NZ613913B2 - Cetp fragments - Google Patents
Cetp fragments Download PDFInfo
- Publication number
- NZ613913B2 NZ613913B2 NZ613913A NZ61391312A NZ613913B2 NZ 613913 B2 NZ613913 B2 NZ 613913B2 NZ 613913 A NZ613913 A NZ 613913A NZ 61391312 A NZ61391312 A NZ 61391312A NZ 613913 B2 NZ613913 B2 NZ 613913B2
- Authority
- NZ
- New Zealand
- Prior art keywords
- seq
- peptide
- amino acid
- cetp
- vaccine
- Prior art date
Links
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6081—Albumin; Keyhole limpet haemocyanin [KLH]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
- C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K9/00—Peptides having up to 20 amino acids, containing saccharide radicals and having a fully defined sequence; Derivatives thereof
Abstract
Discloses a composition including an isolated peptide consisting of 6 to 20 amino acid residues, the peptide being coupled or fused to a pharmaceutically acceptable carrier, wherein the peptide is derived from amino acid sequence VFKGTLKYGYTTAWWLGIDQSIDFEIDSAI, and comprises the amino acid sequence WWLGID. WWLGID.
Description
CETP fragments
The present invention relates to peptides which are able to in-
fluence the in vivo activity of CETP.
Diseases associated with atherosclerosis, such as cardiovas-
cular disease (CVD) as well as stroke and peripheral arterial
occlusion disease, are among the main causes of death in the
United States, Europe, and in large parts of Asia. Compared to
the year 1990, mortality from CVD will have increased by 90% in
2020.
Various risk factors are held responsible for the forming of
atherosclerotic lesions. Circulating lipoprotein profiles, arte-
rial hypertension and abuse of nicotine are of particular sig-
nificance in this respect.
In comprehensive epidemiologic studies, a positive correla-
tion between the level of the serum cholesterol and the occur-
rence of coronary heart disease could be demonstrated. High Low
Density Lipoprotein cholesterol (LDLc) levels constitute a high
cardiovascular risk, and are directly correlating with increased
risk for atherosclerosis. Statins as HMG-CoA Reductase inhibi-
tors are commonly used and are successfully reducing circulating
LDLc levels. However, despite reduction of coronary events due
to aggressive statin treatment, considerable residual cardiovas-
cular risk remains a challenge. Thus, new therapeutics are ur-
gently needed.
Besides the level of the LDL cholesterol, also the level of
the vessel-protecting High Density Lipoprotein cholesterol
(HDLc) plays an important role when estimating the risk profile
for cardiovascular diseases. Epidemiologic studies have demon-
strated an inverse relationship between HDLc levels and athero-
sclerosis. HDLc levels are strong predictors independent from
LDLc values. Therefore it is assumed that HDLc levels have
atheroprotective effects. Elevating HDLc levels therefore is a
major goal and additional target.
Monotherapy with statins does not efficaciously raise HDLc
levels, treatment and prevention of atherosclerosis with this
type of pharmacological compound is not sufficient. Current op-
tions for raising HDLc levels are fibrates and niacin. Especial-
ly niacin is effective, however its use is limited by adverse
effects which lead to low compliance of patients. An efficacious
and safe method for the raise of HDLc levels to complement LDL
reduction by statins is urgently needed. In this respect, inhi-
bition of CETP as monotherapy or as add-on therapy is an attrac-
tive goal.
Cholesterol ester transfer protein (CETP) is a plasma glyco-
protein which is responsible for the transfer of neutral lipids,
including cholesteryl ester and triglyceride (TG), between lipo-
proteins. Cholesteryl esters from atheroprotective HDL are
transferred to pro-atherogenic apolipoprotein (apo) B lipopro-
tein (LDL and VLDL) in exchange for TG. This leads to lower lev-
els of HDL and raises the levels of LDL and VLDL.
It is assumed that CETP, most likely depending on the meta-
bolic background, is a valuable therapeutic target for the goal
of increasing the HDL plasma level.
Many species do not have CETP. In species with susceptibil-
ity to atherosclerosis, such as rabbit and man, CETP activity is
high. Others that are resistant to atherosclerosis do not pos-
sess CETP and have high HDLc levels.
In animal experiments with rabbits and hamsters, the transi-
ent inhibition of CETP with anti-CETP monoclonal antibodies, an-
tisense oligonucleotides or CETP inhibitors led to the increase
in the HDL levels.
Several classes of CETP inhibitors have been described, some
of which are already in advanced clinical trials (e.g. dalce-
trapib (Stein EA, Eur Heart J. 31(4) (2010):480-8) anacetrapib
(Cannon CP, N Engl J Med. 363(25)(2010):2406-15 and torcetrapib
(Nissen SE, N Engl J Med. 356(13)(2007):1304-16).
In US 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 apoC-I of various
sources, wherein especially N-terminal fragments up to amino ac-
id 36 have been identified as CETP inhibitors.
Also in US 5,880,095 A, a CETP-binding peptide is disclosed
which is capable of inhibiting the activity of CETP in an indi-
vidual. The CETP-inhibitory protein comprises an N-terminal
fragment of porcine apoC-III.
In US 2006/0276400 and 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 for-
mation of CETP specific antibodies.
In US 2004/0087481 and US 6,410,022 B1, peptides are dis-
closed which, because of the induction of a CETP-specific immune
response, can be used for the treatment and prevention of cardi-
ovascular diseases, such as, e.g., atherosclerosis. These pep-
tides 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 co-
valently bound to at least one B-cell epitope of CETP. By using
a T helper cell epitope that is alien to the organism, it be-
comes possible to induce antibodies in the body of an individu-
al, which antibodies are directed against that peptide portion
that consists of at least one CETP-B-cell epitope.
In CETP mimotopes to be used for the manufac-
ture of a medicament for the treatment or prevention of athero-
sclerosis is described.
There have already been suggestions for a vaccine approach
with regard to CETP. Rabbits have been treated with a vaccine
which contained a peptide derived from the C-terminus of CETP 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 athero-
sclerosis model also showed reduced atherosclerotic lesions in
comparison with control animals (Rittershaus CW, Arterioscler
Thromb Vasc Biol 20(2000): 2106-12).
The results of a phase II clinical study with the vaccine
CETi-1, which was carried out by the American biotechnology com-
pany Avant, were published (BioCentury Extra For Wednesday, Oc-
tober 22, 2003). In this phase II study, just as in the preced-
ing phase I study, a very good safety profile without any ques-
tionable side effects was proven, allowing the conclusion to be
drawn that 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 in-
creased HDL levels significantly better than those attained by a
placebo treatment.
It is an object of the present invention to provide means
and methods for reducing the activity of CETP in an individual.
The present invention relates to a peptide consisting of 6
to 20 amino acid residues and being derived from amino acid se-
quence VFKGTLKYGYTTAWWLGIDQSIDFEIDSAI (SEQ ID No. 23), wherein
said peptide comprises amino acid sequence WWLGID (SEQ ID No.
24).
It surprisingly turned out that peptides comprising the ami-
no acid sequence WWLGID are able to induce in a mammal, in par-
ticular in mouse, antibodies directed to CETP. These antibodies
are able to bind to CETP and in addition are reducing the in vi-
vo activity of said protein leading to an increase of the HDL
levels in the mammal to which said peptides are administrated.
The increase of the HDL levels in a mammal leads to an allevia-
tion of atherosclerosis and other diseases associated with ath-
erosclerosis, in particular cardiovascular diseases.
The peptides of the present invention comprising the amino
acid sequence WWLGID are derived from the peptide having the
amino acid sequence VFKGTLKYGYTTAWWLGIDQSIDFEIDSAI (SEQ ID No.
23) which is a fragment of human CETP (Protein Data Bank Acces-
sion No. 2OBD_A GI:126031487). The peptide having the amino acid
sequence SEQ ID No. 23 comprises amino acid residues 92 to 121
of the mature CETP protein. This CETP protein fragment and trun-
cated variants thereof which comprise the amino acid sequence
WWLGID induce, as mentioned before, the formation of antibodies
binding specifically to CETP. However, other fragments derived
from CETP including amino acid residues 47 to 55, 152 to 165,
290 to 300, 300 to 316 and 403 to 415 of the mature CETP pro-
tein, although being present on the surface of the full-length
CETP protein, do not lead to the formation of antibodies di-
rected (i.e. binding) to the CETP protein, although the admin-
istration of such fragments to a mammal induces the formation of
fragment specific antibodies. This is surprising since a person
skilled in the art would obviously expect that CETP fragments
exposed on the surface of the CETP protein would be necessary to
induce the formation of antibodies directed to the CETP protein.
It is well known in the art that longer peptides show a
higher probability to comprise one or more T-cell epitopes which
are not desired in such a vaccine. Activation of cytotoxic T
cell epitopes (CD4 and CD8) could lead to the induction of auto-
reactive T cells inducing unwanted adverse events (AEs). There-
fore the skilled artisan – in order to avoid undesired epitopes
in a vaccine – would use peptides as antigens which are as short
as possible in order to induce an antigen specific humoral im-
mune response. Therefore shorter peptides – besides the fact
that shorter peptides can much more easily chemically synthe-
sised - are preferred.
In WO 02/098915 polypeptides of less than 100 amino acids
are disclosed which comprise amino acid sequence SEQ ID No. 23
of the present invention. The polypeptides of WO 02/098915 are
used to produce antibodies directed to CETP. Due to the length
of the peptides disclosed in WO 02/098915 also antibodies are
induced which may not be useful to treat or prevent atheroscle-
rosis and diseases associated with atherosclerosis because such
antibodies are specific for irrelevant parts of CETP.
As used herein, the term "peptide" refers to a polymeric
molecule having at least 6 amino acid residues which are linked
to each other by peptide bonds.
The term "derived from", as used in the context of the pep-
tides of the present invention, means that the peptides of the
present invention are fragments of the peptide having the amino
acid sequence SEQ ID No. 23. Therefore the term "derived from"
can be used interchangeably with "being a fragment of".
According to a preferred embodiment of the present inven-
tionthe peptide consisting of 6 to 20 amino acid residues and
being derived from amino acid sequence VFKGTLKYGYTTAWWLGIDQSID-
FEIDSAI (SEQ ID No. 23) may comprise or consist of 6 to 16 amino
acid residues.
According to a particularly preferred embodiment of the pre-
sent invention the peptide comprises 8 to 20, preferably 8 to
16, amino acid residues. If the peptides of the present inven-
tion comprise a C- and/or N-terminal cysteine residue the pep-
tide may comprise 9 to 21, preferably 9 to 17, or 10 to 22,
preferably 10 to 18, amino acid residues.
In case the peptide of the present invention comprises at
least 8 consecutive amino acid residues of the amino acid se-
quence SEQ ID No. 23, said peptide preferably comprises or con-
sists of amino acid sequence SEQ ID No. 10, SEQ ID No. 11, SEQ
ID No. 18 and/or SEQ ID No. 19.
According to another preferred embodiment of the present in-
vention the amino acid sequence of the peptide is selected from
the group consisting of YTTAWWLGIDQS (SEQ ID No. 1), YGYTTAW-
WLGIDQSID (SEQ ID No. 7), TTAWWLGIDQS (SEQ ID No. 8), TAWWLGIDQS
(SEQ ID No. 9), AWWLGIDQS (SEQ ID No. 10), WWLGIDQS (SEQ ID No.
11), YTTAWWLGIDQ (SEQ ID No. 13), YTTAWWLGID (SEQ ID No. 14),
TTAWWLGIDQ (SEQ ID No. 18) and TTAWWLGID (SEQ ID No. 19), par-
ticularly preferred from the group consisting of TAWWLGIDQS (SEQ
ID No. 9), AWWLGIDQS (SEQ ID No. 10) and WWLGIDQS (SEQ ID No.
11).
The peptides of the present invention can be chemically syn-
thesised by methods which are well known in the art. Of course
it is also possible to produce the peptides of the present in-
vention using recombinant methods. The peptides can be produced
in microorganisms such as bacteria, yeast or fungi, in eukaryot-
ic cells such as mammalian or insect cells, or in a recombinant
virus vector such as adenovirus, poxvirus, herpesvirus, Simliki
forest virus, baculovirus, bacteriophage, sindbis virus or sen-
dai virus. Suitable bacteria for producing the peptides include
E. coli, B. subtilis or any other bacterium that is capable of
expressing such peptides. Suitable yeast cells for expressing
the peptides of the present invention include Saccharomyces
cerevisiae, Schizosaccharomycespombe, Candida, Pichiapastoris or
any other yeast capable of expressing peptides. Corresponding
means and 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. gel filtration, affinity chro-
matography, ion exchange chromatography etc.
To facilitate isolation of the peptides of the present in-
vention, fusion polypeptides may be made wherein the peptides
are translationally fused (covalently linked) to a heterologous
polypeptide which enables isolation by affinity chromatography.
Typical heterologous polypeptides are His-Tag (e.g. His ; 6 his-
tidine residues), GST-Tag (Glutathione-S-transferase) etc. The
fusion polypeptide facilitates not only the purification of the
peptides but can also prevent the degradation of the peptides
during the purification steps. If it is desired to remove the
heterologous polypeptide after purification, the fusion polypep-
tide may comprise a cleavage site at the junction between the
peptide and the heterologous polypeptide. The cleavage site may
consist of an amino acid sequence that is cleaved with an enzyme
specific for the amino acid sequence at the site (e.g. proteas-
es).
The peptides of the present invention may also comprise at
their N- and/or C-terminus a cysteine residue bound thereto.
The provision of a cysteine residue at the N- and/or C-
terminus of a peptide may facilitate its conjugation to a carri-
er and/or may enhance the immunogenicity of the peptide. If a
cystein residue is added to the C- and/or N-terminus of the pep-
tides of the present invention, the number of amino acid resi-
dues mentioned herein increases obviously for 1 or 2 amino acid
residues. Therefore the peptide of consisting of 6 to 20 amino
acid residues and being derived from amino acid sequence
VFKGTLKYGYTTAWWLGIDQSIDFEIDSAI (SEQ ID No. 23), wherein said
peptide comprises amino acid sequence WWLGID (SEQ ID No. 24),
may the consist of 7 to 21 or 8 to 22 amino acid residues.
Particularly preferred peptides of the present invention
having a C- or N-terminal cysteine residue are selected from the
group consisting of C-YTTAWWLGIDQS, C-YGYTTAWWLGIDQSID, C-
TTAWWLGIDQS, C-TAWWLGIDQS, C-AWWLGIDQS, C-WWLGIDQS, C-
YTTAWWLGIDQ, C-YTTAWWLGID, C-TTAWWLGIDQ and TTAWWLGID-C.
According to a preferred embodiment of the present invention
the peptide is used for preventing and/or treating atherosclero-
sis and diseases associated with atherosclerosis.
As outlined above, the peptides of the present invention are
able to induce the formation of antibodies which are able to
bind specifically CETP particularly present in humans. The bind-
ing of these antibodies to CETP leads to a reduction of the ac-
tivity of CETP in vivo. As a consequence thereof the level of
HDL is increased.
The disease associated with atherosclerosis is preferably
selected from the group consisting of peripheral arterial occlu-
sive disease, coronary heart disease, apoplectic cerebral insul-
tus and stroke.
The term "diseases associated with atherosclerosis" refers
to diseases which are a consequence of atherosclerosis. These
diseases include among others peripheral arterial occlusive dis-
ease, coronary heart disease and apoplectic cerebral insultus
(see e.g. Steinberg D., J. Lipid Res. 46 (2005): 179-190; Stein-
berg D et al., J. Lipid Res. 47 (2006): 1339-1351).
According to a preferred embodiment of the present invention
the peptide is administered to an individual in an amount of 0.5
to 500 µg, preferably 1 to 100 µg, per immunization. However,
the peptide of the present invention may alternatively be admin-
istered to an individual in an amount of 0.1 ng to 10 mg, pref-
erably 10 ng to 1 mg, in particular 100 ng to 300 µg/kg body
weight.
The amount of peptides that may be combined with the carrier
materials to produce a single dosage form will vary depending
upon the host treated and the particular mode of administration.
The dose of the vaccine may vary according to factors such as
the disease state, age, sex and weight of the individual, and
the ability of antibody to elicit a desired response in the in-
dividual. Dosage regime may be adjusted to provide the optimum
therapeutic response. For example, several divided doses may be
administered daily or the dose may be proportionally reduced as
indicated by the exigencies of the therapeutic situation. The
dose of the vaccine may also be varied to provide optimum pre-
ventative dose response depending upon the circumstances. For
instance, the peptides and vaccine of the present invention may
be administered to an individual at intervals of several days,
one or two weeks or even months depending always on the level of
antibodies directed to CETP.
In a preferred embodiment of the present invention the pep-
tide/vaccine is applied between 2 and 10, preferably between 2
and 7, even more preferably up to 5 and most preferably up to 3
times. In a particularly preferred embodiment the time interval
between the subsequent vaccinations is chosen to be between 2
weeks and 5 years or more, preferably between 1 month and up to
3 years, more preferably between 2 months and 1.5 years. The re-
peated administration of the peptide/vaccine of the present in-
vention may maximize the final effect of a therapeutic vaccina-
tion.
Another aspect of the present invention relates to a vaccine
comprising at least one peptide as defined above comprising 6 to
amino acid residues and being derived from amino acid se-
quence VFKGTLKYGYTTAWWLGIDQSIDFEIDSAI (SEQ ID No. 23), wherein
said peptide comprises amino acid sequence WWLGID (SEQ ID No.
24).
The vaccine of the present invention may comprise one or
more, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the peptides
selected from the group consisting of YTTAWWLGIDQS (SEQ ID No.
1), YGYTTAWWLGIDQSID (SEQ ID No. 7), TTAWWLGIDQS (SEQ ID No. 8),
TAWWLGIDQS (SEQ ID No. 9), AWWLGIDQS (SEQ ID No. 10), WWLGIDQS
(SEQ ID No. 11), YTTAWWLGIDQ (SEQ ID No. 13), YTTAWWLGID (SEQ ID
No. 14), TTAWWLGIDQ (SEQ ID No. 18) and TTAWWLGID (SEQ ID No.
19).
Particularly preferred combinations of peptides include: SEQ
ID No. 10 and 11; SEQ ID No. 8 and 9; SEQ ID No. 8 and 10; SEQ
ID No. 8 and 11; SEQ ID No. 9 and 10; SEQ ID No. 9 and 11; SEQ
ID No. 8, 9 and 10; SEQ ID No. 8, 9 and 11; SEQ ID No. 8, 9, 10
and 11; SEQ ID No. 10, 11 and 18; SEQ ID No. 8, 9 and 18; SEQ ID
No. 8, 10 and 18; SEQ ID No. 8, 11 and 18; SEQ ID No. 9, 10 and
18; SEQ ID No. 9, 11 and 18; SEQ ID No. 8, 9, 10 and 18; SEQ ID
No. 8, 9, 11 and 18; SEQ ID No. 8, 9, 10, 11 and 18; SEQ ID No.
8 and 18; SEQ ID No. 9 and 18; SEQ ID No. 10 and 18; SEQ ID No.
11 and 18.
The vaccine of the present invention may also comprise other
peptidic fragments of CETP or variants thereof or any other pep-
tides which are able to induce the in vivo formation of antibod-
ies directed to CETP, particularly human CETP. Particularly
suited are the peptides disclosed in . The pep-
tides disclosed therein have amino acid sequence
FX (F) PX HX X X DX X X X X X , wherein X is selected from the
8 o 9 10 11 12 2 3 4 5 6 7 8
group consisting of G, A, F, Y and K, X is selected from the
group consisting of E, Y, A, Q, K and S, X is selected from the
group consisting of H, V, L, F and I, X is selected from the
group consisting of L, W, S, I, F and Y, X is V, T, F or I, X
12 2
is an amino acid residue selected from the group consisting of
F, A, W, R, S, L, Q, V and M, X is an amino acid residue select-
ed from the group consisting of L, A, S, W, E, R, I and H, X is
an amino acid residue selected from the group consisting of Q,
A, H, D, K, R, S and E, X is S or Y, X is L, A or I, X is S, N
6 7
or T, and o is 0 or 1. Preferred combinations comprise the pep-
tides of the present invention and one or more peptides selected
from the group consisting of FGFPEHLLVDFLQSLS, FPEHLLVDFLQSL,
AGFPEHLLVDFLQSLS, FAFPEHLLVDFLQSLS, FGAPEHLLVDFLQSLS, FGFAEH-
LLVDFLQSLS, FGFPAHLLVDFLQSLS, FGFPEALLVDFLQSLS, FGFPEHALVD-
FLQSLS, FGFPEHLAVDFLQSLS, FGFPEHLLADFLQSLS, FGFPEHLLVAFLQSLS,
FGFPEHLLVDALQSLS, FGFPEHLLVDFAQSLS, FGFPEHLLVDFLASLS, FGFPEH-
LLVDFLQALS, FGFPEHLLVDFLQSAS, FGFPEHLLVDFLQSLA, FAFPAHLLVD-
FLQALA, FGFPGHLIWDSLHSLS,FGFPYHHLVDQLHSLS, FGFPYHVQVDVLQSLS,
FGFPSHHLQDSLQSLS, FGFPLHFRSDRIQSLS, FGFPKHLYADMSQSLS,
FGFPAHLSRDLRQSLS, FGFPFHFAQDSWQSLS, FGFPQHLTTDRAQSLS, FGFPQHLTT-
DWAQSLS, FGFPQHLTTDRLQSLS, FGFPQHLTTDWLQSLS, ATPSHLIIDRAQ,
ATPSHLIIDRAQSLS, FGFPSHLIIDRAQSLS, FGFPSHLIIDWAQSLS, FGFPSHLI-
IDWLQSLS, FGFPSHLIIDWSQSLS, FAFPAHVSIDWLQSLS, FGFPAHVSIDWLQLLS,
FGFPAHVSIDWLQWLS, FGFPAHVSIDWLQNLS, FGFPAHVSIDWLQTLS, FGFPAH-
VSIDWLQYLS, FGFPAHVSIDWLQSIS, FGFPAHVSIDWLQSLT, FGFPAHVSID-
WLQSLY, FAFPAHVSIDWLQALA, FGFPAHVSIDRAQSLS, FGFPTHVSIDWLQSLS,
FGFPFHVSIDWLQSLS, FGFPAHISIDWLQSLS, FGFPAHIIIDWLQSLS, FGFPAHLTT-
DWLQSLS, FGFPAHVFIDWLQSLS, FGFPAHVYIDWLQSLS, FGFPAHVSLDWLQSLS,
FGFPAHVSADWLQSLS, FGFPAHVWIDWLQSLS, FGFPAHVFIDWLQSLN, FGFPAH-
FSIDWLQSLS, FGFPAHVSFDWLQSLS, FGFPEHVFIDWLQSLS,
FGFPQHLFTDWLQSLS, FGFPKHLLVDFLQSLS, FGFPAHVSIDWSQSLS, FGFPAH-
VSIDFSQSLS, FGFPSHIIIDWLQSLS, FGFPSHLIIEWLQSLS, FAFPAHVFID-
WLQSLS, FGFPAHVFIDWLQALS, FGFPAHVFIDWLQSLA, FAFPAHVFIDWLQALA,
FGFPEHLFVDFLQSLS, FGFPAHVHIDWLQSLS, FGFPAHVPIDWLQSLS,
FGFPSHLFIDWAQSLS, FGFPAHVYIDWLQ and FGFPAHVFIDWLQ.
The vaccine of the present invention may also comprise anti-
gens derived from other proteins which are also involved in the
regulation of the LDL and/or HDL levels within a human body. For
instance, the CETP fragments of the present invention may be
combined with epitopes derived from PCSK9 (proproteinconvert-
asesubtilisin/kexin type 9) as disclosed in ,
and .
According to a preferred embodiment of the present invention
the at least one peptide is coupled or fused to a pharmaceuti-
cally acceptable carrier, preferably KLH (Keyhole Limpet Hemocy-
anin).
According to a preferred embodiment of the present invention
the peptide is coupled or fused to a pharmaceutically acceptable
carrier, preferably KLH (Keyhole Limpet Hemocyanin), tetanus
toxoid, albumin-binding protein, hepatitis B core antigen, bo-
vine serum albumin, a dendrimer (MAP), peptide linkers (or
flanking regions), or CRM, preferably CRM197, as well as com-
bined with 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 Discov-
ery 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 hetero-
bifunctional 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 formu-
lated with an adjuvant, preferably a low soluble aluminium com-
position, in particular aluminium hydroxide. Of course, also ad-
juvants like MF59 aluminium phosphate, calcium phosphate, cyto-
kines (e.g. IL-2, IL-12, GM-CSF), saponins (e.g. QS21), MDP de-
rivatives, CpG oligonucleotides, LPS, MPL, polyphosphazenes,
emulsions (e.g. Freund's, SAF), liposomes, lipopeptides, viro-
somes, iscoms, cochleates, PLG microparticles, poloxamer parti-
cles, virus-like particles, heat-labile enterotoxin (LT), chol-
era toxin (CT), mutant toxins (e.g. LTK63 and LTR72), micropar-
ticles and/or polymerized liposomes may be used.
The peptides of the present invention are 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 -maleimide).
A vaccine which comprises a peptide of the present invention
and the pharmaceutically acceptable carrier may be administered
by any suitable mode of application, e.g. intradermally (i.d.),
intravenously (i.v.), intraperitoneally (i.p.), intramuscularly
(i.m.), intranasally, orally, subcutaneously (s.c.), etc. and in
any suitable delivery device (O'Hagan et al., Nature Reviews,
Drug Discovery 2(9) (2003): 727-735). The compound of the pre-
sent invention is preferably formulated for s.c., i.d. or i.m.
administration (see e.g. "Handbook of Pharmaceutical Manufactur-
ing Formulations", SarfarazNiazi, CRC Press Inc, 2004).
The vaccine according to the present invention comprises at
least one peptide which is preferably formulated for i.d., s.c.,
or i.m. administration.
At least one peptide in the vaccine is preferably formulated
with an adjuvant, preferably aluminium hydroxide.
Typically, the vaccine contains the peptide according to the
present invention in an amount of 0.5 to 500 µg, preferably 1 to
100 µg and alternatively from 0.1 ng to 10 mg, preferably 10 ng
to 1 mg, in particular 100 ng to 100 µg, or, alternatively, e.g.
100 fmol to 10 µmol, preferably 10 pmol to 1 µmol, in particular
100 pmol to 100 nmol. Typically, the vaccine may also contain
auxiliary substances, e.g. buffers, stabilizers etc.
According to a particularly preferred embodiment ofthe pre-
sent invention the vaccine may comprise two or more of the fol-
lowing components:
antigen: amount of peptide per 0.1 µg to 1 mg, preferably 0.5
dosis µg to 500 µg, more preferably 1
µg to 100 µg, net peptide
carrier anything known to a person
skilled in the art that is
pharmaceutically and medically
acceptable
carrier per dosis 0.1 µg to 50 mg carrier
adjuvant/amount per dosis anything that is medically and
pharmaceutically acceptable
injection volume anything that is medically ac-
ceptable (also depending on
route of application)
buffer anything that is medically and
pharmaceutically acceptable
According to a preferred embodiment of the present invention
the vaccine is used for preventing and/or treating of athero-
sclerosis and diseases associated with atherosclerosis, wherein
the disease associated with atherosclerosis is preferably se-
lected from the group consisting of peripheral arterial occlu-
sive disease, coronary heart disease, apoplectic cerebral insul-
tus and stroke.
Another aspect of the present invention relates to the use
of at least one peptide according to the present invention for
the manufacture of a vaccine for preventing and/or treating of
atherosclerosis and diseases associated with atherosclerosis.
Yet another aspect of the present invention relates to a
method for treating an individual suffering or at risk to suffer
from atherosclerosis or a disease associated with atherosclero-
sis in the course of which a peptide or vaccine according to the
present invention is administered to said individual.
Next to the vaccine of the present invention, the individual
to be treated may receive also other active ingredients known to
influence the LDL and/or HDL levels in humans and mammals such
as statins, fibrates, nicotinic acid, cholesterol uptake inhibi-
tior (e.g. ezetimibe), ApoA1 Milano, delipidated HDL, plant
sterols, PCSK9 inhibitors etc.
“Treating” as used herein means to cure an already present
disease state or condition. Treating can also include inhibit-
ing, i.e. arresting the development of a disease state or condi-
tion, and ameliorating, i.e. causing regression of a disease.
The term “preventing” as used herein means to completely or
almost completely stop a disease state or condition from occur-
ring in a patient or subject, especially when the patient or
subject is predisposed to such a risk of contracting a disease
state or condition.
Another aspect of the present invention relates to antibod-
ies binding to or being directed to a peptide of the present in-
vention consisting of 6 to 20 amino acid residues and being de-
rived from amino acid sequence VFKGTLKYGYTTAWWLGIDQSIDFEIDSAI
(SEQ ID No. 23), wherein said peptide comprises amino acid se-
quence WWLGID (SEQ ID No. 24).
Antibodies directed to the peptides of the present invention
can be used to inhibit the CETP activity in a human or animal
body. Therefore these antibodies can be used for passive vac-
cination of humans and animals.
The antibodies according to the present invention, which may
be monoclonal or polyclonal, can be manufactured as known in the
art. Monoclonal antibodies can be produced by a variety of tech-
niques, including conventional monoclonal antibody methodology,
for example the standard somatic cell hybridization technique of
Kohler and Milstein (1975) Nature 256: 495. Other techniques for
producing monoclonal antibodies can also be employed such as vi-
ral or oncogenic transformation of B lymphocytes.
According to a preferred embodiment the antibody comprises a
heavy chain variable ("VH") region and an antibody light chain
variable ("VL") region, each region comprising complementary de-
termining regions, wherein the complementary determining regions
of the VH region have one or more of the amino acid sequences
NVQLQESGPGLVKPSQSLSLTCTVTGHSITSDYAWNWIRQFPGNKLEWMGYIT-
NSGSTTYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCTRGGPYWGQGTLVTVSA
(SEQ ID No. 104), EVQLVESGGGLVEPGGSLKLSCVASGFTFSTYAMSWFRLTPER-
RLEWVAAISNGGSQNSYPDSVKGRFTVSRDNAK-
NTLYLQMSSLRSEDTAMYYCSRNGNYFDYWGQGTTLTVSS (SEQ ID No. 105),
DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEW-
MGYISYSGTTTYNPSLKSRISITRHTSKNQFFLQLNSVTTEDSATYYC-
TRLGYYFDYWGQGTTLTVSS (SEQ ID No. 106) or
QIQLVQSGPELKKPGETVKISCKASGYTFTDCSMHWVKQAPGQGLK-
WMGWINTKTGEPTYADDFKGRFAFSLETSASTAYLQINILKNEDSATY-
FCAAHSGKDYAIDYWGQGTSVTVSS (SEQ ID No. 107) and the VL region
have one or more of the amino acid sequences DIVMTQSQKFMSTSVG-
DRVSITCKASQNVGTAVVWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTIT-
NMQSEDLADYFCQQYSSYPLTFGAGTKLELK (SEQ ID No 108), QIVLTQSPAIMSAS-
PGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISS-
MEAEDAATYYCHQRSSYPTFGSGTKLEIK (SEQ ID No. 109),
DIVMTQSPASLAMSVGQKVTMNCKSSQSLLSSKNQKNFLAWYQQKPGQSPKVLVY-
FASTRASGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQQYNTPLTFGAGTKLELK (SEQ
ID No. 110) or DVLMTQTPLSLPVSLGDQASISCRSSQSIVHRNGNTYLEW-
YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAQDLGVYFCFQGS-
RVPPTFGGGTKLEIK (SEQ ID No. 111).
Particularly preferred are antibodies comprising a VH region
comprising NVQLQESGPGLVKPSQSLSLTCTVTGHSITSDYAWNWIRQFPGNKLEW-
MGYITNSGSTTYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCTRGGPY-
WGQGTLVTVSA (SEQ ID No. 104) in combination with a VL region
comprising DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAV-
VWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTITNMQSEDLADYFCQQYSSY-
PLTFGAGTKLELK (SEQ ID No 108), or a VH region comprising
EVQLVESGGGLVEPGGSLKLSCVASGFTFSTYAMSWFRLTPERRLEWVAAISNGGSQN-
SYPDSVKGRFTVSRDNAKNTLYLQMSSLRSEDTAMYYCSRNGNYFDYWGQGTTLTVSS (SEQ
ID No. 105), in combination with a VL region comprising QI-
VLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIFDTSKLAS-
GVPARFSGSGSGTSYSLTISSMEAEDAATYYCHQRSSYPTFGSGTKLEIK (SEQ ID No.
109), or a VH region comprising DVQLQESGPGLVKPSQSLSLTCTVTGY-
SITSDYAWNWIRQFPGNKLEWMGYISYSGTTTYNPSLKSRISITRHTSKNQFFLQLNSVTTED-
SATYYCTRLGYYFDYWGQGTTLTVSS (SEQ ID No. 106) in combination with
a VL region comprising DIVMTQSPASLAMSVGQKVTMNCKSS-
QSLLSSKNQKNFLAWYQQKPGQSPKVLVYFASTRASGVPDRFIGSGSGTDFTLTISSVQAED-
LADYFCQQQYNTPLTFGAGTKLELK (SEQ ID No. 110), or a VH region com-
prising QIQLVQSGPELKKPGETVKISCKASGYTFTDCSMHWVKQAPGQGLK-
WMGWINTKTGEPTYADDFKGRFAFSLETSASTAYLQINILKNEDSATY-
FCAAHSGKDYAIDYWGQGTSVTVSS (SEQ ID No. 107) in combination with a
VL region comprising DVLMTQTPLSLPVSLGDQASISCRSSQSIVHRNGNTYLEW-
YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAQDLGVYFCFQGS-
RVPPTFGGGTKLEIK (SEQ ID No. 111).
To create a single chain antibody (scFv), the VH- and VL-
encoding DNA fragments are operatively linked to another frag-
ment encoding a flexible linker, such that the VH and VL se-
quences can be expressed as a contiguous single-chain protein,
with the VL and VH domains joined by the flexible linker (e.g.
Bird et al., Science 242:423-426 (1988); Huston et al., Proc.
Natl. Acad. Sci. USA 85:5879-5883 (1988); McCafferty et al., Na-
ture 348:552-554 (1990)). The single chain antibody may be mono-
valent, if only a single VH and VL are used, bivalent, if two VH
and VL are used, or polyvalent, if more than two VH and VL are
used. Bispecific or polyvalent antibodies may be generated that
bind specifically to CETP and to another molecule.
In another aspect, other modified antibodies may be prepared
using antibody encoding nucleic acid molecules. For instance,
“Kappa bodies” (III et al., Protein Eng. 10: 949-57 (1997)),
“Minibodies” (Martin et al., EMBO J. 13: 5303-9 (1994)),
“Diabodies” (Holliger et al., Proc. Natl. Acad. Sci. USA 90:
6444-6448 (1993)), or “Janusins” (Traunecker et al., EMBO J.
:3655-3659 (1991) and Traunecker et al., Int. J. Cancer
(Suppl.) 7:51-52 (1992)) may be prepared using standard molecu-
lar biological techniques following the teachings of the speci-
fication.
The antibody of the present invention is preferably human-
ised. Methods to obtain such antibodies are well known in the
art. One method is to insert the variable regions disclosed
herein into a human antibody scaffold (see e.g. Hou S, et al. J
Biochem 144 (2008): 115–20).
The present invention is further illustrated in the follow-
ing figures and examples, however, without being restricted
thereto.
Fig. 1 shows median anti-peptide (Fig. 1A) and median anti-
protein titers (n=5 mice per group) (Fig. 1B) for peptides with
SEQ ID Nos 1 to 6 and 22 delineated from the CETP amino acid se-
quence.
Fig. 2 shows median anti-peptide (Fig. 2A) and median anti-
protein titers (Fig. 2B) (n=5 mice per group) for peptides with
SEQ ID No. 1 and 7 to 21 (truncated peptides).
Fig. 3 shows the number of mice with antibodies inhibiting
CETP activity (n=5 mice per group) for peptides with SEQ ID No.
1 and 7 to 21 (truncated peptides).
Fig. 4 shows the percent CETP activity inhibition of sera
from mice (n=5 mice per group) vaccinated with peptides having
amino acid sequence SEQ ID No. 1, 7, 8, 9, 10, 11, 13, 14, 15,
18 and 19.
Fig. 5 shows the recognition of human CETP protein by the
monoclonal antibodies of the present invention.
Fig. 6 shows the inhibition of CETP activity in human serum
by the monoclonal antibodies according to the present invention.
EXAMPLES:
Materials und Methods
Vaccine
The peptides were conjugated via the heterobifunctional
linker GMBS (4-Maleimidobutyric acid N-hydroxysuccinimide ester)
to KLH (Keyhole Limpet Hemocyanin).
µg of the peptides were suspended with aluminum hydroxide
(end concentration of aluminum hydroxide was 0.2 %). As buffer
mannitol/phosphate was used.
Animal experiments
Balb/c mice were subcutaneously immunized. Mice had access
to food and water ad libitum and were kept under a 12 h
light/dark cycle. Age of mice at the beginning of experiments
was usually 8 to 10 weeks.
Mice were injected three times in 2 week intervals with 15
µg of net peptide coupled to KLH and adsorbed to Alum as adju-
vant in a volume of 1 ml in total via the s.c. route.
Blood was taken approximately 2 weeks after the final injec-
tion.
Peptide ELISA
To determine the immunogenicity of the vaccines, 96-well
Nunc-Maxisorb plates were coated with 1 µM of the respective
peptides coupled to bovine serum albumin (BSA) in 0.1 M NaHCO ,
pH 9.2-9.4. An irrelevant peptide was used as negative control.
KLH was included as positive control and coated at a concentra-
tion of 1 µg/ml. Unspecific binding was blocked by incubation
with blocking buffer (5% BSA in PBS). Appropriate serum dilu-
tions were added to the wells, serially diluted 1:2 fold and in-
cubated for approximately 1 hour at 37°C. On every ELISA plate a
standard serum was included as internal control. Bound antibod-
ies were detected by incubation with biotinylated goat anti-
mouse IgG, followed by horseradish peroxidase coupled to Strep-
tavidin. As substrate ABTS was added and the optical density
(OD) at 405 nm was measured in a Microwell plate-reader. The ti-
tres were defined as the dilution of the serum where 50 % of the
ODmax in the assay are reached.
Protein ELISA
The antibodies induced by the vaccination were tested for
their ability to bind to recombinantly expressed human CETP N-
terminally fused to GST ("GST-CETP") using the protocol de-
scribed under "peptide ELISA".
CETP activity inhibition assay
CETP activity was determined with the Roar CETP activity as-
say kit (RB-CETP; Roar Biomedical) by measuring the transfer of
a fluorescently labeled substrate according to the protocol pro-
vided by the manufacturer. Mice do not have endogenous CETP ac-
tivity, therefore a modification of this protocol was intro-
duced. Human serum was used as CETP source and was mixed with
the same amount of serum from vaccinated mice together with do-
nor and acceptor particles (components of the CETP activity as-
say kit).
Mouse sera containing CETP-inhibiting antibodies lead to a
decrease of the signal in this assay. Sera from mice injected
with irrelevant peptide served as negative control.
For testing of inhibition by monoclonal antibodies, indicat-
ed amounts of purified antibodies were added to the human serum.
Example 1: Comparison of several potential CETP epitopes
SEQ ID No. amino acid sequence position within the mature CETP
protein
1 C-YTTAWWLGIDQS AA101-112
C-KAMMLLGQV AA47-55
3 C-LHLQGEREPGWIKQ AA152-165
4 C-DEFKAVLETWG AA290-300
C-GFNTNQEIFQEVVGGFP AA300-316
C-ESIQSFLQSMITA AA403-415
22 C-FPRPDQQHSV AA350-359
Median antibody titers to injected peptide and to CETP (see
Figs. 1A and 1B).
median values (n=5)
SEQ ID No. anti-peptide anti-protein
titers titers
70,000 800
2 66,000 0
3 97,000 50
4 140,000 0
100,000 100
69,000 0
22 229,000 0
These data clearly show that the administration of a peptide
comprising or consisting of amino acid sequence SEQ ID No. 1
leads to the formation of not only antibodies directed to the
peptide itself but also to the mature CETP protein. The admin-
istration of other CETP fragments did only induce the formation
of antibodies directed to the respective fragment and not to the
mature CETP protein.
Example 2: Comparison of immune responseto SEQ ID No. 1 and
truncated versions thereof
SEQ ID No. amino acid sequence position within the mature CETP
protein
1 C-YTTAWWLGIDQS AA101-112
7 C-YGYTTAWWLGIDQSID AA99-114
8 C-TTAWWLGIDQS AA102-112
C-TAWWLGIDQS AA103-112
C-AWWLGIDQS AA104-112
C-WWLGIDQS AA105-112
12 C-WLGIDQS AA106-112
13 C-YTTAWWLGIDQ AA101-111
14 C-YTTAWWLGID AA101-110
C-YTTAWWLGI AA101-109
16 C-YTTAWWLG AA101-108
C-YTTAWWL AA101-107
18 C-TTAWWLGIDQ AA102-111
19 TTAWWLGID-C AA102-110
C-TTAWWLGI AA102-109
21 C-TAWWLGI AA102-108
Median antibody titers to injected peptide and to CETP as well
as number of mice with antibodies decreasing CETP activity of
human serum and percent CETP activity inhibition in human serum
upon addition of sera from single vaccinated mice (see Figs. 2A,
2B, 3 and 4).
median values (n=5)
SEQ ID No. anti-peptide ti- anti-protein ti-
ters ters
1 70,000 800
33,000 500
8 89,000 1,200
9 173,000 2,200
191,000 1,700
11 330,000 1,100
153,000 0
13 143,000 1,200
14 630,000 600
111,000 300
16 74,000 0
94,000 0
18 216,000 900
19 114,000 1,300
124,000 300
21 99,000 0
These data revealed that CETP fragments comprising WWLGID
(SEQ ID No. 24) are able to induce the formation of antibodies
directed to mature CETP. In contrast thereto other CETP frag-
ments derived from SEQ ID No. 23 which do not comprise amino ac-
id sequence SEQ ID No. 24 did not show these effects.
Example 3: Inhibition of the CETP activity
SEQ ID No. no. of mice with
antibodies inhib-
iting CETP activi-
1 3 of 5
7 3 of 5
4 of 5
9 4 of 5
of 5
of 5
12 n.t.
13 1 of 5
1 of 5
0 of 5
n.t.
17 n.t.
18 4 of 5
3 of 5
n.t.
n.t.
n.t. not tested because of low anti-protein titers
Example 4:
Monoclonal antibodies:
Balb/c mice were vaccinated with 15 µg net peptide Seq ID
No. 10 coupled to KLH. Alhydrogel was used as adjuvant. Spleen
cells of mice with high anti-CETP protein titers were fused with
mouse myeloma cells according to standard techniques (protocol
adapted from Köhler, G. and Milstein, C. Nature. 256 (1975):
495–497). Hybridoma clones were tested in standard ELISAs for
the production of antibodies specifically recognizing the in-
jected peptide as wells as recombinantly expressed human CETP
protein. Selected clones were cultured and monoclonal antibodies
were purified from tissue culture supernatants according to
standard protocols.
Sequencing of monoclonal antibodies:
RNA was extracted from hybridoma cells and cDNA was created by
reverse transcription with an oligo(dT) primer. Subsequently PCR
reactions using variable domain primers to amplify both the VH
and VL regions of the monoclonal antibody DNA were performed.
The VH and VL products were extracted and gel purified and
cloned into a sequencing vector and transformed into TOP10. Se-
lected colonies were picked and analyzed through sequencing.
Data from four selected monoclonal antibodies:
clones CJ7B7, 5/C7C8, 12/B3B11, and BTS4-1.
Human CETP protein ELISA and CETP activity inhibition were
performed as described above.
Recognition of human CETP protein by monoclonal antibodies
(see Fig. 5).
These data revealed that all 4 antibodies are recognizing
coated CETP protein. Titration in the ELISA was started with
same amounts for all antibodies (2 mg/ml dilution). As expected,
monoclonal antibodies differ in their ELISA signal which might
be explained by different affinities to the coated protein.
Inhibition of CETP activity in human serum by monoclonal an-
tibodies (see Fig. 6).
These data revealed that all 4 antibodies are not only bind-
ing to CETP but also inhibiting CETP activity. The higher the
amount of antibody added, the more lowering of CETP activity is
observed.
Sequences of monoclonal antibodies (only the variable do-
mains are given):
CJ7B7
Heavy Chain:
NVQLQESGPGLVKPSQSLSLTCTVTGHSITSDYAWNWIRQFPGNKLEWMGYIT-
NSGSTTYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCTRGGPYWGQGTLVTVSA
Light Chain:
DIVMTQSQKFMSTSVGDRVSITCKASQNVGTAVVWYQQKPGQSPKLLIYSASNRYTGVPDRFTG
SGSGTDFTLTITNMQSEDLADYFCQQYSSYPLTFGAGTKLELK
5C7C8
Heavy Chain:
EVQLVESGGGLVEPGGSLKLSCVASGFTFSTYAMSWFRLTPERRLEWVAAISNGGSQN-
SYPDSVKGRFTVSRDNAKNTLYLQMSSLRSEDTAMYYCSRNGNYFDYWGQGTTLTVSS
Light Chain
QIVLTQSPAIMSASPGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIFDTSKLASGVPARFSGS
GSGTSYSLTISSMEAEDAATYYCHQRSSYPTFGSGTKLEIK
12B3B11
Heavy Chain:
DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEW-
MGYISYSGTTTYNPSLKSRISITRHTSKNQFFLQLNSVTTEDSATYYC-
TRLGYYFDYWGQGTTLTVSS
Light Chain
DIVMTQSPASLAMSVGQKVTMNCKSSQSLLSSKNQKNFLAWYQQKPGQSPKVLVYFASTRASGV
PDRFIGSGSGTDFTLTISSVQAEDLADYFCQQQYNTPLTFGAGTKLELK
BTS4-1
Heavy Chain:
QIQLVQSGPELKKPGETVKISCKASGYTFTDCSMHWVKQAPGQGLK-
WMGWINTKTGEPTYADDFKGRFAFSLETSASTAYLQINILKNEDSATY-
FCAAHSGKDYAIDYWGQGTSVTVSS
Light Chain
DVLMTQTPLSLPVSLGDQASISCRSSQSIVHRNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVP
DRFSGSGSGTDFTLRISRVEAQDLGVYFCFQGSRVPPTFGGGTKLEIK
Claims (17)
1. A composition including an isolated peptide consisting of 6 to 20 amino acid residues, the peptide being coupled or fused to a pharmaceutically acceptable carrier, wherein the peptide is derived from amino acid sequence VFKGTLKYGYTTAWWLGIDQSIDFEIDSAI (SEQ ID No. 23), and comprises the amino acid sequence WWLGID (SEQ ID No. 24).
2. A composition according to claim 1, characterized in that the amino acid sequence of the peptide is selected from the group consisting of YTTAWWLGIDQS (SEQ ID No. 1), YGYTTAWWLGIDQSID (SEQ ID No. 7), TTAWWLGIDQS (SEQ ID No. 8), TAWWLGIDQS (SEQ ID No. 9), AWWLGIDQS (SEQ ID No. 10), WWLGIDQS (SEQ ID No. 11), YTTAW- WLGIDQ (SEQ ID No. 13), YTTAWWLGID (SEQ ID No. 14), TTAWWLGIDQ (SEQ ID No. 18) and TTAWWLGID (SEQ ID No. 19).
3. A composition according to claim 1 or 2, characterized in that the peptide comprises at its C- and/or N-terminus a cyste- ine residue.
4. A composition according to any one of claims 1 to 3 to be used for preventing and/or treating of atherosclerosis and dis- eases associated with atherosclerosis.
5. A composition according to claim 4, characterized in that the disease associated with atherosclerosis is selected from the group consisting of peripheral arterial occlusive disease, coro- nary heart disease, apoplectic cerebral insultus and stroke.
6. A composition according to claim 4 or 5, characterized in that the peptide is administered to an individual in an amount of 0.5 to 500 µg, preferably 1 to 100 µg, per immunization.
7. A vaccine including at least one composition according to any one of claims 1 to 3.
8. A vaccine according to claim 7, characterised in that at least one peptide is coupled or fused to a pharmaceutically ac- ceptable carrier.
9. A vaccine as claimed in claim 8 wherein the carrier is KLH (Keyhole Limpet Hemocyanin).
10. A vaccine according to any one of claims 7 to 9, character- ised in that at least one peptide is formulated for intradermal, subcutaneous or intramuscular administration.
11. A vaccine according to any one of claims 7 to 10, character- ised in that at least one peptide is formulated with an adju- vant, preferably aluminium hydroxide.
12. A vaccine according to any one of claims 7 to 11, character- ized in that the vaccine comprises said at least one peptide in an amount of 0.5 to 500 µg per immunisation
13. A vaccine as claimed in claim 12 wherein the peptide is in the amount of 1 to 100 µg, per immunisation.
14. A vaccine according to any one of claims 7 to 13 to be used for preventing and/or treating of atherosclerosis and diseases associated with atherosclerosis.
15. A vaccine according to claim 14, characterized in that the disease associated with atherosclerosis is selected from the group consisting of peripheral arterial occlusive disease, coro- nary heart disease, apoplectic cerebral insultus and stroke.
16. An antibody binding to a peptide according to any one of claims 1 to 3.
17. An antibody according to claim 14, characterized in that the antibody comprises a heavy chain variable ("VH") region and an antibody light chain variable ("VL") region, each region com- prising complementary determining regions, wherein the comple- mentary determining regions of the VH region have one or more of the amino acid sequences NVQLQESGPGLVKP- SQSLSLTCTVTGHSITSDYAWNWIRQFPGNKLEWMGYIT- NSGSTTYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCTRGGPYWGQGTLVTVSA (SEQ ID No. 104), EVQLVESGGGLVEPGGSLKLSCVASGFTFSTYAMSWFRLTPER- RLEWVAAISNGGSQNSYPDSVKGRFTVSRDNAK- NTLYLQMSSLRSEDTAMYYCSRNGNYFDYWGQGTTLTVSS (SEQ ID No. 105), DVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEW- MGYISYSGTTTYNPSLKSRISITRHTSKNQFFLQLNSVTTEDSATYYC- TRLGYYFDYWGQGTTLTVSS (SEQ ID No. 106) or QIQLVQSGPELKKPGETVKISCKASGYTFTDCSMHWVKQAPGQGLK- WMGWINTKTGEPTYADDFKGRFAFSLETSASTAYLQINILKNEDSATY- FCAAHSGKDYAIDYWGQGTSVTVSS (SEQ ID No. 107) and the VL region have one or more of the amino acid sequences DIVMTQSQKFMSTSVG- DRVSITCKASQNVGTAVVWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTIT- NMQSEDLADYFCQQYSSYPLTFGAGTKLELK (SEQ ID No 108), QIVLTQSPAIMSAS- PGEKVTMTCSASSSISYMHWYQQKPGTSPKRWIFDTSKLASGVPARFSGSGSGTSYSLTISS- MEAEDAATYYCHQRSSYPTFGSGTKLEIK (SEQ ID No. 109), DIVMTQSPASLAMSVGQKVTMNCKSSQSLLSSKNQKNFLAWYQQKPGQSPKVLVY- FASTRASGVPDRFIGSGSGTDFTLTISSVQAEDLADYFCQQQYNTPLTFGAGTKLELK (SEQ ID No. 110) or DVLMTQTPLSLPVSLGDQASISCRSSQSIVHRNGNTYLEW- YLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLRISRVEAQDLGVYFCFQGS- RVPPTFGGGTKLEIK (SEQ ID No. 111).
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP11169481A EP2532359A1 (en) | 2011-06-10 | 2011-06-10 | CETP fragments |
EP11169481.6 | 2011-06-10 | ||
PCT/EP2012/061038 WO2012168486A1 (en) | 2011-06-10 | 2012-06-11 | Cetp fragments |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ613913A NZ613913A (en) | 2015-07-31 |
NZ613913B2 true NZ613913B2 (en) | 2015-11-03 |
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