U.S. patent application number 12/394744 was filed with the patent office on 2009-09-10 for peptide-based immunization therapy for treatment of atherosclerosis.
This patent application is currently assigned to FORSKARPATENT I SYD. Invention is credited to Jan Nilsson, Prediman K. Shah.
Application Number | 20090226475 12/394744 |
Document ID | / |
Family ID | 27484534 |
Filed Date | 2009-09-10 |
United States Patent
Application |
20090226475 |
Kind Code |
A1 |
Nilsson; Jan ; et
al. |
September 10, 2009 |
PEPTIDE-BASED IMMUNIZATION THERAPY FOR TREATMENT OF
ATHEROSCLEROSIS
Abstract
The present invention relates to antibodies raised against
fragments of apolipoprotein B, in particular defined peptides
thereof, for immunization or therapeutic treatment of mammals,
including humans, against ischemic cardiovascular diseases, using
one or more of said antibodies.
Inventors: |
Nilsson; Jan; (Genarp,
SE) ; Shah; Prediman K.; (Los Angeles, CA) |
Correspondence
Address: |
GAUTHIER & CONNORS, LLP
225 FRANKLIN STREET, SUITE 2300
BOSTON
MA
02110
US
|
Assignee: |
FORSKARPATENT I SYD
Lund
CA
CEDARS SINAI MEDICAL CENTER
Los Angeles
|
Family ID: |
27484534 |
Appl. No.: |
12/394744 |
Filed: |
February 27, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11485549 |
Jul 12, 2006 |
7537758 |
|
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12394744 |
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10115072 |
Apr 4, 2002 |
|
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11485549 |
|
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60281410 |
Apr 5, 2001 |
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Current U.S.
Class: |
424/185.1 ;
435/7.92; 436/501; 514/1.1; 530/326 |
Current CPC
Class: |
A61K 39/00 20130101;
A61P 9/10 20180101; A61K 38/1709 20130101; C07K 14/775
20130101 |
Class at
Publication: |
424/185.1 ;
530/326; 514/13; 436/501; 435/7.92 |
International
Class: |
A61K 39/00 20060101
A61K039/00; C07K 14/00 20060101 C07K014/00; A61K 38/16 20060101
A61K038/16; G01N 33/566 20060101 G01N033/566; G01N 33/53 20060101
G01N033/53 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 5, 2001 |
SE |
0101232-7 |
Nov 9, 2001 |
SE |
0103754-8 |
Claims
1-11. (canceled)
12. A peptide of apo-lipoprotein B intended for immunization or
therapeutic treatment of mammals, including humans, against
ischemic cardiovascular diseases and having immunogenic or
therapeutic properties against ischemic cardiovascular diseases,
and/or diagnosing the presence or absence of antibodies related to
increased or decreased risk of developing ischemic cardiovascular
diseases, which peptide is encoded by the sequence
FPDLGQEVALNANTKNQKIR (SEQ. ID. NO. 37).
13. The peptide according to claim 12, wherein the peptide is a
hapten of an aldehyde.
14. The peptide according to claim 13, wherein the peptide is
modified using malone dealdehyde or hydroxynonenal.
15. The peptide according to claim 12, in native form.
16. The peptide according to claim 12, in oxidized form.
17. The peptide according to claim 16, wherein the peptide has been
oxidized using copper.
18. The peptide according to claim 12, wherein the peptide is
present in a combination with phospholipid liposomes.
19. The peptide according to claim 12, in the form of a malone
dialdehyde (MDA) derivative thereof.
20. The peptide according to claim 12, in the form of a
hydroxynoneal-derivative thereof.
21. A pharmaceutical preparation comprising a therapeutically
effective amount of the peptide of claim 12, optionally in
combination with one or more pharmaceutically innocuous fillers,
and/or adjuvants.
22. The pharmaceutical preparation according to claim 21, wherein
the peptide is present as linked to cationized bovine serum
albumine, and using aluminium hydroxide as an adjuvant.
23. The pharmaceutical composition according to claim 11, wherein
the composition is present as an injectable composition.
24. A vaccine for immunization of mammals, including humans,
against ischemic cardiovascular diseases comprising a peptide of
claim 12, optionally in combination with an adjuvant
25. The vaccine for immunization according to claim 24, wherein the
peptide present for immunization is present as linked to cationized
bovine serum albumine, and using aluminum hydroxide as an
adjuvant.
26. The method of prophylactic or therapeutic treatment of a
mammal, including a human being, suffering from atheroschlerosis or
facing the risk of developing ischemic cardiovascular diseases,
whereby a therapeutically effective amount of one or more of
peptide of claim 12, either in native form or in the form of a
malone dealdehyde or a hydroxynoneal derivative, is administered to
said mammal suffering from atheroschlerosis, or facing the risk of
developing ischemic cardiovascular diseases, in particular
myocardial infarction.
27. The method according to claim 26, wherein the conditions are
one or more of unstable atherosclerotic plaques in which oxidized
LDL is likely to contribute to inflammation, cell toxicity and risk
for plaque rupture, as well as coronary heart disease in older
individuals.
28. A method of diagnosing the presence or absence of antibodies
related to increased or decreased risk of developing ischemic
cardiovascular diseases, using the peptide of claim 12 in an
assay.
29. The method according to claim 28, wherein the assay is an
immunoassay.
30. The method according to claim 29, wherein the immunoassay is an
ELISA, RIA, Western blotting, Southern blotting
Description
PRIORITY INFORMATION
[0001] This application is a divisional of U.S. patent application
Ser. No. 11/485,549, filed on Jul. 12, 2006 which is a divisional
of U.S. Utility application Ser. No. 10/115,072, filed Apr. 4,
2002, now abandoned, which claims priority to Swedish Application
Nos. 0101232-7, filed on Apr. 5, 2001, and 0103754-8, filed Nov. 9,
2001, and the benefit of U.S. Provisional Application Ser. No.
60/281,410, filed Apr. 5, 2001, the content of which is
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] The present invention relates to new peptides, in particular
peptides to be used for immunization therapy for treatment of
atherosclerosis, and for development of peptide based ELISA for the
determination of immune response against oxidized low density
lipoprotein and the diagnosis of the presence or absence of
atherosclerosis.
[0004] 2. Brief Description of the Art
[0005] In particular the invention includes: [0006] 1) The use of
any of the peptides listed in table 1, alone or in combination,
native or MDA-modified, preferably together with a suitable carrier
and adjuvant as an immunotherapy or "anti-atherosclerosis "vaccine"
for prevention and treatment of ischemic [0007] 2) cardiovascular
disease. [0008] 3) The use of the same peptides in ELISA for
detection of antibodies related to increased or decreased risk of
development of ischemic cardiovascular diseases.
[0009] Atherosclerosis is a chronic disease that causes a
thickening of the innermost layer (the intima) of large and
medium-sized arteries. It decreases blood flow and may cause
ischemia and tissue destruction in organs supplied by the affected
vessel. Atherosclerosis is the major cause of cardiovascular
disease including myocardial infarction, stroke and peripheral
artery disease. It is the major cause of death in the western world
and is predicted to become the leading cause of death in the entire
world within two decades.
[0010] The disease is initiated by accumulation of lipoproteins,
primarily low-density lipoprotein (LDL), in the extracellular
matrix of the vessel. These LDL particles aggregate and undergo
oxidative modification. Oxidized LDL is toxic and cause vascular
injury. Atherosclerosis represents in many respects a response to
this injury including inflammation and fibrosis.
[0011] In 1989 Palinski and coworkers identified circulating
autoantibodies against oxidized LDL in humans. This observation
suggested that atherosclerosis may be an autoimmune disease caused
by immune reactions against oxidized lipoproteins. At this time
several laboratories began searching for associations between
antibody titers against oxidized LDL and cardiovascular disease.
However, the picture that emerged from these studies was far from
clear. Antibodies existed against a large number of different
epitopes in oxidized LDL, but the structure of these epitopes was
unknown. The term "oxidized LDL antibodies" thus referred to an
unknown mixture of different antibodies rather than to one specific
antibody. T cell-independent IgM antibodies were more frequent than
T-cell dependent IgG antibodies.
[0012] Antibodies against oxidized LDL were present in both
patients with cardiovascular disease and in healthy controls.
Although some early studies reported associations between oxidized
LDL antibody titers and cardiovascular disease, others were unable
to find such associations. A major weakness of these studies was
that the ELISA tests used to determine antibody titers used
oxidized LDL particles as ligand. LDL composition is different in
different individuals, the degree of oxidative modification is
difficult both to control and assess and levels of antibodies
against the different epitopes in the oxidized LDL particles can
not be determined. To some extent, due to the technical problems it
has been difficult to evaluate the role of antibody responses
against oxidized LDL using the techniques available so far, but,
however, it is not possible to create well defined and reproducable
reproducible components of a vaccine if one should use intact
oxidized LDL particles.
[0013] Another way to investigate the possibility that autoimmune
reactions against oxidized LDL in the vascular wall play a key role
in the development of atherosclerosis is to immunize animals
against its own oxidized LDL. The idea behind this approach is that
if autoimmune reactions against oxidized LDL are reinforced using
classical immunization techniques this would result in increased
vascular inflammation and progressive of atherosclerosis. To test
this hypothesis rabbits were immunized with homologous oxidized LDL
and then induced atherosclerosis by feeding the animals a
high-cholesterol diet for 3 months.
[0014] However, in contrast to the original hypothesis immunization
with oxidized LDL had a protective effect reducing atherosclerosis
with about 50%. Similar results were also obtained in a subsequent
study in which the high-cholesterol diet was combined with vascular
balloon-injury to produce a more aggressive plaque development. In
parallel with our studies several other laboratories reported
similar observations. Taken together the available data clearly
demonstrates that there exist immune reactions that protect against
the development of atherosclerosis and that these involves
autoimmunity against oxidized LDL.
[0015] These observations also suggest the possibility of
developing an immune therapy or "vaccine" for treatment of
atherosclerosis-based cardiovascular disease in man. One approach
to do this would be to immunize an individual with his own LDL
after it has been oxidized by exposure to for example copper.
However, this approach is complicated by the fact that it is not
known which structure in oxidized LDL that is responsible for
inducing the protective immunity and if oxidized LDL also may
contain epitopes that may give rise to adverse immune
reactions.
[0016] The identification of epitopes in oxidized LDL is important
for several aspects:
[0017] First, one or several of these epitopes are likely to be
responsible for activating the anti-atherogenic immune response
observed in animals immunized with oxidized LDL. Peptides
containing these epitopes may therefore represent a possibility for
development of an immune therapy or "atherosclerosis vaccine" in
man. Further, they can be used for therapeutic treatment of
atheroschlerosis developed in man.
[0018] Secondly, peptides containing the identified epitopes can be
used to develop ELISAs able to detect antibodies against specific
structure in oxidized LDL. Such ELISAs would be more precise and
reliable than ones presently available using oxidized LDL particles
as antigen. It would also allow the analyses of immune responses
against different epitopes in oxidized LDL associated with
cardiovascular disease.
[0019] U.S. Pat. No. 5,972,890 relates to a use of peptides for
diagnosing atherosclerosis. The technique presented in said US
patent is as a principle a form of radiophysical diagnosis. A
peptide sequence is radioactively labelled and is injected into the
bloodstream. If this peptide sequence should be identical with
sequences present in apolipoprotein B it will bind to the tissue
where there are receptors present for apolipoprotein B. In vessels
this is above all atherosclerotic plaque. The concentration of
radioactivity in the wall of the vessel can then be determined
e.g., by means of a gamma camera. The technique is thus a
radiophysical diagnostic method based on that radioactively
labelled peptide sequences will bound to their normal tissue
receptors present in atherosclerotic plaque and are detected using
an external radioactivity analysis. It is a direct analysis method
to identify atherosclerotic plaque. It requires that the patient be
given radioactive compounds.
SUMMARY OF THE INVENTION
[0020] The technique of the present invention is based on quite
different principles and methods. In accordance with claim 1 the
invention relates to fragments of apolipoprotein B for immunisation
against cardiovascular disease as well as a method for diagnosing
immuno reactions against peptide sequences of apolipoprotein B.
Such immuno reactions have in turn showed to be increased in
individuals having a developed atherosclerosis. The present
technique is based in attaching peptide sequences in the bottom of
polymer wells. When a blood sample is added the peptides will bind
antibodies, which are specific to these sequences. The amount of
antibodies bound is then determined using an immunological
method/technique. In contrast to the technique of said US patent
this is thus not a direct determination method to identify and
localise atherosclerotic plaque but determines an immunological
response, which shows a high degree of co-variation with the
extension of the atherosclerosis.
[0021] The basic principle of the present invention is thus quite
different from that of said patent. The latter depends on binding
of peptide sequences to the normal receptors of the lipoproteins
present in atherosclerotic tissue, while the former is based on the
discovery of immuno reactions against peptide sequences and
determination of antibodies to these peptide sequences.
[0022] Published studies (Palinski et al., 1995, and George et al.,
1998) have shown that immunisation against oxidised LDL reduces the
development of atherosclerosis. This would indicate that immuno
reactions against oxidised LDL in general have a protecting effect.
The results given herein have, however, surprisingly shown that
this is not always the case. E.g., immunisation using a mixture of
peptides #10, 45, 154, 199, and 240 gave rise to an increase of the
development of atherosclerosis. Immunisation using other peptide
sequences, e.g., peptide sequences #1, and 30 to 34 lacks total
effect on the development of atherosclerosis. The results are
surprising because they provide basis for the fact that immuno
reactions against oxidised LDL, can protect against the
development, contribute to the development of atherosclerosis, and
be without any effect at all depending on which structures in
oxidised LDL they are directed to. These findings make it possible
to develop immunisation methods, which isolate the activation of
protecting immuno reactions. Further, they show that immunisation
using intact oxidised LDL could have a detrimental effect if the
particles used contain a high level of structures that give rise to
atherogenic immuno reactions.
[0023] WO 99/08109 relates to the use of a panel of monoclonal
mouse antibodies, which bind to particles of oxidised LDL in order
to determine the presence of oxidised LDL in serum and plasma. This
is thus totally different from the present invention wherein a
method for determining antibodies against oxidised LDL is
disclosed.
[0024] U.S. Pat. No. 4,970,144 relates to a method for preparing
antibodies by means of immunisation using peptide sequences, which
antibodies can be used for the determination of apolipoproteins
using ELISA. This is thus something further quite different from
the present invention.
[0025] U.S. Pat. No. 5,861,276 describes a recombinant antibody to
the normal form of apolipoprotein B. This antibody is used for
determining the presence of normal apolipoprotein B in plasma and
serum, and for treating atherosclerosis by lowering the amount of
particles of normal LDL in the circulation.
[0026] Thus in the present invention the use of antibodies are
described for treating atherosclerosis. However, contrary to the
U.S. Pat. No. 5,861,276, these antibodies are directed to
structures present in particles of oxidised LDL and not to the
normal particle of LDL. The advantage is that it is the oxidised
LDL, which is supposed to give rise to the development of
atherosclerosis. The use of antibodies directed to structures being
specific to oxidised LDL is not described in said US patent.
SUMMARY OF THE INVENTION
[0027] Oxidation of lipoproteins, mainly LDL, in the arterial wall
is believed to be an important factor in the development of
atherosclerosis. Products generated during oxidation of LDL are
toxic to vascular cells, cause inflammation and initiate plaque
formation. Epitopes in oxidized LDL are recognized by the immune
system and give rise to antibody formation. Animal experiments have
shown that some of these immune responses have a protective effect
against atherosclerosis. Antibodies are generally almost
exclusively directed against peptide-based structures. Using a
polypeptide library covering the complete sequence of the only
protein present in LDL, apolipoprotein B, the epitopes have been
identified in oxidized LDL that give rise to antibody formation in
man. These peptide-epitopes can be used to develop ELISAs to study
associations between immune responses against oxidized LDL and
cardiovascular disease and to develop an immunotherapy or
anti-atherosclerosis "vaccine" for prevention and treatment of
ischemic cardiovascular disease.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] FIG. 1-6 show antibody response to the different peptides
prepared in accordance with the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0029] A molecular characterization of the epitopes in oxidized LDL
has been performed that give rise to antibody-dependent immune
responses in man. The approach used takes advantage of the fact
that immune reactions almost exclusively are directed against 5-6
amino acid long peptide sequences. LDL only contains one protein,
the 4563 amino acid long apolipoprotein B. During oxidation
apolipoprotein B is fragmented and aldehyde adducts coupled to
positively charged amino acids, in particularly lysine. This means
that peptide sequences not normally exposed because of the three
dimensional structure of apolipoprotein B become accessible to
immune cells and/or that normally exposed peptide sequences becomes
immunogenic because haptenization with aldehydes.
[0030] It has thereby been determined that the following peptides,
native or MDA derivatives possess such an efficiency as producing
an immuno-response, these. These peptides are:
TABLE-US-00001 FLDTVYGNCSTHFTVKTRKG, (SEQ ID NO: 1)
PQCSTHILQWLKRVHANPLL, (SEQ ID NO: 2) VISIPRLQAEARSEILAHWS, (SEQ ID
NO: 3) KLVKEALKESQLPTVMDFRK, (SEQ ID NO: 4) LKFVTQAEGAKQTEATMTFK,
(SEQ ID NO: 5) DGSLRHKFLDSNIKFSHVEK, (SEQ ID NO: 6)
KGTYGLSCQRDPNTGRLNGE, (SEQ ID NO: 7) RLNGESNLRFNSSYLQGTNQ, (SEQ ID
NO: 8) SLTSTSDLQSGIIKNTASLK, (SEQ ID NO: 9) TASLKYENYELTLKSDTNGK,
(SEQ ID NO: 10) DMTFSKQNALLRSEYQADYE, (SEQ ID NO: 11)
MKVKIIRTIDQMQNSELQWP, (SEQ ID NO: 12) IALDDAKINFNEKLSQLQTY, (SEQ ID
NO: 13) KTTKQSFDLSVKAQYKKNKH, (SEQ ID NO: 14) EEEMLENVSLVCPKDATRFK,
(SEQ ID NO: 15) GSTSHHLVSRKSISAALEHK, (SEQ ID NO: 16)
IENIDFNKSGSSTASWIQNV, (SEQ ID NO: 17) IREVTQRLNGEIQALELPQK, (SEQ ID
NO: 18) EVDVLTKYSQPEDSLIPFFE, (SEQ ID NO: 19) HTFLIYITELLKKLQSTTVM,
(SEQ ID NO: 20) LLDIANYLMEQIQDDCTGDE, (SEQ ID NO: 21)
CTGDEDYTYKIKRVIGNMGQ, (SEQ ID NO: 22) GNMGQTMEQLTPELKSSILK, (SEQ ID
NO: 23) SSILKCVQSTKPSLMIQKAA, (SEQ ID NO: 24) IQKAAIQALRKMEPKDKDQE,
(SEQ ID NO: 25) RLNGESNLRFNSSYLQGTNO, (SEQ ID NO: 26)
SLNSHGLELNADILGTDKIN, (SEQ ID NO: 27) WIQNVDTKYQIRIQIQEKLQ, (SEQ ID
NO: 28) TYISDWWTLAAKNLTDFAEQ, (SEQ ID NO: 29) EATLQRIYSLWEHSTKNHLQ,
(SEQ ID NO: 30) ALLVPPETEEAKQVLFLDTV, (SEQ ID NO: 31)
IEIGLEGKGFEPTLEALFGK, (SEQ ID NO: 32) SGASMKLTTNGRFREHNAKF, (SEQ ID
NO: 33) NLIGDFEVAEKINAFRAKVH, (SEQ ID NO: 34) GHSVLTAKGMALFGEGKAEF,
(SEQ ID NO: 35) FKSSVITLNTNAELFNQSDI, (SEQ ID NO: 36)
FPDLGQEVALNANTKNQKIR, (SEQ ID NO: 37) as well as the non
antibody-producing peptide ATRFKHLRKYTYNYQAQSSS, (SEQ ID NO:
38)
or an active site of one or more of these peptides.
Material and Methods
[0031] To determine which parts of apolipoprotein B that become
immunogenic as a result of LDL oxidation a polypeptide library
consisting of 20 amino acid long peptides covering the complete
human apolipoprotein B sequence was produced. The peptides were
produced with a 5 amino acid overlap to cover all sequences at
break points. Peptides were used in their native state, or after
incorporation in phospholipid liposomes, after oxidization by
exposure to copper or after malone dealdehyde dialdehyde
(MDA)--modification to mimic the different modifications of the
amino acids that may occur during oxidation of LDL.
Peptides
[0032] The 302 peptides corresponding to the entire human
apolipoprotein B amino acid sequence were synthesized
(Euro-Diagnostica AB, Malmo, Sweden and K J Ross Petersen A S,
Horsholm, Denmark) and used in ELISA. A fraction of each synthetic
peptide was modified by 0.5 M MDA (Sigma-Aldrich Sweden A B,
Stockholm, Sweden) for 3 h at 37.degree. C. and in presence of
liposomes by 0.5 M MDA for 3 h at 37.degree. C. or by 5 .mu.M
CuCl.sub.2 (Sigma) for 18 h at 37.degree. C. The MDA-modified
peptides were dialyzed against PBS containing 1 mM EDTA with
several changes for 18 h at 4.degree. C. The modification of the
peptides was tested in denatured polyacrylamide gels (Bio-Rad
Laboratories, Hercules, Calif.), suitable for separation of
peptides. Peptides were numbered 1-302 starting at the N-terminal
end of the protein.
[0033] Other aldehydes can be used for preparing derivatives, such
hydroxynonenal and others.
Liposomes
[0034] A mixture of egg phosphatidylcholine (EPC) (Sigma) and
phosphatidylserine (PS) (Sigma) in a chloroform solution at a molar
ratio of 9:1 and a concentration of 3 mM phospholipid (PL) was
evaporated in a glass container under gentle argon stream. The
container was then placed under vacuum for 3 hours. A solution
containing 0.10 mM peptide (5 ml) in sterile filtered 10 mM HEPES
buffer pH 7.4, 145 mM NaCl and 0.003% sodium azide was added to the
EPC/PS dried film and incubated for 15 min at 50.degree. C. The
mixture was gently vortex for about 5 min at room temperature and
then placed in ice-cold water bath and sonicated with 7.5 amplitude
microns for 3.times.3 min (Sonyprep 150 MSE Sanyo, Tamro-Medlab,
Sweden) with 1 min interruptions. The PL-peptide mixture, native or
modified by 0.5 M MDA for 3 h at 37.degree. C. or 5 mM CuCl.sub.2
for 18 h at 37.degree. C., was stored under argon in glass vials at
4.degree. C. wrapped in aluminum foil and used within 1 week. The
MDA-modified mixture was dialyzed against PBS containing 1 mM EDTA
with several changes for 18 h at 4.degree. C. before storage. The
modification of the mixture was tested in denatured polyacrylamide
gels (Bio-Rad Laboratories AB, Sundbyberg, Sweden), suitable for
separation of peptides.
Plasma Samples
[0035] Plasma samples from 10 patients with cardiovascular disease
(AHP) and 50 plasma samples, 25 women and 25 men, from normal blood
donors (NHP) were collected and pooled. The two pools were
aliquoted and stored in -80.degree. C.
ELISA
[0036] Native or modified synthetic peptides diluted in PBS pH 7.4
(20 .mu.g/ml), in presence or absence of liposomes, were absorbed
to microtiter plate wells (Nunc MaxiSorp, Nunc, Roskilde, Denmark)
in an overnight incubation at 4.degree. C. As a reference, one of
the peptides (P6) was run on each plate. After washing with PBS
containing 0.05% Tween-20 (PBS-T) the coated plates were blocked
with SuperBlock in TBS (Pierce, Rockford, Ill.) for 5 min at room
temperature followed by an incubation of pooled human plasma, AHP
or NHP, diluted 1/100 in TBS-0.05% Tween-20 (TBS-T) for 2 h at room
temperature and then overnight at 4.degree. C. After rinsing,
deposition of auto-antibodies directed to the peptides were
detected by using biotinylated rabbit anti-human IgG- or
IgM-antibodies (Dako A/S, Glostrup, Denmark) appropriately diluted
in TBS-T. After another incubation for 2 h at room temperature the
plates were washed and the bound biotinylated antibodies were
detected by alkaline phosphatase conjugated streptavidin (Sigma),
incubated for 2 h at room temperature. The color reaction was
developed by using phosphatase substrate kit (Pierce) and the
absorbance at 405 nm was measured after 1 h of incubation at room
temperature. The absorbance values of the different peptides were
divided with the absorbance value of P6 and compared.
[0037] The sequences in apolipoprotein B that were recognized by
antibodies in human plasma are shown as Seq. Id 1-37 in the
accompanying drawing. Both AHP and NHP contained antibodies to a
large number of different peptides. Antibodies against both native
and modified peptides were identified. Generally antibody titers to
MDA modified peptides were higher or equal to that of the
corresponding native peptide. Comparison between native,
MDA-modified, copper-oxidized peptide showed a high degree of
correlation and that the highest antibody titers were detected
using MDA-modified peptides. The use of peptides incorporated into
liposomes did not result in increased antibody levels. Antibodies
of the IgM subclass were more common than antibodies of the IgG
subtype.
[0038] The peptides against which the highest antibody levels were
detected could be divided into six groups with common
characteristics (Table 1):
(A) High levels of IgG antibodies to MDA-modified peptides (n=3).
(B) High levels of IgM antibodies, but no difference between native
and MDA-modified peptides (n=9). (C) High levels of IgG antibodies,
but no difference between native and MDA-modified peptides (n=2).
(D) High levels of IgG antibodies to MDA-modified peptides and at
least twice as much antibodies in the NHP-pool as compared to the
AHP-pool (n=5). (E) High levels of IgM antibodies to MDA-modified
peptides and at least twice as much antibodies in the NHP-pool as
compared to the AHP-pool (n=11) (F) High levels of IgG antibodies,
but no difference between intact and MDA-modified peptides but at
least twice as much antibodies in the AHP-pool as compared to the
NHP-pool (n=7). (G) No level of IgG or IgM antibodies
TABLE-US-00002 TABLE 1 A. High IgG, MDA-difference P 11.
FLDTVYGNCSTHFTVKTRKG, (SEQ ID NO: 1) P 25. PQCSTHILQWLKRVHANPLL,
(SEQ ID NO: 2) P 74. VISIPRLQAEARSEILAHWS, (SEQ ID NO: 3) B. High
IgM, no MDA-difference P 40. KLVKEALKESQLPTVMDFRK, (SEQ ID NO: 4) P
68. LKFVTQAEGAKQTEATMTFK, (SEQ ID NO: 5) P 94.
DGSLRHKFLDSNIKFSHVEK, (SEQ ID NO: 6) P 99. KGTYGLSCQRDPNTGRLNGE,
(SEQ ID NO: 7) P 100. RLNGESNLRFNSSYLQGTNQ, (SEQ ID NO: 8) P 102.
SLTSTSDLQSGIIKNTASLK, (SEQ ID NO: 9) P 103. TASLKYENYELTLKSDTNGK,
(SEQ ID NO: 10) P 105. DMTFSKQNALLRSEYQADYE, (SEQ ID NO: 11) P 177.
MKVKIIRTIDQMQNSELQWP, (SEQ ID NO: 12) C. High IgG, no MDA
difference P 143. IALDDAKINFNEKLSQLQTY, (SEQ ID NO: 13) P 210.
KTTKQSFDLSVKAQYKKNKH, (SEQ ID NO: 14) D. NHS/AHP, IgG-ak > 2,
MDA-difference P1. EEEMLENVSLVCPKDATRFK, (SEQ ID NO: 15) P 129.
GSTSHHLVSRKSISAALEHK, (SEQ ID NO: 16) P 148. IENIDFNKSGSSTASWIQNV,
(SEQ ID NO: 17) P 162. IREVTQRLNGEIQALELPQK, (SEQ ID NO: 18) P 252.
EVDVLTKYSQPEDSLIPFFE, (SEQ ID NO: 19) E. NHS/AHP, IgM-ak > 2,
MDA-difference P 301. HTFLIYITELLKKLQSTTVM, (SEQ ID NO: 20) P 30.
LLDIANYLMEQIQDDCTGDE, (SEQ ID NO: 21) P 31. CTGDEDYTYKIKRVIGNMGQ,
(SEQ ID NO: 22) P 32. GNMGQTMEQLTPELKSSILK, (SEQ ID NO: 23) P 33.
SSILKCVQSTKPSLMIQKAA, (SEQ ID NO: 24) P 34. IQKAAIQALRKMEPKDKDQE,
(SEQ ID NO: 25) P 100. RLNGESNLRFNSSYLQGTNQ, (SEQ ID NO: 26) P 107.
SLNSHGLELNADILGTDKIN, (SEQ ID NO: 27) P 149. WIQNVDTKYQIRIQIQEKLQ,
(SEQ ID NO: 28) P 169. TYISDWWTLAAKNLTDFAEQ, (SEQ ID NO: 29) P 236.
EATLQRIYSLWEHSTKNHLQ, (SEQ ID NO: 30) F. NHS/AHP, IgG-ak > 0.5,
no MDA-difference P 10. ALLVPPETEEAKQVLFLDTV, (SEQ ID NO: 31) P 45.
IEIGLEGKGFEPTLEALFGK, (SEQ ID NO: 32) P 111. SGASMKLTTNGRFREHNAKF,
(SEQ ID NO: 33) P 154. NLIGDFEVAEKINAFRAKVH, (SEQ ID NO: 34) P 199.
GHSVLTAKGMALFGEGKAEF, (SEQ ID NO: 35) P 222. FKSSVITLNTNAELFNQSDI,
(SEQ ID NO: 36) P 240. FPDLGQEVALNANTKNQKIR, (SEQ ID NO: 37) G. P
2. ATRFKHLRKYTYNYQAQSSS. (SEQ ID NO: 38)
[0039] All of these 38 peptide sequences represent targets for
immune reactions that may be of importance for the development of
atherosclerosis and ischemic cardiovascular diseases. These
peptides may therefor be used to develop ELISAs to determine the
associations between antibody levels against defined sequences of
MDA-modified amino acids in apolipoprotein B and risk for
development of cardiovascular disease.
[0040] These peptides also represent possible mediators of the
protective immunity observed in experimental animals immunized with
oxidized LDL and may be used for testing in further development of
an immunization therapy or "vaccine" against atherosclerosis.
[0041] Thus 38 different sequences in the human apolipoprotein B
protein have been identified that give rise to significant immune
responses in man. These epitopes are likely to represent what has
previously been described as antibodies to oxidized LDL. Since most
immune responses are directed against peptide sequences and
apolipoprotein B is the only protein in LDL the approach used in
this project should be able to identify the specific epitopes for
essentially all antibodies against oxidized LDL-particles. A family
of phospholipid specific antibodies including antibodies against
cardiolipin has been described to react with oxidized LDL but the
specificity and role of these antibodies remain to be fully
characterized.
[0042] In many cases antibody titers were higher to MDA-modified
polypeptides than to native sequences. If antibodies were detected
against a MDA modified sequence it was almost always associated
with presence of antibodies against the native sequence. A likely
explanation to this is that the immune response against an
MDA-modified amino acid sequence in apolipoprotein B (the
MDA-modification occurring as a result of LDL oxidation) leads to a
break of tolerance against the native sequence. For other sequences
there was no difference in antibody titers against MDA-modified or
native sequences. This would suggest that the immune reactions are
directed against the native sequences. There should be no immune
response against amino acid sequences in protein normally exposed
to the immune system. In the native LDL particle large parts of the
apolipoprotein B protein is hidden in phospholipid layer of LDL and
therefore not accessible for the immune system. During oxidation of
LDL the apolipoprotein B amino acid chain is fragmented leading to
changes in the three-dimensional structure. This is likely to lead
to exposure of peptide sequences normally not accessible for the
immune system and to generation of antibodies against these
sequences which may explain the presence of antibodies against
native apolipoprotein B sequences observed. Alternatively, the true
immune response is against MDA-modified sequences but the
cross-reactivity with native sequences is so great that no
difference in binding can be demonstrated.
TABLE-US-00003 TABLE 2 Associations between antibodies to different
peptides and atherosclerosis in the carotid artery assessed as
intima/media thickness in 78 subjects (26 subjects who later
developed myocardial infarction, 26 healthy controls and 26
high-risk individuals without disease). IgG IgM Peptide Native
MDA-modified Native MDA-modified 301 + 10 + + 11 ++ + 25 + + ++ +++
30 ++ 31 ++ ##STR00001## 32 ##STR00002## 33 + 34 + 45 ++ ++ +++ 74
++ + + ++ 99 100 + ++ 102 ##STR00003## 103 + 105 129 ++ +++ 143 + +
++ + 148 + 154 +++ ++ 162 + ++ 199 210 + 240 ++ ##STR00004## +, r
> 0.2 < 0.3, p = <0.05; ++, r > 0.3 < 0.4, p = 0.01;
+++, r > 0.4, p = <0.001, grey, peptide antibody levels
significantly increased in the group suffering from myocardial
infarction.
[0043] The possibility that the ELISAs based on these peptides
(native or MDA-modified) can be used to determine associations
between immune reaction against defined epitopes in oxidized LDL
and presence and/or risk for development of cardio-vascular disease
was investigated in a pilot study. The study was performed on
subjects participating in the Malmo Diet Cancer study a population
based study in which over 30,000 individuals were recruited between
1989 and 1993. Antibody levels against the 24 out of 38 peptides
listed in Table 1 were determined in base line plasma samples of 26
subjects who developed an acute myocardial infarction during the
follow-up period and 26 healthy controls matched for age, gender
and smoking. An additional group of 26 subjects, matched for age,
gender, and smoking, but all with LDL cholesterol levels above 5.0
mmol/L was also included to study antibody levels in a high-risk
group that has not developed cardiovascular disease.
[0044] For 19 out of the 24 peptides analyzed, significant
correlations were identified between IgM antibody levels against
MDA-modified peptides and the severity of atherosclerosis in the
carotid artery (intima/media thickness) as assessed by ultrasound
investigation of common carotid artery, i.e., the higher antibody
levels the more atherosclerosis (Table 2). For many of these
peptides significant correlations also existed between antibody
levels to native peptides and carotid intima/media thickness. Only
4 peptides showed a significant correlation between IgG antibodies
and carotid intima/media thickness. These observations suggest that
ELISA using these MDA-modified peptides (alone or in combination)
may be used to identify subjects with increased
atherosclerosis.
[0045] Four of the peptides tested were not only associated with
increased presence of atherosclerosis but were also significant
elevated in the group of subjects that later suffered from a
myocardial infarction (Table 2). Data for one of these peptides
(peptide 240) is shown in FIG. 7. These observations also
demonstrate that peptide-based ELISA also may be used to identify
subjects with an increased risk to develop myocardial
infarction.
[0046] There were also significant increases in IgG antibody levels
for native peptides 103, 162 and 199, as well as MDA modified 102
in the group that later suffered from myocardial infarction.
However, the IgG antibodies against these peptides were not
significantly associated with the presence of atherosclerosis in
the carotid artery.
[0047] A particularly interesting observation was made with
antibodies against MDA-modified peptide 210 for which there was
significantly higher levels of IgM antibodies in the healthy
controls and the high-risk group (LDL cholesterol above 5.0 mmol/L)
than in the group that developed a myocardial infarction.
Accordingly antibodies against MDA-modified peptide 210 may
represent a marker for individuals with a decreased risk to develop
cardiovascular disease.
[0048] It has now been demonstrated that immunization with native
and MDA-modified apo B-100 peptide sequences results in an
inhibition of atherosclerosis in experimental animals (Nordin
Fredrikson, Soderberg et al, Chyu et al). The mechanisms through
which these athero-protective immune responses operate remain to be
fully elucidated. However, one likely possibility is that the
athero-protective effect is mediated by antibodies generated
against these peptides sequences. These antibodies could, for
example facilitate the removal of oxidatively damaged LDL particles
by macrophage Fc receptors.
[0049] Macrophage scavenger receptors only recognize LDL with
extensive oxidative damage (9). Recent studies have identified the
existence of circulating oxidized LDL (10,11). These particles have
only minimal oxidative damage and are not recognized by scavenger
receptors. Binding of antibodies to these circulating oxidized LDL
particles may help to remove them from the circulation before they
accumulate in the vascular tissue (12).
[0050] Several studies have supported a role for antibodies in
protection against atherosclerosis. B cell reconstitution inhibits
development of atherosclerosis in splenectomized apo E null mice
(13) as well as neointima formation after carotid injury in RAG-1
mice (unpublished observations from our laboratory). Moreover, it
has been shown that repeated injections of immunoglobulins reduce
atherosclerosis in apo E null mice (6).
[0051] As discussed above antibodies against MDA-modified peptide
sequences in apo B-100 may be generated by active immunization
using synthetic peptides. This procedure requires 2-3 weeks before
a full effect on antibody production is obtained.
[0052] In some situations a more rapid effect may be needed. One
example may be unstable atherosclerotic plaques in which oxidized
LDL is likely to contribute to inflammation, cell toxicity and risk
for plaque rupture. Under these circumstances a passive
immunization by injection of purified, or recombinantly produced
antibodies against native and MDA-modified sequences may have a
faster effect.
[0053] Another situation in which a passive immunization by
injection of purified, or recombinantly produced antibodies may be
effective is coronary heart disease in older individuals. Our
studies have shown that a decrease in antibodies against apo B
peptide sequences occurs with increasing age in man and is
associated with an increase in the plasma level of oxidized LDL
(Nordin Fredrikson, Hedblad et al). This may suggest a senescence
of the immune cells responsible for producing antibodies against
antigens in oxidized LDL and result in a defective clearance of
oxidatively damaged LDL particles from the circulation.
Accordingly, these subjects would benefit more from a passive
immunization by injection of purified, or recombinantly produced
antibodies than from an active immunization with apo B-100 peptide
sequences.
[0054] Synthetic native peptides (Euro-Diagnostica AB, Malmo,
Sweden) used in the following were peptide 1, 2 and 301 from the
initially screened polypeptide library. Peptide 1 (amino acid
sequence: EEEMLENVSLVCPKDATRFK, n=10; (SEQ ID NO: 15)) and peptide
301 (amino acid sequence: HTFLIYITELLKKLQSTTVM, n=10; (SEQ ID NO:
20)) were found to have higher IgG or IgM antibody response to MDA
modified form than native peptide, respectively and both titers are
higher in healthy subject. These peptides were chosen based on the
assumption that antibody response to these peptides might be
protective against atherosclerosis.
[0055] Peptide 2 (amino acid sequence: ATRFKHLRKYTYNYQAQSSS, n=10;
(SEQ ID NO: 38)) elicited no antibody response in the initial
antibody screening, hence it was chosen as control peptide. Mice
receiving Alum served as control (n=9).
[0056] Apo E (-/-) mice received subcutaneous primary immunization
at 6-7 weeks of age, followed by an intra-peritoneal booster 3
weeks later. Mice were fed high cholesterol diet from the onset of
immunization and continued until sacrifice at the age of 25 weeks.
At the time of sacrifice, there was no significant difference in
body weight among 4 groups of mice. Nor there was statistically
significant difference in serum cholesterol as measured using a
commercially available kit (Sigma). Their mean serum cholesterol
levels were all above 715 mg/dl.
[0057] The area of the descending aorta covered by atherosclerotic
plaque was measured in an en face preparation after oil red 0
staining. In comparison to the control group, mice immunized with
peptide No.2 and No. 301 had substantially reduced atherosclerosis
(FIG. 2). Immunization with Peptide No 1 did not produce a
significant reduction in atherosclerosis in comparison to control.
In contrast to the descending aorta, extent of atherosclerosis in
the aortic root and aortic arch did not differ among the 4
experimental groups (FIG. 3).
[0058] There were no difference among 4 groups in terms of aortic
sinus plaque size or its lipid content (Table A). Mean plaque sizes
in the aortic arches from 4 groups of mice were not different.
However, en face evaluation of plaque sizes from descending
thoracic and abdominal aorta by oil red 0 staining revealed that
control group and peptide No.1 group had similar amount of
atherosclerotic plaque in the aorta, whereas peptide No.2 and No. 9
groups had a significantly reduced atherosclerotic burden in the
aorta (Table A). The observation that peptide immunization did not
affect aortic sinus or aortic arch plaque size but reduced
descending aortic plaque is intriguing and suggests that peptide
immunization might reduce new plaque formation but does not affect
the progression of plaques.
[0059] It was further tested whether peptide immunization modulates
the phenotype of atherosclerotic plaques. Frozen sections form
aortic sinus plaques were immunohistochemically stained with
monocyte/macrophage antibody (MOMA-2, Serotec). In concordance with
the findings from en face observation, peptide No. 2 significantly
reduced macrophage infiltration in the plaques (FIG. 1). Trichrome
staining revealed a mean collage content of 40.0.+-.7.7% in the
aortic sinus plaques from peptide 2 group; whereas mean collagen
content in alum control group, peptide 1 group and peptide 9 group
were 32.3.+-.5.3%, 35.6.+-.8.5% and 29.4.+-.9.6%, respectively.
[0060] Antibody response against immunized peptide in each group
was determined. Antibody titer after immunization increased
6.1.+-.3.1 fold in peptide 1 group, 2.4.+-.1.0 fold in peptide 2
group and 1.8.+-.0.6 fold in peptide 9 group; whereas alum group
had a 3.9.+-.2.7 fold increase of antibody titer against peptide 1,
2.0.+-.0.5 fold increase against peptide 2 and 2.0.+-.0.9 fold
increase against peptide 9. It is surprising the parallel increase
of antibody titer against immunized peptides both in immunized and
alum treated group. This may mean the following possibilities: (1)
mechanism(s) other than humoral immune response (such as cellular
immune response) may be involved in modulating atherosclerosis; or
(2) this increase of antibody was a by-stander response to
hypercholesterolemia with time.
[0061] Although there is no clear speculative mechanism to explain
why peptide immunization reduced atherosclerosis and/or modulate
plaque phenotype, the novelty of this invention is the concept of
using peptides of LDL as immunogen and its feasibility as an
immunomodulation strategy. This peptide-based immunization strategy
modulates atherosclerotic plaques. Immunization using homologous
oxLDL or native LDL as antigen had been shown to reduce plaque size
1-3, however, the availability, production, infectious
contamination and safety of homologous human LDL make this approach
unappealing for clinical application. Here it is demonstrated that
peptide-based immunotherapy is feasible although our final results
differ from our initial hypothesis that immunization using peptides
with higher IgM or IgG antibody response in normal subjects may
protect experimental animals from developing advanced
atherosclerotic plaques.
[0062] It is surprising to find that immunization using peptide No.
2 protected animal from developing new atherosclerotic lesions in
descending aorta and reduced macrophage infiltration and a higher
collagen content in plaques since this peptide did not render any
antibody response from initial human screen. It may be because (a)
peptide No. 2 may be a part of human apo-B-100 protein structure
that was not exposed to human immune system. Hence, no antibody was
generated and detected from healthy human serum pools; (b) the
amino acid sequence of peptide No. 2 is foreign to mice therefore
mice developed immune response against this peptide, which
modulates new atherosclerotic lesion formation and its
phenotype.
[0063] The effect of homologous LDL immunization on plaque size
varied when plaque sizes were evaluated at different portions of
aortic tree. For example, Ameli et al showed in
hypercholesterolemic rabbit native LDL immunization resulted in a
reduction of plaque formation in aorta.sup.1, whereas Freigang et
al. showed reduction of plaque size in aortic sinus but not in
aorta.sup.2. Taken their findings and the present ones together, it
was speculated that peptide immunization modulates not only plaque
sizes but also plaque composition. The plaque-reducing effect was
only observed in descending aorta. Apo E (-/-) mice are known to
develop atherosclerotic lesions at various stages of evolution in a
single animal, especially when fed high cholesterol diet. The
initial appearance of atherosclerotic lesion in young animal was in
the aortic sinus.sup.6,7 and after 15 weeks on high fat-high
cholesterol diet lesions at aortic sinus were advanced plaques;
whereas earlier stage of atherosclerosis was present in descending
aorta..sup.6 Since the temporal course of plaque maturation and
development in the descending aorta is late compared to that of
aortic sinus, the finding that immunization reduced lesion sizes in
the descending aorta but not in aortic sinus suggested immunization
affects early stage of atherosclerosis formation. It is possible
that as animal aged and in the presence of supra-physiological
level of serum cholesterol the plaque reducing effect of
immunization is overcome by the toxic effect of
hypercholesterolemia. It is also possible that aortic sinus plaques
mature faster and sacrifice at 25 weeks is too late to detect any
difference in plaque size. Though lesion size was not modulated in
the aortic sinus plaque, peptide immunization did modulate plaque
compositions. The present experimental design prevented from
studying the composition of the plaques in their earlier stage of
development in descending aorta.
[0064] The experimental findings highlight the feasibility of using
peptide sequences of LDL associated apo B-100 as immunogens for a
novel approach to preventing atherosclerosis and or favorably
modulating plaque phenotype despite severe hyperlipdemia. This
peptide-based immunization strategy is potentially advantageous
over the use of homologous oxLDL or native LDL as antigen because
such a strategy could eliminate the need for isolation and
preparation of homologous LDL and its attendant risks for
contamination. The plaque-reducing effect of immunization with
Peptide No 2 and 301 was only observed in descending aorta. These
findings are consistent with previous reports where other
therapeutic interventions have also been shown to have a greater
effect on descending aorta compared to the aortic arch.sup.14-17
presumably because lesions develop more rapidly in the aortic root
and the arch than the descending aorta thus creating a smaller
window of opportunity for intervention.sup.14, 15, 16, 18, 19.
Since the temporal course of plaque maturation and development in
the descending aorta is late compared to that of aortic sinus and
the aortic arch, the finding that immunization reduced lesion sizes
in the descending aorta but not in aortic sinus and arch suggest
that immunization preferentially prevents early stage of
atherosclerosis formation. It is possible that as animal aged and
in the presence of supra-physiological level of serum cholesterol
the plaque reducing effect of immunization is overcome by the toxic
effect of severe hypercholesterolemia. Though the lesion size was
not modulated in the aortic sinus or arch, immunization with
Peptide No 2 did modulate plaque composition in a favorable
direction creating a more stable plaque phenotype with reduced
macrophage infiltration and increased collagen content. In summary,
it is demonstrated a novel peptide-based immunomodulatory approach
for inhibition of atherosclerosis in the murine model.
[0065] In summary, it is demonstrated a novel peptide-based
immunomodulatory approach in modulate atherosclerotic plaques.
Although the change in atherosclerosis formation in our model was
only modest, yet this peptide-based immunization may provide an
alternative tool in studying, preventing or treating
atherosclerosis.
Methods
[0066] Peptide preparation. Peptides were prepared using
Imject.RTM. SuperCarrier.RTM. EDC kit (Pierce, Rockford, Ill.)
according to manufacturer's instruction with minor modification.
One mg peptide in 500 .mu.l conjugation buffer was mixed with 2 mg
carrier in 200 .mu.l deionized water. This mixture was then
incubated with 1 mg conjugation reagent (EDC,
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide HCl) in room
temperature for 2 hours. This was then dialyzed against 0.083 M
sodium phosphate, 0.9 M sodium chloride pH 7.2 solution overnight
at 4.degree. C. The dialyzed conjugate was diluted with Imject dry
blend purification buffer to a final volume of 1.5 ml. Alum was
used as immunoadjuvant and mixed with peptide conjugate with 1:1
dilution in volume. The amount of peptide in each immunization was
33 .mu.g/100 .mu.l per injection.
[0067] Animal protocol. Apo E (-/-) mice from the Jackson
Laboratories (Bar Harbor, Me.) received subcutaneous primary
immunization at 6-7 weeks of age, followed by an intra-peritoneal
booster 3 weeks later. Mice were fed high cholesterol diet from the
onset of immunization and continued until sacrifice at the age of
25 weeks. Blood samples were collected 2 weeks after booster and at
the time of sacrifice. Mice receiving Alum served as control.
Experimental protocol was approved by the Institutional Animal Care
and Use Committee of Cedars-Sinai Medical Center. All mice were
housed in an animal facility accredited by the American Association
of Accreditation of Laboratory Animal Care and kept on a 12-hour
day/night cycle and had unrestricted access to water and food. At
the time of sacrifice, mice were anesthetized by inhalation
Enflurane. Plasma was obtained by retro-orbital bleeding prior to
sacrifice.
[0068] Tissue harvesting and sectioning. To evaluate the effect of
peptide immunization on atherosclerosis formation, the plaque size
at aortic sinus was assessed, aortic arch and descending thoracic
and abdominal aorta. After the heart and aortic tree were perfused
with normal saline at physiological pressure, the heart and
proximal aorta were excised and embedded in OCT compound
(Tissue-Tek) and frozen sectioned. Serial 6-.mu.m-thick sections
were collected from the appearance of at least 2 aortic valves to
the disappearance of the aortic valve leaflets for aortic sinus
plaque evaluation. Typically 3 consecutive sections were on one
slide and a total of 25-30 slides were collected from one mouse and
every fifth slide was grouped for staining. Ascending aorta and
aortic arches upto left subclavian artery were also sectioned and
processed similarly. Descending thoracic and abdominal aorta were
processed separately for en face evaluation of plaque formation
after oil red 0 staining. En face preparation of descending
thoracic and abdominal aorta
[0069] Chicken egg albumin (Sigma) in a concentration of 0.8 g/ml
water was mixed 1:1 with glycerol. Sodium azide was added to make a
final concentration of sodium azide 0.2%. After descending thoracic
and abdominal aorta was cleaned off surrounding tissue and fat, the
segment of aorta from left subclavian artery to the level of renal
artery was then carefully removed for overnight fixation in
Histochoice (Amresco). Aorta was then carefully opened
longitudinally and placed with luminal side up on a slide freshly
coated with egg albumin solution. After albumin solution became
dry, the aorta was stained with Oil red O to assess the extent of
atherosclerosis with computer-assisted histomorphometry.
[0070] Immunohistochemistry and Histomorphometry. The sections from
aortic sinus were immunohistochemically stained with MOMA-2
antibody (Serotec) using standard protocol. Trichrome stain to
assess collagen content and oil red O stain for plaque size and
lipid content were done using standard staining protocol.
Computer-assisted morphometric analysis was performed to assess
histomorphometry as described previously..sup.8
[0071] Antibody titer measurement. To measure the antibody response
after peptide immunization, an ELISA was developed. Antibody titer
against immunized peptide was measured using blood collected at 2
weeks after booster and at sacrifice. Antibody response against 3
peptides was also determined in Alum group at the same time-points.
In brief, native synthetic peptides diluted in PBS pH 7.4 (20
.mu.g/ml) were absorbed to microtiter plate wells (Nunc MaxiSorp,
Nunc, Roskilde, Denmark) in an overnight incubation at 4.degree. C.
After washing with PBS containing 0.05% Tween-20 (PBS-T) the coated
plates were blocked with SuperBlock in TBS (Pierce) for 5 min at
room temperature followed by an incubation of mouse serum diluted
1/50 in TBS-0.05% Tween-20 (TBS-T) for 2 h at room temperature and
then overnight at 4.degree. C. After rinsing, deposition of
antibodies directed to the peptides was detected by using
biotinylated rabbit anti-mouse Ig antibodies (Dako A/S, Glostrup,
Denmark) appropriately diluted in TBS-T. After another incubation
for 2 h at room temperature the plates were washed and the bound
biotinylated antibodies were detected by alkaline phosphatase
conjugated streptavidin (Sigma), incubated for 2 h at room
temperature. Using phosphatase substrate kit (Pierce) developed the
colour reaction and the absorbance at 405 nm was measured after 1 h
of incubation at room temperature. Mean values were calculated
after the background was subtracted.
[0072] Other assay models is of course applicable as well, such any
immunoassay detecting an antibody, such as radioactive immunoassay,
Western blotting, and Southern blotting, as well as detection of
antibodies bound to peptides, enzyme electrodes and other methods
for analysis.
Statistics
[0073] Data are presented as mean.+-.Std. Statistical method used
is listed in either text, table or figure legend. P<0.05 was
considered as statistically significant.
TABLE-US-00004 TABLE A Aortic sinus plaque size and its lipid
content, aortic arch plaque size and percent of plaque in
descending aorta. Total plaque Oil red O (+) area Aortic arch % of
plaque size in aortic (% of aortic plaque size in aorta sinus
(mm.sup.2) sinus plaque) (mm.sup.2) (flat prep.) Alum 0.49 .+-.
0.13 21.7 .+-. 4.4 0.057 .+-. 0.040 20 .+-. 4.7 Peptide 1 0.48 .+-.
0.14 32.0 .+-. 8.1 0.054 .+-. 0.027 17 .+-. 4.3 Peptide 301 0.46
.+-. 0.16 23.8 .+-. 4.1 0.050 .+-. 0.024 8.9 .+-. 2.2* *Significant
different from Alum group. ANOVA followed by Tukey-Kramer test was
used for statistical analysis.
[0074] Further data on the effect of immunization with
apolipoprotein B-100 peptide sequences on atherosclerosis in apo E
knockout mice is given below in Table B
TABLE-US-00005 TABLE B Effect of immunization with apolipoprotein
B-100 peptide sequences on atherosclerosis in apo E knockout mice
Effect on atherosclerosis in the aorta Immunizations using mixtures
of several peptide sequences 1. Peptide sequences 143 and 210
-64.6% 2. Peptide sequences 11, 25 and 74 -59.6% 3. Peptide
sequences 129, 148 and 167 -56.8% 4. Peptide sequences 99, 100,
102, 103 and 105 -40.1% 5. Peptide sequences 30, 31, 32, 33 and 34
+6.6% 6. Peptide sequences 10, 45, 154, 199 and 240 +17.8%
Immunizations using a single peptide sequence 1. Peptide sequence 2
-67.7% 2. Peptide sequence 210 -57.9% 3. Peptide sequence 301
-55.2% 4. Peptide sequence 45 -47.4% 5. Peptide sequence 74 -31.0%
6. Peptide sequence 1 -15.4% 7. Peptide sequence 240 0%
[0075] Administration of the peptides is normally carried by
injection, such as subcutaneous injection, intravenous injection,
intramuscular injection or intraperitoneal injection. A first
immunizing dosage can be 1 to 100 mg per patient depending on body
weight, age, and other physical and medical conditions. In
particular situations a local administration of a solution
containing one or more of the peptides via catheter to the coronary
vessels is possible as well. Oral preparations may be contemplated
as well, although particular precautions must be taken to admit
absorption into the blood stream. An injection dosage may contain
0.5 to 99.5% by weight of one or more of the fragments or peptides
of the present invention.
[0076] The peptides are normally administered as linked to
cationized bovine serum albumine, and using aluminium hydroxide as
an adjuvant. Other adjuvants known in the art can be used as
well.
[0077] Solutions for administration of the peptides shall not
contain any EDTA or antioxidants.
[0078] The peptides can also be used as therapeutic agents in
patients already suffering from an atheroschlerosis. Thus any
suitable administration route can be used for adding one or more of
the fragments or peptides of the invention.
[0079] Initial studies focused on determining which type of
oxidative modifications of peptides led to recognition by
antibodies in human plasma. These studies were done using peptides
1-5 and 297-302. During oxidation of LDL polyunsaturated fatty
acids in phospholipids and cholesteryl esters undergo peroxidation
leading to formation of highly reactive breakdown products, such as
malone dialdehyde (MDA). MDA may then form covalent adducts with
lysine and histidine residues in apo B-100 making them highly
immunogenic. Oxidation of LDL also results in fragmentation of apo
B-100 that may lead to exposure of peptide sequences not normally
accessible for the immune system. In these experiments peptides
were used in their native state, after MDA modification or after
incorporation into phospholipid liposomes followed by copper
oxidation or MDA-modification. IgM antibodies were identified
against native, MDA- and liposome oxidized peptides, with antibody
titers MDA-peptide>MDA-modified liposome peptides>liposome
oxidized peptide>native peptide. Specificity testing
demonstrated that binding of antibodies to MDA-modified peptides
was competed by both MDA-LDL and copper oxidized LDL.
[0080] We then performed a screening of the complete peptide
library using pooled plasma derived from healthy control subjects
and native and MDA-modified peptides as antigens. Antibodies to a
large number of sites in apo B-100 were identified. Using twice the
absorbance of the background control as positive titer cut off,
antibodies were detected against 102 of the 302 peptides
constituting the complete apo B-100 sequence. IgM binding was
substantially more abundant than that of IgG. Generally, binding
was higher to MDA modified peptide sequences than to the
corresponding native sequence, but these was a striking correlation
between the two. Binding to both native and MDA modified sequences
was competed by addition of MDA-modified LDL and copper oxidized
LDL, but not by native LDL. These observations suggest that immune
responses against MDA-modified peptide sequences in apo B-100
results in a cross reactivity against native sequences. The
inability of native LDL to compete antibody binding to native apo
B-100 peptide sequences is intriguing, but may indicate that these
sequences only become exposed after the proteolytic degradation of
apo B-100 that occurs as a result of LDL oxidation. Both
hydrophilic and hydrophobic parts of the molecule were recognized
by antibodies. A second screening of the apo B-100 peptide library
was performed using pooled plasma from subjects with clinical signs
of coronary heart disease (CHD, acute myocardial infarction (AMI)
and unstable angina; n=10). Antibodies in pooled CHD plasma bound
to the same sequences and with the same overall distribution as for
antibodies in healthy control plasma. However, antibody titers to
several peptides (#1, 30-34, 100, 107, 148, 149, 162, 169, 236, 252
and 301) were at least twice as high as in control plasma compared
to plasma from CHD subjects, whereas titers against a few peptides
(#10, 45, 111, 154, 199, 222 and 240) were higher in plasma from
CHD patients compared to controls. We then performed a prospective
clinical study to investigate if antibody levels against
MDA-modified peptide sequences in apo B-100 predict risk for
development of CHD. Using a nested case control design we selected
78 subjects with coronary events (AMI or death due to CHD) and 149
controls from the Malmo Diet Cancer Study. Neither cases nor
control individuals had a history of previous MI or stroke. The
median time from inclusion to the acute coronary event was 2.8
years (range 0.1-5.9 years) among cases. Antibody levels were
determined in baseline plasma samples supplemented with
antioxidants. Using the carotid intima-media thickness (IMT) as
assessed by ultrasonography at baseline we also analyzed
associations between antibody levels and degree of existing
vascular disease. We studied 8 MDA-modified peptide sequences that
in the initial screening studies were associated with high plasma
antibody levels (#74, 102 and 210) and/or marked differences
between control and CHD plasma pools (#32, 45, 129, 162 and 240).
Controls were found to have higher IgM levels against MDA peptide
74 (0.258, range 0-1.123 absorbance units versus 0.178, range
0-0.732 absorbance units, p<0.05), otherwise there were no
differences in antibody levels between cases and controls.
Associations between IMT and IgM against MDA-peptides #102, 129,
and 162 (r=0.233, 0.232, and 0.234, respectively, p<0.05) were
observed in cases and between IMT and MDA-peptide 45 (r=0.18,
p<0.05) in controls. Weak correlations were observed between
antibodies to MDA peptide 129 and total and LDL cholesterol (r=0.19
and r=0.19, p<0.01, respectively), otherwise peptide antibody
levels showed no associations with total plasma cholesterol, LDL
cholesterol, HDL cholesterol or plasma triglycerides. There were
strong co-variations between antibody levels to the different
peptides (r values ranging from 0.6 to 0.9). The only exception was
antibodies against MDA-peptide 74 that were weakly or not at all
related to antibodies against the other peptides.
[0081] Antibodies against all sequences except MDA-peptide 74 was
inversely associated with age among cases (r values ranging from
-0.38 to -0.58, p<0.010.001), but not in controls. Plasma levels
of oxidized LDL, in contrast, increased with age. Again this
association was stronger in cases than in controls. To investigate
if the associations between immune responses against MDA-modified
peptide sequences and cardiovascular disease were different in
different age groups a subgroup analysis was performed on cases and
controls under and above the median age (61 years). In the younger
age group cases had increased antibody levels against peptides 32
and 45 and decreased antibody levels against peptide 74 as compared
to controls, whereas no differences were seen in the older age
group. Antibodies against all MDA peptide sequences, except peptide
74, were significantly associated with IMT in the younger age
group, but not in the older (Table).
[0082] These studies identify a number of MDA-modified sequences in
apo B-100 that are recognized by human antibodies. MDA-modification
of apo B-100 occurs as a result of LDL oxidation indicating that
these antibodies belong to the family of previously described
oxidized LDL autoantibodies. This notion is also supported by the
observation that antibody binding to MDA-modified apo B-100
peptides is competed by addition of oxidized LDL. Together with the
oxidized phospholipids identified by Horkko et al, these
MDA-modified peptide sequences are likely to constitute the large
majority of antigenic structures in oxidized LDL. In similarity
with the oxidized LDL antiphospholipid antibodies, antibodies
against MDA-modified apo B-100 sequences were of IgM type. This may
suggest that also the latter antibodies belong to the family of T
15 natural antibodies. T 15 antibodies have been attributed an
important role in the early, T cell independent defense against
bacterial infections as well as in the removal of apoptotic cells.
It remains to be determined if the MDA-peptide antibodies described
here have similar functions. Antibodies were also identified
against a large number of native apo B-100 sequences. However, the
striking co-variation between antibodies to native and MDA-modified
sequences suggests that also these antibodies are formed in
response to LDL oxidation. It is also possible that antibodies
against an MDA-modified peptide sequence cross reacts with the
corresponding native sequence. If antibodies against native apo
B-100 sequences bind also to native LDL particles this is likely to
have a major influence on LDL metabolism. However, the finding that
native LDL does not compete antibody binding to native apo B-100
sequences, as well as the lack of correlation between antibodies
against native apo B-100 sequences and LDL cholesterol levels
against the existence of such a phenomena.
[0083] Antibodies against MDA-modified peptide sequences decreased
progressively with age in the cases, but not in the controls. With
the exception of MDA-peptide 74, IgM antibodies against
MDA-peptides were significantly associated with carotid IMT in the
younger age group (below 62 years), but not in the older age group.
These findings suggest that significant changes in the interactions
between the immune system and the atherosclerotic vascular wall
takes place between ages 50 and 70 years. One possibility is that
in younger individuals the atherosclerotic disease process is at a
more active stage with a more prominent involvement of immune
cells. Another possibility is that the decreased levels of
antibodies against MDA-modified peptide sequences in older subjects
reflect a senescence of the immune cells involved in
atherosclerosis. An impaired function of immune cells due to
immunosenescence have been proposed to contribute to an increased
susceptibility to infection and cancer in the older population.
Interestingly, immunosenescence is inhibited by antioxidants
indicating involvement of oxidative stress. Immune cells that
interact with epitopes in oxidized LDL are likely to be
particularly exposed to oxidative stress. Since oxidized LDL is
present in arteries already at a very early age these immune
response are being continuously challenged for several decades,
which may further contribute to a development of
immunosenescence.
[0084] Increased antibodies against two sites in apo B-100 were
found to predict risk for myocardial infarction and coronary death
in subjects below 62 years of age. Antibodies against these sites
showed a high level of co-variation suggesting that they were
produced in response to the same underlying pathophysiological
processes. The fact that the median time from blood sampling to
coronary event was only 2.8 years makes these antibodies
particularly interesting as makers for increased CHD risk. Antibody
levels against MDA-modified apo B-100 peptide sequences showed no
associations with other CHD risk factors such as hyperlipidemia,
hypertension and diabetes suggesting that they are independent
markers of CHD risk. The CHD cases in the present study were not
extremely high-risk individuals and in this respect representative
of the common CHD patient. The finding that IgM against
MDA-modified apo B-100 sequences predicts short-term risk for
development of acute coronary events in individuals that would not
have been identified as high risk by screening of established risk
factors suggest that it may become a useful instrument in
identifying individuals in need of aggressive preventive treatment.
However, considerably larger prospective studies with multivariate
analysis are required before the clinical value of determining
antibodies against apo B-100 MDA-modified peptide sequences can be
fully established. Another limitation of the present clinical study
is that we have only analysed antibodies against a small number of
the antigenic sites in apo B-100 and that antibody titers against
other sites may be even better markers of cardiovascular risk.
[0085] In subjects below age 60 antibodies against a large number
of MDA-modified sites in apo B-100 were correlated with the extent
of existing vascular disease as assessed by carotid IMT. IgM
antibodies were more closely associated with carotid IMT than IgG
antibodies. Although carotid IMT has obvious limitations as a
measure of general atherosclerotic burden these observations still
suggest that determination of IgM against MDA-modified sequences in
apo B-100 may be one method to assess the severity of existing
atherosclerosis. These observations are also in line with several
previous studies that have reported associations between coronary
and carotid artery disease and IgM antibodies against oxidized
LDL.
[0086] Antibodies against peptide 74 differed against other apo
B-100 peptide antibodies in many respect. They were higher in
controls than in cases, they did not decrease with age and were not
associated with the extent of carotid disease. Accordingly,
antibodies against this peptide sequence represent interesting
candidates for an athero-protective immune response.
[0087] An important question is why these associations occur. They
clearly demonstrate that immune responses against MDA-modified apo
B-100 sites somehow are involved in the atherosclerosic disease
process. Since high antibody levels are associated with more severe
atherosclerosis and increased risk for development of acute
coronary events one obvious possibility is that these immune
responses promote atherogenesis. Studies demonstrating that immune
responses against heat shock proteins, such as HSP 65, are
atherogenic provide some support for this notion. However,
experimental animal studies have shown an athero-protective effect
of oxidized LDL immunization. B cell reconstitution of spleen
ectomized apo E null mice results in a decrease in atherosclerosis.
Reduced atherosclerosis has also been observed in apo E null mice
given repeated injections of immunoglobulin. The present
observations do not necessarily argue against an athero-protective
role of immune responses against oxidized LDL. These immune
responses are activated by pro-atherogenic processes such as LDL
oxidation. Accordingly, they are also likely to be in proportion to
the severity of the disease process and could serve as makers of
disease severity and CHD risk without contributing to disease
progression. The finding that immunization of apo E null mice with
apo B-100 peptide sequences inhibits development of atherosclerosis
reported in two accompanying papers demonstrates that this is
likely to be the case. Indeed, the most important outcome of the
present study may well be the identification of structures that
could be used as components of a vaccine against atherosclerosis.
The observation that the decrease in antibodies against
MDA-modified peptide sequences in apo B-100 that occurs with age is
accompanied by an increase in plasma levels of oxidized LDL suggest
that an increased clearance of minimally oxidized LDL from the
circulation may be one mechanism by which these antibodies could
protect against atherosclerosis.
Methods
Study Population
[0088] The study subjects, borr between 1926-45, belong to the
Malmo "Diet and Cancer (MDC)" study cohort. A random 50% of those
who entered the MDC study between November 1991 and February 1994
were invited to take part in a study on the epidemiology of carotid
artery disease. Routines for ascertainment of information on
morbidity and mortality following the health examination, as well
as definition of traditional risk factors, have been reported.
[0089] Eighty-five cases of acute coronary heart events, i.e. fatal
or non-fatal MI or deaths due to coronary heart disease (CHD) were
identified. Participants who had a history of myocardial infarction
or stroke (n=6) were not eligible for the present study. For each
case two controls without a history of myocardial infarction or
stroke was individually matched for age, sex, smoking habits,
presence of hypertension and month of participation in the
screening examination and duration of follow-up. Due to logistic
reason (blood samples were not available in sufficient quantity for
assessment of peptides) only one control was available for seven
cases and no controls for one case. This case was excluded from
analysis. Thus the study population consists of 227 subjects, 78
cases and 149 controls, aged 49-67 (median 61) years at
baseline.
Laboratory Analyses
[0090] After overnight fasting blood samples were drawn for the
determination of serum values of total cholesterol, triglycerides,
HDL cholesterol, LDL cholesterol and whole blood glucose. LDL
cholesterol in mmol/L was calculated according to the Friedewald
formula. Oxidized LDL was measured by ELISA (Mercordia).
B-Mode Ultrasound Vasculography
[0091] An Acuson 128 Computed Tomography System (Acuson, Mountain
View, Calif.) with a MHz transducer was used for the assessment of
carotid plaques in the right carotid artery as described
previously.
Development of ELISAs Against Apo B-100 Peptide Sequences
[0092] The 302 peptides corresponding to the entire human
apolipoprotein B amino acid sequence were synthesized
(Euro-Diagnostica AB, Malmo, Sweden and K J Ross Petersen A S,
Horsholm, Denmark) and used in ELISA. A fraction of each synthetic
peptide was modified by 0.5 M MDA (Sigma-Aldrich Sweden AB,
Stockholm, Sweden) for 3 h at 37.degree. C. and in presence of
liposomes by 0.5 M MDA for 3 h at 37.degree. C. or by 5 mM
CUCl.sub.2 (Sigma) for 18 h at 37.degree. C. The MDA modified
peptides were dialysed against PBS containing 1 mM EDTA with
several changes for 18 h at 4.degree. C. The modification of the
peptides was tested in denatured polyacrylamide gels (BioRad
Laboratories, Hercules, Calif.), suitable for separation of
peptides.
[0093] A mixture of egg phosphatidylcholine (EPC) (Sigma) and
phosphatidylserine (PS) (Sigma) in a chloroform solution at a molar
ratio of 9:1 and a concentration of 3 mM phospholipid (PL) was
evaporated in a glass container under gentle argon stream. The
container was then placed under vacuum for 3 hours. A solution
containing 0.10 mM peptide (5 ml) in sterile filtered 10 mM HEPES
buffer pH 7.4, 145 mM NaCl and 0.003% sodium azide was added to the
EPC/PS dried film and incubated for 15 min at 50.degree. C. The
mixture was gently vortex for about 5 min at room temperature and
then placed in ice-cold water bath and sonicated with 7.5 amplitude
microns for 3.times.3 min (Sonyprep 150 MSE Sanyo, Tamro-Medlab,
Sweden) with 1 min interruptions. The PL-peptide mixture, native or
modified by 0.5 M MDA for 311 at 37.degree. C. or 5 mM CUCl.sub.2
for 18 h at 37.degree. C., was stored under argon in glass vials at
4.degree. C. wrapped in aluminum foil and used within 1 week. The
MDA-modified mixture was dialyzed against PBS containing 1 mM EDTA
with several changes for 18 h at 4.degree. C. before storage. The
modification of the mixture was tested in denatured polyacrylamide
gels (BioRad Laboratories AB; Sundbyberg, SE), suitable for
separation of peptides.
[0094] Native or modified synthetic peptides diluted in PBS pH 7.4
(20 leg/ml), in presence or absence of liposomes, were absorbed to
microtiter plate wells (Nunc MaxiSorp, Nunc, Roskilde, Denmark) in
an overnight incubation at 4.degree. C. As a reference, one of the
peptides (P6) was ran on each plate. After washing with PBS
containing 0.05% Tween-20 (PBS-T) the coated plates were blocked
with SuperBlock in TBS (Pierce, Rockford, Ill.) for 5 min at room
temperature followed by an incubation of pooled human plasma,
diluted 1/100 in TBS-0.05% Tween-20 (TBS-T) for 2 h at room
temperature and then overnight at 4.degree. C. After rinsing,
deposition of auto-antibodies directed to the peptides were
detected by using biotinylated rabbit anti-human IgG- or
IgM-antibodies (Dako A/S, Glostrup, Denmark) appropriately diluted
in TBS-T. After another incubation for 2 h at room temperature the
plates were washed and the bound biotinylated antibodies were
detected by alkaline phosphatase conjugated streptavidin (Sigma),
incubated for 2 h at room temperature. The color reaction was
developed by using phosphatase substrate kit (Pierce) and the
absorbance at 405 nm was measured after 1 h of incubation at room
temperature. The absorbance values of the different peptides were
divided with the absorbance value of P6 and compared.
Statistics
[0095] SPSS was used for the statistical analyses. The results are
presented as median and range and as proportions when appropriate.
Boxplot and scatterplots were used till illustrate the relationship
between age and selected peptides among cases and corresponding
controls. Corresponding graphs were also used to illustrate the
relationship between age and selected peptides, cases and controls,
respectively, below and above the median age (61 year) at baseline
and separately for cases and controls below the median age. In
cases and controls, separately, partial correlation coefficients,
adjusted for age and sex, were computed between selected peptides
and blood lipid levels and common carotid IMT. Age- and sex
adjusted partial correlation coefficients were also computed
between common carotid IMT and selected peptides in cases and
controls below and over the median age. An independent sample
t-test was used to assess normally distributed continuous variables
and a Chi-square test for proportions between cases and controls.
Non-parametric test (Mann-Whitney) was used to assess non-normally
distributed continuous variables between cases and controls. All
p-values are two-tailed.
TABLE-US-00006 TABLE Age- and sex adjusted correlation coefficient
for different baseline MDA peptides and common carotid artery
intima-media thickness among younger (49-61 years) and older (62-67
years) cases with myocardial infarction and their corresponding
controls matched for age, sex, smoking and hypertension. CASES plus
CONTROLS CASES plus CONTROLS PEPTIDE Aged 49-61 year, n = 116 Aged
62-67 year, n = 111 IGM MDA 32 0.235t -0.101 MDA 45 0.366$ -0.030
MDA 74 0.178 0.063 MDA 102 0.255$ -0.039 MDA 129 0.330$ -0.009 MDA
162 0.2451 0.001 MDA 210 0.254 0.013 MDA 240 0.284$ 0.006 IGG MDA
215 0.119 -0.059 p < 0.05; $/x0.01
Sequence CWU 1
1
38120PRTHuman 1Phe Leu Asp Thr Val Tyr Gly Asn Cys Ser Thr His Phe
Thr Val Lys1 5 10 15Thr Arg Lys Gly 20220PRTHuman 2Pro Gln Cys Ser
Thr His Ile Leu Gln Trp Leu Lys Arg Val His Ala1 5 10 15Asn Pro Leu
Leu 20320PRTHuman 3Val Ile Ser Ile Pro Arg Leu Gln Ala Glu Ala Arg
Ser Glu Ile Leu1 5 10 15Ala His Trp Ser 20420PRTHuman 4Lys Leu Val
Lys Glu Ala Leu Lys Glu Ser Gln Leu Pro Thr Val Met1 5 10 15Asp Phe
Arg Lys 20520PRTHuman 5Leu Lys Phe Val Thr Gln Ala Glu Gly Ala Lys
Gln Thr Glu Ala Thr1 5 10 15Met Thr Phe Lys 20620PRTHuman 6Asp Gly
Ser Leu Arg His Lys Phe Leu Asp Ser Asn Ile Lys Phe Ser1 5 10 15His
Val Glu Lys 20720PRTHuman 7Lys Gly Thr Tyr Gly Leu Ser Cys Gln Arg
Asp Pro Asn Thr Gly Arg1 5 10 15Leu Asn Gly Glu 20820PRTHuman 8Arg
Leu Asn Gly Glu Ser Asn Leu Arg Phe Asn Ser Ser Tyr Leu Gln1 5 10
15Gly Thr Asn Gln 20920PRTHuman 9Ser Leu Thr Ser Thr Ser Asp Leu
Gln Ser Gly Ile Ile Lys Asn Thr1 5 10 15Ala Ser Leu Lys
201020PRTHuman 10Thr Ala Ser Leu Lys Tyr Glu Asn Tyr Glu Leu Thr
Leu Lys Ser Asp1 5 10 15Thr Asn Gly Lys 201120PRTHuman 11Asp Met
Thr Phe Ser Lys Gln Asn Ala Leu Leu Arg Ser Glu Tyr Gln1 5 10 15Ala
Asp Tyr Glu 201220PRTHuman 12Met Lys Val Lys Ile Ile Arg Thr Ile
Asp Gln Met Gln Asn Ser Glu1 5 10 15Leu Gln Trp Pro 201320PRTHuman
13Ile Ala Leu Asp Asp Ala Lys Ile Asn Phe Asn Glu Lys Leu Ser Gln1
5 10 15Leu Gln Thr Tyr 201420PRTHuman 14Lys Thr Thr Lys Gln Ser Phe
Asp Leu Ser Val Lys Ala Gln Tyr Lys1 5 10 15Lys Asn Lys His
201520PRTHuman 15Glu Glu Glu Met Leu Glu Asn Val Ser Leu Val Cys
Pro Lys Asp Ala1 5 10 15Thr Arg Phe Lys 201620PRTHuman 16Gly Ser
Thr Ser His His Leu Val Ser Arg Lys Ser Ile Ser Ala Ala1 5 10 15Leu
Glu His Lys 201720PRTHuman 17Ile Glu Asn Ile Asp Phe Asn Lys Ser
Gly Ser Ser Thr Ala Ser Trp1 5 10 15Ile Gln Asn Val 201820PRTHuman
18Ile Arg Glu Val Thr Gln Arg Leu Asn Gly Glu Ile Gln Ala Leu Glu1
5 10 15Leu Pro Gln Lys 201920PRTHuman 19Glu Val Asp Val Leu Thr Lys
Tyr Ser Gln Pro Glu Asp Ser Leu Ile1 5 10 15Pro Phe Phe Glu
202020PRTHuman 20His Thr Phe Leu Ile Tyr Ile Thr Glu Leu Leu Lys
Lys Leu Gln Ser1 5 10 15Thr Thr Val Met 202120PRTHuman 21Leu Leu
Asp Ile Ala Asn Tyr Leu Met Glu Gln Ile Gln Asp Asp Cys1 5 10 15Thr
Gly Asp Glu 202220PRTHuman 22Cys Thr Gly Asp Glu Asp Tyr Thr Tyr
Lys Ile Lys Arg Val Ile Gly1 5 10 15Asn Met Gly Gln 202320PRTHuman
23Gly Asn Met Gly Gln Thr Met Glu Gln Leu Thr Pro Glu Leu Lys Ser1
5 10 15Ser Ile Leu Lys 202420PRTHuman 24Ser Ser Ile Leu Lys Cys Val
Gln Ser Thr Lys Pro Ser Leu Met Ile1 5 10 15Gln Lys Ala Ala
202520PRTHuman 25Ile Gln Lys Ala Ala Ile Gln Ala Leu Arg Lys Met
Glu Pro Lys Asp1 5 10 15Lys Asp Gln Glu 202620PRTHuman 26Ser Leu
Asn Ser His Gly Leu Glu Leu Asn Ala Asp Ile Leu Gly Thr1 5 10 15Asp
Lys Ile Asn 202720PRTHuman 27Trp Ile Gln Asn Val Asp Thr Lys Tyr
Gln Ile Arg Ile Gln Ile Gln1 5 10 15Glu Lys Leu Gln 202820PRTHuman
28Thr Tyr Ile Ser Asp Trp Trp Thr Leu Ala Ala Lys Asn Leu Thr Asp1
5 10 15Phe Ala Glu Gln 202920PRTHuman 29Glu Ala Thr Leu Gln Arg Ile
Tyr Ser Leu Trp Glu His Ser Thr Lys1 5 10 15Asn His Leu Gln
203020PRTHuman 30Ala Leu Leu Val Pro Pro Glu Thr Glu Glu Ala Lys
Gln Val Leu Phe1 5 10 15Leu Asp Thr Val 203120PRTHuman 31Ile Glu
Ile Gly Leu Glu Gly Lys Gly Phe Glu Pro Thr Leu Glu Ala1 5 10 15Leu
Phe Gly Lys 203220PRTHuman 32Ser Gly Ala Ser Met Lys Leu Thr Thr
Asn Gly Arg Phe Arg Glu His1 5 10 15Asn Ala Lys Phe 203320PRTHuman
33Asn Leu Ile Gly Asp Phe Glu Val Ala Glu Lys Ile Asn Ala Phe Arg1
5 10 15Ala Lys Val His 203420PRTHuman 34Gly His Ser Val Leu Thr Ala
Lys Gly Met Ala Leu Phe Gly Glu Gly1 5 10 15Lys Ala Glu Phe
203520PRTHuman 35Phe Lys Ser Ser Val Ile Thr Leu Asn Thr Asn Ala
Glu Leu Phe Asn1 5 10 15Gln Ser Asp Ile 203620PRTHuman 36Phe Pro
Asp Leu Gly Gln Glu Val Ala Leu Asn Ala Asn Thr Lys Asn1 5 10 15Gln
Lys Ile Arg 203721PRTHuman 37Arg Asn Leu Gly Glu Ser Asn Leu Arg
Phe Asn Ser Ser Tyr Leu Leu1 5 10 15Gln Gly Thr Asn Gln
203820PRTHuman 38Ala Leu Leu Val Pro Pro Glu Thr Glu Glu Ala Lys
Gln Val Leu Phe1 5 10 15Leu Asp Thr Val 20
* * * * *