Use of targeting cpxm2 in treatment of pancreatic cancer

Abstract

This invention provides the application of CPXM2 targeting in the treatment of pancreatic cancer. Specifically, this invention provides the use of a CPXM2 inhibitor in the preparation of compositions or formulations for: (a) the prevention and / or treatment of pancreatic cancer; and / or (b) the inhibition of the growth of pancreatic cancer cells or pancreatic cancer tissue. This invention also provides the use of the CPXM2 target in pancreatic cancer subtyping, corresponding companion diagnostic product combinations, and pharmaceutical compositions for synergistic treatment of pancreatic cancer. This invention provides a new and effective approach to improve the efficacy of pancreatic cancer treatment, which currently lacks satisfactory results, and has broad prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to the application of CPXM2 targeting in the treatment of pancreatic cancer. Background Technology

[0002] Pancreatic cancer is one of the leading causes of global healthcare burden, often referred to as the "king of cancers." Its 5-year relative survival rate is only 8%, with a median survival of just 6-9 months. According to the latest data, in 2018, 1,276,106 people worldwide were diagnosed with pancreatic cancer, and 358,989 died from it. Clearly, pancreatic cancer is a significant public health issue that seriously impacts human life and health.

[0003] Pancreatic cancer is characterized by high malignancy, lack of biomarkers, indistinct clinical features, poor response to systemic therapy, and high recurrence rate after surgery, resulting in an extremely high mortality rate and posing a significant clinical challenge. Due to the lack of reliable blood and urine biomarkers for diagnosis, pancreatic cancer is primarily diagnosed through ultrasound, CT, and MRI. However, by the time of diagnosis, over 80% of patients have invasive progression or distant metastases, mainly to the liver, making surgical eradication difficult at this stage. Even after surgery, 20% of pancreatic cancer cases are found to have nearby or distant metastases two years later. In these cases, chemotherapy is the primary treatment. Currently, representative drugs for pancreatic cancer include gemcitabine (GEM), protein-bound paclitaxel, and FOLFIRINOX (5-fluorouracil, leucovorin, oxaliplatin, and irinotecan). However, most of these drugs are not yet approved or are still in clinical trials, and their efficacy is not entirely satisfactory.

[0004] Given the poor efficacy of current drugs for treating pancreatic cancer, there is an urgent need in the field to develop effective drugs to treat pancreatic cancer and reduce patient mortality. Summary of the Invention

[0005] The purpose of this invention is to provide the application of CPXM2 targeting in the treatment of pancreatic cancer.

[0006] In a first aspect of the invention, there is provided the use of a CPXM2 inhibitor in the preparation of a composition or formulation for: (a) preventing and / or treating pancreatic cancer; and / or (b) inhibiting the growth of pancreatic cancer cells or pancreatic cancer tissue.

[0007] In another preferred embodiment, the pancreatic cancer is pancreatic cancer of mammals (including humans or non-human mammals).

[0008] In another preferred embodiment, the pancreatic cancer is CPXM2-positive pancreatic cancer.

[0009] In another preferred embodiment, the CPXM2 positive sign is defined as a significant increase in the expression and / or activity of CPXM2 compared to normal control tissues or normal control cells.

[0010] In another preferred embodiment, the significant increase refers to: the ratio of CPXM2 expression level E1 in the extracellular matrix of pancreatic cancer cells or pancreatic cancer tissue to the CPXM2 expression level E0 in the extracellular matrix of normal controls (i.e., E1 / E0) ≥ 1.5, preferably ≥ 2, more preferably ≥ 3; and / or the ratio of CPXM2 activity A1 in the extracellular matrix of pancreatic cancer cells or pancreatic cancer tissue to the CPXM2 activity A0 in the extracellular matrix of normal controls (i.e., A1 / A0) ≥ 1.5, preferably ≥ 2, more preferably ≥ 3.

[0011] In another preferred embodiment, the normal control refers to: a non-pancreatic cancer subject or a healthy subject, or a non-pancreatic cancer cell or a normal cell.

[0012] In another preferred embodiment, the pancreatic cancer is selected from the group consisting of pancreatic ductal adenocarcinoma (PDAC), acinar cell carcinoma, adenosquamous carcinoma, small cell pancreatic carcinoma, pancreatic mucinous carcinoma, or combinations thereof.

[0013] In another preferred embodiment, the pancreatic cancer is pancreatic ductal adenocarcinoma (PDAC).

[0014] In another preferred embodiment, the CPXM2 inhibitor targets the CPXM2 protein and / or the CPXM2 gene.

[0015] In another preferred embodiment, the CPXM2 protein includes the complete CPXM2 protein or a fragment thereof or a domain thereof.

[0016] In another preferred embodiment, the CPXM2 inhibitor targets the CPXM2 protein, particularly the DSD domain of the CPXM2 protein.

[0017] In another preferred embodiment, the CPXM2 inhibitor inhibits or blocks the interaction between CPXM2 and collagen.

[0018] In another preferred embodiment, the CPXM2 inhibitor is selected from the group consisting of small molecule compounds, antibodies, nucleic acid molecules, gene editors, or combinations thereof.

[0019] In another preferred embodiment, the CPXM2 inhibitor includes a DSD domain inhibitor that targets the DSD domain of the CPXM2 protein.

[0020] In another preferred embodiment, the DSD domain inhibitor is selected from the group consisting of small molecule compounds, antibodies, nucleic acid molecules, gene editors, or combinations thereof.

[0021] In another preferred embodiment, the antibody is an antibody that targets CPXM2.

[0022] In another preferred embodiment, the antibody is an antibody that targets the DSD domain of the CPXM2 protein.

[0023] In another preferred embodiment, the nucleic acid molecule includes shRNA, siRNA, miRNA, or a combination thereof.

[0024] In another preferred embodiment, the nucleic acid molecule includes antisense nucleic acids (such as antisense oligonucleotides (ASO)).

[0025] In another preferred embodiment, the gene editor includes a DNA gene editor and an RNA gene editor.

[0026] In another preferred embodiment, the gene editor comprises gRNA and gene-editing protein.

[0027] In another preferred embodiment, the gene editor is used to suppress or eliminate the expression of the CPXM2 gene.

[0028] In another preferred embodiment, the gRNA is an RNA that guides the gene-editing protein to specifically bind to the CPXM2 gene.

[0029] In another preferred embodiment, the gene-editing protein is selected from the group consisting of CasRx, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or combinations thereof.

[0030] In another preferred embodiment, the composition or formulation further includes other antitumor drugs (such as other drugs used to treat pancreatic cancer).

[0031] In another preferred embodiment, the other antitumor drugs include: chemotherapy drugs or targeted drugs.

[0032] In another preferred embodiment, the other antitumor drugs include gemcitabine.

[0033] In another preferred embodiment, the other antitumor drugs include immune checkpoint inhibitors.

[0034] In another preferred embodiment, the immune checkpoint is selected from the group consisting of PD-1, PD-L1, CTLA-4, LAG-3, TIM-3, TIGIT, CD28, CD40, CD40L, CD27, B7-1, B7-2, OX40, or combinations thereof.

[0035] In another preferred embodiment, the other antitumor drugs include: antiPD1 antibodies.

[0036] In another preferred embodiment, the composition or formulation is a composition or formulation for the synergistic treatment of pancreatic cancer.

[0037] In another preferred embodiment, the composition or formulation for synergistic treatment of pancreatic cancer comprises:

[0038] (Z1) The primary active ingredient, a CPXM2 inhibitor; and

[0039] (Z2) The second active ingredient gemcitabine and / or the third active ingredient anti-PD1 antibody.

[0040] In a second aspect of the invention, there is provided the use of the CPXM2 gene, mRNA, cDNA, protein, or a detection reagent thereof for preparing a kit for use selected from one or more of the following groups:

[0041] (i) Used to assess whether a patient with pancreatic cancer is suitable for treatment with a CPXM2 inhibitor or a drug or preparation containing a CPXM2 inhibitor; and / or

[0042] (ii) To assess the prognosis of patients with pancreatic cancer treated with CPXM2 inhibitors or drugs or formulations containing CPXM2 inhibitors.

[0043] In another preferred embodiment, the kit is also used for:

[0044] (iii) To aid in the diagnosis of pancreatic cancer or to assess the risk of developing pancreatic cancer; and / or

[0045] (iv) Prognostic assessment of patients with pancreatic cancer.

[0046] In another preferred embodiment, the kit is used to classify pancreatic cancer patients based on CPXM2 expression and / or activity.

[0047] In another preferred embodiment, the detection reagent comprises:

[0048] (a) CPXM2-specific antibodies, CPXM2-specific binding molecules; and / or

[0049] (b) Primers or primer pairs, probes or chips for specific amplification of CPXM2 mRNA or CPXM2 cDNA.

[0050] In another preferred embodiment, the detection reagents include: immunohistochemistry (IHC) reagents, reverse transcription-polymerase chain reaction (RT-PCR) reagents, immunofluorescence reagents, Western blotting (WB) reagents, enzyme-linked immunosorbent assay (ELISA) reagents, real-time PCR reagents, or combinations thereof.

[0051] In another preferred embodiment, the immunohistochemical (IHC) reagent includes: a specific antibody for CPXM2, a substrate, a chromogenic agent, an adhesive, a fixative, an antigen retrieval solution, a blocking solution, a washing solution, a staining solution, a mounting medium, or a combination thereof.

[0052] In another preferred embodiment, the reverse transcription-polymerase chain reaction (RT-PCR) reagent includes: reverse transcriptase, DNA polymerase, reaction buffer, dNTP mixture, primers, RNase inhibitor, positive control RNA, RNase-free dH2O, enhancer, stabilizer, or a combination thereof.

[0053] In another preferred embodiment, the Western Blot (WB) reagent includes: lysis buffer (e.g., RIPA lysis buffer), protease inhibitor (e.g., PMSF), protein quantification kit (e.g., BCA protein quantification kit), electrophoresis-related reagents (e.g., acrylamide, N,N'-methylenebisacrylamide, SDS solution, Tris-HCl buffer, TEMED, 10% ammonium persulfate solution, etc.), transfer-related reagents (e.g., transfer buffer, transfer staining solution (e.g., Ponceau S staining solution)), blocking solution (e.g., 5% skim milk powder or BSA), CPXM2-specific antibody (primary antibody), anti-CPXM2-specific antibody (secondary antibody), Western washing buffer (e.g., TBST or TBS), chemiluminescent or chromogenic reagents, gel staining solutions (e.g., Coomassie Brilliant Blue rapid staining solution, hypersensitive protein rapid staining solution), or combinations thereof.

[0054] In another preferred embodiment, the enzyme-linked immunosorbent assay (ELISA) reagent comprises: a solid-phase carrier, an enzyme-labeled antigen or antibody, an enzyme substrate, a negative control, a positive control, a diluent, a washing buffer, an enzyme reaction termination solution, a blocking solution, a coating buffer, a substrate buffer, or a combination thereof.

[0055] In another preferred embodiment, the real-time PCR reagent includes: DNA polymerase, PCR buffer, deoxynucleoside triphosphates (dNTPs), DNA template, primers, MgCl2, fluorescent dye or probe, stabilizer, ROX, deionized water, or a combination thereof.

[0056] In another preferred embodiment, the CPXM2-specific antibody includes an antibody that targets the DSD domain of the CPXM2 protein.

[0057] In another preferred embodiment, the pancreatic cancer is CPXM2-positive pancreatic cancer.

[0058] In a third aspect of the invention, a product portfolio is provided, the product portfolio comprising:

[0059] (a) a detection reagent or a kit containing said detection reagent, said detection reagent being a detection reagent for detecting the CPXM2 gene, mRNA, cDNA, protein, or combinations thereof; and

[0060] (b) A pharmaceutical composition comprising a CPXM2 inhibitor as an active ingredient and a pharmaceutically acceptable carrier.

[0061] In another preferred embodiment, the kit includes a container containing a detection reagent for detecting the CPXM2 gene, mRNA, cDNA, protein, or combinations thereof, and a label or instruction manual indicating that the kit is used to assess whether a pancreatic cancer patient is suitable for treatment with a CPXM2 inhibitor or a drug or preparation containing a CPXM2 inhibitor.

[0062] In another preferred embodiment, the kit also contains the CPXM2 gene, mRNA, cDNA, and / or protein as a control or quality control.

[0063] In a fourth aspect of the invention, a pharmaceutical composition for the synergistic treatment of pancreatic cancer is provided, the pharmaceutical composition comprising:

[0064] (Z1) The first active ingredient, a CPXM2 inhibitor;

[0065] (Z2) The second active ingredient gemcitabine and / or the third active ingredient anti-PD1 antibody; and

[0066] (Z3) Pharmaceutically acceptable carrier.

[0067] In another preferred embodiment, the pharmaceutical composition comprises:

[0068] (Z1) The first active ingredient, a CPXM2 inhibitor;

[0069] (Z2) The second active ingredient, gemcitabine; and

[0070] (Z3) Pharmaceutically acceptable carrier.

[0071] In another preferred embodiment, the pharmaceutical composition comprises:

[0072] (Z1) The first active ingredient, a CPXM2 inhibitor;

[0073] (Z2) The third active ingredient is anti-PD1 antibody; and

[0074] (Z3) Pharmaceutically acceptable carrier.

[0075] In another preferred embodiment, the pharmaceutical composition comprises:

[0076] (Z1) The first active ingredient, a CPXM2 inhibitor;

[0077] (Z2) The second active ingredient, gemcitabine, and the third active ingredient, anti-PD1 antibody; and

[0078] (Z3) Pharmaceutically acceptable carrier.

[0079] In another preferred embodiment, the dose of the first active ingredient CPXM2 inhibitor is 1-100 mg / kg, more preferably 5-50 mg / kg, and even more preferably 10 mg / kg.

[0080] In another preferred embodiment, the dose of the second active ingredient gemcitabine is 1-500 mg / kg, more preferably 10-250 mg / kg, and even more preferably 50 mg / kg.

[0081] In another preferred embodiment, the dose of the third active ingredient, the anti-PD1 antibody, is 1-100 mg / kg, more preferably 5-50 mg / kg, and even more preferably 10 mg / kg.

[0082] In another preferred embodiment, the dose of the first active ingredient CPXM2 inhibitor is 10 mg / kg, and the dose of the second active ingredient gemcitabine is 50 mg / kg.

[0083] In another preferred embodiment, the dose of the first active ingredient, the CPXM2 inhibitor, is 10 mg / kg, and the dose of the third active ingredient, the anti-PD1 antibody, is 10 mg / kg.

[0084] In another preferred embodiment, the first active ingredient, the CPXM2 inhibitor, is an antibody that targets the DSD domain of the CPXM2 protein.

[0085] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0086] In another preferred embodiment, the pancreatic cancer is CPXM2-positive pancreatic cancer.

[0087] In a fifth aspect of the invention, a medicine box is provided, the medicine box comprising:

[0088] (a) A first formulation comprising a CPXM2 inhibitor and a pharmaceutically acceptable carrier; and

[0089] (b) a second formulation comprising gemcitabine and a pharmaceutically acceptable carrier; and / or a third formulation comprising an anti-PD1 antibody and a pharmaceutically acceptable carrier.

[0090] In another preferred embodiment, the medicine box includes a first formulation and a second formulation.

[0091] In another preferred embodiment, the medicine box includes a first formulation and a third formulation.

[0092] In another preferred embodiment, the medicine box includes a first formulation, a second formulation, and a third formulation.

[0093] In another preferred embodiment, the first formulation, the second formulation, and the third formulation are each independent.

[0094] In another preferred embodiment, the first formulation, the second formulation, and / or the third formulation are lyophilized formulations or liquid formulations.

[0095] In another preferred embodiment, the first formulation, the second formulation, and / or the third formulation are formulated in the same dosage form or different dosage forms.

[0096] In another preferred embodiment, the first formulation, the second formulation, and / or the third formulation are administered simultaneously.

[0097] In another preferred embodiment, the first formulation, the second formulation, and / or the third formulation are administered sequentially.

[0098] In another preferred embodiment, the dosage of the first formulation is 1-100 mg / kg, more preferably 5-50 mg / kg, and even more preferably 10 mg / kg.

[0099] In another preferred embodiment, the dosage of the second formulation is 1-500 mg / kg, more preferably 10-250 mg / kg, and even more preferably 50 mg / kg.

[0100] In another preferred embodiment, the dosage of the third formulation is 1-100 mg / kg, more preferably 5-50 mg / kg, and even more preferably 10 mg / kg.

[0101] In another preferred embodiment, the dosage of the first preparation is 10 mg / kg, and the dosage of the second preparation is 50 mg / kg.

[0102] In another preferred embodiment, the dosage of the first preparation is 10 mg / kg, and the dosage of the third preparation is 10 mg / kg.

[0103] In a sixth aspect of the invention, there is provided the use of a product combination as described in the third aspect of the invention, a pharmaceutical composition as described in the fourth aspect of the invention, or a medicament as described in the fifth aspect of the invention, for the preparation of a medical product for treating CPXM2-positive pancreatic cancer.

[0104] In a seventh aspect of the present invention, a method for in vitro inhibition or synergistic inhibition of the growth of pancreatic cancer cells or pancreatic cancer tissue is provided, the method comprising the steps of: adding a CPXM2 inhibitor or a pharmaceutical composition as described in the fourth aspect of the present invention to a culture system of pancreatic cancer cells or pancreatic cancer tissue, thereby inhibiting or synergistically inhibiting the growth of pancreatic cancer cells or pancreatic cancer tissue.

[0105] Alternatively, the method may include the step of using a product combination as described in the third aspect of the invention or a medicament as described in the fifth aspect of the invention to inhibit or synergistically inhibit the growth of pancreatic cancer cells or pancreatic cancer tissue.

[0106] In another preferred embodiment, the method is for non-disease treatment purposes or non-disease diagnosis purposes.

[0107] In another preferred embodiment, the pancreatic cancer cells or pancreatic cancer tissue are pancreatic cancer cells or pancreatic cancer tissue of humans or non-human mammals (such as mice, rats, etc.).

[0108] In another preferred embodiment, the pancreatic cancer is CPXM2-positive pancreatic cancer.

[0109] In another preferred embodiment, the pancreatic cancer cells are selected from the group consisting of: Panc02, AsPC-1, BxPC-3, SW-1990, Capan-2, Panc 10.05, CFPAC-1, HPAF-II, PANC-1, MIA PaCa-2, or combinations thereof.

[0110] In another preferred embodiment, the pancreatic cancer cells are Panc02 cells.

[0111] In an eighth aspect of the invention, a method for treating pancreatic cancer is provided, the method comprising the steps of administering to a CPXM2-positive pancreatic cancer patient a therapeutically effective amount of a CPXM2 inhibitor, a product combination as described in a third aspect of the invention, a pharmaceutical composition as described in a fourth aspect of the invention, a kit as described in a fifth aspect of the invention, or a combination thereof.

[0112] In another preferred embodiment, the method further includes the step of: classifying pancreatic cancer patients based on CPXM2 expression and / or activity, thereby classifying the patients as CPXM2-positive pancreatic cancer patients or CPXM2-negative pancreatic cancer patients.

[0113] In another preferred embodiment, the method includes the steps of:

[0114] (a) Classifying pancreatic cancer patients based on CPXM2 expression and / or activity, thereby categorizing the patients as CPXM2-positive or CPXM2-negative pancreatic cancer patients; and

[0115] (b) administering a therapeutically effective amount of a CPXM2 inhibitor, a combination of products as described in the third aspect of the invention, a pharmaceutical composition as described in the fourth aspect of the invention, a cassette as described in the fifth aspect of the invention, or a combination thereof to a CPXM2-positive pancreatic cancer patient.

[0116] In another preferred embodiment, step (b) further includes the step of detecting CPXM2 expression and / or activity during treatment.

[0117] In a ninth aspect of the invention, a method for determining a treatment plan is provided, the method comprising:

[0118] (S1) Provide the test sample from the subject or participant;

[0119] (S2) Detect the expression and / or activity of CPXM2 in the test sample; and

[0120] (S3) Determine the treatment regimen based on the expression and / or activity of CPXM2 as determined in step (S2);

[0121] If the measured expression and / or activity of CPXM2 is positive, then the subject or participant is deemed suitable for treatment with a CPXM2 inhibitor or a drug or preparation containing a CPXM2 inhibitor.

[0122] If the measured expression and / or activity of CPXM2 is not CPXM2 positive (e.g., CPXM2 negative), then the subject or participant is determined to be unsuitable for treatment with CPXM2 inhibitors or drugs or preparations containing CPXM2 inhibitors.

[0123] The CPXM2 positive result refers to a significant increase in the expression and / or activity of CPXM2 compared to normal control tissues or normal control cells.

[0124] The significant increase is defined as follows: the ratio of CPXM2 expression level E1 in the extracellular matrix of pancreatic cancer cells or pancreatic cancer tissue to the CPXM2 expression level E0 in the extracellular matrix of normal controls (i.e., E1 / E0) ≥ 1.5, preferably ≥ 2, more preferably ≥ 3; and / or the ratio of CPXM2 activity A1 in the extracellular matrix of pancreatic cancer cells or pancreatic cancer tissue to the CPXM2 activity A0 in the extracellular matrix of normal controls (i.e., A1 / A0) ≥ 1.5, preferably ≥ 2, more preferably ≥ 3.

[0125] In another preferred embodiment, the sample to be tested includes: blood samples (including plasma samples and serum samples), urine samples, tissue samples, cell samples, or combinations thereof.

[0126] In another preferred embodiment, the normal control refers to a non-pancreatic cancer subject or a healthy subject.

[0127] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0128] Figure 1 The results of CPXM2 and α-SMA immunofluorescence staining were shown for adjacent and cancerous tissues of the patient's pancreas.

[0129] Figure 2 The results of CPXM2 and α-SMA immunofluorescence staining were shown for pancreatic tissue from normal mice and pancreatic cancer tissue from KPC mice.

[0130] Figure 3 This study demonstrates the effect of CPXM2 overexpression on the occurrence and progression of pancreatic cancer using in vivo fluorescence imaging.

[0131] Figure 4 The comparison of pancreatic tissue tumor size between the CPXM2-KO group and the WT group is shown.

[0132] Figure 5 The survival time of KPC mice in the IgG antibody group and the CPXM2-DSD antibody group was compared.

[0133] Figure 6 The comparison of tumor size among the experimental groups is shown.

[0134] Figure 7 The comparison of tumor size among the experimental groups is shown. Detailed Implementation

[0135] Through extensive and in-depth research and numerous screenings, the inventors have, for the first time, creatively discovered that targeting CPXM2 (especially the DSD domain) can effectively treat pancreatic cancer. For example, through numerous experiments, the inventors found that CPXM2-KO and antibodies targeting CPXM2-DSD can inhibit the progression of pancreatic cancer and prolong LSL-Kras. G12D / + LSL-Trp53 R172H / + The survival rate of Pdx1-Cre (KPC) mice was improved, enhancing the efficacy of GEM chemotherapy and PD1 monoclonal antibody immunotherapy, resulting in a synergistic effect. These experimental results provide broad prospects for clinical trials and drug development in pancreatic cancer. Based on these findings, this invention was completed.

[0136] the term

[0137] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0138] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0139] The term “administration” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0140] CPXM2-DSD and its inhibitors

[0141] The inventors have observed that pancreatic cancer exhibits a significant proliferative response to connective tissue, with marked activation of fibroblasts and the production of abundant extracellular matrix components. Therefore, we propose a method to target the extracellular matrix components of pancreatic cancer cells to enhance the therapeutic effect of pancreatic cancer.

[0142] Carboxypeptidase X, M14 Family Member 2 (CPXM2), AE-binding protein 1 (AEBP1), and Carboxypeptidase X, M14 Family Member 2 (CPXM1) all belong to the E family of carboxypeptidases and are non-peptidase members with discoid domains. Under physiological conditions, CPXM2, AEBP1, and CPXM1 are expressed only in certain organs and tissues, such as the kidney and colon; however, their expression is upregulated in various tumors, such as pancreatic ductal carcinoma, melanoma, and glioma, and is positively correlated with poor prognosis. Current research indicates that CPXM2, AEBP1, and CPXM1 influence tumorigenesis and development through mechanisms such as promoting epithelial-mesenchymal transition and inhibiting apoptosis and ferroptosis. CPXM2, AEBP1, and CPXM1 are secreted proteins located in the extracellular matrix. Although CPXM2 and AEBP1, CPXM1 do not have the classic carboxypeptidase substrate cleavage activity, they have a discoid domain that can bind to a variety of proteins, such as collagen, growth factors, phospholipids and galactose.

[0143] Single-cell sequencing analysis revealed that CPXM2 was specifically highly expressed in pancreatic cancer fibroblasts, while CPXM1 and AEBP1 expression were not specific. Therefore, we focused on CPXM2. Structural analysis showed that CPXM2 possesses a discoid domain structure similar to discoid domain receptor 1, while COL1A1, COL1A2, COL3A1, and COL4A1 contain the GVMG motif, suggesting that CPXM2 can bind to COL1A1, COL1A2, COL3A1, and COL4A1.

[0144] DSD stands for Discoidin Domain, which is part of the CPXM2 protein and is the functional region of CPXM2 that acts as a molecular glue to arrange collagen. DDR1 / 2, CPXM1, and AEBP proteins also contain Discoidin domains.

[0145] In this invention, we have for the first time creatively proposed that by targeting CPXM2, especially the DSD structure, collagen arrangement can be affected, thereby enhancing the effects of chemotherapy or immunotherapy. Finally, we experimentally confirmed that targeting the CPXM2-DSD structure can effectively treat pancreatic cancer.

[0146] Those skilled in the art will understand that the CPXM2 inhibitor in this invention refers to an inhibitor that targets the CPXM2 protein and / or the CPXM2 gene. Targeting the CPXM2 protein can be targeting the entire CPXM2 protein, or it can be targeting a portion or fragment (e.g., a domain) of the CPXM2 protein, such as the DSD domain of the CPXM2 protein.

[0147] CPXM2 inhibitors can be selected from the group consisting of small molecule compounds, antibodies, nucleic acid molecules, gene editors, or combinations thereof. As long as they can function by targeting the CPXM2 protein and / or the CPXM2 gene, they fall under the definition of CPXM2 inhibitors in this invention. Preferably, CPXM2 inhibitors include DSD domain inhibitors, which target the DSD domain of the CPXM2 protein.

[0148] In a preferred embodiment, the CPXM2 inhibitor is an antibody targeting CPXM2, or an antibody targeting the DSD domain of the CPXM2 protein (i.e., a CPXM2-DSD antibody). It should be understood that using an antibody targeting the entire CPXM2 protein also has the same or similar therapeutic effect on pancreatic cancer as using a CPXM2-DSD antibody. Similarly, when an antibody targeting the entire CPXM2 protein is used in combination with gemcitabine or a PD-1 monoclonal antibody, it also has the same or similar synergistic therapeutic effect on pancreatic cancer as when using a CPXM2-DSD antibody.

[0149] KPC mice

[0150] KPC mice are a spontaneous tumor mouse model used to study pancreatic cancer, particularly pancreatic ductal adenocarcinoma (PDAC). Kras and Trp53 are key factors in pancreatic cancer development; inactivation of the Kras gene combined with inactivation of the Trp53 tumor suppressor gene accelerates the development of pancreatic cancer and promotes its metastasis to other sites. KPC mice carry LSL-Kras. G12D LSL-Trp53 R172H The KPC mouse model, along with the Pdx1-Cre genotype, is one of the most perfect spontaneous pancreatic cancer mouse models, closely resembling key features of human pancreatic cancer. The KPC model can be used as a model of pancreatic ductal adenocarcinoma (PDAC), and because the tumors in this model have a metastatic tendency, KPC mice can be used to study the immunobiology of primary and metastatic lesions.

[0151] Companion diagnostics

[0152] Companion diagnostics is a type of in vitro diagnostics that provides information crucial for the safe and effective use of corresponding therapeutic products (pharmaceuticals or biological products). For the safe and effective use of corresponding therapeutic products, companion diagnostic testing is essential, with objectives including: (1) identifying patients who are likely to benefit from the therapeutic product; (2) identifying patients whose use of the therapeutic product may increase the risk of serious adverse reactions; and (3) monitoring the response of the therapeutic product to treatment in order to adjust the treatment (such as treatment plan, dosage, and discontinuation) to better achieve safety and efficacy. Companion diagnostics comprises reagents for specific tests, quality control samples, and supporting instruments, forming a complete testing system.

[0153] In this invention, the product combination described in the third aspect is one type of companion diagnostic kit. By using a detection reagent that detects the CPXM2 gene, mRNA, cDNA, protein, or combinations thereof, the expression and / or activity of CPXM2 in a patient or subject can be detected and classified. This allows identification of whether the patient or subject is suitable for treatment with a CPXM2 inhibitor or a drug or formulation containing a CPXM2 inhibitor. Such a product combination can also be used to assess the prognosis of a patient or subject treated with a CPXM2 inhibitor or a drug or formulation containing a CPXM2 inhibitor, to assist in the diagnosis of pancreatic cancer, or to detect the risk of developing pancreatic cancer.

[0154] Coefficient of drug interaction (CDI)

[0155] The coefficient of drug interaction (CDI) is a parameter used to calculate whether two drugs have a synergistic effect. The formula for calculating CDI is: CDI = AB / (A × B), where in vitro experiments are calculated based on the number of viable cells (absorbance value), and in vivo therapeutic experiments are calculated based on tumor weight.

[0156] AB represents the ratio of absorbance or tumor weight of the combined two-drug group to the control group, while A or B represents the ratio of absorbance or tumor weight of the individual drug group to the control group. When CDI < 1, it indicates a synergistic effect between the two drugs; when CDI < 0.7, the synergistic effect is very significant.

[0157] Main advantages of the invention

[0158] 1. This invention is the first to develop the use of the CPXM2 target (especially its DSD structure) in the treatment of pancreatic cancer.

[0159] 2. Through extensive testing, this invention has found that CPXM2-KO and antibodies targeting CPXM2-DSD have therapeutic effects on pancreatic cancer.

[0160] 3. This invention is the first to discover that antibodies targeting CPXM2-DSD can significantly enhance the therapeutic effect of gemcitabine, and antibodies targeting CPXM2-DSD also significantly enhance the therapeutic effect of PD1 monoclonal antibodies. These combined treatment regimens all have synergistic effects.

[0161] 4. This invention provides a new method and approach for the treatment of pancreatic cancer, offering a potential solution for improving the prognosis of pancreatic cancer patients.

[0162] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0163] Example 1: Detection of CPXM2 expression level in pancreatic cancer tissue

[0164] In this embodiment, the expression level of CPXM2 in adjacent and cancerous tissues of patients was detected by RT-qPCR, as well as the expression level of CPXM2 in pancreatic cancer tissue of KPC mice relative to that in pancreatic tissue of normal mice. Furthermore, immunofluorescence staining of CPXM2 was performed on adjacent and cancerous tissues of the patient's pancreas.

[0165] The results showed that the expression level of CPXM2 in the cancerous tissue of patients was significantly higher than that in the adjacent normal tissue, as detected by RT-qPCR and immunofluorescence. Similarly, the expression level of CPXM2 in pancreatic cancer tissue of KPC mice was significantly higher than that in pancreatic tissue of normal mice.

[0166] For example, such as Figure 1 As shown, immunofluorescence staining of CPXM2 in adjacent and cancerous tissues of the patient's pancreatic tumor revealed a certain degree of co-localization between CPXM2 and CAF cells (α-SMA as its marker), and the expression level of CPXM2 in cancerous tissue was significantly upregulated compared to that in adjacent tissue. Figure 2 As shown, by performing CPXM2 immunofluorescence staining on pancreatic tissues of normal mice and pancreatic cancer tissues of KPC mice, it was found that CPXM2 co-localizes with CAF cells (α-SMA is its marker), and the expression level of CPXM2 in pancreatic cancer tissues of KPC mice is significantly upregulated compared with that in pancreatic tissues of normal mice.

[0167] Example 2: Observation of the effect of CPXM2 overexpression on the occurrence and progression of pancreatic cancer

[0168] In this embodiment, CPXM2 overexpression was performed using adeno-associated virus, and 2×10^6 Panc02 pancreatic cancer cells were transplanted into mouse pancreatic tissue to observe the effect of CPXM2 overexpression on the occurrence and progression of pancreatic cancer.

[0169] Specifically, normal C57BL / 6J mice were injected orally with 2×10^6 Panc02 cells and 20 μl of control virus or AAV-CPXM2 overexpressing virus. Five days later, 50 μl of virus was added via tail vein. The occurrence and progression of pancreatic cancer were observed by in vivo fluorescence imaging at 12 and 26 days.

[0170] like Figure 3 As shown, the AAV-CPXM2 overexpression group had earlier pancreatic cancer development and faster progression compared to the control group, suggesting that CPXM2 promotes the occurrence and progression of pancreatic cancer. Therefore, it is speculated that using CPXM2 inhibitors to knock out or knock down CPXM2 expression can effectively inhibit the occurrence and progression of pancreatic cancer.

[0171] Example 3: Observation of the effect of CPXM2 knockout on the occurrence and progression of pancreatic cancer.

[0172] In this embodiment, by constructing CPXM2 flox / flox Pdx1-Cre + Pancreatic tissue-specific knockout CPXM2 mice (i.e., CPXM2-KO mice) were used to transplant 2×10^6 Panc02 pancreatic cancer cells into the pancreatic tissue of mice to observe the effect of CPXM2 knockout on the occurrence and progression of pancreatic cancer.

[0173] Specifically, 2×10^6 Panc02 cells were injected orally into the pancreas of normal C57BL / 6J mice and CPXM2-KO mice, and pancreatic tissue tumors were harvested at 45 days to observe tumor size.

[0174] like Figure 4 As shown, tumors in the CPXM2-KO group were significantly smaller than those in the WT group, suggesting that CPXM2 knockout inhibits the occurrence and progression of pancreatic cancer. Therefore, the experiment confirms that using CPXM2 inhibitors to knock out or reduce CPXM2 expression can effectively inhibit the occurrence and progression of pancreatic cancer.

[0175] Example 4: Treatment of two-month-old KPC mice with either a CPXM2-DSD-targeting antibody or a control antibody

[0176] In this embodiment, KPC mice were treated with 10 mg / kg IgG antibody or an antibody targeting the DSD domain of the CPXM2 protein (hereinafter referred to as CPXM2-DSD antibody) via tail vein injection at two months of age, twice a week for two consecutive weeks. The differences in pancreatic cancer progression and survival between the control group (IgG antibody group) and the antibody treatment group (CPXM2-DSD antibody group) were observed.

[0177] like Figure 5 As shown, the study found that the CPXM2-DSD antibody significantly prolonged the survival time of KPC mice, suggesting that the CPXM2-DSD antibody may have a therapeutic effect on pancreatic cancer. It should be understood that antibodies targeting the entire CPXM2 protein also have the same or similar therapeutic effect on pancreatic cancer as the CPXM2-DSD antibody.

[0178] Example 5: Treatment of mice with pancreatic in situ carcinoma with CPXM-DSD antibody in combination with gemcitabine or PD1 monoclonal antibody. treatment

[0179] In this embodiment, mice with pancreatic carcinoma in situ were treated with a combination of CPXM-DSD antibody and gemcitabine, and the difference in efficacy between the combination therapy and gemcitabine alone was observed; mice with pancreatic carcinoma in situ were also treated with a combination of CPXM-DSD antibody and PD1 monoclonal antibody, and the difference in efficacy between the combination therapy and PD1 monoclonal antibody was observed.

[0180] (1) CPXM-DSD antibody combined with gemcitabine:

[0181] Specifically, normal C57BL / 6J mice were injected orally with 2×10^6 Panc02 cells into the pancreas. Treatment was initiated 2 weeks later and divided into (1) control group, which was injected with an equal amount of non-specific IgG antibody; (2) 10 mg / kg CPXM2-DSDAb, injected via tail vein, twice a week for two weeks; (3) GEM, 50 mg / kg, injected intraperitoneally twice every two weeks for one month; (4) CPXM2-DSD Ab combined with GEM, with the same treatment regimen as above (i.e., 10 mg / kg CPXM2-DSD Ab + 50 mg / kg GEM). After treatment, the tumor size was observed.

[0182] like Figure 6 As shown in Table 1, the tumors in the CPXM2-DSD Ab group were smaller than those in the control group, suggesting that CPXM2-DSDAb itself has a therapeutic effect on pancreatic cancer; the tumors in the GEM+CPXM2-DSD Ab combined treatment group were significantly smaller than those in the GEM group, suggesting that CPXM2-DSD Ab enhances the efficacy of GEM.

[0183] Table 1

[0184]

[0185] According to the formula for calculating the coefficient of drug interaction (CDI): CDI = AB / (A×B), where the in vivo treatment trial is calculated based on tumor weight, AB is the ratio of tumor weight in the combined drug group to the control group, and A or B is the ratio of tumor weight in the drug-only group to the control group, the calculated CDI = (0.090 / 2.0735) / [(1.2972 / 2.0735)×(0.408 / 2.0735)]≈0.3526.

[0186] When CDI < 1, it indicates a synergistic effect between the two drugs; when CDI < 0.7, the synergistic effect is very significant. Therefore, the results show that the combination of the CPXM-DSD-targeting antibody and gemcitabine has a very significant synergistic effect.

[0187] (2) CPXM-DSD antibody combined with PD1 monoclonal antibody:

[0188] Specifically, normal C57BL / 6J mice were injected orally with 2×10^6 Panc02 cells into the pancreas. Treatment was initiated 2 weeks later and divided into (1) control group, which was injected with an equal amount of non-specific IgG antibody; (2) 10 mg / kg CPXM2-DSDAb, injected via tail vein, twice a week for two weeks; (3) PD1-mAb, 10 mg / kg, twice a week for two weeks; (4) CPXM2-DSD Ab and PD1-mAb combined treatment, with the same treatment regimen as above (i.e., 10 mg / kg CPXM2-DSD Ab + 10 mg / kg PD1-mAb). After treatment, the tumor size was observed.

[0189] like Figure 7 As shown in Table 2, the tumors in the CPXM2-DSD Ab group were smaller than those in the control group, suggesting that CPXM2-DSDAb itself has a therapeutic effect on pancreatic cancer; the tumors in the PD1-mAb + CPXM2-DSD Ab combination therapy group were significantly smaller than those in the PD1-mAb group, suggesting that CPXM2-DSD Ab enhances the efficacy of PD1-mAb.

[0190] Table 2

[0191]

[0192] According to the formula for calculating the coefficient of drug interaction (CDI): CDI = AB / (A×B), where the in vivo treatment trial is calculated based on tumor weight, AB is the ratio of tumor weight in the combined drug group to the control group, and A or B is the ratio of tumor weight in the drug-only group to the control group, the calculated CDI = (0.029 / 2.0735) / [(1.2972 / 2.0735)×(0.302 / 2.0735)]≈0.1534.

[0193] When CDI < 1, it indicates a synergistic effect between the two drugs; when CDI < 0.7, the synergistic effect is very significant. Therefore, the results show that the combination of the CPXM-DSD antibody and the PD1 monoclonal antibody has a very significant synergistic effect.

[0194] It should be understood that when antibodies targeting the entire CPXM2 protein are used in combination with gemcitabine or PD1 monoclonal antibodies, they have the same or similar synergistic therapeutic effect on pancreatic cancer as when antibodies targeting CPXM2-DSD are used.

[0195] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Use of a CPXM2 inhibitor for the manufacture of a composition or formulation, characterized in that, The composition or formulation is used for: (a) prevention and / or treatment of pancreatic cancer; and / or (b) inhibition of the growth of pancreatic cancer cells or pancreatic cancer tissue.

2. Use according to claim 1, characterized in that, The CPXM2 inhibitor is selected from the group consisting of small molecule compounds, antibodies, nucleic acid molecules, gene editors, or combinations thereof.

3. The use as described in claim 1, characterized in that, The pancreatic cancer is selected from the group consisting of: pancreatic ductal adenocarcinoma (PDAC), acinar cell carcinoma, adenosquamous carcinoma, small cell pancreatic carcinoma, pancreatic mucinous carcinoma, or a combination thereof.

4. The use of a CPXM2 gene, mRNA, cDNA, protein, or a detection reagent thereof, characterized in that, For preparing a kit for use in one or more of the following purposes: (i) Used to assess whether a patient with pancreatic cancer is suitable for treatment with a CPXM2 inhibitor or a drug or preparation containing a CPXM2 inhibitor; and / or (ii) To assess the prognosis of patients with pancreatic cancer treated with CPXM2 inhibitors or drugs or formulations containing CPXM2 inhibitors.

5. A product portfolio, characterized in that, The product portfolio includes: (a) a detection reagent or a kit containing said detection reagent, said detection reagent being a detection reagent for detecting the CPXM2 gene, mRNA, cDNA, protein, or combinations thereof; and (b) A pharmaceutical composition comprising a CPXM2 inhibitor as an active ingredient and a pharmaceutically acceptable carrier.

6. A pharmaceutical composition for synergistic treatment of pancreatic cancer, characterized in that, The pharmaceutical composition comprises: (Z1) The first active ingredient, a CPXM2 inhibitor; (Z2) The second active ingredient gemcitabine and / or the third active ingredient anti-PD1 antibody; and (Z3) Pharmaceutically acceptable carrier.

7. The pharmaceutical composition according to claim 6, characterized in that, The dosage of the first active ingredient, CPXM2 inhibitor, is 1-100 mg / kg, preferably 5-50 mg / kg, and more preferably 10 mg / kg.

8. A medicine box, characterized in that, The medicine box includes: (a) A first formulation comprising a CPXM2 inhibitor and a pharmaceutically acceptable carrier; and (b) a second formulation comprising gemcitabine and a pharmaceutically acceptable carrier; and / or a third formulation comprising an anti-PD1 antibody and a pharmaceutically acceptable carrier.

9. Use of a product combination as claimed in claim 5, a pharmaceutical composition as claimed in claim 6 or 7, or a pillbox as claimed in claim 8, characterized in that, Used to prepare medical products for the treatment of CPXM2-positive pancreatic cancer.

10. A method for inhibiting or synergistically inhibiting the growth of pancreatic cancer cells or pancreatic cancer tissue in vitro, characterized in that, The method includes the step of adding a CPXM2 inhibitor or the pharmaceutical composition as described in claim 6 or 7 to a culture system of pancreatic cancer cells or pancreatic cancer tissue, thereby inhibiting or synergistically inhibiting the growth of pancreatic cancer cells or pancreatic cancer tissue. Alternatively, the method may include the step of using the product combination as described in claim 5 or the kit as described in claim 8 to inhibit or synergistically inhibit the growth of pancreatic cancer cells or pancreatic cancer tissue.

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