IE83756B1 - Apo AI polypeptides, antibodies, and immunoassays - Google Patents
Apo AI polypeptides, antibodies, and immunoassaysInfo
- Publication number
- IE83756B1 IE83756B1 IE1991/1922A IE192291A IE83756B1 IE 83756 B1 IE83756 B1 IE 83756B1 IE 1991/1922 A IE1991/1922 A IE 1991/1922A IE 192291 A IE192291 A IE 192291A IE 83756 B1 IE83756 B1 IE 83756B1
- Authority
- IE
- Ireland
- Prior art keywords
- apo
- polypeptide
- antibody
- amino acid
- acid residue
- Prior art date
Links
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- APFVFJFRJDLVQX-UHFFFAOYSA-N indium Chemical compound [In] APFVFJFRJDLVQX-UHFFFAOYSA-N 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000000977 initiatory Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- PNDPGZBMCMUPRI-UHFFFAOYSA-N iodine Chemical compound II PNDPGZBMCMUPRI-UHFFFAOYSA-N 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 238000001155 isoelectric focusing Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 230000000670 limiting Effects 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 230000002934 lysing Effects 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic Effects 0.000 description 1
- 108010016686 methionyl-alanyl-serine Proteins 0.000 description 1
- 125000004492 methyl ester group Chemical group 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- PSZYNBSKGUBXEH-UHFFFAOYSA-N naphthalene-1-sulfonic acid Chemical compound C1=CC=C2C(S(=O)(=O)O)=CC=CC2=C1 PSZYNBSKGUBXEH-UHFFFAOYSA-N 0.000 description 1
- 230000010807 negative regulation of binding Effects 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 229920001220 nitrocellulos Polymers 0.000 description 1
- 230000036963 noncompetitive Effects 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 239000005022 packaging material Substances 0.000 description 1
- 235000019834 papain Nutrition 0.000 description 1
- 230000036961 partial Effects 0.000 description 1
- 229940111202 pepsin Drugs 0.000 description 1
- 239000008196 pharmacological composition Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000036470 plasma concentration Effects 0.000 description 1
- 229920001308 poly(aminoacid) Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 229920000573 polyethylene Polymers 0.000 description 1
- 229940057838 polyethylene glycol 4000 Drugs 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 229920000379 polypropylene carbonate Polymers 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- OZAIFHULBGXAKX-UHFFFAOYSA-N precursor Substances N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 239000003725 proteoliposome Substances 0.000 description 1
- 230000002797 proteolythic Effects 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 230000002829 reduced Effects 0.000 description 1
- 238000002310 reflectometry Methods 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- YOQDYZUWIQVZSF-UHFFFAOYSA-N sodium borohydride Substances [BH4-].[Na+] YOQDYZUWIQVZSF-UHFFFAOYSA-N 0.000 description 1
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 1
- ODGROJYWQXFQOZ-UHFFFAOYSA-N sodium;boron(1-) Chemical compound [B-].[Na+] ODGROJYWQXFQOZ-UHFFFAOYSA-N 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 229950000244 sulfanilic acid Drugs 0.000 description 1
- 229960000814 tetanus toxoid Drugs 0.000 description 1
- UEUXEKPTXMALOB-UHFFFAOYSA-J tetrasodium;2-[2-[bis(carboxylatomethyl)amino]ethyl-(carboxylatomethyl)amino]acetate Chemical compound [Na+].[Na+].[Na+].[Na+].[O-]C(=O)CN(CC([O-])=O)CCN(CC([O-])=O)CC([O-])=O UEUXEKPTXMALOB-UHFFFAOYSA-J 0.000 description 1
- 238000004809 thin layer chromatography Methods 0.000 description 1
- 229960000984 tocofersolan Drugs 0.000 description 1
- 239000000700 tracer Substances 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N trans-L-hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 125000005270 trialkylamine group Chemical group 0.000 description 1
- VOBHRQFELWTZFS-AWLRYRRCSA-K trisodium;(4Z)-3-oxo-4-[(4-sulfonatonaphthalen-1-yl)hydrazinylidene]naphthalene-2,7-disulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N/N=C3/C4=CC=C(C=C4C=C(C3=O)S(=O)(=O)[O-])S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 VOBHRQFELWTZFS-AWLRYRRCSA-K 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000001262 western blot Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 239000002076 α-tocopherol Substances 0.000 description 1
- 235000004835 α-tocopherol Nutrition 0.000 description 1
Classifications
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/06—Antihyperlipidemics
-
- 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
- C07K14/775—Apolipopeptides
-
- 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
Description
PATENTS ACT,
1922191
APO AI POLYPEPTIDES, ANTIBODIES, AND IMMUNOASSAYS
SCRIPPS CLINIC AND RESEARCH FOUNDATION
Description
Technical Field
The present invention relates to diagnostic
methods and polypeptides useful for immunologically
determining the amount of Apo AI in a vascular fluid
sample. In addition, the polypeptides are useful in
therapeutic methods and compositions for increasing
LCAT-mediated cholesterol esterification and the
formation of cholesterol esters in a human patient.
Background.
Lipoproteins are the primary carriers of plasma
cholesterol. They are micellar lipid—protein
complexes (particles) having a surface film,
comprised of one or more proteins associated with
polar lipids, that surrounds a cholesterol-containing
core. Lipoproteins were originally classified based
on their buoyant densities as.measured by
ultracentrifugation. Accordingly, four major density
classes have been recognized: chylomicrons, very low-
density lipoproteins (VLDL), low—density lipoproteins
LDL and high-density lipoproteins (HDL).
Many studies have now established an inverse
correlation between plasma HDL cholesterol levels and
That is,
elevated levels of plasma cholesterol found in HDL
risk of coronary artery disease (CAD).
particles correlate with a reduced risk of CAD. see,-
for example, Goldbourt et al., Int. J. Epidemiol.,
:51-55 (1986).
ssimilarly, many studies have now shown that
plasma levels of apolipoprotein AI (Apo AI), the
major protein component of HDL, are also inversely
related to the risk of CAD. In addition, Weisweiler
et al., Clin. Chem.,.27:348 (1981) have reported that
knowledge of Apo AI levels may add to the predictive
valve of HDL cholesterol.. I i
Because of its inverse correlation with CAD,
there has been an extensive amount of research into
the structure and function of Apo AI in lipid
metabolism. Functionally, Apo AI is now believed to
mediate the removal of cholesterol from tissues.
structurally, purified Apo AI has been described
as containing a high proportion (55%) of alpha-helix,
which increases to 70% when it is associated with
The lipid
binding properties of Apo AI appear to be a function
phospholipids as in the HDL particle.
of a series of tandemly repeated segments of 22 amino
acid residues punctuated mostly by proline residues
that are alpha—helical and amphophilic.
The amino acid residue sequence of Apo AI,
determined by Edman degradation of cyanogen bromide-
and trypsin—fragments of intact Apo AI, has been
described by Brewer et al., Biochem. Biophvs. Res.
QQmm;’ 80:623-630 (1978). According to Brewer et
al., cyanogen bromide (CNBr) cleavage of Apo AI
produced four major fragments, designated CNBrl,
CNBr2, CNBr3 and CNBr4, in order of their occurrence
along the Apo AI sequence from amino-terminus to
carboxy-terminus. Because it is of particular
interest to the present invention, the amino acid
residue sequence of the region of Apo AI from which
CNBr2 and CNBr3 is produced is illustrated in Figure
1.
Immunochemical characterization of native Apo AI,
i.e., Apo AI as it is found on HDL particles, has
been problematical because it is antigenically
heterogeneous and unstable. The antigenic
heterogeneity of Apo AI appears to be the result of
some epitopes being masked by lipids in the intact
HDL or the antibody—binding ability of some epitopes
being dependent on conformations of Apo AI as
affected by lipids or other HDL associated proteins.
The antigenic instability of Apo AI, as manifest by
its changing immunoreactivity over time with defined
mantisera, appears to be due to such phenomena as self
association and deamidation, both of which have been
shown to occur in vitro. See Curtiss et al.,
Proceeding of the Workshop on Lipoprotein
Heterogeneity, Ed. by Lippel, National Institutes of
Health Publication No. 87-2646, P. 363-377 (1987).
According to Milthorp et al., Arterio., 6:285—296
(1986), the effects of storage and NaOH treatment on
native Apo AI immunoreactivity are similar but not
analogous, suggesting that while loss of Apo AI
immunoreactivity during storage is due in large part
to deamidation, more may be involved.
The antigenic heterogeneity and instability of
Apo AI has made it difficult to produce assay systems
for quantifying Apo AI in patient vascular fluid
samples. This is because, inter alia, such systems
require a reference material (standard) whose
immunoreactivity for the system's primary anti—Apo AI
antibody is consistent, at the very least, and
preferably equivalent to that of the Apo AI in the
patient's sample.
Recently, efforts at overcoming problems
associated with the antigenic heterogenicity and
instability of Apo AI have focused on using
monoclonal antibodies (MAB) to identify epitopes on
native Apo AI whose expression is consistent or
"conserved" under specific isolation and storage
conditions. Such epitopes are referred to herein as
"conserved native epitopes".
An exemplary conserved native Apo AI epitope,
designated epitope A, has been defined by Milthorpe
et al., Arterio., 6:285—296 (1986) as being that
portion of Apo AI CNBr1 that immunoreacts with MAB
4H1. This was in contrast to epitopes designated C,
C‘ and C", all located in the CNBr2 region of Apo AI,
and all of which were found to be "nonconserved"
epitopes.
Monoclonal antibodies AI-4, and AI~11, and AI-18
have been identified as anti—Apo AI pan antibodies;
that is, antibodies that bind all or most species of
Apo AI-containing lipoprotein particles in plasma.
See Hogle et al., J. Lipid. Res., 29:1221-1229
(1988); and Curtiss et al., in "Biotechnology of
Dyslipoproteinemias: Clinical Applications in
Diagnosis and Control", Lenfant et al., eds, pp. 217-
226, Raven Press (New York), 1989.
specific epitopes on Apo AI with which antibodies
However, the
AI-4 and AI-ll immunoreact have not been identified.
Apo AI has also been shown to modulate
lecithin:cholesterol acyltransferase (LCAT)—mediated
cholesterol esterification.
al., Biochem. Biophvs. Acta, 793:l49-156 (1984) have
shown that an interaction between Apo AI and HDL is
Studies by Pownall et
required for LCAT activation that results in
cholesterol esterification. Moreover, specific
regions of Apo AI have been identified that activate
LCAT.
regions have been synthesized and tested for LCAT
Polypeptide fragments corresponding to these
activating potential. According to Sparrow et al.,
Ann. N.Y. Acad. SCi., 3841187-208 (1980), Apo AI
fragment residues 148-185 were involved in both
activation of LCAT and lipid binding. Similar studies of
Fukushima et al., J Biol. Chem. 255:10651—10657 (1980) have
shown that a synthetic polypeptide corresponding to Apo—AI
residues 121-164 was 30% as effective as native Apo AI in
the activation of cholesterol esterification.
More recent studies with synthetic model polypeptides
which mimic the physical properties of native App AI have
shown that LCAT activation results if the polypeptide
associates with HDL. Ponsin et al., Biochem, 23:5337-5342
(1984).
W089/04486 discloses Apo AI polypeptides consisting
essentially of no ‘more than 25 amino acid residues and
including the sequence -AKVQPYLDDFQ-.
said to be useful for determining the amount of Apo AI in a
Such polypeptides are
vascular fluid sample.
J. Biol. Chem. 263 (27), 13779-13785 (1988) discloses
a series of peptides derived from Apo AI which are said to
exhibit specific reactivity with HDL.
Brief Summary of the Invention
epitopes on Apo AI have now been discovered that
to LCAT-mediated
In addition, antibodies to the
define polypeptides useful modulate
cholesterol esterification.
polypeptides which are defined'by these epitopes are useful
in diagnostic methods for detecting Apo AI in vascular fluid
samples.
Thus, the present invention. provides a diagnostic
system, in kit form, comprising, in an amount sufficient to
perform at least one assay, an antibody containing anti—Apo
AI antibody molecules that immunoreact with:
(a) Apo AI/HDL,
(b) isolated Apo AI,
(c) Apo AI CNBr2, and
(d) the polypeptide KVQPYLDDFQKKWQEE, 'but do not
immunoreact with:
(e) Apo AI CNBr1,
(f) Apo AI CNBr3,
(g) the polypeptide SKDLEEVKAKVQPYLDDEQKKWQEE,
(h) the polypeptide SKDLEEVKAKVQPYLDDFQ, and
(i) the polypeptide PYLDDFQKKWQEEMELYRQKVEP.
Also provided by the present invention is a diagnostic
system in kit form, comprising, in an amount sufficient to
perform at least one assay, an antibody containing anti—Ap
AI antibody molecules that immunoreact with: i
(a) Apo AI/HDL,
(b) isolated Apo AI,
(c) Apo AI CNBr2-CNBr3, and
(d) the polypeptide PYLDDFQKKWQEEMEL,
immunoreact with:
(e) Apo AI CNBr1,
(f) the polypeptide SKDLEEVKAKVQPYLDDFQKKWQEE,
(g) the polypeptide LEEVKAKVQPYLDDFQKKWQEE, and
(h) the polypeptide YRQKVEPLRAEL.
but do not
Methods for assaying the amount of Apo AI in a vascular
fluid sample are.also provided by the present invention.
In addition, the invention provides Apo AI polypeptides
for use in increasing esterified cholesterol in a patient.
In one embodiment, such a polypeptide includes an amino acid
the
wherein X is either E or F.
residue formula
-PYLDDXQKKWQEEMEL-,
alternative embodiment,
sequence represented by
In an
an Apo AI polypeptide includes an
amino acid residue sequence represented by the formula
-AKVQPYLDDFQ-.
Brief Description of the Drawings
In the drawings forming a portion of this
disclosure:
Figure 1 illustrates the amino acid residue
sequence of Apo AI, as reported by Brewer et al.,
Biochem. Biophvs. Res. Comm., 80:623-630 (1978), from
amino acid residue positions 85 through 148 using the
Apo AI CNBr2, which is formed by
cleavage at the methionine (M) residues located at
single letter code.
positions 86 and 112, corresponds in sequence to
positions 87 through 111 with the carboxy terminal
methionine being converted to homoserine lactone.
Apo AI CNBr3, which is formed by cleavage at the
methionine (M) residues located at positions 112 and
148, corresponds in sequence to positions 113 through
147 with the carboxy terminal methionine being
converted to homoserine lactone.
The conserved native epitope on Apo AI that is
defined by the MAB AI-4 spans CNBr fragments 2 and 3
and is comprised of amino acid residues sequence 99-
114. The conserved native epitope on Apo AI that is
defined by the MAB AI-11 is confined to CNBr2 from
the amino acid residue sequence 96-111.
Figure 2 illustrates the ability of Apo AI/HDL,
HDL present in fresh plasma, and the polypeptide
AI96-111 to competitively inhibit MAB AI-11 from
immunoreacting with AI96-111. Protein concentrations
were determined according to the method of Markwell
et al., Anal. Biochem., 87:206-220 (1978).
on the X-axis vary linearly with respect to the three
The units
competitors used in the assay. The data points for
Apo AI/HDL correspond to a starting concentration of
1 mg/ml followed by 5 serial 2-fold dilutions. The
data points for HDL present in fresh plasma
correspond to a starting dilution of 1:10 followed by
Polypeptide AI96-111 is
diluted as above for plasma with a starting
serial 2-fold dilutions.
concentration of 1 mg/ml.
Figure 3 illustrates the ability of Apo AI/HDL
and the polypeptides AI94—125 and AI99-121 to
competitively inhibit MAB AI-4 from immunoreacting
with Apo AI/HDL. Polypeptides A190-111, AI93—1l1 and
AI96—111 are not competitive inhibitors.‘ The A
concentration of the competitor is shown in pg
protein/ml determined as described in Figure 2.
Figure 4 illustrates the amino acid residue
sequence of a portion of Apo AI as described in
Figure l. A conserved native epitope is defined by
MAB AI-4 and the epitope is immunologically mimicked
by the polypeptide AI99-121, which is shown as a bold
Polypeptides AI94-125 and AI99—12l, also in
bold, also immunoreacts with MAB AI-4 and mimics the
line.
epitope. Polypeptides derived from Apo AI which do
not immunoreact with MAB AI—4 nor mimic the epitope
are shown as thin lines. Alignments of the thin
lines with actual Apo AI sequences are approximate
with respect to the amino acid residue sequence of
Apo AI, and the actual span of amino acid residues
which comprise the thin line polypeptides are
designated numerically.
Figure 5 illustrates the ability of Apo AI/HDL
and the polypeptides AI90—ll1, AI93-lll, and AI96—lll
to competitively inhibit MAB AI~ll from
immunoreacting with Apo AI/HDL. Polypeptide AI99—12l
is not a competitive inhibitor.
Figure 6 illustrates the amino acid residue
sequence of a portion of Apo AI as described in
Figure l. A conserved native epitope is defined by
MAB AI-ll, and the epitope is immunologically
mimicked by polypeptide AI96-lll, which is shown as a
bold line. Polypeptides AI84-111, AI85-lll, AI90-
lll, AI93-lll, AI94-lll and A194-125, also in bold,
also immunoreact with MAB AI—ll and mimic the
epitope. Polypeptides derived from Apo AI which do
not immunoreact with MAB AI—l1 nor mimic the epitope
are shown as thin lines. Alignments of the
polypeptides which do not react with MAB AI-ll are as
described in Figure 4. '
Figure 7 illustrates the ability of MABS AI-4 and
AI-11 to inhibit LCAT-mediated cholesterol
esterification compared to PBS and MABs AI-9, AI-16
and AI-18.
Detailed Description of the Invention
A. Definitions
Amino Acid Residue: The amino acid residues
described herein are preferred to be in the "L"
isomeric form. However, residues in the "D" isomeric
form can be substituted for any L—amino acid residue,
as long as the desired functional property is
retained by the polypeptide. NH2 refers to the free
amino group present at the amino terminus of a
polypeptide. COOH refers to the free carboxy group
present at the carboxy terminus of a polypeptide. In
keeping with standard polypeptide nomenclature, Q;
Biol. Chem., 243:3552—59 (1969), abbreviations for
amino acid residues are shown in the following Table
of Correspondence:
l—Letter
G
F
M
A
S
I
L
T
V
P
K
H
Q
B
W
R
D
N
C
TABLE OF CORRESPONDENCE
SYMBOL
—Letter
Tyr
Gly
Phe
Met
Ala
Ser
Ile
A Thr
Val
Pro
Lys
His
Gln
Glu
Trp
Arg
Asp
Asn
AMINO ACID
tyrosine
glycine
phenylalanine
methionine
alanine
serine
isoleucine
leucine
threonine
valine
proline
lysine
histidine
glutamine
glutamic acid
tryptophan
arginine
aspartic acid
asparagine
cysteine
It should be noted that all amino acid residue
sequences are represented herein by formulae whose
left and right orientation is in the conventional
direction of amino-terminus to carboxy—terminus.
Furthermore, it should be noted that a dash at the
beginning or end of an amino acid residue sequence
indicates either a peptide bond to a further sequence
of one or more amino acid residues or a covalent bond
to a carboxyl or hydroxyl end group.
Apo AIgHD : Designates Apo AI when it is present
on HDL particles. '
Delipidated Apo AI: Refers to Apo AI that is
substantially free of associated lipids.
Isolated Apo AI: Designates Apo AI that is
substantially free of both associated lipids and
other proteins, such as those, like Apo AII, that are
typically found on HDL in addition to Apo AI.
Polypeptide and Peptide: Polypeptide and peptide
are terms used interchangeably herein to designate a
linear series of amino acid residues connected one to
the other by peptide bonds between the alpha-amino
and carboxy groups of adjacent residues.
Protein: Protein is a term used herein to
designate a linear series of greater than about 50
amino acid residues connected one to the other as in
a polypeptide.
Synthetic peptide: refers to a chemically
produced chain of amino acid residues linked together
by peptide bonds that is free of naturally occurring
proteins and fragments thereof.
B.Polypeptides
As used herein, the phrase "Apo AI polypeptide"
refers to a polypeptide whose amino acid residue
sequence corresponds, and preferably is identical to
a portion of the Apo AI molecule.
Preferred diagnostic kits of the present invention
comprise an Apo AI polypeptide having no more than about 25
amino acid residues, preferably no more than about 22
amino acid residues, and includes an amino acid‘
residue sequence represented by the formula:
-KVQPYLDDFQKKWQEE-.
In
preferred embodiments, the polypeptide has an amino
acid residue sequence represented by a formula
selected from the group consisting of:
QEMSKDLEEVKAKVQPYLDDFQKKWQEE,
EMSKDLEEVKAKVQPYLDDFQKKWQEE,
LEEVKAKVQPYLDDFQKKWQEE ,
VKAKVQPYLDDFQKKWQEE,
KAKVQPYLDDFQKKWQEE,
KAKVQPYLDDFQKKWQEEMELYRQKVEPLRAE, and
KVQPYLDDFQKKWQEE.
In another embodiment, an Apo AI
polypeptide comprises no more than about 60 amino
acid residues and includes an amino acid residue
sequence represented by the formula:
-PYLDDXQKKWQEEMEL—, wherein X is either E or F.
Preferably, the Apo AI polypeptide includes an amino
acid residue sequence represented by the formula: —
PYLDDXQKKWQEEMELYRQKVEP-, wherein X is either E or F.
In preferred embodiments, the polypeptide has an
amino acid residue represented by a formula selected
from the group consisting of:
KAKVQPYLDDXQKKWQEEMEL,
KAKVQPYLDDXQKKWQEEMELYRQKVEPLRAE,
QPYLDDXQKKWQEEMEL,
QPYLDDXQKKWQEEMELYRQKVEP,
PYLDDXQKKWQEEMEL, and
PYLDDXQKKWQEEMELYRQKVEP.
Preferred Apo AI polypeptides, their
designations, and their Apo AI amino acid residue
positions are shown in Table 1.
Table 1
Polypeptide
Designation Amino Acid Residue Seggence
AI84-111 QEMSKDLEEVKAKVQPYLDDFQKKWQEE
AI85-111 EMSKDLEEVKAKVQPYLDDFQKKWQEE
AI87-lll SKDLEEVKAKVQPYLDDFQKKWQEE
AI90-111 LEEVKAKVQPYLDDFQKKWQEE
AI93—1ll VKAKVQPYLDDFQKKWQEE
A194-111 KAKVQPYLDDFQKKWQEE
A194-1141 KAKVQPYLDDXQKKWQEEMEL
A194-1251 KAKVQPYLDDXQKKWQEEMELYRQKVEPLRAE
A196-111 KVQPYLDDFQKKWQEE
A198-114‘ QPYLDDXQKKWQEEMEL
AI98-1211 QPYLDDXQKKWQEEMELYRQKVEP
AI99—114‘ PYLDDXQKKWQEEMEL
AI99-1211 PYLDDXQKKWQEEMELYRQKVEP
An "X" at amino acid residue position 104 indicates that
either an E for glutamic acid or an F for phenylalanine is
present at residue position 104.
Preferably, an Apo AI polypeptide of this invention is
further characterized by its ability to immunologically
mimic an epitope (antigenic determinant) expressed by Apo
AI on substantially all HDL.
As used herein, the phrase "immunologically mimic" in
its various grammatical forms refers to the ability of an
Apo AI polypeptide of this invention to immunoreact with an
antibody of the present invention that recognizes a
conserved native epitope of Apo AI as defined herein.
It should be understood that a subject polypeptide need
not be identical to the amino acid residue sequence of Apo
AI, so long as it includes the required sequence and is
able to immunoreact with antibodies of the present
invention.
A subject polypeptide includes any analog, fragment or
chemical derivative of a polypeptide whose amino acid
residue sequence is shown herein so long as the polypeptide
is capable of immunologically mimicking an Apo AI native
conserved epitope. Therefore, a present polypeptide can be
subject to various changes, substitutions, insertions, and
deletions where such changes provide for certain advantages
in its use.
The term "analog" includes any polypeptide having an
amino acid residue sequence substantially identical to a
sequence specifically shown herein in which one or more
residues have been conservatively substituted with a
functionally similar residue and which displays the ability
to mimic Apo AI as described herein. Examples of
conservative substitutions include the substitution of one
non-polar (hydrophobic) residue such as isoleucine, valine,
leucine or methionine for another, the substitution of one
polar (hydrophilic) residue for another such as between
arginine and lysine, between glutamine and asparagine,
between glycine and serine, the substitution of one basic
residue such as lysine, arginine or histidine for another,
or the substitution of one acidic residue, such as aspartic
acid or glutamic acid for another.
The phrase "conservative substitution" also includes
the use of a chemically derivatized residue in place of a
non-derivatized residue provided that such polypeptide
displays the requisite binding activity.
"Chemical derivative" refers to a subject polypeptide
having one or more residues chemically derivatized by
reaction of a functional side group. Such derivatized
molecules include for example, those molecules in which
free amino groups have been derivatized to form amine
hydrochlorides, p—toluene sulfonyl groups, carbobenzoxy
groups, t—butyloxycarbonyl groups, chloroacetyl groups or
formyl groups. Free carboxyl groups may be derivatized to
form salts, methyl and ethyl esters or other types of
esters or hydrazides. Free hydroxyl groups may be
derivatized to form O—acyl or O—alkyl derivatives. The
imidazole nitrogen of histidine may be derivatized to form
N-im-benzylhistidine. Also included as chemical
derivatives are those peptides which contain one or more
naturally occurring amino acid derivatives of the twenty
standard amino acids. For examples: 4-hydroxyproline may
be substituted for proline; Svhydroxylysine may be
substituted for lysine; 3—methylhistidine may be
substituted for histidine; homoserine may be substituted
for serine; and ornithine may be substituted for lysine.
Polypeptides of the present invention also include any
polypeptide having one or more additions and/or deletions
or residues relative to the sequence of a polypeptide whose
sequence is shown herein, so long as the requisite activity
is maintained.
The term "fragment" refers to any subject polypeptide
having an amino acid residue sequence shorter than that of
a polypeptide whose amino acid residue sequence is shown
herein. X
When a polypeptide of the present invention has a
sequence that is not identical to the sequence of a Apo AI
because one or more conservative or non—conservative
substitutions have been made, usually no more than about 30
number percent, more usually no more than 20 number
percent, and preferably_no more than 10 number percent of
the amino acid residues are substituted, except that the
proline residue at position 99 cannot be substituted or
deleted where additional residues have been added at either
terminus for the purpose of providing a "linker" by which
the polypeptides of this invention can be conveniently
affixed to a label or solid matrix, or carrier, the linker
residues do not form Apo AI epitopes, i.e., are not similar
is structure to the Apo AI. Labels, solid matrices and
carriers that can be used with the polypeptides of this
invention are described hereinbelow.
Amino acid residue linkers are usually at least one
residue and can be 40 or more residues, more often 1 to 10
residues, but do not form Apo AI epitopes. Typical amino
acid residues used for linking are tyrosine, cysteine,
lysine, glutamic and aspartic acid, or the like. In
addition, a subject polypeptide can differ, unless
otherwise specified, from the natural sequence of Apo AI by
the sequence being modified by terminal-NH, acylation,
e.g., acetylation, or thioglycolic acid amidation, by
terminal—carboxlyamidation, e.g., with ammonia,
methylamine, and the like.
when coupled to a carrier to form what is known in the
art as a carrier-hapten conjugate, an Apo AI polypeptide of
the present invention is capable of inducing antibodies
that immunoreact with Apo AI, preferably Apo AI when it is
part of an HDL particle (Apo AI/HDL). In view of the well
established principle of immunologic cross—reactivity, the
present invention therefore contemplates antigenically
related variants of the/polypeptides shown in Table 1. An
"antigenically related variant" is a subject polypeptide
that is capable of inducing antibody molecules that
immunoreact with a polypeptide from Table 1 and Apo AI.
Any peptide of the present invention may be used in the
form of a pharmaceutically acceptable salt. Suitable acids
which are capable of forming salts with the peptides of the
present invention include inorganic acids such as
hydrochloric acid, hydrobromic acid, perchloric acid,
nitric acid, thiocyanic acid, sulfuric acid, phosphoric
acetic acid, propionic acid, glycolic acid, lactic acid,
pyruvic acid, oxalic acid, malonic acid, succinic acid,
maleic acid, fumaric acid, anthranilic acid, cinnamic acid,
naphthalene sulfonic acid, sulfanilic acid or the like.
Suitable bases capable of forming salts with the
peptides of the present invention include inorganic bases
such as sodium hydroxide, ammonium hydroxide, potassium
hydroxide and the like; and organic bases such as mono—,
di- and tri-alkyl and aryl amines (e.g. triethylamine,
diisopropyl amine, methyl amine, dimethyl amine and the
like) and optionally substituted ethanolamines (e.g.
ethanolamine, diethanolamine and the like).
An Apo AI polypeptide of the present invention also
referred to herein as a subject polypeptide, can be
synthesized by any of the techniques that are known to
those skilled in the polypeptide art, including recombinant
DNA techniques. Synthetic chemistry techniques, such as a
solid—phase Merrifield-type synthesis, are preferred for
reasons of purity, antigenic specificity, freedom from
undesired side products, ease of production and the like.
An excellent summary of the many techniques available can
be found in J.M. steward and J.D. Young, "Solid Phase
Peptide Synthesis", W.H. Freeman Co., San Francisco, 1969;
M. Bodanszky, et al., "Peptide Synthesis", John Wiley &
sons, Second Edition, 1976 and J. Meienhofer, "Hormonal
Proteins and Peptides”, Vol. 2, p. 46, Academic Press (New
York), 1983 for solid phase peptide synthesis, and E.
Schroder and K. Kubke, "The Peptides", Vol. 1, Academic
Press (New York), 1965 for classical solution synthesis,
each of which is incorporated herein by reference.
Appropriate protective groups usable in such synthesis are
described in the above texts and in J.F.W. Mcomie,
"Protective Groups in Organic Chemistry", Plenum Press, New
York, 1973, which is incorporated herein by reference.
In general, the solid-phase synthesis methods
contemplated comprise the sequential addition of one or
more amino acid residues or suitably protected amino acid
residues to a growing peptide chain. Normally, either the
amino or carboxyl group of the first amino acid residue is
protected by a suitable, selectively removable protecting
group. A different, selectively removable protecting group
is utilized for amino acids containing a reactive side
group such as lysine.
Using a solid phase synthesis as exemplary, the
protected or derivatized amino acid is attached to an inert
solid support through its unprotected carboxyl or amino
group. The protecting group of the amino or carboxyl group
is then selectively removed and the next amino acid in the
sequence having the complementary (amino or carboxyl) group
suitably protected is admixed and reacted under conditions
suitable for forming the amide linkage with the residue
already attached to the solid support. The protecting
group of the amino or carboxyl group is then removed from
this newly added amino acid residue, and the next amino
acid (suitably protected) is then added, and so forth.
After all the desired amino acids have been linked in the
proper sequence, any remaining terminal and side group
protecting groups (and solid support) are removed
sequentially or concurrently, to afford the final
polypeptide.
An Apo AI polypeptide can be used, inter alia, in the
diagnostic methods and systems of the present invention to
. detect Apo AI present in a body sample, or can be used to
prepare an inoculum as described herein for the preparation
of antibodies that immunoreact with conserved epitopes on
Apo AI.
In addition, an Apo AI polypeptide of this invention
can be used in the therapeutic methods of the present
invention to increase esterification of cholesterol in a
patient.
C. Antibodies and Monoclonal Antibodies
The term "antibody" in its various grammatical forms is
used herein as a collective noun that refers to a
population of immunoglobulin molecules and/or
immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antibody
combining site or paratope.
An "antibody combining site" is that structural portion
of an antibody molecule comprised of heavy and light chain
variable and hypervariable regions that specifically binds
antigen.
The phrase "antibody molecule" in its various
grammatical forms as used herein contemplates both an
intact immunoglobulin molecule and an immunologically
active portion of an immunoglobulin molecule.
Exemplary antibody molecules for use in the diagnostic
methods and systems of the present invention are intact
immunoglobulin molecules, substantially'intact
immunoglobulin molecules and those portions of an
immunoglobulin molecule that contain the.paratope,
including those portions known in the art as Fab, Fab‘,
F(ab')2 and F(v). y
ifab and F(ab')2 portions of antibodies are prepared by
the proteolytic reaction of papain and pepsin,
respectively, on substantially intact antibodies by methods
that are well known.
4,342,566 to Theofilopolous and Dixon.
See for example, U.S. Patent No.
Fab‘ antibody
portions are also well known and are produced from F(ab')2
portions followed by reduction of the disulfide bonds
linking the two heavy chain portions as with
mercaptoethanol, and followed by alkylation of the
resulting protein mercaptan with a reagent such as
iodoacetamide. An antibody containing intact antibody
molecules are preferred, and are utilized as illustrative
herein.
An antibody of the present invention, i.e., an anti-Apo
AI antibody, in one embodiment is characterized as being
capable of immunoreacting with 1)-hpo AI present on HDL
particles (Apo AI/HDL), 2) isolated Apo AI, 3) Apo AI
(CNBr2—CNBr3), and 4) the polypeptide PYLDDFQKKWQEEMELYRQIO/'EP,and
being substantially free of antibody molecules that
immunoreact with 1) Apo AI CNBr1, and 2) the polypeptides:
SKDLEEVKAKVQPYLDDFEKKWQEE,
LEEVKAKVQPYLDDFQKKWQEE, and
YRQKVEPLRAEL.
- In another embodiment, an anti-Apo AI antibody is
characterized as being capable of immunoreacting with 1)
Apo AI/HDL, 2) isolated Apo AI, 3) Apo AI CNBr2, and 4) the
polypeptide KVQPYLDDFQKKWQEE, and being substantially free
of antibody molecules that immunoreact with I) Apo AI
CNBr1, 2) Apo AI CNBr3, and 3) the polypeptides:
SKDLEEVKAKVQPYLDDFQKKWQEE,
SKDLEEVKAKVQPYLDDFQ, and
QPYLDDFQKKWQEEMELYRQKVEP.
Antibody immunoreactivity with Apo AI—containing
antigens can be measured by a variety of immunological
assays known in the art. Exemplary immunoreaction of an
anti-Apo AI antibody with a CNBr fragment is described in
Example 8. Direct binding with Apo AI/HDL, isolated Apo AI
(prepared as described in Example 4), and with Apo AI
polypeptides can be assayed at least by the methods
described in Example 9.
An antibody of the present invention is typically
produced by immunizing a mammal with an inoculum containing
an Apo AI polypeptide of this invention and thereby induce
in the mammal antibody molecules having immunospecificity
for Apo AI polypeptide. The antibody molecules are then
collected from the mammal and isolated to the extent
desired by well known techniques such as, for example, by
using DEAE Sephadex to obtain the IgG fraction.
To enhance the specificity of the antibody, the
antibodies may be purified by immunoaffinity chromatography
using solid phase-affixed immunizing polypeptide. The
antibody is contacted with the solid phase-affixed
immunizing polypeptide for a period of time sufficient for
..the polypeptide to immunoreact with the antibody molecules
The bound
antibodies are separated from the complex by standard
to form a solid phase-affixed immunocomplex.
techniques.
The antibody so produced can be used, igggg alga, in
the diagnostic methods and systems of the present invention
to detect Apo AI present in a body sample. See, for
example, the method described in Example 9.
The word "inoculum" in its various grammatical forms is
used herein to describe a composition containing an Apo AI
polypeptide of this invention as an active ingredient used
for the preparation of antibodies against an Apo AI
polypeptide. when a polypeptide is used in an inoculum to
induce antibodies it is to be understood that the
polypeptide can be used in various embodiments, e.g., alone
or linked to a carrier as a conjugate, or as a polypeptide
polymer.
a polypeptide inoculum, the various embodiments of the
However, for ease of expression and in context of
polypeptides of this invention are collectively referred to
herein by the term "polypeptide", and its various
grammatical forms.
For a polypeptide that contains fewer than about 35
amino acid residues, it is preferable to use the peptide
bound to a carrier for the purpose of inducing the
production of antibodies.
one or more additional amino acid residues can be added
to the amino- or carboxy-termini of the polypeptide to
assist in binding the polypeptide to a carrier. Cysteine
residues added at the amino- or carboxy-termini of the
polypeptide have been found to be particularly useful for
forming conjugates via disulfide bonds. However, other
methods well known in the art for preparing conjugates can
also be used. Exemplary additional linking procedures
include the use of Michael addition reaction products, di-
aldehydes such as glutaraldehyde, Klipstein, et al., J.
Infect. Dis., l47:3l8-326 (1983) and the like, or the use
of carbodiimide technology as in the use of a water—soluble
carbodiimide to form amide links to the carrier. For a
review of protein conjugation or coupling through activated
functional groups, see Aurameas, et al., Scand. J.
Immunol., 1:7-23 (1978).
Useful carriers are well known in the art, and are
generally proteins themselves. Exemplary of such carriers
are keyhole limpet hemocyanin (KLH), edestin,
thyroglobulin, albumins such as bovine serum albumin (BSA)
or human serum albumin (HSA), red blood cells such as sheep
erythrocytes (SRBC), tetanus toxoid, cholera toxoid as well
as polyamino acids such as poly (D—lysine2
acid), and the like.
The choice of carrier is more dependent upon the
D-glutamic
ultimate use of the inoculum and is based upon criteria not
particularly involved in the present invention. For
example, a carrier that does not generate an untoward
reaction in the particular animal to be inoculated should
be selected. X
The present inoculum contains an effective, immunogenic
amount of a polypeptide of this invention, typically as a
conjugate linked to a carrier. The effective amount of
polypeptide per unit dose sufficient to induce an immune
response to the immunizing polypeptide depends, among other
things, on the species of animal inoculated, the body
weight of the animal and the chosen inoculation regimen as
is well known in the art. Inocula typically contain
polypeptide concentrations of about 10 micrograms to about
500 milligrams per inoculation (dose), preferably about 50
micrograms to about 50 milligrams per dose.
The term "unit dose" as it pertains to the inocula
refers to physically discrete units suitable as unitary
dosages for animals, each unit containing a predetermined
quantity of active material calculated to produce the
desired immunogenic effect in association with the required
diluent; i.e., carrier, or vehicle. The specifications for
the novel unit dose of an inoculum of this invention are
dictated by and are directly dependent on (a) the unique
characteristics of the active material and the particular
immunologic effect to be achieved, and (b) the limitations
inherent in the art of compounding such active material for
immunologic use in animals, as disclosed in detail herein,
these being features of the present invention.
Inocula are typically prepared from the dried solid
polypeptide-conjugate by dispersing the polypeptide-
conjugate in a physiologically tolerable (acceptable)
diluent such as water, saline or phosphate-buffered saline
to form an aqueous composition.
Inocula can also include an adjuvant as part of the
diluent. Adjuvants such as complete Freund's adjuvant
(CFA), incomplete Freund's adjuvant (IPA) and alum are
materials well known in the art, and are available
commercially from several sources.
The techniques ofgpolypeptide conjugation or coupling
through activated functional groups presently known in the
art are particularly applicable. See, for example,
Aurameas, et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23
(1973) and U.S. Patent No. 4,493,795, No. 3,791,932 and No.
3,839,153.
can be carried out so that any loss of activity due to
In addition, a site directed coupling reaction
polypeptide orientation after coupling can be minimized.
See, for example, Rodwell et al., Biotech., 3:889—894
(1985), and U.S. Patent No. 4,671,958.
One or more additional amino acid residues may be added
to the amino— or carboxy-termini of the polypeptide to
assist in binding the polypeptide to form a conjugate.
Cysteine residues, usually added at the carboxy-terminus of
the polypeptide, have been found to be particularly useful
for forming conjugates via disulfide bonds, but other
methods well-known in the art for preparing conjugates may
be used.
A preferred anti-Apo AI antibody is a monoclonal
antibody and is used herein as exemplary of an anti-Apo AI
antibody.
‘The phrase "monoclonal antibody" in its various
grammatical forms refers to a population of antibody
molecules that contain only one species of antibody
combining site capable of immunoreacting with a particular
epitope. A monoclonal antibody thus typically displays a
single binding affinity for any epitope with which it
immunoreacts. A monoclonal antibody may therefore contain
an antibody molecule having a plurality of antibody
combining sites, each immunospecific for a different
epitope, e.g., a bispecific monoclonal antibody.
A monoclonal antibody, in one embodiment, is
characterized as being capable of immunoreacting with 1)
Apo AI/HDL, 2) isolated Apo AI, 3) Apo AI CNBr2-CNBr3, and
4) the polypeptide PYLDDFQKKWQEBMELYRQKVEP, and being
substantially free of antibody molecules that immunoreact
with 1) Apo AI CNBrl, and 2) the polypeptides:
SKDLEEVKAKVQPYLDDFQKKWQEE,
LEEVKAKVQPYLDDFQKKWQEE, and
YRQKVEPLRAEL.
Particularly preferred is the monoclonal antibody produced
by the hybridoma AI-4, having an ATCC accession number
HB8744.
In another embodiment, a monoclonal antibody is
characterized as being capable of immunoreacting with 1)
Apo AI/HDL, 2) isolated Apo AI, 3) Apo AI CNBr2, and 4) the
polypeptide KVQPYLDDFQKKWQEE, and being substantially free
of antibody molecules that immunoreact with 1) Apo AI
CNBrl, 2) Apo AI CNBr3, and 3) the polypeptides:
SKDLEEVKAKVQPYLDDFQKKWQEE,
SKDLEEVKAKVQPYLDDFQ, and
QPYLDDFQKKWQEEMELYRQKVEP.
Particularly preferred in this embodiment is the monoclonal
antibody produced by the hybridoma AI~ll, having an ATCC
accession number HB920l. '
A monoclonal antibody is typically composed of
antibodies produced by clones of a single cell called a
hybridoma that secretes (produces) but one kind of antibody
molecule. The hybridoma cell is formed by fusing an
antibody-producing cell and a myeloma or other self-
perpetuating cell line. The preparation of such antibodies
was first described by Kohler and Milstein, Nature 256:495-
497 (1975), which description is incorporated by reference.
The hybridoma supernates so prepared can be screened for
the presence of antibody molecules that immunoreact with an
Apo AI polypeptide, or for inhibition of binding to HDL by
the Apo AI polypeptides of this invention.
Briefly, to form the hybridoma from which the
monoclonal antibody composition is produced, a myeloma or
other self-perpetuating cell line is fused with lymphocytes
obtained from the spleen of a mammal hyperimmunized with an
Apo AI antigen, such as is present in an Apo AI—containing
lipoprotein particles, or with an Apo AI polypeptide of
The polypeptide—induced hybridoma
technology is described by Niman et al.,
this invention.
Proc. Natl. Sci.,
U.S.A., 80:4949—4953 (1983), which description is
incorporated herein by reference.
It is preferred that the myeloma cell line used to
prepare a hybridoma be from the same species as the
lymphocytes.- Typically, a mouse of the strain 129 GlX* is
the preferred mammal. Suitable mouse myelomas for use in
the present invention include the hypoxanthine—aminopterin—
thymidine-sensitive (HAT) cell lines P3X63-Ag8.653, and
Spz/0—Ag14 that are available from the American Type
Culture Collection, Rockville, MD, under the designations
CRL 1580 and CRL 1581, respectively.
Splenocytes are typically fused with myeloma cells
using polyethylene glycol (PEG) 1500.
selected by their sensitivity to HAT.
Fused hybrids are
Hybridomas producing
a monoclonal antibody of this invention are identified
using the radioimmunoassay (RIA) and the enzyme linked
immunosorbent assay (ELISA) described in Examples 10 and 9,
respectively.
A monoclonal antibody of the present invention can also
be produced by initiating a monoclonal hybridoma culture
comprising a nutrient medium containing a hybridoma that
secretes antibody molecules of the appropriate polypeptide
specificity. The culture is maintained under Conditions
and for a time period sufficient for the hybridoma to
secrete the antibody molecules into the medium. The
The antibody
molecules can then be further isolated by well known
antibody-containing medium is then collected.
techniques.
Media useful for the preparation of these compositions
are both well known in/the art and commercially available
and include synthetic culture media, inbred mice and the
like.
essential medium (DMEM; Dulbecco et al., Virol. 8:396
An exemplary synthetic medium is Dulbecco's minimal
(1959)) supplemented with 4.5 gm/1 glucose, 20 mm
glutamine, and 20% fetal calf serum. An exemplary inbred
mouse strain is the Balb/c.
The monoclonal antibodies of this invention can be used
in the same manner as disclosed herein for antibodies of
‘the present invention.
For example, the monoclonal antibody can be used in the
diagnostic methods and systems disclosed herein where
formation of an Apo AI—containing immunoreaction product is
desired.
A hybridoma useful in producing a subject monoclonal
antibody, i.e., MAB AI-4 and MAB AI—ll, are hybridomas
6llAV63C2.lll and HlO308.l0l1, said hybridomas being
deposited pursuant to Budapest Treaty Requirements with the
American Type Culture Collection (ATCC), Rockville, MD
20852 U.S.A. on March 5, 1985 and September 16, 1986,
respectively, and given the ATCC designations HB8744 and
It should be noted that hybridoma
ATCC 1580 can be used, as is well known in the art, to
HB920l, respectively.
produce other immortal cell lines that produce a subject
monoclonal antibody, and thus production of a subject
monoclonal antibody is not dependent on culturing
hybridomas by ATCC HB8744 and HB9201 per se.
Other methods of producing a monoclonal antibody, a
hybridoma cell, or a hybridoma cell culture are also well
known. See, for example, the method of isolating
monoclonal antibodies from an immunological repertoire as
described by sastry, et al., Proc. Natl. Acad. Sci.,
86:5728—5732 (1989); and Huse et al., Science, 246:1275—
1231 (1931).
Also contemplated by this invention is the hybridoma
cell, and cultures obntaining a hybridoma cell that produce
.a monoclonal antibody of this invention.
D. Diagnostic Systems
The present invention also describes a diagnostic
system, preferably in kit form, for assaying for the
presence of Apo Al or an Apo AI polypeptide in a fluid
sample. A diagnostic system includes, in an amount
sufficient for at least one assay, a subject Apo AI
polypeptide and/or a subject antibody or monoclonal
antibody, as a separately packaged immunochemical reagent.
Instructions for use of the packaged reagent are also
typically included.
As used herein, the term "package" refers to a solid
matrix or material such as glass, plastic, paper, foil and
the like capable of holding within fixed limits a
polypeptide, polyclonal antibody or monoclonal antibody of
the present invention. Thus, for example, a package can be
a glass vial used to contain milligram quantities of a
contemplated polypeptide or it can be a microtiter plate
well to which microgram quantities of a contemplated
polypeptide have been operatively affixed, i.e., linked so
as'to be capable of being immunologically bound by an
antibody.
"Instructions for use" typically include a tangible
expression describing the reagent concentration or at least
one assay method parameter such as the relative amounts of
reagent and sample to be admixed, maintenance time periods
for reagent/sample admixtures, temperature, buffer
conditions and the like.
In one embodiment, a diagnostic system for assaying for
the presence of or to quantitate Apo AI in a sample, such
as blood, plasma or serum, comprises a package containing
at least one Apo AI polypeptide of this invention. In
another embodiment, a diagnostic system of the present
invention for assaying/for the presence or amount of Apo Al
or an Apo AI polypeptide in a sample further includes an
anti-Apo AI antibody composition of this invention. An
exemplary diagnostic system is described in Example 9.
In preferred embodiments, a diagnostic system of the
present invention further includes a label or indicating
means capable of signaling the formation of an
immunocomplex containing a polypeptide or antibody molecule
of the present invention.
The word "complex" as used herein refers to the product
of a specific binding reaction such as an antibody-antigen
or receptor-ligand reaction. Exemplary complexes are
immunoreaction products.
As used herein, the terms "label" and "indicating
means" in their various grammatical forms refer to single
atoms and molecules that are either directly or indirectly
involved-in the production of a detectable signal to
indicate the presence of a complex. Any label or
indicating means can be linked to or incorporated in an
expressed protein, polypeptide, or antibody molecule that
is part of an antibody or monoclonal antibody composition
of the present invention, or used separately, and those
atoms or molecules can be used alone or in conjunction with
additional reagents. Such labels are themselves well-known
in clinical diagnostic chemistry and constitute a part of
this invention only insofar as they are utilized with
otherwise novel proteins methods and/or systems.
The labeling means can be a fluorescent labeling agent
that chemically binds to antibodies or antigens without
denaturing them to form a fluorochrome (dye) that is a
Suitable fluorescent
labeling agents are fluorochromes such as fluorescein
useful immunofluorescent tracer.
isocyanate (PIC), fluorescein isothiocyante (FITC), 5-
dimethylamine—l-naphthalenesulfonyl chloride (DANSC),
tetramethylrhodamine isothiocyanate (TRITC), lissamine,
rhodamine 8200 sulphofiyl chloride (RE 200 SC) and the like.
A description of immunofluorescence analysis techniques is
found in DeLuca, "Immunofluorescence Analysis", in Antibody
As a Tool, Marchalonis, et al., eds., John Wiley & Sons,
Ltd., pp. 189-231 (1982), which is incorporated herein by
reference.
In preferred embodiments, the indicating group is an
enzyme, such as horseradish peroxidase (HRP), glucose
oxidase, or the like. In such cases where the principal
indicating group is an enzyme such as HRP or glucose
oxidase, additional reagents are required to visualize the
fact that a receptor-ligand complex (immunoreactant) has
formed. Such additional reagents for HRP include hydrogen
peroxide and an oxidation dye precursor such as
diaminobenzidine. An additional reagent useful with
glucose oxidase is 2,2'—azino-di-(3-ethyl-benzthiazoline-G-
sulfonic acid) (ABTS).
Radioactive elements are also useful labeling agents
and are used illustratively herein. An exemplary
radiolabeling agent is a radioactive element that produces
Elements which themselves emit gamma
125
I
gamma ray emissions.
rays, such as ’“I, ‘”I, , ’”I and “Cr represent one
class of gamma ray emission-producing radioactive element
Another
indicating groups. Particularly preferred is 1”I.
group of useful labeling means are those elements such as
11C, “F, “O and “N which themselves emit positrons. The
positrons so emitted produce gamma rays upon encounters
with electrons present in the animal's body.
111
Also useful
is a beta emitter, such as indium of 3L
The linking of labels, i.e., labeling of, polypeptides
and proteins is well known in the art. For instance,
antibody molecules produced by a hybridoma can be labeled
by metabolic incorporation of radioisotope—containing amino
acids provided as a component in the culture medium. see,
for example, Galfre et al., Meth. Enzymol., 73:3—46 (1981).
The techniques of protein conjugation or coupling through
activated functional groups are particularly applicable.
A See, for example, Aurameas, et al., scand. J. Immunol.,
Vol. 8 Suppl. 7:7—23 (1978), Rodwell et al., Biotech.,
3:889-894 (1984), and U.S. Pat. No. 4,493,795.
The diagnostic systems can also include, preferably as
a separate package, a specific binding agent. A "specific
binding agent" is a molecular entity capable of selectively
binding a reagent species of the present invention or a
complex containing such a species, but is not itself a
polypeptide or antibody molecule composition of the present
invention. Exemplary specific binding agents are second
antibody molecules, complement proteins or fragments
thereof, S. aureus protein A, and the like. Preferably the
specific binding agent binds the reagent species when that
species is present as part of a complex.
In preferred embodiments, the specific binding agent is
labeled.
specific binding agent that is not labeled, the agent is
In these
embodiments, the labeled specific binding agent is capable
However, when the diagnostic system includes a
typically used as an amplifying means or reagent.
of specifically binding the amplifying means when the
amplifying means is bound to a reagent species-containing
complex.
The diagnostic kits of the present invention can be
Vused in an "ELISA" format to detect the quantity of Apo AI
in a vascular fluid sample such as blood, serum, or plasma.
"ELISA" refers to an enzyme-linked immunosorbent assay that
employs an antibody or antigen bound to'a solid phase and
an enzyme-antigen or enzyme—antibody conjugate to detect
and quantify the amount of an antigen present in a sample.
A description of the ELISA technique is found in Chapter 22
of the 4th Edition of Basic and Clinical Immunology by D.P.
Sites et al., published by Lange Medical Publications of
Los Altos, CA in 1982 and in U.S. Patents No. 3,654,090;
No. 3,850,752; and No. 4,016,043, which are all
incorporated herein by reference.
Thus, in preferred embodiments, an Apo AI polypeptide
or a monoclonal antibody of the present invention can be
affixed to a solid matrix to form a solid support that
comprises a package in the subject diagnostic systems.
A reagent is typically affixed to a solid matrix by
adsorption from an aqueous medium although other modes of
affikation applicable to proteins and
polypeptides well known to those skilled in the art, can be
used.
Useful solid matrices are also well known in the art.
Such materials are water insoluble and include the cross-
linked dextran available under the trademark SEPHADEX from
Pharmacia Fine Chemicals (Piscataway, NJ); agarose; beads
of polystyrene beads about 1 micron to about 5 millimeters
in diameter available from Abbott Laboratories of North
Chicago, IL: polyvinyl chloride, polystyrene, cross—linked
polyacrylamide, nitrocellulose— or nylon-based webs such as
sheets, strips or paddles: or tubes, plates or the wells of
a microtiter plate such as those made from polystyrene or
polyvinylchloride.
The reagent species, labeled specific binding agent or
amplifying reagent of any diagnostic system described
herein can be provided in solution, as a liquid dispersion
or as a substantially dry power, e.g., in lyophilized form.
Where the indicating means is an enzyme, the enzyme's
substrate can also be provided in a separate package of a
system. A solid support such as the before-described
microtiter plate and one or more buffers can also be
included as separately packaged elements in this diagnostic
assay system.
The packaging materials discussed herein in relation to
diagnostic systems are those customarily utilized in
diagnostic systems.
The term "package" refers to a solid matrix or material
such as glass, plastic (e.g., polyethylene, polypropylene
and polycarbonate), paper, foil and the like capable of
holding within fixed limits a diagnostic reagent such as a
polypeptide, antibody or monoclonal antibody of the present
invention. Thus, for example, a package can be a bottle,
vial, plastic and plastic—foil laminated envelope or the
like container used to contain a contemplated diagnostic
reagent or it can be a microtiter plate well to which
microgram quantities of a contemplated diagnostic reagent
have been operatively affixed, i.e., linked so as to be
capable of being immunologically bound by an antibody or
polypeptide to be detected.
E. Assay Methods
The present invention contemplates various immunoassay
methods for determining the amount of Apo AI in a
biological fluid sample using a polypeptide, polyclonal
antibody or monoclonal antibody of this invention as an
immunochemical reagent to form an immunoreaction product
whose amount relates, either directly or indirectly, to the
Those skilled in the art
will understand that there are numerous well known clinical
amount of Apo AI in the sample.
diagnostic chemistry procedures in which an immunochemical
reagent of this invention can be used to form an
immunoreaction product whose amount relates to the amount
of Apo AI present in a body sample. Thus, while exemplary
assay methods are described herein, the invention is not so
limited.
Various heterogeneous and homogeneous protocols, either
competitive or noncompetitive, can be employed in
performing an assay method of this invention. For example,
the present invention contemplates a competitive method for
assaying the amount of Apo AI in a vascular fluid sample
which comprises the steps of:
(a) Forming an immunoreaction admixture by admixing a
vascular fluid sample with:
(i) an antibody of the present invention,
preferably-a monoclonal antibody, and
(ii) an Apo AI polypeptide of the present
invention that is able to immunoreact with the antibody
added in step (i).
Preferably, the vascular fluid sample is provided as a
known amount of blood, or a blood derived product such as
serum or plasma. Regardless of the type of sample used, it
is preferably obtained from a person who has fasted at
least about 12 hours as is known in the art. Such a sample
is referred to as a "fasting" sample. It is also noted
that where serum or plasma is used as the sample, that
sample need not be subjected to treatment with a denaturing
or chaotropic agent for purposes of altering the expression
of the Apo AI epitope being assayed.
In one embodiment, the diagnostic method includes
forming an immunoreaction admixture by admixing a vascular
fluid sample with:
(i) an anti-Apo AI antibody molecule that immunoreacts
with 1) Apo AI/HDL, 2) isolated Apo AI, 3) Apo AI CNBr2-
CNBr3, and 4) the polypeptide PYLDDFQKKWQEEMELYRQVEP, and is
substantially free of antibody molecules that immunoreact
with 1) Apo AI CNBr1, and 2) the polypeptides:
SKDLEEVKAKVQPYLDDFQKKWQEE,
LEEVKAKVQPLYDDFQKKWQEE, and
YRQKVEPLRAEL; and
(ii) an Apo AI polypeptide of the present invention
that includes an amino acid residue sequence represented by
the formula: -PYLDDXQKKWQEEMELYRQKVEPe, and preferably’inc1ude5
an amino acid residue sequence represented by the formula:
—PYLDDXQKKWQEEMELYRQKVEP-. In particular preferred
embodiments, the antibody is the monoclonal antibody
produced by the hybridoma cell line having the ATCC
designation HB 8744, and the polypeptide is selected from
the group polypeptides shown in Table 1 consisting of AI99—
121, AI94-125, AI94-114, AI98-114, AI98-121, and AI99-114.
In another embodiment, the immunoreaction admixture is
formed by admixing a vascular fluid sample with:
(i) an anti~Apo AI antibody molecule that immunoreacts
with 1) Apo AI/HDL, 2) isolated Apo AI, 3) Apo AI CNBr2,
and 4) the polypeptide KVQPYLDDFQKKWQEE, and is
substantially free of antibody molecules that immunoreact
with 1) Apo AI CNBrl, 2) Apo AI CNBr3, and 3) the
polypeptides:
SKDLEEVKAKVQPYLDDFQKKWQEE,
SKDLEEVKAKVQPYLDDFQ , and
QPYLDDFQKKWQEEMELYRQKVEP7 and
(ii) an Apo AI polypeptide of the present invention
that includes an amino acid residue sequence represented by
-KVQPYLDDFQKKWQEE-.
preferred embodiments, the antibody is the monoclonal
the formula: In particularly
antibody produced by the hybridoma cell line having the
ATCC designation HB9201, and the polypeptide is selected
from the group of polypeptides shown in Table 1 consisting
of AI96-111, AI93-111, AI84-111, AI85-111, AI87-111, AI94-
111, AI94-125, and AI90-111.
Preferably, the amount of antibody that is admixed is
known. Further preferred are embodiments where the
antibody is labeled, i.e., operatively linked to an
indicating means such as an enzyme, radionuclide and the
like.
Preferably, the Apo AI polypeptide is present as part
of a solid support, i.e., operatively linked to a solid
matrix, so that the immunoreaction admixture formed has a
solid and a liquid phase. Further preferred are
embodiments wherein the amount of polypeptide present in
the immunoreaction admixture is an amount sufficient to
form an excess of epitopes relative to the number of
antibody combining sites present in the immunoreaction
admixture capable of immunoreacting with those epitopes.
(b) The immunoreaction admixture is maintained under
biological assay conditions for a predetermined time period
such as about 10 minutes to about 16-20 hours at a
temperature of about 4 degrees C to about 45 degrees C
that, such time being sufficient for the Apo AI present in
the sample to immunoreact with (immunologically bind) a
portion of the anti-Apo AI antibody combining sites present
in the monoclonal antibody to form an Apo AI-containing
immunoreaction product (immunocomplex). In embodiments
where the polypeptide is in the solid phase, the
immunocomplex formed is also present in the solid phase.
Biological assay conditions are those that maintain the
biological activity of the immunochemical reagents of this
invention and the Apo AI sought to be assayed. Those
conditions include a temperature range of about 4 degrees C
to about 45 degrees C, a pH value range of about 5 to about
9 and an ionic strength varying from that of distilled
Methods
for optimizing such conditions are well known in the art.
water to that of about one molar sodium chloride.
(c) The amount of Apo AI-containing immunoreaction
product that formed in step (b) is determined, thereby
determining the amount of Apo AI present in the sample.
Determining the amount of the Apo AI—containing
immunoreaction product, either directly or indirectly, can
be accomplished by assay techniques well known in the art,
and typically depend on the type of indicating means used.
In preferred competitive assay methods, the amount of
product determined in step (c) is related to the amount of
immunoreaction product similarly formed and determined
using a control sample in place of the vascular fluid
sample, wherein the control sample contains a known amount
of a subject polypeptide from which a standard curve is
determined.
Exemplary of the contemplated competitive diagnostic
assay, wherein an Apo AI polypeptide is operatively linked
to a solid matrix is the ELISA described in Example 9.
In another embodiment, the present invention
contemplates a double antibody or "sandwich" immunoassay
comprising the steps of: A
(a) Forming a first immunoreaction admixture by
admixing a vascular fluid sample with a first antibody,
preferably a monoclonal antibody, wherein the antibody and
Apo AI/HDL present in the sample are capable of forming a
first immunoreaction product that can immunoreact with a
subject monoclonal antibody. Preferably the first antibody
is operatively linked to a solid matrix.
(b) Maintaining the first immunoreaction admixture so
formed under biological assay conditions for a time period
sufficient to form the first immunoreaction product.
Preferably, the first immunoreaction product is then
separated from the sample.
(c) Forming a second immunoreaction admixture by
admixing the first immunoreaction product with a second
antibody, preferably a monoclonal antibody, wherein the
second antibody and Apo AI/HDL present in the first
immunoreaction product are capable of forming a second
immunoreaction product.
(d) Maintaining the second immunoreaction admixture so
formed under biological assay conditions for a time period
sufficient to form the second or "sandwich" immunoreaction
product.
(e) Determining the amount of second immunoreaction
product that formed, and thereby the amount of Apo AI in
the sample.
Preferably, the subject monoclonal antibody of step (c)
is labeled, preferably with an enzyme, and therefore the
second immunoreaction product formed is a labeled product.
In one embodiment, the detection of Apo AI polypeptides
in a body sample is utilized as a means to monitor the fate
of therapeutically administered Apo AI polypeptides
according to the therapeutic methods disclosed herein.
Also contemplated are immunological assays capable of
detecting the presence of immunoreaction product formation
without the use of a label. Such methods employ a
"detection means", which means are themselves.well—known in
clinical diagnostic chemistry and constitute a part of this
invention only insofar as they are utilized with otherwise
novel polypeptides, methods and systems. Exemplary
detection means include methods known as biosensors and
include biosensing methods based on detecting changes in
the reflectivity of a surface, changes in the absorption of
an evanescent wave by optical fibers or changes in the
propagation of surface acoustical waves.
F. Therapeutic Compositions
The present invention contemplates therapeutic
compositions useful for practicing the therapeutic methods
described herein. Therapeutic compositions of the present
invention contain a physiologically tolerable carrier
together with an Apo AI polypeptide, as described herein,
dissolved or dispersed therein as an active ingredient. In
a preferred embodiment, the therapeutic composition is not
immunogenic when administered to a mammal or human patient
for therapeutic purposes.
As used herein, the terms "pharmaceutically
acceptable", "physiologically tolerable" and grammatical
variations thereof, as they refer to compositions,
carriers, diluents and reagents, are used interchangeably
and represent that the materials are capable of
administration to or upon a mammal without the production
of undesirable physiological effects such as nausea,
dizziness, gastric upset and the like.
The preparation of a pharmacological composition that
contains active ingredients dissolved or dispersed therein
is well understood in the art. Typically such compositions
are prepared as injectables either as liquid solutions or
‘ suspensions, however, solid forms suitable for solution, or
suspensions, in liquid prior to use can also be prepared.
The preparation can also be emulsified.
The active ingredient can be mixed with excipients
which are pharmaceutically acceptable and compatible with
the active ingredient and in amounts suitable for use in
Suitable
excipients are, for example, water, saline, dextrose,
the therapeutic methods described herein.
glycerol, ethanol or the like and combinations thereof. In
addition, if desired, the composition can contain minor
amounts of auxiliary substances such as wetting or
emulsifying agents, pH buffering agents and the like which
enhance the effectiveness of the active ingredient.
The therapeutic composition of the present invention
can include pharmaceutically acceptable salts of the
components therein. Pharmaceutically acceptable salts
include the acid addition salts (formed with the free amino
groups of the polypeptide) that are formed with inorganic
acids such as, for example, hydrochloric or phosphoric
acids, or such organic acids as acetic, tartaric, mandelic
and the like. salts formed with the free carboxyl groups
can also be derived from inorganic bases such as, for
example, sodium, potassium, ammonium, calcium or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine
and the like.
Physiologically tolerable carriers are well known in
the art.
solutions that contain no materials in addition to the
Exemplary of liquid carriers are sterile aqueous
active ingredients and water, or contain a buffer such as
sodium phosphate at physiological pH value, physiological
Still
further, aqueous carriers can contain more than one buffer
saline or both, such as phosphate—buffered saline.
salt, as well as salts such as sodium and potassium
chlorides, dextrose, polyethylene glycol and other solutes.
Liquid compositions can also contain liquid phases in
addition to and to the exclusion of water. Exemplary of
such additional liquid phases are glycerin, vegetable oils
such as cottonseed oil, and water-oil emulsions.
G. Therapeutic Methods
It has been discovered that the Apo AI polypeptides of
the present invention have the capacity to modulate the
enzymatic activity of lecithinzcholesterol acyltransferase
(LCAT) and thereby increase the level of esterified
cholesterol in a human patient. The esterification of
cholesterol, where it occurs by the activity of LCAT, is
referred to herein as "LCAT-mediated cholesterol
esterification".
Thus, the present invention provides polypeptides for use in
a method for increasing esterified cholesterol in a patient the
method comprising administering to the patient a therapeutically
effective amount of a physiologically tolerable composition
containing an Apo AI polypeptide of the present invention.
A therapeutically effective amount is a predetermined
amount calculated to achieve the desired effect, i.e., to
increase the amount of esterified cholesterol in a patient.
A therapeutically effective amount is typically an amount
of an Apo AI polypeptide of the present invention that when
administered in a physiologically_tolerable composition is
sufficient to achieve a plasma concentration of from about
0.1 pg/ml to about 100 pg/ml, preferably from about 1.0
pg/ml to about 50 pml, more preferably at least about 2
pg/ml and usually 5 to 10 pg/ml.
The level of esterified cholesterol present in a
patient, particularly in the plasma and associated with
lipoprotein particles, can be readily determined by routine
clinical analysis. Exemplary assays to monitor the level
of esterified cholesterol are described in Example 11. In
addition, changes in esterified cholesterol can be
monitored during a treatment regimen to determine the
effectiveness of the administered Apo AI polypeptide over
time. A
Thus, the present therapeutic method provides a means
for in vivo increasing esterified cholesterol in a human
patient displaying symptoms of elevated serum cholesterol,
or is otherwise at medical risk by the presence of serum
cholesterol, wherein it is beneficial to reduce the levels
of cholesterol by cholesterol esterification.
The therapeutic compositions containing an Apo AI
polypeptide of this invention are conventionally
administered intravenously, as by injection of a unit dose,
for example. The term "unit dose" when used in reference
to a therapeutic composition of the present invention
refers to physically discrete units suitable as unitary
dosage for the subject, each unit containing a
predetermined quantity of active material calculated to
produce the desired therapeutic effect in association with
the required diluent; i.e., carrier, or vehicle.
The compositions are administered in a manner
compatible with the dosage formulation, and in a
therapeutically effective amount. The quantity to be
administered depends on the subject to be treated, capacity
of the subject's immune system to utilize the active
ingredient, and degree of therapeutic effect desired.
Precise amounts of active ingredient required to be
administered depend on the judgement of the practitioner
and are peculiar to each individual. However, suitable
dosage ranges for systemic application are disclosed herein
Suitable
regimes—for initial administration and booster shots are
and depend on the route of administration.
also variable, but are typified by an initial
administration followed by repeated doses at one or more
hour intervals by a subsequent injection or other
administration. Alternatively, continuous intravenous
infusion sufficient to maintain concentrations in the blood
in the ranges specified for in vivo therapies are
contemplated.
As an aid to the administration of effective amounts of
an Apo AI polypeptide, a diagnostic method of this
invention for detecting an APO AI polypeptide in the
subject's blood is useful to characterize the fate of the
administered therapeutic composition. V
The following Examples illustrate, but do not limit,
the present invention.
1. Polypeptides
Polypeptides AI84-lll, AI85-lll, AI87-111, AI90-lll,
AI93-lll, AI94-lll, AI94-114, AI94-125, AI96-lll, AI98-114,
AI98-121, AI99-114, AI99-121,
were synthesized using the classical solid-phase
technique described by Merrifield, Adv. Enzymol., 322221-96
(1969) as adapted for use with a Model 430A automated
peptide synthesizer (Applied Biosystems, Foster City, CA).
Polypeptide resins were cleaved by hydrogen fluoride,
extracted and analyzed for purity by high-performance
liquid chromatograph using a reverse—phase C18 column.
(Waters Associates, Milford, MA).
The amino acid residue sequence of the polypeptides
named above is shown in Table 1.
. Preparation of Apo AI/HDL
HDL was isolated from plasma obtained by plasmaphoresis
of normal fasting—donor blood at the local blood bank
For that
purpose, plasma so obtained was adjusted to contain a final
(San Diego Plasma Center, San Diego, CA).
concentration of 2 millimolar (mM) benzamidine, 14 mM
ethylenediaminetetraacetic acid (disodium salt) (EDTA), 20
micrograms per milliliter (pg/ml) soybean trypsin
inhibitor, 10,000 units per ml aprotinin, 20 pg/ml lima
bean trypsin inhibitor, 25 pg/ml polybrene, and 1uM D-
phenylalanyl-1—prolyl-1—arginine chloromethyl ketone
(PPACK). The HDL was then isolated from this adjusted
plasma by sequential ultracentrifugation using solid
potassium bromide (KBr) for density adjustment.
First, the adjusted plasma was centrifuged at 186,000 x
g for 18 to 24 hours at 4 degrees Centigrade (4'C). The
top layer of the resulting supernatant containing Apo—VLDL
The bottom layer of the
supernatant was recovered and admixed with solid KBr layer
was removed and retained.
until the density was greater than 1.063 grams per
milliliter (g/ml).
layered under a 0.1% EDTA solution containing KBr at
density of 1.063 g/ml and centrifuged at 186,000 x g for 18
The resulting admixture was then
to 24 hours. The bottom layer was again recovered and
admixed with solid KBr until the density was adjusted to
greater than 1.21 g/ml. That adjusted layer was layered
under a 0.1% EDTA solution containing KBr at a density of
1.21 g/ml, and was centrifuged at 186,000 X g for more
about 48 hours at 4'C. -
The resulting top layer was then recovered and admixed
with solid KBr until the density was greater than 1.063
g/ml.
EDTA solution containing KBr at a density of 1.063 g/ml,
That adjusted top layer was layered under a 0.1%
and still further centrifuged at 186,000 X g for 18 to 24
hours at 4°C.
The middle layer was recovered and admixed with solid
KBr until the density was adjusted to greater than 1.21
g/ml.. That adjusted middle layer was layered under a 0.1%
EDTA solution containing KBr at a density of 1.21 g/ml and
centrifuged at 300,000 x g for 18 to 24 hours at 4’C. The
resulting HDL-containing top layer, having a density equal
to 1.063 to 1.21 g/ml, was recovered. The recovered HDL
was dialyzed against lipoprotein buffer (LLB; water
containing 150 mM NaCl, 0.3 mM EDTA, 0.005% alpha-
tocopherol, and 5 mM benzamidine) and the resulting Apo
AI/HDL was stored under sterile conditions for no more than
21 days.
determined to be between 15 and 25 mg/ml by a modification
The protein concentration of Apo AI/HDL was
of the Lowry method [Lowry et al., J. Biol. Chem., 193,
265-275 (1951)) when conducted in the presence of SDS using
a bovine serum albumin standard.
. Preparation of Delipidated Apo AI
Delipidated Apo AI was prepared by organically
extracting the lipids from Apo AI/HDL.
AI/HDL prepared in Example 1B was first dialyzed overnight
A sample of the Apo
(approximately 18 hours) against 0.01 percent EDTA having a
pH value of 7.5, then dialyzed against 0.003 percent EDTA
for approximately 6 hours, and subsequently lyophilized at
to 20 milligrams of protein per tube. To each tube were
admixed 35 ml of absolute ethanolzanhydrous ethyl ether
(1:1) at 4°C.
maintained for 20 minutes at —20°C.
Following admixture, the solution was
The solution was then
centrifuged for 30 minutes at 1000 x g‘at 0°C, the
supernatant was poured off and the Apo AI—containing pellet
was retained.
An ethanol ether extraction was performed twice again
as described above for a total of three extractions.
Subsequently, 35 ml of anhydrous ether at 4°C were admixed
to the sample. The admixture was maintained for 30 minutes
at -20°C, centrifuged at 1000 x g for 30 minutes at -20°C,
and the Apo AI-containing pellet was recovered and dried
It should
be noted that delipidated Apo AI contains not only Apo AI,
using nitrogen gas to form delipidated Apo AI.
but also other proteins associated with the HDL, such as
Apo AII.
. Preparation of Isolated Ago AI
Apo AI was isolated from delipidated Apo AI by size
fractionation using high pressure liquid chromatography
(HPLC) following the procedures of Kinoshita et al., Q;
Biochem., 94:6l5-617 (1983). About 300 pg of delipidated
Apo AI prepared in Example 3 was dissolved in 200
microliters (ul) of 0.1% sodium dodecyl sulfate (SDS), 0.1
M sodium phosphate (pH 7.0) and size fractionated on
Spherogel — TSK 3000 SW HPLC'columns (Beckman Instruments
Inc., Fullerton, CA). Fractions containing the isolated
Apo AI were stored at —20°C.
. Preparation of Polyclonal Antisera to Synthetic
Polypeptides
A. Preparation of Immunoqen
LDL is isolated from plasma obtained by plasmaphoresis
of normal pooled rabbit blood (Scripps Clinic and Research
Foundation Vivarium, La Jolla, Calif:). Plasma so obtained
is treated as described for purification of Apo AI/HDL in
Example 2. After the second centrifugation, the top layer
containing LDL is recovered and the bottom layer containing
HDL is discarded. The top layer is admixed with solid KBr
until the density is adjusted to greater than 1.063 g/ml.
That adjusted layer is layered under a 0.1% EDTA solution
containing KBr at a density of 1.21 g/ml and is centrifuged
at 186,000 x g for 18 to 24 hours at 4'C.
The top layer is then recovered, and solid KBr is
admixed until the density is greater than 1.063 g/ml. That
adjusted top layer is layered under a 0.1% EDTA solution
containing KBr at a density of 1.063 g/ml, and still
further centrifuged at 250,000 x g for 18 to 24 hours at
4'C. ‘The top layer containing concentrated LDL is
recovered and dialyzed against PBS (phosphate—buffered
saline, pH 7.2) and stored at -70°C.
The polypeptides AI84-4111, AI85-111, AI90-lll, AI93-
lll, AIl94+1ll, AI94-ll4, AI94-125, AI96-lll, A198-114,
AI198-121, AI AI99-114, and AI99-121 are synthesized as
described in Example 1. Each polypeptide is individually
dissolved in 1.5 M sodium acetate, pH 7.8, to a final
concentration of 6 mg/ml with a total volume ofu5 mls. A
dissolved polypeptide is admixed with 2.5 mls each of a 2
mg/ml LDL solution and a 3 M sodium acetate solution, pH
7.8, for a peptide:LDL ratio of 1000:1. Added to the
polypeptide and LDL reaction mixture is a 500 mM
glutaraldehyde solution using a 2.7 molar excess of
glutaraldehyde to peptide. The admixture is maintained at
room temperature for 10 minutes, after which a 40 mM
solution of sodium borohydride is added for a final
concentration of 0.2 mM. The admixture is thereafter
maintained at 37’C for 5 to 8 hours, which is followed by
dialysis against PBS for 5 days with two buffer changes per
day using dialysis tubing having a 12,000 to 14f000
molecular weight cut-off. The dialysed solution is
centrifuged at 2500 x g for 10 minutes, and the resulting
pellet is resuspended in 5 mls PBS to form a peptide-LDL
immunogen. A peptide-LDL immunogen is prepared for using
each of the above-described peptides in the above immunogen
preparation protocol.
B. Immunization and Collection
of Polvclonal Antisera
The peptide-LDL immunogen prepared in Example SA is
emulsified using the Ribi Adjuvant System (Ribi Immunochem
Research, Inc., Hamilton, Montana) according to the
manufacturer's instructions, and the peptide-LDL antigens
are incorporated into the emulsion at a concentration of
300 pg/ml.
prepared emulsion after pre-immune serum samples are
Two rabbits are injected with 1 ml of a
collected. The 1 ml emulsion dose is administered as
follows: 0.30 ml intradermal (0.05 ml in each of 6 sites);
0.40 ml intramuscular (0.2 ml into each'hind leg); 0.10 ml‘
subcutaneous (neck region): and 0.20 ml intraperitoneal.
The rabbits are injected 6 times at three—week intervals
following the injection protocol as detailed. At one week
after the second through sixth injections, blood samples
are collected to check antibody titer against the specific
peptide used as an immunogen by the SPRIA assay described
below. The collected blood samples are stirred in a 37°C
oven for 1 hour, after which the samples are centrifuged at
3000 x g for 20 minutes. The interface is collected and
spun in a microfuge at 12,000 x g for 5 minutes. The
supernatant containing anti—peptide antibodies is collected
and stored at -20°C.
The peptide antibody titers are determined by solid
phase radioimmunoassay (SPRIA) essentially as described in
Curtiss and Edgington, J. Biol. Chem., 257:l5213-15221
(1982).
peptides are admixed into the wells of microtiter plates.
Briefly, 50 pl of PBS containing 5 pg/ml synthetic
The plates are maintained overnight (about 16 hours) at 4°C
to permit the peptides to adhere to well walls. After
washing the wells four times with SPRIA buffer (2.68 mM
KCL, 1.47 mM KH2PO,, 137 mM NaCl, 8.03 mM Na2HPO4, 0.05%
Tween-20, 0.1 KIU/ml Traysol, 0.1% BSA, 0.Ol5%”NaN3), 200
pl of SPRIA buffer containing 3% normal goat serum (NGS)
and 3% bovine serum albumin (BSA) are admixed to each well
to block excess protein binding sites. The plates are
maintained for 30 minutes at 20°C, the wells emptied by
shaking, and blotted dry to form a solid—support, i.e., a
solid matrix to which Apo AI/HDL was operatively affixed.
To each well is then admixed 50 pl of serum sample to
form a solid—liquid phase immunoreaction admixture. The
admixture is maintained for 2 hours at 37’C to permit
After
125I_
formation of solid—phase immunoreaction products.
washing the wells as previously described, 50 pl of
labeled goat anti—mouse IgG at 0.25 pg protein per ml are
admixed to each well to form a labeling reaction admixture.
That admixture is maintained for 1 hour at 37°C to permit
formation of 131-labeled solid-phase immunoreaction
products. After washing the wells as previously described,
the amount of ”5I—labeled product bound to each well is
determined by gamma scintillation. Specific anti—peptide
antibody titers in collected serum samples from immunized
rabbits are determined in comparison to pre-immunized
normal rabbit serum samples which are a measure of non-
specific background. Serum samples are considered to
contain anti—peptide polyclonal antibodies if the
radioactive signal is 5 times over that seen with normal
rabbit serum.
. Preparation of Monoclonal Antibodies
A.
The polypeptides designated AI84-111, AI85—lll, AI90-
lll, AI93-lll, AI94-111, AI94-114, AI94-125, AI96-lll,
AI98-114, AI98-121, AI99-114, and AI99-121 are individually
Balb/c ByJ
mice (Scripps Clinic and Research Foundation Vivarium, La
prepared as immunogens according to Example 5A.
Jolla, CA) are immunized intraperitoneally (i.p.) with 50
pg of prepared peptide—LDL immunogens in complete Freund's
adjuvant (CFA) followed by a second and third immunization
using the same peptide—LDL immunogen, each about three
weeks apart, in incomplete Freund's adjuvant (IPA). The
mice receive a boost of 50 pg of prepared peptides
intravenously (i.v.) in normal saline 4 days prior to
fusion and a second similar perfusion boost one day later.
The animals so treated are sacrificed and the spleen of
each mouse is harvested. A spleen cell suspension is then
prepared. Spleen cells are then extracted from the spleen
cell suspension by centrifugation for about 10 minutes at
1000 r.p.m., at 23°C. Following removal of supernatant,
the cell pellet is resuspended in 5 ml cold NH,Cl lysing
buffer, and was incubated for about 10 minutes.
To the lysed cell suspension are admixed 10 ml
Dulbecco's Modified Eagle Medium (DMEM) (GIBCO) and HEPES
[4—(2—hydroxyethyl)-l—piperidineethanesulfonic acid]
buffer, and that admixture is centrifuged for about 10
minutes at 1000 r.p.m. at 23°C.
The supernatant is decanted, the pellet is resuspended
in 15 ml of DMEM and HEPES, and is centrifuged for about 10
minutes at 1000 r.p.m. at 23°C. The above procedure is
repeated. V
The pellet is then resuspended in 5 ml DMEM and HEPES.
An aliquot of the spleen cell suspension is then removed
for counting. Fusions are accomplished in the following
manner using the non-secreting mouse myeloma cell line
P3X63Ag8.653.l, a subclone of line P3X63Ag 8.653 (ATCC
1580). Using a myeloma to spleen cell ratio of about 1 to
or about 1 to 5, a sufficient quantity of myeloma cells
are centrifuged into a pellet, washed twice in 15 ml DMEM
and HEPES, and centrifuged for 10 minutes at 1000 r.p.m. at
23°C. - p
Spleen cells and myeloma cells are combined in round
bottom 15 ml tubes.
minutes at 1000 r.p.m. at 23’C, and the supernatant is
The cell mixture is centrifuged for 10
removed by aspiration. Thereafter, 200 pl of 50 percent
(weight per volume) aqueous polyethylene glycol 4000
molecular weight (PEG; ATCC Baltimore, MD) at about 37°C
are admixed using a 1 ml pipette with vigorous stirring to
disrupt the pellet, and the cells are gently mixed for
between 15 and 30 seconds. The cell mixture is centrifuged
4 minutes at 700 r.p.m.
At about 8 minutes for the time of adding the PEG, 5 ml
of DMEM plus HEPES buffer are admixed slowly to the pellet,
After 1 minute, the
resulting admixture is broken up with a 1 ml pipette, and
without disturbing the cells.
is incubated for an additional 4 minutes. This mixture is
centrifuged for 7 minutes at 1000 r.p.m. The supernatant
is decanted, 5 ml of HT (hypoxanthine/thymidine) medium are
slowly admixed to the pellet, and the admixture is
maintained undisturbed for 5 minutes. The pellet is then
broken into large chunks, and the final cell suspension is
placed into T75 flasks (2.5 ml per flask) into which 7.5 ml
HT medium have been placed previously. The resulting cell
suspension is incubated at 37°C to grow the fused cells.
After 245 hours 10 ml of HT medium are admixed to the
flasks, followed 6 hours later by admixture of 0.3 ml of
0.04 mM aminopterin. 48 hours after fusion, 10 ml of HAT
(hypoxanthine/aminopterin/thymidine) medium are admixed to
the flasks.
Three days after fusion, viable cells are plated out in
96-well tissue culture plates at about 2x10“ viable cells
per well (768 total wells) in HAT buffer medium as
described in Kennett et al., Curr. Top. Microbiol.
Immunol., 81:77 (1978).
fusion with HAT medium and at~approximately 4-5 day
The cells are fed'sevenidays after
intervals thereafter as needed with RT medium. Growth is
followed microscopically, and culture supernatants are
collected about two weeks later and assayed for the
presence of HDL-specific antibody by solid phase
radioimmunoassay (RIA) essentially as described in Curtiss
and Edgington J. Biol. Chem., 257:l52l3-15221 (1982).
Briefly, 50 pl of PBS containing 5 pg/ml of the
prepared peptide-LDL immunogen is admixed into the wells of
microtiter plates. The plates are maintained overnight
(about 16 hours) at 4°C to permit the peptide-LDL immunogen
to adhere to well walls. After washing the wells four
times with SPRIA buffer (2.68 mM KCl, 1.47 mM KHZPOA, 137
mM NaCl, 8.03 mM Na2HP0,, 0.05% Tween-20, 0.1 KIU/ml
Traysol, 0.1% BSA, 0.015% NaN3), 200 pl of SPRIA buffer
containing 3% normal goat serum (NGS) and 3% bovine serum
albumin (BSA) are admixed to each well to block excess
protein binding sites. The plates are maintained for 30
minutes at 20°C, the wells emptied by shaking, and blotted
dry to form a solid-support, i.e., a solid matrix to which
peptide-LDL immunogen is operatively affixed.
To each well is then admixed 50 pl of hybridoma tissue
culture supernatant to form a solid—liquid phase
immunoreaction admixture. The admixture is maintained for
2 hours at 37°C to permit formation of solid—phase
immunofeaction products. After washing the wells as
previously described, 50 pl of lul-labeled goat anti-mouse
IgG at 0.25 pg protein per ml are admixed to each well to
form a labeling reaction admixture. That admixture is
maintained for 1 hour at 37°C to permit formation of l”I-
labeled solid—phase immunoreaction products. After washing
the wells as previously described, the amount of 1“I—
labeled product bound to each well is determined by gamma
scintillation.
Hybridomas are selected from hybridoma cultures that
secrete anti—peptide antibodies into their culture media,
and further characterized as described herein.
B. AI—4
The hybridoma that bears the laboratory designation 611
AV63C2.111 and secretes the paratopic molecule designated
AI-4 was obtained from a fusion of splenocytes from Balb/c
mice (Scripps Clinic and Research Foundation Vivarium, La
Jolla, Calif.) immunized with Apo/VLDL as discussed in
Example 2. The standard fusion protocol was used as
discussed in Example 6. The hybridomas so prepared were
screened and assayed as discussed hereinbefore, with the
one exception that Apo AI/HDL was used as the coated
substrate instead of peptide-LDL immunogen. The hybridoma
was deposited on March 5, 1985 with the American Type
Culture Collection, Rockville, Md. under the ATCC accession
number HB 8744 in accordance with the Budapest Treaty on
International Recognition of the Deposit of Microorganisms
for the Purposes of Patent Procedure.
C. AI-ll
The hybridoma that bears the laboratory designation
H103D8.lDll and secretes the paratopic molecule designated
AI—ll was obtained from a fusion of splenocytes from Balb/c
mice (Scripps Clinic and Research Foundation Vivarium, La
Jolla, Calif.) immunized with Apo AI/HDL as discussed in
Example 2. The standard fusion protocol was used as
discussed in Example 6. The hybridomas so prepared were
screened and assayed as discussed hereinbefore, with the
one exception that Apo AI/HDL was used as the coated
substrate instead of peptide—LDL immunogen. The hybridoma
was deposited on September 16, 1986 with the American Type
Culture Collection, Rockville, Md. under the ATCC accession
number HB 9201 in accordance with the Budapest Treaty on
International Recognition of the Deposit of Microorganisms
for the Purposes of Patent Procedure.
, Monoclonal Antibody Preparation and Purification
Ascites fluids were obtained from separate sets of 10~
week old Balb/c mice, which had been primed with 0.3 ml of
mineral oil and injected intraperitoneally with 5x10‘
hybridoma cells with the designations 611 AV63C2.lll,
Hl03D8.lDll, and HB9570.
of ascites was 9 days.
The average time for development
Following clarification by
centrifugation at 15,000 X g for 15 minutes at 23°C,
ascites fluids produced by hybridomas were pooled and
stored frozen at -20 “C.
Purified AI-4 and AI-11 monoclonal antibodies
from the hybridomas were prepared by fast protein liquid
chromatography (FPLC) using a Pharmacia Mono Q HRS/5 anion
exchange column (Pharmacia Fine Chemicals, Piscataway, NJ)
using a O-0.5 molar (M) Nacl gradient in 10 mM Tris, pH 8.0
following directions supplied with the column. Purified
Mabs were concentrated using an Amicon stirred
ultrafiltration cell (Danvers, MA; PM 30 membrane) to a
concentration of 1 mg/ml, dialyzed into PBS (phosphate-
buffered saline, pH 7.2) and stored at —70°C.
Hybridomas secreting anti—peptide antibodies as
described in Example 6A are injected into 10-week old
Balb/c mice as described hereinbefore to obtain ascites
fluid.
prepared by FPLC as described hereinbefore.
Purified anti—peptide monoclonal antibodies are
Purified Mabs
are concentrated in an Amicon stirred ultrafiltration cell
and stored as described hereinbefore.
. Radioiodination
Radioiodination of HDL, Apo AI and immunochemically
purified goat anti—mouse Ig was performed enzymatically
utilizing the Iodogen iodination procedure and Iodogen
obtained from Pierce Biochemicals. The Iodogen iodination
was utilized to characterize the antigens and antibodies
for the solid phase radioimmunoassay as discussed below.
. Apo AI—Cvanoqen Bromide Fraqment Specificity
The Apo AI CNBr fragment specificity of MAB AI-4 was
determined by Western blot analysis
according to the method in Curtiss et al., Proceeding of
the Workshop on Lipoprotein Heterogeneity, Ed. by Lippel,
NIH Publication NO. 87-2646 p. 363-377 (1987).
CNBr fragmentation was performed on isolated Apo AI
CNBr was added in a 13,000
Briefly
dissolved in 90% formic acid.
molar excess and the reaction mixture was maintained about
hours at about 20 degrees C. Following lyophilization,
the resulting CNBr fragments were solubilized in 1% SDS,
.01 M Tris, pH 8.2 and subjected to isoelectric focusing
in 6% polyacrylamide slab gels containing 8 M urea and 2%
ampholine (pH 4 to pH 6) as described by Curtiss et al., J;
Biol. Chem., 260:2982-93 (1985).
separated proteins were transferred to nitrocellulose for
Electrophoretically
immunoreaction with MAB AI—4.
Production of immunoreaction products was detected by
radioiodinated goat anti—mouse Ig followed by
autoradiography.
The results of these studies indicate that MAB AI—4
does not immunoreact with Apo AI CNBr fragments CNBrl,
CNBr2, CNBr3 and CNBr4 but does immunoreact with CNBr2-
CNBr3.
AI—4 also immunoreacts with isolated Apo AI.
These CNBr immunoreactant results indicate that MAB
The results of these studies indicate that MAB AI-18
does not immunoreact with Apo AI CNBr fragments CNBr1,
These
CNBr immunoreactant results indicate that MAB AI-18 also
CNBr3 and CNBr4 but does immunoreact with CNBR2.
immunoreacts with isolated Apo AI.
9. Solid-Phase Polvoeptide ELISA
Apo AI polypeptides AI84—ll1, AI85-111, AI90-111,
AI93-lll, AI94-lll, AI125, AI96-lll, AI99-111, and
AI99-114 were tested for immunoreactivity with monoclonal
antibody AI—ll in a direct binding ELISA. In the assay, 50
pg/ml of each polypeptide was dissolved in PBS to form a
peptide coating solution, of which 150 pl was admixed into
the wells of a flexible polyvinyl chloride microtiter plate
(Immulon). The wells were then maintained about 16 to 20
at 4°C to permit the peptide to absorb onto (coat) the
walls of the wells.
solution by shaking, the wells were washed once with 350 pl
After removing the peptide coating
of rinsing buffer (PBS containing 1 g/l BSA, 0.5 ml/l Tween
, and 2 pl/1 aprotinin). Excess protein binding sites
were blocked by admixing 200 pl of blocking buffer (PBS
containing 3% BSA) into each well, maintaining the wells
for 1 hour at 37’C, removing the blocking buffer by
shaking, and then washing the wells 3 times as previously
described. The plate was then dried for 1 hour at 37°C
followed by addition of 100 pl of PBS containing 0.5 pg/ml
horseradish peroxidase conjugated AI—ll antibody to form a
solid-liquid phase immunoreaction admixture. The resulting
solid-liquid phase immunoreaction admixture was maintained
at 20°C for 1 hour to permit formation of a solid-phase
The wells
were then washed 3 times with rinsing buffer to remove
po1ypeptide—containing immunoreaction product.
unbound antibody.
The amount of immunoreaction product present in the
solid phase was then determined by admixing two hundred
microliters of OPD substrate into each well to form a
developing-reaction admixture. The admixture was maintained
for 30 minutes at about 20°C. Subsequently, 50 pl of 4 N
IQSO‘ were admixed into each well to stop the developing-
reaction, and the resulting solution was assayed for
absorbance at 450 nanometers using a microtiter plate
reader (Dynatech) to detect the amount of formed
immunoreaction product.
Apo AI polypeptides AI85-lll, AI94-125, and AI96-111
were found to be specifically immunoreactive with the
monoclonal antibody AI—l1 in the above direct binding
ELISA. The Apo AI polypeptides AI90-lll, AI93-111 and
AI99-114 were also recognized by the antibody but with
decreased specificity. The Apo AI polypeptide AI99—l1l was
not bound by the antibody.
To determine the relative effectiveness of AI—l1
binding to Apo AI synthetic polypeptides, a competition
ELISA was performed with AI96-lll as the test synthetic
polypeptide in comparison to Apo AI—containing serum and
purified Apo AI/HDL.
AI96-111 as described hereinbefore.
Microtiter plates were coated with
After the drying step
of the assay described hereinbefore, 50 pl of a fluid
sample (i.e., an Apo AI-containing fluid sample) or
standard (i.e., an Apo AI polypeptide) to be assayed were
admixed into the polypeptide AI96coated well
simultaneously with 50 pl of HRPO-conjugated AI—11 antibody
to form an immunoreaction admixture. In the assay
described herein, 3 competitors were tested for their
ability to compete for binding of AI-ll to the AI96—l1l
polypeptide coated over a range of dilutions. The
polypeptide AI96—l11 was added in separate coated wells at
a starting concentration of 1 mg/ml and diluted 2-fold
serially 6 times down to a final concentration of 0.0156
mg/ml.
at a starting dilution of 1:10 and diluted 2-fold serially
Apo AI/HDL as
described in Example 2 was added at a starting
Serum samples as described in Example 2 were added
6 times down to a final dilution of 1:320.
concentration of 1 mg/ml and diluted 2-fold 5 times down to
a final concentration of 0.031 mg/ml. The plate was then
incubated for 30 minutes at room temperature. The plate
was washed and the assay developed as described
hereinbefore to determine the amount of immunoreaction
product formed, and thereby the amount of competitor
present in the added fluid sample.
The results of this assay, shown in Figure 2, indicate
that MAB AI—11 immunoreacts with AI96—111 present in the
fluid sample but has a greater affinity for native Apo AI
in serum and purified HDL.
l0. MAB Al—4 and Al—ll Immunoreactivity
A. MAB AI-4
The immunoreactivity of MAB AI-4 for native Apo AI/HDL
and various synthetic polypeptides was examined by a
competitive RIA performed as follows:
One hundred pl of PBS (0.15 M NaCl, 0.01 M NaPO,, pH
7.2) containing 10 pg/ml Apo AI/HDL were admixed to the
wells of microtiter plates. The plates were maintained for
1 hour at 20°C on a rotating platform to allow the Apo
AI/HDL to adhere to the wells and form solid supports.
After aspirating excess liquid from the wells, 200 pl of
block solution (3% BSA, 3% NGS in PBS) was admixed to each
well, and the wells were maintained for 30 minutes at 20°C
on a rotating platform. Subsequently, the blocking
solution was removed by aspiration, and the wells were
washed 3 times with SPRIA buffer.
To each well was then admixed first with 50 pl of PBS
containing 3% BSA and various concentrations of competitor
antigen, i.e., Apo AI/HDL peptide, and, second, 50 pl of
MAB AI-4 in the form of clarified ascites diluted 1:11.25 X
105 in PBS containing 3% BSA to form competitive
immunoreaction admixtures. In control wells, either
competing antigen or antibody was replaced by PBS
containing 3% BSA.
The immunoreaction admixtures were maintained about 16
hours at 4°C on a rotating platform to permit formation of
solid-phase immunoreaction products. After washing the
wells as previously described, 100 pl of ”5I—labeled goat
-goat anti—mouse lg diluted to 2 X 105
anti—mouse Ig (
trichloracetic acid precipitable disintegrations per minute
per 100 pl in PBS containing 3% BSA) were admixed to each
well.
were‘maintained for 4 hours at 4°C on a rotating platform.
The labeling immunoreaction admixtures so formed
Subsequently, the wells were washed with SPRIA buffer as
previously described, and the amount of "SI-labeled solid-
phase immunoreaction product formed was determined in a
gamma counter.
The ability of MAB AI-4 to immunoreact with Apo AI/HDL
was compared by using Apo AI/HDL and various synthetic
peptides as competitors in the above-described RIA. The
results of this study are shown in figure 3. B/Bo
represents corrected CPMs which are plotted against
increasing concentrations of competition in pg/ml. B/Bo
values are determined in the following formula:
(Competitor Sample CPM - 0% CPM)
(100% CPM - 0% CPM)
where 0% CPM is a measure of non—specific background based
on CPM obtained in RIAs where wells coated with Apo AI/HDL
are reacted with the labeled secondary antibody in the
absence of primary antibody and competitor, and where 100%
CPM is a measure of the maximum non-competed binding of
primary antibody to the substrate coated to the wells. The
more efficiently a competitor binds to the primary
The results of the
assay in Figure 3 show that peptide AI94-125 is a better
competitor of AI-4 MAB binding to Apo AI/HDL-coated wells
than is peptide AI99-121 or Apo AI/HDL itself. Peptides
AI90-111, AI93-111, and AI96-lll are ineffective in the
competition assay, thus are not immunoreactive with AI-4
MAB. '
other peptides were evaluated for their ability to
antibody, the lower the B/Bo values.
immunoreact with AI—4 MAB in competitive RIAs described
Peptides AI94-114 and AI99—ll4 exhibited partial
reactivity. Peptides AI79-95, AI68-105, AI74-105, AI87-
105, AI87-lll,'AI90-105, AI93-101, AI95-105, AI96-101,
AIIOO-105, AIlOl-lll, AIIOS-116 and AIll5-126 did not react
with AI-4 MAB.
Apo AI polypeptides immunoreactive with MAB AI-4
herein.
according to the above competition RIA are summarized in
Figure 4. The conserved native epitope defined by MAB AI-4
includes the amino acid residues at positions 99-114 at a
minimum, because peptide AI99—1l4 immunoreacted with MAB
AI-4.
AI-4 nearly as well as Apo AI/HDL, indicating that the
residues 99-121 define a preferred epitope on Apo AI. A
However, peptide AI99-121 immunoreacted with MAB
particularly preferred polypeptide for use in diagnostic
competition ELISA or RIA is peptide AI94—l25.
Peptide AI99-121 was prepared having an E in residue
position 104 in place of the usual residue F and was shown
to immunoreact with MAB AI-4. Thus, Apo AI peptides
defined by MAB AI~4 that have either an E or an F residue
at position 104 will immunoreact with MAB AI—4 and are
useful in diagnostic methods when used in conjunction with
MAB AI-4.
B. MAB AI-ll
The immunoreactivity of MAB AI—11 for native Apo AI/HDL
and various synthetic polypeptides was examined by a
The results
Peptides AI96-lll,
AI90—11l, and AI93-111 are competitive inhibitors of AI—l1
MAB binding to Apo AI/HDL but are less effective than the
The peptide AI99—12l does not bind
other peptides were evaluated for their
competitive RIA as described in Example 10A.
of these assays are shown in Figure 5.
native protein itself.
to the AI-ll MAB.
ability to immunoreact with AI-ll MAB in competitive RIAs
Peptides AI84-lll, AI94—lll and AI94—l25
were tested in the above competitive RIA and also inhibited
the immunoreactivity of MAB AI-11 with Apo AI/HDL.
Peptides AI79-95, AI87-105, AI87-lll (with an E instead of
an F residue at position 104), AI90—105, AI93-101, A195-
lO5, AI96-101, AI99-121, AIl0O-105, AIl0l-111, AIlO5-116,
and AIll5—126 do not immunoreact with AI-11 MAB.
‘Apo AI polypeptides immunoreactive with MAB AI-ll
described herein.
according to the above competition RIA are summarized in
Figure 6. The conserved native epitope defined by MAB AI-
includes the amino acid residues at positions 96-111,
because peptide A196-111 immunoreacted with MAB AI-11.
. Inhibition of LCAT-Mediated Cholesterol
Esterification by Apo AI Specific Antibodies
Proteoliposomes containing lecithin, 14-C-
cholesterol and Apo AI were prepared in the following
manner. 7.7 mg of phosphatidylcholine (egg yolk lecithin)
were dried under nitrogen gas in a 13 X 100 glass test
tube.
ethanol and 78.3 pg of 14-C—cholesterol from a 0.29 mg/ml
pg of cholesterol from a 1 mg/ml solution in
solution in benzene with a specific activity of 0.04 mCi/ml
were dried in the same tube without coming in contact with
the dried lecithin. To this tube was added 0.3 ml of a 725
mM solption.of sodium cholate in Tris Hcl buffer (10 mM
Tris, 140 mM Nacl, 1 mM EDTA-tetrasodium salt, pH 7.2), 2.5
ml Tris HCl buffer and 0.8 ml of a 1 mg/ml solution of
purified Apo AI in Tris HCl buffer. The admixture was
vortexed for 60 seconds and then mixed on a rotating wheel
at room temperature for 1 hour. The admixture was dialyzed
overnight against 500 ml of Tris Hcl buffer which was
changed 5 times. The proteoliposomes were adjusted to a
volume of 4 ml after dialysis and stored in the
refrigerator. The 4 mls of proteoliposomes contained 9.78
X 10“ mole lecithin, 3.0 X 10” mole cholesterol, 2.01 X lo"
7 mole 14-C—cholesterol and 3.14 x 10” mole Apo AI.
ratios of lecithin:cholesterol:Apo AI were 250:12.5:O.8.
Molar
The LCAT-mediated cholesterol esterification assay, and
including inhibition of esterification using anti-Apo AI
antibodies, was performed in duplicate. To glass screw—cap
tubes was added 100 pl of the prepared proteoliposome
solution, 125 pl of a 2% solution of human serum albumin in
Tris HCl buffer, monoclonal antibodies in amounts from 15.6
pg to 500 pg/tube and Tris HCl buffer to a final volume of
455 pl.
minutes in a 37°C water bath.
The tubes were capped, vortexed and maintained for
To the tubes was added 25
pl of a 100 mM solution of mercaptoethanol in Tris Hcl
buffer and 30 pl of lipoprotein depleted plasma (LPDP) as
the source of LCAT.
and maintained for 1 hour in a 37°C water bath, after which
The tubes were again capped, vortexed
time the enzyme reaction was stopped by addition of 2 ml of
ethanol. Controls for the assay included duplicate tubes
without LPDP and duplicate tubes with LPDP, but no antibody
which will result in 100% esterification.
Cholesterol and cholesterol esters were extracted from
the reaction by adding to each tube 5 ml hexane containing
16 pg/ml cholesterol and 16 pg/ml cholesteryl linoleate.
The admixture was vortexed for 20 seconds, and the upper
layer was removed to a 13 X 100 glass tube. To the
original reaction mixture was added 3 ml more of the
hexane/cholesterol/cholesteryl linoleate solution. This
admixture was vortexed, and the upper layer was removed and
added to the first extraction. The contents of both
extractions were dried under nitrogen gas or evaporated
overnight.
The dried contents of the tubes containing the
extracted material was dissolved in 50 pl of chloroform and
spotted onto Empore thin layer chromatography sheets (TLC).
The tube was rinsed with an additional 50 pl and added to
the first spot. The TLC sheets were developed in a solvent
of hexanezethyl ether in a 60:40 ratio. The sheets were
air dried and exposed to iodine to visualize the origins.
The sheets were then placed on Kodak X-OMAT autoradiography
Areas on the TLC sheets
corresponding to radioactive signals on the film were cut
for an overnight exposure.
and placed in scintillation vials with 3 ml of scintillant
(PPD-PDPDP in toluene).
detected in a beta counter.
The 14-C radioactive label was
Fractional esterification
rates (FER) were determined from the scintillation counts
where FER is expressed as cholesteryl ester cpm over
cholesterol plus cholesteryl ester cpm.
The results of LCAT—mediated cholesterol esterification
assays in which five monoclonal antibodies, AI—4, AI—9, AI-
ll, AI-16 and AI-18, were tested for their ability to
inhibit cholesterol esterification is shown in Figure 7.
Purified MABS AI-4 and AI—11 were prepared as described
Purified MABs AI-9, AI-16 and AI—l8 were
generated from fusions of splenocytes from mice immunized
with Apo AI/HDL similarly to the methods described in
The MABS AI-9 and AI-16 were used as control
MABs in the LCAT-mediated cholesterol esterification assay
in Example 6C.
Example 6C.
and are known to immunoreact with CNBr4 and the Apo AI
The MAB
AI—18 immunoreacts with the Apo AI amino acid residue
amino acid residues sequence AI1—l5, respectively.
sequence AI95—105.
In this assay, 250 pg of each antibody was evaluated.
, At this amount, the antibodies and Apo AI were in equal
molar concentrations. Data for each monoclonal antibody is
expressed as percent of control where control is the FER in
the absence of antibody but in the presence of LPDP. The
control FERS for the results range from 0.0626 to 0.0948
per hour. Each bar represents data from two experiments,
and each experiment is done in duplicate. Monoclonal
antibody AI—1l was the most effective inhibitor of
cholesterol esterification followed by AI-4. Monoclonal
antibodies AI-16 and AI—l8 inhibited esterification by an
equal amount. Monoclonal AI-9 was ineffective at blocking
cholesterol esterification.
These data show that the Apo AI epitopes
recognized by MAB AI—4, and particularly by
MAB AI—ll comprise a part of the Apo AI molecule that is
involved in the process by which Apo AI normally increases
esterification of cholesterol. Thus, polypeptides which
immunologically mimic the epitope defined by MAB AI—4, MAB
AI—18, or MAB AI—l1 represent useful Apo AI polypeptides
that will function as an analog of Apo AI in the capacity
of Apo AI to increase esterification of cholesterol. That
is, Apo AI polypeptides of this invention can be used
similarly to Apo AI itself to increase cholesterol
esterification.
The foregoing specification, including the specific
embodiments and examples, is intended to be illustrative of
the present invention and is not to be taken as limiting.
Numerous other variations and modifications can be effected
without departing from the true spirit and scope of the
present invention.
Claims (26)
1. A diagnostic system, in kit form, comprising, in an amount sufficient to perform at least one assay, an antibody containing anti-Apo AI antibody molecules that immunoreact with: (a) (b) Apo AI/HDL, isolated Apo AI, (c) Apo AI CNBr2, and (d) the polypeptide KVQPYLDDFQKKWQEE, immunoreact with: (e) Apo AI CNBr1, (f) Apo AI CNBr3, (g) the polypeptide SKDLEEVKAKVQPYLDDEQKKWQEE, (h) the polypeptide SKDLEEVKAKVQPYLDDFQ, and (i) the polypeptide PYLDDFQKKWQEEMELYRQKVEP. but do not and
2. The diagnostic system of claim 1 wherein said antibody molecules are operatively linked to an enzyme indicating means .
3. The diagnostic system of claim 2 wherein said antibody molecules are monoclonal antibody molecules.
4. The diagnostic system of claim 3 wherein said antibody molecules are those produced by the hybridoma having ATCC designation HB9201.
5. The diagnostic system of claim 1 further comprising, inyan amount sufficient to perform at least one assay, an Apo AI polypeptide Comprising no more than 25 amino acid residues and including an amino acid residue sequence -KVQPYLDDFQKKWQEE-, said polypeptide is free of homoserine lactone. represented by the formula: wherein
6. The diagnostic" system of claim. 5 wherein said .polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: QEMSKDLEEVKAKVQPYLDDFQKKWQEE,. EMSKDLEEVKAKVQPYLDDFQKKWQEE, LEEVKAKVQPYLDDFQKKWQEE, VKAKVQPYLDDFQKKWQEE, _ KAKVQPYLDDFQKKWQEE, KAKVQPYLDDFQKKWQEEMELYRQKVEPLRAE, and KVQPYLDDFQKKWQEE.
7. The diagnostic system of claim 5 wherein said polypeptide is operatively linked to a solid matrix.
8. A diagnostic system, in kit form, comprising, in an amount sufficient to perform at least one assay, an antibody containing anti-Apo AI antibody molecules that immunoreact with: (a) Apo AI/HDL, (b). isolated Apo AI, (c) Apo AI CNBr2—CNBr3, and (d) the polypeptide PYLDDFQKKWQEEMELYRQKVEP, but do not immunoreact with: (e) Apo AI CNBrl, (f) the polypeptide SKDLEEVKAKVQPYLDDFQKKWQEE, (g) the polypeptide LEEVKAKVQPYLDDFQKKWQEE, and (h) the polypeptide YRQKVEPLRAEL.
9. The diagnostic system of claim 8 wherein said antibody molecules are operatively linked to an enzyme indicating means .
10. molecules are monoclonal antibody molecules. The diagnostic system of claim 8 wherein said antibody
11. antibody molecules are those produced by the hybridoma having ATCC designation HB8744. The diagnostic system of claim 10 wherein said
12. The diagnostic system of claim 8 further comprising, in an amount sufficient to perform at least one assay, an" Apo AI polypeptide comprising no more than about 60 amino acid residues and including an amino acid residue sequence represented by the formula: —§YLDDXQKKWQEEMEL-, wherein X is either E or F.
13. The diagnostic system of claim 12 fiwherein said polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: KAKVQPYLDDXQKKWQEEMEL, KAKVQPYLDDXQKKWQEEMELYRQKVEPLRAE, QPYLDDXQKKWQEEMEL, QPYLDDXQKKWQEEMELYRQKVEP, PYLDDXQKKWQEEMEL, and PYLDDXQKKWQEEMELYRQKVEP. system of claim 12 wherein said
14. The diagnostic polypeptide is operatively linked to a solid matrix.
15. A method of assaying" the amount of Apo AI in a vascular fluid sample comprising the steps of: (3) a vascular fluid sample with: (i) molecules that immunoreact with: forming an immunoreaction admixture by admixing an anti—Apo AI antibody containing antibody (1) Apo AI/HDL, (2) isolated Apo AI, (3) Apo AI CNBr2, and (4) the polypeptide KVQPYLDDFQKKWQEE but do not immunoreact with: (5) Apo AI CNBr1, (6) Apo AI CNBr3, (7) the polypeptide SKDLEEVKAKVQPYLDDEQKKWQEE, (8) the polypeptide SKDLEEVKAKVQPYLDDFQ, and (9) the polypeptide PYLDDFQKKWQEEMELYRQKVEP; and (ii) an Apo AI polypeptide comprising no more than 25 amino acid residues and including an amino acid 69 residue sequence represented by —KVQPYLDDFQKKWQEE-, said polypeptide being operatively linked to a solid matrix such that the immunoreaction admixture has both a liquid phase and a solid phase; (b) maintaining said immunoreaction admixture for a sufficient to the formula: time period form an Apo KAI-containing immunoreaction product in the solid phase, and (c) determining the amount of product formed in step (b) -
16. has an amino acid residue sequence represented by a formula The method of claim 15 wherein said Apo AI polypeptide selected from the group consisting of: QEMSKDLEEVKAKVQPYLDDFQKKWQEE, EMSKDLEEVKAKVQPYLDDFQKKWQEE, LEEVKAKVQPYLDDFQKKWQEE, VKAKVQPYLDDFQKKWQEE, KAKVQPYLDDFQKKWQEE, KAKVQPYLDlJFQKKWQEEMELYRQKVEPLRAE, and KVQPYLDDFQKKWQEE.
17. operatively linked to an enzyme label, The method of claim 15 wherein said antibody is and said product formed in step (b) is a labeled immunoreaction product.
18. The method of claim 15 wherein said antibody molecules are those produced by the hybridoma having ATCC designation HB920l .
19. vascular fluid sample comprising the steps of: (a) a vascular fluid-sample with: (i) A method of assaying the amount of Apo AI in a forming an immunoreaction admixture by admixing an anti-Apo AI antibody containing antibody ‘molecules that immunoreact with: '(.I) (2) (3) Apo AI/HDL, isolated Apo AI; Apo AI CNBr2—CNBr3, and’ (4) the polypeptide PYLDDFQKKWQEEMELYRQKVEP, but do not immunoreact with: (5) Apo AI CNBr1, (6) the polypeptide SKDLEEVKAKVQPYLDDFQKKWQEE, (7) the polypeptide LEEVKAKVQPYLDDFQKKWQEE, and (8) the polypeptide YRQKVEPLRAEL; (ii) an Apo AI polypeptide comprising no more than about 60 amino acid residues and including an amino acid residue sequence represented by the formula: —PYLDDXQKKWQEEMELYRQKVEP—, wherein X is either E or F, said polypeptide being operatively linked to a solid support such that the immunoreaction admixture has both a liquid phase and a solid phase; (h) period maintaining said immunoreaction admixture for a time sufficient to form an Apo AI-containing immunoreaction product in the solid phase, and (C) determining the amount of product formed in step (b)-
20. The method of claim 19 wherein said Apo AI polypeptide has an amino acid residue sequence represented by a formula selected from the group consisting of: KAKVQPYLDDXQKKWQEEMEL, KAKVQPYLDDXQKKWQEEMELYRQKVEPLRAE, QPYLDDXQKKWQEEEEL, QPYLDDXQKKWQEEMELYRQKVEP, PYLDDXQKKWQEEMEL, PYLDDXQKKWQEEMELYRQKVEP. and
21. y The’ method of claim 19 wherein said antibody is operatively linked to an enzyme label, and said product formed in step (b) is a labeled immunoreaction product.
22. are those produced by the hybridoma having ATCC designation HB8744. The method of claim 19 wherein said antibody molecules 71
23. amino acid residues and including an amino acid residue -KVQBYLDDFQKKWQEE-, for use in increasing esterified cholesterol in a patient. An Apo AI polypeptide comprising no more than 25 sequence represented by the formula:
24. acid residue sequence represented by a formula selected from A polypeptide according to claim 23 having an amino the group consisting of: QEMSKDLEEVKAKVQPYLDDFQKKWQEE, EMSKDLEEVKAKVQPYLDDFQKKWQEE, LEEVKAKVQPYLDDFQKKWQEE, VKAKVQPYLDDFQKKWQEE, KAKVQPYLDDFQKKWQEE, KAKVQPYLDDFQKKWQEEMELYRQKVEPLRAE, KVQPYLDDFQKKWQEE. and
25. acid residues and including an amino acid residue sequence -PYLDDXQKKWQEEMEL-, wherein X increasing esterified An Apo AI polypeptide comprising no more than 60 amino represented by the formula: is either E or F, for use in cholesterol in a patient.
26. acid residue sequence represented by a formula selected from A polypeptide according to claim 25 having an amino the group consisting of: KAKVQPYLDDXQKKWQEEMEL, KAKVQPYLDDXQKKWQEEMELYRQKVEPLRAE, QPYLDDXQKKWQEEMEL, QPYLDDXQKKWQEEMELYRQKVEP, “PYLDDXQKKWQEEMEL, and PYLDDXQKKWQEEMELYRQKVEP. F. R. KELLY & co., AGENTS FOR THE APPLICANTS
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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USUNITEDSTATESOFAMERICA07/06/19905 | |||
US53476190A | 1990-06-07 | 1990-06-07 |
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IE83756B1 true IE83756B1 (en) | |
IE911922A1 IE911922A1 (en) | 1991-12-18 |
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Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
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IE192291A IE911922A1 (en) | 1990-06-07 | 1991-06-05 | Apo ai polypeptides, diagnostic methods and systems for¹quantifying apo ai, and therapeutic methods |
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EP (1) | EP0500826A4 (en) |
JP (1) | JPH06508344A (en) |
CA (1) | CA2084677C (en) |
IE (1) | IE911922A1 (en) |
PT (1) | PT97914B (en) |
WO (1) | WO1991018619A1 (en) |
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US7125678B2 (en) | 2001-11-23 | 2006-10-24 | Nanogen, Inc. | Protein biopolymer markers predictive of type II diabetes |
PT2170956E (en) | 2007-06-15 | 2015-02-05 | Medigene Ag | Treatment of tumors using specific anti-l1 antibody |
NO335243B1 (en) | 2008-01-08 | 2014-10-27 | Isola As | Cradle plate, as well as the application and method of laying a floor covering using such a cradle plate. |
WO2018181935A1 (en) * | 2017-03-30 | 2018-10-04 | シスメックス株式会社 | Anti-apoa1 antibody |
Family Cites Families (2)
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US5055396A (en) * | 1987-11-03 | 1991-10-08 | Scripps Clinic And Research Foundation | Diagnostic methods and systems for quantifying apo ai |
EP0364560A4 (en) * | 1988-03-29 | 1990-12-05 | Scripps Clinic And Research Foundation | Diagnostic methods and systems for quantifying apo ai |
-
1991
- 1991-06-05 IE IE192291A patent/IE911922A1/en not_active IP Right Cessation
- 1991-06-07 EP EP19910912441 patent/EP0500826A4/en not_active Ceased
- 1991-06-07 CA CA002084677A patent/CA2084677C/en not_active Expired - Fee Related
- 1991-06-07 WO PCT/US1991/004038 patent/WO1991018619A1/en not_active Application Discontinuation
- 1991-06-07 JP JP3511924A patent/JPH06508344A/en active Pending
- 1991-06-07 PT PT97914A patent/PT97914B/en not_active IP Right Cessation
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