Antibody for inducing insulin-secreting beta-cell differentiation and composition for preventing or treating metabolic syndromes containing the same

Abstract

The present invention relates to an antibody characterized by inducing the differentiation of bone marrow hematopoietic stem cells into insulin-secreting beta cells, wherein the antibody targets the membrane-binding protein Periostin (POSTN) as a target antigen. Beta cells differentiated using the antibody of the present invention can migrate to the pancreas and secrete insulin, thereby enabling its application as a therapeutic agent for diabetes or related metabolic diseases. Furthermore, the use of an antibody with few side effects and a long half-life in the body can compensate for the shortcomings of conventional treatments and enhance therapeutic efficacy. In addition, the antibody of the present invention can be utilized as a diagnostic kit for the discovery of POSTN agonists.

Description

Technology Field

[0001] The present invention relates to an antibody that induces the differentiation of insulin-secreting beta cells and a composition for the prevention or treatment of metabolic diseases containing the same as an active ingredient. Background Technology

[0002] Metabolic diseases are conditions caused by lifestyle habits such as lack of exercise and overnutrition, and are known to lead to obesity, diabetes, hypertension, hyperlipidemia, fatty liver, and heart disease. Among these, diabetes is a metabolic disease characterized by abnormalities in glucose metabolism resulting from insulin deficiency or insulin resistance. If diabetes persists for several years, it can lead to complications such as glaucoma, vision loss, autonomic neuropathy, renal failure, angina pectoris, myocardial infarction, hypertension, and stroke. Diabetes is classified into Type 1 diabetes, which is insulin-dependent and caused by the destruction of the pancreatic beta cells responsible for insulin secretion; Type 2 diabetes, which is insulin-independent and caused by insulin receptor resistance despite normal beta cells; and gestational diabetes, which involves impaired glucose tolerance and inefficient insulin secretion. In modern society, obesity has risen rapidly due to changes in dietary habits, leading to an increase in Type 2 diabetes patients resulting from insulin resistance caused by obesity.

[0003] Meanwhile, methods to regulate cell differentiation by selecting functional agents from intracellular combinatorial antibody libraries are currently being studied. In particular, controlling the differentiation of cells with self-renewing and multi-efficacy capabilities, such as bone marrow hematopoietic stem cells (HSCs), can be crucial for the treatment of adult diseases, especially infections, the development of autoimmunity, and immunological disorders.

[0004] Accordingly, the present invention was completed by demonstrating the possibility of treating metabolic diseases such as diabetes by confirming the characteristics of said antibody and showing that a novel antibody specifically binding to periostin induces the differentiation of bone marrow hematopoietic stem cells into insulin-secreting beta cells using a library of 108 combination antibodies. Prior art literature

[0005] Republic of Korea Registered Patent No. 10-0726281 (Registered June 1, 2007) Republic of Korea Published Patent No. 10-2021-0108972 (Published September 3, 2021) Republic of Korea Registered Patent No. 10-2201368 (Registered January 5, 2021) The problem to be solved

[0006] The object of the present invention is to provide an antibody that induces the differentiation of insulin-secreting beta cells or an antigen-binding fragment thereof.

[0007] Another objective of the present invention is to provide a method for producing the novel antibody or its antigen-binding fragment.

[0008] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of metabolic diseases.

[0009] Another objective of the present invention is to provide a screening method for therapeutic agents for metabolic diseases. means of solving the problem

[0010] The present invention relates to a heavy chain variable region (V) comprising a complementarity determining region (CDR) 1 represented by the amino acid sequence of SEQ ID NO. 1, a CDR 2 represented by the amino acid sequence of SEQ ID NO. 2, and a CDR 3 represented by the amino acid sequence of SEQ ID NO. 3. H ); and a light chain variable region (V) comprising CDR1 represented by the amino acid sequence of SEQ ID NO. 4, CDR2 represented by the amino acid sequence of SEQ ID NO. 5, and CDR3 represented by the amino acid sequence of SEQ ID NO. 6. JProvides a monoclonal human antibody P1 or its antigen-binding fragment containing );

[0011] In addition, the present invention provides cDNA encoding the monoclonal human antibody P1 or an antigen-binding fragment thereof.

[0012] In addition, the present invention provides a recombinant virus vector comprising the cDNA.

[0013] In addition, the present invention provides a cell transformed with the recombinant virus vector.

[0014] In addition, the present invention provides a method for producing human antibody P1 or an antigen-binding fragment thereof, comprising the steps of: culturing the transformed cells in a culture medium; and recovering and purifying an antibody or an antigen-binding fragment thereof from the cultured cells or the culture medium.

[0015] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of metabolic diseases comprising a Periostin (POSTN) agonist as an active ingredient.

[0016] In addition, the present invention comprises the steps of: treating a cell line with an experimental substance; measuring the degree of expression or activation of periostin in the cell line treated with the experimental substance; and selecting a test substance in which the degree of expression or activation of periostin is increased compared with a control sample.

[0017] A screening method for a metabolic disease treatment is provided, comprising [ ] and characterized by using a monoclonal human antibody P1 for screening the test substance.

[0018] The present invention will be described in more detail below.

[0019] The inventors of the present invention have confirmed that a novel P1 antibody, a Periostin (POSTN) agonist, induces the differentiation of bone marrow stem cells into insulin-secreting beta cells, and have completed the present invention by confirming that it can be utilized for the prevention or treatment of metabolic diseases such as diabetes.

[0020] The term “ScFv (single-chain Fv, single-chain fragment antibody or antibody fragment)” used in the present invention is an antibody in which the variable regions of a light chain and a heavy chain are linked. In some cases, it may include a linker (linking site) composed of a peptide chain with approximately 15 amino acids linked together. In this case, the ScFv may have a structure of light chain variable region-linking site-heavy chain variable region, or heavy chain variable region-linking site-light chain variable region, and has antigen specificity identical or similar to that of the original antibody.

[0021] The term “variable region” used in the present invention refers to a region that performs the function of specifically binding to an antigen and exhibits many variations in sequence, and generally includes complementarity determining regions (CDR) and framework regions (FR).

[0022] The term “antibody (or ScFv) library” as used in the present invention refers to a collection of various antibody genes having different sequences. To isolate specific antibodies against any antigen from an antibody library, very high diversity is required, and a library composed of different antibody clones is constructed and used. The antibody genes constituting such an antibody library can be cloned into, for example, a phagemid vector and transfected into a transformant (E. coli).

[0023] The term “human antibody” as used in the present invention broadly refers to an antibody containing a variable region (CDR and FR) derived from human immunoglobulin, and more narrowly refers to an antibody containing a variable region and a constant region derived from human immunoglobulin. The human antibody may include not only a whole antibody form but also a functional fragment of the antibody molecule. The whole antibody has a structure having two full-length light chains and two full-length heavy chains, and each light chain may be connected to the heavy chain by a disulfide bond; preferably, it may be a monoclonal antibody. Furthermore, the functional fragment of the antibody molecule refers to a fragment possessing an antigen-binding function and may include Fab, Fab', F(ab')2, Fv, double-stranded Fv (dsFv), scFv, etc. Among the above antibody fragments, Fab may have a structure having a variable region of the light chain and heavy chain, a constant region of the light chain and a first constant region (CH1) of the heavy chain, and may include one antigen binding site; Fab' may be distinguished from Fab in that it includes a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain; and the F(ab')2 antibody may be distinguished from other fragments in that the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Meanwhile, the above Fv refers to a minimum antibody fragment having only a heavy chain variable region and a light chain variable region, and is typically distinguished into a single-chain Fv (scFv) and a double-chain Fv (dsFv). The single-chain Fv (scFv) can generally be in a form where the variable region of the heavy chain and the variable region of the light chain are connected by a covalent bond through a peptide linker, and the double-chain Fv (dsFv) can be in a form where the variable region of the heavy chain and the variable region of the light chain are connected by a disulfide bond.These functional fragments can be produced in various ways, typically through the hydrolysis of antibody proteins using enzymes (for example, Fab can be obtained by restricting the whole antibody with papain and F(ab')2 fragment can be obtained by cleaving it with pepsin), and preferably through recombinant technology, which is already known in the art.

[0024] Since all components of the above human antibody are derived from humans, it has the advantage of not causing an unwanted immune response when administered to humans, as the probability of an immunization reaction is lower compared to existing humanized antibodies or mouse antibodies. Therefore, it can be very useful as a therapeutic antibody for humans.

[0025] The term “monoclonal antibody” as used in the present invention refers to a protein molecule directed toward a single antigenic site (single epitope) and specifically binding to it. For the purposes of the present invention, the monoclonal antibody of the present invention specifically binds to Periostin (POSTN), and thus is a protein molecule that recognizes Periostin.

[0026] Since the major part of an antibody that recognizes a specific epitope of an antigen to form an antigen-antibody complex is the variable region of the heavy and light chains, particularly the CDR, which contributes to the formation of such complex, the present invention includes within its scope chimeric antibodies, humanized antibodies, etc., that include the variable region of the monoclonal antibody of the present invention, particularly the CDR. Furthermore, the present invention includes not only a complete form having two full-length light chains and two full-length heavy chains having binding characteristics as described above, but also functional fragments of antibody molecules. A functional fragment of an antibody molecule refers to a fragment that possesses at least an antigen-binding function, and includes Fab, F(ab'), F(ab')2, and Fv, etc.

[0027] Accordingly, the present invention comprises a heavy chain variable region (V) comprising a complementarity determining region (CDR) 1 represented by the amino acid sequence of SEQ ID NO. 1, a CDR 2 represented by the amino acid sequence of SEQ ID NO. 2, and a CDR 3 represented by the amino acid sequence of SEQ ID NO. 3. H ); and a light chain variable region (V) comprising CDR1 represented by the amino acid sequence of SEQ ID NO. 4, CDR2 represented by the amino acid sequence of SEQ ID NO. 5, and CDR3 represented by the amino acid sequence of SEQ ID NO. 6. J Provides a monoclonal human antibody P1 or its antigen-binding fragment containing );

[0028] The above heavy chain variable region may be represented by the amino acid sequence of SEQ ID NO. 7, and the above light chain variable region may be represented by the amino acid sequence of SEQ ID NO. 8.

[0029] The above monoclonal human antibody P1 or its antigen fragment can specifically bind to Periostin (POSTN).

[0030] In addition, the present invention provides cDNA encoding the monoclonal human antibody P1 or an antigen-binding fragment thereof.

[0031] The above cDNA can be represented by the nucleotide sequence of SEQ ID NO. 9 encoding a heavy chain variable region and the nucleotide sequence of SEQ ID NO. 10 encoding a light chain variable region.

[0032] In addition, the present invention provides a recombinant virus vector comprising the cDNA and a cell transformed with the recombinant virus vector.

[0033] In addition, the present invention provides a method for producing human antibody P1 or an antigen-binding fragment thereof, comprising the steps of: culturing the transformed cells in a culture medium; and recovering and purifying an antibody or an antigen-binding fragment thereof from the cultured cells or the culture medium.

[0034] In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of metabolic diseases comprising a periostin agonist as an active ingredient.

[0035] It is specified that the above-mentioned periostin agonist may be one or more selected from the group consisting of the above-mentioned monoclonal human antibody P1, its antigen-binding fragment, and periostin substrate, but is not limited thereto.

[0036] It is specified that the above metabolic disease may be obesity or diabetes, but is not limited thereto.

[0037] The above-mentioned periostin agonist can induce differentiation into insulin-secreting beta cells. Beta cells can prevent or treat metabolic diseases such as diabetes by inducing insulin secretion.

[0038] When the composition of the present invention is a pharmaceutical composition, for administration, it may include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient described above. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0039] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as external preparations, suppositories, or sterile injectable solutions, according to conventional methods. Specifically, when formulating, it may be prepared using diluents or excipients such as fillers, weighting agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, and capsules. In addition to the active ingredient, such solid formulations may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. Furthermore, in addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. In addition to liquids and liquid paraffins for oral administration, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be added. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and the subject. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, macrosol, Tween 61, cacao gelatin, laurin gelatin, glycerogelatin, etc. may be used as bases for suppositories.

[0040] A suitable dosage of the pharmaceutical composition of the present invention may vary depending on the patient's condition and body weight, the degree of disease, the form of the drug, and the time, but can be appropriately selected by a person skilled in the art. The daily dosage of the composition is preferably 0.001 mg / kg to 50 mg / kg, and may be administered once or several times a day as needed.

[0041] In addition, the present invention comprises the steps of: treating a cell line with an experimental substance; measuring the degree of expression or activation of Periostin (POSTN) in the cell line treated with the experimental substance; and selecting a test substance in which the degree of expression or activation of Periostin is increased compared with a control sample.

[0042] A screening method for a metabolic disease treatment agent is provided, characterized by using the monoclonal human antibody P1 of the present invention in the screening of the above test substance.

[0043] The term “test substance” as used in the present invention refers to an unknown candidate substance used in screening to test whether it affects the expression level of a gene or affects the expression or activity of a protein. The sample includes, but is not limited to, chemical substances, nucleotides, antisense RNA, siRNA (small interference RNA), and natural product extracts.

[0044] In addition, the present invention relates to a composition for inducing differentiation of insulin-secreting beta cells, and said composition may be provided as a reagent composition, a pharmaceutical composition, or a health functional food composition. Effects of the invention

[0045] The present invention relates to an antibody characterized by inducing the differentiation of stem cells into insulin-secreting beta cells, wherein the antibody targets the membrane-binding protein Periostin (POSTN) as a target antigen. Beta cells differentiated using the antibody of the present invention can migrate to the pancreas and secrete insulin, thereby enabling its application as a therapeutic agent for diabetes or related metabolic diseases. Furthermore, the use of an antibody with few side effects and a long half-life in the body can compensate for the shortcomings of conventional treatments and enhance therapeutic efficacy. In addition, the antibody of the present invention can be utilized as a diagnostic kit for the discovery of Periostin (POSTN) agonists. Brief explanation of the drawing

[0046] Figure 1 shows a form of 10 that binds to the plasma membrane on the cell surface. 8 This is a schematic diagram illustrating the process of infecting mouse bone marrow cells with lentivirus vectors containing a unique human ScFv antibody gene library. FIG. 2 relates to agonist antibodies that regulate cell migration, FIG. 2a is a schematic diagram showing cell migration from the bone marrow to the pancreas, and FIG. 2b and FIG. 2c show tdTomatoes treated with P1 antibody. + It was observed using an optical microscope that the cells were confirmed in the pancreas recovered. Figure 3 shows the results of immunohistochemical staining analysis of pancreatic cells differentiated by the P1 antibody, where Figure 3a confirms the expression status of Insulin and PDX-1, and Figure 3b confirms the expression status of glucagon. Figure 4 shows human CD34 treated with P1 antibody + As a result of confirming the differentiation status of the cells, Figure 4a shows the differentiation-induced cells observed with an optical microscope, Figure 4b shows the insulin expression status of the differentiation-induced cells confirmed by Western block analysis, and Figure 4c shows the expression status of insulin, PDX-1, and Nkx-6.1 confirmed through flow cytometry analysis. Figure 5 shows the results of identifying the target and signaling proteins of the P1 antibody. Figure 5a confirms by Western blot that Periostin (POSTN) is the target antigen of the P1 antibody, Figure 5b confirms through RNA sequencing the beta cell-related genes POSTN, IGTB1, and ACTB, and Figure 5c confirms by Western blot that the phosphorylation of AKT and ERK is involved in the differentiation of beta cells by the P1 antibody. Figures 6 and 7 show the amino acid sequence and base sequence information of the P1 antibody of the present invention. Specific details for implementing the invention

[0047] Preferred embodiments of the present invention will be described in detail below. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the content introduced herein is provided to fully convey the concept of the present invention to those skilled in the art, so that it may be thorough and complete.

[0048] Example 1: Mouse and cell line

[0049] The mice are C57BL / 6J and B6.Cg- Gt(ROSA)26Sor tm9(CAG-ttdTomato)Hze / J (The Jackson Laboratory) was used. Mice were stored and handled according to protocols approved by the Animal Care and Use Committee of the Scripps Research Institute.

[0050] HEK293T cell lines were cultured in DMEM medium containing 10% (v / v) FCS (fetal calf serum), penicillin, and streptomycin (Gibco-Invitrogen), and Expi293F cell lines were cultured in Expi293 Expression medium (Gibco-Invitrogen). Human CD34 + All-Cell cells and mouse bone marrow cells were cultured in StemSpan serum-free medium containing cytokine cocktail 100 (STEMCELL Technologies), StemSpan serum-free medium, or RPMI medium containing 1% (v / v) FBS (fetal bovine serum).

[0051] Example 2: Combinatorial antibody libraries and transduction

[0052] The single-chain Fv (ScFv) gene is a naive human combination antibody library (1 x 10⁶ 11It was obtained from library diversity and subcloned into a lentivirus vector. Lentiviruses were produced by co-transfecting HEK293T cells with the lentivirus vector at a content ratio of 1:1:1 with pCMVD8.91 and pVSVg virus packaging vectors. Mouse bone marrow cells were cultured with the lentiviruses at 37°C for 3 days.

[0053] Example 3: Bone marrow transplantation

[0054] Bone marrow cells were transfected with a lentivirus antibody library at an infection multiplicity of 2 and transplanted into mice irradiated with a lethal dose of radiation. The bone marrow transplanted mice were reared for 1 week to 6 months. Subsequently, islets of Langerhans from pancreatic tissue were collected from the bone marrow transplanted mice, fixed and recovered, and frozen at -80°C. The antibody genes obtained from the islets of Langerhans were amplified by PCR using primers customized for the lentivirus vector, and then analyzed by electrophoresis.

[0055] Example 4: Purification of scFv-Fc protein

[0056] A vector encoding a ScFv-Fc tag fusion protein was transfected into Expi293F cells. The antibodies from the supernatant were purified using a HiTrap Protein G HP column equipped with an AKTAxpress purifier (GE). The buffer was exchanged with PBS (Dulbecco, pH 7.4), and the purified protein was stored at 4°C.

[0057] Example 5: Western blot

[0058] Cells were washed with PBS and then lysed in lysis buffer (50 mM Hepes, pH 7.2, 150 mM NaCl, 50 mM NaF, 1 mM Na3VO4, 10% (v / v) glycerol, 1% (v / v) Triton X-100). The lysate was centrifuged at 12,000 xg at 4°C for 15 minutes. Proteins were denatured with Laemmli sample buffer (at 95°C for 5 minutes), separated by SDS / PAGE, and then transferred to a nitrocellulose membrane using an iBlot blotting system (Invitrogen). The membrane was then blocked with phosphate-buffered saline (PBST) containing 5% (w / v) BSA and Tween 20 for 30 minutes before incubating with the antibody for 3 hours. After blocking and the primary antibody reaction, a secondary antibody for each was reacted, followed by washing the membrane with PBST and developing with ECL.

[0059] Western blot was performed using Periostin (POSTN) for protein identification against bone marrow cells. Phosphorylation reactions of signaling proteins were also confirmed using phospho-AKT, ERK, and p38 (Cell Signaling Technology). β-actin was used as the housekeeping gene for each reaction.

[0060] Example 6: Flow Cytometry and Cell Classification

[0061] Cells were stained with antibodies against Insulin, PDX-1, and Nkx-6.1, respectively, and each cell was analyzed using an LSRII flow cytometer (Becton Dickinson).

[0062] Example 7: Immunohistochemistry and Immunofluorescence Confocal Microscopy

[0063] Immunohistochemistry was performed on islet of Langerhans sections of horizontally cut frozen pancreatic tissue. Antibodies were diluted with 1x PBS containing 4% horse serum and 0.2% Triton-X100, and Guinea pig anti-Insulin (1:1000, Dako), goat anti-PDX-1 (1:1000, R&D systems), mouse anti-Nkx-6.1 (1:1000, DSHB), and mouse anti-Glucagon-1 (1:1000, Thermo Fisher) were used as primary antibodies for analysis.

[0064] Islet of Langerhans sections were incubated overnight with the primary antibody, washed, and then incubated with the secondary antibody (1:250, Invitrogen) for 1 hour at incubation. Sections and coverslips were mounted on glass slides in anti-fade mounting medium containing DAPI (ThermoFisher). Confocal microscopy was performed using a Zeiss LSM 710 laser scanning confocal microscope.

[0065] Example 8: Mass spectrometry

[0066] Mouse bone marrow cells were prepared and lysed in lysis buffer. The lysate was then reacted with an H3 antibody at 4°C for 2 hours and reacted with 50 μL of protein G-Sepharose beads (Pierce). The eluent was introduced into a linear trap quadrupole mass spectrometer from a nano-ion source with an electric spray voltage of 2 kV. The analysis method consisted of a full MS scan in the 400–2,000 m / z range, followed by data-dependent MS / MS for the three strongest ions in the full MS scan. Raw data from the linear trap quadrupole were analyzed using the IPI human FASTA database with the MASCOT (http: / / www.matrixscience.com / ) search engine.

[0067] Example 9: RNA Sequencing and Data Analysis

[0068] Human CD34 treated with P1 antibody + Total RNA was isolated from cells (cells treated with isotype Ab and vehicle Ab were also tested as a control group).

[0069] Total RNA samples were prepared from an RNAseq library using the Illumina NEBNext Ultra™ Directional RNA Library Prep Kit.

[0070] Briefly, 500 ng of total RNA from each sample was selected as polyA, double-stranded cDNA was synthesized, and fragmentation and extension of the sequencing adapter were performed. Subsequently, the library was amplified for 15 cycles using barcode PCR primers and purified; prior to loading into the Illumina NextSeq500, the size was selected using AMPure XP beads for 75-base single-read sequencing. Human transcript expression levels were measured using Salmon (BioRxiv). Heatmaps were constructed using Cluster3 and JavaTreeView. Functional analysis of differentially expressed genes was performed using Ingenuity Pathway Analysis software (Ingenuity Systems Inc.).

[0071] Experimental Example 1: Regulating cell migration in vivo Antibody screening

[0072] In the present invention To select agonist antibodies that induce bone marrow cells to migrate to pancreatic tissue in vivo A study was conducted. As shown in Fig. 1, 10 of the form binding to the plasma membrane of the cell surface 8 Lentiviral vectors containing a unique human ScFv antibody gene library were infected into mouse bone marrow cells.

[0073] Infected cells were transplanted into C57BL / 6J mice irradiated with a lethal dose to confirm cell migration to the islets of Langerhans in the pancreas. After 7 days, pancreatic tissue from mice perfused with PBS was obtained, and genomic DNA was extracted. PCR analysis was performed to identify human ScFv antibody genes that had migrated to the pancreas, and among them, the P1 antibody was selected. This process is illustrated in Figure 1, and information on the P1 antibody is shown in Figures 6 and 7.

[0074] Experimental Example 2: Confirmation of P1 Antibody Efficacy - Induction of Bone Marrow Cell Migration to Islets of Langerhans

[0075] To determine whether the P1 antibody induces cell migration from the bone marrow to the islets of Langerhans, the P1 antibody gene was cloned into a mammalian expression vector, and the P1 antibody was purified. tdTomato for use as donor cells. + Bone marrow cells were transplanted into irradiated C57BL6 mice, and then P1 antibodies (50 μg / mouse ip twice / week, total 3 weeks) were injected into the transplanted C57BL6 mice.

[0076] Subsequently, the pancreas was harvested as shown in the schematic diagram of Fig. 2a and tdTomato + We checked whether there were cells expressing it in various places.

[0077] To this end, the islets of Langerhans in the pancreas were observed under an optical microscope, and tdTomato, the donor cells of mice injected with the P1 antibody + It is confirmed that the island moved to Langerhans Island as shown in Figs. 2b and 2c.

[0078] Next, to observe the condition of the bone marrow cells that migrated to the Isles of Langerhans, immunohistochemical staining was performed and the results analyzed.

[0079] Accordingly, as shown in Fig. 3a, tdTomato injected with P1 antibody + The cells successfully migrated to the islets of Langerhans, and Insulin + and PDX-1 +It can be confirmed that this is the state. However, in bone marrow cells that reacted with a control antibody rather than the P1 antibody, such expression of Insulin and PDX-1 is not detected.

[0080] In addition, since the cells that migrated to the islets of Langerhans in this way could be alpha cells, they were stained with glucagon as shown in Fig. 3b, but glucagon was not expressed.

[0081] Experimental Example 3: Human hematopoietic stem cell CD34 using antibodies + Induction of differentiation into beta cells

[0082] To confirm the direct function of the purified P1 antibody, human CD34 + cells with P1 antibody and in vitro It was cultured for 2 weeks and the shape of the cells was observed.

[0083] As a result, the purified antibody as shown in Fig. 4a is CD34 + It was confirmed that the cells were induced to differentiate into a form similar to beta cells (Fig. 4a).

[0084] In addition, when the insulin secretion status of the cells in this state was confirmed by Western blot, it was confirmed that insulin secretion was significantly increased in the cells treated with the P1 antibody compared to the control cells, as shown in Fig. 4b. Through the flow cytometry results in Fig. 4c, the expression of insulin, PDX-1, and Nkx-6.1 was also confirmed in the cells treated with the P1 antibody, indicating that they differentiated into a state similar to beta cells.

[0085] Experimental Example 4: Target identification of P1 antibody

[0086] To identify the target proteins of the P1 antibody, recombinant P1 antibodies were produced in Expi293F cells. The purified P1 antibody was administered to mouse bone marrow, and immune complexes from the cell lysates were captured using a protein A / G column. Six candidate proteins reacting with the antibody were identified using SDS gel staining and mass spectrometry.

[0087] Western blot confirmed that among these proteins, Periostin (POSTN) is the target antigen of the P1 antibody. That is, as shown in the results of Figure 5a, it can be seen that the P1 antibody binds not only to mouse bone marrow (M BM) but also to purified Periostin (POSTN) protein.

[0088] Similarly, RNA transcripts were isolated from mouse bone marrow treated with the P1 antibody, sequenced, and compared with control cells that were not treated with the antibody.

[0089] When comparing the genetic analysis results with the results of previous beta cell experiments, as shown in Figure 5b, it was confirmed that the beta cell-related genes POSTN, IGTB1, and ACTB were expressed in significant amounts.

[0090] Western block analysis was used to determine the signaling pathways through which cell differentiation is induced by treatment with P1 antibodies. As shown in Figure 5c, it was found that differentiation is induced through the phosphorylation of AKT and ERK, which are known to be involved in beta-cell differentiation. Of course, this phosphorylation phenomenon was not observed at all in the control group.

[0091] Through these results, it can be proven that the P1 antibody discovered through the present invention induces differentiation of insulin-secreting beta cells.

[0092] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0093] The scope of the present invention is defined by the claims set forth below, and all modifications or variations derived from the meaning and scope of the claims and equivalent concepts thereof should be interpreted as being included within the scope of the present invention.

Claims

Claim 1 A pharmaceutical composition for treating diabetes containing a monoclonal human antibody P1 as an active ingredient, wherein the monoclonal human antibody P1 has the efficacy of inducing the differentiation of bone marrow stem cells into insulin-secreting beta cells, and the heavy chain variable region (V) of the monoclonal human antibody P1 H ) is composed of the amino acid sequence of SEQ ID NO. 7, and the light chain variable region (V J A pharmaceutical composition for treating diabetes containing a monoclonal human antibody P1 as an active ingredient, characterized in that the monoclonal human antibody P1 is an antibody that specifically binds to Periostin (POSTN), wherein the monoclonal human antibody P1 is composed of the amino acid sequence of SEQ ID NO.

8. Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 A pharmaceutical composition for treating diabetes comprising a monoclonal human antibody P1 as an active ingredient, characterized in that, in claim 1, the heavy chain variable region amino acid of SEQ ID NO. 7 is coded as the polynucleotide of SEQ ID NO. 9, and the light chain variable region amino acid of SEQ ID NO. 8 is coded as the polynucleotide of SEQ ID NO.

10. Claim 6 delete Claim 7 delete Claim 8 delete Claim 9 delete Claim 10 delete Claim 11 delete Claim 12 A screening method for a diabetes treatment agent comprising: a step of treating a cell line with a test substance; a step of measuring the degree of expression or activation of Periostin (POSTN) in the cell line treated with the test substance; and a step of selecting a test substance in which the degree of expression or activation of Periostin (POSTN) is increased compared with a control sample; wherein a composition containing the monoclonal human antibody P1 of claim 1 is used for selecting the test substance.

Images