A retinoblastoma protein mutant and its preparation method and application

Abstract

The application belongs to the technical field of biological immunotherapy, and particularly relates to a retinoblastoma protein mutant and a preparation method and application thereof. The retinoblastoma protein mutant comprises a CDK phosphorylation site mutant retinoblastoma protein 1 mutant polypeptide, a CDK phosphorylation site mutant retinoblastoma protein 1 mutant protein or a CDK phosphorylation site mutant retinoblastoma protein 1 mutant analog. The RB1 is improved for long-term binding to E2F family transcription factors, the cell cycle is delayed, the expression of E2F target genes is significantly inhibited, and the proliferation and growth of cancer cells are significantly inhibited.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological immunotherapy, and particularly relates to a retinoblastoma protein mutant and a preparation method and application thereof. BACKGROUND

[0002] Retinoblastoma (RB) is the most common primary intraocular malignancy in children, with an incidence of about 1 / 20000-1 / 15000. In China, about 1100 new RB patients are added every year, and 84% of them are high-risk patients in the late stage. Genetic studies have found that the occurrence of retinoblastoma is usually due to the presence of homozygous mutations, compound heterozygous mutations or gene deletions in RB1 gene (RB transcriptional corepressor 1, RB1), loss of RB1 protein in developing retinal cells, leading to uncontrolled proliferation of cells. Retinoblastoma can involve a single eye or both eyes, and sometimes it can also involve intracranial tumors, i.e. trilateral retinoblastoma. If not treated in time and effectively, retinoblastoma can spread through the optic nerve to the brain, or through the blood to the bone marrow, and cause death. RB generally occurs before the age of 6, but those with a family history or those with bilateral disease usually occur before the age of 1.

[0003] RB1 gene is the first cloned tumor suppressor gene, which encodes RB1 protein as a negative regulator of cell cycle, containing N-terminal domain (DUF3452), pocket domain (Rb-A and Rb-B) and C-terminal domain (Rb-C). The pocket domain composed of Rb-A and Rb-B can bind to E2F transcription factors, hinder the expression of E2F-regulated cell cycle-related proteins, prevent cells from passing through the G1-S checkpoint, and block the cell cycle, playing an indispensable role in inhibiting the occurrence of various tumors. Through DEEP prediction and research, it is found that there are 16 cyclin-dependent kinase (CDK) phosphorylation sites in RB1 protein (A, 1B). When RB1 protein is phosphorylated by CDKs, E2F leaves RB1 protein complex and binds to target genes containing E2F transcription factors, starting the expression of cell cycle-related genes and promoting cell division and proliferation (C). Figure 1 Figure 1

[0004] ​​At present, more than 1000 mutations of RB1 have been found, including missense mutations, nonsense mutations, splice site mutations, frame shift mutations, microdeletions, large fragment deletions and promoter hypermethylation. These mutations lead to inactivation of RB protein function or expression of non-functional protein, loss of cell cycle control and abnormal cell proliferation. SUMMARY

[0005] The present application provides a retinoblastoma protein mutant, a preparation method and application thereof to solve the problems in the prior art. The retinoblastoma protein mutant can improve the long-term binding of RB1 to E2F family transcription factors, delay the cell cycle, significantly inhibit the expression of E2F target genes, and significantly inhibit the proliferation and growth of cancer cells.

[0006] To this end, the present application provides a retinoblastoma protein mutant, including a CDK phosphorylation site mutant polypeptide of retinoblastoma protein 1, a CDK phosphorylation site mutant protein of retinoblastoma protein 1 or a CDK phosphorylation site mutant analog of retinoblastoma protein 1.

[0007] In some embodiments of the present application, the CDK phosphorylation site mutation includes mutation of at least one of the following CDK phosphorylation sites: T5, S230, S249, T252, T356, T373, S567, S608, S612, S780, S788, S795, S807, S811, T821 and T826.

[0008] In some embodiments of the present application, the CDK phosphorylation site mutation includes mutation of the following CDK phosphorylation sites: T5, S230, S249, T252, T356, T373, S608, S612, S780, S788, S795, S807, S811, T821 and T826.

[0009] According to the present application, the retinoblastoma protein mutant is a CDK phosphorylation site full-mutant retinoblastoma protein 1 (RBdel15).

[0010] In some embodiments of the present application, the retinoblastoma protein mutant includes an amino acid sequence as shown in SEQ ID NO. 2 or SEQ ID NO. 4.

[0011] In some embodiments of the present application, the retinoblastoma protein mutant includes an amino acid sequence as shown in SEQ ID NO. 4.

[0012] In some embodiments of the present application, the retinoblastoma protein mutant comprises a retinoblastoma protein mutant encoded by a nucleic acid sequence as set forth in SEQ ID NO. 1 or SEQ ID NO. 3.

[0013] In some embodiments of the present application, the retinoblastoma protein mutant comprises a retinoblastoma protein mutant encoded by a nucleic acid sequence as set forth in SEQ ID NO. 3.

[0014] In some embodiments of the present application, the CDK phosphorylation sites are all mutated to alanine.

[0015] The second aspect of the present application provides an isolated nucleic acid molecule encoding the retinoblastoma protein mutant of the first aspect of the present application.

[0016] The third aspect of the present application provides a vector comprising the nucleic acid molecule of the second aspect of the present application.

[0017] In some embodiments of the present application, the vector comprises an expression vector or a cloning vector.

[0018] In other embodiments of the present application, the vector comprises a viral vector.

[0019] In some embodiments of the present application, the viral vector comprises, but is not limited to, an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, a retroviral vector, a vaccinia vector, or a herpes simplex viral vector, and derivatives thereof.

[0020] The fourth aspect of the present application provides a host cell comprising the nucleic acid molecule of the second aspect of the present application or the vector of the third aspect of the present application.

[0021] The fifth aspect of the present application provides a pharmaceutical composition comprising the retinoblastoma protein mutant of the first aspect of the present application, the nucleic acid molecule of the second aspect of the present application, or the vector of the third aspect of the present application.

[0022] In some embodiments of the present application, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0023] In some embodiments of the present application, the pharmaceutical composition further comprises at least one additive selected from a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressive agent, a growth inhibitory agent, and an active pharmaceutical agent required for the specific indication to be treated.

[0024] The sixth aspect of the present application provides a method for preparing the retinoblastoma protein mutant of the first aspect of the present application, comprising culturing the host cell of the fourth aspect of the present application.

[0025] In some embodiments of the present application, the culture conditions of the host cell in the preparation method are sufficient to enable the host cell to express the retinoblastoma protein mutant.

[0026] The seventh aspect of the present application provides use of the retinoblastoma protein mutant of the first aspect of the present application or the nucleic acid molecule of the second aspect of the present application or the vector of the third aspect of the present application or the pharmaceutical composition of the fifth aspect of the present application in the preparation of a medicament for preventing and / or treating cancer or tumor.

[0027] In some embodiments of the present application, the use includes use in the preparation of a medicament for preventing and / or treating cancer or tumor, which enhances or improves the binding of the retinoblastoma protein mutant to the E2F transcription factor.

[0028] In some embodiments of the present application, the use further includes administration of the retinoblastoma protein mutant, the nucleic acid molecule, the vector or the pharmaceutical composition in combination with other drugs.

[0029] In some embodiments of the present application, the other drugs include diagnostic agents, prophylactic agents and / or therapeutic agents.

[0030] Advantages of the present application:

[0031] The present application forms a CDK phosphorylation site full-mutant retinoblastoma protein 1 (RBdel15) by site-directed mutagenesis of 15 CDK phosphorylation sites of RB1 protein through whole gene synthesis, which can significantly inhibit the expression of E2F target genes and significantly inhibit the proliferation and growth of cancer cells. At the same time, the cells with high expression of RBdel15 of the present application show high-efficiency cell cycle inhibition function. In addition, the RBdel15 of the present application can also significantly inhibit the expression of epigenetic related genes, up-regulate the expression of immune related genes, improve the tumor infiltration of T cells, and thus promote the anti-tumor immune response of the body. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 A schematic diagram of RB1 protein structure and mechanism of action; wherein, A-analysis of RB1 protein phosphorylation sites, B-15 CDK phosphorylation sites, C-mechanism of action of wild-type RB1 and full-mutant RBdel15;

[0033] Figure 2 A graph of the establishment results of RBdel15 high-expression Miapaca2 cell strain in Example 2 of the present application; wherein, A-expression of RBdel15 protein in different cell strains, B-indirect immunofluorescence detection of RBdel15 in different cell strains, C-expression of RBdel15 mRNA in different cell strains;

[0034] Figure 3 Figure for the results of RBdel15 regulating immune and cell cycle protein expression in Example 3 of the present application; A- expression of RBdel15, B- cell proliferation, C- cell cycle, D- heat map analysis of differentially expressed genes, E- functional analysis of differential genes, F- expression of part of E2F target genes, G- expression amount of part of E2F target genes;

[0035] Figure 4 Figure for the results of the influence of RBdel15 on epigenetics in Example 4 of the present application; A- expression of epigenetics related genes, B- mRNA expression of epigenetics related genes, C- protein expression of epigenetics related genes, D- time sequence expression of part of epigenetics and immune related genes, E- influence of EZH2 inhibitor TAZ on gene expression, F- influence of siEZH2 on gene expression, G- influence of 5-Aza treatment on DNMT1 protein expression, H- influence of 5-Aza treatment on mRNA of part of genes;

[0036] Figure 5 Figure for the anti-tumor function of RBdel15; A- tumor cell proliferation, B- cell volume, C- β-galactosidase activity of cells, D- tumor cell colony formation;

[0037] Figure 6 Figure for the establishment of RBdel15 high expression mouse KPC pancreatic cancer cell line; A- indirect immunofluorescence detection of RBdel15 in different cell strains, B- expression of RBdel15 protein in different cell strains, C- number of GFP at treatment time, D- cell proliferation, E- cell cycle distribution;

[0038] Figure 7 Figure for the influence of RBdel15 on mouse pancreatic cancer infiltrating cells; A- tumor volume, B- single cell sequencing of tumor tissue, C- number of myeloid cells, D- number of T cells, E- number of anti-tumor function CAF. DETAILED DESCRIPTION

[0039] In order to make the present application more easily understood, the present application will be described in detail below in combination with examples, which only serve an illustrative purpose and are not limited to the application scope of the present application.

[0040] The term "retinoblastoma protein mutant" in the present invention encompasses any mutant form of retinoblastoma protein 1 in various forms, including full-length retinoblastoma protein 1, truncated forms of retinoblastoma protein 1, and forms of retinoblastoma protein 1 linked to another molecule (such as by fusion or chemical conjugation). "Full-length" as used in reference to retinoblastoma protein 1 refers to the mature, naturally occurring length of retinoblastoma protein 1. For example, full-length retinoblastoma protein 1 is encoded by the nucleic acid sequence set forth in SEQ ID No. 1, or comprises the amino acid sequence set forth in SEQ ID No. 2.

[0041] The term "CDK phosphorylation site mutation" in the present invention refers to an amino acid mutation of at least one of the CDK phosphorylation sites of retinoblastoma protein 1 that affects the interaction of retinoblastoma protein 1 with E2F transcription factor: T5, S230, S249, T252, T356, T373, S567, S608, S612, S780, S788, S795, S807, S811, T821, and T826. "Full mutant" refers to an amino acid mutation of the CDK phosphorylation sites of retinoblastoma protein 1 that affects the interaction of retinoblastoma protein 1 with E2F transcription factor, preferably T5, S230, S249, T252, T356, T373, S608, S612, S780, S788, S795, S807, S811, T821, and T826. Among them, the mutation can involve substitution or replacement of the wild-type amino acid residue normally located at the position. Mutants obtained by amino acid substitution are preferred. Unless otherwise specified, the retinoblastoma protein mutant in the present invention can refer to a retinoblastoma protein 1 mutant polypeptide, a retinoblastoma protein 1 mutant protein, or a retinoblastoma protein 1 mutant analog.

[0042] Various nomenclatures can be used in the present invention to indicate the same mutation. For example, mutation of threonine to alanine at position 5 can be represented as 5A, A5, T5A, or Thr5Ala.

[0043] Genetic or chemical methods known in the art can be used in the present invention to generate amino acid mutations. Genetic methods can include site-directed mutagenesis, PCR, or whole gene synthesis, etc.

[0044] The term "polypeptide" in the present invention refers to a molecule composed of monomers (amino acids) linearly connected by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain having two or more amino acids, and does not refer to a product of a specific length. Therefore, peptides, oligopeptides, proteins, amino acid chains, or any other term used to refer to a chain having two or more amino acids are included in the definition of "polypeptide". Polypeptides can be derived from natural biological sources or prepared by recombinant techniques. Polypeptides can be prepared in any manner, including by chemical synthesis.

[0045] The polypeptide of the present application can be of a size comprising more than 3, more than 5, more than 10, more than 20, more than 25, more than 50, more than 75, more than 100, more than 200, more than 500, more than 1000, or more than 2000 amino acids.

[0046] An "isolated" polypeptide or a variant or derivative thereof refers to a polypeptide that is not in its natural environment. Recombinantly produced polypeptides and proteins expressed in host cells can be considered "isolated", as well as naturally occurring or recombinant polypeptides purified by any suitable technique.

[0047] The present application provides an isolated nucleic acid molecule encoding the retinoblastoma protein mutant of the present application as previously described.

[0048] The present application provides a vector comprising the isolated nucleic acid molecule of the present application as previously described. The vector can be an expression vector or a cloning vector. In some embodiments, the vector is a viral vector. Viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, retroviral vectors, vaccinia vectors, herpes simplex viral vectors, and derivatives thereof.

[0049] The present application provides a host cell comprising a host cell transduced or transfected with the nucleic acid molecule or the vector. Suitable host cells for cloning or expression of DNA are prokaryotic, yeast, or higher eukaryotic cells. Examples of commonly used prokaryotic host cells include E. coli, B. subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, mammalian cells, and the like.

[0050] The present application provides a method of producing the retinoblastoma protein mutant, comprising culturing the host cell as described above. Preferably, the culturing condition of the host cell in the method is sufficient for the host cell to express the retinoblastoma protein mutant.

[0051] It is noted that methods of introducing nucleic acid molecules or vectors into host cells (e.g., mammalian cells) when transducing or transfecting the nucleic acid molecules or vectors, which can be introduced into host cells by physical, chemical, or biological means, are known in the art. Physical methods for introducing nucleic acid molecules or vectors into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Chemical means for introducing nucleic acid molecules or vectors into host cells include colloidal dispersion systems, such as macromolecule complexes, nanocapsules, microspheres, beads, and lipid- based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system for use as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle). Biological means for introducing nucleic acid molecules or vectors into host cells include the use of DNA and RNA vectors. In some embodiments, the transduced or transfected host cells are propagated ex vivo after introduction of the nucleic acid molecules or vectors.

[0052] The present application further provides a pharmaceutical composition comprising the retinoblastoma protein mutant, nucleic acid molecule, or vector of the present application as previously described. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0053] The pharmaceutical composition can be prepared by mixing the retinoblastoma protein mutant, nucleic acid molecule, or vector having the desired degree of purity with optional pharmaceutically acceptable carriers in the form of a lyophilized formulation or an aqueous solution. The pharmaceutically acceptable carriers employed can include at least one of a buffer, an antioxidant, a preservative, an isotonic agent, a stabilizer, and a surfactant, which are non-toxic to recipients at the dosages and concentrations employed. In addition, in order for the pharmaceutical composition to be useful for in vivo administration, they must be sterile. The pharmaceutical composition can be rendered sterile by filtration through a sterile filtration membrane.

[0054] In some embodiments, the pharmaceutical composition can further contain at least one additive of a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active pharmaceutical agent required for the specific indication to be treated. The specific amount of additive to be added can be adjusted as necessary.

[0055] The present application also provides use of the retinoblastoma protein mutant, nucleic acid molecule, vector, or pharmaceutical composition of the present application as previously described in the preparation of a medicament for preventing and / or treating cancer or tumor.

[0056] Preferably, the preventing and / or treating cancer or tumor comprises at least one of the following:

[0057] (1) inhibiting the expression of E2F downstream target genes (such as CCNA2);

[0058] (2) inhibiting the malignant proliferation and growth of cells;

[0059] (3) inhibiting cell cycle, in particular, arresting malignant proliferative cells in G1 phase, and / or reducing the number of cells in S phase;

[0060] (4) up-regulating the expression of immune-related genes, wherein the immune-related genes preferably include CCL28, OAS2, HLA-B, HLA-F, IFI6, ICM1, IFIM10 and MX2;

[0061] (5) inhibiting the expression of epigenetic-related genes, wherein the epigenetic-related genes include genes expressing DNA methyltransferase 1 (DNMT1), Zeste homolog 2 enhancer (EZH2), histone lysine methyltransferase SUV39H1 / 2 (SUV39H1 / 2), chromatin assembly factor 1A (CHAF1A), chromatin assembly factor 1B (CHAF1B), DNA methyltransferase 3B (DNMT3B), and the like;

[0062] (6) inducing tumor cell differentiation;

[0063] (7) enhancing or increasing the binding of retinoblastoma protein 1 to E2F transcription factor.

[0064] In the present application, the cancer or tumor includes but is not limited to pancreatic cancer, myeloid leukemia, acute non-lymphocytic leukemia, acute lymphoblastic leukemia, B-cell lymphoma, breast cancer, cervical cancer, clear cell renal cell carcinoma, dermatofibrosarcoma, gastric sarcoma, gastrointestinal stromal tumor, glioblastoma, leiomyosarcoma, invasive ductal breast carcinoma, malignant fibrous histiocytoma, melanoma, ovarian serous surface papillary carcinoma, prostate cancer, T-cell acute lymphoblastic leukemia, small cell lung cancer or T-cell lymphoma.

[0065] The present application also provides the use of the retinoblastoma protein mutant, nucleic acid molecule, vector or pharmaceutical composition of the present application as previously described in the preparation of a prophylactic and / or therapeutic drug for enhancing or increasing the binding of retinoblastoma protein mutant to E2F transcription factor for treating cancer or tumor.

[0066] In the use for preparing the drug, it includes using a therapeutically effective amount of the retinoblastoma protein mutant, nucleic acid molecule, vector or pharmaceutical composition.

[0067] The term "effective amount" means that amount of a drug or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought, for instance, by a researcher or clinician. Furthermore, the term "therapeutically effective amount" means that amount of an agent that, when administered to a subject in need thereof, will cure or at least partially arrest the disease, condition, or adverse effect being treated, or will provide some other tangible benefit to the subject. The term is also intended to encompass an amount effective to enhance normal physiological function. In general, the effective amount in the present application will vary according to factors such as the given pharmaceutical composition, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the subject being treated, and the like, but can nevertheless be routinely determined by one of skill in the art. An effective amount of a retinoblastoma protein mutant, nucleic acid molecule, vector, or pharmaceutical composition of the present application can be readily determined by one of skill in the art by routine methods known in the art. The terms "subject" or "patient" are used interchangeably to refer to any animal that can be treated with a retinoblastoma protein mutant, nucleic acid molecule, vector, or pharmaceutical composition or related formulation or medicament of the present application. Subjects or patients therefore include, but are not limited to, primates (including humans), canines, felines, murines, and other mammals. Preferably, the subject or patient is a human. The term "treatment" refers to both therapeutic treatment and prophylactic or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progress of cell malignant proliferation. Beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the condition or disorder as well as those in which the condition or disorder is to be prevented.

[0068] The term "E2F transcription factor", used herein, unless otherwise indicated, refers to any native E2F family transcription factor from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (e.g., mice and rats).

[0069] The present application also provides the retinoblastoma protein mutant, nucleic acid molecule, vector, or pharmaceutical composition of the present application as previously described in combination with other drugs. Preferably, the other drugs include diagnostic agents, prophylactic agents, and / or therapeutic agents.

[0070] Example 1

[0071] The embodiment provides a retinoblastoma protein mutant, 15 CDK kinase phosphorylation sites of retinoblastoma protein 1 (RB1) are mutated, wherein the 15 CDK kinase phosphorylation sites are phosphorylated by CDK1, CDK2, CDK3, CDK4, CDK5, CDK6 and CDK9 respectively, as shown in Table 1.

[0072] Table 1 15 CDK kinase phosphorylation site mutations

[0073]

[0074]

[0075] The RB1 protein coding sequence carrying a Flag tag is cloned into a pDONOR vector by means of whole gene synthesis (named as pDONOR-Flag-RB1); in order to avoid the RB1 protein from being modified by CDK kinase phosphorylation, 15 phosphorylation modification sites are all mutated into alanine (A) by means of site-directed mutagenesis, and the whole mutant RB1 gene is named as RBdel15; then the protein coding sequence of Flag-RBdel15 is cloned into a doxycycline (DOX) inducible expression vector plenti-DEST-Tre-3G by means of a Gateway LR system, and named as pDEST-Tre-Flag-RBdel15. (Note: doxycycline is a commonly used broad-spectrum antibiotic, which is a tetracycline analogue, and is commonly used as an inducer or inhibitor in biological experiments for tet-on or tet-off inducible expression or inhibition of target genes, and is widely used for tet-on or tet-off tet system inducible expression in mice).

[0076] Embodiment 2

[0077] The embodiment provides a host cell, pDEST-Tre-Flag-RBdel15 prepared in embodiment 1 and lentivirus packaging plasmids (psPAX2 and pMD2.G) are transfected into HEK293FT cells by means of Lipofectamine 2000, so as to prepare a lentivirus particle expressing a Flag-RBdel15 protein; then the virus particle is used for infecting a pancreatic cancer cell strain MIA PaCa-2, 3 positive clones C1, C2 and C6 cell strains are selected by means of single cell clone screening; dimethyl sulfoxide (DMSO, control group) and doxycycline (DOX, experimental group) are added respectively for treatment. Results are shown in Figure 2 .

[0078] From Figure 2The results show that the host cell prepared in the application has high expression of Flag-RBdel15 under the induction of DOX, and the expression effect of C2 clone is the best, and the expression of E2F target gene CCNA2 is significantly inhibited.

[0079] Example 3

[0080] The C2 cell strain selected in Example 2 and the phosphorylated RB1 protein (pRB1) are respectively added with DOX and DMSO with a final concentration of 1 μg / mL for induction, and Western blot detection is performed with different antibodies, and it is found that Flag-RBdel15 (anti-Flag antibody or anti-RB antibody) is significantly highly expressed, and the phosphorylated RB1 protein (pRB1) is significantly lowly expressed, and the expression of E2F downstream target gene Cyclin A is significantly inhibited Figure 3 A).

[0081] BrdU incorporation experiment is performed, and it is shown that RBdel15 high expression significantly inhibits cell proliferation Figure 3 B); flow cytometry detection is performed, and it is found that RBdel15 high expression cells are significantly arrested in G1 phase, and cells in S phase are significantly reduced Figure 3 C). This shows that RBdel15 has a very high cell cycle inhibition function.

[0082] High-throughput transcriptome sequencing (RNA-seq) is performed on the two groups of cells, and the results show that 1,193 differentially significant genes are found Figure 3 D); functional analysis (GO) shows that high expression genes are significantly enriched in type 1 interferon signaling pathway (GO:0060337, p=9.375E-10), gamma-interferon related signaling pathway (GO:0060333, p=2.449E-08) and antigen processing and presentation (GO:0002480, p=1.005E-04); and low expression genes are significantly enriched in sister chromatid separation (GO:0000070, p=7.533E-17), DNA metabolism (GO:0006259, p=6.747E-16) and mitotic cell cycle phase change (GO:0044772, p=9.911E-15) Figure 3 E).

[0083] Detection of E2F target gene expression revealed that some immune-related genes upregulated by RBdel15 included CCL28, OAS2, HLA-B, HLA-F, IFI6, ICM1, IFIM10, and MX2; while cell cycle-related genes downregulated included MYC, SNAI2, RRM2, MKI67, PTTG1, CDT1, PLK1, CDC7, and TK1. Figure 3 F); subsequently, some E2F target genes (CCNA2, STAT2, IRF9, CD74, and HLA-A) were selected for verification by quantitative real-time PCR. Figure 3 G).

[0084] Example 4

[0085] DOX and DMSO were added to the C2 cell line selected in Example 2 at a final concentration of 1 μg / mL for induction, and the expression of epigenetic-related genes was detected. The results are as follows: Figure 4 As shown.

[0086] Depend on Figure 4 The results showed that RNA-seq sequencing revealed that RBdel15 significantly suppressed the expression of epigenetic-related genes, such as DNA methyltransferase 1 (DNMT1), Zeste enhancer homolog 2 (EZH2), histone lysine methyltransferase SUV39H1 / 2, chromatin assembly factor 1A (CHAF1A), chromatin assembly factor 1B (CHAF1B), and DNA methyltransferase 3B (DNMT3B). Figure 4 A).

[0087] Quantitative real-time PCR and Western blot experiments revealed that RBdel15 almost completely inhibited the expression of the epigenetic genes DNMT1 and EZH1. Figure 4 B,4C). After DOX-induced RBdel15 expression, DNMT1 and EZH2 were persistently suppressed; while the expression of immune-related genes (such as CD74 and HLA-A) increased over time. Figure 4 D).

[0088] Adding the EZH2 inhibitor tazemetostat (TAZ) or the EZH2 siRNA (siEZH2) altered the activity or gene expression of EZH2, but did not significantly affect the function of RBdel15. Figure 4 E,4F).

[0089] 5-Aza-2'-deoxycytidine (5-Aza) is an inhibitor of DNMT1. After treating Miapaca2 cells with 0.25M 5-Aza for a long time (192h), the expression levels of DNMT1 and CCNA2 continuously decreased, while the expression levels of immune-related genes STAT2 and HLA-A significantly increased Figure 4 G,4H). This indicates that RBdel15 has an effect similar to 5-Aza on inhibiting the expression level of DNMT1.

[0090] Example 6

[0091] The Miapaca2 cell line carrying pDEST-Tre-Flag-RBdel15 selected in Example 2 was added with DMSO, DOX, 100nM Palbociclib+50nM Trametinib (P100T50) respectively for treatment, and the anti-tumor function of the cells was detected, and the results are shown in Figure 5 .

[0092] From Figure 5 It can be seen from the results that the cell proliferation speed of the RBdel15 high expression cell induced by DOX is extremely slow, and the level is comparable to the effect of the combination of CDK4 / 6 inhibitor (Palbociclib) and MEK1 / 2 inhibitor (Trametinib) Figure 5 A).

[0093] It was found that the volume of the RBdel15 high expression cell significantly increased by using phalloidin to show the filamentous actin (F-actin) in the cell Figure 5 B); the β-galactosidase activity of the RBdel15 high expression cell significantly increased, and the senescence was presented Figure 5 C).

[0094] The colony formation experiment showed that the RBdel15 high expression significantly inhibited the cell proliferation Figure 5 D). The above results show that the RBdel15 of the application can play an anti-tumor function from the aspects of inhibiting cell cycle, improving tumor immunity, promoting cell senescence and the like.

[0095] Example 7

[0096] To further study the relationship between RBdel15 and in vivo immunity and tumor microenvironment, C57Bl / 6 mouse KPC (Kras G12D / +p53 R172H / +Pdx-1-Cre) derived pancreatic ductal adenocarcinoma (PDAC) cell line 4662 was used as the research object. 4662 cells were infected with lentiviral particles carrying RBdel15 gene, and RBdel15 expression positive cell clones were selected by single cell cloning screening. The cells were treated with dimethyl sulfoxide (DMSO, control group) and doxycycline (DOX, experimental group), respectively, to construct a DOX-induced RBdel15-expressing mouse pancreatic cancer model 4662-RBdel15; the results are shown in Figure 6 .

[0097] From Figure 6 The results show that immunofluorescence examination and Western blot analysis with monoclonal antibody against mouse RB protein (Rb-4H1) found that DOX can significantly induce the expression of Rbdel15 protein Figure 6 (A, 6B), and the expression of E2F target genes such as Cyclin A, DNMT1 and EZH2 was significantly inhibited Figure 6 (B). RBdel15 high expression significantly inhibited the proliferation of 4662-RBdel15 cells Figure 6 (C, 6D); flow cytometry detection found that RBdel15 high expression cells significantly blocked the cells at G1 phase Figure 6 (E).

[0098] Example 8

[0099] 4662-RBdel15 cells were injected subcutaneously into C57 / BL6 mice, and when the tumor grew to about 150 mm 3 , the mice were randomly divided into two groups and fed with DMSO or Dox (2 mg / ml Dox + 1% Sucrose), respectively; the results are shown in Figure 7 .

[0100] From Figure 7 The results show that with the passage of time, it is found that the tumor of the DOX feeding group of mice has almost no change, while the tumor of the DMSO feeding group of mice has significantly increased Figure 7 (A).

[0101] Single-cell sequencing was performed on tumor tissues, and single-cell contour plots (UMAP) were plotted using ggplot2, and these cells were clustered into 6 cell groups: tumor cell group (tumors), T cell group (T cells), myeloid cell group (myeloids), cancer-associated fibroblast group (CAF), endothelial cell group (endothelial), and neutrophil group (neutrophil) Figure 7 B).

[0102] Among the significantly changed cell groups, the myeloid cells of the DOX-fed group mice were significantly reduced ( Figure 7 C), and the number of T cells was increased by about 4 times ( Figure 7 D), which indicated that the RBdel15 of the present application could significantly regulate the immune response of tumor cells.

[0103] At the same time, the RBdel15 could also significantly increase the number of CAFs with anti-tumor function ( Figure 7 E), induce tumor cell differentiation, and achieve inhibition of tumor growth by changing the immune microenvironment.

[0104] It should be noted that the above-described embodiments are only used to explain the present application and do not constitute any limitation on the present application. The present application is described by referring to typical embodiments, but it should be understood that the words used therein are descriptive and explanatory words, rather than limiting words. The present application can be modified as specified within the scope of the claims of the present application, and the present application can be revised without departing from the scope and spirit of the present application. Although the present application described therein relates to specific methods, materials and embodiments, it does not mean that the present application is limited to the specific examples disclosed therein, on the contrary, the present application can be extended to all other methods and applications with the same function.

Claims

1. A retinoblastoma protein mutant, characterized in that, The amino acid sequence of said mutant is shown in SEQ ID NO.

4.

2. An isolated nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the retinoblastoma protein mutant of claim 1.

3. A vector, characterized in that, comprising the nucleic acid molecule of claim 2.

4. A host cell, characterized in that, comprising a host cell transduced or transfected with the nucleic acid molecule of claim 2 or the vector of claim 3.

5. A pharmaceutical composition, characterized by, comprising the retinoblastoma protein mutant of claim 1, the nucleic acid molecule of claim 2 or the vector of claim 3.

6. A method for preparing the retinoblastoma protein mutant according to claim 1, characterized in that, comprising culturing the host cell of claim 4.

7. Use of the retinoblastoma protein mutant of claim 1 or the nucleic acid molecule of claim 2 or the vector of claim 3 or the pharmaceutical composition of claim 5 in the preparation of a medicament for the prevention and / or treatment of pancreatic cancer.

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