Application of adenosine A2AR antagonist in preparation of medicine for enhancing PD-1 antibody and / or PD-L1 antibody immunotherapy tumor

Abstract

The invention belongs to the field of biological treatment, and particularly relates to application of an adenosine A2AR (adenosine A2A receptor) antagonist in preparation of a medicine for enhancing PD-1 antibody immunotherapy. The invention specifically relates to application of the adenosine A2AR antagonist combined with a PD-1 antibody and / or a PD-L1 antibody in treatment of tumors including esophageal squamous cell carcinoma, gastric cancer, colorectal cancer, renal cell carcinoma, melanin and other tumors in enhancement of immunotherapy effects. According to the adenosine A2AR antagonist and PD-1 antibody and / or PD-L1 antibody combined medication scheme, tumor immunotherapy can be conducted on digestive tract tumors and other tumors through the PD-1 antibody and / or PD-L1 antibody, and compared with PD-1 antibody or PD-L1 antibody single-drug treatment and immunity activation, tumor growth can be remarkably inhibited, and the tumor treatment effect can be improved. The method has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotherapy and relates to adenosine A. 2A The application of R-antagonists in the preparation of drugs for enhancing PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, specifically involving adenosine A. 2A Application of R antagonists in the preparation of drugs that enhance the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors and / or inhibit tumor cell growth. Background Technology

[0002] As one of the main types of malignant tumors (such as lung cancer, gastric cancer, esophageal squamous cell carcinoma, colorectal cancer, etc.), tumors have long been a major challenge in the global public health field due to their high incidence and high mortality. Most patients are often in the middle or late stage when diagnosed, at which time the treatment options are extremely limited. At present, the main clinical treatment is surgical resection and radiotherapy and chemotherapy. In recent years, immunotherapy (such as PD-1 / PD-L1 inhibitors) has shown significant efficacy in some patients, but its application in most solid tumors still faces many bottlenecks. In essence, immune escape is the key obstacle leading to the failure of immunotherapy. Its mechanism is complex and diverse, mainly including the following aspects: (1) Tumor cell level: Tumor cells can evade T cell recognition and attack by downregulating the expression of tumor-specific antigens or related antigens and changing the interaction between major histocompatibility complex (MHC) molecules and antigenic peptides. (2) At the tumor microenvironment level: ① Regulatory T cells (Tregs) are recruited into the tumor microenvironment to suppress local immune responses; ② Tumor-associated macrophages not only fail to clear tumor cells, but also promote tumor growth, metastasis, and invasion by secreting pro-inflammatory factors and matrix remodeling; ③ Myeloid-derived suppressor cells further exacerbate the immunosuppressive state by inducing tumor angiogenesis; ④ Immune cell dysfunction: Active factors released by immune cells may trigger chronic inflammatory responses, thereby accelerating tumor progression; (3) In addition, insufficient chemotactic capacity of T cells, low heterogeneity of tumor cell antigen expression, and the inherent immunosuppressive characteristics of the tumor microenvironment all significantly weaken the effect of immunotherapy. In summary, the diversity and complexity of immune escape mechanisms pose a huge challenge to current immunotherapy for solid tumors.

[0003] Studies have shown that a key mechanism of cancer immune escape is the production of high levels of immunosuppressive adenosine in the tumor microenvironment. The abundant adenosine in the tumor microenvironment binds to adenosine receptors on the cell surface, inhibiting the proliferation and maturation of immune cells such as T cells, natural killer (NK) cells, macrophages, and dendritic cells (DCs), as well as the production of immunocytokines, thereby impairing the body's immune system and promoting tumor cell growth. Furthermore, adenosine also has a direct proliferative effect on tumor cells through adenosine receptors. Adenosine A2A receptor (adenosine A) 2A receptor,A 2A R) belongs to the G protein-coupled receptor (GPCR) family, and can be further subdivided into A 2A A 2B There are four subtypes: A1, A3, and A4. In recent years, A... 2A The regulatory function of R in tumor immunology has attracted widespread attention and it has become a highly promising emerging target for tumor immunotherapy. 2A R, by binding to the Gs / Golf protein, promotes increased adenylate cyclase (AC) activity, thereby increasing intracellular cyclic adenosine monophosphate (cAMP) levels. This, in turn, activates protein kinase A (PKA), which... 2A R can activate various ion channels and receptors, transmitting immunosuppressive signals into immune cells. This process inhibits the proliferation, maturation, and infiltration of immune cells such as T cells, NK cells, macrophages, and DC cells, as well as the production of these cytokines, ultimately leading to immune damage and immune escape by tumor cells.

[0004] Therefore, design adenosine A 2A R-type antagonist drugs block the signal transduction mediated by this receptor, thereby preventing the loss of the body's immune system and inhibiting the growth of tumor cells. This is a novel tumor immunotherapy strategy currently in the clinical research stage. Currently, all adenosine receptor antagonists in clinical trials are still in the clinical research stage and have not yet been approved for market. Drugs in phase II clinical trials often have disadvantages such as large molecular weight and poor water solubility, resulting in low efficacy in immunotherapy and limited clinical application. Therefore, there is a need to develop antagonists with better drug-like properties. The development of new adenosine A... 2A R-antagonist drugs, and by combining them with other treatment strategies to enhance the efficacy of immunotherapy for tumors, have significant scientific research value and clinical medical application value for the treatment of tumors. Summary of the Invention

[0005] The purpose of this invention is to address the problems of low efficacy and limited clinical application of immunotherapy for tumors. To this end, this invention provides adenosine A. 2A The use of R antagonists in the preparation of drugs for enhancing PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors (enhancing the efficacy of immunotherapy), wherein the antagonist benzimidazolidine-3-carboxamide is a (S)-1-amino-N-(1-(pyridin-2-yl)ethyl)benzi[4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecule compound with the molecular formula C 18 H 16 N6O, with a molecular weight of 332.36. This invention proposes adenosine A. 2AThe R antagonist benzimidazolidine-3-carboxamide, combined with PD-1 antibody and / or PD-L1 antibody, can enhance the immunotherapeutic efficacy of PD-1 antibody and / or PD-L1 antibody against tumors.

[0006] The innovation of this invention lies in the fact that tumors, especially digestive system tumors, are insensitive to immunotherapy, have low efficacy, and exhibit drug resistance, necessitating new treatment strategies, such as combination therapy regimens, to enhance the efficacy of immunotherapy. Adenosine is an inhibitory neurotransmitter in the human body, and its chemical nature is a nucleoside. Extracellular nucleosides, as a negative immune regulatory mechanism, exert anti-inflammatory effects and prevent autoimmunity by inhibiting the immune system, thus preventing excessive immune damage to the body. In tumor immunology, adenosine receptors bind to adenosine, negatively regulating the immune system's killing of tumor cells; therefore, A... 2A The tumor immunomodulatory function of R has attracted widespread attention, making it a promising emerging target for tumor immunotherapy. Currently, all adenosine receptor antagonists that have entered clinical trials are still in the clinical research stage and have not yet been approved for market. Many drugs in phase II clinical trials have the disadvantages of large molecular weight and poor water solubility, necessitating the development of antagonists with better drug-like properties.

[0007] The above-mentioned objective of this invention is achieved through the following technical solution:

[0008] Currently, several adenosine A ... 2A R-type small molecule antagonists are currently undergoing anti-tumor research. However, adenosine A has not yet been officially approved for marketing. 2A R-antagonist drug molecules consist of only seven small molecules of adenosine A. 2A R-blockers currently in clinical trials include AstraZeneca's AZD4635, Corvus' CPI-444, Novartis' PBF-509, Hengrui Medicine's SHR5126, CStone Pharmaceuticals' CS3005, Exscientia's EXS21546, and Arcus Biosciences' simultaneous adenosine A-targeting inhibitor. 2A R and adenosine A 2B R's AB928. These seven candidate drug molecules are currently undergoing clinical trials for common cancers such as melanoma, squamous cell carcinoma of the lung, colorectal cancer, pancreatic cancer, kidney cancer, bladder cancer, ovarian cancer, and prostate cancer. Specific efficacy and toxicity data for these drugs in humans have not yet been published, and their future marketability remains uncertain. Among them, the small molecule antagonist AZD4635 is an effective and selective oral adenosine A. 2A R antagonist. This invention selects benzimidazolidine-3-carboxamide or AZD4635 for adenosine A antagonism. 2A Research on R antagonists.

[0009] In a specific embodiment of this invention, mouse esophageal squamous cell carcinoma organoids, mouse colorectal cancer CT26 cell line, mouse gastric cancer organoids, mouse renal cell carcinoma Renca cells, and mouse melanoma cell models, respectively, were constructed using chemically induced tumorigenesis. These representative murine tumor models were used to establish subcutaneous tumorigenesis models in immunocompetent C57BL / 6J and Balb / c mice. When the tumors grew to a certain size, they were treated with IgG control therapy, PD-1 monoclonal antibody therapy, and adenosine A, respectively. 2A R-antagonist (benzimidazolidine-3-carboxamide) monotherapy and PD-1 monoclonal antibody and adenosine A 2A R-antagonist (AZD4635 or benzimidazolidine-3-carboxamide) combination therapy. Results showed that, at the given dose, adenosine A alone... 2A R-antagonists (AZD4635 or benzimidazolidine-3-carboxamide) had no significant effect on tumor growth, while the combination of adenosine A with PD-1 monoclonal antibodies... 2A R-antagonists (AZD4635 or benzimidazolidine-3-carboxamide) can significantly enhance the efficacy of PD-1 monoclonal antibodies. Furthermore, this invention measured the body weight of mice during drug treatment and collected mouse hearts, livers, spleens, and kidneys at the experimental endpoint for pathological examination and analysis. The results showed that adenosine A... 2A R-antagonists do not cause toxicity in C57BL / 6J (C57) mice or Balb / c mice when used as monotherapy or in combination with PD-1 monoclonal antibody therapy.

[0010] Therefore, this invention claims protection for:

[0011] Adenosine A 2A The application of R-antagonists in the preparation of drugs that enhance tumor immunotherapy, or in the preparation of drugs for tumor treatment.

[0012] Specifically, the adenosine A 2A R antagonists include one or more of benzimidazolidine-3-carboxamide or its pharmaceutically acceptable salts or solvates, AZD4635, etc.

[0013] And the use of benzimidazolidine-3-carboxamide in the preparation of drugs that enhance tumor immunotherapy, or in the preparation of drugs for tumor treatment.

[0014] And the use of benzimidazolidine-3-carboxamide in the preparation of drugs that enhance PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors that are insensitive.

[0015] In this invention, the molecular formula of the benzimidazolium-3-carboxamide is C2. 18 H16 N6O, with a molecular weight of 332.36, refers specifically to tumors that are insensitive to immunotherapy with PD-1 and / or PD-L1 antibodies, including gastrointestinal tumors and other tumors.

[0016] In this invention, the model used is a mouse tumor model, and the mice used are immune-healthy mice.

[0017] In this invention, the tumor includes gastrointestinal tumors, renal cell carcinoma, and melanoma, etc.

[0018] The digestive tract tumors mentioned include esophageal squamous cell carcinoma, gastric cancer, colorectal cancer, etc.

[0019] Based on the above results, the present invention can provide a therapeutic drug and treatment regimen, wherein the drug is adenosine A. 2A R small molecule antagonists: benzimidazolidine-3-carboxamide, AZD4635. The regimen is benzimidazolidine-3-carboxamide or AZD4635 in combination with PD-1 antibody to treat gastrointestinal tumors and other tumors that are not sensitive to immunotherapy.

[0020] In a specific implementation scheme, in the tumor combination therapy regimen of the present invention, adenosine A 2A R-antagonists and PD-1 monoclonal antibodies can be administered simultaneously, with PD-1 monoclonal antibodies being administered periodically at intervals, while adenosine A... 2A R-antagonist administered daily.

[0021] In the above applications and drug regimens, the subtype-selective adenosine A mentioned above... 2A R antagonists also include pharmaceutically acceptable salts or solvates of small molecule compounds such as benzimidazolium-3-carboxamide (S)-1-amino-N-(1-(pyridin-2-yl)ethyl)benzi[4,5]imidazo[1,2-a]pyrazine-3-carboxamide, AZD4635, etc.

[0022] This invention provides adenosine A 2A R-antagonists are used in the preparation of drugs for enhancing the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibiting tumor cell growth, and / or increasing the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reducing tumor weight, and / or increasing the infiltration of activated cytotoxic T cells in tumor tissue, and / or activating the body's own immune system.

[0023] Specifically, the adenosine A 2A R antagonists include one or more of benzimidazolidine-3-carboxamide or its pharmaceutically acceptable salts or solvates, AZD4635, etc.

[0024] The benzimidazolidine-3-carboxamide is a small molecule antagonist.

[0025] The benzimidazolidine-3-carboxamide is a subtype selective adenosine A. 2A R antagonists are small molecule compounds of the (S)-1-amino-N-(1-(pyridin-2-yl)ethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide class.

[0026] The molecular formula of the benzimidazolium-3-carboxamide is C2. 18 H 16 N6O has a molecular weight of 332.36.

[0027] AZD4635 is a small molecule antagonist, an effective and selective oral adenosine A antagonist. 2A R antagonist.

[0028] Specifically, the tumors include gastrointestinal tumors, renal cell carcinoma, melanoma, etc.

[0029] The digestive tract tumors mentioned include esophageal squamous cell carcinoma, gastric cancer, colorectal cancer, etc.

[0030] Specifically, the immunotherapy promotes the secretion of cytokines by immune cells; and / or the immunotherapy enhances the killing effect of immune cells on tumors.

[0031] The model used in this application is a mouse tumor model.

[0032] The mouse tumor models include models that induce subcutaneous tumor formation in tumor-like organs or cells.

[0033] In one specific embodiment of the application, the model used is a mouse digestive tract tumor model, etc.

[0034] The mouse digestive tract tumor models include mouse esophageal squamous cell carcinoma models, mouse gastric cancer organoid models, and mouse colorectal cancer cell models.

[0035] Among them, mouse digestive tract tumor models include models that use organoids or cells to induce subcutaneous tumor formation.

[0036] This invention also provides a method for targeting adenosine A. 2A R and / or a drug / pharmaceutical composition for tumor immunotherapy and / or inhibition of tumor cell growth, said drug / pharmaceutical composition being adenosine A as described above. 2A R antagonist is the main active ingredient.

[0037] Furthermore, the drug / drug composition may also include a pharmaceutically acceptable carrier, etc.

[0038] Specifically, the drug / drug composition includes liquid dosage forms, gaseous dosage forms, solid dosage forms, and semi-solid dosage forms.

[0039] Specifically, the drug / drug composition is administered orally, by injection, nasal administration, transdermal administration, or mucosal administration.

[0040] Preferably, the pharmaceutically acceptable carrier refers to a carrier that, when properly administered to animals or humans, does not produce adverse, allergic, or other adverse reactions. Pharmaceutically acceptable carriers include, but are not limited to: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium methylcellulose, ethylcellulose, and methylcellulose; tragacanth gum powder; malt; gelatin; talc; solid lubricants such as stearic acid and magnesium stearate; calcium sulfate; vegetable oils such as peanut oil, cottonseed oil, sesame oil, olive oil, corn oil, and cocoa butter; polyols such as propylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; tableting agents; stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; and phosphate buffers, etc. These substances are used as needed to help stabilize the formulation or to improve its activity or bioavailability or to produce an acceptable taste or smell when taken orally.

[0041] Specifically, the drug / drug composition may also contain physiologically compatible excipients, including buffers, diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc.

[0042] Specifically, the drug / drug composition can be formulated into injections, sterile powders for injection, tablets, pills, capsules, lozenges, liniments, powders, granules, syrups, solutions, tinctures, aerosols, powder inhalers, or suppositories, etc. All of the above dosage forms of the drug / drug composition can be prepared according to conventional methods in the pharmaceutical field. In the above pharmaceutical formulation, the dosage form of the drug / drug composition is an injection.

[0043] Specifically, the drug / drug composition can be introduced into the body, such as into muscles, intradermal tissues, subcutaneous tissues, veins, or mucous membranes, via injection, spray, nasal drops, eye drops, penetration, absorption, or physical or chemical mediated methods; or it can be introduced into the body after being mixed with or encapsulated by other substances. Preferably, it is administered by injection. The drug / drug composition can also be used in combination with other treatment methods, including surgery, radiotherapy, chemotherapy, and targeted therapy.

[0044] The dosage level of the drug / drug composition of the present invention can be adjusted according to the amount of composition required to achieve the desired diagnostic or therapeutic outcome. The administration regimen can also be a single injection or multiple injections, or adjustments can be made. The selected dosage level and regimen are rationally adjusted based on various factors including the activity and stability (i.e., half-life) of the cellular drug / drug composition, the formulation, the route of administration, combination with other drugs or treatments, the disease or condition to be detected and / or treated, and the health status and prior medical history of the subject to be treated.

[0045] The therapeutically effective dose of the drug / pharmaceutical composition of the present invention can initially be estimated in cell culture experiments or animal models such as rodents, rabbits, dogs, pigs, and / or primates. Animal models can also be used to determine suitable concentration ranges and routes of administration. These can then be used to determine the useful dose and route of administration in humans. Generally, the determination and adjustment of the effective amount or dose, and the assessment of when and how to make such adjustments, are known to those skilled in the art.

[0046] For further guidance on formulations, dosages, administration regimens, and measurable treatment outcomes, see Berkow et al. (2000) The Merck Manual of Medical Information and Merck & Co. Inc., Whitehouse Station, New Jersey; Ebadi (1998) CRC Desk Reference of Clinical Pharmacology, etc.

[0047] Specifically, the drug / drug composition may be used alone and / or in combination with one or more other therapeutic agents, including PD-1 antibodies and PD-L1 antibodies.

[0048] The present invention also provides an immunotherapy potentiator, said immunotherapy potentiator comprising adenosine A as described above. 2A R antagonists, etc.

[0049] Specifically, the immunotherapy enhancer has the effects of enhancing the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibiting tumor cell growth, and / or increasing the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reducing tumor weight, and / or increasing the infiltration of activated cytotoxic T cells in tumor tissue, and / or activating the body's own immune system.

[0050] The present invention also provides a method for enhancing the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy in mammalian tumors and / or inhibiting the growth of mammalian tumor cells, comprising the steps of administering PD-1 antibody and / or PD-L1 antibody to the desired subject, while simultaneously administering benzimidazolidine-3-carboxamide or a pharmaceutically acceptable salt or solvate thereof as described above, or a drug / drug composition as described above, or an immunotherapy enhancer as described above, for combined treatment.

[0051] Preferably, the object is a person.

[0052] The present invention also provides the use of the drug / drug composition as described above, or the immunotherapy enhancer as described above, or the method as described above in enhancing the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibiting tumor cell growth, and / or tumor immunotherapy enhancer, and / or tumor treatment, and / or inhibiting tumor cell growth or increasing the infiltration of activated cytotoxic T cells in tumor tissue, and / or activating the body's own immune system, etc.

[0053] The present invention also provides the use of the drug / drug composition as described above, or the immunotherapy enhancer as described above, or the method as described above in the preparation of drugs for enhancing the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibiting tumor cell growth, and / or increasing the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reducing tumor weight, and / or increasing the infiltration of activated cytotoxic T cells in tumor tissue, and / or activating the body's own immune system.

[0054] In this invention, in the applications, pharmaceutical / pharmaceutical compositions, immunotherapy enhancers, and methods described above, the adenosine A... 2A R antagonists include one or more of benzimidazolidine-3-carboxamide or its pharmaceutically acceptable salts or solvates, AZD4635, etc.

[0055] Among them, benzimidazolidine-3-carboxamide is a small molecule inhibitor, a subtype selective adenosine A. 2A R antagonist; and / or, the benzimidazolidine-3-carboxamide is a subtype-selective adenosine A antagonist; 2A R antagonist is a (S)-1-amino-N-(1-(pyridin-2-yl)ethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecule compound; and / or, the molecular formula of said benzimidazolium-pyrazine-3-carboxamide is C 18 H 16N6O, with a molecular weight of 332.36; and / or, the benzimidazolidine-3-carboxamide can enhance the therapeutic effect of PD-1 antibody immunotherapy on tumors, or inhibit tumor cell growth, or improve the tumor inhibition rate, etc.

[0056] AZD4635 is a small molecule antagonist, an effective and selective oral adenosine A antagonist. 2A R antagonist; and / or,

[0057] adenosine A 2A R antagonists can enhance the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibit tumor cell growth, and / or increase the tumor suppression rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reduce tumor weight, and / or increase the infiltration of activated cytotoxic T cells in tumor tissue, and / or activate the body's own immune system; and / or,

[0058] The drug can enhance the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibit tumor cell growth, and / or increase the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reduce tumor weight, and / or increase the infiltration of activated cytotoxic T cells in tumor tissue, and / or activate the body's own immune system; and / or,

[0059] The drug / drug composition can enhance the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibit tumor cell growth, and / or increase the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reduce tumor weight, and / or increase the infiltration of activated cytotoxic T cells in tumor tissue, and / or activate the body's own immune system; and / or,

[0060] The immunotherapy enhancer can enhance the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibit tumor cell growth, and / or increase the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reduce tumor weight, and / or increase the infiltration of activated cytotoxic T cells in tumor tissue, and / or activate the body's own immune system; and / or, the tumor includes gastrointestinal tumors, renal cell carcinoma, melanoma, etc.

[0061] The digestive tract tumors mentioned include esophageal squamous cell carcinoma, gastric cancer, colorectal cancer, etc.

[0062] In this invention, preferably, the PD-1 antibody is a PD-1 monoclonal antibody.

[0063] The present invention has the following beneficial effects:

[0064] This invention provides a novel combination therapy for guiding immunotherapy, using adenosine A 2A R-antagonists can enhance the efficacy of tumor immunotherapy, overcoming the drawbacks of low efficacy and limited clinical application. This combination therapy regimen, based on existing immunotherapy, uses benzimidazolidine-3-carboxamide for co-administration, which can effectively enhance the application of immunotherapy in tumors such as gastrointestinal tumors. Attached Figure Description

[0065] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other related drawings can be obtained from these drawings without any creative effort.

[0066] Figure 1 It showed PD-1 monoclonal antibody and adenosine A 2A R-antagonists (AZD4635 or benzimidazolidine-3-carboxamide) have a combination therapeutic effect on esophageal squamous cell carcinoma.

[0067] Figure 2 It showed PD-1 monoclonal antibody and adenosine A 2A R-antagonists (AZD4635 or benzimidazolidine-3-carboxamide) have a combination therapeutic effect on gastric cancer.

[0068] Figure 3 It showed PD-1 monoclonal antibody and adenosine A 2A R-antagonists (AZD4635 or benzimidazolidine-3-carboxamide) have a combination therapeutic effect on colorectal cancer.

[0069] Figure 4 It showed adenosine A 2A The role of R antagonists (benzimidazolidine-3-carboxamide) in combination with PD-1 monoclonal antibodies in other tumor models (renal cell carcinoma).

[0070] Figure 5 It showed adenosine A 2A The role of R antagonists (benzimidazolidine-3-carboxamide) in combination with PD-1 monoclonal antibodies in other tumor models (melanoma).

[0071] Figure 6 It showed adenosine A 2A The combination of R antagonists (AZD4635 or benzimidazolidine-3-carboxamide) with PD-1 monoclonal antibodies showed no significant toxicity in mice during treatment. Detailed Implementation

[0072] The present invention will be further described in detail below with reference to the following embodiments and accompanying drawings. The scope of protection of the present invention is not limited to the following embodiments. All variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in the present invention and are protected by the appended claims.

[0073] The processes, conditions, reagents, and experimental methods used in implementing this invention, except as specifically mentioned below, are all common knowledge and general knowledge in the field, and this invention does not impose any particular limitations. Obviously, the described embodiments are only a part of this invention, not all embodiments. The description of at least one exemplary embodiment is merely illustrative and does not constitute any limitation on this invention or its application or use. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of this invention.

[0074] In all examples shown and discussed herein, any specific values ​​should be interpreted as exemplary only and not as limiting. Therefore, other examples of exemplary embodiments may have different values. Specific embodiments of the invention will be further described below. It should be noted that the description of these embodiments is intended to aid in understanding the invention and does not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the invention described below can be combined with each other as long as they do not conflict with each other.

[0075] This invention belongs to the field of biotherapy, specifically relating to adenosine A. 2A The application of R (adenosine A2A receptor) antagonists in the preparation of drugs for enhancing PD-1 antibody immunotherapy for tumors, specifically targeting the adenosine A2A receptor. 2A The application of R-antagonists combined with PD-1 antibodies and / or PD-L1 antibodies in enhancing the efficacy of immunotherapy for tumors, including esophageal squamous cell carcinoma, gastric cancer, colorectal cancer, renal cell carcinoma, and melanoma. The adenosine A described in this invention... 2A A combination therapy regimen of PD-1 antibody and / or PD-L1 antibody, targeting gastrointestinal tumors and other tumors, can activate the immune system, significantly inhibit tumor growth, and improve the therapeutic effect compared to PD-1 antibody or PD-L1 antibody monotherapy. This invention has broad application prospects.

[0076] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0077] Special note: The reagents and materials used in the following examples include: mouse esophageal squamous cell carcinoma organoids (see Chinese patent application CN202410974482.4), which were not commercially available; mouse gastric cancer organoids (see Chinese patent CN202011266740.1), which were not commercially available; and the drug used in this invention: adenosine A. 2A The R antagonist, benzimidazolidine-3-carboxamide (see Chinese patents CN202210200549.X, CN202210018141.0, and CN202210968765.9), is not commercially available. All other experimental materials in the examples were commercially available.

[0078] Terminology Definition

[0079] In this invention, the terms "comprising" and "including" generally refer to including specific features but do not exclude other elements, and the terms "above" and "below" generally refer to including the number itself.

[0080] In this invention, the term "pharmaceutical composition" generally refers to a mixture of one or more compounds described in this invention, or their physiologically usable salts or prodrugs, with other chemical components, such as pharmaceutically usable carriers and excipients. Pharmaceutical compositions can facilitate administration to organisms, promote the absorption of the active ingredient, and thereby exert its biological activity.

[0081] In this invention, the term "pharmaceutically acceptable" means that a carrier, diluent, or excipient, and / or the salt formed therefrom, is generally chemically or physically compatible with other components constituting a pharmaceutical dosage form and physiologically compatible with receptors.

[0082] In this invention, the term "pharmaceutically acceptable salt" generally refers to a salt of the compounds or antibody-drug conjugates of this invention, or a salt of the compounds described in this invention, which may be safe or effective when used in mammals. The antibody-drug conjugates of this invention can form salts with acids, and non-limiting examples of pharmaceutically acceptable salts include: hydrochloride, hydrobromide, sulfate, hydrogen sulfate, citrate, acetate, succinate, oxalate, hydrogen phosphate, dihydrogen phosphate, salicylate, tartrate, maleate, fumarate, methanesulfonate, and p-toluenesulfonate.

[0083] Unless otherwise specified, all compounds mentioned in this invention include all possible optical isomers, such as compounds with a single chirality, or mixtures of various chiral compounds. In all compounds of this invention, each chiral carbon atom may optionally be in the R configuration or S configuration, or a mixture of R and S configurations.

[0084] As used in this invention, "antibody" is used in its broadest sense and covers monoclonal antibodies, polyclonal antibodies, dimers, multimeric multispecific antibodies, and antibody fragments, provided they exhibit the desired biological activity. Antibodies are proteins produced by the immune system capable of recognizing and binding to specific antigens. Target antigens generally have a CDR composed of multiple antibodies. S Numerous binding sites are identified, i.e., antigenic epitopes. An antigen has more than one corresponding antibody. Such targets include, but are not limited to, cancer cells or cells that produce autoantibodies associated with autoimmune diseases. Immunoglobulins can be derived from any species. However, in one aspect, immunoglobulins are derived from humans, mice, or rabbits. An "antibody fragment" may contain a portion of a full-length antibody, generally its antigen-binding region or variable region. Examples of antibody fragments include: Fab, Fab', F(ab')2, and Fv fragments; biantibodies; nanobodies, etc. Fragments prepared from Fab expression libraries; multispecific antibodies formed from Fab fragments. The antibodies in the antibody-drug conjugates of this application can retain the ability to bind to wild-type antigens. Antigens involved include, for example, tumor-associated antigens (TAAs), cell surface receptor proteins and other cell surface molecules, cell proliferation regulators, lymphokines, cytokines, and molecules related to angiogenesis. Antibodies used in antibody-drug conjugates include, but are not limited to, antibodies against cell surface receptors and tumor-associated antigens. Such tumor-associated antigens are well known to those skilled in the art and can be obtained through antibody preparation methods well known in the industry.

[0085] In this invention, the term "tumor" refers to a tissue mass formed by the abnormal proliferation of body cells under the influence of genetic or environmental factors. Tumors can be classified as benign (localized growth, non-metastatic) or malignant (highly invasive, metastatic). The essence of malignant tumors (cancer) is the uncontrolled growth of cells due to gene mutations (such as activation of proto-oncogenes or inactivation of tumor suppressor genes), which can evade immune clearance and even spread throughout the body via the blood or lymphatic system. Their occurrence is related to multiple factors such as smoking, infection, and radiation, and treatment requires a comprehensive approach combining surgery, radiotherapy, targeted drugs, or immunotherapy.

[0086] In this invention, the term "and / or" includes any one or more of the related listed items and all combinations thereof.

[0087] Example 1 Adenosine A 2A The R-antagonist benzimidazolidine-3-carboxamide has the effect of enhancing PD-1 monoclonal antibody immunotherapy for esophageal squamous cell carcinoma.

[0088] 1. Experimental materials:

[0089] (1) Tumor organoids: SYEC2, a murine esophageal squamous cell carcinoma organoid model

[0090] (2) Experimental mice: C57BL / 6J mice (Guangzhou Yaokang Biotechnology Co., Ltd.).

[0091] (3) Drug: Mouse PD-1 monoclonal antibody (PD-1 monoclonal antibody), clone number G4C2 (BioXcell, BE0146).

[0092] (4) Drug: Adenosine A 2A R-antagonist (AZD4635 or benzimidazolidine-3-carboxamide), AZD4635 is a competitor of benzimidazolidine-3-carboxamide, which is adenosine A. 2A R-chiral small molecule antagonist, S isomer, abbreviated as (S)-E8.

[0093] 2. Experimental steps:

[0094] (1) Four- to six-week-old C57 mice were housed under pathogen-free conditions, and 100 μL of SYEC2 organoids (1 × 10⁻⁶) were injected into both sides of the back of the mice. 6 Suspension. Once the tumor forms a visible mass, measure its size every two days using calipers. When the tumor volume (volume = length (mm) × width (mm)² × 0.5) reaches 1000 mm², the tumor is considered complete. 3 The tumors were removed and transplanted subcutaneously into immunocompetent C57BL / 6J mice to create a tumor model. (Transplantation objective: to increase tumor number and eliminate stromal colloid interference)

[0095] (2) The tumor model volume reached 200 mm after transplantation. 3 Mice were randomly divided into 6 groups for drug treatment, with 5 mice in each group:

[0096] 1) Control group (IgG): IgG (BioXcell, BE0089) was administered as a control treatment in accordance with the PD-1 monoclonal antibody treatment regimen;

[0097] 2) Treatment group (PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody treatment;

[0098] 3) Treatment group (AZD4635): AZD4635 was administered;

[0099] 4) Treatment group ((S)-E8): treated with benzimidazolidine-3-carboxamide;

[0100] 5) Treatment group (AZD4635 + PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody combined with AZD4635 for treatment;

[0101] 6) Treatment group ((S)-E8+PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody combined with benzimidazolidine-3-carboxamide;

[0102] The IgG and PD-1 monoclonal antibodies were administered via intraperitoneal injection on days 1, 4, and 7, respectively, at a dose of 5 mg / kg. The compound AZD4635 (25 mg / kg) or benzimidazolidine-3-carboxamide (25 mg / kg) was administered orally starting on day 1, once a day for 14 consecutive days.

[0103] (3) Measure the size of the tumor every 3 days and calculate the tumor volume. The formula for calculating the tumor volume is length × width × width / 2.

[0104] (4) At the experimental endpoint, mice were euthanized and tumor tissue was dissected. Tumor size and volume were measured every 2–3 days during the drug administration period using calipers. The tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = {1 - (mean volume at endpoint - mean volume at starting point) of the drug administration group / (mean volume at endpoint - mean volume at starting point) of the blank control group} × 100%. After the experiment, mice were anesthetized and euthanized by cervical dislocation, followed by dissection to collect the tumor.

[0105] 3. Result Description:

[0106] Treatment results as Figure 1 The in vivo antitumor activity of the experimental group in the C57 mouse SYEC2 esophageal squamous cell carcinoma model was evaluated. (A) Tumor volume changes, expressed in cubic millimeters, were measured in the single-drug groups of IgG, PD-1 monoclonal antibody, (S)-E8, and AZD4635, and in the groups of (S)-E8 and AZD4635 combined with PD-1 monoclonal antibody, respectively, after 1-14 days of drug treatment. (B) Based on the results of (A), the tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = {1 - (mean volume at endpoint - mean volume at starting point) of the drug-treated group / (mean volume at endpoint - mean volume at starting point) of the blank control group} × 100%. The tumor inhibition rate of each experimental group compared with the IgG group was obtained, reflecting the therapeutic effect of the drug. (C) At the experimental endpoint, tumor tissue was collected and the tumor weight was measured, expressed in grams, accurate to 0.01 units.

[0107] The results showed that AZD4635 or benzimidazolidine-3-carboxamide alone had no significant effect, PD-1 monoclonal antibody monotherapy had no significant effect, and PD-1 monoclonal antibody and adenosine A had no significant effect. 2A The combination of R-antagonists (AZD4635 or benzimidazolidine-3-carboxamide) showed significant effects, with a marked reduction in tumor volume after 14 days of administration. From the perspective of relative changes in tumor volume, the combination of benzimidazolidine-3-carboxamide and PD-1 showed no significant increase in tumor volume relative to the origin of tumor growth, indicating a superior effect compared to AZD4635. Figure 1A). Based on the analysis of tumor inhibition rates, it can be seen that, compared with the normal group, the tumor inhibition rate of the PD-1 monoclonal antibody monotherapy group was 44.40%, the tumor inhibition rate of the AZD4635 monotherapy group was -4.96%, the tumor inhibition rate of the (S)-E8 monotherapy group was 66.36%, the tumor inhibition rate of the AZD4635 + PD-1 monoclonal antibody dual therapy group was 79.94%, and the tumor inhibition rate of the (S)-E8 + PD-1 monoclonal antibody dual therapy group was 96.49%. Figure 1 B). At the experimental endpoint, the isolated tumor tissues were weighed. Statistical analysis showed that the tumor mass in the dual-drug treatment group was smaller than that in other groups, and the (S)-E8+PD-1 monoclonal antibody treatment group was smaller than that in the AZD4635+PD-1 monoclonal antibody treatment group. Figure 1 C). In conclusion, AZD4635 and (S)-E8 significantly improved the tumor-suppressive effect of immunotherapy by more than 50%, indicating that adenosine A... 2A R antagonists enhance the effect of PD-1 monoclonal antibody immunotherapy for esophageal squamous cell carcinoma, and the enhancing effect of benzimidazolidine-3-carboxamide is more significant.

[0108] Example 2 Adenosine A 2A The R-antagonist benzimidazolidine-3-carboxamide enhances the effect of PD-1 monoclonal antibody immunotherapy for gastric cancer.

[0109] 1. Experimental materials:

[0110] (1) Tumor organoids: mouse-derived gastric cancer organoids mGCO.

[0111] (2) Experimental mice: C57BL / 6J mice (Guangzhou Yaokang Biotechnology Co., Ltd.).

[0112] (3) Drug: Mouse PD-1 monoclonal antibody, clone number G4C2 (BioXcell, BE0146).

[0113] (4) Drug: Adenosine A 2A R-antagonist (AZD4635 or benzimidazolidine-3-carboxamide), AZD4635 is a competitor of benzimidazolidine-3-carboxamide, which is adenosine A. 2A R-chiral small molecule antagonist, S isomer, abbreviated as (S)-E8.

[0114] 2. Experimental steps:

[0115] (1) After digesting mouse gastric cancer organoids into single cells, counting them, and taking 2×10⁶ cells... 6 Cells were resuspended in 50% standard matrix gel and injected subcutaneously into immunocompetent C57BL / 6J mice to construct a tumor model. The tumors were cultured until they reached a size of 1000 mm. 3The tumors were removed and transplanted subcutaneously into immunocompetent C57BL / 6J mice to create a tumor model. (Transplantation objective: to increase tumor number and eliminate stromal colloid interference)

[0116] (2) The tumor model volume reached 200 mm after transplantation. 3 The patients were divided into 6 groups for drug administration treatment:

[0117] 1) Control group (IgG): IgG (BioXcell, BE0089) was administered as a control treatment according to the PD-1 treatment regimen;

[0118] 2) Treatment group (PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody treatment;

[0119] 3) Treatment group (AZD4635): AZD4635 was administered;

[0120] 4) Treatment group ((S)-E8): treated with benzimidazolidine-3-carboxamide;

[0121] 5) Treatment group (AZD4635 + PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody combined with AZD4635 for treatment;

[0122] 6) Treatment group ((S)-E8+PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody combined with benzimidazolidine-3-carboxamide;

[0123] IgG and PD-1 were administered via intraperitoneal injection on days 1, 4, and 7, respectively, at a dose of 5 mg / kg. Compound AZD4635 (25 mg / kg) or benzimidazolidine-3-carboxamide (25 mg / kg) was administered orally starting on day 1, once daily for 14 consecutive days.

[0124] (3) Measure the size of the tumor every 3 days and calculate the tumor volume. The formula for calculating the tumor volume is length × width × width / 2.

[0125] (4) At the experimental endpoint, mice were euthanized, and tumor tissue, heart, kidneys, spleen, and liver tissue were dissected. During the drug administration period, tumor size was measured every 2–3 days using calipers, and tumor volume and mouse weight were calculated. The tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = {1 - (mean volume at endpoint - mean volume at starting point) of the drug administration group / (mean volume at endpoint - mean volume at starting point) of the blank control group} × 100%. After the experiment, mice were anesthetized and euthanized by cervical dislocation, and tumors were then dissected and collected.

[0126] 3. Result Description:

[0127] Treatment results as Figure 2Evaluation of in vivo antitumor activity in a C57 mouse gastric cancer model: (A) Tumor volume changes, expressed in cubic millimeters, in the IgG, PD-1 monoclonal antibody, (S)-E8, and AZD4635 monotherapy groups and the (S)-E8 and AZD4635 combined with PD-1 monoclonal antibody groups, treated for 1-14 days. (B) Based on the results of (A), the tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = {1 - (endpoint average volume - starting average volume) of the treated group / (endpoint average volume - starting average volume) of the blank control group} × 100%. This calculation yielded the tumor inhibition rate of each experimental group compared to the IgG group, reflecting the therapeutic effect of the drugs. (C) At the experimental endpoint, tumor tissue was collected and the tumor weight was measured, expressed in grams, accurate to 0.01 units.

[0128] The results showed that AZD4635 or benzimidazolidine-3-carboxamide alone had no significant effect, PD-1 monoclonal antibody monotherapy had no significant effect, and PD-1 monoclonal antibody and adenosine A had no significant effect. 2A The combination of R-antagonists (AZD4635 or benzimidazolidine-3-carboxamide) showed significant effects, with a marked reduction in tumor volume after 14 days of administration. From the perspective of relative changes in tumor volume, the combination of benzimidazolidine-3-carboxamide and PD-1 showed no significant increase in tumor volume relative to the origin of tumor growth, indicating a superior effect compared to AZD4635. Figure 2 A). Based on the analysis of tumor inhibition rates, it can be seen that, compared with the normal group, the tumor inhibition rate of the PD-1 monotherapy group was 36.01%, the tumor inhibition rate of the AZD4635 monotherapy group was 33.52%, the tumor inhibition rate of the (S)-E8 monotherapy group was 5.48%, the tumor inhibition rate of the AZD4635 + PD-1 monoclonal antibody dual therapy group was 78.58%, and the tumor inhibition rate of the (S)-E8 + PD-1 monoclonal antibody dual therapy group was 89.64%. Figure 2 B). At the experimental endpoint, the isolated tumor tissues were weighed. Statistical analysis showed that the tumor mass in the dual-drug treatment group was smaller than that in other groups, and the (S)-E8+PD-1 monoclonal antibody treatment group was smaller than that in the AZD4635+PD-1 monoclonal antibody treatment group. Figure 2 C). In conclusion, AZD4635 and (S)-E8 significantly improved the tumor-suppressive effect of immunotherapy by more than 50%, indicating that adenosine A... 2A R antagonists enhance the effect of PD-1 monoclonal antibody immunotherapy for gastric cancer, and the enhancing effect of benzimidazolidine-3-carboxamide is more significant.

[0129] Example 3 Adenosine A 2A The R-antagonist benzimidazolidine-3-carboxamide enhances the effect of PD-1 monoclonal antibody immunotherapy for colorectal cancer.

[0130] 1. Experimental materials:

[0131] (1) Tumor organoids: murine colorectal cancer cells CT26.

[0132] (2) Experimental mice: C57BL / 6J mice (Guangzhou Yaokang Biotechnology Co., Ltd.).

[0133] (3) Drug: Mouse PD-1 monoclonal antibody, clone number G4C2 (BioXcell, BE0146).

[0134] (4) Drug: Adenosine A 2A R-antagonist (AZD4635 or benzimidazolidine-3-carboxamide), AZD4635 is a competitor of benzimidazolidine-3-carboxamide, which is adenosine A. 2A R-chiral small molecule antagonist, S isomer, abbreviated as (S)-E8.

[0135] 2. Experimental steps:

[0136] (1) After digesting murine colorectal cancer cells CT26 into single cells, they were counted, and 1×10⁶ cells were collected. 6 A tumor model was constructed by subcutaneous injection of cells into immunocompetent C57BL / 6J mice. The tumors were cultured until they reached a size of 50 mm. 3 Dosing was administered in groups on the left and right sides.

[0137] (2) The patients were divided into 6 groups for drug administration:

[0138] 1) Control group (IgG): IgG (BioXcell, BE0089) was administered as a control treatment according to the PD-1 treatment regimen;

[0139] 2) Treatment group (PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody treatment;

[0140] 3) Treatment group (AZD4635): AZD4635 was administered;

[0141] 4) Treatment group ((S)-E8): treated with benzimidazolidine-3-carboxamide;

[0142] 5) Treatment group (AZD4635 + PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody combined with AZD4635 for treatment;

[0143] 6) Treatment group ((S)-E8+PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody combined with benzimidazolidine-3-carboxamide;

[0144] IgG and PD-1 were administered via intraperitoneal injection on days 1, 4, and 7, respectively, at a dose of 5 mg / kg. Compound AZD4635 (25 mg / kg) or benzimidazolidine-3-carboxamide (25 mg / kg) was administered orally starting on day 1, once daily for 14 consecutive days.

[0145] (3) Measure the size of the tumor every 3 days and calculate the tumor volume. The formula for calculating the tumor volume is length × width × width / 2.

[0146] (4) At the experimental endpoint, mice were euthanized, and tumor tissue, heart, kidney, spleen, and liver tissue were dissected. Tumor size was measured every 2–3 days during the drug administration period, and tumor volume and mouse weight were calculated. The tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = {1 - (mean volume at endpoint - mean volume at starting point) of the drug administration group / (mean volume at endpoint - mean volume at starting point) of the blank control group} × 100%. After the experiment, mice were anesthetized and euthanized by cervical dislocation, followed by dissection to collect the tumors.

[0147] 3. Result Description:

[0148] Treatment results as Figure 3 The in vivo antitumor activity of the drug was evaluated in a C57 mouse colorectal cancer model. (A) Tumor volume changes, expressed in cubic millimeters, were measured in the groups treated with IgG, PD-1 monoclonal antibody, (S)-E8, and AZD4635 monotherapy, as well as in the groups treated with (S)-E8 and AZD4635 in combination with PD-1 monoclonal antibody, for 1-14 days. (B) Based on the results of (A), the tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = {1 - (mean volume at endpoint - mean volume at starting point) of the treated group / (mean volume at endpoint - mean volume at starting point) of the blank control group} × 100%. This calculation yielded the tumor inhibition rate of each experimental group compared to the IgG group, reflecting the therapeutic effect of the drug. (C) At the experimental endpoint, tumor tissue was collected and the tumor weight was measured, expressed in grams, accurate to 0.01 units.

[0149] The results showed that AZD4635 or benzimidazolidine-3-carboxamide alone had no significant effect, PD-1 monoclonal antibody monotherapy had no significant effect, and PD-1 monoclonal antibody and adenosine A had no significant effect. 2A The combination of R-antagonists (AZD4635 or benzimidazolidine-3-carboxamide) showed significant effects, with a marked reduction in tumor volume after 14 days of administration. From the perspective of relative changes in tumor volume, the combination of benzimidazolidine-3-carboxamide and PD-1 showed no significant increase in tumor volume relative to the origin of tumor growth, indicating a superior effect compared to AZD4635. Figure 3A). Based on the analysis of tumor inhibition rates, it can be seen that, compared with the normal group, the tumor inhibition rate of the PD-1 monoclonal antibody monotherapy group was 15.25%, the tumor inhibition rate of the AZD4635 monotherapy group was 41.54%, the tumor inhibition rate of the (S)-E8 monotherapy group was 30.03%, the tumor inhibition rate of the AZD4635 + PD-1 monoclonal antibody dual therapy group was 65.59%, and the tumor inhibition rate of the (S)-E8 + PD-1 monoclonal antibody dual therapy group was 77.52%. Figure 3 B). At the experimental endpoint, the isolated tumor tissues were weighed. Statistical analysis showed that the tumor mass in the dual-drug treatment group was smaller than that in other groups, and the (S)-E8+PD-1 monoclonal antibody treatment group was smaller than that in the AZD4635+PD-1 monoclonal antibody treatment group. Figure 3 C). In conclusion, AZD4635 and (S)-E8 significantly improved the tumor-suppressive effect of immunotherapy by more than 50%, indicating that adenosine A... 2A R antagonists enhance the effect of PD-1 monoclonal antibody immunotherapy for colorectal cancer, and the enhancing effect of benzimidazolidine-3-carboxamide is more significant.

[0150] Example 4 Adenosine A 2A The R-antagonist benzimidazolidine-3-carboxamide enhances the effect of PD-1 monoclonal antibody immunotherapy in renal cell carcinoma.

[0151] 1. Experimental materials:

[0152] (1) Tumor organoids: mouse renal cell carcinoma Renca (commercially available model).

[0153] (2) Experimental mice: C57BL / 6J mice (Guangzhou Yaokang Biotechnology Co., Ltd.).

[0154] (3) Drug: Mouse PD-1 monoclonal antibody, clone number G4C2 (BioXcell, BE0146). (4) Drug: Adenosine A 2A R-antagonist (benzimidazolidine-3-carboxamide), benzimidazolidine-3-carboxamide is adenosine A 2A R-chiral small molecule antagonist, S isomer, abbreviated as (S)-E8.

[0155] 2. Experimental steps:

[0156] (1) After digesting mouse colorectal cancer cells (Renca) into single cells, they were counted, and 1×10⁻⁶ cells were collected. 6 A tumor model was constructed by subcutaneous injection of cells into immunocompetent C57BL / 6J mice. The tumors were cultured until they reached a size of 50 mm. 3 Dosing was administered in groups on the left and right sides.

[0157] (2) The patients were divided into 6 groups for drug administration:

[0158] 1) Control group (IgG): IgG (BioXcell, BE0089) was administered as a control treatment according to the PD-1 treatment regimen;

[0159] 2) Treatment group (PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody treatment;

[0160] 3) Treatment group ((S)-E8): treated with benzimidazolidine-3-carboxamide;

[0161] 4) Treatment group ((S)-E8+PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody combined with benzimidazolidine-3-carboxamide;

[0162] IgG and PD-1 were administered via intraperitoneal injection on day 7 of tumor growth, at a dose of 5 mg / kg, twice a week; the compound benzimidazolidine-3-carboxamide (25 mg / kg) was administered orally starting on day 7, once a day for 18 consecutive days.

[0163] (3) Measure the size of the tumor every 4 days and calculate the tumor volume. The formula for calculating the tumor volume is length × width × width / 2.

[0164] (4) At the experimental endpoint, mice were euthanized and tumor tissue was dissected. Tumor size and volume were measured every 2–3 days during the drug administration period using calipers. The tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = {1 - (mean volume at endpoint - mean volume at starting point) of the drug administration group / (mean volume at endpoint - mean volume at starting point) of the blank control group} × 100%. After the experiment, mice were anesthetized and euthanized by cervical dislocation, followed by dissection to collect the tumor.

[0165] 3. Result Description:

[0166] Treatment results as Figure 4 Evaluation of in vivo antitumor activity in a C57 mouse renal cell carcinoma model: (A) Tumor volume changes in the IgG, PD-1 monoclonal antibody, (S)-E8, and (S)-E8 combined with PD-1 monoclonal antibody groups from day 1 to 18 of drug treatment, expressed in cubic millimeters. (B) Based on the results of (A), the tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = {1 - (mean volume at endpoint - mean volume at starting point) of the drug-treated group / (mean volume at endpoint - mean volume at starting point) of the blank control group} × 100%, thus obtaining the tumor inhibition rate of each experimental group compared to the IgG group, reflecting the therapeutic effect of the drugs. (C) At the experimental endpoint, tumor tissue was collected and the tumor weight was measured, expressed in grams, accurate to 0.01 units.

[0167] The results showed that benzimidazolidine-3-carboxamide alone had no significant effect, PD-1 monoclonal antibody alone had no significant effect, but the combination of PD-1 monoclonal antibody and benzimidazolidine-3-carboxamide was significantly effective, significantly reducing tumor volume after two weeks of administration. From the perspective of relative changes in tumor volume, it can be seen that the tumor volume in the benzimidazolidine-3-carboxamide combined with PD-1 monoclonal antibody group did not show significant growth relative to the starting point. Figure 4 A). Based on the analysis of tumor inhibition rates, it can be seen that, compared with the normal group, the tumor inhibition rate of the PD-1 monoclonal antibody monotherapy group was 22.85%, the tumor inhibition rate of the (S)-E8 monotherapy group was 9.48%, and the tumor inhibition rate of the (S)-E8 + PD-1 monoclonal antibody dual therapy group was 51.01%. Figure 4 B). At the experimental endpoint, the isolated tumor tissues were weighed. Statistical analysis showed that the tumor mass in the dual-drug treatment group was smaller than that in other groups, and the (S)-E8+PD-1 monoclonal antibody treatment group was smaller than that in the PD-1 monoclonal antibody single-drug treatment group. Figure 4 C). In conclusion, (S)-E8 significantly improved the tumor-suppressing effect of immunotherapy by more than 50%, indicating that adenosine A... 2A R antagonists can enhance the effect of PD-1 monoclonal antibody immunotherapy for renal cell carcinoma.

[0168] Example 5 Adenosine A 2A The R-antagonist benzimidazolidine-3-carboxamide enhances the effect of PD-1 monoclonal antibody immunotherapy for melanoma.

[0169] 1. Experimental materials:

[0170] (1) Tumor organoids: murine melanoma cells B16-F10 (commercially available model).

[0171] (2) Experimental mice: C57BL / 6J mice (Guangzhou Yaokang Biotechnology Co., Ltd.).

[0172] (3) Drug: Mouse PD-1 monoclonal antibody, clone number G4C2 (BioXcell, BE0146). (4) Drug: Adenosine A 2A R-antagonist (benzimidazolidine-3-carboxamide), benzimidazolidine-3-carboxamide is adenosine A 2A R-chiral small molecule antagonist, S isomer, abbreviated as (S)-E8.

[0173] 2. Experimental steps:

[0174] (1) After digesting mouse melanoma cells B16-F10 into single cells, they were counted, and 1×10⁶ cells were collected. 6 A tumor model was constructed by subcutaneous injection of cells into immunocompetent C57BL / 6J mice. The tumors were cultured until they reached a size of 50 mm. 3Dosing was administered in groups on the left and right sides.

[0175] (2) The patients were divided into 6 groups for drug administration:

[0176] 1) Control group (IgG): IgG (BioXcell, BE0089) was administered as a control treatment according to the PD-1 treatment regimen;

[0177] 2) Treatment group (PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody treatment;

[0178] 3) Treatment group ((S)-E8): treated with benzimidazolidine-3-carboxamide;

[0179] 4) Treatment group ((S)-E8+PD-1 monoclonal antibody): Received murine PD-1 monoclonal antibody combined with benzimidazolidine-3-carboxamide;

[0180] Among them, IgG and PD-1 monoclonal antibodies were administered via intraperitoneal injection on days 7, 10, and 13 of tumor growth, respectively, at a dose of 5 mg / kg; the compound benzimidazolidine-3-carboxamide (25 mg / kg) was administered orally starting on day 7, once a day for 9 consecutive days.

[0181] (3) Measure the size of the tumor every 4 days and calculate the tumor volume. The formula for calculating the tumor volume is length × width × width / 2.

[0182] (4) At the experimental endpoint, mice were euthanized and tumor tissue was dissected. Tumor size and volume were measured every 3 days during the drug administration period using calipers. The tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = {1 - (mean volume at endpoint - mean volume at starting point) of the drug administration group / (mean volume at endpoint - mean volume at starting point) of the blank control group} × 100%. After the experiment, mice were anesthetized and euthanized by cervical dislocation, followed by dissection to collect the tumor tissue.

[0183] 3. Result Description:

[0184] Treatment results as Figure 5The in vivo antitumor activity of the drug was evaluated in a C57 mouse melanoma model. (A) Tumor volume changes in the IgG, PD-1 monoclonal antibody, (S)-E8, and (S)-E8 combined with PD-1 monoclonal antibody groups were measured in cubic millimeters after treatment for 1-16 days. (B) Based on the results of (A), the tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = {1 - (mean volume at endpoint - mean volume at starting point) of the treated group / (mean volume at endpoint - mean volume at starting point) of the blank control group} × 100%. The tumor inhibition rate of each experimental group compared with the IgG group was obtained, reflecting the therapeutic effect of the drug. (C) At the experimental endpoint, tumor tissue was collected and the tumor weight was measured in grams, accurate to 0.01 units.

[0185] The results showed that benzimidazolidine-3-carboxamide alone had no significant effect, while PD-1 monoclonal antibody monotherapy was significantly effective. The combination of PD-1 monoclonal antibody and benzimidazolidine-3-carboxamide was even more effective, significantly reducing tumor volume after one week of administration. From the perspective of relative changes in tumor volume, it could be seen that the tumor volume in the benzimidazolidine-3-carboxamide combined with PD-1 group did not show significant relative proliferation at the origin. Figure 5 A). Based on the analysis of tumor inhibition rates, it can be seen that, compared with the normal group, the tumor inhibition rate of the PD-1 monotherapy group was 11.94%, the tumor inhibition rate of the (S)-E8 monotherapy group was 6.19%, and the tumor inhibition rate of the (S)-E8 + PD-1 monoclonal antibody dual therapy group was 73.14%. Figure 5 B). At the experimental endpoint, the isolated tumor tissues were weighed. Statistical analysis showed that the tumor mass in the dual-drug treatment group was smaller than that in other groups, and the (S)-E8+PD-1 monoclonal antibody treatment group was significantly smaller than that in the PD-1 monoclonal antibody treatment group. Figure 5 C). In conclusion, (S)-E8 significantly improved the tumor-suppressive effect of immunotherapy, increasing it to 73.14%, indicating that adenosine A... 2A R antagonists can enhance the effect of PD-1 monoclonal antibody immunotherapy for melanoma.

[0186] Example 6 Adenosine A 2A The combination of the R-antagonist benzimidazolidine-3-carboxamide and PD-1 monoclonal antibody immunotherapy did not produce significant toxic side effects.

[0187] 1. Based on the results obtained from the experiments in Examples 2 and 3 of the present invention, during the administration of drugs to the gastric cancer tumor model mGCO and the colorectal cancer tumor model CT26 of C57 and Balb / c mice, respectively, the weight of the mice was measured, the surface condition of the mice was observed, and the heart, kidney, spleen and liver tissues of the mice were collected at the end of the experiment.

[0188] 2. Result Description:

[0189] Treatment results as Figure 6Evaluation of toxic side effects in C57 and Balb / C mice before and after administration: (A) Changes in body weight of C57 mice, in grams (g), accurate to 0.1. (B) The three columns on the left, from top to bottom, show the heart, left and right kidneys, and spleen tissues of C57 mice; the three columns on the right, show the liver tissues of C57 mice. Each group contained three mice. (C) Body surface condition of C57 mice. (D) Changes in body weight of Balb / C mice, in grams (g), accurate to 0.1. The three columns on the left, from top to bottom, show the heart, left and right kidneys, and spleen tissues of C57 mice; the three columns on the right, show the liver tissues of Balb / C mice. Each group contained three mice.

[0190] The results showed that benzimidazolidine-3-carboxamide, alone or in combination with PD-1 monoclonal antibody, had no significant effect on body weight in C57 mice. Figure 6 A), and it did not produce toxic effects on the heart, kidneys, spleen, and liver tissues of mice. Figure 6 B) Observation of the body surface revealed that AZD4635 had significant side effects on the ear skin of C57 mice. Figure 6 C). In Balb / c mice, there was no significant effect on body weight. Figure 6 D), and it did not produce toxic effects on the heart, kidneys, spleen, and liver tissues of mice. Figure 6 E). In summary, at the administered dose, (S)-E8 showed no significant toxicity to C57 and Balb / c mice compared to the control group and the competing product AZD4635.

[0191] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Adenosine A 2A The application of R antagonists in the preparation of drugs for enhancing the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibiting tumor cell growth, and / or increasing the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reducing tumor weight, and / or increasing the infiltration of activated cytotoxic T cells in tumor tissue, and / or activating the body's own immune system.

2. The application according to claim 1, characterized in that, The models used in the application include mouse tumor models.

3. The application according to claim 2, characterized in that, The mouse tumor models include models in which tumor organoids or cells undergo subcutaneous tumor formation.

4. A method for targeting adenosine A 2A R and / or a drug / pharmaceutical composition for tumor immunotherapy and / or inhibition of tumor cell growth, characterized in that, The drug / drug composition comprises adenosine A as described in claim 1. 2A R antagonist is the main active ingredient.

5. The drug / drug composition according to claim 4, characterized in that, The drug / drug composition further comprises a pharmaceutically acceptable carrier.

6. The pharmaceutical / pharmaceutical composition according to claim 4 or 5, characterized in that, The drug / drug composition may be used alone and / or in combination with other therapeutic agents, including PD-1 antibodies and PD-L1 antibodies, and with one or more chemotherapy drugs.

7. An immunotherapy enhancer, characterized in that, The immunotherapy enhancer comprises adenosine A as described in claim 1. 2A R antagonist.

8. The immunotherapy enhancer according to claim 7, characterized in that, The immunotherapy enhancer has the effects of enhancing the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibiting tumor cell growth, and / or increasing the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reducing tumor weight, and / or increasing the infiltration of activated cytotoxic T cells in tumor tissue, and / or activating the body's own immune system.

9. The use of the medicament / medical composition according to any one of claims 4-6, or the immunotherapy potentiator according to claim 7 or 8, in the preparation of a medicament for enhancing the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibiting tumor cell growth, and / or increasing the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reducing tumor weight, and / or increasing the infiltration of activated cytotoxic T cells in tumor tissue, and / or activating the body's own immune system.

10. The application, pharmaceutical / pharmaceutical composition, or immunotherapy enhancer according to any one of claims 1-9, characterized in that, adenosine A 2A R antagonists include one or more of benzimidazolidine-3-carboxamide or its pharmaceutically acceptable salts or solvates, and AZD4635; Wherein, the benzimidazolidine-3-carboxamide is a small molecule antagonist; and / or, the benzimidazolidine-3-carboxamide is a subtype-selective adenosine A. 2A R antagonist is a (S)-1-amino-N-(1-(pyridin-2-yl)ethyl)benzo[4,5]imidazo[1,2-a]pyrazine-3-carboxamide small molecule compound; and / or, the molecular formula of said benzimidazolium-pyrazine-3-carboxamide is C 18 H 16 N6O, with a molecular weight of 332.36; AZD4635 is a small molecule antagonist, an effective and selective oral adenosine A antagonist. 2A R antagonist; and / or, The immunotherapy is to promote the secretion of cytokines by immune cells; and / or, The immunotherapy aims to enhance the tumor-killing effect of immune cells; and / or, adenosine A 2A R antagonists can enhance the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibit tumor cell growth, and / or increase the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reduce tumor weight, and / or increase the infiltration of activated cytotoxic T cells in tumor tissue, and / or activate the body's own immune system; and / or, The drug or drug / drug composition is capable of enhancing the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibiting tumor cell growth, and / or increasing the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reducing tumor weight, and / or increasing the infiltration of activated cytotoxic T cells in tumor tissue, and / or activating the body's own immune system; and / or, The immunotherapy enhancer can enhance the therapeutic efficacy of PD-1 antibody and / or PD-L1 antibody immunotherapy for tumors, and / or inhibit tumor cell growth, and / or increase the tumor inhibition rate of PD-1 antibody and / or PD-L1 antibody, and / or significantly reduce tumor weight, and / or increase the infiltration of activated cytotoxic T cells in tumor tissue, and / or activate the body's own immune system; and / or, The tumors include gastrointestinal tumors, renal cell carcinoma, and melanoma.

11. The application, drug / drug composition, and immunotherapy enhancer according to claim 10, characterized in that, The digestive tract tumors include esophageal squamous cell carcinoma, gastric cancer, and colorectal cancer.

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