Fusion protein as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological medicines, discloses a fusion protein as well as a preparation method and application thereof, and particularly discloses a fusion protein which comprises a DDX60 functional structural domain and a tumor-targeted cell-penetrating peptide. The invention provides a fusion protein, the fusion protein has tumor targeted cell penetrability and can activate anti-tumor immunity, and the fusion protein can directly send a DDX60 functional structure domain into tumor cells and maintain the stability of dsRNA; meanwhile, the protein can improve the sensitivity of other anti-tumor immunotherapies by activating the autoimmunity of a body, a new thought is provided for the development of anti-cancer targeted drugs, the efficiency and the application range of the existing anti-tumor immunotherapies are expanded, and the protein has important significance in tumor treatment.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a fusion protein and a preparation method and application thereof. Background Art

[0002] Immune checkpoint blockade (ICB) therapy has been approved for clinical treatment of multiple tumor types and has achieved certain results in solid tumors such as melanoma and lung cancer, improving the prognosis of patients. However, in colorectal cancer (CRC), only some patients with high microsatellite instability (MSI-H) or mismatch repair deficiency (dMMR) show treatment response, while more than 80% of colorectal cancer patients are microsatellite stable (MSS) or mismatch repair proficiency (pMMR) subtypes and cannot benefit from ICB treatment. In addition, most patients with pancreatic cancer, gastric cancer and lung cancer are tolerant to ICB treatment regimens. Therefore, the development of new immunotherapy strategies and combined treatment regimens is of great significance for improving the treatment effect of tumors such as colorectal cancer, pancreatic cancer, gastric cancer and lung cancer, and improving patient survival.

[0003] The innate immune response plays a key role in activating T cell immunity and promoting antigen presentation of tumor cells, and interferons (IFNs) are key cytokines involved in the innate immune response. Studies have shown that IFNs not only play an important role in the regulation of innate immunity, but also directly induce programmed cell death of tumor cells and inhibit tumor progression. In addition, the integrity of the IFN pathway and the activity of the IFN response are closely related to the responsiveness of ICB. The loss of IFN response is the main reason for the failure of ICI treatment. In fact, this response inactivation is common in most tumor patients. Recent studies have shown that nucleic acid-based antiviral innate immunity significantly triggers IFN signaling and anti-tumor immunity through a "viral mimicry" mechanism involving endogenous double-stranded RNA (dsRNA) and its sensors. It is worth noting that the pattern recognition receptors in the cytoplasm, RIG-I-like receptors (RLRs), especially retinoic acid-induced gene I (RIG-I) and melanoma differentiation-associated protein 5 (MDA5), are important sensors of intracellular dsRNA. After recognizing dsRNA, RLRs initiate a strong IFN response, enhance tumor cell immunogenicity, and induce activation of CD8+ T cell infiltration and cytotoxicity. Increasing the levels of dsRNA in cancer cells by genetic and pharmacological approaches has been shown to enhance the infiltration of antitumor T cells and the efficacy of ICB therapy. Therefore, maintaining stable cytoplasmic dsRNA levels may become a tumor-specific therapeutic target that can activate IFN responses and enhance the immunogenicity of tumor cells.

[0004] Studies have shown that there are a variety of endogenous dsRNAs in tumor cells, but these dsRNAs are very unstable and degrade rapidly after generation. Preclinical studies have shown that the use of epigenetic drugs (such as DNA methyltransferase inhibitors, CDK4 / 6 inhibitors, and MEK1 / 2 inhibitors) can increase the abundance of dsRNA in tumor cells, thereby activating anti-tumor immunity associated with interferon (IFN). However, its application in enhancing natural immunity has certain limitations and potential challenges. For example, DNA methyltransferase inhibitors not only affect the production of dsRNA, but may also cause extensive epigenetic changes, which may activate non-specific gene expression, including potential oncogenes, which have not yet achieved ideal results in clinical applications. Therefore, the development of regulatory strategies that can specifically target endogenous dsRNA in tumor cells is particularly important for enhancing the effect of immunotherapy.

[0005] DDX60 is a member of the DEAD-box RNA helicase family. Its protein structure contains a DExD / H box helicase domain that can bind to viral nucleic acids such as dsRNA and their analogs, thereby enhancing the innate immunity mediated by the dsRNA / RIG-I-like receptor pathway. The expression of DDX60 is significantly downregulated in tumors such as colorectal cancer, lung cancer, and pancreatic cancer. In addition, studies have shown that the expression of DDX60 is positively correlated with interferon (IFN) response, antigen presentation, and RLR signaling pathways in a variety of tumors. These data indicate that DDX60 is an important factor in activating IFN response and enhancing anti-tumor immunity, and its functional mechanism may activate RIG-I-like receptor-dependent innate immune pathways by recognizing abnormal nucleic acid components such as dsRNA. Therefore, based on the modification of DDX60 natural protein and improving its tumor targeting and intracellular delivery efficiency, it is expected to achieve the specific supplementation of DDX60 natural protein analogs to tumor cells, which will help enhance the innate immune response, improve the poor effect of anti-tumor immunotherapy in some patients, and expand the scope of application of immunotherapy. Summary of the invention

[0006] The purpose of the first aspect of the present invention is to provide a fusion protein.

[0007] The purpose of the second aspect of the present invention is to provide biological materials related to the fusion protein of the first aspect of the present invention.

[0008] The purpose of the third aspect of the present invention is to provide the use of the fusion protein of the first aspect of the present invention or the biomaterial of the second aspect of the present invention.

[0009] The fourth aspect of the present invention aims to provide a medicine.

[0010] In order to achieve the above object, the technical solution adopted by the present invention is:

[0011] DDX60 is an important factor in maintaining the body's anti-tumor immunity. The fusion protein method is selected to supplement DDX60 in tumor cells because, compared with small molecule drugs, protein drugs have obvious advantages such as high activity, strong specificity, low toxicity, clear biological functions, and convenient production through prokaryotic or eukaryotic expression, which is conducive to large-scale production and clinical application. According to the mechanism of action and structural characteristics of DDX60, the present invention uses fusion expression to construct a protein drug system HL60 that induces the release of tumor-related immune factors, and uses a series of functional experiments to verify the effectiveness of HL60 in activating anti-tumor immunity.

[0012] The first aspect of the present invention provides a fusion protein comprising a DDX60 functional domain and a tumor-targeting cell-penetrating peptide;

[0013] The amino acid sequence of the tumor-targeting cell-penetrating peptide is as shown in SEQ ID NO: 2, or an amino acid sequence shown in SEQ ID NO: 2 that is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

[0014] In some embodiments of the present invention, the tumor includes at least one of bladder cancer, breast cancer, cervical squamous cell carcinoma, pheochromocytoma, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, head and neck tumors, kidney cancer, acute myeloid leukemia, brain glioma, liver cancer, lung cancer, pancreatic cancer, nasopharyngeal carcinoma, kidney cancer, lung cancer, liver cancer, breast cancer, prostate cancer, gastrointestinal cancer, sarcomatoid lung cancer, gastric adenocarcinoma, thyroid cancer, and thymic carcinoma.

[0015] In some embodiments of the present invention, the DDX60 functional domain includes an ATP binding and hydrolysis domain and an RNA binding domain, and the ATP binding and hydrolysis domain and the RNA binding domain are linked by a flexible linker.

[0016] In some embodiments of the present invention, the ATP binding and hydrolysis domain is any one of a1) to a3:

[0017] a1) a protein having an amino acid sequence as shown in LDVVDKNESAVIVAPTSSGKTYASYYCMEKVLKESDDGVVVYVAPTKALVNQVAATV QNRFTKNLPSGEVLCGVFTREYRHDALNCQVLITVPAAFEILLLAPHRQNWVKKIRY VIFDEVHCLGGEIGAEIWEHLLVMIRCPFLALSATISNPEHLTEWLQSVKWYWK (SEQ ID NO: 8);

[0018] a2) a protein having the same or similar function as SEQ ID NO: 8 after one or more amino acids are substituted and / or deleted and / or added;

[0019] a3) a protein that has 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology to SEQ ID NO: 8 and has the same function as SEQ ID NO: 8.

[0020] In some embodiments of the present invention, the RNA binding domain is any one of b1) to b3:

[0021] b1) a protein having an amino acid sequence as shown in YADQKAVDTETLQKVFGRVKFERKGEELKALAERGIGYHHSAMSFKEKQLVEILFRK GYLRVVTATGTLALGVNMPCKSVVFAQNSVYLDALNYRQMSGRAGRRGQDLMGDV YFFDIPFPKIGK (SEQ ID NO: 9);

[0022] b2) a protein having the same or similar function as SEQ ID NO: 9 after one or more amino acids are substituted and / or deleted and / or added;

[0023] b3) a protein that has 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology to SEQ ID NO:9 and has the same function as SEQ ID NO:9.

[0024] In some embodiments of the present invention, the ATP binding and hydrolysis domain is located at the N-terminus or C-terminus of the RNA binding domain.

[0025] In some embodiments of the present invention, a2) a protein having the same or similar function after cysteine ​​in SEQ ID NO: 8 is mutated to alanine or other amino acids.

[0026] In some embodiments of the present invention, the amino acid sequence of the protein described in a2) is as shown in LDVVDKNESAVIVAPTSSGKTYASYYAMEKVLKESDDGVVVYVAPTKALVNQVAATV QNRFTKNLPSGEVLAGVFTREYRHDALNAQVLITVPAAFEILLLAPHRQNWVKKIRY VIFDEVHALGGEIGAEIWEHLLVMIRAPFLALSATISNPEHLTEWLQSVKWYWK (SEQ ID NO: 10).

[0027] In some embodiments of the present invention, b2) a protein having the same or similar function after cysteine ​​in SEQ ID NO: 8 is mutated to alanine or other amino acids.

[0028] In some embodiments of the present invention, the amino acid sequence of the protein described in b2) is as shown in YADQKAVDTETLQKVFGRVKFERKGEELKALAERGIGYHHSAMSFKEKQLVEILFRK GYLRVVTATGTLALGVNMPAKSVVFAQNSVYLDALNYRQMSGRAGRRGQDLMGDV YFFDIPFPKIGK (SEQ ID NO: 11).

[0029] In some embodiments of the present invention, the DDX60 functional domain and the tumor-targeting cell-penetrating peptide are linked via a flexible linker.

[0030] In some embodiments of the present invention, the amino acid sequence of the flexible linker is (GSG)n, wherein n≥1.

[0031] In some embodiments of the present invention, the DDX60 functional domain is located at the N-terminus or C-terminus of the tumor-targeting cell-penetrating peptide.

[0032] In some embodiments of the present invention, the amino acid sequence of the DDX60 functional domain is as shown in SEQ ID NO:3.

[0033] In some embodiments of the present invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO:1 and / or SEQ ID NO:7.

[0034] In some embodiments of the present invention, the fusion protein further comprises a His tag.

[0035] The second aspect of the present invention provides a biological material related to the fusion protein of the first aspect of the present invention, wherein the biological material is any one of c1) to c12):

[0036] c1) a nucleic acid molecule encoding the fusion protein of the first aspect of the present invention;

[0037] c2) an expression cassette containing the nucleic acid molecule described in c1);

[0038] c3) a recombinant vector containing the nucleic acid molecule described in c1);

[0039] c4) a recombinant vector containing the expression cassette described in c2);

[0040] c5) a recombinant microorganism containing the nucleic acid molecule described in c1);

[0041] c6) a recombinant microorganism containing the expression cassette described in c2);

[0042] c7) a recombinant microorganism containing the recombinant vector described in c3);

[0043] c8) a recombinant microorganism containing the recombinant vector described in c4);

[0044] c9) transgenic animal cell lines containing the nucleic acid molecule described in c1);

[0045] c10) a transgenic animal cell line containing the expression cassette described in c2);

[0046] c11) a transgenic animal cell line containing the recombinant vector described in c3);

[0047] c12) a transgenic animal cell line containing the recombinant vector described in c4);

[0048] The transgenic animal cell line contains no reproductive material.

[0049] In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:4 or SEQ ID NO:6.

[0050] In some embodiments of the present invention, the vector comprises a promoter, and the promoter is operably linked to the nucleic acid molecule.

[0051] In some embodiments of the present invention, the vector is independently selected from a non-pathogenic viral vector and a non-viral vector.

[0052] In some embodiments of the present invention, the non-pathogenic viral vector comprises an adenoviral vector or a retroviral vector.

[0053] In some embodiments of the present invention, the non-viral vector comprises a plasmid vector.

[0054] In some embodiments of the present invention, the vector is a plasmid vector, a phagemid, a viral vector, a cell vector, a bacteriophage, a cosmid, a F cosmid, or an artificial chromosome.

[0055] In some embodiments of the present invention, the plasmid vector may be an optional plasmid, and the viral vector may be an optional virus.

[0056] In some embodiments of the present invention, the cells include prokaryotic cells and eukaryotic cells.

[0057] The third aspect of the present invention provides the use of the fusion protein of the first aspect of the present invention or the biomaterial of the second aspect of the present invention in at least one of (1) to (5):

[0058] (1) Preparation of drugs for preventing and / or treating tumors;

[0059] (2) preparing drugs for activating anti-tumor immunity;

[0060] (3) Preparation of drugs that enhance the anti-tumor effects of immune checkpoint inhibitors

[0061] (4) Enhance the expression of IFN effector genes;

[0062] (5) Prepare products that enhance the expression of IFN effector genes.

[0063] In some embodiments of the present invention, the tumor includes at least one of bladder cancer, breast cancer, cervical squamous cell carcinoma, pheochromocytoma, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, head and neck tumors, kidney cancer, acute myeloid leukemia, brain glioma, liver cancer, lung cancer, pancreatic cancer, nasopharyngeal carcinoma, kidney cancer, lung cancer, liver cancer, breast cancer, prostate cancer, gastrointestinal cancer, sarcomatoid lung cancer, gastric adenocarcinoma, thyroid cancer, and thymic carcinoma.

[0064] In some embodiments of the present invention, the drug described in (3) enhances the anti-tumor effect of immune checkpoint inhibitors by enhancing the sensitivity of tumors to immune checkpoint inhibitors.

[0065] In some embodiments of the present invention, the IFN effector gene described in (4) to (5) includes at least one of a cytokine, an immune response regulatory factor, a cell death regulatory factor, and an antigen presentation-related molecule.

[0066] In some embodiments of the present invention, the cytokine includes CXCL10; and / or the immune response regulatory factor includes at least one of IFIT2 and ISG15; and / or the cell death regulatory factor includes CASP9; and / or the antigen presentation-related molecule includes at least one of PSMB8, B2M, and IFI44.

[0067] In some embodiments of the present invention, the immune checkpoint inhibitor includes an immune checkpoint inhibitory antibody, such as at least one of an anti-PD-1 antibody, an anti-PD-L1 antibody, an anti-IDO antibody, an anti-CTLA-4 antibody, an anti-Tim-3 antibody, an anti-GITR antibody, an anti-VISTA antibody, an anti-BTLA antibody, and an anti-OX40 antibody.

[0068] In some embodiments of the present invention, the product described in (5) includes but is not limited to reagents, kits, and drugs.

[0069] The fourth aspect of the present invention provides a drug comprising the fusion protein of the first aspect of the present invention or the biomaterial of the second aspect of the present invention.

[0070] In some embodiments of the present invention, the drug further comprises a pharmaceutically acceptable excipient.

[0071] In some embodiments of the present invention, the pharmaceutically acceptable excipients include at least one of a diluent, a wetting agent, a binder, a disintegrant, a lubricant, a color and flavor regulator, a solvent, a solubilizer, a cosolvent, an emulsifier, an absorption promoter, a surfactant, an antioxidant, an excipient, a stabilizer, a flavoring agent, a sustained-release agent, a metal complexing agent, an inert gas, a preservative, a filler, a local analgesic, a pH regulator, and an isotonic or isotonic regulator.

[0072] In some embodiments of the present invention, the diluent includes but is not limited to: at least one of starch, sugar, cellulose, and inorganic salts. The wetting agent includes but is not limited to: at least one of water, ethanol, and propanol. The adhesive includes but is not limited to: at least one of starch slurry, dextrin, sugar, cellulose derivatives, gelatin, povidone, and polyethylene glycol. The disintegrant includes but is not limited to: at least one of microcrystalline cellulose, cross-linked polyvinyl pyrrolidone, cross-linked sodium carboxymethyl cellulose starch, sodium carboxymethyl starch, low-substituted hydroxypropyl cellulose, cross-linked sodium carboxymethyl cellulose, cross-linked povidone, surfactants (such as sodium dodecylbenzene sulfonate, stearic acid, polyoxyethylene-polyoxypropylene copolymer, fatty acid sorbitan or polysorbate, etc.), and effervescent disintegrants. The lubricant includes but is not limited to: at least one of talc, calcium stearate, magnesium stearate, magnesium lauryl sulfate, micropowder silica gel, calcium carbonate, sodium bicarbonate, and polyethylene glycol. The color and flavor regulator includes but is not limited to: at least one of pigments, flavors, sweeteners, mucilage agents, and deodorants. The solvent includes but is not limited to: at least one of water, ethanol, glycerol, propylene glycol, polyethylene glycol, dimethyl sulfoxide, liquid paraffin, fatty oil, ethyl acetate, and vegetable oil (such as soybean oil, castor oil, peanut oil, blending oil, etc.). The solubilizer includes but is not limited to: at least one of Tweens, benzyl alcohols, polyoxyethylene fatty alcohol ethers, soaps, sulfates, and sulfonates. The cosolvent includes but is not limited to: at least one of organic acids and their salts, amide and amine compounds, inorganic salts, polyethylene glycol, povidone, and glycerol. The emulsifier includes but is not limited to: at least one of Spans, Tweens, benzyl alcohols, benzyl alcohols, glycerol fatty acid esters, higher fatty acid salts, sulfates, sulfonates, gum arabic, tragacanth, gelatin, pectin, phospholipids, agar, sodium alginate, hydroxide, silicon dioxide, and bentonite. The suspending agent includes but is not limited to: at least one of glycerol, syrup, gum arabic, gum tragacanth, agar, sodium alginate, cellulose derivatives, povidone, carbopol, polyvinyl alcohol, and thixotropic glue. The antioxidant includes but is not limited to: at least one of sulfites, pyrosulfites, bisulfites, ascorbic acid, gallic acid and its esters. The metal complexing agent includes but is not limited to: at least one of disodium ethylenediaminetetraacetic acid and polycarboxylic acid compounds; the inert gas includes but is not limited to: at least one of nitrogen and carbon dioxide. The preservative includes but is not limited to: at least one of parabens, organic acids and their salts, quaternary ammonium compounds, chlorhexidine acetate, alcohols, phenols, and volatile oils. The filler includes at least one of starch, sucrose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose or glucose. The flavoring agent includes at least one of aspartame, sucralose, flavor, stevioside, acesulfame potassium, citric acid or saccharin sodium. The local analgesic includes, but is not limited to, at least one of benzyl alcohol, chlorobutanol, lidocaine, and procaine.The pH regulator includes, but is not limited to, at least one of hydrochloric acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, acetic acid, sodium hydroxide, sodium bicarbonate, ethylenediamine, meglumine, phosphate, acetate, and citrate. The isotonic or isotonic regulator includes, but is not limited to, at least one of glucose, sodium chloride, sodium citrate, sorbitol, and xylitol.

[0073] In some embodiments of the present invention, the drug further comprises a combination drug, wherein the combination drug comprises a tumor targeting drug. In this case, at least two active ingredients can be administered together or separately, and as part of the same pharmaceutical preparation or as different preparations.

[0074] In some embodiments of the present invention, the tumor includes at least one of bladder cancer, breast cancer, cervical squamous cell carcinoma, pheochromocytoma, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, head and neck tumors, kidney cancer, acute myeloid leukemia, brain glioma, liver cancer, lung cancer, pancreatic cancer, nasopharyngeal carcinoma, kidney cancer, lung cancer, liver cancer, breast cancer, prostate cancer, gastrointestinal cancer, sarcomatoid lung cancer, gastric adenocarcinoma, thyroid cancer, and thymic carcinoma.

[0075] The beneficial effects of the present invention are:

[0076] The present invention provides a fusion protein, which has tumor-targeted cell penetration and activates anti-tumor immunity. The fusion protein can directly deliver the DDX60 functional domain into tumor cells and maintain the stability of dsRNA; at the same time, the protein can activate the body's own immunity to increase the sensitivity of other anti-tumor immunotherapies, provide new ideas for the development of anti-cancer targeted drugs, expand the efficiency and application scope of existing anti-tumor immunotherapies, and is of great significance in tumor treatment.

[0077] Compared with small molecule drugs, the fusion protein HL60 provided by the present invention is simple to synthesize, and its prokaryotic or eukaryotic expression makes production convenient and its action efficiency high, making it easier to mass produce and commercialize. BRIEF DESCRIPTION OF THE DRAWINGS

[0078] Figure 1 Figure 2 shows the expression of DDX60 gene in different tumors relative to normal tissues. DDX60 is abnormally expressed in multiple tumors.

[0079] Figure 2 Schematic diagram of the construction of HL60 fusion protein.

[0080] Figure 3 This is a diagram of the purification effect of HL60 fusion protein: the target fusion protein is in the box.

[0081] Figure 4 HL60 fusion protein promotes tumor cells to express immune activation-related factors.

[0082] Figure 5 The HL60 fusion protein has tumor targeting properties.

[0083] Figure 6 HL60 fusion protein inhibits tumor growth.

[0084] Figure 7 HL60 fusion protein enhances the effect of PD-1 antibody, and the combination of the two significantly inhibits tumor growth.

[0085] Figure 8 HL60 fusion protein has high safety and has no significant effect on the weight of mice

[0086] Fig. 9 HL60 fusion protein has high safety, and pathological analysis shows that HL60 fusion protein has no obvious side effects on mice.

[0087] Fig.10 This is a diagram of the purification effect of HL60 (CA) fusion protein: the target fusion protein is in the box.

[0088] Fig.11 The HL60 (CA) fusion protein has tumor targeting properties.

[0089] Fig.12 HL60 (CA) fusion protein promotes tumor cells to express immune activation-related factors.

[0090] Fig.13 HL60(CA) fusion protein inhibits tumor growth.

[0091] Fig.14 This is the temperature stability test for HL60 and HL60(CA). DETAILED DESCRIPTION

[0092] The present invention is further described in detail below through specific examples.

[0093] It should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0094] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0095] The general embodiment "comprising" or "including" encompasses the more specific embodiment "consisting of". Furthermore, the singular and plural forms are not used in a limiting manner. As used herein, the singular forms "a", "an", and "the" refer to both the singular and the plural, unless explicitly stated to refer to the singular only.

[0096] The term "protein" is used interchangeably with "amino acid residue sequence" or "polypeptide" and refers to a polymer of amino acids of any length. These terms also include proteins that are post-translationally modified by reactions including, but not limited to, glycosylation, acetylation, phosphorylation, glycation, or protein processing. Modifications and changes can be made in the structure of a polypeptide, such as fusion to other proteins, amino acid sequence substitutions, deletions, or insertions, while the molecule retains its biological functional activity. For example, certain amino acid sequence substitutions can be made in a polypeptide or its underlying nucleic acid coding sequence, and a protein with the same properties can be obtained. The term "polypeptide" generally refers to a sequence having more than 10 amino acids, and the term "peptide" means a sequence having a length of up to 10 amino acids. However, these terms can be used interchangeably.

[0097] The term "fusion protein" is defined as a protein containing the complete sequence or any portion of the sequence of two or more originally separated natural or modified heterologous proteins, or a composition of the complete sequence or any portion of the sequence of two or more originally separated natural or modified heterologous proteins. Fusion proteins can be constructed by genetic engineering methods by fusing two or more genes or portions thereof that originally encoded two or more originally separated natural or heterologous proteins or portions thereof. This produces a fusion protein with functional properties derived from each original protein. Fusion proteins include, but are not limited to, Fc fusion proteins.

[0098] The term "gene" refers to a DNA locus of a heritable genomic sequence that affects the properties of an organism by being expressed as a functional product or by regulating gene expression. Genes and polynucleotides may include introns and exons, as in a genomic sequence, or only include coding sequences, as in cDNA, such as an open reading frame (ORF), which includes a start codon (methionine codon) and a translation stop codon. Genes and polynucleotides may also include regions that regulate their expression, such as transcription initiation, translation, and transcription termination. Therefore, regulatory elements such as promoters are also included.

[0099] The terms "enhance," "increase," and "increase increase" are used interchangeably herein and refer to an increase of at least about 10% compared to a control, such as an increase of at least about 20% or at least about 30% or at least about 40% or at least about 50% or at least about 75% or at least about 80% or at least about 90% or at least about 100% or at least about 200% or at least about 300%, or a decrease of any integer between 10% and 300% compared to a control.

[0100] The term "vector" is a nucleic acid that can be used to introduce a heterologous polynucleotide into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication-defective retroviruses, adenoviruses, and adeno-associated viruses), wherein additional DNA or RNA segments can be introduced into the viral genome.

[0101] The term "encoding" refers broadly to any process whereby the information in the polymer macromolecule is used to guide the production of a second molecule different from the first molecule. In other aspects, a DNA molecule can encode an RNA molecule (e.g., by using a transcription process of a DNA-dependent RNA polymerase). Moreover, as in the translation process, an RNA molecule can encode a polypeptide. When used to describe the translation process, the term "encoding" is also extended to a triplet codon encoding an amino acid. In some aspects, an RNA molecule can encode a DNA molecule, such as by incorporating a reverse transcription process of an RNA-dependent DNA polymerase. On the other hand, a DNA molecule can encode a polypeptide, wherein it should be understood that the "encoding" used in this case incorporates both transcription and translation processes.

[0102] The term "immune checkpoint inhibitory antibody" refers to an antibody as defined above that binds to an inhibitory receptor (IR) or more broadly interferes with the interaction between an inhibitory receptor and its ligand, wherein the inhibitory receptor is essential for balancing co-stimulatory receptor activity and limiting T cell activation. Therefore, this antibody targets immune system checkpoints, such as cytotoxic T lymphocyte antigen 4 (CTLA-4), programmed death factor 1 (PD-1) or its ligand (PD-L1). Examples of immune checkpoint inhibitory antibodies suitable for use in the present invention are anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-GITR antibodies, anti-OX40 antibodies or anti-LAG3 antibodies, but are not limited thereto. In addition, anti-FGL1 antibodies can be considered as immune checkpoint inhibitors. In most cases, immune checkpoint inhibitory antibodies are antagonistic or blocking antibodies, i.e., blocking antibodies selected from the group consisting of anti-PD-1 antibodies, anti-PD-L1 antibodies, anti-IDO antibodies, anti-CTLA-4 antibodies, anti-Tim-3 antibodies, anti-LAG3 antibodies, anti-VISTA antibodies, and anti-BTLA antibodies. However, immune checkpoint inhibitory antibodies may also be agonistic antibodies, such as anti-GITR or anti-OX40 antibodies. Regarding anti-LAG3 antibodies, the antibody may be an antibody that selectively inhibits the interaction of LAG3 with MHC class II molecules or an antibody that selectively inhibits the interaction of LAG3 with FGL1 or an antibody that inhibits the interaction of LAG3 with FGL1 and MHC class II molecules.

[0103] The term "cytokine" refers to a small protein that is released by a cell and acts as an intercellular mediator, for example, affecting the behavior of cells around the secreting cell. Cytokines can be secreted by immune cells such as T cells, B cells, NK cells and macrophages or other cells. Cytokines can participate in intercellular signal transduction events, such as autocrine signal transduction, paracrine signal transduction and endocrine signal transduction. They can mediate a series of biological processes, including but not limited to immunity, inflammation and hematopoiesis. Cytokines can be chemokines, interferons, interleukins, lymphokines or tumor necrosis factors.

[0104] The term "expression" refers to the transcription and / or translation of nucleic acid sequences in host cells. The expression level of the target gene product in cancer or cancer patients can be determined based on the amount of the corresponding mRNA present in the sample provided or by the amount of the protein encoded by the gene of the selected gene, particularly by encoding FGL1. For example, RNA transcribed from genes such as the CXCL10 gene can be quantified by Northern blot hybridization, ribonuclease RNA protection, in situ hybridization with cell RNA, or by PCR (such as qPCR). Proteins encoded by genes such as the CXCL10 gene can be quantified by a variety of methods, for example, by ELISA, by Western blotting, by radioimmunoassay, by immunoprecipitation, by immunohistochemistry, by testing the biological activity of the protein, by the immunostaining of the protein and subsequent FACS analysis, or by homogeneous time-resolved fluorescence (HTRF) assay.

[0105] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0106] Example 1 Construction of HL60 fusion protein and verification of its effect

[0107] This example takes the design and construction of HL60 fusion protein to induce anti-tumor immunity as an example to demonstrate and confirm the feasibility and advantages of the technical solution of the present invention. DDX60 protein is involved in maintaining dsRNA stability and activating anti-tumor immunity. Therefore, designing drugs to bring the functional domain of DDX60 protein into cells to activate anti-tumor immune response is of great significance for the clinical treatment of tumors. However, there is no clinical therapeutic drug based on DDX60 protein so far.

[0108] 1. Preparation of fusion protein HL60 molecule

[0109] (I) Design and construction of TRAP2 molecular tools

[0110] 1. Differential expression of DDX60 protein in different tumors

[0111] According to the public database search (http: / / gepia2.cancer-pku.cn / #general), DDX60 is highly expressed in many tumors, including bladder cancer, breast cancer, cervical squamous cell carcinoma, pheochromocytoma, diffuse large B-cell lymphoma, esophageal cancer, glioblastoma, head and neck tumors, kidney cancer, acute myeloid leukemia, brain glioma, liver cancer, lung cancer, pancreatic cancer, nasopharyngeal carcinoma, breast cancer, prostate cancer, gastrointestinal cancer, thyroid cancer, thymoma, etc. ( Figure 1 ), indicating that DDX60 is a potential tumor marker and the design of anti-tumor drugs based on DDX60 is very feasible.

[0112] 2. Construction of HL60 expression vector

[0113] Figure 2 shown.

[0114] (II) Expression and purification of HL60

[0115] 1.HL60 was induced to express using isopropylthiogalactoside (IPTG), and different IPTG concentrations, action temperatures and action times were used to explore the induction expression conditions. SDS-PAGE and Coomassie Brilliant Blue experiments were then used to determine the optimal expression conditions. In this example, the correctly sequenced bacterial solution was inoculated into TB medium containing kanamycin resistance (50ng / μL) and cultured at 37°C overnight with shaking. The next day, the activated strains were inoculated into fresh culture medium at a ratio of 1:100, and cultured at 37°C with shaking at 180rpm until OD 600 =0.6, cool to 20°C, add isopropylthiogalactoside (IPTG), the final concentration of IPTG is 0.5mM, continue to induce expression under the same conditions for about 18h, collect the cells, centrifuge at 5000rpm at 4°C for 30min, resuspend the cells in 1×PBS buffer and centrifuge again to collect the cells. The collected cells are resuspended again with 1×PBS buffer to 10% bacterial solution, and then use a high-pressure crusher, 900Pa pressure, 4°C cycle crushing for 3min. Centrifuge at 10000rpm at 4°C for 1h, discard the precipitate, filter the supernatant with a 0.22mm pore size filter membrane, and use the supernatant for subsequent purification.

[0116] 2. Resuspend the supernatant obtained above with Ni-NTA agarose gel and incubate at 4°C for 2h (incubate according to 5g of bacteria: 1mL50% Ni-NTA agarose gel). The color of Ni-NTA agarose gel changes from blue to brown during the incubation process. Centrifuge at 1000g, 4°C for 1min, take 20uL of supernatant for subsequent purification effect verification, discard most of the supernatant and keep only a little for resuspending Ni-NTA agarose gel, put it on the column, and wash 3 times with 1×PBS buffer containing 20mM imidazole, 2 times the column volume each time. Collect 20μL of the last washing solution. Then elute with 1×PBS buffer containing 300mM imidazole. If the Ni-NTA agarose gel turns blue again, it means that the elution is basically complete. Take 20uL of the eluate and keep the sample. Use 10% SDS-PAGE to detect the purification efficiency of the fusion protein. The eluate was centrifuged at 10000 g for 10 min, and the supernatant was concentrated using a 10KD ultrafiltration tube (Millipore, catalog number: UFC900396), centrifuged at 4500 g, and ultrafiltered to 500 μL at 4 ° C. The concentrate was then rinsed three times with 1× PBS and centrifuged three times. The concentration of the concentrated protein was measured using a BCA protein concentration detection kit (Thermo Fisher, catalog number: 23227) and stored at -80 ° C.

[0117] The purification effect of HL60 is as follows Figure 3 As shown, the purity reaches more than 90% and can be used for subsequent experimental verification.

[0118] The amino acid sequence of the HL60 fusion protein is:

[0119]

[0120] (The single underline is the amino acid sequence of the tumor-targeting cell-penetrating peptide BR2, recorded as SEQ ID NO: 2; the double underline is the flexible linking sequence between the functional regions, recorded as SEQ ID NO: 5).

[0121] II. Verification of HL60 fusion protein promoting tumor cells to express anti-tumor immune-related factors

[0122] Colorectal cancer, pancreatic cancer, gastric cancer and lung cancer cells were treated with different concentrations of HL60 fusion protein (100 ng / mL and 500 ng / mL) and the cells were collected after 24 h of treatment. RNA was extracted using the TRIzol method. 1 μg RNA and 4 μL 4×g DNA wiper Mix were taken to MicroAmp TM The reaction tube was filled with enzyme-free water to a volume of 20 μL and mixed by pipetting. The mixture was placed in a PCR instrument at 42°C for 2 min and placed on ice for later use. 4 μL 5×HiScript III qRT SuperMix was added, mixed by pipetting, and then placed in a PCR instrument at 50°C for 15 min and 85°C for 5 s to obtain the reverse transcription product. PCR primers were designed using Primer-Blast on the NCBI website, and PCR experiments were performed using the Cham Q SYBR qPCR Master Mix (Q311-02) kit from Novozymes to detect the expression of representative IFN effector genes, including cytokines (CXCL10), immune response regulators (IFIT2, ISG15), cell death regulators (CASP9), and antigen presentation-related molecules (PSMB8, B2M, IFI44).

[0123] The results showed that HL60 fusion protein treatment could significantly increase the expression of these anti-tumor immune-related factors ( Figure 4 ).

[0124] 3. In vivo tumor targeting validation of HL60 fusion protein

[0125] This experiment was conducted in C57BL / 6J mice (4-8 weeks old, purchased from Guangdong Animal Experiment Center). All animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University. Mice were subcutaneously injected with mouse KRAS-overexpressing murine colorectal cancer cells MC38 (MC38K) (3×10 5 / mouse) and inoculated subcutaneously on the left and right sides of the back of the mouse. Three weeks after tumor inoculation, tumors can be observed. At this time, the mouse is injected with Cy5.5-labeled HL60 fusion protein (200μg / mouse) through the tail vein. The main organs and tumor tissues of the mice are removed 8 hours after the injection, and the distribution of HL60 fusion protein in tumor tissues and other important organ tissues is observed using a small animal in vivo imaging system.

[0126] The experimental results showed that after the HL60 fusion protein was injected into the tail vein of tumor-bearing mice, the recombinant protein was mainly enriched in the tumor tissue, indicating that the HL60 fusion protein has good tumor targeting properties ( Figure 5 ).

[0127] IV. Verification of the effect of HL60 fusion protein in inhibiting tumor growth and enhancing the effect of PD-1 antibody

[0128] 1. C57BL / 6J mice (4-8 weeks old) were used in the experiments and were purchased from the Guangdong Animal Experiment Center. All animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University (approval number L025501202407004). The mice were randomly divided into groups, with 6 mice in each group. On day 0, KRAS-overexpressing murine colorectal cancer cells MC38 (MC38K) (3×10 5 One week after tumor inoculation, intraperitoneal injection was started and the groups were divided into: (1) PBS phosphate buffered saline solution; (2) HL60 fusion protein (50 μg); (3) HL60 fusion protein (200 μg).

[0129] Inject twice a week for 4 consecutive weeks. 3 The mice were killed at 14:00 pm and the endpoint of death was recorded. The tumor size was measured with a vernier caliper every other day. The length (L) and width (W) of the tumor were measured respectively and then calculated according to the formula Volume (mm 3 )=LW 2 / 2 to calculate the tumor volume, and weigh the C57 mice every other day with an electronic balance, for a total of 8 measurements. The tumor volume of each group of C57 mice was averaged, and the first measurement was used as the control group, and each measurement after intraperitoneal injection was used as the experimental group. The tumor growth rate was calculated according to the formula (experimental group-control group) / control group×100%, and the growth rate curve was drawn.

[0130] The results showed that HL60 fusion protein treatment could significantly inhibit tumor growth ( Figure 6 ), indicating that HL60 fusion there has a significant anti-tumor effect.

[0131] 2. C57BL / 6J mice (4-8 weeks old) were randomly divided into groups, with 6 mice in each group. On day 0, KRAS-overexpressing murine colorectal cancer cells MC38 (MC38K) (3×10 5 cells). One week after tumor inoculation, the mice were injected intraperitoneally and divided into the following groups: (1) PBS phosphate buffered saline solution + IgG isotype control (BE0089, 2A3); (2) PBS phosphate buffered saline solution + anti-PD-1 monoclonal antibody (BE0146, RMP1-14); (3) IgG isotype control + HL60 (200 μg); (4) anti-PD-1 antibody + HL60 (200 μg). IgG isotype control or anti-PD-1 monoclonal antibody was injected at 200 μg each time, twice a week for 4 consecutive weeks; PBS phosphate buffered saline solution or HL60 fusion protein (200 μg) was injected twice a week for 4 consecutive weeks. When tumor necrosis occurred or the tumor volume reached 1700 mm 3 The mice were killed at 14:00 pm and the endpoint of death was recorded. The tumor size was measured with a vernier caliper every other day. The length (L) and width (W) of the tumor were measured respectively and then calculated according to the formula Volume (mm 3 )=LW 2 / 2 to calculate the tumor volume, and weigh the C57 mice every other day with an electronic balance, for a total of 8 measurements. The tumor volume of each group of C57 mice was averaged, and the first measurement was used as the control group, and each measurement after intraperitoneal injection was used as the experimental group. The tumor growth rate was calculated according to the formula (experimental group-control group) / control group×100%, and the growth rate curve was drawn.

[0132] The results showed that when HL60 fusion protein was used alone, the tumor growth was lower than that of the control group, and when it was used in combination with anti-PD-1 antibody, the tumor growth was further inhibited ( Figure 7 ), indicating that HL60 fusion protein can enhance the sensitivity of tumors to anti-PD-1 antibodies.

[0133] 5. In vivo safety verification of HL60 fusion protein

[0134] This example aims to evaluate the safety of the drug HL60 fusion protein in mice. C57BL / 6J mice (4-8 weeks old) were used in the experiment, and all animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University. Mice were given HL60 fusion protein by intraperitoneal injection, and the dose was set at 100 mg / kg. Within 80 hours after administration, the physiological state and mental state changes of the mice were recorded in detail, and the weight of the mice was measured regularly, and a comprehensive pathological analysis of their visceral tissues was performed after the experiment.

[0135] The results showed that after injection of HL60 fusion protein, the mice maintained a normal mental state during the observation period after administration, and the weight of the mice did not show significant changes ( Figure 8 ); Pathological examination of the main internal organs of mice showed no obvious tissue damage or pathological changes ( Fig. 9 ). The experimental results showed that the HL60 fusion protein had good safety at a dose of 100 mg.

[0136] Example 2 Verification of the effect of cysteine ​​mutation modification of HL60 fusion protein

[0137] "Example 1" describes the design ideas of HL60 fusion protein and verifies the role of HL60 fusion protein in activating anti-tumor immunity and inhibiting tumor growth. Since HL60 protein contains 6 cysteines, its stability is relatively low. Therefore, this example further uses amino acid mutation to improve the protein stability of HL60, increase its drugability, and evaluate the changes in protein stability after cysteine ​​mutation, as well as the changes in the induction of anti-tumor immunity and tumor inhibition effects.

[0138] I. Construction of vector and expression purification of HL60(CA) after cysteine ​​(C) of HL60 fusion protein was mutated to alanine (A)

[0139] (I) Construction of HL60(CA) vector

[0140]

[0141] (II) Expression and purification of HL60(CA) fusion protein

[0142] 1.HL60(CA) was induced to express using isopropylthiogalactoside (IPTG), and different IPTG concentrations, action temperatures and action times were used to explore the induction expression conditions. SDS-PAGE and Coomassie Brilliant Blue experiments were then used to determine the optimal expression conditions. In this example, the correctly sequenced bacterial solution was inoculated into TB medium containing kanamycin resistance (50ng / μL) and cultured at 37°C overnight with shaking. The next day, the activated strains were inoculated into fresh culture medium at a ratio of 1:100 for expansion culture, and cultured at 37°C with shaking at 180rpm until OD 600 =0.6, cool to 20°C, add isopropylthiogalactoside (IPTG), the final concentration of IPTG is 0.5mM, continue to induce expression under the same conditions for about 18h, collect the cells, centrifuge at 5000rpm at 4°C for 30min, resuspend the cells in 1×PBS buffer and centrifuge again to collect the cells. The collected cells are resuspended again with 1×PBS buffer to 10% bacterial solution, and then use a high-pressure crusher, 900Pa pressure, 4°C cycle crushing for 3min. Centrifuge at 10000rpm at 4°C for 1h, discard the precipitate, filter the supernatant with a 0.22mm pore size filter membrane, and use the supernatant for subsequent purification.

[0143] 2. Resuspend the supernatant obtained above with Ni-NTA agarose gel and incubate at 4°C for 2h (incubate according to 5g of bacteria: 1mL50% Ni-NTA agarose gel). The color of Ni-NTA agarose gel changes from blue to brown during the incubation process. Centrifuge at 1000g, 4°C for 1min, take 20μL of supernatant for subsequent purification effect verification, discard most of the supernatant and keep only a little for resuspending Ni-NTA agarose gel, put it on the column, and wash 3 times with 1×PBS buffer containing 20mM imidazole, 2 times the column volume each time. Collect 20μL of the last washing solution. Then elute with 1×PBS buffer containing 300mM imidazole. If the Ni-NTA agarose gel turns blue again, it means that the elution is basically complete. Take 20μL of the eluate and keep the sample. Use 10% SDS-PAGE to detect the purification efficiency of the fusion protein. The eluate was centrifuged at 10000 g for 10 min, and the supernatant was concentrated using a 10KD ultrafiltration tube (Millipore, catalog number: UFC900396), centrifuged at 4500 g, and ultrafiltered to 500 μL at 4 ° C. The concentrate was then rinsed three times with 1× PBS and centrifuged three times. The concentration of the concentrated protein was measured using a BCA protein concentration detection kit (Thermo Fisher, catalog number: 23227) and stored at -80 ° C.

[0144] The purification effect of HL60(CA) is as follows Fig.10 As shown, the purity reaches more than 90% and can be used for subsequent experimental verification.

[0145] The amino acid sequence of the HL60 (CA) fusion protein is:

[0146]

[0147] (The single underline is the amino acid sequence of the tumor-targeting cell-penetrating peptide BR2; the double underline is the flexible linker sequence between the functional regions; the emphasis is the position where cysteine ​​C is mutated to alanine A).

[0148] 2. In vivo tumor targeting validation of HL60(CA) fusion protein

[0149] This experiment was conducted in C57BL / 6J mice (4-8 weeks old, purchased from Guangdong Animal Experiment Center). All animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University. Mice were subcutaneously injected with mouse KRAS-overexpressing murine colorectal cancer cells MC38 (MC38K) (3×10 5 / mouse) and inoculated subcutaneously on the left and right sides of the back of the mouse. Three weeks after tumor inoculation, tumors can be observed. At this time, the mouse is injected with Cy5.5-labeled HL60(CA) fusion protein (200μg / mouse) through the tail vein. The main organs and tumor tissues of the mice are removed 8 hours after the injection, and the distribution of HL60(CA) fusion protein in tumor tissues and other important organ tissues is observed using a small animal in vivo imaging system.

[0150] The experimental results showed that after the HL60 fusion protein was injected into the tail vein of tumor-bearing mice, the recombinant protein was mainly enriched in the tumor tissue, indicating that the fusion protein HL60(CA) after the cysteine ​​(C) of the HL60 fusion protein was mutated to alanine (A) also had good tumor targeting ( Fig.11 ).

[0151] 3. Verification of the effect of HL60(CA) fusion protein in promoting the expression of anti-tumor immune-related factors

[0152] Colorectal cancer, pancreatic cancer, gastric cancer and lung cancer cells were treated with different concentrations of HL60 (CA) fusion protein (100 ng / mL) and the cells were collected after 24 hours of treatment. RNA was extracted using the TRIzol method, and 1 μg RNA and 4 μL 4×g DNA wiper Mix were taken to MicroAmp TMThe reaction tube was filled with enzyme-free water to a volume of 20 μL and mixed by pipetting. The mixture was placed in a PCR instrument at 42°C for 2 min and placed on ice for later use. 4 μL 5×HiScript III qRT SuperMix was added, mixed by pipetting, and then placed in a PCR instrument at 50°C for 15 min and 85°C for 5 s to obtain the reverse transcription product. PCR primers were designed using Primer-Blast on the NCBI website, and PCR experiments were performed using the Cham Q SYBR qPCR Master Mix (Q311-02) kit from Novozymes to detect the expression of representative IFN effector genes, including cytokines (CXCL10), immune response regulators (IFIT2, ISG15), cell death regulators (CASP9), and antigen presentation-related molecules (PSMB8, B2M, IFI44).

[0153] The results showed that HL60(CA) fusion protein treatment could significantly increase the expression of these immune activation-related factors ( Fig.12 ).

[0154] IV. Verification of the effect of HL60(CA) fusion protein in inhibiting tumor growth

[0155] 1. C57BL / 6J mice (4-8 weeks old) were used in the experiments and were purchased from the Guangdong Animal Experiment Center. All animal experiments were approved by the Animal Ethics Committee of Sun Yat-sen University (approval number L025501202407004). The mice were randomly divided into groups, with 6 mice in each group. On day 0, tumor cells (3×10 5 One week after tumor inoculation, intraperitoneal injection was started and the groups were divided into: (1) PBS phosphate buffered saline solution; (2) HL60 fusion protein (50 μg); (3) HL60 (CA) fusion protein (50 μg).

[0156] Inject twice a week for 4 consecutive weeks. 3 The mice were killed at 1:10 pm and the endpoint of death was recorded. The tumor size was measured with a vernier caliper every other day. The length (L) and width (W) of the tumor were measured respectively and then calculated according to the formula Volume (mm 3 )=LW 2 / 2 to calculate the tumor volume, and weigh the C57 mice every other day with an electronic balance, for a total of 8 measurements. The tumor volume of each group of C57 mice was averaged, and the first measurement was used as the control group, and each measurement after intraperitoneal injection was used as the experimental group. The tumor growth rate was calculated according to the formula (experimental group-control group) / control group×100%, and the growth rate curve was drawn.

[0157] The results showed that HL60(CA) fusion protein treatment significantly inhibited tumor growth, and compared with HL60 fusion protein treatment, the tumor growth rate after HL60(CA) fusion protein treatment was significantly slowed down ( Fig.13 ), indicating that HL60(CA) fusion protein has significant anti-tumor effect, and its anti-tumor effect is better than that of HL60 fusion protein.

[0158] 5. Comparison of molecular stability between HL60(CA) and HL60

[0159] This example was conducted using the UNcle protein stability analyzer, and mainly analyzed the changes in the temperature stability of the protein molecule before and after the cysteine-alanine (CA) mutation in the HL60 fusion protein.

[0160] (1) The protein solutions of HL60 and HL60(CA) were diluted with ultrapure water to a final concentration of 0.5 mg / mL, followed by centrifugation at 12,000 rpm for 10 min and the supernatant was collected.

[0161] (2) 9 μL of the above protein dilution solution was respectively added to the sample tube and placed in the instrument. The detection temperature was then set to start from 25°C and increase by 1°C per minute. The Fluorescence detection module was used to record the changes in the melting temperature (Tm) and the center of mass wavelength (BCM) of the endogenous fluorescence emission spectrum. The differences in the stability of the HL60 fusion protein and the HL60 (CA) fusion protein at different temperatures were analyzed.

[0162] The results are as follows Fig.14 As shown, the maximum tolerance temperature (Tm) of HL60(CA) fusion protein is 62.5°C, while the maximum tolerance temperature of HL60 fusion protein is 48°C, indicating that after cysteine ​​is mutated to alanine, the molecular stability of the fusion protein at high temperature is higher.

[0163] The above data show that the HL60 (CA) fusion protein after cysteine ​​mutation to alanine can be successfully expressed and purified, and significantly increases the tumor inhibition and stability of the protein molecule, which will have a significant impact on the application of HL60 fusion protein in pharmaceutical development.

[0164] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A fusion protein comprising a DDX60 functional domain and a tumor-targeting cell-penetrating peptide; in, The amino acid sequence of the tumor-targeting cell-penetrating peptide is as shown in SEQ ID NO: 2, or an amino acid sequence shown in SEQ ID NO: 2 that is modified by substitution, deletion or addition of one or more amino acids and has the same or similar functions.

2. The fusion protein according to claim 1, It is characterized in that The DDX60 functional domain includes an ATP binding and hydrolysis domain and an RNA binding domain, and the ATP binding and hydrolysis domain and the RNA binding domain are connected by a flexible linker; Preferably, the ATP binding and hydrolysis domain is any one of a1) to a3: a1) a protein with an amino acid sequence as shown in SEQ ID NO: 8; a2) a protein having the same or similar function as SEQ ID NO: 8 after one or more amino acids are substituted and / or deleted and / or added; a3) a protein having 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology to SEQ ID NO: 8 and having the same function as SEQ ID NO: 8; Preferably, the RNA binding domain is any one of b1) to b3: b1) a protein with an amino acid sequence as shown in SEQ ID NO:9; b2) a protein having the same or similar function as SEQ ID NO: 9 after one or more amino acids are substituted and / or deleted and / or added; b3) a protein having 99%, 98%, 97%, 96%, 95%, 94%, 93% or 92% homology to SEQ ID NO: 9 and having the same function as SEQ ID NO: 9; Preferably, the ATP binding and hydrolysis domain is located at the N-terminus or C-terminus of the RNA binding domain.

3. The fusion protein according to claim 2, It is characterized in that a2) a protein having the same or similar function after cysteine ​​in SEQ ID NO: 8 is mutated to alanine or other amino acids; and / or, b2) a protein having the same or similar function after cysteine ​​in SEQ ID NO: 8 is mutated to alanine or other amino acids.

4. The fusion protein according to claim 3, It is characterized in that a2) the amino acid sequence of the protein is shown as SEQ ID NO: 10; and / or, b2) the amino acid sequence of the protein is shown as SEQ ID NO:

11.

5. The fusion protein according to any one of claims 1 to 4, It is characterized in that The DDX60 functional domain and the tumor-targeting cell-penetrating peptide are linked via a flexible linker; Preferably, the amino acid sequence of the flexible linker is (GSG)n, wherein n≥1; Preferably, the DDX60 functional domain is located at the N-terminus or C-terminus of the tumor-targeting cell-penetrating peptide.

6. The fusion protein according to any one of claims 1 to 4, It is characterized in that The amino acid sequence of the fusion protein is shown in SEQ ID NO: 1 and / or SEQ ID NO:

7.

7. A biological material related to the fusion protein according to any one of claims 1 to 6, wherein the biological material is any one of c1) to c12): c1) a nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 6; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1); c4) a recombinant vector containing the expression cassette described in c2); c5) a recombinant microorganism containing the nucleic acid molecule described in c1); c6) a recombinant microorganism containing the expression cassette described in c2); c7) a recombinant microorganism containing the recombinant vector described in c3); c8) a recombinant microorganism containing the recombinant vector described in c4); c9) transgenic animal cell lines containing the nucleic acid molecule described in c1); c10) a transgenic animal cell line containing the expression cassette described in c2); c11) a transgenic animal cell line containing the recombinant vector described in c3); c12) a transgenic animal cell line containing the recombinant vector described in c4); The transgenic animal cell line contains no reproductive material.

8. Use of the fusion protein according to any one of claims 1 to 6 or the biomaterial according to claim 7 in at least one of (1) to (5): (1) Preparation of drugs for preventing and / or treating tumors; (2) preparing drugs for activating anti-tumor immunity; (3) Preparation of drugs that enhance the anti-tumor effects of immune checkpoint inhibitors; (4) Enhance the expression of IFN effector genes; (5) Prepare products that enhance the expression of IFN effector genes.

9. The use according to claim 8, It is characterized in that The IFN effector genes described in (4) to (5) include at least one of cytokines, immune response regulatory factors, cell death regulatory factors and antigen presentation-related molecules; preferably, the cytokines include CXCL10; and / or, the immune response regulatory factors include at least one of IFIT2 and ISG15; and / or, the cell death regulatory factors include CASP9; and / or, the antigen presentation-related molecules include at least one of PSMB8, B2M, and IFI44.

10. A drug comprising the fusion protein according to any one of claims 1 to 6 or the biomaterial according to claim 7; Preferably, the drug further comprises a pharmaceutically acceptable excipient; Preferably, the drug further comprises a combination drug, and the combination drug comprises a tumor-targeted drug.

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