KR102261457B1 - A peptide used for Immunotherapeutics - Google Patents

Abstract

The present invention provides a peptide comprising 1) a B-cell epitope, 2) a protective part, and 3) a Th epitope. The B-cell epitope and the protective part combine to form a Th-epitope-protective epitope. The peptide is characterized in that the protective part is linked to the N'-terminus and C'-terminus of the Th epitope, and when injected into a body of a subject, an antibody that specifically binds to an intentionally designed B-cell epitope is uniformly induced. In addition, since the peptide contains a small number of B-cell and Th epitopes, the prsesent invention has advantages of easy production and economical efficiency. The peptide may be used as an immunotherapeutic agent according to its characteristics. Accordingly, a peptide designed to be used as an immunotherapeutic agent for obesity and a pharmaceutical composition thereof are provided.

Description

Peptide for immunotherapy {A peptide used for Immunotherapeutics}

The present invention relates to a peptide that is injected into the body of a subject to induce a humoral immune response, and is a technology in the field of immunotherapy.

The immunotherapeutic agent is introduced into the body of a subject and induces a humoral immune response to the immunotherapeutic agent itself, and the purpose of the immunotherapeutic agent is to treat a specific disease or disease by the resulting antibody. In this case, the "treatment" includes preventing a specific disease or disease. An immunotherapeutic agent is similar to a vaccine in that it induces antibody production through an antigen-antibody reaction in a subject's body, but the antibody has not only the ability to bind to the immunotherapeutic agent itself, but also a specific subject in the body (e.g., a specific tissue in the body, It has the ability to bind to cells or substances produced in the course of metabolism), so it can treat specific diseases or diseases, and it is different in that it can be administered repeatedly.

An object of the present specification is to provide a peptide having a function of inducing a predesigned antibody in the body of a subject.

An object of the present specification is to provide a composition for immunotherapy containing the peptide.

An object of the present specification is to provide the use of the peptide and a composition for immunotherapy containing the peptide.

In order to solve the above problems, the present specification provides a peptide comprising:

helper-T-cell epitopes (Th epitopes);

a first protection unit; and

a Th-epitope-protecting epitope (TEPE) comprising a second protective moiety and a B-cell epitope;

At this time, in the peptide sequence, the sequence of the Th epitope is between the sequence of the first protective part and the sequence of the TEPE,

The length of the Th epitope is 8mer to 30mer,

The TEPE has a sequence selected from the group consisting of SEQ ID NOs: 41 to 75,

The Th epitope has a binding ability to MHC Class II, whereby the Th epitope can be recognized by CD4+ T cells when the peptide is administered to a subject,

The Th epitope is directly linked to the first and second protection groups, and thus the Th epitope can be protected from being cleaved by enzymes in the subject's body, thereby allowing the peptide to bind to the subject. When administered, it can induce a humoral immune response involving CD4+ T cells.

In one embodiment, each of the first protecting part and the second protecting part may include at least one non-natural amino acid.

In another embodiment, each of the first protecting moiety and the second protecting moiety may include at least 6 amino acids.

In one embodiment, the first protective part may have a sequence selected from the group consisting of SEQ ID NOs: 21 to 40 and 78.

In one embodiment, the Th epitope may include a sequence selected from the group consisting of SEQ ID NOs: 17 to 20, and 77.

In one embodiment, the CD4+ T cells may be naive T cells, Th1 cells, Th2 cells, or regulatory T cells (Tregs).

In an embodiment, the Th epitope may be represented by the following [Structural Formula 2]:

[Structural Formula 2]

N'-K-(Cha)-X ₁ -AAX _{2 -TX 3} -X ₄ -AA-C'

In this case, (Cha) is L-cyclohexyllanins;

wherein X ₁ is a hydrophobic amino acid;

wherein X ₂ is an aromatic or cyclic amino acid;

wherein X ₃ is aliphatic long chain amino acids; and

and X ₄ is a charged amino acid.

In one embodiment, the enzyme in the target body may be one or more selected from the group consisting of endopeptidase (endopeptidase) and exopeptidase (exopeptidase).

In one embodiment, as a result of the induction of the humoral immune response, an IgG-type antibody may be induced.

In order to solve the above problems, the present specification provides a peptide comprising:

helper-T-cell epitopes (Th epitopes);

a first Th-epitope-protecting epitope (TEPE) comprising a first protective moiety and a first B-cell epitope; and

a second Th-epitope-protecting epitope (TEPE) comprising a second protective moiety and a second B-cell epitope;

In this case, in the peptide sequence, the sequence of the Th epitope is between the sequence of the first TEPE and the sequence of the second TEPE,

The length of the Th epitope is 8mer to 30mer,

The first TEPE has a sequence selected from the group consisting of SEQ ID NOs: 41 to 75,

The second TEPE has a sequence selected from the group consisting of SEQ ID NOs: 41 to 75,

The Th epitope has a binding ability to MHC Class II, whereby the Th epitope can be recognized by CD4+ T cells when the peptide is administered to a subject,

The Th epitope is directly linked to the first and second protection groups, and thus the Th epitope can be protected from being cleaved by enzymes in the subject's body, thereby allowing the peptide to bind to the subject. When administered, it can induce a humoral immune response involving CD4+ T cells.

In one embodiment, each of the first protecting part and the second protecting part may include at least one non-natural amino acid.

In one embodiment, each of the first protecting portion and the second protecting portion may include at least 4 amino acids.

In one embodiment, the Th epitope may include a sequence selected from the group consisting of SEQ ID NOs: 17 to 20 and 77.

In one embodiment, the CD4+ T cells may be naive T cells, TH1 cells, or TH2 cells.

In an embodiment, the Th epitope may be represented by the following [Structural Formula 2]:

[Structural Formula 2]

N'-K-(Cha)-X ₁ -AAX _{2 -TX 3} -X ₄ -AA-C'

In this case, (Cha) is L-cyclohexyllanins;

wherein X ₁ is a hydrophobic amino acid;

wherein X ₂ is an aromatic or cyclic amino acid;

wherein X ₃ is aliphatic long chain amino acids; and

and X ₄ is a charged amino acid.

In one embodiment, the enzyme in the target body may be one or more selected from the group consisting of endopeptidase (endopeptidase) and exopeptidase (exopeptidase).

In one embodiment, as a result of the induction of the humoral immune response, an antibody of the IgG isotype may be induced.

In order to solve the above problems, the present specification provides a pharmaceutical composition for immunotherapy for obesity comprising:

at least one of the above peptides; and

adjuvants.

In one embodiment, the adjuvant may be at least one selected from the group consisting of aluminum hydroxide [Al(OH)3], AS04 series, MF, liposome, squalene, MF59, and QS21.

In one embodiment, the pharmaceutical composition may additionally include one or more immune enhancers selected from the group consisting of phosphoryl lipid A, saponin, prolactin, growth hormone deoxycholic acid, betaglucan, and polyribonucleotides.

When the peptides provided herein are injected into the body of a subject, they specifically bind to a previously designed antigenic site and have the effect of inducing the production of antibodies with specific physiological functions.

1 is a graph showing changes in body weight for each week according to Experimental Example 6.3 after administering a composition according to the method of Experimental Example 5.1 to the mice of Experimental Group 1-1 of Experimental Example 4.1.
Figure 2 is a graph showing changes in body weight for each week according to Experimental Example 6.3 after administering the composition in the method of Experimental Example 5.1 to the mice of Experimental Group 1-2 of Experimental Example 4.1.
3 is a graph showing changes in body weight according to age of experimental group 1-1 and experimental group 1-2 of Experimental Example 4.1 by week.
FIG. 4 is a graph showing changes in body weight for each week according to Experimental Example 6.3 after each composition was administered to the mice according to Experimental Example 4.2 by the method of Experimental Example 5.2. Lean denotes a normal weight control group, Obesity denotes an obese group induced by a high-fat diet, Mock denotes a placebo-administered group, and 3H-OTP denotes experimental group 2-1.
Figure 5 is a graph showing the measurement of antibody titer (antibody titer) according to Experimental Example 6.1 after administering each composition in the method of Experimental Example 5.2 to the mice to be tested according to Experimental Example 4.2. Obesity represents the obesity group induced by a high-fat diet, Mock represents the placebo-administered group, and 3H-OTP represents the experimental group 2-1.
Figure 6 is after each administration of the composition in the method of Experimental Example 5.2 to the test subject mice according to Experimental Example 4.2, by measuring the lipolytic capacity of hormone sensitive lipase in adipocytes by the method of Experimental Example 7.2. is the graph shown. Lean denotes a normal weight control group, Obesity denotes an obese group induced by a high-fat diet, Mock denotes a placebo-administered group, and 3H-OTP denotes experimental group 2-1. Basal represents no norepinephrine treatment, and Hormone represents norepinephrine treatment. (a) ^{Glycerol secretion concentration per 10 5} adipocytes, (b) Glycerol secretion concentration per 1 g adipocyte.
7 is a photograph showing the size of adipocytes measured by the method of Experimental Example 7.2 after administering the composition in the method of Experimental Example 5.2 to the mice to be tested according to Experimental Example 4.2, respectively, a DAPI staining image, a LipidTOX staining image, and Merge images. Here, Lean represents a normal weight control group, and Obese represents an obese group induced by a high-fat diet.
8 is a photograph showing the size of adipocytes measured by the method of Experimental Example 7.2 after each composition was administered in the method of Experimental Example 5.2 to the mice to be tested according to Experimental Example 4.2. DAPI staining images are shown. In this case, Lean is a normal weight control group, Obese is an obesity group induced by a high-fat diet, Mock is a placebo-administered group, and 3H-OTP indicates experimental group 2-1.
9 is a graph showing the measurement of the blood lipid concentration by the method of Experimental Example 7.1 after administering each composition to the experimental mouse according to Experimental Example 4.2 by the method of Experimental Example 5.2. TG is triglyceride, NEFA is non-esterified fatty acid, CHOL is cholesterol, HDL is high-density lipoprotein, and LDL is low-density lipoprotein. lipoprotein).
10 is a graph showing changes in body weight for each week according to Experimental Example 6.3 after each composition was administered to the mice to be tested according to Experimental Example 4.3 by the method of Experimental Example 5.3. Lean denotes a normal weight control group, Obese denotes an obesity group induced by a high-fat diet, Mock denotes a placebo-administered group, 3H-OTP 30 ug denotes experimental group 3-2, and 3H-OTP 50 ug denotes experimental group 3-1. For reference, a graph for 50 ug of 3H-OTP-2W, which means experimental group 2-1, is also shown.
11 is a graph showing the antibody titer was measured according to Experimental Example 6.1 after each composition was administered in the method of Experimental Example 5.3 to the mice subject to the Experimental Example 4.3. Lean denotes a normal weight control group, Obese denotes an obesity group induced by a high-fat diet, Mock denotes a placebo-administered group, 3H-OTP 30 ug denotes experimental group 3-2, and 3H-OTP 50 ug denotes experimental group 3-1.
12 is a graph showing changes in body weight for each week according to Experimental Example 6.3 after each composition was administered to the mice according to Experimental Example 4.4 by the method of Experimental Example 5.4. Lean means normal weight control group, Obese means obesity group induced by a high-fat diet, and 3H-OTP means experimental group 4-1.
13 shows the weight at 16 weeks of age and 11 weeks of age after each composition was administered to the experimental mouse according to Experimental Example 4.5 by the method of Experimental Example 5.5, and weight changes by week were measured according to Experimental Example 6.3. This is a graph showing the change. Wild(+/+)-Lean means normal weight control group, Wild(+/+) means Experimental group 5-1, Hetero(+/-) means Experimental group 5-2, Homo(-/-) means Experimental group 5-3 do.

Hereinafter, with reference to the accompanying drawings, the content of the present invention will be described in more detail through specific embodiments and examples. It should be noted that the accompanying drawings include some, but not all, embodiments of the invention. The content of the invention disclosed by this specification may be embodied in various forms, and is not limited to the specific embodiments described herein. These embodiments are considered to be provided in order to satisfy the statutory requirements applicable herein. Many modifications and other embodiments of the subject matter disclosed herein will occur to those skilled in the art to which the invention disclosed herein pertains. Accordingly, it is to be understood that the subject matter disclosed herein is not limited to the specific embodiments described herein, and that modifications and other embodiments thereof are also included within the scope of the claims.

DEFINITIONS OF GENERAL TERMS

about

As used herein, the term “about” means approximately approximating any quantity and is 30, 25, 20 for a reference quantity, level, value, number, frequency, percent, dimension, size, amount, weight or length. , 25, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1% means an amount, level, value, number, frequency, percent, dimension, size, amount, weight or length varying by as much as 1%.

peptide

As used herein, the term “peptide” refers to a polymer of amino acids. The term "peptide" refers to a form in which a small number of amino acids are linked, and is mainly used to distinguish it from a protein. There is no clear standard for distinguishing a protein from a peptide, but unless otherwise defined, in the present specification, a polymer of about 200 amino acids is referred to as a peptide, and more than that as a protein. The term "peptide" may include all other meanings recognized by those skilled in the art.

object

As used herein, the term “subject” refers to an organism that is an object exposed to a specific substance (eg, a peptide, etc.). The subject may refer to an independent organism such as a human or an animal, or may refer to a part of the independent organism such as a part of a tissue or a cell. This meaning may be appropriately interpreted according to the context. In addition, the term “object” may include all other meanings recognized by those skilled in the art.

immunotherapeutics

As used herein, the term “immunotherapy” is a concept distinct from a general therapeutic agent or vaccine. The immunotherapeutic agent is injected into the body of a subject and induces a humoral immune response to the immunotherapeutic agent itself is the same as that of a conventional vaccine, but the antibody induced as a result of the humoral immune response is not only the immunotherapeutic agent itself, but also in vivo. By having the ability to bind to specific tissues, cells (eg, receptors present on the cell surface), or specific substances (eg, peptides, lipids, proteins, and/or sugars) produced in the course of metabolism, certain diseases It is different in that it can treat ailments or diseases, and can be administered repeatedly over a period of time. Accordingly, the immunotherapeutic agent generally includes an antigen designed to induce an antibody having the ability to bind to a specific target tissue, cell, or substance in the body. Unless otherwise defined, the term "immunotherapeutic agent" is construed to include all antigens that can be appropriately used by those skilled in the art, such as peptides, proteins, lipids, saccharides, and/or complexes thereof having the above-mentioned functions. The term “immunotherapy” may be more limitedly referred to as “humoral-immunotherapeutics”. In addition, the term “immunotherapy” may include all other meanings recognized by those of ordinary skill in the art.

treatment, or treatment

As used herein, the term “treatment” means to eliminate, alleviate, alleviate, suppress, ameliorate, or eliminate, or alleviate, a disease, disorder, disorder, and/or symptom of a subject having the disease, disorder, disorder, and/or symptom. Refers to any direct or indirect action or action that results in the prevention of symptoms. As used herein, the term “therapeutic agent” refers to various substances (eg, compounds or peptides) that can exhibit the “therapeutic” effect when given in an appropriate way to a subject. In addition, the term "treatment" or "therapeutic agent" may include all other meanings recognized by those skilled in the art.

immunogenicity

As used herein, the term “immunogenicity” refers to “the property of acting as an antigen capable of inducing an immune response” in a dictionary. There are various methods for measuring the immunogenicity of a specific antigen, and the method may be appropriately adopted or designed according to the purpose. For example, 1) a method of determining whether IgG, IgA, and/or IgE type antibodies are produced in the body of the subject when the antigen is administered to the body of a subject, 2) depending on the administration cycle, the IgG , IgA, and / or a method of confirming the time when an IgE type antibody is generated, 3) a method of determining the titer of the induced antibody to the antigen, 4) When the mechanism of action of the induced antibody is found, the There may be a method of measuring the effect according to the mechanism of action, but is not limited thereto. The expression “increased immunogenicity” may be used interchangeably with, for example, “the effect of inducing an immune response is increased”, “the ability to induce an antibody is improved”, or “the effect as an immunotherapeutic agent is increased”. and includes all expressions that a person skilled in the art can properly interpret according to the context.

Background - Humoral Immune Response (BACKGROUND - HUMORAL IMMUNITY)

Humoral Immune Response by IgM (Immunoglobulin M)

Among the humoral immune responses, IgM-induced immune responses are innate immunity and are mainly active in the primary immune response, and occur rapidly in the early stages of infection. IgM is mainly secreted in the form of a pentamer, and theoretically has 10 antigen-binding sites, so that it can bind to a large number of antigens at the same time. IgM can bind to a wide variety of antigens and types, but the affinity and avidity of the binding are limited by the intrinsic affinity of IgM itself. Therefore, the affinity and binding power of IgM to the antigen is significantly lower than that of an antibody such as IgG made with the help of helper T cells.

Limitations of the humoral immune response by IgM

Although the humoral immune response by IgM plays an important role in the initial immune response, 1) the production of IgM produced by B cells is lower than that of other types of antibodies such as IgG produced by differentiated B cells, 2) IgM Since the specific binding ability of the antigen to the antigen is lower than that of IgG and 3) the degree of generating a secondary immune response is weak when exposed to the same antigen again, the dependence on the humoral immune response by IgM is that The effect is limited. Therefore, from the viewpoint of designing an antigen that induces an immune response, when an antigen injected into a subject's body induces only a humoral immune response by IgM, the effect is highly likely to appear insignificant. Therefore, it is very important to design an antigen to induce a humoral immune response by IgG.

Humoral Immune Response 1 by IgG (Immunoglobulin G) - Overview

The humoral immune response that produces IgG mainly occurs in the germinal center of the spleen or lymph node, and is a complex of B cells, helper T cells, and antigen-presenting cells (APCs). proceeds by action. The general process is as follows. 1) B cells recognize antigens (mainly protein or peptide antigens) that have invaded the body. 2) The antigen-presenting cell endocytoses the antigen (or a fragment thereof) and cuts it into smaller fragments within the cell, and then presents some of the fragments to MHC Class II on the surface of the antigen-presenting cell . 3) Helper T cells recognize the antigen fragment presented in the MHC Class II. 4) The helper T cell transmits a differentiation signal to the B cell (antigen-recognized cell). 5) The B cells are activated and some differentiate into plasma cells to produce an IgG antibody having a high specific binding ability to the antigen. 6) As a result of the activation of the B cells, some cells are differentiated into memory B cells and, when the same antigen penetrates again, is stored in the body so that an immune response to produce an IgG antigen can be rapidly generated.

Humoral immune response by IgG 2 - antigen presenting cells

Antigen-presenting cells are a generic term for cells that can present a protein fragment or peptide intracellularly, which is then cut into shorter peptide fragments, placed on MHC Class II and presented on the surface of the antigen-presenting cell. Major antigen-presenting cells include B cells, macrophages, and dendritic cells. Antigen-presenting cells transport the intracellularly entrapped antigen fragments from the site of infection to the lymph nodes, and present the antigen fragments to helper T cells by MHC Class II, thereby activating the recognized helper T cells to induce an immune response. plays a role

Humoral Immune Response by IgG 3 - MHC Class II

MHC Class II is a molecule expressed on the surface of antigen-presenting cells and has a heterodimer structure consisting of α/β chains. MHC Class II can bind to and present a peptide of a certain length in its structure. Antigen-presenting cells bind peptide fragments derived from foreign antigens to MHC Class II and present them on the cell surface. The human leukocyte antigen gene complex is involved in the expression of MHC Class II in humans, and among them, those involved in the expression of MHC Class II cell surface receptors on the surface of antigen-presenting cells are HLA-DP, DQ and It is known as a gene complex such as DR. In humans, the HLA-DR gene is known to have various alleles according to race, and about 12 types of HLA-DR genes are known as the most commonly found alleles.

Humoral immune response by IgG 4 - MHC Class II presentation by antigen presenting cells

The length of the peptide presented in MHC Class II is known to be approximately 17mer to 24mer, although there are slight differences in the literature. Therefore, antigen-presenting cells do not present the antigen protein or peptide fragment that has been incorporated into the cell as it is on MHC Class II, but undergo a process of cleavage into smaller fragments of 17mers to 24mers. The antigen fragment (protein or peptide fragment) that the antigen-presenting cell has entrapped into the cell exists in an endosome, and the endosome fuses with the lysosome of the antigen-presenting cell. Thereafter, the antigen fragment is cleaved into shorter peptides by various kinds of degrading enzymes present in the lysosome. Examples of the degrading enzymes include endopeptidase and exopeptidase. Endopeptidases act to cleave the antigen fragment by acting on the peptide bond inside the antigen fragment, and exopeptidases mainly act on the peptide bond at both ends of the antigen fragment to cleave the antigen fragment. . When the antigen fragment is cleaved into peptides of an appropriate size through the above process, some of them are bound to MHC Class II present in the inner membrane of the lysosome. The lysosome goes back to the cell surface and fuses with the plasma membrane of the cell, and only then MHC Class II and the peptide fragment bound thereto are exposed on the surface of the antigen-presenting cell. Collectively, this process is also called “the process by which antigen-presenting cells present antigens”.

Humoral Immune Response by IgG 5 - Helper T Cells

Helper T lymphocytes are also known as CD4+ cells because they express CD4. Helper T cells express a T cell receptor (TCR, T cell receptor) having the ability to bind to MHC Class II on the surface. The T cell receptor generally forms a complex with CD3. When antigen presenting cells (eg, peptide fragments) delivered to lymph nodes are presented through MHC Class II, helper T cells recognize the antigen fragments presented above. In this recognition process, the T cell receptor-CD3 complex and CD4 are involved. When the helper T cell successfully recognizes the antigen fragment, it is activated and secretes various cytokines or differentiates itself. The secreted cytokines are involved in the differentiation of B cells, which will be described later.

Humoral Immune Response by IgG 6 - B Cell Differentiation

Under the influence of cytokines such as interleukin-4 (IL-4, Interleukin-4) secreted by helper T cells, immunoglobulin class switching of the B cells occurs and the antibody produced by the B cells is isolated. The isotype will change (eg from IgM to IgG). In addition, some of the B cells are differentiated into memory B cells and stored to generate a rapid immune response when the same antigen infiltrates again, and some are differentiated into plasma cells and actively differentiated into plasma cells. Produces IgG antibodies.

Humoral Immune Response by IgG 7 - Conditions for Generation of IgG

For a humoral immune response by IgG to occur, 1) a specific conformational structure of the antigen must be recognized by B cells, and 2) some fragment of the antigen must be recognized by helper T cells through MHC Class II. In general, the part of the antigen recognized by B cells and the part of the antigen recognized by helper T cells are different, and although they activate the immune response through different pathways, the part generally recognized by B cells (B- Cell epitope) and the part recognized by helper T cells (Th epitope) are known to cause the immune response only when there is at least a certain connection relationship. For example, the B-cell epitope and the Th epitope may be included in one molecule, form conjugates, or have other linkages.

LIMITATIONS OF PRIOR ART

Strategies for inducing humoral immune response by IgG to specific antigens

As described above, in order for an IgG antibody to be generated against a specific antigen, 1) a specific B cell must recognize the antigen's three-dimensional structure, and 2) at the same time, a specific site of the antigen is presented in MHC Class II to help the T cell (Helper T cell). Cell), and 3) it must occur simultaneously in the secondary lymphoid organ, for example the spleen or lymph node. This process states that 1) the recognition of an antigen by a specific B cell depends on the probability, and 2) the antigen site presented to the MHC class II of antigen-presenting cells is different because the structure of the cell subunits constituting MHC Class II is different for each person. There is a characteristic. Therefore, if 1) the three-dimensional structure of the antigen is not large enough for B cells to recognize, and/or 2) does not contain various antigen sites that can be presented in MHC Class II, the probability of generating an IgG antibody this lowers For this reason, it is generally known that small molecules (eg, peptides, etc.) have poor immunogenicity that induces a humoral immune response. Therefore, one of the strategies commonly adopted by those skilled in the art to effectively induce the production of IgG antibodies to specific antigens is 1) diversification of antigenic sites recognized by B cells, and 2) the methods to be presented in MHC Class II. Diversification of possible antigenic sites.

Trade-off relationship with this strategy 1 - Uniformity of induced antibodies

Although the above strategy increases the probability of inducing a humoral immune response by IgG, since the antigen sites recognized by B cells are diversified, various types of antibodies are induced. In addition, in the process of diversifying antigen sites that can be presented in MHC Class II, proteins or peptides having a site recognizable by B cells may be inserted into the specific antigen, thereby binding to the site Antibodies can also be induced. In other words, in the process of diversifying antigenic sites that can be presented in MHC Class II, antigenic sites recognized by B cells may be added although not intended. Although the IgG antibody group induced by the above strategy all have a common feature of binding to the same antigen, there is a difference in the structure of the individual antibodies. Therefore, the more diverse the antigenic sites recognized by B cells, the lower the uniformity of the induced antibody. In the worst case, there may be a problem in that the intended effective antibody is culled by clonal selection and thus efficacy does not appear.

Trade-off relationship with the above strategy 2 - Economics

In order to diversify antigenic sites according to the above strategy, a large molecule (eg, protein or lipid complex) is inevitably used, resulting in an increase in molecular weight. If such a large molecule must be produced by an artificial synthetic method (eg, chemical synthesis, protein recombinant technology, etc.), the production cost is high and the yield is low, resulting in low economic feasibility. Therefore, as a larger molecule is used by diversifying an antigenic site, the ease and economical efficiency of manufacturing decreases when chemical synthesis or protein recombination technology is used.

No problem with conventional vaccines

In the case of a conventional vaccine, the main purpose is to induce an IgG antibody against an antigen injected into the body, and then to cause a secondary immune response when the same antigen re-invades the body. Therefore, 1) the uniformity is not a problem as long as the generated antibody recognizes the same antigen, and 2) pathogenic antigens already existing in nature (eg, viruses or fragments thereof) are often used, so these antigens are often used. Since there is little need to manufacture by an artificial synthetic method, the features in the trade-off relationship with the above strategy can be neglected. In conclusion, the aforementioned antigen site diversification strategy is a very useful strategy for increasing the immunogenicity of vaccines.

Immunotherapy can be a problem

In the case of immunotherapy, it is the same as that of a vaccine in that it induces an IgG antibody against the antigen injected into the body of the subject. However, in the case of immunotherapeutic agents, the antigen is intentionally designed so that the antibody generated by the above process can bind to a specific site of a specific tissue, cell, or substance (eg, a specific site of the ApoB-100 protein). , There is a difference in that the purpose of the generated antibody is to produce a therapeutic effect through the action of binding to the specific site. Therefore, in the case of an immunotherapeutic agent, it is not important to induce an antibody that recognizes the injected antigen, but to induce only the antibody to a deliberately designed antigen site is an important task. In addition, since these antigens already exist in nature or are substances that need to be manufactured artificially instead of being processed, in most cases, they are prepared by chemical synthesis, recombinant protein technology, or other artificial synthesis methods. Therefore, when using the above-mentioned strategies to increase the immunogenicity of the humoral immune response by IgG, 1) the homogeneity of the induced antibody is low, and 2) artificial synthesis methods (e.g., chemical synthesis, protein When using recombinant technology, etc.), the problem of economic feasibility is greatly highlighted.

Strategies and limitations for inducing humoral immune response by IgG of existing anti-obesity immunotherapeutic drugs (pB1, B4T, etc.)

As disclosed in prior patent application US10/378,707, PCT/KR2005/000784, and Kim et al. (2016, An apolipoprotein B100 mimotope prevents obesity in mice, Clinical Science 130, 105-116), It is known that a peptide-specific antibody can also bind to the exposed site of the ApoB-100 protein in the LDL molecule, thereby functioning as an immunotherapeutic agent. However, there was a problem that the length of the peptide of the specific sequence was too short to induce sufficient IgG antibody. According to the previously disclosed literature, in order to increase the immunogenicity that causes a humoral immune response to the peptide of the specific sequence, 1) a concatemer is prepared by linking a plurality of the specific sequence to diversify the antigen site recognized by B cells, or , 2) to improve immunogenicity by linking antigen proteins known to be recognized by helper T cells to diversify the corresponding sites. However, as described above, there is a problem in that the generated antibody is not uniform and a large molecule protein is used, so it failed to commercialize despite showing a certain effect as an immunotherapeutic agent. It was a difficult situation.

Peptide (A PEPTIDE)

peptide structure

In one aspect of the invention provided herein, there is provided a peptide. The peptide comprises 1) at least one B-Cell epitope, 2) a helper-T-cell epitope (hereinafter, Th epitope), and 3) at least one protecting moiety, 4) the B-Cell epitope and the protecting moiety bind to Th- Epitope-protecting epitope (hereinafter TEPE, Th-Epitope-Protecting Epitope) can be formed. The Th epitope is protected by connecting its N-terminus and C-terminus to a protecting part. In one embodiment, the peptide may include a first protecting moiety, a Th epitope, and TEPE, and the TEPE may comprise a second protecting moiety and a B-cell epitope. In this case, in the peptide sequence, the sequence of the Th epitope may be between the first protecting moiety and the TEPE, and the Th epitope may be directly linked to the first protecting moiety and the second protecting moiety. In one embodiment, the peptide may include TEPE, a Th epitope, and a protective moiety bound in order from the N-terminus to the C-terminus. In another embodiment, the peptide may include a protective moiety, Th epitope, and TEPE bound in order from the N-terminus to the C-terminus. In another embodiment, the peptide may include a first TEPE, a Th epitope, and a second TEPE bound in order from the N-terminus to the C-terminus.

Peptide Structure - Structural Formula

The peptide may be represented by the following structural formula (1).

[Structural Formula 1]

X ₁ -X ₂ -X ₃ -X ₄ -X ₅

In this case, X ₁ is any peptide;

X ₂ is a first TEPE or a first protection part;

X ₃ is a Th epitope;

X ₄ is a second TEPE or a second protection part; and

X ₅ is defined as any peptide.

In this case, in the [Structural Formula 1], the case where X ₂ is the first protective part and X ₄ is the second protective part is excluded,

wherein the first TEPE is -(first B-cell epitope)-(protection portion)- or -(first B-cell epitope)-(second B-cell epitope)-(protection portion)-,

the second TEPE is -(protection moiety)-(third B-cell epitope)- or -(protection moiety)-(third B-cell epitope)-(fourth B-cell epitope)-;

Wherein X ₁ and / or X ₅ is a peptide that does not affect the immune response in the body,

X ₁ and/or X ₅ may be omitted.

Peptide Feature 1 - Includes B-Cell and Th epitopes

Since the peptide contains a portion having a B-Cell epitope function and a portion having a Th epitope function in one peptide, it is characterized in that it has the minimum conditions for inducing an IgG antibody against the B-Cell epitope portion. Accordingly, the peptide can induce a humoral immune response by IgG in the body of a subject by itself, without the aid of an additional substance or adjuvant.

Peptide feature 2 - contains a small number of B-cell epitopes and Th epitopes

The peptide includes a B-cell epitope and a Th epitope, and the peptide is characterized in that it includes a small number of B-cell epitopes and Th epitopes. In one embodiment, the peptide may include one B-cell epitope. In another embodiment, the peptide may include two B-cell epitopes. In one embodiment, the peptide may include one Th epitope. In another embodiment, the peptide may include two Th epitopes.

Peptide Characteristics 3 - Including Protective Part

The Th epitope included in the peptide is characterized in that it is linked to a protective part. The protective moiety is included so that the Th epitope functions effectively by including the Th epitope in a small number of units in the peptide as described above. When the peptide contains the protective moiety, the immunogenicity of inducing a humoral immune response is higher than that of a peptide that does not include the protecting moiety. The protection unit may be connected to the B-Cell epitope. In one embodiment, the Th epitope may have a protective moiety connected to its N-terminus and/or C-terminus. In one embodiment, the Th epitope may have a peptide having a protective moiety linked to its N-terminus and/or C-terminus. The function of the protection unit is described in detail in the corresponding paragraph, which can be confirmed experimentally through Experimental Examples 6.7, 6.8, and 6.10.

Peptide function

When the peptide is injected into the body of a subject, it functions to induce a humoral immune response to the peptide, in particular, a humoral immune response by IgG.

Advantages of Peptides 1 - Uniform Antibody Generation

When the peptide provided herein is injected into the body of a subject, it induces the generation of an IgG antibody that specifically binds to the peptide. At this time, among the generated IgG antibodies, an IgG antibody that specifically binds to the B-Cell epitope included in the peptide is mainly produced. In other words, when the peptide is injected into the body of a subject, it induces a humoral immune response to uniformly generate an IgG antibody that specifically binds to the B-Cell epitope.

Advantages of Peptides 2 - Relatively Short Sequence Length

The peptides provided herein include a small number (eg, one) of B-cell epitopes and Th epitopes, and contain a minimum protective moiety to allow the function of the Th epitope to be well exerted. Furthermore, the B-Cell epitope was designed in smaller units to generate homogeneous antibodies. Accordingly, the peptide has a relatively small molecular weight among antigenic substances capable of inducing a humoral immune response by IgG, and when the antigenic substance is a protein or peptide, it is characterized in that it has a relatively short sequence length.

Peptide Examples - Possible Sequence Information

In one embodiment, the peptide may be a peptide comprising a sequence selected from the group consisting of SEQ ID NOs: 1 to 16.

Th epitope (HELPER-T-LYMPHOCYTE EPITOPE)

Th Epitope Definition - Functional Definition

The Th epitope contained in the peptides provided herein is expressed on the surface of the antigen-presenting cell by MHC Class II after the peptide is endocytosed by the antigen-presenting cell, MHC Class II and It refers to a portion designed to bind, present on the surface of the antigen-presenting cell, and function as recognized by CD4+ T cells (eg, helper T cells). The process of presenting the antigen by processing the peptide into which the antigen-presenting cell has entered the cell and binding it to MHC Class II has been described above. In other words, the Th epitope is a part that is recognized by the CD4+ T cells when the peptide is injected into the body of a subject, and thus plays a direct role in inducing an IgG-type antibody to the peptide. In one embodiment, the Th epitope may have binding ability to MHC Class II. In one embodiment, the Th epitope can be recognized by CD4+ T cells when the peptide is administered to a subject. For example, the CD4+ T cells may be naive T cells. As another example, the CD4+ T cells may be Th1 cells, Th2 cells, and/or regulatory T cells.

Th epitope length

The Th epitope is characterized in that it has a relatively short length. In one embodiment, the length of the Th epitope is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30mer. In an embodiment, the length of the Th epitope may have a value within two different numerical ranges selected in the immediately preceding sentence. For example, the Th epitope may have a length of about 17mer to 24mer. For another example, the Th epitope may have a length of about 8mer to 30mer.

contains a small number of Th epitopes

The peptides provided herein are characterized in that they contain a small number of Th epitopes. In one embodiment, the peptide may include one Th epitope. In another embodiment, the peptide may comprise 2, 3, or 4 Th epitopes. In one embodiment, the peptide may include one type of Th epitope. In another embodiment, the peptide may include two or more kinds of Th epitopes.

Various Th epitope designs are possible

As described above, due to the diversity of HLA gene complex traits, the structure of MHC Class II may vary according to race and individual. In other words, depending on the trait of the HLA gene complex of the subject, the Th epitope sequence having a high binding ability with MHC Class II of the subject may vary. Since the peptide provided herein is characterized in that it contains a small number of Th epitopes, when the Th epitope is designed according to the trait of the HLA gene complex of the subject, the peptide may better enhance the immune response in the subject's body. can induce In one embodiment, the Th epitope is selected from the HLA-DR1 alleles 2w2b, 2w2a, 3, 4w4, 4w14, 5, 7, 52a, 52b, 52c, and 53 by one or more HLA-DR genes. It may be a peptide sequence having high binding ability with the expressed MHC Class II. If the Th epitope has the ability to bind to various MHC Class II such as various HLA-DP, HLA-DQ, and/or HLA-DR regardless of the trait of the HLA gene complex, the peptide can target more may induce an immune response in In a preferred embodiment, the Th epitope may be a sequence capable of binding to MHC Class II expressed by various HLA gene complexes.

Characteristics of Th epitopes - less likely to act as B-Cell epitopes

The Th epitope was designed to be presented by MHC Class II of antigen-presenting cells and recognized by helper T cells. Therefore, it is known that, in general, since the binding ability to MHC Class II is very high, the probability of acting as a B-Cell epitope is low. In other words, the probability of inducing an antibody that specifically binds to the conformational structure of the Th epitope is very low.

Example 1 of Th epitope

The Th epitope is not particularly limited as long as it satisfies two things: 1) it must have the ability to bind to MHC Class II, and 2) it is unlikely to act as a B-Cell epitope. In one embodiment, the Th epitope may be a sequence named PADRE disclosed in patent application US305,871. In another embodiment, the Th epitope is Cara C. Wilson et al. (2001, Identification and Antigenicity of Broadly Cross-Reactive and Conserved Human Immunodeficiency Virus Type 1-Derived Helper T-Lymphocyte Epitopes, Journal of Virology, 75 (9) ) 4195-4207) and named HA307-312.

Example 2 of Th epitope - PADRE structural formula

In one embodiment, the Th epitope may be represented by Structural Formula 2 as follows.

[Structural Formula 2]

N'-K-(Cha)-X ₁ -AAX _{2 -TX 3} -X ₄ -AA-C'

In this case, (Cha) means L-cyclohexyllanins.

Wherein X ₁ is a hydrophobic amino acid, specifically, may be L or I, but is not limited thereto.

X ₂ is an aromatic or cyclic amino acid, specifically, may be F, Y, or H, but is not limited thereto.

X ₃ is aliphatic long chain amino acids, specifically I and V, but is not limited thereto.

X ₄ is a charged amino acid, and specifically, may be R, L, D, Q, or E, but is not limited thereto.

Example 3 of Th epitope - possible sequence information

In one embodiment, the Th epitope may include a peptide sequence selected from the group consisting of SEQ ID NOs: 17 to 20 and 77.

Limitations and technical solutions of Th epitope function

The peptides provided herein are characterized in that 1) they contain a relatively short Th epitope, and 2) contain only a small number of Th epitopes. Accordingly, there is a high probability that the Th epitope is not completely preserved in the process of the peptide being internalized by antigen-presenting cells and degraded into smaller units by the proteasome, which indicates that the desired level of immune response is not sufficiently exhibited. cause problems Due to the characteristics of the aforementioned immunotherapeutic agents, there is a need for a method capable of increasing immunogenicity without increasing the number of Th epitopes, in addition to a method of increasing the probability of conserving the Th epitope by simply increasing the number of Th epitopes. In accordance with this requirement, the present specification provides a peptide additionally comprising a protective moiety in addition to the B-Cell epitope and the Th epitope in one aspect of the present invention.

PROTECTING PART

Definition of Protective Department - Functional Definition

The peptides provided herein include one or more protecting moieties. The protective moiety is 1) linked to the N-terminus and/or C-terminus of the Th epitope, and 2) the Th epitope is intactly preserved in the process of presentation of the peptide to MHC Class II by antigen-presenting cells. It functions to increase the probability of combining with MHC Class II. Accordingly, the peptide including the protective moiety can induce a desired level of immune response with a higher probability than the peptide without the protecting moiety. In an embodiment, the protecting unit may protect the Th epitope from being cleaved by an enzyme in the subject's body. For example, the enzyme in the body of the subject may be peptidase. Specifically, the peptidase may be an exopeptidase and/or an endopeptidase, but is not limited thereto. In addition, the protection unit may simultaneously have one or more other functions as well as a function of protecting the Th epitope. In one embodiment, the protecting moiety may have a linker function for linking the two moieties included in the peptide. In another embodiment, the protecting part may have a function of increasing the solubility of the peptide in an aqueous solution. In yet another embodiment, the protecting moiety may have both the linker function and the function of increasing the solubility. In another embodiment, the protection unit may have a Th epitope function.

Structure of protective part - degree of hydrophilicity

The protecting moiety may include both hydrophobic amino acids and hydrophilic amino acids. In this case, when the protective moiety contains too much hydrophilic amino acid, the protective moiety covers the B-cell epitope and the Th epitope in an aqueous solution environment (eg, plasma environment) to hide inside the peptide. In other words, the three-dimensional structure of the B-cell epitope in the aqueous solution may be covered by the protective part so as not to be exposed to the outside. In this case, the pathway by which the B cell specifically binds to the B-cell epitope and recognizes the antigen is blocked by the protective moiety, which may result in the induction of an intended immune response. Therefore, the protective moiety should include a certain number or less of hydrophilic amino acids. In one embodiment, the number of hydrophilic amino acids included in the protective part satisfies the following formula.

X _H : the number of hydrophilic amino acids in the protecting group, a: the number of B-cell epitope amino acids, b: the number of Th epitope amino acids, and H: the upper limit of the ratio of hydrophilic amino acids. In this case, the H may vary depending on the type of the B-cell epitope and/or the type of the Th epitope.

In one embodiment, H is about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, or 50%. In one embodiment, H may have a value within a range consisting of two values selected in the immediately preceding sentence. For example, the H may be 30 to 40%. In another example, the H may be 33 to 45%. In a preferred embodiment, the H may be about 35%.

Structure of the protective part - may contain artificial amino acids

It is known that, unlike peptides containing only natural amino acids, peptides containing unnatural amino acids are not cleaved by peptidase. The protecting moiety may include an artificial amino acid in its sequence. In one embodiment, the protecting moiety may include one or more artificial amino acids. In one embodiment, the protecting moiety may include one or more artificial amino acids at the end connected to the Th epitope. In one embodiment, the protecting moiety may include one artificial amino acid at the N-terminus. In another embodiment, the protecting moiety may include two or more artificial amino acids at the N-terminus. In one embodiment, the protecting moiety may include one artificial amino acid at the C-terminus. In another embodiment, the protecting moiety may include two or more artificial amino acids at the C-terminus.

Length of guard - presence of lower limit

Since the protection portion serves to protect the Th epitope from peptidic enzymes, it has a length greater than or equal to the minimum value in order to properly perform this protection function. The minimum value may vary depending on the type and/or length of the Th epitope. In one embodiment, the minimum value may be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15mer. For example, the Th epitope may have a length of about 1 mer or more. As another example, the Th epitope may have a length of about 6mer or more.

Characteristics of the protective part - little effect on the function of the B-cell epitope

The protective part functions to protect the above-described Th epitope, but due to the above-described structural/functional properties, the function of inducing an antibody that specifically binds to the B-cell epitope in the body of the subject is not significantly affected. There is this.

Advantages of introducing a Department of Protection

By including the protective moiety in the peptide, it is possible to improve the immunogenicity of the peptide by compensating for the above-described limitation of Th epitope function. In addition, the protective part does not significantly affect the uniformity of the antibody induced by the peptide in the body. In addition, since it is only a relatively short peptide sequence, it does not act as an obstacle to the production of the peptide. Therefore, including the protective moiety has an important advantage in the design of peptides that can be used as immunotherapeutic agents.

Examples of Protectors - Possible Sequence Information

In one embodiment, the protecting moiety may include a peptide sequence selected from the group consisting of SEQ ID NOs: 21 to 40 and 78.

B-CELL EPITOPE

Definition of B-Cell Epitope - Functional Definition

The peptides provided herein include one or more B-cell epitopes. As used herein, the term "B-cell epitope" refers to a part of a peptide that is intentionally designed to induce a homogeneous antibody of one type. Therefore, when the peptide containing the B-cell epitope is injected into the body of a subject, one type of antibody is predominantly induced per one B-cell epitope.

Structure of B-Cell Epitope

The B-cell epitope is 1) a site that provides all or part of a conformation to which an antibody can specifically bind (hereinafter, a conformational structure forming part), and 2) directly or indirectly affects the conformation of the conformation. It may include an adjacent portion (hereinafter, adjacent portion) that affects In one embodiment, the three-dimensional structure forming part may be specified by a continuous peptide sequence. In one embodiment, the contiguous region may be characterized by a contiguous peptide sequence. In one embodiment, the B-cell epitope may include an adjacent portion and a three-dimensional structure forming portion connected in an N-terminal to C-terminal order. In one embodiment, the B-cell epitope may include a three-dimensional structure forming portion and adjacent portions connected in the order from the N-terminus to the C-terminus. In one embodiment, the B-cell epitope may include a first adjacent portion, a three-dimensional structure forming portion, and a second adjacent portion connected in an N-terminal to C-terminal order. In one embodiment, the B-cell epitope may include one or more three-dimensional structure forming part. In one embodiment, the B-cell epitope may include one or more adjacent regions.

May contain multiple B-Cell epitopes

The peptide may include a plurality of B-cell epitopes. In one embodiment, the peptide comprises one B-cell epitope. In another embodiment, the peptide comprises two B-cell epitopes. In another embodiment, the peptide may include three or more B-cell epitopes.

Length of B-Cell epitope

In one embodiment, the length of the B-cell epitope is about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30mer. In one embodiment, the length of the B-cell epitope may have a value within a range consisting of two values selected in the previous sentence. For example, the length of the B-cell epitope may be 9 to 15mer. As another example, the length of the B-cell epitope may be 15 to 30 mer.

Characteristics of B-Cell epitopes 1 - Contain fixed conformation

The peptide containing the B-cell epitope is characterized in that it forms a three-dimensional structure in which the B-cell epitope region is fixed in an aqueous solution environment (eg, plasma environment). The B-cell epitope may be intentionally designed to form the fixed three-dimensional structure. The fixed conformation may serve to induce an antibody that specifically binds to the conformation. In the case of having a fixed three-dimensional structure in an aqueous solution environment, since the probability that the fixed three-dimensional structure is recognized by B cells is very high, a stable secondary immune response can be induced. In one embodiment, the B-cell epitope may be a peptide that forms a higher-order structure in an aqueous solution environment. In one embodiment, the aqueous solution environment may be the subject's internal environment (eg, plasma environment). In one embodiment, the higher-order structure may be an α-helix structure.

Characteristics of B-Cell Epitope 2 - Induction of Uniform Antibodies

The B-cell epitope has a fixed three-dimensional structure in an aqueous solution environment (eg, plasma environment) as described above, and the sequence length of the three-dimensional structure is several to several tens of peptide units, which is relative to IgM of B cells. Because of its small size, only one or very few types of B cells recognize it. Therefore, when the peptide containing the B-cell epitope is injected into the body of a subject, the probability of inducing the production of one type of antibody per unit of the B-cell epitope is very high. Accordingly, the B-cell epitope is characterized in that the peptide induces the uniform formation of an intended antibody in the body (eg, one type of antibody per one type of B-cell epitope).

Th-Epitope-Protecting Epitope (TEPE, Th-EPITOPE-PROTECTING EPITOPE)

TEPE Definition - Functional Definition

The peptides provided herein include one or more Th-Epitope-Protecting Epitope (TEPE, Th-Epitope-Protecting Epitope). When the peptide is injected into the body of a subject, the TEPE has both 1) the function of the above-described B-cell epitope and 2) the function of the protective part. In general, the TEPE is formed by combining the above-described B-cell epitope and a protective moiety, but is not particularly limited as long as it has both functions.

TEPE structure

The TEPE contains a B-cell epitope and a protective moiety. In one embodiment, the TEPE may include a sequence to which a B-cell epitope and a protective part are bound in an N-terminal to C-terminal order. In another embodiment, the TEPE may include a sequence to which a protective moiety and a B-cell epitope are bound, in the order from N-terminus to C-terminus. In another embodiment, the TEPE may include a sequence to which a first protecting moiety, a B-cell epitope, and a second protecting moiety are bound in an N-terminal to C-terminal order. In one embodiment, the TEPE may include two or more B-cell epitopes. In one embodiment, the TEPE may include a sequence to which a first B-cell epitope, a second B-cell epitope, and a protection part are bound in the order from N-terminus to C-terminus. In one embodiment, the TEPE may include a sequence to which a first B-cell epitope, a second B-cell epitope, and a protective part are bound in the order from C-terminus to N-terminus. In one embodiment, the B-cell epitope and the protective moiety are not specified as a continuous peptide sequence, respectively, and may be discontinuously dispersed throughout the TEPE.

May contain multiple TEPEs

The peptides provided herein may include a plurality of TEPEs. In one embodiment, the peptide may comprise two TEPEs. In another embodiment, the peptide may include 3 or more TEPEs.

TEPE Example - Possible Sequence Information

The TEPE may include a peptide sequence selected from the group consisting of SEQ ID NOs: 41 to 75.

Uses of Peptides

Uses of Peptides - Overview

When the peptides provided herein are injected into the body of a subject, 1) induces the production of an antibody that specifically binds to an intentionally designed B-cell epitope, and 2) induces the production of a uniform antibody. suitable as a therapeutic agent. Therefore, the peptide can be used for immunotherapeutic applications. In one embodiment, the peptides provided herein may be used as immunotherapeutic agents for obesity.

Uses of Peptides for the Treatment of Obesity

The peptides provided herein include B-cell epitopes. In one embodiment, the B-cell epitope may be a B-cell epitope included in SEQ ID NOs: 41 to 75. At this time, the B-cell epitope is known to induce antibodies having binding ability to ApoB-100 (Patent Application No. US10/378,707, PCT/KR2005/000784, and Kim et al, 2016, An apolipoprotein B100 mimotope prevents obesity in mice, Clinical Science 130, 105-116). It is known from the prior literature that when an antibody having binding ability to ApoB-100 is induced by the B-cell epitope in the body of a subject, it has an immunotherapeutic effect on obesity. Accordingly, the present specification discloses the use of the peptide for the treatment of obesity and a method thereof. In order to describe the immunotherapeutic effect on obesity, Patent Application No. US10/378,707, PCT/KR2005/000784, and Kim et al, 2016, An apolipoprotein B100 mimotope prevents obesity in mice, Clinical Science 130, 105-116 The contents described are incorporated herein by reference. In the event of a conflict between the referenced part and the description of the present specification, it is construed that the description in this specification takes precedence.

Pharmaceutical composition comprising a peptide (PHARMACEUTICAL COMPOSITION INCLUDING THE PEPTIDE)

Disclosed herein is a pharmaceutical composition comprising the aforementioned peptide. The peptide can be used as an immunotherapeutic agent and has in common with vaccines in that it induces a humoral immune response when injected into the body. Accordingly, the pharmaceutical composition containing the peptide may include an appropriate composition that a person skilled in the art may add for general vaccine administration and/or to increase the effect of inducing an immune response. For example, the pharmaceutical composition may include, but is not limited to, the formulated peptide, pharmaceutically acceptable carriers, adjuvants and/or adjuvants. Specifically, the pharmaceutical composition includes water, saline, dextrose, ethanol, glycerol, sodium chloride, dextrose, mannitol, sorbitol, lactose, gelatin, albumin, aluminum hydroxide, Freund's Incomplete Adjuvant and Complete Adjuvant (Pifco Laboratories, Detroit, Mich.), Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ.), Alhydrogel (Al(OH) ₃ ), Aluminum Hydroxide Gel (Alum) , or aluminum salts such as aluminum phosphate, AS04 series, MF, squalene, MF59, QS21, calcium, iron or zinc salts, insoluble suspensions of acylated tyrosine, acylated fructose, cationically or anionically derived polysaccharides , polyphosphazenes, biodegradable microspheres, and Quil A, toll-like receptor (TLR) agonists, Avanti polar lipid, Monophosphoryl Lipid A (synthetic) [PHAD], monophosphoryl lipid A (MPL, monophosphoryl Lipid) A), synthetic lipid A, lipid A mimics or analogues, aluminum salts, cytokines, saponins, prolactin, growth hormone deoxycholic acid, betaglucan, polyribonucleotides, muramyl dipeptide (MDP) derivatives, CpG oligos, of Gram-negative bacteria Lipopolysaccharide (LPS), polyphosphazene, emulsion, virosome, cochleate, poly(lactide-co-glycolide) (PLG) microparticles, poloxamer particles, microparticles, liposomes, or these may be an appropriate combination of, but is not limited thereto.

MANUFACTURING METHOD OF THE PEPTIDE

The peptides provided herein may be prepared by a known method that can be adopted by those skilled in the art, and the preparation method is not particularly limited. In one embodiment, the peptide may be prepared by a recombinant protein production method. In another embodiment, the peptide may be chemically synthesized. Specifically, the peptide may be synthesized by a liquid peptide synthesis method, a solid phase peptide synthesis method, or a convergent method of small peptide fragments, but is not limited thereto.

THE PEPTIDE - SUMMARY

The present specification provides a peptide comprising 1) a B-cell epitope, 2) a protective moiety, and 3) a Th epitope. The B-cell epitope and the protecting moiety combine to form a Th-epitope-protecting epitope. The peptide is characterized in that a protective moiety is linked to the N-terminus and C-terminus of the Th epitope, and when injected into a subject's body, it has the advantage of uniformly inducing an antibody that specifically binds to an intentionally designed B-cell epitope. . In addition, since the peptide contains a small number of B-cell and Th epitopes, it has advantages of easy production and economical efficiency. The peptide may be used as an immunotherapeutic agent due to its characteristics. In one embodiment, the present specification provides a peptide designed to be used as an immunotherapeutic agent for obesity and a pharmaceutical composition thereof.

The names of each part of the peptide disclosed herein (eg, protective moiety and TEPE) are given for convenience of description. Accordingly, the scope and name of each part may vary depending on the viewpoint. For example, the protective part may be referred to as an auxiliary part, a dummy part, and/or a linker part, but is not limited thereto. As another example, the Th-epitope-protecting epitope may be referred to as a B-cell epitope, but is not limited thereto.

Experimental example

Hereinafter, the invention provided by the present specification will be described in more detail through experimental examples and examples. These examples are only for illustrating the content disclosed by the present specification, and it is to those of ordinary skill in the art that the scope of the content disclosed by the present specification is not to be construed as being limited by these examples. it will be self-evident

Experimental Example 1 Preparation of peptides

Experimental Example 1.1

The peptides listed in Table 1 below were requested to be synthesized by a peptide synthesis company (Anigen, Gwangju-si, Korea), and the prepared peptides were obtained. The peptide was synthesized using a method of converging small peptide fragments (Convergent).

SEQ ID NO: peptide sequence One RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZ 2 ZaK(Cha)VAAWTLKAAaZRNVPPIFNDVYWIAF 7 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZCRFRGLISLSQVYLS

Here, a represents D-form alanine, Z represents 6-aminohexanoic acid, and (Cha) represents L-cyclohexylalanins. as below.

Experimental Example 1.2

The peptides listed in Table 2 below are requested to be synthesized by an appropriate company (eg, Anygen, Gwangju-si, Korea), and the prepared peptides are obtained. The method for producing the peptide may be appropriately selected according to the characteristics of the sequence, and is not particularly limited.

SEQ ID NO: peptide sequence One RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZ 3 RNVPPIFNDVYWIAFK(Cha)VAAWTLKAA 4 K(Cha)VAAWTLKAARNVPPIFNDVYWIAF

Experimental Example 1.3

The peptides listed in Table 3 below are requested to be synthesized by an appropriate synthesis company (eg, Anygen, Gwangju-si, Korea), and the prepared peptides are obtained. The method for producing the peptide may be appropriately selected according to the characteristics of the sequence, and is not particularly limited.

SEQ ID NO: peptide sequence 3 RNVPPIFNDVYWIAFK(Cha)VAAWTLKAA 5 RNVPPIFNDVYWIAFK(Cha)VAAWTLKAAHHHHHH

Experimental Example 1.4

The peptides listed in Table 4 below are requested to be synthesized by an appropriate synthesis company (eg, Anygen, Gwangju-si, Korea), and the prepared peptides are obtained. The method for producing the peptide may be appropriately selected according to the characteristics of the sequence, and is not particularly limited.

SEQ ID NO: peptide sequence 6 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZGSHHHHHHGSDDDDK 7 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZCRFRGLISLSQVYLS 8 CRFRGLISLSQVYLSZaK(Cha)VAAWTLKAAaZCRFRGLISLSQVYLS 9 KTTKQSFDLSVKAQYKKNKHZaK(Cha)VAAWTLKAAaZCRFRGLISLSQVYLS 10 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZRNVPPIFNDVYWIAF 11 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZKTTKQSFDLSVKAQYKKNKH 12 GSHHHHHHGSDDDDKZaK(Cha)VAAWTLKAAaZRNVPPIFNDVYWIAF

Experimental Example 1.5

The peptides listed in Table 5 below are requested to be synthesized by an appropriate synthesis company (eg, Anygen, Gwangju-si, Korea), and the prepared peptides are obtained. The method for producing the peptide may be appropriately selected according to the characteristics of the sequence, and is not particularly limited.

SEQ ID NO: peptide sequence 13 RNVPPIFNDVYWIAFCRFRGLISLSQVYLSZaK(Cha)VAAWTLKAAaZ 14 RNVPPIFNDVYWIAFCRFRGLISLSQVYLSK(Cha)VAAWTLKAA 15 RNVPPIFNDVYWIAFCRFRGLISLSQVYLSK(Cha)VAAWTLKAAHHHHHH

Experimental Example 1.6

The peptides listed in Table 6 below are requested to be synthesized by an appropriate synthesis company (eg, Anygen, Gwangju-si, Korea), and the prepared peptides are obtained. The method for producing the peptide is appropriately selected according to the characteristics of the peptide, and is not particularly limited.

SEQ ID NO: peptide sequence 16 RNVPPIFNDVYWIAFZPKYVKQNTLKLATZCRFRGLISLSQVYLS

Experimental Example 2 Confirmation of the prepared peptide structure

Experimental Example 2.1 Purity analysis of the prepared peptide

The purity of the peptide prepared in Experimental Example 1.1 was measured by performing HPLC analysis (Shimadzu HPLC LabSolutions) using a C-18 reversed-phase column (SHIMADZU C18 analytical column). At 60° C., the sample was separated and developed into 0.05% Trifluoroactate (TFA) aqueous solution and 0.05% TFA Acetotrile solution, and then the purity was measured by measuring the peak absorbance at 230 nm wavelength.

Experimental Example 2.2 Molecular weight analysis of the prepared peptide

The molecular weight of the peptide prepared in Experimental Example 1.1 was analyzed with a mass spectrometer (AXIMA Assurance, MALDI-TOF, Shimadzu).

Experimental Example 2.3 Quantitative Analysis of the Prepared Peptide

The peptide prepared in Experimental Example 1.1 was quantified by measuring the UV extinction coefficient (Ultrospec 3000 Pro UV/VIS spectrophotometer, Pharmacia). Specifically, it was quantified using the extinction coefficient at 280 nm.

Experimental Example 2.4

For the peptide prepared in Experimental Example 1.2, 1) Purity was measured by the method described in Experimental Example 2.1, 2) Molecular weight was measured by the method described in Experimental Example 2.2, 3) Quantitative analysis by the method described in Experimental Example 2.3 do. According to the characteristics of the prepared peptide, the method may be appropriately modified.

Experimental Example 2.5

For the peptide prepared in Experimental Example 1.3, 1) Purity was measured by the method described in Experimental Example 2.1, 2) Molecular weight was measured by the method described in Experimental Example 2.2, 3) Quantitative analysis by the method described in Experimental Example 2.3 do. According to the characteristics of the prepared peptide, the method may be appropriately modified.

Experimental Example 2.6

For the peptide prepared in Experimental Example 1.4, 1) Purity was measured by the method described in Experimental Example 2.1, 2) Molecular weight was measured by the method described in Experimental Example 2.2, 3) Quantitative analysis by the method described in Experimental Example 2.3 do. According to the characteristics of the prepared peptide, the method may be appropriately modified.

Experimental Example 2.7

For the peptide prepared in Experimental Example 1.5, 1) Purity was measured by the method described in Experimental Example 2.1, 2) Molecular weight was measured by the method described in Experimental Example 2.2, 3) Quantitative analysis by the method described in Experimental Example 2.3 do. According to the characteristics of the prepared peptide, the method may be appropriately modified.

Experimental Example 2.8

For the peptide prepared in Experimental Example 1.6, 1) Purity was measured by the method described in Experimental Example 2.1, 2) Molecular weight was measured by the method described in Experimental Example 2.2, 3) Quantitative analysis by the method described in Experimental Example 2.3 do. According to the characteristics of the prepared peptide, the method may be appropriately modified.

Experimental Example 3 Preparation of a composition for in vivo administration

Experimental Example 3.1 Method for preparing a composition for in vivo administration

A composition for in vivo administration was prepared by mixing the peptide prepared in Experimental Example 1.1, Alhydrogel (Al(OH) ₃ , manufactured by InvivoGe), and PHAD (manufactured by Avanti). The specific process is as follows.

(1) The prepared peptide powder was dissolved in 100% DMSO (dimethyl sulfoxide) to obtain a concentration of 100 mg/ml.

(2) PBS was added to the peptide-DMSO solution of (1) and mixed so that the peptide concentration was 5 mg/mL.

(3) PHAD was dissolved in 100% DMSO to a concentration of 10 mg/mL, and then diluted with distilled water to 1 mg/mL.

(4) Alhydrogel adjuvant (Invivogen, USA) and the PHAD solution were added to the mixture of (2). The concentration of the mixed composition was 50 μg of the peptide, 10 μg of the PHAD, and 10% (v/v) of the Alhydrogel adjuvant per 100 μl of 1 dose.

(5) After mixing the mixture of (4) well, it was reacted overnight (overnight) while stirring with a rotator in a low temperature room (4 ℃).

(6) For DSMO washing, the reaction mass of (5) was centrifuged at 1400 rpm for 15 minutes, and the supernatant except for about 1 mL on the pellet was removed. After that, 10 mL of PBS was added and mixed.

(7) The washing process of (6) was repeated 3 times.

(8) After the last washing process, PBS was added to adjust the final concentration of the composition. At this time, the peptide concentration was 50 μg/100 μL (in this case, 30 μg/100 μL in Example 4), the PHAD concentration was 10 μg/100 μL, and the Alhydrogel adjuvant was 10% (v/v).

(9) The amount of the peptide adsorbed to the aluminum gel was measured according to the method described in Experimental Example 2.3, and as a result, an adsorption rate of 95% or more was confirmed and used in the experiment. Examples prepared according to the above method are shown in Table 7.

Identification number (Label) Peptide sequences included in the composition Peptide SEQ ID NO: Example 1 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZ One Example 2 ZaK(Cha)VAAWTLKAAaZRNVPPIFNDVYWIAF 2 Example 3 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZCRFRGLISLSQVYLS 7 Example 4 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZCRFRGLISLSQVYLS 7

At this time, in the case of Example 4, the final concentration of the peptide was 30 μg/100 μL.

Experimental Example 3.2

A composition for in vivo administration including the peptide prepared in Experimental Example 1.2 was prepared according to the method disclosed in Experimental Example 3.1. According to the characteristics of the prepared peptide, the method may be appropriately modified. The examples in Table 8 were prepared according to the above method.

Identification number (Label) Peptide sequences included in the composition Peptide SEQ ID NO: Example 5 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZ One Example 6 RNVPPIFNDVYWIAFK(Cha)VAAWTLKAA 3 Example 7 K(Cha)VAAWTLKAARNVPPIFNDVYWIAF 4

Experimental Example 3.3

A composition for in vivo administration including the peptide prepared in Experimental Example 1.3 was prepared according to the method disclosed in Experimental Example 3.1. According to the characteristics of the prepared peptide, the method may be appropriately modified. The examples in Table 9 were prepared according to the above method.

Identification number (Label) Peptide sequences included in the composition Peptide SEQ ID NO: Example 8 RNVPPIFNDVYWIAFK(Cha)VAAWTLKAA 3 Example 9 RNVPPIFNDVYWIAFK(Cha)VAAWTLKAAHHHHHH 5

Experimental Example 3.4

A composition for in vivo administration including the peptide prepared in Experimental Example 1.4 was prepared according to the method disclosed in Experimental Example 3.1. According to the characteristics of the prepared peptide, the method may be appropriately modified. The examples in Table 10 were prepared according to the above method.

Identification number (Label) Peptide sequences included in the composition Peptide SEQ ID NO: Example 10 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZGSHHHHHHGSDDDDK 6 Example 11 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZCRFRGLISLSQVYLS 7 Example 12 CRFRGLISLSQVYLSZaK(Cha)VAAWTLKAAaZCRFRGLISLSQVYLS 8 Example 13 KTTKQSFDLSVKAQYKKNKHZaK(Cha)VAAWTLKAAaZCRFRGLISLSQVYLS 9 Example 14 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZRNVPPIFNDVYWIAF 10 Example 15 RNVPPIFNDVYWIAFZaK(Cha)VAAWTLKAAaZKTTKQSFDLSVKAQYKKNKH 11 Example 16 GSHHHHHHGSDDDDKZaK(Cha)VAAWTLKAAaZRNVPPIFNDVYWIAF 12

Experimental Example 3.5

A composition for in vivo administration including the peptide prepared in Experimental Example 1.5 was prepared according to the method disclosed in Experimental Example 3.1. According to the characteristics of the prepared peptide, the method may be appropriately modified. The examples in Table 11 were prepared according to the above method.

Identification number (Label) Peptide sequences included in the composition Peptide SEQ ID NO: Example 17 RNVPPIFNDVYWIAFCRFRGLISLSQVYLSZaK(Cha)VAAWTLKAAaZ 13 Example 18 RNVPPIFNDVYWIAFCRFRGLISLSQVYLSK(Cha)VAAWTLKAA 14 Example 19 RNVPPIFNDVYWIAFCRFRGLISLSQVYLSK(Cha)VAAWTLKAAHHHHHH 15

Experimental Example 3.6

A composition for in vivo administration including the peptide prepared in Experimental Example 1.6 was prepared according to the method disclosed in Experimental Example 3.1. According to the characteristics of the prepared peptide, the method may be appropriately modified. The examples in Table 12 were prepared according to the above method.

Identification number (Label) Peptide sequences included in the composition Peptide SEQ ID NO: Example 20 RNVPPIFNDVYWIAFZPKYVKQNTLKLATZCRFRGLISLSQVYLS 16

Experimental Example 4 Preparation of test subjects

Experimental Example 4.1 Method of preparing the test subject

In order to test the effect of the composition for in vivo administration prepared in Experimental Example 3.1, C57BL/6, Balbc and/or ICR species were used as experimental mice (Purchasing Central Laboratory Animal). Although there are some differences by species, on average, 7-week-old mice were purchased and acclimatized for 1 week, and then used for experiments from 8 weeks of age. Experimental mice were bred in an environment controlled in a constant temperature and humidity environment within the temperature range of 23±1°C and relative humidity of 50±5%, and in a 12-hour light room/12-hour dark room. Drinking water and food were provided ad libitum. In the case of a normal diet (central laboratory animal from the place of purchase), it was composed of 20% protein, 70% carbohydrate, and 10% fat based on total calories. , consisted of a high-fat diet composed of 60% fat. The experimental mice were divided into 5 groups, and the dietary conditions and composition administration conditions were varied, and the experiment was conducted on the number of 6 animals in each experimental group. Detailed conditions for each experimental group are as shown in Table 13 below.

experimental group mouse species feed dosing composition Number of objects per group Experimental group 1-1 C57BL/6, male Up to 13 weeks of age: normal diet,
After 13 weeks of age: an obesity-inducing diet Example 1 6 Experimental group 1-2 C57BL/6, male Up to 13 weeks of age: normal diet, after 13 weeks of age: obesity-induced diet Example 2 6

Experimental Example 4.2

In order to test the effect of the composition for in vivo administration of Example 3, a test subject was prepared according to the method described in Experimental Example 4.1. At this time, the conditions for each experimental group are as described in Table 14.

experimental group mouse species feed dosing composition Number of objects per group normal weight control
(Lean) C57BL/6, male normal diet PBS-Alum 12 Obesity induced by high-fat diet C57BL/6, male Up to 10 weeks of age: normal diet,
After 10 weeks of age: an obesity-inducing diet PBS-Alum 12 Placebo group (Mock) C57BL/6, male Up to 10 weeks of age: normal diet,
After 10 weeks of age: an obesity-inducing diet Alum-PHAD/PBS 12 Experimental group 2-1 (3H-OTP) C57BL/6, male Up to 10 weeks of age: normal diet,
After 10 weeks of age: an obesity-inducing diet Example 3 12

Experimental Example 4.3

In order to test the effects of the compositions for in vivo administration of Examples 3 and 4, test subjects were prepared according to the method described in Experimental Example 4.1. At this time, the conditions for each experimental group are as described in Table 15.

experimental group mouse species feed dosing composition Number of objects per group normal weight control
(Lean) C57BL/6, male normal diet PBS-Alum 8 Obese group induced by high-fat diet (Obese) C57BL/6, male Up to 11 weeks of age: normal diet,
After 11 weeks of age: an obesity-inducing diet PBS-Alum 9 Placebo group (Mock) C57BL/6, male Up to 11 weeks of age: normal diet,
After 11 weeks of age: an obesity-inducing diet Alum-PHAD/PBS 9 Experimental group 3-1 (3H-OTP 50ug) C57BL/6, male Up to 11 weeks of age: normal diet,
After 11 weeks of age: an obesity-inducing diet Example 3 8 Experimental group 3-2 (3H-OTP 30ug) C57BL/6, male Up to 11 weeks of age: normal diet,
After 11 weeks of age: an obesity-inducing diet Example 4 8

Experimental Example 4.4

In order to test the effect of the composition for in vivo administration of Example 3, a test subject was prepared according to the method described in Experimental Example 4.1. At this time, the conditions for each experimental group are as described in Table 16.

experimental group mouse species feed dosing composition Number of objects per group normal weight control
(Lean) C57BL/6, male normal diet PBS-Alum 10 Obese group induced by high-fat diet (Obese) C57BL/6, male Up to 5 weeks of age: normal diet,
After 5 weeks of age: an obesity-inducing diet PBS-Alum 10 Experimental group 4-1 (3H-OTP) C57BL/6, male Up to 5 weeks of age: normal diet,
After 5 weeks of age: an obesity-inducing diet Example 3 10

Experimental Example 4.5

In order to test the effect of the composition for in vivo administration of Example 3, a test subject was prepared according to the method described in Experimental Example 4.1. At this time, the conditions for each experimental group are as described in Table 17.

experimental group mouse species feed dosing composition Number of objects per group normal weight control C57BL/6J-Rag2e ^m1hwl /Korl, female/male
Wild (+/+) normal diet PBS-Alum Female: 3 Experimental group 5-1 (Wild(+/+) C57BL/6J-Rag2e ^m1hwl /Korl, female/male
Wild (+/+) Up to 10 weeks of age: normal diet,
After 10 weeks of age: an obesity-inducing diet PBS-Alum Female: 4
Male: 1 Experimental group 5-2 (Hetero (+/-) C57BL/6J-Rag2e ^m1hwl /Korl, female/male
Hetero (+/-) Up to 10 weeks of age: normal diet,
After 10 weeks of age: an obesity-inducing diet Example 3 Female: 3
Male: 3 Experimental group 5-3 (Homo(-/-) C57BL/6J-Rag2e ^m1hwl /Korl, female/male
Homo(-/-) Up to 10 weeks of age: normal diet,
After 10 weeks of age: an obesity-inducing diet Example 3 Female: 3
Male: 1

* The C57BL/6J-Rag2e ^m1hwl /Korl mouse is a mouse in which the Rag2 gene, which is a gene involved in antibody production in C57BL/6J mice, is knocked out.** Wild (+/+) has no mutated gene. wild-type mice, Hetero (+/-) means heterozygote mice, Homo (-/-) means homozygote mice.

*** Homo (-/-) mice do not have the ability to produce antibodies in the body.

Experimental Example 4.6

In order to test the effect of the composition for in vivo administration prepared in Experimental Example 3.2, a test subject is prepared through the method described in Experimental Example 4.1. At this time, the normal weight control group, the obese group induced by a high-fat diet, and the placebo administration group were prepared under the same conditions, but divided by the number of types of the composition for in vivo administration prepared in Experimental Example 3.2. The conditions for each experimental group are as described in Table 17.

experimental group mouse species feed dosing composition Number of objects per group normal weight control C57BL/6, Balbc and/or ICR normal diet PBS-Alum 6 to 12 Obesity group induced by a high-fat diet C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet,
After administration of the second composition: Obesity-inducing diet PBS-Alum 6 to 12 placebo group C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet,
After administration of the second composition: Obesity-inducing diet Alum-PHAD/PBS 6 to 12 Experimental group 6-1 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 5 6 to 12 Experimental group 6-2 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 6 6 to 12 Experimental group 6-3 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 7 6 to 12

Experimental Example 4.7

In order to test the effect of the composition for in vivo administration prepared in Experimental Example 3.3, a test subject is prepared through the method described in Experimental Example 4.1. At this time, the normal weight control group, the obese group induced by a high-fat diet, and the placebo administration group were prepared under the same conditions, but divided by the number of types of the composition for in vivo administration prepared in Experimental Example 3.3. The conditions for each experimental group are as described in Table 18.

experimental group mouse species feed dosing composition Number of objects per group normal weight control C57BL/6, Balbc and/or ICR normal diet PBS-Alum 6 to 12 Obesity group induced by a high-fat diet C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet PBS-Alum 6 to 12 placebo group C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Alum-PHAD/PBS 6 to 12 Experimental group 7-1 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 8 6 to 12 Experimental group 7-2 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 9 6 to 12

Experimental Example 4.8

In order to test the effect of the composition for in vivo administration prepared in Experimental Example 3.4, a test subject is prepared through the method described in Experimental Example 4.1. At this time, the normal weight control group, the obese group induced by a high-fat diet, and the placebo administration group were prepared under the same conditions, but divided by the number of types of the composition for in vivo administration prepared in Experimental Example 3.4. The conditions for each experimental group are as described in Table 19.

experimental group mouse species feed dosing composition Number of objects per group normal weight control C57BL/6, Balbc and/or ICR normal diet PBS-Alum 6 to 12 Obesity group induced by a high-fat diet C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet PBS-Alum 6 to 12 placebo group C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Alum-PHAD/PBS 6 to 12 Experimental group 8-1 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 10 6 to 12 Experimental group 8-2 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 11 6 to 12 Experimental group 8-3 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 12 6 to 12 Experimental group 8-4 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 13 6 to 12 Experimental group 8-5 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 14 6 to 12 Experimental group 8-6 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 15 6 to 12 Experimental group 8-7 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 16 6 to 12

Experimental Example 4.9

In order to test the effect of the composition for in vivo administration prepared in Experimental Example 3.5, a test subject is prepared through the method described in Experimental Example 4.1. At this time, the normal weight control group, the obese group induced by a high-fat diet, and the placebo administration group were prepared under the same conditions, but divided by the number of types of the composition for in vivo administration prepared in Experimental Example 3.5. The conditions for each experimental group are as described in Table 20.

experimental group mouse species feed dosing composition Number of objects per group normal weight control C57BL/6, Balbc and/or ICR normal diet PBS-Alum 6 to 12 Obesity group induced by a high-fat diet C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet PBS-Alum 6 to 12 placebo group C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Alum-PHAD/PBS 6 to 12 Experimental group 9-1 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 17 6 to 12 Experimental group 9-2 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 18 6 to 12 Experimental group 9-3 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 19 6 to 12

Experimental Example 4.10

In order to test the effect of the composition for in vivo administration prepared in Experimental Example 3.6, a test subject is prepared through the method described in Experimental Example 4.1. At this time, the normal weight control group, the obese group induced by a high-fat diet, and the placebo administration group were prepared under the same conditions, but divided by the number of types of the composition for in vivo administration prepared in Experimental Example 3.6. The conditions for each experimental group are as described in Table 21.

experimental group mouse species feed dosing composition Number of objects per group normal weight control C57BL/6, Balbc and/or ICR normal diet PBS-Alum 6 to 12 Obesity group induced by a high-fat diet C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet PBS-Alum 6 to 12 placebo group C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Alum-PHAD/PBS 6 to 12 Experimental group 10-1 C57BL/6, Balbc and/or ICR Until the second composition administration: normal diet, after the second composition administration: obesity-inducing diet Example 20 6 to 12

Experimental Example 5 Administration of a composition comprising a peptide to a test subject

Experimental Example 5.1 Method of administering drugs to test subjects

For the test subjects prepared in Experimental Example 4.1, different compositions were administered for each experimental group. All administration compositions were administered by intramuscular injection, and after disinfection of both thigh muscles with alcohol swab, 50 μL each, 100 μL in total was injected. Dosing times were as follows: 7 weeks, 9 weeks, 12 weeks, 15 weeks and 18 weeks of age.

Experimental Example 5.2

For the test subject prepared in Experimental Example 4.2, the composition designated for each experimental group was administered by the method described in Experimental Example 5.1. Dosing times were as follows: 8 weeks, 10 weeks, 12 weeks and 14 weeks of age.

Experimental Example 5.3

For the test subject prepared in Experimental Example 4.3, the composition designated for each experimental group was administered by the method described in Experimental Example 5.1. Dosing times were as follows: 8 weeks, 11 weeks, 14 weeks, 17 weeks, and 20 weeks of age.

Experimental Example 5.4

For the test subject prepared in Experimental Example 4.4, the composition designated for each experimental group was administered by the method described in Experimental Example 5.1. Dosing times were as follows: 11 weeks, 13 weeks, 15 weeks, and 17 weeks of age.

Experimental Example 5.5

For the test subject prepared in Experimental Example 4.5, the composition designated for each experimental group was administered by the method described in Experimental Example 5.1. Dosing times were as follows: 8 weeks, 10 weeks, 12 weeks, and 14 weeks of age.

Experimental Example 5.6

For the test subject prepared in Experimental Example 4.6, the composition designated for each experimental group was administered by the method described in Experimental Example 5.1. Depending on the experimental design, the administration cycle, administration timing, and administration frequency may be appropriately modified. For example, it may be administered 4 times at an interval of 2 weeks for an 8-week-old test subject, but is not limited thereto.

Experimental Example 5.7

For the test subject prepared in Experimental Example 4.7, the composition designated for each experimental group is administered by the method described in Experimental Example 5.1. Depending on the experimental design, the administration cycle, administration timing, and administration frequency may be appropriately modified. For example, it may be administered 4 times at an interval of 2 weeks for an 8-week-old test subject, but is not limited thereto.

Experimental Example 5.8

For the test subject prepared in Experimental Example 4.8, the composition designated for each experimental group is administered by the method described in Experimental Example 5.1. Depending on the experimental design, the administration cycle, administration timing, and administration frequency may be appropriately modified. For example, it may be administered 4 times at an interval of 2 weeks for an 8-week-old test subject, but is not limited thereto.

Experimental Example 5.9

For the test subject prepared in Experimental Example 4.9, the composition designated for each experimental group is administered by the method described in Experimental Example 5.1. Depending on the experimental design, the administration cycle, administration timing, and administration frequency may be appropriately modified. For example, it may be administered 4 times at an interval of 2 weeks for an 8-week-old test subject, but is not limited thereto.

Experimental Example 5.10

For the test subject prepared in Experimental Example 4.10, the composition designated for each experimental group is administered in the method described in Experimental Example 5.1. Depending on the experimental design, the administration cycle, administration timing, and administration frequency may be appropriately modified. For example, it may be administered 4 times at an interval of 2 weeks for an 8-week-old test subject, but is not limited thereto.

Experimental Example 6 Effect confirmation of composition for in vivo administration 1 - Antibody titer and weight loss effect

Experimental Example 6.1 Method for confirming potency against B-cell epitope

In order to determine whether the composition administered in the body administered in Experimental Example 5 induced the antibody against the B-cell epitope, the method for determining the titer to the B-cell epitope by separating the serum from the test subject and performing ELISA measurement is as follows. same as

1. Antigen Coating Reaction Process

1-1) Whenever a composition for in vivo administration is injected into a test subject, about 200 μL of blood is collected from the subject's tail vein one week after the injection.

1-2) After leaving the collected blood at 4°C for one hour, centrifugation is performed at 14,000 rpm for 10 minutes to separate the supernatant, the serum.

1-3) Dilute the peptide of SEQ ID NO: 41 to a concentration of 50 μg/100 μL in coating buffer (0.05M, Bicarbonate, pH9.6), add 50 μg per well to a 96-well plate, and overnight ( overnight) so that the peptide is coated on the wall of the well.

1-4) Wash the plate coated with the peptide of SEQ ID NO: 41 three times using 300 μL of PBS-T (Phosphate Buffered Saline, 0.05% with Tween-20) per well.

2. blocking reaction process

2-1) Add 300 μL of 0.5% casein blocking solution per well to the plate, and react at 4° C. overnight.

2-2) Wash the plate 3 times with 300 μL of PBS-T per well.

3. Primary Antibody Reaction Process

3-1) As the primary antibody reaction, the separated serum was diluted to an appropriate concentration, 100 μL per well was added, and the reaction was performed at 37° C. for one hour. At this time, the serum was diluted to 1/20 to 1/1000 in the experiment after the initial injection, and 1/500 to 1/10000 according to the subject during the experiment after the 2nd to 3rd injection, and serial dilution was performed. As a positive control, a purified monoclonal antibody against the peptide of SEQ ID NO: 41 is used.

3-2) After the reaction of 3-1, wash the plate 3 times with 300 μL of PBS-T per well.

4. Secondary Antibody Reaction Process

4-1) As a secondary antibody reaction, 100 μL of HRP (horseradish peroxidase) conjugated to anti-mouse IgG antibody recognizing mouse antibody is added per well, and reacted at 37°C for 1 hour.

4-2) After the reaction of 4-1), wash the plate 3 times with 300 µL of PBS-T per well.

5. Color development and absorbance confirmation process

5-1) Add 100 μL of OPD solution (o-phenylenediamine dihydrochloride solution) per well, react at 37°C for 10 minutes, and measure absorbance at O.D 450nm (Synergy HT microplate reader, BioTek).

5-2) The antibody titer in serum is converted by measuring the extinction coefficient based on the 1 mg/mL concentration of the monoclonal antibody against the peptide of SEQ ID NO: 41 as a positive control.

Experimental Example 6.2 Method for confirming potency against ApoB-100

In order to confirm whether the antibody induced by the in vivo composition administered in Experimental Example 5 has binding ability to ApoB-100, the same method as described in Experimental Example 6.1, but instead of the peptide of SEQ ID NO: 41, Apolipoprotein B-100 (ApoB-100) was used to check the titer of the antibody.

Experimental Example 6.3 Method for confirming weight loss effect

A method for confirming the weight loss effect on the test subject of the in vivo composition administered in Experimental Example 5 is as follows: Body weight and organ weight are measured for each experimental group disclosed in Experimental Example 4.1. 1) From the time of wearing the mouse to the end of the experiment, 3 times a week for the test target mouse, after measuring the weight, calculate the average value to obtain the weight by age. 2) After the end of the experiment, each test subject is anesthetized, the organs are separated through dissection, the weight of each individual is measured, and the average is obtained for each group.

Experimental Example 6.4

In order to investigate the effect of the composition for in vivo administration administered by the method of Experimental Example 5.1 according to the experimental conditions of Experimental Example 4.1, the following experiments were conducted.

(1) In order to confirm the weight loss effect, the weight of the test subject was measured for each experimental group disclosed in Experimental Example 4.1 by the method disclosed in Experimental Example 6.3.

The experimental results are shown in Figs.

Experimental Example 6.5

In order to investigate the effect of the composition for in vivo administration administered by the method of Experimental Example 5.2 according to the experimental conditions of Experimental Example 4.2, the following experiments were conducted.

(1) In order to confirm the antibody-inducing effect, the antibody titer to each B-cell epitope was confirmed according to the method disclosed in Experimental Example 6.1.

(2) In order to confirm the weight loss effect, the body weight and organ weight of the test subject were measured for each experimental group disclosed in Experimental Example 4.2 by the method disclosed in Experimental Example 6.3.

The experimental results are shown in FIGS. 4 to 5 and Tables 23 to 24.

Weight(g) BMI Liver(g) Heart(g) normal weight control 26.68±0.95 0.30±0.01 1.15±0.07 0.13±0.01 Obesity group induced by a high-fat diet 37.10±2.95 0.41±0.01 1.31±0.05 0.13±0.00 placebo group 41.30±4.41 0.46±0.05 1.26±0.1 0.13±0.01 Experimental group 2-1 31.36±2.14 0.37±0.02 1.33±0.07 0.13±0.01

Kidney(g) Spleen(g) Muscle(g) Epididymal (g) Mesenteric (g) normal weight control 0.28±0.01 0.06±0.01 0.25±0.02 0.48±0.04 0.56±0.03 Obesity group induced by a high-fat diet 0.32±0.01 0.07±0.00 0.34±0.02 2.57±0.16 1.22±0.17 placebo group 0.31±0.01 0.07±0.01 0.36±0.03 2.39±0.18 1.36±0.14 Experimental group 2-1 0.33±0.01 0.08±0.01 0.34±0.04 1.42±0.35 0.99±0.17

Experimental Example 6.6

In order to investigate the effect of the composition for in vivo administration administered by the method of Experimental Example 5.3 according to the experimental conditions of Experimental Example 4.3, the following experiment was performed.

(1) In order to confirm the antibody-inducing effect, the antibody titer to each B-cell epitope was confirmed according to the method disclosed in Experimental Example 6.1.

(2) In order to confirm the weight loss effect, the weight of the test subject was measured for each experimental group disclosed in Experimental Example 4.3 by the method disclosed in Experimental Example 6.3.

The experimental results are shown in FIGS. 9 to 10 .

Experimental Example 6.7

In order to investigate the effect of the composition for in vivo administration administered by the method of Experimental Example 5.4 according to the experimental conditions of Experimental Example 4.4, the following experiments were conducted.

(1) In order to confirm the weight loss effect, the weight of the test subject was measured for each experimental group disclosed in Experimental Example 4.4 by the method disclosed in Experimental Example 6.3.

The experimental results are shown in FIG. 11 .

Experimental Example 6.8

In order to examine the effect of the composition for in vivo administration administered by the method of Experimental Example 5.5 according to the experimental conditions of Experimental Example 4.5, the following experiments were conducted.

(1) In order to confirm the weight loss effect, the weight of the test subject was measured for each experimental group disclosed in Experimental Example 4.5 by the method disclosed in Experimental Example 6.3.

The experimental results are shown in FIG. 12 .

Experimental Example 6.9

In order to examine the effect of the composition for in vivo administration administered by the method of Experimental Example 5.6 according to the experimental conditions of Experimental Example 4.6, the experiment was conducted as follows.

(1) To confirm the antibody-inducing effect, the antibody titer to each B-cell epitope is checked according to the method disclosed in Experimental Example 6.1.

(2) To confirm the antibody-inducing effect, the antibody titer to ApoB-100 is checked according to the method disclosed in Experimental Example 6.2.

(3) In order to confirm the weight loss effect, the body weight and organ weight of the test subject are measured for each experimental group disclosed in Experimental Example 4.5 by the method disclosed in Experimental Example 6.3.

In this case, the method may be appropriately modified as necessary.

Experimental Example 6.10

In order to examine the effect of the composition for in vivo administration administered by the method of Experimental Example 5.7 according to the experimental conditions of Experimental Example 4.7, the experiment was conducted as follows.

(1) To confirm the antibody-inducing effect, the antibody titer to each B-cell epitope is checked according to the method disclosed in Experimental Example 6.1.

(2) To confirm the antibody-inducing effect, the antibody titer to ApoB-100 is checked according to the method disclosed in Experimental Example 6.2.

(3) In order to confirm the weight loss effect, by the method disclosed in Experimental Example 6.3, the body weight and organ weight of the test subject for each experimental group disclosed in Experimental Example 4.6 were measured.

In this case, the method may be appropriately modified as necessary.

Experimental Example 6.11

In order to investigate the effect of the composition for in vivo administration administered by the method of Experimental Example 5.8 according to the experimental conditions of Experimental Example 4.8, the experiment was conducted as follows.

(1) To confirm the antibody-inducing effect, the antibody titer to each B-cell epitope is checked according to the method disclosed in Experimental Example 6.1.

(2) To confirm the antibody-inducing effect, the antibody titer to ApoB-100 is checked according to the method disclosed in Experimental Example 6.2.

(3) In order to confirm the weight loss effect, by the method disclosed in Experimental Example 6.3, the body weight and organ weight of the test subject for each experimental group disclosed in Experimental Example 4.7 were measured.

In this case, the method may be appropriately modified as necessary.

Experimental Example 6.12

In order to investigate the effect of the composition for in vivo administration administered by the method of Experimental Example 5.9 according to the experimental conditions of Experimental Example 4.9, the experiment was conducted as follows.

(1) To confirm the antibody-inducing effect, the antibody titer to each B-cell epitope is checked according to the method disclosed in Experimental Example 6.1.

(2) To confirm the antibody-inducing effect, the antibody titer to ApoB-100 is checked according to the method disclosed in Experimental Example 6.2.

(3) In order to confirm the weight loss effect, by the method disclosed in Experimental Example 6.3, the body weight and organ weight of the test subject for each experimental group disclosed in Experimental Example 4.8 were measured.

In this case, the method may be appropriately modified as necessary.

Experimental Example 6.13

In order to investigate the effect of the composition for in vivo administration administered by the method of Experimental Example 5.10 according to the experimental conditions of Experimental Example 4.10, the experiment was conducted as follows.

(1) To confirm the antibody-inducing effect, the antibody titer to each B-cell epitope is checked according to the method disclosed in Experimental Example 6.1.

(2) To confirm the antibody-inducing effect, the antibody titer to ApoB-100 is checked according to the method disclosed in Experimental Example 6.2.

(3) In order to confirm the weight loss effect, by the method disclosed in Experimental Example 6.3, the body weight and organ weight of the test subject for each experimental group disclosed in Experimental Example 4.9 were measured.

In this case, the method may be appropriately modified as necessary.

Experimental Example 7 Effect of the composition for in vivo administration 2 - Comparison of blood lipid concentration, fat resolution, and adipocyte size

Experimental Example 7.1 Method of confirming blood lipid concentration of test subject

The experimental method for examining the effect of the intra-body administration composition administered in Experimental Example 5 on the blood lipid concentration of the test subject is as follows:

(1) One week after administration of each composition, about 200 μL of blood is collected from the subject's tail vein.

(2) Measurement of blood triglyceride (TG, triglyceride) concentration: Triglyzyme-V (Shinyak Chemical Co., Ltd.) was used. i) After mixing 4 μL of blood sample and 300 μL of color reagent, the mixture was reacted at 37° C. for 5 minutes. ii) For the produced red quinone, the absorbance is measured at 505 nm, and the concentration is calculated by comparing with the reference solution.

(3) Measurement of total cholesterol concentration in blood: Cholestezyme-V (Shinyak Chemical Co., Ltd.) was used. i) After mixing 4 μL of blood sample and 300 μL of color reagent, the mixture was reacted at 37° C. for 5 minutes. ii) For the produced red quinone, the absorbance is measured at 505 nm, and the concentration is calculated by comparing with the reference solution.

(4) Measurement of high density lipoprotein (HDL) concentration in blood: HDL-C555 (Shinyak Chemical Co., Ltd.) was used. i) After mixing 10 μL of blood sample and 10 μL of precipitation reagent, it was allowed to react at room temperature for at least 10 minutes. ii) The reaction mass was centrifuged at 300 rpm or higher, and the supernatant was separated. iii) 4 μL of the supernatant and 300 μL of the color reagent were mixed, and then reacted at 37°C for 5 minutes. iv) For the above reaction, measure the absorbance at 555 nm and calculate the concentration compared to the reference solution.

(5) Measurement of blood LDL (low density lipoprotein) concentration: i) The reaction was carried out using the Direct LDL Cholesterol detection kit (Randox). ii) After the reaction of step 2, for the produced quinone, absorbance is measured at 600 nm, and the concentration is calculated by comparing with the reference solution.

Experimental Example 7.2 Comparison of adipocyte resolution and adipocyte size of test subjects

A method for examining the effect of the composition administered in the body in Experimental Example 5 on the adipocyte resolution and adipocyte size of the test subject by hormone sensitive lipase (HSL) is as follows:

1. Adipocyte Isolation

1-1) After cutting the epididymal fat pad with scissors, add 4 ml per 1 g of KRB buffer containing 2% FBS, 2 mM glucose, and 1 mg/ml collagenase, shake and react at 37°C for 1 hour.

1-2) When the reaction is complete, pass through a 300 μm nylon mesh to filter out adipose tissue debris and adipose tissue, and then pass through a 40 μm nylon mesh again to separate adipocytes and macrophages.

1-3) The adipocytes filtered in 1-2) are washed by adding DMEM containing 10% FBS and 1% AA and removing the sublayer with a syringe to obtain adipocytes from which collagenase has been removed.

2. Comparison of fat resolution

2-1) Seeding the adipocytes obtained in 1-3) in a 48 well plate at 1.0 x 10^5 cells per well, and add 1 ml of DMEM (10% FBS, 1% AA) in total for culture. and incubate for 2 hours at 37°C and 5% CO₂ condition.

2-2) For the well to induce HSL activity, norepinephrine is added to a final 10^-5 M.

2-3) After the reaction, 100 µL of the supernatant of each well and 100 µL of the free glycerol reagent are reacted and absorbance at 540 nm is measured.

3. Observation of adipocyte size

3-1) The adipocytes obtained in 1-3) were seeded in a 48 well plate at 1.0 x 10^5 cells/ml per well, treated with 10 μM of DAPI, reacted for 2 hours, and observed under a microscope.

3-2) To confirm that DAPI-stained cells are adipocytes, Lipid and nucleus were stained together and observed. At this time, 10 uM of DAPI and 1:1000 of HCS LipidTOX were treated and reacted for 24 hours, and then observed under a microscope.

Experimental Example 7.3

In order to investigate the effect of the composition for in vivo administration administered by the method of Experimental Example 5.2 according to the experimental conditions of Experimental Example 4.2, the following experiments were conducted.

(1) To confirm the blood lipid concentration of the test subject, according to the method described in Experimental Example 7.1, the blood lipid concentration of the test object was measured for each experimental group disclosed in Experimental Example 4.2.

(2) In order to confirm the adipocyte resolution and adipocyte size of the test subject, according to the method described in Experimental Example 7.2, the adipocyte resolution and adipocyte size of the test subject were measured for each experimental group disclosed in Experimental Example 4.2.

The experimental results are shown in FIGS. 6 to 8 .

Experimental Example 7.4

In order to examine the effect of the composition for in vivo administration administered by the method of Experimental Example 5.6 according to the experimental conditions of Experimental Example 4.6, the experiment was conducted as follows.

(1) In order to confirm the blood lipid concentration of the test subject, by the method disclosed in Experimental Example 7.1, the blood lipid concentration of the test object is measured for each experimental group disclosed in Experimental Example 4.6.

(2) In order to confirm the size of adipocytes in the test subject, the adipocyte resolution and adipocyte size were measured for each experimental group disclosed in Experimental Example 4.6 by the method described in Experimental Example 7.2.

In this case, the method may be appropriately modified as necessary.

Experimental Example 7.5

In order to examine the effect of the composition for in vivo administration administered by the method of Experimental Example 5.7 according to the experimental conditions of Experimental Example 4.7, the experiment was conducted as follows.

(1) In order to confirm the blood lipid concentration of the test subject, by the method disclosed in Experimental Example 7.1, the blood lipid concentration of the test object is measured for each experimental group disclosed in Experimental Example 4.7.

(2) In order to confirm the size of adipocytes in the test subject, adipocyte resolution and adipocyte size were measured for each experimental group disclosed in Experimental Example 4.7 by the method disclosed in Experimental Example 7.2.

In this case, the method may be appropriately modified as necessary.

Experimental Example 7.6

In order to investigate the effect of the composition for in vivo administration administered by the method of Experimental Example 5.8 according to the experimental conditions of Experimental Example 4.8, the experiment was conducted as follows.

(1) In order to confirm the blood lipid concentration of the test subject, the blood lipid concentration of the test subject is measured by the method disclosed in Experimental Example 7.1 for each experimental group disclosed in Experimental Example 4.8.

(2) In order to confirm the size of the adipocytes of the test subject, the adipocyte resolution and adipocyte size were measured for each experimental group disclosed in Experimental Example 4.8 by the method disclosed in Experimental Example 7.2.

In this case, the method may be appropriately modified as necessary.

Experimental Example 7.7

In order to investigate the effect of the composition for in vivo administration administered by the method of Experimental Example 5.9 according to the experimental conditions of Experimental Example 4.9, the experiment was conducted as follows.

(1) In order to confirm the blood lipid concentration of the test subject, the blood lipid concentration of the test subject is measured by the method disclosed in Experimental Example 7.1 for each experimental group disclosed in Experimental Example 4.9.

(2) In order to confirm the size of adipocytes in the test subject, the adipocyte resolution and adipocyte size were measured for each experimental group disclosed in Experimental Example 4.9 using the method disclosed in Experimental Example 7.2.

In this case, the method may be appropriately modified as necessary.

Experimental Example 7.8

In order to examine the effect of the composition for in vivo administration administered by the method of Experimental Example 5.10 according to the experimental conditions of Experimental Example 4.10, the experiment was conducted as follows.

(1) In order to confirm the blood lipid concentration of the test subject, the blood lipid concentration of the test subject is measured by the method disclosed in Experimental Example 7.1 for each experimental group disclosed in Experimental Example 4.9.

(2) To confirm the size of adipocytes in the test subject, the adipocyte resolution and adipocyte size were measured for each experimental group disclosed in Experimental Example 4.9 by the method described in Experimental Example 7.2.

In this case, the method may be appropriately modified as necessary.

<110> 3H Bio <120> A peptide and use thereof <130> CPP20-045 <160> 78 <170> KoPatentIn 3.0 <210> 1 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(30) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. 17th amino acid(Xaa) is a D-form alanine. 19th amino acid (Xaa) is L-cyclohexyllanins. 29th amino acid (Xaa) is a D-form alanine. 30th amino acid (Xaa) is 6-aminohexanoic acid. <400> 1 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa 1 5 10 15 Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Xaa Xaa 20 25 30 <210> 2 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(30) <223> 1st amino acid (Xaa) is a 6-aminohexanoic acid. 2nd amino acid(Xaa) is a D-form alanine. 4th amino acid (Xaa) is L-cyclohexyllanins. 14th amino acid (Xaa) is a D-form alanine. 15th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 2 Xaa Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Xaa Xaa Arg 1 5 10 15 Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 20 25 30 <210> 3 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(26) <223> 17th amino acid (Xaa) is a L-cyclohexyllanins. <400> 3 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Lys 1 5 10 15 Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala 20 25 <210> 4 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(26) <223> 2nd amino acid (Xaa) is a L-cyclohexylalanins. <400> 4 Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Arg Asn Val Pro Pro 1 5 10 15 Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 20 25 <210> 5 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 5 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Lys 1 5 10 15 Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala His His His His His His 20 25 30 <210> 6 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(45) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. 17th amino acid(Xaa) is a D-form alanine. 19th amino acid (Xaa) is L-cyclohexyllanins. 29th amino acid (Xaa) is a D-form alanine. 30th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 6 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa 1 5 10 15 Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Xaa Xaa Gly Ser 20 25 30 His His His His His His Gly Ser Asp Asp Asp Asp Lys 35 40 45 <210> 7 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(45) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. 17th amino acid(Xaa) is a D-form alanine. 19th amino acid (Xaa) is L-cyclohexyllanins. 29th amino acid (Xaa) is a D-form alanine. 30th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 7 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa 1 5 10 15 Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Xaa Xaa Cys Arg 20 25 30 Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 35 40 45 <210> 8 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(45) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. 17th amino acid(Xaa) is a D-form alanine. 19th amino acid (Xaa) is L-cyclohexyllanins. 29th amino acid (Xaa) is a D-form alanine. 30th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 8 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Xaa 1 5 10 15 Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Xaa Xaa Cys Arg 20 25 30 Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 35 40 45 <210> 9 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(50) <223> 21st amino acid (Xaa) is a 6-aminohexanoic acid. 22nd amino acid(Xaa) is a D-form alanine. 24th amino acid (Xaa) is L-cyclohexyllanins. 34th amino acid (Xaa) is a D-form alanine. 35th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 9 Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr Lys 1 5 10 15 Lys Asn Lys His Xaa Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala 20 25 30 Ala Xaa Xaa Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr 35 40 45 Leu Ser 50 <210> 10 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(45) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. 17th amino acid(Xaa) is a D-form alanine. 19th amino acid (Xaa) is L-cyclohexyllanins. 29th amino acid (Xaa) is a D-form alanine. 30th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 10 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa 1 5 10 15 Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Xaa Xaa Arg Asn 20 25 30 Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 35 40 45 <210> 11 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(50) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. 17th amino acid(Xaa) is a D-form alanine. 19th amino acid (Xaa) is L-cyclohexyllanins. 29th amino acid (Xaa) is a D-form alanine. 30th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 11 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa 1 5 10 15 Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Xaa Xaa Lys Thr 20 25 30 Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr Lys Lys Asn 35 40 45 Lys His 50 <210> 12 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(45) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. 17th amino acid(Xaa) is a D-form alanine. 19th amino acid (Xaa) is L-cyclohexyllanins. 29th amino acid (Xaa) is a D-form alanine. 30th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 12 Gly Ser His His His His His His His Gly Ser Asp Asp Asp Asp Lys Xaa 1 5 10 15 Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Xaa Xaa Arg Asn 20 25 30 Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 35 40 45 <210> 13 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 13 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Cys 1 5 10 15 Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Xaa Xaa 20 25 30 Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala Xaa Xaa 35 40 45 <210> 14 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 14 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Cys 1 5 10 15 Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Lys Xaa 20 25 30 Val Ala Ala Trp Thr Leu Lys Ala Ala 35 40 <210> 15 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 15 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Cys 1 5 10 15 Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Lys Xaa 20 25 30 Val Ala Ala Trp Thr Leu Lys Ala Ala His His His His His His 35 40 45 <210> 16 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 16 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Glx 1 5 10 15 Pro Lys Tyr Val Lys Gln Asn Thr Leu Lys Leu Ala Thr Glx Cys Arg 20 25 30 Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 35 40 45 <210> 17 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(10) <223> 2nd amino acid (Xaa) is L-cyclohexyllanins. <400> 17 Lys Val Ala Ala Trp Thr Leu Lys Ala Ala 1 5 10 <210> 18 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 18 Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile 1 5 10 15 <210> 19 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 19 Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala Gln Ala Ile Pro 1 5 10 15 <210> 20 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 20 Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Pro 1 5 10 15 Met Gly Leu Pro Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala Gln 20 25 30 <210> 21 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 1st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 21 Xaa Arg Asn Val One <210> 22 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 1st amino acid (Xaa) is a D-form alanine. 2nd amino acid (Xaa) is a 6-aminohexanoic acid. <400> 22 Xaa Xaa Arg Asn One <210> 23 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 4th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 23 Ile Ala Phe Xaa One <210> 24 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 3rd amino acid (Xaa) is a 6-aminohexanoic acid. 4th amino acid(Xaa) is a D-form alanine. <400> 24 Ala Phe Xaa Xaa One <210> 25 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 25 Arg Asn Val Pro One <210> 26 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 26 Trp Ile Ala Phe One <210> 27 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 1st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 27 Xaa Cys Arg Phe One <210> 28 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 1st amino acid (Xaa) is a D-form alanine. 2nd amino acid (Xaa) is a 6-aminohexanoic acid. <400> 28 Xaa Xaa Cys Arg One <210> 29 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 4th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 29 Tyr Leu Ser Xaa One <210> 30 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 3rd amino acid (Xaa) is a 6-aminohexanoic acid. 4th amino acid(Xaa) is a D-form alanine. <400> 30 Leu Ser Xaa Xaa One <210> 31 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 31 Cys Arg Phe Arg One <210> 32 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 32 Val Tyr Leu Ser One <210> 33 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 1st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 33 Xaa Lys Thr Thr One <210> 34 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 1st amino acid (Xaa) is a D-form alanine. 2nd amino acid (Xaa) is a 6-aminohexanoic acid. <400> 34 Xaa Xaa Lys Thr One <210> 35 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 4th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 35 Asn Lys His Xaa One <210> 36 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(4) <223> 3rd amino acid (Xaa) is a 6-aminohexanoic acid. 4th amino acid(Xaa) is a D-form alanine. <400> 36 Lys His Xaa Xaa One <210> 37 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 37 Gly Ser His His His His His His His Gly Ser Asp Asp Asp Asp Lys 1 5 10 15 <210> 38 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 38 His His His His His His 1 5 <210> 39 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 39 Met Arg Gly Ser His His His His His His Gly Ser Asp Asp Asp Asp 1 5 10 15 Lys Ile Val Asp 20 <210> 40 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 40 Gly Gly Gly Gly Ser Gly Gly Gly Gly Gly Gly Ser Ser 1 5 10 <210> 41 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 41 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 1 5 10 15 <210> 42 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 42 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 1 5 10 15 <210> 43 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 43 Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr Lys 1 5 10 15 Lys Asn Lys His 20 <210> 44 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(16) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 44 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa 1 5 10 15 <210> 45 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(16) <223> 1st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 45 Xaa Arg Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 1 5 10 15 <210> 46 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(16) <223> 16th amino acid (Xaa) is a D-form alanine. <400> 46 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa 1 5 10 15 <210> 47 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(16) <223> 1st amino acid (Xaa) is a D-form alanine. <400> 47 Xaa Arg Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 1 5 10 15 <210> 48 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(17) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. 17th amino acid(Xaa) is a D-form alanine. <400> 48 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa 1 5 10 15 Xaa <210> 49 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(17) <223> 1st amino acid (Xaa) is a D-form alanine. 2nd amino acid (Xaa) is a 6-aminohexanoic acid. <400> 49 Xaa Xaa Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala 1 5 10 15 Phe <210> 50 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(16) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 50 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Xaa 1 5 10 15 <210> 51 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(16) <223> 1st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 51 Xaa Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 1 5 10 15 <210> 52 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(16) <223> 16th amino acid (Xaa) is a D-form alanine. <400> 52 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Xaa 1 5 10 15 <210> 53 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(16) <223> 1st amino acid (Xaa) is a D-form alanine. <400> 53 Xaa Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 1 5 10 15 <210> 54 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(17) <223> 16th amino acid (Xaa) is a 6-aminohexanoic acid. 17th amino acid(Xaa) is a D-form alanine. <400> 54 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Xaa 1 5 10 15 Xaa <210> 55 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(17) <223> 1st amino acid (Xaa) is a D-form alanine. 2nd amino acid (Xaa) is a 6-aminohexanoic acid. <400> 55 Xaa Xaa Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu 1 5 10 15 Ser <210> 56 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(21) <223> 21st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 56 Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr Lys 1 5 10 15 Lys Asn Lys His Xaa 20 <210> 57 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(21) <223> 1st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 57 Xaa Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr 1 5 10 15 Lys Lys Asn Lys His 20 <210> 58 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(21) <223> 21st amino acid (Xaa) is a D-form alanine. <400> 58 Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr Lys 1 5 10 15 Lys Asn Lys His Xaa 20 <210> 59 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(21) <223> 1st amino acid (Xaa) is a D-form alanine. <400> 59 Xaa Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr 1 5 10 15 Lys Lys Asn Lys His 20 <210> 60 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(22) <223> 21st amino acid (Xaa) is a 6-aminohexanoic acid. 22nd amino acid(Xaa) is a D-form alanine. <400> 60 Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln Tyr Lys 1 5 10 15 Lys Asn Lys His Xaa Xaa 20 <210> 61 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(22) <223> 1st amino acid (Xaa) is a D-form alanine. 2nd amino acid (Xaa) is a 6-aminohexanoic acid. <400> 61 Xaa Xaa Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln 1 5 10 15 Tyr Lys Lys Asn Lys His 20 <210> 62 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 62 Xaa Xaa Lys Thr Thr Lys Gln Ser Phe Asp Leu Ser Val Lys Ala Gln 1 5 10 15 Tyr Lys Lys Asn Lys His 20 <210> 63 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(31) <223> 31st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 63 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Cys 1 5 10 15 Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Xaa 20 25 30 <210> 64 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(31) <223> 31st amino acid (Xaa) is a D-form alanine. <400> 64 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Cys 1 5 10 15 Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Xaa 20 25 30 <210> 65 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(32) <223> 31st amino acid (Xaa) is a 6-aminohexanoic acid. 32nd amino acid(Xaa) is a D-form alanine. <400> 65 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Cys 1 5 10 15 Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Xaa Xaa 20 25 30 <210> 66 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(31) <223> 1st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 66 Xaa Arg Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 1 5 10 15 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 20 25 30 <210> 67 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(31) <223> 1st amino acid (Xaa) is a D-form alanine. <400> 67 Xaa Arg Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 1 5 10 15 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 20 25 30 <210> 68 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(32) <223> 1st amino acid (Xaa) is a D-form alanine. 2nd amino acid (Xaa) is a 6-aminohexanoic acid. <400> 68 Xaa Xaa Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala 1 5 10 15 Phe Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 20 25 30 <210> 69 <211> 30 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 69 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Arg 1 5 10 15 Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 20 25 30 <210> 70 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(31) <223> 31st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 70 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Arg 1 5 10 15 Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa 20 25 30 <210> 71 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(31) <223> 31st amino acid (Xaa) is a D-form alanine. <400> 71 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Arg 1 5 10 15 Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa 20 25 30 <210> 72 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(32) <223> 31st amino acid (Xaa) is a 6-aminohexanoic acid. 32nd amino acid(Xaa) is a D-form alanine. <400> 72 Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser Arg 1 5 10 15 Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe Xaa Xaa 20 25 30 <210> 73 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(31) <223> 1st amino acid (Xaa) is a 6-aminohexanoic acid. <400> 73 Xaa Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 1 5 10 15 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 20 25 30 <210> 74 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(31) <223> 1st amino acid (Xaa) is a D-form alanine. <400> 74 Xaa Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 1 5 10 15 Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 20 25 30 <210> 75 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(32) <223> 1st amino acid (Xaa) is a D-form alanine. 2nd amino acid (Xaa) is a 6-aminohexanoic acid. <400> 75 Xaa Xaa Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu 1 5 10 15 Ser Arg Asn Val Pro Ile Phe Asn Asp Val Tyr Trp Ile Ala Phe 20 25 30 <210> 76 <211> 80 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <220> <221> MISC_FEATURE <222> (1)..(80) <223> 36th amino acid (Xaa) is a 6-aminohexanoic acid. 37th amino acid(Xaa) is a D-form alanine. 39th amino acid (Xaa) is L-cyclohexyllanins. 49th amino acid (Xaa) is a D-form alanine. 50th amino acid (Xaa) is a 6-aminohexanoic acid. <400> 76 Met Arg Gly Ser His His His His His His Gly Ser Asp Asp Asp Asp 1 5 10 15 Lys Ile Val Asp Arg Asn Val Pro Pro Ile Phe Asn Asp Val Tyr Trp 20 25 30 Ile Ala Phe Xaa Xaa Lys Xaa Val Ala Ala Trp Thr Leu Lys Ala Ala 35 40 45 Xaa Xaa Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly 50 55 60 Ile Cys Arg Phe Arg Gly Leu Ile Ser Leu Ser Gln Val Tyr Leu Ser 65 70 75 80 <210> 77 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 77 Ile Leu Met Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Pro 1 5 10 15 Met Gly Leu Pro Gln Ser Ile Ala Leu Ser Ser Leu Met Val Ala Gln 20 25 30 <210> 78 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Artificial Sequence <400> 78 Arg Arg Arg Arg Arg Arg 1 5

Claims (5)

A peptide having a length of 45mer, wherein the sequence of the peptide is SEQ ID NO:7.
A pharmaceutical composition for the treatment of obesity, comprising:
a peptide having a length of 45mer, wherein the sequence of the peptide is SEQ ID NO:7; and
adjuvants.
The pharmaceutical composition for treating obesity according to claim 2, wherein the adjuvant is _{at least one selected from the group consisting of aluminum hydroxide (Al(OH) 3} ), liposomes, squalene, MF59, and QS21.
According to claim 2, wherein the pharmaceutical composition is phosphoryl lipid A (phosphoryl lipid A), saponin (saponin), prolactin (prolactin), growth hormone (growth hormone), deoxycholic acid (deoxycholic acid), beta glucan ( Betaglucan), and a pharmaceutical composition for the treatment of obesity, further comprising one or more immune enhancers selected from the group consisting of polyribonucleotides.
The pharmaceutical composition for treating obesity according to claim 4, wherein the adjuvant is aluminum hydroxide (Al(OH) ₃ ), and the immune enhancer is phosphoryl lipid A.

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