Immunogenic compound comprising immunogenic fragment of apolipoprotein B100, immunogenic composition comprising the same and use thereof

Abstract

The present invention relates to an immunogenic compound comprising an ApoB100 epitope, an immunogenic composition comprising the same, and uses thereof. The immunogenic compound can be used to prevent or treat diseases caused by increased ApoB100 levels or increased lipid levels by inducing an immune response to the ApoB100 epitope. The immunogenic compound has a complementary mechanism to GIP-1R agonist obesity treatments, so their combined or concurrent administration can enhance weight loss and improve side effects associated with long-term administration of GIP-1R agonist obesity treatments by improving weight loss plateaus, delaying the yo-yo effect, and improving lean body mass reduction.

Description

Technology Field

[0001] The invention relates to an immunogenic fragment of apolipoprotein B100, an immunogenic composition containing the same, and the use thereof. Background Technology

[0002] Apolipoprotein B100 (ApoB100) is the main protein component of low-density lipoprotein (LDL) and is also present in intermediate-density lipoprotein (IDL) and very low-density lipoprotein (VLDL). Human ApoB100 is translated into a precursor protein consisting of approximately 4,563 amino acid residues, and exists in the form of a mature protein having approximately 4,536 amino acid residues after the signal peptide is cleaved. ApoB100 is synthesized in the liver and is required for the assembly of VLDL.

[0003] Serum LDL is a major risk factor for adult diseases related to lipid metabolism. Recently, research is being attempted to lower cholesterol levels and reduce arteriosclerosis using cholesterol vaccines. Bailey et al. ("Cholesterol vaccines" Science vol 264, p. 1067-1068, 1994) reported that blood cholesterol levels were reduced by immunizing cholesterol-fed rabbits with a synthetic antigen in which a cholesterol ester is covalently linked to a carrier protein.

[0004] However, even with conventional technology, there is still a need for an AopB100 epitope that has immunogenicity to ApoB100 and has weight loss and lipid-reducing effects. The problem to be solved

[0005] One aspect comprises a peptide fragment of the amino acid sequence of SEQ ID NO. 25, comprising residues 2205 to 2210 of SEQ ID NO. 25 and having a length of 10 to 30 amino acids; or a first epitope composed of a peptide fragment having at least 60% sequence identity with said peptide fragment;

[0006] A peptide fragment of the amino acid sequence of SEQ ID NO. 25 comprising residues 3662 to 3688 of SEQ ID NO. 25 and having a length of 27 to 30 amino acids; or a second epitope composed of a peptide fragment having at least 60% sequence identity with said peptide fragment; and

[0007] The present invention provides an immunogenic compound comprising one or more ApoB100 epitopes selected from the group consisting of: a peptide fragment of the amino acid sequence of SEQ ID NO. 25 having residues 3380 to 3394 of SEQ ID NO. 25 and a length of 15 to 30 amino acids; or a third epitope consisting of a peptide fragment having at least 60% sequence identity with said peptide fragment.

[0008] Another aspect provides an immunogenic composition comprising the above-mentioned immunogenic compound.

[0009] Another aspect provides an immunogenic kit containing the above-mentioned immunogenic compound.

[0010] Another aspect provides a method for preventing or treating a disease caused by an increased level of ApoB100, comprising the step of administering the immunogenic compound to an individual. means of solving the problem

[0011] One aspect comprises a peptide fragment of the amino acid sequence of SEQ ID NO. 25, comprising residues 2205 to 2210 of SEQ ID NO. 25 and having a length of 10 to 30 amino acids; or a first epitope composed of a peptide fragment having at least 60% sequence identity with said peptide fragment;

[0012] A peptide fragment of the amino acid sequence of SEQ ID NO. 25 comprising residues 3662 to 3688 of SEQ ID NO. 25 and having a length of 27 to 30 amino acids; or a second epitope composed of a peptide fragment having at least 60% sequence identity with said peptide fragment; and

[0013] The present invention provides an immunogenic compound comprising one or more ApoB100 epitopes selected from the group consisting of: a peptide fragment of the amino acid sequence of SEQ ID NO. 25 having residues 3380 to 3394 of SEQ ID NO. 25 and a length of 15 to 30 amino acids; or a third epitope consisting of a peptide fragment having at least 60% sequence identity with said peptide fragment.

[0014] In this specification, "the amino acid sequence of SEQ ID NO. 25" represents the amino acid sequence of human ApoB100 protein. "The amino acid sequence of SEQ ID NO. 25" corresponds to human ApoB100 protein and is interpreted to include variants of human ApoB100 protein.

[0015] In the above ApoB100 epitope, the “peptide fragment of the amino acid sequence of SEQ ID NO. 25, comprising residues 2205 to 2210 of SEQ ID NO. 25 and having a length of 10 to 30 amino acids” may have an amino acid extending in the N-terminal direction from residue 2205 (hereinafter referred to as “N extension”), an amino acid extending in the C-terminal direction from residue 2210 (hereinafter referred to as “C extension”), or both extensions, having a length of 10 to 30 amino acids. The N extension and C extension may each have 0 to 24 amino acids and 24 to 0 amino acids. The “peptide fragment of the amino acid sequence of SEQ ID NO. 25, comprising residues 2205 to 2210 of SEQ ID NO. 25 and having a length of 10 to 30 amino acids” is also hereinafter referred to as Reference Sequence 1. In this specification, the “fragment” may be composed of a continuous sequence of amino acids.

[0016] In the above ApoB100 epitope, the “peptide fragment of the amino acid sequence of SEQ ID NO. 25, comprising residues 3662 to 3688 of SEQ ID NO. 25 and having a length of 27 to 30 amino acids” may have an amino acid extending in the N-terminal direction from residue 3662 (hereinafter referred to as “N extension”), an amino acid extending in the C-terminal direction from residue 3688 (hereinafter referred to as “C extension”), or both extensions, having a length of 27 to 30 amino acids. The N extension and C extension may each have 0 to 3 and 3 to 0 amino acids, respectively. The “peptide fragment of the amino acid sequence of SEQ ID NO. 25, comprising residues 3662 to 3688 of SEQ ID NO. 25 and having a length of 27 to 30 amino acids” is also hereinafter referred to as Reference Sequence 2.

[0017] In the above ApoB100 epitope, the “peptide fragment of the amino acid sequence of SEQ ID NO. 25, comprising residues 3380 to 3394 of SEQ ID NO. 25 and having a length of 15 to 30 amino acids” may have an amino acid extending in the N-terminal direction from residue 3380 (hereinafter referred to as “N extension”), an amino acid extending in the C-terminal direction from residue 3394 (hereinafter referred to as “C extension”), or both extensions, having a length of 15 to 30 amino acids. The N extension and C extension may each have 0 to 15 and 15 to 0 amino acids, respectively. The “peptide fragment of the amino acid sequence of SEQ ID NO. 25, comprising residues 3380 to 3394 of SEQ ID NO. 25 and having a length of 27 to 30 amino acids” is also referred to as Reference Sequence 3 below.

[0018] Table 1 shows the number of amino acids of the N extension and C extension in the first epitope above, when the length is one of 10 to 30 amino acids. For example, in Table 1, when the length is 30 amino acids, the N extension and C extension may be 0 to 24 and 24 to 0 amino acids, respectively.

[0019] [Table 1]

[0020]

[0021] Tables 2 to 5 show specific examples of N extension and C extension in the first epitope, where the length is any one of 10 to 30 amino acids. For example, in Table 2, when the length is 30 amino acids, N extension and C extension 0 and 24 indicate that 0 and 24 amino acids, respectively, are extended in the N-terminal and C-terminal directions of "residues 2205 to 2210 of sequence number 25".

[0022] [Table 2]

[0023]

[0024] [Table 3]

[0025]

[0026] [Table 4]

[0027]

[0028] Table 5 shows the number of amino acids for the N extension and C extension in the case of any one of the lengths of 27 to 30 amino acids in the second epitope above.

[0029] [Table 5]

[0030]

[0031] Table 6 shows specific examples of N extension and C extension in the second epitope above, when the length is any one of 27 to 30 amino acids. For example, in Table 6, when the length is 30 amino acids, N extension and C extension 0 and 3 indicate that 0 and 3 amino acids are extended in the N-terminal and C-terminal directions, respectively, of "residues 3662 to 3688 of sequence number 25".

[0032] [Table 6]

[0033]

[0034] Table 7 shows the number of amino acids for the N extension and C extension in the case of any one of the lengths of 15 to 30 amino acids in the third epitope above.

[0035] [Table 7]

[0036]

[0037] Tables 8 and 9 show specific examples of N extension and C extension in the third epitope above, where the length is any one of 15 to 30 amino acids. For example, in Table 8, when the length is 30 amino acids, N extension and C extension 0 and 15 indicate that 0 and 15 amino acids are extended in the N-terminal and C-terminal directions, respectively, of "residues 3380 to 3394 of sequence number 25".

[0038] [Table 8]

[0039]

[0040] [Table 9]

[0041]

[0042] The term "peptide fragment having 60% or more sequence identity with the above peptide fragment" refers to a variant of the above reference sequence 1, 2, or 3. The variant comprises a substitution, deletion, or addition of amino acids to the above reference sequence 1, 2, or 3. The variant comprises a human ApoB100 protein variant or a peptide comprising amino acid residues at corresponding positions of the mammalian ApoB100 protein.

[0043] The above "sequence identity" can be calculated as described below. The variant preserves a specific function of the reference sequence, e.g., as an epitope. The variant may be a modified sequence in which one or more amino acids of the reference sequence are deleted, substituted, or inserted. For example, a "variant with 90% or more sequence identity" has no more than 10 modifications, i.e., deletions, insertions, or substitutions, per 100 amino acids of the reference sequence.

[0044] Methods for comparing the identity of two or more sequences are well known in this field. The percentage of sequence identity between two sequences can be determined using mathematical algorithms. An example of a preferred but not limited available mathematical algorithm is the algorithm described by Karlin et al. (1993), PNAS USA, 90:5873-5877. These algorithms are integrated into BLAST-family programs such as BLAST or NBLAST (see Altschul et al., 1990, J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res, 25:3389-3402). Additionally, these algorithms are used in FASTA (Pearson (1990), Methods Enzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci. USA 85, 2444-2448). Sequences that have a certain degree of identity with other sequences can be identified using these programs. Additionally, programs available in the Wisconsin Sequence Analysis Package, version 9.1 (Devereux et al., 1984, Nucleic Acids Res., 387-395), such as BESTFIT and GAP, can also be used to determine the % identity between sequences. BESTFIT uses the "local homology" algorithm of Smith and Waterman (1981), J. Mol. Biol. 147, 195-197, and finds the best single similarity region between two sequences.

[0045] The above ApoB100 epitope may be an immunogenic fragment of ApoB100. The above ApoB100 epitope may be a fragment corresponding to residues 2196-2216 (SEQ No. 1), 2202-2218 (SEQ No. 39), 2202-2216 (SEQ No. 40), 2196-2210 (SEQ No. 45), 2198-2213 (SEQ No. 46), 2202-2210 (SEQ No. 47), 3662-3688 (SEQ No. 2), and 3380-3394 (SEQ No. 43), respectively, in the precursor amino acid sequence of human (Homo Sapiens) ApoB100 (SEQ No. 25). In this specification, the ApoB100 epitope is described based on the precursor amino acid sequence of ApoB100, but even if described based on the mature amino acid sequence of ApoB100, the sequence of the ApoB100 epitope is identical except that the N-terminal signal peptide consisting of 27 amino acid residues has been removed. Additionally, the ApoB100 epitope may have an identity of 60% or more, for example, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more with the amino acid sequence of reference sequence 1, 2, or 3. The ApoB100 epitope may have immunogenicity against ApoB100. The immunogenicity may be immunogenicity against B cells. The above ApoB100 epitope may be a B cell epitope. The above ApoB100 epitope may induce the production of antibodies against ApoB100 in an individual. The antibody may bind to ApoB100 and induce aggregation of ApoB100. The above ApoB100 aggregates may be removed by macrophages. The individual may be homologous or heterologous to the origin of ApoB100.

[0046] The above ApoB100 epitope may be a peptide composed of residues 2196-2216 (Sequence No. 1), 2202-2218 (Sequence No. 39), 2202-2216 (Sequence No. 40), 2196-2210 (Sequence No. 45), 2198-2213 (Sequence No. 46), 2202-2210 (Sequence No. 47), 3662-3688 (Sequence No. 2), or 3380-3394 (Sequence No. 43) of the amino acid sequence of human ApoB100 (Sequence No. 25). Additionally, the ApoB100 epitope may be a fragment of the amino acid sequence of ApoB100 of another animal corresponding to a peptide composed of residues 2196-2216 (Sequence No. 1), 2202-2218 (Sequence No. 39), 2202-2216 (Sequence No. 40), 2196-2210 (Sequence No. 45), 2198-2213 (Sequence No. 46), 2202-2210 (Sequence No. 47), 3662-3688 (Sequence No. 2), and 3380-3394 (Sequence No. 43) of the amino acid sequence of human ApoB100 (Sequence No. 25). The animal may be a mammal. The animal may be a mouse, cat, dog, pig, cow, sheep, or horse. The corresponding ApoB100 epitope may be a corresponding mouse ApoB100 fragment (SEQ NO. 19, SEQ NO. 20, and SEQ NO. 49), a cat ApoB100 fragment (SEQ NO. 21, SEQ NO. 22, and SEQ NO. 50), and a dog ApoB100 fragment (SEQ NO. 23, SEQ NO. 24, and SEQ NO. 51). Examples of amino acid sequences of ApoB100 in humans, mice, cats, and dogs may have SEQ NO. 25, 26, 27, and 28, respectively. The gene encoding recombinant proteins 7 to 15 of Table 10 may have nucleotide sequences of SEQ NO. 30, 31, 32, 33, 34, 35, 36, 37, and 38.

[0047] As used herein, the term “corresponding” refers to the amino acid residue positions of the protein of interest that align with the specified positions of the standard protein (e.g., residues 2196-2216, 3662-3688, and 3380-3394 of SEQ ID NO. 25) when the amino acid sequences of the protein of interest and the standard protein (e.g., the amino acid sequence of human ApoB100 of SEQ ID NO. 25) are aligned using a protein alignment program acceptable in the art, such as BLAST pairwise alignment or the well-known Lipman-Pearson Protein Alignment program. The protein of interest may be an animal, e.g., mammalian ApoB100 protein. FIG. 1 illustrates an example of aligning corresponding first, second, and third epitope regions in the amino acid sequences of human, mouse, cat, and dog ApoB100 proteins. In Fig. 1, the underlined parts are the corresponding parts of the first, second, and third epitopes. Since these corresponding epitope regions are well preserved, the same result can be reasonably predicted from the experimental result of one of them.

[0048] In this specification, the term "T cell epitope" refers to an amino acid sequence capable of binding to an MHC class II molecule with appropriate efficiency, stimulating T cells, or binding to T cells as a complex with MHC class II. It may be recognized by specific receptors present on T cells and serve to provide signals required for B cells to differentiate into antibody-producing cells, or may induce cytotoxic T cells (CTLs) to induce lysis of target cells. In one embodiment, the T cell epitope may be a helper T cell epitope.

[0049] The helper T cell epitope may be a "pan-DR-binding peptide" or a "pan-DR-binding epitope." The terms "pan-DR-binding peptide" or "pan-DR-binding epitope" refer to a member of a group of molecules that binds to one or more MHC class II DR molecules. The helper T cell epitope binds to each of one or more MHC molecules at an IC of less than 100 nM, for example, at least 50 nM. 50 It may be a binding. The helper T cell epitope may be the pan DR-binding epitope disclosed or claimed in U.S. Patent 6,413,935 B1 (hereinafter also referred to as 'PADRE'). The helper T cell epitope may be Padre 1, 2, and 3 having the amino acid sequences of SEQ ID NOs 16, 17, and 18 listed in Table 10, or tetanus toxin (830-844).

[0050] The above immunogenic compound may be linked to a substance that increases immune activity. The substance that increases immune activity comprises a T cell epitope, a carrier protein, or a combination thereof. In this specification, "carrier protein" refers to a protein that is bound to make a small molecule hapten or epitope immunogenic. The above immunogenic compound comprises one of the first, second, and third epitopes, and the epitope may be bound to a T cell epitope, a carrier protein, or a combination thereof. Being bound to a combination thereof includes not only the T cell epitope and a carrier protein being bound to both ends of the epitope, but also the T cell epitope and a carrier protein being bound sequentially to the N-terminus or C-terminus. The above immunogenic compound may be, for example, such as the epitope-T cell epitope-carrier protein, in which the carrier protein is connected to a terminal and the T cell epitope is located between the epitope and the carrier protein, or such as the T cell epitope-the epitope-carrier protein, in which the carrier protein is connected to one end of the epitope and the T cell epitope is located at the other end.

[0051] Additionally, the immunogenic compound may comprise two or more of the first, second, and third epitopes. The epitopes may be connected to each other. The connection may be direct or indirect. The connection may be made by a linker, a T cell epitope, a carrier protein, or a combination thereof. The immunogenic compound comprises two or more of the first, second, and third epitopes, and the epitopes may be bound to a T cell epitope, a carrier protein, or a combination thereof. Being bound to a combination thereof includes not only the T cell epitope and a carrier protein being bound to both ends of the epitope, but also the T cell epitope and a carrier protein being sequentially bound to the N-terminus or C-terminus. The immunogenic compound may be such that the carrier protein is connected to the terminal, as in the epitope-T cell epitope-the epitope-carrier protein, and the T cell epitope is located between the epitopes. The T cell epitope may be a helper T cell epitope.

[0052] The above T cell epitope and carrier protein may be connected to the N-terminus or C-terminus of the epitope, or to the N-terminus or C-terminus of another T cell epitope or carrier protein. The connection may be made by a covalent bond, for example, a peptide bond.

[0053] The above linkage may be direct or via a linker. When linked via a linker, the function of the linked peptide, for example, inducing an immune response, may not substantially affect the function. Such a linker may be known (e.g., GGGSGGG (SEQ No. 29)).

[0054] The immunogenic compound may have the ApoB100 epitope linked to a carrier protein. The immunogenic compound may be prepared by a conventional method for manufacturing a conjugation vaccine. The immunogenic compound may be linked to the carrier protein directly or using a linker. The linkage may be by a covalent bond, for example, a peptide bond.

[0055] The carrier protein may be a known one. The carrier protein may be ferritin, Qβ, avidin, BTG protein, diphtheria toxin, influenza virus hemagglutinin (HA), bovine serum albumin (BSA), CRM197 (cross-reacting material 197), polylysine, HPH (Helix pomatia hemocyanin), human serum albumin, KLH (Keyhole Limpet Hemocyanin), tetanus toxin, tetanus toxin (830-844), or cholera toxin B subunit (CTB). The ferritin may be of rodent or mammalian origin. The ferritin may be of mouse or human origin. The carrier protein may be ferritin, and the ApoB100 epitope may be connected to the N-terminus of the ferritin.

[0056] The above immunogenic compound may have an amino acid sequence of any one of SEQ ID NOs 1 to 14 and 39 to 47.

[0057] The immunogenic compound may induce a B cell response to the ApoB100 epitope, for example, antibody production. The immunogenic compound may induce a B cell response to the first, second, and third epitopes, or a combination thereof, for example, antibody production.

[0058] The immunogenic compound may induce a T cell response to the T cell epitope, for example, T cell proliferation or differentiation. The immunogenic composition may induce a helper T cell response to the helper T cell epitope, for example, helper T cell proliferation or differentiation.

[0059] Another aspect provides a nucleic acid encoding the immunogenic compound. The nucleic acid comprises a gene encoding the immunogenic compound, or a structure containing the same, e.g., a vector. The vector may include a promoter, a regulatory sequence, or a combination thereof. The regulatory sequence may be a transcriptional or translational regulatory sequence. The promoter or regulatory sequence may be derived from the promoter or regulatory sequence of the gene.

[0060] Another aspect provides a composition comprising the above-mentioned immunogenic compound.

[0061] Another aspect provides an immunogenic composition comprising the immunogenic compound described above. The immunogenic compound is as described above.

[0062] The above composition may be intended to induce an immune response to the ApoB100 epitope and the T cell epitope. The immune response may be a B cell response, for example, the production of antibodies against ApoB100 by B cells. In this specification, "immunogenic composition comprising an ApoB100 epitope" is also referred to as "ApoB100 obesity vaccine," which is used interchangeably with "vaccine" and "ApoB100 vaccine." The antibody against ApoB100 may reduce the level of ApoB100 in the body, for example, in the blood. The above composition may reduce lipid levels in the body. The lipid levels may be lipid levels in the blood or lipid levels within cells. The lipids may be one or more of VLDL, IDL, and LDL containing ApoB100. The cells may be cells involved in lipid production, such as mast cells. The above composition may improve obesity. The above composition can reduce the body weight of an individual. The above composition can reduce the number of mast cells. The above composition can induce an immune response to T cell epitopes. The above composition can induce an immune response to help T cell epitopes.

[0063] The above immunogenic composition may be used to reduce the levels of VLDL, IDL, and LDL containing ApoB100 or ApoB100 in an individual.

[0064] In addition, the immunogenic composition may be used to prevent or treat diseases caused by increased levels of VLDL, IDL, and LDL containing ApoB100 or ApoB100.

[0065] The above diseases may be one or more of obesity, fatty liver disease, fatty liver, cardiovascular disease, dyslipidemia, atherosclerosis, increased AST (Aspartate aminotransferase), increased ALT (Alanine aminotransferase), diabetes, hypertension, degenerative arthritis, renal fibrosis, inflammatory vasculitis, and dementia.

[0066] The above cardiovascular disease may be hypertension, coronary artery disease, atherosclerosis, heart failure, myocardial infarction, reperfusion injury, stroke, ischemia, early heart or early cardiovascular disease, left ventricular hypertrophy, cardiomyopathy, angina pectoris, or ischemic cardiovascular disease.

[0067] The above immunogenic composition may be administered in combination with an obesity treatment drug.

[0068] The above obesity treatment drug may be one or more of an agent having one or more activities selected from the group consisting of GLP-1R agonists, GIPR agonists, and GCGR agonists, bupropion / naltrexone, N-lactoyl-phenylalanine, beta-hydroxybutyrate, dapagliflozin, phentermine, phentermine / topiramate, metformin, and orlistat.

[0069] One or more agonists having activity selected from the group consisting of the above GLP-1R agonists, GIPR agonists, and GCGR agonists may be semaglutide, exenatide, liraglutide, dapiglutide, epeglenatide, albiglutide, lixisenatide, tyrzepatide, duraglutide, orforglipron, survodutide, efinopegdutide, mazdutide, pemvidutide, cagrisema, danuglipron, combinations thereof, or derivatives to which albumin or IgG-Fc fusion protein is conjugated.

[0070] The immunogenic composition may be administered before, simultaneously with, or after the administration of the obesity treatment drug. The immunogenic composition may be formulated as a separate formulation or as the same formulation as the obesity treatment drug.

[0071] Currently known obesity treatments cause a rapid weight gain due to the yo-yo effect upon discontinuation of treatment. At this time, lipids accumulate first, inducing a worse form of obesity, which leads to a recurrence of the disease caused by increased blood pressure, blood sugar, and cholesterol (McKenzie Prillaman, Nature, 2024, Vol 628, issue 8008, pp.488-490). Recently, in the case of GLP-1R agonists, which are known for their excellent weight loss effects, GLP-1R agonists, GLP-1R / GIPR dual agonists, and GLP-1R / GIPR / GCGR triple agonists are being developed to enhance weight loss. However, the mechanism of action of GLP-1R agonist drugs causes osteoporosis and sarcopenia due to rapid weight loss through diet, and results in the yo-yo effect upon discontinuation of treatment.

[0072] As described above, when the immunogenic composition of the present invention is administered in combination with an obesity treatment drug, the mechanism of fat removal by the ApoB100-derived vaccine and the weight loss effect through appetite suppression by the GLP-1R agonist can enhance the weight loss effect and prevent muscle loss through a mutually complementary dual mechanism of action.

[0073] In this study, we confirmed the obesity improvement effect resulting from the monotherapy of the ApoB100 obesity vaccine according to the present invention, as well as the dual-action obesity treatment effect resulting from the combined administration of the said ApoB100 obesity vaccine and an obesity treatment drug. The said combined administration improved the problem of sarcopenia and enhanced the weight loss effect, thereby extending the weight maintenance period and delaying the yo-yo effect. Furthermore, it was confirmed that the said combined administration improved the sustainability of the disease treatment effect through the reduction of ApoB100 lipids induced by the said ApoB100 obesity vaccine and the subsequent inhibition of lipid storage.

[0074] The above composition may further comprise a pharmaceutically acceptable adjuvant. The adjuvant may be known. The adjuvant may be CIA06, CIA05, aluminum hydroxide (ALHYDROGEL®), aluminum phosphate, aluminum sulfate, L-Pampo, De-O-acylated monophosphoryl lipid A (MPL), QS-21, Quil A, ECL, complete Freund adjuvant (CFA), incomplete Freund adjuvant (IFA), oil-in-water emulsion, EMULSIGEN-D, PHAD, CpG, AddaVax™, MF59®, or a combination thereof. The adjuvant may induce an immune response synergistically with the epitope.

[0075] The above composition may comprise pharmaceutically acceptable carriers, excipients, diluents, or stabilizers. The above composition may comprise a pH-buffered aqueous solution. The above composition may comprise a buffer, e.g., phosphates, citrates, and other organic acids; an antioxidant, including ascorbic acid; a hydrophilic polymer, e.g., polyvinylpyrrolidone; an amino acid, e.g., glycine, histidine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; a chelating agent, e.g., EDTA; a sugar alcohol, e.g., mannitol or sorbitol; a salt-forming counterion, e.g., sodium; and / or a nonionic surfactant, e.g., polyoxyethylene sorbitan fatty acid ester (TWEEN ® )), polyethylene glycol (PEG), and polyoxyethylene / polyoxypropylene block copolymer (PLURONIC ® It may include ).

[0076] In addition, the above composition may further include fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc.

[0077] The above composition may be formulated using methods well known in the art so as to induce an immune response by one or several administrations after administration to mammals.

[0078] The above composition may be administered via various routes, including oral, transdermal, subcutaneous, intravenous, or intramuscular. The above composition may be administered intradermally, for example.

[0079] For humans, the usual single dose of the above composition may be in the range of 5 μg to 50 mg as the active ingredient, which is a peptide or protein. However, the actual dose of the active ingredient may be determined by taking into account various relevant factors such as immunization conditions, route of administration, patient condition, age, gender, and body weight.

[0080] Another aspect provides an immunogenic kit comprising the above-mentioned immunogenic compound. The kit may further comprise an obesity treatment agent. The obesity treatment agent may be intended for co-administration with the above-mentioned immunogenic compound. The use of the obesity treatment agent and the above-mentioned immunogenic kit is the same as described for the immunogenic composition comprising the above-mentioned immunogenic compound. The above-mentioned immunogenic compound may be formulated in a separate formulation from the obesity treatment agent. The above-mentioned immunogenic compound and the above-mentioned obesity treatment agent may be contained in separate containers.

[0081] Another aspect provides a method for reducing the level of ApoB100 or ApoB100-containing VLDL, IDL, LDL, or a combination thereof in an individual, comprising the step of administering the immunogenic compound to the individual. The method may reduce the body weight of the individual. Additionally, the method may be intended to prevent or treat a disease caused by an increase in the level of ApoB100 or ApoB100-containing VLDL, IDL, LDL, or a combination thereof in an individual.

[0083] Another aspect provides a method for preventing or treating a disease caused by increased ApoB100 levels, comprising the step of administering the immunogenic compound to an individual. The ApoB100 level may be a level in the body, for example, in the blood or within cells.

[0084] In this specification, the term "increase" indicates an increase compared to a control group, for example, a normal person.

[0085] The above-mentioned immunogenic compound and disease are as described above. The above-mentioned immunogenic compound may be in the form of the above-mentioned immunogenic composition containing the same.

[0086] The above individual may be an animal. The above animal may be a mammal. The above individual may be a human, cat, dog, pig, cow, sheep, horse, or mouse. The above individual may be a domestic animal.

[0087] The above method may further include a step of administering an obesity treatment drug.

[0088] The above obesity treatment drug may be one or more of the following: an agent having one or more activities selected from the group consisting of GLP-1R agonists, GIPR agonists, and GCGR agonists, such as semaglutide, exenatide, liraglutide, dapiglutide, epeglenatide, albiglutide, lixisenatide, tyrzepatide, duraglutide, orpogliprone, servodutide, epinopectutide, mazdutide, pembidutide, cagrisema, danuglyprone, combinations thereof, or derivatives to which albumin or IgG-Fc fusion protein is conjugated; bupropion / naltrexone, N-lactoyl-phenylalanine, beta-hydroxybutyrate, dapagliflozin, phentermine, phentermine / topiramate, metformin, and orlistat.

[0089] The above immunogenic compound may be administered before, simultaneously with, or after the administration of the obesity treatment drug. The above immunogenic composition may be formulated as a separate formulation or as the same formulation as the obesity treatment drug.

[0090] The above administration may be oral or parenteral. The above administration may be administered via various routes, including transdermal, subcutaneous, intravenous, or intramuscular. The above administration may be, for example, intradermal.

[0091] The above administration may include one or more boosting administrations after administration to the individual. The administration interval may be an interval commonly known in relation to boosting administration, e.g., 1 to 4 weeks, 2 to 3 weeks, 1 week, 2 weeks, 3 weeks, 4 weeks, or 8 weeks or more. The above administration may involve two or more boosting administrations after the first administration. The above administration may involve 1 to 5 times, 1 to 4 times, 1 to 3 times, 1, 2, 3, 4, or 5 times.

[0092] The above administration may be an effective amount for inducing an immune response to the ApoB100 epitope. The immune response may be a B cell response, for example, the production of antibodies against ApoB100.

[0093] The above administration may be administered as an immunogenic compound, which is the active ingredient, at a dose of 5 μg to 50 mg. However, the actual dosage of the active ingredient may be determined by taking into account various relevant factors such as immunization conditions, route of administration, patient condition, age, gender, and body weight. Effects of the invention

[0094] According to an immunogenic compound according to one aspect, an immune response against the ApoB100 epitope can be efficiently induced in an individual.

[0095] A composition containing an immunogenic compound according to a different aspect can be used to prevent or treat diseases caused by increased ApoB100 or lipid levels.

[0096] According to a method for preventing or treating diseases caused by increased levels of ApoB100 according to different aspects, diseases caused by increased levels of ApoB100 can be efficiently prevented or treated.

[0097] Currently known obesity treatments cause rapid weight gain due to the yo-yo effect when treatment is discontinued. During this process, lipids accumulate preferentially in the body, and this lipid accumulation leads to increases in blood pressure, blood sugar, and cholesterol levels, thereby triggering diseases caused by obesity again.

[0098] In order to further enhance the efficacy of GLP-1R agonists, which have excellent weight loss effects, GLP-1R / GIP dual agonists and GLP-1R / GIPR / GCGR triple agonists are being developed recently. However, since the above-mentioned GLP-1R agonist class of drugs has a mechanism that induces rapid weight loss through dietary suppression, osteoporosis and sarcopenia may occur, and the occurrence of the yo-yo effect upon discontinuation of treatment remains an unresolved problem.

[0099] According to the immunogenic composition of the present invention, it was confirmed that ALT levels are improved, fatty liver is alleviated, and cholesterol, triglycerides (TG), and ApoB levels are reduced due to the sustained lipid scavenging action of macrophages caused by the anti-ApoB100 antibody. Furthermore, it was confirmed that the weight loss effect of the obesity treatment is enhanced by these effects.

[0100] When a GLP-1R agonist was administered alone, body weight decreased by 8.86%, but when an ApoB100 obesity vaccine and a GLP-1R therapeutic agent were administered in combination, body weight decreased by 17.91% (see Example 4 and Table 16). When a GLP-1R agonist is administered alone for a long period, a body weight plateau occurs due to the body's homeostatic mechanism, where further weight loss does not progress. However, it was confirmed that the antibody formed by the ApoB100 obesity vaccine according to the present invention improves this body weight plateau phenomenon. According to the results of previous clinical studies, GLP-1R / GIPR dual agonists and GLP-1R / GIPR / GCGR triple agonists are reported to show improved therapeutic effects compared to GLP-1R monoagonists.

[0101] According to the present invention, combined administration of an ApoB100 obesity vaccine and a GLP-1R agonist improves muscle loss, limited duration of effect, weight plateau, and the occurrence of the yo-yo effect compared to administration of a GLP-1R agonist alone, and it was confirmed that the ApoB100 obesity vaccine enhances the weight loss effect by acting as a dual agonist. In conclusion, it was confirmed that the composition according to the present invention extends the duration of therapeutic effect compared to existing obesity treatments based on dietary control mechanisms.

[0102] In obesity treatments based on dietary control mechanisms, such as GLP-1R agonists, sarcopenia has been reported as a side effect of rapid weight loss. In the case of semaglutide preparations sold by Novo Nordisk, the rate of lean body mass loss during the weight loss process is reported to be about 40%. On the other hand, the combined administration of the ApoB100 obesity vaccine according to the present invention and a GLP-1R agonist was confirmed to increase the lean body mass ratio, and accordingly, it was confirmed that sarcopenia-related side effects observed in existing obesity treatments based on dietary control mechanisms can be alleviated. Brief explanation of the drawing

[0103] Figure 1 shows an example of aligning corresponding first, second, and third epitope regions in the amino acid sequences of ApoB100 proteins in humans, mice, cats, and dogs. Figure 2 shows the results of performing SDS-PAGE on recombinant proteins 7, 9, and 10. Figure 3 is a diagram showing the results of imaging recombinant proteins 9 and 10 with a TEM electron microscope. Figure 4 is a diagram illustrating the process of inducing immunity to a protein immunogenic compound in an individual. Figure 5 is a diagram showing the results of measuring liver weight and ALT after administering recombinant protein 9 (vaccine 2) or protein 11 (vaccine 41) to obese mice. Figure 6 is a graph showing triglyceride (TG), total cholesterol (T-CHO), and LDL levels after administering recombinant protein 9 (vaccine 2) or protein 11 (vaccine 41) to obese mice. Figure 7 is a diagram showing the results of Oil Red (top) and H&E (bottom) staining of liver tissue after administering recombinant protein 9 (vaccine 2) or protein 11 (vaccine 41) to obese mice. Figure 8 is a diagram illustrating the process of inducing immunity to a peptide immunogenic compound in an individual. Figure 9 shows the vaccine, semaglutide, semaglutide and This is a diagram showing body weight over time after vaccination. Fig. 10 shows the vaccine, semaglutide, semaglutide and This is a diagram showing the total body weight gain over the entire period after vaccination. Figure 11 is a figure showing the weight gain at 0, 8, and 12 weeks after the administration of the vaccine, semaglutide, and semaglutide. Figures 12a and 12b respectively show the weight gain during the obesity improvement efficacy test period (0-8 weeks) (Figure 12a) and the entire efficacy test period (0-12 weeks) after the administration of the vaccine, semaglutide, and the vaccine (Figure 12b). Figures 13a and 13b show the body fat percentage (Figure 13a) and lean body mass percentage (Figure 13b) measured by DXA. Figures 14a, 14b, and 14c are drawings showing liver tissue area (Figure 14a), fat (Figure 14b), and lean body mass level (Figure 14c) measured by DXA. Figure 15 shows the change in body weight over time after vaccination. Figure 16 is a diagram showing the body weight gain at 0-8 weeks after vaccination. Figure 17 shows the results of H&E staining analysis showing the fatty liver improvement effects of CN001B, CN001B and CN024, and CN001B and semaglutide. Figure 18 shows the results of administering CN001A and CN024 vaccines to mice along with a high-fat diet and measuring their body weight. Figure 19 is a diagram showing the results of H&E staining of liver tissue after administering CN001A and CN024 vaccines along with a high-fat diet to mice. Specific details for implementing the invention

[0104] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0105] 1. Materials and Methods

[0106] The amino acid sequences of the peptides or proteins used in the following examples are as follows.

[0107] (1) peptide or protein

[0108] [Table 10]

[0109]

[0110]

[0111] In Table 10, for peptides 17 and 18, a represents D-alanine and X represents cyclohexylalanine. Peptides 1 to 6 and 16 to 20 were chemically synthesized by Peptron (Daejeon) GeneScript or Peptide 2.0 (USA). Proteins 7 to 14 were produced using recombinant microorganisms by the following process.

[0112] (2) Production of proteins 7 to 14 using recombinant microorganisms

[0113] (2.1) Protein 7 to 14

[0114] Proteins 7 to 14 are fusion proteins in which immunogenic compounds 1-6 containing ApoB100 epitope peptides are linked to the N-terminus of ferritin. In the present invention, mouse ferritin was used.

[0115] In the above fusion protein, ferritin is present in eukaryotes and some microorganisms and consists of a single subunit composed of 200 amino acids formed into 24 multimers, having a cage shape with an outer diameter of 12 nm and an inner diameter of 8 nm. Due to its high symmetry and cage shape resulting from self-assembly, ferritin can be used as an efficient platform for vaccine development. The N-terminus of the ferritin subunit is located on the outside of the cage, while the C-terminus faces the inner empty space of the cage; thus, when an epitope peptide is attached to the N-terminus of ferritin, the ferritin maintains its ability to form a self-assembly polymer, allowing it to form a structure similar to a virus-like particle (VLP). The VLP structure of the ferritin-epitope fusion protein contains epitope peptides in a repetitive and high concentration that can induce strong T cell and B cell immune responses, making it useful as a vaccine.

[0116] The above fusion proteins were produced by culturing recombinant E. coli. The above fusion proteins can be formed by directly linking each of ApoB100 immunogenic peptides 1 to 6 to mouse ferritin, or by linking them via a linker, for example, GGGSGGG (SEQ ID No. 29). Specifically, the above fusion proteins 7 to 14 were produced as follows.

[0117] (2.2) Preparation and cultivation of recombinant microorganisms

[0118] The vector used was the pET His6 Sumo TEV LIC cloning vector (1S) (Addgene). The recombinant vector was prepared by introducing the respective fusion protein genes into the BamH1 and XhoI regions of the vector. In the vector, Hig6 can be used for the initial purification of the target protein, and the initially purified product can be used to cleave the product using Sumo protease so that only the target protein sequence remains.

[0119] The prepared recombinant vector contains the 6His-TEV-SUMO-target protein gene. When the target protein is peptide1-padre1-ferritin, i.e., protein 10, the gene has the nucleotide sequence of SEQ ID NO. 33. Protein 9 and its gene each have the amino acid sequence of SEQ ID NO. 9 and the nucleotide sequence of SEQ ID NO. 32.

[0120] Transformed E. coli were produced by introducing the above recombinant vector into E. coli. 10 mL of the seed culture of this E. coli was inoculated into a 2 L flask containing 1 L of LB medium containing 50 µg / mL kanamycin and 34 µg / mL chloramphenicol, and the OD was incubated in a stirred incubator at 37°C. 600 It was cultured until the value became 0.6 to 0.8.

[0121] When the target OD value was reached, 400 µl of 1M IPTG was added and incubated for 4 hours at 30°C in the stirred incubator. The OD value was measured before IPTG treatment and after the incubation was completely finished.

[0122] (2.3) Isolation and Purification of Recombinant Protein

[0123] After culture was complete, the cells were transferred to a centrifuge tube and centrifuged using an ultracentrifuge at 6,000 rpm for 20 minutes at 4°C to obtain only the pellet. The pellet was suspended in 9 mL of lysis buffer, transferred to a 50 mL conical tube, and stored overnight in a -70°C deep freezer. The cells stored in the deep freezer were thawed at room temperature. 10 mg of lysozyme dissolved in 1 mL of lysis buffer was added, and the mixture was incubated at room temperature for 1 hour using a rocker stirrer.

[0124] After sonicating for 30 seconds using a sonicator and the lysate became clear, the supernatant was transferred to a new 50 mL conical tube after centrifugation at 12,000 rpm for 20 minutes at 4 °C using an ultracentrifuge. 3 mL of His60 Ni superflow resin slurry was added to the sonicated supernatant according to the manufacturer's instructions and incubated on a rocker stirrer at room temperature for 1 hour to perform the binding reaction.

[0125] After the reaction was finished, centrifuge 700g for 3 minutes using a bulk centrifuge and transfer the supernatant to a tube.

[0126] 20 mL of washing buffer was added to suspend the resin, and the reaction was allowed to proceed for 3 minutes. The mixture was centrifuged at 700 g for 3 minutes, and the supernatant was transferred to a tube.

[0127] 6 mL of elution buffer was added to suspend the resin, and the mixture was reacted for 5 minutes. The mixture was centrifuged at 700 g for 3 minutes, and the supernatant was transferred to a tube.

[0128] The supernatant purified with resin was analyzed using 12% SDS-PAGE to check for protein bands during the elution stage, and the protein concentration was measured using the Bradford assay.

[0129] (2.4) Recombinant protein ulp1 cleavage

[0130] 1 mL of eluted protein (protein concentration 20 mg / mL to 25 mg / mL), 10 mM DTT, 25 μL of ulp1 protease, and 3,950 μL of lysis buffer were placed in a conical tube and reacted overnight in a 30°C water bath. After the reaction was complete, the protein was cleaved by 12% SDS-PAGE. Once cleaving was confirmed, 1 mL of His60 Ni superflow resin slurry was washed according to the manufacturer's instructions and added to the reaction mixture.

[0131] The mixture obtained in the Poly prep chromatography column (Bio-rad, Cat. No. 731-1550) was collected in tubes by flowing it in 1 mL aliquots. The solution obtained at this time was called flow through (FT)4.

[0132] Once all of the reaction mixture from Ulp1 had been flushed through the column, 4 mL of wash buffer was flushed through the column in 1 mL increments and collected in tubes. The resulting solution was designated as wash (W)2.

[0133] 4 mL of elution buffer was passed through the column in 1 mL increments and collected in tubes. The solution obtained at this time was called elution (E)3.

[0134] We confirmed that the column was properly separated using 12% SDS-PAGE and measured the protein concentration using the Bradford assay.

[0135] Figure 2 shows the results of performing SDS-PAGE on recombinant proteins 7, 9, and 10.

[0136] Figure 3 is a diagram showing the results of TEM electron microscopy of recombinant proteins 9 and 10. The purified recombinant proteins 9 and 10 are in the form of nanoparticles.

[0137] The above process is described as an example for recombinant proteins 9 and 10, and other proteins were produced using the same process.

[0138] Example 1: Development of a vaccine composition using synthetic peptides

[0139] In this embodiment, human ApoB100-derived immunogenic peptides were selected, and the immunogenicity of a fusion protein of these and a helper T cell epitope (hereinafter also referred to as 'Th epitope') was confirmed.

[0140] Selected human ApoB100-derived immunogenic peptides are the same as peptides 1 and 2 listed in Table 10.

[0141] Peptide 1 and peptide 2 correspond to residues 2196 to 2216 and 3662 to 3688, respectively, in the amino acid sequence of human ApoB100.

[0142] The above helper T cell epitope is a Pan Dr epitope having the amino acid sequences of peptides Padre 1, 2, and 3 listed in Table 10 ("PADRE TM ")am.

[0143] As a result, antibody titers were measured after chemically synthesizing peptides 3, 4, 5, or 6 and preparing a vaccine formulation.

[0144] Each peptide antigen, depending on its amino acid composition, is 20 mM sodium citrate (pH 6.2), up to 1% TWEEN ® It was dissolved to a final concentration of 10 mg / ml using a solvent selected from 20 mM sodium citrate (pH 6.2) and DMSO (Merck) to which 80 was added. The maximum concentration of the peptide solution used in this analysis was 100 μg / dose on a dosing basis.

[0145] Antigen solutions were first prepared for peptide antigens 3, 4, 5, or 6 at a final concentration of 50 μg / dose, respectively. The above antigen solutions were mixed with CIA06 (I-Gene Co., Ltd., Korea) to a concentration of 100 μg / dose based on the aluminum gel, and then homogenized by adding 20 mM sodium citrate buffer (pH 6.2). The mixture was slowly stirred using a mixer for 4 hours at room temperature, followed by additional stirring at 4°C for 16 hours to adsorb the antigen onto the aluminum gel, thereby preparing an inoculation vaccine formulation with a final inoculation volume of 200 μL / dose. Adjuvant CIA06 is an adjuvant comprising a non-toxic lipopolysaccharide (LPS) derivative derived from E. coli and an aluminum salt (alum), which stimulates the innate immune response to enhance the immune response to the antigen.

[0146] A Bradford assay was performed on the above vaccine formulation to measure the residual protein concentration in the formulation, and the degree of antigen adsorption to the aluminum gel contained in CIA06 was confirmed. As a result, it was confirmed that the peptide antigen was adsorbed to the aluminum gel by more than 95%. In the case of the control formulation, only the adjuvant and buffer were added, excluding the antigen.

[0147] The above vaccine formulations were administered to five C57BL / 6N mice under each condition, with a total of three doses administered at 2-week intervals at a dose of 200 μl / dose (50 μg / dose) via subcutaneous injection. Subsequently, blood samples were collected 2 weeks after the final vaccination, and the anti-ApoB100 antibody titer was measured using the ELISA method with the primary anti-ApoB100 antibody. Table 11 shows the ApoB100 antibody titers measured using serum samples obtained from mice.

[0148] [Table 11]

[0149]

[0150] As shown in Table 11, the groups administered peptides 3, 4, 5, and 6 had higher antibody titers against ApoB100 compared to the control group, and among them, the highest antibody titer was observed in the group administered peptide 3.

[0151] In addition, peptides P3, P3B, and P3C (SEQ No. 48), obtained by conjugating Padre 1 to the peptides P1, P1B, and P1C of Table 10, were prepared using the same process to produce an inoculation vaccine formulation. This vaccine formulation was administered to five C57BL / 6N mice under each condition, and a total of three doses were administered at 2-week intervals by subcutaneous injection at a dose of 200 μl / dose, i.e., 50 μg / dose. Subsequently, blood samples were collected 2 weeks after the final inoculation, and the anti-ApoB100 antibody titer was measured using the ELISA method with the anti-ApoB100 antibody. Table 12 shows the ApoB100 antibody titer measured using serum samples obtained from mice.

[0152] [Table 12]

[0153]

[0154] Example 2: Confirmation of Immunogenicity of Recombinant Proteins 9, 10, 11, and 12 Vaccines

[0155] In this example, the immunogenicity of an ApoB100-derived peptide conjugated with mouse ferritin (SEQ No. 15) was confirmed. The ferritin-conjugated ApoB100-derived recombinant protein is protein 9, 10, 11, or 12. The control is the ferritin recombinant protein of SEQ No. 15. Antibody titers were measured after inducing an immune response in C57BL / 6N mice by administering the peptide antigen.

[0156] Recombinant proteins 9, 10, 11, or 12 were first prepared as antigen solutions at a final concentration of 50 μg / dose, respectively. The antigen solution was mixed with CIA06 (I-Gene Co., Ltd., Korea) to a concentration of 100 μg / dose based on the aluminum gel, and then homogenized by adding 20 mM sodium citrate buffer (pH 6.2). The mixture was slowly stirred using a mixer at room temperature for 4 hours, and then further stirred at 4°C for 16 hours to adsorb the antigen onto the aluminum gel, thereby preparing an inoculation vaccine formulation with a final inoculation volume of 200 μL / dose.

[0157] A Bradford assay was performed on the above vaccine formulation to measure the concentration of residual protein in the formulation, and the degree of antigen adsorption to the aluminum gel contained in CIA06 was confirmed. As a result, it was confirmed that the recombinant proteins 9, 10, 11, or 12 antigens were adsorbed to the aluminum gel by more than 98%.

[0158] The above vaccine formulations were administered to five C57BL / 6N mice under each condition, with a total of three doses administered at 2-week intervals at a dose of 200 μl / dose (i.e., 50 μg / dose) via subcutaneous injection. Subsequently, blood samples were collected 2 weeks after the final vaccination, and the anti-ApoB100 antibody titer was measured using the ELISA method with the anti-ApoB100 antibody. Table 13 shows the ApoB100 antibody titers measured using serum samples obtained from blood collections after administering vaccine formulations of recombinant proteins 9, 10, 11, or 12 to obese mice. The control group was prepared by mixing the ferritin recombinant protein of SEQ ID NO. 15 with the adjuvant in the same way as the vaccine group and inoculated into C57BL / 6N mice.

[0159] [Table 13]

[0160]

[0161] As shown in Table 13, the vaccine groups administered recombinant proteins 9, 10, 11, or 12 showed significantly increased ApoB100 antibody titers compared to the control group. In particular, the vaccine groups administered recombinant proteins 9 or 11 showed significantly increased antibody titers compared to the control group. That is, when combining the ApoB100 immunogenic compound, helper T cell epitope, and ferritin, connecting the helper T cell epitope to the N-terminus of the ApoB100 immunogenic compound induced significantly increased antibody production compared to connecting the helper T cell epitope to the C-terminus of the ApoB100 immunogenic compound.

[0162] Example 3: Using a recombinant protein 9 or 11 vaccine Vibo efficacy in improving obesity test

[0163] After purifying the recombinant protein 9 or 11 with high antibody titer in Example 2, a vaccine formulation was prepared under the conditions of Example 2, and an efficacy test was performed in a diet-induced obese (DIO) C57BL / 6N mouse model.

[0164] Test animals were 5-week-old male C57BL / 6N mice acclimatized for 1 week, then divided into groups fed a normal rodent diet (NIH-41 KO 413125-75-684, Zeigler Bros) and a high-fat diet (60 kcal% fat, D12492, Research Diets) (hereinafter also referred to as 'HFD') and obese for 11 weeks. Obese C57BL / 6N mice were randomly assigned to groups of 8 and used in the study. After preparing vaccine formulations of recombinant proteins 9 or 11, they were administered three times at a dose of 200 μl / dose; blood samples and necropsy were performed 2 weeks later to confirm their potential as therapeutic vaccines. G1 and G2 control samples were prepared by mixing CIA06 with the ferritin recombinant protein (50 μg / head) of SEQ ID NO. 15.

[0165] Figure 4 is a diagram illustrating the process of inducing immunity to an immunogenic compound in an individual.

[0166] The normal diet group and the obesity-induced group were composed of 8 animals per group as follows.

[0167] G1 Control Lean Diet: A group fed a normal diet for rodents as a normal diet control and administered the ferritin recombinant protein vaccine of SEQ ID NO. 15 (50 μg / head).

[0168] G2 HFD: A group fed a high-fat diet as an HFD diet control and administered the ferritin recombinant protein vaccine of SEQ ID NO. 15 (50 μg / head).

[0169] G3 Vaccine 2: A group fed a high-fat diet and administered recombinant protein 9 (P9) vaccine (50 μg / head).

[0170] G4 Vaccine 41: A group fed a high-fat diet and administered recombinant protein 11 (P11) vaccine (50 μg / head).

[0171] Figure 5 and Table 14 show the results of measuring liver weight and ALT after administering recombinant protein 9 (vaccine 2) or protein 11 (vaccine 41) formulations to obese mice.

[0172] As shown in Figure 5, the liver weight of the obese mouse control group G2 significantly increased, while the G3 and G4 groups, administered with recombinant protein 9 (Vaccine 2) or protein 11 (Vaccine 41), showed a decreasing trend (left panel), and ALT levels also showed a decreasing trend (right panel). The decrease in liver weight indicates that recombinant protein 9 (Vaccine 2) or protein 11 (Vaccine 41) improves hepatic steatosis. Additionally, the decrease in ALT indicates that recombinant protein 9 (Vaccine 2) or protein 11 (Vaccine 41) improves liver damage and hepatic metabolic stress. Therefore, the data indicate that recombinant protein 9 (Vaccine 2) or protein 11 (Vaccine 41) can improve obesity-induced hepatic metabolic abnormalities (hepatic steatosis and indicators of liver damage).

[0173] In Figures 5 and 6, values ​​are mean ± SD, compared to the G1 control group * p<0.05, ** p<0.01, *** p<0.001, compared to the G2 HFD. † p<0.05, †† p<0.01, ††† p<0.001, statistics based on ANOVA and Dunnett's multiple comparison test.

[0174] [Table 14]

[0175]

[0176] Figure 6 and Table 15 show the results of measuring triglyceride (TG), total cholesterol (T-CHO), and LDL levels after administering the recombinant protein 9 or 11 vaccine to obese mice. Total cholesterol (T-CHO), LDL, and ApoB were measured using a biochemical analyzer (Hitachi 3500) on blood samples collected from the experimental animals.

[0177] [Table 15]

[0178]

[0179] As shown in Fig. 6, after necropsy 2 weeks following three doses of the vaccine in G3 and G4, an overall decreasing trend in TG, total cholesterol, and LDL levels was confirmed compared to the obese mouse control group G2. Fig. 7 shows the results of Oil Red (top) and H&E (bottom) staining of liver tissues after administering the recombinant protein 9 or 11 vaccine to obese mice. In Fig. 7, vaccine 2 and vaccine 41 represent the G3 and G4 groups of mice administered the recombinant protein 9 or 11 vaccine, respectively. As shown in Fig. 7, the Oil-red O staining analysis revealed a low fat ratio in the normal control group, whereas a high fat ratio was observed in the HFD control group. In the HFD vaccine group, after three doses of vaccine 2 and vaccine 41, the fat ratio decreased significantly compared to the HFD control group, reaching a level similar to that of G1 (normal control group) (top).

[0180] Consistent with the results of H&E staining analysis to identify adipocytes and Oil-red O staining analysis to identify fat, the proportion of adipocytes was high in the HFD control group (bottom). In the HFD vaccine group, after three doses of vaccine 2 and vaccine 41, the proportion of adipocytes decreased significantly compared to the HFD control group, reaching a level similar to that of the normal control group. This indicates that recombinant protein 9 (vaccine 2) or protein 11 (vaccine 41) is effective against fatty liver.

[0181] In this example, protein 9 or 11 is administered at a dose of 50 μg, of which the doses of peptide 1 and peptide 2 are about 5 μg. In the following Example 4, peptide 3 is administered at a dose of 50 μg, of which the dose of peptide 1 corresponds to about 27 μg.

[0182] This indicates that in the case of a conjugate vaccine in which antigen peptides such as peptide 1 and peptide 2 of the present invention are linked to a carrier protein such as ferritin, excellent immunogenicity can be induced with only low-dose administration. Through this, it was confirmed that the obese state in obese mice was improved by the administration of the vaccine of the present invention.

[0183] Example 4: Experiment to confirm the efficacy of ApoB100 peptide 3 vaccine, or ApoB100 peptide 3 vaccine in combination with semaglutide

[0184] 1. Exam Overview

[0185] GLP-1R agonists have side effects in the treatment of obesity, such as muscle loss and the yo-yo effect, along with weight loss. Weight gain resulting from the yo-yo effect causes lipid accumulation in the body, which increases the risk of various diseases, including elevated blood pressure, blood sugar, and cholesterol levels.

[0186] In this example, the effects of administering an ApoB100-derived peptide 3 (SEQ No. 3) vaccine composition (CN001) alone and in combination with semaglutide were evaluated in C57BL / 6N mice that had induced obesity with a high-fat diet. The purpose of the evaluation was to confirm whether there was improvement in obesity, improvement in sarcopenia, and improvement in the yo-yo effect.

[0187] The combination of the ApoB100-derived CN001 vaccine and semaglutide may be more effective than the administration of a GLP-1R agonist alone in the prevention and treatment of the following obesity-induced diseases: weight loss, diabetes and prediabetes, diabetic complications (neuropathy, renal disease), improvement of β-cell function, cardiovascular disease, stroke, hypertension, chronic kidney disease, overall improvement of lipid levels, dyslipidemia, fatty liver, non-alcoholic steatohepatitis, degenerative arthritis, Parkinson's disease, dementia, alcoholism, sleep apnea, arthritis, renal fibrosis, osteoarthritis, polycystic ovary syndrome, and sleep apnea.

[0188] 2. Composition of Peptide 3-Containing Vaccine Formulation

[0189] Peptide 3 was chemically synthesized in Peptron and dissolved in 20 mM sodium citrate buffer (pH 6.2) (TWEEN® 80 1% or less) so that the concentration of peptide 3 was 1 mg / ml.

[0190] The final vaccine formulation was prepared at a dose of 200 μl per dose. Specifically, 50 μg of peptide 3 antigen was mixed with 5 μg of QS21 as an adjuvant, 5 μg of ELS (MLP, manufactured by Eubiologics), and 100 μg of ALHYDROGEL® (Croda), and the vaccine composition was prepared by adjusting the amounts of the NaCl solution and buffer so that the final vaccine formulation was an isotonic solution containing 0.85% NaCl and buffered with 20 mM sodium citrate buffer (pH 6.2). A dose of 50 μg of peptide 3 antigen corresponds to a dose of approximately 27 μg of peptide.

[0191] A vaccine formulation for inoculation was prepared by slowly stirring a homogenized mixture containing peptide 3 antigen and adjuvant using a mixer at room temperature for 3 hours, and then stirring for an additional 16 hours at 4°C to allow the antigen to adsorb onto an aluminum gel.

[0192] Adjuvant QS21 (Invivogen) is a plant cell-derived saponin-based vaccine adjuvant. ELS is a monophosphoryl lipid A (MLP or MPL) known to enhance immune responses, including cellular immune responses, by stimulating the TLR4 pathway.

[0193] This vaccine formulation was named CN001. The control group was prepared by adding only the adjuvant and buffer, excluding the antigen.

[0194] 3. Construction of Obesity Induction Model and Material Processing

[0195] Test animals were 5-week-old male C57BL / 6N mice acclimatized for 1 week and then divided into groups receiving a normal rodent diet (NIH-41 KO 413125-75-684, Zeigler Bros) and a high-fat diet (60 kcal% fat, D12492, Research Diets), and obesity was induced for 11 weeks.

[0196] Figure 8 is a diagram illustrating the process of inducing immunity to an immunogenic compound in an individual.

[0197] The normal diet group and the obesity-induced group were composed of the following test groups, with 8 animals per group randomly assigned.

[0198] G1 Control group (ad): Normal diet + adjuvant administration.

[0199] G2 HFD control group (ad): high-fat diet + adjuvant administration.

[0200] G3 HFD control group (ws): High-fat diet + administration of working solution.

[0201] G4 CN001(ad): High-fat diet + CN001 administration.

[0202] G5 Semaglutide(ws): High-fat diet + Semaglutide administration.

[0203] G6 CN001(ad) + semaglutide(ws): High-fat diet + concomitant administration of CN001 and semaglutide.

[0204] Specifically, the G1 control group (ad) was fed a normal rodent diet and administered only the adjuvant used in the production of CN001, while the G2 HFD control group (ad) and the G3 HFD control group (ws) were each fed a high-fat diet and administered the adjuvant used in the production of CN001 and the working solution used in the production of semaglutide, respectively. The working solution was administered by diluting DMSO four times to PBS pH 7.2.

[0205] G4 CN001(ad) was a group fed a high-fat diet and administered CN001 (50 μg / head), and G5 semaglutide(ws) was a group fed a high-fat diet and administered semaglutide (300 μg / kg), with the number of administrations being the same as that of the G6 group. Semaglutide stock solution was dissolved in DMSO to 1 mg / ml, dispensed into 100 μl aliquots, and stored at -70℃; prior to administration, it was thawed, diluted with PBS, and administered at a dose of 300 μg / kg.

[0206] Semaglutide (CAS No. 910463-68-2) was purchased from Cayman Chemical and used.

[0207] G6 CN001(ad) + semaglutide(ws) is a group that was fed a high-fat diet and administered CN001 (50 μg / head) and semaglutide (300 μg / kg) in combination. CN001 (50 μg / head) was administered once every 2 weeks for a total of 5 times, and based on the total number of weekly administrations of G6 CN001(ad) + semaglutide(ws), the number of administrations was increased by 2 times in weeks 1 to 4 after the start of the study and by 1 time per week in weeks 5 to 8, while the control substance for each administered substance was administered the same number of times.

[0208] Note: The animals were weighed individually on a weekly basis, and the amount of solution administered per individual was calculated based on that weight and administered.

[0209] 4. Weight change

[0210] Individual body weight was measured upon acquisition, once a week during the study period (including body weight before administration), and immediately before necropsy after fasting. Additionally, changes in body weight during the study period were compared based on the individual body weight measurements.

[0211] The results are shown in Figures 9 and 10. Figure 9 is a diagram showing body weight over time after vaccine administration.

[0212] Figure 10 and Table 16 show the weight gain over the entire period after the vaccine, semaglutide, semaglutide, and vaccine administration.

[0213] [Table 16]

[0214]

[0215] Figure 11 and Table 17 are figures showing the weight gain at 0, 8, and 12 weeks after vaccination.

[0216] [Table 17]

[0217]

[0218] As shown in Figures 9 to 11, weight measurements showed that the high-fat diet control groups G2 and G3 maintained significantly higher weights compared to the normal diet group G1 throughout the entire test period, and G4, the CN001 administration group, showed a decrease compared to the high-fat diet group during the obesity improvement efficacy test period (0-8 weeks). G5, the GLP-1R agonist drug control group, showed an 8.86% weight reduction compared to before administration, and G6, the CN001 combination administration group, showed a 17.91% weight reduction. Greater weight loss was observed with the combination of CN001 and the GLP-1R agonist drug than with the combination of the drug alone. Additionally, according to the results in Figure 9, regarding changes in weight gain over the total test period, the time taken to reach the weight level at the start of the test after discontinuation of the drug was longer for the CN001 combination group (G6) compared to the GLP-1R agonist drug control group (G5), and it was confirmed that the slope of weight change between 11 and 12 weeks became flatter, confirming a delay in the yo-yo effect. Figures 12a and 12b are figures showing the weight gain during the efficacy test period (0-8 weeks or 0-12 weeks) after the administration of the vaccine, semaglutide, and the vaccine, respectively.

[0219] As shown in Fig. 12a, during the obesity improvement efficacy test period (0-8 weeks), significant weight loss was observed in the GLP-1R agonist drug control group (G5) and the CN001 combination administration group (G6) compared to the high-fat diet group, and a greater weight loss effect was observed when CN001 was administered in combination than when GLP-1R agonist was administered alone.

[0220] As shown in Fig. 12b, throughout the entire study period, significant weight loss was observed in the CN001 administration group (G4) and the GLP-1R agonist control group (G5) compared to the high-fat diet group, and the CN001 administration group showed lower weight gain. In particular, the greatest weight loss effect was observed in the CN001 combination administration group (G6).

[0221] 5. Body composition and liver analysis

[0222] Each test subject underwent body composition analysis via dual-energy radiation absorptiometry (DXA) measurements before administration and after the completion of the obesity improvement effect test, and the liver extracted during the autopsy underwent additional DXA measurements.

[0223] (1) Proportion of local fat (%)

[0224] Figures 13a and 13b show the body fat percentage (Figure 13a) and lean body mass percentage (Figure 13b) measured by DXA. According to Figure 13a, during the study period, the high-fat diet groups (G2 and G3) showed significantly higher body fat percentages than the normal diet group (G1). At week 8, the body fat percentage was significantly reduced in the GLP-1R agonist control group (G5) and the CN001 combination therapy group (G6).

[0225] (2) Lean body mass ratio (%)

[0226] According to Figure 13b, the lean body mass ratio relative to body weight was significantly lower in the high-fat diet groups (G2 and G3) than in the normal diet group (G1) throughout all study periods. At week 8, there was no significant change in the GLP-1R agonist control group (G5), but a significantly higher lean body mass ratio was observed in the CN001 combination therapy group (G6).

[0227] (3) Liver DXA analysis

[0228] FIGS. 14a, FIG. 14b, and FIG. 14c are drawings showing liver tissue area (Fig. 14a), fat (Fig. 14b), and lean body mass level (Fig. 14c) measured by DXA. It was confirmed that the fat weight of the liver decreased in the CN001 administration group (G4) and the combination administration group (G6).

[0229] (4) Statistical processing

[0230] All test results were expressed as Mean ± SEM, and comparisons between groups were performed using the statistical program GraphPad PRISM ® The study was performed using Version 5.0 (GraphPad Software, USA) via ANOVA and Dunnett's Multiple Comparison Test.

[0231] 6. Conclusion

[0232] In the case of GLP-1R agonists, a body weight plateau occurs after a certain period, even with long-term administration, where further weight loss ceases. Comparing the weight loss of 8.86% in G5 and 17.91% in G6 between weeks 0-8, combination therapy can enhance weight loss efficacy and resolve the issue of body weight plateau with the CN001 vaccine. Although weight loss occurs due to the efficacy of GLP-1R agonists, weight loss plateaus if administration continues beyond a certain period. Significant weight reduction was observed with the combination of GLP-1 and the vaccine between weeks 0-8. To develop obesity treatments with superior efficacy, GLP-1R / GIPR dual agonists and GLP-1R / GIPR / GCGR triple agonists are currently under development. While dual and triple agonists have increased weight loss efficacy, the yo-yo effect has not been resolved. Despite continuous administration of GLP-1R treatment, at the point where weight loss plateaued, the CN001 administration group (G6), which received five doses of the ApoB100 vaccine in combination, showed a decrease in weight compared to G5. Overall, the effect of the ApoB100 vaccine was observed after vaccination.

[0233] During the entire study period (weeks 0-12), a significant level of weight loss was observed in the CN001 combination therapy group (G6), and in the case of the CN001 monotherapy group (G4), weight loss was observed in the last week (Fig. 11).

[0234] In the group administered with semaglutide alone (G5), a GLP-1R agonist, weight loss occurred immediately, but a rebound effect occurred in which the weight reached the starting weight of the study in a short period of time upon discontinuation of administration. Although the weight loss effect of GLP-1R agonists appears rapidly due to dietary effects, in the group administered with the ApoB100-derived vaccine CN001 (G4), significant weight loss was achieved compared to the G3 group at the last 12 weeks depending on the number of vaccinations (Fig. 11). Weight increased rapidly simultaneously with the discontinuation of GLP-1R agonist alone administration (G5), but the administration of semaglutide and the CN001 vaccine (G6) delayed this rebound effect.

[0235] Therefore, the combined administration of a GLP-1R agonist and the CN001 vaccine can improve the yo-yo effect, in which lipid levels increase during rapid weight gain, and sustain the effect of improving obesity.

[0236] In human clinical trials of commercially available semaglutide, semaglutide treatment and lifestyle modifications resulted in an average weight reduction of 17.3%, but approximately two-thirds of the reduced weight was regained one year after discontinuation of the drug. This is the result of efficacy according to the administration of semaglutide treatment, diet, and exercise during the clinical trial; in the present invention, a weight reduction effect of 17.91% was obtained with the administration of semaglutide and the CN001 vaccine under HFD dietary conditions.

[0237] When GLP-1R agonist treatment is discontinued, there is a tendency for previously improved blood pressure, blood sugar, and cholesterol levels to worsen again in addition to weight gain due to the yo-yo effect, indicating that the duration of GLP-1R agonist efficacy is not long (Nature. Vol. 628. 2024). When ApoB100 vaccine and GLP-1R agonist are used in combination therapy, the vaccine can exert its effect within the antibody memory period of the ApoB100 vaccine, which can extend the duration of obesity treatment.

[0238] Among serum lipids, VLDL and LDL are major risk factors for adult diseases related to lipid metabolism. ApoB100 is a major protein component that binds to these low-density lipoproteins (LDL) and is present in IDL and VLDL; therefore, if antibodies in the blood are induced to recognize ApoB100, LDL and TG can be cleared by phagocytes.

[0239] GLP-1R agonists can be used as preventive and therapeutic agents for the following obesity-induced diseases: obesity, appetite suppression and weight loss, improvement of all forms of diabetes and prediabetic conditions, delay of diabetes exacerbation and the onset of complications such as diabetic neuropathy and diabetic nephropathy, improvement of β-cell cellular mass and function, preventive and therapeutic effects on cardiovascular diseases, stroke, hypertension, chronic kidney disease, overall improvement of lipid levels, dyslipidemia, fatty liver, improvement of AST and ALT, non-alcoholic steatohepatitis, degenerative arthritis, Parkinson's disease, dementia, treatment of alcoholism, prevention and treatment of sleep apnea, arthritis, and renal fibrosis, osteoarthritis, polycystic ovary syndrome, and sleep apnea.

[0240] When the above-mentioned GLP-1R agonist and the CN001 vaccine are administered in combination, the preventive and therapeutic effects of the GLP-1R agonist against the above-mentioned disease are enhanced, and the duration of effect can be extended by delaying the yo-yo effect.

[0241] The results of this example demonstrate that co-administration of a GLP-1 agonist and the vaccine of the present invention significantly increases lean body mass (%), thereby improving the side effects of sarcopenia. Antibody therapies are being developed to compensate for sarcopenia caused by long-term administration of GLP-1R agonists. Eli Lilly is conducting clinical trials of bimagrumab in combination with GLP-1R agonists, and Regeneron is developing trevogrumab as an antibody therapy for the prevention of sarcopenia. However, co-administration of GLP-1 agonists and antibodies is relatively expensive.

[0242] In the group administered the ApoB100 vaccine in combination (G6) of the present invention, a significant increase in lean body mass (%) was observed, confirming the effect of improving sarcopenia. Accordingly, when combined with a GLP-1R agonist, the side effects of sarcopenia can be mitigated cost-effectively compared to additional antibody treatments.

[0243] Example 5: Confirmation of obesity improvement and yo-yo effect improvement by combination therapy of ApoB100 peptide P3B vaccine and semaglutide

[0244] 1. Purpose of the test

[0245] In this example, the obesity-improving effects of the CN001B, CN001B and CN024 mixed vaccine and the obesity-improving and yo-yo effect of the CN001B and semaglutide combination therapy were confirmed through a DIO C57BL / 6N mouse model in which obesity was induced by administering a high-fat diet.

[0246] 2. Construction of Obesity Induction Model and Material Processing

[0247] After acclimatizing 5-week-old male C57BL / 6N mice for 1 week, starting from 6 weeks of age, they were divided into groups fed a normal rodent diet (NIH-41 KO 413125-75-684, Zeigler Bros) and a high-fat diet (60 kcal% fat, D12492, Research Diets) to induce obesity for 11 weeks.

[0248] The efficacy evaluation study consisted of a normal diet group (G1), five groups (G2-G6) formed by selecting only obese mice fed a 60 kcal diet and separating them into groups fed a 45 kcal diet, a high-fat diet control group (G2), a CN001B administration group (G3), a CN001B and CN024 administration group (G4), a GLP-1R agonist administration group (G5), and a GLP-1R agonist and CN001B administration group (G6). The test group consisted of 8 mice.

[0249] G1 Control group (ad): Normal diet + administration of Ajuvant (ad: Ajuvant).

[0250] G2 HFD control group (ad): 45 kcal high-fat diet + administration of Ajuvant (Ajuvant).

[0251] G3 CN001B(ad): 45 kcal high-fat diet + CN001B administration.

[0252] G4 CN001B(ad) / CN024(ad): 45 kcal high-fat diet + administration of CN001B and CN024.

[0253] G5 Semaglutide(ws): 45 kcal high-fat diet + administration of semaglutide (ws: working solution).

[0254] G6 CN001B(ad) + Semaglutide(ws): 45 kcal high-fat diet + concomitant administration of CN001B and semaglutide.

[0255] Specifically, the G1 control group (ad) was fed a normal rodent diet, while the vaccine group and HFD control group were fed a 45 kcal HFD high-fat diet. The vaccine groups (G3, G4, and G6) were administered CN001B (50 μg / head) and CN024 (50 μg / head) and were administered subcutaneously once every two weeks for a total of four doses at weeks 0, 2, 4, and 6.

[0256] In groups G5 and G6, semaglutide was administered three times a week (at intervals of 2-3 days) at concentrations of 30 μg / kg in week 1, 60 μg / kg in week 2, 120 μg / kg in week 3, 300 μg / kg in weeks 4, 5, and 6, and 400 μg / kg in weeks 7 and 8.

[0257] The dietary conditions were changed to 45 kcal HFD simultaneously with the start of administration to confirm the efficacy and yo-yo effect for 11 weeks.

[0258] Peptide P3B (Sequence No. 42) and peptide 20 (Sequence No. 44), which are peptides for vaccine production, were chemically synthesized in GeneScript, and peptide P3B and peptide 20 were dissolved in 20 mM phosphate buffer (pH 7.2) containing 0.9% NaCl (TWEEN® 80 ≤ 1%) to obtain a 1 mg / ml solution.

[0259] The final vaccine composition was prepared at a dose of 200 μl per dose. Specifically, 50 μg of peptide antigen was mixed with 5 μg of QS21 as an adjuvant and 5 μg of ELS (MLP, manufactured by Eubiologics), and the vaccine composition was prepared by adjusting the amounts of NaCl solution and buffer so that the final vaccine formulation was a 20 mM phosphate buffer containing 0.9% NaCl (pH 7.2). These vaccine compositions were named CN001B and CN024 (see Table 10). For the control group, only the adjuvant and buffer were added, excluding the antigen.

[0260] 3. Test Results

[0261] (1) Weight measurement : Efficacy evaluation test

[0262] As a result of weight measurement, the high-fat diet control group maintained significantly higher weight values ​​compared to the normal diet group throughout the entire study period. During the obesity improvement efficacy test period (0-8 weeks), the CN001B alone or CN001B and CN024 combination vaccine groups and the GLP-1R agonist and CN001 combination group showed significant weight loss compared to the high-fat diet control group, with the greatest weight loss observed in the GLP-1R agonist and CN001B combination group. During the yo-yo effect improvement efficacy test period (8-11 weeks), the GLP-1R agonist drug control group reached a level similar to the high-fat diet control group after 1 week, while the CN001B and CN024 group (G4) showed a significant difference from the high-fat diet control group (G2) by 2 weeks, and the GLP-1R agonist and CN001B combination group (G6) showed a significant difference by 3 weeks. Figure 15 and Table 18 show changes in body weight over time after vaccination.

[0263] [Table 18]

[0264]

[0265] As shown in Table 18, at 8 weeks, compared to the G2 HFD control group, the G3 group showed a weight loss of 14.88%, the G4 group 12.26%, the G5 group 8.54%, and the G6 group 24.25%, and at the end of the experiment at 11 weeks, the G3 group showed a weight loss of 9.23%, the G4 group 10.90%, the G5 group 2.95%, and the G6 group 10.96%.

[0266] Table 19 shows the weight gain from weeks 0-11 of administration.

[0267] [Table 19]

[0268]

[0269] In Example 4, the vaccine was administered 5 times while following a 60 kcal HFD diet for 0-12 weeks. Example 5 is the result of an obesity efficacy experiment in which the vaccine was administered 4 times while following a 45 kcal HFD diet for 0-11 weeks.

[0270] In Example 4, there was no weight loss from 0 to 12 weeks, but in Example 5, when measuring weight gain from 0 to 11 weeks, there was no weight gain in the G3 vaccine group, while weight loss was confirmed in G4 and G6. The G4 CN001B and CN024 combination vaccine group and the G6 CN001B and semaglutide combination administration group also showed a weight loss effect similar to that of the G4 group.

[0271] Unlike Example 4, in Example 5, to improve GLP-1RA drug compliance similar to human obesity treatment conditions in groups G5 and G6, the semaglutide inoculation dose was increased stepwise while administering it three times a week (at intervals of 2-3 days) at concentrations of 30 μg / kg in week 1, 60 μg / kg in week 2, 120 μg / kg in week 3, 300 μg / kg in weeks 4, 5, and 6, and 400 μg / kg in weeks 7 and 8. In the case of semaglutide alone in group G5, the weight loss effect was negligible up to the treatment concentration of 120 μg / kg in week 3, and although the semaglutide inoculation dose was increased to 300 μg / kg or more starting from week 4, there was no significant weight loss.

[0272] On the other hand, in the group administered the G6 vaccine and semaglutide combination, significant weight loss began to appear after the administration of 120 μg / kg of semaglutide at week 3 and two doses of the vaccine. By week 7, weight decreased similarly to the G1 group. In Example 4, vaccination and semaglutide administration were carried out until week 8, but a rebound effect occurred in the group administered the G6 vaccine and semaglutide combination when discontinued; however, in Example 5, semaglutide was administered for two weeks after the fourth vaccination, delaying the rebound effect, which appeared after two weeks.

[0273] Figure 16 shows body weight gain 0–8 weeks after vaccination. In Figure 16, values ​​are expressed as mean ± SEM (n=6–8). Statistical analysis was performed by ANOVA and Tukey's multiple comparison test. Compared to the HFD control group (G2), *p<0.05, **p<0.01, ***p<0.001

[0274] (2) Lean body mass and body fat ratio

[0275] As a result of measuring the fat and lean body ratios, it was confirmed that the fat (%) was significantly higher in the high-fat diet control group compared to the normal diet group, and at week 8, a significant decrease in the fat ratio compared to the high-fat diet control group was confirmed only in the group administered with a combination of a GLP-1R agonist and CN001B, while a decreasing trend was confirmed in other administration groups.

[0276] The lean body mass ratio was found to be significantly lower in the high-fat diet control group compared to the normal diet group, and at week 8, a significant increase in the lean body mass ratio compared to the high-fat diet control group was observed only in the group administered with a combination of a GLP-1R agonist and CN001B, while an increasing trend was observed in other administration groups. Table 20 shows the results of measuring lean body mass and fat ratios for the CN001B, CN001B and CN024 combined vaccine group, GLP-1R agonist, and GLP-1R agonist and CN001B combination administration group.

[0277] [Table 20]

[0278]

[0279] (3) Fatty liver improvement effect

[0280] H&E staining analysis to identify adipocytes showed that in the HFD vaccine groups, the adipocyte ratio in the CN001B, CN001B and CN024, and CN001B and semaglutide groups was significantly reduced compared to the HFD control group after four doses of the vaccine, resulting in a reduction of adipocytes to a level similar to that of the normal control group. This indicates that the CN001B and CN024 vaccines are effective against fatty liver. For the monotherapy, combination vaccine, or vaccine and semaglutide combination groups, H&E staining analysis after autopsy at 5 weeks post-fourth dose confirmed that, compared to G5 semaglutide, the induction of fatty liver was delayed in all vaccine administration groups even under HFD dietary conditions due to the vaccine effect (Fig. 17).

[0281] Figure 17 shows the results of H&E staining analysis showing the fatty liver improvement effects of CN001B, CN001B and CN024, and CN001B and semaglutide.

[0282] 4. Conclusion

[0283] In the efficacy evaluation test, it was confirmed that when CN001B was administered in combination with low-dose semaglutide, an existing obesity treatment, during the obesity improvement efficacy evaluation period, the effect of improving obesity was superior to that of the same low-dose CN001B obesity treatment alone. It was confirmed that when CN001B was administered in combination with the existing obesity treatment, body weight, fat percentage, muscle percentage, and fatty liver improved to levels similar to the normal diet group, indicating that CN001B combination administration is more effective than existing GLP-1R agonist obesity treatments.

[0284] When CN001B, CN024, and a mixture of these were administered simultaneously with a high-fat diet, significantly lower body weight gain, along with significantly lower body fat percentage and higher lean body mass percentage, were observed compared to the high-fat diet group, and a similar trend of lower body fat percentage and higher lean body mass percentage was also observed in the liver. Therefore, it is believed that CN001B and CN024 inhibit obesity by suppressing the production of body fat caused by dietary intake.

[0285] When comprehensively considering the results of efficacy evaluation tests and metabolic evaluation tests, it is determined that CN001B and CN024 are effective in improving obesity.

[0286] Example 6: Metabolic disease prevention test using CN001A and CN024 vaccines

[0287] In this embodiment, CN001A and CN024 vaccines were administered to mice along with a high-fat diet to treat metabolic diseases The preventive effect was confirmed.

[0288] Mice were divided into groups of 5. 5-week-old male C57BL / 6N mice were received and acclimatized for about 1 week, after which a high-fat diet was applied starting at 6 weeks of age, and vaccines were administered 4 times at 2-week intervals. The peptide sequences used for vaccination were peptide P3A (sequence number 41) and peptide P20 (sequence number 44).

[0289] The formulations of the vaccine group and the control group were prepared under the same conditions as in Example 5, and the vaccine compositions for the peptides P3A and P20 were named CN001A and CN024, respectively (see Table 10). Body weight was measured once a week. Autopsy was performed 2 weeks after the last vaccination. The experimental conditions are as shown in Table 21 below. After the autopsy at week 8, tissue weights were measured, and antibody titers against anti-ApoB100 were measured in blood using the ELISA method. Total cholesterol, LDL, and ApoB content were measured by biochemical analysis. Total cholesterol (T-CHO), LDL, and ApoB were measured in blood samples collected from the experimental animals using a biochemical analyzer (Hitachi 3500).

[0290] H&E staining analysis was performed on liver samples to confirm the preventive effect against fatty liver. Lean mass and fat mass were measured using DXA.

[0291] [Table 21]

[0292]

[0293] 1. Experimental Results

[0294] (1) Measurement of weight change: Metabolic disease prevention test

[0295] As a result of weight measurement, the high-fat diet control group maintained significantly higher body weight compared to the normal diet group starting 2 weeks after the start of the study, and both the CN001A and CN024 administration groups showed significantly lower body weight compared to the high-fat diet control group (G2) starting from 3 or 4 weeks (Fig. 18 and Table 22).

[0296] Figure 18 and Table 22 show the results of administering CN001A and CN024 vaccines to mice fed a high-fat diet and measuring their body weight. In Figure 18, values ​​are mean ± SEM (n=5). Statistical analysis was performed by ANOVA and Dunnett's multiple comparison test. Compared to the control group (G1), †p<0.05, ††p<0.01, and †††p<0.001. Compared to the HFD control group (G2), *p<0.05 and **p<0.05.

[0297] As shown in Figure 18, compared to the G2 group high-fat diet group, weight gain was suppressed by 22.02% and 17.93% in the G3 group (CN001A) and G4 group (CN024), respectively. This indicates that the ApoB100 vaccine has an effect in preventing obesity.

[0298] [Table 22]

[0299]

[0300] (2) Measurement of lean body mass and fat ratio: Metabolic disease prevention test

[0301] Lean mass and fat mass were measured in experimental animals to determine whether the ApoB100 vaccine has a mechanism to inhibit fat accumulation. As a result, it was confirmed that compared to the high-fat diet group, the CN001A and CN024 vaccine groups showed an increased lean mass ratio and a decreased fat ratio.

[0302] Table 23 shows the results of measuring lean body mass and fat percentage after administering CN001A and CN024 vaccines to experimental animals along with a high-fat diet.

[0303] [Table 23]

[0304]

[0305] (3) Fatty liver inhibitory effect

[0306] Liver tissue was obtained from experimental animals and H&E staining analysis was performed. Figure 19 shows the results of H&E staining of liver tissue after administering CN001A and CN024 vaccines to mice along with a high-fat diet. It was confirmed that the ratio of intrahepatic fat increased in the high-fat diet group compared to the normal diet group. It was confirmed that fat accumulation was inhibited in the CN001A and CN024 vaccine groups. This indicates that the CN001A and CN024 vaccines specifically inhibit fat accumulation. Therefore, the present composition can prevent lipid metabolism diseases, including fatty liver.

[0307] (4) Biochemical analysis results

[0308] Total cholesterol (T-CHO), LDL, and ApoB content were measured by biochemical analysis. Total cholesterol (T-CHO), LDL, and ApoB were measured on blood samples collected from experimental animals using a biochemical analyzer (Hitachi 3500).

[0309] Table 24 shows the results of measuring total cholesterol (T-CHO), LDL, and ApoB levels in the blood of mice after administering the CN001A and CN024 vaccines along with a high-fat diet. As shown in Table 24, all three indicators were normal in the G1 group, while they all exceeded the reference values ​​in the G2 group. On the other hand, T-CHO, LDL, and ApoB levels in the G3 CN001A and G4 CN024 vaccine groups were significantly reduced compared to G2. This indicates that the CN001A and CN024 vaccines inhibit the deterioration of lipid metabolism, thereby having a preventive effect against the occurrence of lipid metabolism disorders. The aforementioned lipid metabolism disorder may be hyperlipidemia.

[0310] [Table 24]

[0311]

[0312] (5) Conclusion

[0313] When the CN001A and CN024 vaccines were administered to experimental animals simultaneously with a high-fat diet, respectively, body weight gain was significantly inhibited in the CN001A and CN024 vaccine groups compared to the high-fat diet control group, and the body fat percentage decreased while the lean body mass percentage increased. In addition, a similar trend of decreased body fat percentage and increased lean body mass percentage was confirmed in liver tissue. Furthermore, biochemical analysis results showed that total cholesterol, LDL, and ApoB levels were significantly reduced in the vaccine groups compared to the high-fat diet control group.

[0314] Therefore, it is determined that the CN001A and CN024 vaccines have an effect of preventing the occurrence of obesity by suppressing body fat accumulation induced by a high-fat diet.

Claims

Claim 1 A peptide fragment of the amino acid sequence of SEQ ID NO. 25 comprising residues 2205 to 2210 of SEQ ID NO. 25 and having a length of 10 to 30 amino acids; or a first epitope composed of a peptide fragment having 60% or more sequence identity with said peptide fragment; a peptide fragment of the amino acid sequence of SEQ ID NO. 25 comprising residues 3662 to 3688 of SEQ ID NO. 25 and having a length of 10 to 30 amino acids; or a second epitope composed of a peptide fragment having 60% or more sequence identity with said peptide fragment; and a peptide fragment of the amino acid sequence of SEQ ID NO. 25 comprising residues 3380 to 3394 of SEQ ID NO. 25 and having a length of 10 to 30 amino acids; An immunogenic compound comprising one or more ApoB100 epitopes selected from the group consisting of: a third epitope composed of a peptide fragment having 60% or more sequence identity with the above peptide fragment. Claim 2 The immunogenic compound of claim 1, wherein the ApoB100 epitope is linked to a helper T cell epitope, a carrier protein, or a combination thereof. Claim 3 The immunogenic compound of claim 2, wherein the connection is connected to the N-terminus, C-terminus, or both ends of the ApoB100 epitope by a peptide bond. Claim 4 The immunogenic compound of claim 2, wherein the helper T cell epitope is Padre 1, 2, and 3 having the amino acid sequences of SEQ ID NOs 16, 17, and 18, respectively, or tetanus toxin. Claim 5 The immunogenic compound of claim 2, wherein the carrier protein is ferritin, Qß, avidin, BTG protein, diphtheria toxin, influenza virus hemagglutinin (HA), bovine serum albumin (BSA), CRM197, polylysine, HPH, human serum albumin, KLH, polylysine, tetanus toxin, tetanus toxin (830-844) or cholera toxin B subunit. Claim 6 The immunogenic compound of claim 1, wherein the ApoB100 epitope is composed of the human ApoB100 immunogenic fragment of SEQ ID NOs. 1, 2, 39, 40, 43, 45, 46, and 47, or the corresponding non-human mammalian ApoB100 immunogenic fragment. Claim 7 The immunogenic compound of claim 1, wherein the corresponding ApoB100 immunogenic fragment of a non-human mammal is an ApoB100 immunogenic fragment of a mouse, cat, or dog, and is an immunogenic compound composed of the amino acid sequence of SEQ ID NOs 19, 20, 21, 22, 23, 24, 49, 50, or 54. Claim 8 The immunogenic compound of claim 1, comprising any one of the amino acid sequences of SEQ ID NOs 1 to 14 and 39 to 46. Claim 9 An immunogenic composition comprising any one of claims 1 to 9 immunogenic compounds. Claim 10 The immunogenic composition of claim 9, which is intended to induce an immune response to the ApoB100 epitope. Claim 11 The immunogenic composition of claim 9 for use in preventing or treating diseases caused by increased ApoB100 levels or lipid levels. Claim 12 The immunogenic composition of claim 11, wherein the disease is one or more of coronary artery disease, atherosclerosis, heart failure, myocardial infarction, reperfusion injury, ischemia, early heart or early cardiovascular disease, left ventricular hypertrophy, cardiomyopathy, angina pectoris, and cardiovascular disease including ischemic cardiovascular disease, dyslipidemia, obesity, steatohepatitis, fatty liver, increased AST, increased ALT, diabetes, hypertension, stroke, degenerative arthritis, renal fibrosis, inflammatory vasculitis, and dementia. Claim 13 An immunogenic composition in any one of claims 9 to 12, wherein the immunogenic compound is administered in combination or concurrently with an obesity treatment drug. Claim 14 The immunogenic composition of claim 13, wherein the effect of increasing weight loss by the obesity treatment drug, improvement of weight loss plateau, alleviation of sarcopenia side effects, delay of yo-yo effect, or improvement of weight loss plateau, alleviation of sarcopenia side effects and delay of yo-yo effect. Claim 15 An immunogenic composition according to claim 13 or 14, wherein the obesity treatment agent is one or more of a GLP-1R agonist, a combination of bupropion and naltrexone, N-lactoyl-phenylalanine, beta-hydroxybutyrate, dapagliflozin, phentermine, a combination of phentermine and topiramate, metformin, and orlistat. Claim 16 The immunogenic composition of claim 9, further comprising an adjuvant, an excipient, or a combination thereof. Claim 17 An immunogenic kit comprising any one of claims 1 to 8, an immunogenic compound, and an obesity treatment agent. Claim 18 A method for preventing or treating a disease caused by increased ApoB100 levels or lipid levels, comprising the step of administering an immunogenic compound of any one of claims 1 to 8 to an individual. Claim 19 A method according to claim 18, further comprising the step of administering an obesity treatment agent to the subject.

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