Use of IMMU-132 in combination with IACS010759 in the manufacture of a medicament for treating esophageal squamous cell carcinoma and related medicaments

Abstract

The application discloses application of IMMU-132 and IACS010759 in preparation of an anti-esophageal squamous carcinoma drug and related drugs, relates to the technical field of biological medicine, and the combined drug comprises IMMU-132 and IACS010759. The IMMU-132 and IACS010759 are synergistically combined, PI3K-AKT-mTOR signal pathways are inhibited, and thus, ESCC cell proliferation is inhibited; the combined drug increases the intracellular ROS level and destroys the mitochondrial membrane potential, and thus, the apoptosis of cells in the mitochondrial pathway is synergistically induced. The IMMU-132 and IACS010759 provided by the application exhibit a significant synergistic anti-tumor effect, the PI3K-AKT-mTOR survival signal pathway is inhibited, and thus, the apoptosis of cancer cells is triggered, and the problems of limited curative effect and easy drug resistance of an existing ESCC treatment scheme are reduced, so that a new strategy is provided for clinic.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to the application of IMMU-132 in combination with IACS010759 in the preparation of drugs for treating esophageal squamous cell carcinoma and related drugs. Background Technology

[0002] Esophageal cancer is one of the most common malignant tumors worldwide, and it is divided into esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC). Current standard treatments for ESCC include surgery, radiotherapy, and chemotherapy (such as platinum-based regimens). However, most patients experience recurrence or develop drug resistance, resulting in extremely poor prognosis and a low five-year survival rate. As tumor cells adapt to their microenvironment and rapid growth patterns, their metabolic characteristics undergo numerous changes. Mitochondrial aerobic respiration and oxidative phosphorylation (OXPHOS) are the main processes maintaining the continuous proliferation of cancer cells, and these are most likely to influence the development of drug resistance in malignant tumors. Therefore, the development of novel and effective treatment strategies for ESCC is urgently needed.

[0003] Antibody-drug conjugates (ADCs) have been widely recognized in recent years as a highly effective strategy for treating solid tumors and hematologic malignancies. Their mechanism of action relies on the highly selective recognition of specific antigens on the surface of tumor cells by monoclonal antibodies. After internalization, these antibodies enter the cells and release highly potent cytotoxic drugs, thereby achieving precise killing of tumor cells. This "biological missile" design not only significantly enhances anti-tumor activity but also greatly reduces off-target toxicity to normal tissues, thus combining the synergistic advantages of targeted therapy and chemotherapy.

[0004] Trophoblast surface antigen-2 (TROP2) is a transmembrane glycoprotein, a type I surface antigen, and widely recognized as a pan-cancer target. It exhibits high expression in various solid tumors and is closely associated with poor patient prognosis and increased risk of metastasis. Therefore, TROP2 has become an important molecular target for research into the treatment of various human malignancies.

[0005] Sacituzumab Govitecan (IMMU-132) is an antibody-drug conjugate (ADC) targeting the TROP2 antigen and is approved for the treatment of triple-negative breast cancer and urothelial carcinoma. It works by partially targeting and delivering a highly potent topoisomerase I inhibitor, SN-38, into cancer cells, causing DNA damage. However, its efficacy and application in ESCC have not been fully explored, and monotherapy may eventually lead to drug resistance.

[0006] IACS010759 is a potent and selective inhibitor of mitochondrial respiratory chain complex I. It inhibits OXPHOS and blocks energy production, thus exhibiting a significant killing effect on OXPHOS-dependent cancer cells. It is currently in clinical trials for various solid tumors and hematological malignancies. Summary of the Invention

[0007] To address the aforementioned technical issues, this invention proposes the application of IMMU-132 and IACS010759 in the preparation of anti-esophageal squamous cell carcinoma drugs and related drugs. IMMU-132 and IACS010759 exhibit significant synergistic anti-tumor effects in ESCC models. The mechanism is not a simple additive effect, but rather a synergistic action that deeply inhibits the key PI3K-AKT-mTOR survival signaling pathway, thereby strongly triggering cancer cell apoptosis. This reduces the problems of limited efficacy and easy drug resistance in existing ESCC treatment regimens, achieving highly efficient and safe treatment for ESCC and providing a new strategy for clinical practice.

[0008] To achieve the above objectives, the present invention provides the application of formulations targeting the TROP2 pathway and / or the mitochondrial oxidative phosphorylation pathway in the preparation of antitumor drugs. The formulations targeting the TROP2 pathway are antibody-drug conjugates; the formulations targeting the mitochondrial oxidative phosphorylation pathway are mitochondrial respiratory chain complex I inhibitors.

[0009] Furthermore, the antibody-drug conjugate is IMMU-132, and the mitochondrial respiratory chain complex I inhibitor is IACS010759; the tumors include TROP2-positive esophageal cancer, high-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, higher neuroendocrine tumors, gastric cancer, colon cancer, endometrial cancer, ovarian cancer, cervical cancer, head and neck cancer, salivary cancer, thyroid cancer, breast cancer, bile duct cancer, pancreatic cancer, genitourinary cancer, or prostate cancer.

[0010] Furthermore, the present invention also provides the application of an antibody-drug conjugate targeting the TROP2 pathway combined with a mitochondrial respiratory chain complex I inhibitor targeting the mitochondrial oxidative phosphorylation pathway in the preparation of an antitumor drug, wherein the antibody-drug conjugate targeting the TROP2 pathway is IMMU-132 and the mitochondrial respiratory chain complex I inhibitor targeting the mitochondrial oxidative phosphorylation pathway is IACS010759.

[0011] Furthermore, tumors include TROP2-positive esophageal cancer, high-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, high-grade neuroendocrine tumors, gastric cancer, colon cancer, endometrial cancer, ovarian cancer, cervical cancer, head and neck cancer, salivary cancer, thyroid cancer, breast cancer, bile duct cancer, pancreatic cancer, genitourinary cancer, or prostate cancer.

[0012] Furthermore, when the antibody-drug conjugate IMMU-132 is used in combination with the mitochondrial respiratory chain complex I inhibitor IACS010759:

[0013] ①The synergistic combination of IMMU-132 and IACS010759 inhibits tumor cell proliferation by suppressing the PI3K-AKT-mTOR signaling pathway;

[0014] ②The synergistic combination of IMMU-132 and IACS010759 induces tumor cell apoptosis via the mitochondrial pathway by increasing intracellular ROS levels and disrupting mitochondrial membrane potential.

[0015] ③The synergistic combination of IMMU-132 and IACS010759 inhibits the invasion and metastasis of tumor cells.

[0016] Furthermore, when applying it, ① at the cellular level, the concentration of IMMU-132 is 0~10 μg / mL, and the concentration of IACS010759 is 0~10 μM; ② at the animal level, the concentration of IMMU-132 is 5~10 mg / kg, and the concentration of IACS010759 is 1.0~2.5 mg / kg.

[0017] Furthermore, the present invention also provides an antitumor drug, the active ingredients of which are an antibody-drug conjugate targeting the TROP2 pathway and a mitochondrial respiratory chain complex I inhibitor targeting the mitochondrial oxidative phosphorylation pathway.

[0018] Furthermore, the antitumor drug also includes pharmaceutically acceptable excipients or carriers.

[0019] Furthermore, tumors include TROP2-positive esophageal cancer, high-grade glioma, lung adenocarcinoma, lung squamous cell carcinoma, high-grade neuroendocrine tumors, gastric cancer, colon cancer, endometrial cancer, ovarian cancer, cervical cancer, head and neck cancer, salivary cancer, thyroid cancer, breast cancer, bile duct cancer, pancreatic cancer, genitourinary cancer, or prostate cancer.

[0020] Furthermore, at the cellular level, the concentration of IMMU-132 in the drug was 0-10 μg / mL, and the concentration of IACS010759 was 0-10 μM; at the animal level, the concentration of IMMU-132 in the drug was 5-10 mg / kg, and the concentration of IACS010759 was 1.0-2.5 mg / kg.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] (1) This invention is the first to combine IMMU132, which targets TROP2, with IACS010759. It has shown significant synergistic anti-tumor effects in in vitro cell models, three-dimensional organoid models and in vivo animal models. Its efficacy far exceeds that of single drugs, providing a new solution to the problem of insufficient efficacy or drug resistance of single drug treatment.

[0023] (2) Based on a large-scale clinical sample analysis (94.6% of esophageal squamous cell carcinoma patients were TROP2 positive), the combined strategy proposed in this invention has a broad potential application basis.

[0024] (3) This invention elucidates the unique molecular mechanism of combined drug use. It is not a simple additive effect, but rather induces strong oxidative stress, damages mitochondrial function, and synergistically inhibits the key PI3K-AKT-mTOR tumor survival signaling pathway, thereby triggering tumor cell apoptosis in multiple dimensions and at multiple levels, achieving deep fusion and synergy in mechanism.

[0025] (4) At effective therapeutic doses, the combined drug did not cause significant weight loss or functional and pathological damage to major organs such as liver and kidneys in mice, indicating that the combined regimen has excellent therapeutic window and safety characteristics, providing important safety evidence for clinical translation.

[0026] (5) This invention creatively combines the two strategies of “targeting tumor surface antigens” and “inhibiting tumor energy metabolism” to propose an innovative treatment model of “targeted delivery + energy interruption”. This not only provides a new direction for the treatment of esophageal squamous cell carcinoma, but also provides a reference for the combined treatment of other TROP2 positive solid tumors.

[0027] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0028] Figure 1 In Figure A, the percentage of TROP2 expression in 222 ESCC tumors in Example 1 is shown; in Figure B, the representative images of TROP2 immunohistochemistry (IHC) on human ESCC tumor tissue microarray (TMA) are shown, where ① is a high-expression sample, ② is a moderate-expression sample, ③ is a low-expression sample, and ④ is a TROP2-negative sample.

[0029] Figure 2 In Figure A, the expression level of TROP2 in ESCC cells KYSE30 was detected by immunofluorescence (IF); in Figure B, the expression level of TROP2 in ESCC cells KYSE150 was detected by immunofluorescence (IF).

[0030] Figure 3In Figure A, ESCC KYSE30 cells were treated with different concentrations of IMMU132, and the confluence of ESCC KYSE30 cells during treatment was observed using the IncuCyte® S3 real-time cell analysis system. In Figure B, ESCC KYSE30 cells were treated with different concentrations of IACS010759, and the confluence of ESCC KYSE30 cells during treatment was observed using the IncuCyte® S3 real-time cell analysis system. In Figure C, ESCC KYSE150 cells were treated with different concentrations of IMMU132, and the confluence of ESCC KYSE150 cells during treatment was observed using the IncuCyte® S3 real-time cell analysis system. In Figure D, ESCC KYSE150 cells were treated with different concentrations of IACS010759, and the confluence of ESCC KYSE150 cells during treatment was observed using the IncuCyte® S3 real-time cell analysis system.

[0031] Figure 4 Figure A shows the ATP production level of ESCC cells treated with IMMU132 alone, where ① represents KYSE30 cells and ② represents KYSE150 cells; Figure B shows the ATP production level of ESCC cells treated with IACS010759 alone, where ① represents KYSE30 cells and ② represents KYSE150 cells; *** indicates P<0.001.

[0032] Figure 5 In the middle section, A represents the optimal synergistic concentration screening of IMMI132 and IACS010759 in KYSE30 cells; B represents the optimal synergistic concentration screening of IMMI132 and IACS010759 in KYSE150 cells.

[0033] Figure 6 In Figure A, the proliferation of cells treated with IMMU132, IACS010759, or their COM at different times was detected using the IncuCyte® S3 real-time cell analysis system. ① represents KYSE30 cells, and ② represents KYSE150 cells. Figure B shows representative images of KYSE30 cells daily. Figure C shows representative images of KYSE150 cells daily. *** indicates P < 0.001.

[0034] Figure 7Image A shows the fluorescence graph of ROS levels in KYSE30 cells after treatment with IMMU132, IACS010759, or their COM by flow cytometry; Image B shows the fluorescence graph of ROS levels in KYSE150 cells after treatment with IMMU132, IACS010759, or their COM by flow cytometry; Image C shows the percentage of ROS levels in KYSE30 cells after treatment with IMMU132, IACS010759, or their COM by flow cytometry; Image D shows the percentage of ROS levels in KYSE150 cells after treatment with IMMU132, IACS010759, or their COM by flow cytometry. *** indicates P < 0.001.

[0035] Figure 8 Image A shows the detection of Caspase-3 activity and mitochondrial membrane potential in KYSE30 cells after treatment with IMMU132, IACS010759, or their COM. Increased Caspase-3 activity is indicated by green light, while decreased mitochondrial membrane potential is indicated by red light. Image B shows the detection of Caspase-3 activity and mitochondrial membrane potential in KYSE150 cells after treatment with IMMU132, IACS010759, or their COM. Increased Caspase-3 activity is indicated by green light, while decreased mitochondrial membrane potential is indicated by red light.

[0036] Figure 9 A shows the KEGG pathway analysis of DEGs in RNA-seq of KYSE30 cells after COM treatment; B shows the GSEA gene enrichment analysis of KYSE30 cells after COM treatment using the HALLMARK gene set database in MSigDB, where ① represents PI3K-AKT-mTOR-SIGNALING and ② represents mTORC1-SIGNALING; C shows the PI3K / AKT / mTOR signaling pathway-related proteins in KYSE30 and KYSE150 cells analyzed by Western blotting; D shows the proportion of PI3K / AKT / mTOR signaling pathway-related proteins in KYSE30 and KYSE150 cells compared to the control group, where ① represents KYSE30 cells and ② represents KYSE150 cells; where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001.

[0037] Figure 10Image A shows bright-field plots of ESCC-241-O and ESCC-291-O models; Image B shows representative HE staining images of the primary ESCC tumor, PDX, and their corresponding PDXO; Image C shows IHC staining of TROP2 expression in the primary ESCC tumor, PDX, and their corresponding PDXO; Image D shows ESCC-241-O treated with IMMU132 and IACS010759 alone or with COM for 0-144 hours, and organoid growth calculated using IncuCyte® S3 software; Image E shows ESCC-291-O treated with IMMU132 and IACS010759 alone or with COM for 0-144 hours, and organoid growth calculated using IncuCyte® S3 software; Image F shows PDXO treated with IMMU132 or IACS010759 alone or with COM for 144 hours. Bright-field image of ESCC-241-O; G is the bright-field image of ESCC-291-O processed by IMMU132 or IACS010759 alone or COM; where *** indicates P<0.001;

[0038] Figure 11 Figure A shows the experimental design, illustrating the treatment groups, dosages, and schedules. IMMU132 (red line, 10 mg / kg) was administered intravenously once weekly for 3 weeks; IACS010759 (green line, 2.5 mg / kg) was administered intravenously five times weekly for 4 weeks. Figure B shows the synergistic antitumor effect of IMMU132 and IACS010759 on tumor growth in the ESCC-241 PDX model; Figure C shows the synergistic antitumor effect of IMMU132 and IACS010759 on tumor growth in the ESCC-266 PDX model; Figure D shows the synergistic antitumor effect of IMMU132 and IACS010759 on tumor growth in the ESCC-291 PDX model; Figure E shows the Ki67 staining effect of ESCC-241; Figure F shows the Ki67 staining effect of ESCC-266; Figure G shows the Ki67 staining effect of ESCC-291; Figure H shows the Ki67 staining effect of ESCC-241. Ki67 positive expression level in Ki67-stained tumor tissue; I represents the Ki67 positive expression level in ESCC-266 Ki67-stained tumor tissue; J represents the Ki67 positive expression level in ESCC-291 Ki67-stained tumor tissue; where * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001;

[0039] Figure 12In the ESCC-241 PDX model, A represents the change in body weight of mice treated alone or in combination; B represents the change in body weight of mice treated alone or in combination in the ESCC-266 PDX model; C represents the change in body weight of mice treated alone or in combination in the ESCC-291 PDX model; D represents the serum ALT level, an indicator of liver and kidney function, after combined drug administration; E represents the serum AST level, an indicator of liver and kidney function, after combined drug administration; F represents the Crea level, an indicator of liver and kidney function, after combined drug administration; and G represents the BUN level, an indicator of liver and kidney function, after combined drug administration.

[0040] Figure 13 In Figure A, the pathological changes of each organ (heart, liver, spleen, lung, and kidney) in the ESCC-241 PDX model were evaluated using the H&E method; in Figure B, the pathological changes of each organ (heart, liver, spleen, lung, and kidney) in the ESCC-266 PDX model were evaluated using the H&E method; and in Figure C, the pathological changes of each organ (heart, liver, spleen, lung, and kidney) in the ESCC-291 PDX model were evaluated using the H&E method. Detailed Implementation

[0041] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0042] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0043] Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention. Experimental methods in the following embodiments that do not specify specific conditions are generally determined according to national standards. Experimental instruments, equipment, and reagents in the following embodiments that do not specify their sources are all commercially available materials.

[0044] Unless otherwise defined or stated, all technical and scientific terms used in this invention have the same meaning as those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of this invention. It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined with each other.

[0045] The abbreviation "COM" refers to the simultaneous administration of effective doses of two active ingredients in combination therapy to achieve a synergistic anti-tumor effect.

[0046] NOD / SCID female mice, 5-6 weeks old, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.

[0047] Example 1

[0048] Expression analysis of TROP2 in ESCC:

[0049] To detect the expression level of TROP2 in esophageal squamous cell carcinoma (ESCC) tissues, this invention collected 222 clinical ESCC samples, prepared them into tissue microarrays, and detected them using immunohistochemistry (IHC). Based on staining intensity, TROP2 protein expression was classified into four levels: negative, low expression, intermediate expression, and high expression. To further clarify the TROP2 expression pattern at the cellular level, immunofluorescence analysis was performed on two ESCC cell lines, KYSE30 and KYSE150.

[0050] Statistical analysis of IHC results from 222 samples and plotting an expression distribution pie chart revealed the following: 12 samples (5.4%) showed negative TROP2 expression; 85 samples (38.3%) showed low expression; 65 samples (29.3%) showed moderate expression; and 60 samples (27.0%) showed high expression. The overall TROP2 expression rate in ESCC was 94.6%. Figure 1 Immunofluorescence results showed that TROP2 was highly expressed in the above cell lines and specifically localized in the cell membrane and cytoplasm. Figure 2 ).

[0051] The above results support the IHC results from cell experiments and clinical tissue samples, systematically demonstrating the widespread high expression of TROP2 in ESCC. This indicates that TROP2 is a key factor involved in the development and progression of ESCC and has significant clinical diagnostic and targeted therapeutic value.

[0052] Example 2

[0053] In vitro antitumor effects of IMMU132 and IACS010759 as single drugs:

[0054] To evaluate the in vitro antitumor effects of IMMU132 and IACS010759, IMMU132 concentrations were set at 100 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0.01 μg / mL, and 0 μg / mL; and IACS010759 concentrations were set at 10 μM, 1 μM, 0.1 μM, 10 μM, 1 μM, and 0 μM. KYSE30 and KYSE150 cells were treated for 72 h, and the effects of the two drugs on the proliferation of esophageal squamous cell carcinoma (ESCC) cell lines were analyzed using the IncuCyte® S3 live-cell imaging system. In addition, to investigate the effects of drugs on tumor cell energy metabolism, the effects of IMMU132 and IACS010759 alone on mitochondrial ATP production in ESCC cells were verified. The concentrations of IMMU132 were 10 μg / mL, 1 μg / mL, and 0 μg / mL; the concentrations of IACS010759 were 10 μM, 1 μM, and 0 μM. Cells were seeded in 6-well plates and incubated with the drugs for 48 h. ATP levels were then measured according to the instructions of the ATP assay kit.

[0055] The results showed that within a 72-hour dosing cycle, both IMMU132 and IACS010759 significantly inhibited the growth of both ESCC cell lines in a dose- and time-dependent manner. Compared with the control group (0 μg / mL or 0 μM), the cell proliferation curves of each treatment group showed a clear concentration gradient inhibition effect, indicating that both drugs have significant in vitro antitumor activity. Figure 3 Experiments showed that IACS010759 strongly inhibited ATP synthesis in ESCC cells, demonstrating its expected effect as an inhibitor of mitochondrial respiratory chain complex I. IMMU132 also showed a significant reduction in intracellular ATP levels at different concentrations, suggesting that this antibody-drug conjugate, in addition to its direct killing effect, also has an inhibitory effect on the energy metabolism of tumor cells. Figure 4 ).

[0056] Example 3

[0057] Screening for optimal concentrations of IMMU132 and IACS010759 for synergistic antitumor effects:

[0058] To screen the optimal concentrations for synergistic antitumor effects of combined administration, KYSE30 and KYSE150 cells were used as models, and a concentration gradient of IMMU132 (0, 2, 4, 6, 8, and 10 μg / mL) and IACS010759 (0, 2, 4, 6, 8, and 10 μM) was established for combined administration. Cells were seeded in 96-well plates and incubated with IMMU132 and / or IACS010759 for 72 h. Cell viability was then assessed using the Cell Titer-Glo® luminescent cell viability assay kit. The interaction properties were quantitatively evaluated using the Co-action Index (CDI) model.

[0059] The interaction coefficient (CDI) for the combined treatment of IMMU132 and IACS010759 was calculated using the following formula:

[0060] CDI = AB / A × B;

[0061] The results are calculated based on the number of viable cells (absorbance value). AB is the ratio of the combined drug treatment group to the control group, A is the ratio of IMMU132 to the control group, and B is the ratio of IACS010759 to the control group. If CDI < 1, it indicates that the two drugs have a synergistic effect; if CDI < 0.7, the synergistic effect is very significant. If CDI = 1, the effect of the two drugs is additive; if CDI > 1, the effect of the two drugs is antagonistic.

[0062] The results show that ( Figure 5 At all tested concentration combinations, the CDI value of the combined drug was less than 1, indicating that IMMU132 and IACS010759 have a broad synergistic effect in inhibiting ESCC cell proliferation. Notably, the strength of the synergistic effect was concentration-dependent. The combination of 6 μg / mL IMMU132 and 6 μM IACS010759 showed the most significant synergistic antitumor effect, with the lowest CDI value among all combinations, suggesting that this concentration ratio is the optimal synergistic dosing regimen for subsequent in vitro and in vivo experiments.

[0063] Example 4

[0064] IMMU132 and IACS010759 jointly inhibit ESCC cell proliferation:

[0065] To dynamically verify the antiproliferative effect of the optimized concentration combination, ESCC cells were seeded in 96-well plates and cultured for 12 h, then treated with 6 μg / mL IMMU132 and 6 μM IACS010759, alone or in combination, for 72 h. During drug treatment, the process was continuously monitored using the IncuCyte® S3 live-cell imaging system, with images acquired every 2 h. Data analysis of cell confluence formation curves was performed using IncuCyte® S3 software.

[0066] Compared with the control group, both 6 μg / mL IMMU132 and 6 μM IACS010759 alone significantly inhibited the proliferation of KYSE30 and KYSE150 cells. However, the combined treatment with the two drugs showed the strongest inhibitory effect on cell growth, and its cell confluence curve was significantly lower than that of each single-drug group. Figure 6 Representative microscopic images at different time points (0, 24, 48, 72h) (A). Figure 6 Figures B and C visually demonstrate the difference in the degree of inhibition. With prolonged treatment time, the cell number growth in the combined treatment group almost ceased, indicating that IMMU132 and IACS010759 produced a significant synergistic effect in inhibiting ESCC cell proliferation. This result is consistent with the aforementioned CDI analysis conclusions.

[0067] Example 5

[0068] IMMU132 and IACS010759 induce apoptosis by disrupting mitochondrial membrane potential through increased reactive oxygen species (ROS) levels.

[0069] To further explore the molecular mechanism of drug synergy, the effects of combined treatment on intracellular ROS levels, mitochondrial membrane potential, and apoptosis were investigated. ESCC cells were seeded in 6-well plates and incubated with the drugs for 48 h. Then, the DCFH-DA probe was added to each treatment group, and the cells were incubated at 37°C in the dark for 20 min. After incubation, the cells were washed 1-2 times with pre-warmed PBS to remove any probe that had not entered the cells. The cells were then collected in flow cytometry tubes, and ROS levels were detected using flow cytometry. For the mitochondrial membrane potential assay, after apoptosis induction, cells cultured in 12-well plates were centrifuged at 1000×g for 5 min. The cell culture medium was aspirated, and the cells were washed once with PBS. 300 μl of detection buffer was added and gently mixed. The cells were incubated at room temperature in the dark for 30 min. The cells were then observed under a fluorescence microscope.

[0070] Flow cytometry analysis showed that, compared with the single-treatment group, the combination therapy synergistically increased the ROS levels of both cell lines. Figure 7The results of Caspase-3 activity and mitochondrial membrane potential analysis showed that IMMU132 and IACS01059, respectively, induced apoptosis, increased Caspase-3 enzyme activity (green light), and decreased mitochondrial membrane potential (red light). On the other hand, the combined treatment group showed more pronounced effects in KYSE30 and KYSE150 cells. Figure 8 Therefore, the combined use of these therapies synergistically increases ROS accumulation, thereby disrupting mitochondrial membrane potential and ultimately leading to the activation of Caspase-3-dependent apoptosis, which may be one of the key mechanisms by which they exert their significant anti-tumor effects.

[0071] Example 6

[0072] The combination therapy of IMMU132 and IACS010759 significantly induced mitochondrial apoptosis through the PI3K-AKT-mTOR pathway:

[0073] To further explore the intrinsic molecular mechanism of drug action, KYSE30 cells were selected for transcriptomic analysis. Four groups were established: Control, IMMU132, IACS010759, and COM, each containing three replicates. After 24 hours of drug treatment, changes in total RNA were analyzed using high-throughput whole transcriptome sequencing (RNA-seq). The differential gene thresholds were Padj ≤ 0.05 and |log2fold change| ≥ 0.26. Differential gene enrichment analysis was performed using the Genome Encyclopedia (KEGG). Additionally, gene set enrichment analysis (GSEA) was performed using the Molecular Characteristic Database (MSigDB). Cells were then treated with 6 μg / mL IMMU132 and 6 μM IACS010759, alone or in combination, for 24 hours, and proteins were extracted. Proteins were separated by SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. The membrane was incubated with 5% skim milk to block non-specific binding. The primary antibody was incubated overnight at 4°C, and then bound to the primary antibody-specific enzyme-labeled secondary antibody at room temperature. Finally, protein bands were visualized using ECL reagent, and the signals were captured using the Bio-Rad multifunctional chemiluminescence imaging system.

[0074] KEGG pathway enrichment analysis showed that differentially expressed genes were significantly enriched in multiple key signaling pathways, such as PI3K-AKT-mTOR, p53, MAPK, FOXO, apoptosis, cell cycle, and AMPK pathways. Figure 9 (A); GSEA analysis confirmed that the PI3K-AKT-MTOR-SIGNALING and MTORC1-SIGNALING gene sets were strongly suppressed ( Figure 9(Middle B), suggesting that this pathway is one of the core targets of the combination therapy; to validate at the protein level, Western blot was used to detect the expression of key proteins in the PI3K-AKT-mTOR pathway in KYSE30 and KYSE150 cells. The results were highly consistent with the transcriptomic data, and the combination therapy significantly downregulated the phosphorylation level of key proteins in this pathway ( Figure 9 (C, D)

[0075] These results, from both genomic and proteomic perspectives, confirm that the combined treatment of IMMU132 and IACS010759 synergistically inhibits the activity of the PI3K-AKT-mTOR signaling pathway. Combined with previous findings on mitochondrial dysfunction, the conclusion is that this combined treatment powerfully triggers apoptosis via the mitochondrial pathway by disrupting cellular energy metabolism and blocking key survival signals.

[0076] Example 7

[0077] IMMU132 and IACS010759 synergistically suppress the growth of ESCC PDXOs models:

[0078] To validate the synergistic efficacy of drugs in a more clinically similar model, ESCC organoids were extracted from ESCC PDX xenograft tissue and prepared using an ESCC organoid kit according to standard procedures. 50 μL of Matrigel suspension was added to each well of a 24-well plate, and the plates were incubated at 37°C for 10 min. Then, 500 μL of intact culture medium was added to each well for polymerization. The organoids were cultured in a humidified incubator containing 5% CO2 at 37°C. After the organoids reached a certain size, some were collected, fixed, dehydrated, embedded, and prepared into paraffin sections to examine whether the PDXO model reproduced the biological structure of the PDX model. To assess drug sensitivity, the organoids were digested, resuspended in Matrigel, and seeded in 96-well plates with 150 μL of culture medium (containing 3 μL of Matrigel). After 24 h of culture, the plates were treated with IMMU-132, IACS010759, or a combination of drugs for 6 days. Organoid viability was monitored in real time using the IncuCyte® S3 live cell analysis system.

[0079] Two ESCC PDXO models were constructed from the corresponding PDX tumors. The two PDXO models showed different growth morphologies. Figure 10 (A). H&E staining and immunohistochemical (IHC) analysis confirmed that the PDXO model effectively preserved the histological heterogeneity of the primary tumor and PDX tissue. Figure 10 (Middle B). More importantly, IHC results showed that TROP2 was overexpressed in PDXO, and its subcellular localization was consistent with that of the primary tumor and PDX tissue. Figure 10(C) This indicates that the PDXO model is a reliable platform for evaluating the efficacy of IMMU132 targeting TROP2. Based on validating the model's reliability, the inhibitory effect of the drug on PDXO growth was assessed using a live-cell imaging system. The results showed that, over a treatment period of up to 144 hours, compared to the single-drug treatment group, the combined treatment with IMMU132 and IACS010759 produced a stronger and time-dependent synergistic inhibitory effect on the growth of both PDXO models. Figure 10 (DG). This result further confirms the significant antitumor activity of the two drugs in combination at the three-dimensional organoid level.

[0080] Example 8

[0081] IMMU132 combined with IACS010759 can enhance the antitumor activity of ESCC PDX:

[0082] ESCC xenograft tumor tissue was cut into 2-3 mm pieces. 3 A tumor was implanted on the right side of the mouse. The tumor was allowed to grow to an average size of approximately 100-150 mm. 3 Mice bearing tumors were randomly divided into four groups (n=4-5): a treatment group, a 10 mg / kg IMMU group, a 2.5 mg / kg IACS group, and a COM group (10 mg / kg IMMU + 2.5 mg / kg IACS). IMMU132 was administered intravenously once a week for three weeks; IACS010759 was administered orally five days a week for four weeks. Tumor size and body weight were measured twice weekly using calipers, and tumor volume was calculated using the formula: Tumor volume (mm3) = length × (width)² × 0.5. The tumor growth inhibition (TGI) rate was calculated as: (1 - treatment tumor volume / control tumor volume) × 100%. Mice were euthanized after 29 days of treatment. All ESCC PDX xenograft tumors and major organs were collected for subsequent molecular and pathological analysis. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (Cr) levels were measured to assess liver and kidney function.

[0083] To evaluate the in vivo efficacy of IMMU132 and IACS010759, pharmacodynamic studies were conducted in three different ESCC patient-derived xenograft (PDX) models (ESCC-241, -266, -291). Figure 11 (A) For example Figure 11As shown in the BD results, combination therapy demonstrated superior efficacy compared to either monotherapy in all three models. Particularly in the ESCC-266 model, combination therapy induced significant synergistic antitumor activity, with a tumor growth inhibition rate (TGI) as high as 94.20%, significantly higher than IMMU132 monotherapy (79.62%) and IACS010759 monotherapy (61.77%). In the ESCC-291 model, combination therapy also showed superior efficacy (TGI=73.37%), far exceeding the monotherapy group. Even in the relatively mild ESCC-241 model, the TGI of combination therapy (61.29%) was higher than that of the monotherapy group. Immunohistochemical analysis further confirmed that combination therapy more effectively reduced Ki67 positive expression levels in tumor tissue, indicating that it significantly inhibited tumor cell proliferation. Figure 11 (Zhong EG).

[0084] In terms of safety, the combination therapy regimen demonstrated good tolerability. During the entire 29-day dosing period, no significant decrease in body weight was observed in any group of mice. Figure 12 (AC). After treatment, the serum biochemical indicators (ALT, AST, BUN, Cr) of mice were all within the normal range. Figure 12 The patient underwent DG treatment, and pathological examinations of the major organs (heart, liver, spleen, lungs, and kidneys) revealed no significant damage. Figure 13 The results (including the presence of IMMU132 and IACS010759) indicate that the combination therapy did not produce observable toxicity to liver, kidney function, or major tissues. In vivo experimental results demonstrate that the combination therapy of IMMU132 and IACS010759 synergistically and effectively inhibited tumor growth in various ESCC PDX models, with good safety profiles, providing strong preclinical evidence for the clinical translation of this combination strategy.

[0085] In summary, this invention proposes a novel strategy for treating esophageal squamous cell carcinoma (ESCC) by combining the TROP2-targeting antibody-drug conjugate IMMU132 with the oxidative phosphorylation inhibitor IACS010759. Clinical relevance analysis showed a TROP2 positivity rate as high as 94.6% in ESCC patient samples, providing a basis for its use as a therapeutic target. Systematic preclinical studies have confirmed the superiority of this combination regimen. In vitro, IMMU132 and IACS010759 synergistically inhibited the proliferation of ESCC cell lines in a dose-dependent manner. In three-dimensional organoids and in vivo PDX models, the combination therapy also demonstrated significantly superior synergistic antitumor activity compared to single-agent therapy, with good safety profile. At the mechanism of action level, the synergistic effect of the combination therapy stems from its precise multi-level attack on tumor cells: firstly, it synergistically induces mitochondrial dysfunction, manifested as a sharp increase in ROS levels and damage to mitochondrial membrane potential; secondly, transcriptomic and protein level validations both indicate that the combination therapy significantly inhibits the key PI3K-AKT-mTOR cell survival signaling pathway; ultimately, these effects collectively lead to tumor cell death. In summary, this invention elucidates a novel therapeutic paradigm for overcoming ESCC by targeting TROP2 and synergistically inhibiting oxidative phosphorylation. This strategy not only provides in-depth molecular insights into the synergistic effects of drugs but also holds promise for offering new clinical treatment options for patients with TROP2-positive esophageal squamous cell carcinoma.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. The application of IMMU-132 in combination with IACS010759 in the preparation of antitumor drugs, characterized in that, The tumor is a TROP2-positive esophageal cancer; When applying, ① at the cellular level, the concentration of IMMU-132 is 2~10 μg / mL and the concentration of IACS010759 is 2~10 μM; ② at the animal level, the concentration of IMMU-132 is 10 mg / kg and the concentration of IACS010759 is 2.5 mg / kg.

2. The application according to claim 1, characterized in that, ①The synergistic combination of IMMU-132 and IACS010759 inhibits tumor cell proliferation by suppressing the PI3K-AKT-mTOR signaling pathway; ②The synergistic combination of IMMU-132 and IACS010759 induces tumor cell apoptosis via the mitochondrial pathway by increasing intracellular ROS levels and disrupting mitochondrial membrane potential. ③The synergistic combination of IMMU-132 and IACS010759 inhibits the invasion and metastasis of tumor cells.

3. An antitumor drug, characterized in that, The active ingredients of the drug are the antibody-drug conjugate IMMU-132 targeting the TROP2 pathway and the mitochondrial respiratory chain complex I inhibitor IACS010759 targeting the mitochondrial oxidative phosphorylation pathway. ① At the cellular level, the concentration of IMMU-132 in the drug was 2~10 μg / mL, and the concentration of IACS010759 was 2~10 μM; ② At the animal level, the concentration of IMMU-132 in the drug was 10 mg / kg, and the concentration of IACS010759 was 2.5 mg / kg.

4. The antitumor drug according to claim 3, characterized in that, The antitumor drugs also include pharmaceutically acceptable excipients.

5. The antitumor drug according to claim 3, characterized in that, The tumor is an esophageal cancer that expresses TROP2 positively.

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