Use of IMMU-132 in combination with GSK2606414 in the manufacture of a medicament for treating colorectal cancer and related medicaments

Abstract

The application discloses application of IMMU-132 and GSK2606414 in combination in preparation of an anti-colorectal cancer drug and related drugs, and belongs to the technical field of biological medicines. IMMU-132 and GSK2606414 are synergistically combined, PERK-eIF2alpha signal axis is down-regulated, WNT signal pathway activity is inhibited, and thus tumor cell growth and metastasis are prevented. The application proposes that IMMU-132 and GSK2606414 are synergistic, double regulation of UPR signal pathways and WNT signal pathways is achieved, PERK-eIF2alpha signal axis is regulated, WNT signal pathway activity is inhibited, and a significant synergistic anti-tumor effect is exhibited, so that a new strategy direction is provided for colorectal cancer treatment, and a treatment selection with clinical transformation potential is provided for TROP2 positive colorectal cancer patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, and particularly relates to the application of IMMU-132 and GSK2606414 in the preparation of anti-colorectal cancer drugs and related drugs. Background Technology

[0002] Colorectal cancer is one of the leading causes of cancer-related deaths worldwide, posing significant challenges to its clinical treatment. Currently, the main treatment methods include surgery, radiotherapy, chemotherapy, and molecular targeted therapy (such as anti-VEGF / EGFR drugs) and immune checkpoint inhibitors. Although fluoropyrimidine-based chemotherapy regimens (such as FOLFOX and FOLFIRI) combined with targeted drugs have become the standard therapy for metastatic colorectal cancer, many patients still face limited treatment response rates, drug resistance, and tumor recurrence and metastasis. The efficacy of existing drugs remains unsatisfactory, especially for advanced-stage patients, where prognostic improvement is still insufficient. Therefore, developing effective therapeutic drugs targeting new targets and possessing novel mechanisms remains a crucial issue that urgently needs to be addressed in current clinical research on colorectal cancer.

[0003] Antibody-drug conjugates (ADCs) link highly active cytotoxic drugs to targeting antibodies via linkers, forming biological missiles that specifically target tumors. This approach achieves potent cytotoxicity while reducing systemic toxicity, making it a crucial direction in cancer treatment. Currently, over ten ADC drugs have been approved globally for treating various malignant tumors, including leukemia, lymphoma, and breast cancer. However, no ADC drugs have been successfully marketed for colorectal cancer treatment, and existing ADC drug target development suffers from significant homogeneity. Therefore, exploring novel ADC targets and corresponding drugs with novel mechanisms of action for colorectal cancer, a prevalent malignant tumor, is of great significance for overcoming current treatment bottlenecks and meeting urgent clinical needs.

[0004] Trophoblast surface antigen 2 (TROP2) is a transmembrane glycoprotein highly expressed in various epithelial tumors. Its overexpression is closely related to tumor progression, invasion, and poor prognosis, making it an important pan-cancer target. TROP2-ADC (IMMU-132) has been approved for the treatment of triple-negative breast cancer and urothelial carcinoma, exerting a potent anti-tumor effect by targeting and delivering the topoisomerase I inhibitor SN-38 to tumor cells. However, the potential of IMMU-132 in the treatment of colorectal cancer has not been fully explored, and its specific mechanism of action remains to be elucidated. Furthermore, drug resistance issues arising from monotherapy also limit further improvement in its clinical efficacy. Therefore, in-depth exploration of the therapeutic effects and molecular mechanisms of IMMU-132 in colorectal cancer is of great significance for expanding its indications and optimizing treatment strategies.

[0005] The unfolded protein response (UPR) is a core signaling network in which cells respond to internal and external stresses and maintain protein homeostasis. Its sustained activation is closely related to tumor progression and treatment tolerance. Within the key branches of the UPR, activation of the PERK-eIF2α-ATF4 signaling axis plays a central role in regulating cellular stress adaptation and fate determination. Existing research indicates that intervening in the PERK-eIF2α-ATF4 signaling axis can effectively enhance the sensitivity of cancer cells to chemotherapy and targeted drugs, and has become a potential strategy for improving anti-tumor efficacy. However, the specific role and regulatory mechanism of this signaling axis in the response of colorectal cancer to the TROP2-ADC drug IMMU-132 remain unclear. Summary of the Invention

[0006] To address the aforementioned technical issues, this invention proposes the application of the combined use of IMMU-132 and GSK2606414 in the preparation of anti-colorectal cancer drugs and related drugs. The TROP2-ADC drug IMMU-132 can inhibit tumor growth in the treatment of colorectal cancer by regulating the unfolded protein response and inhibiting the PERK-eIF2α-ATF4 signaling axis. Furthermore, the combined use of IMMU-132 and the PERK inhibitor GSK2606414 was verified. The two drugs showed significant synergistic anti-tumor effects in in vivo and in vitro models, providing a new strategic direction for the treatment of colorectal cancer.

[0007] To achieve the above objectives, the present invention provides the application of formulations targeting TROP2 positive expression and / or the PERK signaling pathway in the preparation of anti-colorectal cancer agents, wherein the formulation targeting TROP2 is an antibody-drug conjugate; and the formulation targeting the PERK signaling pathway is a PERK inhibitor.

[0008] Furthermore, the antibody-drug conjugate is IMMU-132, and the PERK inhibitor is GSK2606414; the 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.

[0009] Furthermore, the present invention also provides the application of an antibody-drug conjugate targeting the TROP2 pathway combined with a PERK inhibitor targeting the PERK signaling pathway in the preparation of an antitumor drug. The antibody-drug conjugate targeting the TROP2 pathway is IMMU-132, and the PERK inhibitor targeting the PERK signaling pathway is GSK2606414.

[0010] 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.

[0011] Furthermore, the synergistic combination of IMMU-132 and GSK2606414 inhibits tumor cell growth and metastasis by downregulating the PERK-eIF2α signaling axis and suppressing the activity of the WNT signaling pathway.

[0012] Furthermore, at the cellular level, the concentration of IMMU-132 was 0-10 μg / mL, and the concentration of GSK2606414 was 0-10 μM; at the animal level, the concentration of IMMU-132 was 2.5-5 mg / kg, and the concentration of GSK2606414 was 50 mg / kg.

[0013] Furthermore, at the cellular level, the concentration of IMMU-132 was 3 μg / mL and the concentration of GSK2606414 was 3 μM; at the animal level, the concentration of IMMU-132 was 2.5 mg / kg or 5 mg / kg and the concentration of GSK2606414 was 50 mg / kg.

[0014] Furthermore, the present invention also provides an anti-tumor drug, the active ingredients of which are an antibody-drug conjugate targeting TROP2 and a PERK inhibitor targeting the PERK signaling pathway.

[0015] Furthermore, antitumor drugs also include pharmaceutically acceptable excipients or carriers.

[0016] 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.

[0017] Furthermore, at the cellular level, the concentration of IMMU-132 was 0~10 μg / mL and the concentration of GSK2606414 was 0~10 μM; at the animal level, the concentration of IMMU-132 was 2.5~5 mg / kg and the concentration of GSK2606414 was 50 mg / kg.

[0018] Furthermore, at the cellular level, the concentration of IMMU-132 was 3 μg / mL and the concentration of GSK2606414 was 3 μM; at the animal level, the concentration of IMMU-132 was 2.5 mg / kg or 5 mg / kg and the concentration of GSK2606414 was 50 mg / kg.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] (1) This invention is the first to discover that the combined use of IMMU-132 and GSK2606414 produces a synergistic effect in the treatment of colorectal cancer, and its anti-tumor effect is significantly better than that of single drug treatment, providing a new and effective strategy for the treatment of this disease;

[0021] (2) The combined treatment of the present invention achieves the effect of multi-target synergistic inhibition of tumor growth by dual regulation of the unfolded protein response (UPR) pathway and the WNT signaling pathway, which surpasses the simple superposition of drug effects;

[0022] (3) Based on the high expression rate of TROP2 in colorectal cancer (89.05%), the combination scheme of the present invention has a wide potential user base;

[0023] (4) The present invention, at a dose that effectively inhibits tumor growth, did not cause significant toxic side effects when used in combination, demonstrating good safety characteristics; at the same time, the multi-level experimental verification system from cell lines, organoids to PDX models provides a solid preclinical basis for the clinical translation of this combination therapy strategy.

[0024] (5) This invention creatively combines targeted therapy with UPR signal axis modulation, providing new ideas and directions for the treatment of colorectal cancer. It not only provides a new and effective treatment plan for colorectal cancer, but also provides a useful reference for the combined treatment of other TROP2 positive tumors.

[0025] 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

[0026] Figure 1 Figure A is a box plot based on the GEPIA database analysis, showing the difference in TROP2 expression between colorectal cancer tissues and normal tissues. ① represents colon adenocarcinoma tissues versus normal tissues, and ② represents rectal adenocarcinoma tissues versus normal tissues. A indicates P < 0.05; B is a survival curve based on the Kaplan-Meier Plotter database, showing the association between TROP2 expression level and prognosis in colorectal cancer patients; C is a representative immunohistochemical (IHC) image of TROP2 protein expression in colorectal cancer tissues, based on a tissue microarray (TMA) containing 539 samples, showing the distribution of TROP2 expression intensity, 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; D is a Western blot image of TROP2 protein expression in DLD-1, CX-1, COLO205, and HCT15 colorectal cancer cell lines, with GAPDH as an internal control; E is the result of detecting TROP2 expression level in four colorectal cancer cell lines by immunofluorescence (IF), where ① is DLD-1 cells, ② is CX-1 cells, ③ is COLO205 cells, and ④ is HCT15 cells;

[0027] Figure 2 Figure A shows the cell survival rate of DLD-1 colorectal cancer cells after 72 hours of treatment with different concentrations of IMMU-132, Capecitabine, or Oxaliplatin; Figure B shows the cell survival rate of CX-1 colorectal cancer cells after 72 hours of treatment with different concentrations of IMMU-132, Capecitabine, or Oxaliplatin; Figure C shows the cell survival rate of COLO205 colorectal cancer cells after 72 hours of treatment with different concentrations of IMMU-132, Capecitabine, or Oxaliplatin; Figure D shows the cell survival rate of HCT15 colorectal cancer cells after 72 hours of treatment with different concentrations of IMMU-132, Capecitabine, or Oxaliplatin.

[0028] Figure 3 The image shows the results of TROP2 expression in four colorectal cancer PDX model xenografts detected by IHC. ① is the CRC036 PDX model, ② is the CRC082 PDX model, ③ is the CRC083 PDX model, and ④ is the CRC196 PDX model.

[0029] Figure 4 Image A shows the tumor growth curve of the xenograft of the colorectal cancer CRC036 PDX model after IMMU-132 treatment; Image B shows the tumor growth curve of the xenograft of the colorectal cancer CRC082 PDX model after IMMU-132 treatment; Image C shows the tumor growth curve of the xenograft of the colorectal cancer CRC083 PDX model after IMMU-132 treatment; Image D shows the tumor growth curve of the xenograft of the colorectal cancer CRC196 PDX model after IMMU-132 treatment. This indicates that P < 0.05;

[0030] Figure 5 The figure shows the results of GSEA-based analysis, illustrating the enrichment of IMMU-132 in the unfolded protein response signaling pathway.

[0031] Figure 6 Western blot images of PERK, eIF2α and ATF4 protein expression in colorectal cancer cells after IMMU-132 treatment, with GAPDH as internal control. Among them, ① are CX-1 cells, ② are DLD-1 cells, ③ are HCT15 cells, and ④ are COLO205 cells.

[0032] Figure 7 Figure A shows the cell survival rate of colorectal cancer cells in CX-1 cells after treatment with different concentrations of IMMU-132, the PERK inhibitor GSK2606414, and their combination for 72 hours; Figure B shows the cell survival rate of colorectal cancer cells in DLD-1 cells after treatment with different concentrations of IMMU-132, the PERK inhibitor GSK2606414, and their combination for 72 hours.

[0033] Figure 8 Figure A shows the results of KEGG pathway enrichment analysis, indicating the signaling pathways enriched by differentially expressed genes after combined drug treatment; Figure B shows the results of GSEA analysis, indicating the enrichment of differentially expressed genes in the WNT signaling pathway and Focal adhesion pathway after combined drug treatment, where ① represents the WNT pathway and ② represents the Focal adhesion pathway; Figure C shows a Western blot of protein expression levels of the PERK-eIF2α pathway and the WNT pathway in CX-1 and DLD-1 cells, with GAPDH as an internal control.

[0034] Figure 9 The graph shows the effects of drug treatments on the growth of colorectal cancer organoids. Figure A shows the growth curves of CRC036-O and CRC082-O organoids treated with IMMU-132, GSK2606414 alone, or in combination, from 0 to 144 hours. These curves were calculated using IncuCyte® S3 software. In the graph, ① represents CRC036-O, and ② represents CRC082-O. This indicates that P < 0.05. This indicates that P < 0.01. P < 0.001; B is a bright-field image of PDXO 144 hours after treatment with IMMU-132 or GSK2606414 alone or in combination, where ① is CRC036-O PDXO and ② is CRC082-O PDXO.

[0035] Figure 10 Image A shows the tumor growth curve of the CRC036 PDXO model xenograft treated with IMMU-132, GSK2606414 monotherapy and combination therapy; Image B shows the weight statistical analysis of the CRC036 PDX model tumor treated with IMMU-132, GSK2606414 monotherapy and combination therapy; Image C shows the tumor growth curve of the CRC082 PDX model xenograft treated with IMMU-132, GSK2606414 monotherapy and combination therapy; Image D shows the weight statistical analysis of the CRC082 PDX model tumor treated with IMMU-132, GSK2606414 monotherapy and combination therapy; Image E shows the tumor growth curve of the CRC083 PDX model xenograft treated with IMMU-132, GSK2606414 monotherapy and combination therapy; Image F shows the tumor growth curve of the CRC083 PDX model xenograft treated with IMMU-132, GSK2606414 monotherapy and combination therapy. The graph shows the statistical analysis of tumor weight in the PDX model; G is the tumor growth curve of the CRC196 PDX model xenograft treated with IMMU-132, GSK2606414 alone and in combination; H is the statistical analysis of tumor weight in the CRC196 PDX model treated with IMMU-132, GSK2606414 alone and in combination; among them, This indicates that P < 0.05. This indicates that P < 0.01. This indicates that P < 0.001;

[0036] Figure 11 Image A shows the results of observing the pathological changes of various organs in the PDX model using H&E staining in the CRC036 PDX model; Image B shows the results of observing the pathological changes of various organs in the PDX model using H&E staining in the CRC082 PDX model; Image C shows the results of observing the pathological changes of various organs in the PDX model using H&E staining in the CRC083 PDX model; Image D shows the results of observing the pathological changes of various organs in the PDX model using H&E staining in the CRC196 PDX model. Detailed Implementation

[0037] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] NOD / SCID female mice, 5-6 weeks old, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd.

[0043] Example 1

[0044] Analysis of TROP2 expression in colorectal cancer:

[0045] To explore the clinical significance of TROP2 in colorectal cancer, this invention first systematically analyzed the expression level of TROP2 in colorectal cancer tissues based on the GEPIA database. Furthermore, using a colorectal cancer patient dataset from the Kaplan-Meier Plotter (www.kmplot.com) database, the correlation between TROP2 expression level and overall patient survival prognosis was evaluated. Subsequently, a tissue microarray (TMA) was constructed using 539 clinical samples from colorectal cancer patients. TROP2 protein expression was detected by IHC, and the samples were classified into four levels based on staining intensity: negative, low expression, intermediate expression, and high expression. Simultaneously, the expression level and subcellular localization of TROP2 in four colorectal cancer cell lines—DLD-1, CX-1, COLO205, and HCT15—were detected by Western blotting and immunofluorescence.

[0046] Experimental results showed that GEPIA database analysis indicated that TROP2 expression levels in colorectal cancer tissues were significantly higher than those in adjacent normal tissues. Figure 1 (A) Survival analysis showed that high TROP2 expression was significantly associated with poor overall survival in colorectal cancer patients (hazard ratio HR=1.51, 95% CI: 1.18–1.94; logrank P=0.0011), and Kaplan-Meier survival curves further indicated a poorer prognosis in the high-expression group. Figure 1(B) IHC test results showed that the positive expression rate of TROP2 in 539 samples was 89.05%, with high, medium, low, and negative expression rates of 13.54%, 17.25%, 58.26%, and 10.95%, respectively. Figure 1 (C). Western blot and immunofluorescence results showed that TROP2 was significantly overexpressed in DLD-1, CX-1, COLO205, and HCT15 cell lines, and it was mainly located in the cell membrane and cytoplasm. Figure 1 middle DE).

[0047] The above results confirm from multiple dimensions that TROP2 is generally highly expressed in colorectal cancer, and its expression level is closely related to patient prognosis, indicating that TROP2 plays a key role in the occurrence and development of colorectal cancer and has important potential as a diagnostic biomarker and targeted therapy target.

[0048] Example 2

[0049] In vitro antitumor effects of IMMU-132 monotherapy:

[0050] To evaluate the in vitro antitumor effect of IMMU-132, this invention compares the efficacy of the marketed anti-TROP2 targeted ADC drug IMMU-132 with the first-line chemotherapy drugs Capecitabine and Oxaliplatin. IMMU-132 concentrations of 100, 30, 10, 3, 1, 0.3, 0.1, and 0 μg / mL, and Capecitabine and Oxaliplatin concentrations of 10, 3, 1, 0.3, 0.1, 0.03, 0.01, and 0 μM were set and applied to four colorectal cancer cell lines for 72 h. The inhibitory effect of the drugs on cell viability was measured using a CellTiter-Glo® (CTG) cell viability assay kit.

[0051] Cell viability assay results showed that after 72 hours of treatment, compared with first-line chemotherapy drugs, IMMU-132 exhibited significantly better proliferation inhibition ability at lower concentrations, and this effect was clearly dose-dependent. Figure 2 ).

[0052] Example 3

[0053] In vivo antitumor effects of IMMU-132 monotherapy:

[0054] IHC was used to detect TROP2 protein expression in four types of PDX tumor tissues to clarify the molecular characteristics of the models and the expression of drug targets. NOD / SCID female mice, 5-6 weeks old, were purchased from Jiangsu Huachuang Xinno Pharmaceutical Technology Co., Ltd. Colorectal cancer xenograft tumor tissues were cut into 2-3 mm sections.3 A tumor was implanted subcutaneously on the right side of the mouse. The tumor was allowed to grow until it reached an average volume of approximately 100-150 mm. 3 Mice bearing tumors were randomly divided into two groups: a control group and a group receiving 10 mg / kg IMMU-132. IMMU-132 was administered intravenously once weekly for three weeks. Tumor size and body weight were measured twice weekly using calipers, and tumor volume was calculated using the following formula:

[0055] Tumor volume (mm) 3 = length × (width) 2 × 0.5.

[0056] Euthanasia was performed 29 days after treatment, and tumor tissue and major organs were collected for subsequent molecular and pathological analysis.

[0057] IHC results showed that TROP2 was positively expressed in all four PDX models, and the protein was mainly located in the cell membrane and cytoplasm. Figure 3 In vivo efficacy results showed that, compared with the blank control group, IMMU-132 exhibited significant antitumor effects in CRC036, CRC082, CRC083, and CRC196 models. Figure 4 ).

[0058] The above results confirm that IMMU-132 exhibited significant anti-tumor activity in all four TROP2-positive colorectal cancer PDX models, providing effective preclinical evidence for its subsequent clinical development and application in the treatment of colorectal cancer.

[0059] Example 4

[0060] IMMU-132 inhibits colorectal cancer by suppressing the PERK-mediated unfolded protein response (UPR) pathway:

[0061] To further explore the antitumor mechanism of IMMU-132, this invention conducted transcriptomic analysis on four colorectal cancer PDX models treated with 10 mg / kg IMMU-132. The experiment included a Vehicel group and an IMMU-132 group. Potential pathways were screened using GSEA analysis of the transcriptomic data. To further validate the analysis results at the protein level, proteins were extracted from colorectal cancer cells treated with the drug for 48 hours, separated by SDS-PAGE gel electrophoresis, and then transferred to PVDF membranes. Non-specific binding sites were blocked with 5% BSA (bovine serum albumin), followed by incubation with primary antibody (overnight incubation at 4°C) and corresponding enzyme-labeled secondary antibody (incubation at room temperature). Finally, the target protein signal was detected using ECL chemiluminescence reagent and a Bio-Rad chemiluminescence imaging system.

[0062] The overall effect of IMMU-132 on the unfolded protein response (UPR) was assessed using GSEA enrichment analysis. The results showed a significant negative enrichment of the UPR pathway. Figure 5 This indicates that IMMU-132 treatment can effectively inhibit the activation of the UPR pathway. Previous studies have shown that inhibiting the PERK-eIF2α-ATF4 signaling axis can block the UPR-dependent stress adaptation process in tumor cells, thereby inducing cell death. To further verify these results at the protein level, Western blot was used to detect the expression levels of key proteins in the PERK-eIF2α-ATF4 pathway in CX-1, DLD-1, HCT15, and COLO205 cells treated with IMMU-132. The experimental results showed that the expression of these key proteins was significantly downregulated, consistent with the results of GSEA analysis. Figure 6 These results indicate that IMMU-132 can simultaneously inhibit the UPR signaling pathway at both the transcriptional and protein levels, particularly its PERK-eIF2α-ATF4 branch, providing an important mechanistic basis for its antitumor activity.

[0063] Example 5

[0064] Screening for the optimal concentration of PERK inhibitor (GSK2606414) in synergistic antitumor activity with IMMU-132:

[0065] To elucidate the antitumor role of the unfolded protein response (UPR) in combination therapy, this invention first screened synergistic drug concentrations using various UPR inhibitors. Based on previous transcriptomic analysis suggesting a potential key role for the PERK pathway, the PERK inhibitor GSK2606414 was further selected for validation. Using CX-1 and DLD-1 cells as models, a concentration gradient of IMMU-132 (0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 μg / mL) and GSK2606414 (0, 0.01, 0.03, 0.1, 0.3, 1, 3, 10 μM) was established for combination drug administration experiments to systematically evaluate their synergistic effect. Cells were seeded in 96-well plates and treated with IMMU-132 alone, GSK2606414 alone, or both drugs in combination for 72 hours. Cell viability was then assessed using the cellTiter-Glo® luminescent cell viability assay kit. To quantitatively assess the interaction properties of the two drugs, the Co-action Index (CDI) model was used for analysis. The formula for calculating the CDI value is:

[0066] CDI = AB / A × B,

[0067] 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 IMMU-132 to the control group, and B is the ratio of GSK2606414 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.

[0068] The results showed that, among different dose combinations, 3 μg / mL IMMU-132 and 3 μM GSK2606414 exhibited the strongest synergistic effect in CX-1 and DLD-1 cells, 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. Figure 7 ).

[0069] Example 6

[0070] Combination therapy inhibits colorectal cancer growth by targeting TROP2 and PERK to suppress the Wnt / β-catenin signaling axis.

[0071] To investigate the molecular mechanism of combined drug therapy, this invention uses CX-1 cells as a model for transcriptomic analysis. Experimental setups included the Vehicel group, IMMU-132 group, GSK2606414 group, and COM (combination) group. KEGG pathway enrichment analysis and GSEA analysis were performed on the transcriptomic data to screen signaling pathways regulated by the combined therapy. To further validate the analysis results at the protein level, proteins were extracted from CX-1 and DLD-1 cells treated with the drugs for 48 hours, and separated by SDS-PAGE gel electrophoresis, followed by transfer to PVDF membranes. Non-specific binding sites were blocked with 5% BSA (bovine serum albumin), and the cells were then incubated with primary antibody (overnight incubation at 4°C) and corresponding enzyme-labeled secondary antibody (incubation at room temperature). Finally, the target protein signal was detected using ECL chemiluminescence reagent and the Bio-Rad chemiluminescence imaging system.

[0072] KEGG analysis showed that the WNT signaling pathway and Focal adhesion pathway were the most significantly enriched. Figure 8 (A). GSEA analysis further showed that the combination therapy significantly inhibited the activity of the WNT signaling pathway (NES=-1.97, P<0.0001) and the Focaladhesion pathway (NES=-2.02, P<0.0001). Figure 8(B) Western blot results showed that in CX-1 and DLD-1 cells, combination therapy reduced the expression levels of β-catenin, a key molecule in the WNT pathway, and its downstream target JUN. Simultaneously, the expression of GSK3B and WNT2B was also downregulated, indicating that WNT pathway activity was inhibited. Furthermore, changes in PERK and eIF2α phosphorylation levels suggested that WNT pathway activity is cross-regulated by the unfolded protein response pathway. Figure 8 (C) The above results collectively indicate that combination therapy inhibits tumor growth and metastasis by downregulating the PERK-eIF2α signaling axis and thereby suppressing the activity of the WNT signaling pathway.

[0073] Example 7

[0074] IMMU-132 combined with GSK20606414 synergistically suppresses the growth of CRC PDXOs models:

[0075] To validate the synergistic efficacy of drugs in a more clinically similar model, this invention extracts tumor cells from CRC PDX xenograft tissue and prepares organoids using a CRC organoid kit according to standard procedures. 50 μL of Matrigel suspension is added to a 24-well plate and incubated at 37°C for 10 minutes to solidify it; then 500 μL of complete culture medium is added to each well. The organoids are cultured in a humidified incubator at 37°C and 5% CO2. After reaching a certain size, some organoids are collected, fixed, dehydrated, and embedded to prepare paraffin sections to verify whether the PDXO model reproduces the biological and structural characteristics of the original PDX model. To assess drug sensitivity, the organoids are digested and resuspended in Matrigel, and seeded in a 96-well plate with 150 μL of culture medium (containing 3 μL of Matrigel). After 24 hours of culture, they are treated with IMMU-132 or GSK2606414 alone or in combination for 144 hours. Organoid viability is monitored in real time using the IncuCyte® S3 viability analysis system.

[0076] Experimental results based on two CRC PDXO models showed that, during the 144-hour drug treatment period, the combined treatment of IMMU-132 and GSK2606414 produced a stronger and time-dependent synergistic inhibitory effect on the growth of both PDXO models compared with the single-drug treatment group. Figure 9 This result further confirms the significant antitumor activity of the two drugs in combination at the three-dimensional organoid level.

[0077] Example 8

[0078] IMMU-132 combined with GSK2606414 can enhance the antitumor activity of CRC PDX:

[0079] Colorectal cancer xenograft tumor tissue was cut into 2-3 mm pieces. 3 A tumor was implanted subcutaneously on the right side of the mouse. The tumor was allowed to grow until it reached an average volume of approximately 100-150 mm. 3 Mice with CRC082, CRC083, and CRC196 were randomly divided into four groups and treated with saline, 2.5 mg / kg IMMU-132 (once a week for three weeks), 50 mg / kg GSK2606414 (five times a week for four weeks), or a combination of IMMU-132 and GSK2606414, respectively. Mice with CRC036 received saline, 5 mg / kg IMMU-132 (once a week for three weeks), 50 mg / kg GSK2606414 (five times a week for four weeks), or a combination of these treatments. Tumor size and body weight were measured twice weekly using calipers, and tumor volume was calculated using the following formula:

[0080] Tumor volume (mm) 3 = length × (width) 2 × 0.5.

[0081] Euthanasia was performed 29 days after treatment, and tumor tissue and major organs were collected for subsequent molecular and pathological analysis.

[0082] In vivo efficacy evaluations conducted in four colorectal cancer PDX models showed that, compared with the blank control group and the IMMU-132 / GSK2606414 monotherapy groups, the combination therapy group of IMMU-132 and GSK2606414 significantly inhibited tumor growth. The combination therapy regimen demonstrated superior therapeutic effects compared to either monotherapy in all models, exhibiting significant synergistic antitumor activity. Figure 10 ).

[0083] In terms of safety, the combination therapy regimen showed good tolerability. Histopathological analysis of the major organs (heart, liver, spleen, lungs, and kidneys) did not reveal significant pathological damage. Figure 11 The results indicate that the combined therapy did not produce detectable toxicity to liver and kidney function or major tissues. The combination therapy of IMMU-132 and GSK2606414 demonstrated synergistic antitumor activity and a favorable safety profile in various colorectal cancer PDX models, providing solid preclinical experimental evidence for the clinical translation of this combination strategy.

[0084] In summary, this invention proposes a novel strategy for the combined treatment of colorectal cancer with the TROP2-targeting antibody-drug conjugate IMMU-132 and the PERK inhibitor GSK2606414. The study first confirmed the clinical relevance of TROP2 in colorectal cancer—its significantly high expression in cancer tissue (89.05% positive rate) and its close association with poor patient prognosis. Further experiments showed that IMMU-132 monotherapy exhibited significant antitumor activity both in vitro and in vivo. Further experiments also demonstrated that IMMU-132 monotherapy showed significant antitumor activity in both in vitro and in vivo models, and its mechanism of action is related to the inhibition of the PERK-eIF2α-ATF4 signaling axis. This novel mechanism provides an important theoretical basis for its antitumor effect. Through screening of UPR pathway inhibitors and concentration gradients, 3 μg / mL IMMU-132 and 3 μM GSK2606414 were determined to be the optimal synergistic concentration combination. Mechanistic studies have shown that the synergistic effect of combination therapy stems from the dual regulation of the UPR and WNT signaling pathways—by modulating the PERK-eIF2α signaling axis, thereby inhibiting WNT signaling pathway activity. Validation results in multiple preclinical models consistently demonstrate that, in three-dimensional organoid models and four different PDX models, the combination therapy regimen exhibits significantly superior antitumor efficacy compared to monotherapy while maintaining good safety. This invention reveals a novel strategy for treating colorectal cancer by synergistically regulating the UPR and WNT signaling pathways, providing a novel treatment option with clinical translational potential for TROP2-positive colorectal cancer patients.

[0085] 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 use of a formulation containing IMMU-132 and GSK2606414 in the preparation of an anti-colorectal cancer drug, characterized in that: ① At the cellular level, the concentration of IMMU-132 was 0.3~10 μg / mL, and the concentration of GSK2606414 was 0.3~10 μM; ②At the animal level, the concentration of IMMU-132 was 2.5 mg / kg, and the concentration of GSK2606414 was 50 mg / kg.

2. The application of the antibody-drug conjugate IMMU-132 in combination with the PERK inhibitor GSK2606414 in the preparation of anti-colorectal cancer drugs, characterized in that: When applying, ① At the cellular level, the concentration of IMMU-132 was 0.3~10 μg / mL, and the concentration of GSK2606414 was 0.3~10 μM; ②At the animal level, the concentration of IMMU-132 was 2.5 mg / kg, and the concentration of GSK2606414 was 50 mg / kg.

3. The application according to claim 2, characterized in that, The synergistic combination of IMMU-132 and GSK2606414 inhibits tumor cell growth and metastasis by downregulating the PERK-eIF2α signaling axis and suppressing the activity of the WNT signaling pathway.

4. An anti-colorectal cancer drug, characterized in that, The active ingredients of the drug are the antibody-drug conjugate IMMU-132, which targets the TROP2 pathway, and the PERK inhibitor GSK2606414, which targets the PERK signaling pathway. ① At the cellular level, the concentration of IMMU-132 was 0.3~10 μg / mL, and the concentration of GSK2606414 was 0.3~10 μM; ②At the animal level, the concentration of IMMU-132 was 2.5 mg / kg, and the concentration of GSK2606414 was 50 mg / kg.

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

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