Note: Descriptions are shown in the official language in which they were submitted.
PCT/CA93/00~9
r~
212~69~
M~T~OD OF TRE~TING R~CURRENT MISCARRIAGE ~ND
IMPROVING THE EFFICACY OF ~SSISTED REP~ODUCTIVE
TECHNIQUES USING P~C'ENT~L PROTEIN 14
'
BACKGROUND OF THE INVENTION
1. Field of the Invention:
l'he present invention relates to inllibitors o~ immune
cell proliferation and function, and more particularly to
the use of Placental Protein 14 ~PP14) to treat recurrent
miscarriage. The invention also relates to methods for
improving the efficacy of assisted reproductive techniques,
using PP14.
2. Baclcqround of the Invention:
l`he human immune system functions to protect the
organism from infection and from ~oreign antigens by
cellular and humoral mechanisms. The immune system
COI-SiStS o~ a comple~ organization of many types of
lymphocytes, and macrophage or other antigen-presenting
cells. l'llese agents regulate each other by means o
multiple cell-cell interactions and by elaborating soluble
~actors, including lymphokirles and antibodies, that have
autocrine, paracrine, and endocrine effects on immune
cells. Disorders of the regulation of this system may
result in the uncontrolled proliferation of immune cells
and eventually to malignancy, -uncontrolled response to .
foreign anti~ens or organisms leading to allergic or
SUBSTlTlJTE SHE:ET -
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inflammatory diseases, aberrant immune responses directed
against host cells leading to organ damage and dysfunction,
or generalized suppression of the immune response leading
to severe and recurrent infections.
Interleukin 1 (IL-1) is a peptide cytokine secreted by
a variety of cell types including accessory cells of the
immune system, and the antigen presenting cells.
Interleukin 1 has a variety of functions including an
involvement in the actlvation of immune system T-cells.
Cells secreting IL-1 include monocytes present in the
circulating blood, macrophages found in interstitial fluid,
and dendritic cells.
It now appears established that IL-1 is a central
mediator of inflammatory reactions and is important in
pathogenesis of chronic inflammatory diseases, of which
rheumatoid arthritis (RA) is one example. Evidence of this
function of IL-1 has been derived from a variety of
experimental approaches and may be summarized as follows:
1. Prostàglandins and leukotrienes are mediators of
inflammatory reactions; hence non-steroidal anti-
inflammatory drugs, which inhibit cyclooxygenase and
prostaglandin synthesis, are useful therapeutically in such
conditions. IL-1 mobilizes free arachidonate, the
¦ precursor of prostaglandins and leucotrienes, by activating
phospholipase, and also induces cyclooxygenase.
2. IL-1 stimulates binding of T-cells to endothelial
cells, thought to be the first step in their influx into
~oints.
¦ 3. Injection of recombinant IL-1 into joints causes
an influx of inflammatory cells, followed by a loss of
proteoglycan from the cartilage.
Treatment of allergies and autoimmune diseases has
been based on modalities which are toxic to immune cells,
that inhibit production of antibodies, or inhibit the
effects of mediators of the immune response, such as
SUBSTITUTE ~HEET
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212~6~
histamine. Over the past several years many soluble
lymphokines which regulate the immune system have been
characterized. Drugs which allow manipulation of the
production or function of such factors would be of use in
5 the treatment of autoimmune diseases and perhaps in the
treatment of diseases resulting from the uncontrolled
proliferation of immune cells.
It is now becoming widely accepted that IL-1 (both IL-
1 alpha and IL-1 beta) are important mediators of
inflammatory responses. IL-1 appears to directly cause
cartilage breakdown in knee joints, and may be central in
the pathogenesis of rheumatoid arthritis. An inhibitor
may, therefore, be of importance in treatment of this
disease.
Placental Protein 14 (PP14) is a natural product
present at elevated levels in the peripheral circulation
early in pregnancy, peaking around week 9-10. There are
reports in the literature that there is a marked
improvement in some sufferers of rheumatoid arthritis in
the first trimester of pregnancy. Similar reports of an
improvement in patients with chronic asthma during the
first trimester of pregnancy can also be found in the
published literature. Chronic asthma is an inflammatory
disease, and although IL-1 has not yet been implicated ln
its etiology, this remains a possibility.
Pregnancy is a normal state in which at least one
aspect of the immune response--reaction to foreign
antigens--is suppressed with regard to paternal antigens
expressed by the fetus. In this regard, certain women
suffer from recurrent miscarriage, which is generally
defined as the loss of three or more consecutive
pregnancies before the 20th week of gestation. Recurrent
miscarriage may arise as a result of problems associated
with implantation of the fertilized egg in the uterus. It
SUBSTITUTE SHEET ~ `
i WO94/098D5 PCr/C~93/00~)
2~2~94
is believed that the failure to implant successfully may
somehow relate to immune system phenomena.
Women may suffer from other reproductive problems or
defects which make it difflcult or impossible to become
pregnant and/or to implant and carry a fetus to term.
Various assisted reproductive techniques have been
developed, such as artificial :insemination ~AI), in-vitro
fertillzation (IVF), and gamete intra-fallopian transfer
(GIFT), which permit women having such problems or defects
to reproduce successfully. The techniques are also
commonly employed to increase the fertility of other
animals. Assisted reproductive techniques do not always
succeed, and it would therefore be desirable to improve the
efficacy of such technlques.
SUMMARY OF THE INVENTION
The present invention is based on the discovery that
women who experience recurrent miscarriage frequently
exhibit greatly reduced levels of PP14. Thus, a method of
preventing miscarriage is provided according to the present
invention, which comprises administering to a female an
active substance selected from the group consisting of
PP14, derivatives of PP14, muteins of PP14, fragments of
PP14, and subunits of PP14, in an amount effective to
prevent future miscarriage. A variety of these natural and
recombinant forms of PP14 may be used according to the
invention, with natural PP14 being the preferred material.
Normal, fertile women have been found to exhibit a ~-
concentration of PP14 in flushings from the uterine lumen
of about 95.1 ng/ml +/- 7.5 ng/ml (means +/- SD after
logarithmic transformation, n=8). The actual luminal
concentration of PP14 is much higher, as the concentration -
of PP14 is diluted in the flushings by washing fluid. -
Accordingly, the invention preferably contemplates the
administration of PP14 in an effective amount so as to ~ ~
'::::
4 ~ ~
SUBSTITIJTE SHEE~:T
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212~6~
provide a concentration of PP14 which exhibits in-vitro
activity when subjected to the testing procedures described
in the present application. In this regard, administration
of PP14 in an amount ranging from about 0.1 ~g/ml to about
5 mg/ml, and preferably from about 5 ~g/ml to about 5
mg/ml, is contemplated by the invention.
The effective amount of PP14 utilized will depend on
such factors as the level of PP14 already present in the
female, the ~orm of PP14 utilized, and method and site of
administration, the stage of the female's menstrual cycle,
and other factors known to those of ordinary skill in the
~ art.
¦ While the invention is particularly intended for the
, prevention of miscarriage in females who suffer from
l 15 recurrent miscarriage, the method of treatment of the
¦ invention is also believed to be useful to prevent
miscarriage in females who have not experienced recurrent
miscarriage.
The Placental Protein 14 (PP14) material may be
administered to the female by any appropriate route,
including intravenous injection, intramuscular injection,
oral administration, intra-vaginal administration, intra-
cervical administration, intra-uterine administration,
intra-tubal administration, topical administration, rectal
administration, and inhalation. In addition, the PP14 may
be delivered to the site of treatment by means of a
controlled-release delivery system, or by means of a gel,
such that the PP14 does not wash out of the site of
administration too easily. The PP14 active substance may
be administered in admixture with a pharmaceutically
acceptable carrier.
According to a second aspect of the invention, a
method of testing a female for the possibility of future
miscarriage is provided, which comprises:
~ -
Sl.lB5TITlJTE~ ~FET
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21~69~
(a) measuring the level of PP14 present in a
predetermined body fluid or tissue sample of a female; and
(b) comparing the level of PPl4 present in the
predetermined body fluid or tissue sample of the female to
the level of PP14 present in a comparable body fluid or
tissue sample of a normal, fertile female suggests the
possibility of future miscarriage.
As PP14 is synthesized in the endometrial glands and
secreted into the uterine lumen, a preferred site in which
to measure level of PP14 is the uterine lumen. A preferred
method according to the invention for measuring PP14 in the
uterine lumen is by flushing the uterus with a saline
solution, collecting the resulting washings, and
subsequently determining the concentration of PP14 at this
site using radioimmunoassay or other techniques.
Measurement of the endometrial production of PP14 is known
to vary depending on the particular stage of the menstrual
cycle; thus, the day of collection of the washings should
be standardized. A preferred day of collection of the
endometrial washings is the seventh day after the midcycle
luteinizing hormone (LH) peak in the female, which date
closely proximates the time of normal implantation.
The above-discussed technique of collecting
endometrial washings is preferably utilized to detect the
level of PP14 present in the uterine lumen of the female.
The particular process for measuring PP14 concentration in
the body fluid or tissue sample may be any known assay,
including those selected from the group consisting of
radioimmunoassay, ELISA assays, and immunoblotting assays.
~ third aspect of the ~.nvention relates to a method of
improving the efficacy of assisted reproductive techniques,
such as artificial insemination (AI), in-vitro
fertilization (IVF), and gamete intra-fallopian transfer
(GIFT), by addlng an effective amount of PP14 to any sample
(embryo, fertilized or unfertilized egg, mixture of eggs
Sl,lB~TIT~JTE SH~ET
WV94/n9805 PCT/CA~3/OU~
,~
~2~6~
and sperm, or sperm alone) that is to be introduced into
the uterus in order to induce pregnancy.
PP14 is found in relatively high concentrations in
seminal plasma. However, sperm used for artificial
insemination (AI) and other known assisted reproductive
techniques are frequently washed in order to remove
prostaglandins present in the seminal plasma before
introducing them into the uterus in the AI procedure. This
conventional washing step results in the removal of PP14
from the seminal plasma. Thus, the invention contemplates
¦ the addition of PP14 to sperm before the AI procedure,
preferably by including PP14 in the medium used to dilute
washed sperm prior to insemination.
In any assisted reproductive technique, the addition
of extra medium into the uterus along with gametes
inevltably dilutes the normal PP14 concentration present in
the uterus. The invention thus contemplates adding an
effective amount of PP14 to the gamete medium or the like,
so as to provide a PP14 concentration having in-vitro
activity when subjected to the testing procedures described
below. In general, adding PP14 to the sample in an amount
ranging from about 0.1 ~g/ml to about 5 mg/ml, and
preferably from about 5 ~g/ml to about 5 mg/ml, is
contemplated by the invention. The PP14 may be added to
the embryo, fertilized or unfertilized egg, mixture of eggs
and sperm, or sperm alone prior to, concurrently with, or
following introduction of the sample into the patient.
DETAILED DESCRIPTION OF THE INVENTION
of the range of proteins which are known to be
associated with the pregnancy state, one has been found to
exhibit an immunosuppressive activity in a variety of in-
vitro tests. Further investigation of the mode of action
of this peptide, PP14, has indicated that it inhibits IL-l
production by peripheral white blood cells (containing both
SUBST~TUTE ~H~;ET
W094/09805 ~'CT/C~93t()U~9
21~4~
T-lymphocytes and monocytes) after stimulation. The
concentration at which PP14 is active appears to be the
levels at which it is found normally during pregnancy. The
time course of this inhibition of IL-1 production closely
relates to immunosuppressive activity of the molecule,
indicating that its primary effect is on monocytes rather
than other immune system cells.
A variety of proteins are expressed at high levels
during pregnancy. One of these, PP14, is a major secretory
protein of decidual tissue, where it comprises about 10~ of
the total soluble protein. ~s increased levels of PP14
appear early ln a normal pregnancy, PP14 is believed to be
associated with the process of implantation and in
maintaining the early conceptus, which may be particularly
prone to im-mune rejection by the maternal immune system.
The present invention is based on the discovery that
PP14 is commonly present in greatly reduced levels in women
who experience recurrent miscarriage. The invention
therefore contemplates a method of preventing miscarriage
by administering to a female an amount of PP14 effective to
prevent future miscarriage.
The PP14 active substance utilized in the inventive
method may be obtained from a variety of sources, including
mammalian placenta, mammalian blood, amniotic fluid,
seminal plasma, cells in tissue culture, decidual cells,
decidual organs, endometrial cells, endometrial organs, as
well as recombinant protein sources, including sources
containing eukaryotic cells or prokaryotic cells engineered
to express PP14, muteins of PP14, fragments of PP14 or
subunits of PP14.
Useful forms of PP14 for purposes of the present
invention therefore include natural and recombinant forms
of PP14 itself, as well as derivatives, muteins, fragments,
and subunits of PP14, provided that the desired therapeutic
activity of the substance (prevention of miscarriage) is
SUBSTITUTE~: SHEET
W094/098~s PCr/CA93/~0~9
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2~2~69~
maintained. Natural PP14 is the preferred material
utilized according to the invention.
Natural PP14 is a glycoprotein comprising about 17.5~
carbohydrate content. The details of this carbohydrate
5 content are not presently known; however, PP14 binds
strongly to the lectin concanavalin-A, which is known to
have an affinity for terminal alpha-D-mannosyl and alpha-D-
glucosyl residues, as well as to wheat germ agglutinin,
which has an affinity for N-acetyl-beta-D-glucosaminyl
residues. The presence of the latter is further evidenced
by the reduction of the interaction of PP14 with specific
antibodies caused by treatment of PP14 with the enzyme
beta-N-acetyl glucosaminidase, which removes these
residues.
The nucleotide and amino acid sequence of PP14 cDNA,
as deduced by Julkunen et al., is shown in accompanying
Sequence Description (SEQ ID N0:1). The entire disclosure
of Julkunen et al., "Complete Amino Acid Sequence of Human
Placental Protein 14: A Progesterone-Regulated Uterine
Protein Homologous to ~-lactoglobulins," Proc. Natl. Acad.
Sci. USA, Vol. 85, pages 8845-8849, December 1988, is
incorporated herein by reference.
It will be understood that the precise chemical
structure of PP14 will depend on a number of factors. For
example, since ionizable amino and carboxyl groups are
present in the molecule, a particular protein may be
obtained as an acidic or basic salt, or in neutral form.
All forms of PP14 which retain their therapeutic activity
for purposes of the instant invention are intended to be
within the scope of the definition of "PP14 " .
It should be noted that the N-terminal amino acid
sequence of PPl4 shows substantial sequence homology with
certain animal ~-lactoglobulins, but the biological
activities of these proteins are not well understood.
SUBSTITUTE SHE~
W~94/09805 rcr/C~3/00~9
2~24~
Furthermore, there is some sequence homology between PP14
and human serum retinol bindin~ protein.
The term "recombinant" used herein refers to PP14
produced by recombinant DNA technlques wherein the gene
coding for the PP14 is clonecl by known recombinant DNA
technology. For example, the human gene for PP14 may be
inserted into a suitable DNA vector, such as a bacterial
plasmid, and the plasmid used to transform a suitable host.
The gene is then expressed in the host to produce the
recombinant protein. The transformed host may be
prokaryotic or eukaryotic, including mammalian, yeast,
Asperyillus and insect cells. One preferred embodiment
employs bacterial cells as the host. -
Therapeutically useful derivatives of PP14 may be
prepared by augmenting the primary amino acid sequence of
the protein PP14 with at least one additional molecule
selected from the group consisting of glucose moieties,
lipids, phosphate groups, acetyl groups, hydroxyl groups,
saccharides, methyl groups, propyl groups, amino acids, and
polymeric molecules. Augmentation may be accomplished
through post-translational processing systems of the
producing host, or it may be carried out in-vitro. Poth
techniques are well-known in the art.
Referring to the Sequence Description of PP14, it
should be noted that the peptide includes three potential
glycosylation sites at amino acid residues 28-30, 63-65,
and 85-87. Glycosylation is a process of forming a protein
derivative, wherein a portion of the protein's amino acid
sequence is augmented by a sugar moiety. It will therefore
be understood that therapeutically useful derivatives of
PP14 may be prepared by addition of one or more sugar
residues to the protein, or alternatively by removal of
some or all of the sugar residues from the sites of
glycosylation on the PP14 molecule.
-
SUE3STlTUlrE ~ EE:T
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2124~94
Other therapeutically useful derivatives of PP14 may
be for~ed by modifying at least one amino acld residue of
PP14 by oxidation, reduction, or other derivatization
processes known in the art.
Muteins of PP14 which do not destroy the activity of
the protein may be used as the active treating substance of
the instance invention. Muteins may be prepared by
modification of the primary structure of the protein
itself, by deletion, addition, or alteration of the amino
acid incorporated into the sequence during translation.
For example, at least one cysteine residue of PP14 may be
replaced with a conservatlve amlno acid, in order to
eliminate sites of undesirable intramolecular disulfide
bond formation. Crosslinkin~ is undesirable if it changes
the conformation of PP14 so as to render the protein
essentially inactive for purposes of treating recurrent
miscarriage. Also, it may be desirable to replace a
methionine which is not essential to bioactivity with a
conservative amino acid. As referred to herein, a
conservative amino acid alteration is defined as one which
does not significantly adversely affect biological activity
and involves substitutions of the amino acid.
The conservative amino acid that may be substituted
for cysteine and methionine include at least: serine,
alanine, glycine, valine, threonine, leucine, isoleucine,
and tyrosine. More preferably they include serine and
alanine. Most preferably, cysteine may be replaced with
serine and methionine replaced with alanine.
Placental Protein 14 (PP14) is believed to exist in
nature as a dimer of two identical, non-covalently linked
protein subunits. Accordingly, since each subunit is
believed to have the amino acid sequence shown in SEQ ID
NO:1, a subunit o~ PP14 could be used as the therapeutic
active substance for treating recurrent miscarriage
according to the instant invention. The invention also
SIJ13~;TITUTI~: ~;HE. T
W094/09805 PCT/CA~3/OOM9
,
2~2~fi~
; encompasses use of subunits of PP14 that are covalently
linked, either naturally or by artificial techniques known
~` in the art.
In addition, it is contemplated that fragments of PP14
would be useful for treating recurrent miscarriage,
provided that such fragments retained their therapeutic
activity. Referring to SEQ ID NO:1, the protein fragment
defined by amino acid residues 63 through 160 is believed
to be a therapeutically active fragment of PP14 for
purposes of the invention. This fragment includes four
cysteine amino acids, which are believed to have biological
activity.
7 The fragment defined by amino acid residues 80 through
105 is also believed to be therapeutically active for
¦ 15 purposes of the invention. The fragment defined by
residues 8C-105 is believed to be therapeutically active
because: it is a linear sequence not involving disulfide
bridges; the fragment contains a glycosylation site (at
residues 85-87); the fragment has two tyrosine residues
which, because of their phenyl side chains, may be involved
in receptor interaction; and the double lysine sequence at
residues 80-81 have double amino acid group activity,
suggesting potential receptor activity.
The above-described forms of PP14 are used in an
effective therapeutic amount, which will vary depending on
the level of PP14 already present in the female, the side
and the method of administration, the form of PP14
utilized, the stage of the menstrual cycle of the female,
and other factors understood to those having ordinary skill
in the art.
In general, one of ordinary skill in the art will be
able to arrive at therapeutically effective dosages of PP14
for treatment of miscarriage, based on the range of
concentrations of PP14 which has been discovered to provide
suitable in-vitro activity. As shown in Example 4 below,
Sl~E35TITUTE SI~EET
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' WO9q/09805 PCr/CA~3/00~9
212469~
normal, fertile women have been found to exhibit a
~ concentration of PP14 in flushings from the uterine lumen
¦ ` of about 95.1 ng/ml ~/- 7.5 ng/ml (means ~/- SD after
! logarithmic transformation, n=8). The actual luminal
I - 5 concentration of PP14 is much higher, as the concentration
I of PP14 is diluted in the flushings by the washing fluid.
~, Accordingly, the invention p:referably contemplates the
administration of PP14 in an effective amount so as to
provide a concentration of PP14 which exhibits in-vitro
activity when subjected to the testing procedures described
¦ below. In this regard, administration of PP14 in an amount
3 ranging from about 0.1 ~g/ml to about 5 mg/ml, and
preferably from about 5 ~g/ml to about 5 mg/ml, is
contemplated by the invention to prevent miscarriage.
¦ 15 The Placental Protein 14 (PP14) material may be
administered to the female by any appropriate route,
including intravenous injection, intramuscular injection,
oral administration, intra-vaginal administration, intra~
¦ cervical administration, intra-uterine administration,
¦ 20 intra-tubal administration, topical administration, rectal
¦ administration, and inhalation. As previously noted, the
~¦ PP14 may be delivered to the site of treatment by means of
a controlled~release delivery system, or by means of a gel,
such that the PP14 does not wash out of the site of
administration too easily. Suitable controlled release
drug delivery systems and gels are well-known in the
pharmaceutical arts. The PP14 active substance may be
administered in admixture with one or more pharmaceutically
acceptable carriers well-known in the art.
The invention is preferably intended for the
prevention of miscarriage in females who suffer from
recurrent miscarriage; however, the method of treatment of
the invention is also believed to be useful to prevent
miscarriage in females who have not previously had a
13
..
SIJBSTIT~JTI~; SHEE:T
~ W094/09~ PCI/CA93/1)~9
21~69~
miscarriage. Also, the method of the invention is intended
for the treatment of humans and other animals.
According to a second aspect of the invention, a
method of testing a female for the possibility of future
j 5 miscarriage is provided, which comprises:
(a) measuring the level of PP14 present in a
predetermined body fluid of tissue sample of a female; and
, (b) comparing the level of PP14 present in the
i predetermined body fluid or tissue sample of the female to
the level of PP14 present in the comparable body fluid or
tissue sample of a normal, fertile female, wherein a level
of PP14 below that present in a normal, fertile female
suggests the possibility of future miscarriage.
As PP14 is synthesized in the endometrial glands and
secreted into the uterine lumen, a preferred site to which
to measure levels of PP14 is the uterine lumen. PP14
levels present in other body fluids or tissues could be
measured and compared with corresponding samples from
fertile individuals.
A preferred method according to the invention for
measuring PP14 in the uterine lumen is by flushing the
uterus with a saline solution, collecting the resulting
washings, and subsequently determining the concentration of
PP14 at this site using radioimmunoassay or other
techniques.
Measurement of the endometrial production of PP14 is
known to vary depending upon the particular stage of the
menstrual cycle; thus, the day of collection of the
washings should be standardized. A preferred day of
collection of the endometrial washings is the seventh day
after the midcycle luteinizing hormone (LH) peak in the
¦ female, which date closely proximates the time of normal
implantation in healthy individuals.
The above-discussed technique of collecting
endometrial washings is preferably utilized to detect the
~: .
14
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W094/09~0~ PCI`/CA93/00~9
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i 212~94
l level of PP14 present in the uterine lumen of the female.
¦ The particular process for measuring PP14 concentration in
the body fluid for tissue samp:Le may be carried out using
any known assay, including those selected from the group
- 5 consisting of radioimmunoassay, ELISA assays, and
immunoblotting assays. As normal, fertile women exhibit a
concentration of PP14 in flushings from the uterine lumen
of about 95.1 ng/ml ~/- 7.5 ng/ml, levels below this are
believed to indicate the possibility of future miscarriage.
1 0
It will be understood that the above-described method
of testing for the possibility of future miscarriage may be
I employed ln conjunction with other techniques for assessing
¦ fertility. For example, hys~erosalpingogram, laparoscopy,
or hysteroscopy techniques well-known in the art may be
used in conjunction with the inventive testing method, to
provide a complete prognosis.
A third aspect of the invention relates to a method of
improving the efficacy of assisted reproductive techniques,
such as artificial insemination (AI), in-vitro
fertilization (IVF), and gamete intra-fallopian transfer
(GIFT), by adding an effective amount of PPl4 to any sample
(embryo, fertilized or unfertilized egg, mixture of eggs
and sperm, or sperm alone) that is to be introduced into
the uterus in order to induce a pregnancy.
A description of assisted reproductive techniques is
found in Tan et al., Infertility~_ Your Questions Answered
(McGraw-Hill Book Co., 1991), the disclosure of which is
incorporated herein by reference.
In-vitro fertilization is a technique whereby one or
more mature eggs (oocytes) are removed from the woman's
ovary, fertilized outside of the body by sperm either from
the husband or from a donor, then transferred back into the
uterus. The fertilized eggs which are transferred into the
uterus are also called "pre-embryos". A similar process is
SLJBSTlTll~E SHEET
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~ WO9f4/09805 PCT/CA~3/00~9
~2~9~
embryo transfer (ET), in which embryos are formed out.side
f of the uterus then transferred into the uterus. An
"emb.ryo" is generally defined clS the state of a Eertilized
~ egg two or more weeks after fertilization.
5 Gamete intra-fallopian transfer (GIFT) is a treatment
for women who have patent (not blocked) fallopian tubes.
The first few steps of GIFT are similar to those in IVF,
namely, stimulation of the ovary with drugs, monitoring
follicular growth, administration of human chorionic
1 10 gonadotrophin (hCG) to induce maturation of the eggs, and
i collection of the eggs. After collection of the eggs, the
maturity of the eggs is observed, then sperm and eggs are
separately drawn into a catheter and deposited into the
patient's fallopian tubes. ~The resulting pre-embryo then
moves down the fallopian tubes and into the uterus where
implantation occurs. Thus, the primary distinction between
GIFT and IVF is that in GIFT, fertilization occurs within
a fallopian tube, whereas in IVF fertilization occurs in a
laboratory.
Direct intra-peritoneal insemination (DIPI) involves
stimulating the patient's ovaries with drugs, observing the
development of the follicles, administering hCG to induce
egg maturation, then injecting a washed sample of semen
through the top of the vagina into the Pouch of Douglas.
Peritoneal oocyte and sperm transfer (POST) is a similar
treatment in which eggs and washed sperm are both
transferred into the Pouch of Douglas proximate to the
opening of the fallopian tubes.
Zygote intra-fallopian tube transfer (ZIFT), also
known as pronuclear stage tubal transfer (PROST), is an in-
vitro fertilization process wherein Eertilized eggs are
transferred into the fallopian tubes by laparoscopy when
the pronuclei form (at about 18 hours following
fertilization). Tubal embryo transfer (TET), also known as
tubal embryo stage transfer (TEST), is a similar process;
16
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~ W094/0980~ f'CT/CA93/~30~9
~- 2124fi9~ -
however, transfer of the fertili~ed eggs into the fallopian
tubes is not carried out until the pre-embryo has divided
`into two or more cells.
In direct oocyte transfer (DOT), eggs are collected
according to IVF, then the eggs are incubated for a few
hours prior to the addition of e:perm. Eggs are transferred
lnto the uterus using IVF once sperm are seen binding to
the eggs.
Trans-uterine fallopian transfer (TUFT) involves the
transfer of a pre-embryo into the fallopian tube using a
catheterizing instrument.
Artificial insemination (AI) is a procedure in which
jsperm is introduced into the patient's genital tract by
artificial means rather than by intercourse. Insemination
15may be either into the cervix or into the uterine cavity
itself (intra-uterine insemination, IUI).
Unwashed semen is generally not employed in IUI
because the protective cervical mucus barrier is bypassed
in this process, and unwashed sperm would create a risk of
20infection or allergic reactions. Also, prostaglandins
present in the semen may cause contractions of the uterus
which may be painful and cause ejection of semen from the
uterus.
Washing of the sperm is carried out by repeated
25extractions in appropriate solvents, followed by
centrifugation to recover the sperm. Alternatively, the
semen may be mixed with a cleaning solvent, and the healthy
sperm permitted to swim to the surface of the solvent where
they are collected for use in the AI or IVF process.
30PP14 is found in relatively high concentrations in
seminal plasma. However, as discussed above, sperm used
for artificial insemination (AI) and other assisted
reproductive techniques are frequently washed prior to use ~-
in order to remove prostaglandins present in the seminal
35plasma. I'he invention therefore contemplates the additlon
17 -
~"'',
SUBSTITUTE ~;H~ T
, ,
W0'~4/~9805 PCT/CA~3/~)0~9
212469~
~l of PP14 to sperm which has been washed, to restore PP14 to
¦ levels which exhibit in-vitro activity.
For example, before sperm is introduced into a patient
~ by AI, PP14 may be added to the sperm sample, preferably by
j 5 including PP14 in the medium used to dilute the washed
I sperm prior to insemination. Similarly, PP14 may be added
to washed sperm before use in other assisted reproductive
techniques, in order to replace PP14 which is lost during
I the conventional washing stage.
¦ lO As discussed above, fertile women have been found to
I exhibit a concentration of PP14 in flushings from the
uterine lumen of about 95.1 ng/ml +/- 7.5 ng/ml. Moreover,
in any assisted reproductive technique, whether or not
washed sperm are being employed, the addition of extra
medium into the uterus along with the gametes inevitably
dilutes the normal PP14 concentration present in the
uterus. The invention thus contemplates adding an
effective amount of PP14 to the gamete medium or the like,
so as to provide a PP14 concentration having in-vitro
~O activity when subjected to the testing procedures described
below. In general, adding PP14 to the sample in an amount
ranging from about 0.1 ~g/ml to about 5 mg/ml, and :~
preferably from about 5 ~g/ml to about 5 mg/ml, is
contemplated by the invention.
It will be understood that the amount of PP14 added to
the sample to be introduced into the patient to induce
pregnancy will vary depending, for example, on the level of
PP14 already present in the patient, the particular
assisted reproductive technique employed, whether or not ;-~
washed sperm are employed, the volume of the sample to be
introduced into the patient, and other factors understood
to those of ordinary skill in the art. ~ ~
As discussed, the PP14 may be added to any sample that ~-
is to be introduced into the reproductive tract of the ~ :
patient. For example, PP14 may be added to an embryo,
SUBSTITUTE SHEET
` W094/09805 PCr/CA93/()0~9
2~2~6~
fertilized or unfertilized egg, mixture of eggs and sperm,
I or sperm alone, and may be added prior to, concurrently
¦ with, or following introduction of the sample into the
~ patient.
I ~ 5 The following additional comments, and Examples 1-3
below, further describe the immuno-inhibitory properties of
I PP14. While not wishing to be bound by any particular
j theory, it is believed that the potential efficacy of PP14
! in preventing miscarriage is related to the immuno-
1 10 suppressive activity of natural PP14.
¦ PP14 has been found to be a natural inhibitory
regulator of the immune response; the protein is present in
the tissues and bloodstream of pregnant women. The
pregnancy-associated protein, PP14, has been found to
inhibit IL-l production by stimulated macrophages and to
¦ inhibit monocytes lymphokine secretion, IL-2 receptor
expression and proliferation of mitogen or allogeneically
stimulated lymphocytes.
PP14 has been found to inhibit mitogenic stimulation
of proliferation and secretion of interferon and IL-2 by
lymphocytes. A similar inhibition of allogeneic
stimulation of lymphocytes has also been measured. These
effects have been found to be accompanied by a reduction in
the affinity of high affinity lymphocyte IL-2 receptors and
an inhibition of the expression of functional IL-2
receptors.
PP14 suitable for purposes of the present invention is
found in extracts of human decidual tissue; the material
binds to the monoclonal antibody disclosed in accordance
with the present invention. The activities observed from
in-vitro test systems correlate with the PP14 content of
tissue extracts and other preparations, as measured in a
radioimmunoassay for PP14 that utilizes a polyclonal
antibody. This radioimmunoassay is described in the
publication by Anthony E. BOLTON et al., entitled "The
SIJ~351-1TUTE: SI~IEET
wo 94/09805 PCr/CA'~3/00'14
`! ,~
~i 2124~9~
.,
i Radioimmunoassay of Human Placental Protein 14 (PP14)",
Clinica Chimica Acta, 135 (1983) 283-291. The PP14
suitable for purposes of the present invention has been
observed to be functional in inhibition of the
proliferakion of mitogen and allogeneically stimulated
peripheral blood mononuclear cells; the inhibition of the
production and/or release of IL-1 by stimulated peripheral
I blood mononuclear cells; and the reduction in the affinity
¦ of binding of IL-2 to stimulated peripheral blood
lo mononuclear cells.
I Activation of the immune regulatory and proliferative
¦ capacity of lymphocytes is a complex process mediated by a
number of lymphokines and requiring the cooperation of
accessory cells. These cells secrete the lymphokine IL-1
in response to antigenic stimulation. This factor is
required for the expression of the IL-2 receptor by
lymphocytes which then respond mutagenically to IL-2.
PP14 inhibits the elaboration of IL-1 by activated or
stimulated peripheral blood monocytes; this may account for
many of the modulating activit.ies of PP14. Similar
suppressive effects are induced by crude decidual extracts;
this activity is abolished by an antibody to PP14. Thus,
PP14 has been discovered to have immunosuppressive activity
related to its ability to decrease expression or secretion
of IL-1 and possibly independent effects on lymphocyte
secretion and proliferation.
Many human diseases result from an abnormal,
unregulated proliferation of lymphocytes, or from an
uncontrolled immune response directed against the patient's
own cells or tissues. Some of these defects may result
from elevated, unregulated secretion of lymphokines. An
example is rheumatoid arthritis, in which IL-1 has been
shown to activate synovial prostaglandin and leukotriene
production, enhance T-cell binding to endothelia, and to
reproduce several aspects of the condition in animal
SUBSTITLITE SHE~:T
W094/098V~ PCr/C~93/~30~9
2~24~9~
models. Similar evidence exists for other chronic
inflammatory diseases. PP14, an inhibitor of IL-1
production, is a potential treatment modality for sueh
diseases, including syndromes such as asthma, which is
- 5 charaeterized by excessive seeretion of leukotrienes in the
bronchial tree, and allergic dermatitis and inflammatory
bowel diseases, which are characterized by ehronie
overproduction of mediators of inflammation.
Other autoimmune diseases, such as systemic lupus
erythematosus, Sjogren's syndrome, and scleroderma are
eharaeterized by abnormal ratios of a variety of different
types of T- and s-lymphocytes which may result from
defective regulation of their proliferation. More extreme
losses of regulation of growth may be accompanied by
further genetic changes in the cells and result in
overgrowth of a malignant elone of eells leading to a
malignaney in the patient. Sueh malignant eells often
still require autoerine or paracrine stimulation by a
lymphokine growth faetor in order to drive their
proliferation.
The aetivation of lymphoeytes, resulting in eell
proliferation, is a eomplex response mediated by a number
of peptide messengers, the cytokines. At a simplified
level, T-lymphoeytes are activated by a se~uential process.
First, there is a requirement for a cytokine Interleukin-1
(IL-l) secreted by accessory cells (e.g., cells of the
monoeyte/maerophage lineage). In the presenee of IL-l the
eytokine Interleukin-2 (IL-2) inereases the expression of
its own reeeptors, making the cell more responsive to IL-2
-- a positive feedbaek eyele. Thus, the proliferation of
T-eells requires the presenee of both IL-l and IL-2.
One approaeh to investigating the mode of aetion of
lymphoproliferation with an inhibitor sueh as PP14 is to
add an exeess of eytokine along with the inhibitor and
determine whether the suppression is reversed. One souree
5UB~TITlJTi~ S~EET
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of a crude mlxture of cytokines is the supernatant taken
, from cultured activated lymphocytes. Such supernatants
¦ were demonstrated to reverse the suppressive action of
PP14, indicating a mode of action relating to the
secretion/activity of cytokines. The addition of
recombinant IL-1 at a single dose significantly reduced the
¦ suppressive action of PP14 on lymphoproliferation, as shown
in the following Table:
TABLE 1
This table shows the effect of the addition of 5U/ml
of recombinant IL-1 on the suppression of tritiated
thymidine uptake by stimulated lymphocytes.
Decidual sample no. PP14 (~q/ml) ~suppression of 3H-Tdr
+IL-1 -IL-1
15DE A 5.0 25 30
DE B 4.8 12 62
DE C 4.0 25 46
DE D 8.0 33 48
DE E 2.0 30 41
20Mean (+/-S.D.)
These data indicate that PP14 may be operating via an
IL-1-mediated mechanism.
To investigate this possibility further, peripheral
blood mononuclear cells (a mixture primary of T-cells and
monocytes) were activated using the mitogen PHA in the
presence and absence of inhibitory amounts of PP14. The
amount of IL-1 released into the supernatants of the
cultured cells was measured after different times of
culture. The results from the two experiments carried out
are shown in the attached Figure, and the data given in
Table 2 below. It can be seen that PP14 significantly
inhibited the release of IL-1 into the supernatant by
activated cells. These data on IL-1 strongly suggest that
PP14 is acting at the IL-1 level of T-cell activation by
inhibiting its synthesis/release.
S~ STITUTE SH~ET ~ - -
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WO 94/0980~s rcr/c~93/0(~449
.~
2~2~694
TABLE 2 `
This Table shows the effect of PP14 ln decidual
- extracts on the release into cell supernatants of IL-1 by
stimulating peripheral blood lymphocytes.
. 5 TA3LE 2
Experiment 1 TIME (hours)
22.5 41 65 89
10Unstimulated 0.2 0.2 0.1 0.1
Immunoabsorbed
extract 1.5831.266 1.232 1.196
15Unabsorbed
extract 0.4060.216 0.212 0.2
~6 Suppression 75~ 83~ 91% 83%
Experiment 2 Time (hours)
18 42 66 80
Unstimulated 0.6 0.2 0.4 0.3
Immunoabsorbed
extract 1.7892.554 2.709 2.807
Unabsorbed
extract 0.7 1.162 I.630 1.967
% Suppression 61% 55% 40% 30%
NOTES
Unstimulated -- spontaneous release of IL-1 from
unstimulated lymphocytes.
Immunoabsorbed extract -- IL-1 release from stimulated
cells` in the presence of a crude decidual extract from
which PP14 had been specifically removed by monoclonal
antibody immunoabsorption.
Unabsorbed extract -- IL-1 release from stimulated
lymphocytes in the presence of a crude decidual extract
containing 8.0 ~g/ml of PP14.
23
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Percent suppression -- the suppression of IL-1
released into the cell culture supernatants by PP14 in
decidual extracts, expressed as a percent of the release of
IL-1 in the presence of decidual extracts from which PP14
had been removed.
Individual results for each experiment are means of
triplicate determinations.
A monoclonal antibody which specifically binds to PP14
has been isolated and characterized. This monoclonal
antibody is designated MAb 14/l/1, and was derived from
hybridomal cell lines produced by fusion, using a
polyethylene glycol method of spleen cells from mice
immunized with crude extracts of deciduum with the myeloma
cell line P3/NS1/1-Ag4-1, which is a standard myeloma cell
line. Clones secreting anti-PP14 were selected using the
radiolabelled PP14 used for radioimmunoassay (BOLTON et
al., 1983, supra). Positive cultures were cloned three
times by limiting dilution and those yielding the highest
titers used to induce tumors in Balb/C mice. IgG was
isolated from ascitic fluid from ion exchange
chromatography or affinity chromatography on protein -A.
The specificity of the antibody was examined in two
ways. First, the two-site immunoradiometric assay was set
up using a polyclonal extracting antibody and the
monoclonal as labelled antibody. Polyclonal antibody,
raised against crude decidual tissue extract, was
covalently linked to Sepharose-4B by standard procedures.
rrhis was incubated in excess in the presence of standard
PP14 or the potentially cross-reacting protein for two
hours at room temperature. Subsequently, radioiodinated
monoclonal antibody was added, incubated a further two
hours, and the solid-coupled antibody washed and counted
for radioactivity. This represents a conventional 2-site
immunoradiometric assay. The cross-reaction of various
24
.
~;UE3STITUTE 5HEET
, W094/09~05 P~T/CA93/()OM9
~l 212~69~
. ~
decidual/placenta proteins was investigated in this system.
The followir.g gave less than 0.1~ cross reaction:
hPL, SP1, PP5, PP12, pregnancy-associated plasma protein-A
(PAPP-A), placental alkaline phosphatase, placental malic
dehydrogenase, placental sphyngomyelinase, placental
I arylamidase, placental choline acetyltransferase, with only
j PP14 having any observed activity in this assay.
¦ The other method involved investigating the binding to
the monoclonal antibody of radioactively-labelled pure
proteins. No significant binding of prolactin, hCG, or
I PAPP-A was detected. This antibody could be used in the
¦ assay, for example by a two-site immunoradiometric
procedure, and purification of PP14. As previously
mentioned, the details of the radioimmunoassay for PP14 are
disclosed in the 1983 Bolton et al. article, identified
above.
The following examples are given to illustrate the
invention, but are not deemed to be limiting thereof.
SU13STlTlJTE SHEET
W094/09805 PCT/CA93/00~9
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;
Example 1
Evidence that PP14 inhibits IL-l release
Method 1 - the mitogenic response of human lymphocytes
Human peripheral blood lymphocytes proliferate in
response to stimulation by the mitogen phytohaemagglutinin
(PHA), the proliferation being measured by the
incorporation of tritiated thymidine into the DNA of the
dividing cells. This is a standard test of lymphocyte
responsiveness. PP14 inhibits lymphocyte proliferation in
response to PHA stimulation. The addition of recombinant
IL-1 partially reverses the inhibition caused by PP14.
The test system used in this example was to
investigate the effect of re$ombinant IL-1 on the mitogen-
induced stimulation of PP14-inhibited peripheral blood
lymphocytes. The use of PP14-inhibited cells in
experiments such as this unique methodology.
The PP14 inhibition of cells in this example was
carried out as follows. Cells were treated with crude
e~tracts of human decidual tissue in which the PP14
concentration was measured by radioimmunoassay. As a
control for each treated cell preparation, a portion of the
cells were treated with the same extract that had been
immunoabsorbed using a monoclonal antibody of PP14,
MAb/PP14/1/1, to remove the PPl4 in a highly specific
manner. The difference between the activity of the cells
treated with these two preparations represented the effect
of PP14. Thus, the effects measured were those of PP14,
which specifically binds to this monoclonal antibody.
Peripheral blood mononuclear cells were isolated from
whole blood obtained from healthy donors by a standard
¦ density gradient centrifugation method. After washing in
a physiological medium, the cells were resuspended at an
appropriate concentration of viable cells and incubated in
26
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the presence of the PP14 preparation and the mitogen at a
stimulatory concentration.
The effects of IL-1, or other test compound, were
assessed by inclusion at appropriate concentrations in this
` 5 incubation, including also the necessary controls. The
cells were incubated for 72 hours at 37- C in an atmosphere
of 5~ carbon dioxlde and 100% humidity under sterile
conditions. Six hours prior to termination of the
cultures, the cells were pulsed with 1 ~Ci tritiated
thymidine, and on termination the cells were harvested
automatically onto glass fiber filters.
The degree of lymphoproliferation was assessed by
measuring the incorporation of tritiated thymidine into the
harvested cells by liq~id scintillation counting.
Inhibition of tritiated thymidine uptake by PPl~, and the
effect of IL-1 were as follows:
inhibition of tritiated
thvmidine uptake
Without IL-1 With 5U/ml IL-l
45.4 +/- 11.6 25.0 ~/- 8.0
These are mean results from 15 different experiments,
each performed in triplicate, using 5 different decidual
extracts as the source of PP14 and cells from 2 separate
donors. The results are significantly different (p
~0.0001), indicating a significant reversal of the
inhibitory effect of PP14 on lymphoproliferation by IL-1.
Example 2
The effect of PP14 on the production of IL-1 by
stimulated peripheral blood mononuclear cells.
Peripheral blood mononuclear cells, as isolated by
density gradient centrifugation, contain not only
lymphocytes (the majority of cells present) but also
monocyte/macrophages, which are necessary accessory cells
27
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W094/09805 PCI~CA93/U0~9
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21~469~
in the lymphoproliferative reaction described above. It is
this latter cell type which synthesizes and secretes IL-l.
These cells can be activated to secrete their specific
products which include IL-1 by both mitogens (e.g., PHA)
and lipopolysaccharide (LPS).
The test system used :in this example was to
investigate the production of macrophage/monocyte products
released from stimulated Ppl4-inhibited peripheral blood
mononuclear cell preparations, measuring the products by
commercially available immunoassay systems. The use of
PP14-inhibition of cells was carried out as in Method 1 of
Example l above, which again is unique methodology.
Peripheral blood mononuclear cells were prepared as
under Method l. Appropriate numbers of cells were
incubated in the presence of the PP14 preparation and the
stimulator for various times under the conditions described ~`
above. At the termination of the culture period the cells
were harvested and the supernatants assayed for IL-1 and
Tumour Necrosis Factor (TNF), both known to be products of
macrophage/monocyte cells. Typical results are summarized
below. ~
Inhibition of IL-1 release by stimulated peripheral ~-`
mononuclear cells by PP14:
~ inhibition of_IL-1 release
PHA stimulated cells LPS stimulated cells
82.5 67.0 `~
These are mean results from 3 experiments.
This inhibition of IL-1 release from stimulated (using
either PHA or LPS) mononuclear cells by PP14 is dose
dependent, as shown in Figure 1, wherein o represents cells
stimulated with mitogen (PHA) and o represents cells
stimulated with LPS.
28
SUBSTITUTE SHIEET
~ W094/09805 PCT/CA93/00~
.~
2~2~9~
Example 3
Comparison of IL-1 and TNF production by PP14-
inhibited stimulation peripheral blood mononuclear cells.
Cells stimulated with mitogen: -
IL-1 produced TNF produced
stimulated control 2.60 ng/ml 1043 pg/ml ~-
-~PP14 0.46 ng/ml 1474 pg/ml
(4 ~g/ml)
Example 4
This example illustrates the measurement of PP14 in
endometrial washings taken from normal, fertile women in
comparison with endometrial washings taken from women
hav1ng recurrent miscarriage.
Methodoloqy:
PP14 is synthesized in the endometrial glands and
secreted into the uterine lumen; therefore the most logical
site in which to measure levels of PP14 in these women is
in the lumen. Measurement of levels of PP14 in the uterine
lumen was achieved by flushing the uterus with 10 ml of
saline solution, and collecting the resulting endometrial
washings.
As it is ~nown that endometrial production of PP14
varies at different stages of the menstrual cycle, the day
of collection of the washings was standardized to be the
7th day after the midcycle LH peak in all of the women
tested. The day of collection of the endometrial washings
therefore was closely related to the normal time of
implantation in the uterus.
All mea~urements of PP14 are expressed in ng/ml,
measured using the radioimmunoassay technique discussed
above.
5UBSTIT~T~: S~EET
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2~2~9~
PP14 concentration in normal fertile women:
Initially, the concentration of PP14 present in
flushings from the uterine lumen of normal, fertile women
was measured. The concentration of PP14 in endometrial
washings at day LH ~ 7 of the menstrual cycle in these
fertile women was found to be 95.1 ng/ml ~/- 7.5 ng/ml
(mean +/- SD after logarithmic transformation, n=8).
PP14 concentration in women with recurrent miscarriaqe:
The concentration of PP14 in flushings from the
uterine lumen of women who had experienced recurrent
miscarriage was measured, using the techni~ue discussed
above. As defined herein, recurrent miscarriage means the
loss of three or more consecutive pregnancles before 20
weeks of gestation.
The concentration of PP14 in the endometrial washings
at day LH ~7 of the menstrual cycle of 9 women with
recurrent miscarriage was as follows:
Patient No.Conc. of PP14 in Endometrial
Washinqs (nq/ml)
1 nd
2 5.3
3 nd
4 nd
nd
S nd
7 nd
8 nd
9 200
nd = not detectable in the assay, i.e., ~ 3 ng/ml
From these data it can be seen that only 1 of the 9
recurrent miscarriage patients had a PP14 level in the
endometrial washings within the normal range.
SUE~STI~UT~: S;~EET
W094/09805 PC~`/C~93/00~9
212~694
The invention being thus described, it will be obvious
that the same may be varied in many ways. Such variations
are not to be regarded as a
departure from the spirit and scope of the invention and
all such modifications are intended to be included within
the scope of the claims.
SLJ13STITUTE SIIEET
:
W094/09805 PCT/CA93/i)0~9
212~69~ ~
. ~.
i26412XX
SEQUENCE LISTING
(1) GENERAL INFORMATION: :
(i) APPLICANT: Bolton, Anthony E.
Drizen, Alan
(ii) TITLE OF INVENIION: METHOD OF TREATING
RECURRENT MISCARRIAGE
AND IMPROVING THE
EFFICACY OF ASSISTED
, REPRODUCTIVE
! TECHNIQUES USING -~.
¦ PLACENTAL PROTEIN 14 :
(iii) NUMBER OF SEQUENCES: 1
(iv) CORRESPONDENCE ADDRESS: ~:
(A) ADDRESSEE: NATH, AMBERLY & ASSOCIATES
(B) STREET: 1835 K Street, N.W., Suite 750
(C) CITY: Washington ::
(D) STATE: D.C.
(E) COUNTRY: U.S.A.
(F) ZIP: 20006
(v) COMPUTER READABLE FORM
(A) MEDIUM TYPE: Diskette, 3.5 inch, 360 Kb storage
(B) COMPUTER: I~M PC/XT/AT compatible
(C) OPERATING SYSTEM: MS-DOS 6.0
(D) SOFTWARE: Word Perfect 5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Nath, Gar~ M.
(B) REGISTRATION NUMBER: 26,965
(C) REFERENCE/DOCKET NUMBER: 26412XX-PC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (202) 775-8383
(B) TELEFAX: (202) 775-8396
(2) INFORMATION FOR SEQ ID NO:l: .
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 819 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: double stranded
(D) TOPOLOGY: linear
(x) PUBLICATION INFORMATION:
(A) AUTHORS: Julkunen, Mervi
Seppala, Markku
Janne, Olli A.
(B) TITLE: Complete Amino Acid
Sequence of Human
Placental Protein 14: a .
Progesterone-
. 32
SUBSTITlJTE SHEET
l W094/09805 PCr/CA93/00~9
1 2~694
I
Regulated Uterine Protein
Homologous To
~ Lactoglobulins
1 (C) JOURNAL: Proc. Natl. Acad. Sci. USA
(D) VOLUME: 85
(F) PAGES: 8845-8849
(G) DATE: DEC-1988
(K) RELEVANT RESIDUES IN SEQ ID NO:l: FROM 1 to 919
33
: ',
SUBSTITUTI~ SHE~
~. .
~,L '
WO 94/09805 PCI'/CA93/00449
:, ,~
212~fi~
i
2i~412XX
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
CATCCCTCTG GCTCCAGAGC TCAGAGCCAC CCACAGCCGC AGCC 44
ATG CTG TGC CTC CTG CTC ACC CTG GGC GTG GCC 77
Met Leu Cys Leu Leu Leu Thr Leu Gly Val Ala
-18 -10
CTG GTC TGT GGT GTC CCG GCC ATG GAC ATC CCC 110
Leu Val Cys Gly Val Pro Ala Met Asp Ile Pro
CAG ACC AAG CAG GAC CTG GAG CTC CCA AAG TTG 143
Gln Thr Lys Gln Asp Leu Glu Leu Pro Lys Leu
GCA GGG ACC TGG CAC TCC ATG GCC ATG GCG ACC 176
Ala Gly Thr Trp His Ser Met Ala Met Ala Thr
AAC AAC ATC TCC CTC ATG GCG ACA CTG AAG GCC 209
Asn Asn Ile Ser Leu Met Ala Thr Leu Lys Ala
CCT CTG AGG GTC CAC ATC ACC TCA CTG TTG CCC 242
Pro Leu Arg Val His Ile Thr Ser Leu Leu Pro
ACC CCC GAG GAC l~C CTG GAG ATC GTT CTG CAC 275
Thr Pro Glu Asp Asn Leu Glu Ile Val Leu His
AGA TGG GAG AAC AAC AGC TGT GTT GAG AAG AAG 308
Arg Trp Glu Asn Asn Ser Cys Val Glu Lys Lys
GTC CTT GGA GAG AAG ACT GGG AAT CCA AAG AAG 341
Val Leu Gly Glu Lys Thr Gly Asn Pro Lys Lys
TTC AAG ATC AAC TAT ACG GTG GCG AAC GAG GCC 374
Phe Lys Ile Asn Tyr Thr Val Ala Asn Glu Ala
ACG CTG CTC GAT ACT GAC TAC GAC AAT TTC CTG 407
Thr Leu Leu Asp Thr Asp Tyr Asp Asn Phe Leu
100
TTT CTC TGC CTA CAG GAC ACC ACC ACC CCC ATC 440
Phe Leu Cys Leu Gln Asp Thr Thr Thr Pro Ile
llo
CAG AGC ATG ATG TGC CAG TAC CTG GCC AGA GTC 473
Gln Ser Met Met Cys Gln Tyr L~u Ala Arg Val
120
CTG GTG GAG GAC GAT GAG ATC ATG CAG GGA TTC 506 -
Leu Val Glu A9p Asp Glu Ile Met Gln Gly Phe
130
34
,
- : '
5LIBSTITUTE SHEET ~ ~
~ WO 94/09805 PCI~CA93/00449
2 ~ 2 ~ 6 9 4
ATC AGG GCT TTC AGG CCC CTG CCC AGG CAC CTA 5 3 9
Ile Arg Ala Phe Arg Pro Leu Pro Arg His Leu
, 140
TGG TAC TTG CTG GAC TTG AAA CAG ATG GAA GAG 5 7 2
Trp Tyr Leu Leu Asp Leu Lys Gln Met Glu Glu
150
CCG TGC CGT TTC TAG CTCACCTCCG CCTCCAGGAA 607
Pro Cys Arg Phe AM
160 162
GACCAGACTC CCACCCTTCC ACACCTCCAG AGCAGTGGGA CTTCCTCCTG 6 5 7
CCCTTTCAAA GAATAACCAC AGCTCAGAAG ACGATGACGT GGTCATCTGT 707
GTCGCCATCC CCTTCCTGCT GCACACCTGC ACC~TTGCCA TGGGGAGGCT 757
GCTCCCTGGG GGCAGAGTCT CTGGCAGAGG TTATTAATAA ACCCTTGGAG 807
CATGAAA~AA AA 819
"
":
. `- '
SUBSTITUT~ SHEET
