U.S. patent application number 12/817718 was filed with the patent office on 2010-11-11 for finger-1 peptide analogs of the tgf-beta superfamily.
This patent application is currently assigned to Wyeth LLC. Invention is credited to Christopher Todd Brown, Wei Liu, Zhijian Lu, Stephane H. Olland, Emily Sheng-ming Shen, Paul John Yaworsky, Jimin Zhang.
Application Number | 20100286071 12/817718 |
Document ID | / |
Family ID | 39197416 |
Filed Date | 2010-11-11 |
United States Patent
Application |
20100286071 |
Kind Code |
A1 |
Lu; Zhijian ; et
al. |
November 11, 2010 |
Finger-1 Peptide Analogs of the TGF-Beta Superfamily
Abstract
Members of the TGF-.beta. superfamily and peptide fragments
based on member proteins are employed to purify solutions
containing member proteins or as therapeutics.
Inventors: |
Lu; Zhijian; (Bedford,
MA) ; Liu; Wei; (Lexington, MA) ; Zhang;
Jimin; (Chestnut Hill, MA) ; Yaworsky; Paul John;
(Boston, MA) ; Olland; Stephane H.; (Arlington,
MA) ; Brown; Christopher Todd; (Lowell, MA) ;
Shen; Emily Sheng-ming; (West Chester, PA) |
Correspondence
Address: |
WYETH LLC;PATENT LAW GROUP
5 GIRALDA FARMS
MADISON
NJ
07940
US
|
Assignee: |
Wyeth LLC
Madison
NJ
|
Family ID: |
39197416 |
Appl. No.: |
12/817718 |
Filed: |
June 17, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
11809383 |
Jun 1, 2007 |
7754689 |
|
|
12817718 |
|
|
|
|
60810798 |
Jun 2, 2006 |
|
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Current U.S.
Class: |
514/21.3 ;
514/21.4; 530/324; 530/325; 530/326; 530/417 |
Current CPC
Class: |
C07K 1/22 20130101; A61P
17/02 20180101; C07K 14/51 20130101; C07K 14/475 20130101; A61K
38/00 20130101; C07K 14/495 20130101 |
Class at
Publication: |
514/21.3 ;
530/325; 530/326; 530/324; 530/417; 514/21.4 |
International
Class: |
A61K 38/16 20060101
A61K038/16; C07K 14/00 20060101 C07K014/00; C07K 7/08 20060101
C07K007/08; C07K 1/16 20060101 C07K001/16; A61K 38/10 20060101
A61K038/10; A61P 17/02 20060101 A61P017/02 |
Claims
1. An isolated polypeptide consisting of an active fragment of at
least 8 consecutive amino acid residues of an amino acid sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11; SEQ
ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17,
SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23, wherein the polypeptide has the ability to
bind to and stabilize a TGF-.beta. superfamily member protein in
solution.
2. An isolated polypeptide consisting of an amino acid sequence
selected from the group consisting of SEQ ID NO:1, SEQ ID NO:4, SEQ
ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11; SEQ
ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:17,
SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID
NO:22, SEQ ID NO:23.
3. An isolated polypeptide consisting of an amino acid sequence
having at least 75% identity to the polypeptide of claim 2, wherein
the polypeptide has the ability to bind to and stabilize a
TGF-.beta. superfamily member protein in solution.
4. A composition comprising the polypeptide of claim 1 and a
pharmaceutically acceptable carrier.
5. The composition of claim 4, wherein the carrier is selected from
the group consisting of at least one of solvent, gel, polymer,
demineralized bone, suture, surgical mesh, ceramic, micelle,
buffer, collagen, collagen sponge, and cellulose.
6. A chromatography media comprising the polypeptide of claim
1.
7. A method of purifying a TGF-.beta. superfamily member protein
from a sample comprising: (a) providing the polypeptide of claim 1
immobilized on a chromatography medium; (b) incubating the medium
with the sample; and (c) eluting the protein from the medium.
8. The method of claim 7, wherein the TGF-.beta. superfamily member
protein is a bone morphogenetic protein (BMP) or a growth
differentiation factor (GDF).
9. The method of claim 8, wherein the BMP is BMP-2.
10. A method of stabilizing a solution containing a TGF-.beta.
superfamily member protein comprising adding a polypeptide of claim
1 to the solution, wherein the ratio of the polypeptide to the
protein is at least about 1:1, 2:1, 5:1, 10:1, 20:1, or 30:1.
11. The method of claim 10, wherein the TGF-.beta. superfamily
member protein is a bone morphogenetic protein (BMP) or a growth
differentiation factor (GDF).
12. The method of claim 11, wherein the BMP is BMP-2.
Description
[0001] This application is a continuation of U.S. application Ser.
No. 11/809,383, filed Jun. 1, 2007, now allowed, which claims
priority to U.S. Provisional Application No. 60,810,798, filed Jun.
2, 2006. The contents of each of the above-referenced applications
are incorporated herein in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the use of peptides and proteins
of the TGF-.beta. superfamily and their mutants.
BACKGROUND OF THE INVENTION
[0003] Natural regulators of cellular growth, differentiation and
function have provided important pharmaceuticals, clinical and
laboratory tools, and targets for therapeutic intervention. A
variety of such regulators have been shown to have profound effects
on basic cellular differentiation and developmental pathways. The
transforming growth factor beta (TGF-.beta.) superfamily is a large
family of multifunctional proteins that regulate a variety of
cellular functions including cellular proliferation, migration,
differentiation and apoptosis. TGF-.beta., the founding member, has
been shown to play a variety of roles ranging from embryonic
pattern formation to cell growth regulation in adult tissues.
TGF-.beta. exerts its biological functions by signal transduction
cascades that ultimately activate and/or suppress expression of a
set of specific genes. Other TGF-.beta. superfamily members include
the TGF-.beta. family, growth differentiation factors (GDFs),
activins, inhibins, Bone Morphogenic Proteins (BMPs), and other
related ligands. BMP-mediated signal transduction is important for
a variety of normal processes, including bone growth and the
function of the nervous system, eyes and organs such as kidneys.
BMPs have diverse biological activities in different biological
contexts, including the induction of cartilage, bone and connective
tissue, and roles in kidney, tooth, gut, skin and hair
development.
[0004] BMPs can be produced in the laboratory, however, there are
few convenient procedures for purification of these materials and
development of purification methods has often been ad hoc and time
consuming, with purification processes sometimes taking up to 6
months to develop. Accordingly it is desired to have more efficient
methods for purifying BMPs and other members of the TGF-.beta.
superfamily.
DEFINITIONS
[0005] As used herein and in the appended claims, the singular
forms "a," "an," and "the" include the plural reference unless the
context clearly indicates otherwise. Thus, for example, a reference
to "a cell" includes a plurality of such cells, and a reference to
"an antibody" is a reference to one or more antibodies and
equivalents thereof known to those skilled in the art, and so
forth.
[0006] The terms "polynucleotide", "nucleotide sequence", "nucleic
acid", "nucleic acid molecule", "nucleic acid sequence", and
"oligonucleotide" refer to a series of nucleotide bases (also
called "nucleotides") in DNA and RNA, and mean any chain of two or
more nucleotides. The polynucleotides can be chimeric mixtures or
derivatives or modified versions thereof, single-stranded or
double-stranded. The oligonucleotide can be modified at the base
moiety, sugar moiety, or phosphate backbone, for example, to
improve stability of the molecule, its hybridization parameters,
etc. The antisense oligonucleotide may comprise a modified base
moiety which is selected from the group including but not limited
to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine,
5-(carboxyhydroxylmethyl)-uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethyl-aminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methyl-guanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N6-adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, wybutoxosine,
pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,
2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid
methylester, uracil-5-oxyacetic acid, 5-methyl-2-thiouracil,
3-(3-amino-3-N-2-carboxypropyl)uracil, and 2,6-diaminopurine. A
nucleotide sequence typically carries genetic information,
including the information used by cellular machinery to make
proteins and enzymes. These terms include double- or
single-stranded genomic and cDNA, RNA, any synthetic and
genetically manipulated polynucleotide, and both sense and
antisense polynucleotides. This includes single- and
double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNA
hybrids, as well as "protein nucleic acids" (PNA) formed by
conjugating bases to an amino acid backbone. This also includes
nucleic acids containing modified bases, for example, thio-uracil,
thio-guanine, and fluoro-uracil, or containing carbohydrate, or
lipids.
[0007] Polynucleotides for use with embodiments of the invention
may be synthesized by standard methods known in the art, e.g., by
use of an automated DNA synthesizer (such as those that are
commercially available from Biosearch, Applied Biosystems, etc.).
As examples, phosphorothioate oligonucleotides may be synthesized
by the method of Stein et al., Nucl. Acids Res., 16, 3209, (1988),
methylphosphonate oligonucleotides can be prepared by use of
controlled pore glass polymer supports (Sarin et al., Proc. Natl.
Acad. Sci. U.S.A. 85, 7448-7451, (1988)), etc. A number of methods
have been developed for delivering antisense DNA or RNA to cells,
e.g., antisense molecules can be injected directly into the tissue
site, or modified antisense molecules, designed to target the
desired cells (antisense linked to peptides or antibodies that
specifically bind receptors or antigens expressed on the target
cell surface) can be administered systemically. Alternatively, RNA
molecules may be generated by in vitro and in vivo transcription of
DNA sequences encoding the antisense RNA molecule. Such DNA
sequences may be incorporated into a wide variety of vectors that
incorporate suitable RNA polymerase promoters such as the T7 or SP6
polymerase promoters. Alternatively, antisense cDNA constructs that
synthesize antisense RNA constitutively or inducibly, depending on
the promoter used, can be introduced stably into cell lines.
However, it is often difficult to achieve intracellular
concentrations of the antisense sufficient to suppress translation
of endogenous mRNAs. Therefore a preferred approach utilizes a
recombinant DNA construct in which the antisense oligonucleotide is
placed under the control of a strong promoter. The use of such a
construct to transfect target cells in the patient will result in
the transcription of sufficient amounts of single stranded RNAs
that will form complementary base pairs with the endogenous target
gene transcripts and thereby prevent translation of the target gene
mRNA. For example, a vector can be introduced in vivo such that it
is taken up by a cell and directs the transcription of an antisense
RNA. Such a vector can remain episomal or become chromosomally
integrated, as long as it can be transcribed to produce the desired
antisense RNA. Such vectors can be constructed by recombinant DNA
technology methods standard in the art. Vectors can be plasmid,
viral, or others known in the art, used for replication and
expression in mammalian cells. Expression of the sequence encoding
the antisense RNA can be by any promoter known in the art to act in
mammalian, preferably human cells. Such promoters can be inducible
or constitutive. Such promoters include but are not limited to: the
SV40 early promoter region (Bernoist and Chambon, Nature, 290,
304-310, (1981), the promoter contained in the 3' long terminal
repeat of Rous sarcoma virus, Yamamoto et al., Cell, 22, 787-797,
(1980), the herpes thymidine kinase promoter, Wagner et al., Proc.
Natl. Acad. Sci. U.S.A. 78, 1441-1445, (1981), the regulatory
sequences of the metallothionein gene Brinster et al., Nature 296,
39-42, (1982), etc. Any type of plasmid, cosmid, yeast artificial
chromosome or viral vector can be used to prepare the recombinant
DNA construct that can be introduced directly into the tissue site.
Alternatively, viral vectors can be used which selectively infect
the desired tissue, in which case administration may be
accomplished by another route (e.g., systemically).
[0008] The polynucleotides may be flanked by natural regulatory
(expression control) sequences, or may be associated with
heterologous sequences, including promoters, internal ribosome
entry sites (IRES) and other ribosome binding site sequences,
enhancers, response elements, suppressors, signal sequences,
polyadenylation sequences, introns, 5'- and 3'-non-coding regions,
and the like. The nucleic acids may also be modified by many means
known in the art. Non-limiting examples of such modifications
include methylation, "caps", substitution of one or more of the
naturally occurring nucleotides with an analog, and internucleotide
modifications such as, for example, those with uncharged linkages
(e.g., methyl phosphonates, phosphotriesters, phosphoroamidates,
carbamates, etc.) and with charged linkages (e.g.,
phosphorothioates, phosphorodithioates, etc.). Polynucleotides may
contain one or more additional covalently linked moieties, such as,
for example, proteins (e.g., nucleases, toxins, antibodies, signal
peptides, poly-L-lysine, etc.), intercalators (e.g., acridine,
psoralen, etc.), chelators (e.g., metals, radioactive metals, iron,
oxidative metals, etc.), and alkylators. The polynucleotides may be
derivatized by formation of a methyl or ethyl phosphotriester or an
alkyl phosphoramidate linkage. Furthermore, the polynucleotides
herein may also be modified with a label capable of providing a
detectable signal, either directly or indirectly. Exemplary labels
include radioisotopes, fluorescent molecules, biotin, and the
like.
[0009] Identity" or "similarity", as known in the art, are
relationships between two or more polypeptide or two or more
polynucleotide sequences as determined by comparing the sequences.
In the art, identity also means the degree of sequence relatedness
between polypeptide sequences as determined by the match between
strings of such sequences. Both identity and similarity can be
readily calculated by known methods such as those described in:
Computational Molecular Biology, Lesk, A. M., ed., Oxford
University Press, New York, 1988; Biocomputing: Informatics and
Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Sequence Analysis in Molecular Biology, von Heinje, G., Academic
Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin,
A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994;
and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds.,
M Stockton Press, New York, 1991. Methods commonly employed to
determine identity or similarity between sequences include, but are
not limited to, those disclosed in Carillo, H., and Lipman, D.,
SIAM J Applied Math., 48:1073 (1988). Methods to determine identity
and similarity are codified in publicly available computer
programs. Preferred computer program methods to determine identity
and similarity between two sequences include, but are not limited
to, GCG program package, Devereux, J., et al., Nucleic Acids
Research, 12(1), 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul,
S. F. et al., J Molec. Biol., 215, 403 (1990)).
[0010] "Homologous" refers to the degree of sequence similarity
between two polymers (i.e. polypeptide molecules or nucleic acid
molecules). The homology percentage figures referred to herein
reflect the maximal homology possible between the two polymers,
i.e., the percent homology when the two polymers are so aligned as
to have the greatest number of matched (homologous) positions.
[0011] The term "percent homology" refers to the extent of amino
acid sequence identity between polypeptides. The homology between
any two polypeptides is a direct function of the total number of
matching amino acids at a given position in either sequence, e.g.,
if half of the total number of amino acids in either of the
sequences are the same then the two sequences are said to exhibit
50% homology.
[0012] The term "fragment", "analog", and "derivative" when
referring to polypeptides refers to a polypeptide which may retain
essentially the same biological function or activity as the
original polypeptide. Thus, an analog may include a precursor
protein that can be activated by cleavage of the precursor protein
portion to produce an active mature polypeptide. The fragment,
analog, or derivative of the polypeptide may be one in which one or
more of the amino acids are substituted with conserved or
non-conserved amino acid residues and such amino acid residues may
or may not be the ones encoded by the genetic code, or the ones in
which one or more of the amino acid residues include a substituent
group, or the ones in which the polypeptide is fused with a
compound such as polyethylene glycol to increase the half-life of
the polypeptide, or the ones in which additional amino acids are
fused to the polypeptide such as a signal peptide or a sequence
such as polyhistidine tag which is employed for the purification of
the polypeptide or the precursor protein. Such fragments, analogs,
or derivatives are deemed to be within the scope of the present
invention.
[0013] The term "polypeptide" refers to a polymer of amino acids
without regard to the length of the polymer; thus, "peptides,"
"oligopeptides", and "proteins" are included within the definition
of polypeptide and used interchangeably herein. This term also does
not specify or exclude chemical or post-expression modifications of
the polypeptides of the invention, although chemical or
post-expression modifications of these polypeptides may be included
or excluded as specific embodiments. Therefore, for example,
modifications to polypeptides that include the covalent attachment
of glycosyl groups, acetyl groups, phosphate groups, lipid groups
and the like are expressly encompassed by the term polypeptide.
Further, polypeptides with these modifications may be specified as
individual species to be included or excluded from the present
invention. The natural or other chemical modifications, such as
those listed in examples above can occur anywhere in a polypeptide,
including the peptide backbone, the amino acid side-chains and the
amino or carboxyl termini. It will be appreciated that the same
type of modification may be present in the same or varying degrees
at several sites in a given polypeptide. Also, a given polypeptide
may contain many types of modifications. Polypeptides may be
branched, for example, as a result of ubiquitination, and they may
be cyclic, with or without branching. Modifications include
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
cross-links, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, pegylation, proteolytic processing, phosphorylation,
prenylation, racemization, selenoylation, sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginylation,
and ubiquitination. (See, for instance, proteins--structure and
molecular properties, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993); posttranslational covalent modification
of proteins, b. c. Johnson, Ed., Academic Press, New York, pgs.
1-12, 1983; Seifter et al., Meth Enzymol 182:626-646, 1990; Rattan
et al., Ann NY Acad Sci 663:48-62, 1992). Also included within the
definition are polypeptides which contain one or more analogs of an
amino acid (including, for example, non-naturally occurring amino
acids, amino acids which only occur naturally in an unrelated
biological system, modified amino acids from mammalian systems,
stereoisomers of various amino acids, etc.), polypeptides with
substituted linkages, as well as other modifications known in the
art, both naturally occurring and non-naturally occurring. The term
"polypeptide" may also be used interchangeably with the term
"protein" or"peptide".
[0014] The term "finger-1 peptide analog" refers to an oligopeptide
that is at least 75% homologous to a portion of the finger-1 region
of a member of the TGF-beta superfamily. In some embodiments, the
finger-1 peptide analog is at least 80%, at least 85%, at least
90%, or at least 95% homologous to the wild type sequence. In
certain embodiments, the oligopeptide includes at least 8 amino
acid residues, at least 16 amino acid residues, or at least 24
amino acid residues. Finger-1 peptide analogs may be mutants in
which one or more amino acids have been altered or deleted and may
include non-natural or modified amino acid residues.
[0015] The term "peptide" refers to any polymer of two or more
amino acids, wherein each amino acid is linked to one or two other
amino acids via a peptide bond (--CONH--) formed between the
NH.sub.2 and the COOH groups of adjacent amino acids. In one
embodiment, the amino acids are naturally occurring amino acids,
particularly alpha-amino acids of the L-enantiomeric form. However,
other amino acids, enantiomeric forms, and amino acid derivatives
may be included in a peptide. Peptides include "polypeptides,"
which, upon hydrolysis, yield more than two amino acids.
Polypeptides may include proteins, which typically comprise 50 or
more amino acids.
[0016] The polypeptides according to embodiments of the present
invention may be provided in an isolated form, and may be purified
to homogeneity. The polypeptides and polynucleotides in certain
instances are at least 90% pure, at least 95% pure, at least 98%
pure, or at least 99% pure.
[0017] The term "polypeptide" refers to a polymer of amino acids
without regard to the length of the polymer; thus, "peptides,"
"oligopeptides", and "proteins" are included within the definition
of polypeptide and used interchangeably herein. This term also does
not specify or exclude chemical or post-expression modifications of
the polypeptides of the invention, although chemical or
post-expression modifications of these polypeptides may be included
or excluded as specific embodiments. Therefore, for example,
modifications to polypeptides that include the covalent attachment
of glycosyl groups, acetyl groups, phosphate groups, lipid groups
and the like are expressly encompassed by the term polypeptide.
Further, polypeptides with these modifications may be specified as
individual species to be included or excluded from the present
invention. The natural or other chemical modifications, such as
those listed in examples above can occur anywhere in a polypeptide,
including the peptide backbone, the amino acid side-chains and the
amino or carboxyl termini. It will be appreciated that the same
type of modification may be present in the same or varying degrees
at several sites in a given polypeptide. Also, a given polypeptide
may contain many types of modifications. Polypeptides may be
branched, for example, as a result of ubiquitination, and they may
be cyclic, with or without branching. Modifications include
acetylation, acylation, ADP-ribosylation, amidation, covalent
attachment of flavin, covalent attachment of a heme moiety,
covalent attachment of a nucleotide or nucleotide derivative,
covalent attachment of a lipid or lipid derivative, covalent
attachment of phosphotidylinositol, cross-linking, cyclization,
disulfide bond formation, demethylation, formation of covalent
cross-links, formation of cysteine, formation of pyroglutamate,
formylation, gamma-carboxylation, glycosylation, GPI anchor
formation, hydroxylation, iodination, methylation, myristoylation,
oxidation, pegylation, proteolytic processing, phosphorylation,
prenylation, racemization, selenoylation, sulfation, transfer-RNA
mediated addition of amino acids to proteins such as arginylation,
and ubiquitination. (See, for instance, proteins--structure and
molecular properties, 2nd Ed., T. E. Creighton, W. H. Freeman and
Company, New York (1993); posttranslational covalent modification
of proteins, b. c. Johnson, Ed., Academic Press, New York, pgs.
1-12, 1983; Seifter et al., Meth Enzymol 182:626-646, 1990; Rattan
et al., Ann NY Acad Sci 663:48-62, 1992). Also included within the
definition are polypeptides which contain one or more analogs of an
amino acid (including, for example, non-naturally occurring amino
acids, amino acids which only occur naturally in an unrelated
biological system, modified amino acids from mammalian systems
etc.), polypeptides with substituted linkages, as well as other
modifications known in the art, both naturally occurring and
non-naturally occurring. The term "polypeptide" may also be used
interchangeably with the term "protein" or "peptide".
[0018] The term "peptide" refers to any polymer of two or more
amino acids, wherein each amino acid is linked to one or two other
amino acids via a peptide bond (--CONH--) formed between the NH2
and the COOH groups of adjacent amino acids. Preferably, the amino
acids are naturally occurring amino acids, particularly
.alpha.-amino acids of the L-enantiomeric form. However, other
amino acids, enantiomeric forms, and amino acid derivatives may be
included in a peptide. Peptides include "polypeptides," which, upon
hydrolysis, yield more than two amino acids. Polypeptides may
include proteins, which typically comprise 50 or more amino
acids.
[0019] The term "isolated" means that the material is removed from
its original or native environment (e.g., the natural environment
if it is naturally occurring). Therefore, a naturally-occurring
polypeptide present in a living animal is not isolated, but the
same polypeptide, separated by human intervention from some or all
of the coexisting materials in the natural system, is isolated.
Polypeptides could be part of a composition, and still be isolated
in that such vector or composition is not part of the environment
in which it is found in nature. Similarly, the term "substantially
purified" refers to a substance, which has been separated or
otherwise removed, through human intervention, from the immediate
chemical environment in which it occurs in nature. Substantially
purified polypeptides may be obtained or produced by any of a
number of techniques and procedures generally known in the
field.
[0020] The term "purification" refers to increasing the specific
activity or concentration of a particular polypeptide or
polypeptides in a sample. In one embodiment, specific activity is
expressed as the ratio between the activity of the target
polypeptide and the concentration of total polypeptide in the
sample. In another embodiment, specific activity is expressed as
the ratio between the concentration of the target polypeptide and
the concentration of total polypeptide. Purification methods
include but are not limited to dialysis, centrifugation, and column
chromatography techniques, which are well-known procedures to those
of skill in the art. See, e.g., Young et al., 1997, "Production of
biopharmaceutical proteins in the milk of transgenic dairy
animals," BioPharm 10(6): 34-38.
[0021] "Associated with": When two entities are "associated with"
one another as described herein, they are linked by a direct or
indirect covalent or non-covalent interaction. Preferably, the
association is covalent. Desirable non-covalent interactions
include hydrogen bonding, van der Waals interactions, hydrophobic
interactions, magnetic interactions, electrostatic interactions,
etc.
[0022] The term "isolated" means that the material is removed from
its original or native environment (e.g., the natural environment
if it is naturally occurring). Therefore, a naturally-occurring
polynucleotide or polypeptide present in a living animal is not
isolated, but the same polynucleotide or polypeptide, separated by
human intervention from some or all of the coexisting materials in
the natural system, is isolated. For example, an "isolated nucleic
acid fragment" is a polymer of RNA or DNA that is single- or
double-stranded, optionally containing synthetic, non-natural or
altered nucleotide bases. An isolated nucleic acid fragment in the
form of a polymer of DNA may be comprised of one or more segments
of cDNA, genomic DNA or synthetic DNA and combined with
carbohydrate, lipid, protein or other materials. Such
polynucleotides could be part of a vector and/or such
polynucleotides or polypeptides could be part of a composition, and
still be isolated in that such vector or composition is not part of
the environment in which it is found in nature. Similarly, the term
"substantially purified" refers to a substance, which has been
separated or otherwise removed, through human intervention, from
the immediate chemical environment in which it occurs in Nature.
Substantially purified polypeptides or nucleic acids may be
obtained or produced by any of a number of techniques and
procedures generally known in the field.
[0023] The terms "substantially pure" and "isolated" are not
intended to exclude mixtures of polypeptides with substances that
are not associated with the polypeptides in nature
[0024] General methods for expressing and recovering foreign
protein produced by a mammalian cell system are provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard techniques for recovering protein produced by a bacterial
system is provided by, for example, Grisshammer et al.,
"Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Transformation of
insect cells and production of foreign polypeptides therein is
disclosed by Guarino et al., U.S. Pat. No. 5,162,222 and WIPO
publication WO94/06463. Methods for isolating recombinant proteins
from a baculovirus system are also described by Richardson (ed.),
"Baculovirus Expression Protocols" (The Humana Press, Inc. 1995).
In one embodiment, the polypeptides of the invention can be
expressed using a baculovirus expression system (see, Luckow et
al., Bio/Technology, 1988, 6, 47, "Baculovirus Expression Vectors:
a Laboratory Manual", O'Rielly et al. (Eds.), W. H. Freeman and
Company, New York, 1992, U.S. Pat. No. 4,879,236, each of which is
incorporated herein by reference in its entirety). In addition, the
MAXBAC.TM. complete baculovirus expression system (Invitrogen) can,
for example, be used for production in insect cells.
[0025] The polypeptides according to various embodiments of the
present invention can also be isolated by exploitation of
particular properties. For example, immobilized metal ion
adsorption (IMAC) chromatography can be used to purify
histidine-rich proteins, including those comprising polyhistidine
tags. Briefly, a gel is first charged with divalent metal ions to
form a chelate (Sulkowski, Trends in Biochem. 3:1 (1985)).
Histidine-rich proteins will be adsorbed to this matrix with
differing affinities, depending upon the metal ion used, and will
be eluted by competitive elution, lowering the pH, or use of strong
chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (M. Deutscher,
(ed.), Meth. Enzymol. 182:529 (1990)). Within additional
embodiments of the invention, a fusion of the polypeptide of
interest and an affinity tag (e.g., maltose-binding protein, an
immunoglobulin domain) may be constructed to facilitate
purification
[0026] The term "in vitro" refers to an artificial environment and
to reactions or processes that occur within an artificial
environment. In vitro environments 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
reaction that occur within a natural environment.
[0027] The term "phenotype" refers to the observable character of a
cell or an organism. Such observable character can involve the
physical appearance, as well as a level of particular physiological
compositions present in the cell or organism.
[0028] The term "signal transduction pathway" refers to the
molecules that propagate an extracellular signal through the cell
membrane to become an intracellular signal. This signal can then
stimulate a cellular response. The polypeptide molecules involved
in signal transduction processes may be receptor and non-receptor
protein tyrosine kinases.
[0029] "Receptor" refers to a molecular structure within a cell or
on the surface of the cell that is generally characterized by the
selective binding of a specific substance.
[0030] The terms "compound" or "agent" are used interchangeably
herein to refer to a compound or compounds or composition of matter
which, when administered to a subject (human or animal) induces a
desired pharmacologic and/or physiologic effect by local and/or
systemic action.
[0031] The term "subject" refers to any mammal, including a human,
or non-human subject. Non-human subjects can include experimental,
test, agricultural, entertainment or companion animals. A subject
may be a human. A subject may be a domesticated animal, such as a
dog, cat, cow, goat, sheep, pig, etc., A subject may be an
experimental animal, such as a mouse, rat, rabbit, monkey, etc.
[0032] As used herein, the term "small molecule" is used to refer
to molecules, whether naturally-occurring or artificially created
(e.g., via chemical synthesis), that have a relatively low
molecular weight. In many embodiments, small molecules are
monomeric and have a molecular weight of less than about 1500
g/mol. Preferred small molecules are biologically active in that
they produce a local or systemic effect in animals, preferably
mammals, more preferably humans. In certain preferred embodiments,
the small molecule is a drug. Preferably, though not necessarily,
the drug is one that has already been deemed safe and effective for
use by the appropriate governmental agency or body. For example,
drugs for human use listed by the FDA under 21 C.F.R.
.sctn..sctn.330.5, 331 through 361, and 440 through 460; drugs for
veterinary use listed by the FDA under 21 C.F.R. .sctn..sctn.500
through 589, incorporated herein by reference, are all considered
acceptable for use in accordance with the present invention.
SUMMARY OF THE INVENTION
[0033] In one aspect, the invention is a chromatography media
comprising a chromatography resin derivatized with a peptide that
is at least 75% homologous to a portion of a protein that is a
member of the TGF-.beta. superfamily. The member may be a
TGF-.beta., a growth differentiation factor (GDF), a bone
morphogenic protein, an activin, or an inhibin. The peptide may be
a substantially complete molecule of the protein. The peptide may
be at least 75%, at least 80%, at least 85%, at least 90%, or at
least 95% homologous to a portion of a finger-1 peptide from the
member.
[0034] In another aspect, the invention is a method of purifying a
sample including a growth factor that is a member of the TGF-.beta.
superfamily. The method includes providing a chromatography column
containing a chromatography resin derivatized with a peptide that
is at least 75% homologous to a portion of a protein that is a
member of the TGF-.beta. superfamily, loading the column with the
sample under conditions in which the growth factor tends to
aggregate, and eluting the growth factor from the column under
conditions in which the growth factor tends to solubilize. The
peptide need not be a portion of the growth factor.
[0035] In another aspect, the invention is a chromatography media
comprising a chromatography resin derivatized with a peptide that
is at least 75% homologous to a portion of a finger-1 peptide of a
member of the TGF-.beta. superfamily.
[0036] In another aspect, the invention is a solution of bone
morphogenic proteins in a solvent comprising a predetermined
concentration of a predetermined bone morphogenic protein, and a
predetermined concentration of a peptide that is at least 75%
homologous to a portion of a finger-1 peptide of a member of the
TGF-.beta. superfamily. The member of the TGF-.beta. superfamily
may, but need not, be the predetermined bone morphogenic
protein.
[0037] In another aspect, the invention is a method of stabilizing
a solution of bone morphogenic protein. The method includes adding
a predetermined concentration of a peptide that is at least 75%
homologous to a portion of a finger-1 peptide of a member of the
TGF-.beta. superfamily to a solution of the bone morphogenic
protein.
[0038] In another aspect, the invention is a composition including
a peptide that is at least 75% homologous to a portion of a
finger-1 peptide of a member of the TGF-.beta. superfamily to a
solution of the bone morphogenic protein and a carrier. The carrier
may be a gel, a polymer, demineralized bone, a suture, a surgical
mesh, a ceramic, a micelle, or any combination of the above. For
example, the carrier may include a buffer solution, collagen, a
collagen sponge, or a cellulose-based material. Alternatively or in
addition, the carrier may include one or more of alkylcellulose
(including hydroxyalkylcellulose), including methylcellulose,
ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose,
hydroxypropyl-methylcellulose, and carboxymethylcellulose. In some
embodiments, the carrier may include polyglyconate, hyaluronic
acid, polylactic acid, poly(ethylene glycol), sodium alginate,
poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer,
and poly(vinyl alcohol). The peptide may be stabilized by one or
more of a conjugated poly(propylene glycol), a conjugated sialyl
group, a conjugated polysialyl chain, incorporation of modified
amino acids, incorporation of non-natural amino acids, interchain
covalent links, intrachain covalent links, interchain non-covalent
links, interchain non-covalent links, and a presentation
enhancer.
[0039] In another aspect, the invention is an assay method
comprising contacting a population of cells with a peptide that is
at least 75% homologous to a portion of a finger-1 peptide of a
member of the TGF-.beta. superfamily and detecting a change in a
characteristic of the cells when the peptide is present versus when
the peptide is absent. The characteristic may be selected from a
stage in a cell cycle, expression of a gene or protein, a visible
or measurable phenotypic characteristic, a level of expression of a
gene or protein, and the transduction of a signal from a receptor.
The method may further include contacting the cells with a protein
that is a member of the TGF-.beta. superfamily. The peptide may be
constrained in a particular configuration.
[0040] In another aspect, the invention is an assay method
comprising contacting a population of receptors with a known
quantity of a peptide that is at least 75% homologous to a finger-1
peptide of a portion of a member of the TGF-.beta. superfamily and
detecting a change in the quantity of peptide that is not bound to
the receptors. The receptors may be contacted with a protein that
is a member of the TGF-.beta. superfamily
[0041] In another aspect, the invention is a composition comprising
a peptide that is at least 75% homologous to a portion of a
finger-1 peptide of a member of the TGF-.beta. superfamily and an
agent that increases the in vivo stability of the peptide with
respect to elimination from an animal.
[0042] In another aspect, the invention is an assay method
comprising contacting a population of receptors with a known
quantity of a peptide that is at least 75% homologous to a finger-1
peptide of a portion of a member of the TGF-.beta. superfamily and
a small molecule and detecting a change in the quantity of peptide
that is not bound to the receptors when the small molecule is
present versus when the small molecule is absent.
BRIEF DESCRIPTION OF THE DRAWINGS
[0043] The invention is described with reference to the several
figures of the drawing, in which,
[0044] FIG. 1 is a schematic of BMP-2, showing the finger regions
and various helices and .beta.-sheets (Adapted from Scheufler, et
al., J. Mol. Bio. (1999), 287: 103-115
[0045] FIG. 2 is a schematic of an affinity chromatography method
according to an exemplary embodiment of the invention.
[0046] FIG. 3 is a chromatogram (A) and a photograph of an SEC gel
(B) showing the elution of BMP-3 from a column employing
immobilized BMP-12.
[0047] FIG. 4 is a chromatogram (A) and a photograph of an SEC gel
(B) showing the elution of BMP-3 from a column employing
immobilized BMP-2.
[0048] FIG. 5 is a chromatogram (A) and a photograph of an SEC gel
(B) showing the elution of BMP-12 from a column employing
immobilized BMP-12.
[0049] FIG. 6 is a chromatogram (A) and a photograph of an SEC gel
(B) showing the elution of BMP-12 from a column employing
immobilized BMP-2
[0050] FIG. 7 is a set of photographs of SEC gels (A: non-reduced,
B: reduced) showing the elution of various BMPs from reversed phase
HPLC column employing immobilized BMPs.
[0051] FIG. 8 is a chromatogram (A) and a photograph of an SEC gel
(B) showing the elution of BMP-3 from a column employing
immobilized BMP-2 mutant fragments.
[0052] FIG. 9 is a chromatogram (A) and a photograph of an SEC gel
(B) showing the elution of BMP-3 from a column employing
immobilized BMP-12 wild-type fragment.
[0053] FIG. 10 is a chromatogram (A) and a photograph of an SEC gel
(B) showing the elution of BMP-12 from a column employing
immobilized BMP-2 mutant fragments
[0054] FIG. 11 is a chromatogram (A) and a photograph of an SEC gel
(B) showing the elution of BMP-12 from a column employing
immobilized BMP-2 mutant fragments.
[0055] FIG. 12 is a chromatogram (A) and a photograph of an SEC gel
(B) showing the elution of GDF-9 from a column employing
immobilized BMP-2 mutant fragments.
[0056] FIG. 13 is a graph illustrating the turbidity of solutions
of 0.5 mg/mL BMP-12 with respect to concentrations of NaCl and
2-methyl-2,4-pentanediol in 50 mM Tris, pH 8.
[0057] FIG. 14 is a photograph of a gel after SEC of the eluate
from resins conjugated with BMP-finger peptides and loaded with
BMP-12 under various conditions.
DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS
[0058] In certain embodiments, a portion of the first finger
peptide of a BMP is employed to perform affinity chromatography.
The secondary structure of members of the TGF-.beta. superfamily is
typified by two antiparallel .beta.-sheets separated by a four turn
alpha-helix about perpendicular to the strands in the
.beta.-sheets. The second of the two .beta.-sheets has a twisted
crossover conformation. The structure is often stabilized by a
cysteine knot formed as pairs of cysteine residues form intrachain
disulfide bridges. Some members of the TGF-.beta. superfamily are
further stabilized by the formation of dimers or higher order
multimers. FIG. 1 shows the finger regions and the various helices
and .beta.-sheets in an exemplary BMP, BMP-2. For many members of
the TGF-.beta. superfamily, the .beta.-sheets can be subdivided
into nine .beta.-strands. Other features of BMP-2, such as the
alpha-2 helix and the .beta.5a strand, are not found in all members
of the TGF-.beta. superfamily, some of which may exhibit additional
helices and .beta.-strands that are not found in BMP-2 (Scheufler,
et al., J. Mol. Bio. (1999), 287: 103-115).
[0059] Exemplary members of the TGF-.beta. superfamily include but
are not limited to BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8A,
BMP8B/OP2, BMP9/GDF2, BMP10, BMP11/GDF11, BMP12/GDF7, BMP13/GDF6,
BMP15, BMP16/nodal, BMP17/LeftyB, BMP18/Lefty2/TGF .beta.4, .alpha.
inhibin, Inhibin .beta.3A, Inhibin .beta.B, Inhibin .beta.C,
Inhibin .beta.E/BMP14/GDF12, TGF .beta.1, TGF .beta.2, TGF .beta.3,
GDF1, GDF3/Vgr-2, GDF5, GDF8, GDF9, GDF10/BMP3B, GDF15, artemin,
GDNF (glial derived neurotrophic factor), Mullerian duct inhibiting
substance, neuturin, and persephin. While the discussion below
focuses on BMP, the teachings may be applied to any member of the
TGF-.beta. superfamily.
[0060] BMPs tend to aggregate and precipitate at physiological pH
(Ruppert, et al., 1996 Eur J Biochem. 1996, 237(1):295-302). In
some embodiments, this tendency was exploited by linking whole BMPs
or finger-1 peptide analogs derived from their first finger domains
as ligands linked to sepharose beads to produce affinity
chromatography resins.
Chromatography
[0061] FIG. 2 illustrates the principles of affinity purification
according to certain embodiments of the invention. A BMP or
finger-1 peptide analog is immobilized on chromatography media.
Exemplary media include agarose (e.g., Sepharose.TM.), polystyrene,
silica, dextran, and acrylamide. The media are then slurried into a
column according to techniques known to those of skill in the art.
The column is loaded with a solution containing a BMP under
conditions that favor precipitation of BMP. In one embodiment, BMPs
are loaded onto the column from CHO-conditioned media containing
either dextran sulfate or heparin, e.g., 100 micrograms/milliliter
at physiological pH. In some embodiments, the solution may also
include 1.2M NaCl. Other impurities can be washed off the column
while the BMP remains aggregated on the column. The BMP are then
washed off the column under conditions that promote solvation. One
skilled in the art will recognize that the ionic strength and pH of
the loading solvent may be optimized for various combinations of
immobilized finger-1 peptide analogs and materials being
purified.
[0062] In some embodiments, additives may be used to optimize the
solubility or insolubility of the material being purified. For
example, dextran sulfate or heparin may be added to a culture
medium to favor solubilization of the BMP while it is being
expressed and secreted by the cells. Prior to loading onto the
column, Conditioned Medium may be adjusted to a high ionic strength
to promote efficient capture and/or aggregation during the
chromatography process.
[0063] Exemplary BMP solubilizers are known to those of skill in
the art and include but are not limited to acetic acid,
trifluoroacetic acid, hydrochloric acid, alcohols, acetonitrile,
propylene glycol, glycerol, Tween-80, CHAPS
(3-((3-Cholamidopropyl)dimethylammonio)-1-Propanesulfonic Acid),
L-arginine, 6M urea, and 6M Guanidine HCl. One skilled in the art
will recognize that these solvents vary in their aggressiveness
with respect to solubilizing BMP. In certain embodiments, BMP are
eluted with a 500 mM solution of arginine or 50 mM acetic acid.
[0064] Exemplary BMP precipitants are known to those of skill in
the art and include but are not limited to solvents with pH greater
than about 5, salt solutions including, for example, sodium
chloride, phosphate salts, sulfate salts of ammonium and sodium,
and citrate. The concentration of sodium chloride to promote
precipitation depends on the pH of the solution. Some TGF-.beta.
proteins will precipitate in solutions of high salt concentration
and low pH, or vice versa. One skilled in the art will recognize
how to optimize the salt concentration and pH to obtain desired
precipitation characteristics for particular proteins.
[0065] Fragments of BMP finger peptides may be employed instead of
the entire finger region. The particular sequence used for
chromatography or the applications described below may have at
least 8, at least 16, or at least 24 amino acid residues. The
sequence may be at least 75%, at least 80%, at least 85%, at least
90%, or at least 95% homologous to the corresponding region of the
native protein. In some embodiments, mutants may be formed with
serine residues in place of cysteine residues in the native
sequence. This can reduce cross-linking between peptide chains on
the sepharose beads, which may interfere with nucleation of BMP
aggregates.
[0066] Modifications to the peptide sequence may also be made to
improve attachment of the peptide to the substrate. For example,
lysine-terminated peptides will easily attach to the chromatography
beads through formation of amide bonds. If the natural sequence of
the desired peptide is not already lysine-terminated, it may be
modified to add the lysine residue. Alternatively or in addition,
the N-terminal of the peptide may be amidated and the C-terminal
acetylated to ensure that only one end of the peptide will attach
to the substrate bead. In an alternative embodiment, a spacer may
be interposed between the peptide and the substrate bead. The
spacer provides additional conformational flexibility to the
peptide, allowing it more freedom to achieve the optimal secondary
structure for nucleating peptide aggregates. The spacer may be a
simple organic chain or may be a non-binding amino acid sequence.
The sequence may be an oligomer of one or two amino acids or may
exhibit at least partial homology to a non-binding portion of a
protein that is a member of the TGF.beta. superfamily or some other
protein. Alternatively or in addition, a spacer may have a portion
that exhibits partial homology to a portion of a protein that is a
member of the TGF.beta. superfamily and a portion that does not. In
some embodiments, the effectiveness of the bound peptide may vary
depending on whether the free end is the C-terminal or the
N-terminal. Individual amino acids may also be deleted, modified as
described herein or according to other methods known to those of
skill in the art, or replaced.
BMP Solution Stabilizers
[0067] In some embodiments, finger-1 peptide analogs may be used to
stabilize solutions of BMPs in various pharmaceutically acceptable
carriers. In some embodiments, BMPs are dissolved in a solution
containing a particular concentration of finger-1 peptide analogs.
In some embodiments, the ratio of concentrations is 1:1, but it may
be less or more. Without being bound by any particular theory, it
is thought that the finger-1 peptide analog will compete with the
first finger regions of the protein, preventing multimerization of
monomeric or dimerized BMPs. As a result, it may be desirable to
have a higher concentration of peptide than protein, for example,
2:1, 5:1, 10:1, 20:1, or 30:1.
[0068] In some embodiments, the concentration of protein to be
delivered is in a range of from about 0.01 to about 4 mg per cc or
ml of carrier, for example, about 0.05 mg to about 1.5 mg per cc.
Exemplary carriers of BMPs include the materials discussed below
for therapeutic compositions, hydrogel-forming materials and other
polymers and carriers such as those disclosed in U.S. Pat. No.
6,620,406, the contents of which are incorporated herein by
reference.
Assays
[0069] In another embodiment, finger-1 peptide analogs are used in
biologic assays. For example, a finger-1 peptide analog may
interact with the BMP receptor and induce a BMP-related biologic
response, for example, signal transduction. Assays may also
determine responses such as a change in the cell cycle, expression
of a particular gene or protein, a visible or measurable alteration
in phenotype, a change in the level or modification state of a
protein, or other cell characteristics. This response may be
measured using commercially available assay or screening test kits.
The finger-1 peptide analog may be used to test particular cells to
simply determine whether they are responsive to BMP or to identify
particular BMPs to which the cell is more or less sensitive. For
example, cells may be identified that respond to the finger-1
peptide analogs by modifying a metabolic activity, for example, by
increasing or decreasing production of a particular protein,
polynucleotide, metabolite, or other cellular component. In an
alternative embodiment, the finger-1 peptide analog is employed as
a BMP competitor. In this embodiment, the finger-1 peptide analog
competes with complete BMP proteins to bind with the cell receptor.
For example, cancer cells, bacteria, and other cells involved in
disease may be assayed for the presence of particular BMP receptors
or other receptors whose binding with finger-1 peptide analogs
inhibits a particular function of the cell. Alternatively or in
addition, assays may be performed with isolated receptors rather
than with cells. In some embodiments, high throughput screening
methods using known receptors may be used to assay receptor-peptide
interactions.
[0070] Exemplary embodiments in which first finger peptide analogs
may be employed as BMP agonists include but are not limited to the
use of BMP-2 first finger peptide analogs to treat osteoporosis,
repair or regenerate bone fractures, and cartilage regeneration,
BMP-12 first finger peptide analogs for tendon repair, BMP-9 first
finger peptide analogs for induction of cholinergic neuronal
differentiation, and GDF-19 first finger peptide analogs for
treatment of heart failure. Exemplary embodiments in which first
finger peptide analogs may be employed as BMP antagonists include
but are not limited to the use of first finger peptide analogs of
BMP3 to treat osteoporosis, bone fracture, and bone diseases, BMP15
and GDF9 for contraception, GDF3 to treat type II diabetes,
obesity, and metabolic syndrome, BMP4 to repair or regenerate CNS
or peripheral nerve tissue, TGF-.beta. to treat various cancers and
tumors, or GDF8 to treat muscular dystrophy, sarcopenia, frailty,
or neurological disorders. In this embodiment, the first finger
peptide fragment competes with the full protein to bind to
receptors but then prevents the cellular or tissue response that
the protein would otherwise promote.
[0071] Peptide binding may be measured or assayed more directly
using a radioreceptor assay, in which labeled peptide in the sample
competes with unlabeled BMP for binding to the particular cell or
receptor, or vice versa. Labeling may be done with .sup.125I,
.sup.35S, .sup.32P, or other suitable radioisotopes. As the binding
of the finger-1 peptide analog increases, it reduces the amount of
BMP that is able to bind. The receptor-BMP complex is then isolated
from free ligand, for example by washing (in the case of an
adherent cell line), rapid filtration or centrifugation (in the
case of a nonadherent cell line or receptor bound to a solid
support), or precipitation of the receptor-ligand complex with
antibodies, polyethylene glycol, or other precipitating agent
followed by filtration or centrifugation (in the case of a soluble
receptor). Comparison with a standard curve prepared with known
concentrations of labeled ligand allows accurate quantitation of
either peptide or protein concentration in the sample. The amount
of labeled ligand in the complex is then quantitated, typically by
gamma counting, and compared to known standards. These methods have
been described in the literature using other receptors (M.
Williams, Med. Res. Rev., 11: 147-184 (1991); M. Higuchi and B. B.
Aggarwal, Anal Biochem., 204: 53-58 (1992); M. J. Cain, R. K.
Garlick and P. M. Sweetman, J. Cardiovasc. Pharm., 17: S150-S151
(1991); each of which are incorporated herein by reference), and
are readily adapted to the present system. Other methods of
quantifying peptides or BMPs include radioimmunoassay and ELISA, as
described in U.S. Pat. No. 4,857,456.
[0072] Alternatively or in addition, once a particular finger-1
peptide analog is identified, it may be further optimized by
replacing individual amino acids to stabilize a particular
secondary structure, prevent the formation interchain cys-cys
linkages, or create a better fit with the cell receptor. Such
mutations may be identified using computer models or using
biological techniques. For example, exemplary methods of protein
mutagenesis may be found in Sambrook et al., Molecular Cloning: A
Laboratory Manual., 2d ed. (Cold Spring Harbor, N.Y.: Cold Spring
Harbor Laboratory Press) (1990). In such an embodiment, it may be
desirable to use the peptide or optimized peptide as a therapeutic
acting as a BMP mimetic. In other embodiments, individual amino
acid residues or peptides may be chemically modified, for example,
by glycosylation or derivitization with poly(ethylene glycol).
[0073] In another embodiment, the finger-1 peptide analog interacts
directly with complete BMP proteins. For example, the finger-1
peptide analog may be used to assay for BMP proteins by preventing
homo- or heterodimerization of the protein. In this embodiment, it
may be desirable to modify the finger-1 peptide analog so that it
can be internalized by a cell, where dimerization often occurs.
Alternatively or in addition, the peptide may be employed to
positively modulate dimerization of BMP proteins. The interaction
of the finger-1 peptide analog and the BMP protein may be assayed
using any of the techniques described above. In addition, assays
for protein size, for example, native gel electrophoresis, mass
spectroscopy, or gel filtration chromatography, may be employed to
characterize the effect of the finger-1 peptide analog. In some
embodiments, the finger-1 peptide analog may be used as a
therapeutic acting as a BMP antagonist by preventing proper BMP
function.
[0074] In another embodiment, finger-1 peptide analogs may be
employed as screens to identify substances, e.g., small molecules,
that positively or negatively modulate BMP-BMP or BMP-receptor
interactions. Exemplary receptors include but are not limited to
serine/threonine kinase receptors, e.g., ACVR1/ALK2, ACVR1B/ALK4,
ACVR1C/ALK7, ACVR2/ACTRII, ACVR2B/ACTRIIB, ACVRL1/ALK1,
BMPR1A/ALK3, BMPR1B/ALK6, BMPR2/T-ALK, TGF.beta.R1/ALK5, and
TGF.beta.R2, and co-receptors such as RGMa, RGMb/DRAGON, RGMc/HFE2,
TGF.beta.R3, Cripto, Cryptic, and Endoglin. For example, high
throughput assays may be employed to identify a small molecule
inhibitor of a BMP. A cell-free assay could be conducted similar to
a FRET assay in which the screen detects disruption of the
BMP-peptide interaction. For example, either the BMP or the
finger-1 peptide analog may be modified with a donor molecule,
while the other is modified with an acceptor molecule.
[0075] In another embodiment, finger-1 peptide analogs may be
modified to be constrained in a particular configuration to retain
a given secondary structure. For example, particular amino acids in
the finger-1 peptide analog may be replaced with cysteines that
will form disulfide bonds and stabilize a particular conformation.
Alternatively or in addition, cysteines may be added to the N- and
C-terminal ends of the finger-1 peptide analog to constrain the
peptide or stabilize a particular conformation. In some
embodiments, it may be desirable to unfold wild type or mutant
finger-1 peptide analogs and allow them to refold in an alternative
configuration. For example, the finger-1 peptide analog may be
solubilized and denatured using a chaotropic agent. Separation of
the finger-1 peptide analog from the chaotropic agent allows it to
refold in a thermodynamically favored configuration. When cysteine
residues are present in the primary amino acid sequence of the
protein, it may be desired to accomplish the refolding in an
environment which allows correct formation of disulfide bonds
(e.g., a redox system). General methods of refolding are disclosed
in Kohno, Meth. Enzym., 185:187-195 (1990). Chaperonins may also be
employed to mediate folding.
Therapeutic Compositions
[0076] Finger-1 peptide analogs identified as having therapeutic
potential may be applied to tissue in the form of a buffer
solution. An exemplary buffer solution is a composition comprising,
in addition to the peptide, about 1.0 to about 10.0% (w/v) glycine,
about 0.1 to about 5.0% (w/v) of a sugar, e.g., sucrose, about 1 to
about 20 mM glutamic acid hydrochloride, and optionally about 0.01
to about 0.1% of a non-ionic surfactant, such as polysorbate 80 or
Tween. Exemplary solutions are from about 1% to about 20% w/v
cellulosic carrier/buffer. If desired, a salt may be added.
[0077] More viscous materials may also be used as carriers. Such
materials may be include pharmaceutically acceptable materials
having viscosity and polarity such that, when combined with the
finger-1 peptide analog, form a composition that possesses
appropriate handling characteristics for the tissue site. For
example, it may be desirable to have relatively less viscous (but
still not runny) material for use in a periodontal site than at a
bone fracture. Adding the carrier to the peptide allows the
finger-1 peptide analog to remain in the disease or lesion site for
a time sufficient to allow the finger-1 peptide analog to act on
local cells by either upregulating or downregulating various
metabolic activities. The carrier may also allow the finger-1
peptide analog to be released from the lesion, defect or disease
site over a time interval.
[0078] An exemplary family of carriers for administration of the
peptides are porous particulate polymers, described in detail in
U.S. Pat. No. 5,171,579, the entire disclosure of which is
incorporated herein by reference. An alternative carrier useful for
the present invention is a formulation of osteogenic protein,
porous particulate polymers and another sequestering or carrier
agent, such as cellulosic material. Other materials that may be
employed as carriers or sequestering agents include
carboxymethylcellulose, hyaluronic acid, sodium alginate,
poly(ethylene glycol), polyoxyethylene oxide, carboxyvinyl polymer
and poly(vinyl alcohol). These compositions are described in the
published PCT application WO 93/00050, the entire disclosure of
which is hereby incorporated herein by reference. The sequestering
agent may be present in a concentration of about 1 to about 10%
(w/v implant). Where porous particulate polymers are employed as
the carrier, porous particulate polymer/cellulosic sequestering
agent may optionally be further combined with aqueous glycerol as a
diluent, for example, in concentrations of about 10 to about 80%
(v/v) with exemplary ratios of sequestering agent/liquid
solution:porous particulate polymers from about 0.1 to about 0.9
(v/v).
[0079] Another exemplary family of carriers is collagenous
materials. Suitable collagen materials include Collastat.TM. and
Helistat.TM. collagen sponges (Integra LifeSciences Corp.,
Plainsboro, N.J.). Other collagen materials which may be suitable
for use in the present invention are described in U.S. Pat. No.
5,206,028; U.S. Pat. No. 5,024,841; U.S. Pat. No. 5,256,418. A
collagen carrier is may be in the form of a sponge. The collagen
sponge may be loaded with peptide prior to administration by
soaking the sponge in the desired volume and concentration of
peptide for a suitable time period. The collagen sponge may soak
loaded with peptide in a range from about 10% to about 150% v/v [ml
protein/cc dry sponge], for example, about 10 to about 60% v/v.
Alternatively, the peptide may be adsorbed to the collagen sponge
during production. In this case, peptide may be added to the
collagen sponge during production and lyophilized to form a unitary
product. The peptide may be added in a ratio of from about 10 to
about 150% v/v, for example, in a range from about 60 to about 80%
v/v.
[0080] Another exemplary family of carriers is cellulose-based
materials such as alkylcellulose (including hydroxyalkylcellulose),
including methylcellulose, ethylcellulose, hydroxyethylcellulose,
hydroxypropylcellulose, hydroxypropyl-methylcellulose, and
carboxymethylcellulose, for example, the cationic salts of
carboxymethylcellulose (CMC). The cross-link and molecular weight
of these materials and any other polymer carrier described herein
may be modified to adjust the delivery rate of the encapsulated
peptide and to stabilize the peptide in vivo.
[0081] In the case of cellulosic carriers, the carrier may be in
the form of a hydrated cellulosic viscous gel. Viscosity of
cellulosic materials may be increased through mechanical means,
such as high agitation for a suitable period of time, followed by
autoclaving. The peptide and cellulosic carrier may be in a
solution of suitable buffer. An exemplary buffer solution is a
composition comprising about 1.0 to about 10.0% (w/v) glycine,
about 0.1 to about 5.0% (w/v) of a sugar, preferably sucrose, about
1 to about 20 mM glutamic acid hydrochloride, and optionally about
0.01 to about 0.1% of a non-ionic surfactant, such as polysorbate
80. Exemplary solutions are from about 1% to about 20% w/v
cellulosic carrier/buffer. If desired, a salt may be added. The
amount of peptide combined with a viscous gel carrier may be in a
range of from about 0.05 to about 1.5 mg, for example, from about
0.1 to about 1.0 mg per cubic centimeter of implant material
required.
[0082] Other materials which may be suitable for use as carriers
for finger-1 peptide analogs according to various embodiments
include but are not limited to demineralized bone, polyglyconate,
hyaluronic acid, surgical mesh or sutures, temperature-sensitive
polymers, and minerals and ceramics, such as calcium phosphates,
hydroxyapatite, etc, and combinations of these. Other potential
carriers include carriers for injectable formulations of BMPs.
Suitable carriers for injectable formulations include, for example,
soluble collagen, hyaluronic acid, polylactic acid/polyethylene
glycol, and polymers.
[0083] Finger-1 peptide analogs for use in this embodiment may
simply be combined with the carrier and complete protein or may be
immobilized on the carrier. For example, amine-terminated peptides
may be attached to carboxylated carriers using standard polymer
chemistry techniques. In another example, peptides may be
terminated with carboxy-NHS and attached to aminated polymers.
Alternatively or in addition, peptides may be biotinylated and
coordinated with carriers that have been functionalized with
streptavidin.
[0084] Alternative carriers for finger-1 peptide analogs include
micelles. For example, double emulsion techniques such as those
described in Gref, et al, Science, 1994, 263:1600-1603 may be used
to encapsulate therapeutic compositions in micelles that may then
be administered to a subject using any of the techniques described
herein or any other delivery techniques known to those of skill in
the art. In some embodiments, the micelle surface may include a
targeting agent. The targeting agent may be covalently attached to
the micelle or may be retained using ligand-receptor interactions
(e.g., biotin-streptavidin) or electrostatic interactions.
Exemplary targeting agents include antibodies and antibody
fragments, nucleic acid ligands (e.g., aptamers), oligonucleotides,
oligopeptides, polysaccharides, low-density lipoproteins (LDLs),
folate, transferrin, asialycoproteins, gp120 envelope protein of
the human immunodeficiency virus (HIV), carbohydrates,
polysaccharides, enzymatic receptor ligands, sialic acid,
glycoprotein, lipid, small molecule, bioactive agent, biomolecule,
immunoreactive fragments such as the Fab, Fab', or F(ab').sub.2
fragments, etc.
[0085] Peptides for use as a therapeutic may be modified to
increase their in vivo stability, increasing their circulation time
and reducing the rate of elimination from the body. For example,
peptides may be functionalized or combined with a poly(alkylene
glycol) chain such as poly(ethylene glycol) (PEG) or poly(propylene
glycol) (PPG). The optimal molecular weight of such polymer chains
may be optimized using techniques known to those of skill in the
art. Alternatively or in addition, peptides may be conjugated with
sialyl groups or polysialyl chains. Peptides may also be modified
by chemically modifying amino acid residues with smaller chemical
groups such as methyl, sulfate, etc. Individual amino acid residues
may also be replaced with non-natural amino acids such as
D-stereoisomers or beta-residues. In some embodiments, peptides may
be stabilized by inter- or intra-chain covalent or non-covalent
links. These links may be promoted by adding appropriate amino acid
residues to the peptide or by modifying amino acids in the peptide
with appropriate chemical groups, such as thiol, hydrogen bond
donors and acceptors, etc. Peptides may also be associated with or
fused (e.g., at the N- or C-terminals or internally) with the full
length or a fragment of a natural or synthetic protein that acts as
a presentation enhancer. Exemplary proteins are known to those of
skill in the art and include bovine serum albumin.
[0086] The finger-1 peptide analogs, in a suitable buffer or
combined with a suitable carrier such as those described above, may
be applied directly to a tissue and/or to a site in need of tissue
repair. For example, the peptide may be physically applied to the
tissue through spraying or dipping, or using a brush or other
suitable applicator, such as a syringe for injection.
Alternatively, or in addition, the peptide may be directly applied
to the site in need of tissue repair.
[0087] In some embodiments, therapeutic compositions according to
an embodiment of the invention may be administered to a subject or
may first be formulated for delivery by any available route
including, but not limited to parenteral (e.g., intravenous),
intradermal, subcutaneous, oral, nasal, bronchial, ophthalmic,
transdermal (topical), transmucosal, rectal, and vaginal routes.
Inventive pharmaceutical compositions may include a purified
polypeptide or protein expressed from a mammalian cell line and a
delivery agent (i.e., a cationic polymer, peptide molecular
transporter, surfactant, etc., as described above) in combination
with a pharmaceutically acceptable carrier. As used herein the
language "pharmaceutically acceptable carrier" includes solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like, compatible
with pharmaceutical administration. Supplementary active compounds
can also be incorporated into the compositions.
[0088] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application may
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes or multiple dose vials made of glass or plastic.
[0089] Pharmaceutical compositions suitable for injectable use
often include sterile aqueous solutions (where water soluble) or
dispersions and sterile powders for the extemporaneous preparation
of sterile injectable solutions or dispersion. For intravenous
administration, suitable carriers include physiological saline,
bacteriostatic water, Cremophor EL.TM. (BASF, Parsippany, N.J.) or
phosphate buffered saline (PBS). In all cases, the composition
should be sterile and should be fluid to the extent that easy
syringability exists. Exemplary pharmaceutical formulations are
stable under the conditions of manufacture and storage and are
preserved against the contaminating action of microorganisms such
as bacteria and fungi. In general, the relevant carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), and suitable mixtures
thereof. The proper fluidity can be maintained, for example, by the
use of a coating such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. Prevention of the action of microorganisms can be
achieved by various antibacterial and antifungal agents, for
example, parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In many cases, it will be desirable to
include isotonic agents, for example, sugars, polyalcohols such as
manitol, sorbitol, or sodium chloride in the composition. Prolonged
absorption of the injectable compositions can be brought about by
including in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
[0090] Sterile injectable solutions can be prepared by
incorporating the purified polypeptide or protein in the required
amount in an appropriate solvent with one or a combination of
ingredients enumerated above, as required, followed by filtered
sterilization. Generally, dispersions are prepared by incorporating
the purified polypeptide or protein expressed from a mammalian cell
line into a sterile vehicle which contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, exemplary methods of preparation are
vacuum drying and freeze-drying which yields a powder of the active
ingredient plus any additional desired ingredient from a previously
sterile-filtered solution thereof.
[0091] Oral compositions may include an inert diluent or an edible
carrier. For the purpose of oral therapeutic administration, the
purified polypeptide or protein can be incorporated with excipients
and used in the form of tablets, troches, or capsules, e.g.,
gelatin capsules. Oral compositions can also be prepared using a
fluid carrier for use as a mouthwash. Pharmaceutically compatible
binding agents, and/or adjuvant materials can be included as part
of the composition. The tablets, pills, capsules, troches and the
like can contain any of the following ingredients, or compounds of
a similar nature: a binder such as microcrystalline cellulose, gum
tragacanth or gelatin; an excipient such as starch or lactose, a
disintegrating agent such as alginic acid, Primogel, or corn
starch; a lubricant such as magnesium stearate or Sterotes; a
glidant such as colloidal silicon dioxide; a sweetening agent such
as sucrose or saccharin; or a flavoring agent such as peppermint,
methyl salicylate, or orange flavoring. Formulations for oral
delivery may advantageously incorporate agents to improve stability
within the gastrointestinal tract and/or to enhance absorption.
[0092] For administration by inhalation, the inventive compositions
comprising a purified polypeptide or protein expressed from a
mammalian cell line and a delivery agent are preferably delivered
in the form of an aerosol spray from a pressured container or
dispenser which contains a suitable propellant, e.g., a gas such as
carbon dioxide, or a nebulizer. The present invention contemplates
delivery of the compositions using a nasal spray, inhaler, or other
direct delivery to the upper and/or lower airway. Intranasal
administration of DNA vaccines directed against influenza viruses
has been shown to induce CD8 T cell responses, indicating that at
least some cells in the respiratory tract can take up DNA when
delivered by this route, and the delivery agents of the invention
will enhance cellular uptake. According to certain embodiments of
the invention the compositions comprising a purified polypeptide
expressed from a mammalian cell line and a delivery agent are
formulated as large porous particles for aerosol administration.
The transepithelial absorption of compositions according to an
embodiment of the invention may be enhanced using the techniques
disclosed in U.S. Patent Publication No. 20030235536, the contents
of which are incorporated herein by reference.
[0093] Systemic administration can also be by transmucosal or
transdermal means. For transmucosal or transdermal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the art,
and include, for example, for transmucosal administration,
detergents, bile salts, and fusidic acid derivatives. Transmucosal
administration can be accomplished through the use of nasal sprays
or suppositories. For transdermal administration, the purified
polypeptide or protein and delivery agents are formulated into
ointments, salves, gels, or creams as generally known in the
art.
[0094] The compositions can also be prepared in the form of
suppositories (e.g., with conventional suppository bases such as
cocoa butter and other glycerides) or retention enemas for rectal
delivery.
[0095] In one embodiment, the compositions are combined with an
agent that will protect the polypeptide or protein against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. The agent may also serve as a carrier for the composition.
Biodegradable, biocompatible polymers can be used, such as ethylene
vinyl acetate, polyanhydrides, polyglycolic acid, collagen,
polyorthoesters, and polylactic acid. Methods for preparation of
such formulations will be apparent to those skilled in the art. The
materials can also be obtained commercially from Alza Corporation
and Nova Pharmaceuticals, Inc. Liposomal suspensions (including
liposomes targeted to infected cells with monoclonal antibodies to
viral antigens) can also be used as pharmaceutically acceptable
carriers. These can be prepared according to methods known to those
skilled in the art, for example, as described in U.S. Pat. No.
4,522,811.
[0096] It is advantageous to formulate oral or parenteral
compositions in dosage unit form for ease of administration and
uniformity of dosage. Dosage unit form as used herein refers to
physically discrete units suited as unitary dosages for the subject
to be treated; each unit containing a predetermined quantity of
active polypeptide or protein calculated to produce the desired
therapeutic effect in association with the required pharmaceutical
carrier.
[0097] The polypeptide or protein according to an embodiment of the
invention can be administered at various intervals and over
different periods of time as required. The skilled artisan will
appreciate that certain factors can influence the dosage and timing
required to effectively treat a subject, including but not limited
to the severity of a disease or disorder, previous treatments, the
general health and/or age of the subject, and other diseases
present. Generally, treatment of a subject with a polypeptide or
protein as described herein can include a single treatment or, in
many cases, can include a series of treatments. It is furthermore
understood that appropriate doses may depend upon the potency of
the polypeptide or protein and may optionally be tailored to the
particular recipient, for example, through administration of
increasing doses until a preselected desired response is achieved.
It is understood that the specific dose level for any particular
animal subject may depend upon a variety of factors including the
activity of the specific polypeptide or protein employed, the age,
body weight, general health, gender, and diet of the subject, the
time of administration, the route of administration, the rate of
excretion, any drug combination, and the degree of expression or
activity to be modulated.
[0098] The present invention includes the use of inventive
compositions for treatment of nonhuman animals. Accordingly, doses
and methods of administration may be selected in accordance with
known principles of veterinary pharmacology and medicine. Guidance
may be found, for example, in Adams, R. (ed.), Veterinary
Pharmacology and Therapeutics, 8th edition, Iowa State University
Press; ISBN: 0813817439; 2001.
[0099] Inventive pharmaceutical compositions can be included in a
container, pack, or dispenser together with instructions for
administration.
EXAMPLES
Example 1
[0100] BMP-12 was immobilized on NHS-activated Sepharose.TM. beads
at a ratio of about 20 mg:4 mL and the resulting media combined
with water or neutral saline buffer to prepare a column.
CHO-conditioned media augmented with 1.2 M NaCl was loaded onto the
column. In some cases, the media was preconcentrated with respect
to BMP before loading. The column was washed with 15 CV (column
volumes) of 1.2 M NaCl in 40 mM Na.sub.2HPO.sub.4 at pH 7.2,
followed by 0.5 M arginine/0.5 M NaCl in 20 mM Tris at pH 7.5 (15
CV) and a low salt wash (50 mM ammonium acetate at pH 7, 15 CV).
The column was further eluted with acetic acid, using a 5 CV
gradient to 50 mM acetic acid at pH 3, followed by a 15 CV chase.
Finally, the column was eluted with 5CV of the acetic acid solution
with 6M guanidine hydrochloride (GuHCl).
[0101] BMP-3 was effectively captured by the chromatography media
and eluted with acetic acid (FIG. 3A). The eluate was analyzed
using HPLC. As seen in FIG. 3B, a propeptide fragment copurifies
along with the mature protein, and trace amounts of the mature
BMP-3 were also observed in the guanidine peak.
Example 2
[0102] BMP-2 was immobilized on sepharose beads as in Example 1 and
used to test the loading and elution of BMP-3. As shown in FIG. 4,
BMP-3 was effectively captured by the high ionic strength solution
and was eluted with acetic acid. As for the BMP-12 column, a
propeptide fragment copurifies along with the mature protein, and
trace amounts of the mature BMP-3 were also observed in the
guanidine peak.
Example 3
[0103] BMP-12 was loaded and eluted from a BMP-12 affinity column
as in Example 1. The BMP-12 was effectively captured by the column
using the high salt solution and eluted with acetic acid and
guanidine (FIG. 5). Both the monomeric and dimeric forms of BMP-12
were captured.
Example 4
[0104] BMP-12 was loaded and eluted from a BMP2 affinity column as
in Examples 1 and 2. The BMP-12 was effectively captured by the
column using the high ionic strength solution and was eluted with
acetic acid and guanidine (FIG. 6).
Example 5
[0105] The BMP affinity resins using BMP-12 and BMP-2 were prepared
as described in Examples 1 and 2 and used to prepare HPLC columns.
These columns were used loaded with BMP-3 and BMP-12. As shown in
FIG. 7, BMP-12 and BMP-2 were equally effective as affinity ligands
for reverse phase HPLC. In some cases, size exclusion or reverse
phase chromatography techniques were employed to enhance
purification.
Example 6
[0106] A mutant of the BMP-2 finger 1 peptide
(SDVGWNDWIVAPPGYHAFYCHGEK) (SEQ ID NO:1) was linked to sepharose
beads via a C-terminal lysine at a ratio of 5 mg peptide/mL resin.
4 mL of the resulting resin (1.6.times.2.0 cm) was used to prepare
a column. The column was loaded with BMP-3 and eluted as in Example
1. As shown in FIG. 8, the BMP-3 was effectively captured by the
column using the high salt solution and eluted, along with a
propeptide fragment, in acetic acid. In general, chromatography
with the immobilized peptides provided higher purity product than
with the whole protein.
Example 7
[0107] A wild-type BMP-12 finger 1 peptide
(KELGWDDWIIAPLDYEAYHCEGLC) (SEQ ID NO:2) was used to purify BMP-3
as in Example 1. As shown in FIG. 9, the BMP-3 was effectively
captured by the column using the high salt solution and eluted in
acetic acid. Less of the propeptide fragment eluted than in Example
6. In general, the immobilized BMP-12 was less effective than the
BMP-2 mutant. SDS-PAGE shows that the immobilized BMP-12 peptide
oligomerized, perhaps due to the free cysteine residues.
Example 8
[0108] BMP-12 was purified using a column prepared with the BMP-2
mutant peptide described in Example 6. As shown in FIG. 10, the
BMP-12 was effectively captured by the column using the high salt
solution and eluted with acetic acid. A small amount of BMP-12
eluted with 6M GuHCl.
Example 9
[0109] BMP-12 was purified using a column prepared with the BMP-12
wild type peptide described in Example 7. As shown in FIG. 11, the
BMP-12 was effectively captured by the column using the high ionic
strength solution and eluted with acetic acid. A small amount of
BMP-12 eluted with 6M GuHCl.
Example 10
[0110] GDF-9 was purified using a column prepared with the BMP-2
first finger mutant peptide described in Example 6. As shown in
FIG. 12, the GDF-9 was effectively captured by the column using a
high salt solution and eluted with both 500 mM arginine and acetic
acid. A lower molecular weight contaminant was also present.
Example 11
[0111] BMP affinity resins are prepared using one or more of the
following peptides:
TABLE-US-00001 TABLE 1 Exemplary Peptide Sequences for Preparation
of Affinity Resins BMP2 Fing1 WT: SDVGWNDWIVAPPGYHAFYCHGEC (SEQ ID
NO: 3) BMP2 Fing1 Mut1: SDVGWNDWIVAPPGYHAFYCHGEK (SEQ ID NO: 1)
BMP2 Fing1 Mut2: SDVGANDWIVAPPGYHAFYCHGEC (SEQ ID NO: 4) BMP2 Fing1
Mut3: SDVGANDWIVAPPGYHAFYCHGEK (SEQ ID NO: 5) BMP2 Fing1 Mut4:
SDVGWNDWIVAPPGYHAFYCHAEC (SEQ ID NO: 6) BMP2 Fing1 Mut5:
SDVGWNDWIVAPPGYHAFYCHAEK (SEQ ID NO: 7) BMP2 Fing1 Mut6:
SDVGANDWIVAPPGYHGFYCHGEC (SEQ ID NO: 8) BMP2 Fing1 Mut7:
SDVGANDWIVAPPGYHGFYCHGEK (SEQ ID NO: 9) BMP2 Fing1 WT:
LYVDFSDVGWNDWIVAPPGYHAFY (SEQ ID NO: 10) BMP2 Fing1 Mut1
LYVDFSDVGANDWIVAPPGYHAFY (SEQ ID NO: 11) BMP2 Fing1 Mut2
LYVDFSDVGWNDWIVAPPGYHGFY (SEQ ID NO: 12) BMP12 Fing1 WT:
KELGWDDWIIAPLDYEAYHCEGLC (SEQ ID NO: 2) BMP12 Fing1 Mut1:
KELGWDDWIIAPLDYEAYHCEGLK (SEQ ID NO: 13) BMP12 Fing1 Mut2:
AELGWDDWIIAPLDYEAYHCEGLK (SEQ ID NO: 14) BMP3 Fing1 WT
ADIGWSEWIISPKSFDAYYCSGAC (SEQ ID NO: 15) BMP3 Mut1:
ADIGWSEWIISPKSFDAYYCSGAK (SEQ ID NO: 16)
Example 12
[0112] BMP Solubility Screen--A Tecan liquid-handling robot was
used to mix a master plate of reagents, 800 .mu.L.times.1.25
concentration (for each solution; see Tables 2 and 3), each in 50
mM Tris buffer at pH 8. An eight-channel pipettor was used to
transfer 80 .mu.L of material from the master plate into a
half-area UV microplate, from which A.sub.320 was read from each
sample to define a blank. A repeat-pipettor was used to add 20
.mu.L of 2.5 mg/mL BMP-12 in 0.1% TFA to make a 0.5 mg/mL solution,
which was then agitated at 14000 rpm. The UV absorption (A.sub.320)
was read and the blank absorption subtracted to determine
solubility. The procedure was repeated with a wider range of
2-methyl-2,4-pentanediol (MPD) concentrations and a finer set of
salt concentrations (Table 4). A graph comparing the turbidity of
BMP-12 solutions with respect to NaCl and MPD concentrations is
shown in FIG. 13.
TABLE-US-00002 TABLE 2 1-Propanol, % NaCl, M 0 2.5 5 10 15 25 0
1.724 1.848 2.134 2.000 3.172 1.419 0.1 1.745 2.135 2.212 1.945
3.199 0.104 0.25 1.804 2.397 2.300 2.105 1.392 0.022 0.5 1.953
2.170 1.629 1.313 0.305 -0.001 0.75 1.911 2.023 1.541 1.032 0.094
-0.003 1 1.988 1.601 1.817 1.000 0.040 -0.010 1.25 1.702 1.376
1.643 1.018 0.033 0.022 1.5 1.981 1.358 1.598 0.973 0.036 0.478
TABLE-US-00003 TABLE 3 2-Methyl-2,4-pentanediol, % NaCl, M 2.5 5 10
15 20 25 0 1.936 2.122 2.148 2.035 2.136 2.631 0.1 1.900 2.338
2.341 2.287 2.573 0.715 0.25 2.061 2.399 2.434 2.433 1.006 0.119
0.5 2.045 2.414 2.258 1.420 0.165 0.020 0.75 1.985 2.076 2.211
0.818 0.099 0.001 1 2.133 1.903 2.110 0.400 0.089 -0.015 1.25 1.819
1.643 1.688 0.257 0.040 -0.011 1.5 1.767 1.521 1.798 0.227 0.045
0.003
TABLE-US-00004 TABLE 4 2-Methyl-2,4-pentanediol, % NaCl, M 2.5 5 10
15 20 25 30 35 40 45 0.00 1.936 2.122 2.148 2.035 2.136 2.631 3.443
3.006 3.519 3.308 0.05 n/a n/a n/a n/a n/a 1.994 0.979 0.376 0.330
0.318 0.10 1.900 2.338 2.341 2.287 2.573 0.626 0.206 0.150 0.083
0.087 0.25 2.061 2.399 2.434 2.433 1.006 0.047 -0.015 -0.065 0.015
0.012 0.50 2.045 2.414 2.258 1.420 0.165 0.038 -0.091 0.026 0.024
-0.008 0.75 1.985 2.076 2.211 0.818 0.099 0.001 1.00 2.133 1.903
2.110 0.400 0.089 -0.015 1.25 1.819 1.643 1.688 0.257 0.040 -0.011
1.50 1.767 1.521 1.798 0.227 0.045 0.003
Example 13
[0113] Affinity resins were coupled with the peptides listed in
Table 5. The coupled resins, plus an unconjugated resin for use as
a control, were suspended in 16% ethanol, 150 mM NaCl, and 20 mM
Tris buffer at pH 7.5 at a 25% v/v ratio (e.g., 25% ethanol, 75%
Tris buffer). A Tecan liquid-handling robot was employed to aliquot
200 .mu.L of each slurry into one of eight rows of a 96 well
polystyrene filter plate (0.45 .mu.m hydrophilic filters, Whatman
catalog number 770-1806). Spinning at 1200 g for 3 min removed the
liquid phase, leaving 50 .mu.L of wet resin on the filters. The
resins were then equilibrated with the loading buffers listed in
Table 6 (K is a coefficient characterizing the strength of protein
binding to a particular resin in a particular buffer). A BMP-12
load plate was prepared by adding 40 .mu.L of low molecular weight
size exclusion chromatography (LMW SEC) solution (BEP, dialyzed in
0.1% TFA and concentrated to 3.76 mg/mL) to 260 .mu.L of each
loading buffer in a 96-well plate, resulting in 0.48 mg/mL of
protein in each well. The resins were loaded by transferring 150
.mu.L from the load plate to the filter plate and then incubating
20 min on a shaking platform to facilitate binding. The filter
plate was spun down and the filtrate collected in a 96 well UV
plate. The protein concentration in the filtrate, or flow-through,
was calculated as (A.sub.280-A.sub.320)/1.3. The filter plate was
then washed with 150 .mu.L of 100 mM acetic acid, followed by 150
.mu.L of 4M guanidine-HCl and 50 mM sodium acetate at pH 4.0. The
protein concentration was measured in the new filtrate as described
above. As shown in Table 6, resins coupled to peptides 747, 747,
748 and 749 bound BMP12 with a high affinity in loading buffers
1-5. However, for peptides 750 and 751, the binding was strong in
25 mM MES at pH 6 but weak in 50 mM acetic acid with 0.1 M NaCl at
pH 3.0, which can allow an efficient binding-elution process
without the need for chaotrops or organic reagents. Flow-through
and acetic acid elution samples from column 4 of the plate (loaded
with MES pH 6) were analyzed on a gel (FIG. 14).
TABLE-US-00005 TABLE 5 Peptide Name ID Peptide BMP2 N-trunc_2
4050-745 Ac-VAPPGYHAFYSHGEK (SEQ ID NO: 17) BMP2 C-trunc_2 4050-746
Ac-LYV**SDVGWNDWIVK (SEQ ID NO: 18) BMP2 Full 4050-747
Ac-LYVDFSDVGWNDWIVAPPGYHAFYSHGEK (SEQ ID NO: 19) BMP5 4050-748
Ac-RDLGWQDWIIAPEGYAAFYSDGEK (SEQ ID NO: 20) BMP12 N- 4050-749
Ac-ELGWDDWIIAPLDYEAYHSEGLK (SEQ ID NO: 21) trun_1S BMP2 N-trun 1
4050-750 Ac-SDVGWNDWIVAPPGYHAFYCHGEK (SEQ ID NO: 22) BMP2 N-trun 1S
4050-751 Ac-SDVGWNUWIVAPPGYHAFYSHGEK (SEQ ID NO: 23)
TABLE-US-00006 TABLE 6 Load conditions* 1 2 10 11 12 50 mM 50 mM 50
mM 50 mM Tris, 50 mM Acetic Acetic 3 4 5 Tris, 0.1 M 0.25 M Tris,
0.1 M Acid, 0.1 Acid, 0.1 M 25 mM 25 mM 25 mM 6 NaCl, 40% NaCl, 30%
NaCl, 20% M NaCl, NaCl, pH NaAcetate MES pH HEPES pH 1M CHES MPD,
pH MPD, pH MPD, pH Resin pH 2.9 4.0 pH 5.0 6.0 7.0 pH 9.7 8.0 8.0
8.0 Flow- Control 0.425 0.408 0.410 0.397 0.396 0.686 0.488 0.482
0.467 through 745 0.397 0.410 0.407 0.426 0.397 0.626 0.464 0.431
0.435 conc, 746 0.150 0.052 0.070 0.029 0.162 0.710 0.505 0.515
0.486 mg/ml 747 0.114 0.029 0.046 0.035 0.103 0.659 0.482 0.450
0.438 748 0.106 0.023 0.024 0.022 0.055 0.626 0.407 0.375 0.383 749
0.175 0.050 0.032 0.025 0.116 0.653 0.434 0.416 0.404 750 0.337
0.164 0.163 0.037 0.290 0.577 0.423 0.342 0.364 751 0.349 0.156
0.124 0.035 0.216 0.647 0.435 0.425 0.411 Flow- Control 0 1 1 1 1
-1 0 0 0 through K 745 1 1 1 0 1 -1 0 0 0 746 7 25 18 47 6 -1 0 0 0
747 10 47 28 38 11 -1 0 0 0 748 11 59 57 62 23 -1 1 1 1 749 5 26 42
56 9 -1 0 0 1 750 1 6 6 36 2 -1 0 1 1 751 1 6 9 38 4 -1 0 0 1 100
mM Control 0.066 0.064 0.070 0.129 0.073 HAc 745 0.058 0.057 0.073
0.104 0.077 elution 746 0.154 0.208 0.296 0.320 0.249 conc, 747
0.123 0.147 0.274 0.324 0.255 mg/ml 748 0.123 0.151 0.264 0.304
0.257 749 0.098 0.175 0.275 0.321 0.280 750 0.096 0.194 0.226 0.311
0.187 751 0.102 0.219 0.285 0.355 0.254 100 mM Control 0 0 0 -1 0
HAc 745 1 1 0 -1 0 elution K 746 3 3 1 1 1 747 6 6 2 1 1 748 6 6 2
2 2 749 6 4 2 1 1 750 1 2 1 1 0 751 1 1 1 1 0 4M Gnd, Control 0.029
0.023 0.025 0.031 0.021 50 mM 745 0.024 0.026 0.026 0.027 0.021
NaAc pH 4 746 0.135 0.162 0.100 0.070 0.068 Strip 747 0.150 0.150
0.111 0.065 0.074 Conc, 748 0.177 0.229 0.140 0.091 0.105 mg/ml 749
0.141 0.196 0.126 0.079 0.093 750 0.044 0.087 0.072 0.060 0.046 751
0.046 0.089 0.070 0.053 0.044 Yield, % Control 108 103 105 116 102
745 100 103 105 116 103 746 91 88 97 87 100 747 81 68 90 88 90 748
85 84 89 87 87 749 86 88 90 88 102 750 100 93 96 85 109 751 104 97
100 92 107 *Load conditions that developed precipitation: 50 mM
HEPES, 0.5M NaCl, 25% 1-propanol, pH 7.0 50 mM Tris. 0.5 M NaCl,
25% IPA, pH 8.6 50 mM HEPES, 0.5M NACl, 25% tert-butanol, pH
7.0
[0114] Other embodiments of the invention will be apparent to those
skilled in the art from a consideration of the specification or
practice of the invention disclosed herein. It is intended that the
specification and examples be considered as exemplary only, with
the true scope and spirit of the invention being indicated by the
following claims.
Sequence CWU 1
1
23124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro
Pro Gly Tyr His1 5 10 15Ala Phe Tyr Cys His Gly Glu Lys
20224PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 2Lys Glu Leu Gly Trp Asp Asp Trp Ile Ile Ala Pro
Leu Asp Tyr Glu1 5 10 15Ala Tyr His Cys Glu Gly Leu Cys
20324PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro
Pro Gly Tyr His1 5 10 15Ala Phe Tyr Cys His Gly Glu Cys
20424PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 4Ser Asp Val Gly Ala Asn Asp Trp Ile Val Ala Pro
Pro Gly Tyr His1 5 10 15Ala Phe Tyr Cys His Gly Glu Cys
20524PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 5Ser Asp Val Gly Ala Asn Asp Trp Ile Val Ala Pro
Pro Gly Tyr His1 5 10 15Ala Phe Tyr Cys His Gly Glu Lys
20624PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro
Pro Gly Tyr His1 5 10 15Ala Phe Tyr Cys His Ala Glu Cys
20724PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 7Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro
Pro Gly Tyr His1 5 10 15Ala Phe Tyr Cys His Ala Glu Lys
20824PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 8Ser Asp Val Gly Ala Asn Asp Trp Ile Val Ala Pro
Pro Gly Tyr His1 5 10 15Gly Phe Tyr Cys His Gly Glu Cys
20924PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Ser Asp Val Gly Ala Asn Asp Trp Ile Val Ala Pro
Pro Gly Tyr His1 5 10 15Gly Phe Tyr Cys His Gly Glu Lys
201024PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp
Trp Ile Val Ala1 5 10 15Pro Pro Gly Tyr His Ala Phe Tyr
201124PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Leu Tyr Val Asp Phe Ser Asp Val Gly Ala Asn Asp
Trp Ile Val Ala1 5 10 15Pro Pro Gly Tyr His Ala Phe Tyr
201224PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 12Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp
Trp Ile Val Ala1 5 10 15Pro Pro Gly Tyr His Gly Phe Tyr
201324PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 13Lys Glu Leu Gly Trp Asp Asp Trp Ile Ile Ala Pro
Leu Asp Tyr Glu1 5 10 15Ala Tyr His Cys Glu Gly Leu Lys
201424PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Ala Glu Leu Gly Trp Asp Asp Trp Ile Ile Ala Pro
Leu Asp Tyr Glu1 5 10 15Ala Tyr His Cys Glu Gly Leu Lys
201524PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Ala Asp Ile Gly Trp Ser Glu Trp Ile Ile Ser Pro
Lys Ser Phe Asp1 5 10 15Ala Tyr Tyr Cys Ser Gly Ala Cys
201624PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 16Ala Asp Ile Gly Trp Ser Glu Trp Ile Ile Ser Pro
Lys Ser Phe Asp1 5 10 15Ala Tyr Tyr Cys Ser Gly Ala Lys
201724PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro
Pro Gly Tyr His1 5 10 15Ala Phe Tyr Ser His Gly Glu Lys
201824PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 18Ser Asp Val Gly Trp Asn Asp Trp Ile Val Ala Pro
Pro Gly Tyr His1 5 10 15Ala Phe Tyr Cys His Gly Glu Lys
201923PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 19Glu Leu Gly Trp Asp Asp Trp Ile Ile Ala Pro Leu
Asp Tyr Glu Ala1 5 10 15Tyr His Ser Glu Gly Leu Lys
202024PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Arg Asp Leu Gly Trp Gln Asp Trp Ile Ile Ala Pro
Glu Gly Tyr Ala1 5 10 15Ala Phe Tyr Ser Asp Gly Glu Lys
202129PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Leu Tyr Val Asp Phe Ser Asp Val Gly Trp Asn Asp
Trp Ile Val Ala1 5 10 15Pro Pro Gly Tyr His Ala Phe Tyr Ser His Gly
Glu Lys 20 252214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 22Leu Tyr Val Ser Asp Val Gly Trp Asn
Asp Trp Ile Val Lys1 5 102315PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 23Val Ala Pro Pro Gly Tyr His
Ala Phe Tyr Ser His Gly Glu Lys1 5 10 15
* * * * *