Composition associated with human cytomegalovirus tegument phosphoprotein 65, and preparation method therefor and use thereof
By mutating the pp65 peptide and combining it with an immune-enhancing sequence and the Fc fragment, a nucleic acid composition was prepared, which solved the safety and efficacy issues of pp65 protein in tumor treatment, achieving safe and efficient tumor immunotherapy and stimulating a strong immune response against a variety of tumors.
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2026-04-02
AI Technical Summary
In the prior art, pp65 protein has strong immunogenicity in tumor treatment, but its phosphorylation activity may affect viral replication efficiency and is associated with systemic lupus erythematosus, posing safety risks and making it difficult to develop safe and effective immunotherapy regimens.
By mutating the pp65 peptide to remove its phosphorylation activity and combining it with an immune-enhancing sequence and Fc fragment, a recombinant pp65 peptide is formed and prepared into a nucleic acid composition for tumor immunotherapy to stimulate an immune response against tumor cells.
It has achieved safe and effective tumor immunotherapy, stimulated a strong T-cell response, reduced the risk of systemic lupus erythematosus, and generated broad-spectrum anti-tumor immunity against various tumor types such as glioblastoma and colorectal cancer.
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Figure CN2025126091_02042026_PF_FP_ABST
Abstract
Description
Composition associated with human cytomegalovirus envelope phosphoprotein 65 and preparation method and application thereof TECHNICAL FIELD
[0001] The present application relates to the field of vaccine technology, in particular to a composition associated with human cytomegalovirus envelope phosphoprotein 65 and preparation method and application thereof. BACKGROUND
[0002] pp65 is a marker protein of HCMV and is one of the most studied proteins in human cytomegalovirus (HCMV). Although pp65 is not a protein essential for viral replication, it has strong immunogenicity. Currently, pp65 has been detected in various malignant tumors, such as glioblastoma, colorectal cancer, prostate cancer, breast cancer, rhabdomyosarcoma, neuroblastoma and hepatoblastoma, and the detection rate in different tumors is expected to open up a new immunotherapy approach for refractory tumors.
[0003] The pp65 protein contains strong T cell epitopes, which can provide strong T cell help for relatively weak tumor-associated antigens. When pp65 is co-presented with tumor antigens to the immune system, activated pp65-specific helper T cells can provide key costimulatory signals and cytokines, thereby amplifying and maintaining cytotoxic T cell responses against tumors. This process ultimately leads to "epitope spreading", i.e., the immune response expands from the initial pp65 and targeted tumor antigens to other multiple antigens against tumor cells, forming a more extensive and more persistent anti-tumor immunity. However, the pp65 protein has autophosphorylation activity, which can regulate the replication environment of the virus itself, facilitate virus assembly and release, and the K436N mutation can destroy the phosphokinase activity of the pp65 protein, destroy normal regulation, and weaken the efficiency of viral replication. In addition, the pp65 protein contains a sequence similar to TAF9, which may be related to human systemic lupus erythematosus, and mutating the related amino acid sites in this sequence can destroy the folding function of histones and eliminate potential risks.
[0004] The present application further analyzes various physiological activities of pp65 in detail, aiming to develop a safe and efficient composition for immunotherapy. SUMMARY
[0005] To achieve the above-mentioned purpose, the present inventors carried out a number of biological experiments on pp65 polypeptide, and through multiple experiments, the antigen modification method was determined. The mutated pp65 of the present application removes the phosphokinase activity, eliminates the potential risk of systemic lupus erythematosus, and can stimulate immune response in the body; a safe and effective tumor immunotherapy scheme is provided.
[0006] A nucleic acid encoding a mutant pp65 polypeptide comprising at least three of A429G, S430G, G434A, R435G and / or K436N mutations compared to a native pp65 polypeptide is provided.
[0007] Preferably, the mutant pp65 polypeptide comprises K436N, S430G, R435G mutations, and one of G434A or A429G compared to a native pp65 polypeptide.
[0008] Preferably, the mutant pp65 polypeptide is any one of SEQ NO. 9-SEQ NO. 11. The native pp65 polypeptide is SEQ NO. 11.
[0009] The present application also provides a polynucleotide comprising a nucleic acid encoding a recombinant pp65 polypeptide comprising a combination of a mutant pp65 polypeptide and an immunopotentiating sequence, the mutant pp65 polypeptide comprising at least three of A429G, S430G, G434A, R435G and / or K436N mutations compared to a native pp65 polypeptide. Optionally, the mutant pp65 polypeptide comprises K436N, S430G, R435G mutations, and one of G434A or A429G compared to a native pp65 polypeptide. Optionally, the mutant pp65 polypeptide is any one of SEQ NO. 9-SEQ NO. 10. The native pp65 polypeptide is SEQ NO. 30.
[0010] Further, the immunopotentiating sequence is one or more of LAMP, MITD, KDEL, Fc. The LAMP comprises a signal, a lumenal domain, and a Lamp transmembrane and cytoplasmic domain, and the native pp65 polypeptide is inserted between the lumenal domain and the Lamp transmembrane and cytoplasmic domain. The LAMP signal-lumenal domain sequence is SEQ NO. 12. The LAMP transmembrane and cytoplasmic domain sequence is SEQ NO. 13. The MITD comprises a signal and a MITD transmembrane and cytoplasmic domain, and the native pp65 polypeptide is inserted between the signal and the MITD transmembrane and cytoplasmic domain. The MITD-signal sequence is SEQ ID NO. 14; the sequence between the MITD transmembrane and cytoplasmic domain is SEQ ID NO. 15.
[0011] Specifically, Fc is an immunoglobulin Fc segment; preferably, the pp65 polypeptide is fused to Fc via a linker sequence; preferably, the linker sequence is selected from the group consisting of one or more of the following: a trimerization motif at the C-terminal of T4 fibritin SEQ NO. 16, a PADRE pan T-cell epitope SEQ NO. 17, a flexible sequence GGGGS, GSG, GGGGSGGGGSGGGGS, IL13Ra2-111~142aa SEQ NO. 18.
[0012] Preferably, the immunoglobulin Fc segment is a human wild-type IgG1 Fc SEQ NO. 37; preferably, the hinge region of the Fc is mutated from cysteine (C) to serine (S) near the N-terminus to avoid non-specific covalent binding; preferably, the IgG1 Fc comprises a mutation site that attenuates Fc-mediated ADCC, ADCP and / or CDC effects; preferably, the end of the immunoglobulin Fc segment is further linked to an IgM μtp tail segment short peptide SEQ NO. 41, and the proline (P) at the end of the Fc is mutated to threonine (T) to mimic the C-terminus of IgM, promoting the formation of hexamerization of the IgG Fc region, i.e. Fc6; preferably, the mutant sequence is selected from one of SEQ NO. 19, SEQ NO. 20, SEQ NO. 21; the N-terminus of the Fc fusion protein is additionally added with a secretion signal peptide, such as a tPA signal peptide SEQ NO. 22.
[0013] Specifically, the recombinant pp65 polypeptide is any one of the following table.
[0014] Specifically, the combination of the recombinant pp65 polypeptide and the immunostimulatory element is as follows
[0015] The nucleic acid is mRNA, viral RNA or replicon RNA.
[0016] The mRNA, viral RNA or replicon RNA is complexed with one or more lipids to form a liposome, a lipid nanoparticle and / or a lipid complex.
[0017] The present application provides a composition comprising a nucleic acid encoding a recombinant pp65 polypeptide, capable of eliciting an immune response to a tumor disease expressing pp65.
[0018] The composition of the present application encoding the above-mentioned recombinant pp65 polypeptide nucleic acid can be administered to a subject to elicit the production of effector T cells against tumor cells.
[0019] The tumor disease includes glioblastoma, colorectal cancer, prostate cancer, breast cancer, rhabdomyosarcoma, neuroblastoma and hepatoblastoma, etc.
[0020] The present application also provides an immune cell loaded with the above-mentioned nucleic acid encoding a recombinant pp65 polypeptide.
[0021] The local delivery of the nucleic acid encoding a recombinant pp65 polypeptide or the nucleic acid encoding a recombinant pp65 polypeptide fragment to the proximal site of a tissue, organ or graft according to the present application can use any available replicable and replication-defective vector, such as a plasmid vector, a retroviral vector, an adenoviral vector, an adeno-associated viral vector, a liposome, a lipid nanoparticle or other vector with appropriate tropism for cells that can be involved in apoptosis.
[0022] The present application also provides the use of a composition of a nucleic acid encoding a recombinant pp65 polypeptide for the preparation of a medicament for the treatment of cancer.
[0023] The composition of a nucleic acid encoding a recombinant pp65 polypeptide according to the present application comprises a pharmaceutically acceptable carrier and / or adjuvant.
[0024] One or more of the pharmaceutically acceptable adjuvants, buffers, protectants, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, inactivators.
[0025] The composition is prepared as a mucosal immunization preparation, a humoral immunization preparation, a cellular immunization preparation, a skin immunization preparation. The mucosal immunization preparation is a liquid dosage form, a solid dosage form, a semi-solid dosage form, a gaseous dosage form, an inhalation dosage form. The administration preparation is an intravenous injection, an intramuscular injection, a subcutaneous injection, an oral administration, an oral administration, a sublingual administration, a rectal administration, a respiratory tract administration, a transdermal administration; preferably, it is a respiratory tract administration preparation, an oral inhalation, a nasal inhalation or an inhalation after atomization by an atomization administration device.
[0026] The pharmaceutically acceptable carrier includes one or more of the following: mineral salt adjuvant, oil-in-water emulsion, saponin, virion and virus-like particle, immunostimulatory oligonucleotide, human immunomodulator, plasmid;
[0027] The mineral salt adjuvant is, for example, an aluminum salt, a calcium salt, a phosphate salt or a sulfate salt or a combination of different mineral salts; the preferred mineral salt adjuvant is aluminum phosphate; the oil-in-water emulsion includes but is not limited to: squalene-water emulsion, complete Freund's adjuvant or incomplete Freund's adjuvant; saponin can also be used as an adjuvant in the present application, saponin is a class of heterogenous sterol glycosides and triterpene glycosides, which exists in the bark, leaves, stems, roots and even flowers of various plants;
[0028] Virosome and virus-like particles can also be used as adjuvants of the present application, virosomes and virus-like particles typically comprise one or more proteins from a virus, optionally in combination or formulated with a phospholipid, suitable viral proteins for virosomes and virus-like particles include those derived from influenza virus (e.g. HA or NA), hepatitis B virus (e.g. core or capsid protein), hepatitis E virus, measles virus, Sindbis virus, rotavirus, foot and mouth disease virus, retroviruses, Norwalk virus, human papilloma virus, HIV, RNA-bacteriophages, Qβ-bacteriophage (e.g. coat protein), GA-bacteriophage, fr-bacteriophage, AP205 bacteriophage. Immunostimulatory oligonucleotides can also be used as adjuvants of the present application, immunostimulatory oligonucleotides include nucleotide sequences containing a CpG motif (a dinucleotide sequence containing an unmethylated cytosine linked by a phosphate bond to a guanosine), double-stranded RNA and oligonucleotides containing palindromic or poly(dG) sequences; human immunomodulators include cytokines, interleukins IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, TGF beta decoy receptor, interferons, macrophage colony-stimulating factor and tumor necrosis factor;
[0029] Adjuvants that can be used in the present application also include cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethyl cellulose, chitosan, microparticles, polyoxyethylene ether and polyoxyethylene ester formulations, imidazoquinolone compounds, muramyl peptides.
[0030] There is provided a mutant pp65 polypeptide comprising at least three of T34A, F27K, C57M, F59Y, E94T and / or T97M mutations compared to a native pp65 polypeptide.
[0031] Preferably, the mutant pp65 polypeptide comprises at least three of F27K, C57M, F59Y, E94T and / or T97M mutations compared to a native pp65 polypeptide.
[0032] Preferably, the mutant pp65 polypeptide comprises one of F27K, C57M or F59Y, one of E94T or T97M compared to a native pp65 polypeptide.
[0033] Preferably, the mutant pp65 polypeptide is any one of SEQ NO. 9-SEQ NO. 10. The native pp65 polypeptide is SEQ NO. 11.
[0034] The present application also provides a polypeptide, comprising a recombinant pp65 polypeptide, wherein the recombinant pp65 polypeptide comprises a combination of a mutant pp65 polypeptide and an immune-enhancing sequence, and the mutant pp65 polypeptide comprises at least three of F27K, C57M, F59Y, E94T and / or T97M mutations compared with a native pp65 polypeptide. Optionally, the mutant pp65 polypeptide comprises one of F27K, C57M or F59Y, one of E94T or T97M compared with the native pp65 polypeptide. Optionally, the mutant pp65 polypeptide is any one of SEQ ID NO. 1-4. The native pp65 polypeptide is SEQ ID NO. 11.
[0035] Further, the immune-enhancing sequence is one or more of LAMP, MITD, KDEL, Fc. The LAMP comprises a signal, a lumenal domain, and a Lamp transmembrane region and cytoplasmic region, and the native pp65 polypeptide is inserted between the lumenal domain and the Lamp transmembrane region and cytoplasmic region. The signal-lumenal domain sequence of the LAMP is SEQ ID NO. 12. The transmembrane region and cytoplasmic region sequence of the LAMP is SEQ ID NO. 13. The MITD comprises a signal and a MITD transmembrane region and cytoplasmic region, and the native pp65 polypeptide is inserted between the signal and the MITD transmembrane region and cytoplasmic region. The MITD-signal sequence is SEQ ID NO. 14; the sequence between the MITD transmembrane region and cytoplasmic region is SEQ ID NO. 15.
[0036] In particular, the Fc is an immunoglobulin Fc fragment; preferably, the pp65 polypeptide is fused to the Fc through a linker sequence; preferably, the linker sequence is selected from one or more of the following: a trimerization motif at the C-terminal end of T4 fibritin SEQ ID NO. 16, a PADRE pan T-cell epitope SEQ ID NO. 17, a flexible sequence GGGGS, GSG, GGGGSGGGGSGGGGS, IL13Ra2-111-142aa SEQ ID NO. 18.
[0037] Preferably, the Fc segment of the immunoglobulin is a human wild-type IgG1 Fc SEQ NO. 37; preferably, the hinge region of the Fc is mutated from cysteine (C) to serine (S) near the N-terminus to avoid non-specific covalent binding; preferably, the IgG1 Fc comprises a mutation site that attenuates Fc-mediated ADCC, ADCP and / or CDC effects; preferably, the Fc segment of the immunoglobulin is further connected to an IgM μtp tail segment short peptide SEQ NO. 41 at the end, and the proline (P) at the end of the Fc is mutated to threonine (T) to mimic the C-terminus of IgM, promoting the formation of a hexamer of the Fc region, i.e. Fc6; preferably, the mutant sequence is selected from one of SEQ NO. 19, SEQ NO. 20, SEQ NO. 21; the N-terminus of the Fc fusion protein is additionally added with a secretion signal peptide, such as tPA signal peptide SEQ NO. 22.
[0038] Specifically, the recombinant pp65 polypeptide is any one of the following Table 1.
[0039] Table 1. Recombinant pp65 polypeptide-immune stimulating combination
[0040] Specifically, the recombinant pp65 polypeptide is any one of the following Table 1.
[0041] The application provides a pharmaceutical composition comprising the recombinant pp65 polypeptide encoding the recombinant pp65 polypeptide of the application, which can induce an immune response to a tumor disease expressing pp65.
[0042] The composition of the application encoding the recombinant pp65 polypeptide can be administered to a subject to induce the production of effector T cells against tumor cells.
[0043] The tumor disease includes glioblastoma, colorectal cancer, prostate cancer, breast cancer, rhabdomyosarcoma, neuroblastoma and hepatoblastoma, etc.
[0044] The application also provides an immune cell loaded with the recombinant pp65 polypeptide encoding the recombinant pp65 polypeptide.
[0045] The recombinant pp65 polypeptide or the recombinant pp65 polypeptide fragment encoding the recombinant pp65 polypeptide of the application can be delivered locally to the proximal site of a tissue, organ or graft using any available replicable and replication-defective vector, such as liposome, lipid nanoparticle or polymer nanoparticle or cell-penetrating peptide.
[0046] The application also provides the use of the composition encoding the recombinant pp65 polypeptide in the preparation of a medicament for treating cancer.
[0047] The composition of the present application encoding recombinant pp65 polypeptide comprises one or more of pharmaceutically acceptable carriers and / or adjuvants.
[0048] one or more of pharmaceutically acceptable adjuvants, buffers, protectors, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, inactivators.
[0049] The composition is prepared as a mucosal immunization preparation, a humoral immunization preparation, a cellular immunization preparation, a skin immunization preparation. The mucosal immunization preparation is a liquid dosage form, a solid dosage form, a semi-solid dosage form, a gaseous dosage form, an inhalation dosage form. The administration preparation is intravenous injection, intramuscular injection, subcutaneous injection, oral administration, buccal administration, sublingual administration, rectal administration, respiratory tract administration, transdermal administration; preferably, it is a respiratory tract administration preparation, an oral inhalation, a nasal inhalation or an inhalation after atomization by an atomization administration device.
[0050] The pharmaceutically acceptable carrier comprises one or more of mineral salt adjuvants, oil-in-water emulsions, saponins, virosomes and virus-like particles, immunostimulatory oligonucleotides, human immunomodulators, plasmids;
[0051] The mineral salt adjuvant is, for example, an aluminum salt, a calcium salt, a phosphate salt or a sulfate salt or a combination of different mineral salts; the preferred mineral salt adjuvant is aluminum phosphate; the oil-in-water emulsion includes but is not limited to squalene-water emulsion, complete Freund's adjuvant or incomplete Freund's adjuvant; the saponin can also be used as an adjuvant of the present application, and the saponin is a kind of heterologous sterol glycoside and triterpene glycoside, which exists in the bark, leaves, stems, roots and even flowers of various plants;
[0052] The virosomes and virus-like particles can also be used as adjuvants of the present application, and the virosomes and virus-like particles usually comprise one or more proteins from viruses, optionally combined or formulated with phospholipids, the viral proteins suitable for virosomes and virus-like particles include those derived from influenza virus (such as HA or NA), hepatitis B virus (such as core or capsid protein), hepatitis E virus, measles virus, Sindbis virus, rotavirus, foot-and-mouth disease virus, retrovirus, Norwalk virus, human papillomavirus, HIV, RNA-phage, Qβ-phage (such as coat protein), GA-phage, fr-phage, AP205 phage. The immunostimulatory oligonucleotide can also be used as an adjuvant of the present application, and the immunostimulatory oligonucleotide includes nucleotide sequences containing CpG motifs (containing a dinucleotide sequence of unmethylated cytosine connected to guanosine by a phosphate bond), double-stranded RNA and oligonucleotides containing palindromic or poly(dG) sequences; human immunomodulators include cytokines, interleukins IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, TGFβ decoy receptor, interferons, macrophage colony-stimulating factor and tumor necrosis factor;
[0053] Adjuvants useful in the present application also include poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, cross-linked derivatives of polysaccharides and carboxymethyl cellulose, chitosan, microparticulates, polyoxyethylene ether and polyoxyethylene ester formulations, imidazoquinolones, muramyl peptides.
[0054] The nucleic acid of the present application is intended to refer to a polymeric form of nucleotides of any length. The polynucleotide can contain deoxyribonucleotides, ribonucleotides, and / or their analogs. The nucleotides can have any three-dimensional structure, and nucleic acids include, for example, single-stranded, double-stranded, and triple-stranded molecules, genes, or gene fragments, exons, introns, mRNA, tRNA, rRNA, ribozymes, antisense molecules, cDNA, recombinant polynucleotides, branched polynucleotides, aptamers, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence.
[0055] The "polypeptide" of the present application refers to a compound of two or more subunit amino acids, amino acid analogs, or peptidomimetics.
[0056] The "coding nucleic acid" is a nucleic acid sequence that is transcribed and translated into a polypeptide when placed under the control of appropriate expression control sequences.
[0057] The "vector" includes plasmids and viruses, whether or not self-replicating, which can be used to transform or transfect cells, and any DNA or RNA molecule. BRIEF DESCRIPTION OF DRAWINGS
[0058] Figure 1. pp65 and K436N mutant protein kinase activity assay
[0059] Figure 2. pp65 and its mutants induced systemic lupus erythematosus related antibody level assay
[0060] Figure 3. pp65 induced cellular immunity evaluation DETAILED DESCRIPTION
[0061] Unless otherwise defined, all scientific and technical terms used in the present application have the same meaning as commonly understood by one of ordinary skill in the art to which the present application pertains.
[0062] The technical solutions of the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0063] Example 1 Preparation of mRNA
[0064] Synthetic plasmid DNA sequence containing RNA transcription related elements and adding His or Flag tag for screening the expression of different antigen design. The plasmid is transformed into E. coli for amplification. The purified plasmid after fermentation is linearized by restriction endonuclease BspQ1. Transcription is carried out by T7 in vitro transcription kit, and capping is carried out during transcription to obtain capped mRNA. The transcription template is digested by DNase I, and the mRNA is purified by LiCl precipitation method. The purified mRNA is dissolved in acidic sodium citrate buffer, and the concentration and integrity of the mRNA are detected by spectrophotometer and capillary electrophoresis method, respectively. The obtained mRNA stock solution is stored at -80°C.
[0065] Table 3. Antigen mutation design
[0066] Example 2 mRNA stock solution encapsulation and LNP preparation
[0067] The cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid is dissolved and mixed in ethanol at a molar ratio of 45:10:43:2. The lipid mixture solution and mRNA stock solution are encapsulated by microfluidic method, and the encapsulated solution is diluted, ultrafiltrated and concentrated with 50 mM sodium acetate buffer containing 435 mg / ml sucrose to prepare mRNA-LNP, and the encapsulation rate, average particle size, PDI and zeta potential of mRNA-LNP are detected. The total flow rate of the nanomedicine manufacturing equipment is set to 12 ml / min. The mRNA solution and the lipid mixture solution are mixed at a flow rate ratio of 3:1.
[0068] Example 3 Detection of kinase activity of pp65 wild type and K436N mutant
[0069] pp65 protein and its mutants are expressed and extracted in HEK293T cells, then pp65 protein and dephosphorylated bovine casein (100 mg / 100 ml, sigma company) are added to 100 ul reaction system (25 mM Tris pH 8.5, 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 30 mM ATP) for 30 minutes at room temperature; after the phosphorylation reaction is completed, 20 ul of 100 mM EDTA is added to terminate the reaction; then the mixed protein solution is denatured and subjected to SDS-PAGE electrophoresis, and the phosphorylation level of the protein is detected using Thr phosphorylation antibody (2 ug / ml, sigma company) to evaluate the kinase activity of pp65 and its variants. The results show that the K436N mutation can effectively remove the protein kinase activity of pp65.
[0070] Example 4 Detection of systemic lupus erythematosus related antibody levels caused by pp65 and its mutants
[0071] The blood of the 6-8 week old female BALB / c mice after the first immunization was taken, and the systemic lupus erythematosus related immune response caused by the wild type and mutant pp65 was detected by Elisa experiment, that is, the antibody titer specific to ASTSAGR polypeptide in the serum of the mice was detected. The results show that the G434A-S430G-R435G and A429G-S430G-R435G mutations can completely remove the antibody production against TAF9, and have good potential safety.
[0072] Example 5 Evaluation of pp65 induced cellular immunity
[0073] The 6-8 week old C57BL / 6 female mice were randomly divided into 11 groups, 5 in each group, and each group was injected once in the hind leg muscle at 0 and 14 days. The blood was taken at 28 days after the first immunization, and pp65 protein was used for each antigen specific antibody detection; the mice were sacrificed, and PBMC cells were taken, and pp65 protein was used for antigen specific IFN-γ ELISPOT detection, respectively; the spleen cells were taken, and pp65 protein was used for antigen specific IFN-γ positive CD8+T cell reaction, respectively. The results show that G434A-S430G-R435G and A429G-S430G-R435G can induce better IFN-γ positive CD8+T cell reaction.
Claims
1. A polynucleotide comprising a nucleic acid encoding a mutated pp65 polypeptide, wherein, The mutant pp65 polypeptide comprises at least three of A429G, S430G, G434A, R435G and / or K436N mutations compared to the native pp65 polypeptide.
2. The polynucleotide of claim 1, wherein The mutant pp65 polypeptide comprises K436N, S430G, R435G mutations, and one of G434A or A429G compared to the native pp65 polypeptide.
3. A polynucleotide according to claim 1 or 2, wherein The mutant pp65 polypeptide is any one of SEQ NO. 9-SEQ NO.
10.
4. A polynucleotide comprising a nucleic acid encoding a recombinant pp65 polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 90% identity to SEQ ID NO:
1. The recombinant pp65 polypeptide comprises any one of the mutant pp65 polypeptide of claims 1-3 and an immunopotentiating sequence, which is one or more of LAMP, MITD, KDEL, Fc; the native pp65 polypeptide is inserted between the lumenal domain and the Lamp transmembrane region and cytoplasmic region; the MITD comprises signal and MITD transmembrane region and cytoplasmic region, and the native pp65 polypeptide is inserted between the signal and MITD transmembrane region and cytoplasmic region.
5. A polynucleotide according to any one of claims 1 to 3, wherein The nucleic acid is mRNA, viral RNA or replicon RNA.
6. A composition characterized in that, The composition comprises the polynucleotide of any one of claims 1-5, which is capable of eliciting an immune response against a tumor disease expressing pp65.
7. The composition of claim 6, wherein, The composition comprises a pharmaceutically acceptable carrier and / or excipient. The excipient comprises one or more of a buffer, a protective agent, a stabilizer, a surfactant, an osmotic pressure adjusting agent, an adjuvant, a preservative, an inactivator.
8. [Amended pursuant to Rule 26 21.10.2025] The composition according to any one of claims 6 or 7, characterized in that, The composition can be prepared as a mucosal immunization preparation, a humoral immunization preparation, a cellular immunization preparation, a skin immunization preparation. The mucosal immunization preparation is a liquid dosage form, a solid dosage form, a semi-solid dosage form, a gaseous dosage form, an inhalation dosage form. The administration preparation is intravenous injection, intramuscular injection, subcutaneous injection, oral administration, buccal administration, sublingual administration, rectal administration, respiratory tract administration, transdermal administration; preferably, it is a respiratory tract administration preparation, which is an inhalation preparation after oral inhalation, nasal inhalation or nebulization by a nebulization administration device.
9. Use of the composition of any one of claims 6-8 in the manufacture of a medicament for immunizing / treating a tumor.
10. Use of the polynucleotide of any one of claims 1-5 in the manufacture of a medicament for immunizing / treating a tumor.
11. A polypeptide comprising a polypeptide encoding a mutant pp65 polypeptide, characterized in that, The mutant pp65 polypeptide comprises at least three of A429G, S430G, G434A, R435G and / or K436N mutations compared to the native pp65 polypeptide.
12. The polypeptide of claim 11, wherein, The mutant pp65 polypeptide comprises K436N, S430G, R435G mutations, and one of G434A or A429G compared to the native pp65 polypeptide.
13. The polypeptide according to claim 11 or 12, characterized in that, The mutant pp65 polypeptide is any one of SEQ NO. 9-SEQ NO.
10. The recombinant pp65 polypeptide comprises any one of the mutant pp65 polypeptide of claims 1-3 and an immunopotentiating sequence, which is one or more of LAMP, MITD, KDEL, Fc; the native pp65 polypeptide is inserted between the lumenal domain and the Lamp transmembrane region and cytoplasmic region; the MITD comprises signal and MITD transmembrane region and cytoplasmic region, and the native pp65 polypeptide is inserted between the signal and MITD transmembrane region and cytoplasmic region. The nucleic acid is mRNA, viral RNA or replicon RNA. The composition comprises the polynucleotide of any one of claims 1-5, which is capable of eliciting an immune response against a tumor disease expressing pp65. The composition comprises a pharmaceutically acceptable carrier and / or excipient. The excipient comprises one or more of a buffer, a protective agent, a stabilizer, a surfactant, an osmotic pressure adjusting agent, an adjuvant, a preservative, an inactivator. The composition can be prepared as a mucosal immunization preparation, a humoral immunization preparation, a cellular immunization preparation, a skin immunization preparation. The mucosal immunization preparation is a liquid dosage form, a solid dosage form, a semi-solid dosage form, a gaseous dosage form, an inhalation dosage form. The administration preparation is intravenous injection, intramuscular injection, subcutaneous injection, oral administration, buccal administration, sublingual administration, rectal administration, respiratory tract administration, transdermal administration; preferably, it is a respiratory tract administration preparation, which is an inhalation preparation after oral inhalation, nasal inhalation or nebulization by a nebulization administration device.
9. Use of the composition of any one of claims 6-8 in the manufacture of a medicament for immunizing / treating a tumor.
10. Use of the polynucleotide of any one of claims 1-5 in the manufacture of a medicament for immunizing / treating a tumor.
14. A polypeptide comprising encoding a recombinant pp65 polypeptide, characterized in that, The recombinant pp65 polypeptide comprises a combination of the mutant pp65 polypeptide of any one of claims 11-13 and an immunopotentiating sequence, which is one or more of LAMP, MITD, KDEL, Fc; the native pp65 polypeptide is inserted between the lumenal domain and the transmembrane and cytoplasmic domains of the Lamp; the MITD comprises a signal and a transmembrane and cytoplasmic domain, and the native pp65 polypeptide is inserted between the signal and the transmembrane and cytoplasmic domain of the MITD.
15. A composition characterized in that, The composition comprises the polypeptide of any one of claims 10-13, and is capable of eliciting an immune response against a tumor disease expressing pp65.
16. The composition of claim 15, wherein, The composition comprises a pharmaceutically acceptable carrier and / or adjuvant. The adjuvant comprises one or more of a buffer, a protective agent, a stabilizer, a surfactant, an osmotic pressure adjusting agent, an adjuvant, a preservative, an inactivator.
17. The composition of any one of claims 15 or 16, wherein, The composition can be prepared as a mucosal immunization preparation, a humoral immunization preparation, a cellular immunization preparation, a cutaneous immunization preparation. The mucosal immunization preparation is a liquid dosage form, a solid dosage form, a semi-solid dosage form, a gaseous dosage form, an inhalation dosage form. The administration preparation is an intravenous injection, an intramuscular injection, a subcutaneous injection, an oral administration, a buccal administration, a sublingual administration, a rectal administration, a respiratory tract administration, a transdermal administration; preferably, it is a respiratory tract administration preparation, an oral inhalation, a nasal inhalation, or an inhalation after nebulization by a nebulization administration device.
18. Use of the composition of any one of claims 15-17 in the manufacture of a medicament for immunizing / treating a tumor.
19. Use of the polypeptide of any one of claims 11-14 in the manufacture of a medicament for immunizing / treating a tumor.
20. An immune cell, comprising: The polynucleotide or polypeptide of any one of claims 1-5, 11-14.