Abstract:
The present invention relates to passive immunization for treating or preventing atherosclerosis using an isolated human antibody directed towards at least one oxidized fragment of apolipoprotein B in the manufacture of a pharmaceutical composition for therapeutical or prophylactical treatment of atherosclerosis by means of passive immunization, as well as method for preparing such antibodies, and a method for treating a mammal, preferably a human using such an antibody to provide for passive immunization.

Description:
TECHNICAL FIELD  
         [0001]    The present invention relates to new isolated human antibodies raised against peptides being derivatives of apolipoprotein B, in particular antibodies to be used for immunization therapy for treatment of atherosclerosis, method for their preparation, and method for passive immunization using said antibodies.  
           [0002]    In particular the invention includes:  
           [0003]    The use of any isolated antibody raised against an oxidized form of the peptides listed in table 1, in particular MDA-modified peptides, preferably together with a suitable carrier and adjuvant as an immunotherapy or “anti-atherosclerosis “vaccine” for prevention and treatment of ischemic cardiovascular disease.  
         BACKGROUND OF THE INVENTION  
         [0004]    The protective effects of humoral immunity are known to be mediated by a family of structurally related glycoproteins called antibodies. Antibodies initiate their biological activity by binding to antigens. Antibody binding to antigens is generally specific for one antigen and the binding is usually of high affinity. Antibodies are produced by B-lymphocytes. Blood contains many different antibodies, each derived from a clone of B-cells and each having a distinct structure and specificity for antigen. Antibodies are present on the surface of B-lymphocytes, in the plasma, in interstitial fluid of the tissues and in secretory fluids such as saliva and mucous on mucosal surfaces.  
           [0005]    All antibodies are similar in their overall structure, accounting for certain similarities in physico-chemical features such as charge and solubility. All antibodies have a common core structure of two identical light chains, each about 24 kilo Daltons, and two identical heavy chains of about 55-70 kilo Daltons each. One light chain is attached to each heavy chain, and the two heavy chains are attached to each other. Both the light and heavy chains contain a series of repeating homologous units, each of about 110 amino acid residues in length which fold independently in a common globular motif, called an immunoglobulin (Ig) domain. The region of an antibody formed by the association of the two heavy chains is hydrophobic. Antibodies, and especially monoclonal antibodies, are known to cleave at the site where the light chain attaches to the heavy chain when they are subjected to adverse physical or chemical conditions. Because antibodies contain numerous cysteine residues, they have many cysteine-cysteine disulfide bonds. All Ig domains contain two layers of beta-pleated sheets with three or four strands of anti-parallel polypeptide chains.  
           [0006]    Despite their overall similarity, antibody molecules can be divided into a small number of distinct classes and subclasses based on physicochemical characteristics such as size, charge and solubility, and on their behavior in binding to antigens. In humans, the classes of antibody molecules are: IgA, IgD, IgE, IgG and IgM. Members of each class are said to be of the same isotype. IgA and IgG isotypes are further subdivided into subtypes called IgA1, IgA2 and IgG1, IgG2, IgG3 and IgG4. The heavy chains of all antibodies in an isotype share extensive regions of amino acid sequence identity, but differ from antibodies belonging to other isotypes or subtypes. Heavy chains are designated by the letters of the Greek alphabet corresponding to the overall isotype of the antibody, e.g., IgA contains .alpha., IgD contains .delta., IgE contains .epsilon., IgG contains .gamma., and IgM contains .mu. heavy chains. IgG, IgE and IgD circulate as monomers, whereas secreted forms of IgA and IgM are dimers or pentamers, respectively, stabilized by the J chain. Some IgA molecules exist as monomers or trimers.  
           [0007]    There are between 10 8  and 10 10  structurally different antibody molecules in every individual, each with a unique amino acid sequence in their antigen combining sites. Sequence diversity in antibodies is predominantly found in three short stretches within the amino terminal domains of the heavy and light chains called variable (V) regions, to distinguish them from the more conserved constant (C) regions.  
           [0008]    Atherosclerosis is a chronic disease that causes a thickening of the innermost layer (the intima) of large and medium-sized arteries. It decreases blood flow and may cause ischemia and tissue destruction in organs supplied by the affected vessel. Atherosclerosis is the major cause of cardiovascular disease including myocardial infarction, stroke and peripheral artery disease. It is the major cause of death in the western world and is predicted to become the leading cause of death in the entire world within two decades.  
           [0009]    The disease is initiated by accumulation of lipoproteins, primarily low-density lipoprotein (LDL), in the extracellular matrix of the vessel. These LDL particles aggregate and undergo oxidative modification. Oxidized LDL is toxic and cause vascular injury.  
           [0010]    Atherosclerosis represents in many respects a response to this injury including inflammation and fibrosis.  
           [0011]    In 1989 Palinski and coworkers identified circulating autoantibodies against oxidized LDL in humans. This observation suggested that atherosclerosis may be an autoimmune disease caused by immune reactions against oxidized lipoproteins. At this time several laboratories began searching for associations between antibody titers against oxidized LDL and cardiovascular disease. However, the picture that emerged from these studies was far from clear. Antibodies existed against a large number of different epitopes in oxidized LDL, but the structure of these epitopes was unknown. The term “oxidized LDL antibodies” thus referred to an unknown mixture of different antibodies rather than to one specific antibody. T cell-independent IgM antibodies were more frequent than T-cell dependent IgG antibodies.  
           [0012]    Antibodies against oxidized LDL were present in both patients with cardiovascular disease and in healthy controls. Although some early studies reported associations between oxidized LDL antibody titers and cardiovascular disease, others were unable to find such associations. A major weakness of these studies was that the ELISA tests used to determine antibody titers used oxidized LDL particles as ligand. LDL composition is different in different individuals, the degree of oxidative modification is difficult both to control and assess and levels of antibodies against the different epitopes in the oxidized LDL particles can not be determined. To some extent, due to the technical problems it has been difficult to evaluate the role of antibody responses against oxidized LDL using the techniques available so far, but, however, it is not possible to create well defined and reproducable components of a vaccine if one should use intact oxidized LDL particles.  
           [0013]    Another way to investigate the possibility that autoimmune reactions against oxidized LDL in the vascular wall play a key role in the development of atherosderosis is to immunize animals against its own oxidized LDL. The idea behind this approach is that if autoimmune reactions against oxidized LDL are reinforced using classical immunization techniques this would result in increased vascular inflammation and progressive of atherosclerosis. To test this hypothesis rabbits were immunized with homologous oxidized LDL and then induced atherosderosis by feeding the animals a high-cholesterol diet for 3 months.  
           [0014]    However, in contrast to the original hypothesis immunization with oxidized LDL had a protective effect reducing atherosderosis with about 50%. Similar results were also obtained in a subsequent study in which the high-cholesterol diet was combined with vascular balloon-injury to produce a more aggressive plaque development. In parallel with our studies several other laboratories reported similar observations. Taken together the available data clearly demonstrates that there exist immune reactions that protect against the development of atherosclerosis and that these involves autoimmunity against oxidized LDL.  
           [0015]    These observations also suggest the possibility of developing an immune therapy or “vaccine” for treatment of atherosclerosis-based cardiovascular disease in man. One approach to do this would be to immunize an individual with his own LDL after it has been oxidized by exposure to for example copper. However, this approach is complicated by the fact that it is not known which structure in oxidized LDL that is responsible for inducing the protective immunity and if oxidized LDL also may contain epitopes that may give rise to adverse immune reactions.  
           [0016]    The identification of epitopes in oxidized LDL is important for several aspects:  
           [0017]    First, one or several of these epitopes are likely to be responsible for activating the anti-atherogenic immune response observed in animals immunized with oxidized LDL. Peptides containing these epitopes may therefore represent a possibility for development of an immune therapy or “atherosclerosis vaccine” in man. Further, they can be used for therapeutic treatment of atheroschlerosis developed in man.  
           [0018]    Secondly, peptides containing the identified epitopes can be used to develop ELISAs able to detect antibodies against specific structure in oxidized LDL. Such ELISAs would be more precise and reliable than ones presently available using oxidized LDL particles as antigen. It would also allow the analyses of immune responses against different epitopes in oxidized LDL associated with cardiovascular disease.  
           [0019]    U.S. Pat. No. 5,972,890 relates to a use of peptides for diagnosing atherosclerosis. The technique presented in said US patent is as a principle a form of radiophysical diagnosis. A peptide sequence is radioactively labelled and is injected into the bloodstream. If this peptide sequence should be identical with sequences present in apolipoprotein B it will bind to the tissue where there are receptors present for apolipoprotein B. In vessels this is above all atherosderotic plaque. The concentration of radioactivity in the wall of the vessel can then be determined e.g., by means of a gamma camera. The technique is thus a radiophysical diagnostic method based on that radioactively labelled peptide sequences will bound to their normal tissue receptors present in atherosclerotic plaque and are detected using an external radioactivity analysis. It is a direct analysis method to identify atherosclerotic plaque. It requires that the patient be given radioactive compounds.  
           [0020]    Published studies (Palinski et al., 1995, and George et al., 1998) have shown that immunisation against oxidised LDL reduces the development of atherosclerosis. This would indicate that immuno reactions against oxidised LDL in general have a protecting effect. The results given herein have, however, surprisingly shown that this is not always the case. E.g., immunisation using a mixture of peptides #10, 45, 154, 199, and 240 gave rise to an increase of the development of atherosderosis. Immunisation using other peptide sequences, e.g., peptide sequences #1, and 30 to 34 lacks total effect on the development of atherosderosis. The results are surprising because they provide basis for the fact that immuno reactions against oxidised LDL, can protect against the development, contribute to the development of atherosclerosis, and be without any effect at all depending on which structures in oxidised LDL they are directed to. These findings make it possible to develop immunisation methods, which isolate the activation of protecting immuno reactions. Further, they show that immunisation using intact oxidised LDL could have a detrimental effect if the particles used contain a high level of structures that give rise to atherogenic immuno reactions.  
           [0021]    The technique of the present invention is based on quite different principles and methods. In accordance with claim  1  the invention relates to antibodies raised against oxidized fragments of apolipoprotein B, which antibodies are used for immunisation against cardiovascular disease.  
           [0022]    As an alternative to active immunisation, using the identified peptides described above, passive immunisation with pre-made antibodies directed to the same peptides is an attractive possibility. Such antibodies may be given desired properties concerning e.g. specificity and crossreactivity, isotype, affinity and plasma halflife. The possibility to develop antibodies with predetermined properties became apparent already with the advent of the monoclonal antibody technology (Milstein and Köhler, 1975 Nature, 256:495-7). This technology used murine hybridoma cells producing large amounts of identical, but murine, antibodies. In fact, a large number of preclinical, and also clinical trials were started using murine monoclonal antibodies for treatment of e.g. cancers. However, due to the fact that the antibodies were of non-human origin the immune system of the patients recognised them as foreign and developed antibodies to them. As a consequence the efficacy and plasma half-lives of the murine antibodies were decreased, and often side effects from allergic reactions, caused by the foreign antibody, prevented successful treatment.  
           [0023]    To solve these problems several approaches to reduce the murine component of the specific and potentially therapeutic antibody were taken. The first approach comprised technology to make so called chimearic antibodies where the murine variable domains of the antibody were transferred to human constant regions resulting in an antibody that was mainly human (Neuberger et al. 1985, Nature 314:268-70). A further refinement of this approach was to develop humanised antibodies where the regions of the murine antibody that contacted the antigen, the so called Complementarity Determining Regions (CDRs) were transferred to a human antibody framework. Such antibodies are almost completely human and seldom cause any harmful antibody responses when administered to patients. Several chimearic or humanised antibodies have been registered as therapeutic drugs and are now widely used within various indications (Borrebaeck and Carlsson, 2001, Curr. Opin. Pharmacol. 1:404-408).  
           [0024]    Today also completely human antibodies may be produced using recombinant technologies. Typically large libraries comprising billions of different antibodies are used. In contrast to the previous technologies employing chimearisation or humanisation of e.g. murine antibodies this technology does not rely on immunisation of animals to generate the specific antibody. In stead the recombinant libraries comprise a huge number of pre-made antibody variants why it is likely that the library will have at least one antibody specific for any antigen. Thus, using such libraries the problem becomes the one to find the specific binder already existing in the library, and not to generate it through immunisations. In order to find the good binder in a library in an efficient manner, various systems where phenotype i.e. the antibody or antibody fragment is linked to its genotype i.e. the encoding gene have been devised. The most commonly used such system is the so called phage display system where antibody fragments are expressed, displayed, as fusions with phage coat proteins on the surface of filamentous phage particles, while simultaneously carrying the genetic information encoding the displayed molecule (McCafferty et al., 1990, Nature 348:552-554). Phage displaying antibody fragments specific for a particular antigen may be selected through binding to the antigen in question. Isolated phage may then be amplified and the gene encoding the selected antibody variable domains may optionally be transferred to other antibody formats as e.g. full length immunoglobulin and expressed in high amounts using appropriate vectors and host cells well known in the art.  
           [0025]    The format of displayed antibody specificities on phage particles may differ. The most commonly used formats are Fab (Griffiths et al., 1994. EMBO J. 13:3245-3260) and single chain (scFv) (Hoogenboom et al., 1992, J Mol Biol. 227:381-388) both comprising the variable antigen binding domains of antibodies. The single chain format is composed of a variable heavy domain (VH) linked to a variable light domain (VL) via a flexible linker (U.S. Pat. No. 4,946,778). Before use as analytical reagents, or therapeutic agents, the displayed antibody specificity is transferred to a soluble format e.g. Fab or scFv and analysed as such. In later steps the antibody fragment identified to have desireable characteristics may be transferred into yet other formats such as full length antibodies.  
           [0026]    Recently a novel technology for generation of variability in antibody libraries was presented (WO98/32845, Soderlind et al., 2000, Nature BioTechnol. 18:852-856).  
           [0027]    Antibody fragments derived from this library all have the same framework regions and only differ in their CDRs. Since the framework regions are of germline sequence the immunogenicity of antibodies derived from the library, or similar libraies produced using the same technology, are expected to be particularly low (Soderlind et al., 2000, Nature BioTechnol. 18:852-856). This property is expected to be of great value for therapeutic antibodies reducing the risk for the patient to form antibodies to the administered antibody thereby reducing risks for allergic reactions, the occurrence of blocking antibodies, and allowing a long plasma half-life of the antibody. Several antibodies derived from recombinant libraries have now reached into the clinic and are expected to provide therapeutic drugs in the near future.  
           [0028]    Thus, when met with the challenge to develop therapeutic antibodies to be used in humans the art teaches away from the earlier hybridoma technology and towards use of modern recombinant library technology (Soderlind et al., 2001, Comb. Chem. &amp; High Throughput Screen. 4:409-416). It was realised that the peptides identified (PCT/SE02/00679), and being a integral part of this invention, could be used as antigens for generation of fully human antibodies with predetermined properties. In contrast to earlier art (U.S. Pat. No. 6,225,070) the antigenic structures i.e. the peptides used in the present invention were identified as being particularly relevant as target sequences for therapeutic antibodies (PCT/SE02/00679). Also, in the present invention the antibodies are derived from antibody libraries omitting the need for immunisation of lipoprotein deficient mice to raise murine antibodies (U.S. Pat. No. 6,225,070). Moreover, the resulting antibodies are fully human and are not expected to generate any undesired immunological reaction when administered into patients.  
           [0029]    The peptides used, and previously identified (PCT/SE02/00679) are the following:  
                                                                                                                                                                                                                       TABLE 1                                       A. High IgG, MDA-difference                    P 11.   FLDTVYGNCSTHFTVKTRKG                           P 25.   PQCSTHILQWLKRVHANPLL                       P 74.   VISIPRLQAEARSEILAHWS                            B. High IgM, no MDA-difference                    P 40.   KLVKEALKESQLPTVMDFRK                           P 68.   LKFVTQAEGAKQTEATMTFK                       P 94.   DGSLRHKFLDSNIKFSHVEK                       P 99.   KGTYGLSCQRDPNTGRLNGE                       P 100.   RLNGESNLRFNSSYLQGTNQ                       P 102.   SLTSTSDLQSGIIKNTASLK                       P 103.   TASLKYENYELTLKSDTNGK                       P 105.   DMTFSKQNALLRSEYQADYE                       P 177.   MKVKIIRTIDQMQNSELQWP                            C. High IgG, no MDA difference                    P 143.   IALDDAKINFNEKLSQLQTY                           P 210.   KTTKQSFDLSVKAQYKKNKH                            D. NHS/AHP, IgG-ak &gt; 2, MDA-difference                    P1.   EEEMLENVSLVCPKDATRFK                           P 129.   GSTSHHLVSRKSISMLEHK                       P 148.   IENIDFNKSGSSTASWIQNV                       P 162.   IREVTQRLNGEIQALELPQK                       P 252.   EVDVLTKYSQPEDSLIPFFE                            E. NHS/AHP, IgM-ak &gt; 2, MDA-difference                    P 301.   HTFLIYITELLKKLQSTTVM                           P 30.   LLDIANYLMEQIQDDCTGDE                       P 31.   CTGDEDYTYKIKRVIGNMGQ                       P 32.   GNMGQTMEQLTPELKSSILK                       P 33.   SSILKCVQSTKPSLMIQKAA                       P 34.   IQKAAIQALRKMEPKDKDQE                       P 100.   RLNGESNLRFNSSYLQGTNQ                       P 107.   SLNSHGLELNADILGTDKIN                       P 149.   WIQNVDTKYQIRIQIQEKLQ                       P 169.   TYISDWWTLAAKNLTDFAEQ                       P 236.   EATLQRIYSLWEHSTKNHLQ                            F. NHS/AHP, IgG-ak &lt;0.5, no               MDA-difference                P 10.   ALLVPPETEEAKQVLFLDTV                           P 45.   IEIGLEGKGFEPTLEALFGK                       P 111.   SGASMKLTTNGRFREHNAKF                       P 154.   NLIGDFEVAEKINAFRAKVH                       P 199.   GHSVLTAKGMALFGEGKAEF                       P 222.   FKSSVITLNTNAELFNQSDI                       P 240.   FPDLGQEVALNANTKNQKIR                            or an active site of one or more of                       these peptides.                                                                                                          
 
         SUMMARY OF THE INVENTION  
         [0030]    The present invention relates to the use of at least one recombinant human antibody or an antibody fragment thereof directed towards at least one oxidized fragment of apolipoprotein B in the manufacture of a pharmaceutical composition for therapeutical or prophylactical treatment of atherosclerosis by means of passive immunization.  
           [0031]    Further the invention relates to the recombinant preparation of such antibodies, as well as the invention relates to method for passive immunization using such antibodies raised using an oxidized apolipoprotein B fragement, as antigen, in particular a fragemnt as identified above.  
           [0032]    The present invention utilises an isolated antibody fragment library to generate specific human antibody fragments against oxidized, in particular MDA modified peptides derived from Apo B100. Identified antibody fragments with desired characteristics may then rebuilt into full length human immunoglobulin to be used for therapeutic purposes. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
       [0033]    Below will follow a detailed description of the invention examplified by, but not limited to, human antibodies derived from an isolated antibody fragment library and directed towards two MDA modified peptides from ApoB 100.  
       EXAMPLE 1  
       [0034]    Selection of scFv Against MDA Modified Peptides IEIGL EGKGF EPTLE ALFGK (P45. Table 1) and KTTKQ SFDLS VKAQY KKNKH (P210, Table 1)  
         [0035]    The target antigens were chemically modified to carry Malone-di-aldehyde (MDA) groups on lysines and histidines. The modified peptides were denoted IEI (P45) and KTT (P210).  
         [0036]    Selections were performed using BioInvent&#39;s n-CoDeR™scFv library for which the principle of construction and production have been described in Soderlind et al. 2000, Nature BioTechnology. 18, 852-856. Briefly, CDRs are isolated from human immunoglobulin genes and are shuffled into a fixed framework. Thus variability in the resulting immunoglobulin variable regions is a consequence of recombination of all six CDRs into the fixed framework. The framework regions are all germline and are identical in all antibodies. Thus variability is restricted to the CDRs which are all natural and of human origin. The library contains approximately 2×10 10  independent clones and a 2000 fold excess of clones were used as input for each selection. Selections were performed in three rounds. In selection round 1, Immunotubes (NUNC maxisorb 444202) were coated with 1.2 ml of 20 μg/ml MDA-modified target peptides in PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na 2 HPO 4 , 1.4 mM KH 2 PO 4 ) with end over end agitation at +4° C. over night. The tubes were then blocked with TPBSB5% (5 % BSA, 0.05% Tween 20, 0.02% sodium Azide in PBS) for 30 minutes and washed twice with TPBSB3% (3% BSA, 0.05% Tween 20, 0.02% sodium Azide in PBS) before use. Each target tube was then incubated with approximately 2×10 13  CFU phages from the n-CodeR™ library in 1.8 ml TPBSB3% for 2 h at room temperature, using end over end agitation. The tubes were then washed with 15×3 ml TPBSB3% and 2×1 ml PBS before the bound phages were eluted with 1 ml/tube of 2 mg/ml trypsin (Roche, 109819) for 30 minutes at room temperature. This procedure takes advantage of a specific trypsin site in the scFv-fusion protein to release the phage from the target. The reaction was stopped by the addition of 100 μl of Aprotein (0.2 mg/ml, Roche, cat.236624), and the immunotubes were washed with 300 ul PBS, giving a final volume of 1.4 ml.  
         [0037]    For amplification of the selected phage  E. Coli  HB101F′ cells were grown exponentially in 10 ml of LB medium (Merck, cat. 1.10285) to OD 600 =0.5 and infected with the selected and eluted phage principally as described (Soderlind et al., 2000, Nature BioTechnol. 18, 852-856. The resulting phage supernatant was then precipitated by addition of ¼ volume of 20% PEG 6000  in 2.5 M NaCl and incubated for 5 h at +4° C. The phages were then pelleted by centrifugation for 30 minutes, 13000×g, re-suspended in 500 μl PBS and used in selection round 2.  
         [0038]    The amplified phagestock was used in selection round 2 in a final volume of 1.5 ml of 5% BSA, 0.05% Tween 20, 0.02% sodium Azide in PBS. Peptide without MDA modification (4×10 −7  M) was also included for competition against binders to MDA-unmodified target peptide. The mixture was incubated in immunotubes prepared with antigen as described above, except that the tubes were blocked with 1% Casein instead of TPBSB3%. The incubations and washing of the immunotubes were as described for selection 1. Bound phages were then eluted for 30 minutes using 600 μl of 100 mM Tris-Glycine buffer, pH 2.2. The tubes were washed with additional 200 μl glycin buffer and the eluates were pooled and then neutralised with 96 μl of 1 M Tris-HCl, pH 8.0. The samples were re-natured for 1 h at room temperature and used for selection round 3.  
         [0039]    For selection round 3, BSA, Tween 20 and Sodium Azide were added to the renaturated phage pool to a final concentration of 3%, 0.05% and 0.02%, respectively. Competitor peptides, MDA modified unrelated peptides as well as native target peptides without modification were added to a concentration of 1×10 −7 M. The phage mixtures (1100 μl) were added to immunotubes coated with target antigen as described in selection 1 and incubated over night at 4° C. with agitation. The tubes were then washed with 3×3 ml TPBSB 3%, 5×3 ml PBS and eventually bound phages were eluted using trypsin as described in selection round 1 above. Each eluate was infected to 10 ml of logarithmically growing HB101F′ in LB containing 100 μg/ml ampicillin, 15 μg/ml tetracycline, 0.1% glucose, and grown over night at 30° C., 200 rpm in a shaker incubator.  
         [0040]    The over night cultures were used for mini scale preparation of plasmid DNA, using Biorad mini prepp Kit (Cat. 732 6100). To remove the phage gene III part from the expression vector, 0.25 μg of the plasmid DNA was cut for 2 h at 37° C. using 2.5 U Eag-1 (New England Biolabs, cat. R050) in the buffer recommended by the supplier. The samples were then heat inactivated for 20 minutes at 65° C. and ligated over night at 16° C. using 1 U T4 DNA ligase in 30 μl of 1× ligase buffer (Gibco/BRL). This procedure will join two Eag-1 sites situated on opposite sides of the phage gene III fragment, thus creating a free scFv displaying a terminal 6×his tag. After ligation the material was digested for 2 h at 37° C. in a solution containing 30 ul ligation mix, 3.6 μl 10×REACT3 stock, 0.4 μl, 1 M NaCl, 5 pi H 2 O 2 , in order to destroy clones in which the phage gene III segment had been religated. Twenty (20) ng of the final product were transformed into chemical competent Top10F′ and spread on 500 cm 2  Q-tray LA-plates (100 μg/ml Amp, 1% glucose), to enable the picking of single colonies for further screening.  
         [0041]    Screening of the n-CoDeR™scFv Library for Specific Antibody Fragments Binding to MDA Modified Peptides from Apolipoprotein B-100  
         [0042]    In order to identify scFv that could discriminate between MDA modified IEI (P45) peptide and native IEI and between MDA modified KTT (P210) and native KTT respectively screenings were performed on bacterial supernatants from selected scFv expressing clones.  
         [0043]    Colony picking of single clones, expression of scFv and screening number 1 was performed on BioInvent&#39;s automatic system according to standard methods. 1088 and 831 single clones selected against the MDA modified 1EI and KTT peptides respectively were picked and cultured and expressed in micro titre plates in 100 μl LB containing 100 μg ampicillin/ml.  
         [0044]    For screening number 1 white Assay plates (Greiner 655074) were coated with 54 pmol peptide/well in coating buffer (0.1 M Sodium carbonate, pH 9.5), either with MDA modified peptide which served as positive target or with corresponding unmodified peptide which served as non target. In the ELISA the expressed scFv were detected through a myc-tag situated C-terminal to the scFv using 1 μg/ml of anti-c-myc monoclonal (9E10 Roche 1667 149) in wash buffer. As a secondary antibody Goat-anti-mouse alkaline phosphatase conjugate (Applied Biosystems Cat #AC32ML) was used at 25000 fold dilution. For luminescence detection CDP-Star Ready to use with Emerald II Tropix (Applied Biosystems Cat #MS100RY) were used according to suppliers recommendation.  
         [0045]    ScFv clones that bound MDA modified peptide but not native peptide were re expressed as described above and to screening another time in a luminescent ELISA (Table 2 and FIG. 1). Tests were run both against directly coated peptides (108 pmol/well coated with PBS) and the more physiological target, LDL particles (1 μg/well coated in PBS+1 mM EDTA) containing the ApoB-100 protein with and without MDA modification were used as targets. Positive clones were those that bound oxidised LDL and MDA modified peptide but not native LDL or peptide. The ELISA was performed as above except that the anti-His antibody (MaBO50 RaD) was used as the detection antibody. Twelve IEI clones and 2 KTT clones were found to give more than three fold higher luminescence signal at 700 nm for the MDA modified form than for the native form both for the peptide and LDL.  
         [0046]    The identified clones were further tested through titration against a fixed amount (1 μg/well) of MDA LDL and native LDL in order to evaluate the dose response of the scFv (FIG. 2).  
                                                                         TABLE 2                           Screening results. The number of clones tested in       each screening step for each target. The scored       hits in percent are shown within brackets.                Target                            IEI   KTT                            Screening   Tested Clones   1088   831           number 1   Scored Hits   64   33               (%)   (5.9%)   (4.0%)           Screening   Tested Clones   64   33           number 2   Scored Hits   12   2               (%)   (1.1%)   (0.2%)           Dose   Tested Clones   12   2           response   Scored Hits   8   2               (%)   (0.7%)   (0.2%)                      
 
         [0047]    The sequences of the chosen scFv clones were determined in order to find unique clones. Bacterial PCR was performed with the Boeringer Mannheim Expand kit using primers (5′-CCC AGT CAC GAC GTT GTA MA CG-3′) and (5′-GAA ACA GCT ATG AAA TAC CTA TTG C-3′) and a GeneAmp PCR system 9700 (PE Applied system) using the temperature cycling program 94° C. 5 min, 30 cycles of 94° C. 30 s, 52° C. for 30 s and 68° C. for 2 min and finally 5 min at 68 min. The sequencing reaction was performed with the bacterial PCR product (five fold diluted) as template, using Big Dye Terminator mix from PE Applied Biosystems and the GeneAmp PCR system 9700 (PE Applied system) and the temperature cycling program 25 cycles of 96° C. 10 s, 50° C. for 5 s and 60° C. for 4 min. The extension products were purified according to the supplier&#39;s instructions and the separation and detection of extension products was done by using a 3100 Genetic analyser (PE Applied Biosystems). The sequences were analysed by the in house computer program. From the sequence information homologous clones and clones with inappropriate restriction sites were excluded, leaving six clones for IgG conversion. The DNA sequence of the variable heavy (VH) and variable light (VL) domains of the finally selected clones are shown in FIG. 3.  
       EXAMPLE 2  
       [0048]    Transfer of Genes Encoding the Variable Parts of Selected scFv to Full Length Human IgG1 Vestors.  
         [0049]    Bacteria containing scFv clones to be converted to Ig-format were grown over night in LB supplemented with 100 μg/ml ampicillin. Plasmid DNA was prepared from over night cultures using the Quantum Prep, plasmid miniprep kit from Biorad (#732-6100). The DNA concentration was estimated by measuring absorbance at 260 nm, and the DNA was diluted to a concentration of 2 ng/μl. VH and VL from the different scFv-plasmids were PCR amplified in order to supply these segments with restriction sites compatible with the expression vectors (see below). 5′ primers contain a BsmI and 3′ primers contain a BsiWI restriction enzyme cleavage site (shown in italics). 3′ primers also contained a splice donor site (shown in bold).  
         [0050]    Primers for amplification of VH-segments:  
                                                                         (SEQ. ID. NO: 13)                    5′VH:   5′-GGTGTGCATTCCGAGGTGCAGCTGTTGGAG                            (SEQ. ID. NO: 14)                    3′VH:   5′-GACGTACG ACTCACCT GAGCTCACGGTGACCAG              
 
         [0051]    Primers for amplification of VL-segments:  
                                                                         (SEQ. ID. NO: 15)                    5′VL:   5′-GGTGTGCATTCCCAGTCTGTGCTGACTCAG                            (SEQ. ID. NO: 16)                    3′VL:   5′-GACGTACGTTCT ACTCACCT AGGACCGTCAGCTT              
 
         [0052]    PCR was conducted in a total volume of 50 μl, containing 10 ng template DNA, 0.4 μM 5′ primer, 0.4 μM 3′ primer and 0.6 mM dNTP (Roche, #1 969 064). The polymerase used was Expand long template PCR system (Roche #1 759 060), 3.5 u per reaction, together with each of the supplied buffers in 3 separate reactions. Each PCR amplification cycle consisted of a denaturing step at 94° C. for 30 seconds, an annealing step at 55° C. for 30 seconds, and an elongating step at 68° C. for 1.5 minutes. This amplification cycle was repeated 25 times. Each reaction began with a single denaturing step at 94° C. for 2 minutes and ended with a single elongating step at 68° C. for 10 minutes. The existence of PCR product was checked by agarose gel electrophoresis, and reactions containing the same amplified material (from reactions with different buffers) were pooled. The PCR amplification products were subsequently purified by spin column chromatography using S400-HR columns (Amersham-Pharmacia Biotech #27-5240-01).  
         [0053]    Four (4) μl of from each pool of PCR products were used for TOPO TA cloning (pCR 2.1 TOPO, InVitrogen #K4550-01) according to the manufacturers recommendations. Bacterial colonies containing plasmids with inserts were grown over night in LB supplemented with 100 μg/ml ampicillin and 20 μg/ml kanamycin. Plasmid DNA was prepared from over night cultures using the Quantum Prep, plasmid miniprep kit from Biorad (#732-6100). Plasmid preparations were purified by spin column chromatography using S400-HR columns (Amersham-Pharmacia Biotech #27-5240-01). Three plasmids from each individual VH and VL cloning were subjected to sequence analysis using BigDye Cycle Sequencing (Perkin Elmer Applied Biosystem, #4303150). The cycle sequencing program consisted of a denaturing step at 96° C. for 10 seconds, an annealing step at 50° C. for 15 seconds, and an elongating step at 60° C. for 4 minutes. This cycle was repeated 25 times. Each reaction began with a single denaturing step at 94° C. for 1 minute. The reactions were performed in a volume of 10 μl consisting of 1 μM primer (5′-CAGGMACAGCTATGAC), 3 μl plasmid DNA and 4 μl Big Dye reaction mix. The reactions were precipitated according to the manufacturers recommendations, and samples were run on a ABI PRISM 3100 Genetic Analyzer. Sequences were compared to the original scFv sequence using the alignment function of the OMIGA sequence analysis software (Oxford Molecular Ltd).  
         [0054]    Plasmids containing VH and VL segments without mutations were restriction enzyme digested. To disrupt the pCR 2.1 TOPO vector, plasmids were initially digested with DraI (Roche #1 417 983) at 37° C. for 2 hours. Digestions were heat inactivated at 70° C. for 20 minutes and purified by spin column chromatography using S400-HR columns (Amersham-Pharmacia Biotech #27-5240-01). The purified DraI digestions were subsequently digested with BsmI (Roche #1 292 307) and BsiWI (Roche #1 388 959) at 55° C. over night. Digestions were purified using phenol extraction and precipitation. The precipitated DNA was dissolved in 10 μl H 2 O and used for ligation.  
         [0055]    The expression vectors were obtained from Lars Norderhaug (J. Immunol. Meth. 204 (1997) 77-87). After some modifications, the vectors (FIG. 4) contain a CMV promoter, an Ig-leader peptide, a cloning linker containing BsmI and BsiWI restriction sites for cloning of VH/VL, genomic constant regions of IgG1(heavy chain (HC) vector) or lambda (light chain (LC) vector), neomycin (HC vector) or methotrexate (LC vector) resistance genes for selection in eukaryotic cells, SV40 and ColEI origins of replication and ampicillin (HC vector) or kanamycin (LC vector) resistance genes for selection in bacteria.  
         [0056]    The HC and LC vectors were digested with BsmI and BsiWI, phosphatase treated and purified using phenol extraction and precipitation. Ligation were set up at 16° C. over night in a volume of 10 μl, containing 100 ng digested vector, 2 μl digested VH/VL-pCR 2.1 TOPO vector (see above), 1 U T4 DNA ligase (Life Technologies, #15224-025) and the supplied buffer. 2 μl of the ligation mixture were subsequently transformed into 50 μl chemocompetent top10F′ bacteria, and plated on selective (100 μg/ml ampicillin or 20 μg/ml kanamycin) agar plates.  
         [0057]    Colonies containing HC/LC plasmids with VH/VL inserts were identified by colony PCR:  
                                           Forward primer:   5′-ATGGGTGACAATGACATC                           Reverse primer:   5′-AAGCTTGCTAGCGTACG          
 
         [0058]    PCR was conducted in a total volume of 20 μl, containing bacterias, 0.5 μM forward primer, 0.5 μM reverse primer and 0.5 mM dNTP (Roche, #1 969 064). The polymerase used was Expand long template PCR system (Roche #1 759 060), 0.7 U per reaction, together with the supplied buffer #3. Each PCR amplification cycle consisted of a denaturing step at 94° C. for 30 seconds, an annealing step at 52° C. for 30 seconds, and an elongating step at 68° C. for 1.5 minutes. This amplification cycle was repeated 30 times. Each reaction began with a single denaturing step at 94° C. for 2 minutes and ended with a single elongating step at 68° C. for 5 minutes. The existence of PCR product was checked by agarose gel electrophoresis. Colonies containing HC/LC plasmids with VH/VL inserts were grown over night in LB supplemented with 100 μg/ml ampicillin or 20 μg/ml kanamycin. Plasmid DNA was prepared from over night cultures using the Quantum Prep, plasmid miniprep kit from Biorad (#732-6100). Plasmid preparations were purified by spin column chromatography using S400-HR columns (Amersham-Pharmacia Biotech #27-5240-01). To confirm the integrity of the DNA sequence, three plasmids from each individual VH and VL were subjected to sequence analysis using BigDye Cycle Sequencing (Perkin Elmer Applied Biosystem, #4303150). The cycle sequencing program consisted of a denaturing step at 96° C. for 10 seconds, an annealing step at 50° C. for 15 seconds, and an elongating step at 60° C. for 4 minutes. This cycle was repeated 25 times. Each reaction began with a single denaturing step at 94° C. for 1 minute. The reactions were performed in a volume of 10 μl, consisting of 1 μM primer (5′-AGACCCAAGCTAGCTTGGTAC), 3 μl plasmid DNA and 4 μl Big Dye reaction mix. The reactions were precipitated according to the manufacturers recommendations, and samples were run on a ABI PRISM 3100 Genetic Analyzer. Sequences were analysed using the OMIGA sequence analysis software (Oxford Molecular Ltd). The plasmid DNA was used for transient transfection of COS-7 cells (see below) and were digested for production of a joined vector, containing heavy- and light chain genes on the same plasmid.  
         [0059]    Heavy and light chain vectors containing VH and VL segments originating from the same scFv were cleaved by restriction enzymes and ligated: HC- and LC-vectors were initially digested with MunI (Roche #1 441 337) after which digestions were heat inactivated at 70° C. for 20 minutes and purified by spin column chromatography using S200-HR columns (Amersham-Pharmacia Biotech #27-5120-01). HC-vector digestions were subsequently digested with NruI (Roche #776 769) and LC-vector digestions with Bst1107I (Roche #1 378 953). Digestions were then heat inactivated at 70° C. for 20 minutes and purified by spin column chromatography using S400-HR columns (Amersham-Pharmacia Biotech #27-5240-01). 5 μl of each digested plasmid were ligated at 16° C. over night in a total volume of 20 μl, contaning 2 U T4 DNA ligase (Life Technologies, #15224-025) and the supplied buffer. 2 μl of the ligation mixture were subsequently transformed into 50 μl chemocompetent top10F′ bacteria, and plated on selective (100 μg/ml ampicillin and 20 μg/ml kanamycin) agar plates.  
         [0060]    Bacterial colonies were grown over night in LB supplemented with 100 μg/ml ampicillin and 20 μg/ml kanamycin. Plasmid DNA was prepared from over night cultures using the Quantum Prep, plasmid miniprep kit from Biorad (#732-6100). Correctly joined vectors were identified by restriction enzyme digestion followed by analyses of fragment sizes by agarose gel-electrophoreses.  
         [0061]    Plasmid preparations were purified by spin column chromatography using S400-HR columns (Amersham-Pharmacia Biotech #27-5240-01) and used for transient transfection of COS-7 cells.  
         [0062]    COS-7 cells (ATCC #CRL-1651) were cultured at 37° C. with 5% CO 2  in Dulbeccos MEM, high glucose+GlutamaxI (Invitrogen #31966021), supplementd with 0.1 mM non-essential amino acids (Invitrogen #11140035) and 10% fetal bovine sera (Invitrogen #12476-024, batch #1128016). The day before transfection, the cells were plated in 12-well plates (Nunc, #150628) at a density of 1.5×10 5  cells per well.  
         [0063]    Prior to transfection, the plasmid DNA was heated at 70° C. for 15 minutes. Cells were transfected with 1 μg HC-plasmid+1 μg LC-plasmid, or 2 μg joined plasmid per well, using Lipofectamine 2000 Reagent (Invitrogen, #11668019) according to the manufacturers recommendations. 24 hours post transfection, cell culture media was changed and the cells were allowed to grow for 5 days. After that, medium was collected and protein production was assayed for using ELISA.  
         [0064]    Ninetysix (96)-well plates (Costar #9018, flat bottom, high binding) were coated at 4° C. over night by adding 100 μl/well rabbit anti-human lamda light chain antibody (DAKO, #A0193) diluted 4000 times in coatingbuffer (0.1M sodium carbonate, pH 9.5). Plates were washed 4 times in PBS containing 0.05% Tween 20 and thereafter blocked with 100 μl/well PBS+3% BSA (Albumin, fraction V, Roche #735108) for 1 h. at room temperature. After washing as above, 100 μl/well of sample were added and incubated in room temperature for 1 hour. As a standard for estimation of concentration, human purified IgG1 (Sigma, #I5029) was used. Samples and standard were diluted in sample buffer (1×PBS containing 2% BSA and 0.5% rabbit serum (Sigma #R4505). Subsequently, plates were washed as described above and 100 μl/well of rabbit anti-human IgG (y-chain) HRP-conjugated antibody (DAKO, #P214) diluted 8000 times in sample buffer was added and incubated at room temperature for 1 hour. After washing 8 times with PBS containing 0.05% Tween 20, 100 μl/well of a substrate solution containing one OPD tablet (10 mg, Sigma #P8287,) dissolved in 15 ml citric acid buffer and 4.5 μl H 2 O 2  (30%) was added. After 10 minutes, the reaction was terminated by adding 150 μl/well of 1M HCl. Absorbance was measured at 490-650 nm and data was analyzed using the Softmax software.  
         [0065]    Bacteria containing correctly joined HC- and LC-vectors were grown over night in 500 ml LB supplemented with ampicillin and kanamycin. Plasmid DNA was prepared from over night cultures using the Quantum Prep, plasmid maxiprep kit from Biorad (#732-6130). Vectors were linearized using PvuI restriction enzyme (Roche #650 129). Prior to transfection, the linearized DNA was purified by spin column chromatography using S400-HR columns (Amersham-Pharmacia Biotech #27-5240-01) and heated at 70° C. for 15 minutes.  
       EXAMPLE 3  
       [0066]    Stable Transfection of NSO Cells Expressing Antibodies Against MDA Modified Peptides frm Apolipoprotein B-100.  
         [0067]    NSO cells (ECACC no. 85110503) were cultured in DMEM (cat nr 31966-021, Invitrogen) supplemented with 10% Fetal Bovine Serum (cat no. 12476-024, lot: 1128016, Invitrogen) and 1×NEAA (non-essential amino acids, cat no. 11140-053, Invitrogen). Cell cultures are maintained at 37° C. with 5% CO 2  in humidified environment.  
         [0068]    DNA constructs to be transfected were four constructs of IEI specific antibodies (IEI-A8, IEI-D8, IEI-E3, IEI-G8), two of KTT specific antibodies (KTT-B8, KTT-D6) and one control antibody (JFPA12).The day before transfection, the cells were trypsinized and counted, before plating them in a T-75 flask at 12×10 6 cells/flask. On the day of transfection, when the cells were 85-90% confluent, the cells were plated in 15 ml DMEM+1×NEAA+10% FBS (as above). For each flask of cells to be transfected, 35-40 μg of DNA were diluted into 1.9 ml of OPTI-MEM I Reduced Serum Medium (Cat no. 51985-026, lot: 3062314, Invitrogen) without serum. For each flask of cells, 114 μl of Lipofectamine 2000 Reagent (Cat nr. 11668-019, lot: 1116546, Invitrogen) were diluted into 1.9 ml OPTI-MEM I Reduced Serum Medium in another tube and incubated for 5 min at room temperature. The diluted DNA was combined with the diluted Lipofectamine 2000 Reagent (within 30 min) and incubated at room temperature for 20 min to allow DNA-LF2000 Reagent complexes to form.  
         [0069]    The cells were washed with medium once and 11 ml DMEM +1×NEM +10% FBS were added. The DNA-LF2000 Reagent complexes (3.8 ml) were then added directly to each flask and gently mixed by rocking the flask back and forth. The cells were incubated at 37° C. in a 5% CO 2  incubator for 24 h.  
         [0070]    The cells were then trypsinized and counted, and subsequently plated in 96-well plates at 2×10 4  cells/well using five 96-well plates/construct. Cells were plated in 100 μl/well of DMEM+1×NEAA+10% FBS (as above) containing G418-sulphate (cat nr.10131-027, lot: 3066651, Invitrogen) at 600 μg/ml. The selection pressure was kept unchanged until harvest of the cells.  
         [0071]    The cells were grown for 12 days and assayed for antibody production using ELISA. From each construct cells from the 24 wells containing the highest amounts of IgG were transferred to 24-well plates and were allowed to reach confluency. The antibody production from cells in these wells was then assayed with ELISA and 5-21 pools/construct were selected for re-screening (Table 3). Finally cells from the best 1-4 wells for each construct were chosen. These cells were expanded successively in cell culture flasks and finally transferred into triple layer flasks (500 cm2) in 200 ml of (DMEM+1×NEAA+10% Ultra low IgG FBS (cat.no. 16250-078, lot.no. 113466, Invitrogen)+G418 (600 μg/ml)) for antibody production. The cells were incubated for 7-10 days and the supernatants were assayed by ELISA, harvested and sterile filtered for purification.  
       EXAMPLE 4  
       [0072]    Production and Purification of Human IgG1  
         [0073]    Supernatants from NSO cells transfected with the different IgG1 antibodies were sterile filtered using a 0.22 μm filter and purified using an affinity medium MabSelect™ with recombinant protein A, (Cat. No. 17519901 Amersham Biosciences).  
         [0074]    Bound human IgG1 was eluted with HCL-glycine buffer pH 2.8. The eluate was collected in 0.5 ml fractions and OD 280  was used to determine presence of protein. The peak fractions were pooled and absorbance was measured at 280 nm and 320 nm. Buffer was changed through dialysis against a large volume of PBS. The presence of endotoxins in the purified IgG-1 preparations was tested using a LAL test (QCL-1000R, cat. No. 50-647U Bio Whittaker). The samples contained between 1 and 12 EU/ml endotoxin. The purity of the preparations were estimated to exceed 98% by PAGE analysis.  
                                                           TABLE 3                           Summary of Production and Purification of human IgG1                Volume                       culture   Total IgG1 in   Total IgG1       Clone   supernatant   supernatant   Purified       name   (ml)   (mg)   (mg)   Yield (%)                    IEI-A8   600   68   42   61.8       IEI-D8   700   45   21   46.7       IEI-E3   700   44.9   25.6   60       IEI-G8   600   74   42.4   57.3       KTT-B8   1790   77.3   37.6   48.6       KTT-D6   1845   47.8   31.8   66.5       JFPA12   2000   32.2   19.2   59.6                  
 
         [0075]    The purified IgG1 preparations were tested in ELISA for reactivity to MDA modified and un-modified peptides (FIG. 5) and were then used in functional in vitro and in vivo studies.  
       EXAMPLE 5  
       [0076]    Analysis of Possible Anti-Atherogenic Effect of Antibodies are Performed both in Experimental Animals and in Cell Culture Studies.  
         [0077]    1. Effect of antibodies on atherosclerosis in apolipoprotein E knockout (apo E−) mice. Five weeks old apo E− mice are fed a cholesterol-rich diet for 15 weeks. This treatment is known to produce a significant amount of atherosclerotic plaques in the aorta and carotid arteries. The mice are then given an intraperitoneal injection containing 500 pg of the respective antibody identified above. Control mice are given 500 μg of an irrelevant control antibody or PBS alone. Treatments are repeated after 1 and 2 weeks. The mice are sacrificed 4 weeks after the initial antibody injection. The severity of atherosclerosis in the aorta is determined by Oil Red O staining of flat preparations and by determining the size of subvalvular atherosclerotic plaques. Collagen, macrophage and T cell content of subvalvular atherosclerotic plaques is determined by Masson trichrome staining and cell-specific immunohistochemistry. Quantification of Oil Red O staining, the size of the subvalvular plaques, trichrome staining and immunohistochemical staining is done using computer-based image analysis.  
         [0078]    In a first experiment the effect of the antibodies on development of atherosclerosis was analysed in apo E−/− mice fed a high-cholesterol diet. The mice were given three intraperitoneal injections of 0.5 mg antibody with week intervals starting at 21 weeks of age, using PBS as control. They were sacrificed two weeks after the last antibody injection, and the extent of atherosclerosis was assessed by Oil Red O staining of descending aorta flat preparations. A pronounced effect was observed in mice treated with the IEI-E3 antibody, with more than 50% reduction of atherosclerosis as compared to the PBS group (P=0.02) and to a control group receiving a human IgG1 antibody (FITC8) directed against a non-relevant fluorescein isothiocynate (FITC) antigen (P=0.03) (FIG. 6). The mice tolerated the human antibodies well and no effects on the general health status of the mice were evident.  
         [0079]    To verify the inhibitory effect of the IEI-E3 antibody on development of atherosclerosis we then performed a dose-response study. The schedule was identical to that of the initial study. In mice treated with IEI-E3 antibodies atherosclerosis was reduced by 2% in the 0.25 mg group (n.s.), by 25% in the 0.5 mg group (n.s.) and by 41% (P=0.02) in the 2.0 mg group as compared to the corresponding FITC antibody-treated groups (FIG. 7).  
         [0080]    2. Effect of antibodies on neo-intima formation following mechanical injury of carotid arteries in apo E− mice. Mechanical injury of arteries results in development of fibro-muscular neo-intimal plaque within 3 weeks. This plaque resembles morphologically a fibro-muscular atherosclerotic plaque and has been used as one model for studies of the development of raised lesion. Placing a plastic collar around the carotid artery causes the mechanical injury. Five weeks old apo E− mice are fed a cholesterol-rich diet for 14 weeks. The mice are then given an intraperitoneal injection containing 500 μg of the respective antibody. Control mice are given 500 μg of an irrelevant control antibody or PBS alone. The treatment is repeated after 7 days and the surgical placement of the plastic collar is performed 1 day later. A last injection of antibodies or PBS is given 6 days after surgery and the animals are sacrificed 15 days later. The injured carotid artery is fixed, embedded in paraffin and sectioned. The size of the neo-intimal plaque is measured using computer-based image analysis.  
         [0081]    3. Effect of antibodies on uptake of oxidized LDL in cultured human macrophages. Uptake of oxidized LDL in arterial macrophages leading to formation of cholesterol-loaded macrophage foam cells is one of the most characteristic features of the atherosclerotic plaque. Several lines of evidence suggest that inhibiting uptake of oxidized LDL in arterial macrophages represent a possible target for treatment of atherosclerosis. To study the effect of antibodies on macrophage uptake of oxidized c are pre-incubated with  125 I-labeled human oxidized LDL for 2 hours. Human macrophages are isolated from blood donor buffy coats by centrifugation in Ficoll hypaque followed by culture in presence of 10% serum for 6 days. The cells are then incubated with medium containing antibody/oxidized LDL complexes for 6 hours, washed and cell-associated radioactivity determined in a gamma-counter. Addition of IEI-E3 antibodies resulted in a five-fold increase in the binding (P=0.001) and uptake (P=0.004) of oxidized LDL compared to FITC-8 into macrophages, but had no effect on binding or uptake of native LDL (FIG. 8a and 8b).  
         [0082]    4. Effect of antibodies on oxidized LDL-dependent cytotoxicity. Oxidized LDL is highly cytotoxic. It is believed that much of the inflammatory activity in atherosclerotic plaques is explained by cell injury caused by oxidized LDL. Inhibition of oxidized LDL cytotoxicity thus represents another possible target for treatment of atherosclerosis. To study the effect of antibodies on oxidized LDL cytotoxicity cultured human arterial smooth muscle cells are exposed to 100 ng/ml of human oxidized LDL in the presence of increasing concentrations of antibodies (0-200 ng/ml) for 48 hours. The rate of cell injury is determined by measuring the release of the enzyme LDH.  
         [0083]    The experiment shown discloses an effect for a particular antibody raised against a particular peptide, but it is evident to the one skilled in the art that all other antibodies raised against the peptides disclosed will behave in the same manner.  
         [0084]    The antibodies of the present invention are used in pharmaceutical compositions for passive immunization, whereby the pharmaceutical compositions primarily are intended for injection, comprising a solution, suspension, or emulsion of a single antibody or a mixture of antibodies of the invention in a dosage to provide a therapeutically or prophylactically active level in the body treated. The compositions may be provided with commonly used adjuvants to enhance absorption of the antibody or mixture of antibodies. Other routes of administration may be the nasal route by inhaling the antibody/antibody mixture in combination with inhalable excipients.  
         [0085]    Such pharmaceutical compositions may contain the active antibody in an amount of 0.5 to 99.5% by weight, or 5 to 90% by weight, or 10 to 90% by weight, or 25 to 80% by weight, or 40 to 90% by weight.  
         [0086]    The daily dosage of the antibody, or a booster dosage shall provide for a therapeutically or prophylactically active level in the body treated to reduce or prevent signs and sympthoms of atherosclerosis by way of passive immunization. A dosage of antibody according to the invention may be 1 μg to 1 mg per kg bodyweight, or more.  
         [0087]    The antibody composition can be supplemented with other drugs for treating or preventing atherosclerosis or heart-vascular diseases, such as blood pressure lowering drugs, such as beta-receptor blockers, calcium antagonists, diurethics, and other antihypertensive agents.  
         [0088]    [0088]FIG. 9 shows binding of isolated scFv to MDA modified ApoB100 derived peptides and to a MDA modified control peptide of irrelevant sequence. Also depicted are the ratios between binding of the scFv to MDA modified and native ApoB100 protein and human LDL respectively. Columns appear in the order they are defined from top to bottom in right hand column of the respective subfigure.  
         [0089]    References  
         [0090]    Dimayuga, P., B. Cercek, et al. (2002). “Inhibitory effect on arterial injury-induced neointimal formation by adoptive B-cell transfer in Rag-1 knockout mice.”  Arteriosclerosis, Thrombosis and Vascular Biology  22: 644-649.  
         [0091]    Jovinge, S., M. Crisby, et al. (1997). “DNA fragmentation and ultrastructural changes of degenerating cells in atherosclerotic lesions and smooth muscle cells exposed to oxidized LDL in vitro.”  Arteriosclerosis, Thrombosis and Vascular Biology  17: 2225-2231.  
         [0092]    Regnström, J., G. Walldius, et al. (1990). “Effect of probucol treatment on suspectibility of low density lipoprotein isolated from hypercholesterolemic patients to become oxidativery modified in vitro.”  Atherosclerosis  82: 43-51.  
         [0093]    Steinberg, D., S. Parthasarathy, et al. (1989). “Beyond cholesterol modifications of low-density lipoprotein that increase its atherogenicity.”  New England Journal of Medicine  320(14): 915-924.  
         [0094]    Zhou, X., G. Paulsson, et al. (1998). “Hypercholesterolemia is associated with a T helper (Th) 1/Th2 switch of the autoimmune response in atherosclerotic apo E-knockout Mice.”  Journal of Clinical Investigation  101: 1717-1725.  
     
       
       
         1 
         
           
             79  
           
           
             1  
             369  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            1 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttcaat aacgcctgga tgagctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac    180 

gcagactcag tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagagtcagt    300 

aggtactact acggaccatc tttctacttt gactcctggg gccagggtac actggtcacc    360 

gtgagcagc                                                            369 

 
           
             2  
             336  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            2 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgctctg gaagcaggtc caacattggg aataattatg tatcctggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat ggtaacaaca atcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggtcattgg    300 

gtgttcggcg gaggaaccaa gctgacggtc ctaggt                              336 

 
           
             3  
             366  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            3 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcgg cctctggatt caccttcagt gactactaca tgagctgggt ccgccaggct    120 

cccgggaagg ggctggagtg ggtatcgggt gttagttgga atggcagtag gacgcactat    180 

gcagactctg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagagcggct    300 

aggtactcct actactacta cggtatggac gtctggggcc aaggtacact ggtcaccgtg    360 

agcagc                                                               366 

 
           
             4  
             327  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            4 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgttctg gaagcagctc caacatcgga aataatgctg taaactggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat gggaatgatc ggcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtcag acctggggca ctggccgggg ggtattcggc    300 

ggaggaacca agctgacggt cctaggt                                        327 

 
           
             5  
             366  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            5 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt cacctttagt agctattgga tgagctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcaagt atcagtggta gtggtcgtag gacatactac    180 

gcagactccg tgcagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagattggtc    300 

tcctatggtt cggggagttt cggttttgac tactggggcc aaggtacact ggtcaccgtg    360 

agcagc                                                               366 

 
           
             6  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            6 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcttgttctg gaagcagctc caatatcgga agtaattatg tatcctggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat ggtaactaca atcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgag tggttgggtg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             7  
             378  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            7 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttcagt aacgcctgga tgagctgggt ccgccaggtt    120 

ccagggaagg ggctggagtg ggtctcaact cttggtggta gtggtggtgg tagcacatac    180 

tacgcagact ccgtgaaggg ccggttcacc atctccagag acaattccaa gaacacgctg    240 

tatctgcaaa tgaacagcct gagagccgag gacactgccg tgtattactg tgcgaagtta    300 

ggggggcgat cccgatatgg gcggtggccc cgccaatttg actactgggg ccaaggtaca    360 

ctggtcaccg tgagcagc                                                  378 

 
           
             8  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            8 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgctctg gaagcagctc caacattgga aataactatg tatcctggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgag tcattggctg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             9  
             372  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            9 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttcagt gactactaca tgagctggat ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcaagt atcagtggcc gtgggggtag ttcctactac    180 

gcagactccg tgaggggccg gttcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagactttcc    300 

tacagctatg gttacgaggg ggcctactac tttgactact ggggccaggg tacactggtc    360 

accgtgagca gc                                                        372 

 
           
             10  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            10 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgctctg gaagcagctc caacattggg aataattatg tatcctggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat aggaataatc agcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc ttagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca acctgggatg acagcctgaa tggttgggtg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             11  
             363  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            11 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt cacctttagc agctatgcca tgagctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcatcc attagtagta gtggtcgttt catttactac    180 

gcagactcaa tgaagggccg cttcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtac gaggctccgg    300 

agagggagct acttctgggc ttttgatatc tggggccaag gtacactggt caccgtgagc    360 

agc                                                                  363 

 
           
             12  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            12 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgttctg gaagcagctc caacattggc ggtgagtctg tatcctggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat agtaataatc agcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggttgggtg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             13  
             30  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            13 

ggtgtgcatt ccgaggtgca gctgttggag                                      30 

 
           
             14  
             33  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            14 

gacgtacgac tcacctgagc tcacggtgac cag                                  33 

 
           
             15  
             30  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            15 

ggtgtgcatt cccagtctgt gctgactcag                                      30 

 
           
             16  
             34  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            16 

gacgtacgtt ctactcacct aggaccgtca gctt                                 34 

 
           
             17  
             18  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            17 

atgggtgaca atgacatc                                                   18 

 
           
             18  
             17  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            18 

aagcttgcta gcgtacg                                                    17 

 
           
             19  
             369  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            19 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt cacctttaga acgtattgga tgacctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcatct attagcagta gcagtaatta catattctac    180 

gcagactcag tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagactcaga    300 

cggagcagct ggtacggggg gtactggttc gacccctggg gccaaggtac actggtcacc    360 

gtgagctca                                                            369 

 
           
             20  
             336  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            20 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgctctg gaagcagctc caacattggg aataattatg tatcctggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat aggaataatc agcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggtcattgg    300 

gtgttcggcg gaggaaccaa gctgacggtc ctaggt                              336 

 
           
             21  
             369  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            21 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttcagt agcaactaca tgagctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac    180 

gcagactcag tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagagtaggc    300 

cggtataact ggaagacggg gcatgctttt gatatctggg gccagggtac actggtcacc    360 

gtgagctca                                                            369 

 
           
             22  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            22 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgctctg gaaggaccta caacattgga aataattatg tatcgtggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat ggtaacatca atcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg tcaggctgaa tggttgggtg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             23  
             381  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            23 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttccgt gactactacg tgagctggat ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcaagt attagtggta gtgggggtag gacatactac    180 

gcagactccg tggagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccatgt attactgtgc cagagtatcc    300 

gcccttcgga gacccatgac tacagtaact acttactggt tcgacccctg gggccaaggt    360 

acactggtca ccgtgagctc a                                              381 

 
           
             24  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            24 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgctctg gaaggagctc caacattggg aatagttatg tctcctggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat aggaataatc agcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca ggatgggatg acaccctgcg tgcttgggtg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             25  
             360  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            25 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttcagt aacgcctgga tgagctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctccgct attagtggta gtggtaacac atactatgca    180 

gactccgtga agggccggtt caccatctcc agagacaatt ccaagaacac gctgtatctg    240 

caaatgaaca gcctgagagc cgaggacact gccgtgtatt actgtgcgag agcctcccac    300 

cgtatattag gttatgcttt tgatatctgg ggccagggta cactggtcac cgtgagctca    360 

 
           
             26  
             328  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            26 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcttgttctg gaagccgctc caacatcggg agaaatgctg ttagttggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat gctaacagca atcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg gcagcctgaa tggttgggtg    300 

ttcggcggag gaaccaagct gacggtcc                                       328 

 
           
             27  
             363  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            27 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttcagt aacgcctgga tgagctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcaagt attagtgttg gtggacatag gacatattat    180 

gcagattccg tgaagggccg gtccaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc acggatacgg    300 

gtgggtccgt ccggcggggc ctttgactac tggggccagg gtacactggt caccgtgagc    360 

tca                                                                  363 

 
           
             28  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            28 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgctctg gaagcaacac caacattggg aagaactatg tatcttggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat gctaatagca atcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgcg tcatgggatg ccagcctgaa tggttgggta    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             29  
             378  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            29 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttcagt aacgcctgga tgagctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac    180 

gcagactcag tgaagggccg atccaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gaggctcaca    300 

aatattttga ctggttatta tacctcagga tatgcttttg atatctgggg ccaaggtaca    360 

ctggtcaccg tgagctca                                                  378 

 
           
             30  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            30 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgctctg gaagcacctc caacattggg aagaattatg tatcctggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat ggtaacagca atcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg ccagcctcag tggttgggtg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             31  
             363  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            31 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttcagt agttcttgga tgagttgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac    180 

gcagactcag tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagagtaggg    300 

aactacggtt tctaccacta catggacgtc tggggccaag gtacactggt caccgtgagc    360 

tca                                                                  363 

 
           
             32  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            32 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcttgttctg gaggcagctc aaacatcgga aaaagaggtg taaattggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat ggtaacagaa atcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgct acatgggatt acagcctcaa tgcttgggtg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             33  
             366  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            33 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt cacctttagt agctattgga tgagctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac    180 

gcagactcag tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagaattaaa    300 

cggttacgat tcggctggac cccttttgac tactggggcc agggtacact ggtcaccgtg    360 

agctca                                                               366 

 
           
             34  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            34 

cagtctgttc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgttctg gaagcagctc caacatcgga aataatggtg taaactggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat ggtaacaaca atcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgcg tggttggctg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             35  
             369  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            35 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttcagt aacgcctgga tgagctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcatcc attagtagta gtagtagtta catatactac    180 

gcagactcag tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagagtcaat    300 

agcaaaaagt ggtatgaggg ctacttcttt gactactggg gccagggtac actggtcacc    360 

gtgagctca                                                            369 

 
           
             36  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            36 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgctctg gaagcagctc caacattggg aataattatg tatcctggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat ggtaacagca atcggccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg acagtctgag tggttgggtg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             37  
             375  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            37 

gaggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgagactc     60 

tcctgtgcag cctctggatt caccttcagt aacgcctgga tgagctgggt ccgccaggct    120 

ccagggaagg ggctggagtg ggtctcatcc attagtacta gtagtaatta catatactac    180 

gcagactcag tgaagggccg gttcaccatc tccagagaca attccaagaa cacgctgtat    240 

ctgcaaatga acagcctgag agccgaggac actgccgtgt attactgtgc gagagtcaag    300 

aagtatagca gtggctggta ctcgaattat gcttttgata tctggggcca aggtacactg    360 

gtcaccgtga gctca                                                     375 

 
           
             38  
             333  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      nucleotide sequence  
             
           
            38 

cagtctgtgc tgactcagcc accctcagcg tctgggaccc ccgggcagag ggtcaccatc     60 

tcctgctctg gaagcagctc cagcattggg aataattttg tatcctggta tcagcagctc    120 

ccaggaacgg cccccaaact cctcatctat gacaataata agcgaccctc aggggtccct    180 

gaccgattct ctggctccaa gtctggcacc tcagcctccc tggccatcag tgggctccgg    240 

tccgaggatg aggctgatta ttactgtgca gcatgggatg acagcctgaa tggttgggtg    300 

ttcggcggag gaaccaagct gacggtccta ggt                                 333 

 
           
             39  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            39 

Phe Leu Asp Thr Val Tyr Gly Asn Cys Ser Thr His Phe Thr Val Lys 
  1               5                  10                  15 

Thr Arg Lys Gly 
             20 

 
           
             40  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            40 

Pro Gln Cys Ser Thr His Ile Leu Gln Trp Leu Lys Arg Val His Ala 
  1               5                  10                  15 

Asn Pro Leu Leu 
             20 

 
           
             41  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            41 

Val Ile Ser Ile Pro Arg Leu Gln Ala Glu Ala Arg Ser Glu Ile Leu 
  1               5                  10                  15 

Ala His Trp Ser 
             20 

 
           
             42  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            42 

Lys Leu Val Lys Glu Ala Leu Lys Glu Ser Gln Leu Pro Thr Val Met 
  1               5                  10                  15 

Asp Phe Arg Lys 
             20 

 
           
             43  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            43 

Leu Lys Phe Val Thr Gln Ala Glu Gly Ala Lys Gln Thr Glu Ala Thr 
  1               5                  10                  15 

Met Thr Phe Lys 
             20 

 
           
             44  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            44 

Asp Gly Ser Leu Arg His Lys Phe Leu Asp Ser Asn Ile Lys Phe Ser 
  1               5                  10                  15 

His Val Glu Lys 
             20 

 
           
             45  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            45 

Lys Gly Thr Tyr Gly Leu Ser Cys Gln Arg Asp Pro Asn Thr Gly Arg 
  1               5                  10                  15 

Leu Asn Gly Glu 
             20 

 
           
             46  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            46 

Arg Leu Asn Gly Glu Ser Asn Leu Arg Phe Asn Ser Ser Tyr Leu Gln 
  1               5                  10                  15 

Gly Thr Asn Gln 
             20 

 
           
             47  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            47 

Ser Leu Thr Ser Thr Ser Asp Leu Gln Ser Gly Ile Ile Lys Asn Thr 
  1               5                  10                  15 

Ala Ser Leu Lys 
             20 

 
           
             48  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            48 

Thr Ala Ser Leu Lys Tyr Glu Asn Tyr Glu Leu Thr Leu Lys Ser Asp 
  1               5                  10                  15 

Thr Asn Gly Lys 
             20 

 
           
             49  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            49 

Asp Met Thr Phe Ser Lys Gln Asn Ala Leu Leu Arg Ser Glu Tyr Gln 
  1               5                  10                  15 

Ala Asp Tyr Glu 
             20 

 
           
             50  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            50 

Met Lys Val Lys Ile Ile Arg Thr Ile Asp Gln Met Gln Asn Ser Glu 
  1               5                  10                  15 

Leu Gln Trp Pro 
             20 

 
           
             51  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            51 

Ile Ala Leu Asp Asp Ala Lys Ile Asn Phe Asn Glu Lys Leu Ser Gln 
  1               5                  10                  15 

Leu Gln Thr Tyr 
             20 

 
           
             52  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            52 

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 

 
           
             53  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            53 

Glu Glu Glu Met Leu Glu Asn Val Ser Leu Val Cys Pro Lys Asp Ala 
  1               5                  10                  15 

Thr Arg Phe Lys 
             20 

 
           
             54  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            54 

Gly Ser Thr Ser His His Leu Val Ser Arg Lys Ser Ile Ser Ala Ala 
  1               5                  10                  15 

Leu Glu His Lys 
             20 

 
           
             55  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            55 

Ile Glu Asn Ile Asp Phe Asn Lys Ser Gly Ser Ser Thr Ala Ser Trp 
  1               5                  10                  15 

Ile Gln Asn Val 
             20 

 
           
             56  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            56 

Ile Arg Glu Val Thr Gln Arg Leu Asn Gly Glu Ile Gln Ala Leu Glu 
  1               5                  10                  15 

Leu Pro Gln Lys 
             20 

 
           
             57  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            57 

Glu Val Asp Val Leu Thr Lys Tyr Ser Gln Pro Glu Asp Ser Leu Ile 
  1               5                  10                  15 

Pro Phe Phe Glu 
             20 

 
           
             58  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            58 

His Thr Phe Leu Ile Tyr Ile Thr Glu Leu Leu Lys Lys Leu Gln Ser 
  1               5                  10                  15 

Thr Thr Val Met 
             20 

 
           
             59  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            59 

Leu Leu Asp Ile Ala Asn Tyr Leu Met Glu Gln Ile Gln Asp Asp Cys 
  1               5                  10                  15 

Thr Gly Asp Glu 
             20 

 
           
             60  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            60 

Cys Thr Gly Asp Glu Asp Tyr Thr Tyr Lys Ile Lys Arg Val Ile Gly 
  1               5                  10                  15 

Asn Met Gly Gln 
             20 

 
           
             61  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            61 

Gly Asn Met Gly Gln Thr Met Glu Gln Leu Thr Pro Glu Leu Lys Ser 
  1               5                  10                  15 

Ser Ile Leu Lys 
             20 

 
           
             62  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            62 

Ser Ser Ile Leu Lys Cys Val Gln Ser Thr Lys Pro Ser Leu Met Ile 
  1               5                  10                  15 

Gln Lys Ala Ala 
             20 

 
           
             63  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            63 

Ile Gln Lys Ala Ala Ile Gln Ala Leu Arg Lys Met Glu Pro Lys Asp 
  1               5                  10                  15 

Lys Asp Gln Glu 
             20 

 
           
             64  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            64 

Arg Leu Asn Gly Glu Ser Asn Leu Arg Phe Asn Ser Ser Tyr Leu Gln 
  1               5                  10                  15 

Gly Thr Asn Gln 
             20 

 
           
             65  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            65 

Ser Leu Asn Ser His Gly Leu Glu Leu Asn Ala Asp Ile Leu Gly Thr 
  1               5                  10                  15 

Asp Lys Ile Asn 
             20 

 
           
             66  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            66 

Trp Ile Gln Asn Val Asp Thr Lys Tyr Gln Ile Arg Ile Gln Ile Gln 
  1               5                  10                  15 

Glu Lys Leu Gln 
             20 

 
           
             67  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            67 

Thr Tyr Ile Ser Asp Trp Trp Thr Leu Ala Ala Lys Asn Leu Thr Asp 
  1               5                  10                  15 

Phe Ala Glu Gln 
             20 

 
           
             68  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            68 

Glu Ala Thr Leu Gln Arg Ile Tyr Ser Leu Trp Glu His Ser Thr Lys 
  1               5                  10                  15 

Asn His Leu Gln 
             20 

 
           
             69  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            69 

Ala Leu Leu Val Pro Pro Glu Thr Glu Glu Ala Lys Gln Val Leu Phe 
  1               5                  10                  15 

Leu Asp Thr Val 
             20 

 
           
             70  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            70 

Ile Glu Ile Gly Leu Glu Gly Lys Gly Phe Glu Pro Thr Leu Glu Ala 
  1               5                  10                  15 

Leu Phe Gly Lys 
             20 

 
           
             71  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            71 

Ser Gly Ala Ser Met Lys Leu Thr Thr Asn Gly Arg Phe Arg Glu His 
  1               5                  10                  15 

Asn Ala Lys Phe 
             20 

 
           
             72  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            72 

Asn Leu Ile Gly Asp Phe Glu Val Ala Glu Lys Ile Asn Ala Phe Arg 
  1               5                  10                  15 

Ala Lys Val His 
             20 

 
           
             73  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            73 

Gly His Ser Val Leu Thr Ala Lys Gly Met Ala Leu Phe Gly Glu Gly 
  1               5                  10                  15 

Lys Ala Glu Phe 
             20 

 
           
             74  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            74 

Phe Lys Ser Ser Val Ile Thr Leu Asn Thr Asn Ala Glu Leu Phe Asn 
  1               5                  10                  15 

Gln Ser Asp Ile 
             20 

 
           
             75  
             20  
             PRT  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      peptide  
             
           
            75 

Phe Pro Asp Leu Gly Gln Glu Val Ala Leu Asn Ala Asn Thr Lys Asn 
  1               5                  10                  15 

Gln Lys Ile Arg 
             20 

 
           
             76  
             23  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            76 

cccagtcacg acgttgtaaa acg                                             23 

 
           
             77  
             25  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            77 

gaaacagcta tgaaatacct attgc                                           25 

 
           
             78  
             17  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            78 

caggaaacag ctatgac                                                    17 

 
           
             79  
             21  
             DNA  
             Artificial Sequence  
             
               Description of Artificial Sequence Synthetic 
      primer  
             
           
            79 

agacccaagc tagcttggta c                                               21