Composition associated with human cytomegalovirus envelope phosphoprotein 65 as well as preparation method and application thereof
By mutating the pp65 peptide and binding it with an immune-enhancing sequence, a recombinant pp65 peptide was formed and delivered locally, solving the safety and efficacy issues of pp65 protein in tumor immunotherapy in existing technologies and achieving a safe and efficient tumor immune response.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-30
- Publication Date
- 2026-03-31
AI Technical Summary
Existing technologies make it difficult to develop a safe and efficient pp65 protein for tumor immunotherapy, and there is a risk that phosphorylation activity may affect viral replication and potentially cause systemic lupus erythematosus.
By mutating the pp65 peptide to remove its phosphokinase activity and combining it with an immune-enhancing sequence, a recombinant pp65 peptide is formed, which encodes a nucleic acid and combines with lipids to form liposomes or lipid nanoparticles, which are then locally delivered to the tumor site to stimulate an immune response.
It has achieved safe and effective tumor immunotherapy, stimulated effector T cell responses against tumor cells, reduced the risk of viral replication, and reduced the potential danger of systemic lupus erythematosus.
Smart Images
Figure CN121759480A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of vaccine technology, and more specifically to a composition associated with human cytomegalovirus envelope phosphoprotein 65, its preparation method, and its application. Background Technology
[0002] pp65 is a hallmark protein of HCMV and one of the most studied proteins in human cytomegalovirus (HCMV). Although pp65 is not essential for viral replication, it possesses strong immunogenicity. Currently, pp65 has been detected in various malignant tumors, such as glioblastoma, colorectal cancer, prostate cancer, breast cancer, rhabdomyosarcoma, neuroblastoma, and hepatoblastoma. Its high detection rate in different tumors holds promise for opening up new immunotherapy avenues for treating refractory tumors.
[0003] The pp65 protein contains a potent T-cell epitope, providing strong T-cell helper antibodies against relatively weak tumor-associated antigens. When pp65 is co-presented to the immune system along with tumor antigens, activated pp65-specific helper T cells provide crucial co-stimulatory signals and cytokines, amplifying and maintaining the cytotoxic T-cell response against tumors. This process ultimately leads to "epitope diffusion," where the immune response expands from the initial pp65 and targeted tumor antigens to multiple other antigens targeting tumor cells, resulting in broader and more durable anti-tumor immunity. However, the pp65 protein possesses autophosphorylation activity, which regulates the viral replication environment, facilitating viral assembly and release. The K436N mutation disrupts the phosphokinase activity of the pp65 protein, impairing normal regulation and weakening viral replication efficiency. Furthermore, the pp65 protein contains a sequence similar to TAF9, which may be associated with systemic lupus erythematosus (SLE). Mutating the relevant amino acid sites in this sequence can disrupt histone folding function, eliminating potential risks.
[0004] This invention further analyzes in detail the various physiological activities of pp65, with the aim of developing a safe and effective composition for immunotherapy. Summary of the Invention
[0005] To achieve the above objectives, the inventors conducted multiple biological experiments on the pp65 peptide. Through these experiments, they clarified the method for modifying the antigen. The mutated pp65 of this invention removes phosphokinase activity, eliminates the potential danger of systemic lupus erythematosus, and can stimulate an immune response in the body; thus providing a safe and effective tumor immunotherapy regimen.
[0006] A nucleic acid encoding a mutant pp65 polypeptide is provided, wherein the mutant pp65 polypeptide comprises at least three of the mutations A429G, S430G, G434A, R435G and / or K436N compared to the natural pp65 polypeptide.
[0007] Preferably, the mutant pp65 peptide includes K436N, S430G, R435G mutations, and one of G434A or A429G, compared to the natural pp65 peptide.
[0008] Preferably, the mutant pp65 polypeptide is any one of SEQ NO. 9-SEQ NO. 11. The natural pp65 polypeptide is SEQ NO. 11.
[0009] The present invention also provides a polynucleotide comprising a nucleic acid encoding 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 the mutations A429G, S430G, G434A, R435G, and / or K436N compared to the natural pp65 polypeptide. Optionally, the mutant pp65 polypeptide comprises the mutations K436N, S430G, and R435G, and one of G434A or A429G compared to the natural pp65 polypeptide. Optionally, the mutant pp65 polypeptide is any one of SEQ NO. 9-SEQ NO. 10. The natural pp65 polypeptide is SEQ NO. 30.
[0010] Further, the immune-enhancing sequence is one or more of LAMP, MITD, KDEL, and Fc. The LAMP includes a signal, a lumenal domain, a transmembrane region, and a cytoplasmic region, with the natural pp65 peptide inserted between the lumenal domain and the transmembrane and cytoplasmic regions. The signal-lumenal domain sequence of the LAMP is SEQ NO. 12. The transmembrane and cytoplasmic region sequences of the LAMP are SEQ NO. 13. The MITD includes a signal, a MITD transmembrane region, and a cytoplasmic region, with the natural pp65 peptide inserted between the signal and the MITD transmembrane and cytoplasmic regions. The MITD-signal sequence is SEQ ID NO. 14; the sequence between the MITD transmembrane and cytoplasmic regions is SEQ ID NO. 15.
[0011] Specifically, Fc is the Fc segment of immunoglobulin; preferably, the pp65 polypeptide is fused to Fc via a linker sequence; preferably, the linker sequence is selected from one or more combinations of: the trimer motif at the C-terminus of T4 fibrin SEQ NO.16, the PADRE pan-T cell epitope SEQ NO.17, the flexible sequence GGGGS, GSG, GGGGSGGGGSGGGGS, and IL13Ra2-111~142aa SEQ NO.18.
[0012] Preferably, the immunoglobulin Fc segment is human wild-type IgG1 Fc SEQ NO.37; preferably, the cysteine (C) near the N-terminus of the hinge region of the Fc is mutated to serine (S) to avoid non-specific covalent binding; preferably, the IgG1 Fc contains mutation sites that weaken the Fc-mediated ADCC, ADCP, and / or CDC effects; preferably, the end of the immunoglobulin Fc segment is also connected to a short IgM μtp tail peptide SEQ NO.41, and the proline (P) at the Fc end is mutated to threonine (T) to mimic the C-terminus of IgM and promote the hexamerization of the IgG Fc region, i.e., Fc6; preferably, the mutant sequence is selected from one of SEQ NO.19, SEQ NO.20, and SEQ NO.21; the N-terminus of the Fc fusion protein is additionally supplemented with a secretion signal peptide, such as the tPA signal peptide SEQ NO.22.
[0013] Specifically, the recombinant pp65 polypeptide is any one of the following in the table.
[0014] 1 LAMP-pp65(G434A-S430G-R435G-K436N) 2 LAMP-pp65(A429G-S430G-R435G-K436N) 5 MITD-pp65(G434A-S430G-R435G-K436N) 6 MITD-pp65(A429G-S430G-R435G-K436N) 9 pp65(G434A-S430G-R435G-K436N)-KDEL 10 pp65(A429G-S430G-R435G-K436N)-KDEL 13 pp65(G434A-S430G-R435G-K436N)-Fc 14 pp65(A429G-S430G-R435G-K436N)-Fc 17 TPA-pp65(G434A-S430G-R435G-K436N)-IL13Ra2-111~142aa-PADRE-Fc6 18 TPA-pp65(A429G-S430G-R435G-K436N)-IL13Ra2-111~142aa-PADRE-Fc6
[0015] Specifically, the combination of the recombinant pp65 peptide and the immunostimulatory element is as follows:
[0016] serial number Specific combination schemes 1 LAMP-pp65 2 MITD-pp65 3 pp65-KDEL 4 pp65-Fc
[0017] The nucleic acid is mRNA, viral RNA, or replicon RNA.
[0018] The mRNA, viral RNA, or replicon RNA is complexed with one or more lipids to form liposomes, lipid nanoparticles, and / or lipid complexes.
[0019] The invention provides a composition comprising a nucleic acid encoding a recombinant pp65 polypeptide capable of triggering an immune response in tumor diseases expressing pp65.
[0020] The present invention provides a composition encoding the above-mentioned recombinant pp65 polypeptide nucleic acid, which can be administered to a subject to induce the generation of effector T cells targeting tumor cells.
[0021] The tumor diseases mentioned include glioblastoma, colorectal cancer, prostate cancer, breast cancer, rhabdomyosarcoma, neuroblastoma, and hepatoblastoma.
[0022] The present invention also provides an immune cell loaded with the nucleic acid encoding the recombinant pp65 polypeptide.
[0023] Local delivery of the nucleic acid encoding the recombinant pp65 polypeptide or the nucleic acid encoding the recombinant pp65 polypeptide fragment described in this invention to the proximal site of a tissue, organ, or graft can be performed using any available reproducible and defective vectors, such as plasmid vectors, retroviral vectors, adenovirus vectors, adeno-associated virus vectors, liposomes, lipid nanoparticles, or other vectors that have appropriate tropism for cells that may be involved in apoptosis.
[0024] The present invention also provides the use of compositions encoding recombinant pp65 polypeptide nucleic acids in the preparation of medicaments for treating cancer.
[0025] The present invention relates to a composition encoding a recombinant pp65 polypeptide nucleic acid, comprising a pharmaceutically acceptable carrier and / or excipients.
[0026] Pharmaceutically acceptable excipients, buffers, protectants, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivators, or one or more of these.
[0027] The composition is prepared as a mucosal immunomodulator, humoral immunomodulator, cellular immunomodulator, or cutaneous immunomodulator. The mucosal immunomodulator is available in liquid, solid, semi-solid, gaseous, or inhaled formulations. The administration formulation is available as an intravenous injection, intramuscular injection, subcutaneous injection, oral administration, oral administration, sublingual administration, rectal administration, respiratory administration, or transdermal administration; preferably, it is a respiratory administration formulation, which is inhaled orally, nasally, or nebulized by a nebulizer.
[0028] Pharmaceutically acceptable carriers include one or more of the following: mineral salt adjuvants, oil-in-water emulsions, saponins, virions and virus-like particles, immunostimulatory oligonucleotides, human immunomodulators, and plasmids;
[0029] Mineral salt adjuvants include aluminum salts, calcium salts, phosphates, or sulfates, or combinations of different mineral salts; the preferred mineral salt adjuvant is aluminum phosphate; oil-in-water emulsions include, but are not limited to: squalene-water emulsions, complete Freund's adjuvants, or incomplete Freund's adjuvants; saponins can also be used as adjuvants in this invention. Saponins are a class of isosterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots, and even flowers of various plants;
[0030] Virisomes and virus-like particles can also be used as adjuvants in this invention. Virisomes and virus-like particles typically contain one or more proteins derived from viruses, optionally combined or formulated with phospholipids. Viral proteins suitable for virisomes and virus-like particles include those derived from influenza viruses (such as HA or NA), hepatitis B viruses (such as core or capsid proteins), hepatitis E viruses, measles viruses, Sindbis viruses, rotaviruses, foot and oral disease viruses, retroviruses, noroviruses, human papillomaviruses, HIV, RNA phages, Qβ phages (such as capsid proteins), GA phages, fr phages, and AP205 phages. Immunostimulatory oligonucleotides can also be used as adjuvants in this invention. Immunostimulatory oligonucleotides include nucleotide sequences containing CpG motifs (dinucleotide sequences containing unmethylated cytosine linked to guanosine via phosphate bonds), double-stranded RNA containing palindromic or multiple (dG) sequences, and oligonucleotides. Human immunomodulators include cytokines, interleukins IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, TGFβ decoy receptors, interferons, macrophage colony-stimulating factor, and tumor necrosis factor.
[0031] The adjuvants that can be used in this invention also include poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, cross-linked derivatives of polysaccharides and carboxymethyl cellulose, chitosan, microparticles, polyoxyethylene ethers and polyoxyethylene ester formulations, imidazole quinolone compounds, and muramyl peptides.
[0032] A mutant pp65 polypeptide is provided, wherein the mutant pp65 polypeptide comprises at least three of the mutations T34A, F27K, C57M, F59Y, E94T and / or T97M compared to the natural pp65 polypeptide.
[0033] Preferably, the mutant pp65 polypeptide contains at least three of the following mutations compared to the natural pp65 polypeptide: F27K, C57M, F59Y, E94T, and / or T97M.
[0034] Preferably, the mutant pp65 polypeptide contains one of F27K, C57M or F59Y, and one of E94T or T97M compared to the natural pp65 polypeptide.
[0035] Preferably, the mutant pp65 polypeptide is any one of SEQ NO.9-SEQ NO.10. The natural pp65 polypeptide is SEQ NO.11.
[0036] The present invention also provides a polypeptide encoding a recombinant pp65 polypeptide, wherein the recombinant pp65 polypeptide comprises a combination of a mutant pp65 polypeptide and an immune-enhancing sequence, wherein the mutant pp65 polypeptide contains at least three of the mutations F27K, C57M, F59Y, E94T, and / or T97M compared to the natural pp65 polypeptide. Optionally, the mutant pp65 polypeptide contains one of F27K, C57M, or F59Y, and one of E94T or T97M compared to the natural pp65 polypeptide. Optionally, the mutant pp65 polypeptide is any one of SEQ NO. 1-SEQ NO. 4. The natural pp65 polypeptide is SEQ NO. 11.
[0037] Further, the immune-enhancing sequence is one or more of LAMP, MITD, KDEL, and Fc. The LAMP includes a signal, a lumenal domain, a transmembrane region, and a cytoplasmic region, with the natural pp65 peptide inserted between the lumenal domain and the transmembrane and cytoplasmic regions. The signal-lumenal domain sequence of the LAMP is SEQ NO. 12. The transmembrane and cytoplasmic region sequences of the LAMP are SEQ NO. 13. The MITD includes a signal, a MITD transmembrane region, and a cytoplasmic region, with the natural pp65 peptide inserted between the signal and the MITD transmembrane and cytoplasmic regions. The MITD-signal sequence is SEQ ID NO. 14; the sequence between the MITD transmembrane and cytoplasmic regions is SEQ ID NO. 15.
[0038] Specifically, Fc is the Fc segment of immunoglobulin; preferably, the pp65 polypeptide is fused to Fc via a linker sequence; preferably, the linker sequence is selected from one or more combinations of: the trimer motif at the C-terminus of T4 fibrin SEQ NO.16, the PADRE pan-T cell epitope SEQ NO.17, the flexible sequence GGGGS, GSG, GGGGSGGGGSGGGGS, and IL13Ra2-111~142aa SEQ NO.18.
[0039] Preferably, the immunoglobulin Fc segment is human wild-type IgG1 Fc SEQ NO.37; preferably, the cysteine (C) near the N-terminus of the hinge region of the Fc is mutated to serine (S) to avoid non-specific covalent binding; preferably, the IgG1 Fc contains mutation sites that weaken the Fc-mediated ADCC, ADCP, and / or CDC effects; preferably, the end of the immunoglobulin Fc segment is also connected to a short IgM μtp tail peptide SEQ NO.41, and the proline (P) at the Fc end is mutated to threonine (T) to mimic the C-terminus of IgM and promote the hexamerization of the IgG Fc region, i.e., Fc6; preferably, the mutant sequence is selected from one of SEQ NO.19, SEQ NO.20, and SEQ NO.21; the N-terminus of the Fc fusion protein is additionally supplemented with a secretion signal peptide, such as the tPA signal peptide SEQ NO.22.
[0040] Specifically, the recombinant pp65 polypeptide is any one of those listed in Table 1 below.
[0041] Table 1. Recombinant pp65 peptide-immunostimulation combinations
[0042]
[0043]
[0044] Specifically, the recombinant pp65 polypeptide is any one of the following in the table.
[0045] 1 LAMP-pp65(G434A-S430G-R435G-K436N) 2 LAMP-pp65(A429G-S430G-R435G-K436N) 5 MITD-pp65(G434A-S430G-R435G-K436N) 6 MITD-pp65(A429G-S430G-R435G-K436N) 9 pp65(G434A-S430G-R435G-K436N)-KDEL 10 pp65(A429G-S430G-R435G-K436N)-KDEL 13 pp65(G434A-S430G-R435G-K436N)-Fc 14 pp65(A429G-S430G-R435G-K436N)-Fc 17 TPA-pp65(G434A-S430G-R435G-K436N)-IL13Ra2-111~142aa-PADRE-Fc6 18 TPA-pp65(A429G-S430G-R435G-K436N)-IL13Ra2-111~142aa-PADRE-Fc6
[0046] The invention provides a pharmaceutical composition comprising the recombinant pp65 polypeptide described herein. This composition is capable of inducing an immune response in tumor diseases expressing pp65.
[0047] The composition of the present invention encoding the above-mentioned recombinant pp65 polypeptide can induce the generation of effector T cells targeting tumor cells in subjects to which it is administered.
[0048] The tumor diseases mentioned include glioblastoma, colorectal cancer, prostate cancer, breast cancer, rhabdomyosarcoma, neuroblastoma, and hepatoblastoma.
[0049] The present invention also provides an immune cell loaded with the above-described encoding recombinant pp65 polypeptide.
[0050] Local delivery of the recombinant pp65 polypeptide or fragment thereof to a tissue, organ, or graft using any available reproducible and defective vector, such as liposomes, lipid nanoparticles, polymer nanoparticles, or cell-penetrating peptides, can be performed using any available reproducible and reproducible vector.
[0051] The present invention also provides the use of compositions encoding recombinant pp65 polypeptides in the preparation of medicaments for treating cancer.
[0052] The present invention relates to compositions encoding recombinant pp65 polypeptides, comprising pharmaceutically acceptable carriers and / or excipients.
[0053] Pharmaceutically acceptable excipients, buffers, protectants, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, and inactivators, or one or more of these.
[0054] The composition is prepared as a mucosal immunomodulator, humoral immunomodulator, cellular immunomodulator, or cutaneous immunomodulator. The mucosal immunomodulator is available in liquid, solid, semi-solid, gaseous, or inhaled formulations. The administration formulation is available as an intravenous injection, intramuscular injection, subcutaneous injection, oral administration, oral administration, sublingual administration, rectal administration, respiratory administration, or transdermal administration; preferably, it is a respiratory administration formulation, which is inhaled orally, nasally, or nebulized by a nebulizer.
[0055] Pharmaceutically acceptable carriers include one or more of the following: mineral salt adjuvants, oil-in-water emulsions, saponins, virions and virus-like particles, immunostimulatory oligonucleotides, human immunomodulators, and plasmids;
[0056] Mineral salt adjuvants include aluminum salts, calcium salts, phosphates, or sulfates, or combinations of different mineral salts; the preferred mineral salt adjuvant is aluminum phosphate; oil-in-water emulsions include, but are not limited to: squalene-water emulsions, complete Freund's adjuvants, or incomplete Freund's adjuvants; saponins can also be used as adjuvants in this invention. Saponins are a class of isosterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots, and even flowers of various plants;
[0057] Virisomes and virus-like particles can also be used as adjuvants in this invention. Virisomes and virus-like particles typically contain one or more proteins derived from viruses, optionally combined or formulated with phospholipids. Viral proteins suitable for virisomes and virus-like particles include those derived from influenza viruses (such as HA or NA), hepatitis B viruses (such as core or capsid proteins), hepatitis E viruses, measles viruses, Sindbis viruses, rotaviruses, foot and oral disease viruses, retroviruses, noroviruses, human papillomaviruses, HIV, RNA phages, Qβ phages (such as capsid proteins), GA phages, fr phages, and AP205 phages. Immunostimulatory oligonucleotides can also be used as adjuvants in this invention. Immunostimulatory oligonucleotides include nucleotide sequences containing CpG motifs (dinucleotide sequences containing unmethylated cytosine linked to guanosine via phosphate bonds), double-stranded RNA containing palindromic or multiple (dG) sequences, and oligonucleotides. Human immunomodulators include cytokines, interleukins IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, TGFβ decoy receptors, interferons, macrophage colony-stimulating factor, and tumor necrosis factor.
[0058] The adjuvants that can be used in this invention also include poly(acrylic acid), polyvinyl alcohol, polyvinylpyrrolidone, cross-linked derivatives of polysaccharides and carboxymethyl cellulose, chitosan, microparticles, polyoxyethylene ethers and polyoxyethylene ester formulations, imidazole quinolone compounds, and muramyl peptides.
[0059] The nucleic acid described in this invention refers to a polymeric form of nucleotides of any length. Polynucleotides may contain deoxyribonucleotides, ribonucleotides, and / or their analogues. Nucleotides may 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-chain polynucleotides, aptamers, plasmids, vectors, isolated DNA sequences of any kind, and isolated RNA sequences of any kind.
[0060] The term "polypeptide" as used in this invention refers to a compound consisting of two or more subunit amino acids, amino acid analogs, or peptide mimics.
[0061] "Encoding nucleic acid" is a nucleic acid sequence that is transcribed and translated into a polypeptide when placed under the control of a suitable expression control sequence.
[0062] "Vectors" include plasmids and viruses, and any DNA or RNA molecules, whether or not they self-replicate, that can be used to transform or transfect cells. Attached Figure Description
[0063] Figure 1 .pp65 and K436N mutant protein kinase activity assay
[0064] Figure 2 Detection of systemic lupus erythematosus-associated antibody levels caused by pp65 and its mutants
[0065] Figure 3 Evaluation of .pp65-induced cellular immunity Detailed Implementation
[0066] Unless otherwise defined, all scientific and technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art.
[0067] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0068] Example 1: Preparation of mRNA
[0069] Artificially synthesized plasmid DNA sequences containing RNA transcription-related elements and tagged with His or Flag tags are used to facilitate screening for expression of different antigen designs. The plasmids are transformed into *E. coli* for amplification. The fermented and purified plasmids are linearized using the restriction endonuclease BspQ1. Transcription is performed using a T7 in vitro transcription kit, with capping occurring during transcription to obtain capped mRNA. The transcription template is digested with DNase I, and the mRNA is purified by LiCl precipitation. The purified mRNA is dissolved in acidic sodium citrate buffer, and the concentration and integrity of the mRNA are detected using spectrophotometry and capillary electrophoresis, respectively. The resulting stock mRNA solution is stored at -80°C.
[0070] Table 3. Antigen Mutation Design
[0071] sequence Mutation / deletion sites SEQ NO.1 Delete 429-436 SEQ NO.2 S430G-R435G SEQ NO.3 R435G-K436N SEQ NO.4 S430G-K436N SEQ NO.5 A429G-K436N SEQ NO.6 G434A-K436N SEQ NO.7 A429G-G434A-K436N SEQ NO.8 S430G-R435G-K436N SEQ NO.9 G434A-S430G-R435G-K436N SEQ NO.10 A429G-S430G-R435G-K436N
[0072] Example 2: Encapsulation of mRNA stock solution and preparation of LNP
[0073] Cationic lipids:neutral phospholipids:steroidal lipids:polyethylene glycol (PEG) lipids were dissolved and mixed in ethanol at a molar ratio of 45:10:43:2. The lipid mixture and the mRNA stock solution were encapsulated using a microfluidic method. The encapsulation solution was diluted, ultrafiltered, and concentrated with 50 mM sodium acetate buffer containing 435 mg / ml sucrose to obtain mRNA-LNP. The encapsulation efficiency, average particle size, PDI, and Zeta potential of the mRNA-LNP were measured. The total flow rate of the nanomedicine manufacturing equipment was set to 12 ml / min. The mRNA solution and lipid mixture were mixed at a flow rate ratio of 3:1.
[0074] Example 3: Detection of kinase activity in wild-type and K436N mutant pp65
[0075] pp65 protein and its mutants were expressed and extracted in HEK293T cells. Then, pp65 protein and dephosphorylated bovine casein (100 mg / 100 ml, Sigma-Aldrich) were added to a 100 μL reaction mixture (25 mM Tris, pH 8.5, 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, 30 mM ATP) and reacted at room temperature for 30 minutes. After phosphorylation, 20 μL of 100 mM EDTA was added to terminate the reaction. The protein mixture was then denatured and subjected to SDS-PAGE electrophoresis. The phosphorylation level of the protein was detected using a Thr phosphorylation antibody (2 μg / ml, Sigma-Aldrich) to assess the kinase activity of pp65 and its variants. The results showed that the K436N mutation effectively eliminated the protein kinase activity of pp65.
[0076] Example 4: Detection of Systemic Lupus Erythematosus-Related Antibody Levels Caused by pp65 and its Mutants
[0077] Blood was collected from 6-8 week old female BALB / c mice after a second immunization. An ELISA assay was used to detect systemic lupus erythematosus-related immune responses induced by wild-type and mutant pp65, specifically by measuring the antibody titer specific to the ASTSAGR peptide in mouse serum. Results showed that the G434A-S430G-R435G and A429G-S430G-R435G mutations completely eliminated antibody production against TAF9, indicating good potential safety.
[0078] Example 5: Evaluation of pp65-induced cellular immunity
[0079] Female C57BL / 6 mice, approximately 6-8 weeks old, were randomly divided into 11 groups of 5 mice each. They were immunized twice, once by intramuscular injection into the hind leg on days 0 and 14. Blood was collected on day 28 post-primary immunization, and pp65 protein was used to detect antigen-specific antibodies. Mice were sacrificed, and PBMCs were collected in tubes for antigen-specific IFN-γ ELISPOT assay using pp65 protein. Spleen cells were also collected for antigen-specific IFN-γ positive CD8+ T cell response using pp65 protein. The results showed that G434A-S430G-R435G and A429G-S430G-R435G could induce a good IFN-γ positive CD8+ T cell response.
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. 9-10.
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 and cytoplasmic domains; the MITD comprises a signal and MITD transmembrane and cytoplasmic domains, and the native pp65 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 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. 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 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. 9-10.
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 Lamp transmembrane and cytoplasmic domains; the MITD comprises a signal and MITD transmembrane and cytoplasmic domains, and the native pp65 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, 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 atomization by an atomization 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.