Conjugate for antigen delivery and use thereof

Abstract

The present invention relates to: a conjugate comprising mRNA of an antigen protein and mRNA encoding a carrier protein linked to the 5' end and the 3' end of the mRNA of the antigen protein; and an immunization composition and / or vaccine composition comprising same. The present invention has the effect of stably increasing the expression of the antigen protein.

Description

Conjugates for antigen delivery and their use

[0001] Cross-citation with related applications

[0002] This application claims the benefit of priority to Republic of Korea Patent Application No. 10-2023-0099234, filed July 28, 2023, and Republic of Korea Patent Application No. 10-2023-0102465, filed August 4, 2023, the entire contents of which are incorporated herein by reference.

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

[0004] Anticancer vaccines treat cancer by enhancing or inducing antigen-specific immune responses. Existing anticancer vaccines do not account for antigen processing, resulting in inconsistent or low expression levels depending on the antigen sequence. Consequently, specific immune responses to different antigen sequences are limited, and consistent therapeutic efficacy is difficult to achieve.

[0005] In order to solve the above problems and express various antigen sequences without problems, an mRNA sequence encoding another protein (carrier protein) was linked to both the 5' and 3' ends of the antigen mRNA sequence, thereby increasing the expression amount when the antigen was delivered into cells in the form of mRNA, reducing the variation in the amount of antigen protein expressed for various antigens, and inducing a high immune response by ensuring that the CD4 / CD8 antigens are respectively delivered to the appropriate processing / presentation pathway.

[0006]

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

[0008] Another example provides an immunizing composition and / or a vaccine composition comprising the conjugate.

[0009] Another example provides a method of immunization comprising administering to a subject the conjugate and an immunizing composition and / or vaccine composition comprising the same.

[0010] One example provides a conjugate comprising mRNA of an antigen protein, a carrier protein linked to the 5' end, the 3' end, or both of the antigen protein,

[0011] Specifically, mRNA of the antigen protein;

[0012] mRNA encoding a first carrier protein linked to the 5' end of the mRNA of the antigen protein; and

[0013] A conjugate is provided, comprising an mRNA encoding a second carrier protein linked to the 3' end of the mRNA of the antigen protein.

[0014] The first carrier protein and the second carrier protein may be the same or different, but are not limited thereto.

[0015] The first carrier protein and the second carrier protein may be at least one selected from 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.

[0016] The above antigen protein may include, but is not limited to, a T cell epitope.

[0017] The above conjugate may have one or more properties selected from the group consisting of (i) and (ii) below by the carrier protein, but is not limited thereto:

[0018] (i) CD4 / CD8 are delivered to the processing and presentation pathways, respectively; and

[0019] (ii) Increased expression of the above antigen protein.

[0020] Another example provides an immunizing composition and / or a vaccine composition comprising the conjugate.

[0021] Another example provides a method for immunizing against cancer, comprising administering to a subject in need of immunization against cancer a conjugate and / or an immunizing composition and / or a vaccine composition comprising the same.

[0022] Another example provides a composition for preventing, improving and / or treating cancer, infectious diseases, autoimmune diseases and / or allergic diseases comprising the conjugate.

[0023] Other examples provide uses of the conjugate and / or composition for use in immunizing against cancer or for use in the manufacture of a composition for use in immunizing against cancer (e.g., an immunizing composition and / or a vaccine composition).

[0024] Other examples provide uses of the conjugate and / or composition for preventing, ameliorating and / or treating cancer, infectious diseases, autoimmune diseases and / or allergic diseases, or for use in the manufacture of a composition for preventing, ameliorating and / or treating cancer, infectious diseases, autoimmune diseases and / or allergic diseases (e.g., a composition for preventing, ameliorating and / or treating cancer, infectious diseases, autoimmune diseases and / or allergic diseases).

[0025]

[0026] Hereinafter, the present invention will be described in more detail.

[0027]

[0028] conjugate

[0029] An example of this specification is mRNA of an antigen protein;

[0030] mRNA encoding a first carrier protein linked to the 5' end of the mRNA of the antigen protein; and

[0031] A conjugate is provided, comprising an mRNA encoding a second carrier protein linked to the 3' end of the mRNA of the antigen protein.

[0032] As used herein, "antigen" means any molecule that induces an immune response in a subject. The antigen means any molecule that can be recognized by a T cell receptor and / or a B cell receptor and that contains an epitope capable of stimulating an immune response, particularly a T cell response and / or a B cell response, in a subject.

[0033] As used herein, “epitope” means a region of an antigen that interacts with a T-cell receptor and / or a B-cell receptor.

[0034] As used herein, the term "carrier protein" refers to a protein that is linked to an antigen protein to improve recombinant expression of the antigen protein (e.g., high expression rate, consistent expression rate, etc.), improve purification, enhance physical properties, stabilize, increase immunogenicity, and / or improve delivery to immune response cells. As used herein, the carrier protein may be, but is not limited to, a full-length protein form and / or a form that includes a portion of the amino acid sequence of the full-length protein. As used herein, the carrier protein may be linked to the N-terminus and the C-terminus of the antigen protein, respectively, and correspond to a first carrier and a second carrier, respectively.

[0035] As used herein, "linked" means that the components are directly or indirectly connected together. The components may be covalently or non-covalently linked. Furthermore, "linked" may mean that the components maintain a chemical or physical bond after contact with and immunization with a cell, such as an antigen-presenting cell or an immune cell. For example, the components may be associated so that they do not freely disperse from each other until contact with an antigen-presenting cell or an immune cell. For example, two components may be covalently linked to each other, such that the two components cannot disperse or diffuse separately.

[0036] A linker may be present between the mRNA of the antigen protein of the conjugate and the mRNA encoding the first carrier protein and / or the mRNA encoding the second carrier protein, but is not limited thereto.

[0037] As used herein, "linker" refers to a molecule or group of atoms that connects, couples, or binds two or more components together. As used herein, each component of an antigen protein, for example, an antigen protein and a carrier protein, may be connected or bound together by any suitable means.

[0038] The above linker may be, but is not limited to, (GS)n, (G2S)n, (G3S)n, (G4S)n, Gn, LE, SSGG or GGGGSGGGGG (wherein G is Gly, S is Ser, L is Leu, E is Glu and n is an integer of at least 1).

[0039]

[0040] carrier protein

[0041] mRNA of the antigen protein provided herein;

[0042] mRNA encoding a first carrier protein linked to the 5' end of the mRNA of the antigen protein; and

[0043] In a conjugate comprising an mRNA encoding a second carrier protein linked to the 3' end of the mRNA of the antigen protein, the first carrier protein and the second carrier protein may be the same or different, but are not limited thereto.

[0044] The first carrier protein and the second carrier protein may be at least one selected from 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.

[0045] The above Ub can be easily degraded when the last sequence alanine (A) of Ub is replaced with arginine (R) when inserted into the N-terminus of the antigen protein, so when an mRNA encoding a carrier protein corresponding to Ub is linked to the 5'-terminus of the mRNA of the antigen protein, an mRNA encoding UbR with R in the last sequence can be linked, but is not limited thereto.

[0046] The above TRXwt-UbR, TRXwt-Lamp, TRXwt-mODC, and TRXwt-cmyc are proteins in which TRXwt is added to the N-terminus of the UbR, Lamp, mODC, and cmyc proteins.

[0047] The above SORT1 w / TM and Lamp w / TM are proteins in which a transmembrane domain is added to the cytosolic domain of each of the above SORT1 and Lamp.

[0048] In the above conjugate,

[0049] (1) The first carrier protein is TRXmt, the second carrier protein is PSBD;

[0050] (2) The first carrier protein is UbR, and the second carrier protein is mODC;

[0051] (3) The first carrier protein is UbR, the second carrier protein is SORT1;

[0052] (4) The first carrier protein is Lamp, and the second carrier protein is mODC;

[0053] (5) The first carrier protein is Lamp, and the second carrier protein is SORT1;

[0054] (6) The first carrier protein is UbR, the second carrier protein is cmyc;

[0055] (7) The first carrier protein is UbR, and the second carrier protein is Lamp;

[0056] (8) The first carrier protein is mODC, the second carrier protein is SORT1;

[0057] (9) The first carrier protein is cmyc, and the second carrier protein is mODC;

[0058] (10) The first carrier protein is cmyc, the second carrier protein is SORT1;

[0059] (11) The first carrier protein is Lamp, and the second carrier protein is Ub;

[0060] (12) The first carrier protein is Lamp, and the second carrier protein is cmyc;

[0061] (13) The first carrier protein is SORT1, the second carrier protein is mODC;

[0062] (14) The first carrier protein is mODC, the second carrier protein is Ub;

[0063] (15) The first carrier protein is mODC, the second carrier protein is cmyc;

[0064] (16) The first carrier protein is mODC, and the second carrier protein is Lamp;

[0065] (17) The first carrier protein is cmyc, the second carrier protein is Ub;

[0066] (18) The first carrier protein is cmyc, and the second carrier protein is Lamp;

[0067] (19) The first carrier protein is SORT1, the second carrier protein is Ub;

[0068] (20) The first carrier protein is SORT1, the second carrier protein is cmyc;

[0069] (21) The first carrier protein is SORT1, and the second carrier protein is Lamp;

[0070] (22) The first carrier protein is TRXwt, the second carrier protein is cmyc;

[0071] (23) The first carrier protein is TRXwt, and the second carrier protein is Lamp;

[0072] (25) The first carrier protein is TRXwt, the second carrier protein is mODC;

[0073] (26) The first carrier protein is TRXwt, the second carrier protein is SORT1;

[0074] (27) The first carrier protein is TRXwt, the second carrier protein is Ub;

[0075] (28) The first carrier protein is cmyc, the second carrier protein is TRXwt;

[0076] (29) The first carrier protein is Lamp, the second carrier protein is TRXwt;

[0077] (30) The first carrier protein is mODC, the second carrier protein is TRXwt;

[0078] (31) The first carrier protein is SORT1, the second carrier protein is TRXwt;

[0079] (32) The first carrier protein is UbR, the second carrier protein is TRXwt;

[0080] (33) The first carrier protein is TRXwt-UbR, the second carrier protein is SORT1;

[0081] (34) The first carrier protein is TRXwt-Lamp, the second carrier protein is mODC;

[0082] (35) The first carrier protein is TRXwt-mODC, the second carrier protein is SORT1;

[0083] (36) The first carrier protein is TRXwt-mODC, the second carrier protein is Ub;

[0084] (37) The first carrier protein is TRXwt-mODC, and the second carrier protein is Lamp;

[0085] (38) The first carrier protein is TRXwt-cmyc, the second carrier protein is Ub;

[0086] (39) The first carrier protein is TRXwt-SORT1, the second carrier protein is Ub;

[0087] (40) The first carrier protein is TRXwt-SORT1, and the second carrier protein is Lamp;

[0088] (41) The first carrier protein is TRXwt, the second carrier protein is SORT1 w / TM; or

[0089] (42) The first carrier protein may be Lamp w / TM, and the second carrier protein may be TRXwt, but is not limited thereto.

[0090] In another example, the first carrier protein and the second carrier protein within the conjugate may be exemplified as follows:

[0091] CP_set N-term (first carrier protein) C-term (second carrier protein) CP01TRXmtPSBDCP11UbRmODCCP12UbRSORT1CP13LampmODCCP14LampSORT1CP21UbRcmycCP22UbRLampCP23mODCSORT1CP24cmycmODCCP25cmycSORT1CP26LampUbCP27LampcmycCP28SORT1mODCCP31mODCUbCP32mODCcmycCP33mODCLampCP34cmycUbCP35cmycLampCP36SORT1UbCP37SORT1cmycCP38SORT1LampCP41TRXwtcmycCP42TRX wtLampCP43TRXwtmODCCP44TRXwtSORT1CP45TRXwtUbCP46cmycTRXwtCP47LampTRXwtCP48mODCTRXwtCP49SORT1TRXwtCP50UbRTRXwtCP112TRXwt-UbRSORT1CP113TRX wt-LampmODCCP123TRXwt-mODCSORT1CP131TRXwt-mODCUbCP133TRXwt-mODCLampCP134TRXwt-cmycUbCP136TRXwt-SORT1UbCP138TRXwt-SORT1LampCP201TRXwtSORT1 w / TMCP202Lamp w / TMTRXwt

[0092] The above UbR may include, but is not limited to, the amino acid sequence of SEQ ID NO: 1 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the amino acid sequence of SEQ ID NO: 1. The gene encoding the above UbR may include, but is not limited to, the base sequence of SEQ ID NO: 21 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the base sequence of SEQ ID NO: 21.

[0093] The above Ub may include, but is not limited to, the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the above amino acid sequence. The gene encoding the above Ub may include, but is not limited to, the base sequence of SEQ ID NO: 22 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the above base sequence.

[0094] The Lamp may include, but is not limited to, the amino acid sequence of SEQ ID NO: 3 or 4 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the amino acid sequence. The gene encoding the Lamp may include, but is not limited to, the base sequence of SEQ ID NO: 23 or 24 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the base sequence. When the Lamp is a first carrier protein, it may have SEQ ID NO: 3 or an amino acid sequence having the sequence homology thereto, and when it is a second carrier protein, it may have SEQ ID NO: 4 or an amino acid sequence having the sequence homology thereto, but is not limited thereto.

[0095] The mODC may include, but is not limited to, the amino acid sequence of SEQ ID NO: 5 or 6 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the amino acid sequence. The gene encoding the mODC may include, but is not limited to, the base sequence of SEQ ID NO: 25 or 26 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the base sequence. When the mODC is a first carrier protein, it may have SEQ ID NO: 5 or an amino acid sequence having the sequence homology thereto, and when the mODC is a second carrier protein, it may have SEQ ID NO: 6 or an amino acid sequence having the sequence homology thereto, but is not limited thereto.

[0096] The cmyc may include, but is not limited to, the amino acid sequence of SEQ ID NO: 7 or 8 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the amino acid sequence. The gene encoding the cmyc may include, but is not limited to, the base sequence of SEQ ID NO: 27 or 28 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the base sequence. When the cmyc is a first carrier protein, it may have the amino acid sequence of SEQ ID NO: 7 or the amino acid sequence having the sequence homology, and when the cmyc is a second carrier protein, it may have the amino acid sequence of SEQ ID NO: 8 or the amino acid sequence having the sequence homology, but is not limited thereto.

[0097] The SORT1 may include, but is not limited to, the 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. The gene encoding the SORT1 may include, but is not limited to, the base sequence of SEQ ID NO: 29 or 30 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 base sequence. When the SORT1 is a first carrier protein, it may have the amino acid sequence of SEQ ID NO: 9 or the amino acid sequence having the sequence homology, and when the SORT1 is a second carrier protein, it may have the amino acid sequence of SEQ ID NO: 10 or the amino acid sequence having the sequence homology, but is not limited thereto.

[0098] The above TRX (specifically, TRXwt) may include, but is not limited to, the amino acid sequence of SEQ ID NO: 11 or 12 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the above amino acid sequence. The gene encoding the TRX (specifically, TRXwt) may include, but is not limited to, the base sequence of SEQ ID NO: 31 or 32 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the above base sequence. When the above TRX (specifically, TRXwt) is a first carrier protein, it may have an amino acid sequence having sequence number 11 or a sequence homology thereto, and when it is a second carrier protein, it may have an amino acid sequence having sequence number 12 or a sequence homology thereto, but is not limited thereto. The above TRXwt refers to a wild type of TRX, a TRX that can be obtained from nature without mutation.

[0099] The PSBD may include, but is not limited to, the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the amino acid sequence of SEQ ID NO: 13. The gene encoding the PSBD may include, but is not limited to, the base sequence of SEQ ID NO: 33 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the base sequence of SEQ ID NO: 33.

[0100] The above TRXwt-UbR may include, but is not limited to, the amino acid sequence of SEQ ID NO: 14 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the amino acid sequence of SEQ ID NO: 14. The gene encoding the TRXwt-UbR may include, but is not limited to, the base sequence of SEQ ID NO: 34 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% sequence homology with the base sequence of SEQ ID NO: 34.

[0101] The above TRXwt-Lamp may include, but is not limited to, the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence having a sequence homology of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% to the amino acid sequence of SEQ ID NO: 15. The gene encoding the TRXwt-Lamp may include, but is not limited to, the base sequence of SEQ ID NO: 35 or an amino acid sequence having a sequence homology of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% to the base sequence of SEQ ID NO: 35.

[0102] The above TRXwt-mODC may include, but is not limited to, the amino acid sequence of SEQ ID NO: 16 or an amino acid sequence having a sequence homology of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% to the amino acid sequence of SEQ ID NO: 16. The gene encoding the TRXwt-mODC may include, but is not limited to, the base sequence of SEQ ID NO: 36 or an amino acid sequence having a sequence homology of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% to the base sequence of SEQ ID NO: 36.

[0103] The above TRXwt-cmyc may include, but is not limited to, the amino acid sequence of SEQ ID NO: 17 or an amino acid sequence having 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 amino acid sequence of the above amino acid sequence. The gene encoding the above TRXwt-cmyc may include, but is not limited to, the base sequence of SEQ ID NO: 37 or an amino acid sequence having 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 of the above amino acid sequence.

[0104] The above TRXwt-SORT1 may include, but is not limited to, the amino acid sequence of SEQ ID NO: 18 or an amino acid sequence having 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 amino acid sequence of the above amino acid sequence. The gene encoding the above TRXwt-SORT1 may include, but is not limited to, the base sequence of SEQ ID NO: 38 or an amino acid sequence having 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 of the above amino acid sequence.

[0105] The above SORT1 w / TM may include, but is not limited to, the amino acid sequence of SEQ ID NO: 19 or an amino acid sequence having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% with the amino acid sequence of SEQ ID NO: 19. The gene encoding the above SORT1 w / TM may include, but is not limited to, the base sequence of SEQ ID NO: 39 or an amino acid sequence having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% with the base sequence of SEQ ID NO: 39.

[0106] The Lamp w / TM may include, but is not limited to, the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% with the amino acid sequence of SEQ ID NO: 20. The gene encoding the Lamp w / TM may include, but is not limited to, the base sequence of SEQ ID NO: 40 or an amino acid sequence having a sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% with the base sequence of SEQ ID NO: 40.

[0107] The above TRXmt may include, but is not limited to, the amino acid sequence of SEQ ID NO: 145 or an amino acid sequence having a sequence homology of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% to the amino acid sequence of SEQ ID NO: 145. The gene encoding the above TRXmt may include, but is not limited to, the base sequence of SEQ ID NO: 146 or an amino acid sequence having a sequence homology of at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% to the base sequence of SEQ ID NO: 146.

[0108] The above TRXmt may include a sequence in which at least one amino acid selected from the group consisting of amino acids corresponding to positions 32nd, 35th, 62nd, 69th, and 73rd from the N-terminus of the TRX amino acid sequence (specifically, the amino acid sequence of SEQ ID NO: 11) is substituted with serine (Serine; S), but is not limited thereto. The substitution may be a substitution of cysteine ​​(C) with serine.

[0109] The protein or polypeptide provided herein may be isolated and / or purified from nature, or may be recombinantly or chemically synthesized. If the amino acid sequence of the protein or polypeptide provided herein includes methionine (Met, M) as the first amino acid residue from the N-terminus, the protein or polypeptide may be recombinantly produced, and the methionine at the first amino acid position from the N-terminus may be encoded by an initiation codon. Accordingly, if the amino acid sequence of the protein or polypeptide provided herein includes a methionine at the N-terminus produced by recombinant production, if the protein or polypeptide is obtained by another method (e.g., chemical synthesis or isolation from nature) or is not located at the N-terminus in a conjugate, it may be interpreted as including an amino acid sequence starting from the second amino acid residue excluding the methionine at the first position from the N-terminus produced by recombinant production.

[0110] 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). The mRNA encoding the first carrier protein may be linked by adding a base sequence encoding methionine (Met, M) to the 5' end, for example, an "atg" sequence may be added and linked, but is not limited thereto.

[0111]

[0112] antigen protein

[0113] mRNA of the antigen protein provided herein;

[0114] mRNA encoding a first carrier protein linked to the 5' end of the mRNA of the antigen protein; and

[0115] In a conjugate comprising an mRNA encoding a second carrier protein linked to the 3' end of the mRNA of the antigen protein, the antigen protein may comprise, but is not limited to, a T cell epitope. Specifically, the antigen protein may comprise an MHC class I and / or II binding motif. For example, the antigen protein may comprise a CD4+ T cell epitope, which is a peptide sequence comprising an MHC class II binding motif and capable of being presented on the surface of an antigen-presenting cell by an MHC class II molecule. The antigen protein may comprise a CD8+ T cell epitope, which is a peptide sequence comprising an MHC class I binding motif and capable of being presented on the surface of a cell by an MHC class I molecule. The antigen protein may also comprise both a CD4+ T cell epitope and a CD8+ T cell epitope.

[0116] In this specification, "MHC (major histocompatibility complex)" or "major histocompatibility complex" refers to a protein that presents antigen fragments to immune cells so that the immune cells can distinguish between self and non-self molecules. There are two types of MHC, MHC class I and MHC class II. MHC class I is present in all cells with a nucleus, while MHC class II is found in antigen-presenting cells. MHC class I molecules interact with CD8+ cytotoxic T cells and play an important role in the rejection of organ transplants and the destruction of infected cells. MHC class II molecules play an important role in inducing cellular immunity by recognizing non-self antigens through interaction with CD4+ helper T cells.

[0117] Typically, in the adaptive immune response, when an antigen enters the body, antigen-presenting cells ingest it and break it down into short peptide fragments. These peptides can then bind to MHC class I or MHC class II molecules within the cell and be transported to the cell surface. When the antigen peptide binds to MHC class I or MHC class II and is presented on the cell surface of an antigen-presenting cell, T cells recognize it through the T cell receptor (TCR), become activated, and initiate an immune response. In this respect, the original antigen protein may correspond to a T cell epitope.

[0118] The antigen protein (e.g., T cell epitope) may be derived from, but is not limited to, a tumor antigen, an infectious agent, an autoantigen, or an allergen. Specifically, the antigen protein (e.g., T cell epitope) may include a portion of a tumor antigen, an infectious agent, an autoantigen, or an allergen that is predicted or known to bind to an MHC class I or MHC class II molecule. The antigens may be, but are not limited to, a CD8+ or CD4+ T cell epitope.

[0119] The antigen protein (e.g., a T cell epitope) may comprise a natural or unnatural amino acid sequence, an amino acid with a post-translational modification, or a peptide mimetic capable of inducing an immune response, such as a T cell or B cell response, in a subject. For example, an antigen 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 may be about 7 to 35 amino acids, for example, but not limited to, 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.

[0120] As used herein, the term "tumor antigen" may be used interchangeably with "cancer antigen," and refers to a molecule expressed by a tumor (cancer) that elicits an immune response. This immune response may involve antibody production, activation of specific immunologically competent cells, or both.

[0121] The above tumor antigen may be derived from an organism containing a tumor, whole tumor cells that have been killed or inactivated, or a lysate, and includes any antigen derived from a tumor. Lysate refers to a substance resulting from the application of a process that causes disruption of the normal structure of a cell. Furthermore, a tumor antigen includes any protein or other substance with antigenic properties that is contained in a tumor cell and expressed differently from that in a normal cell.

[0122] The above tumor antigen may include, but is not limited to, a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor-derived neoantigen.

[0123] The above tumor-associated antigens (TAAs) are antigens that are expressed more abundantly in cancer cells than in normal cells or at different stages of differentiation than in normal cells. They are also tumor-shared antigens, present in trace amounts in normal cells. Therefore, immune responses utilizing these antigens are highly likely to be abrogated by autotolerance, an immunosuppressive mechanism designed to prevent damage to self-cells. Conversely, autoimmunity can lead to unwanted organs being attacked.

[0124] The above tumor-associated antigens (TAAs) include mTrp2, CT (Cancer-testis) antigen, EGFR, Ova, Melan-A, PSMA (Prostate Specific Membrane Antigen), Survivin, ADAbp (adenosine deaminase-binding protein), cyclophilin b, gp100, CRC (Colorectal 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, etc.), 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, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, PRAME, NY-ESO-1, TRP2, Mammaglobin-A, metallopanstimulin-1 (MPS-1), cytochrome P450 isoform 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,may be, but is not limited to, 90K / Mac-2 binding protein, MDM2, or LMNA.

[0125] The above tumor-specific antigen (TSA) refers to an antigen that exists specifically only in cancer cells, and specifically, may be M12, M20, M21, M30, or M44, but is not limited thereto. In particular, when a tumor proliferates in a cancer patient, a cancer cell-specific genetic mutation occurs, creating a new antigen epitope that can stimulate T cells, which is called a neoantigen. That is, a neoantigen may include a cancer cell-specific genetic mutation, and unlike a tumor shared antigen that is expressed in trace amounts in normal cells, a neoantigen is selectively expressed only in cancer cells, so it is recognized as a non-self foreign epitope by the autoimmune system, thereby inducing strong anticancer immune activity.

[0126] When peptides generated from mutated DNA are displayed on MHC molecules on the cell surface, they are recognized by the T cell receptor (TCR). Because mutations do not occur in normal cells or tissues, neoantigen-specific T cells are free from self-tolerance or autoimmunity. Because of this advantage, neoantigens are considered ideal targets for T cell-based cancer immunotherapy.

[0127] The causes of neoantigens include, but are not limited to, frame-shift deletion or insertion, in which one or more nucleotides that make up DNA are added or deleted, thereby disrupting the decoding of the genetic code; point mutation, in which one nucleotide is replaced with another; and other mutations such as missense mutations, splice-site mutations, read-through mutations, or gene-fusion mutations.

[0128] Neoantigens are predicted through analysis of the genome of specific cancer cells from individual cancer patients. For example, cancer cells are obtained from a patient's tumor, DNA is extracted, the base sequence is analyzed, and the DNA is compared with the base sequence of normal cells to select the region where mutations have occurred. Among the base sequences of various regions where mutations have occurred, neoantigens that stimulate T cells can be identified. This can be done using, but is not limited to, big data processing such as next-generation sequencing (NGS), whole-exome sequencing (WES), or RNA sequencing, computer programs that predict MHC binding, or artificial intelligence (AI) for neoantigen prediction. Because mutation patterns are not shared across patients, neoantigens can be developed into personalized cancer vaccines.

[0129] The above tumor-derived neoantigen may include, but is not limited to, a mutation specifically expressed in cancer cells.

[0130] The tumor antigen may include, but is not limited to, an amino acid sequence of SEQ ID NO: 51, 53, 55, 57 or 59 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence homology with the amino acid sequence. The gene encoding the tumor antigen may include, but is not limited to, a base sequence of SEQ ID NO: 52, 54, 56, 58 or 60 or an amino acid sequence having at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% sequence homology with the base sequence.

[0131] The antigen of the above infectious agent may be, but is not limited to, an antigen derived from a virus, bacteria, parasite, or fungus.

[0132] The above virus-derived antigens include chickenpox virus, smallpox 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, rhabdovirus, rotavirus, rhinovirus, adenovirus, papillomavirus, parvovirus, picornavirus, poliovirus, virus causing mumps, virus causing rabies, reovirus, rubella virus, It may be, but is not limited to, an antigen derived from togavirus, orthomyxovirus, retrovirus, hepadnavirus, coxsackievirus, 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.

[0133] Antigens derived from bacteria include Borrelia species, Bacillus anthracis, burgdorferi, Bordetella pertussis, Campylobacter jejuni, Chlamydia species, Chlamydial psittaci, Chlamydial trachomatis, Clostridium species, Clostridium tetani, Clostridium botulinum, Clostridium perfringens, Corynebacterium diphtheriae, Coxiella species, Enterococcus species, Erlichia species, Escherichia coli, Francisella tularensis, Haemophilus species, Haemophilus influenzae, Haemophilus parainfluenzae, Lactobacillus species, Legionella species, Legionella pneumophila, Leptospirosis interrogans, Listeria species, Listeria monocytogenes, Mycobacterium species, Mycobacterium tuberculosis, Mycobacterium leprae, Mycoplasma species,Mycoplasma pneumoniae, Neisseria species, Neisseria meningitidis, Neisseria gonorrhoeae, Pneumococcus species, Pseudomonas species, Pseudomonas aeruginosa, Salmonella species, Salmonella typhi, Salmonella enterica, Rickettsia species, Rickettsia ricketsii, Rickettsia typhi, Shigella species, Staphylococcus species, Staphylococcus aureus, Streptococcus species, Streptococccus pneumoniae pnuemoniae), Streptococcus pyrogenes, Streptococcus mutans, Treponema species, Treponema pallidum, Vibrio species, Vibrio cholerae, or Yersinia pestis.

[0134] The fungal antigen may be a fungal antigen selected from, but not limited to, Candida species, Cryptococcus species, Coccidioides species, Histoplasma species, and Aspergillus species.

[0135] The parasite-derived antigen may be, but is not limited to, an antigen derived from Plasmodium, Trypanosome, Schistosome or Leishmania.

[0136] The above autoantigens may be, but are not limited to, antigens known or suspected to induce autoimmunity.

[0137] The above allergen may be, but is not limited to, an allergen known or suspected to cause an allergy.

[0138]

[0139] Immunization uses of conjugates

[0140] Another example provides an immunizing composition and / or a vaccine composition comprising the conjugate.

[0141] The above composition relates to a composition for preventing, improving and / or treating cancer, infectious diseases, autoimmune diseases and / or allergic diseases.

[0142] As used herein, "immunizing composition" refers to any composition capable of inducing an immune response, and "vaccine" refers to an immunizing composition that induces an immune response to reduce or prevent the risk of a disease or infection, or to improve or treat an existing disease or infection. The immunizing composition may be an immunizing composition for cancer, infectious disease, autoimmune disease, and / or allergic disease.

[0143] Specifically, the above-mentioned immunizing composition may refer to a composition capable of inducing an immune response through the production of antibodies against the above-mentioned antigen.

[0144] The composition may be in a form that can be administered to a subject, including the conjugate provided herein, to induce an immune response. The composition may be conveniently used to prevent, improve, or treat a disease. After being introduced into a subject or host, the composition may induce an immune response, preferably a T-cell-mediated immune response.

[0145] The above vaccine may be an anticancer vaccine comprising a tumor antigen, for example, a tumor antigen sequence obtained from analysis of a patient's tumor, particularly a neoantigen sequence, thereby maximally stimulating the proliferation and activation of patient-specific anticancer T cells to enhance the anticancer immune effect.

[0146] The composition may also include an additional adjuvant to enhance the effectiveness of the vaccine. Suitable adjuvants include (1) aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, and the like; (2) Oil-in-water emulsion formulations (with or without specific immunostimulants such as muramyl peptides or bacterial cell wall components), for example MF59 (WO90 / 14837) comprising (a) 5% squalene, 0.5% Tween 80 and 0.5% Span 85 formulated into submicron particles (optionally but not necessarily including varying amounts of N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1',2'-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE)), (b) 10% squalene, 0.4% Tween 80, 5% pluronic-blocked polymer and agitated to produce microfluidized submicron particles or larger particle size emulsions. RibiTM adjuvant system (RAS) comprising SAF comprising N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), and (c) one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), 2% squalene, and 0.2% Tween 80; (3) saponin adjuvants; (4) Freund's complete adjuvant (CFA) and incomplete adjuvant (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 colony stimulating factor (M-CSF), tumor necrosis factor (TNF), etc.; (6) ADP-ribosylating toxins of bacteria, such as cholera toxin (CT), pertussis toxin (PT), or Escherichia coli (E.Heat-labile toxin (LT) of E. coli, particularly detoxified mutants of LT-R72, CT-S109, PT-K9 / G129 (WO93 / 13302 and WO92 / 19265); and (7) other substances that act as immunostimulants to enhance the effectiveness of vaccines, but are not limited thereto.

[0147] The above composition may additionally contain pharmaceutically acceptable carriers, diluents and / or excipients in commonly used amounts, as needed.

[0148] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above ingredients, the composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0149] The above conjugate and / or composition containing the same as an active ingredient can be manufactured in a unit dosage form or can be manufactured by placing it in a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person skilled in the art.

[0150] For example, the composition may comprise a conventional saline or buffered aqueous medium in which the agent is suspended or dissolved. For example, the composition may typically comprise a diluent, such as water, saline, glycerol, ethanol, etc., and auxiliary substances, such as wetting agents, emulsifiers, pH buffers, etc., may be present in the composition.

[0151] Suitable forms for injectable use include sterile aqueous solutions (water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. These must be stable under the conditions of manufacture and preserved against microbial contamination, such as bacteria or fungi. Microbial contamination can be prevented by the use of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal. In many cases, it is preferable to include isotonic agents, such as sugars or sodium chloride. Prolonged absorption of injectable compositions can be ensured by the use of agents that delay absorption, such as aluminum monostearate or gelatin.

[0152] Sterile injectable solutions are prepared by combining the required amount of the conjugate and various other ingredients listed above in the aforementioned solvent, and filter-sterilizing the remaining ingredients, such as a buffer solution such as PBS, excluding the conjugate and / or heat-sensitive immune adjuvant cytokines, if necessary. Generally, dispersions are prepared by incorporating various sterilizable active ingredients into a sterile vehicle containing a basic dispersion medium and other required ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and lyophilization techniques. By these techniques, a powder of any desired ingredient is obtained from the active ingredient and a sterile-filtered solution thereof as described above.

[0153] The target patient to be administered the conjugate or the composition containing it as an active ingredient may be, but is not limited to, mammals, such as primates including humans and monkeys, rodents including rats and mice, etc.

[0154] Another aspect of the present invention relates to a method of inducing, stimulating, or promoting an immune response to an antigen in a human, e.g., a cancer patient, comprising administering to the patient, e.g., a cancer patient, an effective amount of the composition or conjugate.

[0155] As used herein, "immune response" refers to a change in the activity of cells of the immune system, such as B cells, T cells, or monocytes, as a result of stimulation, either directly or indirectly, through cellular or cytokine mediation. The immune response may be specific (T cell and / or B cell) and / or non-specific.

[0156] Without limiting the action of the present invention in any way, delivery of the conjugate according to the present invention is particularly useful for inducing an immune response, particularly a T cell response, such as a CD4+ T cell response or a CD8+ T cell response to an antigen. The CD4+ T cell response and the CD8+ T cell response may occur together with or independently of a humoral response or other specific or nonspecific immune response.

[0157] Another aspect of the present invention relates to the use of the conjugate in the treatment and / or prevention of disease states. Examples of diseases that can be treated using the methods of the present invention include various cancers, infectious diseases, autoimmune diseases, and allergic diseases.

[0158] The cancer may be a solid cancer or a blood cancer, and specifically, breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, endometrial cancer, uterine cancer, colon cancer, large intestine cancer, colorectal cancer, rectal cancer, kidney cancer, nephroblastoma, skin cancer, oral squamous cell carcinoma, epidermoid cancer, nasopharyngeal cancer, head and neck cancer, bone cancer, esophageal cancer, bladder cancer, lymphatic cancer (e.g., Hodgkin's lymphoma or non-Hodgkin's lymphoma), stomach cancer, pancreatic cancer, testicular cancer, thyroid cancer, thyroid follicular carcinoma, melanoma, myeloma, multiple myeloma, mesothelioma, osteosarcoma, myelodysplastic syndrome, tumor of mesenchymal origin, soft tissue sarcoma, liposarcoma, gastrointestinal stromal sarcoma, malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, It can be rhabdomyosarcoma, teratocarcinoma, neuroblastoma, medulloblastoma, glioma, a benign skin tumor, or leukemia. Lung cancer can be, for example, small cell lung carcinoma (SCLC) or non-small cell lung carcinoma (NSCLC). Leukemia can be, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL). The subject being treated can be a subject receiving secondary anti-hyperproliferative therapy. For example, secondary anti-hyperproliferative therapy can be chemotherapy, radiation therapy, immunotherapy, phototherapy, cryotherapy, toxin therapy, hormone therapy, or surgery.

[0159] Another example provides a method of immunization comprising administering the conjugate and / or the composition to a subject in need of immunization, and specifically, a method of immunizing against cancer by administering the conjugate and / or the composition to a subject in need of immunization against cancer.

[0160] Another example provides a method for preventing or treating a disease, such as cancer, an infectious disease, an autoimmune disease, or an allergic disease, by enhancing an immune response using the composition. In this case, administration of the composition causes, induces, or otherwise promotes an immune response that inhibits, halts, delays, or prevents the onset or progression of the disease state.

[0161] Direct delivery of the composition may generally be systemic, subcutaneous, intradermal, intraperitoneal, intravascular (intravenous), intramuscular, or local, or delivered into tissue spaces. The composition may also be administered intralesionally. Dosage regimens may be single-dose or multiple-dose schedules.

[0162] As used herein, "effective dose" refers to an amount sufficient to achieve a desired result when administered to a subject, including a human, for example, an amount effective in treating or preventing cancer. The effective dose may vary depending on factors such as the subject's disease state, age, sex, and weight. Dosage or treatment regimen can be adjusted to provide the optimal therapeutic response, as will be understood by those skilled in the art.

[0163] A therapeutically effective dose of a vaccine may be administered as a single dose, or alternatively, may comprise a series of applications. The duration of the treatment period will depend on various factors, such as the severity of the disease, the age of the subject, the concentration of the vaccine, the patient's responsiveness to the vaccine, or a combination thereof. It will also be appreciated that the effective dose of the vaccine used for treatment may be increased or decreased over the course of an individual treatment regimen. Dosage variations may occur and can be determined through standard diagnostic assays known in the art. The vaccines of the present invention, such as cancer vaccines, may be administered before, during, or after treatment with conventional anticancer agents, radiation therapy, hormonal therapy, biotherapy, and / or surgical tumor resection.

[0164] The present invention relates to a conjugate comprising mRNA of an antigen protein, mRNA encoding a carrier protein linked to the 5' end and / or 3' end of the mRNA of the antigen protein, and an immunizing composition and / or a vaccine composition comprising the same, which has the effect of stably increasing the expression of the antigen protein.

[0165] Figures 1 and 2 show vector maps of pUC57-T7-MP-A118 and pEGFP, respectively, used for producing mRNA for carrier protein screening.

[0166] Figure 3 shows a schematic diagram of a carrier protein vector used for producing mRNA for carrier protein screening.

[0167] Figure 4 shows the vector map of the pUC57-T7-CP12G carrier protein vector.

[0168] Figure 5 shows the results of confirming the possibility of CD4 / CD8 antigens being transferred to the processing / presentation pathway, respectively, when treated with Proteasome Inhibitor and Lysosomal Inhibitor for the primary screening of carrier proteins.

[0169] Figure 6 shows a schematic diagram of cell expression when a transmembrane domain is added to the cytosolic domain of SORT1 and Lamp.

[0170] Figure 7 shows the results of confirming the possibility of CD4 / CD8 antigens being transferred to the processing / presentation pathway, respectively, when treated with Proteasome Inhibitor and Lysosomal Inhibitor for secondary screening of carrier proteins.

[0171] Figure 8 shows a schematic diagram of a vector used to produce a vector containing a carrier protein and an epitope.

[0172] Figure 9 shows the vector map of pUC57-T7-5epitopes.

[0173] Figure 10 shows a schematic diagram of the pLRK33-empty empty vector used to construct a vector containing a carrier protein and an epitope.

[0174] Figure 11 is a schematic diagram showing the process of inserting fragments including an epitope, carrier protein, etc. into an empty vector during the process of producing a vector including a carrier protein and an epitope.

[0175] Figure 12 shows the vector map of pLRK33-hTrx_WT.

[0176] Figure 13 shows a vector map of the pLRK33-hTrx_m vector used to construct a comparative vector that does not include a carrier protein.

[0177] Figure 14 shows the results of confirming the expression level of epitopes in mRNA depending on the presence or absence of carrier protein in the K562 cell line.

[0178] Figure 15 shows the results of confirming the expression level of epitopes in mRNA depending on the presence or absence of carrier protein in human dendritic cells.

[0179] Figure 16 shows the results of measuring immunogenicity for epitopes in human dendritic cells depending on whether or not carrier proteins are included.

[0180] Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are intended only to illustrate the content of the present invention, and the scope of the present invention is not limited by the following examples.

[0181]

[0182] Example 1. Carrier protein screening

[0183] Example 1-1. Production of mRNA encoding a carrier protein

[0184] Primary screening of carrier proteins was conducted by confirming whether CD4 / CD8 antigens were appropriately delivered to the processing / presentation pathway by mRNA encoding the carrier protein.

[0185] Specifically, for in vitro transcription, pUC57-T7-MP-A118 was produced by requesting GeneWiz to produce the necessary vector based on the pUC57 vector (Cosmogenetech), and the vector map of the vector is shown in Fig. 1.

[0186] The above-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). After the purification process, for the vector strand in which the AscI site is located at the N-terminus and the EcoRI site is located at the C-terminus among the cleaved vector strands, the carrier proteins were positioned at the N-terminus and C-terminus of the vector, respectively, using the T7 oligonucleotides in Table 3 corresponding to the carrier proteins according to the combinations in Table 2 below, and Platinum TM Using SuperFi II Green PCR Master Mix (ThermoFisher; cat#: 12369050), PCR was performed using the EZ-fusion HT Cloning kit (Enzynomics; cat#: EZ015TL) to insert carrier proteins into the AscI site and EcoRI site, thereby constructing pUC57-T7-CP01G to pUC57-T7-CP50G plasmids in Table 2 below. The specific preparation process for each carrier protein is described below:

[0187] (i) mODC

[0188] mODC for insertion into the C-terminus was prepared by mixing all of the oCmODC-BE1-F1, omODC-R2, omODC-F3, and oCmODC-X1-R4 oligonucleotides in Table 3 below and performing PCR. In order to insert mODC used at the C-terminus into the N-terminus, the prepared mODC PCR product was purified and PCR was performed using oNmODC-A1-F1 and oNmODC-BE1-R2 in Table 3 below.

[0189] (ii) cmyc

[0190] For insertion into the N-terminus, cmyc was prepared by mixing all of the oNcmyc-A1-F1 and oNcmyc-BE1-R2 oligonucleotides in Table 3 below and performing PCR. For insertion of cmyc used at the N-terminus into the C-terminus, the prepared cmyc PCR product was purified and PCR was performed using oCcmyc-BE1-F1 and oCcmyc-X1-R2 in Table 3 below.

[0191] (iii) Lamp

[0192] Lamp for insertion into the N-terminus was produced by mixing all of the oNIi-A1-F1 and oNIi-BE1-R2 oligonucleotides in Table 3 below and performing PCR. In order to insert the Lamp used at the N-terminus into the C-terminus, the Lamp PCR product produced was purified and PCR was performed using oCIi-BE1-F1 and oCIi-X1-R2 in Table 3 below.

[0193] (iv) SORT1

[0194] SORT1 for insertion into the C-terminus was produced by mixing all of the oCSORT-BE1-F1, oSORT-R2, oSORT-F3, and oCSORT-X1-R4 oligonucleotides in Table 3 below and performing PCR. In order to insert SORT1 used at the C-terminus into the N-terminus, the produced SORT1 PCR product was purified and PCR was performed using oNSORT-A1-F1 and oNSORT-BE1-R2 in Table 3 below.

[0195] (v) TRX

[0196] All of the 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 were mixed and then PCR was performed to produce the product.

[0197] (vi) PSBD

[0198] The oChPSBD-BE1-F and oChPSBD-X1-R oligonucleotides in Table 3 below were mixed and then PCR was performed to produce them.

[0199] (vii) Ub and UbR

[0200] Since the last sequence alanine (A) of Ub inserted at the N-terminus is easily degraded when replaced with arginine (R), when inserting Ub at the N-terminus, Ub was inserted using the oNUbR-BE1-R5 oligonucleotide in Table 3 so that the last sequence would be R (UbR).

[0201] Specifically, UbR was produced by performing PCR using oNUbR-A1-F1, oUbR-R2, oUbR-F3, and oNUbR-BE1-R5 in Table 3 below, and the UbR PCR product was purified. Ub was produced and inserted by performing PCR using oCUb-BE1-F1 and oCUb-X1-R2 in Table 3 below.

[0202] 플라스미드 종류CP_NCP_CpUC57-T7-CP01GTRXmt (Thioredoxin)PSBD (peripheral subunit-binding domain)pUC57-T7-CP11GUbR (ubiquitin A77R mutant)mODC (mouse ornithine decarboxylase)pUC57-T7-CP12GUbRSORT1 (sortilin 1)pUC57-T7-CP13GLampmODCpUC57-T7-CP14GLampSORT1pUC57-T7-CP21GUbRcmycpUC57-T7-CP22GUbRLamppUC57-T7-CP23GmODCSORT1pUC57-T7-CP24GcmycmODCpUC57-T7-CP25GcmycSORT1pUC57-T7-CP26GLampUb(ubiquitin)pUC57-T7-CP27GLampcmycpUC57-T7-CP28GSORT1mODCpUC57-T7-CP31GmODCUbpUC57-T7-CP32 GmODCcmycpUC57-T7-CP33GmODCLamppUC57-T7-CP34GcmycUbpUC57-T7-CP35GcmycLamppUC57-T7-CP36GSO RT1UbpUC57-T7-CP37GSORT1cmycpUC57-T7-CP38GSORT1LamppUC57-T7-CP41GTRXwtcmycpUC57-T7-CP42GT RXwtLamppUC57-T7-CP43GTRXwtmODCpUC57-T7-CP44GTRXwtSORT1pUC57-T7-CP45GTRXwtUbpUC57-T7-CP46 GCmycTRXwtpUC57-T7-CP47GLampTRXwtpUC57-T7-CP48GmODCTRXwtpUC57-T7-CP49GSORT1TRXwtpUC57-T7- CP50GUbRTRXwtpUC57-T7-CP112GTRXwt-UbRSORT1pUC57-T7-CP113GTRXwt-LampmODCpUC57-T7-CP123GTRX wt-mODCSORT1pUC57-T7-CP131GTRXwt-mODCUbpUC57-T7-CP133GTRXwt-mODCLamppUC57-T7-CP134GTRXwt- cmycUbpUC57-T7-CP136GTRXwt-SORT1UbpUC57-T7-CP138GTRXwt-SORT1LamppUC57-T7-CP201GTRXwtSORT1 w / TMpUC57-T7-CP202GLamp w / TMTRXwt

[0203] (In Table 2 above, TRXwt refers to the wild type of TRX, TRX that can be obtained from nature without mutation, and TRXmt refers to the cysteine ​​(C) at the 32nd, 35th, 62nd, 69th, and 73rd positions from the N-terminus of the TRX amino acid sequence of sequence number 11, which is substituted with serine (Serine; S))

[0204]

[0205] (In Table 3 above, “tccgga” in bold corresponds to the BspEI site, and the BspEI site was inserted simultaneously with the carrier protein insertion.)

[0206] After carrier protein insertion, it was treated with BspEI (NEB, R0540S) restriction enzyme and purified using Expin Gel SV (Gene all, Cat#102-102).

[0207] After that, 10 ng of pEGFP vector (Clontech, cat. no. 6077-1, GenBank U76561.1) containing EGFP reporter as template was prepared, and 10 pmole each of EGFP oligonucleotides (oEGFP-BE1-F and oEGFP-BE1-R) in Table 3 were added, and Platinum TM 100 μl of SuperFi II Green PCR Master Mix (ThermoFisher; cat#: 12369050) was prepared and PCR was performed using it.

[0208] After performing PCR, the product was treated with BspEI (NEB, R0540S) restriction enzyme and sub-cloned into a plasmid capable of expressing each purified carrier protein combination, thereby obtaining a carrier protein vector with EGFP inserted.

[0209] The vector map of the above pEGFP vector is shown in Fig. 2, a schematic diagram of the carrier protein vector produced is shown in Fig. 3, a vector map of the pUC57-T7-CP12G plasmid produced as an example is shown in Fig. 4, and the amino acid and base sequence information of the carrier protein inserted at each of the N-terminus and C-terminus are shown in Tables 4 and 5 below, respectively.

[0210]

[0211] (In the above Table 4, when the carrier protein is inserted into the N-terminus, a methionine (M) having an atg base sequence is added and inserted at the 5' end of each protein, and corresponds to the GS linker sequence, which is the underlined portion above.)

[0212]

[0213] (In the above Table 5, 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, and corresponds to the GS linker sequence, which is the underlined portion above.)

[0214] mMESSAGE mMACHINE for the carrier protein vector manufactured above TM mRNA of the following structural formula 1 was produced by in vitro transcription using the T7 ULTRA Transcription Kit (ThermoFisher, AM1345), and the sequence information corresponding to the 5'UTR and 3'UTR is shown in Table 6 below.

[0215] [Structural formula 1]

[0216] 5'UTR (TE) - (carrier protein I) - GGSGGGGSGG - EGFP - GGSGGGGSGG - (carrier protein II) - 3'UTR (2X HBB)

[0217] (In the above structural formula 1, GGSGGGGSGG represents a linker sequence, carrier protein I (CP #1) corresponds to a carrier protein inserted at the N-terminus of the above-mentioned vector, carrier protein II (CP #2) corresponds to a carrier protein inserted at each of the C-terminus, and the 5'UTR (TE) has a sequence of sequence number 41 in Table 6 below, and the 3'UTR (2X HBB) has a sequence of sequence number 42 in Table 6 below.)

[0218] Division sequence (5' -> 3') SEQ ID NO: 5UTR_TEccggcgggtttctgacatccggcgggtttctgacatccggcgggtttctgacatccggcgggtttctgacatccggcgggtttctgacatccggcgggtttctgacatccggc gggtttctgacatccggcgggtttctgacatccggcgggtttctgacatccggcgggtttctgacattcacaaccaggcctccacaacc413UTR_2X_HBBagctcgctttcttgctgtccaattt ctattaaaggttcctttgttccctaagtccaactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgctgcggatctagctcgcttt cttgctgtccaatttctattaaaagttcctttgttccctaagtccaactactactaaactgggggatattatgaagggccttgagcatctggattctgcctaataaaaaacatttattttcattgctgc42

[0219]

[0220] Example 1-2. Primary screening of carrier proteins

[0221] When the expression pattern is changed by BTZ (bortezomib), a proteasome inhibitor, it can be transferred to the processing / presentation pathway appropriate for the CD8 antigen, and when the expression pattern is changed by CHQ (Chloroquine), a lysosomal inhibitor, it can be transferred to the processing / presentation pathway appropriate for the CD4 antigen. Therefore, BTZ and CHQ treatment were performed to confirm whether processing / presentation of CD8 and CD4 antigens was performed.

[0222] Specifically, each of the above-produced mRNAs was transfected into the K562 Cell line (ATCC, CCL-243) using the 4D-nucleofector system (Lonza). The transfected cells were treated with BTZ or CHQ to confirm the response to each inhibitor using Bortezomib (BTZ), a proteasome inhibitor, and Chloroquine (CHQ), a lysosomal inhibitor. After 16 hours, the cells were subjected to a fluorescence-activated cell sorter (FACS) to confirm whether the reactivity was observed and to confirm whether degradation by the proteasome or lysosome was inhibited, thereby confirming the possibility of transfer to the HLA Class I and Class II pathways. The results confirming the transferability of the combination are shown in Figures 5a to 5k.

[0223] Specifically, in the above FIGS. 5a to 5k, C means CHQ treatment, B means BTZ treatment, BC means treatment with BTZ and CHQ, and X means no treatment. When GFP expression is increased by C compared to X, it means that it is likely to go well with the CD4 antigen, when expression is increased by B compared to X, it means that it is likely to go well with the CD8 antigen, and when expression is increased by BC compared to X, it means that it is likely to go well with both the CD4 and CD8 antigens.

[0224] As a result of confirming the possibility of delivery, it was confirmed that the CP01 (TRXmt-PSBD) combination with inserted mutant TRX showed good expression of EGFP even without the influence of BTZ and CHQ, and 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, confirming that CD4 and CD8 antigens were delivered to the processing / presentation pathway by the CP01 combination, and 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 could not be distinguished from the influences due to BTZ and CHQ.

[0225]

[0226] Example 1-3. Secondary screening of carrier proteins

[0227] In the above Example 1-2, it was confirmed that the CP01 combination allows both CD4 and CD8 antigens to be delivered to the processing / presentation pathway. Therefore, a design was made to add the TRX of the CP01 combination to the CP12, CP13, CP23, CP31, CP33, CP34, CP36 and CP38 combinations, which were confirmed in the above Example 1-2 to allow the CD8 antigen to be delivered to the processing / presentation pathway.

[0228] Specifically, using 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 produced in Example 1-1 as templates, PCR was performed using the oligonucleotides oCCP12G-F1 and oCCPxxG-R1 in Table 7 below as primers, and each product was purified.

[0229] Sequence number (5' -> 3') oCCP12G-F1ggaggaagcggaggacagatcttcgtgaaa43 oCCPxxG-R1gcaagaaagcgagctctcgagttta44

[0230] After purification, the TRX PCR product produced during the plasmid production process in Example 1-1 was combined with AscI (Enzynomics; R068M) and XhoI (Thermo Scientific) into each of the 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 plasmids produced in Example 1-1. TM FastDigest; #FD0694) was purified by treating with restriction enzymes, and the backbone part was inserted into the EZ-fusion method to create a plasmid with TRX added to the N-terminus. The created plasmid was named by adding 1 in front of the number of the original plasmid (e.g., a plasmid with TRX added to pUC57-T7-CP12G: pUC57-T7-CP112G).

[0231] Additionally, the carrier protein at the N terminus was changed to TRX for CP12, CP13, CP21, CP31, and CP33 combinations (CP41 to CP45).

[0232] Specifically, PCR was performed using the pUC57-T7-CP21G, pUC57-T7-CP33G, pUC57-T7-CP-13G, pUC57-T7-CP12G and pUC57-T7-CP31G plasmids of Example 1-1 as templates and the oligonucleotides oGS-EGFP-F and oCCPxxG-R1 of Table 8 below as primers, and each product was purified to secure a fragment in which EGFP and a Carrier protein were linked to the C-terminus.

[0233] Sequence number (5' -> 3') oGS-EGFP-Fggcggatctggcggcggaggaagcggaggagtgagcaagggcgag45 oCCPxxG-R1gcaagaaagcgagctctcgagttta46

[0234] After that, together with the TRX PCR product produced in the above Example 1-1, the backbone portion of each of the plasmids pUC57-T7-CP21G, pUC57-T7-CP33G, pUC57-T7-CP-13G, pUC57-T7-CP12G and pUC57-T7-CP31G produced in the above Example 1-1 was purified by treating them with AscI (Enzynomics; R068M) and XhoI (Thermo ScientificTM FastDigest; #FD0694) restriction enzymes, and was inserted into the purified backbone portion using the EZ-fusion method, thereby finally producing plasmids with TRX as the carrier protein at the N-terminus of each of the above plasmids (pUC57-T7-41G, pUC57-T7-CP42G, pUC57-T7-CP43G, pUC57-T7-44G and pUC57-T7-CP45G).

[0235] Additionally, the C-terminal carrier protein was changed to TRX for the CP34, CP27, CP23, CP36, and CP21 combinations (CP46 to CP50).

[0236] Specifically, PCR was performed using the pUC57-T7-CP34G, pUC57-T7-CP27G, pUC57-T7-CP23G, pUC57-T7-CP36G and pUC57-T7-CP21G plasmids of Example 1-1 as templates and oM13F and oGS-EGFP-R of Table 9 below as primers, and each product was purified to secure a fragment in which EGFP and a Carrier protein were linked to the N-terminus.

[0237] Sequence number (5' -> 3') oM13Fgtaaaacgacggccagt47 oGS-EGFP-Rtcctccgcttcctccgccgccagatccgcccttgtacagctcgtc48

[0238] After that, PCR was performed using pUC57-T7-CP01G produced in Example 1-1 as a template and oligonucleotides oCTRXwt-F1 and oCTRXwt-R1 of Table 10 below as primers to secure a TRX fragment to be linked to the C-terminus, and the fragment linked to the EGFP and the Carrier protein at the N-terminus was inserted into each of pUC57-T7-CP34G, pUC57-T7-CP27G, pUC57-T7-CP23G, pUC57-T7-CP36G and pUC57-T7-CP21G treated with AscI (Enzynomics; R068M) and XhoI (Thermo ScientificTM FastDigest; #FD0694) restriction enzymes by EZ-fusion method, to finally obtain pUC57-T7-46G, pUC57-T7-CP47G, pUC57-T7-CP48G, pUC57-T7-49G, and pUC57-T7-CP50G plasmids were constructed.

[0239] Sequence number (5' -> 3') oCTRXwt-F1ggcggatctggcggcggaggaagcggaggagtcaagcagatcgag49 oCTRXwt-R1gcaagaaagcgagctctcgagtttaaactagttcgttgat50

[0240] Information on carrier proteins inserted into the N-terminus and C-terminus of the designed vector, respectively, is shown in Table 2 above, and information on the amino acid sequence and base sequence of the carrier protein to which TRX is added among the carrier proteins is shown in Tables 4 and 5 above, respectively.

[0241] In addition, since SORT1 and Lamp among carrier proteins are membrane proteins and have a transmembrane domain, a transmembrane domain was added to the existing cytosolic domain of each protein (SORT1 w / TM and Lamp w / TM), and SORT1 w / TM was positioned at the C-term and TRX was positioned at the N-term, or Lamp w / TM was positioned at the N-term and TRX was positioned at the C-term (CP201 ​​and CP202), and a schematic diagram of intracellular expression is shown in Figure 6.

[0242] Specifically, the SORT1 w / TM was produced by performing PCR in substantially the same manner as SORT1 using the oligonucleotides oSORTwTM-1F, oSORTwTM-4R, oSORTwTM-2R, and oSORTwTM-3F of Table 3, and the Lamp w / TM was produced by performing PCR in substantially the same manner as SORT1 using the oligonucleotides oLAMPwTM-1F, oLAMPwTM-4R, oLAMPwTM-2R, and oLAMPwTM-3F of Table 3.

[0243] Information on the carrier protein to which the transmembrane domain of the vector produced above has been added is shown in Table 2, and the amino acid sequence and base sequence information of the carrier protein to which the transmembrane has been added are shown in Tables 4 and 5, respectively.

[0244] The possibility of delivering the carrier protein vector produced above to the HLA Class I and Class II pathways after producing mRNA was confirmed in substantially the same manner as in Example 1-1, and the results are shown in Fig. 7.

[0245] As a result of confirming the possibility of transfer, it was confirmed that the CP136 combination with added TRX strengthened the existing reaction, and in the CP201 ​​and CP202 combinations with added transmembrane, it was confirmed that SORT1 and Lamp showed a reaction to CHQ when the transmembrane was present, confirming that the reaction was strengthened overall.

[0246]

[0247] Example 2. Production of a vector containing an epitope and a carrier protein inserted therein.

[0248] Example 2-1. Production of carrier protein vector

[0249] In order to confirm the effect of the combination of CP12, CP13, CP23, CP31, CP33, CP34, CP36, CP38, CP47, CP48, CP50, CP136, CP201 ​​and CP202, which were confirmed to have the possibility of being transferred to the HLA Class I and Class II pathways in Example 1, on the expression efficiency of the epitope, a new type of vector containing the carrier protein combination was produced.

[0250] Specifically, a vector was constructed in which five epitopes were linked as a linker 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 in the above combination, and EGFP was linked to the C-terminus of carrier protein 2 with a P2A sequence, and a schematic diagram of the constructed vector is shown in Fig. 8. In the vector of Example 1, EGFP is located between two carrier proteins, but in the constructed vector, the epitopes are located as in an actual application example, and EGFP for measuring the expression level is linked to the C-terminus.

[0251] To construct the above vector, inserts corresponding to genes for cloning were constructed by dividing it into four fragments as shown in Fig. 8. The first fragment (CP_N fragment; CP#1) includes carrier protein 1 to a portion of the first linker, the second fragment (Epitope fragment; Epitope_2) includes a portion of the first linker, the epitope and linker, and a portion of the sixth linker, the third fragment (CP_C fragment; CP#3) includes a portion of the sixth linker, carrier protein 2, and a portion of the P2A sequence, and the fourth fragment (P2A-EGFP fragment; GFP_4) includes a portion of the P2A sequence and the EGFP sequence. The second and fourth fragments are commonly used regardless of the carrier protein combination.

[0252] Using the primers in Table 12 that can amplify the carrier proteins in the combinations in Table 11 below in accordance with 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 prepared in Example 1 to amplify the first and third fragments. Using the primers that can amplify the epitope (Epitope_2) and P2A-EGFP (GFP_4) in Table 12 below, PCR was performed on the pUC57-T7-5epitopes plasmid (Fig. 9) to amplify the second fragment, and the fourth fragment was also amplified using the pUC57-T7-CP12G plasmid of Example 1-1 as a template. Additionally, 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 in the combinations in Table 11 (TRX_WT).

[0253] Combination nameCP #1 Primer type used for cloningCP #2 Primer type used for cloninghTrx_WTTrxWT_1_F / TrxWT_1_R-CP12Ubi_1_F / Ubi_1_RSORT1_3_F / SORT1_3_RCP13Lamp_1_F / Lamp_1_RmODC_3_F / mODC_3_RCP23mODC_1_F / mODC_1_RSORT1_3_F / SORT1_3_RCP31mODC_1_F / mODC_1_RUbi_3_F / Ubi_3_RCP33mODC_1_F / mODC_1_RLamp_3_F / Lamp_3_RCP34cMyc_1_F / cMyc_1_RUbi_3_F / Ubi_3_RCP36SORT1_1_F / SORT1_1_RUbi_3_F / Ubi_3_RCP38SORT1_1_F / SORT1_1_RLamp_3_F / Lamp_3_RCP47Lamp_1_F / Lamp_1_RTrxWT_3_F / TrxWT_3_RCP48mODC_1_F / mODC_1_RTrxWT_3_F / TrxWT_3_RCP50Ubi_1_F / Ubi_1_RTrxWT_3_F / TrxWT_3_RCP136TrxWT_1_F / TrxWT_SORT1_1_RUbi_3_F / Ubi_3_RCP201TrxWT_1_F / TrxWT_1_RSORT1_TM_3_F / SORT1_3_RCP202Lamp_1_F / Lamp_TM_1_RTrxWT_3_F / TrxWT_3_R

[0254]

[0255] The three or four types of fragments according to each combination produced above were inserted into the empty vector pLRK33-empty having a BsmBI restriction enzyme site, including a BsmBI site at both ends, using the BsmBI restriction enzyme and T4 DNA Ligase of the NEBridge Golden Gate Assembly-BsmBIv2; NEB # E1602L kit, and the epitope fragments include five types of epitopes, and are located in the order of M44, M30, M21, M12, and mTrp2 based on the CP_N fragment position, and each epitope is connected by a linker of the sequence GGSGGGGSGG. The above M44, M30, M21 and M12 epitopes correspond to B16-M44, B16-M30, B16-M21 and B16-M12 disclosed in the paper https: / www.ncbi.nlm.nih.gov / pmc / articles / PMC4838069.

[0256] The vector map of the above-mentioned empty vector is shown in Fig. 10, a schematic diagram showing the process of inserting each type of fragment into the empty vector is shown in Fig. 11, the composition information of the vector produced by inserting into the empty vector and the amino acid sequence and base sequence information of the five types of inserted epitope fragments and the amino acid sequence and base sequence information of the form in which the five types of epitope fragments are connected by a linker (5 types of Epitope) are shown in Table 14, and the amino acid sequence and base sequence information of the inserted P2A and EGFP are shown in Table 15. For example, the vector map of the produced plasmid pLRK33-hTrx_WT is shown in Fig. 12.

[0257] Plasmid type CP_N fragment Epitope fragment CP_C fragment P2A-EGFP fragment pLRK33-hTrx_WTTrxwt 5 types Epitope-P2A-EGFPpLRK33-CP12_mUbRSORT1pLRK33-CP13_mLampmODCpLRK33-CP23_mmODCSORT1pLRK33-CP31_mmODCUbpLRK33-CP33_mmODCLamppLRK33-CP34_mcMycUbpLRK33-CP36 _mSORT1UbpLRK33-CP38_mSORT1LamppLRK33-CP47_mLampTrxwtpLRK33-CP48_mmODCTrx wtpLRK33-CP50_mUbRTrxwtpLRK33-CP136_mTrxwt-SORT1UbpLRK33-CP201_mTrxwtSORT1 w / TMpLRK33-CP202_mLamp w / TMTrxwt

[0258] 종류서열 (5' -> 3')서열번호M44_PRTEFKHIKAFDRTFANNPGPMVVFATPGM51M44_GENEgagttcaagcacatcaaggccttcgaccggaccttcgccaacaatcccggacctatggtggtgttcgccacacctggaatg52M30_PRTPSKPSFQEFVDWENVSPELNSTDQPFL53M30_GENEcctagcaagcctagcttccaagagttcgtggactgggagaacgtgtcccctgagctgaactctaccgaccagccatttctc54M21_PRTSSPDEVALVEGVQSLGFTYLRLKDNYM55M21_GENEtcttctccagatgaggtggcactggtggaaggcgtgcagtctctgggcttcacctacctgagactgaaggacaactacatg56M12_PRTTPPPEEAMPFEFNGPAQGDHSQPPLQV57M12_GENEacacctccacctgaagaggccatgcctttcgagttcaacggacccgctcagggcgatcactctcagcctccacttcaagtt58mTrp2_PRTPNGTQPQIANCSVYDFFVWLHYYSVRD59mTrp2_GENEcctaatggaacacagccccagatcgccaactgcagcgtgtacgacttcttcgtgtggctgcactactacagcgtcagagat605 Epitopes_PRTGGSGGGGSGGEFKHIKAFDRTFANNPGPMVVFATPGMGGSGGGGSGGPSKPSFQEFVDWENVSPELNSTDQPFLGGSGGGGSGGSSPDEVALVEGVQSLGFTYLRLKDNYMGGSGGGGSGGTPPPEEAMPFEFNGPAQGDHSQPPLQVGGSGGGGSGGPNGTQPQIANCSVYDFFVWLHYYSVRDGGSGGGGSGG615Epitopes_GENEggcggatctggcggcggaggaagcggaggtgagttcaagcacatcaaggccttcgaccggaccttcgccaacaatcccggacctatggtggtgttcgccacacctggaatgggtggaagcggaggaggcggctctggaggccctagcaagcctagcttccaagagttcgtggactgggagaacgtgtcccctgagctgaactctaccgaccagccatttctcggcggatcaggaggcgggggctccggagggtcttctccagatgaggtggcactggtggaaggcgtgcagtctctgggcttcacctacctgagactgaaggacaactacatgggaggatccggcggcggcggaagcggggggacacctccacctgaagaggccatgcctttcgagttcaacggacccgctcagggcgatcactctcagcctccacttcaagttggcggatctggcggcggcggatcaggtggccctaatggaacacagccccagatcgccaactgcagcgtgtacgacttcttcgtgtggctgcactactacagcgtcagagatggcggcagtggcggaggcggctcaggcgga62

[0259]

[0260]

[0261] Comparative Example 1. Production of a vector containing only an epitope without a carrier protein.

[0262] In order to prepare an insert capable of amplifying the N-terminus of the epitope of Example 2 above to the C-terminus of EGFP, the NcoI_Epitope_F and XhoI_EGFP_R oligos of Table 16 below were used to perform PCR on the pLRK33-hTrx_m vector to amplify the corresponding portion, thereby producing an insert. The vector map of the pLRK33-hTrx_m vector is shown in Fig. 13.

[0263] Oligo type sequence (5' -> 3') Sequence number NcoI_Epitope_FCCAAGGCCACCATGGAGTTCAAGCACATCAAGGC143 XhoI_EGFP_RCTGTATTCTCGAGTTTACTTGTACAGCTCGT144

[0264] The above-mentioned manufactured insert was inserted into the NcoI, XhoI site of the pLRK33-empty vector of Fig. 10 (Thermo Scientific TM FastDigest; #FD0574) and XhoI (Scientific TM FastDigest; #FD0694) restriction enzyme and T4 DNA ligase (Thermo Scientific TM ; #EL0012) was used to create a comparative vector pLRK33-No_CP_m in which no carrier protein was inserted.

[0265]

[0266] Example 3. Production of mRNA of carrier protein containing epitope

[0267] In vitro transcription was performed 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) produced in Example 2 and Comparative Example 1 as templates to produce mRNA.

[0268] Specifically, before proceeding with in vitro transcription, the vector was linearized by cutting the rear part of poly A with XbaI (Termo Scientific, FD0684) so ​​that only the desired part could be transcribed into RNA.

[0269] In vitro transcription was performed using 15 linearized vectors under the conditions in Table 17 below, template DNA was removed by treatment with DNase I (Invitrogen, AM2238), and mRNA was produced by purification using the RNeasy Mini kit (Qiagen, 74104).

[0270] [Structural formula 2]

[0271] 5'UTR - (carrier protein 1) - LELELELELEL - (carrier protein 2) - P2A - EGFP - 3'UTR - poly A

[0272] (In the above structural formula 2, L represents the linker sequence GGSGGGGSGG, and carrier protein 1 (CP #1) corresponds to each carrier protein inserted at the N-terminus of the above-mentioned vector, and carrier protein 2 (CP #2) corresponds to each carrier protein inserted at the C-terminus.)

[0273] ComponentVolume or amount10 RNT-107-L)1.8 μl100 mM N1-methyl pseudo UTP (Jena Bioscience #RNT-107-L)1.8 μl1M DTT (Jena Bioscience #RNT-107-L)0.8 μlT7 RNA Polymerase High concentration (NEB #M0460T)0.5 μlMurine RNase Inhibitor (NEB #M0314L)0.5 μlDNA template1 μgDNase / RNase-free waterUp to 20 μlTotal volume20 μl

[0274]

[0275] Test Example 1. Measurement of the expression efficiency of carrier proteins for epitopes in K562 cell lines.

[0276] A portion of the K562 cell line (ATCC, CCL-243) maintained in the tank was taken, and after cell down, resuspended in complete media (RPMI1640, Gibco, 11875-093, 10% FBS, Gibco, 26140-079, and 1% Penicillin-Streptomycin, Gibco, 10378-016), and the cells were counted to 3*10 5 Add more complete media to the above to make the concentration of cells / ml or resuspend the cells in complete media after downing them again to make 3*10 5 20 ml of medium adjusted to cells / ml concentration was cultured in a CO2 incubator.

[0277] The K562 cell line in culture was recovered, and the mRNA prepared in Example 3 was electroporated into the cell line using the SF cell line 4D-Nucleofector X kit S (Lonza, V4XC-2032) 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.

[0278] Cells were harvested from each well, counted, resuspended in FACS buffer (eBioscience, 00-4222-28), and transferred to a FACS tube. Cell analysis was performed with LSRFortessa (BD Biosciences) to determine the expression level of the epitope, and the results are shown in Figure 14 and Table 18. As a negative control, a cell line that was not transfected with mRNA was prepared to determine the expression level of the epitope.

[0279] Mean fluorescence intensityMFI (Fold change)Negative control8420.236849508No CP35551CP12223256.279887482CP13228276.421097046CP23288658.11954993CP313 15358.870604782CP33151084.24978903CP34261887.36652602CP36157464.4292545 71CP38179395.046132208CP47254477.158087201CP48231716.517862166CP5022971 6.461603376CP136221036.217440225CP201102592.885794655CP20294052.64556962

[0280] Cell analysis results confirmed that each CP combination including carrier protein increased the overall EGFP expression efficiency compared to No CP without carrier protein, confirming that the expression of the epitope can be increased by using CP combinations.

[0281]

[0282] Test Example 2. Measurement of the expression efficiency of carrier proteins for epitopes in human dendritic cells.

[0283] Cryopreserved human PBMC (Peripheral Blood Mononuclear Cell) (Stemexpress, PBMNC300C) stored in a liquid nitrogen tank were thawed according to the manufacturer's protocol, and a cell count was performed on the thawed cells. Based on the results, cells were separated using human CD14 microbeads (Miltenyi Biotec, 130-050-201) and LS column (Miltenyi Biotec, 130-042-401) according to the user manual (column activation 2 ml, cell loading 2 ml, wash 3 times 2 ml, cell elution 5 ml each using buffer), and the number of selected cells was measured.

[0284] The monocytes of the above separated cells were suspended in the media (w / differentiation supplement) included in the ImmunoCult dendritic cell culture kit (DC culture kit) (Stemcell, 10985) to a concentration of 1e06 cells / ml to produce monocyte-derived dendritic cells (moDC), and seeded in 8 wells of a 96-well plate with 100 μl per well.

[0285] The medium of cells cultured using the above DC culture kit was removed and replaced with 100 μl of medium containing the differentiation supplement included in the DC culture kit, and the DC maturation supplement included in the DC culture kit was added to the cell medium by calculating the amount according to the user manual.

[0286] According to Table 19 below, the mRNA, DP buffer, and carrier produced in Example 3 were sequentially added and mixed. The carrier is a drug delivery composition including the cationic compound and anionic polymer chemical of Example 1 of Patent Publication No. 10-2024-0035317.

[0287] At each mixing step, pipetting was performed using a pipet to ensure sufficient mixing, and immediately after adding the formulation, pipetting was performed 10 times to prepare a drug product (DP) containing a mixture of mRNA and a carrier. The prepared DP was treated in the cell medium cultured with the DC maturation supplement according to the DP treatment amount in Table 19 below. Additionally, a negative control group not treated with DP was prepared.

[0288] mRNA (1mg / ml stock)DP Buffer carrierDP negative control----No CP10 μl30.4 μl9.6 μl1.46 μlCP231.72 μlCP311.78 μlCP331.67 μlCP381.68 μlCP481.84 μlCP501.92 μl

[0289] After DP treatment, cells were harvested from each well and lysed. Afterwards, the cells were resuspended in a solution containing FACS buffer (eBioscience, 00-4222-28) and 7-AAD (BD biosciences, 559925) at a volume ratio of 100:1, transferred to a FACS tube, and cell analysis was performed three times using LSRFortessa (BD Biosciences). The results are shown in Figure 15 and Table 20.

[0290] MFI (Fold change)Negative control0.173425No CP1CP232.528538CP312.673939CP332.763781CP382.204154CP482.098567CP501.144419

[0291] (The values ​​in Table 20 above are the average values ​​of MFI obtained by performing the above cell analysis three times.)

[0292] Cell analysis results showed that the MFI values ​​were more than twice that of no CP in the CP23, CP31, CP33, CP38, and CP48 combinations, confirming increased human epitope expression.

[0293]

[0294] Test Example 3. Measurement of the immunogenicity-inducing ability of a carrier protein to an epitope in human dendritic cells.

[0295] DP according to Table 19 was prepared in substantially the same manner as in Test Example 2 above, and treated in a medium for culturing the moDC, and cultured for 2 days.

[0296] On the day the above culture was completed, the human PBMC of the above test example 2 used for producing moDC was prepared separately, the initial cell number of PBMC was calculated, and based on this, CD8+ T cells were isolated using a human CD8+ T cell isolation kit (Miltenyi Biotec, 130-096-495) and LS column (Miltenyi Biotec, 130-042-401) according to the user manual.

[0297] The above cultured moDCs and isolated CD8+ T cells were harvested, cell-downed, and resuspended in Immunocult-XF T cell expansion medium (Stemcell, 10981) to a concentration of 5e05 cells / ml for moDCs and 2e06 cells / ml for CD8+ T cells, respectively. Afterwards, each cell suspension was mixed in a volume ratio of 1:1, seeded into a 6-well plate with 2 ml per well, and the moDC and CD8+ T cell mixture was cultured for 7 days.

[0298] After culture, the cells were harvested again and resuspended to 1e06 cells / ml using the CTL media included in the mouse IFN-γ single-color ELISPOT kit (Immunospot, mIFNgp-2M).

[0299] Separately from the above process, each peptide corresponding to the five epitopes in Table 21 below was diluted to 20 μM in CTL media to prepare restimulation peptide media, and 100 μl each of the prepared cell suspension and peptide media was dispensed into each well of the 96-well plate for ELISPOT included in the ELISPOT kit, and cultured for more than 16 hours. As a negative control, culture was performed without adding peptide media.

[0300] Peptide type Peptide sequence Sequence number NY-ESO-1SLLMWITQC147MAGE-A3FLWGPRALV148SurvivinLMLGEFLKL149Multi MAGE-AYLEYRQVPV150Melan AELAGIGILTV151

[0301] (In Table 21 above, Multi MAGE-A refers to an antigen sequence that appears commonly in several subfamily antigens of MAGE-A)

[0302] After culture, the detection reaction was performed according to the user manual and analyzed using an ELISPOT plate analyzer (Immunospot), and the results are shown in Figure 16.

[0303] ELISPOT analysis results confirmed that immunogenicity for multiple epitopes was increased in the combination of CP23, CP31, CP33, and CP38 compared to no CP.

Claims

1. mRNA of antigen protein; mRNA encoding a first carrier protein linked to the 5' end of the mRNA of the antigen protein; and A conjugate comprising an mRNA encoding a second carrier protein linked to the 3' end of the mRNA of the antigen protein.

2. In the first paragraph, the first carrier protein and the second carrier protein are the same or different, and are each at least one selected from 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. In the second paragraph, the first carrier protein and the second carrier protein are selected from the protein combinations shown in Table 1 below, the conjugate: [Table 1] 4. In paragraph 2, The above UbR has an amino acid sequence of sequence number 1; The above Ub has an amino acid sequence of SEQ ID NO: 2; The above Lamp has an amino acid sequence of sequence number 3 or 4; The above mODC has an amino acid sequence of SEQ ID NO: 5 or 6; The above cmyc is an amino acid sequence of sequence number 7 or 8; The above SORT1 has an amino acid sequence of SEQ ID NO: 9 or 10; The above TRXwt has an amino acid sequence of SEQ ID NO: 11 or 12; The above PSBD has an amino acid sequence of SEQ ID NO: 13; The above TRXwt-UbR has an amino acid sequence of SEQ ID NO: 14; The above TRXwt-Lamp has an amino acid sequence of SEQ ID NO: 15; The above TRXwt-mODC has an amino acid sequence of SEQ ID NO: 16; The above TRXwt-cmyc has an amino acid sequence of SEQ ID NO: 17; The above TRXwt-SORT1 has an amino acid sequence of SEQ ID NO: 18; The above SORT1 w / TM has an amino acid sequence of SEQ ID NO: 19; The Lamp w / TM has an amino acid sequence of SEQ ID NO: 20; or The above TRXmt is a conjugate comprising an amino acid sequence of SEQ ID NO:

145.

5. In the third paragraph, the first carrier protein and the second carrier protein are selected from the protein combinations shown in Table 22 below, a conjugate: [Table 22] 6. A conjugate according to claim 2, wherein the antigen protein comprises a T cell epitope.

7. A conjugate according to claim 6, wherein the T cell epitope is derived from a tumor antigen, an antigen of an infectious agent, an autoantigen, or an allergen.

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

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

10. In the 8th paragraph, the tumor-associated antigen (TAA) is mTrp2, CT (Cancer-testis) antigen, EGFR, Ova, Melan-A, PSMA (Prostate Specific Membrane Antigen), Survivin, ADAbp (adenosine deaminase-binding protein), cyclophilin b, gp100, CRC (Colorectal 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, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, PRAME, NY-ESO-1, TRP2, Mammaglobin-A, metallopanstimulin-1 (MPS-1), cytochrome P450 isoform 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, conjugate., 11. A conjugate according to claim 8, wherein the tumor-specific antigen (TSA) is M12, M20, M21, M30 or M44.

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

13. A conjugate according to any one of claims 1 to 12, wherein the conjugate has at least one property selected from the group consisting of (i) and (ii) below by the carrier protein: (i) CD4 / CD8 are delivered to the processing and presentation pathways, respectively; and (ii) Increased expression of the above antigen protein 14. An immunizing composition comprising a conjugate of any one of claims 1 to 12.

15. In the 14th paragraph, the composition for immunization further comprises at least one selected from the group consisting of an antigen adjuvant, an efficacy enhancer, a preservative, a buffer, a surfactant, a carrier, an osmotic agent, an antioxidant, and a stabilizer.

16. An immunizing composition according to claim 14, which is administered by at least one route selected from the group consisting of intranasal, intratracheal, oral, intradermal, intramuscular, intraperitoneal, intravenous, conjunctival, and subcutaneous.

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

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

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