumor grew to a volume of 100-200 mm³, the mice were randomly divided into five groups of five mice each. PBS, ICAM1-MMAE, ICAM1-DXd, ICAM1-MMAE+PD1, and ICAM1-DXd+PD1 were administered via tail vein distribution. The concentration of ICAM1-DXd was 5 mg / kg, and the concentration of PD1 was 10 mg / kg. Administration was once weekly. Mouse weight was recorded, and tumor volume changes were measured using calipers. Tumor volume = 1 / 2 tumor length (mm) × [tumor width (mm)] 2 The experiment was conducted 3-4 times per week. After the experiment, the mice in each group were sacrificed by cervical dislocation, and the tumors were removed and weighed.

[0082] 2.11 Flow Cytometry Multicolor Experiment Procedure

[0083] Red blood cells in tumor single cells were lysed using red blood cell lysis buffer. Cells were stained with Live / Dead cell dye, blocked with CD16 / 32 purified antibody, and the cell surface markers (mCD45, mCD3, mCD4, mCD8, mCD25, mCD11b, mF4 / 80, and mCD206) were stained with a fluorescent dye-conjugated antibody. The staining was performed using CytoFLEX LX (Beckman). CD8 T cells were defined as CD45+, CD3+, CD8+; Treg cells were defined as CD45+, CD3+, CD4+, CD25+; and M2 macrophages were defined as CD45+, CD11b+, F4 / 80+, CD206+.

[0084] 2.12 RNA Sequencing

[0085] Enrichment analysis (GSEA) of RNA-seq data was performed using the OmicStudio tool, available at https: / / www.omicstudio.cn.

[0086] 2.13 Statistical Methods

[0087] All statistical analyses were performed using GraphPad Prism 8. Data were analyzed using either one-way ANOVA or two-way ANOVA. P < 0.05 was considered significant. Error bars represent SD, as shown in each legend.

[0088] Results and Discussion of Example 2

[0089] 1. ICAM1 as a potential target for inflammatory breast cancer and tumor-associated lymphangiogenesis.

[0090] A primary challenge in developing targeted therapies for inflammatory breast cancer is the lack of molecular targets that can differentiate between IBC tumors and normal tissues. To identify novel target molecules (DXd), this application employed flow cytometry to unbiasedly and quantitatively screen 62 relevant cell membrane proteins in human IBC cell lines (KPL4 and SUM149PT) and normal human epithelial 293T cells. Thirteen candidate proteins were found to be overexpressed on the surface of IBC cell membranes, with ICAM1 (intercellular adhesion molecule) showing the highest expression level in IBC cells but not in normal 293T cells. Furthermore, this application compared the expression of ICAM1 with two clinically approved breast cancer DXd targets, HER2 and TROP2. In the KPL4 cell line, ICAM1 expression was 116.4 and 27.8 times higher than that of HER2 and TROP2, respectively, while in SUM149PT, it reached 143.2 and 132.1 times higher, respectively. The results observed by single-photon confocal microscopy were consistent with those by flow cytometry, and showed that ICAM1 was primarily localized on the surface of IBC cell membranes. These findings confirm that ICAM1 could serve as a potential molecular target for developing therapeutics for inflammatory breast cancer DXd. Figure 1 ).

[0091] Based on the above theoretical support, this application utilizes immunohistochemistry to stain tumor samples from patients with inflammatory breast cancer. Figure 2 The results showed that ICAM1 was overexpressed in tumor tissue sections from patients with inflammatory breast cancer. Simultaneously, it was also found in the TCGA-BRCA and GEO databases (…). Figure 2The expression levels of ICAM1 in normal tissues and breast cancer tumor tissues were compared. Compared with normal tissues, ICAM1 was highly expressed in breast cancer tumor tissues, and the expression of ICAM1 in inflammatory breast cancer was also significantly different from that in normal tissues. Further analysis and comparison of the TME composition differences between ICAM1 high- and low-expression groups in IBC samples showed that the ICAM1 high-expression group had a higher proportion of lymphatic endothelial cells.

[0092] 2. Expression of ICAM1 in tumor-associated lymphoid tissue

[0093] Related studies have confirmed that 40%-60% of inflammatory breast cancer patients already have lymph node metastasis at diagnosis. Inflammatory breast cancer exhibits extensive lymph node metastasis within the breast, and reducing tumor lymphangiogenesis could be a potential treatment approach for inflammatory breast cancer. This application first co-cultured human lymphovascular endothelial cells (HLECs) with the supernatant of inflammatory breast cancer. Significant proliferation of HLECs was observed after co-culture. Subsequent in vitro tube formation experiments revealed a more significant trend in the co-culture group compared to the blank control group. Flow cytometry revealed overexpression of ICAM1 in HLECs, and confocal microscopy confirmed the membrane localization of ICAM1. Immunoblot results showed that ICAM1 expression was upregulated after co-culturing lymphovascular endothelial cells with the supernatant of inflammatory breast cancer. Figure 3 ).

[0094] 3. Stable internalization of ICAM1 antibody in inflammatory breast cancer cell lines and lymphoendothelial cells

[0095] Considering that antibody internalization is another key factor in ADC drug design, this application used flow cytometry to quantitatively measure the internalization efficiency of ICAM1 antibody in IBC cells. The results showed that the phycoerythrin (PE)-conjugated ICAM1 antibody could be rapidly and effectively internalized in SUM149PT and KPL cells, reaching a plateau of approximately 45% and 23% after 4 hours of incubation, respectively. This application also used immunofluorescence to visualize the internalization process of ICAM1 antibody in the two cell lines. Figure 4 ).

[0096] Based on the stable internalization of ICAM1 antibody in inflammatory breast cancer cell lines, this example next evaluated the tumor specificity and biodistribution of ICAM1 antibody in an IBC tumor model (SUM149PT) mouse. IBC tumor-bearing mice were injected intravenously with PBS, IgG-Cy3, and ICAM1-Cy3. Forty-eight hours post-injection, ICAM1-Cy3 exhibited strong IBC tumor targeting in vivo. Subsequent fluorescence quantification showed that ICAM1-Cy3 resulted in approximately 3.5 times more tumor accumulation than IgG-Cy3, with no significant changes in normal organs such as the brain, heart, liver, spleen, lung, and kidney. Furthermore, immunofluorescence clearly demonstrated the internalization of ICAM1-Cy3 by tumor cells 48 hours after intravenous injection. Figure 5 These in vitro and in vivo results strongly support the conclusion that ICAM1 exhibits superior tumor specificity and internalization ability, thus making it a potential ADC target for IBC tumors.

[0097] Next, in this embodiment, the internalization efficiency of ICAM1 antibody in human lymphatic endothelial cells was quantified by flow cytometry. The results showed that the internalization efficiency of ICAM1 antibody reached 53% after 4 hours. The internalization process of ICAM1 antibody was also visualized using single-photon confocal microscopy. To better evaluate the targeted internalization ability of ICAM1 antibody on tumor lymphatic vessels, phycoerythrin (PE) conjugated with ICAM1 antibody was injected into mice. Multiplex immunofluorescence was used to locate tumor lymphatic vessels. The results showed that ICAM1 could stably target tumor lymphatic vessels. Figure 6 These results confirm that the ICAM1 antibody has specificity and internalization ability against tumor lymphatic vessels, and the development of ICAM1 ADCs may simultaneously target and kill tumor lymphatic vessels.

[0098] 4. Design, synthesis, and characterization of ICAM1 ADC

[0099] The ICAM1 antibody was diluted to 5 mg / mL with PBS, and an appropriate amount of TCEP was added. The mixture was then incubated at 37°C for 3 hours for reduction. Excess TCEP was removed using PBS containing 4 mM EDTA in an ultrafiltration tube. Ten molar amounts of Mc-VC-PAB-MMAE and 30 molar amounts of Mc-GGFG-DXd were added, and the mixture was reacted with shaking at room temperature for 2 hours. After the reaction, residual small molecules were removed using an ultrafiltration tube at 4°C to obtain ICAM1-MMAE and ICAM1-DXd.

[0100] This embodiment designed and constructed two ICAM1 ADCs. A monoclonal human-mouse chimeric ICAM1 antibody was conjugated with two linker cytotoxic molecules used to treat breast cancer. ICAM1 antibody conjugated with Mc-VC-PAB-MMAE formed ICAM1-MMAE, and ICAM1 antibody conjugated with Mc-GGFG-DXd formed ICAM1-DXd. The drug-to-antibody ratios (DAR) of ICAM1-MMAE and ICAM1-DXd were 4 and 8, respectively. Figure 7 Both Mc-VC-Pab-MMAE and Mc-GGFG-DXd possess enzyme-cleavable linkers, allowing them to be selectively cleaved by cathepsin B in endosomes or lysosomes. This enables the rapid release of the ADC warhead during internalization, while maintaining the integrity of the ADC in the bloodstream. Regarding cytotoxic molecules, MMAE and DXd are clinically used ADC warheads with different mechanisms of action. DXd works by inducing DNA strand breaks, ultimately leading to tumor cell death in the S phase, while MMAE works by inhibiting microtubule polymerization and effectively suppressing cell mitosis.

[0101] Next, the half-maximal inhibitory concentration (IC50) of ICAM1 ADC against IBC cell lines was determined using the CCK-8 assay, with doxorubicin, a standard chemotherapy drug for IBC treatment, used as a control. The results showed that ICAM1-MMAE exhibited strong tumor-specific cytotoxicity against human IBC cells, but had no effect on normal HEK-293T cells. The IC50 values ​​of ICAM1-MMAE in SUM149 cells, KPL4 cells, and HEK-293T cells were 17.02 μg / mL, 5.663 μg / mL, and 5890 μg / mL, respectively. At the given dose of ICAM1-DXd, ICAM1-DXd showed no significant in vitro cytotoxicity against IBC and normal HEK-293T cells, likely due to the low cytotoxicity of DXd itself. In contrast, doxorubicin nonspecifically killed IBC and normal human epithelial cells. Figure 8 Next, the maximum half-maximal inhibitory concentration (MC50) of ICAM1 DXd against lymphatic endothelial cells was determined using the CCK8 assay, with IgG-DXd and IgG-MMAE used as control drugs. The results showed that ICAM1-DXd exhibited potent cytotoxicity against lymphatic endothelial cells compared to the control drugs, with IC50 values ​​of 3.812 μg / mL for ICAM1-MMAE and 4.415 μg / mL for ICAM1-DXd. These in vitro therapeutic data strongly support the ability of ICAM1-DXd to precisely target and kill IBC cells and human lymphatic endothelial cells, which requires further investigation in IBC tumor models.

[0102] 5. ICAM1 DXd significantly inhibits IBC tumors in vivo.

[0103] To evaluate the in vivo efficacy of ICAM1-MMAE and ICAM1-DXd, a mouse IBC xenograft model was established using SUM149 cells. The tumor volume reached 100 mm². 3 Mice were administered ICAM1-MMAE and ICAM1-DXd at doses of 5 mg / kg, respectively, while the control group received phosphate-buffered saline (PBS), IgG-MMAE, and IgG-DXd. Compared with the blank control group (PBS), and the non-targeted IgG-MMAE and IgG-DXd, ICAM1-MMAE and ICAM1-DXd significantly slowed tumor growth. Compared with the PBS control group, the antitumor activity of ICAM1-MMAE and ICAM1-DXd reduced tumor growth by 74.6% and 91.3%, respectively. There was no significant change in body weight in any mouse during the treatment period. Figure 9 ).

[0104] Because immunocompetent tumor models can more realistically reproduce the immune microenvironment of IBC tumors, a robust immunocompetent IBC tumor model was established using the mouse IBC cell line 4T1-hICAM1 expressing human ICAM1 antigen. Among all test groups, ICAM1-DXd showed the highest tumor inhibition rate, approximately 81.5%, significantly higher than ICAM1-MMAE (58.7%) and doxorubicin (46.1%). No significant changes in body weight were observed in any of the mice. These in vivo results indicate that ICAM1-DXd is the optimal targeted therapy for IBC. Figure 10 ).

[0105] 6. ICAM1-DXd selectively removes tumor-associated lymphatic vessels

[0106] Next, to evaluate the effect of ICAM1 ADC on intratumoral lymphangiogenesis, immunohistochemical staining was performed on tumor tissue after treatment. The results showed that in nude mouse models and immunocompetent models, compared with the blank control group, the ICAM1-DXd group had cleared collapsed lymphatic vessels around the tumor, while functional lymphatic vessels were preserved, and a trend of decreasing lymphatic vessel density was observed. No significant difference was found in the number of lymphatic vessels within the tumor after treatment. This indicates that ICAM1 DXd can target and kill collapsed lymphatic vessels around the tumor. Figure 11 ).

[0107] 7. ICAM1-DXd activation of tumor immune progression

[0108] ICAM1-DXd was observed to produce a significant tumor-suppressing effect in animal models, possibly due to the various biological responses it elicited in vivo. To elucidate this phenomenon, RNA-seq analysis was performed on tumor tissues from immune-intact models. GO enrichment analysis was conducted on differentially expressed genes between the ICAM1-DXd treatment group and the PBS and ICAM1-MMAE groups, revealing activation of immune-related pathways.

[0109] Next, flow cytometry was used to analyze immune cells in the tumor. The results showed that, compared with the blank control group (PBS), the ICAM1-DXd treatment group had a significantly increased CD8T cell infiltration and a significantly decreased Treg cell content. Figure 12 ICAM1-DXd can not only directly ablate tumor cells, but also improve prognosis by activating the tumor's immune microenvironment.

[0110] 8. ICAM1 DXd combined with immune checkpoint inhibitor PD1 combination therapy

[0111] Because ICAM1 DXd can regulate chemokines, transforming the immunosuppressive tumor microenvironment into an immune-active state, and can reduce tumor lymphatic vessels to prevent CD8 T cell spillover, this embodiment designed a combined immunotherapy model of ICAM1 DXd and PD1 antibody. In 4T1-hICAM1 tumor-bearing mice, ICAM1 DXd (5 mg / kg) was first administered via tail vein, followed by PD1 antibody (10 mg / kg), while the control group received phosphate-buffered saline (PBS). Notably, ICAM1-DXd combined with PD1 showed significant antitumor activity, significantly superior to other monotherapy treatments. Tumor weight analysis showed a significant difference compared to other groups (…). Figure 13 Throughout the treatment, there was no significant difference in body weight change among all mice. These findings confirm the antitumor activity of ICAM1 DXd in combination with a PD1 inhibitor.

[0112] Given the current lack of effective treatments for inflammatory breast cancer, this application rationally designs and develops two ADCs (ICAM1-DXd and ICAM1-MMAE) as potent candidate drugs for targeted therapy of inflammatory breast cancer. Compared with existing treatment regimens, this application first identifies ICAM1 as a potential cell membrane target for inflammatory breast cancer, which can be used to develop antibody-drug conjugates. Secondly, in vivo experiments demonstrate that cleavable DXd exerts a potent bystander-killing effect in vivo, producing a significant tumor cell killing effect. Finally, this application also found through transcriptome data analysis that ICAM1-DXd activates the immune microenvironment of inflammatory breast cancer, thereby exhibiting a more significant tumor-suppressive effect when combined with the immune checkpoint inhibitor PD1.

[0113] In this study, the expression level of ICAM1 was identified using inflammatory breast cancer cell lines, clinically relevant tumor tissues, and public databases. It was found that ICAM1 expression in inflammatory breast cancer was higher than that of many common cancer targets, such as HER2 and TROP2. Furthermore, ICAM1-specific expression was also observed in tumor-associated lymphoid vessels of inflammatory breast cancer. ICAM1-mediated ICAM1 antibody internalization was directly observed in inflammatory breast cancer cell lines and human lymphatic endothelial cells, leading to the release of cytotoxic substances into the cells.

[0114] This application designed and constructed two types of ICAM1-DXd, ICAM1-MMAE and ICAM1-DXd. MMAE and DXd are clinically used DXd warheads with different mechanisms of action. Both types of ICAM1 DXd have a bystander killing effect and can be selectively cleaved by cathepsin B in endosomes or lysosomes, thereby rapidly releasing the ADC warhead during cell internalization while maintaining the integrity of the ADC in the blood circulation.

[0115] Lymphatic vessels play an important role in inflammatory breast cancer. They are one of the main pathways for cancer cells to travel from the primary tumor site to lymph nodes and other parts of the body. The ICAM1-DXd developed in this application not only targets inflammatory breast cancer but also inhibits the formation of lymphatic vessels in the tumor, which helps improve the poor prognosis of IBC patients.

[0116] In summary, this application identifies ICAM1 as a potential therapeutic target for inflammatory breast cancer and rationally designs and develops two ICAM1-related ADCs: ICAM1-MMAE and ICAM1-DXd. Experimental data confirm that ICAM1-DXd can serve as the optimal ADC for targeted therapy of inflammatory breast cancer, inhibiting lymphangiogenesis, and also demonstrating synergistic therapeutic effects when combined with the immune checkpoint inhibitor PD-1. Finally, this research provides important evidence and perspectives for the development of ADC drugs for the treatment of inflammatory breast cancer.

[0117] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit this application. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of this application. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in this invention should still be covered by the claims of this application.

Claims

1. Use of the ICAM1 antibody-drug conjugate in the preparation of a product, including the product having at least one of the following effects: 1) Treatment of inflammatory breast cancer; 2) Inhibits lymphangiogenesis; 3) Improve the efficacy of immune checkpoint inhibitor therapy for inflammatory breast cancer.

2. The use as described in claim 1, characterized in that, The drug is conjugated to the ICAM1 antibody via a linker to form an ICAM1 antibody-drug conjugate. And / or, the drug in the ICAM1 antibody-drug conjugate is selected from monomethylaurestatin E, monomethylaurestatin F, compuratin A, cytarabine, gemcitabine, maytansine, DM1, DM4, cazithromycin and its derivatives, doxorubicin, pyrrolobenzodiazepines and their derivatives, SN-38, camptothecin analogs, α-ammantin, and tubulin lysin analogs; preferably, the drug is selected from MMAE or DXd; And / or, the linker is Mc-VC-PAB and / or Mc-GGFG.

3. The use as described in claim 2, characterized in that, The ICAM1 antibody is conjugated to four or eight drugs; Preferably, the ICAM1 antibody-drug conjugate is: ICAMI conjugated to MMAE, with a drug-to-antibody ratio of 4, and the linker being Mc-VC-PAB; or ICAMI conjugated to Dxd, with a drug-to-antibody ratio of 8, and the linker being Mc-GGFG.

4. The use as described in claim 1, characterized in that, The immune checkpoint inhibitor is selected from one or more combinations of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors; preferably, the immune checkpoint inhibitor is a PD-1 inhibitor.

5. An antibody-drug conjugate for treating inflammatory breast cancer, characterized in that, This includes drugs that have a cytotoxic effect, which are conjugated to ICAM1 antibodies via a linker to form antibody-drug conjugates.

6. The antibody conjugate as described in claim 5, characterized in that, The drug is selected from monomethylaurestatin E, monomethylaurestatin F, compuratin A, cytarabine, gemcitabine, maytansine, DM1, DM4, chachiin and its derivatives, doxorubicin, pyrrolobenzodiazepines and their derivatives, SN-38, camptothecin analogs, α-ammantin, and tubulin lysin analogs; preferably, the drug is selected from DXd; And / or, the linker is Mc-GGFG.

7. The antibody conjugate as described in claim 6, characterized in that, The antibody conjugate is ICAMI conjugated with Dxd, the drug-to-antibody ratio is 8, and the linker is Mc-GGFG.

8. A pharmaceutical composition comprising an effective amount of an immune checkpoint inhibitor and an ICAM1 antibody-drug conjugate as used in any one of claims 1 to 4.

9. The pharmaceutical composition of claim 8, characterized in that, The immune checkpoint inhibitor is selected from one or more combinations of PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM-3 inhibitors, LAG3 inhibitors, and TIGIT inhibitors; preferably, the immune checkpoint inhibitor is a PD-1 inhibitor.

10. Use of the pharmaceutical composition according to any one of claims 8 to 9 in the preparation of a product for treating inflammatory breast cancer.

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