RNA encoding T-cell co-stimulatory molecules

Abstract

The present invention provides an RNA encoding a T cell co-stimulatory molecule, the RNA comprising at least one nucleic acid sequence encoding: (i) an amino acid sequence comprising a co-stimulatory molecule; (ii) an amino acid sequence comprising an antigen; the co-stimulatory molecule is selected from at least one of B7 family members and at least one of tumor necrosis factor family members.

Description

Technical Field

[0001] The present invention relates to the technical field of gene drugs, and in particular to RNA encoding T cell costimulatory molecules. Background Art

[0002] Since the cowpox vaccination experiment, more and more new vaccines and new ways of using vaccines have been developed. In addition to preventive vaccines, therapeutic vaccines have also been proposed and developed.

[0003] Preventive vaccines stimulate the body's immune response with pathogen antigens, providing protective immunity to the body against subsequent exposure to these pathogens. When the body comes into contact with the harmless pathogen antigens carried in the preventive vaccine, the immune system will produce certain protective substances, such as immune hormones, active physiological substances, special antibodies, etc.; when the body comes into contact with this pathogen again, the body's immune system will follow its original memory and produce more protective substances to prevent the harm of the pathogen. Common preventive vaccines include human papillomavirus vaccine, hepatitis B vaccine, BCG vaccine, polio vaccine, measles vaccine, plague vaccine, pertussis vaccine, typhoid vaccine, meningococcal vaccine, cholera vaccine, influenza vaccine, etc.

[0004] Therapeutic vaccines carry and express specific endogenous or exogenous antigens, mainly by stimulating cytotoxic T cells to induce or enhance cell-mediated immunity, or by activating B cells to produce specific antibodies to induce humoral immunity. Among therapeutic vaccines, therapeutic cancer vaccines are a research hotspot. Therapeutic cancer vaccines aim to enhance the immune system response of patients and help the body recognize and attack existing tumor cells. Therapeutic cancer vaccines can work in various ways, such as promoting T cell immune response, activating antibody response or enhancing the function of immune cells, etc. Currently, therapeutic cancer vaccines for melanoma, prostate cancer, etc. are still in the experimental stage.

[0005] Whether it is a prophylactic vaccine or a therapeutic vaccine, enhancing antigen immunogenicity by adding and screening appropriate adjuvants is a key link in the clinical research and development and use of vaccines. An adjuvant, or immunologic adjuvant, refers to a substance that can non-specifically enhance or alter the specific immune response of the body to a matching antigen, enhance the immunogenicity of the antigen or change the type of immune response, but has no immunogenicity itself. The immunobiological effects of adjuvants are to enhance immunogenicity, increase antibody titers, change the type of antibody production, and induce or enhance delayed hypersensitivity reactions. However, the mechanism of action of adjuvants has not been fully understood, and the mechanisms of action of different adjuvants are also not the same. Commonly used adjuvants include aluminum gels and salts, monophosphoryl lipid A, MF59 water-in-oil emulsion, Freund's complete adjuvant, Freund's incomplete adjuvant, detergents, and plant saponins.

[0006] Different adjuvants have different effects on enhancing the immunogenicity of different vaccines. Aluminum adjuvants are the most commonly used immunological adjuvants and show good safety in both the low-age and high-age groups. However, for the application of some vaccines, their efficacy is not as good as that of new adjuvants. In addition, existing adjuvants are mostly used to enhance the immunogenicity of vaccines such as live attenuated vaccines, subunit vaccines (including polypeptide vaccines), and inactivated vaccines, and their effects on nucleotide vaccines are relatively limited. In view of this, the present invention is proposed. Summary of the Invention

[0007] The present invention provides an RNA encoding a T cell costimulatory molecule, the RNA comprising at least one nucleic acid sequence encoding an amino acid sequence of: (i) an amino acid sequence comprising a costimulatory molecule; (ii) an amino acid sequence comprising an antigen; the costimulatory molecule being selected from at least one of the members of the B7 family and at least one of the members of the tumor necrosis factor family; the members of the B7 family being selected from at least one of CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and HHLA2; in an alternative embodiment, the members of the B7 family are selected from at least one of CD80, CD86, B7-DC, PD-L1, and HHLA2; the members of the tumor necrosis factor family being selected from at least one of TNF-β, TNF-α, LT-β, CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, and GITRL; in an alternative embodiment, the members of the tumor necrosis factor family are selected from at least one of CD252, CD154, CD70, 4-1BBL, and GITRL.

[0008] In an alternative embodiment, the antigen is a pathogen antigen or a tumor antigen.

[0009] In an alternative embodiment, the pathogen antigen is selected from at least one of HIV Env, RSV F, influenza HA, EBV gp350, EBV LMP1, EBV LMP2, EBV EBNA1, EBV EBNA3, CMV gB, CMV UL128, CMV UL130, CMV UL131A, CMV gH, CMV gL, Lyme disease OspA, pertussis toxin, dengue E, SARS S, MERS S, Zaire Ebola virus GP, Sudan Ebola virus GP, Marburg virus GP, hantavirus Gn, hantavirus Gc, measles H, Zika envelope domain III, malaria CSP, malaria Pfs25, MenB fHbp, MenB NadA, MenB NHBA, Nipah virus F, Nipah virus G, rotavirus VP4, rotavirus VP8, human papillomavirus antigen, HBeAg, hepatitis B virus pre-S1 protein, hepatitis B virus pre-S2 protein, hepatitis B virus HBsAg protein, hepatitis B virus HBcAg protein, hand, foot and mouth virus antigen.

[0010] In an alternative embodiment, the tumor antigen is selected from at least one of CLDN6, MAGE-A3, MAGE-A4, PRAME, MAGE-C1, MAGE-C2, NY-ESO-1, Tyrosinase, Gp100, TPTE, PSA, AFP, GPC3, Survivin, Melan-A, Kita-kyushu KK-LC-1, KRAS G12, KRAS G13, KRAS Q61.

[0011] In an alternative embodiment, the nucleic acid sequence encodes an amino acid sequence comprising: (i) an amino acid sequence containing a co-stimulatory molecule; (ii) an amino acid sequence containing an antigen; the co-stimulatory molecules are CD80 and CD70; in an alternative embodiment, the co-stimulatory molecules further include at least one selected from CD252, CD154, 4-1BBL, GITRL.

[0012] In an alternative embodiment, the nucleic acid sequence encodes an amino acid sequence comprising at least one of the following:

[0013] (iii) an amino acid sequence containing a 2A self-cleaving peptide; (iv) an amino acid sequence containing a GS linker polypeptide;

[0014] The 2A self-cleaving peptide is selected from T2A peptide, P2A peptide, E2A peptide or F2A peptide; the nucleic acid sequence encoding the 2A self-cleaving peptide or GS-linked polypeptide is used to link the nucleic acid sequence encoding the co-stimulatory molecule, and / or the nucleic acid sequence encoding the antigen.

[0015] In an alternative embodiment, the CD80 comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16, or a functional fragment thereof, or an extracellular domain composed thereof.

[0016] In an alternative embodiment, the CD70 comprises the amino acid sequence shown in SEQ ID NO.19 or SEQ ID NO.20, or a functional fragment thereof, or is composed thereof.

[0017] In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises a nucleic acid sequence encoding CD80 as shown in at least one of SEQ ID NO.28-31, SEQ ID NO.46-51.

[0018] In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises a nucleic acid sequence encoding CD70 as shown in at least one of SEQ ID NO.52-54.

[0019] In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule is selected from mRNA or circular RNA.

[0020] In an alternative embodiment, the mRNA described in the present disclosure consists of a sequence including a 5' cap, 5' UTR, ORF, 3' UTR, and 3' poly(A) tail in sequence from the 5' end to the 3' end.

[0021] In an alternative embodiment, the 5' cap is selected from ARCA, mCAP, dmCAP, m7G(5'')ppp(5'')(2''OMeA)pG, tmCAP, m7(3''OMeG)(5'')ppp(5'')(2''OMeA)pG, m7(3''OMeG)(5'')ppp(5'')(2''OMeG)pG, dmCAP or m7G(5'')ppp(5'')(2''OMeG)pG.

[0022] In an alternative embodiment, the 5' cap is m7Gppp(5’)(2’-OMeA)pG.

[0023] In an alternative embodiment, the length of the 5’UTR is preferably 10 to 200 nucleotides. In an alternative embodiment, the length of the 5’UTR is 15 to 100 nucleotides. In an alternative embodiment, the nucleotide sequence of the 5’UTR is as shown in SEQ ID NO.1-3. In an alternative embodiment, the nucleotide sequence of the 5’UTR is as shown in SEQ ID NO.1.

[0024] In an alternative embodiment, the 3’UTR sequence is as shown in SEQ ID NO.4-6. In an alternative embodiment, the 3’UTR sequence is as shown in SEQ ID NO.4.

[0025] In an alternative embodiment, one or more uridines in the mRNA are replaced with modified nucleosides. In an alternative embodiment, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ) or 5-methyl-uridine (m5U). In an alternative embodiment, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0026] On the other hand, the present invention provides a protein composition, which comprises a polypeptide encoded by the RNA encoding the T cell co-stimulatory molecule as described above.

[0027] In yet another aspect, the present invention provides a vaccine, which comprises the RNA encoding the T cell co-stimulatory molecule as claimed in claims 1-6, or, the protein composition as claimed in claim 7.

[0028] In an alternative embodiment, the vaccine comprises the RNA encoding the T cell co-stimulatory molecule as claimed in claims 1-6 and a delivery preparation encapsulating the RNA.

[0029] In an alternative embodiment, the delivery preparation is selected from at least one of lipid nanoparticles, cationic liposomes, protamine, exosomes, plasmids.

[0030] In yet another aspect, the present invention provides a biological material, which comprises any one of an expression cassette, a vector, an engineered bacterium or a cell line, and the biological material contains or expresses the RNA encoding the T cell co-stimulatory molecule as claimed in any one of claims 1-6.

[0031] In yet another aspect, the present invention provides a method, which comprises administering to a subject an effective amount of at least one of the RNA encoding the T cell co-stimulatory molecule as described above, the vaccine as described above, the protein composition as described above or the biological material as described above, so as to induce a cellular immune response or a humoral immune response in the subject.

[0032] In alternative embodiments, the co-stimulatory molecule polypeptide contained in the RNA-encoded amino acids encoding a T cell co-stimulatory molecule increases the immunogenicity of the antigen by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or 200%). In alternative embodiments, the co-stimulatory molecule polypeptide contained in the RNA-encoded amino acids encoding a T cell co-stimulatory molecule increases the antigen-encoding immune stimulation and / or immune response by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20%-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100% or 50-200%. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for use in the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0034] Figure 1 Shows the expression of the RNA encoding a T cell co-stimulatory molecule of Example 2 at the in vitro cell level.

[0035] Figure 2 Shows the change in the average tumor volume of mice in Example 3.

[0036] Figure 3 Shows the in vitro function of the engineered nucleotides encoding co-stimulatory molecules of Example 4.

[0037] Figure 4 Shows the in vitro function of the engineered nucleotides encoding co-stimulatory molecules of Example 5.

[0038] Figure 5 Shows the in vitro function of the engineered nucleotides encoding co-stimulatory molecules of Example 6.

[0039] Figure 6 Shows the change in the average tumor volume of mice in Example 7.

[0040] Figure 7 Shows the change in the average tumor volume of mice in Example 8.

[0041] Figure 8 Shows the content of E7 antigen-specific E7 multimer+CD8+T cells in the peripheral blood of mice in Example 9.

[0042] Figure 9 Shows the change in the average tumor volume of mice and the survival curve of mice in Example 10.

[0043] Figure 10 Shows the change in the average tumor volume of mice in Example 11. Detailed implementation mode

[0044] Although the content of the present invention is further described in more detail below, it should be understood that the content of the present invention is not limited to the specific methods, schemes and reagents described herein. It should also be understood that the terms used herein are only for the purpose of describing some specific embodiments, and are not intended to limit the scope of the present invention, which will only be limited by the appended claims.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Hereinafter, the elements of the content of the present invention will be described in more detail. These elements are listed in specific embodiments, however, it should be understood that they can be combined in any way and in any number to form additional embodiments.

[0046] The multiple described embodiments and preferred embodiments should not be construed as limiting the content of the present invention to the explicitly described embodiments. This specification should be understood to support and cover embodiments that combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. In addition, unless the context otherwise indicates, any arrangement and combination of all the elements described in this application should be considered to be disclosed in the specification of this application.

[0047] Unless otherwise specified herein or clearly contradicted by the context, all methods described herein can be performed in any suitable order. The use of any and all examples or exemplary language (e.g., "such as") provided herein is only intended to better illustrate the disclosure of the present invention and does not constitute a limitation on the scope of the disclosure of the present invention that is otherwise claimed. No language in this specification should be construed as indicating any unclaimed element necessary for the practice of the disclosure of the present invention.

[0048] When referring to an amino acid sequence (polypeptide or protein), a "fragment" refers to a part of the amino acid sequence, i.e., a sequence representing an amino acid sequence shortened at the N-terminus and / or C-terminus. A fragment shortened at the C-terminus (N-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 3'-end of the open reading frame. A fragment shortened at the N-terminus (C-terminal fragment) can be obtained, for example, by translating a truncated open reading frame lacking the 5'-end of the open reading frame, provided that the truncated open reading frame contains a start codon for initiating translation. Fragments of an amino acid sequence contain, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% of the amino acid residues from the amino acid sequence. Fragments of an amino acid sequence preferably contain at least 6, particularly at least 8, at least 15, at least 20, at least 30, at least 50 or at least 100 consecutive amino acids from the amino acid sequence.

[0049] According to the disclosure of the present invention, a part or fragment of a peptide or protein preferably has at least one functional property of the peptide or protein from which it is derived. Such functional properties include pharmacological activity, interaction with other peptides or proteins, enzyme activity, interaction with antibodies, and selective binding to nucleic acids. For example, a pharmacological active fragment of a peptide or protein has at least one pharmacological activity of the peptide or protein from which the fragment is derived. A part or fragment of a peptide or protein preferably contains a sequence of at least 6, particularly at least 8, at least 10, at least 12, at least 15, at least 20, at least 30 or at least 50 consecutive amino acids of the peptide or protein. A part or fragment of a peptide or protein preferably contains a sequence of at most 8, particularly at most 10, at most 12, at most 15, at most 20, at most 30 or at most 55 consecutive amino acids of the peptide or protein.

[0050] An object of the present invention is to provide an RNA encoding a T cell co-stimulatory molecule, which contains at least one coding region encoding an immunostimulatory polypeptide and at least one coding region encoding an antigen, and can enhance the immunogenicity of a vaccine.

[0051] The "RNA encoding a T cell co-stimulatory molecule" of the present invention is a nucleotide that does not exist in nature. As a whole, the "RNA encoding a T cell co-stimulatory molecule" of the present invention is a nucleotide that does not exist in nature, but a local or partial sequence of the "RNA encoding a T cell co-stimulatory molecule" of the present invention can be a nucleotide that exists in nature, that is to say, the "RNA encoding a T cell co-stimulatory molecule" in the present invention can include a nucleotide sequence that exists in nature. In an alternative embodiment, the "RNA encoding a T cell co-stimulatory molecule" of the present invention includes nucleotide sequences from different organisms (for example, from different species). For example, in an alternative embodiment, the RNA encoding a T cell co-stimulatory molecule includes a murine nucleotide sequence, a bacterial nucleotide sequence, a human nucleotide sequence, and / or a viral nucleotide sequence.

[0052] The "RNA encoding a T cell co-stimulatory molecule" of the present invention includes recombinant nucleotides and / or synthetic nucleotides. A "recombinant nucleotide" refers to a molecule constructed by ligating nucleotide molecules that can replicate in a vector. A "synthetic nucleotide" refers to a molecule amplified or chemically synthesized or synthesized by other means.

[0053] The RNA encoding a T cell co-stimulatory molecule can express genes using multiple promoters by multiple open reading frames (ORFs), that is, more than one separate mRNA transcript can be produced from a single RNA. For example, a first promoter can be operably linked to a polynucleotide sequence encoding a first polypeptide / amino acid, and a second promoter can be operably linked to a polynucleotide sequence encoding a second polypeptide / amino acid. Generally speaking, any number of promoters can be used to express any number of polypeptides / amino acids.

[0054] The RNA encoding a T cell co-stimulatory molecule can also express genes using one promoter by one open reading frame (ORF), produce one mRNA transcript and translate it into one polypeptide, and then be self-cleaved by an enzyme in the cell to produce multiple polypeptides / amino acids.

[0055] The open reading frame (ORF) described in the present invention is a continuous RNA, generally starting with a start codon (such as AUG) and ending with a stop codon (such as, TGA, or UAA, UAG or UGA).

[0056] In an alternative embodiment, the present invention provides an RNA encoding a co-stimulatory molecule, and the amino acid sequence encoded by the RNA includes at least two functional amino acid sequences. One of the at least two functional amino acid sequences includes a co-stimulatory molecule, and the other includes an antigen.

[0057] "Costimulatory molecules" are cell surface molecules and their ligands involved in costimulatory signal transduction, which provide necessary costimulatory signals for the full activation of T (or B) cells. "Costimulatory molecules" include CD28, ICOS, CTLA-4, members of the B7 family, members of the tumor necrosis factor family, etc. The main biological functions of "costimulatory molecules" include enhancing T cell activation and immune responses, and also participating in the regulation of tumor immune responses. For example, the binding of the B7 family member CD80 to CD28 on T cells can generate a positive signal, thereby enhancing the immune response; while the binding of CTLA-4 to the B7 family member CD80 can generate a negative signal, blocking the T cell activation brought by CD28 and downregulating the immune response. For another example, members of the B7 family include CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, HHLA2, etc. For example, members of the tumor necrosis factor family include TNF-β, TNF-α, LT-β, CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, GITRL, etc.

[0058] In an alternative embodiment, the costimulatory molecule described in the present invention is selected from at least one of members of the tumor necrosis factor family and members of the B7 family. For example, the costimulatory molecule is selected from at least one of CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and HHLA2; for another example, the costimulatory molecule at least includes CD80.

[0059] The native / wild-type costimulatory molecule CD80 has a transmembrane polypeptide structure, including an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain of native / wild-type CD80 is the site that binds to the receptor CD28 or CTLA-4 and participates in signal transduction during the immune regulation process; the transmembrane domain of native / wild-type CD80 is composed of hydrophobic amino acids and is embedded in the cell membrane, ensuring the stable localization of the native / wild-type CD80 protein on the cell membrane and playing a role in supporting protein localization and stability. The intracellular domain of native / wild-type CD80 contains some amino acid sequences related to signal transduction and may interact with intracellular signal transduction pathways to regulate the activation state of T cells.

[0060] The extracellular domain of natural / wild CD80 is further subdivided into two domains, named IgV domain and IgC domain respectively. The IgV domain and IgC domain are considered to play important roles in the binding of CD80 to the receptors CD28 or CTLA-4 (Both Extracellular Immunoglobin-like Domains of CD80 Contain Residues Critical for Binding T Cell Surface Receptors CTLA-4 and CD28, Peach, Robert J. et al. Journal of Biological Chemistry, Volume 270, Issue 36, 21181-21187). The IgV domain is located at positions 35-135 of the extracellular domain of CD80, and the IgC domain is located at positions 145-230 of the extracellular domain of CD80.

[0061] In an alternative embodiment, the co-stimulatory molecule CD80 of the present invention may be a wild-type co-stimulatory molecule CD80, and its amino acid sequence is identical to that of any natural / wild co-stimulatory molecule CD80. In an alternative embodiment, the co-stimulatory molecule CD80 of the present invention may be an engineered co-stimulatory molecule CD80.

[0062] "Engineered co-stimulatory molecule CD80" is a CD80 molecule obtained by modifying or designing and constructing based on the principle of natural / wild CD80 molecules in order to obtain specific functions or improved properties. In an alternative embodiment, the engineered co-stimulatory molecule CD80 of the present invention is obtained by sequence deletion or sequence mutation based on the natural / wild CD80 molecule. In an alternative embodiment, the engineered co-stimulatory molecule CD80 is a truncated form of the natural / wild CD80 molecule. For example, the engineered co-stimulatory molecule CD80 only contains the extracellular domain and transmembrane domain of the natural / wild CD80; for another example, the engineered co-stimulatory molecule CD80 only contains the extracellular domain and transmembrane domain of the natural / wild CD80.

[0063] In an alternative embodiment, the engineered co-stimulatory molecule CD80 of the present invention is a CD80 fusion protein constructed by fusing the extracellular domain of CD80 with other molecules. In an alternative embodiment, the extracellular domain of the CD80 fusion protein may be the full length or truncated form of the extracellular domain of natural / wild-type CD80 (for example, the extracellular domain of the CD80 fusion protein only contains the IgV domain and the IgC domain); in an alternative embodiment, the extracellular domain of the CD80 fusion protein is designed with site mutations based on the extracellular domain of natural / wild-type CD80. In an alternative embodiment, the CD80 fusion protein is obtained by fusing the extracellular domain of CD80 with the intracellular domain of 4-1BB.

[0064] In an alternative embodiment, the CD80 of the present invention comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16, or a functional fragment thereof, or an extracellular domain composed of the same. For example, the CD80 of the present invention comprises an extracellular domain of the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16. For example, the CD80 of the present invention comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO.16, and the functional fragment contains the IgV domain and the IgC domain. For example, the CD80 of the present invention comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO.16, and the functional fragment contains the IgC domain. For example, the CD80 of the present invention comprises an extracellular domain composed of the amino acid sequence shown in SEQ ID NO.16 and a functional fragment of the amino acid sequence shown in SEQ ID NO.16, and the functional fragment contains the IgV domain and the IgC domain. For example, the CD80 of the present invention comprises an extracellular domain composed of the amino acid sequence shown in SEQ ID NO.16 and a functional fragment of the amino acid sequence shown in SEQ ID NO.16, and the functional fragment contains the IgV domain. Again, for example, the CD80 of the present invention comprises an extracellular domain composed of the amino acid sequence shown in SEQ ID NO.16 and a functional fragment of the amino acid sequence shown in SEQ ID NO.16, and the functional fragment contains the IgC domain.

[0065] In an alternative embodiment, the CD80 of the present invention further comprises the amino acid sequence shown in SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, or a functional fragment thereof, or a transmembrane domain composed of the same.

[0066] In an alternative embodiment, the co-stimulatory molecule according to the present invention may also be selected from at least one member of the tumor necrosis factor family and the B7 family. In an alternative embodiment, the co-stimulatory molecule may further include CD70. In an alternative embodiment, the CD70 comprises the amino acid sequence shown in SEQ ID NO.26 or SEQ ID NO.27, or a functional fragment thereof, or an extracellular domain composed thereof. For example, the CD70 according to the present invention comprises an extracellular domain of the amino acid sequence shown in SEQ ID NO.26 or SEQ ID NO.27. For another example, the CD70 according to the present invention comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO.26. For another example, the CD70 according to the present invention comprises an extracellular domain composed of the amino acid sequence shown in SEQ ID NO.26 and a functional fragment of the amino acid sequence shown in SEQ ID NO.26. For another example, the CD70 according to the present invention comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO.27.

[0067] In an alternative embodiment, the CD70 according to the present invention further comprises the amino acid sequence shown in SEQ ID NO.23, or a functional fragment thereof, or a transmembrane domain composed thereof.

[0068] In an alternative embodiment, the co-stimulatory molecule may further include 4-1BBL. In an alternative embodiment, the 4-1BBL comprises the amino acid sequence shown in SEQ ID NO.17 or SEQ ID NO.18, or a functional fragment thereof, or is composed thereof. For example, the 4-1BBL according to the present invention comprises an extracellular domain of the amino acid sequence shown in SEQ ID NO.16 or SEQ ID NO.17. For another example, the 4-1BBL according to the present invention comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO.16. For another example, the 4-1BBL according to the present invention comprises an extracellular domain composed of the amino acid sequence shown in SEQ ID NO.16 and a functional fragment of the amino acid sequence shown in SEQ ID NO.16. For another example, the 4-1BBL according to the present invention comprises an extracellular domain of a functional fragment of the amino acid sequence shown in SEQ ID NO.17.

[0069] In an alternative embodiment, the 4-1BBL according to the present invention further comprises the amino acid sequence shown in SEQ ID NO.23, or a functional fragment thereof, or a transmembrane domain composed thereof.

[0070] In one embodiment, the amino acid sequence encoded by the RNA encoding a T cell costimulatory molecule comprises at least two functional amino acid sequences. One of the at least two functional amino acid sequences comprises a costimulatory molecule and the other comprises an antigen; the costimulatory molecule is CD80, and the CD80 comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16, or a functional fragment thereof, or an extracellular domain composed thereof.

[0071] In one embodiment, the amino acid sequence encoded by the RNA encoding a T cell costimulatory molecule comprises at least two functional amino acid sequences. One of the at least two functional amino acid sequences comprises a costimulatory molecule and the other comprises an antigen; the costimulatory molecules include CD80 and CD70. In an alternative embodiment, the CD80 comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16, or a functional fragment thereof, or an extracellular domain composed thereof. In an alternative embodiment, the CD70 comprises the amino acid sequence shown in SEQ ID NO.26 or SEQ ID NO.27, or a functional fragment thereof, or is composed thereof.

[0072] In one embodiment, the amino acid sequence encoded by the RNA encoding a T cell costimulatory molecule comprises at least two functional amino acid sequences. One of the at least two functional amino acid sequences comprises a costimulatory molecule and the other comprises an antigen; the costimulatory molecules include CD80 and 4-1BBL. In an alternative embodiment, the CD80 comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16, or a functional fragment thereof, or an extracellular domain composed thereof. In an alternative embodiment, the 4-1BBL comprises the amino acid sequence shown in SEQ ID NO.17 or SEQ ID NO.18, or a functional fragment thereof, or is composed thereof.

[0073] In one embodiment, the amino acid sequence encoded by the RNA encoding a T cell costimulatory molecule comprises at least two functional amino acid sequences. One of the at least two functional amino acid sequences comprises a costimulatory molecule and the other comprises an antigen; the costimulatory molecules include CD80, CD70 and 4-1BBL. In an alternative embodiment, the CD80 comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16, or a functional fragment thereof, or an extracellular domain composed thereof. In an alternative embodiment, the 4-1BBL comprises the amino acid sequence shown in SEQ ID NO.17 or SEQ ID NO.18, or a functional fragment thereof, or is composed thereof. In an alternative embodiment, the CD70 comprises the amino acid sequence shown in SEQ ID NO.26 or SEQ ID NO.27, or a functional fragment thereof, or is composed thereof.

[0074] In one embodiment, the amino acid sequence encoded by the RNA encoding the T cell co-stimulatory molecule comprises at least two functional amino acid sequences. One of the at least two functional amino acid sequences comprises a co-stimulatory molecule and the other comprises an antigen; the co-stimulatory molecule is CD80, and the CD80 comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16, or a functional fragment thereof, or an extracellular domain composed thereof; the CD80 further comprises the amino acid sequence shown in SEQ ID NO.21 or SEQ ID NO.22, or a functional fragment thereof, or a transmembrane domain composed thereof.

[0075] In one embodiment, the amino acid sequence encoded by the RNA encoding the T cell co-stimulatory molecule comprises at least two functional amino acid sequences. One of the at least two functional amino acid sequences comprises a co-stimulatory molecule and the other comprises an antigen; the co-stimulatory molecules include CD80 and CD70. In an alternative embodiment, the CD80 comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16, or a functional fragment thereof, or an extracellular domain composed thereof; the CD80 further comprises the amino acid sequence shown in SEQ ID NO.21 or SEQ ID NO.22, or a functional fragment thereof, or a transmembrane domain composed thereof. In an alternative embodiment, the CD70 comprises the amino acid sequence shown in SEQ ID NO.26 or SEQ ID NO.27, or a functional fragment thereof, or is composed thereof.

[0076] In one embodiment, the amino acid sequence encoded by the RNA encoding the T cell co-stimulatory molecule comprises at least two functional amino acid sequences. One of the at least two functional amino acid sequences comprises a co-stimulatory molecule and the other comprises an antigen; the co-stimulatory molecules include CD80 and 4-1BBL. In an alternative embodiment, the CD80 comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16, or a functional fragment thereof, or an extracellular domain composed thereof; the CD80 further comprises the amino acid sequence shown in SEQ ID NO.21 or SEQ ID NO.22, or a functional fragment thereof, or a transmembrane domain composed thereof. In an alternative embodiment, the 4-1BBL comprises the amino acid sequence shown in SEQ ID NO.17 or SEQ ID NO.18, or a functional fragment thereof, or is composed thereof.

[0077] In one embodiment, the amino acid sequence encoded by the RNA encoding the T cell costimulatory molecule comprises at least two functional amino acid sequences. One of the at least two functional amino acid sequences comprises a costimulatory molecule and the other comprises an antigen; the costimulatory molecules include CD80, CD70, and 4-1BBL. In an alternative embodiment, the CD80 comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16 or a functional fragment thereof, or an extracellular domain consisting thereof; the CD80 further comprises the amino acid sequence shown in SEQ ID NO.21 or SEQ ID NO.22 or a functional fragment thereof, or a transmembrane domain consisting thereof. In an alternative embodiment, the 4-1BBL comprises the amino acid sequence shown in SEQ ID NO.17 or SEQ ID NO.18 or a functional fragment thereof, or consists thereof. In an alternative embodiment, the CD70 comprises the amino acid sequence shown in 4-1BBL or a functional fragment thereof, or consists thereof.

[0078] The costimulatory molecules CD70 or 4-1BBL involved in the present invention may comprise CD70 or 4-1BBL amino acid sequences or fragments thereof that exist and / or do not exist in nature.

[0079] In an alternative embodiment, the costimulatory molecules CD70 or 4-1BBL of the present invention may be wild-type costimulatory molecules CD70 or 4-1BBL, the amino acid sequences of which are identical to those of any natural / wild costimulatory molecule CD70 or 4-1BBL.

[0080] In an alternative embodiment, the costimulatory molecules CD70 or 4-1BBL of the present invention may be engineered costimulatory molecules CD70 or 4-1BBL.

[0081] "Engineered costimulatory molecules CD70 or 4-1BBL" are CD70 or 4-1BBL molecules that are modified or designed and constructed using their principles from natural / wild CD70 or 4-1BBL molecules to obtain specific functions or improved properties. For the specific forms of engineered costimulatory molecules CD70 or 4-1BBL, reference may be made to engineered costimulatory molecule CD80.

[0082] The RNA encoding the costimulatory molecule of the present invention is expressed as a polypeptide / amino acid.

[0083] In one embodiment, in the costimulatory molecule polypeptide / amino acid expressed by the RNA encoding the costimulatory molecule, the costimulatory molecule amino acid sequence is linked or unlinked to the antigen amino acid sequence.

[0084] For example, the co-stimulatory molecule amino acid sequence is not linked to the antigen amino acid sequence. In an alternative embodiment, the nucleotide sequence encoding the co-stimulatory molecule and the nucleotide sequence encoding the antigen are located in two separate and unlinked nucleotide sequences. In an alternative embodiment, the nucleotide sequence encoding the co-stimulatory molecule and the nucleotide sequence encoding the antigen are located within the same nucleotide sequence, but in different open reading frames (ORFs). In an alternative embodiment, the nucleotide sequence encoding the co-stimulatory molecule and the nucleotide sequence encoding the antigen are linked by a nucleotide sequence encoding a cleavable linker, and the two nucleotide sequences are located within the same open reading frame (ORF); the cleavable linker is selected from 2A self-cleaving peptides. Specifically, the cleavable linker is selected from T2A peptide, P2A peptide, E2A peptide or F2A peptide.

[0085] For another example, the co-stimulatory molecule amino acid sequence is linked to the antigen amino acid sequence. In an alternative embodiment, the 3' end of the co-stimulatory molecule amino acid sequence is directly linked to the 5' end of the antigen amino acid sequence. In an alternative embodiment, the 3' end of the co-stimulatory molecule amino acid sequence and the 5' end of the antigen amino acid sequence are linked by a GS linker polypeptide.

[0086] In one embodiment, the nucleotide sequence encoding CD80 and the nucleotide sequence encoding the antigen are located in two separate and unlinked nucleotide sequences, and the CD80 amino acid sequence is not linked to the antigen amino acid sequence. In one embodiment, the nucleotide sequence encoding CD80 and the nucleotide sequence encoding the antigen are located within the same nucleotide sequence, but in different open reading frames (ORFs), and the CD80 amino acid sequence is not linked to the antigen amino acid sequence. In one embodiment, the nucleotide sequence encoding CD80 and the nucleotide sequence encoding the antigen are linked by a nucleotide sequence encoding a cleavable linker, and the two nucleotide sequences are located within the same open reading frame (ORF); the cleavable linker is selected from 2A self-cleaving peptides. In one embodiment, the 3' end of the CD80 amino acid sequence is directly linked to the 5' end of the antigen amino acid sequence. In one embodiment, the 3' end of the CD80 amino acid sequence and the 5' end of the antigen amino acid sequence are linked by a GS linker polypeptide.

[0087] In one embodiment, the nucleotide sequence encoding CD70 and the nucleotide sequence encoding the antigen are located in two separate and unlinked nucleotide sequences, and the CD70 amino acid sequence is not linked to the antigen amino acid sequence. In one embodiment, the nucleotide sequence encoding CD70 and the nucleotide sequence encoding the antigen are located in the same nucleotide sequence but in different open reading frames (ORFs), and the CD70 amino acid sequence is not linked to the antigen amino acid sequence. In one embodiment, the nucleotide sequence encoding CD70 and the nucleotide sequence encoding the antigen are linked by a nucleotide sequence encoding a cleavable linker, and the two nucleotide sequences are located in the same open reading frame (ORF); the cleavable linker is selected from 2A self-cleaving peptides. In one embodiment, the 3' end of the CD70 amino acid sequence is directly linked to the 5' end of the antigen amino acid sequence. In one embodiment, the 3' end of the CD70 amino acid sequence is linked to the 5' end of the antigen amino acid sequence by a GS linker polypeptide.

[0088] In one embodiment, the nucleotide sequence encoding 4-1BBL and the nucleotide sequence encoding the antigen are located in two separate and unlinked nucleotide sequences, and the 4-1BBL amino acid sequence is not linked to the antigen amino acid sequence. In one embodiment, the nucleotide sequence encoding 4-1BBL and the nucleotide sequence encoding the antigen are located in the same nucleotide sequence but in different open reading frames (ORFs), and the 4-1BBL amino acid sequence is not linked to the antigen amino acid sequence. In one embodiment, the nucleotide sequence encoding 4-1BBL and the nucleotide sequence encoding the antigen are linked by a nucleotide sequence encoding a cleavable linker, and the two nucleotide sequences are located in the same open reading frame (ORF); the cleavable linker is selected from 2A self-cleaving peptides. In one embodiment, the 3' end of the 4-1BBL amino acid sequence is directly linked to the 5' end of the antigen amino acid sequence. In one embodiment, the 3' end of the 4-1BBL amino acid sequence is linked to the 5' end of the antigen amino acid sequence by a GS linker polypeptide.

[0089] 2A peptides (2A self-cleaving peptides) are a class of peptide fragments 18-22 amino acid residues in length that can induce the self-cleavage of recombinant proteins containing 2A peptides within cells. In an alternative embodiment, the 2A self-cleaving peptide is selected from T2A peptide, P2A peptide, E2A peptide, or F2A peptide. In an alternative embodiment, the amino acid sequence of the F2A peptide is as shown in SEQ ID NO.11; the amino acid sequence of the P2A peptide is as shown in SEQ ID NO.12; the amino acid sequence of the T2A peptide is as shown in SEQ ID NO.13; the amino acid sequence of the E2A peptide is as shown in SEQ ID NO.14.

[0090] In an alternative embodiment, the GS sequence comprises (GnS)m, (GGGGS)o, GGSGGGGSGG, GGSGGGGG, GSGSGSGS, (Gly)p, (EAAAK)q (where n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; m is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20; o is an integer from 1 to 5; p is an integer from 1 to 40; q is an integer from 1 to 5).

[0091] In an alternative embodiment, the 4-1BBL amino acid sequence or the CD70 amino acid sequence is linked to an antigen amino acid sequence. In an alternative embodiment, the amino acid sequence expressed / encoded by the nucleotide sequence encoding the 4-1BBL is, from the 3'-end to the 5'-end, the amino acid sequence as shown in SEQ ID NO. 19 or SEQ ID NO. 20, the Hinge region of CD8a, and the CD8aTM region, such as the CD8a signal peptide. The amino acid sequence of the CD8a signal peptide is as shown in SEQ ID NO. 25, the amino acid sequence of the CD8a Hinge region is as shown in SEQ ID NO. 23, and the amino acid sequence of the CD8a TM region is as shown in SEQ ID NO. 24. In an alternative embodiment, the amino acid sequence expressed / encoded by the nucleotide sequence of the CD70 is, from the 3'-end to the 5'-end, the amino acid sequence as shown in SEQ ID NO. 19 or SEQ ID NO. 20, the Hinge region of CD8a, and the CD8aTM region, such as the CD8a signal peptide. The amino acid sequence of the CD8a signal peptide is as shown in SEQ ID NO. 25, the amino acid sequence of the CD8a Hinge region is as shown in SEQ ID NO. 23, and the amino acid sequence of the CD8a TM region is as shown in SEQ ID NO. 24.

[0092] In one embodiment, the RNA encoding a T cell co-stimulatory molecule in the present invention comprises: (i) an open reading frame nucleotide sequence encoding a co-stimulatory molecule polypeptide / protein, (ii) an open reading frame nucleotide sequence encoding an antigen amino acid sequence; and (iii) an open reading frame nucleotide sequence encoding a 2A self-cleaving peptide, or, (iv) at least one of an open reading frame nucleotide sequence encoding a GS linker polypeptide; if the open reading frame nucleotide sequences of the above (i)-(iv) are connected, as can be understood by those skilled in the art, the connection order and connection method are aimed at not reducing the activity of the co-stimulatory molecule polypeptide / protein or antigen encoded by the open reading frame nucleotide sequence. For example, if the open reading frame nucleotide sequence encoding the co-stimulatory molecule polypeptide / protein is connected with the open reading frame nucleotide sequence encoding the GS linker polypeptide, resulting in a decrease in the transmembrane speed or inability to achieve transmembrane of at least one co-stimulatory molecule polypeptide / protein with transmembrane function in the cell, thereby affecting the activity, this connection method will not be adopted.

[0093] Regarding the selection of antigens

[0094] "Antigen" according to the disclosure of the present invention encompasses any substance or molecular structure that can bind to an antibody or a T cell receptor. The presence of an antigen in the body can trigger an immune response. Thus, "antigen" encompasses any substance against which an immune response or immune mechanism is directed. This also includes cases where the antigen is processed into antigenic peptides, and the immune response or immune mechanism is directed against one or more antigenic peptides, particularly if presented in the context of MHC molecules. In particular, "antigen" refers to any substance that specifically reacts with an antibody or a T lymphocyte (T cell), preferably a peptide or a protein. According to the disclosure of the present invention, the term "antigen" includes any molecule containing at least one epitope (such as a T cell epitope). Preferably, an antigen in the context of the disclosure of the present invention is a molecule that optionally induces an immune response after processing, and the immune response is preferably specifically directed against the antigen (including cells expressing the antigen). In one embodiment, the antigen is a disease-related antigen, such as a tumor antigen, a viral antigen, or a bacterial antigen, or an epitope derived from such an antigen.

[0095] The pathogen antigens described in the present invention are selected from at least one of HIV Env, RSV F, influenza HA, EBV gp350, EBV LMP1, EBV LMP2, EBV EBNA1, EBV EBNA3, CMV gB, CMV UL128, CMV UL130, CMV UL131A, CMV gH, CMV gL, Lyme disease OspA, pertussis toxin, dengue E, SARS S, MERS S, Zaire Ebola virus GP, Sudan Ebola virus GP, Marburg virus GP, hantavirus Gn, hantavirus Gc, measles H, Zika envelope domain III, malaria CSP, malaria Pfs25, MenB fHbp, MenB NadA, MenB NHBA, Nipah virus F, Nipah virus G, rotavirus VP4, rotavirus VP8, human papillomavirus antigen, HBeAg, hepatitis B virus pre-S1 protein, hepatitis B virus pre-S2 protein, hepatitis B virus HBsAg protein, hepatitis B virus HBcAg protein, hand, foot and mouth virus antigen.

[0096] In an alternative embodiment, the antigen comprises at least antigen A and antigen B. In an alternative embodiment, the antigen A is the hepatitis B virus HBsAg protein; the antigen B is HBcAg. In an alternative embodiment, the antigen is selected from influenza A virus hemagglutinin and human respiratory syncytial virus F protein. In an alternative embodiment, the antigen A is the HSV gD protein; the antigen B is the human papillomavirus antigen.

[0097] In an alternative embodiment, the antigen is the human papillomavirus antigen. In an alternative embodiment, the human papillomavirus antigen is selected from at least one of the human papillomavirus early regulatory proteins E1, E2, E6 and E7. In an alternative embodiment, the pathogen in the present invention is the human papillomavirus selected from one or more of human papillomavirus type 6, human papillomavirus type 11, human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 31, human papillomavirus type 33, human papillomavirus type 45, human papillomavirus type 52 and human papillomavirus type 58. The human papillomavirus antigen is the human papillomavirus early regulatory proteins E6 and E7.

[0098] The amino acid sequences of the early regulatory proteins E6 and E7 of human papillomavirus types 6, 11, 16, 18, 31, 33, 45, 52 and 58 are shown in Table 1.

[0099] Table 1 Amino acid and nucleotide sequences of E6 and E7 of human papillomavirus subtypes

[0100] Human papillomavirus subtype E6 amino acid sequence E7 amino acid sequence 6 Uniprot No.P06462 Uniprot No.P06464 11 Uniprot No.P04019 Uniprot No.P04020 16 Uniprot No.P03126 Uniprot No.P03129 18 Uniprot No.P06463 Uniprot No.P06788 31 Uniprot No.P17386 Uniprot No.Q6T377 33 Uniprot No.P06427 Uniprot No.P06429 45 Uniprot No.P21735 Uniprot No.P21736 52 Uniprot No.P36814 Uniprot No.P36831 58 Uniprot No.P26555 Uniprot No.P26557

[0101] In the present invention, "Uniprot" is the English abbreviation of Universal Protein, which is the most information-rich and resource-rich protein database (https: / / www.uniprot.org / ); "UniprotNo." is the number of the protein sequence in this database, corresponding to the unique protein sequence in this database.

[0102] In an alternative embodiment, the antigen A is the human papillomavirus type 18 early regulatory protein E6; the antigen B is the human papillomavirus type 18 early regulatory protein E7. In an alternative embodiment, the antigen A is the human papillomavirus type 16 early regulatory protein E6; the antigen B is the human papillomavirus type 16 early regulatory protein E7. In an alternative embodiment, the antigen A is the human papillomavirus type 18 early regulatory protein E7; the antigen B is the human papillomavirus type 16 early regulatory protein E6. In an alternative embodiment, the antigen A is the human papillomavirus type 16 early regulatory protein E7; the antigen B is the human papillomavirus type 18 early regulatory protein E6.

[0103] In an alternative embodiment, the amino acid sequence of the human papillomavirus type 18 early regulatory protein E6 is as shown in Uniprot NO. P06463; in an alternative embodiment, the amino acid sequence of the human papillomavirus type 18 early regulatory protein E7 is as shown in Uniprot NO. P06788; in an alternative embodiment, the amino acid sequence of the human papillomavirus type 16 early regulatory protein E6 is as shown in Uniprot NO. P03126 (SEQ ID NO. 8); in an alternative embodiment, the amino acid sequence of the human papillomavirus type 16 early regulatory protein E7 is as shown in Uniprot NO. P03129 (SEQ ID NO. 7).

[0104] The tumor antigens of the present invention are selected from at least one of claudin 6 (CLDN6), Melanoma antigen A3 (MAGE-A3), Melanoma antigen 4 (MAGE-A4), Preferentially Expressed Antigen In Melanoma (PRAME), Melanoma antigen C1 (MAGE-C1), MAGE-C2, New York esophageal squamous cell carcinoma-1 (NY-ESO-1), Tyrosinase, gp100, TPTE, PSA, AFP, GPC3, Survivin, Melan-A, Kita-kyushu lung cancer antigen 1 (KK-LC-1), KRAS G12, KRAS G13, and KRAS Q61.

[0105] In an alternative embodiment, the co-stimulatory molecule polypeptide / protein contained in the amino acids encoded by the RNA encoding the T cell co-stimulatory molecule increases the immunogenicity of the antigen by at least 10% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200%). In an alternative embodiment, the co-stimulatory molecule polypeptide / protein contained in the amino acids encoded by the RNA encoding the T cell co-stimulatory molecule increases the immune stimulation and / or immune response of the encoded antigen by 10-20%, 10-30%, 10-40%, 10-50%, 10-60%, 10-70%, 10-80%, 10-90%, 10-100%, 10-200%, 20-30%, 20-40%, 20-50%, 20-60%, 20-70%, 20-80%, 20-90%, 20-100%, 20%-200%, 50-60%, 50-70%, 50-80%, 50-90%, 50-100%, or 50-200%.

[0106] In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises at least one nucleic acid sequence encoding the following amino acid sequences: (i) an amino acid sequence comprising the co-stimulatory molecule CD80 and the co-stimulatory molecule CD70; (ii) an amino acid sequence comprising an antigen; the antigen being the human papillomavirus type 16 early regulatory proteins E6 and E7. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises three nucleic acid sequences, a first nucleic acid sequence encoding the co-stimulatory molecule CD80 and the human papillomavirus type 16 early regulatory protein E7, a second nucleic acid protein encoding the human papillomavirus type 16 early regulatory protein E6, and a third nucleic acid protein encoding the co-stimulatory molecule CD70; the form of connection between the functional regions of the protein encoded by the first nucleic acid sequence is: CD80-E7. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises four nucleic acid sequences, a first nucleic acid sequence encoding the co-stimulatory molecule CD80, a second nucleic acid sequence encoding the human papillomavirus type 16 early regulatory protein E7, a third nucleic acid protein encoding the human papillomavirus type 16 early regulatory protein E6, and a fourth nucleic acid protein encoding the co-stimulatory molecule CD70. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises one nucleic acid sequence, and the form of connection between the functional regions of the protein encoded by the nucleic acid sequence is: CD80-E7…P2A…E6…T2A…CD70.

[0107] In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises at least one nucleic acid sequence encoding the following amino acid sequences: (i) an amino acid sequence comprising the co-stimulatory molecule CD80, the co-stimulatory molecule CD70, and the co-stimulatory molecule 4-1BBL; (ii) an amino acid sequence comprising an antigen; the antigen being the human papillomavirus type 16 early regulatory proteins E6 and E7. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises three nucleic acid sequences, a first nucleic acid sequence encoding the co-stimulatory molecule CD80 and the human papillomavirus type 16 early regulatory protein E7, a second nucleic acid sequence encoding the co-stimulatory molecule 4-1BBL and the human papillomavirus type 16 early regulatory protein E6, and a third nucleic acid sequence encoding the co-stimulatory molecule CD70; the form of connection between the functional regions of the protein encoded by the first nucleic acid sequence is: CD80-E7, and the form of connection between the functional regions of the protein encoded by the second nucleic acid sequence is: 4-1BBL-E6. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises five nucleic acid sequences, a first nucleic acid sequence encoding the co-stimulatory molecule CD80, a second nucleic acid sequence encoding the human papillomavirus type 16 early regulatory protein E7, a third nucleic acid sequence encoding the human papillomavirus type 16 early regulatory protein E6, a fourth nucleic acid sequence encoding the co-stimulatory molecule CD70, and a fourth nucleic acid sequence encoding the co-stimulatory molecule 4-1BBL. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises one nucleic acid sequence, and the form of connection between the functional regions of the protein encoded by the nucleic acid sequence is: CD80-E7…P2A…4-1BBL-E6…T2A…CD70.

[0108] In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises at least one nucleic acid sequence encoding the following amino acid sequences: (i) an amino acid sequence comprising the co-stimulatory molecule CD80 and the co-stimulatory molecule CD70; (ii) an amino acid sequence comprising an antigen; the antigen is selected from the GPC3 protein, a truncated form of the GPC3 protein, or a mutant of the GPC3 protein. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises three nucleic acid sequences, a first nucleic acid sequence encoding the co-stimulatory molecule CD80, a second nucleic acid sequence encoding a truncated form of the GPC3 protein, and a third nucleic acid sequence encoding the co-stimulatory molecule CD70. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises three nucleic acid sequences, a first nucleic acid sequence encoding the co-stimulatory molecule CD80 and a truncated form 1 of the GPC3 protein, a second nucleic acid sequence encoding a truncated form 2 of the GPC3 protein, and a third nucleic acid sequence encoding the co-stimulatory molecule CD70; the amino acid sequences of the truncated form 1 of the GPC3 protein and the truncated form 2 of the GPC3 protein may be the same or different. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises one nucleic acid sequence, and the connection form between the functional regions of the protein encoded by the nucleic acid sequence is: CD80-truncated form 1 of the GPC3 protein…P2A…truncated form 2 of the GPC3 protein…T2A…CD70.

[0109] In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises at least one nucleic acid sequence encoding the following amino acid sequences: (i) an amino acid sequence comprising the co-stimulatory molecule CD80, the co-stimulatory molecule CD70, and the co-stimulatory molecule 4-1BBL; (ii) an amino acid sequence comprising an antigen selected from the GPC3 protein, a truncated form of the GPC3 protein, or a mutant of the GPC3 protein. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises four nucleic acid sequences, a first nucleic acid sequence encoding the co-stimulatory molecule CD80, a second nucleic acid sequence encoding a truncated form of the GPC3 protein, a third nucleic acid sequence encoding the co-stimulatory molecule CD70, and a fourth nucleic acid sequence encoding the co-stimulatory molecule 4-1BBL. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises three nucleic acid sequences, a first nucleic acid sequence encoding the co-stimulatory molecule CD80 and a truncated form 1 of the GPC3 protein, a second nucleic acid sequence encoding 4-1BBL and a truncated form 2 of the GPC3 protein, and a third nucleic acid sequence encoding the co-stimulatory molecule CD70; the amino acid sequences of the truncated form 1 of the GPC3 protein and the truncated form 2 of the GPC3 protein may be the same or different; the connection form between the functional regions of the protein encoded by the first nucleic acid sequence is: CD80 - truncated form 1 of the GPC3 protein; the connection form between the functional regions of the protein encoded by the second nucleic acid sequence is: 4-1BBL - truncated form 2 of the GPC3 protein. In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule comprises one nucleic acid sequence, and the connection form between the functional regions of the protein encoded by the nucleic acid sequence is: CD80 - truncated form 1 of the GPC3 protein…P2A…4-1BBL - truncated form 2 of the GPC3 protein…T2A…CD70.

[0110] In an alternative embodiment, the RNA encoding the T cell co-stimulatory molecule is selected from mRNA or circular RNA.

[0111] In an alternative embodiment, based on the provided mRNA open reading frame sequence, those of ordinary skill in the art will be able to obtain the corresponding circular RNA open reading frame sequence and, in accordance with the content of publicly available documents such as CN202180048567.4, prepare a complete circular RNA sequence capable of encoding the same amino acid sequence. In an alternative embodiment, based on the provided mRNA sequence, those of ordinary skill in the art will be able to obtain the corresponding DNA sequence (e.g., uracil is converted to thymine). Similarly, based on the provided DNA sequence, those of ordinary skill in the art will obtain the corresponding RNA sequence (e.g., thymine is converted to uracil). In an alternative embodiment, based on the provided RNA or DNA sequence, those of ordinary skill in the art will be able to obtain the corresponding amino acid sequence.

[0112] In an alternative embodiment, the mRNA in the present invention comprises a sequence consisting of a 5' cap, a 5' UTR, an ORF, a 3' UTR, and a 3' poly(A) tail in sequence from the 5' end to the 3' end.

[0113] In an alternative embodiment, the mRNA in the present invention comprises at least one ORF sequence. For example, the mRNA comprises two ORF sequences, and the two ORF sequences are arranged in sequence between the 5' UTR and the 3' UTR; specifically, the mRNA comprises a sequence consisting of a 5' cap, a 5' UTR, ORF(1), ORF(2), a 3' UTR, and a 3' poly(A) tail in sequence from the 5' end to the 3' end. For example, the mRNA comprises n ORF sequences (n is a natural number), and the n ORF sequences are arranged in sequence between the 5' UTR and the 3' UTR; specifically, the mRNA comprises a sequence consisting of a 5' cap, a 5' UTR, ORF(1), ORF(2), …, ORF(n - 1), ORF(n), a 3' UTR, and a 3' poly(A) tail in sequence from the 5' end to the 3' end. For example, the mRNA comprises two ORF sequences, and the two ORF sequences are located in two independent nucleotide sequences respectively; specifically, the mRNA comprises two independent (unconnected) nucleotide sequences, denoted as mRNA1 and mRNA2 respectively. mRNA1 comprises a sequence consisting of a 5' cap, a 5' UTR, ORF(1), a 3' UTR, and a 3' poly(A) tail in sequence from the 5' end to the 3' end, and mRNA2 comprises a sequence consisting of a 5' cap, a 5' UTR, ORF(2), a 3' UTR, and a 3' poly(A) tail in sequence from the 5' end to the 3' end; among several independent nucleotide sequences, the 5' cap, the 5' UTR, the 3' UTR, and the 3' poly(A) tail may be the same as each other or different from each other.

[0114] In alternative embodiments, the nucleotide molecule can optimize the mRNA sequence by sequence optimization means to improve the properties related to the expression efficacy after in vivo administration: for example, increasing mRNA stability, increasing the translation efficacy in the target tissue, reducing the number of truncated proteins expressed, improving the folding of the expressed protein or preventing its misfolding, reducing the toxicity of the expression product, reducing cell death caused by the expression product, increasing and / or reducing protein aggregation, to obtain an mRNA with improved properties. The purposes of sequence optimization also include: optimizing the formulation and delivery characteristics of nucleotide-based therapeutic agents while maintaining structural and functional integrity; overcoming the expression threshold; increasing the expression rate; half-life and / or protein concentration; optimizing protein localization; and avoiding adverse biological responses such as immune responses and / or degradation pathways. Sequence optimization means include: (1) codon optimization according to the codon frequency in a specific organ and / or host organism to ensure proper folding and appropriate expression; (2) adjusting the G / C content to increase mRNA stability or reduce secondary structure; (3) minimizing tandem repeat codons or base runs that may damage the gene construct or expression; (4) customizing transcriptional and translational control regions; (5) reducing or eliminating problematic secondary structures within the polynucleotide.

[0115] In alternative embodiments, the 5'-end cap is selected from ARCA, mCAP, dmCAP, m7G(5'')ppp(5'')(2''OMeA)pG, tmCAP, m7(3''OMeG)(5'')ppp(5'')(2''OMeA)pG, m7(3''OMeG)(5'')ppp(5'')(2''OMeG)pG, dmCAP or m7G(5'')ppp(5'')(2''OMeG)pG; in alternative embodiments, the 5'-end cap is m7Gppp(5’)(2’-OMeA)pG.

[0116] In alternative embodiments, the length of the 5'-UTR is preferably 10 to 200 nucleotides. In alternative embodiments, the length of the 5'-UTR is 15 to 100 nucleotides. In alternative embodiments, the nucleotide sequence of the 5'-UTR is as shown in SEQ ID NOs. 1 to 3. In alternative embodiments, the nucleotide sequence of the 5'-UTR is as shown in SEQ ID NO. 1.

[0117] In alternative embodiments, the 3'-UTR sequence is as shown in SEQ ID NOs. 4 to 6. In alternative embodiments, the 3'-UTR sequence is as shown in SEQ ID NO. 4.

[0118] In an alternative embodiment, one or more uridines in the mRNA are replaced with a modified nucleoside. In an alternative embodiment, the modified nucleoside is pseudouridine (ψ), N1-methyl-pseudouridine (m1ψ), or 5-methyl-uridine (m5U). In an alternative embodiment, the modified nucleoside is N1-methyl-pseudouridine (m1ψ).

[0119] In an alternative embodiment, the nucleotide sequence encoding CD80 in the RNA encoding the T cell co-stimulatory molecule is as shown in at least one of SEQ ID NOs: 28-31, 46-51.

[0120] In an alternative embodiment, the nucleotide sequence encoding CD70 in the RNA encoding the T cell co-stimulatory molecule is as shown in at least one of SEQ ID NOs: 52-54.

[0121] In an alternative embodiment, the nucleotide sequence encoding 4-1BBL in the RNA encoding the T cell co-stimulatory molecule is as shown in at least one of SEQ ID NOs: 55-58.

[0122] Regarding the protein composition

[0123] In one embodiment, the present invention provides a protein composition, which includes a polypeptide encoded by the RNA encoding the T cell co-stimulatory molecule as described above.

[0124] In one embodiment, the present invention provides a protein composition, which includes the amino acid sequence of the co-stimulatory molecule;

[0125] The co-stimulatory molecule is selected from at least one of the members of the B7 family and at least one of the members of the tumor necrosis factor family; the members of the B7 family are selected from at least one of CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and HHLA2; preferably, the members of the B7 family are selected from at least one of CD80, CD86, B7-DC, PD-L1, and HHLA2; the members of the tumor necrosis factor family are selected from at least one of TNF-β, TNF-α, LT-β, CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, and GITRL; preferably, the members of the tumor necrosis factor family are selected from at least one of CD252, CD154, CD70, 4-1BBL, and GITRL.

[0126] In one embodiment, the present invention provides a protein composition, which includes the co-stimulatory molecule CD80 and the co-stimulatory molecule CD70.

[0127] In one embodiment, the present invention provides a protein composition, which comprises co-stimulatory molecule CD80, co-stimulatory molecule CD70, and co-stimulatory molecule 4-1BBL.

[0128] In one embodiment, the present invention provides a protein composition, which comprises (i) the amino acid sequence of a co-stimulatory molecule; (ii) the amino acid sequence of an antigen; the co-stimulatory molecule is selected from at least one member of the B7 family and at least one member of the tumor necrosis factor family; the members of the B7 family are selected from at least one of CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6, and HHLA2; preferably, the members of the B7 family are selected from at least one of CD80, CD86, B7-DC, PD-L1, and HHLA2; the members of the tumor necrosis factor family are selected from at least one of TNF-β, TNF-α, LT-β, CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, and GITRL; preferably, the members of the tumor necrosis factor family are selected from at least one of CD252, CD154, CD70, 4-1BBL, and GITRL.

[0129] In one embodiment, the present invention provides a protein composition, which comprises co-stimulatory molecule CD80, co-stimulatory molecule CD70, co-stimulatory molecule 4-1BBL, and an antigen; the antigen is a pathogen antigen or a tumor antigen.

[0130] In one embodiment, the present invention provides a protein composition, which comprises co-stimulatory molecule CD80, co-stimulatory molecule CD70, and an antigen; the antigen is a pathogen antigen or a tumor antigen.

[0131] In one embodiment, the protein composition comprises co-stimulatory molecule CD80, co-stimulatory molecule CD70, and an antigen; the antigen is selected from at least one of the early regulatory proteins E1, E2, E6, and E7 of human papillomavirus. In one embodiment, the antigen is the early regulatory proteins E6 and E7 of human papillomavirus type 16.

[0132] In one embodiment, the protein composition comprises co-stimulatory molecule CD80, co-stimulatory molecule CD70, and an antigen; the antigen is selected from at least one of the early regulatory proteins E1, E2, E6, and E7 of human papillomavirus. In one embodiment, the antigen is the early regulatory proteins E6 and E7 of human papillomavirus type 18.

[0133] In one embodiment, the protein composition comprises a costimulatory molecule CD80, a costimulatory molecule CD70, a costimulatory molecule 4-1BBL, and an antigen; the antigen is selected from at least one of the human papillomavirus early regulatory proteins E1, E2, E6, and E7. In one embodiment, the antigen is the human papillomavirus type 16 early regulatory proteins E6 and E7.

[0134] In one embodiment, the protein composition comprises a costimulatory molecule CD80, a costimulatory molecule CD70, a costimulatory molecule 4-1BBL, and an antigen; the antigen is selected from at least one of the human papillomavirus early regulatory proteins E1, E2, E6, and E7. In one embodiment, the antigen is the human papillomavirus type 18 early regulatory proteins E6 and E7.

[0135] In one embodiment, the protein composition comprises a costimulatory molecule CD80, a costimulatory molecule CD70, and an antigen; the antigen is selected from at least one of GPC3 protein, a truncated form of GPC3 protein, and a mutant of GPC3 protein.

[0136] In one embodiment, the protein composition comprises a costimulatory molecule CD80, a costimulatory molecule CD70, a costimulatory molecule 4-1BBL, and an antigen; the antigen is selected from at least one of GPC3 protein, a truncated form of GPC3 protein, and a mutant of GPC3 protein.

[0137] Regarding the vaccine

[0138] In one embodiment, the present invention provides a vaccine, characterized in that the vaccine comprises the RNA encoding a T cell costimulatory molecule as described above, or the protein composition as described above.

[0139] In one embodiment, the vaccine comprises the RNA encoding a T cell costimulatory molecule as described above.

[0140] In one embodiment, the vaccine comprises the protein composition as described above.

[0141] In one embodiment, the vaccine is a protein vaccine, and the vaccine comprises the protein composition and an antigen as described above. In an alternative embodiment, the vaccine comprises an adjuvant, and the adjuvant is selected from aluminum adjuvant, MF59 adjuvant, AS01 adjuvant, CpG adjuvant, etc. In an alternative embodiment, the vaccine may further comprise a stabilizer, and / or a buffer.

[0142] The stabilizer in the protein vaccine is selected from some saccharides (such as sucrose, lactose, etc.) and amino acids (such as glycine, etc.), etc., which can prevent the antigen protein from aggregating and denaturing during processes such as lyophilization.

[0143] In the field of protein vaccines, phosphate buffered saline (PBS) is a commonly used buffer system. It can maintain the pH stability of the vaccine, keep the vaccine in an appropriate pH environment during storage and transportation, and prevent the denaturation of antigen proteins and other components.

[0144] In one embodiment, the vaccine is a nucleotide vaccine, and the vaccine includes the aforementioned RNA encoding a T cell co-stimulatory molecule. In an alternative embodiment, the vaccine includes the RNA encoding a T cell co-stimulatory molecule and a delivery formulation that encapsulates the RNA.

[0145] In an alternative embodiment, the delivery formulation includes lipid nanoparticles or cationic liposomes.

[0146] In an alternative embodiment, the present invention provides a method for preparing the vaccine according to any one of the aforementioned embodiments, characterized in that it includes: mixing the RNA encoding a T cell co-stimulatory molecule and a delivery formulation to form a vaccine; the delivery formulation includes lipid nanoparticles or cationic liposomes.

[0147] In an alternative embodiment, the RNA encoding a T cell co-stimulatory molecule contains at least 2 independent nucleotide sequences, and the method for preparing the vaccine includes: separately mixing each independent nucleotide with a delivery formulation, and then mixing the delivery formulations encapsulating each independent nucleotide at a mass ratio of (10 - 1):(1 - 10) to form a vaccine.

[0148] In an alternative embodiment, the present invention provides a biomaterial, which includes any one of an expression cassette, a vector, an engineered bacterium, or a cell line, and the biomaterial contains or expresses the RNA encoding a T cell co-stimulatory molecule according to any one of the aforementioned items.

[0149] In an alternative embodiment, the present invention provides a method, which includes administering an effective amount of at least one of the vaccine according to any one of the aforementioned items, the RNA encoding a T cell co-stimulatory molecule according to any one of the aforementioned items, or the biomaterial according to any one of the aforementioned items to a subject, so as to induce a cellular immune response or a humoral immune response in the subject.

[0150] In an alternative embodiment, the method includes administering at least 2 effective amounts of at least one of the vaccine according to any one of the aforementioned items, the RNA encoding a T cell co-stimulatory molecule according to any one of the aforementioned items, or the biomaterial according to any one of the aforementioned items to a subject.

[0151] In an alternative embodiment, the time interval between the first administration and the second administration in the at least 2 administrations is not less than 14 days.

[0152] In an alternative embodiment, the subject has a weakened immune system; in an alternative embodiment, the age of the subject is not higher than 5 years old or not lower than 65 years old.

[0153] In an alternative embodiment, the effective amount of the present invention is at least one of the vaccines as described in any one of the foregoing, the RNA encoding a T cell costimulatory molecule as described in any one of the foregoing, or the biomaterial as described in any one of the foregoing, which is as low as 40 μg, 30 μg, 25 μg, 20 μg, 15 μg, 10 μg, 5 μg, 3 μg or 1 μg.

[0154] Example 1: Process for preparing LNP from RNA

[0155] A lipid nanoparticle comprising RNA encoding an antigen, wherein the lipid nanoparticle comprises 50% Dlin-MC3-DMA, 20% DOPG, 29% cholesterol and 1% PEG-DMG in terms of molar percentage.

[0156] The preparation method is as follows: (a) Dissolve the RNA in a citrate buffer solution with a pH of 4, and adjust the concentration to 0.1 mg / ml to obtain an aqueous phase.

[0157] (b) Dissolve Dlin-MC3-DMA, DOPG, cholesterol and PEG-DMG in anhydrous ethanol according to the formulated amounts, and adjust the concentration of the lipid components in the organic phase to 6 mg / mL to obtain an organic phase.

[0158] (c) Mix the aqueous phase of step (a) and the organic phase of step (b) at a volume ratio of 1:3 using a microfluidic device, and mix at a flow rate of 12 mL / min. Immediately dilute the mixture 100 times with a PBS solution with a pH of 7.4, and use tangential flow filtration (TFF) to remove the ethanol component in the solution, and then concentrate to a concentration of 55 μg / ml of mRNA in the system to obtain a lipid nanoparticle comprising RNA encoding an antigen.

[0159] Example 2

[0160] In this experimental example, the expression of RNA encoding a costimulatory molecule at the in vitro cell level was detected. The specific method is as follows: Digest HEK293 cells cultured for more than 24 hours and seed them into a 6-well plate. After culturing at 37 °C for 24 hours, observe the cell state under a microscope. When the cell confluence reaches more than 80%, mRNA transfection can be carried out. Transfect the corresponding mRNA into HEK293 cells using the lipofectamine2000 kit (5 μg of mRNA is transfected into each well), and the specific operation refers to the kit instructions.

[0161] After adding mRNA, continue to culture at 37 °C for 24 hours, digest HEK293 cells, and stain with fluorescently labeled anti-mouse CD80, 4-1BBL, and CD70 antibodies respectively. Flow cytometry was used to detect the expression of the target protein.

[0162] In this example, the RNA information for detecting the expression at the in vitro cell level is as follows: sample LRIO-094 (the sequence of the RNA encoding the costimulatory molecule is as shown in SEQ ID NO. 34), sample LRIO-097 (the sequence of the RNA encoding the costimulatory molecule is as shown in SEQ ID NO. 37), sample LRIO-098 (the sequence of the RNA encoding the costimulatory molecule is as shown in SEQ ID NO. 38), sample LRIO-099 (the sequence of the RNA encoding the costimulatory molecule is as shown in SEQ ID NO. 39).

[0163] In addition to the above open reading frame sequences, the RNA in this example also includes a 5' cap (m7Gppp(5')), a 5' UTR (as shown in SEQ ID NO. 1), a 3' UTR (as shown in SEQ ID NO. 4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil in the RNA is replaced with 5' pseudouracil.

[0164] The costimulatory molecule polypeptide expressed by sample LRIO-094 includes CD80, and the antigens are the E6 and E7 proteins of HPV16. The connection form between the functional regions in the costimulatory molecule polypeptide expressed by sample LRIO-094 is: CD80-E7…P2A…E6.

[0165] The costimulatory molecule polypeptide expressed by sample LRIO-097 includes CD80 and 4-1BBL, and the antigens are the E6 and E7 proteins of HPV16. The connection form between the functional regions in the costimulatory molecule polypeptide expressed by sample LRIO-097 is: CD80-E7…P2A…4-1BBL-E6.

[0166] The costimulatory molecule polypeptide expressed by sample LRIO-098 includes CD80 and CD70, and the antigens are the E6 and E7 proteins of HPV16. The connection form between the functional regions in the costimulatory molecule polypeptide expressed by sample LRIO-098 is: CD80-E7…P2A…E6…T2A…CD70.

[0167] The costimulatory molecule polypeptide expressed by sample LRIO-099 includes CD70 and 4-1BBL, and the antigens are the E6 and E7 proteins of HPV16. The connection form between the functional regions in the costimulatory molecule polypeptide expressed by sample LRIO-099 is: E7…P2A…4-1BBL-E6…T2A…CD70.

[0168] The above-mentioned "connection form between each functional region" describes the order of each functional region of the polypeptide expressed by RNA from the 3'-end to the 5'-end of the amino acid sequence; for the functional regions connected by "-", not only the nucleotide sequences encoding them are connected respectively, but also the amino acid sequences are connected respectively; for the functional regions connected by "...", the nucleotide sequences encoding them are connected respectively, and the amino acid sequences are not connected.

[0169] In this example, the co-stimulatory molecules CD80, CD70, and 4-1BBL are all murine molecules; among them, CD80 includes the sequences shown in SEQ ID NO.15 and SEQ ID NO.21 in the amino acid sequence; the amino acid sequence of CD70 is as shown in SEQ ID NO.19; the amino acid sequence of 4-1BBL is the sequence composed of SEQ ID NO.25, SEQ ID NO.17, SEQ ID NO.23, and SEQ ID NO.24.

[0170] In this example, the antigens E6 and E7 are from HPV16, and their amino acid sequences are as shown in SEQ ID NO.8 and SEQ ID NO.7 respectively.

[0171] For example, the connection form between each functional region in the co-stimulatory molecule polypeptide expressed by the sample LRIO-099, "E7…P2A…4-1BBL-E6…T2A…CD70", means that the sample LRIO-099 is a polynucleotide sequence, and the functional regions of the co-stimulatory molecule polypeptide expressed by it (which can also be expressed as "the amino acid sequence encoded by the sample LRIO-099") from the 3'-end to the 5'-end are E7, P2A, 4-1BBL, E6, T2A, and CD70 respectively. Specifically, the co-stimulatory molecule polypeptide expressed by the sample LRIO-099 includes two co-stimulatory molecules, 4-1BBL and CD70, two antigens, HPVE6 protein and E7 protein, and two self-cleaving peptides, P2A and T2A. Among them, the polypeptide is divided into three non-connected segments, namely E7, 4-1BBL-E6, and CD70.

[0172] The expression of RNA encoding co-stimulatory molecules at the in vitro cell level is shown in Figure 1 . The results show that more than 40% of the cells express the encoded co-stimulatory molecules.

[0173] Example 3

[0174] C57BL / 6 mice (female, 5-6 weeks old, average body weight 15-20 g, purchased from Zhuhai Biosino Biotechnology Co., Ltd.) were selected for in vivo anti-tumor efficacy evaluation.

[0175] The experimental mice were subcutaneously inoculated with TC-1 tumor cells. When the average tumor volume reached 100 mm3 Around, mice were randomly divided into 7 groups according to tumor volume, with the coefficient of variation (CV) of tumor volume ≤ 1 / 3. The day of grouping was defined as D0. The seven groups were respectively denoted as the NC group (injected with empty LNP), the LRIO-051 group, the LRIO-093 group, the LRIO-097 group, the LRIO-098 group, the LRIO-099 group, and the LRIO-087 group, with 6 mice in each group. On the 0th, 7th, and 14th days of the experiment, each mouse in each group was intramuscularly injected with 5 μg / mouse, and the tumor volume was measured on the 0th, 3rd, 6th, 11th, 13th, 17th, 20th, and 25th days after administration.

[0176] Among them, the LRIO-093 group, the LRIO-097 group, the LRIO-098 group, and the LRIO-099 group were injected with lipid nanoparticles containing RNA encoding co-stimulatory molecules. For the RNA information of the samples used in the LRIO-097 group, the LRIO-098 group, and the LRIO-099 group, refer to Sample LRIO-097, Sample LRIO-098, and Sample LRIO-099 in Example 2; the sample RNA used in the LRIO-093 group is shown as SEQ ID NO.35. The lipid nanoparticles injected into the LRIO-093 group, the LRIO-097 group, the LRIO-098 group, and the LRIO-099 group were prepared according to the method described in Example 1.

[0177] In this example, the co-stimulatory molecules CD80, CD70, and 4-1BBL are all murine molecules; among them, CD80 includes the sequences shown in SEQ ID NO.15 and SEQ ID NO.21; the amino acid sequence of CD70 is shown as SEQ ID NO.19; the amino acid sequence of 4-1BBL includes the sequences composed of SEQ ID NO.25, SEQ ID NO.17, SEQ ID NO.23, and SEQ ID NO.24.

[0178] In this example, the antigens E6 and E7 in the LRIO-093 group, the LRIO-097 group, the LRIO-098 group, and the LRIO-099 group are from HPV16, and their amino acid sequences are shown as SEQ ID NO.8 and SEQ ID NO.7 respectively.

[0179] The co-stimulatory molecule polypeptides expressed in the LRIO-093 group include CD80, 4-1BBL, and CD70, and the antigens are the E6 protein and E7 protein from HPV16. Among them, the connection form between the functional regions in the co-stimulatory molecule polypeptides expressed in Sample LRIO-097 is: CD80-E7…P2A…4-1BBL-E6…T2A…CD70.

[0180] The sample used in the LRIO-087 group was an HPV mRNA vaccine. The mRNA sequence used encoded the E6 and E7 proteins of the HPV16 strain, and its sequence was as shown in SEQ ID NO.40 (refer to Green Leaf Pharmaceutical's 2023-HPV16 E6 E7-based mRNA vaccine is therapeutic in mice bearing aggressive HPV-positive lesions). The lipid nanoparticles injected in the 087 group were prepared according to the method described in Example 1.

[0181] The sample used in the LRIO-051 group was an HPV mRNA vaccine. The mRNA sequence used encoded a fusion protein of MHC I and the E6 and E7 proteins of the HPV16 strain, which was a mixture of the mRNAs shown in SEQ ID NO.9 and SEQ ID NO.10 at a weight ratio of 1:1 (refer to BioNTech's 2019-HPV16 RNA-LPX vaccine mediates complete regression of aggressively growing HPV-positive mouse tumors and establishes protective T cell memory and 2022-Local radiotherapy and E7 RNA-LPX vaccination show enhanced therapeutic efficacy in preclinical models of HPV16+ cancer). The lipid nanoparticles injected in the 051 group were prepared according to the method described in Example 1.

[0182] The experimental results are shown in Figure 2 the following. Compared with the NC group, all other experimental groups could inhibit tumor growth; among them, the groups with the combination of costimulatory molecules CD80 and CD70 (LRIO-098 group and LRIO-093 group) had a better effect on inhibiting tumor growth than the samples of other groups.

[0183] Example 4

[0184] Digest the cultured HEK293 cells and seed them into a 6-well plate. After culturing at 37 °C for 24 hours, observe the cell status under a microscope. When the cell confluence reaches more than 80%, mRNA transfection can be carried out. Transfect the corresponding mRNA into HEK293 cells using the lipofectamine2000 kit (5 μg of mRNA is transfected into each well), and the specific operation refers to the kit instructions.

[0185] After 24 hours of transfection, mouse spleen cells and anti-CD3 antibody (purchased from Bio X Cell) were added. After co-culturing for 48 hours, the expressions of CD25 and CD69 in T cells were detected by flow cytometry.

[0186] Among the LRIO-093 group, LRIO-097 group, LRIO-098 group and LRIO-099 group, the RNA information encoding co-stimulatory molecules can be found in Example 3.

[0187] The measurement results of the expressions of CD25 and CD69 in T cells of samples LRIO-093, LRIO-097, LRIO-098 and LRIO-099 are shown in Figure 3 . The results showed that, compared with the control group NC, samples LRIO-093, LRIO-097, LRIO-098 and LRIO-099 could all promote the expressions of CD25 and CD69 in T cells.

[0188] Example 5

[0189] The cultured HEK293 cells were digested and seeded into 6-well plates. After culturing at 37°C for 24 hours, the cell status was observed under a microscope. When the cell confluence reached more than 80%, mRNA transfection could be carried out. The corresponding mRNA was transfected into HEK293 cells using the lipofectamine2000 kit (5 μg of mRNA was transfected into each well), and the specific operation refers to the kit instructions.

[0190] After 24 hours of transfection, 15 μg / mL of mitomycin was added and incubated for 4 hours to block the growth of HEK293 cells. Then, mitomycin was washed away with PBS and fresh medium was added. Mouse spleen cells were stained with CFSE and co-cultured with the above HEK293 cells, and anti-CD3 antibody (purchased from Bio X Cell) was added. After co-culturing for 72 hours, the staining intensity of CFSE in CD3+ or CD4+ or CD8+ positive T cells was detected by flow cytometry. The cell population with low staining intensity of CFSE reflected the proportion of amplified cells.

[0191] Among the LRIO-051 group, LRIO-093 group, LRIO-097 group, LRIO-098 group and LRIO-099 group, the RNA information encoding co-stimulatory molecules can be found in Example 3.

[0192] The results of samples LRIO-051, LRIO-093, LRIO-097, LRIO-098 and LRIO-099 are shown in Figure 4. The results showed that, compared with the control group NC, the sample LRIO-051 did not significantly promote the expansion of CD4+ or CD8+ positive cell populations; LRIO-093, LRIO-097, and LRIO-098 all significantly promoted the expansion of CD4+ or CD8+ positive cell populations.

[0193] Example 6

[0194] Digest the cultured HEK293 cells and seed them into 6-well plates. After culturing at 37°C for 24 hours, observe the cell status using a microscope. When the cell confluence reaches over 80%, mRNA transfection can be carried out. Transfect the corresponding mRNA into HEK293 cells using the lipofectamine2000 kit (transfect 5 μg of mRNA per well), and the specific operation refers to the kit instructions.

[0195] After 24 hours of transfection, add 15 μg / mL of mitomycin and incubate for 4 hours to block the growth of HEK293 cells. Then wash away the mitomycin with PBS and add fresh medium. Take human PBMC cells, stain them with CFSE, and co-culture them with the above HEK293 cells. Add anti-CD3 antibody (purchased from Bio X Cell). After co-culturing for 72 hours, detect the staining intensity of CFSE in CD4+ or CD8+ positive T cells by flow cytometry. The cell population with low CFSE staining intensity reflects the proportion of expanded cells.

[0196] The co-stimulatory molecule polypeptide expressed by the sample LRIO-118 includes CD80 and CD70, and the antigens are the E6 protein and E7 protein of HPV16. The connection form between the functional regions in the co-stimulatory molecule polypeptide expressed by the sample LRIO-118 is: CD80-E7…P2A…E6…P2A…CD70.

[0197] The co-stimulatory molecule polypeptide expressed by the sample LRIO-067 includes CD80, 4-1BBL, and CD70, and the antigens are the E6 protein and E7 protein of HPV16. The connection form between the functional regions in the co-stimulatory molecule polypeptide expressed by the sample LRIO-067 is: CD80-E7…P2A…4-1BBL-E6…T2A…CD70.

[0198] In this embodiment, the co-stimulatory molecules CD80, CD70, and 4-1BBL are all human-derived molecules; among them, CD80 includes the sequences shown in SEQ ID NO.16 and SEQ ID NO.22 in terms of amino acid sequence; the amino acid sequence of CD70 is as shown in SEQ ID NO.20; the amino acid sequence of 4-1BBL includes the sequence composed of SEQ ID NO.25, SEQ ID NO.18, SEQ ID NO.23, and SEQ ID NO.24.

[0199] In this embodiment, the antigens E6 and E7 are from HPV16, and their amino acid sequences are the same as those in Example 2.

[0200] In this embodiment, the RNA information for detecting the expression at the in vitro cell level is as follows: sample LRIO-118 (the sequence of the RNA encoding the co-stimulatory molecule is as shown in SEQ ID NO.41), sample LRIO-067 (the sequence of the RNA encoding the co-stimulatory molecule is as shown in SEQ ID NO.42). The nucleotide sequence of sample LRIO-051 can be found in Example 3.

[0201] In this embodiment, the co-stimulatory molecules CD80, CD70, and 4-1BBL are all human-derived molecules; among them, CD80 includes the sequences shown in SEQ ID NO.14 and SEQ ID NO.22 in terms of amino acid sequence; the amino acid sequence of CD70 is as shown in SEQ ID NO.18; the amino acid sequence of 4-1BBL includes the sequence composed of SEQ ID NO.25, SEQ ID NO.18, SEQ ID NO.23, and SEQ ID NO.24.

[0202] In addition to the above open reading frame sequences, the RNA in this example also includes a 5' cap (m7Gppp(5')), a 5' UTR (such as SEQ ID NO.1), a 3' UTR (such as SEQ ID NO.4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil in the RNA is replaced with 5' pseudouracil.

[0203] The experimental results are as Figure 5 shown. The results show that compared with the control group NC, LRIO-118 has an obvious promoting effect on the expansion of CD4+ or CD8+ positive T cells, LRIO-067 has an obvious promoting effect on the expansion of CD8+ positive T cells, while LRIO-051 has no obvious promoting effect on the expansion of CD4+ or CD8+ positive T cells.

[0204] Example 7

[0205] Humanized mice targeting three immune checkpoints of CD28, CD27 and 4-1BB (female, 5-6 weeks old, with an average body weight of 15-20 g, purchased from Shanghai Model Organisms Center, Inc.) were selected for in vivo anti-tumor efficacy evaluation.

[0206] The experimental mice were subcutaneously inoculated with TC-1 tumor cells (1.5×10 5 / mouse). When the average tumor volume reached about 100 mm 3 , the mice were randomly divided into 5 groups according to tumor volume, with the coefficient of variation (CV) of tumor volume ≤1 / 3. The day of grouping was defined as D0. The 5 groups were denoted as NC group (injected with empty LNP), LRIO-067(h) group, LRIO-067(l) group, LRIO-118(h) group and LRIO-118(l) group, with 6 mice in each group.

[0207] On days 0, 7 and 14 of the experiment, the drugs were administered by intramuscular injection. The dosage of LRIO-067(h) group and LRIO-118(h) group was 20 μg / mouse / time, and the dosage of LRIO-067(l) group and LRIO-118(l) group was 3 μg / mouse / time. The tumor volume was detected 2-3 times a week after administration.

[0208] Among them, the LRIO-067(h) group, LRIO-067(l) group, LRIO-118(h) group and LRIO-118(l) group were injected with lipid nanoparticles containing RNA encoding costimulatory molecules. For the RNA information of the samples used in the LRIO-067(h) group and LRIO-067(l) group, see sample LRIO-067 in Example 6; for the RNA information of the samples used in the LRIO-118(h) group and LRIO-118(l) group, see sample LRIO-118 in Example 6. The lipid nanoparticles injected into the LRIO-067(h) group, LRIO-067(l) group, LRIO-118(h) group and LRIO-118(l) group were prepared according to the method described in Example 1.

[0209] The experimental results are as Figure 6 shown. The results showed that compared with the control group NC, the LRIO-067(h) group, LRIO-067(l) group, LRIO-118(h) group and LRIO-118(l) group could all significantly inhibit tumor growth. Among them, the high-dose groups of LRIO-067(h) group and LRIO-118(h) group had the most significant tumor inhibitory effect; in the comparison between the low-dose groups, the tumor inhibitory effect of the LRIO-067(l) group was better than that of the LRIO-118(l) group.

[0210] Example 8

[0211] C57BL / 6 mice (female, 5 - 6 weeks old, average body weight 15 - 20 g, purchased from Zhuhai BestBio Technology Co., Ltd.) were selected for in vivo anti - tumor efficacy evaluation. The experimental mice were subcutaneously inoculated with MC38 / GPC3 tumor cells (2×106 cells / mouse). When the average tumor volume reached about 100 mm3, the mice were randomly divided into 4 groups according to tumor volume, with the tumor volume CV ≤ 1 / 3. The day of grouping was defined as D0. The four groups were denoted as the NC group (injected with empty LNP), the LRIO - 060 group, the LRIO - 202 group, and the LRIO - 201 group, with 6 mice in each group. The dosing dose was 25 μg / mouse / time, and the tumor volume was detected 2 - 3 times per week after dosing.

[0212] Among them, the LRIO - 060 group, the LRIO - 202 group, and the LRIO - 201 group were all injected with lipid nanoparticles containing RNA encoding GPC3, and the lipid nanoparticles were prepared according to the method described in Example 1.

[0213] The T - cell co - stimulatory molecule polypeptides encoded by the RNA contained in the sample injected into the LRIO - 060 group included CD80, CD70, and 4 - 1BBL, and the antigen was GPC3 protein (GPC3, also known as DGSX, GTR2 - 2, MXR7, OCI - 5, is a member of the heparan sulfate (HS) proteoglycan family encoded by the GPC3 gene. The GPC3 gene is located on chromosome Xq26, and the protein it encodes contains a 580 - amino - acid core protein and two HS chains at the C - terminus. The GPC3 core protein is anchored to the cell membrane surface through glycosylphosphatidylinositol (GPI) and can be cleaved into an N - terminal soluble protein (sGPC3) of approximately 40 KDa and a 30 KDa C - terminal membrane protein.). Among them, the connection form between the functional regions of the co - stimulatory molecule polypeptides encoded by the RNA contained in the sample injected into the LRIO - 060 group was: CD80 - GPC3(1)…P2A…4 - 1BBL - GPC3(2)…T2A…CD70.

[0214] The T - cell co - stimulatory molecule polypeptides encoded by the RNA contained in the sample injected into the LRIO - 202 group included CD80 and CD70, and the antigen was GPC3 protein. Among them, the connection form between the functional regions of the co - stimulatory molecule polypeptides encoded by the RNA contained in the sample injected into the LRIO - 202 group was: CD80 - GPC3(1)…P2A…GPC3(2)…T2A…CD70.

[0215] The RNA contained in the sample injected into the LRIO - 201 group did not encode T - cell co - stimulatory molecule polypeptides but only encoded the antigen GPC3 protein. Among them, the connection form between the functional regions of the polypeptides encoded by the RNA contained in the sample injected into the LRIO - 201 group was: GPC3(1)…P2A…GPC3(2).

[0216] In this example, the costimulatory molecules CD80, CD70, and 4-1BBL are all murine molecules; among them, CD80 includes the sequences with amino acid sequences as shown in SEQ ID NO.15 and SEQ ID NO.21; the amino acid sequence of CD70 is as shown in SEQ ID NO.19; the amino acid sequence of 4-1BBL is the sequence composed of SEQ ID NO.25, SEQ ID NO.17, SEQ ID NO.23, and SEQ ID NO.24.

[0217] In this example, the amino acid sequence of the antigen GPC3(1) protein is as shown in SEQ ID NO.32, and the amino acid sequence of the antigen GPC3(2) protein is as shown in SEQ ID NO.33.

[0218] In this example, the RNA sequence contained in the sample injected in the LRIO-060 group is as shown in SEQ ID NO.43, the RNA sequence contained in the sample injected in the LRIO-202 group is as shown in SEQ ID NO.44, and the RNA sequence contained in the sample injected in the LRIO-201 group is as shown in SEQ ID NO.45.

[0219] In addition to the above open reading frame sequences, the RNA of this example also includes a 5' cap (m7Gppp(5')), a 5' UTR (such as SEQ ID NO.1), a 3' UTR (such as SEQ ID NO.4), and a 3' polyA tail of 100 adenine nucleotides; and the uracil of the RNA is replaced with 5' pseudouracil. "GPC3(1)" and "GPC3(2)" as described in the present invention are truncated amino acid sequences of GPC3, respectively selected from the full-length GPC3 protein, and there is no sequence overlap between them.

[0220] The results of this example are shown in Figure 7 . The results show that compared with the empty LNP group, all other experimental groups can inhibit tumor growth.

[0221] Example 9

[0222] C57BL / 6 mice (female, 5-6 weeks old, average body weight 15-20 g, purchased from Zhuhai BestBio Technology Co., Ltd.) were selected for vaccine immunogenicity evaluation. The experimental mice were subcutaneously inoculated with TC-1 tumor cells. When the average tumor volume reached 100 mm 3Around, the mice were randomly divided into 2 groups according to tumor volume, with the coefficient of variation (CV) of tumor volume ≤ 1 / 3. The day of grouping was defined as D0. The three groups were denoted as the NC group (injected with empty LNP), the LRIO-045 group, and the LRIO-044 group. There were 6 mice in each group. On days 0, 7, and 14 of the experiment, the mice were administered 30 μg / mouse by intramuscular injection. On days 14 and 21 of the experiment, peripheral blood was collected for flow cytometry to detect the expression of E7 antigen-specific TCR (i.e., E7 multimer+) in CD3+ / CD8+ T lymphocytes.

[0223] Method for detecting the expression of E7 antigen-specific TCR (i.e., E7 multimer+) in CD3+ / CD8+ T lymphocytes by peripheral blood flow cytometry

[0224] Peripheral blood of mice was collected and mixed with freshly prepared 1X red blood cell lysate. After lysis at room temperature, PBS was added, and the supernatant was discarded by centrifugation. After washing once with PBS, specific monoclonal fluorescent antibodies such as CD3, CD4, CD8, E7 multimer, etc. were added and incubated for 30 minutes. Then the cells were washed twice with staining buffer and the cell pellet was resuspended, and then analyzed by flow cytometry. The expression of E7 antigen-specific TCR (i.e., E7 multimer+) in CD3+ / CD8+ T lymphocytes was detected by flow cytometry.

[0225] Among them, the LRIO-045 group was injected with lipid nanoparticles containing RNA encoding co-stimulatory molecules. The sample information injected into the LRIO-045 group was the same as that injected into the LRIO-093 group in Example 3. The LRIO-044 group was injected with lipid nanoparticles containing mRNA encoding a chimeric protein consisting of glycoprotein D (gD) of herpes simplex virus type 1 (HSV-1) and the early regulatory protein E7 of human papillomavirus type 16. The mRNA sequence of the LRIO-044 group was as shown in SEQ ID NO.36. The lipid nanoparticles injected into the LRIO-044 group were prepared according to the method described in Example 1.

[0226] The content of E7 multimer+ CD8+ T cells in peripheral blood was respectively referred to Figure 8 . From Figure 8 it can be seen that E7-specific cytotoxic T cells, namely E7 multimer+ CD8+ T cells, were generated in the peripheral blood of the mice in the LRIO-045 group; the content of E7 multimer+ CD8+ T cells in the peripheral blood of the mice in the LRIO-045 group was stable on days 14 and 21 and showed no obvious changes.

[0227] Example 10

[0228] C57BL / 6 mice (female, 5 - 6 weeks old, average body weight 15 - 20 g, purchased from Zhuhai BestBio Technology Co., Ltd.) were selected for in - vivo anti - tumor efficacy evaluation. The experimental mice were subcutaneously inoculated with TC - 1 tumor cells. When the average tumor volume reached about 100 mm 3 or so, the mice were randomly divided into 5 groups according to tumor volume, and the coefficient of variation (CV) of tumor volume was ≤ 1 / 3. The day of grouping was defined as D0. The five groups were denoted as NC group (injected with empty LNP), LRIO - 044(H) group, LRIO - 044(L) group, LRIO - 045(H) group, and LRIO - 045(L) group, with 5 mice in each group. On the 0th, 7th, and 14th days of the experiment, the LRIO - 044(H) group and LRIO - 045(H) group were administered intramuscularly at a dose of 25 μg / mouse, and the LRIO - 044(L) group and LRIO - 045(L) group were administered intramuscularly at a dose of 1 μg / mouse. The tumor volume was measured on the 0th, 3rd, 6th, 11th, 13th, 17th, 20th, and 25th days after administration.

[0229] Among them, the LRIO - 044 group was injected with lipid nanoparticles containing mRNA encoding a chimeric protein that is a fusion of glycoprotein D (gD) of herpes simplex virus type 1 (HSV - 1) and the early regulatory protein E7 of human papillomavirus type 16. The mRNA sequence of the LRIO - 044 group is shown in SEQ ID NO.36. The lipid nanoparticles injected in the LRIO - 044 group were prepared according to the method described in Example 1. The LRIO - 045 group was injected with lipid nanoparticles containing RNA encoding a co - stimulatory molecule. The sample information injected in the LRIO - 045 group was the same as that injected in the LRIO - 093 group in Example 3.

[0230] Figure 9 The average tumor volume (mm 3 ) of mice in each experimental group at each time point and the survival rate of mice in each experimental group during the experiment are shown. As can be seen from Figure 9 this, the average tumor volume of mice in the LRIO - 044(H) group, LRIO - 044(L) group, LRIO - 045(H) group, and LRIO - 045(L) group was lower than that of mice in the NC group; the average tumor volume of mice in the LRIO - 045(H) group was lower than that of mice in the other 4 groups; the average tumor volume of mice in the LRIO - 045(L) group was lower than that of mice in the LRIO - 044(L) group; and on the 3rd, 6th, 11th, 13th, and 17th days, the average tumor volume of mice in the LRIO - 045(L) group was approximately equal to that of mice in the LRIO - 044(H) group.

[0231] As can be seen from Figure 9As can be seen, all the mice in the LRIO-044(H) group, LRIO-045(H) group, and LRIO-045(L) group survived during the experimental period. Half of the mice in the LRIO-044(L) group died during the 33-day experimental period, and all the mice in the NC group had died by the 25th day.

[0232] Example 11

[0233] C57BL / 6 mice (female, 5 - 6 weeks old, average body weight 15 - 20 g, purchased from Zhuhai BestBio Technology Co., Ltd.) were selected for in vivo anti-tumor efficacy evaluation. The experimental mice were subcutaneously inoculated with TC-1 tumor cells. When the average tumor volume reached about 100 mm 3 or so, the mice were randomly divided into four groups according to tumor volume, with tumor volume CV ≤ 1 / 3. The day of grouping was defined as D0. In the anti-PD-L1 antibody experiment, the four groups were respectively denoted as the NC group (injected with empty LNP), LRIO-045 group, LRIO-045 + α-PD-L1 group, and α-PD-L1 group, with 6 mice in each group. In the anti-PD-1 antibody experiment, the four groups were respectively denoted as the NC group (injected with empty LNP), LRIO-045 group, LRIO-045 + α-PD-1 group, and α-PD-1 group, with 6 mice in each group.

[0234] On the 0th, 7th, and 14th days of the experiment, the LRIO-207 group, LRIO-045 + α-PD-1 group, and LRIO-045 + α-PD-L1 group were intramuscularly injected with the sample LRIO-207 at a dose of 5 μg / mouse. On the 0th, 3rd, 7th, 10th, 14th, and 17th days of the experiment, the α-PD-1 group, α-PD-L1 group, LRIO-045 + α-PD-1 group, and LRIO-045 + α-PD-L1 group were intraperitoneally injected with anti-PD-1 or PD-L1 antibody at a dose of 200 μg / mouse. After administration, the tumor volume was detected 2 - 3 times a week.

[0235] The LRIO-045 group was injected with lipid nanoparticles containing RNA encoding co-stimulatory molecules. The sample information injected into the LRIO-045 group was the same as that injected into the LRIO-093 group in Example 3.

[0236] The experimental results are shown in Figure 10 . Compared with the NC group, all other experimental groups could inhibit tumor growth; the LRIO-045 + α-PD-1 group had a better effect on inhibiting tumor growth than the LRIO-045 group and the α-PD-1 group used alone; the LRIO-045 + α-PD-L1 group had a better effect on inhibiting tumor growth than the LRIO-045 group and the α-PD-L1 group used alone.

[0237] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present disclosure, rather than to limit them; although the present disclosure has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. An RNA encoding a T cell co-stimulatory molecule, characterized in that The RNA comprises at least one nucleic acid sequence encoding the following amino acid sequence: (i) contains the amino acid sequence of a co-stimulatory molecule; (ii) contains the amino acid sequence of an antigen; The co-stimulatory molecule is selected from at least one of the B7 family members and at least one of the tumor necrosis factor family members; The B7 family member is selected from at least one of CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and HHLA2; preferably, the B7 family member is selected from at least one of CD80, CD86, B7-DC, PD-L1 and HHLA2; The tumor necrosis factor family member is selected from at least one of TNF-β, TNF-α, LT-β, ​​CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, and GITRL; preferably, the tumor necrosis factor family member is selected from at least one of CD252, CD154, CD70, 4-1BBL, and GITRL.

2. The RNA according to claim 1, characterized in that The antigen is a pathogen antigen or a tumor antigen. Preferably, the pathogen antigen is selected from HIVEnv, RSV F, influenza HA, EBV gp350, EBV LMP1, EBVLMP2, EBVEBNA1, EBVEBNA3, CMV gB, CMVUL128, CMVUL130, CMV UL131A, CMV gH, CMV gL, Lyme disease OspA, pertussis toxin, dengue E, SARS S, MERS S, Zaire Ebola virus GP, Sudan Ebola virus GP, Marburg virus GP, Handa virus Gn, Handa virus Gc, measles H, Zika envelope domain III, malaria CSP, malaria Pfs25, MenB fHbp, MenB NadA, MenB At least one of NHBA, Nipah virus F, Nipah virus G, rotavirus VP4, rotavirus VP8, human papillomavirus antigen, HBeAg, hepatitis B virus pre-S1 protein, hepatitis B virus pre-S2 protein, hepatitis B virus HBsAg protein, hepatitis B virus HBcAg protein, and hand, foot and mouth disease virus antigen; Preferably, the tumor antigen is selected from at least one of CLDN6, MAGE-A3, MAGE-A4, PRAME, MAGE-C1, MAGE-C2, NY-ESO-1, Tyrosinase, Gp100, TPTE, PSA, AFP, GPC3, Survivin, Melan-A, Kita-kyushu KK-LC-1, KRAS G12, KRAS G13, and KRAS Q61.

3. The RNA according to claim 1 or 2, characterized in that The nucleic acid sequence encodes the following amino acid sequence: (i) contains the amino acid sequence of a co-stimulatory molecule; (ii) contains the amino acid sequence of an antigen; the co-stimulatory molecules are CD80 and CD70; Preferably, the co-stimulatory molecule further comprises at least one selected from CD252, CD154, 4-1BBL, and GITRL.

4. The RNA according to claim 3, characterized in that The nucleic acid sequence encodes at least one of the following amino acid sequences: (iii) an amino acid sequence comprising a 2A self-cleaving peptide; (iv) an amino acid sequence comprising a GS-linked polypeptide; The 2A self-cleaving peptide is selected from T2A peptide, P2A peptide, E2A peptide or F2A peptide; the nucleic acid sequence encoding the 2A self-cleaving peptide or GS connecting polypeptide is used to connect the nucleic acid sequence encoding the co-stimulatory molecule and / or the nucleic acid sequence encoding the antigen.

5. The RNA according to claim 3, characterized in that The CD80 comprises the amino acid sequence shown in SEQ ID NO.15 or SEQ ID NO.16 or a functional fragment thereof, or an extracellular domain consisting thereof; The CD70 comprises the amino acid sequence shown in SEQ ID NO.19 or SEQ ID NO.20 or a functional fragment thereof, or consists of the same.

6. The RNA according to claim 3, characterized in that The RNA comprises a nucleic acid sequence encoding CD80 as shown in at least one of SEQ ID NOs. 28 to 31 and SEQ ID NOs. 46 to 51; the RNA comprises a nucleic acid sequence encoding CD70 as shown in at least one of SEQ ID NOs. 52 to 54.

7. A protein composition, characterized in that The protein composition includes a co-stimulatory molecule; the co-stimulatory molecule is selected from at least one of the B7 family members and at least one of the tumor necrosis factor family members; The B7 family member is selected from at least one of CD80, CD86, B7-DC, PD-L1, B7-H2, B7-H3, B7-H4, B7-H5, B7-H6 and HHLA2; preferably, the B7 family member is selected from at least one of CD80, CD86, B7-DC, PD-L1 and HHLA2; The tumor necrosis factor family member is selected from at least one of TNF-β, TNF-α, LT-β, ​​CD252, CD154, CD95L, CD70, CD153, 4-1BBL, Apo2L, and GITRL; preferably, the tumor necrosis factor family member is selected from at least one of CD252, CD154, CD70, 4-1BBL, and GITRL.

8. A vaccine, characterized in that The vaccine comprises the RNA according to claims 1 to 6, or the protein composition according to claim 7.

9. A biomaterial, characterized in that: The biological material comprises any one of an expression cassette, a vector, an engineered bacterium or a cell line, and the biological material contains or expresses the RNA according to any one of claims 1 to 6, or the protein composition according to claim 7.

10. A method, characterized in that The method comprises administering to a subject an effective amount of at least one of the RNA of claims 1 to 6, the protein composition of claim 7, the vaccine of claim 8, or the biological material of claim 9 to induce a cellular immune response or a humoral immune response in the subject.

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