Note: Descriptions are shown in the official language in which they were submitted.
2~2~ .~2~
POLYPEPTIDE ANALOGS OF APOLIPOPROTEIN E,
DIAGNOSTIC SYSTEMS AND METHODS USING THE ANALOGS
Descri~tion
Technical Field
The present invention relates to
polypeptides capable of mimicking the ability of
apolipoprotein (apo) E to induce differentiated
cellular function. More particularly, the present
invention relates to a polypeptide agonist of Apo E
useful for inhibiting lymphocyte proliferation and/or
ovarian androgen secretion. The present invention
also relates to enhancement of cholesterol clearance
mediated by these receptor-binding (receptor-
competent) polypeptides. In that regard, the presentinvention relates to the use of synthetic tandem
peptides to modulate hepatic cell uptake of
cholesterol-containing proteins and to diagnostic
methods using these peptides and their antibodies to
evaluate efficacy of these therapeutic measures.
Backqround
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.
Original classification of lipoproteins was based on
their buoyant densities as measured by
ultracentrifugation. Accordingly, four major density
classes have been recognized, and subclasses within
these exist.
The first class comprises the chylomicrons.
They are the largest of the lipoproteins and are rich
in triglycerides. The site of origin of the
2 ~ 2 ~
chylomicrons is the intestine. When chylomicrons are
exposed to plasma or high density lipoprotein (HDL) ln
vitro, much of their complement of A apolipoproteins
is lost, and C and E apolipoproteins are acquired.
Chylomicrons also contain apolipoprotein B-48.
The next class of lipoproteins, the very low
density lipoproteins (VLDL), is comprised of particles
made in the liver and involved in triglyceride
metabolism and transport from the liver. The
apolipoproteins, apo B-100 and apo E, are the major
constituents of the VLDL particle.
The third lipoprotein class, comprising low
density lipoproteins (LDL), is a specific product of
the catabolism of VLDL. The predominant
apolipoprotein in LDL particles is apolipoprotein ~-
100, or apo ~-100.
The fourth class, high density lipoprotein
(HDL) contains two major apolipoproteins, apo A-I and
apo A-II. One function of apo A-I is the activation
of the plasma enzyme, lecithin-cholesterol
acyltransferase, which is required for the
esterification of free cholesterol on HDL for
transport to the liver.
Plasma cholesterol is regulated in part by
the LDL receptor and in part on the ability of the
lipoprotein to carry cholesterol and bind the LDL
receptor. Hofmann, et al., Science, 239:1277 (1988).
This receptor is found on the surface of all cells
where it mediates binding and internalization of the
cholesterol-rich lipoproteins that provide membrane
cholesterol: Brown, et al., J. Clin, Invest., 55:783
tl975); Goldstein, et al., Methods Enzymol., 98:241
(1983); and in some specialized cells substrate
cholesterol for the production of bile acids or
steroid hormones; Gwynne, et al., Endocr. Rev., 3:299
2~2~2~
(1982). LDL receptor expression on cells is inversely
regulated by the circulating concentration of LDL,
i.e., the higher the circulating LDL the fewer LDL
receptors on the cell surface Hofmann, et al.,
S Science, 239:1277 (1988). LDL binding to the LDL
receptor has been the focus of much research because
of its importance in regulating the level of plasma
cholesterol, which is considered a major risk factor
for the development of coronary artery disease; Brown,
et al., Scient. Amer., 251:58 (19~4).
LDL binding to the LDL receptor is believed
to be dependent upon the species of apolipoprotein
present in the lipoprotein particle.
Apolipoproteins are lipid-free protein
components of plasma or serum lipoproteins obtained by
treating intact lipoproteins with organic solvents,
detergents, or chaotropic agents. Not all proteins
captured with lipoproteins necessarily have a role in
lipid transport. A pertinent example is the recent
recognition that serum amyloid A proteins, acute phase
reactants, are transported in plasma bound to HDL.
These low molecular weight proteins may comprise up to
30 percent of apo-HDL in inflammatory states, but it
is doubtful that they have specific lipid transport
roles.
Apo B-100 is recognized and bound by cell
LDL receptors. By binding to apo B-100, these
receptors extract LDL pArticles from plasma. The LDL
is thereby taken into a cell and broken down, yielding
its cholesterol to serve the cell'~ needs. The
interaction between apo B-100 and the LDL receptor
thus plays a ma;or role in removal of LDL cholesterol
from the bloodstream. All LDL particles contain apo
B-100.
2 ~ 2 ~ ~ r ~
When compared to apolipoproteins A-l or B,
the relative concentration of apo E in plasma is low.
However, apo E is instrumental in lipoprotein
metabolism in several ways. Mahley, et al., J? Lipid
Res., 25:1277-1294 (1984). It is a recognition site
for several cellular lipoprotein receptors, including
hepatocyte receptors for chylomicron and VLDL remnants
[Hui, et al., J. Biol. Chem., 259:860-869 (1984~;
Shelburne, et al., J. Clin. Invest., 65:652-658
(1980)], receptors for LDL on hepatic and extrahepatic
cells [Hui, et al., J._Biol. Chem., 256:5646-S655
(1981)] and receptors for VLDL on macrophages [Wang-
Iverson et al., Biochem. Bio~hvs. Res. Commun.,
126:578-586 (1985)].
Apo E may also play a role in lipoprotein
lipase-mediated lipolysis of lipoproteins [Yamada, et
al., Biochem. BioPhYs. Res. Com~un., 94:710-715
(1980); Ehnholm, et al., Proc. Natl. Acad. Sci. USA,
81:5566-5570 (1984)] and facilitate transport of
sterols under certain conditions [Fielding et al.,
Metabolism, 31:1023-1028 (1982)].
About half of apo E is associated with VLDL
and the other half with HDL-sized particles. Gibson,
et al., Biochem. BioPhys. Acta., 835:113-123 (1985).
There is virtually no apo E in plasma which is not
associated with lipoproteins.
There are significant differences in the
disposition of certain apo E epitopes on various
lipoproteins, affecting their physiological functions.
The immunochemical data are in accord with
observations that lipoprotein lipids do modulate the
conformation of apo E, as detected by circular
dichroism ~Chen, et al., Bioçhemistrv, 23:6530-6538
(1984)] and optical rotary dispersion [Klimov, et al.,
Mol. Biol., 18:404-409 (1984)]. The heterogeneity of
2~P~ 3~
apo E conformation or disposition on the surfaces of
lipoproteins is also confirmed by the varying
accessibility to thrombin cleavage of apo E on
differently sized VLDL particles. Bradley, et al., J.
5 Biol. Chem., 259:14728-14735 (1984).,
Lipoproteins are cleared from the
plasma by binding to high-affinity receptors on liver
cells and extrahepatic tissues such as the adrenal
glands and ovaries. Kowal, R.C. et al., Proc. Natl.
Acad. Sci. USA, 86:5810-5814, (1989). The LDL
receptor specifically binds apo B and apo E-bearing
lipoproteins. R. W. Mahley, Science, 240:622 (1988).
Thus, apo E is of clinical importance for its role in
binding LDL receptor and facilitating cholesterol
clearance.
The LDL receptor-binding region of apo E has
been mapped to an internal sequence including amino
acid residues 140 to 160. Weisgraber, et al., J.
8iol. Chem. 258:12348 (1983). Additionally, apo E
binds the LDL receptor only when it is associated with
a lipoprotein or phospholipid ~Innerarity, et al., J.
~iol. Chem., 254:4186 (1979)] and 4 apo E molecules
bind the LDL receptor with an affinity that is 10 to
25-fold greater than the binding of a single molecule
of apo B. R. W. Mahley, Science, 240:622 (1988~.
Two distinct sets of receptors bind apo E-
containing lipoproteins. The LDL receptor [Yamamoto
et al., ~ , 39:27-38 (1984)], 70% of which is
thought to be located on hepatic cells, binds VLDL and
apo E-containing remnants o~ chylomlcrons. The
existence o~ a sQcond set of LDL receptors, termed
~remnant receptors~, is inferred from studies showing
that the plasma clearance of apo E-containing
chylomicron remnants occur~ at normal rates in animals
with genetically defective LDL receptors.
2~235~;
Recently, an LDL receptor-related protein
(LRP) has been found on the surface of hepatic cells.
Herz et al., E~BO, 7:4119-4127 (1988). LRP shares
cysteine-repeat sequences with LDL and has been shown
to bind and mediate the extracellular clearance of apo
E-containing lipoproteins. Xowal, R.C. et al. Proc.
Natl. Acad. Sci. USA, 86:5810-5814, (19B9).
Apo E-enriched lipoproteins have also been
described to have a function in the immune system by
inhibiting mitogen-or antigen-stimulated lymphocyte
proliferation in ~itro and in vivo. In the ovaryl apo
E inhibits androgen production by LH-stimulated
cultured theca and interstitial cells; Dyer, et al.,
J Biol. Chem., 263:10965 (1988).
In 1976 it was reported that a discrete
lipoprotein fraction isolated from normal human plasma
inhibited mitogen- and allogenic cell-stimulated human
lymphocyte proliferation in vitro (Curtiss et al., J.
Immunol., 116:1452, (1976)). This inhibitory plasma
lipoprotein wa~ termed LDL-In for Low Density
Lipoprotein-Inhibitor because the active fraction is
localized to a less dense subfraction of total LDL of
density 1.006-1.063 g/ml. The characteristics of LDL-
In-mediated inhibition in vitro are as follows: LDL-
In has comparable inhibitory activity forphytohemagglutinin (PHA), pokeweed mitogen (PWM), and
allogenic cell-stimulated human lymphocyte
proliferation. The inhibitory activity of LDL-In
isnon-toxic and independent o~ mitogen concentration.
Suppression by LDL-In is time dependent and
approximately 18 hr of exposure of the lipoprotein to
the lymphocytes before stimulation is required for
maximum induction of a stable suppressed state. LDL-
In does not inhibit ~H-thymidine uptake when it is
added to the cultures 18-20 hr after stimulation,
2 ~c3~ ~
suggesting that this lipoprotein influences metabolic
events associated with an early inductive phase of
lymphocyte activation.
The immunosuppressive activity of LDL-In has
been studied in a number of systems both in vitro and
in vivo. To summarize, in vitro activities of LDL-In
include suppression of: a) mitoqen stimulated 3H-
thymidine uptake, Cuxtiss et al., J. Immunol.,
116:1452, (1976), b) allogenic cell-stimulated 3H-
thymidine uptake (Curtiss et al., J. Immunol.,
116:1452, (1976), Curtiss et al., J. Immunol.,
118:1966, (1977)), c) the primary generation of
cytotoxic T cells (Edgington et al., Requlatory
Mechanisms in Ly~phQcvte Activation: Proceedinas of
the Eleventh Leukocvte Culture Conference., D.O.
Lucas, ed. Academic Press, New York, pp. 736, (1977)),
d) pokeweed mitogen stimulated immunoglobulin
synthesis (Curtiss et al., J. Clin. Invest., 63:193,
(1979)), and e) B-cell Epstein Barr Virus
transformation (Chisari et al., J. Clin. Invest.,
68:329, (1981)). In vivo LDL-In has been shown to
inhibit: a) the primary humoral immune response to
sheep red blood cells (Curtiss et al., J. Immunol.,
118:648, (1977), DeHeer et al., Immuno~harmacoloqy,
2:9, (1979), Curtiss et al., Cell. Immunol., 49:1,
(1980)), b) the primary generation of cytotoxic T-
cells (Edgington et al., RequlatorY Mechanisms in
LYm~hocvte Activation: Proceedinqs of the Eleventh
LeukocYte Culture Conference., D.O. Lucas, ed.
Academic Press, New York, pp. 736, (1977)), and c)
immunologic attention of tumor growth (Edgington et
al., Cancer Res., 41:3786, (1981), Edgington et al.,
DietarY_Fats and Health., ACOS Monograph No~ 10,
Perkins and Visek, eds., pp. 901, (1981)).
2~23~
The effects of lipoproteins on immune cell
function in vivo are exceedingly complex. A major
finding of the investigation of the physiologic
implications of immunosuppression by LDL-In in vivo is
that the observed functional outcome is strikingly
dose dependent. This important concept is best
illustrated by describing in more detail studies of
the effects of LDL-In on the survival of experimentai
animals challenged with syngeneic tumors (Edgington et
al., Cancer Res., 41:3786, (1981), Edgington et al.,
Dietarv Fats and Health., ACOS Monograph No. 10,
Perkins and Visek, eds., pp. 901, (1981)~. Seemingly
divergent effects of LDL-In are observed on the growth
of the syngeneic SaD2 fibrosarcoma in DBA/2 mice. The
growth of 1 x 105 viable tumor cells in control mice
without immunoprotection (i.e., 10-days prior
immunization with 10-6 irradiated tumor cells) is
detectable at 25 days and proceeds rapidly until death
at about 43 days. In contrast, tumor growth is slower
in immunoprotected mice. This tumor growth is
characterized by a reduction in tumor mass of at least
a half and no deaths by day 60. Intravenous
administration of high doses of LDL-In 24 hr before
immunoprotection with killed tumor cells abolishes the
protective effect of immunization. This dose
corresponds to a dose that is required to abolish both
B-cell and T-cell effector cell functions. The
administration o~ an intermediate dose of LDL-In
before immunoprotection with the killed tumor cells
has no discernable e~fect on the subsequent growth of
the viable tumor cell challenge. In contrast,
intravenous administration of even lower doses of LDL-
In 24 hr before immunoprotection with killed tumor
cells results in the enhancement of tumor rejection
and host survival. This dose of LDL-In is concordant
2~23 ~
with the dose required for selective inhibition of
suppressor cell function in vitro (Curtiss et al., J.
Clin. Invest., 63:193, (1979)). Thucl depending upon
the amount of immunoregulatory lipoprotein that a
particular lymphocyte population is exposed to ln
vivo, very different functional outcomes will result.
Plasma lipoproteins differ from most humoral
immunoregulatory molecules in that they are large
heterogenous non-covalent complexes of lipid and
protein. An important step to understanding the
mechanism of lipoprotein regulation of cell function
is an identification of the constituent(s) of the
lipoprotein particle that mediate the observed
biologic effects. Plasma lipoproteins contain various
amounts of apoproteins, glyceride, free and esterified
cholesterol, phospholipid, glycolipid and free fatty
acid. Many of these constituents of lipoproteins can
by themselves influence cell function.
The heterogeneity of apoproteins in terms of
structure, exposure and function make them likely
candidates as biologically important constituents of
LDL-In, and the contribution of the apoproteins to
biologic activity has been extensively studied. LDL-
In contains Apo B, apo E, and each of the C
apoproteins. The specific role played by Apo B and
apo E in LDL-In was investi~ated immunochemically
using Apo B-specific and apo E-specific monoclonal
antibodies (Curtiss et al., Fed. Proc., 40:348,
(1981), Curtiss et al., Atherosclerosis, 2~S):A111,
(1982)). Some, but not all, o~ the Apo B-antibodies
and each o~ the apo E-speci~ic antibodies bind and
facilitat~ the indirect precipitation and removal of
thQ inhibitory activity of LDL-In from a lipoprotein
fraction. These results indicate that LDL-In contains
both apoprotein~ B and E, but they do not identify
2~23~2~
which apoprotein is important to or required for
activity.
Further substantiation that apo E and Apo B-
containing lipoproteins are important regulators of
lymphocyte function has come from studies of the
inhibitory properties of fetal cord blood plasma
lipoproteins (Curtiss et al., J. Immunol., 133:1379,
(1984)). In these studies a direct correlation
between apo E and inhibition was established. Cord
blood lipoprotein concentrations are lower than those
of adult, i.e., the low density lipoprotein (LDL)
level in cord blood is 30~ that of adult, whereas the
high density lipoprotein (HDL) level is 50% of adult
levels. In contrast, the apo E concentration in fetal
cord blood is 2-fold higher than adult (Curtiss et
al., J. Immunol., 133:1379, (1984)). Therefore, the
capacity of LDL and HDL to inhibit mitogen-stimulated
3H-thymidine uptake in adult peripheral blood
mononuclear cells was used as an in vitro system to
study immunosuppression. Relative to adult
lipoproteins, cord blood LDL and HDL are 2 to 4 times
more potent in inhibiting cellular proliferation.
Radioi~munoassay results demonstrate a strong
correlation between the amount of apo E in cord blood
LDL and HDL and the inhibition of cell proliferation.
Furthermore, selective removal of apo E-containing
lipoproteins decreases the inhibitory capacity of cord
blood LDL and eliminates almost completely inhibition
by HDL. The results indicate that cord blood
lipoproteins containing apo E in as ociation with
either LDL or HDL can suppress the immune response
(Curtiss et al., J. Immunol., 133:1379, (1984)). The
fetus is an allograft to its mother. Therefore the
relatively high fetal levels of apo E may have
functional significance in the establishment of self
2~3~2 ~
11
as well as maintenance of the fetus in_utero.
More recently, the inhibitory ac~ivity of
isolated (lipid-free) apo E has been studied.
ImmunOsuppression was measured as inhibition of 3H-
thymidine uptake by peripheral blsod mononuclear cells(PBM) with phytohemayglutinin (PHA). apo E isolated
from lipoproteins had good activity (i.e.,
approximately 15 ug/ml was required for 50%
inhibition, and maximal inhibition occurred at 20
ug/ml), whereas fractions containing the lipid-free C
apoproteins were not inhibitory at ~ 20 ug~ml (Pepe et
al., J Immunol., 126:3716, (1986)). Suppression of
lymphocyte proliferation by the native lipoprotein,
LDL-In, is irreversible and has distinguishable
temporal requirements (Curtiss et al., J. Immunol.,
116:1452, (1976), Curtiss et al., J. Immunol.,
118:1966, (1977)). Suppression by isolated apo E is
identical. That is, cells exposed to isolated apo E
for 24 hr and washed free of non-cell associated apo E
before mitogen stimulation, remain fully suppressed.
And, maximal inhibition is obtained with either LDL-In
or apo E only after a 24 hr exposure of the cells
before the addition of mitogen. Exposure periods of
18 hr or less result in little or no suppression by
either inhibitor. Furthermore, cells receiving
inhibitors or PHA simultaneously, or cells receiving
either inhibitor after PHA exposure, are fully capable
of responding to mitogen induction, suggesting that
neither LDL-In nor apo E are directly toxic. The
irreversibility and temporal requirements of
suppression confirm that apo E isolated from
lipoproteins has the same characteristics of
immunosuppression as LDL-In and that an active moiety
of LDL-In is apo E ~Pepe et al., J. Immunol.,
126:3716, (1986)).
2~2~
Cardin et al., Biochem. Bio~hvs. Res. Comm.,
154:741-745 (1988) reported that a polypeptide portion
of apo E having an amino acid residue sequence
identical to that of apo E residues 141-lS5 inhibits
lymphocyte proliferation when coupled to bovine serum
albumin (BSA). However, conspicuously absent from the
study of Cardin et al. was any control for cell
viability allowing for a determination of whether or
not the inhibition observed was due to cytotoxicity of
the peptide-BSA conjugate.
By way of further background, Dyer et al.,
J Biol. Chem., 263:10965-10973 (1988) reported that
isolated lipid free rat Apo inhibits androgen
production by the ~larian theca and interstitial cells
induced by the gonadotropin, luteinizing hormone (LH).
Several immunoassays developed for detecting
apo E have been described in recent publications.
Widely differing concentrations of human apo E in
plasma have been reported by researchers using
polyclonal antisera in quantitative immunoassays,
probably because different standards and antisera have
been used. Greg et al., NIH Publication, 83-1266
(1983). Most assays require the use of denaturants or
detergents to nexposen all apo E epitopes in human
plasma and in isolated lipoproteins. This may be
because most of the antisera are produced by
immunization with isolated apo E, which tends to self-
aggregate [Greg et al., NIH Publl~ation, 83-1266
tl983),; Havel, et al., J. ~lin. I~est., 66:1351-1362
(1980)] and to ~orm hetero- and homodimers via
disulfide bridges. Tada, et al., Biochem. Biophys.
Res Comm., 90:297-304 (1979).
Because it is highly likely that these
antisera contain antibody populations directed against
apo E epitopes that are non-existent or nmaskedn in
2 ~ 2 ~
lipid-rich lipoproteins, the use of deter~ents or
denaturants in plasma or intact lipoprotein samples is
necessary, further compounding the problem.
Brief Summary of the Invention
It has now been discovered that the amino
acid residue sequence corresponding to residues 141-
155 of mature apo E can mimic the biological activity
of apo E only when present as a multimeric peptide or
a self-conjugate. It has also been discovered that
the inhibitory effect on lymphocyte proliferation by
peptide-BSA conjugates where the peptide has an amino
acid residue sequence corresponding to residues 141-
155 of apo E is not physiologically specific but
rather due to cytotoxicity. A multimeric apo E
polypeptide is useful for preparing antibodies, and in
diagnostic methodc to monitor the amounts of apo E
antigens in a vascular body fluid.
Thus, the present invention contemplates a
polypeptide analog of apo E characterized by a
plurality of segments each having an amino acid
residue sequence corresponding to residues 141-155 of
apo E, e.g., (LRKLRKRLLRDADDL), where a is an integer
of at least 2 indicating the number of times the
sequence within parenthesis is present within the
primary structure of the polypeptide. In a preferred
embodiment, the present invention contemplates a
tandem peptide representing two repeats of the 141-155
sequence.
In another embodiment, the present invention
contemplates an isolated sel~-con~ugate, i.e.,
polypeptides having corresponding amino acid residue
sequences operatively linked to each other by other
than a peptide bond between the alpha-amino group and
carboxy group of contiguous amino acid residues. The
2 ~ ?~ ~.
conjugate contains a plurality of operatively linked
polypeptides having amino acid residue sequences
corresponding to the sequence of apo E residues 141-
155.
The polypeptides and conjugates of the
present invention are useful agents for modulating
differentiated cell function, such as lymphocyte
proliferation, ovarian androgen production, LDL
binding and degradation, and the like. Therapeutic
compositions containing a polypeptide or conjugate of
this invention in a pharmaceutically acceptable
excipient, typically in unit dose form, are also
contemplated for modulating differentiated cell
function.
Another aspect contemplated by this
invention is a composition comprising antibody
molecules that immunoreact with a polypeptide
containing a plurality of segments each having an
amino acid residue sequence corresponding to the
formula LRKLRKRLLRDADDL (pl41-155) and apo E/VLDL, but
do not immunoreact with the polypeptide p93-112 or
pl72-182 and preferably do not immunoreact with a
polypeptide containing only a monomer of pl41-155.
Further contemplated is a method for
detecting apo E antigens in a vascular fluid sample by
admixing the sample with an anti-apo E antibody to
form an immunoreaction admixture, the antibody
preferably being operatively linked to a solid support
such that the immunoreaction admlxture has both a
liquid phase and a solid phase, and said antibody
containing antibody molecules that immunoreact with a
polypeptide containing a plurality of se~ments each
having an amino acid residue sequence corresponding to
the formula LRXLRXRLLRDADDL and with apo E/VLDL but
does not immunoreact with the polypeptide p93-112 or
~2~ -~2:?i
pl72-182, and prefer~bly does not immunoreact with a
polypeptide containing only a monomer of pl41-155.
This immunoreaction admixture is maintained under
biological assay conditions for a time period
sufficient to form an apo E immunoreaction product.
Thereafter the amount of immunoreaction product thus
formed is detected, and thereby the amount of apo E
antigen present in the vascular fluid sample, i5
determined.
In a more preferred embodiment, the
detecting step is accomplished by admixing a labeled
specific binding agent capable of binding an apo E-
containing lipoprotein particle with the apo E-
containing immunoreaction product to form a labeling
reaction admixture, and maintaining this admixture
under biological assay conditions for a time period
sufficient for the labeled specific binding agent to
bind the apo E-containing immunoreaction product to
form a labeled complex which can be detected.
In a most preferred embodiment, the labeled
specific binding agent is a monoclonal anti-B-100
antibody produced by the hybridoma having ATCC
designation HB 8746.
A method for detecting an apo E antigen in a
vascular fluid sample is also contemplated, comprising
the steps o~ (i) admixing a vascular fluid sample with
a solid phase-bound apo E peptide analog containing a
plurality of segments each having an amino acid
residue sequence corresponding to the formula
LRKLRKRLLRDADDL to form a ~irst solid-liquid phase
admixture; (ii) admixing an antibody composition,
containing a limiting amount of anti-apo E peptide
antibody molecules of this invention that immunoreact
with the apo E antigen, with the first admixture to
form a second admixture; (iii) maintaining the second
~23~
admixture under biological assay conditions for a
period of time sufficient to form an apo E peptide
antigen-containing immunoreaction product in the solid
phase; and (iv) determining the amount of
immunoreaction product present in the solid phase
formed and, thereby, the amount of the apo E antigen
in the fluid.
In a preferred embodiment, the reveal anti-
apo E antibody is operatively linked to an enzyme
indicating means, and the product formed is a labeled
immunoreaction product.
Still further contemplated is a diagnostic
system, in kit form, comprising~ in an amount
sufficient to parform at least one assay, an antibody
composition containing anti-apo E antibody molecules
that immunoreact with a polypeptide containing a
plurality of segments each having an amino acid
residue sequence corresponding to the formula
LRKLRKRLLRDADDL, and with apo E/VLDL, but do not
immunoreact with the polypeptides p73-112 or pl72-182,
and preferably do not immunoreact with a polypeptide
containing only a monomer of pl41-155.
In the case where apo B-100 is to be used as
a marker for detection, a reveal antibody composition
containing anti-apo B-100 antibody molecules is
included.
In a preferred embodiment, the capture
antibody molecules are operatively linked to a solid
matrix, and the reveal antibody molecules are
operatively linked to an enzyme indicating means.
Still further contemplated i5 a method for
monitoring the efficacy o~ a therapeutic regimen for
facilitating cholesterol clearance. This method
comprises (i) determining the total amount of apo E
components in vascular fluid, and (ii) determining the
2~23~;2~
amount of effective apo E associated with cholesterol-
containing lipoprotein particles in vascular fluid.
Thus, the total apo E receptor-competent apo E ratio
i5 determined, and the resultant ratio is related to
predetermined concentration levels that have
previously been correlated to degree of cholesterol
clearance efficiency.
Brief Description of the Drawinqs
Figure 1 illustrates that all Apo E peptide-
BSA conjugates assayed as described in Example 2
inhibited lymphocyte proliferation in an approximately
equivalent manner, thereby indicating a lack of
specificity. Increasing concentrations of Apo E
peptide-BSA conjugates were added to cultures that
contained 1 x 106 peripheral blood mononuclear cells
per ml. The cells were cultured at 37C in RPMI with
5~ fetal bovine serum. All cells were exposed to PHA
at 24 hr. Each point represents the average 3H-
thymidine uptaXe, measured in counts per minute (cpm),
of four wells per treatment and the standard error was
less than 10~ for all points.
Figure 2 illustrates that the inhibition of
lymphocyte proliferation by self-conjugated Apo E-
derived peptide pl41-155 E was conjugate specific when
assayed as described in Example 3. Increasing
concentrations of self-conjugates (pl41-155)-(pl41-
155), (pl72-182)-(pl72-182) and (p93-112)-(p93-112)
were added to cultures that contained 1 x 105
peripheral blood mononuclear cells per ml~ Th~ cells
were cultured at 37C in RPMI with 5% serum. All cells
were exposed to PHA at 24 hr, were labeled with 3H-
thymidine at 48 hr. and were harvested at 72 hr.
Control cells incorporated 124,130 cpm ~ 4539. This
value represents 100%
2 ~ 2 ~
18
Figure 3 illu~trates that the inhibition of
lymphocyte proliferation by self-conjugate tpl41-155)-
(pl41-155) when added to cultures that contained 1 x
106 peripheral blood mononuclear cells per ml was not
due to cytotoxicity. As described in Example 3, the
cells were cultured at 37C in RPMI with 5% fetal
bovine serum. All cells were then exposed to PHA at
24 hr, were labeled with 3H-thymidine at 48 hr and
were harvested at 72 hr. Cell culture supernatants
from duplicate wells were collected for assay of
lactate dehydrogenase (LDH) activity according to the
method of Carney et al., J Immunol., 134:1804,
(1985). Data is expressed as percent of control. 3H-
thymidine uptaXe and LDH activity of PBS-exposed
control lymphocytes was 0.085 and 1.232 change in O.D.
340 nm/min, respectively. LDH activity was measured
following lysis of the cells with H2O. Each point
represents the average value from 4 wells per
treatment and the standard error was less than 10%.
Figure 4 illustrates that all Apo E peptide-
BSA conjugates inhibited ovarian androgen production
in an approximately equivalent manner, thereby
indicating a lack of specificity. Increasing
concentrations of Apo E peptide-BSA conjugates were
added at the indicated concentrations to ovarian cells
(1 x 105/ml) that were cultured at 37C in serum-free
McCoy's 5a modified medium containing 4 ng/ml of LH
and 300 ug/ml of human HDL as described in Example 4.
After 48 hr of culture the SupernatQnts were collected
and the androstenedione concentration measured by
radioimmunoassay. Each point represents the average
androstenedione concentration from 4 wells per
treatment and the standard error was less than 10% for
all points.
2~2~2~
Figure 5 illustrates that the inhibition of
ovarian androstenedione production by self-conjugated
Apo E-derived peptide (pl41-155) was conjugate
specific when measured as described in Example 5.
Increasing concentrations of self-conjugates (pl41-
155) - tpl41-155), (pl72-182) - (pl72-182) and (p93-
112) - (p93-112) were added to ovarian cells (1 x
105/ml) that were cultured at 37C in serum-free
McCoy's 5a medium containing 4 ng/ml of LH and 300
~g/ml or human ~DL. After 48 hours of culture, the
supernatants were collected, the androstenedione
concentration measured by immunoassay, and the data
are expressed as in the legend to Figure 4.
Figure 6 illustrates that ovarian
androstenedione production was specifically inhibited
by self-conjugate (pl41-155)-(pl41-155) without
causing direct cellular toxicity when measured as
described in Example 5. Increasing concentrations of
self-conjugate (pl41-155)-(pl41-155) were added to
ovarian cells (1 x 105/ml) that were cultured at 37C
in serum-free McCoy's 5a medium containing 4 ng/ml of
LH and 300 ug/ml of human ~DL. After 48 hr of culture
the supernatants were collected, androstenedione and
progesterone ~steroid) concentrations measured by
radioimmunoassay, and the data are expressed as in the
legend to Figure 4.
Figure 7 illustrates that the tandem Apo E
peptide p(l41-155)2-(pl41-155)2 affect~ lymphocyte
proliferation in a dose dependent, bi-phasic manner as
described in Example 6. Increase in concentrations o~
tandem Apo E peptide were added to cultures that
contained 8 x 105 peripheral blood mononuclear cells
per ml. The cells were cultured at 37C and RPMI with
5% serum. All cells were exposed to PHA at 24 hours,
labeled with 3~-thymidine at 72 hours and harvested at
2~3~
so hours. The counts per minute (cpm) of 3H-thymidine
in the harvested cells was measured, and the data are
expressed as the mean of four wells per treatment as a
measure of thymidine uptake (x 1000 cpm). The
standard deviation was less than 10% for all means.
Fi~ure 8 illustrates that lymphocyte
proliferation was inhibited by self-conjugate of the
tandem Apo E peptide p(141-155)2 when added to
cultures that contain 8 x 105 peripheral blood
mononuclear cells per ml as described in Example 6.
The cells were cultured, labeled, harvested, and the
thymidine uptake measured as described in the legend
to Figure 7.
Figure 9 illustrates that tandem Apo E
peptide p(l41-155)2 affects ovarian androstenedione
production in a dose-dependent, bi-phasic manner as
described in Example 7. Increasing concentrations of
tandem Apo E peptide were added to ovarian cells (8 x
10~/mil) that were cultured at 37C in serum-free
McCoy's 5a modified medium containing 4 ng/ml of LH
and 300 ~/ml of human HDL. After 48 hours of culture,
the supernatants were collected, the androstenedione
concentration measured by radioimmunoassay, and the
data are expressed as described in the legend to
Figure 4.
Figure 10 illustrates that tandem apo E
peptide p(l41-155)2 affects LDL binding and
degradation in a dose dependent, biphasic manner as
determined in Example 9. In the top panel, increasing
concentrations of competitor comprising tandem apo E
peptide p~l41-155)2, LDL, control monomer pl41-155 or
control p74-105 were added to cultures of THP-l cells
simultaneously with the addition of l25I-LDL. The
disappearance o~ acid soluble 125I-LDL was followed
over a five-hour incubation at 37C. Each point
2 g 2 ~
represents the average radioactivity from 4 wells per
treatment. The results are expressed as B/~o~ where B
equals bound cpm less the TCA soluble counts in
supernatants from cells treated with peptides and Bo
equals bound cpm less the TCA soluble counts in
supernatants from cells treated with PBS. Standard
deviation was less than 10% for all means.
The bottom panel shows the specificity of
tandem peptide binding to LDL receptors. TH~-1 cells
were stimulated with PMA for 4 days. LDL was
acetylated, radiolabeled, and TCA precipitable. After
5 hours at 37 C, the B/Bo ratios were analyzed and
expressed as in the top panel. Neither LDL or tandem
peptide inhibited degradation of 125I-aLDL, but the
$5 tandem peptide caused an 80% inhibition of l25I-LDL
degradation.
Figure 11 illustrates that specific amino
acid substitutions in the tandem peptide alter its
ability to inhibit 125I-LDL binding to fibroblasts when
measured as described in Example 10. Each point is the
average of 3 replicates per treatment with the SEM <
10%. Shown are: Lys 143-Ala; Leu 144-Pro; Arg
150-Ala; and native tandem peptide.
Figure 12 illustrates that the trimer
peptide is a more potent inhibitor of fibroblast LDL
degradation than the tandem peptide when assayed as
described in Example 11. Each point is the average of
5 replicate~ per treatment with SE~ ~ 10% and the
results are expressed as ~/Bo as descrLbed in the
legend in Figure 10.
Figure 13 illustrates a specific, saturable
~2~I-tandem peptide binding to THP-l cells according to
the assay described in Example 12. The points shown
are the average o~ 5 replicates (SEM ~ 10%) and
represent specifically bound pmoles of tandem peptide.
2 ~ 2 ~
The nonspecific binding (i.e. binding in the presence
of 500~g/ml of VLDL) averaged 23.4% of the total
counts bound. Scatchard analysis of the data is shown
in the figure inset.
Figure 14 summarizes the in vitro effects of
tandem Apo E peptide p(l41-155)2 on lymphocyte
proliferation, ovarian androgen production and LDL
binding. These data can serve as a model for the ln
vivo determination of the appropriate amount of
peptide and/or conjugate to be administered based on
the desired therapeutic effect, e.g. hepatic LDL
uptake (degradation).
Detailed DescriPtion of the Invention
A. Definitions
Amino Acid Residue: The amino acid residues
described herein are preferred to be in the nL~
isomeric form. However, residues in the nDn 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.
C00~ refers to the free carboxy group present at the
carboxy terminus of a polypeptide. In keeping with
standard polypeptide nomenclature, J. 8iol. Chem.,
243:3552-59 (1969), abbreviations for amino acid
residues are shown in the following Table of
Correspondence:
TABLE OF CO~RESPONDENCE
SYMBOL AMINO ACI~
l-Letter 3-Letter
Y Tyr tyrosine
G Gly glycine
F Phe phenylalanine
2~23~9~
M Met methionine
A Ala alanine
S Ser serine
~ Ile isoleucine
S L Leu leucine
T Thr threonine
V Val valine
P Pro proline
K Lys lysine
H His histidine
Q Gln glutamine
E Glu glutamic acid
W Trp tryptophan
R Arg arginine
D Asp aspartic acid
N Asn asparagine
C Cys cysteine
It should be noted that all amino acid residue
sequences are represented herein by ~ormulae 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 a peptide bond to a further sequence of one
or more amino acid residues.
Polypeptide: refers to a linear series of
amino acid residues connected to one another by
peptide bonds between the alpha-amino group and
carboxy group of contiguous amino acid residues.
Peptide: as used herein refers to a linear
series of no more than about 50 amino acid residues
connected one to the other as in a polypeptide.
Protein: refers to a linear series o~
greater than 50 amino acid residues connected one to
2~2~ri~
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. A~o E PolvDeptide
The present invention contemplates a
polypeptide capable of substantially mimicking the
ability of apo E to induce differentiated cellular
function, such as hepatic LDL degradation, lymphocyte
proliferation~ androgen secretion by ovarian theca and
interstitial cells, and the like. That is, a subject
polypeptide acts as an analog of apo E at least with
regard to the ability of apo E to inhibit lymphocyte
proliferation and/or ovarian androgen secretion, and
increase the uptaXe of LDL by hepatocytes.
A subject polypeptide is further
characterized by the presence of a plurality of apo E-
derived segments (regions) within the polypeptide's
primary structure, each of the segments being defined
by a sequence of amino acid residues corresponding to
the formula:
Leu-Arg-Lys-Leu-Arg-Lys-Arg-Leu-Leu-Arg-Asp-
Ala-Asp-Asp-Leu, also referred to as p(l41-155),
because the amino acid residue sequence corresponds to
residues 141 through 155 of the native apo E protein.
The apo E-derived segments are capable of
binding to the LD~ receptor and/or LD~ receptor-
related protein [Herz et al., ~MB0 Journal, 7:411~-
4129 (1988)] a~ evidenced by the ability of the
binding to be competitively inhibited.
The apo E-derived segments can be adjacent
and/or contiguous within the polypeptide chain, with
adjacent segments being separated in the amino acid
~23.~2 ~
residue sequence of the polypeptide by one or more
spacing residue. Preferably, the spacing residues
make up a spacing segment in the range of about 1 to
about 20, preferably about 5 to about 15, and more
usually about 10, amino acid residues in len~th.
In addition, a subject polypeptide can
contain a leader segment of 1 conveniently up to about
33, such as about 11, about 18 or about 22, amino acid
residues located amino-terminal to the amino-terminal
apo E-derived or spacing segment.
In a similar manner, a subject polypeptide
need not end with the carboxy-terminal residue of an
apo E-derived segment or spacer segment. A carboxy
terminal tail segment can be present containing 1
conveniently up to about 33, such about 11, about 1
or about 22, amino acid residues.
Preferred polypeptides of the present
invention are therefor defined by formula I:
B-(X~-Leu-Arg-Lys-Leu-Arg-Lys-Arg-
Leu-Leu-Arg-Asp-Ala-Asp-Asp-Leu-
Z~),-J,
In the above formula, B is an amino-terminal NH2 group
or a previously discussed leader segment; J is a
carboxy-terminal COOH group or a previously discussed
tail segment; X and Z are first and second,
respectively, spacing segments whose amino acid
residue sequences can be the same or different; n is
either 1 or 0 such that when n i9 1, ~ i9 present, and
I when n is 0, X is not present; m is elther 1 or 0 suchthat when m is 1, Z is present, and when m is 0, Z is
not present; and a is an integer from 2 to about 10,
more preferably ~ to about 5 and usually 2 to 3,
indicating the number o~ times the amino acid residue
sequence in parenthesis is present (repeated) in the
polypeptide primary structure. Preferably, the
2 ~ 2 ~!
26
sequence in parenthesis corresponds in its entirety,
and preferably is identical to, a portion of the amino
acid residue sequence of apo E. Preferred
polypeptides are those whose formula~ are shown in
Table 1.
Table 1
-
Desianat~nl Amino Acid Residue Sequences
p(141-155)2 LRKLRKRLLRDADDLLRXLRKRLLRDADDL
p(129-163)2 STEELRVRLASHLRKLRKRLLRDADDLQKRLAVYQSTEEL-
RVRLASHLRKLRKRLLRDADDLQKRLAVYQ
1 The designation for each peptide indicates the
position within the amino acid residue sequence of the
mature Apo E protein to which the peptide sequence
corresponds, i.e., is derived from.
It should be noted that p(129-163) 2 contains
a 12 residue leader segment STEELRVRLASH (residues 1-
12), a 20 residue spacing segment QKRLAVYQSTEELR-
VRLASH (residues 28-47) and an 8 residue tail segment
QKRLAVYQ (residues 63-70). The designation p(141-
155)2 defines a tandem apo E peptide which contains
two adjacent sequences of the pl41-155 segment. An
additional preferred polypeptide is a ~trimer~
containing three adjacent sequences of the pl41-155
segment, designated p(141-155)3. Preferred also are
self-conjugates of the tandem apo E peptides
designated p(l41-155)2 - p(l41-155)2 and timer apo E
peptides designated p(141-155)~ - p(141-155)~
A sub~ect polypeptide typically contains a
total of about 30 to about 450 amino acid residues,
preferably about 60 to about 120 residues. Typically,
a subject polypeptide contains no more than about 100,
preferably no more than about 70 and usually no more
than about 30 or 40 amino acid residues in its primary
2~23~J~
27
seguence.
A subject polypeptide includes any analog,
fragment or chemical derivative of a polypeptide whose
amino acid residue sequence is show~ herein so long as
the polypeptide is capable of inducing differentiated
cellular function in a manner corresponding to that of
apo E. 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 nanalog~ 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 E 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 nconservative 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.
nChemical derivative~ refers to a subject
polypeptide having one or more residues chemically
derivatized by reaction of a functional side group.
2 ~
28
Such deri~atized molecules include for example, those
molecules in which free amino groups have been
derivatized to form amine hydrochlorides, p-toluene
sulfonyl groups, carbobenzoxy group~, 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 0-acyl or 0-alkyl derivatives.
The imitazole 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; 5-
hydroxylysine 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 addition3 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 nfragment~ 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.
A sub~ect polypeptide can be prepared using
recombinant nucleic acid methodologies well known in
the art. For instance, DNA sequences useful in
producing a sub;ect polypeptide are described in Paik
et al., Proc. Natl. Acad. Sci. USA, 82:3445-3449,
(1985); McLean et al., J. Biol. Chem., 259:6498-6504,
(1984); and Rall et al., J Biol. Chem., 257:4171-
2 ~3~ 3
29
4178, (1982). A DNA segment coding for a polypeptide
of this invention can be synthesized by chemical
techniques, for example the phosphotriester method of
MatteuC~i et al., J. Am. Chem. Soc., 103:3185, (1981).
The DNA segment can then be ligated into an expression
vector, and a host transformed therewith can be used
to produce the polypeptide. See, for example, Current
Protocols In_Molecular Bioloav, Ausubel et al., eds.,
John Willey & Sons, New York, NY; U.S. Patents No.
4,237,224 and No. 4,356,270.
The recombinant expression vectors capable
of expressing a subject polypeptide and methods of
their use for producing a subject polypeptide are
contemplated as part of the present invention.
A subject polypeptide can also be prepared
using the solid-phase synthetic technique initially
described by Merrifield, in J. Am. Chem. Soc.,
85:2149-2154 (1963). Other polypeptide synthesis
techniques may be found, for example, in M. Bodanszky
et al., Pe~tide Svnthesis, John Wiley & Sons, 2d Ed.,
(1976) as welL as in other reference works known to
those skilled in the art. A summary of polypeptide
synthesis techniques may be found in J. Stuart and
J.D. Young, Solid Phase Pe~tide SYnthesis, Pierce
Chemical Company, Rockford, IL, 3d Ed., Neurath, ~. et
al., Eds., p. 104-237, Academic Press, New York, NY
(1976). Appropriate protective groups for use in such
syntheses will be found in the above text~ as well as
in J.F.W. McOmie, Prot~ctive Grou~ in ~anic
Chemistrv, Plenum Press, New York, NY (1973).
In general, those synthetic methods comprise
the sequential addition of one or more amino acid
residues or suitably protected amino acid residues to
a growing polypeptide chain. Normally, either the
amino or carboxyl group of the first amino acid
2 ~ 2 ~ 34 ~
residue is protected by a suitable, selectively
removable protecting group. A different, selectively
removable protectin~ group is utilized for amino acids
containing a reactive side group such as lysine.
Using a solid phase synthesis as an example,
the protected or derivatized amino acid i~ 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 amid 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 provide
the final polypeptide.
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.
2 ~ 2 3 r~
31
Suitable bases capable of forming salts with
the peptides of the present invention include
inorganic bases such as sodium hydroxide, ammonium
hydroxider potassium hydro~ide and the like; and
organic bases such as mono-, di- and tri-alkyl and
aryl amines (e.g. triethyla~ine, diisopropyl amine,
mPthyl amine, dimethyl amine and the like) and
optionally substituted ethanolamines (e.g.
ethanolamine, diethanolamine and the like).
C. Coniuqates
The present invention further contemplates
an apo E analog in the form of a polypeptide conjugate
comprised of a plurality of polypeptides operatively
linked, by other than a peptide bond between the
alpha-amino group and carboxy group of contiguous
amino acid residues, where at least two of the linked
polypeptides have an amino acid residue sequence
corresponding to that represented by the formula:
B-(Xn-Leu-Arg-Lys-Leu-Arg-Lys-Arg-
Leu-Leu-Arg-Asp-Ala-Asp-Asp-Leu-
Z~)~-J,
wherein B, X, Z, J, n, m and a are defined as
previously discussed except that a can also be the
integer 1.
Preferred self-conjugates are pl41-155
linked to pl41-plS5, designated (pl41-155)-(pl41-155)
and pl29-163 linked to pl29-163, designated (pl29-
163)-(pl29-163).
In preferred embodiments, a conjugate of
this invention has a molecular weight of less than
about 40,000 daltons, preferably less than about
20,000 daltons, and more preferably less than about
10,000 daltons. Typically, a subject conjugate has a
molecular weight of no more than about 15,000 daltons,
2~s3~2 ~
preferably no more than about 8,000 daltons, and
usually no more than about 4,000 daltons. Preferably,
the conjugate is dimeric or trimeric, i.e., consists
essentially of two or three polypeptide chains,
respectively.
A polypeptide conjugate of this invention is
further characterized by its ability to substantially
mimic apo E's ability to induce differentiated
cellular function, such as lymphocyte proliferation,
ovarian androgen secretion, and the like. The subject
conjugates are also substantially free of toxicity
toward lymphocytes and androgen-producing ovarian
(theca/interstitial) cells at concentrations of about
20 micrograms per milliliter (ug/ml).
The techniques of polypeptide conjugation or
coupling through activated functional groups presently
known in the art are particularly applicable. See,
for example, Avrameas, et al., Scand. J. Immunol.,
Vol. 8, Suppl. 7:7-23 (1978) and U.S. Patent No.
4,493,795, No. 3,791,932 and No. 3,839,153. In
addition, a site directed coupling reaction can be
carried out so that any loss of activity due to
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.
2~2~
D. Com~ositions for Modulatinq He~atic LDL
Deqradation
In view of the ability of the polypeptides
and conjugates of the present invention to bind the
LDL receptor present on hepatocytes, the present
invention contemplates a composition for modulating
hepatic uptake of LDL. The composition comprises an
LDL receptor-binding moiety operatively linked to an
LDL binding moiety. The LDL receptor-binding moiety
comprises a polypeptide and/or conjugate of the
present invention. A preferred LDL receptor-binding
moiety comprises the polypeptide segment designated
p(141-lS5)2 whose amino acid residue sequence is shown
in Table 1.
The LDL receptor-binding moiety can be
operatively linked to the LDL binding moiety by a
peptide bond or through a covalent bond that is not a
peptide bond between the alpha-amino group and
carboxyl group of continuous amino acid residues.
An LDL binding moiety can be an anti-LDL
antibody molecule or immunologically active fragment
thereof. Exemplary anti-LDL-antibody molecules are
produced by hybridomas HB8746 and HB8742, which have
been deposited with the American Tissue Culture
Collection (ATCC; Rockville, MD), both of which
produce anti-apo B-100 antibody molecules. The LDL
receptor-binding polypeptide and/or conjugate of this
invention can be chemically coupled as described
hereinbefore to the anti-LDL antibody molecule.
Alternatively, a polypeptide of this invention can be
incorporated into the primary amino acid residue
sequence of the antibody molecule by recombinant DNA
techniques. Typically, the LDL receptor-binding
polypeptide will be incorporated into or substituted
for a portion of one of the antibody molecule's
2 ~ 2 ~
34
constant domains. See U.S. Patent No. 4,816,567, No.
4,816,397 and No. 4,647,334.
In preferred embodiments, the LDL binding
moiety is a lipophilic (hydrophobic) sequence of amino
acid residues. More preferably, the LDL binding
moiety is a polypeptide segment having an amino acid
residue sequence capable of forming an amphipathic
helix.
Of course, when the means for operatively
linking the LDL receptor moiety and LDL binding moiety
is other than a peptide bond, the linking typically
occurs between amino acid residue chains on residues
at or near the carboxy-and/or amino-terminus of the
respective moieties so as to preserve their
activities.
Preferred helical amphipathic polypeptide
seqments of this invention, whether incorporated into
the composition by a peptidic or non-peptidic bond,
include those having an amino acid residue sequence
corresponding to that of an apolipoprotein, such as
apo B-100, apo B-48, apo C-I, apo C-II, apo C-III, apo
A-I, apo A-II, apo D, apo E and the like. See, Fitch,
Genetics, 86:623-644 (1977); Segrest et al.,
Bio~olYmers, 16:2053-2065 (1977): and Chan, Klin
Wochenscher, 67:225-237 (1989). By using a helical
amphipathic polypeptide segment with amino acid
residue sequence derived from apo B-100 or apo B-48,
the polypeptide can be preferentially targeted to LDL
as opposed to other lipoprotein species.
The amphipathic helix is characterized by a
spacial segregation of hydrophobic and hydrophilic
amino acid residues on opposite faces of the helix.
The clustered nonpolar residues can then intercalate
into lipid particles such as LDL. In addition to this
hydrophobic interaction, there may also be specific
2~3~2 ~
charge interactions between lipid and peptide. For
example, it has been demonstrated that an 18-residue
peptide can bind to phospholipid if it has positively
charged residues at the hydrophobic-hydrophilic
interface of an amphipathic helix and negativ~ly
charged residues opposite the hydrophobic face of the
helix. See Epand et al., J. Biol. Chem., 264:4628-
4635 (1989).
A particularly preferred helical amphipathic
polypeptide segment useful in binding the apo E-
derived polypeptide segment LDL receptor-binding
moiety to LDL has an amino acid residue sequence
corresponding to the formula:
EWLKAFYEKVLEKLKELF.
In preferred embodiments, the composition is
a polypeptide according to formula I wherein B and/or
J is a helical amphipathic polypeptide segment as
described above. One preferred polypeptide of this
type has an amino acid residue sequence corresponding
to the formula:
LRKLRKRLLRDADDLLRKLRKRLLRDADDL-
EWLKAFYEKVLEKLKELF.
In preferred embodiments, the subject
polypeptide or conjugate is dispersed in a carrier,
such as a phospholipid. More preferably, the subject
polypeptide or conjugate is removably inserted in a
liposome, i.e., it is incorporated (anchored) into the
liposome bilayer via the LDL binding moiety. See, for
example, Gregoriadis, Trends i~ Biotech., 3:235-241
(1985) and Eriksson et al., pp 141-156 in Li~osome
TechnoloqY Vol. II, ed G. Gregoriadis CRC Press, Boca
Raton, FL.
E. TheraPeutic Methods
~ ~ 2 3 ~J ~ r~
36
The polypeptides, conjugates and
compositions contemplat~d by the present invention are
useful as agents for modulating those physiologic
events induced by native apo E, such as immune
response, steroidogenesis andJor enhance hepatic LDL-
binding. For instance, a polypeptide and/or conjugate
of this invention can be used as an immunosuppressive
agent to inhibit the proliferation of lymphocytes or
as an agent to inhibit ovarian androgen production.
The polypeptide and/or conjugate is administered to
the animal, such as a human, in need of such
treatment, in a predetermined amount calculated to
achieve the desired effect, i.e., in a therapeutically
effective amount.
For instance, when used as an
immunosuppressive agent for inhibiting lymphocyte
proliferation, such as in a patient displaying the
symptoms of an autoimmune disease, the polypeptide
and/or conjugate is administered in an amount
sufficient to achieve a plasma concentration of at
least about 0.8 ug/ml, preferably at least about 1.0
ugJml, more preferably at least about 2 ug/ml, and
usually 3 or 4 ug/ml.
In some cases, it is desirable to apply the
subject polypeptide and/or conjugate locally as an
immunosuppressive agent. For instance, about 10 ug to
about 1 mg can pe applied by injection into an
arthritic joint (e.g., into the synovial ~luid o~ the
joint) to suppress inflammation.
When used as an agent for inhibiting ovarian
androgen production, such as in females having
polycystic ovaries, the polypeptide and/or conjugate
of this invention is administered in an amount
sufficient to achieve a plasma concentration of at
least about a 2 ug/ml, preferably about 5 ug/ml, and
2~3~2~
more preferably about 10 ug/ml.
When the tandem apo E peptide p(l41-155)2 or
its self conjugate is used to inhibit lymphocyte
proliferation, the peptide or its conjugate is
S administered in an amount sufficient to achieve a
plasma concentration of at least about 8 mg/ml,
preferably at least 10 mg/ml, and more preferably at
least 15 mg/ml. When used to enhance lymphocyte
proliferation, the peptide or its conjugate is given
in an amount sufficient to achieve a plasma
concentration of from about 2 mg/ml to about 6 mg/ml,
preferably from about 3 mg/ml to about 5 mg/ml.
When the tandem apo E peptide p(l41-155)2 or
its self conjugate is used to inhibit ovarian androgen
production, the peptide or its conjugate is
administered in an amount sufficient to achieve a
plasma concentration of at least about 0.6 mg/ml,
preferably at least 1.0 mg/ml, and more preferably at
least 8 mg/ml. When used to enhance ovarian androgen
production, the peptide or its conjugate is given in
an amount sufficient to achieve a plasma concentration
of from about 0.1 mg/ml to about 0.4 mg/ml, preferably
from about 0.2 mg/ml to about 0.3 mg/ml.
When a composition contains either tandem
apo E peptide p(l41-~55)2 or its self conjugate
operatively linked to an LDL binding moiety is used to
enhance hepatic LDL binding and uptake, as in subjects
with hyper-cholesterolemia, the composition is
administered in an amount sufficient to achieve a
tandem peptide or con~ugate plasma concentration of at
least about 20 mg/ml, preferably at least about 50
mg/ml, and more preferably at least about 100 mg/ml.
The preparation of therapeutic compositions
which contain polypeptides as active ingredients is
well understood in the art. Typically, such
2~2~2~
38
compositions are prepared as injectables, either as
liquid solutions or suspensions, however, solid forms
suitable for solution in, or suspension in, liquid
prior to injection can also be prepared. The
preparation can also be emulsified. The active
therapeutic ingredient is often mixed with excipients
which are pharmaceutically acceptable and compatible
with the active ingredient. Suitable excipients are,
for example, water, saline, dextrose, 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 which enhance
the effectiveness of the active ingredient.
A polypeptide can be formulated into the
therapeutic composition as neutralized
pharmaceutically acceptable salt forms.
Pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of
the polypeptide or antibody molecule) and which are
formed with inorganic acids such as, for example,
hydrochloric or phosphoric acids,or such organic acids
as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed from 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.
The therapeutic polypeptide-containing
composltions are conventionally administered
intravenously or at the site of autoimmune-induced
inflammation, 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
2~2~
39
refers to physically discrete units suitable as
unitary dosage for humans, 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 inhibition of
lymphoproliferation or androgen production desired.
Precise amounts of active ingredient required to be
administered depend on the judgment of the
practitioner and are peculiar to each individual.
However, suitable dosage ranges for systemic
application are of the order of 0.01 to 10, preferably
one to several, milligrams of active ingredient per
kilogram bodyweight of individual per day and depend
on the route of administration. Suitable regimes for
initial administration and booster shots are 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 o~ ten
nanomolar to ten micromolar in the bloqd are
contemplated.
F. Antibodies and Monoclonal Antibodies
The term nantibody~ 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
~3~
immunoglobulin molecules, i.e., molecules that contzin
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 nantibody moleculen 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 method 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 thos2 portions known
in the art as Fab, Fab', F(ab')2 and F(v).
Fab 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. See, for
example, U.S. Patent No. 4,342,566 to Theofilopolous
and Dixon. 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
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 E antibody, in one embodiment is
characterized as being capable of immunoreacting with
2 ~ 2 3 ~ 2 .`L
41
apo E present on cholesterol containing-lipoprotein
particles such as LDL, VLDL and the like. Apo E in
association with VLDL is referred to as apo E/VLDL.
An anti-apo E antibody of this embodiment is further
characterized as immunoreacting with the polypeptide
of this invention comprising a plurality of segments
having the for~ula ptl41-155), preferably p(l41-155)2,
more preferably p(141-155)3, and still more preferably
self-conjugates of p(l41-155)2 or p(l41-155)3.
In a preferred embodi~ent, an anti-apo E
antibody is characterized as being substantially free
of antibody molecules that immunoreact with the
polypeptide LSKELQAAQARLGADMEDVR corresponding to
residues 93-112 of mature apo E and designated p93-
112, or with the polypeptide RGLSAIRERL corresponding
to residues 172-182 of mature apo E and designated
pl72-182.
Particularly preferred are antibody
molecules that do not immunoreact with a polypeptide
containing only a monomer of the apo E polypeptide
pl41-155. Apo E polypeptides that contain only a
single pl41-155, do not have the capacity to mimic the
LDL receptor-binding capacity of apo E that results in
enhanced hepatic uptake and degradation of apo E-
containing lipoprotein particles as disclosed herein.Polypeptides that mimic the LDL receptor binding
function of apo E are referred to as ~receptor-
competent~ apo E polypeptides. Anti-apo E antibody
molecules that do not immunoreact with monomeric pl41-
15S thus have immunospecificity for receptor-competent
apo E polypeptides, and have immunospecificity for apo
E apolipoprotein that is in a receptor-competent ~orm.
Anti-apo E antibody molecules that are
specific for receptor-competent apo E or apo E
polypeptides have a particular utility in
2~23 ~2.~
42
immunoassays, namely, to monitor the fate of
therapeutically administered apo E polypeptides during
the course of therapeutic regimens as disclosed
herein, or to detect the levels of receptor-competent
apo E in a vascular body fluid sample.
Antibody immunoreactivity with apo E-
containing antigens can be measured by a variety of
immunological assays known in the art. Exemplary
immunoreaction of an anti-apo E antibody of this
invention by direct binding with apo E/VLDL or with
apo E polypeptides can be assayed at least by the
methods described in Example 14.
An antibody of the present invention is
typically produced by immunizing a mammal with an
inoculum containing an apo E polypeptide of this
invention, such as a self-conjugate of p(l41-155)3,
and thereby induce in the mammal antibody molecules
having immunospecificity for apo E polypeptide
Alternatively, apo E/LDL or apo E/VLDL can be used as
the source of immunizing apo E antigen. Exemplary are
the production methods for preparing a polyclonal
anti-apo E polypeptide antisera described in Example
8. 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 antibody specificity,
antibodies that are purified by immunoafflnity
chromatography using solid phase-affixed immunizing
polypeptide are preferred. 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 to form a
solid phase-affixed immunocomplex. The bound
antibodies are separated from the complex by standard
2~23~2Je~
43
techniques.
In a related method to produce an anti-apo E
antibody that does not substantially immunoreact with
monomeric pl41-155, the prepared anti-apo E antibody
can be contacted with solid-phase monomeric pl41-155
so as to allow antibodies that immunoreact with
monomeric pl41-155 to complex in the solid phase, and
the liquid-phase antibody is then collected to form
anti-apo E antibody that is specific for receptor-
competent apo E polypeptides.
The antibody so produced can be used, inter
~ 3, in the diagnostic methods and systems of thepresent invention to detect apo E or apo E polypeptide
present in a body sample.
The word ~inoculum" in its various
grammatical forms is used herein to describe a
composition containing a apo E polypeptide of this
invention as an active ingredient used for the
preparation of antibodies immunoreactive with an apo E
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. However, for
~5 ease of expression and in context of a polypeptide
inoculum, the various embodiments of the polypeptides
of this invention are collectively referred to herein
by the term ~polypeptlde~, and its various grammatical
forms.
For a polypeptide that contains fewer than
about 35 amino acid residues, it ls 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
2 ~ i~ 3 ~ ~ ~
44
polypeptide to assist in binding the polypeptide to a
carrier. Cysteine residues added at the amino~ or
car~oxy-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., 147:318-326 (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
Avrameas, 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-lysine; D-glutamic acid), and the like.
The choice of carrier is more dependent upon
the 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.
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 thing~, on
2~2~
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 S00 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 im~unogenic 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 Peatures 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 ad~uvant (IFA) and
alum are materialq well known in the art, and are
available commercially from several sources.
The techniques of polypeptide conjugation or
coupling throu~h activated functional group~ presently
2 ~ 2 .~'
46
known in the art are particularly applicable. See,
for example, Avrameas, et al., Scand. J. Immunol.,
Vol. 8, Suppl. 7:7-23 (1978) and U.S. Patent No.
4,493,795, No. 3,791,932 and No. 3,839,15~. In
addition, a site directed coupling reaction can be
carried out so that any loss of activity due to
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 ~he 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 particularly preferred anti-apo E antibody
is a monoclonal 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.
Preferred anti-apo E monoclonal antibodies
are prepared as disclosed herein.
Additional monoclonal antibodies useful for
practicing the diagnostic methods of this invention
2~3.3 2~ ~
are those which immunoreact with LDL, VCDL, HDL, and
the like lipoprotein particles. Particularly
preferred are anti-apo B-100 antibodies.
An exemplary hybridoma that secretes
monoclonal antibody molecules that immunoreact with
apo B-100 has been described previously in U.S. Patent
No. 4, 677,057, which is incorporated herein by
reference, and the monoclonal antibody molecules
secreted by the hybridoma is referred to herein as
MB47. The M847 monoclonal antibody immunoreacts with
more than about 90 percent of lZsI-LDL, and with a
distinct and separate conserved antigenic determinant
on apo B-100. As pointed out in Young et al. (198~)
Clin. Chem., 32~8:1484-1490, MB47 reacts only with apo
B-100.
The above hybridoma was deposited with the
American Type Culture Collection (ATCC), Rockville,
MD, in accordance with the Budapest Treaty on the
International Recognition of the Deposit of
Microorganisms for the Purposes of Patent Procedure,
on March 6, 1985, and was assigned the designation HB
8746.
The above deposit was made in compliance
with the Budapest Treaty requirements that the
duration of the deposit or for 5 years after the last
request for the deposit at the depository or for the
enforceable life of a U.S. patent that matures from
this application, whichever is longer. The hybridoma
is replenished should it become non-viable at the
depository, and is made available to the public by the
ATCC upon the issuance of a patent from this
application.
G. Pre~aration of Monoclonal Antibodies
2~3~
48
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
prepara:ion 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 E polypeptide 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 mam~al
hyperimmunized with an apo E polypeptide of this
invention, or with a native apo E molecule, such as is
present in an apo E-containing lipoprotein particle.
The polypeptide-induced hybridoma technology is
descri~ed by Niman, et al., 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-aminoptorin-thymidine-sensitive (HAT)
cell lines P3X63-Ag8.653, and Sp2/0-Agl4 that are
available from the American Type Culture Collection,
Rockville, MD, under the designations CRL 1580 and CR~
1581, respectively.
Splenocytes are typically fused with myeloma
cells using polyethylene glycol (PEG) 1500. Fused
~3~2 1._
hybrids are selected by their sensitivity to HAT.
Hybridomas producing a monoclonal antibody of this
invention are then identified by screening for the
immunospecificities of an anti-apo E antibody as
disclosed herein, for example, the enzyme linked
immunosorbent assay (ELISA) described in Example 16,
or using the solid phase radioimmunoassay (SPRIA)
described in Example 14.
A monoclonal antibody of the present
invention can also be produced by initiating a
monoclonal hybridoma culture compri~ing a nutrient
medium containing a hybridoma that secretes antibody
molecules of the appropriate immunospecificity. The
culture is maintained under conditions and for a time
period sufficient for the hybridoma to secrete the
antibody molecules into the medium. The antibody-
containing medium is then collected. The antibody
molecule~ can then be further isolated by well known
techniques.
Media useful for the preparation of these
compositions are both well known in the art and
commercially a~ailable and include synthetic culture
media, inbred mice, and the like. An exemplary
synthetic medium is Dulbecco's minimal essential
medium (DMEM; Dulbecco, et al., Virol., 8:396 (1959))
supplemented with 4.5 gm/l glucose, 20 mm glutamine,
and 20% fetal calf serum. An exemplary inbred mouse
strain is the Balb/c.
Other methods o~ producing a monoclonal
antibody, a hybridoma cell, or a hybridoma cell
culture are also well known. See, for example, the
method of isolating monoclonal antibodie~ from an
immunological repertoire as described by Sastry, et
al., Proc, Natl. Acad. Sci., 86:5728-5732 (1989); and
Huse, et al., Science, 246:1275-1281 (1981).
2 ~ h ~3 ~ ~ ~
Also contemplated by this invention is the
hybridoma cell, and cultures containing a hybridoma
cell that produce a monoclonal antibody of this
invention.
The monoclonal antibody produced by the
above method can be used, for example, in diagnostic
modalities disclosed herein where formation of an apo
E-containing immunoreaction product is desired.
Hy~ridoma HB 8746 produces M847 monoclonal
antibody molecules and was formed by fusing
splenocytes of mice immunized with LDL and
P3X63Ag8.653.1 myeloma cells~ Detailed preparation of
the MB47 monoclonal antibody and hybridoma was
reported by Young, et al. (1986) Arteriosclerosis,
6:178-188.
H. Assav Methods
Useful solid and liquid phase assay methods
are discussed herein. However, the invention is not
so limited. Further, while the particularly described
assay methods utilize an enzyme-linked indicator
means, the present invention is not specifically
limited to such assays. Additional assay methods are
described herein~elow with particular emphasis on
solid phase immunoassay methods.
Those skilled in the art will understand
that there are numerous methods of solid phase
immunoassays that may be utilized herein. Exemplary,
useful solid phase assays include enzyme multiplied
immunoassay techniques tEMIT) and ~luorescence lmmune
assays (FIA), in addition to the speci~ically
discussed RIA, solid phase radioimmunoassay (SPRIA) of
Exampl~ 8B, and ELISA. However, any method that
results in a signal imparted by the reaction of apo E
with an anti~ody o~ this invention is considered.
2 ~ 2 ~ r~ ~
Each of those assay methods can employ single or
double antibody techniques in which an indicating
means is utilized to signal the immunoreaction, and
thereby the binding of an apo E that is to be assayed
with a receptor of this invention. Exemplary
techniques can be found explained in Maggio, Enzyme
Immunoassay, CRC Pres~, Cleveland, OH (lsal); and in
Goldman, Fluorescent AntibodY Methods, Academic Press,
New York, NY (1980).
A vascular fluid sample is utilized in this
assay method. The sample can be either serum or
plasma. Results obtained using both compositions have
previously been found to be statistically
indistinguishable. Regardless of whether serum or
plasma is used, the vascular fluid sample is
preferably obtained from persons who have fasted for
at least about twelve hours as is known in the art.
Such a blood sample is referred to as a "fasting"
sample.
One contemplated assay method determining
the presence, and preferably the amount of an apo E in
a vascular fluid sample. This method includes the
following steps:
(a) A vascular fluid sample is admixed with
an anti-apo E antibody composition to form an
immunoreaction admixture. The antibody molecules in
the composition preferably are operatively linked to a
solid support such that the immunoreaction admixture
has both a liquid phase and a solid phase. These
antibody molecules immunoreact with:
(i) a polypeptide containing a
plurality o~ segments each having an
amino acid residue sequence
corresponding to the formula:
Leu-Arg-Lys-Leu-Arg-Lys-
2 ~
Arg-Leu-Leu-Arg-Asp-Ala~Asp-Asp-
Leu, and
(ii) apo E/VLDL, but do not
i~munoreact with a polypeptide
corresponding to one of the formulae
p93-112 and pl72-182, and preferably do
not immunoreact with a polypeptide
containing only a monomer of the
polypeptide pl41 18S.
(b) ~he immunoreaction admixture is
maintained under biological assay conditions for a
time period sufficient to form an apo E-containing
immunoreaction product in the solid phase.
(c) The presence, and preferably the amount
of immunoreaction product formed in step (b) and
thereby apo E antigen in the vascular fluid sample is
then determined.
Biological assay conditions are those
conditions that are able to sustain the biological
activity of the immunochemical reagents of this
invention and the antigen sought to be assayed. Those
conditions include a temperature range of about 4
degrees C to about 45 degree~ C, a pH value range of
about 5 to about 9 and an ionic strength varying from
that of distilled water to that of about one molar
sodium chloride. Methods for optimizing such
condition~ are well known in the art.
In a preferred embodiment oS the above
method the amount oS immunoreaction product ls
determined according to step (c) by (i) admixing a
labeled specific binding agent çapable of binding an
apo E-containing particle with the apo E-containing
immunoreaction product to form a labeling reaction
admixture, (ii) maintaining the labelinq reaction
admixture under biological assay condition~ for a time
2~23~2.~
period sufficient for the labeled specific binding
agent to bind the apo E-containing immunoreaction
product to form a labeled complex, and (iii) detecting
the amount of any labeled complex formed, and thereby
detecting the amount of apo E-containing
immunoreaction product.
In a particularly preferred embodiment, the
labeled specific agent is a monoclonal anti-B-100
antibody produced by the hybridoma having ATCC
designation HB 8742.
Another contemplated assay of this invention
is a method for detecting the presence, and preferably
the amount, of an apo E antigen in a vascular fluid
sample. This assay method comprises the following
steps:
(a) A vascular fluid sample is admixed with
a solid phase-bound apo E peptide containing a
plurality of segments each having an amino acid
residue sequence corresponding to the formula:
Leu-Arg-Lys-Leu-Arg-Lys-
Arg-Leu-Leu-Arg-Asp-Ala-Asp-Asp-
Leu,
to form a first solid-liquid phase admixture.
(b) An antibody composition, containing a
limiting amount of anti-apo E peptide antibody
molecules that immunoreact with:
(i) a polypeptide containing a
plurality o~ sQgment~ ~ach havlng an
amino acld residue se~uence
corresponding to the formula:
Leu-Arg-Lys-Leu-Arg-Lys-
Arg-Leu-Leu-Arg-Asp-Ala-Asp-Asp-
Leu, and
(ii) apo E/VLDL, but do not
immunoreact with a polypeptide
2~2~i2~ ~
54
corresponding to one of the formulae
p93-119 and p72-182, and preferably do
not immunoreact with a polypeptide
containing only a monomer of the
polypeptide pl41-155, is admixed with
the first admixture to form a second
admixture.
(c) This second admixture is maintained
under biological assay conditions for a period of time
sufficient to form an apo E-containing immunoreaction
product in the solid phase.
(d) The amount of immunoreaction product
present in the solid phase formed in step ~c) is
determined, and thereby the amount of apo E in the
vascular fluid sample.
In an alternative embodiment, the solid
phase-bound apo E polypeptide is replaced by an apo E-
containing lipoprotein, such as a VLDL particle.
In a more preferred embodiment, the solid
phase-bound apo E polypeptide or apo E containing
component is the above-described receptor-competent
polypeptide analog, and the anti-apo E analog antibody
in step (b) is operatively linked to an enzyme
indicating means, and the product formed in step (c)
is a labeled immunoreaction product.
Insofar as the present diagnostic methods
can be used to monitor the fate of therapeutically
administered apo E polypeptides as disclosed herein,
it is understood that the dlsclosed method~ for
detectlng an apo E antigen can readily be applied to
monitor an analog of an apo E antigen, namely, an apo
E polypeptide. For thi~ reason, the phrase ~apa E
antigen~ refers to antigenic molecules that
immunoreact with the antibodies of the present
3S invention, whether the antigenic molecules are native
~3~ 1
apo E, apo E polypeptides, or combinations of both
analog and native protein.
In embodiments for following the fate of a
therapeutically administered apo E polypeptide, it is
to be understood that native apo E and apo ~
polypeptide may be indistinguishable in a vascular
fluid sample where both components immunoreact with
the anti-apo E antibodies. Thereforel it is useful
and preferred to measure vascular levels of apo E
antigen in a patient prior to the administration of a
therapeutic composition to establish a baseline of apo
E antigen in the patient. At predetermined time
intervals after administration of therapeutic peptide,
the patient's blood is then sampled and the levels of
apo E antigen are again measured to determine the
effect and fate of the therapeutic composition on
circulating apo E antigen.
Also contemplated is a related method for
detecting apo E present in lipoprotein particles in a
vascular fluid sample. The fluid sample is admixed
with a solid-phase anti-8-100 antibody, i.e.,
operatively linked to a solid matrix, to form a
liquid-solid phase immunoreaction admixture. The
admixture i~ maintained under biological assay
conditions for a time period sufficient to form an
immunoreaction product in the 501 id phase. The
immunoreaction product, containing lipoprotein
particles with apo B-100, i5 then admixed with an
anti-apo E antibody of this invention to ~orm a second
liquid-solid phase immunoreaction admixtùre. The
second admixture is maintained as before to allow the
anti-apo E antibodies to immunoreact with the solid-
phase lipoprotein particles and form a second solid-
phase immunoreaction product. The resulting second
product is detected to indicate the presence o~ apo E
2 ~
56
in the vascular fluid.
In preferred embodiments, the anti-apo E is
labeled, and the second product is thereby detected by
detecting the presence of label in the solid phase.
More preferably, the anti-apo E antibody has the
capacity to bind receptor-competent apo E
polypeptides, and can therefore be useful to determine
receptor-competent apo E rather than total apo E.
Techniques for operatively linking an enzyme
to an antibody molecule to form a conjugate are well
known in the art. Exemplary techniques are discussed
in Maggio, EnzYme-Immunoassay, Chapter 4 by Kabakoff,
CRC Press, Boca Ra~on, FL (1980), pages 71-104.
The monoclonal antibody molecules can be
utilized as obtained from hybridoma supernatants or as
ascites. However, it is preferred that purified
monoclonal antibody molecules be utilized.
Several means for purification of monoclonal
antibody molecules are well known in the art and
typically utilize chromatographic techniques. Fast
protein liquid chromatography (FPLC) is the
purification technique of choice herein.
The enzyme-linked monoclonal antibody
molecule conjugates are provided to the admixtures in
the fluid phase. Those molecules are typically
dissolved in an aqueous composition. Typical
compositions contain buffer salts as is the case of
the exemplary purified monoclonal antibody-containing
compo~itions used herein that include phosphate-
buffered saline ~PBg) as a diluent. Diluted ascitesfluid also is useful.
Preferably, non-specific protein binding
sites on the surface of the solid phase support are
blocked. Thus, the solid phase-bound paratopic
molecules are bound as by adsorption or other well
2 ~ 2 3 ~ ~ D
known means of affixation to the solid matrix.
Thereafter, an aqueous solution of a protein free from
interference with the assay such as bovine, horse or
other serum albumin that also is free from
contamination with human apo B-lQ0 or apo E is admixed
with the solid phase to adsorb the admixed protein
onto the surface of the paratopic molecule- containing
solid support at protein binding sites on the surface
that are not occupied by the monoclonal paratopic
molecule.
A typical aqueous protein solution contains
about 3 to about 10 weight percent bovine serum
albumin in PBS at a pH value of 7.1~7.5. The aqueous
protein solution-solid support admixture is typically
maintained for a time period of at least one hour at
37 degrees C, and the resulting solid phase is
thereafter rinsed free of unbound protein.
The vascular fluid sample can be plasma or
serum, as already noted. The sample is preferably
diluted at about 1:500 to about 1:5000, and more
preferably at about 1:1000. The use of le.sser
dilution can provide too much of the apolipoprotein
antigen to the admixture and impair the linearity of
the assay results as well as lower or abolish the
solid phase-bound paratopic molecule excess over the
admixed antigen. Use of greater than about a 1:20,000
dilution tends to decrease precision.
The maintenance times utilized can vary
widely with little variance in result so long as a
minimum time of about 30 minute3 at ambient room
temperature ~about 20-25 degrees C) is utilized.
Where it is desired to use a minimum 30-minute
maintenance time, it i9 preferred that the maintained
admixture be agitated during that time period to
assure substantially complete immunoreaction between
2 ~
58
the apolipoprotein antigen and monoclonal paratopic
molecules. Where longer maintenance times such as one
hour or more at room temperature are utilized,
agitation typically is not required. The desired
agita~ion can be readily supplied by means of a gyro-
shaker operated at about 100 rpm. Each of the assays
used in the method is capable of being carried out
using immunoreaction admixture maintenance times of
about 30 minutes to about 60 minutes at ambient room
temperatures.
The amount of apolipoprotein antigen present
in the assayed immunoreactant is determined by
admixture of the separated enzyme-linked
apolipoprotein-containing solid phase with a
predetermined amount of visualizing reagent or
reagents. Where HRPO is utilized as the enzyme
indicating means, visualizing reagents such as
hydrogen peroxide and an oxidative dye precursor such
as Q-phenylenediamine (OPD) present in an aqueous
medium are admixed with the separated solid phase-
bound immunoreactant. The admixture so formed is
maintained under biological assay conditions for a
predetermined time such as at least about 30 minutes
at ambient temperature for color to develop. Color
development is thereafter stopped by admixture of a
stopping reagent such as sulfuric acid. The optical
density of the composition is thereafter read,
compared to a standard curve value, and the amount of
apolipoprotein is determined, as is well known.
Thus, once the solid support and vascular
fluid ~ample are prepared, each assay can be carried
out at ambient room temperature in a time period of
about one hour; i.e., a 30-minute maintenance time
with agitation for admixtures formed from both
paratopic molecules and the sample aliquot, and
2~2~2~
59
another 30-minute maintenance time for color
development. Indeed, one need not prepare the salid
support just prior to each use, but rather, such
support~ as are described herein can be prepared and
stored damp and covered under usual refrigeration
conditions for a period of at least one month prior to
use.
I. Diaanostlc SYstems
The present invention also contemplates a
diagnostic system, typically in ~it form, that can be
utilized in carrying out the before-described assay
methods. The system includes, in an amount sufficient
for at least one assay, a subject apo E polypeptide
and/or anti-apo E antibody or monoclonal antibody of
this invention as separately packaged immunochemical
reagents. Instructions for use of the packaged
reagent are also typically included.
In one embodiment, a diagnostic system in
kit form includes a solid support comprising a solid
matrix such as a microtiter plate having an anti apo-E
monoclonal antibody of this invention affixed thereto
(operatively linked to the solid matrix) in an amount
sufficient to carry out at least one assay.
In preferred embodiments, the above
diagnostic system further includes, as a separately
packaged reagent, a second antibody, a reveal
antibody, that contains antibody molecules that
immunoreact with apo E-containing lipoprotein
particles. In this embodiment, the reveal antibody
can immunoreact with any component present on the apo
E-containing lipoprotein particle. Such particles
include apo E/LDL, apo E/VLDL, apo E/HDL, and the
li~e. Preferred are reveal antibodies that
immunoreact with apo 8-100 because it is a conserved
2~23~
epitope on apo E-containing lipoprotein particles.
ParticUlarly useful anti B-100 antibody molecules are
thQ monoclonal antibodies secreted by the hybridomas
MB47, which is available from the ATCC and has the
accession number HB8746.
In another embodiment used for assaying a
fluid sample for the presence of apo E polypeptide or
apo E-containing lipoprotein particles, a diagnostic
system includes a solid support comprising a solid
matrix having affixed thereto an apo E polypeptide of
this invention. The system can further include, as a
separately packaged reagent, an anti-apo E polypeptide
antibody of this invention for use in a competition
ELISA format.
` Preferably, a diagnostic system further
includes one or more of the following: (i) a supply of
hydrogen peroxide of known concentration; (ii) a
visualizing oxidative dye precursor such as OPD; (iii)
a solution of a stopping agent such as 4 N sulfuric
acid to quench the color-forming reaction; (iv) one or
more buffers in dry or liquid form for use in the
assay; and (v) materials for preparing standard
reference curves.
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. Thuq,
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.
2~23~
61
~ Instructions or 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 preferred embodiments, a diagnostic
system of the present invention f urther includes a
label or indicating means capable of signaling the
formation of a complex containing a polypeptide or
antibody molecule of the present invention.
The word ~complexn 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 nlabel~ and
"indicating meansn 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 o~ this invention only inso~ar 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
2 ~ ?
62
fluorochrome (dye) that is a useful immunofluorescent
tracer. Suitable fluorescent labeling agents are
fluorochromes such as fluorescein isocyanate (FIC),
fluorescein isothiocyante (FITC~, 5-dimethylamine-1-
naphthalenesulfonyl chloride (DANSC),
tetramethylrhodamine isothiocyanate (TRITC),
lissamine, rhodamine 8200 sulphonyl chloride tRB 200
SC) and the like. A description of immunofluorescence
analysis techniques is found in DeLuca,
"Immunofluorescence Analysisn, 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 em~odiments, the indicating
group is an enzyme, such as horseradish peroxidase
(HRP3, 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)
(A~TS).
Radioactive elements are also useful
labeling agents and are used illustratively herein.
An exemplary radiolabeling agent is a radioactive
element that producQs gamma ray emissions. Elements
which themselves emit gamma rays, such a~ l2~ 2~I,
l28I, ~32I and ~Cr represent one class of gamma ray
emission-producing radioactive element indicating
groups. Particularly preferred i9 12~I. Another group
of useful labeling means are those elements such as
1lC, 18F, 1'o and 13N which themselves emit positrons.
7, ~
The positrons so emitted produce gamma rays upon
encounters with electrons present in the animal's
body. Also useful is a beta emitter, such l~lindium of
3H.
The linking of labels, i.e., labeling of,
polypeptides and proteins is well known in the art.
For instance, antibody molecules produ~ed 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,
&alfre et al., Meth. Enzymol., 73:3-4~ l). The
techniques of protein conjugation or coupling through
activated functional groups are particularly
applicable. See, for example, Aurameas, et al.,
Scand. J. Immunol., Vol. 8 Suppl. 7:7-23 (1978),
Rodwell et al., Bioteçh., 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 nspecific binding agentn 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. However, when the
diagnostic system include~ a specific binding agent
that is not labeled, the agent is typically used as an
amplifying means or reagent. In these embodiments,
2 ~ 2 3 ~ r J . ~
64
the labeled specific binding agent is capable 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 used in an ~ELISA~ format to detect the
quantity of apo E in a vascular fluid sample such as
blood, serum, or plasma. nELISA~ 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 ImmunoloqY
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.
A reagent is typically affixed to a solid
matrix by adsorption from an aqueous medium although
other modes of affixation applicable to proteins and
polypeptides well known to those skilled in the art,
can be used.
Thu~, in preferred embodiments, an apo E
polypeptide, or anti-apo E antibody molecule, of the
present invention can be affixed in a solid matrix to
form a solid support that comprises a package in the
sub;ect diagnostlc system.
Useful solid matrices are also well known in
the art for preparing a solid support containing a
reagent affixed thereto. 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
2 ~
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 thos~ 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. Such materials
include glass and plastic (e~g., polyethylene,
polypropylene and polycarbonate) bottles, vials,
plastic and plastic-foil laminated envelopes and the
like.
J. Exam~les
The following examples are intended to
illustrate, but not limit, the present invention.
1. Polv~ePtide And Coniuqate Preparation
a. Svnthesis
The polypeptides p(141-155)2, referred to
as ~tandem peptide~, p(l41-155)3, the control
2~2~
polypeptides shown in Table 2, and other disclosed
polypeptides were synthesized using the classical
solid-phase technique described by Merrifield, Adv.
Enzvmol., 32:221-96, (1969) as adapted for use with a
model 430A automated peptide synthesizer (Applied
~iosystems, Foster City, California). Polypeptide
resins were cleaved by hydrogen fluoride, extracted
and analyzed for purity by high-performance liquid
chromatography (HPLC) using a reverse-phase C18 column
manufactured by Waters Associates, Milford, MA.
Table 2
Desianation1Amino Acid Residue Sequence
p93-112 LSKELQAAQARLGADMEDVR
pl39-149 SHLRKLRKRLL
pl41-155 LRKLRKRLLRDADDL
pl45-154 RKRLLRDADD
pl50-160 RDADDLQKRLA
pl61-171 VYQ~GAREGAE
pl67-176 REGAERGLSA
pl72-182 RGLSAIRERL
pl74-203 LSAIRE~LGPLVEQGRVRAATVGSLAGQPL
~ The designation for each peptide indicates the
position within the amino acid residue sequence of the
mature Apo E protein to which the peptide sequence
corresponds, i.e., is derived from.
b- ~lf~~ni~aation o~ ~o E Peptide~
~141-155 and pl29-163
The synthetic peptides containing the
amino acid residues 141-155 or 129-163 of apo E or the
peptide p(l41-155)~ were self-conjugated (i.e., pl41-
155 was coupled to pl41-155 and pl29-163 was coupled
to pl29-163) according to the procedure of Hoare et
2 ~ 2 3 !~i L ", ~
67
al., J Biol. Chem., 242:2447, (1967). Briefly, 100
mg of synthetic peptide was dissolved in 10 ml of high
purity water (Nanopure system). One gram of EDG ~1-
ethyl-3-(3-dimethylaminopropyl~ carbodiimide
S hydrochloride~ was admlxed to the peptide solution.
Ths reaction proceeds rapidly at room temperature and
is complete after one hour. During the first five to
ten minutes the pH of the reaction admixture is
monitored. The starting solution, before the addition
of EDG, is at a pH of approximately 3.7. Upon the
addition of EDG, the pH increases to approximately 5.0
in the first five minutes. At five minutes, 50 ul of
0.1 N HCL is added. No further addition of acid is
necessary during the rest of the incubation. The
admixture is rotated while incubating to insure
complete reaction. The reaction is quenched with 10
ml of 2M acetate buffer, pH 4.75.
To isolate conjugated (operatively linked)
peptides from unreacted peptide and chemicals, the
self-conjugation preparation is dialyzed in 2,000
molecular weight cut-off dialysis tubing, 18
millimeter wide and approximately two times the length
of the sample volume. Starting dialysis is done
against 2M acetic acid. A gradient reducing the
concentration of acetic acid from 2M to <0.01M is
achieved by changing the dialysis buffer (one liter)
every hour stepping down the concentration of acetic
acid by 50~ at each change. Once the acetic acid
concentration has been lowered to ~lOmM, the sample is
then dialyzed against one liter highly puri~ied water
over ni~ht at room temperature with at least five one
liter changes. The sample is lyophilized to dryness
and redissolved in phosphate-buffered saline (PBS) and
is ready for addition to cell culture.
2~3~
~8
The yield of self-conjugated peptide from this
procedure is low, usually not greater than 5%. For
instance, in one conjugation starting with 100 mg of
synthetic peptide, 2.82 mg of pl41-155 self-conjugated
peptide was recovered. The polypeptide concentration
of the peptide redissolved in PBS was determined by
the Lowry protein assay method. All additions of
peptide to cell culture of this self-conjugated
preparation were based on the value from Lowry assay.
The activity in the self-conjugated pl41-155
preparation is stable for at least two months when
stored at -20C.
2. Monomeric Pe~tides And Pe~tide-BSA
Coniugates Do Not SpecificallY Inhibit
LvmPhocyte Proliferation
A lymphocyte cell culture system was used to
examine the ability of various polypeptides and
conjugates to mimic the ability of Apo E to inhibit
lymphocyte differentiation as evidence by
proliferation.
Human peripheral blood mononuclear (PBM) cells
are isolated from whole blood on a Ficoll-Hypaque
gradient. The collected PBMs are washed three times
with fresh medium (RPMI-1640 plus 5% fetal bovine
serum, glutamine, penicillin, streptomycin and HEPES
buffer). After washing, the cells are counted and set
at a density o~ 1 x 10~/ml. For culture, 0.2 ml o~
the cells are plated per well of a 96-well microtiter
plate. The peptide or conjugate is added to the wells
in a 50 ul volume that can be either filter or W
sterilized. The typical timing for this experiment is
to expose the cells to the Apo E polypeptide or self-
conjugate over night. PHA, the mitogen that
stimulates lymphocyte growth and proliferation, is
2~3~ 7.;
69
added the next day. Forty-eight hours after the
addition of PHA, 1 uCi of 3H-thymidine is added per
well in a 1 ul volume and 18-24 hours later the cells
are harvested on a mash. The mash cell harvester
functions to collect the cells on filter paper where
they are washed with buffer to remove free 3H-
thymidine. The filter papers are dried, put into
scintillation vials with scintillation cocktail and
the B emissions obtained.
When examined in the above-described assay, the
polypeptides shown in Table 2 had no effect on
lymphocyte proliferation when used in non-conjugated,
monomeric form. However, as shown in Figure 1, when
those same polypeptides were used conjugated to bovine
serum albumin (BSA), lymphocyte proliferation was
apparently inhibited in an equivalent manner by all
peptides studies, as evidenced by decreasing amounts
of thymide uptake with increasing dose of conjugate.
3. Multimeric Peptides And Pe~tide Self-
Coniuqate's S~ecifically Inhibit
Lymphocvte Proliferation In A Non-
Cvtotoxic Manner
The ability of polypeptides and conjugates of
this invention to specifically inhibit lymphocyte
proliferation was examined using the assay described
in Example 2. Specifically, self-conjugates (pl72-
182)-(pl72-182), (p93-112)-(p93-112) and (pl41-155)-
(pl41-155) were compared for the ability to inhibit
lymphocyte proliferation. As shown in Figure 2, self-
conjugates (pl72-182)-(pl72-182) and (p93-112)-(p93-
112) demonstrated no significant capacity to inhibit
lymphocyte proliferation, as evidenced by their
failure to reduce H3-thymidine uptake. In contrast, a
self-conjugate of this invention, (pl41-155)-(pl41-
2~3~ ~
155) began demonstrating significant inhibitory
activity in the concentration range of about 0.8 to
about 1.0 ug/ml.
To examine whether or not tha observed
inhibition of proliferation was due to cell death
(direct cytotoxicity), the treated cell cultures were
subjected to a lactate dehydrogenase (LDH) release
assay as described by Carney et al., J. Immunol.,
134:1084, (1985). The presence of LDH in the cell
culture supernatant indicates toxicity because it is
released by cells that have lysed. According to the
results of this study, shown in Figure 3 for self-
conjugate (pl41-155)-(pl41-155), there is less than 4
release of LDH activity at the concentration of self-
conjugate that inhibits lymphocyte proliferation by
greater than 95%. These results demonstrate that the
polypeptides and self-conjugates of this invention are
not directly cytotoxic and inhibit lymphocyte
proliferation specifically.
Inhibition by Apo E of mitogen-stimulated
lymphocyte proliferation i5 not readily reversible.
The reversibility of the inhibition by the self-
conjugated peptides of this invention was tested by
incubating lymphocytes overnight with the peptides,
then washing the cultures before adding PHA. As shown
in Table 3 the self-conjugate (pl41-155) - (pl41-155),
was maximally inhibitory under these conditions.
Further, this inhibitory activity could not be
reversed by washing away the non-cell-associated self-
con~ugate peptide prior to PHA stimulation. In this
respect, the inhibitory activity o~ self-conjugate
(pl41-155) -(pl41-155) mimicked the irreversible
inhibitory activity observed with natlve Apo E.
2 ~ t~ ; 2 ~
Table 3.
Lymphocyte Proli~eration was Irreversibly Inhibited
by a Self-Conjugate of Peptide 141-155'
3H-Thymidine Uptake
(c~m_+_~D~
Trçatment No Washina
PBS69,106 ~ 6,55557,123 + 8,842
Self-conjugated
apo El4lls~1,035 ~ 170425 + 100
Self-conjugated
apo A - I~5lC5C 83,480 + 4,02764,524 + 4,200
'All cultures contained l x 10~ PBM cells per ml. The
cells were cultured at 37~ C in RPMI 1640 with 5%
fetal bovine serum. All cells were exposed to PHA at
24 hr., labeled with 3H-thymidine at 48 hr. and
harvested at 72 hr.
bAt 24 hr. the cells were washed three times in medium
and resuspended to their original volume in RPMI 1640
containing 5% serum.
CPeptides were used at a final concentration o~ 40
~g/ml.
4 Monomeric Peptides And Peptide-BSA
.
Con~uqates D~ NQt Speci~ically Inhibi~
Ovarian Androaen Production
2~2~
The cell system used to examine the effect of
various peptides and conjugates on ovarian androgen
production wa~ that described by Curtiss et al., J.
Biol. Chem., 263:10965, (1988). Briefly,
hypophysectom~zed (pituitary removed) immature female
rats are sacrificed by cervical dislocation. The
ovaries taken from the rats are trimmed free of fat
and other non ovarian tissues, cut into approximately
six to eight pieces per ovary and dissociated in a
solution of collagenase/ DNAase which degrades the
connective tissue releasing cell clusters.
Approximately one to two hours of 37C incubation is
required to dissociate the tissue. The cells are
washed three times with fresh McCoy'~ 5A modified
medium supplemented with glutamine and
penicillin/streptomycin. No serum is added to this
medium. The cells are cultured over night in 10 ml of
medium in a T-2S flask to allow the non-steroidogenic
cells to adhere. The next day non-adherent cells are
recovered, washed three times with fresh media,
counted and plated at a density of 1 x 103/ml
aliquotting 0.2 ml per well of a 96-well microtiter
plate. All media in the experiment contains
luteinizing hormone (LH) at 4 ng/ml and human high
density lipoprotein (HDL) at 300 ug protein/ml.
Monomeric peptides, peptide-~SA conjugates and self-
conjugated peptide preparations are added in a 50 ~37
~1 volume in PBS. The cells are cultured for 48 hours
then the supernatants are recovered, tranq~erred to a
clean 96-well microtiter plate and kept at -20C until
the concentrations o~ androstenedione and progesterone
are measured by radioimmunoassay.
When examined in the above-described assay, all
of the polypeptides of Table 2 that were studied, when
used in non-conjugated, monomeric form, had no effect
2~2~
on androgen secretion. However, as shown in Figure 4,
when those same peptides were used conjugated to BSA,
androgen production was apparently inhibited in an
equivalent manner by all peptides studied, as
evidenced by decreasing amounts of androstenedione
production with increasing dose of conjugate.
5. A Multimeric Peptide And A Peptide Self-
Coniuate SPecifically Inhibit Ovarian
Androqen Production
The ability of polypeptides and conjugates of
this invention to specifically inhibit ovarian
androgen production was examined using the assay
described in Example 4. As shown in Figure 5, self-
conjugate (pl41-155)-(pl41-155~ demonstrated the
ability to inhibit androgen production in ovarian
theca/interstitial cells at a concentration ~5 low as
that in the range of about 1.5 ~/ml. In contrast,
other self~conjugates not containing the Apo E 141-155
do not inhibit androgen production.
Apo E inhibition of ovarian cell androstenedione
production is reversible. To test if the inhibitory
activity of self-conjugate (P141-155) -tP141-155) was
reversible, ovarian cells were cultured with the
peptide for 48 hours. At the end of this culture, the
medium was removed and replaced with fresh medium
without the self-conjugated peptide. As shown in
Table 4, ovarian androstenedione production was
inhibited during the first 48 hours of culture but,
after the peptide was remaved and the ovarian cells
refed with fresh medium, their androstenedione
production during the subsequent 48 hours returned to
control levels. In this respect, the activity of the
polypeptideA and/or conjugates of the present
invention mimicked the activity of the native APO E.
2~23~2~
74
Table 4.
Ovarian Androgen Production was Reversibly Inhibited
by a Self-Conjugate of Apo E Peptide 141-155'
Androstenedione Accumulation
(na/ml)
First 48 hr. Second 48 hr.
Treatmentof Culture of Culture
PBS12.13 + 0.26 11.27 + 0.84
Self-conjugated
Peptide6.27 + 1.26 13.34 + 2.75
'Ovarian theca/interstitial cells (20,000/0.25 ml)
were cultured in the absence or presence of self-
conjugated peptide. After 48 hr. of culture thesupernatants were removed and the cells refed with
fresh medium without self-conjugated peptide.
To examine whether or not the observed
inhibition of androgen production was due to cell
death (cytotoxicity), the treated cell cultures were
assayed for progesterone production as described by
Dye~ et al., J. Biol. Chem., 263:10965-10973, (1988).
The presence of progesterone in the culture indicates
the cell are viable. According to the results of this
study, also shown in Figure 6 for self-conjugate
(pl41-15S)-(pl41-155), there is no significant
decrease in progesterone production at the
concentration of self-conjugate that inhibits 95% of
androstenedione (Adione) production. This result
indicates that the observed inhibitory activity was
2 ~ 2 ~
specific and not the result of cell death
(cytotoxicity) .
6. A Dimeric PePtide and Sel~-Coniuqates of
the ~imeric PePtide Affect LYm~hocyte
Proliferation
The ability of the dimeric peptide p(l41-l55)2
and self-conjugates of this peptide p(l41 155)2 -
p(l41-155)2 to affect lymphocyte proliferation was
examined using the assay described in Example 2. As
shown in Figure 7, tandem Apo E peptide p(l4l-155)2
enhanced lymphocyte proliferation at low
concentrations and inhibited lymphocyte proliferation
at high concentrations. This ability to both enhance
and inhi~it lymphocyte proliferation, depending upon
the dose of the peptide, mimics the activity of the
native Apo E. Enhancement of lymphocyte proliferatlon
was seen at doses of tandem Apo E peptide from about 2
~g/ml to about 6 ~g/ml, while inhibition of
proliferation was seen at concentrations of at least 8
~g/ml. As shown in Figure 8, self-conjugates of
tandem Apo E peptide were even more potent than tandem
Apo E peptide alone in its ability to affect
lymphocyte proliferation.
7. A Dimeric PeDtide and Self-Coniuqates of
this Pe~tide Affect Ovarian Androaen
Production
The ability of tandem Apo E peptide and sel~-
con~ugates of this peptide to a~ect ovarian androgen
production was examined using the assay described in
Example 4. As shown in Figure ~, when tandem Apo E
peptide was added in concentrations less than about
0.4 ~g/ml, androstenedione production was enhanced.
Concentrations of tandem Apo E peptide greater than
2 v ~
about 0.6 ~g/ml inhibited androstenedione production
in a dose-dependent manner.
8. Apo E poly~ePtide binds ~iPo~roteins
To assess the degree of binding of the tandem
peptide with lipoproteins, ~2~I-tandem peptide was
mixed with very low density lipoprotein (VLDL), LDL,
high density lipoprotein (HDL) or lipoprotein-depleted
serum (LPDS) to measure direct binding. Briefly, the
lipoproteins were isolated by ultracentrifugation
according to Curtiss, et al., J. Biol. Chem.,
257:15213 (1982). The lipoprotein-depleted serum
(LPDS) was obtained as the bottom fraction remaining
after flotation of the ~DL at a density of 1~25 gm/ml.
All fractions were diluted in PBS containing 3% bovine
serum albumin ~BSA). The tandem peptide p(141-155)2
was radiolabeled using the iodine monochloride method
of Brown et al., Methods Enzymol., 98:241 (1983) to a
specific activity of 7.99 x Io6 cpm/ug and was 99%
precipitable in 10% trichloroacetic acid (TCA). The
binding assays were performed in siliconized tubes in
a total volume of 0.3 ml of PBS that contained 3% BSA,
20 ~g of lipoprotein or LPDS and 250 ng of 125I-tandem
peptide. After 1 hr at 37C the lipoprotein bound
peptide was ~eparated from the free peptide. Tubes
containing VLDL, LDL and LPDS were precipitated with
0.3 ml of 555 uM phosphotungstic acid and 25 mM MgCl2
and tubes containing HDL was precipitated with 0.3 ml
of an apa AI-specific antiserum. Each precipitation
condition had been previously optimized with the use
of radioiodinated lipoproteins and was designed to
achieve 100% precipitation of the lipoproteins.
Precipitated radioactivity was measured by detecting
gamma radiation and expressed based on specific
activity as nanograms (ng) of peptide bound per
QJ,~
microgram (ug) of protein.
The results, shown in Table 5, indicate that
less than 0.7% of the added l25I-tandem peptide was
associated with HDL, and that 58% and 39% of the added
peptide was found associated with VLDL and LDL,
respectively. Calculated as the number of tandem
peptide molecules bound per lipoprotein particle,
VLDL, LDL and HDL each contained 1.8, 0.25 and 0.0043
peptide molecules per particle, respectively.
Therefore, the apo E polypeptide p(141-155)2
preferentially binds lipoprotein particles in the
following order VLDL > LDL > HDL.
Table 5
ng of l25I-tandem Molar ratio of
LIPOPROTEIN peptide bound per peptide per lipo-
OR PROTEIN ~g protein protein particle
VLDL 7.25 1.8
LDL 1.87 0.25
HDL 0.07 0-0043
LPDS 0.76
.
9. A Tandem Peptide Affects LDL Bindinq and
De~radation
The LDL receptor binding properties of the
tandem peptide were asses~ed using normal human
fibrobla5ts and a tran~ormed human monocytic-like
cell line, THP-l as a source o~ LDL receptor. THP-1
cells were studied because both the LDL receptor and
another lipoprotein receptor, the scavenger receptor,
are present and binding with those receptors i5 easily
examined. In the undifferentiated state THP-1 cells
express LDL receptors that are regulated by the
2~23.~
78
lipoprotein content of the culture medium; Hara, et
al~, ~iochem. BiophYs. Res. Commun., 146:802 (1987~;
Via, et al., J. Li~id Res., 30:1515 (1989)~ Following
phorbol myristate acetate ester (PMA)-stimulation the
cells stop dividing, differentiate into macrophage-
like cells, eventually lose most of their LDL
receptors and acquire scavenger receptors that bind
acetylated or modified LDL; Hara, et al., 8iochem.
Bio~hvs. Res. Commun., 146:802 (1987); Via, et al., J.
Lipid Res., 30:1515 (1989).
a. Inhibition of de~radation_by
unstimulated THP-l cells
THP-1 cells were acquired from American
Type Culture Collection and cultured in serum-free
RPMI-1640 medium supplemented with 1% Nutridoma-HU for
48 hr to upregulate the LDL receptors. LDL was
isolated by ultracentrifugation and radiolabeled using
the iodine monochloride method as described in Example
8 to a specific activity 200-300 cpm/ng. Greater than
99% of the l25I-~DL ligand was precipitable by 10% TCA.
The peptides indicated in Figure 10 were synthesized,
as described in Example 1, purified to > 95~ by high
pressure liquid chromatography and the amino acid
composition verified. All peptides were solubilized
in PBS.
Binding and degradation of LDL was evaluated as
the disappearance of acid soluble ~2~I-LDL
radioactivity from th~ incubation over a 5-hour
incubation at 37C. Various concentrations o~
unlabeled LDL or tandem apo E peptide were coincubated
with the 125I-LDL to determine their effects on binding
and degradation.
For ass~y, 5 x 105 THP-l cells in O.Sml DMEM
were incubated in a 1.5ml microfuge tube with 2-5
2 ~ g 2 ~_
;79
~g/ml of 125I-LDL at 37'C. After 5 hr the cells were
pelleted at 5000 x g and the supernatants were removed
and extracted with 10% TCA. The results were
expressed as B/Bo where ~ = TCA soluble cpm in
presence of LDL or peptide and ~0 = TCA soluble cpm in
phosphate buffered saline (PBS) control cells. Each
point i5 t~e average of 4 replicates with standard
error of the mean (SEM),10%.
As shown in Figure 10 (top panel), the tandem
141-155 apo E peptide enhanced LDL degradation by
unstimulated THP-l cells at low concentrations and
inhibited LDL degradation at high concentrations. In
this experiment, enhancement of LDL concentration
occurred at tandem apo E peptide concentrations
ranging from about 0.08 to about 1.5 ~M. Inhibition
of LDL degradation began to occur at tandem apo E
peptide levels of 2.0 to 5.0 ~M. Unlabeled LDL
inhibited l25I-LDL degradation, indicating the
specificity of the LDL receptor.
It can be seen that neither a control apo A-I
74-105 peptide or the monomer apo E 141-lS5 peptide
LDL inhibited l25I-LDL degradation. In contrast, the
tandem apo E peptide inhibited l25I-LDL degradation by
50% at 5uM. Approximately a 200-fold molar excess of
the tandem apo E peptide compared with LDL was
required to achieve 50% inhibition of degradation.
b. InhibitiQn_is LD~ re~e~tor-s~eci~L~
To verify that the lnhibition of LDL
degradation was specific for the LDL receptor, the
effect of the tandem peptide on scavenger receptor
processing of acetylated LDL (aLDL) was tested.
THP-l cells (5 x 105 in l.Oml of DMEM medium per
16mm culture dish) were stimulated with PMA (10-'M)
for 4 days. LDL was acetylated (~ara, H. et al.,
2~2~
Biochem. Bio~hvs. Res. Comm., 146(2~:802-808, 19871
and then radio-labeled as described in Example 8 to a
specific activity o~ 300-500 cpm/ng resulting in 12~I-
LDL that was greater than 99% precipitable by 10% TCA.
The cells were washed with PBS and the assay performed
in DMEM medium supplemented with 1% Nutridoma-HU plus
25 ~g/ml of 12~I-aLDL. After 5 hr at 37-C, the
supernatants were removed from th~ wells and extracted
with 10% TC~. The B/Bo ratios were analyzed as
described above. The results are depicted in Figure
lO (bottom panel).
Neither LDL nor the tandem peptide inhibited the
degradation of '2~I-aLDL by PMA-stimulated THP-1 cells.
As can be ceen in Figure 10 (bottom panel), at 12.5 uM
the tandem apo E peptide caused an 80% inhibition of
125I-LDL degradation but had no effect on l2~I-aLDL
degradation.
lO. Confirmation of bindina bY the LDL
rece~tor and Identification of critical
amino acids
The role of specific amino acids within
residues 141-155 of intact apo E has been studied by
Lalazar et al., J. Biol. Chem., 263:3542 (1988) with
human apo E variants containing single amino acid
substitutions. The variants were produced in a
bacterial expression system complexed with the
phospholipid, dimyristoylphosphatidylcholine (DMPC)
and tested ~or their ability to bind to ~ibroblast LDL
receptors. Lalazar et al., J. 3iol. Chem., 263:3542
(1988). To confirm that the tandem peptide was bound
by the LDL receptor and to identify amino acids within
this peptide that were critical for binding, three
tandem peptldes were prepared as described in Example
1 that contained the same amino acid substitutions at
2~2~9~
both positions in the tandem sequence. The changes
included substitutions of the basic amino acids Lys
143 to Ala, Arg 150 to Ala, and Leu 144 with an alpha
helix disrupting amino acid, Pro. ~ LDL at a
specific activity of 700~900 cpm/ng ~> 99%
precipitable by 10% TCA) was used for the binding
assay as described in Example 9. Normal fibrobla~ts
were plated in 16 mm culture dishes, grown for 72-96
hr in DMEM medium with 10% fetal bovine serum tFBS)
and then transferred to D~EM medium with 10%
lipoprotein-depleted serum (LPDS) for 48 hr to
upregulate the LDL receptors. The amount of ~25I-LDL
bound per well was normalized to the protein content
of each well, which was measured with the Bio-Rad
protein assay reagent using bovine serum albumin (BSA)
as a standard.
As illustrated in Figure 11, each of the
substitutions in p(141-155) 2 reduced the ability of
the tandem peptide to inhibit the binding of l25I-LDL
to fibroblasts. The Lys 143 to Ala substitution had
the smallest effect, whereas the Leu 144 to Pro
substitution had the greatest impact. In fact, the
Leu 144 to Pro substituted tandem peptide had no
activity at 60 uM, an effect that was similar to that
observed with the native apo E variant. Lalazar et
al., J. Biol. Chem., 263:3542 (1988).
11. ~iqher ordered multime~ of the 141-155
_equence may_have qreater bindina activitY
A trimer of the pl41-155 peptide, p(l41-155)~,
was synthesized as in Example 1 and its activity
compared with p(141-155) (monomeric~ and with p(141-
155)2 (dimeric) peptide. Normal fibroblasts were
plated in wells of a 96-well culture plate and grown
for 96 hr in DMEM medium with 10% FBS. The cells were
82
transferred to DMEM medium with 10% LPDS for 48 hr to
up regulate the LDL receptors. The LDL degradation
assay was performed according to Example 9 with 2
~g/ml of l25I-LDL (300-500 cpm/ng3 in 0.2 ml/well in
DMEM medium with 10% LPDS and incubated for 5 hr at
37~C. ~he supernatants removed from the wells were
extracted with 10% TCA. The TCA soluble counts were
normalized to protein content per well as measured by
the Bio-Rad protein assay.
Compared on a molar basis for their ability to
inhibit fibroblast '25I~LDL degradation, the trimer
peptide was 15 times more effective than the dimeric
peptide (Figure 12). The results indicate that higher
ordered multimers of the 1~1-155 sequence have
increased receptor binding activity.
12. Total numbeF of bindina_sites ~er cell
The direct binding of the 125I-tandem peptide to
dividing THP-l cells was measured. THP-l cells were
cultured for 48 hours in LDL-free RPMI-1640 medium
containing 1% Nutridoma-Hu. Increasing amounts of
~25I-tandem peptide (17,444 cpm/pmole), labeled as
described in Example 8 were added to 5 x 105
cells/0.05ml of DMEM medium containing 1% Nutridoma-Hu
combined with 0.05 ml of PBS or 0.05ml of VLDL
500~g/ml in siliconized glass tubes. After 20 min at
4-C the free ~25I-tandem peptide was separated from the
bound peptide by sedimenting the cells through
silicone oil. Background counts were ~ 1000 cpm/tube.
l25I-tandem peptide blnding wae found to be
linear with cell number, saturable and reached an
apparent steady state within 20 min at 4'C. ~25I-
tandem peptide binding to THP-1 cells was specifically
inhibited by apo B- and apo E-containing lipoproteins
with the ability to inhibit ordered as follows:
2~2~S2~
VLDL > LDL > HDL.
The binding of the 125I-tandem peptide to the
cells also was dependent on Ca~. A 2-fold increase
in the amount of l25I-tandem peptide bound to the cells
was obtained when the Ca~ concentrations were
increased from 0.1 to 2.0 mM, whereas Mg~ at up to
2.0mM had no effect. Finally, if THP-l cells were
deprived of LDL-containing serum for 96 hr before 125I-
tandem peptide binding was assayed, a 2-3 fold
increase was observed with both ligands, indicating
up-regulation of both the LDL receptor and the tandem
peptide binding site.
When the binding of the l2sI-tandem peptide to
THP-l cells cultured in LDL-free m~dium was studied in
the presence of 2.0 mM Ca~ and in the absence and
presence of 500 ug/ml of VLDL, specific saturable
binding was observed with a Kd of 1.1 x 10-'M (Figure
13). From this data the total number of binding sites
per cell was calculated to be 1500.
Although the data indicates that the tandem
peptide bound the LDL receptor, the LDL receptor may
not be the only fibroblast or THP-l binding site or
the tandem peptide. Low density lipoprotein receptor-
related protein (LRP) is a recently described cell
surface protein that contains reiterated sequences,
which are homologous to similar sequences in the LDL
receptor; Herz et al., EMB0 J., 7:4119 (1988);
Beisiegel, et al., ~3~ , 341:162 ~1989). Recent
studies indicate that LRP may only interact with apo
E-enriched lipoproteins including beta-VLDL; Kowal, et
al., Proc. Natl. Acad. Sci. USA, 86:5810 (1989).
Unlike the LDL receptor, the LRP receptor on
fibroblastq does not appear to be down regulated by
exposure of the cells to LDL; Kowal, et al., Proc.
Natl. Acad. Sci. USA, 86:5810 (1989).
2 ~
84
13. Therapeutic Application
Specific targeting of an apo E polypeptide such
as the tandem peptide to cholesterol-rich lipoproteins
is accomplished by designing a synthetic peptide that
has high affinity lipid and receptor binding
properties. Attachment of the 141-155 tandem sequence
to a lipophilic molecule or peptide facilitates a
specific high affinity association of the tandem
sequence with cholesterol-rich lipoproteins and
increases the hepatic clearance of the lipoprotein
from the circulation. A reproducible and significant
enhancement of LDL degradation by THP-1 cells (Figure
10, top panel) and by fibroblasts (Figure 12) at low
concentrations of the tandem peptide was observed when
tandem peptide was contacted with the cells in vitro.
There are several examples cited in the
literature in which it has been observed that apo E
~unction is dependent on the number of apo E molecules
per lipoprotein particle: (a) reducing the number of
apo E molecules per DMPC complex results in decreased
binding; Mahley, et al., 8iochim. Biophys. Acta,
737:197 (1983); (b) VLDL uptake by the liver is
enhanced by adding additional copies of thrombin-
accessible apo E; Bradley, et al., ~ iQl _5h~m~,259:14728 (1984); (c) an average of greater than one
apo E molecule per particle is required for VLDL to
stimulate macrophage cholesterol esterification;
Soltys, et al., J~ Li~id Re~., 29:191 (1988); (d) VLDL
carrying more apo E are removed ~rom blood more
rapidly; Yamada, et al., Proc. Natl. Acad. Sçi. US~,
86:665 ~1989), and finally (e) only apo E-enriched
beta VLDL is taken up bv the LRP receptor; Kowal, et
al., Proc. Natl. Acad. Sci. USA, 86:5810 (1989). It
is believed herein that this requirement reflects the
2~23~ ~
~5
close association of at least two copies of apo E to
form an LDL receptor-competent apo E ligand.
14. Pre~aration of Polyclonal Antisera to
Synthetic Polypeptides
a. Preparation of Immunogen
LDL was isolated from plasma obtained by
plasmaphoresis of normal pooled rabbit blood (Scripps
Clinic and Research Foundation Vivarium, La Jolla,
Calif.). Plasma so obtained was adjusted to contain a
final concentration of 2 millimolar (mM) benzamidine,
14 mM ethylenediaminetetraacetic acid (disodium salt)
(EDTA), 20 micrograms per milliliter (~g/ml) soybean
trypsin inhibitor, 10,000 units per ml aprotinin, 20
~g/ml lima bean trypsin inhibitor, 25 ~g/ml polybrene,
and l~M D-phenylalanyl-l-prolyl-l-arginine
chloromethyl ketone (PPACK). The LDL 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 was removed and retained. The
bottom layer of the supernatant was recovered and
admixed with solid KBr layer until the density was
greater than 1.063 grams per milliliter (g/ml). The
resulting admixture was then layered under a 0.1% EDTA
solution containing KBr at density of 1.063 g/ml and
centrifuged at 186,000 x g ~or 18 to 24 hours.
After the second centri~ugation, the top layer
containing LDL was recovered and the bottom layer
containing HDL was discarded. The top layer is
admixed with solid KBr until the density was adjusted
to greater than 1.063 g~ml. That adjusted layer wa~
2 0 2 t,j ~ ~ :
~6
layered under a 0.1% EDTA solution containing KBr at a
density of 1.21 g/ml and was centrifuged at 186,000 x
for 18 to 24 hours at 4~C.
The top layer was then recovered, and solid XBr
was admixed until the density was greater than 1.063
g/ml. That adjusted top layer was 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 4C. The top layer containing
lQ concentrated LDL was recovered and dialyzed against
PBS (phosphate-buffered saline, pH 7.2) and stored at
-70'C.
The multimeric polypeptide analogs of apo E,
(pl41-155)2, (pl41-155)3, and self-conjugates of
~5 these, were synthesized as described in Example la and
b. The apo E polypeptide p(141-155)2 was dissolved in
1.5 M sedium acetate, pH 7.8, to a final concentration
of 6 mg/ml in a total volume of S mls. A dissolved
polypeptide was admixed with 2.5 ml 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 was a 500 mM
glutaraldehyde solution using a 2.7 molar excess of
glutaraldehyde to peptide. The admixture was
maintained at room temperature for 10 minutes, after
which a 40 mM solution of sodium borohydride was added
to a final concentration of 0.2 mM. The admixture was
thereafter maintained at 37C for 5 to 8 hours,
followed by dialysis against P~S for 5 days with two
buffer changes per day, using dialysis tubing having a
12,000 to 14,000 molecular weight cut-off. The
dialysed solution was centrifuged at 2500 x g for lO
minutes, and the resulting pellet was resuspended in 5
ml PBS to form a peptide-LDL immunogen. A peptide-LDL
immunogen was prepared using the above-described
2~23~9 i~
87
pQlypeptide, namely p(141-155) 2.
b. Immunization and Collection of Polyclonal
Antisera
The peptide-LDL immunogen prepared in Example
14a above was emulsified using the Ribi Adjuvant
System (Ribi Immunochem Research, Inc., Hamilton,
~ontana) according to the manufacturer's instructions.
The peptide-LDL antigens were incorporated into the
emulsion at a concentration of 300 ~g/ml. After pre-
immune serum samples were collected, two rabbits were
injected with 1 ml of a prepared emulsion. The 1 ml
emulsion dose was administered as follows: 0.30 ml
intradermally (0.05 ml in each of 6 sites); 0.40 ml
intramuscularly (0.2 ml into each hind leg); 0.10 ml
subcutaneously (in the neck region); and 0.20 ml
intraperitoneally. The rabbits were injected 6 times
at three-week intervals following the injection
protocol as detailed. At one week after the second
through sixth injections, blood samples were collected
to check antibody titer against the specific peptide
used as the immunogen by the SPRIA assay described
below. The collected blood samples were stirred in a
37-C oven for 1 hour, after which the samples are
centrifuged at 3000 x g for 20 minutes. The interface
was collected and spun in a microfuge at 12,000 x g
for 5 minutes. The supernatant containing anti-
peptide antibodies was collected and stored at -20C.
The peptide antibody titers were determined by
solid phase radioimmunoa~say ~SPRIA) essentially as
described in Curtiss and Edgington, ~. 3iol. Chem.,
257:15213-15221 (1982). Briefly, 50 ~1 o~ PBS
containing 5 ,ug/ml synthetic peptides were admixed
into the wells of microtiter plates. The plates were
maintained overnight (about 16 hours) at 4C to permit
2$~35~ ~
~8
the peptides to adhere to well walls. After washing
the wells four times with SPRIA buffer (2.68 mM KCL,
1.47 mM KHzPO,, 137 mM NaCl, 8.03 mM Na2~PO~, 0.05%
Tween-20, 0.1 KIU/ml Traysol, 0.1% BSA, 0.015% NaN3),
200 ~1 of SPRIA buffer containing 3% normal goat serum
(NGS) and 3~ bovine serum albumin (BSA) were admixed
to each well to block excess protein binding sites.
The plates were 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 an apo E
polypeptide was operatively affixed.
To each well was then admixed 50 ~1 of a serum
supernatant containing anti-peptide antibodies for
testing by SPRIA form a solid-liquid phase
immunoreaction admixture. The admixture was
maintained for 2 hours at 37 C to permit formation of
solid-phase immunoreaction products. After washing
the wells as previously described, 50 ~1 of a second,
reveal antibody, 12~I-labeled goat anti-mouse I~G, at
0.25 ~g protein per ml were admixed to each well to
form a labeling reaction admixture. That admixture
was maintained for 1 hour at 37-C to permit formation
of l25I-labeled solid-phase immunoreaction products.
After washing the wells as previously described, the
amount of l25I-labeled product bound to each well was
determined by measuring gamma radiation from the
labeled product. Specific anti-peptide antibody
titers in collected serum samples ~rom immunlzed
rabbits were thus determined and compared to titres
measured using pre-immunized normal rabbit serum
samples which are a measure o~ non-speci~ic
background. Se N m samples are c~nsidered to contain
anti-peptide polyclonal antibodies i~ the radioactive
signal was 5 times greater than seen with normal
rabbit serum.
2~3~ .L
89
Anti-apo E polypeptide an~ibodies were obtained
by the above procedure when peptide-LDL immunogens
were used that contains the apo E peptide p(141-155) 2.
Additional anti-apo E polypeptide antibodies are
5 prepared using peptide-LDL immunogens based on the apo
E peptides p(l41-155)3 and self-conjugates of p(l41-
155)2 and p(141-155)3.
The above-described assay i5 utilized to
determine whether this polyclonal antisera binds apo
E/VLDL. Apo E/VLDL is prepared as described in
Example 14a above. Instead of synthetic peptides, 50
ul of PBS containing 5 ug/ml apo E/VLDL is admixed
into the wells of the microtiter plates. SPRIA assays
performed in this manner indicate that the antibodies
bind apo E/VLDL.
15. Monoclonal Antibody Preparation
a. Generation of Hybridomas
Balb/c BvJ mice (Scripps Clinic and Research
Foundation Vivarium, La Jolla, CA) are immunized
intraperitoneally (i.p.) with 50 ug of lipoprotein
(polypeptide-carrier complex is used in the case of
apo E in complete Freund's adjuvant (CFA) followed by
a second immunization in lipoprotein buffer on day 28,
3 days prior to fusion.
The animals immunized with apo E multimeric
polypeptide analog, (pl41-155) 2, conjugated to LDL as
described in Example 14a, 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 degree~ C. Following removal of supernatant, the
cell pellet is resuspended in 5 ml cold NH4Cl lysing
buffer, and is incubated for about 10 minutes.
2~2~
To the lysed cell suspension are admixed 10 ml
~ulbecco's Modified Eagle Medium (DMEMl (GIBCO) and
HEPES [4-(2-hydroxyethyl)-1-piperidineethanesulfonic
acid] buffer, and that admixture is centrifuged for
about 10 minutes at 1000 r.p.m. at 23 degrees 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
degrees C. The above procedure is repeated.
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 P3X63Ag8.653.1, a subclone of line
P3x63Ag 8.653 (ATCC 1580). Using a myeloma to spleen
cell ratio of about 1 to 10 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
degrees C.
Spleen cells and myeloma cells are combined in
round bottom 15 ml tubes. The cell mixture is
centrifuged for 10 minutes at 1000 r.p.m. at 23
degrees C, and the supernatant is removed by
aspiration. Thereafter, 200 ul of 50 percent (weight
per volume) aqueous polyethylene glycol 4000 molecular
weight (PEG; ATCC Baltimore, MD) at about 37 degrees C
are admixed using a 1 ml pipette with vigorous
stirring to disrupt the pellet, and the cells are
gently mixed ~or between lS and 30 seconds. The cell
mixture is centrifuged 4 minutes at 700 r.p.m.
At about 8 minutes from the time of adding the
PEG, 5 ml of DMEM plus HEPES buffer are admixed slowly
to the pellet, without disturbing the cells. After 1
minute, the resulting admixture is broken up with a 1
2 g~ 2 ~
91
ml pipette, and is incubated for an additional 4
minutes. This mixture is centrifuged for 7 minutes at
1000 r.p.m. The supernatant i~ 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 had been placed previously. The
resulting cell suspension is incubated at 37 degrees 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 2x104
viable cells per well (768 total wells) in HAT buffer
medium as described in Kennett et al., 5~r~_~se~
Microbiol. Immunol., 81:77 (1978). The cells are fed
seven days after fusion with HAT medium and at
approximately 4-5 day intervals thereafter as needed
with HT medium. Growth was followed microscopically,
and culture supernatant~ were collected about two
weeks later and assayed for the presence of antibody
molecules that immunoreact with the immunizing
polypeptide by solid phase radioimmunoassay (SPRIA) as
described in Example 14b.
Brie~ly, 50 ul of PBS containing 5 ug/ml o~ the
immunizing apo E polypeptide o~ the immunizing apo E
polypeptide, (pl41-155) 2, are admixed into the wells
o~ microtiter plates. The plates are maintained for 3
hours at room temperature to permit the polypeptide to
adhere to well walls. After washing the wells four
~2~2~
92
times with SPRIA buffer (2.~8 mM XCl, 1.47 mM KH2PO4,
137 mM NaCl, 8.03 mM Na2HPO4, 0.05% Tween-20, 0.1
KIU/ml Traysol, 0.1% BSA, 0.015% NaN3), 200 ul of
SPRIA buffer containing 3% normal goat serum tNGS) 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 degrees C, the wells
emptied by shaking, and blotted dry to form a solid-
support, i.e., a solid matrix to which apo E
polypeptide was operatively affixed.
To each well containing an immunizing
polypeptide was then admixed 50 ul of hybridoma tissue
culture supernatant produced by the corresponding
immunizing polypeptide to form a solid-liquid phase
immunoreaction admixture. The admixture is maintained
for 2 hours at 37 degrees C to permit formation of
solid-phase immunoreaction products. After washing
the wells as previously described, 50 ul of lZ5I-
labeled goat anti-mouse IgG at 0.25 ug protein per ml
are admixed to each well to form a labeling reaction
admixture. That admixture is maintained for 1 hour at
37 degrees C to permit formation of 12~I-laheled solid-
phase immunoreaction products. After washing the
wells as previously described, the amount of 125I-
labeled product bound to each well was determined bymeasuring gamma radiation from the labeled product.
Hybridoma culture supernatant~ are considered to
contain monoclonal anti-apo E polypeptide antibodies
if the immunoreaction product formed produced a
radioactive signal five times greater than a signal
measured using a control hybridoma culture
supernantant.
The anti-B-100 monoclonal antibody MB47 was
prepared essentially as described above, except that
LDL waq used as the immunogen, and the antibody was
~ ~ 2 ~
93
selected by screening for immunoreaction with isolated
apo ~-lnO.
b. Isolati_n of Immunoalobulin
Ascites fluids containing monoclonal antibody
molecules useful herein were prepared using 10-week-
old Balb/c mice. The mice were first primed with 0.3
ml of mineral oil and injected intraperintoneally with
3-50x105 MB47 hybridoma cells prepared as described in
Example 15a. The average time for development of
ascites was 12 days. The resulting ascites fluid was
collected and clarified by centrifugation at 15,000xg
for 1 hour at 4 degrees C to form clarified ascites
fluids, which can be pooled and stored if desired, at
-20 degrees C.
Isolated MB47 monoclonal antibody molecules were
prepared by chromatography of the clarified ascites
fluids on a protein A-Sepharose 4B column (Pharmacia
Fine Chemicals, Piscataway, NJ). Antibody was eluted
from the column with 0.1 molar (~) acetic acid to form
the isolated monoclonal antibody MB47.
Isolated monoclonal antibody molecules were also
prepared by fast protein liquid chromatography (FPLC)
of a clarified ascites fluid on a Pharmacia Mono Q HR
5/5 anion exchange column in a Pharmacia FPLC System
using a 0-0.5M NaCl gradient in 10 mM Tris, pH 8.0,
and following the directions supplied with the column.
The resulting isolated monoclonal antibody molecules
were concentrated using an Amicon stirred
ultrafiltration cell (Danver3, MA; PM 30 membrane) to
a concentration of 1 mg/ml, dlalyzed into P~S
(phosphate-buffered saline, pH 7.2) and stored at -70
degree~ C to form purified monoclonal antibody.
Monoclonal anti-apo E polypeptide antibody
molecules can be purified as above using the hybridoma
2~2~,~2~
94
that can be prepared by the method~ of Example lSa to
form puri~ied anti-apo E polypeptide monoclonal
antibody.
16. Solid-Phase PolY~e~tide ELISA
~po E polypeptides prepared in Example 1
immunoreact with an anti-apo E polypeptide antibody in
a direct binding ELISA. In the assay, 50 ~g/ml of a
polypeptide-containing solution of ~pl41-155) 2 is
dissolved in P~S to form a peptide coating solution,
of which 150 ~1 is admixed into the wells of a
microtiter plate. The wells are then maintained for
about 16 to 20 h at 4'C to permit the peptide to
adsorb onto (coat) the walls of the wells. After
removing the peptide coating solution by shaking, the
wells are washed once with 350 ~1 of rinsing buffer
(PBS containing 1 g/l BSA, 0.5 ml/l Tween 20, and 2
~1/1 aprotinin). Excess protein binding sites are
blocked by admixing 200 ~1 of blocking buffer (PBS
containing 3% BSA) into each well, maintaining the
wells for 1 hour at 371C, removing the blocking buffer
by shaking, and then washing the wells 3 times as
previously described. The plate is then dried for 1
hour at 37~C followed by addition of 100 ~1 of PBS
containing 0.5 ~g/ml horseradish peroxidase~conjugated
anti-(pl41-155) 2 antibody, prepared according to the
method of Nakane, et al., J. Histochem. Cytochem.,
22:1084 (1974), to form a solid-liquid phase
immunoreaction admixture. The resulting solid~ uid
phase immunoreaction admixture is maintained at 20'C
for 1 hour to permit formation of a solid-phase
polypeptide-containing immunoreaction product. The
wells are then washed 3 times with rinsing buffer to
remove unbound antibody.
2~2~2~e
Assays are performed in identical manner using
anti-p(141-15S) 3 polyclonal antibodies, which are
elicited as previously described and conjugated to
horseradish peroxide.
s The amount of immunoreaction product present in
the solid phase is then determined by admixing two
hundred microliters of OPD substrate (3% H202 and 0.67
mg/ml o-phenylene diamine) into each well to form a
developing-reaction admixture. The admixture is
maintained for 30 minutes at about 20C.
Subsequently, SO ~1 of 4 N H2SO4 are admixed into each
well to stop the developing-reaction, and the
resulting solution i5 assayed for absorbance at 450
nanometers using a microtiter plate reader (Dynatech)
to detect the amount of formed immunoreaction product.
In order to determine specificity of the
antibody molecules using the same assay, a monomer
peptide, pl41-155, is prepared as described in Example
1 and tested as above. Additionally, control peptides
p93-112 and pl72-182 are prepared and tested. The
results in all cases indicate that these antibodies do
not bind either the monomer or the control peptides.
A competition ELISA is useful to detect apo E
polypeptide or apo E in a fluid sample such as serum.
~icrotiter plates are coated with the dimer p(141-
155) 2 as described hereinbefore. After the drying
step of the assay described hereinbefore, 50 ~1 of a
fluid sample (i.e., an apo E polypeptide-containing or
an apo E-containing fluid sample) or standard (i.e.,
an apo E polypeptide as a standard) to be assayed are
admixed into the polypeptide-coated well
simultaneously with 50 ~1 of HRPO-con~ugated anti-
peptide antibody to form an immunoreaction admixture.
In the assay described herein, two competitors
(polypeptide standard or the fluid sample) are tested
2~23~
96
in separate immunoreaction admixtures for their
ability ~o compete for binding of the anti-apo E
antibody to the coated polypeptide analog over a range
of antibody dilutions. The polypeptide standard in
solution is added at a starting concentration of 1
mg/ml and serially diluted to a final concentration of
0.0156 mg~ml. Serum or plasma fluid samples are added
at a starting dilution of 1:10 and diluted serially to
a final dilution of 1:320. The plate is then
incubated for 30 minutes at room temperature, 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 competition ELISA is particularly preferred
to measure apo E-containing lipoprotein particles in
bodily fluid, such as total apo E, and can also be
used to monitor the fate of therapeutically
administered apo E polypeptide present in the serum of
a patient after administration of therapeutic apo E
polypeptide compositions.
a. A~o E Sandwich Assay
Described herein is a capture or sandwich assay.
Polystyrene microtiter plates (Nunc-Immuno Plate I)
are coated with 150 ul of sodium bicarbonate buffer,
pH 9.0, containing 1 ug/ml of purified anti-p(141-
155) 3 polyclonal antibody ~or 16 hours at 4-C to
prepare a solid-phase capture antibody. The plates
are washed 3 times with PBS containing 0.1% ~SA, 0.05%
Tween, and then blocked with 3% BSA exactly as
described above. The apo E/VLDL standard (prepared as
in Example 14) is diluted 1:200 in PBS containing 0.5%
(Diluting buffer - LPDP/PBS) to concentrations ranging
from .125 to 4.0 ug/ml. The same controls as
2~23~ .
described above for the competition ELISA are used in
this as~ay. Plasma or serum samples and controls are
diluted 1000-fold .in dilution buffer. Fifty ul of
standardR and unknown samples are added to the wells
in triplicate.
The plates are incubated exactly 30 minutes at
25~C, and the liquid phase is removed from the wells.
Fifty ul of PBS, containing a fixed concentration of
HRPO-linked anti-B100 monoclonal reveal antibody
(MB47), described before, is immediately pipetted into
wells containing aliquot 1 of the plasma samples. The
plates are incubated exactly 30 minutes at 25~C,
washed, and 100 ul of OPD substrate solution are added
for an additional 30 minute incubation at 250C. Color
development is stopped by addition of 50 ul of 4N
H2SO~ and plates are read in a microplate reader as
described before.
Although the present invention has now been
described in terms of certain preferred embodiments,
and exemplified with respect thereto, one skilled in
the art will readily appreciate that various
modifications, changes, omissions and substitutions
may be made without departing from the spirit thereof.
It is intended, therefore, that the present invention
be limited solely by the scope of the following
claims.
