Recombinant fusion proteins for antigen delivery with modified cysteine ​​residues and uses thereof

Abstract

The present invention relates to a fusion protein comprising a peptide antigen and a human thioredoxin protein linked to the N-terminus, C-terminus, or both, of the peptide antigen, wherein all cysteine ​​residues in the amino acid sequence of the human thioredoxin protein have been substituted with non-cysteine ​​residues; a nucleic acid molecule encoding the fusion protein; an expression vector comprising the nucleic acid molecule; a cell transformed with the expression vector; and a composition comprising the fusion protein, nucleic acid molecule, expression vector, or cell.

Description

[Technical Field]

[0001] The present invention relates to a fusion protein comprising a peptide antigen and a human thioredoxin protein linked to the N-terminus, C-terminus, or both, of the peptide antigen, wherein all cysteine ​​residues in the amino acid sequence of the human thioredoxin protein have been substituted with non-cysteine ​​residues; a nucleic acid molecule encoding the fusion protein; an expression vector comprising the nucleic acid molecule; a cell transformed with the expression vector; and a composition comprising the fusion protein, nucleic acid molecule, expression vector, or cell. [Background technology]

[0002] Various technologies have been developed to deliver antigens in various forms to induce T cell immunity, but each form has its own advantages and disadvantages. Delivering antigens in peptide form has the disadvantages of difficult synthesis and purification due to diverse antigen sequences, and low exposure due to solubility and a short half-life. Delivering antigens in mRNA form has the advantage of consistent manufacturing, but the disadvantages of requiring a dosage form due to instability in the body and uncertainty about the delivery of a consistent amount of antigen (consistent expression of diverse sequences in the body). Delivering antigens in DNA form has better in vivo stability than mRNA, but the disadvantages are that it must be delivered to the nucleus, low antigen expression efficiency, and the possibility of DNA being integrated into the genome. Viral vectors have the advantages of in vivo delivery and high immunogenicity, but the disadvantage of generating strong antibodies against the viral surface, which can reduce drug efficacy due to anti-drug antibodies during booster vaccinations.

[0003] Therefore, there is a need to develop vaccines that can more effectively deliver antigens to the body while inducing stronger immune responses. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention provides an antigen delivery system that improves antigen expression, physical properties, stability, and / or immunogenicity, and improves antigen delivery to immune response cells. The present invention also provides an antigen delivery system that can produce, express, purify, and deliver a diverse array of antigens, thereby inducing a stronger immune response in the body.

[0005] One example provides a fusion protein comprising a peptide antigen and a human thioredoxin protein linked to the N-terminus, C-terminus, or both of the peptide antigen, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein have been substituted with non-cysteine ​​residues.

[0006] Another example provides a nucleic acid molecule encoding the fusion protein.

[0007] Another example provides an expression vector comprising the nucleic acid molecule.

[0008] Another example provides a cell transformed with the expression vector.

[0009] Another example provides a method for producing a fusion protein, comprising expressing the expression vector in a cell.

[0010] Other examples provide vaccine or immunogenic compositions comprising the fusion proteins, nucleic acid molecules, expression vectors, and / or cells.

[0011] Another example provides a method for generating, inducing, and / or enhancing an immune response to an antigen, comprising administering an effective amount of the fusion protein, vaccine composition, and / or immunogenic composition to a patient in need of stimulating an immune response.

[0012] Another example provides the use of said fusion protein, vaccine composition and / or immunogenic composition to generate, induce and / or enhance an immune response against an antigen.

[0013] Another example provides the use of said fusion protein for the preparation of a vaccine composition and / or an immunogenic composition.

[0014] Another example provides a composition for preventing or treating a disease, comprising the fusion protein, nucleic acid molecule, expression vector, or cell.

[0015] Another example provides a method for preventing or treating a disease, comprising administering the fusion protein and / or a composition comprising the fusion protein to a patient in need thereof.

[0016] Other examples provide uses of the fusion proteins and / or compositions comprising the fusion proteins for the prevention or treatment of diseases.

[0017] Another example provides the use of the fusion protein for the manufacture of a composition for the prevention or treatment of a disease.

[0018] Another example provides a composition for improving the purification efficiency of a peptide antigen comprising the fusion protein, nucleic acid molecule, expression vector, or cell.

[0019] Another example provides the use of the fusion protein and / or a composition comprising the fusion protein to improve the efficiency of purification of a peptide antigen.

[0020] Another example provides a method for producing a fusion protein, which includes a step of linking a human thioredoxin protein to the N-terminus, C-terminus, or both of the peptide antigen, and in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0021] Another example provides a method for enhancing the immunogenicity of a fusion protein, comprising the step of linking a human thioredoxin protein to the N-terminus, C-terminus, or both, of the peptide antigen to produce a fusion protein, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0022] Another example provides a method for enhancing the disease preventive or therapeutic efficacy of a fusion protein, which comprises linking a human thioredoxin protein to the N-terminus, C-terminus, or both, of the peptide antigen to produce a fusion protein, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0023] Another example provides a method for improving the purification efficiency of a fusion protein, which includes a step of producing a fusion protein by linking a human thioredoxin protein to the N-terminus, C-terminus, or both of the peptide antigen, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0024] Another example provides a thioredoxin mutant polypeptide consisting of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9. [Means for solving the problem]

[0025] One aspect of the present invention provides a fusion protein comprising a peptide antigen and a human thioredoxin protein linked to the N-terminus, C-terminus, or both of the peptide antigen, wherein three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein have been substituted with non-cysteine ​​residues.

[0026] In one embodiment, the human thioredoxin protein may have cysteine ​​residues at amino acid positions 62, 69, and 73 in the amino acid sequence substituted with non-cysteine ​​residues.

[0027] In one embodiment, the human thioredoxin protein may have cysteine ​​residues at amino acid positions 32, 35, 62, 69, and 73 in the amino acid sequence substituted with non-cysteine ​​residues.

[0028] In one embodiment, the fusion protein may additionally comprise a carrier protein other than human thioredoxin at the N-terminus, C-terminus, or both.

[0029] In one embodiment, the fusion protein can have an affinity tag linked to its N-terminus, C-terminus, or both.

[0030] In one embodiment, a linker may be present between the peptide antigen and the human thioredoxin protein.

[0031] In one embodiment, the peptide antigen may comprise a T cell epitope.

[0032] In one embodiment, the peptide antigen may comprise a T cell epitope derived from a tumor antigen, an infectious antigen, an autoantigen, or an allergy-inducing antigen.

[0033] In one embodiment, the carrier protein may be a protein that improves the recombinant expression of the peptide antigen or increases the efficiency of purification of the peptide antigen.

[0034] In one embodiment, the tumor antigen may include a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor-derived neoantigen.

[0035] In one embodiment, tumor-derived neoantigens may contain mutations that are specifically expressed in cancer cells.

[0036] In one embodiment, the tumor-associated antigen (TAA) is a cancer-testis (CT) antigen, EGFR, M12, M20, M21, M30, M44, Ova, Melan-A, Prostate Specific Membrane Antigen (PSMA), Survivin, MAGE-A, adenosine deaminase-binding protein (ADAbp), cyclophilin b, gp100, Colorectal associated antigen (CRC)-C017-1 A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, Prostate Specific Antigen (PSA), or a combination thereof. Antigen), PSA-1, PSA-2, PSA-3, MAGE (melanoma antigen E), GAGE ​​(G antigen), BAGE (melanoma B antigen), RAGE (renal tumor antigen), LAGE (L antigen), NAG, GnT-V, MUM-1, CDK4, p53, tyrosinase, Muc1 (mucin 1), HER2 / neu, p21ras, RCAS1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin, p120ctn, PRAME, NY-ESO-1, TRP2, mammaglobin-A, metallopanstimulin-1 (MPS-1), cytochrome P The protein may be 450 isoform 1B1, 90K / Mac-2 binding protein, Ep-CAM (MK-1), HSP-70, hTERT (TRT), LEA, TAGE-1, 5T4, gp70, SCP-1, c-myc, 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.

[0037] In one embodiment, the infectious antigen may be an antigen derived from a virus, bacteria, parasite, or fungus.

[0038] In one embodiment, the carrier protein may be NDPK (nucleoside diphosphate kinase B), CSTA (cystatin-A), Trx (thioredoxin), RPL7Am (50S ribosomal protein L7Ae), Samp2a (small archaeal modifier protein 2), TE (tenascin), TM1112 (Thermotoga maritima Cupin_3 domain-containing protein), TrxA (thioredoxin1), TTrx (Thermosipho africanus thioredoxin), or PSBD (peripheral subunit-binding domain), or a fragment, mutant, fragment of a mutant, or mutant of a fragment thereof, or one or more of these.

[0039] In one embodiment, the peripheral subunit-binding domain (PSBD) fragment can comprise the amino acid sequence of SEQ ID NO: 13. The peripheral subunit-binding domain (PSBD) fragment comprising the amino acid sequence of SEQ ID NO: 13 may be a variant of a PSBD fragment.

[0040] In one embodiment, the affinity tag may be His or streptavidin.

[0041] In one embodiment, the linker may be (GS)n, (GS)n, (GS)n, (GS), Gn, LE, SSGG, or GGGGSGGGGG (where G is Gly, S is Ser, L is Leu, E is Glu, and n is an integer of at least 1).

[0042] In one embodiment, the fusion protein may be 30 kDa or less in size.

[0043] Another aspect of the present invention provides a nucleic acid molecule encoding the fusion protein.

[0044] Another aspect of the invention provides an expression vector comprising the nucleic acid molecule.

[0045] Another aspect of the invention provides a cell transformed with the expression vector.

[0046] Another aspect of the present invention provides a vaccine composition comprising the fusion protein, nucleic acid molecule, expression vector, or cell.

[0047] Another aspect of the present invention provides an immunogenic composition comprising the fusion protein, nucleic acid molecule, expression vector, or cell.

[0048] In one embodiment, the composition may additionally comprise an immunoadjuvant.

[0049] Another aspect of the present invention provides a method for generating, inducing, and / or enhancing an immune response to an antigen, comprising administering to a patient an effective amount of the fusion protein, vaccine composition, and / or immunogenic composition.

[0050] Another aspect of the present invention provides a method for generating, inducing, and / or enhancing an immune response to an antigen, comprising administering to a patient an effective amount of the fusion protein, vaccine composition, and / or immunogenic composition.

[0051] Another aspect of the invention provides the use of said fusion proteins, vaccine compositions and / or immunogenic compositions to generate, induce and / or enhance an immune response against an antigen.

[0052] Another aspect of the present invention provides the use of the fusion protein for the preparation of a vaccine composition and / or an immunogenic composition.

[0053] Another aspect of the present invention provides a method for preventing or treating a disease, comprising administering the fusion protein and / or a composition containing the fusion protein to a patient in need of such prevention or treatment.

[0054] Another aspect of the present invention provides use of the fusion protein and / or a composition comprising the fusion protein for the prevention or treatment of a disease.

[0055] Another aspect of the present invention provides a use of the fusion protein for the manufacture of a composition for preventing or treating a disease.

[0056] Another aspect of the present invention provides a composition for improving the purification efficiency of a peptide antigen, comprising the fusion protein, nucleic acid molecule, expression vector, or cell.

[0057] Another aspect of the present invention provides a use of the fusion protein and / or a composition comprising the fusion protein for improving the efficiency of purifying a peptide antigen.

[0058] Another aspect of the present invention provides a use of the fusion protein for producing a composition for improving the purification efficiency of a peptide antigen.

[0059] Another example provides a method for producing a fusion protein having enhanced immunogenicity compared to a fusion protein to which a wild-type human thioredoxin protein is linked, the method comprising linking the human thioredoxin protein to the N-terminus, C-terminus, or both of the peptide antigen, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0060] Another example provides a method for producing a fusion protein, which includes a step of linking a human thioredoxin protein to the N-terminus, C-terminus, or both of the peptide antigen, and in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0061] Another example provides a method for enhancing the immunogenicity of a fusion protein, comprising the step of linking a human thioredoxin protein to the N-terminus, C-terminus, or both, of the peptide antigen to produce a fusion protein, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0062] Another example provides a method for enhancing the disease preventive or therapeutic efficacy of a fusion protein, which comprises linking a human thioredoxin protein to the N-terminus, C-terminus, or both, of the peptide antigen to produce a fusion protein, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0063] Another example provides a method for improving the purification efficiency of a fusion protein, which includes a step of producing a fusion protein by linking a human thioredoxin protein to the N-terminus, C-terminus, or both of the peptide antigen, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0064] Another aspect of the present invention provides a thioredoxin mutant polypeptide consisting of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9.

[0065] The present invention will now be described in more detail.

[0066] When the terms "comprise", "comprises", "comprised" or "comprising" are used in this specification (including the claims), they should be interpreted as specifying the presence of stated features, integers, steps or components, but not as excluding the presence of one or more other features, integers, steps, components or groups thereof.

[0067] Discussions of documents, statutes, materials, devices, articles and the like are included in this specification solely for the purpose of providing a context for the present invention, and are not intended, in whole or in part, to form part of the prior art base or to represent or indicate the common general knowledge in the field to which the present invention pertains prior to the priority date of each claim of this application.

[0068] One example of the present application relates to a fusion protein comprising a peptide antigen and a human thioredoxin (Trx) protein linked to the N-terminus, C-terminus, or both, of the peptide antigen, in which all three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein have been substituted with non-cysteine ​​residues.

[0069] As used herein, when a protein (which can be used interchangeably with "polypeptide") or a polynucleotide (which can be used interchangeably with "gene") "comprises a specific amino acid sequence or nucleic acid sequence" or "consists of, consists of, or is represented as a specific nucleic acid sequence or amino acid sequence," this can mean that the polypeptide or polynucleotide essentially contains the specific nucleic acid sequence or amino acid sequence, and can be interpreted as including a "substantially equivalent sequence" in which mutations (deletions, substitutions, modifications, and / or additions) have been made to the specific amino acid sequence or nucleic acid sequence to the extent that the original function and / or intended function of the polypeptide or polynucleotide is maintained (or as not excluding such mutations).

[0070] In one example, a polypeptide or polynucleotide "comprising a specific amino acid sequence or nucleic acid sequence" or "consisting of or expressed as a specific amino acid sequence or nucleic acid sequence" can mean that the polypeptide or polynucleotide (i) essentially contains the specific amino acid sequence or nucleic acid sequence, or (ii) consists of or essentially contains an amino acid sequence or nucleic acid sequence that has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, or 99.9% or more identity to the specific amino acid sequence or nucleic acid sequence, and maintains its original and / or intended function. As used herein, the expression "comprises or is expressed by a specific amino acid sequence" for a polypeptide, peptide antigen, human thioredoxin protein, additional carrier protein, or fusion protein may mean both cases where the amino acid sequence is essentially contained, and cases where meaningless mutations (e.g., substitution, deletion, and / or addition of amino acid residues) have been introduced into the amino acid sequence that do not affect the original activity and / or the desired activity (e.g., immunogenicity, improved purification efficiency, disease prevention or treatment activity, etc.).

[0071] As used herein, the term "identity" refers to the degree of identity with a given nucleic acid or amino acid sequence, and may be expressed as a percentage (%). In the case of nucleic acid sequences, homology can be determined using, for example, the literature-based algorithm BLAST (see Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90, 5873, 1993) or Pearson's FASTA (see Methods Enzymol., 183, 63, 1990). Based on the BLAST algorithm, programs called BLASTN and BLASTX have been developed (see http: / / www.ncbi.nlm.nih.gov).

[0072] The term "antigen" refers to any molecule that induces an immune response in a subject. By way of example, an antigen can refer to any molecule that contains an epitope that can be recognized by a T cell receptor and / or a B cell receptor and stimulate an immune response, particularly a T cell response and / or a B cell response, in a subject.

[0073] The term "epitope" thus refers to the region of an antigen that interacts with a T-cell receptor and / or a B-cell receptor.

[0074] In a preferred example, the peptide antigen of the present application may contain a T cell epitope. For example, the peptide antigen of the present application may contain an MHC class I and / or II binding motif. As a specific example, the peptide antigen of the present application may contain a CD4+ T cell epitope, which is a peptide sequence that contains an MHC class II binding motif and can be presented on the surface of an antigen-presenting cell by an MHC class II molecule. The peptide antigen of the present application may also contain a CD8+ T cell epitope, which is a peptide sequence that contains an MHC class I binding motif and can be presented on the cell surface by an MHC class I molecule. The peptide antigen of the present application may further contain both a CD4+ T cell epitope and a CD8+ T cell epitope.

[0075] The term "MHC (major histocompatibility complex)" refers to proteins that provide antigen fragments to immune cells so they 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 found 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, playing an important role in organ transplant rejection and the destruction of infected cells. MHC class II molecules interact with CD4+ accessory T cells, playing an important role in recognizing non-self antigens and inducing cell-mediated immunity.

[0076] Generally, in adaptive immune responses, when an antigen enters the body, antigen-presenting cells ingest it and break it down into short peptide fragments, which can then bind to MHC class I or MHC class II molecules within the cell and be transported to the cell surface. When antigen peptides bind to MHC class I or MHC class II molecules and are presented on the cell surface of antigen-presenting cells, T cells recognize and activate them via T cell receptors (TCRs), initiating an immune response. In this respect, the peptide antigens of the present application can correspond to T cell epitopes.

[0077] In other preferred embodiments, the peptide antigen may be, but is not limited to, a peptide derived from a tumor antigen, such as a tumor-associated antigen (TAA), tumor-specific antigen (TSA), or tumor-derived neoantigen; an antigen of an infectious source, such as an antigen derived from a virus, bacteria, parasite, or fungus; an autoantigen known or suspected to induce autoimmunity; or an allergy-inducing antigen (allergen) known or suspected to induce allergy.

[0078] For example, peptide antigens can include portions of tumor antigens, infectious antigens, autoantigens, or allergy-inducing antigens that are in silico predicted or known to bind to MHC class I or MHC class II molecules, including, but not limited to, CD8+ or CD4+ T cell epitopes.

[0079] Peptide antigens can include natural or non-natural amino acid sequences, amino acids with post-translational modifications, or peptidomimetics that can induce an immune response, e.g., a T cell or B cell response, in a subject. For example, peptide antigens can be about 5 to about 100 amino acids, or about 5 to about 50 amino acids. For example, peptide antigens can be about 7 to 35 amino acids, such as, 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.

[0080] The term "tumor antigen" can be used interchangeably with "cancer antigen" and refers to a molecule expressed on a tumor (cancer) that elicits an immune response. Such an immune response can involve antibody production, activation of specific immunologically competent cells, or both.

[0081] Tumor antigens can be derived from tumor-bearing organisms, dead or inactivated whole tumor cells, or lysates, and include any antigen derived from a tumor. Lysates are substances resulting from the application of processes that cause the division of normal structures in cells. Tumor antigens also include any protein or other substance with antigenic properties that is contained in tumor cells and expressed differently from normal cells.

[0082] For example, tumor antigens can include tumor-associated antigens (TAA), tumor-specific antigens (TSA), or tumor-derived neoantigens.

[0083] Tumor-associated antigens (TAAs) are antigens that are expressed more in cancer cells than in normal cells, or at a different differentiation stage than normal cells, and are also present in trace amounts in normal cells. Therefore, immune responses using these TAAs are likely to be neutralized by self-tolerance, an immunosuppressive mechanism that prevents damage to self-cells, or conversely, they may lead to autoimmunity and attack of unwanted organs.

[0084] Examples of tumor-associated antigens (TAA) include EGFR, Ova, Melan-A, Prostate Specific Membrane Antigen (PSMA), Survivin, MAGE-A, adenosine deaminase-binding protein (ADAbp), cyclophilin b, gp100, Colorectal associated antigen (CRC)-C017-1 A / GA733, carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, Prostate Specific Antigen (PSA), and the like.Antigen), PSA-1, PSA-2, PSA-3, MAGE (melanoma antigen E) [e.g., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8 , MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4 (MAGE-B4), MAGE-C1, MAGE-C2, M AGE-C3, MAGE-C4, MAGE-C5, etc.], GAGE ​​(G antigen) [e.g., GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9, etc.], BAGE (B melanoma antigen), RAGE (kidney tumor antigen), LAGE (L antigen), NAG, GnT-V, MUM-1, CDK4, p53, tyrosinase, Muc1 (mucin 1), HER2 / neu, p21ras, RCAS1, α-fetoprotein In, 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, g including, but not limited to, p70, SCP-1, c-myc, 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.

[0085] Tumor-specific antigens (TSAs) are antigens that are present specifically in cancer cells. In particular, as tumors grow in cancer patients, cancer cell-specific genetic mutations occur, generating new antigen epitopes that can stimulate T cells; these are called neoantigens. In other words, neoantigens contain cancer cell-specific genetic mutations and, unlike tumor-shared antigens that are expressed in trace amounts in normal cells, are selectively expressed only in cancer cells. Therefore, they are recognized by the immune system as non-self foreign epitopes, eliciting strong anti-cancer immune activity.

[0086] When peptides generated by mutated DNA are presented on the MHC of cells, they are recognized by T cell receptors (TCRs). However, because mutations do not occur in normal cells or tissues, neoantigen-specific T cells are free from the problems of self-tolerance and autoimmunity. Because of these advantages, neoantigens are considered ideal targets for T cell-based cancer immunotherapy.

[0087] The causes of neoantigens include, but are not limited to, frame-shift deletions or insertions, which result in the disruption of the genetic code due to the addition or deletion of one or more nucleotides in DNA; point mutations, which result in the substitution of one nucleotide for another; missense mutations, splice-site mutations, read-through mutations, and gene-fusion mutations.

[0088] Neoantigens are predicted by analyzing specific cancer cell genomes from individual cancer patients. For example, cancer cells are extracted from the patient's tumor, their DNA is sequenced, and then compared with the sequence of normal cells to identify mutated regions. Neoantigens that stimulate T cells can be identified from the various mutated regions. This can be done using, but is not limited to, next-generation sequencing (NGS), whole-exome sequencing (WES), or RNA sequencing, big data processing techniques such as MHC binding prediction computer programs, or artificial intelligence (AI) for neoantigen prediction. Because mutations are not shared between patients, neoantigens can be developed into personalized cancer vaccines.

[0089] In one embodiment, the tumor-derived neoantigen of the present invention may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:5.

[0090] The infectious antigen may be derived from a virus, bacteria, parasite, or fungus.

[0091] For example, antigens derived from viruses include chickenpox virus, smallpox virus, Ebola virus, Marburg virus, dengue virus, influenza virus, parainfluenza virus, respiratory cytoplasmic virus, measles virus, human immunodeficiency virus, human papillomavirus, varicella-zoster virus, and the like. virus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovirus, rotavirus, rhinovirus, adenovirus, papillomavirus, parvovirus, picornavirus, poliovirus, mumps-causing virus, rabies-causing virus, reovirus, rubella virus, togavirus, orthomyxovirus, retrovirus, hepadnavirus, coxsackievirus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus The antigen may be, but is not limited to, an antigen derived from Hepatitis virus, Rift Valley fever virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis D virus, or Hepatitis E virus.

[0092] 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, and species, Enterococcus species, Ehrlichia 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 speciesspecies, Mycoplasma pneumoniae, Neisseria species, Neisseria meningitidis, Neisseria gonorrhoeae, Pneumococcus species, Pseudomonas species, Pseudomonas aeruginosa, Salmonella species, Salmonella typhi, Salmonella enterica, Rickettsia species, Rickettsia rickettsii, Rickettsia typhi, Shigella species, Staphylococcus species, Staphylococcus aureus The antigen may be, but is not limited to, an antigen derived from S. aureus, Streptococcus species, Streptococcus pneumoniae, Streptococcus pyrogenes, Streptococcus mutans, Treponema species, Treponema pallidum, Vibrio species, Vibrio cholerae, or Yersinia pestis.

[0093] The fungal-derived antigen may be, but is not limited to, an antigen derived from a fungus selected from Candida species, Cryptococcus species, Coccidioides species, Histoplasma species, and Aspergillus species.

[0094] The parasite-derived antigen may be, but is not limited to, an antigen from Plasmodium, Trypanosome, Schistosome or Leishmania.

[0095] Thioredoxin (Trx) is a low molecular weight redox protein of approximately 11–12 kDa found in both prokaryotic and eukaryotic cells. It is reversibly reduced to Trx-(SH)2 by NADPH and thioredoxin reductase. Human thioredoxin (hTrx) consists of 105 amino acids (SEQ ID NO: 10) and has five cysteine ​​(Cys) residues at amino acid positions 32, 35, 62, 69, and 73, whereas Escherichia coli thioredoxin (TrxA) consists of 109 amino acids (SEQ ID NO: 11) and has two cysteine ​​(Cys) residues at amino acid positions 32 and 35. Human thioredoxin shares 27% amino acid identity with E. coli thioredoxin. Thioredoxin is known to act as an electron donor for ribonucleotide reductase, methionine sulfoxide reductase, 3'-phosphoadenosine 5'-phosphosulfate reductase, etc., to facilitate the refolding of proteins containing -SS- bonds, and to function as an antioxidant.

[0096] The human thioredoxin protein can be linked to the N-terminus, C-terminus, or both of the peptide antigen, and can be linked to each component of the peptide antigen of the present invention, an additional carrier protein, or an affinity tag via a linker.

[0097] In the present application, the human thioredoxin protein can be linked to the N-terminus, C-terminus, or both, of a peptide antigen to improve recombinant expression of the peptide antigen, increase purification efficiency, improve physical properties, stabilize the peptide, increase immunogenicity, and / or function as a carrier protein to improve delivery to immune response cells. In particular, the present application is characterized in that the human thioredoxin protein is a mutant in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0098] Specifically, the mutants may include, but are not limited to, mutants in which the cysteine ​​residues at amino acid positions 32, 35, and 62; 32, 35, and 69; 32, 35, and 73; 32, 62, and 69; 32, 62, and 73; 32, 69, and 73; 35, 62, and 69; 35, 62, and 73; 35, 69, and 73; 62, 69, and 73; 32, 35, 62, and 69; 32, 35, 62, and 73; 32, 35, 69, and 73; 32, 62, 69, and 73; 35, 62, 69, and 73; or 32, 35, 62, 69, and 73 in the amino acid sequence of human thioredoxin protein are substituted with non-cysteine ​​residues. More preferred examples include, but are not limited to, mutants in which the three cysteine ​​(Cys) residues at amino acid positions 62, 69, and 73 in the amino acid sequence of human thioredoxin protein are all substituted with non-cysteine ​​residues, or the five cysteine ​​(Cys) residues at amino acid positions 32, 35, 62, 69, and 73 in the amino acid sequence of human thioredoxin protein are all substituted with non-cysteine ​​residues.

[0099] The human thioredoxin protein variant, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are replaced with non-cysteine ​​residues, maintains its function as a carrier protein while reducing dimerization due to disulfide bonds. Therefore, a fusion protein prepared by linking the human thioredoxin protein variant to a peptide antigen exhibits reduced multimer formation during purification compared to a fusion protein linked to wild-type human thioredoxin protein, improving purification efficiency and increasing structural stability, ultimately resulting in efficient delivery to immune cells and / or enhanced immunogenicity.

[0100] The cysteine ​​residue may be substituted with another amino acid residue that is not involved in disulfide bond formation. Therefore, it may be substituted with an amino acid other than cysteine ​​(i.e., a non-cysteine ​​residue). More preferably, the cysteine ​​residue may be substituted with an alternative residue that preserves the overall structure, such as serine or alanine, but is not limited thereto. For example, the human thioredoxin protein variant may consist of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 9, but is not limited thereto.

[0101] Other examples of the fusion proteins of the present application may or may not additionally contain a carrier protein other than human thioredoxin at the N-terminus or C-terminus or both of the fusion protein.

[0102] The term "carrier protein" refers to a protein that is linked to a peptide antigen to improve the recombinant expression of the peptide antigen (e.g., higher expression rate, more consistent expression rate, etc.), increase purification efficiency, improve physical properties, stabilize, increase immunogenicity, and / or improve delivery to immune-responsive cells.

[0103] The additional carrier protein may be linked to the N-terminus, C-terminus, or both of the fusion protein, in one or more units. For example, one, two, three, or more additional carrier proteins may be linked to the N-terminus, C-terminus, or both of the fusion protein.

[0104] In one embodiment, when the human thioredoxin protein is linked to the N-terminus of the peptide antigen, one or more of the additional carrier proteins may be linked to one or more positions selected from the group consisting of: N-terminus of human thioredoxin protein; Between the C-terminus of the human thioredoxin protein and the N-terminus of the peptide antigen; and C-terminus of peptide antigen.

[0105] In one embodiment, when the human thioredoxin protein is linked to the C-terminus of the peptide antigen, one or more of the additional carrier proteins may be linked to one or more positions selected from the group consisting of: the N-terminus of the peptide antigen; Between the C-terminus of the peptide antigen and the N-terminus of the human thioredoxin protein; and C-terminus of human thioredoxin protein.

[0106] In one embodiment, when the human thioredoxin protein is linked to the N-terminus and C-terminus of the peptide antigen, one or more of the additional carrier proteins may be linked to one or more positions selected from the group consisting of: N-terminus of the first human thioredoxin protein; between the C-terminus of the first human thioredoxin protein and the N-terminus of the peptide antigen; between the C-terminus of the peptide antigen and the N-terminus of a second human thioredoxin protein; and the C-terminus of the peptide antigen, Here, the first human thioredoxin protein is a human thioredoxin protein linked to or in the N-terminal direction of the peptide antigen, and the second human thioredoxin protein is a human thioredoxin protein linked to or in the C-terminal direction of the peptide antigen.

[0107] Non-limiting examples of carrier proteins additionally contained in the fusion protein of the present invention include nucleoside diphosphate kinase B (NDPK), cystatin-A (CSTA) [e.g., human CSTA (hCSTA) or mouse CSTA (mCSTA)], thioredoxin (Trx) [e.g., mammalian or bacterial Trx], 50S ribosomal protein L7Ae (RPL7Am), small archaeal modifier protein 2 (Samp2a), tenascin (TE) [e.g., TE1, TE2, TE3.1, TE3.2, TE4], Thermotoga maritima Cupin_3 domain-containing protein (TM1112), thioredoxin1 (TrxA), Thermosipho africanus thioredoxin (TTrx), and peripheral subunit-binding domain (PSBD). The polypeptide may be, but is not limited to, a polypeptide having a polypeptide domain (e.g., a polypeptide domain), or one or more of a fragment, a variant, a fragment of a variant, or a variant of a fragment thereof. Furthermore, the polypeptide may be codon-optimized for a host cell, or may include a partial sequence mutation for the purpose of improving function or structural stability, or may be appropriately modified to include an initiating methionine. For example, the modification may be introduced by deleting, substituting, or adding a partial sequence for the purposes of removing residues that may be involved in dimerization or redox reactions, inhibiting nucleic acid binding, inhibiting binding to desampylase (UniProt: Q8U1Y4), reducing excessive binding affinity for human serum albumin, facilitating quantification, or suppressing deamidation, but is not limited to these.

[0108] In a preferred embodiment, the N-terminal carrier protein can be selected to improve antigen expression, taking into account the difficulty of recombinantly expressing tumor antigens depending on their sequence. For example, but not limited to, the N-terminal carrier protein can be selected to express the antigen at a certain level or higher, consistently express it, and / or stabilize it in a host expression system.

[0109] In addition, in a preferred embodiment, a carrier protein located at the C-terminus can be selected to improve the efficiency of antigen purification, taking into consideration the difficulty of purifying the antigen (or removing impurities) depending on the sequence of the tumor antigen. For example, a C-terminal carrier protein can be selected to stabilize the C-terminal structure of the fusion protein in a conventional purification system to improve purification efficiency, but is not limited thereto.

[0110] Carrier proteins can also include, but are not limited to, pan HLA DR binding epitopes (PADRE), tetanus toxin epitope (TT) or diphtheria toxoid or recombinantly produced, genetically detoxified variants thereof, staphylococcal exotoxin or toxoid, or Pseudomonas aeruginosa exotoxin A or derivatives thereof.

[0111] The additional carrier protein can be linked to each component, such as the peptide antigen of the present invention, human thioredoxin protein, other additional carrier protein, or affinity tag, via a linker.

[0112] Other examples of fusion proteins of the present application may or may not additionally contain an affinity tag at the N-terminus, C-terminus, or both, of the fusion protein.

[0113] The term "affinity tag" refers to a substance that provides a site for attachment of the fusion protein to a specific substrate in vitro, i.e., when the fusion protein is experimentally purified. The affinity tag is a part of the fusion protein and should not induce immunogenicity or affect the activity of the fusion protein.

[0114] The fusion protein of the present invention can be purified regardless of the characteristics of the antigen sequence by including an affinity tag, and the fusion protein can be easily purified by affinity purification, allowing for ultra-high speed protein production.

[0115] The affinity tag may be, but is not limited to, polyhistidine (His), polyphenylalanine, polyalanine, streptavidin, maltose-binding protein (MBP), intein, thioredoxin (Trx), protein A, NusA (N utilization substance A), beta-galactosidase, or glutathione-S-transferase (GST). For example, in the case of a polyhistidine tag, a peptide with a sequence of 5 to 8 consecutive histidines (e.g., His-His-His-His-His-His) is used. Because histidine tags have affinity for divalent metal ions, fusion proteins can be purified by affinity chromatography using nickel-immobilized beads, cobalt-immobilized beads, etc.

[0116] In one embodiment, the affinity tag may be 6-His.

[0117] Affinity tags may or may not be cleaved under extracellular or intracellular conditions, and even if they are not cleaved, they are not only safe in vivo but also do not induce structural changes in the fusion protein and do not substantially affect the function of the fusion protein in cells.

[0118] In a preferred embodiment, the affinity tag can be attached to the C-terminus, allowing the antigen to be purified regardless of the sequence characteristics of the antigen, but is not limited thereto.

[0119] The affinity tag can be linked to each component, such as the peptide antigen of the present invention, the human thioredoxin protein, or an additionally contained carrier protein, via a linker.

[0120] Another example of the fusion protein of the present application may be one in which a linker exists between each component such as a peptide antigen, a human thioredoxin protein, a carrier protein, and an affinity tag.

[0121] The term "linker" refers to a molecule or atomic group that links, couples, or binds two or more components together. In the present application, each component of a fusion protein, for example, a peptide antigen and a thioredoxin protein, a peptide antigen and a carrier protein, a thioredoxin protein and a carrier protein, or a carrier protein and an affinity tag, can be linked or bound together by any suitable means. The linker can have additional functions, such as increasing or decreasing aqueous solubility, increasing the distance between the two components to be linked to provide flexibility, or increasing stability, but preferably does not induce immunogenicity or affect the activity of the fusion protein.

[0122] The linker may be a peptide linker and may be, but is not limited to, 1 to 10 or 2 to 10 amino acids in length, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (e.g., 20 or less) amino acids in length, for example, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. As an example, the peptide linker may be composed of neutral amino acids (more specifically, Gly, Ser, Ala, Thr, or a combination of these four amino acids). For example, it may be (GS)n, (GS)n, (GS)n, (GS), Gn, LE, SSGG, or GGGGSGGGGG (wherein G is Gly, S is Ser, L is Leu, E is Glu, and n is an integer from 1 to 10, for example, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10), for example, but not limited to, GS, GGGGS, LE, SSGG, GG, GGGGG, or GGGGSGGGGG.

[0123] In one embodiment, the linker can comprise a degradable peptide sequence that is cleavable by an intracellular enzyme, e.g., a protease, and cleavage of the linker can result in release of any component attached to the linker, e.g., an affinity tag, a thioredoxin protein, or a carrier protein.

[0124] In one specific example, the linker may comprise one or more amino acid sequences selected from the group consisting of SEQ ID NO:51 to SEQ ID NO:52.

[0125] The term "linked" means that components are directly or indirectly linked together. Each component can be covalently or non-covalently linked. "Linked" can also mean that each component maintains a chemical or physical bond after contact with cells, such as antigen-presenting cells or immune cells, and immunization. For example, the components can be linked so that they do not freely disperse from each other until they are contacted with antigen-presenting cells and immune cells. For example, two components can be covalently linked to each other to prevent the two components from dispersing or diffusing separately.

[0126] For example, the size of the fusion protein of the present application is not particularly limited, but a small size may be advantageous, for example, to facilitate the removal of impurities during the purification process. In this aspect, the overall size of the fusion protein may be, but is not limited to, 500 kDa or less, 400 kDa or less, 300 kDa or less, 200 kDa or less, 100 kDa or less, 90 kDa or less, 80 kDa or less, 70 kDa or less, 60 kDa or less, or 50 kDa or less, for example, 10 kDa to 100 kDa, 10 kDa to 90 kDa, 10 kDa to 80 kDa, 10 kDa to 70 kDa, 10 kDa to 60 kDa, 10 kDa to 50 kDa, 10 kDa to 40 kDa, or 10 kDa to 30 kDa.

[0127] As an example, the fusion protein of the present application may comprise an amino acid sequence selected from the group consisting of SEQ ID NO:14 to SEQ ID NO:50.

[0128] Another example of the present application relates to a thioredoxin mutant polypeptide having the amino acid sequence of SEQ ID NO: 7. This corresponds to a mutant polypeptide according to an embodiment of the present application in which three cysteine ​​(Cys) residues at amino acid positions 62, 69, and 73 of human thioredoxin protein are substituted with serine. Another example of the present application relates to a thioredoxin mutant polypeptide having the amino acid sequence of SEQ ID NO: 9. This corresponds to a mutant polypeptide according to an embodiment of the present application in which all five cysteine ​​(Cys) residues at amino acid positions 32, 35, 62, 69, and 73 of human thioredoxin protein are substituted with serine. [Table 1]

[0129] Another example of the present application relates to a nucleic acid molecule encoding the fusion protein. The terms "nucleic acid molecule," "nucleic acid," or "nucleic acid sequence" refer to a polymer of deoxyribonucleotides or ribonucleotides that exists in single-stranded or double-stranded form. The nucleic acid molecule encompasses RNA genomic sequences, cDNA, and RNA sequences transcribed therefrom, and includes analogs of naturally occurring nucleic acids, unless otherwise specified.

[0130] The nucleic acid molecule includes not only a nucleic acid sequence encoding the amino acid sequence of the fusion protein but also a complementary sequence to that sequence. The complementary sequence includes not only a completely complementary sequence but also a substantially complementary sequence, which means a sequence that can hybridize to, for example, a nucleic acid sequence encoding the amino acid sequence of the fusion protein under stringent conditions known in the art.

[0131] The nucleic acid molecule may be an isolated nucleic acid molecule.

[0132] The fusion protein of the present application can be preferably, but not limited to, expressed and purified by recombinant methods. Therefore, the present invention further provides an expression vector containing a nucleic acid molecule encoding the fusion protein, and a cell transformed therewith, for expression and purification of the fusion protein.

[0133] Another example of the present application relates to an expression vector comprising said nucleic acid molecule.

[0134] The term "expression vector" refers to a nucleic acid construct in which a gene insert encoding a protein of interest is operably linked for expression. In one embodiment, the expression vector may be linear or circular, single- or double-stranded DNA, cDNA, RNA, etc., encoding two or more proteins of interest. An expression vector can be part of a vector that can be used to transform, transfect, or transfect a host, but is not limited thereto, and may itself be transcribed and / or translated in vitro.

[0135] The term "operably linked" refers to a binding between nucleic acid sequences that is functionally related. For example, a coding sequence (e.g., a sequence encoding a protein of interest) can be operably linked to appropriate regulatory elements to enable its replication, transcription, and / or translation. For example, a coding sequence is operably linked to a promoter if the promoter is capable of driving the transcription of the coding sequence. Regulatory elements need not be contiguous with the coding sequence, so long as they function properly. For example, intervening sequences that are not translated, but are transcribed, can be present between the promoter sequence and the coding sequence, and the promoter sequence would still be considered "operably linked" to the coding sequence.

[0136] The components within the expression vector must be operably linked to each other, and linking of these component sequences may be accomplished by ligation at convenient restriction enzyme sites or, if such sites do not exist, by the use of synthetic oligonucleotide adapters or linkers by conventional methods.

[0137] An expression vector can contain transcriptional and coding expression control sequences that allow the gene to be expressed in a selected host. Expression control sequences can include a promoter for transcription, an optional operator sequence for regulating the transcription, and / or a sequence for regulating the termination of transcription and coding. Initiation and termination codons are generally considered part of the nucleic acid sequence encoding the protein of interest, and must be functional in an individual when the gene construct is administered, and must be in frame with the coding sequence.

[0138] For example, a promoter refers to a DNA base sequence site to which a transcriptional regulatory factor binds, and for the purposes of the present invention, a promoter capable of inducing strong and stable gene expression can be used to increase the gene expression rate.

[0139] The promoter may be constitutive or inducible. Examples of promoters include, but are not limited to, the early and late promoters of adenovirus, simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoter, the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV) promoter, Epstein-Barr virus (EBV) promoter, Rous sarcoma virus (RSV) promoter, RNA polymerase ± promoter, T3 and T7 promoters, and the major operator and promoter regions of phage lambda.

[0140] Additionally, expression vectors can include adaptors or linkers, enhancers, selection markers (e.g., antibiotic resistance markers), replicative units, polyA sequences, purification tags, or other constitutive and inducible sequences known to regulate gene expression in prokaryotic or eukaryotic cells or viruses, as well as various combinations thereof, as appropriate.

[0141] The expression vector may be in various forms such as a plasmid, a virus vector, a bacteriophage vector, or a cosmid vector.

[0142] Another example of the present application relates to cells transformed with said expression vectors.

[0143] In one embodiment, the transformed cell may be an isolated transformed cell.

[0144] In the present invention, the transformed cells can be any host cells known in the art that can stably and continuously clone or express the expression vector. Prokaryotic cells include E. coli, e.g., E. coli JM109, E. coli BL21, E. coli RR1, E. coli LE392, E. coli B, E. coli X1776, E. coli W3110, Bacillus strains such as Bacillus subtilis and Bacillus thuringiensis, and enterobacteriaceae and strains such as Salmonella typhimurium, Serratia marcescens, and various Pseudomonas species. When transforming eukaryotic cells, host cells include yeast (Saccharomyce cerevisiae), insect cells, plant cells, and animal cells, e.g., CHO cell lines (Chinese hamster ovary (CHO) cell lines). ovary), W138, BHK, COS-7, 293, HepG2, 3T3, RIN and MDCK cell lines can be used, but are not limited to these.

[0145] As is known in the art, expression vectors can be introduced into cells using suitable standard techniques, including, but not limited to, electroporation, electroinjection, microinjection, calcium phosphate co-precipitation, calcium chloride / rubidium chloride, retroviral infection, DEAE-dextran, cationic liposomes, polyethylene glycol-mediated uptake, and gene guns. In this case, circular constructs can be cleaved with appropriate restriction enzymes to introduce them in a linear form.

[0146] The transformed cells can be easily selected by a method well known in the art using the phenotype expressed by the selection marker. For example, when the selection marker is a specific antibiotic resistance gene, the transformants can be easily selected by culturing them in a medium containing the antibiotic.

[0147] The transformed cells can be cultured by various methods known in the art. For example, the transformed cells can be inoculated into a culture medium and cultured. When the cell density reaches a certain level, IPTG is added to the medium to induce protein expression, and the cells can be cultured to obtain the protein secreted into the cells or the medium.

[0148] Proteins secreted into cells or the medium can be purified by various purification methods known in the art, preferably by affinity chromatography using an affinity tag. For example, if the fusion protein is fused to GST, the desired protein can be easily purified using a glutathione-bound resin column, and if it is fused to His, the desired protein can be easily purified using IMAC (immobilized metal affinity chromatography).

[0149] Another example of the present application provides a method for producing a fusion protein, comprising expressing the nucleic acid molecule or the expression vector in a cell.

[0150] In one embodiment, the fusion protein produced by the above production method may have improved purification efficiency, enhanced immunogenicity, and / or enhanced disease prevention or treatment efficacy compared to a fusion protein linked to a wild-type human thioredoxin protein.

[0151] Fusion proteins can be produced by expressing a nucleic acid molecule encoding a fusion protein provided herein in a suitable host cell as described above.

[0152] In one embodiment, the method for producing the fusion protein may include culturing cells containing the nucleic acid molecule or the expression vector under conditions suitable for expression. The culturing may be performed under standard culture conditions as described above. The method may further include, after the culturing step, isolating and / or purifying the fusion protein from the culture.

[0153] Another example of the present application relates to a composition for improving the purification efficiency of a peptide antigen, comprising the fusion protein, nucleic acid molecule, expression vector, or cell. The fusion protein of the present application contains a human thioredoxin protein mutant with a modified cysteine ​​residue, which can reduce the formation of multimers during purification of the fusion protein with the peptide antigen, thereby improving purification efficiency and conferring structural stability, and ultimately enabling the fusion protein to be efficiently delivered to immune cells and / or increasing immunogenicity.

[0154] Another example of the present application relates to a vaccine or immunogenic composition comprising said fusion protein, nucleic acid molecule, expression vector, or cell.

[0155] Yet another example of the present application relates to a composition for preventing or treating cancer, an infectious disease, an autoimmune disease, or an allergic disease, comprising the fusion protein, the nucleic acid molecule, the expression vector, or the cell.

[0156] The term "immunogenic composition" refers to any composition capable of inducing an immune response. The term "vaccine" refers to an immunogenic composition that induces an immune response to reduce or prevent the risk of disease or infection, or to ameliorate or treat an existing disease or infection.

[0157] These compositions may contain the fusion protein of the present application and be in a dosage form that can be administered to a subject to induce an immune response. Therefore, the compositions of the present invention can be conveniently used to prevent, ameliorate, or treat diseases. After being introduced into a subject or host, the compositions can induce an immune response, preferably a T-cell-mediated immune response.

[0158] The vaccine of the present invention may be an anti-cancer vaccine containing tumor antigens, e.g., tumor antigen sequences obtained from patient tumor analysis, particularly neoantigen sequences, which can maximize the stimulation of proliferation and activation of anti-cancer T cells tailored to the patient, thereby enhancing the anti-cancer immune effect.

[0159] The vaccine compositions of the present application may also include additional adjuvants that enhance the effectiveness of the vaccine.Suitable immunopotentiators 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), such as (a) 5% squalene, 0.5% Tween 80, and 0.5% Span 85 formulated into submicron particles (optionally, but not necessarily, containing various amounts of N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1',2'- (b) MF59 (WO 90 / 14837) containing dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), (c) SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic®-blocked polymer and N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP) that has been microfluidized to submicron particles or shaken to produce a large particle size emulsion, and (d) monophosphoryl lipid A (MP (3) a saponin immunopotentiator; (4) Freund's complete immunopotentiator (CFA) and incomplete immunopotentiator (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., cancer (6) bacterial ADP-ribosylating toxins, such as cholera toxin (CT), pertussis toxin (PT), or heat-labile toxin (LT) of Escherichia coli (E. coli), particularly detoxified mutants of LT-R72, CT-S109, and PT-K9 / G129 (WO93 / 13302 and WO92 / 19265); and (7) other substances that act as immunostimulants to enhance vaccine efficacy.

[0160] The compositions of the present application may, if necessary, additionally contain pharmaceutically acceptable carriers, diluents and / or excipients in amounts normally used.

[0161] The pharmaceutically acceptable carriers 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, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition to the above ingredients, the composition may additionally contain lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0162] The fusion protein or a composition containing the same as an active ingredient may be prepared in unit dose form or in multi-dose containers by formulating it with pharmaceutically acceptable carriers and / or excipients by a method that can be easily carried out by a person skilled in the art.

[0163] For example, vaccine compositions can contain a conventional saline or buffered aqueous medium in which the vaccine is suspended or dissolved. For example, conventional diluents, such as water, saline, glycerol, ethanol, etc., can be included, and auxiliary substances, such as wetting agents, emulsifying agents, pH buffering agents, and the like, can also be present in the composition.

[0164] Suitable forms for injection 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 the contamination of microorganisms such as bacteria and fungi. Microbial contamination can be prevented using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. In many cases, it is preferable to include an isotonic agent, for example, sugar or sodium chloride. Prolonged absorption of injectable compositions can be achieved by using agents that delay absorption, for example, aluminum monostearate or gelatin, in the composition.

[0165] Sterile injectable solutions are prepared by combining the required amount of fusion protein and various other ingredients listed above in the aforementioned solvent, and sterilizing the remaining ingredients, excluding the fusion protein and / or heat-sensitive immunoadjuvant cytokines, if necessary, using a buffer solution such as PBS, through filtration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing a basic dispersion medium and the other required ingredients listed above. For sterile powders for the preparation of sterile injectable solutions, preferred methods are vacuum drying and freeze-drying. These methods yield powders of the active ingredient and any desired ingredients from the sterile-filtered solution described above.

[0166] The patient to whom the fusion protein or a composition containing the same as an active ingredient is administered may be a mammal, such as a human, a primate including a monkey, or a rodent including a rat or a mouse, but is not limited thereto.

[0167] Yet another aspect of the present invention relates to a method for generating, inducing and / or enhancing 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 a vaccine composition or fusion protein as described above.

[0168] The term "immune response" refers to a change in the activity of cells of the immune system, e.g., B cells, T cells, or monocytes, as a result of direct or indirect cellular or cytokine-mediated stimulation. The immune response may be a specific (T cell and / or B cell) and / or non-specific immune response.

[0169] Without limiting the operation of the present invention in any way, delivery of fusion proteins according to the present invention is particularly useful for inducing immune responses, particularly T cell responses, e.g., CD4+ T cell responses or CD8+ T cell responses to antigens. CD4+ and CD8+ T cell responses can occur together with or independently of humoral responses or other specific or nonspecific immune responses.

[0170] Yet another aspect of the present invention relates to the use of the fusion proteins of the present invention in connection with the treatment and / or prevention of disease states. Examples of diseases that can be treated by the methods of the present invention include various cancers, infectious diseases, autoimmune diseases, and allergic diseases.

[0171] For example, the cancer may be a solid cancer or a blood cancer, and non-limiting examples include breast cancer, lung cancer, prostate cancer, ovarian cancer, brain cancer, liver cancer, cervical cancer, endometrial cancer, uterine cancer, colon cancer, colorectal cancer, rectal cancer, kidney cancer, nephroblastoma, skin cancer, oral squamous cell carcinoma, epidermal 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, and the like. The cancer may be cancer, testicular cancer, thyroid cancer, thyroid follicular cancer, 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's sarcoma, leiomyosarcoma, mesenchymal chondrosarcoma, lymphosarcoma, fibrosarcoma, rhabdomyosarcoma, teratocarcinoma, neuroblastoma, medulloblastoma, neuroglioma, benign tumor of the skin, or leukemia. The lung cancer may be, for example, small cell lung carcinoma (SCLC) or non-small cell lung carcinoma (NSCLC). The leukemia may be, for example, acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), or chronic lymphocytic leukemia (CLL). The treated subject may be undergoing secondary anti-hyperproliferative therapy. For example, the secondary anti-hyperproliferative therapy can be chemotherapy, radiation therapy, immunotherapy, phototherapy, cryotherapy, toxin therapy, hormone therapy, or surgery.

[0172] Thus, yet another aspect of the present invention provides a method for preventing or treating a disease, e.g., cancer, an infectious disease, an autoimmune disease, or an allergic disease, by enhancing an immune response using the composition, wherein administration of the composition generates, induces, or promotes an immune response that inhibits, halts, delays, or prevents the onset or progression of the disease state.

[0173] The direct delivery of the composition can generally be systemic, subcutaneous, intradermal, intraperitoneal, intravascular (intravenous), intramuscular, or local delivery, or delivered to tissue gaps. The composition can also be administered to a lesion. The administration regimen can be a single dose or a multiple dose schedule.

[0174] The term "effective amount" means an amount sufficient to achieve a desired result when administered to an individual, including a human, e.g., an amount effective to treat or prevent cancer. The effective amount can vary depending on factors such as the disease state, age, sex, and weight of the individual. Dosage or treatment regimen can be adjusted to provide the optimal therapeutic response, as will be appreciated by those skilled in the art.

[0175] A therapeutic regimen for an individual using a therapeutically effective amount may consist of a single administration or, as another example, may include a series of applications. The duration of the treatment period will depend on various factors, such as the severity of the disease, the individual's age, the concentration of the vaccine, the patient's responsiveness to the vaccine, or a combination thereof. It will also be understood that the effective dosage of the vaccine used for treatment may increase or decrease over the course of an individual treatment regimen. Dosage variations may occur and may be determined by standard diagnostic assays known in the art. The vaccines of the present invention, e.g., cancer vaccines, can be administered before, during, or after treatment using conventional anticancer drugs, radiation therapy, hormone therapy, biotherapy, and / or surgical tumor resection.

[0176] Another example of the present application relates to a method for producing a fusion protein, which includes a step of linking a human thioredoxin protein to the N-terminus, C-terminus, or both, of the peptide antigen, and in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0177] In one embodiment, the linking step may be performed ex vivo or in vitro.

[0178] In one embodiment, the fusion protein may have enhanced immunogenicity compared to a fusion protein to which a wild-type human thioredoxin protein is linked.

[0179] In one embodiment, the fusion protein may have improved efficacy in preventing or treating a disease compared to a fusion protein to which a wild-type human thioredoxin protein is linked.

[0180] In one embodiment, the fusion protein may have improved purification efficiency compared to a fusion protein to which a wild-type human thioredoxin protein is linked.

[0181] In one embodiment, the step of linking the human thioredoxin protein to the N-terminus, C-terminus, or both of the peptide antigen may be performed by conventional chemical methods or recombinant methods as described above.

[0182] In one embodiment, the method for producing the fusion protein may additionally include, prior to the step of linking a human thioredoxin protein to the N-terminus, C-terminus, or both, of the peptide antigen, a step of preparing a human thioredoxin protein variant in which three or more cysteine ​​residues in the amino acid sequence of the protein are substituted with non-cysteine ​​residues.

[0183] In the case of the method for producing a fusion protein of the present application, the method for producing a fusion protein according to the example of the present application described above is a method for producing a fusion protein. The overlapping content between the two inventions is also commonly applied to the method for producing a fusion protein, and the description thereof will be omitted to avoid excessive complexity of this specification.

[0184] Another example of the present application provides a method for enhancing the immunogenicity of a fusion protein, comprising the step of linking a human thioredoxin protein to the N-terminus, C-terminus, or both, of the peptide antigen to produce a fusion protein, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0185] Another example provides a method for enhancing the disease preventive or therapeutic efficacy of a fusion protein, which comprises linking a human thioredoxin protein to the N-terminus, C-terminus, or both, of the peptide antigen to produce a fusion protein, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0186] Another example provides a method for improving the purification efficiency of a fusion protein, which includes a step of producing a fusion protein by linking a human thioredoxin protein to the N-terminus, C-terminus, or both of the peptide antigen, in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

[0187] The method for enhancing the immunogenicity of a fusion protein, the method for enhancing the efficacy of disease prevention or treatment, and the method for improving purification efficiency of the present application include the step of producing a fusion protein according to one example of the present application described above. The overlapping content between both inventions is commonly applied, and the description thereof will be omitted to avoid excessive complexity of this specification. [Brief explanation of the drawings]

[0188] [Figure 1] 1 shows the results of confirming peak morphology after two-stage column chromatography during the purification process of TrxA-M30-His(pc0735) according to one example of the present application. [Figure 2] 1 shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of TrxA.v2-M30-His(pc0802) according to one example of the present application. [Figure 3] 1 shows the results of confirming the peak morphology after two-stage column chromatography during the purification process of hTrx.v2-M30-His(pc0763) according to one example of the present application. [Figure 4] 1 shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v1-M30-His(pc0764) according to one example of the present application. [Figure 5]1 shows the results of confirming the peak morphology after two-stage column chromatography during the purification process of hTrx.v3-M30-His(pc0792) according to one example of the present application. [Figure 6] 1 shows the results of confirming the peak morphology after two-stage column chromatography during the purification process of hTrx.v3-M30-PSBD-His(pc0890) according to one example of the present application. [Figure 7] The results of comparing the UV absorbance (280 nm) and the area ratio as a function of time to confirm the tendency for substitution of cysteine ​​residues with serine residues are shown. [Figure 8] This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v1-M44-His(pc0761). [Figure 9] This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v2-M44-His(pc0762). [Figure 10] This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v3-M44-PSBD-His(pc0895). [Figure 11] This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v3-M12-His(pc0811). [Figure 12] This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v3-M12-PSBD-His(pc0883). [Figure 13] This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v3-M21-His(pc0812). [Figure 14] This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v3-M21-PSBD-His(pc0869). [Figure 15]This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v3-CT5-His(pc0824). [Figure 16] This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v3-CT5-PSBD-His(pc0886). [Figure 17] This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.wt-MelanA-His (pc0993). [Figure 18] This shows the results of confirming the peak morphology after two stages of column chromatography during the purification process of hTrx.v3-MelanA-His (pc0992). [Figure 19] 1 shows the results of SDS-PAGE analysis of TrxA-M30-His(pc0735) according to one example of the present application. [Figure 20] 1 shows the results of SDS-PAGE analysis of hTrx.v2-M30-His(pc0763) according to one example of the present application. [Figure 21] 1 shows a comparison of the results of SDS-PAGE analysis of hTrx.v3-M30-His (pc0792) and TrxA.v2-M30-His (pc0802) according to one example of the present application. [Figure 22] 1 shows a comparison of the results of SDS-PAGE analysis of hTrx.v1-M44-His (pc0761) and hTrx.v2-M44-His (pc0762) according to one example of the present application. [Figure 23] 1 shows the results of SDS-PAGE analysis of hTrx.v3-M44-PSBD-His(pc0895) according to one example of the present application. [Figure 24] 1 shows the results of SDS-PAGE analysis of hTrx.v3-M12-His(pc0811) according to one example of the present application. [Figure 25] 1 shows the results of SDS-PAGE analysis of hTrx.v3-M12-PSBD-His(pc0883) according to one example of the present application. [Figure 26]1 shows the results of SDS-PAGE analysis of hTrx.v3-M21-His(pc0812) according to one example of the present application. [Figure 27] 1 shows the results of SDS-PAGE analysis of hTrx.v3-M21-PSBD-His(pc0869) according to one example of the present application. [Figure 28] 1 shows the results of SDS-PAGE analysis of hTrx.v3-CT5-His(pc0824) according to one example of the present application. [Figure 29] 1 shows the results of SDS-PAGE analysis of hTrx.v3-CT5-PSBD-His(pc0886) according to one example of the present application. [Figure 30] 1 shows the results of SDS-PAGE analysis of hTrx.wt-MelanA-His(pc0993) according to one example of the present application. [Figure 31] 1 shows the results of SDS-PAGE analysis of hTrx.v3-MelanA-His(pc0992) according to one example of the present application. [Figure 32] The results of SDS-PAGE analysis of hTrx.v3-MelanA-PSBD-His (pc0984) according to one example of the present application are shown in Figures 19 to 32. In Figures 19 to 32, FT stands for Flow Through, SEC# numbers represent fractionation order numbers during size-exclusion chromatography (SEC), and LB (loading before) represents the starting sample loaded onto the purification column. DETAILED DESCRIPTION OF THE INVENTION

[0189] The present invention will be described in more detail below with reference to the following examples, which are merely for illustrative purposes and are not intended to limit the scope of the present invention.

[0190] Example 1. Epitope Selection To identify the mouse mutations and select the MHC-binding epitopes, we performed whole exome sequencing (WES) of cancer and normal cells, and mRNA sequencing to identify the presence or absence of mutant gene expression. Analysis of the WES results identified mutations that altered the protein sequence (missense mutations, frameshift mutations, and insertion-deletions) and the mouse MHC type.

[0191] Specifically, we used mouse tail tissue as normal cells and mouse melanoma cell line B16-F10 and mouse colon carcinoma cell lines MC38 and CT26 for next-generation sequencing (NGS). NGS was performed using Macrogen, and WES was performed using the Mutect2 algorithm to analyze missense single-nucleotide polymorphism (SNP) mutations. Using SNPs as a reference, we identified 27-mer amino acids by adding 13-mer sequences before and after.

[0192] In mRNA sequencing, only genes with expression levels, i.e., RPKM (Reads Per Kilobase Million) values ​​of 10 or higher, were selected from the WES sequencing results.

[0193] The resulting epitope sequences were entered into the publicly available NetMHCCons and NetMHCPanII programs. The MHC type was entered based on the phenotype appropriate for mouse, and the Strong Binding (SB) and Weak Binding (WB) criteria were set to default. NetMHCCons analyzed 9-mer sequences, and NetMHCPanII analyzed 13-mer sequences. The sequences were then entered into the IEBD Immunogenicity program, where they were analyzed 9-mer sequences and scores were entered. Predicted Score = 10 × NetMHCCons Number of Binders +Number of NetMHCPanII Binders +IEDB Immunogenicity Score

[0194] Assuming that the higher the prediction score, the greater the probability of efficacy, the top 20 were selected and all were confirmed to elicit immune responses. Substances that elicited immune responses or were easy to manufacture were selected for evaluation of their anticancer efficacy. Fusion proteins for anticancer vaccines were then produced using these selected epitopes. The neoantigens and tumor-associated antigens used are listed in Table 2 below. In the table, "CT + number" is an arbitrary designation of the neoantigen number of the CT26 colon cancer cell line, "M + number" is an arbitrary designation of the neoantigen number of the melanoma cell line (B16-F10), and MelanA represents MART-1, a tumor antigen found in melanoma. [Table 2]

[0195] Example 2. Selection of Carrier Protein 2-1. Carrier protein design Neoantigens induce T-cell immunity by binding to MHC-I (Major Histocompatibility Complex-I) or MHC-II proteins, but synthetic long peptides (SLPs) containing neoantigens are generally known to have a short half-life in the body and low solubility depending on the sequence. To overcome these shortcomings of SLPs, we attempted to produce a fusion protein linking neoantigens with thioredoxin protein, which was expressed in E. coli and purified.

[0196] The fusion proteins were expressed in an open reading frame (ORF) consisting of (thioredoxin)-(linker 1)-(neoantigen)-(linker 2)-(His-tag) or (thioredoxin)-(linker 1)-(neoantigen)-(linker 2)-(carrier protein)-(linker 3)-(His-tag) from the N-terminus to the C-terminus. Because the physicochemical properties of the expressed antigen sequences vary, an affinity tag, His-tag (6His), was placed at the C-terminus of the ORF to enable antigen purification regardless of the antigen sequence characteristics. The carrier protein was placed between the antigen and His-tag to ensure that any antigen sequence would not interfere with affinity tag-based purification. The main components of the ORF were connected by a linker composed of glycine and serine. The linkers used are listed in Table 3 below. The types of human thioredoxin and carrier proteins used in this invention are as follows: PSBD refers to a mutant fragment of full-length PSBD (SEQ ID NO: 13), as shown in the following examples.

[0197] [Table 3-1] [Table 3-2] [Table 3-3]

[0198] 2-2. Production results of neoantigen fusion proteins according to the linked proteins Because the protein sequences of neoantigens in anticancer vaccines are different, even when fused to carrier proteins, their pI and hydrophobicity vary depending on the neoantigen protein sequence, making it difficult to purify all antigens using ion exchange chromatography or hydrophobic interaction chromatography. For this reason, we attempted to purify the antigen proteins using affinity chromatography. We selected a His-tag, which has a small tag size of six histidine amino acids and a high protein binding capacity of 20 mg per mL of resin, as the affinity tag for purification. The antigens purified using the affinity tag were then polished using size-exclusion chromatography (SEC). Cloning, culture, and purification methods were as described in Example 3. The results of the preparation of neoantigen fusion proteins conjugated to each carrier protein are shown below. [Table 4]

[0199] Example 3. Vector production and cultivation 3-1. Vector construction When synthesizing fusion proteins, if the insert length was short (up to 400 mer), approximately six 60-90 mer oligos were obtained and then overlap PCR was performed. If the insert length was long, a sequence with restriction enzyme sites at both ends was obtained by gene synthesis. The primers and backbone sequences required for the construction of each construct are summarized in Tables 5 and 6. The sequence and backbone vector were treated with restriction enzymes for 1 hour, followed by gel extraction. The resulting gene fragment was treated with ligase for 1 hour and then transformed into E. coli.

[0200] The fusion protein sequence consists of a thioredoxin protein, a neoantigen, an optional carrier protein, and a tag sequence for purification. The desired sequences were generated by overlap PCR or gene synthesis and then inserted into a vector. To allow for the continual modification of epitope sequences selected by tumor sequencing analysis, the sequence and order of the fusion protein were determined. Additional restriction enzyme sites were inserted before and after the neoantigen insertion site, allowing for convenient insertion of the desired neoantigen sequence by oligonucleotide synthesis. To insert additional sequences, such as neoantigens, two single-stranded oligonucleotide sequences (two complementary single-stranded oligonucleotide sequences, 5'>3' and 3'>5') were added by annealing, followed by insert cloning. If the additional sequence was longer, the number of inserts was increased.

[0201] For PCR, primers, template, polymerase premix, and DW were added and denatured at 95°C for 1 minute, followed by 25 cycles of 95°C for 10 seconds, 60°C for 10 seconds, and 72°C for 30 seconds. Extension was performed at 72°C for 2 minutes and a final cycle of 12°C for 5 minutes in a PCR machine. The sequence and backbone vector were treated with restriction enzymes for 1 hour, followed by gel extraction. The resulting gene fragment was treated with ligase for 1 hour and then transformed into E. coli. For insert generation by oligo annealing, two complementary oligonucleotide sequences were mixed at equal concentrations of 1 μM and incubated at 95°C for 5 minutes with a ramp rate of 0.1°C / sec. The temperature was then lowered to 50°C and incubated for 10 minutes.

[0202] The previously constructed vector backbone was derived from pET28a(+). To change the restriction enzyme site in pET28a(+), PCR was performed using the pET-Duet vector as a template with backbone F1 and backbone R1 primers. The resulting fragment was then digested with the XbaI and XhoI restriction enzyme sites and inserted into pET-28a(+) to create the pET-Backbone vector, which can be used as a different enzyme site from the existing one. The vectors used in the experiments were constructed based on this pET-Backbone vector. TrxA-M30-His was prepared by PCR using pET32a vector as a template and TrxA F SpeI and TrxA BamHI R as primers, followed by cleavage with NdeI-BamHI and ligation with hCSTA-M30-His as a vector. Trx.A2-M30-his was prepared by PCR using TrxA sequence as a template and TrxA F SpeI and TrxA R SS mut as a forward fragment, and PCR using TrxA F SS mut primer and TrxA BamHI R as a reverse fragment. This was then subjected to overlap PCR, followed by cleavage with NdeI-BamHI and ligation with TrxA-M30-His as a vector.hTrx.v1-M30-His and hTrx.v1-M44-His were prepared by PCR using the synthesized hTrx sequence as a template and hT F NdeI and hT R BamHI as primers, followed by cleavage of TrxA-M30-His and TrxA-M44-His as vectors with NdeI-BamHI and ligation. hTrx.v2-M30-His and hTrx.v2-M44-His were prepared by PCR using the hTrx sequence as a template and hT F NdeI and 6269C 2frag F as primers to produce the forward fragment, and the same template and 6269C 1frag R and hT R BamHI as primers to produce the reverse fragment. PCR fragments were prepared by digesting TrxA-M30-His and TrxA-M44-His with NdeI and BamHI and ligating them together. For hTrx.wt-MelanA-His, PCR was performed using hTrx.v2-M30-His as a template and T7 F and hTrx wt R as primers to produce the forward fragment. For hTrx.v3-melanA-PSBD-His as a template and hTrx wt F and MelanA wt R as primers to produce the reverse fragment, overlap PCR was performed using hTrx.v3-MelanA-PSBD-His as a vector and digested with BglII and SacI, followed by ligation.

[0203] For hTrx.v3-neoantigen (M30, M44, M12, M21, or CT5)-His, PCR was performed using the synthesized hTrx sequence as a template and hT F NdeI and hTrx F SS mut as primers. The forward fragment was PCRed using hTrx R SS mut and hT R BamHI as primers. The reverse fragment was PCRed using overlapping PCR. The resulting fragments were digested with NdeI and BamHI and ligated with TrxA-neoantigen-His as a vector. For hTrx.v3-MelanA-His, PCR was performed using hTrx.v3-MelanA-PSBD-His as a template and T7 F and MelanA wt R as primers. The resulting fragment was digested with BglII and SacI and ligated with hTrx.v3-MelanA-PSBD-His as a vector.

[0204] To construct the hTrx.v3-neoantigen-PSBD-His vector, we created a backbone vector designed to allow neoantigen insertion using two BsaI sites located between hTrx.v3 and PSBD in the order hTrx-BsaI enzyme site-PSBD-His, and then inserted the neoantigen. Overlap PCR was performed to construct the vector. PCR was performed using the nucleotide sequence of hTrx.v1 synthesized by Cosmogenetech as a template and hT NdeI F and hTrx.v3 R as primers to generate the first template. PCR was performed using the forward hTrx.v1 sequence as a template and hTrx.v3 F and hT BsaI R1 as primers to generate the second template. Overlap PCR was performed using the forward hTrx.v3 F and hT BsaI R as primers to generate the first template. The overlap PCR product was then used as the first template. PCR was performed using the PSBD sequence synthesized by Cosmogenetech as a template and BsaI F and PSBD R as primers to generate the second template. Overlap PCR was performed using hT NdeI F, the forward primer for the forward PCR product, and PSBD R, the reverse primer for the reverse PCR product, as primers. The PCR product was inserted into pET-28 and pET-Duet-based backbone vectors, which were then digested with NdeI and SacI and ligated to generate the hTrx-BsaI enzyme site-PSBD-His vector. To generate hTrx.v3-neoantigen-PSBD-His, the hTrx-BsaI-PSBD-His vector was digested with BsaI and annealed with two F / R nucleotide oligomers (primers SEQ ID NOS: 48-49 and 58-77, respectively, each named for the corresponding neoantigen) and then ligated to the digested vector.

[0205] DH5alpha (Engynomics) cells were used for transformation. After heat shock for 1 minute 30 seconds, the cells were stabilized in LB media for 1 hour and then spread onto solid medium containing antibiotic marker for selection. One or two colonies were grown in 2 ml of LB media overnight (12-16 hours) at 37°C. After 6-8 hours of growth, mini-preps were performed to obtain vectors, and an aliquot was used for sequence analysis. [Table 5] [Table 6]

[0206] 3-2.Culture The sequence-confirmed vector was transformed into ClearColi BL21(DE3) for cultivation. Since the strain's comp. cells were electroporated, the DNA was transferred to a separate cuvette and subjected to electric shock. After stabilization in LB media for 1 hour, the cells were spread on solid medium containing antibiotic marker for selection. One colony grown in the medium incubated overnight (16-20 h) at 37°C was inoculated into 3 ml of LB media containing antibiotics and grown overnight. The following morning, a secondary inoculation was performed on 0.8 L of medium. When the OD reached approximately 0.7-1.2, IPTG 0.2 mM induction was performed, and the culture temperature was lowered from 37°C to 25°C for overnight cultivation. The cells were then harvested the following day.

[0207] 3-3. Collection and disruption of bacterial cells The culture medium expressing the recombinant fusion protein was placed in a 1 L centrifuge bottle and centrifuged at 7000 rpm for 5 minutes at 5 ± 3°C. The supernatant was discarded and the precipitated bacterial cells were collected. The collected bacterial cells were suspended in a disruption solution (20 mM sodium phosphate, 0.3 M NaCl, 10 mM imidazole, 1 mM PMSF, pH 8.0) and then disrupted using an ultrasonic homogenizer. After disruption, the cell lysate was placed in a 50 mL centrifuge bottle and centrifuged at 40,000 x g for 30 minutes at 5 ± 3°C. The supernatant was purified using an Äkta pure system.

[0208] Example 4. Purification 4-1. Purification of fusion proteins Purification of the fusion protein was performed using filtration and two steps of column chromatography. Step 1: Metal ion affinity chromatography (IMAC) A metal ion affinity column (Cobalt immobilized column) was equilibrated with 20 mM sodium phosphate / 0.3 M sodium chloride / 10 mM imidazole buffer. After disruption, the centrifuged supernatant was filtered through a 0.22 μm filter and loaded onto the column. The column was washed with 20 mM sodium phosphate / 0.3 M sodium chloride / 10 mM imidazole buffer (pH 8.0). The recombinant anti-cancer vaccine was then eluted with 20 mM sodium phosphate / 0.3 M sodium chloride / 150 mM imidazole buffer (pH 8.0).

[0209] Step 2: Size Exclusion Chromatography (SEC) A size-exclusion chromatography column (Hiload Superdex G75) was equilibrated with phosphate-buffered saline. After loading the eluate from the metal ion affinity column onto the column, UV absorbance (280 nm) was monitored, and peak fractions with an absorbance of 50 mAU or higher were collected.

[0210] 4-2. Analysis of chromatogram results The SEC chromatograms of each carrier protein variant were analyzed to confirm the morphology of the multimer and monomer peaks of the variants (FIGS. 1 to 18).

[0211] Figure 1 shows the chromatogram of TrxA-M30-His (pc0735, wild-type), and Figure 2 shows the chromatogram of TrxA.v2-M30-His (pc0802, two Cys substitutions). Comparing the chromatograms in Figures 1 and 2, it was confirmed that the retention time peak corresponding to the multimer of wild-type TrxA (TrxA-M30-His, pc0745) was not observed in the cysteine-substituted TrxA mutant (TrxA.v2-M30-His, pc802), indicating that the purification efficiency of the mutant was higher.

[0212] Figure 3 shows the chromatogram of hTrx.v2-M30-His (pc0763, one Cys substitution), Figure 4 shows the chromatogram of hTrx.v1-M30-His (pc0764, three Cys substitution), Figure 5 shows the chromatogram of hTrx.v3-M30-His (pc0792, five Cys substitution), and Figure 6 shows the chromatogram of hTrx.v3-M30-PSBD-His (pc0890, five Cys substitution, additional carrier protein PSBD included). As shown in Figures 3 to 6, the peak ratio of multimers (peak #1) and monomers (peak #2) in the size exclusion chromatography step varied depending on the number of cysteine ​​substitutions in hTrx and whether or not the carrier protein PSBD was included. As a result, it was found that the ratio of the monomer peak was highest when the cysteine ​​residue present in the wild type was replaced with a serine residue, and that the ratio of the monomer peak was even higher when PSBD was included at the C-terminus.

[0213] In addition, the tendency of cysteine ​​residues to serine residues was confirmed by comparing the area ratio over time with UV absorbance (280 nm) (Figure 7). The monomer peak area was quantified and compared according to the number of cysteine ​​residues substituted. The peak area was 54% for a single cysteine ​​substitution and 84-87% for all five cysteine ​​residues. This demonstrates that the structural stability of the monomer increases with the number of cysteine ​​residue substitutions, and that the structural stability of the monomer is further enhanced when the carrier protein PSBD is additionally included.

[0214] Figure 8 shows the chromatograms of hTrx.v1-M44-His (pc0761, three Cys substitutions), Figure 9 shows the chromatograms of hTrx.v2-M44-His (pc0762, one Cys substitution), and Figure 10 shows the chromatograms of hTrx.v3-M44-PSBD-His (pc0895, five Cys substitutions, additional carrier protein PSBD). As shown in Figures 8 to 10, when the number of cysteine ​​residues in hTrx replaced with serine residues increases or when PSBD is added, the multimer peak decreases and purification efficiency increases.

[0215] Figure 11 shows the chromatogram of hTrx.v3-M12-His (pc0811, five Cys substitutions), Figure 12 shows the chromatogram of hTrx.v3-M12-PSBD-His (pc0883, five Cys substitutions, additionally containing carrier protein PSBD), Figure 13 shows the chromatogram of hTrx.v3-M21-His (pc0812, five Cys substitutions), Figure 14 shows the chromatogram of hTrx.v3-M21-PSBD Figure 11 shows the chromatogram of hTrx.v3-CT5-His (pc0869, five Cys substitutions, containing the additional carrier protein PSBD), Figure 15 shows the chromatogram of hTrx.v3-CT5-His (pc0824, five Cys substitutions), and Figure 16 shows the chromatogram of hTrx.v3-CT5-PSBD-His (pc0886, five Cys substitutions, containing the additional carrier protein PSBD). As shown in Figures 11 to 16, regardless of the type of antigen, when the additional carrier protein PSBD was included, the multimer peak decreased and the monomer peak increased, confirming that the purification efficiency of the fusion protein increased.

[0216] Figure 17 shows the chromatograms of hTrx.wt-MelanA-His (pc0993, wild-type), and Figure 18 shows the chromatograms of hTrx.v3-MelanA-His (pc0992, five Cys substitutions). As shown in Figures 17 and 18, when the cysteine ​​residues of hTrx were replaced with serine residues, the multimer peak was reduced, and the purification efficiency of the fusion protein was increased.

[0217] 4-3. Analysis of adjustment confirmation test results during manufacturing process Meanwhile, confirmation tests (SDS-PAGE) and protein content (UV method) were carried out during the manufacturing process (in-process control, IPC), and the results are shown in FIGS. 19 to 32 and Table 4.

[0218] The confirmation test (SDS-PAGE) was performed as follows. While preparing for electrophoresis, the heating block (Cat. No. Iso-Block) was first heated to 95°C. The sample, distilled water (DW), and loading dye (3X with beta-mercaptoethanol) were mixed appropriately, and the mixed sample to be electrophoresed was boiled for 5 minutes in a heating block heated to 95°C. While boiling, a gel tank was assembled with Tris-Glycine-PAGE and SDS gel (Cat. No. KG75355). SDS solution was poured into the assembled gel tank, which was then connected to an electrophoresis power supply (Cat. No. EPS1001) and warmed up to 90V. After boiling, the sample was cooled to room temperature and then spun down using a microcentrifuge (13,000 rpm, 1 min). To determine protein size, approximately 5 μL of protein marker (Precision Plus Protein Dual Color Standards, Cat. No.: #161-0394 or EzWay™ Protein-PreBlue Ladder, Cat. No.: #K18010) was injected into the SDS-Polyacrylamide gel well, followed by approximately 15 μL of sample. The power of the electrophoresis power supply was adjusted to 90 V for 20 min and 200 V for 1 hr. After electrophoresis, an appropriate amount of Coomasse protein gel stain solution was poured onto the SDS-Gel and it was rotated at 70 rpm on an orbital shaker (Cat. No.: NB-101S) for at least 30 minutes.After gel staining, the Coomassie protein gel stain solution was removed, and the gel was then spun in Fixing Solution on an ORBITAL SHAKER (Cat No.: NB-101S) at 70 rpm. The SDS-Gel, from which the CBG staining had been removed, was photographed and saved using the Image Lab 6.0 program on a BIO-RAD Gel Doc EZ Imager.

[0219] Protein content (UV method) was measured as follows. A 1 cm path length cuvette (quartz) was thoroughly washed with distilled water, then dehydrated with a tissue that generates few particles, such as Kimtex wipes, and blown with an air gun. 200 μL of reference material (water for injection or PBS) was placed in the cuvette, and the absorbance at 280 nm was measured using a UV / Visible Spectrophotometer. After repeating the distilled water washing, dehydration, and air gun blowing process, 200 μL of sample was placed in the cuvette, and the absorbance at 280 nm was measured using a UV / Visible Spectrophotometer. The content was calculated by multiplying the dilution factor of the sample and dividing by an Abs. 0.1% coefficient constant, which varies depending on the protein sequence.

[0220] Reducing SDS-PAGE of the peak fractions corresponding to the multimers confirmed that they were not completely dissolved into monomers despite treatment with a reducing agent. This was observed for both TrxA (wild type, lanes 6 to 9) and hTrx.v2 (one cysteine ​​substitution) (Figures 19 and 20). In contrast, in the cases of TrxA.v2 and hTrx.v3, in which all cysteine ​​residues were substituted, no bands corresponding to the multimers were observed in reducing SDS-PAGE analysis (Figure 21).

[0221] Figure 22 shows the results of SDS-PAGE of hTrx.v1-M44-His (pc0761, triple Cys substitution) and hTrx.v2-M44-His (pc0762, single Cys substitution), and Figure 23 shows the results of SDS-PAGE of hTrx.v3-M44-PSBD-His (pc0895, five Cys substitution, additionally containing the carrier protein PSBD). Multimer bands were observed in hTrx.v2-M44-His (pc0762, single Cys substitution), but not in hTrx.v1-M44-His (pc0761, triple Cys substitution) or hTrx.v3-M44-PSBD-His (pc0895, five Cys substitution, additional carrier protein PSBD). In particular, in the case of hTrx.v3-M44-PSBD-His (pc0895, five Cys substitution, additional carrier protein PSBD), a thicker monomer band appeared, confirming that the more cysteine ​​substitutions there were and the more PSBD there was, the less multimer formation there was, and the more efficient the purification of the fusion protein.

[0222] Figure 24 shows the results of SDS-PAGE of hTrx.v3-M12-His (pc0811, five Cys substitutions), Figure 25 shows the results of SDS-PAGE of hTrx.v3-M12-PSBD-His (pc0883, five Cys substitutions, additionally containing the carrier protein PSBD), Figure 26 shows the results of SDS-PAGE of hTrx.v3-M21-His (pc0812, five Cys substitutions), and Figure 27 shows the results of SDS-PAGE of hTrx.v3-M21-PSBD Figure 24 shows the SDS-PAGE results for hTrx.v3-CT5-His (pc0869, five Cys substitutions, containing the additional carrier protein PSBD), Figure 28 shows the chromatograms for hTrx.v3-CT5-His (pc0824, five Cys substitutions), and Figure 29 shows the chromatograms for hTrx.v3-CT5-PSBD-His (pc0886, five Cys substitutions, containing the additional carrier protein PSBD). As shown in Figures 24 to 29, regardless of the type of antigen, when the additional carrier protein PSBD was included, the monomer band appeared more clearly, confirming that the purification efficiency of the fusion protein increased.

[0223] Figure 30 shows the results of SDS-PAGE for hTrx.wt-MelanA-His (pc0993, wild-type), Figure 31 shows hTrx.v3-MelanA-His (pc0992, five-Cys substitution), and Figure 32 shows hTrx.v3-MelanA-PSBD-His (pc0984, five-Cys substitution, additionally containing the carrier protein PSBD). As shown in Figures 30 to 32, bands corresponding to multimers were observed in the case of hTrx.wt-MelanA-His (pc0993, wild-type), while a faint multimer band was observed in the case of hTrx.v3-MelanA-His (pc0992, five-Cys substitution). In contrast, in the case of hTrx.v3-MelanA-PSBD-His (pc0984, five Cys substitutions, and the additional carrier protein PSBD), no multimer band was observed, and a clear monomer band was observed. This indicates that when cysteines are substituted, the formation of multimers during purification is reduced, improving the purification efficiency of the fusion protein, and that when PSBD is included as an additional carrier protein, the purification efficiency of the fusion protein is further improved.

[0224] The sterilized final anti-cancer vaccine active substance was stored at -70±10°C.

[0225] From the above description, those skilled in the art will understand that the present invention can be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. Therefore, it should be understood that the above-described embodiments are illustrative in all respects and not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, rather than the above detailed description, and equivalent concepts thereof.

Claims

1. A peptide antigen; a human thioredoxin protein linked to the N-terminus, C-terminus, or both of the peptide antigen; Including, A fusion protein in which three or more cysteine ​​residues in the amino acid sequence of the human thioredoxin protein are substituted with non-cysteine ​​residues.

2. The fusion protein according to claim 1, wherein the cysteine ​​residues at amino acid positions 62, 69 and 73 in the amino acid sequence of human thioredoxin protein are substituted with non-cysteine ​​residues.

3. The fusion protein according to claim 1, wherein the cysteine ​​residues at amino acid positions 32, 35, 62, 69 and 73 in the amino acid sequence of human thioredoxin protein are substituted with non-cysteine ​​residues.

4. The fusion protein comprises: The fusion protein according to claim 1, which additionally comprises a carrier protein other than human thioredoxin at the N-terminus, C-terminus, or both.

5. The fusion protein of claim 1, further comprising an affinity tag at its N-terminus, C-terminus, or both.

6. The fusion protein of claim 1 , wherein a linker is present between the peptide antigen and the human thioredoxin protein.

7. The fusion protein of claim 1 , wherein the peptide antigen comprises a T cell epitope.

8. The fusion protein of claim 1, wherein the peptide antigen comprises a T cell epitope derived from a tumor antigen, an infectious antigen, an autoantigen, or an allergy-inducing antigen.

9. The fusion protein of claim 8, wherein the tumor antigen comprises a tumor-associated antigen (TAA), a tumor-specific antigen (TSA), or a tumor-derived neoantigen.

10. The fusion protein of claim 9, wherein the tumor-derived neoantigen contains a mutation that is specifically expressed in cancer cells.

11. Tumor-associated antigens (TAA) include CT (Cancer-testis) antigen, EGFR, Melan-A, PSMA (Prostate Specific Membrane Antigen), survivin, MAGE-A, 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 (melanoma B antigen), RAGE (kidney tumor antigen), LAGE (L antigen), NAG, GnT-V, MUM-1, CDK4, p53, tyrosinase, Muc1 (mucin 1), HER2 / neu, p21ras, 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 isoforms (isoform) 1B1, 90K / Mac-2 binding protein, Ep-CAM (MK-1), HSP-70, hTERT (TRT), LEA, TAGE-1, 5T 4, gp70, SCP-1, c-myc, 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.

12. The fusion protein of claim 8 , wherein the infectious antigen is an antigen derived from a virus, bacterium, parasite, or fungus.

13. The fusion protein of claim 4, wherein the carrier protein is a protein that improves recombinant expression of the peptide antigen or improves the purification efficiency of the peptide antigen.

14. The fusion protein according to claim 4, wherein the additional carrier protein is a protein that improves recombinant expression of the peptide antigen or enhances purification efficiency of the peptide antigen.

15. The additional carrier protein may be selected from the group consisting of NDPK (nucleoside diphosphate kinase B), CSTA (cystatin-A), Trx (thioredoxin), RPL7Am (50S ribosomal protein L7Ae), Samp2a (small archaeal modifier protein 2), TE (tenascin), TM1112 (Thermotoga maritima Cupin_3 domain-containing protein), TrxA (thioredoxin 1), TTrx (Thermosiphos africanus 5. The fusion protein of claim 4, which is one or more of thioredoxin, PSBD (peripheral subunit-binding domain), or fragments thereof.

16. The fusion protein of claim 5 , wherein the affinity tag is His or streptavidin.

17. The linker may be (GS)n, (G ２ S)n, (G ３ S)n, (G ４ 7. The fusion protein of claim 6, wherein G is Gly, S is Ser, L is Leu, E is Glu, and n is an integer of at least 1.

18. The fusion protein of claim 1 , wherein the fusion protein is 30 kDa or less in size.

19. A nucleic acid molecule encoding the fusion protein of any one of claims 1 to 18.

20. 20. An expression vector comprising the nucleic acid molecule of claim 19.

21. A cell transformed with the expression vector of claim 20.

22. A fusion protein according to any one of claims 1 to 18; a nucleic acid molecule encoding the fusion protein; an expression vector comprising the nucleic acid molecule; or a cell transformed with the expression vector, Immunogenic composition.

23. 23. The immunogenic composition of claim 22, additionally comprising an adjuvant.

24. A fusion protein according to any one of claims 1 to 18; a nucleic acid molecule encoding the fusion protein; an expression vector comprising the nucleic acid molecule; or a cell transformed with the expression vector, A composition for improving the purification efficiency of peptide antigens.

25. A fusion protein according to any one of claims 1 to 18; a nucleic acid molecule encoding the fusion protein; an expression vector comprising the nucleic acid molecule; or a cell transformed with the expression vector, A composition for preventing or treating cancer.

26. A thioredoxin mutant polypeptide consisting of the amino acid sequence of SEQ ID NO:7 or SEQ ID NO:9.

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