Note: Descriptions are shown in the official language in which they were submitted.
W094/07366 ~ 7 G ~ - PCT/US93/09~16
DESCRIPTION
PP14-Based Thera~y
Backqround of the Invention
This invention relates to placental protein 14
("PP14") and monoclonal antibodies thereto, and their
uses.
5~l~m~n PP14 is a 28 kilodalton glycoprotein expressed
during the first and second trimesters of pregnancy by the
endometrium. During this period, it constitutes up to
5-10~ of the total secreted protein from the endometrial
decidua (Julkunen et al., 92 Br. J. Obstet. Gynaecol.
101145, 1985). PP14 accumulates to significant levels in
serum from pregnant women as well. In addition, PP14 is
found at high concentrations in male sem;nAl fluid (19-515
mg/l) (Pockley et al., 43 Biochem. Soc. Trans. 317, 1989),
although the cellular source of PP14 in the male reproduc-
tive tract has not been determined.
PP14 belongs to a class of proteins called ~-lacto-
globulins, which include lactoferrin (Rado et al., 64
Blood 1103, 1984) and the retinoic acid receptor (Papiz
et al., 324 Nature 383, 1986). A PP14 cDNA has been
cloned from endometrium, and analysis of this endometrial
PP14 cDNA has shown the PP14-coding region to be approxi-
mately 70~ homologous to other ~-lactoglobulin family
members, and their genomes are similar in size and organ-
ization (Vaisse et al., 9 DNA Cell. Biol 401, 1990).
25PP14 has been shown to inhibit lymphocyte prolifera-
tion (Bolton et al., in Lancet 593, 1987). Extracts of
hllm~n decidual tissue were added to mixed lymphocyte
cultures (MLCs), and a linear relationship was observed
between the quantity of PP14 present, and the inhibition
of lymphocyte proliferation observed. Id. Moreover, an
anti-PP14 monoclonal antibody added to the MLCs inhibited
the anti-proliferative effect, indicating a functional
link between PP14 and anti-proliferative activity. Subse-
W094/07366 PCT/US93/09216 -
2~ ~7~2
quent studies have noted an inhibitory effect of PPl4 on
the synthesis of IL-l, IL-2 and soluble IL-2 receptors by
peripheral blood mononuclear cells (Pockley et al., 16
Biochem. Soc. Trans. 794, 1988 ; Pockley et al., 77 Clin.
5 EXP. Immunol. 252, 1989; and Pockley & Bolton, 69 Immunol-
oay 277, 1990). Okamoto et al., 26 Amer. J. Reprod. Immu-
nol. 137, 1991 indicate that PPl4 suppresses natural kil-
ler cell activity.
It has been proposed that PP14 be used for non-
specific ;mmnnosuppression in patients in need of such;mmnnosuppression, such as those suffering from autoimmune
conditions, inflammatory conditions and allergic condi-
tions. Bolton et al., U.S. Patent 5,039,521. Specific-
ally, Bolton et al. propose use of PP14 to treat arthri-
15 tis, rheumatoid arthritis, asthma, graft-versus-host dis-
ease, organ rejection, osteoarthritis, systemic lupus
erythematosus, atopic allergy, multiple sclerosis, aller-
gic dermatitis, inflammatory bowel disease, psoriasis,
sarcoidosis, and other inflammatory disorders. In addi-
20 tion, they suggest treatment of a lymphoproliferativedisorder, such as malignant non-Hodgkin's lymphoma,
Hodgkin's disease, or malignant histiocystotis. Sugges-
tion is also made to treat inflammatory and autoimmune
diseases, and to treat infertility and a neoplastic
25 disorder such as leukemia. Finally, it is stated that
monoclonal antibodies to PPl4 can be used to treat an
immune system disorder, specifically, acquired ;mm1lno-
deficiency syndrome (AIDS).
SummarY of the Invention
Applicant has discovered that PPl4 is expressed in
cells outside of the female and male reproductive tracts,
and specifically, that PPl4-encoding mRNA and PPl4 protein
is expressed in hematopoietic cells. In particular, when
the human myelogenous leukemia cell line K562 (which has
35 bipotential differentiative capacities, as it can be chem-
ically induced to diff-erentiate along erythroid and mega-
W094/07366 PCT/US93/09216
21~5762
karyocytic lineages) is caused to differentiate along the
megakaryocytic lineage, PP14 cDNAs are detectable.
This hematopoietically expressed PP14 has two poly-
peptide isoforms, encoded by two mRNA species that arise
5 through alternative splicing of the PP14 gene. These two
polypeptide isoforms, and their corresponding mRNAs, are
designated " PP14.1" and " PP14.2" herein. Previous studies
of PP14 in the female and male reproductive tracts (dis-
cussed above), recognized only one PP14 isoform, corres-
ponding to PP14.1.
The unexpected finding of PP14 in cells of the mega-
karyocytic lineage, and particularly in the end-cell of
that lineage, the platelet, indicates the use of PP14 in
otherwise unexpected areas. These discoveries are evi-
lS dence of PP14's pathophysiological role. Specifically,the release of PP14 from platelets in the course of plate-
let disorders, including coagulopathies (such as dissem-
inated intravascular coagulation), will lead to general-
ized ;mmllnosuppression in patients suffering from such
disorders, with all of PP14's attendant untoward (; mmllno-
suppressive) effects. Thus, Applicant has discovered that
it is important to reduce the effect that PP14 release
from platelets has on pathogenesis. This indicates that,
for a variety of disease conditions, PP14 blockade, as
opposed to potentiation, is the desired therapeutic end-
point for certain clinical conditions. To this end, App-
licant discloses several methods for treatment of patients
suffering from untoward effects of platelets, and for
blocking ;mmllnosuppression associated with PP14 release
from platelets in such patients. These methods are based
upon blocking PP14 effects. This comprises, but is not
limited to, the therapeutic use of anti-PP14 antibodies,
peptides which block PP14 activity, solubilized PP14
receptor, and anti-PP14 receptor antibodies, or their
equivalent (e.a., active fragments thereof, which can be
identified by standard procedures). Compositions or
W094/07366 2 1 ~ 5 7 ~ 2 PCT/US93/09216 -
; ~..
therapeutic formulations, including these therapeutic
agents are also disclosed.
The invention also includes ~m; n; stration, to a
patient suffering from platelet-induced;mml~nosuppression,
of a reagent that will inhibit PP14 production by hemato-
poietic cells of the patient. Antisense PP14 oligonucleo-
tides and ribozymes are suitable reagents for interfering
with PP14 transcription and/or translation. Methods for
identifying additional reagents for blocking PP14 produc-
tion at the m~RNA or protein levels are straightforward,and readily practiced by those familiar with the art.
Specifically, the invention features methods for
treating a patient suffering from leukemia, where the
leukemic cells of the patient have megakaryocytic differ-
entiative potential and produce a PP14 polypeptide with;mmllnosuppressive activity. The methods for blocking PP14
function and reducing PP14 production cited above can also
be applied in this clinical setting.
The invention also features production of anti-PP14.1
and PP14.2-specific antibodies and PP14 receptors for
therapeutic and diagnostic applications. The discovery of
a second PP14 isoform in hematopoietic cells permits the
generation of well-characterized anti-PP14 monoclonal
antibodies with specificity for one or both isoforms.
Methods for producing both anti-PP14.1-specific and anti-
PP14.2-specific antibodies are disclosed, and make use of
the precise determination in the present invention of the
amino acid sequence difference between the two isoforms.
This knowledge permits the production and use as ;mmnno-
gens of peptides corresponding to junctional and internalamino acid sequences that distinguish between PP14.1 and
PP14.2 polypeptides.
Such isoform-specific anti-PP14 antibodies are useful
for detection of PP14 isoforms in the serum of patients,
to determine which patients can benefit from PP14 blockade
therapy, and to monitor therapeutic responses to such
therapy. In addition,-PP14 isoforms can serve as a marker
~ W094/07366 PCT/US93/09216
214~762
for certain platelet disorders, such as disseminated
intravascular coagulation, and PP14 isoform-specific or
non-specific diagnostic assays are useful in this clinical
setting. In pregnant women, PP14-isoform-specific anti-
5 bodies allow for the discriminative analysis of hemato-
poietic cell- and endometrial cell-derived PP14 poly-
peptides.
This invention also allows production and use of a
polypeptide derivative of a PP14 receptor (and/or anti-
bodies with specificity for such a receptor) for blockinga functional interaction between a PP14 polypeptide and
its receptor. Methods for cloning receptors for known
ligands, such as ones based upon the use of readily puri-
fied ligand:;mml~noglobulin Fc conjugates, are well-estab-
lished and can be expeditiously carried out by those fam-
iliar with the art.
Thus, this invention features any method by which the
activity of either PP14 polypeptide isoform is reduced to
thereby interfere with PP14 -induced ;mmllnosuppression.
Such reduction of PP14 activity is useful for treatment of
disease states (other than AIDS) characterized by elevated
PP14 levels. Those in the art will recognize that all of
the various methods discussed above, as well as others
(such as use of blocking PP14 peptides or blocking anti-
idiotypic antibody mimics of PP14), achieve the same endas one another, that is, the prevention of PP14-induced
immunosuppression, by in some way affecting the end-
function or production of PP14 in the body.
The invention also features therapeutic methods tar-
geted at various ;mmllnological diseases. Unlike diseasesthat are the subject of PP14 blockade therapy, in which
there is a need for reversal of immunosuppression, these
other immunological diseases require exogenous immunosupp-
ressive agents. The PP14.2 polypeptide isoform, according
to the present invention, provides a distinct immunosupp-
ressive agent for pharmaceutical use which can be used
independently of (or ~ointly with) PP14.1, or other immu-
W094/07366 ~ PCT/US93/09216 -
7 ~ 2
nosuppressive polypeptides. PP14 therapeutic preparations
having the hematopoietic PP14.2 polypeptide have advan-
tages over PP14 therapeutic preparations consisting exclu-
sively of the PP14.1 polypeptide, with respect to both
stability (in blood and other tissue fluids) and thera-
peutic efficacy for diseases described by Bolton, suPra.
Thus, PP14 polypeptides can be used in known therapeutic
methods which target cytokine circuits in the ;mmllne sys-
tem, such as those predicated upon soluble interleukin-l
receptors for blocking interleukin-l:interleukin-l recep-
tor interaction, for treating acute inflammatory condi-
tions. PP14.2 can also be used to suppress natural killer
cell function.
The invention also features use of hematopoietic
cells as a source of PPl4 with ;mmllnosuppressive activity,
providing the possibility of isolating both PPl4.l and
PPl4.2 polypeptides concurrently. Furthermore, hematopoi-
etic cells, as well as other cells, can be used as cellu-
lar transfection targets for the production of recombinant
PP14.1 and PP14.2 polypeptides which will process and
secrete the desired PP14 isoform in an appropriate fash-
ion. Both intact versions of these polypeptides, as well
as polypeptide derivatives of these polypeptides having a
desired biological activity, can be readily generated. A
preferred composition includes a PP14 polypeptide sequence
linked to a glycophosphatidylinositol (GPI) moiety. This
membrane-anchored PPl4 variant is expressed at the cell
surface and can be preferentially cleaved from the surface
by virtue of the GPI membrane anchor.
The invention also concerns production of PPl4.1:
PPl4.2 heterodimers and PPl4.2 homodimers. Dimers com-
prising PPl4.2 polypeptide are more stable than PPl4.1:
PPl4.l homodimers, and provide a more optimal therapeutic
and diagnostic reagent. Standard cotransfection strate-
gies can be effectively used to produce the heterodimers.
Thus, in a first aspect, the invention features a
method for treating a patient suffering from a non-AIDS
W094/07366 PCT/US93J09216
~1~571i~
, . ;, .
immunosuppression condition, by administering to the
patient a reagent that specifically binds to at least one
isoform of PP14, or to a receptor for a PP14 isoform,
under physiological conditions, to thereby prevent or
5 reduce binding of PP14 to its receptor.
By "non-AIDS immunosuppression" is meant a condition
which is caused by (or adversely affected by, or related
to) an elevated level of a PP14 isoform. Provision of the
reagent will allow the reagent to bind to the PP14 iso-
form(s), or its receptors, and thus neutralize the PP14activity. Examples of such conditions include platelet
disorders, such as disseminated intravascular coagulation,
platelet-induced immunosuppression seco~ry to platelet
transfusion, and thrombocytosis, and leukemia.
In preferred embodiments, the reagent comprises, con-
sists of, or consists essentially of, a compound selected
from an antibody specific for PP14.1; an antibody (mono-
clonal or polyclonal, which may be a hllm~n; zed murine or
non-primate antibody) specific for PP14.2, and antibody
specific for PP14.1 and PP14.2, a receptor for a PP14
isoform, a portion of a receptor for a PP14 isoform, an
antibody to a PP14 receptor, a polypeptide portion of a
PP14 isoform, or any other reagent which competitively
inhibits binding of PP14 to a PP14 receptor in vivo or
ln vitro, e.a., a PP14 receptor antibody or anti-idiotypic
antibody. Such antibodies may be hllm~n;zed, that is the
framework may be derived from a human antibody and the
complementarily determining regions from another organism,
e.g., a mouse or non-human primate. The reagent prefer-
ably competitively inhibits binding of PP14 and itsreceptor.
In one example, the polypeptide portion of PP14
includes that portion of the polypeptide defined by amino
acids 33 through 54 of PP14.1 (i.e., specific to PP14.1
and not PP14. 2); the reagent may be (a) an anti-idiotypic
antibody mimic of PP14 which competes with PP14 for bind-
ing to (but does not-activate) a cellular receptor for
W094/07366 , PCT/US93/09216 -
214!~i7162 ' `
PPl4; (b) a soluble polypeptide derivative of a receptor
for PPl4 having the extracellular domain of a PPl4 recep-
tor free of the receptor's native transmembrane and cyto-
plasmic domains; or (c) a soluble polypeptide derivative
of a PPl4 receptor:immunoglobulin Fc (or other useful tar-
geting polypeptide) chimeric polypeptide.
In a second aspect, the invention features a method
for cloning a PP14 receptor, by determining an amino acid
sequence o the receptor and screening a library for a
clone of the receptor using an oligonucleotide probe cor-
responding to the amino acid sequence.
I,n a preferred embodiment, the method is a method for
cloning a complementary DNA corresponding to a PP14 recep-
tor including providing a ch;m~ric polypeptide having a
lS PP14 polypeptide linked to the sequence of an Fc region of
an immunoglobulin (or its equivalent); contacting the
ch;meric polypeptide with a cellular extract from a cell
expressing a PPl4 receptor under conditions suitable for
forming a complex of the ch;meric polypeptide bound to the
PPl4 receptor; precipitating complex by contacting protein
A or protein G (or its equivalent) conjugated to an insol-
uble matrix with the complex; recovering the PP14 receptor
from the matrix; determining the amino acid sequence of a
portion of the PP14 receptor; and screening a cDNA library
for a PP14 receptor CDNA using an oligonucleotide probe
corresponding to the amino acid sequence of the PP14
receptor.
In a third aspect, the invention features a method
for producing an antibody with specificity for a receptor
for a PP14 polypeptide, by immunizing a host with a por-
tion of a receptor for a PPl4 polypeptide.
In a fourth aspect, the invention features a method
for blocking immunosuppression in a patient'by administer-
ing to the patient a reagent that blocks transcription of
a PP14 gene and/or translation of a PPl4 transcript.
In preferred embodiments, an antisense oligonucleo-
tide, ribozyme, or a t-riplex-forming nucleic acid is used
w094/07366 PCT/US93/09216
~1~57~62-
~ 1 ' ' tl ; ;
to block transcription and/or translation of a PP14 poly-
peptide.
- In a fifth aspect, the invention features a method
for identifying a reagent that blocks transcription of
5 PP14, by screening a chosen compound for its capacity to
block a PP14 isoform promoter-driven transcription of a
reporter gene.
In a sixth aspect, the invention features a method
for identifying a patient with a platelet disorder, by
contacting a sample from the patient with an antibody or
other reagent with specificity for PP14.1 and/or PP14.2,
and determining the amount of reaction of the antibody
with the sample, compared to the amount of reaction
observed in a normal patient not having a disorder.
In a seventh aspect, the invention features a method
for preparing PP14.1-specific antibodies, by immunizing a
host with a polypeptide having an antigenic portion of the
polypeptide defined by amino acids 33 to 54 of PP14.1.
In related aspects, the invention features a method
for preparing PP14.2-specific antibodies, by ;mmlln;zing a
host with a polypeptide having a sequence of amino acids
overlapping the junctional site of amino acids 32-33 of
PP14.2; a pharmaceutical or other composition including an
antibody specific for PP14.1 or PP14.2 in a pharmaceutic-
ally acceptable buffer; a pharmaceutical or other composi-
tion, including a portion of a PP14 polypeptide which com-
petitively inhibits the binding of a native PP14 polypep-
tide to its receptor in a pharmaceutically acceptable buf-
fer; a pharmaceutical or other composition, including an
anti-idiotypic antibody mimic of PP14 which competes for
binding to, but does not activate, a cellular receptor for
PP14; a pharmaceutical or other composition including a
soluble polypeptide derivative of a receptor for PP14 hav-
ing the extracellular domain of a PP14 receptor free of
the receptor's native transmembrane and cytoplasmic
domains; and a pharmaceutical or other composition includ-
ing a Ppl4-binding portion of an extracellular domain of
WO 94/07366 r .- i~ .. PCI'/US93/09216--
2 ~ ~ ~ 7 ~ 2
a receptor for PP14 linked to a Fc domain of an immuno-
globulin heavy chain.
By "pharmaceutical" is meant a composition containing
as its active reagent at least the noted compound in a
buffer which is suitable for administration to a human or
other ~n;m~l, The term is used in its art-recognized
m~nner/ and includes those standard reagents known in the
art.
In another related aspect, the invention features a
method for treating a patient in need of ;mml~nosuppression
by administering to the patient a PP14.2 polypeptide.
In preferred embodiments, the patient suffers from an
autoimmune disease, rheumatoid arthritis, an allergic dis-
order, transplant rejection, or graft-versus-host disease.
In another aspect, the invention features a method
for preparing a PP14 polypeptide by isolating PP14 from a
hematopoietic cell, e.a., a phorbol myristate acetate-
induced cell, or a platelet; and the invention features
the resulting purified PP14 polypeptide PP14.1, or PP14.2.
In related aspects, the invention features a method
for preparing a PP14.2 polypeptide by introducing a trans-
criptional cassette having a promoter transcriptionally
linked to a portion of a coding sequence for PP14.2; a
composition having a transcriptional cassette having a
eukaryotic or prokaryotic promoter linked to a portion of
a coding sequence for PP14.2; and a composition including
a PP14 receptor-binding portion of a PP14 polypeptide
linked to a glycosylphosphatidylinositol (GPI) moiety or
its equivalent cell surface binding portion.
In still another related aspect, the invention fea-
tures a method for producing a PP14 polypeptide, by trans-
fecting a cell with a transcriptional cassette including
nucleic acid encoding a portion of a PP14 polypeptide
linked to a cell targeting signal sequence (e.a., glyco-
sylphosphatidylinositol (GPI)), to express a PP14 poly-
peptide linked to the signal sequence at the cell surface;
and cleaving the signal sequence with a cleaving reagent,
W094/07366 214 a 7 6 2 PCT/US93/09216
thereby releasing the PP14 polypeptide into the medium for
subsequent recovery.
- The invention also features a composition having a
portion of a PP14 receptor-binding polypeptide linked to
5 a portion of a member of a specific-binding pair (e.q.,
streptavidin or avidin with a biotin-binding domain); and
a purified or recombinant PP14 receptor, PP14 receptor
antibody, anti-idiotypic antibody, and purified nucleic
acids encoding therefore; and a cell having a transcrip-
tional cassette including nucleic acid encoding a portion
of a PP14 polypeptide linked to a cell surface resident
specific signal sequence, such as a glycosylphosphatidyl-
inositol (GPI) signal sequence, to express a PP14 polypep-
tide linked to a GPI moiety at the cell surface.
By "purified" is meant that the composition is dif-
ferent from that naturally occurring in nature in that the
active ingredient is at higher purity relative to one or
more other compounds naturally associated with it. By
~'recombinant" is meant produced by genetic engineering
procedures, e.q., it is expressed from a cloned gene or
its equivalent.
The invention also features a composition including
a heteromultimer of PP14.1 and PP14. 2 polypeptide chains,
or a homomultimer of PP14.2 polypeptide ch~; n~, and a
method for producing such a heteromultimer by cotransfect-
ing PP14.1 and PP14.2 transcriptional cassettes into a
cell.
In other aspects, the invention features a method for
suppressing natural killer cell activity by contacting
such cells with a PP14.2 polypeptide; a PP14 polypeptide
modified to target a cell surface (e.g., an antigen-
presenting cell), e.q., by covalent bonding of a polypep-
tide moiety, such as GPI or its equivalent; and a method
for coating an antigen-presenting cell by use of such a
targeted PP14 polypeptide. Specifically, a PP14 -GPI chim-
eric polypeptide will target the cell surface of an anti-
gen-presenting cell, or a PP14-streptavidin polypeptide
W O 94/07366 P~r/US93/09216 -
21~7~
12
will target a cell coated with biotin. Such cells are
useful for therapeutic or diagnostic procedures based upon
detection of or binding to PP14, as discussed herein.
Other features and advantages of the invention will
be apparent from the following description of the pre-
ferred embodiments of the invention, and from the claims.
~escri~tion of the Preferred Embodiments
The drawings will first briefly be described.
Drawinqs
Fig. 1 is a RNA transfer blot demonstrating that PP14
mRNA expression is induction- and lineage-specific in leu-
kemic cells differentiating along the megakaryocytic line-
age. Total RNA from lln;n~llced and induced K562, HL-60,
U937, and PLB-985 cell lines were probed with a purified
PP14 probe. K562 cells were induced along the megakaryo-
cytic lineage for 0, 24, 48, and 72h with PMA (lOnM), or
along the erythroid lineage for 0, 24, 48, and 72h with
hemin (50~M). HL-60 cells were induced along the macro-
phage lineage for O or 48h with PMA (lOnM), or along the
neutrophil lineage for O or 48h with DMSO (2~). U937
cells were induced along the macrophage lineage for O or
48h with PMA (lOnM), and PLB-985 were also induced along
the macrophage lineage for O or 48h with PMA (lOnM).
Peripheral blood mononuclear cells (PBL) stimulated with
anti-CD3 antibody for 3 days serves as a negative control.
The approximately 800bp band corresponding to PP14 mRNA is
indicated by an arrowhead.
Fig. 2 is a RNA transfer blot demonstrating the kine-
tics of PP14 expression after PMA-induction and showing
that PP14 mRNA is detectable after 6 hours of inducer
treatment. Total RNA from K562 cells induced with PMA
(lOnM) for 0, 0.5, 1, 2, 3, 6, 12, or 24 hours was iso-
lated and hybridized to a purified PP14 probe.
Fig. 3 is a copy of an autoradiogram showing PP14
immunoprecipitates from PMA-induced K562 cultured medium.
W094/07366 PCT/US93~09216
21~57;62
..
13
K562 cells were metabolically labeled by culturing in cys-
teine and methionine free-medium with the addition of
35S-cysteine and 35S-methionine for 24 or 48h. Cells were
removed and supernatants were immunoprecipitated with pro-
5 tein G and an anti-PP14 polyclonal or monoclonal antibody
(Ab). Bound product was run under reducing conditions on
a 12~ PAGE-SDS gel. The expected mobility of PP14.1 and
PP14.2 are noted. Upon longer exposure, a 28kD band
(representing PP14.1) is visible in the 24 hour lane as
well.
Fig. 4 is a two-way mixed lymphocyte culture showing
that a potent inhibitory effect of PMA-induced K562 condi-
tioned medium upon alloantigen-stimulated proliferation
can be neutralized with anti-PP14 antibodies. K562 cells
were induced with PMA (lOnM) for 24 or 48h to induce PP14
expression. Conditioned medium was added to the mixed
lymphocyte culture (MLC) at 50~ v/v ratio with or without
a rabbit polyclonal anti-PP14 antiserum (1:100 dilution).
The MLC was harvested at days 5, 6, or 7, and incorporated
3H-thymidine cpm were counted. Values represent the
average of triplicate cultures, and the experiment was
repeated two times with similar results. No effect on the
observed inhibition by conditioned medium was seen with
normal rabbit serum or anti-TGF~ Ab (data not shown).
Figs. 5a-5c show a schematic representation of PP14.1
and PP14.2 mRNAs and highlight the nucleotide and amino
acid sequence differences between the two. Fig. 5a:
Exon-l and exon-2, along with intron-l, of the PP14 gene
are schematized, with differential shading of the two
halves of exon-2; the upstream portion of exon-2 is elimi-
nated from PP14.2 mRNA by alternative splicing. The
dashed line indicates the stretch of sequence deleted by
such alternative splicing. Fig. 5b: The nucleotide
sequence of the portion of the PP14 gene involved in the
alternative splicing event is shown. The common splice
donor (SD) site and the two alternative splice acceptor
(SA) sites are highlighted by arrows, and the dinucleo-
W094/07366 PCT/US93/09216
., ~,; ..
7 ~ 2 14
tides at the splice sites are boxed. Fig. 5c: The aminoacid sequence of the portion of PP14.1 that is deleted in
the PP14.2 polypeptide is boxed. Numbers indicate the
boundaries of the deletion, based upon numbering of the
PP14.1 amino acid sequence.
Fig. 6 is a reverse transcriptase-polymerase chain
reaction (RT-PCR) analysis using PP14-specific primers
able to distinguish between the PP14.1 and PP14.2 mRNA
species in PMA-induced K562 cells of the megakaryocytic
lineage and placenta cells. PP14.2 mRNA is present in
significant amounts only in K562 cells, not in placenta
cells taken from 18, 28, 36 and 37 weeks old placenta.
Molecular weight numbers are included on the right-hand
side.
Fig. 7 is a RT-PCR analysis using PP14-specific pri-
mers showing that PP14 expression is restricted to tissues
of the hematopoietic and reproductive systems, and that
whereas both PP14.1 and PP14.2 isoforms are expressed at
significant, and apparently equimolar, levels in hemato-
poietic cells, the PP14.1 isoform. 2.5~g total RNA was
reverse transcribed using a 3'-end specific primer for
PP14. The resulting product was used directly in a PCR
reaction with a 5'-end specific primer for PP14. Lane 1:
nn;n~llced Ks62 cells. Lanes 2 and 3: PMA-induced K562
cells at 24 and 48h, respectively. Lane 4: brain.
Lane 5: spleen. Lane 6: small intestine. Lane7:
liver. Lane 8: kidney. Lane 9: brain meninges.
Lane 10: KM-102 bone marrow fibroblastoid stromal cells.
Lane 11: placenta at 18 weeks. Lane 12: placenta at
term. Lane 13: fibroblasts. Lane 14: primers alone.
Lane M - molecular weight markers. Arrows indicate the
relative portions of Ppl4.1 and PP14.2 as well as molecu-
lar weight markers.
Platelet-derived PP14 Isoforms and Dimers
Placental protein 14 (PP14), originally named after
the placental tissue it was thought to derive from, was
W094/07366 PCT/US93~09216
21~762
. . ,
later, shown to originate from associated endometrial
tissue (Vaisse et al., 9 DNA Cell. Biol. 401, 1990). Sub-
- sequent studies indicated that PP14 was present not only
in the endometrial decidua and serum of pregnant women,
- 5 but also in the s~m; n~l fluid of men. Applicant has dis-
covered that PP14 is also produced in cells outside of the
reproductive tract. PP14 mRNA and protein has been local-
ized not only to a human leukemic cell line induced to
differentiate along the megakaryocytic lineage, but also
to the end-cell of that lineage, namely, the platelet.
Cloning and hybridization analyses of PP14 mRNA, and
immunoprecipitation analyses of PP14 protein, have further
established that whereas endometrial PP14 is composed of
a single dominant species, hematopoietic PP14 ~as detected
in PMA-induced K562) is comprised of two codominant spe-
cies. One of the PP14 mRNA species comigrates with endo-
metrial PP14 mRNA, whereas the second one is shorter. The
latter contains an internal deletion that is predicted to
yield a 22 amino acid deletion in the encoded protein. To
simplify considerations of these two mRNA species and
their encoded protein products, the undeleted and deleted
variants have been here given the designations PP14.1 and
PP14.2, respectively.
The identification of a suitable splice acceptor con-
sensus sequence at the site within exon-2 where the dele-
tion of PP14. 2 terminates indicates that this variant
arises through alternative splicing of the PP14 gene.
The observation that PP14.1 and PP14. 2 polypeptides,
as well as their corresponding mRNAs, occur in equimolar
amounts in PMA-induced K562 cells is consistent with the
notion that in the case of cells of the megakaryocytic
lineage, the two form heterodimers. Previous work has
suggested that endometrial PP14, which consists of PP14.1,
consists of weak homodimers which are readily dissociated
by antibody. The appearance of both PP14.1 and PP14.2 in
;mmllnoprecipitates from célls of the megakaryocytic line-
age suggests either that the two form a relatively stable
W094/07366 PCT/US93J09216 -
214~7 ~2
16
heterodimer which is not dissociated by antibody, as is
the PP14.1:PP14.1 homodimer, or that the monoclonal anti-
body cross-reacts with the two isoforms. Since there is
evidence that PP14.1 may not function as a monomer and the
PP14.1 homodimer is easily dissociated, a PP14.1:PP14.2
heterodimer, as disclosed in this invention, provides an
alternative to the homodimer which is more stable for use
in ;mml~notherapeutic applications. Moreover, the data do
not rule out the presence of PP14.2 homodimers which also
may have higher stability than PP14.1 homodimers.
K562 (available from the American type culture col-
lection) human myeloid leukemic cells, can be induced to
differentiate along the megakaryocytic lineage by phorbol
12-myristate 13-acetate (PMA), and provides a model cellu-
lar system for addressing molecular issues in megakaryo-
cytopoiesis. E~uivalent cell lines can be isolated by
methods known in the art. Megakaryocytic markers known to
be expressed by such cells include platelet glycoprotein
IIIa (gpIIIa), platelet-derived growth factor (PDGF) alpha
and ~ ch~~n~, and transforming growth factor ~ (TGF~)
(Alitalo, 14 Leukemia Res. 501, 1990). In order to study
changes in the mRNA expression profile that accompany K562
differentiation along the megakaryocyte lineage, and to
clone and identify novel genes associated with megakaryo-
cyte differentiation and platelets, differential cDNAscreening was applied to clone mRNAs that are differen-
tially expressed post-PMA induction of the K562 cell line.
Exam~le 1: MYeloqenous Cell Differentiation
The human myelogenous leukemia cell line K562 (ATCC
243) was maintained in RPMI medium (Whittaker Bioproducts)
supplemented with 10~ heat-inactivated fetal calf serum
(FCS), lOmM HEPES, pH 7.2, 40~g/ml gentamycin, and 2mM
glutamine. Cells were grown at 37-C with 5~ CO2. The
phorbol ester used to cause differentiation was phorbol
12-myristate 13-acetate (PMA, Sigma) which was diluted in
dimethyl sulfoxide (DMSO) and stored at -20C at lmM-3mM
W094/07366 PCT/US93/09216
21~762
, ~ i.....
17
until use. Final concentrations of PMA ranged between
lOOnM and lOnM. To determine the effect of PMA on K562
- differentiation, K562 cells were cultured at 2x105 cells/ml
in tissue culture flasks. PMA was added directly to the
flask and cells were harvested 24, 48 or 72h after
addition.
Exam~le 2: Cloninq PP14 Genes
A phage cDNA library was constructed from a pool of
three groups of K562 cells alternatively induced with PMA
for 24, 48, and 72 hour. Such a pool was chosen to maxi-
mize chances of finding different genes activated through-
out the stochastic K562 differentiation program. Dupli-
cate lifts of the "induced" K562 cDNA library were differ-
entially screened with subtracted (induced minuslln;n~l~ced
K562) and nonsubtracted (lln;n~llced K562) single-stranded
probes.
Total RNA was isolated from cells using a standard
guanidinium isothiocyanate/cesium chloride method. Poly
(A)+ RNA was isolated using oligo (dT) cellulose. A cDNA
library was constructed using 1.5~g poly (A)+ RNA from
K562 cells treated for 24, 48 and 72h (4.5~g poly (A)+ RNA
total) with PMA (lOOnM). The library was constructed
using the Uni-ZAP XR cloning kit according to manufac-
turer's recomm~n~tions (Stratagene, La Jolla, CA).
To differentially screen the induced K562 cDNA lib-
rary, 60,000 individual primary clones were used to infect
PLK-F' bacteria (Stratagene). Duplicate membrane lifts
were taken using Stratagene Duralon U.V. membranes. The
DNA was denatured by incubating the membranes in 1.5M
NaCl-0.5M NaOH for 4 min., neutralized for 5 min. in a
solution of 1.5M NaCl-0.5M Tris-HCl (pH 8.0), and finally
rinsed in 0.2M Tris-Cl (pH 7.2)-2x SSC (lx SSC: 150mM
NaCl-15mM sodium citrate, pH 7.0). The DNA was W cross-
linked using the W Stratalinker 1800 (Stratagene) and
filters were allowed to dry.
W094/07366 . PCT/US93/09216 -
f; .,, ~ _~
s~2
Membranes for the primary screens were hybridized
with non-subtracted single-stranded cDNA probe from
untreated K562 cells (lln;n~llced) and compared to duplicate
lifts hybridized with subtracted single-stranded cDNA
probe from PMA-treated K562 cells (induced). Secondary
and tertiary screens were hybridized with single-stranded
cDNA probe from untreated K562 cells and compared to
duplicate lifts hybridized with non-subtracted single-
stranded cDNA probe from PMA-treated K562 cells. To gen-
erate the uninduced cDNA probe and the non-subtracted
induced cDNA probe, l~g poly (A)+ RNA from untreated K562
cells or from PMA-treated K562 cells, respectively, was
reverse transcribed for lh at 37 C in a buffer containing
0.lmM oligo (dT) 12-18 primer, 0.5mM each dATP, dGTP,dTTP,
with 100~Ci {~-32P}dCTP (Amersham, Arlington Heights, IL),
10U Superscript reverse transcriptase (Bethesda Research
Labs, Gaithersburg, MD), 50mM Tris-HCl (pH 8.3), 75mM KCl,
3mM MgCl2, and 0.lmM DTT. Unlabeled dCTP was added to
0.5mM for the last 15 min. Following this RT reaction,
RNA was hydrolyzed in 100mM NaOH for 30 min. at 65C and
the single-stranded cDNA passed over a Bio-Spin 30 column
(Bio-Rad, Richmond, CA) to eliminate free nucleotides.
To generate the subtracted, induced cDNA probe,
0.33~g poly(A)+ RNA from K562 cells treated with PMA for
24, 48, and 72 was pooled (1.0~g of poly (A)+ RNA total)
and reverse transcribed as described above, but with
250~Ci {~-32P)dCTP. Following RNA hydrolysis and cDNA
precipitation, the single-stranded cDNA was annealed with
a 30-fold excess of photobiotinylated poly (A)+ RNA from
untreated K562 cells and subtracted according to manufac-
turer's suggestions (Invitrogen, San Diego, CA).
Membranes were prehybridized at 42C overnight in 50
deionized formamide, 1~ sodium dodecyl sulfate (SDS), 10~
dextran sulfate (MW 300,000), lM NaCl, and 150~g/ml of
denatured sheared salmon sperm DNA (Sigma). Single-
stranded cDNA probe was added and incubated for 48h at
42C in a rotating oven (Hybaid, Middlesex, UK). Lifts
W O 94/07366 ~ 1 4 5 7 6 2 Pt~r/US93~09216
were washed 2x in 2x SSC, 1~ SDS at 22C for 10 min.; then
3x in O.lx SSC, 0.1~ SDS at 65C for 30 min. Membranes
- were exposed to Kodak AR film as necessary using inten-
sifying screens at -70C.
Approximately sixty-thousand cDNA clones were ana-
lyzed, and 127 putative positives were selected after the
first round of screening. Two of the 127 cDNA clones were
identified as PP14, after analysis and sequencing of all
the clones in a random fashion and subsequent sequence
comparisons with the GenhAnk sequence database (Pearson
et al., 85 Proc. Natl. Acad. Sci. USA 2444, 1988).
Exam~le 3: PP14 mRNA Expression
Verification of PP14 mRNA expression in the K562
line, and confirmation of its PMA-inducibility, was accom-
plished by RNA transfer blot analyses (see figures 1and 2).
Total RNA (lO~g) was isolated as described above,
heated to 65C for 15 min. in 50~ formamide, 6~ formalde-
hyde, lx EPPS (N-(2-hydroxyethyl)- piperazine-N'-3-pro-
panesulfonic acid) buffer (lx EPPS buffer: 20mM EPPS(pH 8.2), lOmM Na-acetate (pH 5.2), 2 mM EDTA) and sepa-
rated on 1.2~ agarose gels containing lx EPPS buffer and
6~ formaldehyde. The RNA was passively transferred to
Duralon W membranes (Stratagene) and W crosslinked.
Membranes were prehybridized as described above. Probe
was generated by random priming 20ng of purified PP14 DNA.
The DNA was denatured by heating to 100C for 10 min.,
then incubated for 30 min. at 37C in 50~Ci {32P}dCTP
(Amersham), 0.2mM each dGTP, dTTP, dATP and lOU Klenow
enzyme in a buffer containing random hexanucleotides
(Boehringer M~nnheim~ Indianapolis, IN). Probe was
hybridized overnight at 42C. Membranes were washed and
exposed as described above.
Specifically, cellular RNA from ln;n~llced K562 cells
or from K562 cells induced with PMA (lOnM) for 0.5, 1, 2,
- 3, 6, 12, and 24 hours was hybridized with a labeled PP14
W094/07366 2 1 ~ ~ 7 ~ 2 . PCT/USg3/092l6 -
cDNA probe. PP14 mRNA was undetectable in l~n1n~ced K562
cells and became apparent within 6 hours of PMA treatment
(Fig. 1). PP14 mRNA reached plateau levels at 24h and
remained relatively constant out to at least 72h (Figs. 1
and 2). A single broad PP14 mRNA band corresponding to a
length of approximately 0.8 kilobases was observed in all
cases, coinciding with the size reported for endometrial
PP14 mRNA.
Exam~le 4: Cell Specificity of PP14
In order to determine whether the PP14 induction
event is cell-, lineage-, and/or inducer-specific, several
leukemic lines were induced with alternative chemical
inducers (Fig. 2). Whereas PMA-induction of K562 along
the megakaryocytic lineage was associated with high levels
of PP14 mRNA accumulation, hemin (50uM)-induction of K562
cells along the erythroid lineage (Andersson et al., 278
Nature 364, 1979) ~e~o~trated no similar PP14 activation.
HL-60, another bipotential leukemic line, was tested for
PP14 inducibility. Neither PMA-induced HL-60 cells, which
differentiate along the myelo-monocytic lineage (Rovera et
al., 76 Proc. Natl. Acad. Sci. USA 2779, 1979) or DMSO-
treated HL-60 cells, which differentiate along the neutro-
philic lineage, displayed detectable PP14 expression.
Similarly, PMA induction of two other leukemic lines, U937
and PLB-985, along the myelo-monocytic lineage failed to
elicit PP14 mRNA expression. The intactness of the mRNA
used from these various leukemic lines was confirmed using
an actin probe (data not shown). Hence, PP14 does not
seem to simply be a phorbol ester-responsive gene. More-
over, these data suggest that PP14 is not promiscuouslyexpressed in leukemic cells. Instead, these data are
consistent with the notion that there is a specific asso-
ciation between PP14 and the megakaryocytic lineage of
PMA-induced K562 cells.
WO 94/07366 2 1 4 ~ 7 6 2 ~usg3~092l6
Exam~le 5: PP14 Protein Expression
To determine whether hematopoietic PP14 mRNA is in
fact translated into PP14 protein, ;mmllnoprecipitation
analyses were performed.
K562 cells were cultured at 5X106 cells/ml and treated
with PMA as described above. Cells were labeled with
0.5mCi 35S-cysteine and 35S-methionine (ICN Biomedicals,
Inc., Irvine, CA) in cysteine- and methionine-free media
supplemented with 10~ FCS which had been extensively dia-
lyzed in PBS. The next day, cells were collected by cen-
trifugation and conditioned media were saved. Cells were
lysed in 1~ triton X-100, 1~ bovine serum albumin (BSA)
and lmM phenylmethylsulfonylfluoride (Sigma). Conditioned
media were used directly. After pre-clearing cell lysates
or conditioned media with protein-G Sepharose (Pharmacia,
Piskataway, NJ), 100~1 of a 50~ slurry of protein-G Sepha-
rose was added with 2~1 of mouse monoclonal anti-PP14
antibody (105DHlFl; Riittinen et al., 136 J. Immunol.
Meth. 85, 1991). After incubating overnight at 4C with
gentle rotation the beads were pelleted by centrifugation
and washed as follows: 5x in 0.1~ triton X-100, 0.1~ BSA;
then lx in 0.01M Tris-HCl (pH 8.0), 0.14M NaCl, 0.025~
NaN3; and finally lx in 0.01M Tris-HCl (pH 7.5). Bound
protein was eluted by boiling and electrophoresed on a 3~
stacking, 15~ resolving SDS-PAGE gel. The gel was dried
and exposed to Kodak XR film at -70C with an intensifying
screen.
K562 cells were metabolically labeled with a combina-
tion of 35S-cysteine and 3~S-methionine. Conditioned medium
and cell lysis extracts were then immunoprecipitated using
a mouse monoclonal anti-PP14 antibody, or a polyclonal
antibody. Only conditioned medium from PMA-induced, but
not lln;nclllced, K562 cells ~emo~trated PP14 protein
(Fig. 3). Significantly, two protein species were noted
in the 28 kilodalton size range, with the upper one
comigrating with purified endometrial PP14 (data not
shown). The two PP14 isoforms were present in approxi-
W094/07366 ~ . PCT/US93/09216 -
2l4~7~2
mately equivalent amounts. No PP14 was seen in cell lysis
extracts of uninduced K562 cells and only small amounts of
PP14 were detected in the extracts of induced K562 cells,
consistent with PP14's being a secreted protein (data not
shown). Hence, PP14 protein parallels PP14 mRNA in its
PMA-inducibility in K562 cells.
Example 6: Bioloqical Effect of PP14
In order to assess the effect of conditioned medium
from K562 cells on peripheral blood lymphocyte prolifera-
tion, two-way mixed lymphocyte cultures (MLC) were set up
as follows. The blood from two unrelated donors was col-
lected in heparin (lOU/ml blood). Blood was diluted two
times in lx PBS; 0.3 vol. ficoll-paque (Pharmacia, Upp-
sala, Sweden) was under-layered and then centrifuged for
30 min. (1000 x g). The leukocyte layer was removed,
washed three times with lx PBS, and lymphocytes counted in
a hemocytometer. Cells were cultured in 96 well plates at
2 X105 cells/well in triplicate. Supernatants, where used,
were added to 50~ v/v in cell culture wells. Antibody was
added directly at a 1:100 dilution. Cells were cultured
for 5-7 days as described above. Twelve to fifteen hours
before harvesting 0.5~Ci [3H]thymidine (New England
Nuclear, Boston, MA) was added to each well. Cells were
harvested onto filters with a MiniMash II cell harvester,
the filters were dried, and counted in lml scintillation
cocktail with a Beckman (Fullerton, CA) LS3801 beta
counter.
In previous studies, endometrial PP14 has been shown
to be a potent suppressor of lymphocyte proliferation. In
order to determine whether the PP14 originating from hema-
topoietic cells shared this immunoregulatory function,
conditioned medium from PMA-induced K562 cells was added
to two-way mixed lymphocyte cultures, and the effect on
proliferation, as measured by3H-thymidine incorporation on
days 5, 6, and 7, was monitored. In these experiments,
conditioned medium was added to the MLC at a 1:2 ratio.
W094/07366 ~1~ 5 7 6 2 PCT/US93/09~16
In positive control MLCs, when lymphocytes from two
unrelated individuals were cocultured, a high degree of
proliferation, peaking on days 6 and 7, was detected (data
not shown). Addition of conditioned medium from 24h
(Fig. 4) or 48h (data not shown) PMA-induced K562, but not
from nn;n~l~ced K562, resulted in significant inhibition of
proliferation.
Exam~le 7: AntibodY blockin~ of PP14
In order to verify that the lymphocyte proliferative
inhibition effected by PMA-induced K562 conditioned medium
was indeed due to the presence of PP14 protein, antibody-
blocking experiments were performed. A rabbit polyclonal
antiserum against purified PP14 and which is known to
effectively immunoprecipitate PP14 protein was added to
the cocultures containing K562 conditioned medium. Anti-
PP14 antiserum (1:100 dilution) completely reversed the
anti-proliferative effect of the conditioned medium by day
7 for both 24h- (Fig.4) and 48h-induced (data not shown)
K562 conditioned medium. Hence, PP14 is clearly impli-
cated in the K562 ;mmnnosuppressive effect.
The anti-proliferative activity of PMA-induced K562
conditioned medium was unaffected by normal rabbit serum
or a neutralizing anti-transforming growth factor (TGF)
monoclonal antibody (R&D Systems, Minneapolis, MN) (data
not shown). Since TGF~ has been reported to be produced
by K562 cells, and furthermore, since TGF~ is known to
inhibit lymphocyte proliferation, one can presume that the
TGF~ that is present has not been cleaved into its active
immunomodulatory form. Significantly, PP14 does not
require proteolytic cleavage for functional activation.
Exam~le 8: mRNA sPecies of PP14 isoforms
While the ;mmllnoprecipitation analyses clearly dis-
tinguished between two PP14 polypeptide isoforms, RNA
transfer blot analyses did not clearly resolve distinct
PP14 mRNA species corresponding to the polypeptide iso-
W O 94/07366 . ~ P~r/US93~09216
24
forms. Re~x~mln~tion of weaker exposures of the RNAtransfer blots, however, suggested the presence of a tight
doublet hybridizing to the PP14 probe. In order to inves- -
tigate this in more detail, reverse transcriptase polymer-
ase chain reaction (RT-PCR) cloning and analyses were
performed.
For PCR analyses of PP14 mRNA, total RNA was reverse
transcribed as described above using a PP14 specific pri-
mer (5'-GGATCCCATGCTCCAAGGGTTTATTAATAACCTCTGC-3'; Seq.
I.D. No. 1). Resulting product was PCR amplified with a
5' primer (5'-GGTACCGCTCCAGAGCTCAGAGCCACCCACAG-3'; Seq.
I.D. No. 2) and a 3' (5'-GTGCAGAACGATCTCCAGGTTG-3'; Seq.
I.D. No. 3) primer in a buffer containing 25mM TAPS-HCl
tpH 9.3), 50mM KCl, 2mM MgCl2, lmM DTT, 200~M each dATP,
dCTP, dGTP, dTTP, and 2.5U Taq polymerase (Perkin Elmer,
Norwalk, CT). Amplification was performed on a PTC-100
thermal cycler (MJ Research, Watertown, MA) for 25 cycles
of 1 min. at 94C, 45 sec. at 65C, and 1 min. at 72C.
PCR products were analyzed on a 1.2~ agarose gel stained
with EtBr.
The initial two PP14 cDNA clones obtained through
differential cDNA screening (vide supra) proved to be only
partial clones encompassing the 3'-end of PP14 mRNA only.
PCR cloning of full-length PP14 cDNA was performed by
annealing a PP14 3'-end specific primer (5'- CATGCTCCAAGGG
TTTATTAATAACCTCTGC-3'; Seq. I.D. No. 4) and reverse tran-
scribing as described above. The resulting product was
used directly in a PCR (described above) using the 3'-end
specific primer and a 5' -end specific primer (5'-AGCTCAGA
GCCACCCACAGCCGCAG-3'; Seq. I.D. No 5). The PCR product
was gel purified and cloned into a T-vector.
A full-length PP14 cDNA was RT-PCR cloned from PMA-
induced K562 mRNA using primers based upon the 3'-end
sequence of K562 PP14 mRNA (identical to the endometrial
PP14 mRNA sequence) and the published 5'-end sequence of
endometrial PP14 mRNA. DNA sequencing was performed
using the Tabor and Ri-chardson sequencing method, and the
W094/07366 ~11 5 7 6 2 PCT/US93~09216
SEQUENASE~ sequencing kit as per the manufacture's recom-
mendations (United States Biochemicals, Cleveland).
Sequence analysis of the K562 PP14 clone revealed an
encoded PP14 protein identical to endometrial PP14, with
the significant difference of a 66 nucleotide long in-
frame deletion resulting in a predicted 22 amino acid
deletion in the encoded protein (Fig. 5). When the gen-
omic PP14 structure was analyzed, this deletion corres-
ponded to the upstream end of exon-2, and a consensus
splice acceptor site was detected at the appropriate site
within exon-2. This points to alternative splicing as the
operative mechanism in the generation of the shorter PP14
mRNA variant.
When the PCR product using PP14 end primers was size-
fractionated and visualized on agarose gels, two distinctPCR products were noted (Fig. 6). The two mRNA species
were designated PP14.1 and PP14.2, with the latter corres-
ponding to the smaller RNA species. The size difference
between these two PCR products was consistent with the 66
nucleotide deletion documented in the K562 PP14 mRNA that
had been PCR-cloned. Notably, only the PP14.1 mRNA var-
iant could be readily detected in endometrial tissue.
Exam~le 9: Detection of PP14 Proteins in Platelets
Immunoprecipitation from platelets was performed from
platelets isolated using a modified citrate buffer.
Briefly, whole blood was collected in a citrate/phosphate/
dextrose/adenine anticoagulant 1.4ml CPDA/lOml whole blood
(CPDA contains 15.8 mM citric acid, 91.3mM sodium citrate,
16.6mM dextrose, 1.7mM a~en;ne, and 16.6mM monobasic
sodium phosphate). Cells were spun at 200 x g, for 10
min. at 22C. The upper platelet rich plasma (PRP) was
removed and the platelets were washed two times in regular
ringers/citrate/dextrose, pH 6.5 (RCD, containing 71.9mM
NaCl, 0.7mM KCl, 0.6mM CaCl2, 0.8mM NaHCO3, 20mM trisodium
citrate, 27.8mM dextrose, 43ng/ml prostaglandin E1 (PGE
Sigma Chemical Co, St. Louis)). Between washes, platelets
W094/07366 PCT/USg3/09216 -
214~ 7 ~2~. e;
26
were spun at 1,100 x g for 15 min. at 22C. Reductive
methylation of platelet extracts was performed by resus-
pending the platelet pellet after the final spin in a
modified lysis buffer containing 50mM sodium phosphate
(pH 7.0), 1~ NP-40,150mM NaCl, 2~g/ml leupeptin, 2~g/ml
aprotinin, 20~g/ml PMSF. NaCNBrH3 (Sigma) was added to
50mM and [l4C]formaldehyde (Amersham) was added to lOmM.
The mixture was allowed to reductively methylate for lh at
37C. Following methylation the reaction was dialyzed
overnight in lx PBS, then again the next morning for lh in
lx PBS to remove rem~;n;ng NaCNBrH3 and [14C]formaldehyde.
After dialysis, the radioactive extracts were immunopre-
cipitated and analyzed as described above.
As stated above, the finding of PP14 in PMA-induced,
but not hemin-induced K562 leukemic cells, along with the
absence of PP14 in other PMA-induced leukemic lines,
together substantiated that PP14 expression in PMA-induced
K562 cells reflects an underlying association between PP14
and the platelet lineage. To confirm this point, immuno-
precipitation/SDS-PAGE analysis was performed using poly-
clonal anti-PP14 antibody and platelet extracts. Evidence
for the presence of the two PP14 polypeptides in the
platelet extract was obtained (data not shown).
Exam~le 10: Screeninq for PP14 by PCR
The finding of PP14 mRNA and protein in hematopoietic
cells of the megakaryocytic lineage prompted screening of
additional tissue sites outside of the reproductive tract
for the presence of PP14 mRNA. PCR analysis was used to
optimize sensitivity. Total cellular RNA, isolated from
autopsy tissues, was reverse-transcribed using PP14-
specific primer (Seq. I.D. No. 1) and amplified using the
same oligonucleotide as 3' primer, and a 5' primer (Seq.
I.D. No. 2) designed to allow resolution of the two PP14
mRNA variants. As seen in Fig. 7, PP14.1 and PP14.2 were
detected, and clearly resolved, in the PMA-induced K562
cells serving as controls (lanes 2 and 3). Moreover, the
W094/07366 PCT/US93~09216
21 45762
27
placental tissue (contaminated with PP14-containing con-
t~m; n~nt endometrial decidua) showed, as expected, only
the larger PP14.1 mRNA variant (lanes 11 and 12). In
contrast, the panel of other tissues ~x~m;ned were all
- 5 negative for PP14 transcript. These tissues included
brain (lane 4), spleen (lane 5), small intestine (lane 6),
liver (lane 7), kidney (lane 8), and meninges (lane 9).
Similarly, two fibroblastic cell lines, KM-102 (human bone
marrow stromal cells; lane 10) and dermal fibroblasts
(lane 11) were also negative. Hence, PP14 mRNA expression
seems to be restricted to the reproductive and hematopoi-
etic systems.
Methods
The presence of a constitutively active ;mmllnosupp-
ressive molecule in cells of the platelet lineage is ofconsiderable physiological significance, and indicates
previously unsuspected roles for PP14. These data indi-
cate that this potent ;mml~nosuppressive molecule is con-
centrated in tissue sites where coagulation occurs. Thus,
PP14 may play a role in the resolution of inflammatory
processes at wound healing sites. This discovery thus
shows a previously unknown critical molecular link between
the coagulation and immune systems.
The discovery that PP14 is produced by cells of the
platelet lineage leads to novel therapies for reversing
platelet-driven immunosuppression. PP14 was previously
thought to be a beneficial molecule, in its suggested
physiological role as a blocker of alloresponses in the
female and male reproductive tracts. The present inven-
tion discloses that PP14 can also be a deleterious mole-
cule, in its pathophysiological role as a potent general
immunosuppressive agent released by platelets in certain
clinical settings, such as coagulopathies. This discovery
provides the first motivation for developing methods for
blocking PP14 and its immunosuppressive effects. Methods
are disclosed for carrying out this PP14 blockade. These
W 0 94/07366 ~ .. P~r/US93/09216 -
2~ ~a~;2
28
methods involve both methods for interfering with PP14
protein interaction with a PP14 receptor, and methods for
preventing PP14 transcription and/or translation (i.e.,
PP14 blockade therapies).
The survey of human leukemic cell lines reported in
this study indicates that PP14 is not promiscuously
expressed in leukemic lines. In fact, even in K562, it is
only expressed following chemical induction with one par-
ticular chemical inducer. Nonetheless, it is possible
that under ln vivo conditions, myeloid leukemic cells that
share K562's megakaryocytic differentiative potential, may
be triggered into a more differentiated state wherein they
can express PP14. This could occur spontaneously or in
response to therapeutic agents. Under such conditions,
PP14, by virtue of its potent immunosuppressive function,
is expected to play a pathogenic role in blocking effec-
tive anti-tumor immune responses. As was shown here for
K562 leukemic cells, the secreted PP14 is in its biologic-
ally active form. The present invention provides methods
for detecting PP14.2 in serum of patients. Such methods
can readily be applied to patients with leukemia or
related diseases in order to determine which patients are
candidates for PP14 blockade therapy.
There follow examples of the therapeutic and diagnos-
tic methods of the invention below. These examples are
not limiting in the invention and those of ordinary skill
in the art will recognize that many equivalent methods and
reagents can be discovered within the scope of the claims.
PP14 Blockade Therapy
Patients with elevated levels of one or both of the
PP14 polypeptide isoforms in their serum are candidates
for PP14 blockade therapy. Such elevated levels of PP14
may occur in a variety of disease conditions where plate-
lets release excess PP14 into the bloodstream. For exam-
ple, sepsis is often associated with disseminated intra-
vascular coagulation, a condition which leads to the
W094/07366 ~1~3 7 6 2 PCT/US93/09216
release of platelet contents into blood as the platelets
coagulate. PP14 that is released in this clinical setting
contributes to generalized immunosuppression which, in
turn, further aggravates the sepsis. Hence, PP14 blockade
serves to interfere with this pathogenic cycle and aid
recovery.
The first step is to identify a patient in need of
PP14 blockade therapy. Preferred diagnostic methods for
accomplishing this are described below. Generally, those
individuals having higher than normal tissue or serum
levels of PP14.1 or PP14.2 or both are treated by methods
of this invention. Such levels can be determined by use
of antibody-based assays.
The present invention discloses that PP14 blockade
can have therapeutic benefit. Methods for blocking PP14
action include standard approaches for blocking proteins
to achieve therapeutic endpoints. For example, monoclonal
and/or polyclonal antibodies can be used with specificity
for the two isoforms of PP14 as PP14 blocking agents.
Antibodies with specificity for PP14 have previously been
described, but, the only suggested therapeutic benefit was
for treatment of an immune system disorder, the only given
example of which was AIDS, and in the absence of knowledge
about the two PP14 isoforms, the isoform-specificity of
the antibodies r~;n~d unknown. Knowledge of the PP14.2
isoform in the present invention permits the development
of antibodies with PP14.1 or PP14.2-specificity and their
therapeutic use. Thus, this invention features methods to
treat all other non-AIDS- diseases (or even other non-
immune system disorders) characterized by excess amountsof PP14 in a patient or tissue. It also features use of
PP14.2-specific antibodies for treatment of all diseases
associated with elevated PP14 levels.
A preferred strategy for producing anti-PP14.1-speci-
fic antibodies is to ;mmlln;ze with a peptide largely lim-
ited to the amino acid sequence within the PP14.1-specific
sequence that is absent from PP14.2. Alternatively, pep-
W094/07366 2 1 ~ ~ ~ 6 2; PCT/US93/09216 _
tides overlapping the junctions between this sequence andthe rest of PPl4.l can be used, as well as other PPl4.l
sequences. In the latter case, antibodies with cross-
reactivity to both PPl4.l and PPl4.2 can be eliminated by
conventional approaches.
A preferred strategy for producing anti-PPl4.2-speci-
fic antibodies is to immunize with a peptide spanning the
junction corresponding to the site where the PP14.1
sequence insertion is present. Standard peptide ;mml]n;za
tion protocols can be employed.
PPl4 peptides having only a portion of the native
PPl4 polypeptides can be used to competitively inhibit
interaction of PPl4 with a receptor for PP14 (a PPl4
receptor) to thereby interfere with the nonspecific immu-
nosuppression mediated by the native PPl4 polypeptide.Straightforward in vitro experiments based upon competi-
tive peptide inhibition and/or site-specific mutagenesis
can be used to localize the appropriate PPl4 subsequences
for effecting such blockade, and can be carried out based
upon standard protocols by those familiar with the art.
Preferred subsequences are generally those coinciding with
hydrophilic amino acid sequence stretches. Moreover,
strategies for optimizing in vivo dosing schedules for
patients to be treated are well-known.
Similarly, anti-idiotypic antibodies that mimic PPl4
in its capacity to bind to its receptor can be used for
competitive blockade of a PP14 receptor. Methods for
preparing such anti-idiotypic antibodies and their use are
well-known to those familiar with the art, and are parti-
cularly well-documented in the infectious disease
literature.
The PPl4 receptor, instead of PP14 itself, can also
be focussed upon for purpoæes of blocking the PPl4: PP14
receptor interaction. The purification and cloning of
receptors, in situations where the ligand is available in
purified form, has become a straightforward exercise for
those familiar with the art. Prior to this invention, the
W094/07366 ~ 7 ~ PCT/US93/09216
art provided no compelling therapeutic reason to purify
and clone the PPl4 receptor. Moreover, in the absence of
prior knowledge of the existence of two distinct PPl4
isoforms, the issue of isoform-specificity for PPl4 recep-
tors could not be addressed. The present invention, byteaching that PPl4 is not simply a byproduct of pregnancy
or restricted to a distal site of the male reproductive
tract, but instead can be derived from platelets which
contribute to pathophysiology, provides a compelling rea-
son to purify and clone receptors for PPl4. Structuralcharacterization of the PPl4 receptor allows, in turn, the
generation of agents for blocking PPl4:PPl4 receptor
interaction.
A preferred method for isolation and cloning the PPl4
receptor involves generation and use of a PPl4:;mm1~noglob-
ulin Fc ch;me~ic polypeptide. This chimeric polypeptide
has the complete sequence of PPl4, or a functional poly-
peptide derivative thereof, linked to the Fc region of
imm1~noglobulin Gl (IgGl). The latter serveæ as a useful
tag for detecting and isolating the ~h;meriC polypeptide,
since the Fc region of ;mm1~noglobulin binds well to pro-
tein A or protein G. Analogous ligand:Fc ~h;m~ric poly-
peptides have been used by a number of investigative
groups to isolate specific receptors. Hematopoietic
cells, such as monocytes and lymphocytes, respond to PPl4
and hence the mononuclear cell pool is a suitable cellular
source from which to isolate PPl4 receptors. A detergent
extract is prepared from said cells, and the PP14:;mm1~no-
globulin Fc ch;me~a, produced by recombinant DNA methods
in standard NOS cells or their equivalent, is added to the
extract. Protein A-sepharose chromatography is performed
to isolate a complex in which the PPl4 receptor complexed
to the PPl4:;mml~noglobulin Fc chimera. pH elution is used
to recover the purified PPl4 receptor. Amino acid
sequence for the amino-terminus, a~ well as for defined
sub-peptides generated by peptidase cleavage, is deter-
mined by conventional amino acid sequencing methods.
W094/07366 2 ~ ~ 5 7 ; ~ ~ r PCT/US93/09216 -
Based upon this amino acid sequence, degenerate oligo-
nucleotides encoding this amino acid sequence are syn-
thesized using an oligonucleotide synthesizer. These
degenerate oligonucleotides are then labeled and used as
probes to screen a cDNA library from the same cells from
which the receptor was originally purified. Hence, purif-
ication and cloning of PP14 receptors can be carried out
in a straightforward way using conventional methods.
While the example of using PP14: Fc chimeras as a
receptor trap is provided here, there are multiple alter-
native approaches that can be employed to the same end.
Alternative polypeptide tags can be appended to PP14 for
detection and isolation purposes. In addition, the
sequence of steps outlined here can be repeated for both
PPl4.l and PP14.2, as well as heterodimeric PP14, poly-
peptides in order to isolate and clone isoform-specific
receptors. Moreover, native PPl4 can be used in unmodi-
fied form for binding to the receptor, and anti-PPl4
antibodies can then be used to recover the PPl4:PPl4
receptor complex.
According to one therapeutic method for effecting
PPl4 blockade via the PPl4 receptor, anti-PPl4 receptor
antibodies are infused into a patient in need of PPl4-
induced generalized or localized ;mml~nosuppression. Anti-
PPl4 receptor antibodies can be generated using PPl4
receptor, recombinant PPl4 receptor or PP14 receptor pep-
tides as immunogens. Methods for preparing monoclonal
antibodies useful for human therapeutics are well-
described in the scientific literature.
According to another therapeutic method for effecting
PPl4 blockade via the PPl4 receptor, a soluble derivative
of the PP14 receptor is infused into a patient in need of
PPl4-induced generalized ;mmtlnosuppression. This soluble
derivative of the PPl4 receptor can be readily generated
through standard recombinant DNA methods. For example, a
site-specific mutagenesis method can be employed to intro-
duce a stop codon at the carboxy-terminus of the PPl4
~ W094/07366 2 I ~ 5 7 6 2 PCT/US93~09216
receptor extracellular domain. This mutagenized coding
sequence for a soluble PP14 receptor can be subcloned into
any one of a number of available eukaryotic or prokaryotic
expression vectors for quantitative production of this
molecule. An alternative therapeutic soluble derivative
of PP14 receptor comprises the extracellular domain of the
PP14 receptor linked to the Fc domain of immunoglobulin G.
This latter molecule has the advantage of being a more
stable molecule in vivo, as has been shown for other
;mmllnoglobulin Fc ch;meriC polypeptides, such as CD4:
;mml~noglobulin Fc.
Therapy Based Upon Inhibition of PPl4 Production
In addition to therapeutic methods based upon binding
of the PP14 protein in the blood and tissues of a patient,
and thereby interfering with its ;mmllnosuppressive activ-
ity, therapeutic methods can also be used which are based
upon reducing levels of PP14 production. Such a reduction
can be at transcriptional and post-transcriptional levels.
Thus, genetic therapeutic agents that interfere with
PP14 expression by cells of the platelet lineage can be
used. A preferred agent for reducing PP14 production is
an antisense PP14 oligonucleotide that has been covalently
derivatized through known methods to enhance n vivo sta-
bility and cell membrane penetration. Isoform-specific
antisense PP14 oligonucleotides can readily be designed
based upon the PP14.1 and PP14. 2 nucleotide sequence
information disclosed herein, along with general guide-
lines that are well-known to those familiar with the art
for optimizing functional antisense oligonucleotides. In
the case of PP14.1, the antisense oligonucleotide can be
directed against the 66 nucleotide stretch that is absent
in PP14. 2. In the case of PP14. 2, the antisense oligo-
nucleotide can span the junctional site that overlaps the
66 nucleotide deletion of PP14.1 sequence such that the
oligonucleotide binds only to this junction. Other regu-
latory polynucleotides can be readily designed, such as
W094/07366 PCT/US93/09216 ~
6 2
.
34
ones that incorporate ribozymes or that function as tri-
plex-forming regulatory elements. There are well-estab-
lished procedures for developing such agents and for
screening their efficacies and toxicities.
Other therapeutic methods can be developed that are
based upon chemical agents that function as PP14 gene-
specific transcriptional inhibitors. There are currently
well-described methods for screening banks of chemicals
for ones that will block transcription from a specific
gene promoter. One experimental approach involves gener-
ating a reporter gene construct including the upstream
sequence of the PP14 gene, with the PP14 promoter element,
linked to a gene sequence encoding a readily detectable
promoter, such as luciferase, beta-galactosidase, or
chloramphenicol acetyltransferase. This transcriptional
cassette is stably transfected into a target cell that is
capable of supporting active transcription from the PP14
promoter. K562 cells are one example of a suitable cell
line for this purpose. Alternative cell lines with mega-
karyocytic differentiation potential, as well as endo-
metrial and testicular lines, can be readily identified.
In the case of K562 cells transfected with the ch;meric
reporter, the cells are distributed into multi-well tissue
culture plates, and chemicals to be screened are added to
individual wells, along with PMA to activate the PP14 pro-
moter. Wells containing cells failing to express the
reporter are identified. This represents a relatively
rapid method for screening large numbers of chemicals for
relevant drug candidates. Once a candidate is identified,
procedures well-established in the field of pharmacology
are used to study efficacy and toxicity and to optimize
dosage regimens.
Treatment of Leukemia
PP14 characterizes cells of the platelet lineage, and
leukemic cells corresponding to this lineage also express
PP14. Leukemic cells producing this potent immunosuppres-
W094/07366 ~ PCT/US93/09216
7 6 2
sive molecule in vivo may be protected from effectiveanti-leukemic T-cell responses. Moreover, PP14 derived
from these cells will lead to generalized immunosuppres-
sion in the patient, rendering the patient susceptible to
5 other diseases, including infectious diseases. Hence, the
discovery that certain leukemic cells produce PP14 pro-
vides motivation for screening patients with leukemia for
PP14 expression in blood and leukemic cells. Those
patients who are PP14-positive, can be treated by PP14
blockade therapy, by one of the methods discussed above.
Patients with megakaryocytic leukemia or chronic myeloid
leukemia cells with megakaryocytic differentiation poten-
tial, are particularly suitable candidates for this mode
of therapy.
The present invention discloses the existence of the
PP14.2 isoform. This disclosure is significant in that it
teaches those skilled in the art how to effectively block
platelet-derived PP14, in that both isoforms must be tar-
geted with antibody, PP14 peptide, or PP14 receptor deriv-
atized therapeutic compositions. Cloning of the PP14.2
isoforms has provided critical sequence information which
allows the generation of PP14 isoform-specific reagents.
Such reagents can be used either in combination or inde-
pendently. For example, therapeutic benefit may be
obtained in a pregnant patient who is in need of ameliora-
tion of platelet-induced generalized ;mml~nosuppression, as
for example in the clinical context of a pregnancy-associ-
ated coagulopathy. Since in such a patient, PP14.1 block-
ade is undesirable due to potential adverse effects upon
the developing fetus, selective PP14. 2 blockade is prefer-
able. A therapeutic benefit will be derived by decreasing
the PP14 load in the patient, even if there is residual
PP14.1 from endometrium and platelets in the patient.
PP14 Diaqnostic Assays
The disclosure of PP14 in cells of the platelet lin-
eage has diagnostic implications. Monoclonal and/or poly-
W094/07366 ~ PCT/US93/09216 -
.... .
2 ~
36
clonal antibodies, prepared by the methods described
above, permit the simple development of ELISA assays for
measuring PP14 expression in serum. PP14.1- and/or
PP14.2-specific antibodies can be used for this purpose.
A sensitive ELISA can be used for PP14 detection in serum
samples. Two anti-PP14 antibodies with specificities for
distinct, non-overlapping portions of PP14 protein, can be
combined in a conventional double-antibody sandwich ELISA.
The present invention teaches that two isoforms of PP14
are produced by platelets, and hence, informs those
skilled in the art how to develop suitable diagnostic
assays. The ability to distinguish between the PP14.1 and
PP14.2 polypeptide isoforms is of particular utility when
diagnosing platelet disorders in pregnant women, since the
PP14 polypeptides deriving from platelet and endometrial
cells can be resolved.
Not only is this assay useful for precisely defining
candidates for PP14 blockade therapy, but in addition,
this assay can serve as a sensitive diagnostic tool for
determining whether there is in fact a coagulopathy in a
patient where the diagnosis is uncertain. According to
the latter, PP14 detection can serve as a diagnostic test
for clinical disorders such as disseminated intravascular
coagulation which are often difficult to diagnose. Thus,
PP14.2 polypeptide is a useful diagnostic marker for
platelets.
Immunosu~ression Thera~Y with PP14.2 Polype~tide
This invention provides a novel immunosuppressive
agent that can be used for the treatment of patients with
diseases where immunosuppression is the desired endpoint.
Such diseases include, but are not limited to, autoimmune
diseases, rheumatoid arthritis, allergic disorders such as
allergic dermatitis, transplant rejection, and graft-
versus-host disease in the context of bone marrow trans-
plantation. The present invention provides PP14.2 as an
W O 94/07366 Pt~r/US93/09216
2145762
;
37
alternative to PP14.1 for achieving immunosuppression inpatients in need of this.
A patient is identified who is in need of immunosupp-
ression. A composition including a PP14 polypeptide, or
a functional polypeptide derivative thereof, is adminis-
tered to the patient. The route of administration and the
precise components of the pharmaceutical preparation are
dictated by the particular disease entity being treated.
For example, in the treatment of a systemic autoimmune
disease, intravascular or intramuscular administration is
preferable. In contrast, topical application is prefer-
able for allergic dermatitis, whereas intra-articular
injection may be necessary in the context of rheumatoid
arthritis. Methods for optimizing clinical protocols
involving therapeutic polypeptides are well-established,
and the methods to be used for PP14 polypeptides parallel
these. For example, an amount of PP14 between 1 and 1000
for kg ~n;m~l per day is suitable. PP14.1 and/or PP14.2
serum levels in treated patients can be monitored in order
to provide one measure of therapeutic efficacy.
Methods for PP14 Poly~eptide Production
The present invention discloses a significant set of
new potential sources for both small and large scale pro-
duction of not only the novel PP14.2 isoform, but also the
PP14.1 isoform. Previously there have been no natural
cellular sources from which human PP14 polypeptide might
be readily derived. Clearly, the availability of first
and second trimester endometrium from cases of abortion is
limited by several factors. According to the present
invention, platelets can be used as a source for native
PP14. The finding of PP14 polypeptide in PMA-induced K562
cells, which proliferate rapidly in cell culture, provides
another potential source for native (i.e., non-recombi-
nant) PP14. Significantly, both PP14 isoforms can be
obtained from these sources, and these further serve as a
source for functional dimeric molecules. Moreover, other
W094/07366 PCT/US93~09216 -
2~4~7 ~2
38
cell lines corresponding to the megakaryocytic lineage are
alternative sources for native PP14.
Alternatively, expression constructs for PP14.2 can
be introduced into K562 or other megakaryocytic lines for
purposes of PP14 overexpression. Recombinant monomeric
PP14. 2, homodimeric PP14.2, and heterodimeric PP14.1:
PP14.2 can all be produced in this way. Such cells pro-
vide optimal cellular backgrounds in which proper post-
transcriptional processing of PP14 mRNA and protein can
take place. Methods for gene transfer and the expression
and purification of recombinant proteins are well-
established in the art.
Alternative methods can be devised for producing
soluble PP14.1 and PP14.2 polypeptides. One preferred
method involves the expression of ch;meric PP14.1:GPI and
PP14.2:GPI polypeptides, including monomeric and hetero-
dimeric forms of these polypeptides, on the surfaces of
adherent cells and the recovery of soluble PP14.1 or
PP14.2 polypeptides by cleavage of the GPI membrane
anchor. This type of method has been used successfully
for the production of other soluble molecules and offers
the particular advantage of being compatible with con-
tinuous-flow cell culture systems.
Other embodiments are within the following claims.
W094/07366 PCT/US93/09216
~5762
39
(1) GENERAL INFORMATION:
(i) APPLICANT: MARK L. TYKOCINSKI
(ii) TITLE OF INVENTION: PP14-BASED THERAPY
(iii) NUMBER OF SEQUENCES: 5
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storage
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(Version 5.0)
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(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
Prior applications total, including application
described below:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Warburg, Richard J.
(B) REGISTRATION NUMBER: 32,327
(C) REFERENCE/DOCKET NUMBER: 199/187
(ix) TELECOMMUNICATION INFORMATION:
(A) TEL~E~O~: (213) 489-1600
(B) TELEFAX: (213) 955-0440
(C) TELEX: 67-3510
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37
(B) TYPE: NUCLEIC ACID
W094/07366 ~ - .Y . PCT/US93/09216 -
21~5~
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:
GGATCCCATG CTCCAAGGGT TTATTAATAA CCTCTGC 37
(3) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTX: 32
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: Single
(D) TOPOhOGY: T,; neAr
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
GGTACCGCTC CAGAGCTCAG AGCCACCCAC AG 32
(4) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: Single
(D) TOPOLOGY: T.i neAr
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
20 GTGCAGAACG ATCTCCAGGT TG 22
(5) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: Single
(D) TOPOLOGY: T,; neAr
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
CATGCTCCAAG ~lllATTAA TAACCTCTGC 30
(6) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
AGCTCAGAGC CACCCACAGC CGCAG 25
