Composition associated with baculoviral inhibitor of apoptosis protein 5, and preparation method therefor and use thereof

Abstract

A composition comprising a nucleic acid encoding a human survivin polypeptide, which contains at least three mutations compared with a natural human survivin polypeptide. The composition is capable of inducing tumor-specific humoral or cellular immune responses.

Description

A composition associated with baculovirus apoptosis inhibitor 5 and its preparation method and application TECHNICAL FIELD

[0001] The present application relates to the field of vaccine technology, in particular to a composition associated with baculovirus apoptosis inhibitor 5 and its preparation method and application. BACKGROUND

[0002] Survivin belongs to the recently identified members of the inhibitor of apoptosis protein family. It is not detected in normal adult tissues, but is overexpressed in many types of cancer, including lung cancer, colon cancer, breast cancer, pancreatic cancer and prostate cancer, as well as hematopoietic malignancies, etc. Studies have also found that the expression of survivin is associated with poor prognosis. In further studies, it was found that survivin is not a single-function protein, it controls two vital life processes in cancer cells at the same time: inhibiting apoptosis and promoting cell classification. Inhibiting survivin can remove the blockade of apoptosis, reactivating the suicide program of cancer cells, thereby directly inducing cancer cell death; inhibiting survivin will disrupt the division process of cancer cells, leading to their division failure and death. In the study of using survivin as immunotherapy, it was found that injecting specific antigen fragments of survivin into patients can activate the patient's own T cells to recognize and attack tumor cells expressing survivin. Survivin has become a highly concerned tumor treatment target due to its "high expression in tumors, multifunctional, pro-survival" characteristics, and "low expression in normal tissues" safety.

[0003] The present application further analyzes various physiological activities of survivin in detail, aiming to develop a safe and efficient tumor vaccine. SUMMARY

[0004] To achieve the above-mentioned purpose, the present inventors carried out a number of biological experiments on human survivin polypeptide, and through multiple experiments, the antigen modification method was determined, and a nucleic acid encoding a mutant human survivin polypeptide was provided, wherein the mutant human survivin polypeptide comprises at least three of T34A, F27K, C57M, F59Y, E94T and / or T97M mutations compared to the natural human survivin polypeptide.

[0005] Preferably, the mutant human survivin polypeptide comprises at least three of F27K, C57M, F59Y, E94T and / or T97M mutations compared to the natural human survivin polypeptide.

[0006] Preferably, the mutant human survivin polypeptide comprises F27K, and at least one of C57M or F59Y, and at least one of E94T or T97M, as compared to the native human survivin polypeptide.

[0007] Preferably, the mutant human survivin polypeptide comprises peptide SEQ ID NO. 1, and at least one of peptide SEQ ID NO. 2 or SEQ ID NO. 3, and at least one of peptide SEQ ID NO. 4 or SEQ ID NO. 5; the copy number of any one of peptide SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4 or SEQ ID NO. 5 is 1 or 2; preferably, the mutant human survivin polypeptide is any one of SEQ ID NO. 26-SEQ ID NO. 35.

[0008] Preferably, the mutant human survivin polypeptide is any one of SEQ ID NO. 6-SEQ ID NO. 9 or SEQ ID NO. 26-SEQ ID NO. 35. The native human survivin polypeptide is SEQ ID NO. 25.

[0009] The present application also provides a polynucleotide comprising a nucleic acid encoding a recombinant human survivin polypeptide, the recombinant human survivin polypeptide comprising a combination of a mutant human survivin polypeptide and an immunopotentiating sequence, the mutant human survivin polypeptide comprising at least three of F27K, C57M, F59Y, E94T and / or T97M mutations, as compared to the native human survivin polypeptide. Optionally, the mutant human survivin polypeptide comprises F27K, and at least one of C57M or F59Y, and at least one of E94T or T97M, as compared to the native human survivin polypeptide.

[0010] Preferably, the mutant human survivin polypeptide comprises F27K, and at least one of C57M or F59Y, and at least one of E94T or T97M, as compared to the native human survivin polypeptide.

[0011] Further, the immune-enhancing sequence is one or more of LAMP, MITD, KDEL, Fc. The LAMP includes signal, lumenal domain, and Lamp transmembrane region and cytoplasmic region, and the natural human survivin 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 NO. 42. The transmembrane region and cytoplasmic region sequence of the LAMP is SEQ NO. 43. The MITD includes signal and MITD transmembrane region and cytoplasmic region, and the natural human survivin polypeptide is inserted between the signal and the MITD transmembrane region and cytoplasmic region. MITD-signal sequence SEQ ID NO. 44-SEQ ID NO. 46; sequence between MITD transmembrane region and cytoplasmic region SEQ ID NO. 47-SEQ ID NO. 49.

[0012] Specifically, the Fc is an immunoglobulin Fc segment; preferably, the survivin polypeptide is fused to the Fc through a linker sequence; preferably, the linker sequence is selected from the group consisting of one or more of the following: the trimerization motif at the C-terminal end of T4 fibritin SEQ NO. 50, the PADRE pan T-cell epitope SEQ NO. 51, the flexible sequences GGGGS, GSG, GGGGSGGGGSGGGGS, IL13Ra2-111~142aa SEQ NO. 52.

[0013] Preferably, the immunoglobulin Fc segment is a human wild-type IgG1 Fc SEQ NO. 53; 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 weakens the Fc-mediated ADCC, ADCP, and / or CDC effect; preferably, the end of the immunoglobulin Fc segment is further linked to an IgM μtp tail segment short peptide SEQ NO. 54, and the proline (P) at the end of the Fc is mutated to threonine (T) to mimic the C-terminal end 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. 55, SEQ NO. 56, SEQ NO. 57; the N-terminus of the Fc fusion protein is additionally added with a secretion signal peptide, such as a tPA signal peptide SEQ NO. 58.

[0014] Specifically, the recombinant human survivin polypeptide is any one of the following table.

[0015] Recombinant human survivin polypeptide-immune stimulating element combination

[0016] Specifically, the recombinant human survivin polypeptide is any one of the following table, the following table is only as an exemplary combination, those skilled in the art can understand that the various mutations of the human survivin polypeptide are suitable for combination with the immune stimulating element.

[0017] Table recombinant human survivin polypeptide (containing mutations) - immune stimulating element combination

[0018] The nucleic acid is a non-replicative linear mRNA, a self-replicating mRNA, or a circular RNA.

[0019] The non-replicative linear mRNA, the self-replicating mRNA, or the circular RNA is complexed with one or more lipids and / or polymers to form a liposome, a lipid nanoparticle, a lipoplex polyplex particle, and / or a lipid-polyplex complex.

[0020] The present application provides a composition comprising a nucleic acid encoding a recombinant human survivin polypeptide capable of eliciting an immune response specific to a native human survivin polypeptide.

[0021] The composition encoding the recombinant human survivin polypeptide / nucleic acid provided by the present application can express the recombinant human survivin polypeptide in vivo after being used by a subject, and generate effector T cells and specific antibodies having tumor cell killing effects.

[0022] The tumor includes melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, colon cancer, pancreatic cancer, prostate cancer, liver cancer, gastric cancer, brain tumor, etc.

[0023] The present application also provides an immune cell loaded with the nucleic acid encoding the recombinant human survivin polypeptide.

[0024] The nucleic acid encoding the recombinant human survivin polypeptide or the nucleic acid encoding the recombinant human survivin polypeptide fragment can be delivered locally to a proximal site of a tissue, an organ, or a graft using 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 vectors with appropriate tropism for cells that can be involved in apoptosis.

[0025] The present application also provides the use of the composition encoding the recombinant human survivin polypeptide / nucleic acid in the preparation of a medicament for treating cancer.

[0026] The present application is a composition of nucleic acid encoding recombinant human survivin polypeptide, comprising pharmaceutically acceptable carriers and / or adjuvants.

[0027] One or more of pharmaceutically acceptable adjuvants, buffers, protective agents, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, inactivators.

[0028] The composition is prepared as a mucosal immune preparation, a humoral immune preparation, a cellular immune preparation, a skin immune preparation. The mucosal immune 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, oral administration, sublingual administration, rectal administration, respiratory tract administration, transdermal administration; preferably, it is a respiratory tract administration preparation, an inhalation preparation after atomization by oral inhalation, nasal inhalation or atomization by a drug delivery device.

[0029] 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;

[0030] 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; saponins can also be used as adjuvants in the present application, saponins are a class of heterogenous sterol glycosides and triterpene glycosides, which exist in the bark, leaves, stems, roots and even flowers of various plants;

[0031] Virosomes and virus-like particles can also be used as adjuvants in the present application, virosomes and virus-like particles usually contain 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. Immunostimulatory oligonucleotides can also be used as adjuvants in the present application, immunostimulatory oligonucleotides include nucleotide sequences containing CpG motifs (containing a dinucleotide sequence of unmethylated cytosine linked 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;

[0032] Adjuvants useful in the present application also include poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, cross-linked derivatives of polysaccharides and carboxymethyl cellulose, chitosan, microparticles, polyoxyethylene ether and polyoxyethylene ester formulations, imidazoquinolones, muramyl peptides.

[0033] Provided is a mutant human survivin polypeptide comprising at least three of T34A, F27K, C57M, F59Y, E94T and / or T97M mutations compared to a native human survivin polypeptide.

[0034] Preferably, the mutant human survivin polypeptide comprises at least one of F27K and C57M or F59Y and at least one of E94T or T97M compared to a native human survivin polypeptide.

[0035] Preferably, the mutant human survivin polypeptide comprises peptide segment SEQ NO. 1 and one of peptide segment SEQ NO. 2 or SEQ NO. 3 and one of peptide segment SEQ NO. 4 or SEQ NO. 5; the copy number of any one of the peptide segment SEQ NO. 1, SEQ NO. 2, SEQ NO. 3, SEQ NO. 4 or SEQ NO. 5 is 1 or 2; preferably, the mutant human survivin polypeptide is any one of SEQ NO. 26-SEQ NO. 35.

[0036] Preferably, the mutant human survivin polypeptide is any one of SEQ NO. 6-SEQ NO. 9 or SEQ NO. 26-SEQ NO. 35. The native human survivin polypeptide is SEQ NO. 25.

[0037] The present application also provides a polypeptide comprising a recombinant human survivin polypeptide comprising a combination of a mutant human survivin polypeptide and an immune-enhancing sequence, the mutant human survivin polypeptide comprising at least three of F27K, and C57M, F59Y, E94T and / or T97M mutations compared to a native human survivin polypeptide. Alternatively, the mutant human survivin polypeptide comprises one of F27K, and C57M or F59Y, one of E94T or T97M compared to a native human survivin polypeptide. Alternatively, the mutant human survivin polypeptide is any one of SEQ NO. 6-SEQ NO. 9 or SEQ NO. 26-SEQ NO. 35. The native human survivin polypeptide is SEQ NO. 25.

[0038] Further, the immune-enhancing sequence is one or more of LAMP, MITD, KDEL, Fc. The LAMP includes signal, lumenal domain, and Lamp transmembrane region and cytoplasmic region, and the natural human survivin 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 NO. 42. The transmembrane region and cytoplasmic region sequence of the LAMP is SEQ NO. 43. The MITD includes signal and MITD transmembrane region and cytoplasmic region, and the natural human survivin polypeptide is inserted between the signal and the MITD transmembrane region and cytoplasmic region. MITD-signal sequence SEQ ID NO. 44-SEQ ID NO. 46; sequence between MITD transmembrane region and cytoplasmic region SEQ ID NO. 47-SEQ ID NO. 49.

[0039] Specifically, the Fc is an immunoglobulin Fc segment; preferably, the survivin polypeptide is fused to the Fc through a linker sequence; preferably, the linker sequence is selected from the group consisting of one or more of the following: the trimerization motif at the C-terminal end of T4 fibrous protein SEQ NO. 50, PADRE pan T cell epitope SEQ NO. 51, flexible sequences GGGGS, GSG, GGGGSGGGGSGGGGS, IL13Ra2-111~142aa SEQ NO. 52.

[0040] Preferably, the immunoglobulin Fc segment is a human wild-type IgG1 Fc SEQ NO. 53; preferably, the hinge region of the Fc is mutated from cysteine (C) near the N-terminus to serine (S) to avoid non-specific covalent binding; preferably, the IgG1 Fc contains a mutation site that weakens the Fc-mediated ADCC, ADCP, and / or CDC effect; preferably, the end of the immunoglobulin Fc segment is further connected to an IgM μtp tail segment short peptide SEQ NO. 54, and the proline (P) at the end of the Fc is mutated to threonine (T) to mimic the C-terminal end 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. 55, SEQ NO. 56, SEQ NO. 57; the N-terminus of the Fc fusion protein is additionally added with a secretion signal peptide, such as tPA signal peptide SEQ NO. 58.

[0041] Specifically, the recombinant human survivin polypeptide is any one of the following table.

[0042] Recombinant human survivin polypeptide-immune stimulating combination

[0043] Specifically, the recombinant human survivin polypeptide is any one of Table 2 below. Table 2 below is only an exemplary combination, and those skilled in the art can understand that various mutations of the human survivin polypeptide are suitable for combination with the immunostimulatory element.

[0044] The present application provides a pharmaceutical composition comprising the recombinant human survivin polypeptide encoding the recombinant human survivin polypeptide, which can induce an immune response to a tumor disease expressing survivin.

[0045] The nucleic acid composition encoding the recombinant human survivin polypeptide provided by the present application can express the recombinant human survivin polypeptide in vivo after being used by a subject, and produce effector T cells and specific antibodies with tumor cell killing effect.

[0046] The tumor includes melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, colon cancer, pancreatic cancer, prostate cancer, liver cancer, gastric cancer, brain tumor, etc.

[0047] The present application also provides an immune cell loaded with the recombinant human survivin polypeptide.

[0048] The recombinant human survivin polypeptide or the recombinant human survivin polypeptide fragment encoding the recombinant human survivin polypeptide can be delivered locally to the proximal site of the tissue, organ or graft using any available replicable and replication-defective vector, such as liposome, lipid nanoparticle, polymer nanoparticle or cell penetrating peptide.

[0049] The present application also provides the use of the recombinant human survivin polypeptide encoding composition in the preparation of a medicament for treating cancer.

[0050] The recombinant human survivin polypeptide encoding composition of the present application comprises a pharmaceutically acceptable carrier and / or adjuvant.

[0051] One or more of the pharmaceutically acceptable adjuvants, buffers, protectors, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, inactivators.

[0052] The composition is prepared as a mucosal immune preparation, a humoral immune preparation, a cellular immune preparation, a skin immune preparation. The mucosal immune 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, oral 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.

[0053] The pharmaceutically acceptable carrier includes one or more of mineral salt adjuvants, oil-in-water emulsions, saponins, virosomes and virus-like particles, immunostimulatory oligonucleotides, human immunomodulators, plasmids;

[0054] 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; saponins can also be used as adjuvants in the present application, saponins are a class of heterogenous sterol glycosides and triterpene glycosides, which exist in the bark, leaves, stems, roots and even flowers of many plants;

[0055] Virosomes and virus-like particles can also be used as adjuvants in the present application, virosomes and virus-like particles usually contain 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. Immunostimulatory oligonucleotides can also be used as adjuvants in the present application, immunostimulatory oligonucleotides include nucleotide sequences containing CpG motifs (containing a dinucleotide sequence of unmethylated cytosine linked 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;

[0056] The adjuvants that can be used in the present application also include cross-linked derivatives of poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, polysaccharides and carboxymethylcellulose, chitosan, microparticles, polyoxyethylene ether and polyoxyethylene ester formulations, imidazoquinolone compounds, muramyl peptides.

[0057] The nucleic acid of the present application refers to the polymeric form of nucleotides of any length. The polynucleotide can contain deoxyribonucleotides, ribonucleotides, and / or their analogs. A nucleotide 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.

[0058] The polypeptide of the present application refers to a compound of two or more subunits of amino acids, amino acid analogs or peptidomimetics.

[0059] 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.

[0060] 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

[0061] Figure 1. IFN-γ positive CD8+ T cell responses induced by different mutant designs of survivin

[0062] Figure 2. IFN-γ positive CD8+ T cell responses induced by different ROP designs of survivin DETAILED DESCRIPTION

[0063] 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.

[0064] The technical solutions of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, but not 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.

[0065] Example 1 Screening of mutant site combination

[0066] Preparation of mRNA: The plasmid DNA sequence was artificially synthesized, and the DNA sequence contained RNA transcription-related elements. The plasmid was transformed into E. coli for amplification. The purified plasmid after fermentation was linearized by restriction endonuclease BspQl. Transcription was performed using a T7 in vitro transcription kit, and capping was performed during transcription to obtain capped mRNA. The transcription template was digested with DNase I, and the mRNA was purified by LiCl precipitation method. The purified mRNA was dissolved in an acidic sodium citrate buffer, and the concentration and integrity of the mRNA were detected by spectrophotometer and capillary electrophoresis, respectively. The obtained mRNA stock solution was stored at -80°C.

[0067] Table 1. Antigen mutation design

[0068] Encapsulation of mRNA stock solution and preparation of LNP: The cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid was dissolved and mixed in ethanol at a molar ratio of 50:10:48.5:1.5. The lipid mixture solution and mRNA stock solution were encapsulated by microfluidic method, and the encapsulated solution was diluted, ultrafiltrated and concentrated with 50 mM sodium acetate buffer containing 435 mg / ml sucrose to prepare mRNA-LNP. The encapsulation efficiency, average particle size, PDI and zeta potential of mRNA-LNP were detected. The total flow rate of the nanomedicine manufacturing equipment was set to 12 ml / min. The mRNA solution and the lipid mixture solution were mixed at a flow rate ratio of 3:1.

[0069] Immunogenicity evaluation: 105 SPF female C57BL / 6 mice aged 6-8 weeks were randomly divided into 21 groups, 5 mice in each group. The immunization was performed by intramuscular injection, and the mice were immunized on days 0, 14 and 28. The mice were sacrificed on day 42, and the splenocytes were detected by ICS to detect the IFN-γ positive CD8+ T cell response. The results showed that SEQ NO. 6, SEQ NO. 7, SEQ NO. 8 and SEQ NO. 9 could induce higher specific IFN-γ positive CD8+ T cell response in all group designs.

[0070] Example 2 Screening of overlapping mutation site combination

[0071] Preparation of mRNA: The plasmid DNA sequence was artificially synthesized, and the DNA sequence contained RNA transcription-related elements. The plasmid was transformed into E. coli for amplification. The purified plasmid after fermentation was linearized by restriction endonuclease BspQl. Transcription was performed using a T7 in vitro transcription kit, and capping was performed during transcription to obtain capped mRNA. The transcription template was digested with DNase I, and the mRNA was purified by LiCl precipitation method. The purified mRNA was dissolved in an acidic sodium citrate buffer, and the concentration and integrity of the mRNA were detected by spectrophotometer and capillary electrophoresis, respectively. The obtained mRNA stock solution was stored at -80°C.

[0072] Table 3. Overlapping antigen mutation design

[0073] Encapsulation of mRNA stock solution and preparation of LNP: The cationic lipid: neutral phospholipid: steroidal lipid: polyethylene glycol (PEG)-lipid were dissolved and mixed in ethanol at a molar ratio of 50:10:48.5:1.5. The lipid mixture solution and mRNA stock solution were encapsulated by microfluidic method, and the encapsulated solution was diluted, ultrafiltrated and concentrated with 50 mM sodium acetate buffer containing 435 mg / ml sucrose to prepare mRNA-LNP. The encapsulation efficiency, average particle size, PDI and zeta potential of mRNA-LNP were detected. The total flow rate of the nanomedicine manufacturing equipment was set to 12 ml / min. The mRNA solution and the lipid mixture solution were mixed at a flow rate ratio of 3:1.

[0074] Immunogenicity evaluation: 90 SPF female C57BL / 6 mice aged 6-8 weeks were randomly divided into 18 groups, 5 mice in each group. The immunization was performed by intramuscular injection, and the mice were immunized on days 0, 14 and 28. The mice were sacrificed on day 42, and the splenocytes were detected by ICS to detect the IFN-γ positive CD8+ T cell response. The results showed that the groups containing at least one of F27K and C57M, F59Y mutations and one of E94T and T97M could induce higher IFN-γ positive CD8+ T cell response.

Claims

1. A polynucleotide comprising a nucleic acid encoding a mutant human survivin polypeptide, characterized in that, The mutant human survivin polypeptide comprises at least three of F27K, C57M, F59Y, E94T and / or T97M mutations compared to the native human survivin polypeptide.

2. The polynucleotide of claim 1, wherein The mutant human survivin polypeptide comprises one of F27K and C57M or F59Y, and one of E94T or T97M compared to the native human survivin polypeptide. Preferably, the mutant human survivin polypeptide is any one of SEQ NO. 6-SEQ NO.

9.

3. A polynucleotide according to claim 1 or 2, wherein The mutant human survivin polypeptide comprises peptide segment SEQ NO. 1, and one of peptide segment SEQ NO. 2 or SEQ NO. 3, and one of peptide segment SEQ NO. 4 or SEQ NO. 5; the copy number of any one of the peptide segment SEQ NO. 1, SEQ NO. 2, SEQ NO. 3, SEQ NO. 4 or SEQ NO. 5 is 1 or 2; preferably, the mutant human survivin polypeptide is any one of SEQ NO. 26-SEQ NO.

35.

4. A polynucleotide comprising a nucleic acid encoding a recombinant human survivin polypeptide, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:

2. The recombinant human survivin polypeptide comprises any one of the mutant human survivin polypeptide of claims 1-3 and an immunopotentiating sequence, which is one or more of LAMP, MITD, KDEL, Fc; the native human survivin polypeptide is inserted between the lumenal domain and the Lamp transmembrane and cytoplasmic domains; the MITD comprises a signal and MITD transmembrane and cytoplasmic domains, and the native human survivin polypeptide is inserted between the signal and MITD transmembrane and cytoplasmic domains.

5. A polynucleotide according to any one of claims 1 to 3, wherein The nucleic acid is a non-replicative linear mRNA, a self-replicating mRNA, or a circular 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 specific to the native human survivin polypeptide.

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 delivery vehicle, a buffer, a protective agent, a stabilizer, a surfactant, an osmotic pressure regulator, 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 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, 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 preventing / treating a tumor.

10. Use of the polynucleotide of any one of claims 1-5 in the manufacture of a medicament for preventing / treating a tumor.

11. A polypeptide comprising a mutant human survivin polypeptide, wherein the mutant human survivin polypeptide comprises the amino acid sequence of SEQ ID NO:

2. The mutant human survivin polypeptide comprises at least three of F27K, C57M, F59Y, E94T and / or T97M mutations compared to the native human survivin polypeptide.

12. The polypeptide of claim 11, wherein The mutant human survivin polypeptide comprises one of F27K and C57M or F59Y, and one of E94T or T97M compared to the native human survivin polypeptide. Preferably, the mutant human survivin polypeptide is any one of SEQ NO. 6-SEQ NO.

9.

13. The polypeptide according to claim 11 or 12, characterized in that, The mutant human survivin polypeptide comprises peptide segment SEQ NO. 1, and one of peptide segment SEQ NO. 2 or SEQ NO. 3, and one of peptide segment SEQ NO. 4 or SEQ NO. 5; the copy number of any one of the peptide segment SEQ NO. 1, SEQ NO. 2, SEQ NO. 3, SEQ NO. 4 or SEQ NO. 5 is 1 or 2; preferably, the mutant human survivin polypeptide is any one of SEQ NO. 26-SEQ NO.

35.

14. A polypeptide comprising a polypeptide encoding a recombinant human survivin polypeptide, characterized in that, The recombinant human survivin polypeptide comprises the combination of any one of the mutant human survivin polypeptides of claims 11-13 and an immunopotentiating sequence, which is one or more of LAMP, MITD, KDEL, Fc; the native human survivin polypeptide is inserted between the lumenal domain and the Lamp transmembrane and cytoplasmic domains; the MITD comprises a signal and MITD transmembrane and cytoplasmic domains, and the native human survivin polypeptide is inserted between the signal and MITD transmembrane and cytoplasmic domains.

15. A composition characterized in that, The composition comprises the polypeptide of any one of claims 10-13, which is capable of inducing an immune response specific to the native human survivin polypeptide.

16. The composition of claim 15, 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 regulator, 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, which is an inhalation preparation after oral inhalation, nasal inhalation or nebulization by a nebulization administration device.

18. Use of the composition of any one of claims 15-17 in the preparation of a medicament for immunizing / treating a tumor.

19. Use of the polypeptide of any one of claims 11-14 in the preparation 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 is loaded. The polynucleotide or polypeptide of any one of claims 1-5, 11-14 is loaded.

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