NZ612783B2 - Stem cell factor inhibitor - Google Patents
Stem cell factor inhibitor Download PDFInfo
- Publication number
- NZ612783B2 NZ612783B2 NZ612783A NZ61278312A NZ612783B2 NZ 612783 B2 NZ612783 B2 NZ 612783B2 NZ 612783 A NZ612783 A NZ 612783A NZ 61278312 A NZ61278312 A NZ 61278312A NZ 612783 B2 NZ612783 B2 NZ 612783B2
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- antibody
- scf
- fibrosis
- disease
- stem cell
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
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Abstract
Use of a monoclonal antibody or an antigen-binding antibody fragment in the preparation of a medicament for treating or preventing a disease that is a fibrotic disease or a tissue remodelling disease in a subject, wherein the monoclonal antibody or antigen-binding antibody fragment specifically binds to stem cell factor isoform b relative to stem cell factor isoform a. ds to stem cell factor isoform b relative to stem cell factor isoform a.
Description
STEM CELL FACTOR INHIBITOR
This application claims priority to US. Patent Application Serial Number 61/431,246
filed on January 10, 2011, which is orated herein by reference in its entirety for all
purposes.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR
DEVELOPMENT
This invention was made with government support under 78 d by the
National Institutes of Health. The government has certain rights in the invention.
FIELD OF INVENTION
Provided herein are methods, compositions, and uses relating to inhibitors of stem cell
factor. For example, provided herein are antibodies targeting stem cell factor and methods for
treating fibrotic and tissue remodeling diseases.
OUND
Diseases involving tissue remodeling and fibrosis are a leading cause of death
worldwide. Nearly 45 percent of all natural deaths in the western world are utable to
some type of chronic fibroproliferative disease and the associated health care costs are in the
billions of dollars. Tissue ling is the reorganization or tion of existing tissues,
which can either change the characteristics of a tissue (e.g., blood vessel remodeling) or
ipate in establishing the dynamic equilibrium of a tissue (e.g., bone remodeling).
Fibrosis is the formation or development of excess fibrous connective tissue in an organ or
tissue as a reparative or ve process, as opposed to formation of fibrous tissue as a
normal tuent of an organ or tissue. Fibrosis affects nearly all tissues and organ systems,
and fibrotic tissue remodeling can influence cancer metastasis and accelerate c graft
rejection in transplant recipients. Diseases in which fibrosis is a major cause of morbidity and
mortality include the interstitial lung diseases, liver cirrhosis, kidney disease, heart e,
and systemic sclerosis, among others.
Stem cell factor (SCF) and its receptor c—Kit have been implicated in fibrotic and
tissue remodeling diseases (El-Koraie, et al., Kidney Int. 60: 167 (2001); Powell, et al., Am.
J. Physiol. 289: G2 (2005); El Kossi, et al., Am. J. Kidney Dis. 41: 785 (2003); Powell, et al.,
Am. J. Physiol. 277: C183 (1999)). c-Kit is a type III receptor-tyrosine kinase that is present
in many cell types (Orr-Urtreger et al., Development 109: 911 (1990)). It is also expressed in
the early stages of entiation (Andre et al., ne 4: 1047 (1989)) and certain tumors
exhibit elevated expression of c-kit. SCF is a ligand specific for the c-Kit or kinase.
Binding causes dimerization of c-Kit and activation of its kinase activity. SCF was first
isolated from the supernatant of murine fibroblasts. At the time, SCF was called mast cell
growth factor (MGF) (Williams et al., Cell 63: 167 (1990)) or hematopoietic growth factor
KL (Kit ligand) (Huang et al., Cell 63: 225 (1990)). A homologue was subsequently isolated
from rat liver cells and designated stem cell factor (SCF) (Zsebo et al., Cell 63: 195 (1990)).
The corresponding human protein is designated variously as SCF, MGF, or Steel Factor (SF)
(Cell 63: 203 (1990)).
Previous studies have suggested that an inhibitor of c-Kit receptor tyrosine kinase can
significantly inhibit aberrant tissue fibrosis (see, e.g., Aono, Am. J. Respir. Crit. Care Med.
171: 1279 (2005); Vuorinen, et al., Exp. Lung Res. 33: 357 (2007); Vittal, et al., J.
Pharmacol. Exp. Ther. 321: 35 (2007); Distler, et al., Arthritis Rheum 56: 311 (2007)).
However, this inhibitor has several disadvantages. It needs to be given systemically by oral
administration, it has some toxicity associated with its use, and the compound must be
delivered intracellularly for efficacy. Consequently, ative therapies are needed.
SUMMARY
Provided herein are methods, compositions, and uses relating to inhibitors of stem cell
factor. For example, provided herein are antibodies targeting stem cell factor and methods for
treating fibrotic and tissue remodeling diseases as well as for research and diagnostic uses.
In a first , the present invention provides use of a onal dy or an
antigen-binding antibody fragment in the preparation of a medicament for treating or
preventing a disease that is a fibrotic disease or a tissue ling disease in a subject,
wherein the monoclonal antibody or antigen-binding dy nt specifically binds to
stem cell factor isoform b relative to stem cell factor isoform a.
In a second aspect, the present invention es use of a onal antibody or an
antigen-binding antibody fragment that specifically binds to stem cell factor isoform b
relative to stem cell factor isoform a in the preparation of a medicament for administering to a
cell or a tissue
In some embodiments, the compositions, methods, and uses herein provide
ies relating to inhibiting stem cell factor (SCF). Some embodiments provide an
9351045-1
isolated antibody that s SCF. In some embodiments, inhibiting SCF s the
activity of c-Kit. The compositions, methods, and uses provided herein find use in
treating fibrotic diseases and maladies associated with tissue remodeling. Unlike some
other therapies that produce undesirable side effects due to interfering with general
intracellular signaling pathways, the embodiments provided herein eliminate or
minimize such side effects by ting the activity of SCF. Consequently, toxicity is
minimized. Moreover, targeting an extracellular ligand s the need to deliver a
composition into a cell to interact with an ellular target. In some embodiments,
the compositions are delivered into the airway, thus providing
9351045-1
an advantage over previous technologies that require oral stration and, as such,
resulting in ic bioavailability.
In some embodiments, provided herein are methods comprising providing an inhibitor
of stem cell factor and stering a eutically effective amount of the inhibitor to a
subject. In some embodiments the inhibitor is an isolated antibody or an antigen-binding
fragment thereof (e.g., Fab, Fab’, F(ab')2, and Fv fragments, etc.). In some embodiments the
inhibitor is a small interfering RNA. In more specific embodiments, the antibody is a
monoclonal antibody or a polyclonal antibody. Some embodiments provide that the antibody
or antigen-binding fragment f specifically binds to stem cell factor. Some embodiments
provide that the antibody or antigen—binding fragment thereof specifically binds to a peptide
comprising amino acid sequence SEQ ID NO: 1 or SEQ ID NO: 8.
In some embodiments of the methods provided herein, the subject has a disease.
Accordingly, some embodiments provide that administering the inhibitor prevents or reduces
the severity of at least one sign or symptom of the disease. In some ments, the t
has an abnormal activity of stem cell factor or the subject has abnormal collagen production.
In some embodiments, the subject has a disease including, but not limited to, fibrosis or a
remodeling disease. In additional embodiments, the disease is a ary disease. Some
embodiments provide that a subject has a pulmonary disease including, but not limited to,
idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory
distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, or
asthma. In on, some embodiments provide that a subject has a e including, but not
limited to, sclerodoma, inflammation, liver sis, renal fibrosis, hymal fibrosis,
endomyocardial fibrosis, inal fibrosis, nodular subepidermal fibrosis, fibrous
histiocytoma, fibrothorax, c fibrosis, fibromyalgia, gingival fibrosis, or radiation-
induced fibrosis.
While not limited in the mode of administration, in some embodiments of the method,
the antibody is delivered into an airway of the t, e.g., by intranasal administration.
In some embodiments, administering the inhibitor reduces the activity of a receptor.
Some embodiments provide that administering the inhibitor reduces an interaction of stem
cell factor with a receptor. In more specific embodiments, the receptor is a receptor tyrosine
kinase, and in yet more specific embodiments, the receptor is c-Kit. Importantly, the methods
are not limited in the location of the targeted or or the origin of stem cell factor. For
e, in some embodiments the receptor is found on a hematopoietic progenitor cell, a
melanocyte, a germ cell, an eosinophil, a lymphocyte, a fibroblast, a myofibroblast, or a mast
cell. Additionally, in some embodiments, stem cell factor originates from a bone marrow cell,
a liver cell, an epithelial cell, a smooth muscle cell, or a fibroblast. In some embodiments,
administering the inhibitor to a subject results in a direct inhibition of fibroblast tion.
Some embodiments provide a composition comprising an isolated dy or
antigen-binding fragment thereof that specifically binds to stem cell factor (e.g., a protein or a
peptide fragment thereof (e.g., an epitope)). For example, some embodiments e a
composition comprising an isolated antibody or antigen-binding fragment f that
specifically binds to a peptide of amino acid sequence SEQ ID NO: 1. Additional
embodiments provide an antibody or antigen—binding fragment than binds to the SCF isoform
b sor (e.g., a protein or peptide fragment of the sequence available at GenBank
accession number NP_000890 (SEQ ID NO: 4)), or a variant or modified form thereof, or to
the SCF m a precursor (e. g., a protein or peptide fragment of the sequence available at
k accession number 985 (SEQ ID NO: 6)), or a variant or modified form
thereof. Some embodiments provide an antibody or antigen-binding fragment that binds to a
protein or peptide, or variants or modified forms f, that is a translation product of the
NCBI Reference Gene Sequence for SCF (e.g., accession number NG_012098 (SEQ ID NO:
7)) or ts or fragments thereof. Some embodiments provide an antibody or antigenbinding
nt that binds to a peptide sing the first 11 amino acids of the mature
form of SCF (e.g., EGICRNRVTNN (SEQ ID NO: 8)).
Some embodiments provide an antibody or antigen-binding fragment than binds to the
translation product (e.g., a protein or peptide), or a variant or modified form thereof, of a
nucleic acid encoding SCF, or a variant or a modified form thereof. For example,
ments provide an antibody or antigen—binding nt than binds to the translation
product (e. g., a protein or peptide), or a variant or modified form thereof, of the nucleic acids
having sequences comprising a sequence as defined by k accession numbers
NM_000899 (SEQ ID NO: 3), 994 (SEQ ID NO: 5), and NG_012098 (SEQ ID NO:
7), or fragments or variants thereof (e.g., mutants, cDNAs, expression-optimized variants,
operably linked to a regulatory element (e.g., er, enhancer, rase binding site,
etc.), etc.). In some embodiments, the antibody or antigen-binding fragment binds to a protein
or peptide, or a variant or modified form thereof, that is the translation product of a
nucleotide sequence that encodes the peptide sequence EGICRNRVTNN (SEQ ID NO: 8).
The peptides and proteins (and fragments and variants thereof) and the nucleic acids (and
fragments and variants thereof) that encode the peptides and proteins (and fragments and
variants thereof) are used in some embodiments to raise antibodies. Also contemplated are
vectors, plasmids, expression constructs, cells, cell lines, hybridomas, and organisms used to
e the dies as provided herein.
Some embodiments provide a monoclonal antibody and some embodiments provide a
humanized antibody. In some embodiments, the composition is used for a medicament or is
used for the manufacture of a ment. In some embodiments, the medicament is used to
treat disease. Use of the composition as a medicament is not limited in the disease that can be
treated. For example, in some embodiments, the disease is idiopathic ary fibrosis,
chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis,
peribronchial fibrosis, hypersensitivity pneumonitis, asthma, sclerodoma, inflammation, liver
cirrhosis, renal s, parenchymal s, ocardial fibrosis, mediatinal fibrosis,
nodular subepidermal fibrosis, fibrous histiocytoma, fibrothorax, hepatic fibrosis,
fibromyalgia, gingival fibrosis, or radiation-induced fibrosis. In some embodiments, the
composition is used to study disease in vitro or in a model system (e.g., in vivo).
Embodiments provide herein a method ofpreparing an antibody (e.g., a monoclonal
antibody) targeting stem cell factor comprising the steps of providing a peptide comprising or
consisting of an immunogenic n of SCF (e.g., as provided by SEQ ID NO: 1 or 8),
immunizing a host with the peptide, isolating an immune cell from the host, preparing a
hybridoma using the immune cell, and isolating the dy or antigen-binding fragment
thereof. Some embodiments provide a method ofpreparing an antibody (e.g., a monoclonal
antibody) targeting stem cell , wherein the dy or antigen-binding fragment thereof
specifically binds to stem cell factor (e.g., a protein or a peptide fragment thereof (e. g., an
epitope)). For example, some embodiments provide a method of preparing an isolated
antibody or antigen-binding fragment thereof that specifically binds to a peptide of amino
acid sequence SEQ ID NO: 1. Additional embodiments provide a method of preparing an
antibody or antigen-binding fragment than binds to the SCF isoform b precursor (e.g., a
n or peptide fragment of the sequence available at GenBank accession number
NP_000890 (SEQ ID NO: 4)), or a variant or modified form thereof, or to the SCF isoform a
precursor (e.g., a protein or peptide fragment of the ce ble at GenBank accession
number NP_003985 (SEQ ID NO: 6)), or a variant or modified form f. Some
embodiments provide a method of preparing an antibody or n-binding fragment that
binds to a protein or peptide, or variants or modified forms f, that is a translation
product of the NCBI Reference Gene Sequence for SCF (e. g., accession number NG_012098
(SEQ ID NO: 7)) or variants or fragments thereof. Some embodiments provide a method of
preparing an antibody or antigen-binding fragment that binds to a peptide comprising the first
11 amino acids of the mature form of SCF (e.g., EGICRNRVTNN (SEQ ID NO: 8)).
Some embodiments provide a method of preparing an antibody or antigen-binding
fragment than binds to the translation product (e.g., a protein or peptide), or a variant or
modified form thereof, of a nucleic acid encoding SCF, or a variant or a modified form
f. For example, embodiments provide a method of preparing an antibody or antigen-
binding fragment than binds to the translation product (e.g., a protein or peptide), or a variant
or modified form f, of the nucleic acids having sequences comprising a sequence as
defined by GenBank ion numbers 899 (SEQ ID NO: 3), NM_003994 (SEQ
ID NO: 5), and NG_012098 (SEQ ID NO: 7), or fragments or variants thereof (e.g., s,
cDNAs, expression-optimized variants, operably linked to a regulatory element (e.g.,
promoter, er, rase binding site, etc.), etc.). In some embodiments, the antibody
or antigen-binding fragment binds to a protein or peptide, or a variant or modified form
f, that is the translation product of a nucleotide sequence that encodes the peptide
sequence EGICRNRVTNN (SEQ ID NO: 8). The peptides, proteins, and fragments and
variants thereof; and nucleic acids, and fragments and variants thereof, that encode the
peptides, proteins, and fragments and ts thereof, find use in some embodiments in a
method of preparing antibodies as provided by the technology ed. Also contemplated
are methods of producing vectors, plasmids, expression constructs, cells, cell lines,
hybridomas, and organisms that find use in producing the antibodies as ed herein.
Some embodiments e a method sing the steps of providing an inhibitor
of stem cell factor and administering the inhibitor to a cell or tissue.
In addition, some embodiments provide a kit comprising a composition comprising an
isolated antibody or antigen-binding fragment f that specifically binds to stem cell
factor, a means for administering the composition to a subject, and/or instructions for use.
Additional embodiments will be apparent to persons skilled in the relevant art based
on the teachings contained .
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present technology will
become better understood with regard to the following drawings:
Figure 1 shows a series of plots demonstrating that inhibiting SCF with an antibody
reduces the expression of tissue remodeling mediators. Figure 1A shows a plot trating
that an anti-SCF antibody reduces the amount of yproline in bleomycin treated lung;
Figure 1B shows a plot demonstrating that an anti-SCF antibody reduces the amount of IL-25
mRNA; Figure 1C shows a plot demonstrating that an anti-SCF antibody reduces the amount
of IL-13 mRNA; Figure 1D shows a plot demonstrating that an anti—SCF antibody reduces
the amount of soluble SCF present in plasma. Figure 1E shows a plot demonstrating that an
anti-SCF dy reduces the amount of IL-25 receptor.
Figure 2 shows a plot demonstrating that IL-4 stimulates c-kit expression in human
asts.
Figure 3 shows an amino acid sequence and the corresponding nucleotide sequence
of an immunogenic peptide used to produce antibodies c for SCF.
Figure 4 shows a plot demonstrating that a monoclonal antibody specific for SCF
inhibits the activation of HMC-l cells for MCP-l production.
Figure 5 shows a plot demonstrating that a lower amount of hydroxyproline is
detected in a mouse deficient in SCF production after bleomycin injury.
DETAILED DESCRIPTION
Provided herein are methods, compositions, and uses relating to tors of stem cell
. For example, provided herein are antibodies ing stem cell factor, s of
producing antibodies targeting stem cell factor, and methods for treating fibrotic and tissue
remodeling es as well as for research and diagnostic uses. In some embodiments, the
compositions, methods, and uses herein provide therapies relating to inhibiting stem cell
factor (SCF). Some embodiments provide an isolated antibody that targets SCF. In some
embodiments, inhibiting SCF affects the activity of c-Kit. The compositions, methods, and
uses provided herein find use in treating c diseases and maladies associated with tissue
remodeling.
ions
To facilitate an understanding of embodiments of the present technology, a number of
terms and phrases are defined below. Additional definitions are set forth throughout the
detailed description.
Throughout the specification and , the following terms take the meanings
explicitly associated herein, unless the context clearly dictates otherwise. The phrase “in one
embodiment” as used herein does not necessarily refer to the same embodiment, though it
may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily
refer to a different embodiment, although it may. Thus, as described below, various
embodiments of the invention may be readily combined, without departing from the scope or
spirit of the invention.
In addition, as used herein, the term “or” is an ive “or” operator and is
equivalent to the term “and/or” unless the context y dictates otherwise. The term “based
on” is not exclusive and allows for being based on additional factors not described, unless the
context clearly dictates ise. In addition, hout the specification, the g of
“a”, “an”, and “the” include plural references. The meaning of “in” es “in” and “on.”
The terms “protein” and “polypeptide” refer to compounds comprising amino acids
joined Via peptide bonds and are used interchangeably. A “protein” or “polypeptide” encoded
by a gene is not limited to the amino acid ce encoded by the gene, but includes post-
translationalmodifications of the protein.
Where the term “amino acid sequence” is recited herein to refer to an amino acid
sequence of a protein molecule, “amino acid sequence” and like terms, such as “polypeptide”
or in” are not meant to limit the amino acid sequence to the complete, native amino
acid sequence associated with the d protein molecule. Furthermore, an “amino acid
sequence” can be deduced from the nucleic acid sequence encoding the protein.
The term “nascent” when used in reference to a protein refers to a newly synthesized
protein, which has not been subject to post-translational modifications, which es but is
not limited to glycosylation and polypeptide shortening. The term “mature” when used in
reference to a protein refers to a protein which has been subject to post-translational
processing and/or which is in a cellular location (such as within a membrane or a multi-
molecular complex) from which it can perform a particular function which it could not if it
were not in the location.
The term “portion” when used in nce to a protein (as in “a portion of a given
protein”) refers to fragments of that protein. The fragments may range in size from four
amino acid residues to the entire amino sequence minus one amino acid (for example, the
range in size includes 4, 5, 6, 7, 8, 9, 10, or 11 . . . amino acids up to the entire amino acid
sequence minus one amino acid).
The term og” or “homologous” when used in reference to a polypeptide refers
to a high degree of sequence identity between two ptides, or to a high degree of
similarity between the three-dimensional structure or to a high degree of similarity between
the active site and the mechanism of . In a preferred embodiment, a homolog has a
greater than 60% sequence identity, and more preferably greater than 75% sequence identity,
and still more preferably greater than 90% sequence identity, with a reference sequence.
The terms “variant” and “mutant” when used in nce to a polypeptide refer to an
amino acid sequence that differs by one or more amino acids from another, usually related
polypeptide. The variant may have “conservative” changes, wherein a substituted amino acid
has similar structural or chemical properties. One type of conservative amino acid
substitutions refers to the interchangeability of residues having similar side chains. For
e, a group of amino acids having aliphatic side chains is glycine, alanine, valine,
leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is
serine and threonine; a group of amino acids having amide-containing side chains is
gine and glutamine; a group of amino acids having ic side chains is
phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is
, arginine, and histidine; and a group of amino acids having sulfur-containing side
chains is cysteine and methionine. Preferred conservative amino acids substitution groups
are: valine-leucine-isoleucine, alanine—tyrosine, lysine-arginine, alanine-valine, and
asparagine-glutamine. More rarely, a variant may have “non-conservative” changes (e.g.,
replacement of a glycine with a tryptophan). Similar minor variations may also include amino
acid deletions or insertions (i.e., additions), or both. Guidance in ining which and how
many amino acid residues may be substituted, inserted or deleted without abolishing
biological activity may be found using computer programs well known in the art, for
example, DNAStar software. ts can be tested in functional assays. Preferred ts
have less than 10%, and preferably less than 5%, and still more preferably less than 2%
changes (whether substitutions, deletions, and so on).
The term “domain” when used in reference to a polypeptide refers to a subsection of
the polypeptide which possesses a unique structural and/or fianctional characteristic;
typically, this characteristic is similar across diverse polypeptides. The subsection typically
comprises contiguous amino acids, although it may also comprise amino acids which act in
concert or which are in close proximity due to g or other configurations. Examples of a
protein domain include the embrane s, and the glycosylation sites.
The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises
coding sequences necessary for the production of an RNA, or a ptide or its precursor
(e. g., proinsulin). A functional polypeptide can be d by a filll length coding sequence
or by any portion of the coding sequence as long as the desired activity or functional
properties (e. g., enzymatic activity, ligand binding, signal transduction, etc.) of the
polypeptide are retained. The term “portion” when used in reference to a gene refers to
nts of that gene. The fragments may range in size from a few nucleotides to the entire
gene sequence minus one nucleotide. Thus, “a nucleotide comprising at least a portion of a
gene” may comprise fragments of the gene or the entire gene.
The term “gene” also encompasses the coding regions of a structural gene and
es sequences located adjacent to the coding region on both the 5' and 3' ends for a
distance of about 1 kb on either end such that the gene corresponds to the length of the full-
length mRNA. The sequences which are located 5' of the coding region and which are present
on the mR,\IA are referred to as 5' non-translated ces. The sequences which are located
3' or downstream of the coding region and which are present on the mRNA are referred to as
3' non-translated sequences. The term “gene” encompasses both cDNA and genomic forms of
a gene. A genomic form or clone of a gene contains the coding region interrupted with non-
coding sequences termed “introns” or “intervening regions” or “intervening sequences.”
Introns are ts of a gene which are transcribed into nuclear RNA (hnRNA); introns
may contain regulatory elements such as enhancers. Introns are removed or “spliced out”
from the nuclear or primary transcript; introns therefore are absent in the ger RNA
(mRNA) transcript. The mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
In addition to containing introns, genomic forms of a gene may also include
sequences d on both the 5' and 3' end of the sequences which are present on the RNA
transcript. These sequences are referred to as “flanking” ces or regions (these flanking
sequences are located 5' or 3' to the anslated sequences present on the mRNA
transcript). The 5' flanking region may n regulatory sequences such as ers and
enhancers which control or influence the transcription of the gene. The 3' flanking region
may contain sequences which direct the termination of transcription, posttranscriptional
cleavage and enylation.
The terms “oligonucleotide” or “polynucleotide” or “nucleotide” or “nucleic acid”
refer to a molecule comprised of two or more deoxyribonucleotides or ribonucleotides,
preferably more than three, and usually more than ten. The exact size will depend on many
factors, which in turn depends on the ultimate on or use of the oligonucleotide. The
oligonucleotide may be ted in any manner, including chemical synthesis, DNA
replication, reverse ription, or a combination thereof.
The terms “an oligonucleotide having a nucleotide sequence encoding a gene” or “a
nucleic acid sequence encoding” a specified polypeptide refer to a nucleic acid sequence
comprising the coding region of a gene or in other words the nucleic acid sequence which
encodes a gene product. The coding region may be present in either a cDNA, genomic DNA
or RNA form. When present in a DNA form, the ucleotide may be single-stranded (i.e.,
the sense strand) or double—stranded. Suitable control ts such as enhancers/promoters,
splice junctions, polyadenylation signals, etc. may be placed in close proximity to the coding
region of the gene if needed to permit proper initiation of transcription and/or correct
processing of the primary RNA transcript. Alternatively, the coding region ed in the
expression vectors of the present invention may contain endogenous enhancers/promoters,
splice ons, intervening sequences, polyadenylation signals, etc. or a ation of
both endogenous and exogenous l elements.
The term “recombinant” when made in reference to a nucleic acid le refers to a
nucleic acid molecule which is sed of segments of nucleic acid joined together by
means of lar ical techniques. The term “recombinant” when made in reference
to a protein or a polypeptide refers to a n molecule which is expressed using a
recombinant nucleic acid molecule.
The terms “complementary” and “complementarity” refer to polynucleotides (i.e., a
sequence of nucleotides) related by the base—pairing rules. For example, for the sequence “5'-
A-G-T-3',” is complementary to the sequence C-A-5'.” Complementarity may be
“partial,” in which only some of the nucleic acids' bases are matched according to the base
pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic
acids. The degree of complementarity between nucleic acid strands has significant effects on
the efficiency and strength of hybridization between nucleic acid strands. This is of particular
importance in amplification reactions, as well as detection methods which depend upon
binding between nucleic acids.
The term “wild-type” when made in reference to a gene refers to a gene that has the
characteristics of a gene isolated from a naturally occurring source. The term “wild-type”
when made in reference to a gene product refers to a gene product that has the characteristics
of a gene product isolated from a naturally occurring source. The term “naturally-occurring”
as applied to an obj ect refers to the fact that an object can be found in nature. For example, a
polypeptide or polynucleotide sequence that is present in an organism (including s) that
can be isolated from a source in nature and which has not been intentionally modified by man
in the laboratory is naturally-occurring. A wild—type gene is frequently that gene which is
most frequently observed in a population and is thus arbitrarily designated the “normal” or
“wild-type” form of the gene. In contrast, the term “modified” or “mutant” when made in
reference to a gene or to a gene product refers, tively, to a gene or to a gene product
which displays modifications in sequence and/or functional properties (i.e., altered
characteristics) when compared to the wild—type gene or gene product. It is noted that
naturally-occurring s can be isolated; these are identified by the fact that they have
d characteristics when ed to the wild—type gene or gene product.
The term “allele” refers to different variations in a gene; the variations include but are
not limited to variants and mutants, polymorphic loci and single nucleotide polymorphic loci,
frameshift and splice mutations. An allele may occur naturally in a population, or it might
arise during the lifetime of any particular individual of the population.
Thus, the terms “variant” and “mutant” when used in reference to a nucleotide
sequence refer to an nucleic acid sequence that differs by one or more nucleotides from
r, usually d nucleotide acid ce. A “variation” is a difference between two
different nucleotide sequences; typically, one sequence is a reference sequence.
The term “antisense” refers to a deoxyribonucleotide sequence whose sequence of
deoxyribonucleotide residues is in reverse 5' to 3' orientation in on to the sequence of
deoxyribonucleotide residues in a sense strand of a DNA duplex. A “sense strand” of a DNA
duplex refers to a strand in a DNA duplex which is transcribed by a cell in its natural state
into a “sense mRNA.” Thus an ense” sequence is a sequence having the same sequence
as the non-coding strand in a DNA duplex. The term “antisense RNA” refers to a RNA
transcript that is complementary to all or part of a target primary transcript or mRNA and that
blocks the expression of a target gene by interfering with the processing, transport and/or
translation of its primary transcript or mRNA. The complementarity of an antisense RNA
may be with any part of the c gene transcript, i.e., at the 5' non-coding sequence, 3'
non-coding sequence, s, or the coding sequence. In addition, as used , antisense
RNA may contain regions of ribozyme ces that increase the efficacy of antisense RNA
to block gene expression. “Ribozyme” refers to a catalytic RNA and es sequence-
specific endoribonucleases. “Antisense inhibition” refers to the production of antisense RNA
transcripts capable of preventing the sion of the target protein.
The term “primer” refers to an oligonucleotide, whether occurring naturally as in a
d restriction digest or produced synthetically, which is e of acting as a point of
initiation of synthesis when placed under conditions in which synthesis of a primer extension
product which is complementary to a nucleic acid strand is induced, (e.g., in the presence of
nucleotides and an inducing agent such as DNA rase and at a suitable temperature and
pH). The primer is preferably single stranded for maximum efficiency in amplification, but
may alternatively be double stranded. If double stranded, the primer is first treated to separate
its strands before being used to prepare extension products. Preferably, the primer is an
oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of
extension products in the presence of the inducing agent. The exact lengths of the primers
will depend on many factors, including temperature, source of primer and the use of the
The term ” refers to an oligonucleotide (i.e., a sequence of nucleotides),
whether occurring naturally as in a purified restriction digest or produced synthetically,
recombinantly or by PCR amplification, that is capable of hybridizing to another
oligonucleotide of interest. A probe may be single-stranded or double-stranded. Probes are
useful in the ion, identification and isolation of particular gene sequences. It is
contemplated that any probe used in the present invention will be labeled with any “reporter
molecule,” so that is detectable in any detection system, including, but not limited to enzyme
(e. g., ELISA, as well as enzyme-based histochemical ), fluorescent, radioactive, and
scent systems. It is not intended that the present invention be limited to any particular
detection system or label.
The term “isolated” when used in relation to a nucleic acid, as in “an isolated
oligonucleotide” refers to a nucleic acid sequence that is identified and separated from at least
one contaminant nucleic acid with which it is ordinarily ated in its natural .
Isolated nucleic acid is present in a form or setting that is different from that in which it is
found in nature. In contrast, non—isolated nucleic acids, such as DNA and RNA, are found in
the state they exist in . Examples of non-isolated nucleic acids include: a given DNA
sequence (e.g., a gene) found on the host cell chromosome in ity to neighboring genes;
RNA sequences, such as a specific mRNA ce encoding a specific protein, found in the
cell as a mixture with numerous other mRNAs which encode a multitude of proteins.
However, isolated nucleic acid encoding a particular protein includes, by way of example,
such nucleic acid in cells ordinarily expressing the protein, where the nucleic acid is in a
chromosomal location different from that of natural cells, or is otherwise flanked by a
ent nucleic acid sequence than that found in nature. The isolated nucleic acid or
oligonucleotide may be present in single—stranded or -stranded form. When an ed
nucleic acid or oligonucleotide is to be utilized to express a protein, the ucleotide will
contain at a minimum the sense or coding strand (i.e., the oligonucleotide may single-
stranded), but may contain both the sense and anti-sense strands (i.e., the oligonucleotide may
be double-stranded).
The term “purified” refers to molecules, either nucleic or amino acid sequences, that
are d from their natural environment, isolated or separated. An “isolated nucleic acid
sequence” may therefore be a purified nucleic acid sequence. “Substantially purified”
molecules are at least 60% free, preferably at least 75% free, and more preferably at least
90% free from other components with which they are naturally associated. As used herein,
the term “purified” or “to purify” also refer to the removal of contaminants from a sample.
The removal of contaminating ns results in an increase in the percent of polypeptide of
interest in the sample. In r example, recombinant polypeptides are expressed in plant,
bacterial, yeast, or mammalian host cells and the polypeptides are purified by the removal of
host cell proteins; the percent of recombinant polypeptides is y increased in the sample.
The term “composition sing” a given polynucleotide sequence or polypeptide
refers broadly to any composition containing the given polynucleotide sequence or
polypeptide. The composition may se an aqueous solution. Compositions comprising
cleotide sequences or fragments thereofmay be employed as ization . In
some embodiments, polynucleotide sequences are employed in an aqueous solution
containing salts (e.g., NaCl), detergents (e.g., SDS), and other components (e.g., Denhardt's
solution, dry milk, salmon sperm DNA, etc.).
The term “test compound” refers to any chemical entity, pharmaceutical, drug, and
the like that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily
function, or otherwise alter the physiological or cellular status of a sample. Test nds
comprise both known and potential therapeutic compounds. A test compound can be
determined to be therapeutic by ing using the screening methods of the present
invention. A “known eutic compound” refers to a therapeutic compound that has been
shown (e.g., through animal trials or prior experience with administration to humans) to be
effective in such treatment or prevention.
As used , the term “antibody” is used in its broadest sense to refer to whole
antibodies, monoclonal antibodies (including human, humanized, or chimeric antibodies),
polyclonal antibodies, and antibody fragments that can bind antigen (e.g., Fab’, F’ (ab)2, FV,
single chain dies), sing complementarity determining regions (CDRs) of the
foregoing as long as they exhibit the d biological activity.
As used herein, “antibody fragments” comprise a portion of an intact dy,
preferably the antigen binding or variable region of the intact antibody. Examples of dy
fragments include Fab, Fab', 2, and FV fragments; diabodies; linear antibodies (Zapata
et al., Protein Eng. 8(10): 1057—1062 ); single-chain antibody molecules; and
multispecific antibodies formed from antibody fragments.
An dy that “specifically binds to” or is “specific for” a particular polypeptide or
an epitope on a particular polypeptide is one that binds to that ular polypeptide or
epitope on a particular polypeptide without substantially binding to any other polypeptide or
polypeptide epitope.
As used herein, “active” or “activity” refers to native or naturally occurring biological
and/or logical activity.
As used herein the term, “in Vitro” refers to an artificial environment and to processes
or reactions that occur within an artificial environment. In Vitro environments may include,
but are not limited to, test tubes and cell cultures. The term “in Vivo” refers to the natural
environment (e.g., an animal or a cell) and to processes or reactions that occur within a
natural environment.
As used herein, “inhibitor” refers to a molecule which eliminates, minimizes, or
decreases the activity, e.g., the biological, tic, chemical, or immunological activity, of
a target.
As used herein the term “disease” refers to a deviation from the condition regarded as
normal or average for members of a species, and which is detrimental to an affected
individual under conditions that are not inimical to the majority of individuals of that species
(e.g., diarrhea, , fever, pain, inflammation, etc).
As used herein, the term “administration” refers to the act of giving a drug, prodrug,
antibody, or other agent, or eutic treatment to a physiological system (e.g., a subject or
in Vivo, in Vitro, or ex Vivo cells, tissues, and organs). Exemplary routes of administration to
the human body can be through the eyes (ophthalmic), mouth , skin (transdermal), nose
(nasal), lungs (inhalant), oral mucosa (buccal), ear, by injection (e. g., enously,
subcutaneously, intratumorally, intraperitoneally, etc.) and the like. “Coadministration” refers
to administration of more than one chemical agent or therapeutic treatment (e.g., radiation
therapy) to a logical system (e.g., a subject or in vivo, in vitro, or ex vivo cells, tissues,
and organs). As used herein, administration “in combination wit ”
one or more further
therapeutic agents includes aneous (concurrent) and consecutive administration in any
order. “Coadministration” of therapeutic treatments may be concurrent, or in any temporal
order or al combination.
As used herein, the term “treating” includes reducing or alleviating at least one
adverse effect or symptom of a disease or disorder through introducing in any way a
therapeutic composition of the present technology into or onto the body of a subject.
“Treatment” refers to both eutic treatment and prophylactic or tative measures,
wherein the object is to t or slow down (lessen) the targeted pathologic condition or
disorder. Those in need of treatment include those already with the disorder as well as those
prone to have the disorder or those in whom the disorder is to be prevented.
As used herein, “therapeutically effective dose” refers to an amount of a therapeutic
agent sufficient to bring about a beneficial or desired clinical effect. Said dose can be
administered in one or more administrations. However, the precise ination of what
would be considered an effective dose may be based on factors individual to each patient,
including, but not limited to, the patient’s age, size, type or extent of disease, stage of the
disease, route of administration, the type or extent of supplemental therapy used, ongoing
disease process, and type of treatment desired (e.g., sive vs. conventional treatment).
As used herein, the term “effective amount” refers to the amount of a composition
sufficient to effect ial or d results. An effective amount can be administered in
one or more administrations, applications, or dosages and is not intended to be limited to a
particular formulation or administration route.
As used herein, the term “pharmaceutical composition” refers to the ation of
an active agent with, as desired, a r, inert or active, making the composition especially
suitable for diagnostic or therapeutic use in vitro, in Vivo, or ex vivo.
As used herein, the terms “pharmaceutically acceptable” or “pharmacologically
acceptable” refer to compositions that do not substantially produce adverse reactions, e.g.,
toxic, allergic, or immunological ons, when administered to a subject.
As used , “carriers” include pharmaceutically acceptable carriers, excipients, or
stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and
trations employed. Often the physiologically acceptable carrier is an aqueous pH-
buffered solution. Examples of physiologically acceptable carriers include s such as
phosphate, citrate, and other organic acids; antioxidants ing ascorbic acid; low
molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin,
gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino
acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides,
disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating
agents such as EDTA; sugar alcohols such as ol or sorbitol; salt-forming rions
such as sodium; and/or nonionic surfactants.
As used herein, the terms “patient” or “subject” refer to organisms to be treated by the
compositions of the present technology or to be subject to various tests provided by the
technology. The term “subject” includes animals, preferably mammals, including humans. In
a preferred embodiment, the t is a primate. In an even more preferred ment, the
subject is a human.
As used herein, the term “sample” is used in its broadest sense. In one sense it can
refer to animal cells or tissues. In r sense, it is meant to include a specimen or culture
obtained from any source, such as biological and environmental samples. ical samples
may be obtained from plants or animals (including humans) and encompass fluids, solids,
tissues, and gases. nmental samples include environmental material such as surface
matter, soil, water, and industrial samples. These examples are not to be construed as limiting
the sample types applicable to the present logy.
Embodiments of the technology
Although the disclosure herein refers to certain illustrated embodiments, it is to be
understood that these embodiments are presented by way of e and not by way of
limitation.
1. Inhibitors of SCF
Stem cell factor (SCF) is a ligand that is c for the c-Kit receptor kinase.
Binding of SCF to c-Kit causes dimerization of c—Kit and activation of its kinase activity,
which is important for hemopoiesis, melanogenesis, and fertility. Through c-Kit, SCF acts to
promote cell survival, proliferation, differentiation, on, and onal activation.
nt activation of c-Kit can result in disease, including fibrosis and tissue remodeling
defects. In ular, there are multiple pulmonary diseases with known remodeling defects
as well as other chronic tissue remodeling diseases affecting other organs and tissues.
Specific examples of diseases involving fibrosis or tissue remodeling defects are idiopathic
pulmonary fibrosis, chronic obstructive pulmonary disease, acute respiratory distress
syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity nitis, asthma,
doma, inflammation, liver cirrhosis, renal fibrosis, hymal fibrosis,
endomyocardial fibrosis, inal fibrosis, nodular subepidermal fibrosis, fibrous
histiocytoma, fibrothorax, c s, fibromyalgia, gingival fibrosis, and radiation-
induced fibrosis.
ingly, ering with the interaction n SCF and c-Kit can be used to
treat or study es involving aberrant activation of c—Kit that causes fibrosis and tissue
remodeling defects. The c-Kit receptor is found on hematopoietic progenitor cells,
melanocytes, germ cells, eosinophils, lymphocytes, and mast cells. Thus, preventing SCF
interaction with c-Kit can alter the activation of several disease-associated cell populations
that have been implicated in fibrosis and tissue remodeling disease phenotypes.
Additionally, SCF induces key mediators in the fibrotic response, IL-25 and IL-l3.
Data suggest that IL-25 can drive IL-l3 expression in a T-cell and antigen-independent
manner. Therefore, these processes can progress without an antigen-specific response and
consequently chronically perpetuate remodeling and fibrotic disease. It is contemplated that a
complex cascade is established in which SCF induces IL-25, which in turn induces
tion of IL-l3, myofibroblast differentiation, and collagen production. IL-4 has also
been identified as a fibrosis-associated cytokine.
2. Antibodies
In some embodiments, inhibiting the ability of SCF to interact with c-Kit is
accomplished by means of an antibody that recognizes SCF. The antibody can be a
monoclonal antibody or a polyclonal antibody, and may be, for example, a human,
humanized, or chimeric antibody. Monoclonal antibodies against target antigens are produced
by a variety of ques including conventional onal antibody methodologies such
as the somatic cell hybridization techniques of Kohler and Milstein (Nature, 256:495 (1975)).
Although in some embodiments, somatic cell hybridization procedures are preferred, other
techniques for producing monoclonal antibodies are contemplated as well (e.g., viral or
oncogenic ormation of B lymphocytes).
It is contemplated that antibodies against SCF find use in the experimental,
diagnostic, and therapeutic methods described herein. In certain ments, the antibodies
ed herein are used to detect the expression of SCF in biological samples. For example,
a sample comprising a tissue biopsy can be sectioned and protein detected using, for example,
immunofluorescence or immunohistochemistry. atively, individual cells from a sample
can be isolated, and protein expression detected on fixed or live cells by FACS analysis.
Furthermore, the antibodies can be used on n arrays to detect expression of SCF. In
other embodiments, the antibodies provided herein are used to decrease the activity of cells
expressing c-Kit by inhibiting SCF either in an in vitro cell—based assay or in an in vivo
animal model. In some embodiments, antibodies are used to treat a human patient by
administering a therapeutically ive amount of an antibody t SCF.
For the production of antibodies, various host animals can be immunized by injection
with the e ponding to the desired epitope (e.g., a fragment of SCF, e.g., a
fragment sing the sequence provided by SEQ ID NO: 1 or 8 or immunogenic portions
thereof) including, but not limited to, rabbits, mice, rats, sheep, goats, etc. dies to SCF
can be raised by immunizing (e.g., by ion) with an antigen comprising a peptide, a
portion, or the full protein of the SCF isoform b sor (e.g., a protein or peptide fragment
of the sequence available at GenBank accession number NP_000890 (SEQ ID NO: 4)), or a
variant or modified version thereof, or a peptide, a portion, or the fiill protein of the SCF
isoform a precursor (e.g., a protein or peptide fragment of the sequence available at GenBank
accession number NP_003985 (SEQ ID NO: 6)), or a variant or modified version thereof.
Antibodies can also be raised by immunization with a translation product of the NCBI
Reference Gene Sequence for SCF (e.g., accession number NG_012098 (SEQ ID NO: 7)) or
ts or fragments thereof.
In some embodiments, the peptide is conjugated to an immunogenic carrier (e. g.,
eria toxoid, bovine serum albumin (BSA), or keyhole limpet hemocyanin (KLH)).
Various adjuvants are used to increase the immunological response, depending on the host
species, including, but not limited to, Freund’s (complete and incomplete), mineral gels such
as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and
potentially useful human adjuvants such as BCG (Bacille te-Guerin) and
Corynebacterium parvum.
Polyclonal antibodies can be ed by any known method. Polyclonal dies
can be raised by immunizing an animal (e.g., a rabbit, rat, mouse, donkey, etc) by le
subcutaneous or intraperitoneal injections of the relevant antigen (a purified peptide
fragment, full-length recombinant protein, fusion protein, etc.) optionally conjugated to KLH,
serum albumin, etc., diluted in sterile saline, and combined with an adjuvant to form a stable
emulsion. The polyclonal antibody is then recovered from blood, s, and the like, of an
animal so immunized. ted blood is clotted, and the serum decanted, clarified by
centrifugation, and assayed for dy titer. The polyclonal antibodies can be purified from
serum or ascites according to standard methods in the art including affinity chromatography,
ion-exchange chromatography, gel ophoresis, dialysis, etc.
For preparation of monoclonal antibodies, any technique that provides for the
production of antibody molecules by continuous cell lines in culture may be used (see e.g.,
Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, NY). These include, but are not limited to, the oma technique
originally developed by Kohler and Milstein and the trioma technique, the human B-cell
hybridoma technique (See, e.g., Kozbor et al., l. Today, 4:72 (1983)), and the EBV-
hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal
Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)).
In some embodiments provided herein, the antibodies are ed from a hybridoma.
Using the hybridoma method, a mouse, hamster, or other appropriate host animal, is
zed as described above to elicit the production by cytes of antibodies that will
specifically bind to an immunizing antigen. Alternatively, lymphocytes can be immunized in
vitro. Following immunization, the lymphocytes are isolated and fused with a suitable
myeloma cell line using, for example, polyethylene glycol, to form hybridoma cells that can
then be selected away from unfused lymphocytes and myeloma cells. Hybridomas that
produce monoclonal antibodies directed specifically against a chosen antigen as determined
by immunoprecipitation, immunoblotting, or by an in vitro g assay such as
mmunoassay (RIA) or enzyme—linked immunosorbent assay (ELISA) can then be
propagated in vitro (e.g., in culture) using standard methods (Goding, Monoclonal
Antibodies: Principles and Practice, Academic Press, 1986) or in vivo as ascites tumors in an
animal. The monoclonal dies can then be purified from the culture medium or ascites
fluid as described for polyclonal antibodies above.
The preferred animal system for preparing hybridomas is the murine system.
Hybridoma production in the mouse is a well-established procedure. Immunization protocols
and techniques for isolation of zed splenocytes for fiasion are known in the art. Fusion
partners (e.g., murine myeloma cells) and fusion procedures are also known. Embodiments of
the technology herein provide antibodies (e.g., monoclonal antibodies) produced from a
hybridoma prepared by immunizing mice with a peptide that is a n or fragment of the
SCF protein. For example, some embodiments provide an antibody or antigen-binding
fragment than binds to SCF by immunizing with, e.g., a protein or peptide fragment of the
sequence available at GenBank accession number NP_000890 (SEQ ID NO: 4)), or a variant
or modified version thereof, or by immunizing with, e.g., a protein or peptide fragment of the
sequence available at GenBank accession number NP_003985 (SEQ ID NO: 6)), or a variant
or modified version thereof. Some embodiments provide an antibody or n-binding
fragment that binds to a protein or peptide, or variants or modified versions thereof, that is a
translation t of the NCBI Reference Gene Sequence for SCF (e.g., accession number
NG_Ol2098 (SEQ ID NO: 7)) or variants or fragments thereof.
For e, embodiments of the technology herein e monoclonal dies
produced from a hybridoma prepared by immunizing mice with a e of amino acid
sequence SEQ ID NO: 1 or 8. Also contemplated are methods and compositions related to
dies prepared using a variant of SEQ ID NO: 1 or 8 comprising one or more
substitutions, deletions, insertions, or other changes, as long as said variant produces an
antibody specific for SCF. Producing polypeptides of SEQ ID NO: 1 or 8 and similar
sequences thereto can be accomplished according to s techniques well known in the art.
For example, a polypeptide of SEQ ID NO: 1 or 8 or a t f can be produced using
a bacterial expression system and a nucleic acid encoding a polypeptide of SEQ ID NO: 1 or
8 or a variant thereof. As an example, a polypeptide ing to SEQ ID NO: 1 can be
produced using the nucleotide sequence according to SEQ ID NO: 2.
Moreover, human monoclonal antibodies directed against human proteins can be
generated using transgenic mice carrying the complete human immune system rather than the
mouse system. Splenocytes from the transgenic mice are immunized with the antigen of
interest, which are used to produce hybridomas that e human onal antibodies
with specific affinities for epitopes from a human protein.
Monoclonal antibodies can also be generated by other methods known to those skilled
in the art of recombinant DNA technology. For instance, combinatorial antibody y has
can be ed to produce monoclonal antibodies (see, e.g., Sastry et al., Proc. Nat. Acad. Sci.
USA, 86: 5728 (1989); Huse et al., Science, 246: 1275 (1989); Orlandi et al., Proc. Nat.
Acad. Sci. USA, 86:3833 (1989)). After immunizing an animal with an immunogen as
described above, the dy repertoire of the resulting B—cell pool is cloned. Methods are
generally known for obtaining the DNA sequence of the le regions of a diverse
population of immunoglobulin molecules by using a e of oligomer primers and PCR.
For instance, mixed oligonucleotide primers corresponding to the 5' leader l peptide)
sequences and/or framework 1 (FRI) sequences, as well as primers to a conserved 3' region
can be used to amplify and e the heavy and light chain variable regions from a number
of murine antibodies (see. e. g., Larrick et al., Biotechniques, 11: 152 (1991)). A similar
gy can also been used to amplify human heavy and light chain variable s from
human antibodies (see, e. g., Larrick et al., Methods: Companion to Methods in Enzymology,
2: 106 (1991)).
Alternatively, monoclonal antibodies can also be made using recombinant DNA
methods as described in US. Patent 4,816,567. The polynucleotides encoding a monoclonal
antibody are isolated (e. g., from mature B-cells or hybridoma cells), by, e.g., RT-PCR using
oligonucleotide primers that specifically amplify the genes encoding the heavy and light
chains of the antibody, and their sequences are determined using conventional procedures.
The isolated polynucleotides encoding the heavy and light chains are then cloned into
suitable expression vectors, which, when transfected into host cells such as E. 0012' cells,
simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not
otherwise produce immunoglobulin protein, cause monoclonal antibodies to be generated by
the host cells. Also, recombinant monoclonal antibodies or fragments thereof of the d
species can be ed from phage display libraries as described (McCafferty et al., 1990,
Nature, 348:552-554; Clackson et al., 1991, Nature, 352:624-628; and Marks et al., 1991, J.
Mol. Biol, 222:581-597).
The polynucleotide encoding a monoclonal antibody can fiarther be modified in a
number of ent manners using recombinant DNA technology to generate alternative
antibodies. In one embodiment, the constant domains of the light and heavy chains of, for
example, a mouse monoclonal dy can be substituted 1) for those s of, for
example, a human antibody to generate a chimeric antibody or 2) for a non-immunoglobulin
polypeptide to generate a fusion antibody. In other embodiments, the constant regions are
truncated or removed to generate the desired antibody fragment of a monoclonal antibody.
Furthermore, site-directed or high-density mutagenesis of the variable region can be used to
optimize specificity, affinity, etc. of a monoclonal antibody.
For example, also contemplated are chimeric mouse—human monoclonal antibodies,
which can be produced by recombinant DNA techniques known in the art. For example, a
gene encoding the constant region of a murine (or other species) monoclonal antibody
molecule is digested with restriction enzymes to remove the region encoding the murine
constant region, and the equivalent portion of a gene encoding a human constant region is
substituted (see, e.g., Robinson et al., PCT/USS6/02269; European Patent Application
184,187; European Patent Application 171,496; European Patent Application 173,494; WO
86/01533; US 4,816,567; European Patent Application 125,023 (each of which is herein
incorporated by reference in its entirety); Better et al., Science, 240:1041-1043 (1988); Liu et
al., Proc. Nat. Acad. Sci. USA, 84:3439—3443 (1987); Liu et al., J. Immunol, 139:3521-3526
(1987); Sun et al., Proc. Nat. Acad. Sci. USA, —218 (1987); Nishimura et al., Canc.
Res., 47999-1005 (1987); Wood et al., Nature, 314:446-449 (1985); and Shaw et al., J. Natl.
Cancer Inst., 3-1559 (1988)).
The chimeric dy can be further humanized by replacing sequences of the
variable region that are not ly ed in antigen g with equivalent sequences
from human variable s. General reviews of humanized chimeric antibodies are
ed by S.L. Morrison, Science, 229:1202-1207 (1985) and by Oi et al., Bio ques,
4:214 (1986). Those methods include isolating, manipulating, and expressing the c acid
sequences that encode all or part of immunoglobulin variable regions from at least one of a
heavy or light chain. Sources of such nucleic acid are well known to those skilled in the art.
The inant DNA encoding the chimeric antibody, or fragment thereof, can then be
cloned into an appropriate expression vector.
le humanized antibodies can alternatively be produced by CDR substitution
(see, e.g., US 5,225,539; Jones et al., Nature, 321:552-525 (1986); Verhoeyan eta1., Science,
34 (1988); and Beidler et al., J. Immunol, 14124053 (1988)). All ofthe CDRs ofa
particular human antibody may be replaced with at least a portion of a non-human CDR or
only some of the CDRs may be ed with non—human CDRs. It is only necessary to
replace the number of CDRs important for binding of the zed antibody to the Fc
receptor.
An antibody can be humanized by any method that is capable of replacing at least a
portion of a CDR of a human antibody with a CDR derived from a non-human antibody. The
human CDRs may be replaced with non-human CDRs using oligonucleotide site-directed
mutagenesis.
Also contemplated are chimeric and humanized antibodies in which specific amino
acids have been substituted, deleted, or added. In particular, red humanized antibodies
have amino acid substitutions in the framework region, such as to improve binding to the
antigen. For example, in a humanized antibody having mouse CDRs, amino acids located in
the human framework region can be ed with the amino acids located at the
corresponding positions in the mouse antibody. Such substitutions are known to improve
binding of humanized antibodies to the antigen in some instances.
In n embodiments provided herein, it is desirable to use an antibody fragment.
Various techniques are known for the production of antibody fragments. Traditionally, these
fragments are derived via proteolytic ion of intact antibodies (for example Morimoto et
al., 1993, Journal of Biochemical and Biophysical Methods 24: 107-1 17 and Brennan et al.,
1985, Science, 229:81). For example, papain digestion of antibodies produces two identical
antigen-binding fragments, called Fab fragments, each with a single n-binding site, and
a residual Fc fragment. Pepsin treatment yields an F(ab’)2 fragment that has two antigen-
combining sites and is still capable of cross—linking antigen.
r, these fragments are now typically produced directly by recombinant host
cells as bed above. Thus Fab, Fv, and scFv antibody fragments can all be expressed in
and secreted from E. coli or other host cells, thus allowing the production of large amounts of
these fragments. Alternatively, such antibody fragments can be isolated from the antibody
phage libraries discussed above. The antibody fragment can also be linear antibodies as
described in US. Patent 5,641,870, for example, and can be ecific or ific. Other
techniques for the production of antibody fragments will be apparent to the skilled
practitioner.
Fv is the minimum dy fragment which contains a complete antigen-recognition
and antigen-binding site. This region consists of a dimer of one heavy-chain and one light-
chain variable domain in tight, non—covalent association. It is in this configuration that the
three CDRs of each variable domain ct to define an n-binding site on the surface
of the VH-VL dimer. tively, the six CDRs confer antigen-binding specificity to the
dy. However, even a single variable domain (or half of an Fv comprising only three
CDRs specific for an antigen) has the ability to recognize and bind n, although at a
lower affinity than the entire binding site.
The Fab nt also contains the constant domain of the light chain and the first
constant domain (CH1) of the heavy chain. Fab fragments differ from Fab’ nts by the
addition of a few residues at the carboxy terminus of the heavy chain CH1 domain including
one or more cysteines from the antibody hinge region. F(ab')2 antibody nts originally
were produced as pairs of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of dy fragments are also known to the skilled n.
The technology herein provided also contemplates modifying an antibody to increase
its serum half—life. This can be achieved, for example, by incorporating a salvage receptor
binding e into the dy fragment by mutation of the appropriate region in the
antibody fragment or by incorporating the epitope into a peptide tag that is then fused to the
dy fragment at either end or in the middle (e.g., by DNA or peptide synthesis).
The technology embraces variants and equivalents which are substantially
gous to the chimeric, humanized, and human antibodies, or antibody fragments
thereof, provided herein. These can contain, for example, vative substitution mutations,
i.e. the substitution of one or more amino acids by similar amino acids. For example,
conservative substitution refers to the substitution of an amino acid with another within the
same general class such as, for example, one acidic amino acid with another acidic amino
acid, one basic amino acid with another basic amino acid, or one neutral amino acid by
another neutral amino acid. What is intended by a conservative amino acid substitution is
well known in the art.
An additional embodiment utilizes the techniques known in the art for the
construction of Fab expression libraries (Huse et al., Science, 246:1275-1281 (1989)) to
allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity.
Also, this logy encompasses bispecific antibodies that specifically recognize
SCF. Bispecific antibodies are antibodies that are capable of specifically recognizing and
binding at least two different epitopes. Bispecific antibodies can be intact antibodies or
antibody fragments. Techniques for making bispecific dies are common in the art
(Millstein et al., 1983, Nature 305:537—539; Brennan et al., 1985, Science 229:81; Suresh et
a1, 1986, Methods in Enzymol. 121 : 120; Traunecker et al., 1991, EMBO J. 5-3659;
Shalaby et al., 1992, J. Exp. Med. 175:217—225; Kostelny et al., 1992, J. Immunol. 148: 1547-
1553; Gruber et al., 1994, J. Immunol. 68; and US. Patent 5,731,168).
Techniques described for the production of single chain antibodies (US. 4,946,778;
herein incorporated by reference) can be adapted to produce specific single chain dies
as desired. Single-chain Fv antibody nts comprise the VH and VL domains of an
antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv
polypeptide further comprises a ptide linker between the VH and VL domains that
enables the single-chain Fv antibody fragments to form the desired ure for antigen
binding. For a review of single-chain Fv antibody fragments, see Pluckthun in The
Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-
Verlag, New York, pp. 269-315 (1994).
3. Other SCF inhibitors
It is also contemplated that inhibiting SCF can be accomplished by a variety of other
types of inhibitors. For example, in some embodiments a small interfering RNA (siRNA) can
be designed to target and e SCF mRNA. siRNAs are double-stranded RNA molecules
of 20—25 nucleotides in length. While not limited in their features, typically an siRNA is 21
nucleotides long and has 2-nt 3’ overhangs on both ends. Each strand has a 5’ phosphate
group and a 3’ hydroxyl group. In vivo, this structure is the result of processing by dicer, an
enzyme that converts either long dsRNAs or small hairpin RNAs into siRNAs. r,
siRNAs can also be synthesized and exogenously introduced into cells to bring about the
specific knockdown of a gene of interest. Essentially any gene of which the sequence is
known can be targeted based on sequence complementarity with an riately tailored
siRNA. For example, those of ordinary skill in the art can synthesize an siRNA (see, e.g.,
Elbashir, et al., Nature 411: 494 (2001); Elbashir, et a1. Genes Dev 15 :188 (2001); Tuschl T,
et al., Genes Dev 13 23191 ).
In some embodiments, RNAi is utilized to inhibit SCF. RNAi represents an
evolutionarily ved cellular defense for controlling the expression of n genes in
most eukaryotes, including humans. RNAi is typically triggered by double-stranded RNA
(dsRNA) and causes sequence-specific degradation of single-stranded target RNAs (e. g., an
mRNA). The mediators ofmRNA ation are small ering RNAs (siRNAs), which
are normally produced from long dsRNA by enzymatic cleavage in the cell. siRNAs are
lly approximately twenty—one nucleotides in length (e.g. 21-23 nucleotides in length)
and have a base-paired structure characterized by two tide 3' overhangs. Following the
introduction of a small RNA, or RNAi, into the cell, it is believed the sequence is delivered to
an enzyme complex called RISC (RNA-induced silencing complex). RISC recognizes the
target and cleaves it with an endonuclease. It is noted that if larger RNA sequences are
delivered to a cell, an RNase III enzyme (e.g., Dicer) converts the longer dsRNA into 21-23
nt double-stranded siRNA fragments. In some embodiments, RNAi oligonucleotides are
designed to target the on region of fusion proteins. Chemically synthesized siRNAs
have become powerful reagents for genome-wide analysis ofmammalian gene fimction in
cultured somatic cells. Beyond their value for validation of gene function, siRNAs also hold
great potential as gene-specific therapeutic agents (see, e.g., Tuschl and Borkhardt, Molecular
Intervent. 2002; 2(3): 158-67, herein incorporated by reference).
The transfection of siRNAs into animal cells results in the potent, long-lasting post-
transcriptional silencing of specific genes (Caplen et al, Proc Natl Acad Sci USA. 2001; 98:
9742—47; Elbashir et al., Nature. 2001; 411:4 94—98; Elbashir et al., Genes Dev. 2001; 15:
188—200; and ir et al., EMBO J. 2001; 20: 8, all of which are herein
incorporated by nce). Methods and compositions for performing RNAi with siRNAs are
described, for example, in US. Pat. 6,506,559, herein orated by reference.
siRNAs are rdinarily effective at lowering the amounts of targeted RNA and
their protein products, frequently to undetectable . The silencing effect can last several
months, and is extraordinarily specific — a cleotide mismatch between the target RNA
and the central region of the siRNA is frequently sufficient to prevent silencing
(Brummelkamp et al, Science 2002; 296: 550—53; and Holen et al, Nucleic Acids Res. 2002;
: 1757—66, both of which are herein incorporated by nce).
An important factor in the design of siRNAs is the presence of accessible sites for
siRNA binding. Bahoia et al., (J. Biol. Chem, 2003; 278: 15991—97; herein incorporated by
reference) describe the use of a type ofDNA array called a scanning array to find accessible
sites in mRNAs for designing effective siRNAs. These arrays comprise oligonucleotides
ranging in size from rs to a certain maximum, usually Co—mers, synthesized using a
physical r (mask) by stepwise addition of each base in the sequence. Thus the arrays
ent a full oligonucleotide ment of a region of the target gene. Hybridization of
the target mRNA to these arrays provides an exhaustive accessibility profile of this region of
the target mRNA. Such data are useful in the design of antisense oligonucleotides (ranging
from 7mers to 25mers), where it is important to achieve a compromise between
oligonucleotide length and binding affinity, e.g., to retain efficacy and target specificity
(Sohail et al, c Acids Res., 2001; 29(10): 2041—45). Additional methods and concerns
for selecting siRNAs are described, for example, in WO 05054270, WOO5038054Al,
WOO3070966A2, J Mol Biol. 2005 May l3;348(4):883-93, J Mol Biol. 2005 May
l3;348(4):87 l -8 l, and Nucleic Acids Res. 2003 Aug 1,3 1(15):44l7-24, each of which is
herein incorporated by reference in its entirety. In addition, software (e.g., the MWG online
siMAX siRNA design tool) is commercially or ly available for use in the ion and
design of siRNAs and RNAi reagents.
In some embodiments, the present invention utilizes siRNA including blunt ends (See
e. g., US20080200420, herein incorporated by reference in its entirety), overhangs (See e.g.,
US20080269147A1, herein incorporated by reference in its entirety), locked nucleic acids
(See e.g., WO2008/006369, /043753, and W02008/051306, each ofwhich is herein
incorporated by reference in its entirety). In some embodiments, siRNAs are delivered Via
gene expression or using bacteria (See e.g., Xiang et al., Nature 24: 6 (2006) and
WOO6066048, each ofwhich is herein incorporated by reference in its entirety).
In other embodiments, shRNA techniques (See e.g., 20080025958, herein
incorporated by reference in its enterety) are utilized. A small hairpin RNA or short hairpin
RNA (shRNA) is a sequence ofRNA that makes a tight hairpin turn that can be used to
silence gene expression via RNA interference. shRNA uses a vector introduced into cells and
es the U6 er to ensure that the shRNA is always expressed. This vector is usually
passed on to daughter cells, allowing the gene silencing to be ted. The shRNA hairpin
structure is cleaved by the ar machinery into siRNA, which is then bound to the RNA-
induced silencing complex (RISC). This complex binds to and cleaves mRNAs which match
the siRNA that is bound to it. shRNA is transcribed by RNA polymerase III.
The present ion also includes pharmaceutical compositions and formulations
that include the RNAi compounds of the present invention as described below.
SCF exists in both transmembrane and soluble forms. Upon cleavage of the SCF
soluble domain from the embrane form, SCF is released from the cell surface to
function as the ligand of c-Kit. Thus, it is contemplated that SCF ty can be altered by
inhibiting the release of soluble SCF from the membrane-bound form, for example, by
ting or otherwise ng the activity of a protease that cleaves the soluble domain
from the membrane-bound form.
In addition, it is contemplated that SCF can be inhibited by chemicals (e.g., a small
molecule, e.g., a pharmacological agent) or other biological agents that bind or modify SCF.
For example, one of ordinary skill in the art can design and produce RNA aptamers or other
nucleic acids that specifically recognize and bind to SCF, for ce by using SELEX or
other in Vitro evolution methods known in the art. Furthermore, SCF activity can be inhibited
by specifically ing SCF or inducing an altered conformation of SCF such that it is less
effective in interacting with c-Kit. In some embodiments, the SCF inhibitor is a “designed
ankyrin repeat protein” (DARPin) (see, e.g., Stumpp MT & z P, “DARPins: a true
alternative to antibodies”, Curr Opin Drug Discov Devel 2007, 10(2): 153—59, orated
herein in its ty for all purposes). In some embodiments, SCF is inhibited by a small
molecule, e.g., a small molecule that binds to SCF and blocks its function (e. g., inhibits its
binding and/or other interaction (e.g., an activating ction) with the c-Kit receptor).
It is contemplated that altering SCF activity can be effected by inhibiting the
expression of SCF, for instance, by inhibiting the transcription of SCF, by inhibiting the
translation of SCF, by ting the processing of the SCF mRNA, by inhibiting the
sing of the SCF ptide, by inhibiting the folding of the SCF polypeptide, by
inhibiting king of SCF within a cell, or by inhibiting the insertion of SCF into the
plasma membrane. SCF ty can be altered by changes in chromatin structure or other
means of epigenetic regulation of SCF (e.g., changes in DNA methylation). Also, SCF
actiVity may be altered by specifically sequestering SCF in a vesicle or other cellular
compartment that hinders its action upon c-Kit.
4. Therapies using inhibitors of SCF
Inhibiting SCF finds use in therapies to treat disease. Accordingly, provided herein
are therapies comprising inhibiting SCF to benefit individuals suffering from disease. In
ular, as shown herein, disease states involving fibrosis and tissue remodeling
demonstrate aberrant SCF activity. For e, fibroblasts isolated from diseased
individuals with fibrotic or tissue remodeling phenotypes directly respond to SCF, which
results in the tion of a more severe phenotype that includes increased collagen
production. As such, as shown herein, inhibiting SCF can significantly affect the generation
of severe disease consequences including ation and remodeling of target tissue. Also
contemplated are therapies targeting SCF during the generation of fibrosis associated with
acute and chronic disorders that have either a dynamic disease course or a more predictable
disease . Indications that can benefit from therapy inhibiting SCF include, but are not
limited to, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease, acute
respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity
pneumonitis, asthma, sclerodoma, inflammation, liver cirrhosis, renal fibrosis, parenchymal
fibrosis, endomyocardial fibrosis, mediatinal fibrosis, nodular subepidermal s, fibrous
histiocytoma, fibrothorax, hepatic fibrosis, fibromyalgia, gingival s, and radiation-
induced fibrosis.
Importantly, therapies targeting SCF reduce or eliminate toxic effects associated with
other similar therapies, for example those targeting c-Kit. These undesirable toxic effects are
ated with targeting an intracellular, rather than extracellular, target, and the more
widespread and general changes in cell signaling that result. While the therapies are not
limited in their route of administration, embodiments of the logy provided herein
deliver the SCF inhibitor via the airway by intranasal administration. Such administration
allows direct delivery of the eutic agent to target tissues in pulmonary diseases
involving fibrosis and tissue remodeling, rather than relying on systemic delivery via an
orally administered composition.
In certain embodiments, a physiologically riate solution containing an effective
concentration of an antibody specific for SCF can be administered lly, intraocularly,
parenterally, orally, intranasally, enously, intramuscularly, subcutaneously, or by any
other effective means. In particular, the dy may delivered into an airway of a subject by
intranasal administration. Alternatively, a tissue can receive a physiologically riate
composition (e.g., a solution such as a saline or phosphate buffer, a suspension, or an
emulsion, which is sterile) containing an ive concentration of an antibody specific for
SCF via direct injection with a needle or via a catheter or other delivery tube. Any effective
imaging device such as X-ray, sonogram, or fiber-optic visualization system may be used to
locate the target tissue and guide the ration. In another alternative, a physiologically
appropriate solution containing an effective concentration of an antibody specific for SCF can
be administered systemically into the blood circulation to treat tissue that cannot be directly
reached or anatomically isolated. Such lations have in common the goal of placing an
effective concentration of an antibody c for SCF in sufficient contact with the target
tissue to permit the antibody specific for SCF to contact the tissue.
With respect to stration of a SCF inhibitor (e.g., an antibody specific for SCF)
to a subject, it is contemplated that the SCF inhibitor be administered in a pharmaceutically
effective amount. One of ordinary skill izes that a pharmaceutically effective amount
varies depending on the therapeutic agent used, the subject’s age, condition, and sex, and on
the extent of the disease in the subject. Generally, the dosage should not be so large as to
cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive
heart failure, and the like. The dosage can also be adjusted by the individual physician or
veterinarian to achieve the d therapeutic goal.
As used herein, the actual amount encompassed by the term “pharmaceutically
effective amount” will depend on the route of administration, the type of subject being
treated, and the physical characteristics of the specific t under consideration. These
factors and their relationship to determining this amount are well known to skilled
practitioners in the medical, veterinary, and other related arts. This amount and the method of
administration can be tailored to achieve optimal efficacy but will depend on such s as
weight, diet, concurrent medication, and other factors that those skilled in the art will
recognize.
In some embodiments, a SCF inhibitor (e.g., an dy specific for SCF) according
to the technology provided herein is administered in a pharmaceutically effective amount. In
some embodiments, a SCF inhibitor (e.g., an antibody specific for SCF) is administered in a
therapeutically effective dose. The dosage amount and frequency are selected to create an
effective level of the SCF inhibitor without substantially harmful effects. When administered,
the dosage of a SCF inhibitor (e.g., an antibody specific for SCF) will lly range from
0.001 to 10,000 mg/kg/day or dose (e.g., 0.01 to 1000 mg/kg/day or dose; 0.1 to 100
mg/kg/day or dose).
Pharmaceutical compositions preferably comprise one or more compounds of the present
invention associated with one or more pharmaceutically able carriers, diluents, or
excipients. Pharmaceutically acceptable carriers are known in the art such as those described
in, for e, Remingtons Pharmaceutical es, Mack Publishing Co. (A. R. Gennaro
ed., 1985).
In some embodiments, a single dose of a SCF inhibitor (e.g., an antibody specific for
SCF) according to the technology provided herein is administered to a t. In other
embodiments, multiple doses are stered over two or more time points, separated by
hours, days, weeks, etc. In some embodiments, compounds are administered over a long
period of time (e.g., chronically), for example, for a period of months or years (e.g., 1, 2, 3, 4,
5, 6, 7, 8, 9, 10, ll, 12, or more months or years; e.g., for the lifetime ofthe subject). In such
embodiments, compounds may be taken on a regular led basis (e.g., daily, ,
etc.) for the duration of the extended period.
In some embodiments, a SCF inhibitor (e.g., an antibody c for SCF) according
to the technology provided herein is inistered with another compound or more than
one other compound (e.g., 2 or 3 or more other compounds).
. Kits
Some embodiments provide herein kits for the treatment of a t. In some
embodiments, the kits include an tor of SCF and appropriate solutions and buffers.
Embodiments e all controls and instructions for use.
Examples
Materials and methods
SCF nucleotide sequences andproteins
The human gene encoding Stem Cell Factor (SCF) is also known as kit ligand and has
the official symbol KITLG and HGNC number HGNC26343. SCF is also known as SF;
MGF; SCF; FPH2; KL-l; Kitl; SHEP7; and kit-ligand. Two transcript variants encoding
different isoforms have been found for this gene. The SCF (kit ligand) isoform b precursor is
available at GenBank accession numbers NM_000899 (mRNA transcript; SEQ ID NO: 3)
and NP_000890 (protein sequence; SEQ ID NO: 4). The SCF (kit ligand) isoform a precursor
is available at GenBank accession numbers NM_003994 (mRNA transcript; SEQ ID NO: 5)
and NP_003985 (protein sequence; SEQ ID NO: 6). The NCBI Reference Gene Sequence has
accession number NG_012098 (SEQ ID NO: 7). For both ms, the first 25 amino acids
comprise the signal peptide and the mature form begins at amino acid 26. The first 11 amino
acids of the mature form are EGICRNRVTNN (SEQ ID NO: 8).
Bleomycz'n model
Interstitial pulmonary fibrosis was induced in specific pathogen-free (SPF) ,
CBA/J mice (6—8 weeks old; The Jackson tory, Bar Harbor, ME) by the it. injection of
0.003 U of bleomycin (Blenoxane, sterile cin sulfate; Bristol-Meyers Pharmaceuticals,
Evansville, IN; 0.15 U/Kg of mouse body weight) dissolved in 60 ul of phosphate-buffered
saline (PBS). Controls received 60 ul of PBS by the same route. All procedures were
conducted in a sterile environment and were ed by the institutional animal care and use
committee.
Whole lung histology
Following anesthesia-induced euthanasia, whole lungs from bleomycin-challenged
mice were fully inflated with 10% formalin, ted, and placed in fresh formalin for 24
hours. Routine histological techniques were used to embed the entire lung in paraffin, and 5-
um sections of whole lung were d with hematoxylin and eosin.
Production and administration ofanti—SCFpolyclonal antibodies
Anti-SCF antibodies were generated by immunizing rabbits with recombinant (whole
protein) SCF and ting polyclonal SCF-specific antibodies. Polyclonal antibodies were
isolated from the serum using a protein G column. The isolated IgG portion was quantified
and used at the specified concentrations suspended in saline. IgG from pre-immune serum
was isolated in a similar fashion for use as a control. Briefly, 100, 150 or 200 ug of control or
anti-SCF was given to mice by intranasal administration 7 days after treatment with
bleomycin. This treatment was repeated on a daily basis until 12 days after bleomycin
administration. Thus, the treatment protocol is considered therapeutic.
Generation ofmouse anti-human monoclonal antibodies
After identifying an immunogenic human peptide (e. g., SEQ ID NO: I or 8), mice
were immunized with a standard ol. The ination of high titer serum antibodies
indicated the riate immunization and fission hybridomas were made. Culture
supematants were analyzed from dual clones for SCF-specific antibody and chosen
based upon specificity. Five hybridomas producing specific monoclonal antibodies against
the peptide were propagated and the onal with the t titer was subsequently
tested in biologically relevant cultures. In some ments, a peptide having the sequence
EGICRNRVTNN (SEQ ID NO: 8) was used to generate an antibody (e.g., a monoclonal
antibody). In some embodiments, any e fragment (e.g., an antigenic nt) of the
SCF protein sequence (e.g., as provided by SEQ ID NO: 4 and/or SEQ ID NO: 6) is used to
generate antibodies. In some embodiments, mutant or variant forms (e.g., comprising one or
more amino acid substitutions with respect to the sequences provided by SEQ ID NO: 4 and
SEQ ID NO: 6) of SCF are used to provide a peptide for generating antibodies. It is to be
understood that these embodiments comprise additions, ons, substitutions, post-
translational modifications (e. g., glycosylation, cyclization, N— and C-terminal modification,
etc.) and other variations of proteins and peptides that are known in the art of molecular
biology as applied to provide a peptide for antibody generation.
Testing mouse anti-human monoclonal antibodies
To demonstrate that onal antibodies inhibit SCF, mast cell lines that are
sensitive to SCF were . The HMC-l cell line, a ytoma cell line that expresses 0-
Kit and responds to SCF was first used. In brief, HMC-l cells were cultured in specific
growth media and plated in 24—well tissue culture plates at a concentration of l X 106
ml. Recombinant human SCF (1—100 ng/ml) was mixed with monoclonal anti-SCF
antibody (12 ug/ml) and incubated at 37°C for 30 minutes. Afier incubation, the
antibody/SCF or SCF alone was added to the HMC-l cells. Afier 1 hour or 24 hours, the
cultured HMC-l cells were harvested and mRNA and protein levels were measured as an
indication of SCF inhibition by the monoclonal antibodies.
Analysis ofmRNA expression by quantitative PCR
Cells or tissue to be tested were dispersed in 1 ml of Trizol t (Invitrogen). RNA
was isolated as described (Invitrogen), and 5 ug ofmRNA was reverse-transcribed to assess
gene sion. ion of ne mRNA was determined using usly available
primer/probe sets (PE Biosystems, Foster City, CA) and analyzed using an ABI Prism 7500
Sequence Detection System (Applied Biosystems, Foster City, CA). GAPDH mRNA was
measured as a control for normalizing mRNA expression. Changes in gene expression were
calculated relative to gene expression in unchallenged mice.
ination ofcytokine production
Protein levels of cytokines were quantified using a Bio-Plex bead-based cytokine
assay purchased from Bio-Rad Laboratories (Hercules, CA). Using standard protocols, the
level of cytokines can be y and consistently assessed with this methodology.
Statistical analysis
Data were analyzed using Prism GraphPad software. Unless otherwise specified, data
shown are representative of two or more experiments. Statistical significance in all
experiments was determined by one-way ANOVA, followed by a Newman-Keuls post test.
Significant differences were regarded asp < 0.05.
[salarian andpropagation ofpulmonaryfibroblastsfrom patientpopulations
The utional Review Board at the University of Michigan Medical School
approved this study. All patients underwent al evaluation, including chest radiography,
lung function measurements, and thin—section computed tomography before fiber optic
bronchoscopy. In these patients, interstitial pneumonia was determined from a compilation of
ms, physiological ms, and radiographical findings. Surgical lung biopsies were
obtained Via the Clinical Core at the University of Michigan Medical School from patients
ted of having interstitial pneumonia between May 2000 and May 2002. Histologically
normal lung was obtained from resected specimens in patients undergoing thoracic resection.
Each biopsy was processed separately using sterile technique in a r flow hood and
processed for ing primary fibroblast lines. Two pathologists who were unaware of any
other clinical findings independently reviewed each biopsy and histological classification was
based on previously published criteria for idiopathic interstitial pneumonia.
Interstitial pneumonia and normal biopsies were finely minced and the dispersed
tissue pieces were placed into 150-cm2 cell culture flasks (Corning Inc., g, NY)
ning co’s modified Eagle’s medium (DMEM, ttaker, Walkersville, MD)
supplemented with 15% fetal bovine serum (DMEM-15, BioWhittaker), 1 mmol/L glutamine
(BioWhittaker), 100 U/ml penicillin (BioWhittaker), 100 ug/ml streptomycin (BioWhittaker),
and 0.25 ug amphotericinB (Fungizone; BioWhittaker). All primary lung cell lines were
maintained in DMEM-l5 at 37°C in a 5% C02 incubator and were serially ed a total of
five times to yield pure populations of lung fibroblasts. All primary fibroblast cell lines were
used at passages 6 to 10 in the experiments outlined below and all of the experiments were
performed under comparable conditions.
1. Anti-SCF antibody reduces fibrosis and inflammation
Experiments conducted while developing embodiments of the technology
demonstrated that anti-SCF antibody reduced fibrosis and inflammation. Pulmonary fibrosis
was induced in mice as described. On day 7 following cin injury, mice were subjected
to treatment with anti-SCF antibodies delivered into the airway by asal administration.
Treatment ued until day 12 ing bleomycin exposure. Lungs were harvested on
day 16 and examined by microscopy and a series ofmicrographs were taken. Lung histology
demonstrated that anti-SCF antibodies d l inflammation. In addition, Masson’s
trichrome staining, which designates collagen deposition, was reduced.
2. Anti-SCF antibody reduces levels of SCF, hydroxyproline, IL-25, and IL-13
Levels of hydroxyproline and ular cytokines were monitored while developing
embodiments of the technology. Lung tissue sections from the above experiment were
examined for the ce of hydroxyproline, a collagen precursor. The data demonstrated
that the anti-SCF antibody reduced the tion of hydroxyproline and plasma levels of
SCF in a dose—dependent manner (Figure 1A and D). Also, IL-25 and IL-13 expression,
ed as a on ofmRNA levels, were reduced, as was expression of IL-25 receptor
e 1B, C, and E).
In particular, the experiments tested the effect of anti-SCF antibody treatment in the
BLM model (Figure 1). Mice were treated with saline (Figure 1, “SAL”) or BLM (Figure 1,
“BLM”) on day 0. On days 8 and 12, different groups were also treated intratracheally with
non-immune (Figure 1, “IgG”) or anti-SCF antibodies (Figure 1, “aSCF”) at the indicated
doses. H&E stained lung tissue sections from each treatment group were ed and
examined. is was quantified biochemically as lung hydroxyproline content (Figure 1A).
Lungs were then analyzed for IL-13 mRNAs by real time PCR (Figure 1C). Plasma and lung
tissue collected from SAL- or BLM-treated mice were then analyzed for soluble SCF by
ELISA (Figure 1D) or IL-25 mRNA by real time PCR (Figure 1B). Values ent the
means +/— the standard error with an n = 7. A single asterisk (*) indicates statistical
significance (P < 0.05) when compared to the saline control group, while double asterisks
(**) indicate significance with respect to the BLM + IgG l group.
3. IL-4 stimulates c-kit expression in human fibroblasts
Experiments conducted while developing embodiments of the technology
demonstrated that IL-4 stimulated c-kit expression in human fibroblasts. In addition to the
mouse model of pulmonary inflammation, SCF receptor is expressed in fibroblast populations
from patients diagnosed with hypersensitivity pneumonitis and who thus have a pro-fibrotic
environment. Pulmonary fibroblasts were grown from normal areas of lungs from patients
l) and those diagnosed with hypersensitivity pneumonitis. Expression of c-kit was
measured after stimulation with IL—4 at 1 or 10 ng/ml. Individual cell lines (133, 131, 173,
177A, 177B) were assessed using real-time PCR. Compared to lung fibroblasts grown from
ts with non-fibrotic disease, fibroblasts from the hypersensitivity pneumonitis patients
displayed significant upregulation of c—kit when stimulated with IL-4, a fibrosis-associated
cytokine. The data demonstrated that SCF activated fibroblasts from inflammatory lesions,
but not those from normal tissue, and promoted the expression of fibrosis-associated genes
including collagen (Figure 2).
4. A mouse anti-human monoclonal antibody blocks SCF-induced HMC mast cell
activation.
Experiments conducted while developing embodiments of the technology
demonstrated that the monoclonal antibody specific for SCF inhibited the activation of HMC-
1 cells for MCP-l production. The activation ofmast cells is a classic SCF-induced response
that can be used to r antibody neutralization of SCF-mediated cytokine responses.
Previous studies have demonstrated that monocyte actic protein (MCP)-l is ly
upregulated by SCF in mast cells. A onal antibody was produced against SEQ ID NO:
1 (Figure 3). The efficacy of this antibody was tested using a human mast cell line, HMC-l,
stimulated with 100 ng/ml of SCF. The monoclonal antibody (6 ug/ml) was preincubated
with the recombinant SCF for 5 minutes prior to placing the SCF or the SCF plus CF
onto the cultured HMC-l cells (l X 106 cells/ml). The cells were subsequently incubated for
12 hours, after which the cell-free supernatant was collected and MCP-l was analyzed by
Bio-Plex. The data illustrate that the monoclonal antibody specific for SCF inhibited the
activation ofHMC-l cells for MCP-l production (Figure 4).
. SCF-deficient mice subjected to BLM-induced injury have reduced fibrosis.
During the development of embodiments of the technology provided herein, the
effects of SCF deficiency in KitlSI/KitlSI‘d mutant mice were examined (Figure 5). These
mice have a complete deletion of the SCF gene in one allele (8]) and a deletion of the
membrane-bound ligand in the other (Sld), which significantly decreases the expression of
soluble SCF. When these mice and their wild type controls (WT) were subjected to BLM-
induced lung , there was a significantly reduced s in the mutant mice compared
to wild type mice, both morphologically n ome stain) and mically by
hydroxyproline is (Figure 5).
Wild-type and SCF deficient mice were d with saline (“SAL”) or BLM
(“BLM”) on day 0 and lungs were harvested 21 days later. Fibrosis was quantified
biochemically as lung hydroxyproline content. Values represent the mean +/— standard
deviation with an n = 3. A single asterisk (*) indicates statistical cance (P < 0.05) when
compared to the WT saline-treated control mean, while double asterisks (**) indicate
significance when compared to the WT BLM-treated group.
Similar suppression of cytokine expression and rase induction was also noted
in S 1/Sld mice. These data taken together indicated an essential role for the SCF/c-Kit
signaling induced pulmonary s.
All publications and patents mentioned in the above specification are herein
incorporated by nce in their entirety for all purposes. s modifications and
variations of the described compositions, methods, and uses of the technology will be
nt to those skilled in the art without departing from the scope and spirit of the
logy as described. Although the technology has been described in connection with
specific ary embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed, various modifications of
the described modes for carrying out the invention that are obvious to those skilled in
pharmacology, biochemistry, medical science, or related fields are intended to be within the
scope of the following claims.
Claims (22)
1. Use of a monoclonal antibody or an antigen-binding antibody nt in the preparation of a medicament for ng or preventing a e that is a fibrotic disease or a tissue remodeling disease in a subject, wherein the onal antibody or antigen-binding antibody nt specifically binds to stem cell factor isoform b relative to stem cell factor isoform a.
2. The use as claimed in claim 1, wherein the antibody or antigen-binding antibody fragment specifically binds to a protein comprising an amino acid sequence provided by SEQ ID NO: 4 or a peptide fragment thereof.
3. The use as claimed in claim 1, n the antibody or antigen-binding antibody fragment specifically binds to a polypeptide comprising an amino acid sequence provided by SEQ ID NO: 1.
4. The use as d in claim 1, wherein stem cell factor isoform a comprises an amino acid sequence provided by SEQ ID NO: 6.
5. The use as claimed in any one of claims 1 to 4, wherein the antibody or antigenbinding antibody fragment prevents or reduces the severity of at least one symptom of the disease.
6. The use as claimed in any one of claims 1 to 5, wherein the antibody is a humanized antibody.
7. The use as claimed in any one of claims 1 to 6, wherein the subject has an abnormal activity of stem cell factor.
8. The use as claimed in any one of claims 1 to 7, wherein the subject has abnormal collagen production.
9. The use as claimed in any one of claims 1 to 8, wherein the disease is is. 9351045-1
10. The use as d in any one of claims 1 to 8, wherein the disease is a remodeling disease.
11. The use as claimed in any one of claims 1 to 8, wherein the disease is a pulmonary disease.
12. The use as claimed in any one of claims 1 to 8, n the disease is thic pulmonary fibrosis, c obstructive ary disease, acute respiratory distress syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity pneumonitis, or asthma.
13. The use as d in any one of claims 1 to 8, wherein the disease is sclerodoma, inflammation, liver cirrhosis, renal fibrosis, parenchymal fibrosis, endomyocardial is, mediastinal fibrosis, retinal fibrosis, nodular subepidermal fibrosis, fibrous histiocytoma, fibrothorax, c fibrosis, fibromyalgia, gingival fibrosis, or radiation-induced fibrosis.
14. The use as claimed in any one of claims 1 to 13, wherein the antibody or antigenbinding antibody fragment is formulated for delivery into an airway of the subject.
15. The use as claimed in any one of claims 1 to 14, wherein said antibody or antigenbinding antibody fragment reduces the activity of a receptor.
16. The use as claimed in claim 15, wherein said antibody or antigen-binding antibody fragment reduces an interaction of stem cell factor with the receptor.
17. The use as claimed in claim 15 or claim 16, wherein the receptor is a receptor tyrosine kinase.
18. The use as d in claim 15 or claim 16, wherein the receptor is c-Kit.
19. The use as claimed in any one of claims 15 to 18, wherein the receptor is found on a hematopoietic progenitor cell, a melanocyte, a germ cell, an eosinophil, a lymphocyte, a fibroblast, a myofibroblast, or a mast cell. 9351045-1
20. The use as claimed in any one of claims 1 to 19, wherein the antibody or antigenbinding antibody fragment inhibits a stem cell factor that ates from a bone marrow cell, a liver cell, an epithelial cell, a smooth muscle cell, a kidney cell, or a fibroblast.
21. The use as claimed in any one of claims 1 to 20, wherein the antibody or antigenbinding antibody fragment ly inhibits fibroblast activation.
22. Use of a monoclonal antibody or an n-binding antibody fragment that specifically binds to stem cell factor isoform b relative to stem cell factor isoform a in the preparation of a medicament for administering to a cell or a tissue. The Regents of the sity of Michigan By the Attorneys for the Applicant
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NZ702818A NZ702818B2 (en) | 2011-01-10 | 2012-01-10 | Stem cell factor inhibitor |
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201161431246P | 2011-01-10 | 2011-01-10 | |
US61/431,246 | 2011-01-10 | ||
PCT/US2012/020782 WO2012096960A2 (en) | 2011-01-10 | 2012-01-10 | Stem cell factor inhibitor |
Publications (2)
Publication Number | Publication Date |
---|---|
NZ612783A NZ612783A (en) | 2014-12-24 |
NZ612783B2 true NZ612783B2 (en) | 2015-03-25 |
Family
ID=
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