Use of a kat5 inhibitor in enhancing the efficacy of natural killer cell immunotherapy drugs, drugs

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of a KAT5 inhibitor in a medicine for enhancing the immunotherapy effect of natural killer cells, and a medicine for enhancing the immunotherapy effect of natural killer cells. The KAT5 inhibitor comprises at least one of Nu9056, TH1834 and MG149. The KAT5 inhibitor can inhibit the recruitment of a transcription factor SP1 to an ADAM10 promoter, reduce the transcriptional activity of ADAM10, reduce the expression of NKG2D ligands on the cell surface, and thus enhance the sensitivity of tumor cells to NK cell killing. The application first discloses a new mechanism in which KAT5 regulates an ADAM10-NKG2D signal axis through an epigenetic mechanism, and proves that the KAT5 inhibitor can significantly enhance the immunotherapy effect of NK cells in vitro and in vivo. The application provides a brand-new target and strategy for developing a new immunosensitizer and a combined cell therapy.

Description

Technical Field

[0001] This application belongs to the field of biomedical technology, and in particular relates to the application of a KAT5 inhibitor in a drug that enhances the immunotherapeutic efficacy of natural killer cells, and a drug that enhances the immunotherapeutic efficacy of natural killer cells. Background Technology

[0002] Natural killer (NK) cells are the core effector cells of the body's innate immune system, serving as a bridge between innate and adaptive immunity. They can directly recognize and kill tumor cells and exert immunomodulatory functions by secreting various cytokines, thus playing a crucial role in the body's immune surveillance and early defense. The killing activity of NK cells is finely regulated by a series of activating and inhibitory receptors on their surface, maintaining a balance between self-tolerance and effective killing. Activating receptors can recognize specific ligands on target cells. When these receptors are activated, they transmit signals to the NK cell interior, promoting NK cell activation and enhancing its killing ability. For example, NKG2D (natural killer cell receptor 2D) is an important activating receptor that can recognize various stress-induced ligands, such as MICA (major histocompatibility complex class I chain-associated protein A) and MICB (major histocompatibility complex class I chain-associated protein B). These ligands are expressed at increased levels on tumor cells and virus-infected cells, thereby activating NK cells to attack these abnormal cells.

[0003] Unfortunately, tumor cells have evolved various mechanisms to evade the immune surveillance of NK cells. The most common of these is the downregulation or complete loss of expression of their surface NKG2D ligands. Furthermore, some tumor cells cleave membrane-bound MICA / B into soluble forms (sMICA / B) through protease-mediated proteolysis. These soluble ligands not only competitively bind to the NKG2D receptor on the NK cell surface, blocking its recognition, but also induce NKG2D receptor endocytosis and degradation, leading to NK cell functional exhaustion and severely weakening the NK cell's killing ability. Summary of the Invention

[0004] The purpose of this application is to provide an application of a KAT5 inhibitor in a drug that enhances the efficacy of natural killer cell immunotherapy, and a drug that enhances the efficacy of natural killer cell immunotherapy, aiming to solve the problem of insufficient NK cell killing activity in existing tumor immunotherapy.

[0005] To achieve the above-mentioned objectives, the technical solution adopted in this application is as follows: In a first aspect, this application provides the use of a KAT5 inhibitor in a drug that enhances the immunotherapeutic efficacy of natural killer cells, including applying the KAT5 inhibitor to the drug that enhances the immunotherapeutic efficacy of natural killer cells, wherein the KAT5 inhibitor includes at least one of Nu9056, TH1834 and MG149.

[0006] In some possible implementations, the KAT5 inhibitor downregulates ADAM10 transcription by inhibiting the recruitment of transcription factor SP1 to the ADAM10 gene promoter region, thereby reducing the expression of NKG2D ligands on the cell surface and enhancing the sensitivity of tumor cells to natural killer cells.

[0007] In some possible implementations, the KAT5 inhibitor includes Nu9056.

[0008] In some possible implementations, the natural killer cells include at least one of the NK-92 cell line, primary NK cells, IPSC-NK cells, and CAR-NK cells.

[0009] In some possible implementations, the natural killer cells include the NK-92 cell line.

[0010] In some possible implementations, the KAT5 inhibitor is used in combination with the natural killer cell adoptive therapy.

[0011] In some possible implementations, the amount of the KAT5 inhibitor is such that, in in vitro cytotoxicity experiments, the natural killer cells increase the killing rate of tumor cells by ≥50% when the effector-target ratio is 5:1 to 10:1.

[0012] Secondly, this application provides a drug that enhances the immunotherapeutic effect of natural killer cells, comprising a KAT5 inhibitor and an excipient, wherein the KAT5 inhibitor comprises at least one of Nu9056, TH1834 and MG149.

[0013] In some possible implementations, the effective in vivo concentration of the KAT5 inhibitor is 1 mg / kg / day to 3 mg / kg / day.

[0014] In some possible implementations, the effective in vitro concentration of the KAT5 inhibitor is 25 μM to 50 μM.

[0015] In some possible implementations, the excipients include at least one of 0.85% to 0.9% sodium chloride solution, dimethyl sulfoxide, phosphate buffer, Tween 80, or glucose injection.

[0016] In some possible implementations, the drug is in the form of an injection.

[0017] The first aspect of this application discloses the application of KAT5 inhibitors in drugs that enhance the immunotherapeutic efficacy of natural killer (NK) cells. This application, through research, discovered that KAT5 promotes the recruitment of transcription factor SP1 to the ADAM10 gene promoter and drives its transcription. ADAM10 expression directly regulates the abundance of NKG2D ligands on the cell surface, thereby determining the sensitivity of tumor cells to NK cell killing. Inhibiting KAT5 activity downregulates this pathway, significantly enhancing the sensitivity of tumor cells to NK cells. In vitro and in vivo experiments have demonstrated that KAT5 inhibitors can effectively enhance the killing ability of NK cells. This application reveals for the first time a novel mechanism by which KAT5 regulates the ADAM10-NKG2D signaling axis through epigenetic mechanisms, providing a novel target and strategy for developing novel immunosensitizers and combined cell therapies.

[0018] The drug for enhancing the immunotherapeutic effect of natural killer cells provided in the second aspect of this application contains a KAT5 inhibitor. This small molecule KAT5 inhibitor can inhibit the recruitment of transcription factor SP1 to the ADAM10 promoter, reduce ADAM10 transcriptional activity, and decrease the expression of NKG2D ligand on the surface of tumor cells, thereby enhancing the sensitivity of tumor cells to NK cell killing. Therefore, the pharmaceutical composition of this application has excellent therapeutic effects on a variety of solid tumors, especially tumors that are insensitive to traditional immunotherapy, and has significant pharmaceutical value and market benefits. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 This is a graph showing the results of an experimental analysis of the correlation between KAT5 expression levels and the proportion of various immune cells in the microenvironment, performed using CIBERSORTx, as provided in the embodiments of this application. Figure 2 This is a diagram showing the results of gene set enrichment analysis of the high KAT5 expression group and the low KAT5 expression group provided in the embodiments of this application; Figure 3 This is a graph showing the experimental results of enrichment scores of specific immune-related pathways in samples with high KAT5 expression and low KAT5 expression, provided in an embodiment of this application, through enrichment analysis of single-sample gene sets. Figure 4 This is a graph showing the experimental results of transcriptomics analysis after KAT5 knockdown provided in the embodiments of this application; Figure 5This is a graph showing the results of gene set enrichment analysis provided in the embodiments of this application; Figure 6 This is a graph showing the cytotoxicity experiment results of in vitro experimental cells after KAT5 knockdown, co-cultured with NK-92 cells, primary NK cells, IPSC-NK cells and CAR-NK cells respectively, according to the embodiments of this application. Figure 7 These are experimental results from embodiments of this application showing how KAT5 gene knockdown enhances the in vitro activation function of NK-92 cells, primary NK cells, IPSC-NK cells, and CAR-NK cells, respectively. Figure 8 This is a diagram showing the experimental results of KAT5 gene knockdown enhancing the in vitro IFN-γ secretion function of NK-92 cells, primary NK cells, IPSC-NK cells, and CAR-NK cells, as provided in the embodiments of this application. Figure 9 This is a diagram showing the experimental results of KAT5 gene knockdown downregulating the transcription level of ADAM10 gene, as provided in the embodiments of this application. Figure 10 This is a Western blot result of the KAT5 gene knockdown downregulating the expression level of ADAM10 protein provided in the embodiments of this application; Figure 11 This is a diagram showing the results of predicting SP1 as a potential upstream transcription factor of the ADAM10 gene using bioinformatics analysis, as provided in the embodiments of this application. Figure 12 This is a graph showing the results of quantitative real-time PCR detection, provided in the embodiments of this application, demonstrating that SP1 gene knockdown significantly downregulates ADAM10 mRNA expression levels. Figure 13 This is a diagram showing the experimental results of KAT5 gene knockdown inhibiting MICA ligand secretion in tumor cells, as provided in the embodiments of this application. Figure 14 This is a diagram showing the experimental results of the KAT5 inhibitor combined with NK-92 cell therapy, as provided in the embodiments of this application, significantly inhibiting tumor growth in vivo. Detailed Implementation

[0021] To make the technical problems, technical solutions, and beneficial effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application.

[0022] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0023] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "at least one of a, b or c", or "at least one of a, b and c", can both mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0024] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0025] The terminology used in the embodiments of this application is for the purpose of describing particular embodiments only and is not intended to be limiting of this application. The singular forms "a" and "the" as used in the embodiments of this application and the appended claims are also intended to include the plural forms, unless the context clearly indicates otherwise.

[0026] The weights of the relevant components mentioned in the embodiments of this application can refer not only to the specific content of each component, but also to the proportional relationship between the weights of the components. Therefore, any scaling up or down of the content of the relevant components according to the embodiments of this application is within the scope disclosed in the embodiments of this application. Specifically, the mass described in the embodiments of this application can be a mass unit known in the chemical industry, such as µg, mg, g, or kg.

[0027] The terms "first" and "second" are used for descriptive purposes only, to distinguish objects, such as substances, from one another, and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. For example, without departing from the scope of the embodiments of this application, "first XX" may also be referred to as "second XX," and similarly, "second XX" may also be referred to as "first XX." Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of that feature.

[0028] The cytotoxic activity of natural killer (NK) cells is finely regulated by a series of activating and inhibitory receptors on their surface, maintaining a balance between self-tolerance and effective killing. Activating receptors can recognize specific ligands on target cells. When these receptors are activated, they transmit signals into the NK cell interior, promoting NK cell activation and enhancing their cytotoxic ability. Currently, tumor cells have evolved various mechanisms to evade NK cell immune surveillance. Among these, the most common is the downregulation or complete loss of expression of their surface NKG2D ligand. In addition, some tumor cells cleave membrane-bound MICA / B into a soluble form (sMICA / B) through protease-mediated proteolysis. These soluble ligands not only competitively bind to the NKG2D receptor on the NK cell surface, blocking its recognition, but also induce NKG2D receptor endocytosis and degradation, leading to NK cell functional exhaustion and severely weakening the NK cell's cytotoxic ability.

[0029] Histone acetyltransferases (KATs) play a crucial role in gene transcription regulation by catalyzing histone acetylation, and their abnormalities are closely related to tumorigenesis and development. KAT5 is one of the important acetyltransferases, involved in various processes such as DNA damage repair and apoptosis, but its function in regulating immunogenicity, particularly NK cell recognition, remains unclear. Currently, there are no reports or drugs on specifically enhancing NK cell killing ability by targeting KAT5.

[0030] Based on this, the first aspect of the present application provides the application of a KAT5 inhibitor in a drug that enhances the immunotherapeutic efficacy of natural killer cells, including applying the KAT5 inhibitor to the drug that enhances the immunotherapeutic efficacy of natural killer cells, wherein the KAT5 inhibitor includes at least one of Nu9056, TH1834 and MG149.

[0031] The application of KAT5 inhibitors in enhancing the immunotherapeutic efficacy of natural killer cells (NK cells) according to the first aspect of this application reveals that KAT5 promotes the recruitment of transcription factor SP1 to the ADAM10 gene promoter and drives its transcription. ADAM10 expression directly regulates the abundance of NKG2D ligands on the cell surface, thereby determining the sensitivity of tumor cells to NK cell killing. Inhibiting KAT5 activity downregulates this pathway, significantly enhancing the sensitivity of tumor cells to NK cells. In vitro and in vivo experiments have demonstrated that KAT5 inhibitors can effectively enhance the killing ability of NK cells. This application reveals for the first time a novel mechanism by which KAT5 regulates the ADAM10-NKG2D signaling axis through epigenetic mechanisms, providing a new target and strategy for developing novel immunosensitizers and combined cell therapies.

[0032] It should be noted that NU9056, TH1834, and MG149 are all effective selective Tip60 (KAT5) histone acetyltransferase inhibitors. NU9056, chemically known as 5,5'-dithiobis(isothiazolium) or 1,2-di(isothiazol-5-yl)dithioethane, contains two isothiazole rings linked by disulfide bonds and is typically a yellow to brown liquid at room temperature; its CAS number is 1450644-28-6, its molecular weight is 232.37, and its molecular formula is C6H4N2S4. MG149, chemically known as 2-[2-(4-heptylphenyl)ethyl]-6-hydroxybenzoic acid, is a white to off-white powder, an analogue of chalky acid; its CAS number is 1243583-85-8, its molecular weight is 340.46, and its molecular formula is C6H4N2S4. 22 H 28 O3. TH1834 has the chemical name 2-[5-(4-{[(2-phenylethyl)({4-[4-(pyrrolid-1-ylmethyl)phenoxy]butyl})amino]methyl}phenyl)-2H-1,2,3,4-tetrazol-2-yl]acetic acid dihydrochloride. It is a more complex organic compound with a larger molecular weight than the previous two, containing multiple nitrogen atoms in its structure, and belonging to a specific heterocyclic organic molecule. Its CAS number is 2108830-08-4, its molecular weight is 568.71, and its molecular formula is C 33 H 40 N6O3. This application's embodiments revealed that knocking down KAT5 gene expression significantly enhances the immunotherapeutic effect of NK cells. This result mechanistically confirms that as long as KAT5 activity can be effectively inhibited, regardless of the method used, it can ultimately enhance the sensitivity of target cells to NK cell killing by downregulating SP1-mediated ADAM10 transcription and reducing NKG2D ligand expression. Therefore, Nu9056, TH1834, and MG149 are all effective and selective KAT5 histone acetyltransferase inhibitors. These KAT5 inhibitors can all enhance the efficacy of NK cell immunotherapy. They share the same target and downstream regulatory pathways and can all serve as candidate drugs for KAT5-targeting strategies to enhance NK cell immunotherapy.

[0033] It should also be noted that NU9056, TH1834, and MG149, these KAT5 histone acetyltransferase inhibitors, are all small molecule drugs. Due to their small molecular weight and strong cell membrane penetration, they can efficiently target multiple intracellular and extracellular targets (such as kinases, receptors, and nucleic acids). Based on their mature synthesis processes and controllable costs, developing small molecule drugs targeting novel targets is an important direction for promoting precise and efficient treatment. Nu9056, TH1834, and MG149 are all small molecule inhibitors that can specifically target KAT5 activity. The studies in this application found that after knocking down KAT5, the RNA and protein levels of ADAM10 were significantly reduced, and the binding of SP1 to the ADAM10 promoter was significantly decreased. This indicates that KAT5 can regulate the expression of NKG2D ligands by modulating the SP1-ADAM10 axis, thereby regulating the sensitivity of tumor cells to NK cell killing. Elucidating this mechanism provides a theoretical basis for enhancing the therapeutic effect of NK cell immunotherapy by targeting KAT5. Therefore, the KAT5 inhibitor in this application inhibits the recruitment of transcription factor SP1 to the promoter region of the ADAM10 gene, ultimately leading to a decrease in the transcriptional activity of ADAM10. Downregulation of ADAM10 further reduces the expression of NKG2D ligands on the cell surface. This key change makes target cells more sensitive to NK cell attack, thereby enhancing the sensitivity of tumor cells to natural killer cells.

[0034] The mechanism described in this application breaks through the limitations of traditional immunotherapy, which mainly focuses on T cell activation or immune checkpoint blockade, by directly linking epigenetic regulation with the function of innate immune effector cells. Specifically, KAT5, as a histone acetyltransferase, not only participates in classical pathways such as DNA damage repair, but also enhances the transcriptional activity of the ADAM10 promoter by acetylation modifying chromatin structure, providing an open chromatin environment for transcription factors such as SP1. ADAM10, as a key metalloproteinase, mediates the extracellular domain cleavage of membrane-bound NKG2D ligands (such as MICA / B), causing them to detach from the tumor cell surface and form soluble ligands (sMICA / sMICB). The latter not only weakens the NK cell recognition ability but also induces NKG2D receptor internalization, leading to NK cell functional exhaustion. Therefore, targeting KAT5 not only inhibits ADAM10 transcription at its source and reduces ligand detachment but also maintains the "immune visibility" of tumor cells, reshaping a microenvironment conducive to NK cell attack. This discovery not only explains the molecular basis of natural resistance to NK cell therapy in some tumors, but also provides a druggable intervention point for overcoming immune escape in solid tumors. More importantly, this strategy is independent of the patient's HLA (human leukocyte antigen) background or T cell functional status, and has broad applicability, especially suitable for "cold tumor" types with insufficient T cell infiltration or no response to PD-1 / PD-L1 inhibitors. It provides theoretical support and practical pathways for synergistic combinations of next-generation cell immunotherapy.

[0035] In some possible implementations, KAT5 inhibitors include Nu9056. In this embodiment, Nu9056, as a highly selective, cell-membrane-penetrating small-molecule KAT5 inhibitor, can more effectively block KAT5-mediated histone acetylation activity, thereby significantly inhibiting the recruitment of transcription factor SP1 to the ADAM10 promoter region, downregulating ADAM10 expression, reducing the shedding of NKG2D ligands such as MICA / B from the surface of tumor cells, and ultimately enhancing the ability of natural killer (NK) cells to recognize and kill tumor cells.

[0036] In some embodiments, in vivo experiments have demonstrated that Nu9056, a KAT5 inhibitor, can significantly enhance NK cell killing rate (e.g., from 36% to over 72%) within a concentration range of 1 mg / kg / day to 3 mg / kg / day, while maintaining good cell viability and animal tolerability. Compared to other KAT5 inhibitors such as TH1834 and MG149, Nu9056 possesses superior pharmacological properties. It has a stable chemical structure, moderate water solubility, and strong cell permeability. Furthermore, its specific regulatory effect on the ADAM10-NKG2D pathway in the context of immune regulation has been clearly verified in existing literature and in the experiments of this application. Therefore, Nu9056 is preferred as the core KAT5 inhibitor of this application, possessing both a clear mechanism, reliable efficacy, and clinical translational potential.

[0037] Among possible implementations, natural killer cells include at least one of the NK-92 cell line, primary NK cells, IPSC-NK cells, and CAR-NK cells. This application reveals that regardless of NK cell origin or genetic engineering, their cytotoxic activity depends on the recognition of NKG2D ligands on the surface of target cells. KAT5 inhibitors, by downregulating ADAM10 expression in tumor cells, stabilize the level of NKG2D ligands on their surface, thereby universally enhancing the tumor recognition and clearance capabilities of various NK cell types. Experiments have demonstrated that this strategy is not only applicable to the commonly used NK-92 cell line in the laboratory but also significantly enhances the natural killer efficacy of primary NK cells, further amplifying the anti-tumor potential of novel therapeutic NK cells such as IPSC-NK and CAR-NK. It exhibits a broad-spectrum synergistic effect, particularly in overcoming immune escape in the solid tumor microenvironment, providing a universal sensitization strategy for diverse clinical NK cell therapies.

[0038] In some possible implementations, natural killer cells include the NK-92 cell line. In this embodiment, the NK-92 cell line, as an immortalized NK cell line with high cytotoxic activity and capable of in vitro expansion, exhibits cytotoxic function dependent on the expression level of NKG2D ligands on the surface of target cells. By combining the use of a KAT5 inhibitor, ADAM10-mediated NKG2D ligand shedding from tumor cells can be effectively inhibited, thereby maintaining or upregulating the ligand density on the cell membrane surface, significantly enhancing the recognition efficiency and cytotoxic activity of NK-92 cells against tumor cells. Both in vivo and in vitro experiments showed that pretreatment with KAT5 inhibitors significantly increased the NK-92 cell-mediated tumor cell lysis rate and demonstrated a stronger tumor growth inhibitory effect in a mouse xenograft model, validating the feasibility and synergistic value of this combination strategy in NK-92-based adoptive immunotherapy.

[0039] It should be noted that the NK-92 cell line is an immortalized cell line derived from human NK / T-cell lymphoma. It possesses highly homogeneous, potent, and stable tumor-killing activity and can be expanded on a large scale in vitro. It has been approved by the US FDA for use in multiple clinical trials and is one of the most mature "off-the-shelf" NK cell therapy products currently available. In the specific implementation scheme of this application, the combined use of a KAT5 inhibitor (such as Nu9056) with NK-92 cells can effectively overcome the recognition barrier caused by the shedding of NKG2D ligands from tumor cells in the solid tumor microenvironment of NK-92 cells.

[0040] In some possible implementations, KAT5 inhibitors are used in combination with natural killer cell adoptive therapy. It should be noted that "adoptive therapy" refers to isolating immune cells (specifically natural killer cells) from a patient (autologous) or a healthy donor (allogeneic), activating and expanding them in vitro (even genetically modifying them to reach hundreds of millions in number and extremely high combat effectiveness), and then reinfusing them into the patient. "Combined use of adoptive therapy" in this application refers to the strategy of reinfusing in vitro expanded or engineered natural killer (NK) cells into the patient (i.e., NK cell adoptive immunotherapy), simultaneously or sequentially administering KAT5 inhibitors to synergistically enhance anti-tumor efficacy. Specifically, NK cell adoptive therapy itself relies on the ability of the infused NK cells to effectively recognize and kill tumor cells. However, many solid tumors highly express proteases such as ADAM10, causing the shedding of NKG2D ligands (such as MICA / B) from the cell surface, forming an immune escape barrier, thus limiting the function of the adopted NK cells. The combination therapy group of KAT5 inhibitor and NK-92 cells in the embodiments of this application showed the most significant tumor growth inhibition effect, which was significantly better than the single therapy, indicating that the two have a significant synergistic anti-tumor effect.

[0041] In some possible implementations, the dosage of KAT5 inhibitors increases the killing rate of natural killer cells against tumor cells by ≥50% in in vitro cytotoxicity experiments when the effector-to-target ratio is between 5:1 and 10:1. For example, the effector-to-target ratio can be any typical but non-limiting point value or any value between any two points, such as 5:1, 6:1, 7:1, 8:1, 9:1, and 10:1. Under these effective concentration conditions, the NK cell killing rate increased from 36% to 72% after KAT5 knockdown, an increase of 100%, demonstrating the significant effect of KAT5 inhibition on enhancing the killing ability of NK cells.

[0042] In some possible implementations, treatment with KAT5 inhibitors increases the positivity rate of CD107a on the surface of NK cells by 46.1% to 58.0%, thereby enhancing the cytotoxic activity of NK cells. For example, the increase in the positivity rate of CD107a on the surface of NK cells after treatment with KAT5 inhibitors can be any typical but non-limiting point value or any value between any two points, such as 46.1%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, or 58%. The technical effects of this application stem from the fact that KAT5 inhibitors, by inhibiting KAT5-mediated epigenetic regulation, block the recruitment of transcription factor SP1 to the ADAM10 promoter region, thereby downregulating ADAM10 expression and reducing the protein hydrolysis and shedding of NKG2D ligands (such as MICA / B) on the surface of tumor cells. Stable, highly expressed membrane-bound ligands more effectively activate NKG2D receptors on the surface of NK cells, triggering downstream signaling pathways and promoting the polarization and release of cytotoxic granules (containing perforin and granzymes) towards the immune synapse. CD107a (LAMP-1), as a lysosome-associated membrane protein, is transiently expressed on the surface of NK cells during degranulation, and its positivity rate directly reflects the cell's degranulation activity and cytotoxic potential. Experimental data show that the CD107a positivity rate increased by nearly 50%, indicating that KAT5 inhibitors significantly enhanced the functional activation state of NK cells, providing direct and quantifiable functional evidence for this strategy to enhance anti-tumor immune responses.

[0043] A second aspect of this application provides a drug for enhancing the immunotherapeutic effect of natural killer cells, comprising a KAT5 inhibitor and an excipient, wherein the KAT5 inhibitor comprises at least one of Nu9056, TH1834 and MG149.

[0044] The drug for enhancing the immunotherapeutic effect of natural killer cells provided in the second aspect of this application contains a KAT5 inhibitor. This small molecule KAT5 inhibitor can inhibit the recruitment of transcription factor SP1 to the ADAM10 promoter, reduce ADAM10 transcriptional activity, and decrease the expression of NKG2D ligand on the surface of tumor cells, thereby enhancing the sensitivity of tumor cells to NK cell killing. Therefore, the pharmaceutical composition of this application has excellent therapeutic effects on various solid tumors, especially tumors that are insensitive to traditional immunotherapy, and has significant pharmaceutical value and market benefits.

[0045] In some possible implementations, the effective in vivo concentration of the KAT5 inhibitor is between 1 mg / kg / day and 3 mg / kg / day. For example, the effective in vivo concentration of the KAT5 inhibitor in the drug can be any typical but non-limiting point value, such as 1 mg / kg / day, 2 mg / kg / day, or 3 mg / kg / day, or any value between any two points. Under these effective concentration conditions, the KAT5 inhibitor can efficiently and specifically inhibit KAT5 acetyltransferase activity, significantly reduce the recruitment of SP1 to the ADAM10 promoter region, thereby downregulating ADAM10 expression and stabilizing the level of NKG2D ligand on the surface of tumor cells; while avoiding the off-target cytotoxicity caused by high concentrations.

[0046] In some possible implementations, the effective in vitro concentration of the KAT5 inhibitor is 25 μM to 50 μM. For example, the effective in vitro concentration of the KAT5 inhibitor in the drug can be any typical but non-limiting point value or any value between any two points, such as 25 μM, 30 μM, 35 μM, 40 μM, 45 μM, and 50 μM. Under these effective concentration conditions, the KAT5 inhibitor can effectively inhibit the acetyltransferase activity of KAT5, blocking its mediated histone H4K16 acetylation, thereby reducing the recruitment of transcription factor SP1 to the ADAM10 gene promoter region and significantly downregulating the mRNA and protein expression of ADAM10. After the ADAM10 level decreases, the protein hydrolysis and shedding of NKG2D ligands (such as MICA / B) on the surface of tumor cells decreases, membrane expression stability is enhanced, and thus more effectively activates the NKG2D receptor pathway of NK cells, improving their degranulation ability (increased CD107a positivity rate) and cytotoxic activity.

[0047] In some possible implementations, excipients include at least one of 0.85%–0.9% sodium chloride solution, dimethyl sulfoxide (DMSO), phosphate buffer, Tween 80 (polysorbate 80 or polyoxyethylene sorbitan monooleate), or glucose injection. These excipients have good solubility for KAT5 inhibitors, facilitating the preparation of stable and homogeneous injection solutions. DMSO, in particular, is soluble and permeable to many drugs and possesses anti-inflammatory, analgesic, blood circulation-promoting, and wound-healing properties, as well as diuretic and sedative effects. It can increase drug absorption and improve efficacy. Furthermore, it can increase the local drug concentration in the lesion area and reduce the harmful effects of other drugs on the body. Studies have found that the half-maximum LG50 value of DMSO is (22.4±1.4) g / kg, which is non-toxic and consistent with pathological findings. In animal experiments, the local drug concentration was 2 to 8 times higher than that of other drugs, enhancing the absorption capacity of drugs dissolved in DMSO. Simultaneously, DMSO has the ability to dissolve and penetrate the keratin of cancerous tissue, improving efficacy. In the embodiments of this application, DMSO is used as a solvent in the injection, which can increase the component content of the KAT5 inhibitor NU9056, thereby improving the efficacy.

[0048] Among some possible implementations, the dosage form of drugs that enhance the immunotherapeutic effect of natural killer cells is injection. Injection ensures that the drug enters the bloodstream rapidly, avoids the first-pass effect, improves bioavailability, and achieves effective concentrations in the tumor microenvironment, thereby maximizing the killing effect of NK cells.

[0049] In some embodiments, the solvent in the injection includes 0.85%~0.9% sodium chloride solution and dimethyl sulfoxide. When diluted for use, solvents such as phosphate buffer, Tween-80 (polysorbate 80 or polyoxyethylene sorbitan monooleate) and 5% glucose injection can be used.

[0050] Among some possible implementations, drugs that enhance the immunotherapeutic effect of natural killer cells are suitable for treating solid tumors, including but not limited to ovarian cancer, endometrial cancer, breast cancer, or colon cancer. The embodiments of this application validated the immunosensitizing effect of KAT5 inhibitors in an ovarian cancer model; however, based on its mechanism of action, this strategy should be applicable to various solid tumors expressing NKG2D ligands, particularly tumor types that are insensitive to conventional immunotherapy.

[0051] In some possible implementations, drugs that enhance the immunotherapeutic efficacy of natural killer (NK) cells are incorporated into antitumor drugs. By integrating KAT5 inhibitors with natural killer (NK) cells into the same drug system, a ready-to-use therapeutic strategy of "targeted sensitization + effector cells" synergistic effect is achieved. Specifically, the KAT5 inhibitor can act on the tumor microenvironment pre- or simultaneously, stabilizing the expression of NKG2D ligands on the tumor cell surface by inhibiting the SP1-ADAM10 signaling axis, significantly enhancing its immunogenicity against NK cells; while the included NK cells directly exert highly efficient and specific cytotoxic effects at local high concentrations. This application's research found that this drug achieves superior tumor suppression effects compared to its individual components in a subcutaneous xenograft model, and the administration regimen is simpler, providing a novel combined immunotherapy product with clear efficacy advantages and ease of operation for clinical translation.

[0052] To ensure that the above-described implementation details and operations of this application are clearly understood by those skilled in the art, and to demonstrate the significant advancements in the application of KAT5 inhibitors in enhancing the immunotherapeutic efficacy of natural killer cells, the following examples illustrate the above technical solutions. This application's examples investigated the role of KAT5 in regulating NK cell recognition and verified the effect of the KAT5 inhibitor Nu9056 in enhancing the anti-tumor activity of NK cells in vitro and in vivo. Details are as follows: Example 1 This application's embodiment is a correlation analysis of KAT5 expression and immune cells: Transcriptome data and corresponding clinical sample information were downloaded from TCGA (Cancer Genome Atlas). The CIBERSORTx algorithm (an extended version of the cell type identification algorithm) was used, with the LM22 feature matrix (a leukocyte feature matrix (22 types)) as a reference, to deconvolve the immune cell composition of each sample and calculate the relative proportions of immune cell subsets. Subsequently, Pearson correlation analysis was performed between the calculated immune cell proportions and the KAT5 gene expression levels of the corresponding samples. Statistical significance was assessed using a t-test, with p < 0.05 considered statistically significant. CIBERSORTx is a computational analysis tool based on gene expression data, primarily used to analyze the cell composition and state in mixed cell samples (such as tumor tissue). Through an algorithm called "deconvolution," it can infer the relative proportions of different cell types in a sample and even assess the gene expression profiles of specific cell types.

[0053] Based on the median KAT5 expression value, all samples were divided into a high KAT5 expression group and a low KAT5 expression group. Using GSEA (Gene Set Enrichment Analysis) software, with the Hallmark gene set as a reference, enrichment analysis was performed on the differentially expressed gene lists between the high and low expression groups. Single-sample GSEA analysis was used to calculate the enrichment score for each sample on a specific immune pathway, and differences were shown by comparing the score distributions between the two groups.

[0054] The test results are attached. Figures 1-3 As shown, where Figure 1 The results showed that correlation analysis between KAT5 expression level and the proportion of various immune cells in the microenvironment was performed using CIBERSORTx, and KAT5 expression was positively correlated with resting NK cells (Pearson correlation coefficient r = 0.224, p < 0.05). Figure 2 The results showed that gene set enrichment analysis of the high KAT5 expression group and the low KAT5 expression group revealed a significant enrichment of pathways related to NK cell activation and mediated immunity. Figure 3 Enrichment analysis of single-sample gene sets assessed the enrichment scores of specific immune-related pathways in samples with high and low KAT5 expression. Further analysis confirmed significant differences in enrichment scores between high and low KAT5 expression samples in pathways related to NK cell activation and antigen processing and presentation. These results suggest that KAT5 may be involved in regulating NK cell-related immune responses.

[0055] Example 2 The embodiments of this application demonstrate through in vitro experiments that KAT5 expression is negatively correlated with NK cell-mediated immune responses.

[0056] A. Using lentiviral transduction, specific short hairpin RNA targeting KAT5 was introduced into tumor cell lines. Cells from the knockdown group and control group were collected, and total RNA was extracted for high-throughput RNA sequencing. After quality control, alignment, and quantification, the sequencing data were analyzed for differentially expressed genes using the DESeq2 software package (differential expression sequence analysis software package (version 2)) (screening condition set to: adjusted p-value < 0.05). The obtained differentially expressed gene list was subjected to GO (Gene Ontology) biological process enrichment analysis, and the enrichment of NK cell-related pathways was further verified using GSEA software.

[0057] The test results are attached. Figure 4 and 5 As shown, where Figure 4The results of GO biological process (GO-BP) enrichment analysis of differentially expressed genes are shown. The x-axis represents the enrichment score (-log10 (P-value)), indicating the statistical confidence that the biological process was significantly enriched. The y-axis represents the biological process, listing the specific biological functions enriched. Gene enrichment analysis showed that the experimental treatment not only increased NK cell maturity and cytotoxic potential phenotypically (flow cytometry results), but also confirmed significant changes in gene expression profiles related to NK cell function and immune response at the gene function level (GO analysis results). This indicates that after KAT5 knockdown, transcriptomic analysis revealed that KAT5 expression was significantly associated with multiple biological processes, including endogenous antigen processing and presentation, NK cell-mediated cytotoxicity, and immune regulation.

[0058] Figure 5 The diagram shows typical results from Gene Set Enrichment Analysis (GSEA), illustrating the distribution of predefined gene sets (such as specific biological pathways) within a differentially expressed gene sequence list. GSEA further confirmed significant enrichment of pathways related to NK cell activation, NK cell-mediated immunity, and antigen processing and presentation. Genes associated with this pathway (the blue squares in the middle) are primarily concentrated in the first half of the sequence list (left side), indicating an overall upregulation trend of genes involved in antigen processing and presentation in the experimental group (KAT5 knockdown group). This aligns with previous GO enrichment bubble plot findings, further confirming the core role of this pathway. The "Regulation of Cell Killing" (orange curve) below the blue line also shows an upward trend, suggesting an upregulation trend for genes related to "cell killing," although not statistically significant, but potentially biologically important. The "Natural Killer Cell-Mediated Immunity" (green curve) is positioned lower, corroborating previous NK cell maturity experiments (increased CD107a expression), indicating potential activation of NK cell-related immune functions.

[0059] B. Cytotoxicity Assay: KAT5 knockdown (labeled as shKAT5) or control target cells (shRNA) were co-cultured with NK-92 cells, primary NK cells, IPSC-NK cells, and Car-NKG2D-92 cells at different effector-to-target ratios (5:1, 10:1) for 4 hours. After co-culture, the supernatant was collected, and the NK cell killing rate was calculated using a lactate dehydrogenase release kit. The test results are attached. Figure 6This bar chart shows the changes in cytotoxic activity (killing ability) of four different types of NK cells (NK-92, primary NK, IPSC-NK, and Car-NKG2D NK-92) against target cells after KAT5 gene knockdown (shKAT5). The structure shows that KAT5 gene knockdown significantly enhanced the killing ability of NK-92 cells, and the effect was dose-dependent (the higher the proportion, the higher the killing rate). Consistent with NK-92 cells, KAT5 gene knockdown significantly enhanced the killing ability of primary NK cells; KAT5 gene knockdown also significantly enhanced the killing ability of IPSC-NK cells; even in CAR-modified NK cells, KAT5 gene knockdown still significantly enhanced their killing ability. In vitro experiments confirmed that after KAT5 knockdown, co-culturing NK cells with these cells showed that KAT5 knockdown significantly enhanced the killing ability of NK cells. After KAT5 knockdown, the killing ability of NK cells was significantly enhanced, with the killing rate increasing from 36% to 72%, an increase of 100%.

[0060] C. Detection of NK cell activation markers: KAT5 knockdown (labeled as shKAT5) or control target cells (shRNA) were co-cultured with NK-92 cells, primary NK cells, IPSC-NK cells, and Car-NKG2D-92 cells at a ratio of 10:1 for 4 hours. During co-culture, CD107a-FITC fluorescent antibody was added to mark degranulation. After co-culture, cells were collected, washed, and resuspended, and the proportion of CD107a-positive cells in the NK cell population was immediately detected by flow cytometry. The test results are as follows: Figure 7 As shown, in the NK-92 cell line, KAT5 knockdown significantly increased the proportion of mature cells (P<0.01, marked with **). In primary NK cells isolated from human peripheral blood, KAT5 knockdown also significantly promoted cell maturation (P<0.05, marked with *). In NK cells derived from induced pluripotent stem cells, KAT5 knockdown also significantly increased maturity (P<0.05, marked with *). In NK-92 cells modified with CAR (chimeric antigen receptor), KAT5 knockdown also showed a maturation-promoting effect (P<0.01, marked with **). This indicates that after KAT5 knockdown, co-culturing NK cells with these cells, as analyzed by flow cytometry, significantly increased the level of CD107a, a cell surface activation marker, indicating enhanced NK cell activation.

[0061] D. IFN-γ secretion assay: Collect the supernatant after co-culturing NK cells and target cells. Use a human IFN-γ enzyme-linked immunosorbent assay kit, following the manufacturer's instructions, and detect the concentration of IFN-γ in the supernatant using a microplate reader. Figure 8 The results show the changes in the ability of four different types of NK cells (NK-92, primary NK, IPSC-NK, and Car-NKG2D NK-92) to secrete interferon-γ (IFN-γ) after KAT5 gene knockdown (shKAT5). The results showed that KAT5 gene knockdown significantly enhanced the ability of NK-92 cells to secrete IFN-γ (P<0.001, indicated by ***); in primary NK cells, KAT5 gene knockdown also significantly promoted IFN-γ secretion (P<0.001, indicated by ***); in IPSC-NK cells, KAT5 gene knockdown also significantly promoted IFN-γ secretion (P<0.001, indicated by ***); even in CAR-modified NK cells, KAT5 gene knockdown still significantly promoted IFN-γ secretion (P<0.01 or P<0.001, indicated by *** or *). This indicates that KAT5 gene knockdown significantly enhanced the ability of various NK cell types to secrete interferon-γ (IFN-γ) in co-culture, further confirming the increased activation level of NK cells.

[0062] Example 3 This embodiment investigates the mechanism by which KAT5 affects NKG2D ligands through SP1 regulation of ADAM10: A. ADAM10 Expression Level Detection: Total RNA was extracted from KAT5 or SP1 knockdown cells and corresponding controls using the TRIzol method and reverse transcribed into cDNA. Using 18S as an internal reference gene, qRT-PCR was performed using the SYBR Green method to detect the relative mRNA expression level of ADAM10 (2^-ΔΔCt method). Simultaneously, total protein was extracted from lysed cells, quantified using the BCA method, and then subjected to SDS-PAGE electrophoresis and transferred to a membrane. Western blot analysis was performed using specific anti-ADAM10 and anti-β-actin antibodies, and the images were visualized using a chemiluminescence imaging system. The test results are attached. Figure 9 and 10 As shown; where, Figure 9 The results showed that in three different ovarian cancer cell lines (Kuramochi, OVCAR3, Caov3), shKAT5 gene knockdown (#1 and #2) significantly reduced ADAM10 mRNA levels compared to the control group (shRNA) (P<0.0001). This indicates that knockdown of KAT5 resulted in a decrease in ADAM10 RNA levels as shown by quantitative real-time PCR. Figure 10The results showed that, at the protein level, KAT5 knockdown also led to a significant reduction in ADAM10 protein expression, as demonstrated by Western blot analysis. This indicates a positive correlation between KAT5 gene expression and ADAM10 expression, and that KAT5 knockdown inhibits ADAM10 transcription and translation.

[0063] B. Transcription Factor Prediction and Validation: Using the JASPAR public bioinformatics database, transcription factors that may bind to the ADAM10 gene promoter region were retrieved and predicted, and candidate factors with high binding scores were screened. For example... Figure 11 As shown, bioinformatics analysis revealed a highly conserved SP1 binding motif (GGGCGG) in the promoter region of the ADAM10 gene, and its binding to the transcription factor SP1 showed extremely high statistical significance (P = 1e-28). This indicates that SP1 is likely a key transcription factor regulating ADAM10 gene expression. Therefore, SP1 is predicted to be a possible transcription factor for ADAM10. After independently knocking down the transcription factor SP1, changes in ADAM10 mRNA levels were verified by qRT-PCR, as shown... Figure 12 As shown, knocking down the SP1 gene (SP1 knockdown shRNA) in the experiment resulted in a significant decrease in the mRNA level of ADAM10 (P<0.001). This indicates that knocking down SP1 significantly reduces the RNA level of ADAM10, confirming the transcriptional regulatory role of SP1 in ADAM10.

[0064] C. NKG2D Ligand Detection: Target cell supernatant was collected after co-culturing with NK cells. The concentration of soluble NKG2D ligand (sMICA) in the supernatant was detected using a human sMICA kit, following the manufacturer's instructions. Figure 13 As shown, ADAM10 is a metalloproteinase, one of whose known functions is to cleave MICA molecules on the cell membrane surface, releasing soluble MICA (sMICA). Experimental results showed that after KAT5 knockdown, co-culturing NK cells with ADAM10 significantly reduced the secretion of NKG2D ligand MICA (P<0.001). This was due to the decreased expression of ADAM10, which reduced its ability to cleave MICA, leading to a decrease in the production and secretion of sMICA. This indicates that KAT5 regulates the sensitivity of tumor cells to NK cell killing by modulating ADAM10 and thus affecting NKG2D ligand expression.

[0065] Example 4 This study investigated the in vivo evaluation of the antitumor efficacy of KAT5 inhibitors combined with NK cells: Mouse subcutaneous xenograft tumor model and treatment: Eight-week-old immunodeficient NOD mice were selected, and 5×10⁻⁶ tumor cells were injected into the ovaries and fat pads. 5 One target tumor cell per tumor. Target tumor cells until the tumor volume reaches approximately 100 mm. 3 At that time, tumor-bearing mice were randomly divided into four groups (n=5): control group (PBS treatment), NK cell therapy group (intraperitoneal injection of NK-92 cells, 2×10⁻⁶ cells), and NK cell therapy group (intraperitoneal injection of NK-92 cells, 2×10⁻⁶ cells). 6 The treatment groups include: NK-92 cells (2 mg / kg / day, administered twice a week), KAT5 inhibitor treatment group (which is further divided into 3 subgroups, receiving intraperitoneal injections of Nu9056, TH1834, and MG149, respectively), and combination therapy group (which is further divided into 3 subgroups, where NK-92 cells are treated in combination with Nu9056, TH1834, and MG149, respectively).

[0066] like Figure 14 As shown, tumor growth in mice was compared among the control group (Ctrl), the group treated with NK-92 cells only (NK-92), the group treated with KAT5 inhibitors (Nu9056, TH1834, and MG149), and the group treated with both NK-92 cells and KAT5 inhibitors (Nu9056, TH1834, and MG149) (NK-92 + KAT5 inhibitor). The results showed that the group treated with NK-92 cells only exhibited a certain tumor growth inhibition effect compared to the control group. The group treated with KAT5 inhibitors alone also showed tumor growth inhibition. The most significant tumor growth inhibition effect appeared in the group treated with both NK-92 cells and KAT5 inhibitors, indicating that the combination therapy had a synergistic effect and significantly enhanced the anti-tumor effect of NK cells.

[0067] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The application of a KAT5 inhibitor in drugs that enhance the immunotherapeutic efficacy of natural killer cells, characterized in that, This includes the application of KAT5 inhibitors to drugs that enhance the immunotherapeutic effect of natural killer cells, wherein the KAT5 inhibitors include at least one of Nu9056, TH1834, and MG149.

2. The application of the KAT5 inhibitor according to claim 1 in drugs that enhance the immunotherapeutic efficacy of natural killer cells, characterized in that, The KAT5 inhibitor downregulates ADAM10 transcription by inhibiting the recruitment of transcription factor SP1 to the ADAM10 gene promoter region, thereby reducing the expression of NKG2D ligands on the cell surface and enhancing the sensitivity of target cells to natural killer cells.

3. The application of the KAT5 inhibitor according to claim 1 or 2 in drugs that enhance the immunotherapeutic efficacy of natural killer cells, characterized in that, The KAT5 inhibitor includes Nu9056.

4. The application of the KAT5 inhibitor according to claim 3 in drugs that enhance the immunotherapeutic efficacy of natural killer cells, characterized in that, The natural killer cells include at least one of the NK-92 cell line, primary NK cells, IPSC-NK cells, and CAR-NK cells.

5. The application of the KAT5 inhibitor according to claim 4 in drugs that enhance the immunotherapeutic efficacy of natural killer cells, characterized in that, The natural killer cells include the NK-92 cell line; And / or, the KAT5 inhibitor is used in combination with the natural killer cell adoptive therapy.

6. The use of the KAT5 inhibitor according to any one of claims 1-2 or 4-5 in drugs that enhance the immunotherapeutic efficacy of natural killer cells, characterized in that, When the dosage of the KAT5 inhibitor is such that the effector-to-target ratio is 5:1 to 10:1, the natural killer cells increase the killing rate of tumor cells by ≥50%.

7. A drug that enhances the immunotherapeutic effect of natural killer cells, characterized in that, It includes a KAT5 inhibitor and excipients, wherein the KAT5 inhibitor includes at least one of Nu9056, TH1834 and MG149.

8. The drug for enhancing the immunotherapeutic effect of natural killer cells according to claim 7, characterized in that, The effective in vivo concentration of the KAT5 inhibitor is 1 mg / kg / day to 3 mg / kg / day; And / or, the effective in vitro concentration of the KAT5 inhibitor is 25 μM to 50 μM.

9. The drug for enhancing the immunotherapeutic effect of natural killer cells according to claim 8, characterized in that, The excipients include at least one of 0.85%~0.9% sodium chloride solution, dimethyl sulfoxide, phosphate buffer, Tween 80, or glucose injection.

10. The drug for enhancing the immunotherapeutic effect of natural killer cells according to claim 9, characterized in that, The drug is in the form of an injection.

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