U.S. patent application number 14/086159 was filed with the patent office on 2014-06-26 for treatment of atherosclerosis with cholesterol ester transport protein mimotopes.
This patent application is currently assigned to AFFIRIS AG. The applicant listed for this patent is AFFIRIS AG. Invention is credited to Sylvia BRUNNER, Petra Luehrs, Frank Mattner, Walter Schmidt, Barbara Wittmann.
Application Number | 20140179900 14/086159 |
Document ID | / |
Family ID | 39865273 |
Filed Date | 2014-06-26 |
United States Patent
Application |
20140179900 |
Kind Code |
A1 |
BRUNNER; Sylvia ; et
al. |
June 26, 2014 |
TREATMENT OF ATHEROSCLEROSIS WITH CHOLESTEROL ESTER TRANSPORT
PROTEIN MIMOTOPES
Abstract
The present invention relates to the use of compounds for
producing a medicament for preventing and/or treating
atherosclerosis, atherosclerosis risk diseases and atherosclerosis
sequelae.
Inventors: |
BRUNNER; Sylvia; (Vienna,
AT) ; Luehrs; Petra; (Vienna, AT) ; Mattner;
Frank; (Vienna, AT) ; Schmidt; Walter;
(Vienna, AT) ; Wittmann; Barbara; (Traiskirchen,
AT) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AFFIRIS AG |
Vienna |
|
AT |
|
|
Assignee: |
AFFIRIS AG
Vienna
AT
|
Family ID: |
39865273 |
Appl. No.: |
14/086159 |
Filed: |
November 21, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12673081 |
Feb 11, 2010 |
8618046 |
|
|
PCT/AT2008/000281 |
Aug 8, 2008 |
|
|
|
14086159 |
|
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Current U.S.
Class: |
530/326 ;
530/327; 530/328; 530/329; 530/395 |
Current CPC
Class: |
A61P 3/06 20180101; A61K
2039/55505 20130101; A61K 47/646 20170801; A61P 9/10 20180101; C07K
16/18 20130101; A61K 47/643 20170801; A61K 39/0012 20130101; A61P
43/00 20180101; A61K 2039/6081 20130101; C07K 7/06 20130101; A61K
2039/55566 20130101; A61P 9/00 20180101; C07K 7/08 20130101; A61K
2039/55555 20130101; A61K 38/1709 20130101 |
Class at
Publication: |
530/326 ;
530/329; 530/328; 530/327; 530/395 |
International
Class: |
C07K 7/08 20060101
C07K007/08; A61K 47/48 20060101 A61K047/48; C07K 7/06 20060101
C07K007/06 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 10, 2007 |
AT |
A 1258/2007 |
Claims
1. Peptide consisting of at least one amino acid sequence selected
from the group consisting of SYHATFL, TMAFPLN, HYHGAFL, EHHDIFL,
SSLELFL, TGLSVFL, WMPSLFY, SMPWWFF, TMPLLFW, DTWPGLE, SMPPIFY,
MPLWWWD, SMPNLFY, RMPPIFY, NPFEVFL, TLPNWFW, SMPLTFY, SFLDTLT,
NFLKTLS, DFLRTLT, AFLDTLV, TFLSSLA, GFLDSLM, SPHPHFL, NFMSIGL,
SQFLASL, SNFLKTL, TGFLATL, WSWPGLN, IAWPGLD, SKFMDTL, SDFLRAL,
SMPMVFY, YEWVGLM, KGFLDHL, SANPRDFLETLF, RMFPESFLDTLW,
TIYDSFLDSLAS, HQSDDKMPWWFF, KPYLLKDFLEAL, AMGPYDALDLFL,
TWNPIESFLESL, YVWQDPSFTTFF, QYQTPLTFLEAL, RHISPATFLEAL,
HTDSFLSTFYGD, YVWQDPSFTTFF, ADSTFTSFLQTL, GPVSIYADTDFL,
DSNDTLTLAAFL, NGSPALSHMLFL, TDYDPMWVFFGY, IFPLDSQWQTFW,
NESMPDLFYQPS, DWGDKYFSSFWN, VSAYNNV, WPLHLWQ, TPTHYYADFSQL,
LPGHLIWDSLHY, LPQTHPLHLLED, IPYHHLVDQLHH, YPYHVQVDVLQN,
IPSHHLQDSLQL, EYAHHTSLDLRQ, EPLHFRSDRIQA, ATPSHLIIDRAQ,
APKHLYADMSQA, FKPAHVSIDWLQ, MPAHLSRDLRQS, NPKHYSIDRHQA,
SPQHLTTDRAQA, TPFHFAQDSWQW, TPTHYYADFSQLLS, TPTHYYADFSQSLS,
GTPTHYYADFSQLL, GTPTHYYADFSQSL, FGTPTHYYADFSQSLS, FGFPTHYYADFSQSLS,
LPGHLIWDSLHY, LPGHLIWDSLHYL, LPGHLIWDSLHYLS, LPGHLIWDSLHSL,
LPGHLIWDSLHSLS, GLPGHLIWDSLHYL, GLPGHLIWDSLHSL, FGLPGHLIWDSLHSLS,
FGFPGHLIWDSLHSLS, LPQTHPLHLLED, IPYHHLVDQLHH, IPYHHLVDQLHLS,
IPYHHLVDQLHSLS, FGIPYHHLVDQLHHLS, FGFPYHHLVDQLHSLS, YPYHVQVDVLQN,
YPYHVQVDVLQNLS, YPYHVQVDVLQSLS, FGYPYHVQVDVLQNLS, FGFPYHVQVDVLQSLS,
IPSHHLQDSLQL, IPSHHLQDSLQLLS, IPSHHLQDSLQSLS, GIPSHHLQDSLQLL,
FGIPSHHLQDSLQLLS, FGFPSHHLQDSLQSLS, EYAHHTSLDLRQ, EPLHFRSDRIQA,
EPLHFRSDRIQALS, EPLHFRSDRIQSLS, GEPLHFRSDRIQAL, FGEPLHFRSDRIQALS,
FGFPLHFRSDRIQSLS, APKHLYADMSQA, APKHLYADMSQALS, APKHLYADMSQSLS,
GAPKHLYADMSQAL, FGFPKHLYADMSQSLS, MPAHLSRDLRQS, MPAHLSRDLRQSL,
MPAHLSRDLRQSLS, GMPAHLSRDLRQSL, FGFPAHLSRDLRQSLS, NPKHYSIDRHQA,
TPFHFAQDSWQW, TPFHFAQDSWQWLS, TPFHFAQDSWQSLS, GTPFHFAQDSWQWL,
FGFPFHFAQDSWQSLS, ACSFAYLYRC, ACFMGDKWVC, ACVLYPKAIC, ACYMGQQFVC,
ACLTAYLHWC, ACTLFPVAYC, ACWLFPYAHC, ACKSINMWLC, ACQTINRWLC,
FGFPEHLLVDFLQSLS, FGFPEHLLVDFLQSLS, FPEHLLVDFLQSL,
AGFPEHLLVDFLQSLS, FAFPEHLLVDFLQSLS, FGAPEHLLVDFLQSLS,
FGFAEHLLVDFLQSLS, FGFPAHLLVDFLQSLS, FGFPEALLVDFLQSLS,
FGFPEHALVDFLQSLS, FGFPEHLAVDFLQSLS, FGFPEHLLADFLQSLS,
FGFPEHLLVAFLQSLS, FGFPEHLLVDALQSLS, FGFPEHLLVDFAQSLS,
FGFPEHLLVDFLASLS, FGFPEHLLVDFLQALS, FGFPEHLLVDFLQSAS,
FGFPEHLLVDFLQSLA, FAFPAHLLVDFLQALA, AAFPAHLLADFLQALA, SPQHLTTDRAQA,
SPQHLTTDRAQALS, SPQHLTTDRAQALS, GSPQHLTTDRAQAL, FGFPQHLTTDRAQSLS,
FGFPQHLTTDWAQSLS, FGFPQHLTTDRLQSLS, FGFPQHLTTDWLQSLS, ATPSHLIIDRAQ,
ATPSHLIIDRAQSLS, FGFPSHLIIDRAQSLS, FGFPSHLIIDWAQSLS,
FGFPSHLIIDWLQSLS, FGFPSHLIIDWSQSLS, FATPSHLIIDWLQSLS, FKPAHVSIDWLQ,
FKPAHVSIDWLQSLS, FGFPAHVSIDWLQSLS, AGFPAHVSIDWLQSLS,
FAFPAHVSIDWLQSLS, FGAPAHVSIDWLQSLS, FGFAAHVSIDWLQSLS,
FGFPAHVSADWLQSLS, FGFPAHVSIDWLQALS, FGFPAHVSIDWLQSLA,
FAFPAHVSIDWLQALA, FGFAAHVSIDWLQSLS, FGFFAHVSIDWLQSLS,
FGFPAHVSIRWLQSLS, FGFPAHVSIEWLQSLS, FGFPAHVSIDWLNSLS,
FGFPAHVSIDWLHSLS, AGFPAHVSIDWLQSLS, PGFPAHVSIDWLQSLS,
WGFPAHVSIDWLQSLS, FAFPAHVSIDWLQSLS, FGFPAHVSIDWLQSLS,
FYFPAHVSIDWLQSLS, FDFPAHVSIDWLQSLS, FGAPAHVSIDWLQSLS,
FGFPAHVSIDWLQLLS, FGFPAHVSIDWLQWLS, FGFPAHVSIDWLQNLS,
FGFPAHVSIDWLQTLS, FGFPAHVSIDWLQYLS, FGFPAHVSIDWLQSIS,
FGFPAHVSIDWLQSLT, FGFPAHVSIDWLQSLY, FAFPAHVSIDWLQALA,
FGFPAHVSIDRAQSLS, FGFPTHVSIDWLQSLS, FGFPFHVSIDWLQSLS,
FGFPAHISIDWLQSLS, FGFPAHIIIDWLQSLS, FGFPAHLTTDWLQSLS,
FGFPAHVFIDWLQSLS, FGFPAHVYIDWLQSLS, FGFPAHVSLDWLQSLS,
FGFPAHVSADWLQSLS, TPTHYYADFSQSLS, FGFPAHVSIDWSQSLS,
FGFPAHVSIDFSQSLS, FGFPSHIIIDWLQSLS, FGFPSHLIIEWLQSLS,
AAFPAHLLADAAQALA, AAFPAHAAADFLQALA, AAFAAHLLADFLQAAA,
AAAPAHLLVDAAQAAA, FAFPAHVFIDWLQSLS; FGFPAHVFIDWLQALS,
FGFPAHVFIDWLQSLA, GFPAHVFIDWLQSLS, FPAHVFIDWLQSLS, PAHVFIDWLQSLS,
FAFPAHVFIDWLQALA, FGFPEHLFVDFLQSLS, FGFPAHVHIDWLQSLS,
FGFPAHVPIDWLQSLS, FGFPSHLFIDWAQSLS, PGFPAHVFIDWLQLIT,
PAHVYIDWLQSLS, FGFPAHVYIDWLQ, FGFPAHVFIDWLQ, DFGFPSHLIIDWLQSLS,
DFGFPAHVFIDWLQSLN, PSHLIIDWLQ, PAHVFIDWLQ, DFGFPAHVTIDWLQSLN,
DFGFPAHVLIDWLQSLN, FGFPAHVYIDWLQSLS, FGFPAHVFIDWLQSLN and
FGFPAHVFIDWLQSLA.
2. Pharmaceutical formulation comprising at least one peptide
according to claim 1.
3. Formulation according to claim 2, characterised in that the
peptide is coupled to a pharmaceutically acceptable carrier,
preferably KLH (Keyhole Limpet Hemocyanin).
Description
[0001] This application is a divisional of U.S. Ser. No. 12/673,081
filed Feb. 11, 2010, incorporated herein by reference, which was a
National Stage of PCT/AT08/000,281 filed Aug. 8, 2008 and claims
the benefit of Austrian application A1 258/2007 filed Aug. 10,
2007.
[0002] The invention relates to the prevention and treatment of
atherosclerosis, atherosclerosis risk diseases and atherosclerosis
sequelae.
[0003] Atherosclerotic sequelae, such as the peripheral arterial
occlusion disease, coronary heart disease as well as the apoplectic
cerebral insultus, are still among the main causes of death in the
United States, Europe, and in large parts of Asia. The development
of the atherosclerosis is considered to be a chronic progressive
inflammation of the arterial vessel wall which is characterized by
a complex interaction of growth factors, cytokines and cell
interactions. According to the "response-to-injury" hypothesis, the
"injury" of the endothelium constitutes the initial event of the
disease, leading to an endothelial dysfunction which triggers a
cascade of cellular interactions culminating in the formation of
the atherosclerotic lesions. As risk factors promoting such an
"injury", exogenous and endogenous influences are mentioned which
correlate statistically significantly with atherosclerosis.
Increased and modified LDL, Lp(a), arterial hypertension, Diabetes
mellitus and hyperhomocysteinaemia are, for instance, counted among
the most important ones of these endothelium-damaging factors.
Since the endothelium does not constitute a rigid, but much rather
an extremely dynamic barrier, a plurality of molecular changes
occur in the course of the endothelial dysfunction in addition to
an increased permeability for lipoproteins, which molecular changes
have a decisive influence on the interaction of monocytes,
T-lymphocytes and endothelial cells. By the expression of
endothelial adhesion molecules of the type of the E, L and P
selectins, integrins, ICMA-1, VCAM-1 and platelet-endothelial-cell
adhesion molecule-1, adhesion of monocytes and T-lymphocytes at the
lumen side occurs. The subsequent migration of the leukocytes over
the endothelium is mediated by MCP-1, interleukin-8, PDGF, MCSF and
osteopontin. Via the so-called scavenger receptor, macrophages and
monocytes resident in the intima are capable of taking up the
penetrated LDL particles and to deposit them as vacuoles of
cholesterol esters in the cytoplasma. The foam cells formed in this
manner accumulate mainly in groups in the region of the vessel
intima and form the "fatty streak" lesions occurring already in
childhood. LDL are lipoproteins of low density and are formed by
catabolic effects of lipolytic enzymes from VLDL particles rich in
triglyceride. Besides their damaging properties on endothelial
cells and smooth muscle cells of the media, LDL moreover has a
chemotactic effect on monocytes and is capable of increasing the
expression of MCSF and MCP-1 of the endothelial cells via gene
amplification. In contrast to LDL, HDL is capable of taking up
cholesterol esters from loaded macrophages mediated by
apolipoprotein E, under formation of so-called HDLc complexes. By
the interaction of SR-B1 receptors, these cholesterol ester-loaded
particles are capable of binding to hepatocytes or to cells of the
adrenal cortex and delivering cholesterol for the production of
bile acids and steroids, respectively. This mechanism is called
reverse cholesterol transport and elucidates the protective
function of HDL. Activated macrophages are capable of presenting
antigens via HLA-DR and thereby activate CD4 and CD8 lymphocytes
which, consequently, are stimulated to secrete cytokines, such as
IFN-gamma and TNF-alpha, and moreover, contribute to increasing the
inflammatory reaction. In the further course of the disease, smooth
muscle cells of the media start to grow into the region of the
intima which has been altered by inflammation. By this, the
intermediary lesion forms at this stage. Starting from the
intermediary lesion, the progressive and complicated lesion will
develop over time, which is morphologically characterized by a
necrotic core, cellular detritus and a fibrinous cap rich in
collagen on the side of the lumen. If the cell number and the
portion of the lipoids increase continuously, tears in the
endothelium will occur, and surfaces with thrombotic properties
will be exposed. Due to the adhesion and activation of thrombocytes
at these tears, granules will be released which contain cytokines,
growth factors and thrombin. Proteolytic enzymes of the macrophages
are responsible for the thinning of the fibrinous cap which, at
last, will lead to a rupture of the plaques with consecutive
thrombosis and stenosing of the vessels and an acute ischemia of
the terminal vessels.
[0004] Various risk factors are held responsible for the forming of
atherosclerotic lesions. Hyperlipoproteinemia, arterial
hypertension and abuse of nicotine are of particular significance
in this respect. A disease which involves an excessive increase in
the total and LDL cholesterol is the familial hypercholesterinemia
(FH). It belongs to the most frequent monogenetically inherited
metabolic diseases. The moderate heterozygous form occurs with a
frequency of 1:500, the homozygous form with 1:1 million clearly
more rarely. Causes of the familial hypercholesterinemia are
mutations in the LDL receptor gene on the short arm of chromosome
19. These mutations may be deletions, insertions or point
mutations. The characteristic finding of the lipoproteins in
familial hypercholesterinemia is an increase in the total and LDL
cholesterol at mostly normal triglyceride and VLDL concentrations.
Often the HDL is lowered. Phenotypically, there is a type
IIAa-hyperlipoproteinemia. In the heterozygous form, the total
cholesterol is increased by the two to three-fold, in the
homozygous form it is increased by the five to six-fold as compared
to the normal level. Clinically the familial hypercholesterinemia
manifests itself by an early coronary sclerosis. As a rule, in
heterozygous men the first symptoms of a coronary heart disease
(CHD) occur between their 30.sup.th and the 40.sup.th year of age,
in women on an average 10 years later. 50% of the afflicted men die
of the consequences of their coronary sclerosis before they are 50
years old. Besides the massively increased LDL levels, also lowered
HDL concentrations are responsible for the rapid progress of
atherosclerosis. Atherosclerotic changes may become manifest also
on extra-cardiac vessels, such as the aorta, the carotid arteries
and peripheral arteries. With the homozygous form of the disease,
the coronary sclerosis develops already in early childhood. The
first myocardial infarction often occurs before the 10.sup.th year
of age, and in most cases the afflicted persons die before they are
20 years old. The development of xanthomas is a function of the
level of the serum cholesterol and the duration of the disease.
Approximately 75% of the heterozygous individuals afflicted who are
more than 20 years old exhibit tendinous xanthomas. The homozygous
individuals have skin and tendon xanthomas in nearly 100%. Lipid
deposits may also occur on the eye lid and in the cornea
(xanthelasmas; Arcus lipoides). These are, however, not a specific
sign of a hypercholesterinemia, since they are also found with
normal cholesterol levels. Furthermore, with the FH, acute
arthritides and tendosynovitides occur frequently. The individual
lipoproteins differ with respect to size and density, since they
contain differently large portions of lipids and proteins,
so-called apoproteins. The density increases with increasing
protein and decreasing lipid portion. Due to their different
densities, they can be separated into different fractions by
ultracentrifugation. This is the basis for the classification of
the lipoproteins into their main groups: chylomicrones,
very-low-density lipoproteins (VLDL), intermediate-density
lipoproteins (IDL), low-density lipoproteins (LDL), high-density
lipoproteins (HDL), lipoprotein (a) (Lp(a)). Among the lipoproteins
with a high atherogenic potential there are primarily the LDL, the
Lp(a) and the VLDL. LDL has a density of approximately
d=1.006-1.063 g/ml. The core is formed by esterified cholesterol
molecules. This highly hydrophobic core is surrounded by an
envelope of phospholipids, non-esterified cholesterol and one
single Apo B100 molecule. Besides, Apoprotein E is found on the
surface of the LDL particles. The function of the LDL consists in
transporting cholesterol to peripheral tissues where--mediated by
the apoprotein B-100--it is taken up into the cells via the LDL
receptor. In comprehensive epidemiologic studies, a positive
correlation between the level of the serum cholesterol and the
occurrence of a coronary heart disease could be demonstrated. LDL
cholesterol levels of higher than 160 mg/dl constitute a high
cardiovascular risk. Besides the level of the LDL cholesterol, also
the level of the vessel-protecting HDL cholesterol plays an
important role when estimating the risk profile for cardiovascular
diseases. Levels of below 35 mg/dl are associated with an increased
risk. VLDL are lipoproteins with a low density (d=0.94-1.006 g/ml)
and a high triglyceride portion. Substantially, VLDL contain
apoprotein C, and small portions of apoproteins B-100 and E.
Different from chylomicrons, VLDL do not consist of food lipids,
but are synthesized in the liver from endogenously formed
triglycerides and secreted into circulation. As with the
chylomicrons, the triglycerides are hydrolyzed by the aproprotein
C-II-activated lipoprotein-lipase, and the free fatty acids are
supplied to the muscle and fat tissue. The remaining
cholesterol-rich VLDL remnants are called intermediate density
lipoproteins because of their higher density. Lipoprotein(a)
(Lp(a)) has a density of 1.05 to 1.12 g/ml and resembles LDL in its
composition. Besides apoprotein B-100, its protein portion consists
of the apoprotein(a) which is characteristic of Lp(a). To date,
very little is known about the physiology and function of the
Lp(a). Since the apoprotein(a) molecule has a high sequence
homology to plasminogen, it is assumed that Lp(a) both promotes the
formation of thrombi on atherosclerotic plaques and also has an
atherogenic effect. Lp(a) is found together with apoprotein B in
atherosclerotic lesions. Retrospective studies have shown a
correlation between increased Lp(a) and a CHD. Likewise, the
metaanalysis of numerous prospective studies has shown that Lp(a)
is an independent risk factor for the occurrence of a CHD. Levels
of between 15 and 35 mg/dl are considered to be normal. So far,
Lp(a) can be influenced neither by diet nor by medicaments.
Therefore, therapy measures are restricted to reducing further risk
factors. In particular, a lowering of the LDL cholesterol seems to
lower the cardiovascular risk of Lp(a). In the pathogenesis of
atherosclerosis, considerable pathophysiologic importance is,
moreover, attributed to coagulation factors. Epidemiologic findings
suggest a correlation between the fibrinogen concentration in
plasma and the development of a coronary heart disease, and,
primarily, a myocardial infarction. In this context, increased
fibrinogen levels (>300 mg/dl) proved to be an independent
indicator and risk factor for cardiovascular diseases. Yet also
high concentrations of the tissue plasminogen activator inhibitor
tPA-I are associated with the occurrence of CHD. The relationship
between hypertriglyceridemia and coronary risk is a different one
in each case, depending on the cause of the elevation of the blood
lipids. Despite the discussion whether or not triglycerides are to
be considered as an independent risk factor it is undisputed that
they play an important role in the pathogenesis of coronary heart
diseases. Incidence of the disease is the highest in patients who
exhibit high LDL cholesterol and a high triglyceride level.
[0005] The cholesterol ester transfer protein (CETP) is a stable
plasma glycoprotein which is responsible for the transfer of
neutral lipids and phospholipids between lipoproteins and which
down-regulates the plasma concentration of HDL. The inhibition of
the CETP lipid transfer activity has already been suggested as a
therapeutic approach for increasing the HDL plasma level. There are
numerous reasons which suggest that the reduction of CETP activity
in plasma should lead to an increase in the HDL levels. Thus, CETP
lowers the HDL concentration by the transfer of cholesterol esters
from HDL to LDL and VLDL. In animal experiments with rabbits and
hamsters, the transient inhibition of CETP with anti-CETP
monoclonal antibodies, antisense oligonucleotides or CETP
inhibitors led to the increase in the HDL levels. Lasting CETP
inhibition with antisense oligonucleotides increased the HDL levels
and, thus, led to a reduction of the atherosclerotic lesions in the
rabbit animal model for atherosclerosis.
[0006] In the literature several CETP inhibitors are described,
some of which are in clinical trials (e.g. Anacetrapib (Krishna R.,
Lancet 370 (9603) (2007): 1907-14) and Torcetrapib (Sikorski, J.
A., J. Med. Chem. 49 (1) (2006): 1-22)).
[0007] In U.S. Pat. No. 5,512,548 and in WO 93/011782, polypeptides
and their analogues are described which are capable of inhibiting
CETP that catalyses the transfer of cholesterol esters from HDL to
VLDL and LDL, and, therefore, have anti-atherosclerotic activity if
administered to a patient. According to these documents, such a
CETP polypeptide inhibitor is derived from apolipoprotein C-I of
various sources, wherein especially N-terminal fragments up to
amino acid 36 have been identified as CETP inhibitors.
[0008] Also in U.S. Pat. No. 5,880,095 A, a CETP-binding peptide is
disclosed which is capable of inhibiting the activity of CETP in an
individual. The CETP-inhibitory protein comprises an N-terminal
fragment of porcine apolipoprotein C-III.
[0009] In the US 2006/0276400 and the WO 96/034888 peptides are
disclosed, which are derived from CETP and comprise T-cell and/or
B-cell epitopes. These peptides are able to induce in vivo the
formation of CETP specific antibodies.
[0010] In US 2004/0087481 and U.S. Pat. No. 6,410,022 B1, peptides
are disclosed which, because of the induction of a CETP-specific
immune response, can be used for the treatment and prevention of
cardiovascular diseases, such as, e.g., atheroslerosis. These
peptides comprise a T helper cell epitope which is not derived from
CETP, and at least one B-cell epitope that comes from CETP and can
be derived directly from the latter. The T helper cell epitope
advantageously is derived from tetanus toxoid and is covalently
bound to at least one B-cell epitope of CETP. By using a T helper
cell epitope that is alien to the organism, it becomes possible to
induce antibodies in the body of an individual, which antibodies
are directed against that peptide portion that consists of at least
one CETP-B-cell epitope.
[0011] In Mao D et al (Vaccine 24 (2006): 4942-4950) the use of a
plasmid comprising a nucleic acid molecule encoding for a B cell
epitope of CETP as vaccine is described.
[0012] In the WO 2006/029982 CETP mimotopes to be used for the
manufacture of a medicament for the treatment or prevention of
atherosclerosis is described.
[0013] Most recently, there have already been suggestions for a
vaccine approach with regard to CETP. Thus, e.g., rabbits have been
treated with a vaccine which contained that peptide of CETP
responsible for the cholesterol-ester transfer as an antigen. The
immunized rabbits had a reduced CETP activity and altered
lipoprotein levels with increased HDL and reduced LDL values.
Moreover, the treated test animals of the atherosclerosis model
also showed reduced atherosclerotic lesions in comparison with
control animals.
[0014] The results of a phase II-clinical study were published,
which study had been carried out by the American biotechnology
company Avant with the vaccine CETi-1 (BioCentury Extra For
Wednesday, Oct. 22, 2003). In this phase II-study, just as in the
preceding phase I-study, a very good safety profile without any
questionable side effects was proven, allowing the conclusion to be
drawn that basically no side effects are to be expected from an
anti-CETP vaccination approach. With regard to efficacy, however,
the Avant vaccine was disappointing since it did not lead to
increased HDL levels significantly better than those attained by a
placebo treatment.
[0015] The problem with the CETi-1 vaccine is that it uses
endogenous antigen. The human immune system is tolerant relative to
endogenous structures, since with most of the endogenous
molecules--other than with CETP--it is vital that no autoantibodies
be formed. Thus, it was the object of the CETi-1 vaccine to break
the endogenous tolerance which, apparently, it has not achieved to
a sufficient extent.
[0016] Thus, it is the object of the present invention to provide
antigens for an anti-CETP vaccine which are selected such that they
are considered as foreign by the immune system and therefore need
not break a self-tolerance. These antigens may be used for
preventing and/or treating atherosclerosis, atherosclerosis risk
diseases and atherosclerosis sequelae.
BRIEF DESCRIPTION OF THE DRAWINGS
[0017] FIG. 1 shows the result of a representative competition
ELISA after screening phage display library Ph.D. 7 with monoclonal
antibody "Paula".
[0018] FIGS. 2a and 2b show the results of 2 typical competition
ELISAs after screening phage display library Ph.D. 12 with
monoclonal antibody "Paula".
[0019] FIGS. 3a and 3b show the results of 2 representative
competition ELISAs after screening phage display library Ph.D. 7
with mAb Frida.
[0020] FIG. 4a shows the result of a representative competition
ELISA after screening phage display library Ph.D. 12 with
monoclonal antibody "Frida".
[0021] FIG. 4b shows binding of monoclonal antibody "Frida" to
ELISA plates coated with mimotope-BSA.
[0022] FIGS. 5a and 5b show the results of a representative
competition ELISA after screening phage display library Ph.D. 12
with monoclonal antibody "Frida".
[0023] FIG. 6 shows the results of a competition ELISA of two
mimotopes after screening phage display library Ph.D. 12 with
monoclonal antibody "Frida".
[0024] FIGS. 7a, 7b, 7c and 7d show the antibody titer (anti mouse
IgG) of in vivo experiments, whereby the following mimotope-BSA
conjugates were injected into mice.
[0025] FIGS. 8a and 8b show the results of two representative
competition ELISA after screening phage display library Ph.D. 7C7
with monoclonal antibody "Frida".
[0026] FIG. 9 shows an in vitro ELISA test for the detection of the
binding between "Frida" and cyclic mimotopes.
[0027] FIGS. 10a and 10b show the results of an inhibition ELISA
assay with FGFPSHLIIDWLQSLS (SEQ ID NO. 179), FGFPAHVFIDWLQSLS (SEQ
ID NO. 222) and FGFPAHVYIDWLQSLS (SEQ ID NO. 223).
[0028] FIG. 11 shows the in vivo induction of antibodies directed
to CETP by mimotopes of the invention that are administered to
mice.
[0029] FIGS. 12a and 12b show the in vivo induction of CETP
specific antibodies by the administration of the mimotopes of the
invention.
[0030] FIG. 13 shows the in vivo induction of antibodies directed
to CETP by mimotopes of the invention that are administered to
mice.
[0031] FIG. 14 shows a CETP activity assay, wherein 0.6 .mu.l human
serum (with endogenous CETP activity) is mixed with serum from
wild-type mice (not containing CETP activity) vaccinated with
KLH/Alum (negative control group), p4703-KLH/Alum (original CETP
epitope), or p4361 (or p4362 or p 4325) mimotope, respectively.
[0032] FIG. 15 shows that the addition of p4325-KLH/Alum to human
serum inhibits significantly CETP activity.
[0033] FIG. 16 shows that the addition of p4361-KLH/Alum to human
serum inhibits significantly CETP activity.
[0034] FIG. 17 shows that the addition of p4362-KLH/Alum to human
serum inhibits significantly CETP activity.
[0035] FIG. 18a shows an inhibition ELISA with mimotopes (Coat. 1
.mu.M 4073 peptide, detection .alpha. IgG1).
[0036] FIG. 18b shows an inhibition ELISA with mimotopes (Coat. 1
.mu.M 4073 peptide, detection .alpha. IgG1).
[0037] FIG. 18c shows a inhibition ELISA with mimotopes screen
PhD12 Frida and Ala-exchange for mimotope characterisation/mAb
Frida (Coat 1 .mu.M 4073. Detection .alpha.IgG1.)
[0038] FIG. 19a shows a peptide ELISA, immunisation with
C-DFGFPAHVYIDWLQSLS (p4628-KLH/Alum) (SEQ ID NO. 236), titre to
original epitope.
[0039] FIG. 19b shows a peptide ELISA, immunisation with
C-FGFPAHVFIDWLQSLN (p4474-KLH/Alum) (SEQ ID NO. 230), titre to
original epitope.
[0040] FIG. 19c shows a peptide ELISA, immunisation with
C-FGFPAHVFIDWLQSLN (p4474-KLH/Alum) (SEQ ID NO. 230), titre to
injected mimotope.
[0041] FIG. 19d shows an anti-protein ELISA. Mice were injected 3
times with 30 .mu.g of the indicated mimotopes coupled to KLH with
Alum as adjuvant. Sera from each group (comprising 5 mice) were
pooled, diluted 1:100 and tested on ELISA plates coated with
purified rabbit CETP.
[0042] FIG. 19e shows an anti-protein ELISA, wherein mice were
injected 3 times with 30 .mu.g of the indicated mimotopes coupled
to KLH with Alum as adjuvant. Mouse sera (from single mice) were
diluted 1:100 and tested on ELISA plates coated with purified
rabbit CETP.
[0043] Therefore the present invention relates to the use of a
compound comprising the amino acid sequence
[0044] (Z.sub.1).sub.nX.sub.1X.sub.2X.sub.3X.sub.4(Z.sub.2).sub.m,
(SEQ ID NO. 1)
wherein Z.sub.1 is an amino acid residue other than C, X.sub.1 is
an amino acid residue selected from the group consisting of D, A,
R, E, S, N, T and G, X.sub.2 is an amino acid residue selected from
the group consisting of F, A, W, R, S, L, Q, V and M, X.sub.3 is an
amino acid residue selected from the group consisting of L, A, S,
W, E, R, I and H, X.sub.4 is an amino acid residue selected from
the group consisting of Q, A, H, D, K, R, S and E, Z.sub.2 is an
amino acid residue other than C, n is an integer between 0 and 10,
preferably between 0 and 9, m is an integer between 0 and 3, is
not, or does not comprise, a 4- to 16-mer polypeptide fragment of
the cholesterol ester transport protein (CETP) or a CETP-epitope,
said compound having a binding capacity to an antibody which is
specific for the natural CETP glycoprotein, or comprising an amino
acid sequence selected from the group consisting of SYHATFL (SEQ ID
NO. 2), TMAFPLN (SEQ ID NO. 3), HYHGAFL (SEQ ID NO. 4), EHHDIFL
(SEQ ID NO. 5), TGLSVFL (SEQ ID NO. 6), WMPSLFY (SEQ ID NO. 7),
SMPWWFF (SEQ ID NO. 8), TMPLLFW (SEQ ID NO. 9), DTWPGLE (SEQ ID NO.
10), SMPPIFY (SEQ ID NO. 11), MPLWWWD (SEQ ID NO. 12), SMPNLFY (SEQ
ID NO. 13), RMPPIFY (SEQ ID NO. 14), NPFEVFL (SEQ ID NO. 15),
TLPNWFW (SEQ ID NO. 16), SMPLTFY (SEQ ID NO. 17), SPHPHFL (SEQ ID
NO. 18), NFMSIGL (SEQ ID NO. 19), SQFLASL (SEQ ID NO. 20), WSWPGLN
(SEQ ID NO. 21), IAWPGLD (SEQ ID NO. 22), SKFMDTL (SEQ ID NO. 23),
SMPMVFY (SEQ ID NO. 24), YEWVGLM (SEQ ID NO. 25), KGFLDHL (SEQ ID
NO. 26), HQSDDKMPWWFF (SEQ ID NO. 27), YVWQDPSFTTFF (SEQ ID NO.
28), YVWQDPSFTTFF (SEQ ID NO. 29), LPQTHPLHLLED (SEQ ID NO. 30),
GPVSIYADTDFL (SEQ ID NO. 31), DSNDTLTLAAFL (SEQ ID NO. 32),
NGSPALSHMLFL (SEQ ID NO. 33), TDYDPMWVFFGY (SEQ ID NO. 34),
IFPLDSQWQTFW (SEQ ID NO. 35), NESMPDLFYQPS (SEQ ID NO. 36),
DWGDKYFSSFWN (SEQ ID NO. 37), VSAYNNV (SEQ ID NO. 38) and WPLHLWQ
(SEQ ID NO. 39) for producing a medicament for preventing and/or
treating atherosclerosis, atherosclerosis risk diseases and
atherosclerosis sequelae.
[0045] The present invention provides CETP mimotopes for these
purposes. These mimotopes are able to induce antibodies which are
able to inhibit CETP enzyme activity. The CETP mimotopes according
to the present invention preferably are antigenic polypeptides
which in their amino acid sequence vary from the amino acid
sequence of CETP or of fragments of CETP. In this respect, the
inventive mimotopes may comprise one or more non-natural amino
acids (i.e. not from the 20 "classical" amino acids) or they may be
completely assembled of such non-natural amino acids. Moreover, the
inventive antigens which induce anti-CETP antibodies may be
assembled of D- or L-amino acids or of combinations of DL-amino
acids and, optionally, they may have been changed by further
modifications, ring closures or derivatizations. Suitable
anti-CETP-antibody-inducing antigens may be provided from
commercially available peptide libraries. Preferably, these
peptides are at least 4 amino acid residues in length, in
particular at least 7 amino acids, and preferred lengths may be up
to 16, preferably up to 14 or 20 amino acids (e.g. 5 to 16 amino
acid residues). According to the invention, however, also longer
peptides may very well be employed as anti-CETP-antibody-inducing
antigens. Furthermore the mimotopes of the present invention may
also be part of a polypeptide and consequently comprising at their
N- and/or C-terminus at least one further amino acid residue.
[0046] The mimotopes of the present invention are capable to bind
to antibodies which may be obtained by administration of
C-FGFPEHLLVDFLQSLS (SEQ ID NO. 146) (16 C-terminal amino acids of
CETP protein) coupled to KLH or other carriers to mammals. Once
administered to a mammal the mimotopes are able to induce a
corresponding immune response, so that antibodies directed against
CETP are produced in said mammal.
[0047] The CETP-mimotopes (i.e. anti-CETP-antibody-inducing
antigens) of the present invention can be identified and prepared
by various methods, including phage libraries or peptide libraries.
They can be produced and identified for instance by means of
combinatorial chemistry or by means of high throughput screening
techniques for the most varying structures (Display: A Laboratory
Manual by Carlos F. Barbas (Editor), et al.; Willats W G Phage
display: practicalities and prospects. Plant Mol. Biol. 2002;
50(6):837-54).
[0048] Furthermore, according to the invention also
anti-CETP-antibody-inducing antigens based on nucleic acids
("aptamers") may be employed, and these, too, may be found with the
most varying (oligonucleotide) libraries (e.g. with 2-180 nucleic
acid residues) (e.g. Burgstaller et al., Curr. Opin. Drug Discov.
Dev. 5(5) (2002), 690-700; Famulok et al., Acc. Chem. Res. 33
(2000), 591-599; Mayer et al., PNAS 98 (2001), 4961-4965, etc.). In
anti-CETP-antibody-inducing antigens based on nucleic acids, the
nucleic acid backbone can be provided e.g. by the natural
phosphor-diester compounds, or also by phosphorotioates or
combinations or chemical variations (e.g. as PNA), wherein as
bases, according to the invention primarily U, T, A, C, G, H and mC
can be employed. The 2'-residues of the nucleotides which can be
used according to the present invention preferably are H, OH, F,
Cl, NH.sub.2, O-methyl, O-ethyl, O-propyl or O-butyl, wherein the
nucleic acids may also be differently modified, i.e. for instance
with protective groups, as they are commonly employed in
oligonucleotide synthesis. Thus, aptamer-based
anti-CETP-antibody-inducing antigens are also preferred
anti-CETP-antibody-inducing antigens within the scope of the
present invention.
[0049] According to the present invention the term "mimotope"
refers to a molecule which has a conformation that has a topology
equivalent to the epitope of which it is a mimic. The mimotope
binds to the same antigen-binding region of an antibody which binds
immunospecifically to a desired antigen. The mimotope will elicit
an immunological response in a host that is reactive to the antigen
to which it is a mimic. The mimotope may also act as a competitor
for the epitope of which it is a mimic in in vitro inhibition
assays (e.g. ELISA inhibition assays) which involve the epitope and
an antibody binding to said epitope. However, a mimotope of the
present invention may not necessarily prevent or compete with the
binding of the epitope of which it is a mimic in an in vitro
inhibition assay although it is capable to induce a specific immune
response when administered to a mammal.
[0050] As used herein, the term "epitope" refers to an immunogenic
region of an antigen which is recognized by a particular antibody
molecule. In general, an antigen will possess one or more epitopes,
each capable of binding an antibody that recognizes the particular
epitope.
[0051] The abbreviations for the amino acid residues disclosed in
the present invention follow the IUPAC recommendations:
TABLE-US-00001 Amino Acid 3-Letter Code 1-Letter Code Alanine Ala A
Arginine Arg R Asparagine Asn N Aspartic Asp D Cysteine Cys C
Glutamic Glu E Glutamine Gln Q Glycine Gly G Histidine His H
Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M
Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T
Tryptophan Trp W Tyrosine Tyr Y Valine Val V
[0052] The mimotopes of the present invention can be synthetically
produced by chemical synthesis methods which are well known in the
art, either as an isolated peptide or as a part of another peptide
or polypeptide. Alternatively, the peptide mimotope can be produced
in a microorganism which produces the peptide mimotope which is
then isolated and if desired, further purified. The peptide
mimotope can be produced in microorganisms such as bacteria, yeast
or fungi, in eukaryote cells such as a mammalian or an insect
cells, or in a recombinant virus vector such as adenovirus,
poxvirus, herpesvirus, Simliki forest virus, baculovirus,
bacteriophage, sindbis virus or sendai virus. Suitable bacteria for
producing the peptide mimotope include E. coli, B. subtilis or any
other bacterium that is capable of expressing peptides such as the
peptide mimotope. Suitable yeast types for expressing the peptide
mimotope include Saccharomyces cerevisiae, Schizosaccharomyces
pombe, Candida, Pichia pastoris or any other yeast capable of
expressing peptides. Corresponding methods are well known in the
art. Also methods for isolating and purifying recombinantly
produced peptides are well known in the art and include e.g. as gel
filtration, affinity chromatography, ion exchange chromatography
etc.
[0053] To facilitate isolation of the peptide mimotope, a fusion
polypeptide may be made wherein the peptide mimotope is
translationally fused (covalently linked) to a heterologous
polypeptide which enables isolation by affinity chromatography.
Typical heterologous polypeptides are His-Tag (e.g. His.sub.6; 6
histidine residues), GST-Tag (Glutathione-S-transferase) etc. The
fusion polypeptide facilitates not only the purification of the
mimotopes but can also prevent the mimotope polypeptide from being
degraded during purification. If it is desired to remove the
heterologous polypeptide after purification the fusion polypeptide
may comprise a cleavage site at the junction between the peptide
mimotope and the heterologous polypeptide. The cleavage site
consists of an amino acid sequence that is cleaved with an enzyme
specific for the amino acid sequence at the site (e.g.
proteases).
[0054] The mimotopes of the present invention may also modified at
or nearby their N- and/or C-termini so that at said positions a
cysteine residue is bound thereto. In a preferred embodiment
terminally positioned (located at the N- and C-termini of the
peptide) cysteine residues are used to cyclize the peptides through
a disulfide bond.
[0055] The mimotopes of the present invention may also be used in
various assays and kits, in particular in immunological assays and
kits. Therefore, it is particularly preferred that the mimotope may
be part of another peptide or polypeptide, particularly an enzyme
which is used as a reporter in immunological assays. Such reporter
enzymes include e.g. alkaline phosphatase or horseradish
peroxidase.
[0056] The term "atherosclerosis sequelae" or "sequelae of
atherosclerosis" refers to the diseases which are a consequence of
atherosclerose. These diseases include among others peripheral
arterial occlusive disease, coronary heart disease and apoplectic
cerebral insultus (see e.g. Steinberg D. J. Lipid Res. (2005) 46:
179-190; Steinberg D et al. J. Lipid Res (2006) 47: 1339-1351).
[0057] According to another preferred embodiment of the present
invention X.sub.1 is D and X.sub.4 is Q or H, preferably Q. Such a
molecule preferably comprises at its N-terminus further amino acid
residues having the sequence X.sub.a X.sub.b X.sub.c X.sub.d
X.sub.e X.sub.f (SEQ ID NO. 41), wherein X.sub.a is P, Y, T or K,
X.sub.b is an amino acid residue other than C, X.sub.c is H,
X.sub.d is Y, L, H, V, T, I or F, X.sub.e is Y, I, P, L, Q, S, R,
T, F or A and X.sub.f is A, W, V, Q, L, S, I, R or T.
[0058] According to a preferred embodiment of the present invention
n is 7, 8 or 9, Z.sub.1 is an amino acid residue other than C or
selected from the group consisting of F, G, F, A, P, W, Y, S, G, D,
L, E, K, T, P, I and M, preferably from the group consisting of F,
G, F, A, P, Y, T, S, G, K and D, and Z.sub.2 is selected from the
group consisting of S, L, A, W, L, N, T, I, Y and H.
[0059] According to a further preferred embodiment of the present
invention X.sub.1 is selected from the group consisting of D, A, R,
E and L, X.sub.2 is selected from the group consisting of F, A, W,
Q and R, X.sub.3 is selected from the group consisting of L, A and
S, and X.sub.4 is selected from the group consisting of Q, A and
H.
[0060] According to a preferred embodiment of the present invention
X.sub.1 is D, X.sub.2 is selected from the group consisting of F, Q
and W, X.sub.3 is L or S and X.sub.4 is Q or H.
[0061] According to a preferred embodiment of the present invention
the compound comprises the amino acid sequence FX.sub.8
(F).sub.oPX.sub.9HX.sub.10X.sub.11X.sub.12DX.sub.2X.sub.3X.sub.4X.sub.5X.-
sub.6X.sub.7 (SEQ ID NO. 42), wherein
X.sub.8 is selected from the group consisting of G, A, F, Y and K,
X.sub.9 is selected from the group consisting of E, Y, A, Q, K and
S, X.sub.10 is selected from the group consisting of H, V, L, F and
I, X.sub.11 is selected from the group consisting of L, W, S, I, F
and Y,
X.sub.12 is V, T, F or I,
X.sub.5 is S or Y,
X.sub.6 is L, A or I,
X.sub.7 is S, N or T, and
[0062] o is 0 or 1.
[0063] The compound of the present invention comprises preferably
the amino acid sequence
X.sub.1X.sub.2X.sub.3X.sub.4X.sub.5X.sub.6X.sub.7 (SEQ ID NO. 43),
wherein X.sub.1 is selected from the group consisting of D, S, N, T
and G, X.sub.2 is F, X.sub.3 is L, X.sub.4 is selected from the
group consisting of Q, D, K, R, S and E, X.sub.5 is S or T, X.sub.6
is L and X.sub.7 is an amino acid residue other than C, preferably
selected from the group consisting of S, T, A, M, F and W.
[0064] According to a preferred embodiment of the present invention
the amino acid sequence is selected from the group consisting of
SSLELFL (SEQ ID NO. 44), SFLDTLT (SEQ ID NO. 45), NFLKTLS (SEQ ID
NO. 46), DFLRTLT (SEQ ID NO. 47), AFLDTLV (SEQ ID NO. 48), TFLSSLA
(SEQ ID NO. 49), GFLDSLM (SEQ ID NO. 50), SPHPHFL (SEQ ID NO. 51),
SNFLKTL (SEQ ID NO. 52), TGFLATL (SEQ ID NO. 53), SDFLRAL (SEQ ID
NO. 54), SANPRDFLETLF (SEQ ID NO. 55), RMFPESFLDTLW (SEQ ID NO.
56), TIYDSFLDSLAS (SEQ ID NO. 57), KPYLLKDFLEAL (SEQ ID NO. 58),
AMGPYDALDLFL (SEQ ID NO. 59), TWNPIESFLESL (SEQ ID NO. 60),
QYQTPLTFLEAL (SEQ ID NO. 61), RHISPATFLEAL (SEQ ID NO. 62),
HTDSFLSTFYGD (SEQ ID NO. 63), ADSTFTSFLQTL (SEQ ID NO. 64),
GPVSIYADTDFL (SEQ ID NO. 65), DSNDTLTLAAFL (SEQ ID NO. 66),
TPTHYYADFSQL (SEQ ID NO. 67), LPGHLIWDSLHY (SEQ ID NO. 68),
LPQTHPLHLLED (SEQ ID NO. 69), IPYHHLVDQLHH (SEQ ID NO. 70),
YPYHVQVDVLQN (SEQ ID NO. 71), IPSHHLQDSLQL (SEQ ID NO. 72),
EYAHHTSLDLRQ (SEQ ID NO. 73), EPLHFRSDRIQA (SEQ ID NO. 74),
ATPSHLIIDRAQ (SEQ ID NO. 75), APKHLYADMSQA (SEQ ID NO. 76),
FKPAHVSIDWLQ (SEQ ID NO. 77), MPAHLSRDLRQS (SEQ ID NO. 78),
NPKHYSIDRHQA (SEQ ID NO. 79), SPQHLTTDRAQA (SEQ ID NO. 80),
TPFHFAQDSWQW (SEQ ID NO. 81), TPTHYYADFSQLLS (SEQ ID NO. 82),
TPTHYYADFSQSLS (SEQ ID NO. 83), GTPTHYYADFSQLL (SEQ ID NO. 84),
GTPTHYYADFSQSL (SEQ ID NO. 85), FGTPTHYYADFSQSLS (SEQ ID NO. 86),
FGFPTHYYADFSQSLS (SEQ ID NO. 87), LPGHLIWDSLHY (SEQ ID NO. 88),
LPGHLIWDSLHYL (SEQ ID NO. 89), LPGHLIWDSLHYLS (SEQ ID NO. 90),
LPGHLIWDSLHSL (SEQ ID NO. 91), LPGHLIWDSLHSLS (SEQ ID NO. 92),
GLPGHLIWDSLHYL (SEQ ID NO. 93), GLPGHLIWDSLHSL (SEQ ID NO. 94),
FGLPGHLIWDSLHSLS (SEQ ID NO. 95), FGFPGHLIWDSLHSLS (SEQ ID NO. 96),
LPQTHPLHLLED (SEQ ID NO. 97), IPYHHLVDQLHH (SEQ ID NO. 98),
IPYHHLVDQLHLS (SEQ ID NO. 99), IPYHHLVDQLHSLS (SEQ ID NO. 100),
FGIPYHHLVDQLHHLS (SEQ ID NO. 101), FGFPYHHLVDQLHSLS (SEQ ID NO.
102), YPYHVQVDVLQN (SEQ ID NO. 103), YPYHVQVDVLQNLS (SEQ ID NO.
104), YPYHVQVDVLQSLS (SEQ ID NO. 105), FGYPYHVQVDVLQNLS (SEQ ID NO.
106), FGFPYHVQVDVLQSLS (SEQ ID NO. 107), IPSHHLQDSLQL (SEQ ID NO.
108), IPSHHLQDSLQLLS (SEQ ID NO. 109), IPSHHLQDSLQSLS (SEQ ID NO.
110), GIPSHHLQDSLQLL (SEQ ID NO. 111), FGIPSHHLQDSLQLLS (SEQ ID NO.
112), FGFPSHHLQDSLQSLS (SEQ ID NO. 113), EYAHHTSLDLRQ (SEQ ID NO.
114), EPLHFRSDRIQA (SEQ ID NO. 115), EPLHFRSDRIQALS (SEQ ID NO.
116), EPLHFRSDRIQSLS (SEQ ID NO. 117), GEPLHFRSDRIQAL (SEQ ID NO.
118), FGEPLHFRSDRIQALS (SEQ ID NO. 119), FGFPLHFRSDRIQSLS (SEQ ID
NO. 120), APKHLYADMSQA (SEQ ID NO. 121), APKHLYADMSQALS (SEQ ID NO.
122), APKHLYADMSQSLS (SEQ ID NO. 123), GAPKHLYADMSQAL (SEQ ID NO.
124), FGFPKHLYADMSQSLS (SEQ ID NO. 125), MPAHLSRDLRQS (SEQ ID NO.
126), MPAHLSRDLRQSL (SEQ ID NO. 127), MPAHLSRDLRQSLS (SEQ ID NO.
128), GMPAHLSRDLRQSL (SEQ ID NO. 129), FGFPAHLSRDLRQSLS (SEQ ID NO.
130), NPKHYSIDRHQA (SEQ ID NO. 131), TPFHFAQDSWQW (SEQ ID NO. 132),
TPFHFAQDSWQWLS (SEQ ID NO. 133), TPFHFAQDSWQSLS (SEQ ID NO. 134),
GTPFHFAQDSWQWL (SEQ ID NO. 135), FGFPFHFAQDSWQSLS (SEQ ID NO. 136),
ACSFAYLYRC (SEQ ID NO. 137), ACFMGDKWVC (SEQ ID NO. 138),
ACVLYPKAIC (SEQ ID NO. 139), ACYMGQQFVC (SEQ ID NO. 140),
ACLTAYLHWC (SEQ ID NO. 141), ACTLFPVAYC (SEQ ID NO. 142),
ACWLFPYAHC (SEQ ID NO. 143), ACKSINMWLC (SEQ ID NO. 144),
ACQTINRWLC (SEQ ID NO. 145), FGFPEHLLVDFLQSLS (SEQ ID NO. 146),
FGFPEHLLVDFLQSLS (SEQ ID NO. 147), FPEHLLVDFLQSL (SEQ ID NO. 148),
AGFPEHLLVDFLQSLS (SEQ ID NO. 149), FAFPEHLLVDFLQSLS (SEQ ID NO.
150), FGAPEHLLVDFLQSLS (SEQ ID NO. 151), FGFAEHLLVDFLQSLS (SEQ ID
NO. 152), FGFPAHLLVDFLQSLS (SEQ ID NO. 153), FGFPEALLVDFLQSLS (SEQ
ID NO. 154), FGFPEHALVDFLQSLS (SEQ ID NO. 155), FGFPEHLAVDFLQSLS
(SEQ ID NO. 156), FGFPEHLLADFLQSLS (SEQ ID NO. 157),
FGFPEHLLVAFLQSLS (SEQ ID NO. 158), FGFPEHLLVDALQSLS (SEQ ID NO.
159), FGFPEHLLVDFAQSLS (SEQ ID NO. 160), FGFPEHLLVDFLASLS (SEQ ID
NO. 161), FGFPEHLLVDFLQALS (SEQ ID NO. 162), FGFPEHLLVDFLQSAS (SEQ
ID NO. 163), FGFPEHLLVDFLQSLA (SEQ ID NO. 164), FAFPAHLLVDFLQALA
(SEQ ID NO. 165), AAFPAHLLADFLQALA (SEQ ID NO. 166), SPQHLTTDRAQA
(SEQ ID NO. 167), SPQHLTTDRAQALS (SEQ ID NO. 168), SPQHLTTDRAQSLS
(SEQ ID NO. 169), GSPQHLTTDRAQAL (SEQ ID NO. 170), FGFPQHLTTDRAQSLS
(SEQ ID NO. 171), FGFPQHLTTDWAQSLS (SEQ ID NO. 172),
FGFPQHLTTDRLQSLS (SEQ ID NO. 173), FGFPQHLTTDWLQSLS (SEQ ID NO.
174), ATPSHLIIDRAQ (SEQ ID NO. 175), ATPSHLIIDRAQSLS (SEQ ID NO.
176), FGFPSHLIIDRAQSLS (SEQ ID NO. 177), FGFPSHLIIDWAQSLS (SEQ ID
NO. 178), FGFPSHLIIDWLQSLS (SEQ ID NO. 179), FGFPSHLIIDWSQSLS (SEQ
ID NO. 180), FATPSHLIIDWLQSLS (SEQ ID NO. 181), FKPAHVSIDWLQ (SEQ
ID NO. 182), FKPAHVSIDWLQSLS (SEQ ID NO. 183), FGFPAHVSIDWLQSLS
(SEQ ID NO. 184), AGFPAHVSIDWLQSLS (SEQ ID NO. 185),
FAFPAHVSIDWLQSLS (SEQ ID NO. 186), FGAPAHVSIDWLQSLS (SEQ ID NO.
187), FGFAAHVSIDWLQSLS (SEQ ID NO. 188), FGFPAHVSADWLQSLS (SEQ ID
NO. 189), FGFPAHVSIDWLQALS (SEQ ID NO. 190), FGFPAHVSIDWLQSLA (SEQ
ID NO. 191), FAFPAHVSIDWLQALA (SEQ ID NO. 192), FGFAAHVSIDWLQSLS
(SEQ ID NO. 193), FGFFAHVSIDWLQSLS (SEQ ID NO. 194),
FGFPAHVSIRWLQSLS (SEQ ID NO. 195), FGFPAHVSIEWLQSLS (SEQ ID NO.
196), FGFPAHVSIDWLNSLS (SEQ ID NO. 197), FGFPAHVSIDWLHSLS (SEQ ID
NO. 198), AGFPAHVSIDWLQSLS (SEQ ID NO. 199), PGFPAHVSIDWLQSLS (SEQ
ID NO. 200), WGFPAHVSIDWLQSLS (SEQ ID NO. 201), FAFPAHVSIDWLQSLS
(SEQ ID NO. 202), FSFPAHVSIDWLQSLS (SEQ ID NO. 203),
FYFPAHVSIDWLQSLS (SEQ ID NO. 204), FDFPAHVSIDWLQSLS (SEQ ID NO.
205), FGAPAHVSIDWLQSLS (SEQ ID NO. 206), FGFPAHVSIDWLQLLS (SEQ ID
NO. 207), FGFPAHVSIDWLQWLS (SEQ ID NO. 208), FGFPAHVSIDWLQNLS (SEQ
ID NO. 209), FGFPAHVSIDWLQTLS (SEQ ID NO. 210), FGFPAHVSIDWLQYLS
(SEQ ID NO. 211), FGFPAHVSIDWLQSIS (SEQ ID NO. 212),
FGFPAHVSIDWLQSLT (SEQ ID NO. 213), FGFPAHVSIDWLQSLY (SEQ ID NO.
214), FAFPAHVFIDWLQALA (SEQ ID NO. 215), FGFPAHVSIDRAQSLS (SEQ ID
NO. 216), FGFPTHVSIDWLQSLS (SEQ ID NO. 217), FGFPFHVSIDWLQSLS (SEQ
ID NO. 218), FGFPAHISIDWLQSLS (SEQ ID NO. 219), FGFPAHIIIDWLQSLS
(SEQ ID NO. 220), FGFPAHLTTDWLQSLS (SEQ ID NO. 221),
FGFPAHVFIDWLQSLS (SEQ ID NO. 222), FGFPAHVYIDWLQSLS (SEQ ID NO.
223), FGFPAHVSLDWLQSLS (SEQ ID NO. 224), FGFPAHVSADWLQSLS (SEQ ID
NO. 225), TPTHYYADFSQSLS (SEQ ID NO. 226), FGFPAHVSIDWSQSLS (SEQ ID
NO. 227), FGFPAHVSIDFSQSLS (SEQ ID NO. 228), FGFPSHIIIDWLQSLS (SEQ
ID NO. 239), FGFPSHLIIEWLQSLS (SEQ ID NO. 240), AAFPAHLLADAAQALA
(SEQ ID NO. 241), AAFPAHAAADFLQALA (SEQ ID NO. 242),
AAFAAHLLADFLQAAA (SEQ ID NO. 243), AAAPAHLLVDAAQAAA (SEQ ID NO.
244), FAFPAHVFIDWLQSLS (SEQ ID NO. 245); FGFPAHVFIDWLQALS (SEQ ID
NO. 246), FGFPAHVFIDWLQSLA (SEQ ID NO. 247), GFPAHVFIDWLQSLS (SEQ
ID NO. 248), FPAHVFIDWLQSLS (SEQ ID NO. 249), PAHVFIDWLQSLS (SEQ ID
NO. 250), FAFPAHVFIDWLQALA (SEQ ID NO. 251), FGFPEHLFVDFLQSLS (SEQ
ID NO. 252), FGFPAHVHIDWLQSLS (SEQ ID NO. 253), FGFPAHVPIDWLQSLS
(SEQ ID NO. 254), FGFPSHLFIDWAQSLS (SEQ ID NO. 255),
PGFPAHVFIDWLQLIT (SEQ ID NO. 256), PAHVYIDWLQSLS (SEQ ID NO. 257),
FGFPAHVYIDWLQ (SEQ ID NO. 258), FGFPAHVFIDWLQ (SEQ ID NO. 259),
DFGFPSHLIIDWLQSLS (SEQ ID NO. 235), DFGFPAHVFIDWLQSLN (SEQ ID NO.
260), PSHLIIDWLQ (SEQ ID NO. 261), PAHVFIDWLQ (SEQ ID NO. 262),
DFGFPAHVTIDWLQSLN (SEQ ID NO. 263), DFGFPAHVLIDWLQSLN (SEQ ID NO.
264), FGFPAHVFIDWLQSLN (SEQ ID NO. 230) and FGFPAHVFIDWLQSLA (SEQ
ID NO. 265).
[0065] Particularly preferred mimotopes to be used according to the
present invention are SANPRDFLETLF (SEQ ID NO. 55), RMFPESFLDTLW
(SEQ ID NO. 56), SFLDTLT (SEQ ID NO. 45), NFLKTLS (SEQ ID NO. 46),
DFLRTLT (SEQ ID NO. 47), TFLSSLA (SEQ ID NO. 49), GFLDSLM (SEQ ID
NO. 50), FGFPYHVQVDVLQSLS (SEQ ID NO. 107), FGFPSHLIIDRAQSLS (SEQ
ID NO. 177), FKPAHVSIDWLQSLS (SEQ ID NO. 183), FGFPAHVSIDWLQSLS
(SEQ ID NO. 184), FGFPQHLTTDRAQSLS (SEQ ID NO. 171),
FGFPTHYYADFSQSLS (SEQ IN NO. 87), FGFPGHLIWDSLHSLS (SEQ ID NO. 96),
FGFPYHHLVDQLHSLS (SEQ ID NO. 102), FGFPSHHLQDSLQSLS (SEQ ID NO.
113), FGFPLHFRSDRIQSLS (SEQ ID NO. 120), FGFPKHLYADMSQSLS (SEQ ID
NO. 125), FGFPAHLSRDLRQSLS (SEQ ID NO. 130) and FGFPFHFAQDSWQSLS
(SEQ ID NO. 136).
[0066] Especially preferred mimotopes of the present invention are
FGFPSHLIIDWLQSLS (SEQ ID NO. 179), FGFPAHVFIDWLQSLS (SEQ ID NO.
222) and FGFPAHVYIDWLQSLS(SEQ ID NO. 223).
[0067] Further preferred mimotopes are FGFPAHVWIDWLQSLS (SEQ ID NO.
229), FGFPAHVFIDWLQSLN (SEQ ID NO. 230), FGFPAHFSIDWLQSLS (SEQ ID
NO. 231), FGFPAHVSFDWLQSLS (SEQ ID NO. 232), FGFPEHVFIDWLQSLS (SEQ
ID NO. 233), DFGFPAHVFIDWLQSLS (SEQ ID NO. 234), DFGFPSHLIIDWLQSLS
(SEQ ID NO. 235), DFGFPAHVYIDWLQSLS (SEQ ID NO. 236),
FGFPQHLFTDWLQSLS (SEQ ID NO. 237) and FGFPKHLLVDFLQSLS (SEQ ID NO.
238).
[0068] According to a preferred embodiment of the present invention
the compound is coupled to a pharmaceutically acceptable carrier,
preferably KLH (Keyhole Limpet Hemocyanin), tetanus toxoid,
albumin-binding protein, bovine serum albumin, a dendrimer (MAP;
Biol. Chem. 358: 581), peptide linkers (or flanking regions) as
well as the adjuvant substances described in Singh et al., Nat.
Biotech. 17 (1999), 1075-1081 (in particular those in Table 1 of
that document), and O'Hagan et al., Nature Reviews, Drug Discovery
2 (9) (2003), 727-735 (in particular the endogenous
immuno-potentiating compounds and delivery systems described
therein), or mixtures thereof. The conjugation chemistry (e.g. via
heterobifunctional compounds such as GMBS and of course also others
as described in "Bioconjugate Techniques", Greg T. Hermanson) in
this context can be selected from reactions known to the skilled
man in the art. Moreover, the vaccine composition may be formulated
with an adjuvant, preferably a low soluble aluminium composition,
in particular aluminium hydroxide. Of course, also adjuvants like
MF59 aluminium phosphate, calcium phosphate, cytokines (e.g., IL-2,
IL-12, GM-CSF), saponins (e.g., QS21), MDP derivatives, CpG oligos,
LPS, MPL, polyphosphazenes, emulsions (e.g., Freund's, SAF),
liposomes, virosomes, iscoms, cochleates, PLG microparticles,
poloxamer particles, virus-like particles, heat-labile enterotoxin
(LT), cholera toxin (CT), mutant toxins (e.g., LTK63 and LTR72),
microparticles and/or polymerized liposomes may be used.
[0069] The compound of the present invention is preferably bound to
the carrier or adjuvant via a linker, which is selected from the
group consisting of NHS-poly (ethylene oxide) (PEO) (e.g.
NHS-PEO.sub.4-maleimide).
[0070] A vaccine which comprises the present compound (mimotope)
and the pharmaceutically acceptable carrier may be administered by
any suitable mode of application, e.g. i.d., i.v., i.p., i.m.,
intranasally, orally, subcutaneously, etc. and in any suitable
delivery device (O'Hagan et al., Nature Reviews, Drug Discovery 2
(9), (2003), 727-735). The compound of the present invention is
preferably formulated for intravenous, subcutaneous, intradermal or
intramuscular administration (see e.g. "Handbook of Pharmaceutical
Manufacturing Formulations", Sarfaraz Niazi, CRC Press Inc,
2004).
[0071] Typically, the vaccine contains the compound according to
the invention in an amount of from 0.1 ng to 10 mg, preferably 10
ng to 1 mg, in particular 100 ng to 100 .mu.g, or, alternatively,
e.g. 100 fmol to 10 .mu.mol, preferably 10 pmol to 1 .mu.mol, in
particular 100 pmol to 100 nmol. Typically, the vaccine may also
contain auxiliary substances, e.g. buffers, stabilizers etc.
[0072] Another aspect of the present invention relates to a peptide
consisting of at least one amino acid sequence selected from the
group consisting of SYHATFL (SEQ ID NO. 2), TMAFPLN (SEQ ID NO. 3),
HYHGAFL (SEQ ID NO. 4), EHHDIFL (SEQ ID NO. 5), SSLELFL (SEQ ID NO.
44), TGLSVFL (SEQ ID NO. 6), WMPSLFY (SEQ ID NO. 7), SMPWWFF (SEQ
ID NO. 8), TMPLLFW (SEQ ID NO. 9), DTWPGLE (SEQ ID NO. 10), SMPPIFY
(SEQ ID NO. 11), MPLWWWD (SEQ ID NO. 12), SMPNLFY (SEQ ID NO. 13),
RMPPIFY (SEQ ID NO. 14), NPFEVFL (SEQ ID NO. 15), TLPNWFW (SEQ ID
NO. 16), SMPLTFY (SEQ ID NO. 17), SFLDTLT (SEQ ID NO. 45), NFLKTLS
(SEQ ID NO. 46), DFLRTLT (SEQ ID NO. 47), AFLDTLV (SEQ ID NO. 48),
TFLSSLA (SEQ ID NO. 49), GFLDSLM (SEQ ID NO. 50), SPHPHFL (SEQ ID
NO. 51), NFMSIGL (SEQ ID NO. 19), SQFLASL (SEQ ID NO. 20), SNFLKTL
(SEQ ID NO. 52), TGFLATL (SEQ ID NO. 53), WSWPGLN (SEQ ID NO. 21),
IAWPGLD (SEQ ID NO. 22), SKFMDTL (SEQ ID NO. 23), SDFLRAL (SEQ ID
NO. 54), SMPMVFY (SEQ ID NO. 24), YEWVGLM (SEQ ID NO. 25), KGFLDHL
(SEQ ID NO. 26), SANPRDFLETLF (SEQ ID NO. 55), RMFPESFLDTLW (SEQ ID
NO. 56), TIYDSFLDSLAS (SEQ ID NO. 57), HQSDDKMPWWFF (SEQ ID NO.
27), KPYLLKDFLEAL (SEQ ID NO. 58), AMGPYDALDLFL (SEQ ID NO. 59),
TWNPIESFLESL (SEQ ID NO. 60), YVWQDPSFTTFF (SEQ ID NO. 28),
QYQTPLTFLEAL (SEQ ID NO. 61), RHISPATFLEAL (SEQ ID NO. 62),
HTDSFLSTFYGD (SEQ ID NO. 63), YVWQDPSFTTFF (SEQ ID NO. 29),
ADSTFTSFLQTL (SEQ ID NO. 64), GPVSIYADTDFL (SEQ ID NO. 65),
DSNDTLTLAAFL (SEQ ID NO. 66), NGSPALSHMLFL (SEQ ID NO. 33),
TDYDPMWVFFGY (SEQ ID NO. 34), IFPLDSQWQTFW (SEQ ID NO. 35),
NESMPDLFYQPS (SEQ ID NO. 36), DWGDKYFSSFWN (SEQ ID NO. 37), VSAYNNV
(SEQ ID NO. 38), WPLHLWQ (SEQ ID NO. 39), TPTHYYADFSQL (SEQ ID NO.
67), LPGHLIWDSLHY (SEQ ID NO. 68), LPQTHPLHLLED (SEQ ID NO. 69),
IPYHHLVDQLHH (SEQ ID NO. 70), YPYHVQVDVLQN (SEQ ID NO. 71),
IPSHHLQDSLQL (SEQ ID NO. 72), EYAHHTSLDLRQ (SEQ ID NO. 73),
EPLHFRSDRIQA (SEQ ID NO. 74), ATPSHLIIDRAQ (SEQ ID NO. 75),
APKHLYADMSQA (SEQ ID NO. 76), FKPAHVSIDWLQ (SEQ ID NO. 77),
MPAHLSRDLRQS (SEQ ID NO. 78), NPKHYSIDRHQA (SEQ ID NO. 79),
SPQHLTTDRAQA (SEQ ID NO. 80), TPFHFAQDSWQW (SEQ ID NO. 81),
TPTHYYADFSQLLS (SEQ ID NO. 82), TPTHYYADFSQSLS (SEQ ID NO. 83),
GTPTHYYADFSQLL (SEQ ID NO. 84), GTPTHYYADFSQSL (SEQ ID NO. 85),
FGTPTHYYADFSQSLS (SEQ ID NO. 86), FGFPTHYYADFSQSLS (SEQ ID NO. 87),
LPGHLIWDSLHY (SEQ ID NO. 88), LPGHLIWDSLHYL (SEQ ID NO. 89),
LPGHLIWDSLHYLS (SEQ ID NO. 90), LPGHLIWDSLHSL (SEQ ID NO. 91),
LPGHLIWDSLHSLS (SEQ ID NO. 92), GLPGHLIWDSLHYL (SEQ ID NO. 93),
GLPGHLIWDSLHSL (SEQ ID NO. 94), FGLPGHLIWDSLHSLS (SEQ ID NO. 95),
FGFPGHLIWDSLHSLS (SEQ ID NO. 96), LPQTHPLHLLED (SEQ ID NO. 97),
IPYHHLVDQLHH (SEQ ID NO. 98), IPYHHLVDQLHLS (SEQ ID NO. 99),
IPYHHLVDQLHSLS (SEQ ID NO. 100), FGIPYHHLVDQLHHLS (SEQ ID NO. 101),
FGFPYHHLVDQLHSLS (SEQ ID NO. 102), YPYHVQVDVLQN (SEQ ID NO. 103),
YPYHVQVDVLQNLS (SEQ ID NO. 104), YPYHVQVDVLQSLS (SEQ ID NO. 105),
FGYPYHVQVDVLQNLS (SEQ ID NO. 106), FGFPYHVQVDVLQSLS (SEQ ID NO.
107), IPSHHLQDSLQL (SEQ ID NO. 108), IPSHHLQDSLQLLS (SEQ ID NO.
109), IPSHHLQDSLQSLS (SEQ ID NO. 110), GIPSHHLQDSLQLL (SEQ ID NO.
111), FGIPSHHLQDSLQLLS (SEQ ID NO. 112), FGFPSHHLQDSLQSLS (SEQ ID
NO. 113), EYAHHTSLDLRQ (SEQ ID NO. 114), EPLHFRSDRIQA (SEQ ID NO.
115), EPLHFRSDRIQALS (SEQ ID NO. 116), EPLHFRSDRIQSLS (SEQ ID NO.
117), GEPLHFRSDRIQAL (SEQ ID NO. 118), FGEPLHFRSDRIQALS (SEQ ID NO.
119), FGFPLHFRSDRIQSLS (SEQ ID NO. 120), APKHLYADMSQA (SEQ ID NO.
121), APKHLYADMSQALS (SEQ ID NO. 122), APKHLYADMSQSLS (SEQ ID NO.
123), GAPKHLYADMSQAL (SEQ ID NO. 124), FGFPKHLYADMSQSLS (SEQ ID NO.
125), MPAHLSRDLRQS (SEQ ID NO. 126), MPAHLSRDLRQSL (SEQ ID NO.
127), MPAHLSRDLRQSLS (SEQ ID NO. 128), GMPAHLSRDLRQSL (SEQ ID NO.
129), FGFPAHLSRDLRQSLS (SEQ ID NO. 130), NPKHYSIDRHQA (SEQ ID NO.
131), TPFHFAQDSWQW (SEQ ID NO. 132), TPFHFAQDSWQWLS (SEQ ID NO.
133), TPFHFAQDSWQSLS (SEQ ID NO. 134), GTPFHFAQDSWQWL (SEQ ID NO.
135), FGFPFHFAQDSWQSLS (SEQ ID NO. 136), ACSFAYLYRC (SEQ ID NO.
137), ACFMGDKWVC (SEQ ID NO. 138), ACVLYPKAIC (SEQ ID NO. 139),
ACYMGQQFVC (SEQ ID NO. 140), ACLTAYLHWC (SEQ ID NO. 141),
ACTLFPVAYC (SEQ ID NO. 142), ACWLFPYAHC (SEQ ID NO. 143),
ACKSINMWLC (SEQ ID NO. 144), ACQTINRWLC (SEQ ID NO. 145),
FGFPEHLLVDFLQSLS (SEQ ID NO. 146), FGFPEHLLVDFLQSLS (SEQ ID NO.
147), FPEHLLVDFLQSL (SEQ ID NO. 148), AGFPEHLLVDFLQSLS (SEQ ID NO.
149), FAFPEHLLVDFLQSLS (SEQ ID NO. 150), FGAPEHLLVDFLQSLS (SEQ ID
NO. 151), FGFAEHLLVDFLQSLS (SEQ ID NO. 152), FGFPAHLLVDFLQSLS (SEQ
ID NO. 153), FGFPEALLVDFLQSLS (SEQ ID NO. 154), FGFPEHALVDFLQSLS
(SEQ ID NO. 155), FGFPEHLAVDFLQSLS (SEQ ID NO. 156),
FGFPEHLLADFLQSLS (SEQ ID NO. 157), FGFPEHLLVAFLQSLS (SEQ ID NO.
158), FGFPEHLLVDALQSLS (SEQ ID NO. 159), FGFPEHLLVDFAQSLS (SEQ ID
NO. 160), FGFPEHLLVDFLASLS (SEQ ID NO. 161), FGFPEHLLVDFLQALS (SEQ
ID NO. 162), FGFPEHLLVDFLQSAS (SEQ ID NO. 163), FGFPEHLLVDFLQSLA
(SEQ ID NO. 164), FAFPAHLLVDFLQALA (SEQ ID NO. 165),
AAFPAHLLADFLQALA (SEQ ID NO. 166), SPQHLTTDRAQA (SEQ ID NO. 167),
SPQHLTTDRAQALS (SEQ ID NO. 168), SPQHLTTDRAQSLS (SEQ ID NO. 169),
GSPQHLTTDRAQAL (SEQ ID NO. 170), FGFPQHLTTDRAQSLS (SEQ ID NO. 171),
FGFPQHLTTDWAQSLS (SEQ ID NO. 172), FGFPQHLTTDRLQSLS (SEQ ID NO.
173), FGFPQHLTTDWLQSLS (SEQ ID NO. 174), ATPSHLIIDRAQ (SEQ ID NO.
175), ATPSHLIIDRAQSLS (SEQ ID NO. 176), FGFPSHLIIDRAQSLS (SEQ ID
NO. 177), FGFPSHLIIDWAQSLS (SEQ ID NO. 178), FGFPSHLIIDWLQSLS (SEQ
ID NO. 179), FGFPSHLIIDWSQSLS (SEQ ID NO. 180), FATPSHLIIDWLQSLS
(SEQ ID NO. 181), FKPAHVSIDWLQ (SEQ ID NO. 182), FKPAHVSIDWLQSLS
(SEQ ID NO. 183), FGFPAHVSIDWLQSLS (SEQ ID NO. 184),
AGFPAHVSIDWLQSLS (SEQ ID NO. 185), FAFPAHVSIDWLQSLS (SEQ ID NO.
186), FGAPAHVSIDWLQSLS (SEQ ID NO. 187), FGFAAHVSIDWLQSLS (SEQ ID
NO. 188), FGFPAHVSADWLQSLS (SEQ ID NO. 189), FGFPAHVSIDWLQALS (SEQ
ID NO. 190), FGFPAHVSIDWLQSLA (SEQ ID NO. 191), FAFPAHVSIDWLQALA
(SEQ ID NO. 192), FGFAAHVSIDWLQSLS (SEQ ID NO. 193),
FGFFAHVSIDWLQSLS (SEQ ID NO. 194), FGFPAHVSIRWLQSLS (SEQ ID NO.
195), FGFPAHVSIEWLQSLS (SEQ ID NO. 196), FGFPAHVSIDWLNSLS (SEQ ID
NO. 197), FGFPAHVSIDWLHSLS (SEQ ID NO. 198), AGFPAHVSIDWLQSLS (SEQ
ID NO. 199), PGFPAHVSIDWLQSLS (SEQ ID NO. 200), WGFPAHVSIDWLQSLS
(SEQ ID NO. 201), FAFPAHVSIDWLQSLS (SEQ ID NO. 202),
FSFPAHVSIDWLQSLS (SEQ ID NO. 203), FYFPAHVSIDWLQSLS (SEQ ID NO.
204), FDFPAHVSIDWLQSLS (SEQ ID NO. 205), FGAPAHVSIDWLQSLS (SEQ ID
NO. 206), FGFPAHVSIDWLQLLS (SEQ ID NO. 207), FGFPAHVSIDWLQWLS (SEQ
ID NO. 208), FGFPAHVSIDWLQNLS (SEQ ID NO. 209), FGFPAHVSIDWLQTLS
(SEQ ID NO. 210), FGFPAHVSIDWLQYLS (SEQ ID NO. 211),
FGFPAHVSIDWLQSIS (SEQ ID NO. 212), FGFPAHVSIDWLQSLT (SEQ ID NO.
213), FGFPAHVSIDWLQSLY (SEQ ID NO. 214), FAFPAHVSIDWLQALA (SEQ ID
NO. 215), FGFPAHVSIDRAQSLS (SEQ ID NO. 216), FGFPTHVSIDWLQSLS (SEQ
ID NO. 217), FGFPFHVSIDWLQSLS (SEQ ID NO. 218), FGFPAHISIDWLQSLS
(SEQ ID NO. 219), FGFPAHIIIDWLQSLS (SEQ ID NO. 220),
FGFPAHLTTDWLQSLS (SEQ ID NO. 221), FGFPAHVFIDWLQSLS (SEQ ID NO.
222), FGFPAHVYIDWLQSLS (SEQ ID NO. 223), FGFPAHVSLDWLQSLS (SEQ ID
NO. 224), FGFPAHVSADWLQSLS (SEQ ID NO. 225), TPTHYYADFSQSLS (SEQ ID
NO. 226), FGFPAHVWIDWLQSLS (SEQ ID NO. 229), FGFPAHVFIDWLQSLN (SEQ
ID NO. 230), FGFPAHFSIDWLQSLS (SEQ ID NO. 231), FGFPAHVSFDWLQSLS
(SEQ ID NO. 232), FGFPEHVFIDWLQSLS (SEQ ID NO. 233),
DFGFPAHVFIDWLQSLS (SEQ ID NO. 234), DFGFPSHLIIDWLQSLS (SEQ ID NO.
235), DFGFPAHVYIDWLQSLS (SEQ ID NO. 236), FGFPQHLFTDWLQSLS (SEQ ID
NO. 237), FGFPKHLLVDFLQSLS (SEQ ID NO. 238), FGFPAHVSIDFSQSLS (SEQ
ID NO. 227), FGFPAHVSIDFSQSLS (SEQ ID NO. 228), FGFPSHIIIDWLQSLS
(SEQ ID NO. 239), FGFPSHLIIEWLQSLS (SEQ ID NO. 240),
AAFPAHLLADAAQALA (SEQ ID NO. 241), AAFPAHAAADFLQALA (SEQ ID NO.
242), AAFAAHLLADFLQAAA (SEQ ID NO. 243), AAAPAHLLVDAAQAAA (SEQ ID
NO. 244), FAFPAHVFIDWLQSLS (SEQ ID NO. 245); FGFPAHVFIDWLQALS (SEQ
ID NO. 246), FGFPAHVFIDWLQSLA (SEQ ID NO. 247), GFPAHVFIDWLQSLS
(SEQ ID NO. 248), FPAHVFIDWLQSLS (SEQ ID NO. 249), PAHVFIDWLQSLS
(SEQ ID NO. 250), FAFPAHVFIDWLQALA (SEQ ID NO. 251),
FGFPEHLFVDFLQSLS (SEQ ID NO. 252), FGFPAHVHIDWLQSLS (SEQ ID NO.
253), FGFPAHVPIDWLQSLS (SEQ ID NO. 254), FGFPSHLFIDWAQSLS (SEQ ID
NO. 255), PGFPAHVFIDWLQLIT (SEQ ID NO. 256), PAHVYIDWLQSLS (SEQ ID
NO. 257), FGFPAHVYIDWLQ (SEQ ID NO. 258), FGFPAHVFIDWLQ (SEQ ID NO.
259), DFGFPAHVFIDWLQSLN (SEQ ID NO. 260), PSHLIIDWLQ (SEQ ID NO.
261), PAHVFIDWLQ (SEQ ID NO. 262), DFGFPAHVTIDWLQSLN (SEQ ID NO.
263), DFGFPAHVLIDWLQSLN (SEQ ID NO. 264), and FGFPAHVFIDWLQSLA (SEQ
ID NO. 265).
[0073] The peptides of the present invention turned out to be
mimotopes for CETP and, hence, the mimotopes were able to bind to
antibodies binding to the CETP fragment C-FGFPEHLLVDFLQSLS (SEQ ID
NO. 146) (16 C-terminal amino acids of CETP protein).
[0074] Yet, another aspect of the present invention relates to a
pharmaceutical formulation comprising at least one peptide
according to the present invention.
[0075] The peptides of the present invention may be formulated in a
pharmaceutical formulation which may be administered to an
individual. These formulations may be used, e.g., for preventing
and/or treating atherosclerosis, atherosclerosis risk diseases and
atherosclerosis sequelae.
[0076] The peptides in the formulation can be combined from the
pool of peptides disclosed herein. Furthermore is also possible to
provide pharmaceutical formulations, which comprise one or more of
the peptides of the present invention, and which can be
administered separately or together to an individual in need
thereof.
[0077] The peptides of the present invention can be mixed into one
single pharmaceutical formulation or in a combination of two or
three. The resulting formulation can be administered at the same or
the different moments in time. According to a preferred embodiment
of the present invention the peptide present in the formulation is
coupled to a pharmaceutically acceptable carrier, preferably KLH
(Keyhole Limpet Hemocyanin).
[0078] The present invention is further illustrated by the
following figures and examples, however, without being restricted
thereto.
[0079] FIG. 1 shows the result of a representative competition
ELISA after screening phage display library Ph.D. 7 with monoclonal
antibody "Paula".
TABLE-US-00002 FGFPEHLLVDFLQSLS SEQ ID NO. 146 FGFPEHLLVDFLQSLS SEQ
ID NO. 147 SYHATFL SEQ ID NO. 2 TMAFPLN SEQ ID NO. 3 HYHGAFL SEQ ID
NO. 4 EHHDIFL SEQ ID NO. 5 SSLELFL SEQ ID NO. 44 TGLSVFL SEQ ID NO.
6 WMPSLFY SEQ ID NO. 7 SMPWWFF SEQ ID NO. 8 TMPLLFW SEQ ID NO. 9
DTWPGLE SEQ ID NO. 10 SMPPIFY SEQ ID NO. 11 MPLWWWD SEQ ID NO. 12
SMPNLFY SEQ ID NO. 13 RMPPIFY SEQ ID NO. 14 NPFEVFL SEQ ID NO. 15
TLPNWFW SEQ ID NO. 16
[0080] FIGS. 2a and 2b show the results of 2 typical competition
ELISAs after screening phage display library Ph.D. 12 with
monoclonal antibody "Paula".
TABLE-US-00003 FGFPEHLLVDFLQSLS SEQ ID NO. 146 FGFPEHLLVDFLQSLS SEQ
ID NO. 147 RHISPATFLEAL SEQ ID NO. 62 TIYDSFLDSLAS SEQ ID NO. 57
HTDSFLSTFYGD SEQ ID NO. 63 ADSTFTSFLQTL SEQ ID NO. 64 GPVSIYADTDFL
SEQ ID NO. 65 DSNDTLTLAAFL SEQ ID NO. 66 NGSPALSHMLFL SEQ ID NO. 33
TDYDPMWVFFGY SEQ ID NO. 34 IFPLDSQWQTFW SEQ ID NO. 35 NESMPDLFYQPS
SEQ ID NO. 36 DWGDKYFSSFWN SEQ ID NO. 37 HQSDDKMPWWFF SEQ ID NO. 27
KPYLLKDFLEAL SEQ ID NO. 58 SANPRDFLETLF SEQ ID NO. 55 RMFPESFLDTLW
SEQ ID NO. 56 AMGPYDALDLFL SEQ ID NO. 59 TWNPIESFLESL SEQ ID NO. 60
YVWQDPSFTTFF SEQ ID NO. 29 QYQTPLTFLEAL SEQ ID NO. 61
[0081] FIGS. 3a and 3b show the results of 2 representative
competition ELISAs after screening phage display library Ph.D. 7
with mAb Frida.
TABLE-US-00004 FGFPEHLLVDFLQSLS SEQ ID NO. 146 FGFPEHLLVDFLQSLS SEQ
ID NO. 147 WPLHLWQ SEQ ID NO. 39 VSAYNNV SEQ ID NO. 38
[0082] FIG. 4a shows the result of a representative competition
ELISA after screening phage display library Ph.D. 12 with
monoclonal antibody "Frida".
TABLE-US-00005 FGFPEHLLVDFLQSLS SEQ ID NO. 146 FGFPEHLLVDFLQSLS SEQ
ID NO. 147 TPTHYYADFSQL SEQ ID NO. 67 LPGHLIWDSLHY SEQ ID NO. 68
LPQTHPLHLLED SEQ ID NO. 69
[0083] FIG. 4b shows binding of monoclonal antibody "Frida" to
ELISA plates coated with mimotope-BSA
[0084] FIGS. 5a and 5b show the results of a representative
competition ELISA after screening phage display library Ph.D. 12
with monoclonal antibody "Frida".
TABLE-US-00006 FGFPEHLLVDFLQSLS SEQ ID NO. 146 FGFPEHLLVDFLQSLS SEQ
ID NO. 147 GTPTHYYADFSQLL SEQ ID NO. 84 GTPTHYYADFSQSL SEQ ID NO.
85 FGTPTHYYADFSQSLS SEQ ID NO. 86 FGFPTHYYADFSQSLS SEQ ID NO. 87
GLPGHLIWDSLHYL SEQ ID NO. 93 GLPGHLIWDSLHSL SEQ ID NO. 94
FGLPGHLIWDSLHSLS SEQ ID NO. 95 FGFPGHLIWDSLHSLS SEQ ID NO. 96
FGIPYHHLVDQLHHLS SEQ ID NO. 101 FGFPYHHLVDQLHSLS SEQ ID NO. 102
FGYPYHVQVDVLQNLS SEQ ID NO. 106 FGFPYHVQVDVLQSLS SEQ ID NO. 107
GIPSHHLQDSLQLL SEQ ID NO. 111 FGIPSHHLQDSLQLLS SEQ ID NO. 112
FGFPSHHLQDSLQSLS SEQ ID NO. 113 GEPLHFRSDRIQAL SEQ ID NO. 118
FGEPLHFRSDRIQALS SEQ ID NO. 119 FGFPLHFRSDRIQSLS SEQ ID NO. 120
ATPSHLIIDRAQSLS SEQ ID NO. 176 FGFPSHLIIDRAQSLS SEQ ID NO. 177
GAPKHLYADMSQAL SEQ ID NO. 124 FGFPKHLYADMSQSLS SEQ ID NO. 125
FKPAHVSIDWLQSLS SEQ ID NO. 183 FGFPAHVSIDWLQSLS SEQ ID NO. 184
GMPAHLSRDLRQSL SEQ ID NO. 129 FGFPAHLSRDLRQSLS SEQ ID NO. 130
GSPQHLTTDRAQAL SEQ ID NO. 170 FGFPQHLTTDRAQSLS SEQ ID NO. 171
GTPFHFAQDSWQWL SEQ ID NO. 135 FGFPFHFAQDSWQSLS SEQ ID NO. 136
[0085] FIG. 6 shows the results of a competition ELISA of two
mimotopes after screening phage display library Ph.D. 12 with
monoclonal antibody "Frida".
TABLE-US-00007 FGFPEHLLVDFLQSLS SEQ ID NO. 146 FGFPEHLLVDFLQSLS SEQ
ID NO. 147 FGFPSHLIIDWAQSLS SEQ ID NO. 178 FGFPSHLIIDWLQSLS SEQ ID
NO. 179
[0086] FIGS. 7a to 7d show the antibody titer (anti mouse IgG) of
in vivo experiments, whereby the following mimotope-BSA conjugates
were injected into mice:
TABLE-US-00008 Fr12/3/26/65 ext4 C-FGFPYHVQ (SEQ ID NO. 107) p4286
VDVLQSLS Fr12/3/55 ext2 C-FGFPSHLIIDRA (SEQ ID NO. 177) p4294 QSLS
Fr12/3/55 ext2 W instead of R (SEQ ID NO. 178) p4324
C-FGFPSHLIIDWAQSLS Fr12/3/55 ext2 WL instead of (SEQ ID NO. 179)
p4325 RAC-FGFPSHLIIDWLQSLS Fr12/3/84 ext2 C-FGFPAHVSIDWL (SEQ ID
NO. 184) p4298 QSLS Fr12/3/40 ext4 C-FGFPQHLTTDRA (SEQ ID NO. 171)
p4302 QSLS Fr12/2/6 ext6 C-FGFPTHYYADFS (SEQ ID NO. 87) p4278 QSLS
Fr12/2/11 ext7 C-FGFPGHLIWDSL (SEQ ID NO. 96) p4282 HSLS
Fr12/3/1/19/88 ext4 C-FGFPYHHL (SEQ ID NO. 102) p4284 VDQLHSLS
Fr12/3/68 ext5 C-FGFPSHHLQDSL (SEQ ID NO. 113) p4289 QSLS Fr12/3/83
ext5 C-FGFPLHFRSDRI (SEQ ID NO. 120) p4292 QSLS Fr12/3/63 ext4
C-FGFPKHLYADMS (SEQ ID NO. 125) p4296 QSLS Fr12/3/47 ext4
C-FGFPAHLSRDL (SEQ ID NO. 130) p4300 RQSL Fr12/3/35 ext4
C-FGFPFHFAQDSW (SEQ ID NO. 136) p4304 QSLS
[0087] FIGS. 8a and 8b show the results of two representative
competition ELISA after screening phage display library Ph.D. 7C7
with monoclonal antibody "Frida".
TABLE-US-00009 FGFPEHLLVDFLQSLS SEQ ID NO. 146 FGFPEHLLVDFLQSLS SEQ
ID NO. 147 ACSFAYLYRC SEQ ID NO. 137 ACYMGQQFVC SEQ ID NO. 140
ACLTAYLHWC SEQ ID NO. 141 ACTLFPVAYC SEQ ID NO. 142 ACWLFPYAHC SEQ
ID NO. 143 ACQTINRWLC SEQ ID NO. 145
[0088] FIG. 9 shows an in vitro ELISA test for the detection of the
binding between "Frida" and cyclic mimotopes.
TABLE-US-00010 FGFPEHLLVDFLQSLS SEQ ID NO. 147 ACSFAYLYRC SEQ ID
NO. 137 ACYMGQQFVC SEQ ID NO. 140 ACLTAYLHWC SEQ ID NO. 141
ACTLFPVAYC SEQ ID NO. 142 ACWLFPYAHC SEQ ID NO. 143 ACQTINRWLC SEQ
ID NO. 145
[0089] FIGS. 10a and 10b show the results of an inhibition ELISA
assay with FGFPSHLIIDWLQSLS (SEQ ID NO. 179), FGFPAHVFIDWLQSLS (SEQ
ID NO. 222) and FGFPAHVYIDWLQSLS (SEQ ID NO. 223).
[0090] FIG. 10a (Coat 1 .mu.M peptide. Detection .alpha.IgG1)
TABLE-US-00011 2.5 ng mAb Frida Frida 2 .mu.g 20 .mu.g pept
N.degree. peptide peptide buffer only -- 1.05 0.96 p4073 original
epitope C-FGFPEHLLVDFLQSLS 0.44 0.1 p1358 irrelevant peptide
irrelevant peptide 1.08 0.91 p4361 FGFPAHVFIDWLQSLS Fr12/3/84 ext2
VSI VFI 0.82 0.16 p4362 FGFPAHVYIDWLQSLS Fr12/3/84 ext2 VSI VYI
0.75 0.15
[0091] FIG. 10b (Coat 1 .mu.M peptide. Detection .alpha.IgG1)
TABLE-US-00012 2.5 ng mAb Firda Frida 2 .mu.g 20 .mu.g pept N.sup.o
peptide peptide buffer only -- 0.84 0.75 p4073 original epitope
C-FGFPEHLLVDFLQSLS 0.64 0.15 p1358 irrelevant peptide irrelevant
peptide 0.88 0.77 p4325 FGFPSHLIIDWLQSLS Fr12/3/55 ext2 RA WL 0.42
0.1
[0092] FIG. 11 shows the in vivo induction of antibodies directed
to CETP by mimotopes of the invention that are administered to
mice. Balb/c mice/30 .mu.g Peptide, 2 injections in 2 week
intervals. S3=2 weeks after 3rd injection. Alum as adjuvant. Titers
against original epitope (p4073) induced by injection of mimotopes.
Well coating: 50 .mu.l of 1 .mu.M p4073-BSA or 1 .mu.g/ml activated
KLH. Detection: .alpha.IgG:
TABLE-US-00013 injected original irrelevant peptide-
epitope-peptide- BSA BSA BSA group 1 KLH KLH 2.040 400 group 2
original epitope p4073-KLH 8.600 10 group 3 C-FGFPQHLTTDWLQSLS
p4369-KLH 14.000 12.900 10 (SEQ ID NO. 174) group 4
C-FGFPSHLIIDWAQSLS p4324-KLH 12.570 7.600 10 (SEQ ID NO. 178) group
5 C-FGFPSHLIIDWLQSLS p4325-KLH 2.930 1.820 10 (SEQ ID NO. 179)
group 6 C-FGFPSHLIIDWSQSLS p4366-KLH 4.700 3.600 10 (SEQ ID NO.
180) group 7 C-FATPSHLIIDWLQSLS p4345-KLH 8.380 1.270 10 (SEQ ID
NO. 181) group 8 C-FAFPAHVSIDWLQALA p4328-KLH 10.100 2.740 400 (SEQ
ID NO. 186) group 9 C-PGFPAHVSIDWLQSLS p4340-KLH 18.100 15.640 10
(SEQ ID NO. 200) group C-WGFPAHVSIDWLQSLS p4341-KLH 10.350 5.500 10
10 (SEQ ID NO. 201) group C-FSFPAHVSIDWLQSLS p4342-KLH 4.620 1.610
10 11 (SEQ ID NO. 203) group C-FYFPAHVSIDWLQSLS p4343-KLH 5.580
2.900 10 12 (SEQ ID NO. 204) group C-FDFPAHVSIDWLQSLS p4344-KLH
12.200 3.580 10 13 (SEQ ID NO. 205) group C-FGFPAHVSIDWLQLLS
p4347-KLH 12.000 9.160 10 14 (SEQ ID NO. 207) group
C-FGFPAHVSIDWLQYLS p4351-KLH 2.950 2.400 10 15 (SEQ ID NO. 211)
group C-FGFPAHVSIDWLQSIS p4352-KLH 19.680 12.070 10 16 (SEQ ID NO.
212) group C-FGFPAHVSIDWLQSLT p4353-KLH 11.200 8.650 10 17 (SEQ ID
NO. 213) group C-FGFPAHISIDWLQSLS p4358-KLH 16.500 12.940 10 18
(SEQ ID NO. 219) group C-FGFPAHIIIDWLQSLS p4359-KLH 8.540 5.340 10
19 (SEQ ID NO. 220) group C-FGFPAHVFIDWLQSLS p4361-KLH 17.940 9.530
10 20 (SEQ ID NO. 222)
[0093] FIGS. 12a and 12b show the in vivo induction of CETP
specific antibodies by the administration of the mimotopes of the
invention. Titers to p4073 and its correlation to titers to CETP of
selected groups (which show high titers against p4073): gr.4, gr.9,
gr.10, gr.14, gr.16-20/gr.1 (KLH), gr.2 (original epitope) as
controls. Coating: recombinant GST-CETP or purified rabbit CETP,
respectively:
FIG. 12a
TABLE-US-00014 recombinant rabbit GST-CETP CETP group 1 KLH KLH/
0.35 0.19 Alum group 2 original epitope p4073- 1.49 1.25 KLH/ Alum
group 3 C-FGFPQHLTTDWLQSLS p4369- 0.45 0.21 (SEQ ID NO. 174) KLH/
Alum group 4 C-FGFPSHLIIDWAQSLS p4324- 0.58 0.28 (SEQ ID NO. 178)
KLH/ Alum group 9 C-PGFPAHVSIDWLQSLS p4340- 0.49 0.21 (SEQ ID NO.
200) KLH/ Alum group 10 C-WGFPAHVSIDWLQSLS p4341- 0.39 0.18 (SEQ ID
NO. 201) KLH/ Alum group 14 C-FGFPAHVSIDWLQLLS p4347- 0.35 0.2 (SEQ
ID NO. 207) KLH/ Alum group 16 C-FGFPAHVSIDWLQSIS p4352- 0.48 0.28
(SEQ ID NO. 212) KLH/ Alum group 17 C-FGFPAHVSIDWLQSLT p4353- 0.57
0.39 (SEQ ID NO. 213) KLH/ Alum group 18 C-FGFPAHISIDWLQSLS p4358-
0.68 0.58 (SEQ ID NO. 219) KLH/ Alum group 19 C-FGFPAHIIIDWLQSLS
p4359- 0.79 0.54 (SEQ ID NO. 220) KLH/ Alum group 20
C-FGFPAHVFIDWLQSLS p4361- 1.64 1.51 (SEQ ID NO. 222) KLH/ Alum
FIG. 12b
TABLE-US-00015 recombinant rabbit GST-CETP CETP group 1 KLH KLH/
0.18 0.47 Alum group 2 original epitope p4073- 1.26 1.42 KLH/ Alum
group 5 C-FGFPSHLIIDWLQSLS p4325- 0.59 0.85 (SEQ ID NO. 179) KLH/
Alum group 6 C-FGFPSHLIIDWSQSLS p4366- 0.4 0.65 (SEQ ID NO. 180)
KLH/ Alum group 7 C-FATPSHLIIDWLQSLS p4345- 0.39 0.46 (SEQ ID NO.
181) KLH/ Alum group 8 C-FAFPAHVSIDWLQALA p4328- 0.45 0.43 (SEQ ID
NO. 186) KLH/ Alum group 11 C-FSFPAHVSIDWLQSLS p4342- 0.38 0.41
(SEQ ID NO. 203) KLH/ Alum group 12 C-FYFPAHVSIDWLQSLS p4343- 0.61
1.05 (SEQ ID NO. 204) KLH/ Alum group 13 C-FDFPAHVSIDWLQSLS p4344-
0.35 0.43 (SEQ ID NO. 205) KLH/ Alum group 15 C-FGFPAHVSIDWLQYLS
p4351- 0.54 0.59 (SEQ ID NO. 211) KLH/ Alum
[0094] FIG. 13 shows the in vivo induction of antibodies directed
to CETP by mimotopes of the invention that are administered to
mice.
[0095] Sera of each group (5 Balb/c mice each) were combined,
diluted 1:100 and tested on ELISA plates coated with recombinant
GST-CETP or rabbit CETP, respectively. Detection of bound
antibodies was with algG.
TABLE-US-00016 recombinant rabbit GST-CETP CETP group 1 KLH KLH/
0.23 0.17 Alum group 2 original epitope p4073- 1.08 0.46 KLH/ Alum
group 3 C-FGFAAHVSIDWLQSLS p4335- 0.26 0.14 (SEQ ID NO. 188) KLH/
Alum group 4 C-FGFPAHVSIDWLQWLS p4348- 0.33 0.16 (SEQ ID NO. 208)
KLH/ Alum group 5 C-FGFPAHLTTDWLQSLS p4360- 0.4 0.23 (SEQ ID NO.
221) KLH/ Alum group 6 C-FGFPAHVYIDWLQSLS p4362- 0.86 0.94 (SEQ ID
NO. 223) KLH/ Alum group 7 C-FGFPAHVSIDWLQSLY P4354- 0.29 0.23 (SEQ
ID NO. 214) KLH/ Alum group 8 C-FGFPAHVSIRWLQSLS p4337- 0.24 0.14
(SEQ ID NO. 195) KLH/ Alum
[0096] FIG. 14 shows a CETP activity assay, wherein 0.6 .mu.l human
serum (with endogenous CETP activity) is mixed with serum from
wild-type mice (not containing CETP activity) vaccinated with
KLH/Alum (negative control group), p4703-KLH/Alum (original CETP
epitope), or p4361 (or p4362 or p 4325) mimotope, respectively. It
could be demonstrated that the addition of 1.2 .mu.l and 0.6 .mu.l
serum from p4361-KLH/Alum vaccinated mice completely inhibits CETP
activity and the addition of 0.2 .mu.l serum reduces significantly
said activity in contrast to the addition of serum from mice
vaccinated with KLH/Alum-control only or with the original epitope
(p4073-KLH/Alum).
TABLE-US-00017 SEQ ID NO. 146 FGFPEHLLVDFLQSLS SEQ ID NO. 222
FGFPAHVFIDWLQSLS
[0097] FIG. 15 shows that the addition of p4325-KLH/Alum to human
serum inhibits significantly CETP activity.
TABLE-US-00018 SEQ ID NO. 179 FGFPSHLIIDWLQSLS
[0098] FIG. 16 shows that the addition of p4361-KLH/Alum to human
serum inhibits significantly CETP activity.
TABLE-US-00019 SEQ ID NO. 146 FGFPEHLLVDFLQSLS SEQ ID NO. 222
FGFPAHVFIDWLQSLS
[0099] FIG. 17 shows that the addition of p4362-KLH/Alum to human
serum inhibits significantly CETP activity.
TABLE-US-00020 SEQ ID NO. 146 FGFPEHLLVDFLQSLS SEQ ID NO. 221
FGFPAHLTTDWLQSLS SEQ ID NO. 223 FGFPAHVYIDWLQSLS
[0100] FIG. 18a shows an inhibition ELISA with mimotopes (Coat. 1
.mu.M 4073 peptide, detection .alpha. IgG1).
TABLE-US-00021 Frida 2.5 ng mAB Frida pept N.sup.o low high buffer
only buffer only buffer only 1.084 1.079 4% DMSO 4% DMSO 4% DMSO
1.180 1.201 p4073 C-FGFPEHLLVDFLQSLS p4073 0.537 0.094 (SEQ ID NO.
147), positive control peptide p1208 positive control p1208 0.712
0.093 peptide FGFPEH- LLVDFLQSLS-C (SEQ ID NO. 146) p1358 negative
control p1358 1.158 1.050 peptide p4474 C-PAVVYIDWLQSLS Fr12/3/84
ext2 1.452 0.179 (SEQ ID NO. 257) VSI VFI SLS SLN p4475
C-FGFPAHFSIDWLQSLS Fr12/3/84 ext2 2.211 1.429 (SEQ ID NO. 231) VSI
FSI p4476 C-FGFPAHVSFDWLQSLS Fr12/3/84 ext2 2.000 1.417 (SEQ ID NO.
232) VSI VSF p4477 C-FGFPEHVFIDWLQSLS Fr12/3/84 ext2 0.808 0.116
(SEQ ID NO. 233) VSI VFI PAH PEH p4478 C-FKPAHVFIDWLQSLS Fr12/3/84
ext1 2.231 1.206 (SEQ ID NO. 266) VSI VFI p4479 C-GFKPAHVFIDWLQSLS
Fr12/3/84 ext1 2.165 1.591 (SEQ ID NO. 267) VSI VFI plus G on
N-terminus p4480 C-DFGPAHVFIDWLQSLS Fr12/3/84 ext2 0.521 0.103 (SEQ
ID NO. 234) VSI VFI plus D on N-terminus; = 4361 plus D p4481
C-FGFPQHLFTDWLQSLS Fr12/3/40 ext4 0.551 0.156 (SEQ ID NO. 237) RA
WL LTT LFT = p4369 with exchange T F
[0101] FIG. 18b shows an inhibition ELISA with mimotopes (Coat. 1
.mu.M 4073 peptide, detection .alpha. IgG1).
TABLE-US-00022 p1208 positive control p1208 0.264 0.079 peptide
p1358 negative control p1358 1.902 1.661 peptide p4629
C-PAHVYIDWLQSLS C-terminus of p4362; 0.313 0.118 (SEQ ID NO. 257)
p4362 minus 3 aa on N-terminus p4630 C-FGFPAHVYIDWLQ N-terminus of
p4362 2.131 2.115 (SEQ ID NO. 258) (minus 3 aa on C-terminus) p4631
C-FGFPAHVFIDWLQ N-terminus of p4361 2.111 2.147 (SEQ ID NO. 259)
(minus 3 aa on C-terminus) p4642 C-DFGFPHSHLIIDWLQSLS Fr12/3/55
ext2 RA .fwdarw. 0.171 0.082 (SEQ ID NO. 235) WL plus D; p4325 plus
D on N-terminus p4818 C-DFGFPAHVFIDWLQSLN FR12/3/84 ext 2 VSI
.fwdarw. 0.332 0.091 (SEQ ID NO. 260) VFI SLS .fwdarw. SLN plus D;
= 4361 N hinten plus D vorne p4819 C-PSHLIIDWLQ = 4325 minus 3AA am
N 2.226 2.158 (SEQ ID NO. 261) und am C-Terminus p4820 C-PAHVFIDWLQ
= 4361 minus 3AA am N 2.310 2.374 (SEQ ID NO. 262) und am
C-Terminus p4989 C-DFGFPAHVTIDWLQSLN Fr12/3/84 ext2 VSI .fwdarw.
0.932 0.274 (SEQ ID NO. 263) VTI; = p4361 F replaced by T, plus D
on N-term and N instead of S on C-term p4990 C-DFGFPAHVLIDWLQSLN
Fr12/3/84 ext2 VSI .fwdarw. 0.263 0.073 (SEQ ID NO. 264) VLI; =
p4361 F replaced by L, plus D on N-term and N instead of S on
C-term p5067 FGFPAHVYIDWLQSLS-C p4362 C on C-terminus 0.563 0.217
(SEQ ID NO. 223) p5068 FGFPAHVFIDWLQSLN-C p4474 C on C-terminus
0.757 0.271 (SEQ ID NO. 230)
[0102] FIG. 18c shows a inhibition ELISA with mimotopes screen
PhD12 Frida and Ala-exchange for mimotope characterisation/mAb
Frida (Coat 1 .mu.M 4073. Detection .alpha.IgG1.)
TABLE-US-00023 Frida 2.5 ng mAb Frida pept N.sup.o low high buffer
only buffer only 0.964 0.964 4% DMSO 4% DMSO 0.973 0.923 positive
control p4073 0.554 0.088 peptide p1208 p1208 0.942 0.101 negative
control p1358 0.986 0.93 peptide p4432 C-FGFPSHIIIDWLQSLS Fr12/3/55
ext2exch2 0.635 0.096 (SEQ ID NO. 239) L -> I p4433
C-FGFPSHLIIEWLQSLS Fr12/3/55 ext2exch2 1.114 0.672 (SEQ ID NO. 240)
D -> E p4434 C-AAFPAHLLADAAQALA Ala-exchange for 1.74 1.461 (SEQ
ID NO. 241) mimotope characterisation p4435 C-AAFPAHAAADFLQALA
Ala-exchange for 1.281 1.969 (SEQ ID NO. 242) mimotope
characterisation p4436 C-AAFAAHLLADFLQAAA Ala-exchange for 1.632
1.691 (SEQ ID NO. 243) mimotope characterisation p4437
C-AAAPAHLLVDAAQAAA Ala-exchange for 1.84 1.674 (SEQ ID NO. 244)
mimotope characterisation
[0103] FIG. 19a shows a peptide ELISA, immunisation with
C-DFGFPAHVYIDWLQSLS (p4628-KLH/Alum) (SEQ ID NO. 236), titre to
original epitope.
[0104] FIG. 19b shows a peptide ELISA, immunisation with
C-FGFPAHVFIDWLQSLN (p4474-KLH/Alum) (SEQ ID NO. 230), titre to
original epitope.
[0105] FIG. 19c shows a peptide ELISA, immunisation with
C-FGFPAHVFIDWLQSLN (p4474-KLH/Alum) (SEQ ID NO. 230), titre to
injected mimotope.
[0106] FIG. 19d shows an anti-protein ELISA. Mice were injected 3
times with 30 .mu.g of the indicated mimotopes coupled to KLH with
Alum as adjuvant. Sera from each group (comprising 5 mice) were
pooled, diluted 1:100 and tested on ELISA plates coated with
purified rabbit CETP.
TABLE-US-00024 SEQ ID NO. 245 FAFPAHVFIDWLQSLS SEQ ID NO. 246
FGFPAHVFIDWLQALS SEQ ID NO. 247 FGFPAHVFIDWLQSLA SEQ ID NO. 248
GFPAHVFIDWLQSLS SEQ ID NO. 249 FPAHVFIDWLQSLS SEQ ID NO. 250
PAHVFIDWLQSLS SEQ ID NO. 251 FAFPAHVFIDWLQALA
[0107] FIG. 19e shows an anti-protein ELISA, wherein mice were
injected 3 times with 30 .mu.g of the indicated mimotopes coupled
to KLH with Alum as adjuvant. Mouse sera (from single mice) were
diluted 1:100 and tested on ELISA plates coated with purified
rabbit CETP.
TABLE-US-00025 SEQ ID NO. 252 FGFPEHLFVDFLQSLS SEQ ID NO. 253
FGFPAHVHIDWLQSLS SEQ ID NO. 254 FGFPAHVPIDWLQSLS SEQ ID NO. 269
FGFPAHVWIDWLQSL SEQ ID NO. 255 FGFPSHLFIDWAQSLS SEQ ID NO. 268
FPGFPAHVFIDWLQLIT
EXAMPLES
[0108] There exists a strong inverse relationship between the
plasma concentration of cholesterol in high density lipoproteins
(HDLs) and the development of coronary heart disease (CHD). Thus,
the risk for CHD is higher when HDLs decrease. Although 33% of
patients with CHD have low plasma levels of HDLs, there is
currently no effective therapy for increasing the plasma
concentration of HDLs. Diet and moderate exercise are ineffective,
statins only achieve a low 5 to 7% increase in HDL, and niacin has
side efects and compliance profiles limiting its use.
[0109] The inhibition of CETP activity has been suggested as
therapeutic approach to increase plasma HDL levels. CETP is a
plasma glycoprotein that facilitates transfer of neutral lipids and
phospholipids between lipoproteins and regulates the concentration
of plasma HDL. The inhibition of CETP activity is expected to
increase plasma HDL concentrations for several reasons. CETP lowers
HDL concentrations by moving cholesteryl esters from HDLs to VLDLs
and LDLs. Transient inhibition of CETP in rabbits and hamsters by
monoclonal antibodies, small molecules (Sikorski, J. A., J. Med.
Chem. 49 (1) (2006): 1-22), or antisense oligonucleotides causes
HDL increase. Sustained CETP inhibition with antisense nucleotides
increased plasma HDL and reduced atherosclerotic lesions in a
rabbit model of atherosclerosis. CETP-transgenic mice and rats show
decreased plasma HDL. Humans with reduced CETP activity have
elevated plasma HDL.
[0110] Recently, a vaccine approach has been proposed. Rabbits were
immunized with a human CETP-derived peptide containing a region of
CETP critical for neutral lipid transfer function. Vaccinated
rabbits had reduced CETP activity and an altered lipoprotein
profile with lower LDL and higher HDL concentration. Furthermore,
CETP-vaccinated rabbits were shown to have smaller atherosclerotic
lesions than control animals.
[0111] The problem of the anti-CETP vaccine approach discussed
above is that the vaccine formulation comprises a self peptide and
therefore must break natural tolerance against self antigens. The
invention describes a CETP mimotope that can be used for
vaccination: The mimotope shall induce the production of antibodies
against CETP. The CETP mimotope does not have a self sequence and
therefore does not need to break tolerance. Thus, the induction of
an anti-CETP antibody response is greatly facilitated. The mimotope
is identified with a monoclonal antibody (mAb) and (commercially
available) peptide libraries. An anti-CETP monoclonal antibody is
used that neutralizes CETP activity. This mAb detects a sequence
within the C-terminal 26 amino acids of CETP necessary for neutral
lipid transfer activity.
Example 1
Generation of Monoclonal Antibodies to be Used for Screening of
Phage Display Libraries
[0112] A.) 2 Antibodies Derived from "Fusion F":
[0113] Balb/c mouse were immunized with original CETP epitope
C-FGFPEHLLVDFLQSLS (SEQ ID NO. 147) (16 C-terminal amino acids of
CETP protein) coupled to KLH and Alum as adjuvant.
[0114] 2 hybridoma clones (both IgG1) were purified and used for
screening: F5AF9G4 ("Paula") and F6F11D1 ("Felix").
[0115] These 2 monoclonal antibodies recognize the injected epitope
as well as CETP protein in ELISA. They can also be used in Western
Blot to detect CETP protein (recombinant protein expressed in
bacteria as well as protein isolated from rabbit serum). Both
antibodies do not inhibit CETP enzyme activity (tested with Roar
CETP Activity Assay Kit, see e.g. U.S. Pat. No. 5,585,235; U.S.
Pat. No. 5,618,683; U.S. Pat. No. 5,770,355).
[0116] B.) 2 Antibodies Derived from "Fusion I":
[0117] Balb/c mouse were immunized with original CETP epitope
C-FGFPEHLLVDFLQSLS (SEQ ID NO. 147) (16 C-terminal amino acids of
CETP protein) coupled to KLH and Alum as adjuvant.
[0118] 2 hybridoma clones (both IgG1) were purified and used for
screening: 12G6H5 ("Frida") and 12G6H7 ("James").
[0119] These 2 monoclonal antibodies recognize the injected epitope
as well as CETP protein in ELISA. They can also be used in Western
Blot to detect CETP protein (recombinant protein expressed in
bacteria as well as protein isolated from rabbit serum). In
contrast to the antibodies derived from "Fusion F" (see A.)) both
antibodies "Frida" and "James" inhibit CETP enzyme activity (tested
with Roar CETP Activity Assay Kit).
Example 2
Phage Display, In Vitro Inhibition ELISA and In Vivo Testing of
Mimotopes
[0120] Phage Display libraries used in this example were:
[0121] Ph.D. 7: New England BioLabs E8102L (linear 7mer
library)
[0122] Ph.D. C7C: New England BioLabs E8121L (7mer library,
cyclized peptides)
[0123] Ph.D. 12: New England BioLabs E8111L (linear 12mer
library)
[0124] Phage Display was done according to manufacturer's protocol
(www.neb.com).
[0125] After 2 or 3 subsequent rounds of panning, single phage
clones were picked and phage supernatants were subjected to ELISA
on plates coated with the antibody that was used for the panning
procedure. Phage clones that were positive in this ELISA (strong
signal for the target, but no signal for unspecific control) were
sequenced. From DNA sequences, peptide sequences were deduced.
These peptides were synthesized and characterised in inhibition
ELISA.
[0126] 1. In Vitro Inhibition Assay (ELISA)
[0127] Different amounts of peptides (2 and 20 .mu.g, as indicated
in the respective figures) derived from Phage Display were
incubated with the monoclonal antibody that was used for the
screening procedure. Peptides diminishing subsequent binding of the
antibody to the original CETP epitope (C-terminal 16 amino acids of
CETP protein) coated on ELISA plates were considered as inhibiting.
(Results see i.a. FIGS. 19a to 19c)
[0128] 2. In Vivo Testing of Mimotopes
[0129] Inhibiting as well as some non-inhibiting peptides were
coupled to KLH and injected into mice (wildtype or CETP-transgenic
mice; subcutaneously into the flank or intra-dermaly into the ears)
or rabbits (subcutaneously into the flank) together with an
appropriate adjuvant (aluminium hydroxide and Gerbu 100 for mice
and aluminium hydroxide or CFA/IFA for rabbits).
[0130] Titers to injected peptides as well as to the original CETP
epitope were determined. In addition, for selected sera also immune
response to CETP protein was measured (Results see FIGS. 7a to 7d
and FIGS. 19a to 19e).
[0131] 3. Results
[0132] 3.1. Screening with 2 Antibodies Derived from "Fusion F":
"Paula" and "Felix"
[0133] 3.1.1. Phage Display Library Ph.D. 7
[0134] 3.1.1.1. Screening with Monoclonal Antibody "Paula"
[0135] 17 Sequences were identified in this screen:
TABLE-US-00026 (SEQ ID NO. 2) P2_8 SYHATFL (SEQ ID NO. 3) P2_9
TMAFPLN (SEQ ID NO. 4) P2_11 HYHGAFL (SEQ ID NO. 5) P2_12 EHHDIFL
(SEQ ID NO. 44) P2_15 SSLELFL (SEQ ID NO. 6) P2_16 TGLSVFL (SEQ ID
NO. 7) P3_2 WMPSLFY (SEQ ID NO. 8) P3_6, 14, 28 SMPWWFF (SEQ ID NO.
9) P3_9 TMPLLFW (SEQ ID NO. 10) P3_13 DTWPGLE (SEQ ID NO. 11) P3_16
SMPPIFY (SEQ ID NO. 12) P3_17 MPLWWWD (SEQ ID NO. 13) P3_18 SMPNLFY
(SEQ ID NO. 14) P3_19 RMPPIFY (SEQ ID NO. 15) P3_21 NPFEVFL (SEQ ID
NO. 16) P3_25 TLPNWFW (SEQ ID NO. 17) P3_26 SMPLTFY
[0136] The result of a representative competition ELISA is shown in
FIG. 1.
[0137] 3.1.1.2. Screening with Monoclonal Antibody "Felix"
[0138] 6 sequences were identified that inhibit binding of
monoclonal antibody "Felix" in in vitro competition
experiments:
TABLE-US-00027 F2-9 C SFLDTLT (SEQ ID NO. 45) F3-6 C NFLKTLS (SEQ
ID NO. 46) F3-18 C DFLRTLT (SEQ ID NO. 47) F3-23 C AFLDTLV (SEQ ID
NO. 48) F3-34 C TFLSSLA (SEQ ID NO. 49) F3-38 C GFLDSLM (SEQ ID NO.
50)
[0139] Additional 12 sequences were identified that do not inhibit
binding of monoclonal antibody "Felix" in in vitro competition
experiments:
TABLE-US-00028 F2-2 + 5 SPHPHFL (SEQ ID NO. 51) F2-6 NFMSIGL (SEQ
ID NO. 19) F2-16/F3-30 SQFLASL (SEQ ID NO. 20) F2-29 SNFLKTL (SEQ
ID NO. 52) F3-1-_ TGFLATL (SEQ ID NO. 53) F3-11-_ WSWPGLN (SEQ ID
NO. 21) F3-17- IAWPGLD (SEQ ID NO. 22) F3-32- SKFMDTL (SEQ ID NO.
23) F3-41- SDFLRAL (SEQ ID NO. 54) F3-44-_ SMPMVFY (SEQ ID NO. 24)
F3-49- YEWVGLM (SEQ ID NO. 25) F3-64- KGFLDHL (SEQ ID NO. 26
[0140] All mimotopes inhibiting the binding of monoclonal antibody
"Felix" in vitro were coupled to KLH and injected subcutaneously
(into the flank; s.c.) or intradermally (i.d.) into wild-type mice
(mice do not have CETP protein), CETP-tg mice, or rabbits,
respectively, and induced immune response to the injected peptide
with all adjuvants that were tested (Alum and CFA (Complete
Freund's adjuvant); Gerbu).
[0141] For all in vitro inhibiting mimotopes listed above,
antibodies reacting to the original CETP epitope could be detected
in mice and in rabbits.
[0142] For 5 out of 6 mimotopes (see below and Table 1) antibodies
reacting with purified human CETP and recombinantly expressed human
CETP could be detected in ELISAs from rabbit sera:
TABLE-US-00029 F2-9 C SFLDTLT (SEQ ID NO. 45) F3-6 C NFLKTLS (SEQ
ID NO. 46) F3-18 C DFLRTLT (SEQ ID NO. 47) F3-34 C TFLSSLA (SEQ ID
NO. 49) F3-38 C GFLDSLM (SEQ ID NO. 50)
[0143] Subcutaneous injections in the flank were performed in week
1, week 3 and week 7 with 30 .mu.g peptide-KLH per mouse.
Intradermal injections in the ear were performed in week 1, week 3
and week 6 with 10 .mu.g peptide-KLH per mouse. Sera were taken 2
weeks after the 3rd injection. Vaccine formulation with Alum
(always 1 mg per mouse): up to 250 .mu.l, injected into one flank.
The Alum formulation with 1 ml per mouse (500 .mu.l into each
flank) was in 1.times.PBS as buffer.
[0144] Vaccine formulation with Gerbu Adjuvant 100 (Gerbu Cat. Nr.
#3100; always 50 .mu.l adjuvant per mouse): 200 .mu.l, 100 .mu.l
injected into each flank comprising 1.times.HEPES as buffer.
TABLE-US-00030 TABLE 1 Results of the titer determination P4073
injected (FGFPEH- p irrel- Adjuvant KLH mimotope LLVDFLOSLS) evant
Alum s.c. (30 KLH 1:20.000 n.a. 1:400 no titer .mu.g peptide)
p4073-KLH C- 1:70.000 n.a. 1:20.000 no titer FGFPEHLLVD- FLQSLS
(SEQ ID NO. 147) p4223-KLH F2-9; C- 1:15.000 1:15.000 1:6.400 no
titer SFLDTLT (SEQ ID NO. 45) p4181-KLH F3-6 C- 1:8.000 1:6.400
1:800 no titer NFLKTLS (SEQ ID NO. 46) p4184-KLH F3-18 C- 1:5.000
1:10.000 1:3.000 1:2.500 DFLRTLT (SEQ ID NO. 47) p4187 F3-34 C-
1:3.200 1:9.000 1:4.000 no titer TFLSSLA (SEQ ID NO. 49) p4188-KLH
F3-38 C- 1:10.000 1:9.000 1.5.000 no titer GFLDSLM (SEQ ID NO. 50)
p4227-KLH P12-19; C- 1:12.800 1:10.000 1:5.000 no titer
SANPRDFLETLF (SEQ ID NO. 55) p4228-KLH P12-21; C- 1:10.000 1:4.000
1:1.000 1:400 RMFPESFLDTLW (SEQ ID NO. 56) KLH/Gerbu s.c. KLH
1:70.000 n.a. 1:6.000 1:800 (30 .mu.g pep- tide) p4073-KLH C-
1:25.000 n.a. 1:15.000 1:200 FGFPEHLLVD- FLQSLS (SEQ ID NO. 147)
p4223-KLH F2-9; C- 1:40.000 1:25.000 1:50.000 1:1.000 SFLDTLT (SEQ
ID NO. 45) p4181-KLH F3-6 C- 1:20.000 1.20.000 1:8.000 1:400
NFLKTLS (SEQ ID NO. 46) p4184-KLH F3-18 C- 1:27.000 1.35.000
1:15.000 1:6.000 DFLRTLT (SEQ ID NO. 47) p4187-KLH F3-34 C-
1.20.000 1.20.000 1:15.000 no titer TFLSSLA (SEQ ID NO. 49)
p4188-KLH F3-38 C- 1:40.000 1:35.000 1:35.000 1:400 GFLDSLM (SEQ ID
NO. 50) p4227-KLH P12-19; C- 1.20.000 1:30.000 1.3.000 1:400
SANPRDFLETLF (SEQ ID NO. 55) p4228-KLH P12-21; C- 1:27.000 1:8.000
1:5.000 no titer RMFPESFLDTLW (SEQ ID NO. 56) p4073-KLH C- 1:10.000
1:10.000 no titer FGFPEHLLVD- FLQSLS (SEQ ID NO. 147) KLH/Alum i.d.
KLH 1:12.800 n.a. no titer no titer (10 .mu.g pep- tide) p4073-KLH
C- 1:10.000 n.a. 1:3.200 no titer FGFPEHLLVD- FLQSLS (SEQ ID NO.
147) p4223-KLH F2-9; C- 1:6.400 1:3.200 SFLDTLT (SEQ ID NO. 45)
p4181-KLH F3-6 C- 1:10.000 1:1.500 1:600 no titer NFLKTLS (SEQ ID
NO. 46) p4184-KLH F3-18 C- 1:15.000 1:5.000 1:1.500 no titer
DFLRTLT (SEQ ID NO. 47) p4187-KLH F3-34 C- 1:50.000 1:6.400 1:3.200
1:500 TFLSSLA (SEQ ID NO. 49) p4188-KLH F3-38 C- 1:12.000 1:5.000
1:2.000 no titer GFLDSLM (SEQ ID NO. 50) p4227-KLH P12-19; C-
1:6.400 1:6.400 no titer no titer SANPRDFLETLF (SEQ ID NO. 55)
p4228-KLH P12-21; C- 1:20.000 1:2.000 1:1.600 no titer RMFPESFLDTLW
(SEQ ID NO. 56) p4298-KLH Fr12/3/84ext2; 1:25.000 1:3.200 1:1.600
no titer C- FGFPAHVSID- WLQSLS (SEQ ID NO. 184)
[0145] 3.1.2. Phage Display Library Ph.D. 12
[0146] 3.1.2.1. Screening with Monoclonal Antibody "Paula"
[0147] Out of 20 amino acid sequences derived from this screen, 3
were inhibiting in in vitro inhibition experiments:
TABLE-US-00031 P12-19 SANPRDFLETLF (SEQ ID NO. 55) P12-21
RMFPESFLDTLW (SEQ ID NO. 56) P12-37 TIYDSFLDSLAS (SEQ ID NO.
57)
[0148] Not inhibiting peptides were:
TABLE-US-00032 P12-5/44/46/49 HQSDDKMPWWFF (SEQ ID NO. 27) P12-9
KPYLLKDFLEAL (SEQ ID NO. 58) P12-24/43-_ AMGPYDALDLFL (SEQ ID NO.
59) P12-25 TWNPIESFLESL (SEQ ID NO. 60) P12-28 + 42 YVWQDPSFTTFF
(SEQ ID NO. 28) P12-30 QYQTPLTFLEAL (SEQ ID NO. 61) P12-35-
RHISPATFLEAL (SEQ ID NO. 62) P12-39- HTDSFLSTFYGD (SEQ ID NO. 63)
P12-42- YVWQDPSFTTFF (SEQ ID NO. 29) P12-45- ADSTFTSFLQTL (SEQ ID
NO. 64) P12-50-_ GPVSIYADTDFL (SEQ ID NO. 65) P12-51-_ DSNDTLTLAAFL
(SEQ ID NO. 66) P12-52-_ NGSPALSHMLFL (SEQ ID NO. 33) P12-53-
TDYDPMWVFFGY (SEQ ID NO. 34) P12-56- IFPLDSQWQTFW (SEQ ID NO. 35)
P12-58- NESMPDLFYQPS (SEQ ID NO. 36) P12-61- DWGDKYFSSFWN (SEQ ID
NO. 37)
[0149] Results of 2 typical competition ELISAs are shown in FIGS.
2A and 2b.
[0150] All 3 mimotopes were coupled to KLH and injected into
wildtype mice (mice do not have CETP protein), CETP-tg mice, or
rabbits, respectively, and induced immune response to the injected
peptide with all adjuvants that were tested (Alum and CFA;
Gerbu).
[0151] Mimotope P12-19; C-SANPRDFLETLF (SEQ ID NO. 55) and P12-21;
C-RMFPESFLDTLW (SEQ ID NO. 56) induced an immune response to the
original CETP epitope in wt mice and in rabbits.
[0152] In contrast thereto, mimotope P12-37 C-TIYDSFLDSLAS (SEQ ID
NO. 57) did not induce an antibody response to the original
epitope.
[0153] 3.2 Screening with 2 Antibodies Derived from "Fusion I":
"Frida" and "James"
[0154] 3.2.1. Phage Display Library pH.D. 7
[0155] 3.2.1.1. Screening with Monoclonal Antibodies "Frida" and
"James"
[0156] Two different peptide sequences were identified in these
screens, 11 of 12 clones that were sequenced had identical
sequences. These peptides are not inhibiting in in vitro
competition experiments.
TABLE-US-00033 Fr7-2-2 Fr7-2B-65 Fr7-3-7 Fr7-3-13 Fr7-3-26 Fr7-3-32
Ja7-2-22 Ja7-3-28 Ja7-3-41 Ja7-3-52 Ja7-3-56 VSAYNNV (SEQ ID NO.
38) Ja7-3-89 WPLHLWQ (SEQ ID NO. 39)
[0157] The results of 2 representative competition ELISAs with mAb
"Frida" are shown in FIGS. 3A and 3b. The same pattern was seen
with mAb "James".
[0158] 3.2.2. Phage Display Library pH.D. 12
[0159] 3.2.2.1. Screening with Monoclonal Antibody "Frida"
TABLE-US-00034 Fr12/2/6 TPTHYYADFSQL (SEQ ID NO. 67) Fr12/2/11
LPGHLIWDSLHY (SEQ ID NO. 68) Fr12/2/27 LPQTHPLHLLED (SEQ ID NO. 69)
Fr12/3/1 Fr12/3/19 Fr12/3/88 IPYHHLVDQLHH (SEQ ID NO. 70) Fr12/3/26
Fr12/3/65 YPYHVQVDVLQN (SEQ ID NO. 71) Fr12/3/68 IPSHHLQDSLQL (SEQ
ID NO. 72) Fr12/3/12 EYAHHTSLDLRQ (SEQ ID NO. 73) Fr12/3/83
EPLHFRSDRIQA (SEQ ID NO. 74) Fr12/3/55 ATPSHLIIDRAQ (SEQ ID NO. 75)
Fr12/3/63 APKHLYADMSQA (SEQ ID NO. 76) Fr12/3/84 FKPAHVSIDWLQ (SEQ
ID NO. 77) Fr12/3/47 MPAHLSRDLRQS (SEQ ID NO. 78) Fr12/3/80
NPKHYSIDRHQA (SEQ ID NO. 79) Fr12/3/40 SPQHLTTDRAQA (SEQ ID NO. 80)
Fr12/3/35 TPFHFAQDSWQW (SEQ ID NO. 81)
[0160] None of the 15 amino acid sequences identified in this
screen were inhibiting in in vitro competition experiments.
However, sequence analysis revealed rather high homology to the
original protein sequence for many of the mimotopes. On the other
hand, for some peptides binding of monoclonal antibody "Frida" to
ELISA plates coated with mimotope-BSA could be shown (see FIGS. 4a
and 4b).
[0161] This shows that binding of monoclonal antibody to
immobilised mimotopes does not necessarily allow to predict
inhibition in in vitro competition ELISA.
[0162] In vitro inhibition experiments with variations of the
original sequence FGFPEHLLVDFLQSLS (SEQ ID NO. 147) (16 C-terminal
AA of CETP protein) showed that removing more than 2 amino acids
from the N-terminus or more than 1 amino acid from the C-terminus
abolishes inhibition (for monoclonal antibodies "Frida" and
"James". "Paula" and "Felix" recognise a different part of the
original sequence).
[0163] In addition, simultaneously removing 2 amino acids from the
N-terminus and 1 amino acid from the C-terminus also results in a
peptide that is not inhibiting in vitro any more.
TABLE-US-00035 (SEQ ID NO. 147) C-FGFPEHLLVDFLQSLS "original"
sequence (peptide derived from CETP)/inhibiting in vitro
C-GFPEHLLVDFLQSLS sequence N-1/ inhibiting in vitro
C-FPEHLLVDFLQSLS sequence N-2/ inhibiting in vitro C-PEHLLVDFLQSLS
sequence N-3/ evtl. slightly inhibiting in vitro C-FGFPEHLLVDFLQSL
sequence C-1/ inhibiting in vitro C-FGFPEHLLVDFLQS sequence C-2/
not inhibiting in vitro C-FPEHLLVDFLQSL sequence N-2 and C-1/not
inhibiting in vitro! "original" FGFPEHLLVDFLQSLS Fr12/2/6
TPTHYYADFSQL (SEQ ID NO. 67) Fr12/2/11 LPGHLIWDSLHY (SEQ ID NO. 68)
Fr12/2/27 LPQTHPLHLLED (SEQ ID NO. 69) Fr12/3/1 IPYHHLVDQLHH (SEQ
ID NO. 70) Fr12/3/19 IPYHHLVDQLHH (SEQ ID NO. 70) Fr12/3/88
IPYHHLVDQLHH (SEQ ID NO. 70) Fr12/3/26 YPYHVQVDVLQN (SEQ ID NO. 71)
Fr12/3/65 YPYHVQVDVLQN (SEQ ID NO. 71) Fr12/3/68 IPSHHLQDSLQL (SEQ
ID NO. 72) Fr12/3/12 EYAHHTSLDLRQ (SEQ ID NO. 73) Fr12/3/83
EPLHFRSDRIQA (SEQ ID NO. 74) Fr12/3/55 ATPSHLIIDRAQ (SEQ ID NO. 75)
Fr12/3/63 APKHLYADMSQA (SEQ ID NO. 76) Fr12/3/84 FKPAHVSIDWLQ (SEQ
ID NO. 77) Fr12/3/47 MPAHLSRDLRQS (SEQ ID NO. 78) Fr12/3/80
NPKHYSIDRHQA (SEQ ID NO. 79) Fr12/3/40 SPQHLTTDRAQA (SEQ ID NO. 80)
Fr12/3/35 TPFHFAQDSWQW (SEQ ID NO. 81)
[0164] Consequently, using the original CETP sequence as a
template, peptide sequences obtained in this Phage Display
procedure were elongated on the N-terminus and/or C-terminus to
check whether in vitro inhibition is possible with longer
peptides.
[0165] 3.2.2.2. Mimotopes Frida pH.D.12 and Variations Thereof:
TABLE-US-00036 Fr12/2/6 TPTHYYADFSQL (SEQ ID NO. 67) Fr12/2/6 ext1
TPTHYYADFSQLLS (SEQ ID NO. 82) Fr12/2/6 ext2 TPTHYYADFSQSLS (SEQ ID
NO. 83) Fr12/2/6 ext3 GTPTHYYADFSQLL (SEQ ID NO. 84) Fr12/2/6 ext4
GTPTHYYADFSQSL (SEQ ID NO. 85) Fr12/2/6 ext5 FGTPTHYYADFSQSLS (SEQ
ID NO. 86) Fr12/2/6 ext6 FGFPTHYYADFSQSLS (SEQ ID NO. 87) Fr12/2/11
LPGHLIWDSLHY (SEQ ID NO. 68) Fr12/2/11 ext1 LPGHLIWDSLHYL (SEQ ID
NO. 89) Fr12/2/11 ext2 LPGHLIWDSLHYLS (SEQ ID NO. 90) Fr12/2/11
ext3 LPGHLIWDSLHSL (SEQ ID NO. 91) Fr12/2/11 ext4 LPGHLIWDSLHSLS
(SEQ ID NO. 92) Fr12/2/11 ext5 GLPGHLIWDSLHYL (SEQ ID NO. 93)
Fr12/2/11 ext5 GLPGHLIWDSLHSL (SEQ ID NO. 94) Fr12/2/11 ext6
FGLPGHLIWDSLHSLS (SEQ ID NO. 95) Fr12/2/11 ext7 FGFPGHLIWDSLHSLS
(SEQ ID NO. 96) Fr12/2/27 LPQTHPLHLLED (SEQ ID NO. 69)
Fr12/3/1/19/88 IPYHHLVDQLHLS (SEQ ID NO. 99) ext1 Fr12/3/1/19/88
IPYHHLVDQLHSLS (SEQ ID NO. 100) ext2 Fr12/3/1/19/88
FGIPYHHLVDQLHHLS (SEQ ID NO. 101) ext3 Fr12/3/1/19/88
FGFPYHHLVDQLHSLS (SEQ ID NO. 102) ext4 Fr12/3/26/65ext1
YPYHVQVDVLQNLS (SEQ ID NO. 104) Fr12/3/26/65ext2 YPYHVQVDVLQSLS
(SEQ ID NO. 105) Fr12/3/26/65ext3 FGYPYHVQVDVLQNLS (SEQ ID NO. 106)
Fr12/3/26/65ext4 FGFPYHVQVDVLQSLS (SEQ ID NO. 107) Fr12/3/68 ext1
IPSHHLQDSLQLLS (SEQ ID NO. 109) Fr12/3/68 ext2 IPSHHLQDSLQSLS (SEQ
ID NO. 110) Fr12/3/68 ext3 GIPSHHLQDSLQLL (SEQ ID NO. 111)
Fr12/3/68 ext4 FGIPSHHLQDSLQLLS (SEQ ID NO. 112) Fr12/3/68 ext5
FGFPSHHLQDSLQSLS (SEQ ID NO. 113) Fr12/3/83 ext1 EPLHFRSDRIQALS
(SEQ ID NO. 116) Fr12/3/83 ext2 EPLHFRSDRIQSLS (SEQ ID NO. 117)
Fr12/3/83 ext3 GEPLHFRSDRIQAL (SEQ ID NO. 118) Fr12/3/83 ext4
FGEPLHFRSDRIQALS (SEQ ID NO. 119) Fr12/3/83 ext5 FGFPLHFRSDRIQSLS
(SEQ ID NO. 120) Fr12/3/55 ext1 ATPSHLIIDRAQSLS (SEQ ID NO. 176)
Fr12/3/55 ext2 FGFPSHLIIDRAQSLS (SEQ ID NO. 177) Fr12/3/55 ext2
FGFPSHLIIDWAQSLS (SEQ ID NO. 178) R->W Fr12/3/55 ext2
FGFPSHLIIDWLQSLS (SEQ ID NO. 179) RA->WL Fr12/3/63 ext1
APKHLYADMSQALS (SEQ ID NO. 122) Fr12/3/63 ext2 APKHLYADMSQSLS (SEQ
ID NO. 123) Fr12/3/63 ext3 GAPKHLYADMSQAL (SEQ ID NO. 124)
Fr12/3/63 ext4 FGFPKHLYADMSQSLS (SEQ ID NO. 125) Fr12/3/84 ext1
FKPAHVSIDWLQSLS (SEQ ID NO. 183) Fr12/3/84 ext2 FGFPAHVSIDWLQSLS
(SEQ ID NO. 184) Fr12/3/47 ext1 MPAHLSRDLRQSL (SEQ ID NO. 127)
Fr12/3/47 ext2 MPAHLSRDLRQSLS (SEQ ID NO. 128) Fr12/3/47 ext3
GMPAHLSRDLRQSL (SEQ ID NO. 129) Fr12/3/47 ext4 FGFPAHLSRDLRQSLS
(SEQ ID NO. 130) Fr12/3/40 ext1 SPQHLTTDRAQALS (SEQ ID NO. 168)
Fr12/3/40 ext2 SPQHLTTDRAQSLS (SEQ ID NO. 169) Fr12/3/40 ext3
GSPQHLTTDRAQAL (SEQ ID NO. 170) Fr12/3/40 ext4 FGFPQHLTTDRAQSLS
(SEQ ID NO. 171) Fr12/3/35 ext1 TPFHFAQDSWQWLS (SEQ ID NO. 133)
Fr12/3/35 ext2 TPFHFAQDSWQSLS (SEQ ID NO. 134) Fr12/3/35 ext3
GTPFHFAQDSWQWL (SEQ ID NO. 135) Fr12/3/35 ext4 FGFPFHFAQDSWQSLS
(SEQ ID NO. 136)
[0166] Representative examples of inhibition ELISA are shown in
FIGS. 5A and 5b. The elongated peptides Fr12/3/84 ext2 and
Fr12/3/55 ext3 showed a significant inhibition:
TABLE-US-00037 C-FGFPSHLIIDRAQSLS Fr12/3/55 ext3 (SEQ ID NO. 177)
C-FGFPAHVSIDWLQSLS Fr12/3/84 ext2 (SEQ ID NO. 184)
[0167] Three additional peptides were also inhibiting in this
assay:
TABLE-US-00038 C-FGFPYHVQVDVLQSLS Fr12/3/26/65ext4 (SEQ ID NO. 107)
C-FKPAHVSIDWLQSLS Fr12/3/84 ext1 (SEQ ID NO. 183)
C-FGFPQHLTTDRAQSLS Fr12/3/40 ext4 (SEQ ID NO. 171)
[0168] After sequence analysis comparing the original epitope and
all mimotopes derived from Phage Display screens additional 2
peptides were created.
[0169] For mimotope Fr12/3/55 ext3 C-FGFPSHLIIDRAQSLS (SEQ ID NO.
177) (inhibiting in ELISA, see above) amino acid exchanges were
tested in inhibition ELISA:
TABLE-US-00039 Strongly inhibiting: C-FGFPAHVSIDWLQSLS (SEQ ID NO.
184) Fr12/3/84 ext2 Slightly inhibiting: C-FGFPSHLIIDRAQSLS (SEQ ID
NO. 177) Fr12/3/55 ext3 Peptides with altered sequences
(inhibiting, see FIG. 6): C-FGFPSHLIIDWAQSLS (SEQ ID NO. 178)
Fr12/3/55 ext2 W instead of R C-FGFPSHLIIDWLQSLS (SEQ ID NO. 179)
Fr12/3/55 ext2 WL instead of RA
Further preferred mimotopes have been characterised by the
following example-set-up:
TABLE-US-00040 Exp. Nr. C42-1 KLH/Alum -- CETP-42 C42-2 p4073-KLH/
C-FGFPEHLLVDFLQSLS Alum (SEQ ID NO. 147 C42-3 p4073 LLV->LFV
p4468-KLH/ C-FGFPEHLFVDFLQSLS Alum (SEQ ID NO. 252) C42-4 Fr12/3/84
ext2 VSI->VFI P4361-KLH/ C-FGFPAHVFIDWLQSLS Alum (SEQ ID NO.
222) C42-5 Fr12/3/84 ext2 VSI->VHI p4469- C-FGFPAHVHIDWLQSLS
KLH/Alum (SEQ ID NO. 253) C42-6 Fr12/3/84 ext2 VSI->VPI p4470-
C-FGFPAHVPIDWLQSLS KLH/Alum (SEQ ID NO. 254) C42-7 Fr12/3/84 ext2
VSI->VWI p4471- C-FGFPAHVWIDWLQSLS KLH/Alum (SEQ ID NO. 229)
C42-8 Fr12/3/55 ext2 R->W LII->LFI p4472- C-FGFPSHLFIDWAQSLS
KLH/Alum (SEQ ID NO. 255) C42-9 Fr12/3/84 ext2 VSoVFI FGF- p4473-
C-PGFPAHVFIDWLQLIT >PGF SLS->LIT KLH/Alum (SEQ ID NO. 256)
C42-10 Fr12/3/84 ext2 VSI->VYI P4362-KLH/ C-FGFPAHVYIDWLQSLS
Alum (SEQ ID NO. 223) Exp. Nr. C45-1 KLH/Alum -- CETP-45 C45-2
p1358-KLH/ neg. control peptide Alum C45-3 p4073-KLH/
C-FGFPEHLLVDFLQSLS Alum (SEQ ID NO. 147) C45-4 Fr12/3/84 ext2
VSI->VFI SLS- p4474-KLH/ C-FGFPAHVFIDWLQSLN >SLN Alum (SEQ ID
NO. 230) C45-5 Fr12/3/84 ext2 VSI->FSI p4475-KLH/
C-FGFPAHFSIDWLQSLS Alum (SEQ ID NO. 231) C45-6 Fr12/3/84 ext2
VSI->VSF p4476-KLH/ C-FGFPAHVSFDWLQSLS Alum (SEQ ID NO. 232)
C45-7 Fr12/3/84 ext2 VSI->VFI PAH- p4477-KLH/ C-FGFPEHVFIDWLQSLS
>PEH Alum (SEQ ID NO. 233) C45-8 Fr12/3/1/19/88 ext4 p4284-KLH/
C-FGFPYHHLVDQLHSLS Alum (SEQ ID NO. 102) C45-9 Fr12/3/84 ext1
VSI->VFI plus p4479-KLH/ C-GFKPAHVFIDWLQSLS G on N-terminus Alum
(SEQ ID NO. 270) C45-10 Fr12/3/84 ext2 VSI->VFI plus p4480-KLH/
C-DFGFPAHVFIDWLQSLS D on N-terminus; =4361 plus D Alum (SEQ ID NO.
234) C45-11 Fr12/3/40 ext4 RA->WL LTT->LFT p4481-KLH/
C-FGFPQHLFTDWLQSLS =p4369 with exchange ToF Alum (SEQ ID NO. 237)
C45-12 Fr12/3/55 ext2 RA->WL (see C- p4325-KLH/
C-FGFPSHLIIDWLQSLS 31 and C-33; sera inhibiting Alum (SEQ ID NO.
179) activity) C45-13 Fr12/3/84 ext2 FGF->FYF (see p4343-KLH/
C-FYFPAHVSIDWLQSLS C-33: recogn. protein/not in- Alum (SEQ ID NO.
204) hibiting activity) C45-14 rabbit sequence p4125-KLH/
C-FGFPKHLLVDFLQSLS Alum (SEQ ID NO. 238)
[0170] 3.2.2.3. In Vivo Testing of Mimotopes
[0171] Female Balb/c mice, five mice per group, were subcutaneously
immunized with 30 .mu.g peptide coupled to KLH. Control groups were
administered KLH or C-FGFPEHLLVDFLQSLS (SEQ ID NO. 147). As
adjuvant alum was used. The peptides administered were all able to
bind to "Frida" and to induce an immune response for CETP, although
some of these peptides did not inhibit the binding of CETP to
"Frida" in vitro (in an in vitro inhibition assay). The in vitro
ELISA assay to determine the antibody titer was performed with
pooled sera after two vaccinations in a two week interval (S2; see
FIGS. 7a to 7d). The wells of the ELISA plate were coated with KLH
(positive control), mimotope-BSA conjugate, C-FGFPEHLLVDFLQSLS (SEQ
ID NO. 147) and a irrelevant peptide-BSA conjugate (negative
control). The detection was performed with anti-mouse IgG.
[0172] 3.2.3. Phage Display Library pH.D. 7C7
[0173] 3.2.3.1. Screening with Monoclonal Antibodies "Frida" and
"James"
TABLE-US-00041 Fr2-1 ACSFAYLYRC (SEQ ID NO. 137) Fr2-5 Fr2-6 Fr2-18
Fr2-19 Fr2-28 Ja2-5 Ja2-20 Ja2-23 Ja2-24 Ja2-30 ACFMGDKWVC (SEQ ID
NO. 138) Fr2-7 Fr2-9 ACVLYPKAIC (SEQ ID NO. 139) Fr2-11 Ja2-19
ACYMGQQFVC (SEQ ID NO. 140) Fr2-16 ACLTAYLHWC (SEQ ID NO. 141)
Fr2-20 ACTLFPVAYC (SEQ ID NO. 142) Fr2-25 ACWLFPYAHC (SEQ ID NO.
143) Fr2-26 ACKSINMWLC (SEQ ID NO. 144) Fr2-27 ACQTINRWLC (SEQ ID
NO. 145)
[0174] Due to their cyclic nature of these mimotope-peptides their
synthesis is more complicated than the synthesis of linear
peptides. Seven out of 9 cyclic sequences were chosen for in vitro
analysis in inhibition ELISA (see FIGS. 8a and 8b). None of these
sequences inhibited binding of the monoclonal antibody that was
used for Phage Display Screening to the original CETP epitope. In
addition, when these peptides were coupled to BSA and coated onto
ELISA plate they were not detected by the monoclonal antibody (see
FIG. 9). This was in contrast to data with mimotopes derived from
the Ph.D.7 or Ph.D.12 libraries, where the monoclonal antibodies
bound to most of the identified mimotopes when these peptides were
coupled to BSA and coated onto ELISA plates.
Example 3
CETP Activity Assay
[0175] The CETP activity assay was performed with assays
commercially available (e.g. ROAR CETP Activity Assay) and
described, for instance, in the U.S. Pat. No. 5,585,235, U.S. Pat.
No. 5,618,683 and U.S. Pat. No. 5,770,355. The assay is performed
according to the manufacturers' recommendations.
Sequence CWU 1
1
27016PRTArtificial SequenceCETP mimotope 1Xaa Xaa Xaa Xaa Xaa Xaa 1
5 27PRTArtificial SequenceCETP mimotope 2Ser Tyr His Ala Thr Phe
Leu 1 5 37PRTArtificial SequenceCETP mimotope 3Thr Met Ala Phe Pro
Leu Asn 1 5 47PRTArtificial SequenceCETP mimotope 4His Tyr His Gly
Ala Phe Leu 1 5 57PRTArtificial SequenceCETP mimotope 5Glu His His
Asp Ile Phe Leu 1 5 67PRTArtificial SequenceCETP mimotope 6Thr Gly
Leu Ser Val Phe Leu 1 5 77PRTArtificial SequenceCETP mimotope 7Trp
Met Pro Ser Leu Phe Tyr 1 5 87PRTArtificial SequenceCETP mimotope
8Ser Met Pro Trp Trp Phe Phe 1 5 97PRTArtificial SequenceCETP
mimotope 9Thr Met Pro Leu Leu Phe Trp 1 5 107PRTArtificial
SequenceCETP mimotope 10Asp Thr Trp Pro Gly Leu Glu 1 5
117PRTArtificial SequenceCETP mimotope 11Ser Met Pro Pro Ile Phe
Tyr 1 5 127PRTArtificial SequenceCETP mimotope 12Met Pro Leu Trp
Trp Trp Asp 1 5 137PRTArtificial SequenceCETP mimotope 13Ser Met
Pro Asn Leu Phe Tyr 1 5 147PRTArtificial SequenceCETP mimotope
14Arg Met Pro Pro Ile Phe Tyr 1 5 157PRTArtificial SequenceCETP
mimotope 15Asn Pro Phe Glu Val Phe Leu 1 5 167PRTArtificial
SequenceCETP mimotope 16Thr Leu Pro Asn Trp Phe Trp 1 5
177PRTArtificial SequenceCETP mimotope 17Ser Met Pro Leu Thr Phe
Tyr 1 5 187PRTArtificial SequenceCETP mimotope 18Ser Pro His Pro
His Phe Leu 1 5 197PRTArtificial SequenceCETP mimotope 19Asn Phe
Met Ser Ile Gly Leu 1 5 207PRTArtificial SequenceCETP mimotope
20Ser Gln Phe Leu Ala Ser Leu 1 5 217PRTArtificial SequenceCETP
mimotope 21Trp Ser Trp Pro Gly Leu Asn 1 5 227PRTArtificial
SequenceCETP mimotope 22Ile Ala Trp Pro Gly Leu Asp 1 5
237PRTArtificial SequenceCETP mimotope 23Ser Lys Phe Met Asp Thr
Leu 1 5 247PRTArtificial SequenceCETP mimotope 24Ser Met Pro Met
Val Phe Tyr 1 5 257PRTArtificial SequenceCETP mimotope 25Tyr Glu
Trp Val Gly Leu Met 1 5 267PRTArtificial SequenceCETP mimotope
26Lys Gly Phe Leu Asp His Leu 1 5 2712PRTArtificial SequenceCETP
mimotope 27His Gln Ser Asp Asp Lys Met Pro Trp Trp Phe Phe 1 5 10
2812PRTArtificial SequenceCETP mimotope 28Tyr Val Trp Gln Asp Pro
Ser Phe Thr Thr Phe Phe 1 5 10 2912PRTArtificial SequenceCETP
mimotope 29Tyr Val Trp Gln Asp Pro Ser Phe Thr Thr Phe Phe 1 5 10
3012PRTArtificial SequenceCETP mimotope 30Leu Pro Gln Thr His Pro
Leu His Leu Leu Glu Asp 1 5 10 3112PRTArtificial SequenceCETP
mimotope 31Gly Pro Val Ser Ile Tyr Ala Asp Thr Asp Phe Leu 1 5 10
3212PRTArtificial SequenceCETP mimotope 32Asp Ser Asn Asp Thr Leu
Thr Leu Ala Ala Phe Leu 1 5 10 3312PRTArtificial SequenceCETP
mimotope 33Asn Gly Ser Pro Ala Leu Ser His Met Leu Phe Leu 1 5 10
3412PRTArtificial SequenceCETP mimotope 34Thr Asp Tyr Asp Pro Met
Trp Val Phe Phe Gly Tyr 1 5 10 3512PRTArtificial SequenceCETP
mimotope 35Ile Phe Pro Leu Asp Ser Gln Trp Gln Thr Phe Trp 1 5 10
3612PRTArtificial SequenceCETP mimotope 36Asn Glu Ser Met Pro Asp
Leu Phe Tyr Gln Pro Ser 1 5 10 3712PRTArtificial SequenceCETP
mimotope 37Asp Trp Gly Asp Lys Tyr Phe Ser Ser Phe Trp Asn 1 5 10
387PRTArtificial SequenceCETP mimotope 38Val Ser Ala Tyr Asn Asn
Val 1 5 397PRTArtificial SequenceCETP mimotope 39Trp Pro Leu His
Leu Trp Gln 1 5 4017PRTArtificial SequenceCETP mimotope 40Cys Phe
Gly Phe Pro Glu His Leu Leu Val Asp Phe Leu Gln Ser Leu 1 5 10 15
Ser 416PRTArtificial SequenceCETP mimotope 41Xaa Xaa His Xaa Xaa
Xaa 1 5 4216PRTArtificial SequenceCETP mimotope 42Phe Xaa Phe Pro
Xaa His Xaa Xaa Xaa Asp Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15
437PRTArtificial SequenceCETP mimotope 43Xaa Phe Leu Xaa Xaa Leu
Xaa 1 5 447PRTArtificial SequenceCETP mimotope 44Ser Ser Leu Glu
Leu Phe Leu 1 5 457PRTArtificial SequenceCETP mimotope 45Ser Phe
Leu Asp Thr Leu Thr 1 5 467PRTArtificial SequenceCETP mimotope
46Asn Phe Leu Lys Thr Leu Ser 1 5 477PRTArtificial SequenceCETP
mimotope 47Asp Phe Leu Arg Thr Leu Thr 1 5 487PRTArtificial
SequenceCETP mimotope 48Ala Phe Leu Asp Thr Leu Val 1 5
497PRTArtificial SequenceCETP mimotope 49Thr Phe Leu Ser Ser Leu
Ala 1 5 507PRTArtificial SequenceCETP mimotope 50Gly Phe Leu Asp
Ser Leu Met 1 5 517PRTArtificial SequenceCETP mimotope 51Ser Pro
His Pro His Phe Leu 1 5 527PRTArtificial SequenceCETP mimotope
52Ser Asn Phe Leu Lys Thr Leu 1 5 537PRTArtificial SequenceCETP
mimotope 53Thr Gly Phe Leu Ala Thr Leu 1 5 547PRTArtificial
SequenceCETP mimotope 54Ser Asp Phe Leu Arg Ala Leu 1 5
5512PRTArtificial SequenceCETP mimotope 55Ser Ala Asn Pro Arg Asp
Phe Leu Glu Thr Leu Phe 1 5 10 5612PRTArtificial SequenceCETP
mimotope 56Arg Met Phe Pro Glu Ser Phe Leu Asp Thr Leu Trp 1 5 10
5712PRTArtificial SequenceCETP mimotope 57Thr Ile Tyr Asp Ser Phe
Leu Asp Ser Leu Ala Ser 1 5 10 5812PRTArtificial SequenceCETP
mimotope 58Lys Pro Tyr Leu Leu Lys Asp Phe Leu Glu Ala Leu 1 5 10
5912PRTArtificial SequenceCETP mimotope 59Ala Met Gly Pro Tyr Asp
Ala Leu Asp Leu Phe Leu 1 5 10 6012PRTArtificial SequenceCETP
mimotope 60Thr Trp Asn Pro Ile Glu Ser Phe Leu Glu Ser Leu 1 5 10
6112PRTArtificial SequenceCETP mimotope 61Gln Tyr Gln Thr Pro Leu
Thr Phe Leu Glu Ala Leu 1 5 10 6212PRTArtificial SequenceCETP
mimotope 62Arg His Ile Ser Pro Ala Thr Phe Leu Glu Ala Leu 1 5 10
6312PRTArtificial SequenceCETP mimotope 63His Thr Asp Ser Phe Leu
Ser Thr Phe Tyr Gly Asp 1 5 10 6412PRTArtificial SequenceCETP
mimotope 64Ala Asp Ser Thr Phe Thr Ser Phe Leu Gln Thr Leu 1 5 10
6512PRTArtificial SequenceCETP mimotope 65Gly Pro Val Ser Ile Tyr
Ala Asp Thr Asp Phe Leu 1 5 10 6612PRTArtificial SequenceCETP
mimotope 66Asp Ser Asn Asp Thr Leu Thr Leu Ala Ala Phe Leu 1 5 10
6712PRTArtificial SequenceCETP mimotope 67Thr Pro Thr His Tyr Tyr
Ala Asp Phe Ser Gln Leu 1 5 10 6812PRTArtificial SequenceCETP
mimotope 68Leu Pro Gly His Leu Ile Trp Asp Ser Leu His Tyr 1 5 10
6912PRTArtificial SequenceCETP mimotope 69Leu Pro Gln Thr His Pro
Leu His Leu Leu Glu Asp 1 5 10 7012PRTArtificial SequenceCETP
mimotope 70Ile Pro Tyr His His Leu Val Asp Gln Leu His His 1 5 10
7112PRTArtificial SequenceCETP mimotope 71Tyr Pro Tyr His Val Gln
Val Asp Val Leu Gln Asn 1 5 10 7212PRTArtificial SequenceCETP
mimotope 72Ile Pro Ser His His Leu Gln Asp Ser Leu Gln Leu 1 5 10
7312PRTArtificial SequenceCETP mimotope 73Glu Tyr Ala His His Thr
Ser Leu Asp Leu Arg Gln 1 5 10 7412PRTArtificial SequenceCETP
mimotope 74Glu Pro Leu His Phe Arg Ser Asp Arg Ile Gln Ala 1 5 10
7512PRTArtificial SequenceCETP mimotope 75Ala Thr Pro Ser His Leu
Ile Ile Asp Arg Ala Gln 1 5 10 7612PRTArtificial SequenceCETP
mimotope 76Ala Pro Lys His Leu Tyr Ala Asp Met Ser Gln Ala 1 5 10
7712PRTArtificial SequenceCETP mimotope 77Phe Lys Pro Ala His Val
Ser Ile Asp Trp Leu Gln 1 5 10 7812PRTArtificial SequenceCETP
mimotope 78Met Pro Ala His Leu Ser Arg Asp Leu Arg Gln Ser 1 5 10
7912PRTArtificial SequenceCETP mimotope 79Asn Pro Lys His Tyr Ser
Ile Asp Arg His Gln Ala 1 5 10 8012PRTArtificial SequenceCETP
mimotope 80Ser Pro Gln His Leu Thr Thr Asp Arg Ala Gln Ala 1 5 10
8112PRTArtificial SequenceCETP mimotope 81Thr Pro Phe His Phe Ala
Gln Asp Ser Trp Gln Trp 1 5 10 8214PRTArtificial SequenceCETP
mimotope 82Thr Pro Thr His Tyr Tyr Ala Asp Phe Ser Gln Leu Leu Ser
1 5 10 8314PRTArtificial SequenceCETP mimotope 83Thr Pro Thr His
Tyr Tyr Ala Asp Phe Ser Gln Ser Leu Ser 1 5 10 8414PRTArtificial
SequenceCETP mimotope 84Gly Thr Pro Thr His Tyr Tyr Ala Asp Phe Ser
Gln Leu Leu 1 5 10 8514PRTArtificial SequenceCETP mimotope 85Gly
Thr Pro Thr His Tyr Tyr Ala Asp Phe Ser Gln Ser Leu 1 5 10
8616PRTArtificial SequenceCETP mimotope 86Phe Gly Thr Pro Thr His
Tyr Tyr Ala Asp Phe Ser Gln Ser Leu Ser 1 5 10 15 8716PRTArtificial
SequenceCETP mimotope 87Phe Gly Phe Pro Thr His Tyr Tyr Ala Asp Phe
Ser Gln Ser Leu Ser 1 5 10 15 8812PRTArtificial SequenceCETP
mimotope 88Leu Pro Gly His Leu Ile Trp Asp Ser Leu His Tyr 1 5 10
8913PRTArtificial SequenceCETP mimotope 89Leu Pro Gly His Leu Ile
Trp Asp Ser Leu His Tyr Leu 1 5 10 9014PRTArtificial SequenceCETP
mimotope 90Leu Pro Gly His Leu Ile Trp Asp Ser Leu His Tyr Leu Ser
1 5 10 9113PRTArtificial SequenceCETP mimotope 91Leu Pro Gly His
Leu Ile Trp Asp Ser Leu His Ser Leu 1 5 10 9214PRTArtificial
SequenceCETP mimotope 92Leu Pro Gly His Leu Ile Trp Asp Ser Leu His
Ser Leu Ser 1 5 10 9314PRTArtificial SequenceCETP mimotope 93Gly
Leu Pro Gly His Leu Ile Trp Asp Ser Leu His Tyr Leu 1 5 10
9414PRTArtificial SequenceCETP mimotope 94Gly Leu Pro Gly His Leu
Ile Trp Asp Ser Leu His Ser Leu 1 5 10 9516PRTArtificial
SequenceCETP mimotope 95Phe Gly Leu Pro Gly His Leu Ile Trp Asp Ser
Leu His Ser Leu Ser 1 5 10 15 9616PRTArtificial SequenceCETP
mimotope 96Phe Gly Phe Pro Gly His Leu Ile Trp Asp Ser Leu His Ser
Leu Ser 1 5 10 15 9712PRTArtificial SequenceCETP mimotope 97Leu Pro
Gln Thr His Pro Leu His Leu Leu Glu Asp 1 5 10 9812PRTArtificial
SequenceCETP mimotope 98Ile Pro Tyr His His Leu Val Asp Gln Leu His
His 1 5 10 9913PRTArtificial SequenceCETP mimotope 99Ile Pro Tyr
His His Leu Val Asp Gln Leu His Leu Ser 1 5 10 10014PRTArtificial
SequenceCETP mimotope 100Ile Pro Tyr His His Leu Val Asp Gln Leu
His Ser Leu Ser 1 5 10 10116PRTArtificial SequenceCETP mimotope
101Phe Gly Ile Pro Tyr His His Leu Val Asp Gln Leu His His Leu Ser
1 5 10 15 10216PRTArtificial SequenceCETP mimotope 102Phe Gly Phe
Pro Tyr His His Leu Val Asp Gln Leu His Ser Leu Ser 1 5 10 15
10312PRTArtificial SequenceCETP mimotope 103Tyr Pro Tyr His Val Gln
Val Asp Val Leu Gln Asn 1 5 10 10414PRTArtificial SequenceCETP
mimotope 104Tyr Pro Tyr His Val Gln Val Asp Val Leu Gln Asn Leu Ser
1 5 10 10514PRTArtificial SequenceCETP mimotope 105Tyr Pro Tyr His
Val Gln Val Asp Val Leu Gln Ser Leu Ser 1 5 10 10616PRTArtificial
SequenceCETP mimotope 106Phe Gly Tyr Pro Tyr His Val Gln Val Asp
Val Leu Gln Asn Leu Ser 1 5 10 15 10716PRTArtificial SequenceCETP
mimotope 107Phe Gly Phe Pro Tyr His Val Gln Val Asp Val Leu Gln Ser
Leu Ser 1 5 10 15 10812PRTArtificial SequenceCETP mimotope 108Ile
Pro Ser His His Leu Gln Asp Ser Leu Gln Leu 1 5 10
10914PRTArtificial SequenceCETP mimotope 109Ile Pro Ser His His Leu
Gln Asp Ser Leu Gln Leu Leu Ser 1 5 10 11014PRTArtificial
SequenceCETP mimotope 110Ile Pro Ser His His Leu Gln Asp Ser Leu
Gln Ser Leu Ser 1 5 10 11114PRTArtificial SequenceCETP mimotope
111Gly Ile Pro Ser His His Leu Gln Asp Ser Leu Gln Leu Leu 1 5 10
11216PRTArtificial SequenceCETP mimotope 112Phe Gly Ile Pro Ser His
His Leu Gln Asp Ser Leu Gln Leu Leu Ser 1 5 10 15
11316PRTArtificial SequenceCETP mimotope 113Phe Gly Phe Pro Ser His
His Leu Gln Asp Ser Leu Gln Ser Leu Ser 1 5 10 15
11412PRTArtificial SequenceCETP mimotope 114Glu Tyr Ala His His Thr
Ser Leu Asp Leu Arg Gln 1 5 10 11512PRTArtificial SequenceCETP
mimotope 115Glu Pro Leu His Phe Arg Ser Asp Arg Ile Gln Ala 1 5 10
11614PRTArtificial SequenceCETP mimotope 116Glu Pro Leu His Phe Arg
Ser Asp Arg Ile Gln Ala Leu Ser 1 5 10 11714PRTArtificial
SequenceCETP mimotope 117Glu Pro Leu His Phe Arg Ser Asp Arg Ile
Gln Ser Leu Ser 1 5 10 11814PRTArtificial SequenceCETP mimotope
118Gly Glu Pro Leu His Phe Arg Ser Asp Arg Ile Gln Ala Leu 1 5 10
11916PRTArtificial SequenceCETP mimotope 119Phe Gly Glu Pro Leu His
Phe Arg Ser Asp Arg Ile Gln Ala Leu Ser 1 5 10 15
12016PRTArtificial SequenceCETP mimotope 120Phe Gly Phe Pro Leu His
Phe Arg Ser Asp Arg Ile Gln Ser Leu Ser 1 5 10 15
12112PRTArtificial SequenceCETP mimotope 121Ala Pro Lys His Leu Tyr
Ala Asp Met Ser Gln Ala 1 5 10 12214PRTArtificial SequenceCETP
mimotope 122Ala Pro Lys His Leu Tyr Ala Asp Met Ser Gln Ala Leu Ser
1 5 10 12314PRTArtificial SequenceCETP mimotope 123Ala Pro Lys His
Leu Tyr Ala Asp Met Ser Gln Ser Leu Ser 1 5 10 12414PRTArtificial
SequenceCETP mimotope 124Gly Ala Pro Lys His Leu Tyr Ala Asp Met
Ser Gln Ala Leu 1 5 10 12516PRTArtificial SequenceCETP mimotope
125Phe Gly Phe Pro Lys His Leu Tyr Ala Asp Met Ser Gln Ser Leu Ser
1 5 10 15 12612PRTArtificial SequenceCETP mimotope 126Met Pro Ala
His Leu Ser Arg Asp Leu Arg Gln Ser 1 5 10 12713PRTArtificial
SequenceCETP mimotope 127Met Pro Ala His Leu Ser Arg Asp Leu Arg
Gln Ser Leu 1 5 10 12814PRTArtificial SequenceCETP mimotope 128Met
Pro Ala His Leu Ser Arg Asp Leu Arg Gln Ser Leu Ser 1 5 10
12914PRTArtificial SequenceCETP mimotope 129Gly Met Pro Ala His Leu
Ser Arg Asp Leu Arg Gln Ser Leu 1 5 10 13016PRTArtificial
SequenceCETP mimotope 130Phe Gly Phe Pro Ala His Leu Ser Arg Asp
Leu Arg Gln Ser Leu Ser 1 5 10 15 13112PRTArtificial SequenceCETP
mimotope 131Asn Pro Lys His Tyr Ser Ile Asp Arg His Gln Ala 1 5 10
13212PRTArtificial SequenceCETP mimotope 132Thr Pro Phe His Phe Ala
Gln Asp Ser Trp Gln Trp 1 5 10 13314PRTArtificial SequenceCETP
mimotope 133Thr Pro Phe His Phe Ala Gln Asp Ser Trp Gln Trp Leu Ser
1 5 10 13414PRTArtificial SequenceCETP mimotope 134Thr Pro Phe His
Phe Ala Gln Asp Ser Trp Gln Ser Leu Ser 1 5 10 13514PRTArtificial
SequenceCETP
mimotope 135Gly Thr Pro Phe His Phe Ala Gln Asp Ser Trp Gln Trp Leu
1 5 10 13616PRTArtificial SequenceCETP mimotope 136Phe Gly Phe Pro
Phe His Phe Ala Gln Asp Ser Trp Gln Ser Leu Ser 1 5 10 15
13710PRTArtificial SequenceCETP mimotope 137Ala Cys Ser Phe Ala Tyr
Leu Tyr Arg Cys 1 5 10 13810PRTArtificial SequenceCETP mimotope
138Ala Cys Phe Met Gly Asp Lys Trp Val Cys 1 5 10
13910PRTArtificial SequenceCETP mimotope 139Ala Cys Val Leu Tyr Pro
Lys Ala Ile Cys 1 5 10 14010PRTArtificial SequenceCETP mimotope
140Ala Cys Tyr Met Gly Gln Gln Phe Val Cys 1 5 10
14110PRTArtificial SequenceCETP mimotope 141Ala Cys Leu Thr Ala Tyr
Leu His Trp Cys 1 5 10 14210PRTArtificial SequenceCETP mimotope
142Ala Cys Thr Leu Phe Pro Val Ala Tyr Cys 1 5 10
14310PRTArtificial SequenceCETP mimotope 143Ala Cys Trp Leu Phe Pro
Tyr Ala His Cys 1 5 10 14410PRTArtificial SequenceCETP mimotope
144Ala Cys Lys Ser Ile Asn Met Trp Leu Cys 1 5 10
14510PRTArtificial SequenceCETP mimotope 145Ala Cys Gln Thr Ile Asn
Arg Trp Leu Cys 1 5 10 14616PRTArtificial SequenceCETP mimotope
146Phe Gly Phe Pro Glu His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser
1 5 10 15 14716PRTArtificial SequenceCETP mimotope 147Phe Gly Phe
Pro Glu His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 1 5 10 15
14813PRTArtificial SequenceCETP mimotope 148Phe Pro Glu His Leu Leu
Val Asp Phe Leu Gln Ser Leu 1 5 10 14916PRTArtificial SequenceCETP
mimotope 149Ala Gly Phe Pro Glu His Leu Leu Val Asp Phe Leu Gln Ser
Leu Ser 1 5 10 15 15016PRTArtificial SequenceCETP mimotope 150Phe
Ala Phe Pro Glu His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 1 5 10
15 15116PRTArtificial SequenceCETP mimotope 151Phe Gly Ala Pro Glu
His Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 1 5 10 15
15216PRTArtificial SequenceCETP mimotope 152Phe Gly Phe Ala Glu His
Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 1 5 10 15
15316PRTArtificial SequenceCETP mimotope 153Phe Gly Phe Pro Ala His
Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 1 5 10 15
15416PRTArtificial SequenceCETP mimotope 154Phe Gly Phe Pro Glu Ala
Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 1 5 10 15
15516PRTArtificial SequenceCETP mimotope 155Phe Gly Phe Pro Glu His
Ala Leu Val Asp Phe Leu Gln Ser Leu Ser 1 5 10 15
15616PRTArtificial SequenceCETP mimotope 156Phe Gly Phe Pro Glu His
Leu Ala Val Asp Phe Leu Gln Ser Leu Ser 1 5 10 15
15716PRTArtificial SequenceCETP mimotope 157Phe Gly Phe Pro Glu His
Leu Leu Ala Asp Phe Leu Gln Ser Leu Ser 1 5 10 15
15816PRTArtificial SequenceCETP mimotope 158Phe Gly Phe Pro Glu His
Leu Leu Val Ala Phe Leu Gln Ser Leu Ser 1 5 10 15
15916PRTArtificial SequenceCETP mimotope 159Phe Gly Phe Pro Glu His
Leu Leu Val Asp Ala Leu Gln Ser Leu Ser 1 5 10 15
16016PRTArtificial SequenceCETP mimotope 160Phe Gly Phe Pro Glu His
Leu Leu Val Asp Phe Ala Gln Ser Leu Ser 1 5 10 15
16116PRTArtificial SequenceCETP mimotope 161Phe Gly Phe Pro Glu His
Leu Leu Val Asp Phe Leu Ala Ser Leu Ser 1 5 10 15
16216PRTArtificial SequenceCETP mimotope 162Phe Gly Phe Pro Glu His
Leu Leu Val Asp Phe Leu Gln Ala Leu Ser 1 5 10 15
16316PRTArtificial SequenceCETP mimotope 163Phe Gly Phe Pro Glu His
Leu Leu Val Asp Phe Leu Gln Ser Ala Ser 1 5 10 15
16416PRTArtificial SequenceCETP mimotope 164Phe Gly Phe Pro Glu His
Leu Leu Val Asp Phe Leu Gln Ser Leu Ala 1 5 10 15
16516PRTArtificial SequenceCETP mimotope 165Phe Ala Phe Pro Ala His
Leu Leu Val Asp Phe Leu Gln Ala Leu Ala 1 5 10 15
16616PRTArtificial SequenceCETP mimotope 166Ala Ala Phe Pro Ala His
Leu Leu Ala Asp Phe Leu Gln Ala Leu Ala 1 5 10 15
16712PRTArtificial SequenceCETP mimotope 167Ser Pro Gln His Leu Thr
Thr Asp Arg Ala Gln Ala 1 5 10 16814PRTArtificial SequenceCETP
mimotope 168Ser Pro Gln His Leu Thr Thr Asp Arg Ala Gln Ala Leu Ser
1 5 10 16914PRTArtificial SequenceCETP mimotope 169Ser Pro Gln His
Leu Thr Thr Asp Arg Ala Gln Ser Leu Ser 1 5 10 17014PRTArtificial
SequenceCETP mimotope 170Gly Ser Pro Gln His Leu Thr Thr Asp Arg
Ala Gln Ala Leu 1 5 10 17116PRTArtificial SequenceCETP mimotope
171Phe Gly Phe Pro Gln His Leu Thr Thr Asp Arg Ala Gln Ser Leu Ser
1 5 10 15 17216PRTArtificial SequenceCETP mimotope 172Phe Gly Phe
Pro Gln His Leu Thr Thr Asp Trp Ala Gln Ser Leu Ser 1 5 10 15
17316PRTArtificial SequenceCETP mimotope 173Phe Gly Phe Pro Gln His
Leu Thr Thr Asp Arg Leu Gln Ser Leu Ser 1 5 10 15
17416PRTArtificial SequenceCETP mimotope 174Phe Gly Phe Pro Gln His
Leu Thr Thr Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
17512PRTArtificial SequenceCETP mimotope 175Ala Thr Pro Ser His Leu
Ile Ile Asp Arg Ala Gln 1 5 10 17615PRTArtificial SequenceCETP
mimotope 176Ala Thr Pro Ser His Leu Ile Ile Asp Arg Ala Gln Ser Leu
Ser 1 5 10 15 17716PRTArtificial SequenceCETP mimotope 177Phe Gly
Phe Pro Ser His Leu Ile Ile Asp Arg Ala Gln Ser Leu Ser 1 5 10 15
17816PRTArtificial SequenceCETP mimotope 178Phe Gly Phe Pro Ser His
Leu Ile Ile Asp Trp Ala Gln Ser Leu Ser 1 5 10 15
17916PRTArtificial SequenceCETP mimotope 179Phe Gly Phe Pro Ser His
Leu Ile Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
18016PRTArtificial SequenceCETP mimotope 180Phe Gly Phe Pro Ser His
Leu Ile Ile Asp Trp Ser Gln Ser Leu Ser 1 5 10 15
18116PRTArtificial SequenceCETP mimotope 181Phe Ala Thr Pro Ser His
Leu Ile Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
18212PRTArtificial SequenceCETP mimotope 182Phe Lys Pro Ala His Val
Ser Ile Asp Trp Leu Gln 1 5 10 18315PRTArtificial SequenceCETP
mimotope 183Phe Lys Pro Ala His Val Ser Ile Asp Trp Leu Gln Ser Leu
Ser 1 5 10 15 18416PRTArtificial SequenceCETP mimotope 184Phe Gly
Phe Pro Ala His Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
18516PRTArtificial SequenceCETP mimotope 185Ala Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
18616PRTArtificial SequenceCETP mimotope 186Phe Ala Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
18716PRTArtificial SequenceCETP mimotope 187Phe Gly Ala Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
18816PRTArtificial SequenceCETP mimotope 188Phe Gly Phe Ala Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
18916PRTArtificial SequenceCETP mimotope 189Phe Gly Phe Pro Ala His
Val Ser Ala Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
19016PRTArtificial SequenceCETP mimotope 190Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ala Leu Ser 1 5 10 15
19116PRTArtificial SequenceCETP mimotope 191Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ala 1 5 10 15
19216PRTArtificial SequenceCETP mimotope 192Phe Ala Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ala Leu Ala 1 5 10 15
19316PRTArtificial SequenceCETP mimotope 193Phe Gly Phe Ala Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
19416PRTArtificial SequenceCETP mimotope 194Phe Gly Phe Phe Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
19516PRTArtificial SequenceCETP mimotope 195Phe Gly Phe Pro Ala His
Val Ser Ile Arg Trp Leu Gln Ser Leu Ser 1 5 10 15
19616PRTArtificial SequenceCETP mimotope 196Phe Gly Phe Pro Ala His
Val Ser Ile Glu Trp Leu Gln Ser Leu Ser 1 5 10 15
19716PRTArtificial SequenceCETP mimotope 197Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Asn Ser Leu Ser 1 5 10 15
19816PRTArtificial SequenceCETP mimotope 198Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu His Ser Leu Ser 1 5 10 15
19916PRTArtificial SequenceCETP mimotope 199Ala Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
20016PRTArtificial SequenceCETP mimotope 200Pro Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
20116PRTArtificial SequenceCETP mimotope 201Trp Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
20216PRTArtificial SequenceCETP mimotope 202Phe Ala Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
20316PRTArtificial SequenceCETP mimotope 203Phe Ser Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
20416PRTArtificial SequenceCETP mimotope 204Phe Tyr Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
20516PRTArtificial SequenceCETP mimotope 205Phe Asp Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
20616PRTArtificial SequenceCETP mimotope 206Phe Gly Ala Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
20716PRTArtificial SequenceCETP mimotope 207Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Leu Leu Ser 1 5 10 15
20816PRTArtificial SequenceCETP mimotope 208Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Trp Leu Ser 1 5 10 15
20916PRTArtificial SequenceCETP mimotope 209Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Asn Leu Ser 1 5 10 15
21016PRTArtificial SequenceCETP mimotope 210Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Thr Leu Ser 1 5 10 15
21116PRTArtificial SequenceCETP mimotope 211Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Tyr Leu Ser 1 5 10 15
21216PRTArtificial SequenceCETP mimotope 212Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Ile Ser 1 5 10 15
21316PRTArtificial SequenceCETP mimotope 213Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Thr 1 5 10 15
21416PRTArtificial SequenceCETP mimotope 214Phe Gly Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ser Leu Tyr 1 5 10 15
21516PRTArtificial SequenceCETP mimotope 215Phe Ala Phe Pro Ala His
Val Ser Ile Asp Trp Leu Gln Ala Leu Ala 1 5 10 15
21616PRTArtificial SequenceCETP mimotope 216Phe Gly Phe Pro Ala His
Val Ser Ile Asp Arg Ala Gln Ser Leu Ser 1 5 10 15
21716PRTArtificial SequenceCETP mimotope 217Phe Gly Phe Pro Thr His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
21816PRTArtificial SequenceCETP mimotope 218Phe Gly Phe Pro Phe His
Val Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
21916PRTArtificial SequenceCETP mimotope 219Phe Gly Phe Pro Ala His
Ile Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
22016PRTArtificial SequenceCETP mimotope 220Phe Gly Phe Pro Ala His
Ile Ile Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
22116PRTArtificial SequenceCETP mimotope 221Phe Gly Phe Pro Ala His
Leu Thr Thr Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
22216PRTArtificial SequenceCETP mimotope 222Phe Gly Phe Pro Ala His
Val Phe Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
22316PRTArtificial SequenceCETP mimotope 223Phe Gly Phe Pro Ala His
Val Tyr Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
22416PRTArtificial SequenceCETP mimotope 224Phe Gly Phe Pro Ala His
Val Ser Leu Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
22516PRTArtificial SequenceCETP mimotope 225Phe Gly Phe Pro Ala His
Val Ser Ala Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
22614PRTArtificial SequenceCETP mimotope 226Thr Pro Thr His Tyr Tyr
Ala Asp Phe Ser Gln Ser Leu Ser 1 5 10 22716PRTArtificial
SequenceCETP mimotope 227Phe Gly Phe Pro Ala His Val Ser Ile Asp
Trp Ser Gln Ser Leu Ser 1 5 10 15 22816PRTArtificial SequenceCETP
mimotope 228Phe Gly Phe Pro Ala His Val Ser Ile Asp Phe Ser Gln Ser
Leu Ser 1 5 10 15 22916PRTArtificial SequenceCETP mimotope 229Phe
Gly Phe Pro Ala His Val Trp Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10
15 23016PRTArtificial SequenceCETP mimotope 230Phe Gly Phe Pro Ala
His Val Phe Ile Asp Trp Leu Gln Ser Leu Asn 1 5 10 15
23116PRTArtificial SequenceCETP mimotope 231Phe Gly Phe Pro Ala His
Phe Ser Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
23216PRTArtificial SequenceCETP mimotope 232Phe Gly Phe Pro Ala His
Val Ser Phe Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
23316PRTArtificial SequenceCETP mimotope 233Phe Gly Phe Pro Glu His
Val Phe Ile Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
23417PRTArtificial SequenceCETP mimotope 234Asp Phe Gly Phe Pro Ala
His Val Phe Ile Asp Trp Leu Gln Ser Leu 1 5 10 15 Ser
23517PRTArtificial SequenceCETP mimotope 235Asp Phe Gly Phe Pro Ser
His Leu Ile Ile Asp Trp Leu Gln Ser Leu 1 5 10 15 Ser
23617PRTArtificial SequenceCETP mimotope 236Asp Phe Gly Phe Pro Ala
His Val Tyr Ile Asp Trp Leu Gln Ser Leu 1 5 10 15 Ser
23716PRTArtificial SequenceCETP mimotope 237Phe Gly Phe Pro Gln His
Leu Phe Thr Asp Trp Leu Gln Ser Leu Ser 1 5 10 15
23816PRTArtificial SequenceCETP mimotope 238Phe Gly Phe Pro Lys His
Leu Leu Val Asp Phe Leu Gln Ser Leu Ser 1 5 10 15
23916PRTArtificial SequenceCETP mimotope 239Phe Gly Phe Pro Ser His
Ile Ile Ile Asp Trp Leu Gln Ser Leu Ser1 5 10 15 24016PRTArtificial
SequenceCETP mimotope 240Phe
Gly Phe Pro Ser His Leu Ile Ile Glu Trp Leu Gln Ser Leu Ser1 5 10
15 24116PRTArtificial SequenceCETP mimotope 241Ala Ala Phe Pro Ala
His Leu Leu Ala Asp Ala Ala Gln Ala Leu Ala1 5 10 15
24216PRTArtificial SequenceCETP mimotope 242Ala Ala Phe Pro Ala His
Ala Ala Ala Asp Phe Leu Gln Ala Leu Ala1 5 10 15 24316PRTArtificial
SequenceCETP mimotope 243Ala Ala Phe Ala Ala His Leu Leu Ala Asp
Phe Leu Gln Ala Ala Ala1 5 10 15 24416PRTArtificial SequenceCETP
mimotope 244Ala Ala Ala Pro Ala His Leu Leu Val Asp Ala Ala Gln Ala
Ala Ala1 5 10 15 24516PRTArtificial SequenceCETP mimotope 245Phe
Ala Phe Pro Ala His Val Phe Ile Asp Trp Leu Gln Ser Leu Ser1 5 10
15 24616PRTArtificial SequenceCETP mimotope 246Phe Gly Phe Pro Ala
His Val Phe Ile Asp Trp Leu Gln Ala Leu Ser1 5 10 15
24716PRTArtificial SequenceCETP mimotope 247Phe Gly Phe Pro Ala His
Val Phe Ile Asp Trp Leu Gln Ser Leu Ala1 5 10 15 24815PRTArtificial
SequenceCETP mimotope 248Gly Phe Pro Ala His Val Phe Ile Asp Trp
Leu Gln Ser Leu Ser1 5 10 15 24914PRTArtificial SequenceCETP
mimotope 249Phe Pro Ala His Val Phe Ile Asp Trp Leu Gln Ser Leu
Ser1 5 10 25013PRTArtificial SequenceCETP mimotope 250Pro Ala His
Val Phe Ile Asp Trp Leu Gln Ser Leu Ser1 5 10 25116PRTArtificial
SequenceCETP mimotope 251Phe Ala Phe Pro Ala His Val Phe Ile Asp
Trp Leu Gln Ala Leu Ala1 5 10 15 25216PRTArtificial SequenceCETP
mimotope 252Phe Gly Phe Pro Glu His Leu Phe Val Asp Phe Leu Gln Ser
Leu Ser1 5 10 15 25316PRTArtificial SequenceCETP mimotope 253Phe
Gly Phe Pro Ala His Val His Ile Asp Trp Leu Gln Ser Leu Ser1 5 10
15 25416PRTArtificial SequenceCETP mimotope 254Phe Gly Phe Pro Ala
His Val Pro Ile Asp Trp Leu Gln Ser Leu Ser1 5 10 15
25516PRTArtificial SequenceCETP mimotope 255Phe Gly Phe Pro Ser His
Leu Phe Ile Asp Trp Ala Gln Ser Leu Ser1 5 10 15 25616PRTArtificial
SequenceCETP mimotope 256Pro Gly Phe Pro Ala His Val Phe Ile Asp
Trp Leu Gln Leu Ile Thr1 5 10 15 25713PRTArtificial SequenceCETP
mimotope 257Pro Ala His Val Tyr Ile Asp Trp Leu Gln Ser Leu Ser1 5
10 25813PRTArtificial SequenceCETP mimotope 258Phe Gly Phe Pro Ala
His Val Tyr Ile Asp Trp Leu Gln1 5 10 25913PRTArtificial
SequenceCETP mimotope 259Phe Gly Phe Pro Ala His Val Phe Ile Asp
Trp Leu Gln1 5 10 26017PRTArtificial SequenceCETP mimotope 260Asp
Phe Gly Phe Pro Ala His Val Phe Ile Asp Trp Leu Gln Ser Leu1 5 10
15 Asn26110PRTArtificial SequenceCETP mimotope 261Pro Ser His Leu
Ile Ile Asp Trp Leu Gln1 5 10 26210PRTArtificial SequenceCETP
mimotope 262Pro Ala His Val Phe Ile Asp Trp Leu Gln1 5 10
26317PRTArtificial SequenceCETP mimotope 263Asp Phe Gly Phe Pro Ala
His Val Thr Ile Asp Trp Leu Gln Ser Leu1 5 10 15
Asn26417PRTArtificial SequenceCETP mimotope 264Asp Phe Gly Phe Pro
Ala His Val Leu Ile Asp Trp Leu Gln Ser Leu1 5 10 15
Asn26516PRTArtificial SequenceCETP mimotope 265Phe Gly Phe Pro Ala
His Val Phe Ile Asp Trp Leu Gln Ser Leu Ala1 5 10 15
26615PRTArtificial SequenceCETP mimotope 266Phe Lys Pro Ala His Val
Phe Ile Asp Trp Leu Gln Ser Leu Ser1 5 10 15 26716PRTArtificial
SequenceCETP mimotope 267Gly Phe Lys Pro Ala His Val Phe Ile Asp
Trp Leu Gln Ser Leu Ser1 5 10 15 26817PRTArtificial SequenceCETP
mimotope 268Phe Pro Gly Phe Pro Ala His Val Phe Ile Asp Trp Leu Gln
Leu Ile1 5 10 15 Thr26915PRTArtificial SequenceCETP mimotope 269Phe
Gly Phe Pro Ala His Val Trp Ile Asp Trp Leu Gln Ser Leu1 5 10 15
27016PRTArtificial SequenceCETP mimotope 270Gly Phe Lys Pro Ala His
Val Phe Ile Asp Trp Leu Gln Ser Leu Ser1 5 10 15
* * * * *