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CA 02584690 2011-10-25
PIP PEPTIDES BIOLOGIC ACTIVITIES, SITE OF ACTION, AND THE ANTIBODY
TO DETECT FIF
BACKGROUND
[0002] Fertilization requires the proper interaction between the egg and
sperm. Such a
targeted event, even under natural conditions, is random, and hence the
genetic make up of the
nascent genome is unpredictable. The impending pregnancy has to prepare the
maternal
environment towards acceptance of a semi- or oven a total allograft. This
preparation can be
divided to four distinct phases; the first is the pre-fertilization period,
the second is the
fertilization/post fertilization, the third phase is trophoblast development,
and the final fourth
phase is the implantation period.
[00031 The first phase, which is the pre-fertilization period, takes place
during follicular
development. The egg is surrounded by the cumulus oophorus and it is bathed in
the rich
follicular fluid. This fluid has some immune suppressive activity that may
facilitate the
fertilization process as well as post-fertilization development. This inunune
suppressive activity
is required, due to the fact that shortly after fertilization, expression of
foreign antigens, caused
by the spenn, may be present. This mechanism, however, is not a necessary
requirement. In
cases of in vitro fertilization, no such fluid is available, and fertilization
takes place without
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difficulty in an artificial environment. Moreover, women who have no ovaries
can get pregnant
by donor embryo transfer.
[0004] The next phase is the fertilization/post fertilization process. Once
the sperm
penetrates the egg it becomes "non visible" by the maternal environment.
During the process of
egg and sperm head fusion, as long as the egg surface membrane does not change
its
characteristics and become recognizable by the maternal immune system, no
immune reaction
would be expected. To safeguard the fertilized egg, it is rapidly surrounded
by the zona
pellucida, which is a hard and impenetrable shell designed to ward off immune
cells. A further
protection is due to the presence of the maternal cumulus cells. Those cells
may further prevent
direct access of immune cells to the embryo. The cumulus cells persist only
for the first few
days after fertilization because they facilitate the fallopian tube's cilia to
propagate the zygote
towards the uterus. Following fertilization, it is not excluded that small
proteins derived from
the maternal environment, such as cytokines, will reach the zygote and early
embryo. So far,
there has been no evidence for such an occurrence. Following the few initial
embryonic cell
divisions, to the eight-cell stage, the trophoblast phase is initiated.
[0005] The trophoblast phase that occurs by the sixteen-cell stage leads to
embryoblast
and trophoblast differentiation. While the trophoblast's genome is principally
paternally derived,
the embryoblast's genome is principally maternally derived. However, since the
zona pellucida
still surrounds the embryo, it provides a major protection against maternal
immune onslaught.
Therefore, it appears that the early embryo, during the pen-implantation
phase, is rather well
protected from maternal immune system. This is despite the fact that the
embryo is a semi-
antigen. This period in in vitro fertilization/embryo transfer (IVF/ET)
procedures does not
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occur. Consequently, the development of maternal tolerance remains permissive
until
implantation, which is where direct embryo/maternal contact become a necessary
prerequisite.
[0006] The final preparation phase is implantation, which occurs when the
embryo
reaches the uterus and intimate maternal contact is initiated. During
implantation the zona
pellucida opens, and the trophoblast cells are extruded. This is the time that
the embryo is most
vulnerable. The embryo is not yet attached to the maternal surface, and it is
still exposed to
endometrial maternal immune cells as well as potentially hostile cytokines. Of
all phases of
reproduction, the implantation phase is the most crucial. Specifically, in the
case of embryo
transfer, following IVF, the embryo has to sojourn in the endometrial cavity
for 4-5 days until
the maternal organism will accept it. This is a period of endometrial priming
by embryonic
signaling that leads to maternal tolerance, which is the pre-requisite for
successful pregnancy.
[0007] Mammalian reproduction was the last to evolve and it required a major
shift in the
immune system. This is because it allowed a sperm, which might be regarded as
a parasite, to
invade the maternal organism, and impose, in part, its own genome expression.
This suggests
that the embryo must have an active role in allowing the initiation of
pregnancy. This suggestion
is supported by the following observations:
(i) Donor embryos can implant without difficulty, therefore sharing of
maternal antigens is not required;
(ii) The site of embryo implantation is not obligatory, although the uterus
is
preferred. Occasionally, implantation can be found in the fallopian tubes,
ovaries, and even
inside the abdominal cavity;
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(iii) Under certain circumstances, embryos from one species can be implanted
and delivered by another species. Genetic mismatch also does not prevent a
successful
reproduction (i.e.: mule);
(iv) Only viable embryos will implant. Therefore, the implantation is an
active
act that requires a passive accommodation by the maternal recipient, upon
which the embryo can
impose its will, in order for the pregnancy to develop;
(v) Sick mammals can also get pregnant, which indicates that the maternal
organism does not need to be in good health in order for pregnancy to
initiate. This reiterates the
passive role of the maternal organism and supports a specific embryo effect;
(vi) The chances of multiple embryos to implant are higher than that of a
single embryo. Consequently, an enhanced embryo derived signaling is likely to
lead to
maternal acceptance; and
(vii) Although a window of opportunity for implantation does exist, it is not
strict. Therefore the embryo can implant in less than favorable endometrium,
as well.
[0008] In conclusion, it appears that the embryo, to a large degree, controls
it own
destiny. This destiny is irrespective of timing in cycle, site of
implantation, the sharing of genes,
species, or the health of the mother.
[0009] Early work on pregnancy suggested that shortly after fertilization,
certain changes
that favor tolerance take place in the maternal environment. Those changes
were believed to be
due to early pregnancy factor (EPF) and platelet activating factor (PAF).
However, these factors
are not specific to pregnancy and are found in a non-pregnant state as well.
More recent work
indicates that the embryo's presence, and the products that it secretes,
creates a favorable and
tolerant environment for a successful pregnancy. Several reports have shown
that the embryo-
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conditioned media has immune-modulatory effects on the maternal organism. The
addition of
the conditioned culture media, from human and mouse embryos, affects human
immune cells
activity. This immune modifying activity occurs very early, at the two- cell
stage embryo. The
activity is dependent on embryo viability, since the media of cultured atretic
eggs does not exert
such immune-modulatory features. Therefore, shortly after fertilization, the
embryo starts
actively to emit signals that create maternal recognition of pregnancy which
lead to immune
tolerance. The cumulus cells, which surround the segregated embryo, may serve
as a relay
system, since they contain active immune cells that secrete cytokines. Such an
intimate contact
between putative embryo-derived compounds, and the maternal immune system,
would allow for
a rapid diffusion of signals from the embryo. This would lead to a local
immune response, due
to the embryo presence, followed by a systemic maternal immune recognition.
Such changes in
maternal immunity are shown via a variety of bioassays, including a pre-
implantation factor
(PIF) assay that measures immune changes in the maternal systemic circulation.
This immune
change occurs within the first few days after fertilization. Additionally,
using IVF cycles, it has
been shown that within three days after embryo transfer, PIP activity can be
found already in
maternal circulation. This indicates that embryo-initiated signaling will
rapidly create a systemic
immune system tolerance to the embryo. Without wishing to be bound by theory,
increased PIP
activity may explain why implantation does not necessarily take place in the
uterus, but it can
occur elsewhere within the organism, and suggest that for embryo transfer to
be successful, a
similar PIP signal has to exist.
SUMMARY
[0010] Embodiments of the present invention relate to biological effects
induced in vitro
and/or in vivo by pre-implantation factor, (PIP), peptides, peptidomimetics,
and compounds
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derived from pre-implantation embryos that harbors in part, is identical to,
or is homologous to
the amino acid sequence of PIF peptides or to the scrambled amino acid
sequence of PIF
peptides. The invention also relates to the development of antibodies to
quantitatively detect
PlFs peptides in biological fluids. In particular, the present invention
relates to use of PIF
peptides or peptidomimetics to effect changes on the immune system of a
patient. More
specifically, the addition of PIF peptides creates specific changes both in
cellular immunity as
well as in a patient's secreted cytokine profile. Namely, following exposure
or stimulation to
diverse mitogens, phytohemagglutinin (PHA), CD3 antibody, and the mixed
lymphocyte
reaction (MLR), PIF causes a shift towards immune tolerance reducing
peripheral mononuclear
blood cells (PBMC) proliferation while un-stimulated cells remain unresponsive
to the effect of
the PT peptides. Moreover, addition of nP1F-115 (SEQ ID NO:1) to stimulated
PBMC caused an
increase in TH2 (ml 0) type cytokine while reducing TH1 (INF-7) type cytokine
secretion.
Synthetic scrambled PIE' peptide (scrPIF-115), (SEQ ID NO: 5), had no effect
on these cells;
while addition of synthetic PT, sPIF-115 (SEQ ID NO:13) together with the
scrPIF-1 (SEQ ID
NO: 5) blocked completely the effect of nPIF-115 (SEQ ID NO:1) on the immune
system.
[0011] Additional embodiments include peptides derived from pre-implantation
embryos
found in gestational biological fluids or tissues that have a PIF like
activity and binds to PIF
receptors on cell such as but not limited to monocytes, macrophages, as well
as those that bind to
activated lymphocytes.
[0012] Another embodiment of the present invention includes receptors
expressed on the
surface of cells which interact with the PT peptides. These cells may be PBMC
including T or
B cells which appears in response to activation and responds to PT peptides
secreted by the
embryo, they may include receptors on monocytes and macrophages that affect
the cell biology
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of these cells upon interaction with embryo derived compounds or they may be
receptors that
transduce PIT effect on the immune system and elsewhere in the body.
[0013] The effects of PIF on the immune response may be measured using flow
cytometry and ELISA techniques. Without wishing to be bound by theory, based
on the use of
fluoroscence labeled PIE' peptides, it is believed PIF binds to specific and
likely novel cell
surface receptor sites. These PIF receptors appear to be distributed on
monocytes, >90% of B
cells, and on 4% of un-stimulated and 80% of mitogen-stimulated T cells.
Antibodies generated
against PIE peptides can be used to develop ELISA and other assays to
determine quantitatively
or semi-quantitatively levels of PIF in biologic fluids. Any diagnostic
procedure whereby
compounds of pre-implantation embryo origin that decrease the TH1/TH2 ratio in
lymphocytes
are used to assess embryo quality in animals and humans. Such assays may be
used as part of a
diagnostic procedure whereby compounds of pre-implantation embryo origin that
decrease the
TH1/TH2 ratio in lymphocytes are used to assess the quality of pregnancy in
animal and humans
or to determine receptivity of the immune system and endometrium prior to
pregnancy.
[0014] The present invention further relates to PIP peptides effect on
endometrial cells
leading to an increase in a major marker of endometrial receptivity, beta
integrin, an adhesion
molecule. The present invention farther relates to generation of specific
antibodies against the
PIF by injecting KLH bound peptide and determining the titer of the antibody
produced the basis
for ELISA development. High titer polyclonal antibodies were generated against
synthetic nPIF'-
115 (SEQ ID NO:1), nPIF-213 (SEQ ID NO:7) and nPIF-318 (SEQ ID NO:10) and
ELISA assays
to measure PIF in biological fluids has been developed, showing differences
between pregnant
and non-pregnant samples for PIF-1 ELISA (SEQ ID NO:1); determining presence
of pregnancy,
its viability and outcome in human as well as in other mammals.
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[0015] PIP peptides and their receptors could be used beyond pregnancy for
diagnostic
and therapeutic uses. As a diagnostic, monitoring changes in the PIP receptor
that are present on
immune cells as well as elsewhere in the body. PIP receptor also could serve
as an assay for PIP,
where PIP would bind the receptor when present in biologic fluids to determine
prenanacy and
viability. In addition PIP peptides, since they modulate the immune system and
do not cause
basal immune suppression, could be applied to prevent transplant rejection.
This is based on the
observation that in the MLR system PIP -1 (SEQ ID NO:1) blocked the reaction.
This is viewed
as an important assay in assessing tolerance development. Further the present
invention relates to
treatment of autoimmune diseases (including, but not limited to, lupus,
arthritis, diabetes) where
activated inappropriate immunity plays a key role. The ability to suppress
that portion of the
immune system that attacks various elements in the body may reduce or prevent
these serious
debilitating conditions.
[0016] In a further non-limiting embodiment, the present invention relates to
the
development of a novel non-steroid based contraceptive method, since PIP-1
(SEQ ID NO:1)
activity was blocked by scrPIF-1 (SEQ ID NO:5) both on the immune cells and
the
endometrium, likely acting through the same receptor, since unlabeled scrPIF-1
(SEQ ID NO:5)
displaced FITC labeled PIP-1 (SEQ ID NO:1) from the receptor. Therefore scrPIF-
1 (SEQ ID
NO:5) could be administered to women or other mammals to prevent conception
since it would
not allow further embryo development, and will not interfere with the hormonal
cycle. As such it
could be devoid of the side effects that are associated with the use of
current steroid based
contraceptives. Data in vivo shows non toxic contraceptive effect of scrPIF-1.
The scrPIF-1
(SEQ ID NO:5), since it is an inducer of TH1 activity, could have therapeutic
use for stimulating
the immune system in cases of immune suppression due to cancer, HIV for a non-
limiting
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example. The present invention also relates to the use of PIF antibodies for
immunocytochemical
and Western blot to identify PIF related proteins in pregnant tissues, fetus
and placenta. This
allows identifying pregnancy pathologies like premature labor and growth
restriction as non-
limiting examples. The PIF antibodies used as affinity column can identify
associated functional
proteins in pregnant tissues as seen by identification of 10 distinct proteins
in the term placenta,
several of them novel for that tissue. In such embodiment, other biomarkers
can be identified that
may be modified by pregnancy disorders. Identification of these proteins allow
the examination
of the genes that are associated with these proteins highly relevant for
blastocyst development.
These proteins using mass spectrometry or antibodies can also aid together
with PIF peptides to
determine embryo viability following in vitro fertilization thereby increasing
the chances for
pregnancy following transfer.
DESCRIPTION OF THE DRAWINGS
[0017] In part, other aspects, features, benefits and advantages of the
embodiments of the
present invention will be apparent with regard to the following description,
appended claims and
accompanying drawings where:
[0018] FIG. 1 shows that 5PIF-1 (is) (SEQ ID NO:13), binds to PBMC, which
forms
rosettes in their P-L assay. (A) Fluorescence- labeled sPIF-1(15) (SEQ ID
NO:13), binding to
human Lymphocytes in a dose dependent manner. (B) Binding of FITC 5PIF-1 (15)
(SEQ ID
NO:13), to the total PBMC population (C) Binding to lymphocytes that form
rosettes with
platelets, P-L bioassay, documents presence of nPIF-1(I5) (SEQ ID NO:1),
receptors on the
PBMC surface
[0019] Fig 2. 5PIF-1(l5) (SEQ ID NO:13) increases the percentage of PBMC that
contain
TH2 type cytokines (IL1 0, IL 4 while scrPIF-1(15) (SEQ ID NO: 5) has a TH1
bias (INFgamma).
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Isolated PBMC were stimulated by PHA (lug/ml) and cultured 2-4 days with 5PIF-
1(l5) (SEQ ID
NO:13) or scrPIF-1(l5) (SEQ ID NO:5) 30 nM. PBMC cytokines content was
determined by
specific staining using flow cytometry.
[0020] FIG 3. shows preferential binding of FITC ( SEQ ID NO:13) to
subpopulation of PBMC. sPlF-1 binds monocytes and macrophages (primary antigen
presenting
cells) at the basal state. FITC labeled PIF-1 was added to unstimulated PBMC.
Binding of the
labeled peptide to PBMC subpopulation was determined by using specific CD
markers. CD14 +
cells represents monocytes.
[0021] FIG. 4 shows effects of binding by FITC 5PIF-1(15) (SEQ ID NO:13) to
basal and
PHA stimulated PBMC PIF receptors. Expression of PIF-1(15) (SEQ ID NO: 1),
receptor
fo.11owing PHA induced PBMC proliferation CD-4 T cells; CD-8 T cells; NK cell
and B cells
(unstimulated-top); (PHA stimulated- bottom). Flow cytometry analysis of PBMC
following
exposure to FITC sPIF-1(I5) (SEQ ID NO:13), % expression was determined in
PBMC subtypes
before and after exposure to 1 ug/ml PHA for 24 hours. There was a major
increase in PIF-1
(SEQ ID NO:1) receptors expression on T ( CD4, CD8 cells, 60-80%) and B cells
( >90%)
while no changes in the NK cells receptors was noted. This indicates that both
T cells (cellular
immunity) and B cells (antigen presenting cells) increase PEF-1 receptor
expression markedly
within 24 hours. While macrophages/monocytes have already a full complement of
PIF
receptors (first responders). In contrast, minimal receptors expression could
be induced by the
mitogen on NK ( natural killer) cells that are supposed to be protect the
body's basal immune
response.The delay seen by PIF effects on mitogen induced PBMC cytokines
secretion (24h
following exposure) may be explained by requirement for receptor induction
(takes 24 hours).
Therefore an only 4 hours exposure had no effect (see Table 1).
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[0022] FIG 5 a sPIF-1(l5) (SEQ ID NO: 13) ELISA standard curve PIF antibody
detects
low sPIF levels (pg). Polyclonal antibodies AbP1F-1(15) were generated against
sP1F-1(15) (SEQ
ID NO:13) in rabbits (Covance Inc.). High titers 50% at 1:50,000 were
achieved. Serial
dilutions of synthetic sPIF-1(15) (SEQ ID NO:13) were plated, blocked and then
washed off. PIE-
1 antibody (1:5000) was added incubated and washed off. Goat anti-rabbit
antibody was added,
incubated and washed off. Reaction was stopped by SDS and counted in plate
reader
(Biosynthesis Inc, G Vandydriff). The antibody affinity was also confirmed by
using a
competition analysis between biotin labeled and unlabeled sPIF-1(15) (SEQ ID
NO:13) (data not
shown). Also, when scrPlF-1 (SEQ ID NO 5) was tested in the assay the antibody
did not
recognize it attesting to the high specificity of the antibody that was
generated. Similar dose
dependent results in the ELISA were obtained with affinity purified PIF-2 and
PIF-3 antibodies
(dilutions of the antibody up to 25,600) with linearity to the 30 pM of the
peptide.
[0023] FIG. 5 b shows ELISA profile of high affinity PIF-1 IgY antibodies
(sandwich
assay). Chicken were injected with KLH bound PIF-1 and the eggs were collected
and affinity
purified on a PIF column.
[0024] FIG. 6 shows ELISA profile of nPIF-1(15) and scrP1F-1(15) using Biotin
labeled
versus unlabeled peptide where the antibody captures the peptide in the
unknown samples and
compares it to standards (see Figure 7).
[00251 FIG 7 depicts an example of a PIF'-based diagnostic of the present
invention.
Four clones of monoclonal antibodies to PIF-1(15) were developed as well in
mice and ascites
fluid was generated with high affinity antibodies as hybridomas with sustained
MAb production.
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[0026] Fig 8nPIF-1(l5) (SEQ ID NO: 1) is present in the ovine placenta, as
demonstrated
by immunocytochemistry methods. AbPIF-1(15) was exposed to placental tissue
derived from a
midtrimester ovine fetus. Compared to the non specific staining by rabbit IgG,
the PIF has show
intense staining of the fetal portion of the placenta.
[0027] Figure 9. Human term placental extracts were prepared (Dr Jerry
Feitelson,
GenWay, Inc) and were exposed to PIF antibodies using Western blot analysis,
PIF like
molecules were stained and the bands obtained were compared to a serial
molecular weight
standards run in parallel. Results showed that a number of PIF-1 related
proteins are present at
the15-40 kDa range PIF-3 had a lower intensity with and was associated with
different molecular
weight bands. Finally, PIF-2 expression was minimal. This supports the notion
that the human
placenta may have precursor proteins from which by cleavage PIP peptides are
produced. This
also supports that view that PIF like molecules are present throughout
pregnancy. Finally, it
documents that in terms of intensity of expression in human by far PIF-1 is
the most relevant at
term.
[0028] FIG. 10. PIF purification from pregnant porcine serum (PPS). A) HPLC
profile
of first trimester PPS in a preparative column. B) HPLC profile of PPS-310a
previously purified
by MabCD2 affinity chromatography. C) HPLC profile of a PIF + peak from PPS-
310a
previous purified by MabCD2 chromatography affinity and HPLC.
[0029] FIG 11. Mass spectrum based identification of PIF peptides in pregnant
pig
serum as compared to non-pregnant serum following the use of CD2 based
affinity column.
Mass of the peptides in the non-pregnant v. pregnant samples revealed
significant differences at
the 900-1100 molecular weight region, where three distinct peptides were
identified.
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[0030] FIG. 12. PIF-1 IgG staining of human placenta during the first
trimester, second
trimester high in the trophoblastic layer while it declined at term.
100311 FIG. 13 reflects expression of PIF in various human tissues using IgG
in 14-18
weeks human fetus tissue way.
DETAILED DESCRIPTION
100321 Before the present compositions and methods are described, it is to be
understood
that this invention is not limited to the particular molecules, compositions,
methodologies or
protocols described, as these may vary. It is also to be understood that the
terminology used in
the description is for the pmpose of describing the particulai versions or
embodiments only, and
is not intended to limit the scope of the present invention which will be
limited only by the
appended claims.
[00331 It must also be noted that as used herein and in the appended claims,
the singular
forms "a", "an", and "the" include plural reference unless the context clearly
dictates otherwise.
Thus, for example, reference to a "cell" is a reference to one or more cells
and equivalents
thereof known to those skilled in the art, and so forth. Unless defined
otherwise, all technical
and scientific tenns used herein have the same meanings as commonly understood
by one of
ordinary skill in the art. Although any methods and materials similar or
equivalent to those
described herein can be used in the practice or testing of embodiments of the
present invention,
the preferred methods, devices, and materials are now described.
Nothing herein is to be construed as an admission that the
invention is not entitled to antedate such disclosure by virtue of prior
invention.
100341 The terms "mimetic," "peptide mimetic" and "peptidomimetic" are used
interchangeably herein, and generally refer to a peptide, partial peptide or
non-peptide molecule
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that mimics the tertiary binding structure or activity of a selected native
peptide or protein
functional domain (e. g., binding motif or active site). These peptide
mimetics include
recombinantly or chemically modified peptides, as well as non-peptide agents
such as small
molecule drug mimetics, as further described below.
[0035] In one embodiment, the PIF peptides of the invention are modified to
produce
peptide mimetics by replacement of one or more naturally occurring side chains
of the 20
genetically encoded amino acids (or D amino acids) with other side chains, for
instance with
groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6-, to 7 membered alkyl,
amide, amide lower
alkyl, amide di (lower alkyl), lower alkoxy, hydroxy, carboxy and the lower
ester derivatives
thereof, and with 4-, 5-, 6-, to 7 membered heterocyclics. For example,
proline analogs can be
made in which the ring size of the proline residue is changed from 5 members
to 4, 6, or 7
members. Cyclic groups can be saturated or unsaturated, and if unsaturated,
can be aromatic or
nonaromatic. Heterocyclic groups can contain one or more nitrogen, oxygen,
and/or sulphur
heteroatoms. Examples of such groups include the furazanyl,furyl,
imidazolidinyl, imidazolyl,
imidazolinyl, isothiazolyl, isoxazolyl, moipholinyl (e.g. morpholino),
oxazolyl, piperazinyl (e.g.
1-piperazinyl), piperidyl (e.g. 1-piperidyl, piperidino), pyranyl, pyrazinyl,
pyrazolidinyl,
pyrazolinyl, pyrazolyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolidinyl (e.g.
1-pyrrolidinyl),
pyrrolinyl, pyrrolyl, thiadiazolyl, thiazolyl, thienyl, thiomorpholinyl (e.g.
thiomorpholino), and
triazolyl. These heterocyclic groups can be substituted or unsubstituted.
Where a group is
substituted, the substituent can be alkyl, alkoxy, halogen, oxygen, or
substituted or unsubstituted
phenyl. Peptidomimetics may also have amino acid residues that have been
chemically modified
by phosphorylation, sulfonation, biotinylation, or the addition or removal of
other moieties.
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[0036] A variety of techniques are available for constructing peptide mimetics
with the
same or similar desired biological activity as the corresponding native but
with more favorable
activity than the peptide with respect to solubility, stability, and/or
susceptibility to hydrolysis or
proteolysis (see, e.g., Morgan & Gainor, Arm. Rep. Med. Chem. 24,243-
252,1989). Certain
peptidomimetic compounds are based upon the amino acid sequence of the
peptides of the
invention. Often, peptidomimetic compounds are synthetic compounds having a
three-
dimensional structure (i.e. a "peptide motif') based upon the three-
dimensional structure of a
selected peptide. The peptide motif provides the peptidomimetic compound with
the desired
biological activity, i.e., binding to PIF receptors, wherein the binding
activity of the mimetic
compound is not substantially reduced, and is often the same as or greater
than the activity of the
native peptide on which the mimetic is modeled. Peptidomimetic compounds can
have additional
characteristics that enhance their therapeutic application, such as increased
cell permeability,
greater affinity and/or avidity and prolonged biological half-life.
[0037] Peptidomimetic design strategies are readily available in the art (see,
e.g., Ripka
& Rich, Curr. Op. Chem. Biol. 2,441-452,1998; Hruby et al., CUIT. Op.Chem.
Biol. 1,114-
119,1997; Hruby & Balse, Curr.Med. Chem. 9,945-970,2000). One class of
peptidomimetics a
backbone that is partially or completely non-peptide, but mimics the peptide
backbone atom-for
atum and comprises side groups that likewise mimic the functionality of the
side groups of the
native amino acid residues. Several types of chemical bonds, e.g., ester,
thioester, thioamide,
retroamide, reduced carbonyl, dimethylene and ketomethylene bonds, are known
in the art to be
generally useful substitutes for peptide bonds in the construction of protease-
resistant
peptidomimetics. Another class of peptidomimetics comprises a small non-
peptide molecule that
binds to another peptide or protein, but which is not necessarily a structural
mimetic of the native
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peptide. Yet another class'of peptidomimetics has arisen from combinatorial
chemistry and the
generation of massive chemical libraries. These generally comprise novel
templates which,
though structurally unrelated to the native peptide, possess necessary
functional groups
positioned on a nonpeptide scaffold to serve as "topographical" mimetics of
the original peptide
(Ripka & Rich,1998, supra).
[0038] The PIF assay, as disclosed in U.S. Patent No, 5,646,003 to Bamea et
al., entitled
"Preimplantation Factor" issued July 9, 1997, and in U.S. Patent 5,981,198 to
Barnea et al.,
entitled "Preimplantation Factor" granted November 9, 1999,
imay be used to measure the response of the
immune system to pregnancy specific preimplantation factors. Studies employing
the PIF assay
for culture media of human or mouse embryos that were grown, show that PlFs
were able to
increase the in-vitro formation of rosettes between donor lymphocytes and
platelets in the
presence of monoclonal anti-CD2 (type T11-1). Lymphocyte-platelet rosettes
result from the
interaction of the T cell surface protein CD2 with its ligand CD58 expressed
on the platelet
membrane. Anti-CD2, by binding to the CD2 antigen on the T cells, inhibits
their interaction
with platelets. However, the embryo-derived factor(s), PlFs, present in the
culture medium or
pregnant peripheral sera appears to counteract this inhibition. The PIF
activity was already
apparent in the viable two-cell stage embryo. Thus both of those compounds
properties are very
likely to be similar. This observation strongly suggests that there are
several putative
compounds that may be very potent, and create an environment that is favorable
for pregnancy.
[0039] Using this assay, it has been determined that the presence of PIF
activity in
maternal serum within four days after embryo transfer indicates a >70% chance
of successful
pregnancy outcome. In contrast, absence of PIF activity indicated that
pregnancy would not
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develop in 97% of cases. PIF is detectable 5-6 days after intrauterine
insemination and is absent
in non-pregnant serum and in culture media of non viable embryos, present in
the sera of various
mammals including horse, cow, pig and humans. Without wishing to be bound by
theory, the
PIF assay results indicate that if the embryo is able to secrete these
immunomodulatory PIF
compounds, it is capable of implanting and achieving a good pregnancy outcome.
The
importance of PIF as a marker of a good quality pregnancy is further
illustrated by the fact that if
a pregnancy ends in miscarriage, the PIF activity progressively declines until
it reaches non-
detectable levels. In contrast in the case of a poor quality pregnancy, Human
Chronic
Gonadotropin (hCG) levels do not change significantly for the next 3 weeks
until the miscarriage
is clinically evident.
[0040] PIF activity is found in several mammalian species, including humans,
horse,
cow, pig, and mouse and sheep. Human immune cells used for the HP assay
(homologous
lymphocytes and platelets) interacted well with the human sera, as well as
with sera from
different species and embryo culture media. This cross-species interaction
indicates that similar
compounds are involved in the different species. PIP activity is due to the
presence of similar
low molecular weight peptides, both in mouse embryo culture media and in
pregnant porcine
serum. A PIF assay was used as a test to identify and characterize the PE
related compounds
within a conditioned mouse embryo culture media. Using a multi-step
chromatographic
technique, coupled with the MY bioassay, a group of a putative PIP embryo
derived peptides
with 9-18 amino acids in length were identified and sequenced. These sequences
are disclosed in
WO/2003/004601 to Bar/lea et al., entitled "New Assays for Preimplantation
Factor and
Preimplantation Peptides," filed June 28, 2002.
Based on the sequences derived, synthetic peptides were generated.
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[0041] The first natural PIE compound identified, termed nPIF-1(l5) (SEQ ID
NO:1), is a
15 amino acid peptide. A synthetic version of this peptide, 5PlF-1(l5) (SEQ ID
NO:13), showed
activity that was similar to the native peptide, nPIF-1(15) (SEQ ID NO:1).
This peptide is
homologous to a small region of the Circumsporozoite protein, a malaria
parasite. The second
PIF peptide, nPIF-2(13) (SEQ ID NO:7), includes 13 amino acids and shares
homology with a
short portion of a large protein named thyroid and retinoic acid transcription
co-repressor, which
is identified as a receptor-interacting factor, (SMRT); the synthetic version
is sPIE-2 (SEQ ID
NO:14). The third distinct peptide, nPLF-3(I8) (SEQ ID NO:10), consists of 18
amino acids and
matches a small portion of reverse transcriptase; the synthetic version of
this peptide 5PIF-3(l8)
is (SEQ ID NO:15). nPIF'-4(0) (SEQ ID NO:12) shares homology with a small
portion of reverse
transcriptase..
[0042] A composition comprising a synthetic PIF peptide and an excipient. In
further
embodiments, the synthetic PIF peptide corresponds to the amino acid sequence
of SEQ ID
NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:7, SEQ ID
NO:10, or SEQ ID NO:12 or comprises the amino acid sequence of SEQ ID NO:13,
SEQ ID
NO:14, SEQ ID NO:15, SEQ ID NO:16, or SEQ ID NO:17 or a peptidomimetic
thereof. In
another embodiment, the composition may further include a cell having an
expressed PIE
receptor bonded to said PIF peptide, for example, embryo cells.
[0043] In a further embodiment a compound of the formula R1-R2-R3-R4-R5-R6-R7-
R8-
R9-R10-R11-R12-R13-R14-R15, wherein R1 is Met or a mimetic of Met, R2 is Val
or a mimetic of
Val, R3 is Arg or a mimetic of Arg, R4 is Ile or a mimetic of Ile, R5 is Lys
or a mimetic of Lys,
R6 is Pro or a mimetic of Pro, R7 is Gly or a mimetic of Gly, R8 is Ser or a
mimetic of Ser, R9 is
Ala or a mimetic of Ala, R10 is Asn or a mimetic of Asn, Rii is Lys or a
mimetic of Lys, R12 is
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Pro or a mimetic of Pro, R13 is Ser or a mimetic of Ser, R14 is Asp or a
mimetic of Asp and R15 is
Asp or a mimetic of Asp is provided. In a further embodiment, a compound
comprising the
formula R1-R2-R3-R4-R5-R6-R7-R8- R9-R10, wherein R1 is Ser or a mimetic of
Ser, R2 is Gin or a
mimetic of Gin, R3 is Ala or a mimetic of Ala, R4 is Val or a mimetic of Val,
R5 is Gin or a
mimetic of Gin, R6 is Glu or a mimetic of Glu, R7 is His or a mimetic of His,
Rg is Ala or a
mimetic of Ala, R9 is Ser or a mimetic of Ser, and R10 is Thr or a mimetic of
Thr; a compound
comprising the formula R1-R2-R3-R4-R5-R6-R7-R8- R9-R10-R11-R12-R13-R14-R15-R16-
R17-R18,
wherein R1 is Ser or a mimetic of Ser, R2 is Gly or a mimetic of Gly, R3 is
Ile or a mimetic of Ile,
R4 is Val or a mimetic of Val, R5 is Ile or a mimetic of Ile, R6 is Tyr or a
mimetic of Tyr, R7 is
Gin or a mimetic of Gin, Rg is Tyr or a mimetic of Tyr, R9 is Met or a mimetic
of Met, R10 is Asp
or a mimetic of Asp, Rii is Asp or a mimetic of Asp, R12 is Arg or a mimetic
of Arg, R13 is Tyr
or a mimetic of Tyr, R14 is Val or a mimetic of Val, R15 is Gly or a mimetic
of Gly, R16 is Ser or
a mimetic of Ser, R17 is Asp or a mimetic of Asp and R18 is Leu or a mimetic
of Leu; and a
compound comprising the formula R1-R2-R3-R4-R5-R6-R7-R8- R9, wherein R1 is Val
or a mimetic
of Val, R2 is Ile or a mimetic of Ile, R3 is Ile or a mimetic of Ile, R4 is
Ile or a mimetic of Ile, R5
is Ala or a mimetic of Ala, R6 is Gin or a mimetic of Gin, R7 is Tyr or a
mimetic of Tyr, R8 is
Met or a mimetic of Met, and R9 is Asp or a mimetic of Asp is provided.
[0044] Without wishing to be bound by theory, present evidence suggests that
both
within the embryos immediately surrounding (found in the fallopian tube, and
endometrium in
vivo), and in the peripheral circulation, similar PEF peptides may be
responsible for both the
immune effects, and for the creation of a pro-pregnancy environment.
[0045] Considerable evidence exists that impaired maternal immune tolerance to
the
semi-allogeneic conceptus is a cause of implantation failure and pregnancy
loss. The distribution
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of T-helper cell (TH) sub-populations and the resulting local and systemic
cytokine balance may
play an important role in pregnancy viability. Following antigenic
stimulation, TH cells respond
by differentiating into one of two cell types: TH1 which produces mainly
interleukin 2 (IL-2) and
interferon y (IFN-y ) as well as tumor necrosis factor a (TNF-a); and TH2,
which produces IL-
4, IL-5 and IL-10. TH-specific cytokines tend to both stimulate proliferation
of the TH cell subset
from which they are derived and inhibit development of the opposite TH cell
subset. TH1 cells
are involved in cell-mediated immune reactions while TH2 cells are involved in
humoral
immunity. A predominance of TH2 cytokines is found in normal pregnancies, and
IL-4 and IL-
released by these cells appear to support pregnancy. In contrast, the TH1
cytokines, IL2,
IFN-y and TNF-a are associated with reproductive failure in both humans and
mice. However
both types of cytokines are required to maintain pregnancy, since the maternal
system must be
able to fight aginst infection while tolerating the fetus. It has been
postulated that the pre-
implantation embryo may play a role in protecting itself from maternal immune
rejection by
secretion of factors that would promote the shift of TH cells towards the TH2
phenotype. These
PIF compounds may be to be used for treatment of inflammatory or other
immunological
diseases, and preferably the drug or biological is derived from- or its
structure is based on the
structure of the circumsporosoite protein of malaria.
[0046] In one embodiment, embryo-derived compounds, PIF peptides or
peptidomimetics thereof, can be used for both diagnosis and therapy. Non-
limiting examples of
the effects of such PIF peptides include modulation of the immune system while
not causing
basal immune suppression, and use of PIF peptide to enhance endometrial
receptivity. Such
methods of treatment may involve increased expression of endometrial
receptivity markers,
including, but not limited to beta 3-integrin.
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[0047] In another embodiment, a method of detecting a PIF petptide is
provided. The
PIF peptide may include, for example, SEQ ID NO. 1, SEQ ID NO. 2, SEQ ID NO.
3, SEQ ID
NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9,
SEQ ID
NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO.
15,
SEQ ID NO. 16, SEQ ID NO. 17. In a further embodiment, a method of detecting a
PIF peptide
which includes a fragment of nPIF-1(l5), nPIF-2(l3), nPIF-3(18) or nPIF-4(9).
[0048] PIF peptides and peptidemimetics of the present invention may be
coupled to
produce labeled peptides, for example but not limited to FITC, biotin,
rhodamine, radioactive
labels, fluorescent nanocrystals, and other labels known to those skilled in
the art, that may be
used to identify PIF receptor sites present on immune cells, endometrium, on
the embryo itself,
as well as elsewhere within the body where PIF peptides specifically bind.
[0049] Embodiments of the present invention may be used to identify and clone
the
genes that are responsible for PIF peptides expression. cDNA library is
prepared from human
placenta (Invitrogen) that have libraries of 1-2.5kb size inserts which
represent even the rarest
sequences. Oligonucleotides are generated based on the peptides sequences and
are probed
against the cDNA library using plate screening procedures. The PIF peptide
presence in the
placenta was adding previously documented using immunohistochemical technics
by labeled
PIF-1 antibody. The species of PIF peptides present in the placenta are
confirmed with affinity
purified and labeled PIF-1, PIF-2, and PIF-3 antibodies using a Western blot.
The present
invention may be used to generate specific antibodies polyclonal and
monoclonal for assay
development to measure PIF levels and activity in biologic fluids and tissues
such as but not
limited to serum, blood, urine, milk, and saliva as well as embryo culture
media, gestational
tissue, and fetal tissue.
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[0050] Embodiments of the present invention include those peptides derived
from pre-
implantation embryos that induces TH2 type cytokines like IL-10 synthesis or
secretion from
lymphocytes or other white blood cells and pharmacophores that binds
specifically to PIE'
receptors (such but not limited to (A)VRIKPGSANKPSDD or (Q)VRIICPGSANKPSDD) or
by
substituting with L amino acids or by adding PEG. Preferably such peptides are
from pre-
implantation embryos and increases TH2/TH1 ratio through increased number of
lymphocytes
containing the desired cytokines and or by preferential secretion or TH2 over
TH1 cytokines into
the media. Such pre-implantation embryo-derived peptide may be used to cause a
shift from pro-
inflammatory to anti-inflammatory activities in lymphocytes.
[0051] In a further embodiment isolated or synthetic PIF peptides may be used
in a
method of identifying cellular or tissue binding sites for PIP peptides in a
patient, the method
comprising administering labeled PIF peptides to said tissue and detecting the
label. The binding
sites may include, for example, immune cells, endometrial cells, epithelial
cells, gestational
tissues, embryos and the like.
[0052] In another embodiment, a method of identifying PIF receptors on cells
is
provided. The method may involve combining labeled PIF peptides with activated
immune cells
membranes and further detecting the presence of labeled PIF peptides on
activated cells.
[0053] PT peptides or peptidomimetics may be used to treat a patient by
administering
to the mother a therapeutic amount of one or more PIFs to create tolerance for
the embryo and
therefore pregnancy acceptance by the mother. In this embodiment PT peptides
can be used for
the treatment of infertility disorders and for the enhancement of pregnancy.
Other non-limiting
examples where such PIFs may be used include preventing miscarriage and
premature labor in
mammals such as women, farm, and non-farm animals. The PIF peptides,
peptidomimetics or
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compositions thereof may be administered using transdermal methods including
patch, by
injection, or pill, and may include liposome or carbohydrate coated
formulations, for example.
[0054] In another non- limiting embodiment PlF peptide could be added to
embryo
culture media in order to enhance the ability of the transferred embryos to
implant thereby
reducing the number of embryos that are needed in order to have a high rate of
implantation, and
successful pregnancy. P1F could also enhance embryonal viability by acting in
an autocrine
manner on the embryo itself.
[0055] In further embodiments, methods of involving compounds of pre-
implantation
embryo origin that decrease the TH1/TH2 ratio are used as drugs (biologics) to
improve the
immune system of the mother to be able to better receive the embryo, as a
treatment of infertile
women (parenterally or as a co-additive to the embryo cultures in the ET
procedure) are
provided.
[0056] Pre-implantation embryo origin compounds or analogs may be used in
procedures
that decrease the TH1/TH2 ratio in lymphocytes. In these procedures the PIFs
are used as drugs
or biologics to treat immunological diseases that benefit from a reduction in
the pro-
inflammatory activity or enhancement of anti inflammatory activity of the
immune system in
animals and humans. Alternatively, these compounds and their analogs can block
a decrease in
the TH 1 /TH2 ratio in lymphocytes in which case they are used as drugs or
biologics to treat
immunological diseases where antibodies are over-produced and inhibition
thereof is beneficial
in humans and animals. For example, PIP peptides or peptidomimetics may be
administered to
non-pregnant patients that have autoimmune diseases like lupus and rheumatoid
arthritis where
the aim is to reduce the rate of activated immunity while maintaining the
basal immunity that is
required for defense of the organism. In another example, the compounds of pre-
implantation
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embryo origin are used to decrease the TH1/TH2 ratio in lymphocytes are used
to control the
function of other proteins that do same effects (e.g.. Progesterone Induced
Blocking Factor). The
administration can be made through non-limiting examples such as a patch,
injection, or pill
form.
[0057] The PIFs may have their molecular structure and/or of amino acid
sequence
modified to form antagonists. In one embodiment, PIF antagonists may include
scrambled PIF
peptides, including SEQ ID NO:5, SEQ 1D NO:8 and SEQ ID NO;9. In a further
embodiment,
compositions comprising a scrambled PIF peptide that blocks PIF recognition by
cell receptors
and an excipient are provided. The composition may further include a cell
having an expressed
PIF receptor bonded to the scrambled PIF peptide.
[0058] Another embodiment of the invention is the use of scrambled PIFs,
including but
not limited to scrPIF-115 (SEQ ID NO:5), to antagonize endogenous PIP pro-
fertility effect,
thereby preventing pregnancy initiation thus serving as a contraceptive for
mammals including
women. The scrPIF-115 (SEQ ID NO:5) administration can be made through a non-
limiting
example, a patch, injection or in a pill form. Another non-limiting
application may be
administration of scrPIF-115 (SEQ ID NO:5) at term to induce labor. Modified
PIF peptides may
also be used to negates PIF activity on any cell, tissues and exert
contraceptive effects, or lead to
premature delivery, or induce delivery. Any procedure whereby compounds of pre-
implantation
embryo origin that decrease the TH1/TH2 ratio in lymphocytes are used as drugs
or biologics to
prepare the endometrium (uterus) to maintain a healthy implantation window by
inducing
implantation window specific gene product(s) expression.
[0059] Another embodiment of the present invention provides for a method of
identifying the site of action, the cell receptors, to which PIF peptides have
to bind in order to
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exert their biological effects. This binding may be on immune cells,
endometrium, and
elsewhere in the organism, including the embryo itself. The method may include
administering a
labeled PIE peptide and further detecting the labeled PIP peptide. Further
embodiments provide
for a composition for identifying PIP receptor sites comprising a PIP peptide
and a label. The
label may include, for example, FITC, biotin, rhodamine, radioactive isotopes
and fluorescent
labels, such as nanocrystals. A further embodiment includes isolation and
cloning of these
receptors. cDNA library of PBMC (Invitrogen) is used for expression screening.
Binding of PIP-
1,2,3 (FITC) to COS-M6 cells is examined and positive clones are sequenced.
This method also
provides for identifying the intracellular mechanisms including the
transcription factors that lead
to the changes noted in cytokines secretion but not limited to the immune
system's function.
Also the method allows for the identification of the secretory products, such
as but not limited to
cytokines, and growth factors, that are modified following exposure to the PIP
peptides. It also
provides the method for identifying the genes' expression that is modified
secondary to PIP
peptides' effect.
[0060] Another embodiment of the present invention provides for making
polyclonal or
monoclonal antibodies that were raised against PIP. In one non-limiting
embodiment, polyclonal
or monoclonal antibodies may be raised against PIP in mice and rabbits. In
another embodiment,
antibodies to PIP may be created by providing a hybridoma cell that produces a
monoclonal
antibody specific for a PIF peptide and culturing the cell.
[0061] Such antibodies provide a method for determining the presence of PIP
levels in
samples by using but not limited to ELISA, ETA, lateral flow assay,
microfluidics or mass
spectometry. Such a method and antibodies may allow precise measurements of
PIP levels in
fluids such as but not limited to maternal blood, urine, saliva, milk, and
embryo culture media
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and gestational tissues. The method is applicable for all PIF peptides and may
be used to provide
an early diagnostic method that reflects pregnancy and its viability in
various patients starting at
the pre-implantation period. The patients may include women, to monitor
results of infertility
therapy and pregnancy well being, as well as other mammals, including farm and
non-farm
animals, and non-mammals. In the embryo culture media the ELISA assay using
such antibodies
provides a method for assessing the presence of PIE' peptides to assess embryo
viability before
transfer. Various aspects of the present invention will be illustrated with
reference to the
following non-limiting examples.
Table 1. PIF Peptides
(SEQ BD NO) Peptide Amino Acid Sequence
SEQ ID NO:1 nPIF-115 MVRlKPGSANKPSDD
isolated native, matches region of
Circumsporozoite protein (Malaria)
SEQ ID NO:2 nPIF-1(15- alter) MVRlKYGSYNNKPSD
isolated native, matches region of
Circumsporozoite protein (Malaria)
SEQ ID NO:3 nPIF-1(13) MVRIKPGSANKPS
isolated native, matches region of
Circumsporozoite protein (Malaria)
SEQ ID NO:4 nPLF-1(9) MVRIKPGSA
isolated native, matches region of
Circumsporozoite protein (Malaria)
SEQ ID NO:5 scrPlF-115 GRVDPSNKSMPKDIA
synthetic, scrambled amino acid sequence
from region of Circumsporozoite protein
Malaria
SEQ ID NO:6 nPIF-2(lo) SQAVQEHAST
isolated native, matches region of human
retinoid and thyroid hormone receptor-SMRT
SEQ ID NO:7 nPIF-2(13) SQAVQEHASTNMG
isolated native, matches region of human
retinoid and thyroid hormone receptor
(SMRT)
SEQ ID NO:8 scrPLF-2(13) EVAQHSQASTMNG
synthetic, scrambled amino acid sequence
from region of human retinoid and thyroid
hormone receptor SMRT
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SEQ ID NO:9 scrPIF-2(14) GQASSAQMNSTGVH
SEQ ID NO:10 nPIF-3(l8) SGIVIYQYMDDRYVGSDL
isolated native, matches region of Rev Trans
SEQ BD NO:11 Neg control GMRELQRSANK
synthetic, scrambled amino acid sequence for negPIF-
from region of Circumsporozoite protein 1(15)
Malaria
SEQ ID NO: 12 nPT-4(9) VIIIAQYMD
isolated native, matches region of Rev Trans
antibody of native isolated nPIF-115 AbPIF-1(l5)
(SEQ ID NO: 13) sPW-1(15) MVRIKPGSANKPSDD
synthetic, amino acid sequence from region of
Circumsporozoite protein Malaria
(SEQ NO: 14) sPIF-2(l3) SQAVQEHASTNMG
synthetic, amino acid sequence from of human
retinoid and thyroid hormone receptor SMRT
(SEQ ID NO: 15) sPIF-3(18) SGIVIYQYMDDRYVGSDL
synthetic, amino acid sequence from region of
Circumsporozoite protein Malaria
(SEQ ID NO: 16) sPIF-1(9) MVRIKPGSA
synthetic, amino acid sequence from region of
Circumsporozoite protein Malaria
antibody of native isolated nPIF-2(l3) AbPIF-2(13)
antibody of native isolated nil-F-3(ls) AbPIF-3(l8)
(SEQ ID NO: 17) sPIF-4(9) VIIIAQYMD
synthetic
n=native, s= synthetic, scr =scrambled, same AA, ( )= number of AA,
Ab=antibody
EXAMPLE BIOLOGICAL EFFECTS OF PIF PEPTIDES
[0062] Preimplantion factor peptides were synthesized by solid-phase peptide
synthesis
(SPPS, Applied Biosystems Peptide Synthesizer, Model 433) employing Fmoc (9-
fluorenylmethoxycarbonyl) chemistry in which the a-amino nitrogen of each
amino acid is
blocked with Fmoc. Upon completion of the synthesis, final purification is
carried out by
reversed-phase HPLC and identity is verified by MALDI-TOF mass spectrometry
and amino
acid analysis. sPIF-115 (SEQ ID NO:13) and scrPIF-115(SEQ ID NO:5) scrambled
peptide
(GRVDPSNKSMPKDIA) and an irrelevant 11 amino acid negative control peptide
negPIF-1(l5)
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(SEQ ID NO: 11) (GMRELQRSANK) containing a similar carboxyl-terminal sequence,
were
synthesized. Peptides were labeled on their N-termini with fluorescein
isothyocyanate (FITC) in
the solid phase. A spacer group (b-alanine) was inserted between the
fluorophor and the peptide.
sP1F-115(SEQ ID NO:13) was also labeled by adding Lysine at the C terminal. In
addition,
SMRT (SQAVQEHASTNMG) 5PIF-2(l3) (SEQ ID NO: 14), FITC and Biotin labeled were
generated on the N terminal. Scrambled SMRT, scrPIF-2(l4) (SEQ ID NO: 9),
GQASSAQMNSTGVH; scrP1F-2(13) (SEQ ID NO: 8), EVAQHSQASTMNG; and
SGIVIYQYMDDRYVGSDL peptide (reverse transcriptase homologue- RTH) sPIF-3(l8)
(SEQ
ID NO:15) were also generated synthetically.
[0063] While the present invention is described with reference to PIF's
derived from
mammals like mice or humans, it is to be understood that the invention is not
limited to these
peptides. For example, PIF peptides or their antagonists which are cloned,
synthesized, or
isolated from mammals like horses, cows, or swine or substituted variants of
these peptides may
be used in the practice of various embodiments of the present invention. It is
also contemplated
that substitutions of amino acids in the peptide sequence of these PIFs can be
made and used as
would be known to those skilled in the art in the practice of various
embodiments of the present
invention. Such PIF variants may be characterized by their ability to alter
the TH1/TH2 ratio of
antigen stimulated cells or by their ability interact with PIF receptors on
cells.
PIF-1 PEPTIDES ENHANCE ROSETTES FORMATION.
[0064] Figure 1 shows that 5PIF-1(l5) (SEQ ID NO: 13), binds to PBMC, which
forms
rosettes in their P-L assay. Figure 1(A) depicts fluorescence- labeled sPIF-
1(15) (SEQ ED NO: 13)
binding to human lymphocytes in a dose dependent manner. Figure 1(B) shows
binding of FITC
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nPIF-105) (SEQ 1D NO: 1) to the total PBMC population and Figure 1(C) shows
binding to
lymphocytes that form rosettes with platelets, P-L bioassay, documents
presence of nPIF-1(15)
(SEQ B3 NO: 1), receptors on the PBMC surface.
100651 Human PBMC were isolated by the Ficoll method. FTTC-labeled nPIF-1 is
(SEQ
ID NO: 1) and (FITC)-labeled scrP1F-1 (SEQ ID NO:5) and match-size irrelevant
peptide
(FTTC)-labeled negative controls negPIF-105) (SEQ ID NO:11), were dissolved in
PBS at
concentrations ranging from 0.75 to 48.19 pM, and were incubated with 500,000
lymphocytes
for 1 h at room temperature. The cells were washed 6 times with PBS and
resuspended in 500 ill
of the same buffer. T-cells and monocytes identification was performed in
independent
experiments by incubation with anti-CD3 and anti-CD14 antibody-phycoerithryn
(PE) labeled.
nPIF-115 (SEQ ID NO: 1) binding to PBMC was determined by flow cytometry. In
addition,
rosettes formed by T cell-platelets were detected by flow cytometry using anti-
CD3 antibody-PE
and anti-CD41a antibody-FITC (all antibodies were from Pharmingen Inc.). These
rosettes were
not labeled by the control peptides (FITC)-labeled scrPIF-1(,5) (SEQ ID NO:5)
and match-size
irrelevant peptide (FITC)-labeled negative controls negP1F-1(I5) (SEQ ID
NO:11). This indicates
that both embryo culture media and serum contain similar peptides and provides
evidence for the
utility of biologic effects of the peptides and for the diagnostic potential
using antibodies against
the same.
[0066] The biological characteristics of preimplantation factors in vitro were
determined
employing the synthetic versions of both 15-residue sPIF-1 (SEQ ID NO: 13) and
9-residue
sPIF-1(9) (SEQ ID NO: 16) isoforms, which exhibit similar biological
activities in vitro. Flow
cytometric determination of lymphocyte/platelet rosette formation shows that
both sP1F- 1 is
(SEQ ID NO: 13) and sP1F-1(9) (SEQ ID NO: 16) isoforms induce a four-fold
increase in the
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number of rosettes in the presence of the anti-CD2 antibody (data not shown),
demonstrating that
they exhibit the same anti-CD2-blocking effect manifested by the embryo-
conditioned culture
medium and maternal serum.
[0067] In a further example, PIFs effect on PIF bioassay and flow cytometry
was
observed. Compared to control, the addition of PIF increased
platelet/lymphocyte rosette
formation as follows:
Table 2.
PIF Increase in Rosette Formation
1 nM >370%
nM >288%
100 nM >208%
Results indicate that synthetic PIF replicates the effects seen by pregnant
serum and embryo
culture media results.
PIF-1 IMMUNE EFFECTS
[0068] Human PBMC were isolated and cultured for 2-4 days in AIM-V medium
containing 0 to 200 nM of sPIF-115 (SEQ ID NO:13) or scrPIF-115 (SEQ ID NO:5).
The
following proliferation-activating agents were used: anti-CD3 antibody (10
g/ml solution)
bound on the plate wells in the presence or absence of IL-2 at 10 ptg/m1;
phytohemmaglutinin
(PHA) at 4 g/ml. The effects of sPIF-115 (SEQ ID NO:13) or scrPIF-115 (SEQ ID
NO:5)
peptides were also determined by the mixed lymphocyte reaction (MLR) after 3
days in
heterologous cultures of PBMC with cells previously treated with mitomycin C
(100 g/ml for 4
h). Cell proliferation was determined by tritiated thymidine incorporation (16
h) of 72-h culture.
Cytokine release (IL-4, IL-10, IFN-y and TNF-a) into culture supernatants was
determined at 72
h of culture by ELISA (R&D System).
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[0069] Overall, sPIF-115 (SEQ ID NO:13) decreases the proliferation rate of
human
lymphocytes stimulated with diverse reagents and provokes a shift toward a TH2
cytokine
phenotype. sPIF-115 (SEQ ID NO:13) negatively affects the proliferation of
activated
lymphocytes. Lower rates of lymphocyte proliferation was found at 250, 62.5
and 1nM of sPlF-
115 (SEQ ID NO:13) for PHA, anti-CD3 antibody and MLR, respectively. The
results were
compared with CD3 antibody stimulated lymphocytes without sPIF-115 (SEQ ID
NO:13) or with
scrPIF-115 (SEQ ID NO:5) used as controls. IL-10 release is significantly
increased in the
culture supernatants and IFN-y release is significantly decreased by exposure
to sPIF-115 (SEQ
ID NO:13). In contrast, sPlF-115 (SEQ ID NO:13) does not have a significant
effect on IL-4 or
TNF-a release As show in Table 3, sPIF-115 (SEQ ID NO:13) increases the
TH2/TH1 cytokine
ratio in PBMC more than five-fold, owing mainly to the substantial increase of
IL-10 coupled
with a decrease in IFN-y. Number of IL-10 secreting cells also increased (50-
60%) starting at
day 2 and peaking at days 3-4 and preceding the IFNI decreases (30%) by one
day, suggesting
that a causal relationship is likely in place with respect to the dynamics of
these cytokines, as
show in Figure 2. In contrast, same concentrations of scrPIF-115 (SEQ ID NO:5)
had no effect,
as demonstrated by intracellular staining and flow cytometry (Fig. 2).
[0070] These results demonstrate that sPIF-115 (SEQ ID NO: 13) has
immunomodulatory
effects that may lead to the development of an immune environment that is
favorable to or at
least tolerant for the presence of the early embryo. The TH2/TH1 cytokine
ratio was determined
at different concentrations of sPIF-115 (SEQ ID NO: 13). Similar dose
dependent results at the
1-500 nM range were found in PHA and MLR activated lymphocytes by effects of
sPIF-l15
(SEQ ID NO: 13) and 5PIF-1 (9) (SEQ ID NO: 16). Similar results were obtained
with sPLF-2
(SEQ ID NO: 14), compared to scrPIF-2(13) (SEQ ID NO: 8).
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[00711 The time dependent ELF-1 inununomodulatory effects are demonstrated in
Figure 2.
A detailed time dependent and multiple controls were used the secretion of
several cytolcines by
PBMC (IL-2, IL-5, IL-8, GM-CSF, IL-4, 1L-10, TNF-et, IFN-13, IL-1 and IL-6)
were measured.
Using CD3MAb as mitogen, PIF-1 stimulated mitogen-induced but not basal immune
response
=
in a time dependent manner most effects were noted at 24-48 hours. By 4 hours
sPTF-1 blocked
TNF alpha with recovery by 24 hours. Also INFgamma secretion initiated only
after 48 hours.
Major increase in a Th2 cytokine (IL10) after 24 hours. was Thus, sPIF-115
(SEQ ID NO:13)
while scrambled peptide, scrPIF-115 (SEQ ID NO:5) had an early and consitent
stimulatory effect
only on TNFalpha. Effects on cytokines was already noted at 0.7 nM. Similar
effects were found
also with other mitogens LPS and PHA when PIF-1 was tested.
PIF CORRELATES WITH AN IN-VIVO PRO-INFLAMMATORY RESPONSE
[0072] PIE activity was measured in normal (n=4) and thrombophilic pregnancies
(n=4)
using a FC bioassay based upon CD2 binding to Jurkat leukemia cell line. A
correlation between
PIP activity and 1L-1 in normal pregnancies was observed, but not in
thrombophilic conditions
(p7.1.02). No correlation was observed between PIP and TNF a, IL-8 or thrombus
precursor
protein (TpP) in either normal pregnancies or thrombophilic conditions.
Results appear to
indicate that the embryo directs maternal immune response, rather than playing
a passive role
and that PIP may be an early indicator of pregnancy well-being.
DYNAMICS OF SYNTHETIC PIF-1 INTERACTION WITH RESTING/ACTIVATED
PBMC
[0073] The expression of PIP binding sites was examined utilizing flow
cytometry
studies employing FITC labeled sPIF-115 (SEQ ID NO: 13) and PBMC from normal
human
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donors showed that it binds to a small number of naive T cells. As shown in
Figure 3, sPIF-115
(SEQ ID NO: 13) binds to monocytes and macrophages, the primary antigen
presenting cells, at
basal state. In contrast, sPIF-115 (SEQ ID NO: 13) binds all monocytes but not
to platelets.
Without wishing to be bound by theory, this suggests that monocytes and a sub-
set of T cells
express prior to pregnancy receptors, and also indicates that although sPIF-
115 (SEQ ID NO: 13)
is somehow similar enough to CD2 that it binds to anti-CD2, it does not bind
to the platelet
marker CD58, as does CD2 itself. PIF-1 sequence has no homology to that of
CD2.
[0074] Using flow cytometry fluoroscein- labeled sPIF-115 (SEQ ID NO: 13)
binding to
immune cells was examined on resting PBMC. Monocytes (CD14+ cells) express
binding sites
on most cells, while the expression on resting T cells (CD4+ and CD8+), B
cells (CD19+) or NK
cells (CD56) populations remained very low. As shown in Figure 4, within 24
hrs of activation
by PBMC cultured with the T-cell mitogen, phytohemmaglutinin (PHA)), 60-80% of
T
lymphocytes (T helper cells, T cytotoxic cells), and >90% of B cells became
positive for the
fluorescent sPIF-115 (SEQ ID NO: 13) binding, demonstrating that lymphocytes
can recognize
preimplantation factors and may respond to them but only if activated. NK
cells did not appear
to bind sPIF-115 (SEQ ID NO: 13) even after several days in culture with PHA.
SCRAMBLED PIF-1 SYNTHETIC PEPTIDE ACTS AS ANTAGONIST TO PIF-1.
[0075] scrPIF-1(15) (SEQ ID NO:5) has an identical amino acids composition as
sPIF-1(j5)
(SEQ ID NO:13) but the sequence is in random order. When used as a control for
sPIF-115 (SEQ
ID NO:13) the FITC scrambled peptide scrPIF-1(I5) (SEQ ID NO:5) did bind to
PBMC as
determined by flow cytometry. Increasing concentrations of scrPIF-1(l5) (SEQ
ID NO:5)
reduced the binding of fluorescent sPIE-1(15) (SEQ ID NO:13) peptide
suggesting that scrPIF-
1(15) (SEQ ID NO:5) acts on the same receptor as sPIF-1(l5) (SEQ ID NO:13).
This was further
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confirmed by the antagonistic effect noted by scrPIF-1 (15) (SEQ ID NO:5)
effect on PBMC
proliferation. PHA or anti-CD3 antibody induced proliferation was dramatically
blocked by low
concentrations of scrPIF-1(l5) (SEQ ID NO:5) (125-1000 nM). Addition of the
antagonist
scrPIF-1 (15) (SEQ ID NO:5) (125 nM) blocked also sPEF-1(15) (SEQ ID NO:1)
effect on IL10
secretion. scrPIF-1(15) (SEQ ID NO:5) also blocked the effect of sPLF-1(l5)
(SEQ ID NO:13)
induced increase in beta integrin expression by human endometrial cells.
[0076] scrPIF-115 (SEQ ID NO: 5) reverses sPIF-115 (SEQ ID NO: 14) induced
inhibition
of PHA triggered PBMC proliferation. Isolated PBMC were stimulated by PHA ( 4
ug/ml) and
cultured 2-4 days with sPIF-19 (SEQ ID NO: 16) ( 0-125 nM) +/- scrPIF-115 (SEQ
ID NO: 5)
125/nM. PBMC proliferation was determined by 3H Thymidine incorporation. *.=
p<0.05.
[0077] scrPIF-115 (SEQ ID NO: 5) also reverses 5PIF-1 (9) (SEQ ID NO: 19)
induced
inhibition of mAbCD3 triggered PBMC proliferation. Isolated PBMC were
stimulated by plate
bound antiCD3 antibody ( 10 ug/ml) and cultured 2-4 days with sPIF-19 (SEQ ID
NO: 16) ( 0-
125 nM) +/- scrPIF-115 (SEQ ID NO: 5) 125/nM. PBMC proliferation was
determined by 3H
Thymidine incorporation. *= p<0.05.
[0078] scrPIF-1 (15) (SEQ ID NO: 5) also reverses sPIF-1 (9) (SEQ ID NO: 16)
induced
IL10 secretion by PBMC following PHA exposure. Isolated PBMC were stimulated
by PHA (4
ug/ml) and cultured 2-4 days with sPIF-1 (9) (SEQ ID NO: 16) ( 0-15 nM) +/-
scrPIF-1(l5) (SEQ
ID NO: 5) 125/nM. IL10 secretion was determined by ELISA *= p<0.05.
[0079] In scrPIF-1 inhibitory action appears to be exerted on the PIF binding
site. Both
PIF-1 and scrPIF'-1 bind specifically to isolated mouse splenocytes receptors.
Based on flow
cytometry data it appears that the binding of both peptides was for the same
site therefore
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explaining the clear antagonistic effect of scrPIF-1 on PIF-1 on both cytokine
secretion and
PBMC proliferation (see below).
[0080] Overall, the results from PIF effects on PBMC proliferation, cytokine
release and
cytokine content of PBMC indicate a major inhibitory effects on cell
proliferation and
modulation of cytokines secretion and their cellular content. This only noted
only under mitogen
activated conditions, while PIF alone had no effect. In addition, an increase
in some Thl type of
cytokines is also required to protect the maternal organism against infection.
The examples of
mitogens that were used are highly potent. In contrast, the embryo as it
develops on a practical
basis until hatching is surrounded by the protective zona pellucida. Therefore
the immune
reaction towards the embryo by the mother until implantation (where direct
contact is established
is minimal, and is gradual allowing for development of tolerance in gentle
manner. In contrast
PIF-lscr has practically no effect except for stimulating TNFalpha, a Thl type
cytokine.
PIF LIKELY ACTS THROUGH AN INDUCIBLE UNIQUE IMMUNE CELL SURFACE
RECEPTOR
[0081] Fluorescent PIF peptides bind to immune cell membranes following
activation. In
order to better evaluate the receptor site that is an activation marker, flow
cytometry (FC) using
specific CD marker antibodies was performed. CD69 is expressed during
activation of
lymphocytes and monocytes, and is a marker of NK cells activation. CD25 is the
receptor for
IL-2, and a known activation marker as well. The size of these molecules'
positive cell
population did not correlate with the sPIF-115 (SEQ ID NO:13) positive
populations. This
confirmed that PIF receptors in the immune system are unique and selectively
expressed on
subpopulation of immune cells and they are inducible by mitogenic activation.
scrPIF-1(1 5) (SEQ
ID NO:5) inhibitory action appears to be exerted on the PIP binding site.
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100821 We examined how PIF and PIF scrambled bind specifically to immune
cells.
Splenocytes from virgin female C57BIJ6 mice were stained with 300 nM of PIF-1-
FITC or
scrPIF-1-FITC. Filled histograms indicated PIF-FITC or scrPIF-FITC alone.
Empty histograms
=
represent PIF-FITC or scrPIF-FITC staining in the presence of 100-fold excess
of corresponding
unlabeled peptide. Histograms represent both monocytes and lymphocytes
according to scatter
gating. These results show that fluorescent-labeled PIF and scrPIF binding is
competed out by
unlabeled PIF and scrPIF. scrP1F binding is specific,however, the immune
effect is negated
(see above).
PIF-1 ENHANCES ENDOMETRIAL RECEPTIVITY
[0083] Human endometrial tissues were digested and stromal and epithelial
cells (hFPC)
isolated and cultured with a fetal bovine serum (FBS)-enriched medium until
confluent layers
were obtained. After that the cells were cultured for two more days in a PBS
free -medium
followed by a further 2-day culture in the same medium containing PIF peptides
(1-500 nM).
The expression of P-3 integrin by hEEC was qualitatively determined by
immunocytochemistry
and quantitatively measured by flow cytometry using a specific anti-13-3
integrin monoclonal
antibody (SS A6). sPIF-115 (SEQ ID NO: 13) and 9-residue sPIF-1(9) (SEQ ID NO:
16) isoforms
and sPIF-2(13) (SEQ ID NO: 8) treatment of hEEC exhibits up to a four-fold
increase in the
expression of P-3-epithelial integrin. These data indicate that preimplantion
factor peptides
released by the preimplantation embryo up-regulate the expression of an
important marker for
endometrial receptivity facilitating pregnancy initiation and maintenance.
[0084] In addition, to compare the recovery of -3 integrin expression with PIF
effects
described after cultured hEEC in a free-fetal bovine serum (FBS) medium some
cells were
cultured again in PBS-medium. Interestingly, these cells expressed higher
levels of13-3-integrin
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than did cells growing in free FBS- medium but still levels of13-3-integrin
expression triggered
by PIF effects were higher. In contrast, PIF did not interact with cultures of
human endometrial
stromal cells. These results could imply that PIF effects on [3-3-integrin
expression, which
indicate an increase of endometrial receptivity, are higher that those
triggered by a medium
containing progesterone and an undefined variety and concentration of growth
factors and
hormones. Data using FITC PIF-1 showed that PIF binds to specific sites on
epithelial cells.
PIF IDENTIFIES ABNORMAL IMMUNE RESPONSE OF A PATIENT WITH
RECURRENT PREGNANCY LOSS
[0085] sPIF-115 (SEQ ID NO:13) effect was tested on a patient with over 14
miscarriages
and no live birth. Her immune cells were examined in the non- pregnant state,
by exposure to
sP1F-115 (SEQ ID NO:13) using PBMC preparations. Induction of PIF receptor
expression, and
binding to lymphocytes were used to characterize the treated cells. Table 3
illustrates the ability
of PIF assay to predict premature labor.
Table 3.
Specimen PHA (PIF+/CD4+)/CD4+ (PIF+/CD8+)/CD8+ (P11F+/CD19+)/CD19+
- Control 22.1 ( 0% ) 24.5 46.2
Patient 16.2 (-27%) 16.3 (-33%) 48.2
[0086] The patient's PIP receptors appear to be inducible by PHA as are those
of the
control subject. No differences were observed on B cells, but patient's T
cells PIF receptor were
¨30% less than the control. Table 4 shows ability of PIT assay to correlate
with
proinflammatory cytokines in coagulation disorder associated with pregnancy
(thrombophylia).
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Table 4. PIF-1 Effects on Cytokine Expression
Specimen PHA PIF-115 TNFa* IL-10* TH1/TH2
patient 7.98 7.77 1.03
patient 14.23 24.76 0.57
patient 18.32 24.32 0.75(+32%)
control 4.76 4.71 0.99
control 17.43 25.72 0.68
control 15.46 28.18 0.55 (-19%)
*) All cytokine numbers are % of total PBMC in scatter gate (including CD14)
and 4 in table
above).
[0087] Under sPIF-1(15) SEQ ID NO:13) influence, the TH 1 frH2 ratio decreases
(as
expected) in the control specimen; but the same did not decrease in the
patient's PBMC culture
media (see bold numbers in table above).
[0088] The results obtained with this illustrative clinical case indicate that
changes in the
immune system of patients with poor obstetric history can be identified prior
pregnancy by using
PlFs as an embryo surrogate. Such testing can allow for pre-pregnancy
identification of patients
with poor ability to mount an immune change or tolerance to initiate and
maintain pregnancy.
Such an insight could help in screening patients at risk of miscarriage and
lead to correction of
the underlying pathologic condition perhaps by PIF administration. Such
testing can be applied
during pregnancy as well. Moreover, adding PIF to test the patient's
endometrial cells properties
(beta integrin or such) , following collection by biopsy may help to determine
whether the
mother is able to respond to the presence of the embryo by increased
receptivity.
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GENERATE POLYCLONAL ANTIBODIES TO THE SYNTHETIC PIF-1, PIF -2 AND
PIF-3 PEPTIDES
[0089] To generate specific antibodies against sPIF-115 (SEQ ID NO:13)
conjugation to
carrier, Keyhole Limpet hemocyanin (KLH) was carried out. sPIF-115 (SEQ ID
NO:13) was
conjugated to KLH either on the carboxy or amine terminus of the molecule to
cover potential
differences in immunogenicity related to peptide presentation. The two peptide-
carrier
conjugates generated were injected into two rabbits. Within a 5-week
immunization protocol all
4 rabbits responded by generating a high titer serum, with a titer of 1:50,000-
1:150,000. The
titer strength appeared to increase with the second bleeding. These rabbits
may serve as a long-
term reservoir of serum for antibody generation AbPIF-1(15). The rabbits may
continue to be
injected with immunogens on a monthly basis, collecting sera periodically and
testing for titer
and affinity. Antibodies to other PIN, including AbPIF-2(13) and AbPIF-3(l8),
were generated
with the same method using KLH bound peptide in the amine terminal. Rabbits
bled 8 weeks
after immunization yielded 1:25,000 titers for both peptides with detection of
the PIF peptides to
the nanomolar region. These antibodies were affinity purified using PIF-1, PIF-
2 and PIF-3
bound affinity columns. The purified antibodies were conjugated each to a
separate affinity
column and they will serve for isolation of PIF peptides from various
biological fluids.
[0090] Monoclonal antibodies to PIF-1 were developed as well. A hybridoma cell
that
produces a monoclonal antibody specific for a PIF polypeptide, and culturing
the cell under
conditions that permit production of the monoclonal antibody.
[0091] Such PIF antibodies may be used in assay as well as in therapeutic
treatment
(vaccination) of patients. For example, PIT peptide conjugates may be used as
antigen (vaccine)
to fight malaria. PIF itself, being a minimal unit might behave as a better
antigen than the when
its sequence is embedded in the intact, full length circumsporozoite protein
in the malaria outer
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cell membrane. In another example, PIF antagonist (a peptide or other chemical
shown to bind
to PIE receptors, and block PIF function) or any procedure whereby such
compound is used as
drug, may be useful to treat malaria or block malaria propagation in the human
body (by
blocking the sites through/by which the parasite controls and paralyses the
immune system and
allows it to proliferate). Similarly, any humanized or horse antibodies to PIE
or a procedure
whereby these are used as agents may be used for passive immunization for
malaria. (assuming
such antibodies must recognize the circumsporozoite protein on the malaria
parasite).
[0092] In one example, polyclonal antibodies AbPIF-1(l5) were generated
against sPIF-
1(15) (SEQ ID NO: 13) in rabbits (Covance Inc.). High titers 50% at 1:50,000
were achieved.
Serial dilutions of synthetic sPlF-1(15) (SEQ ID NO: 13) were plated, blocked
and then washed
off. PIE-1 antibody (1:5000) was added incubated and washed off. Goat anti-
rabbit antibody was
added, incubated and washed off. Reaction was stopped by SDS and counted in
plate reader
(Biosynthesis Inc, G Vandydriff). As shown in the ELISA standard curve of
Figure 4, PIE
antibody detects low sPIF levels (pg). The antibody affinity was also
confirmed by using a
competition analysis between biotin labeled and unlabeled nPIF-1(15) (SEQ ID
NO: 1) (data not
shown). Also when scrPIF-1 (SEQ ID NO 5) was tested in the assay the antibody
did not
recognize it attesting to the high specificity of the antibody that was
generated.
[0093] Figure 3a demonstrates the affinity of PIE-1 IgY antibodies. Peptide as
test
antigen. Affi-pure IgY as the primary antibody and goat anti-Ig-Y as the
secondary antibody. A
fixed amount of antigen (5 ug/ml) and serial dilution of IgY.
[0094] Figure 3b demonstrates the specificity of PIE-1 polyclonal antibody. At
4.5 ug/ml
pAb coating concentration, PIE-1 15 was detectable at 10-30 pM in a dose
response curve with an
IC50 of 500-700 pm, and linearly up to 30 nM. scrPlF-1 did not compete with
biotinylated
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peptide, as the native. No binding appears to occur on uncoated plates,
yielding a good
background. Results demonstrate that PIF-1 polyclonal antibody appears to
avoid false positives
and negatives.
[0095] In one non-limiting embodiment, a PIF-based pregnancy diagnosis
utlilizing an
ELISA or yes/no stick in the form of a kit is provided. The components of the
PIF ELISA kit
may include, for example, HRP-Avidin, PIF-Biotin and anti-PIF-115 antibody. In
the absence of
PIF in the test sample, HRP enzyme would bind to the antibody through the PIF-
biotin complex,
generating a maximum color. In the presence of PIF in the test sample, PIF
binds to the PIF
antibody and prevents the HRP enzyme complex from binding, generating a
minimum color.
ISOLATION AND IDENTIFICATION OF PIF LIKE PROTEINS IN HUMAN
PLACENTA AND OTHER FETAL TISSUES
Using affinity purified PIF IgG 1,2 and 3 and Igy PIF-1, PIFs were identified
in human
term placenta using Western blot. Human placenta was the test antigen. Lanes 1
and 3 were
loaded with 50 ug of antigen per lane and lanes 2, 4 and 5 were loaded with
100 ug of antigen
per lane. Lanes 1 and 2 were incuabeted with affi-pure anti-PIF-1 igY in a
1:50 dilution, goat
anti-IgY-HRP in a 1:1000 dilution. Lane 3 was incubated with anti-PIF-1
antibody in 1:200
dilution. Lane 4 was incubated with anti-PIF-2 antibody in 1:50 dilution. Lane
5 was incubated
with anti-PIF-3 antibody in 1:50 dilution and goat/anti-rabbit-HRP in a
dilution of 1:1000.
Results of western blot are shown in Figure 6.
[0096] Expression of PIP-1 in human pregnancy tissues was examined using
affinity
purified IgG using immunohistochemistry methods. Intense trophoblastic
expression was found
in first and second trimester placenta while expression was low at term. With
respect to the 14-
18 weeks fetus using a tissue array (60 samples, covering practically all
organs). The highest
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expression was in the spleen and liver, with lesser in the adrenal, stomach
and small bowel with
no detectable expression in the esophagus and several other organs. The
presence of PlF was
also measured in the adrenal tissue, stomach, small bowel, thyroid and other
organs (not shown).
Non relevant IgG was used as controls.
100971 Overall this indicates that PIP-1 is expressed in the human placenta
and fetus
across gestation where it declines at term to facilitate the process of
delivery by lowering
maternal tolerance for the fetus. With respect for the fetus highest
expression are found in
hemopoietic organs where immune reaction is expected to be the highest.
[0098] In a placental sample derived from a patient with premature
labor at mid-trimester, PIP expression decreases significantly as examined
with the affinity
purified PLF-1 IgG antibody (data not shown). The decline correlated with that
of IL10, a major
Th2 cytokine. When placental explants are placed in culture and interferon
gamma is added, then
PIE is reexpressed back to the intensity that was observed in mid trimester.
This suggest a
reciprocal relationship between cytokines (seen in Table 1) and PIF.
[0099] In another example, PIP-1 associated proteins were identified in human
placental
tissue. Term human placental homogenates were passed through an affinity
column of PIF-1
antibody. The mass spectrometry profile following elution by PIP-1 antibody
affinity column.
Various PIP-1 associated proteins were identified and sequenced following
affinity
chromatography, including (NM 000039) apolipoprotein A-I precursor [Homo
sapiens],
electron-transferring-flavoprotein dehydrogenase, (BC017165) similar to
triosephosphate
isomerase 1 [Homo sapiens], (NM_052925) leukocyte receptor cluster (LRC)
member [Homo
sapiens], (NM 018141) mitochondrial ribosomal protein SW; mitochondrial 28S
ribosomal
protein S10 [Homo sapiens], (NM 000518) beta globin [Homo sapiens], (BC012292)
heat shock
=
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27kDa protein 1, stress responsive, estrogen regulated [Homo sapiens]
P04792.40, Estradiol beta
1 dehydrogenase 1 P14061.13, Fetal Beta MHC binding factor Q 14297.01,
microtubule-
associated protein IA (proliferation-related protein p80 P78559.40). Some of
those proteins
were not previously described in the placenta. The proteins sequenced appear
to show roles in
immune function, cytoskeleton, enzyme function, and protein synthesis and cell
proliferation.
None of the sequenced proteins have sequence homology with PIF-1, therefore it
likely reflects,
in some cases, that PIF is attached to these proteins reflecting a protein-
protein interaction
related to the peptides function.
DEMONSTRATION THAT PIF IS PRESENT IN THE PLACENTA OF SHEEP
[00100] Placental tissues were collected from a mid-gestation sheep
fetus. The
placenta was embedded in paraffin and slides were prepared. Representative
slides exposed to
the 1/100 dilution of rabbit AblIF-1(l5) antibody. Compared with the non
immunized serum,
AbP1F-1(l5) antibody intensely stained the placenta, as shown in Figure 8. The
DAKO
Chemmate system on the auto stainer with DAB as the substrate was used.
Moreover, the
binding was highly specific since no adjacent maternal tissues appeared to be
stained by the
antibody (Fig. 14). As such, AbPIF-1(15) antibody as well as AbP1F-2(l3) and
AbPIF-3(18) could
be useful in identifying presence of PIF in pregnant tissues. Gestational
changes: Visual analysis
was carried out on day 50 (n = 4), day 80 (n = 7), day 100 (n = 7), day 128 (n
= 4) and day 135
(n = 8) 9Term = 145 days). Based on visual assessment of percent staining,
placental levels were
highest at day 50 and then declined to day 80 after which they remained
constant. This pattern
reflected the observations that were made with the human placenta.
[00101] In terms of localization, PIF-1 is localized to the ovine
maternal-fetal
interface which is comprised of fetal trophoblast and maternal epithelium.
Interestingly, PIF-1 is
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localized to the binucleate trophoblast cells. These are non-proliferative
migratory cells which
fuse with the maternal epithelium to form a hybrid maternal-fetal syncytium.
On many of the
slides there appears to be epithelial staining. Some of this may come from the
fetal contribution
to this layer. Later in gestation, the staining becomes more restricted to the
binucleate
trophoblastic cell population.
[001021 PIP -1 expression decreases at term of growth restricted
fetuses. A
comparison of the placental growth restricted group (n = 14) to the controls
(n = 14) at day 80
(placental growth period) gave no significant differences. In late gestation,
there is evidence of a
decrease in PIF1 in the growth restricted group compared with controls.
ISOLATION OF PIF PEPTIDES FROM SERUM OF PREGNANT MAMMALS
[00103] Blood was collected from known pregnant sow. Serum was
separated by
centrifugation and the serum was stored at 20C until use. The collected serum
was filtered
through a <31(Da Amicon membrane. The filtrate was further subjected to a CD2
affinity
column, as shown in Figure 10. The collected samples were prepared and mass
spectroscopy
used to determine the molecular weight of samples by MALDI-TOF workstation.
The molecular
weight of samples was compared with non-pregnant porcine samples prepared in
parallel. A
number of peptides unique to pregnancy were isolated molecular weight range of
about 1030-
1100 daltons as shown in Figure 11.
[001041 In another example, PIP-1 ELISA was used to detect pregnancy
in pigs
and cows. Using the PT-1 ELISA competitive Biotin assay, known pregnant and
non-pregnant
samples were tested. Heat inactivated serum samples were diluted 1/30 and
1/100 and a low
dilution difference between pregnant and non-pregnant samples were found for
sows and a cow.
The estimated PT-1 in the pregnant sera are 10-30 nM. The heat inactivation
helped to reduce
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the background of the assay generated by some high molecular weight proteins
that were
recognized by the PIP-1 antibody seen be Western blot (data not shown) .
[00105] In a thither example, serum samples were obtained at weekly
intervals
from twenty (20) women in a cross-sectional study (eight (8) in the first
trimester, seven (7) in
the second trimester, and five (5) in the third trimester) and two (2) women
from 5-40 weeks
gestation. All women had measurable PIP activity present in serum samples
using bioassay. No
PIP activity was detected in umbilical cord serum using bioassay. Results
indicated that P1F is
present throughout various stages of pregnancy.
GENOME ANALYSIS
[00106] Ten genes isolated from human placenta were analyzed. Seven
of the 10
genes were annotated for both human (Hs) and mouse (Mm) genomes. When these
were
researched on the mpuse microarray dataset for preimplantation development,
using either Mm
or Hs unigene identifiers retuned the same result. Genes were coded
as green down-regulated, red up-regulated, and black no change relative to the
oocyte. The
arrays represent four replicated samples averaged from 2AFFY chip platforms.
Three of the
genes (HSP1, BHSDH, MAF'l A) did not change; however, four genes (mRP-S10,
ApoA-1,
FDH, TP1) were all upregulated in the blastocyst and there may be a weak
tendency for this
increase as early as the 8.-cell stage.
100107] Although the present invention has been described in
considerable detail
with reference to certain preferred embodiments thereof, other versions are
possible. For
example any use of affinity column generated by using PIP-1, P1F-2 and P1F-3
antibodies for the
isolation of PIF peptides from biological samples, any peptide derived from
pre-implantation
embryos that causes enhanced lymphocyte death by any mechanism (apoptosis,
etc.), any
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modified PIF peptides that negate PIF activity and exert biological effects in
order to increase
immune reaction to enhance immune response in cancer, other immune suppressed
conditions,
like HIV; and any use of PIF-3 peptide to block HIV infection. A non limiting
example is since
PIF-3 and PIF-4 share homology with a region of HIV-1 RNA dependent DNA
polymerase and
the PIF-3 antibody recognizes such a domain on such a major region of HIV -1
virus such
compounds could help negate or reduce infectivity of the virus by neutralizing
and interdicting
its penetration into the cell. The PIF-1 antagonist on the other hand could
create a strong Till
type immunity thereby overcome immune suppressive conditions that are present
for example in
cancer. Therefore the spirit and scope of the appended claims should not be
limited to the
description and the preferred versions contain within this specification.
PIF-1 AND SCR PIF-1 CONTRACEPTIVE EFFECTS IN MICE
[00108] Preliminary in vivo assessment of PIF-1 antagonist by using
PIF1scr/amide. With the view that PIP-lscr is a peptide, which, in general has
a short half- life,
estimated to be 30 minutes, a PIF-lscr was generated that had an additional
amide group at the C
terminus. This was an attempt to make the molecule more stable and have a
longer half-life.
Following the protocol below: (Dr Hoberman, Argus, Inc) PIF-1 scr/amide was
introduced two
days after estrus to mice through an osmotic mini-pump (Alzet Model 2001; 1
ul/hr for 7 days)
containing either 0.9% saline or 150 ug or 800 ug PIP-1 scr/amide/day release
in saline. The
pumps were inserted subcutaneously under ketamine/xylazine anesthesia. Female
mice were
placed with male mice on the 3rd day afternoon of the expected estrus. Four
different groups 5
each were studied, low and high dose PIF-lscr/amide, saline control, and one
group with PIF-1.
Mating was confirmed by the presence of sperm in the vagina or a copulatory
plug the next
morning. Pregnancy (or lack thereof) was determined by sacrificing on day 10
after breeding
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and the uterine horns were examined for implantation sites by using Chicago
Blue solution.
Results showed a trend towards lower number of implantation sites at the lower
PIF-lscr/amide
dose v. control group. This however did not reach statistical significance. No
differences
between high dose PLF-lscr/amide and controls were noted.
[00109] PLF-1 has no toxic effects following administration in early
pregnancy to
pregnant mice. This is evidenced by no significant effect on mouse fetuses
number as well there
was no effect on maternal body weights were noted among all the tested groups.
SCRAMBLED PIF-1 BY INTRAVAGINAL ADMINISTRATION MAY HAVE A NON-
TOXIC CONTRACEPTIVE EFFECT
[00110] Female mice were mated with mice from the same strain.
Subsequently,
on day 0 (presumed day of mating) PIF-lscr in saline solution was administered
intravaginally
twice daily for seven days (Dr Alan Hoberman, Argus, Inc). The dose was
800ug/day, 15Oug/m1
or saline vehicle only (5 animals in each group). Subsequently, mice were
sacrificed at day 12 of
presumed gestation and Caesarean-sectioned. Corpora lutea, implantation sites
and live and dead
embryos were recorded. A total of 2 animals in each of the treatment groups
and one mouse in
the control group failed to conceive. 4/10 of treated while 1/5 control
animals conceived, the
effect actually being all or none. No toxic or teratogenic effects were noted
following the PIF
administration since the number of implantation sites and viable embryos were
unaffected in
those mice that conceived
PRELIMINARY STUDY: PIF-1 ANTIBODY AND SCRAMBLED PIF-1 INTRAVENOUS
ADMINISTRATION IS NON TOXIC
[00111] The contraceptive effect of either 150 ug of PIF-lscr in DMSO
or 10 ug
affinity purified PIF-1 (10 animals per group) or 20% DMS0 solution (used as
controls) was
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tested using single daily intravenous injections (Dr Alan Hoberman, Argus,
Inc). Female mice
were placed with male mice on the 3rd day afternoon of the expected estrus.
Mating was
confirmed by the presence of sperm in the vagina or a copulatory plug the next
morning.
Subsequently, were injected for 5 days one injection/day. Mice were sacrificed
at day 12 of
presumed gestation and Caesarean-sectioned. Corpora lutea, implantation sites
and number of
live and dead embryos were recorded. 2/10 mice in the PIF-la and PIF-1
antibody group did not
get pregnant, while in the control group all mice were pregnant. The effect
was all or none since
no toxic or teratogenic effects were noted in the mice that conceived.
[00112]
Although the present invention has been described in considerable detail
with reference to certain preferred embodiments thereof, other versions are
possible. Therefore
the scope of the appended claims should not be limited to the description and
the
preferred versions contain within this specification.
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