Use of exosomes in reversing tumor resistance to immune checkpoint inhibitors

Abstract

This invention relates to the application of exosomes in reversing tumor resistance to immune checkpoint (PD-1) inhibitors. A novel application of exosomes in the preparation of pharmaceutical compositions reversing tumor resistance to immune checkpoint inhibitors is disclosed. Exosomes generated from tumor cells recombinantly expressing adenylate cyclase 7 (ADCY7) can significantly reverse tumor resistance to PD-1 inhibitors, and the exosomes exhibit a synergistic effect with the PD-1 inhibitors.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the application of exosomes in reversing tumor resistance to immune checkpoint inhibitors. Background Technology

[0002] Although significant progress has been made in tumor surgery and drug therapy in recent years, many patients still do not experience a marked improvement in prognosis. Immunotherapy has demonstrated potent anti-tumor activity in the treatment of various cancers; for example, PD-1 / PD-L1 antibodies have shown significant efficacy in patients with advanced melanoma and non-small cell lung cancer. Hepatocellular carcinoma (HCC), for instance, remains a leading cause of cancer death worldwide, characterized by low early diagnosis rates, high recurrence rates after surgery, and high metastasis rates. Currently, tumor immunotherapy primarily targets CD8+ in the tumor microenvironment. + T cells suppress tumors by activating or enhancing their tumor-killing ability. However, disappointingly, immunotherapy is only effective for a small percentage of patients in clinical practice, and the disease continues to progress in most patients with prolonged treatment. This is because, on the one hand, tumor cells can continuously evolve to evade immune recognition; on the other hand, tumor cells can suppress CD8 by reshaping the tumor microenvironment. + T cell infiltration and function, such as suppressing CD8 by recruiting regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs). + T cell function is impaired, thereby accelerating tumor progression. Therefore, in-depth research into the molecular mechanisms by which cancer cells regulate T cell immunity, discovering new drug targets, and finding new ways to reverse tumor resistance to existing drugs are of significant clinical importance for improving the efficacy of immunotherapy.

[0003] The tumor microenvironment (TME) is composed of tumor cells, immune cells, stromal cells, and extracellular components. A key characteristic of the TME is the interaction between various cell types. Crosstalk between different cell types plays a significant role not only in the occurrence, development, and metastasis of tumors but also influencing the effectiveness of clinical treatment.

[0004] Immunotherapy primarily works by activating the body's immune system to eliminate tumor cells. Unlike traditional surgery, chemotherapy, radiotherapy, and targeted therapy, which target tumor cells and tissues, immunotherapy targets the body's own immune system. For example, it can utilize PD-1 / PD-L1 antibodies to regulate CD8+ in the tumor microenvironment (TME). +T cell killing activity or reduced recruitment of immunosuppressive cells (such as regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSC) cells) can significantly enhance the function of the patient's immune system and improve the patient's prognosis. Summary of the Invention

[0005] The purpose of this invention is to provide the application of ADCY7 overexpression in promoting T cell infiltration in the immune microenvironment of liver cancer.

[0006] In a first aspect of the invention, there is provided an application of exosomes for preparing a pharmaceutical composition that reverses tumor resistance to immune checkpoint (PD-1) inhibitors; said exosomes containing adenylate cyclase 7 (ADCY7) protein.

[0007] In one or more embodiments, the exosomes are exosomes isolated from tumor cells, and the tumor cells recombinantly express (overexpress) exogenous adenylate cyclase 7; preferably, the method of recombinantly expressing (overexpressing) exogenous adenylate cyclase 7 includes: introducing an expression construct (such as an expression vector) into the tumor cells, the expression construct containing an expression cassette of adenylate cyclase 7; more preferably, the tumor cells are liver cancer cells.

[0008] In one or more embodiments, the method for preparing the exosomes includes: amplifying and culturing tumor cells that recombinantly express (overexpress) exogenous adenylate cyclase 7, then washing the cells, culturing them in serum-free culture medium to release the exosomes, and separating and purifying the exosomes.

[0009] In one or more embodiments, in the preparation method: after amplification culture to a confluence of 70-95% (preferably 75-95%, more preferably 80-92%, more preferably 85-90%), the cells are washed.

[0010] In one or more embodiments, amplification culture is performed using high-glucose DMEM medium containing 10±3% serum; and / or.

[0011] In one or more embodiments, the culture medium is incubated for 36-60 hours, preferably 42-55 hours, and more preferably 45-52 hours.

[0012] In one or more embodiments, exosomes are separated and purified by ultracentrifugation.

[0013] In one or more embodiments, the immune checkpoint (PD-1) inhibitor includes an anti-PD-1 antibody.

[0014] In one or more embodiments, the application of the exosomes is for non-therapeutic purposes.

[0015] In one or more embodiments, the amino acid sequence of the adenylate cyclase 7 is shown in SEQ ID NO:2.

[0016] In one or more embodiments, the exosomes are used in combination with or in combination with the immune checkpoint inhibitors.

[0017] In one or more embodiments, the tumor includes: liver cancer, pancreatic cancer, and lung cancer.

[0018] In another aspect of the invention, a pharmaceutical combination for reversing tumor drug resistance and inhibiting tumors is provided, the pharmaceutical combination comprising: exosomes and an immune checkpoint (PD-1) inhibitor; wherein the exosomes contain adenylate cyclase 7 protein.

[0019] In one or more embodiments, the exosomes are exosomes isolated from tumor cells, and the tumor cells recombinantly express (overexpress) adenylate cyclase 7; preferably, the method for preparing the exosomes includes: expanding and culturing tumor cells, then washing the cells, culturing them in a serum-free culture medium to release the exosomes, and separating and purifying the exosomes; more preferably, the tumor cells are liver cancer cells.

[0020] In one or more embodiments, the weight ratio of the exosomes to the immune checkpoint (PD-1) inhibitor in the drug combination is 1:(5-200); preferably 1:(10-100).

[0021] In one or more embodiments, the weight ratio of the exosomes to the immune checkpoint (PD-1) inhibitor is, for example, 1:8, 1:15, 1:20, 1:30, 1:40, 1:50, 1:60, 1:80, 1:100, 1:120, or 1:150.

[0022] In another aspect of the invention, the use of a pharmaceutical combination in a kit prepared to reverse tumor drug resistance and inhibit tumors is provided.

[0023] In one or more embodiments, the kit may further include one or more of the following: chemotherapy drugs for tumors; radiotherapy drugs for tumors; adjuvant therapy drugs; nutritional preparations; and / or instructions for use.

[0024] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0025] Figure 1 These are control cells (Hepa 1-6) NC ADCY7 overexpressing cells (Hepa 1-6) ADCY7ADCY7 protein expression level and Hepa 1-6 NC and Hepa 1-6 ADCY7 Detection of cell proliferation capacity. Among them... Figure 1 A represents the ADCY7 expression level in Hepa 1-6ADCY7 cells compared to Hepa 1-6NC cells; Figure 1 B is the CCK-8 result; Figure 1 C represents the subcutaneous tumor-bearing results in NSG mice. NC: The negative control is a control virus carrying an empty plasmid.

[0026] Figure 2 It is Hepa 1-6 NC and Hepa 1-6 ADCY7 The influence of cells on the immune microenvironment. Figure 2 A is an image of a subcutaneous tumor in a mouse. Figure 2 B is the growth curve of subcutaneous tumors in mice.

[0027] Figure 3 The assay was performed using flow cytometry to verify the presence of Hepa 1-6. NC and Hepa 1-6 ADCY7 Changes in the infiltration and function of immune cells in the group (AB).

[0028] Figure 4 The immunohistochemical staining method was used to verify the presence of Hepa 1-6. NC and Hepa 1-6 ADCY7 Changes in immune cells in the group (AB).

[0029] Figure 5 Electron microscopy and Western blot methods were used to verify the results from Hepa 1-6. NC and Hepa 1-6 ADCY7 Exosomes obtained by ultracentrifugation in cells (named NC-exo and ADCY7-exo, respectively) and verification of ADCY7 presence in Hepa 1-6 ADCY7 In cell-derived exosomes (AB).

[0030] Figure 6 The study investigated the changes in tumor growth and intratumoral immune microenvironment (AE) after injection of NC-exo and ADCY7-exo into a subcutaneous tumor-bearing model in C57BL / 6J mice.

[0031] Figure 7This study established a subcutaneous tumor-bearing model in C57BL / 6J mice using the PD-1-tolerant Hepa 1-6R cell line and the LLC and Pan02 cell lines, which were poorly responsive to PD-1. Mice were injected with NC-exo and ADCY7-exo, respectively, and some mice were given a combination of PD-1 inhibitors to observe tumor growth under different treatment conditions (AC). Detailed Implementation

[0032] Through in-depth research, the inventors have revealed a novel application of exosomes in the preparation of pharmaceutical compositions that reverse tumor resistance to immune checkpoint (PD-1) inhibitors. The inventors have also discovered that exosomes generated from tumor cells recombinantly expressing adenylate cyclase 7 (ADCY7) can significantly reverse tumor resistance to PD-1 inhibitors, and that the exosomes exhibit a synergistic effect with the PD-1 inhibitors. Therefore, based on this and other aspects, the inventors have proposed this invention.

[0033] As used herein, an "expression cassette" refers to a gene expression system containing all the necessary elements required to express a target gene (ADCY7 in this invention), typically including the following elements: a promoter, the target gene sequence, and a terminator; additionally, it may optionally include a signal peptide coding sequence, etc. These elements are operatively linked.

[0034] As used herein, “operationally linked,” “operationally connected,” or “operationally constructed” refers to the functional spatial arrangement of two or more nucleic acid regions or sequences. For example, a promoter region is placed at a specific position relative to the nucleic acid sequence of a target gene, such that transcription of the nucleic acid sequence is guided by the promoter region, thereby the promoter region is “operationally linked” to the nucleic acid sequence.

[0035] As used herein, “overexpression” refers to an intracellular ADCY7 level (e.g., expression level) that significantly exceeds the level in the naïve cells (cells without the foreign gene); such as a 20% increase, preferably 50% increase, more preferably over 100% increase, such as 200%, 300%...500% or higher, compared to the naïve cells. One form of “overexpression” is the introduction of the gene encoding a foreign transcription factor into the cell and its expression.

[0036] As used in this article, "exogenous" or "heterogeneous" refers to the relationship between two or more nucleic acid or protein sequences from different sources, or the relationship between nucleotides or proteins from different sources and cells or organisms. For example, if a particular sequence is not naturally present in the cell or organism into which it is inserted, then it is "exogenous" to that cell or organism.

[0037] As used in this invention, "exosome" refers to a nanoscale body, a type of extracellular vesicle (EV), released from cells after specific treatment. The membrane of an exosome is mainly composed of lipids and proteins, and its contents may include proteins, DNA, mRNA, microRNA, and ncRNA, etc. The types and quantities of various components contained in exosomes are related to their source cells, but may also be affected by physiological stress or other factors.

[0038] As used in this invention, a "pharmaceuticalally acceptable" ingredient is a substance suitable for human and / or mammalian use without excessive adverse side effects (such as toxicity), i.e., a reasonable benefit / risk ratio. The term "pharmaceuticalally acceptable carrier" refers to a carrier used for the administration of a therapeutic agent, including various excipients and diluents. This term refers to pharmaceutical carriers that are not themselves essential active ingredients and do not exhibit excessive toxicity after administration.

[0039] As used in this invention, the term “treatment” here includes preventive (e.g., prophylactic), curative, or palliative treatment of a mammal (particularly a human); and includes (1) preventing, treating, or alleviating an individual’s morbidity for a disease (e.g., cancer), wherein the individual is in a high-risk group for the disease or has the disease but has not yet been diagnosed; (2) suppressing a disease (e.g., inhibiting its occurrence); or (3) alleviating a disease (e.g., alleviating symptoms associated with the disease).

[0040] As used in this invention, "individual" or "subject" means an animal including a human being who is eligible for treatment with the exosomes of this invention and the PD-1 inhibitor used in combination therewith.

[0041] As used in this invention, "effective amount" refers to an amount of active ingredient (in this invention, a combination of exosomes and a PD-1 inhibitor used in conjunction therewith) sufficient to produce the desired therapeutic response. Effective amount also includes situations where the therapeutic benefit of the agent outweighs its toxic or adverse effects. An effective amount of an agent may not necessarily cure a disease or condition, but may delay, inhibit, or prevent its occurrence, or may alleviate symptoms associated with the disease or condition. The effective amount may be divided into one, two, or more doses and administered once, twice, or more times in an appropriate dosage form over a specified period. The specific effective amount depends on various factors, such as the specific condition to be treated, the individual's physiological conditions (e.g., body weight, age, or sex), the type of individual receiving treatment, the duration of treatment, the nature of any concurrent treatments, and the specific formulation and structure of the compound or its derivatives used.

[0042] As used in this invention, the "immune checkpoint inhibitor" is a major drug in tumor immunotherapy. Its mechanism of action is to enhance the anti-tumor immune response of T cells by inhibiting the function of negative immunoregulatory molecules on the surface of T cells, thereby producing an anti-tumor immune effect.

[0043] As used in this invention, the term "tumor" can include tumors treated with "immune checkpoint inhibitors" in the art, especially tumors that have developed resistance to "immune checkpoint inhibitors." Examples include, for instance, liver cancer, lung cancer, or pancreatic cancer.

[0044] The inventors unexpectedly discovered during their research that exosomes produced by tumor cells under specific conditions can synergistically reverse tumor resistance to PD-1 inhibitors by acting on immune checkpoint inhibitors (PD-1). Based on this new discovery, a drug combination is provided, comprising a PD-1 inhibitor and exosomes containing adenylate cyclase 7 (ADCY7) protein. Preferably, the exosomes are isolated from tumor cells, and the tumor cells recombinantly express (overexpress) exogenous adenylate cyclase 7. In a preferred embodiment, the tumor cells are liver cancer cells.

[0045] Adenylate cyclase 7 (ADCY7) is a member of the adenylate cyclase family. As a key downstream signaling molecule of G protein-coupled receptors (GPCRs), adenylate cyclases (ACs) influence the function of various cells and participate in the regulation of pathophysiological processes by controlling the synthesis of cyclic adenosine-3',5'-monophosphate (cAMP). The AC family currently comprises ten members, including nine transmembrane AC subtypes (AC1–9, mACs) and one soluble AC (sAC, also known as AC10), and the expression levels of each subtype vary in different tissue cell membranes. Studies have reported that adenylate cyclases play important roles in physiological and various pathological processes, such as being significantly associated with the development of the central nervous system, brain development and function, alcohol addiction, and depression from adolescence to adulthood.

[0046] The ADCY7 described herein comprises the full-length ADCY7 amino acid sequence. This invention may also employ modified or altered ADCY7, for example, ADCY7 modified or altered to enhance its half-life, efficacy, metabolism, and / or potency. The modified or altered ADCY7 may share minor similarities with naturally occurring ADCY7, but still perform the same or substantially the same functions as the wild type without introducing other adverse effects. In other words, any variation that does not affect the biological activity of ADCY7 can be applied in this invention.

[0047] This invention also includes isolated nucleic acids encoding the bioactive fragment of ADCY7, which may also be its complementary strand. As a preferred embodiment, the coding sequence of ADCY7 can be codon-optimized to improve expression efficiency. The DNA sequence encoding the bioactive fragment of ADCY7 can be synthesized in its entirety artificially or obtained by PCR amplification. After obtaining the DNA sequence encoding the bioactive fragment of ADCY7, it is ligated into a suitable expression construct (such as an expression vector) and then transformed into a suitable host cell. Finally, the desired protein is obtained by culturing the transformed host cells.

[0048] This invention also includes expression constructs comprising nucleic acid molecules encoding a bioactive fragment of said ADCY7. The expression construct may include one or more gene expression cassettes encoding said ADCY7, and may also include expression regulatory sequences operatively linked to the sequence of said nucleic acid molecule to facilitate protein expression. The design of said expression regulatory sequences is well known in the art. Depending on different needs, inducible or constitutive promoters can be used in the expression regulatory sequences. Inducible promoters enable more controllable protein expression and compound production, which is beneficial for industrial applications.

[0049] The creation of expression constructs is already a technique familiar to those skilled in the art. Therefore, once the sequence of ADCY7 is obtained, those skilled in the art can easily create the expression construct.

[0050] Methods for overexpressing a foreign gene (ADCY7 in this invention) in cells are well known to those skilled in the art. The polynucleotide sequence encoding ADCY7 can be inserted into an expression construct such as a recombinant expression vector. The term "recombinant expression vector" refers to viruses (such as lentiviruses, adenoviruses, retroviruses), bacterial plasmids, bacteriophages, yeast plasmids, or other vectors well known in the art. Any plasmid and vector can be used as long as it can replicate and remain stable in the host. A vector containing the polynucleotide sequence encoding ADCY7 and a suitable promoter or control sequence can be used to transform cells to express ADCY7. Preferably, the recombinant expression vector is a viral vector, more preferably a lentiviral vector.

[0051] In a preferred embodiment, the exosomes are derived from tumor cells. First, tumor cells expressing recombinant ADCY7 are cultured to obtain a sufficient quantity of cell culture. The cells are then washed and cultured in serum-free medium to release the exosomes, which are then isolated and purified. This method effectively encapsulates ADCY7 within the exosomes. More preferably, the tumor cells are liver cancer cells.

[0052] Exosomes used to achieve the technical effects claimed in this invention are distinct from vesicles produced by cells under normal conditions. In other words, the exosomes obtained by this invention include exosomes derived from tumor cells as well as bioactive components encapsulated / attached / derived from / on the surface of the exosomes.

[0053] In some embodiments, the PD-1 inhibitor is an anti-PD-1 antibody. For example, the PD-1 antibody includes GoInVivo. TM Purified anti-mouse CD279 (PD-1) antibody (BioLegend).

[0054] This invention also provides a kit for reversing drug resistance in tumors (including liver cancer, pancreatic cancer, and lung cancer) and inhibiting tumor growth. The kit includes a PD-1 inhibitor and exosomes. More preferably, the kit also includes an instruction manual to guide clinicians in administering the medication correctly and appropriately.

[0055] The PD-1 inhibitor and exosomes can be individually packaged and placed in the kit. Alternatively, for convenient administration, the PD-1 inhibitor and exosomes can be mixed to form a single-dose formulation and placed in the kit. "Single-dose formulation" refers to a dosage form prepared for a single dose, including but not limited to various solid dosage forms (such as lyophilized preparations), liquid dosage forms, capsules, and sustained-release formulations. Furthermore, the PD-1 inhibitor and exosomes can also be individually placed in different containers and mixed as needed for application.

[0056] As a preferred embodiment of the present invention, the ratio of the PD-1 inhibitor to the exosomes (by weight) is 1:(5-200); more preferably, it is 1:(10-100).

[0057] In a specific embodiment of the present invention, the test subjects were mice. However, it should be understood that converting the dosage for animals such as mice into a dosage suitable for humans is easily done by those skilled in the art, for example, it can be calculated using the Meeh-Rubner formula: Meeh-Rubner formula: A = k × (W 2 / 3 ) / 10,000. Where A is the body surface area, expressed in meters. 2 Calculations: W represents body weight in grams; K is a constant that varies depending on the animal species, generally 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans. It should be understood that dosage conversions can vary depending on the drug and clinical circumstances, and are based on the assessment of an experienced pharmacist.

[0058] 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 that do not specify specific conditions are generally performed according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Science Press, or according to the manufacturer's recommendations.

[0059] Example 1: Construction of ADCY7 overexpression lentivirus

[0060] ADCY7 overexpression lentivirus was purchased from Shanghai Jikai Gene Co., Ltd., with the gene name ADCY7 (NM_007406). The target gene vector used was GV705, the cloning site was BamHI / BamHI, and the target gene sequence is (SEQ ID NO:1):

[0061]

[0062] The ADCY7 polypeptide encoded by the above nucleic acid sequence is (SEQ ID NO:2):

[0063]

[0064] After plasmid construction and purification, the virus was coated using 293T cells, followed by virus isolation, purification, and titer determination.

[0065] Example 2: Hepa 1-6 NC and Hepa 1-6 ADCY7 Cell line construction, identification, and detection of cell proliferation capacity

[0066] 1. Hepa 1-6 NC and Hepa 1-6 ADCY7 Cell preparation

[0067] 2×10 5 Hepa cell carcinoma cells (Hepa 1-6) were cultured in 6-well cell culture plates and infected with either control virus (NC) or ADCY7-overexpressing lentivirus at an MOI of 10 for 24 hours to obtain Hepa 1-6 cells. NC Or Hepa1-6 ADCY7 Cells. Proteins were collected after screening with puromycin.

[0068] The expression level of ADCY7 was verified using Western blotting, and the amplified culture was used for subsequent detection.

[0069] 2. Detection of cell proliferation capacity

[0070] Hepa 1-6 to be cultured NC and Hepa 1-6 ADCY7 After cell digestion, centrifugation, and counting, the cell concentration was adjusted to 1×10⁻⁶. 7 / mL.

[0071] (1) CCK-8 experiment

[0072] After appropriate dilution of the cells, 3,000 tumor cells were seeded into 96-well cell culture plates, and the medium was changed every 24 hours. 10% volume of CCK-8 detection solution was added to each well, and after incubation for 2 hours, the absorbance at 450 nm was detected using an ELISA reader. The CCK-8 detection data were collected and the cell proliferation curve was plotted after 3 consecutive days of detection.

[0073] like Figure 1 A. Compared with Hepa 1-6NC cells, Hepa 1-6ADCY7 cells showed significantly increased ADCY7 expression levels. In vitro CCK-8 assays showed ( Figure 1 B) After ADCY7 overexpression, there was no significant difference in the proliferation capacity of liver cancer cells.

[0074] (2) In vivo mouse experiments

[0075] Prepare 4-6 week old male mice, 8 mice per group; take 100 μL of cell suspension and inject it into the back of the mouse with an insulin needle; start measuring after 8 days, and then measure every two days thereafter, record and plot the growth curve; after 4 measurements, sacrifice the mice by cervical dislocation, remove the tumor tissue, and take pictures for recording.

[0076] like Figure 1 Subcutaneous tumor-bearing experiments in C, NSG mice also showed that ADCY7 overexpression does not affect the in vivo growth of liver cancer cells.

[0077] Example 3: Hepa 1-6 NC and Hepa 1-6 ADCY7 The influence of cells on the immune microenvironment

[0078] Hepa 1-6 to be cultured NC and Hepa 1-6 ADCY7 After cell collection, the cell concentration was adjusted to 1×10⁻⁶. 7 / mL. Prepare 4-6 week old male mice (9 in the NC group and 10 in the ADCY7 overexpression group); take 100μL of cell suspension and inject it into the back of the mouse with an insulin needle; start measuring after 8 days, and then measure every two days thereafter, record and plot the growth curve; after 5 measurements, sacrifice the mouse by cervical dislocation, remove the tumor tissue and take pictures for recording.

[0079] Tumors of different groups were divided into several portions according to experimental needs for RNA extraction, protein extraction, flow cytometry detection, or immunohistochemical detection.

[0080] like Figure 2 As shown, compared with the NC treatment group, the ADCY7 overexpression cell treatment group had significantly slower tumor growth rate and significantly smaller tumor volume.

[0081] like Figure 3 As shown, flow cytometry results indicated that, compared to the NC treatment group, the ADCY7 overexpression cell treatment group had significantly more T cell infiltration, with CD3+ being the predominant type of T cells. + T cells, CD8 + T cells.

[0082] like Figure 4 As shown, immunohistochemical results indicated that, compared to the NC treatment group, the ADCY7 overexpression cell treatment group had significantly more T cell infiltration, with CD3+ being the predominant type of T cells. + T cells, CD8 + T cells.

[0083] Example 4: Hepa 1-6 NC and Hepa 1-6 ADCY7 Identification of cell-derived exosomes

[0084] Hepa 1-6 in good condition NC and Hepa 1-6 ADCY7 Cells were seeded into 10cm dishes and cultured in high-glucose DMEM medium containing 10% serum. When the cell confluence reached about 90%, the liver cancer cells were washed with PBS and cultured in serum-free medium (high-glucose DMEM medium) for 48 hours to release exosomes.

[0085] Next, exosomes were isolated and purified using ultracentrifugation: centrifuged at 300g for 10 minutes at 4°C; centrifuged at 2,000g for 10 minutes; centrifuged at 10,000g for 30 minutes; then, the supernatant was purified by centrifugation at 120,000g for 70 minutes, and the supernatant was discarded; the previous step was repeated; the exosomes were resuspended in 50 μL of PBS solution and stored at -80°C for further transmission scanning electron microscopy analysis and Western blotting detection.

[0086] like Figure 5 As shown, ADCY7 exists in Hepa 1-6 ADCY7 In the exosomes of cells. From Hepa1-6 NC and Hepa 1-6 ADCY7 The exosomes obtained by ultracentrifugation in the cells were named NC-exo and ADCY7-exo, respectively.

[0087] Example 5: Effects of exosomal ADCY7 on the immune microenvironment of liver cancer

[0088] Hepa 1-6 liver cancer cells in logarithmic growth phase were harvested, digested with trypsin, centrifuged, resuspended in PBS, and counted. The cell concentration was adjusted to 1×10⁶ cells / year. 7 / mL; Prepare 4-6 week old male mice (9 in the NC group and 7 in the ADCY7 overexpression group); Take 100μL of cell suspension and inject it into the back of the mouse using an insulin needle. After 7 days, when the tumor volume reaches 100mm... 3 Around 5 μg each of pre-prepared NC-exo and ADCY7-exo were injected intratumorally. Measurements were taken every three days, and growth curves were recorded and plotted. After five measurements, mice were euthanized by cervical dislocation, tumor tissue was dissected, and photographs were taken. Tumors from different groups were divided into several portions according to experimental needs for RNA extraction, protein extraction, flow cytometry, or immunohistochemical detection, etc. Figure 6 A).

[0089] like Figure 6 As shown in BC, the size of the tumor was measured and it was found that the subcutaneous tumor in the control group grew rapidly. Compared with the control group, the exosome ADCY7 group significantly inhibited the growth of liver cancer (****P<0.0001).

[0090] like Figure 6 As shown in the DE assay, flow cytometry and immunohistochemical results indicated that the exosomal ADCY7 group had greater T cell infiltration. Furthermore, the exosomal ADCY7 group was able to produce more IFN-γ (**P<0.01).

[0091] The above results indicate that exosomal ADCY7 can remodel the immune microenvironment of liver cancer, resulting in a higher concentration of CD8+ in the microenvironment. + T and IFN-γ + CD8 + T-cell infiltration inhibits tumor progression.

[0092] Example 6: Exosomal ADCY7 can enhance the efficacy of PD-1 inhibitors and reverse PD-1 inhibitor resistance.

[0093] Preparation of hepa1-6 resistant tumor cell line: After subcutaneous implantation of primary hepa1-6 cells, PD-1 antibody was injected to obtain residual tumor cells, which were then isolated and subcutaneously implanted again until the same dose of PD-1 was ineffective, thus obtaining hepa1-6R.

[0094] Three tumor cell lines were subcutaneously inoculated into normal wild-type C57BL / 6J mice: PD-1 resistant Hepa 1-6R, the mouse pancreatic cancer cell line Pan02 (which did not respond well to PD-1 treatment), and the mouse lung cancer cell line LLC. After 5–7 days, when the tumor volume reached 100–200 mm², the inoculation was completed. 3 Randomly divide the left and right sides into 4 groups:

[0095] 1) NC-exo (5μg);

[0096] 2) ADCY7-exo (5μg);

[0097] 3) NC-exo (5 μg) combined with a PD-1 inhibitor (200 μg);

[0098] 4) ADCY7-exo (5μg) combined with PD-1 inhibitor (200μg).

[0099] Among them, exosomes are administered via intratumoral injection (once every two days), and PD-1 inhibitors are administered via intraperitoneal injection (once every three days).

[0100] Among them, the PD-1 inhibitor is GoInVivo TM Purified anti-mouse CD279(PD-1) antibody, purchased from BioLegend.

[0101] Subsequently, measurements were taken every three days, and growth curves were recorded and plotted.

[0102] like Figure 7 As shown, PD-1 inhibitors could not significantly inhibit the growth of PD-1 resistant tumors, while the ADCY7-exo group could slightly inhibit tumor growth; however, the tumor volume in the ADCY7-exo combined with PD-1 inhibitor group was significantly reduced (P<0.0001), indicating that ADCY7-exo and PD-1 inhibitors have a significant synergistic effect.

[0103] 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. Application of exosomes for the preparation of pharmaceutical compositions that reverse tumor resistance to immune checkpoint inhibitors; said exosomes containing adenylate cyclase 7 protein.

2. The application as described in claim 1, characterized in that, The exosomes are exosomes isolated from tumor cells, and the tumor cells recombinantly express exogenous adenylate cyclase 7; preferably, the method of recombinantly expressing exogenous adenylate cyclase 7 includes: introducing an expression construct into tumor cells, the expression construct containing an expression cassette of adenylate cyclase 7; more preferably, the tumor cells are liver cancer cells.

3. The application as described in claim 2, characterized in that, The method for preparing the exosomes includes: amplifying and culturing the recombinant tumor cells expressing exogenous adenylate cyclase 7, then washing the cells, culturing them in a serum-free culture medium to release the exosomes, and separating and purifying the exosomes.

4. The application as described in claim 3, characterized in that, In the preparation method: After amplification and culture to a confluence of 70-95%, wash the cells; and / or Amplification culture was performed using high-glucose DMEM medium containing 10±3% serum; and / or Incubate in serum-free medium for 36-60 hours, preferably 42-55 hours, more preferably 45-52 hours; and / or Exosomes were isolated and purified by ultracentrifugation.

5. The application as described in claim 1 or 2, characterized in that, The exosomes are used in combination or in combination with the immune checkpoint inhibitors; and / or The tumors mentioned include: liver cancer, pancreatic cancer, and lung cancer.

6. A drug combination for reversing tumor drug resistance and inhibiting tumors, characterized in that, The drug combination includes exosomes and immune checkpoint inhibitors; the exosomes contain adenylate cyclase 7 protein.

7. The pharmaceutical combination as described in claim 6, characterized in that, The exosomes are exosomes isolated from tumor cells, and the tumor cells recombinantly express adenylate cyclase 7; preferably, the method for preparing the exosomes includes: expanding and culturing tumor cells, then washing the cells, culturing them in a serum-free culture medium to release the exosomes, and separating and purifying the exosomes; more preferably, the tumor cells are liver cancer cells.

8. The pharmaceutical combination as described in claim 6, characterized in that, In the drug combination, the weight ratio of the exosomes to the immune checkpoint inhibitor is 1:(5-200); preferably 1:(10-100).

9. Use of the pharmaceutical combination of claim 7 or 8 in a kit for reversing tumor drug resistance and inhibiting tumors.

10. The application as described in claim 9, characterized in that, The medicine box also includes one or more items selected from the group consisting of: Chemotherapy drugs for tumors; Cancer radiotherapy drugs; Adjunctive therapy drugs; Nutritional preparations; and / or Instruction manual.

Images