Histone lactylation blocker and use thereof in treatment of tumors

Abstract

Provided are a histone lactylation blocker and the use thereof in the treatment of tumors. The histone lactylation blocker has an amino acid sequence as shown in SEQ ID NO: 1, and exhibits the effect of blocking histone lactylation. Further disclosed are a polynucleotide encoding the histone lactylation blocker, a corresponding vector, host cell, pharmaceutical composition, composition, product combination, kit, etc., and the use thereof and a production method therefor. The histone lactylation blocker has an anti-tumor effect and exhibits a synergistic effect with immune checkpoint blockade therapy (such as an anti-PD-1 antibody).

Description

A histone lactation inhibitor and its application in tumor treatment Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically to a histone lactation inhibitor and its application in the treatment of tumors. Background Technology

[0002] Lactic acid, a major end product of intracellular glycolysis, has long been considered a metabolic waste product. However, the abnormal proliferation and metabolic reprogramming of tumor cells lead to excessive consumption of oxygen and nutrients, causing hypoxia and nutrient deficiency in the tumor microenvironment (TME), thus triggering an abnormally high level of lactate. Recent studies have shown that lactate is not only an important signaling regulator in normal tissues and tumors, but also shuttles between various cells (such as tumor cells, tumor-associated fibroblasts, tumor-associated macrophages, and tumor-infiltrating lymphocytes), regulating the microenvironment conducive to tumor growth and immune escape.

[0003] In 2019, a novel lactate-induced lactation modification was first reported, revealing that lysine residues at the tail of histones can undergo lactation. Subsequent studies confirmed that lactation is widespread in various cancers and is closely related to the development of malignant tumors. For example, in ocular melanoma, histone lactation levels are significantly elevated and associated with poor patient prognosis. Research has shown that the use of glycolysis inhibitors (such as sodium oxalate or 2-deoxy-D-glucose) can effectively inhibit histone lactation, thereby inhibiting the growth and invasion of melanoma cells and tumor growth in mouse xenograft models. These findings reveal the important role of histone lactation in carcinogenesis and provide new targets for the treatment of ocular melanoma.

[0004] In summary, targeting lactate metabolism and lactation modification is gradually emerging as a potential therapeutic strategy. Exploring lactation modification and its regulatory sites can provide new directions for finding effective cancer therapeutic targets and combination therapies. Although increasing evidence suggests that lactate plays an important role in anti-tumor activity and tumor sensitization, the specific mechanisms of lactation modification still need further investigation. Currently, research on interventions in lactation modification mainly focuses on lactate generation, transport, and signal transduction, and its specificity and effectiveness still need improvement.

[0005] Therefore, there is an urgent need in this field to continue exploring and identifying specific proteins related to lactation modification in order to precisely target lactation modification and develop new therapeutic targets. Summary of the Invention

[0006] The purpose of this invention is to provide a histone lactation inhibitor and its application in the treatment of tumors.

[0007] In a first aspect of the invention, a histone lactation inhibitor is provided, said histone lactation inhibitor specifically inhibiting the binding of ACSS2 to KAT2A, said histone lactation inhibitor being selected from the group consisting of:

[0008] (a) Antagonists that specifically inhibit ACSS2 expression and / or activity;

[0009] (b) Antagonists that specifically inhibit KAT2A expression and / or activity;

[0010] (c) Structural analogues of ACSS2;

[0011] (d) Structural analogues of KAT2A;

[0012] (e) Any combination of the above items.

[0013] In another preferred embodiment, the histone lactation blocker comprises a polypeptide having an amino acid sequence as shown in SEQ ID NO:1.

[0014] In another preferred embodiment, the histone lactation blocker is a polypeptide, wherein the polypeptide is selected from the group consisting of:

[0015] (P1) comprises polypeptide A, which has the amino acid sequence shown in SEQ ID NO:1;

[0016] (P2) A polypeptide B that has the same function as polypeptide A, which is formed by adding, deleting, modifying and / or substituting at least one amino acid, or cyclizing it.

[0017] (P3) The combination of (P1) and (P2) above.

[0018] In another preferred embodiment, the histone lactation blocker is a structural analog of ACSS2.

[0019] In another preferred embodiment, the histone lactation inhibitor is a structural analog of KAT2A.

[0020] In another preferred embodiment, the histone lactation inhibitor comprises, from the N-terminus to the C-terminus, a structure as shown in Formula A or Formula B:

[0021] Z1-Z2 (A)

[0022] Z2-Z1 (B)

[0023] In the formula,

[0024] Z1 is a structurally similar polypeptide to ACSS2 or KAT2A;

[0025] Z2 is a non-membrane-penetrating peptide;

[0026] "-" indicates a linking peptide or peptide bond.

[0027] In another preferred embodiment, the structurally similar polypeptide of ACSS2 is selected from the group consisting of:

[0028] (i) A polypeptide having the amino acid sequence shown in SEQ ID NO:1;

[0029] (ii) A polypeptide obtained by adding, deleting, modifying and / or substituting at least one amino acid based on the polypeptide described in (i), and having the same function as the polypeptide described in (i);

[0030] (iii) The combination of (i) and (ii) above.

[0031] In another preferred embodiment, the sequence of the structurally similar polypeptide of the ACSS2 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the sequence shown in SEQ ID NO:1.

[0032] In another preferred embodiment, the structurally similar polypeptide of KAT2A is selected from the group consisting of:

[0033] (i) A polypeptide having the amino acid sequence shown in SEQ ID NO:1;

[0034] (ii) A polypeptide obtained by adding, deleting, modifying and / or substituting at least one amino acid based on the polypeptide described in (i), and having the same function as the polypeptide described in (i);

[0035] (iii) The combination of (i) and (ii) above.

[0036] In another preferred embodiment, the sequence of the structurally similar polypeptide of KAT2A has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the sequence shown in SEQ ID NO:1.

[0037] In another preferred embodiment, the transmembrane peptide is a nuclear localization signal.

[0038] In another preferred embodiment, Z2 is the SV40 core positioning signal.

[0039] In another preferred embodiment, the amino acid sequence of the SV40 nuclear localization signal is shown in SEQ ID NO:2.

[0040] In another preferred embodiment, the histone includes histone H3.

[0041] In another preferred embodiment, the lactation includes lactation at histone H3K14 and / or H3K18 sites.

[0042] In another preferred embodiment, the antagonist includes: microRNA, siRNA, shRNA, antisense oligonucleotide (ASO), or a combination thereof.

[0043] In another preferred embodiment, the antagonist includes: peptides, antibodies (preferably monoclonal antibodies), small molecule compounds, or combinations thereof.

[0044] In another preferred embodiment, the antagonist includes a gene editor.

[0045] In another preferred embodiment, the gene editor includes: a DNA gene editor and an RNA gene editor.

[0046] In another preferred embodiment, the gene editor comprises gRNA and gene-editing protein.

[0047] In another preferred embodiment, the gRNA is an RNA that guides the gene editing protein to specifically bind to the gene corresponding to the ACSS2 protein or the KAT2A protein.

[0048] In another preferred embodiment, the gene-editing protein is selected from the group consisting of CasRx, Cpf1, Cas9, Cas13a, Cas13b, Cas13c, or combinations thereof.

[0049] In a second aspect of the invention, a polynucleotide is provided that encodes a histone lactation inhibitor as described in one aspect of the invention.

[0050] In another preferred embodiment, the polynucleotide comprises DNA or RNA.

[0051] In a third aspect of the invention, a carrier is provided, the carrier comprising the polynucleotide as described in the second aspect of the invention.

[0052] In a fourth aspect of the invention, a host cell is provided, the host cell comprising a vector as described in the third aspect of the invention, or having a genome incorporating polynucleotides as described in the second aspect of the invention.

[0053] In another preferred embodiment, the host cell includes: prokaryotic cells and eukaryotic cells.

[0054] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0055] In a fifth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0056] (a) a histone lactation inhibitor as described in the first aspect of the invention, a polynucleotide as described in the second aspect of the invention, a carrier as described in the third aspect of the invention, a host cell as described in the fourth aspect of the invention, or a combination thereof; and

[0057] (b) Pharmaceutically acceptable carriers.

[0058] In another preferred embodiment, the pharmaceutical composition further includes other drugs used for: (i) treating tumors; (ii) inhibiting tumor growth; (iii) inhibiting histone lactation; and / or (iv) increasing the sensitivity of tumors to anti-immune checkpoint therapy.

[0059] In another preferred embodiment, the other drugs include: other drugs for blocking histone lactation or drugs for treating tumors.

[0060] In another preferred embodiment, the pharmaceutical composition is an injectable formulation.

[0061] In another preferred embodiment, the pharmaceutically acceptable carrier is a lipid particle.

[0062] In another preferred embodiment, the pharmaceutically acceptable carrier is a lipid nanoparticle (LNP).

[0063] In another preferred embodiment, the pharmaceutical composition is lipid nanoparticles.

[0064] In another preferred embodiment, the lipid nanoparticles are encapsulated with components selected from the group consisting of: histone lactation inhibitors as described in the first aspect of the invention, polynucleotides as described in the second aspect of the invention, carriers as described in the third aspect of the invention, host cells as described in the fourth aspect of the invention, or combinations thereof.

[0065] In another preferred embodiment, the lipid nanoparticles also contain elements that target tumor cells.

[0066] In another preferred embodiment, the lipid nanoparticles include: ionizable lipids, lecithin, phospholipids, and PEG esters.

[0067] In another preferred embodiment, the lipid nanoparticles are ionizable lipids.

[0068] In another preferred embodiment, the pharmaceutical composition further comprises pharmaceutically acceptable excipients.

[0069] In another preferred embodiment, the pharmaceutically acceptable excipient is selected from the group consisting of diluents, excipients, surfactants, lubricants, disintegrants, or combinations thereof.

[0070] In a sixth aspect of the invention, a composition is provided, the composition comprising:

[0071] (i) Histone lactation blocking agents as described in the first aspect of the present invention;

[0072] (ii) Inhibitors of immune checkpoints, wherein the immune checkpoints are selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3, TIGIT / ICOS, LAG3, OX40, CD47, VISTA, 4-1BB, or combinations thereof; and

[0073] (iii) Pharmaceutically acceptable carriers.

[0074] In another preferred embodiment, the components (i) and (ii) constitute 0.01-99.99 wt% of the total weight of the composition, more preferably 0.1-90 wt%, and even more preferably 1-80 wt%.

[0075] In another preferred embodiment, the composition further includes other drugs used for: (i) treating tumors; (ii) inhibiting tumor growth; (iii) inhibiting histone lactation; and / or (iv) increasing the sensitivity of tumors to anti-immune checkpoint therapy.

[0076] In another preferred embodiment, the other drugs include: other drugs for blocking histone lactation or drugs for treating tumors.

[0077] In another preferred embodiment, the other drugs used to block histone lactation include glycolysis inhibitors.

[0078] In another preferred embodiment, the inhibitor of the immune checkpoint is selected from the group consisting of antibodies or antigen-binding fragments thereof, small molecule compounds, or combinations thereof.

[0079] In another preferred embodiment, the immune checkpoint is PD-1.

[0080] In another preferred embodiment, the inhibitor of the immune checkpoint is an anti-PD-1 antibody.

[0081] In another preferred embodiment, the tumor includes: a solid tumor or a hematoma.

[0082] In another preferred embodiment, the tumor is selected from the group consisting of: brain tumors, lung cancer, melanoma, colon cancer, liver cancer, stomach cancer, non-Hodgkin lymphoma, prostate cancer, ovarian cancer, breast cancer, or combinations thereof.

[0083] In another preferred embodiment, the tumor includes a tumor associated with histone lactation.

[0084] In another preferred embodiment, the tumor associated with histone lactation is the tumor associated with elevated levels of histone lactation.

[0085] In another preferred embodiment, tumors associated with histone lactation include, but are not limited to: brain tumors (such as gliomas), melanomas (such as ocular melanomas), lung cancers (such as non-small cell lung cancer), breast cancers, liver cancers (such as hepatocellular carcinomas), colorectal cancers, gastric cancers, cervical cancers, or combinations thereof.

[0086] In another preferred embodiment, the tumor includes a glioma.

[0087] In a seventh aspect of the invention, a product portfolio is provided, the product portfolio comprising:

[0088] (I) A first pharmaceutical composition, the first pharmaceutical composition comprising:

[0089] (Ia) A first active ingredient, wherein the first active ingredient is a histone lactation blocker as described in the first aspect of the present invention, and

[0090] (Ib) Pharmaceutically acceptable carriers; and

[0091] (II) A second pharmaceutical composition, the second pharmaceutical composition comprising:

[0092] (IIa) A second active ingredient, wherein the second active ingredient is an inhibitor of an immune checkpoint, said immune checkpoint being selected from the group consisting of: PD-1, PD-L1, CTLA-4, TIM3, TIGIT / ICOS, LAG3, OX40, CD47, VISTA, 4-1BB, or combinations thereof; and

[0093] (IIb) Pharmaceutically acceptable carriers;

[0094] Wherein, the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions or the same pharmaceutical composition.

[0095] In another preferred embodiment, the weight ratio of the first pharmaceutical composition to the second pharmaceutical composition is 1:10-10:1, more preferably 1:5-5:1, and even more preferably 1:2-2:1.

[0096] In another preferred embodiment, the content of the histone lactation inhibitor in the product combination is 1%-99%, more preferably 10%-90%, and even more preferably 30%-70%.

[0097] In another preferred embodiment, the product combination contains 1%-99% of the inhibitor of the immune checkpoint, more preferably 10%-90%, and even more preferably 30%-70%.

[0098] In another preferred embodiment, in the product mix, components (I) and (II) account for 0.01-99.99 wt% of the total weight of the product mix, more preferably 0.1-90 wt%, and even more preferably 1-80 wt%.

[0099] In another preferred embodiment, the dosage form of the pharmaceutical composition includes: an injectable dosage form, a topical dosage form, and an oral dosage form.

[0100] In another preferred embodiment, the pharmaceutical composition is administered by subcutaneous injection, intravenous injection, or intramuscular injection.

[0101] In another preferred embodiment, the oral dosage form includes: tablets, capsules, films, and granules.

[0102] In another preferred embodiment, the dosage form of the pharmaceutical composition includes: a sustained-release dosage form and a non-sustained-release dosage form.

[0103] In another preferred embodiment, the dosage form of the pharmaceutical composition includes liposome nanoparticles.

[0104] In another preferred embodiment, the dosage form of the first pharmaceutical composition is peptide-loaded liposome nanoparticles.

[0105] In an eighth aspect of the invention, a medicine box is provided, the medicine box comprising:

[0106] (a1) A first container, and a histone lactation blocker as described in the first aspect of the invention, or a drug containing a histone lactation blocker, located within the first container;

[0107] (a2) A second container, and an inhibitor of an immune checkpoint located within the second container, or a drug containing an inhibitor of an immune checkpoint, wherein the immune checkpoint is selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3, TIGIT / ICOS, LAG3, OX40, CD47, VISTA, 4-1BB, or combinations thereof.

[0108] In another preferred embodiment, the kit further comprises (a3) ​​a third container and other drugs located within the third container, the other drugs being used to (i) treat tumors; (ii) inhibit tumor growth; (iii) inhibit histone lactation; and / or (iv) increase the sensitivity of tumors to anti-immune checkpoint therapy.

[0109] In another preferred embodiment, the other drugs include: other drugs for blocking histone lactation or drugs for treating tumors.

[0110] In another preferred embodiment, the other drugs used to block histone lactation include glycolysis inhibitors.

[0111] In another preferred embodiment, the first container, the second container, and / or the third container are the same or different containers.

[0112] In another preferred embodiment, the drug in the first container is a single-ingredient preparation containing a histone lactation blocker.

[0113] In another preferred embodiment, the drug in the second container is a single-component preparation containing antibodies against immune checkpoints.

[0114] In another preferred embodiment, the third container is a single-drug formulation containing other drugs.

[0115] In another preferred embodiment, the dosage form of the drug is an oral dosage form or an injectable dosage form.

[0116] In another preferred embodiment, the medicine box also contains instructions.

[0117] In another preferred embodiment, the specification includes one or more descriptions selected from the group consisting of:

[0118] (a) A method of treating tumors by combining histone lactation blockers and inhibitors of immune checkpoints;

[0119] (b) A method of inhibiting tumor growth by combining histone lactation blockers and inhibitors of immune checkpoints;

[0120] (c) A method of inhibiting the tumor-promoting function of histone lactation by combining histone lactation blockers with inhibitors of immune checkpoints;

[0121] (d) A method to enhance tumor sensitivity to anti-immune checkpoint therapy by combining histone lactation blockers with inhibitors of anti-immune checkpoints.

[0122] In a ninth aspect of the invention, there is provided the use of a histone lactation inhibitor as described in the first aspect of the invention, a polynucleotide as described in the second aspect of the invention, a carrier as described in the third aspect of the invention, a host cell as described in the fourth aspect of the invention, a pharmaceutical composition as described in the fifth aspect of the invention, a composition as described in the sixth aspect of the invention, a product combination as described in the seventh aspect of the invention, or a cassette as described in the eighth aspect of the invention, for the preparation of formulations or compositions selected from the group consisting of:

[0123] (a) Preparations or compositions that inhibit or block histone lactation;

[0124] (b) Formulations or compositions that competitively inhibit the binding of ACSS2 and KAT2A;

[0125] (c) Formulations or compositions that competitively bind to the ACSS2 protein;

[0126] (d) Formulations or compositions that competitively bind to the KAT2A protein;

[0127] (e) Preparations or compositions for the prevention and / or treatment of tumors;

[0128] (f) Formulations or compositions that enhance the sensitivity of tumors to immunotherapy (such as T-cell vaccines, tumor vaccines, immune checkpoint inhibitors); and / or

[0129] (g) Preparations or compositions that enhance the sensitivity of tumors to conventional treatments (such as targeted therapy, chemotherapy, and radiotherapy).

[0130] In another preferred embodiment, the histone includes histone H3.

[0131] In another preferred embodiment, the lactation includes lactation at histone H3K14 and / or H3K18 sites.

[0132] In a tenth aspect of the invention, a method for treating tumors is provided, comprising the steps of administering to a subject in need a histone lactation inhibitor as described in the first aspect of the invention, a polynucleotide as described in the second aspect of the invention, a carrier as described in the third aspect of the invention, a host cell as described in the fourth aspect of the invention, a pharmaceutical composition as described in the fifth aspect of the invention, a composition as described in the sixth aspect of the invention, a product combination as described in the seventh aspect of the invention, or a combination thereof.

[0133] In another preferred embodiment, the method includes the step of providing a medicine box as described in the eighth aspect of the invention.

[0134] In another preferred embodiment, the object includes a human or a non-human mammal.

[0135] In another preferred embodiment, the non-human mammals include rodents and primates, preferably mice, rats, rabbits, and monkeys.

[0136] In another preferred embodiment, the histone lactation blocker is administered simultaneously or sequentially with an inhibitor of the immune checkpoint.

[0137] In an eleventh aspect of the present invention, a reagent combination is provided, the reagent combination comprising:

[0138] (a) a histone lactation inhibitor as described in the first aspect of the invention, a polynucleotide as described in the second aspect of the invention, a carrier as described in the third aspect of the invention, a pharmaceutical composition as described in the fifth aspect of the invention, or a combination thereof; and

[0139] (b) Detection reagents for detecting histone lactation.

[0140] In another preferred embodiment, the reagent combination further includes: (c) a detection-acceptable carrier.

[0141] In another preferred embodiment, the detectably acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0142] In another preferred embodiment, the reagent combination includes one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence assay reagents, magnetic nanoparticles, and imaging agents.

[0143] In another preferred embodiment, the reagent combination is used for in vivo or in vitro detection.

[0144] In another preferred embodiment, the dosage form of the reagent combination is liquid or powder (such as aqueous solution, injection, lyophilized powder, tablet, lozenge, inhaler).

[0145] In another preferred embodiment, the reagent combination is used to detect histone lactation levels.

[0146] In a twelfth aspect of the invention, a kit is provided comprising a reagent combination as described in an eleventh aspect of the invention.

[0147] In a thirteenth aspect of the present invention, a method for inhibiting histone lactation in vitro is provided, comprising the steps of:

[0148] Histones or samples containing histones are contacted with histone lactation inhibitors as described in the first aspect of the invention, polynucleotides as described in the second aspect of the invention, carriers as described in the third aspect of the invention, pharmaceutical compositions as described in the fifth aspect of the invention, or combinations thereof, thereby inhibiting histone lactation.

[0149] In another preferred embodiment, the method is non-diagnostic and non-therapeutic.

[0150] In another preferred embodiment, the method is an in vitro method.

[0151] In another preferred embodiment, the histone includes histone H3.

[0152] In another preferred embodiment, the lactation includes lactation at histone H3K14 and / or H3K18 sites.

[0153] In a fourteenth aspect of the invention, a method for generating a histone lactation inhibitor as described in the first aspect of the invention is provided, wherein the histone lactation inhibitor is a polypeptide, the method comprising the steps of:

[0154] (a) Culturing host cells as described in the fourth aspect of the invention under conditions suitable for polypeptide production, thereby obtaining a culture containing said polypeptide; and

[0155] (b) Isolate or recover the polypeptide from the culture to obtain the histone lactation blocker.

[0156] In a fifteenth aspect of the invention, a method for screening histone lactation inhibitors is provided, the method comprising the steps of:

[0157] (S1) Contacting the candidate with a histone lactation blocker as described in the first aspect of the invention, a polynucleotide as described in the second aspect of the invention, a carrier as described in the third aspect of the invention, a host cell as described in the fourth aspect of the invention, a pharmaceutical composition as described in the fifth aspect of the invention, or a combination thereof; and

[0158] (S2) Detect whether histone lactation level decreases. If a decrease is detected, it indicates that the candidate is a histone lactation blocker.

[0159] In another preferred embodiment, the method further includes performing one or more detections selected from the group consisting of:

[0160] (a) Detect whether ACSS2 expression and / or activity are decreased;

[0161] (b) Detect whether KAT2A expression and / or activity are decreased.

[0162] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0163] Figure 1 shows the design of the control peptide and the ACSS2 / KAT2A blocking peptide.

[0164] Figure 2 shows the effect of treatment of CT2A mouse glioma cells with the control peptide and the ACSS2 / KAT2A blocking peptide on the interaction between ACSS2 and KAT2A.

[0165] Figure 3 shows the effect of treatment of CT2A mouse glioma cells with the control peptide and the ACSS2 / KAT2A blocking peptide on the level of H3 histone lactation.

[0166] Figure 4 shows the effects of the control peptide and the ACSS2 / KAT2A blocking peptide on EGF-induced PD-L1 at the mRNA and protein levels in CT2A mouse glioma cells.

[0167] Figure 5 shows the tissue fluorescence staining results of the accumulation of ACSS2 / KAT2A blocking peptide in the nucleus of tumor cells.

[0168] Figure 6 shows the changes in tumor volume in mice in each experimental group.

[0169] Figure 7 shows the survival curves of mice in each experimental group.

[0170] Figure 8 shows the proportion of CD8-positive and Granzyme B-positive immune cells in each experimental group. Detailed Implementation

[0171] Through extensive and in-depth research and screening, the inventors have made a groundbreaking discovery for the first time: the interaction between ACSS2 and KAT2A proteins leads to histone lactation. Based on this, the inventors have developed histone lactation inhibitors (e.g., peptides), polynucleotides encoding these inhibitors, corresponding carriers, host cells, pharmaceutical compositions, compositions, product combinations, and kits. The histone lactation inhibitors of this invention exhibit significant antitumor activity and synergistic effects with immune checkpoint blockade therapy (such as anti-PD-1 antibodies), providing a new avenue for improving cancer treatment. This invention was completed based on these findings.

[0172] the term

[0173] To facilitate a clearer understanding of this disclosure, certain terms are first defined. As used herein, unless otherwise expressly specified herein, each of the following terms shall have the meaning given below.

[0174] The term “about” can refer to a value or composition within an acceptable range of error for a particular value or composition as determined by a person skilled in the art, which will depend in part on how the value or composition is measured or determined.

[0175] The term “administration” means the physical introduction of the product of the present invention into a subject using any of the various methods and delivery systems known to those skilled in the art, including intravenous, intratumoral, intramuscular, subcutaneous, intraperitoneal, spinal, or other parenteral routes of administration, such as by injection or infusion.

[0176] As used herein, the terms “containing” or “including (comprise)” can be open-ended, semi-closed, or closed. In other words, the terms also include “consistently made of” or “composed of”.

[0177] As used herein, unless otherwise stated, any concentration range, percentage range, proportion range, or integer range shall be understood to include any integer value within the range and, where appropriate, its fractional value (e.g., one-tenth and one-hundredth of an integer).

[0178] As used herein, the term “and / or” refers to and covers any and all possible combinations of one or more of the related listed items.

[0179] ACSS2 protein

[0180] ACSS2 belongs to the acetyl-CoA synthase family, which also includes ACSS1 and ACSS3, initially isolated and identified from yeast. The main function of this family is to catalyze the conversion of short-chain fatty acids into acetyl-CoA, which then participates in the carbon metabolism cycle and drives lipid synthesis. Specifically, ACSS1 and ACSS2 prefer acetic acid as their primary substrate, but can also utilize β-hydroxybutyrate and β-ketoglutarate with lower affinity. As a key enzyme in acetyl-CoA synthesis, abnormalities in the activity and expression levels of ACSS2 have been scientifically proven to be closely related to tumor cell proliferation, invasion, metastasis, anti-apoptotic properties, and drug resistance.

[0181] This invention is the first to discover the link between the interaction between ACSS2 and KAT2A and histone lactation.

[0182] KAT2A protein

[0183] Lysine acetyltransferase 2A (KAT2A), also known as GCN5, is an important member of the histone acetyltransferase (HAT) family, with its gene precisely located in the q21.2 region of human chromosome 17. Studies have shown that abnormal expression of KAT2A is closely related to tumorigenesis and development. It effectively enhances the transcriptional activity of downstream genes by regulating histone acetylation levels, promoting histone succinylation, and recruiting transcriptional co-stimulatory factors.

[0184] This invention is the first to discover the link between the interaction between ACSS2 and KAT2A and histone lactation.

[0185] The pharmaceutical compositions of the present invention and their applications

[0186] In one aspect of the invention, a pharmaceutical composition is also provided. In one embodiment of the invention, the pharmaceutical composition is a protein (peptide) and / or nucleic acid pharmaceutical composition.

[0187] There are many methods for delivering protein (peptide) or nucleic acid drugs. Scientists have established methods such as liposome delivery, polymer delivery, peptide chain delivery, virus-like replicon particle delivery, and cationic nanoemulsion delivery. In addition, naked proteins or nucleic acids can also be directly injected into cells. The most commonly used delivery method is lipid nanoparticle delivery, which has advantages such as low toxicity and high delivery efficiency. In a preferred embodiment, the peptide of the present invention can be encapsulated in lipid nanoparticles for delivery to tumor cells. In a preferred embodiment, the lipid nanoparticles further include a targeting element for targeting tumor cells.

[0188] The "active ingredient" in the pharmaceutical composition of this invention refers to a histone lactation inhibitor as described in the first aspect of this invention, a polynucleotide as described in the second aspect of this invention, or a carrier as described in the third aspect of this invention. In a preferred embodiment, the "active ingredient" further comprises other drugs for inhibiting histone lactation or drugs for treating tumors.

[0189] The "active ingredients," formulations, and / or compositions described in this invention can be used to prevent and / or treat diseases or conditions such as tumors, obesity, diabetes, pancreatitis, non-alcoholic fatty liver disease, lipid metabolism disorders, atherosclerosis, and cardiovascular diseases; preferably, for the prevention and / or treatment of tumors and diseases related to histone lactation. "Safe and effective amount" means that the amount of the active ingredient is sufficient to significantly improve the condition or symptoms without causing serious side effects. "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid fillers or gel substances suitable for human use, and must have sufficient purity and sufficiently low toxicity. "Compatibility" here refers to the ability of the components in the composition to interact with and incorporate the active ingredients of this invention without significantly reducing the efficacy of the active ingredients.

[0190] The pharmaceutical composition can be a liquid or a solid, such as a powder, gel, or paste. Preferably, the composition is a liquid, and more preferably an injectable liquid.

[0191] Pharmaceutically acceptable examples of carrier components include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), and emulsifiers (such as... Wetting agents (such as sodium dodecyl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0192] Pharmaceutical compositions may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions, or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents, or excipients include water, ethanol, polyols, and suitable mixtures thereof.

[0193] The pharmaceutical compositions of the present invention can be formulated into dosage forms such as injections, lyophilized formulations, nebulized inhalers, oral formulations, and topical formulations. The pharmaceutical compositions of the present invention can be delivered (administered) by any suitable method, including oral, parenteral, and topical methods. The pharmaceutical compositions of the present invention can also be administered by injection, i.e., intravenous, intratumoral, intramuscular, intradermal, subcutaneous, or intraperitoneal injection. Furthermore, the pharmaceutical compositions of the present invention can be administered transdermally. Transdermal administration via a local route can be formulated into medicated sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, gels, paints, powders, and aerosols. In addition, the pharmaceutical compositions of the present invention can be actively administered to intradermal, subcutaneous, intramuscular, tumor, tissue, organ, and central nervous system sites via electrodes / electric fields / potential differences.

[0194] The pharmaceutical compositions of the present invention can be co-administered with another active agent. Co-administration includes administering the compound and active agent of the present invention within 0.5, 1, 2, 4, 6, 8, 10, 12, 16, 20, or 24 hours of each other. Co-administration also includes administering the compound and active agent of the present invention simultaneously, substantially simultaneously (e.g., within about 1, 5, 10, 15, 20, or 30 minutes of each other), or sequentially in any order. In some embodiments, co-administration can be accomplished by co-formulation, i.e., preparing a single pharmaceutical composition comprising both the active ingredient of the present invention (the mRNA transcription template construct or optimized mRNA described in the present invention) and the active agent. In other embodiments, the active ingredient and active agent of the present invention can be formulated separately.

[0195] The present invention also provides the use of the pharmaceutical composition for preparing a medicament for the prevention and / or treatment of diseases, including (but not limited to) diseases or tumors associated with histone lactation. In a preferred embodiment, the tumor includes: gastrointestinal cancer, central or peripheral nervous system tissue cancer, endocrine or neuroendocrine system cancer or hematopoietic system cancer, glioma, sarcoma, epithelial carcinoma, lymphoma, melanoma, fibroma, meningioma, brain cancer, kidney cancer, thyroid cancer, parathyroid cancer, pituitary adenoma, adrenal adenoma, bone-derived sarcoma, neuroendocrine system tumor, breast cancer, lung cancer, head and neck cancer, prostate cancer, esophageal cancer, tracheal cancer, liver cancer, bladder cancer, stomach cancer, pancreatic cancer, ovarian cancer, uterine cancer, cervical cancer, testicular cancer, colon cancer, rectal cancer, skin cancer, leukemia, or combinations thereof. Preferably, the tumor is selected from the group consisting of: brain tumors (such as glioma), melanoma (such as ocular melanoma), lung cancer (such as non-small cell lung cancer), breast cancer, liver cancer (such as hepatocellular carcinoma), colorectal cancer, gastric cancer, cervical cancer, or combinations thereof.

[0196] Main advantages of the invention

[0197] (1) The histone lactation blocking agent (such as a polypeptide) of the present invention can inhibit tumor cell growth by inhibiting lactation at histone H3K14 and H3K18 sites, and can be used to prepare targeted drug delivery for efficient treatment of tumors.

[0198] (2) The histone lactation blocking agent (such as a polypeptide) of the present invention can be administered simultaneously or sequentially with the inhibitor of the immune checkpoint to sensitize the inhibitor of the immune checkpoint and enhance the tumor inhibition effect.

[0199] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0200] Sequence information

[0201] The key region for the combination of ACSS2 and KAT2A (SEQ ID NO:1)

[0202] SV40 core positioning signal (SEQ ID NO:2)

[0203] Control polypeptide sequence (SEQ ID NO:3)

[0204] Example 1: Histone lactation inhibitors suppress the interaction between ACSS2 and KAT2A and inhibit histone lactation.

[0205] To explore the potential effects of blocking the ACSS2-KAT2A interaction in cancer treatment, we designed a specific peptide sequence. This sequence integrates the key region for ACSS2-KAT2A binding (LMQEAGDECEPEWCDAEDP, SEQ ID NO:1) and the SV40 nuclear localization signal (KKKRKV, SEQ ID NO:2). Figure 1 shows the design of the control peptide (SEQ ID NO:3) and the ACSS2-KAT2A interaction blocking peptide (referred to as the ACSS2 / KAT2A blocking peptide).

[0206] After culturing CT2A cells in a medium containing EGF (100 ng / mL) for 6 hours, 20 μM of control peptide or ACSS2-KAT2A interaction blocking peptide was added to the medium and the cells were cultured for another 24 hours. Cells from different experimental groups were then collected for total protein lysis or RNA extraction. The total protein lysate was co-incubated with anti-ACSS2 antibody and Protein A / G magnetic beads for immunoprecipitation experiments. The levels of KAT2A binding to ACSS2 protein in different experimental groups, histone lactation and acetylation expression, and PD-L1 protein expression were detected by Western blotting experiments. After total RNA extraction, reverse transcription was performed, and the expression level of PD-L1 mRNA was detected by real-time PCR.

[0207] As shown in Figure 2, when CT2A mouse glioma cells were treated with the ACSS2 / KAT2A blocking peptide, we observed that the interaction between ACSS2 and KAT2A was weakened, while the control peptide did not have the above function.

[0208] Meanwhile, as shown in Figure 3, compared with the control peptide, the lactation level of H3 histones treated with the ACSS2 / KAT2A blocking peptide was also significantly reduced at the K18 and K14 sites.

[0209] Of particular importance, as shown in Figure 4, this ACSS2 / KAT2A blocking peptide effectively inhibited EGF-induced upregulation of PD-L1 at both the mRNA and protein levels. This indicates that its inhibitory effect on PD-L1 depends on lactation of H3 histones at K18 and K14 sites.

[0210] Example 2: Application of histone lactation blocking agents in the treatment of glioma

[0211] In this embodiment, immunocompetent C57BL / 6 mice were treated with a control peptide, an ACSS2-KAT2A interaction-blocking peptide, an anti-PD-1 antibody or an IgG antibody, or a combination of peptide and antibody. Specifically, 5 × 10 5 The mouse glioma cell line CT2A was injected subcutaneously into C57BL / 6 mice, and tumor growth was observed. Eighteen days after injection, tumor growth was examined, and tumor volume and mouse survival time were recorded. Flow cytometry was used to analyze the proportion of CD8-positive T cells and granzyme B / CD8 double-positive T cells in the CT2A xenografts.

[0212] Preparation of peptide-loaded liposome nanoparticles: 8 mg of ACSS2 / KAT2A blocking peptide or control peptide was dissolved in 4 mL of PBS (pH 7.2), while 7 mg of DSPE-PEG2000 was dissolved in 0.5 mL of dimethyl thionamide (DMF). After mixing, the mixture was magnetically stirred at room temperature for 2 hours, avoiding light exposure. After the reaction was complete, the solution was transferred to a dialysis bag to remove excess peptide and DMF. The target product, DSPE-PEG2000-peptide, was obtained by freeze-drying. Then, 10 mL of chloroform containing 6 mg of phospholipid and 2 mg of DSPE-PEG2000-peptide was evaporated to prepare for the formation of a second membrane in the same flask. Finally, 1 mL of ultrapure water containing 1 mg C16-TAT (C16-Gly-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Pro-Gln) and 1 mg DSPE-PEG2000-peptide was added, and the mixture was sonicated for 20 minutes to prepare peptide-loaded liposome nanoparticles.

[0213] For combination therapy with anti-PD-1 antibody or IgG antibody and peptide: ACSS2 / KAT2A blocking peptide or control peptide (both 10 mg / kg, 100 μL PBS) was administered intraperitoneally on days 6, 8, 10, 12, 14, and 16. Anti-PD-1 antibody (100 μg / 100 μL) or control IgG (100 μg / 100 μL) was administered intraperitoneally to mice on days 6, 9, 12, and 15 post-vaccination. Tumor volume was calculated using length a and width b: V = ab 2 / 2. Another group of mice was used for survival monitoring. The use of animals was approved by the Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine.

[0214] As shown in Figure 5, after administering the ACSS2 / KAT2A blocking peptide to C57BL / 6 mice via intraperitoneal injection, we used tissue fluorescence staining to find that the peptide accumulated in the nuclei of tumor cells.

[0215] As shown in Figure 6, compared to the control peptide, the ACSS2 / KAT2A blocking peptide significantly reduced tumor volume when used in combination with either anti-PD-1 antibody or IgG antibody. On Day 18, the mean tumor volume in the IgG antibody + control peptide group was approximately 618.69 mmHg. 3 The mean tumor volume in the IgG antibody + ACSS2 / KAT2A blocking peptide group was approximately 409.31 mm. 3 The average tumor volume in the anti-PD-1 antibody + control peptide group was approximately 326.68 mm. 3 The average tumor volume in the anti-PD-1 antibody + ACSS2 / KAT2A blocking peptide group was approximately 105.33 mm. 3 It is evident that the ACSS2 / KAT2A blocking peptide and the anti-PD-1 antibody produced a synergistic effect.

[0216] As shown in Figure 7, compared to the control peptide, the ACSS2 / KAT2A blocking peptide significantly prolonged the survival time of mice when used in combination with anti-PD-1 antibody or IgG antibody. Furthermore, the ACSS2 / KAT2A blocking peptide sensitized the therapeutic effect of PD-1.

[0217] As shown in Figure 8, during this process, the administration of ACSS2 / KAT2A blocking peptide was accompanied by an increase in the infiltration of CD8+ T cells in the tumor tissue and an increase in the secretion of granzyme B.

[0218] discuss

[0219] Given the widespread application of immune checkpoint blockade therapy in clinical cancer treatment, the inventors further combined ACSS2-KAT2A interaction-blocking peptide therapy with anti-immune checkpoint inhibitors (such as anti-PD-1 antibodies). Notably, this combination therapy exhibited significant synergistic effects in inhibiting tumor growth, prolonging survival time in mice, and promoting CD8+ T cell infiltration and granzyme B secretion in tumor tissue. These results suggest that combining strategies that disrupt ACSS2-KAT2A interaction with immune checkpoint blockade provides a new approach to improving cancer treatment.

[0220] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A histone lactylation blocker, characterized in that, The histone lactation blocker specifically inhibits the binding of ACSS2 to KAT2A, and the histone lactation blocker is selected from the following group: (a) Antagonists that specifically inhibit ACSS2 expression and / or activity; (b) Antagonists that specifically inhibit KAT2A expression and / or activity; (c) Structural analogues of ACSS2; (d) Structural analogues of KAT2A; (e) Any combination of the above items.

2. The histone lactamization blocker of claim 1, wherein, The histone lactation blocker is a polypeptide, and the polypeptide is selected from the following group: (P1) comprises polypeptide A, which has the amino acid sequence shown in SEQ ID NO:1; (P2) A polypeptide B that has the same function as polypeptide A, which is formed by adding, deleting, modifying and / or substituting at least one amino acid, or cyclizing it. (P3) The combination of (P1) and (P2) above.

3. A polynucleotide (including DNA and RNA), characterized in that, The polynucleotide encodes the histone lactation blocker as described in claim 1 or 2.

4. A vector, characterized in that, The carrier comprises the polynucleotide as described in claim 3.

5. A host cell, characterized in that, The host cell includes the vector as described in claim 4, or its genome is integrated with the polynucleotide as described in claim 3.

6. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: (a) the histone lactation inhibitor of claim 1 or 2, the polynucleotide of claim 3, the carrier of claim 4, the host cell of claim 5, or a combination thereof; and (b) Pharmaceutically acceptable carriers.

7. A composition characterized in that, The composition comprises: (i) the histone lactation blocker as described in claim 1 or 2; (ii) Inhibitors of immune checkpoints, wherein the immune checkpoints are selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3, TIGIT / ICOS, LAG3, OX40, CD47, VISTA, 4-1BB, or combinations thereof; and (iii) Pharmaceutically acceptable carriers.

8. A product combination, characterized by The product portfolio includes: (I) A first pharmaceutical composition, the first pharmaceutical composition comprising: (Ia) A first active ingredient, wherein the first active ingredient is a histone lactation blocker as described in claim 1 or 2, and (Ib) Pharmaceutically acceptable carriers; and (II) A second pharmaceutical composition, the second pharmaceutical composition comprising: (IIa) A second active ingredient, wherein the second active ingredient is an inhibitor of an immune checkpoint, said immune checkpoint being selected from the group consisting of: PD-1, PD-L1, CTLA-4, TIM3, TIGIT / ICOS, LAG3, OX40, CD47, VISTA, 4-1BB, or combinations thereof; and (IIb) Pharmaceutically acceptable carriers; Wherein, the first pharmaceutical composition and the second pharmaceutical composition are different pharmaceutical compositions or the same pharmaceutical composition.

9. A kit characterized in that, The medicine box includes: (a1) A first container, and a histone lactation blocker as described in claim 1 or 2, or a drug containing a histone lactation blocker, located within the first container; (a2) A second container, and an inhibitor of an immune checkpoint located within the second container, or a drug containing an inhibitor of an immune checkpoint, wherein the immune checkpoint is selected from the group consisting of PD-1, PD-L1, CTLA-4, TIM3, TIGIT / ICOS, LAG3, OX40, CD47, VISTA, 4-1BB, or combinations thereof.

10. Use of a histone lactamization blocker of claim 1 or 2, a polynucleotide of claim 3, a vector of claim 4, a host cell of claim 5, a pharmaceutical composition of claim 6, a composition of claim 7, a product combination of claim 8, or a kit of claim 9, characterized in that, Used for the preparation of formulations or compositions selected from the following group: (a) Preparations or compositions that inhibit or block histone lactation; (b) Formulations or compositions that competitively inhibit the binding of ACSS2 and KAT2A; (c) Formulations or compositions that competitively bind to the ACSS2 protein; (d) Formulations or compositions that competitively bind to the KAT2A protein; (e) Preparations or compositions for the prevention and / or treatment of tumors; (f) Formulations or compositions that enhance the sensitivity of tumors to immunotherapy (such as T-cell vaccines, tumor vaccines, immune checkpoint inhibitors); and / or (g) Preparations or compositions that enhance the sensitivity of tumors to conventional treatments (such as targeted therapy, chemotherapy, and radiotherapy).

11. A method of treating a tumor, comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-10. The method includes the steps of administering to a subject in need the histone lactation blocker as described in claim 1 or 2, the polynucleotide as described in claim 3, the carrier as described in claim 4, the host cell as described in claim 5, the pharmaceutical composition as described in claim 6, the composition as described in claim 7, the product combination as described in claim 8, or a combination thereof; Alternatively, the method may include the step of providing a medicine box as described in claim 9.

12. A reagent combination, characterized in that The reagent combination comprises: (a) the histone lactation blocker of claim 1 or 2, the polynucleotide of claim 3, the carrier of claim 4, the pharmaceutical composition of claim 6, or a combination thereof; and (b) Detection reagents for detecting histone lactation.

13. The agent combination of claim 12, wherein The reagent combination includes one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence assay reagents, magnetic nanoparticles, and imaging agents.

14. A method of inhibiting histone lactylation in vitro, characterized in that, The method includes the following steps: Contacting histones or samples containing histones with the histone lactation inhibitor of claim 1 or 2, the polynucleotide of claim 3, the carrier of claim 4, the pharmaceutical composition of claim 6, or a combination thereof, thereby inhibiting histone lactation.

15. A method for screening histone lactation blocking agents, characterized in that, The method includes the following steps: (S1) Contacting the candidate with a histone lactation inhibitor as described in claim 1 or 2, a polynucleotide as described in claim 3, a carrier as described in claim 4, a host cell as described in claim 5, a pharmaceutical composition as described in claim 6, or a combination thereof; and (S2) Detect whether histone lactation level decreases. If a decrease is detected, it indicates that the candidate is a histone lactation blocker.

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