Note: Descriptions are shown in the official language in which they were submitted.
W095/18858
2 1 (8 `t 0 2 PCT/US94/14553
THROMBOPOIETIN
FIELD OF THE INVENTION
This invention relates to the isolation, purification and recombinant or
chemical synthesis of proteins that influence survival, proliferation,
differentiation
or maturation of hematopoietic cells, especially platelet progenitor cells.
This
invention specifically relates to the cloning and expression of nucleic acids
encoding a
protein ligand capable of binding to and activating mpl, a member of the
cytokine
receptor superfamily. This invention further relates to the use of these
proteins alone
or in combination with other cytokines to treat immune or hematopoietic
disorders
including thrombocytopenia.
BACKGROUND OF THE INVENTION
I . The Hematopoietic System
The hematopoietic system produces the mature highly specialized blood cells
known to be necessary for survival of all mammals. These mature cells include;
erythrocytes, specialized to transport oxygen and carbon dioxide, T- and B-
lymphocytes, responsible for cell- and antibody-mediated immune responses,
platelets or thrombocytes, specialized to form blood clots, and granulocytes
and
macrophages, specialized as scavengers and as accessory cells to combat
infection.
Granulocytes are further subdivided into; neutrophils, eosinophils, basophils
and mast
cells, specialized cell types having discrete functions. Remarkably, all of
these
specialized mature blood cells are derived from a single common primitive cell
type,
referred to as the pluripotent (or totipotent) stem cell, found primarily in
bone
marrow (Dexter et at, Ann. Rev. Cell Biol., 3:423-441 [1987]).
The mature highly specialized blood cells must be produced in large numbers
continuously throughout the life of a mammal. The vast majority of these
specialized
blood cells are destined to remain functionally active for only a few hours to
weeks
(Cronkite et at, Blood Cells, 2:263-284 [1976]). Thus, continuous renewal of
the
mature blood cells, the primitive stem cells themselves, as well as any
intermediate
or lineage-committed progenitor cell lines lying between the primitive and
mature
cells, is necessary in order to maintain the normal steady state blood cell
needs of the
mammal.
At the heart of the hematopoietic system lies the pluripotent stem cell(s).
These cells are relatively few in number and undergo self-renewal by
proliferation to
produce daughter stem cells or are transformed, in a series of differentiation
steps,
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WO 95/18858 2178482 PCT/US94/14553
into increasingly mature lineage-restricted progenitor cells, ultimately
forming the
highly specialized mature blood cell(s).
For example, certain multipotent progenitor cells, referred to as CFC-Mix,
derived from stem cells undergo proliferation (self-renewal) and development
to
produce colonies containing all the different myeloid cells; erythrocytes,
neutrophils,
megakaryocytes (predecessors of platelets), macrophages, basophils,
eosinophils, and
mast cells. Other progenitor cells of the lymphoid lineage undergo
proliferation and
development into T-cells and B-cells.
Additionally, between the CFC-Mix progenitor cells and myeloid cells lie
another rank of progenitor cells of intermediate commitment to their progeny.
These
lineage-restricted progenitor cells are classified on the basis of the progeny
they
produce. Thus, the known immediate predecessors of the myeloid cells are:
erythroid
colony-forming units (CFU-E) for erythrocytes, granulocyte/macrophage colony-
forming cells (GM-CFC) for neutrophils and macrophages, megakaryocyte colony-
1 5 forming cells (Meg-CFC) for megakaryocytes, eosinophil colony-forming
cells (Eos-
CFC) for eosinophils, and basophil colony-forming cells (Bas-CFC) for mast
cells.
Other intermediate predecessor cells between the pluripotent stem cells and
mature
blood cells are known (see below) or will likely be discovered having varying
degrees
of lineage-restriction and self-renewal capacity.
The underlying principal of the normal hematopoietic cell system appears to be
decreased capacity for self-renewal as multipotency Is lost and lineage-
restriction and
maturity is acquired. Thus, at one end of the hematopoietic cell spectrum lies
the
pluripotent stem cell possessing the capacity for self-renewal and
differentiation into
all the various lineage-specific committed progenitor cells. This capacity is
the basis
of bone marrow transplant therapy where primitive stem cells repopulate the
entire
hematopoietic cell system. At the other end of the spectrum lie the highly
lineage-
restricted progenitors and their progeny which have lost the ability of self-
renewal
but have acquired mature functional activity.
The proliferation and development of stem cells and lineage-restricted
progenitor cells is carefully controlled by a variety of hematopoietic growth
factors or
cytokines. The role of these growth factors in vivo is complex and
incompletely
understood. Some growth factors, such as interleukin-3 (IL-3), are capable of
stimulating both multipotent stem cells as well as committed progenitor cells
of
several lineages, including for example, megakaryocytes. Other factors such as
granulocyte/macrophage colony-stimulating factor (GM-CSF) was initially
thought to
be restricted in its action to GM-CFC's. Later, however, it was discovered GM-
CSF
also influenced the proliferation and development of interalia megakaryocytes.
Thus,
IL-3 and GM-CSF were found to have overlapping biological activities, although
with
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WO 95/18858 178482 PCT/US94/14553
differing potency. More recently, both interleukin-6 (IL-6) and interleukin-11
(IL-11), while having no apparent influence on meg-colony formation alone, act
synergistically with IL-3 to stimulate maturation of megakaryocytes (Yonemura
et
al., Exp. Hematol., 20:1011-1016 [1992]).
Thus, hematopoietic growth factors may influence growth and differentiation of
one or more lineages, may overlap with other growth factors in affecting a
single
progenitor cell line, or may act synergistically with other factors.
It also appears that hematopoietic growth factors can exhibit their effect at
different stages of cell development from the totipotent stem cell through
various
committed lineage-restricted progenitors to the mature blood cell. For
example,
erythropoietin (epo) appears to promote proliferation only of mature erythroid
progenitor cells. IL-3 appears to exert its effect earlier influencing
primitive stem
cells and intermediate lineage-restricted progenitor cells. Other growth
factors such
as stem cell factor (SCF) may influence even more primitive cell development.
It will be appreciated from the foregoing that novel hematopoietic growth
factors that affect survival, proliferation, differentiation or maturation of
any of the
blood cells or predecessors thereof would be useful, especially to assist in
the re-
establishment of a diminished hematopoietic system caused by disease or after
radiation- or chemo-therapy.
II. Megakaryocytopoiesis - Platelet Production
Regulation of megakaryocytopoiesis and platelet production has been reviewed
by: Mazur, Exp. Hematot., 15:248 [1987] and Hoffman, Blood, 74:1196-1212
[1989]. Briefly, bone marrow pluripotent stem cells differentiate into
megakaryocytic, erythrocytic, and myelocytic cell lines. It is believed there
is a
hierarchy of committed megakaryocytic progenitor cells between stem cells and
megakaryocytes. At least three classes of megakaryocytic progenitor cells have
been
identified, namely; burst forming unit megakaryocytes (BFU-MK), colony-forming
unit megakaryocytes (CFU-MK), and light density megakaryocyte progenitor cells
(LD-CFU-MK). Megakaryocytic maturation itself is a continuum of development
that
has been separated into stages based on standard morphologic criteria. The
earliest
recognizable member of the megakaryocyte (MK or meg) family are the
megakaryoblasts. These cells are initially 20 to 30 m in diameter having
basophilic
cytoplasm and a slightly irregular nucleus with loose, somewhat reticular
chromatin
and several nucleoli. Later, megakaryoblasts may contain up to 32 nuclei
(ployploid),
but the cytoplasm remains sparse and immature. As maturation proceeds, the
nucleus
becomes more lobulate and pyknotic, the cytoplasm increases in quantity and
becomes
more acidophilic and granular. The most mature cells of this family may give
the
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WO 95/18858 2178482 PCT/US94114553
appearance of releasing platelets at their periphery. Normally, less than 10%
of
megakaryocytes are in the blast stage and more than 50% are mature. Arbitrary
morphologic classifications commonly applied to the megakaryocyte series are
megakaryoblast for the earliest form; promegakaryocyte or basophilic
megakaryocyte
for the intermediate form; and mature (acidophilic, granular, or platelet-
producing)
megakaryocyte for the late forms. The mature megakaryocyte extends filaments
of
cytoplasm into sinusoidal spaces where they detach and fragment into
individual
platelets (Williams at at, Hematology, 1972).
Megakaryocytopoiesis is believed to involve several regulatory factors
(Williams et at, Br. J. Haematot., 52:173 [1982] and Williams at al., J. Cell
Physiol., 110:101 [1982]). The early level of megakaryocytopoiesis is
postulated as
being mitotic, concerned with cell proliferation and colony initiation from
CFU-MK
but is not affected by platelet count (Burstein at at, J. Cell Physiol.,
109:333
[1981] and Kimura at at, Exp. Hematof., 13:1048 [1985]). The later stage of
maturation is non-mitotic, involved with nuclear polyploidization and
cytoplasmic
maturation and is probably regulated in a feedback mechanism by peripheral
platelet
number (Odell at at, Blood, 48:765 [1976] and Ebbe at at, Blood, 32:787
[1968]).
The existence of a distinct and specific megakaryocyte colony-stimulating
factor (MK-CSF) has been disputed (Mazur, Exp. Hematol., 15:340-350 [1987]).
However most authors believe that a process so vital to survival as platelet
production
would be regulated by cytokine(s) exclusively responsible for this process.
The
hypothesis that megakaryocyte/platelet specific cytokine(s) exist has provided
the
basis for more than 30 years of search - but to date no such cytokine has been
purified, sequenced and established by assay as a unique MK-CSF (TPO).
Although it has been reported that MK-CSF's have been partly purified from
experimentally produced thrombocytopenia (Hill at at, Exp. Hematol., 14:752
[1986]) and human embryonic kidney conditioned medium [CM] (McDonald et al.;
J.
Lab. Clin. Med., 85:59 [1975]) and in man from a plastic anemia and idiopathic
thrombocytopenic purpura urinary extracts (Kawakita at at, Blood, 6:556
[1983])
and plasma (Hoffman at at, J. Clin. Invest., 75:1174 [1985]), their
physiological
function is as yet unknown in most cases.
The conditioned medium of pokeweed mitogen-activated spleen cells (PWM-
SpCM) and the murine myelomonocyte cell line WEHI-3 (WEHI-3CM) have been used
as megakaryocyte potentiators. PWM-SpCM contains factors enhancing CFU-MK
growth (Metcalf et at, Pro. Natl. Acad. Sci., USA, 72:1744-1748 [1975];
Quesenberry at at, Blood, 65:214 [1985]; and Iscove, N.N., in Hematopoietic
Cell
Differentiation, !CN-UCLA Symposia on Molecular and Cellular Biology, Vol. 10,
Golde
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2178482
WO 95/18858 PCTIUS94114553
at at., eds. [New York, Academy Press] pp 37-52 [1978]), one of which is
interleukin-3 (IL-3), a multilineage colony stimulating factor (multi-CSF
[Burstein, Blood Cells, 11:469 [1986]). The other factors in this medium have
not
yet been identified and isolated. WEHI-3 is a murine myelomonocytic cell line
secreting relatively large amounts of IL-3 and smaller amounts of GM-CSF. IL-3
has
been found to potentiate the growth of a wide range of hematopoietic cells
(Ihle at at, J.
Immunol., 13:282 [1983]). IL-3 has also been found to synergize with many of
the
known hematopoietic hormones or growth factors (Bartelmez at a!., J. Cell
PhysioL,
122:362-369 [1985] and Warren et at, Cell, 46:667-674 [1988]), including
both erythropoietin (EPO) and interleukin-1 (IL-1), in the induction of very
early
multipotential precursors and the formation of very large mixed hematopoietic
colonies.
Other sources of megakaryocyte potentiators have been found in the conditioned
media of murine lung, bone, macrophage cell lines, peritoneal exudate cells
and human
embryonic kidney cells. Despite certain conflicting data (Mazur, Exp.
Hematol.,
15:340- 350 [19871), there is some evidence (Geissler et at, Br. J. Heematol.,
60:233-238 [1985]) that activated T lymphocytes rather than monocytes play an
enhancing role in megakaryocytopoiesis. These findings suggest that activated
T-
lymphocyte secretions such as interleukins may be regulatory factors in MK
development (Geissler at al., Exp. Hematol., 15:845-853 11987]). A number of
studies on megakaryocytopoiesis with purified erythropoietin EPO (Vainchenker
et al.,
Blood, 54:940 [1979]; McLeod at at, Nature, 261:492-4 [1976]; and Williams of
at, Exp. Hematol., 12:734 [19841) indicate that this hormone has an enhancing
effect on MK colony formation. This has also been demonstrated in both serum-
free
and serum-containing cultures and in the absence of accessory cells (Williams
at at,
Exp. Hematol., 12:734 [1984]). EPO was postulated to be involved more in the
single
and two-cell stage aspects of megakaryocytopolesis as opposed to the effect of
PWM-
SpCM which was involved in the four-cell stage of megakaryocyte development.
The
interaction of all these factors on both early and late phases of
megakaryocyte
development remains to be elucidated.
Data produced from several laboratories suggests that the only multi-lineage
factors that individually have MK-colony stimulating activity are GM-CSF and
IL-3
and, to a lesser extent, the B-cell stimulating factor IL-6 (Ikebuchi at at,
Proc. Natl.
Acad. Sci. USA, 84:9035 [1987]). More recently, several authors have reported
that
IL-11 and leukemia inhibitory factor (LIF) act synergistically with IL-3 to
increase
megakaryocyte size and ploidy (Yonemura at at, British Journal of Hematology,
84:16-23 [1993]; Burstein et at, J. Cell. PhysioL, 153:305-312 [1992]; Metcalf
at at, Blood, 76:50-56 [1990]; Metcalf at at, Blood, 77:2150-2153 [1991];
-5-
%
WO 95/18858 PCTIUS94/14553 2178482 Bruno at at, Exp. Hematol., 19:378-381
[1991]; and Yonemura at at, Exp.
Hematol., 20:1011-1016 [1992]).
Other documents of interest include: Eppstein at at, U.S. Patent No.
4,962,091; Chong, U.S. Patent No. 4,879,111; Fernandes at at, U.S. Patent No.
4,604,377; Wissler at at, U.S. Patent No. 4,512,971; Gottlieb, U.S. Patent No.
4,468,379; Bennett at at, U.S. Patent No. 5,215,895; Kogan at al., U.S. Patent
No.
5,250,732; Kimura at at, Eur. J. Immunol., 20(9):1927-1931 11990]; Secor at
at, J. of Immunol., 144(4):1484-1489 [1990]; Warren at at, J. of Immunot,
140(1):94-99 [1988]; Warren at at, Exp. Hematol., 17(11):1095-1099
[1989]; Bruno at at, Exp. Hematol., 17(10):1038-1043 [1989]; Tanikawa at at,
Exp. Hematol., 17(8):883-888 [1989]; Koike at at, Blood, 75(12):2286-2291
[1990]; Lotem, Blood, 75(5):1545-1551 [1989]; Rennick at at, Blood,
73(7):1828-1835 [1989]; and Clutterbuck at at, Blood, 73(6):1504-1512
[1989].
Ill. Thrombocytopenla
Platelets are critical elements of the blood clotting mechanism. Depletion of
the circulating level of platelets, called thrombocytopenia, occurs in various
clinical
conditions and disorders. Thrombocytopenia is commonly defined as a platelet
count
below 150 X 109 per liter. The major causes of thrombocytopenia can be broadly
divided into three categories on the basis of platelet life span, namely; (1)
impaired
production of platelets by the bone marrow, (2) platelet sequestration in the
spleen
(splenomegaly), or (3) increased destruction of platelets in the peripheral
circulation (e.g., autoimmune thrombocytopenia or chemo- and radiation-
therapy).
Additionally, in patients receiving large volumes of rapidly administered
platelet-poor
blood products, thrombocytopenia may develop due to dilution.
The clinical bleeding manifestations of thrombocytopenia depend on the
severity
of thrombocytopenia, its cause, and possible associated coagulation defects.
In general,
patients with platelet counts between 20 and 100 X 109 per liter are at risk
of
excessive post traumatic bleeding, while those with platelet counts below 20 X
109
per liter may bleed spontaneously. These latter patients are candidates for
platelet
transfusion with attendant immune and viral risk. For any given degree of
thrombocytopenia, bleeding tends to be more severe when the cause is decreased
production rather than increased destruction of platelets. In the latter
situation,
accelerated platelet turnover results in the circulation of younger, larger
and
hemostatically more effective platelets. Thrombocytopenia may result from a
variety
of disorders briefly described below. A more detailed description may be found
in
Schafner; A. I., "Thrombocytopenia and Disorders of Platelet Function,"
Internal
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WO 95/18858 PCT/US94/14553
Medicine, 3rd Ed., John J. Hutton et at., Eds., Little Brown and Co.,
Boston/Toronto/
London [1990].
(a) Thrombocytopenia due to impaired platelet production
Causes of congenital thrombocytopenia include constitutional aplastic anemia
(Fanconi syndrome) and congenital amegakaryocytic thrombocytopenia, which may
be
associated with skeletal malformations. Acquired disorders of platelet
production are
caused by either hypoplasia of megakaryocytes or ineffective thrombopoiesis.
Megakaryocytic hypoplasia can result from a variety of conditions, including
marrow
aplasia (including idiopathic forms or myelosuppression by chemotherapeutic
agents
or radiation therapy), myelfibrosis, leukemia, and invasion of the bone marrow
by
metastatic tumor or granulomas. In some situations, toxins, infectious agents,
or
drugs may interfere with thrombopoiesis relatively selectively; examples
include
transient thrombocytopenias caused by alcohol and certain viral infections and
mild
thrombocytopenia associated with the administration of thiazide diuretics.
Finally,
ineffective thrombopoiesis secondary to megaloblastic processes (folate or B12
deficiency) can also cause thrombocytopenia, usually with coexisting anemia
and
leukopenia.
Current treatment of thrombocytopenias due to decreased platelet production
depends on identification and reversal of the underlying cause of the bone
marrow
failure. Platelet transfusions are usually reserved for patients with serious
bleeding
complications, or for coverage during surgical procedures, since
isoimmunization may
lead to refractoriness to further platelet transfusions. Mucosal bleeding
resulting
from severe thrombocytopenia may be ameliorated by the oral or intravenous
administration of the antifibrinolytic agents. Thrombotic complications may
develop,
however, if antifibrinolytic agents are used in patients with disseminated
intravascular coagulation (DIC).
(b) Thrombocytopenia due to splenic sequestration
Splenomegaly due to any cause may be associated with mild to moderate
thrombocytopenia. This is a largely passive process (hypersplenism) of splenic
platelet sequestration, in contrast to the active destruction of platelets by
the spleen in
cases of immunomediated thrombocytopenia discussed below. Although the most
common cause of hypersplenism is congestive splenomegaly from portal
hypertension
due to alcoholic cirrhosis, other forms of congestive, infiltrative, or
lymphoproliferative splenomegaly are also associated with thrombocytopenia.
Platelet
counts generally do not fall below 50 X 109 per liter as a result of
hypersplenism
alone.
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(c) Thrombocytopenia due to nonimmune-mediated platelet destruction
Thrombocytopenia can result from the accelerated destruction of platelets by
various nonimmunologic processes. Disorders of this type include disseminated
intravascular coagulation, prosthetic intravascular devices, extra corporeal
circulation of the blood, and thrombotic microangiopathies such as thrombotic
thrombocytic purpura. In all of these situations, circulating platelets that
are exposed
to either artificial surfaces or abnormal vascular intima either are consumed
at these
sites or are damaged and then prematurely cleared by the reticuloendothelial
system.
Disease states or disorders in which disseminated intravascular coagulation
(DIC) may
arise are set forth in greater detail in Braunwald et al. (eds), Harrison's
Principles of
Internal Medicine, 11th Ed., p.1478, McGraw Hill [1987]. Intravascular
prosthetic
devices, including cardiac valves and intra-aortic balloons can cause a mild
to
moderate destructive thrombocytopenia and transient thrombocytopenia In
patients
undergoing cardiopulmonary bypass or hemodialysis may result from consumption
or
damage of platelets in the extra corporeal circuit.
(d) Drug-induced immune thrombocytopenia
More than 100 drugs have been implicated in immunologically mediated
thrombocytopenia. However, only quinidine, quinine, gold, sulfonamides,
cephalothin,
and heparin have been well characterized. Drug-induced thrombocytopenia is
frequently very severe and typically occurs precipitously within days while
patients
are taking the sensitizing medication.
(e) Immune (autoimmune) thrombocytopenic purpura (ITP)
ITP in adults is a chronic disease characterized by autoimmune platelet
destruction. The autoantibody is usually IgG although other immunoglobulins
have also
been reported. Although the autoantibody of ITP has been found to be
associated with
platelet membrane GPllbllla, the platelet antigen specificity has not been
identified in
most cases. Extravascular destruction of sensitized platelets occurs in the
reticuloendothelial system of the spleen and liver. Although over one-half of
all cases
of ITP are idiopathic, many patients have underlying rheumatic or autoimmune
diseases (e.g., systemic lupus erythematosus) or lymphoproliferative disorders
(e.g.,
chronic lymphocytic leukemia).
(f) HIV-Induced ITP
ITP is an increasingly common complication of HIV infection (Morris et at,
Ann. Intern. Med., 96:714-717 [1982]), and can occur at any stage of the
disease
progression, both in patients diagnosed with the Acquired Immune Deficiency
Syndrome (AIDS), those with AIDS-related complex, and those with HIV infection
but
without AIDS symptoms. HIV infection is a transmissible disease ultimately
characterized by a profound deficiency of cellular immune function as well as
the
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occurrence of opportunistic infection and malignancy. The primary immunologic
abnormality resulting from infection by HIV is the progressive depletion and
functional impairment of T lymphocytes expressing the CD4 cell surface
glycoprotein
(Lane et at, Ann. Rev. ImmunoL, 3:477 [1985]). The loss of CD4 helper/inducer
T
cell function probably underlies the profound defects in cellular and humoral
immunity leading to the opportunistic infections and malignancies
characteristic of
AIDS (H. Lane supra).
Although the mechanism of HIV-associated ITP is unknown, it is believed to be
different from the mechanism of ITP not associated with HIV infection. (Walsh
et at,
N. Eng. J. Med., 311:635-639 [1984]; and Ratner, Am. J. Med., 86:194-198
[1989]).
IV. Current Therapy for Thrombocytopenla
The therapeutic approach to the treatment of patients with thrombocytopenia is
dictated by the severity and urgency of the clinical situation. The treatment
is similar
for HIV-associated and non-HIV-related thrombocytopenia, and although a number
of
different therapeutic approaches have been used, the therapy remains
controversial.
Platelet counts in patients diagnosed with thrombocytopenia have been
successfully increased by glucocorticoid (e.g., prednisolone) therapy, however
in most
patients, the response is incomplete, or relapse occurs when the
glucocorticoid dose is
reduced or its administration is discontinued. Based upon studies with
patients having
HIV-associated ITP, some investigators have suggested that glucocorticoid
therapy may
result in predisposition to AIDS. Glucocorticoids are usually administered if
platelet
count falls below 20 X 109/liter or when spontaneous bleeding occurs.
For patients refractory to glucocorticoids, the compound:
4-(2-chlorphenyl)-9-methyl-2-[3-(4-morpholinyl)-3-propanon-l-yl]6H-
thieno[3,2,f][1,2,4]triazolo[4,3,a,][1,4]diazepin (WEB 2086)
has been successfully used to treat a severe case of non HIV-associated ITP. A
patient
having platelet counts of 37,000-58,000/ I was treated with WEB 2086 and after
1-2 weeks treatment platelet counts increased to 140,000-190,000/ l. (EP
361,077 and Lohman et at, Lancet, 1147 [1988]).
Although the optimal treatment for acquired amegakaryocytic thrombocytopenia
purpura (AATP) is uncertain, antithymocyte globulin (ATG), a horse antiserum
to
human thymus tissue, has been shown to produce prolonged complete remission
(Trimble et at, Am. J. Hematol., 37:126-127 [1991]). A recent report however,
indicates that the hematopoietic effects of ATG are attributable to
thimerosal, where
presumably the protein acts as a mercury carrier (Panella et at, Cancer
Research,
50:4429-4435 [1990]).
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WO 95/18858 2 1 7 8 4 8 2 PCTIUS94/14553
Good results have been reported with splenectomy. Splenectomy removes the
major site of platelet destruction and a major source of autoantibody
production in
many patients. This procedure results in prolonged treatment-free remissions
in a
large number of patients. However, since surgical procedures are generally to
be
avoided in immune compromised patients, splenectomy is recommended only in
severe
cases of thrombocytopenia (e.g. severe HIV-associated ITP), in patients who
fail to
respond to 2 to 3 weeks of glucocorticoid treatment, or do not achieve
sustained
response after discontinuation of glucocorticoid administration. Based upon
current
scientific knowledge, it is unclear whether splenectomy predisposes patients
to AIDS.
In addition to prednisolone therapy and splenectomy, certain cytotoxic agents,
e.g., vincristine, and azidothimidine (AZT, zidovudine) also show promise in
treating
HIV-induced ITP; however, the results are preliminary.
It will be appreciated from the foregoing that one way to treat
thrombocytopenia would be to obtain an agent capable of accelerating the
differentiation and maturation of megakaryocytes or precursors thereof into
the
platelet-producing form. Considerable efforts have been expended on
identifying such
an agent, commonly referred to as "thrombopoietin" (TPO). Other names for TPO
commonly found in the literature include; thrombocytopoiesis stimulating
factor
(TSF), megakaryocyte colony-stimulating factor (MK-CSF), megakaryocyte-
2 0 stimulating factor and megakaryocyte potentiator. TPO activity was
observed as early
as 1959 (Rak at at, Med. Exp., 1:125) and attempts to characterize and purify
this
agent have continued to the present day. While reports of partial purification
of TPO-
active polypeptides exist (see, for example, Tayrien at at, J. Biol. Chem.,
262:3262
[1987] and Hoffman at at, J. Clin. Invest. 75:1174 [1985]), others have
postulated
that TPO is not a discrete entity in its own right but rather is simply the
polyfunctional manifestation of a known hormone (IL- 3, Sparrow et at., Prog.
Clin.
Biol. Res., 215:123 [1986]). Regardless of its form or origin, a molecule
possessing
thrombopoietic activity would be of significant therapeutic value. Although no
protein
has been unambiguously identified as TPO, considerable interest surrounds the
recent
discovery that mpl, a putative cytokine receptor, may transduce a
thrombopoietic
signal.
V. Mpi Is a Megakaryocytopoletic Cytokine Receptor
It is believed that the proliferation and maturation of hematopoietic cells is
tightly regulated by factors that positively or negatively modulate
pluripotential stem
cell proliferation and multilineage differentiation. These effects are
mediated through
the high-affinity binding of extracellular protein factors to specific cell
surface
receptors. These cell surface receptors share considerable homology and are
generally
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WO 95118858 21 7 8 4 O 2 PCT/US94114553
classified as members of the cytokine receptor superfamily. Members of the
superfamily include receptors for: IL-2 ((3 and y chains) (Hatakeyama at at,
Science,
244:551-556 [1989]; Takeshita at at., Science, 257:379-382 [1991]); IL-3
(Itch at at, Science, 247:324-328 [1990]; Gorman at al., Proc. Natl. Acad.
Sci. USA,
87:5459-5463 [1990]; Kitamura at al., Cell, 66:1165-1174 [1991 a]; Kitamura
at at, Proc. Natl. Acad. Sci. USA, 88:5082-5086 [1991b]), IL-4 (Mosley at at.,
Cell, 59:335-348 [1989], IL-5 (Takaki at at, EMBO J., 9:4367-4374 [1990];
Tavernier at at., Cell, 66:1175-1184 [1991]), IL-6 (Yamasaki at at, Science,
241:825-828 [1988]; Hibi at at, Cell, 63:1149-1157 [1990]), IL-7 (Goodwin
at at., Cell, 60:941-951 [1990]), IL-9 (Renault at at, Proc. Natt. Acad. Sci.
USA,
89:5690-5694 [1992]), granulocyte-macrophage colony-stimulating factor (GM-
CSF) (Gearing at at, EMBO J., 8:3667-3676 [1991]; Hayashida at at, Proc. Natl.
Acad. Sci. USA, 244:9655-9659 [1990]), granulocyte colony-stimulating factor
(G-CSF) (Fukunaga at at, Cell, 61:341-350 [1990a]; Fukunaga at at, Proc. Natl.
Acad. Sci. USA, 87:8702-8706 [1990b]; Larsen at at, 'J. Exp. Med., 172:1559-
1570 [1990]), EPO (D'Andrea at at, Cell, 57:277-285 [1989]; Jones at at,
Blood,
76:31-35 [1990]), Leukemia inhibitory factor (LIF) (Gearing at at, EMBO J.,
10:2839-2848 [1991]), oncostatin M (OSM) (Rose at at, Proc. Natl. Acad. Sci.
USA, 88:8641-8645 [1991]) and also receptors for prolactin (Boutin at at,
Proc.
Natl. Acad. Sci. USA, 88:7744-7748 [1988]; Edery at at, Proc. Natl. Acad. Sci.
USA,
86:2112-2116 [1989]), growth hormone (GH) (Leung at at, Nature, 330:537-
543 [1987]) and ciliary neurotrophic factor (CNTF) (Davis at at, Science,
253:59-63 [1991].
Members of the cytokine receptor superfamily may be grouped into three
functional categories (for review see Nicola at at, Cell, 67:1-4 [1991]). The
first
class comprises single chain receptors, such as erythropoietin receptor (EPO-
R) or
granulocyte colony stimulating factor receptor (G-CSF-R), which bind ligand
with
high affinity via the extracellular domain and also generate an intracellular
signal. A
second class of receptors, so called a-subunits, includes interleukin-6
receptor
(IL6-R), granulocyte-macrophage colony stimulating factor receptor (GM-CSF-R),
interleukin-3 receptor (IL3-Ra) and other members of the cytokine receptor
superfamily. These a-subunits bind ligand with low affinity but cannot
transduce an
intracellular signal. A high affinity receptor capable of signaling is
generated by a
heterodimer between an a-subunit and a member of a third class of cytokine
receptors, termed a-subunits, e.g., Pc, the common p-subunit for the three a-
subunits IL3-Ra and GM-CSF-R.
Evidence that mpl is a member of the cytokine receptor superfamily comes
from sequence homology (Gearing, EMBO J., 8:3667-3676 [1988]; Bazan, Proc.
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WO 95/18858 21 7 8 4 8 2 PCTIUS94/14553
Natl. Acad. Sci. USA, 87:6834-6938 [1990]; Davis et at, Science, 253:59-63
[1991] and Vigon et at, Proc. Natl. Acad. Sci. USA, 89:5640-5644 [1992]) and
its
ability to transduce proliferative signals.
Deduced protein sequence from molecular cloning of murine c-mpl reveals this
protein is homologous to other cytokine receptors. The extracellular domain
contains
465 amino acid residues and is composed of two subdomains each with four
highly
conserved cysteines and a particular motif in the N-terminal subdomain and in
the C-
terminal subdomain. The ligand-binding extracellular domains are predicted to
have
similar double p-barrel fold structural geometries. This duplicated
extracellular
domain Is highly homologous to the signal transducing chain common to IL-3, IL-
5 and
GM-CSF receptors as well as the low-affinity binding domain of LIF (Vigon et
at,
Oncogene, 8:2607-2615 [1993]). Thus mpi may belong to the low affinity ligand
binding class of cytokine receptors.
A comparison of murine mpl and mature human mpl P, reveals these two
proteins show 81% sequence identity. More specifically, the N-terminus and C-
terminus extracellular subdomains share 75% and 80% sequence identity
respectively. The most conserved mpl region is the cytoplasmic domain showing
91%
amino acid identity, with a sequence of 37 residues near the transmembrane
domain
being identical in both species. Accordingly, mpl is reported to be one of the
most
conserved members of the cytokine receptor superfamily (Vigon supra).
Evidence that mpl is a functional receptor capable of transducing a
proliferative signal comes from construction of chimeric receptors containing
an
extracellular domain from a cytokine receptor having high affinity for a known
cytokine with the mpl cytoplasmic domain. Since no known ligand for mp/ has
been
reported, it was necessary to construct the chimeric high affinity ligand
binding
extracellular domain from a class one cytokine receptor such as IL-4R or G-
CSFR.
Vigon et al., supra fused the extracellular domain of G-CSFR with both the
transmembrane and cytoplasmic domain of c-mpl. An IL-3 dependent cell line,
BAF/B03 (Ba/F3) was transfected with the G-CSFR/mp/ chimera along with a full
length G-CSFR control. Cells transfected with the chimera grew equally well in
the
presence of cytokine IL-3 or G-CSF. Similarly, cells transfected with G-CSFR
also
grew well in either IL-3 or G-CSF. All cells died in the absence of growth
factors. A
similar experiment was conducted by Skoda et al., EMBO J., 12(7):2645-2653
[1993] in which both the extracellular and transmembrane domains of human IL-4
receptor (hIL-4-R) were fused to the murine mpl cytoplasmic domain, and
transfected into a murine IL-3 dependent Ba/F3 cell line. Ba/F3 cells
transfected
with wild type hIL-4-R proliferated normally in the presence of either of the
species
specific IL-4 or IL-3. Ba/F3 cells transfected with hIL-4RImp! proliferated
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normally in the presence of hIL-4 (in the presence or absence of IL-3)
demonstrating
that in Ba/F3 cells the mpl cytoplasmic domain contains all the elements
necessary to
transduce a proliferative signal.
These chimeric experiments demonstrate the proliferation signaling capability
of the mpl cytoplasmic domain but are silent regarding whether the mp!
extracellular
domain can bind a ligand. These results are consistent with at least two
possibilities,
namely, mpl is a single chain (class one) receptor like EPO-R or G-CSFR or it
is a
signal transducing p-subunit (class three) requiring an a-subunit like IL-3
(Skoda
et a!. supra).
VI. Mpl Ligand is a Thrombopoietin (TPO)
As described above, it has been suggested that serum contains a unique factor,
sometimes referred to as thrombopoietin (TPO), that acts synergistically with
various
other cytokines to promote growth and maturation of megakaryocytes. No such
natural
factor has ever been isolated from serum or any other source even though
considerable
effort has been expended by numerous groups. Even though it is not known
whether
mpl is capable of directly binding a megakaryocyte stimulating factor, recent
experiments demonstrate that mpl is involved in proliferative signal
transduction
from a factor or factors found in the serum of patients with aplastic bone
marrow
(Methia et at, Blood, 82(5):1395-1401 [1993]).
Evidence that a unique serum colony-forming factor distinct from IL-1a, IL-
3, IL-4, IL-6, IL-11, SCF, EPO, G-CSF, and GM-CSF transduces a proliferative
signal through mpl comes from examination of the distribution of c-mpl
expression in
primitive and committed hematopoietic cell lines and from mp! antisense
studies in one
of these cell lines.
Using reverse transcriptase (RT)-PCR in immuno-purified human
hematopoietic cells, Methia of at, supra demonstrated that strong mpl mRNA
messages
were only found in CD34+ purified cells, megakaryocytes and platelets. CD34+
cells
purified from bone marrow (BM) represents about 1% of all BM cells and are
enriched in primitive and committed progenitors of all lineages (e.g.,
erythroid,
granulomacrophage, and megakaryocytic).
Mp! antisense oligodeoxynucleotides were shown to suppress megakaryocytic
colony formation from the pluripotent CD34+ cells cultured in serum from
patients
with aplastic marrow (a rich source of megakaryocyte colony-stimulating
activity
[MK-CSA]). These same antisense oligodeoxynucleotides had no effect on
erythroid or
= granulomacrophage colony formation.
Whether mp/ directly bound a ligand and whether the serum factor shown to
cause megakaryocytopoiesis acted through mpl was still unknown. It had been
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1
suggested, however, that if mpl did directly bind a ligand, its amino acid
sequence was
likely to be highly conserved and have species cross-reactivity owing to the
considerable sequence identity between human and murine mpl extracellular
domains
(Vigon et al., supra [1993]).
VII. Objects
In view of the foregoing, it will be appreciated there is a current and
continuing need in the art to isolate and identify molecules capable of
stimulating
proliferation, differentiation and maturation of hematopoietic cells,
especially
megakaryocytes or their predecessors for therapeutic use in the treatment of
thrombocytopenia. It is believed such a molecule is a mpl ligand and thus
there exists
a further need to isolate such ligand(s) to evaluate their role(s) in cell
growth and
differentiation.
Accordingly, it is an object of this invention to obtain a pharmaceutically
pure
molecule capable of stimulating proliferation, differentiation and/or
maturation of
megakaryocytes into the mature platelet-producing form.
It is another object to provide the molecule in a form for therapeutic use in
the
treatment of a hematopoietic disorder, especially thrombocytopenia.
It is a further object of the present invention to isolate, purify and
specifically
identify protein ligands capable of binding in vivo a cytokine superfamily
receptor
known as mpl and to transduce a proliferative signal.
It is still another object to provide nucleic acid molecules encoding such
protein ligands and to use these nucleic acid molecules to produce mpl binding
ligands
In recombinant cell culture for diagnostic and therapeutic use.
It is yet another object to provide derivatives and modified forms of the
protein
ligands including amino acid sequence variants, variant glycoprotein forms and
covalent derivatives thereof.
It is an additional object to provide fusion polypeptide forms combining a mpl
ligand and a heterologous protein and covalent derivatives thereof.
It is still an additional object to provide variant polypeptide forms
combining a
mpl ligand with amino acid additions and substitutions from the EPO sequence
to
produce a protein capable of regulating proliferation and growth of both
platelets and
red blood cell progenitors.
It is yet an additional object to prepare immunogens for raising antibodies
against mpl ligands or fusion forms thereof, as well as to obtain antibodies
capable of
binding such ligands.
These and other objects of the invention will be apparent to the ordinary
artisan upon consideration of the specification as a whole.
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SUMMARY OF THE INVENTION
The objects of the invention are achieved by providing an isolated mammalian
megakaryocytopoietic proliferation and maturation promoting protein,
denominated
the "mpl ligand" (ML) or "thrombopoietin" (TPO), capable of stimulating
proliferation, maturation and/or differentiation of megakaryocytes into the
mature
platelet-producing form.
This substantially homogeneous protein may be purified from a natural source
by a method comprising; (1) contacting a source plasma containing the mpl
ligand
molecules to be purified with an immobilized receptor polypeptide,
specifically mpl or
a mpl fusion polypeptide immobilized on a support, under conditions whereby
the mp!
ligand molecules to be purified are selectively adsorbed onto the immobilized
receptor
polypeptide, (2) washing the immobilized receptor polypeptide and its support
to
remove non-adsorbed material, and (3) eluting the mpi ligand molecules from
the
immobilized receptor polypeptide to which they are adsorbed with an elution
buffer.
Preferably the natural source is mammalian plasma or urine containing the mpl
ligand. Optionally the mammal is aplastic and the immobilized receptor is a
mpl-IgG
fusion.
Optionally, the prefered megakaryocytopoietic proliferation and maturation
promoting protein is an isolated substantially homogeneous mpi ligand
polypeptide
made by synthetic or recombinant means.
The "mpl ligand" polypeptide or "TPO" of this invention preferably has at
least
70% overall sequence identity with the amino acid sequence of the highly
purified
substantially homogeneous porcine mpl ligand polypeptide and at least 80%
sequence
identity with the "EPO-domain" of the porcine mpl ligand polypeptide.
Optionally, the
mp! ligand of this invention is mature human mpi ligand (hML), having the
mature
amino acid sequence provided in Fig. 1 (SEQ ID NO: 1), or a variant or
posttranscriptionally modified form thereof or a protein having about 80%
sequence
identity with mature human mpl ligand. Optionally the 'mpl ligand variant is a
fragment, especially an amino-terminus or "EPO-domain" fragment, of the mature
human mpl ligand (hML). Preferably the amino terminus fragment retains
substantially all of the human ML sequence between the first and forth
cysteine
residues but may contain substantial additions, deletions or substitutions
outside that
region. According to this embodiment, the fragment polypeptide may be
represented by
the formula:
X-hML(7-151)-Y
Where hML(7-151) represents the human TPO (hML) amino acid sequence from Cys7
through Cys151 inclusive; X represents the amino group of Cys7 or one or more
of the
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amino-terminus amino acid residue(s) of the mature hML or amino acid residue
extensions thereto such as Met, Tyr or leader sequences containing, for
example,
proteolytic cleavage sites (e.g. Factor Xa or thrombin); and Y represents the
carboxy
terminal group of Cys151 or one or more carboxy-terminus amino acid residue(s)
of
the mature hML or extensions thereto.
Optionally the mpl ligand polypeptide or fragment thereof may be fused to a
heterologous polypeptide (chimera). A preferred heterologous polypeptide is a
cytokine, colony stimulating factor or interleukin or fragment thereof,
especially kit-
ligand (KL), IL-1, IL-3, IL-6, IL-11, EPO, GM-CSF or LIF. An optional
preferred
heterologous polypeptide is an immunoglobin chain, especially human IgG1,
IgG2,
IgG3, IgG4, IgA, IgE, IgD, IgM or fragment thereof, especially comprising the
constant
domain of an IgG heavy chain.
Another aspect of this invention provides a composition comprising an isolated
mpl agonist that is biologically active and is preferably capable of
stimulating the
incorporation of labeled nucleotides (e.g., 3H-thymidine) into the DNA of IL-3
dependent Ba/F3 cells transfected with human mot. Optionally the mpl agonist
is
biologically active mpl ligand and is preferably capable of stimulating the
incorporation of 35S into circulating platelets in a mouse platelet rebound
assay.
Suitable mpl agonist include hML153, hML(R153A, R154A), hML2, hML3, hML4,
mML, mML2, mML3, pML, and pML2 or fragments thereof.
In another embodiment, this invention provides an isolated antibody capable of
binding to the mpl ligand. The isolated antibody capable of binding to the mpl
ligand
may optionally be fused to a second polypeptide and the antibody or fusion
thereof may
be used to isolate and purify mpl ligand from a source as described above for
immobilized mpl. In a further aspect of this embodiment, the invention
provides a
method for detecting the mpl ligand in vitro or in vivo comprising contacting
the
antibody with a sample, especially a serum sample, suspected of containing the
ligand
and detecting if binding has occurred.
In still further embodiments, the invention provides an isolated nucleic acid
molecule, encoding the mpl ligand or fragments thereof, which nucleic acid
molecule
may optionally be labeled with a detectable moiety, and a nucleic acid
molecule having a
sequence that is complementary to, or hybridizes under moderate to highly
stringent
conditions with, a nucleic acid molecule having a sequence encoding a mpl
ligand.
Preferred nucleic acid molecules are those encoding human, porcine, and murine
mpl
ligand, and include RNA and DNA, both genomic and cDNA. In a further aspect of
this
embodiment, the nucleic acid molecule is DNA encoding the mpi ligand and
further
comprises a replicable vector in which the DNA is operably linked to control
sequences
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WO 95/18858 2.1 78482 PCT/US94114553
recognized by a host transformed with the vector. Optionally the DNA is cDNA
having
the sequence provided in Fig. 1 5'-3' (SEQ ID NO: 2), 3'-5' or a fragment
thereof.
This aspect further includes host cells, preferably CHO cells, transformed
with the
vector and a method of using the DNA to effect production of mpl ligand,
preferably
comprising expressing the cDNA encoding the mpl ligand in a culture of the
transformed host cells and recovering the mpl ligand from the host cells or
the host
cell culture. The mpl ligand prepared in this manner is preferably human mpl
ligand.
The invention further includes a method for treating a mammal having a
hematopoietic disorder, especially thrombocytopenia, comprising administering
a
therapeutically effective amount of a mpl ligand to the mammal. Optionally the
mpl
ligand is administered in combination with a cytokine, especially a colony
stimulating
factor or interleukin. Preferred colony stimulating factors or interleukins
include;
kit-ligand (KL), LIF, G-CSF, GM-CSF, M-CSF, EPO, IL-1, IL-3, IL-6, and IL-11.
The invention further includes a process for isolating and purifying TPO (ML)
from a TPO producing microorganism comprising:
(1) disrupting or lysing cells containing TPO,
(2) optionally seperating soluble material from insoluble material
containing TPO,
(3) solublizing TPO in the insoluble material with a solublizing buffer,
(4) seperating solublized TPO from other soluble and insoluble material,
(5) refolding TPO in a redox buffer, and
(6) separating properly folded TPO from misfolded TPO.
The process provides for solubilizing the insoluble material containing TPO
with a chaotropic agent where the chaotropic agent is selected from a salt of
guanidine,
sodium thiocyanate, or urea. The process further provides that solublized TPO
is
seperated from other soluble and insoluble material by one or more steps
selected
from centrafugation, gel filtration and reverse phase chromotography. The
refolding
step of the process provides for a redox buffer containing both an oxidizing
and
reducing agent. Generally, the oxidizing agent is oxygen or a compound
containing at
least one disulfide bond and the reducing agent is a compound containing at
least one
free sulfhydryl. Preferably, the oxidizing agent is selected from oxidized
glutathione(GSSG) and cystine and the reducing agent is selected from reduced
glutathione(GSH) and cysteine. Most preferably the oxidizing agent is oxidized
glutathione(GSSG) and the reducing agent is reduced glutathione(GSH). It is
also
prefered that the molar ratio of the oxidizing agent is equal to or greater
then that of
the reducing agent. The redox buffer additionally contains a detergent,
preferably
selected from CHAPS and CHAPSO, present at a level of at leastl%. The redox
buffer
additionally contains NaCl preferably at a concentration range of about 0.1-
0.5M, and
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glycerol preferably at a concentration greater than 15%. The pH of the redox
buffer
preferably ranges from about pH 7.5-pH 9Ø and the refolding step Is
conducted at 4
degrees for 12.4Bhr. The refolding step produces biologically active TPO In
which a
disulfide bond Is formed between the Cys nearest the amino-terminus with the
Cys
6 nearest the carboxy-terminus of the EPO domain.
The Invention further Includes a process for purifying biologically active TPO
from a microorganism comprising:
(1) lysing at least the extracellular membrane of the
microorganism,
(2) treating the lyeate containing TPO with a chaotroplc agent,
(3) refolding the TPO, and
(4) separating Impurities and mlstolded TPO from properly folded
TPO
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows the deduced amino add sequence (SEQ iD NO: 1) of human mpf Ilgand
(hML) cDNA and the coding nucleotide sequence (SEQ ID NO: 2). Nucleotides are
numbered at the beginning of each line. The 6' and 3' untranslated regions are
Indicated in lower case letters. Amino acid residues are numbered above the
sequence
starting at Ser 1 of the mature mpl Ilgand (ML) protein sequence. The
boundrles of
presumed axon 3 are Indicated by the arrows and the potential N-glycosylation
sites
are boxed. Cystelne residues are Indicated by a dot above the sequence. The
underlined
sequence corresponds to the N-terminal sequence determined from mpl ligand
purified
from porcine plasma.
Fig. 2 shows the procedure used for the mpl ligand 3H-thymidine Incorporation
assay. To determine the presence of mpl Ilgand from various sources, the mpl P
Ba/F3
cells were starved of IL-3 for 24 hours In a humidified Incubator at 37 C In
5% CO2
and air. Following IL-3 starvation the cells were plated out In 96 well
culture dishes
with or without diluted samples and cultured for 24 hra In a call culture
Incubator.
20 id of serum tree RPMI media containing 1 Cl of 3H-thymidine was added to
each
well for the last 6-8 hours. The cells were then harvested on 96 well filter
plates and
washed with water. The filters were then counted.
Fig. 3 shows the effect of pronase, DTT and heat on the ability of APP to
stimulate
Ba/F3-mpl cell proliferation. For pronase digestion of APP, pronase
(Boehringer
Mannheim) or bovine serum albumin was coupled to Affi-gelid' (Blorad) and
Incubated Individually with APP for 18hrs. at 37 C. Subsequently, the resins
were
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removed by centrifugation and supernatants assayed. APP was also heated to 80
C for
4 min. br made 100 pM OTT followed by dialysis against PBS.
Fig. 4 shows the elution of mpi Iigand activity from Phenyl-Toyopeart Blue-
Sepharost and Ultrallnltmpi columns. Fractions 4-8 from the mpi affinity
column
were the peak activity fractions eluted from the column.
Fig. 5 shows the SDS-PAGE of eluted Ultralink-mpi fractions. To 200 pt of each
fraction 2-8, 1 ml of acetone containing 1mM MCI at -20 C was added. After
Shrs. at
-20 C samples were centrifuged and resultant pellets were washed 2x with
acetone at
-20 C. The acetone pellets were subsequently dissolved in 30 pl of SOS-
solubtllzadon
buffer, made 100 pM DTT and heated at 90 C for 5 min. The samples were than
resolved on a 4-20% SDS-poyacrylamide gel and proteins were visualized by
silver
staining.
Fig. 6 shows elution of mpi ligand activity from SDS-PAGE. Fraction 5 from the
mpl-affinity column was resolved on a 4-20% SDS-polyacrylamlde gel under non-
reducing conditions. Following electrophoresis the gel was sliced Into 12
equal regions
and electroeiuted as described In the examples. The electroeluted samples were
dialyzed Into PBS and assayed at a 1/20 dilution. The Mr standards used to
calibrate
the gel were Novex Mark 12 standards.
Fig. 7 shows the effect of mpi Iigand depleted APP on human
megakaryocytopolesls.
mpi Iigand depleted APP was made by passing 1 ml over a 1 ml mp/-affinity
column
(700 pg mpl-IgGIml NHS-superose* Pharmacla). Human peripheral atom call
cultures were made 10% APP or 10% mpi Iigand depleted APP and cultured for 12
days. Megakaryocytopolesls was quantitated as described In the examples.
Fig. 8 shows the effect of mpi-IgG on the stimulation of human
megakaryocytopoiesis
So by APP. Human peripheral stem cell cultures were made 10% with APP and
cultured
for 12 days. At day 0. 2 and 4, mpldgG (0.5 pg) or ANP-R-190 (0.5 pg) was
added.
After 12 days megakaryocytopoiesis was quantitated as described In the
examples. The
average of duplicate samples Is graphed with the actual duplicate data In
parenthesis.
Fig. 9 shows both strands of a 390 bp fragment of human genomic DNA encoding
the
mpi Iigand. The deduced amino acid sequence of 'axon 3' (SEQ ID NO: 3), the
coding
sequence (SEQ ID NO; 4), and Its compliment (SEQ ID NO: 5) are shown.
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WO 95/18858 2178482 PCT/US94114553
Fig. 10 shows deduced amino acid sequence of mature human mpl ligand (hML)
(SEQ
ID NO: 6) and mature human erythropoietin (hEPO) (SEQ ID NO: 7). The predicted
amino acid sequence for the human mp! ligand is aligned with the human
erythropoietin
sequence. Identical amino acids are boxed and gaps introduced for optimal
alignment
are indicated by dashes. Potential N-glycosylation sites are underlined with a
plain
line for the hML and with a broken line for hEPO. The two cysteines important
for
erythropoietin activity are indicated by a large dot.
Fig. 11 shows deduced amino acid sequence of mature human mpl ligand isoforms
hML
(SEQ ID NO: 6), hML2 (SEQ ID NO: 8), hML3 (SEQ ID NO: 9), and hML4 (SEQ ID NO:
10). Identical amino acids are boxed and gaps introduced for optimal alignment
are
indicated by dashes.
Figs. 12A, 12B and 12C show the effect of human mp! ligand on Ba/F3-mpi cell
proliferation (A), in vitro human megakaryocytopoiesis quantitated using a
radiolabeled murine IgG monoclonal antibody specific to the megakaryocyte
glycoprotein GPllbllla (B), and murine thrombopoiesis measured in a platelet
rebound
assay (C).
Two hundred ninety-three cells were transfected by the CaPO4 method
(Gorman, C in DNA Cloning : A New Approach 2:143-190 [1985]) with pRK5 vector
alone, pRK5-hML or with pRK5-ML153 overnight (pRK5-ML153 was generated by
Introducing a stop codon after residue 153 of hML by PCR). Media was then
conditioned for 36h and assayed for stimulation of cell proliferation of Ba/F3-
mp/ as
described in Example 1 (A) or in vitro human megakaryocytopoiesis (B).
Megakaryocytopoiesis was quantitated using a 1251 radiolabeled murine IgG
monoclonal antibody (HP1-1D) to the megakaryocyte specific glycoprotein
GPllbllla
as described (Grant et al., Blood 69:1334-1339 [1987]). The effect of
partially
purified recombinant ML (rML) on in vivo platelet production (C) was
determined
using the rebound thrombocytosis assay described by McDonald, T.P. Proc. Soc.
Exp.
Biol. Med. 144:1006-10012 (1973). Partially purified rML was prepared from
200ml of conditioned media containing the recombinant ML. The media was passed
through a 2m1 Blue-Separose column equilabrated in PBS and the column was
washed
with PBS and eluted with PBS containing 2M each of urea and NaCl. The active
fraction
was dialyzed into PBS and made 1 mg/ml with endotoxin free BSA. The sample
contained less than one unit of endotoxin /ml. Mice were injected with either
64,000,
32,000 or 16,000 units of rML or excipient alone. Each group consisted of six
mice.
The mean and standard deviation of each group is shown. p values were
determined by a
2 tailed T-test comparing medians.
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Fig. 13 compares the effect of human mpl ligand isoforms and variants in the
Sa/F3-
mpl cell proliferation assay. hML, mock, hML2, hML3, hML(R153A, R154A), and
hML153 were assayed at various dilutions as described in Example 1.
Figs. 14A, to 14E show the deduced amino acid sequence (SEQ ID NO: 1) of
human mpl ligand (hML) or human TPO (hTPO) and the human genomic DNA coding
sequence (SEQ ID NO: 11). Nucleotides and amino acid residues are numbered at
the
beginning of each line.
Fig. 15 shows a SDS-PAGE of purified 293-rhML332 and purified 293-rhML1 g3.
Fig. 16 shows the nucleotide sequence: cDNA coding (SEQ ID NO: 12) and deduced
amino acid sequence (SEQ ID NO: 13) of the open reading frame of a murine ML
isoform. This mature murine mpl ligand isoform contains 331 amino acid
residues,
four fewer than the putative full length mML, and is therefore designated
mML2.
Nucleotides are numbered at the beginning of each line. Amino acid residues
are
numbered above the sequence starting with Ser 1. The potential N-glycosylation
sites
are underlined. Cystelne residues are indicated by a dot above the sequence.
Fig. 17 shows the cDNA sequence (SEQ ID NO: 14) and predicted protein sequence
(SEQ ID NO: 15) of this murine ML isoform (mML). Nucleotides are numbered at
the
beginning of each line. Amino acid residues are numbered above the sequence
starting
with Ser 1. This mature murine mpl ligand isoform contains 335 amino acid
residues
and is believed to be the full length mpl ligand, designated mML. The signal
sequence is
indicated with a dashed underline and the likely cleavage point is denoted
with an
arrow. The 5' and 3' untranslated regions are indicated with lower case
letters. The
two deleations found as a result of alternative splicing (mML2 and mML3) are
underlined. The four cysteine residues are indicated by a dot The seven
potential N-
glycosylation sites are boxed.
Fig. 18 compares the deduced amino acid sequence of the human ML isoform hML3
(SEQ ID NO: 9) and a murine ML isoform designated mML3 (SEQ ID NO: 16). The
predicted amino acid sequence for the human mpl ligand is aligned with the
murirre mpl
ligand sequence. Identical amino acids are boxed and gaps Introduced for
optimal
alignment are indicated by dashes. Amino acids are numbered at the beginning
of each
fine.
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Fig. 19 compares the predicted amino acid sequences of mature ML isoforms from
mouse-ML (SEQ ID NO: 17), porcine-ML (SEQ ID NO: 18) and human-ML (SEQ ID NO:
6). Amino acid sequences are aligned with gaps, indicated by dashes,
introduced for
optimal alingment. Amino acids are numbered at the beginning of each line with
Identical residues boxed. Potential N-glycosylation sites are indicated by a
shaded box
and cysteine residues are designated with a dot. The conserved di-basic amino
acid
motif that presents a potential protease cleavage site is underlined. The four
amino
acid deletion found to occur in all three species (MI-2) is outlined with a
bold box.
Fig. 20 shows the cDNA sequence (SEQ ID NO: 19) and predicted mature protein
sequence (SEQ ID NO: 18) of a porcine ML isoform (pML). This porcine mpl
ligand
isoform contains 332 amino acid residues and is believed to be the full length
porcine
mpl ligand, designated pML. Nucleotides are numbered at the beginning of each
line.
Amino acid residues are numbered above the sequence starting with Ser 1.
Fig. 21 shows the cDNA sequence (SEQ ID NO: 20) and predicted mature protein
sequence (SEQ ID NO: 21) of a porcine ML isoform (pML2). This porcine mpl
ligand
isoform contains 328 amino acid residues and is a four residues deletion form
of the
full length porcine mpl ligand, designated pML2. Nucleotides are numbered at
the
beginning of each line. Amino acid residues are numbered above the sequence
starting
with Ser 1.
Fig. 22 compares the deduced amino acid sequence of the full length porcine ML
isoform pML (SEQ ID NO: 18) and a porcine ML isoform designated pML2 (SEQ ID
NO:
21). The predicted amino acid sequence for the pML is aligned with pML2
sequence.
Identical amino acids are boxed and gaps introduced for optimal alignment are
indicated
by dashes. Amino acids are numbered at the beginning of each line.
Fig. 23 shows the pertinent features of plasmid pSV15.ID.LL.MLORF ("full
length" or
TPO332) used to transfect host CHO-DP12 cells for production of CHO-rhTPO332=
Fig. 24 shows the pertinent features of plasmid pSVI5.ID.LL.MLEPO-D
("truncated"
or TPO153) used to transfect host CHO-DP12 cells for production of CHO-
rhTPO153=
Figs. 25A, 25B, and 25C show the effect of E. coli-rhTPO(Met-1, 153) on
platelets (A), red blood cells (B) and (C) white blood cells in normal mice.
Two
groups of 6 female C57 B6 mice were injected daily with either PBS buffer or
0.3 g
E. coli-rhTPO(Met 1, 153) (1O091 sc.). On day 0 and on days 3-7 40 I of blood
was
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WO 95/18858 217 8 4 8 2 PCTIUS94l14553
taken from the orbital sinus. This blood was immediately diluted in 10 ml of
commercial diluant and complete blood counts were obtained on a Serrono Baker
Hematology Analyzer 9018. The data are presented as means t Standard error of
the
mean.
Figs. 26A, 26B and 26C show the effect of E. co/i-rhTPO(Met-1, 153) on
platelets
(A), red blood cells (B) and (C) white blood cells in sublethally irradiated
mice. Two
groups of 10 female C57 B6 mice were sublethally irradiated with 750 cGy of
gamma
radiation from a 137Cs source and injected daily with either PBS buffer or 3.0
g E.
coli-rhTPO(Met 1, 153) (1O091 sc.). On day 0 and at subsequent intermediate
time
points 40 I of blood was taken from the orbital sinus. This blood was
immediately
diluted in 10 ml of commercial diluant and complete blood counts were obtained
on a
Serrono Baker Hematology Analyzer 9018. The data are presented as means t
Standard error of the mean.
Figs. 27A, 27B and 27C show the effect of CHO-rhTPO332 on (A) platelets
(thrombocytes), (B) red blood cells (erythrocytes) and (C) white blood cells
(leukocytes) in normal mice. Two groups of 6 female C57 B6 mice were injected
daily with either PBS buffer or 0.3 g CHO-rhTPO832 (100 I sc.). On day 0 and
on
days 3-7 40 I of blood was taken from the orbital sinus. This blood was
immediately
diluted in 10 ml of commercial diluant and complete blood counts were obtained
on a
Serrono Baker Hematology Analyzer 9018. The data are presented as means t
Standard error of the mean.
Fig. 28 shows dose response curves for various forms of rhTPO obtained from
various cell lines. Dose response curves were constructed to rhTPO from the
following cell lines: hTPO332 from CHO (full length from Chinese hamster ovary
cells); hTPOMet-1 153 (E. coil-derived truncated form with an N-terminal
methionine); hTP0332 (full length TPO from human 293 cells); Met-less 155 E-
Coli
(the truncated form [rhTPO155] without the terminal methionine from E. coll.
Groups of 6 female C57B6 mice were injected daily for 7 days with rhTPO
depending
upon group. Each day 40 I of blood was taken from the orbital sinus for
complete blood
counts. The data presented above are the maximal effects seen with the various
treatments and with the exception of (met 153 E-Coil) this occurred on day 7
of
treatment. In the aforementioned "met 153 E-CoiP group the maximal effect was
seen
on day 5. The data are presented as means Standard error of the mean.
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CA 02178482 2009-07-20
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Fig. 29 shows dose response curves comparing the activity of full length and
"clipped"
forms of rhTPO produced In CHO cells with the truncated form from E. colt
Groups of
8 female C57136 mice were Injected daily with 0.3 p rhTPO of various types. On
days
2-7 401iI of blood was taken from the orbital sinus for complete blood counts.
6 Treatment groups were TPO153 the truncated form of TPO from E. colt, TPO332
(Mix
fraction) Full length TPO containing approximately 80-90% full length and
10.20%
clipped forms; TP0332(30K traction) = purified clipped fraction from the
original =
"mix" preparation; TP0332(70K fraction) = purified full length TPO fraction
from
the original "mix" preparation. The data are presented as means t Standard
error of
the mean.
Fig. 30 Is a cartoon showing the KIRA ELISA assay for measuring TPO. The
figure
shows the MPURse.gD chimera and relevant parts of the parent receptors as well
as
the final construct (right portion of the figure) and a flow diagram (left
portion of the
figure) showing relevant steps of the assay.
Fig. 31 is a flow chart for the KIRA ELISA assay showing each step In the
procedure.
Figs. 32A-32L provide the nucleotide sequence (SEQ ID NO: 22) of the
pSVIl7.ID.LL
expression vector used for expression of Rse.gD In Example 17.
Fig. 33 Is a schematic representation of the preparation of piasmid pMP1.
Fig. 34 Is a schematic representation of the preparation of plasmid pMP21.
Fig. 35 is a schematic representation of the preparation of plasmld pMP161.
Fig. 36 is a schematic representation of the preperation of plasmid pMP202.
Fig. 37 is a schematic representation of the preparation of plasmld pMP172.
Fig. 38 Is a schematic representation of the preparation of plasmld pMP210.
Fig. SO is a table of the five best expressing TPO clones from the pMP210
plasmid
bank (SEO ID NOS: 23, 24, 26, 26, 27 and 28).
Fig. 40 Is a schematic representation of the preparation of piasmid pMP41.
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Fig. 41 is a schematic representation of the preperation of plasmid pMP57.
Fig. 42 is a schematic representation of the preperation of plasmid pMP251.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
In general, the following words or phrases have the indicated definition when
used in the description, examples, and claims.
"Chaotropic agent" refers to a compound which, in aqueous solution and in
suitable concentrations, can cause a change in the spatial configuration or
conformation of a protein by at least partially disrupting the forces
responsible for
maintaining the normal secondary and tertiary structure of the protein. Such
compounds include, for example, urea, guanidine-HCl, and sodium thiocyanate.
High
concentrations, usually 4-9M, of these compounds are normally required to
exert the
conformational effect on proteins.
"Cytokine" is a generic term for proteins released by one cell population
which
act on another cell as intercellular mediators. Examples of such cytokines are
lymphokines, monokines, and traditional polypeptide hormones. Included among
the
cytokines are growth hormone, insulin-like growth factors, human growth
hormone,
N-methionyl human growth hormone, bovine growth hormone, parathyroid hormone,
thyroxine, insulin, proinsulin, relaxin, prorelaxin, glycoprotein hormones
such as
follicle stimulating hormone (FSH), thyroid stimulating hormone (TSH), and
leutinizing hormone (LH), hematopoietic growth factor, hepatic growth factor,
fibroblast growth factor, prolactin, placental lactogen, tumor necrosis factor-
a
(TNF-a and TNF-p) mullerian-inhibiting substance, mouse gonadotropin-
associated
peptide, inhibin, activin, vascular endothelial growth factor, integrin, nerve
growth
factors such as NGF-0, platelet-growth factor, transforming growth factors
(TGFs)
such as TGF-a and TGF-(3, insulin-like growth factor-I and -II, erythropoietin
(EPO), osteoinductive factors, interferons such as interferon-a, -p, and -y,
colony
stimulating factors (CSFs) such as macrophage-CSF (M-CSF), granulocyte-
macrophage-CSF (GM-CSF), and granulocyte-CSF (G-CSF), interleukins (IL's) such
as IL-1, IL-1a, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12
and
other polypeptide factors including LIF, SCF, and kit-ligand. As used herein
the
foregoing terms are meant to include proteins from natural sources or from
recombinant cell culture. Similarly, the terms are intended to include
biologically
active equivalents; e.g., differing in amino acid sequence by one or more
amino acids or
in type or extent of glycosylation.
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"mp! ligand", "mp! ligand polypeptide", "ML", "thrombopoietin" or 7130" are
used interchangeably herein and comprise any polypeptide that possesses the
property
of binding to mpl, a member of the cytokine receptor superfamily, and having a
biological property of the ML as defined below. An exemplary biological
property is
the ability to stimulate the incorporation of labeled nucleotides (e.g., 3H-
thymidine)
Into the DNA of IL-3 dependent Ba/F3 cells transfected with human mpl P.
Another
exemplary biological property is the ability to stimulate the incorporation of
35S into
circulating platelets in a mouse platelet rebound assay. This definition
encompasses
the polypeptide isolated from a mpl ligand source such as aplastic porcine
plasma
1 0 described herein or from another source, such as another animal species,
including
humans or prepared by recombinant or synthetic methods and includes variant
forms
including functional derivatives, fragments, alleles, isoforms and analogues
thereof.
A "mpl ligand fragment" or "TPO fragment" is a portion of a naturally
occurring mature full length mpl ligand or TPO sequence having one or more
amino
acid residues or carbohydrate units deleted. The deleted amino acid residue(s)
may
occur anywhere in the peptide including at either the N-terminal or C-terminal
end or
internally. The fragment will share at least one biological property in common
with
mpl ligand. Mpl ligand fragments typically will have a consecutive sequence of
at least
10, 15, 20, 25, 30, or 40 amino acid residues that are identical to the
sequences of
the mpl ligand isolated from a mammal including the ligand isolated from
aplastic
porcine plasma or the human or murine ligand, especially the EPO-domain
thereof.
Representative examples of N-terminal fragments are hML153 or TPO(Met-11-153).
"Mpl ligand variants" or "mpl ligand sequence variants" as defined herein
means a biologically active mpl ligand as defined below having less than 100%
sequence identity with the mpl ligand isolated from recombinant cell culture
or
aplastic porcine plasma or the human ligand having the deduced sequence
described in
Fig. 1 (SEQ ID NO: 1). Ordinarily, a biologically active mpl ligand variant
will have
an amino acid sequence having at least about 70% amino acid sequence identity
with the
mpl ligand isolated from aplastic porcine plasma or the mature murine or human
ligand or fragments thereof (see Fig. 1 [SEQ ID NO: 1]), preferably at least
about
75%, more preferably at least about 80%, still more preferably at least about
85%,
even more preferably at least about 90%, and most preferably at least about
95%.
A "chimeric mpl ligand" is a polypeptide comprising full length mpl ligand or
one or more fragments thereof fused or bonded to a second heterologous
polypeptide or
one or more fragments thereof. The chimera will share at least one biological
property in common with mpl ligand. The second polypeptide will typically be a
cytokine, immunoglobin or fragment thereof.
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WO 95118858 2 1 7 8 4 8 2 PCTIUS94/14553
"Isolated mpl ligand", "highly purified mpl ligand" and "substantially
homogeneous mpl ligand" are used interchangeably and mean a mpl ligand that
has been
purified from a mp! ligand source or has been prepared by recombinant or
synthetic
methods and is sufficiently free of other peptides or proteins (1) to obtain
at least 15
and preferably 20 amino acid residues of the N-terminal or of an internal
amino acid
sequence by using a spinning cup sequenator or the best commercially available
amino
acid sequenator marketed or as modified by published methods as of the filing
date of
this application, or (2) to homogeneity by SDS-PAGE under non-reducing or
reducing
conditions using Coomassie blue or, preferably, silver stain. Homogeneity here
means
less than about 5% contamination with other source proteins.
"Biological property" when used in conjunction with either the "mpl ligand" or
"Isolated mpl ligand" means having thrombopoietic activity or having an in
vivo
effector or antigenic function or activity that is directly or indirectly
caused or
performed by a mpl ligand (whether in its native or denatured conformation) or
a
fragment thereof. Effector functions includemp/ binding and any carrier
binding
activity, agonism or antagonism of mpl, especially transduction of a
proliferative
signal including replication, DNA regulatory function, modulation of the
biological
activity of other cytokines, receptor (especially cytokine) activation,
deactivation,
up- or down regulation, cell growth or differentiation and the like. An
antigenic
function means possession of an epitope or antigenic site that is capable of
cross-
reacting with antibodies raised against the native mpl ligand. The principal
antigenic
function of a mpl ligand polypeptide is that it binds with an affinity of at
least about
106 Umole to an antibody raised against the mpl ligand isolated from aplastic
porcine
plasma. Ordinarily, the polypeptide binds with an affinity of at least about
107
I/mole. Most preferably, the antigenically active mpl ligand polypeptide is a
polypeptide that binds to an antibody raised against the mpl ligand having one
of the
above described effector functions. The antibodies used to define
"biologically activity"
are rabbit polyclonal antibodies raised by formulating the mp! ligand isolated
from
recombinant cell culture or aplastic porcine plasma in Freund's complete
adjuvant,
subcutaneously injecting the formulation, and boosting the immune response by
intraperitoneal injection of the formulation until the titer of mp/ ligand
antibody
plateaus.
"Biologically active" when used in conjunction with either the "mpl ligand" or
"Isolated mpl ligand" means a mpl ligand or polypeptide that exhibits
thrombopoietic
activity or shares an effector function of the mpl ligand isolated from
aplastic porcine
plasma or expressed in recombinant cell culture described herein. A principal
known
effector function of the mpl ligand or polypeptide herein is binding to mp!
and
stimulating the incorporation of labeled nucleotides (3H-thymldine) into the
DNA of
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2178482
IL-3 dependent Ba/F3 cells transfected with human mp! P. Another known
effector
function of the mpi ligand or polypeptide herein is the ability to stimulate
the
incorporation of 35S into circulating platelets in a mouse platelet rebound
assay. Yet
another known effector function of mp! ligand is the ability to stimulate in
vitro
human megakaryocytopoiesis that may be quantitated by using a radio labeled
monoclonal antibody specific to the megakaryocyte glycoprotein GPIIbilla.
"Percent amino acid sequence identity" with respect to the mpl ligand sequence
is defined herein as the percentage of amino acid residues in the candidate
sequence that
are identical with the residues in the mpl ligand sequence isolated from
aplastic
porcine plasma or the murine or human ligand having the deduced amino acid
sequence
described in Fig. 1 (SEQ ID NO: 1), after aligning the sequences and
introducing gaps,
if necessary, to achieve the maximum percent sequence identity, and not
considering
any conservative substitutions as part of the sequence identity. None of N-
terminal,
C-terminal, or internal extensions, deletions, or insertions into the mpl
ligand
sequence shall be construed as affecting sequence identity or homology. Thus
exemplary biologically active mpl ligand polypeptides considered to have
identical
sequences include; prepro-mpl ligand, pro-mpl ligand, and mature mpi ligand.
"Mp! ligand microsequencing" may be accomplished by any appropriate
standard procedure provided the procedure is sensitive enough. In one such
method,
highly purified polypeptide obtained from SDS gels or from a final HPLC step
are
sequenced directly by automated Edman (phenyl isothiocyanate) degradation
using a
model 470A Applied Biosystems gas phase sequencer equipped with a 120A
phenylthiohydantion (PTH) amino acid analyzer. Additionally, mpl ligand
fragments
prepared by chemical (e.g., CNBr, hydroxylamine, 2-nitro-5-thiocyanobenzoate)
or
enzymatic (e.g., trypsin, clostripain, staphylococcal protease) digestion
followed by
fragment purification (e.g., HPLC) may be similarly sequenced. PTH amino acids
are
analyzed using the ChromPerfect data system (Justice Innovations, Palo Alto,
CA).
Sequence interpretation is performed on a VAX 11/785 Digital Equipment Co.
computer as described by Henze] at al., J. Chromatography, 404:41-52 [1987].
Optionally, aliquots of HPLC fractions may be electrophoresed on 5-20% SDS-
PAGE,
electrotransferred to a PVDF membrane (ProBlott, AIB, Foster City, CA) and
stained
with Coomassie Brilliant Blue (Matsurdiara, J. Biol. Chem., 262:10035-10038
[1987]. A specific protein identified by the stain is excised from the blot
and N-
terminal sequencing is carried out with the gas phase sequenator described
above. For
internal protein sequences, HPLC fractions are dried under vacuum (SpeedVac),
resuspended in appropriate buffers, and digested with cyanogen bromide, the
Lys-
specific enzyme Lys-C (Wako Chemicals, Richmond, VA), or Asp-N (Boehringer
Mannheim, Indianapolis, IN). After digestion, the resultant peptides are
sequenced as a
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2178482
mixture or after HPLC resolution on a C4 column developed with a propanol
gradient in
0.1% TFA prior to gas phase sequencing.
"Thrombocytopenia" is defined as a platelet count below 150 X 109 per liter of
blood.
"Thrombopoietic activity" is defined as biological activity that consists of
accelerating the proliferation, differentiation and/or maturation of
megakaryocytes or
megakaryocyte precursors into the platelet producing form of these cells. This
activity may be measured in various assays including an in vivo mouse platelet
rebound synthesis assay, induction of platelet cell surface antigen assay as
measured
by an anti-platelet immunoassay (anti-GPllbIlia) for a human leukemia
megakaryoblastic cell line (CMK), and induction of polyploidization in a
megakaryoblastic cell line (DAMI).
"Thrombopoietin" (TPO) is defined as a compound having thrombopoietic
activity or being capable of increasing serum platelet counts in a mammal. TPO
is
preferably capable of increasing endogenous platelet counts by at least 10%,
more
preferably by 50%, and most preferably capable of elevating platelet counts in
a
human to greater that 150X109 per liter of blood.
"Isolated mpl ligand nucleic acid" is RNA or DNA containing greater than 16
and
preferably 20 or more sequential nucleotide bases that encode biologically
active mpl
ligand or a fragment thereof, is complementary to the RNA or DNA, or
hybridizes to the
RNA or DNA and remains stably bound under moderate to stringent conditions.
This
RNA or DNA is free from at least one contaminating source nucleic acid with
which it is
normally associated in the natural source and preferably substantially free of
any
other mammalian RNA or DNA. The phrase "free from at least one contaminating
source
nucleic acid with which it is normally associated" includes the case where the
nucleic
acid is present in the source or natural cell but is in a different
chromosomal location
or is otherwise flanked by nucleic acid sequences not normally found in the
source cell.
An example of isolated mpl ligand nucleic acid is RNA or DNA that encodes a
biologically
active mpl ligand sharing at least 75% sequence identity, more preferably at
least
80%, still more preferably at least 85%, even more preferably 90%, and most
preferably 95% sequence identity with the human, murine or porcine mpl ligand.
"Control sequences" when referring to expression means DNA sequences
necessary for the expression of an operably linked coding sequence in a
particular host
organism. The control sequences that are suitable for prokaryotes, for
example,
include a promoter, optionally an operator sequence, a ribosome binding site,
and
possibly, other as yet poorly understood sequences. Eukaryotic cells are known
to
utilize promoters, polyadenylation signals, and enhancers.
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"Operably linked" when referring to nucleic acids means that the nucleic acids
are placed in a functional relationship with another nucleic acid sequence.
For
example, DNA for a presequence or secretory leader is operably linked to DNA
for a
polypeptide if it is expressed as a preprotein that participates in the
secretion of the
polypeptide; a promoter or enhancer is operably linked to a coding sequence if
it
affects the transcription of the sequence; or a ribosome binding site is
operably linked
.to a coding sequence if it is positioned so as to facilitate translation.
Generally,
"operably linked" means that the DNA sequences being linked are contiguous
and, in the
case of a secretory leader, contiguous and in reading phase. However,
enhancers do not
have to be contiguous. Linking is accomplished by ligation at convenient
restriction
sites. If such sites do not exist, the synthetic oligonucleotide adaptors or
linkers are
used in accord with conventional practice.
"Exogenous" when referring to an element means a nucleic acid sequence that
is foreign to the cell, or homologous to the cell but in a position within the
host cell
nucleic acid in which the element is ordinarily not found.
"Cell," "cell line," and "cell culture" are used interchangeably herein and
such
designations include all progeny of a cell or cell line. Thus, for example,
terms like
"transformants" and "transformed cells" include the primary subject cell and
cultures
derived therefrom without regard for the number of transfers. It is also
understood
that all progeny may not be precisely identical in DNA content, due to
deliberate or
Inadvertent mutations. Mutant progeny that have the same function or
biological
activity as screened for in the originally transformed cell are included.
Where
distinct designations are intended, it will be clear from the context.
"Plasmids" are autonomously replicating circular DNA molecules possessing
Independent origins of replication and are designated herein by a lower case
"p"
preceded and/or followed by capital letters and/or numbers. The starting
plasmids
herein are either commercially available, publicly available on an
unrestricted basis,
or can be constructed from such available plasmids in accordance with
published
procedures. In addition, other equivalent plasmids are known in the art and
will be
apparent to the ordinary artisan.
"Restriction enzyme digestion" when referring to DNA means catalytic cleavage
of internal phosphodiester bonds of DNA with an enzyme that acts only at
certain
locations or sites in the DNA sequence. Such enzymes are called "restriction
endonucleases". Each restriction endonuclease recognizes a specific DNA
sequence
called a "restriction site" that exhibits two-fold symmetry. The various
restriction
enzymes used herein are commercially available and their reaction conditions,
cofactors, and other requirements as established by the enzyme suppliers are
used.
Restriction enzymes commonly are designated by abbreviations composed of a
capital
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WO 95/18858 2 1 78482 PCTIUS94/14553
letter followed by other letters representing the microorganism from which
each
restriction enzyme originally was obtained and then a number designating the
particular enzyme. In general, about 1 g of plasmid or DNA fragment is used
with
about 1-2 units of enzyme in about 20 l of buffer solution. Appropriate
buffers and
substrate amounts for particular restriction enzymes are specified by the
manufacturer. Incubation of about 1 hour at 37 C is ordinarily used, but may
vary In
accordance with the supplier's instructions. After incubation, protein or
polypeptide
is removed by extraction with phenol and chloroform, and the digested nucleic
acid Is
recovered from the aqueous fraction by precipitation with ethanol. Digestion
with a
restriction enzyme may be followed with bacterial alkaline phosphatase
hydrolysis of
the terminal 5' phosphates to prevent the two restriction-cleaved ends of a
DNA
fragment from "circularizing" or forming a closed loop that would impede
insertion of
another DNA fragment at the restriction site. Unless otherwise stated,
digestion of
plasmids is not followed by 5' terminal dephosphorylation. Procedures and
reagents
for dephosphorylation are conventional as described in sections 1.56-1.61 of
Sambrook et al., Molecular Cloning: A Laboratory Manual [New York: Cold Spring
Harbor Laboratory Press, 1989].
"Recovery" or "isolation" of a given fragment of DNA from a restriction digest
means separation of the digest on polyacrylamide or agarose gel by
electrophoresis,
identification of the fragment of interest by comparison of its mobility
versus that of
marker DNA fragments of known molecular weight, removal of the gel section
containing the desired fragment, and separation of the gel from DNA. This
procedure is
known generally. For example, see Lawn et at, Nucleic Acids Res., 9:6103-6114
[1981], and Goeddel et at, Nucleic Acids Res., 8:4057 [1980].
"Southern analysis" or "Southern blotting" is a method by which the presence
of DNA sequences in a restriction endonuclease digest of DNA or DNA-containing
composition is confirmed by hybridization to a known, labeled oligonucleotide
or DNA
fragment. Southern analysis typically involves electrophoretic separation of
DNA
digests on agarose gels, denaturation of the DNA after electrophoretic
separation, and
transfer of the DNA to nitrocellulose, nylon, or another suitable membrane
support
for analysis with a radiolabeled, biotinylated, or enzyme-labeled probe as
described in
sections 9.37-9.52 of Sambrook et at, supra.
"Northern analysis" or "Northern blotting" is a method used to identify RNA
sequences that hybridize to a known probe such as an oligonucleotide, DNA
fragment,
cDNA or fragment thereof, or RNA fragment. The probe is labeled with a
radioisotope
such as 32P, or by biotinylation, or with an enzyme. The RNA to be analyzed is
usually
electrophoretically separated on an agarose or polyacrylamide gel, transferred
to
nitrocellulose, nylon, or other suitable membrane, and hybridized with the
probe,
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PCT/US94/14553
WO 95118858 2 1 78482
using standard techniques well known in the art such as those described in
sections
7.39-7.52 of Sambrook et at, supra.
"Ligation" is the process of forming phosphodiester bonds between two nucleic
acid fragments. For ligation of the two fragments, the ends of the fragments
must be
compatible with each other. In some cases, the ends will be directly
compatible after
endonuclease digestion. However, it may be necessary first to convert the
staggered
ends commonly produced after endonuclease digestion to blunt ends to make them
compatible for ligation. For blunting the ends, the DNA is treated in a
suitable buffer
for at least 15 minutes at 15 C with about 10 units of the Klenow fragment of
DNA
polymerase I or T4 DNA polymerase in the presence of the four
deoxyribonucleotide
triphosphates. The DNA is then purified by phenol-chloroform extraction and
ethanol
precipitation. The DNA fragments that are to be ligated together are put in
solution in
about equimolar amounts. The solution will also contain ATP, ligase buffer,
and a
ligase such as T4 DNA ligase at about 10 units per 0.5 g of DNA. If the DNA
is to be
ligated into a vector, the vector is first linearized by digestion with the
appropriate
restriction endonuclease(s). The linearized fragment is then treated with
bacterial
alkaline phosphatase or calf intestinal phosphatase to prevent self-ligation
during the
ligation step.
"Preparation" of DNA from cells means isolating the plasmid DNA from a
culture of the host cells. Commonly used methods for DNA preparation are the
large-
and small-scale plasmid preparations described in sections 1.25-1.33 of
Sambrook et
at., supra. After preparation of the DNA, it can be purified by methods well
known in
the art such as that described in section 1.40 of Sambrook et at., supra.
"Oligonucleotides" are short-length, single- or double-stranded
polydeoxynucleotides that are chemically synthesized by known methods (such as
phosphotiester, phosphite, or phosphoramidite chemistry, using solid-phase
techniques such as described in EP 266,032 published 4 May 1988, or via
deoxynucleoside H-phosphonate intermediates as described by Froehler et at,
Nucl.
Acids Res., 14:5399-5407 [1986]). Further methods include the polymerase chain
reaction defined below and other autoprimer methods and oligonucleotide
syntheses on
solid supports. All of these methods are described in Engels et at, Agnew.
Chem. Int.
Ed. Engl., 28:716-734 (1989). These methods are used if the entire nucleic
acid
sequence of the gene is known, or the sequence of the nucleic acid
complementary to the
coding strand is available. Alternatively, if the target amino acid sequence
is known,
one may infer potential nucleic acid sequences using known and preferred
coding
residues for each amino acid residue. The oligonucleotides are then purified
on
polyacrylamide gels.
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WO 95/18858 21 / 0 4 0 2 PCT/US94114553
"Polymerase chain reaction" or "PCR" refers to a procedure or technique in
which minute amounts of a specific piece of nucleic acid, RNA and/or DNA, are
amplified as described in U.S. Patent No. 4,683,195 issued 28 July 1987.
Generally,
sequence information from the ends of the region of interest or beyond needs
to be
available, such that oligonucleotide primers can be designed; these primers
will be
identical or similar in sequence to opposite strands of the template to be
amplified. The
5' terminal nucleotides of the two primers may coincide with the ends of the
amplified
material. PCR can be used to amplify specific RNA sequences, specific DNA
sequences
from total genomic DNA, and cDNA transcribed from total cellular RNA,
bacteriophage
or plasmid sequences, etc. See generally Mullis et at, Cold Spring Harbor
Symp.
Quant. Biol., 51:263 [1987]; Erlich, ed., PCR Technology, (Stockton Press, NY,
1989). As used herein, PCR is considered to be one, but not the only, example
of a
nucleic acid polymerase reaction method for amplifying a nucleic acid test
sample
comprising the use of a known nucleic acid as a primer and a nucleic acid
polymerase
to amplify or generate a specific piece of nucleic acid.
"Stringent conditions" are those that (1) employ low ionic strength and high
temperature for washing, for example, 0.015 M NaCI/0.0015 M sodium
citrate/0.1%
NaDodSO4 (SDS) at 50 C, or (2) employ during hybridization a denaturing agent
such
as formamide, for example, 50% (vol/vol) formamide with 0.1% bovine serum
albumin/0.1% Ficoll/0.1%. polyvinylpyrrolidone/50 mM sodium phosphate buffer
at
pH 6.5 with 750 mM NaCl, 75 mM sodium citrate at 42 C. Another example is use
of
50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium
phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution,
sonicated
salmon sperm DNA (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42 C, with
washes at 42 C in 0.2 x SSC and 0.1% SDS.
"Moderately stringent conditions" are described in Sambrook et at, supra, and
include the use of a washing solution and hybridization conditions (e.g.,
temperature,
Ionic strength, and %SDS) less stringent than described above. An example of
moderately stringent conditions are conditions such as overnight incubation at
37 C in
a solution comprising: 20% formamide, 5 X SSC (150 mM NaCl, 15 mM trisodium
citrate), 50 mM sodium phosphate (pH 7.6), 5 X Denhardt's solution, 10%
dextran
sulfate, and 20 ld/ml denatured sheared salmon sperm DNA, followed by washing
the
filters in 1 X SSC at about 37-50 C. The skilled artisan will recognize how to
adjust
the temperature, ionic strength etc. as necessary to accommodate factors such
as probe
length and the like.
"Antibodies" (Abs) and "immunoglobulins" (Igs) are glycoproteins having the
same structural characteristics. While antibodies exhibit binding specificity
to a
specific antigen, immunoglobulins include both antibodies and other antibody-
like
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WO 95118858 21 7 8 4 8 2 PCTIUS94114553
molecules which lack antigen specificity. Polypeptides of the latter kind are,
for
example, produced at low levels by the lymph system and at increased levels by
myelomas.
"Native antibodies and immunoglobulins" are usually heterotetrameric
glycoproteins of about 150,000 daltons, composed of two identical light (L)
chains and
two identical heavy (H) chains. Each light chain is linked to a heavy chain by
one
covalent disulfide bond, while the number of disulfide linkages varies between
the
heavy chains of different immunoglobulin isotypes. Each heavy and light chain
also has
regularly spaced intrachain disulfide bridges. Each heavy chain has at one end
a
variable domain (VH) followed by a number of constant domains. Each light
chain has a
variable domain at one and NO and a constant domain at its other end; the
constant
domain of the light chain is aligned with the first constant domain of the
heavy chain,
and the light chain variable domain is aligned with the variable domain of the
heavy
chain. Particular amino acid residues are believed to form an interface
between the
light and heavy chain variable domains (Clothia at at, J. Mol. Biot, 186:651-
663
[1985]; Novotny and Haber, Proc. Natl. Acad. Sci. USA, 82:4592-4596 [1985]).
The term "variable" refers to the fact that certain portions of the variable
domains differ extensively in sequence among antibodies and are used in the
binding and
specificity of each particular antibody for its particular antigen. However,
the
variability is not evenly distributed through the variable domains of
antibodies. It is
concentrated in three segments called complementarity determining regions
(CDRs) or
hypervarlable regions both in the light chain and the heavy chain variable
domains.
The more highly conserved portions of variable domains are called the
framework
(FR). The variable domains of native heavy and light chains each comprise four
FR
regions, largely adopting a n-sheet configuration, connected by three CDRs,
which
form loops connecting, and in some cases forming part of, the n-sheet
structure. The
CDRs in each chain are held together in close proximity by the FR regions and,
with the
CDRs from the other chain, contribute to the formation of the antigen binding
site of
antibodies (see Kabat et at, Sequences of Proteins of Immunological Interest,
National
Institute of Health, Bethesda, MD [1987]). The constant domains are not
involved
directly in binding an antibody to an antigen, but exhibit various effector
functions,
such as participation of the antibody in antibody-dependent cellular toxicity.
Papain digestion of antibodies produces two identical antigen binding
fragments,
called "Fab" fragments, each with a single antigen binding site, and a
residual "Fc"
fragment, whose name reflects its ability to crystallize readily. Pepsin
treatment
yields an F(ab')2 fragment that has two antigen combining sites and is still
capable of
cross-linking antigen.
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WO 95/18858 8 4 82 PCT/US94114553
217
"Fv" is the minimum antibody fragment which contains a complete antigen
recognition and binding site. This region consists of a dimer of one heavy and
one light
chain variable domain in tight, non-covalent association. It is in this
configuration
that the three CDRs of each variable domain interact to define an antigen
binding site on
the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen
binding
specificity to the antibody. However, even a single variable domain (or half
of an Fv
comprising only three CDRs specific for an antigen) has the ability to
recognize and
bind antigen, although at a lower affinity than the entire binding site.
The Fab fragment also contains the constant domain of the light chain and the
first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab
fragments by the addition of a few residues at the carboxy terminus of the
heavy chain
CH1 domain including one or more cysteines from the antibody hinge region.
Fab'-SH
is the designation herein for Fab' in which the cysteine residue(s) of the
constant
domains bear a free thiol group. F(ab')2 antibody fragments originally were
produced
as pairs of Fab' fragments which have hinge cysteines between them. Other,
chemical
couplings of antibody fragments are also known.
The "light chains" of antibodies (immunoglobulins) from any vertebrate
species can be assigned to one of two clearly distinct types, called kappa and
lambda
(?,), based on the amino acid sequences of their constant domains.
Depending on the amino acid sequence of the constant domain of their heavy
chains, immunoglobulins can be assigned to different classes. There are five
major
classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these
may be
further divided into subclasses (isotypes), e.g., IgG-1, IgG-2, IgG-3, and IgG-
4; IgA-
1 and IgA-2. The heavy chain constant domains that correspond to the different
classes
of immunoglobulins are called a, delta, epsilon, y, and , respectively. The
subunit
structures and three-dimensional configurations of different classes of
immunoglobulins are well known.
The term "antibody" is used in the broadest sense and specifically covers
single
monoclonal antibodies (including agonist and antagonist antibodies), antibody
compositions with polyepitopic specificity, as well as antibody fragments
(e.g., Fab,
F(ab')2, and Fv), so long as they exhibit the desired biological activity.
The term "monoclonal antibody" as used herein refers to an antibody obtained
from a population of substantially homogeneous antibodies, i.e., the
individual
antibodies comprising the population are identical except for possible
naturally
occurring mutations that may be present in minor amounts. Monoclonal
antibodies are
highly specific, being directed against a single antigenic site. Furthermore,
in
contrast to conventional (polyclonal) antibody preparations which typically
include
different antibodies directed against different determinants (epitopes), each
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PCT/US94/14553
W 95/18858 2178482
monoclonal antibody is directed against a single determinant on the antigen.
In addition
to their specificity, the monoclonal antibodies are advantageous in that they
are
synthesized by the hybridoma culture, uncontaminated by other immunoglobulins.
The
modifier "monoclonal" indicates the character of the antibody as being
obtained from a
substantially homogeneous population of antibodies, and is not to be construed
as
requiring production of the antibody by any particular method. For example,
the
monoclonal antibodies to be used in accordance with the present invention may
be made
by the hybridoma method first described by Kohler & Milstein, Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S. Patent No.
4,816,567 [Cabilly et at]).
The monoclonal antibodies herein specifically include "chimeric" antibodies
(immunoglobulins) in which a portion of the heavy and/or light chain is
identical with
or homologous to corresponding sequences in antibodies derived from a
particular
species or belonging to a particular antibody class or subclass, while the
remainder of
the chain(s) is identical with or homologous to corresponding sequences in
antibodies
derived from another species or belonging to another antibody class or
subclass, as
well as fragments of such antibodies, so long as they exhibit the desired
biological
activity (U.S. Patent No. 4,816,567 (Cabilly at at); and Morrison et a!.,
Proc. Natf.
Acad. Sci. USA, 81:6851-6855 [1984]).
"Humanized" forms of non-human (e.g., murine) antibodies are chimeric
immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab,
Fab',
F(ab')2 or other antigen-binding subsequences of antibodies) which contain
minimal
sequence derived from non-human immunoglobulin. For the most part, humanized
antibodies are human immunoglobulins (recipient antibody) in which residues
from a
complementary determining region (CDR) of the recipient are replaced by
residues
from a CDR of a non-human species (donor antibody) such as mouse, rat or
rabbit
having the desired specificity, affinity and capacity. In some instances, Fv
framework
residues of the human immunoglobulin are replaced by corresponding non-human
residues. Furthermore, humanized antibody may comprise residues which are
found
neither in the recipient antibody nor in the imported CDR or framework
sequences.
These modifications are made to further refine and optimize antibody
performance. In
general, the humanized antibody will comprise substantially all of at least
one, and
typically two, variable domains, in which all or substantially all of the CDR
regions
correspond to those of a non-human immunoglobulin and all or substantially all
of the
FR regions are those of a human immunoglobulin consensus sequence. The
humanized
antibody optimally also will comprise at least a portion of an immunoglobulin
constant
region (Fc), typically that of a human immunoglobulin. For further details
see: Jones
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W095118858 t i 21 7 8 4 82 PCTIVS94114553
at at, Nature, 321:522-525 [1986]; Reichmann at at., Nature, 332:323-329
[1988]; and Presta, Curr. Op. Struct. Biol., 2:593-596 [1992]).
"Non-immunogenic in a human" means that upon contacting the polypeptide in a
pharmaceutically acceptable carrier and in a therapeutically effective amount
with the
appropriate tissue of a human, no state of sensitivity or resistance to the
polypeptide
is demonstratable upon the second administration of the polypeptide after an
appropriate latent period (e.g., 8 to 14 days).
If. Preferred Embodiments of the Invention
Preferred polypeptides of this invention are substantially homogeneous
poiypeptide(s), referred to as mpt ligand(s) or thrombopoietin (TPO), that
possesse
the property of binding to mpl, a member of the receptor cytokine superfamily,
and
having the biological property of stimulating the incorporation of labeled
nucleotides
(3H-thymidine) into the DNA of IL-3 dependent Ba/F3 cells transfected with
human
mpl P. More preferred mpl ligand(s) are isolated mammalian protein(s) having
hematopoietic, especially megakaryocytopoietic or thrombocytopoietic activity -
namely, being capable of stimulating proliferation, maturation and/or
differentiation
of immature megakaryocytes or their predecessors into the mature platelet-
producing
form. Most preferred polypeptides of this invention are human mpl ligand(s)
including fragments thereof having hematopoietic, megakaryocytopoietic or
thrombopoietic activity. Optionally these human mpl ligand(s) lack
glycosylation.
Other prefered human mpl ligands are the "EPO-domain" of hML refered to as
hML153
or hTPO153, a truncated form of hML refered to as hML245 or hTPO245 and the
mature full length polypeptide having the amino acid sequence shown in Fig. 1
(SEQ
ID NO: 1), refered to as hML, hML332 or hTPO332 and the biolocically active
substitutional variant hML(R153A, R154A).
Optional preferred polypeptides of this invention are biologically or
immunologically active mpl ligands variants selected from hML2, hML3, hML4,
mML,
mML2, mML3, pML and pML2.
Optional preferred polypeptides of this invention are biologically active mpl
ligand variant(s) that have an amino acid sequence having at least 70% amino
acid
sequence identity with the human mpl ligand (see Fig. 1 [SEQ ID NO: 1]), the
murine
mp/ ligand (see Fig. 16 [SEQ ID NOS: 12 & 13]), the recombinant porcine mpl
ligand
(see Fig. 19 [SEQ ID NO: 18]) or the porcine mpl ligand isolated from aplastic
porcine plasma, preferably at least 75%, more preferably at least 80%, still
more
preferably at least 85%, even more preferably at least 90%, and most
preferably at
least 95%.
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WO 95118858 2 7 p A p '} PCTI1JS94114553
The mpl ligand isolated from aplastic porcine plasma has the following
characteristics:
(1) The partially purified ligand elutes from a gel filtration column run in
either PBS, PBS containing 0.1% SDS or PBS containing 4M MgCl2 with Mr of
60,000-70,000;
(2) The ligand's activity is destroyed by pronase;
(3) The ligand is stable to low pH (2.5), SDS to 0.1%, and 2M urea;
(4) The ligand is a glycoprotein, based on its binding to a variety of lectin
columns;
(5) The highly purified ligand elutes from non-reduced SDS-PAGE with a
Mr of 25,000-35,000. Smaller amounts of activity also elute with Mr of -18,000-
22,000 and 60,000;
(6) The highly purified ligand resolves on reduced SDS-PAGE as a doublet
with Mr of 28,000 and 31,000;
(7) The amino-terminal sequence of the 18,000-22,000, 28,000 and
31,000 bands is the same - SPAPPACDPRLLNKLLRDDHVLHGR (SEQ ID NO: 29); and
(8) The ligand binds and elutes from the following affinity columns
Blue-Sepharose,
CM Blue-Sepharose,
MONO-Q,
MONO-S,
Lentil lectin-Sepharose,
WGA-Sepharose,
Con A-Sepharose,
Ether 650m Toyopearl,
Butyl 650 m Toyopearl,
Phenyl 650m Toyopearl, and
Phenyl-Sepharose.
More preferred mpl ligand polypeptides are those encoded by human genomic or
cDNA having an amino acid sequence described in Fig. 1 (SEQ ID NO: 1).
Other preferred naturally occurring biologically active mpl ligand
polypeptides of this invention include prepro-mp! ligand, pro-mpl ligand,
mature mpl
ligand, mpl ligand fragments and glycosylation variants thereof.
Still other preferred polypeptides of this invention include mpl ligand
sequence
variants and chimeras. Ordinarily, preferred mpl ligand sequence variants and
chimeras are biologically active mpl ligand variants that have an amino acid
sequence
having at least 70% amino acid sequence identity with the human mpl ligand or
the mpl
ligand isolated from aplastic porcine plasma, preferably at least 75%, more
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W0 95õ8858 2 1 7 8 4 8 PCT/US94114553
2
preferably at least 80%, still more preferably at least 85%, even more
preferably at
least 90%, and most preferably at least 95%. An exemplary preferred mpl ligand
variant is a N-terminal domain hML variant (refered to as the "EPO-domain"
because
of its sequence homology to erythropoietin). The prefered hML EPO-domain
comprises about the first 153 amino acid residues of mature hML and is refered
to as
hML153. An optionally prefered hML sequence variant comprises one in which one
or
more of the basic or dibasic amino acid residue(s) in the C-terminal domain is
substituted with a non-basic amino acid residue(s) (e.g., hydrophobic,
neutral, acidic,
aromatic, Gly, Pro and the like). A prefered hML C-terminal domain sequence
variant
comprises one in which Arg residues 153 and 154 are replaced with Ala
residues.
This variant is refered to as hML332(R153A, R154A). An alternative prefered
hML
variant comprises either hML332 or hML153 in which amino residues 111-114
(QLPP or LPPQ) are deleted or replaced with a diferent tetrapeptide
sequence(e.g.
AGAG or the like). The foregoing deletion mutants are refered to as A4hML332
or
A4hML153.
A preferred chimera is a fusion between mp! ligand or fragment (defined
below) thereof with a heterologous polypeptide or fragment thereof. For
example,
hML153 may be fused to an IgG fragment to improve serum half-life or to IL-3,
G-
CSF or EPO to produce a molecule with inhanced thrombopoietic or chimeric
hematopoietic activity.
An alternative preferred human mpt ligand chimera is a "ML-EPO domain
chimera" that consists of the N-terminus 153 to 157 hML residues substituted
with
one or more, but not all, of the human EPO residues approximately aligned as
shown in
Fig. 10 (SEQ ID NO: 7). In this embodiment, the hML chimera would be about 153-
166 residues in length in which individual or blocks of residues from the
human EPO
sequence are added or substituted into the hML sequence at positions
corresponding to
the alignment shown in Fig. 10 (SEQ ID NO: 6). Exemplary block sequence
inserts
into the N-terminus portion of hML would include one or more of the N-
glycosylation
sites at positions (EPO) 24-27, 38-40, and 83-85; one or more of the four
predicted amphipathic a-helical bundles at positions (EPO) 9-22, 59-76, 90-
107,
and 132-152; and other highly conserved regions including the N-terminus and C-
terminus regions and residue positions (epo) 44-52 (see e.g., Wen et at.,
Blood,
82:1507-1516 [1993] and Boissel et at, J. Blot Chem., 268(21):15983-15993
[1993]). It is contemplated this "ML-EPO domain chimera" will have mixed
thrombopoietic-erythropoietic (TEPO) biological activity.
Other preferred polypeptides of this invention include mpl ligand fragments
having a consecutive sequence of at least 10, 15, 20, 25, 30, or 40 amino acid
residues that are identical to the sequences of the mp! ligand isolated from
aplastic
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CA 02178482 2009-07-20..-- ..w........,_. n __.__.._. .
WO 95118858 PCr/US94/14553
porcine plasma or the human mpl ligand described herein (see e.g. Table 14,
Example 24). A preferred mpl ligand fragment is human ML[1-X] where X is 153,
164, 161, 205, 207, 217, 229, or 245 (see Fig. 1 [SEQ ID NO: 11 for the
sequence
of residues I-X). Other preferred mpi ligand fragments Include those produced
as a
6 result of chemical or enzymatic hydrolysis or digestion of the purified
ligand.
Another preferred aspect of the invention Is a method for purifying mpl ligand
molecules comprises contacting a mpl ligand source containing the mpl Ilgand
molecules with an Immobilized receptor polypeptide, specifically mpl or a mpl
fusion
polypeptide, under conditions whereby the mpl ligand molecules to be purified
are
selectively adsorbed onto the immobilized receptor polypeptide, washing the
immobilized support to remove non-adsorbed material, and eluting the molecules
to be
purified from the Immobilized receptor polypeptide with an elution buffer. The
source
containing the mpl ligand may be plasma where the Immobilized receptor Is
preferably
a mpl-IgG fusion.
1 5 Alternatively, the source containing the mpl ligand Is recombinant cell
culture
where the concentration of mpl ligand In either the culture medium or in cell
lysates
Is generally higher than In plasma or other natural sources. In this case the
above
described mpl-lgG immunoaffinity method, while stilt useful, is usually not
necessary
and more traditional protein purification methods known in the art may be
applied.
Briefly, the preferred purification method to provide substantially
homogeneous mpl
Ngand comprises: removing particulate debris, either host cells or lysed
fragments
by, for example, centrifugation or ultrafiltration; optionally, proteen may be
concentrated with a commercially available protein concentration filter;
followed by
separating the Ilgand from other Impurities by one or more steps selected
from;
26 immunoaffinity, Ion-exchange (e.g., DEAE or matricles containing
carboxymethyi or
sulfopropyl groups), Blue-Sepharose, CM Blue-Sepharose, MONO-Q, MONO-S, lentil
lecttn-Sepharose, WGA-Sepharose, Con A-Sepharose, Ether Toypearl, Butyl
Toypearl,
Phenyl Toypearl, protein A Sepharose, SOS-PAGE, reverse phase HPLC (e.g.,
silica get
with appended aliphatic groups) or Sophadext molecular salve or size exclusion
chromatography, and ethanol or ammonium sulfate precipitation. A protease
inhibitor
such as methylsulfonylfluoride (PMSF) may be included In any of the foregoing
steps
to Inhibit proteolysis.
In another preferred embodiment, this Invention provides an Isolated antibody
capable of binding to the mpl ligand. A preferred mpl Ilgand Isolated antibody
Is
monoclonal (Kohler and Milstein, Nature, 256:495.497 [1975]; Campbell,
Laboratory Techniques In Blochem/stry and Molecular Biology, Burdon of al.,
Edo,
Volume 13, Elsevier Science Pubiisrers, Amsterdam [1995]; and Huse at at.,
Science,
246:1275-1281 (1989]). Preferred mpl ligand isolated antibody Is one that
binds
*-trademark -40-
= WO 95/18858 21 7 8 4 8 2 PCT/US94114553
to mpl ligand with an affinity of at least about 106 I/mole. More preferably
the
antibody binds with an affinity of at least about 107 I/mole. Most preferably,
the
antibody is raised against the mpl ligand having one of the above described
effector
functions. The isolated antibody capable of binding to the mpl ligand may
optionally be
fused to a second polypeptide and the antibody or fusion thereof may be used
to isolate
and purify mpl ligand from a source as described above for immobilized mpl
polypeptide. In a further preferred aspect of this embodiment, the invention
provides
a method for detecting the mpl ligand in vitro or in vivo comprising
contacting the
antibody with a sample, especially a serum sample, suspected of containing the
ligand
and detecting if binding has occurred.
In still further preferred embodiments, the invention provides an isolated
nucleic acid molecule encoding the mpl ligand or fragments thereof, which
nucleic acid
molecule may be labeled or unlabeled with a detectable moiety, and a nucleic
acid
molecule having a sequence that is complementary to, or hybridizes under
stringent or
moderately stringent conditions with, a nucleic acid molecule having a
sequence
encoding a mpl ligand. A preferred mpl ligand nucleic acid is RNA or DNA that
encodes
a biologically active mpl ligand sharing at least 75% sequence identity, more
preferably at least 80%, still more preferably at least 85%, even more
preferably
90%, and most preferably 95% sequence identity with the human mp! ligand. More
preferred isolated nucleic acid molecules are DNA sequences encoding
biologically
active mpl ligand, selected from: (a) DNA based on the coding region of a
mammalian
mpl ligand gene (e.g., DNA comprising the nucleotide sequence provided in Fig.
1 (SEQ
ID NO: 2), or fragments thereof); (b) DNA capable of hybridizing to a DNA of
(a)
under at least moderately stringent conditions; and (c) DNA that is degenerate
to a DNA
defined in (a) or (b) which results from degeneracy of the genetic code. It is
contemplated that the novel mpl ligands described herein may be members of a
family
of ligands or cytokines having suitable sequence identity that their DNA may
hybridize
with the DNA of Fig. 1 (SEQ ID NO: 2) (or the complement or fragments thereof)
under low to moderate stringency conditions. Thus a further aspect of this
invention
includes DNA that hybridizes under low to moderate stringency conditions with
DNA
encoding the mpl ligand polypeptides.
In a further preferred embodiment of this invention, the nucleic acid molecule
is cDNA encoding the mpl ligand and further comprises a replicable vector in
which the
cDNA is operably linked to control sequences recognized by a host transformed
with the
vector. This aspect further includes host cells transformed with the vector
and a
method of using the cDNA to effect production of mpl ligand, comprising
expressing the
cDNA encoding the mpl ligand in a culture of the transformed host cells and
recovering
the mp! ligand from the host cell culture. The mpl ligand prepared in this
manner is
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WO 95118858 21 7 8 4 8 2 PCTIUS94114553 =
preferably substantially homogeneous human mpt ligand. A preferred host cell
for
producing mpl ligand is Chinese hamster ovary (CHO) cells.
The invention further includes a preferred method for treating a mammal
having an immunological or hematopoietic disorder, especially thrombocytopenia
comprising administering a therapeutically effective amount of a mpl ligand to
the
mammal. Optionally, the mpl ligand is administered in combination with a
cytokine,
especially a colony stimulating factor or interleukin. Preferred colony
stimulating
factors or interleukins include; kit-ligand, LIF, G-CSF, GM-CSF, M-CSF, EPO,
IL-1,
IL-2, IL-3, IL-5, IL-6, IL-7, IL-8, IL-9 or IL-11.
III. Methods of Making
Platelet production has long been thought by some authors to be controlled by
multiple lineage specific humoral factors. It has been postulated that two
distinct
cytokine activities, referred to as megakaryocyte colony-stimulating factor
(meg-
CSF) and thrombopoietin, regulate megakaryocytopoiesis and thrombopoiesis
(Williams at at, J. Cell Physiol., 110:101-104 [1982]; Williams et at, Blood
Cells, 15:123-133 [1989]; and Gordon et al., Blood, 80:302-307 [1992]).
According to this hypothesis, meg-CSF stimulates the proliferation of
progenitor
megakaryocytes while thrombopoietin primarily affects maturation of more
differentiated cells and ultimately platelet release. Since the 1960's the
induction and
appearance of both meg-CSF and thrombopoietin activities in the plasma, serum
and
urine of animals and humans following thrombocytopenic episodes has been well
documented (Odell et at, Proc. Soc. Exp. Biol. Med., 108:428-431 [1961];
Nakeff at
at, Acta Haematol., 54:340-344 [1975]; Specter, Proc. Soc. Exp. Biol., 108:146-
149 [1961]; Schreiner et at, J.Clin.lnvest., 49:1709-1713 [1970]; Ebbe, Blood,
44:605-608 [1974]; Hoffman et at, N. Engl. J. Med., 305:533 [1981]; Straneva
et
at, Exp. Hematol., 17:1122-1127 [1988]; Mazur et at, Exp. Hematol., 13:1164
[1985]; Mazur et at, J.Clin. Invest., 68:733-741 [1981]; Sheiner et at, Blood,
56:183-188 [1980]; Hill et at, Exp. Hematot, 20:354-360 [1992]; and Hegyi et
at, Int. J. Cell Cloning, 8:236-244 [1990]). These activities were reported to
be
lineage specific and distinct from known cytokines (Hill R.J. of at, Blood
80:346
(1992); Erickson-Miller C.L. et at, Brit. J. Haematol., 84:197-203 (1993);
Straneva J.E. et at, Exp. HematoL 20:4750(1992); and Tsukada J. et at, Blood
81:866-867 [1993]). Heretofore, attempts to purify meg-CSF or thrombopoietin
from thrombocytopenic plasma or urine have been unsuccessful.
Consistent with the above observations describing thrombocytopenic plasma,
we have found that aplastic porcine plasma (APP) obtained from irradiated pigs
stimulates human megakaryocytopoiesis in vitro. We have found that this
stimulatory
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= WO 95/18858 2 1 7 8 4 82 PCT/US94114553
activity is abrogated by the soluble extracellular domain of c-mpl, confirming
APP as
a potential source of the putative mpl ligand (ML). We have now successfully
purified
the mpl ligand from APP and amino acid sequence information was used to
isolate
murine, porcine and human ML cDNA. These ML's have sequence homology to
erythropoietin and have both meg-CSF and thrombopoietin-like activities.
1. Purification and Identification of mp! Ligand from Plasma
As set forth above, aplastic plasma from a variety of species has been
reported
to contain activities that stimulate hematopoiesis in vitro, however no
hematopoietic
stimulatory factor has previously been reported isolated from plasma. One
source of
aplastic plasma is that obtained from Irradiated pigs. This aplastic porcine
plasma
(APP) stimulates human hematopoiesis in vitro. To determine if APP contained
the
mpl ligand, its effect was assayed by measuring 3H-thymidine incorporation
into
Ba/F3 cells transfected with human mpl P (Ba/F3-mpl ) by the procedure shown
in
Fig. 2. APP stimulated 3H-thymidine incorporation into Ba/F3-mp/ cells but not
Ba/F3 control cells (Le., not transfected with human mpl P). Additionally, no
such
activity was observed in normal porcine plasma. These results indicated that
APP
contained a factor or factors that transduced a proliferative signal through
the mpl
receptor and therefore might be the natural ligand for this receptor. This was
futher
supported by the finding that treatment of APP with soluble mpl-IgG blocked
the
stimulatory effects of APP on Ba/F3-mpl cells.
The activity in APP appeared to be a protein since pronase, DTT, or heat
destroy the activity in APP (Fig. 3). The activity was also non-dialyzable.
The
activity was, however, stable to low pH (pH 2.5 for 2 hrs.) and was shown to
bind and
elute from several lectin-affinity columns, indicating that it was a
glycoprotein. To
further elucidate the structure and identity of this activity it was affinity
purified
from APP using a mpl-IgG chimera.
APP was treated according to the protocol set forth in Examples 1 and 2.
Briefly, the mpl ligand was purified using hydrophobic interaction
chromatography
(HIC), immobilized dye chromatography, and mp/-affinity chromatography. The
recovery of activity from each step is shown in Fig. 4 and the fold
purification is
provided in Table 1. The overall recovery of activity through the mpl-affinity
column was approximately 10%. The peak activity fraction (F6) from the mpl-
affinity column has an estimated specific activity of 9.8 x106 units/mg. The
overall
purification from 5 liters of APP was approximately 4 x106 fold (0.8 units/mg
to
3.3 x 106 units/mg) with a 83 x 106-fold reduction in protein (250 gms to 3
g).
We estimated the specific activity of the ligand eluted from the mpl-affinity
column to
be -3x106 units/mg.
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W o 95118858 2 1 78482 PCT/US94/14553 =
TABLE 1
Purification of m l Li and
Specific
Sample Volume Protein Units/ml Units Acitivity Yield Fold
mis m /ml Units/m %
Purification
APP 5000 50 40 200 000 0.8 - 1
Phenyl 4700 0.8 40 200.00 0 50 94 62
Blue-Sep. 640 0.93 400 256,000 430 128 538
mpl ( l)
(Fxns 5-7) 12 5x10-4 1666 20,000. 3 300 000 10 4 100 000
Protein was determined by the Bradford assay. Protein concentration of mpl-
eluted fractions
5-7 are estimates based on staining intensity of a silver stained SIDS-gel.
One unit is defined
as that causing 50% maximal stimulation of Ba/F3-mpl cell proliferation.
Analysis of eluted fractions from the mpl affinity column by SDS-PAGE (4-
1 0 20%, Novex gel) run under reducing conditions, revealed the presence of
several
proteins (Fig. 5). Proteins that silver stained with the strongest intensity
resolved
with apparent Mr of 66,000, 55,000, 30,000, 28,000 and 18,000-22,000. To
determine which of these proteins stimulated proliferation of Ba/F3-mpl cell
cultures, the proteins were eluted from the gel as described in Example 2.
The results of this experiment showed that most of the activity eluted from a
gel slice that included proteins with Mr 28,000-32,000, with lesser activity
eluting
in the 18,000-22,000 region of the gel (Fig. 6). The only proteins visible in
these
regions had Mr of 30,000, 28,000 and 18,000-22,000. To identify and obtain
protein sequence for the proteins resolving in this region of the gel (i.e.
bands at 30,
28 and 18-22 kDa), these three proteins were electroblotted to PVDF and
sequenced as
described in Example 3. Amino-terminus sequences obtained are provided in
Table
2.
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TABLE 2
Mot Li and Amino-Terminus Sequences
30 kDa
1 5 10 15 20 25
S PAPPACDPRLLNKLLRDD H/S V LH (G) R L (SEQ ID NO: 3
28 kDa
1 5 10 15 20 25
S PAP PAXDPRLLNKLLRDD H VL H GR SEQIDNO:31
18-22 kDa
1 5 10
XPAPPAXDPRLX N K SEQIDNO:32
Computer-assisted analysis revealed these amino acid sequences to be novel.
Because all three sequences were the same, it was believed the 30 kDa, 28 kDa
and
18-22 kDa proteins were related and might be different forms of the same novel
protein. Futhermore, this protein(s) was a likely candidate as the natural mpl
ligand
because the activity resolved on SDS-PAGE in the same region (28,000-32,000)
of a
4-20% gel. In addition, the partially purified ligand migrated with a Mr of
17,000-
30,000 when subjected to gel filtration chromatography using a Superose 12
(Pharmacia) column. It is believed the different Mr forms of the ligand are a
result of
proteolysis or glycosylation differences or other post or pre-translational
modifications.
As described earlier, antisense human mp! RNA abrogated
megakaryocytopoiesis in human bone marrow cultures enriched with CD 34+
progenitor cells without affecting the differentiation of other hematopoietic
cell
lineages (Methia et al., supra). This result suggested that the mpl receptor
might play
a role in the differentiation and proliferation of megakaryocytes in vitro. To
further
elucidate the role of the mp! ligand in megakaryocytopoiesis, the effects of
APP and mpl
ligand depleted APP on in vitro human megakaryocytopoiesis was compared. The
effect
of APP on human megakaryocytopoiesis was determined using a modification of
the
liquid suspension megakaryocytopoiesis assay described in Example 4. In this
assay,
human peripheral stem cells (PSC) were treated with APP before and after mpl-
IgG
affinity chromatography. GPIIbIIIa stimulation of megakaryocytopoiesis was
quantitated with an 1251-anti-llbllla antibody (Fig. 7). Shown in Fig. 7, 10%
APP
caused approximately a 3-fold stimulation while APP depleted of mp/ ligand had
no
effect. Significantly, the mpl ligand depleted APP did not induce
proliferation of the
Ba/F3-mpl cells.
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CA 02178482 2009-07-20
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In another experiment, soluble human mpl-IgG added at days 0, 2 and 4 to
cultures containing 10% APP neutralized the stimulatory effects of APP on
human
megakaryooytopoiests (Pig. 8). These results Indicate that the mpl Ilgand
plays a
role in regulating human megakaryocytopoiesis and therefore may be useful for
the
6 treatment of thrombocytopenia.
2. Molecular Cloning of the mpl Ligand
Based on the amino-terminal amino acid sequence obtained from the 30 kDa, 28
kDa and 18-22 kDa proteins (see Table 2 above), two degenerate oligonucleotide
primer pools were designed and used to amplify porcine genomic DNA by FOR. It
was
reasoned that If the amino-terminal amino acid sequence was encoded by a
single exon
then the correct PCR product was expected to be 89 bp long. A DNA fragment of
this
size was found and subcloned into pGEMT. The sequences of the ollgonucleotide
PCR
primers and the three clones obtained are shown in Example 5. The amino acid
sequence (PRLLNKLLR [BED ID NO: 33)) of the peptide encoded between the PCR
primers was identical to that obtained by amino-terminal protein sequencing of
the
porcine Ilgand (see residues 9-17 for the 28 and 30 kDa porcine protein
sequences
above).
A synthetic oligonucleotlde based on the sequence of the PCR fragment was used
to screen a human genomtc DNA library. A 45-mar oligonucleotide, designated
pR45,
was designed and synthesized based on the sequence of the PCR fragment. This
ollgonucleotlde had the following sequence:
5' GCC-GTG-AAG-GAC-GTG-GTC=GTC-ACG-AAG-CAG-TTT-ATTTAG-GAG-TCG 3'
(BED ID NO: 34)
This deoxyollgonucleotide was used to screen a human genomic DNA library in
kgeml2 under tow stringency hybridization and wash conditions according to
Example 6. Positive clones were picked, plaque purified and analyzed by
restriction
mapping and southern blotting. A 390 bp EcoRI-Xbel fragment that hybridized to
the
45-mer was subcloned Into pB)uesorlp? SK-. DNA sequencing of this clone
confirmed
that DNA encoding the human homolog of the porcine mpl Ilgand had been
Isolated. The
human DNA sequence and deduced amino acid sequence are shown in Fig. 9 (SEQ ID
NOS: 3 & 4). The predicted positions of Introns in the genomic sequence are
also
Indicated by arrows, and define a putative axon (axon 3").
Based on the human "axon 3' sequence (Example 8) oligonucleotides
corresponding to the 3' and 5' ends of the axon sequence were synthesized.
These 2
primers were used in PCR reactions employing as a template cDNA prepared from
various human tissues. The expected size of the correct PCR product was 140
bp.
After analysis of the RCA products on a 12% potyacrylamide gel, a DNA fragment
of the
*-trademark -46-
= WO 95/18858 { 217802 PCTIUS94114553
expected size was detected in cDNA libraries prepared from human adult kidney,
293
fetal kidney cells and cDNA prepared from human fetal liver.
A fetal liver cDNA library (7x106 clones) in lambda DR2 was next screened
with the same 45-mer oligonucleotide used to screen the human genomic library
and
the fetal liver cDNA library under low stringency hybridization conditions.
Positive
clones were picked, plaque purified and the insert size was determined by PCR.
One
clone with a 1.8 kb insert was selected for further analysis. Using the
procedures
described in Example 7 the nucleotide and deduced amino acid sequence of the
human
mpl ligand (hML) were obtained. These sequences are presented in Fig. 1 (SEQ
ID
NOS: 1 & 2).
3. Structure of the Human mpl Ligand (hML)
The human mpl ligand (hML) cDNA sequence (Fig. 1 [SEQ ID NO: 2])
comprises 1774 nucleotides followed by a poly(A) tail. It contains 215
nucleotides of
5' untranslated sequence and a 3' untranslated region of 498 nucleotides. The
presumed initiation codon at nucleotide position (216-218) is within a
consensus
sequence favorable for eukaryotic translation initiation. The open reading
frame is
1059 nucleotides long and encodes a 353 amino acid residue polypeptide,
beginning at
nucleotide position 220. The N-terminus of the predicted amino acid sequence
is
highly hydrophobic and probably corresponds to a signal peptide. Computer
analysis of
the predicted amino acid sequence (von Heijne et at, Eur. J. Biochem., 133:17-
21
[1983]) indicates a potential cleavage site for signal peptidase between
residues 21
and 22. Cleavage at that position would generate a mature polypeptide of 332
amino
acid residues beginning with the amino-terminal sequence obtained from mpl
ligand
purified from porcine plasma. The predicted non-glycosylated molecular weight
of the
332 amino acid residue ligand is about 38 kDa. There are 6 potential N-
glycosylation
sites and 4 cysteine residues.
Comparison of the mpl ligand sequence with the Genbank sequence database
revealed 23% identity between the amino terminal 153 residues of mature human
mpl
ligand and human erythropoietin (Fig. 10 [SEQ ID NOS: 6 & 7]). When
conservative
substitutions are taken into account, this region of hML shows 50% similarity
to
human erythropoietin (hEPO). Both hEPO and the hML contain four cysteines.
Three of
the 4 cysteines are conserved in hML, including the first and last cysteines.
Site-
directed mutagenesis experiments have shown that the first and last cysteines
of
erythropoietin form a disulfide bond that is required for function (Wang,
F.F.et aL,
Endocrinology 116:2286-2292 [1983]). By analogy, the first and last cysteines
of
hML may also form a critical disulfide bond. None of the glycosylation sites
are
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WO 95118858 2 1 7 8 4 8 2 PCT1US94/14553
conserved in hML. All potential hML N-linked glycosylation sites are located
in the
carboxy-terminal half of the hML polypeptide.
Similar to hEPO, the hML mRNA does not contain the consensus polyadenylation
sequence AAUAAA, nor the regulatory element AUUUA that is present in 3'
untranslated
regions of many cytokines and is thought to influence mRNA stability (Shawet
at, Cell,
46:659-667 [1986]). Northern blot analysis reveals low levels of a single 1.8
kb
hML RNA transcript in both fetal and adult liver. After longer exposure, a
weaker
band of the same size could be detected in adult kidney. By comparison, human
erythropoietin is expressed in fetal liver and, in response to hypoxia, the
adult kidney
and liver (Jacobs at at, Nature, 313:804-809 [1985] and Bondurant et al.,
Molec.
Cell. Biol., 6:2731-2733 [1986]).
The importance of the C-terminal region of the hML remains to be elucidated.
Based on the presence of the six potential sites for N-linked glycosylation
and the
ability of the ligand to bind lectin-affinity columns, this region of the hML
is likely
glycosylated. In some gel elution experiments, we observed activity resolving
with a
Mr around 60,000 which may represent the full length, glycosylated molecule.
The
C-terminal region may therefore act to stabilize and increase the half-life of
circulating hML. In the case of erythropoietin, the non-glycosylated form has
full in
vitro biological activity, but has a significantly reduced plasma half-life
relative to
glycosylated erythropoietin (Takeuchi at at, J. Biol. Chem., 265:12127-12130
[1990]; Narhi et at, J. Biol. Chem., 266:23022-23026 [1991] and Spivack et at,
Blood, 7:90-99 [1989]). The C-terminal domain of hML contains two di-basic
amino acid sequences [Arg-Arg motifs at positions 153-154 and 245-246] that
could
serve as potential processing sites. Cleavage at these sites may be
responsible for
generating the 30, 28 and 18-22 kDa forms of the ML isolated from APP.
Significantly, the Ar9153-Arg154 sequence occurs immediately following the
erythropoietin-like domain of the ML. These observations indicate that full
length ML
may represent a precursor protein that undergoes limited proteolysis to
generate the
mature ligand.
4. Isoforms and Variants of the Human mpl Ligand
Isoforms or alternatively spliced forms of human mpl ligand were detected by
PCR in human adult liver. Briefly, primers were synthesized corresponding to
each
end as well as selected internal regions of the coding sequence of hML. These
primers
were used in RT-PCR to amplify human adult liver RNA as described in Example
10.
In addition to the full length form, designated hML, three other forms,
designated
hML2, hML3 and hML4, were observed or deduced. The mature deduced amino acid
sequences of all four isoforms is presented in Fig. 11 (SEQ ID NOS: 6, 8, 9 &
10).
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= WO 95118858 21, 7 8 4 8 2 PCTIUS94/14553
hML3 has a 116 nucleotide deletion a position 700 which results in both an
amino acid
deletion and a frameshift. The cDNA now encodes a mature polypeptide that is
265
amino acid long and diverges from the hML sequence at amino acid residue 139.
Finally, hML4 has both a 12 nucleotide deletion following nucleotide position
618
(also found in the mouse and the pig sequences [see below]) and the 116 bp
deletion
found in hML3. Altough no clones with only the 12 bp deletion (following
nucleotide
619) have been isolated in the human (designated hML2), this form is likely to
exist
because such a isoform has been identified in both the mouse and pig (see
below), and
because it has been identified in conjunction with the116 nucleotide deletion
in hML4.
Both a substitutional variant of hML in which the dibasic Arg153-Arg154
sequence was replaced with two alanine residues and a "EPO-domain" truncated
form of
hML were constructed to determine whether the full length ML was necessary for
biological activity. The Ar9153-Ar9154 dibasic sequence substitutional
variant,
refered to as hML(R153A, R154A), was constructed using PCR as described in
Example 10. The "EPO-domain" truncated form, hML153, was also made using PCR
by introducing a stop codon following Arg153.
5. Expression of Recombinant Human mpl Ligand (rhML) In
Transiently Transfected Human Embryonic Kidney (293)
Cells
To confirm that the cloned human cDNA encoded a ligand for mpl, the ligand was
expressed in mammalian 293 cells under the control of the cytomegalovirus
immediate
early promoter using the expression vectors pRK5-hML or pRK5-hML153=
Supernatants from transiently transfected human embryonic kidney 293 cells
were
found to stimulate 3H-thymidine incorporation in Ba/F3-mp/ cells, but not in
parental Ba/F3 cells (Fig. 12A). Media from the 293 cells transfected with the
pRK
vector alone did not contain this activity. Addition of mpl-IgG to the media
abolished
the stimulation (data not shown). These results show that the cloned cDNA
encodes a
functional human ML (hML).
To determine if the "EPO-domain" alone could bind and activate mpl, the
truncated form of hML, rhML153, was expressed in 293 cells. Supernatants from
transfected cells were found to have activity similar to that present in
supernatants
from cells expressing the full length hML (Fig. 12A), indicating that the C-
terminal
domain of ML is not required for binding and activation of c-mpl.
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6. mpl Ligand Stimulates Megakaryocytopoiesis and
Thrombopolesis
Both the full length rhML and the truncated rhML153 forms of recombinant
hML stimulated human megakaryocytopoiesis in vitro (Fig. 12B). This effect was
observed in the absence of other exogenously added hematopoietic growth
factors. With
the exception of IL-3, the ML was the only hematopoietic growth factor tested
that
exhibited this activity. IL-11, IL-6, IL-1, erythropoietin, G-CSF, IL-9, LIF,
kit
ligand (KL), M-CSF, OSM and GM-CSF had no effect on megakaryocytopoiesis when
tested separately in our assay (data not shown). This result demonstrates that
the ML
has megakaryocyte-stimulating activity, and indicates a role for ML in
regulating
megakaryocytopoiesis.
Thrombopoietic activities present in plasma of thrombocytopenic animals have
been shown to stimulate platelet production in a mouse rebound thrombocytosis
assay
(McDonald, Proc. Soc. Exp. Biol. Med., 14:1006-1001 [1973] and McDonald at at,
Scand. J. Haematol., 16:326-334 [1976]). In this model mice are made acutely
thrombocytopenic using specific antiplatelet serum, resulting in a predictable
rebound
thrombocytosis. Such immuno-thrombocythemic mice are more responsive to
exogenous thrombopoietin-like activities than are normal mice (McDonald, Proc.
Soc.
Exp. Biol. Med., 14:1006-1001 [1973]), just as exhypoxic mice are more
sensitive
to erythropoietin than normal are mice (McDonald, at at, J. Lab. Clin. Med.,
77:134-
143 [1971]). To determine whether the rML stimulates platelet production in
vivo,
mice in rebound thrombocytosis were injected with partially purified rhML.
Platelet
counts and incorporation of 35S into platelets were then quantitated.
Injection of mice
with 64,000 or 32,000 units of rML significantly increased platelet
production, as
evidenced by a -20% increase in platelet counts (p=0.0005 and 0.0001,
respectively) and a -40% increase in 35S incorporation into platelets
(p=0.003) in
the treated mice versus control mice injected with excipient alone (Fig. 12C).
This
level of stimulation is comparable to that which we have observed with IL-6 in
this
model (data not shown). Treatment with 16,000 units of rML did not
significantly
stimulate platelet production. These results indicate that ML stimulates
platelet
production in a dose-dependent manner and therefore possesses thrombopoietin-
like
activity.
293 cells were also transfected with the other hML isoform constructs
described above and the supernatants were assayed using the Ba/F3-mpi
proliferation
assay (see Fig. 13). hML2 and hML3 showed no detectable activity in this
assay,
however the activity of hML(R153A, R154A) was similar to hML and hML153
Indicating that processing at the Arg153-Arg154 di-basic site is neither
required for
nor detrimental to activity.
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PCTIUS94/14553
= WO 95/18858 i' 2178482
7. Megakaryocytopoiesis and the mpl Ligand
It has been proposed that megakaryocytopoiesis is regulated at multiple
cellular levels (Williams et at, J.Celf Physiof., 110:101-104 [1982] and
Williams
et at, Blood Cells, 15:123-133 [1989]). This is based largely on the
observation
that certain hematopoietic growth factors stimulate proliferation of
megakaryocyte
progenitors while others appear to primarily affect maturation. The results
presented
here suggest that the ML acts both as a proliferative and maturation factor.
That ML
stimulates proliferation of megakaryocyte progenitors is supported by several
lines of
evidence. First, APP stimulates both proliferation and maturation of human
megakaryocytes in vitro, and this stimulation is completely inhibited by mpl-
IgG
(Figs. 7 and 8). Furthermore, the inhibition of megakaryocyte colony formation
by
c-mpl antisense oligonucleotides (Methia et at, Blood, 82:1395-1401 [1993])
and
the finding that c-mpl can transduce a proliferative signal in cells into
which it is
transfected (Skoda et at, EMBO, 12:2645-2653 [1993] and Vigon of at, Oncogene,
8:2607-2615'[1993]) also indicate that ML stimulates proliferation. The
apparent
expression of c-mpl during all stages of megakaryocyte differentiation (Methia
et at,
Blood, 82:1395-1401 [1993]) and the ability of recombinant ML to rapidly
stimulate platelet production in vivo indicate that ML also affects
maturation. The
availability of recombinant ML makes possible a careful evaluation of its role
in
regulating megakaryocytopolesis and thrombopoiesis as well as its potential to
influence other hematopoietic lineages.
8. Isolation of the Human mp1 Ligand (TPO) Gene
Human genomic DNA clones of the TPO gene were isolated by screening a human
genomic library in ?.-Gem12 with pR45, under low stringency conditions or
under
high stringency conditions with a fragment corresponding to the 3' half of
human cDNA
coding for the mpl ligand. Two overlapping lambda clones spanning 35 kb were
isolated. Two overlapping fragments (BamHl and EcoRl) containing the entire
TPO
gene were subcloned and sequenced (see Figs. 14A, 14B and 14C).
The structure of the human gene is composed of 6 exons within 7 kb of genomic
DNA. The boundaries of all exon/intron junctions are consistent with the
consensus
motif established for mammalian genes (Shapiro, M. B., et at, Nucl. Acids Res.
15:7155 [1987]). Exon 1 and exon 2 contain 5' untranslated sequence and the
initial
four amino acids of the signal peptide. The remainder of the secretory signal
and the
first 26 amino acids of the mature protein are encoded within exon 3. The
entire
carboxyl domain and 3' untranslated as well as -50 amino acids of the
erythropoietin-
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W095/18858 2178482 PCT/U594/14553
like domain are encoded within exon 6. The four amino acids involved in the
deletion
observed within hML-2 (hTPO-2) are encoded at the 5' end of exon 6.
Analysis of human genomic DNA by Southern blot indicated the gene for TPO is
present in a single copy. The chromosomal location of the gene was determined
by
fluorescent in situ hybridization (FISH) which mapped to chromosome 3q27-28.
9. Expression and Purification of TPO from 293 Cells
Preperation and purification of ML or TPO from 293 cells is described in
detail
in Example 19. Briefly, cDNA corresponding to the TPO entire open reading
frame
was obtained by PCR using pRK5-hmp! I. The PCR product was purified and cloned
between the restriction sites Cial and Xbal of the plasmid pRK5tkneo (a pRK5
derived
vector modified to express a neomycin resistance gene under the control of the
thymidine kinase promote) to obtain the vector pRK5tkneo.ORF(a vector coding
for the
entire open reading frame).
A second vector coding for the EPO homologous domain was generated the same
but using different PCR primers to obtain the final construct called pRK5-
tkneoEPO-
D.
These two constructs were transfected into Human Embryonic Kidney cells by
the CaPO4 method and neomycin resistant clones were selected and allowed to
grow to
confluency. Expression of ML153 or ML332 in the conditioned media from these
clones was assessed using the Ba/F3-mpl proliferation assay.
Purification of rhML332 was conducted as described in Example 19.
Briefly, 293-rhML832 conditioned media was applied to a Blue-Sepharose
(Pharmacia) column that was subsequently washed with a buffer containing 2M
urea.
The column was eluted with a buffer containing 2M urea and 1M NaCl. The Blue-
Sepharose elution pool was then directly applied to a WGA-Sepharose column,
washed
with 10 column volumes of buffer containing 2M urea and 1 M NaCl and eluted
with the
same buffer containing 0.5M N-acetyl-D-glucosamine. The WGA-Sepharose eluate
was applied to a C4-HPLC column (Synchrom, Inc.) and eluted with a
discontinuous
propanol gradient. By SDS-PAGE the purified 293-rhML332 migrates as a broad
band
in the 68-80 kDa region of the gel (see Fig. 15).
Purification of rhML153 was also conducted as described in Example 19.
Briefly, 293-rhML153 conditioned media was resolved on Blue-Sepharose as
described for rhML332. The Blue Sepharose eluate was applied directly to a mp!-
affinity column as described above. RhML153 eluted from the mp!-affinity
column
was purified to homogeneity using a C4-HPLC column run under the same
conditions
used for rhML332. By SDS-PAGE the purified rhML153 resolves into 2 major and 2
minor bands with Mr of -18,000-22,000 (see Fig. 15).
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10. The Murine mpl Ligand
A DNA fragment corresponding to the coding region of the human mpl ligand was
obtained by PCR, gel purified and labeled in the presence of 32P-dATP and 32P-
dCTP.
This probe was used to screen 106 clones of a mouse liver cDNA library in
%GT10. A
murine clone (Fig. 16 [SEQ ID NOS: 12 & 13]) containing a 1443 base pair
insert
was isolated and sequenced. The presumed initiation codon at nucleotide
position 138-
141 was within a consensus sequence favorable for eukaryotic translation
initiation
(Kozak, M. J.Cell Biol., 108:229-241 [1989]). This sequence defines an open
1 0 reading frame of 1056 nucleotides, which predicts a primary translation
product of
352 amino acids. Flanking this open reading frame are 137 nucleotides of 5'
and 247
nucleotides of 3' untranslated sequence. There is no poly(A) tail following
the 3'
untranslated region indicating that the clone is probably not complete. The N-
terminus
of the predicted amino acid sequence is highly hydrophobic and probably
represents a
signal peptide. Computer analysis (von Heijne, G. Eur. J. Biochem. 133:17-21
[1983]) indicated a potential cleavage site for signal peptidase between
residues 21
and 22. Cleavage at that position would generate a mature polypeptide of 331
amino
acids (35 kDa) identified as mML331 (or mML2 for reasons described below). The
sequence contains 4 cysteines, all conserved in the human sequence, and seven
potential N-glycosylation sites, 5 of which are conserved in the human
sequence.
Again, as with hML, all seven potential N-glycosylation sites are located in
the C-
terminal half of the protein.
When compared with the human ML, considerable identity for both nucleotide
and deduced amino acid sequences were observed in the "EPO-domains" of these
ML's.
However, when deduced amino acid sequences of human and mouse ML's were
aligned,
the mouse sequence appeared to have a tetrapeptide deletion between residues
111-
114 corresponding to the 12 nucleotide deletion following nucleotide position
618 seen
in both the human (see above) and pig (see below) cDNA's. Accordingly,
additional
clones were examined to detect possible murine ML isoforms. One clone encoded
a 335
amino acid deduced sequence polypeptide containing the "missing" tetrapeptide
LPLQ.
This form is believed to be the full length murine ML and is refered to as mML
or
mML335. The nucleotide and deduced amino acid sequence for mML are provided in
Fig. 17 (SEQ ID NOS: 14 & 15). This cDNA clone consists of 1443 base pairs
followed by a poly(A) tail. It possesses an open reading frame of 1068 bp
flanked by
134 bases of 5' and 241 bases of 3' untranslated sequence. The presumed
initiation
codon lies at nucleotide position 138-140. The open reading frame encodes a
predicted
protein of 356 amino acids, the first 21 of which are highly hydrophobic and
likely
function as a secretion signal.
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Finally, a third murine clone was isolated, sequenced and was found to
contained
the 116 nucleotide deletion corresponding to hML3. This murine isoform is
therefore
denominated mML3. Comparison of the deduced amino acid sequences of these two
isoforms is shown in Fig. 18 (SEQ ID NOS: 9 & 16).
The overall amino acid sequence identity between human and mouse ML (Fig.
19 [SEQ ID NOS: 6 & 17]) is 72% but this homology is not evenly distributed.
The
region defined as the "EPO-domain" (amino acids 1-153 for the human sequence
and
1-149 for the mouse) is better conserved (86% homology) than the carboxy-
terminal region of the protein (62% homology). This may further indicate that
only
the "EPO-domain" is important for the biological activity of the protein.
Interestingly, of the two di-basic amino acid motifs found in hML, only the di-
basic
motif immediately following the "EPO-domain" (residue position 153-154) in the
human sequence is present in the murine sequence. This is consistent with the
possibility that the full length ML may represent a precursor protein that
undergoes
limited proteolysis to generate the mature ligand. Alternatively, proteolysis
between
Ar9153-Arg154 may facilitate hML clearance
An expression vector containing the entire coding sequence of mML was
transiently transfected into 293 cells as described in Example 1. Conditioned
media
from these cells stimulated 3H-thymidine incorporation into Ba/F3 cells
expressing
either murine or human mpl but had no effect on the parental (mpl-less) cell
line.
This indicates that the cloned murine ML cDNA encodes a functional ligand that
is able
to activate both the murine and human ML receptor (mpt).
11 . The Porcine mpl Ligand
Porcine ML (pML) cDNA was isolated by RACE PCR as described in Example
13. A PCR cDNA product of 1342 bp was found in kidney and subcloned. Several
clones were sequenced and found to encode a pig mpl ligand of 332 amino acid
resudues
referred to as pML (or pML332) having the nucleotide and deduced amino acid
sequence shown in Fig. 20 (SEQ ID NOS: 18 & 19).
Again, a second form, designated pML2, encoding a protein with a 4 amino acid
residue deletion (228 amino acid residues) was identified (see Fig. 21 [SEQ ID
NO:
21]). Comparison of pML and pML2 amino acid sequences shows the latter form is
identical except that the tetrapeptide QLPP corresponding to residues 111-114
inclusive have been deleted (see Fig. 22 [SEQ ID NOS: 18 & 21]). The four
amino
acid deletions observed in both murine and porcine ML cDNA occur at precisely
the
same position within the predicted proteins.
Comparison of the predicted amino acid sequences of the mature ML from
human, mouse, and pig (Fig. 19 [SEQ ID NOS: 6, 17 & 18]) indicates that
overall
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sequence identity is 72 percent between mouse and human, 68 percent between
mouse
and pig and 73 percent between pig and human. The homology is substantially
greater
in the amino-terminal half of the ML (EPO homologous domain). This domain is
80 to
84 percent identical between any two species whereas the carboxy-terminal half
(carbohydrate domain) is only 57 to 67 percent identical. A di-basic amino
acid motif
that could represent a protease cleavage site is present at the carboxyl end
of the
erythropoeitin homology domain. This motif is conserved between the three
species at
this position (Fig. 19 [SEQ ID NOS: 6, 17 & 18]). A second di-basic site
present at
position 245 and 246 in the human sequence is not present in the mouse or pig
sequences. The murine and the pig ML sequence contain 4 cysteines, all
conserved in
the human sequence. There are seven potential N-glycosylation sites within the
mouse
ligand and six within the porcine ML, 5 of which are conserved within the
human
sequence. Again, all the potential N-glycosylation sites are located in the C-
terminal
half of the protein.
12. Expression and Purification of TPO from Chinese Hamster
Ovary (CHO) Cells
The expression vectors used to transfect CHO cells are designated:
pSVI5.ID.LL.MLORF (full length or TPO332), and pSVI5.ID.LL.MLEPO-D (truncated
or
TPO153). The pertinent features of these plasmids are presented in Fig. 23 and
24.
The transfection procedures are described in Example 20. Briefly, cDNA
corresponding to the entire open reading frame of TPO was obtained by PCR. The
PCR
product was purified and cloned between two restriction sites (Clal and Sall)
of the
plasmid pSVI5.ID.LL to obtain the vector pSVIS.ID.LL.MLORF. A second construct
corresponding to the EPO homologous domain was generated the same way but
using a
different reverse primer(EPOD.Sal). The final construct for the vector coding
for the
EPO homologous domain of TPO is called pSV15.ID.LL.MLEPO-D.
These two constructs were linearized with Noll and transfected into Chinese
Hamster Ovary Cells (CHO-DP12 cells, EP 307,247 published 15 March 1989) by
electroporation. 107 cells were electroporated in a BRL electroporation
apparatus
(350 Volts, 330 mF, low capacitance) in the presence of 10, 25 or 50 mg of DNA
as
described (Andreason, G.L. J. Tissue Cult. Meth. 15,56 [1993]). The day
following
transfection, cells were split in DHFR selective media (High glucose DMEM-F12
50:50 without glycine, 2mM glutamine, 2-5% dialyzed fetal calf serum). 10 to
15
days later individual colonies were transferred to 96 well plates and allowed
to grow to
confluency. Expression of ML153 or ML332 in the conditioned media from these
clones
was assessed using the Ba/F3-mpl proliferation assay (described in Example I).
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The process for purifying and Isolating TPO from harvested CHO cell culture
fluid Is described In Example 20. Briefly, harvested cell culture fluid (HCCF)
Is
applied to a Blue Sepharose column (Phamacla) at a ratio of approximately 100E
of
HCCF per liter of resin. The column Is then washed with 3 to 5 column volumes
of
6 buffer followed by 3 to 5 column volumes of a buffer containing 2.OM urea.
TPO Is
then eluted with 3 to 5 column volumes of buffer containing both 2.OM urea and
1.0M
NsCI.
The Blue Sepharose eluaee pool containing TPO Is then applied to a Wheat Germ
Lectin Sepharose column (Pharmacla) equilibrated in the Blue Sepharose eluting
buffer at a ratio of from 8 to 16 ml of Blue Sepharose eluate per ml of resin.
The
column is then washed with 2 to 3 column volumes of equilibration buffer. TPO
Is then
eluted with 2 to 5 column volumes of a butler containing 2.OM urea and 0.5M N-
acetyi-D-glucosamine.
The Wheat Germ Lectin eluate containing TPO is then acidified and C12EB Is
16 added to a final concentration of 0.04%. The resulting pool Is applied to a
04 reversed
phase column equilibrated In 0.1% TFA, 0.04% 012E8 at a load of approximately
0.2
to 0.5 mg protein per ml of resin.
The protein Is eluted In a two phase linear gradient of acetonltrile
containing
0.1% TFA and 0.04% C12EB and a pool is made on the basis of SOS-PAGE.
The C4 Pool is then diluted and dlafilitered versus approximately 6 volumes of
buffer on an Amlcon YM or like ultrafiltration membrane having a 10,000 to
30,000
Dalton molecular weight cut-off. The resulting disfiitrate may be then
directly
processed or further concentrated by ultrafiltration. The
dlafiltrate/concentrate is
usually adjusted to a final concentration of 0.01% Tweeen-80.
26 All or a portion of the dlafiltrate/concentrate equivalent to 2 to 5% of
the
calculated column volume Is then applied to a Sephaoryt S=300 HR column
(Pharmacia) equilibrated In a buffer containing 0.01% Tween-8O* and
chromatographed. The TPO containing fractions which are free of aggregate and
proteotytic degradation products are then pooled on the basis of SDS-PAGE. The
resulting pool Is filtered and stored at 2-80C.
13. Methods for Transforming and Inducing TPO Synthesis In a
Microorganism and Isolating, Purifying and Refolding TPO
Made Therein
Construction of E. cop TPO expression vectors Is described In detail in
Example 21. Briefly. plasmids pMP21, pMP151, pMP41, pMP57 and pMP202
were all designed to express the first 155 amino acids of TPO downstream of a
small
leader which varies among the different constructs. The leaders provide
primarily for
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high level translation initiation and rapid purification. The plasmids pMP210-
1,
-T8, -21, -22, -24, -25 are designed to express the first 153 amino acids of
TPO
downstream of an initiation methionine and differ only in the codon usage for
the first
6 amino acids of TPO, while the plasmid pMP251 is a derivative of pMP210-1 in
which the carboxy-terminal end of TPO is extended by two amino acids. All of
the
above plasmids will produce high levels of intracellular expression of TPO in
E. coil
upon induction of the tryptophan promoter (Yansura, D. G. et. at Methods in
Enzymology ( Goeddel, D. V., Ed.) 185:54-60, Academic Press, San Diego
[1990]).
The plasmids pMP1 and pMP172 are intermediates in the construction of the
above
TPO intracellular expression plasmids.
The above TPO expression plasmids were used to transform the E. coil using the
CaCl2 heat shock method (Mandel, M. et a!. J. Mot Blot, 53:159-162, [1970])
and
other procedures described in Example 21. Briefly, the transformed cells were
grown first at 37 C until the optical density (600nm) of the culture reached
approximately 2-3. The culture was then diluted and, after growth with
aeration, acid
was added. The culture was then allowed to continue growing with aeration for
another
15 hours after which time the cells were harvested by centrifugation.
The Isolation, Purification and Refolding procedures given below for
production
of biologically active, refolded human TPO or fragments thereof is described
in
Examples 22 and 23 can be applied for the recovery of any TPO variant
including N
and C terminal extended forms. Other procedures suitable for refolding
recombinant
or synthetic TPO can be found in the following patents; Builder et at, U.S.
Patent
4,511,502; Jones et at, U.S. Patent 4,512,922; Olson U.S. Patent 4,518,526 and
Builder et at, U.S. Patent 4,620,948; for a general description of the
recovery and
refolding process for a variety of recombinant proteins expressed in an
insoluble form
in E. coll.
A Recovery of non-soluble TPO
A microorganism such as E. coil expressing TPO encoded by any suitable
plasmid is fermented under conditions in which TPO is deposited in insoluble
"refractile bodies". Optionally, cells are first washed in a cell disruption
buffer.
Typically, about 100g of cells are resuspended in about 10 volumes of a cell
disruption buffer (e.g. 10 mM Tris, 5 mM EDTA, pH 8) with, for example, a
Polytron
homogenizer and the cells centrifuged at 5000 x g for 30 minutes. Cells are
then lysed
using any conventional technique such as tonic shock, sonication, pressure
cycling,
chemical or enzymatic methods. For example, the washed cell pellet above may
be
resuspended in another 10 volumes of a cell disruption buffer with a
homogenizer and
the cell suspension is passed through an LH Cell Disrupter (LH Inceltech,
Inc.) or
through a Microfluidizer (Microfluidics International) according to the
manufactures'
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Instructions. The particulate matter containing TPO Is than separated from the
liquid
phase and optionally washed with any suitable liquid. For example, a
suspension of cell
lysate may be centrifuged at 5,000 X g for 30 minutes, resuspended and
optionally
centrifuged a second time to make a washed retractile body pellet. The washed
pellet
6 may be used Immediately or optionally stored frozen (at e.g. -70 0).
B. Solubilizatkn and Purification of Monomeric TPO
Insoluble TPO In the retractile body pellet Is then solubilized with a
solublizing
buffer. The solubiizing buffer contains a chaotropic agent and Is usually
buffered at a
basic pH and contains a reducing agent to improve the yield of monomeric TPO.
Representative chaotropic agents Include urea, guanldlne=HCI, and sodium
thlocyanate.
A preferred chaotropic agent Is guanldlne.HCi. The concentration of chaotroplc
agent Is
usually 4-9M, preferably 6-8M. The pH of the sofublizing buffer is maintained
by
any suitable butter in a pH range of from about 7.6-9.6, preferably 8.0-9.0
and most
preferably 8Ø Preferably the solubllizing buffer also contains a reducing
agent to
aid formation of the monomeric form of TPO. Suitable reducing agents Include
organic
compounds containing a free Not (RSH). Representative reducing agents Include
dithlothreitol (OTT), dithloerythritol (DTE), mercaptoethanol, glutathione
(GSH),
cysteamine and cysteine. A preferred reducing agent is dithlothreitol (OTT).
Optionally, the solublllzing buffer may contain a mild oxidizing agent (e.g.
molecular
oxygen) and a sulfite salt to form monomeric TPO via sulfitoysis. In this
embodiment,
the resulting TPO-S-suifonate is later refolded In the presence of the redox
buffer
(e.g. GSWGSSG) to form the property folded TPO.
The TPO protein Is usually further purified using, for example,
centrifugation,
gel filtration chromatography and reversed phase column chromatography.
26 By way of Illustration, the following procedure has produced suitable
yields of
monomeric TPO. The retractile body pellet Is resuspended in about 5 volumes by
weight of the solubfllzing buffer (20 mM Tris, pH 8, with 6.8 M guanidine and
26
mM DTT) and stirred for 1-3 hr., or overnight, at 4 C to effect solubilization
of the
TPO protein. High concentrations of urea (6-8M) are also useful but generally
result
in somewhat lower yields compared to guanidine. After solubllization, the
solution is
centrifuged at 30,000 x g for 30 min. to produce a clear supernatant
containing
denatured, monomeric TPO protein. The supernatant Is then ohromatographed on a
Superde `200 gel filtration column (Pharmacla, 2.8 x 60 cm) at a flow rate of
2
mi/min. and the protein eluted with 20 mM Na phosphate, pH 8,0, with 10 mM
DTT.
Fractions containing monomeric, denatured TPO protein eluting between 180 and
200
ml are pooled. The TPO protein is further purified on a semi-preparative C4
reversed
phase column (2 x 20 cm VYDAC). The sample is applied at 5 ml/min. to a column
equilibrated In 0.1% TFA (trifiuoroscetic acid) with 30% acetonitrile. The
protein Is
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eluted with a linear gradient of acetonitrlle (30.60% In 60 min.). The
purified
reduced protein elutes at approximately 50% acetonitrlle. This material Is
used for
refolding to obtain biologically active TPO variant.
C Refolding TPO to Generate the Blologicalty Active Form
Following solubilizatlon and further purification of TPO, the biologically
active
form is obtained by refolding the denatured monomeric TPO In a redox buffer.
Because
of the high potency of TPO (half maximal stimulation In the Ba/F3 assay Is
achieved at
approximately 3 pg/mi), it Is possible to obtain biologically active material
utilizing
many different buffer, detergent and redox conditions. However, under most
conditions
only a small amount of properly folded material (<10%) Is obtained. For
commercial
manufacturing processes, it is desirable to have refolding yields at least
10%, more
preferably 30-50% and most preferably >50%. Many different detergents
Including
Triton X-10( dodecyl-beta-maltosida, CHAPS, CHAPSO, SDS, sarkoeyl, Tween 2(and
Tween 80, Zwittergent 3-14 and others were found suitable for producing at
least
some properly folded material. Of these however, the most preferred detergents
were
those of the CHAPS family (CHAPS and CHAPSO) which were found to work beat In
the
refolding reaction and to limit protein aggregation and Improper disulfide
formation.
Levels of CHAPS greater than about 1% were most preferred. Sodium chloride was
required for the best yields, with the optimal levels between 0.1 M and 0.5M.
The
presence of EDTA (1-5 mM) In the redox buffer was preferred to limit the
amount of
metal-catalyzed oxidation (and aggregation) which was observed with some
preparations. Glycerol concentrations of greater than 15% produced the optimal
refolding conditions. For maximum yields, It was essential to have a redox
pair in the
redox buffer consisting of both an oxidized and reduced organic thiol (RSH).
Suitable
redox pairs Include mercaptoethanol, glutathione (GSH), cysteamine, cysteine
and
their corresponding oxidized forms. Preferred redox pairs were
glutathione(GSH):oxidized glutathlone(GSSG) or cystelne:cystine. The most
preferred
redox pair was glutathlone(GSH):oxldlzed glutathtone(GS8G). Generally higher
yields
were observed when the mote ratio of oxidized member of the redox pair was
equal to
or In excess over the reduced member of the redox pair. pH values between 7.5
and
about 9 were optimal for refolding of these TPO variants. Organic solvents
(e.g.
ethanol, acetonitrlle, methanol) were tolerated at concentrations of 10-15% or
lower.
Higher levels of organic solvents Increased the amount of Improperly folded
forms.
The and phosphate buffers were generally useful. Incubation at 4 C also
produced
higher levels of properly folded TPO.
Refolding yields of 4060% (based on the amount of reduced and denatured TPO
used In the refolding reaction) are typical for preparations of TPO that have
been
purified through the first C4 step. Active material can be obtained when less
pure
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preparations (e.g. directly after the Superdex 200 column or after the initial
refractile body extraction) although the yields are less due to extensive
precipitation
and interference of non-TPO proteins during the TPO refolding process.
Since TPO contains 4 cysteine residues, it is possible to generate three
different disulfide versions of this protein:
version 1: disulfides between cysteine residues 1-4 and 2-3
version 2: disulfides between cysteine residues 1-2 and 3-4
version 3: disulfides between cysteine residues 1-3 and 2-4.
During the initial exploration in determining refolding conditions, several
different peaks containing the TPO protein were separated by C4 reversed phase
chromatography. Only one of these peaks had significant biological activity as
determined using the Ba/F3 assay. Subsequently, the refolding conditions were
optimized to yield preferentially that version. Under these conditions, the
misfolded
versions were less than 10-20% of the total monomeric TPO obtained from the
solubilizing step.
The disulfide pattern for the biologically active TPO has been determined to
be
1-4 and 2-3 by mass spectrometry and protein sequencing, where the cysteines
are
numbered sequentially from the amino-terminus. This cysteine cross-linking
pattern
is consistent with the known disulfide bonding pattern of the related molecule
erythropoietin.
D. Biological Activity of Recombinant, Refolded TPO
Refolded and purified TPO has activity in both in vitro and in vivo assays.
For
example, in the Ba/F3 assay, half-maximal stimulation of thymidine
incorporation
into the Ba/F3 cells for TPO (Merl 1-153) was achieved at 3.3 pg /ml (0.3 pM).
In the mpl receptor-based ELISA, half-maximal activity occurred at 1.9 ng/ml
(120
pM). In normal and myelosuppressed animals produced by near-lethal X-
radiation,
refolded TPO (Met-1 1-153) was highly potent (activity was seen at doses as
low as
ng/mouse) to stimulate the production of new platelets. Similar biological
activity
was observed for other forms of TPO refolded in accordance with the above
described
30 procedures (see Figs. 25, 26 and 28).
14. Methods for Measurement of Thrombopoletic Activity
Thrombopoietic activity may be measured in various assays including the
Ba/F3 mpi ligand assay described in Example 1, an in vivo mouse platelet
rebound
synthesis assay, induction of platelet cell surface antigen assay as measured
by an
anti-platelet immunoassay (anti-GPllbilla) for a human leukemia
megakaryoblastic
cell line (CMK) (see Sato et at, Brit. J. Heamatol., 72:184-190 [1989])(see
also
the liquid suspension megakaryocytopoiesis assay described in Example 4), and
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induction of polyploidization in a megakaryoblastic cell line (DAMI) (see
Ogura et at,
Blood, 72(1):49-60 [1988]). Maturation of megakaryocytes from immature,
largely non-DNA synthesizing cells, to morphologically identifiable
megakaryocytes
involves a process that includes appearance of cytoplasmic organelles,
acquisition of
membrane antigens (GPllbllla), endoreplication and release of platelets as
described
In the background. A lineage specific promoter (i.e., the mpl ligand) of
megakaryocyte
maturation would be expected to induce at least some of these changes in
immature
megakaryocytes leading to platelet release and alleviation of
thrombocytopenia. Thus,
assays were designed to measure the emergence of these parameters in immature
megakaryocyte cell lines, i.e., CMK and DAMI cells. The CMK assay (Example 4)
measures the appearance of a specific platelet marker, GPIIbIIIa, and platelet
shedding.
The DAMI assay (Example 15) measures endoreplication since increases in ploidy
are hallmarks of mature megakaryocytes. Recognizable megakaryocytes have
ploidy
values of 2N, 4N, 8N, 16N, 32N, etc. Finally, the in vivo mouse platelet
rebound
assay (Example 16) is useful in demonstrating that administration of the test
compound (here the mpl ligand) results in elevation of platelet numbers.
Two additional in vitro assays have been developed to measure TPO activity.
The first is a kinase receptor activation (KIRA) ELISA in which CHO cells are
transfected with a mpl-Rse chimera and tyrosine phosphorylation of Rse is
measured
by ELISA after exposure of the mpl portion of the chimera to mpl ligand (see
Example
17). The second is a receptor based ELISA in which ELISA plate coated rabbit
anti-
human IgG captures human chimeric receptor mpl-IgG which binds the mpl ligand
being assayed. A biotinylated rabbit polyclonal antibody to mpl ligand
(TPO158) is
used to detect bound mpl ligand which is measured using streptavidin-
peroxidase as
described in Example 18.
15. In Vivo Biological Response of Normal and Sublethally
Irradiated Mice Treated with TPO
Both normal and sublethally irradiated mice were treated with truncated and
full length TPO isolated from Chinese hamster ovary (CHO) cells, E. coli, and
human
embryonic kidney (293) cells. Both forms of TPO produced in these three hosts
stimulated platelet production in mice, however, full length TPO isolated from
CHO
speared to produce the greatest in vivo response. These results indicate that
proper
glycosylation of the carboxy-terminal domain may be necessary for optimal in
vivo
activity.
(a) E. coii-rhTPO(Met-1,153)
The "Met" form of the EPO domain (Met in the -1 position plus the first 153
residues of human TPO) produced in E. coli (see Example 23) was injected daily
into
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normal female C57 B6 mice as described in the legends to Figs. 25A, 25B and
25C.
These figures show that the non-glycosylated truncated form of TPO produced in
E. coli
and refolded as described above is capable of stimulating about a two-fold
increase in
platelet production in normal mice with out effecting the red or white blood
cell
population.
This same molecule injected daily into sublethally irradiated (137Cs ) female
C57 B6 mice as described in the legends to Figs. 26A, 26B and 26C stimulated
platelet recovery and diminished nadir but had no effect on erythrocytes or
leukocytes.
(b) CHO-rhTPO332
The full length form of TPO produced in CHO and injected daily into normal
female C57 B6 mice as described in the legends to Figs. 27A, 27B and 27C
produced
about a five-fold increase in platelet production in normal mice with out
effecting the
erythrocyte or leukocyte population.
(c) CHO-rhTPO332; E. coli-rhTPO(Met 1,153); 293-rhTPO332; and E.
coli-rhTPO155
Dose response curves were constructed for treatment of normal mice with
rhTPO from various cell lines (CHO-rhTPO332; E. coli-rhTPO(Met-1,153); 293-
rhTPO332; and E. coli-rhTPO155) as described in the legend to Fig. 28. This
figure
shows that all tested forms of the molecule stimulate platelet production,
however the
full length form produced in CHO has the greatest in vivo activity.
(d) CHO-rhTPO153, CHO-rhTPO"clipped" and CHO-rhTPO332
Dose response curves were also constructed for treatment of normal mice with
various forms of rhTPO produced in CHO (CHO-rhTPO153, CHO-rhTPO"clipped" and
CHO-rhTPO332) as described in the legend to Fig. 29. This figure shows that
all
tested CHO forms of the molecule stimulate platelet production,but that the
full length
70 Kda form has the greatest in vivo activity.
16. General Recombinant Preparation of mpl Ligand and
Variants
Preferably mpl ligand is prepared by standard recombinant procedures which
involve production of the mpl ligand polypeptide by culturing cells
transfected to
express mpl ligand nucleic acid (typically by transforming the cells with an
expression vector) and recovering the polypeptide from the cells. However, it
is
optionally envisioned that the mpl ligand may be produced by homologous
recombination, or with recombinant production methods utilizing control
elements
introduced into cells already containing DNA encoding the mpl ligand. For
example, a
powerful promoter/enhancer element, a suppressor, or an exogenous
transcription
modulatory element may be inserted in the genome of the intended host cell in
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proximity and orientation sufficient to influence the transcription of DNA
encoding the
desired mpl ligand polypeptide. The control element does not encode the mpi
ligand,
rather the DNA is indigenous to the host cell genome. One next screens for
cells making
the receptor polypeptide of this invention, or for increased or decreased
levels of
expression, as desired.
Thus, the invention contemplates a method for producing mpl ligand comprising
inserting into the genome of a cell containing the mpl ligand nucleic acid
molecule a
transcription modulatory element in sufficient proximity and orientation to
the
nucleic acid molecule to influence transcription thereof, with an optional
further step
comprising culturing the cell containing the transcription modulatory element
and the
nucleic acid molecule. The invention also contemplates a host cell containing
the
indigenous mp! ligand nucleic acid molecule operably linked to exogenous
control
sequences recognized by the host cell.
A. Isolation of DNA Encoding mpl ligand Polypeptide
The DNA encoding mpl ligand polypeptide may be obtained from any cDNA
library prepared from tissue believed to possess the mp/ ligand mRNA and to
express
it at a detectable level. The mpl ligand gene may also be obtained from a
genomic DNA
library or by in vitro oligonucleotide synthesis from the complete nucleotide
or amino
acid sequence.
Libraries are screened with probes designed to identify the gene of interest
or
the protein encoded by A. For cDNA expression libraries, suitable probes
include
monoclonal or polyclonal antibodies that recognize and specifically bind to
the mpl
ligand. For cDNA libraries suitable probes include oligonucleotides of about
20-80
bases in length that encode known or suspected portions of the mpl ligand cDNA
from
the same or different species; and/or complementary or homologous cDNAs or
fragments thereof that encode the same or a similar gene. Appropriate probes
for
screening genomic DNA libraries include, but are not limited to,
oligonucleotides,
cDNAs, or fragments thereof that encode the same or a similar gene, and/or
homologous
genomic DNAs or fragments thereof. Screening the cDNA or genomic library with
the
selected probe may be conducted using standard procedures as described in
Chapters
10-12 of Sambrook at at, supra.
An alternative means to isolate the gene encoding mpl ligand is to use PCR
methodology as described in section 14 of Sambrook at at, supra. This method
requires the use of oligonucleotide probes that will hybridize to DNA encoding
the mp!
ligand. Strategies for selection of oligonucleotides are described below.
A preferred method of practicing this invention is to use carefully selected
oligonucleotide sequences to screen cDNA libraries from various tissues,
preferably
human or porcine kidney (adult or fetal) or liver cell lines. For example,
human fetal
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liver cell line cDNA libraries are screened with the oligonucleotide probes.
Alternatively, human genomic libraries may be screened with the
oligonucleotide
probes.
The oligonucleotide sequences selected as probes should be of sufficient
length
and sufficiently unambiguous that false positives are minimized. The actual
nucleotide
sequence(s) is usually designed based on regions of the mpl ligand which have
the least
codon redundancy. The oligonucleotides may be degenerate at one or more
positions.
The use of degenerate oligonucleotides is of particular importance where a
library is
screened from a species in which preferential codon usage is not known.
The oligonucleotide must be labeled such that it can be detected upon
hybridization to DNA in the library being screened. The preferred method of
labeling is
to use ATP (e.g., '32P) and polynucleotide kinase to radiolabel the 5' end of
the
oligonucleotide. However, other methods may be used to label the
oligonucleotide,
Including, but not limited to, biotinylation or enzyme labeling.
Of particular interest is the mpl ligand nucleic acid that encodes a full-
length
mpl ligand polypeptide. In some preferred embodiments, the nucleic acid
sequence
includes the native mpl ligand signal sequence. Nucleic acid having all the
protein
coding sequence is obtained by screening selected cDNA or genomic libraries
using the
deduced amino acid sequence.
B. Amino Acid Sequence Variants of Native mpl ligand
Amino acid sequence variants of mpl ligand are prepared by introducing
appropriate nucleotide changes into the mpl ligand DNA, or by in vitro
synthesis of the
desired mpl ligand polypeptide. Such variants include, for example, deletions
from, or
insertions or substitutions of, residues within the amino acid sequence for
the porcine
mpl ligand. For example, carboxy terminus portions of the mature full length
mpl
ligand may be removed by proteolytic cleavage, either in vivo or in vitro, or
by
cloning and expressing a fragment or the DNA encoding full length mpi ligand
to
produce a biologically active variant. Any combination of deletion, insertion,
and
substitution is made to arrive at the final construct, provided that the final
construct
possesses the desired biological activity. The amino acid changes also may
alter post-
translational processes of the mpl ligand, such as changing the number or
position of
glycosylation sites. For the design of amino acid sequence variants of the mpl
ligand,
the location of the mutation site and the nature of the mutation will depend
on the mpl
ligand characteristic(s) to be modified. The sites for mutation can be
modified
individually or in series, e.g., by (1) substituting first with conservative
amino acid
choices and then with more radical selections depending upon the results
achieved, (2)
deleting the target residue, or (3) inserting residues of the same or a
different class
adjacent to the located site, or combinations of options 1-3.
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A useful method for identification of certain residues or regions of the mpi
ligand polypeptide that are preferred locations for mutagenesis is called
"alanine
scanning mutagenesis," as described by Cunningham and Wells, Science, 244:1081-
1085 [1989]. Here, a residue or group of target residues are identified (e.g.,
charged
residues such as arg, asp, his, lys, and glu) and replaced by any, but
preferably a
neutral or negatively charged, amino acid (most preferably alanine or
polyalanine) to
affect the interaction of the amino acids with the surrounding aqueous
environment in
or outside the cell. Those domains demonstrating functional sensitivity to the
substitutions then are refined by introducing further or other variants at or
for the
sites of substitution. Thus, while the site for introducing an amino acid
sequence
variation is predetermined, the nature of the mutation per se need not be
predetermined. For example, to optimize the performance of a mutation at a
given
site, ale scanning or random mutagenesis is conducted at the target codon or
region and
the expressed mpl ligand variants are screened for the optimal combination of
desired
activity.
There are two principal variables in the construction of amino acid sequence
variants: the location of the mutation site and the nature of the mutation.
For example,
variants of the mpl ligand polypeptide include variants from the mpl ligand
sequence,
and may represent naturally occurring alleles (which will not require
manipulation of
the mpl ligand DNA) or predetermined mutant forms made by mutating the DNA,
either
to arrive at an allele or a variant not found in nature. In general, the
location and
nature of the mutation chosen will depend upon the mpl ligand characteristic
to be
modified.
Amino acid sequence deletions generally range from about 1 to 30 residues,
more preferably about 1 to 10 residues, and typically are contiguous.
Alternatively,
amino acid sequence deletions for the mpl ligand may include a portion of or
the entire
carboxy-terminus glycoprotein domain. Amino acid sequence deletions may also
include one or more of the first 6 amino-terminus residues of the mature
protein.
Optional amino acid sequence deletions comprise one or more residues in one or
more
of the loop regions that exist between the 'helical bundels". Contiguous
deletions
ordinarily are made in even numbers of residues, but single or odd numbers of
deletions are within the scope hereof. Deletions may be introduced into
regions of low
homology among the mpl ligands that share the most sequence identity to modify
the
activity of the mpl ligand. Or deletions may be introduced into regions of low
homology
among human mpl ligand and other mammalian mpl ligand polypeptides that share
the
most sequence identity to the human mpl ligand. Deletions from a mammalian mpl
ligand polypeptide in areas of substantial homology with other mammalian mpl
ligands
will be more likely to modify the biological activity of the mpi ligand more
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significantly. The number of consecutive deletions will be selected so as to
preserve
the tertiary structure of mpl ligands in the affected domain, e.g., beta-
pleated sheet or
alpha helix.
Amino acid sequence insertions include amino- and/or carboxyl-terminal
fusions ranging in length from one residue to polypeptides containing a
hundred or
more residues, as well as intrasequence insertions of single or multiple amino
acid
residues. Intrasequence insertions (i.e., insertions within the mature mot
ligand
sequence) may range generally from about 1 to 10 residues, more preferably 1
to 5,
most preferably 1 to 3. An exemplary preferred fusion is that of mpl ligand or
fragment thereof and another cytokine or fragment thereof. Examples of
terminal
insertions include mature mpl ligand with an N-terminal methionyl residue, an
artifact of the direct expression of mature mpl ligand in recombinant cell
culture, and
fusion of a heterologous N-terminal signal sequence to the N-terminus of the
mature
mpl ligand molecule to facilitate the secretion of mature mp/ ligand from
recombinant
hosts. Such signal sequences generally will be obtained from, and thus
homologous to,
the intended host cell species. Suitable sequences include STII or Ipp for E.
coli, alpha
factor for yeast, and viral signals such as herpes gD for mammalian cells.
Other insertional variants of the mpl ligand molecule include the fusion to
the
N- or C-terminus of mpl ligand of immunogenic polypeptides (i.e., not
endogenous to
the host to which the fusion is administered), e.g., bacterial polypeptides
such as beta-
lactamase or an enzyme encoded by the E. coli trp locus, or yeast protein, and
C-
terminal fusions with proteins having a long half-life such as immunoglobulin
constant regions (or other immunoglobulin regions), albumin, or ferritin, as
described in WO 89/02922 published 6 April 1989.
A third group of variants are amino acid substitution variants. These variants
have at least one amino acid residue in the mpl ligand molecule removed and a
different
residue inserted in its place. The sites of greatest interest for
substitutional
mutagenesis include sites identified as the active site(s) of mpi ligand and
sites where
the amino acids found in other analogues are substantially different in terms
of side-
chain bulk, charge, or hydrophobicity, but where there is also a high degree
of
sequence identity at the selected site among various mpl ligand species and/or
within
the various animal analogues of one mpl ligand member.
Other sites of interest are those in which particular residues of the mpi
ligand
obtained from various family members and/or animal species within one member
are
identical. These sites, especially those falling within a sequence of at least
three other
identically conserved sites, are substituted in a relatively conservative
manner. Such
conservative substitutions are shown in Table 3 under the heading of preferred
substitutions. If such substitutions result in a change in biological
activity, then more
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substantial changes, denominated exemplary substitutions in Table 3, or as
further
described below in reference to amino acid classes, are introduced and the
products
screened.
TABLE 3
Original Exemplary Preferred
Residue Substitutions Substitutions
Ala (A) Val; Leu; Ile Val
Arg (R) Lys; Gin; Asn Lys
Asn (N) Gin; His; Lys; Arg Gin
Asp (D) Glu Glu
Cys (C) Ser Ser
Gin (Q) Asn Asn
Glu (E) Asp Asp
Gly (G) Pro Pro
His (H) Asn; Gin; Lys; Arg Arg
Ile (I) Leu; Val; Met; Ala; Phe;
norleucine Leu
Leu (L) norleucine; Ile; Val;
Met; Ala; Phe Ile
Lys (K) Arg; Gin; Asn Arg
Met (M) Leu; Phe; Ile Leu
Phe (F) Leu; Val; Ile; Ala Leu
Pro (P) Gly Gly
Ser (S) Thr Thr
Thr (T) Ser Ser
Trp (W) Tyr Tyr
Tyr (Y) Trp; Phe; Thr; Ser Phe
Val (V) Ile; Leu; Met; Phe;
Ala; norleucine Leu
Substantial modifications in function or immunological identity of the mpl
ligand are accomplished by selecting substitutions that differ significantly
in their
effect on maintaining (a) the structure of the polypeptide backbone in the
area of the
substitution, for example, as a sheet or helical conformation, (b) the charge
or
hydrophobicity of the molecule at the target site, or (c) the bulk of the side
chain.
Naturally occurring residues are divided into groups based on common side-
chain
properties:
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(1) hydrophobic: norleucine, Met, Ala, Val, Leu, lie;
(2) neutral hydrophilic: Cys, Ser, Thr;
(3) acidic: Asp, Glu;
(4) basic: Asn, Gin, His, Lys, Arg;
(5) residues that influence chain orientation: Gly, Pro; and
(6) aromatic: Trp, Tyr, Phe.
Non-conservative substitutions will entail exchanging a member of one of these
classes for another. Such substituted residues also may be introduced into the
conservative substitution sites or, more preferably, into the remaining (non-
1 0 conserved) sites.
In one embodiment of the invention, it is desirable to inactivate one or more
protease cleavage sites that are present in the molecule. These sites are
identified by
inspection of the encoded amino acid sequence, in the case of trypsin, e.g.,
for an
arginyl or lysinyl residue. When protease cleavage sites are identified, they
are
rendered inactive to proteolytic cleavage by substituting the targeted residue
with
another residue, preferably a basic residue such as glutamine or a hydrophobic
residue such as serine; by deleting the residue; or by inserting a prolyl
residue
Immediately after the residue.
In another embodiment, any methionyl residues other than the starting
methionyl residue of the signal sequence, or any residue located within about
three
residues N- or C-terminal to each such methionyl residue, is substituted by
another
residue (preferably in accordance with Table 3) or deleted. Alternatively,
about 1-
3 residues are inserted adjacent to such sites.
Any cysteine residues not involved in maintaining the proper conformation of
the mpl ligand also may be substituted, generally with serine, to improve the
oxidative
stability of the molecule and prevent aberrant crosslinking. It has been found
that the
first and forth cysteines in the epo domain, numbered from the amino-terminus,
are
necessary for maintaining proper conformation but that the second and third
are not.
Accordingly, the second and third cysteines in the epo domain may be
substituted.
Nucleic acid molecules encoding amino acid sequence variants of mp/ ligand are
prepared by a variety of methods known in the art. These methods include, but
are not
limited to, isolation from a natural source (in the case of naturally
occurring amino
acid sequence variants) or preparation by oligonucleotide-mediated (or site-
directed)
mutagenesis, PCR mutagenesis, and cassette mutagenesis of an earlier prepared
variant or a non-variant version of mpl ligand polypeptide.
Oligonucleotide-mediated mutagenesis is a preferred method for preparing
substitution, deletion, and insertion variants of mp/ ligand DNA. This
technique is well
known in the art as described by Adelman et aL, DNA, 2:183 [1983]. Briefly,
mpl
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ligand DNA is altered by hybridizing an oligonucleotide encoding the desired
mutation to
a DNA template, where the template is the single-stranded form of a plasmid or
bacteriophage containing the unaltered or native DNA sequence of mpl ligand.
After
hybridization, a DNA polymerase is used to synthesize an entire second
complementary
strand of the template that will thus incorporate the oligonucleotide primer,
and will
code for the selected alteration in the mpl ligand DNA.
Generally, oligonucleotides of at least 25 nucleotides in length are used. An
optimal oligonucleotide will have 12 to 15 nucleotides that are completely
complementary to the template on either side of the nucleotide(s) coding for
the
mutation. This ensures that the oligonucleotide will hybridize properly to the
single-stranded DNA template molecule. The oligonucleotides are readily
synthesized
using techniques known in the art such as that described by Crea at at, Proc.
Natl.
Acad. Sol. USA, 75:5765 [1978].
The DNA template can be generated by those vectors that are either derived
from bacteriophage M13 vectors (the commercially available M13mp18 and
M13mp19 vectors are suitable), or those vectors that contain a single-stranded
phage
origin of replication as described by Viera at at, Meth. Enzymol., 153:3
[1987].
Thus, the DNA that is to be mutated may be inserted into one of these vectors
to
generate single-stranded template. Production of the single-stranded template
is
described in Sections 4.21-4.41 of Sambrook et al., Molecular Cloning: A
Laboratory
Manual (Cold Spring Harbor Laboratory Press, NY 1989).
Alternatively, single-stranded DNA template may be generated by denaturing
double-stranded plasmid (or other) DNA using standard techniques.
For alteration of the native DNA sequence (to generate amino acid sequence
variants, for example), the oligonucleotide is hybridized to the single-
stranded
template under suitable hybridization conditions. A DNA polymerizing enzyme,
usually the Klenow fragment of DNA polymerase I, is then added to synthesize
the
complementary strand of the template using the oligonucleotide as a primer for
synthesis. A heteroduplex molecule is thus formed such that one strand of DNA
encodes
the mutated form of the mpl ligand, and the other strand (the original
template)
encodes the native, unaltered sequence of the mpl ligand. This heteroduplex
molecule is
then transformed into a suitable host cell, usually a prokaryote such as E.
coil JM101.
After the cells are grown, they are plated onto agarose plates and screened
using the
oligonucleotide primer radiolabeled with 32-phosphate to identify the
bacterial
colonies that contain the mutated DNA. The mutated region is then removed and
placed
in an appropriate vector for protein production, generally an expression
vector of the
type typically employed for transformation of an appropriate host.
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The method described immediately above may be modified such that a
homoduplex molecule is created wherein both strands of the plasmid contain the
mutation(s). The modifications are as follows: The single-stranded
oligonucleotide is
annealed to the single-stranded template as described above. A mixture of
three
deoxyribonucleotides, deoxyriboadenosine (dATP), deoxyriboguanosine (dGTP),
and
deoxyribothymidine (dTTP), is combined with a modified thio-deoxyribocytosine
called dCTP-(aS) (which can be obtained from the Amersham Corporation). This
mixture is added to the template-oligonucleotide complex. Upon addition of DNA
polymerase to this mixture, a strand of DNA identical to the template, except
for the
mutated bases is generated. In addition, this new strand of DNA will contain
dCTP-(aS)
Instead of dCTP, which serves to protect it from restriction endonuclease
digestion.
After the template strand of the double-stranded heteroduplex is nicked with
an
appropriate restriction enzyme, the template strand can be digested with
Exolll
nuclease or another appropriate nuclease past the region that contains the
site(s) to be
mutagenized. The reaction is then stopped to leave a molecule that is only
partially
single-stranded. A complete double-stranded DNA homoduplex is then formed
using
DNA polymerase in the presence of all four deoxyribonucleotide triphosphates,
ATP,
and DNA ligase. This homoduplex molecule can then be transformed into a
suitable host
cell such as E. coil JM101, as described above.
DNA encoding mpl ligand mutants with more than one amino acid to be
substituted may be generated in one of several ways. If the amino acids are
located
close together in the polypeptide chain, they may be mutated simultaneously
using one
oligonucleotide that codes for all of the desired amino acid substitutions.
If, however,
the amino acids are located some distance from each other (separated by more
than
about ten amino acids), it is more difficult to generate a single
oligonucleotide that
encodes all of the desired changes. Instead, one of two alternative methods
may be
employed.
In the first method, a separate oligonucleotide is generated for each amino
acid
to be substituted. The oligonucleotides are then annealed to the single-
stranded
template DNA simultaneously, and the second strand of DNA that is synthesized
from
the template will encode all of the desired amino acid substitutions.
The alternative method involves two or more rounds of mutagenesis to produce
the desired mutant. The first round is as described for the single mutants:
wild-type
DNA is used for the template, an oligonucleotide encoding the first desired
amino acid
substitution(s) is annealed to this template, and the heteroduplex DNA
molecule is then
generated. The second round of mutagenesis utilizes the mutated DNA produced
in the
first round of mutagenesis as the template. Thus, this template already
contains one or
more mutations. The oligonucleotide encoding the additional desired amino acid
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4b
substitution(s) is then annealed to this template, and the resulting strand of
DNA now
encodes mutations from both the first and second rounds of mutagenesis. This
resultant
DNA can be used as a template in a third round of mutagenesis, and so on.
PCR mutagenesis is also suitable for making amino acid variants of mpi ligand
polypeptide. While the following discussion refers to DNA, it is understood
that the
technique also finds application with RNA. The PCR technique generally refers
to the
following procedure (see Erlich, supra, the chapter by R. Higuchi, p. 61-70):
When
small amounts of template DNA are used as starting material in a PCR, primers
that
differ slightly in sequence from the corresponding region in a template DNA
can be
used to generate relatively large quantities of a specific DNA fragment that
differs
from the template sequence only at the positions where the primers differ from
the
template. For introduction of a mutation into a plasmid DNA, one of the
primers is
designed to overlap the position of the mutation and to contain the mutation;
the
sequence of the other primer must be identical to a stretch of sequence of the
opposite
strand of the plasmid, but this sequence can be located anywhere along the
plasmid
DNA. It is preferred, however, that the sequence of the second primer is
located within
200 nucleotides from that of the first, such that in the end the entire
amplified region
of DNA bounded by the primers can be easily sequenced. PCR amplification using
a
primer pair like the one just described results in a population of DNA
fragments that
differ at the position of the mutation specified by the primer, and possibly
at other
positions, as template copying is somewhat error-prone.
If the ratio of template to product material is extremely low, the vast
majority
of product DNA fragments incorporate the desired mutation(s). This product
material
is used to replace the corresponding region in the plasmid that served as PCR
template
using standard DNA technology. Mutations at separate positions can be
introduced
simultaneously by either using a mutant second primer, or performing a second
PCR
with different mutant primers and ligating the two resulting PCR fragments
simultaneously to the vector fragment in a three (or more)-part ligation.
In a specific example of PCR mutagenesis, template plasmid DNA (1 g) is
linearized by digestion with a restriction endonuclease that has a unique
recognition
site in the plasmid DNA outside of the region to be amplified. Of this
material, 100 ng
Is added to a PCR mixture containing PCR buffer, which contains the four
deoxynucleotide triphosphates and is included in the GeneAmp kits (obtained
from
Perkin-Elmer Cetus, Norwalk, CT and Emeryville, CA), and 25 pmole of each
oligonucleotide primer, to a final volume of 50 l. The reaction mixture is
overlayed
with 35 gi mineral oil. The reaction mixture is denatured for five minutes at
100 C,
placed briefly on ice, and then 1 I Thermus aquaticus (Taq) DNA polymerase (5
units/gi, purchased from Perkin-Elmer Cetus) is added below the mineral oil
layer.
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The reaction mixture is then inserted into a DNA Thermal Cycler (purchased
from
Perkin-Elmer Cetus) programmed as follows:
2 min. 55 C
30 sec. 72 C, then 19 cycles of the following:
30 sec. 94 C
30 sec. 55 C, and
30 sec. 72 C.
At the end of the program, the reaction vial is removed from the thermal
cycler
and the aqueous phase transferred to a new via], extracted with
phenol/chloroform
(50:50 vol), and ethanol precipitated, and the DNA is recovered by standard
procedures. This material is subsequently subjected to the appropriate
treatments for
insertion into a vector.
Another method for preparing variants, cassette mutagenesis, is based on the
technique described by Wells et at, Gene, 34:315 [1985]. The starting material
is
the plasmid (or other vector) comprising the mpl ligand DNA to be mutated. The
codon(s) in the mpl ligand DNA to be mutated are identified. There must be a
unique
restriction endonuclease site on each side of the identified mutation site(s).
If no such
restriction sites exist, they may be generated using the above-described
oligonucleotide-mediated mutagenesis method to introduce them at appropriate
locations in the mpl ligand DNA. After the restriction sites have been
introduced into
the plasmid, the plasmid is cut at these sites to linearize it. A double-
stranded
oligonucleotide encoding the sequence of the DNA between the restriction sites
but
containing the desired mutation(s) is synthesized using standard procedures.
The two
strands are synthesized separately and then hybridized together using standard
techniques. This double-stranded oligonucleotide is referred to as the
cassette. This
cassette is designed to have 3' and 5' ends that are compatible with the ends
of the
linearized plasmid, such that it can be directly ligated to the plasmid. This
plasmid
now contains the mutated mpl ligand DNA sequence.
C. Insertion of Nucleic Acid into a Replicable Vector
The nucleic acid (e.g., cDNA or genomic DNA) encoding native or variant mpl
ligand polypeptide is inserted into a replicable vector for further cloning
(amplification of the DNA) or for expression. Many vectors are available, and
selection of the appropriate vector will depend on (1) whether it is to be
used for DNA
amplification or for DNA expression, (2) the size of the nucleic acid to be
inserted into
the vector, and (3) the host cell to be transformed with the vector. Each
vector
contains various components depending on its function (amplification of DNA or
expression of DNA) and the host cell with which it is compatible. The vector
components generally include, but are not limited to, one or more of the
following: a
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signal sequence, an origin of replication, one or more marker genes, an
enhancer
element, a promoter, and a transcription termination sequence.
(i) Signal Sequence Component
The mpl ligand of this invention may be expressed not only directly, but also
as
a fusion with a heterologous polypeptide, preferably a signal sequence or
other
polypeptide having a specific cleavage site at the N-terminus of the mature
protein or
polypeptide. In general, the signal sequence may be a component of the vector,
or it
may be a part of the mpl ligand DNA that is inserted into the vector. The
heterologous
signal sequence selected should be one that is recognized and processed (i.e.,
cleaved by
a signal peptidase) by the host cell. For prokaryotic host cells that do not
recognize
and process the native mpl ligand signal sequence, the signal sequence is
substituted by
a prokaryotic signal sequence selected, for example, from the group of the
alkaline
phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II leaders. For
yeast
secretion the native signal sequence may be substituted by, e.g., the yeast
invertase,
alpha factor, or acid phosphatase leaders, the C. albicans glucoamylase leader
(EP
362,179 published 4 April 1990), or the signal described in WO 90/13646
published 15 November 1990. In mammalian cell expression the native signal
sequence (i.e., the mpl ligand presequence that normally directs secretion of
mot
ligand from its native mammalian cells in vivo) is satisfactory, although
other
2 0 mammalian signal sequences may be suitable, such as signal sequences from
other mpl
ligand polypeptides or from the same mpl ligand from a different animal
species,
signal sequences from a mpl ligand, and signal sequences from secreted
polypeptides of
the same or related species, as well as viral secretory leaders, for example,
the
herpes simplex gD signal.
(ii) Origin of Replication Component
Both expression and cloning vectors contain a nucleic acid sequence that
enables
the vector to replicate in one or more selected host cells. Generally, in
cloning vectors
this sequence is one that enables the vector to replicate independently of the
host
chromosomal DNA, and includes origins of replication or autonomously
replicating
sequences. Such sequences are well known for a variety of bacteria, yeast, and
viruses. The origin of replication from the plasmid pBR322 is suitable for
most
Gram-negative bacteria, the 2 t plasmid origin is suitable for yeast, and
various viral
origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors
in
mammalian cells. Generally, the origin of replication component is not needed
for
mammalian expression vectors (the SV40 origin may typically be used only
because it
contains the early promoter).
Most expression vectors are "shuttle" vectors, i.e., they are capable of
replication in at least one class of organisms but can be transfected into
another
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organism for expression. For example, a vector is cloned in E. coli and then
the same
vector is transfected into yeast or mammalian cells for expression even though
it is
not capable of replicating independently of the host cell chromosome.
DNA may also be amplified by insertion into the host genome. This is readily
accomplished using Bacillus species as hosts, for example, by including in the
vector a
DNA sequence that is complementary to a sequence found in Bacillus genomic
DNA.
Transfection of Bacillus with this vector results in homologous recombination
with the
genome and insertion of mpl ligand DNA. However, the recovery of genomic DNA
encoding mpl ligand is more complex than that of an exogenously replicated
vector
because restriction enzyme digestion is required to excise the mpl ligand DNA.
(iii) Selection Gene Component
Expression and cloning vectors should contain a selection gene, also termed a
selectable marker. This gene encodes a protein necessary for the survival or
growth of
transformed host cells grown in a selective culture medium. Host cells not
transformed with the vector containing the selection gene will not survive in
the
culture medium. Typical selection genes encode proteins that (a) confer
resistance to
antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or
tetracycline,
(b) complement auxotrophic deficiencies, or (c) supply critical nutrients not
available from complex media, e.g., the gene encoding D-alanine racemase for
Bacilli.
One example of a selection scheme utilizes a drug to arrest growth of a host
cell.
Those cells that are successfully transformed with a heterologous gene express
a
protein conferring drug resistance and thus survive the selection regimen.
Examples
of such dominant selection use the drugs neomycin (Southern et at, J. Molec.
Appl.
Genet., 1:327 [1982]) mycophenolic acid (Mulligan of at, Science, 209:1422
[1980]) or hygromycin Sugden et at, Mol. Cell. Biol., 5:410-413 [1985]). The
three examples given above employ bacterial genes under eukaryotic control to
convey
resistance to the appropriate drug G418 or neomycin (geneticin), xgpt
(mycophenolic
acid), or hygromycin, respectively.
Examples of other suitable selectable markers for mammalian cells are those
that enable the identification of cells competent to take up the mpl ligand
nucleic acid,
such as dihydrofolate reductase (DHFR) or thymidine kinase. The mammalian cell
transformants are placed under selection pressure that only the transformants
are
uniquely adapted to survive by virtue of having taken up the marker. Selection
pressure is imposed by culturing the transformants under conditions in which
the
concentration of selection agent in the medium is successively changed,
thereby leading
to amplification of both the selection gene and the DNA that encodes mpl
ligand
polypeptide. Amplification is the process by which genes in greater demand for
the
production of a protein critical for growth are reiterated in tandem within
the
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chromosomes of successive generations of recombinant cells. Increased
quantities of
mpl ligand are synthesized from the amplified DNA.
For example, cells transformed with the DHFR selection gene are first
identified by culturing all of the transformants in a culture medium that
contains
methotrexate (Mtx), a competitive antagonist of DHFR. An appropriate host cell
when
wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line
deficient in
DHFR activity, prepared and propagated as described by Urlaub and Chasin,
Proc. Natl.
Acad. Scf. USA, 77:4216 [1980]. The transformed cells are then exposed to
increased
levels of Mtx. This leads to the synthesis of multiple copies of the DHFR
gene, and,
concomitantly, multiple copies of other DNA comprising the expression vectors,
such
as the DNA encoding mp! ligand. This amplification technique can be used with
any
otherwise suitable host, e.g., ATCC No. CCL61 CHO-K1, notwithstanding the
presence of
endogenous DHFR if, for example, a mutant DHFR gene that is highly resistant
to Mtx is
employed (EP 117,060). Alternatively, host cells [particularly wild-type hosts
that
contain endogenous DHFR] transformed or co-transformed with DNA sequences
encoding mpl ligand, wild-type DHFR protein, and another selectable marker
such as
aminoglycoside 3' phosphotransferase (APH) can be selected by cell growth in
medium
containing a selection agent for the selectable marker such as an
aminoglycosidic
antibiotic, e.g., kanamycin, neomycin, or G418. See U.S. Patent No. 4,965,199.
A suitable selection gene for use in yeast is the trpl gene present in the
yeast
plasmid YRp7 (Stinchcomb at at, Nature, 282:39 [1979]; Kingsman at at, Gene,
7:141 [1979]; or Tschemper at at, Gene, 10:157 [1980]). The trpi gene provides
a selection marker for a mutant strain of yeast lacking the ability to grow in
tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12
[1977]). The presence of the trpl lesion in the yeast host cell genome then
provides
an effective environment for detecting transformation by growth in the absence
of
tryptophan. Similarly, Leu2-deficient yeast strains (ATCC No. 20,622 or
38,626)
are complemented by known plasmids bearing the Leu2 gene.
(iv) Promoter Component
Expression and cloning vectors usually contain a promoter that is recognized
by
the host organism and is operably linked to the mpl ligand nucleic acid.
Promoters are
untranslated sequences located upstream (5') to the start codon of a
structural gene
(generally within about 100 to 1000 bp) that control the transcription and
translation of particular nucleic acid sequence, such as the mpl ligand
nucleic acid
sequence, to which they are operably linked. Such promoters typically fall
into two
classes, inducible and constitutive. Inducible promoters are promoters that
initiate
increased levels of transcription from DNA under their control in response to
some
change in' culture conditions, e.g., the presence or absence of a nutrient or
a change in
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WO 95118858 21 7 8 4 8 2 PCT/US94/14553 0
temperature. At this time a large number of promoters recognized by a variety
of
potential host cells are well known. These promoters are operably linked to
mpl ligand
encoding DNA by removing the promoter from the source DNA by restriction
enzyme
digestion and inserting the isolated promoter sequence into the vector. Both
the native
mpl ligand promoter sequence and many heterologous promoters may be used to
direct
amplification and/or expression of the mpl ligand DNA. However, heterologous
promoters are preferred, as they generally permit greater transcription and
higher
yields of expressed mpl ligand as compared to the native mpl ligand promoter.
Promoters suitable for use with prokaryotic hosts include the 13-lactamase and
lactose promoter systems (Chang et at, Nature, 275:615 [1978]; and Goeddel et
at,
Nature, 281:544 [1979]), alkaline phosphatase, a tryptophan (trp) promoter
system (Goeddel, Nucleic Acids Res., 8:4057 [1980] and EP 36,776) and hybrid
promoters such as the tac promoter (deBoer et at, Proc. Natl. Acad. Scl. USA,
80:21-
25 [1983]). However, other known bacterial promoters are suitable. Their
nucleotide sequences have been published, thereby enabling a skilled worker
operably
to ligate them to DNA encoding mpl ligand (Siebenlist et at, Cell, 20:269
[1980])
using linkers or adaptors to supply any required restriction sites. Promoters
for use
In bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence
operably
linked to the DNA encoding mpl ligand polypeptide.
Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes
have an AT-rich region located approximately 25 to 30 bases upstream from the
site
where transcription is initiated. Another sequence found 70 to 80 bases
upstream
from the start of transcription of many genes is a CXCAAT region where X may
be any
nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that
may be
the signal for addition of the poly A tail to the 3' end of the coding
sequence. All of these
sequences are suitably inserted into eukaryotic expression vectors.
Examples of suitable promoting sequences for use with yeast hosts include the
promoters for 3-phosphoglycerate kinase (Hitzeman et at, J. Biol. Chem.,
255:2073
[1980]) or other glycolytic enzymes (Hess et at, J. Adv. Enzyme Reg., 7:149
[1968]; and Holland, Biochemistry, 17:4900 [1978]), such as enolase,
glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase,
phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase,
pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and
glucokinase.
Other yeast promoters, which are inducible promoters having the additional
advantage of transcription controlled by growth conditions, are the promoter
regions
for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative
enzymes
associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-
phosphate
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2178482
= WO 95/18858 PCT/US94/14553
1: k' F
dehydrogenase, and enzymes responsible for maltose and galactose utilization.
Suitable
vectors and promoters for use in yeast expression are further described in
Hitzeman
at al., EP 73,657A. Yeast enhancers also are advantageously used with yeast
promoters.
Mpl ligand transcription from vectors in mammalian host cells is controlled,
for example, by promoters obtained from the genomes of viruses such as polyoma
virus, fowlpox virus (UK 2,211,504 published 5 July 1989), adenovirus (such as
Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a
retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), from
heterologous mammalian promoters, e.g., the actin promoter or an
immunoglobulin
promoter, from heat-shock promoters, and from the promoter normally associated
with the mpl ligand sequence, provided such promoters are compatible with the
host
cell systems.
The early and late promoters of the SV40 virus are conveniently obtained as an
SV40 restriction fragment that also contains the SV40 viral origin of
replication.
Fiers at at, Nature, 273:113 [1978]; Mulligan and Berg, Science, 209:1422-
1427 [1980]; Pavlakis at at, Proc. Natl. Acad. Sci. USA, 78:7398-7402 [1981].
The immediate early promoter of the human cytomegalovirus is conveniently
obtained
as a Hindlll E restriction fragment. Greenaway at at, Gene, 18:355-360 [1982].
A
system for expressing DNA in mammalian hosts using the bovine papilloma virus
as a
vector is disclosed in U.S. Patent No. 4,419,446. A modification of this
system is
described in U.S. Patent No. 4,601,978. See also Gray at at, Nature, 295:503-
508
[1982] on expressing cDNA encoding immune interferon in monkey cells; Reyes et
al.,
Nature, 297:598-601 [1982] on expression of human 13-interferon cDNA in mouse
cells under the control of a thymidine kinase promoter from herpes simplex
virus;
Canaani and Berg, Proc. Natl. Acad. Sci. USA, 79:5166-5170 [1982] on
expression of
the human interferon 131 gene in cultured mouse and rabbit cells; and Gorman
at at,
Proc. Nat/. Acad. Sci. USA, 79:6777-6781 [1982] on expression of bacterial CAT
sequences in CV-1 monkey kidney cells, chicken embryo fibroblasts, Chinese
hamster
ovary cells, HeLa cells, and mouse NIH-3T3 cells using the Rous sarcoma virus
long
terminal repeat as a promoter.
(v) Enhancer Element Component
Transcription of a DNA encoding the mpl ligand of this invention by higher
eukaryotes is often increased by inserting an enhancer sequence into the
vector.
Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp,
that act
on a promoter to increase its transcription. Enhancers are relatively
orientation and
position independent, having been found 5' (Laimins at at, Proc. Nat. Acad.
Sol. USA,
78:993 [1981]) and 3' (Lusky at at, Mol. Cell Bio., 3:1108 [1983]) to the
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WO 95/18858 2178482 PCT1US94/14553 =
transcription unit, within an intron (Banerji et at, Cell, 33:729 [1983]), as
well
as within the coding sequence itself (Osborne et at, Mol. Cell Bio., 4:1293
[1984]).
Many enhancer sequences are now known from mammalian genes (globin, elastase,
albumin, a-fetoprotein, and insulin). Typically, however, one will use an
enhancer
from a eukaryotic cell virus. Examples include the SV40 enhancer on the late
side of
the replication origin (bp 100-270), the cytomegalovirus early promoter
enhancer,
the polyoma enhancer on the late side of the replication origin, and
adenovirus
enhancers. See also Yaniv, Nature, 297:17-18 [1982] on enhancing elements for
activation of eukaryotic promoters. The enhancer may be spliced into the
vector at a
position 5' or 3' to the mpl ligand encoding sequence, but is preferably
located at a site
5' from the promoter.
(vi) Transcription Termination Component
Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant,
animal, human, or nucleated cells from other multicellular organisms) will
also
contain sequences necessary for the termination of transcription and for
stabilizing
the mRNA. Such sequences are commonly available from the 5' and, occasionally
3'
untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions
contain
nucleotide segments transcribed as polyadenylated fragments in the
untranslated
portion of the mRNA encoding mpl ligand.
(vii) Construction and Analysis of Vectors
Construction of suitable vectors containing one or more of the above listed
components employs standard ligation techniques. Isolated plasmids or DNA
fragments
are cleaved, tailored, and religated in the form desired to generate the
plasmids
required.
For analysis to confirm correct sequences in plasmids constructed, the
ligation
mixtures are used to transform E. coli K12 strain 294 (ATCC No. 31,446) and
successful transformants selected by ampicillin or tetracycline resistance
where
appropriate. Plasmids from the transformants are prepared, analyzed by
restriction
endonuclease digestion, and/or sequenced by the method of Messing et at,
Nucleic Acids
Res., 9:309 [1981] or by the method of Maxam et al., Methods in Enzymology,
65:499 [1980].
(viii) Transient Expression Vectors
Particularly useful in the practice of this invention are expression vectors
that provide for the transient expression in mammalian cells of DNA encoding
the mp!
ligand polypeptide. In general, transient expression involves the use of an
expression
vector that is able to replicate efficiently in a host cell, such that the
host cell
accumulates many copies of the expression vector and, in turn, synthesizes
high levels
of a desired polypeptide encoded by the expression vector. Sambrook et at,
supra, pp.
-78-
2178482
WO 95/18858 PCT/US94/14553
16.17 - 16.22. Transient expression systems, comprising a suitable expression
vector and a host cell, allow for the convenient positive identification of
polypeptides
encoded by cloned DNAs, as well as for the rapid screening of such
polypeptides for
desired biological or physiological properties. Thus, transient expression
systems are
particularly useful in the invention for purposes of identifying analogues and
variants
of mpl ligand polypeptide that have mpl ligand polypeptide biological
activity.
(ix) Suitable Exemplary Vertebrate Cell Vectors
Other methods, vectors, and host cells suitable for adaptation to the
synthesis of
mpl ligand in recombinant vertebrate cell culture are described in Gething et
at,
Nature, 293:620-625 [1981]; Mantel et at, Nature, 281:40-46 [1979];
Levinson et at; EP 117,060; and EP 117,058. A particularly useful plasmid for
mammalian cell culture expression of mpl ligand is pRK5 (EP 307,247 U. S.
patent
no. 5,258,287) or pSV16B (PCT Publication No. WO 91/08291).
D. Selection and Transformation of Host Cells
Suitable host cells for cloning or expressing the vectors herein are the
prokaryote, yeast, or higher eukaryotic cells described above. Suitable
prokaryotes
include eubacteria, such as Gram-negative or Gram-positive organisms, for
example,
E. coli, Bacilli such as B. subtilis, Pseudomonas species such as P.
aeruginosa,
Salmonella typhimurium, or Serratia marcescans. One preferred E. coli cloning
host
is E. coli 294 (ATCC No, 31,446), although other strains such as E. coil B, E.
coli
X1776 (ATCC No. 31,537), and E. coli W3110 (ATCC No. 27,325) are suitable.
These examples are illustrative rather than limiting. Preferably the host cell
should
secrete minimal amounts of proteolytic enzymes. Alternatively, in vitro
methods of
cloning, e.g., PCR or other nucleic acid polymerase reactions, are suitable.
In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or
yeast are suitable hosts for mpl ligand encoding vectors. Saccharomyces
cerevisiae, or
common baker's yeast, is the most commonly used among lower eukaryotic host
microorganisms. However, a number of other genera, species, and strains are
commonly available and useful herein, such as Schizosaccharomyces pombe (Beach
and
Nurse, Nature, 290:140 [1981]; EP 139,383 published 2 May 1985),
Kluyveromyces hosts (U.S. Patent No. 4,943,529) such as, e.g., K. lactis
(Louvencourt et al., J. Bacteriol., 737 [1983]), K. fragilis, K. bulgaricus,
K.
thermotolerans, and K. marxianus, yarrowia [EP 402,226], Pichia pastoris (EP
183,070; Sreekrishna at at, J. Basic Microbiol., 28:265-278 [1988]), Candida,
Trichoderma reesia (EP 244,234), Neurospora crassa (Case at al., Proc. Natl.
Acad.
Sci. USA, 76:5259-5263 [1979]), and filamentous fungi such as, e.g,
Neurospora,
Penicillium, Tolypocladium (WO 91/00357 published 10 January 1991), and
Aspergillus hosts such as A. nidulans (Ballance et at, Biochem. Biophys. Res.
-79-
WO 95/18858 2 1 7 8 4 8 2 PCT/US94/14553
Commun., 112:284-289 [1983]; Tilburn et at., Gene, 26:205-221 [1983]; Melton
at at, Proc. Natl.. Acad. Sci. USA, 81:1470-1474 [1984]) and A. niger (Kelly
and
Hynes, EMBO J., 4:475-479 [1985]).
Suitable host cells for the expression of glycosylated mpl ligand are derived
from multicellular organisms. Such host cells are capable of complex
processing and
glycosylation activities. In principle, any higher eukaryotic cell culture is
workable,
whether from vertebrate or invertebrate culture. Examples of invertebrate
cells
include plant and insect cells. Numerous baculoviral strains and variants and
corresponding permissive insect host cells from hosts such as Spodoptera
frugiperda
(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),
Drosophila
melanogaster (fruitfly), and Bombyx mori have been identified. See, e.g.,
Luckow at
at, Bio/Technology, 6:47-55 [1988]; Miller at at, Genetic Engineering, Setlow
at
at, eds., Vol. 8 (Plenum Publishing, 1986), pp. 277-279; and Maeda at at,
Nature,
315:592-594 [1985]. A variety of viral strains for transfection are publicly
available, e.g., the L-1 variant of Autographs califomica NPV and the Bm-5
strain of
Bombyx mori NPV, and such viruses may be used as the virus herein according to
the
present invention, particularly for transfection of Spodoptera frugiperda
cells.
Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, and
tobacco can be utilized as hosts. Typically, plant cells are transfected by
incubation
with certain strains of the bacterium Agrobacterium tumefaciens, which has
been
previously manipulated to contain the mpl ligand DNA. During incubation of the
plant
cell culture with A. tumefaciens, the DNA encoding the mpl ligand is
transferred to the
plant cell host such that it is transfected, and will, under appropriate
conditions,
express the mpl ligand DNA. In addition, regulatory and signal sequences
compatible
with plant cells are available, such as the nopaline synthase promoter and
polyadenylation signal sequences. Depicker at a!., J. Mot. App!. Gen., 1:561
[1982].
In addition, DNA segments isolated from the upstream region of the T-DNA 780
gene
are capable of activating or increasing transcription levels of plant-
expressible genes
in recombinant DNA-containing plant tissue. EP 321,196 published 21 June 1989.
However, interest has been greatest in vertebrate cells, and propagation of
vertebrate cells in culture (tissue culture) has become a routine procedure in
recent
years (Tissue Culture, Academic Press, Kruse and Patterson, editors [1973]).
Examples of useful mammalian host cell lines are monkey kidney CV1 line
transformed
by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells
subcloned for growth in suspension culture, Graham at at, J. Gen Virol., 36:59
11977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary
cells/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216
[1980]);
mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 [1980]); monkey
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PCTIUS94/14553
WO 95/18858 = 2178482
kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76,
ATCC
CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney
cells
(MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human
lung
cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary
tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et at, Annals N.Y. Acad.
Sci.,
383:44-68 [1982]); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
Host cells are transfected and preferably transformed with the above-described
expression or cloning vectors of this invention and cultured in conventional
nutrient
media modified as appropriate for inducing promoters, selecting transformants,
or
amplifying the genes encoding the desired sequences.
Transfection refers to the taking up of an expression vector by a host cell
whether or not any coding sequences are in fact expressed. Numerous methods of
transfection are known to the ordinarily skilled artisan, for example, CaPO4
and
electroporation. Successfultransfection is generally recognized when any
indication of
the operation of this vector occurs within the host cell.
Transformation means introducing DNA into an organism so that the DNA is
replicable, either as an extrachromosomal element or by chromosomal integrant.
Depending on the host cell used, transformation is done using standard
techniques
appropriate to such cells. The calcium treatment employing calcium chloride,
as
described in section 1.82 of Sambrook et al., supra, is generally used for
prokaryotes
or other cells that contain substantial cell-wall barriers. Infection with
Agrobacterium tumefaciens is used for transformation of certain plant cells,
as
described by Shaw et at, Gene, 23:315 [1983] and WO 89/05859 published 29 June
1989. In addition, plants may be transfected using ultrasound treatment as
described
in WO 91/00358 published 10 January 1991. For mammalian cells without such
cell walls, the calcium phosphate precipitation method of Graham and van der
Eb,
Virology, 52:456-457 [1978] is preferred. General aspects of mammalian cell
host
system transformations have been described by Axel in U.S. Patent No.
4,399,216
issued 16 August 1983. Transformations into yeast are typically carried out
according to the method of Van Solingen et al., J. Bact., 130:946 [1977] and
Hsiao et
al., Proc. Natl. Acad. Sci. (USA), 76:3829 [1979]. However, other methods for
introducing DNA into cells such as by nuclear injection, electroporation, or
protoplast
fusion may also be used.
E. Culturing the Host Cells
Prokaryotic cells used to produce the mpl ligand polypeptide of this invention
are cultured in suitable media as described generally in Sambrook at at,
supra.
The mammalian host cells used to produce the mpl ligand of this invention may
be cultured in a variety of media. Commercially available media such as Ham's
F10
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WO 95/18858 F- n r F "' 21 7 8 4 8 2 PCT/US94/14553
(Sigma), Minimal Essential Medium ([MEM], Sigma), RPMI-1640 (Sigma), and
Dulbecco's Modified Eagle's Medium ([DMEM], Sigma) are suitable for culturing
the
host cells. In addition, any of the media described in Ham and Wallace, Meth.
Enz.,
68:44 [1979], Barnes and Sato, Anal. Biochem., 102:255 [1980], U.S. Patent No.
4,767,704; 4,657,866; 4,927,762; or 4,560,655; WO 90/03430; WO
87/00195; U.S. Patent Re.' 30,985; or copending U.S.S.N. 07/592,107 or
07/592,141, both filed on 3 October 1990, the disclosures of all of which are
incorporated herein by reference, may be used as culture media for the host
cells. Any
of these media may be supplemented as necessary with hormones and/or other
growth
factors (such as insulin, transferrin, or epidermal growth factor), salts
(such as
sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES),
nucleosides (such as adenosine and thymidine), antibiotics (such as Gentamycin
drug), trace elements (defined as inorganic compounds usually present at final
concentrations in the micromolar range), and glucose or an equivalent energy
source.
Any other necessary supplements may also be included at appropriate
concentrations
that would be known to those skilled in the art. The culture conditions, such
as
temperature, pH, and the like, are those previously used with the host cell
selected for
expression, and will be apparent to the ordinarily skilled artisan.
The host cells referred to in this disclosure encompass cells in in vitro
culture
as well as cells that are within a host animal.
F. Detecting Gene Amplification/Expression
Gene amplification and/or expression may be measured in a sample directly,
for example, by conventional Southern blotting, northern blotting to
quantitate the
transcription of mRNA (Thomas, Proc. Natl. Acad. Sci. USA, 77:5201-5205
[1980]), dot blotting (DNA analysis), or in situ hybridization, using an
appropriately labeled probe, based on the sequences provided herein. Various
labels
may be employed, most commonly radioisotopes, particularly 32P. However, other
techniques may also be employed, such as using biotin-modified nucleotides for
introduction into a polynucleotide. The biotin then serves as the site for
binding to
avidin or antibodies, which may be labeled with a wide variety of labels, such
as
radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies
may be
employed that can recognize specific duplexes, including DNA duplexes, RNA
duplexes,
and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn
may
be labeled and the assay may be carried out where the duplex is bound to a
surface, so
that upon the formation of duplex on the surface, the presence of antibody
bound to the
duplex can be detected.
Gene expression, alternatively, may be measured by immunological methods,
such as immunohistochemical staining of tissue sections and assay of cell
culture or
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WO 95/18858 21 / 8 4 8 2 PCT/US94114553
body fluids, to quantitate directly the expression of gene product. With
immunohistochemical staining techniques, a cell sample is prepared, typically
by
dehydration and fixation, followed by reaction with labeled antibodies
specific for the
gene product coupled, where the labels are usually visually detectable, such
as
enzymatic labels, fluorescent labels, luminescent labels, and the like. A
particularly
sensitive staining technique suitable for use in the present invention is
described by
Hsu et at, Am. J. Clin. Path., 75:734-738 [1980].
Antibodies useful for immunohistochemical staining and/or assay of sample
fluids may be either monoclonal or polyclonal, and may be prepared in any
mammal.
Conveniently, the antibodies may be prepared against a native mpl ligand
polypeptide
or against a synthetic peptide based on the DNA sequences provided herein as
described
further below.
G. Purification of mpl ligand Polypeptide
Mpl ligand preferably is recovered from the culture medium as a secreted
polypeptide, although it also may be recovered from host cell lysates when
directly
expressed without a secretory signal.
When mpl ligand is expressed in a recombinant cell other than one of human
origin, the mpl ligand is completely free of proteins or polypeptides of human
origin.
However, it is still usually necessary to purify mpl ligand from other
recombinant
cell proteins or polypeptides to obtain preparations that are substantially
homogeneous as to the mpl ligand per se. As a first step, the culture medium
or lysate
is centrifuged to remove particulate cell debris. The membrane and soluble
protein
fractions are then separated. Alternatively, a commercially available protein
concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration
units) may be
used. The mpl ligand may then be purified from the soluble protein fraction
and from
the membrane fraction of the culture lysate, depending on whether the mp1
ligand is
membrane bound. Mpl ligand thereafter is purified from contaminant soluble
proteins
and polypeptides by salting out and exchange or chromatographic procedures
employing
various gel matrices. These matrices include; acrylamide, agarose, dextran,
cellulose
and others common to protein purification. Exemplary chromatography procedures
suitable for protein purification include; immunoaffinity (e.g., anti-hmp/
ligand
Mab), receptoraffinity (e.g., mpl-IgG or protein A Sepharose), hydrophobic
interaction chromatography (HIC) (e.g., ether, butyl, or phenyl Toyopearl),
lectin
chromatography (e.g., Con A-Sepharose, lentil-lectin-Sepharose), size
exclusion
(e.g., Sephadex G-75), cation- and anion-exchange columns (e.g., DEAE or
carboxymethyl- and sulfopropyl-cellulose), and reverse-phase high performance
liquid chromatography (RP-HPLC) (see e.g., Urdal of at, J. Chromatog., 296:171
[1984] where two sequential RP-HPLC steps are used to purify recombinant human
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WO 95/18858 2 1 7 8 4 8 2 PCT/US94114553 =
IL-2). Other purification steps optionally include; ethanol precipitation;
ammonium
sulfate precipitation; chromatofocusing; preparative SDS-PAGE, and the like.
Mpl ligand variants in which residues have been deleted, inserted, or
substituted are recovered in the same fashion as native mpl ligand, taking
account of
any substantial changes in properties occasioned by the variation. For
example,
preparation of a mpl ligand fusion with another protein or polypeptide, e.g.,
a
bacterial or viral antigen, facilitates purification; an immunoaffinity column
containing antibody to the antigen can be used to adsorb the fusion
polypeptide.
Immunoaffinity columns such as a rabbit polyclonal anti-mp/ ligand column can
be
employed to absorb the mpl ligand variant by binding it to at least one
remaining
immune epitope. Alternatively, the mpl ligand may be purified by affinity
chromatography using a purified mpl-IgG coupled to a (preferably) immobilized
resin
such as Affi-Gel 10 (Bio-Rad, Richmond, CA) or the like, by means well known
in the
art. A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF) also
may be
useful to inhibit proteolytic degradation during purification, and antibiotics
may be
included to prevent the growth of adventitious contaminants. One skilled in
the art will
appreciate that purification methods suitable for native mp! ligand may
require
modification to account for changes in the character of mp! ligand or its
variants upon
expression in recombinant cell culture.
H. Covalent Modifications of mpt ligand Polypeptide
Covalent modifications of mpl ligand polypeptides are included within the
scope
of this invention. Both native mpl ligand and amino acid sequence variants of
the mpl
ligand may be covalently modified. One type of covalent modification included
within
the scope of this invention is a mpl ligand fragment. Variant mpl ligand
fragments
having up to about 40 amino acid residues may be conveniently prepared by
chemical
synthesis or by enzymatic or chemical cleavage of the full-length or variant
mpl
ligand polypeptide. Other types of covalent modifications of the mpl ligand or
fragments thereof are introduced into the molecule by reacting targeted amino
acid
residues of the mpl ligand or fragments thereof with an organic derivatizing
agent that
is capable of reacting with selected side chains or the N- or C-terminal
residues.
Cysteinyl residues most commonly are reacted with a-haloacetates (and
corresponding amines), such as chloroacetic acid or chloroacetamide, to give
carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are
derivatized by reaction with bromotrifluoroacetone, a-bromo-(i-(5-
imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-
pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-
chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.
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Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH
5.5-7.0 because this agent is relatively specific for the histidyl side chain.
Para-
bromophenacyl bromide also is useful; the reaction is preferably performed in
0.1M
sodium cacodylate at pH 6Ø
Lysinyl and amino terminal residues are reacted with succinic or other
carboxylic acid anhydrides. Derivatization with these agents has the effect of
reversing the charge of the lysinyl residues. Other suitable reagents for
derivatizing
-amino-containing residues include imidoesters such as methyl picolinimidate;
pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic
acid; 0-
methylisourea; 2,4-pentanedione; and transaminase-catalyzed reaction with
glyoxylate.
Arginyl residues are modified by reaction with one or several conventional
reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and
ninhydrin. Derivatization of arginine residues requires that the reaction be
performed in alkaline conditions because of the high pKa of the guanidine
functional
group. Furthermore, these reagents may react with the groups of lysine as well
as the
arginine epsilon-amino group.
The specific modification of tyrosyl residues may be made, with particular
interest in introducing spectral labels into tyrosyl residues by reaction with
aromatic
diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and
tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro
derivatives,
respectively. Tyrosyl residues are iodinated using 1251 or 1311 to prepare
labeled
proteins for use in radioimmunoassay, the chloramine T method described above
being
suitable.
Carboxyl side groups (aspartyl or glutamyl) are selectively modified by
reaction with carbodiimides (R-N=C=N-R'), where R and R' are different alkyl
groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-
ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and
glutamyl residues are converted to asparaginyl and glutaminyl residues by
reaction
with ammonium ions.
Derivatization with bifunctional agents is useful for crosslinking mpl ligand
to
a water-insoluble support matrix or surface for use in the method for
purifying anti-
mp/ ligand antibodies, and vice versa. Commonly used crosslinking agents
include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide
esters, for example, esters with 4-azidosalicylic acid, homobifunctional
imidoesters,
including disuccinimidyl esters such as 3,3'-
dithiobis(succinimidylpropionate), and
bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing
agents
such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable
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WO 95/18858 2178482 PCT/ITS94114553
intermediates that are capable of forming crosslinks in the presence of light.
Alternatively, reactive water-insoluble matrices such as cyanogen bromide-
activated
carbohydrates and the reactive substrates described in U.S. Patent Nos.
3,969,287;
3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for
protein immobilization.
Glutaminyl and asparaginyl residues are frequently deamidated to the
corresponding glutamyl and aspartyl residues, respectively. These residues are
deamidated under neutral or basic conditions. The deamidated form of these
residues
falls within the scope of this invention.
Other modifications include hydroxylation of proline and lysine,
phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation
of the
a-amino groups of lysine, arginine, and histidine side chains (T.E. Creighton,
Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San
Francisco,
pp. 79-86 [1983]), acetylation of the N-terminal amine, and amidation of any C-
terminal carboxyl group.
Another type of covalent modification of the mpl ligand polypeptide included
within the scope of this invention comprises altering the native glycosylation
pattern
of the polypeptide. By altering is meant deleting one or more carbohydrate
moieties
found in native mpl ligand, and/or adding one or more glycosylation sites that
are not
present in the native mpl ligand.
Glycosylation of polypeptides is typically either N-linked or 0-linked. N-
linked refers to the attachment of the carbohydrate moiety to the side chain
of an
asparagine residue. The tripeptide sequences asparagine-X-serine and
asparagine-X-
threonine, where X is any amino acid except proline, are the recognition
sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine side chain.
Thus,
the presence of either of these tripeptide sequences in a polypeptide creates
a potential
glycosylation site. O-linked glycosylation refers to the attachment of one of
the sugars
N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most
commonly
serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be
used.
Addition of glycosylation sites to the mpl ligand polypeptide is conveniently
accomplished by altering the amino acid sequence such that it contains one or
more of
the above-described tripeptide sequences (for N-linked glycosylation sites).
The
alteration may also be made by the addition of, or substitution by, one or
more serine
or threonine residues to the native mpl ligand sequence (for O-linked
glycosylation
sites). For ease, the mpi ligand amino acid sequence is preferably altered
through
changes at the DNA level, particularly by mutating the DNA encoding the mpl
ligand
polypeptide at preselected bases such that codons are generated that will
translate into
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Wo 95/18858 f 2178482
the desired amino acids. The DNA mutation(s) may be made using methods
described
above under the heading of "Amino Acid Sequence Variants of mpl Ligand."
Another means of increasing the number of carbohydrate moieties on the mpl
ligand is by chemical or enzymatic coupling of glycosides to the polypeptide.
These
procedures are advantageous in that they do not require production of the
polypeptide
in a host cell that has glycosylation capabilities for N- or O-linked
glycosylation.
Depending on the coupling mode used, the sugar(s) may be attached to (a)
arginine and
histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those
of
cysteine, (d) free hydroxyl groups such as those of serine, threonine, or
hydroxyproline, (e) aromatic residues such as those of phenylalanine,
tyrosine, or
tryptophan, or (f) the amide group of glutamine. These methods are described
in WO
87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev.
Biochem., pp. 259-306 [1981].
Removal of carbohydrate moieties present on the mpt ligand polypeptide may be
accomplished chemically or enzymatically. Chemical deglycosylation requires
exposure of the polypeptide to the compound trifluoromethanesulfonic acid, or
an
equivalent compound. This treatment results in the cleavage of most or all
sugars
except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while
leaving the polypeptide intact. Chemical deglycosylation is described by
Hakimuddin, et
at, Arch. Biochem. Biophys., 259:52 [1987] and by Edge at al., Anal. Biochem.,
118:131 [1981]. Enzymatic cleavage of carbohydrate moieties on polypeptides
can
be achieved by the use of a variety of endo- and exo-glycosidases as described
by
Thotakura et at, Meth. Enzymot, 138:350 [1987].
Glycosylation at potential glycosylation sites may be prevented by the use of
the
compound tunicamycin as described by Duskin et at, J. Biol. Chem., 257:3105
[1982]. Tunicamycin blocks the formation of protein-N-glycoside linkages.
Another type of covalent modification of mpl ligand comprises linking the mpl
ligand polypeptide to one of a variety of nonproteinaceous polymers, e.g.,
polyethylene
glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in
U.S.
Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337. Mpl ligand polypeptides covalently linked to the forgoing polymers
are
refered to herein as pegylated mpl ligand polypeptides
It will be appreciated that some screening of the recovered mpt ligand variant
will be needed to select the optimal variant for binding to a mpl and having
the
immunological and/or biological activity defined above. One can screen for
stability in
recombinant cell culture or in plasma (e.g., against proteolytic cleavage),
high
affinity to a mpl member, oxidative stability, ability to be secreted in
elevated yields,
and the like. For example, a change in the immunological character of the mpl
ligand
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polypeptide, such as affinity for a given antibody, is measured by a
competitive-type
immunoassay. Other potential modifications of protein or polypeptide
properties such
as redox or thermal stability, hydrophobicity, or susceptibility to
proteolytic
degradation are assayed by methods well known in the art.
17. General Methods for Preparation of Antibodies to mpl Ligand
Antibody Preparation
(1) Polyclonal antibodies
Polyclonal antibodies to mpl ligand polypeptides or fragments are generally
raised in animals by multiple subcutaneous (se) or intraperitoneal (ip)
injections of
the mpl ligand and an adjuvant. It may be useful to conjugate the mpl ligand
or a
fragment containing the target amino acid sequence to a protein that is
immunogenic in
the species to be immunized, e.g., keyhole limpet hemocyanin, serum albumin,
bovine
thyroglobulin, or soybean trypsin inhibitor using a bifunctional or
derivatizing agent,
for example maleimidobenzoyl sulfosuccinimide ester (conjugation through
cysteine
residues), N-hydroxysuccinimide (through lysine residues), glytaraldehyde,
succinic
anhydride, SOCI2, or R1 N=C=NR, where R and R1 are different alkyl groups.
Animals are immunized against the mpl ligand polypeptide or fragment,
immunogenic conjugates or derivatives by combining 1 mg of 1 g of the peptide
or
conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's
complete
adjuvant and injecting the solution intradermally at multiple sites. One month
later
the animals are boosted with 1/5 to 1/10 the original amount of peptide or
conjugate
in Freund's complete adjuvant by subcutaneous injection at multiple sites.
Seven to
14 days later the animals are bled and the serum is assayed for mpl ligand
antibody
titer. Animals are boosted until the titer plateaus. Preferably, the animal
boosted
with the conjugate of the same mpl ligand, but conjugated to a different
protein and/or
through a different cross-linking reagent. Conjugates also can be made in
recombinant
cell culture as protein fusions. Also, aggregating agents such as alum are
used to
enhance the immune response.
(ii) Monoclonal antibodies
Monoclonal antibodies are obtained from a population of substantially
homogeneous antibodies, he., the individual antibodies comprising the
population are
identical except for possible naturally occurring mutations that may be
present in
minor amounts. Thus, the modifier "monoclonal" indicates the character of the
antibody as not being a mixture of discrete antibodies.
For example, the mpl ligand monoclonal antibodies of the invention may be
made using the hybridoma method first described by Kohler & Milstein, Nature,
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WO 95/18858 217 8 4 8 2 PCT/US94/14553
256:495 [1975], or may be made by recombinant DNA methods (U.S. Patent No.
4,816,567 [Cabilly at al.]).
In the hybridoma method, a mouse or other appropriate host animal, such as
hamster is immunized as hereinabove described to elicit lymphocytes that
produce or
are capable of producing antibodies that will specifically bind to the protein
used for
immunization. Alternatively, lymphocytes may be immunized in vitro.
Lymphocytes
then are fused with myeloma cells using a suitable fusing agent, such as
polyethylene
glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles
and
Practice, pp.59-103 [Academic Press, 1986]).
The hybridoma cells thus prepared are seeded and grown in a suitable culture
medium that preferably contains one or more substances that inhibit the growth
or
survival of the unfused, parental myeloma cells. For example, if the parental
myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically will include
hypoxanthine, aminopterin, and thymidine (HAT medium), which substances
prevent
the growth of HGPRT-deficient cells.
Preferred myeloma cells are those that fuse efficiently, support stable high
level expression of antibody by the selected antibody-producing cells, and are
sensitive to a medium such as HAT medium. Among these, preferred myeloma cell
lines
are murine myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse
tumors available from the Salk Institute Cell Distribution Center, San Diego,
California USA, and SP-2 cells available from the American Type Culture
Collection,
Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell
lines also have been described for the production of human monoclonal
antibodies
(Kozbor, J. Immunol., 133:3001 [1984]; Brodeur et a!., Monoclonal Antibody
Production Techniques and Applications, pp.51-63, Marcel Dekker, Inc., New
York,
1987).
Culture medium in which hybridoma cells are growing is assayed for
production of monoclonal antibodies directed against mpl ligand. Preferably,
the
binding specificity of monoclonal antibodies produced by hybridoma cells is
determined
by immunoprecipitation or by an in vitro binding assay, such as
radioimmunoassay
(RIA) or enzyme-linked immunoabsorbent assay (ELISA).
The binding affinity of the monoclonal antibody can, for example, be
determined
by the Scatchard analysis of Munson & Pollard, Anal. Biochem., 107:220 [1980].
After hybridoma cells are identified that produce antibodies of the desired
specificity, affinity, and/or activity, the clones may be subcloned by
limiting dilution
procedures and grown by standard methods (Goding, supra). Suitable culture
media
for this purpose include, for example, Dulbecco's Modified Eagle's Medium or
RPMI-
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WO 95/18858 2178482 PCT/US94/14553
1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites
tumors in an animal.
The monoclonal antibodies secreted by the subclones are suitably separated
from the culture medium, ascites fluid, or serum by conventional
immunoglobulin
purification procedures such as, for example, protein A-Sepharose,
hydroxylapatite
chromatography, gel electrophoresis, dialysis, or affinity chromatography.
DNA encoding the monoclonal antibodies of the invention is readily isolated
and
sequenced using conventional procedures (e.g., by using oligonucleotide probes
that are
capable of binding specifically to genes encoding the heavy and light chains
of murine
antibodies). The hybridoma cells of the invention serve as a preferred source
of such
DNA. Once isolated, the DNA may be placed into expression vectors, which are
then
transfected into host cells such as simian COS cells, Chinese hamster ovary
(CHO)
cells, or myeloma cells that do not otherwise produce immunoglobulin protein,
to
obtain the synthesis of monoclonal antibodies in the recombinant host cells.
The DNA
also may be modified, for example, by substituting the coding sequence for
human
heavy and light chain constant domains in place of the homologous murine
sequences,
(Cabilly et al., supra; Morrison, of at, Proc. Nat. Acad. Sci., 81:6851
[1984]), or
by covalently joining to the immunoglobulin coding sequence all or part of the
coding
sequence for a non-immunoglobulin polypeptide.
Typically such non-immunoglobulin polypeptides are substituted for the
constant domains of an antibody of the invention, or they are substituted for
the
variable domains of one antigen-combining site of an antibody of the invention
to
create a chimeric bivalent antibody comprising one antigen-combining site
having
specificity for a mpt ligand and another antigen-combining site having
specificity for a
different antigen.
Chimeric or hybrid antibodies also may be prepared in vitro using known
methods in synthetic protein chemistry, including those involving crosslinking
agents.
For example, immunotoxins may be constructed using a disulfide exchange
reaction or
by forming a thioether bond. Examples of suitable reagents for this purpose
include
iminothiolate and methyl-4-mercaptobutyrimidate.
For diagnostic applications, the antibodies of the invention typically will be
labeled with a detectable moiety. The detectable moiety can be any one which
is capable
of producing, either directly or indirectly, a detectable signal. For example,
the
detectable moiety may be a radioisotope, such as 3H, 14C, 32p 35S, or 1251, a
fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate,
rhodamine, or luciferin; radioactive isotopic labels, such as, e.g., 1251, 32p
14C, or
3H, or an enzyme, such as alkaline phosphatase, beta-galactosidase or
horseradish
peroxidase.
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WO 95/18858 2 1 7 8 4 8 2 PCTIUS94/14553
Any method known in the art for separately conjugating the antibody to the
detectable moiety may be employed, including those methods described by
Hunter, at
at, Nature, 144:945 [1962]; David, et at, Biochemistry, 13:1014 [1974]; Pain,
et at, J. lmmunol. Meth., 40:219 [1981]; and Nygren, J. Histochem. and
Cytochem.,
30:407 [1982].
The antibodies of the present invention may be employed in any known assay
method, such as competitive binding assays, direct and indirect sandwich
assays, and
immunoprecipitation assays. Zola, Monoclonal Antibodies: A Manual of
Techniques,
pp.147-158 (CRC Press, Inc., 1987).
Competitive binding assays rely on the ability of a labeled standard (which
may
be a mpl ligand or an immunologically reactive portion thereof) to compete
with the
test sample analyte (mpl ligand) for binding with a limited amount of
antibody. The
amount of mpl ligand in the test sample is inversely proportional to the
amount of
standard that becomes bound to the antibodies. To facilitate determining the
amount of
standard that becomes bound, the antibodies generally are insolubilized before
or after
the competition, so that the standard and analyte that are bound to the
antibodies may
conveniently be separated from the standard and analyte which remain unbound.
Sandwich assays involve the use of two antibodies, each capable of binding to
a
different immunogenic portion, or epitope, of the protein (mpl ligand) to be
detected.
In a sandwich assay, the test sample analyte is bound by a first antibody
which is
immobilized on a solid support, and thereafter a second antibody binds to the
analyte,
thus forming an insoluble three part complex. David & Greene, U.S. Patent No.
4,376,110. The second antibody may itself be labeled with a detectable moiety
(direct
sandwich assays) or may be measured using an anti-immunoglobulin antibody that
is
labeled with a detectable moiety (indirect sandwich assay). For example, one
type of
sandwich assay is an ELISA assay, in which case the detectable moiety is an
enzyme
(e.g., horseradish peroxidase).
(iii) Humanized and human antibodies
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized antibody has one or more amino acid residues introduced
into it
from a source which is non-human. These non-human amino acid residues are
often
referred to as "import" residues, which are typically taken from an "import"
variable
domain. Humanization can be essentially performed following the method of
Winter
and co-workers (Jones at at, Nature, 321:522-525 [1986]; Riechmann et at,
Nature, 332:323-327 [1988]; Verhoeyen et at, Science, 239:1534-1536
[1988]), by substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized" antibodies are
chimeric
antibodies (Cabilly at at, supra), wherein substantially less than an intact
human
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WO 95118858 PCrIUS94114553 =
variable domain has been substituted by the corresponding sequence from a non-
human
species. In practice, humanized antibodies are typically human antibodies In
which
some CDR residues and possibly some FR residues are substituted by residues
from
analogous sites In rodent antibodies.
The choice of human variable domains, both light and heavy, to be used in
making the humanized antibodies 1s very important In order to reduce
antigenicity.
According to the so called "best-1It= method, the sequence of the variable
domain of a
rodent antibody Is screened against the entire library of known human variable
domain
sequences. The human sequence which Is closest to that of the rodent is then
accepted as
the human framework (FR) for the humanized antibody (Sims of at, J. ImmunoL,
151:2296 (1993]; Chothla and Leak, J. Mol. Blot., 196:901 (1987]). Another
method uses a particular framework derived from the concensus sequence of all
human
antibodies of a particular subgroup of light or heavy chains. The same
framework may
be used for several different humanized antibodies (Carter of al., Proc. Nat!.
Acad. Sol.
USA, 89:4285 (19921; Presto of a!., J. ImmnoL, 151:2823 (19931).
It Is further Important that antibodies be humanized with retention of high
affinity for the antigen and other favorable biological properties. To achieve
this goal,
according to a preferred method, humanized antibodies are prepared by a
process of
analysis of the parental sequences and various conceptual humanized products
using
three dimensional models of the parental and humanized sequences. Three
dimensional
immunoglobulin models are commonly available and are familiar to those skilled
In the
art. Computer programs are available which Illustrate and display probable
three-
dimensional conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the likely role of
the
residues In the functioning of the candidate immunoglobulin sequence, to,, the
analysis
of residues that Influence the ability of the candidate immunoglobulin to bind
its
antigen. In this way, FR residues can be selected and combined from the
consensus and
import sequence so that the desired antibody characteristic, such as Increased
affinity
for the target antigen(s), Is achieved. In general, the CDR residues are
directly and
most. substantially involved in influencing antigen binding. For further
details see U.S.
Patent 5,821,337 issued October 13, 1998.
Alternatively, it is now possible to produce tranagenic animals (e.g., mice)
that are capable, upon Immunization, of producing a full repertoire of human
antibodies in the absence of endogenous immunoglobulin production. For
example, it
has been described that the homozygous deletion of the antibody heavy chain
Joining
region (JH) gene In chimeric and germ-line mutant mice results In complete
inhibition of endogenous antibogy_praduQ bw-TMnsfar of thg-human-garmJina.
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21 7 8 4 8 2 PCTIUS94/14553
WO 95/18858
immunoglobulin gene array in such germ-line mutant mice will result in the
production of human antibodies upon antigen challenge. See, e.g., Jakobovits
et at,
Proc. Natl. Acad. Sci. USA, 90:2551-255 [1993]; Jakobovits et at, Nature,
362:255-258 [1993]; Bruggermann et at, Year in Immuno., 7:33 [1993]. Human
antibodies can also be produced in phage display libraries (Hoogenboom and
Winter, J.
Mol. Biol. 227, 381 [1991]; Marks et at, J. Mol. Biol. 222, 581 [1991]).
(iv) Bispecific antibodies
Bispecific antibodies are monoclonal, preferably human or humanized,
antibodies that have binding specificities for at least two different
antigens. Methods
for making bispecific antibodies are known in the art.
Traditionally, the recombinant production of bispecific antibodies is based on
the coexpression of two immunoglobulin heavy chain-light chain pairs, where
the two
heavy chains have different specificities (Millstein and Cuello, Nature,
305:537-
539 [1983]). Because of the random assortment of immunoglobulin heavy and
light
chains, these hybridomas (quadromas) produce a potential mixture of 10
different
antibody molecules, of which only one has the correct bispecific structure.
The
purification of the correct molecule, which is usually done by affinity
chromatography
steps, is rather cumbersome, and the product yields are low. Similar
procedures are
disclosed in PCT publication No. WO 93/08829 (published 13 May 1993), and in
Traunecker et at, EMBO, 10:3655-3659 [1991].
According to a different and more preferred approach, antibody variable
domains with the desired binding specificities (antibody-antigen combining
sites) are
fused to immunoglobulin constant domain sequences. The fusion preferably is
with an
immunoglobulin heavy chain constant domain, comprising at least part of the
hinge,
CH2 and CH3 regions. It is preferred to have the first heavy chain constant
region
(CH1) containing the site necessary for light chain binding, present in at
least one of
the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if
desired, the
immunoglobulin light chain, are inserted into separate expression vectors, and
are
cotransfected into a suitable host organism. This provides for great
flexibility in
adjusting the mutual proportions of the three polypeptide fragments in
embodiments
when unequal ratios of the three polypeptide chains used in the construction
provide
the optimum yields. It is, however, possible to insert the coding sequences
for two or
all three polypeptide chains in one expression vector when the expression of
at least
two polypeptide chains in equal ratios results in high yields or when the
ratios are of
no particular significance. In a preferred embodiment of this approach, the
bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with a first
binding
specificity in one arm, and a hybrid immunoglobulin heavy chain-light chain
pair
(providing a second binding specificity) in the other arm. It was found that
this
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CA 02178482 2009-07-20 õr .. _,~ .._ ,..._.. _ . .
WO 95/18858 PCTiUS94/14553 =
asymmetric structure facilitates the separation of the desired blapecific
compound
from unwanted Immunoglobulln chain combinations, as the presence of an
Immunoglobulln fight chain in only one half of the biapeclfic molecule
provides for a
facile way of separation.
For further details of generating bispeclfic antibodies see, for example,
Suresh
of al., Methods In Enzymology, 121:210 11986).
(v) Heteroconjugate antibodies
Heteroconjugate antibodies are also within the scope of the present Invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such
antibodies have, for example, been proposed to target Immune system cells to
unwanted
cells (U.S. Patent No. 4,676,980), and for treatment of HIV Infection (PCT
publication Nos. WO 91/00360 and WO 92100373: EP 03089). Heteroconjugate
antibodies may be made using any convenient cross-linking methods. Suitable
cross-
linking agents are well known In the an, and are disclosed in U.S. Patent No.
4,676,980, along with a number of cross-linking techniques.
IV. Therapeutic Use of the Megakaryocytopoietlc Protein mpl Ligand
The biologically active mpl iigand having hematopoletic effector function and
referred to here as a megakaryocytopoletic or thrombocytopoletic protein (TPO)
may
be used in a sterile pharmaceutical preparation or formulation to stimulate
megakaryocytopoletic or thrombopoletlc activity in patients suffering from
thrombocytopenla due to Impaired production, sequestration, or Increased
destruction
of platelets. Thrombocytopenia-associated bone marrow hypopiasia (e.g.,
apiastic
anemia following chemotherapy or bone marrow transplant) may be effectively
treated
with the compounds of this Invention as well as disorders such as disseminated
Intravascular coagulation (DIC), Immune thrombocytopenia (including HIV-
Induced
ITP and non HIV-Induced ITP), chronic idiopathic thrombocytopenia, congenital
thrombocytopenia, myelodysplasia, and thrombotic thrombocytopenia.
Additionally,
these megakaryocytopoletic proteins may be useful in treating
myeloproliferatlve
thrombocytotic diseases as well as thrombocytosis from inflammatory conditions
and
in Iron deficiency.
Preferred uses of the megakaryocytopoletic or thrombocytopoletlc protein
(TPO) of this invention are In: myeiotoxic chemotherapy for treatment of
leukemia or
solid tumors, myeloablatlve chemotherapy for autologous or allogenetc bone
marrow
transplant, myelodysplasia, Idiopathic apfaatio anemia, congenital
thrombocytopenia,
and immune thrombocytopenis.
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Still other disorders usefully treated with the megakaryocytopoietic proteins
of
this invention include defects or damage to platelets resulting from drugs,
poisoning or
activation on artificial surfaces. In these cases, the instant compounds may
be
employed to stimulate "shedding" of new "undamaged" platelets. For a more
complete
list of useful applications, see the "Background" supra, especially section
(a)-(f) and
references cited therein.
The megakaryocytopoietic proteins of the instant invention may be employed
alone or in combination with other cytokines, hematopoietins, interleukins,
growth
factors, or antibodies in the treatment of the above-identified disorders and
conditions.
Thus, the instant compounds may be employed in combination with other protein
or
peptide having thrombopoietic activity including; G-CSF, GM-CSF, LIF, M-CSF,
IL-1,
IL-3, erythropoietin (EPO), kit ligand, IL-6, and IL-11.
The megakaryocytopoietic proteins of the instant invention are prepared in a
mixture with a pharmaceutically acceptable carrier. This therapeutic
composition can
be administered intravenously or through the nose or lung. The composition may
also
be administered parenterally or subcutaneously as desired. When administered
systematically, the therapeutic composition should be pyrogen-free and in a
parenterally acceptable solution having due regard for pH, isotonicity, and
stability.
These conditions are known to those skilled in the art. Briefly, dosage
formulations of
the compounds of the present invention are prepared for storage or
administration by
mixing the compound having the desired degree of purity with physiologically
acceptable carriers, excipients, or stabilizers. Such materials are non-toxic
to the
recipients at the dosages and concentrations employed, and include buffers
such as
phosphate, citrate, acetate and other organic acid salts; antioxidants such as
ascorbic
acid; low molecular weight (less than about ten residues) peptides such as
polyarginine, proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidinone; amino acids such as
glycine,
glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and
other
carbohydrates including cellulose or its derivatives, glucose, mannose, or
dextrins;
chelating agents such as EDTA; sugar alcohols such as mannitol or sobitol;
counterions
such as sodium and/or nonionic surfactants such as Tween, Pluronics or
polyethyleneglycol.
About 0.5 to 500 mg of a compound or mixture of the megakaryocytopoietic
protein as the free acid or base form or as a pharmaceutically acceptable
salt, is
compounded with a physiologically acceptable vehicle, carrier, excipient,
binder,
preservative, stabilizer, flavor, etc., as called for by accepted
pharmaceutical
practice. The amount of active ingredient in these compositions is such that a
suitable
dosage in the range indicated is obtained.
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Sterile compositions for injection can be formulated according to conventional
pharmaceutical practice. For example, dissolution or suspension of the active
compound in a vehicle such as water or naturally occurring vegetable oil like
sesame,
peanut, or cottonseed oil or a synthetic fatty vehicle like ethyl oleate or
the like may
be desired. Buffers, preservatives, antioxidants and the like can be
incorporated
according to accepted pharmaceutical practice.
Suitable examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the polypeptide, which
matrices
are in the form of shaped articles, e.g., films, or microcapsules. Examples of
sustained-release matrices include polyesters, hydrogels [e.g., poly(2-
hydroxyethyl-
methacrylate) as described by Langer et al., J. Blomed. Mater. Res., 15:167-
277
[1981] and Langer, Chem. Tech., 12:98-105 [1982] or poly(vinylalcohol)],
polylactides (U.S. Patent No. 3,773,919, EP 58,481), copolymers of L-glutamic
acid
and gamma ethyl-L-glutamate (Sidman et at, Biopolymers, 22:547-556 [1983]),
non-degradable ethylene-vinyl acetate (Langer et al., supra), degradable
lactic acid-
glycolic acid copolymers such as the Lupron Depot' (injectable microspheres
composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-
(-)-3-hydroxybutyric acid (EP 133,988).
While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid
enable release of molecules for over 100 days, certain hydrogels release
proteins for
shorter time periods. When encapsulated proteins remain in the body for a long
time,
they may denature or aggregate as a result of exposure to moisture at 37 C,
resulting
in a loss of biological activity and possible changes in immunogenicity.
Rational
strategies can be devised for protein stabilization depending on the mechanism
Involved. For example, if the aggregation mechanism is discovered to be
intermolecular S-S bond formation through disulfide interchange, stabilization
may be
achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and developing
specific
polymer matrix compositions.
Sustained-release megakaryocytopoietic protein compositions also include
liposomally entrapped megakaryocytopoietic protein. Liposomes containing
megakaryocytopoietic protein are prepared by methods known per se: DE
3,218,121;
Epstein et at, Proc. Natl. Acad. Sol. USA, 82:3688-3692 [1985]; Hwang et at,
Proc.
Natl. Acad. Sci. USA, 77:4030-4034 [1980]; EP 52,322; EP 36,676; EP 88,046;
EP 143,949; EP 142,641; Japanese patent application 83-118008; U.S. Patent
Nos.
4,485,045 and 4,544,545; and EP 102,324. Ordinarily the liposomes are of the
small (about 200-800 Angstroms) unilamellar type in which the lipid content is
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WO 95/18858 21 7 8 4 8 2 PCT/US94/14553
greater than about 30 mol. % cholesterol, the selected proportion being
adjusted for
the optimal megakaryocytopoietic protein therapy.
The dosage will be determined by the attending physician taking into
consideration various factors known to modify the action of drugs including
severity
and type of disease, body weight, sex, diet, time and route of administration,
other
medications and other relevant clinical factors. Typically, the daily regimen
will
range from 0.1-100 g/kg body weight. Preferably the dosage will range from
0.1-
50 g/kg body weight. More preferably, the initial dosage will range from 1 to
5
g/kg/day. Optionally, the dosage range will be the same as that of other
cytokines,
especially G-CSF, GM-CSF, and EPO. Therapeutically effective dosages may be
determined by either in vitro or in vivo methods.
EXAMPLES
Without further description, it is believed that one of ordinary skill in the
art
can, using the preceding description and illustrative examples, make and
utilize the
present invention to the fullest extent. The following working examples
therefore
specifically point out preferred embodiments of the present invention, and are
not to
be construed as limiting in any way of the remainder of the disclosure.
EXAMPLE 1
Partial Purification of the Porcine mpl Ligand
Platelet-poor plasma was collected from normal or aplastic anemic pigs. Pigs
were rendered aplastic by irradiation with 900 cGy of total body irradiation
using a
4mEV linear accelerator. The irradiated pigs were supported for 6-8 days with
intramuscular injections of cefazolin. Subsequently, their total blood volume
was
removed under general anesthesia, heparinized, and centrifuged at 1800 x g for
30min. to make platelet-poor plasma. The megakaryocyte stimulating activity
was
found to peak 6 days after irradiation.
Aplastic porcine plasma obtained from irradiated pigs is made 4M with NaCI
and stirred for 30 min. at room temperature. The resultant precipitate is
removed by
centrifugation at 3800 rpm in a Sorvall RC3B and the supernatant is loaded
onto a
Phenyl-Toyopearl column (220 ml) equilibrated in 10 mM NaPO4 containing 4M
NaCl. The column is washed with this buffer until A280 is <0.05 and eluted
with
dH2O. The eluted protein peak is diluted with dH20 to a conductivity of 15mS
and
loaded onto a Blue-Sepharose column equilibrated (240 ml) in PBS.
Subsequently,
the column is washed with 5 column volumes each of PBS and 10mM NaPO4 (pH 7.4)
containing 2M urea. Proteins are eluted from the column with 10mM NaPO4 (pH
7.4)
containing 2M urea and 1M NaCl. The eluted protein peak is made 0.01% octyl
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glucoside(n-octyi p-D-glucopyranoside) and 1 mM each with EDTA and Pefabloc
(Boehinger Mannheim) and loaded directly onto tandemly linked CD4-IgG (Capon,
D.J.
at at. Nature 337:526-531 (19891) and mpl-IgG Ultralink (Pierce) columns (see
below). The CD4-IgG (2 ml) column is removed after the sample is loaded and
the
6 mp/-IgG (4 ml) column Is washed with 10 column volumes each of PBS and PBS
containing 2 M NaCI and eluted with O.1M giyclne-HCI pH 225. Fractions are
collected Into 1/10th volume /M Trls-HCI (pH 8.0).
Analysis of eluted fractions from the mpl-afflnity column by SDS-PAGE (4-
20%, Novex gel) run under reducing conditions, revealed the presence of
several
proteins (Fig. 5). Proteins that silver stain with the strongest Intensity
resolve
with apparent Mr of 66,000, 55,000, 30,000, 28,000 and 14,000. To determine
which of these proteins stimulate proliferation of Ba/F3-mpl cell cultures
these
proteins were eluted from the gel as described in Example 2 below.
Ultralink Affinity Columns
1 5 10-20 mg of mpl-IgG or CD4-IgG in PBS are coupled to 0.5 grams of
Ultralink
resin (Pierce) as described by the manufacturer's instructions.
Construction and Expression of mpl-IgG
A chimeric molecule comprising the entire extraceilular domain of human mpl
(amino acids 1-491) and the Fc region of a human IgG1 molecule was expressed
in
293 cells. A cDNA fragment encoding amino adds 1.491 of human mp/ was obtained
by PCR from a human megakaryocytic CMK cell cDNA library and sequenced. A Clai
site was Inserted at the 5' end and a BstEli site at the 3' end. This fragment
was cloned
upstream of the IgGI Fc coding region in a Bluescript vector between the Clal
and the
BstEli sites after partial digestion of the PCR product with BstEll because of
two other
BstEfl sites present in the DNA encoding the extraceilular domain of mpl. The
BstEli
site Introduced at the 3' end of the mpl PCR product was designed to have the
Fe region
In frame with the mpl extracellular domain. The construct was subcloned into
pRKS-
tkneo vector between the Clal and Xbal sites and transfected Into 293 human
embryonic
kidney cells by the calcium phosphate method. The cells were selected M 0.4
mg/ml
G418 and individual clones were Isolated. Mpi-IgG expression from Isolated
clones
was determined using a human Fc specific ELISA. The best expression clone had
an
expression level of 1-2 mg/ml of mpl-IgG.
Ba/F3 mpl P Expressing Cells
A cDNA corresponding to the entire coding region of human mpl P was cloned
Into pRK5-tkneo which was subsequently linearized with Notl and transfected
Into the
IL-3 dependent cell line Ba/F3 by electroporation (1 x 107 cells, 9605F,
250Volts).
Three days later selection was started In the presence of 2 mg/ml of G418. The
cello
were selected as pools or Individual clones were obtained by limiting dilution
In 96
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217 8 4 8 2 PCT/US94/14553
well plates. Selected cells were maintained in RPMI containing 15% FBS, 1mg
/ml
G418, 20mM Glutamine, 10mM HEPES and 100 g/ml of Pen-Strep. Expression of
mp! P in selected clones was determined by FACS analysis using a anti-mp/ P
rabbit
polyclonal antibody.
Ba/F3 mpl ligand Assay
The mpt ligand assay was conducted as shown is Fig. 2. To determine the
presence of mpl ligand from various sources, the mpl P Ba/F3 cells were
starved of
IL-3 for 24 hours at a cell density of 5 x 105 cells/ml in a humidified
incubator at
37 C in 5% CO2 and air. Following IL-3 starvation the cells were plated out in
96
well culture dishes at a density of 50,000 cells in 200 l of media with or
without
diluted samples and cultured for 24 hrs in a cell culture incubator. 20 l of
serum
free RPMI media containing 1 RCi of 3H-thymidine was added to each well for
the last
6-8 hours. The cells were then harvested on 96 well GF/C filter plates and
washed 5
times with water. The filters were counted in the presence of 40 l of
scintillation
fluid (Microscint 20) in a Packard Top Count counter.
EXAMPLE 2
Highly Purified Porcine mpl Ligand
Gel Elution Protocol
Equal amounts of affinity purified mpi ligand (fraction 6 eluted from the mpl-
IgG column) and 2X Laemmli sample buffer were mixed at room temperature
without
reducing agent and loaded onto a Novex 4-20% polyacrylamide gel as quickly as
possible. The sample was not heated. As a control, sample buffer without
ligand was
run in an adjacent lane. The gel was run at 4-6 C at 135 volts for
approximately 2
1/4 hours. The running buffer was initially at room temperature. The gel was
then
removed from the gel box and the plate on one side of the gel removed.
A replica of the gel was made on nitrocellulose as follows: A piece of
nitrocellulose was wet with distilled water and carefully laid on top of the
exposed gel
face so air bubbles were excluded. Fiducial marks were placed on the
nitrocellulose
and the gel plate so the replica could be accurately repositioned after
staining. After
approximately 2 minutes, the nitrocellulose was carefully removed, and the gel
was
wrapped in plastic wrap and placed in the refrigerator. The nitrocellulose was
stained
with Biorad's gold total protein stain by first agitating it in 3 x 10 ml 0.1%
Tween 20
+ 0.5 M NaCl + 0.1 M Tris-HCI pH 7.5 over approximately 45 minutes followed by
3
x 10 ml purified water over 5 minutes. The gold stain was then added and
allowed to
develop until the bands in the standards were visible. The replica was then
rinsed with
water, placed over the plastic wrap on the gel and carefully aligned with the
fiducial
marks. The positions of the Novex standards were marked on the gel plate and
lines
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were drawn to indicate the cutting positions. The nitrocellulose and plastic
wrap were
then removed and the gel cut along the Indicated lines with a sharp razor
blade. The
cuts were extended beyond the sample lanes so they could be used to determine
the
positions of the slices when the gel was stained. After the slices were
removed, the
remaining gel was silver stained and the positions of the standards and the
cut marks
were measured. The molecular weights corresponding to the cut positions were
determined from the Novex standards.
The 12 gel slices were placed Into the cells In two Blared model 422 electro-
eluters. 12-14K molecular weight cutoff membrane caps were used In the cells.
50
mM ammonium bicarbonate + 0.05% SOS (approximately pH 7.8) was the elution
buffer. One liter of buffer was chilled approximately 1 hour In a 4-6 C
coldroom
before use. Gel slices were eluted at 10 ma/cell (40 v Initially) In a 4.6 C
ooidroom.
Elution took approximately 4 hours. The cells were then carefully removed and
the
liquid above the irk removed with a pipet. The elution chamber was removed and
any
16 liquid above the membrane cap removed with a pipet. The liquid In the
membrane cap
was removed with a Pipetmai' and saved. Fifty pl aliquots of purified water
were then
placed In the cap, agitated and removed until all the SOS crystals dissolved.
These
washes were combined with the saved liquid above. Total elution sample volume
was
300-500 pl per gel slice. Samples were placed in 10 mm Spectrapor 4 12.14K
cutoff dialysis tubing which had been soaked several hours In purified water.
They
were dialyzed overnight at 4-6 C against 800 ml of phosphate buttered saline
(PBS Is
approximately 4 mM In potassium) per 6 samples. The butter was replaced the
next
morning and dialysis continued for 2.5 hours. Samples were then removed from
the
dialysis bags and placed in microfuge tubes. The tubes were placed on be for 1
hour.
microfuged at 14K rpm for 3 min. and the supernatants carefully removed from
the
precipitated SOS. The supernatants were then placed on Ice for approximately 1
hour
more and microfuged again for 4 min. The supernatants were diluted In
phosphate
buffered saline and submitted for the activity assay. Remaining samples were
frozen
at -70 C.
EXAMPLE 3
Porcine mpl Ligand Mlcrosequencing
Fraction 6 (2.8 ml) from the mpl-IgG affinity column was concentrated on a
Microcort*-1b (Amicon). In order to prevent the mpl ilgand from absorbing to
the
Mlcrocon, the membrane was rinsed with 1% SOS and 5 id of 10 % SOS was added
to
fraction 6. Sample buffer (20 pl) of 2X was added to the fraction #6 after
Microcon
concentration (20 pl) and the total volume (40 id) was loaded on a single lane
of a 4-
20 % gradient acrylamide gel (Novex). The gel was run following Nov" protocol.
The
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gel was then equilibrated for 5 min. prior to electroblotting in 10 mM
3-(oyciohexylamino)-1-propanesulfonic acid (CAPS) buffer, pH 11.0, containing
10% methanol. Eiectroblotting onto ImmoblloriPSQ membranes (Millipore) was
carried out for 45 min. at 250 mA constant current in a BloRad Trans-Blot
transfer
cell (32). The PVDF membrane was stained with 0.1% Coomassie Blue R-250 In 40%
methanol, 0.1% acetic acid for I min. and destained for 2-3 min. with 10%
acetic acid
in 50% methanol. The only proteins that were visible in the Mr 18,000.35,000
region of the blot had Mr of 30,000, 28,000 and 22,000.
Bands at 30, 28 and 22 kDa were subjected to protein sequencing. Automated
protein sequencing was performed on a model 470A Applied Biosyslem sequencer
equipped with an on-line PTH analyzer. The sequencer was modified to inject 80-
90%
of the sample (Rodriguez, J. Chromatogr., 350:217.225 11985D. Acetone (-12
hi) was added to solvent A to balance the UV absorbance. Electroblotted
proteins
were sequenced in the Blott cartridge. Peaks were Integrated with Justice
Innovation
software using Nelson Analytical 970 Interfaces. Sequence Interpretation was
performed on a VAX 5900 (Hemel at al., J. Chromatogr., 404:41-52 (1987)). N-
terminal sequences (using one letter code with uncertain residues In
parenthesis) and
quantity of material obtained (in brackets) is presented In Table 2'.
TABLE 2'
Mgt LI and Amino-Terminus Sequences
kDa 11.8 pmoll
1 5 10 15 20 25
S PAP PACOPRLLNKLLRDDHIS VLH G RL SEQIDNO:30
28 kPa (0.6 pmol)
1 5 10 15 20 25
S PAPPAXDPRLLNKLLRDD H VL H QR SEQIDNO:31
18-22 kDa (0.5 pmolj
1 5 10
XPAPPAXDPRLX N SEQIDNO:32
EXAMPLE 4
Liquid Suspension Megakaryocytopolesls Assay
26 Human peripheral stem cells (PSC) (obtained from consenting patients) were
diluted 6 told with IMDM media (Glbco) and centrifuged for 16 min. at room
temp. at
800 x g. The cell pellets were resuspended in IMOM and layered onto 80%
Perooll
(density 1.077 gm/ml ) (Pharmacla) and centrifuged at 800 x g for 30 min. The
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light density mononuclear cells were aspirated at the interface and washed 2x
with
IMDM and plated out at 1-2 x 106 cells/ml In IMDM containing 30% FBS (1 ml
final
volume) In 24 well tissue culture clusters (Costar). APP or mpl Ilgand
depleted APP
was added to 10% and cultures were grown for 12-14 days In a humidified
Incubator
6 at 37 C In 6% CO2 and air. The cultures were also grown In the presence of
10% APP
with 0.5 g of mpl-Igd added at days 0, 2 and 4. APP was depleted of mpl
Ilgand by
passing APP through a mpl IgG affinity column.
To quantitate megakaryocytopolesls In these liquid suspension cultures, a
modification of Solberg at al. was used and employs a radloiabeled murlne IgG
monoclonal antibody (HP1-1D) to GPIfbHIa (provided by Dr. Nichols, Mayo
Clinic).
100 g of HP1-1D (see Grant, B. at al., Blood 09:1334-1339 11987]). was
radiolabeled with lmCi of Na1251 using Enzymobeads (Biorad, Richmond, CA) as
described by the manufacturer's Instructions. Radloiabeled HPI.1D was stored
at
-70 C in PBS containing 0.01% octyl-giucoslde. Typical specific activities
were 1-2
x 106 cpml g (>95% precipated by 12.5% tdchloroacello sold ).
Liquid suspension cultures were set up In triplicate for each experimental
point. After 12-14 days In culture the I ml cultures were transferred to 1.5ml
eppendort tubes and centrifuged at 800 x g for 10 min. at room temp. and the
resultant
cell pellets were resuspended In 100 pi of PBS containing 0.02% EDTA and 20%
bovine call serum. long of 1251-HPi-1 D In 50 i of assay buffer was added to
the
resuspended cultures and Incubated for 60 min. at room temperature (RT) with
occasional shaking. Subsequently, cells were collected by centrifugation at
800 x g for
10 min. at RT and washed 2x with assay buffer. The pellets were counted for 1
min. In
a gamma counter (Packard). Non-specific binding was determined by adding I g
of
unlabeled HP1.1D for 60 min. before the addition of labeled HPi-ID. Specific
binding was determined as the total 1251-HPi-1D bound minus that bound in the
presence of excess unlabeled HPI-1D.
EXAMPLE 5
Oligonucleotlds PCR Primers
Based on the amino-terminal amino acid sequence obtained from the 30 kDa, 28
kDa and 18-22 kDa proteins, degenerate ollgonucleotides were designed for use
as
poiymerase chain reaction (PCR) primers (see Table 4). Two primer pools were
synthesized, a positive sense 20 mer pool encoding amino acid residues 2-8
(mpl 1)
and an anti-sense 21-mer pool complimentary to sequences encoding amino acids
18-
24 (mpl 2).
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TABLE 4 r U L
Degenerate Oligonucleotide Primer Pools
m 11:5' CCN GCN CCN CCN GCN TGY GA 3' (2,048-fold degenerate) SEQ ID NO: 35
mpl 2:5' NCC RTG NAR NAC RTG RTC RTC 3' (2,048-fold degenerate) (SEQ ID NO:
36)
Porcine genomic DNA, isolated from porcine peripheral blood lymphocytes, was
used as a template for PCR. The 50 gl reaction contained: 0.8 g of porcine
genomic
DNA in 10mM Tris-HCI (pH 8.3), 50mM KCI, 3mM MgCI2, 100 g/ml BSA, 400 M
dNTPs, 1 M of each primer pool and 2.5 units of Taq polymerase. Initial
template
denaturation was at 94 C for 8 min. followed by 35 cycles of 45 seconds at 94
C, 1
min. at 55 C and 1 min. at 72 C. The final cycle was allowed to extend for 10
min. at
72 C. PCR products were separated by electrophoresis on a 12% polyacrylamide
gel
and visualized by staining with ethidium bromide. It was reasoned that If the
amino-
terminal amino acid sequence was encoded by a single exon then the correct PCR
product was expected to be 69 bp. A DNA fragment of this size was eluted from
the gel
and subcloned into pGEMT (Promega). Sequences of three clones are shown below
in
Table 5.
TABLE 5
69 bp Porcine Genomic DNA Fragments
gemT3
5'CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA
3'GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG ACGGAGCAC
TGACCACGTT CAGCACGGC [69 bp] (SEQ ID NO: 37)
ACTGGTGCAA GTCGTACCG (SEQ ID NO: 38)
gemT7
5'CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA
3'GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCA.Qj
CGACCACGTC CATCACGGC [69 bp] (SEQ ID NO: 39)
GCTGGTGCAG GTAGTGCCG (SEQ ID NO: 40)
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gemT9
P R L L N K L L R (SEQ ID
NO: 32)
5' CCAGCACCGCCGGCATGTGACCCCCGACTCCTAAATAAACTGCTTCGTGACG
3' GGTCGTGGCGGCCGTACACTGGGGGCTGAGGATTTATTTGACGAAGCACTGC
ATCATGTCTATCACGGT 3' (SEQ ID NO: 41)
TAGTACAGATAGTGCCA 5' (SEQ ID NO: 42)
The position of the PCR primers is indicated by the underlined bases. These
results
verify the N-terminal sequence obtained for amino acids 9-17 for the 30 kDa,
28 kDa
and 18-22 kDa proteins and indicated that this sequence is encoded by a single
axon of
porcine DNA.
EXAMPLE 6
Human mpl Ligand Gene
Based on the results from Example 5, a 45-mer deoxyoligonucleotide, called
pR45, was designed and synthesized to screen a genomic library. The 45-mer had
the
following sequence:
5' GCC-GTG-AAG-GAC-GTG-GTC-GTC-ACG-AAG-CAG-TTT-ATT-TAG-GAG-TCG 3'
(SEQ ID NO: 28)
This oligonucleotide was 32P-labeled with (y32P)-ATP and T4 kinase and used
to screen a human genomic DNA library in %gem12 under low stringency
hybridization
and wash conditions (see Example 7). Positive clones were picked, plaque
purified
and analyzed by restriction mapping and southern blotting. Clone #4 was
selected for
additional analysis.
A 2.8 kb BamHl-Xbal fragment that hybridized to the 45-mer was subcloned
into pBluescript SK-. Partial DNA sequencing of this clone was preformed using
as
primers oligonucleotides specific to the porcine mpl ligand DNA sequence. The
sequence obtained confirmed that DNA encoding the human homolog of the porcine
mpl
ligand had been isolated. An EcoRl restriction site was detected in the
sequence
allowing us to isolate a 390 bp EcoRl-Xbal fragment from the 2.8 kb BamHl-Xbal
and
to subclone it in pBluescript SK-.
Both strands of this fragment were sequenced. The human DNA sequence and
deduced amino acid sequence are shown in Fig. 9 (SEQ ID NOS: 3 & 4). The
predicted
positions of introns in the genomic sequence are also indicated by arrows, and
define a
putative exon ("exon 3").
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Examination of the predicted amino acid sequence confirms that a serine
residue
is the first amino acid of the mature mpl ligand, as determined from direct
amino acid
sequence analysis. Immediately upstream from this codon the predicted amino
acid
sequence is highly suggestive of a signal sequence Involved in secretion of
the mature
mpl iigand. This signal sequence coding region Is probably Interrupted at
nucleotide
position 88 by an intron.
In the 3' direction the axon appears to terminate at nucleotide 198. This axon
therefore encodes a sequence of 42 amino acids, 16 of which are likely to be
part of a
signal sequence and 26 of which are part of the mature human mpl itgand.
EXAMPLE 7
Full Length Human mpl Llgand cDNA
Based on the human *exon 3' sequence (Example 6) two non-degenerate
ollgonucleotldes corresponding to the 3' and 5' ends of the 'exon 3' sequence
were
synthesized (Table 6).
TABLE 6
Human cDNA Non-de enerate PCR Oll onucleotld Primers
Fwd primer. 5' GCT AGC TOT AGA AAT TGC TCC TCG TGG TCA TGC TTC T 3' (SEQ ID
NO:
43
Rvs primer. 5' CAG TCT GCC GTG AAG GAC ATG G 3' (SEO ID NO'
44
These two primers were used in PCR reactions employing as a template DNA
from various human cDNA libraries or I ng of Quick Clone cDNA (Clonetech) from
various tissues using the conditions described in the Example S. The expected
size of
the correct PCR product was 140 bp. After analysis of the PCR products on a
12%
polyacrylamide gel, a DNA fragment of the expected size was detected In cDNA
libraries
prepared from adult kidney, 293 fetal kidney cells and cDNA prepared from
human
fetal liver (Clonetech cat. 87171-1).
A fetal liver cDNA library In I DR2 (Clonetech cat. ii HL1151x) was screened
with the same 45 mar oligonucleotlde used to screen the human genomic library.
The
otigonucleotide was labelled with (y32P)=ATP using T4 polymucleotide kinase.
The
library was screened under low stringency hybridization conditions. The
filters were
prehybridlzed for 2hr then hybridized with the probe overnight at 421C in 20%
formamlde, SxSSC, loxDenhardt's, 0.OSM sodium phosphate (pH 8.5), 0.1% sodium
pyrophosphate, 50 "/ml of sonicated salmon sperm DNA for 16hr. Fitters were
then
rinsed In 2xSSC and then washed once In 0.5xSSC, 0.1% SDS at 4210. Filters
were
exposed overnight to KodaK X-Ray film. Positive clones were picked, plaque
purified
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and the insert size was determined by PCR using oligonucleotides flanking the
BamHl-
Xbal cloning in % DR2 (Clonetech cat. #6475-1). 5 sl of phage stock was used
as a
template source. Initial denaturation was for 7 min. at 94 C followed by 30
cycles of
amplification (1 min. at 94 C, 1 min. at 52 C and 1.5 min. at 72 C). Final
extention
was for 15 min. at 72 C. Clone # FL2b had a 1.8kb insert and was selected for
further analysis.
The plasmid pDR2 (Clonetech, A.DR2 & pDR2 cloning and Expression System
Library Protocol Handbook, p 42) contained within the XDR2 phage arms, was
rescued
as described per manufacturer's instructions (Clonetech, %DR2 & pDR2 cloning
and
Expression System Library Protocol Handbook, p 29-30). Restriction analysis of
the
plasmid pDR2-FL2b with BamHl and Xbal indicated the presence of an internal
BamHl
restriction site in the insert approximately at position 650. Digestion of the
plasmid
with BamHl-Xbal cut the insert in two fragments, one of 0.65 kb and one of
1.15 kb.
DNA sequence was determined with three different classes of template derived
from the
plasmid pDR2-FL2b. DNA sequencing of double-stranded plasmid DNA was carried
out
with the ABi373 (Applied Biosystems, Foster City, California) automated
fluorescent
DNA sequencer using standard protocols for dye-labeled dideoxy nucleoside
triphosphate terminators (dye-terminators) and custom synthesized walking
primers
(Sanger et al., Proc. Natl. Acad. Sc!. USA, 74:5463-5467 [1977]; Smith et at,
Nature, 321:674-679 [1986]). Direct sequencing of polymerase chain reaction
amplified fragments from the plasmid was done with the AB1373 sequencer using
custom primers and dye-terminator reactions. Single stranded template was
generated
with the M13 Janus vector (DNASTAR, Inc., Madison, Wisconsin) (Burland et at,
Nucl. Acids Res., 21:3385-3390 [1993]). BamHI-Xbal (1.15 kb) and BamHl (0.65
kb) fragments were isolated from the plasmid pDR2-FL2b, the ends filled in
with T4
DNA polymerase in the presence of deoxynucleotides, and then subcloned into
the Smal
site of M13 Janus. Sequencing was carried out with standard protocols for dye-
labeled
M13 universal and reverse primers, or walking primers and dye-terminators.
Manual sequencing reactions were carried out on single strand M13 DNA using
walking
primers and standard dideoxy-terminator chemistry (Sanger et at, Proc. Natl.
Acad.
Sci. USA, 74:5463-5467 [1977]), 33P-labeled a-dATP and Sequenase (United
States Biochemical Corp., Cleveland, Ohio). DNA sequence assembly was carried
out
with Sequencher V2.1b12 (Gene Codes Corporation, Ann Arbor, Michigan). The
nucleotide and deduced sequences of hML are provided in Fig. 1 (SEQ ID NO: 1).
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EXAMPLE 8
Isolation of the Human mpl Ligand (TPO) Gene.
Human genomic DNA clones of the TPO gene were isolated by screening a human
genomic library in k-Gem12 with pR45, a previously described oligonucleotide
probe
under low stringency conditions (see Example 7) or under high stringency
conditions with a fragment corresponding to the 3' half of human cDNA coding
for the
mpl ligand (from the BamH1 site to the 3'end). Two overlapping lambda clones
spanning 35 kb were isolated. Two overlapping fragments (BamH1 and EcoRI)
containing the entire TPO gene were subcloned and sequenced. The structure of
the
human gene is composed of 6 exons within 7 kb of genomic DNA (Fig. 14 A, B and
C).
The boundaries of all exon/intron junctions are consistent with the consensus
motif
established for mammalian genes (Shapiro, M. B., et at, Nucl. Acids Res.
15:7155
[1987]). Exon 1 and exon 2 contain 5' untranslated sequence and the initial
four
amino acids of the signal peptide. The remainder of the secretory signal and
the first
26 amino acids of the mature protein are encoded within exon 3. The entire
carboxyl
domain and 3' untranslated as well as -50 amino acids of the erythropoietin-
like
domain are encoded within exon 6. The four amino acids involved in the
deletion
observed within hML-2 (hTPO-2) are encoded at the 5' end of exon 6.
EXAMPLE 9
Transient Expression of Human mpl Ligand (hML)
In order to subclone the full length insert contained in pDR2-FL2b, the
plasmid was digested with Xbal to completion, then partially digested with
BamHl. A
DNA fragment corresponding to the 1.8 kb insert was gel purified and subcloned
in
pRK5 (pRK5-hmpl I) (see U.S. Patent No. 5,258,287 for construction of pRK5)
under the control of the cytomegalovirus immediate early promoter. DNA from
the
construct pRK5-hmp/ I was prepared by the PEG method and transfected in Human
embryonic kidney 293 cells maintained in Dulbecco's modified Eagle's medium
(DMEM) supplemented with F-12 nutrient mixture, 20 mM Hepes (pH 7.4) and 10%
fetal bovine serum. Cells were transfected by the calcium phosphate method as
described (Gorman, C. [1985] in DNA Cloning: A Practical Approach (Glover, D.
M.,
ed) Vol. II, pp. 143-190, IRL Press, Washington, D. C.). 36 h after
transfection, the
supernatant of the transfected cells was assayed for activity in the
proliferation assay
(see Example I). Supernatant of 293 cells transfected with pRK vector only
gave no
stimulation of the Ba/F3 or Ba/F3-mpl cells (Fig. 12A). Supernatant of cells
transfected with pRK5-hmp/ I had no effect on the Ba/F3 cells but dramatically
stimulates the proliferation of Ba/F3-mpl cells (Fig. 12A), indicating that
this
cDNA encodes a functionally active human mpi ligand.
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EXAMPLE 10
Human Mpl Ligand Isoforms
hML2, hML3, and hML4
In order to identify alternatively spliced forms of hML, primers were
synthesized corresponding to each end of the coding sequence of hML. These
primers
were employed in RT-PCR to amplify human adult liver RNA. Additionally,
internal
primers flanking selected regions of interest (see below) were constructed and
similarly employed. Direct sequencing of the ends of the PCR product revealed
a single
sequence corresponding exactly to the sequence of the cDNA isolated from the
human
fetal liver library (see Fig. 1 [SEQ ID NO: 1]). However, a region near the C-
terminus of the EPO-domain (in the middle of the PCR product) exhibited a
complex
sequence pattern sugesting the existence of possible splice variants in that
region. To
Isolate these splice variants, the primers provided in Table 7 flanking the
region of
interest were used in a PCR as templates for human adult liver cDNA.
TABLE 7
Human ML Isoform PCR Primers
hm llcdna.3e1: 5'TGTGGACTT7AGCTTGGGAGAATG3' (SEQ ID NO: 45
pbx4.f2: 5'GGTCCAGGGACCTGGAGGTTTG3' (SEQ ID NO: 46
The PCR products were suboloned blunt into M13. Sequencing of individual
subclones revealed the existence of at least 3 ML isoforms. One of them, hML
(also
refered to as hML332), is the longest form and corresponds exactly to the
sequence
isolated from the fetal liver library. Sequences of the four human mpl ligand
isoforms
listed from longest (hML) to shortest (hML-4) are provided in (Fig. 11 [SEQ ID
NOS: 6, 8, 9 & 10]).
EXAMPLE 11
Construction and Transient Expression of Human Mpl Ligand
Isoforms and Substitutional Variants
hML2, hML3, and hML(R153A, R154A)
Isoforms hML2 and hML3 and substitutional variant hML(R153A, R154A)
were reconstituted from hML using the recombinant PCR technique described by
Russell Higuchi, in PCR Protocols, A guide to Methods and Applications, Acad.
Press,
M.A.Innis, D.H. Gelfand, J.J. Sninsky & T.J. White Editors.
In all contructs, the "outside" primers used are shown in Table 8 and the
"overlapping" primers are shown in Table 9.
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TABLE 8
Outside Primers
Cla.FL.F2:
5'ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G3' (SEQ ID NO:
47)
HMPLL-R:
5'GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA3' (SEQ ID NO:
48)
TABLE 9
Overlapping Primers
h M L-2: - --
MLA4.F: 5'CTC CTT GGA ACC CAG GGC AGG ACC 3' (SEQ ID NO: 49)
MLA4.R 5'GGT CCT GCC CTG GGT TCC AAG GAG 3' (SEQ ID NO: 50)
hML-3:
hMLe116+: 5'CTG CTC CGA GGA AAG GAC TTC TGG ATT 3' (SEQ ID NO: 51)
hMLA1 16-: 5'AAT CCA GAA GTC CTT TCC TCG GAG CAG 3' (SEQ ID NO: 52
hML(R153A. R154A):
RR-KO-F: 5'000 TCT GCG TCG CGG CGG CCC CAC CCA C 3' (SEQ ID NO: 53)
RR-KO-R: 5'GTG GGT GGG GCC GCC GCG ACG CAG AGG G 3' (SEQ ID NO: 54
All PCR amplifications were performed with cloned Pfu DNA polymerase
(Stratagene) using the following conditions: Initial template denaturation was
at 94 C
for 7 min. followed by 30 cycles of 1 min. at 94 C, 1 min. at 55 C and 1.5
min. at
72 C. The final cycle was allowed to extend for 10 min. at 72 C. The final PCR
product
was digested with Clal-Xbal, gel purified and cloned in pRK5tkneo. 293 cells
were
transfected with the various constructs as described above and the supernatant
was
assayed using the Ba/F3-mp/ proliferation assay. hML-2 and hML-3 showed no
detectable activity in this assay, however the activity of hML(R153A, R154A)
was
similar to hML indicating that processing at this di-basic site is not
required for
activity (see Fig. 13).
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EXAMPLE 12
Murine mpl Ligand cDNA
mML, mML-2 and mML-3
Isolation of mML cDNA.
A DNA fragment corresponding to the entire coding region of the human mpi
ligand was obtained by PCR, gel purified and labeled by random priming in the
presence of 32P-dATP and 32P-dCTP. This probe was used to screen 106 clones of
a
mouse liver cDNA library in XGT10 (Clontech cat# ML3001a). Duplicate filters
were hybridized in 35% formamide, 5xSSC, 1oxDenhardt's, 0.1% SDS, 0.05M
sodium phosphate (pH 6.5), 0.1% sodium pyrophosphate, 100 g/ml of sonicated
salmon sperm DNA overnight in the presence of the probe. Filters were rinsed
in
2xSSC and then washed once in 0.5xSSC, 0.1% SDS at 42 C. Hybridizing phage
were
plaque-purified and the cDNA inserts were subcloned into the Eco Ri site of
Bluescript
SK- plasmid. Clone "LD" with a 1.5 kb insert was chosen for further analysis
and both
strands were sequenced as descibed above for the human ML cbNA. The nucleotide
and
deduced amino acid sequences from clone LD are provided in Fig. 14 (SEQ ID
NOS: 1 &
11). The deduced mature ML sequence from this clone was 331 amino acid
residues
long and identified as mML331 (or mML-2 for reasons described below).
Considerable identity for both nucleotide and deduced amino acid sequences
were
observed in the EPO-like domains of these ML's. However, when deduced amino
acid
sequences of human and mouse ML's were aligned, the mouse sequence appeared to
have
a tetrapeptide deletion between human residues 111-114 corresponding to the 12
nucleotide deletion following nucleotide position 618 seen in both the human
(see
above) and pig (see below) cDNA's. Accordingly, additional clones were
examined to
detect possible murine ML isoforms. One clone, "L7", had a 1.4 kb insert with
a 335
amino acid deduced sequence containing the "missing" tetrapeptide LPLQ. This
form is
believed to be the full length murine ML and is refered to as mML or mML335.
The
nucleotide and deduced amino acid sequence for mML are provided in Fig. 16
(SEQ ID
NOS: 12 & 13). Finally, clone "L2" was isolated and sequenced. This clone has
the
116 nucleotide deletion corresponding to hML3 and is therefore denominated mML-
3.
Comparison of the deduced amino acid sequences of these two isoforms is shown
in Fig.
16.
Expression of recombinant mML. Expression vectors for murine ML were
prepared essentially as described in Example 8. Clones encoding mML and mML-2
were subcloned into pRKStkneo, a mammalian expression vector that provides
expression under the control of the CMV promoter and an SV40 polyadenylation
signal.
The resulting expression vectors, mMLpRKtkneo and mML2pRKtkneo were
transiently
transfected into 293 cells using the calcium phosphate method. Following
transient
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transfection, media was conditioned for five days. The cells were maintained
in high
glucose DMEM media supplemented with 10% fetal calf serum.
Expression of murine-mpl (mmpl) in Ba/F3 cells. Stable cell lines
expressing c-mpl were obtained by transfection of mmpl pRKtkneo, essentially
as
described for human mpl in Example 1. Briefly, an expression vector (20 g;
linearized) containing the entire coding sequence of murine mpf (Skoda, R. C.,
at at,
EMBO J. 12:2645-2653 [1993]) was transfected into Ba/F3 cells by
electroporation (5 X 106 cells, 250 volts, 960 pF) followed by selection for
neomycine resistance with 2 mg/ml G418. Expression of mpl was assessed by flow
cytometry analysis using rabbit anti-murine mpl-IgG antisera. Ba/F3 cells were
maintained in RPMI 1640 media from WEHI -3B cells as a source of IL-3.
Supernatants from 293 cells transiently transfected with both mML and mML-2
were
assayed in BaF3 cells transfected with both mmpl and hmpl as described in
Example
1.
EXAMPLE 13
Porcine mpl Ligand cDNA
pML and pML-2
Porcine ML (pML) cDNA was isolated by RACE PCR. Briefly, an oligo dT
primer and 2 specific primers were designed based on the sequence of the exon
of the
porcine ML gene encoding the amino terminus of the ML purified from the
aplastic pig
serum. cDNA prepared from various aplastic pig tissues was obtained and
amplified. A
PCR cDNA product of 1342 bp was found in kidney and subcloned. Several clones
were
sequenced and found to encode the mature pig mpl ligand (not including a
complete
secretion signal). The cDNA was found to encode a 332 amino acid mature
protein
(pML332) having the sequence shown in Fig. 18 (SEQ ID NOS: 9 & 16).
Method:
Isolation of pML gene and cDNA. Genomic clones of the porcine ML gene were
isolated by screening a pig genomic library in EMBL3 (Clontech Inc.) with
pR45. The
library was screened essentially as described in Example 7. Several clones
were
isolated and the exon encoding amino acid sequence identical to that obtained
from the
purified ML was sequenced. Porcine ML cDNA were obtained using a modification
of the
RACE PCR protocol. Two specific ML primers were designed based on the sequence
of
the pig ML gene. Polyadenylated mRNA was isolated from the kidney of aplastic
pigs
essentially as previously described. cDNA was prepared by reverse
transcription with
the BamdT primer
(BamdT: 5' GACTCGAGGATCCATCGA 3')
(SEQ ID NO: 55)
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directed against the polyadenosine tall of the mRNA. An Initial round of PCR
amplification (28 cycles of 95 C for 60 seconds, 58 C for 60 seconds, and 72 C
for
ninety seconds) was conducted using the ML specific h-forward-1 primer
(h-forward-1: 5' GCTAGCTCTAGAAATTGCTCCTCGTGGTCATGCTTCT 3')
(SEQ ID NO: 43)
and the BAMAD primer
(BAMAD: 5' GACTCGAGGATCCATCG 3')
(SEQ ID NO; 58)
in a 100 ml reaction (50 mM KCI, 1.5 mM MgCI, 10 mM Tris pH 8.0, 02 mM
dNTPs,with 0.05 U/mi Amplltaq polymerese (Perkin Elmer Inc.)) The PCR product
was then digested with Cial, extracted with phenol-chloroform (1:1), ethanol
precipitated, and ilgated to 0.1 mg of Bluescript SK- vector (Stratagens Inc.)
that had
been cut with Clal and Kpn 1. After Incubation for two hours at room
temperature,
one fourth of the ligation mixture was added directly to a second round of PCR
(22
16 cycles as described above) using a second ML specific forward-1 primer
(forward-1: 5' GCTAGCTCTAGAAGCCCGGCTCCTCCTGCCTG 3')
(SEQ ID NO: 57)
and T3-21 (an oligonucleotide that binds to a sequence adjacent to the
multiple cloning
region within the Bluescript SK- vector):
(5' CGAAATTAACCCTCACTAAAG 3')
(SEQ ID NO: 58).
The resulting PCR product was digested with Xbal and Clal and subcioned Into
Bluescript SK-. Several clones from indepedent PCR reactions were sequenced.
Again, a second form, designated pML-2, encoding a protein with a 4 amino acid
26 residue deletion (328 amino sold residues) was Identified (see Fig. 21 (SEQ
ID NO:
21)). Comparison of pML and pML-2 amino acid sequences shows the latter form
is
identical except that the tetrapeptide QLPP corresponding to residues 111-114
inclusive have been deleted (see Fig. 22 [SEQ ID NOS: 18 & 211). The four
amino
acid deletions observed In murine, human and porcine ML cDNA occur at
precisely the
same position within the predicted proteins.
EXAMPLE 14
CMK Assay for Thrombopoletin (TPO) induction of Platelet Antigen
GPllbllls Expression
CMK cells are maintained In RMPI 1640 medium (Sigma) supplemented with
10% fetal bovine serum and 10mM giutamine. In preparation for the assay, the
calls
are harvested, washed and resuspended at 5x105 cefis/ml in serum-free GIF
medium
supplemented with 5mg/i bovine Insulin, 10mgA apo-transferrin, 1 X trace
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elements. In a 96-well flat-bottom plate, the TPO standard or experimental
samples
are added to each well at appropriate dilutions in 100 I volumes. 100 I of
the CMK
cell suspension is added to each well and the plates are incubated at 37 C, in
a 5% C02
incubator for 48 hours. After incubation, the plates are spun at 1000rpm at 4
C for
five minutes. Supernatants are discarded and 100 l of the FITC-conjugated
GPllbllla
monoclonal 2D2 antibody is added to each well. Following incubation at 4 C for
1 hour,
plates are spun again at 1000rpm for five minutes. The supernatants containing
unbound antibody are discarded and 200 l of 0.1% BSA-PBS wash is added to
each
well. The 0.1% BSA-PBS wash step is repeated three times. Cells are then
analyzed
on a FASCAN using standard one parameter analysis measuring relative
fluorescence
intensity.
EXAMPLE 15
DAMI Assay for Thrombopoietin (TPO) by Measuring Endomitotic
Activity of DAMI Cells on 96-well Microtiter Plates
DAMI cells are maintained in IMDM + 10% horse serum (Gibco) supplemented
with 10mM glutamine, 100ng/ml Penicillin G, and 50 g/ml streptomycin. In
preparation for the assay, the cells are harvested, washed, and resuspended at
ix106cells/ml in IMDM + 1% horse serum. In a 96-well round-bottom plate, 100
p1 of the TPO standard or experimental samples is added to DAMI cell
suspension. Cells
are then incubated for 48 hours at 37 C in a 5% C02 incubator. After
incubation,
plates are spun in a Sorvall 6000B centrifuge at 1000rpm for five minutes at 4
C.
Supernatants are discarded and 200 l of PBS-0.1% BSA wash step is repeated.
Cells
are fixed by the addition of 200 gi ice-cold 70% Ethanol-PBS and resuspended
by
aspiration. After incubation at 4 C for 15 minutes, the plates are spun at
2000 rpm
for five minutes and 150 l of 1mg/ml RNAse containing 0.1mg/ml propidium
iodide
and 0.05% Tween-20 is added to each well. Following a one hour incubation at
37 C
the changes in DNA content are measured by flow cytometry. Polyploidy is
measured
and quantitated as follows:
Normalized Polyploid Ratio (NPR) = (%Cells in >G2+Mi%Cells in <G2+M) with TPO
(%Cells in >G2+M/%Cells in <G2+M) in control
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EXAMPLE 16
Thrombopoietin (TPO) In Vivo Assay
(Mouse Platelet Rebound Assay)
In Vivo Assay for 35S Determination of Platelet Production
C57BL6 mice (obtained from Charles River) are injected intraperitoneally
(IP) with 1 ml goat anti-mouse platelet serum (6 amps) on day 1 to produce
thrombocytopenia. On days 5 and 6, mice are given two IP injections of the
factor or
PBS as the control. On day 7, thirty Ci of Na235SO4 in 0.1 ml saline are
injected
intravenously and the percent 35S incorporation of the injected dose into
circulating
platelets is measured in blood samples obtained from treated and control mice.
Platelet
counts and leukocyte counts are made at the same time from blood obtained from
the
retro-orbital sinus.
EXAMPLE 17
KIRA ELISA for Thrombopoietin (TPO)
by Measuring Phosphorylation of the mpl-Rse.gD Chimeric Receptor
The human mpl receptor has been disclosed by Vigon et at, PNAS, USA
89:5640-5644 (1992). A chimeric receptor comprising the extracellular domain
(ECD) of the mpl receptor and the transmembrane (TM) and intracellular domain
(ICD) of Rse (Mark et at, J. of Biol. Chem. 269(14):10720-10728 [1994]) with
a carboxyl-terminal flag polypeptide (Le. Rse.gD) was made for use in the KIRA
ELISA
described herein. See Fig. 30 and 31 for a diagrammatic description of the
assay.
(a) Capture agent preparation
Monoclonal anti-gD (clone 5B6) was produced against a peptide from Herpes
simplex virus glycoprotein D (Paborsky et at, Protein Engineering 3(6):547-553
[1990]). The purified stock preparation was adjusted to 3.0mg/ml in phosphate
buffered saline (PBS), pH 7.4 and 1.Oml aliquots were stored at -20' C.
(b) Anti-phosphotyrosine antibody preparation
Monoclonal anti-phosphotyrosine, clone 4G10, was purchased from UBI (Lake
Placid, NY) and biotinylated using long-arm biotin-N-hydroxysuccinamide
(Biotin-
X-NHS, Research Organics, Cleveland, OH).
(c) Ligand
The mpl ligand was prepared by the recombinant techniques described herein.
The purified mpl ligand was stored at 4 'C. as a stock solution.
(d) Preparation of Rse.gD nucleic acid
Synthetic double stranded oligonucleotides were used to reconstitute the
coding
sequence for the C-terminal 10 amino acids (880 - 890) of human Rse and add an
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additional 21 amino acids containing an epitope for the antibody 5B6 and a
stop codon.
Table 10 presents the final sequence of the synthetic portion of the fusion
gene.
TABLE 10
S nthetic Double Stranded Portion of Human Rse Fusion Gene
coding strand:
5'-TGCAGCAAGGGCTACTGCCACACTCGAGCTGCGCAGATGCTAGCCTCAAGA
TGGCTG ATCCAAATCGATTCCGCGGCAAAGATCTTCCGGTCCTGTAGAAGCT-3' (SEQ ID
NO: 59)
noncoding (anti-sense) strand:
5'-AGCTTCTACAGGACCGGAAGATCTTTGCCGCGGAATCGA7TTGGATCAGCCA
TCTTG AGGCTAGCATCTGCGCAGCTCGAGTGTGGCAGTAGCCCTTGCTGCA-3' (SEQ ID
NO: 60)
The synthetic DNA was ligated with the cDNA encoding amino acids 1-880 of
human Rse at the Pstl site beginning at nucleotide 2644 of the published human
Rse
cDNA sequence (Mark et at, Journal of Biological Chemistry 269(14):10720-
10728 [19941) and Hindlll sites in the polylinker of the expression vector
pSV17.ID.LL (See Fig. 32 A-L; SEQ ID NO: 22) to create the expression plasmid
pSV.ID.Rse.gD. Briefly, the expression plasmid comprises a dicistronic primary
transcript which contains sequence encoding DHFR bounded by 5' splice donor
and 3'
splice acceptor intron splice sites, followed by sequence that encodes the
Rse.gD. The
full length (non-spliced) message contains DHFR as the first open reading
frame and
therefore generates DHFR protein to allow selection of stable transformants.
(e) Preparation of mp!-Rse.gD nucleic acid
The expression plasmid pSV.ID.Rse.gD produced as described above was modified
to produce plasmid pSV.ID.M.tmRd6 which contained the coding sequences of the
ECD of
human mpl (amino acids 1-491) fused to the transmembrane domain and
intracellular domain of Rse.gD (amino acids 429-911). Synthetic
oligonucleotides
were used to join the coding sequence of a portion of the extracellular domain
of human
mpl to a portion of the Rse coding sequence in a two step PCR cloning reaction
as
described by Mark et at, J. Biol. Chem. 267:26166-26171 (1992). Primers used
for the first PCR reaction were M1
(5'-TCTCGCTACCGTTTACAG-3')
(SEQ ID NO: 61)
and M2
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(5'-CAG GTACCCACCAG G CGGTCTCG GT-3')
(SEQ ID NO: 62)
with a mpl cDNA template and R1
(5'-G G G C CATG A C A C TGTCAA-3' )
(SEQ ID NO: 63)
and R2
(5'-GACCGCCACCGAGACCGCCTGGTGGGTACCTGTGGTCCTT-3')
(SEQ ID NO: 64)
with a Rse cDNA template. The Pvull-Smal portion of this fusion junction was
used for
the construction of the full-length chimeric receptor.
(f) Cell transformation
DP12.CHO cells (EP 307,247 published 15 March 1989) were electroporated
with pSV.ID.M.tmRd6 which had been linearized at a unique Notl site in the
plasmid
backbone. The DNA was ethanol precipitated after phenol/chloroform extraction
and
was resuspended in 20 I 1/10 Tris EDTA. Then, 10 g of DNA was incubated with
107
CHO DP12 cells in 1 ml of PBS on ice for 10 min. before electroporation at 400
volts
and 330 f. Cells were returned to ice for 10 min. before being plated into non-
selective medium. After 24 hours cells were fed nucleoside-free medium to
select for
stable DHFR+ clones.
(g) Selection of transformed cells for use in the K/RA ELISA
Clones expressing MPL/Rse.gD were identified by western-blotting of whole
cell lysates post-fractionation by SDS-PAGE using the antibody 5B6 which
detects the
gD epitope tag.
(h) Media
Cells were grown in F12/DMEM 50:50 (Gibco/BRL, Life Technologies, Grand
Island, NY). The media was supplemented with 10% diafiltered FBS (HyClone,
Logan,
Utah), 25mM HEPES and 2mM L-glutamine.
(I) KIRA ELISA
Mpl-Rse.gD transformed DP12.CHO cells were seeded (3x104 per well) in the
wells of a flat-bottom-96 well culture plate in 100 gl media and cultured
overnight
at 37 'C in 5% CO2. The following morning the well supernatants were decanted,
and
the plates were lightly tamped on a paper towel. 50 I of media containing
either
experimental samples or 200, 50, 12.5, 3.12, 0.78, 0.19, 0.048 or 0 ng/ml mpl
ligand was then added to each well. The cells were stimulated at 37'C for 30
min., the
well supernatants were decanted, and the plates were once again lightly tamped
on a
paper towel. To lyse the cells and solubilize the chimeric receptors, 100 l
of lysis
buffer was added to each well. Lysis buffer consisted of 150 mM NaCl
containing 50
mM HEPES (Gibco), 0.5 % Triton-X 100 (Gibco), 0.01 % thimerosal, 30 KIU/ml
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aprotinin (ICN Biochemicals, Aurora, OH), 1mM 4-(2-aminoethyl)-benzenesulfonyl
fluoride hydrochloride (AEBSF; ICN Biochemicals), 50 M leupeptin (ICN
Biochemicals), and 2 mM sodium orthovanadate (Na3VO4; Sigma Chemical Co, St.
Louis, MO), pH 7.5. The plate was then agitated gently on a plate shaker
(Bellco
Instruments, Vineland, NJ) for 60 min. at room temperature.
While the cells were being solubilized, an ELISA microtiter plate (Nunc
Maxisorp, Inter Med, Denmark) coated overnight at 4'C with the 5B6 monoclonal
anti-
gD antibody (5.0 gg/ml in 50 mM carbonate buffer, pH 9.6, 100 l/well) was
decanted, tamped on a paper towel and blocked with 150 l/well of Block Buffer
[PBS
containing 0.5 % BSA (Intergen Company, Purchase, NY) and 0.01 % thimerosal]
for
60 min. at room temperature with gentle agitation. After 60 minutes, the anti-
gD 5B6
coated plate was washed 6 times with wash buffer (PBS containing 0.05 % Tween-
20
and 0.01 % thimerosal) using an automated plate washer (ScanWasher 300,
Skatron
Instruments, Inc, Sterling, VA).
The lysate containing solubilized MPURse.gD from the cell-culture microtiter
well was transferred (85 l/well) to anti-gD 5B6 coated and blocked ELISA well
and
was incubated for 2 h at room temperature with gentle agitation. The unbound
mpl-
Rse.gD was removed by washing with wash buffer and 100 l of biotinylated 4G10
(anti-phosphotyrosine) diluted 1:18000 in dilution buffer (PBS containing 0.5
%
BSA, 0.05 % Tween-20, 5 mM EDTA, and 0.01 % thimerosal), i.e. 56 ng/ml was
added to each well. After incubation for 2 hr at room temperature the plate
was washed
and 100 gl of horseradish peroxidase (HRPO)-conjugated streptavidin (Zymed
Laboratories, S. San Francisco, CA) diluted 1:60000 in dilution buffer was
added to
each well. The plate was incubated for 30 minutes at room temperature with
gentle
agitation. The free avidin-conjugate was washed away and 100 p1 freshly
prepared
substrate solution (tetramethyl benzidine [TMB]; 2-component substrate kit;
Kirkegaard and Perry, Gaithersburg, MD) was added to each well. The reaction
was
allowed to proceed for 10 minutes, after which the color development was
stopped by
the addition of 100 l/well 1.0 M H3P04. The absorbance at 450 nm was read
with a
reference wavelength of 650 nm (ABS450/650), using a vmax plate reader
(Molecular Devices, Palo Alto, CA) controlled with a Macintosh Centris 650
(Apple
Computers, Cupertino, CA) and DeltaSoft software (BioMetallics, Inc,
Princeton, NJ).
The standard curve was generated by stimulating dpl2.trkA,B or C.gD cells
with 200, 50, 12.5, 3.12, 0.78, 0.19, 0.048 or 0 ng/ml mpl ligand and
presented as
ng/ml TPO vs. mean ABS450/650 sd using the DeltaSoft program. Sample
concentrations were obtained by interpolation of their absorbance on the
standard
curve and are expressed in terms of ng/ml TPO activity.
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The mpl-ligand was found to be able to activate the mpl-Rse.gD chimeric
receptor in a concentration-dependent and ligand-specific manner. Further, the
mpl-
Rse.gD KIRA-ELISA was found to be tolerant of up to 100% human serum (shown)
or
100% plasma (not shown), allowing the assay to be used to readily screen
patient and
pK samples.
293-produced TPO332 Std Curve
0.9
0.8
B Media
0.7
J 100% Hu Ser
0.6
0.5
2 0.4--
0.3-
0.2.:
0.1
0
0.01 0.1 1 10 100 1000
TPO Conc
(ng/ml)
Summary of TPO EC50's
EC50 ECSO
TPO Form (cells) wt/Vol molarit
Hu TPO 332 (293) 2.56 no/ml 67.4 M
Mu TPO 332 (293) 3.69 n /ml 97.1 M
Hu TPO 153 (293) -41 n /ml -1.08 nM
Hu TPO 155 E. coil 0.44 n /ml 11.6 M
Hu TPO 153met E. coil 0.829 no/ml 21.8 M
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EXAMPLE 18
Receptor Based ELISA for Thrombopoletin (TPO)
ELISA plates were coated with rabbit F(ab')2 anti-human IgG (Fc) in pH 9.6
carbonate buffer at 4 C overnight. Plates were blocked with 0.5 % bovine serum
albumin in PBS at room temperature for one hour. Fermenter harvest containing
the
chimeric receptor, mpl--IgG, was added to the plates and incubated for 2
hours. Twofold
a
serial dilutions (0.39-25 ng/ml) of the standard (TP0332 produced in 293 cells
with the concentration determined by quantitative amino acid analysis) and
serially
diluted samples in 0.5% bovine serum albumin, 0.05% tween 20 were added to the
plates and incubated for 2 hours. Bound TPO was detected with protein A
purified,
biotinylated rabbit antibodies to TPO155 which was produced in E. coli (1 hour
incubation), followed by streptavidin-peroxidase (30 min. incubation) and
3,3',5,5'-tetramethyl benzidine as the substrate. The absorbance was read at
450 nm.
Plates were washed between steps. For data analysis, the standard curve is
fitted using
a four-parameter curve fitting program by Kaleidagraph. Concentrations of the
samples were calculated from the standard curve.
EXAMPLE 19
Expression and Purification of TPO from 293 Celis
1. Preperation of 293 Cell Expression Vectors
A cDNA corresponding to the TPO entire open reading frame was obtained by
PCR using the following oligonucleotides as primers:
TABLE 11
293 PCR Primers
CIa.FL.F: 5' ATC GAT ATC GAT CAG CCA GAC ACC CCG GCC AG 3' (SEQ ID NO:
65)
hmpll-R: 5' GCT AGC TCT AGA CAG GGA AGG GAG CTG TAC ATG AGA 3' (SEQ ID NO:
48)
PRK5-hmp/ I (described in Example 9) was used as template for the reaction
in the presence of pfu DNA polymerase (Stratagene). Initial denaturation was
for 7
min. at 94 C followed by 25 cycles of amplification (1 min. at 94 C, 1 min. at
55 C
and 1 min. at 72 C). Final extension was for 15 min. at 72 C). The PCR product
was
purified and cloned between the restriction sites Clal and Xbal of the plasmid
pRK5tkneo, a pRKS derived vector modified to express a neomycin resistance
gene
under the control of the thymidine kinase promote, to obtain the vector
pRK5tkneo.ORF. A second construct corresponding to the epo homologous domain
was
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generated the same way but using Cla.FL.F as forward primer and the following
reverse
primer:
Arg.STOP.Xba: 5' TCT AGA TCT AGA TCA CCT GAC GCA GAG GGT GGA CC 3'
(SEQ ID NO: 66)
The final construct is called pRK5-tkneoEPO-D. The sequence of both constructs
was
verified as described in Example 7.
2. Transfection of Human Embryonic Kidney cells
These 2 constructs were transfected into Human Embryonic Kidney cells by the
CaPO4 method as described in Example 9. 24 hours after transfection selection
of
neomycin resistant clones was started in the presence of 0.4 mg/ml G418.10 to
15
days later individual colonies were transferred to 96 well plates and allowed
to grow to
confluency. Expression of ML153 or ML332 in the conditioned media from these
clones was assessed using the Ba/F3-mpl proliferation assay (described in
Example
I) .
3. Purification of rhML332
293-rhML332 conditioned media was applied to a Blue-Sepharose
(pharmacia) column that was equilibrated in 10mM sodium phosphate pH 7.4
(buffer
A). The column was subsequently washed with 10 column volumes each of buffer A
and
buffer A containing 2M urea. The column was then eluted with buffer A
containing 2M
urea and 1M NaCl. The Blue-Sepharose elution pool was then directly applied to
a
WGA-Sepharose column equilibrated in buffer A. The WGA-Sepharose column was
then washed with 10 column volumes of buffer A containing 2M urea and 1 M NaCl
and
eluted with the same buffer containing 0.5M N-acetyl-D-glucosamine. The WGA-
Sepharose eluate was applied to a C4-HPLC column (Synchrom, Inc.) equilibrated
in
0.1% TFA. The C4-HPLC column was eluted with discontinuous propanol gradient
(0-
25%, 25-35%, 35-70%). rhML332 was found to elute in the 28-30% propanol
region of the gradient. By SDS-PAGE the purified rhML332 migrates as a broad
band
in the 68-80 kDa region of the gel(see Figure 15).
4. Purification of rhML153
293-rhML153 conditioned media was resolved on Blue-Sepharose as described
for rhML332. The Blue Sepharose eluate was applied directly to a mpi-affinity
column as described above. RhML153 eluted from the mpl-affinity column was
purified to homogeneity using a C4-HPLC column run under the same conditions
as
described for rhML332. By SDS-PAGE the purified rhML153 resolves into 2 major
and 2 minor bands with Mr of _18,000-21,000(see Figure 15).
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EXAMPLE 20
Expression and Purification of TPO from CHO
1. Description of CHO Expression Vectors
The expression vectors used in the electroporation protocols described below
have been designated:
pSV15.ID.LL.MLORF (full length or hTPO332), and
pSVI5.ID.LL.MLEPO-D (truncated or hTPO153)=
The pertinent features of these plasmids are presented in Fig. 23 and 24.
2. Preperation of CHO Expression Vectors
A cDNA corresponding to the hTPO entire open reading frame was obtained by
PCR using the oligonucleotide primers of Table 12.
TABLE 12
CHO Expression Vector PCR Primers
CIa.FL.F2 5' ATC GAT ATC GAT AGC CAG ACA CCC CGG CCA G 3' (SEQ ID NO:
47)
ORF. Sal 5' AGT CGA CGT CGA CGT CGG CAG TGT CTG AGA ACC 3' (SEQ ID NO:
67)
PRK5-hmp/ I (described in Example 7 and 9) was used as template for the
reaction in the presence of pfu DNA polymerase (Stratagene). Initial
denaturation was
for 7 min. at 94 C followed by 25 cycles of amplification (1 min. at 94 C, 1
min. at
55 C and 1 min. at 72 C). Final extension was for 15 min. at 72 C). The PCR
product was purified and cloned between the restriction sites Clal and Sall of
the
plasmid pSVIS.ID.LL to obtain the vector pSV15.ID.LL.MLORF. A second construct
corresponding to the EPO homologous domain was generated the same way but
using
Cla.FL.F2 as forward primer and the following reverse primer:
EPOD.Sal 5' AGT CGA CGT CGA CTC ACC TGA CGC AGA GGG TGG ACC 3'
(SEQ ID NO: 68)
The final construct is called pSV15.ID.LL.MLEPO-D. The sequence of both
constructs
was verified as described in Example 7 and 9.
In essence, the coding sequences for the full length and truncated ligand were
introduced into the multiple cloning site of the CHO expression vector
pSV15.ID.LL.
This vector contains the SV40 early promoter/enhancer region, a modified
splice unit
containing the mouse DHFR cDNA, a multiple cloning site for the introduction
of the
gene of interest (in this case the TPO sequences described) an SV40
polyadenylation
signal and origin of replication and the beta-lactamase gene for plasmid
selection and
amplification in bacteria.
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3. Methodology for Establishing Stable CHO Cell Lines Expressing Recombinant
Human TP0332 and TPO153
a. Description of CHO parent cell line
The host CHO (Chinese Hamster Ovary) cell line used for the expression of the
TPO molecules described herein is known as CHO-DP12 (see EP 307,247 published
March 1989). This mammalian cell line was clonally selected from a
transfection
of the parent line (CHO-K1 DUX-B11(DHFR-)- obtained from Dr. Frank Lee of
Stanford University with the permission of Dr.L. Chasin) with a vector
expressing
preproinsulin to obtain clones with reduced insulin requirements. These cells
are also
10 DHFR minus and clones can be selected for the presence of DHFR cDNA vector
sequences
by growth on medium devoid of nucleoside supplements (glycine, hypoxanthine,
and
thymidine). This selection system for stably expressing CHO cell lines is
commonly
used.
b. Transfection method (electroporation)
15 TPO332 and TP0153 expressing cell lines were generated by transfecting
DP12 cells via electroporation (see e.g. Andreason, G.L. J. Tiss. Cult. Meth.,
15,56
[19931) with linearized pSV15.ID.LL.MLORF or pSVI5.ID.LL.MLEPO-D plasmids
respectively. Three (3) restriction enzyme reaction mixtures were set up for
each
plasmid cutting; 10 g, 25 g and 50 g of the vector with the enzyme NOTI by
standard
molecular biology methods. This restriction site is found only once in the
vector in the
linearization region 3' and outside the TPO ligand transcription units (see
Fig. 23).
The 100 I reactions were set up for overnight incubation at 37 degrees. The
next day
the mixes were phenol-chloroform-isoamyl alcohol (50:49:1) extracted one time
and
ethanol precipitated on dry ice for approximately one hour. The precipitate
was then
collected by a 15 minute microcentrifugation and dried. The linearized DNA was
resuspended into 50 I of Ham's DMEM-F12 1:1 medium supplemented with standard
antibiotics and 2mM glutamine.
Suspension growing DP12 cells were collected, washed one time in the medium
described for resuspending the DNA and finally resuspended in the same medium
at a
concentration of 107 cells per 7504l. Aliquots of cells (750 I) and each
linearized
DNA mix were incubated together at room temperature for one hour and then
transferred to a BRL electroporation chamber. Each reaction mix was then
electroporated in a standard BRL electroporation apparatus at 350 volts set at
33O F
and low capacitance. After electroporation, the cells were allowed to sit in
the
apparatus for 5 minutes and then on ice for an additional 10 minute incubation
period.
The electroporated cells were transferred to 60mm cell culture dishes
containing 5 ml
of standard, complete growth medium for CHO cells (High glucose DMEM-F12 50:50
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without glycine supplemented with 1X GHT, 2mM glutamine, and 5% fetal calf
serum)
and grown overnight in a 5% CO2 cell culture incubator.
c. Selection and screening method
The next day, cells were trypsinized off the plates by standard methods and
transferred to 150mm tissue culture dishes containing DHFR selective medium
(Ham's
DMEM-F12, 1:1 medium described above supplemented with either 2% or 5%
dialyzed
fetal calf serum but devoid of glycine, hypoxanthine and thymidine this is the
standard
DHFR selection medium we use). Cells from each 60mm dish were subsequently
replated into 5 /150 mm dishes. Cells were then incubated for 10 to 15 days(
with
one medium change) at 37 degrees/5% CO2 until clones began to appear and
reached
sizes amenable to transfer to 96 well dishes. Over a period of 4-5 days, cell
lines
were transferred to 96 well dishes using sterile yellow tips on a pipettman
set at
50ml. The cells were allowed to grow to confluency (usually 3-5 days) and then
the
trays were trypsinized and 2 copies of the original tray were reproduced. Two
of these
copies were short term stored in the freezer with cells in each well diluted
into 50 I
of 10%FCS in DMSO. 5 day conditioned serum free medium samples were assayed
from
confluent wells in the third tray for TPO expression via the Ba/F cell based
activity
assay. The highest expressing clones based on this assay were revived from
storage
and scaled up to 2 confluent 150mm T-flasks for transfer to the cell culture
group for
suspension adaptation, re-assay and banking.
d. Amplification Protocol
Several of the highest titer cell lines from the selection described above
were
subsequently put through a standard methotrexate amplification regime to
generate
higher titer clones. CHO cell clones are expanded and plated in 10cm dishes at
4
concentrations of methotrexate (i.e.. 50nM, 100nM, 200nM and 400nM) at two or
three cell numbers (105, 5x105, and 106 cells per dish). These cultures are
then
incubated at 37 degree/5% CO2 until clones are established and amenable to
transfer
to 96 well dishes for further assay. Several high titer clones from this
selection were
again subjected to greater concentrations of methotrexate (i.e. 600nM, 800 nM,
1000nM and 1200nM) and as before resistant clones are allowed to establish and
then
transferred to 96 well dishes and assayed.
4. Culturing Stable CHO Cell Lines Expressing Recombinant Human TP0332 and
TP0 1 53
Banked cells are thawed and the cell population is expanded by standard cell
growth methods in either serum free or serum containing medium. After
expansion to
sufficient cell density, cells are washed to remove spent cell culture media.
Cells are
then cultured by any standard method including; batch, fed-batch or continuous
culture at 25-40 C, neutral pH, with a dissolved 02 content of at least 5%
until the
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constitutively secreted TPO is accumulated. Cell culture fluid is then
separated from
the cells by mechanical means such as centrifugation.
Purification of Recombinant Human TPO from CHO Culture Fluids
Harvested cell culture fluid (HCCF) is directly applied to a Blue Sepharose 6
5 Fast Flow column (Phamacia) equilibrated in 0.01M Na Phosphate pH7.4, 0.15M
NaCl
at a ratio of approximately 100L of HCCF per liter of resin and at a linear
flow rate of
approximately 300 ml/hr/cm2. The column is then washed with 3 to 5 column
volumes of equilibration buffer followed by 3 to 5 column volumes of 0.01 M Na
Phosphate pH7.4, 2.OM urea. The TPO is then eluted with 3 to 5 column volumes
of
0.01M Na Phosphate pH7.4, 2.OM urea, 1.OM NaCl.
The Blue Sepharose Pool containing TPO is then applied to a Wheat Germ Lectin
Sepharose 6MB column (Pharmacia) equilibrated in 0.01 M Na Phosphate pH7.4,
2.OM urea, and 1.OM NaCl at a ratio of from 8 to 16 ml of Blue Sepharose Pool
per ml
of resin at flow rate of approximately 50 ml/hr/cm2. The column is then washed
with
2 to 3 column volumes of equilibration buffer. The TPO is then eluted with 2
to 5
column volumes of 0.01M Na Phosphate pH7.4, 2.0M urea, 0.5M N-acetyl-D-
glucosamine.
The Wheat Germ Lectin Pool is then adjusted to a final concentration of 0.04%
C12E8 and 0.1% trifluroacetic acid (TFA). The resulting pool is applied to a
C4
reverse phase column (Vydac 214TP1022) equilibrated in 0.1% TFA, 0.04% C12E8
at a load of approximately 0.2 to 0.5 mg protein per ml of resin at a flow
rate of 157
ml/hr/cm2.
The protein is eluted in a two phase linear gradient of acetonitrile
containing
0.1% TFA, 0.04% C12E8. The first phase is composed of a linear gradient from 0
to
30% acetonitrile in 15 minutes, The second phase is composed of a linear
gradient
from 30 to 60% acetonitrile in 60 minutes. The TPO elutes at approximately 50%
acetonitrile. A pool is made on the basis of SDS-PAGE.
The C4 Pool is then diluted with 2 volumes of 0.01M Na Phosphate pH7.4,
0.15M NaCl and diafilitered versus approximately 6 volumes of 0.01M Na
Phosphate
pH7.4, 0.15M NaCl on an Amicon YM or like ultrafiltration membrane having a
10,000 to 30,000 Dalton molecular weight cut-off. The resulting diafiltrate
may be
then directly processed or further concentrated by ultrafiltration. The
diafiltrate/concentrate is adjusted to a final concentration of 0.01% Tween-
80.
All or a portion of the diafiltrate/concentrate equivalent to 2 to 5% of the
calculated column volume is then applied to a Sephacryl S-300 HR column
(Pharmacia) equilibrated in 0.O1M Na Phosphate pH7.4, 0.15M NaCl, 0.01% Tween-
80 and chromatographed at a flow rate of approximately 17 ml/hr/cm2. The TPO
containing fractions which are free of aggregate and proteolytic degradation
products
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are pooled on the basis of SDS-PAGE. The resulting pool is filtered on a 0.22
filter,
Millex-GV or like, and stored at 2-8 C.
EXAMPLE 21
Transformation and Induction of TPO Protein Synthesis in E. coli
1. Construction of E. coil TPO expression vectors
The plasmids pMP21, pMP151, pMP41, pMP57 and pMP202 are all designed
to express the first 155 amino acids of TPO downstream of a small leader which
varies
among the different constructs. The leaders provide primarily for high level
translation initiation and rapid purification. The plasmids pMP210-1, -T8, -
21,
-22, -24, -25 are designed to express the first 153 amino acids of TPO
downstream
of an initiation methionine and differ only in the codon usage for the first 6
amino acids
of TPO, while the plasmid pMP251 is a derivative of pMP210-1 in which the
carboxy
terminal end of TPO is extended by two amino acids. All of the above plasmids
will
produce high levels of intracellular expression of TPO in E. coli upon
induction of the
tryptophan promoter (Yansura, D. G. et. at Methods in Enzymology ( Goeddel, D.
V.,
Ed.) 185:54-60, Academic Press, San Diego [1990]). The plasmids pMP1 and
pMP172 are intermediates in the construction of the above TPO intracellular
expression plasmids.
(a) Plasmid pMPf
The plasmid pMP1 is a secretion vector for the first 155 amino acids of TPO,
and was constructed by ligating together 5 fragments of DNA as shown in Fig.
33. The
first of these was the vector pPho2l in which the small Mlul-BamHl fragment
had
been removed. pPho2l is a derivative of phGH1 (Chang, C. N. et. at, Gene
55:189-
196 [19871) in which the human growth hormone gene has been replaced with the
E.
coli phoA gene, and a Mlul restriction site has been engineered into the
coding sequence
for the Sill signal sequence at amino acids 20-21.
The next two fragments, a 258 base pair Hinfl-Pstl piece of DNA from pRK5-
hmplI (Example 9) encoding TPO amino acids 19-103, and the following synthetic
DNA encoding amino acids 1-18
5'-CGCGTATGCCAGCCCGGCTCCTCCTGCTTGTGACCTCCGAGTCCTCAGTAAACTGCTTCG
TG
ATACGGTCGGGCCGAGGAGGACGAACACTGGAGGCTCAGGAGTCATTTGACGAAGC
ACTGA-5'
(SEQ ID NO: 69)
(SEQ ID NO: 70)
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were preligated with T4-DNA ligase, and second cut with Pstl. The fourth was a
152
base pair Pstl-Haelll fragment from pRK5hmpll encoding amino acids 104-155 of
TPO. The last was a 412 base pair Stul-BamHl fragment from pdh108 containing
the
lambda to transcriptional terminator as previously described (Scholtissek, S.
at. al.,
NAR 15:3185 [1987]).
(b) Plasmid pMP21
The plasmid pMP21 is designed to express the first 155 amino acids of TPO
with the aid of a 13 amino acid leader comprising part of the STII signal
sequence. It
was constructed by ligating together three (3) DNA fragments as shown in Fig.
34,
the first of these being the vector pVEG31 in which the small Xbal-Sphl
fragment had
been removed. The vector pVEG31 is a derivative of pHGH2O7-1 (de Boer, H. A.
of. at.
, In Promoter Structure and Function (Rodriguez, R. L. and Chamberlain, M. J.
, Ed),
462, Praeger, New York [1982]) in which the human growth hormone gene has been
replaced by the gene for vascular endothelial growth factor ( this identical
vector
fragment can be obtained from this latter plasmid).
The second part in the ligation was a synthetic DNA duplex with the following
sequence:
5'-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAA
TTAATACTTTTTCTTATAGCGTAAAGAAGAATTGCGC-5'
(SEQ ID NO: 71)
(SEQ ID NO: 72)
The last piece was a 1072 base pair Mlul-Sphl fragment from pMP1 encoding 155
amino acids of TPO.
(c) Plasmid pMP151
The plasmid pMP151 is designed to express the first 155 amino acids of TPO
downstream of a leader comprising 7 amino acids of the STII signal sequence, 8
histidines, and a factor Xa cleavage site. As shown in Fig. 35, pMP151 was
constructed by ligating together three DNA fragments, the first of these being
the
previously described vector pVEG31 from which the small Xbal-Sphl fragment had
been removed. The second was a synthetic DNA duplex with the following
sequence:
5'-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACATCGAAG
GTCGTAGCC
TTAATACTfTfTCTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTTC
CAGCAT-5'
(SEQ ID NO: 73)
(SEQ ID NO: 74)
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The last was a 1064 base pair Bgll-Sphl fragment from pMP11 encoding 154 amino
acids of TPO. The plasmid pMP11 is identical to pMP1 with the exception of a
few
codon changes in the STII signal sequence( this fragment can be obtained from
pMP1).
(d) Plasmid pMP202
The plasmid pMP202 is very similar to the expression vector pMP151 with
the exception that the factor Xa cleavage site in the leader has been replaced
with a
thrombin cleavage site. As shown in Fig. 36, pMP202 was constructed by
ligating
together three DNA fragments. The first of these was the previously described
pVEG31
in which the small Xbal-Sphl fragment had been removed. The second was a s
thetic
DNA duplex with the following sequence:
5'-CTAGAATTATGAAAAAGAATATCGCATTTCATCACCATCACCATCACCATCACATCGAA
CCACGTAGCC
TTAATACTTTTI-CTTATAGCGTAAAGTAGTGGTAGTGGTAGTGGTAGTGTAGCTT
GGTGCAT-5'
(SEQ ID NO: 75)
(SEQ ID NO: 76)
The last piece was a 1064 base pair Bgll-Sphl fragment from the previously
described
plasmid pMP1 1.
(e) Plasmid pMP172
The plasmid pMP172 is a secretion vector for the first 153 amino acids of
TPO, and is an intermediate for the construction of pMP210. As shown in Fig.
37,
pMP172 was prepared by ligating together three DNA fragments, the first of
which
was the vector pLS321amB in which the small EcoRl-Hindlll section had been
removed.
The second was a 946 base pair EcoRl-Hgal fragment from the previously
described
plasmid pMP11. The last piece was a synthetic DNA duplex with the following
sequence:
5'-TCCACCCTCTGCGTCAGGT (SEQ ID NO: 77)
GGAGACGCAGTCCATCGA-5' (SEQ ID NO: 78)
3 0 (f) Plasmid pMP210
The plasmid pMP210 is designed to express the first 153 amino acids of TPO
after a translational initiation methionine. This plasmid was actually made as
a bank
of plasmids in which the first 6 codons of TPO were randomized in the third
position of
each codon, and was constructed as shown in Fig. 38 by the ligation of three
DNA
3 5 fragments. The first of these was the previously described vector pVEG31
in which the
small Xbal-Sphl fragment had been removed. The second was a synthetic DNA
dupex
shown below treated first with DNA polymerasel (Klenow) followed by digestion
with
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Xbal and Hinfl, and encoding the initation methionine and the randomized first
6 codons
of TPO.
5'-GCAGCAGTTCTAGAATTATGTCNCCNGCNCCNCCNGCNTGTGACCTCCGA
ACACTGGAGGCT
GTTCTCAGTAAA (SEQ ID NO: 79)
CAAGAGTCAT7TGACGAAGCACTGAGGGTACAGGAAG-5' (SEQ ID NO: 80)
The third was a 890 base pair Hinfl-Sphl fragment from pMP172 encoding amino
acids 19-153 of TPO.
The plasmid pMP210 bank of approximately 3700 clones was retransformed
onto high tetracycline (50 g/ml) LB plates to select out high translational
initiation
clones (Yansura, D. G. et. at, Methods: A Companion to Methods in Enzymology
4:151-
158 [1992]). Of the 8 colonies which came up on high tetracycline plates, five
of the
best in terms of TPO expression were subject to DNA sequencing and the results
are
shown in Fig. 39 (SEQ ID NOS: 23, 24, 25, 26, 27 and 28).
(g) Plasmid pMP41
The plasmid pMP41 is designed to express the first 155 amino acids of TPO
fused to a leader consisting of 7 amino acids of the STII signal sequence
followed by a
factor Xa cleavage site. The plasmid was constructed as shown in Fig. 40 by
ligating
together three pieces of DNA, the first of which was the previously described
vector
pVEG31 in which the small Xbal-Sphl fragment had been removed. The second was
the
following synthetic DNA duplex:
5'-CTAGAATTATGAAAAAGAATATCGCATTTATCGAAGGTCGTAGCC (SEQ ID NO: 81)
TTAATACTTTTTCTTATAGCGTAAATAGCTTCCAGCAT-5' (SEQ ID NO: 82)
The last piece of the ligation was the 1064 base pair Bgil-SphI fragment from
the
previously described plasmid pMP11.
(h) Plasmid pMP57
The plasmid pMP57 expresses the first 155 amino acids of TPO downstream of
a leader consisting of 9 amino acids of the STII signal sequence and the
dibasic site Lys-
Arg. This dibasic site provides for a means of removing the leader with the
protease
ArgC. This plasmid was constructed as shown in Fig. 41 by ligating together
three
DNA pieces. The first of these was the previously described vector pVEG31 in
which
the small Xbal-Sphl fragment had been removed. The second was the following
synthetic DNA duplex:
5'-CTAGAATTATGAAAAAGAATATCGCATTTCTTCTTAAACGTAGCC (SEQ ID NO: 83)
TTAATACTTTTTCTTATAGCGTAAAGAAGAATTTGCAT-5' (SEQ ID NO: 84)
-128-
CA 02178482 2009-07-20 ~...~._ .__.,...._
= WO 95/18858 PCTIVS94114553
The last part of the ligation was the 1064 base pair Bgll-Sphl fragment from
the
previously described plasmid pMP11.
(1) Plasmid pMP251
The plasmid pMP251 is a derivative of pMP210-1 in which two additional
amino acids of TPO are Included on the carboxy terminal end. As shown In
F1g.42,
this plasmid was constructed by ligating together two pieces of DNA, the first
of these
being the previously described pMP21 In which the small Xbal-Apal fragment had
been removed. The second part of the ligation was a 318 base pair Xbal-Apal
fragment
from pMP210.1.
2. Transformation and Induction of E. coil with TPO egression vectors
The above TPO expression plasmids were used to transform the E colt strain
4406 (w3110 tonAs rpoHts long cIpPA gatE) using the CaCi2 heat shock method
(Mandel, M. of a/., J. Mol. 8101., 53:159-162, 11970]). The transformed cells
were
grown first at 3710 In LB media containing 50 pg/ml carbeniclliin until the
optical
density (600nm) of the culture reached approximately 2-3. The LB culture was
then
diluted 20x Into M9 media containing 0.49% casamino acids (w/v) and 50 g/ml
carbenicillin. After growth with aeration at 306C for 1 hour, indole-3-acrylic
acid
was added to a final concentration of 50 g/ml. The culture was then allowed
to
continue growing at 301C with aeration for another 15 hours at which time the
cells
were harvested by centrifugation.
EXAMPLE 22
Production of Biologically Active rPO (Not-11-153) In E. colt
The procedures given below for production of biologically active, refolded TPO
(met-1 1-153) can be applied In analogy for the recovery of other TPO variants
Including N and C terminal extended forms (see Example 23).
A Recovery of non-soluble TPO (Metl 1-153)
E. coil cells expressing TPO (Met-1 1-153) encoded by the plasmid pMP210-
1 are fermented as described above. Typically, about 10og of cells are
resuspended In
1 L (10 volumes) of cell disruption buffer (10 mM Trio, 6 mM EDTA, pH 8) with
a
Polytron homogenizer and the cells centrifuged at 5000 x g for 30 minutes. The
washed cell pellet Is again resuspended in 1 L cell disruption buffer with the
Polytron
homogenizer and the cell suspension Is passed through an LH Cell Disrupter (1-
H
Inceltech, Inc.) or through a Microfluldlzer (Mlcrofluldics international)
according to
36 the manufactures' Instructions. The suspension is centrifuged at 5,0008 for
30 min.
and resuspended and centrifuged a second time to make a washed retractile body
pellet.
The washed pellet Is used Immediately or stored frozen at -70 C.
*-trademark -129-
2178482
WO 95/18858 PCTIUS94114553 =
B. Solubilization and purification of monomeric TPO (Met-1 1-153)
The pellet from above is resuspended in 5 volumes by weight of 20 mM Tris,
pH 8, with 6-8 M guanidine and 25 mM DTT (dithiothreitol) and stirred for 1-3
hr.,
or overnight, at 4 C to effect solubilization of the TPO protein. High
concentrations of
urea (6-8M) are also useful but generally result in lower yields compared to
guanidine. After solubilization, the solution is centrifuged at 30,000 x g for
30 min.
to produce a clear supernatant containing denatured, monomeric TPO protein.
The
supernatant is then chromatographed on a Superdex 200 gel filtration column
(Pharmacia, 2.6 x 60 cm) at a flow rate of 2 ml/min. and the protein eluted
with 20
mM Na phosphate, pH 6.0, with 10 mM DTT Fractions containing monomeric,
denatured TPO protein eluting between 160 and 200 ml are pooled. The TPO
protein is
further purified on a semi-preparative C4 reversed phase column (2 x 20 cm
VYDAC). The sample is applied at 5 ml/min. to a column equilibrated in 0.1%
TFA(trifluoroacetic acid) with 30% acetonitrile. The protein is eluted with a
linear
gradient of acetonitrile (30-60% in 60 min.). The purified reduced protein
elutes at
approximately 50% acetonitrile. This material is used for refolding to obtain
biologically active TPO variant.
C Generation of biologically active TPO (Met-1 1-153)
Approximately 20 mg of monomeric, reduced and denatured TPO protein in 40
ml 0.1% TFA/50% acetonitrile is diluted into 360 ml of refolding buffer
containing
optimally the following reagents:
50 mM Tris
0.3 M NaCl
5 mM EDTA
2% CHAPS detergent
25% glycerol
5 mM oxidized glutathione
1 mM reduced glutathione
pH adjusted to 8.3
After mixing, the refolding buffer is gently stirred at 4 C for 12-48 hr to
effect maximal refolding yields of the correct disulfide-bonded form of TPO
(see
below). The solution is then acidified with TFA to a final concentration of
0.2%,
filtered through a 0.45 or 0.22 micron filter, and 1/10 volume of acetonitrile
added.
This solution is then pumped directly onto a C4 reversed phase column and the
purified, refolded TPO (Met-1 1-153) eluted with the same gradient program as
above. Refolded, biologically active TPO is eluted at approximately 45%
acetonitrile
under these conditions. Improper disulfide-bonded versions of TPO are eluted
earlier.
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= WO 95/18858 2178482
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The final purified TPO (Met-1 1-153) is greater than 95% pure as assessed by
SDS
gels and analytical C4 reversed phase chromatography. For animal studies, the
C4
purified material was dialyzed into physiologically compatible buffers.
Isotonic
buffers (10 mM Na acetate, pH 5.5, 10 mM Na succinate, pH 5.5 or 10 mM Na
phosphate, pH 7.4) containing 150 mM NaCl and 0.01% Tween 80 were utilized.
Because of the high potency of TPO in the Ba/F3 assay (half maximal
stimulation is achieved at approximately 3 pg/ml), it is possible to obtain
biologically
active material utilizing many different buffer, detergent and redox
conditions.
However, under most conditions only a small amount of properly folded material
(<10%) is obtained. For commercial manufacturing processes, it is desirable to
have
refolding yields at least 10%, more preferably 30-50% and most preferably
>50%.
Many different detergents (Triton X-100, dodecyl-beta-maltoside, CHAPS,
CHAPSO,
SDS, sarkosyl, Tween 20 and Tween 80, Zwittergent 3-14 and others) were
assessed
for efficiency to support high refolding yields. Of these detergents, only the
CHAPS
family (CHAPS and CHAPSO) were found to be generally useful in the refolding
reaction to limit protein aggregation and improper disulfide formation. Levels
of
CHAPS greater than 1% were most useful. Sodium chloride was required for best
yields, with the optimal levels between 0.1 M and 0.5M. The presence of EDTA
(1-5
mM) limited the amount of metal-catalyzed oxidation (and aggregation) which
was
observed with some preparations. Glycerol concentrations of greater than 15%
produced the optimal refolding conditions. For maximum yields, it was
essential to
have both oxidized and reduced glutathione or oxidized and reduced cysteine as
the redox
reagent pair. Generally higher yields were observed when the mole ratio of
oxidized
reagent is equal to or in excess over the reduced reagent member of the redox
pair. pH
values between 7.5 and about 9 were optimal for refolding of these TPO
variants.
Organic solvents (e.g. ethanol, acetonitrile, methanol) were tolerated at
concentrations
of 10-15% or lower. Higher levels of organic solvents increased the amount of
improperly folded forms. Tris and phosphate buffers were generally useful.
Incubation at 4 C also produced higher levels of properly folded TPO.
Refolding yields of 40-60% (based on the amount of reduced and denatured TPO
used in the refolding reaction) are typical for preparations of TPO that have
been
purified through the first C4 step. Active material can be obtained when less
pure
preparations (e.g. directly after the Superdex 200 column or after the initial
refractile body extraction) although the yields are less due to extensive
precipitation
and interference of non-TPO proteins during the TPO refolding process.
Since TPO (Met-1 1-153) contains 4 cysteine residues, it is possible to
generate three different disulfide versions of this protein:
version 1: disulfides between cysteine residues 1-4 and 2-3
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WO 95/18858 21 7 8 `f S 2 PCT/US94114553
version 2: disulfides between cysteine residues 1-2 and 3-4
version 3: disulfides between cysteine residues 1-3 and 2-4.
During the initial exploration in determining refolding conditions, several
different peaks containing the TPO protein were separated by C4 reversed phase
chromatography. Only one of these peaks had significant biological activity as
determined using the Ba/F3 assay. Subsequently, the refolding conditions were
optimized to yield preferentially that version. Under these conditions, the
misfolded
versions are less than 10-20% of the total monomer TPO obtained.
The disulfide pattern for the biologically active TPO has been determined to
be
1-4 and 2-3 by mass spectrometry and protein sequencing(i.e. version 1).
Aliquots
of the various C4-resolved peaks (5-10 nmoles) were digested with trypsin
(1:25
mole ratio of trypsin to protein). The digestion mixture was analyzed by
matrix-
assisted laser desorption mass spectrometry before and after reduction with
DTT. After
reduction, masses corresponding to most of the larger tryptic peptides of TPO
were
detected. In the un-reduced samples, some of these masses were missing and new
masses were observed. The mass of the new peaks corresponded basically to the
sum of
the individual tryptic peptides involved in the disulfide pair. Thus it was
possible to
unequivocally assign the disulfide pattern of the refolded, recombinant,
biologically
active TPO to be 1-4 and 2-3. This is consistent with the known disulfide
pattern of
the related molecule erythropoletin.
D. Biological activity of recombinant, refolded TPO (met 1-153)
Refolded and purified TPO (Met-1 1-153) has activity in both in vitro and in
vivo assays. In the Ba/F3 assay, half-maximal stimulation of thymidine
incorporation
Into the Ba/F3 cells was achieved at 3.3 pg /ml (0.3 pM). In the mpl receptor-
based
ELISA, half-maximal activity occurred at 1.9 ng/ml (120 pM). In normal and
myelosuppressed animals produced by near-lethal X-radiation, TPO (Met-1 1-153)
was highly potent (activity was seen at doses as low as 30 ng/mouse) to
stimulate the
production of new platelets.
EXAMPLE 23
Production of Other Biologically Active TPO Variants In E. coli
Three different TPO variants produced in E. coli, purified and refolded into
biological active forms are provided below.
(1) MLF - 13 residues from the bacterial-derived signal sequence STII are
fused to the N-terminal domain of TPO (residues 1-155). The resulting sequence
is
MKKNIAFLLNAYASPAPPAC.....CVRRA (SEQ ID NO: 85)
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= WO 95/18858 2 1 7 8 4 8 2 PCTIUS94/14553
where the leader sequence is underlined and C.....C represents Cys7 through
Cys151.
This variant was constructed to provide a tyrosine for radio-iodination of TPO
for
receptor and biological studies.
(2 ) H8MLF - 7 residues from the STII sequence, 8 histidine residues and
the Factor Xa enzymatic cleavage sequence IEGR are fused to the N-terminal
domain
(residues 1-155) of TPO. The sequence is
MKKNIAFHHHHHHHHIE QRSPAPPAC.....CVRRA (SEQ ID NO: 86)
where the leader sequence is underlined and C.....C represents Cys7 through
Cys151.
This variant, when purified and refolded, can be treated with the enzyme
Factor Xa
which will cleave after the arginine residue of the sequence IEGR yielding a
TPO
variant of 155 residues in length with a natural serine N-terminal amino acid.
(3 ) T-H8MLF - is prepared as described above for variant (2), except a
thrombin sensitive sequence IEPR is fused to the N-terminal domain of TPO. The
resulting sequence is
MKKNIAFHHHHHHHHIEPRSPAPPAC..... CVRRA (SEQ ID NO: 87)
where the leader sequence is underlined and and C.....C represents Cys7
through
Cys151. This variant, after purification and refolding can be treated with the
enzyme
thrombin to generate a natural N-terminal variant of TPO of 155 residues in
length.
A Recovery, solubilization and purification of monomeric, biologically active
TPO
variants (1), (2),and (3).
All of the variants were expressed in E. coli. The majority of the variants
were
found in refractile bodies, as observed in Example 22 for TPO (Met-1 1-153).
Identical procedures for the recovery, solubilization and purification of
monomeric
TPO variants was achieved as described in Example 22. Identical refolding
conditions to those used for TPO (Met-1 1-153) were used with overall yields
of 30-
50%. After refolding, the TPO variants were purified by C4 reversed phase
chromatography in 0.1% TFA utilizing an acetonitrile gradient as described
previously. All of the TPO variants (in their unproteolyzed forms) had
biological
activity as assessed by the Ba/F3 assay, with half-maximal activities of 2-5
pM.
3 0 B. Proteolytic processing of Variants (2) and (3) to generate authentic N-
terminal TPO (1-155).
TPO variants (2) and (3) above were designed with an enzymatically-
cleavable leader peptide before the normal N-terminal amino acid residue of
TPO.
After refolding and purification of variants (2) and (3) as described above,
each was
subjected to digestion with the appropriate enzyme. For each variant, the
acetonitrile
from the C4 reversed phase step was removed by blowing a gentle stream of
nitrogen
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WO 95/18858 21 7 84 82 PCT/U594114553
on the solution. Thereafter the two variants were treated with either Factor
Xa or
thrombin as described below.
For TPO variant (2), 1 M Tris buffer, pH 8, was added to the acetonitrile-free
solution to a final concentration of 50 mM and the pH was adjusted to 8 if
necessary.
NaCl and CaCl2 were added to 0.1 M and 2 mM, respectively. Factor Xa (New
England
Biolabs) was added to achieve about a 1:25 to 1:100 mole ratio of enzyme to
variant.
The sample was incubated at room temperature for 1-2 hr. to achieve maximal
cleavage as assessed by a change in migration on SDS gels representing the
loss of the
leader sequence. Thereafter, the reaction mixture was purified by C4 reversed
phase
chromatography using the same gradient and conditions as described above for
the
purification of properly folded variants. Uncleaved variant B was separated
from
cleaved variant (2) by these conditions. The N-terminal amino acids were shown
to be
SPAPP, indicating that removal of the N-terminal leader sequence was
successful.
Factor Xa also generated variable amounts of an internal cleavage within the
TPO
domain; cleavage was observed after the arginine residue at position number
118
generating an additional N-terminal sequence of TTAHKDP=(SEQ ID NO: 88). On
non-
reducing SDS gels, a single band at approximately 17000 daltons was observed
for the
Factor Xa cleaved variant; on reducing gels two bands were seen of molecular
weight of
approximately 12000 and 5000 daltons, consistent with cleavage at arginine
118.
This observation also confirmed that the two parts of the molecule were held
together
by a disulfide bond between the 1st and 4th cysteine residues, as deduced from
the
tryptic digestion experiments described above. In the Ba/F3 biological assay,
the
purified TPO (1-155) variant, after removal of the N-terminal leader sequence
and
with the internal cleavage, had a half-maximal activity of 0.2 to 0.3
picomolar. The
intact variant with the leader sequence had a half-maximal activity of 2-4
picomolar.
For variant (3), the digestion buffer consisted of 50 mM Tris, pH 8, 2%
CHAPS, 0.3 M NaCl, 5 mM EDTA and human or bovine thrombin (Calbiochem) at a
1:25 to 1:50 by weight of enzyme to TPO variant protein. Digestion was
conducted at
room temperature for 2-6 hours. The progress of the digestion was assessed by
SDS
gels as described above for the Factor Xa cleavage reaction. Generally, more
than 90%
cleavage of the leader sequence was achieved in this time. The resultant TPO
was
purified on C4 reversed phase columns as described above and was shown to have
the
desired N-terminal by amino acid sequencing. Only very minor (<5%) amounts of
an
Internal cleavage at the same arginine-threonine bond as observed above with
Factor
Xa was obtained. The resultant TPO protein had high biological activity with
half-
maximal responses in the Ba/F3 assay at 0.2-0.4 picomolar protein. In the mp/
receptor based ELISA, this protein had a half-maximal response at 2-4 ng/ml
purified
protein (120-240 picomolar) while the intact variant containing the leader
sequence
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t S" ! WO 95/18858 2 1 7 8 `f 8 2 PCT/US94/14553
was less potent in both assays by 5-10 fold. For animal studies, the HPLC-
purified
cleaved protein was dialyzed into physiological acceptable buffers, with 150
mM NaCl,
0.01% Tween 80 and 10 mM sodium succinate, pH 5.5, or 10 mM sodium acetate, pH
5.5, or 10 mM sodium phosphate, pH 7.4. By HPLC and SDS gels, the purified
protein
was stable for several weeks when stored at 4 C. In normal and myelosuppressed
mice,
this purified TPO with the authentic N-terminal sequence was highly active,
stimulating the production of platelets at doses as low as 30 ng/mouse.
EXAMPLE 24
Synthetic mp! Ligand
Although Human mpl ligand (hML) is usually made using recombinant methods,
it can also be synthesized via enzymatic ligation of synthetic peptide
fragments using
methods described below. Synthetic production of hML allows the incorporation
of
unnatural amino acids or synthetic functionalities such as polyethylene
glycol.
Previously, a mutant of the serine protease subtilisin BPN, subtiligase
(S221C/P225A) was engineered to efficiently ligate peptide esters in aqueous
solution
(Abrahmsen et at, Biochem., 30:4151-4159 [1991]). It has now been shown that
synthetic peptides can be enzymatically ligated in a sequential manor to
produce
enzymatically active long peptides and proteins such as ribonuclease A
(Jackson et at,
Science, [1994]). This technology, described in more detail below, has enabled
us to
chemically synthesize long proteins that previously could be made only with
recombinant DNA technology.
A general strategy for hML153 synthesis using subtiligase is shown (Scheme
1). Beginning with a fully deprotected peptide corresponding to the C-terminal
fragment of the protein, an N-terminal protected, C-terminal activated ester
peptide
is added along with subtiligase. When the reaction is complete, the product is
isolated
by reverse phase HPLC and the protecting group is removed from the N-terminus.
The
next peptide fragment is ligated, deprotected and the process is repeated
using
successive peptides until full length protein is obtained. The process is
similar to
solid phase methodology in that an N-terminal protected C-terminal activated
petide is
ligated to the N-terminus of the preceding peptide and protein is synthesized
in a C->N
direction. However because each coupling results in addition of up to 50
residues and
the products are isolated after each ligation, much longer highly pure
proteins can be
synthesized in reasonable yields.
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21 78 4 8 2 PCT/US94/14553
WO 95/18858
Scheme 1. Strategy for Synthesis of hML Using Subtiligase
R-NH-Peptide2-CO-R' + H2N-Peptides-CO2
1) Subtiligase
R-NH-Peptide2-CO-NH-Peptide1-CO2
2) Zn/CH3CO2H
H2N-Peptide2-CO-NH-Peptide1-CO2
1 3) repeat 1 + 2
H2N-Peptide3-CO-N H-Peptide2-CO-N H-Peptides -CO2
O O
R= i \ O R'= ISO NH 2
NH
~
)A 0 (CH2)4
NH2
Based on our knowledge of the sequence specificity of the subtiligase as well
as
the amino acid sequence of the biologically active "epo-domain" of hML, we
divided
hML153 into seven fragments 18-25 residues in length. Test ligation
tetrapeptides
were synthesized to determine suitable ligation junctions for the 18-25mer's.
Table
13 shows the results of these test ligations.
TABLE 13
hML Test Ligations. Donor and nucleophile peptides were dissolved at 10 mM in
100 mM tricine (pH 7.8) at 22 C. Ligase was added to a final concentration of
10 M
from a 1.6 mg/mL stock (-70 M) and the ligation allowed to proceed overnight.
Yields are based on % ligation vs. hydrolysis of the donor peptides.
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WO 95/18858 PCT/US94114553
Site Donor (glc-K-NH2) Nucleophile-NH2 %H drol sis %Li ation
1 (23/24) HVLH SRLS 92 08
(SEQ ID NO: 89) (SEQ ID NO: 90)
(22/23) SHVL HSRL 48 52
(SEQ ID NO: 91) (SEQ ID NO: 92)
2 (46/47) AVDF SLGE 22 78
(SEQ ID NO: 93) (SEQ ID NO: 94)
3 (69/70) AVTL LLEG 53 47
(SEQ ID NO: 95) (SEQ ID NO: 96)
4 (89/90) LSSL LGQL 95 05
(SEQ ID NO: 97) (SEQ ID NO: 98)
(88/89) C(acm)LSS LLGQ 00 00
(SEQ ID NO: 99) (SEQ ID NO: 100)
(90/91) SSLL GQLS 45 55
(SEQ ID NO: 101) (SEQ ID NO: 102)
(88/89) CLSS LLGQ 90 10
(SEQ ID NO: 103) (SEQ ID NO: 100)
5(107/108) LQSL LGTQ 99 01
(SEQ ID NO: 104) (SEQ ID NO: 105)
(106/107) ALQS LLGT 70 30
(SEQ ID NO: 106) (SEQ ID NO: 107)
6(128/129) NAIF LSFQ 60 40
(SEQ ID NO: 108) (SEQ ID NO: 109)
Based on these experiments, the ligation peptides indicated in Table 14 should
be efficiently ligated by the subtiligase. A suitable protecting group for the
N-
terminus of each donor ester peptide was needed to prevent self-ligation. We
chose an
isonicotinyl (iNOC) protecting group (Veber et at, J. Org. Chem., 42:3286-3289
[1977]) because it is water soluble, it can be incorporated at the last step
of solid
phase peptide synthesis and it is stable to anhydrous HF used to deprotect and
cleave
peptides from the solid phase resin. In addition, it can be removed from the
peptide
after each ligation under mild reducing conditions (Zn/CH3CO2H) to afford a
free N-
terminus for subsequent ligations. A glycolate-lysyl-amide (gIc-K-NH2) ester
was
used for C-terminal activation based on previous experiments which showed this
to be
efficiently acylated by subtiligase (Abrahmsen et at, Biochem., 30:4151-4159
[1991]). The iNOC-protected, glc-K-amide activated peptides can be synthesized
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WO 95118858 21 7 8 4 8 2 PCT/US94114553
using standard solid phase methods as outlined (Scheme 2). The peptides are
then
seqentially ligated until the full protein is produced and the final product
refolded in
vitro. Based on homology with EPO, disulfide pairs are believed to be formed
between
cysteine residues 7 and 151 and between 28 and 85. Oxidation of the disulfides
may be
accomplished by simply stirring the reduced material under an oxygen
atmosphere for
several hours. The refolded material can then be purified by HPLC and
fractions
containing active protein pooled and lyophilized. As an alternative,
disulfides can be
differentially protected to control sequential oxidation between specific
disulfide pairs.
Protection of cysteines 7 and 151 with acetamidomethyl (acm) groups would
ensure
oxidation of 28 and 85. The acm groups could then be removed and residues 7
and 151
oxidized. Conversely, residues 28 and 85 could be acm protected and oxidized
in case
sequential oxidation is required for correct folding. Optoinally, Cysteins 28
and 85
may be substituted with another natural or unnatural residue other than Cys to
insure
proper oxidation of cysteins 7 and 151.
TABLE 14.
Peptide Fragments Used For Total Synthesis of h-ML Using Subtiligase
Fragment
Sequence
1 (SEQ ID NO: 110)
iNOC-HN-SPAPPACDLRVLSKLLRDSHVL-gIc-K-NH2 (1-22)
2 (SEQ ID NO: 111)
INOC-HN-HSRLSQCPEVHPLPTPVLLPAVDF-gIc-K-NH2 (23-46)
3 (SEQ ID NO: 112)
iNOC-HN-SLGEWKTQMEETKAQDILGAVTL-gIc-K-NH2 (47-69)
4 (SEQ ID NO: 113)
iNOC-HN-LLEGVMAARGQLGPTCLSSLL-gIc-K-NH2 (70-90)
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PCT/US94/14553
WO 95118858 2178482
(SEQ ID NO: 114)
iNOC-HN-GQLSGQVRLLLGALQS-glc-K-NH2 (90-106)
5 6 (SEQ ID NO: 115)
1NOC-HN-LLGTQLPPQGRTTAHKDPNAIF-glc-K-NH2 (107-128)
7 (SEQ ID NO: 116)
H2N-LSFQHLLRGKVRFLMLVGGSTLCVR-CO2 (129-153)
Peptide ligations are carried out at 25 C in 100mM tricine, pH 8 (freshly
prepared and degassed by vacuum filtration through a 5 M filter). Typically
the C-
1 5 terminal fragment is dissolved in buffer (2-5 mM peptide) and a 10x stock
solution of
subtiligase (1 mg/ml in 100mM tricine, pH 8) is added to bring the final
enzyme
concentration to - 5 M. A 3-5 molar excess of the glc-K-NH2 activated donor
peptide is then added as a solid, dissolved, and the mixture allowed to stand
at 25 C.
The ligations are monitored by analytical reverse phase C18 HPLC (CH3CN/H2O
gradient with 0.1% TFA). The ligation products are purified by preparative
HPLC and
lyophilized. Isonicotinyl (iNOC) deprotection was performed by stirring HCl
activated
zinc dust with the protected peptide in acetic acid. The zinc dust is removed
by
filtration and the acetic acid evaporated under vacuum. The resulting peptide
can be
used directly in the next ligation and the process is repeated. Synthetic
hML153 can
be ligated by procedures analogous to those described above to synthetic or
recombinant hML154-332 to produce synthetic or semisynthetic full length hML.
Synthetic hML has many advantages over recombinant. Unnatural side chains
can be introduced in order to improve potency or specificity. Polymer
functionalities
such as polyethylene glycol can be incorporated to improve duration of action.
For
example, polyethylene glycol can be attached to lysine residues of the
individual
fragments (Table 14) before or after one or more ligation steps have been
performed. Protease sensitive peptide bonds can be removed or altered to
improve
stability in vivo. In addition, heavy atom derivatives can be synthesized to
aid in
structure determination.
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WO 95/18858 21 78482 PCT/US94/14553
Scheme 2. Solid Phase Synthesis of Peptide Fragments for Segment
Ligation.
O O
HA b
H2N mBIIA NH ~MPESIN
RESIN a Br
R O
R
2
O o
BoC-NH` Ao~NH ,, c , d (automated synthesis)
R R
3
O O
H .,(Peptide NH ~,^ _NH t,,ffi , e , f (cleavage)
2N RESIN
O R O R
4
0 0 0
O)NH (Peptide) \j~ _NH O--~NH_T& NH2
N 5 0 R O (CH2,)4
I NHz ~
Isonicotinyl (iNOC) glycolate-lysyl-amide (glc-K-NH2)
a) Lysyl-paramethylbenzhydrylamine (MBHA) resin 1 (0.63 meq./gm., Advanced
ChemTech) is stirred with bromoacetic acid (5 eq.) and diisopropyl
carbodiimide (5
eq.) for 1 h. at 25 C in dimethylacetamide (DMA) to afford the bromoacetyl
derivative
2. b) The resin is washed extensively with DMA and individual Boc-protected
amino
acids (3 eq., Bachem) are esterified by stirring with sodium bicarbonate (6
eq.) in
dimethylformamide (DMF) for 24 h. at 50 C to afford the corresponding
glycolate-
phenylalanyl-amide-resin 3. The amino acetylated resin 3 is washed with DMF
(3x)
and dichloromethane (CH2CI2) (3x) and can be stored at room temperature for
several
months. The resin 3 can then be loaded into an automated peptide synthesizer
(Applied
Biosystems 430A) and the peptides elongated using standard solid phase
procedures
(5). c) The N-a-Boc group is removed with a solution of 45% trifluoroacetic
acid in
CH2CI2. d) Subsequent Boc-protected amino acids (5 eq.) are preactivated using
benzotriazol-1-yl-oxy-tris-(dimethylamino) phosphonium hexafluorophosphate
(BOP, 4 eq.) and N-methylmorpholine (NMM, 10 eq.) in DMA and coupled for 1-2
h.
e) The final N-a-Boc group is removed (TFA/CH2CI2) to afford 4 and the
isonicotinyl
(iNOC) protecting group is introduced as described previously (4) via stirring
with of
-140-
CA 02178482 2011-03-21
W045118858 PCT/US94114553
2178482
4-isonicotinyl-2-4-dinitrophenyl carbonate (3 eq.) and NMM (6 eq.) in DMA at
25 C for 24 h. f) Cleavage and deprotection of the peptide via treatment with
anhydrous HF (5% anisole/ 5% ethytmethyl sufide) at 0 C for 1 h. affords the
iNOC-
protected, glycolate-lys-amide activated peptide 5 which is purified by
reverse phase
C18 HPLC (CH3CN/H20 gradient, 0.1% TFA). The identity of all substrates is
confirmed by mass spectrometry.
SUPPLEMENTALENABLEMENT
The invention as claimed is enabled in accordance with the above specification
and readily available references and starting materials. Nevertheless,
Applicants have
deposited with the American Type Culture Collection, Rockville, Md., USA
(ATCC) the
cell line listed below:
Escherichia coli, DH10B-pBSK-hmpl 1 1.8, ATCC accession no. CRL 69575,
deposited February 24, 1994.
Plasmid, pSVl5.ID.LL.MLORF, ATCC accession no. CRL 75958, deposited
December 2, 1994; and
CHO DP-12 cells, ML 1/50 MCB (labeled #1594), ATCC accession no. CRL
11770, deposited December 6, 1994.
This deposit was made under the provisions of the Budapest Treaty on the
International Recognition of the Deposit of Microorganisms for the Purposes of
Patent
Procedure and the regulations thereunder (Budapest Treaty). This assures
maintenance of a viable culture for 30 years from date of deposit. The
organisms will
be made available by ATCC under the terms of the Budapest Treat, and subject
to an
agreement between Applicants and ATCC which assures unrestricted availability
upon
issuance of the pertinent U.S. patent. Availability of the deposited strain is
not to be
construed as a license to practice the invention in contravention of the
rights granted
under the authority of any government in accordance with its patent laws.
While the invention has necessarily been described in conjunction with
preferred embodiments and specific working examples, one of ordinary skill,
after
reading the foregoing specification, will be able to effect various changes,
substitutions of equivalents, and alterations to the subject matter set forth
herein,
without departing from the spirit and scope thereof. Hence, the invention can
be
practiced in ways other than those specifically described herein. It is
therefore
intended that the protection granted by letters patent hereon be limited only
by the
appended claims and equivalents thereof.
-141-
CA 02178482 2010-06-29
Sequence Listing
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Genentech, Inc.
Eaton, Dan L.
de Sauvage, Frederic J.
(ii) TITLE OF INVENTION: THROMBOPOIETIN
(iii) NUMBER OF SEQUENCES: 144
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Genentech, Inc.
(B) STREET: 1 DNA Way
(C) CITY: South San Francisco
(D) STATE: California
(E) COUNTRY: USA
(F) ZIP: 94080
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch, 1.44 Mb floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: WinPatin (Genentech)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/176553
(B) FILING DATE: 03-JAN-1994
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/348657
(B) FILING DATE: 02-DEC-1994
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/185607
(B) FILING DATE: 21-JAN-1994
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/348658
(B) FILING DATE: 02-DEC-1994
(vii) PRIOR APPLICATION DATA:
142
CA 02178482 2010-06-29
(A) APPLICATION NUMBER: 08/196689
(B) FILING DATE: 15-FEB-1994
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/223263
(B) FILING DATE: 04-APR-1994
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/249376
(B) FILING DATE: 25-MAY-1994
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Winter, Daryl B.
(B) REGISTRATION NUMBER: 32,637
(C) REFERENCE/DOCKET NUMBER: P0871P5PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 650/225-1249
(B) TELEFAX: 650/952-9881
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 353 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Met Glu Leu Thr Glu Leu Leu Leu Val Val Met Leu Leu Leu Thr
1 5 10 15
Ala Arg Leu Thr Leu Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu
20 25 30
Arg Val Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser
35 40 45
Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val
50 55 60
Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln
65 70 75
Met Glu Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu
80 85 90
Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr
95 100 105
Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu
110 115 120
Leu Leu Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro
125 130 135
143
CA 02178482 2010-06-29
Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu
140 145 150
Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu
155 160 165
Val Gly Gly Ser Thr Leu Cys Val Arg Arg Ala Pro Pro Thr Thr
170 175 180
Ala Val Pro Ser Arg Thr Ser Leu Val Leu Thr Leu Asn Glu Leu
185 190 195
Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Thr Ala Ser
200 205 210
Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys Trp Gln Gln Gly Phe
215 220 225
Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln Thr Ser Arg Ser Leu
230 235 240
Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile His Glu Leu Leu Asn
245 250 255
Gly Thr Arg Gly Leu Phe Pro Gly Pro Ser Arg Arg Thr Leu Gly
260 265 270
Ala Pro Asp Ile Ser Ser Gly Thr Ser Asp Thr Gly Ser Leu Pro
275 280 285
Pro Asn Leu Gln Pro Gly Tyr Ser Pro Ser Pro Thr His Pro Pro
290 295 300
Thr Gly Gln Tyr Thr Leu Phe Pro Leu Pro Pro Thr Leu Pro Thr
305 310 315
Pro Val Val Gln Leu His Pro Leu Leu Pro Asp Pro Ser Ala Pro
320 325 330
Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn Thr Ser Tyr Thr His
335 340 345
Ser Gln Asn Leu Ser Gln Glu Gly
350
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1795 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
144
CA 02178482 2010-06-29
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TCTTCCTACC CATCTGCTCC CCAGAGGGCT GCCTGCTGTG CACTTGGGTC 50
CTGGAGCCCT TCTCCACCCG GATAGATTCC TCACCCTTGG CCCGCCTTTG 100
CCCCACCCTA CTCTGCCCAG AAGTGCAAGA GCCTAAGCCG CCTCCATGGC 150
CCCAGGAAGG ATTCAGGGGA GAGGCCCCAA ACAGGGAGCC ACGCCAGCCA 200
GACACCCCGG CCAGAATGGA GCTGACTGAA TTGCTCCTCG TGGTCATGCT 250
TCTCCTAACT GCAAGGCAAA CGCTGTCCAG CCCGGCTCCT CCTGCTTGTG 300
ACCTCCGAGT CCTCAGTAAA CTGCTTCGTG ACTCCCATGT CCTTCACAGC 350
AGACTGAGCC AGTGCCCAGA GGTTCACCCT TTGCCTACAC CTGTCCTGCT 400
GCCTGCTGTG GACTTTAGCT TGGGAGAATG GAAAACCCAG ATGGAGGAGA 450
CCAAGGCACA GGACATTCTG GGAGCAGTGA CCCTTCTGCT GGAGGGAGTG 500
ATGGCAGCAC GGGGACAACT GGGACCCACT TGCCTCTCAT CCCTCCTGGG 550
GCAGCTTTCT GGACAGGTCC GTCTCCTCCT TGGGGCCCTG CAGAGCCTCC 600
TTGGAACCCA GCTTCCTCCA CAGGGCAGGA CCACAGCTCA CAAGGATCCC 650
AATGCCATCT TCCTGAGCTT CCAACACCTG CTCCGAGGAA AGGTGCGTTT 700
CCTGATGCTT GTAGGAGGGT CCACCCTCTG CGTCAGGCGG GCCCCACCCA 750
CCACAGCTGT CCCCAGCAGA ACCTCTCTAG TCCTCACACT GAACGAGCTC 800
CCAAAAAGGA CTTCTGGATT GTTGGAGACA AACTTCACTG CCTCAGCCAG 850
AACTACTGGC TCTGGGCTTC TGAAGTGGCA GCAGGGATTC AGAGCCAAGA 900
TTCCTGGTCT GCTGAACCAA ACCTCCAGGT CCCTGGACCA AATCCCCGGA 950
TACCTGAACA GGATACACGA ACTCTTGAAT GGAACTCCTG GACCCTTTCC 1000
TGGACCCTCA CGCAGGACCC TAGGAGCCCC GGACATTTCC TCAGGAACAT 1050
CAGACACAGG CTCCCTGCCA CCCAACCTCC AGCCTGGATA TTCTCCTTCC 1100
CCAACCCATC CTCCTACTGG ACAGTATACG CTCTTCCCTC TTCCACCCAC 1150
CTTGCCCACC CCTGTGGTCC AGCTCCACCC CCTGCTTCCT GACCCTTCTG 1200
CTCCAACGCC CACCCCTACC AGCCCTCTTC TAAACACATC CTACACCCAC 1250
TCCCAGAATC TGTCTCAGGA AGGGTAAGGT TCTCAGACAC TGCCGACATC 1300
145
CA 02178482 2010-06-29
AGCATTGTCT CATGTACAGC TCCCTTCCCT GCAGGGCGCC CCTGGGAGAC 1350
AACTGGACAA GATTTCCTAC TTTCTCCTGA AACCCAAAGC CCTGGTAAAA 1400
GGGATACACA GGACTGAAAA GGGAATCATT TTTCACTGTA CATTATAAAC 1450
CTTCAGAAGC TATTTTTTTA AGCTATCAGC AATACTCATC AGAGCAGCTA 1500
GCTCTTTGGT CTATTTTCTG CAGAAATTTG CAACTCACTG ATTCTCTACA 1550
TGCTCTTTTT CTGTGATAAC TCTGCAAAGG CCTGGGCTGG CCTGGCAGTT 1600
GAACAGAGGG AGAGACTAAC CTTGAGTCAG AAAACAGAGA AAGGGTAATT 1650
TCCTTTGCTT CAAATTCAAG GCCTTCCAAC GCCCCCATCC CCTTTACTAT 1700
CATTCTCAGT GGGACTCTGA TCCCATATTC TTAACAGATC TTTACTCTTG 1750
AGAAATGAAT AAGCTTTCTC TCAGAAAAAA AAAAAAAAAA AAAAA 1795
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 42 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Leu Leu Leu Val Val Met Leu Leu Leu Thr Ala Arg Leu Thr Leu
1 5 10 15
Ser Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys
20 25 30
Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu
35 40
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 390 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
GAATTCCTGG AATACCAGCT GACAATGATT TCCTCCTCAT CTTTCAACCT 50
CACCTCTCCT CATCTAAGAA TTGCTCCTCG TGGTCATGCT TCTCCTAACT 100
146
CA 02178482 2010-06-29
GCAAGGCTAA CGCTGTCCAG CCCGGCTCCT CCTGCTTGTG ACCTCCGAGT 150
CCTCAGTAAA CTGCTTCGTG ACTCCCATGT CCTTCACAGC AGACTGGTGA 200
GAACTCCCAA CATTATCCCC TTTATCCGCG TAACTGGTAA GACACCCATA 250
CTCCCAGGAA GACACCATCA CTTCCTCTAA CTCCTTGACC CAATGACTAT 300
TCTTCCCATA TTGTCCCCAC CTACTGATCA CACTCTCTGA CAAGAATTAT 350
TCTTCACAAT ACAGCCCGCA TTTAAAAGCT CTCGTCTAGA 390
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 390 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CTTAAGGACC TTATGGTCGA CTGTTACTAA AGGAGGAGTA GAAAGTTGGA 50
GTGGAGAGGA GTAGATTCTT AACGAGGAGC ACCAGTACGA AGAGGATTGA 100
CGTTCCGATT GCGACAGGTC GGGCCGAGGA GGACGAACAC TGGAGGCTCA 150
GGAGTCATTT GACGAAGCAC TGAGGGTACA GGAAGTGTCG TCTGACCACT 200
CTTGAGGGTT GTAATAGGGG AAATAGGCGC ATTGACCATT CTGTGGGTAT 250
GAGGGTCCTT CTGTGGTAGT GAAGGAGATT GAGGAACTGG GTTACTGATA 300
AGAAGGGTAT AACAGGGGTG GATGACTAGT GTGAGAGACT GTTCTTAATA 350
AGAAGTGTTA TGTCGGGCGT AAATTTTCGA GAGCAGATCT 390
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu
1 5 10 15
Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro
20 25 30
147
CA 02178482 2010-06-29
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp
35 40 45
Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala
50 55 60
Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met
65 70 75
Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu
80 85 90
Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln
95 100 105
Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala
110 115 120
His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu
125 130 135
Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu
140 145 150
Cys Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr
155 160 165
Ser Leu Val Leu Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly
170 175 180
Leu Leu Glu Thr Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser
185 190 195
Gly Leu Leu Lys Trp Gln Gln Gly Phe Arg Ala Lys Ile Pro Gly
200 205 210
Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly Tyr
215 220 225
Leu Asn Arg Ile His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe
230 235 240
Pro Gly Pro Ser Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser
245 250 255
Gly Thr Ser Asp Thr Gly Ser Leu Pro Pro Asn Leu Gln Pro Gly
260 265 270
Tyr Ser Pro Ser Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu
275 280 285
Phe Pro Leu Pro Pro Thr Leu Pro Thr Pro Val Val Gln Leu His
290 295 300
148
CA 02178482 2010-06-29
Pro Leu Leu Pro Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser
305 310 315
Pro Leu Leu Asn Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln
320 325 330
Glu Gly
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 166 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Ala Pro Pro Arg Leu Ile Cys Asp Ser Arg Val Leu Glu Arg Tyr
1 5 10 15
Leu Leu Glu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Ala
20 25 30
Glu His Cys Ser Leu Asn Glu Asn Ile Thr Val Pro Asp Thr Lys
35 40 45
Val Asn Phe Tyr Ala Trp Lys Arg Met Glu Val Gly Gln Gln Ala
50 55 60
Val Glu Val Trp Gln Gly Leu Ala Leu Leu Ser Glu Ala Val Leu
65 70 75
Arg Gly Gln Ala Leu Leu Val Asn Ser Ser Gln Pro Trp Glu Pro
80 85 90
Leu Gln Leu His Val Asp Lys Ala Val Ser Gly Leu Arg Ser Leu
95 100 105
Thr Thr Leu Leu Arg Ala Leu Gly Ala Gln Lys Glu Ala Ile Ser
110 115 120
Pro Pro Asp Ala Ala Ser Ala Ala Pro Leu Arg Thr Ile Thr Ala
125 130 135
Asp Thr Phe Arg Lys Leu Phe Arg Val Tyr Ser Asn Phe Leu Arg
140 145 150
Gly Lys Leu Lys Leu Tyr Thr Gly Glu Ala Cys Arg Thr Gly Asp
155 160 165
Arg
149
CA 02178482 2010-06-29
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 328 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu
1 5 10 15
Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro
20 25 30
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp
35 40 45
Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala
50 55 60
Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met
65 70 75
Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu
80 85 90
Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln
95 100 105
Ser Leu Leu Gly Thr Gln Gly Arg Thr Thr Ala His Lys Asp Pro
110 115 120
Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val
125 130 135
Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val Arg Arg
140 145 150
Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser Leu Val Leu
155 160 165
Thr Leu Asn Glu Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr
170 175 180
Asn Phe Thr Ala Ser Ala Arg Thr Thr Gly Ser Gly Leu Leu Lys
185 190 195
Trp Gln Gln Gly Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln
200 205 210
Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly Tyr Leu Asn Arg Ile
215 220 225
150
CA 02178482 2010-06-29
His Glu Leu Leu Asn Gly Thr Arg Gly Leu Phe Pro Gly Pro Ser
230 235 240
Arg Arg Thr Leu Gly Ala Pro Asp Ile Ser Ser Gly Thr Ser Asp
245 250 255
Thr Gly Ser Leu Pro Pro Asn Leu Gin Pro Gly Tyr Ser Pro Ser
260 265 270
Pro Thr His Pro Pro Thr Gly Gln Tyr Thr Leu Phe Pro Leu Pro
275 280 285
Pro Thr Leu Pro Thr Pro Val Val Gln Leu His Pro Leu Leu Pro
290 295 300
Asp Pro Ser Ala Pro Thr Pro Thr Pro Thr Ser Pro Leu Leu Asn
305 310 315
Thr Ser Tyr Thr His Ser Gln Asn Leu Ser Gln Glu Gly
320 325
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 265 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu
1 5 10 15
Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro
20 25 30
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp
35 40 45
Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala
50 55 60
Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met
65 70 75
Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu
80 85 90
Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln
95 100 105
Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala
110 115 120
151
CA 02178482 2010-06-29
His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe Gin His Leu Leu
125 130 135
Arg Gly Lys Asp Phe Trp Ile Val Gly Asp Lys Leu His Cys Leu
140 145 150
Ser Gln Asn Tyr Trp Leu Trp Ala Ser Glu Val Ala Ala Gly Ile
155 160 165
Gln Ser Gln Asp Ser Trp Ser Ala Glu Pro Asn Leu Gln Val Pro
170 175 180
Gly Pro Asn Pro Arg Ile Pro Glu Gln Asp Thr Arg Thr Leu Glu
185 190 195
Trp Asn Ser Trp Thr Leu Ser Trp Thr Leu Thr Gln Asp Pro Arg
200 205 210
Ser Pro Gly His Phe Leu Arg Asn Ile Arg His Arg Leu Pro Ala
215 220 225
Thr Gln Pro Pro Ala Trp Ile Phe Ser Phe Pro Asn Pro Ser Ser
230 235 240
Tyr Trp Thr Val Tyr Ala Leu Pro Ser Ser Thr His Leu Ala His
245 250 255
Pro Cys Gly Pro Ala Pro Pro Pro Ala Ser
260 265
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 261 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu
1 5 10 15
Leu Arg Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro
20 25 30
Glu Val His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp
35 40 45
Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala
50 55 60
Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met
65 70 75
152
CA 02178482 2010-06-29
Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu
80 85 90
Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln
95 100 105
Ser Leu Leu Gly Thr Gln Gly Arg Thr Thr Ala His Lys Asp Pro
110 115 120
Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly Lys Asp
125 130 135
Phe Trp Ile Val Gly Asp Lys Leu His Cys Leu Ser Gln Asn Tyr
140 145 150
Trp Leu Trp Ala Ser Glu Val Ala Ala Gly Ile Gln Ser Gln Asp
155 160 165
Ser Trp Ser Ala Glu Pro Asn Leu Gln Val Pro Gly Pro Asn Pro
170 175 180
Arg Ile Pro Glu Gln Asp Thr Arg Thr Leu Glu Trp Asn Ser Trp
185 190 195
Thr Leu Ser Trp Thr Leu Thr Gln Asp Pro Arg Ser Pro Gly His
200 205 210
Phe Leu Arg Asn Ile Arg His Arg Leu Pro Ala Thr Gln Pro Pro
215 220 225
Ala Trp Ile Phe Ser Phe Pro Asn Pro Ser Ser Tyr Trp Thr Val
230 235 240
Tyr Ala Leu Pro Ser Ser Thr His Leu Ala His Pro Cys Gly Pro
245 250 255
Ala Pro Pro Pro Ala Ser
260
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7849 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CCCAGCCTCC TTTCTCTTGT TCCCTGGTCA TGCCTGCCTC CCTGTCTCCT 50
GTCTCTCCCT CCCACACACA CCCACTATCC TCCCAGCTAT CCCTACACCC 100
153
CA 02178482 2010-06-29
TCCTTCCTAA TCTTGGGAGA CATCTCGTCT GGCTGGACGG GAAAATTCCA 150
GGATCTAGGC CACACTTCTC AGCAGACATG CCCATCCTTG GGGAGGAGGA 200
ACAGGAGAGA GCCTGAGGAA GTTCTGGGGG ACAGGGGGAT GATGGGATCA 250
AGGTCAGGCC AGGAAGCCCC TGAGGACAGA GACTGTGGGG AGACTGGGAC 300
TGGGAAGAAA GCAAAGGAGC TAGAGCCAGG GCCAAAGGAA AAGGGGGGCC 350
AGCAGGGAGG TATTTGCGGG GGAGGTCCAG CAGCTGTCTT TCCTAAGACA 400
GGGACACATG GGCCTGGTTA TTCCTCTTGT CACATGTGGA ACGGTAGGAG 450
ATGGAAGACG GAGACAGAAC AAGCAAAGAA GGGCCCTGGG CACAGAGGTC 500
TGTGTGTGTA GCCATCCAAG CCACTGGACC CCAGCAGACG AGCACCTAAG 550
CTCAGGCTTA ACCCAGTGCA CGTGTGCGCA CATACATGTG CCCCGCACCT 600
GACAGTCCAC TCAACCCGTC CAAACCCTTT CCCCATAACA CCAACCCATA 650
ACAGGAGATT TCTCTCATGT GGGCAATATC CGTGTTCCCA CTTCGAAAGG 700
GGGAATGACA AGATAGGACT CCCTAGGGGA TTACAGAAAG AAAAGCAGGA 750
AAGCAAGCAT CCTGTTGGAT TTCAGCAGCA GGTATGATGT CCAGGGAAAA 800
GAAATTTGGA TAGCCAGGGA GTGAAAACCC CACCAATCTT AAACAAGACC 850
TCTGTGCTTC TTCCCCAGCA ACACAAATGT CCTGCCAGAT TCCTCCTGGA 900
AAAAAACTTC TGCTCCTGTC CCCCTCCAGG TCCCAGGTTG CCCATGTCCA 950
GGAAAAGATG GATCCCCCTA TCCAAATCTT CTCCGTGGTG TGTGTGGGTG 1000
GAGGAGTGGA CCCTGGTCCA GGCAGGGGCT CCAGGGAAGA GAAGGCGTCA 1050
CTTCCGGGGG CCTTCACCAG TGTCTGGTGG CTCCCTTCTC TGATTGGGCA 1100
GAAGTGGCCC AGGCAGGCGT ATGACCTGCT GCTGTGGAGG GGCTGTGCCC 1150
CACCGCCACA TGTCTTCCTA CCCATCTGCT CCCCAGAGGG CTGCCTGCTG 1200
TGCACTTGGG TCCTGGAGCC CTTCTCCACC CGGTGAGTGG CCAGCAGGGT 1250
GTGGGGTTAT GTGAGGGTAG AAAGGACAGC AAAGAGAAAT GGGCTCCCAG 1300
CTGGGGGAGG GGCAGGCAAA CTGGAACCTA CAGGCACTGA CCTTTGTCGA 1350
GAAGAGTGTA GCCTTCCCAG AATGGGAGGA GCAGGGCAGA GCAGGGGTAG 1400
GGGGTGGGGT GCTGGTTTCT GAGGGACTGA TCACTTACTT GGTGGAATAC 1450
154
CA 02178482 2010-06-29
AGCACAGCCC TGGCTGGCCC TAAGGAAAGG GGACATGAGC CCAGGGAGAA 1500
AATAAGAGAG GGAGCTGCAC TTAGGGCTTA GCAAACACAG TAGTAAGATG 1550
GACACAGCCC CAATCCCCAT TCTTAGCTGG TCATTCCTCG TTAGCTTAAG 1600
GTTCTGAATC TGGTGCTGGG GAAGCTGGGC CAGGCAAGCC AGGGCGCAAG 1650
GAGAGGGTAA TGGGAGGAGG GCCCACTCAT GTTGACAGAC CTACAGGAAA 1700
TCCCAATATT GAATCAGGTG CAAGCCTCTT TGCACAACTT GTGAAAGGAG 1750
GAGGAAGCCA TGTGGGGGGT CCTGTGAAGG AACCGGAAGG GGTTCTGCCA 1800
AGGGGGCAGG GAGGCAGGTG TGAGCTATGA GACAGATATG TTAGTGGGCG 1850
CCTAAGACAA GGTAAGCCCC TAAGGTGGGC ATCACCCAGC AGGTGCCCGT 1900
TCCTGGGCAG CTGGTCTCAG GAAGGAAGTC CCAGAACTGT TAGCCCATCT 1950
CTTGGCCTCA GATAATGGAG TATTTCAGGA CTTGGAGTCC AAAGAAAAGC 2000
TCCAGTGGCT TTATGTGTGG GGGTAGATAG GGAAAGAATA GAGGTTAATT 2050
TCTCCCATAC CGCCTTTTAA TCCTGACCTC TAGTGGTCCC AGTTACAGCT 2100
TTGTGCAGTT CCCCTCCCCA GCCCCACTCC CCACCGCAGA AGTTACCCCT 2150
CAACATATTG CGCCCGTTTG CCAGTTCCTC ACCCAGGCCC TGCATCCCAT 2200
TTTCCACTCT CTTCTCCAGG CTGAAGCCAC AATACTTTCC TTCTCTATCC 2250
CCATCCCAGA TTTTCTCTGA CCTAACAACC AAGGTTGCTC AGAATTTAAG 2300
GCTAATTAAG ATATGTGTGT ATACATATCA TGTCCTGCTG CTCTCAGCAG 2350
GGGTAGGTGG CACCAAATCC GTGTCCGATT CACTGAGGAG TCCTGACAAA 2400
AAGGAGACAC CATATGCTTT CTTGCTTTCT TTCTTTCTTT CTTTCTTTTT 2450
TTTTTTTTGA GACGGAGTTT CACTCTTATT GCCCAGGCTG GAGTGCAATG 2500
GTGCGATCTC GGCTCACCAC AAACCTCCGC CTCCCAGGTA CAAGCGATTC 2550
TCCTGTCTCA GCCTCCCAAG TAGCTTGGAT TACAGGCATG AGCCACCACA 2600
CCCTGCTAGT TTTTTTGTAT TTCGTAGAGC CGGGGTTTCA CCATGTTAGT 2650
GAGGCTGGTG GCGAACTCCT GACCTCAGGT GATCCACCCG CCTTGGACTC 2700
CCAAAGTGCT GGGATTACAG GCATGAGCCA CTGCACCCGG CACACCATAT 2750
GCTTTCATCA CAAGAAAATG TGAGAGAATT CAGGGCTTTG GCAGTTCCAG 2800
155
CA 02178482 2010-06-29
GCTGGTCAGC ATCTCAAGCC CTCCCCAGCA TCTGTTCACC CTGCCAGGCA 2850
GTCTCTTCCT AGAAACTTGG TTAAATGTTC ACTCTTCTTG CTACTTTCAG 2900
GATAGATTCC TCACCCTTGG CCCGCCTTTG CCCCACCCTA CTCTGCCCAG 2950
AAGTGCAAGA GCCTAAGCCG CCTCCATGGC CCCAGGAAGG ATTCAGGGGA 3000
GAGGCCCCAA ACAGGGAGCC ACGCCAGCCA GACACCCCGG CCAGAATGGA 3050
GCTGACTGGT GAGAACACAC CTGAGGGGCT AGGGCCATAT GGAAACATGA 3100
CAGAAGGGGA GAGAGAAAGG AGACACGCTG CAGGGGGCAG GAAGCTGGGG 3150
GAACCCATTC TCCCAAAAAT AAGGGGTCTG AGGGGTGGAT TCCCTGGGTT 3200
TCAGGTCTGG GTCCTGAATG GGAATTCCTG GAATACCAGC TGACAATGAT 3250
TTCCTCCTCA TCTTTCAACC TCACCTCTCC TCATCTAAGA ATTGCTCCTC 3300
GTGGTCATGC TTCTCCTAAC TGCAAGGCTA ACGCTGTCCA GCCCGGCTCC 3350
TCCTGCTTGT GACCTCCGAG TCCTCAGTAA ACTGCTTCGT GACTCCCATG 3400
TCCTTCACAG CAGACTGGTG AGAACTCCCA ACATTATCCC CTTTATCCGC 3450
GTAACTGGTA AGACACCCAT ACTCCCAGGA AGACACCATC ACTTCCTCTA 3500
ACTCCTTGAC CCAATGATGA TTCTTCCCAT ATTGTCCCCA CCTACTGATC 3550
ACACTCTCTG ACAAGAATTA TTCTTCACAA TACAGCCCGC ATTTAAAAGC 3600
TCTCGTCTAG AGATAGTACT CATGGAGGAC TAGCCTGCTT ATTAGGCTAC 3650
CATAGCTCTC TCTATTTCAG CTCCCTTCTC CCCCAACCAA TCTTTTTCAA 3700
CAGAGCCAGT GCCCAGAGGT TCACCCTTTG CCTACACCTG TCCTGCTGCC 3750
TGCTGTGGAC TTTAGCTTGG GAGAATGGAA AACCCAGATG GTAAGAAAGC 3800
CATCCCTAAC CTTGGCTTCC CTAAGTCCTG TCTTCAGTTT CCCACTGCTT 3850
CCCATGGATT CTCCAACATT CTTGAGCTTT TTAAAAATAT CTCACCTTCA 3900
GCTTGGCCAC CCTAACCCAA TCTACATTCA CCTATGATGA TAGCCTGTGG 3950
ATAAGATGAT GGCTTGCAGG TCCAATATGT GAATAGATTT GAAGCTGAAC 4000
ACCATGAAAA GCTGGAGAGA AATCGCTCAT GGCCATGCCT TTGACCTATT 4050
CCYGTTCAGT CTTCTTAAAT TGGCATGAAG AAGCAAGACT CATATGTCAT 4100
CCACAGATGA CACAAAGCTG GGAAGTACCA CTAAAATAAC AAAAGACTGA 4150
156
CA 02178482 2010-06-29
ATCAAGATTC AAATCACTGA AAGACTAGGT CAAAAACAAG GTGAAACAAC 4200
AGAGATATAA ACTTCTACAT GTGGGCCGGG GGCTCACGCC TGTAATCCCA 4250
GCACTTTGGG AGGCCGAGGC AGGCAGATCA CCTGAGGGCA GGAGTTTGAG 4300
AGCAGCCTGG CCAACATGGC GAAACCCCGT CTCTACTAAG AATACAAAAT 4350
TAGCCGGGCA TGGTAGTGCA TGCCTGTAAT CCCAGCTACT TGGAAGGCTG 4400
AAGCAGGAGA ATCCCTTGAA CCCAGGAGGT GGAGGTTGTA GTGAGCTGAG 4450
ATCATGGCAA TGCACTCCAG CCTGGGTGAC AAGAGCAAAA CTCCGTCTCA 4500
AAAAGAAAAA AAAATTCTAC ATGTGTAAAT TAATGAGTAA AGTCCTATTC 4550
CAGCTTTCAG GCCACAATGC CCTGCTTCCA TCATTTAAGC CTCTGGCCCT 4600
AGCACTTCCT ACGAAAAGGA TCTGAGAGAA TTAAATTGCC CCCAAACTTA 4650
CCATGTAACA TTACTGAAGC TGCTATTCTT AAAGCTAGTA ATTCTTGTCT 4700
GTTTGATGTT TAGCATCCCC ATTGTGGAAA TGCTCGTACA GAACTCTATT 4750
CCGAGTGGAC TACACTTAAA TATACTGGCC TGAACACCGG ACATCCCCCT 4800
GAAGACATAT GCTAATTTAT TAAGAGGGAC CATATTAAAC TAACATGTGT 4850
CTAGAAAGCA GCAGCCTGAA CAGAAAGAGA CTAGAAGCAT GTTTTATGGG 4900
CAATAGTTTA AAAAACTAAA ATCTATCCTC AAGAACCCTA GCGTCCCTTC 4950
TTCCTTCAGG ACTGAGTCAG GGAAGAAGGG CAGTTCCTAT GGGTCCCTTC 5000
TAGTCCTTTC TTTTCATCCT TATGATCATT ATGGTAGAGT CTCATACCTA 5050
CATTTAGTTT ATTTATTATT ATTATTTGAG ACGGAGTCTC ACTCTATCCC 5100
CCAGGCTGGA GTGCAGTGGC ATGATCTCAA CTCACTGCAA CCTCAGCCTC 5150
CCGGATTCAA GCGATTCTCC TGCCTCAGTC TCCCAAGTAG CTGGGATTAC 5200
AGGTGCCCAC CACCATGCCC AGCTAATTTT TGTATTTTTG GTAGAGATGG 5250
GGTTTCACCA TGTTGGCCAG GCTGATCTTG AACTCCTGAC CTCAGGTGAT 5300
CCACCTGCCT CAGCCTCCCA AAGTGCTGGG ATTACAGGCG TGAGCCACTG 5350
CACCCAGCCT TCATTCAGTT TAAAAATAAA ATGATCCTAA GGTTTTGCAG 5400
CAGAAAGAGT AAATTTGCAG CACTAGAACC AAGAGGTAAA AGCTGTAACA 5450
GGGCAGATTT CAGCAACGTA AGAAAAAAGG AGCTCTTCTC ACTGAAACCA 5500
157
CA 02178482 2010-06-29
AGTGTAAGAC CAGGCTGGAC TAGAGGACAC GGGAGTTTTT GAAGCAGAGG 5550
CTGATGACCA GCTGTCGGGA GACTGTGAAG GAATTCCTGC CCTGGGTGGG 5600
ACCTTGGTCC TGTCCAGTTC TCAGCCTGTA TGATTCACTC TGCTGGCTAC 5650
TCCTAAGGCT CCCCACCCGC TTTTAGTGTG CCCTTTGAGG CAGTGCGCTT 5700
CTCTCTTCCA TCTCTTTCTC AGGAGGAGAC CAAGGCACAG GACATTCTGG 5750
GAGCAGTGAC CCTTCTGCTG GAGGGAGTGA TGGCAGCACG GGGACAACTG 5800
GGACCCACTT GCCTCTCATC CCTCCTGGGG CAGCTTTCTG GACAGGTCCG 5850
TCTCCTCCTT GGGGCCCTGC AGAGCCTCCT TGGAACCCAG GTAAGTCCCC 5900
AGTCAAGGGA TCTGTAGAAA CTGTTCTTTT CTGACTCAGT CCCACTAGAA 5950
GACCTGAGGG AAGAAGGGCT CTTCCAGGGA GCTCAAGGGC AGAAGAGCTG 6000
ATCTACTAAG AGTGCTCCCT GCCAGCCACA ATGCCTGGGT ACTGGCATCC 6050
TGTCTTTCCT ACTTAGACAA GGGAGGCCTG AGATCTGGCC CTGGTGTTTG 6100
GCCTCAGGAC CATCCTCTGC CCTCAGCTTC CTCCACAGGG CAGGACCACA 6150
GCTCACAAGG ATCCCAATGC CATCTTCCTG AGTTTCCAAC ACCTGCTCCG 6200
AGGAAAGGTG CGTTTCCTGA TGCTTGTAGG AGGGTCCACC CTCTGCGTCA 6250
GGCGGGCCCC ACCCACCACA GCTGTCCCCA GCAGAACCTC TCTAGTCCTC 6300
ACACTGAACG AGCTCCCAAA CAGGACTTCT GGATTGTTGG AGACAAACTT 6350
CACTGCCTCA GCCAGAACTA CTGGCTCTGG GCTTCTGAAG TGGCAGCAGG 6400
GATTCAGAGC CAAGATTCCT GGTCTGCTGA ACCAAACCTC CAGGTCCCTG 6450
GACCAAATCC CCGGATACCT GAACAGGATA CACGAACTCT TGAATGGAAC 6500
TCGTGGACTC TTTCCTGGAC CCTCACGCAG GACCCTAGGA GCCCCGGACA 6550
TTTCCTCAGG AACATCAGAC ACAGGCTCCC TGCCACCCAA CCTCCAGCCT 6600
GGATATTCTC CTTCCCCAAC CCATCCTCCT ACTGGACAGT ATACGCTCTT 6650
CCCTCTTCCA CCCACCTTGC CCACCCCTGT GGTCCAGCTC CACCCCCTGC 6700
TTCCTGACCC TTCTGCTCCA ACGCCCACCC CTACCAGCCC TCTTCTAAAC 6750
ACATCCTACA CCCACTCCCA GAATCTGTCT CAGGAAGGGT AAGGTTCTCA 6800
GACACTGCCG ACATCAGCAT TGTCTCATGT ACAGCTCCCT TCCCTGCAGG 6850
158
CA 02178482 2010-06-29
GCTCCCCTGG GAGACAACTG GACAAGATTT CCTACTTTCT CCTGAAACCC 6900
AAAGCCCTGG TAAAAGGGAT ACACAGGACT GAAAAGGGAA TCATTTTTCA 6950
CTGTACATTA TAAACCTTCA GAAGCTATTT TTTTAAGCTA TCAGCAATAC 7000
TCATCAGAGC AGCTAGCTCT TTGGTCTATT TTCTGCAGAA ATTTGCAACT 7050
CACTGATTCT CTACATGCTC TTTTTCTGTG ATAACTCTGC AAAGGCCTGG 7100
GCTGGCCTGG CAGTTGAACA GAGGGAGAGA CTAACCTTGA GTCAGAAAAC 7150
AGAGAAAGGG TAATTTCCTT TTCTTCAAAT TCAAGGCCTT CCAACGCCCC 7200
CATCCCCTTT ACTATCATTC TCAGTGGGAC TCTGATCCCA TATTCTTAAC 7250
AGATCTTTAC TCTTGAGAAA TGAATAAGCT TTCTCTCAGA AATGCTGTCC 7300
CTATACACTA GACAAAACTG AGCCTGTATA AGGAATAAAT GGGAGCGCCG 7350
AAAAGCTCCC TAAAAAGCAA GGGAAAGATG TTCTTCGAGG GTGGCAATAG 7400
ATCCCCCTCA CCCTGCCACC CCAAACAAAA AAGCTAACAG GAAGCCTTGG 7450
AGAGCCTCAC ACCCCAGGTA AGGCTGTGTA GACAGTTCAG TAAAGACAGG 7500
ACCTGGATGT GACAGCTGAG CAAACAGCTA GAGCTTTGGC AGCTCAGCAG 7550
GAGGCTTTGC CAGGCATGGA CGCCTGCCTC CCTCCTGTGG AGGTCAGGAG 7600
GAAGTGCAGG AAGTGGCATG AGTCAGGCTC CTTGAGCTCA CACAGCAGGA 7650
GAACAAGTAC AAGTCAAGTA CAAGTTGAAG GCTCATTTCC CAGTTCCCGC 7700
AAATGCATCT AAAAAGCAGC TCTGTGTGAC CACCATAAAC TCTGCTAGGG 7750
GATCTCTAAA AAGGAGTCAG GCTTATGGGG CTTTGCAAAT AAGTGCTGCC 7800
TTGGTGCTCA GGAAAAGGTT TGTGTTGCAC AAAACACAAA TTCCACTGC 7849
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1443 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GAGTCCTTGG CCCACCTCTC TCCCACCCGA CTCTGCCGAA AGAAGCACAG 50
AAGCTCAAGC CGCCTCCATG GCCCCAGGAA AGATTCAGGG GAGAGGCCCC 100
159
CA 02178482 2010-06-29
ATACAGGGAG CCACTTCAGT TAGACACCCT GGCCAGAATG GAGCTGACTG 150
ATTTCCTCCT GGCGGCCATG CTTCTTGCAG TGGCAAGACT AACTCTGTCC 200
AGCCCCGTAG CTCCTGCCTG TGACCCCAGA CTCCTAAATA AACTGCTGCG 250
TGACTCCCAC CTCCTTCACA GCCGACTGAG TCAGTGTCCC GACGTCGACC 300
CTTTGTCTAT CCCTGTTCTG CTGCCTGCTG TGGACTTTAG CCTGGGAGAA 350
TGGAAAACCC AGACGGAACA GAGCAAGGCA CAGGACATTC TAGGGGCAGT 400
GTCCCTTCTA CTGGAGGGAG TGATGGCAGC ACGAGGACAG TTGGAACCCT 450
CCTGCCTCTC ATCCCTCCTG GGACAGCTTT CTGGGCAGGT TCGCCTCCTC 500
TTGGGGGCCC TGCAGGGCCT CCTAGGAACC CAGGGCAGGA CCACAGCTCA 550
CAAGGACCCC AATGCCCTCT TCTTGAGCTT GCAACAACTG CCTCGGGGAA 600
AGGTGCGCTT CCTGCTTCTG GTAGAAGGTC CCACCCTCTG TGTCAGACGG 650
ACCCTTCCAA CCACAGCTGT CCCAAGCAGT ACTTCTCAAC TCCTCACACT 700
AAACAAGTTC CCAAACAGGA CTTCTGGATT GTTGGAGACG AACTTCAGTG 750
TCACAGCCAG AACTGCTGGC CCTGGACTTC TGAGCAGGCT TCAGGGATTC 800
AGAGTCAAGA TTACTCCTGG TCAGCTAAAT CAAACCTCCA GGTCCCCAGT 850
CCAAATCTCT GGATACCTGA ACAGGACACA CGGACCTGTG AATGGAACTC 900
ATGGGCTCTT TGCTGGAACC TCACTTCAGA CCCTGGAACC CTCAGACATC 950
TCGCCCGGAG CTTTCAACAA AGGCTCCCTG GCATTCAACC TCCAGGGTGG 1000
ACTTCCTCCT TCTCCAAGCC TTGCTCCTGA TGGACACACA CCCTTCCCTC 1050
CTTCACCTGC CTTGCCCACC ACCCATGGAT CTCCACCCCA GCTCCACCCC 1100
CTGTTTCCTG ACCCTTCCAC CACCATGCCT AACTCTACCG CCCCTCATCC 1150
AGTCACAATG TACCCTCATC CCAGGAATTT GTCTCAGGAA ACATAGCGCG 1200
GGCACTGGCC CAGTGAGCGT CTGCAGCTTC TCTCGGGGAC AAGCTTCCCC 1250
AGGAAGGCTG AGAGGCAGCT GCATCTGCTC CAGATGTTCT GCTTTCACCT 1300
AAAAGGCCCT GGGGAAGGGA TACACAGCAC TGGAGATTGT AAAATTTTAG 1350
GAGCTATTTT TTTTTAACCT ATCAGCAATA TTCATCAGAG CAGCTAGCGA 1400
TCTTTGGTCT ATTTTCGGTA TAAATTTGAA AATCACTAAT TCT 1443
160
CA 02178482 2010-06-29
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 352 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Met Glu Leu Thr Asp Leu Leu Leu Ala Ala Met Leu Leu Ala Val
1 5 10 15
Ala Arg Leu Thr Leu Ser Ser Pro Val Ala Pro Ala Cys Asp Pro
20 25 30
Arg Leu Leu Asn Lys Leu Leu Arg Asp Ser His Leu Leu His Ser
35 40 45
Arg Leu Ser Gln Cys Pro Asp Val Asp Pro Leu Ser Ile Pro Val
50 55 60
Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln
65 70 75
Thr Glu Gln Ser Lys Ala Gin Asp Ile Leu Gly Ala Val Ser Leu
80 85 90
Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Glu Pro Ser
95 100 105
Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu
110 115 120
Leu Leu Gly Ala Leu Gln Gly Leu Leu Gly Thr Gln Gly Arg Thr
125 130 135
Thr Ala His Lys Asp Pro Asn Ala Leu Phe Leu Ser Leu Gln Gln
140 145 150
Leu Leu Arg Gly Lys Val Arg Phe Leu Leu Leu Val Glu Gly Pro
155 160 165
Thr Leu Cys Val Arg Arg Thr Leu Pro Thr Thr Ala Val Pro Ser
170 175 180
Ser Thr Ser Gln Leu Leu Thr Leu Asn Lys Phe Pro Asn Arg Thr
185 190 195
Ser Gly Leu Leu Glu Thr Asn Phe Ser Val Thr Ala Arg Thr Ala
200 205 210
Gly Pro Gly Leu Leu Ser Arg Leu Gln Gly Phe Arg Val Lys Ile
215 220 225
161
CA 02178482 2010-06-29
Thr Pro Gly Gln Leu Asn Gln Thr Ser Arg Ser Pro Val Gln Ile
230 235 240
Ser Gly Tyr Leu Asn Arg Thr His Gly Pro Val Asn Gly Thr His
245 250 255
Gly Leu Phe Ala Gly Thr Ser Leu Gln Thr Leu Glu Ala Ser Asp
260 265 270
Ile Ser Pro Gly Ala Phe Asn Lys Gly Ser Leu Ala Phe Asn Leu
275 280 285
Gln Gly Gly Leu Pro Pro Ser Pro Ser Leu Ala Pro Asp Gly His
290 295 300
Thr Pro Phe Pro Pro Ser Pro Ala Leu Pro Thr Thr His Gly Ser
305 310 315
Pro Pro Gln Leu His Pro Leu Phe Pro Asp Pro Ser Thr Thr Met
320 325 330
Pro Asn Ser Thr Ala Pro His Pro Val Thr Met Tyr Pro His Pro
335 340 345
Arg Asn Leu Ser Gin Glu Thr
350
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1536 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GAGTCCTTGG CCCACCTCTC TCCCACCCGA CTCTGCCGAA AGAAGCACAG 50
AAGCTCAAGC CGCCTCCATG GCCCCAGGAA AGATTCAGGG GAGAGGCCCC 100
ATACAGGGAG CCACTTCAGT TAGACACCCT GGCCAGAATG GAGCTGACTG 150
ATTTGCTCCT GGCGGCCATG CTTCTTGCAG TGGCAAGACT AACTCTGTCC 200
AGCCCCGTAG CTCCTGCCTG TGACCCCAGA CTCCTAAATA AACTGCTGCG 250
TGACTCCCAC CTCCTTCACA GCCGACTGAG TCAGTGTCCC GACGTCGACC 300
CTTTGTCTAT CCCTGTTCTG CTGCCTGCTG TGGACTTTAG CCTGGGAGAA 350
TGGAAAACCC AGACGGAACA GAGCAAGGCA CAGGACATTC TAGGGGCAGT 400
162
CA 02178482 2010-06-29
GTCCCTTCTA CTGGAGGGAG TGATGGCAGC ACGAGGACAG TTGGAACCCT 450
CCTGCCTCTC ATCCCTCCTG GGACAGCTTT CTGGGCAGGT TCGCCTCCTC 500
TTGGGGGCCC TGCAGGGCCT CCTAGGAACC CAGCTTCCTC TACAGGGCAG 550
GACCACAGCT CACAAGGACC CCAATGCCCT CTTCTTGAGC TTGCAACAAC 600
TGCTTCGGGG AAAGGTGCGC TTCCTGCTTC TGGTAGAAGG TCCCACCCTC 650
TGTGTCAGAC GGACCCTGCC AACCACAGCT GTCCCAAGCA GTACTTCTCA 700
ACTCCTCACA CTAAACAAGT TCCCAAACAG GACTTCTGGA TTGTTGGAGA 750
CGAACTTCAG TGTCACAGCC AGAACTGCTG GCCCTGGACT TCTGAGCAGG 800
CTTCAGGGAT TCAGAGTCAA GATTACTCCT GGTCAGCTAA ATCAAACCTC 850
CAGGTCCCCA GTCCAAATCT CTGGATACCT GAACAGGACA CACGGACCTG 900
TGAATGGAAC TCATGGGCTC TTTGCTGGAA CCTCACTTCA GACCCTGGAA 950
GCCTCAGACA TCTCGCCCGG AGCTTTCAAC AAAGGCTCCC TGGCATTCAA 1000
CCTCCAGGGT GGACTTCCTC CTTCTCCAAG CCTTGCTCCT GATGGACACA 1050
CACCCTTCCC TCCTTCACCT GCCTTGCCCA CCACCCATGG ATCTCCACCC 1100
CAGCTCCACC CCCTGTTTCC TGACCCTTCC ACCACCATGC CTAACTCTAC 1150
CGCCCCTCAT CCAGTCACAA TGTACCCTCA TCCCAGGAAT TTGTCTCAGG 1200
AAACATAGCG CGGGCACTGG CCCAGTGAGC GTCTGCAGCT TCTCTCGGGG 1250
ACAAGCTTCC CCAGGAAGGC TGAGAGGCAG CTGCATCTGC TCCAGATGTT 1300
CTGCTTTCAC CTAAAAGGCC CTGGGGAAGG GATACACAGC ACTGGAGATT 1350
GTAAAATTTT AGGAGCTATT TTTTTTTAAC CTATCAGCAA TATTCATCAG 1400
AGCAGCTAGC GATCTTTGGT CTATTTTCGG TATAAATTTG AAAATCACTA 1450
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1500
AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAA 1536
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 356 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
163
CA 02178482 2010-06-29
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Met Glu Leu Thr Asp Leu Leu Leu Ala Ala Met Leu Leu Ala Val
1 5 10 15
Ala Arg Leu Thr Leu Ser Ser Pro Val Ala Pro Ala Cys Asp Pro
20 25 30
Arg Leu Leu Asn Lys Leu Leu Arg Asp Ser His Leu Leu His Ser
35 40 45
Arg Leu Ser Gln Cys Pro Asp Val Asp Pro Leu Ser Ile Pro Val
50 55 60
Leu Leu Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln
65 70 75
Thr Glu Gln Ser Lys Ala Gln Asp Ile Leu Gly Ala Val Ser Leu
80 85 90
Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Glu Pro Ser
95 100 105
Cys Leu Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu
110 115 120
Leu Leu Gly Ala Leu Gln Gly Leu Leu Gly Thr Gln Leu Pro Leu
125 130 135
Gln Gly Arg Thr Thr Ala His Lys Asp Pro Asn Ala Leu Phe Leu
140 145 150
Ser Leu Gln Gln Leu Leu Arg Gly Lys Val Arg Phe Leu Leu Leu
155 160 165
Val Glu Gly Pro Thr Leu Cys Val Arg Arg Thr Leu Pro Thr Thr
170 175 180
Ala Val Pro Ser Ser Thr Ser Gln Leu Leu Thr Leu Asn Lys Phe
185 190 195
Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr Asn Phe Ser Val Thr
200 205 210
Ala Arg Thr Ala Gly Pro Gly Leu Leu Ser Arg Leu Gln Gly Phe
215 220 225
Arg Val Lys Ile Thr Pro Gly Gln Leu Asn Gln Thr Ser Arg Ser
230 235 240
Pro Val Gln Ile Ser Gly Tyr Leu Asn Arg Thr His Gly Pro Val
245 250 255
Asn Gly Thr His Gly Leu Phe Ala Gly Thr Ser Leu Gln Thr Leu
164
CA 02178482 2010-06-29
260 265 270
Glu Ala Ser Asp Ile Ser Pro Gly Ala Phe Asn Lys Gly Ser Leu
275 280 285
Ala Phe Asn Leu Gln Gly Gly Leu Pro Pro Ser Pro Ser Leu Ala
290 295 300
Pro Asp Gly His Thr Pro Phe Pro Pro Ser Pro Ala Leu Pro Thr
305 310 315
Thr His Gly Ser Pro Pro Gln Leu His Pro Leu Phe Pro Asp Pro
320 325 330
Ser Thr Thr Met Pro Asn Ser Thr Ala Pro His Pro Val Thr Met
335 340 345
Tyr Pro His Pro Arg Asn Leu Ser Gln Glu Thr
350 355
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 241 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Ser Pro Val Ala Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu
'1 5 10 15
Leu Arg Asp Ser His Leu Leu His Ser Arg Leu Ser Gln Cys Pro
20 25 30
Asp Val Asp Pro Leu Ser Ile Pro Val Leu Leu Pro Ala Val Asp
35 40 45
Phe Ser Leu Gly Glu Trp Lys Thr Gln Thr Glu Gln Ser Lys Ala
50 55 60
Gln Asp Ile Leu Gly Ala Val Ser Leu Leu Leu Glu Gly Val Met
65 70 75
Ala Ala Arg Gly Gln Leu Glu Pro Ser Cys Leu Her Ser Leu Leu
80 85 90
Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln
95 100 105
Gly Leu Leu Gly Thr Gln Leu Pro Leu Gln Gly Arg Thr Thr Ala
110 115 120
His Lys Asp Pro Asn Ala Leu She Leu Ser Leu Gln Gln Leu Leu
165
CA 02178482 2010-06-29
125 130 135
Arg Gly Lys Asp Phe Trp Ile Val Gly Asp Glu Leu Gln Cys His
140 145 150
Ser Gln Asn Cys Trp Pro Trp Thr Ser Glu Gln Ala Ser Gly Ile
155 160 165
Gln Ser Gln Asp Tyr Ser Trp Ser Ala Lys Ser Asn Leu Gln Val
170 175 180
Pro Ser Pro Asn Leu Trp Ile Pro Glu Gln Asp Thr Arg Thr Cys
185 190 195
Glu Trp Asn Ser Trp Ala Leu Cys Trp Asn Leu Thr Ser Asp Pro
200 205 210
Gly Ser Leu Arg His Leu Ala Arg Ser Phe Gln Gln Arg Leu Pro
215 220 225
Gly Ile Gln Pro Pro Gly Trp Thr Ser Ser Phe Ser Lys Pro Cys
230 235 240
Ser
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 335 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l7:
Ser Pro Val Ala Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu
1 5 10 15
Leu Arg Asp Ser His Leu Leu His Ser Arg Leu Ser Gln Cys Pro
20 25 30
Asp Val Asp Pro Leu Ser Ile Pro Val Leu Leu Pro Ala Val Asp
35 40 45
Phe Ser Leu Gly Glu Trp Lys Thr Gln Thr Glu Gln Ser Lys Ala
50 55 60
Gln Asp Ile Leu Gly Ala Val Ser Leu Leu Leu Glu Gly Val Met
65 70 75
Ala Ala Arg Gly Gln Leu Glu Pro Ser Cys Leu Ser Ser Leu Leu
80 85 90
Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln
166
CA 02178482 2010-06-29
95 100 105
Gly Leu Leu Gly Thr Gln Leu Pro Leu Gln Gly Arg Thr Thr Ala
110 115 120
His Lys Asp Pro Asn Ala Leu Phe Leu Ser Leu Gln Gln Leu Leu
125 130 135
Arg Gly Lys Val Arg Phe Leu Leu Leu Val Glu Gly Pro Thr Leu
140 145 150
Cys Val Arg Arg Thr Leu Pro Thr Thr Ala Val Pro Ser Ser Thr
155 160 165
Ser Gln Leu Leu Thr Leu Asn Lys Phe Pro Asn Arg Thr Ser Gly
170 175 180
Leu Leu Glu Thr Asn Phe Ser Val Thr Ala Arg Thr Ala Gly Pro
185 190 195
Gly Leu Leu Ser Arg Leu Gln Gly Phe Arg Val Lys Ile Thr Pro
200 205 210
Gly Gln Leu Asn Gln Thr Ser Arg Ser Pro Val Gln Ile Ser Gly
215 220 225
Tyr Leu Asn Arg Thr His Gly Pro Val Asn Gly Thr His Gly Leu
230 235 240
Phe Ala Gly Thr Ser Leu Gln Thr Leu Glu Ala Ser Asp Ile Ser
245 250 255
Pro Gly Ala Phe Asn Lys Gly Ser Leu Ala Phe Asn Leu Gln Gly
260 265 270
Gly Leu Pro Pro Ser Pro Ser Leu Ala Pro Asp Gly His Thr Pro
275 280 285
Phe Pro Pro Ser Pro Ala Leu Pro Thr Thr His Gly Ser Pro Pro
290 295 300
Gln Leu His Pro Leu Phe Pro Asp Pro Ser Thr Thr Met Pro Asn
305 310 315
Ser Thr Ala Pro His Pro Val Thr Met Tyr Pro His Pro Arg Asn
320 325 330
Leu Ser Gln Glu Thr
335
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 332 amino acids
167
CA 02178482 2010-06-29
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu
1 5 10 15
Leu Arg Asp Ser His Val Leu His Gly Arg Leu Ser Gln Cys Pro
20 25 30
Asp Ile Asn Pro Leu Ser Thr Pro Val Leu Leu Pro Ala Val Asp
35 40 45
Phe Thr Leu Gly Glu Trp Lys Thr Gln Thr Glu Gln Thr Lys Ala
50 55 60
Gln Asp Val Leu Gly Ala Thr Thr Leu Leu Leu Glu Ala Val Met
65 70 75
Thr Ala Arg Gly Gln Val Gly Pro Pro Cys Leu Ser Ser Leu Leu
80 85 90
Val Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln
95 100 105
Asp Leu Leu Gly Met Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala
110 115 120
His Lys Asp Pro Ser Ala Ile Phe Leu Asn Phe Gln Gln Leu Leu
125 130 135
Arg Gly Lys Val Arg Phe Leu Leu Leu Val Val Gly Pro Ser Leu
140 145 150
Cys Ala Lys Arg Ala Pro Pro Ala Ile Ala Val Pro Ser Ser Thr
155 160 165
Ser Pro Phe His Thr Leu Asn Lys Leu Pro Asn Arg Thr Ser Gly
170 175 180
Leu Leu Glu Thr Asn Ser Ser Ile Ser Ala Arg Thr Thr Gly Ser
185 190 195
Gly Phe Leu Lys Arg Leu Gln Ala Phe Arg Ala Lys Ile Pro Gly
200 205 210
Leu Leu Asn Gln Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly His
215 220 225
Gln Asn Gly Thr His Gly Pro Leu Ser Gly Ile His Gly Leu Phe
230 235 240
Pro Gly Pro Gln Pro Gly Ala Leu Gly Ala Pro Asp Ile Pro Pro
168
CA 02178482 2010-06-29
245 250 255
Ala Thr Ser Gly Met Gly Ser Arg Pro Thr Tyr Leu Gln Pro Gly
260 265 270
Glu Ser Pro Ser Pro Ala His Pro Ser Pro Gly Arg Tyr Thr Leu
275 280 285
Phe Ser Pro Ser Pro Thr Ser Pro Ser Pro Thr Val Gln Leu Gln
290 295 300
Pro Leu Leu Pro Asp Pro Ser Ala Ile Thr Pro Asn Ser Thr Ser
305 310 315
Pro Leu Leu Phe Ala Ala His Pro His Phe Gln Asn Leu Ser Gln
320 325 330
Glu Glu
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1026 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
AGCCCGGCTC CTCCTGCCTG TGACCCCCGA CTCCTAAATA AACTGCTTCG 50
TGACTCCCAT GTCCTTCACG GCAGACTGAG CCAGTGCCCA GACATTAACC 100
CTTTGTCCAC ACCTGTCCTG CTGCCTGCTG TGGACTTCAC CTTGGGAGAA 150
TGGAAAACCC AGACGGAGCA GACAAAGGCA CAGGATGTCC TGGGAGCCAC 200
AACCCTTCTG CTGGAGGCAG TGATGACAGC ACGGGGACAA GTGGGACCCC 250
CTTGCCTCTC ATCCCTGCTG GTGCAGCTTT CTGGACAGGT TCGCCTCCTC 300
CTCGGGGCCC TGCAGGACCT CCTTGGAATG CAGCTTCCTC CACAGGGAAG 350
GACCACAGCT CACAAGGATC CCAGTGCCAT CTTCCTGAAC TTCCAACAAC 400
TGCTCCGAGG AAAGGTGCGT TTCCTGCTCC TTGTAGTGGG GCCCTCCCTC 450
TGTGCCAAGA GGGCCCCACC CGCCATAGCT GTCCCGAGCA GCACCTCTCC 500
ATTCCACACA CTGAACAAGC TCCCAAACAG GACCTCTGGA TTGTTGGAGA 550
CAAACTCCAG TATCTCAGCC AGAACTACTG GCTCTGGATT TCTCAAGAGG 600
169
CA 02178482 2010-06-29
CTGCAGGCAT TCAGAGCCAA GATTCCTGGT CTGCTGAACC AAACCTCCAG 650
GTCCCTAGAC CAAATCCCTG GACACCAGAA TGGGACACAC GGACCCTTGA 700
GTGGAATTCA TGGACTCTTT CCTGGACCCC AACCCGGGGC CCTCGGAGCT 750
CCAGACATTC CTCCAGCAAC TTCAGGCATG GGCTCCCGGC CAACCTACCT 800
CCAGCCTGGA GAGTCTCCTT CCCCAGCTCA CCCTTCTCCT GGACGATACA 850
CTCTCTTCTC TCCTTCACCC ACCTCGCCCT CCCCCACAGT CCAGCTCCAG 900
CCTCTGCTTC CTGACCCCTC TGCGATCACA CCCAACTCTA CCAGTCCTCT 950
TCTATTTGCA GCTCACCCTC ATTTCCAGAA CCTGTCTCAG GAAGAGTAAG 1000
GTGCTCAGAC CCTGCCAACT TCAGCA 1026
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1014 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
AGCCCGGCTC CTCCTGCCTG TGACCCCCGA CTCCTAAATA AACTGCTTCG 50
TGACTCCCAT GTCCTTCACG GCAGACTGAG CCAGTGCCCA GACATTAACC 100
CTTTGTCCAC ACCTGTCCTG CTGCCTGCTG TGGACTTCAC CTTGGGAGAA 150
TGGAAAACCC AGACGGAGCA GACAAAGGCA CAGGATGTCC TGGGAGCCAC 200
AACCCTTCTG CTGGAGGCAG TGATGACAGC ACGGGGACAA GTGGGACCCC 250
CTTGCCTCTC ATCCCTGCTG GTGCAGCTTT CTGGACAGGT TCGCCTCCTC 300
CTCGGGGCCC TGCAGGACCT CCTTGGAATG CAGGGAAGGA CCACAGCTCA 350
CAAGGATCCC AGTGCCATCT TCCTGAACTT CCAACAACTG CTCCGAGGAA 400
AGGTGCGTTT CCTGCTCCTT GTAGTGGGGC CCTCCCTCTG TGCCAAGAGG 450
GCCCCACCCG CCATAGCTGT CCCGAGCAGC ACCTCTCCAT TCCACACACT 500
GAACAAGCTC CCAAACAGGA CCTCTGGATT GTTGGAGACA AACTCCAGTA 550
TCTCAGCCAG AACTACTGGC TCTGGATTTC TCAAGAGGCT GCAGGCATTC 600
170
CA 02178482 2010-06-29
AGAGCCAAGA TTCCTGGTCT GCTGAACCAA ACCTCCAGGT CCCTAGACCA 650
AATCCCTGGA CACCAGAATG GGACACACGG ACCCTTGAGT GGAATTCATG 700
GACTCTTTCC TGGACCCCAA CCCGGGGCCC TCGGAGCTCC AGACATTCCT 750
CCAGCAACTT CAGGCATGGG CTCCCGGCCA ACCTACCTCC AGCCTGGAGA 800
GTCTCCTTCC CCAGCTCACC CTTCTCCTGG ACGATACACT CTCTTCTCTC 850
CTTCACCCAC CTCGCCCTCC CCCACAGTCC AGCTCCAGCC TCTGCTTCCT 900
GACCCCTCTG CGATCACACC CAACTCTACC AGTCCTCTTC TATTTGCAGC 950
TCACCCTCAT TTCCAGAACC TGTCTCAGGA AGAGTAAGGT GCTCAGACCC 1000
TGCCAACTTC AGCA 1014
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 328 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu
1 5 10 15
Leu Arg Asp Ser His Val Leu His Gly Arg Leu Ser Gln Cys Pro
20 25 30
Asp Ile Asn Pro Leu Ser Thr Pro Val Leu Leu Pro Ala Val Asp
35 40 45
Phe Thr Leu Gly Glu Trp Lys Thr Gln Thr Glu Gln Thr Lys Ala
50 55 60
Gln Asp Val Leu Gly Ala Thr Thr Leu Leu Leu Glu Ala Val Met
65 70 75
Thr Ala Arg Gly Gln Val Gly Pro Pro Cys Leu Ser Ser Leu Leu
80 85 90
Val Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln
95 100 105
Asp Leu Leu Gly Met Gln Gly Arg Thr Thr Ala His Lys Asp Pro
110 115 120
Ser Ala Ile Phe Leu Asn Phe Gln Gln Leu Leu Arg Gly Lys Val
125 130 135
171
CA 02178482 2010-06-29
Arg Phe Leu Leu Leu Val Val Gly Pro Ser Leu Cys Ala Lys Arg
140 145 150
Ala Pro Pro Ala Ile Ala Val Pro Ser Ser Thr Ser Pro Phe His
155 160 165
Thr Leu Asn Lys Leu Pro Asn Arg Thr Ser Gly Leu Leu Glu Thr
170 175 180
Asn Ser Ser Ile Ser Ala Arg Thr Thr Gly Ser Gly Phe Leu Lys
185 190 195
Arg Leu Gln Ala Phe Arg Ala Lys Ile Pro Gly Leu Leu Asn Gln
200 205 210
Thr Ser Arg Ser Leu Asp Gln Ile Pro Gly His Gln Asn Gly Thr
215 220 225
His Gly Pro Leu Ser Gly Ile His Gly Leu Phe Pro Gly Pro Gln
230 235 240
Pro Gly Ala Leu Gly Ala Pro Asp Ile Pro Pro Ala Thr Ser Gly
245 250 255
Met Gly Ser Arg Pro Thr Tyr Leu Gln Pro Gly Glu Ser Pro Ser
260 265 270
Pro Ala His Pro Ser Pro Gly Arg Tyr Thr Leu Phe Ser Pro Ser
275 280 285
Pro Thr Ser Pro Ser Pro Thr Val Gln Leu Gln Pro Leu Leu Pro
290 295 300
Asp Pro Ser Ala Ile Thr Pro Asn Ser Thr Ser Pro Leu Leu Phe
305 310 315
Ala Ala His Pro His Phe Gln Asn Leu Ser Gln Glu Glu
320 325
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5141 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Double
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
TTCGAGCTCG CCCGACATTG ATTATTGACT AGAGTCGATC GACAGCTGTG 50
GAATGTGTGT CAGTTAGGGT GTGGAAAGTC CCCAGGCTCC CCAGCAGGCA 100
172
CA 02178482 2010-06-29
GAAGTATGCA AAGCATGCAT CTCAATTAGT CAGCAACCAG GTGTGGAAAG 150
TCCCCAGGCT CCCCAGCAGG CAGAAGTATG CAAAGCATGC ATCTCAATTA 200
GTCAGCAACC ATAGTCCCGC CCCTAACTCC GCCCATCCCG CCCCTAACTC 250
CGCCCAGTTC CGCCCATTCT CCGCCCCATG GCTGACTAAT TTTTTTTATT 300
TATGCAGAGG CCGAGGCCGC CTCGGCCTCT GAGCTATTCC AGAAGTAGTG 350
AGGAGGCTTT TTTGGAGGCC TAGGCTTTTG CAAAAAGCTA GCTTATCCGG 400
CCGGGAACGG TTCATTGGAA CGCGGATTCC CCGTGCCAAG AGTGACGTAA 450
GTACCGCCTA TAGAGCGATA AGAGGATTTT ATCCCCGCTG CCATCATGGT 500
TCGACCATTG AACTGCATCG TCGCCGTGTC CCAAAATATG GGGATTGGCA 550
AGAACGGAGA CCTACCCTGG CCTCCGCTCA GGAACGAGTT CAAGTACTTC 600
CAAAGAATGA CCACAACCTC TTCAGTGGAA GGTAAACAGA ATCTGGTGAT 650
TATGGGTAGG AAAACCTGGT TCTCCATTCC TGAGAAGAAT CGACCTTTAA 700
AGGACAGAAT TAATATAGTT CTCAGTAGAG AACTCAAAGA ACCACCACGA 750
GGAGCTCATT TTCTTGCCAA AAGTTTGGAT GATGCCTTAA GACTTATTGA 800
ACAACCGGAA TTGGCAAGTA AAGTAGACAT GGTTTGGATA GTCGGAGGCA 850
GTTCTGTTTA CCAGGAAGCC ATGAATCAAC CAGGCCACCT TAGACTCTTT 900
GTGACAAGGA TCATGCAGGA ATTTTAAAGT GACACGTTTT TCCCAGAAAT 950
TGATTTGGGG AAATATAAAC CTCTCCCAGA ATACCCAGGC GTCCTCTCTG 1000
AGGTCCAGGA GGAAAAAGGC ATCAAGTATA AGTTTGAAGT CTACGAGAAG 1050
AAAGACTAAC AGGAAGATGC TTTCAAGTTC TCTGCTCCCC TCCTAAAGCT 1100
ATGCATTTTT ATAAGACCAT GGGACTTTTG CTGGCTTTAG ATCCCCTTGG 1150
CTTCGTTAGA ACGCGGCTAC AATTAATACA TAACCTTATG TATCATACAC 1200
ATACGATTTA GGTGACACTA TAGATAACAT CCACTTTGCC TTTCTCTCCA 1250
CAGGTGTCCA CTCCCAGGTC CAACTGCACC TCGGTTCTAA GCTTCTGCAG 1300
GTCGACTCTA GAGGATCCCC GGGGAATTCA ATCGATGGCC GCCATGGCCC 1350
AACTTGTTTA TTGCAGCTTA TAATGGTTAC AAATAAAGCA ATAGCATCAC 1400
AAATTTCACA AATAAAGCAT TTTTTTCACT GCATTCTAGT TGTGGTTTGT 1450
173
CA 02178482 2010-06-29
CCAAACTCAT CAATGTATCT TATCATGTCT GGATCGATCG GGAATTAATT 1500
CGGCGCAGCA CCATGGCCTG AAATAACCTC TGAAAGAGGA ACTTGGTTAG 1550
GTACCTTCTG AGGCGGAAAG AACCAGCTGT GGAATGTGTG TCAGTTAGGG 1600
TGTGGAAAGT CCCCAGGCTC CCCAGCAGGC AGAAGTATGC AAAGCATGCA 1650
TCTCAATTAG TCAGCAACCA GGTGTGGAAA GTCCCCAGGC TCCCCAGCAG 1700
GCAGAAGTAT GCAAAGCATG CATCTCAATT AGTCAGCAAC CATAGTCCCG 1750
CCCCTAACTC CGCCCATCCC GCCCCTAACT CCGCCCAGTT CCGCCCATTC 1800
TCCGCCCCAT GGCTGACTAA TTTTTTTTAT TTATGCAGAG GCCGAGGCCG 1850
CCTCGGCCTC TGAGCTATTC CAGAAGTAGT GAGGAGGCTT TTTTGGAGGC 1900
CTAGGCTTTT GCAAAAAGCT GTTACCTCGA GCGGCCGCTT AATTAAGGCG 1950
CGCCATTTAA ATCCTGCAGG TAACAGCTTG GCACTGGCCG TCGTTTTACA 2000
ACGTCGTGAC TGGGAAAACC CTGGCGTTAC CCAACTTAAT CGCCTTGCAG 2050
CACATCCCCC CTTCGCCAGC TGGCGTAATA GCGAAGAGGC CCGCACCGAT 2100
CGCCCTTCCC AACAGTTGCG TAGCCTGAAT GGCGAATGGC GCCTGATGCG 2150
GTATTTTCTC CTTACGCATC TGTGCGGTAT TTCACACCGC ATACGTCAAA 2200
GCAACCATAG TACGCGCCCT GTAGCGGCGC ATTAAGCGCG GCGGGTGTGG 2250
TGGTTACGCG CAGCGTGACC GCTACACTTG CCAGCGCCCT AGCGCCCGCT 2300
CCTTTCGCTT TCTTCCCTTC CTTTCTCGCC ACGTTCGCCG GCTTTCCCCG 2350
TCAAGCTCTA AATCGGGGGC TCCCTTTAGG GTTCCGATTT AGTGCTTTAC 2400
GGCACCTCGA CCCCAAAAAA CTTGATTTGG GTGATGGTTC ACGTAGTGGG 2450
CCATCGCCCT GATAGACGGT TTTTCGCCCT TTGACGTTGG AGTCCACGTT 2500
CTTTAATAGT GGACTCTTGT TCCAAACTGG AACAACACTC AACCCTATCT 2550
CGGGCTATTC TTTTGATTTA TAAGGGATTT TGCCGATTTC GGCCTATTGG 2600
TTAACAAATG AGCTGATTTA ACAAAAATTT AACGCGAATT TTAACAAAAT 2650
ATTAACGTTT ACAATTTTAT GGTGCACTCT CAGTACAATC TGCTCTGATG 2700
CCGCATAGTT AAGCCAACTC CGCTATCGCT ACGTGACTGG GTCATGGCTG 2750
CGCCCCGACA CCCGCCAACA CCCGCTGACG CGCCCTGACG GGCTTGTCTG 2800
174
CA 02178482 2010-06-29
CTCCCGGCAT CCGCTTACAG ACAAGCTGTG ACCGTCTCCG GGAGCTGCAT 2850
GTGTCAGAGG TTTTCACCGT CATCACCGAA ACGCGCGAGG CAGTATTCTT 2900
GAAGACGAAA GGGCCTCGTG ATACGCCTAT TTTTATAGGT TAATGTCATG 2950
ATAATAATGG TTTCTTAGAC GTCAGGTGGC ACTTTTCGGG GAAATGTGCG 3000
CGGAACCCCT ATTTGTTTAT TTTTCTAAAT ACATTCAAAT ATGTATCCGC 3050
TCATGAGACA ATAACCCTGA TAAATGCTTC AATAATATTG AAAAAGGAAG 3100
AGTATGAGTA TTCAACATTT CCGTGTCGCC CTTATTCCCT TTTTTGCGGC 3150
ATTTTGCCTT CCTGTTTTTG CTCACCCAGA AACGCTGGTG AAAGTAAAAG 3200
ATGCTGAAGA TCAGTTGGGT GCACGAGTGG GTTACATCGA ACTGGATCTC 3250
AACAGCGGTA AGATCCTTGA GAGTTTTCGC CCCGAAGAAC GTTTTCCAAT 3300
GATGAGCACT TTTAAAGTTC TGCTATGTGG CGCGGTATTA TCCCGTGATG 3350
ACGCCGGGCA AGAGCAACTC GGTCGCCGCA TACACTATTC TCAGAATGAC 3400
TTGGTTGAGT ACTCACCAGT CACAGAAAAG CATCTTACGG ATGGCATGAC 3450
AGTATGAGAA TTATGCAGTG CTGCCATAAC CATGAGTGAT AACACTGCGG 3500
CCAACTTACT TCTGACAACG ATCGGAGGAC CGAAGGAGCT AACCGCTTTT 3550
TTGCACAACA TGGGGGATCA TGTAACTCGC CTTGATCGTT GGGAACCGGA 3600
GCTGATTGAA GCCATACCAA ACGACGAGCG TGACACCACG ATGCCAGCAG 3650
CAATGGCAAC AACGTTGCGC AAACTATTAA CTGGCGAACT ACTTACTCTA 3700
GCTTCCCGGC AACAATTAAT AGACTGGATG GAGGCGGATA AAGTTGCAGG 3750
ACCACTTCTG CGCTCGGCCC TTCCGGCTGG CTTGTTTATT GCTGATAAAT 3800
CTGGAGCCGG TGAGCGTGGG TCTCGCGGTA TCATTGCAGC ACTGGGGCCA 3850
GATGGTAAGC CCTCCCGTAT CGTAGTTATC TACACGACGG GGAGTCAGGC 3900
AACTATGGAT GAACGAAATA GACAGATCGC TGAGATAGGT GCCTCACTGA 3950
TTAAGCATTG GTAACTGTCA GACCAAGTTT ACTCATATAT ACTTTAGATT 4000
GATTTAAAAC TTCATTTTTA ATTTAAAAGG ATCTAGGTGA AGATCCTTTT 4050
TGATAATCTC ATGACCAAAA TCGCTTAACG TGAGTTTTCG TTCCACTGAG 4100
CGTCAGACCC CGTAGAAAAG ATCAAAGGAT CTTCTTGAGA TCCTTTTTTT 4150
175
CA 02178482 2010-06-29
CTGCGCGTAA TCTGCTGCTT GCAAACAAAA AAACCACCGC TACCAGCGGT 4200
GGTTTGTTTG CCGGATCAAG AGCTACCAAC TCTTTTTCCG AAGGTAACTG 4250
GCTTCAGCAG AGCGCAGATA CCAAATACTG TCCTTCTAGT GTAGCCGTAG 4300
TTAGGCCACC ACTTCAAGAA CTCTGTAGCA CCGCCTACAT ACCTCGCTCT 4350
GCTAATCCTG TTACCAGTGG CTGCTGCCAG TGGCGATAAG TCGTGTCTTA 4400
CCGGGTTGGA CTCAAGACGA TAGTTACCGG ATAAGGCGCA GCGGTCGGGC 4450
TGAACGGGGG GTTCGTGCAC ACAGCCCAGC TTGGAGCGAA CGACCTACAC 4500
CGAACTGAGA TACCTACAGC GTGAGCATTG AGAAAGCGCC ACGCTTCCCG 4550
AAGGGAGAAA GGCGGACAGG TATCCGGTAA GCGGCAGGGT CGGAACAGGA 4600
GAGCGCACGA GGGAGCTTCC AGGGGGAAAC GCCTGGTATC TTTATAGTCC 4650
TGTCGGGTTT CGCCACCTCT GACTTGAGCG TCGATTTTTG TGATGCTCGT 4700
CAGGGGGGCG GAGCCTATGG AAAAACGCCA GCAACGCGGC CTTTTTACGG 4750
TTCCTGGCCT TTTGCTGGCC TTTTGCTCAC ATGTTCTTTC CTGCGTTATC 4800
CCCTGATTCT GTGGATAACC GTATTACCGC CTTTGAGTGA GCTGATACCG 4850
CTCGCCGCAG CCGAACGACC GAGCGCAGCG AGTCAGTGAG CGAGGAAGCG 4900
GAAGAGCGCC CAATACGCAA ACCGCCTCTC CCCGCGCGTT GGCCGATTCA 4950
TTAATCCAGC TGGCACGACA GGTTTCCCGA CTGGAAAGCG GGCAGTGAGC 5000
GCAACGCAAT TAATGTGAGT TACCTCACTC ATTAGGCACC CCAGGCTTTA 5050
CACTTTATGC TTCCGGCTCG TATGTTGTGT GGAATTGTGA GCGGATAACA 5100
ATTTCACACA GGAAACAGCT ATGACCATGA TTACGAATTA A 5141
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
ATGTCNCCNG CNCCNCCNGC N 21
(2) INFORMATION FOR SEQ ID NO:24:
176
CA 02178482 2010-06-29
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
ATGTCTCCAG CGCCGCCAGC G 21
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
ATGTCGCCTG CTCCACCTGC T 21
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
ATGTCGCCAG CGCCACCAGC C 21
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
ATGTCTCCAG CCCCACCCGC A 21
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
177
CA 02178482 2010-06-29
(A) LENGTH: 21 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
ATGTCGCCAG CGCCGCCAGC G 21
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu
1 5 10 15
Leu Arg Asp Asp His Val Leu His Gly Arg
20 25
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Ser Pro Ala Pro Pro Ala Cys Asp Pro Arg Leu Leu Asn Lys Leu
1 5 10 15
Leu Arg Asp Asp Xaa Val Leu His Gly Arg Leu
20 25
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Ser Pro Ala Pro Pro Ala Xaa Asp Pro Arg Leu Leu Asn Lys Leu
1 5 10 15
Leu Arg Asp Asp His Val Leu His Gly Arg
20 25
178
CA 02178482 2010-06-29
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Xaa Pro Ala Pro Pro Ala Xaa Asp Pro Arg Leu Xaa Asn Lys
1 5 10
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Pro Arg Leu Leu Asn Lys Leu Leu Arg
1 5
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
GCCGTGAAGG ACGTGGTCGT CACGAAGCAG TTTATTTAGG AGTCG 45
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
CCNGCNCCNC CNGCNTGYGA 20
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
179
CA 02178482 2010-06-29
(A) LENGTH: 21 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
NCCRTGNARN ACRTGRTCRT C 21
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
CCAGCGCCGC CAGCCTGTGA CCCCCGACTC CTAAATAAAC TGCCTCGTGA 50
TGACCACGTT CAGCACGGC 69
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
GGTCGCGGCG GTCGGACACT GGGGGCTGAG GATTTATTTG ACGGAGCACT 50
ACTGGTGCAA GTCGTGCCG 69
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
CCAGCACCTC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 50
CGACCACGTC CATCACGGC 69
180
CA 02178482 2010-06-29
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
GGTCGTGGAG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCACT 50
GCTGGTGCAG GTAGTGCCG 69
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
CCAGCACCGC CGGCATGTGA CCCCCGACTC CTAAATAAAC TGCTTCGTGA 50
CGATCATGTC TATCACGGT 69
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
GGTCGTGGCG GCCGTACACT GGGGGCTGAG GATTTATTTG ACGAAGCACT 50
GCTAGTACAG ATAGTGCCA 69
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
181
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
GCTAGCTCTA GAAATTGCTC CTCGTGGTCA TGCTTCT 37
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
CAGTCTGCCG TGAAGGACAT GG 22
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
TGTGGACTTT AGCTTGGGAG AATG 24
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
GGTCCAGGGA CCTGGAGGTT TG 22
(2) INFORMATION FOR SEQ ID NO:47:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 31 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:
182
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ATCGATATCG ATAGCCAGAC ACCCCGGCCA G 31
(2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
GCTAGCTCTA GACAGGGAAG GGAGCTGTAC ATGAGA 36
(2) INFORMATION FOR SEQ ID NO:49:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:
CTCCTTGGAA CCCAGGGCAG GACC 24
(2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:
GGTCCTGCCC TGGGTTCCAA GGAG 24
(2) INFORMATION FOR SEQ ID NO:51:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:
CTGCTCCGAG GAAAGGACTT CTGGATT 27
183
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(2) INFORMATION FOR SEQ ID NO:52:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:
AATCCAGAAG TCCTTTCCTC GGAGCAG 27
(2) INFORMATION FOR SEQ ID NO:53:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:
CCCTCTGCGT CGCGGCGGCC CCACCCAC 28
(2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:
GTGGGTGGGG CCGCCGCGAC GCAGAGGG 28
(2) INFORMATION FOR SEQ ID NO:55:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
GACTCGAGGA TCCATCGATT TTTTTTTTTT TTTTT 35
(2) INFORMATION FOR SEQ ID NO:56:
184
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:
GACTCGAGGA TCCATCG 17
(2) INFORMATION FOR SEQ ID NO:57:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:
GCTAGCTCTA GAAGCCCGGC TCCTCCTGCC TG 32
(2) INFORMATION FOR SEQ ID NO:58:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
CGAAATTAAC CCTCACTAAA G 21
(2) INFORMATION FOR SEQ ID NO:59:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 103 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:
TGCAGCAAGG GCTACTGCCA CACTCGAGCT GCGCAGATGC TAGCCTCAAG 50
ATGGCTGATC CAAATCGATT CCGCGGCAAA GATCTTCCGG TCCTGTAGAA 100
GCT 103
185
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(2) INFORMATION FOR SEQ ID NO:60:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 103 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:
AGCTTCTACA GGACCGGAAG ATCTTTGCCG CGGAATCGAT TTGGATCAGC 50
CATCTTGAGG CTAGCATCTG CGCAGCTCGA GTGTGGCAGT AGCCCTTGCT 100
GCA 103.
(2) INFORMATION FOR SEQ ID NO:61:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:
TCTCGCTACC GTTTACAG 18
(2) INFORMATION FOR SEQ ID NO:62:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:
CAGGTACCCA CCAGGCGGTC TCGGT 25
(2) INFORMATION FOR SEQ ID NO:63:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:
186
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GGGCCATGAC ACTGTCAA 18
(2) INFORMATION FOR SEQ ID NO:64:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:
GACCGCCACC GAGACCGCCT GGTGGGTACC TGTGGTCCTT 40
(2) INFORMATION FOR SEQ ID NO:65:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:
ATCGATATCG ATCAGCCAGA CACCCCGGCC AG 32
(2) INFORMATION FOR SEQ ID NO:66:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 35 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:
TCTAGATCTA GATCACCTGA CGCAGAGGGT GGACC 35
(2) INFORMATION FOR SEQ ID NO:67:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:
AGTCGACGTC GACGTCGGCA GTGTCTGAGA ACC 33
187
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(2) INFORMATION FOR SEQ ID NO:68:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 36 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:
AGTCGACGTC GACTCACCTG ACGCAGAGGG TGGACC 36
(2) INFORMATION FOR SEQ ID NO:69:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:
CGCGTATGCC AGCCCGGCTC CTCCTGCTTG TGACCTCCGA GTCCTCAGTA 50
AACTGCTTCG TG 62
(2) INFORMATION FOR SEQ ID NO:70:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 61 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:
ATACGGTCGG GCCGAGGAGG ACGAACACTG GAGGCTCAGG AGTCATTTGA 50
CGAAGCACTG A 61
(2) INFORMATION FOR SEQ ID NO:71:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:
188
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CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTAA 37
(2) INFORMATION FOR SEQ ID NO:72:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 37 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:
TTAATACTTT TTCTTATAGC GTAAAGAAGA ATTGCGC 37
(2) INFORMATION FOR SEQ ID NO:73:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:
CTAGAATTAT GAAAAAGAAT ATCGCATTTC ATCACCATCA CCATCACCAT 50
CACATCGAAG GTCGTAGCC 69
(2) INFORMATION FOR SEQ ID NO:74:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:
TTAATACTTT TTCTTATAGC GTAAAGTAGT GGTAGTGGTA GTGGTAGTGT 50
AGCTTCCAGC AT 62
(2) INFORMATION FOR SEQ ID NO:75:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 69 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear.
189
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:
CTAGAATTAT GAAAAAGAAT ATCGCATTTC ATCACCATCA CCATCACCAT 50
CACATCGAAC CACGTAGCC 69
(2) INFORMATION FOR SEQ ID NO:76:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:
TTAATACTTT TTCTTATAGC GTAAAGTAGT GGTAGTGGTA GTGGTAGTGT 50
AGCTTGGTGC AT 62
(2) INFORMATION FOR SEQ ID NO:77:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:
TCCACCCTCT GCGTCAGGT 19
(2) INFORMATION FOR SEQ ID NO:78:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:
GGAGACGCAG TCCATCGA 18
(2) INFORMATION FOR SEQ ID NO:79:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 62 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
190
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(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:
GCAGCAGTTC TAGAATTATG TCNCCNGCNC CNCCNGCNTG TGACCTCCGA 50
GTTCTCAGTA AA 62
(2) INFORMATION FOR SEQ ID NO:80:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 49 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:
ACACTGGAGG CTCAAGAGTC ATTTGACGAA GCACTGAGGG TACAGGAAG 49
(2) INFORMATION FOR SEQ ID NO:81:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:
CTAGAATTAT GAAAAAGAAT ATCGCATTTA TCGAAGGTCG TAGCC 45
(2) INFORMATION FOR SEQ ID NO:82:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:
TTAATACTTT TTCTTATAGC GTAAATAGCT TCCAGCAT 38
(2) INFORMATION FOR SEQ ID NO:83:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
191
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(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:
CTAGAATTAT GAAAAAGAAT ATCGCATTTC TTCTTAAACG TAGCC 45
(2) INFORMATION FOR SEQ ID NO:84:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 38 base pairs
(B) TYPE: Nucleic Acid
(C) STRANDEDNESS: Single
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:
TTAATACTTT TTCTTATAGC GTAAAGAAGA ATTTGCAT 38
(2) INFORMATION FOR SEQ ID NO:85:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 168 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:
Met Lys Lys Asn Ile Ala Phe Leu Leu Asn Ala Tyr Ala Ser Pro
1 5 10 15
Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu Leu Arg
20 25 30
Asp Ser His Val Leu His Ser Arg Leu Ser Gln Cys Pro Glu Val
35 40 45
His Pro Leu Pro Thr Pro Val Leu Leu Pro Ala Val Asp Phe Ser
50 55 60
Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln Asp
65 70 75
Ile Leu Gly Ala Val Thr Leu Leu Leu Glu Gly Val Met Ala Ala
80 85 90
Arg Gly Gln Leu Gly Pro Thr Cys Leu Ser Ser Leu Leu Gly Gln
95 100 105
Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln Ser Leu
110 115 120
Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His Lys
192
CA 02178482 2010-06-29
125 130 135
Asp Pro Asn Ala Ile Phe Leu Ser Phe Gln His Leu Leu Arg Gly
140 145 150
Lys Val Arg Phe Leu Met Leu Val Gly Gly Ser Thr Leu Cys Val
155 160 165
Arg Arg Ala
168
(2) INFORMATION FOR SEQ ID NO:86:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:
Met Lys Lys Asn Ile Ala Phe His His His His His His His His
1 5 10 15
Ile Glu Gly Arg Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val
20 25 30
Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu
35 40 45
Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu
50 55 60
Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu
65 70 75
Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu
80 85 90
Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu
95 100 105
Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu
110 115 120
Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly
125 130 135
Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe
140 145 150
Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly
155 160 165
Gly Ser Thr Leu Cys Val Arg Arg Ala
193
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170 174
(2) INFORMATION FOR SEQ ID NO:87:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 174amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:
Met Lys Lys Asn Ile Ala Phe His His His His His His His His
1 5 10 15
Ile Glu Pro Arg Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val
20 25 30
Leu Ser Lys Leu Leu Arg Asp Ser His Val Leu His Ser Arg Leu
35 40 45
Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr Pro Val Leu Leu
50 55 60
Pro Ala Val Asp Phe Ser Leu Gly Glu Trp Lys Thr Gln Met Glu
65 70 75
Glu Thr Lys Ala Gln Asp Ile Leu Gly Ala Val Thr Leu Leu Leu
80 85 90
Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr Cys Leu
95 100 105
Ser Ser Leu Leu Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu
110 115 120
Gly Ala Leu Gln Ser Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly
125 130 135
Arg Thr Thr Ala His Lys Asp Pro Asn Ala Ile Phe Leu Ser Phe
140 145 150
Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met Leu Val Gly
155 160 165
Gly Ser Thr Leu Cys Val Arg Arg Ala
170 174
(2) INFORMATION FOR SEQ ID NO:88:
194
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:
Thr Thr Ala His Lys Asp Pro
1 5
(2) INFORMATION FOR SEQ ID NO:89:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:
His Val Leu His
1
(2) INFORMATION FOR SEQ ID NO:90:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:
Ser Arg Leu Ser
1
(2) INFORMATION FOR SEQ ID NO:91:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:
Ser His Val Leu
1
(2) INFORMATION FOR SEQ ID NO:92:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
195
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:
His Ser Arg Leu
1
(2) INFORMATION FOR SEQ ID NO:93:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:
Ala Val Asp Phe
1
(2) INFORMATION FOR SEQ ID NO:94:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:
Ser Leu Gly Glu
1
(2) INFORMATION FOR SEQ ID NO:95:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:
Ala Val Thr Leu
1
(2) INFORMATION FOR SEQ ID NO:96:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:
Leu Leu Glu Gly
1
(2) INFORMATION FOR SEQ ID NO:97:
196
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:
Leu Ser Ser Leu
1
(2) INFORMATION FOR SEQ ID NO:98:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:
Leu Gly Gln Leu
1
(2) INFORMATION FOR SEQ ID NO:99:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:
Cys Xaa Leu Ser Ser
1 5
(2) INFORMATION FOR SEQ ID NO:100:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:
Leu Leu Gly Gln
1
(2) INFORMATION FOR SEQ ID NO:101:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
197
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:
Ser Ser Leu Leu
1
(2) INFORMATION FOR SEQ ID NO:102:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:
Gly Gln Leu Ser
1
(2) INFORMATION FOR SEQ ID NO:103:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:
Cys Leu Ser Ser
1
(2) INFORMATION FOR SEQ ID NO:104:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:
Leu Gln Ser Leu
1
(2) INFORMATION FOR SEQ ID NO:105:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:
Leu Gly Thr Gln
1
(2) INFORMATION FOR SEQ ID NO:106:
198
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:
Ala Leu Gln Ser
1
(2) INFORMATION FOR SEQ ID NO:107:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:
Leu Leu Gly Thr
1
(2) INFORMATION FOR SEQ ID NO:108:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:
Asn Ala Ile Phe
1
(2) INFORMATION FOR SEQ ID NO:109:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:
Leu Ser Phe Gln
1
(2) INFORMATION FOR SEQ ID NO:110:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:
Ser Pro Ala Pro Pro Ala Cys Asp Leu Arg Val Leu Ser Lys Leu
1 5 10 15
Leu Arg Asp Ser His Val Leu
(2) INFORMATION FOR SEQ ID NO:111:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:
His Ser Arg Leu Ser Gln Cys Pro Glu Val His Pro Leu Pro Thr
1 5 10 15
Pro Val Leu Leu Pro Ala Val Asp Phe
(2) INFORMATION FOR SEQ ID NO:112:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:
Ser Leu Gly Glu Trp Lys Thr Gln Met Glu Glu Thr Lys Ala Gln
1 5 10 15
Asp Ile Leu Gly Ala Val Thr Leu
(2) INFORMATION FOR SEQ ID NO:113:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:
Leu Leu Glu Gly Val Met Ala Ala Arg Gly Gln Leu Gly Pro Thr
1 5 10 15
Cys Leu Ser Ser Leu Leu
(2) INFORMATION FOR SEQ ID NO:114:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:
Gly Gln Leu Ser Gly Gln Val Arg Leu Leu Leu Gly Ala Leu Gln
1 5 10 15
Ser
(2) INFORMATION FOR SEQ ID NO:115:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:
Leu Leu Gly Thr Gln Leu Pro Pro Gln Gly Arg Thr Thr Ala His
1 5 10 15
Lys Asp Pro Asn Ala Ile Phe
(2) INFORMATION FOR SEQ ID NO:116:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:
Leu Ser Phe Gln His Leu Leu Arg Gly Lys Val Arg Phe Leu Met
1 5 10 15
Leu Val Gly Gly Ser Thr Leu Cys Val Arg
20 25
(2) INFORMATION FOR SEQ ID NO:117:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D),TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:
Met Pro Pro Ala
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1
(2) INFORMATION FOR SEQ ID NO:118:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:
Met Ala Pro Pro Ala
1 5
(2) INFORMATION FOR SEQ ID NO:119:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:
Met Pro Ala Pro Pro Ala
1 5
(2) INFORMATION FOR SEQ ID NO:120:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:120:
Met Ser Pro Ala Pro Pro Ala
1 5
(2) INFORMATION FOR SEQ ID NO:121:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:121:
Ala Pro Pro Ala
1
(2) INFORMATION FOR SEQ ID NO:122:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
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(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:122:
Pro Ala Pro Pro Ala
1 5
(2) INFORMATION FOR SEQ ID NO:123:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear.
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:123:
Ser Pro Ala Pro Pro, Ala
1 5
(2) INFORMATION FOR SEQ ID NO:124:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:124:
Val Arg Arg Ala
1
(2) INFORMATION FOR SEQ ID NO:125:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:125:
Val Arg Arg Ala Pro
1 5
(2) INFORMATION FOR SEQ ID NO:126:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:126:
Val Arg Arg Ala Pro Pro
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1 5
(2) INFORMATION FOR SEQ ID NO:127:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:127:
Val Arg Arg Ala Pro Pro Thr
1 5
(2) INFORMATION FOR SEQ ID NO:128:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:128:
Val Arg Arg Ala Pro Pro Thr Thr
1 5
(2) INFORMATION FOR SEQ ID NO:129:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:129:
Val Arg Arg Ala Pro Pro Thr Thr Ala
1 5
(2) INFORMATION FOR SEQ ID NO:130:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:130:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val
1 5 10
(2) INFORMATION FOR SEQ ID NO:131:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 11 amino acids
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(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:131:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:132:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:132:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser
1 5 10
(2) INFORMATION FOR SEQ ID NO:133:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 13 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:133:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO:134:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:134:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr
1 5 10
(2) INFORMATION FOR SEQ ID NO:135:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:135:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser
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1 5 10 15
(2) INFORMATION FOR SEQ ID NO:136:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 16 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:136:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser
1 5 10 15
Leu
(2) INFORMATION FOR SEQ ID NO:137:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:137:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser
1 5 10 15
Leu Val
(2) INFORMATION FOR SEQ ID NO:138:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:138:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser
1 5 10 15
Leu Val Leu
(2) INFORMATION FOR SEQ ID NO:139:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:139:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser
1 5 10 15
Leu Val Leu Thr
(2) INFORMATION FOR SEQ ID NO:140:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:140:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser
1 5 10 15
Leu Val Leu Thr Leu
(2) INFORMATION FOR SEQ ID NO:141:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:141:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser
1 5 10 15
Leu Val Leu Thr Leu Asn
(2) INFORMATION FOR SEQ ID NO:142:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:142:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser
1 5 10 15
Leu Val Leu Thr Leu Asn Glu
(2) INFORMATION FOR SEQ ID NO:143:
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(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:143:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser
1 5 10 15
Leu Val Leu Thr Leu Asn Glu Leu
(2) INFORMATION FOR SEQ ID NO:144:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: Amino Acid
(D) TOPOLOGY: Linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:144:
Val Arg Arg Ala Pro Pro Thr Thr Ala Val Pro Ser Arg Thr Ser
1 5 10 15
Leu Val Leu Thr Leu Asn Glu Leu Pro
208
