Composition for immunization / treatment of tumors, preparation method therefor and use thereof

Abstract

A polynucleotide and a composition thereof, the composition comprising a nucleic acid that encodes a human IL13Rα2 polypeptide, a nucleic acid that encodes an immunoglobulin Fc segment, and at least one nucleic acid that encodes other tumor antigens. The composition can induce immune responses against IL13Rα2 and other tumor antigens, and is thus used for preventing / treating tumors.

Description

A composition for preventing / treating tumors and a preparation method and application thereof TECHNICAL FIELD

[0001] The present application relates to the field of vaccine technology, in particular to a composition for preventing / treating tumors. BACKGROUND

[0002] IL13Rα2 as a tumor antigen target, its core value comes from the unique expression pattern and functional characteristics. The receptor is lowly expressed or absent in normal tissues, but is specifically highly expressed in various solid tumors such as glioblastoma (expression rate 58%-74.8%), pancreatic cancer (65%-80%), ovarian cancer (60%-70%) and the like, and is a tumor-associated antigen that has been clearly identified. As a high-affinity decoy receptor for IL-13, it regulates cytokine signaling by competitive binding to IL-13, and promotes TGF-β secretion by activating the AP-1 pathway in the tumor microenvironment, and is involved in the process of tumor-related fibrosis. Its membrane-bound and soluble forms also provide dual markers for clinical detection.

[0003] In terms of therapeutic applications, IL13Rα2-targeted therapy has shown significant clinical potential. In the field of CAR-T cell therapy, Mustang Bio's MB-101 achieved the longest 66-month complete remission in patients with recurrent glioblastoma in clinical trials, demonstrating the effectiveness of the target; antibody drug conjugates such as IL13-PE38 showed a 22% objective remission rate in refractory glioma in early trials; and multi-target combination strategies (such as IL13Rα2 / B7-H3 bispecific CAR-T) effectively reduce the risk of antigen escape by 30%. These advances have driven IL13Rα2 to become an important breakthrough in the immunotherapy of solid tumors.

[0004] With the approval of the new crown mRNA vaccine and the development of mRNA technology, the development of mRNA tumor vaccine has also become a hot topic. The advantage of mRNA tumor vaccine technology is that it can efficiently and quickly stimulate the immune system to produce specific anti-tumor immune response. Currently, mRNA-DC vaccine (dendritic cell vaccine) therapy for glioblastoma has been reported, but there are few clinical studies on mRNA vaccine for the treatment of glioblastoma. One of the difficulties in the development of mRNA tumor vaccine is the selection of target points, which is closely related to the treatment effect. Generally, molecules with high expression levels in tumor cells and relatively low expression in normal cells are selected as targets for immunotherapy. In order to further improve the effect of tumor treatment, existing technologies develop combination therapy of multiple targets, and in further research, the inventors found that different targets directly interfere and inhibit each other, which directly affects the design of multi-target treatment programs. SUMMARY

[0005] To achieve the above object, the present inventors have carried out a plurality of biological experiments on IL13Rα2, and through a plurality of experiments, have determined an improved scheme for IL13Rα2, and provided a polynucleotide comprising a nucleic acid encoding the 111-142 aa region of IL13Rα2 polypeptide, which has been proved through experiments that the 111-142 aa region of IL13Rα2 polypeptide can improve immunogenicity, and the 111-142 aa region of IL13Rα2 polypeptide can also solve the problem of immunosuppression when IL13Rα2 and other tumor antigens are used in combination, thereby facilitating the combined use of IL13Rα2 and other tumor antigens.

[0006] The present application provides a polynucleotide comprising a nucleic acid encoding the 111-142 aa region of IL13Rα2 polypeptide.

[0007] To further improve the immunogenicity of IL13Rα2, the present application also provides a polynucleotide comprising a nucleic acid encoding the 111-142 aa region of human IL13Rα2 polypeptide and a nucleic acid encoding the Fc segment of immunoglobulin.

[0008] To further improve the immunogenicity of tumor treatment, the present inventors have combined the 111-142 aa region of IL13Rα2 polypeptide with a plurality of tumor antigens, and provided a polynucleotide comprising a nucleic acid encoding human IL13Rα2 polypeptide and at least one nucleic acid encoding a tumor antigen.

[0009] To further improve the immunogenicity of tumor treatment, the present inventors have combined the 111-142 aa region of IL13Rα2 polypeptide with a plurality of tumor antigens, and provided a polynucleotide comprising a nucleic acid encoding human IL13Rα2 polypeptide, a nucleic acid encoding the Fc segment of immunoglobulin, and at least one nucleic acid encoding a tumor antigen.

[0010] Preferably, the nucleic acid encoding human IL13Rα2 polypeptide is located between the nucleic acid encoding the Fc segment of immunoglobulin and the nucleic acid encoding a tumor antigen.

[0011] The nucleic acid encoding a tumor antigen comprises human survivin, EGFRvIII, CEA, HER2, MUC1, and MUC16.

[0012] The nucleic acid encoding human IL13Rα2 polypeptide comprises the 111-142 aa region of IL13Rα2, such as SEQ NO. 3.

[0013] The N-terminus of the nucleic acid encoding a tumor antigen is a secretion signal peptide, which can use the secretion signal peptide of the original tumor antigen, or a commonly used signal peptide such as tPA signal peptide SEQ NO. 35.

[0014] Preferably, the immunoglobulin Fc segment is human wild-type IgG1 Fc SEQ NO. 42; 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 to attenuate Fc-mediated ADCC, ADCP and / or CDC effects; preferably, the end of the immunoglobulin Fc segment is further connected to a short peptide of IgM μtp tail segment SEQ NO. 36, while 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. 32, SEQ NO. 33, SEQ NO. 34.

[0015] In some embodiments, the nucleic acid encoding the human IL13Rα2 polypeptide and the nucleic acid encoding the immunoglobulin Fc segment further comprise a nucleic acid encoding a helper T cell epitope, which is a flexible segment comprising PADRE, Tetanus Toxin (TT)-P2, TT-P21, TT-P23, TT-P30, TT-P32, TT-P7, TT-P17, TT-P28, HBsAg-aa 19-33, HBVnc-aa 50-69, Influenza Matrix Protein-aa 17-31.

[0016] Preferably, the polynucleotide of the present application is designed as follows, and the following is only an exemplary combination, and those skilled in the art can understand that different tumor antigens are applicable.

[0017] Table 1. Polynucleotide design scheme

[0018] In some embodiments, the human survivin polypeptide is a native human survivin polypeptide or a mutant human survivin polypeptide comprising at least two of T34A, F27K, C57M, F59Y, E94T, and / or T97M mutations compared to the native human survivin polypeptide. The mutant human survivin polypeptide is any one of SEQ NO. 37-SEQ NO. 40. The native human survivin polypeptide is SEQ NO. 41.

[0019] Those skilled in the art can understand that various mutations of the human survivin polypeptide are applicable. The following is only an example.

[0020] The nucleic acid is a non-replicative linear mRNA, a self-replicating mRNA, or a circular RNA.

[0021] The non-replicative linear mRNA, self-replicating mRNA, or circular RNA is complexed with one or more lipids to form a liposome, a lipid nanoparticle, and / or a lipoplex.

[0022] The polynucleotide of the present application can be delivered locally to a tissue, organ or graft at a proximal site using any available replicable and replication-defective vectors, such as plasmid vectors, retroviral vectors, adenoviral vectors, adeno-associated viral vectors, liposomes, lipid nanoparticles or other vectors with appropriate tropism for cells that can be involved in apoptosis.

[0023] The present application provides a composition comprising the polynucleotide as described above.

[0024] The present application provides a composition comprising a nucleic acid encoding a human IL13Rα2 polypeptide, a nucleic acid encoding an immunoglobulin Fc segment and at least one nucleic acid encoding a tumor antigen.

[0025] The present application provides a composition comprising a nucleic acid encoding a human IL13Rα2 polypeptide and at least one nucleic acid encoding a tumor antigen.

[0026] Preferably, the present application provides a composition comprising a nucleic acid encoding a human IL13Rα2 polypeptide, a nucleic acid encoding an immunoglobulin Fc segment and at least one nucleic acid encoding a tumor antigen, wherein the nucleic acid encoding a human IL13Rα2 polypeptide is located between the nucleic acid encoding an immunoglobulin Fc segment and the at least one nucleic acid encoding a tumor antigen.

[0027] Preferably, no linker is located between the nucleic acid encoding a human IL13Rα2 polypeptide and the at least one nucleic acid encoding a tumor antigen.

[0028] The nucleic acid encoding a tumor antigen comprises a human survivin, EGFRvIII, CEA, HER2, MUC1, MUC16.

[0029] The nucleic acid encoding a human IL13Rα2 polypeptide comprises a 111-142 aa region of IL13Rα2, such as SEQ NO. 1.

[0030] The nucleic acid encoding a tumor antigen comprises an additional secretion signal peptide at the N-terminus, such as a tPA signal peptide SEQ NO. 2.

[0031] Preferably, the immunoglobulin Fc segment is human wild-type IgGl Fc SEQ NO. 42; 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 IgGl Fc comprises a mutation site that attenuates Fc-mediated ADCC, ADCP and / or CDC effects; preferably, the immunoglobulin Fc segment is further linked to an IgM μtp tail short peptide SEQ NO. 4 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 hexamerization of the IgG Fc region, i.e. Fc6; preferably, the mutant sequence is selected from one of SEQ NO. 5, SEQ NO. 6, SEQ NO. 7.

[0032] In some embodiments, the nucleic acid encoding the human IL13Rα2 polypeptide and the nucleic acid encoding the immunoglobulin Fc segment further comprise a nucleic acid encoding a helper T cell epitope, which is a flexible fragment. Preferably, the helper T cell epitope is PADRE.

[0033] Preferably, the polynucleotides in the composition of the present application are designed as follows,

[0034] As only exemplary combinations, one skilled in the art can understand that different tumor antigens are all applicable.

[0035] Polynucleotide design scheme

[0036] In some embodiments, the human survivin polypeptide is a native human survivin polypeptide or a mutant human survivin polypeptide comprising at least two of T34A, F27K, C57M, F59Y, E94T and / or T97M mutations compared to the native human survivin polypeptide. The mutant human survivin polypeptide is any one of SEQ NO. 8-SEQ NO. 11. The native human survivin polypeptide is SEQ NO. 18.

[0037] One skilled in the art can understand that various mutations of the human survivin polypeptide are all applicable. The following are only exemplary.

[0038] To further improve the effect of tumor treatment, the application further provides a composition comprising: two or more polynucleotide fragments, wherein each polynucleotide fragment is a tandem polynucleotide encoding a tumor antigen, a nucleic acid encoding a human IL13Rα2 polypeptide 111-142 aa region, and a nucleic acid encoding an immunoglobulin Fc segment; and each polynucleotide fragment carries a different nucleic acid encoding a tumor antigen. The tumor antigen includes any one of human survivin, EGFRvIII, CEA, HER2, MUC1, and MUC16. The immunoglobulin Fc segment is introduced to solve the problem of immunosuppression when different tumor antigens are used in combination.

[0039] Preferably, the immunoglobulin Fc segment is a human wild-type IgG1 Fc SEQ NO. 3; 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 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 connected to an IgM μtp tail short peptide SEQ NO. 4, and the proline (P) at the end of the Fc is mutated to threonine (T) to mimic the C-terminus of IgM and promote the formation of a hexamer of the Fc region, i.e., Fc6; preferably, the mutant sequence is selected from one of SEQ NO. 5, SEQ NO. 6, and SEQ NO. 7.

[0040] In some embodiments, the nucleic acid encoding the human IL13Rα2 polypeptide and the nucleic acid encoding the immunoglobulin Fc segment further comprise a nucleic acid encoding a helper T cell epitope, which is a flexible fragment. Preferably, the helper T cell epitope is PADRE.

[0041] In some embodiments, the human survivin polypeptide is a native human survivin polypeptide or a mutant human survivin polypeptide comprising at least two of T34A, F27K, C57M, F59Y, E94T, and / or T97M mutations compared to the native human survivin polypeptide. The mutant human survivin polypeptide is any one of SEQ NO. 8-SEQ NO. 11. The native human survivin polypeptide is SEQ NO. 18.

[0042] As understood by those skilled in the art, various mutations of the human survivin polypeptide are applicable. The following are only examples.

[0043] The above-mentioned composition of the present application can induce the production of effector T cells and B cells having an effect on tumor cells in a subject to which the composition is administered.

[0044] The tumor disease includes melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, colon cancer, pancreatic cancer, prostate cancer, liver cancer, gastric cancer, brain tumor, etc.

[0045] The present application also provides the use of a composition comprising the above-mentioned polynucleotide in the preparation of a medicament for treating cancer.

[0046] The composition comprises a pharmaceutically acceptable carrier and / or adjuvant.

[0047] One or more of pharmaceutically acceptable adjuvants, buffers, protectors, stabilizers, surfactants, osmotic pressure regulators, adjuvants, preservatives, inactivators.

[0048] 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, a muscle injection, a subcutaneous injection, an oral administration preparation, an oral administration preparation, a sublingual administration preparation, a rectal administration preparation, a respiratory administration preparation, a transdermal administration preparation; preferably, it is a respiratory administration preparation, an oral inhalation, a nasal inhalation or an inhalation preparation after atomization by a nebulization administration device.

[0049] 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;

[0050] 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;

[0051] Virus-like particles can also be used as adjuvants in the present application, and typically comprise one or more proteins from a virus, optionally in combination or formulated with a phospholipid, suitable viral proteins for use in virus-like particles include those derived from influenza virus (e.g. HA or NA), hepatitis B virus (e.g. core or capsid proteins), 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 in the present application, and 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;

[0052] 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, imidazoquinolones, muramyl peptides.

[0053] The present application also provides an immune cell, which is loaded with the above-mentioned nucleic acid encoding a human IL13Rα2 polypeptide, a nucleic acid encoding an immunoglobulin Fc segment, and at least one nucleic acid encoding a tumor antigen

[0054] The present application provides a polypeptide, which comprises a 111-142 aa region of a human IL13Rα2 polypeptide.

[0055] The present application provides a polypeptide, which comprises a 111-142 aa region of a human IL13Rα2 polypeptide and an immunoglobulin Fc segment.

[0056] The present application provides a polypeptide, which comprises a 111-142 aa region of a human IL13Rα2 polypeptide and at least one tumor antigen.

[0057] The present application provides a polypeptide, which comprises a 111-142 aa region of a human IL13Rα2 polypeptide, an immunoglobulin Fc segment and at least one tumor antigen.

[0058] Preferably, the 111-142 aa region of a human IL13Rα2 polypeptide is located between the immunoglobulin Fc segment and the at least one tumor antigen.

[0059] Preferably, the 111-142 aa region of the human IL13Rα2 polypeptide is not connected to the tumor antigen by any other linker.

[0060] The tumor antigen includes human survivin, EGFRvIII, CEA, HER2, MUC1, MUC16.

[0061] The human IL13Rα2 polypeptide includes the 111-142 aa region of IL13Rα2, such as SEQ NO. 3.

[0062] The tumor antigen is additionally added with a secretion signal peptide at the N-terminus, such as tPA signal peptide SEQ NO. 2.

[0063] Preferably, the immunoglobulin Fc segment is human wild-type IgG1 Fc SEQ NO. 3; 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 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 a short peptide of IgM μtp tail segment SEQ NO. 4, 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. 5, SEQ NO. 6, SEQ NO. 7.

[0064] In some embodiments, the human IL13Rα2 polypeptide and the immunoglobulin Fc segment are further connected by a helper T cell epitope, which is a flexible fragment. Preferably, the helper T cell epitope is PADRE.

[0065] Preferably, the polypeptide of the present application is designed as follows, which is only an exemplary combination, and those skilled in the art can understand that different tumor antigens are applicable.

[0066] Polypeptide design scheme

[0067] In some embodiments, the human survivin polypeptide is a native human survivin polypeptide or a mutant human survivin polypeptide comprising at least two of T34A, F27K, C57M, F59Y, E94T, and / or T97M mutations compared to the native human survivin polypeptide. The mutant human survivin polypeptide is any one of SEQ NO. 8-SEQ NO. 11. The native human survivin polypeptide is SEQ NO. 18.

[0068] As will be appreciated by those skilled in the art, various mutations of the human survivin polypeptide are suitable. The following are merely exemplary.

[0069] The present application provides a composition comprising the polypeptide described above.

[0070] The present application provides a composition comprising, an immunoglobulin Fc segment and at least one tumor antigen, wherein the human IL13Rα2 polypeptide is encoded.

[0071] Preferably, the human IL13Rα2 polypeptide is located between the immunoglobulin Fc segment and the at least one tumor antigen.

[0072] Preferably, the human IL13Rα2 polypeptide and the tumor antigen are not connected by any linker.

[0073] The tumor antigen comprises: human survivin, EGFRvIII, CEA, HER2, MUC1, MUC16I.

[0074] The human IL13Rα2 polypeptide comprises the 111-142 aa region of IL13Rα2, such as SEQ NO. 1.

[0075] The N-terminal of the tumor antigen is additionally added with a secretion signal peptide, such as the tPA signal peptide SEQ NO. 2.

[0076] Preferably, the immunoglobulin Fc segment is a human wild-type IgG1 Fc SEQ NO. 3; preferably, the hinge region of the Fc is mutated from cysteine (C) to serine (S) near the N-terminal 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 connected with a short peptide of IgM μtp tail segment SEQ NO. 4, and the proline (P) at the end of the Fc is mutated to threonine (T) to mimic the C-terminal 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. 5, SEQ NO. 6, SEQ NO. 7.

[0077] In some embodiments, the human IL13Rα2 polypeptide and the immunoglobulin Fc segment further comprise a helper T cell epitope, which is a flexible fragment. Preferably, the helper T cell epitope is PADRE.

[0078] Preferably, the polypeptides in the composition of the present application are designed as follows, which are only exemplary combinations, and those skilled in the art can understand that different tumor antigens are applicable.

[0079] Table 2. Polypeptide design scheme

[0080] In some embodiments, the human survivin polypeptide is a native human survivin polypeptide or a mutant human survivin polypeptide comprising at least two of T34A, F27K, C57M, F59Y, E94T and / or T97M mutations compared to the native human survivin polypeptide. The mutant human survivin polypeptide is any one of SEQ NO. 8-SEQ NO. 11. The native human survivin polypeptide is SEQ NO. 18.

[0081] Those skilled in the art can understand that various mutations of the human survivin polypeptide are applicable. The following are only exemplary.

[0082] In some embodiments, the HER2 includes a native HER2 or a mutant HER2, and the mutant HER2 polypeptide comprises K436N, S430G, R435G mutations, and one of G434A or A429G compared to the native HER2 polypeptide. The mutant HER2 polypeptide is any one of SEQ NO. 18-SEQ NO. 19. The native HER2 polypeptide is SEQ NO. 20.

[0083] To further improve the effect of tumor treatment, the present application also provides a composition comprising: two or more polypeptide fragments, and each polypeptide fragment is a tandem polypeptide of tumor antigen-human IL13Rα2 polypeptide 111-142 aa region-immunoglobulin Fc segment. Each polypeptide fragment carries a different tumor antigen. The tumor antigen includes any one of human survivin, EGFRvIII, CEA, HER2, MUC1 and MUC16. The immunoglobulin Fc segment is introduced to solve the problem of immune suppression when different tumor antigens are used together.

[0084] Preferably, the immunoglobulin Fc segment is human wild-type IgG1 Fc SEQ NO. 3; 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 to attenuate Fc-mediated ADCC, ADCP and / or CDC effects; preferably, the immunoglobulin Fc segment is further linked to an IgM μtp tail short peptide SEQ NO. 4 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 hexamerization of the IgG Fc region, i.e. Fc6; preferably, the mutant sequence is selected from one of SEQ NO. 5, SEQ NO. 6, SEQ NO. 7.

[0085] In some embodiments, the nucleic acid encoding the human IL13Rα2 polypeptide and the nucleic acid encoding the immunoglobulin Fc segment further comprise a nucleic acid encoding a helper T cell epitope, which is a flexible segment. Preferably, the helper T cell epitope is PADRE.

[0086] In some embodiments, the human survivin polypeptide is a native human survivin polypeptide or a mutant human survivin polypeptide comprising at least two of T34A, F27K, C57M, F59Y, E94T and / or T97M mutations compared to the native human survivin polypeptide. The mutant human survivin polypeptide is any one of SEQ NO. 8-SEQ NO. 11. The native human survivin polypeptide is SEQ NO. 18.

[0087] As understood by those skilled in the art, various mutations of the human survivin polypeptide are applicable. The following are only examples.

[0088] The above composition of the present application can induce the production of effector T cells and B cells with tumor cell specificity in the subject to which it is administered.

[0089] The tumor disease includes melanoma, breast cancer, cervical cancer, ovarian cancer, lung cancer, colon cancer, pancreatic cancer, prostate cancer, liver cancer, gastric cancer, brain tumor, etc.

[0090] Local delivery of the polypeptide or polypeptide fragment of the present application to a proximal site of a tissue, organ or graft can use any available replicable and replication-defective vectors, such as liposomes, lipid nanoparticles or

[0091] The present application also provides the use of a composition comprising the above-mentioned polypeptide in the preparation of a medicament for treating cancer.

[0092] The composition, including a pharmaceutically acceptable carrier and / or adjuvant.

[0093] One or more of a pharmaceutically acceptable adjuvant, buffer, protective agent, stabilizer, surfactant, osmotic pressure regulator, adjuvant, preservative, inactivation agent.

[0094] 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.

[0095] The pharmaceutically acceptable carrier includes one or more of a mineral salt adjuvant, an oil-in-water emulsion, a saponin, a virosome and a virus-like particle, an immunostimulatory oligonucleotide, a human immunomodulator, a plasmid;

[0096] 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, the saponin is a class of heterogenous sterol glycosides and triterpene glycosides, which exist in the bark, leaves, stems, roots and even flowers of various plants;

[0097] The virosome and virus-like particle can also be used as an adjuvant of the present application, the virosome and virus-like particle usually contains 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, the immunostimulatory oligonucleotide includes 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;

[0098] 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.

[0099] 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. 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.

[0100] The "polypeptide" of the present application refers to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics.

[0101] 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.

[0102] 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

[0103] Figure 1. Humoral immune responses induced by IL13Ra2 and its epitopes

[0104] Figure 2. Humoral immune responses after IL13Ra2 is concatenated with different antigens

[0105] Figure 3. Cellular immune responses after IL13Ra2 is concatenated with different antigens

[0106] Figure 4. Humoral immune responses induced by single component mRNA-LNP vaccines

[0107] Figure 5. Cellular immune responses induced by single component mRNA-LNP vaccines

[0108] Figure 6. Humoral immune responses induced by two component mRNA-LNP vaccine combinations

[0109] Figure 7. Cellular immune responses induced by two component mRNA-LNP vaccine combinations DETAILED DESCRIPTION

[0110] 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.

[0111] 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 the other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0112] Example 1 B cell epitope screening of IL13Rα2

[0113] The polypeptides of SEQ NO. 1-SEQ NO. 6 were synthesized, mixed with aluminum adjuvant, and 6-8 weeks old C57BL / 6 female mice were immunized, once at day 0, 14 and 28, by intramuscular injection in the hind leg, with a dose of 30 μg. The antibody response level against IL13Rα2 protein was detected by blood sampling at day 42.

[0114] The results showed that the antibody level induced by IL13Rα2-111-142aa was the highest, and the response levels of IL13Rα2-23-44aa and IL13Rα2-314-343aa were the second. IL13Rα2-111-142aa, IL13Rα2-23-44aa and IL13Rα2-314-343aa were selected for subsequent study.

[0115] Example 2 Evaluation of immune response of IL13Rα2 and other tumor antigens in tandem design

[0116] The plasmid DNA sequence was artificially synthesized, and the DNA sequence contained RNA transcription related elements. The mRNA encoded the antigens in the following table.

[0117] The plasmid was transformed into E. coli for amplification. The purified plasmid after fermentation was linearized by restriction endonuclease BspQl. Transcription was performed by using T7 in vitro transcription kit, and capping was performed during the transcription to obtain capped mRNA. The transcription template was digested by DNase I, and the mRNA was purified by LiCl precipitation method. The purified mRNA was dissolved in acid 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. 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 mRNA stock solution and the lipid mixture solution were encapsulated by microfluidic method, and the encapsulated solution was diluted, ultrafiltrated and concentrated by using 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 the 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.

[0118] The C57BL / 6 female mice at the age of 6-8 weeks were immunized by using mRNA-LNP at a dose of 10 μg by intramuscular injection at the back legs at day 0 and day 14. The blood was collected at day 28 (two weeks after the second immunization), and the survivin protein, HER2 protein, EGFRvIII-1-25 aa polypeptide and IL13Rα2 protein were used for the detection of the antibody specific to each antigen; the mice were sacrificed, and the splenocytes were collected, and the survivin protein overlapping peptide library, HER2 protein overlapping peptide library and EGFRvIII-1-25 aa polypeptide were used for the detection of the antigen-specific IFN-γ ELISPOT.

[0119] The results showed that the combination of IL13Rα2-111-142 aa with different antigens did not affect the humoral and cellular immune responses of the other antigens and IL13Rα2.

[0120] Example 4 Evaluation of immune responses of different combinations of tumor antigens

[0121] C57BL / 6 female mice of 6-8 weeks old were immunized with mRNA-LNP at day 0 and 14, by intramuscular injection in the hind leg, at a dose of 10 μg. Blood was collected at day 28 (two weeks post-2ndimmunization) and survivin protein, HER2 protein, EGFRvIII-1-25aa polypeptide, IL13Rα2 protein were used for each antigen-specific antibody detection. Mice were sacrificed and splenocytes were collected, and survivin protein overlapping peptide library, HER2 protein overlapping peptide library, and EGFRvIII-1-25aa polypeptide were used for antigen-specific IFN-γ ELISPOT detection, respectively.

[0122] Group 1-12 mice were inoculated with single-component mRNA-LNP vaccine as follows:

[0123] Group 13-24 mice were inoculated with two-component mRNA-LNP vaccine as follows:

[0124] The results show that the combination of different antigen designs containing Fc6 does not produce immune interference compared with the single-component vaccine.

Claims

1. A polynucleotide, comprising, The nucleic acid encoding the 111-142 aa region of the IL13Rα2 polypeptide.

2. A polynucleotide, comprising, The nucleic acid encoding the 111-142 aa region of the human IL13Rα2 polypeptide and at least one nucleic acid encoding a tumor antigen.

3. The polynucleotide of any one of claims 2, wherein, The nucleic acid encoding the human IL13Rα2 polypeptide, the nucleic acid encoding the Fc segment of immunoglobulin and at least one nucleic acid encoding a tumor antigen, the nucleic acid encoding the human IL13Rα2 polypeptide is located in the middle of the nucleic acid encoding the Fc segment of immunoglobulin and the nucleic acid encoding the tumor antigen.

4. The polynucleotide of any one of claims 2-4, wherein, The tumor antigen includes any one of human survivin, EGFRvIII, CEA, HER2, MUC1, MUC16.

5. The polynucleotide of any one of claims 1-4, wherein The nucleic acid encoding the human IL13Rα2 polypeptide includes the 111-142 aa region of IL13Rα2, such as SEQ NO.

3.

6. The polynucleotide according to any one of claims 2-5, wherein the Fc segment of immunoglobulin is human wild type IgG1 Fc SEQ NO. 42; or contains a mutation site weakening Fc-mediated ADCC, ADCP and / or CDC effect, and the mutant sequence is selected from one of SEQ NO. 32, SEQ NO. 33, SEQ NO.

34.

7. A polynucleotide according to any one of claims 1 to 6, wherein The nucleic acid is non-replicative linear mRNA, self-replicative mRNA, or circular RNA.

8. A composition characterized in that, The composition includes the polynucleotide according to any one of claims 1-7, which can induce an antibody response specific to IL13Rα2.

9. A composition characterized in that, The nucleic acid encoding the 111-142 aa region of the IL13Rα2 polypeptide. The nucleic acid encoding the 111-142 aa region of the human IL13Rα2 polypeptide and at least one nucleic acid encoding a tumor antigen.

10. The composition of claim 9, wherein, The nucleic acid encoding the human IL13Rα2 polypeptide, the nucleic acid encoding the Fc segment of immunoglobulin and at least one nucleic acid encoding a tumor antigen, the nucleic acid encoding the human IL13Rα2 polypeptide is located in the middle of the nucleic acid encoding the Fc segment of immunoglobulin and the nucleic acid encoding the tumor antigen. The tumor antigen includes any one of human survivin, EGFRvIII, CEA, HER2, MUC1, MUC16. The nucleic acid encoding the human IL13Rα2 polypeptide includes the 111-142 aa region of IL13Rα2, such as SEQ NO.

3.

13. A polypeptide, characterized in that, 6. The polynucleotide according to any one of claims 2-5, wherein the Fc segment of immunoglobulin is human wild type IgG1 Fc SEQ NO. 42; or contains a mutation site weakening Fc-mediated ADCC, ADCP and / or CDC effect, and the mutant sequence is selected from one of SEQ NO. 32, SEQ NO. 33, SEQ NO.

34.

14. The polypeptide of claim 12, wherein The nucleic acid is non-replicative linear mRNA, self-replicative mRNA, or circular RNA.

15. The polypeptide of claim 14, wherein The composition includes the polynucleotide according to any one of claims 1-7, which can induce an antibody response specific to IL13Rα2.

16. A polypeptide according to any one of claims 14-15, wherein The nucleic acid encoding the 111-142 aa region of the IL13Rα2 polypeptide.

17. A polypeptide according to any one of claims 12 to 16, wherein The nucleic acid encoding the 111-142 aa region of the human IL13Rα2 polypeptide and at least one nucleic acid encoding a tumor antigen. The nucleic acid encoding the human IL13Rα2 polypeptide, the nucleic acid encoding the Fc segment of immunoglobulin and at least one nucleic acid encoding a tumor antigen, the nucleic acid encoding the human IL13Rα2 polypeptide is located in the middle of the nucleic acid encoding the Fc segment of immunoglobulin and the nucleic acid encoding the tumor antigen. The tumor antigen includes any one of human survivin, EGFRvIII, CEA, HER2, MUC1, MUC16. The nucleic acid encoding the human IL13Rα2 polypeptide includes the 111-142 aa region of IL13Rα2, such as SEQ NO.

3.

18. A polypeptide according to any one of claims 13 to 17, wherein The immunoglobulin Fc segment is human wild type IgG1 Fc SEQ NO. 42; or contains a mutation site weakening Fc-mediated ADCC, ADCP and / or CDC effect, and the mutant sequence is selected from one of SEQ NO. 32, SEQ NO. 33, SEQ NO.

34.

19. A composition characterized in that, The composition comprises the polypeptide of any one of claims 13-18, and is capable of inducing an antibody response specific to IL13Rα2.

20. A composition characterized in that, Comprise: Two or more polypeptide fragments, and each polypeptide fragment is a tandem polypeptide of tumor antigen-human IL13Rα2 polypeptide 111-142aa region-immunoglobulin Fc segment; and each polypeptide fragment carries a different tumor antigen.

21. The composition of claim 20, wherein, The tumor antigen comprises any one of human survivin, EGFRvIII, CEA, HER2, MUC1, MUC16.

22. Use of the composition of any one of claims 19-21 in the preparation of a medicament for preventing / treating tumors.

23. Use of the polypeptide of any one of claims 13-18 in the preparation of a medicament for preventing / treating tumors.

24. An immune cell, comprising, The polynucleotide or polypeptide of any one of claims 1-7, 13-18 is loaded. The polynucleotide or polypeptide of any one of claims 1-7, 13-18 is loaded.

Images