Conjugates for antigen delivery and uses thereof

Abstract

The present invention relates to a conjugate comprising an mRNA of an antigen protein and an mRNA encoding a carrier protein linked to the 5'terminal and the 3 'terminal of the mRNA of the antigen protein, and an vaccination composition and / or a vaccine composition comprising the conjugate, the present invention having an effect of stably increasing the expression of an antigen protein.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2023-0099234, filed on July 28, 2023, and Korean Patent Application No. 10-2023-0102465, filed on August 4, 2023, and the entire contents of the files of the respective Korean Patent Applications are incorporated herein by reference as part of the present specification. TECHNICAL FIELD

[0003] The present specification relates to a conjugate including mRNA of an antigen protein and mRNA encoding a carrier protein linked to the 5' end and / or 3' end of the mRNA of the antigen protein, and an immunization composition and / or vaccine composition including the same. BACKGROUND

[0004] An anticancer therapeutic vaccine is a method of treating cancer by enhancing or inducing an antigen-specific immune response. A conventional anticancer therapeutic vaccine does not reflect an antigen processing process, and thus the expression level is not constant or high, depending on the antigen sequence, and thus the specific immune response against antigen sequences different from each other is not high, or it is difficult to expect constant therapeutic efficacy.

[0005] In order to express various antigen sequences without problems by solving the above-described problems, an attempt is made to induce a high immune response by increasing the expression level by linking mRNA sequences encoding different proteins (carrier proteins) at both the 5' and 3' ends of the antigen mRNA sequence, to reduce the deviation in the amount of antigen protein expressed by various different antigens, and to appropriately deliver CD4 / CD8 antigens to the processing / presentation pathway, respectively, when the antigen is delivered to a cell in the form of mRNA. SUMMARY

[0006] TECHNICAL PROBLEM

[0007] The present specification provides a conjugate including mRNA of an antigen protein and mRNA encoding a first carrier protein linked to the 5' end and / or 3' end of the mRNA of the antigen protein, and improves the expression level of the antigen protein.

[0008] Another embodiment provides an immunization composition and / or vaccine composition including the conjugate.

[0009] Other embodiments provide a method for immunization, the method including administering the conjugate and the immunization composition and / or vaccine composition including the conjugate into a subject.

[0010] TECHNICAL SOLUTION

[0011] One embodiment provides a conjugate comprising an mRNA of an antigenic protein and an mRNA encoding a carrier protein linked to the 5' end, the 3' end, or both of the mRNA of the antigenic protein, and

[0012] In particular, provided is a conjugate comprising: an mRNA of an antigenic protein; an mRNA encoding a first carrier protein linked to the 5' end of the mRNA of the antigenic protein; and an mRNA encoding a second carrier protein linked to the 3' end of the mRNA of the antigenic protein.

[0013] The first carrier protein and the second carrier protein can be the same or different, but are not limited thereto.

[0014] The first carrier protein and the second carrier protein can each be at least one selected from the group consisting of Ub (ubiquitin), UbR (ubiquitin A77R mutant), Lamp, mODC (mouse ornithine decarboxylase), cmyc, SORT1 (sortilin 1), TRX (thioredoxin) (e.g., TRXwt and / or TRXmt), PSBD (peripheral subunit binding domain), TRX-UbR (e.g., TRXwt-UbR), TRX-Lamp (e.g., TRXwt-Lamp), TRX-mODC (e.g., TRXwt-mODC), TRX-cmyc (e.g., TRXwt-cmyc), TRX-SORT1 (e.g., TRXwt-SORT1), SORT1 w / TM, and Lamp w / TM, but are not limited thereto.

[0015] The antigenic protein can comprise a T cell epitope, but is not limited thereto.

[0016] The conjugate can have at least one characteristic selected from the group consisting of (i) and (ii) by virtue of the carrier protein, but is not limited thereto: (i) delivery of CD4 / CD8 through the processing and presentation pathway, respectively; and (ii) improvement of expression of the antigenic protein.

[0017] Another embodiment provides an immunization composition and / or a vaccine composition comprising the conjugate.

[0018] Other embodiments provide an immunization method against cancer, the method comprising administering the conjugate and / or an immunization composition and / or a vaccine composition comprising the conjugate to a subject in need of immunization against cancer.

[0019] Other embodiments provide a composition for preventing, ameliorating, and / or treating cancer, infectious disease, autoimmune disease, and / or allergic disease.

[0020] Other embodiments provide use of the conjugate and / or composition for immunization against cancer, or use for preparing a composition for immunization against cancer (e.g., an immunization composition and / or a vaccine composition).

[0021] Other embodiments provide use of the conjugate and / or composition for preventing, ameliorating, and / or treating cancer, infectious disease, autoimmune disease, and / or allergic disease, or use for preparing a composition for preventing, ameliorating, and / or treating cancer, infectious disease, autoimmune disease, and / or allergic disease (e.g., a composition for preventing, ameliorating, and / or treating cancer, infectious disease, autoimmune disease, and / or allergic disease).

[0022] Hereinafter, the present application will be described in more detail below.

[0023] Conjugates

[0024] One embodiment of the present specification provides a conjugate comprising: mRNA of an antigen protein; mRNA encoding a first carrier protein linked to a 5' end of the mRNA of the antigen protein; and mRNA encoding a second carrier protein linked to a 3' end of the mRNA of the antigen protein.

[0025] In the present specification, "antigen" means any molecule that induces an immune response in a subject. Antigen means any molecule that can be recognized by a T cell receptor and / or a B cell receptor, and can stimulate an immune response, particularly a T cell response and / or a B cell response, in a subject.

[0026] In the present specification, "epitope" means a region of an antigen that interacts with a T cell receptor and / or a B cell receptor.

[0027] In the present specification, "carrier protein" means a protein that is linked to an antigen protein and improves the recombinant expression (e.g., high expression rate, uniform expression rate, etc.) of the antigen protein, improves its purification, enhances its physical properties, stabilizes it, increases its immunogenicity, and / or improves its delivery to immune response cells. In the present specification, the carrier protein can take the form of a full-length protein and / or take the form of containing some sequences in the amino acid sequence of the full-length protein, but is not limited thereto. In the present specification, the carrier protein can be linked to the N-terminus and C-terminus of the antigen protein, respectively, and each corresponds to the first carrier protein and the second carrier protein.

[0028] In the present specification, "connection" means that elements are connected together directly or indirectly. Each element can be connected covalently or non-covalently. In addition, "connection" can mean that chemical or physical bonds of each of the elements are maintained after contact with a cell (e.g., an antigen-presenting cell or an immune cell) and immunization. For example, when the antigen-presenting cell and the immune cell are in contact, they can associate so as not to be freely dispersed from each other. For example, two components can be covalently connected to each other so that the two components cannot be individually dispersed or diffused.

[0029] The connection can exist between the mRNA of the antigen protein and the mRNA encoding the first carrier protein and / or the mRNA encoding the second carrier protein of the conjugate, but is not limited thereto.

[0030] In the present specification, "linker" means a molecule or a group of atoms that connects, couples, or binds two or more components together. In the present specification, each component of the antigen protein, such as an antigen protein and a carrier protein, can be connected or bound together, respectively, by any appropriate means.

[0031] The linker can be (GS)n, (G2S)n, (G3S)n, (G4S)n, Gn, LE, SSGG, or GGGGSGGGGG (here, G is Gly, S is Ser, L is Leu, E is Glu, and n is an integer of at least 1), but is not limited thereto.

[0032] Carrier proteins

[0033] In the conjugate provided in the present specification, the conjugate comprises: mRNA of an antigen protein; mRNA encoding a first carrier protein connected to the 5' end of the mRNA of the antigen protein; and mRNA encoding a second carrier protein connected to the 3' end of the mRNA of the antigen protein, The first carrier protein and the second carrier protein can be the same or different, but are not limited thereto.

[0034] The first and second carrier proteins can be at least one selected from the group consisting of Ub (ubiquitin), UbR (ubiquitin A77R mutant), Lamp, mODC (mouse ornithine decarboxylase), cmyc, SORT1 (sortilin 1), TRX (thioredoxin) (e.g., TRXwt and / or TRXmt), PSBD (peripheral subunit binding domain), TRXwt-UbR, TRXwt-Lamp, TRXwt-mODC, TRXwt-cmyc, TRXwt-SORT1, SORT1 w / TM, and Lamp w / TM, but are not limited thereto.

[0035] When the last sequence alanine (A) of Ub is replaced with arginine (R), Ub can be well degraded in the case of being inserted into the N-terminus of the antigen protein, and thus, when mRNA encoding a carrier protein corresponding to Ub is linked to the 5' end of the mRNA of the antigen protein, mRNA encoding UbR in which the last sequence is R can be linked, but is not limited thereto.

[0036] The TRXwt-UbR, TRXwt-Lamp, TRXwt-mODC, and TRXwt-cmyc are proteins in which TRXwt is added to the N-terminus of UbR, Lamp, mODC, and cmyc proteins.

[0037] The SORT1 w / TM and Lamp w / TM are proteins in which a transmembrane domain is added to each cytoplasmic domain of SORT1 and Lamp.

[0038] In the conjugate, (1) The first carrier protein can be TRXmt, and the second carrier protein can be PSBD; (2) The first carrier protein can be UbR, and the second carrier protein can be mODC; (3) The first carrier protein can be UbR, and the second carrier protein can be SORT1; (4) The first carrier protein can be Lamp, and the second carrier protein can be mODC; (5) The first carrier protein can be Lamp, and the second carrier protein can be SORT1; (6) The first carrier protein can be UbR, and the second carrier protein can be cmyc; (7) The first carrier protein can be UbR, and the second carrier protein can be Lamp; (8) the first carrier protein can be mODC and the second carrier protein can be SORT1; (9) the first carrier protein can be cmyc and the second carrier protein can be mODC; (10) the first carrier protein can be cmyc and the second carrier protein can be SORT1; (11) the first carrier protein can be Lamp and the second carrier protein can be Ub; (12) the first carrier protein can be Lamp and the second carrier protein can be cmyc; (13) the first carrier protein can be SORT1 and the second carrier protein can be mODC; (14) the first carrier protein can be mODC and the second carrier protein can be Ub; (15) the first carrier protein can be mODC and the second carrier protein can be cmyc; (16) the first carrier protein can be mODC and the second carrier protein can be Lamp; (17) the first carrier protein can be cmyc and the second carrier protein can be Ub; (18) the first carrier protein can be cmyc and the second carrier protein can be Lamp; (19) the first carrier protein can be SORT1 and the second carrier protein can be Ub; (20) the first carrier protein can be SORT1 and the second carrier protein can be cmyc; (21) the first carrier protein can be SORT1 and the second carrier protein can be Lamp; (22) the first carrier protein can be TRXwt and the second carrier protein can be cmyc; (23) the first carrier protein can be TRXwt and the second carrier protein can be Lamp; (25) the first carrier protein can be TRXwt and the second carrier protein can be mODC; (26) the first carrier protein can be TRXwt and the second carrier protein can be SORT1; (27) the first carrier protein can be TRXwt and the second carrier protein can be Ub; (28) the first carrier protein can be cmyc, and the second carrier protein can be TRXwt; (29) the first carrier protein can be Lamp, and the second carrier protein can be TRXwt; (30) the first carrier protein can be mODC, and the second carrier protein can be TRXwt; (31) the first carrier protein can be SORT1, and the second carrier protein can be TRXwt; (32) the first carrier protein can be UbR, and the second carrier protein can be TRXwt; (33) the first carrier protein can be TRXwt-UbR, and the second carrier protein can be SORT1; (34) the first carrier protein can be TRXwt-Lamp, and the second carrier protein can be mODC; (35) the first carrier protein can be TRXwt-mODC, and the second carrier protein can be SORT1; (36) the first carrier protein can be TRXwt-mODC, and the second carrier protein can be Ub; (37) the first carrier protein can be TRXwt-mODC, and the second carrier protein can be Lamp; (38) the first carrier protein can be TRXwt-cmyc, and the second carrier protein can be Ub; (39) the first carrier protein can be TRXwt-SORT1, and the second carrier protein can be Ub; (40) the first carrier protein can be TRXwt-SORT1, and the second carrier protein can be Lamp; (41) the first carrier protein can be TRXwt, and the second carrier protein can be SORT1 w / TM; or (42) the first carrier protein can be Lamp w / TM, and the second carrier protein can be TRXwt, but not limited thereto.

[0039] In another embodiment, the first carrier protein and the second carrier protein in the conjugate can be shown as follows: [Table 1]

[0040] The UbR can include the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the amino acid sequence, but is not limited thereto. A gene encoding the UbR can have the base sequence of SEQ ID NO: 21 or have 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the base sequence, but is not limited thereto.

[0041] The Ub can include the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the amino acid sequence, but is not limited thereto. A gene encoding the Ub can have the base sequence of SEQ ID NO: 22 or have 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the base sequence, but is not limited thereto.

[0042] The Lamp can include the amino acid sequence of SEQ ID NO: 3 or 4 or an amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the amino acid sequence, but is not limited thereto. A gene encoding the Lamp can have the base sequence of SEQ ID NO: 23 or 24 or have 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the base sequence, but is not limited thereto. When the Lamp is the first carrier protein, it can have the amino acid sequence of SEQ ID NO: 3 or have the above-described sequence homology, and when it is the second carrier protein, it can have the amino acid sequence of SEQ ID NO: 4 or have the above-described homology, but is not limited thereto.

[0043] The mODC can comprise an amino acid sequence of SEQ ID NO: 5 or 6, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the amino acid sequence, but is not limited thereto. A gene encoding the mODC can have a base sequence of SEQ ID NO: 25 or 26, or a sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the base sequence, but is not limited thereto. When the mODC is the first carrier protein, it can have SEQ ID NO: 5 or an amino acid sequence having the above sequence homology, and when it is the second carrier protein, it can have SEQ ID NO: 6 or an amino acid sequence having the above homology, but is not limited thereto.

[0044] The cmyc can comprise an amino acid sequence of SEQ ID NO: 7 or 8, or an amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the amino acid sequence, but is not limited thereto. A gene encoding the cmyc can have a base sequence of SEQ ID NO: 27 or 28, or a sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the base sequence, but is not limited thereto. When the cmyc is the first carrier protein, it can have SEQ ID NO: 7 or an amino acid sequence having the above sequence homology, and when it is the second carrier protein, it can have SEQ ID NO: 8 or an amino acid sequence having the above homology, but is not limited thereto.

[0045] The SORT1 can include an amino acid sequence of SEQ ID NO: 9 or 10 or an amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the amino acid sequence, but is not limited thereto. A gene encoding the SORT1 can have a base sequence of SEQ ID NO: 29 or 30, or a sequence homology of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with the base sequence, but is not limited thereto. When the SORT1 is the first carrier protein, it can have SEQ ID NO: 9 or an amino acid sequence having the above sequence homology, and when it is the second carrier protein, it can have SEQ ID NO: 10 or an amino acid sequence having the above homology, but is not limited thereto.

[0046] The TRX (in particular, TRXwt) can include an amino acid sequence of SEQ ID NO: 11 or 12 or an amino acid sequence having 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence homology with the amino acid sequence, but is not limited thereto. A gene encoding the TRX (in particular, TRXwt) can have a base sequence of SEQ ID NO: 31 or 32, or a sequence homology of 50% or more, 60% or more, 70% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with the base sequence, but is not limited thereto. When the TRX (in particular, TRXwt) is the first carrier protein, it can have SEQ ID NO: 11 or an amino acid sequence having the above sequence homology, and when it is the second carrier protein, it can have SEQ ID NO: 12 or an amino acid sequence having the above homology, but is not limited thereto. The TRXwt refers to a wild type of TRX, which can be obtained from nature without mutation.

[0047] The PSBD may comprise the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the amino acid sequence, but is not limited thereto. The gene encoding the PSBD may have the base sequence of SEQ ID NO: 33, or a sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the base sequence, but is not limited thereto.

[0048] The TRXwt-UbR may comprise the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the amino acid sequence, but is not limited thereto. The gene encoding the TRXwt-UbR may have the base sequence of SEQ ID NO: 34, or a base sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the base sequence, but is not limited thereto.

[0049] The TRXwt-Lamp may contain the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the amino acid sequence, but is not limited thereto. The gene encoding the TRXwt-Lamp may contain the base sequence of SEQ ID NO: 35, or have 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the base sequence, but is not limited thereto.

[0050] The TRXwt-mODC may contain the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the amino acid sequence, but is not limited thereto. The gene encoding the TRXwt-mODC may contain the base sequence of SEQ ID NO: 36, or have 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the base sequence, but is not limited thereto.

[0051] The TRXwt-cmyc may contain the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the amino acid sequence, but is not limited thereto. The gene encoding the TRXwt-cmyc may contain the base sequence of SEQ ID NO: 37, or have 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the base sequence, but is not limited thereto.

[0052] The TRXwt-SORT1 may comprise the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the amino acid sequence, but is not limited thereto. The gene encoding the TRXwt-SORT1 may have the base sequence of SEQ ID NO: 38, or a base sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the base sequence, but is not limited thereto.

[0053] The SORT1 w / ™ may contain the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the amino acid sequence, but is not limited thereto. The gene encoding the SORT1 w / ™ may contain the base sequence of SEQ ID NO: 39, or have 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the base sequence, but is not limited thereto.

[0054] The Lamp w / TM may contain the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the amino acid sequence, but is not limited thereto. The gene encoding the Lamp w / TM may contain the base sequence of SEQ ID NO: 40, or a base sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the base sequence, but is not limited thereto.

[0055] The TRXmt may contain the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the amino acid sequence, but is not limited thereto. The gene encoding the TRXmt may contain the base sequence of SEQ ID NO: 146, or a base sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the base sequence, but is not limited thereto.

[0056] The TRXmt may include, but is not limited to, a sequence in which at least one amino acid selected from the amino acids corresponding to positions 32, 35, 62, 69, and 73 from the N-terminus of the TRX amino acid sequence (specifically, the amino acid sequence of SEQ ID NO: 11) is replaced by a serine (S). The substitution may be the replacement of cysteine ​​with serine.

[0057] The proteins or peptides provided in this specification can be isolated and / or purified from nature, or recombined or chemically synthesized. When the amino acid sequence of the protein or peptide provided in this specification contains methionine (Met, M) as the first amino acid residue from the N-terminus, the protein or peptide can be recombinantly produced, and the methionine at the first amino acid position from the N-terminus can be encoded by a start codon. Therefore, when the amino acid sequence of the protein or peptide provided in this specification contains methionine at the N-terminus through recombinant production, it can be interpreted as containing an amino acid sequence starting from the second amino acid residue, excluding the methionine at the first N-terminus when produced through recombinant production, provided that the protein or peptide is obtained by another method (e.g., chemical synthesis or isolation from nature) or is not located at the N-terminus of the conjugate.

[0058] The mRNA encoding the first carrier protein is linked to the 5' end of the mRNA of the antigen protein (corresponding to the N-terminus of the antigen protein), and the mRNA encoding the first carrier protein can be linked by adding a base sequence encoding methionine (Met, M) at the 5' end, for example, adding and linking the "atg" sequence, but not limited thereto.

[0059] Antigenic proteins

[0060] The couplings provided in this specification include: mRNA of antigenic proteins; The mRNA encoding the first carrier protein is attached to the 5' end of the mRNA of the antigen protein; and The mRNA encoding the second carrier protein is attached to the 3' end of the mRNA of the antigen protein. The antigenic protein may contain, but is not limited to, T-cell epitopes. Specifically, the antigenic protein may contain MHC class I and / or class II binding motifs. For example, the antigenic protein may contain an MHC class II binding motif and may contain a CD4+ T-cell epitope, which is a peptide sequence that can be presented on the surface of an antigen-presenting cell by MHC class II molecules. The antigenic protein may contain an MHC class I binding motif and may contain a CD8+ T-cell epitope, which is a peptide sequence that can be presented on the surface of a cell by MHC class I molecules. The antigenic protein may also contain both CD4+ T-cell epitopes and CD8+ T-cell epitopes.

[0061] In this specification, "MHC (major histocompatibility complex)" or "major histocompatibility complex" refers to a protein that delivers antigen fragments to immune cells to distinguish non-self molecules. There are two types: MHC class I and MHC class II. MHC class I is present in all nucleated cells, while MHC class II is present in antigen-presenting cells. MHC class I molecules interact with CD8+ cytotoxic T cells and play a crucial role in organ transplant rejection or the destruction of infected cells. MHC class II molecules recognize non-self antigens by interacting with CD4+ helper T cells and play a vital role in inducing cell-mediated immunity.

[0062] Generally, in an adaptive immune response, when an antigen enters the body, antigen-presenting cells take up it and break it down into short peptide fragments. These peptides can bind to MHC class I or MHC class II molecules and be transported to the cell surface. In this way, when the peptides of the antigen bind to MHC class I or MHC class II molecules and are presented on the cell surface of antigen-presenting cells, T cells recognize them via T cell receptors (TCRs) and are activated, triggering an immune response. In this respect, the antigen proteins described herein can correspond to epitopes on T cells.

[0063] The antigenic protein (e.g., a T-cell epitope) may be derived from tumor antigens, infectious antigens, autoantigens, or sensitizing antigens, but is not limited thereto. Specifically, the antigenic protein (e.g., a T-cell epitope) is expected to bind to MHC class I or MHC class II molecules, but may contain a subset of known tumor antigens, infectious antigens, autoantigens, or sensitizing antigens. The antigen may be a CD8+ or CD4+ T-cell epitope, but is not limited thereto.

[0064] The antigenic protein (e.g., a T-cell epitope) may comprise a natural or non-natural amino acid sequence, an amino acid with post-translational modifications, or a peptide mimic capable of inducing an immune response (e.g., a T-cell or B-cell response) in a subject. For example, the antigenic protein (e.g., a T-cell epitope) may be about 5 to about 100 amino acids or about 5 to about 50 amino acids, or it may be about 7 to 35 amino acids, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35 amino acids, but is not limited thereto.

[0065] In this specification, "tumor antigen" may be used interchangeably with "cancer antigen" and refers to an antigen expressed in a tumor (cancer) that elicits an immune response. This immune response may be accompanied by the production of antibodies or the activation of specific immune cells, or both.

[0066] The tumor antigen may be derived from an organism with a tumor, completely killed or inactivated tumor cells, or lysates, and includes any antigen derived from the tumor. Lysates refer to substances produced by applying processes that cause the normal cellular structure to rupture. Furthermore, the tumor antigen includes any protein or other substance with antigenic characteristics that is contained in tumor cells and expressed differentially from normal cells.

[0067] The tumor antigen may include, but is not limited to, tumor-associated antigens (TAAs), tumor-specific antigens (TSAs), or tumor-derived neoantigens.

[0068] The tumor-associated antigen (TAA) is an antigen that appears more frequently in cancer cells than in normal cells or at a different differentiation stage than in normal cells, and is also a tumor-shared antigen present in trace amounts in normal cells. Therefore, it is highly likely that an immune response to its use may be ineffective due to autotolerance, an immunosuppressive mechanism that prevents damage to one's own cells, or conversely, unwanted organs may be attacked by autoimmunity.

[0069] The tumor-associated antigen (TAA) may be mTrp2, CT (testicular cancer) antigen, EGFR, Ova, Melan-A, PSMA (prostate-specific membrane antigen), survivin, ADAbp (adenosine deaminase-binding protein), cyclophilin b, gp100, CRC (colorectal cancer-associated antigen) -C017-1 A / GA733, CEA (carcinoembryonic antigen), CAP-1, CAP-2, etv6, AML1, PSA (prostate-specific antigen), PSA-1, PSA-2, PSA-3, MAGE (melanoma antigen E) (e.g., MAGE-A, MAGE-A3), GAGE ​​(G antigen), BAGE (B melanoma antigen), RAGE (renal tumor antigen), LAGE (L antigen), NAG, GnT-V, MUM-1, CDK4, p53, tyrosinase, Muc1 (mucin 1), HER2 / neu, p21ras, N-RAS, K-RAS, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, p120ctn, PRAME, NY-ESO-1, TRP2, mammary globin- A. Metallo-Plasmin-1 (MPS-1), Cytochrome P450 subtype 1B1, 90K / Mac-2 binding protein, Ep-CAM (MK-1), HSP-70, hTERT (TRT), LEA, TAGE-1, 5T4, gp70, SCP-1, cmyc, Cyclin B1, MDM2, p62, Koc, IMP1, TA90, OA1, CT-7, HOM-MEL-40 / SSX-2, SSX-1, SSX-4, HOM-TES-14 / SCP-1, HOM-TES-85, HDAC5, MBD2, TRIP4, NY-CO-45, KNSL6, HIP1R, Seb4D, KIAA1416, IMP1, 90K / Mac-2 binding protein, MDM2, or LMNA, but not limited to these.

[0070] The tumor-specific antigen (TSA) refers to an antigen that is specifically present only in cancer cells, and specifically, it can be M12, M20, M21, M30, or M44, but is not limited to these. In particular, when tumors proliferate in cancer patients, cancer cell-specific gene mutations occur, producing neoantigen epitopes that can stimulate T cells; these are called neoantigens. In other words, a neoantigen may contain cancer cell-specific gene mutations and is selectively expressed only in cancer cells, unlike tumor-shared antigens that are also expressed in trace amounts in normal cells. Therefore, it is recognized by the autoimmune system as a non-self exogenous epitope and induces strong anti-cancer immune activity.

[0071] When peptides derived from mutated DNA are displayed on the MHC layer on the cell surface, T cell receptors (TCRs) recognize them. However, mutations do not occur in normal cells or tissues, so neoantigen-specific T cells do not have self-tolerance or autoimmune problems. Due to these advantages, neoantigens are considered ideal targets for T cell-based cancer immunotherapy.

[0072] The causes of neoantigens include, but are not limited to, frameshifts (frameshift deletions or insertions) (where the translation of the genetic code is misdirected when one or more nucleotides that make up DNA are added or deleted), point mutations that occur by replacing one nucleotide with another, other missense mutations, splice site mutations, readthrough mutations, or gene fusion mutations.

[0073] Neoantigens are predicted through specific genomic analysis of cancer cells from individual cancer patients. For example, cancer cells are obtained from a patient's tumor, DNA is extracted, and the base sequence is analyzed and compared with the base sequence of normal cells. Neoantigens stimulating T cells can then be identified in the base sequence of various mutated regions. For this purpose, big data processing techniques such as next-generation sequencing (NGS), whole-exome sequencing (WES), or RNA sequencing can be used, along with computer programs for predicting MHC binding, or artificial intelligence (AI) for predicting neoantigens, but not limited to these. Because the forms of mutations are not shared between patients, neoantigens can be produced as personalized cancer vaccines.

[0074] The tumor-derived neoantigens may include, but are not limited to, mutations specifically expressed in cancer cells.

[0075] The tumor antigen may comprise the amino acid sequence of SEQ ID NO: 51, 53, 55, 57, or 59, or an amino acid sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the stated amino acid sequence, but is not limited thereto. The gene encoding the tumor antigen may have the base sequence of SEQ ID NO: 52, 54, 56, 58, or 60, or a base sequence having 50% or higher, 60% or higher, 70% or higher, 80% or higher, 85% or higher, 90% or higher, 95% or higher, or 99% or higher sequence homology with the stated amino acid sequence, but is not limited thereto.

[0076] The infectious antigen may be an antigen derived from a virus, bacteria, parasite, or fungus, but is not limited thereto.

[0077] The virus-derived antigen may be derived from, but is not limited to, antigens derived from poxvirus, varicella virus, Ebola virus, Marburg virus, dengue virus, influenza virus, parainfluenza virus, respiratory syncytial virus, measles virus, human immunodeficiency virus, human papillomavirus, varicella-zoster virus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, JC virus, rhinovirus, rotavirus, rhinovirus, adenovirus, papillomavirus, parvovirus, piconemavirus, poliovirus, mumps virus, rabies virus, reovirus, rubella virus, cloacal virus, orthomyxovirus, retrovirus, hepatotropic virus, Coxsackie virus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, or hepatitis E virus.

[0078] The bacterial-derived antigens may be derived from species of the genus *Borrelia*, *Bacillus anthracis*, *Borrelia burgdorferi*, *Bordetella pertussis*, *Campylobacter jejuni*, *Chlamydia*, *Chlamydia psittaci*, *Chlamydia trachomatis*, *Clostridium*, *Clostridium tetani*, *Clostridium botulinum*, and *Clostridium perfringens*. The following bacteria are listed: * perfringens*, *Corynebacterium diphtheriae*, *Coxiella*, *Enterococcus*, *Erlichia*, *Escherichia coli*, *Francisella tularensis*, *Haemophilus*, *Haemophilus influenzae*, *Haemophilus parainfluenzae*, *Lactobacillus*, *Legionella*, *Legionella pneumophila*, *Leptospirosis interrogans*, *Listeria*, *Listeria monocytogenes*, *Mycobacterium*, and *Mycobacterium tuberculosis*. tuberculosis), Mycobacterium leprae, Mycoplasma, Mycoplasma pneumoniae, Neisseria, Neisseria meningitidis, Neisseria gonorrhoeae, Pneumococcus, Pseudomonas, Pseudomonas aeruginosa* Salmonella* species, *Salmonella typhi*, *Salmonella enterica*, *Rickettsia* species, *Rickettsia rickettsii*, *Rickettsia typhi*, *Shigella* species, *Staphylococcus* species, *Staphylococcus aureus*, *Streptococcus* species, *Streptococccus pnuemoniae*, *Streptococcus pyrogenes*, *Streptococcus mutans*, *Treponema* species, *Treponema pallidum*, *Vibrio* species, *Vibrio cholerae*. Antigens of *Yersinia pestis* (or *Yersinia cholerae*), but not limited to these.

[0079] The fungal-derived antigen may be an antigen derived from fungi, wherein the fungi are selected from, but are not limited to, species of the genera *Candida*, *Cryptococcus*, *Coccidioides*, *Histoplasma*, and *Aspergillus*.

[0080] The parasite-derived antigens may be antigens derived from the genera Plasmodium, Trypanosome, Schistosoma, or Leishmania, but are not limited to these.

[0081] The self-antigen may be an antigen known or suspected of causing autoimmunity, but is not limited to this.

[0082] The sensitizing antigen (allergen) can be an antigen that is known or suspected of causing an allergy, but is not limited to this.

[0083] Immunization uses of conjugates

[0084] Other embodiments provide an immunization composition and / or vaccine composition comprising the conjugate.

[0085] The composition relates to a composition for the prevention, improvement and / or treatment of cancer, infectious diseases, autoimmune diseases and / or allergic diseases.

[0086] In this specification, "immunization composition" means any composition capable of inducing an immune response, and "vaccine" means an immunization composition that reduces or prevents the risk of disease or infection or improves or treats an existing disease or infection by inducing an immune response. The immunization composition may be an immunization composition against cancer, infectious diseases, autoimmune diseases, and / or allergic diseases.

[0087] Specifically, the immunization composition may refer to a composition that can induce an immune response by generating antibodies against the antigen.

[0088] The composition may be a formulation comprising the conjugates provided in this specification and may be administered to a subject to elicit an immune response. The composition may be conveniently used to prevent, improve, or treat a disease. Upon introduction into a subject or host, the composition may elicit an immune response, preferably a T-cell-mediated immune response.

[0089] The vaccine may be an anticancer vaccine containing tumor antigens, such as tumor antigen sequences obtained from, for example, patient tumor analysis, particularly neoantigen sequences, which can maximize the personalized proliferation and activation of anticancer T cells to enhance anticancer immune efficacy.

[0090] The composition may also contain additional adjuvants to enhance vaccine efficacy. Suitable adjuvants include (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc.; and (2) oil-in-water emulsion formulations (containing or not containing specific immunostimulants such as muramyl peptides or bacterial cell wall components), such as (a) MF59 (WO90 / 14837) formulated into submicron particles, which contains 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (not necessarily but selectively containing varying amounts of N-acetylmuramyl-L-alanyl-D-isoglutamine). (a) L-alanine-2-(1',2'-dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), (b) SAFs microfluidized into submicron particles or shaken to produce large-particle emulsions, comprising 10% squalene, 0.4% Tween 80, 5% Prönnicke block polymer and N-acetyl-muramycyl-L-threonyl-D-isoglutamine (thr-MDP), and (c) the Ribi™ adjuvant system (RAS), comprising (2) At least one bacterial cell wall component selected from monophosphoryl lipid A (MPL), trehalose dimethicone ester (TDM) and the cell wall skeleton, 2% squalane and 0.2% Tween 80; (3) saponin adjuvants; (4) complete Freund's adjuvants and incomplete Freund's adjuvants (IFA); (5) cytokines, such as interleukins (e.g. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (e.g. gamma interferon), macrophage colonies (6) Detoxified mutants of bacterial ADP ribosylated toxins such as cholera toxin (CT), pertussis toxin (PT) or heat-labile toxin (LT) of Escherichia coli, particularly LT-R72, CT-S109, PT-K9 / G129 (WO93 / 13302 and WO92 / 19265); and (7) other substances used as adjuvants to enhance vaccine efficacy, but not limited thereto.

[0091] If desired, the composition may also contain commonly used amounts of pharmaceutically acceptable carriers, diluents, and / or excipients.

[0092] Pharmaceutically acceptable carriers are typically used in the preparation of, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil. In addition to the above components, the composition may further comprise lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0093] The conjugate and / or composition containing it as an active ingredient can be prepared in unit dose form or in a multi-dose container, using a method readily applicable to those skilled in the art and by using pharmaceutically acceptable carriers and / or excipients.

[0094] For example, the composition may contain a suspended or dissolved ordinary saline or buffered aqueous solution medium. For example, it may typically contain diluents such as water, saline, glycerol, ethanol, etc., and auxiliary substances such as wetting agents, emulsifiers, pH buffers, etc., may be present in the composition.

[0095] Suitable forms for injection include sterile aqueous solutions (aqueous) or dispersions, as well as sterile powders for the immediate preparation of sterile injectable solutions or dispersions. They should be stable under the preparation conditions and should be preservative-resistant against microbial contamination such as bacteria or fungi. Microbial contamination can be prevented using various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, isotonic agents, such as sugars or sodium chloride, are preferably included. Prolonged absorption of the injectable composition can be achieved by using a delayed-absorption agent, such as aluminum monostearate or gelatin, in the composition.

[0096] The desired amount of the conjugate and the various other components described above are combined in the solvent, and if necessary, other components, such as buffer solutions (e.g., PBS), besides the conjugate and / or thermosensitive adjuvants and cytokines, are filtered and sterilized to prepare a sterile injectable solution. Generally, various sterile active ingredients are incorporated from the basic dispersion medium and the aforementioned media into a sterile medium containing the desired other components to prepare a dispersion. In the case of sterile powders used to prepare sterile injectable solutions, vacuum drying and freeze-drying methods are preferred. These methods yield powders of the active ingredients and any desired components from the sterile filtered solution described above.

[0097] Patients who receive the conjugate or a composition containing it as an active ingredient may be mammals, such as humans, primates (including monkeys), rodents (including rats, mice, etc.), but are not limited thereto.

[0098] Another aspect of the invention relates to a method for inducing, generating, or promoting an immune response against an antigen in a human being (e.g., a cancer patient), and the method includes administering an effective dose of the composition or conjugate to the patient (e.g., a cancer patient).

[0099] In this specification, "immune response" refers to the result of direct or indirect stimulation mediated by cells or cytokines, and specifically to changes in the activity of immune system cells such as B cells, T cells, or monocytes. The immune response can be specific (T cell and / or B cell) and / or non-specific.

[0100] Without limiting the scope of the invention in any way, delivery of the conjugate according to the invention induces an immune response, and specifically, it is particularly useful for inducing T cell responses, such as CD4+ T cell responses or CD8+ T cell responses against antigens. These CD4+ and CD8+ T cell responses may occur in conjunction with humoral responses or other specific or nonspecific immune responses, or they may occur independently.

[0101] Other aspects of the invention relate to the use of the conjugate in the treatment and / or prevention of disease states. Examples of diseases that can be treated according to the methods of the invention include various cancers, infectious diseases, autoimmune diseases, and allergic diseases.

[0102] The cancers mentioned can be solid cancers or blood cancers, and specifically, they can be breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, endometrial cancer, uterine cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, nephroblastoma, skin cancer, oral squamous cell carcinoma, epidermoid carcinoma, nasopharyngeal carcinoma, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, lymphoma (such as Hodgkin's lymphoma or non-Hodgkin's lymphoma), stomach cancer, pancreatic cancer, testicular cancer, etc. The treatment may include thyroid cancer, follicular carcinoma, melanoma, myeloma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, mesenchymal tumor, soft tissue sarcoma, liposarcoma, gastrointestinal stromal sarcoma, malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma, leiomyosarcoma, stromal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, testicular cancer, neuroblastoma, myeloma, glioma, benign skin tumors, or leukemia. The lung cancer may be, for example, small cell lung cancer (SCLC) or non-small cell lung cancer (NSCLC). The leukemia may be, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), or chronic lymphoblastic leukemia (CLL). The subject to treatment may be a subject who has received two doses of anti-proliferative therapy. For example, the secondary anti-excessive proliferation therapy can be chemotherapy, radiotherapy, immunotherapy, phototherapy, cryotherapy, tomography, hormone therapy, or surgery.

[0103] Other embodiments provide an immunization method comprising administering the conjugate and / or composition to a subject requiring immunization, and more specifically, provide an immunization method for cancer, the method comprising administering it to a subject requiring immunization against cancer.

[0104] Other embodiments provide a method for preventing or treating diseases such as cancer, infectious diseases, autoimmune diseases, or allergic diseases by using the composition to enhance the immune response. Here, the application of the composition induces, promotes, or enhances an immune response that inhibits, stops, delays, or prevents its occurrence or progression.

[0105] The composition can be delivered directly, typically systemically, subcutaneously, intradermally, intraperitoneally, intravascularly (intravenously), intramuscularly, or locally, or into the interstitial space. The composition can also be applied to the lesion. The administration regimen can be a single-dose or multiple-dose regimen.

[0106] In this specification, "effective dose" means an amount sufficient to achieve the desired outcome when administered to subjects, including humans, such as an amount effective in treating or preventing cancer. The effective dose may vary depending on factors such as an individual's disease condition, age, sex, and weight. As will be understood by those skilled in the art, the dose or treatment regimen can be adjusted to provide an optimal therapeutic response.

[0107] Treatment regimens for subjects using therapeutically effective doses can consist of a single administration or, as another example, include a series of applications. The duration of a treatment phase depends on various factors, such as disease severity, the subject's age, vaccine concentration, patient responsiveness to the vaccine, or combinations thereof. Furthermore, it is understood that the effective dose of the vaccine used for treatment may increase or decrease during an individual treatment regimen. Dosage variations may occur and can be elucidated by standard diagnostic assays known in the art. The vaccines of the present invention, such as cancer vaccines, can be administered before, during, or after treatment with common anticancer agents, radiotherapy, hormone therapy, biotherapy, and / or surgical tumor resection.

[0108] Beneficial effects

[0109] This invention relates to a conjugate comprising mRNA of an antigen protein and mRNA encoding a carrier protein linked to the 5' and / or 3' ends of the mRNA of the antigen protein, and also to an immunization composition and / or vaccine composition comprising the conjugate, having the effect of stably increasing the expression of the antigen protein. Attached Figure Description

[0110] Figure 1 and 2The vector diagrams for constructing pUC57-T7-MP-A118 and pEGFP for protein screening are shown respectively.

[0111] Figure 3 A schematic diagram is shown of a delivery protein vector used to construct mRNA for protein screening.

[0112] Figure 4 A vector diagram of the pUC57-T7-CP12G protein delivery vector is shown.

[0113] Figure 5 shows the results confirming that CD4 / CD8 antigens were delivered to the processing / presentation pathways, respectively, under the conditions of proteasome inhibitor and lysosomal inhibitor treatments used for preliminary screening of the delivery proteins.

[0114] Figure 6 This diagram illustrates cellular expression when transmembrane domains are added to the cytoplasmic domains of SORT1 and Lamp.

[0115] Figure 7 The results show that, under the treatment of proteasome inhibitors and lysosome inhibitors used for secondary screening of the delivery proteins, the CD4 / CD8 antigens were confirmed to be delivered to the processing / presentation pathways, respectively.

[0116] Figure 8 A schematic diagram of a vector used to construct a vector containing a delivery protein and an epitope is shown.

[0117] Figure 9 A vector diagram of the pUC57-T7-5 epitope is shown.

[0118] Figure 10 A schematic diagram of the pLRK33-empty vector used to construct a vector containing a transport protein and an epitope is shown.

[0119] Figure 11 This diagram illustrates the process of inserting a fragment containing an epitope, a carrier protein, etc., into an empty vector during the construction of a vector containing a carrier protein and an epitope.

[0120] Figure 12 The vector diagram of pLRK33-hTrx_WT is shown.

[0121] Figure 13 A vector diagram of the pLRK33-hTrx_m vector used to construct a comparative example vector without the carrier protein is shown.

[0122] Figure 14 The results show the results of confirming the expression level of epitopes in mRNA based on whether the carrier protein is included in the K562 cell line.

[0123] Figure 15 The results show the results of confirming the expression level of epitopes in mRNA based on whether the carrier protein is included in human dendritic cells.

[0124] Figure 16 The results show the immunogenicity of the target epitope in mRNA based on whether the carrier protein is contained in human dendritic cells. Detailed Implementation

[0125] The invention will be described in more detail below through embodiments. However, the following embodiments are intended to illustrate the content of the invention only, and the scope of the invention is not limited to the following embodiments.

[0126] Example 1. Screening of transport proteins

[0127] Example 1-1. Construction of mRNA encoding carrier proteins

[0128] Preliminary screening of delivery proteins is performed by confirming whether the mRNA encoding the delivery protein appropriately delivers the CD4 / CD8 antigen to the processing / presentation pathway.

[0129] Specifically, for in vitro transcription, the required vector based on the pUC57 vector (Cosmogenetech) was constructed by requesting GeneWiz. The resulting vector, pUC57-T7-MP-A118, is illustrated in the diagram below. Figure 1 middle.

[0130] The constructed vector was treated with AscI (Enzynomics; R068M) and EcoRI (Enzynomics; R002M) restriction enzymes and purified using Expin Gel SV (Gene all, Cat#102-102). During this purification process, for vector chains where the AscI site is located at the N-terminus and the EcoRI site at the C-terminus, to place the carrier protein at each of the N-terminus and C-terminus of the vector according to the combinations in Table 2, the T7 oligonucleotides corresponding to the carrier proteins according to the combinations in Table 3 of Table 2 were used, along with Platinum. TM The SuperFi II Green PCR master mix (ThermoFisher; Cat#: 12369050) was used for PCR via the EZ-fusion HT cloning kit (Enzynomics; Cat#: EZ015TL). The carrier proteins were inserted into the AscI and EcoRI sites to construct the pUC57-T7-CP01G to pUC57-T7-CP50G plasmids listed in Table 2 below. The specific preparation procedures for each carrier protein are described below: (i)mODC The mODC fragments for C-terminal insertion were generated by PCR after mixing the oligonucleotides oCmODC-BE1-F1, omODC-R2, omODC-F3, and oCmODC-X1-R4 (Table 3). The mODC fragments for C-terminal insertion were generated by purifying the previously generated mODC fragments for N-terminal insertion and performing PCR using the oligonucleotides oNmODC-A1-F1 and oNmODC-BE1-R2 (Table 3).

[0131] (ii)cmyc

[0132] The CMYC fragment for N-terminal insertion was generated by PCR after mixing the oligonucleotides oNcmyc-A1-F1 and oNcmyc-BE1-R2 (Table 3). The CMYC fragment for N-terminal insertion was generated by purifying the previously generated CMYC fragment for C-terminal insertion and performing PCR using the oligonucleotides oCcmyc-BE1-F1 and oCcmyc-X1-R2 (Table 3).

[0133] (iii) Lamp

[0134] Lamp fragments for N-terminal insertion were generated by PCR after mixing oligonucleotides oNIi-A1-F1 and oNIi-BE1-R2 (Table 3). Lamp fragments for N-terminal insertion were generated by purifying previously generated Lamp fragments for C-terminal insertion and performing PCR using oligonucleotides oCIi-BE1-F1 and oCIi-X1-R2 (Table 3).

[0135] (iv) SORT1

[0136] The SORT1 fragment for C-terminal insertion was generated by PCR after mixing the oligonucleotides oCSORT-BE1-F1, oSORT-R2, oSORT-F3, and oCSORT-X1-R4 (Table 3). The SORT1 fragment for C-terminal insertion was generated by purifying the previously generated SORT1 fragment for N-terminal insertion and performing PCR using the oligonucleotides oNSORT-A1-F1 and oNSORT-BE1-R2 (Table 3).

[0137] (v)TRX

[0138] It is generated by PCR after mixing all oNhTRX-A1-F, oNhTRX-BE1-R, oTRXwt-1F, oTRXwt-2R, oTRXwt-3F, oTRXwt-4R, oTRXwt-5F, oTRXwt-6R and oTRXwt-3F1 oligonucleotides in Table 3 below.

[0139] (vi) PSBD

[0140] It is generated by PCR after mixing all oChPSBD-BE1-F and oChPSBD-X1-R oligonucleotides from Table 3 below.

[0141] (vii) Ub and UbR

[0142] Because the last sequence of Ub inserted at the N-terminus, alanine (A), is prone to degradation when replaced by arginine (R), the oNUbR-BE1-R5 oligonucleotide (UbR) from Table 3 below is used to insert Ub so that the last sequence is R when inserting Ub at the N-terminus.

[0143] Specifically, PCR was performed using oNUbR-A1-F1, oUbR-R2, oUbR-F3, and oNUbR-BE1-R5 from Table 3 below to generate UbR, and Ub was generated and inserted by purifying the UbR PCR product and performing PCR using oCUb-BE1-F1 and oCUb-X1-R2 from Table 3 below.

[0144] [Table 2]

[0145] (In Table 2 above, TRXwt refers to the wild type of TRX, which can be obtained from nature without mutation, and TRXmt refers to TRX in which the cysteine ​​(C) corresponding to the N-terminus of the TRX amino acid sequence from SEQ ID NO: 11 is replaced by serine (S).)

[0146] [Table 3]

[0147] (In Table 3 above, the bold "tccgga" corresponds to the BspEI site, and the BspEI site is inserted simultaneously with the insertion of the carrier protein.)

[0148] After insertion of the carrier protein, the protein was treated with BspEI (NEB, R0540S) restriction enzyme and purified with Expin Gel SV (Gene all, Cat#102-102).

[0149] Then, 10 ng of a pEGFP vector containing an EGFP reporter (Clontech, catalog number 6077-1, GenBank U76561.1) was prepared as a template, and 10 pmole of each of the EGFP oligonucleotides (oEGFP-BE1-F and oEGFP-BE1-R) listed in Table 3 and 100 μl of Platinum were prepared. TM SuperFi II Green PCR master mix (ThermoFisher; cat#: 12369050) was used for PCR.

[0150] By treating with BspEI (NEB, R0540S) restriction enzyme after PCR and subcloning into plasmids capable of expressing each purified carrier protein combination, carrier protein vectors containing inserted EGFP were obtained.

[0151] The vector diagram of the pEGFP vector is shown in Figure 2 The schematic diagram of the generated protein carrier is shown in the diagram. Figure 3 The exemplary vector diagram of the generated pUC57-T7-CP12G plasmid is shown in the figure. Figure 4 The amino acid and base sequence information of the carrier proteins, and those inserted into the N-terminus and C-terminus respectively, are shown in Tables 4 and 5 below.

[0152] [Table 4]

[0153] (In Table 4 above, when the carrier protein is inserted into the N-terminus, a methionine (M) with an atg base sequence is added and inserted at the 5' end of each protein, and it corresponds to the GS linker sequence indicated by the underline above.)

[0154] [Table 5]

[0155] (In Table 5 above, when the carrier protein is inserted into the N-terminus, an atg base sequence is added and inserted at the 5' end of each protein, which corresponds to the GS adapter sequence indicated by the underline above.)

[0156] By using mMESSAGE mMACHINE TM The T7 ULTRA transcription kit (ThermoFisher, AM1345) was used to transcribe the generated delivery protein vector in vitro, producing mRNA of the following structural formula 1. The sequence information corresponding to the 5'UTR and 3'UTR is shown in Table 6 below.

[0157] [Structure 1]

[0158] 5'UTR (TE) - (Carrier Protein I) - GGSGGGGSGG - EGFP - GGSGGGGSGG - (Carrier Protein II) - 3'UTR (2X HBB)

[0159] (In Formula 1, GGSGGGGSGG represents the adapter sequence, carrier protein I (CP#1) corresponds to the carrier protein inserted into the N-terminus of the resulting vector, carrier protein II (CP#2) corresponds to the carrier protein inserted into the C-terminus, the 5'UTR (TE) has the sequence of SEQ ID NO: 41 in Table 6 below, and the 3'UTR (2X HBB) has the sequence of SEQ ID NO: 42 in Table 6 below.)

[0160] [Table 6]

[0161] Example 1-2. Primary screening of carrier proteins

[0162] When the expression pattern is altered by the proteasome inhibitor BTZ (bortezomib), the CD8 antigen can be appropriately delivered to the processing / presentation pathway, and when the expression pattern is altered by the lysosomal inhibitor CHQ (chloroquine), the CD4 antigen can be appropriately delivered to the processing / presentation pathway. Therefore, BTZ and CHQ treatments can be used to determine whether the processing / presentation of CD8 and CD4 antigens is carried out.

[0163] Specifically, each generated mRNA was transfected into the K562 cell line (ATCC, CCL-243) using the 4D Nucleofector system (Lonza). For transfected cells, to confirm response to each inhibitor used—the proteasome inhibitor bortezomib (BTZ) and the lysosomal inhibitor chloroquine (CHQ)—cells were treated with either BTZ or CHQ, and responsiveness was confirmed 16 hours later using a fluorescence-activated cell sorting system (FACS) to determine whether proteasome or lysosomal degradation was inhibited, thereby confirming the potential for delivery to HLA class I and II pathways. The results confirming the delivery potential of the above combinations are shown in... Figure 5a to 5k middle.

[0164] Specifically, in the above Figure 5a to 5k In the diagram, C represents CHQ treatment, B represents BTZ treatment, BC represents both BTZ and CHQ treatment, and X represents no treatment. When C increases GFP expression compared to X, it means the likelihood of CD4 antigen is high. When B increases expression compared to X, it means the likelihood of CD8 antigen is high. When BC increases expression compared to X, it means the likelihood of both CD4 and CD8 antigen is high.

[0165] As a result confirming the delivery probability, the CP01 (TRXmt-PSBD) combination with inserted mutated TRX was shown to express EGFP well without the influence of BTZ and CHQ. Furthermore, the CP12, CP13, CP23, CP31, CP33, CP34, CP36, and CP38 combinations in Table 1 were confirmed to be affected by BTZ, and no combination was confirmed to be affected by CHQ. Therefore, it was confirmed that CD4 and CD8 antigens were delivered to the processing / presentation pathway by the CP01 combination, and that the CD8 antigen was delivered to the processing / presentation pathway by the CP12, CP13, CP23, CP31, CP33, CP34, CP36, and CP38 combinations. In addition, the CP21 (UbR-cmyc) and CP27 (Lamp-cmyc) combinations showed no indistinguishable effects of BTZ and CHQ.

[0166] Example 1-3. Secondary screening of carrier proteins

[0167] Since the CP01 combination was demonstrated in Examples 1-2 above to allow both CD4 and CD8 antigens to be delivered to the processing / presentation pathway, a design was made to add the TRX of the CP01 combination to the CP12, CP13, CP23, CP31, CP33, CP34, CP36 and CP38 combinations that were demonstrated in Examples 1-2 above to allow the delivery of CD8 antigen to the processing / presentation pathway.

[0168] Specifically, each of the plasmids pUC57-T7-CP12G, pUC57-T7-CP13G, pUC57-T7-CP23G, pUC57-T7-CP31G, pUC57-T7-CP33G, pUC57-T7-CP34G, pUC57-T7-CP36G, and pUC57-T7-38G generated in Examples 1-1 above was used as a template, and the oligonucleotides oCCP12G-F1 and oCCPxxG-R1 in Table 7 below were used as primers to perform PCR and purify each product.

[0169] [Table 7]

[0170] After purification, the TRX PCR product generated during the plasmid production process in Examples 1-1 above was inserted into the purified backbone portion via EZ fusion, ultimately producing a plasmid in which TRX was added to the N-terminus. The backbone portion was then processed using AscI (Enzynomics; R068M) and XhoI (Thermo Scientific). TM FastDigest (#FD0694) generates each of the following plasmids produced in Examples 1-1 by restriction enzyme treatment: pUC57-T7-CP12G, pUC57-T7-CP13G, pUC57-T7-CP23G, pUC57-T7-CP31G, pUC57-T7-CP33G, pUC57-T7-CP34G, pUC57-T7-CP36G, and pUC57-T7-38G. The generated plasmids are named by adding 1 before the number of the original plasmid (e.g., the plasmid in which TRX is added to pUC57-T7-CP12G: pUC57-T7-CP112G).

[0171] In addition, for the combination of CP12, CP13, CP21, CP31 and CP33, the carrier protein at the N-terminus is changed to TRX (CP41 to CP45).

[0172] Specifically, the pUC57-T7-CP21G, pUC57-T7-CP33G, pUC57-T7-CP-13G, pUC57-T7-CP12G, and pUC57-T7-CP31G plasmids from Examples 1-1 were used as templates, and the oligonucleotides oGS-EGFP-F and oCCPxxG-R1 from Table 8 below were used as primers for PCR. Each product was purified to obtain fragments in which EGFP and a carrier protein at the C-terminus were linked.

[0173] [Table 8]

[0174] Subsequently, the TRX PCR products generated in Examples 1-1 above were inserted into the purified backbone portion via EZ fusion, ultimately producing plasmids (pUC57-T7-41G, pUC57-T7-CP42G, pUC57-T7-CP43G, pUC57-T7-44G, and pUC57-T7-CP45G) in which the N-terminus of each plasmid is a TRX carrier protein. The backbone portion was then processed using AscI (Enzynomics; R068M) and XhoI (Thermo Scientific). TMFastDigest (#FD0694) generates each of the plasmids pUC57-T7-CP21G, pUC57-T7-CP33G, pUC57-T7-CP13G, pUC57-T7-CP12G, and pUC57-T7-CP31G produced in Examples 1-1 by restriction enzyme treatment.

[0175] In addition, for the combination of CP34, CP27, CP23, CP36 and CP21, the carrier protein at the C-terminus is changed to TRX (CP46 to CP50).

[0176] Specifically, the pUC57-T7-CP34G, pUC57-T7-CP27G, pUC57-T7-CP23G, pUC57-T7-CP36G, and pUC57-T7-CP21G plasmids from Examples 1-1 were used as templates, and oM13F and oGS-EGFP-R from Table 9 were used as primers for PCR. Each product was purified to obtain fragments containing EGFP and a carrier protein at the N-terminus.

[0177] [Table 9]

[0178] Subsequently, using pUC57-T7-CP01G generated in Example 1-1 as a template, PCR was performed using oligonucleotides oCTRXwt-F1 and oCTRXwt-R1 as primers (Table 10) to obtain a TRX fragment for C-terminal insertion. This fragment, along with a fragment containing EGFP and a carrier protein at the N-terminus, was then inserted into a pre-treated TRX fragment using AscI (Enzynomics; R068M) and XhoI (Thermo Scientific) via EZ fusion. TM FastDigest (#FD0694) treated each of the following plasmids with restriction enzymes: pUC57-T7-CP34G, pUC57-T7-CP27G, pUC57-T7-CP23G, pUC57-T7-CP36G, and pUC57-T7-CP21G, thereby producing the final plasmids pUC57-T7-46G, pUC57-T7-CP47G, pUC57-T7-CP48G, pUC57-T7-49G, and pUC57-T7-CP50G.

[0179] [Table 10]

[0180] Information about the carrier protein inserted into each of the N-terminus and C-terminus of the designed vector is shown in Table 2 above, while the amino acid and base sequence information of the carrier protein with added TRX is shown in Tables 4 and 5 above, respectively.

[0181] Furthermore, since SORT1 and Lamp, as transport proteins, possess transmembrane domains as membrane proteins, these transmembrane domains were added to the existing cytoplasmic domains of each protein (SORT1 w / TM and Lamp w / TM), with SORT1 w / TM located at the C-terminus and TRX at the N-terminus, or with Lamp w / TM located at the N-terminus and TRX at the C-terminus (CP201 ​​and CP202). A schematic diagram of intracellular expression is shown in [the diagram]. Figure 6 middle.

[0182] Specifically, SORT1 w / TM is generated by PCR using oligonucleotides of oSORTwTM-1F, oSORTwTM-4R, oSORTwTM-2R, and oSORTwTM-3F from Table 3 above, using essentially the same method as SORT1. Similarly, Lamp w / TM is generated by PCR using oligonucleotides of oLAMPwTM-1F, oLAMPwTM-4R, oLAMPwTM-2R, and oLAMPwTM-3F from Table 3 above, using essentially the same method as SORT1.

[0183] Information on the transport proteins with added transmembrane domains is shown in Table 2 above, while the amino acid and base sequences of the transport proteins with added transmembrane domains are shown in Tables 4 and 5 above, respectively.

[0184] The resulting delivery protein vector was tested using essentially the same method as in Examples 1-1 above, demonstrating its potential for delivery to HLA class I and II pathways after mRNA production. Results showed that... Figure 7 middle.

[0185] As a result of confirming delivery probability, it was confirmed that the existing response was enhanced in the CP136 combination with added TRX, and that a response to CHQ was observed when the transmembrane domain was present in SORT1 and Lamp in the CP201 ​​and CP202 combinations with added transmembrane domain, thus confirming that the overall response was enhanced.

[0186] Example 2. Construction of a vector containing a delivery protein and epitopes.

[0187] Example 2-1. Construction of carrier protein vectors

[0188] To confirm the potential for delivery to HLA class I and II pathways, the combination of CP12, CP13, CP23, CP31, CP33, CP34, CP36, CP38, CP47, CP48, CP50, CP136, CP201, and CP202, which was demonstrated in Example 1 above, had an effect on epitope expression efficiency, resulting in a novel vector containing a combination of transport proteins.

[0189] Specifically, in the above combination, a vector was constructed in which five epitopes were used as linkers between a carrier protein (CP_N; carrier protein 1) inserted at the N-terminus and a carrier protein (CP_C; carrier protein 2) inserted at the C-terminus, and EGFP was ligated to the C-terminus of carrier protein 2 using the P2A sequence. A schematic diagram of the constructed vector is shown in [the diagram]. Figure 8 In the vector of Example 1, EGFP is located between two carrier proteins, while in the generated vector, as a practical application example, an epitope is placed and EGFP for measuring expression levels is linked to the C-terminus.

[0190] In order to construct the carrier, as Figure 8 As shown, insert fragments corresponding to the gene used for cloning were generated by splitting the gene into four segments. The first segment (CP_N segment; CP#1) contains a portion of the first adapter from carrier protein 1; the second segment (epitope segment; epitope_2) contains a portion of the first adapter, the epitope, the adapter, and a portion of the sixth adapter; the third segment (CP_C segment; CP#3) contains a portion of the sixth adapter, carrier protein 2, and a portion of the EGFP sequence; and the fourth segment (P2A-EGFP segment; GFP_4) contains a portion of the P2A sequence and the EGFP sequence. The second and fourth segments are used interchangeably regardless of the carrier protein combination.

[0191] Based on the combinations of CP12, CP13, CP23, CP31, CP33, CP34, CP36, CP38, CP47, CP48, CP50, CP136, CP201, and CP202 in Table 2 above, PCR was performed on each vector manufactured in Example 1 to amplify the first and third fragments using primers from Table 12 that can amplify the carrier proteins listed in Table 11 below. Using primers from Table 12 that can amplify the epitope (epitope_2) and P2A-EGFP (GFP_4), PCR was performed on the pUC57-T7-5 epitope plasmid (…). Figure 9PCR was performed to amplify the second fragment, and the fourth fragment was also amplified using the pUC57-T7-CP12G plasmid from Example 1-1 as a template. Furthermore, to prepare a plasmid with TRX inserted at the N-terminus and no carrier protein inserted at the C-terminus, PCR was performed using the primers in Table 12 below with the combination (TRX_WT) in Table 11.

[0192] [Table 11]

[0193] [Table 12]

[0194] Each combination produces three or four fragments containing BsmBI sites at both ends. Therefore, these fragments are inserted into the empty vector pLRK33-empty using the BsmBI restriction enzyme and T4 DNA ligase of the NEBridge Golden Gate Assembly-BsmBIv2 (NEB # E1602L kit). The epitope fragments contain five epitopes, arranged in the order M44, M30, M21, M12, and mTrp2 according to the CP_N fragment position, and each epitope is ligated via a GGSGGGGSGG sequence adapter. The M44, M30, M21, and M12 epitopes correspond to B16-M44, B16-M30, B16-M21, and B16-M12 as disclosed in the paper https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4838069.

[0195] The empty carrier is illustrated in the diagram. Figure 10 The diagram illustrates the process of inserting each type of fragment into an empty vector. Figure 11 The configuration information of the vectors generated by insertion into empty vectors is shown in Table 13 below. The amino acid and base sequences of the five inserted epitope fragments, as well as the amino acid and base sequences of the five epitope fragments linked to adapters (five epitopes), are shown in Table 14 below. The amino acid and base sequences of the inserted P2A and EGFP are shown in Table 15 below. For example, the vector diagram of the generated plasmid pLRK33-hTrx_WT is shown in... Figure 12 middle.

[0196] [Table 13]

[0197] [Table 14]

[0198] [Table 15]

[0199] Comparative Example 1. Construction of a vector containing only epitopes and no carrier proteins.

[0200] To prepare an insert fragment capable of amplifying from the N-terminus of the epitope in Example 2 to the C-terminus of EGFP, the insert fragment was prepared by PCR amplification of the corresponding portion using the NcoI_epitope_F and XhoI_EGFP_R oligonucleotides listed in Table 16 below. The vector diagram of the pLRK33-hTrx_m vector is shown in [the diagram]. Figure 13 middle.

[0201] [Table 16]

[0202] Using the restriction enzyme NcoI (Thermo Scientific) TM FastDigest; #FD0574) and XhoI (Scientific TM FastDigest (#FD0694) and T4 DNA ligase (Thermo Scientific) TM (#EL0012), insert the generated insert fragment into Figure 10 The pLRK33-empty vector was inserted into the NcoI and XhoI sites to generate a comparative vector pLRK33-No_CP_m in which no carrier protein was inserted.

[0203] Example 3. Construction of mRNA containing epitopes and transport proteins

[0204] mRNAs were constructed by in vitro transcription using 16 vectors (pLRK33-hTrx_WT, pLRK33-CP12_m, pLRK33-CP13_m, pLRK33-CP23_m, pLRK33-CP31_m, pLRK33-CP33_m, pLRK33-CP34_m, pLRK33-CP36_m, pLRK33-CP38_m, pLRK33-CP47_m, pLRK33-CP48_m, pLRK33-CP50_m, pLRK33-CP136_m, pLRK33-CP201_m, pLRK33-CP202_m, and pLRK33-No_CP_m) generated in Example 2 and Comparative Example 1 above.

[0205] Specifically, prior to in vitro transcription, the vector was linearized by cutting the posterior part of poly A with XbaI (Termo Scientific, FD0684), so that only the desired region could be transcribed into RNA.

[0206] Fifteen linearized vectors were used for in vitro transcription under the conditions shown in Table 17 below. The template DNA was removed by treatment with DNase I (Invitrogen, AM2238) and purified using the RNeasy Mini kit (Qiagen, 74104) to complete the construction of mRNA.

[0207] [Structure 2]

[0208] 5'UTR - (Carrier protein 1) - LELELELELEL - (Carrier protein 2) - P2A - EGFP - 3'UTR - poly A

[0209] (In Formula 2, L refers to the linker sequence GGSGGGGGSGG, and carrier protein 1 (CP#1) and carrier protein 2 (CP#2) correspond to each carrier protein inserted into the N-terminus and C-terminus of the resulting vector, respectively.)

[0210] [Table 17]

[0211] Example 1. Measuring the expression efficiency of transport proteins to epitopes in the K562 cell line.

[0212] Some K562 cell lines (ATCC, CCL-243) were obtained and maintained in the jar. After cell sedimentation, they were resuspended in complete medium (RPMI 1640, Gibco, 11875-093; 10% FBS, Gibco, 26140-079; and 1% penicillin-streptomycin, Gibco, 10378-016), and cell counts were performed. Further addition of complete medium or re-sedimentation was then performed to achieve a concentration of 3... 10 5 10 cells / ml, then resuspended in complete culture medium, adjusting the concentration to 3 in 20 ml. 10 5 Cultured at 1 cell / ml in a CO2 incubator.

[0213] The K562 cell line cultured as described above was obtained. Using the SF Cell Line 4D Nucleofector X Kit S (Lonza, V4XC-2032), the mRNA prepared in Example 3 was electroporated into the cell line according to the Amaxa 4D-Nucleofector protocol for K562, and cell healing was performed. The amount of mRNA used for transfection was 1 pmol per sample.

[0214] Cells were harvested from each well and allowed to settle. They were then resuspended in FACS buffer (eBioscience, 00-4222-28) and transferred to FACS tubes. Cell analysis was performed using LSR Tortessa (BD Biosciences) to confirm epitope expression levels. Results showed that... Figure 14 And in Table 18. As a negative control, cell lines without transfected mRNA were prepared, and epitope expression levels were confirmed.

[0215] [Table 18]

[0216] As a result of cell analysis, it was confirmed that EGFP expression efficiency was generally improved in each CP combination containing the carrier protein compared to CP without the carrier protein, and that the use of CP combinations can enhance epitope expression.

[0217] Experimental Example 2. Measuring the expression efficiency of transport proteins to epitopes in human dendritic cells.

[0218] According to the manufacturer's instructions, the cryopreserved human PBMCs (peripheral blood mononuclear cells) (Stemexpress, PBMNC300C) stored in liquid nitrogen were thawed, and the thawed cells were counted. Based on the results, human CD14 microbeads (Miltenyi Biotec, 130-050-201) and LS columns (Miltenyi Biotec, 130-042-401) were used (2 ml of buffer was used for column activation, 2 ml for cell loading, 2 ml for washing 3 times, and 5 ml for cell elution, respectively) to separate the cells according to the user manual, and the number of selected cells was measured.

[0219] The isolated mononuclear cells were suspended in the culture medium included in the ImmunoCult Dendritic Cell Culture Kit (DC Culture Kit) (Stemcell, 10985) containing culture medium (without differentiation supplement) to achieve a concentration of 1e06 cells / ml to generate mononuclear cell-derived dendritic cells (moDCs). These were then seeded in 8 wells of a 96-well plate at a dose of 100 μl per well.

[0220] Remove the culture medium from the cells being cultured using the DC culture kit, and replace it with 100 μl of culture medium containing the differentiation supplement included in the DC culture kit. Add the DC maturation supplement included in the DC culture kit to the cell culture medium according to the calculations in the user manual.

[0221] According to Table 19 below, the mRNA, DP buffer, and carrier generated in Example 3 above were added sequentially and mixed. The carrier is a composition for drug delivery, comprising the cationic compound and anionic polymer compound of Example 1 of Japanese Patent Publication No. 10-2024-0035317, etc.

[0222] In each mixing step, pipetting was performed using a pipette to ensure thorough mixing, and the drug product (DP) mixed with mRNA and carrier was prepared by performing 10 pipettings immediately after the addition of the formulation. The prepared DP was added to cell culture medium supplemented with DC maturation enhancers for treatment and culture, according to the amount of DP treated as shown in Table 19 below. A negative control group without DP treatment was also prepared.

[0223] [Table 19]

[0224] DP treatment was performed, cells were harvested from each well and subjected to sedimentation. They were then resuspended in a solution of FACS buffer (eBioscience, 00-4222-28) and 7-AAD (BD Biosciences, 559925) at a volume ratio of 100:1 and transferred to FACS tubes. Cell analysis was performed three times using LSR Tortessa (BD Biosciences). Results are shown in... Figure 15 And in Table 20.

[0225] [Table 20]

[0226] (The values ​​in Table 20 above are the average of the MFI obtained from three cell analyses.)

[0227] As a result of cell analysis, the MFI values ​​in the combination of CP23, CP31, CP33, CP38 and CP48 were shown to be 2-fold or more compared with those without CP, thus confirming increased human epitope expression.

[0228] Experimental Example 3. Ability to measure the immunogenicity of transport protein-induced epitopes in human dendritic cells.

[0229] The DPs according to Table 19 above were generated using a method substantially the same as that used in Experiment 2 above. They were then added to the culture medium for culturing moDCs for treatment and cultured for 2 days.

[0230] On the day the culture was completed, human PBMCs for moDC production in Experiment Example 2 were prepared separately, and the cell count of the initial PBMCs was counted. Based on this, CD8+ T cells were isolated according to the user manual using a human CD8+ T cell isolation kit (Miltenyi Biotec, 130-096-495) and an LS column (Miltenyi Biotec, 130-042-401).

[0231] Cultured moDCs and isolated CD8+ T cells were harvested, then subjected to cell sedimentation and resuspended in Immunocult-XF T cell expansion medium (Stemcell, 10981) to achieve a moDC density of 5e05 cells / ml and a CD8+ T cell density of 2e06 cells / ml, respectively. Subsequently, each cell suspension was mixed at a 1:1 volume ratio and seeded at 2 ml per well in 6-well plates, and the moDC / CD8+ T cell mixture was cultured for 7 days.

[0232] The cultured cells were harvested again and resuspended to 1e06 cells / ml using CTL medium included in the Mouse IFN-γ Monochrome ELISPOT Kit (Immunospot, mIFNgp-2M).

[0233] Separately from the above process, each peptide corresponding to one of the five epitopes in Table 21 below was diluted to 20 μM with CTL medium to prepare restimulation peptide medium. The prepared cell suspension and peptide medium were then aliquoted into each well of a 96-well plate included in the ELISPOT kit, at 100 μl each, and cultured for 16 hours or longer. As a negative control, they were cultured without the addition of peptide medium.

[0234] [Table 21]

[0235] (In Table 21 above, Multi MAGE-A refers to the antigen sequence that is typically displayed in various subfamily antigens of MAGE-A.)

[0236] After incubation, the reaction was tested according to the user manual and analyzed using an ELISPOT plate analyzer (Immunospot). The results are shown in... Figure 16 middle.

[0237] The results of the ELISPOT analysis confirmed that the immunogenicity of multiple epitopes was enhanced in the combination of CP23, CP31, CP33 and CP38 compared with no CP.

Claims

1. A coupling comprising: mRNA of antigenic proteins; The mRNA encoding the first carrier protein is attached to the 5' end of the mRNA of the antigen protein; and The mRNA encoding the second carrier protein is attached to the 3' end of the mRNA of the antigen protein.

2. The conjugate according to claim 1, wherein the first and second carrier proteins are the same or different, and are each selected from at least one of Ub (ubiquitin), UbR (ubiquitin A77R mutant), Lamp, mODC (mouse ornithine decarboxylase), cmyc, SORT1 (sortilin 1), TRX (thioredoxin), PSBD (peripheral subunit binding domain), TRXwt-UbR, TRXwt-Lamp, TRXwt-mODC, TRXwt-cmyc, TRXwt-SORT1, SORT1 w / TM, and Lamp w / TM.

3. The conjugate according to claim 2, wherein the first and second carrier proteins are selected from the protein combinations shown in Table 1 below: [Table 1] 。 4. The coupling according to claim 2, wherein The UbR contains the amino acid sequence of SEQ ID NO: 1; The Ub contains the amino acid sequence of SEQ ID NO: 2; The Lamp contains the amino acid sequence of SEQ ID NO: 3 or 4; The mODC contains the amino acid sequence of SEQ ID NO: 5 or 6; The cmyc contains the amino acid sequence of SEQ ID NO: 7 or 8; SORT1 contains the amino acid sequence of SEQ ID NO: 9 or 10; The TRXwt contains the amino acid sequence of SEQ ID NO: 11 or 12; The PSBD contains the amino acid sequence of SEQ ID NO: 13; The TRXwt-UbR contains the amino acid sequence of SEQ ID NO: 14; The TRXwt-Lamp contains the amino acid sequence of SEQ ID NO: 15; The TRXwt-mODC contains the amino acid sequence of SEQ ID NO: 16; The TRXwt-cmyc contains the amino acid sequence of SEQ ID NO: 17; The TRXwt-SORT1 contains the amino acid sequence of SEQ ID NO: 18; The SORT1 w / TM contains the amino acid sequence of SEQ ID NO: 19; The Lamp w / ™ contains the amino acid sequence of SEQ ID NO: 20; or The TRXmt contains the amino acid sequence of SEQ ID NO:

145.

5. The conjugate according to claim 3, wherein the first and second carrier proteins are selected from the protein combinations shown in Table 22 below: [Table 22] 。 6. The conjugate according to claim 2, wherein the antigen protein comprises a T-cell epitope.

7. The conjugate according to claim 6, wherein the T cell epitope is derived from a tumor antigen, an infectious antigen, an autoantigen, or a sensitizing antigen.

8. The conjugate according to claim 7, wherein the tumor antigen includes tumor-associated antigen (TAA), tumor-specific antigen (TSA), or a tumor-derived neoantigen.

9. The conjugate of claim 8, wherein the tumor-derived neoantigen comprises a mutation specifically expressed in cancer cells.

10. The conjugate according to claim 8, wherein the tumor-associated antigen (TAA) is mTrp2, CT (testicular cancer) antigen, EGFR, Ova, Melan-A, PSMA (prostate-specific membrane antigen), survivin, ADAbp (adenosine deaminase-binding protein), cyclophilin b, gp100, CRC (colorectal cancer-associated antigen)-C017-1 A / GA733, CEA (carcinoembryonic antigen), CAP-1, CAP-2, etv6, AML1, PSA (prostate-specific antigen), PSA-1, PSA-2, PSA-3, MAGE (melanoma antigen E), GAGE ​​(G antigen), BAGE (B melanoma antigen), RAGE (renal tumor antigen), LAGE (L antigen), NAG, GnT-V, MUM-1, CDK4, p53, tyrosinase, Muc1 (mucin 1), HER2 / neu, p21ras, N-RAS, K-RAS, RCAS1, alpha-fetoprotein, E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, p120ctn, PRAME, NY-ESO-1, TRP2, mammary globin-A, metalloidin Protein-1 (MPS-1), cytochrome P450 subtype 1B1, 90K / Mac-2 binding protein, Ep-CAM (MK-1), HSP-70, hTERT (TRT), LEA, TAGE-1, 5T4, gp70, SCP-1, cmyc, cyclin B1, MDM2, p62, Koc, IMP1, TA90, OA1, CT-7, HOM-MEL-40 / SSX-2, SSX-1, SSX-4, HOM-TES-14 / SCP-1, HOM-TES-85, HDAC5, MBD2, TRIP4, NY-CO-45, KNSL6, HIP1R, Seb4D, KIAA1416, IMP1, 90K / Mac-2 binding protein, MDM2 or LMNA.

11. The conjugate according to claim 8, wherein the tumor-specific antigen (TSA) is M12, M20, M21, M30 or M44.

12. The conjugate according to claim 7, wherein the infectious antigen is an antigen derived from a virus, bacteria, parasite, or fungus.

13. The conjugate according to any one of claims 1 to 12, wherein the conjugate has at least one feature selected from (i) and (ii) by virtue of the carrier protein: (i) Delivery of CD4 / CD8 via processing and presentation pathways, respectively; and (ii) Increase the expression of the antigen protein.

14. An immunization composition comprising the conjugate according to any one of claims 1 to 12.

15. The immunization composition according to claim 14, wherein the immunization composition further comprises at least one selected from the group consisting of antigen adjuvants, efficacy enhancers, preservatives, buffers, surfactants, carriers, penetrants, antioxidants, and stabilizers.

16. The immunization composition according to claim 14, administered via at least one route selected from intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, conjunctival, and subcutaneous routes.

17. The immunization composition according to claim 14, wherein the immunization composition is an immunization composition for cancer.

18. A method of immunization against cancer, the method comprising administering a conjugate or a vaccine composition comprising any one of claims 1 to 12 to a subject.

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