Note: Descriptions are shown in the official language in which they were submitted.
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Therapeutic Compositions and Methods for Treating Disease
States with Myeloid Progenitor Inhibitory Factor-1 (MPIF-1),
Monocyte Colony Inhibitory Factor (M-CIF), and
Macrophage Inhibitory Factor-4 (MIP-4)
Background of the Invention
- Field oJtf:e Invention
The present invention relates to novel chemokine polypeptides and encoding
nucleic acids. More specifically) therapeutic compositions and methods are
provided
using isolated nucleic acid molecules encoding a human myeloid progenitor
inhibitory
factor-1 (MPIF-1) polypeptide (previously termed MIP-3 and chemokine Q38
(CK~38 or
ckb-8)); a human monocyte-colony inhibitory factor (M-CIF) polypeptide
(previously
termed MIP 1-y and chemokine Q31 (CK~i 1 or ckb-1 )), and a macrophage
inhibitory
protein-4 (MIP-4), as well as MPIF-1, M-CIF and/or MIP-4 polypeptides
themselves,
as are vectors, host cells and recombinant methods for producing the same.
Related Art
Chcmokines, also referred to as intercrine cytokincs) are a subfamily of
structura115~ and functionally related cytokines. These molecules are 8-14 kd
in size. In
general chemokines exhibit 20% to 75% homology at the amino acid level and are
characterized by four conserved cysteine residues that form two disulfide
bonds. Based
on the arrangement of the first two cysteine residues, chemokines have been
classified
into two subfamilies, alpha and beta. In the alpha subfamily, the first two
cysteines arc
separated by one amino acid and hence are referred to as the "C-X--C"
subfamily. In
the beta subfamily, the two cysteines are in an adjacent position and are,
therefore,
referred to as the -C-C- subfamily. Thus far, at least eight different members
of this
family have been identified in humans.
The intercrine cytokines exhibit a wide variety of functions. A hallmark
feature
is their ability to elicit chemotactic migration of distinct cell types,
including monocytes,
neutrophils) T lymphocytes, basophils and fibroblasts. Many chemokines have
proinflammatory activity and are involved in multiple steps during an
inflammatory
reaction. These activities include stimulation of histamine release, lysosomal
enzyme
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and leukotriene release, increased adherence of target immune cells to
endothelial cells,
enhanced binding of complement proteins, induced expression of granulocyte
adhesion
molecules and complement receptors, and respiratory burst. In addition to
their
involvement in inflammation, certain chemokines have been shown to exhibit
other
activities. For example, macrophage inflammatory protein I (MIP-1 ) is able to
suppress
hematopoietic stem cell proliferation, platelet factor-4 (PF-4) is a potent
inhibitor of
endothelial cell growth, Interleukin-8 (IL-8) promotes proliferation of
keratinocytes, and
GRO is an autocrine growth factor for melanoma cells.
In light of the diverse biological activities, it is not surprising that
chemokines
have been implicated in a number of physiological and disease conditions,
including
lymphocyte trafficking, wound healing, hematopoietic regulation and
immunological
disorders such as allergy, asthma and arthritis. An example of a hematopoietic
lineage
regulator is MIP-1. MIP-1 was originally identified as an endotoxin-induced
proinllammatory cytokine produced from macrophages. Subsequent studies have
shown
1 S that MIP-1 is composed of two different, but related, proteins MIP-1 a and
MIP-1 ~3.
Both MIP-1 a and MIP-Iii are chemo-attractants for macrophages, monocytes and
T
lymphocytes. Interestingly, biochemical purification and subsequent sequence
analysis
of a multipotent stem cell inhibitor (SCI) revealed that SCI is identical to
MIP-1~3.
Furthermore, it has been shown that MIP-1 ~i can counteract the ability of MIP-
1 a to
suppress hematopoietic stem cell proliferation. This finding leads to the
hypothesis that
the primary physiological role of MIP-1 is to regulate hematopoiesis in bone
marrow,
and that the proposed inflammatory function is secondary. The mode of action
of
MIP-1 a as a stem cell inhibitor relates to its ability to block the cell
cycle at the GAS
interphase. Furthermore, the inhibitory effect of MIP-la seems to be
restricted to
immature progenitor cells and it is actually stimulatory to late progenitors
in the
presence of granulocyte macrophage-colony stimulating factor (GM-CSF).
Marine MIP-1 is a major secreted protein from lipopolysaccharide stimulated
RA W 264.7, a marine macrophage tumor cell line. It has been purified and
found to
consist of two related proteins, MIP-1 a and MIP-1 (3.
Several groups have cloned what are likely to be the human homologs of MIP-1 a
and MIP-1 (3. In all cases, cDNAs were isolated from libraries prepared
against activated
T-cell RNA.
MIP-1 proteins can be detected in early wound inflammation cells and have been
shown to induce production of IL-1 and IL-6 from wound fibroblast cells. In
addition,
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purified native MIP-1 (comprising MIP-l, MIP-1 a and MIP-1 ~i polypeptides)
causes
acute inflammation when injected either subcutaneously into the footpads of
mice or
intracisternally into the cerebrospinal fluid of rabbits (Wolpe and Cerami,
FASEB J.
3:2565-73 ( 1989)). In addition to these proinflammatory properties of MIP-1,
which can
S be direct or indirect, MIP-1 has been recovered during the early
inflammatory phases of
wound healing in an experimental mouse model employing sterile wound chambers
(Fahey, et al. Cytokine, 2:92 ( l990)). For example, PCT application U.S.
92/05198 filed
by Chiron Corporation, discloses a DNA molecule which is active as a template
for
producing mammalian macrophage inflammatory proteins (MIPs) in yeast.
The marine MIP-1 a and MIP-1 p are distinct but closely related cytokines.
Partially purified mixtures of the two proteins affect neutrophil function and
cause local
inflammation and fever. MIP-1 a has been expressed in yeast cells and purified
to
homogeneity. Structural analysis confirmed that MIP-1 a has a very similar
secondary
and tertiary structure to platelet factor 4 (PF-4) and interleukin 8 (IL-8)
with which it
1 S shares limited sequence homology. It has also been demonstrated that MIP-1
a is active
in vivn to protect mouse stem cells from subsequent in vitro killing by
iritiated
thymidine. MIP-1 a was also shown to enhance the proliferation of more
committed
progenitor granulocyte macrophage colony-forming cells in response to
granulocyte
macrophage colony-stimulating factor. (Clemens, J.M. et al., C:vtokine -t:76-
82 ( 1992)).
The polypeptides of the present invention, M-CIF originally referred to as
MIP-1 y and Ckp-1 in the parent patent application, is a new member of the (3
chemokine
family based on amino sequence homology. The MPIF-1 polypeptide, originally
referred to as MIP-3 and Ck~3-8 in the parent application, is also a new
member of the
~3 chemokine family based on the amino acid sequence homology.
Summary of the Invention
In accordance with one aspect of the present invention, there are provided
novel
full length or mature polypeptides which are MPIF-1, MIP-4 and/or M-CIF, as
well as
biologically active, diagnostically useful or therapeutically useful
fragments, analogs
and derivatives thereof. The MPIF-1, MIP-4 and M-CIF of the present invention
are
preferably of animal origin, and more preferably of human origin.
In accordance with another aspect of the present invention, there are provided
polynucleotides (DNA or RNA) which encode such polypeptides and isolated
nucleic
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acid molecules encoding such polypeptides, including mRNAs, DNAs, cDNAs,
genomic
DNA as well as biologically active and diagnostically or therapeutically
useful
fragments, analogs and derivatives thereof.
MPIF 1 Polynucleotides. The present invention also provides isolated nucleic
acid molecules comprising a polynucleotide encoding the MPIF-1 polypeptide
having
the amino acid sequence shown in Figure 1 (SEQ ID N0:4) or the amino acid
sequence
encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number
75676 on February 9, 1994. The nucleotide sequence determined by sequencing
the
deposited MPIF-1 clone, which is shown in Figure 1 (SEQ ID N0:3), contains an
open
reading frame encoding a polypeptide of 120 amino acid residues, with a leader
sequence of about 21 amino acid residues, and a predicted molecular weight for
the
mature protein of about 11 kDa in non-glycosylated form, and about 11-14 kDa
in
glycosylated form, depending on the extent of glycoslyation. The amino acid
sequence
of the mature MPIF-1 protein is shown in Figure 1 (SEQ ID N0:4).
Thus, one aspect of the invention provides an isolated nucleic acid molecule
comprising a polynucleotide having a nucleotide sequence selected from the
group
consisting of: ( 1 )(a) a nucleotide sequence encoding an MPIF-1 polypeptide
having the
complete amino acid sequence in Figure 1 (SEQ ID N0:4); (1)(b) a nucleotide
sequence
encoding the MPIF-1 polypeptide having the complete amino acid sequence in
Figure
1 (SEQ ID N0:4} but minus the N-terminal methionine residue; (1)(c) a
nucleotide
sequence encoding the mature MPIF-1 polypeptide having the amino acid sequence
at
positions 22-120 in Figure 1 (SEQ ID N0:4}; ( 1 )(d) a nucleotide sequence
encoding the
MPIF-1 polypeptide having the complete amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 75676; ( 1 )(e) a nucleotide sequence
encoding the
mature MPIF-1 polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 75676; and ( 1 )(f) a nucleotide sequence
complementary to any of the nucleotide sequences in ( 1 }- (a), (b), (c), (d),
or (e) above.
M CIF Polynucleotides. In one aspect, the present invention provides isolated
nucleic acid molecules comprising a polynucleotide encoding the M-CIF
polypeptide
having the amino acid sequence shown in Figure 2 (SEQ ID N0:2) or the amino
acid
sequence encoded by the eDNA clone deposited in a bacterial host as ATCC
Deposit
Number 75572 on October 13, 1993. The nucleotide sequence determined by
sequencing the deposited M-CIF clone, which is shown in Figure 2 (SEQ ID NO: I
),
contains an open reading frame encoding a polypeptide of 93 amino acid
residues, with
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a leader sequence of about 19 amino acid residues, and a predicted molecular
weight of
about 9 kDa in non-glycosylated form, and about 9-14 kDa in glycosylated form,
depending on the extent of glycoslyation. The amino acid sequence of the
mature M-
CIF protein is shown in Figure 2 (SEQ ID N0:2).
S Thus, one aspect of the invention provides an isolated nucleic acid molecule
comprising a polynucleotide having a nucleotide sequence selected from the
group
consisting of: (2)(a) a nucleotide sequence encoding the M-CIF polypeptide
having the
complete amino acid sequence in Figure 2 (SEQ ID N0:2); (2)(b) a nucleotide
sequence
encoding the M-CIF polypeptide having the complete amino acid sequence in
Figure 2
(SEQ ID N0:2) but minus the N-terminal methionine residue: (2)(c) a nucleotide
sequence encoding the mature M-CIF polypeptide having the amino acid sequence
at
positions 20-93 in Figure 2 (SEQ ID N0:2); (2)(d) a nucleotide sequence
encoding the
M-CIF polypeptide having the complete amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 75572; (2)(e) a nucleotide sequence
encoding the
1 S mature M-CIF polypeptide having the amino acid sequence encoded by the
cDNA clone
contained in ATCC Deposit No. 75572; and (2)(f) a nucleotide sequence
complementary
to any of the nucleotide sequences in (2)- (a), (b), (c), (d), or (e) above.
MIP-4 Polynucleotides. The present invention further provides isolated nucleic
acid molecules comprising a polynucleotide encoding the MIP-4 polypeptide
having the
amino acid sequence shown in Figure 3 (SEQ ID N0:6) or the amino acid sequence
encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number
75675 on February 9, 1994. The nucleotide sequence determined by sequencing
the
deposited MIP-4 clone, which is shown in Figure 3 (SEQ ID NO:S), contains an
open
reading frame encoding a polypeptide of 89 amino acid residues, with a leader
sequence
of about 20 amino acid residues, and a predicted molecular weight of about 8
kDa in
non-glycosylated form, and about 8-14 kDa in glycosylated form, depending on
the
extent of glycoslyation. The amino acid sequence of the mature MIP-4 protein
is shown
in Figure 2 (SEQ ID N0:6).
Another aspect of the invention provides an isolated nucleic acid molecule
comprising a polynucleotide having a nucleotide sequence selected from the
group
consisting of: (3)(a) a nucleotide sequence encoding the MIP-4 polypeptide
having the
complete amino acid sequence in Figure 3 (SEQ ID N0:6); (3)(b) a nucleotide
sequence
encoding the MIP-4 polypeptide having the complete amino acid sequence in
Figure 3
(SEQ ID N0:6) but minus the N-terminal methionine residue; (3)(c) a nucleotide
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sequence encoding the mature MIP-4 polypeptide having the amino acid sequence
at
positions 21-89 in Figure 3 (SEQ ID N0:6); (3)(d) a nucleotide sequence
encoding the
MIP-4 polypeptide having the complete amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 75675; (3)(e) a nucleotide sequence
encoding the
mature MIP-4 polypeptide having the amino acid sequence encoded by the cDNA
clone
contained in ATCC Deposit No. 75675; and (3)(f) a nucleotide sequence
complementary
to any of the nucleotide sequences in (3)- (a), (b), (c), (d), or (e) above.
MPIF l, M CIF and MIP 4 Polynucleotide Variants. The present invention
further relates to variants of the hereinabove described polynucleotides which
encode
for fragments, analogs and derivatives of the polypeptide having the deduced
amino
acid sequence of Figures 1, 2 and 3 (SEQ ID NOS:2, 4 and 6) or the
polypeptides
encoded by the cDNA of the deposited clone(s). The variants of the
polynucleotides can
be a naturally occurring allelic variant of the polynucleotides or a non-
naturally
occurring variant of the polynucleotides.
Homologous MPIF I, M CIF and MIR4 Poly nucleotides. Further
embodiments of the invention include isolated nucleic acid molecules that
comprise a
polynucleotide having a nucleotide sequence at least 95%, 96%, 97%, 98% or 99%
identical, to any of the nucleotide sequences in (1 )-, (2)- or (3)- (a), (b),
(e), (d), (e), or
(~, above, or a polynucleotide which hybridizes under stringent hybridization
conditions
to a polynucleotide in (1)-, (2)- or (3)- (a), (b), (c), (d), (e), or (f),
above. These
polynucleotides which hybridize do not hybridize under stringent hybridization
conditions to a polynucleotide having a nucleotide sequence consisting of only
A
residues or of only T residues.
Nucleic Acid Probes. In accordance with yet another aspect of the present
invention, there are also provided nucleic acid probes comprising nucleic acid
molecules
of sufficient length to specifically hybridize to the MPIF-I , M-CIF and/or
MIP-4 nucleic
acid sequences.
Recombinant Vectors, Host Cells and Expression. The present invention also
relates to recombinant vectors, which include the isolated nucleic acid
molecules of the
present invention, and to host cells containing the recombinant vectors, as
well as to
methods of making such vectors and host cells and for using them for
production of
MPIF-1, M-CIF or MIP-4 polypeptides or peptides by recombinant techniques.
MPIF 1 Polypeptides. The invention further provides an isolated MPIF-1
polypeptide having an amino acid sequence selected from the group consisting
of: (I)(a)
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the amino acid sequence of the MPIF-1 polypeptide having the complete 120
amino acid
sequence, including the leader sequence shown in Figure 1 (SEQ ID N0:4);
(I)(b) the
amino acid sequence of the MPIF-1 polypeptide having the complete 120 amino
acid
sequence, including the leader sequence shown in Figure 1 (SEQ ID N0:4) but
minus
the N-terminal methionine residue; (I)(c) the amino acid sequence of the
manure MPIF-1
polypeptide (without the leader) having the amino acid sequence at positions
22-120 in
Figure 1 (SEQ ID N0:4); (I)(d) the amino acid sequence of the MPIF-1
polypeptide
having the complete amino acid sequence, including the leader, encoded by the
cDNA
clone contained in ATCC Deposit No. 75676; and (I)(e) the amino acid sequence
of the
mature MPIF-1 polypeptide having the amino acid sequence encoded by the cDNA
clone contained in ATCC Deposit No. 7S676.
M CIF Polypeptides. The invention further provides an isolated M-CIF
polypeptide having an amino acid sequence selected from the group consisting
of: (II)(a)
the amino acid sequence of the M-CIF polypeptide having the complete 93 amino
acid
sequence, including the leader sequence shown in Figure 2 (SEQ ID N0:2);
(I)(b) the
amino acid sequence of the M-CIF polypeptide having the complete 93 amino acid
sequence, including the leader sequence shown in Figure 2 (SEQ ID N0:2) but
minus
the N-terminal methionine residue; (II)(c) the amino acid sequence of the
mature M-CIF
polypeptide (without the leader) having the amino acid sequence at positions
20-93 in
Figure 2 (SEQ ID N0:2); (II)(d) the amino acid sequence of the M-CIF
polypeptidc
having the complete amino acid sequence, including the leader, encoded by the
cDNA
clone contained in ATCC Deposit No. 75572; and (II)(e) the amino acid sequence
of the
mature M-CIF polypeptide having the amino acid sequence encoded by the cDNA
clone
contained in ATCC Deposit No. 75572.
MIP-4 Polypeptides. The invention further provides an isolated MIP-4
polypeptide having an amino acid sequence selected from the group consisting
of:
(III)(a) the amino acid sequence of the MIP-4 polypeptide having the complete
89 amino
acid sequence, including the leader sequence shown in Figure 3 (SEQ ID N0:6);
(III)(b)
the amino acid sequence of the MIP-4 polypeptide having the complete 89 amino
acid
sequence, including the leader sequence shown in Figure 3 (SEQ ID N0:6) but
minus
the N-terminal methionine residue; (III)(c) the amino acid sequence of the
mature MIP-4
polypeptide (without the leader) having the amino acid sequence at positions
2I -89 in
Figure 3 (SEQ ID N0:6); (III)(d) the amino acid sequence of the MIP-4
polypeptide
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having the complete amino acid sequence, including the leader, encoded by the
cDNA
clone contained in ATCC Deposit No. 75675; and (III)(e) the amino acid
sequence of
the mature MIP-4 polypeptide having the amino acid sequence encoded by the
cDNA
clone contained in ATCC Deposit No. 7S675.
$ Homologous MPIF l, M CIF and MIP 4 Polypeptides. Polypeptides of the
present invention also include homologous polypeptides having an amino acid
sequence
with at least 95% identity to those described in (I)-, (II)- and (III)(a),
(b), (c), (d), or (e)
above, as well as polypeptides having an amino acid sequence at least 95%,
96%, 97%,
98% or 99% identical to those above.
MPIF l, M CIF and MIP-4 Epitope Bearing Polypeptides and Encoding
Polvnucleotides. An additional embodiment of this aspect of the invention
relates to a
peptide or polypeptide which has the amino acid sequence of an epitope-bearing
portion
of an MPIF-l, M-CIF or MIP-4 polypeptide having an amino acid sequence
described
in (I)-, (II)-, or (III)- (a), (b), (c), (d), or (e), above. Peptides or
polypeptides having the
1 S amino acid sequence of an epitope-bearing portion of an MPIF- I , M-CIF or
MIP-4
polypeptide of the invention include portions of such polypeptides with at
least six or
seven, preferably at least nine, and more preferably at least about 30 amino
acids to
about 50 amino acids, although epitope-bearing polypeptides of any length up
to and
including the entire amino acid sequence of a polypeptide of the invention
described
above also are included in the invention.
An additional nucleic acid embodiment of the invention relates to an isolated
nucleic acid molecule comprising a polynucleotide which encodes the amino acid
sequence of an epitope-bearing portion of an MPIF-l, M-CIF or MIP-4
polypeptide
having an amino acid sequence in (I)-, (II)- or (III)-(a), (b), (c), (d), or
(e), above.
MPIF I, M CIF and MIP-4 Antibodies. In accordance with yet a further aspect
of the present invention, there is provided an antibody against such
polypeptides. In
another embodiment, the invention provides an isolated antibody that binds
specifically
to an MPIF-1, M-CIF or MIP-4 polypeptide having an amino acid sequence
described
in (I)-, (II)-, and/or (III)- (a), (b), (c), (d), or (e), above.
The invention further provides methods for isolating antibodies that bind
specifically to an MPIF-l, M-CIF or MIP-4 polypeptide having an amino acid
sequence
as described herein. Such antibodies are useful diagnostically or
therapeutically as
described below.
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MPIF l, M CIFand MIP-4Antagonists and Methods. In accordance with yet
another aspect of the present invention, there are provided antagonists or
inhibitors of
such polypeptides, which can be used to inhibit the action of such
polypeptides, for
example, in the treatment of arteriosclerosis, autoimmune and chronic
inflammatory and
S infective diseases, histamine-mediated allergic reactions, hyper-
eosinophilic syndrome,
silicosis, sarcoidosis, inflammatory diseases of the lung, inhibition of IL-1
and TNF,
aplastic anaemia, and myelodysplastic syndrome. Alternatively, such
polypeptides can
be used to inhibit production of IL-1 and TNF-a, to treat aplastic anemia,
myelodysplastic syndrome, asthma and arthritis.
Diagnostic Assays. In accordance with still another aspect of the present
invention, there are provided diagnostic assays for detecting diseases related
to the
underexpression and overexpression of the polypeptides and for detecting
mutations in
the nucleic acid sequences encoding such polypeptides.
In accordance with yet another aspect of the present invention, there is
provided
a process for utilizing such polypeptides, or polynucleotides encoding such
polypeptides, as research reagents for in vitro purposes related to scientific
research,
synthesis of DNA and manufacture of DNA vectors, for the purpose of developing
therapeutics and diagnostics for the treatment of human disease.
The present invention also provides a screening method for identifying
compounds capable of enhancing or inhibiting a cellular response induced by an
MPIF-
1, M-CIF or MIP-4 polypeptide, which involves contacting cells which express
the
MPIF-1, M-CIF or MIP-4 polypeptide with the candidate compound, assaying a
cellular
response, and comparing the cellular response to a standard cellular response,
the
standard being assayed when contact is made in absence of the candidate
compound;
whereby, an increased cellular response over the standard indicates that the
compound
is an agonist and a decreased cellular response over the standard indicates
that the
compound is an antagonist.
For a number of disorders, it is believed that significantly higher or lower
levels
of MPIF-1, M-CIF or MIP-4 gene expression can be detected in certain tissues
or bodily
fluids (e.g., serum, plasma, urine, synovial fluid or spinal fluid) taken from
an individual
having such a disorder, relative to a "standard" MPIF-1, M-CIF or MIP-4 gene
expression level, i.e., the MPIF-1, M-CIF or MIP-4 expression level in tissue
or bodily
fluids from an individual not having the disorder. Thus, the invention
provides a
diagnostic method useful during diagnosis of a disorder, which involves: (a)
assaying
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MPIF-1, M-CIF or MIP-4 gene expression level in cells or body fluid of an
individual;
(b) comparing the MPIF-l, M-CIF or MIP-4 gene expression level with a standard
MPIF-1, M-CIF or MIP-4 gene expression level, whereby an increase or decrease
in the
assayed MPIF-1, M-CIF or MIP-4 gene expression level compared to the standard
expression level is indicative of a disorder. Such disorders include leukemia,
chronic
inflammation, autoimmune diseases, solid tumors.
Pharmaceutical Compositions. The present invention also provides, in another
aspect, pharmaceutical compositions comprising at least one of an MPIF-l, M-
CIF or
MIP-4: polynucleotide, probe, vector, host cell, polypeptide, fragment,
variant,
derivative, epitope bearing portion, antibody, antagonist, or agonist.
Therapeutic Methods. In accordance with yet a further aspect of the present
invention, there is provided a process for utilizing such polypeptides, or
polynucleotides
encoding such polypeptides for therapeutic purposes, for example, to protect
bone
marrow stem cells from chemotherapeutic agents during chemotherapy, to remove
leukemic cells, to stimulate an immune response, to regulate hematopoiesis and
lymphocyte trafficking, treatment of psoriasis, solid tumors, to enhance host
defenses
against resistant and acute and chronic infection, and to stimulate wound
healing.
An additional aspect of the invention is related to a method for treating an
individual in need of an increased level of MPIF-1, M-CIF or MIP-4 activity in
the body
comprising administering to such an individual a composition comprising a
therapeutically effective amount of an isolated MPIF-1, M-CIF or MIP-4
polypeptide
of the invention or an agonist thereof, respectively.
A still further aspect of the invention is related to a method for treating an
individual in need of a decreased level of MPIF-1, M-CIF or MIP-4 activity in
the body
comprising, administering to such an individual a composition comprising a
therapeutically effective amount of an MPIF-l, M-CIF or MIP-4 antagonist.
Preferred
antagonists for use in the present invention are M-CIF-specific antibodies,
respectively.
These and other aspects of the present invention should be apparent to those
skilled in the art from the teachings herein.
Brief Description of the Figures
The following drawings are illustrative of embodiments of the invention and
are
not meant to limit the scope of the invention as encompassed by the claims.
-10-
~ . _: i: ~: ~-4 1 w,.; . ;i I p
m\ , . ~'.:~\ \~1;.1,~~(.. m.~ .:m. 7 ;v5 ~(< 7m _ a ~-... i=J rV , .~V
-nJ i ~ L tJ
FIG. : displays the eDNA sequence encoding MPIF-1 (SEQ LD NG:~) and the
~:arresponding deduced amino acid sequc;nce (SEQ ID N0;4). The initial 21
amino acids
represents the putative leader sc;qu.ence. All the signal sequences were as
determined by
N-terminal peptide sequencing of the baculovizus expressed protein.
FIG. ? displays the cDNA sequence encoding M-CIF (SEQ ID NO: I) and the
corresponding deduced amino acid sequence (SEQ ID N0:2). The zzitial 19 amino
acids
represents a leader sequence.
FIG. 3 displays the cDifA sequence encoding ~IIP-4 (SEQ ID NG:~) and the
~rresponding deduced amino acid sequence (SEQ ID ~0:6). Tne initial 20 amino
acids
represents a leader sequence.
FIG. 4 illustrates the amino acid homology between I~IPIF-1 (top) (SEQ ID
N0:4) and hurnarz :~:IP-1 a (bottom) {SEQ ID NO:S S). The four cysteines
characteristic
of a11 chemokines are showzz.
FICi. ~ displays two amino acid sequences wherein, the top sequenc: is thLe
human :'vlzP~ 3naino acid sequence c;SEQ ID NQ:6) and the bottom sequenc;, is
human
MIP-la (HurczanTonsillar lymphocyte 1,D78 L~cta protein precursor) (SEQ ID
NG:~S),
FIG. 6 illustrates the amino acid sequence alignment between hi-CIF (topl and
h~.irnac~ MIP-la (bottom) (SEQ 1D NG:55).
FIG. 7 is a photoyaph of a gel in which M-CiF has been eiectrophoresed after
the expression of IiA-tagged M-CII~ in CAS cells.
FICr. 8 is a photograph of a SDS-P:~GE geI after expression and purification
of
M-CIF in a baculovirus expression system.
FIG. 9:~-9B is a photograph of an SDS-PAGE gel after exaression and a threc-
step puritcation of MPIF-1 in a bacutovizvs expression system.
FIG. I0. The chemoattractant activity of MPIF-1 was determined with
cheznotaxis assays using a 4$-well tnicrochamber device (Neuro Probe, Inc.).
'1''ne
exp erimental procedure tugs as described in the manufacturers manual. For
each
concentration o~MPIF-I tested, migratioa in 5 high-power fields was examiwd.
The
results presented represent the average values obtained fxom two independent
experiments. The chemoattractant activity on TI-Ip-I {~,) cells and hum~acz
PBMCs {B)
is shown..
FIG. 11. Change in intracellular calciazrn concentration in response to MPIF-I
was determined using a F~itachi F-2000 fluorescence spectrophotometer.
Bacterial
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WO 98114582 PCT/US97117505
expressed MPIF-1 was added to Indo-1 loaded THP-1 cells to a final
concentration of
50 nM and the intracellular level of calcium concentration was monitored.
FIG. 12. A low density population of mouse bone marrow cells was plated
(1,500 cells/dish) in agar containing-medium with or without the indicated
chemokines
(100 ng/ml), but in the presence of IL-3 (S ng/ml), SCF (100 ng/ml), IL-la (10
ng/ml),
and M-CSF (S ng/ml). The data shown represents the average obtained from two
independent experiments (each performed in duplicate). Colonies were counted
14 days
after plating. The number of colonies generated in the presence of chemokines
is
expressed as a mean percentage of those produced in the absence of any added
chemokines.
FIG. 13 illustrates the effect of MPIF-1 and M-CIF on mouse bone marrow
colony formation by HPP-CFC (A) and LPP-CFC (B).
FIG. 14 illustrates the effect of baculovirus-expressed M-CIF and MPIF-1 on M-
CFS and SCF-stimulated colony formation of freshly isolated bone marrow cells.
FIG. 15 illustrates the effect of MPIF-1 and M-CIF on IL3 and SCF-stimulated
proliferation and differentiation of the lim population of bone marrow cells.
FIG. 16A-B. Figures 16A-B show the effect of MPIF-1 and M-CIF on the
generation of Gr. l and Mac-1 (surface markers) positive population of cells
from lineage
depleted population of bone marrow cells. liri cells were incubated in growth
medium
supplemented with IL-3 (5 ng/ml) and SCF ( 100 ng/ml) alone (a) with MPIF-1
(SO
ng/ml) (b) or M-CIF (50 ng/ml) (c). Cells were then stained with Monoclonal
antibodies
against myeloid differentiation Gr. l , Mac-1, Sca-1, and CD45R surface
antigens and
analyzed by FACScan. Data is presented as percentage of positive cells in both
large
(Figure 16A) and small (Figure 16B) cell populations.
FIG. 17 illustrates that the presence of MPIF-1 protein inhibits bone marrow
cell
colony formation in response to IL3, M-CSF and GM-CSF.
FIG. 18. Dose response of MPIF-1 inhibits bone marrow cell colony formation.
Cells were isolated and treated as in Figure 19. The treated cells were plated
at a density
of l,000 cells/dish in agar-based colony formation assays in the presence of
IL-3, GM-
CSF or M-CSF (5 ng/ml) with or without MPIF-1 at 1, 10, SO and 100 ng/ml. The
data
is presented as colony formation as a percentage of the number of colonies
formed with
the specific factor alone. The data is depicted as the average of duplicate
dishes with
error bars indicating the standard deviation.
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FIG. 19. Expression of RNA encoding MPIF-1 in human monocytes. Total
RNA from fresh elutriated monocytes was isolated and treated with 100 U/ml hu
rIFN-g,
l00 ng/ml LPS, or both. RNA (8 fig) from each treatment was separated
electrophoretically on a 1.2% agarose gel and transferred to a nylon membrane.
MPIF-1
mRNA was quantified by probing with 32P-labeled cDNA and the bands on the
resulting
autoradiograph were quantified densitometrically.
FIG. 20A-B. Figure 20A shows an analysis of the MPIF-1 amino acid sequence
(SEQ ID N0:4). Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicity;
amphipathic regions; flexible regions; antigenic index and surface probability
are shown.
In the "Antigenic Index - Jameson-Wolf' graph, amino acid residues 21-30, 31-
44, 49-
55, 59-67, 72-83, 86-103 and 110-120 in Figure I (SEQ ID N0:4), or any range
or value
therein, in Figure 1 (SEQ ID N0:4) correspond to the shown highly antigenic
regions
of the MPIF-1 protein. Figure 20B shows an analysis of the M-CIF amino acid
sequence (SEQ ID N0:2). Alpha, beta, turn and coil regions; hydrophilicity and
hydrophobicit5~; amphipathic regions; flexible regions; antigenic index and
surface
probability are shown. In the "Antigenic Index - Jameson-Wolf' graph, amino
acid
residues 20-36, 42-52, 52-64, 67-75, 75-84 and/or 86-93 in Figure 2 (SEQ ID
N0:2), or
any range or value therein, in Figure 2 (SEQ ID N0:2) correspond to the shown
highly
antigenic regions of the M-CIF protein.
FIG. 21A-B. Figure 21A shows the myeloprotective effect of MPIF-I on the 5-
Fu-induced killing of LPP-CFC cells. Figure 21 B shows the myeloprotective
effect of
MPIF-1 on the Ara-C induced killing of LPP-CFC cells.
FIG. 22 shows the effect of MPIF-1 pre-treatment of mice on the 5-Fu-induced
reduction in the circulating WBC counts.
FIG. 23 shows the experimental design involving three groups of mice (6
animals per group) that were treated as follows: Group-l, injected with saline
on days
1, 2, and 3; Group-2, injected with 5-Fu on days 0 and 3; and Group-3,
injected with 5-
Fu on days 0 and 3 and MPIF-1 on days 1, 2, and 3. Bone marrow was harvested
on
days 6 and 9 to determine HPP-CFC and LPP-CFC frequencies using a clonogenic
assay.
FIG. 24 shows the effect of administration of MPIF-I prior to the second dose
of 5-Fu on the HPP-CFC and LPP-CFC frequencies in the bone marrow.
FIG. 25 shows MPIF-1 variants. The first 80 out of 120 amino acids sequence
of MPIF-1 (FIG. 1 (SEQ ID N0:4)) is shown using a single amino acid letter
code of
-13-
,JmJ i .- i U i .J ~ J~ a I a V - V ' . L V
y\ \O\-l.l'\-\ll 1:'.C:111-.~', n:? :W- E-:ui I;i I I - -IJ) ~i;~ ~:3;~:LI
It~: ~ l~~
which the first 21 residues show characteristics of a signal sequence that is
craved to
;ive rise to a mature, wild type protein. Mutants-1 and -b contain methioninc
as the N-
terminal residue which is not present in the wild type. Also, the first four
ar;zino acids
(HAAG) of Mutant-9 aze not present in the wild type bfPlF-1 protein. Mutants-
I, -(i
and, -9 correspond to SEQ ID NOS:7, 8 and 9, respectively. Mutant-2
corresponds to
amino acid residues 46-120 in SEQ ID N0:4. Mutant-3 corresponds to amino acid
residues 45-120 in SEQ ID N0:4. Mutant-4 corresponds to amino acid residues 48-
120
in SEQ ID N0:4. Mutant-5 corresponds to amino acid residues 49-120 :n SEQ ID
N0:4. Mutant-7 corresponds to amino acid residues 39-120 in SEQ ID NOv4:
Mutant-8
corresponds to amino acid residues 44-120 in SEQ ID N0:4.
FIG. 26A-2bB. Figure 26A shows the nucleotide sequence of a human MPIF-i
splice variant cDNA (SEQ ID N0:10). This cDNA sequence is shown along with ;he
npen reading fi-ame encoding for a protein of 137 amino acids {SEQ ID N0:11)
using
a single letter amino acid code. The N-terminal 21 amine acids which are
underlined
represent the putative leader sequence. The insertion of 18 amino acids
sequcmc~ not
represented in the ~fPIF-1 sequence but unique to the splice variant are high-
lighted in
italics. Figure 26B shawl the comparison of the amino acid sequence of the
i~LfIF-1
v~xiant (SEQ ID N0:11 ) with that of the wild typo MP1F-1 malee,ile (SEQ ID
N0:4).
FIG. 27 shows the concentrations of MPIF-1 mutant proteins required for 50~ra
of maximal calcium mobilizatian response induced'oy MIP-1 c~ i_n human
:nonoeytes.
FIG. 28A-28B shows the charges in the intracellular Exec calcium concentration
was measured in human monocvtes in response to the indicated proteins at 100
ng'rrzl
as described in the legend to Figure 27.
FIG. 29 shows the ability of MPIF-1 mutants to desensitize I~fIP- I a
stimulated
2S calcium mobilization in human monacytes ( summary).
FIG. 30 shows the chemotactic responses of human peripheral blood
mononuclear cells (PBMC) to MPIF-L mutants. Numbers within the parenthesis
reflect
Cold stirrnilation of chemotaxis above background observed at the indicate
concxntration
range.
FIG. 31 shows the effect of MPIF-1 variants on the trrowth and differentiation
of L,ow Proliferative Potential Colony-forming Cells (LPP-CFC) in vitro.
FIG. 32 shows protection against LPS-induced septic shock in mice by
pretreatment with recombinant human M-CIF. Groups of Balblc mice (n=7) ~~ere
injected i.p. with 25 mg/kg of LPS on day 0. M-CIF was biven i.p. daily at 3
mglkg of
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body weight of for 3 consecutive days ftom one day before, on the same day,
and one
day after LPS challenge (-1, 0, +1). Mice receiving buffer only served as
disease
control. The kinetic of lethality was followed for 56 hours after LPS
challenge.
FIG. 33 shows the protective effect of M-CIF on lethal shock is dependent on
LPS dose. Crroups of Balb/c mice (n=9) were injected i.p. with 25 mg/kg of LPS
on day
0 for different degrees of sepsis induction. 10 mg/kg of M-CIF was given i.p.
daily for
3 consecutive day to each group of LPS-treated mice. The kinetic of lethality
was
followed for 56 hours after LPS challenge.
FIG. 34 shows protection against LPS-induced lethal shock in mice is dependent
on M-CIF dose. Groups of Balb/c mice (n=8) were challenged i.p. with 25 mg/kg
of
LPS on day 0 and treated daily with different doses (1, 3 or 10 mg/kg) of M-
CIF for 3
consecutive days (-1, 0, +I ). Mice receiving buffer only served as a disease
control.
The kinetic of lethality was followed for 120 hours after LPS challenge.
FIG. 35A-B shows the protective effect of M-CIF on LPS-induced shock in
Balb/c SCID mice. Groups of Balb/c SCID mice (n=5-7) were challenged i.p. with
20,
30 or 40 mg/kg of LPS on day 0; and M-CIF treatment was given to each group of
LPS-
injected mice at 3 mg/kg daily dosing for 3 consecutive days (-1, 0, +1 ). The
kinetic of
lethality was followed for 120 hours after LPS challenge. The result of M-CIF
pretreatment on 20 mg/kg of LPS-injected mice is the same as that of LPS-
injection
alone with no death occurring.
FIG. 36 shows the protective effect of M-C1F protein from E. coli and CHO
expression vectors on sepsis. Groups of Balb/c mice (n=8) were injected with
25 mg/kg
of LPS on day 0; and treated with two different batches (E 1 and C 1 ) of M-
CIF at 1
mg/kg for 3 consecutive days (-1, 0, +I ). Mice receiving buffer only served
as a disease
control. The kinetic of lethality was followed for 120 hours after LPS
challenge.
FIG. 37. Efficacy of M-CIF in reducing paw edema in adjuvant-induced arthritis
model. Groups of Lewis rats (n = S) were injected intradermally at the base of
the tail
with 100 ul/rat of Freund's complete adjuvant containing 5 mg/ml Mycobacterium
butyricum on day 0. Preventative treatment started on day 0 and continued
daily (M-
CIF, 5 times/week) for 16 days with i.p. M-CIF at 1 or 3 mg/kg in buffer (40
mM
sodium acetate; 500 mM NaCI) or with p.o. indomethacin at 1 mg/kg in methyl
cellulose, as drug control, daily dose (5 times / week) for 16 days. Rats
receiving buffer
or methyl cellulose only served as disease control. Swelling of both hind paws
were
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WO 98/14582 PCT/CTS97/I7505
monitored on the days as indicated using a plethysmometer chamber, and
percentage of
efficacy of testing drugs on paw volume were calculated.
FIG. 38. Protective effect of M-CIF on total joint inflammation. At the end of
the same experiment as FIG. 40, which was 40 days after adjuvant immunization,
both
hind limbs from two rats per group were collected for histopathological
analysis. The
results are expressed as mean of total histological score.
FIG. 39. Protective effect of M-CIF on chronic features of arthritis. A
similar
experiment as FIG. 40 with prolonged daily treatment of M-CIF or indomethacin
to 40
days post adjuvant immunization, was conducted to further analyze chronic
histopathological changes including hypertrophy, fibrosis, blood vessel
dilation and
lymphoid aggregates around blood vessels. The results were expressed as mean
{n = S)
of the total features mentioned above. An unpaired T test was employed for
obtaining
assessing statistical significance.
FIG. 40. Protective effect of M-CIF on bone and cartilage erosion. In the same
experiment as FIG. 39, pannus formation, bone and cartilage destruction were
evaluated.
The results were expressed as mean (n = S) of the total features mentioned
above. An
unpaired T test was employed for assessing statistical significance.
FIG. 41. M-CIF treatment prevents developing type 11 collagen-induced
arthritis
in DBA/I mice. Female DBA/ilacJ mice were immunized i.d. at the base of the
tail with
Bovine type 11 collagen emulsified in complete Freund's adjuvant. 20 days
later, the
mice were challenged with a s.c. injection of 60 mg/100 of LPS. Two days
preceding
LPS injection, 3 groups of animals (n=10 per group) were i.p. treated with 3
mg/ml of
indomethacin, M-CIF, or their buffer controls respectively. These treatments
continued
daily for 14 days. The animals were examined and their clinical presentation
semiquantified. The % incidence is shown in this FIG.
FIG. 42 Animals were immunized with bovine type II collagen as described in
FIG. 44. The results are expressed as the mean severity.
FIG. 43 shows the suppressive effect of M-CIF on systemic TNF-A production.
Groups of female Balb/c mice were challenged with 25 mg/kg of
lipopolysaccharide
(LPS) from E. toll serotype 0127:B8 (Sigma) in saline on Day 0. M-CIF or
buffer was
administered one day before and the same day (I hour before) of LPS injection.
Serum
was collected at various time points after LPS administration and the TNF-A
level
determined. The results were analyzed with an unpaired T test and the data
expressed
as the mean + SEM.
-16-
rt'.> a:J _l'.J:J4-1":~:.rl
hl\ lm\.i.l'1-\ll l'..~ill'.'. n:; :t11- -,li; : 12>: I l
J'HV ~ ~ J i ;J 1 ' 1 G.J ~-~ ~C: W . --- 1 V
fIG. 44 shows the dcxre3se in TNF-(a) production from peritoneal cells
isolatc?d
frarn M-C:IF treated mice. Mic:, were treated with M-CIF 3t 3 rng,'kg for tlwv
days. One
hour after the second M-CIF injection, the peritoneal cctls were harvested and
put into
culture to assay for cytokine production in the presence or abse: ce of LPS.
'i"~F-(~~
levels were measured by ELISA.
c IG. 45 siZOws the increased total cell number in the peritone:ri cavitrr of
M-CIF
treated mice. 'v~ice were u.-ttreated, treated with vehicle control or treated
with M-C;IF
at I m~~Cg and 3 mgr'kg daily for six consecutive days. Gn the seventh day)
mice were
sac:ificed and the p.,riton~;~.l :;ells harvested are qtrantitated.
t 0 FIG. 45 snows the specific inorease it CD4 positive '1'-ly mphocVtes in
the
peritoneal cavity of ;yi-CIF treated trice. Ivlice wre treated as described in
FIC~_ ~8A-
48$. Each a.~imaI is reprefentod by a different symboE from tl-~e untreated,
vehicle
treated, 1 rny'itG M-C'IF) :end 3 ;-ng,~'k~ ~1-C1F groups. Each group
contair_ed :0 animals
each) vnth the cells from each anirnsi ~.:.zaly~~d by cell surface staining
using antibodies
directed at C174, C'D5, s_Dg, l~tacl. >~iHC ci:~.,s II, B220, IgM. Gr I and CD
1~4.
FG. 47 shows an izicrease n total T iyznphocyto cell numbers (CD:,~Ib.~s-.
CI?=~,
and CI~$1 in the peri:anea; cavity of M-CIF treated mi:,e.
t~IG. 4a,~.=s8E shows a decrease in the percenragc of :vsacl-~/Ml-.C clas,;
II3- aclls
in the peritoneal eaviry oz M-CIF :.;,aced mice ~.vith a corr~spor~din~
increase in the
?0 percentage of Macl-~i'~I:~C cl:~,s II c~lis.
;~IG. 49 shows an increase in trio weal number of Macl+,~~-IC class Tr-cells
in
thv p~ritore:~l cavity cf M-CIF treated :rsiC?.
FICr. 50 snows the stem cell mobilization in normal mii~.in response to '~e
administration of ~yLpIF-1.
Ir IG . 5 I s;aows a comparison o. the effect ;~f M..PIF -1 with G-CSF on the
recovery
of platelets foIlowinc two cyi;~ of 5-Fu Crew~.ment as deterained by F ACS
Vantage
method.
FIG. 52 shows acomparison of the effect ofMFtF-t with C-CSF on the recovery
ef Gra.l a.>ld Nlac.l double positive cells in the bleed following two cycles
of S-Fu
3G treatment.
FIG. 53 shows a comparison of the effect of ~IPFF-1 with G-(:al~ on the
recovery
of Gra. L and Mac.: double positive cells iu the brne marrow following nvo
cycles o~ 5-
Fu tr,:atment as determined by FRCS Vantage method.
1
1'..
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FIG. 54 shows a comparison of the effect of MPIF-1 with G-CSF on the recovery
of hematopoietic progenitors in the bone marrow during following two cycles of
S-Fu
treatment.
FIG. 55. Time course of survival rates of MRL, lpr mice (8 weeks old) in 3
S different groups treated i.p. daily (S days/week) with 1 or S mg/kg of M-CIF
(n = 8) or
buffer (n = 9) for 14 weeks.
FIG. S6. Time course of survival rates of MRL lpr mice (8 weeks old) in 3
different groups treated i.p. once per day, 3 days/week with 1 or S mg/kg of M-
CIF (n
= 8) or buffer (n = 9) for 14 weeks.
FIG. S7. Changes in the protein cast formation by multi-blinded histological
evaluation of both kidneys of 23 week-old MRL lpr/lpr mice treated with buffer
control,
rhM-CIF or methotrexate (MTX) at 1 mg/kg, daily (S days each week) for 14
weeks
since mice were 8 weeks old. Values are the mean of 4-S mice per group ~ SEM.
FIG. S8. Changes in the glomerular lesions by multi-blinded histological
1 S evaluation of both kidneys of 23 week-old MRL lpr/lpr mice treated with
buffer control,
rhM-CIF or methotrexate (MTX) at 1 mg/kg, daily (S days each week) for 14
weeks
since mice were 8 weeks otd. Glomerular lesions assessed by multi-blinded
histological
evaluation represent the sum of basement membrane thickening, crescent
formation and
scarring, hypercellularity, fibrosis and karyorrhexis. Values are the mean of
4-5 mice
per group ~ SEM.
FIG. S9. Changes of renal sclerosis by multi-blinded histological evaluation
of
both kidneys of 23 week-old MRL lpr/lpr mice treated with buffer control, rhM-
CIF or
methotrexate (MTX) at lmg/kg, daily (S days each week) for 14 weeks since mice
were
8 weeks old. Values are the mean of 4-S mice per group ~ SEM.
FIG. 60. Changes of macrophage infiltration by immunohistological evaluation
of both kidneys of 23 week-old MRL lpr/lpr mice treated with buffer control,
rhM-CIF
or methotrexate (MTX) at 1 mg/kg, daily (S days each week) for 14 weeks since
mice
were 8 weeks old. Values are the mean of 4-S mice per group ~ SEM.
FIG. 61. Changes of lymphocyte infiltration and perivasculitis by multi-
blinded
histological evaluation of both kidneys of 23 week-old MRL lpr/lpr mice
treated with
buffer control, rhM-CIF or methotrexate (MTX) at 1 mg/kg, daily (S days each
week)
-18-
IW \. \~\.;-.!'-\-\II i.;.~IIL'. ~~:p :tm- 4-:i2i t23 I_ , _. J . ti :i:) -
:SSl:l4.in;yFI23
J~-.V ~ ~J-~.- -~J ,'1n , -Cn .V - . 11
for I4 weeks since mice were 8 weeks old. Values are the mean of 4-5 mice per
group
T SEM.
FIG. 62 shows a schematic representation of the pHE4-S expression vector (SEQ
ID ~IU:Sd) and the subcioned MPIF-1 d2.3 cDNA coding sequence. The Io~cior~ of
the
kanamycin resistance marker gene) tl-.e MPTF-i Q23 coding sequence, the oriC
sequence,
and the Iaclq coding sequence are indicated.
F1G. 63 shows an overview of the fermentation process for the production of
MPIF-1 Q23.
FIG- 64 shows a flow diagram of the methods used to recover W'IF-I23
I 0 produced by the process shown in FIG. 63.
FIG. 65 shows the process for the purification of MPIF-IA23 produced and
recovered by the processes shown in FIGS. b3 and 64.
FIG. 66 shows the nucleotide sequence of the regulatory elements of the pHE
promoter (SEQ ID N0:57). 'Ii'te two lac operator sequences, the Shine-BelSatro
1 ~ sequence (SID), and the terminal Hi~itII and lVdel restriction sites
(ital;ei~ed) are
:ndicatcd.
rIU. 67:i-67G shows the complete nucleotide sequence of the pI-lE4-~ vector
(SEQ ID N0:6).
Description of .Embodiments
20 The present invention provides diagnostic or therapeutic compositions and
methods that utiIiae isolated polynucleotide tnolecules Encoding polypeptides,
or the
polypeptides themselves, as: (i) a hunczan monocvte-colony inhibitory factor
(M-CIA')
polypeptides (previously termed MIPi-y and chemokine ~31(CK~31 or ckb-I));
(ii) human myeloid progenitor inhibitory factor-1 (MPIF-I) polypeptides
(previously
25 termed MIP-3 and chemokine j~8(CKpB or ckb-8)); andlor (iii) macrophage
inhibitory
protein-4 (MIP-4~}, as are vectozs, host cells and recombinant or synthetic
methods for
producing she same.
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MPIF 1, M CIF and MIP 9 Polynucleotides
In accordance with an aspect of the present invention, there are provided
isolated
nucleic acids (polynucleotides) which encode for the full-length or mature
MPIF-1, M-
CIF or MIP-4 polypeptide having the deduced amino acid sequence of,
respectively,
Figures l, 2 or 3 (SEQ ID NOS:4, 2, and 6) and for the mature MPIF-1
polypeptide
encoded by the cDNA of the clones) deposited as ATCC Deposit No. 75676 on
February 9, 1994, and for the mature MIP-4 polypeptide encoded by the cDNA of
the
clone deposited as ATCC Deposit No. 75675 on February 9, 1994 and for the
mature M-
CIF polypeptide encoded by the cDNA of the clone deposited as ATCC No. 75572,
deposited on October 13, 1993. The address of the American Type Culture
Collection
is 1230l Park Lawn Drive, Rockville, Maryland 20852. The deposited clones are
contained in the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA).
The deposits} referred to herein will be maintained under the terms of the
Budapest Treaty on the International Recognition of the Deposit of Micro-
Organisms
for Purposes of Patent Procedure. These deposits are provided merely as
convenience
to those of skill in the art and are not an admission that a deposit is
required under 35
U.S.C. ~ 112. The sequence of the polynucleotides contained in the deposited
materials,
as well as the amino acid sequence of the polypeptides encoded thereby, are
incorporated herein by reference and are controlling in the event of any
conflict with
description of sequences herein. A license can be required to make, use or
sell the
deposited materials, and no such license is hereby granted.
Polynucleotides encoding polypeptides of the present invention are
structurally
related to the pro-inflammatory supergene "inteTCrine" which is in the
cytokine or
chemokine family. Both MPIF-l and MIP-4 are M-CIF homologues and are more
homologous to MIP-1 a than to MIP-1 ~3. The polynucleotide encoding for MPIF-1
was
derived from an aortic endothelium cDNA library and contains an open reading
frame
encoding a polypeptide of 120 amino acid residues, which exhibits significant
homology
to a number of chemokines. The top match is to the human macrophage
inflammatory
protein 1 alpha, showing 36% identity and 66% similarity (Figure 4).
The polynucleotide encoding MIP-4 (SEQ ID NO:S) was derived from a human
adult lung cDNA library and contains an open reading frame encoding a
polypeptide of
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WO 98/14582 PCTNS97/17505
89 amino acid residues (SEQ ID N0:6), which exhibits significant homology to a
number of chemokines. The top match is to the human tonsillar lymphocyte LD78
beta
protein (SEQ ID NO:55), showing 60% identity and 89% similarity (Figure 5).
Furthermore, the four cysteine residues occurring in all chemokines in a
characteristic
motif are conserved in both clone(s). The fact that the first two cysteine
residues in the
genes are in adjacent positions classifies them as "C-C" or ~3 subfamily of
chemokines.
In the other subfamily, the "CXC" or a subfamily, the first two cysteine
residues are
separated by one amino acid.
The polynucleotide encoding from M-CIF (SEQ ID NO:1 ) contains and open
reading frame encoding a polypeptide of 93 amino acids (SEQ ID N0:2), of which
the
first about 19 are a leader sequence such that the mature peptide contains
about 74
amino acid residues. M-CIF exhibits significant homology to human macrophage
inhibitory protein-a, with 48% identity and 72% similarity over a stretch of
80 amino
acids. Further, the four cysteine residues comprising a characteristic motif
are
conserved.
The polynucleotides of the present invention can be in the form of RNA or in
the
form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA. The
DNA can be double-stranded or single-stranded, and if single stranded can be
the coding
strand or non-coding (anti-sense) strand. The coding sequence which encodes
the
mature polypeptides can be identical to the coding sequence shown in Figures
1, 2 and
3 (SEQ ID NOS:3, 1, and 5, respectively) or that of the deposited clones) or
can be a
different coding sequence which coding sequence, as a result of the redundancy
or
degeneracy of the genetic code, encodes the same, mature polypeptides as the
DNA of
Figure 1, 2 and 3 (SEQ ID NOS:3) l and 5) or the deposited cDNAs.
The polynucleotides which encode for the mature polypeptides of Figures 1, 2
and 3 (SEQ ID NOS:4, 2, and 6) or for the mature polypeptides encoded by the
deposited cDNA can include: only the coding sequence for the mature
polypeptide; the
coding sequence for the mature polypeptides and additional coding sequence
such as a
leader or secretory sequence or a proprotein sequence; the coding sequence for
the
mature polypeptides (and optionally additional coding sequence) and non-coding
sequence, such as introns or non-coding sequence 5' and/or 3' of the coding
sequence for
the mature polypeptides.
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Thus, the term "polynucleotide encoding a polypeptide" encompasses a
polynucleotide which includes only coding sequence for the polypeptide as well
as a
polynucleotide which includes additional coding and/or non-coding sequence.
Unless otherwise indicated, all nucleotide sequences determined by sequencing
a DNA molecule herein were determined using an automated DNA sequencer (such
as
the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of
polypeptides encoded by DNA molecules determined herein were predicted by
translation of a DNA sequence determined as above. Therefore, as is known in
the art
for any DNA sequence determined by this automated approach, any nucleotide
sequence
determined herein may contain some errors. Nucleotide sequences determined by
automation are typically at least about 90% identical, more typically at least
about 95%
to at least about 99.9% identical to the actual nucleotide sequence of the
sequenced
DNA molecule. The actual sequence can be more precisely determined by other
approaches including manual DNA sequencing methods well known in the art. As
is
also known in the art, a single insertion or deletion in a determined
nucleotide sequence
compared to the actual sequence will cause a frame shift in translation of the
nucleotide
sequence such that the predicted amino acid sequence encoded by a determined
nucleotide sequence will be completely different from the amino acid sequence
actually
encoded by the sequenced DNA molecule, beginning at the point of such an
insertion
or deletion.
Unless otherwise indicated, each "nucleotide sequence" set forth herein is
presented as a sequence of deoxyribonucleotides (abbreviated A, G , C and T).
However, by "nucleotide sequence" of a nucleic acid molecule or polynucleotide
is
intended, for a DNA molecule or polynucleotide, a sequence of
deoxyribonucleotides,
and for an RNA molecule or polynucleotide, the corresponding sequence of
ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T)
in the
specified deoxyribonucleotide sequence is replaced by the ribonucleotide
uridine (U).
For instance, reference to an RNA molecule having the sequence of SEQ ID NO:1,
3 or
5, as set forth using deoxyribonucleotide abbreviations, is intended to
indicate an RNA
molecule having a sequence in which each deoxyribonucleotide A, G or C of SEQ
ID
NO:1 has been replaced by the corresponding ribonucleotide A, G or C, and each
deoxyribonucleotide T has been replaced by a ribonucleotide U.
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Using the information provided herein, such as the nucleotide sequence in
Figures l, 2, or 3, a nucleic acid molecule of the present invention encoding
an MPIF-I,
M-CIF or MIP-4 (respectively) polypeptide may be obtained using standard
cloning and
screening procedures, such as those for cloning cDNAs using mRNA as starting
material.
The present invention further relates to variants of the hereinabove described
polynucleotides which encode for fragments, analogs and derivatives of the
polypeptide
having the deduced amino acid sequence of Figures 1, 2 and 3 (SEQ ID NOS:4, 2,
and
6) or the polypeptides encoded by the cDNA of the deposited clone(s). The
variants of
the polynucleotides can be a naturally occurring allelic variant of the
polynucleotides
or a non-naturally occurnng variant of the polynucleotides.
The present invention also includes polynucleotides encoding the same mature
polypeptides as shown in Figures 1, 2 and 3 (SEQ ID NOS:4, 2 and 6) or the
same
mature polypeptides encoded by the cDNA of the deposited clones) as well as
variants
of such polynucleotides which variants encode for a fragment, derivative or
analog of
the polypeptides of Figures 1, 2 and 3 (SEQ ID NOS:4, 2 and 6) or the
polypeptides
encoded by the cDNA of the deposited clone(s). Such nucleotide variants
include
deletion variants, substitution variants and addition or insertion variants.
As hereinabove indicated, the polynucleotide can have a coding sequence which
is a naturally occurring allelic variant of the coding sequence shown in
Figures 1, 2 and
3 (SEQ ID NOS:4, 2 and 6) or of the coding sequence of the deposited clone(s).
As
known in the art, an allelic variant is an alternate form of a polynucleotide
sequence
which can have a substitution, deletion or addition of one or more
nucleotides, which
does not substantially alter the function of the encoded polypeptide.
The present invention also includes polynucleotides, wherein the coding
sequence for the mature polypeptides can be fused in the same reading frame to
a
polynucleotide sequence which aids in expression and secretion of a
polypeptide from
a host cell, for example, a leader sequence which functions as a secretory
sequence for
controlling transport of a polypeptide from the cell. The polypeptide having a
leader
sequence is a preprotein and can have the leader sequence cleaved by the host
cell to
form the mature form of the polypeptide. The polynucleotides can also encode
for a
proprotein which is the mature protein plus additional 5' amino acid residues.
A mature
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protein having a prosequence is a proprotein and is an inactive form of the
protein.
Once the prosequence is cleaved an active mature protein remains.
Thus, for example, the polynucleotides of the present invention can encode for
a mature protein, or for a protein having a prosequence or for a protein
having both a
prosequence and a presequence (leader sequence).
The polynucleotides of the present invention can also have the coding sequence
fused in frame to a marker sequence which allows for purification of the
polypeptides
of the present invention. The marker sequence can be a hexa-histidine tag
supplied by
a pQE-9 vector to provide for purification of the mature polypeptides fused to
the
marker in the case of a bacterial host, or, for example, the marker sequence
can be a
hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used. The
HA tag
corresponds to an epitope derived from the influenza hemagglutinin protein
(Wilson, I.,
et al., Cell, 37:767 (1984)).
The term "gene" means the segment of DNA involved in producing a
polypeptide chain; it includes regions preceding and following the coding
region (leader
and trailer) as well as intervening sequences (introns) between individual
coding
segments (exons).
As indicated, nucleic acid molecules of the present invention may be in the
form
of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and
genomic DNA obtained by cloning or produced synthetically. The DNA may be
double-stranded or single-stranded. Single-stranded DNA or RNA may be the
coding
strand, also known as the sense strand, or it may be the non-coding strand,
also referred
to as the anti-sense strand.
The term "isolated" means that the material is removed from its original
environment (e.g., the natural environment if it is naturally occurring). For
example, a
naturally-occurring polynucleotide or polypeptide present in a living animal
is not
isolated, but the same polynucleotides or DNA or polypeptides, separated from
some or
a11 of the coexisting materials in the natural system, is isolated. Such
polynucleotides
could be part of a vector and/or such polynucleotides or polypeptides could be
part of
a composition, and still be isolated in that such vector or composition is not
part of its
natural environment. Isolated RNA molecules include in vivo or in vitro RNA
transcripts of the DNA molecules of the present invention. Isolated nucleic
acid
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molecules according to the present invention further include such molecules
produced
synthetically.
Isolated nucleic acid molecules of the present invention include DNA molecules
comprising an open reading frame (ORF) for a MPIF-l, M-CIF or MIP-4 cDNA; DNA
molecules comprising the coding sequence for a mature M-CIF, MPIF-1 or MIP-4
protein; and DNA molecules which comprise a sequence substantially different
from
those described above but which, due to the degeneracy of the genetic code,
still encode
an MPIF-1, M-CIF or MIP-4 polypeptide. Of course, the genetic code is well
known
in the art. Thus, it would be routine for one skilled in the art to generate
the degenerate
variants described above.
The present invention further relates to polynucleotides which hybridize to
the
hereinabove-described sequences if there is at least 95% identity between the
sequences.
The present invention particularly relates to polynucleotides which hybridize
under
stringent conditions to the hereinabove-described polynucleotides. As herein
used, the
term "stringent conditions" means hybridization will occur only if there is at
least 95%
and preferably at least 97% identity between the sequences. The
polynucleotides which
hybridize to the hereinabove described polynucleotides in a preferred
embodiment
encode polypeptides which retain substantially the same biological function or
activity
as the mature polypeptide encoded by the cDNAs of Figure l, 2 and 3 (SEQ ID
N0:3,
1, and 5) or the deposited cDNA(s).
Alternatively, the polynucleotide may have at least 20 bases, preferably 30
bases,
and more preferably at least 50 bases which hybridize to a polynucleotide of
the present
invention and which has an identity thereto, as hereinabove described) and
which may
or may not retain activity. For example, such polynucleotides may be employed
as
probes for the polynucleotide of SEQ ID NO:I, 3 and 5, for example, for
recovery of the
polynucleotide or as a diagnostic probe or as a PCR primer.
In another aspect, the invention provides an isolated nucleic acid molecule
comprising a polynucleotide which hybridizes under stringent hybridization
conditions
to a portion of the polynucleotide in a nucleic acid molecule of the invention
described
above, for instance, the eDNA clone contained in ATCC Deposit 75S72 (M-CIF);
ATCC Deposit 75676 (MPIF-1 ); or ATCC Deposit 75675 (MIP-4). By "stringent
hybridization conditions" is intended overnight incubation at 42 ~ C in a
solution
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WO 98I14582 PCTIUS97/17505
comprising: 50% formamide, 5x SSC (150 mM NaCI, lSmM trisodium citrate), 50 mM
sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20
g/ml
denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1x
SSC at
about 65 ~C.
By a polynucleotide which hybridizes to a "portion" of a polynucleotide is
intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15
nucleotides (nt), and more preferably at least about 20 nt, still more
preferably at least
about 30 nt, and even more preferably about 30-70 nt of the reference
polynucleotide.
These are useful as diagnostic probes and primers as discussed above and in
more detail
below.
Of course, polynucleotides hybridizing to a larger portion of the reference
polynucleotide (e.g. the deposited cDNA clone), for instance, a portion 50-750
nt in
length, or even to the entire length of the reference polynucleotide, are also
useful as
probes according to the present invention, as are polynucleotides
corresponding to most,
if not a11, of the nucleotide sequence of the deposited cDNA or the nucleotide
sequence
as shown in SEQ ID NO:I (M-CIF); SEQ ID N0:3 (MPIF-1); or SEQ ID N0:5 (MIP-
4). By a portion of a polynucleotide of "at least 20 nt in length," for
example, is
intended 20 or more contiguous nucleotides from the nucleotide sequence of the
reference polynucleotide. As indicated, such portions are useful
diagnostically either
as a probe according to conventional DNA hybridization techniques or as
primers for
amplification of a target sequence by the polymerase chain reaction (PCR), as
described,
for instance, in Molecular Cloning, A Laboratory Manual, 2nd. edition,
Sambrook, J.,
Fritsch, E. F. and Maniatis, T., eds., Cold Spring Harbor Laboratory Press,
Cold Spring
Harbor, N.Y. ( 1989), the entire disclosure of which is hereby incorporated
herein by
reference.
Since a MPIF-1, M-CIF and MIP-4 cDNA clones have been deposited and its
determined nucleotide sequence provided, generating polynucleotides which
hybridize
to a portion of the MPIF-1, M-CIF or MIP-4 cDNA molecules would be routine to
the
skilled artisan. For example, restriction endonuclease cleavage or shearing by
sonication
of a MPIF-1, M-CIF or MIP-4 cDNA clone could easily be used to generate DNA
portions of various sizes which are polynucleotides that hybridize,
respectively, to a
portion of the MPIF-1, M-CIF or MIP-4 cDNA molecules.
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Alternatively, the hybridizing polynucleotides of the present invention could
be
generated synthetically according to known techniques. Of course, a
polynucleotide
which hybridizes only to a poly A sequence (such as the 3' terminal poly(A)
tract of a
cDNA, or to a complementary stretch of T (or U) residues, would not be
included in a
S polynucleotide of the invention used to hybridize to a portion of a nucleic
acid of the
invention, since such a polynucleotide would hybridize to any nucleic acid
molecule
containing a poly (A) stretch or the complement thereof (e.g. practically any
double-stranded cDNA clone).
As indicated, nucleic acid molecules of the present invention which encode an
IO MPIF-I, M-CIF or MIP-4 polypeptide may include, but are not limited to
those
encoding the amino acid sequence of the mature polypeptide, by itself; the
coding
sequence for the mature polypeptide and additional sequences, such as those
encoding
the leader or secretory sequence, such as a pre-, or pro- or prepro- protein
sequence; the
coding sequence of the mature polypeptide, with or without the aforementioned
15 additional coding sequences, together with additional, non-coding
sequences, including
for example, but not limited to introns and non-coding 5' and 3' sequences,
such as the
transcribed, non-translated sequences that play a role in transcription, mRNA
processing, including splicing and polyadenylation signals, for example -
ribosome
binding and stability of mRNA; an additional coding sequence which codes for
20 additional amino acids, such as those which provide additional
functionalities. Thus,
the sequence encoding the polypeptide may be fused to a marker sequence, such
as a
sequence encoding a peptide which facilitates purification of the fused
polypeptide. In
certain preferred embodiments of this aspect of the invention, the marker
amino acid
sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector
(Qiagen)
25 Inc.), among others, many of which are commercially available. As described
in Gentz
et al., Proc. Natl. Acad. Sci. USA 86:821-824 (l989), for instance, hexa-
histidine
provides for convenient purification of the fusion protein. The "HA" tag is
another
peptide useful for purification which corresponds to an epitope derived from
the
influenza hemagglutinin protein, which has been described by Wilson et al.,
Cell 37:
30 767 ( I 984). As discussed below, other such fusion proteins include at
least one of an
MPIF-1, M-CIF or MIP-4 polypeptide or fragment fused to Fc at the N- or C-
terminus.
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The present invention further relates to variants of the nucleic acid
molecules of
the present invention, which encode portions, analogs or derivatives of an
MPIF-1, M-
CIF or MIP-4 polypeptide. Variants may occur naturally, such as a natural
allelic
variant. By an "allelic variant" is intended one of several alternate forms of
a gene
occupying a given locus on a chromosome of an organism. Genes V, Lewin, B.,
ed.,
Oxford University Press, New York ( 1994). Non-naturally occurring variants
may be
produced using art-known mutagenesis techniques.
Such variants include those produced by nucleotide substitutions, deletions or
additions. The substitutions, deletions or additions may involve one or more
nucleotides. The variants may be altered in coding regions, non-coding
regions, or both.
Alterations in the coding regions may produce conservative or non-conservative
amino
acid substitutions, deletions or additions. Especially preferred among these
are silent
substitutions, additions and deletions, which do not alter the properties and
activities of
an MPIF-1, M-CIF or MIP-4 polypeptide or portions thereof. Also especially
preferred
in this regard are conservative substitutions. Most highly preferred are
nucleic acid
molecules encoding the mature protein or the mature amino acid sequence
encoded by
the deposited cDNA clone, as described herein.
MPIF l, M CIF and MIP-4 HomoJog Pnlvnucleotides. The present invention
is further directed to polynucleotides having at least 95% identity to a
polynucleotide
which encodes the polypeptide of SEQ ID N0:2, 4 and 6 as well as fragments
thereof,
which fragments have at least 30 bases and preferably at least 50 bases and to
polypeptides encoded by such polynucleotides.
Further embodiments of the invention include isolated nucleic acid molecules
comprising a polynucleotide having a nucleotide sequence at least 95%, 96%,
97%, 98%
or 99% identical to (a) a nucleotide sequence encoding an MPIF-1, M-CIF or MIP-
4
polypeptide or fragment, having an amino acid sequence of SEQ ID N0:4, SEQ ID
N0:2, or SEQ ID N0:6, respectively, including the predicted leader sequence;
(b) a
nucleotide sequence encoding an MPIF-1, M-CIF or MIP-4 polypeptide or
fragment,
having an amino acid sequence of SEQ ID N0:4, SEQ ID N0:2, or SEQ ID N0:6,
respectively, including the predicted leader sequence, but minus the N-
terminal
methionine residue; (c) a nucleotide sequence encoding the mature MPIF-1, M-
CIF or
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WO 98/14582 PCT/US97/17505
MIP-4 polypeptide (full-length polypeptide with the leader removed); (d) a
nucleotide
sequence encoding the full-length polypeptide having the complete amino acid
sequence
including the leader encoded by the deposited cDNA clone; (e) a nucleotide
sequence
encoding the mature polypeptide having the amino acid sequence encoded by the
deposited cDNA clone; or (f) a nucleotide sequence complementary to any of the
nucleotide sequences in (a), (b), (c), (d), or (e).
By a polynucleotide having a nucleotide sequence at least, for example, 95%
"identical" to a reference nucleotide sequence encoding an MPIF-I, M-CIF or
MIP-4
polypeptide is intended that the nucleotide sequence of the polynucleotide is
identical
to the reference sequence except that the polynucleotide sequence may include
up to five
point mutations per each 100 nucleotides of the reference nucleotide sequence
encoding
the polypeptide. In other words, to obtain a polynucleotide having a
nucleotide
sequence at least 95% identical to a reference nucleotide sequence, up to 5%
of the
nucleotides in the reference sequence may be deleted or substituted with
another
nucleotide, or a number of nucleotides up to 5% of the total nucleotides in
the reference
sequence may be inserted into the reference sequence. These mutations of the
reference
sequence may occur at the 5' or 3' terminal positions of the reference
nucleotide
sequence or anywhere between those terminal positions, interspersed either
individually
among nucleotides in the reference sequence or in one or more contiguous
groups within
the reference sequence.
As a practical matter, whether any particular nucleic acid molecule is at
least
95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence
shown
in Figures 1, 3 or 5, or to the nucleotides sequence of the deposited cDNA
clone can be
determined conventionally using known computer programs such as the Bestfit
program
(Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
Group,
University Research Park, 57S Science Drive, Madison, WI 53711. Bestfit uses
the
local homology algorithm of Smith and Waterman, Advances in Applied
Mathematics
2: 482-489 ( 198 I ), to find the best segment of homology between two
sequences. When
using Bestfit or any other sequence alignment program to determine whether a
particular
sequence is, for instance, 95% identical to a reference sequence according to
the present
invention, the parameters are set, of course, such that the percentage of
identity is
calculated over the full length of the reference nucleotide sequence and that
gaps in
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WO 98/14582 PCT/US97/17505
homology of up to 5% of the total number of nucleotides in the reference
sequence are
allowed.
As one of ordinary skill would appreciate, due to the possibilities of
sequencing
errors discussed above, as well as the variability of cleavage sites for
leaders in different
known proteins, the mature M-CIF polypeptide encoded by the deposited cDNA
comprises about 74 amino acids, but may be anywhere in the range of 69-93
amino
acids; and the actual leader sequence of this protein is about 19 amino acids,
but may be
anywhere in the range of about 15 to about 24 amino acids.
As one of ordinary skill would appreciate, due to the possibilities of
sequencing
errors discussed above, as well as the variability of cleavage sites for
leaders in different
known proteins, the mature MPIF-1 polypeptide encoded by the deposited cDNA
comprises about 99 amino acids, but may be anywhere in the range of 75-120
amino
acids; and the actual leader sequence of this protein is about 21 amino acids,
but may be
anywhere in the range of about 15 to about 35 amino acids.
I 5 As one of ordinary skill would appreciate, due to the possibilities of
sequencing
errors discussed above, as well as the variability of cleavage sites for
leaders in different
known proteins, the mature MIP-4 polypeptide encoded by the deposited cDNA
comprises about 69 amino acids, but may be anywhere in the range of 60-89
amino
acids; and the actual leader sequence of this protein is about 20 amino acids,
but may be
anywhere in the range of about 15 to about 30 amino acids.
Nucleic Acid Probes. Such isolated molecules, particularly DNA molecules, are
useful as probes for gene mapping, by in sim hybridization with chromosomes,
and for
detecting expression of a MPIF-1, M-CIF and/or MIP-4 gene in human tissue, for
instance, by Northern blot analysis. The present invention is further directed
to
fragments of the isolated nucleic acid molecules described herein. By a
fragment of an
isolated nucleic acid molecule having the nucleotide sequence of the deposited
MPIF-I,
M-CIF or MIP-4 cDNAs, or a nucleotide sequence shown in any or all of Figures
1, 2
and 3 (SEQ ID NOS:3, I and S), respectively, is intended fragments at least
about 15
nt, and more preferably at least about 20 nt, still more preferably at least
about 30 nt, and
even more preferably, at least about 40 nt in length which are useful as
diagnostic probes
and primers as discussed herein. Of course, larger fragments 50-500 nt in
length are also
useful according to the present invention as are fragments corresponding to
most, if not
-30-
.o : ,rs . . .,',) ;s ,','.i',ll I~ ..:y'.I
~ ,II i ~.:i , I -.
,. . . ~ - _ _ mL_~ ~__ rn1~ _.e~ ~~: W. m
a11- of a nucleotide sequence of the deposited MPIF-I, PvI-CIF or ivfIP--~
cDNAs, or as
shown in Figures 1, 2 and 3 (SEQ Il7 NOS:3, 1 and 5). By a f~ag~tent at lease
20 nt in
length) for example, is intended fr~ments which include 20 or rr~o:e
conti;uous baszs
from the nucleotide sequence of the deposited cDNA or the nucleotide sequence
as
sho~.ur~ in Figures 1, 2 and 3 (SEQ ID NOS:3, 1 arid 5). Since the gene has
lien
deposited and the nuclwtide sequences shown in Figures I, 2 and 3 (SEQ ID
NOS:3,
l and 51 are provided, generating such DNA fragments would be routine to the
skilled
artisan. Ir or example, restriction endonucle'ase cleavage or shearing by
sonication could
easily be used to generate Fragments of various sues. Alternatively, su~.h
!~a'~ments
LU coald be generated synthetically.
Fragments of the full lea th gene of the present invention may be used as a
hybrdiz3tion_ probe for a cDN:~ library to isolate the full lengt<n cDNA and
to isolate
other cDNAS which have a high sequence similarity to the gene or similar
biological
activity. Probes of this type preferably have at least 30 bases and rnav
eontailz, for
1 ~ ~~ample, 50 or ;pore bases. The probe may also be used to identify a cDN:~
clone
co:responding to a full length transcript and a gnomic clone or clones that
contain ~h~
complete gene including regulatory and promotor regions) exons, and introns.
An
e:cample of a screen comprises isolating the coding region of the gene by
using the
known DNA sequercc to synthesize an oIibonucleotide probe. Labeled
c~iibonucleotides
?0 hawing a sequence complementary to that of the gene of the pr;,sent
invention are used
to scze~n a library of human cDNA, genonuc DNA or mP.NA to determine which
members of the library the probe hybridizes to.
Vectors) Host Cells, and Protein Expression. The present i_nventic-,n aisc~ ;-
eiat~s
to vectors containing the isolated nucleic acid molecules of the present
invention,
25 genetically engineered host cells containing the recombinant vector, and
the production
of MPIF-1, M-CIF or MIP-4 polypeptides or fragments thereof by recombinant
techniques: The present invention further relaxes to novel expression vectors
useful for
the production of proteins in bacterial systems. These novel vectors are
exemplified by
the pa7~4 series of vectors and, in particular, the pldE4-5 vector (FIGs. 62
and 67A
30 67G).
The pol~-nu~elebtides encoding the proteins of the present invention rxay be
joined
to a vector containing a selectable marker for propagation in a host- As
discussed in
d~ tail below) gcner3.liy, a plasmid vector is introduced into a host cell in
a p.rec:ipit~ae,
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WO 98/14582 PCT/US97/17505
such as a calcium phosphate precipitate, or in a complex with a charged lipid.
If the
vector is a virus, it may be packaged in vitro using an appropriate packaging
cell line
and then transduced into host cells.
Preferred for use in the practice of the present invention are vectors
comprising
cis-acting control regions operatively linked to the polynucleotide of
interest. Cis-acting
control regions include operator and enhancer sequences. As used herein, the
term
"operator" refers to a nucleotide sequence, usually composed of DNA, which
controls
the transcription of an adjacent nucleotide sequence. Operator sequences are
generally
derived from bacterial chromosomes.
Transcription of the nucleotide sequences encoding the polypeptides of the
present invention by higher eukaryotes may be increased by inserting an
enhancer
sequence into the vector. Enhancers are cis-acting elements usually about from
10 to
300 by that act to increase transcriptional activity of a promoter in a given
host cell-type.
Examples of enhancers include the SV40 enhancer, which is located on the late
side of
1 S the replication origin at by 100 to 270, the cytomegalovirus early
promoter enhancer, the
polyoma enhancer on the late side of the replication origin, and adenovirus
enhancers.
Appropriate traps-acting factors may be supplied by the host, supplied by a
complementing vector, or supplied by the vector itself upon introduction into
the host.
In certain preferred embodiments in this regard, the vectors provide for
specific
expression, which may be inducible and/or cell type-specific. Particularly
preferred
among such vectors are those inducible by environmental factors that are easy
to
manipulate, such as temperature and nutrient additives. Also preferred for the
expression of MPIF-1 is the pHE4-5 vector described in Example 30.
Additional expression vectors useful in the present invention include
chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from
bacterial
plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses
such as
baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox
viruses,
pseudorabies viruses and retroviruses, and vectors derived from combinations
thereof,
such as cosmids and phagemids.
The appropriate nucleic acid sequence can be inserted into the vector by a
variety
of procedures. In general, the nucleic acid sequence is inserted into an
appropriate
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WO 98/14582 PCT/US97/17505
restriction endonuclease sites) by procedures known in the art. Such
procedures and
others are deemed to be within the scope of those skilled in the art.
The nucleic acid insert should be operatively linked to an appropriate
promoter,
such as the phage lambda PL promoter, the E. coli lac) trp and tac promoters,
the SV40
early and late promoters and promoters of retroviral LTRs, and other promoters
known
to control expression of genes in prokaryotic or eukaryotic cells or their
viruses. Other
suitable promoters will be known to the skilled artisan. As used herein, the
term
"promoter" refers to a nucleotide sequence or group of nucleotide sequences
which, at
a minimum, provides a binding site or initiation site for RNA polymerase
action. The
expression constructs will further contain sites for transcription initiation,
termination
and, in the transcribed region, a ribosome binding site for translation. The
coding
portion of the mature transcripts expressed by the constructs will preferably
include a
translation initiating at the beginning and a termination codon (UAA, UGA or
UAG)
appropriately positioned at the end of the polypeptide to be translated. The
vector can
1 S also include appropriate sequences for amplifying expression.
As used herein, the phrase "operatively linked" refers to a linkage in which a
nucleotide sequence is connected to another nucleotide sequence (or sequences)
in such
a way as to be capable of altering the functioning of the sequence (or
sequences). For
example, a protein coding sequence which is operatively linked to a
promoter/operator
places expression of the protein coding sequence under the influence or
control of these
sequences. Two nucleotide sequences (such as a protein encoding sequence and a
promoter region sequence linked to the 5' end of the encoding sequence) are
said to be
operatively linked if induction of promoter function results in the
transcription of the
protein encoding sequence mRNA and if the nature of the linkage between the
two
nucleotide sequences does not ( 1 ) result in the introduction of a frame-
shift mutation nor
(2) prevent the expression regulatory sequences to direct the expression of
the mRNA
or protein. Thus, a promoter region would be operatively linked to a
nucleotide
sequence if the promoter were capable of effecting transcription of that
nucleotide
sequence.
As used herein, the phrase "cloning vector" refers to a plasmid or phage
nucleic
acid or other nucleic acid sequence which is able to replicate autonomously in
a host
cell, and which is characterized by one or a small number of endonuclease
recognition
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WO 98I14582 PCT/US97/17505
sites at which such nucleic acid sequences may be cut in a determinable
fashion without
loss of an essential biological function of the vector, and into which nucleic
acid may
be spliced in order to bring about its replication and cloning. The cloning
vector may
further contain a marker suitable for use in the identification of cells
transformed with
the cloning vector. Markers, for example, are erythromycin and kanamycin
resistance.
The term "vehicle" is sometimes used for "vector."
As used herein, the phrase "expression vector" refers to a vector similar to a
cloning vector which is capable of expressing a structural gene cloned into
the
expression vector, after transformation of the expression vector into a host.
In an
expression vector, the cloned structural gene (any coding sequence of
interest) is placed
under the control of (i.e., operatively linked to) certain sequences which
allow such gene
to be expressed in a specific host. In the pHE4-5 vector, for example, the
structural gene
is operatively linked to a TS phage promoter sequence and two lac operator
sequences.
Expression control sequences will vary, and may additionally contain
transcriptional
elements such as termination sequences and/or translational elements such as
initiation
and termination sites.
As indicated above, the expression vectors will preferably include at least
one
selectable marker. Such markers include dihydrofolate reductase or neomycin
resistance
for eukaryotic cell culture and tetracycline, kanamycin, or ampicillin
resistance genes
for culturing in E. col i and other bacteria. Representative examples of
appropriate hosts
include, but are not limited to, bacterial cells, such as E. col i,
Streptomyces and
Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells
such as
Drosophila S2 and Spodoptera Sf~ cells; animal cells such as CHO, COS and
Bowes
melanoma cells; and plant cells. Appropriate culture mediums and conditions
for the
above-described host cells are known in the art.
In addition to the use of expression vectors in the practice of the present
invention, the present invention further includes novel expression vectors
comprising
operator and promoter elements operatively linked to nucleotide sequences
encoding a
protein of interest. One example of such a vector is pHE4-5 (SEQ ID N0:56)
which is
~ described in detail both below and in Example 30. A bacterial host
containing the
pHE4-5 vector was deposited on September 30, I997 at the American Type Culture
-34-
~~\ w: . i W. -',U I ~ ill. n:-, ~ .~;, irj I _ . _. -I ; ;iii ~:i:~;Ll-.I
5.;: y i)
~..,u i - _ i em- _._ ~~ v _u ~u m. m
Collection, 12.301 Park Lawn Drive, Roekville, l~iaryland 20852, and given
ATCC
accession number ?0931 I .
t'$ Slunmaa'lZed in FIGS. 52 and 67A-67G, components of the pHE=1-5 vector
(SEQ ID'_V O:J6) include: 1 ). a neotuycinphesphotrans:erwe gene as a
selection marker,
2). an E. toll origin of replication, 3). a TS phage promoter sequence, 4).
rivo lac
operator Sequences, ~). a nucleotide ;,equence encoding MPIF-123 (SEQ ID
N0:27),
5). a Shine-Delaarnc sequence. 7). the lactose opexonrepressor gene (JacIq).
The origin
of replication (ariC) is dxived from pUCl9 (LTI, Gaithersburg, MD). The
promoter
sequence was and operator sequences were made synthetically. Synthetic
production of
i 0 nucleic acid sequences is well hno;am in the art. CLOt~ta~C~i 95I96
Catalog, pages 21 ~-
21 b, C~oNw::~i, 1020 East Meadow Circle, Palo ~Itv, C.~ 94303.
As noted abo~r, the pHr4-5 wc~or c; ntains a IacIq gene. LacIq is ~ allele of
the lacl ?ezze which confers tight regulation of the lac operator. ~unann, F.
er al., Gehe
59.301-31~ (1988); Stark, Vii., Gene A:255-267 (198m. The IacIq gene encodes a
1 ~ repressor protein which binds to lac operator sequences and blocks
transcription ox
down-stream (l. e., =') sequences. However, the laciq gene product dissociates
fiom the
lac operator in the pres~yne~ of either lactose or certain lac;tosc analogs,
e.~. , isopropyl
B-D-thiogalactopyranoside (TPTCT). r~iPIF-1,~3 thus is r_ot produced in
appreciable
quantities in uninduc~d >zost cells containir<g the pHE4-~ vector. Induction
of these host
20 cells by the additional onan agent such as IPTG, however, results in the
expression of the
i~iPIF-1~'?3 coding sequet3ce.
T'.ae promoter.:ope~tor sequences of the pI-~4-5 rector (SEQ ID a0:57)
comprise a -fJ phage promoter and two lac cperator sequznees. Ore operator is
looated
5' to the transcriptio~-tal start site and the ocher is located 3' to the same
site. These
25 operators, when present in combination with the iacIq gene product confer
tight
repression of down-stream sequences in the aosenec of a lac operon inducer, e.
g. , IPTG.
E.cpression of operatively linked sequences located down-stream from the lac
operators
may be induced by the addition of a lac operon induces, such as IPTG. Binding
of a lac
inducts to the IacIq proteins results in their rolease from the lac operator
sequences and
30 the initiation of transcription of operatively linked sequences. Lac operon
regulation of
gene expression is reviewed in Devlin, T., TEX'1'~OUK OF BIOCHE\-tISTR~I WITH
CLN(CAL
CoRRFS,aTIONS, ~th Fdicion ( I997 ), pages 8A2-807
CA 02267193 1999-03-30 AMENDED SHEET
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WO 98/14582 PCTIUS97/17505
The pHE4 series of vectors contain all of the components of the pHE4-5 vector
except for the MPIF-1023 coding sequence. Features of the pHE4 vectors include
optimized synthetic TS phage promoter, lac operator, and Shine-Delagarno
sequences.
Further, these sequences are also optimally spaced so that expression of an
inserted gene
may be tightly regulated and high level of expression occurs upon induction.
Among known bacterial promoters suitable for use in the production of proteins
of the present invention include the E. col i IacI and IacZ promoters, the T3
and T7
promoters, the gpt promoter, the lambda PR and PL promoters and the trp
promoter.
Suitable eukaryotic promoters include the CMV immediate early promoter, the
HSV
thymidine kinase promoter, the early and late SV40 promoters, the promoters of
retroviral LTRs, such as those of the Rous Sarcoma Virus (RSV), and
metallothionein
promoters, such as the mouse metallothionein-I promoter.
The pHE4-S vector also contains a Shine-Delgarno sequence 5' to the AUG
initiation codon. Shine-Delgarno sequences are short sequences generally
located about
10 nucleotides up-stream (i.e., 5') from the AUG initiation codon. These
sequences
essentially direct prokaryotic ribosomes to the AUG initiation codon.
Thus, the present invention is also directed to expression vector useful for
the
production of the proteins of the present invention. This aspect of the
invention is
exemplified by the pHE4-5 vector (SEQ ID N0:56).
Additional vectors preferred for use in the expression of the proteins of the
present invention in bacteria include pQE70, pQE60 and pQE-9, (Qiagen); pBS
vectors,
pD 10, Phagescript vectors, pBluescript vectors, pNHBA, pNH 16a, pNH 18A,
pNH46A,
available from Stratagene; and ptrc99a, pKK233-3, pDR540, pRITS available from
Pharmacia. Among preferred eukaryotic vectors~are pWLNEO, pSV2CAT, pOG44)
pXTI and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL
available from Pharmacia.
Other suitable vectors will be readily apparent to the skilled artisan and
include
pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and
GEM1 (Promega Biotec, Madison, WI, USA). These pBR322 "backbone" sections are
combined with an appropriate promoter and the structural sequence to be
expressed.
Following transformation of a suitable host strain and growth of the host
strain to an
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appropriate cell density, the selected promoter is induced by appropriate
means (e.g.,
temperature shift or chemical induction) and cells are cultured for an
additional period.
In a further embodiment, the present invention relates to host cells
containing the
above-described construct. The host cell can be a higher eukaryotic cell, such
as a
mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host
cell can be
a prokaryotic cell, such as a bacterial cell. Introduction of the construct
into the host cell
can be effected by calcium phosphate transfection, DEAF-dextran mediated
transfection,
cationic lipid-mediated transfection, electroporation, transduction, infection
or other
methods. Such methods are described in many standard laboratory manuals, such
as
Davis e! al., BASIC METHODS IN MOLECULAR BIOLOGY ( 1986).
Recombinant constructs may be introduced into host cells using well known
techniques such infection, transduction, transfection, transvection,
electroporation and
transformation. The vector may be, for example, a phage, plasmid, viral or
retroviral
vector. Retroviral vectors may be replication competent or replication
defective. In the
latter case, viral propagation generally will occur only in complementing host
cells.
Host cells are genetically engineered (transduced or transformed or
transfected)
with the vectors of this invention which can be) for example, a cloning vector
or an
expression vector. The vector can be, for example, in the form of a plasmid, a
viral
particle, a phage, etc. The engineered host cells can be cultured in
conventional nutrient
media modified as appropriate for activating promoters, selecting
transformants or
amplifying the MPIF-1, MIP-4 and M-CIF genes. The culture conditions, such as
temperature, pH and the like, are those previously used with the host cell
selected for
expression, and will be apparent to the ordinarily skilled artisan.
The polynucleotides of the present invention can be employed for producing
polypeptides by recombinant techniques. Thus, for example, the polynucleotide
sequence can be included in any one of a variety of expression vehicles, in
particular
vectors or plasmids for expressing a poiypeptide. Such vectors include
chromosomal,
nonchromosomal and synthetic nucleic acid sequences, e.g., derivatives of
SV40;
bacterial plasmids; phage nucleic acid; yeast plasmids; vectors derived from
combinations of plasmids and phage nucleic acid, viral nucleic acid such as
vaccinia,
adenovirus, fowl pox virus, alphaviruses and pseudorabies. However, any other
plasmid
or vector can be used as long they are replicable and viable in the host.
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WO 98I14582 PCTIUS97117505
As noted above, the vector containing the appropriate nucleic acid sequence as
hereinabove described, as well as an appropriate promoter or control sequence,
can be
employed to transform an appropriate host to permit the host to express the
protein.
As representative examples of appropriate hosts, there can be mentioned:
bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium; fungal
cells, such
as yeast; insect cells such as Drosophila and Sf9; animal cells such as CHO,
COS or
Bowes melanoma; plant cells, etc. The selection of an appropriate host is
deemed to be
within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention also includes recombinant constructs
comprising one or more of the sequences as broadly described above. The
constructs
comprise a vector, such as a plasmid or viral vector, into which a sequence of
the
invention has been inserted, in a forward or reverse orientation. In a
preferred aspect of
this embodiment, the construct further comprises regulatory sequences,
including, for
example, a promoter, operatively linked to the sequence. Large numbers of
suitable
vectors and promoters are known to those of skill in the art, and are
commercially
available. The following vectors are provided by way of example. Bacterial:
pQE70,
pQE60, pQE-9 (Qiagen), pBS, pD 10, phagescript, psiX 174, pBluescript SK,
pbsks,
pNHBA, pNH 16a, pNH 18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3,
pDR540, pRITS (Pharmacia). Eukaryotic: pWLNEO, pSV2CAT, pOG44, pXTI, pSG
(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). However, any other plasmid
or vector can be used as long as they are replicable and viable in the host.
The constructs in host cells can be used in a conventional manner to produce
the
gene product encoded by the recombinant sequence. Alternatively, the
polypeptides of
the invention can be synthetically produced by conventional peptide
synthesizers.
Mature proteins can be expressed in mammalian cells, yeast, bacteria, or other
cells under the control of appropriate promoters. Cell-free translation
systems can also
be employed to produce such proteins using RNAs derived from the nucleic acid
constructs of the present invention. Appropriate cloning and expression
vectors for use
with prokaryotic and eukaryotic hosts are described by Sambrook, et al.,
Molecular
Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,
(1989), the
disclosure of which is hereby incorporated by reference.
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Generally, recombinant expression vectors vrill include origins of replication
and
selectable markers permitting transformation of the host cell, e.g., the
ampicillin
resistance gene of E. coli and S. cerevisiae TRP 1 gene, and a promoter
derived from a
highly-expressed gene to direct transcription of a downstream structural
sequence. Such
promoters can be derived from operons encoding glycolytic enzymes such as
3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock
proteins,
among others. The heterologous structural sequence is assembled in appropriate
phase
with translation initiation and termination sequences, and preferably, a
leader sequence
capable of directing secretion of translated protein into the periplasmic
space or
extracellular medium. Optionally, the heterologous sequence can encode a
fusion
protein including an N-terminal identification peptide imparting desired
characteristics,
e.g., stabilization or simplified purification of expressed recombinant
product.
Useful expression vectors for bacterial use are constructed by inserting a
structural nucleic acid sequence encoding a desired protein together with
suitable
translation initiation and termination signals in operable reading phase with
a functional
promoter. The vector will comprise one or more phenotypic selectable markers
and an
origin of replication to ensure maintenance of the vector and to, if
desirable, provide
amplification within the host. Suitable prokaryotic hosts for transformation
include E.
coli, Bacillus subtilis, Salmonella typhimurium and various species within the
genera
Pseudomonas, Streptomyces, and Staphylococcus, although others can also be
employed
as a matter of choice.
Cells are typically harvested by centrifugation, disrupted by physical or
chemical
means, and the resulting crude extract retained for further purification.
Microbial cells employed in expression of proteins can be disrupted by any
convenient method, including freeze-thaw cycling, sonication, mechanical
disruption,
or use of cell lysing agents, such methods are well known to those skilled in
the art.
Various mammalian cell culture systems can also be employed to express
recombinant protein. Examples of mammalian expression systems include the COS-
7
lines of monkey kidney fibroblasts, described by Gluzman, Cell 23:175 ( 1981
), and
other cell lines capable of expressing a compatible vector, for example, the C
127, 3T3,
CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprise an
origin
of replication, a suitable promoter and enhancer, and also any necessary
ribosome
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WO 98I14582 PCT/US97/17505
binding sites, polyadenylation site, splice donor and acceptor sites,
transcriptional
termination sequences, and S' flanking nontranscribed sequences. Nucleic acid
sequences derived from the SV40 splice, and polyadenylation sites can be used
to
provide the required nontranscribed genetic elements.
S Polypeptides and Polypeptide Fragments. The invention further provides an
isolated MPIF-1, M-CIF, or MIP-4 polypeptide having the amino acid sequence
encoded by the deposited cDNA, or the amino acid sequence in Figure l, 2 or 3
(SEQ
ID NOS:4, 2 or 6, respectively), or a peptide or polypeptide comprising a
portion of the
above polypeptides. The terms "peptide" and "oligopeptide" are considered
synonymous (as is commonly recognized) and each term can be used
interchangeably
as the context requires to indicate a chain of at least two amino acids
coupled by
peptidyl linkages. The word "polypeptide" is used herein for chains containing
more
than ten amino acid residues. A11 oligopeptide and polypeptide formulas or
sequences
herein are written from left to right and in the direction from amino terminus
to carboxy
1 S terminus.
By "a polypeptide having MPIF-1 activity" is intended polypeptides exhibiting
activity similar, but not necessarily identical, to an activity of the MPIF-1
protein of the
invention (either the full-length protein or, preferably, the mature protein),
as measured
in a particular biological assay. MPIF-1 protein activity can be measured by
the assays
set forth in Examples 15, 16, as well as Figure 11. For example, MPIF-1
protein activity
measured using the in vitro myeloprotection assay disclosed in Example 15,
infra.
Briefly, lineage-depleted populations of cells (Liri cells) are isolated from
mouse
bone marrow and incubated in the presence of multiple cytokines with or
without MPIF-
1. After 48 hours, one set of each culture receives S-Fu and the incubation is
then
2S continued for additional 24 hours, at which point the numbers of surviving
low
proliferative potential colony-forming cells (LPP-CFC) are determined by any
suitable
clonogenic assay known to those of skill in the art. A large percentage (e.g.,
> 30-50%,
such as >40%) of LPP-CFC are protected from the S-Fu-induced cytotoxicity in
the
presence of MPiF-l, whereas little protection (c5%) of LPP-CFC will be
observed in
the absence of MPIF-1 or in the presence of an unrelated protein. In such an
assay, high
proliferative potential colony-forming cells (HPP-CFC) can additionally be
protected
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WO 98/14582 PCT/US97/17505
from the 5-Fu-induced cytotoxicity in the presence of MPIF-1, but in some
cases are not.
HPP-CFC are generally not protected when LPP-CFC are not protected.
Thus, "a polypeptide having MPIF-1 protein activity" includes polypeptides
that
exhibit MPIF-1 activity, in the above-described assay. Although the degree of
activity
need not be identical to that of the MPIF-1 protein, preferably, "a
polypeptide having
MPIF-1 protein activity" will exhibit substantially similar activity as
compared to the
MPIF-1 protein (i.e., the candidate polypeptide will exhibit greater activity
or not more
than about twenty-fold less and, preferably, not more than about ten-fold less
activity
relative to the reference MPIF-1 protein).
By "a polypeptide having M-CIF activity" is intended polypeptides exhibiting
activity similar, but not necessarily identical) to an activity of the M-CIF
protein of the
invention (either the full-length protein or, preferably, the mature protein),
as measured
in a particular biological assay. For example, M-CIF protein activity can be
measured
using the in vitro inhibition of M-CSF-induced colony formation by animal
cells, such
as bone marrow cells, in an assay as described in Example 25, infra.
Thus, "a polypeptide having M-CIF protein activity" includes polypeptides that
exhibit M-CIF activity, in the above-described assay. Although the degree of
activity
need not be identical to that of the M-CIF protein, preferably. "a polypeptide
having M-
CIF protein activity" will exhibit substantially similar activity as compared
to the M-CIF
protein (i.e., the candidate polypeptide will exhibit greater activity or not
more than
about twenty-fold less and, preferably, not more than about ten-fold less
activity relative
to the reference M-CIF protein).
The present invention further relates to MPIF-1, M-CIF and MIP-4 polypeptides
which have the deduced amino acid sequence of Figures I , 2 and 3 (SEQ ID
NOS:4, 2
and 6) or which have the amino acid sequence encoded by the deposited cDNA, as
well
as fragments, analogs and derivatives of such polypeptide.
The terms "fragment," "derivative" and "analog" when referring to the
polypeptides of Figures l, 2 and 3 (SEQ ID NOS:4, 2 and 6) or that encoded by
the
deposited cDNA, means a polypeptide which retains essentially the same
biological
function or activity as such polypeptide. Thus, an analog includes a
proprotein which
can be activated by cleavage of the proprotein portion to produce an active
mature
polypeptide.
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The polypeptides of the present invention can be a recombinant polypeptide, a
natural polypeptide or a synthetic polypeptide, preferably a recombinant
polypeptide.
The fragment, derivative or analog of the polypeptides of Figures l, 2 and 3
(SEQ ID NOS:4, 2 and 6) or that encoded by the deposited cDNA can be (i) one
in
which one or more of the amino acid residues are substituted with a conserved
or non-
conserved amino acid residue (preferably a conserved amino acid residue) and
such
substituted amino acid residues is or is not be one encoded by the genetic
code, or (ii)
one in which one or more of the amino acid residues includes a substituent
group, or (iii)
one in which the mature polypeptides are fused with another compound, such as
a
compound to increase the half life of the polypeptide (for example,
polyethylene glycol),
or (iv) one in which the additional amino acids are fused to the mature
polypeptides,
such as a leader or secretory sequence or a sequence which is employed for
purification
of the mature polypeptides or a proprotein sequence. Such fragments,
derivatives and
analogs are deemed to be within the scope of those skilled in the art from the
teachings
herein.
The polypeptides of the present invention are preferably provided in an
isolated
form, and preferably are purified to homogeneity.
The polypeptides of the present invention include the polypeptide of SEQ ID
NOS:2, 4 and 6 (in particular the mature polypeptide) as well as polypeptides
which
have at least 95% similarity (still more preferably at least 95% identity) to
the
polypeptide of SEQ ID NOS:2, 4 and 6 and also include portions of such
polypeptides
with such portion of the polypeptide generally containing at least 30 amino
acids and
more preferably at least 50 amino acids.
As known in the art "similarity" betweeci two polypeptides is determined by
comparing the amino acid sequence and its conserved amino acid substitutes of
one
polypeptide to the sequence of a second polypeptide.
Of course, due to the degeneracy of the genetic code, one of ordinary skill in
the
art will immediately recognize that a large number of the nucleic acid
molecules having
a sequence at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence
of the deposited cDNA (ATCC 7S676) or the nucleic acid sequence shown in
Figure 1
(SEQ ID N0:3) will encode a polypeptide "having MPIF-1 protein activity." One
of
ordinary skill in the art will also immediately recognize that a large number
of the
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WO 98/14582 PCT/US97/17505
nucleic acid molecules having a sequence at least 95%, 96%, 97%, 98%, or 99%
identical to the nucleic acid sequence of the deposited cDNA (ATCC 75572) or
the
nucleic acid sequence shown in Figure 2 (SEQ ID NO:1 ) will encode a
polypeptide
"having M-CIF protein activity." Additionally, one of ordinary skill in the
art will
immediately recognize that a large number of the nucleic acid molecules having
a
sequence at least 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid
sequence
of the deposited cDNA (ATCC 75675) or the nucleic acid sequence shown in
Figure 3
(SEQ ID NO:S) will encode a polypeptide "having MIP-4 protein activity." In
fact, since
degenerate variants of these nucleotide sequences all encode the same
polypeptide, this
will be clear to the skilled artisan even without performing the above
described
comparison assay. It will be further recognized in the art that, for such
nucleic acid
molecules that are not degenerate variants, a reasonable number will also
encode a
polypeptide having MPIF-1, M-CIF or MIP-4 protein activity. This is because
the
skilled artisan is fully aware of amino acid substitutions that are either
less likely or not
likely to significantly effect protein function (e.g. replacing one aliphatic
amino acid
with a second aliphatic amino acid).
For example, guidance concerning how to make phenotypically silent amino acid
substitutions is provided in Bowie, J. U. et al., "Deciphering the Message in
Protein
Sequences: Tolerance to Amino Acid Substitutions," Science 2t7: 1306-1310
(1990),
wherein the authors indicate that there are two main approaches for studying
the
tolerance of an amino acid sequence to change. The first method relies on the
process
of evolution, in which mutations are either accepted or rejected by natural
selection. The
second approach uses genetic engineering to introduce amino acid changes at
specific
positions of a cloned gene and selections or screens to identify sequences
that maintain
functionality. As the authors state, these studies have revealed that proteins
are
surprisingly tolerant of amino acid substitutions. The authors further
indicate which
amino acid changes are likely to be permissive at a certain position of the
protein. For
example, most buried amino acid residues require nonpolar side chains, whereas
few
features of surface side chains are generally conserved. Other such
phenotypically silent
substitutions are described in Bowie, J.U. et al., supra, and the references
cited therein.
Fragments or portions of the polypeptides of the present invention may be
employed for producing the corresponding full-length polypeptide by peptide
synthesis;
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therefore, the fragments may be employed as intermediates for producing the
full-length
polypeptides. Fragments or portions of the polynucleotides of the present
invention may
be used to synthesize full-length polynucleotides of the present invention.
For secretion of the translated protein into the lumen of the endoplasmic
S reticulum, into the periplasmic space or into the extracellular environment,
appropriate
secretion signals may be incorporated into the expressed polypeptide. The
signals may
be endogenous to the polypeptide or they may be heterologous signals.
The polypeptide may be expressed in a modified form, such as a fusion protein,
and may include not only secretion signals, but also additional heterologous
functional
regions. For instance, a region of additional amino acids, particularly
charged amino
acids, may be added to the N-terminus of the polypeptide to improve stability
and
persistence in the host cell, during purification, or during subsequent
handling and
storage. Also, peptide moieties may be added to the polypeptide to facilitate
purification. Such regions may be removed prior to final preparation of the
poiypeptide.
The addition of peptide moieties to polypeptides to engender secretion or
excretion, to
improve stability and to facilitate purification, among others, are familiar
and routine
techniques in the art. A preferred fusion protein comprises a heterologous
region from
immunoglobulin that is useful to solubilize proteins. For example, EP-A-O 464
533
(Canadian counterpart 2045869) discloses fusion proteins comprising various
portions
of constant region of immunoglobin molecules together with another human
protein or
part thereof. In many cases, the Fc part in a fusion protein is thoroughly
advantageous
for use in therapy and diagnosis and thus results, for example, in improved
phanmacokinetic properties (EP-A 0232 262). On the other hand, for some uses
it would
be desirable to be able to delete the Fc part after the fusion protein has
been expressed,
detected and purified in the advantageous manner described. This is the case
when Fc
portion proves to be a hindrance to use in therapy and diagnosis, for example
when the
fusion protein is to be used as antigen for immunizations. In drug discovery,
for
example, human proteins, such as, hILS-receptor has been fused with Fc
portions for the
purpose of high-throughput screening assays to identify antagonists of hIL-5.
See, D.
Bennett et al., Journal of Molecular Recognition, Vol. 8:52-58 ( 1995) and K.
Johanson
et al., The Journal ofl3iological Chemistry, Vol. 270, No. 16:9459-9471
(1995).
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The MPIF-1, M-CIF or MIP-4 protein can be recovered and purified from
recombinant cell cultures by well-known methods including ammonium sulfate or
ethanol precipitation, acid extraction, anion or canon exchange
chromatography,
phosphocellulose chromatography, hydrophobic interaction chromatography,
affinity
chromatography, hydroxylapatite chromatography and lectin chromatography. Most
preferably, high performance liquid chromatography ("HPLC") is employed for
purification. Polypeptides of the present invention include naturally purified
products,
products of chemical synthetic procedures, and products produced by
recombinant
techniques from a prokaryotic or eukaryotic host, including, for example,
bacterial,
yeast, higher plant) insect and mammalian cells. Depending upon the host
employed in
a recombinant production procedure, the polypeptides of the present invention
may be
glycosylated or may be non-glycosylated. In addition, polypeptides of the
invention
may also include an initial modified methionine residue, in some cases as a
result of
host-mediated processes.
MPIF l, M CIF and MIP-4 Polypeptide Variants. It will be recognized in the
art that some amino acid sequences of the MPIF-1, M-CIF or MIP-4 polypeptide
can
be varied without significant effect of the structure or function of the
protein. If such
differences in sequence are contemplated, it should be remembered that there
will be
critical areas on the protein which determine activity. In general, it is
possible to replace
residues which form the tertiary structure, provided that residues performing
a similar
function are used. In other instances, the type of residue may be completely
unimportant
if the alteration occurs at a non-critical region of the protein.
Thus, the invention further includes variations of an MPIF-1, M-CIF or MIP-4
polypeptide which show, respectively, substantial IvIPIF-1, M-CIF or MIP-4
polypeptide
activity or which include regions, respectively, of an MPIF-1, M-CIF or MIP-4
protein
such as the protein portions discussed below. Such mutants include deletions,
insertions, inversions, repeats, and type substitutions (for example,
substituting one
hydrophilic residue for another, but not strongly hydrophilic for strongly
hydrophobic
as a rule). Small changes or such "neutral" amino acid substitutions will
generally have
little effect on activity.
Typically seen as conservative substitutions are the replacements, one for
another, among the aliphatic amino acids Ala, Val, Leu and Ile; interchange of
the
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hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and Glu,
substitution
between the amide residues Asn and Gln, exchange of the basic residues Lys and
Arg
and replacements among the aromatic residues Phe, Tyr.
Of additional special interest are also substitutions of charged amino acids
with
another charged amino acid or with neutral amino acids. This may result in
proteins
with improved characteristics such as less aggregation. Prevention of
aggregation is
highly desirable. Aggregation of proteins cannot only result in a reduced
activity but be
problematic when preparing pharmaceutical formulations because they can be
immunogenic (Pinckard et al., Clin. Exp. Immunol. 2:331-340 ( 1967), Robbins
et al.,
Diabetes 36: 838-845 (l987), Cleland et al., Crit. Rev. Therapeutic Drug
Carrier
Systems 10:307-377 (1993).
The replacement of amino acids can also change the selectivity of the binding
to cel l surface receptors. Ostade et al., Nature 36 I : 266-268 ( 1993 ),
described certain
TNF alpha mutations resulting in selective binding of TNF alpha to only one of
the two
known TNF receptors.
As indicated in detail above, further guidance concerning which amino acid
changes are likely to be phenotypically silent (i. e., are not likely to have
a significant
deleterious effect on a function) can be found in Bowie, J.U., et al.,
"Deciphering the
Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science
2a7:1306-13l0 (l990) (see Table 1).
As indicated, changes are preferably of a minor nature, such as conservative
amino acid substitutions that do not significantly affect the folding or
activity of the
protein (see Table 1 ).
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TABLE 1. Conservative Amino Acid Substitutions.
Aromatic Phenylalanine
Tryptophan
Tyrosine
Hydrophobic Leucine
Isoleucine
Vaiine
Pol~ ~ Glutamine
Asparagine
Basic Arginine
Lysine
Histidine
Acidic Aspartic Acid
Glutamic Acid
Small Alanine
Serine
Threonine
Methionine
Glycine
Of course, the number of amino acid substitutions a skilled artisan would make
depends on many factors, including those described above and below. Generally
speaking, the number of substitutions for any given MPIF-1 or MCIF poypeptide
or
mutant thereof will not be more than S0, 40, 30, 20) 10, 5, or 3, depending on
the
objective. Specific MPIF-1 and MCIF amino acid substitutions are described
below.
MPIF 1 Variants. In addition, variants of MPIF-1 have been identified and
characterized. Several of these analogs comprise amino terminal truncations.
In
addition, an MPIF-1 analog apparently resulting from an alternative splice
site has also
been identified and characterized (Figure 26 (SEQ ID NO:11 ). Example 17
discloses
the biological activities of these MPIF-1 analogs. The sequences of these
analogs are
shown in Figure 25 (SEQ ID NOS:7, 8, and 9, as well amino acid residues 46-
l20, 45-
120, 48-l20, 49-120, 39-120, and 44-120 in SEQ ID N0:4).
In order to improve or alter the characteristics of the MPIF-1 polypeptide(s),
protein engineering may be employed. Recombinant DNA technology known to those
skilled in the art can be used to create novel proteins. Muteins and deletions
or fusion
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proteins can show, e.g., enhanced activity or increased stability. In
addition, they could
be purified in higher yields and show better solubility at least under certain
purification
and storage conditions. Set below are additional examples of mutations that
can be
constructed.
$ MPIF I Aminoterminal and carboxyterminal deletions: Interferon gamma shows
up to ten times higher activities by deleting 8-10 amino acid residues from
the carboxy
terminus of the protein (Dobeli et al., J. of Biotechnology 7:199-216 ( 1988).
Ron et al.,
J. Biol. Chem., 268(4):2984-2988 (l993) reported modified KGF proteins that
had
heparin binding activity even if 3, 8, or 27 amino terminal amino acid
residues were
missing. Many other examples are known to anyone skilled in the art.
Particularly preferred MPIF-1 polypeptides of the amino acid sequence shown
in Figure 1 (SEQ ID N0:4) are shown below:
Val (23) --- Asn (120)Val (23) ---
Lys
( 119)
Thr (24) --- Asn ( Thr (24) ---
120) Arg
( 1
I8)
1$ Lys (25) --- Asn (120)Lys (25) ---
Thr
(I
17)
Asp (26) ---Asn (120) Asp(26) ---
Lys
(116)
Ala (27) --- Asn (l20)Ala (27) ---
Ile
( 115)
Glu (28) --- Asn ( Glu (28) ---
120) Arg
( 114)
Thr(29) -- Asn ( 120)Thr (29) ---
- Thr
( 113
)
Glu (30) ---Asn (120) Thr (29) ---
Asp
(I
12)
Phe (31 --- Asn ( Thr (29) ---
) I 20) Leu
( 111
)
Met (32) --- Asn (120)Thr (29) ---
Lys
(I
10)
Met (33) --- Asn (I20)Met (33) ---Leu
(I09)
Ser (34) Ser (34) ---
--- Asn Met
(120) (108)
2$ Lys (35) --- Asn ( Ser (34) ---
I20) Arg
( 107)
Leu (36) --- Asn ( Ser (34) ---
120) Met
( I06)
Pro (37) --- Asn (120)Ser (34) ---
Cys(105)
Leu (38) --- Asn (120)Ser (34) ---
Val
(104)
Glu (39) --- Asn (120)Ser (34) ---
Gln
(103)
Asn (40) --- Asn ( Ser (34) ---
120) Val(
102)
Pro (41 --- Asn ( Ser (34) ---
) 120) Gln
( 101
)
Val (42) --- Asn (120)Ser (34) ---
Lys
(100)
Leu (43) --- Asn (I20)Ser (34) ---
Asp(99)
Leu (44) --- Asn (120)Ser (34) ---
Ser
(98)
3$ Asp (45) --- Asn (120)Ser (34) ---
Pro
(97)
Arg (46) --- Asn (120)Ser (34) ---
Asn
(96)
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Phe (47) --- Asn (t20) Ser (34) Ala
--- (95)
His (48) --- Asn (120) Ser (34) Cys
--- (94)
Ala (49) --- Asn (120) Ser (34) Phe
--- (93)
Thr (50) --- Asn (120) Ser (34) Arg
--- (92)
Ser (51 ) --- Asn ( 120) Ser (34) Arg
--- (91
)
Ala (52) --- Asn (120) Ser (34) Gly
--- (90)
Asp (53) --- Asn Ser (34) Lys
(120) --- (89)
Ser (34) Ile
--- (84)
Ser (34) Ser
--- (79)
] ~ Ser (34) Asn
--- (75)
Ser (34) --- Phe (72)
Ser (34) --- Leu (68)
Thus, in one aspect, MPIF-1 N-terminal deletion mutants are provided by the
present invention. Such mutants include those comprising an amino acid
sequence
shown in Figure 1 (SEQ ID N0:4) having a deletion of at least the first 22 N-
terminal
amino acid residues (i.e., a deletion of at least Met ( 1 ) -- Arg (22)) but
not more than the
first 60 N-terminal amino acid residues of Figwe 1 (SEQ ID N0:4).
Alternatively) the
deletion will include at least the first 22 N-terminal amino acid residues but
not more
than the first 53 N-terminal amino acid residues of Figure 1 (SEQ ID N0:4).
Alternatively, the deletion will include at least the first 33 N-terminal
amino acid
residues but not more than the first 53 N-terminal amino acid residues of
Figwe 1 (SEQ
ID N0:4). Alternatively, the deletion will include at least the first 37 N-
terminal amino
acid residues (i.e., a deletion of at least Met (1) -- Pro (37)) but not more
than the first
53 N-terminal amino acid residues of Figure 1 (SEQ ID N0:4). Alternatively,
the
deletion will include at least the first 48 N-terminal amino acid residues but
not more
than the first 53 N-terminal amino acid residues of Figure 1 (SEQ ID N0:4).
In addition to the ranges of MPIF-1 N-terminal deletion mutants described
above, the present invention is also directed to all combinations of the above
described
ranges, e.g., deletions of at least the first 22 N-terminal amino acid
residues but not more
than the first 48 N-terminal amino acid residues of Figure 1 (SEQ ID N0:4);
deletions
of at least the first 37 N-terminal amino acid residues but not more than the
first 48 N-
terminal amino acid residues of Figure 1 (SEQ ID N0:4); deletions of at least
the first
22 N-terminal amino acid residues but not more than the first 37 N-terminal
amino acid
residues of Figwe 1 (SEQ ID N0:4); deletions of at least the first 22 N-
terminal amino
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acid residues but not more than the first 33 N-terminal amino acid residues of
Figure 1
(SEQ ID N0:4); deletions of at least the first 33 N-terminal amino acid
residues but not
more than the first 37 N-terminal amino acid residues of Figure 1 (SEQ ID
N0:4); and
deletions of at least the first 33 N-terminal amino acid residues but not more
than the
first 48 N-terminal amino acid residues of Figure 1 (SEQ ID N0:4).
In another aspect, MPIF-1 C-terminal deletion mutants are provided by the
present invention. Preferably, the N-terminal amino acid residue of said MPIF-
I C-
terminal deletion mutants is amino acid residue 1 (Met) or 22 (Arg) of Figure
I (SEQ
ID N0:4). Such mutants include those comprising an amino acid sequence shown
in
Figure 1 (SEQ ID N0:4) having a deletion of at least the last C-terminal amino
acid
residue (Asn (120)) but not more than the last 52 C-terminal amino acid
residues (e.g.,
a deletion of amino acid residues Glu (69) - Asn (120) of Figure 1 (SEQ ID
N0:4)).
Alternatively, the deletion will include at least the last 10 or I S C-
terminal amino acid
residues but not more than the last 52 C-terminal amino acid residues of
Figure 1 (SEQ
1 S ID N0:4). Alternatively, the deletion will include at least the last 20 C-
terminal amino
acid residues but not more than the last 52 C-terminal amino acid residues of
Figure 1
(SEQ ID N0:4}. Alternatively, the deletion will include at least the last 30 C-
terminal
amino acid residues but not more than the last 52 C-terminal amino acid
residues of
Figure 1 (SEQ ID N0:4). Alternatively, the deletion will include at least the
last 36 C-
terminal amino acid residues but not more than the last 52 C-terminal amino
acid
residues of Figure 1 (SEQ ID N0:4). Alternatively, the deletion will include
at least the
last 41 C-terminal amino acid residues but not more than the last 52 C-
terminal amino
acid residues of Figure 1 (SEQ ID N0:4). Alternatively, the deletion will
include at
least the last 45 C-terminal amino acid residues but not more than the last 52
C-terminal
amino acid residues of Figure 1 (SEQ ID N0:4). Alternatively, the deletion
will include
at least the last 48 C-terminal amino acid residues but not more than the last
52 C-
terminal amino acid residues of Figure 1 (SEQ ID N0:4).
In addition to the ranges of C-terminal deletion mutants described above, the
present invention is also directed to all combinations of the above described
ranges, e.g.,
deletions of at least the last C-terminal amino acid residue but not more than
the last 48
C-terminal amino acid residues of Figure 1 (SEQ ID N0:4); deletions of at
least the last
C-terminal amino acid residue but not more than the last 45 C-terminal amino
acid
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residues of Figure 1 (SEQ ID N0:4); deletions of at least the last C-terminal
amino acid
residue but not more than the last 41 C-terminal amino acid residues of Figure
1 (SEQ
ID N0:4); deletions of at least the last C-terminal amino acid residue but not
more than
the last 36 C-terminal amino acid residues of Figure 1 (SEQ ID N0:4);
deletions of at
least the last C-terminal amino acid residue but not more than the last 10 C-
terminal
amino acid residues of Figure 1 (SEQ ID N0:4); deletions of at least the last
10 C-
terminal amino acid residues but not more than the last 20 C-terminal amino
acid
residues of Figure 1 (SEQ ID N0:4); deletions of at least the last 10 C-
terminal amino
acid residues but not more than the last 30 C-terminal amino acid residues of
Figure 1
(SEQ ID N0:4); deletions of at least the last 10 C-terminal amino acid
residues but not
more than the last 36 C-terminal amino acid residues of Figure 1 (SEQ ID
N0:4);
deletions of at least the last 20 C-terminal amino acid residues but not more
than the last
30 C-terminal amino acid residues of Figure 1 (SEQ ID N0:4); etc. etc. etc. .
. . .
In yet another aspect, also included by the present invention are MPIF-1
deletion
mutants having amino acids deleted from both the N- terminal and C-terminal
residues.
Such mutants include a11 combinations of the N-terminal deletion mutants and C-
terminal deletion mutants described above. Such mutants include those
comprising an
amino acid sequence shown in Figure 1 (SEQ ID I'0:4) having a deletion of at
least the
first 22 N-terminal amino acid residues but not more than the first 52 N-
terminal amino
acid residues of Figure 1 (SEQ ID N0:4) and a deletion of at least the last C-
terminal
amino acid residue but not more than the last 52 C-terminal amino acid
residues of
Figure 1 (SEQ ID N0:4). Alternatively, a deletion can include at least the
first 33, 37,
or 48 N-terminal amino acids but not more than the first 52 N-terminal amino
acid
residues of Figure 1 (SEQ ID N0:4) and a deletion of at least the last 10, 20,
30, 36, 41,
45, or 48 C-terminal amino acid residues but not more than the last 52 C-
terminal amino
acid residues of Figure 1 (SEQ ID N0:4). Further included are all combinations
of the
above described ranges.
Substitution of amino acids: A further aspect of the present invention also
includes the substitution of amino acids. Of special interest are conservative
amino acid
substitutions that do not significantly affect the folding of the protein.
Examples of
conservative amino acid substitutions known to those skilled in the art are
set forth
Table 1, above.
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Of additional special interest are also substitutions of charged amino acids
with
another charged amino acid or with neutral amino acids. This may result in
proteins
with improved characteristics such as less aggregation. Prevention of
aggregation is
highly desirable. Aggregation of proteins cannot only result in a reduced
activity but be
problematic when preparing pharmaceutical formulations because they can be
immunogenic (Pinckard et al., Clin. Exp. Immunol. 2:33l -340 ( 1967), Robbins
et al.,
Diabetes 36:838-845 (1987), Cleland et al.. Crit. Rev. Therapeutic Drug
Carrier
Systems 10:307-377 (1993).
The MPIF-1 protein may contain one or several amino acid substitutions,
deletions or additions, either from natural mutation or human manipulation.
Examples
of some preferred mutations of the amino acid sequence shown in Figure 1 (SEQ
ID
N0:4) are provided below. (By the designation, for example, Ala (21 ) Met is
intended
that the Ala at position 21 of Figure 1 (SEQ ID N0:4) is replaced by Met.)
Ala (21 ) Met Asp (53) Ser
1 S Thr (24) Ala Asp (53) Thr
Lys (25) Asn Asp (53) Met
Asp (26) Ala Ser (51 ) G!y
Asp (45) Ala Ser (34) Gly
Asp (45) Gly Glu (30) Gln
Asp (45) Ser Glu (28) Gln
Asp (45) Thr Pro (60) Thr
Asp (45) Met Ser (70) Ala
Asp (53) Ala
Asp (53) Gly
Aminoterminal deletions of the MPIF I 13 i amino acid splice variant:
As indicated above, the present invention further provides a human MPIF-1
splice variant. The cDNA sequence and the I37 amino acid sequence are shown in
Figure 26A (SEQ ID NOs:lO and 11, respectively). Using eukaryotic expression
systems, the present inventions have discovered three N-terminal deletion
mutants of
this MPIF-1 splice variant. These include His (60) - Asn (137); Met (46) - Asn
(137);
and Pro (54) - Asn (137). Thus, in a further aspect, MPIF-1 splice variant N-
terminal
deletion mutants are provided by the present invention. Such mutants include
those
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comprising an amino acid sequence shown in Figure 26A (SEQ ID NO:11 ) having a
deletion of at least the first 45 N-terminal amino acid residues but not more
than the first
59 N-terminal amino acid residues of Figure 26A (SEQ ID NO: l I ).
Alternatively, the
deletion will include at least the first S 3 N-terminal amino acid residues
but not more
than the first 59 N-terminal amino acid residues of Figure 26A (SEQ ID NO:11
).
Alternatively, the deletion will include at least the first 45 N-terminal
amino acid
- residues but not more than the first 53 N-terminal amino acid residues of
Figure 26A
(SEQ ID NO:1 I).
M CIF Variants. In order to improve or alter the characteristics of the M-CIF
polypeptide(s), protein engineering may be employed. Recombinant DNA
technology
known to those skilled in the art can be used to create novel proteins.
Muteins and
deletions or fusion proteins can show, e.g., enhanced activity or increased
stability. In
addition, they could be purified in higher yields and show better solubility
at least under
certain purification and storage conditions. Set below are examples of
mutations that
can be constructed.
M CIFAmino terminal and carboxvterminul deletions: Interferon gamma shows
up to ten times higher activities by deleting 8-10 amino acid residues from
the carboxy
terminus of the protein (Dobeli et al.) J. of Biotechnology 7:199-216 (l988).
Ron et al.,
J. Biol. Chem., 268(4):2984-2988 ( I 993 ) reported modified KGF proteins that
had
heparin binding activity even if 3, 8, or 27 amino terminal amino acid
residues were
missing. Many other examples are known to anyone skilled in the art.
Particularly preferred variants of M-CIF polypeptides of some preferred
mutations of the amino acid sequence sho~~n in Figure 2 (SEQ ID N0:2) are:
Gly ( 19) Asn (93) Glu (23) --- Asn (93)
---
Gly (19) Glu (92) Thr (20) --- Cys (75)
---
Thr (20) --- Asn (93) Ser (24) --- Asn {93)
Thr (20) --- Glu (92) Lys (21 ) --- Glu
(92)
Lys (21 ) Asn (93) Ser (25) --- Asn {93)
---
Thr (20) --- Lys (91 ) Thr (22) --- Lys (91
)
Thr (22) Asn (93) Ser (26) --- Asn (93)
---
Thr (20) --- Lys (81) Glu (23) --- Lys (91)
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Arg (27) --- Asn (93) His (31 ) --- Asn (93)
Ser (24) --- Lys (91) Ser (25) --- Lys (88)
Gly (28) --- Asn {93) Pro (32) --- Asn (93)
Ser (25) --- Glu (92) Ser (25) --- Lys (81)
Pro (29) Asn {93) Ser (33) --- Asn (93)
---
Ser (25) --- Lys (91) Ser (25) --- Cys(75)
Tyr (30) --- Asn (93) Glu (34) ---
Asn (93)
Ser (25) --- Met (90) Ser (26) --- Cys (75)
Thus, in one aspect, M-CIF N-terminal deletion mutants are provided by the
present invention. Such mutants include those comprising an amino acid
sequence
shown in Figure 2 (SEQ ID N0:2) having a deletion of at least the first 20 N-
terminal
amino acid residues (i.e., a deletion of at least Met ( 1 ) -- Thr (20)) but
not more than the
first 40 N-terminal amino acid residues of Figure 2 (SEQ ID N0:2).
Alternatively, the
deletion will include at least the first 20 N-terminal amino acid residues but
not more
than the first 33 N-terminal amino acid residues of Figure 2 (SEQ ID N0:2).
Alternatively, the deletion will include at least the first 23 N-terminal
amino acid
residues but not more than the first 33 N-terminal amino acid residues of
Figure 2 (SEQ
ID N0:2). Alternatively, the deletion will include at least the first 28 N-
terminal amino
acid residues but not more than the first 33 N-terminal amino acid residues of
Figure 2
(SEQ ID N0:2).
In addition to the ranges of M-CIF N-terminal deletion mutants described
above,
the present invention is also directed to all combinations of the above
described ranges,
e.g., deletions of at least the first 20 N-terminal amino acid residues but
not more than
the first 28 N-terminal amino acid residues of Figure 2 (SEQ ID N0:2);
deletions of at
least the first 20 N-terminal amino acid residues but not more than the first
23 N-
terminal amino acid residues of Figure 2 (SEQ ID N0:2); and deletions of at
least the
first 28 N-terminal amino acid residues but not more than the first 33 N-
terminal amino
acid residues of Figure 2 (SEQ ID N0:2).
In another aspect, M-CIF C-terminal deletion mutants are provided by the
present
invention. Preferably, the N-terminal amino acid residue of said M-CIF C-
terminal
deletion mutants is amino acid residue I (Met) or 20 (Thr) of Figure 2 (SEQ ID
N0:2).
Such mutants include those comprising an amino acid sequence shown in Figure 2
(SEQ
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ID N0:2) except for a deletion of at least the last C-terminal amino acid
residue (Asn
(93)) but not more than the last 25 C-terminal amino acid residues (e.g., a
deletion of
amino acid residues Lys (69) - Asn (93)) of Figure 2 (SEQ ID N0:2).
Alternatively, the
deletion will include at least the last C-terminal amino acid residue but not
more than
the last 18 C-terminal amino acid residues of Figure 2 (SEQ ID N0:2).
Alternatively,
the deletion will include at least the last 3 C-terminal amino acid residues
but not more
than the last 18 C-terminal amino acid residues of Figure 2 (SEQ ID N0:2).
Alternatively, the deletion will include at least the last 5 C-terminal amino
acid residues
but not more than the last 18 C-terminal amino acid residues of Figure 2 (SEQ
ID
N0:2). Alternatively, the deletion will include at least the last 12 C-
terminal amino acid
residues but not more than the last 18 C-terminal amino acid residues of
Figure 2 (SEQ
ID N0:2). Alternatively, the deletion will include at least the last S C-
terminal amino
acid residues but not more than the last 12 C-terminal amino acid residues of
Figure 2
(SEQ ID N0:2).
I 5 In yet another aspect, also included by the present invention are M-CIF
deletion
mutants having amino acids deleted from both the N- terminal and C-terminal
residues.
Such mutants include all combinations of the N-terminal deletion mutants and C-
terminal deletion mutants described above. Such mutants include those
comprising an
amino acid sequence shown in Figure 2 (SEQ ID N0:2) having a deletion of at
least the
first 20 N-terminal amino acid residues but not more than the first 33 N-
terminal amino
acid residues of Figure 2 (SEQ ID N0:2) and a deletion of at least the last C-
terminal
amino acid residue but not more than the last 18 C-terminal amino acid
residues of
Figwe 2 (SEQ ID N0:2). Alternatively, a deletion can include at least the
first 23 or 28
N-terminal amino acids but not more than the first 33 N-terminal amino acid
residues
of Figure 2 (SEQ ID N0:2) and a deletion of at least the last 3, 5, or 12 C-
terminal
amino acid residues but not more than the last 18 C-terminal amino acid
residues of
Figure 2 (SEQ ID N0:2). Further included are all combinations of the above
described
ranges.
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An M-CIF polypeptide can contain one or several amino acid substitutions,
deletions or additions, either from natural mutation or human manipulation.
Examples
of some preferred mutations of the amino acid sequence shown in Figure 2 (SEQ
ID
N0:2) are:
Gly (19) Met Asp (51) Thr
Thr (20) Ala Asp (51 ) Met
Lys (21 ) Asn Lys (8I ) Asn
Glu (23) Gln Lys (81 ) Ala
Ser (24) Ala Lys (88) Asn
Ser (24) Met Lys (88) Ala
Ser (25) Ala Lys (91) Ala
Ser (25) Gly Pro (32) Glu
Glu (34) Gln Ser (33) Leu
Lys (43) Ala Glu (34) Arg
Asp (51) Ala
Asp (51 ) Gly
Asp (51 ) Ser
The polypeptides of the present invention are preferably provided in an
isolated
form, and preferably are substantially purified. A recombinantly produced
version of
the MPIF-1, M-CIF or MIP-4 polypeptide can be substantially purified by the
one-step
method described in Smith and Johnson, Gene 67:31-40 (I988).
The polypeptides of the present invention include the polypeptide encoded by
the deposited cDNA including the leader, the mature polypeptide encoded by the
deposited the cDNA minus the leader (i. e. , the mature protein)) the
polypeptide of Figure
I (SEQ ID N0:4)) Figure 2 (SEQ ID N0:2) or Figure 3 (SEQ ID N0:6) including
the
leader) the polypeptide of Figure 1 (SEQ ID N0:4), Figure 2 (SEQ ID N0:2) or
Figure
3 (SEQ ID N0:6) including the leader but minus the N-terminal methionine
residue, the
polypeptide of Figure 1 (SEQ ID N0:4), Figure 2 (SEQ ID N0:2) or Figure 3 (SEQ
ID
N0:6) minus the leader, as well as polypeptides which have at least 95%
similarity, and
still more preferably at least 96%, 97%, 98% or 99% similarity to those
described above.
Further polypeptides of the present invention include polypeptides at least
95%
identical, still more preferably at least 96%, 97%, 98% or 99% identical to
the
polypeptide encoded by the deposited cDNA, to the polypeptide of Figure 1 (SEQ
ID
N0:4), Figure 2 (SEQ ID N0:2) or Figure 3 (SEQ ID N0:6) and also include
portions
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of such polypeptides with at least 30 amino acids and more preferably at least
50 amino
acids.
By "% similarity" for two polypeptides is intended a similarity score produced
by comparing the amino acid sequences of the two polypeptides using the
Bestfit
program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711
)
and the default settings for determining similarity. Bestfit uses the local
homology
algorithm of Smith and Waterman (Advances in Applied Mathematics 2:482-489,
1981 )
to find the best segment of similarity between two sequences.
By a polypeptide having an amino acid sequence at least, for example, 95%
"identical" to a reference amino acid sequence of an MPIF-1, M-CIF or MIP-4
polypeptide is intended that the amino acid sequence of the polypeptide is
identical to
the reference sequence except that the polypeptide sequence may include up to
five
amino acid alterations per each 100 amino acids of the reference amino acid of
the
MPIF-1, M-CIF or MIP-4 polypeptide. In other words, to obtain a polypeptide
having
an amino acid sequence at least 95% identical to a reference amino acid
sequence, up
to 5% of the amino acid residues in the reference sequence may be deleted or
substituted
with another amino acid, or a number of amino acids up to 5% of the total
amino acid
residues in the reference sequence may be inserted into the reference
sequence. These
alterations of the reference sequence may occur at the amino or carboxy
terminal
positions of the reference amino acid sequence or anywhere between those
terminal
positions, interspersed either individually among residues in the reference
sequence or
in one or more contiguous groups within the reference sequence.
As a practical matter, whether any particular polypeptide is at least 95%,
96%)
97%, 98% or 99% identical to, for instance, the amino acid sequence shown in
Figure
1 (SEQ ID N0:4)) Figure 2 (SEQ ID N0:2) or Figure 3 (SEQ ID N0:6) or to the
amino
acid sequence encoded by deposited eDNA clones can be determined
conventionally
using known computer programs such the Bestfit program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group, University
Research
Park, 575 Science Drive, Madison, WI 53711. When using Bestfit or any other
sequence alignment program to determine whether a particular sequence is, for
instance,
95% identical to a reference sequence according to the present invention, the
parameters
are set, of course, such that the percentage of identity is calculated over
the full length
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of the reference amino acid sequence and that gaps in homology of up to S% of
the total
number of amino acid residues in the reference sequence are allowed.
The polypeptide of the present invention could be used as a molecular weight
marker on SDS-PAGE gels or on molecular sieve gel filtration columns using
methods
S well known to those of skill in the art.
As described in detail below, the polypeptides of the present invention can
also
be used to raise polyclonal and monoclonal antibodies, which are useful in
assays for
detecting MPIF-1, M-CIF or MIP-4 protein expression as described below or as
agonists
and antagonists capable of enhancing or inhibiting MPIF-1, M-CIF or MIP-4
protein
function. Further, such polypeptides can be used in the yeast two-hybrid
system to
"capture" MPIF-1, M-CIF or MIP-4 protein binding proteins which are also
candidate
agonist and antagonist according to the present invention. The yeast two
hybrid system
is described in Fields and Song, Nature 340:24S-246 (1989).
MPIF 1, M CIF and MIP-4 Epitope-Bearing Polypeptides. In another aspect,
the invention provides a peptide or polypeptide comprising an epitope-bearing
portion
of a polypeptide of the invention. The epitope of this polypeptide portion is
an
immunogenic or antigenic epitope of a polypeptide of the invention. An
"inununogenic
epitope" is defined as a part of a protein that elicits an antibody response
when the whole
protein is the immunogen. These immunogenic epitopes are believed to be
confined to
a few loci on the molecule. On the other hand, a region of a protein molecule
to which
an antibody can bind is defined as an "antigenic epitope." The number of
immunogenic
epitopes of a protein generally is less than the number of antigenic epitopes.
See, for
instance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998- 4002 ( 1983).
As to the selection of peptides or polypeptides bearing an antigenic epitope
(i.e.,
that contain a region of a protein molecule to which an antibody can bind), it
is well
known in that art that relatively short synthetic peptides that mimic part of
a protein
sequence are routinely capable of eliciting an antiserum that reacts with the
partially
mimicked protein. See, e.g., Sutcliffe, J. G., Shinnick, T. M., Green, N. and
Learner,
R.A., Science 219:660-666 (1983).
Peptides capable of eliciting protein-reactive sera are frequently represented
in
the primary sequence of a protein, can be characterized by a set of simple
chemical rules,
and are confined neither to immunodominant regions of intact proteins (i.e.,
immunogenic epitopes) nor to the amino or carboxyl terminals. Peptides that
are
extremely hydrophobic and those of six or fewer residues generally are
ineffective at
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inducing antibodies that bind to the mimicked protein; longer, peptides,
especially those
containing proline residues, usually are effective. Sutcliffe et al., supra,
at 661. For
instance, 18 of 20 peptides designed according to these guidelines, containing
8-39
residues covering 75% of the sequence of the influenza virus hemagglutinin HA1
polypeptide chain, induced antibodies that reacted with the HA 1 protein or
intact virus;
and 12/12 peptides from the MuLV polymerase and 18/18 from the rabies
glycoprotein
induced antibodies that precipitated the respective proteins.
Antigenic epitope-bearing peptides and polypeptides of the invention are
therefore useful to raise antibodies, including monoclonal antibodies, that
bind
specifically to a polypeptide of the invention. Thus, a high proportion of
hybridomas
obtained by fusion of spleen cells from donors immunized with an antigen
epitope-bearing peptide generally secrete antibody reactive with the native
protein.
Sutcliffe et al., supra, at 663. The antibodies raised by antigenic epitope-
bearing
peptides or polypeptides are useful to detect the mimicked protein, and
antibodies to
different peptides may be used for tracking the fate of various regions of a
protein
precursor which undergoes post-translational processing. The peptides and anti-
peptide
antibodies may be used in a variety of qualitative or quantitative assays for
the
mimicked protein, for instance in competition assays since it has been shown
that even
short peptides (e.g. about 9 amino acids) can bind and displace the larger
peptides in
immunoprecipitation assays. See, for instance, Wilson et al., Cell 37:767-778
(l984)
at 777. The anti- peptide antibodies of the invention also are useful for
purification of
the mimicked protein, for instance, by adsorption chromatography using methods
well
known in the art.
Antigenic epitope-bearing peptides and polypeptides of the invention designed
according to the above guidelines preferably contain a sequence of at least
seven, more
preferably at least nine and most preferably between about 15 to about 30
amino acids
contained within the amino acid sequence of a polypeptide of the invention.
However,
peptides or polypeptides comprising a larger portion of an amino acid sequence
of a
polypeptide of the invention, containing about 30 to about 50 amino acids, or
any length
up to and including the entire amino acid sequence of a polypeptide of the
invention,
also are considered epitope-bearing peptides or polypeptides of the invention
and also
are useful for inducing antibodies that react with the mimicked protein.
Preferably, the
amino acid sequence of the epitope-bearing peptide is selected to provide
substantial
solubility in aqueous solvents (i.e., the sequence includes relatively
hydrophilic residues
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and highly hydrophobic sequences are preferably avoided); and sequences
containing
proline residues are particularly preferred.
Non-limiting examples of antigenic polypeptides or peptides that can be used
to
generate MPIF-1-specific antibodies include: a polypeptide comprising amino
acid
S residues from about 21 to about 30 in SEQ ID N0:4; a polypeptide comprising
amino
acid residues from about 31 to about 44 in SEQ ID N0:4; a polypeptide
comprising
amino acid residues from about 49 to about 55 in SEQ ID N0:4; a polypeptide
comprising amino acid residues from about 59 to about 67 in SEQ ID N0:4; a
polypeptide comprising amino acid residues from about 72 to about 83 in SEQ ID
N0:4;
a polypeptide comprising amino acid residues from about 86 to about 103 in SEQ
ID
N0:4; a polypeptide comprising amino acid residues from about 110 to about 120
in
SEQ ID N0:4. As indicated above, the inventors have determined that the above
polypeptide fragments are antigenic regions of the MPIF-1 protein.
Non-limiting examples of antigenic polypeptides or peptides that can be used
to
generate M-CIF-specific antibodies include: a polypeptide comprising amino
acid
residues from about 20 to about 36 in SEQ ID N0:2; a polypeptide comprising
amino
acid residues from about 42 to about 52 in SEQ ID N0:2; a polypeptide
comprising
amino acid residues from about 52 to about 64 in SEQ ID N0:2; a polypeptide
comprising amino acid residues from about 67 to about 7~ in SEQ ID N0:2; a
polypeptide comprising amino acid residues from about 75 to about 84 in SEQ ID
N0:2;
and a polypeptide comprising amino acid residues from about 86 to about 93 in
SEQ ID
N0:2. As indicated above, the inventors have determined that the above
polypeptide
fragments are antigenic regions of the M-CIF protein.
The epitope-bearing peptides and polypeptides of the invention may be produced
by any conventional means for making peptides or polypeptides including
recombinant
means using nucleic acid molecules of the invention. For instance, a short
epitope-bearing amino acid sequence may be fused to a larger polypeptide which
acts
as a earner during recombinant production and purification, as well as during
immunization to produce anti-peptide antibodies. Epitope-bearing peptides also
may
be synthesized using known methods of chemical synthesis. For instance,
Houghten has
described a simple method for synthesis of large numbers of peptides, such as
10-20 mg
of 248 different 13 residue peptides representing single amino acid variants
of a segment
of the HA 1 polypeptide which were prepared and characterized (by ELISA-type
binding
studies) in less than four weeks. Houghten, R. A. (I985) General method for
the rapid
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solid-phase synthesis of large numbers of peptides: specificity of antigen-
antibody
interaction at the level of individual amino acids. Proc. Natl. Acad. Sci. USA
82:S 131-5l35. This "Simultaneous Multiple Peptide Synthesis (SMPS)" process
is
further described in U.S. Patent No. 4,63I,211 to Houghten et al. (1986). In
this
procedure the individual resins for the solid-phase synthesis of various
peptides are
contained in separate solvent-permeable packets, enabling the optimal use of
the many
identical repetitive steps involved in solid-phase methods. A completely
manual
procedure allows 500-l000 or more syntheses to be conducted simultaneously.
Houghten et al., supra, at 5134.
Preferred nucleic acid fragments of the present invention include nucleic acid
molecules encoding epitope-bearing portions of the MPIF-I, M-CIF or MIP-4
protein.
In particular, such nucleic acid fragments of the MPIF-1 of the present
invention
include nucleic acid molecules encoding: a polypeptide comprising amino acid
residues
from about 21 to about 30 in SEQ ID N0:4; a polypeptide comprising amino acid
residues from about 3I to about 44 in SEQ ID N0:4; a polypeptide comprising
amino
acid residues from about 49 to about 55 in SEQ ID N0:4; a polypeptide
comprising
amino acid residues from about 59 to about 67 in SEQ ID N0:4; a polypeptide
comprising amino acid residues from about 72 to about 83 in SEQ ID N0:4; a
polypeptide comprising amino acid residues from about 86 to about 103 in SEQ
ID
N0:4; a polypeptide comprising amino acid residues from about 1 10 to about
l20 in
SEQ ID N0:4, or any range or value therein.
In particular, such nucleic acid fragments of the M-CIF of the present
invention
include nucleic acid molecules encoding: a polypeptide comprising amino acid
residues
from about 20 to about 36 in SEQ ID N0:2; a polypeptide comprising amino acid
residues from about 42 to about 52 in SEQ ID N0:2; a polypeptide comprising
amino
acid residues from about 52 to about 64 in SEQ ID N0:2; a polypeptide
comprising
amino acid residues from about 67 to about 75 in SEQ ID N0:2; a polypeptide
comprising amino acid residues from about 75 to about 84 in SEQ ID N0:2; and a
polypeptide comprising amino acid residues from about 86 to about 93 in SEQ ID
N0:2,
or any range or value therein.
The inventors have determined that the above polypeptide fragments are
antigenic regions of the MPIF-1 and M-CIF proteins. Methods for determining
other
such epitope-bearing portions of the MPIF-l, M-CIF or MIP-4 protein are
described in
detail below.
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Epitope-bearing peptides and polypeptides of the invention are used to induce
antibodies according to methods well known in the art. See, for instance,
Sutcliffe et al.,
supra; Wilson et al., supra; Chow, M. et al., Proc. Natl. Acad. Sci. USA
82:9I0-914;
and Bittle, F. J. et al., J. Gen. Virol. 66:2347-2354 (1985). Generally,
animals may be
immunized with free peptide; however, anti-peptide antibody titer may be
boosted by
coupling of the peptide to a macromolecular carrier, such as keyhole limpet
hemacyanin
(KLH) or tetanus toxoid. For instance, peptides containing cysteine may be
coupled to
earner using a linker such as m-maleimidobenzoyl-N-hydroxysuccinimide ester
(MBS),
while other peptides may be coupled to carrier using a more general linking
agent such
as glutaraldehyde. Animals such as rabbits, rats and mice are immunized with
either
free or carrier-coupled peptides) for instance, by intraperitoneal and/or
intradermal
injection of emulsions containing about 100 g peptide or carrier protein and
Freund's
adjuvant. Several booster injections may be needed, for instance, at intervals
of about
two weeks, to provide a useful titer of anti-peptide antibody which can be
detected, for
example, by ELISA assay using &ee peptide adsorbed to a solid surface. The
titer of
anti-peptide antibodies in serum from an immunized animal may be increased by
selection of anti-peptide antibodies, for instance, by adsorption to the
peptide on a solid
support and elution of the selected antibodies according to methods well known
in the
art.
Immunogenic epitope-bearing peptides of the invention, i.e., those parts of a
protein that elicit an antibody response when the whole protein is the
immunogen, are
identified according to methods known in the art. For instance, Geysen et al.,
supra,
discloses a procedure for rapid concurrent synthesis on solid supports of
hundreds of
peptides of sufficient purity to react in an enzyme-linked immunosorbent
assay.
Interaction of synthesized peptides with antibodies is then easily detected
without
removing them from the support. In this manner a peptide bearing an
immunogenic
epitope of a desired protein may be identified routinely by one of ordinary
skill in the
art. For instance, the immunologically important epitope in the coat protein
of
foot-and-mouth disease virus was located by Geysen et al. with a resolution of
seven
amino acids by synthesis of an overlapping set of all 208 possible
hexapeptides covering
the entire 213 amino acid sequence of the protein. Then, a complete
replacement set of
peptides in which all 20 amino acids were substituted in turn at every
position within the
epitope were synthesized, and the particular amino acids conferring
specificity for the
reaction with antibody were determined. Thus, peptide analogs of the epitope-
bearing
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peptides of the invention can be made routinely by this method. U.S. Patent
No.
4,708,781 to Geysen ( 1987) further describes this method of identifying a
peptide
bearing an immunogenic epitope of a desired protein.
Further still, U.S. Patent No. 5,l94,392 to Geysen (1990) describes a general
method of detecting or determining the sequence of monomers (amino acids or
other
compounds) which is a topological equivalent of the epitope (i.e., a
"mimotope") which
is complementary to a particular paratope (antigen binding site) of an
antibody of
interest. More generally, U.S. Patent No. 4,433,092 to Geysen (1989) describes
a
method of detecting or determining a sequence of monomers which is a
topographical
equivalent of a ligand which is complementary to the ligand binding site of a
particular
receptor of interest. Similarly, U.S. Patent No. 5,480,97l to Houghten, R. A.
et al.
( 1996) on Peralkylated Oligopeptide Mixtures discloses linear C,-C,-alkyl
peralkylated
oligopeptides and sets and libraries of such peptides, as well as methods for
using such
oligopeptide sets and libraries for determining the sequence of a peralkyiated
oligopeptide that preferentially binds to an acceptor molecule of interest.
Thus,
non-peptide analogs of the epitope-bearing peptides of the invention also can
be made
routinely by these methods.
The entire disclosure of each document cited in this section on "Polypeptides
and
Peptides" is hereby incorporated herein by reference.
As one of skill in the art will appreciate, MP1F-1, M-CIF or M1P-4
polypeptides
of the present invention and the epitope-bearing fragments thereof described
above can
be combined with parts of the constant domain of immunoglobulins (IgG),
resulting in
chimeric polypeptides. These fusion proteins facilitate purification and show
an
increased half life in vivo. This has been shown, e.g. for chimeric proteins
consisting
of the first two domains of the human CD4-polypeptide and various domains of
the
constant regions of the heavy or light chains of mammalian immunoglobulins
(EPA
394,827; Traunecker et al., Nature 33l:84-86 ( 1988)). Fusion proteins that
have a
disulfide-linked dimeric structure due to the IgG part can also be more
efficient in
binding and neutralizing other molecules than the monomeric MPIF-1, M-CIF or
MIP-4
protein or protein fragment alone (Fountoulakis et al., (I Biochem 270:3958-
3964
(1995)).
Polypeptide Purification and Isolation. MPIF-1, MIP-4 and M-CIF are
recovered and purified from recombinant cell cultures by methods including
ammonium
sulfate or ethanol precipitation, acid extraction, anion or cation exchange
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chromatography, phosphocellulose chromatography, hydrophobic interaction
chromatography, affinity chromatography hydroxylapatite chromatography and
lectin
chromatography. Protein refolding steps can be used, as necessary, in
completing
configuration of the mature protein. Finally, high performance liquid
chromatography
(HPLC) can be employed for final purification steps.
The polypeptides of the present invention can be a naturally purified product,
or
a product of chemical synthetic procedures, or produced by recombinant
techniques
from a prokaryotic or eukaryotic host (for example, by bacterial, yeast,
higher plant,
insect and mammalian cells in culture). Depending upon the host employed in a
recombinant production procedure, the polypeptides of the present invention
can be
glycosylated with mammalian or other eukaryotic carbohydrates or can be non-
glycosylated. Polypeptides of the invention can also include an initial
methionine amino
acid residue.
Antibodies. MPIF-1, M-CIF or MIP-4-protein specific antibodies for use in the
present invention can be raised against the intact MPIF-l, M-CIF or MIP-4
protein or
an antigenic polypeptide fragment thereof, which may presented together with a
carrier
protein, such as an albumin, to an animal system (such as rabbit or mouse) or,
if it is
long enough (at least about 25 amino acids), without a carrier.
As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is
meant to include intact molecules as well as antibody fragments (such as, for
example, Fab and F(ab')z fragments) which are capable of specifically binding
to MPIF-
1, M-CIF or MIP-4 protein. Fab and F(ab')~ fragments lack the Fc fragment of
intact
antibody, clear more rapidly from the circulation, and may have less non-
specific tissue
binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)).
Thus,
these fragments are preferred.
The polypeptides, their fragments or other derivatives, or analogs thereof, or
cells
expressing them can be used as an immunogen to produce antibodies thereto.
These
antibodies can be, for example, polyclonal or monoclonal antibodies. The
present
invention also includes chimeric, single chain and humanized antibodies, as
well as Fab
fragments, or the product of an Fab expression library. Various procedures
known in
the art can be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of
the present invention or its in vivo receptor can be obtained by direct
injection of the
polypeptides into an animal or by administering the polypeptides to an animal,
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WO 98I14582 PCT/US97117505
preferably a nonhuman. The antibody so obtained will then bind the
polypeptides itself.
In this manner, even a sequence encoding only a fragment of the polypeptides
can be
used to generate antibodies binding the whole native polypeptides. Such
antibodies can
then be used to isolate the polypeptides from tissue expressing that
polypeptide.
For preparation of monoclonal antibodies, any technique which provides
antibodies produced by continuous cell line cultures can be used. Examples
include the
hybridoma technique (Kohler and Milstein, l975, Nature, 256:49S-497), the
trioma
technique, the human B-cell hybridoma technique (Kozbor et al., 1983,
Immunology
Today 4:72), and the EBV-hybridoma technique to produce human monoclonal
antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy,
Alan R.
Liss, Inc., pp. 77-96).
Techniques described for the production of single chain antibodies (U.S.
Patent
4,946,778) can be adapted to produce single chain antibodies to immunogenic
polypeptides products of this invention.
1 S The antibodies of the present invention may be prepared by any of a
variety of
methods. For example, cells expressing the MPIF-1, M-CIF or MIP-4 protein or
an
antigenic fragment thereof can be administered to an animal in order to induce
the
production of sera containing polyclonal antibodies. In a preferred method, a
preparation of MPIF-1, M-CIF or MIP-4 protein is prepared and purified to
render it
substantially free of natural contaminants. Such a preparation is then
introduced into an
animal in order to produce polyclonal antisera of greater specific activity.
In the most preferred method, the antibodies of the present invention are
monoclonal antibodies (or MP1F-1, M-CIF or MIP-4 protein binding fragments
thereof).
Such monoclonal antibodies can be prepared using, hybridoma technology (Kohler
et al.,
Nature 26:495 ( 1975); Kohler et al., Eur. J Immunol. 6:511 ( 1976); Kohler et
al., Eur.
(I. Immunol. 6:292 (1976); Hammerling et ul.) In: Alonoclonal Antibodies and T
Cell
Hybridomas, Elsevier, N.Y., ( 1981 ) pp. 563-681 ). In general, such
procedures involve
immunizing an animal (preferably a mouse) with an MPIF-1, M-CIF or MIP-4
protein
antigen or, more preferably, with an MPIF-l, M-CIF or MIP-4 protein-expressing
cell.
Suitable cells can be recognized by their capacity to bind anti-MPIF-1, M-CIF
or MIP-4
protein antibody. Such cells may be cultured in any suitable tissue culture
medium;
however, it is preferable to culture cells in Earle's modified Eagle's medium
supplemented with 10% fetal bovine serum (inactivated at about 56~C), and
supplemented with about 10 g/1 of nonessential amino acids, about 1,000 U/ml
of
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penicillin, and about 100 glml of streptomycin. The splenocytes of such mice
are
extracted and fused with a suitable myeloma cell line. Any suitable myeloma
cell line
may be employed in accordance with the present invention; however, it is
preferable to
employ the parent myeloma cell line (SP20), available from the American Type
Culture
S Collection, Rockville, Maryland. After fusion, the resulting hybridoma cells
are
selectively maintained in HAT medium, and then cloned by limiting dilution as
described by Wands et al. (Gastroenterology 80:225-232 ( 1981 )). The
hybridoma cells
obtained through such a selection are then assayed to identify clones which
secrete
antibodies capable of binding the MPIF-1, M-CIF or MIP-4 protein antigen.
Alternatively, additional antibodies capable of binding to the MPIF-1, M-CIF
or
MIP-4 protein antigen may be produced in a two-step procedure through the use
of
anti-idiotypic antibodies. Such a method makes use of the fact that antibodies
are
themselves antigens, and that, therefore, it is possible to obtain an antibody
which binds
to a second antibody. In accordance with this method, MPIF-1, M-CIF or MIP-4-
protein
specific antibodies are used to immunize an animal, preferably a mouse. The
splenocytes of such an animal are then used to produce hybridoma cells, and
the
hybridoma cells are screened to identify clones which produce an antibody
whose ability
to bind to the MPIF-1, M-CIF or MIP-4 protein-specific antibody can be blocked
by the
MPIF-1, M-CIF or MIP-4 protein antigen. Such antibodies comprise anti-
idiotypic
antibodies to the MPIF-1, M-CIF or MIP-4 protein-specific antibody and can be
used
to immunize an animal to induce formation of further MPIF-l, M-CIF or MIP-4
protein-specific antibodies.
It will be appreciated that Fab and F(ab')Z and other fragments of the
antibodies
of the present invention may be used according to the methods disclosed
herein. Such
fragments are typically produced by proteolytic cleavage, using enzymes such
as papain
(to produce Fab fragments) or pepsin (to produce F(ab')z fragments).
Alternatively,
MPIF-1, M-CIF or MIP-4 protein-binding fragments can be produced through the
application of recombinant DNA technology or through synthetic chemistry.
It may be preferable to use "humanized" chimeric monoclonal antibodies. Such
antibodies can be produced using genetic constructs derived from hybridoma
cells
producing the monoclonal antibodies described above. Methods for producing
chimeric
antibodies are known in the art. See, for review, Morrison, Science 229:1202
(1985);
Oi et al., BioTechni9ues 4:214 (1986); Cabilly et al., U.S. Patent No.
4,8l6,567;
Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO
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8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984);
Neuberger et al.) Nature 3l4:268 (1985).
Further suitable labels for the MPIF-1, M-CIF or MIP-4 protein-specific
antibodies of the present invention are provided below. Examples of suitable
enzyme
labels include malate dehydrogenase, staphylococcal nuclease, delta-5-steroid
isomerase,
yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose
phosphate
isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,
beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate
dehydrogenase,
glucoamylase, and acetylcholine esterase.
Examples of suitable radioisotopic labels include 3H, "'In,'zsI, "'I, 3zp,
ssSyaC,
s'Cr, s'To, sBCo, s9Fe,'sSe, 'szEu, 9~Y, 6'Cu, z"Ci, z"At, z'zPb, "'Sc, '~9Pd,
etc. "'In is a
preferred isotope where in vivo imaging is used since its avoids the problem
of
dehalogenation of the'zsI or "'I-labeled monoclonal antibody by the liver. In
addition,
this radionucleotide has a more favorable gamma emission energy for imaging
(Perkins
et al., Eur. J. Nucl. Med. I0:296-301 ( l985); Carasquillo et al., J. Nucl.
Med.
28:28l -287 ( 1987)).
Examples of suitable non-radioactive isotopic labels include 's'Gd, ssMn,
'6zDy,
szTr, and s6Fe.
Examples of suitable fluorescent labels include an'szEu label, a fluorescein
label,
an isothiocyanate label, a rhodamine label, a phycoerythrin label, a
phycocyanin label,
an allophycocyanin label, an o-phthaldehyde label, and a fluorescamine label.
Examples of suitable toxin labels include diphtheria toxin, ricin, and cholera
toxin.
Examples of chemiluminescent labels include a luminal label, an isoluminal
label, an aromatic acridinium ester label) an imidazole label) an acridinium
salt label, an
oxalate ester label, a luciferin label, a luciferase label, and an aequorin
label.
Examples of nuclear magnetic resonance contrasting agents include heavy metal
nuclei such as Gd, Mn, and iron.
Typical techniques for binding the above-described labels to antibodies are
provided by Kennedy et al., Clin. Chim. Acta 70:l-31 (1976), and Schurs et
al., Clin.
Chim. Acta 81:1-40 ( 1977). Coupling techniques mentioned in the latter are
the
glutaraldehyde method, the periodate method, the dimaleimide method, the
m-maleimidobenzyl-N-hydroxy-succinimide ester method, all of which methods are
incorporated by reference herein.
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Chromosome Assays. The nucleic acid molecules of the present invention are
also valuable for chromosome identification. The sequence is specifically
targeted to
and can hybridize with a particular location on an individual human
chromosome.
Moreover, there is a current need for identifying particular sites on the
chromosome.
Few chromosome marking reagents based on actual sequence data (repeat
polymorphisms) are presently available for marking chromosomal location. The
mapping of DNAs to chromosomes according to the present invention is an
important
first step in correlating those sequences with genes associated with disease.
In certain preferred embodiments in this regard, the cDNA herein disclosed is
used to clone genomic DNA of an MPIF-1, M-CIF or MIP-4 protein gene. This can
be
accomplished using a variety of well known techniques and libraries, which
generally
are available commercially. The genomic DNA then is used for in situ
chromosome
mapping using well known techniques for this purpose. Typically) in accordance
with
routine procedures for chromosome mapping, some trial and error may be
necessary to
1 S identify a genomic probe that gives a good in situ hybridization signal.
Briefly, sequences can be mapped to chromosomes by preparing PCR primers
(preferably 15-25 bp) from the cDNA. Computer analysis of the cDNA is used to
rapidly select primers that do not span more than one exon in the genomic DNA,
thus
complicating the amplification process. These primers are then used for PCR
screening
of somatic cell hybrids containing individual human chromosomes. Only those
hybrids
containing the human gene corresponding to the primer will yield an amplif ed
fragment.
PCR mapping of somatic cell hybrids is a rapid procedure for assigning a
particular DNA to a particular chromosome. Using the present invention with
the same
oligonucleotide primers, sublocalization can be achieved with panels of
portions from
specific chromosomes or pools of large genomic clones in an analogous manner.
Other
mapping strategies that can similarly be used to map to its chromosome include
in situ
hybridization, prescreening with labeled flow- sorted chromosomes and
preselection by
hybridization to construct chromosome specific-cDNA libraries.
Fluorescence in situ hybridization ("FISH") of a cDNA clone to a metaphase
chromosomal spread can be used to provide a precise chromosomal location in
one step.
This technique can be used with probes from the cDNA as short as SO or 60 bp.
For a
review of this technique, see Verma et al., Human Chromosomes: A Manual Of
Basic
Technigues, Pergamon Press, New York (l988).
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Once a sequence has been mapped to a precise chromosomal location, the
physical position of the sequence on the chromosome can be correlated with
genetic map
data. Such data are found, for example, in V. McKusick, Mendelian Inheritance
In Man,
available on-line through Johns Hopkins University, Welch Medical Library. The
relationship between genes and diseases that have been mapped to the same
chromosomal region are then identified through linkage analysis (coinheritance
of
physically adjacent genes).
Next, it is necessary to determine the differences in the cDNA or genomic
sequence between affected and unaffected individuals. If a mutation is
observed in some
or all of the affected individuals but not in any normal individuals, then the
mutation is
likely to be the causative agent of the disease.
With current resolution of physical mapping and genetic mapping techniques,
a cDNA precisely localized to a chromosomal region associated with the disease
could
be one of between 50 and 500 potential causative genes. This assumes 1
megabase
mapping resolution and one gene per 20 kb.
Comparison of affected and unaffected individuals generally involves first
looking for structural alterations in the chromosomes, such as deletions or
translocations
that are visible from chromosome spreads or detectable using PCR based on that
cDNA
sequence. Ultimately, complete sequencing of genes from several individuals is
required to confirm the presence of a mutation and to distinguish mutations
from
polymorphisms.
The present invention is further directed to inhibiting MPIF-1, MIP-4 and M-
CIF
in vivo by the use of antisense technology. Antisense technology can be used
to control
gene expression through triple-helix formation or antisense DNA or RNA, both
of which
methods are based on binding of a polynucleotide to DNA or RNA. For example,
the
5' coding portion of the polynucleotide sequence, which encodes for the
polypeptides
of the present invention, is used to design an antisense RNA oligonucleotide
of from
about 10 to 40 base pairs in length. A DNA oligonucleotide is designed to be
complementary to a region of the gene involved in transcription (triple helix -
see Lee
et al., Nucl. Acids Res., 6:3073 ( 1979); Cooney et al, Science, 241:456 (
1988); and
Dervan et al., Science, 251: 1360 ( 1991 )), thereby preventing transcription
and the
production of MPIF-1, MIP-4 and M-CIF. The antisense RNA oligonucleotide
hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule
into the
MPIF-1, MIP-4 and M-CIF (antisense - Okano, J. Neurochem., 56:S60 (1991);
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Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press,
Boca
Raton, FL (1988)).
Alternatively, the oligonucleotides described above can be delivered to cells
by
procedures in the art such that the antisense RNA or DNA can be expressed in
vivo to
inhibit production of MPIF-l, MIP-4 and M-CIF in the manner described above.
Accordingly, antisense constructs to the MPIF-1, MIP-4 and M-CIF can be used
to treat disorders which are either MPIF-I -, MIP-4- and/or M-CIF-induced or
enhanced,
for example, atherosclerosis, auto-immune, e.g. multiple sclerosis and insulin-
dependent
diabetes, and chronic inflammatory and infective diseases, histamine-mediated
allergic
reactions, rheumatoid arthritis, silicosis, sarcoidosis, idiopathic pulmonary
fibrosis and
other chronic inflammatory diseases of the lung, idiopathic hyper-eosinophilic
syndrome, endotoxic shock, histamine-mediated allergic reactions,
prostaglandin-
independent fever, and aplastic anemia and other cases of bone marrow failure.
Ar:tagonists, Agonists and Metl:ods. This invention further provides methods
for screening compounds to identify agonists and antagonists to the chemokine
polypeptides of the present invention. An agonist is a compound which has
similar
biological functions, or enhances the functions, of the polypeptides, while
antagonists
block such functions. Chemotaxis may be assayed by placing cells, which are
chemoattracted by either of the polypeptides of the present invention, on top
of a filter
with pores of sufficient diameter to admit the cells (about 5 pm). Solutions
of potential
agonists are placed in the bottom of the chamber with an appropriate control
medium
in the upper compartment, and thus a concentration gradient of the agonist is
measured
by counting cells that migrate into or through the porous membrane over time.
When assaying for antagonists, the chemokine polypeptides of the present
invention are placed in the bottom chamber and the potential antagonist is
added to
determine if chemotaxis of the cells is prevented.
Alternatively, a mammalian cell or membrane preparation expressing the
receptors of the polypeptides would be incubated with a labeled chemokine
polypeptide,
e.g. radioactivity, in the presence of the compound. The ability of the
compound to
block this interaction could then be measured. When assaying for agonists in
this
fashion, the chemokines would be absent and the ability of the agonist itself
to interact
with the receptor could be measured.
Examples of potential MPIF-l, MIP-4 and M-CIF antagonists include antibodies,
or in some cases, oligonucleotides, which bind to the polypeptides. Another
example
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of a potential antagonist is a negative dominant mutant of the polypeptides.
Negative
dominant mutants are polypeptides which bind to the receptor of the wild-type
polypeptide, but fail to retain biological activity.
Antisense constructs prepared using antisense technology are also potential
antagonists. Antisense technology can be used to control gene expression
through triple-
helix formation or antisense DNA or RNA, both of which methods are based on
binding
of a polynucleotide to DNA or RNA. For example, the 5' coding portion of the
polynucleotide sequence, which encodes for the mature polypeptides of the
present
invention, is used to design an antisense RNA oligonucleotide of from about 10
to 40
base pairs in length. A DNA oligonucleotide is designed to be complementary to
a
region of the gene involved in transcription (triple- helix, see Lee et al.,
Nucl. Acids Res.
6:3073 (l979); Cooney et al, Science 24l:456 (1988); and Dervan et al.,
Science
2~ 1:1360 ( 1991 )), thereby preventing transcription and the production of
the chemokine
polypeptides. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo
and
1 S blocks translation of the mRNA molecule into the polypeptides (antisense -
Okano, J.
Neurochem. 56:560 (1991 ); oligodeoxynucleotides as Antisense Inhibitors of
Gene
Expression, CRC Press, Boca Raton, FL (1988)). The oligonucleotides described
above
can also be delivered to cells such that the antisense RNA or DNA may be
expressed in
vivo to inhibit production of the chemokine polypeptides.
Another potential chemokine antagonist is a peptide derivative of the
polypeptides which are naturally or synthetically modified analogs of the
polypeptides
that have lost biological function yet still recognize and bind to the
receptors of the
polypeptides to thereby effectively block the receptors. Examples of peptide
derivatives
include, but are not limited to, small peptides or peptide-like molecules.
The antagonists may be employed to treat disorders which are either MPIF-1-,
MIP-4- and M-CIF -induced or enhanced, for example, auto-immune and chronic
inflammatory and infective diseases. Examples of auto-immune diseases include
multiple sclerosis, and insulin-dependent diabetes.
The antagonists may also be employed to treat infectious diseases including
silicosis, sarcoidosis, idiopathic pulmonary fibrosis by preventing the
recruitment and
activation of mononuclear phagocytes. They may also be employed to treat
idiopathic
hyper-eosinophilic syndrome by preventing eosinophil production and migration.
Endotoxic shock may also be treated by the antagonists by preventing the
migration of
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macrophages and their production of the chemokine polypeptides of the present
invention.
The antagonists may also be employed for treating atherosclerosis, by
preventing
monocyte infiltration in the artery wall.
The antagonists may also be employed to treat histamine mediated allergic
reactions and immunological disorders including late phase allergic reactions,
chronic
urticaria, and atopic dermatitis by inhibiting chemokine-induced mast cell and
basophil
degranulation and release of histamine. IgE-mediated allergic reactions such
as allergic
asthma, rhinitis, and eczema may also be treated.
The antagonists may also be employed to treat chronic and acute inflammation
by preventing the attraction of monocytes to a wound area. They may also be
employed
to regulate normal pulmonary macrophage populations, since chronic and acute
inflammatory pulmonary diseases are associated with sequestration of
mononuclear
phagocytes in the lung.
Antagonists may also be employed to treat rheumatoid arthritis by preventing
the
attraction of monocytes into synovial fluid in the joints of patients.
Monocyte influx and
activation plays a significant role in the pathogenesis of both degenerative
and
inflammatory arthropathies.
The antagonists may be employed to interfere with the deleterious cascades
attributed primarily to IL-1 and TNF, which prevents the biosynthesis of other
inflammatory cytokines. In this way, the antagonists may be employed to
prevent
inflammation. The antagonists may also be employed to inhibit prostaglandin-
independent fever induced by chemokines.
The antagonists may also be employed to treat cases of bone marrow failure,
for
example, aplastic anemia and myelodysplastic syndrome.
The antagonists may also be employed to treat asthma and allergy by preventing
eosinophil accumulation in the lung. The antagonists may also be employed to
treat
subepitheIial basement membrane fibrosis which is a prominent feature of the
asthmatic
lung.
Agonists. M-CIF, MPIF-1 and/or MIP-4 agonists include any small molecule
that has an activity similar to any one or more of these polypeptides, as
described herein.
For example, MPIF-1 agonists can be used to enhance MPIF-1 activity. For
example,
to enhance MPIF-1 induced myeloprotection in patients undergoing chemotherapy
or
bone marrow transplantation. As another example, M-CIF agonists can provide
one or
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more of antiinflammatory activity, anti-TNFa activity, and the like, as
described herein
for various functional activities of M-CIF.
Disease Diagnosis and Prognosis. Certain diseases or disorders, as discussed
below, may be associated with enhanced levels of the MPIF-1, M-CIF or MIP-4
protein
and mRNA encoding the MPIF-1, M-CIF or MIP-4 protein when compared to a
corresponding "standard" mammal, i. e. , a mammal of the same species not
having the
disease or disorder. Further, it is believed that enhanced levels of the MPIF-
I, M-CIF
or MIP-4 protein can be detected in certain body fluids (e. g. sera, plasma,
urine, and
spinal fluid) from mammals with a disease or disorder when compared to sera
from
mammals of the same species not having the disease or disorder. Thus, the
invention
provides a diagnostic method, which involves assaying the expression level of
the gene
encoding the MPIF-1, M-CIF or MIP-4 protein in mammalian cells or body fluid
and
comparing the gene expression level with a standard MPIF-1, M-CIF or MIP-4
gene
expression level, whereby an increase in the gene expression level over the
standard is
indicative of certain diseases or disorders.
Where a disease or disorder diagnosis has already been made according to
conventional methods, the present invention is useful as a prognostic
indicator, whereby
patients exhibiting enhanced MPIF-1, M-CIF or MIP-4 gene expression will
experience
a worse clinical outcome relative to patients expressing the gene at a lower
level.
By "assaying the expression level of the gene encoding the MPIF-l, M-CIF or
MIP-4 protein" is intended qualitatively or quantitatively measuring or
estimating the
level of the MPIF-1, M-CIF or MIP-4 protein or the level of the mRNA encoding
the
MPIF-1, M-CIF or MIP-4 protein in a first biological sample either directly
(e.g. by
determining or estimating absolute protein level or mRNA level) or relatively
(e.g. by
comparing to the MPIF-1, M-CIF or MIP-4 protein level or mRNA level in a
second
biological sample).
Preferably, the MPIF-1, M-CIF or MIP-4 protein level or mRNA level in the
first biological sample is measured or estimated and compared to a standard
MPIF-1, M-
CIF or MIP-4 protein level or mRNA level, the standard being taken from a
second
biological sample obtained from an individual not having the disease or
disorder. As
will be appreciated in the art, once a standard MPIF-1, M-CIF or MIP-4 protein
level or
mRNA level is known, it can be used repeatedly as a standard for comparison.
By "biological sample" is intended any biological sample obtained from an
individual, cell line, tissue culture, or other source which contains MPIF-1,
M-CIF or
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MIP-4 protein or mRNA. Biological samples include mammalian body fluids (such
as
sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted
mature
MPIF-1, M-CIF or MIP-4 protein, and ovarian, prostate, heart, placenta,
pancreas,
ascites, muscle, skin, glandular, kidney, liver, spleen, lung, bone, bone
marrow, ocular,
S peripheral nervous, central nervous, breast and umbilical tissue. Methods
for obtaining
tissue biopsies and body fluids from mammals are well known in the art. Where
the
biological sample is to include mRNA, a tissue biopsy is the preferred source.
The present invention is useful for detecting disease in mammals. In
particular
the invention is useful during useful for diagnosis or treatment of various
immune
system-related disorders in mammals, preferably humans. Such disorders include
tumors, cancers, and any disregulation of immune cell function including, but
not
limited to, autoimmunity, arthritis, leukemias, lymphomas, immunosuppression,
sepsis,
wound healing, acute and chronic infection, cell mediated immunity, humoral
immunity,
inflammatory bowel disease, myelosuppression, and the like. Preferred mammals
include monkeys, apes, cats, dogs, cows, pigs, horses, rabbits and humans.
Particularly
preferred are humans.
Total cellular RNA can be isolated from a biological sample using any suitable
technique such as the single-step guanidinium-thiocyanate-phenol-chloroform
method
described in Chomcrynski and Sacchi, Anal. Biochem. 16?:156-l59 (l987). Levels
of
mRNA encoding the MPIF-1, M-CIF or MIP-4 protein are then assayed using any
appropriate method. These include Northern blot analysis, S I nuclease
mapping, the
polymerase chain reaction (PCR), reverse transcription in combination with the
polymerase chain reaction (RT-PCR), and reverse transcription in combination
with the
ligase chain reaction (RT-LCR).
Northern blot analysis can be performed as described in Harada et al.) Cell
63: 303-312 ( 1990). Briefly, total RNA is prepared from a biological sample
as
described above. For the Northern blot, the RNA is denatured in an appropriate
buffer
(such as glyoxal/dimethyl sulfoxide/sodium phosphate buffer), subj ected to
agarose gel
electrophoresis, and transferred onto a nitrocellulose filter. After the RNAs
have been
linked to the filter by a UV linker, the filter is prehybridized in a solution
containing
formamide, SSC, Denhardt's solution, denatured salmon sperm, SDS, and sodium
phosphate buffer. MPIF-1, M-CIF or MIP-4 protein cDNA labeled according to any
appropriate method (such as the 3zP-multiprimed DNA labeling system
(Amersham)) is
used as probe. After hybridization overnight, the filter is washed and exposed
to x-ray
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film. cDNA for use as probe according to the present invention is described in
the
sections above and will preferably at least 15 by in length.
S 1 mapping can be performed as described in Fujita et al., Cell 49: 357- 367
( 1987). To prepare probe DNA for use in S 1 mapping, the sense strand of
above-described cDNA is used as a template to synthesize labeled antisense
DNA. The
antisense DNA can then be digested using an appropriate restriction
endonuclease to
generate further DNA probes of a desired length. Such antisense probes are
useful for
visualizing protected bands corresponding to the target mRNA (i.e., mRNA
encoding
the MPIF-1, M-CIF or MIP-4 protein). Northern blot analysis can be performed
as
described above.
Preferably, levels of mRNA encoding the MPIF-l, M-CIF or MIP-4 protein are
assayed using the RT-PCR method described in Makino et al., Technique 2:295-
301
{ 1990). By this method, the radioactivities of the "amplicons" in the
polyacrylamide gel
bands are linearly related to the initial concentration of the target mRNA.
Briefly, this
method involves adding total RNA isolated from a biological sample in a
reaction
mixture containing a RT primer and appropriate buffer. After incubating for
primer
annealing, the mixture can be supplemented with a RT buffer, dNTPs, DTT, RNase
inhibitor and reverse transcriptase. After incubation to achieve reverse
transcription of
the RNA, the RT products are then subject to PCR using labeled primers.
Alternatively,
rather than labeling the primers, a labeled dNTP can be included in the PCR
reaction
mixture. PCR amplification can be performed in a DNA thermal cycler according
to
conventional techniques. After a suitable number of rounds to achieve
amplification,
the PCR reaction mixture is electrophoresed on a polyacrylamide gel. After
drying the
gel, the radioactivity of the appropriate bands (corresponding to the mRNA
encoding the
MPIF-I, M-CIF or MIP-4 protein)) is quantified using an imaging analyzer. RT
and
PCR reaction ingredients and conditions, reagent and gel concentrations, and
labeling
methods are well known in the art. Variations on the RT-PCR method will be
apparent
to the skilled artisan.
Any set of oligonucleotide primers which will amplify reverse transcribed
target
mRNA can be used and can be designed as described in the sections above.
Assaying MPIF-1, M-CIF or MIP-4 protein levels in a biological sample can
occur using any art-known method. Preferred for assaying MPIF-1, M-CIF or MIP-
4
protein levels in a biological sample are antibody-based techniques. For
example,
MPIF-1, M-CIF or MIP-4 protein expression in tissues can be studied with
classical
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immunohistological methods. In these, the specific recognition is provided by
the
primary antibody (polyclonal or monoclonal) but the secondary detection system
can
utilize fluorescent, enzyme, or other conjugated secondary antibodies. As a
result, an
immunohistological staining of tissue section for pathological examination is
obtained.
S Tissues can also be extracted, e.g. with urea and neutral detergent, for the
liberation of
MPIF-1, M-CIF or MIP-4 protein for Western-blot or dot/slot assay (Jalkanen,
M., et
al., J. Cell. Biol. 10l: 976-98S (1985); Jalkanen, M., et al., J. Cell. Biol.
10S:3087-3096
(1987)). In this technique, which is based on the use of cationic solid
phases,
quantitation of MPIF-I, M-CIF or MIP-4 protein can be accomplished using
isolated
MPIF-1, M-CIF or MIP-4 protein as a standard. This technique can also be
applied to
body fluids. With these samples, a molar concentration of MPIF-I, M-CIF or MIP-
4
protein will aid to set standard values of MPIF-1, M-CIF or MIP-4 protein
content for
different body fluids, like serum, plasma, urine, spinal fluid, etc. The
normal appearance
of MPIF-1, M-CIF or MIP-4 protein amounts can then be set using values from
healthy
individuals, which can be compared to those obtained from a test subject.
Other antibody-based methods useful for detecting MPIF-1, M-CIF or MIP-4
protein gene expression include immunoassays, such as the enzyme linked
immunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example, an
MPIF-1, M-CIF or MIP-4 protein-specific monoclonal antibodies can be used both
as
an immunoabsorbent and as an enzyme-labeled probe to detect and quantify the
MPIF-1,
M-CIF or MIP-4 protein. The amount of MPIF-1, M-CIF or MIP-4 protein present
in
the sample can be calculated by reference to the amount present in a standard
preparation using a linear regression computer algorithm. In another ELISA
assay, two
distinct specific monoclonal antibodies can be used to detect MPIF-l, M-CIF or
MIP-4
2S protein in a body fluid. In this assay, one of the antibodies is used as
the
immunoabsorbent and the other as the enzyme-labeled probe.
The above techniques may be conducted essentially as a "one-step" or "two-
step"
assay. The "one-step" assay involves contacting MPIF-I, M-CIF or MIP-4 protein
with
immobilized antibody and, without washing, contacting the mixture with the
labeled
antibody. The "two-step" assay involves washing before contacting the mixture
with the
labeled antibody. Other conventional methods may also be employed as suitable.
It is
usually desirable to immobilize one component of the assay system on a
support,
thereby allowing other components of the system to be brought into contact
with the
component and readily removed from the sample.
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Suitable enzyme labels include, for example, those from the oxidase group,
which catalyze the production of hydrogen peroxide by reacting with substrate.
Glucose
oxidase is particularly preferred as it has good stability and its substrate
(glucose) is
readily available. Activity of an oxidase label may be assayed by measuring
the
concentration of hydrogen peroxide formed by the enzyme-labeled
antibody/substrate
reaction. Besides enzymes, other suitable labels include radioisotopes, such
as iodine
('25I, 'z'I), carbon ('4 C), sulphur (35 S), tritium (3 H), indium ("z In),
and technetium
(9~"'Tc), and fluorescent labels, such as fluorescein and rhodamine, and
biotin.
The polypeptides of the present invention, and polynucleotides encoding such
polypeptides, may be employed as research reagents for in vitro purposes
related to
scientific research, synthesis of DNA and manufacture of DNA vectors, and for
the
purpose of developing therapeutics and diagnostics for the treatment of human
disease.
For example, M-CIF and MPIF-1 may be employed for the expansion of immature
hematopoietic progenitor cells, for example, granulocytes, macrophages or
monocytes,
by temporarily preventing their differentiation. These bone marrow cells may
be
cultured in vitro.
Fragments of the full length MPIF-l, MIP-4 or M-CIF genes may be used as a
hybridization probe for a cDNA library to isolate the full length gene and to
isolate other
genes which have a high sequence similarity to the gene or similar biological
activity.
Preferably, however, the probes have at least 30 bases and may contain, for
example, 50
or more bases. The probe may also be used to identify a cDNA clone
corresponding to
a full length transcript and a genomic clone or clones that contain the
complete genes
including regulatory and promotor regions, exons, and introns. An example of a
screen
comprises isolating the coding region of the genes by using the known DNA
sequence
to synthesize an oligonucleotide probe. Labeled oligonucleotides having a
sequence
complementary to that of the genes of the present invention are used to screen
a library
of human cDNA, genomic DNA or mRNA to determine which members of the library
the probe hybridizes to.
This invention is also related to the use of the MPIF-1, MIP-4 and M-CIF gene
as part of a diagnostic assay for detecting diseases or susceptibility to
diseases related
to the presence of mutations in the nucleic acid sequences. Such diseases are
related to
under-expression of the chemokine polypeptides.
Individuals carrying mutations in the MPIF-1, MIP-4 and M-CIF may be
detected at the DNA level by a variety of techniques. Nucleic acids for
diagnosis may
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be obtained from a patient's cells, such as from blood, urine, saliva, tissue
biopsy and
autopsy material. The genomic DNA may be used directly for detection or may be
amplified enzymatically by using PCR (Saiki et al., Nature 324:163-166 (1986))
prior
to analysis. RNA or cDNA may also be used for the same purpose. As an example,
PCR primers complementary to the nucleic acid encoding MPIF-1, MIP-4 and M-CIF
can be used to identify and analyze MPIF-1, MIP-4 and M-CIF mutations. For
example,
deletions and insertions can be detected by a change in size of the amplified
product in
comparison to the normal genotype. Point mutations can be identified by
hybridizing
amplified DNA to radiolabeled MPIF-1, MIP-4 and M-CIF RNA or alternatively,
radiolabeled MPIF-l, MIP-4 and M-CIF antisense DNA sequences. Perfectly
matched
sequences can be distinguished from mismatched duplexes by RNase A digestion
or by
differences in melting temperatures.
Genetic testing based on DNA sequence differences may be achieved by
detection of alteration in electrophoretic mobility of DNA fragments in gels
with or
I S without denaturing agents. Small sequence deletions and insertions can be
visualized
by high resolution gel electrophoresis. DNA fragments of different sequences
may be
distinguished on denaturing formamide gradient gels in which the mobilities of
different
DNA fragments are retarded in the gel at different positions according to
their specific
melting or partial melting temperatures (see, e.g. Myers et al., Science 230:
l242 { 1985)).
Sequence changes at specific locations may also be revealed by nuclease
protection assays, such as RNase and Sl protection or the chemical cleavage
method
(e.g. Cotton et al., PNAS, USA 85:4397-4401 (l985)).
Thus, the detection of a specific DNA sequence may be achieved by methods
such as hybridization, RNase protection, chemical cleavage, direct DNA
sequencing or
the use of restriction enzymes, (e.g. Restriction Fragment Length
Polymorphisms
(RFLP)) and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA sequencing,
mutations can also be detected by in situ analysis.
The present invention also relates to a diagnostic assay for detecting altered
levels of MPIF-l, MIP-4 and M-CIF protein in various tissues since an over-
expression
of the proteins compared to normal control tissue samples may detect the
presence of
a disease or susceptibility to a disease, for example, a tumor. Assays used to
detect
levels of MPIF-1, MIP-4 and M-CIF protein in a sample derived from a host are
well-
known to those of skill in the art and include radioimmunoassays, competitive-
binding
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assays, Western Blot analysis, ELISA assays and "sandwich" assay. An ELISA
assay
(Coligan, et al., Current Protocols in Immunology 1(2), Chapter 6, (1991))
initially
comprises preparing an antibody specific to the MPIF-1 MIP-4 and M-CIF
antigens,
preferably a monoclonal antibody. In addition a reporter antibody is prepared
against
the monoclonal antibody. To the reporter antibody is attached a detectable
reagent such
as radioactivity, fluorescence or, in this example, a horseradish peroxidase
enzyme. A
sample is removed from a host and incubated on a solid support, e.g. a
polystyrene dish,
that binds the proteins in the sample. Any free protein binding sites on the
dish are then
covered by incubating with a non-specific protein like BSA. Next, the
monoclonal
antibody is incubated in the dish during which time the monoclonal antibodies
attach to
any MPIF-1, MIP-4 and M-CIF proteins attached to the polystyrene dish. All
unbound
monoclonal antibody is washed out with buffer. The reporter antibody linked to
horseradish peroxidase is now placed in the dish resulting in binding of the
reporter
antibody to any monoclonal antibody bound to MPIF-1, MIP-4 and M-CIF.
Unattached
1 S reporter antibody is then washed out. Peroxidase substrates are then added
to the dish
and the amount of color developed in a given time period is a measurement of
the
amount of MPIF-I, MIP-4 and M-CIF protein present in a given volume of patient
sample when compared against a standard curve.
A competition assay may be employed wherein antibodies specific to MPIF-1,
MIP-4 and M-CIF are attached to a solid support and labeled MPIF-1, MIP-4 and
M-CIF
and a sample derived from the host are passed over the solid support and the
amount of
label detected, for example by liquid scintillation chromatography, can be
correlated to
a quantity of protein in the sample.
A "sandwich" assay is similar to an ELISA assay. In a "sandwich" assay MPIF-
1, MIP-4 and M-CIF is passed over a solid support and binds to antibody
attached to a
solid support. A second antibody is then bound to the MPIF-1, MIP-4 and M-CIF.
A
third antibody which is labeled and specific to the second antibody is then
passed over
the solid support and binds to the second antibody and an amount can then be
quantified.
This invention provides a method for identification of the receptors for the
chemokine polypeptides. The gene encoding the receptor can be identified by
numerous
methods known to those of skill in the art, for example, ligand panning and
FACS
sorting (Coligan, et al. , Current Protocols in Immun. 1 (2), Chapter S, (
1991 )).
Preferably, expression cloning is employed wherein polyadenylated RNA is
prepared
from a cell responsive to the polypeptides, and a cDNA library created from
this RNA
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is divided into pools and used to transfect COS cells or other cells that are
not
responsive to the polypeptides. Transfected cells which are grown on glass
slides are
exposed to the labeled polypeptides. The polypeptides can be labeled by a
variety of
means including iodination or inclusion of a recognition site for a site-
specific protein
S kinase. Following fixation and incubation, the slides are subjected to
autoradiographic
analysis. Positive pools are identified and sub-pools are prepared and
retransfected
using an iterative sub-pooling and rescreening process, eventually yielding a
single
clones that encodes the putative receptor.
As an alternative approach for receptor identification, the labeled
polypeptides
can be photoaffmity linked with cell membrane or extract preparations that
express the
receptor molecule. Cross-linked material is resolved by PAGE analysis and
exposed to
X-ray film. The labeled complex containing the receptors of the polypeptides
can be
excised, resolved into peptide fragments, and subjected to protein
microsequencing. The
amino acid sequence obtained from microsequencing would be used to design a
set of
1 S degenerate oligonucleotide probes to screen a cDNA library to identify the
genes
encoding the putative receptors.
Therapeutics. Polypeptides of the present invention can be used in a variety
of
immunoregulatory and inflammatory functions and also in a number of disease
conditions. MPIF-1, MIP-4 and M-CIF are in the chemokine family and therefore
they
are a chemo-attractant for leukocytes (such as monocytes, neutrophils, T
lymphocytes,
eosinophils, basophils, etc.).
Northern Blot analyses show that MPIF-1) MIP-4 and M-CIF are expressed
predominantly is tissues of hemopoietic origin.
MPIF-1 TherapeuticlDiagnostic Applications. MPIF-1 is shown to play an
important role in the regulation of the immune response and inflammation. In
Figure
19, it is shown that lipopolysaccharide induces the expression of MPIF-1 from
human
monocytes. Accordingly, in response to the presence of an endotoxin, MPIF-1 is
expressed from monocytes and, therefore, administration of MPIF-1 may be
employed
to regulate the immune response of a host. MPIF-1 could be used as an anti
inflammatory agent.
As illustrated in Figure 10, the chemoattractant activity of MPIF-1 on TFIP-1
cells (A) and PBMCs (B) is significant. MPIF-1 also induces significant
calcium
mobilization in THP-1 cells (Figure 11) showing that MPIF-1 has a biological
effect on
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monocytes. Further, MPIF-1 produces a dose dependent chemotactic and calcium
mobilization response in human monocytes.
Further, the polypeptides of the present invention can be useful in anti-tumor
therapy since there is evidence that chemokine expressing cells injected into
tumors have
caused regression of the tumor, for example, in the treatment of Karposi
sarcoma.
MPIF-1 may induce cells to secrete TNF-a, which is a known agent for
regressing
tumors, in which case this protein could be used to induce tumor regression.
MPIF-1
may also induce human monocytes to secrete other tumor and cancer inhibiting
agents
such as IL-6, IL-1 and G-CSF. Also, MPIF-l, MIP-4 and M-CIF stimulate the
invasion
and activation of host defense (tumoricidal) cells, e.g., cytotoxic T-cells
and
macrophages via their chemotactic activity, and in this way can also be used
to treat
solid tumors.
The polypeptides can also be employed to inhibit the proliferation and
differentiation of hematopoietic cells and therefore may be employed to
protect bone
marrow stem cells from chemotherapeutic agents during chemotherapy. Figures 12
and
13 illustrate that MPIF-1 inhibits colony formation by low proliferative
potential colony
forming cells (LPP-CFC). Figure 14 illustrates that M-CIF specifically
inhibits M-CSF-
stimulated colony formation, while MPIF-1 does not. Since, both MPIF-1 and M-
CIF
significantly inhibit growth and/or differentiation of bone marrow cells, this
antiproliferative effect may allow administration of higher doses of
chemotherapeutic
agents and, therefore, more effective chemotherapeutic treatment.
The inhibitory effect of the M-CIF and MPIF-1 polypeptides on the
subpopulation of committed progenitor cells, (for example granulocyte, and
macrophage/monocyte cells) may be employed therapeutically to inhibit
proliferation
of leukemic cells.
Further, the inventors have found that MPIF-1, and variants thereof (e.g.,
MPIF-1D23), inhibit in vitro proliferation and differentiation of human
myeloid and
granulocyte precursors. Similarly, animal studies have shown that MPIF-123,
for
example, specifically inhibits the development of low proliferative potential-
colony
forming cells (LPP-CFCs) and granulocyte/monocyte committed progenitors both
in
vitro and in vivo. These findings indicate that MPIF-1 has therapeutic
application as a
chemoprotective agent that may spare early myeloid progenitors from the
cytotoxic
effects of commonly used chemotherapeutic drugs.
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Because MPIF-1, and variants thereof, has the ability to selectively inhibit
myeloid progenitor cells, MPIF-I can be used to treat myeloproliferative
disorders such
as essential thrombocytosis (ET), polycythemia vera (PV), or agnogenic myeloid
metaplasia (AMM), which are clinically closely related. Each disorder results
from an
acquired mutation of a single hematopoietic stem cell that gives the progeny
of that stem
cell a growth advantage. The pathophysiology of these disorders is distinct in
that there
is an overproduction of different cell types. In PV, there is an
overproduction of
erythrocytes, granulocytes, and megakaryocyte. In ET, there is, by definition,
overproduction of platelets as well as leukocytes. AMM also shows
thrombocytosis or
leukocytosis in addition to bone marrow fibrosis.
Stabilization of PV patients can be addressed by removal of red cells by
phlebotomy. However, there is no comparable therapy for elevated platelet
levels in ET
patients. Several myelosuppressive therapies have been studied for lowering
the risk of
thrombocytosis) Treatment with radioactive phosphorus, hydroxyurea, alkylating
agents
(busulfan and chlorambucil), interferons, or anagreIide have all shown
significant side
effects. In particular, there is an increased risk of acute leukemia with each
myelosuppressive therapy except anagrelide. Anagrelide is a promising therapy.
However, adverse reactions to anagrelide are a concern and its chronic
toxicity potential
has not been established. Interferons are, at present, considered second-line
therapy
because of expense, side effects, and the inconvenience of parenteral
administration.
These findings indicate that there is still a substantial need for therapy in
these diseases.
In vivo studies in mice pretreated with MPIF-1023 and then treated with 5-FU
demonstrate an inhibition of platelet progenitor cell proliferation.
The present invention further encompasses the use of MPIF-l, and variants
thereof, in combination with other myelosuppressive therapies and agents.
In Figures 1 S, I 6 and 17 the committed cells of the cell lineages utilized
were
removed and the resulting population of cells were contacted with M-CIF and
MPIF-1
causes a decrease in the Mac-1 positive population of cells by nearly 50%,
which is
consistent with the results of Figure 14 which shows M-CIF induces inhibition
of M-
CSF responsive colony-forming cells. MPIF-1, as shown in Figure 17, inhibits
the
ability of committed progenitor cells to form colonies in response to IL-3, GM-
CSF and
M-CSF. Further, as shown in Figure 18, a dose response of MPIF-1 is shown to
inhibit
colony formation. This inhibition could be due to a specific blockage of the
differentiative signal mediated by these factors or to a cytotoxic effect on
the progenitor
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cells. In addition, Examples 15 and 16 demonstrate that MPIF-1 has in vitro
and in vivo
myeloprotection activity against cytotoxicity of chemotherapeutic drugs. Thus,
MPIF-1
can be useful as a myeloprotectant for patients undergoing chemotherapy.
As noted above, one major complication resulting from chemotherapy and
radiation therapy is the destruction of non-pathological cell-types. The
present invention
provides methods for myeloprotection from radiation and chemotherapeutic
agents by
suppressing myeloid cell proliferation in an individual. These methods involve
administering a myelosuppressive amount of MPIF-1 either alone or together
with one
or more chemokines selected from the group consisting of Macrophage
Inflammatory
Protein-1 a (MIP-1 a), Macrophage Inflammatory Protein-2a (MIP-2a), Platelet
Factor
4 (PF4), Interleukin-8 (IL-8), Macrophage Chemotactic and Activating Factor
(MCAF),
and Macrophage Inflammatory Protein-Related Protein-2 (MRP-2) to an individual
as
part of a radiation treatment or chemotherapeutic regimen. The
myelosuppressive
compositions of the present invention thus provide myeloprotective effects and
are
1 S useful in conjunction with therapies that have an adverse affect on
myeloid cells. This
is because the myelosuppressive compositions of the present invention place
myeloid
cells in a slow-cycling state thereby providing protection against cell damage
caused by,
for example, radiation therapy or chemotherapy using cell-cycle active drugs,
such as
cytosine arabinoside, hydroxyurea, 5-Fu and Ara-C. Once the chemotherapeutic
drug
has cleared the individual's system, it would be desirable to stimulate rapid
amplification and differentiation of progenitor cells that were protected by
MPIF-1
using, for example, myelostimulators, such as Interleukin-11 (IL-1 1 ),
erythropoietin
(EPO), GM-CSF, G-CSF, stern cell factor (SCF), and thrombopoietin (Tpo).
The ability of MPIF-1 to confer in vivo myeloprotection in the presence of a
chemotherapeutic agent is demonstrated in Example 28. Example 28 shows that
the
administration of MPIF-1 to an individual prior to the administration of a
chemotherapeutic agent accelerates the recovery of platelets in the blood even
after
multiple cycles of 5-Fu treatment. The experiments set forth in Example 28
also
demonstrate that MPIF-1 treatment during multiple cycles of 5-Fu treatment
results in
the faster recovery of granulocytes. In addition, the results of Experiment 28
also
suggest that MPIF-1 and G-CSF exert additive effects when co-administered.
As indicated, the inventors have found that MPIF-1, and variants thereof,
exhibit
potent in vitro suppression of low proliferation potential-colony forming
cells
(LPP-CFCs) from bone marrow. LPP-CFCs are bipotential hematopoietic
progenitors
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that give rise to granulocyte and monocyte lineages. MPIF-I also reversibly
inhibits
colony formation by human CD34+ stem cell derived granulocyte and monocyte
colony
forming cells. The inventors' in vitro chemoprotection experiments have shown
protection of these hematopoietic progenitors by MPIF-1023 from the cytotoxic
effects
S of the drugs S-fluorouracil (5-Fu), cytosine arabinoside, and Taxol~.
The use of a MPIF-1 variant (O23} in an in vivo chemotherapeutic model has
shown that it produces a more rapid recovery of both bone marrow progenitor
cells and
peripheral cell populations of neutrophils and platelets. Further, as shown in
Examples
I6 and 28, the administration of MPIF-1 results in the accelerated recovery
from
neutropenia and thrombocytopenia in experimental animals treated with 5-Fu.
Thus,
MPIF-l, and variants thereof, shorten the period of bone marrow aplasia,
granulopenia,
and thrombocytopenia associated with the chemotherapeutic agents and thereby
reducing the likelihood of infection in patients undergoing treatment with
such agents.
Thus, the invention relates to methods for protecting myeloid progenitor cells
I S and to accelerating recovery of platelets and granulocytes which comprise
the
administration of MPIF-1 to an individual undergoing therapy which
preferentially kills
dividing cells (e.g., radiation therapy or treatment with a cell-cycle active
drug). MPIF-
1 is administered in sufficient quantity to provide in vivo myeloprotection
against
treatments and agents which preferentially kill dividing cells. By "MPIF-1 is
administered" is meant that MPIF-l, an analog of MPIF-I, or combination
thereof is
administered in a therapeutically effective amount. Modes of administration of
MPIF-1
are discussed in detail below.
MPIF-1 may be administered prior to, after, or during the therapy in which
dividing cells are preferentially killed. In a preferred embodiment, MPIF-1 is
administered prior to radiation therapy or administration of a cell-cycle
active drug and
sufficient time is allowed for MPIF-1 to suppress the proliferation of myeloid
cells.
Further contemplated by the present invention is the use of MPIF-I to protect
myeloid
cells during multiple rounds of therapy in which dividing cells are
preferentially killed.
In such a case, MPIF-1 may be administered in either a single dose or multiple
doses at
different time points in the therapy or treatment regimen.
As indicated above, MPIF-1 may be used alone or in conjunction with one or
more myelostimulators. Myelostimulators are currently used in the art to
stimulate the
proliferation of myeloid cells after their depletion in an individual
undergoing radiation
therapy or treatment with a cell-cycle active drug. See, e.g., Kannan, V. et
al., Int. J.
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WO 98I14582 PCT/US97t17505
Radiat. ~ncol. Biol. Phys. 3 7:1005-1010 ( 1997); Engelhardt, M. et al., Bone
Marrow
Transplant l9:529-537 (1997); Vadhan-Raj, S. et al., Ann Intern Med. l26:673-
681
(1997); Hacker, L. et al., Blood 89:155-I65 (1997); Basser, R, et al.) Lancet
348:1279-
1281 ( 1996); Grossman, A. et al., Blood 88:3363-3370 ( 1996); Gordon, M. et
al., Blood
87:3615-3624 (1996). MPIF-1 may, for example, be administered prior to therapy
which
kills dividing cells and one or more myelostimulators administered after or
during the
course of such therapy. In such a case, MPIF-I will protect myeloid cells from
the
therapy and administration of the myelostimulator(s) will then result in
expansion of the
protected myeloid cell population.
Myelostimulators are typically administered to patients undergoing treatment
with a chemotherapeutic agent in therapeutically effective amounts. Dosage
formulation
and mode of administration may vary with a number of factors including the
individual
being treated, the condition of the cells being stimulated, the stage of
treatment in the
chemotherapeutic regimen, and the myelostimulator(s) being used. GM-GSF and
G-CSF, for examples, are therapeutically effective at dosages of about 1
~g/kilogram
and 5 to 10 ~g/kilogram of body weight, respectively, and may be administered
daily
by subcutaneous injection. See, e.~., Kannan, V. et ul., Int. J. Radiat.
Oncol. Biol. Phys.
3 7:1005-1010 ( I 997); Engelhardt, M. et al., Bone Marro~s~ Transplant l9:529-
537
( l997); Sniecinski, I. et al., Blood 89:1521-1528 ( 1997). IL-11 may be
administered by
daily subcutaneous injection at a dosage range of up to l00 pg/kilogram of
body weight.
Gordon, M. et al., supra. Doses of IL-11 below 10 pg/kilogram, however, are
believed
to be effective in reducing chemotherapy-induced thrombocytopenia. Tpo may be
administered by intravenous injection at a dosage range of 0.3 to 2.5
~g/kilogram of
body weight. See, e.g., Vadhan-Raj, S. et al., Ann. Interrz Med. l26:673-681 {
1997);
Hacker, L. et al., Blood 89:155-165 ( 1997). As one skilled in the art would
recognize,
the optimal dosage formulation and mode of administration will vary with a
number of
factors including those noted above. Dosage formulation and mode of
administration
for additional myelostimulators are known in the art.
The timing of administration of myelostimulators as part of a treatment
protocol
involving therapy which preferentially kill dividing cells may also vary with
the factors
described above for dosage formulation and mode of administration. A number of
reports have been published which disclose the administration of
myelostimulators to
individuals as part of treatment protocols involving radiation therapy or cell-
cycle active
drugs. Vadhan-Raj, S. et al., supra, for example, report the use of a single
intravenous
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WO 98I14582 PCT/US97/17505
dose of Tpo three weeks prior to the administration of a chemotherapeutic
agent.
Papadimitrou, C. et al., Cancer 79:2391-2395 (1997) and Whitehead, R. et al.,
J. Clin.
Oncol. l5:2414-2419 ( 1997) report chemotherapeutic treatment methods which
involve
the administration of chemotherapeutic agents over the course of several
weeks. In each
of these cases, doses of G-CSF are administered at multiple time points after
the first
day and before the last day of treatment with the chemotherapeutic agent.
Similar usage
of both IL-11 and GM-CSF are reported in Gordon, M. et al., supra, and
Michael, M.,
et al., Am. J. Clin. Oncol. 20:259-262 (1997). One skilled in the art would
recognize,
however, that optimal timing of administration of myelostimulators will vary
with the
particular myelostimulators used and the conditions under which they are
administered.
Thus, the administration of myelostimulators to alleviate cytotoxic effects
that
therapies which preferentially kill dividing cells have on myeloid cells is
known in the
art. The myelostimulators may be administered by several routes, including
intravenous
and subcutaneous injection. The concentrations of myelostimulators
administered vary
widely with numerous factors but generally range between 0.1 to l00
pg/kilogram of
body weight and may be administered in a single dose or in multiple doses at
various
time points in the chemotherapeutic or radiological treatment regimen.
Myelostimulators are generally administered, however, prior to or after
administration
of the chemotherapeutic agent or radiological treatment. As one skilled in the
art would
understand, the conditions under which myelostimulators are used will vary
with both
the particular myelostimulator and the treatment regimen.
As the skilled artisan will appreciate, MPIF-1 can be used as described above
to
enhance the effectiveness of hematopoietic growth factors generally. Such
hematopoietic growth factors include erythropoietin, which stimulates
production of
erythrocytes, and IL-3, a multilineage growth factor that stimulates more
primitive stem
cells, thus increasing the number of a11 blood cell types. Others include stem
cell factor;
GM-CSF; and hybrid molecules of G-CSF and erythropoietin; IL-3 and SCF; and GM-
CSF and G-CSF.
The myelosuppressive pharmaceutical compositions of the present invention are
also useful in the treatment of leukemia, which causes a hyperproliferative
myeloid cell
state. Thus, the invention further provides methods for treating leukemia,
which involve
administering to a leukemia patient a myelosuppressive amount of MPIF-1 either
alone
or together with one or more chemokines selected from the group consisting of
MIP-1 a,
MIP-2a, PF4, IL-8, MCAF, and MRP-2.
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By "suppressing myeloid cell proliferation" is intended decreasing the cell
proliferation of myeloid cells and/or increasing the percentage of myeloid
cells in the
slow-cycling phase. As above, by "individual" is intended mammalian animals,
preferably humans. Preincubation of the myelosuppressive compositions of the
present
invention with acetonitrile (ACN) significantly enhances the specific activity
of these
chemokines for suppression of myeloid progenitor cells. Thus, preferably,
prior to
administration, the myelosuppresive compositions of the present invention are
pretreated
with ACN as described in Broxmeyer H. E., et al., Ann-Hematol. 71 (5): 23 S-
46( 1995)
and PCT Publication WO 94/l3321, the entire disclosures of which are hereby
incorporated herein by reference.
The myelosuppressive compositions of the present invention may be used in
combination with a variety of chemotherapeutic agents including alkylating
agents such
as nitrogen mustards, ethylenimines, methylmelamines, alkyl sulfonates,
nitrosuoureas,
and triazenes; antimetabolites such as folic acid analogs, pyrimidine analogs,
in
1 S particular fluorouracil and cytosine arabinoside, and purine analogs;
natural products
such as vinca alkaloids, epipodophyllotoxins, antibiotics, enzymes and
biological
response modifiers; and miscellaneous products such as platinum coordination
complexes, anthracenedione, substituted urea such as hydroxyurea, methyl
hydrazine
derivatives, and adrenocorticoid suppressant.
Chemotherapeutic agents can be administered at known concentrations according
to known techniques. The myelosuppressive compositions of the present
invention can
be co-administered with a chemotherapeutic agent, or administered separately,
either
before or after chemotherapeutic administration.
Certain chemokines, such as MIP-1f3, MIP-213 and GRO-a, inhibit (at least
partially block) the myeloid suppressive affects of the myelosuppresive
compositions
of the present invention. Thus, in a further embodiment, the invention
provides methods
for inhibiting myelosuppression, which involves administering an effective
amount of
a myelosuppressive inhibitor selected from the group consisting of MIP-113,
MIP-2f3 and
GRO-a to a mammal previously exposed to the myelosuppresive agent MPIF-1
either
alone or together with one or more of MIP-1 a, MIP-2a, PF4, IL-8, MCAF, and
MRP-2.
One of ordinary skill will appreciate that effective amounts of the MPIF-1
polypeptides for treating an individual in need of an increased level of MPIF-
1 activity
(including amounts of MPIF-1 polypeptides effective for myelosuppression with
or
without myelosuppressive agents or myelosuppressive inhibitors) can be
determined
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empirically for each condition where administration of MPIF-1 is indicated.
The
polypeptide having MPIF-1 activity my be administered in pharmaceutical
compositions
in combination with one or more pharmaceutically acceptable excipients.
MPIF-1 may also be employed to treat leukemia and abnormally proliferating
cells, for example tumor cells, by inducing apoptosis. MPIF-1 induces
apoptosis in a
population of hematopoietic progenitor cells.
MPIF-1 may be employed for the expansion of immature hematopoietic
progenitor cells, for example, granulocytes, macrophages or monocytes, by
temporarily
preventing their differentiation. These bone marrow cells may be cultured in
vitro.
Thus, MPIF-1 can also be useful as a modulator of hematopoietic stem cells in
vitro for
the purpose of bone marrow transplantation and/or gene therapy. Since stem
cells are
rare and are most useful for introducing genes into for gene therapy, MPIF can
be used
to isolate enriched populations of stem cells. Stem cells can be enriched by
culturing
cells in the presence of cytotoxins, such as 5-Fu, which kills rapidly
dividing cells,
where as the stem cells will be protected by MPIF-1. These stem cells can be
returned
to a bone marrow transplant patient or can then be used for transfection of
the desired
gene for gene therapy. In addition, MPIF-1 can be injected into individuals
which
results in the release of stem cells from the bone marrow of the individual
into the
peripheral blood. These stem cells can be isolated for the purpose of
autologous bone
marrow transplantation or manipulation for gene therapy. After the patient has
finished
chemotherapy or radiation treatment, the isolated stem cells can be returned
to the
patient.
In addition, since MPIF-1 has effects on T-lymphocytes as well as macrophages,
MPIF-1 may enhance the capacity of antigen presenting cells (APCs) to take up
virus,
bacteria or other foreign substances, process them and present them to the
lymphocytes
responsible for immune responses. MPIF-1 may also modulate the interaction of
APCs
with T-lymphocytes and B-lymphocytes. MPIF-1 may provide a costimulatory
signal
during antigen presentation which directs the responding cell to survive,
proliferate,
differentiate, secrete additional cytokines or soluble mediators, or
selectively removes
the responding cell by inducing apoptosis or other mechanisms of cell death.
Since
APCs have been shown to facilitate the transfer of HIV to CD4+ T-lymphocytes,
MPIF-1 may also influence this ability and prevent infection of lymphocytes by
HIV or
other viruses mediated through APCs. This is also true for the initial
infection of APCs,
T-lymphocytes or other cell types by HIV, EBV, or any other such viruses.
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In addition, recent demonstration that the MIP-1 a receptor serves as a
cofactor
in facilitating the entry of HIV into human monocytes and T-lymphocytes raises
an
interesting possibility that MPIF-1 or its variants might interfere with the
process of HIV
entry into the cells. (See, Example 17). Thus, MPIF-1 can be useful as an
antiviral agent
for viruses and retroviruses whose entry is facilitated by the MIP-1 a
receptor.
MPIF-1 may act as an immune enhancement factor by stimulating the intrinsic
activity of T-lymphocytes to fight bacterial and viral infection as well as
other foreign
bodies. Such activities are useful for the normal response to foreign antigens
such as
infection of allergies as well as immunoresponses to neoplastic or benign
growth
including both solid tumors and leukemias.
For these reasons the present invention is useful for diagnosis or treatment
of
various immune system-related disorders in mammals, preferably humans. Such
disorders include tumors, cancers) and any disregulation of immune cell
function
including, but not limited to, autoimmunity, arthritis, leukemias, lymphomas,
immunosuppression, sepsis, wound healing, acute and chronic infection, cell
mediated
immunity, humorai immunity, inflammatory bowel disease, myelosuppression, and
the
like.
M-CIF TherapeuticlDia~;nostic Applications. M-CIF activity is useful for
immune enhancement or suppression, myeloprotection. stem cell mobilization,
acute and
chronic inflammatory control and treatment of leukemia. In addition, since M-
CIF has
effects on T-lymphocytes as well as macrophages, M-CIF enhances the capacity
of
antigen presenting cells (APCs) to take up virus, bacteria or other foreign
substances,
process them and present them to the lymphocytes responsible for immune
responses.
In addition, M-CIF also modulates the interaction of APCs with T-lymphocytes
and B-
lymphocytes. For instance, M-CIF provides a costimulation signal during
antigen
presentation which directs the responding cell to survive, proliferate,
differentiate,
secrete additional cytokines or soluble mediators, or selectively removes the
responding
cell by inducing apoptosis or other mechanisms of cell death. Since APCs have
been
shown to facilitate the transfer of HIV to CD4+ T-lymphocytes, M-CIF also
influences
this ability and prevents infection of lymphocytes by HIV or other viruses
mediated
through APCs. This is also true for the initial infection of APCs, T-
lymphocytes or
other cell types by HIV, EBV, or any other such viruses.
M-CIF suppresses the immune system. As one mechanism, it is believed that M-
CIF down regulates the activity of T-lymphocytes via CTLA-4. The activation
and
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subsequent differentiation of T-cells requires two types of signals from APCs.
One of
these two signals is an antigen-independent signal mediated by engagement of
the T cell
surface molecule CD28 with members of the B7 family on the APC. Allison, Curr.
Opin. Immunol. 6:414 (1994); June et al., Immunol. Today I5: 32l (l994). In
contrast
to CD28, CTLA-4 is critical for the negative regulation of T cell responses.
Waterhouse
et al., Science 270: 985 (1995). Recent studies suggest that the outcome of T
cell
activation is determined by a delicately balanced interplay between positive
signals from
CD28 and negative signals from CTLA-4. Waterhouse et al.) Science 270: 985 (
1995).
The cumulative results of a number studies suggest that the blockade of CTLA-4
removed, whereas aggregation of CTLA-4 provided, inhibitory signals that down
regulate T cell responses. Allison et al., Science 270: 932-933 (1995). In
addition, the
phenotype of CTLA-4 knock-out mice supports an inhibitory signaling role for
CTLA-4
in the regulation of T cell responses. Allison et al., Science 270: 932-933
(1995). M-CIF
appears to induce CTLA-4 cells which, as discussed above, is a known down
regulator
of T cells. In addition, M-CIF directly inhibits CD8+ T cells which is also a
known
down regulator of T cells.
The ability of M-CIF to down regulate T cells is useful for modulating the
immune response to foreign antigens from infection by bacteria or viruses and
allergies
as well as immunoresponses to neoplastic or benign growth including both solid
tumors
and leukemias.
For these reasons the present invention is useful for diagnosis or treatment
of
various immune system-related disorders in mammals, preferably humans. Such
disorders include tumors, cancers, and any disregulation of immune cell
function
including, but not limited to, autoimmunity, arthritis, asthma, leukemias,
lymphomas,
immunosuppression, sepsis) wound healing, acute and chronic infection, cell
mediated
immunity) humoral immunity, inflammatory bowel disease, myelosuppression, and
the
like.
M-CIF, as an antiinflammatory, can be used to treat such disorders as, but not
limited to, those involving abnormal production of TNFa. Such disorders
include, but
are not limited to, sepsis syndrome, including cachexia, circulatory collapse
and shock
resulting from acute or chronic bacterial infection, acute and chronic
parasitic or
infectious processes, including bacterial, viral and fungal infections, acute
and chronic
immune and autoimmune pathologies, such as systemic lupus erythematosus (SLE)
and
rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatory
pathologies such
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as sarcoidosis and Crohn's pathology, vascular inflammatory pathologies such
as
disseminated intravascular coagulation, graft-versus-host pathology,
Kawasaki's
pathology; malignant pathologies involving TNF-secreting tumors and
neurodegenerative diseases.
Neurodegenerative diseases include, but are not limited to, AIDS dementia
complex, demyelinating diseases, such as multiple sclerosis and acute
transverse
myelitis; extrapyramidal and cerebellar disorders' such as lesions of the
corticospinal
system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic
movement
disorders such as Huntington's Chorea and senile chorea; drug-induced movement
disorders, such as those induced by drugs which block CNS dopamine receptors;
hypokinetic movement disorders, such as Parkinson's disease; Progressive
supranucleo
Palsy; structural lesions of the cerebellum; spinocerebellar degenerations,
such as spinal
ataxia, Friedreich's ataxia, cerebellar cortical degenerations) multiple
systems
degenerations (Mencel, Dejerine-Thomas, Shi-Draper, and Machado-Joseph);
systemic
disorders (Refsum's disease, abetalipoprotemia, ataxia, telangiectasia) and
mitochondria)
multisystem disorder); demyelinating core disorders, such as multiple
sclerosis, acute
transverse myelitis; and disorders of the motor unit' such as neurogenic
muscular
atrophies (anterior horn cell degeneration, such as amyotrophic lateral
sclerosis, infantile
spinal muscular atrophy and juvenile spinal muscular atrophy); Alzheimer's
disease:
Down's Syndrome in middle age; Diffuse Ley~ body disease; Senile Dementia of
Lewy
body type; Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt-Jakob
disease; Subacute sclerosing panencephalitis Hallerrorden-Spatz disease; and
Dementia
pugilistica. One preferred neurodegenerative disease is multiple sclerosis.
See, e.g., Berkow et al, eds., The Merck Manual) 16th edition, Merck and Co.,
Rahway, N.J., l992, which reference, and references cited therein, are
entirely
incorporated herein by reference.
As noted above, M-CIF may also be used to treat SLE and other disease-states
involving immune responses and inflammation. SLE is an autoimmune disease
which
results in the formation of complement-fixing immune aggregates capable of
inducing
glomerulonephritis and vasculitis. Steinberg, A.D. and Klinman, D.M., Rheum.
Dis.
Clinics of No. Amer. l4:25 {1988). A number of agents are currently in use, or
are
proposed for use, in treating SLE and both lupus associated glomerulonephritis
and
vasculitis. Among these agents are antibodies with specificity for cell
surface receptors
required for the induction of immune responses. Anti-CD 11 a and anti-CD-54
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monoclonal antibodies, for example, have been shown to be effective in the
treatment
of experimental lupus nephritis. Koostra, C.J. et al., Clin. Exp. Immunol.
108:324-332
{1997). Blocking the interaction between CD28/CTLA-4 and their ligands (e.g.,
CD80
and CD86) has also been proposed as a means for suppressing immune responses
associated with lupus, and the administration of CD80 and CD86 specific
monoclonal
antibodies has been shown to prevent the development and progression of lupus
in an
experimental animal model. Nakajirna, A. et al., Eur. J. Immunol. 25:3060-3069
(l995).
Chemical agents have also been shown to have therapeutic effect in the
treatment of SLE
and both lupus associated glomerulonephritis and vasculitis. These agents
include
antifolate compounds (e.g., methotrexate and MX-68) and immunosuppressants
(e.g.,
corticosteroids, cyclophosphamide, mycophenolate mofetil, azathioprine).
Corna, D.
et al., Kidney Int. 5l:1583-1 S89 ( 1997); Mihara, M. et al., Int. Arch.
Allergy Immunol.
1l3:454-459 (1997); Gansauge, S. et al., Ann. Rheum. Dis. S6:382-385 (I997).
Additional agents have therapeutic effect for the treatment of afflictions
associated with
non-lupus associated immune complexes. Two classes of such compounds are free
radical scavengers (e.g., OPC-15161 ) and angiotensin-converting enzyme
inhibitors
(e.g., quinapril). Sanaka, T. et al., Nephron 76:3l5-322 (1997); Ruiz-Ortega,
M. et al.,
J. Am. Soc. Nephrol. 8:7S6-768 ( 1997).
As shown in Examples 19 and 29, M-CIF suppresses renal inflammation
associated with cell mediated immunity and ameliorates the progression of
lupus
associated nephritis. The present invention thus provides a method for
treating SLE, as
well as other diseases involving immune responses (e.g., those resulting from
cell
mediated immunity and the formation of immune complexes), comprising the
administration of M-CIF to a patient in need thereof. M-CIF may be
administered as the
sole immune response modulator or may be administered in conjunction with one
or
more additional agents which modulate immune responses.
Accordingly, MPIF-1, MIP-4 and M-CIF can be used to facilitate wound healing
by controlling infiltration of target immune cells to the wound area. In a
similar fashion,
the polypeptides of the present invention can enhance host defenses against
chronic
infections, e.g. mycobacterial, via the attraction and activation of
microbicidal
leukocytes.
The polypeptides of the present invention, and polynucleotides encoding such
polypeptides, may be employed as research reagents for in vitro purposes
related to
scientific research, synthesis of DNA and manufacture of DNA vectors, and for
the
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purpose of developing therapeutics and diagnostics for the treatment of human
disease.
For example, M-CIF and MPIF-1 may be employed for the expansion of immature
hematopoietic progenitor cells, for example, granulocytes, macrophages or
monocytes,
by temporarily preventing their differentiation. These bone marrow cells may
be
cultured in vitro.
Another use of the polypeptides is the inhibition of T-cell proliferation via
inhibition of IL-2 biosynthesis, for example, in auto-immune diseases and
lymphocytic
leukemia.
MPIF-1, MIP-4 and M-CIF can also be useful for inhibiting epidermal
keratinocyte proliferation which has utility in psoriasis {keratinocyte hyper-
proliferation)
since Langerhans cells in skin have been found to produce MIP-1 a.
MPIF-1, MIP-4 and M-CIF can be used to prevent scarring during wound
healing both via the recruitment of debris-cleaning and connective tissue-
promoting
inflammatory cells and by its control of excessive TGFp-mediated fibrosis, in
addition
these polypeptides can be used to treat stroke, thrombocytosis, pulmonary
emboli and
myeloproliferative disorders, since MPIF-1, MIP-4 and M-CIF increase vascular
permeability.
Pfrarmaceutical Compositions. The MPIF- I , M-CIF or MIP-4 polypeptide
pharmaceutical composition comprises an effective amount of an isolated MPIF-
l, M-
CIF or MIP-4 polypeptide of the invention, particularly a mature form of the
MPIF-1,
M-CIF or MIP-4, effective to increase the MP1F-1, M-CIF or MIP-4 activity
level in
such an individual. Such compositions can be formulated and dosed in a fashion
consistent with good medical practice, taking into account the clinical
condition of the
individual patient (especially the side effects of treatment with MPIF-1, M-
CIF or MIP-4
polypeptide alone), the site of delivery of the MPIF-1, M-CIF or M1P-4
polypeptide
composition, the method of administration, the scheduling of administration,
and other
factors known to practitioners. The "effective amount" of MPIF-1, M-CIF or MIP-
4
polypeptide for purposes herein is thus determined by such considerations.
Polypeptides, antagonists or agonists of the present invention can be employed
in combination with a suitable pharmaceutical carrier. Such compositions
comprise a
therapeutically effective amount of the protein, and a pharmaceutically
acceptable carrier
or excipient. Such a carrier includes but is not limited to saline, buffered
saline,
dextrose, water, glycerol, ethanol, and combinations thereof. The formulation
should
suit the mode of administration.
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By "pharmaceutically acceptable earner" is meant a non-toxic solid, semisolid
or liquid filler, diluent, encapsulating material or formulation auxiliary of
any type. The
term "parenteral" as used herein refers to modes of administration which
include
intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and
intraarticular
injection and infusion.
The MPIF-1, M-CIF or MIP-4 polypeptide is also suitably administered by
sustained-release systems. Suitable examples of sustained-release compositions
include
semi-permeable polymer matrices in the form of shaped articles, e. g. films,
or
mirocapsules. Sustained-release matrices include polylactides (tl.S. Pat. No.
3,773,919,
EP 58,481 ), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate
(Sidman, U.
et al., Biopolymers 22:547-556 ( 1983)), poly (2- hydroxyethyl methacrylate)
(R. Larger
et al., J. Biomed Mater. Res. l5:167-277 (1981), and R. Larger, Chem. Tech.
12:98-l05
( 1982)), ethylene vinyl acetate (R. Larger et al., Id. ) or poly-D- (-)-3-
hydroxybutyric
acid (EP 133,988). Sustained-release MPIF-l, M-CIF or MIP-4 polypeptide
compositions also include liposomally entrapped MPIF-1, M-CIF or MIP-4
polypeptide.
Liposomes containing MPIF-1, M-CIF or MIP-4 polypeptide are prepared by
methods
known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad Sci. (USA)
82:3688-3692
( 1985 ); Hwang et al., Proc. Natl. Acad Sci. (USA) 77:4030-4034 ( 1980); EP
52,322; EP
36,676; EP 88,046; EP 143,949; EP l42,641; 3apanese Pat. Appl. 83-1 l8008;
U.S. Pat.
Nos. 4,485,04 and 4,544,545; and EP l02,324. Ordinarily, the liposomes are of
the
small (about 200-800 Angstroms) unilamellar type in which the lipid content is
greater
than about 30 mol. percent cholesterol, the selected proportion being adjusted
for the
optimal MPIF-1, M-CIF or MIP-4 polypeptide therapy.
For parenteral administration, in one embodiment, the MPIF-1, M-CIF or MIP-4
polypeptide is formulated generally by mixing it at the desired degree of
purity, in a unit
dosage injectable form (solution, suspension, or emulsion)) with a
pharmaceutically
acceptable carrier, i.e., one that is non-toxic to recipients at the dosages
and
concentrations employed and is compatible with other ingredients of the
formulation.
For example, the formulation preferably does not include oxidizing agents and
other
compounds that are known to be deleterious to polypeptides.
Generally, the formulations are prepared by contacting the MPIF-1, M-CIF or
MIP-4 polypeptide uniformly and intimately with liquid carriers or finely
divided solid
carriers or both. Then, if necessary, the product is shaped into the desired
formulation.
Preferably the carrier is a parenteral earner, more preferably a solution that
is isotonic
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with the blood of the recipient. Examples of such carrier vehicles include
water, saline,
Ringer's solution, and dextrose solution. Non-aqueous vehicles such as fixed
oils and
ethyl oleate are also useful herein, as well as liposomes.
The carrier suitably contains minor amounts of additives such as substances
that
enhance isotonicity and chemical stability. Such materials are non-toxic to
recipients
at the dosages and concentrations employed, and include buffers such as
phosphate,
citrate, succinate, acetic acid, and other organic acids or their salts;
antioxidants such as
ascorbic acid; low molecular weight (less than about ten residues)
polypeptides, e.g.
polyarginine or tripeptides; proteins, such as serum albumin, gelatin, or
immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids,
such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides,
disaccharides, and other carbohydrates including cellulose or its derivatives,
glucose,
mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as
mannitol
or sorbitol; counterions such as sodium; and/or nonionic surfactants such as
1 S polysorbates, poloxamers, or PEG.
The MPIF-1, M-CIF or MIP-4 polypeptide is typically formulated in such
vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferabl y 1-10
mg/ml, at
a pH of about 3 to 8. It will be understood that the use of certain of the
foregoing
excipients, carriers, or stabilizers will result in the formation of MPIF-1, M-
CIF or MIP-
4 polypeptide salts.
When MPIF-l, and/or variants thereof, is administered as a myeloprotectant as
part of a chemotherapeutic regimen for the treatment of hyperproliferative
disorders in
humans, a suitable dosage range for intravenous administration is 0.01 pg/kg
to 10 ~g/kg
of body weight. Further, MPIF-1 may be administered intravenously at doses of
0.1,
1.0, 10, and l00 ~g/kg of body weight. Extrapolation of data from animal
studies
indicates that a dosage of MPIF-1 suitable for myeloprotection in humans is
0.016 ~g/kg
of body weight.
Further, MPIF-1, and/or a variant thereof, may be administered once daily for
a specified number of days (e.g., three days). In addition, when used in a
chemotherapeutic regimen, MPIF-1 may be administered to a human prior to the
administration of the chemotherapeutic agent(s). For example, MPIF-1 may be
administered two days before, one day before and the day of administration of
a
chemotherapeutic agent(s).
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When MPIF-l, and/or a variant thereof, is administered to a human for the
treatment of myeloproliferative disorders the dosages administered may be the
same as
when MPIF-1 is used as a myeloprotectant. When administered to a human for the
treatment of myeloproliferative disorders, MPIF-I may be administered
subcutaneously.
MPIF-1, M-CIF or MIP-4 polypeptide to be used for therapeutic administration
must be sterile. Sterility is readily accomplished by filtration through
sterile filtration
membranes (e. g. , 0.2 micron membranes). Therapeutic MPIF-1, M-CIF or MIP-4
polypeptide compositions generally are placed into a container having a
sterile access
port, for example, an intravenous solution bag or vial having a stopper
pierceable by a
hypodermic injection needle.
MPIF-l, M-CIF or MIP-4 polypeptide ordinarily will be stored in unit or multi-
dose containers, for example, sealed ampules or vials, as an aqueous solution
or as a
lyophilized formulation for reconstitution. As an example of a lyophilized
formulation,
10-ml vials are filled with 5 ml of sterile-filtered 1 % (w/v) aqueous MPIF-1,
M-C1F or
1 ~ MIP-4 polypeptide solution, and the resulting mixture is lyophilized. The
infusion
solution is prepared by reconstituting the lyophilized MPIF-1, M-CIF or MIP-4
polypeptide using bacteriostatic Water-for-Injection.
The invention aiso provides a pharmaceutical pack or kit comprising one or
more
containers filled with one or more of the ingredients of the pharmaceutical
compositions
of the invention. Associated with such containers) can be a notice in the form
prescribed by a governmental agency regulating the manufacture, use or sale of
pharmaceuticals or biological products, which notice reflects approval by the
agency of
manufacture, use or sale for human administration. In addition, the
polypeptides of the
present invention may be employed in conjunction with other therapeutic
compounds.
Modes of administration. It will be appreciated that conditions caused by a
decrease in the standard or normal level of MPIF-I, M-CIF or MIP-4 activity in
an
individual, can be treated by administration of MPIF-l, M-CIF or MIP-4
protein. Thus,
the invention further provides a method of treating an individual in need of
an increased
level of MPIF-1, M-CIF or MIP-4 activity comprising administering to such an
individual a pharmaceutical composition comprising an effective amount of an
isolated
MPIF-1, M-CIF or MIP-4 polypeptide of the invention, particularly a mature
form of
the MPIF-I, M-CIF or MIP-4, effective to increase the MPIF-1, M-CIF or MIP-4
activity level in such an individual.
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The amounts and dosage regimens of MPIF-1, MIP-4 and M-CIF administered
to a subject will depend on a number of factors such as the mode of
administration, the
nature of the condition being treated and the judgment of the prescribing
physician. The
pharmaceutical compositions are administered in an amount which is effective
for
treating and/or prophylaxis of the specific indication. In general, the
polypeptides will
be administered in an amount of at least about 10 ~cg/kg body weight and in
most cases
they will be administered in an amount not in excess of about 10 mg/kg body
weight per
day and preferably the dosage is from about 10 ~g/kg body weight daily, taking
into
account the routes of administration, symptoms, etc.
As a general proposition, the total pharmaceutical ly effective amount of MPIF-
1,
M-CIF or MIP-4 polypeptide administered parenterally per dose will more
preferably
be in the range of about 1 ~g/kg/day to 10 mg/kg/day of patient body weight,
although,
as noted above, this will be subject to therapeutic discretion. Even more
preferably, this
dose is at least 0.0l mg/kg/day, and most preferably for humans between about
0.01 and
1 S 1 mg/kg/day. If given continuously, the MPIF-1, M-CIF or MIP-4 polypeptide
is
typically administered at a dose rate of about 1 ug/kg/hour to about 50
ug/kg/hour,
either by 1-4 injections per day or by continuous subcutaneous infusions, for
example,
using a mini-pump. An intravenous bag solution may also be employed. The
length of
treatment needed to observe changes and the interval following treatment for
responses
to occur appears to vary depending on the desired effect.
Pharmaceutical compositions containing the MPIF-l, M-CIF or MIP-4 of the
invention may be administered orally, rectally, parenterally, intracistemally,
intravaginally, intraperitoneally, topically (as by powders, ointments, drops
or
transdermal patch), bucally, or as an oral or nasal spray.
Gene Therapy. The chemokine polypeptides, and agonists or antagonists which
are polypeptides, may be employed in accordance with the present invention by
expression of such polypeptides in vivo, which is often referred to as "gene
therapy."
Thus, for example, cells from a patient can be engineered with a
polynucleotide
(DNA or RNA) encoding a polypeptide ex vivv, with the engineered cells then
being
provided to a patient to be treated with the polypeptides. Such methods are
well-known
in the art. For example, cells can be engineered by procedures known in the
art by use
of a retroviral particle containing RNA encoding the polypeptides of the
present
invention.
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Similarly, cells can be engineered in vivo for expression of a polypeptides in
vivo
by, for example, procedures known in the art. As known in the art, a producer
cell for
producing a retroviral particle containing RNA encoding the polypeptides of
the present
invention can be administered to a patient for engineering the cells in vivo
and
S expression of the polypeptides in vivo. These and other methods for
administering
polypeptides of the present invention by such method should be apparent to
those skilled
in the art from the teachings of the present invention. For example, the
expression
vehicle for engineering cells can be other than a retrovirus, for example, an
adenovirus
which can be used to engineer cells in vivo after combination with a suitable
delivery
vehicle.
The retroviral plasmid vectors may be derived from retroviruses which include,
but are not limited to, Moloney Marine Sarcoma Virus, Moloney Marine Leukemia
Virus, spleen necrosis virus, Rous Sarcoma Virus and Harvey Sarcoma Virus.
In a preferred embodiment the retroviral expression vector, pMV-7, is flanked
by the long terminal repeats (LTRs) of the Moloney marine sarcoma virus and
contains
the selectable drug resistance gene neo under the regulation of the herpes
simplex virus
(HSV) thymidine kinase (tk) promoter. Unique EcoRI and HindIII sites
facilitate the
introduction of coding sequence (Kirschmeier, P.T. et al., DNA 7:219-25 (
1988)).
The vectors include one or more suitable promoters which include, but are not
limited to, the retroviral LTR; the SV40 promoter; and the human
cytomegalovirus
(CMV) promoter described in Miller, et al., l3iotechniques, Vol. 7, No. 9:980-
990
( 1989), or any other promoter (e.g. cellular promoters such as eukaryotic
cellular
promoters including, but not limited to, the histone, pol III, and p-actin
promoters). The
selection of a suitable promoter will be apparent to those skilled in the art
from the
teachings contained herein.
The nucleic acid sequence encoding the polypeptide of the present invention is
under the control of a suitable promoter which includes, but is not limited
to, viral
thymidine kinase promoters, such as the Herpes Simplex thymidine kinase
promoter;
retroviral LTRs, the ~3-actin promoter, and the native promoter which controls
the gene
encoding the polypeptide.
The retroviral plasmid vector is employed to transduce packaging cell lines to
form producer cell lines. Examples of packaging cells which may be transfected
include, but are not limited to, the PE501, PA317 and GP+aml2. The vector may
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transduce the packaging cells through any means known in the art. Such means
include,
but are not limited to, electroporation, the use of liposomes, and CaP04
precipitation.
The producer cell line generates infectious retroviral vector particles which
include the nucleic acid sequences) encoding the polypeptides. Such retroviral
vector
particles then may be employed, to transduce eukaryotic cells, either in vitro
or in vivo.
The transduced eukaryotic cells will express the nucleic acid sequences)
encoding the
polypeptide. Eukaryotic cells which may be transduced, include but are not
limited to,
fibroblasts and endothelial cells.
The present invention will be further described with reference to the
following
examples; however, it is to be understood that the present invention is not
limited to
such examples. All parts or amounts, unless otherwise specified, are by
weight.
In order to facilitate understanding of the following examples certain
frequently
occurring methods and/or terms will be described.
"Plasmids" are designated by a lower case p preceded and/or followed by
capital
letters and/or numbers. The starting plasmids herein are either commercially
available,
publicly available on an unrestricted basis, or can be constructed from
available
plasmids in accord with published procedures. In addition, equivalent plasmids
to those
described are known in the art and will be apparent to the ordinarily skilled
artisan.
"Digestion" of DNA refers to catalytic cleavage of the DNA with a restriction
enzyme that acts only at certain sequences in the DNA. The various restriction
enzymes
used herein are commercially available and their reaction conditions,
cofactors and other
requirements were used as would be known to the ordinarily skilled artisan.
For
analytical purposes, typically 1 pg of plasmid or DNA fragment is used with
about 2
units of enzyme in about 20 pl of buffer solution. For the purpose of
isolating DNA
fragments for plasmid construction, typically 5 to 50 ~g of DNA are digested
with 20
to 250 units of enzyme in a larger volume. Appropriate buffers and substrate
amounts
for particular restriction enzymes are specified by the manufacturer.
Incubation times
of about 1 hour at 37~C are ordinarily used, but can vary in accordance with
the
supplier's instructions. After digestion the reaction is electrophoresed
directly on a
polyacrylamide gel to isolate the desired fragment.
Size separation of the cleaved fragments is performed using 8 percent
polyacrylamide gel described by Goeddel, D. et al. , Nucleic Acids Res.,
8:4057 ( 1980).
"Oligonucleotides" refers to either a single stranded polydeoxynucleotide or
two
complementary polydeoxynucleotide strands which can be chemically synthesized.
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Such synthetic oligonucleotides have no 5' phosphate and thus will not ligate
to another
oligonucleotide without adding a phosphate with an ATP in the presence of a
kinase.
A synthetic oligonucleotide will ligate to a fragment that has not been
dephosphorylated.
"Ligation" refers to the process of forming phosphodiester bonds between two
double stranded nucleic acid fragments (Maniatis, T., et al., Id., p. l46).
Unless
otherwise provided, ligation can be accomplished using known buffers and
conditions
with 10 units to T4 DNA ligase ("ligase") per 0.5 ~g of approximately
equimolar
amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as described in the
method of Graham, F. and Van der Eb, A., Virology, 52:456-4S7 (I973).
Having now generally described the invention, the same will be more readily
understood through reference to the following example which is provided by way
of
illustration, and is not intended to be limiting of the present invention.
Exampl a 1
Bacterial Expression and Purification of MPIF 1
The DNA sequence encoding for MPIF-1, ATCC # 75676 is initially amplified
using PCR oligonucleotide primers corresponding to the 5' and sequences of the
processed MPIF-I protein (minus the signal peptide sequence) and the vector
sequences
3' to the MPIF-1 gene. Additional nucleotides corresponding to Bam HI and XbaI
were
added to the 5' and 3' sequences respectively. The 5' oligonucleotide primer
has the
sequence 5'-TCAGGATCCGTCACAAAAGATGCAGA-3' (SEQ ID N0:12) contains
a BamHI restriction enzyme site followed by I 8 nucleotides of MPIF-1 coding
sequence
starting from the presumed terminal amino acid of the processed protein codon.
The 3'
sequence 5'-CGCTCTAGAGTAAAACGACGGCCAGT-3' (SEQ ID N0:13) contains
complementary sequences to an XbaI site.
The restriction enzyme sites correspond to the restriction enzyme sites on the
bacterial expression vector pQE-9 {Qiagen, Inc. Chatsworth, CA). pQE-9 encodes
antibiotic resistance (Amp'), a bacterial origin of replication (ori), an IPTG-
regulatable
promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and
restriction
enzyme sites. pQE-9 is then digested with BamHI and XbaI. The amplified
sequences
are ligated into pQE-9 and are inserted in frame with the sequence encoding
for the
histidine tag and the RBS. The ligation mixture is then used to transform E.
coli strain
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M 15/rep4 available from Qiagen. M 1 S /rep4 contains multiple copies of the
plasmid
pREP4, which expresses the lacI repressor and also confers kanamycin
resistance (Kanr).
Transformants are identified by their ability to grow on LB plates and
ampicillin/kanamycin resistant colonies are selected. Plasmid DNA is isolated
and
S confirmed by restriction analysis overnight (O/N) in liquid culture in LB
media
supplemented with both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N culture is
used
to inoculate a large culture at a ratio of 1:100 to 1:250. The cells are grown
to an optical
density 600 (O.D.boo) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto
pyranoside") is then added to a final concentration of 1 mM. IPTG induces by
inactivating the lacI repressor, clearing the P/O leading to increased gene
expression.
Cells are grown an extra 3 to 4 hours. Cells are then harvested by
centrifugation. The
cell pellet is solubilized in the chaotropic agent 6 M Guanidine HC1. After
clarification,
solubilized MPIF-1 is purified from this solution by chromatography on a
Nickel-
Chelate column under conditions that allow for tight binding by proteins
containing the
6-His tag. Hochuli, E. et al., J. Chromatography 411:177-184 (l984). MPIF-1
(95%
pure) is eluted from the column in 6 M guanidine HC1 pH 5.0 and for the
purpose of
renaturation adjusted to 3 M guanidine HC1, 100 mM sodium phosphate, 10 mM
glutathione (reduced) and 2 mM glutathione (oxidized). After incubation in
this solution
for 12 hours the protein is dialyzed to 10 mM sodium phosphate.
Alternatively, the following non-tagged primers were used to clone the gene
into
plasmid pQE70:
5' primer: 5' CCC GCA TGC GGG TCA CAA AAG ATG CAG 3' (SEQ ID N0:14)
Sphl
3' primer: 5' AAA GG T T~,~ ATT CTT CCT GGT CTT 3' (SEQ ID NO:15)
BamHl Stop
Construction of E. coli optimized MPIF-1
In order to increase expression levels of MPIF-1 in an E. coli expression
system,
the codons of the gene were optimized to highly used E. coli codons. For the
synthesis
of the optimized region of MPIF-1, a series of 4 oligonucleotides were made:
mpif 1
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oligo numbers I-4 (set forth below). These overlapping oligos were used in a
PCR
reaction for seven rounds at the following conditions:
Denaturation 95 degrees 20 seconds
Annealing 58 degrees 20 seconds
Extension 72 degrees 60 seconds
Following the seven rounds of synthesis, a 5' primer to this region, (ACA T
ATG CGU GUU ACC AAA GAC GCU GAA ACC GAA UUC AUG AUG UCC (SEQ
ID N0:16)) and a 3' primer to this entire region, (GCC CAA GCT TTC AGT TTT TAC
GGG TTT TGA TAC GGG (SEQ ID N0:17)), were added to a PCR reaction,
I 0 containing 1 microliter from the initial reaction of the six
oligonucleotides. This product
was amplified for 30 rounds using the following conditions:
Denaturation 95 degrees 20 seconds
Annealing 55 degrees 20 seconds
Extension 72 degrees 60 seconds
The product produced by this final reaction was restricted with Sph I and
HindIII, and
cloned into pQE70, which was also cut with Sph I and HindIII. These clones
were
expressed and found to have superior expression levels that without the above
mutations.
mpif oligo number 1:
5' GCA TGC GUG UUA CCA AAG ACG CUG AAA CCG AAU UCA UGA UGU
CCA AAC UGC CGC UGG AAA ACC CGG UUC UGC UGG ACC GUU UCC ACG
C 3' (SEQ ID N0:18)
mpif 1 oligo number 2:
5' GCU GGA AUC CUA CUU CGA AAC CAA CUC CGA AUG CUC CAA ACC
GGG UGU UAU CUU CCU GAC CAA AAA AGG UCG UCG UUU CUG CGC UAA
CCC GUC CGA CAA ACA GG 3' (SEQ ID N0:19)
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mpifl oligo number 3:
S' AAG CTT TCA GTT TTT ACG GGT TTT GAT ACG GGT GTC CAG TTT CAG
CAT ACG CAT ACA AAC CTG AAC CTG TTT GTC GGA CGG GTT AGC GC 3'
(SEQ ID N0:20)
mpif 1 oligo number 4:
5' GGT TTC GAA GTA GGA TTC CAG CAG GGA GCA CGG GAT GGA ACG
CGG GGT GTA GGA GAT GCA GCA GTC AGC GGA GGT AGC GTG GAA ACG
GTC CAG C 3' (SEQ ID N0:21 )
Construction of MPIF 1 Deletion Mutants
Deletion mutants were constructed from the 5' terminus of the MPIF-1 gene
using the E. coli optimized MPIF-1 construct set forth above. The primers used
to
construct the 5' deletions are set forth below. The PCR amplification was
performed as
set forth above for the E coli optimized MPIF-1 construct. The products for
the Delta
17-A qe6, Delta 23, Delta 28 were restricted with NcoI for the 5' site and
HindIII for the
3' site and cloned into plasmid pQE60 that was digested with NcoI and HindIII.
All
other products were restricted with Sph1 for the 5' site and HindIII for the
3' site and
cloned into plasmid pQE70 that was digested with Sphl and HindIIl.
The 5' primers used are as follows:
Delta 17-A qe6 (pQE60)
5' NcoI gc gca g cca~g as aac ccg gtt ctg ctg gac 3' (SEQ ID N0:22)
The resulting amino acid sequence of this deletion mutant:
MENPVLLDRFHATSADCCISYTPRSIPCSLLESYFE'I'NSECSKPGVIFLTKKGR
RFCANPSDKQVQVCMRMLKLDTRIKTRKN (SEQ ID N0:23)
Delta 16-A qe7 (pQE70)
5' Sphl gc cat g gc~c tg gaa aac ccg gtt ctg ctg gac (SEQ ID N0:24)
The resulting amino acid sequence of this deletion mutant:
MLENPVLLDRFHATSADCCISYTPRSIPCSLLESYFETNSECSh.PGVIFLTKKG
RRFCANPSDKQVQVCMRMLKLDTRIKTRKN (SEQ ID N0:25)
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Delta 23 (pQE60)
5' NcoI gc gca g cc~gg ac cgt ttc cac get acc tcc (SEQ ID N0:26)
The resulting amino acid sequence of this deletion mutant:
MDRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRFCANP
SDKQVQVCMRMLKLDTRIKTRKN (SEQ ID N0:27)
Delta 24 (pQE70)
5' SphI gcc atg gca~c gtt tcc acg cta cct cc (SEQ ID N0:28)
The resulting amino acid sequence of this deletion mutant:
MRFHATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRFCANPS
DKQVQVCMRMLKLDTRIKTRKN (SEQ ID N0:8)
Delta 28 (pQE60)
5' NcoI gcg cag cc~gg cta cct ccg cig act get gc (SEQ ID N0:29)
The resulting amino acid sequence of this deletion mutant:
MATSADCCISYTPRSIPCSLLESYFETNSECSKPGVIFLTKKGRRFCANPSDKQ
VQVCMRMLKLDTRIKTRKN (SEQ ID N0:30)
S70 to A mutant (Ser at position 70 was mutated to Ala) (pQE70)
antisense ttc gaa gta ggc ttc cag cag (SEQ ID N0:31 )
sense ctg ctg gaa gcc tac ttc gaa (SEQ ID N0:32)
5' Sphl full gcc atg gc~c gtg tta cca aag acg ctg aaa cc (SEQ ID N0:33)
The resulting amino acid sequence of this deletion mutant:
MRVTKDAETEFMMSKLPLENPVLLDRFHATSADCCISYTPRSIPCSLLEaYFE
TNSECSKPGVIFLTKKGRRFCANPSDKQVQVCMRMLKLDTRIKTRKN (SEQ ID
N0:34)
The 3' primer used for all constructs:
3' Hind III
gcc c aagctt ~ gt ttt tac ggg ttt tga tac ggg (SEQ ID N0:35}
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The full length MPIF-1 sequence (from E. coli biased nt's)
MRVTKDAETEFMMSKLPLENPVLLDRFHATSADCCISYTPRSIPCSLLESYFE
TNSECSKPGVIFLTKKGRRFCANPSDKQVQVCMRMLKLDTRIKTRKN (SEQ ID
N0:7)
Example 2
Bacterial Expression and Purification of MIP 4
The DNA sequence encoding for MIP-4 ATCC # 75675 was initially amplified
using PCR oligonucleotide primers corresponding to the 5' sequences of the
processed
MIP-4 protein (minus the signal peptide sequence). Additional nucleotides
corresponding to Bam HI and XbaI were added to the 5' and 3' sequences
respectively.
The 5' oligonucleotide primer has the sequence S'-TCAGGATCCTGTGCACAAGT
TGGTACC -3' (SEQ ID N0:36) contains a BarnHI restriction enzyme site followed
by
18 nucleotides of MIP-4 coding sequence starting from the presumed terminal
amino
acid of the processed protein codon; The 3' sequence 5'-CGCTCTAGAGTAAAACG
ACGGCCAGT-3' (SEQ ID N0:13) contains complementary sequences to an XbaI site.
The restriction enzyme sites correspond to the restriction enzyme sites on the
bacterial expression vector pQE-9 (Qiagen, Inc., Chatsworth, CA). pQE-9
encodes
antibiotic resistance (Amp'), a bacterial origin of replication (ori), an IPTG-
regulatable
promoter operator (P/O), a ribosome binding site (RBS), a 6-His tag and
restriction
enzyme sites. pQE-9 was then digested with BamHI and XbaI The amplified
sequences
were ligated into pQE-9 and were inserted in frame with the sequence encoding
for the
histidine tag and the RBS. The ligation mixture was then used to transform E.
coli
strain 1 S/rep4 available from Qiagen. M 1 S/rep4 contains multiple copies of
the plasmid
pREP4, which expresses the lacl repressor and also confers kanamycin
resistance (Kan').
Transformants are identified by their ability to grow on LB plates and
ampicillin/kanamycin resistant colonies were selected. Plasmid DNA was
isolated and
confirmed by restriction analysis. Transformants are identified by their
ability to grow
on LB plates and ampicillin/kanamycin resistant colonies were selected.
Plasmid DNA
was isolated and confirmed by restriction analysis. Clones containing the
desired
constructs were grown overnight (O/N) in liquid culture in LB media
supplemented with
both Amp ( 100 ug/mI) and Kan (25 ug/ml). The O/N culture is used to inoculate
a large
culture at a ratio of 1:l00 to 1:250. The cells were grown to an optical
density 600
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(O.D.~~~) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto
pyranoside") was
then added to a final concentration of 1 mM. IPTG induces by inactivating the
lacI
repressor, clearing the P/O leading to increased gene expression. Cells were
grown an
extra 3 to 4 hours. Cells were then harvested by centrifugation. The cell
pellet was
solubilized in the chaotropic agent 6 M Guanidine HCI. After clarification,
solubilized
MIP-4 was purified from this solution by chromatography on a Nickel-Chelate
column
under conditions that allow for tight binding by proteins containing the 6-His
tag.
Hochuli, E. et al., J. Chromatography 411:177-184 (1984). MIP-4 (95% pure) was
eluted from the column in 6 M guanidine HCI pH 5.0 and for the purpose of
renaturation
adjusted to 3 Mr guanidine HCI, 100 mM sodium phosphate, 10 mM glutathione
(reduced) and 2 mM glutathione (oxidized). After incubation in this solution
for 12
hours the protein was dialyzed to 10 mM sodium phosphate.
Alternatively, the following non-tagged primers were used to clone the gene
into
plasmid pQE60:
5' AAA AAG CTT TCA GGC ATT CAG CTT CAG 3' (SEQ ID N0:37) pQE60
HindlIl (3' primer)
5' AAA CCA TGG CAC AAG TTG GTA CCA AC 3' (SEQ ID N0:38) pQE60
NcoI (5' primer)
Example 3
Bacterial Expression and Purification of M-CIF
The DNA sequence encoding for M-CIF (ATCC # 75572) is initially amplified
using PCR oligonucleotide primers corresponding to the 5' and 3' sequences of
the
processed M-CIF protein (minus the signal peptide sequence) and additional
nucleotides
corresponding to Bam HI and XbaI were added to the 5' and 3' sequences
respectively.
The 5' oligonucleotide primer has the sequence 5'-GCCCGCGGATCCTCCTCACG
GGGACCTTAC-3' (SEQ ID N0:39) contains a BamHI restriction enzyme site followed
by 15 nucleotides of M-CIF coding sequence starting from the presumed terminal
amino
acid of the processed protein codon; The 3' sequence 5'-GCCTGCTCTAGATCAAAG-
CAGGGAAGCTCCAG-3' (SEQ ID N0:40) contains complementary sequences to XbaI
site a translation stop codon and the last 20 nucleotides of M-CIF coding
sequence.
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The restriction enzyme sites correspond to the restriction enzyme sites on the
bacterial expression vector pQE-9. (Qiagen, Inc. 9259 Eton Avenue, Chatsworth,
CA,
91311 ). pQE-9 encodes antibiotic resistance (Ampr), a bacterial origin of
replication
(ori), an IPTG-regulatable promoter operator (P/O), a ribosome binding site
(RBS), a 6-
His tag and restriction enzyme sites. pQE-9 was then digested with BamHI and
XbaI.
The amplified sequences were ligated into pQE-9 and were inserted in frame
with the
sequence encoding for the histidine tag and the RBS. Figwe 6 shows a schematic
representation of this arrangement. The ligation mixture was then used to
transform E.
col i strain available from Qiagen under the trademark M 15/rep4. M 1 S/rep4
contains
multiple copies of the plasmid pREP4, which expresses the lacI repressor and
also
confers kanamycin resistance (Kan'). Transformants are identified by their
ability to
grow on LB plates and ampicillin/kanamycin resistant colonies were selected.
Plasmid
DNA was isolated and confirmed by restriction analysis. Clones containing the
desired
constructs were grown overnight (O/N) in liquid cultwe in LB media
supplemented with
both Amp (100 ug/ml) and Kan (25 ug/ml). The O/N cultwe is used to inoculate a
large
culture at a ratio of 1:100 to 1:250. The cells were grown to an optical
density 600
(O.D.6~~) of between 0.4 and 0.6. IPTG ("Isopropyl-B-D-thiogalacto
pyranoside") was
then added to a final concentration of 1 mM. IPTG induces by inactivating the
lacI
repressor, clearing the P/O leading to increased gene expression. Cells were
grown an
extra 3 to 4 bows. Cells were then harvested by centrifugation. The cell
pellet was
solubilized in the chaotropic agent 6 Molar Guanidine HC1. After
clarification,
solubilized M-CIF was purified from this solution by chromatography on a
Nickel-
Chelate column under conditions that allow for tight binding by proteins
containing the
6-His tag Hochuli, E. et al., J. Chromatography 41l: l 77-184 ( 1984). M-CIF
(95%
pwe) was eluted from the column in 6 M guanidine HC1 pH 5.0 and for the
purpose of
renaturation adjusted to 3 M guanidine HCI) 100 mM sodium phosphate, 10 mM
glutathione (reduced) and 2 mM glutathione (oxidized). After incubation in
this solution
for 12 hows the protein was dialyzed to 10 mM sodium phosphate. The presence
of a
new protein corresponding to 14 kDa following induction demonstrated
expression of
the M-CIF (Figwe 7).
Alternatively, the following non-tagged primers were used to insert the gene
into
plasmid pQE60:
S' primer: 5' AAA TC T A CCA AGA CTG AAT CCT CCT 3' (SEQ ID N0:41 )
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BspHI
3' primer: 5' AAA AAG CTT TCA GTT CTC CTT CAT GTC 3' (SEQ ID N0:42)
HindIII
Example 4
Most of the vectors used for the transient expression of the MPIF-1, M-CIF or
MIP-4 protein gene sequence in mammalian cells should carry the SV40 origin of
replication. This allows the replication of the vector to high copy numbers in
cells (e.g.,
COS cells) which express the T antigen required for the initiation of viral
DNA
synthesis. Any other mammalian cell line can also be utilized for this
purpose.
A typical mammalian expression vector contains the promoter element) which
mediates the initiation of transcription of mRNA, the protein coding sequence,
and
signals required for the termination of transcription and polyadenylation of
the
transcript. Additional elements include enhancers, Kozak sequences and
intervening
sequences flanked by donor and acceptor sites for RNA splicing. Highly
efficient
1 S transcription can be achieved with the early and late promoters from SV40,
the long
terminal repeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the
early
promoter of the cytomegalovirus (CMV). However, cellular signals can also be
used
(e.g., human actin promoter). Suitable expression vectors for use in
practicing the
present invention include, for example, vectors such as pSVL and pMSG
(Pharmacia,
Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC 12MI
(ATCC 67109). Mammalian host cells that could be used include, human HeLa,
283,
H9 and Jurkari cells, mouse NIH3T3 and C127 cells, Cos l, Cos 7 and CV1,
African
green monkey cells, quail QC 1-3 cells, mouse L cells and Chinese hamster
ovary cells.
Alternatively, the gene can be expressed in stable cell lines that contain the
gene
integrated into a chromosome. The eo-transfection with a selectable marker
such as dhfr,
gpt, neomycin, hygromycin allows the identification and isolation of the
transfected
cells.
The transfected gene can also be amplified to express large amounts of the
encoded protein. The DHFR (dihydrofolate reductase) is a useful marker to
develop cell
lines that carry several hundred or even several thousand copies of the gene
of interest.
Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy
et al.,
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Biochem J. 227:277-279 (1991); Bebbington et al., BiolTechnology 10:169-175
(1992)).
Using these markers, the mammalian cells are grown in selective medium and the
cells
with the highest resistance are selected. These cell lines contain the
amplified genes)
integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used
for
the production of proteins.
The expression vectors pC 1 and pC4 contain the strong promoter (LTR) of the
Rous Sarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447
(March,
1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell 41:52l-S30
(l985)).
Multiple cloning sites, e.g., with the restriction enzyme cleavage sites
BamHI, Xbal and
Asp718, facilitate the cloning of the gene of interest. The vectors contain in
addition the
3' intron, the polyadenylation and termination signal of the rat preproinsulin
gene.
A. Expression ojRecombinant MPIF 1 in COS cells
The expression of plasmid, CMV-MPIF-1 HA is derived from a vector
pcDNAI/Amp (Invitrogen) containing: 1 ) SV40 origin of replication, 2)
ampicillin
resistance gene, 3) E. coli replication origin, 4) CMV promoter followed by a
polylinker
region, a SV40 intron and polyadenylation site. A DNA fragment encoding the
entire
MPIF-I precursor and a HA tag fused in frame to its 3' end is cloned into the
polylinker
region of the vector, therefore, the recombinant protein expression is
directed under the
CMV promoter. The HA tag correspond to an epitope derived from the influenza
hemagglutinin protein as previously described (Wilson, H., et al., Cell 3
7:767 ( 1991 )).
The infusion of HA tag to our target protein allows easy detection of the
recombinant
protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence, ATCC # 75676, encoding for MPIF-1 is constructed by
2S PCR on the original EST cloned using two primers: the 5' primer 5'-
GGAAAGCTTATGAAGGTCTCCGTGGCT-3' (SEQ ID N0:43) contains a HindIII
site followed by 18 nucleotides of MPIF-1 coding sequence starting from the
initiation
codon; the 3' sequence 5'-CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTA-
TGGGTAATTCTTCCTGGTCTTGATCC-3' (SEQ ID N0:44) contains complementary
sequences to Xba I site, translation stop codon, HA tag and the last 20
nucleotides of the
MPIF-1 coding sequence (not including the stop codon). Therefore, the PCR
product
contains a HindIII site, MPIF-1 coding sequence followed by HA tag fused in
frame, a
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translation termination stop codon next to the HA tag, and an XbaI site. The
PCR
amplified DNA fragment and the vector, pcDNAI/Amp, are digested with HindIII
and
XbaI restriction enzyme and ligated. The ligation mixture is transformed into
E. col i
strain SURE (available from Stratagene Cloning Systems, 1 l099 North Torrey
Pines
Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin
media plates
and resistant colonies are selected. Plasmid DNA is isolated from
transformants and
examined by restriction analysis for the presence of the correct fragment. For
expression
of the recombinant MPIF-1, COS cells are transfected with the expression
vector by
DEAR-DEXTRAN method. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning:
A Laboratory Manual, Cold Spring Laboratory Press, ( 1989)). The expression of
the
MPIF-1-HA protein is detected by radiolabelling and immunoprecipitation
method. (E.
Harlow, D. Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor
Laboratory
Press, (1988)). Cells are labeled for 8 hours with 35S-cysteine two days post
transfection. Culture media are then collected and cells are lysed with
detergent (RIPA
buffer ( 150 mM NaCI, 1 % NP-40, 0.1 % SDS, 1 % NP-40, 0.5% DOC, 50mM Tris, pH
7.5). (Wilson, I. et al., Id. 37:767 (1984)). Both-cell lysate and culture
media are
precipitated with a HA specific monoclonal antibody. Proteins precipitated are
analyzed
on 15% SDS-PAGE gels.
B. Cloning and Expression in CHO Cells
The vector pC 1 is used for the expression of MPIF-1 protein. Plasmid pC 1 is
a
derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). Both plasmids
contain the mouse DHFR gene under control of the SV40 early promoter. Chinese
hamster ovary- or other cells lacking dihydrofolate activity that are
transfected with
these plasmids can be selected by growing the cells in a selective medium
(alpha minus
MEM, Life Technologies) supplemented with the chemotherapeutic agent
methotrexate.
The amplification of the DHFR genes in cells resistant to methotrexate (MTX)
has been
well documented (see, e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and
Schimke, R.T.,
1978, J. Biol. Chem. 253:1357-1370, Hamlin, J.L. and Ma, C. l990, Biochem. et
Biophys. Acta, 1097:107-143, Page, M.J. and Sydenham, M.A. 1991, Biotechnology
Vol. 9:64-68). Cells grown in increasing concentrations of MTX develop
resistance to
the drug by overproducing the target enzyme, DHFR, as a result of
amplification of the
DHFR gene. If a second gene is linked to the DHFR gene it is usually co-
amplified and
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over-expressed. It is state of the art to develop cell lines carrying more
than 1,000
copies of the genes. Subsequently, when the methotrexate is withdrawn, cell
lines
contain the amplified gene integrated into the chromosome(s).
Plasmid pC 1 contains for the expression of the gene of interest a strong
promoter
S of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al.
, Molecular
and Cellular Biology, March 1985:438-4470) plus a fragment isolated from the
enhancer
of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al.,
Cell
41:521-530, l985). Downstream of the promoter are the following single
restriction
enzyme cleavage sites that allow the integration of the genes: BamHI, followed
by the
3' intron and the polyadenylation site of the rat preproinsulin gene. Other
high efficient
promoters can also be used for the expression, e.g., the human ~i-actin
promoter, the
SV40 early or late promoters or the long terminal repeats from other
retroviruses, e.g.,
HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g., from
the
human growth hormone or globin genes can be used as well.
I S Stable cell lines carrying a gene of interest integrated into the
chromosomes can
also be selected upon co-transfection with a selectable marker such as gpt,
G418 or
hygromycin. It is advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
The plasmid pCl is digested with the restriction enzyme BamHI and then
dephosphorylated using calf intestinal phosphates by procedures known in the
art. The
vector is then isolated from a 1 % agarose gel.
The DNA sequence encoding MPIF-1, ATCC No. 75676, is amplified using
PCR oligonucleotide primers corresponding to the 5' and 3' sequences of the
gene:
The 5' primer has the sequence:
S' AAA GGA TCC GCC ACC ATG AAG GTC TCC GTG GTC 3'
BamHl KOZAK
(SEQ ID N0:45) containing the underlined BamH 1 restriction enzyme site and a
portion
of the sequence encoding the MPIF-1 protein of Figure 1 (SEQ ID N0:3).
Inserted into
an expression vector, as described below, the 5' end of the amplified fragment
encoding
human MPIF-1 provides an efficient signal peptide. An efficient signal for
initiation
of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol.
196:947-950
(l987) is appropriately located in the vector portion of the construct.
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The 3' primer has the sequence:
5' AAA GGA TCC TCA ATT CTT CCA GGT CTT 3'
BamHI Stop
(SEQ ID N0:46) containing the Asp718 restriction site and a portion of
nucleotides
S complementary to the MPIF-1 coding sequence set out in Figure 1 (SEQ ID
N0:3),
including the stop codon.
The amplified fragments are isolated fiom a 1% agarose gel as described above
and then digested with the endonucleases BamHI and Asp718 and then purified
again
on a 1 % agarose gel.
The isolated fragment and the dephosphorylated vector are then ligated with T4
DNA ligase. E. coli HB 1 O 1 cells are then transformed and bacteria
identified that
contained the plasmid pC 1 inserted in the correct orientation using the
restriction
enzyme BamHI. The sequence of the inserted gene is confirmed by DNA
sequencing.
Transfection of CHO-DHFR-cells
Chinese hamster ovary cells lacking an active DHFR enzyme are used for
transfection. 5 ~tg of the expression plasmid C 1 are cotransfected with 0.5
~tg of the
plasmid pSVneo using the lipofecting method (Felgner et al., supra). The
plasmid
pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding
an
enzyme that confers resistance to a group of antibiotics including G418. The
cells are
seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the
cells
are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and
cultivated from 10-14 days. After this period, single clones are trypsinized
and then
seeded in 6-well petri dishes using different concentrations of methotrexate
(25 nM, 50
nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of
methotrexate are then transferred to new 6-well plates containing even higher
concentrations of methotrexate (500 nM, 1 ~tM, 2 uM, 5 ~tM). The same
procedure is
repeated until clones grow at a concentration of 100 ~tM.
The expression of the desired gene product is analyzed by Western blot
analysis
and SDS-PAGE.
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Example 5
A. Expression of Recombinant MIP 4 in COS cells
The expression of plasmid, CMV-MIP-4 HA is derived from a vector
pcDNAI/Amp (Invitrogen) containing: 1 ) SV40 origin of replication, 2)
ampicillin
resistance gene, 3) E. coli replication origin, 4) CMV promoter followed by a
polylinker
region, a SV40 intron and polyadenylation site. A DNA fragment encoding the
entire
MIP-4 precursor and a HA tag fused in frame to its 3' end is cloned into the
polylinker
region of the vector, therefore, the recombinant protein expression is
directed under the
CMV promoter. The HA tag correspond to an epitope derived from the influenza
hemagglutinin protein as previously described (Wilson, H., et al.) Cell 37:767
( 1984)).
The infusion of HA tag to the target protein allows easy detection of the
recombinant
protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence ATCC No. 75675 encoding for MIP-4 is constructed by PCR
using two primers: the 5' primer:
5'- GGAAAGCTTATGAAGGGCCTTGCAGCTGCC-3' (SEQ ID N0:47) contains a
HindIII site followed by 20 nucleotides of MIP-4 coding sequence starting from
the
initiation codon; the 3' sequence 5'-CGCTCTAGATCAABCGTAGTCTGGGACGT
CGTATGGGTAGGCATTCAGCTTCAGGTC-3' (SEQ ID N0:48) contains
complementary sequences to Xba I site, translation stop codon, HA tag and the
last 19
nucleotides of the MIP-4 coding sequence (not including the stop codon).
Therefore,
the PCR product contains a HindIII site, MIP-4 coding sequence followed by HA
tag
fused in frame, a translation termination stop codon next to the HA tag, and
an XbaI site.
The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with
HindIII and XbaI restriction enzyme and ligated. The ligation mixture is
transformed
into E. coli strain SURE (available from Stratagene Cloning Systems, La Jolla,
CA) the
transformed culture is plated on ampicillin media plates and resistant
colonies are
selected. Plasmid DNA is isolated from transformants and examined by
restriction
analysis for the presence of the correct fragment. For expression of the
recombinant
MIP-4, COS cells are transfected with the expression vector by DEAE-DEXTRAN
method. (J. Sambrook, E. Fritsch, T. Maniatis, Molecular Cloning: A Laboratory
Manual, Cold Spring Laboratory Press, ( 1989)). The expression of the MIP-4-HA
protein is detected by radiolabelling and immunoprecipitation method. (E.
Harlow, D.
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Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
(1988)).
Cells are labelled for 8 hours with 35S-cysteine two days post transfection.
Culture
media are then collected and cells are lysed with detergent (RIPA buffer (150
mM NaCI,
1 % NP-40, 0.1 % SDS, 1 % NP-40, 0.5% DOC, SOmM Tris, pH 7.5). (Wilson, H., et
al.,
S Cell 37:767 (l984)). Both cell lysate and culture media are precipitated
with a HA
specific monoclonal antibody. Proteins precipitated are analyzed on 15% SDS-
PAGE
gels.
B. Cloning and Expression in CHO Cells
The vector pC 1 is used for the expression of MIP-4 protein. Plasmid pC 1 is a
derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). Both plasmids
contain the mouse DHFR gene under control of the SV40 early promoter. Chinese
hamster ovary- or other cells lacking dihydrofolate activity that are
transfected with
these plasmids can be selected by growing the cells in a selective medium
{alpha minus
MEM, Life Technologies) supplemented with the chemotherapeutic agent
methotrexate.
The amplification of the DHFR genes in cells resistant to methotrexate (MTX)
has been
well documented (see, e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and
Schimke, R.T.,
1978, J. Biol. Chem. 2S3:1357-l370, Hamlin, J.L. and Ma, C. 1990, Biochem. et
Biophys. Acta, 1097:I07-143, Page, M.J. and Sydenham, M.A. 199l, Biotechnology
Vol. 9:64-68). Cells grown in increasing concentrations of MTX develop
resistance to
the drug by overproducing the target enzyme, DHFR, as a result of
amplification of the
DHFR gene. If a second gene is linked to the DHFR gene it is usually co-
amplified and
over-expressed. It is state of the art to develop cell lines carrying more
than 1,000
copies of the genes. Subsequently, when the methotrexate is withdrawn, cell
lines
contain the amplified gene integrated into the chromosome(s).
Plasmid pC 1 contains for the expression of the gene of interest a strong
promoter
of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al.,
Molecular
and Cellular Biology, March 1985:438-4470) plus a fragment isolated from the
enhancer
of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al.,
Cell
4l:521-530, l985). Downstream of the promoter are the following single
restriction
enzyme cleavage sites that allow the integration of the genes: BatnHI,
followed by the
3' intron and the polyadenylation site of the rat preproinsulin gene. Other
high efFcient
promoters can also be used for the expression, e.g., the human (3-actin
promoter, the
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5V40 early or late promoters or the long terminal repeats from other
retroviruses, e. g. ,
HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g., from
the
human growth hormone or globin genes can be used as well.
Stable cell lines carrying a gene of interest integrated into the chromosomes
can
also be selected upon co-transfection with a selectable marker such as gpt,
G418 or
hygromycin. It is advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
The plasmid pCl is digested with the restriction enzyme BamHI and then
dephosphorylated using calf intestinal phosphates by procedures known in the
art. The
vector is then isolated from a 1 % agarose gel.
The DNA sequence encoding MIP-4, ATCC No. 7567S, is amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
The 5' primer has the sequence:
5' AAA GGA TCC GCC ACC ATG AAG GGC CTT GCA AGC 3'
1 S BamH1 KOZAK
(SEQ ID N0:49) containing the underlined BamH 1 restriction enzyme site and a
portion
of the sequence encoding the MIP-4 protein of Figure 3 (SEQ ID NO:S). Inserted
into
an expression vector, as described below) the 5' end of the amplified fragment
encoding
human MIP-4 provides an efficient signal peptide. An efficient signal for
initiation of
translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol.
l96:947-950
(1987) is appropriately located in the vector portion of the construct.
The 3' primer has the sequence:
5' AAA GGA TCC ~ GGC ATI~ CAG CTI~ CAG 3'
BamH1 Stop
(SEQ ID NO:50) containing the Asp718 restriction site followed by nucleotides
complementary to a portion of the MIP-4 coding sequence set out in Figure 3
(SEQ ID
NO:S), including the stop codon.
The amplified fragments are isolated from a 1 % agarose gel as described above
and then digested with the endonucleases BamHI and Asp718 and then purified
again
on a 1% agarose gel.
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The isolated fragment and the dephosphorylated vector are then ligated with T4
DNA ligase. E. coli HB101 cells are then transformed and bacteria identified
that
contained the plasmid pC 1 inserted in the correct orientation using the
restriction
enzyme BamHI. The sequence of the inserted gene is confirmed by DNA
sequencing.
S Transfection of CHO-DHFR-cells
Chinese hamster ovary cells lacking an active DHFR enzyme are used for
transfection. Five gg of the expression plasmid C 1 are cotransfected with 0.5
pg of the
plasmid pSVneo using the lipofecting method (Felgner et al., supra). The
plasmid
pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding
an
enzyme that confers resistance to a group of antibiotics including G418. The
cells are
seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the
cells
are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and
cultivated from 10-14 days. After this period, single clones are trypsinized
and then
seeded in 6-well petri dishes using different concentrations of methotrexate
(25 nM, SO
nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of
methotrexate are then transferred to new 6-well plates containing even higher
concentrations of methotrexate (500 nM, 1 ~M, 2 ~M, 5 pM). The same procedure
is
repeated until clones grow at a concentration of 100 ~M.
The expression of the desired gene product is analyzed by Western blot
analysis
and SDS-PAGE.
Example 6
A. Expression of Recombinant M CIF in COS cells
The expression of plasmid, CMV-M-CIF HA is derived from a vector
pcDNAI/Amp (Invitrogen) containing: 1 ) SV40 origin of replication, 2)
ampicillin
resistance gene, 3) E. coli replication origin, 4) CMV promoter followed by a
polylinker
region, a SV40 intron and polyadenylation site. A DNA fragment encoding the
entire
M-CIF precursor and a HA tag fused in frame to its 3' end was cloned into the
polylinker
region of the vector, therefore, the recombinant protein expression is
directed under the
CMV promoter. The HA tag correspond to an epitope derived from the influenza
hemagglutinin protein as previously described (Wilson, H., et al., Cell 37:767
(1984)).
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The infusion of HA tag to our target protein allows easy detection of the
recombinant
protein with an antibody that recognizes the HA epitope.
The plasmid construction strategy is described as follows:
The DNA sequence encoding for M-CIF, ATCC # 75572, was constructed by
PCR using two primers: the S' primer 5'- GGAAAGCTTATGAAGATTCCGT
GGCTGC-3' (SEQ ID NO:S 1 ) contains a HindIII site followed by 20 nucleotides
of M-
CIF coding sequence starting from the initiation codon; the 3' sequence S'-CGC
TCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAGTTCTCCTTCATGTCC-
TTG -3' (SEQ ID N0:52) contains complementary sequences to Xba I site,
translation
stop codon, HA tag and the last 19 nucleotides of the M-CIF coding sequence
(not
including the stop codon). Therefore, the PCR product contains a HindIII site,
M-CIF
coding sequence followed by HA tag fused in frame, a translation termination
stop
codon next to the HA tag, and an XbaI site. The PCR amplified DNA fragment and
the
vector, pcDNAI/Amp, were digested with HindIII and XbaI restriction enzyme and
ligated. The ligation mixture was transformed into E. coli strain SURE
(Stratagene
Cloning Systems, La Jolla, CA) the transformed culture was plated on
ampicillin media
plates and resistant colonies were selected. Plasmid DNA was isolated from
transformants and examined by restriction analysis for the presence of the
correct
fragment. For expression of the recombinant M-CIF, COS cells were transfected
with
the expression vector by DEAE-DEXTRAN method. (J. Sambrook, E. Fritsch, T.
Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Laboratory
Press,
( 1989)). The expression of the M-CIF-HA protein was detected by
radiolabelling and
immunoprecipitation method. (E. Harlow, D. Lane, Antibodies: A Laboratory
Manual,
Cold Spring Harbor Laboratory Press, (1988)). Cells were labelled for 8 hours
with'SS-
cysteine two days post transfection. Culture media were then collected and
cells were
lysed with detergent (RIPA buffer ( 150 mM NaCI, 1 % NP-40, 0.1 % SDS, 1 % NP-
40,
0.5% DOC, SOmM Tris, pH 7.5). (Wilson, H., et al., Cell 37:767 (1984)). Both
cell
lysate and culture media were precipitated with a HA specific monoclonal
antibody.
Proteins precipitated were analyzed on 15% SDS-PAGE gels.
B. Cloning and Expression in CHD Cells
The vector pC 1 is used for the expression of M-CIF protein. Plasmid pC 1 is a
derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). Both plasmids
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WO 98I14582 PCT/US97/17505
contain the mouse DHFR gene under control of the SV40 early promoter. Chinese
hamster ovary- or other cells lacking dihydrofolate activity that are
transfected with
these plasmids can be selected by growing the cells in a selective medium
(alpha minus
MEM, Life Technologies) supplemented with the chemotherapeutic agent
methotrexate.
The amplification of the DHFR genes in cells resistant to methotrexate (MTX)
has been
well documented (see, e.g., Alt, F.W., Kellems, R.M., Bertino, J.R., and
Schimke, R.T.,
1978, J. Biol. Chem. 253:13S7-1370, Hamlin, J.L. and Ma, C. 1990, Biochem. et
Biophys. Acta, l097:107-143, Page, M.J. and Sydenham, M.A. l991, Biotechnology
Vol. 9:64-68). Cells grown in increasing concentrations of MTX develop
resistance to
the drug by overproducing the target enzyme, DHFR, as a result of
amplification of the
DHFR gene. If a second gene is linked to the DHFR gene it is usually co-
amplified and
over-expressed. It is state of the art to develop cell lines carrying more
than l,000
copies of the genes. Subsequently, when the methotrexate is withdrawn, cell
lines
contain the amplified gene integrated into the chromosome(s).
Plasmid pC 1 contains for the expression of the gene of interest a strong
promoter
of the long terminal repeat (LTR) of the Rouse Sarcoma Virus (Cullen, et al.,
Molecular
and Cellular Biology, March 1985:438-4470) plus a fragment isolated from the
enhancer
of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al.,
Cell
4I:521-530, 1985). Downstream of the promoter are the following single
restriction
enzyme cleavage sites that allow the integration of the genes: BamHI, followed
by the
3' intron and the polyadenylation site of the rat preproinsulin gene. Other
high efficient
promoters can also be used for the expression, e.g., the human (3-actin
promoter, the
SV40 early or late promoters or the long terminal repeats from other
retroviruses, e.g.,
HIV and HTLVI. For the polyadenylation of the mRNA other signals, e.g., from
the
human growth hormone or globin genes can be used as well.
Stable cell lines carrying a gene of interest integrated into the chromosomes
can
also be selected upon co-transfection with a selectable marker such as gpt,
G418 or
hygromycin. It is advantageous to use more than one selectable marker in the
beginning, e.g., G418 plus methotrexate.
The plasmid pC 1 is digested with the restriction enzyme BamHI and then
dephosphorylated using calf intestinal phosphates by procedures known in the
art. The
vector is then isolated from a 1% agarose gel.
The DNA sequence encoding M-CIF, ATCC No. 75572, is amplified using PCR
oligonucleotide primers corresponding to the 5' and 3' sequences of the gene:
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The 5' primer has the sequence:
5' AAA G A GQC ACC ATG AAG ATC TCC GTG GCT 3'
BamHI KOZAK
(SEQ ID N0:53) containing the underlined BamH 1 restriction enzyme site and
the
sequence of M-CIF of Figure 2 (SEQ ID NO: l ). Inserted into an expression
vector, as
described below, the 5' end of the amplified fragment encoding human M-CIF
provides
an efficient signal peptide. An efficient signal for initiation of translation
in eukaryotic
cells, as described by Kozak, M., J. Mol. Bivl. l96:947-9S0 ( 1987) is
appropriately
located in the vector portion of the construct.
The 3' primer has the sequence:
5' AAA GGA TCC ~ GTT CTC CTT CAT GTC CTT 3'
BamH1 Stop
(SEQ ID N0:54) containing the Asp718 restriction site and a portion of the M-
CIF
coding sequence set out in Figure 2 (SEQ ID NO: l ), including the stop codon.
The amplified fragments are isolated from a 1 % agarose gel as described above
and then digested with the endonucleases BamHI and Asp718 and then purified
again
on a 1 % agarose gel.
The isolated fragment and the dephosphorylated vector are then ligated with T4
DNA ligase. E. coli HB101 cells are then transformed and bacteria identified
that
contained the plasmid pC 1 inserted in the correct orientation using the
restriction
enzyme BamHI. The sequence of the inserted gene is confirmed by DNA
sequencing.
Transfection of CHO-DHFR-cells
Chinese hamster ovary cells lacking an active DHFR enzyme are used for
transfection. Five ~tg of the expression plasmid C 1 are cotransfected with
0.5 pg of the
plasrnid pSVneo using the lipofecting method (Felgner et al., supra). The
plasmid
pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding
an
enzyme that confers resistance to a group of antibiotics including G418. The
cells are
seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the
cells
are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and
cultivated from 10-14 days. After this period, single clones are trypsinized
and then
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seeded in 6-well petri dishes using different concentrations of methotrexate
(25 nM, 50
nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of
methotrexate are then transferred to new 6-well plates containing even higher
concentrations of methotrexate (500 nM, 1 ~M, 2 ~M, 5 pM). The same procedure
is
repeated until clones grow at a concentration of 100 pM.
The expression of the desired gene product is analyzed by Western blot
analysis
and SDS-PAGE.
Example 7
Expression pattern of M CIF in human tissue
Northern blot analysis was carried out to examine the levels of expression of
M-
CIF in human tissues. Total cellular RNA samples were isolated with RNAzoITM B
system (Biotecx Laboratories, Inc. Houston, TX). About 10 ug of total RNA
isolated
from each human tissue specified was separated on 1 % agarose gel and blotted
onto a
nylon filter. (Sambrook, Fritsch, and Maniatis, Molecular Cloning, A
Laboratory
1 S Manual, Cold Spring Harbor Press, ( 1989)). The labeling reaction was done
according
to the Stratagene Prime-It kit with 50 ng DNA fragment. The labeled DNA was
purified
with a Select-G-50 column. (5 Prime - 3 Prime, Inc., Boulder, CO). The filter
was then
hybridized with radioactive labeled full length M-CIF gene at l,000,000 cpm/ml
in 0.5
M NaP04, pH 7.4 and 7% SDS overnight at 65~C. After wash twice at room
temperature and twice at 60~C with 0.5 x SSC, 0.1% SDS, the filter was then
exposed
at -70~C overnight with an intensifying screen.
Example 8
Expression pattern of MPIF 1 in l:uman tissue
Northern blot analysis was carried out to examine the levels of expression of
MPIF-1 in human tissues. Total cellular RNA samples were isolated with
RNAzoIT"'
B system (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033).
About 10 ug of total RNA isolated fi om each human tissue specified is
separated on 1
agarose gel and blotted onto a nylon filter. (Sambrook, Fritsch, and Maniatis,
Molecular
Cloning, Cold Spring Harbor Press, (1989)). The labeling reaction is done
according
to the Stratagene Prime-It kit with SO ng DNA fragment. The labeled DNA is
purified
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WO 98I14582 PCT/US97l17505
with a Select-G-50 column. (5 Prime-3 Prime, Inc. 5603 Arapahoe Road, Boulder,
CO
80303). The filter is then hybridized with radioactive labeled full length
MPIF-1 gene
at 1,000,000 cpm/ml in 0.5 M NaP04, pH 7.4 and 7% SDS overnight at 65~C. After
wash twice at room temperature and twice at 60~C with 0.5 x SSC, 0.1 % SDS,
the filter
is then exposed at -70~C overnight with an intensifying screen.
Example 9
Expression pattern of MIR4 in human cells
Northern blot analysis was carried out to examine the levels of expression of
MIP-4 in human cells. Total cellular RNA samples were isolated with RNAzoITM B
system (Biotecx Laboratories, Inc. 6023 South Loop East, Houston, TX 77033).
About
10 ug of total RNA isolated from each human tissue specified was separated on
1
agarose gel and blotted onto a nylon filter. (Sambrook, Fritsch, and Maniatis,
Molecular
Cloning, Cold Spring Harbor Press, (1989)). The labeling reaction was done
according
to the Stratagene Prime-It kit with 50 ng DNA fragment. The labeled DNA was
purif ed
with a Select-G-50 column. (5 Prime - 3 Prime, Inc. 5603 Arapahoe Road.
Boulder, CO
80303). The filter was then hybridized with radioactive labeled full length
M1P-4 gene
at 1,000,000 cpm/ml in 0.5 M NaP04, pH 7.4 and 7% SDS overnight at 65~C. After
wash twice at room temperature and twice at 60~C with G.5 x SSC, 0.1 % SDS,
the filter
was then exposed at -70~C overnight with an intensifying screen. See Figure 6.
2o Example 10
Expression and Purification oJChemokine MPIF 1 using a baculovirus expression
system
SF9 cells were infected with a recombinant baculovirus designed to express the
MPIF-1 cDNA. Cells were infected at an MOI of 2 and cultured at 28~C for 72-96
hours. Cellular debris from the infected culture was removed by low speed
centrifugation. Protease inhibitor cocktail was added to the supernatant at a
final
concentration of 20 ~g/ml Pefabloc SC, 1 ~g/ml leupeptin, 1 ~g/mI E-64 and I
mM
EDTA. The level of MPIF-1 in the supernatant was monitored by loading 20-30 gl
of
supernatant only 15% SDS-PAGE gels. MPIF-1 was detected as a visible 9 Kd
band,
corresponding to an expression level of several mg per liter. MPIF-1 was
further purified
-121-
KW. \o\:I:I'.\-\II I~\c.llL::\ n;; ::W - l--:7a : i:? I:? . I - +I:> >?:7
_;?:7~3-i~+-E7:,_:~'=I
V'~V I .- V I VG- i3J r1i \ .rV _V s ~ 4L
through a three-step purification procedure: Heparin binding affnity
chromatography.
Supernatant of baculovinxs culture was-mixed with 1I3 volume of buffer
containing I00
mM HEPESlMESINaOAc pH 6 and filtered through 0.22 Vim. membrane. The sample
was then applied to a heparin binding column (HEl poros 20, Bi-Perceptive
Syster.~
Inc.}. MPIF-1 was eluted at approximately 300 mM NaCI in a linear gradient of
50 to
500 ~nM NaCl in 50 mNl I-iEPESIMES/NaOAc at pH 6; Cation exchange
chromatography. The IvIPIF-1 enriched from heparin chromatography was
subjected
to a 5-fold dilution with a buffer containing 50 mM T-IEPES/MES/Na4Ac pH 6.
The
resultant raixture was then applied to a canon exchange column (S/M poros 20,
Bio-
Percxptive System Inc.). MPIF-1 was eluted at 250 mM NaCI in a linear gzadient
of 25
to 300 mM NaCI in ~0 mM HEPESIMESINaOAc at pH 6; Size exclusion
chromatography. Following the canon exchanbe chromatography, MPIF-1 was
further
purified by applying to a size exclusion column (HW50, TOSO HAAS, I.4 x 45
ctn).
MPG'-1 fractionated at a position close to a 13.? Kd molecular weight standard
(R.:'~Tase
A), correspondiag to the dimeric form of the protein.
Following the three-step purification described above, the resultant MPIF-1
was
judged to be greater than 90% pure as determined from commassie blue staining
of an
SDS-PAGE gei (Figure 9A-9B).
The purified ?Lff'IF-1 was also tcstea. for endotoxin/LPS contami nation_ The
LPS
content was less than 0.1 ng/mI according to LAh assays {Bio~Vhittaker).
Example ll
Effect a~'baculovirus-expressed M CIF arxd :yIPIF 1 on !N-CSC" and SCF
stimulated
colony formation of frashly isolated bone marrow celLr
A Iow density population of mouse bone marrow cells were incubated in a
treated tissue culture dish for one hour at 37~C to remove monocytes,
macrophages, and
other cells that adhere to the plastic surface. The non-adherent population of
cells were
then plated (10,004 cellsldish) in agar containing growth medium in the
presence or
absence ofthe factors shown in Figure 14. Cultures were incubated for 10 days
at 37~C
{88% N2, 5% C02, and 7~ia O~ aad colonies were scored under an inverted
microscope.
Data is expressed as mean number of colonies and was obtained from assays
performed
in triplicate.
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Example 1 Z
Effect of MPIP 1 and M CIF on IL-3 and SCF stimulated proliferation and
differentiation of lin population of bone marrow cells
A population of mouse bone marrow cells enriched in primitive hematopoietic
progenitors was obtained using a negative selection procedure, where the
committed
cells of most of the lineages were removed using a panel of monoclonal
antibodies (anti
cdllb, CD4, CDB, CD45R, and Gr-1 antigens) and magnetic beads. The resulting
population of cells (lineage depleted cells) were plated (5 x 10a cells/ml) in
the presence
or absence of the indicated chemokine (50 ng/ml) in a growth medium
supplemented
with IL-3 (5 ng/ml) plus stem cell factor (SCF) ( 100 ng/ml). After seven days
of
incubation at 37~C in a humidified incubator (5% CO,, 7% O2, and 88% NZ
environment), cells were harvested and assayed for the I-IPP-CFC, and immature
progenitors. In addition, cells were analyzed for the expression of certain
differentiation
antigens by FACScan. Colony data are expressed as mean number of colonies -+-/-
SD)
and were obtained from assays performed in six dishes for each population of
cells
(Figure 15).
Example 13
MPIF I inhibits colony formation in response to IL-3, M CSF, and GM CSF
Mouse bone marrow cells were flushed from both the femur and tibia, separated
on a ficoll density gradient and monocytes removed by plastic adherence. The
resulting
population of cells were incubated overnight in an. MEM-based medium
supplemented
with IL-3 (5 ng/ml), GM-CSF (5 ng/ml), M-CSF ( 10 ng/ml) and G-CSF ( 10
ng/ml).
These cells were plated at 1,000 cells/dish in agar-based colony formation
assays in the
presence of IL-3 (S ng/ml), GM-CSF (5 ng/ml) or M-CSF (5 ng/ml) with or
without M-
CIF at 50 ng/ml. The data is presented as colony formation as a percentage of
the
number of colonies formed with the specific factor alone. Two experiments are
shown
with the data depicted as the average of duplicate dishes with error bars
indicating the
standard deviation for each experiment (Figure 17).
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Example 14
Expression via Gene Therapy
Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue
is
placed in tissue-culture medium and separated into small pieces. Small chunks
of the
tissue are placed on a wet surface of a tissue culture flask, approximately
ten pieces are
placed in each flask. The flask is turned upside down, closed tight and left
at room
temperature over night. After 24 hours at room temperature, the flask is
inverted and
the chunks of tissue remain fixed to the bottom of the flask and fresh media
(e.g. Ham's
F 12 media, with 10% FBS, penicillin and streptomycin, is added. This is then
incubated
at 37~C for approximately one week. At this time) fresh media is added and
subsequently changed every several days. After an additional two weeks in
culture, a
monolayer of fibroblasts emerge. The monolayer is trypsinized and scaled into
larger
flasks.
pMV-7 (Kirschmeier, P.T. et al, DNA 7:219-25 (1988) flanked by the long
1 S terminal repeats of the Moloney marine sarcoma virus, is digested with
EcoRI and
HindIII and subsequently treated with calf intestinal phosphatase. The linear
vector is
fractionated on agarose gel and purified, using glass beads.
The cDNA encoding a polypeptide of the present invention is amplified using
PCR primers which correspond to the 5' and 3' end sequences respectively. The
5'
primer containing an EcoRI site and the 3 ' primer having contains a HindIII
site. Equal
quantities of the Moloney marine sarcoma virus linear backbone and the EcoRI
and
HindIII fragment are added together, in the presence of T4 DNA ligase. The
resulting
mixture is maintained under conditions appropriate for ligation of the two
fragments.
The ligation mixture is used to transform bacteria HB101, which are then
plated onto
agar-containing kanamycin for the purpose of confirming that the vector had
the gene
of interest properly inserted.
The amphotropic pA317 or GP+aml2 packaging cells are grown in tissue culture
to confluent density in Dulbeccol's Modified Eagles Medium (DMEM) with 10%
calf
serum (CS), penicillin and streptomycin. The MSV vector containing the gene is
then
added to the media and the packaging cells are transduced with the vector. The
packaging cells now produce infectious viral particles containing the gene
(the
packaging cells are now referred to as producer cells).
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Fresh media is added to the transduced producer cells, and subsequently, the
media is harvested from a 10 cm plate of confluent producer cells. The spent
media,
containing the infectious viral particles, is filtered through a millipore
filter to remove
detached producer cells and this media is then used to infect fibroblast
cells. Media is
removed from a sub-confluent plate of fibroblasts and quickly replaced with
the media
from the producer cells. This media is removed and replaced with fresh media.
If the
titer of virus is high, then virtually a11 fibroblasts will be infected and no
selection is
required. If the titer is very low, then it is necessary to use a retroviral
vector that has
a selectable marker, such as neo or his.
The engineered fibroblasts are then injected into the host, either alone or
after
having been grown to confluence on cytodex 3 microcarrier beads. The
fibroblasts now
produce the protein product.
Example 15
In Vitro Myeloproteetion
As demonstrated above, MPIF-1 is a potent inhibitor of the Low Proliferative
Potential Colony-Forming Cell (LPP-CFC), a myeloid progenitor that gives rise
to
granulocyte and monocyte lineages. To demonstrate that MPIF-1 provides
protection
for LPP-CFC from the cytotoxicity of the cell cycle acting chemotherapeutic
drug,
lineage-depleted populations of cells (Lin- cells) were isolated from mouse
bone marrow
and incubated in the presence of multiple cytokines with or without MPIF-1.
After 48
hours, one set of each culture received 5-Fu and the incubation was then
continued for
additional 24 hours, at which point the numbers of surviving LPP-CFC were
determined
by a clonogenic assay. As shown in Figure 21 A, ---40% of LPP-CFC were
protected from
the 5-Fu-induced cytotoxicity in the presence of MPIF-l, whereas little
protection (<5%)
of LPP-CFC was observed in the absence of MPIF-1 or in the presence of an
unrelated
protein. High Proliferative Potential Colony-Forming Cells (HPP-CFC) were not
protected by MPIF-1 under the same culture conditions, demonstrating
specificity of
the MPIF-1 protective effect.
Similar experiments were performed using the chemotherapeutic agent, Ara-C
instead of 5-Fu. As shown in Figure 21 B, dramatic protection of LPP-CFC by
both
from wild type MPIF-l and a mutant MPIF-1 (i.e., mutant-1, see Example 17
below for
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description of this mutant). Thus, MPIF-1 is able to protect LPP-CFC from the
cytotoxicity induced by both chemotherapeutic drugs, 5-Fu and Ara-C.
Example 16
In Vivo Myeloprotection
S The in vitro myeloprotection results suggest that myelotoxicity elicited by
the
cytotoxic drugs, a severe side effect observed in cancer patients undergoing
chemotherapy, might be ameliorated if the critical cell types within the bone
marrow
could be protected by MPIF-1 during the period of action of the
chemotherapeutic drugs.
To demonstrate in vivo myeloprotection, two types of experiments were
performed in
mice. In one experiment, a group of mice (Group-4) were injected (LP.) daily
for three
days, at 24 hour intervals, with 1.0 mg/Kg MPIF-1, and on the third day these
mice were
also injected (LP.) with 5-Fu at 150 mg/Kg. Animals injected with either
saline (Group-
1 ), MPIF-1 alone (Group-2), or 5-Fu alone (Group-3) served as controls. Then,
four
animals from each of the groups were sacrificed at 3, 6, and 10 days post S-Fu
1 S administration to determine White Blood Cell (WBC) counts in the
peripheral blood.
As shown in the Figure 22, injection of MPIF-1 alone had little effect on the
WBC
counts. As expected, S-Fu treatment resulted in a dramatic reduction in the
circulating
WBC counts on day 6 post 5-Fu. Significantl5-, animals treated with MP1F-1
prior to
5-Fu administration exhibited about two fold higher WBC counts in the blood
compared
to animals treated with 5-Fu alone. Thus, treatment of mice with MPIF-1 prior
to 5-Fu
results in the accelerated recovery from neutropenia.
Hematopoietic stem and multipotential progenitor cells in the bone marrow are
responsible for restoring all the hematopoietic lineages following
chemotherapy. In
normal individuals, these cells divide less frequently, and are, therefore,
spared from a
single dose of the chemotherapeutic drug. However, these cells are killed if a
second
dose of the drug is administered within three days after the first dose
because the critical
progenitor cell types in the marrow are rapidly dividing during this period.
To demonstrate that MPIF-1 is able to protect these cell types in the bone
marrow, the following experiment was performed. The experimental was performed
using three groups of mice (6 animals per group) that were treated as follows:
Group-1,
injected with saline on days 1, 2, and 3; Group-2, injected with 5-Fu on days
0 and 3;
and Group-3, injected with S-Fu on days 0 and 3 and MPIF-1 on days 1, 2, and
3. (See
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Figure 23.) Bone marrow was harvested on days 6 and 9 to determine HPP-CFC and
LPP-CFC frequencies using a clonogenic assay well known to those of skill in
the art.
The results demonstrate that administration of MPIF-1 prior to the second dose
of 5-Fu
results in a rapid recovery of the HPP-CFC and LPP-CFC frequencies by day 9
compared to animals treated with 5-Fu alone. (See, Figure 24.)
Example 17
Studies with the MPIF 1 mutants
A number of MPIF-1 variants that are truncated from the N-terminus have been
identified and characterized. The amino terminal sequences of these variants
as
determined by Edman degradation are presented in the Figure 25. For example,
Mutants-2, -3, -7, and -8 arose spontaneously during the purification of the
mature form
of MPIF-I and this preparation is called Preparation K0871. Similarly, Mutants-
2, -3,
-4, and -5 were discovered in another batch of the purified MPIF-1 preparation
(Preparation HG00300-B7). Since it was not possible to purify these variants
from one
another, Preparations K0871 and HG00300-B7 were used as is in the experiments
described below. Mutant-6, which is identical to Mutant-3 with respect to the
amino
terminal sequence except for the N-terminal methionine, was generated by in
vitro
mutagenesis. Mutant-1, which is identical to the wild type except for the N-
terminal
methionine, was also generated by mutagenesis. In addition, an alternatively
spliced
form of MPIF-1 (Mutant-9), the cDNA clone of which encodes for a protein of
137
amino acids (Figure 26A) was discovered (See, Figure 25). Comparison of the
amino
acid sequence for Mutant-9's with that of MPIF-1 reveals an insertion of 18
amino acids
between residues 45 and 46 in the MPIF-1 sequence and a loss of argininc 46 of
MPIF-1
(Figure 26B). The following summarizes the biological activities of these MPIF-
1
mutant proteins.
Intracellular Calcium mobilization. In the foregoing Examples, MPIF-1 protein
has been shown to mobilize calcium in monocytes. The wild type and mutant MPIF-
1
proteins were tested for their ability to induce mobilization of intracellular
calcium in
human monocytes using human MIP-1 a as a positive control. The experiment was
performed as follows: Human monocytes were isolated by elutriation and loaded
with
Indo-l/acetoxymethylester by incubating 1 x 106 cells in 1 ml of in HBSS
containing 1
mM CaCl2, 2 mM MgS04, 5 mM glucose and 10 mM HEPES, pH 7.4 plus 2.5 mM
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Indo-1/acetoxymethylester for 30 min at 37~C. Cells were then washed with HBSS
and
resuspended in the same buffer at 5 x 105 cells/ml and stimulated with various
concentrations of the indicated proteins at 37~C. The fluorescent signal
induced in
response to changes in intracellular calcium ((Ca++)i) was measured on a
Hatchi F-2000
fluorescence spectrophotometer by monitoring Indo-1 excitation at 330 nm and
emission
at 405 and 485 nm. The results are shown in Figure 27.
The results demonstrate that preparations K0871, HG00300-B7, and Mutant-9
are ten-fold more active than the wild type, whereas Mutants-6 is
indistinguishable from
the wild type and Mutant-1 is about two-fold more active than the wild type.
(See,
Figure 27). Since MIP-1 a and MPIF-1 are 45% identical with respect to the
primary
amino acid sequence, it was of interest to determine whether they interacted
with the
same receptor. To explore this possibility, the ability of MPIF-1 to
desensitize MIP-1 a-
induced calcium mobilization was studied. Figures 28A and 28B show that MIP-la
and
the MPIF-1 wild type protein can desensitize each others ability to mobilize
calcium in
monocytes, but not MCP-4 (another beta-chemokine).
In similar experiments, preparations K0871, HG00300-B7, and Mutants-1, -6,
and -9 were able to block MIP-1 a induced calcium mobilization. This
experiment was
performed as follows: Calcium mobilization response of human monocytes to the
indicated proteins at 100 ng/ml was measured as indicated above for the
experiment
disclosed in Figure 27. For desensitization studies, monocytes were first
exposed to one
factor and when the response to the first treatment returned to baseline a
second factor
was added to the same cells. No response to the second factor is indicated by
the (-) sign
and a stimulatory response to the first factor by a (+) sign. (See, Figure
29).
Thus, MPIF-1 and its mutant variants appear to interact with or share a
component of the cell surface receptor for MIP-1 a. Recent demonstration that
the MIP-
1 a receptor serves as a cofactor in facilitating the entry of HIV into human
monocytes
and T-lymphocytes raises an interesting possibility that MPIF-1 or its
variants might
interfere with the process of HIV entry into the cells.
Chemotaxis. Chemotaxis of human peripheral blood mononuclear cell (PBMC)
fraction (consisting mainly of lymphocytes and monocytes) was measured in
response
to various concentrations of MPIF-l and its variants in a 96-well neuroprobe
chemotaxis
chambers. The experiment was performed as follows: cells were washed three
times in
HBSS with 0.1% BSA (HBSSBSA) and resuspended at 2x106/ml for labeling. Calcein-
AM (Molecular Probes) was added to a final concentration of 1 mM and the cells
were
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incubated at 37~C for 30 minutes. Following this incubation, the cells were
washed
three times in HBSSBSA. Labeled cells were then resuspended to 8x106/ml and 25
ml
of this suspension (2x105 cells) dispensed into each upper chamber of a 96
well
chemotaxis plate. The chemotactic agent was distributed at various
concentrations in
S the bottom chamber of each well. The upper and the bottom chambers are
separated by
a polycarbonate filter (3-5 mm pore size; PVP free; NeuroProbe, Inc.). Cells
were
allowed to migrate for 45-90 minutes and then the number of migrated cells
(both
attached to the bottom surface of the filter as well as in the bottom chamber)
were
quantitated using a Cytofluor 11 fluorescence plate reader (PerSeptive
Biosystems).
Values represent concentrations at which peak activity was observed with the
relative
fold induction over background indicated in parentheses.
The results, shown in Figure 30, demonstrate that preparations K0871 and
HG00300-B7 are more potent inducers of chemotaxis than the wild type, whereas
Mutants-1 and -6 were indistinguishable from the wild type.
Effects on colony formation by LPP CFC. To determine the impact of MPIF-1
variants on colony formation by LPP-CFC, a limiting number of mouse bone
marrow
cells were plated in soft agar containing medium supplemented with multiple
cyokines
with or without various concentrations of MPIF-I variants. The experiment was
performed as follows: a low density population of mouse bone marrow cells were
plated
( 1,500 cells/3.5 cm diam. dish) in agar containing median: with or without
the indicated
MPIF- I vzriants at various concentrations, but in the presence of the
following
recombinant marine cytokines IL-3 (5 ng/ml), SCF (100 ng/ml), IL-1 alpha (10
ng/ml),
and M-CSF (5 ng/ml). Dishes were then incubated in a tissue culture incubator
for 14
days at which point LPP-CFC colonies were scored under an inverted microscope.
Data
presented in Figure 31 are pooled from several different experiments where
each
condition was assayed in duplicates.
The results demonstrate that the effective concentration required for 50% of
maximal inhibition in the case of preparations K0871 and HG00300-B7 were 20-
to 100-
fold lower than that of the wild type and for Mutant-6 it was 2- to 10- fold
lower. (See,
Figure 31 ). Thus, deletion of the N-terminal amino acids of MPIF-1 protein
results in
an increased potency of the molecule.
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Example 18
M CIF Protection of Lipopolysaccharide-Induced Lethal Sepsis
Septic shock, a disease with significant morbidity and mortality in humans,
results from uncontrollable release of cytokines in response to blood-borne
bacterial
infection. Bacterial endotoxins are recognized as a major factor in the
pathogenesis of
Gram-negative septic shock (Morrison & Ryan, Annu. Rev. Med. 38:417 ( 1987);
Wolff
& Benett, N. Engl. J. Med 29l:733 (l974)), which appears to be mediated by
macrophages in response to endotoxins for the production of TNF-a and other
cytokines
(Freudenberg et al., Infect. Immu~z 51:891 ( 1986), Tracey et al., Nature
(Lond). 330:662
( 1987)).
M-CIF is a new member of the beta-chemokine family with no in vitro
chemotactic activity to monocvtes/macrophages and some degree of chemotactic
activiy
to T lymphocytes. It is inactive on most leukocytes except that it induces
monocyte/macrophages for intracellular Ca" change via receptors shared with
MIP-1 a
and RANTES (Schulz-Knappe et al., J. Exp. Med. I83:295 ( 1996)). In addition,
M-CIF
has been shown to have a strong inhibitory effect on M-CSF- induced
promonocytic
colony formation (Kreider et al., Abstract,for The International.Society.for
Interferon
and C.ytokine Research. Geneva, Switzerland, 1996).
In the present study, we examine the effect of M-CIF on endotoxin-induced
septic shock in animal models. In some experiments, to bypass the known
natural
resistance of mice to the effect of bacterial toxins (Peavy et al., J.
Immunol. l05: l453
(l970)), we increased their sensitivity by pretreatment with D-galactosamine
(Galanos
et al., Proc. Natl. Acad. Sci. USA. 76:5939 ( 1979): Lehmann et al., J. Exp.
Med l65:657
(1987)). We show that systemic treatment of potentially septic mice with M-CIF
significantly prevented LPS-induced lethal shock.
Materials and Methods
Chemicals and reagents. The endotoxins LPS (derived from E. coli 0127:B8)
and D-galactosamine were purchased from Sigma Chemical Co. (St Louis, MO).
Recombinant human M-CIF was produced utilizing three different vector systems:
baculovirus, E. coli and CHO cells, for protein expression and purification.
Final
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protein preparations for in vivo usage contained more than 90% M-CIF as
determined
by SDS-PAGE analysis and had an endotoxin level less than 4.0 EU/mg.
Table 1
Batches
and vectors
oJ'lll
CIF used
in experiments
$ M-CIF Vector Batch % Purity Endotoxin Buffer content
No. level
(SDS-PAGE)(EU/mg) (NaOAc; NaCI)
1. Baculovirus B8 >95 4.0 40 mM; pH 5.5;
500 mM
2. Baculovirus B9 >95 0.2 40 mM; pH 5.5;
I50 mM
3. Baculovirus B I 1 >90 2.4 40 mM; pH 5.5;
l50 mM
4. E. coli E1 95 0.04 40 mM; pH 6.0;
400 mM
5. CHO C1 >95 0.75 SO mM; pH 6.5;
500 mM
Animals. These experiments were conducted with Balb/c and CF-1 mice
purchased from Harlan Sprague Dawley (Indianapolis, IN ) and Balb/c scid/scid
(SCID)
mice purchased from the Animal Production Facility at National Cancer
I S Institute/Charles River (Frederick, MD). All mice were used at 8-12 weeks
of age and
were maintained on a standard lab diet with free access to tap water. Animals
were
housed under controlled conditions in plastic microisolator cages with filter
tops in a
room with a 12 hour light cycle (6 am to 6 pm, light] and monitored 22~C
temperature
and 65% humidity for at least one week before use in experiments. SCID mice
had all
bedding and water autoclaved and food irradiated before use.
Experimental design. Lethal sepsis was induced in mice with i.p. injection of
LPS at various doses dissolved in normal saline on day 0 with or without prior
( 1 hour
before LPS) D-gal sensitization. M-CIF from various vectors/batches at
different doses
was given i.p. daily for 3 consecutive days on day -l, day 0 (1 hour before
LPS) and day
1. Mice receiving buffer (40 mM sodium acetate, pII 5.5; 150 mM NaCI) sen~e as
the
disease control. Animals were monitored for morbundity and morbidity 3 times /
day
after LPS challenge for as long as 120 hours after LPS challenge. Percent
surviving
mice is calculated as: number of living mice / total mice x I00%.
Results
Effect of M CIF in two animal models of septic shock in Balblc mice. The first
model of lethal shock was induced in mice with LPS (25 mg/kg, i.p.). In this
model,
85% of the animals died 52 hours after LPS injection. M-CIF (3 mg/kg, i.p.)
daily
treatment for 3 days prevented lethality as much as 40% compared with the
buffer
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control (Figure 32). The second model of lethal sepsis was induced by
injecting mice
with LPS (1 ug/mouse, i.p.) one hour after D-gal (20 mg/mouse, i.p.)
sensitization and
all animals died within 8 hours after LPS administration. Pretreatment of mice
with M-
CIF ( 1 mg/kg, i.p.) for 3 days in a similar dosing regiment prevented 50%
lethality in
comparison with saline control, and single dosing treatment only prevented
lethality in
25% of the mice. In addition, the combination treatment of M-CIF with either
LPS
( 1 ug/mouse) or D-gal (20 mg/mouse) caused no sign of morbidity and
moribundity in
animals suggesting that the endotoxin level in M-CIF preparation is negligible
(Table
2).
Table 2
Group StrainM-CIF ip LPS ip NaCI Survival
D-gal ip within
ip
1 mgJkg I ug 0.1 ml B I I hr 22
20 mg hr hr
living/total
1 BALBrc- + + -1,0.+1 0l4 0l4 ND
2 BALB/c0 + _ 2!4 ~~ ND
3 BALB/c-1,0,+1 + - 2I4 2l4 ND
+
4 BALB/c-1.0,+ + _ 4I4 4/4 ND
I -
5 BALBIc-1,0I + _ - 4l4 4/4 ND
Mice were injected for 3 consecutive days I day prior to LPS on day -1, 1 hr
prior to 1.PS on day 0 and I day post LI'S on day I (-1,0,+ 1 ) or I hr
prior to LPS on day 0 only (0). ND=not done.
Preventive effect of M CIF on sepsis is independent of animal strains. CF-1
mice were also used in the D-gal-sensitized LPS-induced lethal shock model.
Unlike
Balb/c mice, only 50% of the CF-1 mice suffered from lethality by 11 hours
post LPS
in the saline control group and additional M-CIF daily dosing for 3
consecutive days
prevented all of the mice from dying (Table 3). These results suggest that
human M-CIF
may be very close to the murine homologue and the protective effect M-CIF on
sepsis
is a broad phenomenon rather than animal strain-selective.
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Table 3
Group Strain M-CIF ip D-gal ip LPS ip NaCI ip Survival within
1 mg/kg 20 mg 1 ug 0.1 ml 8 hr 11 hr 22 hr
living/total
1 CF-1 - + + -2; 1,0 4/4 2l4 2I4
2 CF-1 -2; 1,0 + + - 4l4 4l4 4/4
3 CF-1 -2; 1.0 - + - 5I5 5l5 5l5
Mice were injected for 3 consecutive days 2 days prior to LPS on day -2, I day
prior to LPS on day -1 and 1 hr prior to LPS on day 0 (-2; 1,0).
Preventive effect oJM CIF on septic shock is dependent on LPS dose. In a
large scale experiment, Balb/c mice were challenged i.p. one dose of LPS (25
mg/kg),
and the degrees of lethality in this group was 90% (Figure 33). Pretreatment
of M-CIF
daily at 10 mg/kg for 3 consecutive days protected as much as 70% (Figure 36).
Dose-dependent effect oJM CIF on lethal sepsis. This large scale experiment
was based on 25 mg/kg of LPS in Balb/c mice. 100% lethality was induced in the
buffer
control group within 48 hours after LPS injection In contrast, there was still
40%
survival in the mice treated with 1 mg/kg of M-C1F in the same period of time
and by
day 5 a11 mice died in this group. Moreover, M-CIF at 3 and 10 mg/kg doses
prevented
50% and 65% of mice from lethal shock, respectively (Figure 34).
M CIFis capable of preventing sepsis in Balblc SCID mice. SCID mice, which
have a deficiency in B and T lymphocytes, were injected i.p. with 20, 30, 40
or 50
mg/kg of LPS to determine the optimal degree of lethality. Unlike the normal
Balb/c
mice, no deaths occurred in the mice injected with 20 mg/kg LPS with or
without M-CIF
treatment (n = 8). Only 30% lethality was observed in the 30 mg/kg LPS group
and
additional treatment with 3 mg/kg of M-CIF protected all of the SCID mice from
shock.
As the LPS dose was further increased to 40 mg/kg, 80% mortality was induced
in the
buffer control group of the immunodeficient mice and additional treatment of M-
CIF at
3 mg/kg for three consecutive days protected 40% of the mice from lethality
(Figure
35A and 35B). Once the LPS dose was given at SO mg/kg, just like normal Balb/c
mice,
all of the SCID mice died in the buffer control group within 24 hours; and
none of the
5 animals could be protected by additional M-CIF treatment.
Consistent protective effect oJM CIF from different vector preparations on
sepsis. M-CIF proteins, prepared from E. coli and CHO expression vectors were
tested
in LPS-induced lethal sepsis in Balb/c mice. Compared with the buffer control
which
showed 100% lethality within 48 hours after 25 mg/kg LPS challenge, M-CIF (1
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mg/kg) derived from the CHO vector saved as much as 60% of the mice from death
during the same time period and 50% 3 days after LPS injection. Moreover, the
same
dose of the protein from the E. coli vector also prevented 25% of the mice
from lethal
shock. However, this preparation of M-CIF seems Iess potent than the materials
derived
from the other two vectors, suggesting that there may be a significant change
during the
protein expression and purification process (Figure 36).
Example 19
M CIF Modulation in Renal Injury
TNF-a has been shown to be involved in the pathogenesis of several types of
glomerular injury (Martin, et al., Clin. Exp. Immunol. 2:283-288 (1995);
Ortiz, et al.,
Adv. Nephrol. Necker. Hosp. 24:53-77 (1995); Karkar, et al., h'idney Int.
44:967-973
(l993); Nikolic-Paterson, et al., Kidney Int. 45:S79-S82 (1994); Egido, et
al., Kidney
Int. 43:S59-S64 (1993)) and may play a role in tubulointerstitial nephritis,
fibrosis, and
renal allograft rejection (Baud, et al., Miner. Electrolyte Metab. 2l:336-341
(1995);
Tang, et al., Lab. Invest. 70:631-638 (1994); Wilson, in The Kidney, Brenner,
ed.,
Philadelphia, W.B. Saunders Company, p.1253 (1996); Perkins, et al., in The
Kidney,
Brenner, ed., Philadelphia, W.B. Saunders Company, p.2576 ( 1996)). To
investigate the
efficacy of M-CIF in modifying the onset and progression of renal diseases.
animal
models are utilized for crescentic glomerulonephritis, focal and segmental
glomerulosclerosis (FSGS), and drug induced interstitial nephritis.
A model of anti-GBM disease is induced in a strain of rats (WKY) particularly
prone to the development of glomerular crescents (Huang et al., Kidney Int.
46:69-78
(1994); Bolton et al., Kidney Int. 44:294-306, ( l993)). The antibody used in
this study
is produced in female New Zealand White rabbits. The rabbits are immunized
repeatedly with the basement membrane-rich sediment of kidney (Schreiner, et
al.) J.
Exp. Med. l47:369-384 (l978)). The immune serum are heat-inactivated at 56~ C
for
min and absorbed with rat red blood cells and the resultant serum called
nephrotoxic
serum (NTS). Normal male WKY rats (125-150g) receive a single intravenous
injection
of a subnephritogenic dose of NTS. The dose is chosen such that immediate
glomerular
30 injury is not caused in Lewis rats.
According to known methods, administration of NTS to WKY rats causes
macrophages to infiltrate the glomeruli within 30 minutes and to increase in
number
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over a 10 day period. Glomerular hypercellularity is apparent within 48 hours
and by
day 6 there is necrosis and the presence of early crescent formation. Ten days
after
administration of NTS the majority of the glomeruli will exhibit a diffuse and
proliferative glomerulonephritis.
To test the efficacy of M-CIF to alter disease progression, rats receive NTS
and
then are treated daily with an intraperitoneal injection of M-CIF daily or
placebo. The
disease progression is monitored by daily collection of urine and serum for
assessment
of proteinuria and TNF-a levels, respectively. At various time points ranging
from 30
minutes to 10 days after NTS administration, rats are sacrificed and the
identity of the
infiltrating cells is assessed by immunohistological examination of frozen
sections using
commercially available monoclonal antibodies specific for macrophages and T
cells.
A model of chronic aminonucleoside nephrosis is used as a prototype of
progressive focal and segmental glomerulosclerosis. In this model) macrophages
infiltrate the renal cortex in which are found increased levels of TNF-a and
elevated
expression of the endothelin receptor gene (Diamond, et al., Am. J. Pathol.
14I:887-894
(l992); Diamond et al., Lab. Invest. 64:21-28 (I991); Nakamura, et al., J. Am.
Soc.
Nephrnl. S:1585-l590 (199S)). Male Sprague-Dawley rats weighing 125-150g are
used
for these studies. These rats receive a single intravenous injection of
puromycin
aminonucleoside (SOmg/kg; Sigma Chemical Co, St. Louis, MO) through the right
jugular vein over a period of 3 minutes. Within 2 weeks the animals develop
proteinuria, severe tubulointerstitial abnormalities, and exhibit an influx of
macrophages. This period of proteinuria will abate and then reappear by 18
weeks at
which time 44% of the glomeruli will exhibit focal and segmental
glomerulosclerosis
(Diamond, et al., Kidney Int. 32:67l -677 ( I 987)).
To test the ability of M-CIF to prevent this progressive renal injury, rats
are
injected intravenously with puromycin aminonucleoside and then treated with a
daily
intraperitoneal injection of either M-CIF or placebo. Proteinuria and serum
levels of
TNF-a are monitored at selected intervals over the I8 week study. At various
time
points rats are sacrificed and the renal cortical infiltrate examined on
sections of kidneys
using commercially available monoclonal antibodies to macrophages and T cells.
The
degree of morphologic abnormalities are assessed on standard paraffin sections
stained
with hematoxylin and eosin by two individuals in a blinded fashion and by
using a
computerized morphometric unit.
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A model of cell-mediated immune injury to the renal tubules leading to
granuloma formation is used to evaluate the efficacy of M-CIF to ameliorate
drug-
induced interstitial nephritis. Male Brown Norway rats weighing 140-180g are
used in
this model as previously reported (Rennke, et al., Kidney Int. 45:1044-1056
(1994)). A
haptenic molecule (ABA) is used as the target antigen. To produce the
immunogen
(ABA-KLH), 31.4 mg of p-Arsanilic acid (Eastman Kodak Co., Rochester, NY) are
dissolved in 2.5 ml of 1N HCl and then diazotized by the slow addition of
sodium
nitrite, resulting in activated ABA. A solution of keyhole limpet hemocyanin
(KLH)
(Calbiochem Corp, La Jolla, CA) is prepared by dissolving. SOOmg in 20m1 of
borate
buffered saline and the pH is adjusted to 9.2. The diazotized arsanilic acid
is added
slowly and after 60 minutes the mixture dialyzed against phosphate buffered
saline. The
resultant ABA-KLH is frozen in aliquots at -20 ~C until use.
Rats are immunized subcutaneously at the base of tail with 1 mg of ABA-KLI-I
emulsified in complete Freund's adjuvant containing Smg/ml of H37Ra
mycobacterium
tuberculosis (Difco laboratories, Detroit, MI). Ten days after this
immunization, the left
kidney is perfused through the renal artery successively with 1-2m1 of
phosphate
buffered saline, containing O.OSmglml verapamil, 2 ml of activated ABA (4mM
solution
in borate buffered saline solution at pH 8.1 ), and 1 ml of phosphate buffered
saline
containing O.OSmg/ml of verapamil.
To accomplish this, rats are anesthetized, placed on a heated operating table,
and
a laparotomy performed. The left renal vessels are isolated and loose snares
placed
around the left renal vein and the abdominal aorta. The left renal artery is
cannulated
with a 30 gauge needle and the snares around the aorta and renal vein closed.
Ex vivo
perfusion of the left kidney then occurs at a rate of 1.1 ml/min and the
effluent is then
drained through a puncture of the temporarily ligated left renal vein. After
hemostasis
is restored and the ligatures released, re-perfusion of the kidney occurs
within 1-2 min.
Within 24 hours a mild but diffuse inflammatory cell infiltrate is produced
that is
composed of polymorphonuclear leukocytes and mononuclear cells. By day 5
monocytes and macrophages predominate. At this time (day 5), 75% of the renal
cortex
is involved by a granulomatous inflammation.
To test the efficacy of M-CIF in this model, M-CIF or placebo is administered
intraperitoneally daily. Rats are sacrificed at various time points, their
serum levels of
TNF-a quantitated, and the amount of renal cortex involved in the inflammatory
process
estimated on standard paraffin sections stained with hematoxylin and eosin
using a
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computerized morphometric unit. The identity of the infiltrating inflammatory
cells are
identified on histological sections using commercially available monoclonal
antibodies
to monocytes/macrophages and T cells. M-CIF is expected to provide reduced
inflammation in renal injuries.
Example 20
Protection oJchronic joint inflammation in adjuvant artirritis
in rats by M CIF
In rheumatoid arthritis, pain and swelling can generally be controlled by
currently available drugs, but it has been difficult to halt the progressive
joint destruction
associated with this disease. Therefore, much effort has been directed at more
specific
inhibition of the cellular and molecular mechanisms underlying bone and
cartilage
destruction. The Freund's adjuvant-induced arthritis model in rats shares a
number of
features with the arthritis patient, from the presence of a proliferative
synovitis and
swelling of the extremities ultimately leading to cartilage and bone erosion
(Pearson 8:
Wood, Arthritis Rheum. 2:440 (1959); Jones R. Ward, Arthritis Rheum. 6:23
(1963)).
As in rheumatoid arthritis in humans, macrophages are abundantly present in
the
inflamed synovial membrane of rats with adjuvant arthritis (Johnson et al.,
Arthritis
Rheum. 29:1122 ( 1986)). Macrophages are thought to play a major role in
arthritis,
either as effector cells of tissue destruction, by secreting tissue-degrading
enzymes or
pro-inflammatory cytokines (Lopex-Bote et al., Arthritis Rheum. 3l:769 (
1988)), or by
virtue of their immunoregulatory functions in the course of antigen-driven
responses
(Unanue & Allen, Science 236:5S1 (1987). This animal model has been used for
the
detection of anti-inflammatory and immunosuppressive drugs by quantitating
hind-paw
swelling (as a measure of acute inflammation)) and histopathological
alterations in
cartilage and bone for chronic joint damage. In this study, we have tested the
effect of
M-CIF on both acute and chronic inflammatory arthritis in the adjuvant
arthritis rat
model.
On day 0 adult male Lewis rats (l20-l50 g) were injected intradermally at the
base of the tail with Freund's complete adjuvant, which was prepared by adding
Mycobacterium butyricum (Difco Lab, Detroit, MI) into mineral oil at a
concentration
of 5 mg/mI. M-CIF or its buffer were injected intraperitoneally to rats daily
from day
0 to day 16 or from day 0 to day 40 as described below. Indomethacin at a dose
of 1
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mg/kg or its methylcellulose vehicle were orally administered daily in other
groups of
rats. Swelling of the hindpaws were measured using a plethysmometer chamber
(Baxco
EIectronics, Troy, NIA. The hindpaw volume was expressed as the mean of the
volumes
of both hindpaws and as a percent change in paw volume.
At the end of experiment, the ankle and tarsal joints were excised and
processed
for histological evaluation. Two investigators evaluated the pathological
changes and
alterations of bone and cartilage in a blinded fashion using the following
parameters:
blood vessel dilation, fibrosis/fibroplasia, hyperplasia/hypertrophy,
perivascular
lymphoid aggregates, pannus formation, cartilage destruction, and bone
destruction. A
subjective semiquantitive scoring system, used to differentiate the degree and
distribution of the changes, was defined as follows: 0 = normal; 0.5 = slight;
1 =
moderate; 2 = severe; and 3 = very severe.
In the first experiment, the animals were treated from day 0 to day 16. Their
ankles were swollen by day 14 (the first time period tested) and reached their
maximal
severity between day 16 and 20. After this time the acute inflammation
gradually
subsided. The effect of M-CIF on ankle swelling is shown in Figure 37. Both
doses
of M-CIF showed moderate reduction in paw swelling, however indomethacin was
much more effective in reducing the edema. In a pilot study the limbs from two
animals
from each group were processed for histopathological scoring and the results
are shown
in Figure 38. Taking both the acute and chronic features into account, animals
treated
with M-CIF from day 0 to day 16 showed a significant reduction in total joint
inflammation compared with the buffer control group.
Based on these results, a second experimental protocol was utilized in which
the
rats were treated daily throughout the experiment (day 0 to day 40). At the
end of the
study, limbs from five animals per groups were processed for histological
evaluation.
When M-CIF was given daily at a dose of 3 mg/kg, there was significant
reduction in
the chronic synovitis (Figure 39) and the bone and cartilage erosion (Figure
40) when
compared with its buffer controls. Indomethacin failed to show any efficacy in
the
histopathology of chronic arthritis. Therefore, M-CIF showed a significant
protective
effect on the chronic features of arthritis, most importantly the bone and
cartilage
erosion, although only a mild effect on acute edema.
M CIF treatment prevents developing type II Collagen-induced arthritis in
DBAlI mice. An emulsion was prepared using equal volumes of a 2mg/ml solution
of
bovine type II collagen and complete Freund's adjuvant. Female DBA/lLac3 mice,
5-6
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weeks old were immunized intradermally at the base of the tail with l00 ~1 of
the
emulsion. Eighteen days later, the mice were divided into 3 groups of 10 mice
and
injected intraperitoneally with 3 mg/ml of indomethacin, M-CIF, or a control
buffer.
This injection was repeated for 14 days. Two days after the start of this
treatment
(which is 20 days after the start of the experiment), the mice were challenged
with a s.c.
injection of 60 ~cg of LPS in a total volume of l 00 ~cl. The animals were
examined and
their clinical presentation semiquantified for development of the arthritis by
the
following scoring system:
Incidence = number of mice with at least one affected paw x 100
total number of mice
Clinical severity score Description
0.5 - One or more swollen digits.
I .0 - Entire paw swollen
2.0 - Deformity observed after inflammation subsides.
3.0 - Ankylosis: total loss of joint function in the paw.
As shown in Figure 41, about 70% mice developed acute paw edema by 4-10
days post LPS challenge in both M-CIF and its buffer treated groups. However,
the
severity of this acute inflammation is less pronounced in M-CIF treated mice
than that
in the buffer group (Figure 42). Over time, the buffer treated group's
incidence and
severity increased while M-CIF treated animals improved. Indomethacin, used as
positive control, was also effective in reducing both the incidence and
severity as
expected.
Discussion. Adjuvant and collagen induced arthritis are widely used
experimental models of rheumatoid arthritis with common clinical and
histological
features. In rheumatoid arthritis, pain and swelling can generally be
controlled by
currently available drugs, but it has been difficult to halt the progressive
joint destruction
associated with this disease. Therefore, much effort has been directed at more
specific
inhibition of the cellular and molecular mechanisms underlying bone and
cartilage
destruction. The protective effect of M-CIF on chronic features of arthritis,
most
importantly the bone and cartilage erosion which leads to joint deformity and
destruction
strongly suggests that M-CIF has good potential as a therapeutic agent for
chronic
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inflammatory arthritis such as rheumatoid arthritis in human. Although M-CIF
only has
a mild effect on acute edema, combinational treatment of M-CIF and NSAID may
be
beneficial for both acute phase arthritis such as pain and swelling and the
progressive
joint destruction. Thus, M-CIF is shown to provide protection against the
chronic
features of arthritis, such as inflammation and pain.
Example 21
Suppressive Effect of M CIF on Systemic TNF a Production
Septic shock is a disease with significant morbidity and mortality in humans,
which results from uncontrollable release of cvtokines in response to blood-
borne
bacterial infection. Bacterial endotoxins are recognized as a major factor in
the
pathogenesis of Gram-negative septic shock (Morrison & Ryan, Annu. Rev. Med
38:417
1987; Wolff & Benett, N. Engl. J. Med 29l :733 ( 1974). It appears to be
mediated by
macrophages in response to endotoxins for the production of TNF-a and other
cytokines
(Freudenberg et al, Infect. Immun. 51:891 ( 1986); Tracey et al., Nature
(Loud). 330:662
( 1987)).
Earlier work showed that systemic treatment of mice with M-CIF significantly
prevented LPS-induced lethal shock in two animal models. Since TNF-a
production is
central in causing septic shock we asked whether M-CIF interferes with the
production
of TNF-a and thereby protects against TNF-mediated endotoxic shock in vivo.
hr Vivo. Female Balb/c mice, 7-8 weeks old, were challenged with 25 mg/kg
of lipopolysaccharide (LPS) from E. coli serotype 0127:B8 (Sigma Chemical Co.,
St.
Louis, MO) in saline on Day 0. M-CIF or its buffer were administered
intraperitoneally
1 day before and 1 hour before the LPS injection. Groups of 4 mice were
sacrificed at
1, 2, and 4 hours after LPS administration. Sera was obtained from the
retrorbital plexus
and the TNF-a levels determined using an ELISA kit purchased from Genzyme
Corp.,
Cambridge, MA. The assay was performed as described by the manufacturer. Each
sample was diluted 1:4 and assayed in duplicate wells and the results analyzed
with an
unpaired T test. Data are expressed as mean values + SEM.
As shown in Figure 43, serum TNF-a levels in the buffer control group is
highest
at one hour post LPS injection and then quickly declines afterwards. In
contrast, mice
given 3mg/kg of M-CIF had significantly less TNF-a in their serum at one hour
post
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LPS than the buffer control group. Animals treated with lmg/kg of M-CIF had
reduced
levels but this did not attain statistical significance.
The inhibitory effect of M-CIF on systemic TNF-a production is expected to be
one aspect of the mechanism by which M-CIF protects mice from LPS-induced
septic
shock, and this effect would be beneficial for treating autoimmune
inflammatory
diseases such as rheumatoid arthritis and osteoarthritis.
In vitro. Female Balb/c mice, 4-6 weeks old were put into 2 groups of ten
animals per group. The groups were either injected intraperitonealy with
vehicle control
or injected with M-CIF at 3 mg/kg for 2 consecutive days. One hour after the
second
injection, the mice were sacrificed and peritoneal cavity lavage performed to
collect the
resident cells. The cells were then washed and resuspended at a density of I x
10~
cells/ml in culture medium (RPMI 1640/ 20% FBS). The cells were then plated in
48
well plates and incubated overnight in the presence or absence of LPS (1 and
10 ng/ml).
After 18 hours, the supernatants from each well were collected and stored
frozen until
use. The ELISA for the determination of TNF-a content in the supernatants was
performed as specified by the manufacturer (Genzyme Diagnostics, Cambridge,
MA).
As seen in Figure 44, cells isolated from M-CIF treated animals and then
treated with
LPS in vitro secrete statistically significant lower amounts of TNF-a than do
cells
isolated from control mice.
M-CIF thus has the capacity to inhibit TNF-a production in vivo. This activity
would be beneficial for both acute and chronic inflammation. Taken together
with the
data on the circulating TNF-a levels presented above, this can explain one
aspect of the
mechanism by which M-CIF protects from LPS induced sepsis. Since increased
levels
of TNF-a have been correlated with a wide variety of immune cell diseases or
reactions,
M-CIF treatment could be used on such disease states, as described herein.
Recent studies have shown the efficacy of inhibiting TNF-a activity with the
use
of antibodies to TNF-a or soluble TNF-a receptors. These diseases include
acute
pancreatitis, allograft rejection, non-insulin dependent diabetes mellitus
(NIDDM),
asthma, delayed hypersensitivity reactions in the skin, pulmonary fibrosis,
and
ischemia/reperfusion injury. In contrast, TNF-a plays a paracrine role in
liver
regeneration and in some circumstances suppresses skin and cardiac allograft
rejection.
Thus, M-CIF or its agonists are expected to be beneficial in such disease
situations.
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w,
-1-;l ti;) _:i'!;nlp.~'~-.;i__~
J .. J i n W ~. JL ~V a :.J
KC~\.\c\,n.t'\-~III~..'.~Iii~::. o:i :W - 4-.)z~ . 1is l:1 ~ _ ~ .._
.E.rample .?2
M-CIF as a Clremoattrarta~stJ'or T lyrtrphorytes in viva.
Female Balblc mice, 4-5 weeks vld were put Irxto 4 groups of tar. animals per
group. T'ne groups were either untreated, injected inttaperitoneally with
vehicle control
S or injected w~ch M-CIF a! 1 mg!kg, or 3 mg/kg for 6 consecutive days. On day
seven,
the mice were sacritsced and peritonea.( cavity lavage performed to collect
the resident
cells- Total cell numbers werz; calculated and the Lells suojecte3 to cell
surface sta;ning
using the folio~ving panel of monoclonal antibodies: CD3, CD4, CDB, Mac 1, GR
1,
32;0, MHC class II, CD14, CD4S, and Cr?5 (Pharmingen, San Diego) CA).
As shown in Figure 45, the total cell nurn.bers within the peritoneal cavity
increased '-3 old wee untreated or ~rehicie treated controls. This appears to
be due :o
an ir~'~ux of T-lymphocytes as deterrrzined by cell surface stai~~:ing for
CIa4. CD3, ~d
CD8_ There; is a dramatic il~crease in CD4 positive cells (Figure 46) as welt
as CD5 arid
CD8 cells resulring in a net increase in the relatiie number of T-lymphocyte
(Figure
1 S 47 (2)). In addition, th~rt is a significant increase in Mac 1 positive,
MH C class I I
negative;, subporulation of cells within the peritoneal cao-ity with a
corre.spording
decrease in the percenta5e of ~IHC class II positive, Macl positve
subpopulation of
cells (Figure 48A-48B). 'Ibis is also reflected in the tctal number of VIHC
CIasS I3
negative, hiac I positive coils within the peritoneal cavity (Figs a :19).
0 'vi-CIF is t~'_~1S S~U WT1 to tx a chemoatEractant for 'F-lymphocytes in
vivo. T:Zis
could be for CD4, CDS or both subpopulations of T-cells. Based on this, M-CiF
may
beneficial for daease states rvhich would benefit from the attraction andior
acaivation
of this population of immune cells. This would include bacterial or viral
infecaor.;
cancer, and the like. Also, if M-CIF has a specific effect on the T'nl or T~n2
subclass of
25 CD4 lymphocytes; it could bias the normal production of cytokines from
these cells and
dramatica~Iy influence other immune cells such as monocytes, macrophages,
eosinophils, and other immune calls.
The fact that the MI-IC class II negative subpopulation of Macl positive cells
increases in the M-CTF treated animals suggests that the monocyte population
within
30 these animals consists of a higher percentage ~f non-activated cells. This
is consistent
with the data showing that the peritoneal cells from the M-CIF treated animals
produce
less 'T~-F-a in response to LPS.
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Example 23
In Vivo Stem Cell Mobilization Induced by MPIF 1
To demonstrate that MPIF-1 stimulates stem cell mobilization in vivo, the
following experiment was performed. Six mice were used for each treatment
group
(C57Black 6/J , female, about 6 weeks old). The mice were injected (I.P.) with
either
saline (vehicle control) or MPIF-1 at 5 pg/mouse. After 30 minutes, mice were
bled and
analyzed for WBC by Coulter counter. Then, blood from all six animals of each
group
was pooled and analyzed for the Gr.l+ cells and CD34.Sca-1+ double positive
cells by
FACScan. WBC counts are expressed as Mean + S.D. and FACScan data as % of
total
cells. Since CD34.Sca-1+ double positive cells are thought to exhibit
properties
expected of the hematopoietic stem cells, the results shown in Figure SO
illustrate that
MPIF-1 can be used as stem cell mobilizer.
Example 24
Purification ojM CIF
Purification Jronr CHO Expression Svstenr
Following expression of M-CIF in Chinese hamster ovary cells, the protein was
purified using the following procedure. All of the purification procedures
were
performed at 5-10~C, unless otherwise specified. The transfected CHO cells
were
grown in HGS-CHO-3 medium using the microcarrier culture system (cytodex I,
Pharmacia) for 4 days. The conditioned media were harvested using low speed
centrifugation to remove cells and cell debris. After pH was adjusted to 7.0
with acetic
acid, the conditioned media was loaded onto a strong cation exchange column
{Poros
HS-S0, Perseptive Biosystems Inc.) pre-equilibrated with phosphate buffered
saline
(PBS), pH 7Ø The column was then washed with same buffer until the
absorbance at
280 nm was less than 0.01 O.D. (10 CV). The desired protein was eluted by
washing
the column with I M NaCI in phosphate buffered saline, pH 7Ø Fractions were
then
analyzed by SDS-PAGE through 4-20% gradient gels to confirm the presence of
the
desired polypeptide.
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Those fractions containing M-CIF were then pooled and loaded onto a gel
filtration column of Superdex-75 resin (Pharmacia) equilibrated in "sizing
buffer"
comprising 50 mM sodium acetate and 150 mM NaCI, pH 6Ø The sample loaded was
less than 10% {V/V) of the column volume. After allowing the sample to run
into the
column, the protein was eluted from the gel filtration matrix using the same
buffer.
Fractions were collected and the absorbance at 280 nm of the effluent was
continuously
monitored. Fractions identified by A280 as containing eluted material were
then
analyzed by SDS-PAGE. Fractions containing M-CIF was eluted in a peak centered
at
0.62 column volumes and pooled.
The pooled fractions from gel filtration chromatography was applied onto a set
of strong anion (Poros HQ-50, Perseptive Biosystems) and weak anion (Poros CM-
20)
exchange columns in a tandem mode. Both columns were pre-equilibrated and
washed
with 50 mM sodium acetate buffer, pH 6.0 after sample loading. The cation
exchange
column (CM-20) was then washed with 0.3M NaCI followed by a 0.3M to 0.8M NaCI
gradient elution in the same buffer system. The eluted fractions were analyzed
through
SDS-PAGE and fractions containing protein of interest were combined.
Following the purification steps described above, the resultant M-CIF was of
greater than 95% purity as determined from Commassie blue staining of a SDS-
PAGE
gel. The purified protein was also tested for endotoxin/LPS contamination. The
LPS
content was less than 0.1 ng/mg of purified protein according to LAL assays.
An alternative purification procedure was also used to purify M-CIF. The
procedure involves the following steps, and unless otherwise specified, all
procedures
were conducted at 5-10~C.
Upon completion of the production phase of a CHO culture, the conditioned
media were obtained after cells/cell debris removal using low speed
centrifugation.
Following pH of the media being adjusted to pH 7.0 by adding acetic acid, the
media
were loaded onto a strong cation exchange column (Poros HS-S0, Perspective
Biosystems, Inc.) pre-equilibrated with phosphate buffered saline (PBS), pH
7Ø The
column was then washed with same buffer until the absorbance at 280 nm was
less than
0.0l O.D. (10 CV). The desired protein was eluted by washing the column with
1M
NaCI in phosphate buffered saline, pH 7Ø Fractions were then analyzed by SDS-
PAGE
through 4-20% gradient gels to confirm the presence of the M-CIF.
Those fractions containing M-CIF were then pooled, followed by the addition
of 4 volumes of 10 mM sodium acetate, pH 6.5. The diluted sample was then
loaded
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onto a previously prepared set of tandem columns of strong anion (Poros HQ-50,
Perceptive Biosystems) and weak anion (Poros CM-20, Perceptive Biosystems)
exchange resin. The columns were equilibrated with 50 mM sodium acetate pH
6.5.
The CM-20 column was washed with S column volumes of 0.2 M NaCI, 50 mM sodium
acetate, pH 6.5 and eluted using a 10 column volume linear gradient ranging
from 0.2M
NaCI, 50 mM sodium acetate, pH 6.5 to 1.0M NaCI 50 mM sodium acetate, pH 6.5.
Fractions were collected under constant A280 monitoring of the effluent. Those
fractions containing the protein of interest (determined by 4-20% SDS-PAGE)
were then
pooled.
The combined fractions containing M-CIF were then loaded (V/V, 5% of the
column volume) onto a sizing exclusion column (Superdex-75, Phanmacia)
equilibrated
with l00 mM NaCI, 50 mM sodium acetate, pH 6.~. After allowing the sample to
run
into the column, the protein was eluted from the gel filtration matrix using
100 mM
NaCI, 50 mM sodium acetate, pH 6.5. Fractions were collected and the
absorbance at
280 nm of the effluent was continuously monitored. Fractions identified to
AzBO as
containing the eluted material were then analyzed by SDS-PAGE. Fractions
containing
M-CIF was then pooled.
Following the three step purification procedure described above, the resultant
M-CIF was of greater than 95% purity as determined from Commassie blue
staining of
a SDS-PAGE gel. The purified protein was also tested for endotoxin/LPS
contamination. The LPS content was less than 0.1 ng/mg of purified protein
according
to LAL assays.
Purification of M CIFfrom E. coli
The purification involves the following steps, and unless otherwise specified,
all
procedures were conducted at 4-10~C.
Upon completion of the production phase of the E. coli fermentation, the cell
culture was cooled to 4-10~C and the cells were harvested by continuous
centrifugation
at 15,000 rpm (Heraeus Sepatech). On the basis of the expected yield of
protein per unit
weight of cell paste and the amount of purified protein required, an
appropriate amount
of cell paste, by weight, was suspended in a buffer solution containing 100 mM
Tris, 50
mM EDTA, pH 7.4. The cells were dispersed to a homogeneous solution using a
high
shear mixer.
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The cells were then lysed by passing the solution through microfluidizer
(Microfuidics, Corp. or APV Gaulin, Inc.) twice at 4000-6000 psi. The
homogenate was
then mixed with NaCI solution to a final concentration of 0.5 M NaCI, followed
by
centrifugation at 7000 g f or 15 min. The resulted pellet was washed again
using 0.5M
NaCI, 100 mM Tris, 50 mM EDTA, pH 7.4.
The washed inclusion body was solubilized with 1.5 M Guanidine hydrochloride
(GuHCI) for 2-4 hours. After 7000 g centrifugation for 15 min., pellet was
discarded
and the M-CIF-containing supernatant was placed at 4 ~ C overnight for further
GuHCI
extraction.
Following high speed centrifugation (30000 g) to remove the insoluble
particles,
the GuI-ICl solubilized proteins were refolded by quickly mixing the GuHCI
extraction
with 20 volumes of buffer containing SO mM sodium, pH 4.5, 150 mM NaCI, 2 mM
EDTA by vigorous stirring. The refolded diluted protein solution was set kept
at 4~C
without mixing for 12 hours prior to further purification steps.
To clarify the refolded M-CIF solution, a previously prepared tangential
filtration
unit equipped with 0.16 um membrane filter with appropriate surface area
(Filtron),
equilibrated with 40 mM sodium acetate, pH 6.0 was employed. The filtered
sample
was loaded onto a cation exchange of poros HS-50 resin (Perceptive
Biosystems). The
column was washed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM,
500
mM, 1000 mM, and 1 S00 mM NaCI in the same buffer, in a stepwise manner. The
absorbance at 280 mm of the effluent was continuously monitored. Fractions
were
collected and further analyzed by SDS-PAGE.
Those fractions contained desired protein was then pooled and mixed with 4
volumes of water. The diluted sample was then loaded onto a previously
prepared set
of tandem columns of strong anion (Poros HQ-50, Perseptive Biosystems) and
weak
anion (Poros CM-20, Perceptive Biosystems) exchange resin. The columns were
equilibrated with 40 mM sodium acetate, pH 6Ø Both columns were washed with
40
mM sodium acetate, pH 6.0, 200 mM NaCI. The CM-20 column was then eluted using
a 10 column volume linear gradient ranging from 0.2 M NaCI, 50 mM sodium
acetate,
pH 6.0 to 1.0M NaCI, 50 mM sodium acetate, pH 6.5. Fractions were collected
under
constant A280 monitoring of the effluent. Those fractions containing the
protein of
interest (determined by 16% SDS-PAGE) were then pooled.
The resultant M-CIF was of greater than 95% purity after the above refolding
and purification steps. No major contaminant bands was observed from the
Commassie
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blue stained 16% SDS-PAGE gel when 5 ug of purified protein was loaded. The
purified protein was also tested for endotoxin/LPS contamination. The LPS
content was
less than 0.1 ng/ml according to LAL assays.
Example 25
M CIF inhibits M CSF stimulated colony formation of human and mouse cells in a
dose dependent manner.
Progenitor cells are isolated and processed as described herein. Marine bone
marrow cells are isolated from the femur and tibia, ficoll separated and
depleted of
plastic adherent cells. Both cell populations are plated in agar containing
medium in the
presence of M-CSF (5 ng/ml) with or without M-CIF at the concentrations
indicated.
Data is expressed as mean number of colonies +/- S.D. from samples done in
duplicate.
Clonogenic assays on mouse bone marrow cells. CFU-M colony formation
assays is performed in a two-layered agar culture system. The bottom layer is
prepared
in 3.5 cm diameter tissue culture dishes with 1 ml of MEM medium supplemented
with
20% FBS (Sigma Tissue Culture Products, St. Louis, MO), 0.5% Difco agar and 15
ng/ml of M-CSF in the presence or absence of the indicated concentrations of M-
CIF
or a control beta-family chemokine. This layer is then overlayed with 0.5 ml
of marine
bone marrow cell suspension ( 1 Oa cells/ dish) prepared in the agar medium
described
above except that it contained 0.3% agar and no cytokines. The dishes are then
incubated for seven days in a tissue culture incubator (37~C, 88% Nz, 5% C02,
and
7%0,) and CFU-M colonies are scored under an inverted microscope.
Clonogenic assays on human CD34' derived cells. Freshly purified CD34'
cells (5x 10~ cells/ml) are cultured for four days in Myelocult H5100 growth
medium
(Stem Cell Technologies Inc., Vancouver, Canada) supplemented with human IL-3
( 10
ng/ml) and human SCF (50 ng/ml). The resulting populations of committed
hematopoietic progenitors are counted and l,000 cells in 1 ml of MethoCult
medium
(Stem Cell Technologies Inc., Vancouver, BC, Canada are plated in 3.5 cm
diameter
tissue culture dishes with supplemented M-CSF ( 10 ng/mI) in the presence or
absence
of the indicated concentrations of M-CIF or a control beta-family chemokine.
After
fourteen days in incubator (37~C, 88% Nz, 5% CO2, and 7%OZ), the colonies are
scored
under an inverted microscope.
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Example 26
Evaluation of M CIF in a Surgically-Induced Model Osteoarthritis in Guinea
Pigs
To demonstrate that M-CIF slows the onset and progression of osteoarthritis
(OA), a surgically-induced model of OA in Hartley guinea pig is used. The use
of the
guinea pig in experimental OA is a well-characterized, relevant and
reproducible model
of OA. This strain has been shown to develop spontaneous osteoarthritis with
age.
Surgically-induced joint instability creates altered biomechanical loads in
the knee joint,
leading to OA. Pathologic changes observed in this model are similar to those
observed
in human OA (Meacock, S.C. et al., J. Exp. Pathol. 71 (2):279-93 (1990),
Bendele, A.M.
et al., Vet Pathol. 28:207-215 (1991), Jimenez, P.A. et al., Inflam. Res.
4=I(2):129-130
(1995)).
Surgery is performed on eight week old male Hartley guinea pigs (n=5)
anesthetized subcutaneously with ketamine (40 mg/kg), xylazine (S mg/kg),
fentanyl
(0.06 mg/kg) and post-operative buprenorphine (0.05 mg/kg). Prior to surgery,
guinea
1 S pigs are fasted for 12 hours. Animals are kept on a heating pad during
skin disinfection,
surgery and post-surgery. An incision is made with a # 10 blade trough the
joint capsule
of the right knee. The fascia over the medial meniscus is dissected, and the
medial
collateral ligament and medial incision retracted. The anterior medial
meniscus is
isolated with a Tyrel micro-dissecting hook and the anterior portion excised
with a # 15
blade. The joint capsule is sutured with continuous S-0 Vicrylt. Two wound
clips are
used to close the skin and are then removed at 4 days post-surgery. The
weights of the
animals are determined at the beginning of the experiment and every two weeks
thereafter.
M-CIF and placebo are administered daily (i.p) for six weeks commencing on
the day of surgery. Used are: an untreated control, a placebo group and M-CIF
treated
groups. Radiographs are taken at the end of the study prior to euthanasia. At
the end of
the experiment, all animals are euthanized with an overdose of sodium
pentobarbital
(300 mg/kg). The knee joints are harvested, fixed in 10% formalin for 4 days
and
decalcified in 20% formic acid in PBS (pH 7.2) for 4 days. Sections are cut at
5
intervals and stained with Safranin 0, Fast Green and Hematoxylin.
Histopathologic evaluation is performed using the Mankin scoring system
(Mankin H.J., Orth. Clin. North America 2:l9-30 (1971).
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Example 27
Evaluation of M CIF in a Peptidoglycan-Polysaccharide Polymer Model of
Granulomatous Enterocolitis in Rats
To demonstrate that M-CIF would slow the onset and progression of
granulomatous enterocolitis in a surgically-induced model of colitis in Lewis
rats is
used. The use of the Lewis rat in experimental colitis is a well
characterized, relevant
and reproducible model of enterocolitis. The Lewis stain of rats has been
shown to be
susceptible to the enterocolitis following surgical implantation of
peptidoglycan-
polysaccharide (PG-PS) in various areas of the distal ileum, peyer's patches,
cecum and
distal colon. Surgically-implanted PG-PS creates an acute enterocolitis which
peaks at
1-2 days, remains quiescent for 7-9 days, and spontaneously reactivates by 12-
17 days
with an active inflammation which can persist for up to four months. (Elson et
al.,
Gastroenterol. l09:1344-1367 ( 1995)). Development of chronic inflammation is
dependent on a T-cell mediated immune response, poorly degradable PG-PS, and
1 S genetic host susceptibility (Sartor et al., Methods: A Companion to
Methods in
Enrymolo~ 9:233-247 ( 1996)). Immune responses observed in this model are
similar
to those observed in human enterocolitis.
Surgery is performed on l30-170 g Lewis rats (n=10) anesthetized
subcutaneously with ketamine (40 mg/kg), xylazine (~ mg/kg), fentanyl (0.06
mg/kg)
and post-operative buprenorphine (0.05 mg/kg). Animals are kept on a heating
pad
during skin disinfection, surgery and post-surgery. A 6-8 cm incision is made
with a
# 10 blade through the abdomen to expose the ileum, cecum and colon. Rats are
injected
intramurally (subserosally) with PG-APS (4~ mg dry weight and 15 mg rhamnose/g
body wt). At each site 0.05 ml ( 1 / 10 of the total dose) is injected 2 and 4
cm proximal
to the ileocecal valve, two distal peyers patches, four midcecal sites,
lymphoid aggregate
at the cecal tip, and removed at 4 days post-surgery. The weights of the
animals are
determined at the beginning of the experiment and every five days thereafter.
The extent
of inflammation is assessed by morphological scoring of the extent of swelling
of the
ankle joint. Size of the ankle joint has been shown to be a reliable indicator
of the
presence of inflammation in the intestines.
M-CIF and placebo will be administered (i.p.) daily for four weeks commencing
on the day of surgery. There will be an untreated control, a placebo group and
M-CIF
groups.
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Two hours prior to euthanasia, rats are injected with BrdU (100 mg/kg i.p.).
At
the end of the experiment, all animals are killed using COz asphyxiation.
Samples taken
from distal ileum, cecum and distal colon are fixed in 10% formalin. Sections
are cut
and stained with H & E, mucicarmine, trichrome, and anti-BrdU antibodies.
Histopathologic evaluation is performed using the Sartor scoring system.
(Sartor, et al.,
Methods: A Companion to Methods in Enrymology 9:233-247 (1996).
Example 28
MPIF 1 Treatment During S-Fu Treatment Results in Faster Recovery of Platelets
and Granulocytes.
Two of the major complications resulting from chemotherapy are neutropenia
(reduced blood neutrophil counts) and thrombocytopenia (reduced platelet
counts).
Granulocyte-Colony Simulating Factor (G-CSF) is currently used in the clinic
to
mitigate neutropenia. G-CSF is known to stimulate colony formation by the
Colony
Forming Unit-Granulocyte (CFU-G) in vitro and stimulate granulocyte production
in
animal models. Thrombopoietin (Tpo) is in clinical trials for the purpose of
alleviating
thrombocytopenia. Tpo is known to stimulate colony formation by Colony Forming
Unit-Megakaryocyte (CFU-Meg) in vitro and stimulate platelet production in
experimentally induced thrombocytopenia in animals. One of the major
limitations of
G-CSF in the clinic is that it is not effective in alleviating neutropenia in
patients that
are subjected to multiple cycles of chemotherapy. This is likely due to the
depletion of
CFL1-G in the bone marrow, a target cell upon which G-CSF acts. Tpo might also
suffer
from the same fate as indicated by the initial clinical trial results. Any
agent that can
prevent the depletion of G-CSF and Tpo target cells during chemotherapy would
be of
great clinical value. The data shown below suggests that MPIF-I could meet
this
clinical need.
In the previous Examples, MPIF-I has been shown to inhibit colony formation
by bipotential, granulocyte/monocyte myeloid progenitors in vitro. In
particular,
Examples 15-16 provide data demonstrating that MPIF-1 protects primitive,
multipotential myeloid progenitors from 5-Fu induced cytotoxicity in vitro and
in vivo.
These multipotential progenitors are expected to give rise to more committed
progenitors of all the myeloid lineages including CFU-G and CFU-Meg. The
following
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experiment was performed to demonstrate that MPIF-1 treatment during two or
three
cycles of 5-Fu treatment results in faster recovery of platelets and
granulocytes.
Materials and Methods: C57BL6 female mice (7-10 weeks old) with a mean body
weight 19.4g (~ 1.1 S.D., n=1 SO) were used. A11 mice were housed under
standard diet
S and housing conditions of dark/light cycle and temperature throughout the
course of the
experiment. MPIF-1 preparation (HG00304-E6) was made in E. coli and represents
the
truncated form of MPIF-1 lacking 23 N-terminal amino acids of the mature
protein (i.e.,
MPIF-1 Mutant-3 in Figure 25 with an N-terminal Met added thereto). Clinical
grade
of G-CSF (Neupogen~) was purchased from the Shady Grove Pharmacy, Rockville,
MD 20850 (Neupogen~ is manufactured by Amgen lnc., Amgen Center, Thousand
Oaks, CA 91320). 5-Fluorouracil (5-Fu) was purchased from Sigma Chemicals and
it
was freshly prepared by dissolving in warm water just prior to use. MPIF-1
solution
was freshly prepared by dilution in normal saline. Likewise, G-CSF was diluted
in a
buffer consisting of 10 mM sodium acetate, 5% (wt/v) mannitol, 0.004% (v/v)
Tween
80, pH 4Ø Appropriate fluorochrome conjugated rat monoclonal antibodies
against
mouse CD41 a, Gra. l , and Mac. l antigens were purchased from Pharmingen.
Five groups of mice (30 mice per group) were treated as follows:
Group 1 was injected I.P. with 0.1 ml of normal saline on -2, -l, 0, 6, 7, and
8
days to serve as normal control.
Group 2 was injected with LP. with 0.2 ml of 5-Fu solution (at 100 mg/kg body
weight) on days 0 and 8.
Group 3 was injected with 5-Fu as in Group 2 and in addition 0.1 ml of MPIF-1
solution (at 1.0 mg/Kg body weight) was injected I.P. on -2, -1, 0, 6, 7, and
8 days.
Group 4 was injected with 5-Fu as in Group 2 and in addition 0.1 ml of G-CSF
solution (at 0.5 mg/Kg body weight) was injected I.P. on 1, 2, 3, 9, 10, and
11 days.
Group 5 was injected with 5-Fu as in Group 2, MPIF-1 as in Group 3, and
G-CSF as in Group 4.
Six animals from each of the groups were then analyzed on the indicated days
for monitoring platelet and granulocyte recovery at the level of the
peripheral blood and
the bone marrow. It should be noted that the mice analyzed on 6 and 8 days
post first
5-Fu did not receive second treatment with MPIF-1 or 5-Fu.
Peripheral blood was collected from the retroorbital sinus in EDTA-coated
tubes
and was immediately analyzed by FACS Vantage to determine platelet (CD4la
positive
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events) and granulocyte (Gra.l and Mac.l double positive cells) counts. It
should be
noted that the method of analysis and the species of animal employed here does
not
permit obtaining absolute counts. Instead, granulocytes are expressed as
percentage of
total white blood cells and platelets were estimated as CD41 a positive events
per 15
seconds on the sorter. Mice were then sacrificed to obtain bone marrow cells
using
standard methods. Bone marrow cells were also analyzed by FACS to monitor
percentage of Gra.l and Mac. l double positive populations of cells in the
bone marrow.
Since the stage at which these antigens begin to be expressed in the
granulocyte lineage
is not precisely known, Gra.l and Mac.l double positive cells in the bone
marrow are
expected to be heterogenous with regards to the stage of their development and
maturation potential.
Bone marrow was also analyzed to determine the frequency of clonogenic
progenitors using an in vitro clonogenic assay. Briefly, High Proliferative
Potential
Colony forming Cell (HPP-CFC) and Low Proliferative Potential Colony Forming
Cell
(LPP-CFC) assay was performed in a two-layered agar culture system. The bottom
layer
was prepared in 3.5 cm diameter dishes with 1 ml of MEM supplemented with 20%
FBS, 0.5% Difco agar, 7.5 ng/ml mIL-3, 75 ng/ml mSCF, 7.5 ng/ml hM-CSF and 15
ng/ml mIL-1 a. This layer was then overlayed with 0.5 ml of murine hone marrow
cell
suspension to have 2,000 cells/dish in MEM with 20% FBS and 0.3% agar. The top
agar was allowed to solidify at room temperature for about I 5 minutes. The
dishes were
then incubated for 14 days in a tissue culture incubator (37~C, 88% N2, 5%
COz, and 7%
Oz) and colonies were scored under an inverted microscope. In this experiment
total
colony counts are reported.
FACS data were generated by analyzing material obtained from three animals
of each of the groups per time point, whereas the clonogenic assay was
performed with
cells obtained from six animals of each of the groups per time point. Finally,
data points
for the day 1 group of the experiment represents values obtained from the
saline injected
normal mice (Group 1 ).
Results: To monitor the recovery of platelets in the peripheral blood, the
steady state
levels of CD41 a positive cells was determined by FACS Vantage. As shown in
Figure
51, MPIF-1 treatment prior to 5-Fu (Group 3) resulted in a much faster and
stronger
recovery of platelets than that observed in mice treated with 5-Fu + saline
(Group 2).
As expected the kinetics of platelet recovery in mice treated with G-CSF
(Group 4) was
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indistinguishable from that observed in mice treated 5-Fu + saline. Also,
administering
G-CSF plus MPIF-1 to 5-Fu treated mice (Group 5) had little effect on the
overall steady
state levels of platelets when compared to that observed in mice treated with
MPIF-1
alone (Group 3). Thus, MPIF pre-treatment of mice prior to the 5-Fu treatment
resulted
in a rapid recovery of platelets in the peripheral blood.
The recovery of granulocytes in the peripheral blood was monitored by
quantitating the steady state levels of Gra. l and Mac. l double positive
cells in the blood.
As illustrated in the Figure 52, 5-Fu treatment of mice resulted in a sharp
decrease in the
steady state levels of Gra.l and Mac.l double positive cells in the blood at
six days after
the first as well as the second 5-Fu treatments. MPIF-1 pre-treatment had two
beneficial
effects; the degree of neutropenia (the extent of depletion of Gra.l and Mac.l
double
positive cells) was much smaller and the rate of recovery was much faster
compared to
that observed in mice treated with 5-Fu + saline (Group 2). As expected, the
administration of G-CSF after 5-Fu treatment (Group 4) resulted in a rapid
recovery of
Gra. I and Mac. I double positive cells in the blood. However, the extent of
the recovery
from neutropenia in the G-CSF treated mice was notably smaller than that
observed in
the MPIF-1 treated mice on day 8 (Group 3). The effect of administering MPIF-1
plus
G-CSF (Group 5) on the granulocyte depletion and recovery was quite dramatic
in that
these mice displayed much higher steady state levels of Gra. l and Mac. l
double positive
cells in the blood than that observed in mice treated with either MPIF-1 or G-
CSF alone.
Thus, as ir_dicated in FIG. 52, it appears that MPIF-1 and G-CSF may exert
additive
effects when they are co-administered.
As indicated above, recovery at the level of the bone marrow was monitored by
FACS Vantage method and clonogenic assay. Results obtained with FACS are
illustrated in Figure 53. As expected, the level of Cira.l and Mac.l double
positive
population of cells in the 5-Fu treated marrows (Group 2) remained remarkably
depressed from days 6 through 14 and then recovered to normal level by day 16.
This
effect of 5-Fu mediated depletion of Gra.l and Mac.l double positive cells was
completely abrogated when mice were treated with MPIF-1 prior to 5-Fu (Group
3).
Surprisingly, G-CSF (Group 4) was able to prevent the depletion of the Gra. l
and Mac. l
double positive cells in response to the first S-Fu dose, but not the second.
This is likely
due to the availability of G-CSF target cells and the timing of G-CSF
administration.
A similar response was evident in mice that were treated with MPIF-I plus G-
CSF
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(Group 5), although the extent of recovery on day 8 post first 5-Fu was much
higher than
that observed in mice treated with either MPIF-1 or G-CSF alone.
Data from the clonogenic assay are presented in Figure 54. The frequency of
progenitors in the bone marrow remained depressed in response to 5-Fu
throughout the
fourteen days of the experiment period with a hint of recovery on day 16. This
reduction
in the frequency of the progenitors was abrogated in mice that were treated
with MPIF-1
prior to 5-Fu. In contrast, G-CSF treatment of mice was not effective in
sustaining the
frequency of progenitors found either in normal or MPIF-1 treated marrows. The
effect
of administering G-CSF plus MPIF-1 on the progenitor frequency in the bone
marrow
appears to be complex.
Example 29
Amelioration oJlupus nephritis in MRL Iprllpr mice by rhlll CIF treatnlent
Systemic lupus erythematosus (SLE) is a mufti-organ-associated autoimmune
disease characterized by the overproduction of pathogenic autoantibodies, and
the
formation of complement-fixing immune aggregates capable of inducing life-
threatening
glomerulonephritis and vasculitis (Steinberg, A.D. and Klinman, D.M., Rheum.
Dis.
Clinics oJNo. Amen. l4:25 (1988)).
MRL lpr/lpr mice, due to a mutation in the apoptotic fas receptor (Watanabe-
Fukunaga, R., et al., Nature 356:314-317 (1992)), spontaneously develop an
autoimmune disease with important similarities serologically and
immunopathologically
to human SLE (Andrews, B.S., et al., J. Exp. Med. 148:1198 ( 1978)). Extensive
characterization of this marine model has provided many insights into the
pathology of
human lupus, including high autoantibody titers to a variety of autoantigens,
glomerulonephritis, arthritis, vasculitis and premature death (Theofilopoulos,
A.N. and
Dixon, F.J., Immunol. Rev. 55:179 ( 1981 ); Tarkowski, A. et al., Cl in. Exp.
Immunol.
72:91 (1988)).
Abnormal macrophages and other cellular and molecular defects in MRL lpr/lpr
mice are implicated in the pathogenesis of autoimmune disease (Cohen, P.L. and
Eisenberg, R.A., Annu. Rev. Immunol. 9:243-269 (1991)). MRL Ipr/lpr mice have
an
increased number of peritoneal macrophages, which are in a more activated
stage than
the macrophages from normal mice (Kelly, V.E. and Roths, J.B., J. Immunol.
129:923
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(l982); Dang-Vu, A.P., et al., J. Immunol. 138:1757 (l987)}. In addition, MRL
lpr/lpr
macrophages produce higher levels of the proinflammatory cytokines such as IL-
1 and
TNF-a. Macrophages are rarely present in normal renal glomeruIi, but can be
found in
MRL lpr/lpr glomeruli before proteinuria and are more prominent as the
S glomerulonephritis progresses (Boswell, J.M., et al., J. Immunol. 141:3050
(1988)).
rhM-CIF, a new beta-chemokine, has weak chemotactic activity and is inactive
on most leukocytes except that it induces monocyte intracellular Ca~ flux via
receptors
shared with MIP-1 a and RANTES (Schulz-Knappe, P., et al., J. Exp. Med.
183:295
( I 996)). In addition, rhM-CIF has a strong selective inhibitory effect on M-
CSF-
induced promonocytic colony formation (Kreider, B.L., et al., "A beta-family
chemokine which specifically inhibits M-CSF mediated colony formation." Oral
presentation at First Joint Meeting of the International Cytokine Society and
the
International Society for Interferon and Cytokine Research ( 1996)). Our early
in vivo
work demonstrated that rhM-CIF has a significant protective effect on LPS- or
live
E coli bacteria-induced macrophage-mediated lethal sepsis, which is at least
in part due
to its reduction of TNFa and increase of IL-10 serum levels in mice (Zhang,
J., et al.,
"Selective modulation of TNF-a and IL-10 by rhM-CIF (HCC-1 ) correlated with
its
protective effect on LPS-mediated lethal sepsis in SCID mice. Oral and poster
presentations at Keystone Symposia, The Role of Chemokines in Leukocyte
Trafficking
and Disease ( 1997)). A significant ameliorative effect of rhM-CIF on moderate
and
progressive joint damage has also been observed in both murine collagen-
induced
arthritis and rat adjuvant arthritis models (Zhang, J., et al., "Protection of
Progressive
Joint Destruction by rhM-CIF (HCC-1 ) in a Rat Model of Adjuvant Arthritis."
Poster
presentation at ILAR Congress of Rheumatology ( 1997); Sturm, B., et al. ,
2S "Ameliorative effect of rhM-CIF (HCC-1) on collagen-induced arthritis in
mice."
Abstract submitted for 61 st National Meeting of American College of
Rheumatology
( 1997)).
In the present study, we examined the possible effect of rhM-CIF on this
spontaneous lupus model in MRL lpr/lpr mice. Preventive treatment with rhM-CIF
for
the entire course of lupus nephritis development significantly ameliorated the
glomerular
lesions and nephrosclerosis, and may have protected kidney function by
reducing protein
cast formation. Neither rhM-CIF nor methotrexate had any significant effect on
premature death, probably as a consequence of the severity of disease in other
organs
beside kidney.
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Materials and Methods
Animals
Female MRL lpr/lpr mice were purchased from The Jackson Laboratory (Bar
Harbor, ME) and maintained according to recommended standards at the HGS
animal
facility for at least one week before being employed in experiments.
Chemicals
Amethopterin (methotrexate) was purchased from Sigma Chemicals (St. Louis,
MO). Saline solution was obtained from Abbott Labs (North Chicago, IL).
Recombinant human M-CIF (batch B9) was expressed in baculovirus vector and
purified
by SDS-PAGE with a molecular weight of 8.677 Kd. The protein was then
dissolved
in a buffer consisting of 40 mM NaOAc, 150 mM NaCI, pH 5.5 with an endotoxin
level
less than 0.2 EU/mg.
Experimental design
Fifty MRL lpr/lpr mice, 8-9 per group, were administered rhM-CIF in buffer or
methotrexate in saline ( 1 or 5 mg/kg, i.p.) daily, Monday-Friday, each week
for 14
weeks beginning at 8 weeks of age when there is no sign of disease. Mice
receiving
buffer or saline served as the disease control. Animals were monitored for
clinical
symptoms and morbidity weekly or biweekly until the lethality rate reached 50%
in the
buffer treated group. All of the remaining animals were sacrificed at the end
of the
experiment for the histopathological evaluation of kidneys.
Histopathology analysis
Both kidneys were removed and immediately placed in 10% neutral buffered
formalin for the procedure of paraffin embedding and sectioning. Tissue
sections were
stained with hematoxylin and eosin, PAS and trichrome for comprehensive
examination.
The pathological evaluation of the kidney lesions were conducted with a mufti-
blinded
procedure. According to the general impression after viewing all the slides, a
subjective
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scoring system was employed that allowed differentiation of degree and
distribution of
the changes by giving 0, +/-, +, ++, +++, ++++ to the following
histopathological
features:
1 ) glomerular lesions such as irregular hypercellularity and enlargement, the
S basement membrane thickening, karyorrhexis, fibrosis and hyalinization,
crescent
formation and fibrosis of the Bowman capsule;
2) tubular lesions including the formation of protein casts in the lumens of
the
tubules (for the convenience of assessment of the lesion severity, protein
cast formation
was classified here, even though it also contributed to the increased
permeability of the
basement membrane of glomerular capillary tufts) and substantial atrophy and
compensatory hypertrophy and dilatation of the tubules;
3) the interstitial lesions including inflammatory infiltration, lymphocytic
perivasculitis, interstitial fibrosis;
4) gross appearance such as granular nephrosclerosis.
I S The semiquantification analysis was done by using the following scoring
systems:
0=0;+/-=l; +=2;++=4;+++=6;and+++-~=8.
Upon completion of individual histopathological evaluation, the slides were
decoded and matched to each group for interpretation of the patological
changes with
respect to the regimen of tratement.
Macrophage immunohutocl:emical analysis
Paraffin sections from MRL lpr/Ipr kidneys were stained for macrophages using
a specific rat monoclonal antibody against mouse (F4/80) macrophage antigen
(Caltag
Labs, San Francisco, CA) followed by a standard immunohistochemistry
technique. The
criteria for evaluating the degree of macrophages infiltration in the kidney
were similar
to those mentioned above (0 to ++++).
Statistical analysis
Percentage of surviving mice was calculated as number of living mice/total
mice
x l00%. Analysis of the histopathology data was performed only for the groups
with
no less than 4 surviving mice at the end of the experiment. Mean scores, SEM
and P
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values for different pathological features were calculated individually or in
combination
by InStat statistical software.
Results
Effect of rhM CIF on the survival of MRL lprllpr mice
Spontaneous autoimmune MRL lpr/Ipr mice were treated with buffer or
methotrexate in saline ( 1 or 5 mg/kg, i.p.) daily, Monday-Friday each week
for 14 weeks
when the B 11 batch of rhM-CIF was exhausted. Compared with the buffer treated
group, which served as a disease development control, rhM-CIF treatment showed
a
similar survival rate during the 16-week-experiment period, except that at the
end of the
experiment when the buffer treated group reach 56% lethality, there was a 63%
survival
rate in the rhM-CIF 1 mg/kg treated group (FIG. 55). Similar to rhM-CIF
treatment, the
group treated with the low dose of methotrexate ( I mg/kg) showed a parallel
pattern to
the saline control group until the last week of the experiment when there was
about 20%
protection. However, the group treated with the high dose of methotrexate (5
mg/kg)
showed an accelerated death rate, which may be due to its accumulated toxicity
(FIG.
56).
Reduction of protein casts in MRL Iprllpr kidney by rltM CIF treatment
Protein cast information as measured by histopathological evaluation was
selected as an alternative measure for proteinuria to assess renal function.
Only three
groups (buffer, 1 mg/kg of rhM-CIF and methotrexate) had 4 mice surviving at
the end
of this pilot experiment, which qualified for their subsequent histology
analysis. As
shown in FIG. 57, the buffer control group revealed a great amount of the
protein and
cellular casts in Henle's loops, the distal convoluted tubules, the collecting
tubules and
the tubular lumens of MRL lpr/lpr kidneys. In contrast, only a small amount of
protein
casts was found in the rhM-CIF (1 mg/kg) treated mice; and one mouse in the
methotrexate ( 1 mg/kg) group showed a severe protein cast formation while the
other
three mice remained totally free of this pathological feature. The reduction
in the
formation of protein casts by rhM-CIF is statistically significant (p=0.02)
when
compared with the buffer group.
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Ameliorative effect of rhM CIF on the glomerular lesions
The glomerular lesions, especially the lesions of capillary tufts and the
basement
membrane of the glomeruli, are the essential features seen in this lupus
model. Half of
the surviving animals (two mice) in the buffer control group showed extremely
severe
damage in almost all of the visible glomeruli, which were represented by hyper-
proliferation of macrophages in the Bowman's capsule and extensive formation
of
crescents; mesangial cell proliferation in the tufts demonstrated by PAS
staining;
karyorrhexis due to nuclear breakdown, thickening of the basement membrane
resulting
in "wire-loop lesion", leakage of red blood cells to the Bowman's capsule, and
fibrosis
and/or hyalinization. In addition, complete or partially dysfunctional
glomeruli
extensively spread in the cortex region. Crescent formation, adhesion of the
parietal and
visceral layers, and fibrosis of the Bowman's capsule were as severe as the
glomerular
lesions in terms of percentage. The other two mice in this group showed much
less
severe lesions. In contrast, most of the surviving mice (3/5) treated with rhM-
CIF (I
mg/kg) showed about 50% severity of the buffer controls, and the others (2/5)
showed
only very mild damage or were without obvious glomerular lesions. Similar mild
lesions were also seen in most of the surviving mice ('/4) with methotrexate
treatment,
except that only one animal showed relatively severe glomerular lesions. The
reduced
glomerular lesions in rhM-CIF treated mice are statistically significant
(p=0.0l ) in
comparison with the buffer control (FIG. 58).
rhM CIF retarded the development of nephrosclerosis
The progressive and long standing process of lupus glomerulonephritis
eventually leads to nephrosclerosis due to focal atrophy and compensated
hypertrophy
of glomeruli and tubules. This late stage feature was severe in two of the
four mice
treated with buffer control, and the surface of their kidneys showed punctate
scarnng
resembling grained leather. The other two mice did not show such advanced
changes.
Comparatively, none of the mice in rhM-CIF treated group showed obvious
nephrosclerosis, except that milder atrophy of tubules was observed
occasionally.
Furthermore, only one mouse in the methotrexate group (n=4) developed an
advanced
state of the sclerotic feature with the other three mice remaining free of
nephrosclerosis.
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Statistical analysis indicates that the severity of nephrosclerosis in the
buffer control was
significantly higher than that of rhM-CIF and methotrexate treated groups
(FIG. 59).
rhM CIF moderately inhibited the macrophage infiltration in MRL lprllpr
kidney
The presence of macrophages in MRL lpr/lpr kidneys is an important sign of
progressive glomerulonephritis. Immunohistochemical analysis showed that
macrophages extensively infiltrated the kidney of the buffer treated mice. The
severity
was parallel to the other pathological features as described above. The two
mice with
significant kidney lesions showed most severe macrophage infiltration. They
appeared
in the glomerular crescents mixed with the proliferating epithelial cells of
Bowman's
capsule, in the periglomerular area, interstitium of the nephrons and the
areas of
perivascular infiltrates. The other three mice in this group showed moderate
or mild
macrophage infiltration which was also parallel to their lesion severity.
However, two
mice with mild histopathological lesions from the methotrexate treated group
revealed
1 S relatively higher scores of macrophage infiltration. The most severe mouse
in this group
also showed the most severe infiltration of macrophages. In the rhM-CIF group,
the
appearance of the macrophage seems moderately suppressed compared to the
buffer
group, even though the p value was 0.08, marginally significant statistically.
Lack of effect of rhM CIF on lymphocyte infiltration and perivasculitis in
MRL lprllpr kidney
Massive and diffuse infiltration of lymphoid cells in the interstitial tissue
of the
kidney is another prominent pathological feature in this MRL lpr/lpr model.
These
infiltrates were seen mainly in the perivascular (larger arteries such as
interlobular
arteries or even the arcuate arteries) area, the periglomerular area, and the
interstitium.
The lymphoid cell infiltrates in the interstitium seem to be more severe in
the buffer
control because of massive atrophic tubules. However, a large amount of
infiltrate was
found in the periglomerular areas of rhM-CIF and methotrexate treated mice,
although
their glomerular lesions were very mild. The cell types of the infiltrates
vary. In the
perivascular area (especially around the larger arteries), most cells were
lymphoblasts
and mononuclearblasts. The whole picture of the infiltrates showed multiple
cellular
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WO 98I14582 PCTIUS97I17505
types of lymphoid cells, which were similar to the cellular classifications
seen in the
enlarged lymph nodes (data not shown). Semiquantification and comparison among
buffer, rhM-CIF and methotrexate treated groups showed no obvious difference
of
perivasculitis and periglomerulitis (FIG. 61).
Discussion
In this pilot experiment, protein cast formation evaluated by renal
histopathology
at the end of experiment was selected as an alternative feature to proteinuria
to assess
renal function. The preliminary results showed that preventive treatment of
rhM-CIF
for 14 weeks during the course of lupus nephritis development, resulted in
significant
reduction of protein cast formation, and amelioration of the glomerular
lesions and
nephrosclerosis in the 5-6 month old MRL lpr/lpr kidney. However, rhM-CIF
showed
little or no effect on nephritis and/or vasculitis-induced premature death.
The presence of abnormal activated macrophages in the MRL Ipr/lpr renal
glomeruli has been implicated in the pathogenesis of lupus nephritis; and
increased level
1 S of M-CSF mRNA transcripts in the kidney and M-CSF protein in the
circulation of
MRL lpr/lpr mice may be responsible for macrophage infiltration and activation
(Yui,
M.A., et al. ( Amer. J. Path. l39:255 ( 199l )). Although rhM-CIF treatment
showed a
moderate inhibition on macrophage infiltration in MRL lpr/lpr kidney, the
possible
effect of rhM-CIF on M-CSF-mediated renal macrophage function which causes
tissue
destruction remains unclear. However, previous studies indicated that rhM-CIF
is
effective in ( 1 ) inhibiting M-CSF-induced promonocytic colony formation, (2)
protecting LPS-induced macrophage mediated lethal sepsis, and (3) down-
regulating
TNF-a and up-regulating IL-10 systematically in vivo. All these consequences
may
provide supporting evidence for the ameliorative effect of rhM-CIF on lupus
nephritis.
Massive infiltration of lymphocytes in the interstitial tissue of the kidney
is a
unique pathological feature of MRL lpr/lpr mice from human SLE, which is
caused by
~s receptor deficiency in the clonal deletion of lymphocytes during any immune
or
autoimmune responses. Lack of effect of rhM-CIF on lymphocyte infiltration in
the
MRL lpr/lpr kidney suggests that rhM-CIF has no inhibiting effect on the
migration,
activation and clonal expansion of MRL lpr/lpr lymphocytes. Indeed, rhM-CIF
has been
shown to be chemotactic for activated T lymphocytes in vitro.
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Summary
Preliminary study showed that prolonged treatment of rhM-CIF, like
methotrexate, during the development of spontaneous autoimmune disease,
significantly
ameliorated the progression of lupus nephritis by reducing protein cast
formation, the
glomerular lesions and eventually nephrosclerosis in MRL lpr/lpr kidney.
However,
rhM-CIF, like methotrexate, showed little or no effect on nephritis and/or
vasculitis-
induced premature death.
Summary Preclinical Pharmacology Tables
The following tables (Tables 4, 5, and 6) summarize the in vitro and in vivo
primary and secondary pharmacology studies.
Table Key for Batches Referenced in Tables 5, 6, and 8. MPIF-1 batches are
designated by a multi-component code which indicates the organism the protein
was
expressed in and the form of the expressed product (e.g., mature, full-length,
or a
variant). Letters after a hyphen at the end of the designation indicate either
the organism
the protein was expressed in or the vector used for expression (i.e.,
B=baculovirus,
C=CHO cells, E=E. coli). The last three digits preceding the hyphen indicate
the form
or variant of the protein expressed (i.e., 300=full-length MPIF-1, 30l=the
MPIF-1D17
variant, 302=mature MPIF-1 with a methionine residue added to the amine
terminus of
the mature amino acid sequence, 304=the MPIF-1D23 variant, 31 I=full-length
MPIF-I).
Thus, the batch designation indicates the form of the expressed MPIF-1
protein, whether
the protein will be secreted from the host cell, and the form of the secreted
protein, if
any. For example, HG00300-BS indicates that the full-length MPIF-1 protein was
expressed using a baculovirus vector. Further, since MPIF-1 expressed using
this
system is processed by the insect host cells, the secreted form of this
protein is mature
MPIF-1.
One exception to the above noted nomenclature occurs with batch HG00300-B7.
This batch contains a mixture of four different MPIF-1 polypeptides. The
inventors
believe that these polypeptides were produced as a result of proteolytic
cleavage of
MPIF-1 which occurred during the purification process. The MPIF-1 variants
present
in batch HG00300-B7 are discussed in Example 17.
-162-
Table 4~ Primary Pharmacology In Vitro
Ezperimental Design ~ Cell MPIF-1D23 Chemical Results
Type Dose ~ Agent '
~ ~
o
o
Effect of MPIF-1 or MPIF-102311PP-CFC0.0l - I00 NA Both MP1F-I and
MPIF-1023 caused
on colony ng/mL a dose dependent
formation using mouse LPP-CFC reduction of the
frequency of LPP-CFCs.
bone marrow
MPIF-1d23 was significantly more
efFective than MPIF-I at
all concentrations tested.
Neither isofortn had a significant
effect on the frequency of
HPP-CFCs.
Effect of MPIF-1D23 on CD34', I - 1000 ng/mLNA MP1F-1023 treatment
resulted in
the proliferation of 20% to 40% inhibition
of
human hematopoietic progenitorhuman cell survival.
cells cord
blood The results suggest that MPIF-1
O23 is a myeloid progenitor
inhibitory factor.
Determination of the specificCD34', 50 ng/mL NA MPIF-1023 inhibits
(50% to 64,6) a
progenitor cell the formation of CFU-
GM
types targeted by MPIF-IA23human and CFU-A~lix.
Formation of BFU-E, CFU-G, CFU-M,
and CFU-Meg were
not inhibited.
The results define MPIF-1 d23 as
an inhibitor of human
o,~
granulocyte/monocyte precursor '''
cells.
i
(..
Characterization of the Mouse 50 ng/mL NA MPIF-I A23 reduced the
frequency
inhibitory effects of bone of myeloid CFU-GM
1 ~ MPIF-1 A23 on mouse bone marrow colonies to
30'0 of control.
marrow
The frequency of LPP-CFC colonies
was reduced to 24% of
control.
w
MPIF-1 D23 did not inhibit the
formation of CFU-E, BFU-E
and HPP-CFC colonies.
Determined the ability Mouse NA 5-FU MP1F-1 A23 protects
40% to 50,6
of MPIF-1 A23 to protect bone of LPP-CFC from
lineage-depleted populationsmarrow cytotoxicity
induced by 5-FU.
of bone marrow
cells from the cytotoxic MPIF-1D23 did not
protect HPP-CFC.
effects of 5-FU
'b
f7
~o
Dose) 0
MPIF-1 MPIF-1 ChemicaSchedule) o~~o
Dose,
Ex erimental DesiS Batch Schedule) I A Route End oint
'"'
n ecies Route ent
a
In vivo effects MouseHG00300-BS0.5 mg/hg!NA NA MPIF-1 D23
significantly reduced
of MPIF-1 or the frequency of LPP-
CFC in
MPIF-1D23 on the IIG00304-E2injection bone marrow.
frequency twice
a
$ of HPP-CFC and day at The effects
of MPIF-1023 on the
LPP-CFC in 8 hour frequency of LPP-CFC in
peripheral blood intervals blood were variable.
and bone for
marrow 2 days, MPIF-1423 had no effect
on the frequency
i.p. of HPP-CFC.
Determination MouseHG00304-E21 mg/kg, 5-FU l50 MPIF-1023 given on
Days -2, -1,
of the optimal variable mg/I:g,and 0 was most
effective in
MPIF-1023 dosing between Day protecting bone marrow
against the
schedule Days -3 0, cytotoxic effects of 5-
FU.
i.p.
1 ~ for protection and 0,
against the i.p.
cytotoxic effects
of 5-FU
Determination MouseHG00304-E60.01 to 5-FU 150 A dose-dependent
response was observed
of dose 10 mg/kg, mg/kg, on Day 4, with the best
dependency of i.p. on i.p. recovery occurring at
the lowest
MPIF-1 D23 on dose tested (0.01
mg/kg).
i bone marrow recovery Days -2, No dose
response was observed on
after -1, 0 Day 6.
I S 5-FU A bell shaped
dose-response curve
was obtained on Day 8, with
i
optimal activity observed at 0.1
mg/kg.
Determination MouseHG00304-E6I mg/kg; 5-FU I50 Colony formation from
bone marrow
of the ability mg/I:g,of mice treated with
of
MPIF-1023 to protect Days -2, Day MPIF-1023 returned
to normal 7 days
myeloid -I, 0, 0, after treatment with 5-
FU.
i.p. i.p.
progenitors in Bone marrow colony
formation from
vivo from mice treated with 5-FU
cytotoxic therapy alone showed no
recovery at this
time.
Determination MouseHG00304-E6I mg/lcg, 5-FU I00 MPIF-1 Q23 protected
progenitor
of the protective mg/kg, cells after two cycles
of 5-FU.
effect of MPIF-1023 Days -2, Days The most dramatic
protection was
against -1, 0. 0 and seen after the second
cycle of
6,
multiple cycles 7, 8 8, i.p.5-FU.
of
chemotherapy The chemoprotective
effect of MPIF-1023
was manifest in the
periphery by increased numbers of
hematopoietic-derived CD45'
cells in blood.
b
C7
H
~o
0
Table 5. Primary Pharmacology-In Vivo
Dose,
MPIF-1 lItPIF-1 ChemicaSchedule,
Dose,
Ex erimental DesiS Batch Schedule, I A Route End oint
n ecies Route ent
00
Determination Mouse IiG00304-E61 5-FU 100 mg/kg,The degree
p
of the ability mg/kg; of neutropenia
of as measured
by the
depletion
of Gr-I
MPIF-1423 to accelerate Days Days and lvlac-I
-2, 0 and double
-1, positive
0, cells was
6, significantly
less and
the
recovery of bone 7, 8, i.p. rate of
marrow 8, recovery
i.p. more rapid
in mice
treated
with MPIF-I
and
colonies, neutrophils 5-FU compared
and with that
in mice
treated
with 5-FU
alone.
platelets after G-CSF 0.5 mg/l:g,Treatment
multiple cycles with G-CSF
after 5-FU
resulted
in a rapid
recovery
of
of chemotherapy Days double positive
1) 2, cells in
3, the blood.
The extent
of recovery
in
Determination 9. 10, G-CSF treated
of the activity and mice was
of 1 1 markedly
less than
that observed
in
MPIF-1423 in combination MPIF-I treated
mice on
Day 8.
with G-CSF Mice treated
with MPIF-1
and G-CSF
had higher
steady
state
levels of
positive
cells in
the blood
than those
treated
with either
MPIF-I or
G-CSF alone.
There was
a marked
decrease
in
colony formation
from the
bone marrow
of mice
treated
with
5-FU.
hiPIF-1
treatment
prior to
5-FU abrogated
the effect
of 5-FU
on
colony formation.
There was
a more
rapid and
stronger
recovery
of plateleu ''
in mice
treated
with MPIF-l
and 5-FU
relative
to that
seen in
mice treated
with 5-FU
alone.
Addition
of G-CSF
had no
t further
effect.
~~' 10 Table 6. Secondaryarmacol oy-In ro
Ph Vit
Ex erimental Desi Cell I~IPIF-1 MPIF-1 Results
n Tv Batch Dose
w
a Ran
a
Determination T fI(i00300-f37 I to
Detectable responses o
of calcium mobilization cells, 1000
were observed in
f3 ng,~mL
rells,
by MPIF-1 or MPIF-1423 monocytes, IIGt)0302-E2
monocytes and dendritic
neutrophils, cells at
bwsophils, f Iti00302-E3 l00 ng/mL.
dendritic
cells,
'~K IIG0030a-E2 The monocy1ie cell line
cells THP-1 responded
THP-1
cells
IiG0030~1-E3 to MPIF-1423 with a
maximal effect at
HG0030a-E6 100 ng/mL.
IfG0030-t-E7
IiG00301-CI
"d
HG0031 C7
I
-C
I
~3
~o
U
0
O
Experimental Design ~ Cell Type ~ MPIF-1 Batch~ MPIF-I Dose Range~ Results
Determination of the T cells, monocytes,HG00300-BS 0.1 to I000 ng/mL MPIF-
1023 stimulated
chemotactic chemotaxis
in
activity of MPIF-1023.neutrophils, HG00300-B7 resting T
cells with
lymphocytes, a maximal response
at
eosinophils, HG00302-E 10 ng/mL.
basophils, I
NK
cells, plateletsHG00302-E2 MPIF-1 Q23 was chemotactic
for freshly
HG00303-E isolated monocy~tes
I with a maximal effect
(1G00304-E2 at 100 nglmL.
HG00304-E6 A weak chemotactic response
was
HG00304-E7 observed in neutrophils.
There was no
response in the other
cells tested.
Effect of MPIF-1 or Monocytes HG00300-B7 0.5 to 1000 ng/mL MPIF-1023
induced a
MPIF-1023 on low but
variable
monocytes HG00302-E release of
lysosomal
1 N-acetyl-(i-D-
IiG00302-E2 glucosidase from freshly
isolated
HG00302-E3 monocytes.
((G00304-E3 MPIF-1023 had no effect
on the release
HG00304-E6 of the lysosonal enzymes
elastase,
i HG00301-CI
glucuromidase, and myleperoxidase.
HG0031 I-CI MPIF-1023 does not induce
monocytes
o~, to
secrete IL-I Vii,
TNF-a, IL-10, or IL-12.
MPIF-1023 had no effect
on oxidative
burst or cyrtotoxic
activity of activated
macrophages.
$ Effect of MPIF-1 or Basophils, HG00300-BS 1 to 1000 ng/mL
MPIF-1 and MPIF-123
MPIF-1023 on human did not
induce
histamine release HG00300-B7 histamine
release from
basophils.
HG00302-E2
HG00304-E6
Effect of MPIF-1 or NK cells, humanHG00302-E 1 to 100 ng/mL MPIF-1 and
MPIF-1 D23
MPIF-I D23 on t had no
effect on
NK cell-mediated killing HG00300-B7 IL-2
stimulated NK cell-mediated
killing
of K562 cells.
n
H
Effect of MPIF-1 or Platelets, HG00302-E 0. I to 100 ng/mL MPIF-1 Q23
did not induce
MPIF-I023 on human 1 or modulate
1 ~ platelet aggregation HG00300-B7
platelet aggregation.
0
Table 6. Secondary Pharmacology-Irt Vitro (continued)
Ex erimental Desi Cell T a MPIF-I Batch MPIF-1 Dose Ran Results
n a
Effect of MPIF-1 Q23 Fibroblasts, HG00300-B7 0.1 to 1000 ng/mL MPIF-l D23
did not induce,0
on the growh of astrocytes, enhance, or
non-transformed humanSchwann cells,HG00300-BS inhibit the
proliferationo~~o
cells smooth of the
cells Listed
muscle cells, HG00302-E1 studied.
epithelia)
cells, vein ~o
and
microvascular
endothelial
cells, bone
marrow, B
cells) T cells,
monocytes,
neutrophils,
keratinocytes
Effect of MPIF-1 or human primary f IG00300-BS 0.1 to I000 ng/mL MPIF-1 and
MPIF-1023
MPIF-I023 on had no
effect on
the release of IL-6 endothelial HG00300-B7 release of
IL-6 or prostaglandins.
and prostaglandins cells, lung
fibroblasts) HG00302-EI
and aortic
smooth muscle HG00300-E2
cells
HG00304-E2 n
HG00301-C
I
0
$ Effect of MPIF-1D23 Primary microvascularEIG00304-E2 0.1 to I00
ng/mt. MPIF-IA23 did not induce
on formation of the
formation
capillaries endothelial of
capillaries in vitro.
cells y .
cr Effect of MPIF-1 on Primary endothelial11G00300-B7 0.1 to 10 nglmL
No effect.
ability of tumor cells
cells to infiltrate
through a confluent
monolayer of endothelial
cells
1 ~ Effect of MPIF-1 or Priri~an~ endothelialHG00300-B5 0.1 to 100
mg/m1. No effect. o
MPIF-1023 on cells
w
adhesion of peripheral HG00300-BS
blood
mononuclear cells IIG00304-E6
0
or granulocytes to
IL-1 activated endothelium IIG00304-E7
HG00301-C
1
b
n
0
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Example 30
Production, Recovery, and Purification of MPIF 1d23 Using the pHE4-5
Expression
Vector
MPIF-1 is a novel human (3-chemokine. The mature form of MPIF-1 is
secreted as a 99 amino acid peptide, with a molecular mass of I 1.2 kDa. A
truncated
form (MPIF-1023) 76 amino acids in length was also identified during initial
expression
studies of MPIF-1. In a baculovirus expression system, MPIF-1023 was
subsequently
isolated and subcloned. Biological assays indicate that the truncated form is
more active
than the full length counterpart.
Cloning and Expression
The MPIF-1O23 gene originally isolated from an aortic endothelial
complementary deoxyribonucleic acid library has been subcloned into the
expression
vector pHE4 at the single restriction enzyme cleavage sites NdeI and Asp 718
(FIG. 62)
and has been transformed into the K12 derived E. coli strain SG I3009
(available from
Susan Gottesman, National Institutes of Health, Bethesda, MD.). Additional
strains of
E. col i which may serve as suitable hosts for protein expression using pHE4
include
strains DHSa and W3110 (ATCC Accession No. 27325). The pHE4 vector contains a
strong synthetic promoter with two lac operators. Expression from this
promoter is
regulated by the presence of a lac repressor, and is induced using isopropyl B-
D-
thiogalactopyranoside (IPTG) or lactose. The plasmid also contains an
efficient
ribosomal binding site and a synthetic transcriptional terminator downstream
of the
MPIF-l O23 gene. The vector also contains the replication region of pUC
plasmids and
the neomycinphosphotransferase gene resulting in kanamycin resistance in
transformed
bacteria.
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CA 02267193 1999-03-30
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Method of Manufacture
Overview of Fermentation Process
The fermentation process for MPIF-1023 is outlined in following stages and
is illustrated in FIG. 63.
Master Seed Bank
A master cell bank (MCB) of E. col i transformed with the plasmid expressing
MPIF-1023 was prepared under current Good Manufacturing Practices. The bank
was
prepared in media containing glycerol as a cryopreservative, and frozen at -
80~ C. After
preparation, the MCB was tested to assure the absence of phage or
contamination with
other micro-organisms.
First Seed Stage
First seed stage culture is prepared in a baffled shake flask containing
inoculum preparation medium. The shake flask is inoculated at a 1:2000
dilution with
thawed seed stock and is placed in a shaker maintained at 225 rpm and 3 7 ~ C
for 12
hours.
Production Phase
Production Phase culture is prepared in a 100 liter Fed-Batch fermenter
equipped with DO2, pH, temperature and nutrient feed control. The production
medium
(37~C) is inoculated with first seed stage culture to provide an optical
density (OD) of
0.20 units per milliliter at 600 nm. When the culture reaches an OD of 10 plus
or minus
. 2 units per milliliter at 600 nm, protein expression is induced with the
addition of IPTG
(final concentration 20 mM). Cells are harvested 4 hours after induction.
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CA 02267193 1999-03-30
WO 98I14582 PCT/US97117505
Cell Harvest Phase
Bacteria are recovered by centrifugation at 18,000 g using a continuous flow
centrifuge. The resulting cell paste is stored at -80 ~ C.
Recovery of MPIF 1 d23
The recovery of MPIF-1O23 is outlined in FIG. 64.
Cell Lysis
The E. coli cell paste is thawed and resuspended in ten volumes of
resuspension buffer. Cells are then disrupted following their passage (twice)
through
a homogenizer at 7000 psi.
Inclusion Body Wash
NaCI is added to the cell lysate to a final concentration of 0.5 M and then
concentrated two-fold by tangential flow filtration using a 0.45-pm membrane.
The
remaining retentate is diafiltered against three volumes wash-2 buffer (100 mM
Tris-
HCI, S00 mM NaCI, and 25 mM EDTA-Na2), followed by one volume wash-1 (100 mM
Tris-HCI, 25 mM EDTA-Naz). The retentate is diluted two-fold with wash-1
buffer, and
the insoluble fraction is collected by continuous centrifugation.
Alternatively, inclusion
bodies can be washed by centrifugation.
Inclusion Body Solubilization
The resulting pellet obtained following centrifugation is suspended in an
equivalent of nine packed inclusion body volumes of solubilization buffer (
100 mM
Tris-HCI, 1.75 M Guanidine-HCI, and 25 mM EDTA-Naz). The suspension is stirred
initially for 2 to 4 hours at room temperature, and then for 12 to 18 hours at
2 ~ to 10 ~ C .
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WO 98/14582 PCT/US97117505
Refold
The suspension is centrifuged, and the supernatant is collected and mixed
with nine volumes of refold buffer ( 100 mM Sodium Acetate, 125 mM NaCI, and 2
mM
EDTA-Naz). The diluted material is kept for about two hours (2 ~ to 10 ~ C) to
allow the
precipitate to settle. The material is filtered and then may be processed
immediately or
stored for up to 72 hours and then processed.
Purification
HS SO Cation Exchange Chromatography
The purification of MPIF-1D23 is outlined in FIG. 65. The filtrate is loaded
onto a POROS HS-50 column equilibrated with 50 mM NaOAc, 1 SO mM NaCI, pH 5.8
to 6.2. The protein is eluted in a stepwise manner with NaCI (300 to 1500 mM).
Fractions are eluted with S00 mM NaCI are pooled and are diluted two-fold with
water
for injection.
HQ-SOlCM 20 AnionlCation Exchange Chromatography
Pooled fractions obtained following HS-50 chromatography are loaded onto
a tandem set of columns (HQ-50 column followed by CM-20 column) equilibrated
with
CM-1 buffer. MPIF-1 D23 is eluted from the Ctvi-20 column with NaCI ( 100 to
900
mM). Eluted fractions are analyzed by sodium dodecyl sulfate-polyacrylamide
gel
electrophoresis (SDS-PAGE) and reverse-phase high-performance liquid
chromatography (HPLC), those fractions containing MPIF-1 D23 are pooled and
concentrated by ultrafiltration or passage through an additional HS-SO column.
-171-
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WO 98/14582 PCT/US97/17505
Size Exclusion Chromatography
The CM-20 eluate is loaded onto a Sephacryl-100 HR equilibrated with S-
l00 buffer. Fractions are collected and analyzed by SDS-PAGE and reverse-phase
HPLC. Fractions containing MPIF-1023 are pooled, sterile-filtered using a 0.2
pm filter
and stored at 2 ~ to 10 ~ C.
Specifications for Bulk Substance
The following specifications, listed in Table 7, have been established for
bulk
MPIF-1023.
Table 7: Tests and Tentative Specifications for Release of
Bulk MPIF-1023
Description Specification
Appearance Clear, colorless
solution
pH 5.8 t 0.2
Protein concentration by i-5 mg/mL
BCA
Purity*
Reverse-phase HPLC z 90%
Size-exclusion HPLC z 90%
SDS-PAGE (Coomassie blue
staning)
Reducing conditions s 90%
Non-reducing conditions s 90%
Residual DNA s 100 pg per mg
protein
Endotoxin s 10 EU per mg
protein
Limulus amoebocyte lysate
gel
clot
Bioassay (Assessed by Caz'Report results
mobilization assay)
* The purity of MPIF-1023 preparations will be compared to a standard
reference, the
specifications for which are currently being defined.
- I 72-
CA 02267193 1999-03-30
WO 98I14582 PCT/US97/17505
Specifications for Drug Product
The finished drug product meets all of the specifications as described for the
bulk
substance in Table 7, and is also tested for sterility (21 CRF610.12).
MPIF l d23 Mediated Inhibition of Colony Formation Correlates With the Ability
of
MPIF 1 to Mobilize Intracellular Ca1+ in Monocytes
MPIF-123 inhibits LPP-CFC colony formation in in vitro soft agar assays and
induces mobilization of intracellular calcium in monocytes including THP-1
cells
(human myelomonocytic cell line). Both assays have been used to assess
biological
activity of MPIF-1O23 in purification and stability studies. In the LPP-CFC
assay,
freshly isolated marine bone marrow cells are plated in soft agar in the
presence of
multiple cytokines (5 ng/mL IL-3, 50 ng/mL SCF, 5 ng/mL M-CSF, and 10 ng/mL
IL-1 a). Cultures are incubated for 14 days, after which time, colonies are
scored using
an inverted microscope.
Calcium mobilization assays use freshly isolated human monocytes or THP-1
cells
loaded with Fura-2 (0.2 nM per million). When cells are stimulated with MPIF-1
O23,
Caz' mobilization is assessed by a fluorimeter. The Ca'i mobilization assay
provides a
rapid indicator regarding the activity of the MPIF-1O23 preparation (Table 8).
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CA 02267193 1999-03-30
WO 98/14582 PCT/US97/17505
Table 8: MPIF-1O23 Mediated Inhibition of Colony Formation Correlates
With the Ability of MPIF-1 to Mobilize Intracellular CaZ+ in
Monocytes
Ca2+ mobilizationLPP-CFC
MPIF-1 Construct/Batch/Condition(n inhibition
/mL)* (n
/mL)t
g g
MPIF-1/HG00300-BS 1000 20-40
MPIF-1~23/HG00304-E2, 100 5-10
stored at
4C for 3 months
MPIF-1023/HG00304, storedl00 5-10
for 1
week
MPIF-1023/HG00304, stored100 5-10
for 4
weeks
MPIF-1 /I-IG00302-E2, 1000 > 100
stored at 4 C
for 3 months
MPIF-1~23/HG00304-E3, 100 5-10
first peak
from CM column
MPIF-1023/HG00304-E4, 100 5-10
second
peak from CM column
MPIF-1023/HG00304-E3, >l000 >I000
third peak
from CM column
* Minimum concentration required to mobilize calcium in human monocytes and/or
THP-1
cells.
~ Concentration producing SO% inhibition of LPP-CFC colony formation compared
to the
control.
Formulation and Storage
Bulk MPIF-1023 is manufactured aseptically, and the liquid formulation is a
sterile, single-use, product. The protein is buffered in 50 mM sodium acetate,
12S mM
NaCI, pH 5.8, filled into a 5-mL Wheaton Type 1 glass vials and stored at 2 ~
to 8 ~C.
Stability
The stability study was performed using a protein concentration of 1.0 mg/mL
buffered with sodium acetate at pH 5, 6, and 7 at temperatures of - 80 ~ C, 2
~ to 8 ~ C, 20 ~
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CA 02267193 1999-03-30
WO 98I14582 PCT/US97/17505
to 25 ~ C, and 2 ~ to 8 ~ C. MPIF-1 D23 has been found to be stable for at
least six months
when stored at or below 2 ~ to 8 ~ C in a solution of 10 mM sodium acetate,
125 mM
NaCI at pH 5 to 7. In currently ongoing studies, samples will be assayed for
appearance,
protein concentration, purity (SDS-PAGE (reduced and nonreduced); reverse-
phase and
size-exclusion HPLC), and activity (Ca2+ mobilization bioassay) to meet the
specif cations previously outlined.
A stability study for the MPIF-1D23 batch (HG00304-E10) used in the
preclinical
toxicology studies was initiated. The MPIF-1023 batch used in these studies
was
formulated at a protein concentration of 4.0 mg/mL in 50 mM NaOAc, 12S mM
NaCI,
pH 5 .9. The storage conditions are - 80 ~ C, 2 ~ to 8 ~ C, 2 5 ~ C, and 3 7 ~
C, at a rel alive
humidity of 60%, and at 45 ~C, at a relative humidity of 75%. The stability
study
duration is 12 months for temperatures up to 25 ~ C, 6 months at 37 ~ C, and 1
month at
45 ~C. The stability will be assayed for appearance, pH, protein
concentration, purity
(SDS-PAGE (reduced and non-reduced); reverse-phase and size-exclusion HPLC),
and
activity (Ca'-' mobilization bioassay). Endotoxin assay and bioburden tests
will be
performed at selected time points.
It will be clear that the invention may be practiced otherwise than as
particularly
described in the foregoing description and examples.
Numerous modifications and variations of the present invention are possible in
light of the above teachings and, therefore, are within the scope of the
appended claims.
The disclosures of all patents) patent applications, and publications referred
to
herein are hereby incorporated by reference.
-175-
z,. a. : ,-h., , . ,
hW W \ I,I'\-Q11 Ii:.t.ltl.~. ~n~~ :W- 1-:;ti . ~.t ,.i , ~ ~ ~~._~ . ~_ r,n
.=~3'I:)~L.~~;:;.-,y
Jry _ _ _ .nV _:V lv~ L~
SEQUENC"'~.' LISTING
ii) GENERAL INFORMATION:
APPLICANT: Human Gez7ome Sciences, Inc
9410 Kev 'test Avenue
Rockville, N~ 20850
United Status cf ~cnerica
A_~PLIC~'I'S/IIv'~JENTORS: G~ITZ, REINER L.
rAT$L; VICKRR.'~1
E3EIDER, BRENT L.
ZIiADIG, .TUN
?:~ITONAC=0, MTCFiAEL
ZIEZSDRICK, DONNA
vT_lT~Lr'~TE'z., PA.BLO
( ii) T=,I. OF INV"~'9TION: THERAPEUTIC CGMPCSxTIOidS ArJD ME='aODS FOR
TREATING 'L'IjEASE STtlTE5 WITH MXEL:vID PRCGE:TITOR iNEI3ITOFc:,~
FiIC - -~~~J.,-~~ _ , I tdp r F _ 1 ) , ~~:ONC C'~"'z' CCTrCDT'~ INH 3 ' T OR
_' FC _TOR
(:~1-CIF) , ANC MACROPHA:ac INHIHITOR'= =ACTOR-9: ('4IP-41
(iiij NUMBER OF S.c.QUENC~9: 57
(iv) CORRESPONDENCE A,D.;.R.~SS
('a.) ADDRESSEE ~. ST3RNE, KESSLcR, G'JT,DSTEIN ~ FOX P .:~ _ L _ ' .
(B) STRE$: : 1100 NEW YORK AVENUE) Zi . W . , SJ:':.'E' 6C0
(C) CITY: WASH_Iv'GT:N
(D) STATE'': DC
(E) COUN_"RY: LSA
(F) ZIP: 20005-3934
;'r) COP'LPQTER READA$L:. FORM_
(A) KEDTJM T~?E: F,',oppy disk
(Hf COMPUT~R: IBM PC ccn~patibl=
(C) OPERATING SYSTELVI: PC-rJOS jMS-DOS
(DI SOFTWARE: Patentln Release #~1_0, ~%ersion =1.30
',Vi) CURRENT ApPLICATIQN DA: A:
(A) APPLICATION NLZdHER: PC'/U537/1?505
(8) FI~I:7G DATE: 30-5EP-97
(C) CLASSIFICATION:
(viii ?RxOR APPLICATION DATA:
' (A) APPLCATION '.~1UI~EP: U5 60/027r299
(8) FILING DA2'.~.: 30-SEP-1996
(viii PRIOR APPLICATION DATA:
(A) APPLICATxON VUM~ER: US a0/02~" 300
(B) FILING DATE: 30-3F.P-I996
(viii) ATTORNEY/AGEL''1T INFORMATION:
(A) NAME: STEFPE, 3RIC K.
(B) REGISTRATION i~''Lr'rIBER: 36, 538
(C) REF~RgICE/DOCKc.T rTTJMBER: 14d8.033PC09
-176-
CA 02267193 1999-03-30 AMENDED SHEET
133HS ~3aN3W'd 0~-~0-666l ~6iL9ZZ0 ~a
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pT~E ou-we :BdTt,:. (6)
spto2 o~~zwe B6 'F-:3.~1Z3: ('d)
sJI3.sI2~$.i.Jti2TtfH~ 5.?t~''~11Z5s ( ~ )
Z v 0!~ QI 53S ?IGd IdOLZKW~Cats_T ( Z )
05 SS
use nT~ ~it~ ~,ay~ d9~ sh~r s2I .v~I. dstt uTE ~~n ~S. sI i
Z B Z '~Z J'Y~ JE J ~' JZ'd Jtf~~ 9~S J,Ltf ~~LI. JtItJ ~J J,:.~ JOZ Jtiti
C'o 5~ OL 59
~sa aac o.xd us~1 z;;y et.J TQ~1 :.y sT~ ALT, b~Td s~Z wu,;, ayI a'~d i'~l:
O~Z J~ .:.tJtT J:a:~ J~' JJ'd Z~J.:. J.i.J ;~JS. .L':J J':?'.~ Jr~ C~'ti OJ~d
J.i.ii JS.S. J..'.
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~~',T~ ~~J ~JS'2 ,T..I.
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zaS
,z. ' ~~ ~~ ~~.1 vl~J v.u\ ~~ JC~~ ~~JW i J~ ~~u .~~~ r)u.Z .~J... ~WL ~~ ~Z
.7rC SZ Cr
cwd sir .xi~,I, csd ~I~ ~.xt ~aS aaS xaS r tJ ~.; Si=~T ~uZ f -=~ ne i 2T~
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(~~wo~,,-2~; qua :sa.;~ ~zno~zo~a (zT)
~;zoq ~~oZOao~, ro
~'T~'~o~ : SS3I~3Qi~.LS (J)
p:~F c-aianu :3~Z iB)
8.:z24 2St?C Z B r ~ Y'..L'~JL~"I ('h )
' a~~.i3.SI2i~.vIJ~'h~ 5J1~3f7~;3S ( Z )
T = :~t aI ~S~s ~ca ~oz~~o~~;z c z)
~~~5"-4Li. (F.OZ; ~5.~d:?23~ (H)
009Z-TL~ (ZOZ) v3NOHdS'I$,:, (~)
: tZOI3.~'ht2~Od?~I ts'JIS,~I~~rIY~TQJ,~ I~T, ( 7;T )
s
rr .-~T n~ rar .. ~.~ ~-. ;non ~ , _ ~ -~...n n
wi -f;l fic',-1 -~~f: ''~' ~..-III~', 1 1i1'\~f l:\~~\ \W
fl~.di~.~y)Vwl(;(;f:- r;f; (;i- - _
rl.\ \W\i~i'\ ~pl I~.~.t~lll:~'. ~.~ im ~ ! -,~2s . ,:i I o ~ i , - _.1~.~
to.! _ ;:::J~I~~Lv;.i: nr'!;l
.. ~ V n V.=._ m_ ~f '~ _V ~V lv.~ .J
( ii ) iHCLECL'I,,c T'i'Pc~ : protein
(xi) SEQLTcr7CE DESCQ=p: ION: $EC~' ID N0:3:
Met Lys I'-a Sex' Val Ala Ala Ile B,ro Phe Pho Leu Leu Ile Tsar Ile
1 5 10 'S
Ala Leu 31y Thr Lys Thr Glu Sir Ser Se1 A~g Gly Pro Tyr His Pro
?0 :S 30
Ser Giu Cys Cys Phe Thr Tyr Thr Thr Tyr Lys Ila P=o Arg Gln Arg
35 40 45
I12 :bet Asp T-jr Tyr Glu Thr Asn 5er Gln Cys SeY Lys pro Gly ale
50 55 0'0
Val Phe ie Thr Lys Arg G=f His Ser Va1 Cars Thr Asn Pro Ser ~p
'05 70 75 3p
Lys Trp val Gln Asp Tvx Ile Lys AsQ Met Lys G1u Asn
85 90
( 2 ) =D1PORMATION FOR 5';~ .D .d0 : 3
(=i S.'-.'.~Jr,NCB CAARF~CTERISTC~CS
(Ai LENGTH: 363 bast ~aira
($% TYPE: :ucleic aCiti
C ; STRP.hIDEDNESS : ~ou~l a
(D) TOPOLO~l: both
i i ) 'riOLECV=.i. 'I".: PE : :.Pi.=~ (g2ncmi c )
( i:c ) FEATURE
v ; ,---, c
,Ai NAME/K:.".
iE) LOCATIGN: 1..360
;~ci) ScQUENCE DF'SCRIPTICN: SEQ ID N0:3.
ATG AAG GTC TCC GTG GCT GCC CTC TCC TGC CTC A~G CTT G'I"T' ACT GCC 4$
Met Lys Val Ser Val Ala Ala L.~u Ser Cys Leu Met Leu Vai Thr Ala
1 5 10 :.5
CTT GGA TCC CAG GCC CC,G GTC ACA :~r'~F, GAT GCA GAG rICA GA3 ?TC ATG 96
Leu Gly Ser Gln Ala A_~., Val :':~r L~rs Asp Ala ~;,lu Thr Clu Phe ~Iet
20 25 30
ATG .~Ci. AAG CTT CCA T:G GAA AAT CCA GTA CTT CTG GAC AGA TTC CAT i44
?4et Ser Lys Leu Pro Lzu Glu Asn 8ro Val Leu Leu Asp Arg Phe His
35 40 45
GCT ACT AGT GCT GAC TGC TGC ATC .TCC TAC ACC CC.a CGA AGC ATC CC3 192
Ala Thr Ser Ala Asp Cys Cya Ilp .rer Tyr T?~r Pro Arg Ser Ila Brc
50 55 60
CA 02267193 1999-03-30 AM ENDED SHEET
KO\ 1~.;\ I.t'\-\I~ n.:~.C:lll~.'. o,~ .:~,m ; -:)t~ . I:f: Im~ . - -E.:l ~;~!
~ '.:~:yl~~ ~:.~;1;~
J'~V ~ v. a ~ J'.L ...JJ I~.1, . .~V .J .... uJ
T GT T CA CTC C"I'G GAG rIGT TAC TTT GAA ACG AAC AGC GAG T3C TCC e'~r'~G 24 0
Cfs Sex Leu Leu Glu Ser :'~.~ Phe G'u Thr Asn Sar G1u Cys Ser Lys
b5 70 75 ap
CCG GGT GTC ATC '~'C CiC aCC AAG AAG GGG CGA CGT T'='C TGT GCC AAC 398
?ro Gly Val Ile Phe Lau Thr :~ys Lys Gly Arg Arg Phe Cys Ala Asn
85 30 95
CCC AGT_ Crr~.T e'~AG CAA GTT CAG G:'.' .'~C'sC ATG AGA A: G CTG '.~' G C i G
GAC 3 ? o'
Pra Ser Asp L;~s Glr1 'Tal Gln 'Jal C.ys MeC Arg Het Leu Lys Leu A3p
100 :.05 110
ACA CGG ATC 14AG t:CC AGG AAG AAT TGr1 303
Thr Arg Ile Lys T'_L Arg ~ys A.sn
15 1? 0 _ ..
;2 ) IPIFCRrIATION FOR SEQ ID :~TO:-!
l _; ScQUEa.':E C~ARAC'='E2I$TICS
;A) LENGTH: 1C. amino adds
18) T~?5: amir_p aCld
(Di TO?OLOG'I: l.near
(ii) MOLcWJLE TYP~: protein
( 1) SEQ(.'ENCE .~,E.SCRIPTICN: SC ID rT0:4:
Met Lys ':~al SFr Val Aia Ala Leu Ser Cys Leu Met Leu Val Trr Ala
S 7.0 15
Leu Gly Ser GJ.:~ Lla Arg 'Ja'_ T:~r Lys asp Ala Glu Thr Gnu Phe Met
20 25 30
MeL Ser Lys Leu Pro Lsu Gla Asn Pra Vdl L2u Leu Asp Arg Fne ui3
:;5 40 45
,Ia '~f:r Ser Aia Asp ors C~rs ==s Ser '~yr 'T:y.Y pro Arg Sex Lie ?ro
50 5. b0
Cys ser Leu Leu Glu Ser Tyr Pha G1u Thr Asn Se. Glu Cars Ser Lys
65 70 a5 80
Pro Gly V31 Ile Phe Leu Thr L~rs Li~s Gly Arg Arg Dre Cys Ala asr.
_ 85 90 SS
Pro Ser Asp Lys G_n Val Gln '3a1 ~~s Met Arg Met Leu Lys Leu Asp
l00 105 110
Thr Arg :le Lys Thr A:g Lys Asn
11a 12G
( 2 ; INFORMATwO:i FOR SEQ ID P1C : S
-179-
AMENDED SHEET
CA 02267193 1999-03-30
rW \. \v:\.i.l'\-~Il i. .Lilli. ~~v ::Jr~G_:)t: ' I;i I i - rl;l ti:)
_.'i:1:J41-tiai:!/:Sl
U'~V ~ ~ 'J ~ UU- i,-.J "1~ '~ ..iV
.U w.lJ
(1) SEQUENCE CHARACTERIST=CS:
(A) LENGTH: ?70 case pairs
(B) TY9E: nuclaic acid
(~~) STRAIYDEDNESS: double
~ TopoLOCY: ~ocr.
i i ) MOLaCULE T"P~ : CrIA ; genc:~.ic )
( i:c ) FEA'I".;RE
C A) NAME/KE'I: C:.S
(H) L?CATION: 1..267
(xi) SEQL'ENCB JFSCRIPTT_CN: SEQ I:,r N0:5:
ATG :~~G GGC CTT GCA GCT C-CC CTC CT'_~ CrTC CTC GTC Tr'aC ACC ATG GCC -~8
Met Lys Gly Lau Al.a :~,:;.a ?=a Lau :.eu Va'~ Leu val Cys T2:x ~;et ?lia
r. 5
C'I'C TGC TCC TiT GCA Ctl~ GTT GGT ACC AAC AAA .~sAG C'x''.". :'GC T;:zC CTC
36
Leu Cys Ser Cys Ala Gln 'Jal Gly Trr Asn Lys Glu Leu Cys C-~s Lau
20 25 30
,_.TC TAT ACC T CC TGG C:.G aTT ~. Cr'>, L ~.z1 AAG TT C i:': A GTT Gr'lC T'AT
TC T 1=
oral Tyr Thr 3er Trp Gln I;e P=o G1~ Lys FY:e :.le ~Ta1 AsF Tyr Ssr
3~ 4C 45
J.=,a :,CC AGC CCC CAG TC-C CCC AAG CCA Gv': G:C r~TC CTC CTP. ACC AFsG 192
:~lu Thx Sex prc Glr. Cys ?ro Ly= Fro Gly Val I1' Leu Las T2:r Lys
54 5~ 00
ACA GGC CGG CAG ATC TGT GCT ;AC CCC A.~T AAG AAG TGG GTC CAG .r~A 240
Arg Gly Arg Gln Il a Cys ~l.a Asp Frc Asn Lys Lys :.p va-_ Gln Lys
F5 7v~ 75 30
TAC ATC AGC GAC C T G FaAG C T G A lT GCC TGA 2 7 0
'_~-rr Ile Ser Asp Lat; L~rs Leu rs:1 Aia
(2) INr~OF.MATICN FOR SEQ ID NC~.6:
( i j SEQL1F'I1CE CHAR~~CTER:STT_CS
_ (A) LENGT3: 89 amino acids
c~) ~_'YP~: ami:a.o acid
C? 'y'ODGLCGY ~. i inear
!ii) ;~loLEC'JLE TYFE: p,rotain
(xi) SEQL1~JCE DoSCR'F'"T_GDt: aEQ XD N0:5:
Met Lys Gly Leu Ala Ala Ala Leu Leu Val L2u Vai Cys Thr Met A~.a
1 5 1;i Z5
-13Q-
AMENDED SHEET
CA 02267193 1999-03-30
iW \ 1O\ 1:'t'\-\Il 1.~~<III.'. ~.~ :f11- I -.)2i !ti: I ~ _ _ n j (v~ ,_,~'1\
='3'):Ll~l~i:j. y:)_'
_ _...~. _ .%CJ JV 1 ~. y V
Leu ~y s Wr Cys Ala GLn vial VI f :'hr Asn Lys Glu Leu Cys Cys Leu
20 35 ~0
Val Tyy Thr Ser Ti'p Glz ILa Pro Gln :.ys phe Ile Va1 ~p Tyr Ser
~S 4C 4~
Glu Thr Ser Pro GIn C:rs Prc Lws Pro Gly Val ILe Leu Leu Thr Lye
50 5~ 50
Arg Giy Arg Gln Ile C-,rs Ala Asp >: ra asn Lys Lys Trp Val G~.r. L.rs
65 70 75 $p
Tyr I'_.e Ser Aep Leu Lys T_eu As:1 A_a
! 2 ) iIQPOFtt~GlT ION FOF~ .EQ ID NO : 7
( i ) SsQ~NCE CHaRt~CT~'c=S T ICS
yiVGT~- 100 znino acics
(Hl TYFE: amine acid
t C ) ST_TL~NDr~N~.'SS : riot relew:~nL
TOPOLOGY: liaeax
I.ii) vIOLEC'uLE TYPE: peptide
lxi! SEQU~rF' 1CE DESCR.I?T='~N: SeQ ID N0: 7:
hl~2L Z1.X'g Val Thr L.rs :,;~ A1 a Glu 'Ihr Glu Ph? MeC f~iet Ser Lys Lcu
5 i0 15
?=o Leu Glu Asn Pro '~al Leu Leu :.sp A=g phe His ~7.a T.zr Ser Ala
20 .~5 30
abp Cys Cys I'_e Ser T',Ir '"hr Px'o A::g Ser Ile Pro Cys Ser Leu _T.au
35 40 45
Glu 52x Tyr Phe G';u Thr A3n Ser Gi.u Cys Ser Lys ?ro G_y '~al ILe
50 55 60
DI=re Leu Thr Lyy Lys G:.t Arg Ara ? a C'ys Ala Asri pro Se. Asp Lys
;;= 7~ 'S 30
c3,a.n Val Gln Val Cys Met ."-.r3 MeC Leu Lys Leu .Fssp 'r'hr ~g Z:.e Ljs
8~ 90 95
i11. Arg Lys A821
100
(2) INFORMATION c'Ol~ SEQ ID 2d0:8:
(i1 SEQUENCE CIiAFc.4CT~RI~TT_CS:
' (A? LENGTH: 76 anyno acids
-181-
AMENDED SHEET
CA 02267193 1999-03-30
f; l:\ W~. I-!' \ -11I I..~..~Ill.'. ~ " W - k -:~;, . ; ~ I ~ , ,J-"~ ~ _. ..
~ ' yl ;t:l '_;i;~:l l U;;,: n:;:i
U~J.. _ r-11 ~, J,V W J 1G.~ 1
(s) TY~S: amino scid -
(C) STRANDEDNESS: got relevant
(D) TCPOLCGY: linear
(ii) MOLECULE TYPE: peptide
(xi) S~QQENCE DESCRIPTION: 8EQ ID :10:8:
i~et Arg Fhe His Ala T_hr Ser A:.a Asp Cys Cy s Ile Ser ~I'y_ T:zr pro
1 S 1~ I~
Arg Se. Ile Pro Cys Ser ~eu Leu !=iu Ser "'.rr Pie r~a Thr Asr. Ser
20 2~ 3Q
Glu Cys Sa: Lys 2ro G1f '!al Iie r~_~_e L2u T:~..r :.ys :.ys G1_~ Arg Arg
35 '-_0 43
Phe Cys A:.a Asn Pro Ser Asp L~rs G1= V~.1 Gin val ~Y s :y!e t At g ?bet
50 S~~ eQ
:~=a Lys Leu :.sp T.: r Arg T_le ,T. rs Thr Arg Ls Asn
65 74 7~
( 2 ) .r.~ORM.ATIGD1 FOR SEQ I~ ?~10 v
( ?. i S~cQUEi~ICE CFinRACT ER I S"_~' CS v
(A) i.ENGTf~: 78 ami.: c acids
v g i T~~PE.' : alttR110 dCid
r; ST~ANDED?~'ESS : rot ralevwn:.
;D) TOPOLOGY: l.near
.i 1 MOLfiCG~E T'!2E : nr ote;A
(xi i SEQLz'NCE D2SCRI?TIO:~T ~ SEQ ID NO : 3 v
Hi.s Ala A.la Gly 2hE cIis Tsl.a Thr Sar Ala Asp Cys Cfs Ila 5er ~h~-
1 S 10 15
:'hr Prc Ara Ser Ila Pro Cya Sar Leu Lest tsiu Ser Ty= 2he Glu T~~
20 25 30
A.sn Ser G:.u Cys Ser L_rs P=c G~y Val. Ile Phe :,au Thr Lf3 Lls Gly
35 4d 45
Arg Arch Phe ~-s Ala Asri Prc Ser ~;p Lys Gln 1,7a1 Gln ~Ia1 C'~rs Mec
S0 55 6~:
Arg Met Lau Lye Leu Asp Thr Arg I.~ Ly8 Thr A.~_-g Lys As~
5S 70 75
~ ( 2 ) ILIFURMF,TION . OR SEQ ID NO : i0
-18~-
CA 02267193 1999-03-30 AMEnIDED SHEET
KO\ \o'~ I.1'\-Q11 i.'.~ill '. m:; :s..I -.lri !;; I ; _ -!:) ~i:s ,;:1'.1I
!v;.;..~:3,~
i J..,u ~ '. .. vJ...~-~ ~_~ ~ ~u _u l . 1 i
(i) SEQUENCE CHAR.aCTE.2ISTICS:
(A) Lb~IGTH: 599 base pairs
(B) T'iPE: nucleic acid
(C) STRAND:~NESS: dOUble
(b) TOFOLOGY: bot::
( ii ) ;HOLECULE TY9E : ~N:a (g~nOm.iC)
ix; c fiATL~B
f, A) NAME/Ke.'Y : CDS
(B) i,OG~T1CIV: 35, .9~~5
(xi) SEQUENCE DESCRIPTION: SEQ ID VO::O:
G'_"CCTCCGGC CAGCCCTGCC ~CCG~.CCAG G1GG ATG AG GTC TCC GTG 'CT 52
Met Lys Val Ser ~Tal Aia
_ 5
GCC CTC TCC TC-C ~'TC A;;, C?: GTT :,CT 3CC C'I'T GGC TCC C.~G GCC CGG 00
Aia :,eu Ser Cys Lea Me t leu Val Thz' .~la Leu Gly S2r Gh~ ~.la Arg
.5 20
GTC FAG? A.aF: Ga'~ GGA GAG ACA GAG TTG ACG A'LG TCA AAG CTT CCA '=TG 148
,:al Thr Lvs ~>p ~.la Glu Thr Glu Leu Thr Met Ser Lys Lpu ?rc Lei:
25 30 35
.GAA AAT CCA GTA CTT vTG GAC ATG CTC TCG AGG AGA AAG AT~_' GGT CC'.' 196
Gl a ?.siz 3ro v'al Leu Leu Asp ale ~ Leu Tip Arg Arg Lys Ile Gl.r Pxo
40 4_'- 50
CAG A'_'G ACC CTT '.~CT CAT GC C GCA GGA TTC CA';' GCT P CT AGT Gv':' GFiC "a
4-3
31n r'.ec Thr z~eu Ser ais Ala Ala GIy Phe Iiis A1a ~hr Ser Aia asp
55 50 &. '; 0
TGC TGC ATG TCC T_AC ACC CGa CGA AGC AT:. CCG TGT TCA CTC CTG GAG 292
;.'~s Cys Met Ser T~r- Thr Pro 3:g Ser Iie Pro Cys Ser Leu Leu Glu
75 80 35
AG: TAC TTT GAA ACG AAC AGC s.,AG TGC TCC AAG CCG GG= GTC ATC T"_'C 340
''r Tyr Phe Glu Th?- Asn Ser Glu Cys Ser Lys Prc Gly Val Ile Phe
90 95 1C0
t.TC s'1CC AAG AAG GGG CGA CG: TTC TGT GCC FsAC CCC AGT GAT AAG CAA 388
Leu Thr Lys Lys Gly Arg Arg Phe cys Ala Asn Pro Ser Asp Lys Gln
205 110 115
GTT CAG GTT TGC A' s AGA ATG CTG Ar'1G CTG Gi..C ACA CGG ATC i~AG ACC 436
;?al Gln Val Cys Met Arg Mez Leu Lys Leu Asp Thr Arc ~le Lls ~Y:r
12C y~5 13C
AC-G AAG AAT TGAACTTGTC P,AGGTGAAGG GGACACAAGT TGCCAGCCAC 485
Arg Lys e'~sn
135
-~ s3-
CA 02267193 1999-03-30 AMENDED SHEET
lily \m'~ I f';1-\II I'~.llll'.\ n:p ;31- f.-:~:3 ;;3 113 . - -.f:) 13;)
'=:3;);)-I'1-~~:i, rt:i
t 'J~ll_.~ '- ~ t~ I tJ~J.~ --_ r11- '. ~1:. _ J 1 ~ a V
C.AACTT'1'CTT GCCTCAAC'i'u ~CTTCCTGAA TTCTT':'TT?T AAGAAGCATT T ~T : C'_'T'G 3
54 5
TTCTGGr~:".: i AGAGC.~ATTC ATCTTT.TCTC ACCTT""~1F,.~A a.:=.AAAt~AAA:a r~AA 599
(2) I:7FORMATICN rO:Z SEQ ID ND:11:
('_ ) SEQUENCE CHARAC?ERIS T zCS
(A) Le~,NG'_T~H: 13'' aat_ro acids
(B) TYPE: amino acid
(D) TCPOLGGY: linear
('-i) WOLECLTLE '1"':PE: prcte_:~
(xi) SEQU~CE :~ESC:~IF:'xON: SSQ IO N0:11:
Mec Lys Val Ser Va1 Ala F~l,a Lau Ser C~fa Leu Plet Leu ''a1 Th: ~,a
1 S 10 1 5
Lau G1y Ser Gln ~sla ~ig JG.1 T~Y Id JS Asp A:.a Glu '."hr Jlu Leu ':'hr~
20 ~5 30
Met Sex Lys Leu Pro Lau Gla .ysr~ Pro Jal Leu Lau Asp MeL :leu
35 i~ 'si
Arg Arg Lyrs Il a GI f Pro Gln filet Thr Lew Ser Eis h1a a.la GLy Pile
50 55 00
his Ala Trr Se: Ala Asp Cya ,'s llet Ser Tyr T':r ?ro ~rg Ser rle
65 ?Q 7~ fi0
Pro Cys Ser Leu Leu Glu Ser T~jr Pne G1'.: Tar Asn Ser GIs C'~rs Se=
85 95 9~
Lys B=c G? y rTaL Ile Phe Leu T:xr Lys Lys ~~Ly P.rg arg Phe Cys al.a
10d 105 1.l0
~.sr_ P=o Ser asp Lys 31n Val Gln Val Cys ;Het arg Met Lau T_,ys Leu
115 120 125
Asp Thr Arg Ile Lys Thr Arg Lys fir.
i30 1~5
(2) INFORMaTT_ON r0'2 SEND N0:12:
(i) SEQVENCG LHARACTE.;2T_STTCS:
(A) L)~7GTFi: 26 base pairs
(H) ~YpE: nucle.~'.c acid
(C) ST~NDEDNESS: s_ngle
(D) TOPOLOGY: 1-near
(iit MOLEC-JLF. TYPE: CDNA
-1 a~-
CA 02267193 1999-03-30 A~IE~IDED SHE~I
W ~ \w-. ;:1'v-v( ~:v.Wll.\ m;p :W _ ~ -,jt; Iri. ~:? . - r.;.;1 :;:) _: in.
. ~~.t.t:;:~: u:it;
I ~~'-rlJ ' - ~ I r.'~lJ.~ m_. -'I '. JJ _:lJ 1G~ 1V
(ri) SEQUENCE DESCRIPTIJN: 3EQ ID NO:.2:
TC~GG?vTCCG TCACr~.ArllG~ "_'C-CAC 2 E
2 ) =?~TFORI~.AT_ ICN FCR S EQ ID V0 : 13 :
i i'1 SEQ:7T~L,1'VC'S' CF.ARAC~_'E~2ISTICS
(A} LENGTH: 26 base pairs
(B) TYPE: nuclzic acid
(C} S'~Rr~.NDEDNcSS: single
(D} TOPOLOGY: linear
i i ; MCLECULE T': PE : CL'Nr1
;xi) SSQU'ENCE OESCRIPTICN: SF,Q ID N0:13:
CGCTCTAGAG TA~CGACG GCCAGT 26
(i; INFOR.'HF.TION FCR SEQ ID ~1C):=~
(i; SEQUET7CE CIaAR.~CTEi.IST=CS:
(Ai LL2iGTH: 2 r ease pairs
(Bi TY?E: nucleic acid
( C i S :T~.~1NF7EDNE5 S : s ing=, a
(D? TOPOLOGY: l:.near
( i i ) MOLEC;JLE TYPE . cL2PA
ixi; SEQTJENCE D.ESCRiPTIGN: SEQ ID ~IO~i4:
CCCGC::'GCG GGTCACaAAA GATGCAG ~?
( 2 ) =NFOFuu.A, T =QN FOR S c Q I ~J NO : l S
'_ i SEQUENCE CHP.RAC'I'ERIS'T_'T_CS :
(Al LENGTH~_ 27 base pasrs
(3) TY?E: nucleic acid
( C 1 STR~~TDEDNESS : s ingl a
iD} TQPOL4GY: li:~ear
( ii } :~1~7LECULc, TYPE : cDNA
xi ) S:.QLTENC~ :.ESCRIPTICN : SEQ ID NQ : z S
ArIAGGATC C? CAJi_'Z'CTTCC TGG T CTT 2 7
-185-
CA 02267193 1999-03-30 AMEnIDED SHEET
hL\. \m'. I.I vy ;-'.L111-.~. m.- ;;~!- [._:.ty . it',: ~,) _ _i:i ;il'~
_':;;1;!1 ly.u: r:i-
iJ.mJ i ~ _. i _ _ 1.__ ~I v JCJ ~V lv. yiJ
(.2i ::'FORMATION FOR SEQ ZD NC : z6
(ii SEQUENCE CHaR.AC':ERISTICS
(A) LENGTH: 46 base pairs
yHi TY?L: nuc?e~c acid
(C; STRANDEDNESS: single
(b) 'TOPOLOGY: '~irea_-
(ii) MOi.ECULE TYPW c~NA
(:Cl) ScQLTENCE DESCR_TP'TT~:~N: SBQ ID N0:16:
ACATGC~.TGC GUGUUAC CAA AGACG~~GAA ACCG~WCA .1GALTG'GCC .~8
2 ) INFORMATION FQR SEQ rD ?~O : 1''
I i! SEQL"a~'..TCE Ciiyec..~sCTERIST;CS
(A) L~rGTT.~: 36 ease pairs
(D) Tl'PE: nucle_c ac:ri
;C) STF~fD317NESS: Siag;e
D) 20FCLCGY: Ii_~ear
(ii:~ MOLECL~E '~'f?E: cDVA
(:ci) SE(~UEVCE OESCRapTCCN: oEQ ID NO:'_7
~'aC: G~:1GCT'_' TCAIiT i'TTTA CGGG T T T TGA T'ACGS-a 3 6
Z' INFORI~TSC!V FOR SEQ Ii :~C : 13
i ) SEQJ~1~' CSARACT'RIS'_'ICS
(A) LE~iGTH: 88 base hairs
(3) TYFE: nucleic acid
(c) sTR~:~~sLr~ss : sinalz
(D) TOPOLOGY= ;inear
( i i ) MOLECL:.E TY?E : cbNF.
(xi) SEQUE'iCE DESCRIPTION: S~Q ,D h0:18:
GCATC-CGUGU UACVAAr~Gr'1C Cri.UC:F~AACCV AACTUCALT3AU GUCC.'~isr~C;JG
~CCGCCJGG:.is~s 6 G
nCCCGG~3CU GCBGGa:CCGU r.JUCC.'~G:~C 88
! 2 ; i-7FCR.''7ATION c~CR 5 );u T_C NC = '_ 9 :
-lss-
CA 02267193 1999-03-30 AME~IOED SHE~i
KO', . \W \ I I'\-III I ~~.1 lll.'. ms :SO- t ;,ti . ~i ;;) . - -.L:~ 2i:1
~:i;l:il li; i: g:523
~r'~G '~ -' W i nL,_Jn i.-... nl- ~ W J ~V 1L. 1 J
t i ) sEQBEVCE c-.3aaACx~~:s: Ics .
(A) LENGTH: 1C4 basR pairs
(B) TYF6: nucleic a~:id
(C) ST'R.ANLEDNESS : singlQ
(D> TOPOLOGY. l i:~ear
( =i ) MOLcC'JLE TYPE : CDNA
(:.1;' SEQUENCE DESCRIPTION: S'EQ ID NC:19:
GC'JGL~.-AAUCC UACUUCGAAA CCAACCCCGA AJGCG C.~.AAA CCGGC3UGUQaI TJCL'UCCL-GRC
o' O
CT.~.=~C-GU CGUCGL'U'JC'J GCGC'JAACCC G-JCCGACARrI C~.GG lC4
t~! IN~OR.~"~EATICN FCR SEQ ID ',JO: ~C
( ; ) SEQL'EiICS CHARACTERIST'C3
(A) L=NG"'H: 89 base pairs
fB) TYPE; nucleic ac-d
(C) ST2aNZEDNESS; single
(D' TOBCLOGY: :in~a~
:.:. ) ,~o~.~cL~Ls T ~~ : coirA
t%ti) SHil(7ENCE DESC_?IPTIO~J: Sc~Q ID NC:2C:
.~.GC T TTC AG TTTT'='<'-.CGGG TGGGCA,~,ACG uGi'GTCC.aGT T TCAGCA~-'AC
GG.~_~AC "..,e;:..C S 0
c.~'GF~.CCTGT TTGTCGGACG CirCTTAGCGC 89
( 2 ) TNFC_~2MATT_~DN FOR SEQ ID 1T0 : 21
( i) SEQUENCE '',.I' IARAC':ERIST.ICS
(A) LENGTH. 94 base pairs
(H) T'IPE: ruclei~ acid
(C) STRANDEDNESS: s'_ngle
(D) TOPOLOGY: lin9dx
( i i )_ MOLECULE T': PE : CLNA
(:r~;) SEQUE:JCE DESCRIPTION: SEQ ID NC:21:
G~3TTTCCa.Fu'a.G T L1GGAT T CCA GCAGGGAGCA CGGGi.TGGA.A CGCGC-GGTGT
.:.GGAGATGC3~ 5 U
GCAGTCAGCG ~3AGGTAGCGT r~G :AACGGTC CAGC 94
s
-1 s~-
AMENDED SHEE?
CA 02267193 1999-03-30
W \ \t:\=I.I'\ -\II I..'.L!I1.:. .~:> :;~~- I -:)i! - I;i ;;l - --I:1 ;;:)
_:i;):p I~~.i: u: s:)
nJ'-'V ~ ~ ~ ~. J .. ~ ~ .-J ~11 ' ...V
-'V rte. LJ
(2) INFORMATIOl~i FCR SEQ zD No:zb:
( i 1 SEQUErICE CHAR.ACTERi S'I ICS
(A} Lc,"2tG'~i: 32 base hairs
(B} TYPE: nucleic acid
(C} STR."~,s7DEDNESS: s_ngla
(D} TCPCLCGY: li:~ear
( ii MCLECL'LE T'12E : cDNA
(xi} SEQL'aNCF DESCRIPTION: SEQ :: N0:22:
GCGC.~C~CCAT GGAAAACCCCy GTTC?GCTGG AC 32
(2) INFCR~"2~TICN FCFc Sc'Q r1~ :I0:23:
(i;~ SEQL~c2tCE CHF~e2a.CT~IS.'ICS:
;.~i :..ENGTH: 8.3 amino acids
;B} TYPE' : aciiro acid
( C; STRA2iDEDD:ESS : _toc rA'_e~rant
;D} TCPOLv.'~GY: linear
(ii) MCLE'.::~ TYPE: pep~ide
(xi > SEQ~CE DBSCRIPTI02J : SEQ ID :~C : 2 3
biet Glu Asa Pro Val Lei: ;:eu aSn Arg Pre his :-.ia Th; Se~ Ala err:
I 5 1C -5
C~~rs Cys Ile Ser Tyr Thr Pro Arg 52z' ..e Pro C~:~s Ser Leu i.=t. G1u
20 25 30
Ser :'yr Phe Giu Thr Asr..Ser G1u Cys ~ar Lys Pro Gly Va1 ale Phe
35 ~0 45
Lau Thr Lys Lys ~Wy :,rg Arg Phe Cys F~l.a ash Pro Ser As~o L-; s Glc
SG ~~ SO
~Jal 3i:1 vat Cys N:et a=~ Met Leu Lys Leu Asp Thr A~-g Ile Lys Thr
65 .0 %5 80
Arg Lys Ash
i 2 ) TNFCRMA'r~.ON FOR SEQ ID u0 : 2~ :
! i ; SEQtIENCE CHAR.~.CTERISTICS
(A) TE_tGTH: 35 i7ase pair
(H) TYD6: nuclai.c acid
-Igg-
ANIEi~IDED Si~EE ~
CA 02267193 1999-03-30
Ivl \ . \ v', . I j' \-\11 I:\l l it.~. O i :jn - l -:32i j ;-3 : _m . - r 1
:3 ;~:1 _:5; ~:3 1 3n ;: > : ~i.l.m
' . ,J..y _ L n CJyJ: ~. ~.r r~I v .;.J . V 1._ . L J
(C) STRANDEDNESS: siag'_e
(A) TOPOLOGY: linear
( i 1 ) MOL,c.C'JLE TYPE : cDNA
(xi) SEQUENCE DESCRIrTT_GN: SEQ ID NC:24:
GCCATGGCAT GCTGG.~i.SAA.C CCGG':':CTGC TGGAC 35
i 2 ) INFOR."~'.ATIO?~1 OR 8EQ ID NO : 2 5 :
i ; SEQtJE;ITCE ~~~-IAR_AC'~'P IST _S
(A) L3NGTE: 84 ami: o aci3s
(B) i".'PE: amino acid
(C) S:RBNDED:VES3. r.ot re~~.e~rar_t
(D) TGPOLOGY: iirear
(:.i; MOrr.C'JLE TYPc: pegcide
(xi ) SEQtJ~TCE D~SCRFTT_OIv : SEQ ID NC : 2
Mec :.eu o:.a Asn Pro gal Lea Leu asp ~~g ?he e?is ~':.a ':h= Ser A.ra
_ 5 10 1F
Asp Cys ~ys I=a Ser T~~r Thr Pro Arg Ser Iia 2ro Cys Sew Lau aeu
2C 25 30
J1::. Se_- 3'y. Pre Glu T: r :~sn Ser Glu Cys Ser Lys Yrc Gl y '~'a1 I.e
35 ~0 45
21:e Leu 'I'?~.r Lys Lys Gly .~,rg axg Phe Cye Ala Asn Pro Se= asp Lyg
50 55 60
Gln Val Gln Val Cys Met Axg Met Leu Lys Leu Asp ~':~r Arg =le Ly~
65 70 ~~ 8C
Thr Arg Lys Asn
(2) :NFORMA2ION 'FOR SEQ XD NC:26:
( i7 SEQQENCE CHAR~.CTERISTICS
;F,) L=,.~~1GTH~. 32 bdsa paira
(B) TYFE: nucleic acid
( C ) S2RAI3DEDiYESS : s a.rg l a
;D) TOPOLOGY: Linear
(zi) MOLECVLc TYPE: cDNA
-189-
AMENDED SHEET
CA 02267193 1999-03-30
hL\ \~: . ~n'\-\II I. .~ iil , , :,gym J -'. ;t I:i _~r . - _i.1 .~:J _;i:!:3I
Ir~.;. p.3 I
~~-It.. ~ .. L : CJ~11 1~... nl \ JY~ ~V 1L. L J
(~cil SEQCT~~;CE DESCRIFTIvV: SEQ ID :.0:?.6:
GCGCAGC~;.~? GGACCGTTTC r..ACGCTACCT ,_C 32
( 2 ) INFORiyIATiCI\T : CR SEQ IC i'IO: 2 7
( ii SEQUENCE C~iAF,AC'~'RIS'I ICS
(A) L~7GTH: 77 ami.~.c acids
(H) TYPB: a~inc aci3
(C) STRANIrEDNFSS: not reievanz
(D) ;CPOLOGY: l.:ear
(_i) MOLECULE TYFB: pepG:.de
(::ii ~E(~eGcPICT's DE$CL~_PiIC1 : SEQ _C~ DIQ:2 r :
Me= :gyp Arg Phe H~~s L-a I'hr S?r Ala A.sp Cars C-~s I've Sex Tyr T!~.
_ 5 1.~ 15
Pro A.rg Ser I_e Prc Cs Ser Leu Les Glu Ser Tyz' :he Glu Thr :-.sn
20 ~~ ~u
Ser Giu Cys 5er Lys Pro ,sl;~ vz_ Ila Phe Leu Thr Lv s Lyo G1_r r~'~g
35 4U ~~5
A.rg r>ve Cys nla Asn Fro Se= =ap Lys Gla Ja:. G1:1 Val Cys '~Iet Arg
5C 55 . 50
r9et Lau Lys Leu Asp T_~r Ara Ile Lys Thr Arg :,ys ?Sr
65 70 75
( 2 l INFCr!'~'.ATiCN ~OP 3EQ ID lIC : 28
~; i ) SEQUr~WC.~.' CHARFLCTERISTICS
(A} LENGTH~ 29 base pairs
(B) TY?E: Zucieic acid
(C} S'I'RAN~EyNESS: single
(D; TG2GLGGY: =inear
( ii j' MOLECULE TYPE : cDISA
ixi) SEQUEtICE DESCRIFTICN: SEQ ID N0:29:
GCCATGGCAT GCG':TrCCAC GCTACCTCC 29
(2) INFORMATICIF FCR SEQ ID NC:29:
_1 gU_
AUIE(~JDED SHEET
CA 02267193 1999-03-30
iW\ \I7'_.i-!'\-1I1' _l.111'. ~rs :1- ~-:in' - .:i.=m _
r l'.) ;i;! _:i:);L~_p;:i. ut I
i ~~-v _ i _ ~ I ~ ._. ~ _~ rin '. ~t_ ~ _ 1L . .V
(i1 SECUENCB CHARACTERIS~; CS:
(A) LENG'~N: 3? base e~a::rs
(3) TYPE: nucleic acid
(C) STT~.A,N~F.DNESS: ~,ingle
() TOPOL..'~GY: linear
1 ii ' NiCLECL.'T_E T'iPE : CDh'.zi
lxi; SEt2Ur.."NCE' DES:~T_PTT_GN: SEQ ID 1C:29:
GCGCAGCt=AT GGCTACCTCC GCTGr~C'_'~iCT ~3C
" ; IN. OFMkTION FCR SEQ I : '_lQ : 3 0
G i ; Sr.QUr.,'~TCE CEAFi~,CTER_ 3'~ W S
(A) Lr.'~21G'~H: 73 ami nc acids
(H) TYPL: dTlnC 3C:~
(C) STF~ANDEDNi;SS: rat releva.:c
(~ ) TOPC=~CGY : i'_ne ar
(:.l ) NlOxrE~rL:F.. i x~?~ : _~..2DtldE
(xi; SEQU~~..~iCB DES.~_.?IF'T_'=CN: S3Q ID b10:30:
:oleo Al.a ='hr Ser Aia Asp C::s Cys .le Ser Ty= T. hr P.ro ?~rc Ser I1e
., 5 10 i 5
Pro C.'ys Ser Lau -au Gl~,~ Ser Ty=- Phe GI-_ Thr Ash Se. Clu Cys Ser
2b 25 30
Lye yrc Gly ;lal Il a Fhe L'u T'_ir Lys Ly.; G:.y A,rg Arg Fhe Cys rL' a
35 4D 45
:.sn Pxo Ser Asp :~ys Glr. 'Ial Gla val C,~s Met Arg Met Lz~~ Lys Leu
50 55 SO
Asp Th= Arc Ila Lys Thr Ayg L_fs Asn
65 ~0
( Z ) IPiFOTcM~~TION f~F SEQ Iu t70: 31 :
(ii S=QU~~CE ~iA~~~cACxEI~aSTICS:
(A) LENGT'd. 21 base pair3
(E) TYPE: nucia=c acid
(C) STR.F,NDc~~;:~7~nSS : single
!L) xOF~LOGX: linear
iii; IvlCLECtJhE i'Pr: .cD2:n.
-19I-
CA 02267193 1999-03-30 AMENDED SHEEP
KC'\ 1,;'~, fa'\-\II f.~.L!II,'. v:i :in- E-~i;i : ~,i:-n
-I:~ ~i;~ _V~.!:,i.lc'~:,.".I;y
~~-rnl , .- ~ a _ ~ 1.~J ~ _'U -~ ~tJ
~xi) SBQUENC~ DESCRI?TION: SEQ ID W :31:
"~'CGAAG TizG GCTTC ~AGC~1 G 21
( ~ ) INFCRNL3.TION F02 SEQ ID NC : 3
f i) Sc.Q~=ICE ~~RrICTERSS'."ICS
(a) LENGTH: 2. base pairs
(8) '_'1'Pls: nucleic acid
(C) STR,~NDEDNESS: ai.ngla
(D) TOPOLOGY: linear
~, ;, ) MOLEC;JLE T': PE : cD~1_:,
s: i) SEQ~'~'C8 CES'~_F'-"ICi'~: ScQ TD Pr0~32:
_TC-CTGGA~,G CCTACTTC.:A A ~--
( 2 ) IPFFOf2I~IATIC:1 FOR SEQ ID :JO : 3 3
( i ) SEQUENCE CFir~icyC':'ER=STICS
(~,) LEaTGT~i: 35 ;ase Pairs
~B) ~YP~: r~~cleic acid
', C ) S T~AL3D:~NL S S ; s i:~g 1 a
;'L') T~PCi:~CGY: l~:Lcar
i i ~ ! MG:.EC'~ E :'YP3 : c~~11.
( xi ) SEQLTENCy DBSCRI2TIOT~1: 58Q ID NO: 3 3 :
GCC?TGGC.i.T irCGTuTTACC ?~T~AGAC3CTG A~.CC
( 2 ) T_IFGRI~IF~T~ON FOR SEQ ID :rC : 34
c i; sEQveiac~ Cxa.~.~cmsTxcs
(A) LENGTH: Z00 amino aci~
3 ) TY2E : a~;inc acid
(C) STQRNDEDYESS: nCC relevant
(O ) mnpOZ,pGY : 1 inear
(;i) "~OLBCULc T'iPE: peptide
::Ci) SBQLTENCE DESCc2T_PT=uN: SGQ ID N0:34:
-~92-
CA 02267193 1999-03-30 AMENDED SHEET
i<CV 1cW-:I-.I'1 ,II I.',ulll - '~:~ :;,,- l.-:):i 1:4 '_i
~W :1 :i:l _:i:l:it~ll;:n.,~.tl.
i ~ ~ 1 ~~I . ~.-~ fuel ', ~.V ~V
L~....CJ
Met ,rg Val T:-~ Lys A~P .~Li i Glu Thr G'_u Phe Met :bet Sar i~ys Leu
1 5 i0 15
Fro Leu Giu Asz1 Pio val Leu L2u Fsp Arg Phe His Rla T :_ Ser A.la
20 25 30
Ash Cys Cys Ile Sey ':~~'r.' !'rr Pro Arg SeT T_'-c Fro C'ys Sc_ Le~~: T_eu
35 4U
Glu p~.a T',jr Phe Glu The Asn Sar Gls Cys Se: Lys Pro G1.: V41 I'_e
50 55 60
hhe Leu Thr Lys Lys Gw;~ Arg ?.zg Fh.e C ys r~la :,sn rro Ser asp Lys
55 ~.0 75 SQ
G1.~_~- ~Ta:: Gln Val Cys stet Arg '.~!et Leu LyE Let: Ash '=~hr Arg Iie Lv~s
BS 90 SS
:h.: :gig Ly= ?.:~
'~ C G
i,7.) TPIPORMATICTt FGFt SEQ IE' N~:35:
1 i SE(1L'ENCE G~T.AR.A,CT~R=ST;CS
tA) L.cNG~r: 36 ~:ase wa.i;s
iB} 'i'Y~E: ~luC~:ei: acid
'C; S~I'R.~1DEL~NESS: si:~ale
(D) TOPCLCGY: J.~.~.ear
i ! MGLEC~ E TY~E : CD:~~.
~; ~<y ) SEQITLW.~ ~ESC'R=3?'I0.1: ~rQ .D NC : 3 ~
G ~ ~ ~-~C~3'T~T TCnG: TTTTA CG~vGTTT ~ Ge3 'CAL=L~.-GG 3 s
l 2 ) Iar'O?.:~IFx210I3 r OR SEQ ID NC : s 6
r-.> s~,~v~rrc~ r'HAR.ACTER_STICS:
CA; LENGTH: 27 base pai~s
(Bi TIpE: nucleic acid
( C ~ S y'RANDEDNES 5 : s ir~.gle
- CD) TOPCLC~1: linear
i ii ) MGLECL~_.8 TYPE : CSNA
(ri) SEQL'~1~ DESCRI?TICDI: SEQ ID N0:3o'
TCAGGATCC'_' CTGC.'.CAAG~T TGGTACC 27
-193-
AMENDED SHEET
CA 02267193 1999-03-30
KO\ . \ o\ v L:f'.\-',II I:',~ Ili~:.\ W ::W - I -:?ei : 1,i _ 1
;i;J _:i;J;rl 4m:i: y.4~i
r u"WJ i ~ _ ~ .J~~ ~ ~J, rl, ~, ~~i
JL, _G. GU
( 2. ) ~.NFORMAT ICN ?CR SBQ ID N0 : 3 7
(i) SEQUENCa CHARACT:RISTwCS:
(A) L~NGTFi: 2"' base pairs
(3) '~:'PE: Rucleic acid
;C) STR,ANDEDNySS: si~g?e
TCFOLOGY : l i.~.aar
( ii) MOLSCLTr.~E TYPE: cDNA
(xi) ScQL7ENCJ DESCRIPT=ON: SEQ ID :7G::7:
AGCTTT CAGC~~C~.TTG~ GC~'TCAG 27
( 2 f :1'LT~"Gfil~l.'a.': I02~i FOR SEQ I D ~1C~ : 3 8
SEQL~1C8 CF.FRr~.CI'E:.ISTICS
(A} LEN sTh: 36 ~ase pal rs
(E) :"YPE: nucleic acid
(C) STRAi'v"D~N~c.~.S: single
(D} TOPOLOGY: linear
( i i ; MCLECLTLE TYPE : cDNA
(xi; SExL"F:TdCE DESVRT_PTION: S?Q ID d0:38:
:-,~1CCATGGC ACAdtGTT~~ciT ~.CG"..AC 2 6
( 2 ) I27=CRI~i~:.TION 'C12 SLQ ID NO : 3 9
i i SSQLWCE CFiARACTERISI'iCS
;?s) LENGTH: 30 oase pairs
(8) TYPE: T~C1~.C aC_d
(C} STR.A3~1D~NESS: single
(D) TOPOLGGY: linear
(ii ) MGLECUI:E TYPn : cDIrTA.
(Yi) aEQUr'~JCE DESCRIPTION: S$Q T_D NC:3j:
GCCCGC ;IIAT CCTCCTCACG G:NrICCT~_':iC = 0
( (~, ) I~,'~'GR.MATICN FOR SEQ ID NC' ~. ?0
(ii SEQUe v-CE CF..a,F.D,CTERiSTICS :
(A) LEN3TH: 32 ~as2 pairs
.1 g4_
CA 02267193 1999-03-30 ~~iENDED SHEEP
!sL\ ',O'. I.i'v-~II I.~~.~;II,\ mr' :~m- I -.lti : iri _. - . ; 1 :,:!
_:i;n.nl. iw_
~. ~ . :1 W ;
' ._ !J v ~ .u.~ 1~.W~I .J CJ ~V 1._. ~1
(By TYrT:: nuc;eic acid
( C ) STRANI=EDNE55 : s irgl2
(D) TOPOLOGY: 1_near
( 1..1. ) MOLEC'TT:~ T'IFE : CDhTA
(all $QUENC& DHS=RIPTICN: SEQ I~ :7C:40:
GCCT3CTCTA Gr',TC.:.~i GGw'~AGCTCC .'-.G 3
( 2 ) z:YF012:'"iAT IOTA' FCiZ S&Q ID NO: -~ 1
(i) SEQU"a.~~TCE Cx~.xAC':ERISTI~S:
(Ay '-~wGTH: 27 base pairs
(B) TYPE: nucleic acid
tC) STR~NDEDNESS: sincle
(Dy TOPOLOGY: ?in<'a.'
iii) i'iC:,~BC'J:~c TIPS: CGNA
(:ci) SY.QL'~1CE DESCRIPTION: SEn =D 2~0:~11:
~.=~iITCATCrIC Cr'~:.GAC~_'G.'~A TCCTCt:'T 27
(2) INFORMATION FOR SEQ _D N0:42:
( i i SHQU'~1CE Cr,ARAC:'ERIST:.CS
(1) _ENGT~i : ' 7 base pairs
(B) T'1PE: nucleic acid
(C) STRANDEDNESS: single
iDl ;CPGLOGY: _inear
( .~) . ) MOLECULE TY&E : cDNA
(xi) SEQUENCE DESCRIP'~=ON: SEQ .D N0:42:
~,~AA.G~TTT CAGTTCTCCT TCATGTC 2
(2j T~1FORMATION FOI~ SEQ iD N0:43:
(i) SEQLENCE ~C~ARACTERISTICS:
(A) LE:1GTF~: 27 base pairs
(B) TYT~E: nucleic acid
(C) STRANDEDNESS: single
tD) TOPOLOGY: =inear
-19S-
CA o2267193 1999-o3-3o AMENDED SHEET
!~:C'\ . \i>\.I.l' \-111 1.-,C;111.\ O,i ::n- I -:)ti : lii: ~ I , - ~-1:!
:i:) _:f:):i-I Iti:,: 11 E i
~J'-~W - V 1 V~ 1 _~ 11 v JCJ ~V 1G. Gl
(ii) MOLECLi.3 :YpE: cCNA
(Xi) SE(,d'UENCE DESCRIPTION: cEQ iD 11C:43.
ci(;i,i~sGCTTA TGAAGGTCTC CGT''..rirCT 27
(2) IN~ORMATION FOR SEQ _TD N0:4-~:
(i) SEQUENCE CIi~AC:'ERISTI~"S:
(A) LENGTfI: 59 ease pairs
iB) TYPE: nwcleic a:;i3
(C) STRANDEDNSS: single
(p) TOPOLOGY: lirea~
(ii! MCLECZE TYPE: cDNa
(x3.) SEQQFS1C= DESCRI2TION: ScIQ D N0:4~4:
s,GCTCTAGAT Cai:GCvT.aG T CTGGGrICG'x'C GTA'='C-GGTAe~ TTCi _ CCTGv T
C'I'TGATCC 5
( 3 ? INb'OR.'~Ic~TICN FOR SE,~~ XD NC : 4 S
i ) Sc'IQUENC?~ C3aR~CT.SRISTICS
(Ai LE23GTFi: 33 'case pairs
(E; T~t'?E : nucleic acid
(Ci S'CRs~.VDEDNESS: sl.~_cle
(D) ='CPOL~GY: _~r_ear
i i ) MCLEC'~'LE TYPE : =DNa
(xi) SEQTJr~ICE DESCRIPTION: SEQ ID NO:.l:5:
AAAGGATCCG CCACCATvF~ vGTCTCCGTG G:C 33
(2) T_NFORMATION SCR Sc,2 ID DiU:4n:
( i ) SEQU~1C8 CFiAt~'sC_'SRISTICS :
(A) LENGTH: 27 ~as$ fairs
(B) T'irB: nucleic aci~?
(C) STQANDEDNESS: single
(D) TOPOLGGY: linear
(ii) M~LECL~~ Ti'1~E: C~LJR
-196-
CA 02267193 1999-03-30 AMENDED SHEET
vC_\ \; '~ I..;~~ vll n. .~lll.'. O:> :ip- E._;y
I:i'-_ _I:) o:! '_:i;l;lll~i:~:miti
n .;~.r~ i _ : n u.~m.m_ ri , ..a
-4 1G~11
i xi~ SEQ~:ENCE DESCRIPTIG:T: SEQ ID N0: 46:
A~.:~ATCCT CAATTCTTCC AGGTCTT ~ 7
l2) _TNFCRMA':IOI~i cOR SIrQ wD LC:4?:
(i) SE('',,I1ENCB CFI~R~CTEkIrTICS:
(~) LENGTH: 3o base paira
(H) n~PE: rucisic acid
;C; STR~Iu'DEDNSSS: single
(D) TOPOLc7GY: :.:.near
~, ': i ) MOLBCCT'~E TYPE : cDNA
(vi) SEQL'ENIC3 DESCRIPTSCN: SEQ ID N0:47:
GG_~GCT':A '~G:ru':Cav"GCCT TG~~C-L_~3CC ;p
( 2 i I~:. O:~MAT I ON FOxs S EQ T-D NU : 4 fi
', i! SEQC,'ENCE 4I~AR~.CTE=tIST_ ICS
~A) LE~~TGT3; S? base pairs
(E) TYFEwucleic acid
!C7 3:'RAb~~rNESS: single
(~; TOFOLCSG'~l: linear
( _ i ; r9G:.~F.~'~ =E T't?8 : cDrL4
(ai; SGQL-ELVCE DESCRIP':iON: S3Q ID :10:48:
C~'T_'CTAGA'y CAABC3TAGT C'~GG:~ACs''C GT~.TGGGTAG GCATTCxGCT TC.w-.GGT~ 57
(~) ITFFORMA'.'.'_TON FGR ,~EQ .I? NC:49:
(:.) SEQL'~1CE CFL'~RACTEnIa'x'ICS:
(A) LF,NGTF3: 33 base pairs
(H) T"t'?E: nucleic acid
(Ci STRANDEDNESS: single
(D) TOPOLQGY: li:.eax
-=1 MOLECQL :Ys~E : CDFZA
(:C1) SSQ(JE.'JCE D~SCAT_P'.~T_ON: SEQ ID N0:49:
F,.~GC3A'Z'CCCy C~j''<C:.:'-~TGA.A ~3GGCCTTGCr~a a~GC 33
-197-
CA 02267193 1999-03-30 AMENDED SHEET
K~'. \W \_ Itl~\.11i I.,1.11L~. m:~ .:im- ! .~;i !ti ._ - :i ;i:~ ' _
r
I ,JHIU v ._ L I ~ _ r ~ _ of y ~~ ~: )~! V :. )
1~. '
(2) iVF'CR~IATIOI~I FOR SEQ ID N0:50:
( i i SEQLJ:~~1'CE ~,RAC'TrRISTICS
(,~) LENGTB_ 27 ~Hase pains
i :rpE : :Z1C 2iC 3C1'
(C) STQANL1ET;NESS: 5~:.ngle
(D; TOPOLOGY: ii:~ear
( i i l hfOLECL~,E :"PE : cCDtA
(y:i ) SEQUE~TCE DESC_RI?TIOrl: SEQ TD NO:- SQ
rWAGGAT,=CT CAv~GCATTCr'1 GC'I~_ C:~G 2 7
t 2 ) IaFOR.'~1i IJrF FO~2 88Q .D Nv : =1
;i) SBQUEaCE CJ.~CTE<_TSTICS;
(A) :.~GTIi: 28 base pairs
(3) T:FE: r.~cl We aciv
(:C) ST~aD,IEBS: si.~.gie
(D) 'T_'C,npLCG'~: linger
i i i '~GLEC'JL= T'TPE : cDI~A
;~i) SEQ'J~-CE ESCrcID'~TC~i: SEQ ID NOv5'_:
GGr~.e=~L:C'_T"_~A '-'GA :GAT:CC GTGG.:.'I'GC 20
~,'3; Iilr'JP_MATIC"-7 F(:R SoQ ID NQ : S."'.
( i ~ SEQCELTCE CTE:?ISTICS
(A) Lr'~IG~~F: 50 bas~ g3:.~s
T'IPE: nucleic acid
(~) ~~~'J~V~ ~ J3..1
(D) TOPOLOGY: lir_ea-
ii ) MOL.GC~1LE TY?E : cJNA
( xz ) S~cQTJENC.J DESC_~-.IP T ICN r SEQ IIJ 270 : 52
CGCTCTAGAT CAAGCGTAG? CTC-GGRC GTC GTATG~3GTAG TTCTCC T TC~'. TGT C':': "_'G 5
8
(Z; INFORMATION r''OR SE'Q ID N0:53:
;i) SEQLT~VCE vHARACTERISTICS:
tA) LENGTE: ~~ aase pairs
-198-
AMENDED SHEET
CA 02267193 1999-03-30
RO\. \c7\=I~.f'\ ~Il I.\c.:lll_v ,1:, :3n- L-:~2j : I;; __ .
- -6;1 ;~:) _;3:):)l lt'~ i::,:~~7
_ _ ~ _~e~- ;__ m ,
m .~~
(B) TYPE: nucleic aci~
(C) STRrIt7DEDIr'ESS : sy:.gle
(D) TOPOLOGY: linear
( i i ) MOLECULE '~~I PE : cD~la
',ai) Sr.QL'EI3:,E DESCRIPTION: SEQ ID N0:53:
aaAGGA~~CCG CCACCATGAA GATCTCCGTr, GCT 33
i2) rNFORMATION FOR SEQ ID 'T0:5:
(i) SEQLn"'I,VCE CFT~ARA~TERI~TICS:
(A) LE:TGTE: 30 base pairs
($) T:rPS: nucleic acid
(CJ STRANDELNBSS: s~ng~=
( J ) TOPGLOGY : l:.-~-a ar
~:ii) MOLECULE TY?E; CDN1
f,:~i) SEQTJ"iiTdCE DBSCR=PTT_CN: a"&Q .0 N0: S4:
a~AGGiy"-'CCT CAG'_TCTCCT TG:TGTC~~"T_' 30
( 2 ) =biFORM.~'I TON FOR SEC ID Q70 : 5 S
!i) BEQUE:1CE C:i~IFcACTEI~.ISTICS:
(A) LENGTH: 92 atninc aCSds
(B) TYPE: ami_~.o ac-d
(C) ST:Rr'1ND8DNESS : noL relevaa
(DJ TOFOLOGY: linear
(i1) hIOLEC'G2.E TYPE: prct4in
ixi) SEQT1EPICr DESCRIPT:CN: Sc,C,~ ID r70: 5S :
Met u'ln 'Jal Ser Thr Ala A:.a Leu Ala Val Leu Leu Cps Thr Met Ala
1 5 10 :.5
eu c.'',~s Asn GIu Phe ae; A'_~ Sex Leu Ala Ala Asp ::~ Pro Th= Ala
20 3S 30
C'~s Cys Fhe Ser Tyr Thr Ser Arg G1n ile P;;o G1n :..sn Phe I'_e A.a
3S 40 45
A3p T'r- Pre Glu Thx Ser Ser Gln Cys Ser Lys Pro Gly 'Ja1 ila Phe
-199-
AMENDED SHEET
CA 02267193 1999-03-30
..tm ;~ -:i:l:L/..to.a: ~tf.l
RC:\.\W', I.I'\ \lli.~.u!I! ~,, in- !-',Ri I~i __ ~ _._ . _ _
_ _ . _ _ _ _ ~ ~~~ -m
5u ~s ~o
Leu T'.zr Ly9 Elr~ Sir :yrg Gln Val :".ys A? a A.sp Pro Se- G1~ Giu Trp
55 70 75
.'r11 Gi:: ::V3 Tj,rr ~1~.1 $2I' :, D "~11 Gi:l L211 5eL Aid
85 3t7
( 2 i INFG~~LAi T_ON FGR SE,~ ID NC : 56
:1; SE~CTENCB Cs.-T~3.~~CTEFcT_STIC3:
~;Ai ~ENGT~:: Q205 base .airs
(H) T'IPE: :luciei= ac_d
!C) STR~~2:ES5: doub? ~
(Di TGPOLGGY: hear
(iii MGLE,~"~LE 'TYPE: rrlr'1 (gzncmic!
,';:i.)
SEQQ~~1C~,'
DF.SCRIPT;CN:
S,T,4
ID .:C:S6:
A.~GCTT-~.1-1r".CTGCAAAAAATAGT'="_'a1-i~GTGAC-
C~3~~,it.~ae~CA~?T':n:~,CATGT.wnCCc0
C.~'='='GTGAGCC~? TAi-iCr~~' '_"'_' TA~Gl.',iSliACr~ i.~'~'~_T GV.~C
T :.r'lCi..C~.T W_AT. tGTTT
A
Cr' ~C _~CCGCTGAC';'Gv:uCATCTCCTRCr'1CCCCGCGTT. C:~TGCTCGCT.8
G CTACC CC.'~TCC C
G':': T ACT C t~.a..~.Ca': GC i_ CGG:c'T_'GTT~sT CTTCCTG.=.:,?.
GGi~s?TCC.TCGr'is~.a-s::: CGr'1x C =Arlt~. ~
t)
Cr'~~Cv';'.~:.TC.~LTTC'TGCG~.Ir~.A~CCGTC'3.ACrlr~lCAGGTTC.n.Gu"~'_s':'?TG:G
TY'r GCTG'Ø::.AC'=''',~GAC.A.CCCGTA'".~_yCCGTAp~AAC': ;::AT~.:,~TACC :
A.AGT::AG3
~1C ~
G
'T:.fC-CSGCGTCCGATCG.AC3a.:..CGCCT"'~'_'TmrT'~Ja'~A='_'C,iii-a,.~Tt"'.l-
~.TGGTCATAGC"rG'~TT4~i:
C C'T ?~TTGTTATCC~~C'_"CA~T":CCAC~r.C_?': =.C:~uCCGG:,.-.~C.~':~
G T G': ~.C_'~ ~ ~~~ 2
G?.a 0
TGTA.AAGL.'r"T_'GGGGTGCCTAATG:
G':Ga=.~'I'.~AC':t=AC~a_~'T.'a.'~'=TGC:~TTGCGC"fG~CTG5~0
CCCGCTTTCCi~GTCGGGr~s~~~CTGTCGTGCCrGC'TC-CI3TTAnTGAATCGC:CCAeaCGCGCG60G
VGGA~:AG~.tCGGTTTGCG~'hTT:rGGC~.;CTC'"TCCGC'L_'CCTCGCTCs-
1CTG.e~.CTC,~sC'!'GCGC6fiG
TCGGTCG:TCGGCTGC'.CiGCGAGCGGTAT".A3C'=Ca,.CT.'C.~,AGGCGGT~'.,T:~CGG'T:'ATCC7~0
.~G.GAnTCAGG;aC,ATn.=iCGCAGGA.~',.rt=.ACATGT,~~-
AGCF~F,:~GGCCAGGA_Ai,S,~;f:CCAG;790
:iaCCGTAA:,AAGGCC.~rc'.'GTTGC':G~G'~i'T~_'C:~;?GGCTCCGtCCCCC'r'GP~:GAGC:~Tfi4~
I.AC:~AATCGACGCTC.~~AGTCAG:aGGTCG~CC CnGGF.CTAT',~AAG.A T 9
~G CGF. y? ~~ Q
__~,AG 0
GC:,TTTCCCC~=TGGPAGCTCCC?CGTGCGCTCTCCTGT.TCCGACCCTGCCGCTTACCGC:,y,.9EJ
CA 02267193 1999-03-30 Av~E~~flED SHEEI
KC:\ \~\=I.;'1-\11 I.',OIII.'~, n:i :im- l-:!:i . Itf _:1 . - +1J .3:)
=:):i:i~l-11,
I 'J~ll.1 I _ i U n J,JG' 1G.- f'If v JC.: ) i
JV 1L. '
TACCTGTCC.~r CCTTTCTCCCTTC~~~GG~L~GCGTGGCGCTTTCTCATAGCTCACGCTGTAGCx1020 _"
- _,._,._
T:~TCTC~'1G'fT CC-
G1'GTAGGTCGTTCGCTCCAAGC':'GGGCTG~_'G'T_'GC.~.CGAACCCCCCGTT10S0
CAGCCCGACC GCTGCCCC ATCCGGT:-~.sCTATCGTCTTGAGTCCAACCCGGTIaAGACAC1110
TT
lx'~.CTTt1":::GC C.rsCTtiGCAC-CAGCCA :-L~CALiGriTTAGC.~IGACvCGr'S,GGTAT
;TAGGCl2 0 0
~: T
GGT
GGTGCTACAG AG :'TCTTGAAGTGGTC~CCTAACTACGGCT'.CACTAGAA.G.AACAGTA'~TT12 5
0
GGTATCTGCG GTCTGCTG_~AGCCAGTTACCTTCGGAA.'~AGAGTTGGTAGCTCTTGATCC1320 ..
GGCA~CA.~ CCACCGC'':'t_TAGCGGTGGTTTT'~ GCP.AGCAGCAGATTACGCGC13 8 0
G T'TGTTT
~~r'~A.AF,G .rATCTC.~.:'CaAAGiITCCT:'."GATCi-
T'T_'C';'AC3GGGTCTGACGCTCAG':'C-.i1440
A~.CGaAAaCT CACGTTAAC-GGATTTTGGT'CATGAGATTAT~CGTCGA'(,~,TTCGCGCGCGAl500
AGGCGAAGCG rCATGCATT~-'AC3TT3nCi-
.CCATCGAA:GGTGCa~AAACCTTTCGC;~C,T=,'~156 C
;3Gi.ATGA'_T'~G CGCCCGG_~GAGt.GTC:,A':TCAGGGTGGTGAA')
~TGaAACCAGT:~ACGrTTa.620
:..TACGA'_'GTC GCa.G=. CC,sGT.G'7C'='CTTATCACaACCG~''CCCGCGTGGTGAACn 1680
GT:~TG
GGCC_yGCCaC v'-
"~TCTGSG.=vA.'~CGCsG:aAAFsAP~GTGGAAGCGGCGATGGC3GAGCTG.'~lis174C
TaC:~: TC':'C AACCGCGTGGCA C~.IC~u:.CTGGCGGGCAAACAG T TGATTGGCGT~. 8 0 0
CGTTGC
:'GCCrICCTCC FiGTCTGGCCCTGCACG~GCCGTCGC~'.>rAATTGTCGCGGCGATiAPtITCTCG,36C
CC-CC;GATCr'\A CT. GC-:JTGCCAGC:~TGG~-GGTGTCGATGGTAGAFsCG:~GCGGCGTCGA~aGC'i
92 C
CTGTAnAGC.~a s.-:~C-
GTGCACAF.'TCTT!:'=CGCGCAt~CGCGTCAGTGGGC~.'CaATCe'1TTAACT.'~1980
TCCGCTGCHT C-aCCAC3GATGCC-ATTGCTGTGGAAGC'TGCCTGC
AC'='AATGTTCC~CGT'_"2040
.':TTTCTTGA= G T CTCTGACCAGP.Ct'lCC'l.~aAi-
,".a~u.ATTTTCTCCC?iTGA.'~GACGVi;10 0
'C~1T Tai :
TACGC.~sAC-'~G uGCGTuGAGCATCTGGTCGCATIGGGT~CACCAGCA.:.ATCGCGCT'GT:.''~GC2160
GGGC CG~T':A AGTTC='G CGGC,~~ GCGTCTGGC'='GGCTGGCATAAA2'ATCTCAC2 2 2 0
T C'r CG :
CT
TCG'"~'I'CFsxs .'~.T TaC,CGGe':~CGGGAAGC-CGACTGGAGi'rCCA':GTCCGGTTT2 28 C
: CrGCCGP~
TCAACAAACC~
ATGCAAATGCTGA.4':Gs.,GGGC.ATCGTTCCC.ACTGCGATGCTGG'_'TGCC.~.2340
CsATCAGA'_'G GCGC~_'GGGCGCAATGCGCGCC:.TTACCGAGTCCGGGCTGCGCGTTGGT 2 4 0 0
GC
C,r',~ATA':'CTCCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTT2450
AACCAC.~.A~"C ~.~.CAGGAT'=TTCGCC"GCTGGGGC~~ACCAGCGTGGACCGCTTGCTGCA2520
;.,CTC~_'C':'C GGCCAGGCG::TGAAGGGCAATCAGCTGTTGCCCGTCTCACTGG'='GAF~AAG2 5 8 0
AG
AAAAACCACC CTG~',~GCCC'.AA='ACGC~.C~CGCCTCTCC.~_CiCGCGTTGGCCGATTCATT2540
-?al-
AMENDED SHEET
CA 02267193 1999-03-30
W :\ . \m\ f:l' \ -\II I.~,.l:llt:.'. O5 :sm - 4-:tri . I vS .:S : I - _ 5;~
;;:) ...;:1:1.i-t,c:) . n ;;S
.r - ._ ... V.....'~ 1-.- t~I '. ~V .:V 1...~ _J
3.~,TGCAGCTGGCACGACAGGTTTCCCSr?~C:GG.a.~IGCGGGC.~.GTGAGCGC.<',.r.CCiC:yA'~'"A2
%
00
A~''GTAAGTTAGCGCGAATTGTCGACCrIF~..aGCGGCCRTCGTGCCTCCCC:ACTCCTGG'ai,T.''.
T ~
60
CGGGGGCATGGATGCGCGGATAGCCGCTGCTGGTT'rCC':GGATGCCG?CGGATTTGCACV'B30
GCCGGTAGAACTCCGCGAGG'_"C3TCC.~.G~CC':CAGGCAGCAGCTGAACCAA~~CaCG?GGG2880
GRTCGAGCCCGGGG TGGGi.GAFsGAACTCCAGCA'~'GAG.'~.TCCCCGC AGGATCATCC~
GCTGG 9=3
0
AGCCGGC CCGGAAA.~CGAT'.~CC~.aAGCCCAACC A'?'AGAAGGCGGC G'sT~zCAAT3
rTC T TTC 00
Q
CC's.~'ar~:1'CTCGTGrlTGGCAvGTTGC-GCG"_'C.~~CT': GG C'aTTTC CCG$GAG'"CC3
T .~.3GT ~.~. 06
C
CGCTCAGA.tIGA.ACTCGTCAAGAAGCaCGATr'1''CG.'LTGCC-CTGCGFiATCG:~Gi.~GCGGC3
.2
G
i3aTACCGTAAAGCACGAGGAAGCGGTCAGCtC.'~T': ~CCAAGCTC'I'T~...r'~GCAATATC3
CGCC ip
G G
ACGGGTAGCCe~?~CGCTt~.TG'=CCTGATAC-CG~GTCCGCC CCCAC-CCGGCC:.C.~GTUGA':y
AC.A 2
i
C
GF~TCC?.G'?.~AAGCGGCCA=TTTCCrlC~'.~TGAT:aTTCCIiAAGCAvGCATCGCCAT 3
C GGGT 3
0
G
CrICGACGAGATCCTCGCCGTC~GC..~'"GCGCGCCTTGAGCC!'C,GCGe;ACAGTT.r..~xsCTGG3360
CGCGAGCCCCTGATGCTC"."TCGTCCAGATCATCCTGATCG:~C.~AGACCGGCTTC~...ATCCG32r
cIGTACGTGCTCGCTCGATGCGr~TGTTTC:n:TTC-GTGGTCGr'~ATGGGCA:aGTALC C 3
G;~A':'C i
B
0
A.=.CCGTA'TGCAGCC JCCC-~.ATTG~~T~-AVC=~.TGi.TGGATACT'"TC CAGGaG~G 3
T CGG 54
~G
aTGAGAT3ACAC-GAGATCC.'TGCCCC:fiCAC'rTC iCCCrIATAG:.P.GCr'AGTC'CCTT:j 3
CCGC 6
~~
0
""TCnGTGAC:-.A~CGTCGrIGC~1C.AGC:G:.rCr_:~GGAACC-CCCG:CGTGGCCAGCC.'rICGA=AG3660
CCGCGCT. TCGTCCTGCe,GT T C: GGCACCuGACAGGTC TGasCiIAAF~'~:G3
GCC >.TT iGTCT 72
CAG 0
4ACCGGGCGCCCCTGC3CTGACAGCCGG.~ACACGGCGGCA'1'C.~iJAGCAGCCGAa'TGTCTG:?90
T T GTGCCCAGTCATAGCCG.:,sTAGC CACCCAAGCGGCCCrGAGAACCTGC;~'~G~3
=T=TC ~A 8
a
0
~~.'CC.'~'1'CTTGTTC.'-
IFaTC.~ITGCGAAACGA:CCTCe'~TCCTGTCTC'fTGATCAGATCTC'~'.u'I.TS.C390G
C C';"GCGCCATCAGATCC GCC~C.'~:,GAAAGCC.'~TCCAGTTTA CTTTGCAGGGCTTCCC3
: i G 96
a
AhCCTTACCAGAGGGCGCCCCAGCTGG~...sA'_"'TCCGGTTCGCT~'xCTGTCCATAAAAC::GC02G
yCAGTCTi~GCTATC~aCC.~'~'3T.'>aP.GCCCACTGCa.AGCTACC2'GCTTTCTCT'TTGCGCTTGC4080
:,TTTTCCCTTGTCCAGAT.~GCC CAGTAGCTGACATTCATCCGGGGTCAGCACCGTT 4
_'CTG i
4
G
CGGAC:GGCTTTCTACGTGT'~CC iC~_'TCCT'~TAGC.~GCCCTTGCGCCCTGAGTGCTTGCG420G
GG'?~.~CGTG 4
2
0
8
-2U2-
CA 02267193 1999-03-30 AMC~1DED SHEET
iW\ '. W~. I L' \ -\ll l .~ l l ll .~. ~ o~ . Sn - I -l!ii . ~ ri _ I I r J _ -
. _JY r.l ; r >i:? ':;::; l l i ~ ~. ~y ; l
a _J .11 '. ~CJ _ V ~ LJ
(2) T_NFQRMATION FOR SEQ iD :ICvS%:
( i 1 ScQ(TENCE CP.AR~,CTERISTTCS
(A) LrNGTFI: 11Z base pairs
(3) TYPE: nucleic ac'_d
lC) STRANDEDITES3: dcub'_~
(D) TC2-.L~Y: linear
( ii) hIC~CGZ.~ T''P~ : DNA ;~encTi~;
(xi} SnQL'ENCy DESCEtI?TICN: SEQ ID NO:57:
:~GCT'~"?.rsAA ~CTG~.~A ~': rlri'L T"'GA CTTGTsAC~CG U~! T."-.AC A.~:T ~
~lGiaTG~'=,CC 6 0
C.~.TTG:GAG CC-GAT~rlCesfi ':'~'i'C~CACAT T~C'We",G r~TTAC'1:A ''_'U' __..
_~~3_
AMENDED SHEET
CA 02267193 1999-03-30
