Composition for chemotherapy of intrahepatic cholangiocarcinoma and application thereof

Abstract

The invention particularly discloses a composition for chemotherapy of intrahepatic cholangiocarcinoma and application of the composition, and relates to the technical field of biological medicine. The invention provides a pharmaceutical composition. The pharmaceutical composition comprises: (1) a therapeutically effective amount of an LCN2 inhibitor and a chemotherapeutic drug; and (2) a pharmaceutically or immunologically acceptable carrier or excipient. The LCN2 inhibitor is combined with gemcitabine and cis-platinum for use, so that the infiltration of killer CD8 + T lymphocytes can be remarkably improved, the growth and multiplication capacities of tumors are slowed down, and the chemical treatment effect of the intrahepatic cholangiocarcinoma is further enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a composition for intrahepatic bile duct cancer chemotherapy and application thereof. Background Art

[0002] Intrahepatic cholangiocarcinoma (ICC) is a malignant tumor originating from the epithelial cells of the intrahepatic bile ducts. It is highly invasive and has a poor prognosis. Its incidence has gradually increased in recent years. Statistics show that the five-year survival rate of intrahepatic cholangiocarcinoma is less than 10%, and most patients are already in the advanced stage when diagnosed, which makes treatment more difficult.

[0003] Current treatments include surgery, radiotherapy, and chemotherapy, but surgical resection is usually only suitable for early-stage patients, and the postoperative recurrence rate is high. For advanced patients who cannot undergo surgery, chemotherapy is usually the main treatment option. At present, gemcitabine and cisplatin are commonly used drugs in chemotherapy for intrahepatic cholangiocarcinoma. As an anti-metabolite drug, gemcitabine can inhibit DNA synthesis in tumor cells, thereby exerting an anti-tumor effect. However, when gemcitabine is used alone, it often encounters drug resistance problems, which limits its therapeutic effect. Cisplatin is a platinum-based chemotherapeutic agent that can inhibit cell division and induce cell apoptosis by forming DNA cross-linking substances. However, in clinical applications, cisplatin also has high toxic side effects and drug resistance problems.

[0004] In recent years, targeted therapy and immunotherapy have received increasing attention, among which treatment strategies targeting FGFR2 and IDH1 have become mature. However, single therapy often fails to achieve the desired therapeutic effect, so there is an urgent need to explore new treatment methods or combine other treatments to further improve clinical efficacy and thus improve the prognosis of intrahepatic cholangiocarcinoma. Summary of the invention

[0005] 1. Technical issues to be solved

[0006] In view of this, one of the main purposes of the present invention is to provide a use of a lipocalin 2 (LCN2) inhibitor and a chemotherapeutic drug in the preparation of a drug for preventing and / or treating cholangiocarcinoma and / or diseases and / or symptoms associated with cholangiocarcinoma, by targeting the lipocalin LCN2, combined with chemotherapeutic drugs gemcitabine and cisplatin, to solve the problems of poor treatment effect and chemotherapy resistance of intrahepatic cholangiocarcinoma. Compared with the use of chemotherapeutic drugs alone, the combined use can significantly increase the number of cytotoxic CD8 +The number of T lymphocytes, thereby effectively slowing down the growth and proliferation ability of tumors. In addition, the combined use significantly enhances the efficacy of chemotherapy for intrahepatic cholangiocarcinoma, providing new treatment ideas and intervention strategies for the systemic treatment of intrahepatic cholangiocarcinoma. This discovery has important significance for the clinical treatment of intrahepatic cholangiocarcinoma.

[0007] (II) Technical solution

[0008] To achieve the above object, the present invention provides a pharmaceutical composition, which comprises:

[0009] (1) A therapeutically effective amount of an LCN2 inhibitor and a chemotherapeutic drug;

[0010] (2) A pharmaceutically or immunologically acceptable carrier or excipient.

[0011] In one embodiment, the LCN2 inhibitor is administered before, simultaneously with, or after the administration of the chemotherapeutic drug.

[0012] In one embodiment, the LCN2 inhibitor comprises an antibody, siRNA, miRNA, gRNA, and antisense oligonucleotide against LCN2 or a nucleic acid molecule encoding the protein, or a combination thereof.

[0013] In one embodiment, the LCN2 inhibitor is an LCN2 antibody.

[0014] In one embodiment, the LCN2 antibody comprises one or a combination of OACA01413, MHF70701, and RD78709A.

[0015] In one embodiment, the LCN2 antibody is Cat#AF1857, Novus Biologicals.

[0016] In one embodiment, the chemotherapeutic drug comprises one or a combination of gemcitabine, cisplatin, fluorouracil, oxaliplatin, irinotecan, capecitabine, tegafur, paclitaxel, docetaxel, raltitrexed, and liposomal doxorubicin.

[0017] In one embodiment, the chemotherapeutic drug is gemcitabine and cisplatin.

[0018] The present invention also provides, in another aspect, the use of the above pharmaceutical composition in the preparation of a drug for preventing and / or treating cholangiocarcinoma and / or a disease and / or symptom associated with cholangiocarcinoma.

[0019] The present invention also provides, in another aspect, a pharmaceutical preparation, which comprises the above pharmaceutical composition.

[0020] The present invention also provides, in another aspect, the use of the above-mentioned pharmaceutical preparation in the preparation of a drug for preventing and / or treating cholangiocarcinoma and / or diseases and / or symptoms related to cholangiocarcinoma.

[0021] In one embodiment, the cholangiocarcinoma in the above use includes one or a combination of intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma, and extrahepatic cholangiocarcinoma.

[0022] In one embodiment, the cholangiocarcinoma is intrahepatic cholangiocarcinoma.

[0023] In one embodiment, the dosage ratio of the drugs in the above use is LCN2 inhibitor: cisplatin: gemcitabine = (1 - 2): (30 - 40): (1 - 2).

[0024] In one embodiment, the dosage of the LCN2 inhibitor is 5 - 10 mg / kg.

[0025] In one embodiment, the dosage of the LCN2 inhibitor is 5 mg / kg.

[0026] In one embodiment, the dosage of gemcitabine is 150 - 200 mg / kg.

[0027] In one embodiment, the dosage of gemcitabine is 200 mg / kg.

[0028] In one embodiment, the dosage of cisplatin is 5 - 10 mg / kg.

[0029] In one embodiment, the dosage of cisplatin is 5 mg / kg.

[0030] In one embodiment, the LCN2 inhibitor and the chemotherapeutic drug are administered by intraperitoneal injection.

[0031] In one embodiment, the subject to be administered is healthy.

[0032] In one embodiment, the subject to be administered is non - healthy.

[0033] In one embodiment, the subject to be administered has cholangiocarcinoma.

[0034] In one embodiment, the subject to be administered does not have cholangiocarcinoma.

[0035] In one embodiment, the subject to be administered is resistant to the above chemotherapeutic drugs.

[0036] In one embodiment, the subject to be administered is sensitive to the above chemotherapeutic drugs.

[0037] In one embodiment, the subjects include mammals and non - mammals.

[0038] Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cows, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs. Examples of non-mammals include, but are not limited to, birds, fish, or other non-mammals, etc.

[0039] In one embodiment, the subject is a mouse.

[0040] Beneficial effects

[0041] The present invention provides a pharmaceutical composition, which comprises: (1) a therapeutically effective amount of an LCN2 inhibitor and a chemotherapeutic drug; (2) a pharmaceutically or immunologically acceptable carrier or excipient. Compared with the prior art, the following beneficial effects are achieved:

[0042] 1. Enhanced anti-tumor effect: The combined use of the lipocalin LCN2 monoclonal antibody and chemotherapeutic drugs (gemcitabine and cisplatin) can significantly enhance the anti-tumor effect of chemotherapy.

[0043] 2. Improvement of the tumor immune microenvironment: The combination therapy can increase the infiltration of cytotoxic CD8 + T lymphocytes in the tumor microenvironment, thereby enhancing the immune surveillance of tumor cells and contributing to the long-term anti-tumor process.

[0044] 3. Reduction of chemotherapy resistance and side effects: The combination therapy helps to reduce the phenomenon of chemical resistance and drug side effects in drug treatment, providing new treatment ideas and intervention means for the systemic treatment of intrahepatic cholangiocarcinoma.

[0045] Terms and definitions

[0046] As used herein, the terms "LCN2" and "LCN2 protein" are used interchangeably and refer to lipocalin 2. The proteins disclosed in the present invention can be natural purified products, or chemically synthesized products, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (e.g., bacteria, yeast, higher animals, insects, and mammalian cells). In the present disclosure, the LCN2 protein is preferably encoded by the human LCN2 gene or its homologous gene or family gene.

[0047] As used herein, the terms "inhibitor" or "inhibitor of LCN2 or its coding nucleic acid molecule" are used interchangeably and refer to a substance that can reduce the level or activity of LCN2 or its coding nucleic acid molecule. In some embodiments, the LCN2 inhibitor includes, but is not limited to, antibodies, siRNA, miRNA, gRNA, and antisense oligonucleotides against LCN2 or the nucleic acid molecule encoding this protein.

[0048] As used herein, the term "antibody" includes glycosylated and non-glycosylated immunoglobulins of any isotype or subclass mentioned, or antigen-binding regions thereof that compete with intact antibodies for specific binding, including monoclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies, antibody mimetics, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, antibody conjugates, single-chain antibodies, antibody derivatives, antibody analogs, and their corresponding fragments. Also included are immunological fragments of antibodies (e.g., Fab, Fab’, F(ab’)2 or scFv), whether such antibodies are produced in whole or in part by immunization, by recombinant techniques, by in vitro synthetic means, or by other methods. Thus, as used herein, the term "antibody" includes antibodies prepared, expressed, produced, or isolated by recombinant means, such as (a) antibodies isolated from transgenic animals (e.g., mice) with human immunoglobulin genes or from hybridomas prepared with human immunoglobulin genes; (b) antibodies isolated from host cells transfected to express antibodies (e.g., from transfectomas); (c) antibodies isolated from recombinant combinatorial antibody libraries; and (d) antibodies prepared, expressed, produced, or isolated by any other means involving splicing of immunoglobulin gene sequences to other DNA sequences. These antibodies have variable and constant regions of germline immunoglobulin sequences from two different animal species. However, in certain embodiments, these antibodies may be subjected to in vitro mutagenesis (or, when using transgenic animals with human immunoglobulin sequences, in vivo somatic mutagenesis), and thus, the amino acid sequences of the VH and VL regions of these antibodies, although derived from and related to the germline VH and VL sequences of a particular species (e.g., human), may not be sequences that occur naturally in the antibody germline repertoire of that species in vivo. Unless otherwise indicated, the term "antibody" includes, in addition to antibodies containing two full-length heavy chains and two full-length light chains, their derivatives, variants, fragments, and mutant proteins. In some instances, an "antibody" may include fewer chains, such as antibodies that naturally occur in camels and may contain only heavy chains.

[0049] As used herein, the terms "patient" or "subject" are used interchangeably and refer to mammals and non-mammals. Examples of mammals include, but are not limited to, any member of the class Mammalia: humans, non-human primates such as chimpanzees and other apes and monkeys; farm animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs, and cats; laboratory animals, including rodents such as rats, mice, and guinea pigs, etc. Examples of non-mammals include, but are not limited to, birds, fish, or other non-mammals, etc.

[0050] As used herein, the term "pharmaceutical composition" refers to a composition comprising an immune checkpoint inhibitor and / or a histone acetyltransferase inhibitor formulated with one or more pharmaceutically acceptable carriers.

[0051] The formulation of the pharmaceutical composition can be adjusted according to the application. In particular, the pharmaceutical composition can be formulated by methods known in the art to provide rapid, continuous, or delayed release of the active ingredient after administration to a mammal.

[0052] The dosage forms of the pharmaceutical composition disclosed in the present invention can be granules, tablets, lyophilized powders, suppositories, capsules, sublingual tablets, liquid solutions, nasal drops, sprays, metered spray forms.

[0053] Any known method can be used to administer the pharmaceutical composition of the present invention. One of the various methods known to those skilled in the art can be used to effect the term "administering" or "applying" a substance, compound, or agent to a subject.

[0054] For example, the compound or agent can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ophthalmically, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). The compound or reagent can also be appropriately introduced through a rechargeable or biodegradable polymeric device or other device (e.g., patches and pumps or formulations) that provide extended, slowed, or controlled release of the compound, reagent. Administration can also be effected, for example, once, multiple times, and / or over one or more extended periods.

[0055] As used herein, the term "pharmaceutically acceptable" ingredient is a substance that is suitable for use in humans and / or animals without undue adverse reactions (such as toxicity, irritation, and allergic response), i.e., a substance having a reasonable benefit / risk ratio.

[0056] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" is a carrier for the administration of a therapeutic agent, including various excipients and diluents. The term refers to those pharmaceutical carriers that are not necessarily the active ingredient itself and that are not unduly toxic when administered. Suitable carriers are well known to those of ordinary skill in the art, and a thorough discussion of pharmaceutically acceptable excipients can be found in Remington’s Pharmaceutical Sciences, Mack Pub. Co., N.J. 1991.

[0057] Pharmaceutically acceptable carriers in the composition include any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH regulators, buffers, enhancers, wetting agents, solubilizers, surfactants, antioxidants, etc. that are compatible with drug administration. The use of such media and agents for active pharmaceutical substances is well known in the art. The composition may contain other active compounds that provide supplementary, additional or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch or talc, or liquid carriers, such as water, fatty oils or liquid paraffin. Other examples of the carrier include culture media, such as DMEM or RPMI; and cryopreservation media, containing components that scavenge free radicals, provide pH buffering, osmotic / osmolarity support, energy substrates and ion concentrations, which can balance the intracellular state at low temperatures; and mixtures of organic solvents and water.

[0058] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and effective dose levels include factors such as the type and severity of the subject, age, gender, drug activity, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.

[0059] As used herein, the term "treating" a condition or patient means taking steps to achieve a beneficial or desired result, including a clinical result. Beneficial or desired clinical results include, but are not limited to, alleviating, substantially inhibiting, slowing or reversing the progression of a disease, condition or disorder, substantially improving or alleviating the clinical or aesthetic symptoms of a condition, substantially preventing the clinical or aesthetic symptoms of a disease, condition or disorder, and avoiding adverse or unwanted symptoms. Treatment also refers to accomplishing one or more of the following: (a) reducing the severity of the condition; (b) limiting the development of characteristic symptoms of the condition being treated; (c) limiting the worsening of characteristic symptoms of the condition being treated; (d) limiting the recurrence of the condition in patients previously suffering from the condition; and / or (e) limiting the recurrence of symptoms in patients previously without symptoms of the condition.

[0060] As used herein, the term "preventing" means reducing the likelihood of the onset (or recurrence) of a disease, disorder, condition or associated symptoms. Description of the Drawings

[0061] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other accompanying drawings can be obtained based on these drawings.

[0062] Figure 1 is the growth of tumors in combination therapy.

[0063] Figure 2 is the proportion of CD8 + T lymphocytes, the proportion of secreted Granzyme B and IFN-γ shown by tissue flow cytometry in combination therapy.

[0064] Figure 3 is the infiltration of cytotoxic CD8 + T cells in different treatment groups of combination therapy.

[0065] Figure 4 is the combination index (CI) value of combination therapy.

[0066] Figure 5 is the growth of tumors in combination therapy with half the dose.

[0067] Figure 6 is the proportion of CD8 + T lymphocytes, the proportion of secreted Granzyme B and IFN-γ shown by tissue flow cytometry in combination therapy with half the dose.

[0068] Figure 7 is the infiltration of cytotoxic CD8 + T cells in different treatment groups of combination therapy with half the dose. Detailed implementation manners

[0069] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.

[0070] As used herein, "comprising", "having", or "including" includes "containing", "consisting essentially of...", "consisting substantially of...", and "consisting of..."; "consisting essentially of...", "consisting substantially of...", and "consisting of..." are subordinate concepts of "comprising", "having", or "including".

[0071] The experimental methods used in the following examples are all conventional methods unless otherwise specified. The reagents, methods, and equipment used are all conventional reagents, methods, and equipment in the technical field unless otherwise specified.

[0072] Example 1

[0073] Effect of combination therapy on tumor growth:

[0074] Mix 50 μL of Matrigel with 50 μL of medium containing 3×10 6 mIC-22 cells and inject the mixture subcutaneously into the right side of C57BL / 6J mice. Two weeks after cell injection, the mice are randomly divided into four groups: the first group is the control group, which is injected with the same volume of normal saline every 72 hours; the second group is injected with 5 mg / kg of Cat#AF1857 every 3 days; the third group is injected with 200 mg / kg of gemcitabine and 5 mg / kg of cisplatin every 4 days; the fourth group is injected with 5 mg / kg of Cat#AF1857 every 3 days and 200 mg / kg of gemcitabine and 5 mg / kg of cisplatin every 4 days. The experiment is continued until the tumor volume of one group of mice reaches 1500 mm 3 at which point the injection of each group of mice is stopped. The tumor length (L) and width (W) of each group of mice are measured weekly, and the tumor volume is calculated using the formula V = (L × W 2 ) / 2.

[0075] The results are as Figure 1 shown. In the experimental group with combined drug use, the tumor growth rate is significantly slowed down and the tumor volume is significantly reduced.

[0076] To further verify the synergistic effect of Cat#AF1857, gemcitabine, and cisplatin, CD8 T cells, neutrophils, and cholangiocarcinoma cells are co-cultured in vitro at a ratio of 1:1:1, and Anti-LCN2 and Gem + Cis are added. The activity of cholangiocarcinoma cells is detected by CCK8, and the combination index (CI) value is calculated.

[0077] The results are as Figure 4 shown. Using the Chou-Talalay method to calculate the combination index (CI) value, there is a synergistic interaction between Anti-LCN2 and Gem + Cis in cholangiocarcinoma cells (the CI at ED50 is 0.48232), rather than an additive effect.

[0078] Example 2

[0079] Effect of combination therapy on the proportion of cytotoxic CD8 + T cells in mouse tumor tissues:

[0080] The mouse tumor tissues of each test group and control group in Example 1 were cut into pieces and ground, and the resulting mixtures were filtered through a 70 μm cell filter, and then centrifuged at 700g for 5 minutes. The supernatant was discarded, 10 ml of red blood cell lysis solution (White Shark, Catalog No. BL503B) was added, and the mixture was shaken slowly for 10 minutes at 4°C. After sufficient lysis, it was centrifuged at 700g for 10 minutes. The supernatant was discarded, and the cell suspension was washed in PBS containing 2% fetal bovine serum (Gibco). Next, the cell suspension was treated with an Fc receptor antibody to reduce nonspecific binding. For extracellular staining, antibodies CD45 Biolegend cat: 103138; CD3 Biolegend cat: 100248; CD8 Biolegend cat: 100734 were used, incubated at 4°C for 60 minutes, then washed twice with PBS, and then analyzed. For intracellular staining, cells were first fixed with 4% paraformaldehyde for 15 minutes, then permeabilized with 1% Triton X-100 buffer, and finally stained with intracellular marker antibodies gzmb Biolegend cat:372222; IFN-r Biolegend cat:113606. After staining, cells were suspended in cell staining buffer and then analyzed by flow cytometry.

[0081] The results are as follows Figure 2 Compared with the use of LCN2 monoclonal antibody or gemcitabine and cisplatin chemotherapy alone, the combination of LCN2 and gemcitabine and cisplatin chemotherapy significantly increased CD8 + The proportion of T lymphocytes increased significantly, and the proportion of Granzyme B and IFN-γ secreted by them also increased significantly.

[0082] Example 3

[0083] Combination therapy for CD8 + Effects of T cell infiltration:

[0084] The tumor tissues of the mice in each experimental group and the control group in Example 1 were cut into slices 5 mm thick and baked at 60 °C for 30 minutes. Then, the slices were incubated in xylene I for 5 minutes, xylene II for 5 minutes, xylene III for 5 minutes, absolute ethanol for 1 minute, 95% ethanol for 1 minute, 75% ethanol for 1 minute, and distilled water for 5 minutes to complete dewaxing and dehydration. Then, the slices were subjected to antigen retrieval in a 10 mM citrate buffer at pH 6.0 at 98 °C for 10 minutes. Subsequently, the slices were treated with 3% hydrogen peroxide to quench the activity of endogenous peroxidase for 10 minutes; then washed three times with PBST, 5 minutes each time. Next, diluted CD8 (cat: 98941 1:1000) was incubated at 4 °C overnight. After washing three times with PBST (3 minutes each time), the residual washing solution on the glass slide was removed, and the working solution of the secondary antibody corresponding to the primary antibody (HRP-labeled) was directly added dropwise and incubated at room temperature with moisture for 30 minutes. Again, washed three times with PBST, 5 minutes each time, and after removing the residual washing solution, TSA working solution was added dropwise and incubated at room temperature with moisture for 10 - 15 minutes.

[0085] If multiple staining (to detect multiple targets) was performed, the above experimental procedure was repeated after antigen retrieval, and finally observed and photographed with a confocal microscope.

[0086] The results were as Figure 3 shown, and combination therapy significantly increased the infiltration of cytotoxic CD8 + T cells, indicating that combination therapy improved the therapeutic effect on intrahepatic cholangiocarcinoma.

[0087] Example 4:

[0088] Effect of combination therapy with halved doses on tumor growth:

[0089] 50 μL of Matrigel was mixed with 50 μL of the culture medium containing 3×10 6 mIC-22 cells and subcutaneously injected into the right side of C57BL / 6J mice. Two weeks after cell injection, the mice were randomly divided into four groups: the first group was the control group, and the same volume of normal saline was injected every 72 hours; the second group was injected with 100 μg Cat#AF1857 every 3 days; the third group was injected with 200 mg / kg of gemcitabine and 5 mg / kg of cisplatin every 4 days, and the fourth group was injected with 50 μg Cat#AF1857 every 3 days and 100 mg / kg of gemcitabine and 2.5 mg / kg of cisplatin every 4 days. The experiment was continued until the tumor volume of one group of mice reached 1500 mm 3 at which time the injection of each group of mice was stopped. The tumor length (L) and width (W) of each group of mice were measured weekly, and the formula V = (L×W 2) / 2 to calculate the tumor volume.

[0090] result Figure 5 As shown, the tumor growth rate in the experimental group with half the dose and combined drug use was significantly slowed down and the tumor volume was significantly reduced.

[0091] Example 5

[0092] Effect of half-dose combined therapy on cytotoxic CD8 in tumor tissue of mice + Effect of T cell ratio:

[0093] The mouse tumor tissues of each test group and control group in Example 4 were cut into pieces and ground, and the resulting mixtures were filtered through a 70 μm cell filter, and then centrifuged at 700g for 5 minutes. The supernatant was discarded, 10 ml of red blood cell lysis solution (White Shark, Catalog No. BL503B) was added, and the mixture was shaken slowly for 10 minutes at 4°C. After sufficient lysis, it was centrifuged at 700g for 10 minutes. The supernatant was discarded, and the cell suspension was washed in PBS containing 2% fetal bovine serum (Gibco). Next, the cell suspension was treated with an Fc receptor antibody to reduce nonspecific binding. For extracellular staining, antibodies CD45 Biolegend cat: 103138; CD3 Biolegend cat: 100248; CD8 Biolegend cat: 100734 were used, incubated at 4°C for 60 minutes, then washed twice with PBS, and then analyzed. For intracellular staining, cells were first fixed with 4% paraformaldehyde for 15 minutes, then permeabilized with 1% Triton X-100 buffer, and finally stained with intracellular marker antibodies gzmb Biolegend cat:372222; IFN-r Biolegend cat:113606. After staining, cells were suspended in cell staining buffer and then analyzed by flow cytometry.

[0094] result Figure 6 Compared with the use of LCN2 monoclonal antibody or gemcitabine and cisplatin chemotherapy alone, the CD8 + The proportion of T lymphocytes increased significantly, and the proportion of Granzyme B and IFN-γ secreted by them also increased significantly.

[0095] Example 6

[0096] Effect of halved dose combination therapy on CD8 + Effects of T cell infiltration:

[0097] The tumor tissues of the mice in each experimental group and the control group in Example 1 were cut into 5-mm thick slices and baked at 60 °C for 30 minutes. Subsequently, the slices were incubated in xylene I for 5 minutes, xylene II for 5 minutes, xylene III for 5 minutes, absolute ethanol for 1 minute, 95% ethanol for 1 minute, 75% ethanol for 1 minute, and distilled water for 5 minutes to complete dewaxing and dehydration. Then, the slices were subjected to antigen retrieval in a 10 mM citrate buffer at pH 6.0 at 98 °C for 10 minutes. Subsequently, the slices were treated with 3% hydrogen peroxide to quench the activity of endogenous peroxidase for 10 minutes; then washed three times with PBST, 5 minutes each time. Next, diluted CD8 (cat: 98941 1:1000) was incubated overnight at 4 °C. After washing three times with PBST (3 minutes each time), the residual washing solution on the glass slide was removed, and the working solution of the secondary antibody corresponding to the primary antibody (HRP-labeled) was directly added dropwise and incubated at room temperature for 30 minutes with moisturization. Again, washed three times with PBST, 5 minutes each time, and after removing the residual washing solution, the TSA working solution was added dropwise and incubated at room temperature for 10 - 15 minutes with moisturization.

[0098] If multiple staining was performed (to detect multiple targets), the above experimental procedure was repeated after antigen retrieval was completed, and finally observed and photographed with a confocal microscope.

[0099] The results were as Figure 7 shown, and combination therapy significantly increased the infiltration of cytotoxic CD8 + T cells, indicating that combination therapy improved the therapeutic effect on intrahepatic cholangiocarcinoma.

[0100] It should be noted that in this article, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device. Without further limitation, an element defined by the statement "comprising an..." does not exclude the existence of additional identical elements in the process, method, article or device comprising the element.

[0101] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (1) a therapeutically effective amount of an LCN2 inhibitor and a chemotherapeutic agent; (2) a pharmaceutically or immunologically acceptable carrier or excipient.

2. The pharmaceutical composition according to claim 1, wherein The LCN2 inhibitor is administered before, simultaneously with, or after the administration of the chemotherapeutic agent.

3. The pharmaceutical composition according to claim 2, wherein The LCN2 inhibitor comprises one or a combination of OACA01413, MHF70701, RD78709A, Cat#AF1857.

4. The pharmaceutical composition according to claim 3, wherein The LCN2 inhibitor is Cat#AF1857.

5. The pharmaceutical composition according to claim 2, characterized in that, The chemotherapeutic agent comprises one or a combination of gemcitabine, cisplatin, fluorouracil, oxaliplatin, irinotecan, capecitabine, tegafur, paclitaxel, docetaxel, raltitrexed, and liposomal doxorubicin.

6. The pharmaceutical composition according to claim 5, wherein, The chemotherapeutic agent is gemcitabine and cisplatin.

7. Use of the pharmaceutical composition according to any one of claims 1-6 in the preparation of a medicament for preventing and / or treating cholangiocarcinoma and / or a disease and / or symptom associated with cholangiocarcinoma.

8. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the pharmaceutical composition according to any one of claims 1-6 or the use according to claim 7.

9. Use of the pharmaceutical preparation according to claim 8 in the preparation of a medicament for preventing and / or treating cholangiocarcinoma and / or a disease and / or symptom associated with cholangiocarcinoma.

10. The application according to claim 7 or 9, characterized in that The cholangiocarcinoma comprises one or a combination of intrahepatic cholangiocarcinoma, hilar cholangiocarcinoma, and extrahepatic cholangiocarcinoma.

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