AU2007200834B2 - Mimetic peptides for epitope of apolipoprotein B-100, concatemer and modified peptides thereof, and the vaccine composition comprising the same - Google Patents

Description

AUSTRALIA Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant: HYO-JOON KIM Invention Title: MIMETIC PEPTIDES FOR EPITOPE OF APOLIPOPROTEIN B-100, CONCATEMER AND MODIFIED PEPTIDES THEREOF, AND THE VACCINE COMPOSITION COMPRISING THE SAME The following statement is a full description of this invention, including the best method for performing it known to me: - la MIMETIC PEPTIDES FOR EPITOPE OF APOLIPOPROTEIN B-100, CONCATEMER AND MODIFIED PEPTIDES THEREOF, AND THE VACCINE COMPOSITION COMPRISING THE SAME s The entire disclosure in the complete specification of our Australian Patent Application No. 86286/01 is by this cross-reference incorporated into the present specification. 10 Technical Field The present invention relates to a concatemer comprising 2 to 15 mimetic peptides of an epitope of apolipoprotein B-100, a method of treatment for obesity comprising administering the concatemer, a process for is preparing the concatemer and isolated DNA encoding the concatemer. Background Art All references, including any patents or patent 20 applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and 25 pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in 30 Australia or in any other country. Blood serum lipid is composed of cholesterol, triglycerides(TG), free fatty acid, phospholipid and the like, and exists in blood stream in the form of lipoproteins which are complexes of lipid and 35 apolipoprotein. Of these lipoproteins, low density lipoprotein 2377301_1 (GHMauers) 18/08/10 - 2 (LDL) is the major carrier for TG and cholesterol. The number of patients suffering from arteriosclerosis, coronary artery disease, or cardiac infarction caused by elevated LDL-cholesterol level in blood has been 5 considerably increased due to the change of dietary life or other factors. Therefore, various approaches for lowering the level of LDL-cholesterol and for showing the cause of above diseases, have been tried to treat patients 10 suffering from the above diseases. LDL-cholesterol, a major etiological factor for adult disease related to lipid metabolism, may be converted to the form of high density lipoprotein(HDL) by macrophage. In addition, LDL-cholesterol may also 15 be converted to another material or be converted to a bile acid in the liver. (Brown, M.S. and Goldstein, J.L., 1983, Annu. Rev. Biochem., 52:223-261). Apolipoprotein B-100 is a major protein component of LDL and exists also in very low density lipoprotein 20 (VLDL) and chylomicron. LDL-cholesterol in blood may be removed through phagocytosis by macrophages whose an antibody in the bloodstream is induced by recognizing the apolipoprotein B-100, since apolipoprotein B-100 leads LDL particles to bind to 25 LDL-receptors exposed on the cell surface (Dalum I., et al., 1997, Mol. Immunol., 34(16-17): 1113-20). When a macromolecule such as an antibody has been bound to apolipoprotein B-100 which exists on the surface of LDL, a lipase such as lipoprotein lipase 30 cannot hydrolize TG and the like due to the steric hindrance caused by the macromolecule bound to apolipoprotein B-100. Consequently, the formation of - 3 free fatty acid, a major factor for obesity, can be inhibited by means of the antibody which can bind to apolipoprotein B-100. Recently, several approaches for lowering LDL 5 cholesterol level and for inhibiting the outbreak of arteriosclerosis by using a vaccine, have been tried in various animal models such as mouse and rabbit. For example, C.R. Alving reported that cholesterol may be modified by metabolites or its oxidation, and that 10 the modified cholesterol can be a strong antigenic determinants in some cases (Alving, C.R., et al., 1989, Biochem. Soc. Trans., 17(4): 637-9; Alving, C.R., et al., 1996, J. Lab. Clin. Med. , 127: 40-49; Alving, C.R., et al., 1996, Curr. Top. Microbiol. Immunol., 15 210: 181-6). Furthermore, it has been reported that an endogenous antibody for cholesterol exists in blood serum (Wu, J.T., L.L., 1997, Clin. Lab. Med., 17(3): 595-604, Review). It has also been reported that, in 20 the experiment wherein arteriosclerosis and hypercholesterolemia are induced in rabbits by feeding cholesterol-containing meal, the occurrence of hypercholesterolemia and arteriosclerosis in the rabbit immunized by injecting cholesterol-containing 25 liposome, is suppressed or lowered remarkably compared to those in the control group. Such an antibody induced by cholesterol vaccine, is immunoglobin M (IgM) which binds to VLDL, intermediate density lipoprotein (IDL) and LDL. Based 30 on the above, it is believed that a vaccine for treatment or prevention of hyperlipoidemia or arteriosclerosis caused by high level of cholesterol, -4 will be possible (Bailey J.M., 1994, Science, 264: 1067 1068; Palinski, W. et al., 1995, Proc. Natl. Acad. Sci. U.S.A., 92(3): 821-5; Wu, R. et al., 1999, Hypertension, 33(1): 53-9). 5 The present inventors have found that obesity can be effectively prevented by a mimetic peptide epitope of apolipoprotein B-100, and based on the above, have developed a vaccine composition for treatment of obesity. 10 Disclosure of the Invention Therefore, in a first aspect there is provided a concatemer comprising 2 to 15 mimetic peptides of an epitope of apolipoprotein B-100, in which each of the peptides has the sequence set out in SEQ ID NO:1. 15 In a second aspect, there is provided a concatemer comprising 2 to 15 mimetic peptides of an epitope of apolipoprotein B-100, in which each of the peptides has the sequence set out in SEQ ID NO:2. In a third aspect, there is provided a concatemer 20 comprising 2 to 15 mimetic peptides of an epitope of apolipoprotein B-100, in which each of the peptides has the sequence set out in SEQ ID NO:3. In a fourth aspect there is provided a method for treating obesity comprising administering the concatemer 25 of any of the first, second and third aspects. In a fifth aspect, there is provided a process for preparing the concatemer of any of the first, second and third aspects. A peptide library system of phage was employed in 30 the present invention in order to screen an epitope of human apolipoprotein B-100 which is bound by monoclonal 2377301_1 (GHMattcrs) 18/08/10 - 5 antibody (MabB23). The screened peptides in the above were mimetic peptides structurally similar to an antigenic determinant which can be recognized by the antibody, and these mimetic peptides were synthesized according to the s amino acid sequence of the thus-identified peptide. A peptide library system is one process for searching a three-dimensional form of antigenic determinant. That is, DNA fragments which encode random sequenced peptides are inserted into DNAs which encodes lo the minor coat protein of the phage, and then the above DNAs are inserted into RF (Reading frame) DNA and transformed to E.coli in order to express them. The peptides expressed on the surface of E.coli, are reacted with antigen in order to identify the peptides which are 15 structurally similar to the antigenic determinant. In order to prepare anti-serum, mice were immunized by introducing the above mimetic peptides. It was confirmed that the anti-serum thus obtained recognizes the original apolipoprotein B-100, mimetic peptides and LDL at 20 the same time (Identification of Antigenic Determinants for the Murine Monoclonal Antibodies Against Apolipoprotein A-1 and Apolipoprotein B-100 by using Phage-displayed Random Peptide library, Chi-Hoon Kim, Hanyang Univ., 1997). 25 The mimetic peptide of the epitope of apolipoprotein B-100 of the present invention may be selected from the group consisting of peptides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 or the mixtures thereof. The mimetic peptides disclosed herein may be used in 30 the form of a concatemer in order to improve their antigenic determinant. As an embodiment of the present 2377301_1 (GHMaucrs) 18/08/10 - 6 invention, two or more mimetic peptides may be linked with each other. Preferably the concatemer comprises three(3) to fifteen(15) peptides. More preferably the concatemer of the present invention comprises four(4) peptides of SEQ 5 ID NO:l. The term "Concatemer" of the above mimetic peptide of the present invention refers to a polymer wherein the ends of the above mimetic peptides are linked with each other. 10 "The modified peptide" of the above mimetic peptide, refers to mimetic peptide variants which can be recognized by monoclonal or polyclonal antibody for apolipoprotein B-100. Such variants include substitutions, deletions, addition, and chemical substitutions of one or more amino 15 acid. The present invention also provides a method for preparing the concatemer of any one of the first, second and third aspects, which comprises: (i) a step for inserting DNAs which encodes the concatemer into a vector, 20 (ii) a step for transforming the above vector to host cells, and then incubating them, and (iii) a step for isolating the concatemer from the above host cells. Formulations of a vaccine composition comprising the concatemer of the present invention may be prepared by any 25 conventional method. In the process for preparing the above formulation, preferably, the composition wherein the active compound is mixed or diluted with immune adjuvant, drug for reinforcing immunity, carrier, excipient and diluent, is selected from the group consisting of tablet, 30 pill, granule, powder, cachet, suspension, emulsion, liquid, syrup, aerosol, soft or hard gelatin capsule, 2377301_ (GHMatters) 18/08/10 -7 sterilized liquid for injection, sterilized powder and the like. Immune adjuvants which may be employed in the composition of the present invention include proteins s comprising the T cell epitope (e.g. surface protein of hepatitis B virus), inert carriers such as aluminium salt, bentonite, latex, acrylic particle and the like; hydrophobic antigens (e.g. lipids), water-oil and oil water emulsions, depot formers (e.g. polysaccharides) 10 T cell activators such as PPD, polyadenine, polyuracil and the like; B cell activator (e.g. B cell mitogen), surfactants such as saponin, lysolecithin, retinal, quil A, liposomes and the like; materials which stimulate the activity of macrophage; and alternative pathway is complement activators such as inulin, zymosan, endotoxin, levamisole, Corynelsaderium parvum and the like. "Carrier protein" as used herein means a pharmaceutically acceptable material such as a protein or aluminium salt which can transport the concatemer of the 20 present invention through the blood stream. Aluminium salt, phenoxyethyl ethanol, water, physiological salt solution, lactose, dextrose, sucrose, sorbitol, mannitol, calcium silicate, cellulose, methyl cellulose, amorphous cellulose, polyvinylpyrolidon, 25 methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate and a mineral oil may be used as suitable carriers, excipients or diluents. In addition, the vaccine compositions disclosed herein may further comprise one or more components 30 selected from the group consisting of a filler, an anti cohesive agent, a lubricant, a moisturizer, a perfume, an 2377301_1 (GH Matters) 18/08/10 - 8 emulsifier and an antiseptic. The vaccine compositions disclosed herein may be formulated by conventional methods well-known in this field to induce immune response in a mammal through one(1) 5 or more inoculations. The vaccine compositions disclosed herein for the treatment of obesity may be administered by various routes such as oral, dermal, intradermal, venous or muscular administration. Preferably the administration is 10 intradermal. The effective dose of the vaccine composition of the present invention is 0.1 to 10 pg (active peptide) per kg of body weight, preferably, 0.5 to 1.0 ptg per kg. However, the actual dosage of the active principle of the 15 vaccine composition may be determined depending on factors such as immune status, administration route, condition of patient, age, sex, body weight and the like. Therefore the ranges of the said dosage amount do not limit the scope of the present invention in any way. 20 The primary pharmaceutical effect of the vaccine compositions disclosed herein is to prevent or treat obesity through the mechanism that human antibody induced by the concatemer binds to the epitope of apolipoprotein B-100 on the surface of LDL, and thereby hinders 25 sterically and inhibits lipase from generating of fatty acids, the major etiological factor for obesity. In addition, the vaccine compositions disclosed herein have an effect also on suppressing hyperlipidemia through the mechanism wherein LDL is detected and removed 30 easily by macrophages through the opsonization caused by the human antibody induced by the concatemer and bound to 2377301_1 (GH Matters) 18/08/10 - 9 the epitope of apolipoprotein B-100 on the surface of LDL. Another pharmaceutical effect of the vaccine compositions disclosed herein is to prevent or treat obesity by inhibiting accumulation of lipid as cholesterol s of free fatty acid in the cell through the mechanism wherein human antibody induced by the concatemer binds to the epitope of apolipoprotein B-100 on the surface of LDL, and thereby prevents LDL from binding specifically to LDL receptors exposed on the cell surface. 10 In the claims of this application and in the description of the invention, except where the context requires otherwise due to express language or necessary implication, the words "comprise" or variations such as "comprises" or "comprising" are used in an inclusive 15 sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention. Brief Description of the Drawings 2377301_1 (GiHMatters) 18/08/10 - 10 The above aspects and other advantages of the present invention will become more apparent by describing in detail a non-limiting preferred embodiment thereof with reference to the attached 5 drawings, in which: Fig.la to ld represent the structures and compositions of vector for expressing the mimetic peptide of the present invention.. Fig la represents the structure of leader cassette, Fig lb represents 10 the structure of LB cassette, Fig ic represents the structure of BL cassette and Fig ld represents the structure of pBX4 expression vector. Fig.2 represents the procedures for preparation of pBX1 and pBX4 vector for expressing the mimetic 15 peptide of the present invention. Fig.3 represents the result of polyacrylamide gel electrophoresis(PAGE) for the identification of LB cassette. Fig.4 represents the result of PAGE for the 20 identification BL cassette incorporated in plasmid pBlue-BL. Fig.5 represents the result of PAGE for the confirmation of the direction and the number of copy of DNA inserted in plasmid pBX1 and pBX3. 25 Fig. 6 represents the result of western-blotting for identification of the expressed PB1 4 peptide. Fig. 7 represents the result of sodiumdodecyl sulfate(SDS) PAGE for confirmation of the purified PB14 peptide. 30 Fig. 8 represents the result of western-blotting - 11 for confirmation of the reactivity of the purified PB1 4 peptide against anti-PB1 4 serum. Fig. 9 represents the result of ELISA for measurement of the avidity of the antibody of mouse 5 induced by PB1 4 peptide. Fig. 10 is a graph which illustrates the suppressing effect of PB1 4 on the increase of body weight of mouse. Fig. lla and llb are graphs illustrating the 10 change of body weight of mice depends on administration of PB1 4 vaccine of the present invention in 20 weeks after injection of drug which can destroy of hypothalamus. Fig. 12 is the graph which represents the effect 15 on the concentration of lipid in blood serum according to the injection of PB1 4 vaccine. Best mode for carrying out the present invention Hereinafter, the present invention will be 20 described in more details. However, the present invention explained in below, is given only for the explanation of embodiment of the present invention and not intended to limit the scope of the present invention. 25 Example 1: synthesis and annealing of oligonucleotide The oligonucleotides were chemically synthesized at Genemed Synthesis Inc. (San Francisco, CA, USA) in 30 accordance with the sequence requested from the - 12 present inventor. In order to phosphorylate the 5' end of oligonucleotides, 50ml of lOOpmol/ml oligonucleotide was incubated with 10ml of 10mM ATP, 3ml of 1OU/ml T4 polynucleotide kinase (Takara, Otsu, 5 Japan) and 7ml of 1oX kinase buffer for two(2) hours at 37 0 C. Each of 10ml aliquots of above phosphorylated oligonucleotides were mixed together, heated at 80 0 C for 5 min. and then chilled very slowly to room 10 temperature thus were annealed to the specific pairing between complementary strands. Example 2: ligation Ligation mixture was prepared by mixing 1ml of 15 vector DNA, 5ml of insert DNA, 1ml of T4 DNA ligase (NEB, Beverly, MA, USA), 1ml of 1OX enzyme reaction buffer solution(NEB, Beverly, MA) and 2ml of distilled water, and then incubated at 16 0 C during the night and then incubated. 20 Example 3: construction of pBX expression vector for expression of mimetic peptide of apolipoprotein B 100 Step 1: design of the vector The plasmid vector for expression of mimetic 25 peptide generally comprises a leader cassette and one or more PB1 peptide genes. As depicted in Fig. 1, the plasmid pBX1 which comprises one(l) PB1 gene was prepared by cloning the leader cassette (Fig.la) at the multicloning site of pQE30 plasmid (Qiagen, Hilden, 30 Germany). The resulting plasmid was digested with HindTI and SalI and the small fragment was replaced - 13 by the LB cassette (Fig.lb) to give the plasmid pBX1 which is good for the insertion of multiple numbers of BL cassettes (Fig.lc) easily. Meanwhile, the plasmid pBluescriptII SK+ was cut 5 with SalI and XhoI and ligated with BL cassettes in which single to multiple BL cassette insertions occurred randomly. The expected repeats of the PB1 peptide genes in the plasmid pBlue-BL were selected and cleaved out and subcloned into pBX1 (Fig.ld). 10 Step 2: Preparation of vector for expression of PB1 mono-peptide Leader cassette was prepared by annealing SEQ ID NO:10 and SEQ ID NO:1l oligonucleotide synthesized 15 through the same process as Example 1 and 2. Then, the pQE30 vector (Qiagen, Hilden, Germany), which had been cleaved by SalI and BamHI, was ligated with the above leader cassette in order to prepare pQE-Leader plasmid. As a result of expression of the above pQE30 20 vector, six histidine residues were additionally incorporated to the N-terminus of the expressed protein in order for the protein to be purified easily. The above leader cassette was designed to comprise a recognition site (DDDDKI; SEQ ID NO:12) for 25 enterokinase in order to reduce the additional amino acid to a minimum. According to the process of Example 1, four oligonucleotides of which sequences were represented by SEQ ID NO:4 to 7, were synthesized, phosphorylated, 30 and then, annealed with complementary oligonucleotides respectively in order to synthesize the LB cassette of Fig. lb (SEQ ID NO:13 and 14) . Forty micro-litter of - 14 the annealed oligonucleotides were mixed with 3ml of 1 U/ml T4 DNA ligase, 5ml of 1OX enzyme buffer and 2ml of distilled water to prepare ligation mixture. Then, the ligation mixture was incubated during the night to 5 link the oligonucleotides each other. After the reaction being completed, the reaction mixture was loaded onto 20% polyacrylamide gel and electrophoresed. The LB cassette (52 bp oligonucleotides) (Fig. 3) was identified by dying the 10 gel with ethidium bromide (EtBr). In Fig. 3, lane M was a mark for 20 bp ladder DNA, and lane 1 was the reaction solution. LB cassette was obtained from the gel by means of a QIAEX II gel extraction kit (Qiagen, Hilden, Germany). 15 The above pQE-Leader was cleaved by HindIII and SalI, and then ligated with the above LB cassette according to Example 2 in order to prepare an expression vector for PB1 mimetic peptide. The prepared expression vector was named pBX1, and the 20 peptide expressed by the vector was named PBl 1 peptide (see Fig.2). Step 3: preparation of expression vector for PB1 1 peptide concatemer According to the process of Example 1, four 25 oligonucleotides whose sequences are shown in SEQ ID Nos:4, 5, 8, and 9, were synthesized, phosphorylated and then, annealed with complementary oligonucleotides respectively in order to synthesize the BL cassette of Fig. 1c (SEQ ID NO:15 and 16). Subsequently such 30 oligonucleotides were ligated with each other as in the process of Step 2 and then were loaded onto 20% polyacrylamide gel for electrophoresis. A fifty-five - 15 bp oligonucleotide (leader cassette) was identified by staining the gel with EtBr. BL cassette was obtained from the gel using a QIAEX II gel extraction kit, and cleaved by SalI and XhoI. 5 Meanwhile, a p~luescript II SK(Stratagene, La Jolla, CA, U.S.A) was cleaved by SailI and XhoI. The vector was electrophoresed on 0.8% agarose gel, and obtained by means of QIAEX II gel extraction kit (Qiagen, Hilden, Germany). 10 The same ligation reaction as in Example 2 was carried out to obtain pBlue-BL plasmid by using 5ml of BL cassette DNA and lml of the above cleaved-vector DNA. The pBlue-BL was cleaved by SalI and XhoI, and 15 the BL cassette was extracted. Plasmid pBX2 was prepared by inserting the BL cassette into the SalI site of the pBX1 vector prepared in Step 2. In addition, pBX3 and pBX4 vectors were prepared by changing the number of the BL cassette which inserted 20 into the SalI site of the pBX1 vector from two(2) to three(3) (see Fig. 2). The peptides expressed from the pBX2, pBX3 and pBX4 vector, were a concatemer which comprises two(2) to four(4) PB1 peptides. They were named PB1 2 , PBl 3 25 and PB1 4 respectively. Step 4: Identification of the insert Host cell (E. coli M15 [pREP4] ; Qiagen, Hilden, Germany) was transformed with the pBlue-BL plasmid and spread on to a 1% agar plate, and then incubated for 30 16 hours at 37 0 C so that colonies of E. coli could be formed. One of the colonies which had been formed on the agar plate was inoculated in 10ml of LB medium and - 16 incubated with shaking at 37 0 C for sixteen(16) hours, and then the plasmid was isolated using a DNA purification system (Wizard PLUS SV DNA miniprep DNA purification system; Promega, Madison, W1, U.S.A). 5 The plasmid harvested from the transformed E. coli was incubated with SalI and XhoI restriction enzyme in order to be cleaved at 37'C for one(1) hour, and analyzed through 20% PAGE (Fig. 4). In Fig. 4, lane M represents 20 bp ladder DNA, lane 1 represents 10 oligonucleotide product obtained from Step 3, lane 2 represents BL cassette DNA isolated by 20% PAGE from step 3, and lane 3 represents recombinant pBlue-BL plasmid treated with restriction enzyme. As shown in Fig. 4, it was confirmed that the pBlue-BL plasmid 15 contained BL cassette. E.coli (M15[pREP4]) was transformed with pBX1 or pBX3 plasmid, and the plasmid DNA was isolated as explained above in order to confirm the number and orientation of the DNA cassette inserts. The isolated 20 plasmid was cleaved by SalI and HindIII restriction enzyme, and analyzed using 20% PAGE (Fig. 5). In Fig. 5, lane M represents 20 bp ladder DNA, lane 1 and 3 represent the pBX1 plasmid containing an LB but not a BL cassette, lane 2 represents the plasmid harboring 25 one LB and two BL cassettes with the right direction. On the other hand, lane 4 represents the plasmid having one LB and two BL cassettes, but with reversed orientation. As shown in Fig. 5, the number of B cassettes (BL or LB cassette) which were inserted and 30 the direction in which they were inserted into the pBX vector could be identified by restriction enzyme mapping.

- 17 In addition, the DNA sequence of the B cassettes incorporated into the plasmid which had been harvested from the transformed E. coli, was confirmed to be identical to the designed sequences. The plasmids 5 were prepared by Wizard PLUS DNA miniprep kit and were sequenced by using the Sequenase (Ver. 2.1) DNA sequencing kit (Amersham, Cleveland, UK). Example 4: Expression of PB14 peptide in E.coli and its purification. 10 Step 1: confirmation of expression of PBl4 peptide To confirm the expression of PB1 4 peptide, three kinds of transformed E.coli M15[pREP4] were cultivated on LB agar broth containing amphicillin and kanamycin. 15 One sample of E.coli M15[pREP4] was transformed with the plasmid pBX4, another sample was mock-transformed with pQE30, and the third sample was untransformed E.coli M15[pREP4]. Each colony formed from the solid culture was inoculated respectively in liquid LB 20 culture medium which contained 100pl/ml amphicillin and 25pl/ml kanamycin, and incubated overnight. The culture was incubated at 37 0 C for one(l) hour with shaking until the O.D value reached approximately 0.5 to 0.7 at 600nm. Thereafter, 1 mM isopropyl-thio-0 25 galactopyranoside (IPTG) was added to the culture medium to facilitate the expression of the recombinant protein, and the samples were further incubated at 37 0 C for five(5) hours. 1ml of the culture medium was taken and centrifuged at 14,000rpm for two(2) min. to 30 precipitate the bacterial cells. The cell pellet was - 18 suspended in 50ml of 2X SDS solution[100mM Tris-Cl pH 6.8, 20% glycerol(w/v), 4% SDS(w/v), 2% 2 mercaptoethanol, 0.001% bromophenol blue] to apply to SDS-PAGE gels. The suspended solution was heated at 5 95*C for five(5) min., and then 10ml of the solution was loaded on the well of the cast gel and electrophoresed at 20mA for five(5) hours (Mighty Small II, Hoefer, USA) . Acrylamide concentrations of the stacking gel and resolution gel which were 10 employed, were 5% and 15% respectively, and pre stained standard SeeBlue (250Kda to 4kDa; NOVEX, San Diego, CA, U.S.A) or wide-ranging standard Markl2 (200kDa to 2.5kDa) were used as a standard size marker protein. After electrophoresis, the gel was stained 15 with Coomassie brilliant blue R-250 for one(l) hour and destained with decolorizing solution (5% Methanol and 7% acetic acid) for ten(10) hours. To confirm that the expressed protein was PB14 peptide, the proteins in the electrophoresis gel, 20 western-blotting was carried out using anti-PBl rabbit antibody (Fig.6). Antiserum was produced by immunizing ovalbumin conjugated with PB1 peptide which was chemically synthesized by Bio-Synthesis, Inc. (Lewisville, TX, USA) . In Fig. 6, lane M represents 25 the pre-stained standard SeeBlue label, lane 1 represents medium used for incubation of E.coli M15 [pREP4] which was not transformed, lane 2 represents medium used for incubation of E.coli M15 [pREP4] which was transformed with pQE30 vector, lane 3 represents 30 medium used for incubation of E.coli MiS [pREP4] which was transformed with pBX4 vector. As depicted in Fig. 6, only the pBX4 transformed - 19 E.coli expressed the recombinant PB1 4 peptide represented a specific immunity with anti-PB1 mouse serum. Step 2: Identification of solubility of the 5 expressed peptide E.coli M15 (pREP4] which had been transformed with pBX4 vector was incubated using the method of Step 1. 10ml of the culture medium was taken and centrifuged to harvest the cells. The cell pellet was 10 suspended in 5ml of cell lysis solution (300mM NaCl, 50mM NaH 2

PO

4 , 10mM imidazole pH 8.0) to obtain natural protein from the cell. After being chilled, the pellet-suspended solution was sonicated 20 cycles with ultrasonic wave in order to lyse the cells. The 15 supernatant was taken by centrifuge at 4 0 C, 10,000rpm for 30 min. The same volume of 2X SDS solution was mixed with solution and SDS-PAGE was carried out as the same method described in Step 1. After boiling each solution at 95'C for 5 min. As a result of SDS 20 PAGE, it was confirmed that PB1 4 peptide could be isolated and purified from the soluble extract A in that it was contained in the insoluble crude extract B. Step 3: purification of PB1 4 peptide Step 3-1: affinity chromatography 25 Ni-NTA resin for purifying His-tagged protein, was used to purify the recombinant peptide in Step 1. The affinity chromatography using attractive force between Ni' saturated in resin and histidine residues at the end of the expressed protein, is a well known 30 method for purifying interest protein easily. First of all, E.coli M15[pREP4] which had been transformed with pBX4, was inoculated in 11 of LB - 20 culture medium and incubated at 37'C to the extent that the O.D value was over 0.6 at 600nm. The ratio of LB culture medium to pBX4 vector was fifty (50) to one(l). IPTG was added in a final concentration of 1mM and 5 incubated again for five (5) hours. After incubation, the cell pellet was obtained by centrifuging the culture medium at 6,000 Xg for 30 min, and the pellet was stored at -70'C overnight. The pellet thawed in ice was suspended in dissolving solution (300mM NaCl, 10 50mM NaH 2

PO

4 , 10mM imidazol pH 8.0) wherein 5ml of dissolving solution per 1 g of the pellet, was used. Cells were lysed by sonication by the method of Step 2, and then centrifuged at room temperature at 10,000 Xg for 30 min. The same volume of buffer (8M urea, 0.1M 15 NaH 2

PO

4 , 0.01M Tris-HCl pH 8.0) as the pellet, was added for re-suspending the cellular debris and for denaturing proteins therein, and the pellet-suspended solution was treated with brief ultrasonic wave so that more proteins could be dissolved in buffer. The 20 suspension was centrifuged at 8,000rpm for 30 min. to remove cellular debris which had not been solubilized in 8M urea. To the 4ml of the supernatant prepared above, lml of Ni-NTA resin was added at 4*C and shaken at 200rpm for 2 hours in order proteins containing 25 His-tag to be captured. The supernatant containing protein/Ni-NTA complex was poured carefully into the chromatography column (size 2cm(id)X 2.7cm(h)) . Excess buffer was drained by opening the cap after the resin had been sedenated. 30 The column was washed with 20ml of buffer(8M urea, 0.1M NaH 2

PO

4 0.01M Tris-HCl pH 8.0) and subsequently - 21 20ml of medium pH buffer(8M urea, 0.1M NaH 2

PO

4 0.01M Tris-HCl pH 6.3) in order to wash out proteins which had been non-specifically bound to the Ni-NTA resin. The target proteins containing His-tag were eluted by 5 pouring 5ml of low pH buffer (8M urea, O.1M NaH 2

PO

4 0.01M Tris-HCl pH 5.9) two(2) times and subsequently 5ml of strong acid buffer (8M urea, O.1M NaH 2

PO

4 0.01M Tris-HCl pH 4.5) four(4) times, and then SDS-PAGE was used to confirm the eluted target proteins by using 10 15% acrylamide gel (Fig. 7). In Fig. 7, lane M represents pre-stained SeeBlue size marker and lane 1 represents the purified PB1 4 peptide. The above purified proteins were dialyzed against PBS (8g/L NaCl, 0.2g/L KCl, 1.44g/L Na 2

HPO

4 and 0.24g/L 15 KH 2

PO

4 ) in order to regain their original conformations. The dialysis tube employed was 3,500 Da in molecular weight cut-off size. During dialysis, 3L of PBS containing 2M urea was used first for 5 hours, and then 5L of PBS without urea was used two(2) times 20 overnight. Step 3-2: Hydrophobic chromatography Hydrophobic chromatography was carried out in order to improve the purity of the PB1 4 peptide which had been obtained in Step 3-1. 25 Ammonium sulfate was gradually added up to final 20% concentration to the solution containing PB14 peptide, which was eluted from Ni-NTA resin in Step 3 1, and then adjusted to pH 7.0. The solution was left for three or more hours after 10% of ammonium sulfate 30 had been dissolved completely, and then the solution was loaded on the phenyl sepharose column [Phenyl sepharose Fast Flow resin (Phamacia, Sweden) ; column - 22 size: 1 cm(id) x 3cm(h)]. Each fraction which was eluted from the column by pouring eluting solution (8M urea, 0.1M NaH 2

PO

4 , 0.01M Tris-HCl pH 6.3) into the column at the flow rate of 5 0.5ml/min under the reverse gradient of ammonium sulfate from 10% to 0%, was loaded on the gel for SDS PAGE. The fraction containing PB1 4 peptide was collected and dialyzed in a buffer solution to be desalted, and urea which had been used as a 10 denaturating agent, was removed at the same time. Step 3-3: Removal of His-tag 2M urea was added to buffer solution (50mM NaCl, 20mM Tris-HCl, 2mM CaCl 2 pH7.4) which was effective for removing denaturating agent and imidazol etc. from the 15 purified his-tagged protein and also for activating enterokinase. The dialyzed PB1 4 peptide which had been obtained from Step 3-2 was dialyzed again by using the above urea containing buffer to desalt the PBl 4 peptide, during which the concentration of urea was lowered 20 gradually by repeated dialysis against urea depleted buffer. 3U/ml of enterokinase was added to the PB14 peptide-containing solution of which buffer was changed with the above second buffer, and incubated at 23 0 C. The solution which was taken at every hour, then 25 was analyzed by SDS-PAGE in order to check the amount of his-tag removal from the his-tagged PB1 4 (PB1 4 +his peptide. Step 3-4: Ion exchange chromatography Unwanted proteins and peptides which had been 30 produced as a result of treatment of enterokinase, were removed through ion exchange chromatography. The solution containing PB1 4 +his peptide which had - 23 been obtained in Step 3-3, was dialyzed in dialysis buffer (2M urea, 0.1M NaH2 P04 , 0.01M Tris-HCl, pH 7.0), and the buffer was exchanged sufficiently. The dialyzed solution was loaded on to DEAE Sepharose 5 resin (Phamacia, Uppsala, Sweden). Thereafter, the column was equilibrated with equilibrating buffer (50mM sodium phosphate buffer, 2M urea, pH 7.0) and the peptide was eluted with a NaCl concentration gradient from 0 to 1M by using another buffer (50mM 10 sodium phosphate buffer, 2M urea, 1M NaCl) (flow rate:0.5ml/min). Each fraction was recovered and target protein-containing fractions were pooled. The presence of PB1 4 -his peptide was confirmed by SDS-PAGE after concentrating the compartments. 15 Step 4: Quantitative analysis of PB14 The purified PB1 4 peptide which had been obtained by the method of Step 3 was quantitatively analyzed by colorimetric analysis using micro BCA reagent (Pierce, Rockford, USA). 20 Step 5: Confirmation of characteristics of the recombinant PB1 4 peptide The purity of the PB1 4 peptides which had been purified in Step 3 and their immunogenicity against anti-serum which had been obtained by using synthetic 25 PB1 4 peptide as an antigen, were confirmed through western-blot assay by ECL (Amersham, Cleveland, UK). After SDS-PAGE (Example 2, Step 1), the gel was incubated with a PVDF membrane in buffer (0.3% Tris, 1.5% glycine, 20% methanol) at a constant voltage of 30 60V for three(3) hours in order for the protein in the gel to be transferred into the PVDF membrane. Then, - 24 the blotted membrane was incubated with a 5ml of blocking solution (TBS pH 7.5, 5% skim milk powder(w/v), 0.02% Tween 20) for 1.5 hours, and then was washed three times with TTBS (Tris-buffered saline 5 solution containing 0.1% Tween 20) for 15 min., 5 min. and 5 min. respectively. The antiserum against the peptide PB1 (refers to Step 1 of Example 2) was diluted with the TTBS solution in ratio of one(l) to five thousand (5,000), and then incubated with the 10 membrane for 1.5 hours. To confirm the purity of the PB1 4 peptide, anti-serum against PB1 4 peptide (Example 3) was used. After washing the gel with TTBS three times for 15 min., 5 min. and 5 min. in turn, the membrane was incubated for 1.5 hours at room 15 temperature with the solution in which alkaline phosphatase-F(ab)' 2 -goat anti-mouse IgG (H+L) (Zymed, San Francisco, CA) was diluted with the TTBS solution in ratio of one(l) to one thousand(1000). The membrane was washed again with TTBS three times, and 20 then colorized by adding BCIP/NBT (5-bromo-4-chloro-3 indolyl phosphate/nitro blue tetrazolium(Sigma)). BCIP/NBT solution was removed using TTBS solution after staining. As a result of western-blotting analysis, the expressed PB1 4 peptide could be 25 recognized with the anti-PB1 4 serum. In the case of ECL, PVDF membrane (Gelman Science, BioTraceR) was used instead of nitrocellulose membrane. In addition, the first antibody was used in ratio of one(l) to ten thousand(10,000) and HRP-conjugated 30 rabbit anti-mouse IgG (Pierce, Rockford, IL, U.S.A.) was used as the second antibody in ratio of one(1) to ten thousand(10,000). 1 ml of solution A of ECL+Plus - 25 western-blot agent (Amersham) per 25 ml of solution B, was used in color reaction. When color was generated sufficiently, the membrane was inserted into a film cassette for 5, 10, 20 and 30 seconds respectively to 5 be exposed to the film so that the bands on the gel could be detected (Fig. 8). In Fig. 8, lane M represents a ECL detecting label (Gibco BRL) and lane 1 represents the PB1 4 peptide. As the result from Fig. 8, the expressed PB1 4 peptide could be recognized with 10 the anti-PB1 4 serum. In addition, the result of western-blot analysis in which the PB1 4 peptide was detected using polyclonal antibody isolated from rabbit serum by using Protein G column (Bio-Rad, USA), gave the same result. 15 Example 5: Preparation of anti-PBl 4 peptide mouse antibody -his The PB1 4 peptide used herein was the PB1 4 -hS peptide from which his-tag was removed, in step 3-3 of example 2. 20 Step 1: Ligation between PB1 4 peptide and OVA As a carrier protein, ovalbumin(OVA), was added to the purified PB1 4 peptide in Step 3 in Example 2, in molar ration of one(1) to ten(10), and was incubated for one (1) hour at 4*C. To the P21 4 peptide-ovalbumin 25 solution, 2%(v/v) glutaraldehyde was added with the same volume, and incubated for one(l) hour with continuous shaking. Then glycine was added to the reaction mixture until final concentration is to be 0.2M to stop the reaction proceeding therein. 30 After the reaction, the remaining glutaraldehyde glycine in reaction mixture, were removed by dialysis using MWCO 12,000-14,000 dialysis membrane (SpectrumR, - 26 Dominguez, CA, USA). Step 2. Immunization of mice The peptide with which OVA was linked in Step 1, 5 was concentrated and used to immunize mice. The amount of the antigen to be injected to the mouse, was 5pg, which was the amount of PB1 4 peptide before conjugation with OVA. The antigen, which was emulsified with the same amount of an adjuvant, was 10 injected intraperitoneally into the mouse in a volume of 0.2ml. Complete Freund's Adjuvant(CFA) was used as the adjuvant for the first injection, and Incomplete Freund's Adjuvant (IFA) was used as the adjuvant for 15 the two(2) boosting immunizations at two(2) week intervals. In the control mouse, BSA (bovine serum albumin) was injected. After five (5) days from the final injection, lml of blood was taken from mouse by cardiac puncture and 20 the blood was clotted for 30 min. at 37 0 C. Then the blood was centrifuged for 30min. at 4 0 C, 2500xg and the clot was removed from the blood. The supernatant (i.e., blood serum) was incubated overnight at 4*C for the remaining blood coagulants to be concentrated 25 completely, and centrifuged for 20min. at 10,000xg. The resulting supernatant was aliquoted into several tubes. The blood serum which was to be used in experiment, was stored at 4 0 C, and the remainders were stored at -20 0 C. 30 Step 3. Measurement of avidity of anti-PB14 antibody by indirect ELISA - 27 The avidity of the antibody was measured by using blood serum obtained in Step 2. 100pl of PBl 4 peptide was distributed into each well of a 96 well micro titer plate(Flacon: pro-binding) and left at 4 0 C for 6 5 hours or more, and then washed three(3) times with TTBS (Tirs buffer saline solution containing 0.05% Tween 20). 200ptl of blocking solution (1% BSA in TTBS) was added to each well and incubated at 37 0 C for one (1) hour, and then washed three(3) times with TTBS. 10 100pl of the isolated serum which had been diluted with the blocking solution in a ratio of one(1) from 102 to 105 was added to the reaction solution and incubated at 37'C for 1 hour, and then washed three (3) times with 200ptl of TTBS. 100ptl of HRP-conjugated goat anti 15 rabbit IgG antibody (Pierce, Rockford, IL) which had been diluted with the blocking solution in a ratio of one(1) to 103, was added to the reaction solution and incubated at 37 0 C for 1 hour, and washed three (3) times with 20011 of TTBS. Solution A of the HRP 20 substrate kit (Bio-Rad) was mixed with solution B of the same in ratio of nine(9) to one(1). 100pil of the resultant mixture was added to the reaction solution and colorized for thirty(30) min., and then optical absorbance for the reaction mixture was measured at 25 405nm by using ELISA leader (EL312e, Bio-Tek Ins.) (Fig.9). In Fig. 9, it was confirmed that the mouse antibody specific for PB1 4 peptide could be applied to western-blot and ELISA analysis by one(1) thousand fold (3.0 of the X axis in the figure).

- 28 Example 6: Anti-obesity effect of PB1 4 vaccine in a mouse model. Step 1:Induction of obesity in mice 5 5 week-old ICR mice (Korea Center for Animal Experiment Ltd., Seoul, Korea) were used herein. The mice were raised in a breeding farm in which the temperature was kept from 17 0 C to 25'C, and fed with a mixed feed (Sam Yang Feed Ltd., Seoul, Korea, 10 [ingredient: water 11.8% or more, protein 20.0% or more, crude lipid 3.0% or more, crude fiber 10.0% or less, crude ash 10.0% or less, calcium 0.6% or less and phosphorus 0.4% or more]). Goldthioglucose (GTG) was administered to the mice to induce obesity. GTG 15 induces desensitization of the ventero- medial hypothalamic nucleus (VMH) . Therefore the mice administered with GTG did not feel satiated and always had a desire to eat. The GTG used herein is a very unstable compound, which is easily degraded in water 20 or moisture. Therefore, 100mg of GTG (Sigma, Inc.) was diluted with 1ml of sesame oil (Sigma Inc.), and was used according to the method of Brecher et al (Brechere G. and Waxler, S. H. Proc. Soc. Exp. Biol. Med., 70: 498-501(1949)) in order for a proper amount 25 of GTG to be administered. The mice were allocated a test group (twenty(20) mice) and a control group (four(4) mice) , and 25ml of GTG was injected to the test group whereas the control was injected with nothing. 30 Body weight of the mice of the test group was measured prior to experiment and the mice of which deviation of body weight was not significant, were - 29 selected and used in the experiment. Body weight of the mice measured after one(l) week after GTG injection, was in the range from 26.5 to 29.5grams. Seven mice of the GTG-injected group were induced 5 to be obese whereas the remainder was not. The mice which were not induced to be obese were injected again with GTG, then all the mice were induced to be obese. All of the obesity-induced mice were distributed into three(3) groups. A week after the second GTG 10 injection the PB1 4 peptide was injected into the mice of test group 1, consisting of seven(7) mice, used the method of Step 2 of Example 3. In addition, the mice (test group 2 containing seven(7) mice) of another group of the three, were injected with ovalbumin 15 instead of PB1 4 peptide as a mock experiment, and vaccine was not injected into the other group (test group 3 containing six(6) mice) to induce obesity. On the other hand, 0.2ml of PBS was injected to the control group to be compared with the test groups to 20 confirm the effect of the vaccine of the present invention. In addition, feed used herein was mixed with the yolk of an egg and dried at 500C to induce intake of cholesterol so that the level of cholesterol could 25 increase in mouse serum. Feed was also provided enough for disease related to the level of cholesterol to be caused. Body weight of the mice was measured everyday. As depicted in Fig. 10, the body weight of the 30 vaccine-injected mice of test group 1(-A -A -), increased from 27.7±0.4g to 52.2±1.7g after twelve - 30 weeks(12) of GTG injection. The data justify a conclusion that there was no significant difference of an increase in body weight between the test group 1 and the control group (-0 -0 -). However, body weight 5 of the mice of both the test group 2 (-0 -0 -) in which ovalbumin was injected after being obese and the test group (-N -0 -) in which no vaccine was injected after the induction of obesity, increased continuously from 28.3±0.5g to 68.9±2.8g. Therefore, it was confirmed 10 that obesity could be inhibited by injection of PBl4 peptide vaccine. In Fig. 10, G1 and G2 represent the time of GTG injection and V1, V2 and V3 represent the time of injection of PB14 peptide vaccine. 15 Fig. 11 represents the appearance of the obesity induced mice. A 20-week old mouse of the test group 1 (Fig.lla: normal mouse) was compared with a 20-week old mouse of the test group 3 (Fig. llb:obese mouse). As depicted in Fig. 11, it was confirmed that the 20 vaccine of the present invention was effective in inhibiting obesity. Step 2: Measurement of the level of cholesterol in blood. After the first GTG injection, the blood 25 cholesterol level of 12-week old mice of the control group was compared with that of 12-week old GTG injected mice of test groups 1, 2 and 3. The concentration of total cholesterol, triglyceride, HDL cholesterol and LDL-cholesterol was measured by an - 31 enzymatic method by using Cholestezyme-V, Triglyzyme-V, HDL-C555 (Shin Yang Chemicals, Seoul, Korea) and LDL EX kit (Denka Bio-Research, Ltd., Tokyo, Japan). In each experiment, a standard curve for O.D value was 5 prepared by using standard Calibrater-D (Denka Bio Research, Ltd., Tokyo, Japan) to decrease experimental error. The O.D value for the interest was calculated based on the calibration curve to confirm the concentration and content of the lipid, and the 10 results are depicted in Table 1 and Fig. 12. Table 1. Total TG HDL-C cholesterol Control 79±3.7 180±26 59±3.4 6±1.2 Test group 118±3.6 217±47 92±4.7 20±1.7 1 Test group 131±8.8 218±70 119±7.5 30±4.5 2 and 3 TG: triglyceride, HDL-C: HDL-cholesterol LDL-C: LDL-cholesterol As depicted in Table 1 and Fig. 12, as the result 15 of the induction of obesity, it was confirmed that there was no significant difference in the content of cholesterol of both the test group and the control did not increase, whereas the overall blood concentration of total cholesterol, HDL-C and LDL-C increased by a 20 small amount (Fig.12).

- 32 Industrial Applicability The vaccine compositions disclosed herein which contain a concatemer comprising 2 to 15 mimetic peptides of an epitope of apolipoprotein B-100, can inhibit s occurrence of obesity without causing auto-immunity in the organism. Therefore, LDL-related circulatory disease can be treated by the vaccine compositions disclosed herein more effectively than the transitory and expensive conventional 10 method, in which a cholesterol metabolism-related enzyme was inhibited. While the present invention has been particularly shown and described with reference to particular examples thereof, it will be understood by those skilled in the art 15 that various changes in form and details may be conceived, therefrom without departing from the spirit and scope of the present invention as defined by the appended claims. 2377301_1 (GHMauers) 18/08/10

Claims (17)

1. A concatemer comprising 2 to 15 mimetic peptides 5 of an epitope of apolipoprotein B-100, in which each of the peptides has the sequence set out in SEQ ID NO:1.

2. A concatemer according to claim 1, wherein said concatemer comprises 4 said mimetic peptides linked 10 consecutively.

3. A concatemer comprising 2 to 15 mimetic peptides of an epitope of apolipoprotein B-100, in which each of the peptides has the sequence set out in SEQ ID NO:2. 15

4. A concatemer according to claim 3, wherein said concatemer comprises 4 said mimetic peptides linked consecutively. 20

5. A concatemer comprising 2 to 15 mimetic peptides of an epitope of apolipoprotein B-100, in which each of the peptides has the sequence set out in SEQ ID NO:3.

6. A concatemer according to claim 5, wherein said 25 concatemer comprises 4 said mimetic peptides linked consecutively.

7. A method of treating or preventing obesity, comprising administering the concatemer according to any 30 one of claims 1 to 6.

8. A method according to claim 7, in which the administering is by intradermal injection. 35

9. A method according to claim 7, wherein the concatemer is formulated as a tablet, pill, granule, cachet, elixir, suspension, emulsion, solution, syrup, 2377301_1 (GHMatters) 18/08/10 - 34 aerosol, soft or hard gelatin capsule, sterilized injectable solution or sterilized powder.

10. A process for preparing the concatemer according s to any one of claims 1 to 6 comprising: (i) inserting DNA which encodes the concatemer into a vector; (ii) transforming host cells with the vector obtained in step i) and incubating the same under 10 conditions; and (iii) isolating the concatemer from the host cell.

11. An isolated DNA molecule encoding a polypeptide composed of 4 mimetic peptides of an epitope of 15 apolipoprotein B-100 linked consecutively, in which each of the peptides has the sequence set out in SEQ ID NO:1.

12. An expression vector which comprises a DNA fragment encoding a polypeptide composed of 4 mimetic 20 peptides of an epitope of apolipoprotein B-100 of SEQ ID NO:1 linked consecutively, in which each of the peptides has the sequence set out in SEQ ID NO:1.

13. A concatemer according to claim 1, 3 or 5, 25 substantially as herein described with reference to the examples and drawings.

14. A method according to claim 7, substantially as herein described with reference to the examples and 30 drawings.

15. A process according to claim 10, substantially as herein described with reference to the examples and drawings. 35

16. An isolated DNA molecule according to claim 11, substantially as herein described with reference to the 2377301_1 (GilMatters) 18/08/10 - 35 examples and drawings.

17. An expression vector according to claim 12, substantially as herein described with reference to the s examples and drawings. 2377301_1 (GHMatters) 18/08/10