AU2003267905B2 - Peptide-based passive immunization therapy for treatment of atherosclerosis - Google Patents
Peptide-based passive immunization therapy for treatment of atherosclerosis Download PDFInfo
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- 229960002180 tetracycline Drugs 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Heart & Thoracic Surgery (AREA)
- Public Health (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Cardiology (AREA)
- Pharmacology & Pharmacy (AREA)
- Vascular Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Urology & Nephrology (AREA)
- General Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Peptides Or Proteins (AREA)
Description
00 -1-
O
TITLE
C.)
0 PEPTIDE-BASED PASSIVE IMMUNIZATION THERAPY FOR TREATMENT OF ATHEROSCLEROSIS
DESCRIPTION
Technical field The present invention relates to new isolated human antibodies raised against peptides being derivatives ofapolipoprotein B. In particular, antibodies to be used for N, 10 immunization therapy for treatment ofatherosclerosis, methods for their preparation, and methods for passive immunization using said antibodies.
In particular, the invention includes: 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 Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.
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 Blymphocytes. Blood contains many different antibodies, each derived from a clone of Bcells 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.
00 -la- All antibodies are similar in their overall structure, accounting for certain similarities in ophysico-chemical features such as charge and solubility. All antibodies have a common
O
core structure of two identical light chains, each about 24 kilo Daltons, and two identical
(N
O 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- WO 2004/030607 PCT/SE2003/001547 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 antiparallel polypeptide chains.
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, 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.
There are between 108 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 regions, to distinguish them from the more conserved constant regions.
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.
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.
WO 2004/030607 PCTISE2003/001547 Atherosclerosis represents in many respects a response to this injury including inflammation and fibrosis.
In 1989 Palinski and coworkers identified circulating autoantibodies against oxidized LDL in s 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 cellindependent IgM antibodies were more frequent than T-cell dependent IgG antibodies.
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 reproducible components of a vaccine if one should use intact oxidized LDL particles.
Another way to investigate the possibility that autoimmune reactions against oxidized LDL in the vascular wall play a key role in the development of atherosclerosis 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 progression of atherosclerosis. To test this hypothesis rabbits were immunized with homologous oxidized LDL and then induced atherosclerosis by feeding the animals a high-cholesterol diet for 3 months.
However, in contrast to the original hypothesis immunization with oxidized LDL had a protective effect reducing atherosclerosis with about 50%. Similar results were also obtained in a subsequent study in which the high-cholesterol diet was combined with vascular ballooninjury 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.
WO 2004/030607 PCTISE2003/001547 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.
The identification of epitopes in oxidized LDL is important for several aspects: First, one or several of these epitopes are likely to be responsible for activating the antiatherogenic 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.
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.
US patent 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 atherosclerotic plaque. The concentration of radioactivity in the wall of the vessel can then be determined 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.
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 WO 2004/030607 PCT/SE2003/001547 increase of the development of atherosclerosis. Immunisation using other peptide sequences, peptide sequences and 30 to 34 lacks total effect on the development of atherosclerosis. 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.
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.
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 cross reactivity, isotype, affinity and plasma half-life. The possibility to develop antibodies with predetermined properties became apparent already with the advent of the monoclonal antibody technology (Milstein and Kohler, 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.
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 WO 2004/030607 PCT/SE2003/001547 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).
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.
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 (US 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 desirable characteristics may be transferred into yet other formats such as full length antibodies.
Recently a novel technology for generation of variability in antibody libraries was presented (W098/32845, Soderlind et al., 2000, Nature BioTechnol. 18:852-856).
WO 2004/030607 PCT/SE2003/001547 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 libraries 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.
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. 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 (US 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 (US 6,225,070). Moreover, the resulting antibodies are fully human and are not expected to generate any undesired immunological reaction when administered into patients.
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 WO 20)04/030607 P 103. TASLKYENYELTLKSDTNGK P 105. DMTFSKQNALLRSEYQADYE P 177. MKVKIIRTIDQMQNSELQWP C. High IgG, no MDA difference P 143. IALDDAKINFNEKLSQLQTY P 210. KTTrKQSFDLSVKAQYKKNKH D. NHS/AHP, [gG-ak 2, MDA-difterence P1. EEEMLENVSLVCPKDATRFK P 129. GSTSHHLVSRKSISMALEHK P 148. IENIDFNKSGSSTASWIQNV P 162. IREVTQRLNGEIQALELPQK P 252. EVDVLTKYSQPEDSLIPFFE E. NHS/AHP, IgM-ak 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 O.5,no MDA-difference P i0. ALLVPPETEEAKQVLFLDTV P 45. [EIGLEGKGFEPTLEALFGK p ill. SGASMKLTTNGRFREHNAKF P 154. NLIGDFEVAEKINAFRAKVH p 199. GHSVLTAKGMALFGEGKAEF P 222. FKSSVITLNTNAELFNQSDI P 240. FPDLGQEVALNANTKNQKIR or an active site of one or more of these peptidles.
In Table 1 above, the following is due: PCTSE2003OO 1547 -9- Fragments that produce high levels of IgG antibodies to MDA-modified peptides S(B) Fragments that produce high levels of IgM antibodies, but no difference between native and MDA-modified peptides C Fragments that produce high levels of IgG antibodies, but no difference between native and MDA-modified peptides Fragments that produce high levels of IgG antibodies to MDA-modified peptides and at least twice as much antibodies in the NHP-pool as compared to the AHP-pool N Fragments that produce high levels of IgM antibodies to MDA-modified peptides and at least twice as much antibodies in the NHP-pool as compared to the AHP-pool (n=l and Fragments that produce high levels of IgG antibodies, but no difference between intact and MDA-modified peptides but at least twice as much antibodies in the AHP-pool as compared to the NHP-pool Summary of the invention Use of at least one isolated human antibody or antibody fragment 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, wherein the oxidized fragment is IEIGLEGKGFEPTLEALFGK and wherein the antibody comprises a variable heavy region (VH) selected from the group of nucleic acid sequences consisting of:
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAATAACGCCTGGATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTA
GTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGTCAGTAGGTACTACT
ACGGACCATCTTTCTACTTTGACTCCTGGGGCCAGGGTACACTGGTCACCGT
GAGCAGC (SEQ. ID. NO. 1);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTCAGTGACTACTACATGA
9a
GCTGGGTCCGCCAGGCTCCCGGGAAGGGGCTGGAGTGGGTATCGGGTGTTA
GTTGGAATGGCAGTAGGACGCACTATGCAGACTCTGTGAAGGGCCGATTCA
CCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCT
c-I GAGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGCGGCTAGGTACTC
CTACTACTACTACGGTATGGACGTCTGGGGCCAAGGTACACTGGTCACCGTG
AGCAGC (SEQ. ID. NO. 3);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAG
c-i 1 CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATCAG
TGGTAGTGGTCGTAGGACATACTACGCAGACTCCGTGCAGGGCCGGTTCACC
ATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACTGCCGTGTATTACTGTGCGAGATTGGTCTCCTATGGTTC
GGGGAGTTTCGGTTTTGACTACTGGGGCCAAGGTACACTGGTCACCGTGAGC
AGC(S EQ. ID. NO.
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAciTGGGTCTCAACTCTTGG
TGGTAGTGGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTC
ACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC
TGAGAGCCGAGGACACTGCCGTGTATTACTGTGCGAAGTTAGGGGGGCGAT
CCCGATATGGGCGGTGGCCCCGCCAATTTGACTACTGGGGCCAAGGTACACT
GGTCACCGTGAGCAGC (SEQ. ID. NO. 7);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGAACGTATTGGATGA
CCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAG
CAGTAGCAGTAATTACATATTCTACGCAGACTCAGTGAAGGGCCGATTCACC
ATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACTGCCGTGTATTACTGTGCGAGACTCAGACGGAGCAGCT
GGTACGGGGGGTACTGGTTCGACCCCTGGGGCCAAGGTACACTGGTCACCGT
GAGCTCA (SEQ. ID. NO. 19); 9b
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCAACTACATGA
GCTGGGTCCGCCAGGCTCGAGGGAAGGGGCTGGAGTGGGTCTCATCCATTA
GTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGTAGGCCGGTATAAC
TGGAAGACGGGGCATGCTTTTGATATCTGGGGCCAGGGTACACTGGTCACCG
TGAGCTCA (SEQ. ID. NO. 21);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCCGTGACTACTACGTGAG
CTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTAG
TGGTAGTGGGGGTAGGACATACTACGCAGACTCCGTGGAGGGCCGGTTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGGAAATGAACAGCCTG
AGAGCCGAGGACACTGCCATGTATTAGTGTGCCAGAGTATCCGCCCTTCGGA
GACCCATGACTACAGTAACTACTTACTGGTTCGACCCCTGGGGCCAAGGTAC
ACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 23);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCCGCTATTA
GTGGTAGTGGTAACACATACTATGCAGACTCGGTGAAGGGCCGGTTCACCAT
CTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAG
AGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGCCTCCCACCGTATATTA
GGTTATGCTTTTGATATCTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA
(SEQ. ID. NO.
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTA
GTGTTGGTGGACATAGGACATATTATGCAGATTCCGTGAAGGGCCGGTCCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
9c
AGAGCCGAGGACACTGCCGTGTATTACTGTGCACGGATACGGGTGGGTCCGT
CCGGCGGGGCCTTTGACTACTGGGGCCAGGGTACACTGGTCACCGTGAGCTC
A (SEQ. ID. NO. 27);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTA
GTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATCCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGGCTCACAAATATTTTGA
CTGGTTATTATACCTCAGGATATGCTTTTGATATCTGGGGCCAAGGTACACT
GGTCACCGTGAGCTCA (SEQ. ID. NO. 29);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTCTTGGATGAG
TTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGT
AGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCA
TCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAG
AGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGTAGGGAACTACGGTTTC
TACCACTACATGGACGTCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA
(SEQ. ID. NO. 3 1);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAG
CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGT
AGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCA
TCTCCAGAGACAATTCCAAGAACAGGCTGTATCTGCAAATGAACAGCCTGAG
AGCCGAGGACACTGCCGTGTATTACTGTGCGAGAATTAAACGGTTACGATTC
GGCTGGACCCCTTTTGACTACTGGGGCCAGGGTACACTGGTCACCGTGAGCT
CA (SEQ. ID. NO. 33);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
9d
GCTGGGTCGGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTA
GTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGTCAATAGCAAAAAG
TGGTATGAOGGCTACTTCTTTGACTACTGGGGCCAGGGTACACTGGTCACCG
TGAGCTCA (SEQ. ID. NO. 35); and
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTA
GTACTAGTAGTAATTACATATACTACGCAGACTCAGTGAAGGGCCGGTTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCGAGGACACTGCCGTGTATTACTGTGCGAGAGTCAAGAAGTATAGCA
GTGGCTGGTACTCGAATTATGCTTTTGATATCTGGGGCCAAGGTACACTGGT
CACCGTGAGCTCA (SEQ. ID. NO. 37) and/or a variable light region (VL) selected from the group of nucleic acid sequences consisting of:
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTGTGGAAGCAGGTCCAACATTGGGAATAATTATGTATC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
AACAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGACAGCCTGAATGGTCATTGGGTGTTCGGCGGA
GGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 2); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGcjGCAGAGGG
TCACCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGCTGTAAA
CTGGTATCAGCAGGTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGGAAT
GATCGGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTGAGCCTCCCTGGGCATCAGTGGGCTCCGGTCCGAcjGATGAGGCTGATTA 9e
TTAGTGTCAGACCTGGGGCACTGGCCGGGGGGTATTCGGCGGAGGAACCAA
GCTGACGGTCCTAGGT (SEQ. ID. NO. 4);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTGTTGTTCTGGAAGCAGCTCCAATATCGGAAGTAATTATGTATCC
TGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACT
ACAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCAC
CTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTAT
TACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 6);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGAAATAACTATGTATC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTAAT
AATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGACAGCCTGAGTCATTGGCTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 8);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGGAGAGGG
TCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATC
GTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAAT
AATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGACAGCCTGAATGGTCATTGGGTGTTCGGCGGA
GGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO.
CAGTCTGTGCTGACTCAGCGACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCGTGCTCTGGAAGGACCTACAACATTGGAAATAATTATGTATC
GTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
ATCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
9f
TTACTGTGCAGCATGGGATGTCAGGCTGAATGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 22);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGGAGCTCCAACATTGGGAATAGTTATGTCTC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAAT
AATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGGATGGGATGACACCCTGCGTGCTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 24);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCTTGTTCTGGAAGCCGCTCCAACATCGGGAGAAATGCTGTTAG
TTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGCTAAC
AGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGGCAGCCTGAATGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCC (SEQ. ID. NO. 26);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCAACACCAACATTGGGAAGAACTATGTATC
TTGGTATCAGCAGCTCCCAGGAAGGGCCCCGAAACTCCTGATCTATGCTAAT
AGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCGTCATGGGATGCCAGCCTGAATGGTTGGGTATTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 28);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCACCTCCAACATTGGGAAGAATTATGTATC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
AGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
9g
TTACTGTGCAGCATGGGATGCCAGCCTCAGTGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO.
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCTTGTTCTGGAGGCAGCTCAAACATCGGAAAAAGAGGTGTAAA
TTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
AGAAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCTACATGGGATTACAGCCTCAATGCTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 32);
CAGTCTGTTCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGGTGTAAA
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
1 5 AACAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGACAGCCTGCGTGGTTGGCTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 34);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCAGGTCCAACATTGGGAATAATTATGTATC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
AGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGACAGTCTGAGTGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 36); and
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCAGCTCCAGCATTGGGAATAATTTTGTATGC
TGGTATCAGCAGCTCCCAGGAACGGCCCCCAAAGTCCTCATCTATGACAATA
ATAAGCGACCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCAC
CTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTAT
9h
TACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 38).
According to a second aspect, the present invention provides an antibody or antibody fragment thereof.
According to a third aspect, the present invention provides a method for preparation of an isolated antibody according to the invention, using a fragment library to generate specific human antibody fragments against an oxidized, MDA modified peptide derived from Apo B100 as defined in the invention, whereupon identified antibody fragments having the desired characteristics are rebuilt into a full length human immunoglobulin to be used for therapeutic purposes.
According to a fourth aspect, the present invention provides a method for amplification of an isolated human antibody according to the invention, using a recombinant technology, comprising transfer of genes encoding the variable parts of a selected scFv into full length human IgG1 vectors using a PCR-amplification wherein the primers used are: primers for amplification of VH-segments: 5'-GGTGTGCATTCCGAGGTGCAGCTGTTGGAG (SEQ. ID. NO: 13), and 3'VH: 5'-GACGTACGACTCACCTGAGCTCACGGTGACCAG (SEQ. ID. NO: 14) and primers for amplification of VL-segments: 5'-GGTGTGCATTCCCAGTCTGTGCTGACTCAG (SEQ. ID. NO: 15), and 3'VL: 5'-GACGTACGTTCTACTCACCTAGGACCGTCAGCTT (SEQ. ID. NO: 16).
According to a fifth aspect, the present invention provides a method for passive immunization of a mammal comprising the administration of a therapeutically or prophylactically effective amount of an isolated human antibody according to the invention to a mammal in need thereof.
9i- According to a sixth aspect, the present invention provides a pharmaceutical composition comprising an isolated human antibody directed towards at least one oxidized fragment of apolipoprotein B according to the invention for therapeutical or prophylactical treatment of atherosclerosis by means of passive immunization, which antibody is present in combination with a pharmaceutical excipient.
According to a seventh aspect, the present invention provides an antibody or antibody fragment thereof according to the invention for use in medicine.
According to an eighth aspect, the present invention provides an antibody or antibody fragment thereof according to the invention for the therapeutic or prophylactic treatment of atherosclerosis by means of passive immunisation.
According to a ninth aspect, the present invention provides a method for the therapeutical or prophylactical treatment of atherosclerosis by means of passive immunization, comprising administering to a patient in need thereof an effective amount of at least one isolated human antibody or antibody fragment directed towards at least one oxidized fragment of apolipoprotein B, wherein the oxidized fragment is IEIGLEGKGFEPTLEALFGK and wherein the antibody comprises a variable heavy region (VH) selected from the group of nucleic acid sequences consisting of:
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAATAACGCCTGGATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTA
GTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGTCAGTAGGTACTACT
ACGGACCATCTTTCTACTTTGACTCCTGGGGCCAGGGTACACTGGTCACCGT
GAGCAGC (SEQ. ID. NO. 1);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCGGCCTCTGGATTCACCTTCAGTGACTACTACATGA
GCTGGGTCCGCCAGGCTCCCGGGAAGGGGCTGGAGTGGGTATCGGGTGTTA
9j
GTTGGAATGGCAGTAGGACGCACTATGCAGACTCTGTGAAGGGCCGATTCA
CCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCT
GAGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGCGGCTAGGTACTC
CTACTACTACTACGGTATGGACGTCTGGGGCCAAGGTACACTGGTCACCGTG
AGCAGC (SEQ. ID. NO. 3);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAG
CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATCAG
TGGTAGTGGTCGTAGGACATACTACGCAGACTCCGTGCAGGGCCGGTTCACC
ATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACTGCCGTGTATTACTGTGCGAGATTGGTCTCCTATGGTTC
GGGGAGTTTCGGTTTTGACTACTGGGGCCAAGGTACACTGGTCACCGTGAGC
AGC(SEQ. ID. NO.
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTCTTGG
TGGTAGTGGTGGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTC
ACCATGTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCC
TGAGAGCCGAGGACACTGCCGTGTATTACTGTGCGAAGTTAGGGGGGCGAT
CCCGATATGGGCGGTGGCCGGGCCAATTTGACTACTGGGGCGAAGGTAGACT
GGTCACCGTGAGCAGC (SEQ. ID. NO. 7);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGAACGTATTGGATGA
CCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAG
CAGTAGCAGTAATTACATATTCTACGCAGACTCAGTGAAGGGCCGATTCACC
ATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGA
GAGCCGAGGACACTGCCGTGTATTACTGTGCGAGACTCAGACGGAGCAGCT
GGTACGGGGGGTACTGGTTCGACCCCTGGGGCCAAGGTACACTGGTCACCGT
GAGCTCA (SEQ. ID. NO. 19); 9k
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCAACTACATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTA
GTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGGCGATTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACTGCCGTGTATTACTGTGGGAGAGTAGGCCGGTATAAC
TGGAAGACGGGGCATGCTTTTGATATCTGGGGCCAGGGTACACTGGTCACCG
TGAGCTCA (SEQ. ID. NO. 21); io GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCCGTGACTACTACGTGAG
CTGGATCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTAG
TGGTAGTGGGGGTAGGACATACTACGCAGACTCCGTGGAGGGCCGGTTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACTGCCATGTATTACTGTGCCAGAGTATCCGCCCTTCGGA
GACCCATGACTACAGTAACTACTTACTGGTTCGACCCCTGGGGCCAAGGTAC
ACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 23);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCCGCTATTA
GTGGTAGTGGTAACACATACTATGCAGAGTCCGTGAAGGGCCGGTTGACCAT
CTCGAGAGACAATTCCAAGAACACGGTGTATCTGCAAATGAACAGCCTGAG
AGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGCCTCCCACCGTATATTA
GGTTATGCTTTTGATATCTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA
(SEQ. ID. NO.
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTGC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTA
GTGTTGGTGGACATAGGACATATTATGCAGATTCCGTGAAGGGCCGGTCCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACTGCCGTGTATTACTGTGCACGGATACGGGTGGGTCCGT
91
CCGGCGGGGCCTTTGACTACTGGGGCCAGGGTACACTGGTCACCGTGAGCTC
A (SEQ. ID. NO. 27);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGGCTGGGGGGTCC
CTGAGACTCTGCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTA
GTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATCCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGGCTCACAAATATTTTGA
0 CTGGTTATTATACCTCAGGATATGCTTTTGATATCTGGGGCCAAGGTACACT GGTCACCGTGAGCTCA (SEQ. ID. NO. 29);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTCTTGGATGAG
TTGGGTCCGCGAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGT
AGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCA
TCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAG
AGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGTAGGGAACTACGGTTTC
TACCACTACATGGACGTCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA
(SEQ. ID. NO. 3 1);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAG
CTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGT
AGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCA
TCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAG
AGCCGAGGACACTGCCGTGTATTACTGTGCGAGAATTAAACGGTTACGATTC
GGCTGGACCCCTTTTGACTACTGGGGCCAGGGTACACTGGTCACCGTGAGCT
CA (SEQ. ID. NO. 33);
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTA
-9m-
GTAGTAGTAGTAGTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCCGAGGACACTGCCGTGTATTACTGTGCGAGAGTCAATAGCAAAAAG
TGGTATGAGGGCTACTTCTTTGACTAGTGGGGCCAGGGTACACTGGTCACCG
TGAGCTCA (SEQ. ID. NO. 35); and
GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCC
CTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGA
GCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTA
GTACTAGTAGTAATTACATATACTACGCAGACTCAGTGAAGGGCCGGTTCAC
CATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTG
AGAGCGAGGACACTGCCGTGTATTACTGTGCGAGAGTCAAGAAGTATAGCA
GTGGCTGGTACTCGAATTATGCTTTTGATATCTGGGGCCAAGGTACACTGGT
CACCGTGAGCTCA (SEQ. ID. NO, 37) and/or a variable light region (VL) selected from the group of nucleic acid sequences consisting of:
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCAGGTCCAACATTGGGAATAATTATGTATC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
AACAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGGA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGACAGCCTGAATGGTCATTGGGTGTTCGGCGGA
GGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGcGG
TCACCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAA-ATAATGCTGTAAA
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGGAAT
GATCGGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTCAGACCTGGGGCACTGGCCGGGGGGTATTCGGCGGAGGAACCAA
GCTGACGGTCCTAGGT (SEQ. ID. NO. 4); 9n
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCTTGTTCTGGAAGCAGCTCCAATATCGGAAGTAATTATGTATCC
TGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACT
ACAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCAC
CTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTAT
TACTGTGCAGCATGGGATGACAGCCTGAGTGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 6);
CAGTCTGTGCTGACTCAGCCACCGTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCAGCTCCAAGATTGGAAATAACTATGTATC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTAAT
AATCAGCGGCCCTCAGGGGTCCGTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGACAGCCTGAGTCATTGGCTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 8);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAAT
AATCAGCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGACAGCCTGAATGGTCATTGGGTGTTCGGCGGA
GGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO.
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGGACCTACAACATTGGAAATAATTATGTATC
GTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
ATCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGTCAGGCTGAATGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 22); 90
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGGAGCTCCAACATTGGGAATAGTTATGTCTC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAAT
AATCAGCGGCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGGCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGGATGGGATGACACCCTGCGTGCTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 24);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCTTGTTCTGGAAGCCGCTCCAACATCGGGAGAAATGCTGTTAG
TTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGCTAAC
AGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGGCAGCCTGAATGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCC (SEQ. ID. NO. 26);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCAACACCAACATTGGGAAGAACTATGTATC
TTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGCTAAT
AGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCGTCATGGGATGCCAGCCTGAATGGTTGGGTATTCGGCGGAGGA
ACCAAGCTGACGGTGCTAGGT (SEQ. ID. NO. 28);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCACCTCCAACATTGGGAAGAATTATGTATC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
AGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGCCAGCCTCAGTGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 9 p
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCTTGTTCTGGAGGCAGCTCAAACATCGGAAAAAGAGGTGTAAA
TTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
AGAAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCTACATGGGATTACAGCCTCAATGCTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 32);
CAGTCTGTTCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGGTGTAAA
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
AACAATCGGCCCTGAGGGGTCCGTGACCGATTGTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCGTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGACAGCCTGCGTGGTTGGCTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 34);
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATC
CTGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAAC
AGCAATCGGCCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCA
CCTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTA
TTACTGTGCAGCATGGGATGACAGTCTGAGTGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 36); and
CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGG
TCACCATCTCCTGCTCTGGAAGCAGCTCCAGCATTGGGAATAATTTTGTATCC
TGGTATCAGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGACAATA
ATAAGCGACCCTCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCAC
CTCAGCCTCCCTGGCCATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTAT
TACTGTGCAGCATGGGATGACAGCCTGAATGGTTGGGTGTTCGGCGGAGGA
ACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 38).
9q Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to".
Detailed description of the invention Below will follow a detailed description of the invention exemplified 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.
Selection of scFv against MDA modified peptides IEIGL EGKGF EPTLE ALFGK WO 2004/030607 PCT/SE2003/001547 The target antigens were chemically modified to carry Malone-dialdehyde (MDA) groups on lysines and histidines. The modified peptides were denoted IEI (P45) and KTT (P210).
Selections were performed using Biolnvent's n-CoDeRTMscFv 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 x 1010 independent clones and a 2000 fold excess of clones was 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 pg/ml MDA-modified target peptides in PBS (137 is mM NaCI, 2.7 mM KCI, 4.3 mM Na 2
HPO
4 1.4 mM KH 2
PO
4 with end over end agitation at +4 0 Cover night. The tubes were then blocked with TPBSB5% (5 BSA, 0.05% Tween 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 x 1013 CFU phages from the n-CodeRTM library in 1.8 ml TPBSB3% for 2 h at room temperature, using end over end agitation. The tubes were then washed with 15 x 3 ml TPBSB3% and 2 x 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 pl of Aprotein (0.2 mg/ml, Roche, cat.236624), and the immunotubes were washed with 300 pi PBS, giving a final volume of 1.4 ml.
For amplification of the selected phage E. Coli HB101F' cells were grown exponentially in ml of LB medium (Merck, cat. 1.10285) to OD 6 oo 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 1/4 volume of 20% PEG 600 0 in 2.5 M NaCI and incubated for 5 h at +4 0 C. The phages were then pelleted by centrifugation for 30 minutes, 13000 x g, re-suspended in 500 pl PBS and used in selection round 2.
The amplified phagestock was used in selection round 2 in a final volume of 1.5 ml of BSA, 0.05% Tween 20, 0.02 sodium Azide in PBS. Peptide without MDA modification (4 x 10 7 M) was also included for competition against binders to MDA- WO 2004/030607 PCT/SE2003/001547 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 pl of 100 mM Tris- Glycine buffer, pH 2.2. The tubes were washed with additional 200 pl glycin buffer and the eluates were pooled and then neutralised with 96 pi of 1 M Tris-HCI, pH 8.0. The samples were re-natured for 1 h at room temperature and used for selection round 3.
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 x 10 7 M. The phage mixtures (1100 pl) were added to immunotubes coated with target antigen as described in selection 1 and incubated over night at 4 0 C with agitation. The tubes were then washed with 3 x 3 ml TPBSB 5 x 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 pg/ml ampicillin, 15 pg/ml tetracycline, 0.1% glucose, and grown over night at 30 0 C, 200 rpm in a shaker incubator.
The over night cultures were used for mini scale preparation of plasmid DNA, using Biorad mini prep Kit (Cat. 732 6100). To remove the phage gene III part from the expression vector, 0.25 pg of the plasmid DNA was cut for 2 h at 37 0 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 0 C and ligated over night at 16 0 C using 1 U T4 DNA ligase in 30 pl of 1 x 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 6xhis tag. After ligation the material was digested for 2 h at 37 0 C in a solution containing 30 pl ligation mix, 3.6 pl 10 x REACT3 stock, 0.4 pl 1 M NaCI, 5 pl H 2 0 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 ToplOF' and spread on 500 cm 2 Q-tray LA-plates (100 pg/ml Amp, 1% glucose), to enable the picking of single colonies for further screening, Screening of the n-CoDeRTMscFv library for specific antibody fragments binding tO MDA modified peptides from Apolipoprotein B-100 WO 2004/030607 PCT/SE2003/001547 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.
Colony picking of single clones, expression of scFv and screening number 1 was performed on Biolnvent's automatic system according to standard methods. 1088 and 831 single clones selected against the MDA modified IEI and KTT peptides respectively were picked and cultured and expressed in micro titre plates in 100 pl LB containing 100 pg ampicillin/ml.
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 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 pg/ml of anti-c-myc monoclonal (9E10 Roche 1667 149) in wash buffer. As a secondary antibody Goat-antimouse 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 supplier's recommendation.
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 Figure Tests were run both against directly coated peptides (108 pmol/well coated with PBS) and the more physiological target; LDL particles (1 pg/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 (MaB050 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.
The identified clones were further tested through titration against a fixed amount (1 pg/well) of MDA LDL and native LDL in order to evaluate the dose response of the scFv (Figure 2).
WO 2004/030607 PCT/SE2003/001547 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 Tested Clones 1088 831 Screening Scored Hits 64 33 number 1 Tested Clones 64 33 Screening Scored Hits 12 2 number 2 Tested Clones 12 2 Dose Scored Hits 8 2 response 1(0.2%) 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 CCC AGT CAC GAC GTT GTA AAA CG-3') and (5'-GAA ACA GCT ATG AAA TAC CTA TTG Cand a GeneAmp PCR system 9700 (PE Applied system) using the temperature cycling program 94 0 C 5 min, 30 cycles of 94 0 C 30s, 52 0 C for 30s and 68 0 C for 2min and finally 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 0 C 10s, 50°C for 5s and 60°C for 4 min. The extension products were purified according to the supplier'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 sequences of the variable heavy (VH) and variable light (VL) domains of the finally selected clones are shown in Figure 3.
Example 2.
Transfer of genes encoding the variable parts of selected scFv to full length human IgG1 vectors.
Bacteria containing scFv clones to be converted to Ig-format were grown over night in LB supplemented with 100 pg/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 260nm, and the DNA was diluted to a concentration of 2 ng/pl.
WO 2004/030607 PCT/SE2003/001547 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). 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).
Primers for amplification of VH-segments: 5'-GGTGTGCATTCCGAGGTGCAGCTGTTGGAG (SEQ. ID. NO: 13) 3'VH: 5'-GACGTACGACTCACCTGAGCTCACGGTGACCAG (SEQ. ID. NO: 14) 1o Primers for amplification of VL-segments: 5'-GGTGTGCATTCCCAGTCTGTGCTGACTCAG (SEQ. ID. NO: 3'VL: 5'-GACGTACGTTCTACTCACCTAGGACCGTCAGCTT (SEQ. ID. NO: 16) PCR was conducted in a total volume of 50 pl, containing 10ng template DNA, 0.4 pM primer, 0.4 pM 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 0 C for 30 seconds, an annealing step at 55 0 C for 30 seconds, and an elongating step at 68 0 C for 1.5 minutes. This amplification cycle was repeated 25 times. Each reaction began with a single denaturing step at 94 0 C for 2 minutes and ended with a single elongating step at 68 0 C for 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).
Four pl 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 pg/ml ampicillin and 20 pg/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 0 C for 10 seconds, an annealing step at 50 0 C for 15 seconds, and an elongating step at 60 0 C for 4 minutes.
This cycle was repeated 25 times. Each reaction began with a single denaturing step at WO 2004/030607 PCT/SE2003/001547 94 0 C for 1 minute. The reactions were performed in a volume of 10 pl consisting of 1 pM primer (5'-CAGGAAACAGCTATGAC), 3 pl plasmid DNA and 4 pi Big Dye reaction mix.
The reactions were precipitated according to the manufacturers recommendations, and samples were run on an ABI PRISM 3100 Genetic Analyzer. Sequences were compared s to the original scFv sequence using the alignment function of the OMIGA sequence analysis software (Oxford Molecular Ltd).
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 Dral (Roche 1 417 983) at 37 0 C for 2 hours. Digestions were heat inactivated at 70 0 C for minutes and purified by spin column chromatography using S400-HR columns (Amersham-Pharmacia Biotech 27-5240-01). The purified Dral digestions were subsequently digested with BsmI (Roche 1 292 307) and BsiWI (Roche 1 388 959) at 55 0 C over night. Digestions were purified using phenol extraction and precipitation.
The precipitated DNA was dissolved in 10 pl H 2 0 and used for ligation.
The expression vectors were obtained from Lars Norderhaug Immunol. Meth. 204 (1997) 77-87). After some modifications, the vectors (Figure 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.
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 0 C over night in a volume of 10 pl, containing 100 ng digested vector, 2 pl digested VH/VL-pCR 2.1 TOPO vector (see above), 1 U T4 DNA ligase (Life Technologies, 15224-025) and the supplied buffer. 2 pl of the ligation mixture were subsequently transformed into ul chemo competent toplOF' bacteria, and plated on selective (100 pg/ml ampicillin or pg/ml kanamycin) agar plates.
Colonies containing HC/LC plasmids with VH/VL inserts were identified by colony PCR: Forward primer: Reverse primer: WO 2004/030607 PCT/SE2003/001547 PCR was conducted in a total volume of 20 pl, containing bacteria, 0.5 pM forward primer, 0.5 pM 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 Each PCR amplification cycle consisted of a denaturing step at 94 0 C for 30 seconds, an annealing step at 52 0 C for 30 seconds, and an elongating step at 68 0 C for 1.5 minutes. This amplification cycle was repeated times. Each reaction began with a single denaturing step at 94 0 C for 2 minutes and ended with a single elongating step at 68 0 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 pg/ml ampicillin or pg/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 0 C for 10 seconds, an annealing step at 50 0 C for 15 seconds, and an elongating step at 60 0 C for 4 minutes. This cycle was repeated 25 times. Each reaction began with a single denaturing step at 94 0 C for 1 minute. The reactions were performed in a volume of 10 pl consisting of 1 pM primer 3pl plasmid DNA and 4pl Big Dye reaction mix. The reactions were precipitated according to the manufacturers recommendations, and samples were run on an 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 was digested for production of a joined vector, containing heavy- and light chain genes on the same plasmid.
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 700C 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 Bstll07I (Roche 1 378 953). Digestions were then heat inactivated at 700C for minutes and purified by spin column chromatography using S400-HR columns (Amersham-Pharmacia Biotech 27-5240-01). 5 pl of each digested plasmid were ligated at 160C over night in a total volume of 20 pl, containing 2 U T4 DNA ligase (Life WO 2004/030607 PCTISE2003/001547 Technologies, 15224-025) and the supplied buffer. 2 pl of the ligation mixture were subsequently transformed into 50 pl chemo competent toplOF' bacteria, and plated on selective (100 pg/ml ampicillin and 20 pg/ml kanamycin) agar plates.
s Bacterial colonies were grown over night in LB supplemented with 100 pg/ml ampicillin and 20 pg/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 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.
COS-7 cells (ATCC CRL-1651) were cultured at 37 0 C with 5% CO 2 in Dulbeccos MEM, high glucose Glutamaxl (Invitrogen 31966021), supplemented with 0.1 mM nonessential amino acids (Invitrogen 11140035) and 10% fetal bovine sera (Invitrogen 12476-024, batch 1128016). The day before transfection, the cells were plated in 12well plates (Nunc, 150628) at a density of 1.5x10 5 cells per well.
Prior to transfection, the plasmid DNA was heated at 70 0 C for 15 minutes. Cells were transfected with 1 pg HC-plasmid 1 pg LC-plasmid, or 2 pg 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.
Ninetysix (96)-well plates (Costar 9018, flat bottom, high binding) were coated at 4 0
C
over night by adding 100 pl/well rabbit anti-human lambda light chain antibody (DAKO, A0193) diluted 4000 times in coating buffer (0.1M sodium carbonate, pH Plates were washed 4 times in PBS containing 0.05% Tween 20 and thereafter blocked with 100 pl/well PBS+3% BSA (Albumin, fraction V, Roche 735108) for 1 h. at room temperature. After washing as above, 100 pl/well of sample were added and incubated in room temperature for 1 hour. As a standard for estimation of concentration, human purified IgG1 (Sigma, 15029) was used. Samples and standard were diluted in sample buffer (lx PBS containing 2% BSA and 0.5% rabbit serum (Sigma R4505).
Subsequently, plates were washed as described above and 100 pl/well of rabbit antihuman IgG (y-chain) HARP-conjugated antibody (DAKO, P214) diluted 8000 times in WO 2004/030607 PCT/SE2003/001547 sample buffer was added and incubated at room temperature for 1 hour. After washing 8 times with PBS containing 0.05% Tween 20, 100 pl/well of a substrate solution containing one OPD tablet (10 mg, Sigma P8287,) dissolved in 15 ml citric acid buffer and 4.5 pl H 2 0 2 was added. After 10 minutes, the reaction was terminated by s adding 150 pl/well of 1M HCI. Absorbance was measured at 490-650 nm and data was analyzed using the Softmax software.
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 0 C for minutes.
Example 3 Stable transfection of NSO cells expressing antibodies against MDA modified peptides form Apolipoprotein B-100.
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 1X NEAA (non-essential amino acids, cat no. 11140-053, Invitrogen).
Cell cultures are maintained at 37 0 C with 5% CO 2 in humidified environment.
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 12xl0 6 cells/flask. On the day of transfection, when the cells were 85-90% confluent, the cells were plated in 15 ml DMEM 1X NEAA 10 FBS (as above). For each flask of cells to be transfected, pg 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 pl 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.
WO 2004/030607 PCT/SE2003/001547 The cells were washed with medium once and 11 ml DMEM 1X NEAA 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 OC in a 5% CO 2 incubator for 24 h.
The cells were then trypsinized and counted, and subsequently plated in 96-well plates at 2x10 4 cells/well using five 96-well plates/construct. Cells were plated in 100 pl/well of DMEM 1X NEAA 10 FBS (as above) containing G418-sulphate (cat nr.10131-027, lot: 3066651, Invitrogen) at 600 pg/ml. The selection pressure was kept unchanged until harvest of the cells.
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 is production from cells in these wells was then assayed with ELISA and 5-21 pools/construct were selected for re-screening (Table 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 1xNEAA 10% Ultra low IgG FBS (cat. no. 16250-078, lot. no. 113466, Invitrogen) G418 (600 pg/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.
Production and purification of human IgG1 Supernatants from NSO cells transfected with the different IgG1 antibodies were sterile filtered using a 0.22 pm filter and purified using an affinity medium MabSelectTM with recombinant protein A, (Cat. No. 17519901 Amersham Biosciences).
Bound human IgG1 was eluted with HCL-glycine buffer pH 2.8. The eluate was collected in 0.5 ml fractions and OD2sz was used to determine presence of protein. The peak fractions were pooled and absorbance was measured at 280nm and 320nm. 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. 647U Bio Whittaker). The samples contained between 1 and 12 EU/ml endotoxin. The purity of the preparations was estimated to exceed 98% by PAGE analysis.
WO 2004/030607 PCT/SE2003/001547 Table 3 Summary of Production and Purification of human IqG1 Clone Volume Total IgG1 in Total IgG1 Yield name culture supernatant Purified supernatant (mg) (mg) (ml) IEI-A8 600 68 42 61.8 IEI-D8 700 45 21 46.7 IEI-E3 700 44.9 25.6 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 The purified IgG1 preparations were tested in ELISA for reactivity to MDA modified and un-modified peptides (Figure 5) and were then used in functional in vitro and in vivo studies.
Example Analysis of possible anti-atherogenic effect of antibodies is performed both in experimental animals and in cell culture studies.
1. Effect of antibodies on atherosclerosis in apolipoprotein E knockout (apo 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 pg 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 0 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.
In a first experiment the effect of the antibodies on development of atherosclerosis was analysed in apo 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 WO 2004/030607 PCT/SE2003/001547 injection, and the extent of atherosclerosis was assessed by Oil Red 0 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 isothiocyanate (FITC) antigen (P=0.03) (Fig. The mice tolerated the human antibodies well and no effects on the general health status of the mice were evident.
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 by 25% in the mg group and by 41% (P=0.02) in the 2.0 mg group as compared to the corresponding FITC antibody-treated groups (Fig. 7).
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 fibromuscular 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 pg of the respective antibody. Control mice are given 500 pg 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 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.
3, Effect of antibodies on uptake of oxidized LDL in cultured human macrophages.
Uptake of oxidized LDL in arterial macrophages leading to formation of cholesterolloaded 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 12 5 I-labeled human oxidized LDL for 2 hours. Human macrophages are isolated from blood donor buffy coats by centrifugation in Ficoll WO 2004/030607 PCT/SE2003/001547 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).
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.
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.
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.
Such pharmaceutical compositions may contain the active antibody in an amount of 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.
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 symptoms of atherosclerosis by way of passive immunization. A dosage of antibody according to the invention may be 1 pg to 1 mg per kg bodyweight, or more.
WO 2004/030607 PCTISE2003/001547 23 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.
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.
WO 2004/030607 PCT/SE2003/001547
REFERENCES
Dimayuga, B. Cercek, et al. (2002).
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Jovinge, 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.
Regnstrom, 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.
Steinberg, 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.
Zhou, 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.
Claims (25)
- 2. The use according to claim 1, wherein the fragment has been oxidized using malone dialdehyd e.
- 3. The use according to claim 1, wherein the fragment has been oxidized using copper.
- 4. The use according to any one of claimrs 1 to 3, wherein the antibody or fragment thereof comprises a variable heavy region (VH) selected from the group of nucleic acid sequences consisting of: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAATAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATFAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGAGTCAGTAGGTACTACTACGGACCATCTTTCTACTTTGACT CCTGGGGCCAGGGTACACTGGTCACCGTGAGCAGC (SEQ. ID. NO. 1); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCGGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGGTCC GCCAGGCTCCCGGGAAGGGGCTGGAGTGGGTATCGGGTGTTAGTTGGAATGGCAG TAGGACGCACTATGGAGACTCTGTGAAGGGCCGATTCACGATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGT GTATTACTGTGCGAGAGCGGCTAGGTACTCCTACTACTACTACGGTATGGACGTCT GGGGCCAAGGTACACTGGTCACCGTGAGCAGC (SEQ. ID. NO. 3); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATCAGTGGTAGTGGTCG TAGGACATACTACGCAGACTCCGTGCAGGGCCGGTTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGT GTATTACTGTGCGAGATTGGTCTCCTATGGTTCGGGGAGTTTCGGTTTTGACTACTG GGGCCAAGGTACACTGGTCACCGTGAGCAGC(SEQ. ID. NO. 34 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTCTTGGTGGTAGTGGTGG TGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCAGCATCTCCAGAGACA ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGC CGTGTATTACTGTGCGAAGTTAGGGGGGCGATCCCGATATGGGCGGTGGCCCCGCC AATTTGACTACTGGGGCCAAGGTACACTGGTCACCGTGAGCAGC (SEQ. ID. NO. 7); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGAACGTATTGGATGACCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGCAGTAGCAGTAA TTACATATTCTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGT GTATTACTGTGCGAGACTCAGACGGAGCAGCTGGTACGGGGGGTACTGGTTCGACC CCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 19); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCAACTACATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTA GTTACATATACTAGGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGAGTAGGCCGGTATAACTGGAAGACGGGGCATGCTTTTGAT ATCTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 21); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTGTCCTGTGCAGCCTCTGGATTCACCTTCCGTGACTACTACGTGAGCTGGATCC GCCAGGCTGCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTAGTGGTAGTGGGGG TAGGACATAGTACGCAGACTCCGTGGAGGGCCGGTTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCAT GTATTACTGTGCCAGAGTATCCGCCCTTCGGAGACCCATGACTACAGTAACTACTT ACTGGTTCGACCCCTGGGGCCAAGGTAGACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 23); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCCGCTATTAGTGGTAGTGGTA ACACATACTATGGAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCC AAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGT ATTACTGTGCGAGAGCCTGCCACGGTATATTrAGGFFATGCTTTTGATATCTGGGGCC AGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGOAGTGGGTCTCAAGTATTAGTGTTGGTGGAC ATAGGACATATTATGCAGATTCCGTGAAGGGCCGGTCCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCACGGATAGGGGTGGGTCCGTCCGGCGGGGCCTTTGACTACTGG GGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 27); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATCCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGGCTCACAAATATTTTGACTGGTTATTATACGTCAGGATATG CTTTTGATATCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 29); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTCTTGGATGAGTTGGGTCC -36- GCCAGGCTCCAGGGAAGGGGGTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGT TACATATACTAGGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTC CAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTG TATTACTGTGCGAGAGTAGGGAACTACGGTTTCTACCACTACATGGACGTCTGGGG CCAAGGTACAGTGGTCACCGTGAGCTGA (SEQ. ID. NO. 3 1); GAGGTGGAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGT TACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTC CAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTG TATTACTGTGCGAGAATTAAACGGTTACGATTCGGCTGGACCCCTTTTGACTAGTG GGGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 33); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCAccTrTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGAGTCAATAGCAAAAAGTGGTATGAGGGCTACTTCTTTGAC TACTGGGGGCAGGGTACACTGGTCACCGTGAGGTCA (SEQ. ID. NO. 35); and GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCGTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTACTAGTAGTAA TTACATATACTACGCAGACTCAGTGAAGGGCCGGTTCACCATCTCCAGAGACAATT GCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGT GTATTACTGTGCGAGAGTCAAGAAGTATAGCAGTGGCTGGTACTCGAATTATGCTT TTGATATCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 37), -37- in combination with at least one variable light region selected from the group of nucleic acid sequences consisting of: CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGGTCCAAGATTGGGAATAATTATGTATCCTGGTATC AGCAGCTCGCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAACAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCGCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGGCTGAATGGTCATTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 2); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGTTCTGGAAGCAGGTCCAACATCGGAAATAATGCTGTAAACTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGGAATGATCGGCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTCAGACCTGGGGCAC TGGCCGGGGGGTATTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 4); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCTTGTTCTGGAAGCAGCTCCAATATCGGAAGTAATTATGTATCCTGGTATCA GCAGCTCCCAGGAACGGCGCCCAAACTCCTCATCTATGGTAACTACAATCGGCCCT CAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAGTGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 6); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGCTCCAACATTGGAAATAACTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTAATAATCAGCGGCGC 39 TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAGTCATTGGCTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 8); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCGTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTATA GCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCCT CAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCGTGAATGGTCATTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGGACCTACAACATTGGAAATAATTATGTATCGTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACATCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGT CAGGCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 22); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGGAGCTCCAACATTGGGAATAGTTATGTCTCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGGATGGGATGA CACCCTGCGTGCTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 24); -39- CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCTTGTTCTGGAAGCCGCTCCAACATFCGGGAGAAATGCTGTTAGTTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGCTAACAGCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTGCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGG CAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCC (SEQ. ID. NO. 26); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAACACCAACATTGGGAAGAACTATGTATCTTGGTATC AGCAGCTCCCAGGAAGGGCCCCCAAACTCCTCATCTATGCTAATAGCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGGGTCATGGGATGC CAGCCTGAATGGTTGGGTATTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 28); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCACCTCCAACATTGGGAAGAATTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGC CAGCCTCAGTGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCTTGTTCTGGAGGCAGCTCAAACATCGGAAAAAGAGGTGTAAATFTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGAAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCTACATGGGATTA 40 CAGCCTCAATGCTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 32); CAGTCTGTTCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGGTGTAAAGTGGTATG AGCAGCTCGCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAACAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGGGTGGTTGGCTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 34); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCC TCAGGGGTGCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGTCTGAGTGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 36); and CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC GATCTCCTGCTCTGGAAGCAGCTCGAGCATTGGGAATAATTTTGTATCCTGGTATCA GCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGACAATAATAAGCGACCCT CAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 38). The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region (V 11 C1 -41- GAGGTGCAGGTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAATAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGAGTCAGTAGGTACTACTACGGACCATCTTTCTACTTTGACT CI CCTGGGGCCAGGGTACACTGGTCACCGTGAGCAGC (SEQ. ID. NO. 1) CK1 and the variable light region (VL): I0 CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGGTCCAACATTGGGAATAATTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAACAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAATGGTCATTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 2).
- 6. The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region (VH): GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCGGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGGTCC GCCAGGCTCCCGGGAAGGGGCTGGAGTGGGTATCGGGTGTTAGTTGGAATGGCAG TAGGACGCACTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGT GTATTACTGTGCGAGAGCGGCTAGGTACTCCTACTACTACTACGGTATGGACGTCT GGGGCCAAGGTACACTGGTCACCGTGAGCAGC (SEQ. ID. NO. 3) and the variable light region 42 CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGCTGTAAACTGGTATC AGCAGCTCCCAGGAACGGCGCCCAAACTCCTCATCTATGGGAATGATGGGCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTCAGACCTGGGGCAC NI TGGCCGGGGGGTATTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 4).
- 7. The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region (V 11 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCGTGTGCAGCCTCTGGATTCACC7FFAGTAGCTATTGGATGAGCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATCAGTGGTAGTGGTCG TAGGACATACTACGCAGACTCGGTGCAGGGCCGGTTCACCATCTCCAGAGACAATI CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGT GTATTACTGTGCGAGATTGGTCTCCTATGGTTCGGGGAGTTTCGGTTTTGACTACTG GGGCCAAGGTACACTGGTCACCGTGAGCAGC (SEQ. ID. NO. and the variable light region (VL): CAGTCTGTGCTGACTCAGCCAGCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCTTGTTCTGGAAGCAGCTCCAATATCGGAAGTAATTATGTATCCTGGTATCA GCAGCTCCCAGGAACGGCGCCCAAACTCCTCATCTATGGTAACTACAATCGGCCCT CAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAGTGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 6). -43-
- 8. The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region (VI.1): GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTCTTGGTGGTAGTGGTGG TGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGC CGTGTATTACTGTGCGAAGTTAGGGGGGCGATCCCGATATGGGCGGTGGCCCCGCC AATTTGACTACTGGGGCCAAGGTACACTGGTCACCGTGAGCAGC (SEQ. ID. NO. 7) and the variable light region CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGCTCCAACATTGGAAATAACTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTAATAATCAGCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAGTCATTGGCTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 8).
- 9. The use according to claim 4, wherein the antibody or fragment comprises the variable heavy region (VFI): GAGGTGCACCTGTTGGAGTCTGGGGGAGGCUTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGAACGTATTGGATGACCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGCAGTAGCAGTAA TTACATATTCTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGGCTGAGAGCCGAGGACACTGCCGT 44 GTAYFACTGTGCGAGACTCAGACGGAGCAGCTGGTACGGGGGGTACTGGTTCGACC CCTGGGGCCAAGGTACAGTGGTCACCGTGAGCTCA SEQ. ID. NO. 19) and the variable light region CAGTCTGTGCTGACTCAGCCACCCTCAGGGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAATGGTCATTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region (V 11 GAGGTGCAGCTGTTOGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCAACTACATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCAFFAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGAGTAGGCCGGTATAACTGGAAGAGGGGGCATGGTTTTGAT ATGTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 2 1) and the variable light region CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGGACCTACAACATTGGAAATAATTATGTATCGTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACATCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCGTCCCTGGCC c-I ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATI'ATTACTGTGCAGCATGGGATGT CAGGCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT c-I (SEQ. ID. NO. 22).
- 11. The use according to claim 4, wherein the antibody or fragment thereof comprises the S 5 variable heavy region (V11): c-I GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCCGTGACTACTACGTGAGCTGGATCC c-I GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTAGTGGTAGTGGGGG TAGGACATACTACGCAGACTCCGTGGAGGGCCGGTTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCAT GTA'FFACTGTGCCAGAGTATCCGCCCTTCGGAGACCCATGACTACAGTAACTACTT ACTGGTTCGACCCCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 23) and the variable light region (VL): CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGGAGCTCCAACATTGGGAATAGTTATGTGTCCTGGTATC AGCAGGTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCC TCAGGGGTCCCTGACCGATTCTGTGGCTCCAAGTGTGGCAGCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGGATGGGATGA CACCCTGCGTGCTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 24).
- 12. The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region (V GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC 46 CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCCGCTATTAGTGGTAGTGGTA ACACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCC AAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTGT ATTACTGTGCGAGAGCCTCCCACCGTATATTAGGTTATGCTTTTGATATCTGGGGCC AGGGTACACTGGTCACCGTGAGCTCA(SEQ. ID. NO. and the variable light region (VL): CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCTTGTTCTGGAAGCCGCTCCAACATCGGGAGAAATGCTGTTAGTTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATGTATGCTAACAGCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCOGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGG CAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCC (SEQ. ID. NO. 26).
- 13. The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region (Vj-j): GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTAGTGTTGGTGGAC ATAGGACATATTATGCAGATTCCGTGAAGGGCCGGTCCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCACGGATACGGGTGGGTCCGTCCGGCGGGGCCTTTGACTACTGG GGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 27) and the variable light region (VL): -47- CAGTCTGTGCTGACTCAGCCACCCTCAGGGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAACACCAACATTGGGAAGAACTATGTATCTTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGCTAATAGCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCGTCATGGGATGC CAGCCTGAATGGTTGGGTATTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 28).
- 14. The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region (V1 1 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCAUTAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATCCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGGCTCACAAATATTTTGACTGGTTATTATACCTCAGGATATG CTTTTGATATCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 29) and the variable light region (VL): CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCACCTCCAACATTGGGAAGAATTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGC CAGCCTCAGTGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 48 The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region (VI I): GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTCTTGGATGAGTTGGGTCC CI GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGT TACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTC CI CAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTG TATTACTGTGCGAGAGTAGGGAACTACGGTTTCTACCACTACATGGACGTCTGGGG CCAAGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 3 1) and the variable light region (VL): CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCTTGTTCTGGAGGCAGCTCAAACATCGGAAAAAGAGGTGTAAATTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGAAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCTACATGGGATTA CAGCCTCAATGCTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 32).
- 16. The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region (V 1 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGT TACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTC CAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTG 49 TATTACTGTGCGAGAATTAAACGGTTACGATTCGGCTGGACCCCTTTTGACTACTG GGGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 33) and the variable light region (Vj: CAGTCTGTTCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGGTGTAAACTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAACAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATOA CAGCCTGCGTGGTTGGCTGT[CGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 34).
- 17. The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGAGTCAATAGCAAAAAGTGGTATGAGGGCTACTTCTTTGAC TACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. and the variable light region (VL): CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC -so- ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGTCTGAGTGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 36).
- 18. The use according to claim 4, wherein the antibody or fragment thereof comprises the variable heavy region GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTACTAGTAGTAA TTACATATACTACGGAGAGTCAGTGAAGGGCCGGTTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGT GTATTACTGTGCGAGAGTCAAGAAGTATAGCAGTGGCTGGTACTCGAATTATGCTT TTGATATCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 37) and the variable light region (VL): CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCGCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGCTCCAGCATTGGGAATAATTTTGTATCCTGGTATCA GCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGACAATAATAAGCGACCCT CAGGGGTCCCTGACCGATTCTCTGGGTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 38).
- 19. The use according to any one of claims 5 to 18 wherein the antibody or antibody fragment has CDR sequences from the variable heavy region (V 11 and/or the variable light region (VL) nucleic acid sequences defined in claim 4.
- 20. 30 20. An antibody or antibody fragment thereof as defined in any of claims I to 19. -51 _21. The antibody or antibody fragment thereof according to claim 20, wherein the antibody or antibody fragment has CDR sequences from the variable heavy region (Va) and/or the variable light region (VL) nucleic acid sequences defined in claim 4. 0
- 22. A method for preparation of an isolated antibody according to claim 20 or 21, using a C(N fragment library to generate specific human antibody fragments against an oxidized, MDA 0 modified peptide derived from Apo B 100 as defined in claim 1, whereupon identified antibody N1 fragments having the desired characteristics are rebuilt into a full length human immunoglobulin to be used for therapeutic purposes.
- 23. A method for amplification of an isolated human antibody according to claim 20 or 21, using a recombinant technology, comprising transfer of genes encoding the variable parts of a selected scFv into full length human IgGI vectors using a PCR-amplification wherein the primers used are: primers for amplification of VH-segments: 5'-GGTGTGCATTCCGAGGTGCAGCTGTTGGAG (SEQ. ID. NO: 13), and 3'VH: 5'-GACGTACGACTCACCTGAGCTCACGGTGACCAG (SEQ. ID. NO: 14) and primers for amplification of VL-segments: 5'-GGTGTGCATTCCCAGTCTGTGCTGACTCAG (SEQ. ID. NO: 15), and 3'VL: 5'-GACGTACGTTCTACTCACCTAGGACCGTCAGCTT (SEQ. ID. NO: 16).
- 24. The method according to claim 23, wherein colonies containing heavy chain region and light chain region plasmids with variable heavy chain region and variable light chain region inserts are identified by colony PCR using: Forward primer: 5'-ATGGGTGACAATGACATC (SEQ. ID. NO: 17), and Reverse primer: 5'-AAGCTTGCTAGCGTACG (SEQ. ID. NO: 18). -52- A method for passive immunization of a mammal comprising the administration of a therapeutically or prophylactically effective amount of an isolated human antibody according to claim 20 or 21 to a mammal in need thereof.
- 26. The method according to claim 25, wherein the mammal is a human.
- 27. A pharmaceutical composition comprising an isolated human antibody directed towards at least one oxidized fragment of apolipoprotein B according to claim 20 or 21 for therapeutical or prophylactical treatment of atherosclerosis by means of passive immunization, which antibody is present in combination with a pharmaceutical excipient.
- 28. An antibody or antibody fragment thereof according to claim 20 or 21 for use in medicine.
- 29. An antibody or antibody fragment thereof according to claim 20 or 21 for the therapeutic or prophylactic treatment of atherosclerosis by means of passive immunisation. A method for the therapeutical or prophylactical treatment of atherosclerosis by means of passive immunization, comprising administering to a patient in need thereof an effective amount of at least one isolated human antibody or antibody fragment directed towards at least one oxidized fragment of apolipoprotein B, wherein the oxidized fragment is IEIGLEGKGFEPTLEALFGK and wherein the antibody comprises a variable heavy region (VI) selected from the group of nucleic acid sequences consisting of: GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAATAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG -53 TGTATTACTGTGCGAGAGTCAGTAGGTACTACTACGGACCATCTTTCTACTTTGACT CCTGGGGCCAGGGTACACTGGTCACCGTGAGCAGC (SEQ. ID. NO. 1); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCGGCCTCTGGATTCACCTTCAGTGACTACTACATGAGCTGGGTCC GCCAGGCTCCCGGGAAGGGGCTGGAGTGGGTATCGGGTGTTAGTTGGAATGGCAG TAGGACGCACTATGCAGACTCTGTGAAGGGCCGATTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGT GTATTACTGTGCGAGAGCGGCTAGGTACTCCTACTACTACTACGGTATGGACGTCT GGGGCCAAGGTACACTGGTCACCGTGAGCAGC (SEQ. ID. NO. 3); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATCAGTGGTAGTGGTCG TAGGACATACTACGCAGACTCCGTGCAGGGCCGGTTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGT GTATTACTGTGCGAGATTGGTCTCCTATGGTTCGGGGAGTTTCGGTTTTGACTACTG GGGCCAAGGTACACTGGTCACCGTGAGCAGC(SEQ. ID. NO. GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGTTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTCTTGGTGGTAGTGGTGG TGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACA ATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGC CGTGTATTACTGTGCGAAGTTAGGGGGGCGATCCCGATATGGGCGGTGGCCCCGCC AATT[GACTACTGGGGCCAAGGTACACTGGTCACCGTGAGCAGC (SEQ. ID. NO. 7); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGAACGTATTGGATGACCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAGCAGTAGCAGTAA 54 TTACATATTCTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGT GTATTACTGTGCGAGACTCAGACGGAGCAGCTGGTACGGGGGGTACTGGTTCGACC CCTGGGGCGAAGGTACACTGGTCACCGTGAGGTCA (SEQ. ID, NO. 19); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCAACTACATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGAGTAGGCCGGTATAACTGGAAGACGGGGCATGCTTTTGAT ATCTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 2 1); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCCGTGACTACTACGTGAGCTGGATCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTAGTGGTAGTGGGGG TAGGACATACTACGCAGACTCCGTGGAGGGCCGGTTGACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCAT GTATTACTGTGCCAGAGTATCCGCCCTTCGGAGACCCATGACTACAGTAACTACTT ACTGGTTCGACCCCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 23); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCUTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGAFITCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCCGCTA'ITAGTGGTAGTGGTA ACACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCC AAGAACACGCTGTATCTGCAAATGAAGAGCCTGAGAGCCGAGGACACTGCCGTGT ATTACTGTGCGAGAGCCTCCCACCGTATATTAGGTTATGCTTFTGATATCTGGGGCC AGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 55 GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTAGTGTTGGTGGAC ATAGGACATATTATGCAGATTCCGTGAAGGGCCGGTCCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCACGGATACGGGTGGGTCCGTCCGGCGGGGCCTTTGACTACTGG GGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 27); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATCCACCATCTCCAGAGACAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGGCTCACAAATATTTTGACTGGTTATTATACCTCAGGATATG CTTTTGATATCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 29); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTCTTGGATGAGTTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGT TACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTC CAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTG TATTACTGTGCGAGAGTAGGGAACTACGGTTTCTACCACTACATGGACGTCTGGGG CCAAGGTACAGTGGTCACCGTGAGCTCA (SEQ. ID. NO. 3 1); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGTAGCTATTGGATGAGCTGGGTCC GCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTAGT TACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGACAATTC CAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCGTG -56- TATTACTGTGCGAGAATTAAACGGTTACGATTCGGCTGGACCCCTTTTGACTACTG GGGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 33); GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTCTGGATTCACCITCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTAGTAGTAGTA GTTACATATACTACGCAGACTCAGTGAAGGGCCGATTCACCATCTCCAGAGAGAAT TCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACTGCCG TGTATTACTGTGCGAGAGTCAATAGCAAAAAGTGGTATGAGGGCTACTTCTTTGAC TACTGGGGCCAGGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 35); and GAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGA GACTCTCCTGTGCAGCCTGTGGATTCACCTTCAGTAACGCCTGGATGAGCTGGGTC CGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCCATTAGTACTAGTAGTAA TTACATATACTACGCAGACTCAGTGAAGGGCCGGTTCACCATCTCCAGAGACAATT CCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCGAGGACACTGCCGTG TATTACTGTGCGAGAGTCAAGAAGTATAGCAGTGGCTGGTACTCGAATTATGCTTT TGATATCTGGGGCCAAGGTACACTGGTCACCGTGAGCTCA (SEQ. ID. NO. 37) and/or a variable light region (VL) selected from the group of nucleic acid sequences consisting of: CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGGTGCAACATTGGGAATAATTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAACAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCGTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAATGGTCATTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 2); 57 CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTGAC CATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGCTGTAAACTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGGAATGATCGGCGGCCC TCAGGGGTCCCTGAGCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTCAGACCTGGGGCAC TGGCCGGGGGGTATTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 4); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCTTGTTCTGGAAGCAGCTCCAATATCGGAAGTAATTATGTATCCTGGTATCA GCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACTACAATCGGCCCT CAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAGTGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 6); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGCTCCAACATTGGAAATAACTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGTAATAATCAGCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAGTCATTGGCTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 8); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGCTCCAACATTGGGAATAATTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTGCAAGTCTGGCACCTCAGCCTCCCTGGGC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAATGGTCATTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. -58- CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGGACCTACAACATTGGAAATAATTATGTATCGTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACATCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGGC ATCAGTGGGCTGCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGT CAGGCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 22); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGGAGCTCCAACATTGGGAATAGTTATGTCTCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATAGGAATAATCAGCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGGATGGGATGA CACCCTGCGTGCTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. NO. 24); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCTTG'ITCTGGAAGCCGCTCCAACATCGGGAGAAATGCTGTTAGTTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGCTAACAGCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGG CAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCC (SEQ. ID. NO. 26); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAACACCAACATTGGGAAGAACTATGTATCTTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGCTAATAGCAATGGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCGTCCCTGGCC ATCAGTGGGCTGCGGTCCGAGGATGAGGCTGATTATTACTGTGCGTCATGGGATGC c-I -59- ct CAGCCTGAATGGTTGGGTATTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 28); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCACCTCCAACATTGGGAAGAATTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGCAATGGGCCC c-I TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACGTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGC c-I CAGCCTCAGTGGTTGGGTG1TCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTGAC CATCTCTTGTTCTGGAGGCAGCTCAAACATCGGAAAAAGAGGTGTAAATTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGAAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCTACATGGGATTA CAGCCTCAATGCTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 32); CAGTCTGTTCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGTTCTGGAAGCAGCTCCAACATCGGAAATAATGGTGTAAACTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAACAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGGTCCAAGTGTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGCGTGGTTGGCTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 34); CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGCTGCAACATTGGGAATAATTATGTATCCTGGTATC AGCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGGTAACAGCAATCGGCCC TCAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGTCTGAGTGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 36); and CAGTCTGTGCTGACTCAGCCACCCTCAGCGTCTGGGACCCCCGGGCAGAGGGTCAC CATCTCCTGCTCTGGAAGCAGCTCCAGCATTGGGAATAATTTTGTATCCTGGTATCA GCAGCTCCCAGGAACGGCCCCCAAACTCCTCATCTATGACAATAATAAGCGACCCT CAGGGGTCCCTGACCGATTCTCTGGCTCCAAGTCTGGCACCTCAGCCTCCCTGGCC ATCAGTGGGCTCCGGTCCGAGGATGAGGCTGATTATTACTGTGCAGCATGGGATGA CAGCCTGAATGGTTGGGTGTTCGGCGGAGGAACCAAGCTGACGGTCCTAGGT (SEQ. ID. NO. 38).
- 31. A method according to any one of claims 22 to 26 or 30 or use according to any one of claims I to 19, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.
- 32. An antibody or antibody fragment thereof according to any one of claims 20, 21, 28 or 29, or a pharmaceutical composition according to claim 27, substantially as herein described with reference to any one or more of the examples but excluding comparative examples.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2009201260A AU2009201260B2 (en) | 2002-10-04 | 2009-03-31 | Peptide-based passive immunization therapy for treatment of atherosclerosis |
Applications Claiming Priority (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| SE0202959-3 | 2002-10-04 | ||
| SE0202959A SE0202959D0 (en) | 2002-10-04 | 2002-10-04 | Peptide-Base passive immunization therapy for the treatment of atherosclerosis |
| SE0302312A SE0302312D0 (en) | 2002-10-04 | 2003-08-27 | Peptide-based passive immunization therapy for the treatment of atherosclerosis |
| SE0302312-4 | 2003-08-27 | ||
| AU2003263712 | 2003-09-22 | ||
| PCT/SE2003/001469 WO2004030698A1 (en) | 2002-10-04 | 2003-09-22 | Peptide-based passive immunization therapy for treatment of atherosclerosis |
| PCT/SE2003/001547 WO2004030607A2 (en) | 2002-10-04 | 2003-10-06 | Peptide-based passive immunization therapy for treatment of atherosclerosis |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2009201260A Division AU2009201260B2 (en) | 2002-10-04 | 2009-03-31 | Peptide-based passive immunization therapy for treatment of atherosclerosis |
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| Publication Number | Publication Date |
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| AU2003267905A1 AU2003267905A1 (en) | 2004-04-23 |
| AU2003267905B2 true AU2003267905B2 (en) | 2009-02-05 |
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| Application Number | Title | Priority Date | Filing Date |
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| AU2003267905A Ceased AU2003267905B2 (en) | 2002-10-04 | 2003-10-06 | Peptide-based passive immunization therapy for treatment of atherosclerosis |
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| Country | Link |
|---|---|
| AU (1) | AU2003267905B2 (en) |
| CA (1) | CA2500895A1 (en) |
| WO (1) | WO2004030607A2 (en) |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US8703126B2 (en) | 2000-10-12 | 2014-04-22 | Genentech, Inc. | Reduced-viscosity concentrated protein formulations |
| BRPI0403964B8 (en) | 2003-04-04 | 2021-05-25 | Genentech Inc | stable liquid formulations, article of manufacture and use of these formulations for the treatment of ige-mediated dysfunction |
| GB0517878D0 (en) | 2005-09-02 | 2005-10-12 | Bioinvent Int Ab | Immunotherapeutic treatment |
| WO2008104194A1 (en) * | 2007-02-28 | 2008-09-04 | Bioinvent International Ab | Oxidized ldl and antibodies thereto for the treatment of atheroscleroti c plaques |
| US20110182851A1 (en) * | 2008-07-11 | 2011-07-28 | Jan Nilsson | Oxidized ldl specific antibody-fusion and conjugated proteins |
| ES2828721T3 (en) | 2008-10-14 | 2021-05-27 | Genentech Inc | Immunoglobulin variants and their uses |
| JP2012519706A (en) * | 2009-03-06 | 2012-08-30 | ジェネンテック， インコーポレイテッド | Antibody preparation |
| US10858422B2 (en) | 2016-05-31 | 2020-12-08 | Abcentra, Llc | Methods for treating systemic lupus erythematosus with an anti-apolipoprotein B antibody |
| WO2021097146A1 (en) * | 2019-11-12 | 2021-05-20 | Abcentra, Llc | Methods and compositions for treating cancer |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999008109A2 (en) * | 1997-08-07 | 1999-02-18 | The Regents Of The University Of California | Antibodies to oxidation-specific epitopes on lipoprotein and methods for their use in detecting, monitoring and inhibiting the growth of atheroma |
| WO2001032070A2 (en) * | 1999-10-26 | 2001-05-10 | The Regents Of The University Of California | Reagents and methods for diagnosing, imaging and treating atherosclerotic disease |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US5972890A (en) * | 1988-05-02 | 1999-10-26 | New England Deaconess Hospital Corporation | Synthetic peptides for arterial imaging |
| US5196324A (en) * | 1989-12-15 | 1993-03-23 | Eli Lilly And Company | Monoclonal antibodies reactive with a human atheroma associated antigen |
| WO1994000592A1 (en) * | 1992-06-26 | 1994-01-06 | Exocell, Inc. | Monoclonal antibodies against glycated low density lipoprotein |
| KR0185334B1 (en) * | 1995-11-02 | 1999-04-01 | 김은영 | Cdna coding mouse antibody of apolipoprotein b-100 |
| GB9705831D0 (en) * | 1997-03-20 | 1997-05-07 | Univ Leicester | Oxidised ldl |
| CN1418106A (en) * | 2000-03-03 | 2003-05-14 | 史密丝克莱恩比彻姆生物有限公司 | Vaccine |
| SE0103754L (en) * | 2001-04-05 | 2002-10-06 | Forskarpatent I Syd Ab | Peptides from apolipoprotein B, use thereof immunization, method of diagnosis or therapeutic treatment of ischemic cardiovascular diseases, and pharmaceutical composition and vaccine containing such peptide |
-
2003
- 2003-10-06 WO PCT/SE2003/001547 patent/WO2004030607A2/en active Application Filing
- 2003-10-06 CA CA002500895A patent/CA2500895A1/en not_active Abandoned
- 2003-10-06 AU AU2003267905A patent/AU2003267905B2/en not_active Ceased
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999008109A2 (en) * | 1997-08-07 | 1999-02-18 | The Regents Of The University Of California | Antibodies to oxidation-specific epitopes on lipoprotein and methods for their use in detecting, monitoring and inhibiting the growth of atheroma |
| WO2001032070A2 (en) * | 1999-10-26 | 2001-05-10 | The Regents Of The University Of California | Reagents and methods for diagnosing, imaging and treating atherosclerotic disease |
Also Published As
| Publication number | Publication date |
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| WO2004030607A3 (en) | 2004-06-24 |
| CA2500895A1 (en) | 2004-04-15 |
| WO2004030607A2 (en) | 2004-04-15 |
| AU2003267905A1 (en) | 2004-04-23 |
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