U.S. patent application number 16/708877 was filed with the patent office on 2020-08-13 for therapeutic antibodies and uses thereof.
The applicant listed for this patent is THE REGENTS OF THE UNIVERSITY OF MICHIGAN. Invention is credited to CORY HOGABOAM, STEVEN L. KUNKEL, NICHOLAS W. LUKACS.
Application Number | 20200255509 16/708877 |
Document ID | 20200255509 / US20200255509 |
Family ID | 1000004794526 |
Filed Date | 2020-08-13 |
Patent Application | download [pdf] |
United States Patent
Application |
20200255509 |
Kind Code |
A1 |
LUKACS; NICHOLAS W. ; et
al. |
August 13, 2020 |
THERAPEUTIC ANTIBODIES AND USES THEREOF
Abstract
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.
Inventors: |
LUKACS; NICHOLAS W.;
(BRIGHTON, MI) ; KUNKEL; STEVEN L.; (ANN ARBOR,
MI) ; HOGABOAM; CORY; (ANN ARBOR, MI) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
THE REGENTS OF THE UNIVERSITY OF MICHIGAN |
ANN ARBOR |
MI |
US |
|
|
Family ID: |
1000004794526 |
Appl. No.: |
16/708877 |
Filed: |
December 10, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15163391 |
May 24, 2016 |
10501535 |
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16708877 |
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14328024 |
Jul 10, 2014 |
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15163391 |
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15058918 |
Mar 2, 2016 |
9790272 |
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15163391 |
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13937852 |
Jul 9, 2013 |
9353178 |
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15058918 |
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13347459 |
Jan 10, 2012 |
8911729 |
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13937852 |
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61844728 |
Jul 10, 2013 |
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61431246 |
Jan 10, 2011 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/52 20130101;
C07K 2317/56 20130101; C07K 16/24 20130101 |
International
Class: |
C07K 16/24 20060101
C07K016/24 |
Claims
1-20. (canceled)
21. A method of treating a subject, said method comprising
administering an anti-stem cell factor (SCF) antibody to a subject,
wherein said anti-SCF antibody comprises: a light chain variable
domain comprising an amino acid sequence comprising: a first amino
acid sequence comprising the CDR1 sequence provided by amino acids
42-53 of SEQ ID NO: 4; a second amino acid sequence comprising the
CDR2 sequence provided by amino acids 69-79 of SEQ ID NO: 4; and a
third amino acid sequence comprising the CDR3 sequence provided by
amino acids 112-124 of SEQ ID NO: 4; and a heavy chain variable
domain comprising an amino acid sequence comprising: a first amino
acid sequence comprising the CDR1 sequence provided by amino acids
45-56 of SEQ ID NO: 2; a second amino acid sequence comprising the
CDR2 sequence provided by amino acids 71-86 of SEQ ID NO: 2; and a
third amino acid sequence comprising the CDR3 sequence provided by
amino acids 119-123 of SEQ ID NO: 2.
22. The method of claim 21 wherein said subject has a disease that
is fibrosis.
23. The method of claim 21 wherein said subject has a disease that
is a tissue remodeling disease.
24. The method of claim 21 wherein said subject has a disease that
is idiopathic pulmonary fibrosis, pulmonary arterial hypertension,
chronic obstructive pulmonary disease, acute respiratory distress
syndrome, cystic fibrosis, peribronchial fibrosis, hypersensitivity
pneumonitis, asthma, scleroderma, inflammation, liver cirrhosis,
renal fibrosis, parenchymal fibrosis, endomyocardial fibrosis,
mediastinal fibrosis, nodular subepidermal fibrosis, fibrous
histiocytoma, fibrothorax, hepatic fibrosis, fibromyalgia, gingival
fibrosis, or radiation-induced fibrosis.
25. The method of claim 21 wherein said antibody is administered
into an airway of said subject.
26. The method of claim 21 wherein said antibody is administered
intranasally, topically, intraocularly, parenterally, orally,
intranasally, intravenously, intramuscularly, or
subcutaneously.
27. The method of claim 21 wherein said antibody is provided in a
physiologically appropriate solution comprising a pharmaceutically
effective amount of said antibody.
28. The method of claim 21 wherein said antibody is administered in
multiple doses.
29. The method of claim 21 wherein said antibody is co-administered
with another compound.
30. The method of claim 21 wherein said antibody is a monoclonal
antibody.
31. The method of claim 21 wherein said antibody is a humanized
antibody.
32. The method of claim 21 wherein said antibody is a recombinant
antibody.
33. The method of claim 21 wherein said antibody is a chimeric
antibody.
34. The method of claim 21, wherein the antibody is monovalent and
comprises an Fc region.
35. The method of claim 21, wherein the antibody is bivalent or
bispecific.
36. The method of claim 21, wherein the antibody is a Fab, Fab'-SH,
Fv, scFv, or a (Fab').sub.2 antibody fragment.
37. The method of claim 21, wherein the antibody comprises a single
Fab region linked to an Fc region.
38. The method of claim 21 further comprising detecting SCF in a
biological sample from said subject.
Description
[0001] The present application is a continuation of, and claims
priority to, U.S. patent application Ser. No. 14/328,024, filed
Jul. 10, 2014, which claims priority to U.S. Provisional Patent
Application Ser. No. 61/844,728, filed Jul. 10, 2013; and is a
continuation-in-part of, and claims priority to, U.S. patent
application Ser. No. 15/058,918, filed Mar. 2, 2016, which is a
divisional of U.S. patent application Ser. No. 13/937,852, filed
Jul. 9, 2013, which is a divisional of U.S. patent application Ser.
No. 13/347,459, filed Jan. 10, 2012, now U.S. Pat. No. 8,911,729,
issued on Dec. 16, 2014, which claims priority to U.S. Provisional
Patent Application Ser. No. 61/431,246, filed on Jan. 10, 2011, the
disclosures of each of which is herein incorporated by reference in
its entirety.
FIELD OF INVENTION
[0002] Provided herein are methods, compositions, and uses relating
to antibody inhibitors of stem cell factor. For example, provided
herein are antibodies targeting stem cell factor and methods for
treating fibrotic and tissue remodeling diseases.
BACKGROUND
[0003] 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 attributable to some type of
chronic fibroproliferative disease and the associated health care
costs are in the billions of dollars. Tissue remodeling is the
reorganization or renovation of existing tissues, which can either
change the characteristics of a tissue (e.g., blood vessel
remodeling) or participate 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 reactive process, as opposed to formation
of fibrous tissue as a normal constituent of an organ or tissue.
Fibrosis affects nearly all tissues and organ systems, and fibrotic
tissue remodeling can influence cancer metastasis and accelerate
chronic 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
disease, and systemic sclerosis, among others.
[0004] 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 differentiation (Andre et
al., Oncogene 4: 1047 (1989)) and certain tumors exhibit elevated
expression of c-kit. SCF is a ligand specific for the c-Kit
receptor 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)).
[0005] 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, alternative therapies are needed.
SUMMARY
[0006] 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.
[0007] Embodiments of the present invention provide an isolated
recombinant monoclonal anti-stem cell factor (SCF) antibody
comprising: (a) a light chain variable domain comprising the amino
acid sequence of SEQ ID NO:4 or sequences with at least 80% (e.g.,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or fractions thereof) identity to SEQ
ID NO:4; and (b) a heavy chain variable domain comprising the amino
acid sequence of SEQ ID NO:2 or sequences with at least 80% (e.g.,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or fractions thereof identity to SEQ
ID NO:2. In some embodiments, the light chain variable region has
the amino acid sequence of SEQ ID NO:4 and the heavy chain variable
region has the amino acid sequence of SEQ ID NO:2. In some
embodiment, the antibody is monovalent and comprises an Fc region.
In some embodiments, the antibody is bivalent. In some embodiments,
the antibody is bispecific. In some embodiments, the antibody is an
antibody fragment selected from, for example a Fab, a Fab'-SH, an
Fv, an scFv, or a (Fab').sub.2 fragment. In some embodiments, the
antibody comprises a single Fab region linked to an Fc region.
Antibodies of the invention can further comprise any suitable
framework and/or light chain variable domain sequences, provided
SCF binding activity is substantially retained. For example, in
some embodiments, these antibodies further comprise a human
subgroup III heavy chain framework consensus sequence. In one
embodiment, these antibodies further comprise a human xI light
chain framework consensus sequence.
[0008] Further embodiments provide a pharmaceutical composition
comprising any of the aforementioned antibodies and a
pharmaceutically acceptable carrier.
[0009] Additional embodiments provide a nucleic acid encoding any
of the aforementioned antibodies. In some embodiments, the nucleic
acid comprises a nucleic acid encoding a light chain variable
region comprising SEQ ID NO:3 or sequences that are at least 80%
(e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, or fractions thereof or
alternative codons that encode the amino acids of SEQ ID NO:4)
homologous to SEQ ID NO:3 and a nucleic acid encoding a heavy chain
variable region comprising SEQ ID NO:1 or sequences that are at
least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or fractions thereof or
alternative codons that encode the amino acids of SEQ ID NO:2)
homologous to SEQ ID NO:1. In some embodiments, the nucleic acid
encoding a light chain variable region comprises SEQ ID NO:3 and
the nucleic acid encoding a heavy chain variable region comprises
SEQ ID NO:1.
[0010] In some embodiments, the present invention provides the use
of the aforementioned pharmaceutical composition or antibodies in
the treatment of a lung, fibrotic or tissue remodeling disease.
[0011] In some embodiments, the present invention provides a method
of treating a fibrotic or tissue remodeling disease comprising
administering the aforementioned pharmaceutical composition or
antibodies to a subject with or at risk for a fibrotic or tissue
remodeling disease. In some embodiments, the subject has an
abnormal activity of stem cell factor or abnormal collagen
production. In some embodiments, the disease is fibrosis, a
remodeling disease, or a pulmonary disease (e.g., idiopathic
pulmonary fibrosis, chronic obstructive pulmonary disease, acute
respiratory distress syndrome, cystic fibrosis, peribronchial
fibrosis, hypersensitivity pneumonitis, asthma, pulmonary arterial
hypertension (PAH), sclerodoma, inflammation, liver cirrhosis,
renal fibrosis, parenchymal fibrosis, endomyocardial fibrosis,
mediatinal fibrosis, nodular subepidermal fibrosis, fibrous
histiocytoma, fibrothorax, hepatic fibrosis, fibromyalgia, gingival
fibrosis, or radiation-induced fibrosis). In some embodiments, the
pharmaceutical composition is delivered into an airway of the
subject by e.g., intranasal or inhalational (e.g. dry powder or
nebulizer) administration. In some embodiments, the administering
reduces an activity of a receptor and/or reduces an interaction of
stem cell factor with a receptor (e.g., a receptor tyrosine kinase
such as c-Kit). In some embodiments, the administering results in a
direct inhibition of fibroblast activation. In some embodiments,
the administering results in inhibition of progression of signs or
symptoms of a disease.
[0012] Additional embodiments will be apparent to persons skilled
in the relevant art based on the teachings contained herein.
DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows exemplary CDR and Framework regions of the
antibodies of embodiments of the present disclosure.
DETAILED DESCRIPTION
[0014] Provided herein are methods, compositions, and uses relating
to inhibitors of stem cell factor. For example, provided herein are
antibodies targeting stem cell factor, methods of producing
antibodies targeting stem cell factor, and methods for treating
fibrotic and tissue remodeling diseases 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 fibrotic diseases and maladies
associated with tissue remodeling.
Definitions
[0015] 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.
[0016] Throughout the specification and claims, 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.
[0017] In addition, as used herein, the term "or" is an inclusive
"or" operator and is equivalent to the term "and/or" unless the
context clearly dictates otherwise. The term "based on" is not
exclusive and allows for being based on additional factors not
described, unless the context clearly dictates otherwise. In
addition, throughout the specification, the meaning of "a", "an",
and "the" include plural references. The meaning of "in" includes
"in" and "on."
[0018] 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 sequence encoded by the gene, but
includes post-translational modifications of the protein.
[0019] 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 "protein" are
not meant to limit the amino acid sequence to the complete, native
amino acid sequence associated with the recited protein molecule.
Furthermore, an "amino acid sequence" can be deduced from the
nucleic acid sequence encoding the protein.
[0020] 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 includes 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.
[0021] The term "portion" when used in reference 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).
[0022] The term "homolog" or "homologous" when used in reference to
a polypeptide refers to a high degree of sequence identity between
two polypeptides, 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 action. 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.
[0023] The terms "variant" and "mutant" when used in reference 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
example, 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 asparagine and glutamine; a group of amino acids having
aromatic side chains is phenylalanine, tyrosine, and tryptophan; a
group of amino acids having basic side chains is lysine, 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,
phenylalanine-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
determining 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. Variants can be tested in
functional assays. Preferred variants have less than 10%, and
preferably less than 5%, and still more preferably less than 2%
changes (whether substitutions, deletions, and so on).
[0024] The term "domain" when used in reference to a polypeptide
refers to a subsection of the polypeptide which possesses a unique
structural and/or functional 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 folding or other configurations. Examples of
a protein domain include the transmembrane domains, and the
glycosylation sites.
[0025] 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 polypeptide or its precursor (e.g.,
proinsulin). A functional polypeptide can be encoded by a full
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 fragments 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.
[0026] The term "gene" also encompasses the coding regions of a
structural gene and includes 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 mRNA are referred to as
5' non-translated sequences. 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 segments 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 messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
[0027] In addition to containing introns, genomic forms of a gene
may also include sequences located on both the 5' and 3' end of the
sequences which are present on the RNA transcript. These sequences
are referred to as "flanking" sequences or regions (these flanking
sequences are located 5' or 3' to the non-translated sequences
present on the mRNA transcript). The 5' flanking region may contain
regulatory sequences such as promoters 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
polyadenylation.
[0028] 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 function or
use of the oligonucleotide. The oligonucleotide may be generated in
any manner, including chemical synthesis, DNA replication, reverse
transcription, or a combination thereof.
[0029] 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
oligonucleotide may be single-stranded (i.e., the sense strand) or
double-stranded. Suitable control elements 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 utilized in the expression vectors can contain
endogenous enhancers/promoters, splice junctions, intervening
sequences, polyadenylation signals, etc. or a combination of both
endogenous and exogenous control elements.
[0030] The term "recombinant" when made in reference to a nucleic
acid molecule refers to a nucleic acid molecule which is comprised
of segments of nucleic acid joined together by means of molecular
biological techniques. The term "recombinant" when made in
reference to a protein or a polypeptide refers to a protein
molecule which is expressed using a recombinant nucleic acid
molecule. In some embodiments, recombinant nucleic acids are in an
expression vector (e.g., plasmid), optionally joined to nucleic
acids useful for driving expression of the nucleic acid (e.g.,
promoter or enhancer sequences).
[0031] 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 "3'-T-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.
[0032] 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
object 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 viruses) 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, respectively, 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 mutants can be isolated; these are identified
by the fact that they have altered characteristics when compared to
the wild-type gene or gene product.
[0033] The term "isolated" when used in relation to a nucleic acid
or polypeptide, refers to a nucleic acid or polypeptide that is
substantially free of other proteins or nucleic acids (e.g.,
suitable for pharmaceutical administration).
[0034] The terms "antibody" and "immunoglobulin" are used
interchangeably in the broadest sense and include monoclonal
antibodies (for e.g., full length or intact monoclonal antibodies),
polyclonal antibodies, multivalent antibodies, multi specific
antibodies (e.g., bispecific antibodies so long as they exhibit the
desired biological activity) and may also include certain antibody
fragments (as described in greater detail herein). An antibody can
be human, humanized and/or affinity matured.
[0035] An antibody that "specifically binds to" or is "specific
for" a particular polypeptide or an epitope on a particular
polypeptide is one that binds to that particular polypeptide or
epitope on a particular polypeptide without substantially binding
to any other polypeptide or polypeptide epitope.
[0036] "Binding affinity" generally refers to the strength of the
sum total of noncovalent interactions between a single binding site
of a molecule (e.g., an antibody) and its binding partner (e.g., an
antigen). Unless indicated otherwise, as used herein, "binding
affinity" refers to intrinsic binding affinity which reflects a 1:1
interaction between members of a binding pair (e.g., antibody and
antigen). The affinity of a molecule X for its partner Y can
generally be represented by the dissociation constant (Kd).
Affinity can be measured by common methods known in the art,
including those described herein. Low-affinity antibodies generally
bind antigen slowly and tend to dissociate readily, whereas
high-affinity antibodies generally bind antigen faster and tend to
remain bound longer. A variety of methods of measuring binding
affinity are known in the art, any of which can be used. Specific
illustrative embodiments are described in the following.
[0037] 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.
[0038] As used herein, "inhibitor" refers to a molecule which
eliminates, minimizes, or decreases the activity, e.g., the
biological, enzymatic, chemical, or immunological activity, of a
target.
[0039] 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, nausea, fever, pain, inflammation,
etc.).
[0040] As used herein, the term "administration" refers to the act
of giving a drug, prodrug, antibody, or other agent, or therapeutic
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 (oral), skin (transdermal), nose (nasal), lungs
(inhalant), oral mucosa (buccal), ear, by injection (e.g.,
intravenously, 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 physiological system (e.g., a subject or in
vivo, in vitro, or ex vivo cells, tissues, and organs). As used
herein, administration "in combination with" one or more further
therapeutic agents includes simultaneous (concurrent) and
consecutive administration in any order. "Coadministration" of
therapeutic treatments may be concurrent, or in any temporal order
or physical combination.
[0041] As used herein, the term "treating" includes reducing or
alleviating at least one adverse effect, sign, 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 therapeutic treatment and
prophylactic or preventative measures, wherein the object is to
prevent 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.
[0042] 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
determination 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., aggressive vs. conventional
treatment).
[0043] As used herein, the term "effective amount" refers to the
amount of a composition sufficient to effect beneficial or desired
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.
[0044] As used herein, the term "pharmaceutical composition" refers
to the combination of an active agent with, as desired, a carrier,
inert or active, making the composition especially suitable for
diagnostic or therapeutic use in vitro, in vivo, or ex vivo.
[0045] 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 reactions, when administered to a subject.
[0046] As used herein, "carriers" include pharmaceutically
acceptable carriers, excipients, or stabilizers which are nontoxic
to the cell or mammal being exposed thereto at the dosages and
concentrations employed. Often the physiologically acceptable
carrier is an aqueous pH-buffered solution. Examples of
physiologically acceptable carriers include buffers such as
phosphate, citrate, and other organic acids; antioxidants including
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 mannitol or sorbitol; salt-forming
counterions such as sodium; and/or nonionic surfactants.
[0047] 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 subject
is a primate. In an even more preferred embodiment, the subject is
a human. In some embodiments, the subject is a companion animal
(e.g., dog, cats, etc.), an agricultural animal (e.g., cow, sheep,
goat, pig, etc.), or an equine.
[0048] As used herein, the term "sample" is used in its broadest
sense. In one sense it can refer to animal cells or tissues. In
another sense, it is meant to include a specimen or culture
obtained from any source, such as biological and environmental
samples. Biological samples may be obtained from plants or animals
(including humans) and encompass fluids, solids, tissues, and
gases. Environmental 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 technology.
Embodiments of the Technology
[0049] Although the disclosure herein refers to certain illustrated
embodiments, it is to be understood that these embodiments are
presented by way of example and not by way of limitation.
[0050] 1. Inhibitors of SCF
[0051] Stem cell factor (SCF) is a ligand that is specific 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,
adhesion, and functional activation. Aberrant activation of c-Kit
can result in disease, including fibrosis and tissue remodeling
defects. In particular, 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, pulmonary arterial hypertension (PAH), asthma, 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 fibrosis, fibrous histiocytoma, fibrothorax, hepatic
fibrosis, fibromyalgia, gingival fibrosis, and radiation-induced
fibrosis.
[0052] Accordingly, interfering with the interaction between SCF
and c-Kit can be used to treat or study diseases 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.
[0053] Additionally, SCF induces key mediators in the fibrotic
response, IL-25 and IL-13. Data suggest that IL-25 can drive IL-13
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 production of
IL-13, myofibroblast differentiation, and collagen production. IL-4
has also been identified as a fibrosis-associated cytokine.
[0054] 2. Antibodies
[0055] In some embodiments, inhibiting the ability of SCF to
interact with c-Kit is accomplished by means of an antibody that
recognizes SCF. In some embodiments, the antibody is a recombinant
antibody (See e.g., Example 1).
[0056] It is contemplated that antibodies against SCF find use in
the experimental, diagnostic, and therapeutic methods described
herein. In certain embodiments, the antibodies provided 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. Alternatively, 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
protein 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 subject (e.g., human
patient) by administering a therapeutically effective amount of an
antibody against SCF.
[0057] In some embodiments, the present disclosure provides
antibodies having the sequences of SEQ ID NOs:1-4 and variants
thereof. For example, in some embodiments, the light chain variable
domain comprises the amino acid sequence of SEQ ID NO:4 or
sequences with at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
fractions thereof) identity to SEQ ID NO:4; and the heavy chain
variable domain comprises the amino acid sequence of SEQ ID NO:2 or
sequences with at least 80% (e.g., 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
fractions thereof identity to SEQ ID NO:2. In some embodiments, the
light chain variable region is encoded by the nucleic acid of SEQ
ID NO:3 or sequences with at least 80% (e.g., 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, 99% or fractions thereof) homology to SEQ ID NO:3. In some
embodiments, the heavy chain variable region is encoded by the
nucleic acid of SEQ ID NO:1 or sequences with at least 80% (e.g.,
81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99% or fractions thereof) homology to SEQ
ID NO:1.
[0058] In certain embodiments, antibodies are engineered, for
example by including modifications of the Fc region which can alter
serum half-life, complement fixation, Fc receptor binding and/or
antigen dependent cellular cytotoxicity.
[0059] In some embodiments, modified or variant antibodies as
described herein retain binding affinity to SCF (e.g., within 10%,
5%, 4%, 3%, 2%, or 1%) of the unmodified antibody. In some
embodiments, binding affinity is increased or decreased relative to
the wild-type or unmodified antibody.
[0060] In certain embodiments, modifications in the biological
properties of an antibody (e.g., those described herein) are
accomplished by selecting substitutions that affect (a) the
structure of the polypeptide backbone in the area of the
substitution, for example, as a sheet or helical conformation, (b)
the charge or hydrophobicity of the molecule at the target. The
polynucleotide encoding a monoclonal antibody can further be
modified in a site, or (C) the bulk of the side chain. Amino acids
may be grouped according to similarities in the properties of their
side chains (A. L. Lehninger, in Biochemistry, 2nd Ed., 73-75,
Worth Publishers, New York (1975)): (1) non-polar: Ala (A), Val
(V), Leu (L), lIe (I), Pro (P), Phe (F), Trp (W), Met (M); (2)
uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn
(N), GIn (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg
(R), His(H). Alternatively, naturally occurring residues may be
divided into groups based on common side-chain properties: (1)
hydrophobic: Norleucine, Met, Ala, Val, Leu, lIe; (2) neutral
hydrophilic: Cys, Ser, Thr, Asn, GIn; (3) acidic: Asp, Glu; (4)
basic: His, Lys, Arg; (5) residues that influence chain
orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.
Non-conservative substitutions will entail exchanging a member of
one of these classes for another class. Such substituted residues
also may be introduced into the conservative substitution sites or,
into the remaining (e.g., non-conserved) sites in a number of
different manners using recombinant DNA.
[0061] In some embodiments, antibodies or antibody fragments are
provided that can be produced which have altered glycosylation
patterns. In certain embodiments, an antibody is altered to
increase or decrease the extent to which the antibody is
glycosylated. Glycosylation of polypeptides is typically either
N-linked or O-linked. N-linked refers to the attachment of a
carbohydrate moiety to the side chain of an asparagine residue. The
tripeptide sequences asparagine-X-serine and asparagine-X
-threonine, where X is any amino acid except proline, are the
recognition sequences for enzymatic attachment of the carbohydrate
moiety to the asparagine side chain. Thus, the presence of either
of these tripeptide sequences in a polypeptide creates a potential
glycosylation site. O-linked glycosylation refers to the attachment
of one of the sugars N-aceylgalactosamine, galactose, or xylose to
a hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
[0062] In some embodiments, the antibodies are PEGylated by
reacting the antibody with a polyethylene glycol (PEG) derivative.
In certain embodiments, the antibody is defucosylated and therefore
lacks fucose residues.
[0063] In some embodiments, humanized anti-SCF antibodies are
generated. For example, also contemplated are chimeric mouse-human
monoclonal antibodies, which are 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/US86/02269; European Patent Application
184,187; European Patent Application 171,496; European Patent
Application 173,494; WO 86/01533; U.S. Pat. No. 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, 84:214-218 (1987); Nishimura et al., Canc.
Res., 47:999-1005 (1987); Wood et al., Nature, 314:446-449 (1985);
and Shaw et al., J. Natl. Cancer Inst., 80:1553-1559 (1988)).
[0064] The chimeric antibody can be further humanized by replacing
sequences of the variable region that are not directly involved in
antigen binding with equivalent sequences from human variable
regions. General reviews of humanized chimeric antibodies are
provided by S. L. Morrison, Science, 229:1202-1207 (1985) and by Oi
et al., Bio Techniques, 4:214 (1986). Those methods include
isolating, manipulating, and expressing the nucleic 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 recombinant DNA
encoding the chimeric antibody, or fragment thereof, can then be
cloned into an appropriate expression vector.
[0065] Suitable humanized antibodies can alternatively be produced
by CDR substitution (see, e.g., U.S. Pat. No. 5,225,539; Jones et
al., Nature, 321:552-525 (1986); Verhoeyan et al., Science,
239:1534 (1988); and Beidler et al., J. Immunol., 141:4053 (1988)).
All of the CDRs of a particular human antibody may be replaced with
at least a portion of a non-human CDR or only some of the CDRs may
be replaced with non-human CDRs. It is only necessary to replace
the number of CDRs important for binding of the humanized antibody
to the Fc receptor.
[0066] 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.
[0067] A humanized antibody may comprise one or more human and/or
human consensus non-hypervariable region (e.g., framework)
sequences in its heavy and/or light chain variable domain. In some
embodiments, one or more additional modifications are present
within the human and/or human consensus non-hypervariable region
sequences. In one embodiment, the heavy chain variable domain of an
antibody comprises a human consensus framework sequence, which in
one embodiment is the subgroup III consensus framework sequence. In
one embodiment, an antibody comprises a variant subgroup III
consensus framework sequence modified at least one amino acid
position. In one embodiment, the light chain variable domain of an
antibody comprises a human consensus framework sequence, which in
one embodiment is the id consensus framework sequence. In one
embodiment, an antibody comprises a variant id consensus framework
sequenced modified at least one amino acid position.
[0068] As is known in the art, and as described in greater detail
herein below, the amino acid position/boundary delineating a
hypervariable region of an antibody can vary, depending on the
context and the various definitions known in the art (as described
below). Some positions within a variable domain may be viewed as
hybrid hypervariable positions in that these positions can be
deemed to be within a hypervariable region under one set of
criteria while being deemed to be outside a hypervariable region
under a different set of criteria. One or more of these positions
can also be found in extended hypervariable regions (as further
defined below). Embodiments of the invention provide antibodies
comprising modifications in these hybrid hypervariable positions.
In one embodiment, an antibody comprises a human variant human
subgroup consensus framework sequence modified at one or more
hybrid hypervariable positions.
[0069] An antibody as described herein can comprise any suitable
human or human consensus light chain framework sequences, provided
the antibody exhibits the desired biological characteristics (e.g.,
a desired binding affinity). In one embodiment, an antibody of
embodiments of the disclosure comprises at least a portion (or all)
of the framework sequence of human .kappa. light chain. In one
embodiment, an antibody of embodiments of the disclosure comprises
at least a portion (or all) of human kappa subgroup I framework
consensus sequence.
[0070] In some embodiments, antibodies are humanized antibodies or
human antibodies. Humanized forms of non-human (e.g., murine)
antibodies are chimeric immunoglobulins, immunoglobulin chains or
fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other
antigen-binding subsequences of antibodies) which contain minimal
sequence derived from non-human immunoglobulin. Humanized
antibodies include human immunoglobulins (recipient antibody) in
which residues from a complementary determining region (CDR) of the
recipient are replaced by residues from a CDR of a non-human
species (donor antibody) such as mouse, rat or rabbit having the
desired specificity, affinity and capacity. In some instances, Fv
framework residues of the human immunoglobulin are replaced by
corresponding non-human residues. Humanized antibodies may also
comprise residues which are found neither in the recipient antibody
nor in the imported CDR or framework sequences. In general, the
humanized antibody comprises substantially all of at least one, and
typically two, variable domains, in which all or substantially all
of the CDR regions correspond to those of a non-human
immunoglobulin and all or substantially all of the FR regions are
those of a human immunoglobulin consensus sequence. The humanized
antibody optimally also comprises at least a portion of an
immunoglobulin constant region (Fc), typically that of a human
immunoglobulin (Jones et al., Nature, 321:522-525 (1986); Riechmann
et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct.
Biol., 2:593-596 (1992)).
[0071] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting rodent CDRs or CDR sequences for the corresponding
sequences of a human antibody. Accordingly, such "humanized"
antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567),
wherein substantially less than an intact human variable domain has
been substituted by the corresponding sequence from a non-human
species. In practice, humanized antibodies are typically human
antibodies in which some CDR residues and possibly some FR residues
are substituted by residues from analogous sites in rodent
antibodies.
[0072] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is important to
reduce antigenicity and HAMA response (human anti-mouse antibody)
when the antibody is intended for human therapeutic use. Reduction
or elimination of a HAMA response is a significant aspect of
clinical development of suitable therapeutic agents. See, e.g.,
Khaxzaeli et al., J. Natl. Cancer Inst. (1988), 80:937; Jaffers et
al., Transplantation (1986), 41:572; Shawler et al., J. Immunol.
(1985), 135:1530; Sears et al., J. Biol. Response Mod. (1984),
3:138; Miller et al., Blood (1983), 62:988; Hakimi et al., J.
Immunol. (1991), 147:1352; Reichmann et al., Nature (1988),
332:323; Junghans et al., Cancer Res. (1990), 50:1495. As described
herein, in some embodiments, the invention provides antibodies that
are humanized. Variants of these antibodies can further be obtained
using routine methods known in the art, some of which are further
described below. According to the so-called "best-fit" method, the
sequence of the variable domain of a rodent antibody is screened
against the entire library of known human variable domain
sequences. The human V domain sequence which is closest to that of
the rodent is identified and the human framework region (FR) within
it accepted for the humanized antibody (Sims et al., J. Immunol.
151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987)).
Another method uses a particular framework region derived from the
consensus sequence of all human antibodies of a particular subgroup
of light or heavy chains. The same framework may be used for
several different humanized antibodies (Carter et al., Proc. Natl.
Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol. 151:2623
(1993)).
[0073] For example, an amino acid sequence from an antibody as
described herein can serve as a starting (parent) sequence for
diversification of the framework and/or hypervariable sequence(s).
A selected framework sequence to which a starting hypervariable
sequence is linked is referred to herein as an acceptor human
framework. While the acceptor human frameworks may be from, or
derived from, a human immunoglobulin (the VL and/or VH regions
thereof), preferably the acceptor human frameworks are from, or
derived from, a human consensus framework sequence as such
frameworks have been demonstrated to have minimal, or no,
immunogenicity in human patients.
[0074] Where the acceptor is derived from a human immunoglobulin,
one may optionally select a human framework sequence that is
selected based on its homology to the donor framework sequence by
aligning the donor framework sequence with various human framework
sequences in a collection of human framework sequences, and select
the most homologous framework sequence as the acceptor.
[0075] In one embodiment, human consensus frameworks herein are
from, or derived from, VH subgroup III and/or VL kappa subgroup I
consensus framework sequences.
[0076] While the acceptor may be identical in sequence to the human
framework sequence selected, whether that be from a human
immunoglobulin or a human consensus framework, embodiments of the
present invention contemplate that the acceptor sequence may
comprise pre-existing amino acid substitutions relative to the
human immunoglobulin sequence or human consensus framework
sequence. These pre-existing substitutions are preferably minimal;
usually four, three, two or one amino acid differences only
relative to the human immunoglobulin sequence or consensus
framework sequence.
[0077] Hypervariable region residues of the non-human antibody are
incorporated into the VL and/or VH acceptor human frameworks. For
example, one may incorporate residues corresponding to the Kabat
CDR residues, the Chothia hypervariable loop residues, the Abm
residues, and/or contact residues. Optionally, the extended
hypervariable region residues as follows are incorporated: 24-34
(L1), 50-56 (L2) and 89-97 (L3), 26-35B (H1), 50-65, 47-65 or 49-65
(H2) and 93-102, 94-102, or 95-102 (H3).
[0078] While "incorporation" of hypervariable region residues is
discussed herein, it will be appreciated that this can be achieved
in various ways, for example, nucleic acid encoding the desired
amino acid sequence can be generated by mutating nucleic acid
encoding the mouse variable domain sequence so that the framework
residues thereof are changed to acceptor human framework residues,
or by mutating nucleic acid encoding the human variable domain
sequence so that the hypervariable domain residues are changed to
non-human residues, or by synthesizing nucleic acid encoding the
desired sequence, etc.
[0079] Phage(mid) display (also referred to herein as phage display
in some contexts) can be used as a convenient and fast method for
generating and screening many different potential variant
antibodies in a library generated by sequence randomization.
However, other methods for making and screening altered antibodies
are available to the skilled person.
[0080] Phage(mid) display technology has provided a powerful tool
for generating and selecting novel proteins which bind to a ligand,
such as an antigen. Using the techniques of phage(mid) display
allows the generation of large libraries of protein variants which
can be rapidly sorted for those sequences that bind to a target
molecule with high affinity. Nucleic acids encoding variant
polypeptides are generally fused to a nucleic acid sequence
encoding a viral coat protein, such as the gene III protein or the
gene VIII protein. Monovalent phagemid display systems where the
nucleic acid sequence encoding the protein or polypeptide is fused
to a nucleic acid sequence encoding a portion of the gene III
protein have been developed. (Bass, S., Proteins, 8:309 (1990);
Lowman and Wells, Methods: A Companion to Methods in Enzymology,
3:205 (1991)). In a monovalent phagemid display system, the gene
fusion is expressed at low levels and wild type gene III proteins
are also expressed so that infectivity of the particles is
retained. Methods of generating peptide libraries and screening
those libraries have been disclosed in many patents (e.g. U.S. Pat.
Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908 and 5,498,530).
[0081] Libraries of antibodies or antigen binding polypeptides have
been prepared in a number of ways including by altering a single
gene by inserting random DNA sequences or by cloning a family of
related genes. Methods for displaying antibodies or antigen binding
fragments using phage(mid) display have been described in U.S. Pat.
Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197,
5,580,717, and 5,658,727. The library is then screened for
expression of antibodies or antigen binding proteins with the
desired characteristics.
[0082] Additional methods of preparing and screening libraries are
described, for example, in Geyer et al., Methods Mol Biol.
2012;901:11-32; and de Marco A. Crit Rev Biotechnol. 2013 March;
33(1):40-8; each of which is herein incorporated by reference in
its entirety.
[0083] Methods of substituting an amino acid of choice into a
template nucleic acid are well established in the art, some of
which are described herein. For example, hypervariable region
residues can be substituted using the Kunkel method. See, e.g.,
Kunkel et al., Methods Enzymol. 154:367-382 (1987).
[0084] According to another method, antigen binding may be restored
during humanization of antibodies through the selection of repaired
hypervariable regions (See US20060122377). The method includes
incorporating non-human hypervariable regions onto an acceptor
framework and further introducing one or more amino acid
substitutions in one or more hypervariable regions without
modifying the acceptor framework sequence. Alternatively, the
introduction of one or more amino acid substitutions may be
accompanied by modifications in the acceptor framework
sequence.
[0085] According to another method, a library can be generated by
providing upstream and downstream oligonucleotide sets, each set
having a plurality of oligonucleotides with different sequences,
the different sequences established by the codon sets provided
within the sequence of the oligonucleotides. The upstream and
downstream oligonucleotide sets, along with a variable domain
template nucleic acid sequence, can be used in a polymerase chain
reaction to generate a "library" of PCR products. The PCR products
can be referred to as "nucleic acid cassettes", as they can be
fused with other related or unrelated nucleic acid sequences, for
example, viral coat proteins and dimerization domains, using
established molecular biology techniques.
[0086] The sequence of the PCR primers includes one or more of the
designed codon sets for the solvent accessible and highly diverse
positions in a hypervariable region. As described above, a codon
set is a set of different nucleotide triplet sequences used to
encode desired variant amino acids.
[0087] Antibody selectants that meet the desired criteria, as
selected through appropriate screening/selection steps are isolated
and cloned using standard recombinant techniques. As is
well-established in the art, binding affinity of a ligand to its
receptor can be determined using any of a variety of assays, and
expressed in terms of a variety of quantitative values.
Accordingly, in one embodiment, the binding affinity is expressed
as Kd values and reflects intrinsic binding affinity (e.g., with
minimized avidity effects). Generally and preferably, binding
affinity is measured in vitro, whether in a cell-free or
cell-associated setting. As described in greater detail herein,
fold difference in binding affinity can be quantified in terms of
the ratio of the monovalent binding affinity value of a humanized
antibody (e.g., in Fab form) and the monovalent binding affinity
value of a reference/comparator antibody (e.g., in Fab form) (e.g.,
a murine antibody having donor hypervariable region sequences),
wherein the binding affinity values are determined under similar
assay conditions. Thus, in one embodiment, the fold difference in
binding affinity is determined as the ratio of the Kd values of the
humanized antibody in Fab form and said reference/comparator Fab
antibody. For example, in one embodiment, if an antibody has an
affinity that is "3-fold lower" than the affinity of a reference
antibody (M), then if the Kd value for A is 3.times., the Kd value
of M would be 1.times., and the ratio of Kd of A to Kd of M would
be 3:1. Conversely, in one embodiment, if an antibody has an
affinity that is "3-fold greater" than the affinity of a reference
antibody (R), then if the Kd value for C is 1.times., the Kd value
of R would be 3.times., and the ratio of Kd of C to Kd of R would
be 1:3. Any of a number of assays known in the art, including those
described herein, are used to obtain binding affinity measurements,
including, for example, Biacore, radioimmunoassay (RIA) and ELISA
technology to generate alternative antibodies. In one embodiment,
the constant domains of the light and heavy chains of, for example,
a mouse monoclonal antibody can be substituted 1) for those regions
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.
[0088] It may be desirable to modify the antibody with respect to
effector function, e.g., so as to enhance or decrease
antigen-dependent cell-mediated cyotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) of the antibody. This may
be achieved by introducing one or more amino acid substitutions in
an Fc region of the antibody. Alternatively or additionally,
cysteine residue(s) may be introduced in the Fc region, thereby
allowing interchain disulfide bond formation in this region. The
homodimeric antibody thus generated may have improved
internalization capability and/or increased complement-mediated
cell killing and antibody-dependent cellular cytotoxicity (ADCC).
See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, B.
J. Immunol. 148:2918-2922 (1992). Homodimeric antibodies with
enhanced anti-tumor activity may also be prepared using
heterobifunctional cross-linkers as described in Wolff et al.,
Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can
be engineered which has dual Fc regions and may thereby have
enhanced complement lysis and ADCC capabilities. See Stevenson et
al., Anti-Cancer Drug Design 3:219-230 (1989). To increase the
serum half life of the antibody, one may incorporate a salvage
receptor binding epitope into the antibody (especially an antibody
fragment) as described in U.S. Pat. No. 5,739,277, for example. As
used herein, the term "salvage receptor binding epitope" refers to
an epitope of the Fc region of an IgG molecule (e.g., IgG.sub.1,
IgG.sub.2, IgG.sub.3, or IgG.sub.4) that is responsible for
increasing the in vivo serum half-life of the IgG molecule.
[0089] DNA encoding the Fv clones can be combined with known DNA
sequences encoding heavy chain and/or light chain constant regions
(e.g. the appropriate DNA sequences can be obtained from Kabat et
al., supra) to form clones encoding full or partial length heavy
and/or light chains. It will be appreciated that constant regions
of any isotype can be used for this purpose, including IgG, IgM,
IgA, IgD, and IgE constant regions, and that such constant regions
can be obtained from any human or animal species. An Fv clone
derived from the variable domain DNA of one animal (such as human)
species and then fused to constant region DNA of another animal
species to form coding sequence(s) for "hybrid," full length heavy
chain and/or light chain is included in the definition of
"chimeric" and "hybrid" antibody as used herein. In certain
embodiments, an Fv clone derived from human variable DNA is fused
to human constant region DNA to form coding sequence(s) for full-
or partial-length human heavy and/or light chains.
[0090] It is further useful that antibodies be humanized with
retention of high binding affinity for the antigen and other
favorable biological properties. To achieve this goal, according to
a preferred method, humanized antibodies are prepared by a process
of analysis of the parental sequences and various conceptual
humanized products using three-dimensional models of the parental
and humanized sequences. Three-dimensional immunoglobulin models
are commonly available and are familiar to those skilled in the
art. Computer programs are available which illustrate and display
probable three-dimensional conformational structures of selected
candidate immunoglobulin sequences. Inspection of these displays
permits analysis of the likely role of the residues in the
functioning of the candidate immunoglobulin sequence, i.e., the
analysis of residues that influence the ability of the candidate
immunoglobulin to bind its antigen. In this way, FR residues can be
selected and combined from the recipient and import sequences so
that the desired antibody characteristic, such as increased
affinity for the target antigen(s), is achieved. In general, the
hypervariable region residues are directly and most substantially
involved in influencing antigen binding.
[0091] Also contemplated are chimeric and humanized antibodies in
which specific amino acids have been substituted, deleted, or
added. In particular, preferred 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 replaced 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.
[0092] In certain 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 digestion of intact antibodies (for
example Morimoto et al., 1993, Journal of Biochemical and
Biophysical Methods 24:107-117 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 antigen-binding site, and a residual Fc fragment.
Pepsin treatment yields an F(ab').sub.2 fragment that has two
antigen-combining sites and is still capable of cross-linking
antigen.
[0093] However, these fragments are now typically produced directly
by recombinant host cells as described 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
U.S. Pat. No. 5,641,870, for example, and can be monospecific or
bispecific. Other techniques for the production of antibody
fragments will be apparent to the skilled practitioner.
[0094] Fv is the minimum antibody 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 interact
to define an antigen-binding site on the surface of the
V.sub.H-V.sub.L dimer. Collectively, the six CDRs confer
antigen-binding specificity to the antibody. However, even a single
variable domain (or half of an Fv comprising only three CDRs
specific for an antigen) has the ability to recognize and bind
antigen, although at a lower affinity than the entire binding
site.
[0095] The Fab fragment also contains the constant domain of the
light chain and the first constant domain (CH1) of the heavy chain.
Fab fragments differ from Fab' fragments by the addition of a few
residues at the carboxy terminus of the heavy chain CH1 domain
including one or more cysteines from the antibody hinge region.
F(ab').sub.2 antibody fragments originally were produced as pairs
of Fab' fragments which have hinge cysteines between them. Other
chemical couplings of antibody fragments are also known to the
skilled artisan.
[0096] 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 epitope
into the antibody 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 antibody fragment at either
end or in the middle (e.g., by DNA or peptide synthesis).
[0097] The technology embraces variants and equivalents which are
substantially homologous to the chimeric, humanized, and human
antibodies, or antibody fragments thereof, provided herein. These
can contain, for example, conservative 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.
[0098] 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 of monoclonal Fab fragments with the desired
specificity.
[0099] Also, this technology 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
antibodies are common in the art (Millstein et al., 1983, Nature
305:537-539; Brennan et al., 1985, Science 229:81; Suresh et al,
1986, Methods in Enzymol. 121:120; Traunecker et al., 1991, EMBO J.
10:3655-3659; 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. 152:5368; and U.S. Pat. No. 5,731,168).
[0100] Techniques described for the production of single chain
antibodies (U.S. Pat. No. 4,946,778; herein incorporated by
reference) can be adapted to produce specific single chain
antibodies as desired. Single-chain Fv antibody fragments comprise
the V.sub.H and V.sub.L domains of an antibody, wherein these
domains are present in a single polypeptide chain. Preferably, the
Fv polypeptide further comprises a polypeptide linker between the
V.sub.H and V.sub.L domains that enables the single-chain Fv
antibody fragments to form the desired structure 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).
[0101] Antibodies with the desired properties can be generated and
purified using any suitable method. In some embodiments, the
expressed polypeptides are secreted into and recovered from the
periplasm of the host cells. Protein recovery typically involves
disrupting the microorganism, generally by such means as osmotic
shock, sonication or lysis. Once cells are disrupted, cell debris
or whole cells may be removed by centrifugation or filtration. The
proteins may be further purified, for example, by affinity resin
chromatography.
[0102] Alternatively, proteins can be transported into the culture
media and isolated therein. Cells may be removed from the culture
and the culture supernatant being filtered and concentrated for
further purification of the proteins produced. The expressed
polypeptides can be further isolated and identified using commonly
known methods such as polyacrylamide gel electrophoresis (PAGE) and
Western blot assay.
[0103] In some embodiments, antibody production is conducted in
large quantity by a fermentation process. Various large-scale
fed-batch fermentation procedures are available for production of
recombinant proteins. Large-scale fermentations have at least 1000
liters of capacity, preferably about 1,000 to 100,000 liters of
capacity. These fermentors use agitator impellers to distribute
oxygen and nutrients, especially glucose (the preferred
carbon/energy source). Small scale fermentation refers generally to
fermentation in a fermentor that is no more than approximately 100
liters in volumetric capacity, and can range from about 1 liter to
about 100 liters. Medium scale reactors are reactors of 100 L-1000
L.
[0104] In a fermentation process, induction of protein expression
is typically initiated after the cells have been grown under
suitable conditions to a desired density, e.g., an OD550 of about
180-220, at which stage the cells are in the early stationary
phase. A variety of inducers may be used, according to the vector
construct employed, as is known in the art and described above.
Cells may be grown for shorter periods prior to induction. Cells
are usually induced for about 12-50 hours, although longer or
shorter induction time may be used.
[0105] To improve the production yield and quality of the
antibodies, various fermentation conditions can be modified. For
example, to improve the proper assembly and folding of the secreted
antibody polypeptides, additional vectors overexpressing chaperone
proteins, such as Dsb proteins (DsbA, DsbB, DsbC, DsbD and or DsbG)
or FkpA (a peptidylprolyl cis,trans-isomerase with chaperone
activity) can be used to co-transform the host prokaryotic cells.
The chaperone proteins have been demonstrated to facilitate the
proper folding and solubility of heterologous proteins produced in
bacterial host cells. Chen et al. (1999) J Bio Chem
274:19601-19605; Georgiou et al., U.S. Pat. No. 6,083,715; Georgiou
et al., U.S. Pat. No. 6,027,888; Bothmann and Pluckthun (2000) J.
Biol. Chem. 275:17100-17105; Ramm and Pluckthun (2000) J. Biol.
Chem. 275:17106-17113; Arie et al. (2001) Mol. Microbiol.
39:199-210.
[0106] To minimize proteolysis of expressed heterologous proteins
(especially those that are proteolytically sensitive), certain host
strains deficient for proteolytic enzymes can be used for the
present invention. For example, host cell strains may be modified
to effect genetic mutation(s) in the genes encoding known bacterial
proteases such as Protease III, OmpT, DegP, Tsp, Protease I,
Protease Mi, Protease V, Protease VI and combinations thereof. Some
E. coli protease-deficient strains are available and described in,
for example, Joly et al. (1998), supra; Georgiou et al., U.S. Pat.
No. 5,264,365; Georgiou et al., U.S. Pat. No. 5,508,192; Hara et
al., Microbial Drug Resistance, 2:63-72 (1996).
[0107] In one embodiment, E. coli strains deficient for proteolytic
enzymes and transformed with plasmids overexpressing one or more
chaperone proteins are used as host cells in the expression
system.
[0108] In one embodiment, the antibody protein produced herein is
further purified to obtain preparations that are substantially
homogeneous for further assays and uses. Standard protein
purification methods known in the art can be employed. The
following procedures are exemplary of suitable purification
procedures: fractionation on immunoaffinity or ion-exchange
columns, ethanol precipitation, reverse phase HPLC, chromatography
on silica or on a cation-exchange resin such as DEAE,
chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, and gel
filtration using, for example, Sephadex G-75.
[0109] In one aspect, Protein A immobilized on a solid phase is
used for immunoaffinity purification of the full length antibody
products of the invention. Protein A is a 41 kD cell wall protein
from Staphylococcus aureas which binds with a high affinity to the
Fc region of antibodies. Lindmark et al (1983) J. Immunol. Meth.
62:1-13. The solid phase to which Protein A is immobilized is
preferably a column comprising a glass or silica surface, more
preferably a controlled pore glass column or a silicic acid column.
In some applications, the column has been coated with a reagent,
such as glycerol, in an attempt to prevent nonspecific adherence of
contaminants.
[0110] As the first step of purification, the preparation derived
from the cell culture as described above is applied onto the
Protein A immobilized solid phase to allow specific binding of the
antibody of interest to Protein A. The solid phase is then washed
to remove contaminants non-specifically bound to the solid phase.
Finally the antibody of interest is recovered from the solid phase
by elution.
[0111] 2. Therapies Using Inhibitors of SCF
[0112] Inhibiting SCF finds use in therapies to treat disease.
Accordingly, provided herein are therapies comprising inhibiting
SCF to benefit individuals suffering from disease. In particular,
as shown herein, disease states involving fibrosis and tissue
remodeling demonstrate aberrant SCF activity. For example,
fibroblasts isolated from diseased individuals with fibrotic or
tissue remodeling phenotypes directly respond to SCF, which results
in the generation 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 inflammation 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 course. Indications that can benefit from therapy
inhibiting SCF include, but are not limited to, idiopathic
pulmonary fibrosis, pulmonary arterial hypertension (PAH), 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 fibrosis, fibrous
histiocytoma, fibrothorax, hepatic fibrosis, fibromyalgia, gingival
fibrosis, and radiation-induced fibrosis.
[0113] 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
associated 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 technology
provided herein deliver the SCF inhibitor via the airway by
intranasal administration. Such administration allows direct
delivery of the therapeutic agent to target tissues in pulmonary
diseases involving fibrosis and tissue remodeling, rather than
relying on systemic delivery via an orally administered
composition.
[0114] In certain embodiments, a physiologically appropriate
solution containing an effective concentration of an antibody
specific for SCF can be administered topically, intraocularly,
parenterally, orally, intranasally, intravenously, intramuscularly,
subcutaneously, or by any other effective means. In particular, the
antibody may delivered into an airway of a subject by intranasal
administration. Alternatively, a tissue can receive a
physiologically appropriate composition (e.g., a solution such as a
saline or phosphate buffer, a suspension, or an emulsion, which is
sterile) containing an effective 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 admistration. 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 manipulations have in common the goal of placing an effective
concentration of an antibody specific for SCF in sufficient contact
with the target tissue to permit the antibody specific for SCF to
contact the tissue.
[0115] With respect to administration 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 recognizes 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 desired therapeutic goal.
[0116] 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 subject 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
factors as weight, diet, concurrent medication, and other factors
that those skilled in the art will recognize.
[0117] In some embodiments, a SCF inhibitor (e.g., an antibody
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 generally 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).
[0118] Pharmaceutical compositions preferably comprise one or more
antibodies as described herein associated with one or more
pharmaceutically acceptable carriers, diluents, or excipients.
Pharmaceutically acceptable carriers are known in the art such as
those described in, for example, Remingtons Pharmaceutical
Sciences, Mack Publishing Co. (A. R. Gennaro ed., 1985).
[0119] For administration by inhalation, the compounds are
delivered in the form of an aerosol spray or dry powder from
pressured container or dispenser which contains a suitable
propellant, e.g., a gas such as carbon dioxide, or a nebulizer.
Compositions for inhalation or insufflation include solutions and
suspensions in pharmaceutically acceptable, aqueous or organic
solvents, or mixtures thereof, and powders. The liquid or solid
compositions may contain suitable pharmaceutically acceptable
excipients as described supra. In some embodiments, the
compositions are administered by the oral or nasal respiratory
route for local or systemic effect. Compositions in can be
nebulized by use of inert gases. Nebulized solutions may be
breathed directly from the nebulizing device or the nebulizing
device can be attached to a face masks tent, or intermittent
positive pressure breathing machine. Solution, suspension, or
powder compositions can be administered orally or nasally from
devices which deliver the formulation in an appropriate manner.
[0120] 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 subject. In other embodiments, multiple
doses are administered 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, 11, 12, or more months or years; e.g., for the
lifetime of the subject). In such embodiments, compounds may be
taken on a regular scheduled basis (e.g., daily, weekly, etc.) for
the duration of the extended period.
[0121] In some embodiments, a SCF inhibitor (e.g., an antibody
specific for SCF) according to the technology provided herein is
co-administered with another compound or more than one other
compound (e.g., 2 or 3 or more other compounds). Examples include,
but are not limited to, immunosuppressive therapy such as
corticosteroids and immunosuppressants, such as cyclophosphamide,
azathioprine, methotrexate, penicillamine, and cyclosporine.
[0122] 3. Kits
[0123] Some embodiments provide herein kits for the treatment of a
subject. In some embodiments, the kits include an antibody that
binds to SCF and appropriate solutions and buffers. Embodiments
include all controls and instructions for use. In some embodiments,
kits include delivery systems (e.g., injectors, inhalers,
nebulizers, etc.).
EXAMPLES
Example 1
Isolation and Sequencing of Monoclonal Antibodies to SCF
A. Methods
[0124] Total RNA was extracted from fresh hybridoma cells recovered
by GenScript and cDNA was synthesized from the RNA. RT-PCR was then
performed to amplify the variable regions (heavy and light chains)
of the antibody, which were then cloned into a standard cloning
vector separately and sequenced.
[0125] Hybridoma cells recovered by GenScript; TRIzol.RTM. Plus RNA
Purification System (Invitrogen, Cat. No.: 15596-026);
SuperScript.TM. III First-Strand Synthesis System (Invitrogen, Cat.
No.: 18080-051).
[0126] Total RNA was isolated from the hybridoma cells following
the technical manual of TRIzol.RTM. Plus RNA Purification System.
The total RNA was analyzed by agarose gel electrophoresis.
[0127] Total RNA was reverse transcribed into cDNA using
isotype-specific anti-sense primers or universal primers following
the technical manual of SuperScript.TM. III First-Strand Synthesis
System. The antibody fragments of VH and VL were amplified
according to the standard operating procedure of RACE of
GenScript.
[0128] Amplified antibody fragments were separately cloned into a
standard cloning vector using standard molecular cloning
procedures.
[0129] Colony PCR screening was performed to identify clones with
inserts of correct sizes. No less than five single colonies with
inserts of correct sizes were sequenced for each antibody
fragment.
B. Results
[0130] Five single colonies with correct VH and VL insert sizes
were sequenced. The VH and VL genes of five different clones were
found nearly identical. The consensus sequence, listed below, is
the sequence of the antibody produced by the hybridoma 2G8D3.
TABLE-US-00001 Heavy chain: DNA sequence (402 bp; SEQ ID NO: 1)
Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGACAGGCTTACTTCTTCATTCCTGCTGCTGATTGTCCCTGCATATGT
CTTATCCCAAGTTTCTCTAAAAGAGTCTGGCCCTGGGATATTGAGGCCCT
CACAGACCCTCATTCTGACTTGTTCTTTCTCTGGGTTTTCACTGAGTACT
TCTGGTATGGGTGTGGGCTGGATTCGTCAGCCTTCAGGGAAGGGTCTGGA
GTGGCTGGCACACATTTGGTGGGATGATGAGAAGTCCTATAACCCATCCC
TGAAGAGCCGGCTCACGATCTCCAAGGATGCCTCCCGAGACCAGGTTTTC
CTCAAGATCACCAATGTGGACACTACAGATACTGCCACTTACTTCTGTGC
TCGAAGCGGCTTGGACTACTGGGGTCAAGGAATTTCAGTCACCGTCTCCT CA Heavy chain:
Amino acids sequence (134 AA; SEQ ID NO: 2) Leader
sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MDRLTSSFLLLIVPAYVLSQVSLKESGPGILRPSQTLILTCSFSGFSLST
SGMGVGWIRQPSGKGLEWLAHIWWDDEKSYNPSLKSRLTISKDASRDQVF
LKITNVDTTDTATYFCARSGLDYWGQGISVTVSS Light chain: DNA sequence (402
bp; SEQ ID NO: 3) Leader sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGCCTGGACTCCTCTCTTCTTCTTCTTTGTTCTTCATTGCTCAGGTTC
TTTCTCCCAACCTGTGCTCACTCAGTCATCTTCAGCCTCTTTCTCCCTGG
GAGCCTCAGCAAAAATCACGTGCACCTTGAGTAGTCAGCACAGGACGTAC
ACCATTGAATGGTATCAGCAACAGCCACTCAAGCCTCCTAAGTATGTGAT
GGAACTTAAGAGAGATGGAAGTCACAGAACAGGTGATGGGATTCCTGATC
GCTTCTCTGGATCCAGCTCTGGTGCTGATCGCTACCTAACCATTGCCAAC
ATCCAGCCTGAAGATGAAGCAATGTACATCTGTGGTGCTGATGATACAAT
TCAGGAACAATTTGTGTATGTTTTCGGCGGTGGAACCAAAGTCACTGTCC TC Light chain:
Amino acids sequence (134 AA; SEQ ID NO: 4) Leader
sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MAWTPLFFFFVLHCSGSFSQPVLTQSSSASFSLGASAKITCTLSSQHRTY
TIEWYQQQPLKPPKYVMELKRDGSHRTGDGIPDRFSGSSSGADRYLTIAN
IQPEDEAMYICGADDTIQEQFVYVFGGGTKVTVL
[0131] All publications and patents mentioned in the above
specification are herein incorporated by reference in their
entirety for all purposes. Various modifications and variations of
the described compositions, methods, and uses of the technology
will be apparent to those skilled in the art without departing from
the scope and spirit of the technology as described. Although the
technology has been described in connection with specific exemplary
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.
Sequence CWU 1
1
41402DNAHomo sapiens 1atggacaggc ttacttcttc attcctgctg ctgattgtcc
ctgcatatgt cttatcccaa 60gtttctctaa aagagtctgg ccctgggata ttgaggccct
cacagaccct cattctgact 120tgttctttct ctgggttttc actgagtact
tctggtatgg gtgtgggctg gattcgtcag 180ccttcaggga agggtctgga
gtggctggca cacatttggt gggatgatga gaagtcctat 240aacccatccc
tgaagagccg gctcacgatc tccaaggatg cctcccgaga ccaggttttc
300ctcaagatca ccaatgtgga cactacagat actgccactt acttctgtgc
tcgaagcggc 360ttggactact ggggtcaagg aatttcagtc accgtctcct ca
4022134PRTHomo sapiens 2Met Asp Arg Leu Thr Ser Ser Phe Leu Leu Leu
Ile Val Pro Ala Tyr1 5 10 15Val Leu Ser Gln Val Ser Leu Lys Glu Ser
Gly Pro Gly Ile Leu Arg 20 25 30Pro Ser Gln Thr Leu Ile Leu Thr Cys
Ser Phe Ser Gly Phe Ser Leu 35 40 45Ser Thr Ser Gly Met Gly Val Gly
Trp Ile Arg Gln Pro Ser Gly Lys 50 55 60Gly Leu Glu Trp Leu Ala His
Ile Trp Trp Asp Asp Glu Lys Ser Tyr65 70 75 80Asn Pro Ser Leu Lys
Ser Arg Leu Thr Ile Ser Lys Asp Ala Ser Arg 85 90 95Asp Gln Val Phe
Leu Lys Ile Thr Asn Val Asp Thr Thr Asp Thr Ala 100 105 110Thr Tyr
Phe Cys Ala Arg Ser Gly Leu Asp Tyr Trp Gly Gln Gly Ile 115 120
125Ser Val Thr Val Ser Ser 1303402DNAHomo sapiens 3atggcctgga
ctcctctctt cttcttcttt gttcttcatt gctcaggttc tttctcccaa 60cctgtgctca
ctcagtcatc ttcagcctct ttctccctgg gagcctcagc aaaaatcacg
120tgcaccttga gtagtcagca caggacgtac accattgaat ggtatcagca
acagccactc 180aagcctccta agtatgtgat ggaacttaag agagatggaa
gtcacagaac aggtgatggg 240attcctgatc gcttctctgg atccagctct
ggtgctgatc gctacctaac cattgccaac 300atccagcctg aagatgaagc
aatgtacatc tgtggtgctg atgatacaat tcaggaacaa 360tttgtgtatg
ttttcggcgg tggaaccaaa gtcactgtcc tc 4024134PRTHomo sapiens 4Met Ala
Trp Thr Pro Leu Phe Phe Phe Phe Val Leu His Cys Ser Gly1 5 10 15Ser
Phe Ser Gln Pro Val Leu Thr Gln Ser Ser Ser Ala Ser Phe Ser 20 25
30Leu Gly Ala Ser Ala Lys Ile Thr Cys Thr Leu Ser Ser Gln His Arg
35 40 45Thr Tyr Thr Ile Glu Trp Tyr Gln Gln Gln Pro Leu Lys Pro Pro
Lys 50 55 60Tyr Val Met Glu Leu Lys Arg Asp Gly Ser His Arg Thr Gly
Asp Gly65 70 75 80Ile Pro Asp Arg Phe Ser Gly Ser Ser Ser Gly Ala
Asp Arg Tyr Leu 85 90 95Thr Ile Ala Asn Ile Gln Pro Glu Asp Glu Ala
Met Tyr Ile Cys Gly 100 105 110Ala Asp Asp Thr Ile Gln Glu Gln Phe
Val Tyr Val Phe Gly Gly Gly 115 120 125Thr Lys Val Thr Val Leu
130
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