Note: Descriptions are shown in the official language in which they were submitted.
CA 02370129 2001-10-15
INTEKIVA'11V1VAL
JL~AtctaWCrrVlc~ In ~tlonal
Application
No
PCT/US
00/10340
C.(Continuation)
DOCUMENTS
CONSIDERED
TO
BE
RELEVANT
CategoryCitation of document, with indication,where Relevant to
appropriate, of the relevant passages claim No.
A WEBER GEORG F ET AL: "The immunology 1-70
of
Eta-1/Osteopontin."
CYTOKINE & GROWTH FACTOR REVIEWS,
vol. 7, no. 3, 1996, pages 241-248,
XP000982501
ISSN: 1359-6101
the whole document
P,X ASHKAR SAMY ET AL: "Eta-1 (osteopontin): 1-70
An early component of type-1
(cell-mediated) immunity."
SCIENCE (WASHINGTON D C).,
vol. 287, no. 5454,
4 February 2000 (2000-02-04), pages
860-864, XP002161278
ISSN: 0036-8075
the whole document
Form PCT/ISA/210 (continuation of second sheet) (July 1992)
page 2 of 2
CA 02370129 2001-10-15
~..~.~~w v mT/1'T ~ t (T T1 ~ T1117T TITTIf~ItIT
Form PCT/ISA/210 (patent family annex) (July 1992)
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METHODS AND COMPOSITIONS FOR
MODULATING AN IMMUNE RESPONSE
Related Applications
This application claims the benefit of previously filed U.S. Provisional
Application Serial No. 60/129,772, filed April 15, 1999, the content of which
is hereby
incorporated by reference. This invention was made with government support
from the
National Institutes of Health. Accordingly, the government may have certain
rights in
the invention.
Background of the Invention
Efficient development of inflammatory responses and protection against
most infectious pathogens depends, in part, on monocytes as the final effector
cells. The
participation of monocytes in inflammation entails emigration of these cells
from
peripheral blood into infected tissues, where they produce cytokines that
regulate
diverse processes including anti-microbial activity, cell growth,
differentiation and
wound healing (Singer et al. ( 1995) J. Clin. Invest. 95:2178-2186. In acute
reactions,
monocytes may be attracted by neutrophils whereas, in delayed responses, they
act in a
neutrophil-independent manner. Secretion of T-cell cytokines plays a pivotal
role in
recruitment of monocytes to sites of infection and activation of these
emigrant cells to
express bacteriocidal activity. The mechanism of this process bears intensely
on wound
healing and delayed-type immune responses but its molecular basis is not
understood.
An important component of this T-cell dependent response is a protein
known as Eta-1 (for early T lymphocyte activation-1 )/osteopontin, which
mediates
macrophage chemotaxis in vitro (Weber et al. ( 1996) Science 271:509-512,
recruits
monocytes to inflammatory sites in vivo (Singh et al. ( 1990) J. Exp. Med.
171:1931-
1942) and regulates immunological resistance to several intracellular
pathogens (Patarca
et al. ( 1989) J. Exp. Med. 170:145-161; Lampe et al. ( 1991 ) J. Immunol.
147:2902-
2906. Inbred mouse strains that carry an allele of Eta-1/osteopontin which
allows high
level expression in activated T-cells are resistant to lethal effects of
infection by-the
intracellular parasite Rickettsia tsutsugamushi while inbred strains carrying
a low
expression allele do not develop a population of bacteriocidal monocytic
migrants at the
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area of infection and succumb to systemic bacteremia (Patarca et nl. ( 1990)
Immunol.
Rev. ( 116:1-16). Eta-1/osteopontin expression has also been linked to
granuloma
formation, where it may regulate the chronic cellular response associated with
tuberculosis infection and silicosis l Nau et al. ( 1997) Proc. Natl. Acad.
Sci. USA
94:6414-6419. Moreover. in experimental glomerulonephritis. neutralizing
antibodies to
osteopontin greatly diminish the influx of macrophages and T-cells and reduce
kidney
damage (Yu et al. ( 1998) Proc. Am. Assoc. Physicians 110:50-64). While Eta-
1/ostepontin has been implicated in at least certain immunological reactions,
its precise
role in the immune system has not previously been established. Moreover, Eta-1
is a
multifunctional protein having diverse biological roles including , but not
limited to, bone
resorption, neoplastic transformation. atheromatous plaque formation,
dystrophic
calcification of inflamed and/or damased tissues and resistance to certain
bacterial
infections. (See e.g., Oldberg et al. ( 1986) Proc. Natl. Acad. Sci. USA
83:8819; Ross et al.
(1993) J. Biol. Chem. 268:9901; Giachelli et al. (1995) Ann. NYAcad. Sci.
760:109; Senger
et al. ( 1983) Nature 302:714: and Srivatsa at al. ( 1997) J. Clin. Invest.
99:996). With
regards in particular, to understanding the role of Eta-1/osteopontin in
immunity, there
exists a need to understand the precise role that Eta-1/osteopontin plays in
regulating
immune responses in order to develop new approaches to treating immune
disorders and
diseases.
Summary of the Invention
The present invention establishes that Eta-1/osteopontin is a critical
regulator of type-1 (cell-mediated) immunity and that this molecule includes a
domain
that promotes the production of the type 1 cytokine IL-12 and a domain that
inhibits the
production of the type 2 cytokine IL-10. Thus, the invention provides for the
use of Eta-
1/osteopontin modulatory agents (i.e, agents that stimulate or inhibit Eta-
1/osteopontin
activity) to bias an immune response either toward type 1 or type 2 immunity,
depending
on the clinical situation. The present invention identifies Eta-1/osteopontin
as a critical
cytokine in type 1 immune responses, in particular, in delayed. type
hypersensitivity
responses. The invention defines Eta-1/osteopontin as a multifunctional
molecule which
acts as both a stimulator of IL-12 secretion by macrophages and an inhibitor
of IL-10
expression. As such. Eta-1/osteopontin serves to bias an organism's cytokine
pattern
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towards that of a type 1 immune response. In. particular, induction of IL-12
and
inhibition of IL-10 reflect differential engagement of macrophage receptors: a
phosphorylation-dependent interaction between the N-terminal portion of Eta-
1/osteopontin and its integrin receptor on macrophages leads to IL-12
expression, while a
phosphorylation-independent interaction of a C-terminal domain of Eta-
1/osteopontin with
CD44 mediates IL-10 inhibition. Moreover, cleavage of Eta-1 /osteopontin by
thrombin
results in a C-terminal fragment of the cytokine which interacts with CD44 and
induces
macrophage chemotaxis, while engagement of integrin receptors by a non-
overlapping
N-terminal fragment leads to macrophage spreading and activation.
Based, at least in part, on a detailed understanding of the role this
multifunctional
cytokine plays in type 1 immune responses, the present invention features
novel
approaches to modulating immune responses, in particular, in potentiating type
1
immune responses. The invention further features new methods of treating
disorders
that may benefit from either a type 1 or type 2 immune response. More
specifically, the
identification of Eta-1/osteopontin as a critical regulator of type 1 immunity
allows for
selective manipulation of T cell subsets in a variety of clinical situations
using the
modulatory methods of the invention. The stimulatory methods of the invention
(i.e.,
methods that use an Eta-1/osteopontin stimulatory agent) upregulate the
production of
the Thl-associated cytokine IL-12 and/or dowregulate the production of the Th2-
associated cytokine IL-10, with concomitant promotion of a type 1 immune
responses
and downregulation of type 2 immune responses. These stimulatory methods that
promote a type 1 response can be used, for example, in the treatment of
infections (e.g.,
bacterial, viral), cancer, allergy, burn-associated sepsis and
immunodeficiency disorders.
In contrast, the inhibitory methods of the invention (i.e., methods that use
an Eta-
1/osteopontin inhibitory agent) downregulate the production of the Thl-
associated
cytokine IL-12 and/or upregulate the production of the Th2-associated cytokine
IL-10,
with concomitant downregulation of a type 1 immune responses and promotion of
type 2
immune responses. These inhibitory methods that promote type 2 responses can
be
used, for example, in the treatment of autoimmune disorders, transplant
rejection.
granulomatous disorders. herpes simplex keratitis and bacterial arthritis.
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Moreover, based on a detailed-understanding of the functional domains of
Eta-1/osteopontin, the present invention provides biosynthetic molecules which
mimic
distinct functions of Eta-1/osteopontin for use in a variety of therapeutic
applications. in
particular, in wound healing, enhancement of the immune response and in
treatment of
granulomatous disease. In particular, the biosynthetic molecules of the
present
invention are useful in biasing an immune response towards a delayed type
hypersensitivity response, i.e., towards type 1 immunity. A preferred IL-12
stimulatory
domain of Eta-1/osteopontin comprises amino acids 71-168 of SEQ ID NO: 2. A
preferred IL-10 inhibitory domain of Eta-1/osteopontin comprises amino acids
169-266
of SEQ ID NO: 2. Other features and advantages of the invention will be
apparent from
the following detailed description, and from the claims.
Brief Description of the Drawings
Figure IA-C demonstrates gramuloma formation in normal, cytokine-
deficient and Eta-1/osteopontin-deficient mice. Figure lA depicts the data as
the mean
number of granulomas per high-power field (HPF) (X200 magnification), mean
number
of cells per granuloma, and as the product of these two indices, termed
"granuloma
burden". (Error bars indicate 1 SEM.). Figure 1B depicts an analysis of
surface
antigens expressed by cells within granulomas in the indicated mouse strains.
Figure
1 C depicts cytokine expression by cells from lymph nodes draining the site of
aranulomas. Data are representative of three independent experiments.
Figure 2A-E demonstrates HSV-1-specific delayed-type hypersensitivity
(DHT) reactions in normal and Eta-1/opn-~- mice. Figure 2A depicts footpad
swelling in
Eta-1/opn-~- vs. Eta-1/opn+~+ mice inoculated with HSV-1. The right (control,
~) and left
(HSV-1, ~) footpads of each mouse were measured 24h later using a micrometer.
Each
data point represents the mean and standard error of three micelgroup. Figure
2B depicts
inhibition of the HSV-1 DHT response in Eta-1/opn+~+ mice by acute depletion
of Eta-
1/opn. Figure 2C depicts HSK in Eta-1/opn-~- (open circles) vs. Eta-1/opn+~+
(closed
circles) mice inoculated with HSV-1. Figure 2D depicts HSK in BALBIcByJ (open
circles). Eta-1/opn-~- (closed circles), Eta-1/opn+~+ (open squares). and CB-
17 (closes
squares) mice inoculated with HSV-1. Figure2E depicts the cvtokine response
after
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restimulation of draining lymph node cells (from mice 1~ days after HSV-1
infection in
vivo) or splenic macrophages.
Figure 3A-D demonstrates the differential regulation of macrophage IL-12
and IL-10 responses by purified Eta-1/opn. Figure 3A depicts the dose-
dependent
induction of IL.-12 secretion, but not IL-10 secretion, from macrophages by
Eta-1/opn.
Assays were performed in quadruplets and each data point represents the mean
and
standard error of two independent experiments. Figure 3B depicts selective IL-
12
secretion as stimulated by Eta-1/opn as compared to LPS and/or IL-4 which
stimulate both
IL-12 and IL-10 secretion. Assays were performed in quadruplets and each data
point
represents the mean and standard error of two independent experiments. Figure
3C
demonstrates that the inhibitory effects of Eta-1/opn on IL-4-induced IL-10
production by
macrophage are unaffected by the presence of anti-IL-12 antibody. Assays were
performed
in quadruplets and each point represents the mean and standard error of two
independent
experiments. Figure 3D depicts the inhibitory effect of Eta-1 on LPS-activated
macrophage IL-10 production. Assays were performed in quadruplets, and each
point
represents the mean and standard error (error bars) or two independent
experiments.
Figure 4 depicts the attachment and spreading of MH-S macrophages on
phosphorylated Eta-1/opn, Eta-1/opn, Eta-1/opn-N-terminal fragment, and Eta-
1/opn-C-
terminal fragment. Figure 4A depicts the attachment and spreading of MH-S
cells on
phosphorylated Eta-1/opn, Eta-1/opn, Eta-1/opn-NT and Eta-1/opn-CT in the
presence
or absence of the peptide GRGDS (SEQ ID NO:11 ). Figure 4B depicts the
correlation
between PI-3K activation and spreading of cells on Eta-1/opn, dephosphorylated
Eta-
1/opn, C- or N-terminal fragment, or NK10 fragment.
Figure SA-C demonstrates that induction of IL-12 and inhibition of 1L-10
occur via distinct receptors on macrophages. Figure ~A demonstrates that
secretion of IL-
12 by macrophages is mediated by a 10 kD (NK10) peptide derived from the N-
terminal
fragment of Eta-1/opn (NT) and is inhibited by a blocking anti-integrin 13~
antibody but is
unaffected by antibody to CD44. Figure ~B demonstrates that Eta-1/opn-
dependent
inhibition of IL-4-induced Il-10 production is reversed by anti-CD44 but not
by anti-
integrin antibodies. Figure ~C demonstrates that macrophages from CD44-'- mice
are
resistant to OEta-1/opn inhibition of the IL-10 response as compared to
control mice in
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which Eta-1/opn inhibits IL-4 induced IL-10 production. In all panels. mean
values and
standard errors from at least four data points are shown.
Figure 6A-B demonstrates that that phosphorylation of Eta-1/opn is
necessary for engagement of integrin receptors leading to IL-12 production but
not for
ligation of CD44 leading to IL-10 inhibition by macrophages. Figure 6A depicts
IL-12
secretion resulting from phosphorylated vs. unphosphorylated Eta-1/opn. Figure
6B
demonstrates that dephosphorylation of native Eta-1/opn results in loss of IL-
12
inducing activity, while phosphorylation of (inactive) recombinant Eta-1/opn
restores
this function.
Figure 7 is a bar graph demonstrating that ligation of integrin receptors
on macrophages (e.g., via Eta-1/osteopontin, recombinant phosphorylated Eta-
1/osteopontin. N terminal fragment or NK10) causes predominantly IL-12, TGF(3,
and
TNFa secretion.
Figure 8 is a bar graph demonstrating that ligation of integrin receptors
on macrophages causes predominantly IL-12, TGF(3, and TNFa secretion and
depicts
the effect of various inhibitors (e.g., wortmanin, genestein, chelerythine,
pertussis toxin,
cytochalasin D, and N-(2-metylpiperazyl)-5-isoquinolinesulfoamide(H-7)) on the
cytokine secretion profile. The data are represented as cytokine concentration
in media
harvested from appropriately treated cells.
Figure 9 is a bar graph representing the data of Figure 8 as a fold-
induction of cvtokine secreted.
Figure 10 is a schematic diagram of a biosynthetic immunomodulatory
molecule of the present invention, termed "immunomodulin-2".
Figure 11 is a schematic diagram of a biosynthetic immunomodulatory
molecule of the present invention, termed "immunomodulin-1".
Figure 12 is a bar graph depicting the effect of the biosynthetic
immunomodulatory molecules immunomodulin-1 and immunomodulin-2 on IL-12 and
IL-10 secretion by macrophages. Data are represented as cytokine concentration
in
media harvested from appropriately treated cells.
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Figure 13 is a bar graph depicting the effect of the biosynthetic
immunomodulatory molecules immunomodulin-1 and immunomodulin-2 on IL-12 and
IL-10 secretion by macrophages stimulated with IL--1 or LPS. Data are
represented as
cytokine concentration in media harvested from appropriately treated cells.
Figure 14 is a bar graph depicting the effect of immunomodulin-2
administration in an in vivo model of allergy.
Detailed Description of the Invention
The present invention is based, at least in part, on the elucidation of a new
role for Eta-1/osteopontin in regulating immune responses, in particular, as a
modulator of
type-1 immunity. It has been discovered that Eta-1/osteopontin plays a dual
role in
activation of, for example, the type-1 cytokine IL-12, as well as in the
inhibition of the
type-2 cytokine IL-10. As such, Eta-1/osteopontin is capable of biasing an
immune
response in favor of a type-1 response, or a cellular immune response, as
compared with a
type-2 response, or humoral response. It has been further discovered that the
IL-12
stimulatory and IL-10 inhibitory functions of Eta-1/osteopontin can be
localized to a
specific domains of the naturally-occurring protein. Identification of these
biologically
active domains of Eta-1/osteopontin has led to the development of new
approaches to and
therapeutics useful for the treatment of various immune response-related
diseases and
disorders. Moreover, the role of Eta-1/osteopontin in processes including
monocvte
recruitment, adhesion and activation (i.e., cytokine secretion) has been
analyzed in detail
and new mechanisms for performing such functions have been disclosed.
In one aspect, the invention features methods of modulating immune
responses. in particular, methods of modulating type-1 immune responses in a
subject or
patient (e.g., a human subject or patient) which involve administering to the
subject or
patient an Eta-1/osteopontin modulator such that the immune response (e.g.,
the type-1
immune response) is modulated. In one embodiment, the Eta-1/osteopontin
modulator
stimulates Eta-1/osteopontin activity and the type-1 immune response is
potentiated. In
another embodiment, the Eta-1/osteopontin modulator inhibits Eta-1/osteopontin
activity
and the type-1 immune response is downregulated. In another embodiment. the
Eta-
1/osteopontin modulator is administered_in a therapeutically effective amount.
In
another embodiment, the method also includes monitoring the type-1 response in
the
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subject (e.g., determining the level of a detectable indicator of the type-1
response)
and/or comparing the level of the detectable indicator to a control.
In another embodiment, the invention features methods of potentiatin~
type-1 immune responses in a subject or patient that include selecting a
patient or
subject suffering from a disease or disorder that would benefit from a
potentiated type-1
immune response (e.g., selecting ar_ individual patient or subject from the
human
population) and administering to that patient an Eta-1/osteopontin stimulatory
modulator
such that the type-1 immune response is potentiated. In a preferred
embodiment, the
disease or disorder that would benefit from a potentiated type-1 immune
response is at
least one of the following: (1) burn-associated sepsis, (2) bacterial
infection, (3) viral
infection. (4) cancer, (5) immunodeficiency disorders, (6) AIDS, (7) bone
marrow
transplant-related immunodeficiency, (7) chemotherapy-related immunodeficiency
and
(7) allergy.
In another embodiment, the invention features methods of
downregulating type-1 immune responses in a subject or patient that include
selecting a
patient or subject suffering from a disorder that would benefit from a
downregulated
type-1 immune response (e.g., selecting an individual patient or subject from
the human
population) and administering to the patient or subject an Eta-1/osteopontin
inhibitory
modulator such that the type-1 immune response is downregulated. In a
preferred
embodiment, the disease or disorder that would benefit from a downregulated
type-1
immune response is at least one of the following: ( 1 ) bacterial arthritis,
(2)
granulomatous disorder, (3) herpes simplex keratitis, and (4) autoimmune
diseases.
In yet another embodiment, the present invention features methods for
enhancing production of a type-1 immune response-associated cytokine (e.g,
interleukin-2 (IL-2), interleukin-12 (IL-12) and/or interferon-y (IFN-y)) by
an immune
cell (e.g., a human immune cell) that include contacting the cell with an Eta-
1/osteopontin stimulatory modulator such that production of the cytokine is
enhanced.
In yet another embodiment, the invention features methods for downregulating
production of a type-2 immune response-associated cytokine (e.g., interleukin-
4 (IL-4).
interleukin-~ (IL-~), interleukin-6 (IL-6), and/or interleukin-10 (IL,-10)) by
an immune
cell that include contacting (e.g., in vivo or in vitro) the cell with an Eta-
1/osteopontin
inhibitory modulator such that production of the cytokine is downregulated.
Exemplary
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immune cells include macrophages, dendritic _cells, T cells. B cells,
monocytes and/or
neutrophils. In yet another embodiment, the invention features methods for
stimulating
interleukin-12 (IL-12) production by macrophages that include contacting the
macrophages with an Eta-1/osteopontin stimulatory modulator such that
production of
IL-12 is stimulated. In yet another embodiment, the invention features a
method for
inhibiting interleukin-10 (IL-10) production by macrophages that includes
contacting the
macrophages with an Eta-1/osteopontin stimulatory modulator such that
production of
IL-10 is inhibited.
The present invention also features methods for potentiating type-1
immune responses in a subject or patient that include culturing immune
effector cells
isolated from the subject or patient in the presence of an Eta-1/osteopontin
stimulatory
modulator and administering the cultured cells to the subject such that the
type-1
immune response in the subject is potentiated. Also featured are modified
tumor cells,
for example, irradiated tumor cells transduced with Eta-llosteopontin and such
modified
tumor cells further transduced with GMCSF.
Preferred Eta-1/osteopontin modulators of the present invention include,
but are not limited to, isolated Eta-1/osteopontin polypeptides and
biologically active
fragments thereof, isolated nucleic acid molecules that encodes Eta-
1/osteopontin
polypeptides and that encode biologically active fragments thereof. In one
embodiment,
the Eta-1/osteopontin modulator is an Eta-1/osteopontin polypeptide at least
90%
identical to a polypeptide having the amino acid sequence of SEQ ID N0:2. In
another
embodiment, the Eta-1/osteopontin modulator is an Eta-1/osteopontin
polypeptide
having the amino acid sequence of SEQ ID N0:2, SEQ ID N0:4 or SEQ ID N0:6.
In another embodiment, the Eta-1/osteopontin modulator is an isolated
nucleic acid molecule at least 90% identical to a nucleic acid molecule having
the
nucleotide sequence of SEQ ID NO:1. In yet another embodiment, the Eta-
1/osteopontin modulator is an isolated nucleic acid molecule having the
nucleotide
sequence of SEQ ID NO:I, SEQ ID N0:3 or SEQ ID NO:S.
In another embodiment, the Eta-1/osteopontin modulator is a biologically
active fragment of Eta-1/osteopontin, or a nucleic acid molecule encoding such
a
biologically active fragment. Preferred biologically active fragments include
IL-12
stimulatory domains and/or IL-10 inhibitory domains of Eta-1/osteopontin. In
one
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embodiment, an IL-12 stimulatory domain includes an amino acid sequence
between 65
and 160 amino acids in length and is at least 90% identical to amino acids 71
to 168 of
SEQ ID N0:2. In another embodiment. an IL-10 inhibitory domain includes an
amino
acid sequence between 65 and 160 amino acids in length and is at least 90%
identical to
amino acids 169-266 of SEQ ID N0:2.
Additional preferred Eta-1/osteopontin modulators of the present
invention include compounds that specifically bind to Eta-1/osteopontin
polypeptides,
compounds that specifically binds to Eta-1/osteopontin target molecules,
compounds
that specifically modulate the activity of Eta-1/osteopontin polypeptides and
compounds
that specifically modulate the activity of Eta-1/osteopontin target molecules.
In one
embodiment, the Eta-l/osteopontin modulator is an antibody that specifically
binds to
Eta-1/osteopontin. In another embodiment, the Eta-1/osteopontin modulator is
an
antibody that specifically binds an Eta-1/osteopontin target molecule (e.g.,
an antibody
that specifically binds to an integrin or a CD44 molecule).
In yet another embodiment, the Eta-1/osteopontin modulator is a
biosynthetic immunomodulatory molecule. Preferred biosynthetic
immunomodulatory
molecules include an IL-12 stimulatory component (e.g., an IL-12 stimulatory
component derived from Eta-1/osteopontin) and a biomodular component, forming
a
molecule which modulates an immune response. For example, an IL-12 stimulatory
component can be an Eta-1/osteopontin-derived polypeptide (e.g., a polypeptide
that has
an amino acid sequence between 65 and 160 amino acid residues in length and is
at least
90% identical to amino acids 71 to 168 of SEQ ID N0:2).
Additional preferred biosynthetic immunomodulatory molecules include
an IL,-10 inhibitory component (e.g., an IL-10 inhibitory component derived
from Eta-
1/osteopontin) and a biomodular component. forming a molecule which modulates
an
immune response. For example, an IL-10 inhibitory component can be an Eta-
1/osteopontin-derived polypeptide (e.g., a polypeptide that has between 65 and
160
amino acid residues in length and is at least 90% identical to amino acids 169
to 266 of
SEQ ID N0:2). Exemplary biomodular component include signal peptides.
calcium/apatite binding domains and/or heparin binding domains. Additional
preferred
biosynthetic immunomodulatory molecules include at least two biomodular
components.
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A preferred biosynthetic immunomodulatory molecule includes [Picture
claim for Immunomodulin-2] (e.g., the biosynthetic immunomodulatory molecule
comprising the amino acid sequence of SEQ ID N0:8 and/or the molecule encoded
by
nucleic acid molecule comprising the nucleotide sequence of SEQ ID N0:7).
Another
preferred biosynthetic immunomodulatory molecule includes an IL-10 inhibitory
component, a signal peptide, a calcium/apatite binding domain and a heparin
binding
domain (e.g., the biosynthetic immunomodulatory molecule comprising the amino
acid
sequence of SEQ ID NO:10 and/or the molecule encoded by nucleic acid molecule
comprising the nucleotide sequence of SEQ ID N0:9).
Preferred biosynthetic immunomodulatory molecules of the present
invention are useful for modulating an immune response (e.g., in a subject or
patient, for
example, a human subject or patient) for example, in modulating cytokine
secretion,
regulation of chemotaxis, regulation of hapotaxis, and regulation of cell
spreading. Also
featured are isolated nucleic acid molecules that encode the biosynthetic
immunomodulatory molecules of the present invention, expression vectors that
include
such nucleic acid molecules, and host cell including such vectors. The present
invention
also features methods of producing biosynthetic immunomodulatory molecule that
include culturing such host cell under conditions such that the
immunomodulatory
molecule is produced. Pharmaceutical composition that include the biosynthetic
immunomodulatory molecules of the present invention are also featured.
The present invention also features method of modulating an immune
response in a cell that include contacting the cell with a featured
biosynthetic
immunomodulatory molecule such that an immune response is modulated. In a
preferred embodiment the cell is present within a subject or patient and the
immunomodulatory molecule is administered to the subject.
In order that the present invention may be more readily understood, certain
terms are first defined herein.
The term ''Eta-1/osteopontin" refers to a protein known in the art and
referred to herein interchangeably as "early T lymphocyrte activation-1", "Eta-
1".
"osteopontin", "opn" and "Eta-1/opn". Eta-llosteopontin was originally
identified in bone
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and is now also known to be secreted T cells early during their activation by
a variety of
stimuli. Eta-1/osteopontin is a noncollagenous adhesive matrix protein
normally found in
bone and at epithelial cell surfaces. Eta-1/osteopontin contains an arginine-
glycine-
aspartate (RGDI-binding motif common to many extracellular matrix proteins.
Eta-
I/osteopontin also contains a thrombin cleavage site, cleavage of which alters
the proteins
adhesive properties. Eta-1 has at least two cellular receptors including
integrin and CD44
(Weber et al. ( 1996) Science 271:509-512). As described herein. Eta-1 has
muliple
biological functions. In particular, Eta-1/osteopontin can function as an
immune response
modulator. (See e.g., U.S. Patent No. 5,049,659 and WO 98/08379). A preferred
biological function of Eta-1, as described herein, is in potentiating a type 1
immune
response. For a detailed review of the structure and biological functions of
Eta-
I/osteopontin, see e.g., Denhardt and Guo (1993) 7:1475-1482 and Patarca et
al. (1993)
Crit. Revs. Imunol. 13:225-246, and the referenced cites therein.
The term "immune response" includes any response associated with
immunity including, but not limited to, increases or decreases in cytokine
expression,
production or secretion (e.g., IL-12, IL-10, TGF(3 or TNFa expression,
production or
secretion), cytotoxicity, immune cell migration, antibody production and/or
immune
cellular responses. The phrase "modulating an immune response" or "modulation
of an
immune response" includes upregulation, potentiating, stimulating, enhancing
or
increasing an immune response, as defined herein. For example, an immune
response
can be upregulated, enhanced, stimulated or increased directly by use of a
modulator of
the present invention (e.g., a stimulatory modulator). Alternatively, a
modulator can be
used to "potentiate" an immune response, for example, by enhancing,
stimulating or
increasing immune responsiveness to a stimulatory modulator. The phrase
"modulating
an immune response" or "modulation of an immune response" also includes
downregulation, inhibition or decreasing an immune response as defined herein.
Immune responses in a subject or patient can be further characterized as being
either
type-I or type-2 immune responses.
A "type-1 immune response", also referred to herein as a "type-1
response" or a "T helper type I (Th I ) response" includes a response by CD4+
T cells
that is characterized by the expression,_production or secretion of one or
more type-1
cytokines and that is associated with delayed type hypersensitivity responses.
The
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phrase "type-1 cytokine" includes a cytokine that is preferentially or
exclusively
expressed, produced or secreted by a Thl cell, that favors development of Thl
cells
and/or that potentiates, enhances or otherwise mediates delayed type
hypersensitivity
reactions. Preferred type-1 cytokines include. but are not limited to,
interleukin-2 (IL-
2), interleukin-12 (IL-12), interferon-y (IFN-y) and tumor necrosis factor-(3
(TNF-/3).
A "type-2 immune response", also referred to herein as a ''type-2
response or a "T helper type 2 (Th2) response" refers to a response by CD4+ T
cells
that is characterized by the production of one or more type-2 cytokines and
that is
associated with humoral or antibody-mediated immunity (e.g., efficient B cell,
"help"
provided by Th2 cells, for example, leading to enhanced IgG 1 and/or IgE
production).
The phrase "type-2 cytokine" includes a cytokine that is preferentially or
exclusively
expressed, produced or secreted by a Th2 cell, that favors development of Th2
cells
and/or that potentiates, enhances or otherwise mediates antibody production by
B
lymphocytes. Preferred type-2 cytokines include, but are not limited to,
interleukin-4
(IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6), interleukin-10 (IL-10) and
interleukin-
13 (IL-13).
Cytokine expression, secretion or production modulates or further
enhances or upregulates an immune response, for example, a type-1 of type-2
immune
response. For example, it is known that cvtoktnes play a dominant role in
controlling
the differentiation of T helper precursors (Th0) to either the Thl or Th2
lineage. Type-1
cvtoktnes, such as IFN-'y, can enhance the development of Thl cells and
inhibit the
development of Th2 cells, whereas type-2 cytokines> such as IL-4 and IL-10,
can
enhance the development of Th2 cells and inhibit the development of Th 1
cells. Thus.
cytokines can reciprocally regulate the development and/or progression of
either a type-
1 or a type-2 response.
Cytokine expression, secretion or production can also be an indicator of
an immune response, for example, an indicator of a type-1 or type-2 immune
response.
For example, a "cytokine profile" can be indicative of a type-1 or type-2
immune response. The term "cytokine profile" includes expression, production
or
secretion of at least one cvtokine associated with a particular type of immune
response
(e.g., a type-1 or type-2 immune response) and/or includes diminished or
reduced
expression, production or secretion of at least one cytokine associated with a
mutually
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exclusive type of immune response (e.g.. a type-2 or type-1 immune response.
respectively). For example, a type-1 cytokine profile can include enhanced or
increased
expression, production or secretion of at least one of interleukin-2 (IL-2).
interleukin-12
(IL-12). interferon-y (IFN-y) and tumor necrosis factor-~ (TNF-(3) and/or can
include
reduced or decreased expression, production or secretion of at least one of
interleukin-4
(IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6) and interleukin-10 (IL-10).
Likewise, a
type-2 cvtokine profile can include expression, production or secretion of at
least one of
interleukin-4 (IL-4), interleukin-5 (IL-5), interleukin-6 (IL-6) and
interleukin-10 (IL-10)
and/or can include reduced or decreased expression, production or secretion of
at least
one of interleukin-2 (IL-2), interleukin-12 (IL-12), interferon-y (IFN-y) and
tumor
necrosis factor-~ (TNF-(3).
The phrase "type-1 immunity" includes immunity characterized
predominantly by type-1 immune responses (e.g., delayed type hypersensitivity,
macrophage activation and or cellular cytotoxicity), by expression, production
or
secretion of at least one type-1 cytokine and/or expression of a type-1
immunity
cytokine profile. The phrase "type-2 immunity" includes immunity characterized
predominantly by type-2 immune responses (e.g., B cell help, IgGI and/or IgE
production, eosinophil activation, mast cell stimulation and/or macrophage
deactivation), by expression, production or secretion of at least one type-2
cytokine
and/or expression of a type-2 immunity cytokine profile.
The course of certain disease states is influenced by whether a
predominant type-1 or type-2 response is mounted. For example, in experimental
leishmania infections in mice, animals that are resistant to infection mount
predominantly a type-1 immune response, whereas animals that are susceptible
to
progressive infection mount predominantly a type-2 immune response (Heinzel et
al.
( 1989) J. Exp. Med. 169:59-72: and Locksley and Scott ( 1992)
Immunoparasitology
Today 1:A58-A61 ). In murine schistosomiasis, a switch from type-1 to type-2
immunity
is observed coincident with the release of eggs into the tissues by female
parasites and is
associated with a worsening of the disease condition (Pearce et al. ( 1991 )
J. E.rp. Med.
173:159-166; Grzych et al. (19911 J. Irnmunol. 141:1322-1327; and Kullberg et
al.
(1992) J. Irnmunol. 148:3264-3270). Many human diseases, including chronic
infections (such as with human immunodeficiency virus (HIV) or tuberculosis)
and
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certain metastatic carcinomas, also are characterized by a type-1 to type-2
switch. (see
e.g., Shearer and Clerici ( 1992) Prog. Chem. Immccnol. 54:21-43; Clerici and
Shearer
( 1993) Imnusnol. Today 14:107-111; Yamamura et ail. ( 1993) J. Clin. Invest.
91:1005-
1010: Pisa et al. ( 1992) Proc. Natl. Acad. Sci. USA 89:7708-7712; Fauci (
1988) Science
239:617-623). Furthermore, certain autoimmune diseases have been shown to be
associated with a predominant type-1 response. For example, patients with
rheumatoid
arthritis have predominantly Th 1 cells in synovial tissue (Simon et al. (
1994) Proc. Natl.
Acad. Sci. USA 91:8562-8566) and experimental autoimmune encephalomyelitis
(EAE)
can be induced by autoreactive Thl cells (Kuchroo et al. (1993) J. Immunol.
151:4371-
4381).
The phrase "potentiating or potentiation of a type-1 or type-2 immune
response" includes upregulation, stimulation or enhancement of a type-1 or
type-2
response, respectively (e.g., commitment of T helper precursors to either a
Thl or Th2
lineage, further differentiation of cells to either the Th 1 or Th2 phenotype
and/or
continued function of Thl or Th2 cells during an ongoing immune response). For
a
review of Th l and Th2 subsets see, for example, Seder and Paul ( 1994) Ann.
Rev.
Immunol. 12:635-673.
The phrase "potentiating or potentiation of a type-1 immune response"
also includes downregulation or inhibition of a type-2 immune response. The
phrase
"potentiating or potentiation of a type-2 immune response" also includes
downregulation or inhibition of a type-1 immune response.
The term "immunomodulatory molecule", used interchangeably herein
with the term "immunomodulatory "agent" includes a molecule or agent which has
a
modulatory or regulatory activity which is normally associated with an immune
response in an organism, for example, higher animals and humans. An activity
(e.g., a
biological or functional activity) associated with an immune response can be
any
activity associated with resistance of the organism to infection with
microorganisms.
response to infection or response to disease. The term "activity", "biological
activity' or
"functional activity", refers to an activity exerted by a molecule of the
invention (e.g., a
immunomodulatory molecule. for example, a protein, polypeptide, fragment,
nucleic
acid molecule, antibody, biosynthetic immunomodulatory molecule, or the like )
as
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determined in vivo, or in vitro, according to standard techniques and/or
methods such as
those described in the Examples.
The term "immune cell" includes cells of the immune system which are
capable of expressing, producing or secreting cytokines that regulate an
immune
response, for example a type-1 or type-2 immune response. Preferred immune
cells
include human immune cells. Exemplary preferred immune cells include, but are
not
limited to, macrophages, dendritic cells, T cells, B cells and neutrophils.
Immune cells
are also referred to herein as "immune effector cells". The term "macrophage"
includes
all cells within the macrophage lineage, including monocytes, circulating
macrophages,
tissue macrophages, activated macrophages, and the like, from mammals (e.g.,
from
humans). The term "T cell" (i.e., T lymphocyte) is intended to include all
cells within
the T cell lineage, including thymocytes, immature T cells, mature T cells and
the like,
from mammals (e.g., from humans).
The phrase "contacting" (e.g., contacting a cell, for example, with an
agent or modulator) is intended to include incubating the agent and the cell
together in
vitro (e.g., adding the agent or modulator to cells in culture) or
alternatively,
administering the agent or modulator to a subject or patient such that the
agent or
modulator is capable or contacting the cells of the subject or patient in
vivo.
"Administering" an agent or modulator includes any routine means known in the
art or
described herein of providing a subject or patient with an agent or modulator.
"Coadministering" agents includes administering a first and second agent or
modulator,
for example, sequentially or coincidentally. In addition to administering
agents and/or
modulators (e.g., immunomodulatory molecules), certain aspects of the present
invention feature administering cells to a subject or patient. For example,
cells of a
patient (e.g., immune cells or immune effector cells) can be isolated from a
subject,
contacted with an agent or modulator in vitro (e.g., culturing the cells in
the presence of
the agent or modulator), and administered or readministered to the subject or
patient.
Routine means can be utilized for isolating immune cells, for example,
isolating and/or
separating plasma from a subject or patient, isolating bone marrow from a
patient or
subject. as well as for administering or readministering cells, for example.
plasmaphoresis or bone marrow transplants.
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The term "subject'' includes a liking animal, preferably a human subject.
The term "patient'' includes a subject, preferably a human subject, in need
of treatment (e.g., treatment according to the methodologies of the present
invention),
potentially in need of treatment, presently undergoing treatment. having or
suffering from a
disease or disorder which would benefit from at least one methodology of the
present
invention. Preferably a "patient'' is a human patient.
Exemplary diseases and/or disorders from which a patient, as defined
herein, may be at risk for, have or be suffering from include but are not
limited to burn-
associated sepsis, infectious diseases or disorders (e.g., bacterial
infection, viral
infection, HIV and tuberculosis) cancer, immunodeficiency disorders, AIDS,
bone
marrow transplant-related immunodeficiency, chemotherapy-related
immunodeficiency,
allergy, bacterial arthritis, granulomatous disorder, herpes simplex
keratitis, autoimmune
disease, and various forms of glomerulonephritis including, but not limited
to, rhematoid
arthritis and multiple sclerosis. Diseases and disorders are to be given their
accepted, art-
recognized definitions, for example, as set forth in The Physicians Desk
Reference.
The phrase ''monitoring and immune response", for example,
"monitoring a type-1 immune response" includes monitoring the ability of an
agent or
modulator of the invention to enhance, potentiate, stimulate, upregulate or
downregulate
or inhibit an immune response, for example, a type-1 or type-2 immune
response. In
one embodiment, monitoring a type-1 or type-2 response includes determining
the level
of a detectable indicator of the type-1 or type-2 response. Preferred
detectable
indicators include cytokines associated with a particular response, cytokine
profiles
associated with a particular response and/or phenotypic responses. Exemplary
detectable indicators of a type-1 response include expression, production or
secretion to
type-1 cytokines, type-1 cytokine profiles, as well as any other type-1
phenotypic
response, as described herein. Exemplary detectable indicators of a type-2
response
include expression, production or secretion to type-2 cytokines, type-2
cytokine profiles.
as well as any other type-2 phenotypic response, as described herein. In
another
embodiment, monitoring an immune response further comprises comparing the
detectable indicator to a control (e.g., a control profile or control
phenotype, for
example, the profile or phenotype of the_ subject or patient prior to
treatment or at a
previous stage of treatment with an agent or modulator, the profile or
phenotype) of a
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normal or average subject, or an expected or target profile or phenotype
(e.g., a
theoretical, desired or predicted profile or phenotype ).
Various aspects of the invention are described in further detail in the
following subsections.
I. Immunomodulator~A~ents
In the immunomodulatory methods of the invention, for example, type-1
immunomodulatory methods, an Eta-1/osteopontin modulator is administered to a
subject (e.g., a human subject) or a cell (e.g., a human immune cell) is
contacted with
the modulator such that an immune response, for example, a type-1 immune
response is
modulated. In one embodiment. the Eta-1/osteopontin modulator is a
"stimulatory
agent" (e.g., an agent or modulator that stimulates Eta-1/osteopontin
activity), which
enhances, potentiated, increases or upregulates a type-1 immune response in a
cell or
subject. Preferred "stimulatory agents" or "stimulatory modulators" include
isolated
Eta-1/osteopontin proteins or polypeptides and biologically active fragments
thereof,
isolated nucleic acid molecules encoding such Eta-l/osteopontin proteins or
polypeptides and biologically active fragments thereof, biosynthetic
immunomodulatory
molecules, Eta-1 peptides, peptidomimetics and small molecule agonists (e.g.,
Eta-1
peptides, peptidomimetics and small molecule agonists capable of specifically
binding
to an Eta-1/osteopontin receptor. for example, integrin or CD44, and/or
upregulating the
activity of the Eta-1/osteopontin receptor as described in further detail
below. In
another embodiment, the Eta-1/osteopontin modulator is an "inhibitory agent"
(e.g., an
agent or modulator that inhibits Eta-1/osteopontin activity), which decreases
or
downregulates a type-1 immune response in a cell or subject. Preferred
"inhibitory
agents" or "inhibitory modulators'' include antisense Eta-1/osteopontin
nucleic acid
molecules, Eta-1/osteopontin antibodies and/or Eta-1/osteopontin receptor
antibodies
(e.g. compounds capable of specifically binding to an Eta-1/osteopontin
receptor. for
exart~ple. integrin or CD44. and/or downregulating the activity of the Eta-
1/osteopontin
receptor). as described in further detail below. Additional preferred
modulatory agents
modulate selected activities of Eta-1/osteopontin, for example, modulate
activities
resulting from ligation of CD44 and/or integrin by Eta-1/osteopontin.
Particularly
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preferred modulatory agents modulate immune responses specific for Eta-
1/osteopontin
interacting with CD44 and/or integrin.
A Isolated Eta-1/Osteopontin Proteins. Biolo~ically-active Fragments
Thereof and
One aspect of the invention pertains to isolated Eta-1/osteopontin
proteins and biologically active portions thereof. Eta-1/osteopontin proteins
can be
isolated from cells or tissue sources by an appropriate purification scheme
using
standard protein purification techniques, can produced by recombinant DNA
techniques
or can be synthesized chemically using standard peptide synthesis techniques.
Biologically active portions of Eta-1/osteopontin polypeptides can be further
generated
by enzymatic digestion of full-length Eta-1/osteopontin polypeptides, can be
produced
by recombinant DNA techniques or can be synthesized chemically using standard
peptide synthesis techniques.
An "isolated" or "purified" Eta-1/osteopontin protein or biologically
active portion thereof is substantially free of cellular material or other
contaminating
proteins from the cell or tissue source from which the Eta-1/osteopontin is
derived, or
substantially free from chemical precursors or other chemicals when chemically
synthesized. The language "substantially free of cellular material" includes
preparations
of Eta-1/osteopontin in which the protein is separated from cellular
components of the
cells from which it is isolated or recombinantly produced. The language
"substantially
free of cellular material" includes preparations in which the recombinant
molecule is
separated from cellular components of the cells from which it is isolated or
recombinantly produced. In one embodiment, the language "substantially free of
cellular material" includes preparations having less than about 30% (by dry
weight) of
contaminating cellular material, more preferably less than about 20% of
contaminating
material, still more preferably less than about 10% of contaminating material,
and most
preferably less than about 5% contaminating material. When Eta-1/osteopontin
is
recombinantly produced, it is also preferably substantially free of culture
medium, i.e..
culture medium represents less than about 20%. more preferably less than about
10%.
and most preferably less than about 5%_of the volume of the preparation.
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The language "substantially free of chemical precursors or other
chemicals" includes preparations in which the chemically synthesized molecule
is
separated from chemical precursors or other chemicals which are involved in
the
synthesis of the molecule. In one embodiment, the language "substantially free
of
chemical precursors or other chemicals" includes preparations having less than
about
30% (by dry weight) of chemical precursors or contaminating chemicals, more
preferably less than about 20% chemical precursors or contaminating chemicals,
still
more preferably less than about 10% chemical precursors or contaminating
chemicals.
and most preferably less than about 5% chemical precursors or contaminating
chemicals.
In a preferred embodiment, an Eta-1/osteopontin protein for use in the
present invention is a human Eta-1/osteopontin. For example, any of the Eta-
1/osteopontin proteins set forth as SEQ ID N0:2, SEQ ID N0:4 or SEQ ID N0:6
are
suitable for use in the immunomodulatory methods of the present invention
(e.g., Eta-
1/osteopontin protein-a, Eta-1/osteopontin protein-b or Eta-1/osteopontin
protein-c,
respectively). Also suitable for use in the immunomodulatory methods of the
present
invention are Eta-1/osteopontin homologues or variants which vary at the amino
acid
sequence level when compared, for example, to the Eta-1/osteopontin proteins
set forth
as SEQ >D N0:2, SEQ ID N0:4 or SEQ ID N0:6 but which retain the biological
activity of the Eta-1/osteopontin proteins set forth as SEQ ID N0:2. SEQ ID
N0:4 or
SEQ 117 N0:6. For example, Eta-1/osteopontin homologues or variants having 85-
90%.
90-95%, 96%. 97%, 98%, 99% or more sequence identity to the Eta-1/osteopontin
proteins set forth as SEQ ID N0:2, SEQ ID N0:4 or SEQ ID N0:6 but which retain
biological activity are suitable for use in the immunomodulatory methods of
the present
invention. Eta-1/osteopontin homologues or variants can have amino acid
substitutions
(particularly conservative amino acid substitutions) at "non-essential" amino
acid
residues in the sequence of SEQ ID N0:2, SEQ ID N0:4 or SEQ ID N0:6. A "non-
essential" amino acid residue is a residue that can be altered from the
sequence set forth
in SEQ B~ N0:2, SEQ ID N0:4 or SEQ ID N0:6 without altering the biological
activity, whereas an "essential" amino acid residue is required for biological
activity.
For example, amino acid residues that are conserved among proteins or domains
of
proteins from different species are predicted to be particularly unamenable to
alteration.
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Alternatively, Eta-1/osteopontin homologues _or variants can have a
conservative amino
acid substitutions at one or more predicted essential or non-essential amino
acid
residues. A "conservative amino acid substitution" is one in which the amino
acid
residue is replaced with an amino acid residue having a similar side chain.
Families of
amino acid residues having similar side chains have been defined in the art.
These
families include amino acids with basic side chains (e.g., lysine, arginine,
histidine),
acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side
chains (e.g.,
glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),
nonpolar side
chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine,
tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine)
and aromatic
side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a
predicted
essential or nonessential amino acid residue is preferably replaced with
another amino
acid residue from the same side chain family.
Also suitable for use in the immunomodulatory methods of the present
invention are Eta-1/osteopontin homologues or variants which are encoded by
nucleic
acid molecules comprising the nucleotide sequences of SEQ ID NO:I, SEQ ID N0:3
or
SEQ ID N0:5, as well as Eta-1/osteopontin homologues or variants encoded by
nucleic
acid molecules having 85-90%, 90-95%, 96%, 97%, 98%, 99% or more sequence
identity to the Eta-1/osteopontin nucleotide sequences of SEQ ID NO:I, SEQ ID
N0:3
or SEQ ID N0:5 and/or isolated nucleic acid molecules which hybridize under
stringent
hybridization conditions to the Eta-1/osteopontin nucleotide sequences of SEQ
ID NO:1,
SEQ m N0:3 or SEQ ID N0:5. As used herein, the term "hybridizes under
stringent
conditions" is intended to describe conditions for hybridization and washing
under
which nucleotide sequences at least 90% homologous to each other typically
remain
hybridized to each other. Such stringent conditions are known to those skilled
in the art
and can be found in Current Protocols in Molecular Biology, John Wiley & Sons,
N.Y.
( 1992). A preferred, non-limiting example of stringent hybridization
conditions are
hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C,
followed by
one or more washes in 0.2 X SSC. 0.1 % SDS at 50-65°C.
Biologically active portions of Eta-1/osteopontin include fragments or
portions sufficiently homologous to Eta-1/osteopontin, e.g., the amino acid
sequence
shown in SEQ ID N0:2. which include less amino acids than the full length
polypeptide,
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and exhibit at least one activity of full-length Eta-1/osteopontin. Typically.
biologically
active portions comprise a domain with at least one activity of the full-
length
polypeptide. A biologically active portion can be a polypeptide which is. for
example,
~0-100. 100-1~0. 150-200, 200-250, 250-300 or more amino acids in length.
In one embodiment, a biologically active portion of Eta-1/osteopontin
comprises an IL-12 stimulatory domain of Eta-1/osteopontin. As used herein, an
IL-12
stimulatory domain is a domain of Eta-1/osteopontin capable of ligating
integrin (e. g.,
av~3 integrin) such that IL-12 production is stimulated by the cell. In a
preferred
embodiment, an IL-12 stimulatory domain is incapable of ligating CD44
expressed on
the cell. In one embodiment, an IL-12 stimulatory domain is about 50-60, 60-
70, 70-80,
80-90 or 90-100 amino acid residues in length. In another embodiment, an IL-12
stimulatory domain is of a size sufficient to induce IL-12 production by a
cell but
includes insufficient amino acid residues to inhibit II.-10 production by the
cell. A
particularly preferred IL-12 stimulatory domain includes the residues of the
fragment
NK10 described herein.
In another embodiment, a biologically active portion of Eta-1/osteopontin
comprises an IL-10 inhibitory domain of Eta-1/osteopontin. As used herein, an
IL-10
inhibitory domain is a domain of Eta-1/osteopontin capable of ligating CD44
such that
IL-10 production is inhibited by the cell. In a preferred embodiment, an IL-10
inhibitory domain is incapable of ligating integrin (e.g., av~i3 integrin)
expressed on the
cell. In one embodiment, an IL-10 inhibitory domain is about 50-60, 60-70, ?0-
80. 80-
90 or 90-100 amino acid residues in length. In another embodiment, an IL-10
inhibitory
domain is of a size sufficient to inhibit IL-10 production by a cell but
includes
insufficient amino acid residues to stimulate IL-12 production by the cell. A
particularly
preferred IL-10 inhibitory domain includes about amino acid residues 169-200.
169-220,
169-240, 169-260, or 169-280 of SEQ ID N0:2.
To determine the percent homology of two amino acid sequences or of
two nucleic acid sequences, the sequences are aligned for optimal comparison
purposes
(e.g., gaps can be introduced in the sequence of a first amino acid or nucleic
acid
sequence for optimal alignment with a second amino or nucleic acid sequence).
The
amino acid residues or nucleotides at corresponding amino acid positions or
nucleotide
positions are then compared. When a position in the first sequence is occupied
by the
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J _
same amino acid residue or nucleotide as the corresponding position in the
second
sequence, then the molecules are homologous at that position (i.e., as used
herein amino
acid or nucleic acid "homology" is equivalent to amino acid or nucleic acid
"identity" ).
The percent homology between the two sequences is a function of the number of
identical positions shared by the sequences (i.e., % homology = # of identical
positions/total # of positions x 100). The determination of percent homology
between
two sequences can be accomplished using a mathematical algorithm. A preferred,
non-
limiting example of a mathematical algorithm utilized for the comparison of
two
sequences is the algorithm of Karlin and Altschul ( 1990) Proc. Natl. Acad.
Sci. USA
87:2264-68, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci.
USA
90:5873-77. Such an algorithm is incorporated into the NBLAST and XBLAST
programs of Altschul et al. ( 1990) J. Mol. Biol. 215:403-10. BLAST nucleotide
searches can be performed with the NBLAST program, score = 100, wordlength =
12 to
obtain nucleotide sequences homologous to the nucleic acid molecule sequences
of the
invention. BLAST protein searches can be performed with the XBLAST program,
score
= 50, wordlength = 3 to obtain amino acid sequences homologous to the protein
molecules of the invention. To obtain gapped alignments for comparison
purposes,
Gapped BLAST can be utilized as described in Altschul et al. ( 1997) Nucleic
Acids
Research 25( 17):3389-3402. When utilizing BLAST and Gapped BLAST programs,
the default parameters of the respective programs (e.g., XBLAST and NBLAST)
can be
used. See http://www.ncbi.nlm.nih.gov. Another preferred, non-limiting example
of a
mathematical algorithm utilized for the comparison of sequences is the
algorithm of
Myers and Miller ( 1988) Comput Appl Biosci. 4:11-17. Such an algorithm is
incorporated into the ALIGN program available, for example, at the GENESTREAM
network server, IGH Montpellier, FRANCE (http://vega.igh.cnrs.fr) or at the
ISREC
server (http://www.ch.embnet.org). When utilizing the ALIGN program for
comparing
amino acid sequences, a PAM 120 weight residue table, a gap length penalty of
12, and a
gap penalty of 4 can be used.
The invention also provides chimeric or fusion proteins, for example.
recombinant chimeric or fusion proteins designed to facilitate the
purification of Eta-
1/osteopontin (e.g., GST-fusion proteins or HA-tagged fusion proteins). Also
provided
are chimeric or fusion proteins (e.g., Eta-1/osteopontin containing a
heterologous signal
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_ 2,~ _
sequence at its N-terminus ) to enhance expression and/or secretion
recombinant Eta-
1/osteopontin. Chimeric or fusion proteins of the invention are produced by
standard
recombinant DNA techniques as described. for example, in Current Protocols in
Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992). Moreover,
many
expression vectors are commercially available that already encode a fusion
moiety (e.g..
a GST polypeptide). An Eta-1/osteopontin-encoding nucleic acid, as described
herein.
can be cloned into such an expression vector such that the fusion moiety is
linked in-
frame to the Eta-1/osteopontin protein.
Also featured are Eta-1/osteopontin proteins and biologically active
portions which are incorporated into pharmaceutical compositions as described
herein.
B. Isolated Nucleic Acid Molecules. Vectors, Host Cells
Another aspect of the invention pertains to isolated nucleic acid
molecules that encode Eta-1/osteopontin proteins or portions or biologically
active
fragments thereof. The term "nucleic acid molecule" includes DNA molecules
(e.g.,
cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or
RNA generated using nucleotide analogs. The nucleic acid molecule can be
single-
stranded or double-stranded, but preferably is double-stranded DNA.
An "isolated" nucleic acid molecule is one which is separated from other
nucleic acid molecules which are present in the natural source of the nucleic
acid.
Preferably, an "isolated" nucleic acid is free of sequences which naturally
flank the
nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic
acid) in the
genomic DNA of the organism from which the nucleic acid is derived. For
example, in
various embodiments. the isolated nucleic acid molecule can contain less than
about 5
kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which
naturally flank
the nucleic acid molecule in genomic DNA of the cell from which the nucleic
acid is
derived. Moreover. an "isolated" nucleic acid molecule, such as a cDNA
molecule, can
be substantially free of other cellular material, or culture medium when
produced by
recombinant techniques, or substantially free of chemical precursors or other
chemicals
when chemically synthesized.
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In another preferred embodiment, an isolated nucleic acid molecule of the
invention comprises a nucleotide sequence shown in SEQ ID NO:1, SEQ ID N0:3,
SEQ
ID N0:5 or a portion thereof. Also included within the scope of the invention
are
isolated nucleic acid molecules which are complementary to the nucleotide
sequences
shown in SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:5 or a portion thereof. Also
included within the scope of the present invention are isolated nucleic acid
molecules
which hybridize (e.g., under stringent hybridization conditions) to a
complement of the
nucleotide sequences shown in SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:5 or a
portion
thereof, thereby forming a stable duplex. Also included within the scope of
the present
invention are isolated nucleic acid molecules having 80-85%, 90-95%, 96%, 97%,
98%,
99% or more homology or identity to the nucleotide sequences shown in SEQ ID
NO:1,
SEQ 117 N0:3, SEQ ID N0:5 or a portion thereof. Also included within the scope
of the
present invention are isolated nucleic acid molecules which are antisense to
the nucleic
acid molecules shown in SEQ ID NO:1, SEQ ID N0:3, SEQ ID N0:5. The invention
further encompasses nucleic acid molecules that differ from the nucleotide
sequence
shown in SEQ ID NO:1, SEQ ID N0:3 or SEQ ID N0:5 yet due to degeneracy of the
genetic code encode the same molecules as those encoded by the nucleotide
sequence
shown in SEQ ID NO:1, SEQ ID N0:3 or SEQ ID N0:5.
A nucleic acid molecule of the invention, or portion thereof, can be
amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and
appropriate oligonucleotide primers according to standard PCR amplification
techniques. The nucleic acid so amplified can be cloned into an appropriate
vector and
characterized by DNA sequence analysis. Furthermore, oligonucleotides (e.g.,
probes
andlor primers) and antisense nucleic acid molecules can be prepared by
standard
synthetic techniques, e.g., using an automated DNA synthesizer.
Oligonucleotides for
use in the present invention typically comprises substantially purified
oligonucleotide.
The oligonucleotide typically comprises a region of nucleotide sequence that
hybridizes
under stringent conditions to at least about 12. preferably about 25, more
preferably
about 40. 50 or 75 consecutive nucleotides of the nucleotide sequence of SEQ
ID NO:1,
SEQ ID N0:3 or SEQ ID N0:5 of or of the complement of the nucleotide sequence
of
SEQ ID NO:1, SEQ ID N0:3 or SEQ ID N0:5. Also included within the scope of the
present invention are oligonucleotides at least 15, 30. 50. 100. 250 or 500
nucleotides in
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length which hybridizes under stringent conditions to the nucleic acid
molecule
comprising the nucleotide sequence of SEQ ID NO:1. SEQ ID N0:3 or SEQ ID NO:~.
A nucleic acid fragment encoding a "biologically active" portion of an
Eta-1/osteopontin molecule of the present invention can be prepared by
isolating a
portion of SEQ ID NO: l, SEQ ID N0:3 or SEQ ID NO:S which encodes a
polypeptide
having a biological activity of the naturally-occurring protein from which the
portion
was derived, expressing the encoded portion of the naturally-occurring protein
(e.g., by
recombinant expression in vitro) and assessing the activity of the encoded
portion of the
naturally-occurring protein. As used herein, a "naturally-occurring" nucleic
acid
molecule or protein molecule refers to a molecule having a nucleotide or amino
acid
sequence that occurs in nature (e.g., a nucleic acid molecule that encodes a
natural
protein).
Another aspect of the invention pertains to vectors, preferably expression
vectors, containing a nucleic acid molecule of the present invention. As used
herein, the
term "vector" refers to a nucleic acid molecule capable of transporting
another nucleic
acid to which it has been linked. One type of vector is a "plasmid", which
refers to a
circular double stranded DNA loop into which additional DNA segments can be
ligated.
Another type of vector is a viral vector, wherein additional DNA segments can
be
ligated into the viral genome. Certain vectors are capable of autonomous
replication in a
host cell into which they are introduced (e.g., bacterial vectors having a
bacterial origin
of replication and episomal mammalian vectors). Other vectors (e.g., non-
episomal
mammalian vectors) are integrated into the genome of a host cell upon
introduction into
the host cell, and thereby are replicated along with the host genome.
Moreover, certain
vectors are capable of directing the expression of genes to which they are
operatively
linked. Such vectors are referred to herein as "expression vectors". In
general,
expression vectors of utility in recombinant DNA techniques are often in the
form of
plasmids. In the present specification, "plasmid" and "vector" can be used
interchangeably as the plasmid is the most commonly used form of vector.
However.
the invention is intended to include such other forms of expression vectors,
such as viral
vectors (e.g., replication defective retroviruses. adenoviruses and adeno-
associated
viruses ), which serve equivalent functions.
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The recombinant expression vectors of the invention comprise a nucleic
acid of the invention in a form suitable for expression of the nucleic acid in
a host cell.
which means that the recombinant expression vectors include one or more
regulatory
sequences, selected on the basis of the host cells to be used for expression.
which is
operatively linked to the nucleic acid sequence to be expressed. Within a
recombinant
expression vector, "operably linked" means that the nucleotide sequence of
interest is
linked to the regulatory sequences) in a manner which allows for expression of
the
nucleotide sequence (e.g., in an in vitro transcription/translation system or
in a host cell
when the vector is introduced into the host cell). The term "regulatory
sequence"
includes promoters, enhancers and other expression control elements (e.g.,
polyadenylation signals). Such regulatory sequences are described, for
example, in
Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic
Press.
San Diego, CA ( 1990). Regulatory sequences include those which direct
constitutive
expression of a nucleotide sequence in many types of host cell and those which
direct
expression of the nucleotide sequence only in certain host cells (e.g., tissue-
specific
regulatory sequences). It will be appreciated by those skilled in the art that
the design of
the expression vector can depend on such factors as the choice of the host
cell to be
transformed, the level of expression of protein desired, etc. The expression
vectors of
the invention can be introduced into host cells to thereby produce proteins or
peptides.
including fusion proteins or peptides, encoded by nucleic acids as described
herein.
The recombinant expression vectors of the invention can be designed for
expression in prokaryotic or eukaryotic cells. For example, recombinant
proteins can be
expressed in bacterial cells such as E. coli, insect cells (using baculovirus
expression
vectors) yeast cells or mammalian cells. Suitable host cells are discussed
further in
Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic
Press.
San Diego, CA ( 1990). Alternatively, the recombinant expression vector can be
transcribed and translated in vitro. for example using T7 promoter regulatory
sequences
and T7 polymerase.
Expression of proteins in prokaryotes is most often carried out in E. coli
with vectors containing constitutive or inducible promotors directing the
expression of
either fusion or non-fusion proteins. Fusion vectors add a number of amino
acids to a
protein encoded therein. usually to the amino terminus of the recombinant
protein. Such
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_~g_
fusion vectors typically serve three purposes: _ 1 ) to increase expression of
recombinant
protein; 2) to increase the solubility of the recombinant protein: and 3) to
aid in the
purification of the recombinant protein by acting as a ligand in affinity
purification.
Often, in fusion expression vectors, a proteolytic cleavage site is introduced
at the
function of the fusion moiety and the recombinant protein to enable separation
of the
recombinant protein from the fusion moiety subsequent to purification of the
fusion
protein. Such enzymes, and their cognate recognition sequences, include Factor
Xa,
thrombin and enterokinase. Typical fusion expression vectors include pGEX
(Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S. ( 1988) Gene 67:31-40),
pMAL
(New England Biolabs, Beverly, MA) and pRITS (Pharmacia, Piscataway, NJ) which
fuse glutathione S-transferase (GST), maltose E binding protein, or protein A,
respectively, to the target recombinant protein.
Examples of suitable inducible non-fusion E. coli expression vectors
include pTrc (Amann et al., (1988) Gene 69:301-315) and pET 1 ld (Studier et
al., Gene
Expression Technology: Methods in Enzymology 185, Academic Press, San Diego,
California ( 1990) 60-89). Target gene expression from the pTrc vector relies
on host
RNA polymerase transcription from a hybrid trp-lac fusion promoter. Target
gene
expression from the pET l ld vector relies on transcription from a T7 gnl0-lac
fusion
promoter mediated by a coexpressed viral RNA polymerise (T7 gn 1 ). This viral
polymerise is supplied by host strains BL21 (DE3) or HMS 174(DE3) from a
resident ~.
prophage harboring a T7 an 1 gene under the transcriptional control of the
lacUV 5
promoter.
One strategy to maximize recombinant protein expression in E. coli is to
express the protein in a host bacteria with an impaired capacity to
proteolytically cleave
the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in
Enzymology 185, Academic Press. San Diego, California (1990) 119-128). Another
strategy is to alter the nucleic acid sequence of the nucleic acid to be
inserted into an
expression vector so that the individual codons for each amino acid are those
preferentially utilized in E. coli (Wada et al., ( 1992) Nucleic Acids Res.
20:2111-2118).
Such alteration of nucleic acid sequences of the invention can be carried out
by standard
DNA synthesis techniques.
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In another embodiment, the expression vector is a yeast expression
vector. Examples of vectors for expression in yeast S. cerivisae include
pYepSec 1
(Baldari, et al., ( 1987) Embo J. 6:229-234), pMFa f Kurjan and Herskowitz. (
1982) Cell
30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2
(Invitrogen
Corporation, San Diego, CA), and picZ (InVitrogen Corp, San Diego, CA).
Alternatively, recombinant proteins can be expressed in insect cells using
baculovirus expression vectors. Baculovirus vectors available for expression
of proteins
in cultured insect cells (e.g., Sf 9 cells) include the pAc series (Smith et
al. ( 1983) Mol.
Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers ( 1989)
Virology
170:31-39).
In yet another embodiment, a nucleic acid of the invention is expressed in
mammalian cells using a mammalian expression vector. Examples of mammalian
expression vectors include pCDM8 (Seed, B. ( 1987) Nature 329:840) and pMT2PC
(Kaufman et al. (1987) EMBO J. 6:187-195). When used in mammalian cells, the
expression vector's control functions are often provided by viral regulatory
elements.
For example, commonly used promoters are derived from polyoma, Adenovirus 2,
cytomegalovirus and Simian Virus 40. For other suitable expression systems for
both
prokaryotic and eukaryotic cells see chapters 16 and 17 of Sambrook, J.,
Fritsh, E. F.,
and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
NY,
1989.
In another embodiment, the recombinant mammalian expression vector is
capable of directing expression of the nucleic acid preferentially in a
particular cell type
(e.g., tissue-specific regulatory elements are used to express the nucleic
acid). Tissue-
specific regulatory elements are known in the art. Non-limiting examples of
suitable
tissue-specific promoters include the albumin promoter (liver-specific;
Pinkert et al.
( 1987) Genes Dev. 1:268-277), lymphoid-specific promoters (Calame and Eaton (
1988)
Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto
and
Baltimore ( 1989) EMBO J. 8:729-733) and immunoglobulins (Banerji et al. (
1983) Cell
33:729-740; Queen and Baltimore ( 1983) Cell 33:741-748), neuron-specific
promoters
(e.g., the neurofilament promoter: Byrne and Ruddle ( 1989) PNAS 86:473-5477),
pancreas-specific promoters (Edlund et al. ( 1985) Science 230:912-916), and
mammary
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gland-specific promoters (e.g., milk whey promoter: U.S. Patent No. 4.873.316
and
European Application Publication No. 264,166). Developmentally-regulated
promoters
are also encompassed. for example the murine hox promoters (Kessel and Gruss (
1990)
Science 249:374-379) and the a-fetoprotein promoter (Campes and Tilghman (
1989)
Genes Dev. 3:537-546).
The invention further provides a recombinant expression vector
comprising a DNA molecule of the invention cloned into the expression vector
in an
antisense orientation. That is, the DNA molecule is operatively linked to a
regulatory
sequence in a manner which allows for expression (by transcription of the DNA
molecule) of an RNA molecule which is antisense to an mRNA corresponding to a
nucleic acid molecule of the present invention. Regulatory sequences
operatively linked
to a nucleic acid cloned in the antisense orientation can be chosen which
direct the
continuous expression of the antisense RNA molecule in a variety of cell
types, for
instance viral promoters andlor enhancers, or regulatory sequences can be
chosen which
direct constitutive, tissue specific or cell type specific expression of
antisense RNA.
The antisense expression vector can be in the form of a recombinant plasmid,
phagemid
or attenuated virus in which antisense nucleic acids are produced under the
control of a
high efficiency regulatory region, the activity of which can be determined by
the cell
type into which the vector is introduced. For a discussion of the regulation
of gene
expression using antisense genes see Weintraub, H. et al., Antisense RNA as a
molecular tool for genetic analysis, Reviews - Trends in Genetics, Vol. 1(1)
1986.
Another aspect of the invention pertains to host cells into which a
recombinant expression vector of the invention has been introduced. The terms
"host
cell" and "recombinant host cell" are used interchangeably herein. It is
understood that
such terms refer not only to the particular subject cell but to the progeny or
potential
progeny of such a cell. Because certain modifications may occur in succeeding
Qenerations due to either mutation or environmental influences, such progeny
may not,
in fact, be identical to the parent cell, but are still included within the
scope of the term
as used herein.
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A host cell can be any prokaryotic or eukaryotic cell. For example,
Immunomodulin protein can be expressed in bacterial cells such as E. coli,
insect cells.
yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS
cells).
Other suitable host cells are known to those skilled in the art.
Vector DNA can be introduced into prokaryotic or eukaryotic cells via
conventional transformation or transfection techniques. As used herein, the
terms
"transformation" and "transfection" are intended to refer to a variety of art-
recognized
techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell,
including
calcium phosphate or calcium chloride co-precipitation, DEAF-dextran-mediated
transfection, lipofection, or electroporation. Suitable methods for
transforming or
transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A
Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
For stable transfection of mammalian cells, it is known that, depending
upon the expression vector and transfection technique used, only a small
fraction of cells
may integrate the foreign DNA into their genome. In order to identify and
select these
integrants, a gene that encodes a selectable marker (e.g., resistance to
antibiotics) is
generally introduced into the host cells along with the gene of interest.
Preferred
selectable markers include those which confer resistance to drugs, such as
6418,
hygromycin and methotrexate. Nucleic acid encoding a selectable marker can be
introduced into a host cell on the same vector as that encoding recombinant
proteins or
can be introduced on a separate vector. Cells stably transfected with the
introduced
nucleic acid can be identified by drug selection (e.g., cells that have
incorporated the
selectable marker gene will survive, while the other cells die).
A host cell of the invention, such as a prokaryotic or eukaryotic host cell
in culture, can be used to produce (i.e., express) recombinant protein.
Accordingly, the
invention further provides methods for producing recombinant protein using the
host
cells of the invention. In one embodiment, the method comprises culturing the
host cell
of invention (into which a recombinant expression vector encoding recombinant
protein
has been introduced) in a suitable medium such that the recombinant protein is
produced. In another embodiment, the method further comprises isolating the
recombinant protein from the medium or the host cell.
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_;
C. Activating. Neutralizing and/or Blocking Antibodies
As described herein, preferred Eta-1 modulators are agents that are
capable of modulating select Eta-1/osteopontin-mediated activities, in
particular. select
activities associated with potentiation of a type-1 immune response.
Accordingly, in
one embodiment, the invention fea~ures methods of modulating an immune
response
which include administering an Eta-1 antibody, for example, an antibody which
specifically block or neutralizes the interaction of Eta-1/osteopontin with a
cell surface
receptor (e.g., CD44 and/or integrin av~3). In one embodiment. an antibody is
specific
for the N-terminal, e.g., IL-12 stimulatory domain of Eta-1/osteopontin, as
defined
herein. In another embodiment, the antibody is specific for the C-terminal,
e.g., IL-10
inhibitory domain if Eta-1/osteopontin. In yet another embodiment, the
antibody is
specific for the RGD sequence of Eta-1/osteopontin (e.g., the integrin binding
domain).
In a preferred embodiment, the antibody is LF 123, as described herein. Also
included
within the scope of the present invention are fragments of such antibodies,
e.g., Fab'
fragments, humanized antibodies, and the like, for use as therapeutic agents.
D. Peptides. Peptidomimetics and Small Molecule Modulators
The present invention also pertains to Eta-1/osteopontin peptides, Eta-
1/osteopontin peptidomimetics and or small molecule modulators of Eta-
1/osteopontin
which function as either Eta-1/osteopontin agonists (mimetics) or as Eta-
1/osteopontin
antagonists. An Eta-1/osteopontin agonists can retain substantially the same,
or a
subset, of the biological activities of the naturally occurring form of Eta-
1/osteopontin.
An Eta-1/osteopontin antagonist can inhibit one or more of the activities of
the naturally
occurring form of Eta-1/osteopontin. Thus, specific biological effects can be
elicited by
treatment with an Eta-1/osteopontin agonist or antagonist of limited function.
In one
embodiment, treatment of a subject with an Eta-1/osteopontin agonist or
antagonist
having a subset of the biological activities of the naturally occurring form
of the protein
has fewer side effects in a subject relative to treatment with the full length
Eta-
1/osteopontin.
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Eta-1/osteopontin agonist or antagonist can be identified by screening
libraries of Eta-1 peptides, combinatorial libraries based on Eta-1 peptides
or small
molecule libraries for Eta-1 agonist or antagonist activity. In one
embodiment, a
variegated library is generated by combinatorial mutagenesis at the Eta-
1/osteopontin
nucleic acid level and is encoded by a variegated gene library. Variegated
libraries of
compounds can be produced by, for example, enzymatically ligating a mixture of
synthetic oligonucleotides into gene sequences such that a degenerate set of
potential
Eta-1/osteopontin sequences is expressible as individual polypeptides, or
alternatively,
as a set of larger fusion proteins (e.g., for phage display) containing the
set of Eta-
1/osteopontin sequences therein. There are a variety of methods which can be
used to
produce libraries of potential Eta-1/osteopontin variants from a degenerate
oligonucleotide sequence. Chemical synthesis of a degenerate gene sequence can
be
performed in an automatic DNA synthesizer, and the synthetic gene then ligated
into an
appropriate expression vector. Use of a degenerate set of genes allows for the
provision,
in one mixture, of all of the sequences encoding the desired set of potential
Eta-
1/osteopontin sequences. Methods for synthesizing degenerate oligonucleotides
are
known in the art (see, e.g., Narang, S.A. (1983) Tetrahedron 39:3; Itakura et
al. (1984)
Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al.
(1983)
Nucleic Acid Res. 11:477.
In addition, libraries of fragments of Eta-1/osteopontin coding sequence
can be used to generate a variegated population of Eta-1/osteopontin fragments
for
screening and subsequent selection of variants of Eta-1/osteopontin. In one
embodiment, a library of coding sequence fragments can be generated by
treating a
double stranded PCR fragment of an Eta-1/osteopontin coding sequence with a
nuclease
under conditions wherein nicking occurs only about once per molecule,
denaturing the
double stranded DNA, renaturing the DNA to form double stranded DNA which can
include senselantisense pairs from different nicked products, removing single
stranded
portions from reformed duplexes by treatment with S 1 nuclease, and ligating
the
resulting fragment library into an expression vector. By this method, an
expression
library can be derived which encodes N-terminal. C-terminal and internal
fragments of
various sizes of the Eta-1/osteopontin protein.
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Additional exemplary methods for the synthesis of molecular libraries
can be found in the art, for example in: DeWitt et al. ( 1993 ) Proc. Natl.
Acad. Sci.
U.S.A. 90:6909: Erb et al. ( 1994) Proc. Natl. Acad. Sci. USA 91:11422;
Zuckermann et
al. ( 1994). J. Med. Chem. 37:2678: Cho et al. ( 1993) Science 261:1303;
Carrell et al.
( 1994) Angew. Chem. Int. Ed. Engl. 33:2059; Carell et al. ( 1994) Angew.
Chem. Int. Ed.
Engl. 33:2061; and in Gallop et al. ( 1994) J. Med. Chem. 37:1233.
Libraries of compounds may be presented in solution (e.g., Houghten
( 1992) Biotechniques 13:412-421 ), or on beads (Lam ( 1991 ) Nature 34:82-
84), chips
(Fodor ( 1993) Nature 364:555-556), bacteria (Ladner USP 5.223,409), spores
(Ladner
USP '409), plasmids (Cull et al. ( 1992) Proc Natl Acad Sci USA 89:1865-1869)
or on
phage (Scott and Smith ( 1990) Science 249:386-390); (Devlin ( 1990) Science
249:404-
406); (Cwirla et al. ( 1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici (
1991 ) J. Mol.
Biol. 222:301-310); (Ladner supra.).
Additional test compounds can be obtained using any of the numerous
approaches in combinatorial library methods known in the art, including:
biological
libraries; spatially addressable parallel solid phase or solution phase
libraries; synthetic
library methods requiring deconvolution; the 'one-bead one-compound' library
method;
and synthetic library methods using affinity chromatography selection. The
biological
library approach is limited to peptide libraries, while the other four
approaches are
applicable to peptide, non-peptide oligomer or small molecule libraries of
compounds
(Lam. K.S. ( 1997) Anticancer Drug Des. 12:145).
A. Screening_Assays:
Several techniques are known in the art for screening libraries (e.g.,
combinatorial libraries or small molecule libraries) for compounds having a
selected
property. Such techniques are preferably adaptable for rapid screening of the
libraries
described herein. Particularly preferred techniques are those which are
amenable to high
through-put analysis.
For example, candidate or test compounds can be screened for their
ability to modulate the interaction of Eta-1/osteopontin with a CD44 or
intearin
receptor. In one embodiment, an assay-fs a cell-based assay in which a cell
which
expresses CD44 or integrin on the cell surface is contacted with a test
compound.
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optionally in the presence of Eta-1/osteopontin, and the ability of the test
compound to
modulate the interaction of Eta-1/osteopontin with a CD44 or integrin receptor
is
determined. The cell can be of mammalian origin, for example. a macrophage.
Determining the ability of the test compound to modulate the interaction of
Eta-
1/osteopontin with a CD44 or integrin receptor can be accomplished, for
example, by
coupling the test compound (or Eta-1/osteopontin) with a radioisotope or
enzymatic
label such that binding to CD44 or integrin can be determined by detecting the
labeled
compound in a complex. For example, test compounds can be labeled with 12 5I,
3 5 S.
14C, or 3H, either directly or indirectly, and the radioisotope detected by
direct
counting of radioemmission or by scintillation counting. Alternatively,
reagents can be
enzymatically labeled with, for example, horseradish peroxidase, alkaline
phosphatase,
or luciferase, and the enzymatic label detected by determination of conversion
of an
appropriate substrate to product.
The ability of a test compound to modulate the interaction of Eta-
1/osteopontin with a CD44 or integrin receptor can also be determined without
the
labeling of any of the interactants. For example, a microphysiometer can be
used to
detect the interaction of components without the labeling of either the test
compound or
the receptor. McConnell, H. M. et al. (1992) Science 257:1906-1912.
Determining the
ability to modulate the interaction of Eta-1/osteopontin with a CD44 or
integrin receptor
can also be accomplished by determining, for example, induction of a cellular
second
messenger (i.e. intracellular Ca2+, diacylglycerol, IP3, etc.), detecting the
induction of a
reporter gene, or detecting a cellular response, for example, a proliferative
response or
an inflammatory response.
In yet another embodiment, an assay of the present invention is a cell-free
assay in which an Eta-1/osteopontin protein or biologically active portion
thereof is
contacted with a test compound and the ability of the test compound to bind to
the Eta-
1/osteopontin protein or biologically active portion thereof is determined.
Binding of the
test compound to the Eta-1/osteopontin protein can be determined either
directly or
indirectly as described above. Binding of the test compound to the Eta-
1/osteopontin
protein can also be accomplished using a technology such as real-time
Biomolecular
Interaction Analysis (BIA). Sjolander,_S. and Urbaniczky, C. ( 1991 ) Anal.
Chem.
63:2338-2345 and Szabo et al. ( 1995) Curr. Opin. Struct. Biol. x:699-705. As
used
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herein, "BIA" is a technology for studying biospecific interactions in real
time. without
labeling any of the interactants (e.g., BIAcoreT""). Changes in the optical
phenomenon of
surface plasmon resonance (SPR) can be used as an indication of real-time
reactions
between biological molecules.
In a preferred embodiment, the assay includes contacting the Eta-1/osteopontin
protein or biologically active portion thereof with a known ligand which binds
Eta-
1/osteopontin to form an assay mixture, contacting the assay mixture with a
test
compound, and determining the ability of the test compound to interact with an
Eta-
1/osteopontin protein, wherein determining the ability of the test compound to
interact
with an Eta-1/osteopontin protein comprises determining the ability of the
test
compound to preferentially bind to Eta-1/osteopontin or biologically active
portion
thereof as compared to the known ligand.
In another embodiment, the assay is a cell-free assay in which a CD44
receptor or integrin receptor is contacted with a test compound (and
optionally with Eta-
1/osteopontin) and the ability of the test compound to modulate (e.g.,
stimulate or
inhibit) the resulting interaction is determined. Cell-free assays of the
present invention
are amenable to use of both soluble and/or membrane-bound forms of receptors.
In the
case of cell-free assays in which a receptor is used it may be desirable to
utilize a
solubilizing agent such that the membrane-bound form of the isolated protein
is
maintained in solution. Examples of such solubilizing agents include non-ionic
detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside.
octanoyl-
N-methylglucamide, decanoyl-N-methylglucamide, Triton~ X-100, Triton~ X-114,
Thesit~, Isotridecypoly(ethylene glycol ether)n, 3-[(3-
cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS), 3-[(3-
cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate (CHAPSO), or N-
dodecyl=N,N-dimethyl-3-ammonio-1-propane sulfonate.
In more than one of the above assay methods, it may be desirable to immobilize
at least
one assay reagent to facilitate separation of complexed from uncomplexed forms
of one
or both of the reagents, as well as to accommodate automation of the assay,
for example,
glutathione-S-transferase/ fusion proteins can be adsorbed onto glutathione
sepharose
beads (Sigma Chemical. St. Louis, MO) or glutathione derivatized microtitre
plates.
Alternatively at least one reagent can be immobilized utilizing conjugation of
biotin and
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streptavidin. Alternatively, antibodies reactive at least one reagent can be
derivatized to
wells or plates to immobilize reagents.
Novel agents identified by the above-described screening assays can be
tested in an appropriate animal model, for example, to determine the efficacy,
toxicity.
or side effects of treatment with such an agent. Alternatively, agent can be
tested in at
least one of the in vitro or in situ assays described herein.
II. Biosynthetic Immunomodulatory Molecules
Based on the discovery of an immunomodulatory function of Eta-
Ilosteopontin, and in particular, the discovery of the IL-12 stimulatory and
IL-10 inhibitory
domains of Eta-1/osteopontin, the present invention features biosynthetic
molecules which
are modeled after these key functional domains. The biosynthetic molecules are
useful in
regulating a variety of cellular processes as well as in modulating immune
responses. In
particular, the biosynthetic immunomodulatory molecules are useful in biasing
an immune
response from a type-1 to a type-2 immune response.
As used herein, the term "biosynthetic molecule" includes molecules
which have a biological activity and which are built or synthesized by the
combination
or union of components or elements that are simpler than the biosynthesized
molecule.
A biosynthetic molecule of the present invention is made or built by the hand
of man
(including automated processes) and accordingly, is distinguishable from a
naturally-
occurring molecule which is results from a naturally-occurring biological
process.
Alternatively, an organism can be used to produce a biosynthetic molecule of
the present
invention, provided that at least at one step in the synthesis, there is the
intervention of
man.
Accordingly, in one embodiment, the present invention features
biosynthetic immunomodulatory molecules which include an IL-12 stimulatory
component
and a biomodular component, forming a molecule which modulates an immune
response.
The term "IL-12 stimulatory component" includes a piece or constituent of a
molecule
(e.g., a fragment of Eta-1/osteopontin) which is smaller than the molecule of
which it is
a part, which functions to stimulate, enhance, upregulate the expression,
production
and/or secretion of the cytokine, IL-12,_from a cell. A molecule which
includes an IL-
12 stimulatory component, for example, is capable of causing a cell capable of
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expressing, producing and/or secreting IL-12._to express and/or secrete more
of the
cytokine in the presence of the IL-12 stimulatory component than in the
absence of the
IL-12 stimulatory component.
In another embodiment, the present invention features biosynthetic
immunomodulatory molecules which include an IL-10 inhibitory component and a
biomodular component, forming a molecule which modulates an immune response.
The
term "IL-10 inhibitory component" includes a piece or constituent of a
molecule which
is smaller than the molecule of which it is a part, which functions to inhibit
the
expression and/or secretion of the cytokine, IL-10, from a cell. A molecule
which
includes an IL-10 inhibitory component, for example, is capable of causing a
cell
capable of expressing and/or secreting IL-10, to express and/or secrete less
of the
cytokine in the presence of the IL-10 inhibitory component than in the absence
of the
IL-10 inhibitory component.
In addition to the IL-12 stimulatory component or the IL-10 inhibitory
component defined herein, the biosynthetic immunomodulatory molecules of the
present
invention can include a biomodular component. The term "biomodular component"
includes a piece or constituent of a molecule which is smaller than the
molecule of
which it is a part, which has either a biological function which is distinct
from that of the
IL-12 stimulatory component, the IL-10 inhibitory component or has a
biological
structure which is distinct from that of the IL-12 stimulatory component or
the IL-10
inhibitory component. A biomodular component is a piece or constituent that
either is
not found in a naturally-occurring molecule which includes an IL-12
stimulatory
component or an IL.-10 inhibitory component (e.g., Eta-1/osteopontin) or is
not found in
the same proximal relation to an IL-12 stimulatory component or an IL-10
inhibitory
component as it exists within a naturally-occurring molecule. In one
embodiment, a
biomodular component is a polypeptide. Polypeptide biomodular components of
the
present invention include, but are not limited to signal peptides, a
calcium/apatite
binding domains and a heparin binding domains.
The term "signal peptide" or "signal sequence" refers to a peptide
containing about 20 amino acids which occurs at the N-terminus of secretory
and
integral membrane proteins and which contains a large number of hydrophobic
amino
acid residues. For example, a signal sequence contains at least about 14-28
amino acid
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_,
residues, preferably about 16-26 amino acid residues, more preferably about 18-
24
amino acid residues, and more preferably about 20-22 amino acid residues, and
has at
least about 40-70%. preferably about 50-6~%, and more preferably about ~5-60%
hydrophobic amino acid residues (e.g., Alanine, Valine, Leucine. Isoleucine,
Phenylalanine, Tyrosine, Tryptophan, or Proline). Such a "signal sequence",
also
referred to in the art as a "signal peptide", serves to direct a protein
containing such a
sequence from the endoplasmic reticulum of a cell to the golgi apparatus and
ultimately
to a lipid bilayer (e.g., for secretion).
The term "calcium/apatite binding domain" includes a domain which,
when included within a protein, polypeptide, or biosynthetic molecule of the
present
invention, functions to bind calcium, bind metal ions, or bind apatite (e.g.,
hydroxyapatite). A "calcium/apatite binding domain" can also be referred to as
a "6-
Asp" domain. Also preferred is a 6-His domain. 6-Asp and 6-His domains are
particularly useful for purification of the biosynthetic molecules of the
preset invention.
The term "heparin binding domain" includes a domain which, when
included within a protein, polypeptide, or biosynthetic molecule of the
present
invention, functions to bind the protein, polypeptide, or biosynthetic
molecule to
heparin. A "heparin binding domain" further includes a domain which has within
it at
least one, preferably two, three, four, five, six, or more "heparin binding
domain
minimum repeating units". The term "heparin binding domain minimum repeating
unit"
includes the consensus motif basic residue - basic residue - any residue -
basic residue.
Preferably, a "heparin binding domain minimum repeating unit" has the sequence
arginine - arginine - any residue - arginine. Also preferred are collagen
binding
domains. Heparin binding domains and/or collagen binding domains are
particularly
useful for stabilizing the biosynthetic molecules of the present invention,
e.g., for
anchoring or adhering the molecules to ECM surrounding target cells of the
invention.
Accordingly, a biosynthetic immunomodulatory molecule of the present
invention is formed by the combination of at least an IL-12 stimulatory
component or an
IL-10 inhibitory domain and a biomodular component. The term ''formed" or
"forming"
includes the bringing together of at least two components into a structural
and/or
functional association. For example, a recombinant nucleic acid molecule can
be
formed by the bringing together of at least two nucleic acid components.
Alternatively,
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a recombinant protein can be formed by the bringing together of at least two
protein
components. Moreover. a composition can be formed by the bringing together of
at
least two compositions.
In a preferred embodiment, the present invention features biosynthetic
molecules which include an IL-10 inhibitory component which is derived from
Eta-
1/osteopontin. A component "derived from''. for example, Eta-1/osteopontin,
includes a
component which has certain features which originate from Eta-1/osteopontin
and are
recognizable as such, but which is not identical to Eta-1/osteopontin. In one
embodiment, an IL-10 inhibitory component is a polypeptide which is derived
from Eta-
1/osteopontin. Accordingly, the IL-10 inhibitory component has features of Eta-
1/osteopontin (e.g., functions to inhibit IL-10 secretion) but is not
identical to
osteopontin. In one embodiment, an IL-10 inhibitory component includes a
polypeptide
which has at least 50% identity to an IL-10 inhibitory domain of Eta-
1/osteopontin. In
yet another embodiment, an IL-10 inhibitory component is at least 55%, 65%,
70%,
75%, 80%. 85%, 90%, 95%, or more identical to an IL.-10 inhibitory domain of
Eta-
1/osteopontin IL-10. In yet another embodiment, an IL-10 inhibitory component
includes a polypeptide which has at least 80-85%, 85-90%, 90-95%, 96%, 97%,
98%,
99% or more identity to about amino acids 169-266 of SEQ ID N0:2 In another
embodiment, an IL-10 inhibitory component includes a polypeptide which is at
least 65-
160 amino acids in length. In another embodiment, an IL-10 inhibitory
component
includes a polypeptide which is between 30-35, 40-45, 45-50, 50-55, 55-60, 60-
65, 65-
70, 70-75, 75-80, 80-85, 85-90 90-100 or more amino acids in length. In
another
embodiment, an IL-10 inhibitory component includes a polypeptide which is
greater
than 115 amino acids in length.
Another embodiment of the present invention features biosynthetic
molecules which include an IL-10 inhibitory component having an amino acid
sequence
sufficiently homologous to the an IL-10 inhibitory domain of a protein having
the amino
acid sequence of Eta-1/osteopontin (e.g., SEQ ID N0:2), as defined herein. In
a
preferred embodiment, an IL-10 inhibitory component retains an IL-10
inhibitory,
preferably an IL-10 inhibitory activity of Eta-1/osteopontin. In another
embodiment. a
molecule has an immunomodulatory activity. In another embodiment, an IL-10
inhibitory component includes an amino acid sequence selected from the group
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consisting of amino acids 71-180 of SEQ ID N0:4, amino acids 58-166 of SEQ ID
N0:6, or amino acids 44-153 of SEQ ID N0:8.
The present invention further features isolated nucleic acid molecules which
encode the biosynthetic immunomodulatory molecules of the present invention.
In one
embodiment, an isolated nucleic acid molecule of the present invention
includes a nucleic
acid sequence which encodes an IL-10 inhibitory domain. In another embodiment,
an
isolated nucleic acid molecule of the present invention includes a nucleic
acid sequence
which encodes a biomodulatory domain. In another embodiment, an isolated
nucleic acid
molecule of the present invention includes a nucleic acid sequence (SEQ ID
N0:9) which
encodes Immunomodulin-1 (SEQ ID NO:10).
A. Isolated Biosynthetic Molecules
"Isolated" or "purified" biosynthetic molecules are also features
according to the present invention. "Isolated" or "purified" biosynthetic
molecules are
substantially free of cellular material or other contaminating proteins from
the cell or
tissue source from which the molecule is derived, or substantially free from
chemical
precursors or other chemicals when chemically synthesized. The phrases
"substantially
free of cellular material" and "substantially free of chemical precursors or
other
chemicals" are as defined herein for isolated Eta-1/proteins or polypeptides.
Also featured are isolated nucleic acid molecules encoding the
biosynthetic molecules of the present invention, vectors including such
nucleic acid
molecules, as well as host cells into which such vectors have been
incorporated, as
defined herein. Also featured are methods of making the biosynthetic molecules
of the
present invention, as described herein for making Eta-1/osteopontin proteins
or
polypeptides.
Biologically active portions of a biosynthetic molecules of the present
invention are also featured and include molecules sufficiently homologous to
or derived
from the biosynthetic molecules of the present invention which include less
amino acids
than the full biosynthetic molecules, and exhibit at least one activity of the
biosynthetic
molecules. Typically, biologically active portions at least one domain or
motif with at
least one activity of the biosynthetic molecules. A biologically active
portion can be a
polypeptide which is, for example, 10. 25, 50, 100 or more amino acids in
length.
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III. Pharmaceutical Compositions
The nucleic acid molecules, proteins, and biosynthetic molecules (also
referred to herein as "active compounds") of the invention can be incorporated
into
pharmaceutical compositions suitable for administration. Such compositions
typically
comprise the nucleic acid molecule, protein, or antibody and a
pharmaceutically
acceptable carrier. As used herein the language "pharmaceutically acceptable
carrier" is
intended to include any and all solvents, dispersion media, coatings,
antibacterial and
antifungal agents, isotonic and absorption delaying agents, and the like,
compatible with
pharmaceutical administration. The use of such media and agents for
pharmaceutically
active substances is well known in the art. Except insofar as any conventional
media or
agent is incompatible with the active compound, use thereof in the
compositions is
contemplated. Supplementary active compounds can also be incorporated into the
compositions.
A pharmaceutical composition of the invention is formulated to be
compatible with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral
(e.g., inhalation), transdermal (topical), transmucosal, and rectal
administration.
Solutions or suspensions used for parenteral, intradermal, or subcutaneous
application
can include the following components: a sterile diluent such as water for
injection,
saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol
or other
synthetic solvents; antibacterial agents such as benzyl alcohol or methyl
parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such
as
ethylenediaminetetraacetic acid; buffers such as acetates, citrates or
phosphates and
agents for the adjustment of tonicity such as sodium chloride or dextrose. pH
can be
adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
The
parenteral preparation can be enclosed in ampoules, disposable syringes or
multiple dose
vials made of glass or plastic.
Pharmaceutical compositions suitable for injectable use include sterile
aqueous solutions (where water soluble) or dispersions and sterile powders for
the
extemporaneous preparation of sterile injectable solutions or dispersion. For
intravenous administration, suitable carriers include physiological saline,
bacteriostatic
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water. Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline
(PBS).
In all cases, the composition must be sterile and should be fluid to the
extent that easy
syringability exists. It must be stable under the conditions of manufacture
and storage
and must be preserved against the contaminating action of microorganisms such
as
bacteria and fungi. The carrier can be a solvent or dispersion medium
containing, for
example, water, ethanol, polyol (for example, glycerol, propylene glycol, and
liquid
polyetheylene glycol, and the like), and suitable mixtures thereof. The proper
fluidity
can be maintained, for example, by the use of a coating such as lecithin, by
the
maintenance of the required particle size in the case of dispersion and by the
use of
surfactants. Prevention of the action of microorganisms can be achieved by
various
antibacterial and antifungal agents, for example, parabens, chlorobutanol,
phenol,
ascorbic acid, thimerosal, and the like. In many cases, it will be preferable
to include
isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol,
sodium
chloride in the composition. Prolonged absorption of the injectable
compositions can be
brought about by including in the composition an agent which delays
absorption, for
example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by incorporating the active
compound (e.g., a Immunomodulin protein or anti-Immunomodulin antibody) in the
required amount in an appropriate solvent with one or a combination of
ingredients
enumerated above, as required, followed by filtered sterilization. Generally,
dispersions
are prepared by incorporating the active compound into a sterile vehicle which
contains
a basic dispersion medium and the required other ingredients from those
enumerated
above. In the case of sterile powders for the preparation of sterile
injectable solutions,
the preferred methods of preparation are vacuum drying and freeze-drying which
yields
a powder of the active ingredient plus any additional desired ingredient from
a
previously sterile-filtered solution thereof.
Oral compositions generally include an inert diluent or an edible carrier.
They can be enclosed in gelatin capsules or compressed into tablets. For the
purpose of
oral therapeutic administration, the active compound can be incorporated with
excipients
and used in the form of tablets, troches. or capsules. Oral compositions can
also be
prepared using a fluid carrier for use as a mouthwash, wherein the compound in
the fluid
carrier is applied orally and swished and expectorated or swallowed.
Pharmaceutically
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compatible binding agents. and/or adjuvant materials can be included as part
of the
composition. The tablets, pills. capsules, troches and the like can contain
anv of the
following ingredients. or compounds of a similar nature: a binder such as
microcrystalline cellulose, gum tragacanth or gelatin: an excipient such as
starch or
lactose, a disintegrating agent such as alginic acid, Primogel, or corn
starch: a lubricant
such as magnesium stearate or Sterotes; a glidant such as colloidal silicon
dioxide; a
sweetening agent such as sucrose or saccharin; or a flavoring agent such as
peppermint,
methyl salicylate, or orange flavoring.
For administration by inhalation, the compounds are delivered in the
form of an aerosol spray from pressured container or dispenser which contains
a suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Systemic administration can also be by transmucosal or transdermal
means. For transmucosal or transdermal administration, penetrants appropriate
to the
barrier to be permeated are used in the formulation. Such penetrants are
generally
known in the art, and include, for example, for transmucosal administration,
detergents,
bile salts, and fusidic acid derivatives. Transmucosal administration can be
accomplished through the use of nasal sprays or suppositories. For transdermal
administration, the active compounds are formulated into ointments, salves,
gels, or
creams as generally known in the art.
The compounds can also be prepared in the form of suppositories (e.g.,
with conventional suppository bases such as cocoa butter and other glycerides)
or
retention enemas for rectal delivery.
In one embodiment, the active compounds are prepared with carriers that
will protect the compound against rapid elimination from the body, such as a
controlled
release formulation, including implants and microencapsulated delivery
systems.
Biodegradable, biocompatible polymers can be used, such as ethylene vinyl
acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic
acid.
Methods for preparation of such formulations will be apparent to those skilled
in the art.
The materials can also be obtained commercially from Alza Corporation and Nova
Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to
infected
cells with monoclonal antibodies to viral antigens j can also be used as
pharmaceutically
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acceptable carriers. These can be prepared according to methods known to those
skilled
in the art, for example, as described in U.S. Patent No. 4,522,811.
It is especially advantageous to formulate oral or parenteral compositions
in dosage unit form for ease of administration and uniformity of dosage.
Dosage unit
form as used herein refers to physically discrete units suited as unitary
dosages for the
subject to be treated; each unit containing a predetermined quantity of active
compound
calculated to produce the desired therapeutic effect in association with the
required
pharmaceutical carrier. The specification for the dosage unit forms of the
invention are
dictated by and directly dependent on the unique characteristics of the active
compound
and the particular therapeutic effect to be achieved, and the limitations
inherent in the art
of compounding such an active compound for the treatment of individuals.
Toxicity and therapeutic efficacy of such compounds can be determined
by standard pharmaceutical procedures in cell cultures or experimental
animals, e.g., for
determining the LD50 (the dose lethal to 50% of the population) and the ED50
(the dose
therapeutically effective in 50% of the population). The dose ratio between
toxic and
therapeutic effects is the therapeutic index and it can be expressed as the
ratio
LD50/ED50. Compounds which exhibit large therapeutic indices are preferred.
While
compounds that exhibit toxic side effects may be used, care should be taken to
design a
delivery system that targets such compounds to the site of affected tissue in
order to
minimize potential damage to uninfected cells and, thereby, reduce side
effects.
The data obtained from the cell culture assays and animal studies can be
used in formulating a range of dosage for use in humans. The dosage of such
compounds lies preferably within a range of circulating concentrations that
include the
ED50 with little or no toxicity. The dosage may vary within this range
depending upon
the dosage form employed and the route of administration utilized. For any
compound
used in the method of the invention, a "therapeutically effective" dose can be
estimated
initially from cell culture assays. A "therapeutically effective" dose can be
further
formulated in animal models to achieve a circulating plasma concentration
range that
includes the IC50 (i.e., the concentration of the test compound which achieves
a half-
maximal inhibition of symptoms) as determined in cell culture. Such
information can be
used to more accurately determine useful doses in humans. Levels in plasma may
be
measured, for example, by high performance liquid chromatography.
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The nucleic acid molecules of the invention can be inserted into vectors
and used as gene therapy vectors. Gene therapy vectors can be delivered to a
subject by,
for example, intravenous injection, local administration (see U.S. Patent
x,328,470) or
by stereotactic injection (see e.g., Chen et al. ( 1994) PNAS 91:3054-3057).
The
pharmaceutical preparation of the gene therapy vector can include the gene
therapy
vector in an acceptable diluent, or can comprise a slow release matrix in
which the gene
delivery vehicle is imbedded. Alternatively, where the complete gene delivery
vector
can be produced intact from recombinant cells, e.g., retroviral vectors, the
pharmaceutical preparation can include one or more cells which produce the
gene
delivery system.
The pharmaceutical compositions can be included in a container, pack, or
dispenser together with instructions for administration.
IV. Uses and Methods Featuring-B'~osynthetic Immunomodulatory Molecules of the
Present invention
A. Assays
The ability of an stimulatory or inhibitory agent of the invention (e.g., an
Eta-1/osteopontin modulator or a biosynthetic immunomodulatory molecule) to
modulate an immune response (e.g., to bias an immune response from a type-1 to
a type-
2 immune response or from a type-2 to a type-1 immune response) can be
evaluated
using an in vitro culture system such as that described herein in the
Examples. For
example, expression, production or secretion of a cytokine can be determined
(e.g., of a
type-1 or a type-2 cytokine) or a cytokine profile can be determined (e.g., a
type-1 or a
type-2 cytokine profile). Immune effector cells (e.g., peripheral blood
mononuclear
cells) can be cultured in the presence of an stimulatory or inhibitory agent
of the
invention as described in the examples in a medium suitable for culture of the
chosen
cells. In the case of assaying for the ability of an inhibitory agent of the
invention to
modulate an immune response (e.g., an Eta-1/osteopontin inhibitory modulator
or an IL-
10 component-containing biosynthetic immunomodulatory molecule) it may be
necessary to also stimulate cells with a known stimulatory agent. After a
period of time
(e.g., 24-72 hours), production of cytokine(s) (e.g., at least one type-1
cytokine, at least
one type-2 cytokine, a type-1 cvtokine profile or a type-2 cvtokine profile)
is assessed
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by determining the level of the cytokine in the_culture supernatant as
described in the
examples. The ability of a stimulatory agent to stimulate cytokine production
is
evidenced by a higher level of cytokine in the supernatants of cells cultured
in the
presence of the stimulatory agent compared to the level of cytokine in the
supernatant of
cells cultured alone or in the presence of a control. The ability of an
inhibitory agent to
inhibit cytokine production is evidenced by a lower level of cytokine in the
supernatants
of cells cultured in the presence of both the inhibitory agent and the
stimulatory agent
compared to the level of cytokine in the supernatant of cells cultured only in
the
presence of the stimulatory agent.
B. Therapeutic Uses
The present invention provides for both prophylactic and therapeutic
methods of treating subjects (e.g., human subjects). In one aspect, the
invention
provides a method for preventing or treating a disease or a disorder in a
subject
prophylactically or therapeutically. Administration of a agent
prophylactically can
occur prior to the manifestation of symptoms of an undesired disease or
disorder, such
that the disease or disorder is prevented or, alternatively, delayed in its
progression. The
prophylactic methods of the present invention can be carned out in a similar
manner to
therapeutic methods described herein, although dosage and treatment regimes
may
differ.
Another aspect of the invention pertains to methods for treating a subject
therapeutically. In one embodiment, the present invention includes methods of
modulating an immune response. In particular, modulation of an immune response
includes, but is not limited to, modulation of cellular toxicity, modulation
of cytokine
expression, production or secretion (e.g., enhancement or inhibition of
cytokine
expression, production or secretion). A preferred embodiment of the invention
involves
modulation of IL-12, in particular, stimulation of IL-12 using an Eta-
1/osteopontin
stimulatory modulator or, alternatively, inhibition of IL-12 using an Eta-
1/osteopontin
inhibitory modulator. Another preferred embodiment of the invention involves
modulation of IL-10. in particular, inhibition of IL-10 using an Eta-
1/osteopontin
stimulatory modulator or, alternatively, stimulation of IL-10 using an Eta-
1/osteopontin
inhibitory modulator. Accordingly, the present method has therapeutic utility
in biasing
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an immune response towards, or away from, a_type-1 immune response depending
upon
the desired therapeutic regimen. Such modulatory methods are particularly
useful in
diseases such as cancer, in immunology, for example. in allergy, organ
transplantation
and organ rejection. Moreover, the immunomodulatory methods of the present
invention
can be used to treat an immunocompromized individual to enhance immunity. Uses
to
increase resistance to viral infection and enhance the rejection of foreign
molecules are
also within the scope of the present invention. The immunomodulatory methods
of the
present invention are further useful in wound healing. For example, an
enhancement of
type-1 immunity in a burn victim, or alternatively, at the burn or wound site.
can result
in a more rapid immune response, thus preventing infection. The
immunomodulatory
methods of the present invention are further useful in treating asthma. These
various
immunomodulatory therapeutic applications are described in further detail in
the
following subsection.
V. Clinical Applications of the Modulator~Methods of the Invention
The identification of Eta-1/osteopontin as a critical regulator of type 1
immunity
allows for selective manipulation of T cell subsets in a variety of clinical
situations
using the modulatory methods of the invention. The stimulatory methods of the
invention (i.e., methods that use an Eta-1/osteopontin stimulatory agent)
upregulate the
production of the Thl-associated cytokine IL-12 andlor dowregulate the
production of
the Th2-associated cytokine IL-10, with concomitant promotion of a type 1
immune
responses and downregulation of type 2 immune responses. In contrast, the
inhibitory
methods of the invention (i.e., methods that use an Eta-1/osteopontin
inhibitory agent)
downregulate the production of the Thl-associated cytokine IL-12 and/or
upregulate the
production of the Th2-associated cytokine IL-10, with concomitant
downregulation of a
type 1 immune responses and promotion of type 2 immune responses.
Thus, to treat a disease condition wherein a type 1 immune response is
beneficial, a stimulatory method of the invention is selected such that type 1
immune
responses are promoted while downregulating type 2 immune responses.
Alternatively,
to treat a disease condition wherein a type 2 immune response is beneficial.
an inhibitory
method of the invention is selected such_that type 1 immune responses are
downregulated while promoting type 2 immune responses. Application of the
methods
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of the invention to the treatment of disease conditions may result in cure of
the
condition, a decrease in the type or number of symptoms associated with the
condition.
either in the long term or short term (i.e., amelioration of the condition) or
simply a
transient beneficial effect to the subject.
Numerous disease conditions associated with a predominant type 1 or type
immune responses have been identified and could benefit from modulation of the
type of
response mounted in the individual suffering from the disease condition.
Application of
the immunomodulatory methods of the invention to such diseases is described in
further
detail below.
A. Allergies
Allergies are mediated through IgE antibodies whose production is
regulated by the activity of Th2 cells and the cytokines produced thereby. In
allergic
reactions, IL-4 is produced by Th2 cells, which further stimulates production
of IgE
antibodies and activation of cells that mediate allergic reactions, i.e., mast
cells and
basophils. IL-4 also plays an important role in eosinophil mediated
inflammatory
reactions. Accordingly, the Eta-1/osteopontin stimulatory methods of the
invention,
which promote type 1 responses and inhibit type 2 responses, can be used to
inhibit the
type 2 responses (e.g., production of Th2-associated cytokines) in allergic
patients as a
means to downregulate production of pathogenic IgE antibodies. A stimulatory
agent
may be directly administered to the subject or cells (e.g., Th0 cells or Thl
cells) may be
obtained from the subject, contacted with a stimulatory agent ex vivo, and
readministered to the subject. Moreover, in certain situations it may be
beneficial to
coadminister to the subject the allergen together with the stimulatory agent
or cells
treated with the stimulatory agent to inhibit (e.g., desensitize) the allergen-
specific type
2 response. The treatment may be further enhanced by administering other Thl-
promoting agents, such as the cytokine IL-12 or antibodies to Th2-associated
cytokines
(e.g., anti-IL-4 or anti-IL-10 antibodies), to the allergic subject in amounts
sufficient to
further stimulate a type 1 immune response.
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B. Cancer
The expression of Th2-promoting cytokines has been reported to be
elevated in cancer patients (see e.g.. Yamamura, M., et al. ( 1993) J. Clin.
Invest.
91:1005-1010: Pisa, P., et al. ( 1992) Proc. Natl. Acad. Sci. USA 89:7708-
7712) and
malignant disease is often associated with a shift from Th 1 type responses to
Th2 type
responses along with a worsening of the course of the disease. Accordingly,
the
stimulatory methods of the invention can be used to promote type 1 responses
and
inhibit type 2 responses (e.g., the production of Th2-associated cytokines) in
cancer
patients, as a means to counteract the Thl to Th2 shift and thereby promote an
ongoing
Thl response in the patients to ameliorate the course of the disease. The
stimulatory
methods can involve either direct administration of a stimulatory agent to a
subject with
cancer or ex vivo treatment of cells obtained from the subject (e.g., Th0 or
Thl cells)
with a stimulatory agent followed by readministration of the cells to the
subject. The
treatment may be further enhanced by administering other Thl-promoting agents,
such
as the cytokine IL-12 or antibodies to Th2-associated cytokines (e.g., anti-IL-
4 or anti-
IL-10 antibodies), to the recipient in amounts sufficient to further stimulate
a Thl-type
response.
C. Infectious Diseases (e.g., Bacterial or Viral)
The expression of Th2-promoting cytokines also has been reported to
increase during a variety of infectious diseases (including viral and
bacterial infectious
diseases), including HIV infection, tuberculosis, leishmaniasis,
schistosomiasis, filarial
nematode infection and intestinal nematode infection (see e. g.; Shearer, G.M.
and
Clerici, M. ( 1992) Prog. Chem. Immunol. 54:21-43; Clerici, M and Shearer,
G.M.
(1993) Immunology Today 14:107-111; Fauci, A.S. (1988) Science 239:617-623;
Locksley, _R. M. and Scott, P. (1992) Immunoparasitology Today 1:A58-A61;
Pearce,
E.J., et al. ( 1991 ) J. Exp. Med. 173:159-166; Grzych, J-M., et al. ( 1991 )
J. Immunol.
141:1322-1327; Kullberg, M.C., et al. (1992) J. Immunol. 148:3264-3270;
Bancroft,
A.J., et al. (1993) J. Immunol. 150:1395-1402; Pearlman, E., et al. (1993)
Infect. Immun.
61:1105-1112; Else. K.J., et al. ( 1994) J. Exp. Med. 179:347-351 ) and such
infectious
diseases are also associated with a Th 1 to Th2 shift in the immune response.
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Accordingly, the stimulatory methods_of the invention can be used in
infectious
diseases (caused by bacterial, viral or other pathogenic origins) to promote a
type 1
response and inhibit a type 2 response ie.~.. the production of Th?-associated
cytokines)
in subjects with infectious diseases, as a means to counteract the Thl to Th2
shift and
thereby promote an ongoing Th 1 response in the patients to ameliorate the
course of the
infection. The stimulatory method can involve either direct administration of
a
stimulatory agent to a subject with an infectious disease or ex vivo treatment
of cells
obtained from the subject (e.g., Th0 or Thl cells) with a stimulatory agent
followed by
readministration of the cells to the subject. The treatment may be further
enhanced by
administering other Thl-promoting agents. such as the cytokine IL-12 or
antibodies to
Th2-associated cytokines (e.g., anti-IL-4 or anti-IL-10 antibodies), to the
recipient in
amounts sufficient to further stimulate a Thl-type response.
D. Autoimmune Diseases
The Eta-1/osteopontin inhibitory methods of the invention can be used
therapeutically in the treatment of autoitnmune diseases that are associated
with a Th2-
type dysfunction. Many autoimmune disorders are the result of inappropriate
activation
of T cells that are reactive against self tissue and that promote the
production of
cytokines and autoantibodies involved in the pathology of the diseases.
Modulation of T
helper-type responses can have an effect on the course of the autoimmune
disease. For
example, in experimental allergic encephalomyelitis (EAE), stimulation of a
Th2-type
response by administration of IL-4 at the time of the induction of the disease
diminishes
the intensity of the autoimmune disease (Paul, W.E., et al. ( 1994) Cell
76:241-251 ).
Furthermore, recovery of the animals from the disease has been shown to be
associated
with an increase in a Th2-type response as evidenced by an increase of Th2-
specific
cytokines (Koury, S. J., et al. ( 1992) J. Exp. Med. 176_ 1355-1364).
Moreover, T cells
that can suppress EAE secrete Th2-specific cytokines (Chen, C., et al. ( 1994)
Immunity
1:147-154). Since stimulation of a Th2-type response in EAE has a protective
effect
against the disease, stimulation of a Th2 response in subjects with multiple
sclerosis (for
which EAE is a model) is likely to be beneficial therapeutically.
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Similarly, stimulation of a Th2-type response in type I diabetes in mice
provides a protective effect against the disease. Indeed. treatment of NOD
mice with
IL-4 (which promotes a Th2 response) prevents or delays onset of type I
diabetes that
normally develops in these mice (Rapoport. M.J., et al. (1993) J. Exp. Med.
178:87-99).
Thus, stimulation of a Th2 response in a subject suffering from or susceptible
to diabetes
may ameliorate the effects of the disease or inhibit the onset of the disease.
Yet another autoimmune disease in which stimulation of a Th2-type
response may be beneficial is rheumatoid arthritis (RA). Studies have shown
that
patients with rheumatoid arthritis have predominantly Th 1 cells in synovial
tissue
(Simon, A.K., et al., ( 1994) Proc. Natl. Acad. Sci. USA 91:8562-8566). By
stimulating
a Th2 response in a subject with RA, the detrimental Thl response can be
concomitantly
downmodulated to thereby ameliorate the effects of the disease.
Accordingly, the Eta-1/osteopontin inhibitory methods of the invention,
which downregulate type 1 responses (e.g., by inhibition of IL-12 production)
can be
used to shift the immune response to a type 2 immune response (e.g.,
stimulating
production of Th2-associated cytokines) in subjects suffering from, or
susceptible to, an
autoimmune disease in which a Th2-type response is beneficial to the course of
the
disease. The inhibitory method can involve either direct administration of an
inhibitory
agent to the subject or ex vivo treatment of cells obtained from the subject
with an
inhibitory agent followed by readministration of the cells to the subject. The
treatment
may be further enhanced by administering other Th2-promoting agents, such as
IL-4 or
IL-10 itself or antibodies to Thl-associated cytokines (e.g., anti-IL-12
antibodies) to the
subject in amounts sufficient to further stimulate a Th2-type response.
In contrast to the autoimmune diseases described above in which a Th2
response is desirable, other autoimmune diseases may be ameliorated by a Thl-
type
response. Such diseases can be treated using an Eta-1/osteopontin stimulatory
agent of
the invention (as described above for cancer and infectious diseases). The
treatment
may be further enhanced by administrating a Thl-promoting cytokine (e.g.. IFN-
~) to
the subject in amounts sufficient to further stimulate a Thl-type response.
The efficacy of agents for treating autoimmune diseases can be tested in
the above described animal models of human diseases (e.g., EAE as a model of
multiple
sclerosis and the NOD mice as a model for diabetes ) or other well
characterized animal
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models of human autoimmune diseases. Such_animal models include the
mrlllprllpr
mouse as a model for lupus erythematosus, murine collagen-induced arthritis as
a model
for rheumatoid arthritis, and murine experimental myasthenia gravis ( see Paul
ed.,
Fundamental Immunology, Raven Press, New York. 1989, pp. 840-856). A
modulatory
(i.e., stimulatory or inhibitory) agent of the invention is administered to
test animals and
the course of the disease in the test animals is then monitored by the
standard methods
for the particular model being used. Effectiveness of the modulatory agent is
evidenced
by amelioration of the disease condition in animals treated with the agent as
compared
to untreated animals (or animals treated with a control agent).
Non-limiting examples of autoimmune diseases and disorders having an
autoimmune component that may be treated according to the invention include
diabetes
mellitus, arthritis (including rheumatoid arthritis, juvenile rheumatoid
arthritis,
osteoarthritis, psoriatic arthritis, bacterial arthritis), multiple sclerosis,
myasthenia
gravis, systemic lupus erythematosis, autoimmune thyroiditis, dermatitis
(including
atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome,
including
keratoconjunctivitis sicca secondary to Sjogren's Syndrome, alopecia areata,
allergic
responses due to arthropod bite reactions, Crohn's disease, aphthous ulcer,
iritis,
conjunctivitis, keratoconjunctivitis, ulcerative colitis, asthma, allergic
asthma, cutaneous
lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions,
leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis,
allergic
encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic
bilateral
progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia,
idiopathic
thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active
hepatitis.
Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Crohn's disease,
Graves
ophthalmopathy, sarcoidosis, primary biliary cirrhosis, uveitis posterior, and
interstitial
lung fibrosis.
E. Transplantation Rejection
While graft rejection or graft acceptance may not be attributable
exclusively to the action of a particular T cell subset (i.e., Th1 or Th2
cells) in the graft
recipient (for a discussion see Dallman, M.J. ( 1995) Curr. Opin. Immunol.
7:632-638).
numerous studies have implicated a predominant Th2 response in prolonged graft
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survival or a predominant Th 1 response in graft rejection. For example, graft
acceptance has been associated with production of a Th2 cytokine pattern
and/or Qraft
rejection has been associated with production of a Th1 cytokine pattern (see
e.g..
Takeuchi, T. et al. ( 1992) Transplantation 53:1281-1291: Tzakis. A.G. et al.
( 1994) J.
Pediatr. Surg. 29:754-756; Thai, N.L. et al. ( 1995) Transplantation 59:274-
281 ).
Additionally, adoptive transfer of cells having a Th2 cytokine phenotype
prolongs skin
graft survival (Maeda, H. et al. ( 1994) Int. Immunol. 6:855-862) and reduces
graft-
versus-host disease (Fowler, D.H. et al. ( 1994) Blood 84:3540-3549; Fowler,
D.H. et al.
( 1994) Prog. Clin. Biol. Res. 389:533-540). Still further, administration of
IL-4, which
promotes Th2 differentiation, prolongs cardiac allograft survival (Levy, A.E.
and
Alexander, J.W. (1995) Transplantation 60:405-406), whereas administration of
IL-12
in combination with anti-IL-10 antibodies, which promotes Thl differentiation,
enhances skin allograft rejection (Gorczynski, R.M. et al. ( 1995)
Transplantation
60:1337-1341).
Accordingly, the Eta-1/osteopontin inhibitory methods of the invention, which
inhibit type 1 immune responses, can be used to shift the bias toward type 2
immune
responses in transplant recipients to prolong survival of the graft. The
inhibitory
methods can be used both in solid organ transplantation and in bone marrow
transplantation (e.g., to inhibit graft-versus-host disease). The inhibitory
method can
involve either direct administration of an inhibitory agent to the transplant
recipient or
ex vivo treatment of cells obtained from the subject with an inhibitory agent
followed by
readministration of the cells to the subject. The treatment may be further
enhanced by
administering other Th2-promoting agents, such asIL-4 or IL-10 itself or
antibodies to
Thl-associated cytokines (e.g., anti-IL-12 antibodies), to the recipient in
amounts
sufficient to further stimulate a Th2-type response.
F. Other Disorders for I~pregulation of Type 1 Immune Responses
In addition to the foregoing, there are numerous other disorders in which
it can be beneficial to upregulate (i.e., bias toward) type 1 immune responses
using the
Eta-1/osteopontin stimulatory methods of the invention, as follows:
Burn associated sepsis is_associated with the excess production of the
type 2 cytokine IL-10. Accordingly, use of an Eta-1/osteopontin stimulatory
method of
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the invention to promote type 1 responses (e.g., by upregulating IL-12
production and/or
downregulating IL-10 production) can be beneficial in the treatment of burn-
associated
sepsis.
Immunodeficiency disorders often are associated with a lack of, or
insufficient, type 1 immunity. Accordingly, immunodeficiency disorders such as
AIDS,
bone marrow transplant-associated immunodeficiency, and chemotherapy-
associated
immunodeficiencies, can be treated using an Eta-1/osteopontin stimulatory
method of
the invention to promote type 1 responses (e.g., by upregulating IL-12
production and/or
downregulating IL-10 production).
Any of the foregoing treatments may be further enhanced by
administering other Thl-promoting agents, such as the cytokine IL-12 or
antibodies to
Th2-associated cytokines (e.g., anti-IL-4 or anti-IL-10 antibodies), to the
recipient in
amounts sufficient to further stimulate a Thl-type response.
G. Other Disorders for Downregulation of Type 1 Immune Responses
In addition to the foregoing, there are numerous other disorders in which
it can be beneficial to downregulate (i.e., bias away from) type 1 immune
responses (and
bias toward type 2 immune responses) using the Eta-1/osteopontin inhibitory
methods of
the invention, as follows:
Granulomatous disorders result from excessive type 1 responses
(discussed further in Example 1 ) and experiments have demonstrated that in
the absence
of Eta-1 (e.g., in an Eta-1 deficient animal) sarcoid-type granulomas fail to
form.
Accordingly, use of an Eta-1/osteopontin inhibitory method of the invention to
downregulate type 1 responses (e.g., by downregulating IL-12 production and/or
upregulating IL-10 production) can be beneficial in the treatment of
granulomatous
disorders.
Herpes Simplex Virus Keratitis (HSK) results from corneal infection by
Herpes Simplex Virus-1 (HSV-1) that leads to a destructive autoimmune
inflammatory
reaction that depends on the production of IL-12 and that is inhibited by IL-
10
(discussed further in Example 2). Accordingly, use of an Eta-1/osteopontin
inhibitory
method of the invention to downregulate type 1 responses (e.g., by
downregulating IL-
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12 production and/or upregulating IL-10 production) can be beneficial in the
treatment
of HSK.
Bacterial arthritis is associated with excessive type 1 responses
subsequent to bacterial infection. Accordingly, use of an Eta-1/osteopontin
inhibitory
method of the invention to downregulate type 1 responses (e.g., by
downregulating IL-
12 production and/or upregulating IL-10 production) can be beneficial in the
treatment
of bacterial arthritis.
Any of the foregoing treatments may be further enhanced by
administering other Th2-promoting agents, such asIL-4 or IL-10 itself or
antibodies to
Thl-associated cytokines (e.g., anti-IL-12 antibodies), to the recipient in
amounts
sufficient to further promote a Th2-type response.
In addition to the foregoing disease situations, the modulatory methods of
the invention also are useful for other purposes. For example, the stimulatory
methods
of the invention (i.e., methods using a stimulatory agent) can be used to
stimulate
production of Thl-promoting cytokines (e.g., IL-12) in vitro for commercial
production
of these cytokines (e.g., cells can be contacted with the stimulatory agent in
vitro to
stimulate IL.-12 production and the IL-12 can be recovered from the culture
supernatant,
further purified if necessary, and packaged for commercial use).
Furthermore, the modulatory methods of the invention can be applied to
vaccinations to promote either a Th 1 or a Th2 response to an antigen of
interest in a
subject. That is, the agents of the invention can serve as adjuvants to direct
an immune
response to a vaccine either to a Th 1 response or a Th2 response. For
example, to
stimulate an antibody response to an antigen of interest (i.e., for
vaccination purposes),
the antigen and an Eta-1 inhibitory agent of the invention can be
coadministered to a
subject to bias the response towards type 2 responses (e.g., antibody
production) to the
antigen in the subject, since Th2 responses provide efficient B cell help and
promote
IgG 1 production. Alternatively, to promote a cellular immune response to an
antigen of
interest, the antigen and a stimulatory agent of the invention can be
coadministered to a
subject to promote a Th 1 response to the antigen in a subject, since Th 1
responses favor
the development of cell-mediated immune responses (e.g., delayed
hypersensitivity
responses). The antigen of interest and the modulatory agent can be formulated
together
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into a single pharmaceutical composition or in separate compositions. In a
preferred
embodiment, the antigen of interest and the modulatory agent are administered
simultaneously to the subject. Alternatively, in certain situations it may be
desirable to
administer the antigen first and then the modulatory agent or vice versa (for
example, in
the case of an antigen that naturally evokes a Th2 response, it may be
beneficial to first
administer the antigen alone to stimulate a Th2 response and then administer
an Eta-
1/osteopontin stimulatory agent, alone or together with a boost of antigen, to
shift the
immune response to a Thl response).
VI. Tumor Immunity Irradiated Tumor Cells
The present invention also features methods of modulating tumor
immunity. Such methods are based, at least in part, on the understanding that
tumor
cells are capable of escaping destruction by a subject's immune system, i.e.,
are capable
of escaping the subject's natural immune responses. Accordingly, in one
embodiment,
the invention features a method of modulating tumor immunity which involves
contacting a tumor cell with an Eta-1/osteopontin modulator such that tumor
immunity
is modulated. A preferred embodiment features a method of enhancing a type 1
response to a tumor cell which involves contacting the cell with an Eta-1
stimulatory
agent such that a type 1 response against the cell is enhanced. Another
preferred
embodiment features a method of enhancing a type 1 response to a tumor cell
which
involves contacting the cell with an Eta-1 stimulatory agent such that a type
1 response
against the cell is stimulated (e.g., is stimulated by the tumor cell so
contacted). In one
embodiment, the tumor cell is contacted in vivo. In another embodiment, the
tumor cell
is contacted ex vivo. For example, tumor cells can be isolated from the
subject and
cultures in the presence of an Eta-1/osteopontin modulator (e.g., an Eta-1
stimulatory
agent). In another embodiment, the method can further include administering
(e.g.,
readministering) the cells to the patient. In yet another embodiment, the
cells are
transfected in culture with an isolated nucleic acid Eta-1/osteopontin
modulator (e.g., a
nucleic acid molecule encoding Eta-1/osteopontin or a biologically active
fragment
thereof, or encoding a biosynthetic molecule of the present invention. Methods
for
transfecting cells, vectors and the like are as described herein (for example,
in section
LB.)
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In yet another embodiment. the_invention features methods of modulating
tumor immunity (e.g., methods of enhancing a type 1 response against a tumor
or tumor
cells) which further includes the step of irradiating the tumor cells (e.g.,
before or after
contacting with an Eta-1 modulator) such that the cells are incapable of
replicating once
administered to the patient. In yet another embodiment, the method further
features the
step of transfecting the cells with GMCSF. Also featured are tumor cells
treated with an
Eta-1/osteopontin modulator of the present invention. In one embodiemnt, the
invention
features tumor cells transfected with an Eta-1-encoding nucleic acid molecule
or nucleic
acid molecule encoding a biologically active fragment of Eta-1/osteopontin. In
another
embodiment, the invention features tumor cells transfected with Eta-
1/osteopontin and
GMCSF. In yet another embodiemnt, the invention features a tumor cells
transfected
with a nucleic acid molecule which encodes a biosynthetic immunomodulatory
molecule
of the preset invention.
The present invention is further illustrated by the following Examples
which in no way should be construed as further limiting. The entire contents
of all of
the references (including literature references, issued patents, and published
patent
applications) cited throughout this application are hereby expressly
incorporated by
reference.
Exemplification
Examples 1-4 demonstrate an essential role for Eta-1/osteopontin in regulating
immune
responses (e.g., type-1 immune responses) in vivo. These examples further
demonstrate
the applicability of administering Eta-1/osteopontin as an in vivo approach to
regulating
such immune responses.
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Example 1 ~ Eta-1/opn-dependent modulation of type-1 immunity (e.Q., in a
classical
aranulomatous response) in vivo in control. nude, cvtokine-deficient and
Eta-1/o~n-deficient mice
An early and essential step in type-1 immunity is the migration of
macrophages/dendritic cells to the site of infection, and subsequent
activation of the
recruited macrophages, a process that is controlled by CD4 T-cells. Eta-
1/osteopontin is
the most abundantly expressed mRNA transcript after activation of CD4 cells
(Patarca et
al. ( 1989) J. Exp. Med. 170:145-161; Weber et al. ( 1997) Proc. Assoc. Am.
Physicians
109:1-9; Rittling and Denhardt (1999) Exp. Nephrol. 7:103). Production of IL-
12 by
activated macrophages/dendritic cells and reception of the IL-12 signal by CD4
cells are
subsequent critical steps in this process. Although an interaction between
CD40 ligand on
activated T-cells and CD40 on macrophages can induce IL-12 expression
(Scheicher et al.
( 1995) Eur. J. Immunol. 25, 1566; Macatonia et al. ( 1995) J. Immunol.
154:5071;
Murphy ( 1998) Curr. Opin. Immunol. 10:226) this interaction also induces the
inhibitory
IL-10 cytokine and may not suffice for induction of IL-12 in vitro (Ria et al.
( 1998) Eur.
J. Immunol. 28:2003) or for sustained levels of IL-12 that follow viral
infection in vivo
(Sharma et al. ( 1998) J. Immunol. 161:5357).
The Eta-1 gene is expressed in T cells early in the course of bacterial
infections (within 48 hours), and interaction of its protein product with
macrophages can
induce inflammatory responses (Singh et al. ( 1990) Exp. Med. 171:1931; Yu et
al.
( 1998) Proc. Assoc. Am. Physicians 110:50; Denhardt and Noda ( 1998) J. Cell.
Biochem. Suppl. 30/31:92). Genetic resistance to infection by certain strains
of
Rickettsia may depend on Eta-1-dependent attraction of monocytes into
infectious sites
and acquisition of bacteriocidal activity (Patarca et al. ( 1993) Crit. Rev.
Immunol.
13:225; Jerrells and Osterman ( 1981 ) Infect. Immun. 31:1014); the
granulomatous
responses characteristic of sarcoidosis and tuberculosis are associated with
high levels of
Eta-1 expression (Nau et al.(1997) Proc. Natl. Acad. Sci. U.S.A. 94:6414;
O'Regan et al.
( 1999) J. Immunol. 162:1024).
Granuloma formation in these human diseases is a cellular consequence
of type-1 immunity (Patarca et al. (1993) Crit. Rev. Immunol. 13:225; Jerrells
and
Osterman ( 1981 ) Infect. Immun. 31:1014). Accordingly, a valuable in vivo
animal model
for studying type-1 immune responses involves inducing sarcoid-type granulomas
in mice
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by injection of polyvinyl pyrrolidone (PVP) (van den Bogert et al. ( 1986)
Virchows
Arch. ~ 1:39). Because certain murine models of parasite-induced granulomas
may
reflect a mixture of type-2 and type-1 immunity (O'Garra ( 1998) Immunity
8:?75), the
importance of IL-12-dependent type-1 immunity in this murine model of
granuloma
formation was first established.
The granulomatous response was first measured in control (C57BL/6
(+/+)) and nude (C57BL/6 nulnu) mice. PVP-dependent granulomas were formed by
injecting mice subcutaneously above the right hind limb with 500 pl of 0.5%
PVP.
After 5 days, mice were killed, and tissue was extracted for histologic
analysis. Figure
1 A depicts the data as ( 1 ) the mean number of granulomas per high-power
field (HPF)
(X200 magnification); (2) as the mean number of cells per granuloma after
examination
of 50 to 80 HPF per mouse; and (3) as the product of these two indices, termed
"granuloma burden". (Error bars indicate 1 SEM.). An intense granulomatous
response
was provoked shortly after subcutaneous injection of PVP into C57BL/6 (+/+)
but not
C57BL/6 nulnu strains of mice (see e.g., top two bars of Figure lA).
It was next determined whether Eta-1 administration could reconstitute
the granulomatous response in C57BL/6 nulnu strains of mice. For experiments
using
purified Eta-1/opn to modulate immunity, Eta-1 is prepared as follows. To
generate
naturally-produced (native) Eta-1/opn, MC3T3E1 cells or ArSv T-cells were
grown in
defined media (consisting of DME/H 12 supplemented with pyruvate, insulin,
transferrin, selenium and ethanolmine) in 5% CO, at 37°C. Media was
dialized against
PBS and concentrated using a Millipore tangential flow system applied to
Millipore
LC 100 equipped with a DEAE-Memsep 1000 cartridge and developed in a
discontinuous gradient of 0 to 1 M NaCI in phosphate buffer, pH 7.4. Eta-1/opn-
containing fractions were pooled (the major Eta-1/opn peak eluted at 0.26 M
salt),
concentrated by ultrafiltration, chromatofocused on mono P columns (Pharmacia)
at pH
8.2, developed with polybuffer 74 (Pharmacia) and the major Eta-1/opn fraction
eluted
from monobeads at pH 4.6. The protein was judged pure by several criteria
including
SDS electrophoresis and amino acid sequence analysis (both N-terminal and
internal
peptide analysis). Mass spectroscopic analysis revealed a peak centered around
a mass
of 35,400 that was highly phosphorylated ( 11 mots of phosphate/mol of
protein), O-
~lycosylation but not N-glycosylation, and no measurable sulfate. For
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dephosphorylated. naturally-produced Eta-1/opn. ~ mg of purified Eta-1/opn was
incubated with 6 units (60 units/mg> type II potato acid phosphatase in 20 mM
phosphate buffer pH 4.8 at 37 C for ? h. After adjusting the pH to 8.2.
dephosphorylated protein was applied to a chromatofocusing column and the
major peak
~ eluted at a pH of 5.1; amino acid analysis of the protein revealed a
phosphate content of
less than 1 mollmol protein.
When C57BL/6 nulnu mice were coinjected with PVP inoculum plus 10
pg of Eta-l, the granulomatous response was partially restored, demonstrating
that the
Eta-1/opn gene product can partially substitute for activated T lymphocytes in
this
setting. The granulomatous response was likewise determined in cytokine-
deficient
(C57BL/6 IL-12-x- and C57BL/6 IL-10-~-) mice. The granulomatous response was
diminished by 70 to 80% in C57BL/6 IL-12-'- mice and was enhanced two- to
three-fold
in C57BL/6 IL-10-~- mice.
It was then asked whether mice deficient in Eta-1 secondary to targeted
gene mutation formed granulomas after PVP injection. C57BL/6 x 129/SV Eta-1~'~
mice
generated as described by Rittling et al. ( 1998) J. Bone Miner. Res. 13:1101,
were
compared to either Eta-1+~+ littermates or age-matched C57BL/6 x 129/SV mice
as
controls. Histological analysis was performed on tissue sections from PVP
injection
sites. Briefly, samples were fixed in 10% buffered formalin and embedded in
paraffin.
Embedded sampled were sectioned into 4- to ~-pm serial sections and stained
with
hematoxylin and eosin. Images were captured with a Sony DXC-970MD video camera
and Optima 5.2 Histomorphometric analysis software.
Histological analysis of tissue sections at PVP injection sites at 20X.
100X and 400X magnifications showed granulomatous infiltrations of mononuclear
cells in subcutaneous dermal and subdermal areas in Eta-1+~+ mice 5 days after
injection
of PVP, PBS, or PVP + ~ pg purified Eta-1. By contrast, Eta-1-~- mice did not
develop a
detectable granulomatous response after challenge with PVP. However,
coinjection of
purified Eta-1 with PVP the partially restored the granulomatous response in
Eta-1-'-
mice (these experiments and Figure lA).
Analysis of surface antigens expressed by cells within ~ranulomas in the
various mouse strains was done with monoclonal antibodies to Mac-1. B220. CD3.
and
BP-5~ (a neutrophil marker). Histologic analysis of granulomas formed in Eta-
1T'- mice
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and in Eta-1-'- mice reconstituted with purified Eta-1 revealed a similar
macrophage-
dominant cellular infiltrate. About 85 r'o of aranulomatous cells in both
cases were vlac-
1'. whereas 5 to 10% were CD3~ T cells or B220~' B cells. BP-55' neutrophils.
which
were only a minor component ( 1 to 2%) of granulomas in these mice. increased
5- to 10-
fold in granulomas firmed in IL-10-'- mice. Eta-1-'- mice also displayed
defective
aranulomatous responses to injection of collagen and latex. consistent with
reports that
human T cells resident in sterile granulomas have high expression of Eta-1
(Nau et nl.
( 1997) Proc. Natl. Acad. Sci. U.S.A. 94:6414: O'Regan et al. ( 1999) J.
Immunol.
162:1024).
Lastly, the cytokine expression profiles were determined for cells from
lymph nodes draining the site of granulomas in Eta-1+'+ and Eta-1-'- mice.
Briefly, PVP-
dependent granulomas were formed as described above. After 5 days, mice were
killed,
and local lymph nodes were obtained for cvtokine expression. Cytokine
expression was
measured 48 hours after incubation with PVP (2 x 106 cells per well).
Restimulation of
lymph nodes draining subcutaneous sites of PVP injection in Eta-1-'- mice and
control
mice with PVP revealed impaired IL-12 and interferon-y (IFN-y) responses. The
IL-12
response was reduced by ~95%. and the IFN-y response of Eta-1-'- mice was
reduced by
90% in comparison to Eta-1+'+ controls (Figure 1C).
Example 2: Eta-1/opn-dependent modulation of type-1 immunity and destructive
type-1 autoimmune responses in viva in herpes simplex virus-type 1
( HS V-1 ) infected control and Eta-1 /opn-deficient mice
A second valuable in viva animal model for studying type-1 immune
responses involves inoculating mice (e.g., corneal inoculation) with herpes
simplex virus-1
("HSV-1"). Inoculation with HSV-1 leads to delayed type immune responses in
mice
that can manifest as classical footpad swelling (Foster et al ( 1986) Clira.
Irnmunol.
Irnmunopathol. 40:313-325). Corneal HS V-1 infection can also lead to a
destructive
autoimmune inflammatory reaction, Herpes Simplex Keratitis (HSK), initiated by
CD4
cells that recognize a viral peptide mimic of a murine corneal self-antigen
(Zhao et al.
( 1998) Science 279:1344: Avery et al. (19951 Nature 376:431 ). This
inflammatory
response depends on the production of IL-12 and is inhibited by IL-10
(Streilein et czl.
( 1997) Immunol. Today 18:443: Daheshia et al. ( 1997) J. Lnmunol. 159:1945 ).
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Eta-1 '~- mice were infected in the right eye with 4x106 plaque-forming units
(PFUI HSV-1 (KOS strain) and challenged five days later in the left footpad
with 1x10'
PFU of UV-inactivated HSV-1 (KOS). Eta-1-~-(opn-'-) mice infected by HSV-1
(4x106
PFU via the cornea) fail to develop a significant DTH response after footpad
challenge with
10' pfu HSV-1. in contrast to the strong DTH response of Eta-1+~T(opn+'+)
controls (Figure
2A).
The numbers of T cells and proportions of T cell subsets in the thymus
and peripheral lymphoid tissues of Eta-1-~- mice were similar to Eta-1T~+
littermates. T
and B cell subsets in Eta-1-'- and Eta-1'~+ littermates were as follows:
C57BL/6 x 129
Eta-1+~+ spleen, 93.7 x 106 total cells (30.8% CD3, 19.8% CD4, 11% CDB, and
49.7%
B220); C57BL/6 x 129 Eta-1-'- spleen. 82.6 x 106 cells (27.8% CD3, 18.8% CD4,
9.0%
CD8. and 55.5% B220); C57BL/6 x 129 Eta-1+~+ lymph node, 32.0 x 106 cells
(82.4%
CD3, 42.8% CD4, 34.2% CD8, and 12.8% B220); and C57BL/6 x 129 Eta-1-'- lymph
node, 21.9 x 106 cells (82.8% CD3, 49.3% CD4, 28.4% CDB, and 11.2% B220).
Moreover. T cells from Eta-l~~- and Eta-1+~+ mice expressed levels of CD44 and
CD62
that were not distinguishable. Although the T cell numbers and proportions
were
similar in Eta-1-'- and Eta-1+~+ mice, the possibility existed that defective
antiviral DTH
response in Eta-1-~- mice might reflect a subtle alteration in lymphocyte or
macrophage
development. Accordingly, the effects of acute in vivo depletion of Eta-1 with
a
neutralizing antibody were tested in the Eta-1-~- mice. The neutralizing
antisera LF-123
(Fisher et al. ( 1995) Acta Orthop. Scand. 66:61 ) or control normal rabbit
serum were
injected at 25 qg per dose per day, starting 2 days before injection. On day
0, mice were
infected with HSV-1 (KOS) and rechallenged 5 days later. The right and left
footpads
of each mouse were measured 24 hours after rechallenge, and specific swelling
(left
versus right footpad) is shown in Figure 2B. Administration of antibody to Eta-
1 (LF-
123) immediately before and repeatedly after HSV-1 infection efficiently
inhibited the
DTH response upon rechallenge.
In a second experiment. Eta-1-~- and control mice (Eta-1 "') were
subjected to ocular challenge with virus. As shown in Figure 2C, Eta-1-'- mice
failed to
develop significant HSK within 2 weeks after corneal inoculation with HSV-1 in
contrast to the severe HSK developed within this period by control littermates
(Eta-1T'')
(i.e.. 690 of control Eta-1+'~ mice developed HSK. Figure 2C). Similar results
were
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obtained when the experiment was repeated using BALB/cB~/J mice and CB-17 mice
in
addition to Eta-1-'- and Eta-1~'t mice, as demonstrated in Figure 2D.
Furthermore.
skewing of the cell numbers in Eta-1/opn knockout mice after challenge with
HSV-1
was diminished compared to control mice in which the increase of CD8+ cells is
consistent with a Th-1 response.
Moreover. T-cells from Eta-1-~- mice are not impaired in their
proliferative response to irradiated virus plus antigen presenting cells. The
right
superficial cervical draining lymph nodes of Eta-1-'- mice and Eta-1+~+
littermate controls
were harvested l~ days after infection of the right eye with 4 x 106 PFU of
HSV-1
(KOS). Cells from these lymph nodes (2 x 106 cells per well) were incubated in
the
presence of 4 x 107 PFU of ultraviolet (UV)-inactivated HSV-1 (KOS). The
proliferative response of lymph node cells from HS V-1-infected Eta-1 T'+ and
Eta-1-~- mice
measured by 'H-thymidine incorporation at 72 h was 20.9x 103 and 18.7x 10'
cpm,
respectively. Furthermore, the absence of DTH does not reflect a general
impairment of
the immune system in these mice since clonal expansion followed by apoptosis
after
superantigen (SEB) injection were indistinguishable from wild-type mice. T
cell
expansion followed by apoptosis after superantigen (50 pg of staphylococcal
enterotoxin
B) intraperitoneal injection into Eta-I~~- and Eta-1+~+ mice was
indistinguishable at 3
days: +/+ Vp8+ CD4 cells (percentage of total spleen) increased from 3.6 to
~%: -/- Vp8+
CD4 cells increased from 3.2 to 5.5%; +/+ Vp6+ CD4 cells increased from 2.3 to
2.6%: -
/- Vp6T CD4 cells increased from 2.5 to 2.6/0.
Although cells from the draining lymph nodes of virus-infected Eta-1-'- and
Eta-1+~+ mice respond equally well to HSV-1 according to [3H]-thymidine
incorporation
after viral restimulation in vitro, they differed conspicuously according to
their cytokine
profiles. Briefly, cells were isolated and restimulated with HSV-1 (KOS) as
described
above. Supernatants were harvested 48h later and IL-10 and IL-12 p40 cytokine
levels
were measured by sandwich ELISA using OptIEA antibody sets (Pharmingen. La
Jolla
CA). IL,-4 was measured after stimulation of draining lymph node cells by
plate-bound
anti-CD3. Cells from Eta-1 '~- mice produced high levels of IL-10 and IL-4 but
markedly
reduced levels of IL-12, compared with Eta-1t~+ controls (Figure 2E) and
splenic
macrophages from virus-infected Eta-1 t'+ but not Eta-1-'- mice continued to
produce IL-12
ten days after infection. In contrast with the sterile granulomatous response.
IFN-v levels
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were not reduced in Eta-1-'- mice after HSV-1-viral infection, consistent with
an IL-12-
independent pathway to IFN-y production that may depend on virally induced IFN-
ai (3
production (Oxenius et al. ( 1999) J. Immunol. 162-965: Cousens et al. ( 1999)
J. E~p.
ll~led. 189:1315). Moreover, expression of IL-2 by lymph node and spleen T
lymphocytes from Eta-1-'- and Eta-1*~* littermates in response to immobilized
antibody
to CD3 was indistinguishable between the C57BL/6 x 129/SV Eta-1~~- and C57BL/6
x
129/SV Eta-1*'* mice. These cytokine profiles suggest that Eta-1/osteopontin
expression
normally may imprint the in vivo ratio of IL-12 and IL-10 cytokines that
dictates a type-1
immunity.
Example 3~ Treatment of HSV-1 infected mice with anti-Eta-1/osteopontin
antibodies
significantly downre~ulates type-1 immunity and destructive type-1
autoimmune responses
In a similar experiment to those described above, the levels of HSV-1-
specific DTH reactions were measured 24 hours after footpad challenge in Ca120
mice
that had been primed five days earlier by corneal inoculation of 4 x 10~ to 4
x 10' plaque
forming units ("pfu") of UV-inactivated HSV-1 and treated with anti-
osteopontin
antibody. LF-123 (Fisher et al. (1995) Acta Orthop. Scand. 66:61), or control
serum
every 48 hours. Mice treated only with control serum exhibited classical
footpad
swelling when footpads were measured 24 hours after challenge. By contrast,
mice
treated with LF-123 serum exhibited significantly diminished footpad swelling.
indicating that neutralization of osteopontin significantly inhibited footpad
swelling.
Table 1: Specific footpad swelling in Ca120 mice in the presence of rabbit
serurn or
anti-osteopontin antibody following HSV-1 inoculation (increasing pfus).
HS V-KOS rabbit serum ' LF-123
'~4x 10~ 0.04 mm ~ 0.0?
2 x 10' 0.1 T 0.05
14x10' X0.42 '0.03
~ =IxlO~ ~ 0.57 0.13
4x10' (24 hr) ~ 1.114 _ I 0.6175
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Yloreover, as described above_HSV-1 infection of murine cornea leading
to HSK results in corneal inflammation and destruction within 14 days after
viral
inoculation. Ca120 mice that were treated with anti-osteouontin antibody every
48 hours
after challenge had reduced severity and incidence of HSK compared to mice
injected
with rabbit serum.
Table II: HSK in Ca120 rnice in the presence of rabbit serum or anti-
osteopontin
antibody following HSV-I inoculation
'; Incidence* Severity*
Ca120 77.77% x.11
100% 3.1
~; Ca120 rabbit ~0% ~ _~
serum
50% 2 25
~ Cal 20 LF-123 60% ~ 1
6 i
60% ~
'
*day 11/day 14
Example 4: Eta-1/osteopontin-dependent modulation of protective immune
responses
following infection (e.g.. listeria monocvtogenes infection) in vivo in
control and Eta-1/osteopontin-deficient mice
The murine response to Listeria monocytogenes is an experimental
cornerstone of our understanding of the early events leading to type-1
immunity after
microbial infection (Unanue ( 1997) Immunol. Rev. 158:11 ) and depends on
early
macrophage production of IL-12 and downstream expression of IFN-y (Tripp et
al.
( 1993) Proc. Natl. Acad. Sci. U.S.A. 90:3725: C. S. Tripp, Gately et al. (
1994) J.
Immunol. 152:1883; Tripp et al. ( 1995) J. Immunol. 155:3427). Accordingly,
the ability
of Eta-1-'- mice to mount a protective immune response after Listeria
infection was
investigated.
Listeria infection and cvtokine production were as follows. Virulent L.
monocvtogenes (strain 1778, American Type Culture Collection (ATCC)
designation
43251 ) was grown in trypticase soy broth. and lOr colony-forming units
(CFLT). a
sublethal dose for this strain of L. monocwoeenes, were injected intravenously
into
C57BL/6 (B61. B6-IL-12-~~. B6-IL-10-'-._B6 x 129-Eta-1-~-. and B6 x 129-Eta-
1~'~ mice
(Unanue ( 1997) Imrm~nol. Rev. 158:11: Stordeur et al. (19951 Mol. hnmunol.
32:233
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( 19951: Stordeur and Goldman ( 19981 Int. Rev Immccnol. 16:501). The titer of
viable
bacteria in the inoculum and in organ homogenates Ue.g., liver and spleen) was
determined by plating 10-fold serial dilutions on trypticase soy agar plates.
Plates were
incubated at 37°C, and the numbers of CFU were counted after 24 hours.
Eta-1-~- mice
contained liver-associated Listeria-infected cysts that were apparent 4 (first
experiment)
and 5 (second experiment) days after infection (also seen in IL-12-~~ mice).
At 5 days
(second experiment) Listeria infection was also evident in spleen homogenate
of Eta-1-'-
mice. These data demonstrate that Eta-1-~- mice were defective in their
ability to clear L.
rnonocytogenes after systemic infection, similar to the defect in IL-12-~-
mice.
Restimulation of spleen cells from Eta-1-~- and Eta-1+~+ mice with heat-
killed bacteria revealed that cells from the former mice had reduced IFN-v
responses.
Briefly, Spleen cells (4 x 106/ml) from four to five C57BL/6 x 129 Eta-1+~' or
four to
five C57BL/6 x 129 Eta-1-~- mice that had been intravenously inoculated 5 days
earlier
with 103 CFU were stimulated with heat-killed L. monocytogenes (2 x 10g
CFU/ml) 96
hours before IFN-y measurement by an OptEIATM ELISA kit (PharMingen). 25.5 ~
6.5
ng/ml of IFN-y were produced by spleen cells from Eta-1+~+ mice in comparison
with 3.2
t 1.2 ng/ml of IFN-y from Eta-1-~- mice.
The data presented in Examples 1-4 clearly demonstrate a role for Eta-
1/osteomodulin in a variety of type-1 immune responses and demonstrate that
type-1
immunity can be modulated by administration of purified Eta-1. The data
presented in
Examples 1-4 further indicate that Eta-1/osteopontin expression potentially
effects type-
1 immunity through regulation of the IL-12 and IL-10 cytokine ratio.
Example 5: Eta-1/osteopontin-dependent modulation of type-1 immunity cytokine
profiles in vitro
This example further defines the role of Eta-1/opn in modulating immune
effector cells, in particular, by demonstrating the ability of Eta-1/opn to
modulate type-1
cvtokine secretion in vitro (e.g., in isolate peritoneal macrophages).
Resident peritoneal
macrophages were isolated from normal mice and treated with increasing amounts
of
purified Eta-1/opn. Briefly, peritoneal macrophages were obtained by
peritoneal lavage
(2x10 ml PBS) of C57BL/6 mice. Contaminating red cells were eliminated by
hypotonic
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lysis with ACK buffer. Cells were plated at 10'/100 ~tl in 96-well plates and
non-
adherent cells were washed off after 2 hours. Adhered cells were then
incubated for 48
hours with increasing concentrations of purified Eta-1/opn in serum-free
medium and
levels of IL-10 and IL-12 p75 in the supernatant were determined by ELISA
(Figure 3A).
Briefly, supernatant was withdrawn at the indicated time points for analysis
of IL-10 or
IL-12 p70 using commercial ELISA kits (R & D Systems). At the end of the
incubation,
the cells were tested for viability by propidium iodide incorporation (>98%)
and their
purity was confirmed by staining with fluorescence-conjugated anti-Mac 1
antibody
(>98%).
Treatment of cells with Eta-1/opn resulted in the secretion of as much as
400 pg/ml of IL.-12 at 48 hours whereas IL-10 production was not detected
(Figure 3A).
Eta-1/opn-dependent induction of IL-12 secretion from macrophages was not due
to
contamination with endotoxin as Limulus ameboid lysate assay indicated that
purified Eta-
1/opn contained less than lng/g endotoxin. Moreover, quantities of endotoxin
that escape
detection in the limulus ameboid lysate assay do not contribute to biologic
activity of Eta-
1/opn because the IL-12 response of macrophages derived from C3H.HeJ mice
(which are
defective in endotoxin receptor-mediated signaling) was not impaired compared
to other
strains.
Next, resident peritoneal macrophages (Sx 10'/ml) were treated with either 5
pmollml Eta-1/opn, 30 ng/ml LPS or 500 U/ml IL-4 and IL.-12/IZ,-10 detected by
ELISA at
increasing times post-induction (Figure 3B). While LPS stimulation of these
resident
peritoneal macrophages induced both IL-12 (about 250 pg/ml) and IL-10 (about
100 pg/ml)
and while IL-4 predominantly caused production of IL-10, Eta-1/opn selectively
lead to
secretion of IL-12. The failure of Eta-1/opn to induce IL-10 was somewhat
surprising
since other cytokines that activate macrophages (e.g., TNFa, IL.-1,IL-2,IL-3
and IL-6 all
stimulate IL-10 secretion (Stordeur et al. ( 1995 Mol. Immonol. 32:233:
Stordeur and
Goldman ( 1998) Int. Rev. Immunol. 16:501 ), and lippopolysaccharide (LPS)
stimulation of
these resident peritoneal macrophages induced both IL.-12 0250 pJml) and IL-10
0100
pg/ml).
Further analysis showed that Eta-1/opn actively suppressed IL.-10 secretion
by resident peritoneal macrophages stimulated with IL-4 (Figure 3C). Briefly.
macrophages were activated with IL-4 (500 U/ml x 1 hour) before addition of
Eta-1/opn (~
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pmol/ml) for an additional 48 h before measurement of IL-12 and IL-10 by
ELISA. In
some groups, anti-IL-12 (R & D Systems, Minneapolis, MN) was added at a final
concentration of 2 ~g/ml. As shown in Figure 3C, IL-4-dependent induction of
macrophage IL-10 was inhibited by the addition of Eta-1/opn, but this effect
was not
altered by anti-IL-12 neutralizing antibody, suggesting a direct mode of
action.
Moreover, Eta-1 actively suppressed the LPS dependent IL-10 response
of resident peritoneal macrophages (Figure 3D). Briefly, macrophages were
activated
with LPS (30 ng/ml) for 1 hour before addition of Eta-1 (5 nM) for an
additional 48
hours and consecutive measurement of IL-12 and IL-10 by ELISA. Assays were
performed in quadruplets, and each point represents the mean and standard
error (error
bars) of two independent experiments.
Examples 6-12 define the functional domains of Eta-1/osteopontin and map
various
Eta-1/osteopontin-dependent activities to their respective domains. These
Examples
also define various bioactive fragments of Eta-1 for modulating immune
effector cell
activation (e.g., cell motility, spreading, cytokine and metalloproteinase
secretion).
These examples also describe the phosphorylation dependence of various Eta-
1/osteopontin-dependent activities.
Additional Materials and Methods for Examples 6-12
Cell lines: A31 is an integrin a,,(3~, CD44 murine embryonic fibroblast
clone derived from Balb 3T3 cells (CCL-163, ATCC). A31 cells transfected with
CD44
(A31.C 1 ) or A31 mock-transfectants were generated as described (Weber et al.
( 1996)
Science 26:271:509-512). MH-S is a macrophage cell line that was derived by
SV40
transformation from an adherent cell enriched population of alveolar
macrophages
(CRL-2019, ATCC). MT-2/1 is a thymus-derived macrophage from a Balb/c mouse
that was immortalized by infection with retroviral vector. It expresses CD44
and
integrin a,,(3~.
Eta-1/opn purification and cleavage: To generate recombinant Eta-1/opn,
GST-Eta-1/opn fusion protein was expressed in E. coli. digested with factor
Xa. and
purified by affinity chromatography as_described (3 refs). For phosphorylated
recombinant Eta-1/opn. GST-Eta-1/opn (~ mg) was incubated withl0 ~g of Golai
kinase
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for ? h before passage through a GSH-Sepharose column and elution from GSH-
Beads
with 100 U of factor Xa. The eluate was applied to a chromatofocusing column
and
eluted from the resin with polybuffer 7-~ as described above. The major peak
eluted at
pH =L6 and phospho-amino acid analysis of the recovered protein revealed a
phosphoserine content of 16 mol of phosphate/mol protein and 0.8 mots of
phosphothreonine/mol protein. Native Eta-1/opn were prepared as described
above.
Thrombin cleaves Eta-1/opn into two fragments following the arginine in the
sequence
VVYGLR in Eta-1/opn (e.g., amino acid residues 162-168 of SEQ ID N0:2). an
N-terminal fragment ("Eta-1/opn NT) containing the RGD motif and a C-terminal
fragment ("Eta-1/opn CT"). Thrombin cleavage and phosphorylation of either the
dephosphorylated native protein or recombinant Eta-1/opn was accomplished by
human
thrombin (Sigma Chemicals), Golgi kinases or purified casein kinase II or
casein kinase
I.
Chemotaxis: Directed migration of cells was determined in mufti-well
chemotaxis chambers as described (Weber et al. ( 1996) Science 26:271:509-~
12).
Briefly, two-well culture plates (Transwell) with polycarbonate filters (pore
size 8-12
Vim) separating top and bottom wells were coated with 5~g fibronectin. 2 X 10'
cells
were added to the upper chamber and incubated at 37° C in the presence
or absence of
Eta-1/opn in the lower chamber. After 4 h, the filters were removed, fixed in
methanol,
stained with hematoxylin and eosin and cells that had migrated to various
areas of the
lower surface were counted microscopically. Controls for chemokinesis included
200
ng of the appropriate form of osteopontin in the top well. All assays were
done in
triplicates and are reported as mean ~ standard deviation.
Haptotaxis: Haptotaxis of monocytic cell lines to Eta-1/opn or fragments
of Eta-1/opn was assayed using a Boyden chamber. The lower surface or both
sides of
polvcarbonate filters with 8 ~m pore size were coated with the indicated
amounts of Eta-
1/opn. 2 X 10' cells were added to the upper chamber, and incubated at
37° C in the
absence of any factors in the lower chamber. After 4 h. the filters were
removed, fixed
in methanol and stained with hematoxvlin and eosin. Cells that had migrated to
the
lower surface were counted under a microscope. All assays were done in
triplicates and
are reported as mean ~ standard deviation.
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Cell attachment and spreading:- 24-well plates were coated over night at
-1°C with 10 qg/ml of the indicated ligand then blocked for 1 h at room
temperature with
1-10 mg/ml BSA in PBS. To preserve the integrity of adhesion receptors, MH-S
monocytic cells were harvested from subconfluent cultures by non-enzymatic
cell
~ dissociation solution (Sigma, St Louis MOI. Cells were washed twice with PBS
and
resuspended at a concentration of 1 X 10' cell/ml of sterile Ca'+ and Mg''+-
free PBS
supplemented with 0.1 % BSA and 1 mM sodium pyruvate. 5 x 103 to 5 x 10~ cells
were
incubated in each well and. after 1 h at 37°C, the wells were washed 3
times with 0.5 ml
PBS to remove non-adherent cells, fixed in 10% buffered formalin, 1 %
paraformaldehyde, or 1 % glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) at
room
temperature for -1 hour then stained with toluidene blue and hematoxylin. The
total
number of attached or spread cells in each well were counted microscopically
using a
Nikon Eclipse microscope equipped with a Sony digital Camera. Total number of
attached or spread cells were quantitated using Optima 5.2 image analysis
system. Each
experiment was done in triplicates and is reported as mean ~ SEM. To minimize
variability inherent to cell attachment studies, cells were scored as attached
only when a
defined nucleus was observed, accompanied by a transition from round to
cuboidal cell
morphology. Round cells that are loosely attached with no defined nucleus were
scored
as non-attached. These cells can be removed with repeated washes. The
viability of the
cells was measured before and after the termination of the experiments and
only data
from experiments with greater than 95 % cel l viability were used. Further.
under the
conditions used in these experiments, cell attachment was temperature
dependent,
inhibitable by trypsin treatment and not affected by inhibitors of protein
synthesis or
secretion. Cell spreading was determined by membrane contour analysis and was
scored
according to increase in cell volume/surface area. In some experiments, cell
spreading
was also assessed by the formation of stress fibers. Each experiment was
performed in
quadruplicate wells and repeated 3 times.
Example 6: Effects of Various Eta-1/opn Domains on Cellular Chemotaxis
This example describes the domain-specific effects of Eta-1/osteopontin
on chemotaxis of immune effector cells_(e.g., monocytes).
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Stable CD44 transfectants of ad~3~ fibroblasts (Weber et al. ( 1996) Science
X6:271:509-5121 were used to examine the interaction of CD44 with osteopontin.
Chemotactic activity of Eta-1/opn or Eta-1/opn fragments was tested in a
modified
Boyden chamber (Weber et al. ( 1996) Science 26:271:509-512). Purified natural
osteopontin exerted chemotactic activity for the MH-S monocyte cell line.
Moreover,
the C-terminal thrombin cleavage product but not the N-terminal cleavage
fragment
mediated chemotaxis of A31.C 1 (CD44 stably transfected cells), but not
mock-transfected A31.MLV cells, confirming the dependence of cell migration on
the
expression of CD44. The C-terminal fragment of Eta-1/opn also induced
chemotaxis of
macrophage cell line MH-S as efficiently as intact Eta-1/opn, whereas the N-
terminal 30
kDa Eta-1/opn fragment was inactive.
Table 111: Chemotactic Response of MH-S Cells to Eta-Ilopn
LOWER CHAMBER
UPPER
CHAMBER pBS Eta-1/opn Eta-1/opn Eta-1/opn
CT NT
PBS 5610 31256* 47898* 7121
I Eta-1/o 347 ~ 16824 ~ 30550 ~ 3619
n
I Eta-1/o 94 8824 ~ 22038 265~
n CT
I Eta-1/opn6311 28760 i 40955 ; 145
NT ~
* P<0.01
Moreover, equimolar mixtures of both fragments displayed activity
similar to that of the 28 kDa C-terminal fragment alone. These results, taken
together,
indicate that the chemotactic domain of Eta-1/opn resides in the 28 kDa C-
terminal part
of the molecule. This C-terminal fragment-mediated activity could further be
inhibited
by Eta-1/opn fragments and various modulators of receptor interaction.
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_ "_
Table IV: Inhibition of Monocvte Chemotaxis
yII ' MI
I Eta-1/opn Eta-llo n
CT
Control I 13.3 1.9 9.6 1.9
+ GRGDS' ( 1mM) 10.6 1.3 7.7 1.6
+ anti CD44 (0.17.8 0.7** 4.6 0.7*
)
+anti 3 (0.1 9.80.6 12.1 2.1
) I
+ Eta-1/o n NT 12.3 2.0 9.6 1.9
+ Eta-1/o n CT 6.3 0.7* 3.7 1.6*
+Eta-1/o n 4.40.6* 2.60.2*
* P<0.01 ** P<0.05 ' (SEQ m NO:11)
Previous investigations of stable transfectants of A31 cells showed that
the interaction of Eta-1/opn with CD44 depended on expression of CD44 splice
variants
3-6 (Weber et al. ( 1996) Science 26:271:509-512), which characterize
activated
lymphocytes (Arch et al. ( 1992) Science 257:682-685). More recent studies
indicate
that A31 cells transfected with the standard form of CD44 (lacking variant
exons) do not
bind Eta-1/opn.
Example 9: Effects of Various Eta-1/opn Domains of Hapotaxis
This example describes the domain-specific effects of Eta-1/osteopontin
on haptotaxis of immune effector cells (e.g., monocytes).
Cells can move up a gradient of immobilized ligand, a process referred to
as haptotaxis. This cell crawling may occur on vessel walls or in the
interstitium.
Therefore, the contribution to cell motility of interactions between
immobilized Eta-
1/opn, Eta-1/opn fragments, and integrin receptors was assessed as follows.
The ability
of the immobilized ligand to induce monocs~te haptotaxis was judged by cell
migration
through poly-carbonate filters. Eta-1/opn induced monocvte migration that was
mainly
directional (i.e., the cells responded to apositive gradient of bound Eta-
1/opn>. and thus
haptotactic and was inhibited by GRGDS (SEQ ID NO:11) and antibody to the ~3~
chain
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of integrins but not by antibody to CD44. Data are expressed as migratory
index (cells
migrating in response to Eta-1/opn/cells migration in response to buffer).
Values are
expressed as mean ~ SEM.
Table V.~ Hapotactic Response of Monocvtes to Etn-1/opn
Eta-1/onn Bound to Unner Side
Eta-1/opn Bound
to Lower Side ~ 0 pmol 30 pmol 90 pmol 150 pmol
0 1.0 0.15 1.5 0.2 1.8 0.6 0.2
0.1
1 ~ 5.20.35 1.50.3
3 7.8 0.4 2.1
0.3
9.3 0.8 ~ ~ 2 0.1
Table VI: Inhibition of Monocvte Hapotaxis
MI MI
i Eta-1/o n Eta-1/o n-NT
control 9.8 0.9 6.1 0.7
+GRGDS' (1mM) 3.6 1* 2.1 0.2*
+ anti CD44 (0.1 7.8 0.7 5.5 0.3
~ )
~ + anti rote rin 4.8 0.6* 1.6 0.1
3
* P<0.01 ' (SEQ ID NO:11 )
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Table VII: Effects of Eta-Ilopn Phosphon~lation on Hapotaxis artd Chemotaxis
Phosphorvlated ~ Lnphosphorylated
Eta-1/opn Eta-1/opn
I
', j Hapotactic ~ ChemotacticHapotacticChemotactic
i ~ Index i Index j
Index Index
I Control' 1 0.1 1.3 0.3 1 0.3 1 0.2
~
Eta-1/on 9.8 0.9* 13.3 1.9*3:6 0.6**11.4 1.8*
Eta-1/on NT 1.8 0.7 0.9 0.1 0.6 0.2 0.9 0.2
Eta-1/onCT ~ 1.60.5 9.61.9* 1.30.6 9.61.9*
NTIOk 4.21.1** 1.10.2 1.80.9 1.10.1
~
rEta-1/on ~ - - 1.5 0.1 10.5 2.2*
rEta-1/on (GK) 12.6 2.1 10.4 1.6*- -
~ *
rEta-1/on (CKII)8 1.8* 10.3 1.6*-
~
rEta-1/on (CKI) 10 1.9* 9.9 2.3* - -
~
~ rEta-1/opn (PKG)0.8 0.4 8.7 2.0* - I -
I ~ I
Example 8: Effects of Eta-1/opn and Various Eta-1/opn Domains on Cellular
Spreading
This example describes the domain-specific and phosphorylation-
dependent effects of Eta-1/osteopontin on the spreading of immune effector
cells (e.g.,
the spreading of monocytes j.
Macrophage spreading on extracellular matrix proteins depends, in part.
on engagement of their integrin receptors. MH-S cells attached and spread on
immobilized phosphorylated Eta-1/opn whereas MH-S cells plated on
unphosphorylated
Eta-1/opn did not spread (as determined microscopically). Spreading of the MH-
S
macrophage cell line on immobilized native Eta-1/opn is mediated by the RGD-
containing N-terminal thrombin cleavage fragment but not by the C-terminal
fragment
and is reversed by addition of soluble GRGDS (SEQ ID NO:11 ) but not control
GRGES
(SEQ ID N0:12) peptide (Figure 4A).
Moreover, phosphorylation of recombinant Eta-1/opn is required for this
activity. rEta-1/opn was phosphorylated with the indicated kinases as
previously
described (Ashkar, 1993, 1993b. 1995, ~alih,1997). rEta-1/opn (GKI,
recombinant Eta-
1/opn phosphorylated with golgi kinases isolated for mouse calvarial cells (
14 mol of
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phosphate/mol protein); rEta-1/opn (CKII) rEta-llopn phosphorylated with
casein
kinase II (9 mol phosphate/mol protein) rEta-1/opn (CKI) phosphorylated with
casein
kinase I (11 mol phosphate/mol protein) rEta-1/opn (PKG) recombinant Eta-1/opn
phosphorylated with cGMP dependent protein kinase (3 mol phosphate/mol protein
).
Since none of the sites are phosphorylated 100% the mol phophate/mol protein
does not
reflect the total number of sites phosphorylated.
Table Vlll: Spreading Indices Indicate the Effect of Phosphorvlation of
Recombinant
Eta-1/opn (rEta-1/opn) and Dephosphorvlation of Native Eta-1/opn
Phosphorylated Unphosphorylated
Eta-1/o n '~,
~ Eta-1/o n
~ Control 1 1 0.1
0.3
Eta-1/o n ~ 10 2.1* 2 0.7
Eta-1/o nNT ~ 111.6* 111.5
Eta-1/o n CT 2 0.7 2 0.8
~
NTlOk 52.4** 70.5
REta-1/o n - 2.4 0.3
rEta-1/o n (GK) 11 1.7* -
rOEta-1/o n (CKII)12 3.1 * -
I, rEta-1/opn 8 2.6* -
(CKII) ~
i
' Eta-1/opn(PKG) -
' 1 0.1
Cleavage of Eta-1/opn with thrombin exposes the RGD motif and may
enhance its cell attachment properties (Senger et al. (1995)). Mutagenesis of
the RGD
sequence substantially reduced attachment of melanoma cells (Smith et al. (
1998)),
1~ tumor cells, and gingival fibroblasts (Xuan et al. (1995)) demonstrating
the necessity of
this motif.
While the RGD sequence is necessary for integrin binding, it is not
specific for a particular integrin receptor. Eta-1/opn may be secreted in
nonphosphorylated (Kubota et al. ( 1989) Biochem. Biophys. Res. Cornm.
162:1453-
1459: Chambers et al. ( 1992) Anticancer Res. 12:43-47: Barak-Shalom et al. (
1995)
Cornp. Bioche»t. Physiol. 111:49-~9: and Chang and Prince ( 1993) Cancer Res.
X3:2? 17-2220) and phosphorylated forms that contain up to 28 phosphate
residues
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(Sorensen and Peterson ( 1994) Biochem. Biophys. Res Comrn. 198:200-205; Salih
et al.
( 1995) Aran. NYAcad. Sci. 760:357-360; Salih et al. ( 1997) J. Biol. Chem.
272:13966-
13973. Phosphorylation is functionally important because it may determine
whether
Eta-1/opn associates with the cell surface or with the extracellular matrix
and it may be
essential for integrin-mediated cell adhesion. Therefore. phosphorylation of
the
molecule may provide selectivity of integrin binding. Phosphorylation has to
occur at
specific sites because Golgi kinases and casein kinases I or II can activate
Eta-1/opn
whereas protein kinases A or G phosphorylate the recombinant molecule but do
not
confer integrin binding.
In a second experiment, MH-S cells attached to, but did not spread on
phosphorylated and unphosphorylated PNGRGDSLAYGLR (SEQ ID N0:13) synthetic
peptides. In an attempts to define an N-terminal peptide capable of support
attachment
and spreading, partial tryptic, chemotryptic and Asp-N endopeptidase digestion
of Eta-
1/opn was performed. None of these, however, resulted in the isolation of an
active
peptide. A 10-kD fragment isolated from a Lys-C digest was found to be active.
NK10
has the NHS-terminal sequence QETLPSN (SEQ 1D N0:14) and is predicted to
terminate at the thrombin cleavage site. This 10-kD fragment also contains -~
mol of
phosphate per 1 mol of peptide at seven potential phosphorylation sites. NK10
was
capable of mediating the spreading of macrophages at approximately 40 %
(mol/mol)
the activity of the larger N-terminal thrombin fragment (Figure 4B). Upon
dephosphorylation of this peptide spreading activity is lost, but can be
regained by
rephosphorylation with Golgi kinases. Earlier studies which showed that RGD-
containing peptides can confer function may have induced non-specific effects
through
multiple integrin receptors. These data demonstrate that the RGD motif is
necessary but
not sufficient to mediate specific Eta-1/opn activity, phosphorylation in
defined sites is
also needed.
Example 9: Eta-1/osteopontin-dependent modulation of type-1 immunity cvtokines
via distinct receptors on immune effector cells l e. ~.. macrophages i
As shown in Examples 6-8. Eta-1/opn interaction with macrophages is
mediated through two distinct functional receptors. Engagement of CD44
mediates
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chemotactic migration and interaction with av(~~ integrin causes haptotaxis,
adhesion and
spreading.
To determine whether distinct macrophage receptors were responsible for
the type-1 cvtokine production by Eta-1/opn stimulated macrophages, fragments
from a
~ Lys-C digest of Eta-1/opn were analyzed for the ability to stimulate IL-12
secretion. The
kDa NK10 proteolytic fragment from the N-terminal portion of Eta-1/opn
containing
the inte~rin binding site was found to be sufficient to induce macrophage IL-
12 expression
(Figure SA).
In contrast to IL-12 induction, inhibition of IL-10 depends on engagement
10 of the CD44 receptor: Figure SB shows that Eta-1/opn-dependent inhibition
of lL-4-
induced production of IL-10 was reversed by anti-CD44 (KM81, purified from
ATCC
hybridoma TIB 241, described in Mayake et al. ( 1990) J. Exp. Med. 171:477-
488) but
not anti-integrin (33 antibody (Pharmingen, described in Schultz and Armant (
1995) J.
Biol. Chem. 270:11522). Moreover, macrophages from CD44~~- mice are resistant
to Eta-
1/opn inhibition of the IL-10 response. Figure SC shows that secretion of IL-
12 in
response to Eta-1/opn was not impaired in macrophages from mice that are
deficient in the
CD44 gene and cells from C3H.HeJ mice (which do not respond to endotoxin)
displayed
the same levels of induction as control mice. Conversely, while the inhibition
of IL-10
secretion was not affected in C3H.HeJ mice or in C57B1/6 mice, it was
abrogated in CD44
~- mice.
Example 10' Phosphorylation of Eta-1/osteopontin is necessary for enQa~ement
of
inte~irin receptors on macrophages leading to IL-12 production but not
for ligation of CD44 leading to IL-10 inhibition
Eta-1/opn is secreted in nonphosphorylated and phosphorylated forms
(ref.). Phosphorylation may allow Eta-1/opn to associate with the cell surface
rather than
the extracellular matrix (refs) through a contribution to integrin binding. In
contrast, serine
phosphorylation of recombinant Eta-1/opn is not required for CD44-dependent
interactions
leading to chemotactic migration (ref.). To determine whether phosphorylation
of Eta-
1/opn might affect its ability to regulate cvtokine expression. phosphorylated
and
dephosphorylated Eta-1/opn were tested-for their ability to affect IL-12/IL-10
secretion.
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Figure 6A demonstrates that dephosphorylated purified naturally-produced
Eta-1/opn abolished IL-12 stimulatory activity whereas phosphorylation of
recombinant
Eta-1/opn at specific sites restores activity. Secretion of IL-12 was measured
by ELISA
after culture of resident peritoneal macrophages with 6 pmol/ml of
dephosphorylated
natural Eta-1/opn (dpEta-1/osteopontin), recombinant Eta-1/opn (rEta-
1/osteopontin) or
recombinant phosphorylated Eta-1/opn (rEta-1/osteopontin-P) for 6 h in defined
medium
at 37°C. Dephosphorylated native Eta-1/opn and recombinant
(unphosphorylated) Eta-
1 /opn does not induce IL-12 production but retains inhibitory activity for IL-
10.
Recombinant Eta-1/opn phosphorylation with Golgi kinases (rEta-1/osteopontin--
P)
conferred IL-12 inducing activity while similar levels of phosphorylation by
PKA and PKC
did not restore this activity.
Although recombinant Eta-1/opn lacking phosphate groups cannot induce
IL-12, this molecule retains inhibitory activity for the macrophage IL-10
response (Figure
6B). Dephosphorylation of native Eta-1/opn resulted in loss of IL-12 inducing
activity,
while phosphorylation of (inactive) recombinant Eta-1/opn restored this
function. There is
abundant evidence that phosphorylation can regulate the biological activity of
intracellular
enzymes and their substrates; these results indicate that serine
phosphorylation can also
provide molecular information that regulates the biological activity of a
secreted protein.
Figure 7 depicts cytokine profiles for macrophages after engagement by
phosphorylated versus unphosphorylated Eta-1/opn, as well as by Eta-1/opn
fragments.
TNFa, TGF~3, and the type-1 cvtokine IL-12 as well as the type-1 cvtokine IL-
10 were
determined by commercial ELISA kits. (For induction of TGF~ cells were
cultured on the
indicated ligand for 6 h in defined media at 37° C in a humidified
atmosphere.) Cytokine
secretion data is presented as fold induction over resting values. Ligation of
integrin
receptors (e.g., by native Eta-1/opn, recombinant phosphorylated Eta-1/opn, N-
terminal
fragment or NK10) on macrophages caused predominantly secretion of IL-12,
TNFa.
TGF(3 but not IL-10 or IL-la (e.g., a type-1 cytokine profile).
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Example 1''' Si~nal Transduction Pathways Associated with Eta-1/opn-Mediated
Functions Including Chemotaxis. Hapotaxis and Cell Spreading
This example demonstrates that distinct cellular signaling mechanisms
are activated by association of the two key functional domains of Eta-
1/osteopontin with
their respective receptors on macropages.
Three biological response phenotypes have been observed in association
with ligation of CD44 and integrin a"~i3: chemotaxis, cell crawling, and
activation (after
spreading on substrate). Accordingly, it was tested whether these functions
were
distinguishable on the level of intracellular signal transduction. Signal
transduction
mechanisms were initially examined through the use of specific chemical
inhibitors at
the following final concentrations: 50 mM for cycloheximide, PKA inhibitors
H89 at
1mM and. H7 at 20 uM, Inhibitors of PI pathway Wortmannin at 10 nM, tyrosine
kinase
inhibitors genistein at 25 uM for, PKC inhibitor chelerythrine at 20 uM,
Casein Kinase
II inhibitor quercetin at 6 mM. In all experiments using these compounds cells
were
preincubated for 0.5 hours with the inhibitors before start of the experiment.
Cell
viability was determined by trypan blue exclusion on cell samples before and
after the
termination of the experiments. Cell viability in all reported experiments was
> 95 %.
Microfilament disruption was carried out by preincubation of the cell cultures
for one
hour in 50 uM cytochalasin D. Microtubule dissociation was carried out by pre-
incubation of the cultures for 6 hours in 1 ~tM colchicine. All compounds were
suspended in either DMSO or absolute ethanol and were added to the culture
media at
1:1000 dilution. Controls were carried out with the corresponding vehicle. In
separate
experiments in which PKC and PKA were chemically activated, 50 ng/ml phorbol
12-
myristate 13-acetate and 10' M of forskolin were used respectively. In these
experiments treatments were for 2 hours.
In order to delineate the mechanisms mediating differential activities of
Eta-1/opn following ligation of its two groups of receptors, the effects of
protein kinase
inhibitors, cytoskeletal disrupting agents. and toxins were tested on the
macrophage
responses. Eta-1/opn mediated chemotaxis is diminished by the G-protein
inhibitor
pertussis toxin but not by inhibitors of protein kinase C or A. in contrast
haptotaxis is
not affected by pertussis toxin or protein. kinase A inhibitors but is
inhibited by the
protein kinase C inhibitor chelerythrine.
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Table IX: Inl2ibition of Monocvte HapotaxiS
MI Eta-1/opn I MI Eta-1/opn
~
Control 9.8 0.9 ' 6.1 - 0.7
~ + Wortmanin ( l OnMI10.1 2 ~ 8.8 1.3
~ + Chelervthrine (20~tM)3.3 0.2* 1.3 0.1
~
+ Genistein (25 M) 3.2 0.5** 2.0 0.3
~
+PT ~ 10.6 1.1* 9.4 1.5*
~ +H7(20 M) I 10.81.7 6.9~.0
+ Cytocholasin D ( 1.1 0.2* 2.7 0.1
l~tM) ~
* P<0.01
Table X: Inhibition of Monocyte Chemotaxis
MI Eta-1/o MI Eta-1/o n
n CT
control ~ 13.3 1.9 9.6 1.9
+ Wortmanin ( l OnM) 12.6 2.5 10.5 1.6
~
+ Cheler thrine (20 3.1 0.5* 1.9 0.8*
M)
+ Genistein (25~M) - 6.6 1.1 4.3 0.7
I **
+PT . 2.30.21* 1.60.3*
I + H 7 (20~.M) I 8.7 1.2
14.1 2.7 ~
+ Cytocholasin D ( ? 2 0.3*
l~tM) '~ 1.8 0.9*
*P<0.01 **P<0.05
Ligation of integrin receptors by Eta-1/opn may lead to
dephosphorylation of Src in chicken osteoblasts and recombinant Eta-1/opn may
phosphorylate paxillin, tensin, and p125 focal adhesion kinase in ras-
transformed
NIH3T3 cells. G proteins are linked to ligation of CD44 by Eta-1/osteopontin.
Ligation
of integrin a"(3~ initially leads to activation of PKC (see e.g., Figures 8
and 9). After cell
spreading. the cytoskeleton rearranges and a second integrin-associated signal
transduction component, phosphatidylinositol 3-kinase, is activated (Figure
4B1.
Spreading of macrophages on Eta-1/opn is inhibited by chelerythrine and by
inhibitors
of the phosphatidylinositol pathway consistent with earlier reports that
engagement of
integrin a,.~3 on osteoclasts by Eta-1/opn leads to activation of
phosphatidylinositol 3-
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hydroxyl kinase . Direct measurement of phosphorylation of
phosphatidylinositol 3-
kinase is in accord with the inhibitor-based observations. The selective
inhibition of
macrophage responses argues against a toxic effect of these inhibitors.
The data in Example 12 demonstrate that CD44-dependent chemotaxis is
associated with a signal transduction pathway that involves G-protein, while
integrin-
dependent haptotaxis is mediated by a pathway involving protein kinase C. Once
a cell
has spread. phosphatidylinositol signaling is integrated as a second component
into
integrin-dependent signaling. Distinct macrophage phenotypes induced by Eta-
1/opn
can be separated on the level of signal transduction using G-protein, protein
kinase C,
and phosphatidylinositol 3-kinase as biochemical markers.
Example 11: Domain-Specificity and Phosphorvlation-Dependence ofInduction of
Metallo~rotease Secretion by Eta-1/osteopontin
Because cell spreading is often associated with cellular activation, an
investigation was made into whether the interaction between phosphorylated Eta-
1/opn
and macrophages leads to additional signs of macrophage activation including
secretion
of metalloproteinases and cytokines.
For metalloprotease secretion assays, MH-S cells were stimulated for 6
hours with either phosphorylated or unphosphorylated Eta-1/opn at a
concentration of 10
pg/ml in serum-free defined medium. In order to visualize the secreted
metalloproteases, gelatin zymograms were performed. Briefly, cell culture
supernatant
was collected after 6 hours of culture, concentrated 5 times and resuspended
in 200 pl
zymogram buffer (40 mM Tris, pH 7.5) before addition to Laemmli sample buffer
and
electrophoresis in 10% polyacrylamide gels impregnated with lmg/ml gelatin.
Following electrophoresis, gels were incubated for 30 min at 37°C in 50
ml of 50 mM
Tris-HCl buffer, pH 8.0, containing 2% Triton-X 100 and 10 mM CaCh to remove
the
SDS, followed by incubation for 18 h in 50 mM Tris-HCl buffer, pH 8.0,
containing ~
mM CaCI~. After staining the gels with Coomassie Brilliant Blue, gelatin and
casein
degrading enzymes were identified as clear bands against a dark blue
background.
MMP-9 and MMP-2 were both visible in the samples stimulated with
natural Eta-1/opn or with phosphorylated rEta-1/opn. Control MH-S cells were
incubated
with serum-free defined medium. MMP9 but not MMP2 was stimulated by the N-
terminal
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fragment of osteopontin, while the C-terminal fragment of Eta-1/opn had little
or no
stimulatory activity. MMP-9 induction could be inhibited by GRGDS (SEQ m NO:11
but not GRGES (SEQ ID N0:12). Dephosphorylation of Eta-1/opn with acid
phosphatase
abolished the stimulatory activity of Eta-1/opn. Similarly. rEta-1/opn had no
stimulatory
activity. The results demonstrate that only phosphorylated Eta-1/opn had a
stimulatory
effect the gelatinolytic activity secreted by MH-S cells.
Definition of the functional domains of Eta-1/opn in the examples described
above represents an important step in understanding this process and is
critical for the
rational development of Eta-1/opn analogs that antagonize or mimic discrete
biological
activities of the parent molecule. Examples 13-14 describe the generation and
testing of
such analogs.
Example 13' Generation of Biosynthetic Immunomodulator~Molecules That
Stimulate
IL-12
A first generation osteopontin-derived biosynthetic molecule was
engineered based on the isolation of a domain of osteopontin sufficient to
impart IL-12
stimulatory activity when isolated from the naturally-occurring polypeptide.
Figure 10
depicts the amino acid and encoding nucleic acid sequence of such a molecule,
termed
immunomodulin-1, based on its ability to modulate immune responses. In
particular, the
biosynthetic immunomodulin-1 molecule depicted in Figure 10 has the ability to
bias an
immune response from a type-2 response to a type-1 response.
Example 14~ Generation of Biosynthetic Immunomodulatory Molecules That Inhibit
IL-10
A first generation osteopontin-derived biosynthetic molecule was
engineered based on the isolation of a domain of osteopontin sufficient to
impart IL-10
inhibitory activity when isolated from the naturally-occurring polypeptide.
Figure 11
schematically depicts the structure of such a molecule, termed immunomodulin-
2, based on
its ability to modulate immune responses. In particular, the biosynthetic
immunomodulin-2
molecule depicted in Figure 11 has the ability to bias an immune response from
a type-2
response to a type-1 response.
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Example l~: Testing of Immunomodulin-1 and Immunomodulin-2 in vitro and in
vivo
Immunomodulin-1 and Immunomodulin-2 were tested for their ability to
stimulate and/or inhibit cytokine secretion. in particular for their ability
to stimulate and/or
inhibit secretion of IL-12 and/or IL-10. As shown in Figure 12, Immunomodulin-
1 is
capable of stimulating IL-12 secretion by macrophages to levels greater than
those induced
by LPS. Moreover, as demonstrated in Figure 13. IL-10 is capable of inhbiting
IL-4
induced IL-10 secretion by macrophages. The data demonstrate that
Immunomodulin-1
and -2 are capable of biasing an immune response towards a type-1 response in
vitro.
In order to test the ability of Immunomodulin-2 to bias an immune response
in vivo from a type-2 to a type-1 response, C57blk mice were sensitized with a
single
intraperitoneal injection of 0.1 ~g/ml poke weed adsorbed to 2 mg aluminum
hydroxide.
Animals were challenged at 7 or 14 days following sensitization with either
aerosol poke
weed ( 1 ~g/ml in an atomizer) or via subcutaneous injection of 0.05 qgiml
poke weed in
phosphate-buffered saline (PBS). Plasma levels of IgE were determined by ELISA
using
antibodies to mouse IgE. Immunomodulin-2 was injected intraperitoneally ( 100
ql at a
concentration of 10 qg/ml in PBS). Plasma concentrations of IgE were
determined 3 and
14 days after injection (Figure 14).
Equivalents
Those skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, many equivalents to the specific
embodiments of the
invention described herein. Such equivalents are intended to be encompassed by
the
following claims.
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SEQUENCE LISTING
<110> DANA-FARBER CANCER INSTITUTE, INC.
CHILDREN'S MEDICAL CENTER CORPORATION
<120> METHODS AND COMPOSITIOPdS FOR MODULATING AN IMMUNE
RESPONSE
<130> CMZ-121CPPC
<140>
<141>
<150> USSN 60/129,772
<151> 1999-04-15
<160> 6
<170> PatentIn Ver. 2.0
<210>
1
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945
<212>
DNA
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Homo
sapiens
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CDS
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(1)..(942)
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1
atg attgca gtgatttgc ttttgcctc ctaggcatc acctgt gcc 48
aga
Met IleAla ValIleCys PheCysLeu LeuGlyIle ThrCys Ala
Arg
1 5 10 15
ata gttaaa caggetgat tctggaagt tctgaggaa aagcag ctt 96
cca
Ile ValLys GlnAlaAsp SerGlySer SerGluGlu LysGln Leu
Pro
20 25 30
tac aaatac ccagatget gtggccaca tggctaaac cctgac cca 144
aac
Tyr LysTyr ProAspAla ValAlaThr TrpLeuAsn ProAsp Pro
Asn
35 40 45
tct aagcag aatctccta gccccacag aatgetgtg tcctct gaa 192
cag
Ser LysGln AsnLeuLeu AlaProGln AsnAlaVal SerSer Glu
Gln
50 55 60
gaa aatgac tttaaacaa gagaccctt ccaagtaag tccaac gaa 240
acc
Glu AsnAsp PheLysGln GluThrLeu ProSerLys SerAsn Glu
Thr
65 70 75 80
agc gaccac atggatgat atggatgat gaagatgat gatgac cat 288
cat
Ser AspHis MetAspAsp MetAspAsp GluAspAsp AspAsp His
His
g5 90 95
gtg agccag gactccaLt gacLcgaac gactctgat gatgta gat 336
gac
Val SerGln AspSerIle AspSerAsn AspSerAsp AspVal Asp
Asp
100 105 110
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gac act gat gat tct cac cag tct gat gag tct cac cat tct gat gaa 384
Asp Thr Asp Asp Ser His Gln Ser Asp Glu Ser His His Ser Asp Glu
115 120 125
tctgat gaactg gtcactgat tttccc acggacctg ccagcaacc gaa 432
SerAsp GluLeu ValThrAsp PhePro ThrAspLeu ProAlaThr Glu
130 135 140
gttttc actcca gttgtcccc acagta gacacatat gatggccga ggt 480
ValPhe ThrPro ValValPro ThrVal AspThrTyr AspGlyArg Gly
145 150 155 160
gatagt gtggtt tatggactg aggtca aaatctaag aagtttcgc aga 528
AspSer ValVal TyrGlyLeu ArgSer LysSerLys LysPheArg Arg
165 170 175
cctgac atccag taccctgat getaca gacgaggac atcacctca cac 576
ProAsp IleGln TyrProAsp AlaThr AspGluAsp IleThrSer His
180 185 190
atg gaa agc gag gag ttg aat ggt gca tac aag gcc atc ccc gtt gcc 624
Met Glu Ser Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro Val Ala
195 200 205
cag gac ctg aac gcg cct tct gat tgg gac agc cgt ggg aag gac agt 672
Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys Asp Ser
210 215 220
tat gaa acg agt cag ctg gat gac cag agt get gaa acc cac agc cac 720
Tyr Glu Thr Ser Gln Leu Asp Asp Gln Ser Ala Glu Thr His Ser His
225 230 235 240
aag cag tcc aga tta tat aag cgg aaa gcc aat gat gag agc aat gag 768
Lys Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser Asn Glu
245 250 255
cattccgatgtg attgatagt caggaactt tccaaagtc agccgt gaa 816
HisSerAspVal IleAspSer GlnGluLeu SerLysVal SerArg Glu
260 265 270
ttccacagccat gaatttcac agccatgaa gatatgctg gttgta gac 864
PheHisSerHis GluPheHis SerHisGlu AspMetLeu ValVal Asp
275 280 285
cccaaaagtaag gaagaagat aaacacctg aaatttcgt atttct cat 912
ProLysSerLys GluGluAsp LysHisLeu LysPheArg IleSer His
290 295 300
gaattagatagt gcatcttct gaggtcaat taa 945
GluLeuAspSer AlaSerSer GluValAsn
305 310
<210> 2
<211> 314
<212> PRT
<213> Homo Sapiens
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<400> 2
Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala
1 5 l0 15
Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Leu
20 25 30
Tyr Asn Lys Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn Pro Asp Pro
35 40 45
Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Asn Ala Val Ser Ser Glu
50 55 60
Glu Thr Asn Asp Phe Lys Gln Glu Thr Leu Pro Ser Lys Ser Asn Glu
65 70 75 80
Ser His Asp His Met Asp Asp Met Asp Asp Glu Asp Asp Asp Asp His
85 90 95
Val Asp Ser Gln Asp Ser Ile Asp Ser Asn Asp Ser Asp Asp Val Asp
100 105 110
Asp Thr Asp Asp Ser His Gln Ser Asp Glu Ser His His Ser Asp Glu
115 120 125
Ser Asp Glu Leu Val Thr Asp Phe Pro Thr Asp Leu Pro Ala Thr Glu
130 135 140
Val Phe Thr Pro Val Val Pro Thr Val Asp Thr Tyr Asp Gly Arg Gly
145 150 155 160
Asp Ser Val Val Tyr Gly Leu Arg Ser Lys Ser Lys Lys Phe Arg Arg
165 170 175
Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr Ser His
180 185 190
Met Glu Ser Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro Val Ala
195 200 205
Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys Asp Ser
210 215 220
Tyr Glu Thr Ser Gln Leu Asp Asp Gln Ser Ala Glu Thr His Ser His
225 230 235 240
Lys Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser Asn Glu
245 250 255
His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser Lys Val Ser Arg Glu
260 265 270
Phe His Ser His Glu Phe His Ser His Glu Asp Met Leu Val Val Asp
275 280 285
Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe Arg Ile Ser His
290 295 300
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Glu Leu Asp Ser Ala Ser Ser Glu Val Asn
305 310
<210> 3
<211> 903
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(900)
<400> 3
atg aga att gca gtg att tgc ttt tgc ctc cta ggc atc acc tgt gcc 48
Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala
1 5 10 15
ata cca gtt aaa cag get gat tct gga agt tct gag gaa aag cag ctt 96
Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Leu
20 25 30
tac aac aaa tac cca gat get gtg gcc aca tgg cta aac cct gac cca 144
Tyr Asn Lys Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn Pro Asp Pro
35 40 45
tctcagaagcag aatctccta gccccacag accctt ccaagtaag tcc 192
SerGlnLysGln AsnLeuLeu AlaProGln ThrLeu ProSerLys Ser
50 55 60
aacgaaagccat gaccacatg gatgatatg gatgat gaagatgat gat 240
AsnGluSerHis AspHisMet AspAspMet AspAsp GluAspAsp Asp
65 70 75 80
gaccatgtggac agccaggac tccattgac tcgaac gactctgat gat 288
AspHisValAsp SerGlnAsp SerIleAsp SerAsn AspSerAsp Asp
85 90 95
gtagatgacact gatgattct caccagtct gatgag tctcaccat tct 336
ValAspAspThr AspAspSer HisGlnSer AspGlu SerHisHis Ser
100 105 110
gatgaatctgat gaactggtc actgatttt cccacg gacctgcca gca 384
AspGluSerAsp GluLeuVal ThrAspPhe ProThr AspLeuPro Ala
115 120 125
accgaagttttc actccagtt gtccccaca gtagac acatatgat ggc 432
ThrGluValPhe ThrProVal ValProThr ValAsp ThrTyrAsp Gly
130 135 140
cgaggtgatagt gtggtttat ggactgagg tcaaaa tctaagaag ttt 480
ArgGlyAspSer ValValTyr GlyLeuArg SerLys SerLysLys Phe
145 150 155 160
cgcagacctgac atccagtac cctgatget acagac gaggacatc acc 528
ArgArgProAsp IleGlnTyr ProAspAla ThrAsp GluAspIle Thr
165 170 175
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tcacac atggaaagc gaggag ttgaatggt gcatacaag gccatc ccc 576
SerHis MetGluSer GluGiu LeuAsnGly AlaTyrLys AlaIle Pro
180 185 190
gttgcc caggacctg aacgcg ccttctgat tgggacagc cgtggg aag 624
ValAla GlnAspLeu AsnAla ProSerAsp TrpAspSer ArgGly Lys
195 200 205
gacagt tatgaaacg agtcag ctggatgac cagagtget gaaacc cac 672
AspSer TyrGluThr SerGln LeuAspAsp GlnSerAla GluThr His
210 215 220
agccac aagcagtcc agatta tataagcgg aaagccaat gatgag agc 720
SerHis LysGlnSer ArgLeu TyrLysArg LysAlaAsn AspGlu Ser
225 230 235 240
aatgag cattccgat gtgatt gatagtcag gaactttcc aaagtc agc 768
AsnGlu HisSerAsp ValIle AspSerGln GluLeuSer LysVal Ser
245 250 255
cgtgaa ttccacagc catgaa tttcacagc catgaagat atgctg gtt 816
ArgGlu PheHisSer HisGlu PheHisSer HisGluAsp MetLeu Val
260 265 270
gtagac cccaaaagt aaggaa gaagataaa cacctgaaa tttcgt att 864
ValAsp ProLysSer LysGlu GluAspLys HisLeuLys PheArg Ile
275 280 285
tctcat gaattagat agtgca tcttctgag gtcaattaa 903
SerHis GluLeuAsp SerAla SerSerGlu ValAsn
290 295 300
<210> 4
<211> 300
<212> PRT
<213> Homo sapiens
<400> 4
Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala
1 5 10 15
Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Leu
20 25 30
Tyr Asn Lys Tyr Pro Asp Ala Val Ala Thr Trp Leu Asn Pro Asp Pro
35 40 45
Ser Gln Lys Gln Asn Leu Leu Ala Pro Gln Thr Leu Pro Ser Lys Ser
50 55 60
Asn Glu Ser His Asp His Met Asp Asp Met Asp Asp Glu Asp Asp Asp
65 70 75 80
Asp His Val Asp Ser Gln Asp Ser Ile Asp Ser Asn Asp Ser Asp Asp
85 90 95
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Val Asp Asp Thr Asp Asp Ser His Gln Ser Asp Glu Ser His His Ser
100 105 110
Asp Glu Ser Asp Glu Leu Val Thr Asp Phe Pro Thr Asp Leu Pro Ala
115 120 125
Thr Glu Val Phe Thr Pro Val Val Pro Thr Vai Asp Thr Tyr Asp Gly
130 135 140
Arg Gly Asp Ser Val Val Tyr Gly Leu Arg Ser Lys Ser Lys Lys Phe
145 150 155 160
Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp Glu Asp Ile Thr
165 170 175
Ser His Met Glu Ser Glu Glu Leu Asn Gly Ala Tyr Lys Ala Ile Pro
180 185 190
Val Ala Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser Arg Gly Lys
195 200 205
Asp Ser Tyr Glu Thr Ser Gln Leu Asp Asp Gln Ser Ala Glu Thr His
210 215 220
Ser His Lys Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn Asp Glu Ser
225 230 235 240
Asn Glu His Ser Asp Val Ile Asp Ser Gln Glu Leu Ser Lys Val Ser
245 250 255
Arg Glu Phe His Ser His Glu Phe His Ser His Glu Asp Met Leu Val
260 265 270
Val Asp Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys Phe Arg Ile
275 280 285
Ser His Glu Leu Asp Ser Ala Ser Ser Glu Val Asn
290 295 300
<210> 5
<211> 864
<212> DNA
<213> Homo sapiens
<220>
<221> CDS
<222> (1)..(861)
<400> 5
atg aga att gca gtg att tgc ttt tgc ctc cta ggc atc acc tgt gcc 48
Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala
1 5 10 15
ata cca gtt aaa cag get gat tct gga agt tct gag gaa aag cag aat 96
Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Asn
20 25 30
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getgtgtcctct gaagaaacc aatgacttt aaacaa gagaccctt cca 144
AlaValSerSer GluGluThr AsnAspPhe LysGln GluThrLeu Pro
35 40 45
agtaagtccaac gaaagecat gaccacatg gatgat atggatgat gaa 192
SerLysSerAsn GluSerHis AspHisMet AspAsp MetAspAsp Glu
50 55 60
gatgatgatgac catgtggac agccaggac tccatt gactcgaac gac 240
AspAspAspAsp HisValAsp SerGlnAsp SerIle AspSerAsn Asp
65 70 75 80
tctgatgatgta gatgacact gatgattct caccag tctgatgag tct 288
SerAspAspVal AspAspThr AspAspSer HisGln SerAspGlu Ser
85 90 95
caccattctgat gaatctgat gaactggtc actgat tttcccacg gac 336
HisHisSerAsp GluSerAsp GluLeuVal ThrAsp PheProThr Asp
100 105 110
ctgccagcaacc gaagttttc actccagtt gtcccc acagtagac aca 384
LeuProAlaThr GluValPhe ThrProVal ValPro ThrValAsp Thr
115 120 125
tatgatggccga ggtgatagt gtggtttat ggactg aggtcaaaa tct 432
TyrAspGlyArg GlyAspSer ValValTyr GlyLeu ArgSerLys Ser
130 135 140
aagaagtttcgc agacctgac atccagtac cctgat getacagac gag 480
LysLysPheArg ArgProAsp IleGlnTyr ProAsp AlaThrAsp Glu
145 150 155 160
gacatcacctca cacatggaa agcgaggag ttgaat ggtgcatac aag 528
AspIleThrSer HisMetGlu SerGluGlu LeuAsn GlyAlaTyr Lys
165 170 175
gccatccccgtt gcccaggac ctgaacgcg ccttct gattgggac agc 576
AlaIleProVal AlaGlnAsp LeuAsnAla ProSer AspTrpAsp Ser
180 185 190
cgtgggaaggac agttatgaa acgagtcag ctggat gaccagagt get 624
ArgGlyLysAsp SerTyrGlu ThrSerGln LeuAsp AspGlnSer Ala
195 200 205
gaaacccacagc cacaagcag tccagatta tataag cggaaaget aat 672
GluThrHisSer HisLysGln SerArgLeu TyrLys ArgLysAla Asn
210 215 220
gatgagagcaat gagcattcc gatgtgatt gatagt caggaactt tcc 720
AspGluSerAsn GluHisSer AspValIle AspSer GlnGluLeu Ser
225 230 235 240
aaagtcagccgt gaattccac agccatgaa tttcac agccatgaa gat 768
LysValSerArg GluPheHis SerHisGlu PheHis SerHisGlu Asp
245 250 255
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atg ctg gtt gta gac ccc aaa agt aag gaa gaa gat aaa cac ctg aaa 816
Met Leu Val Val Asp Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys
260 265 270
ttt cgt att tct cat gaa tta gat agt gca tct tct gag gtc aat taa 864
Phe Arg Ile Ser His Glu Leu Asp Ser Ala Ser Ser Glu Val Asn
275 280 285
<210> 6
<211> 287
<212> PRT
<213> Homo Sapiens
<400> 6
Met Arg Ile Ala Val Ile Cys Phe Cys Leu Leu Gly Ile Thr Cys Ala
1 5 10 15
Ile Pro Val Lys Gln Ala Asp Ser Gly Ser Ser Glu Glu Lys Gln Asn
20 25 30
Ala Val Ser Ser Glu Glu Thr Asn Asp Phe Lys Gln Glu Thr Leu Pro
35 40 45
Ser Lys Ser Asn Glu Ser His Asp His Met Asp Asp Met Asp Asp Glu
50 55 60
Asp Asp Asp Asp His Val Asp Ser Gln Asp Ser Ile Asp Ser Asn Asp
65 70 75 80
Ser Asp Asp Val Asp Asp Thr Asp Asp Ser His Gln Ser Asp Glu Ser
85 90 95
His His Ser Asp Glu Ser Asp Glu Leu Val Thr Asp Phe Pro Thr Asp
100 105 110
Leu Pro Ala Thr Glu Val Phe Thr Pro Val Val Pro Thr Val Asp Thr
115 120 125
Tyr Asp Gly Arg Gly Asp Ser Val Val Tyr Gly Leu Arg Ser Lys Ser
130 135 140
Lys Lys Phe Arg Arg Pro Asp Ile Gln Tyr Pro Asp Ala Thr Asp Glu
145 150 155 160
His Ile Thr Ser His Met Glu Ser Glu Glu Leu Asn Gly Ala Tyr Lys
165 170 175
Ala Ile Pro Val Ala Gln Asp Leu Asn Ala Pro Ser Asp Trp Asp Ser
180 185 190
Arg Gly Lys Asp Ser Tyr Glu Thr Ser ~ln Leu Asp Asp Gln Ser Ala
195 200 205
Glu Aia :iis Ser His Lys Gln Ser Arg Leu Tyr Lys Arg Lys Ala Asn
210 215 220
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Asp Glu Ser Asn Glu His Ser Asp Val I_le Asp Ser Gln Glu Leu Ser
225 230 235 240
Lys Val Ser Arg Glu Phe His Ser His Glu Phe His Ser His Glu Asp
245 250 255
Met Leu Val Val Asp Pro Lys Ser Lys Glu Glu Asp Lys His Leu Lys
260 265 270
Phe Arg Ile Ser His Glu Leu Asp Ser Ala Ser Ser Glu Val Asn
275 280 285
