Note: Descriptions are shown in the official language in which they were submitted.
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METHODS AND COMPOSITIONS FOR STIIVIULATING
AND INHIBITING TGF- (3 ACTIVITY
BACKGROUND OF THE IIWENTION
FIELD OF THE INVENTION
The present invention relates to a method of regulating TGF-P activity.
In particular, the present invention relates to a method of stimulating TGF-p
activity
by the application of TSP or specific peptides from TSP which stimulate TGF-R
activity or inhibiting TGF-0 activity by the application of antibodies against
TSP or
specific peptides from TSP which inhibit TGF-P activity.
BACKGROUND ART
Transforming growth factor-a (TGF-(3) is a member of a family of
growth, differentiation, and morphogenesis autocrine and paracrine factors
(3,26).
TGF-P can affect diverse cellular functions in virtually all cell types.
Depending on the
cell type and its extracellular environment, these effects can be either
positive or
negative. TGF-P inhibits the proliferation of endothelial cells in vitro (31),
but
stimulates angiogenesis in vivo (39). TGF-R has also been shown to enhance or
inhibit
the proliferation of fibroblasts depending on the nature of the substrate and
the
mitogens present (3). Myoblast differentiation can also be induced or blocked
by
TGF-P depending on the availability of mitogens (25, 45).
TGF-P 1 is a disulfide-linked homodimer that is synthesized as part of a
latent precursor molecule (26). The latent precursor molecule is 390 amino
acids in
length and consists of an N-terminal 278 amino acid latency associated peptide
(LAP)
and a C terminal 112 amino acid active domain (15-17). The proregion of TGF-R
is
unique in that it remains non-covalently attached to the active region after
intracellular
proteolytic processing and secretion (15). Association of the LAP with the
mature
peptide region confers latency: the LAP-associated growth factor is unable to
interact
with its cellular receptors. The LAP contains three N-linked glycosylation
sites, two of
which have mannose-6-phosphate residues (8,28,38). These carbohydrate
structures
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2
may be important for latency since endoglycosidase F treatment leads to
activation of
TGF-P (28). The disulfide-bonded dimeric structure of LAP is critical for
latency,
since site-directed mutagenesis of critical cysteine residues (cys 223, 225)
in the LAP
abolishes the latency function (9). The active domain contains nine conserved
cysteine
residues that participate in inter-and intrachain disulfide bonding (27).
TGF-R is secreted by most cell types as a latent complex (27,37).
Since TGF- P synthesis and TGF- P receptor expression are not highly
regulated,
primary regulation of TGF- P activity occurs by controlling conversion of the
latent
TGF- R complex to the active molecule. Physicochemical activation can occur by
extremes of pH, heat, chaotropic agents, and deglycosylation (6,27,28,37).
Activation
in vivo is more complex and not well understood. There is evidence from cell
culture
models that activation may occur through binding of the latent molecule to
mannose
6-phosphate receptors (12,21), by plasmin-mediated proteolytic processing
(4,23,40,41), and/or by processing in acidic cellular microenvironments (20).
In some
systems, activation of latent TGF-P by plasmin is relatively inefficient (41).
In
addition, there are reports of TGF-(3 activation occuning independently of
these
mechanisms (19). These results suggest that additional mechanisms of latent
TGF-P
activation may exist.
TGF-P has been demonstrated, through numerous studies, to play a
significant role in wound healing and fibrosis. The three phases of
inflammation,
granulation tissue formation and biosynthesis of the extracellular matrix, are
identical
in both the wound healing process and the development of fibrosis. A fine
balance in
the biosynthetic and degradative pathways involved in extracellular matrix
biosynthesis
appears to be determinative of whether proper wound healing or fibrosis
results. Due
to its function of regulating genes critically involved in extracellular
matrix formation,
TGF-P significantly influences this phase of tissue regeneration, the final
outcome of
which is either wound healing or fibrosis. (46). Thus, sensitive regulation of
TGF-R
activity in this process will permit control of the wound healing and fibrotic
processes.
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The thrombospondins (TSP) are a growing family of multidomain
glycoproteins (5,30,48,49). TSPI is the best characterized and serves as the
prototypical TSP molecule. TSP, a disulfide-linked trimer (450,000 daltons)
preseiit in
connective tissues and in platelet alpha granules, is associated with TGF-P as
an active
complex in the releasate of stimulated platelets (5,14,30,34). TSP is secreted
and
incorporated into the extracellular matrix of a number of cells in culture (1-
5). TSP,
like TGF-(3, has diverse effects on cellular functions that vary with cell
type. TSP can
inhibit endothelial proliferation and migration (2,34,42,5 1), but stimulates
the growth
of smooth muscle cells and dermal fibroblasts (36,52). TSP may also serve as
both an
attachment protein and an anti-adhesive molecule as shown by its ability to
cause
disassembly of focal adhesions in endothelial cells (33). TSP also plays a
role in
angiogenesis, fibrinolysis, platelet aggregation, and inflammation (1-5).
TSP is present transiently in wound environments and its synthesis is
rapidly induced by growth factors, including TGF-P (50). TSP is detectable in
incisional wound margins for 2-7 days, after which it localizes around
vascular
channels near the wound. Although the role of TSP is not yet clearly
understood, it
has been speculated that TSP may facilitate cell migration into the wound site
or
possibly act as a localized growth promoting agent (49).
There are three sequences in TSP known as type 1 repeats. Each
repeat consists of approximately 60 amino acids, has six conserved cysteine
residues
and has approximately 47% sequence homology to similar repeats found in the
human
complement component, properdin. Within the type 1 repeats of TSP, there are
two
wel! defined consensus sequences, CSVTCG (SEQ ID NO: 1) and WSXW (SEQ ID
NO:2). CSVTCG (SEQ ID NO:1) inhibits metastasis of melanoma cells in a murine
lung colonization assay (47), and promotes cell adhesion (53,54). Tolsma et
al.
showed that CSVTCG (SEQ ID NO:1) inhibits angiogenesis in vivo using a corneal
neovascularization assay (55).
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The sequence WSXW (SEQ ID NO:2) binds specifically to sulfated
glycoconjugates and promotes cell adhesion and chemotaxis (56). The binding of
TSP
to the gelatin-binding domain of fibronectin can be blocked using the peptide
GGWSHW (SEQ ID NO:3) (57). This sequence is also conserved within members of
the TGF-R and cytokine receptor superfamilies (58,59). The present invention
provides a method for activation of latent TGF-P
by TSP and a method for inhibiting the activation of latent TGF-P by binding
TSP with
a specific ligand. This invention also provides specific TSP peptides which
activate
latent TGF-R and TSP peptides which inhibit activation of latent TGF- j3.
These
peptides from TSP can both positively and negatively modulate TGF-R levels at
nanomolar to micromolar concentrations, and, therefore, can be used as
therapeutic
agents in vivo for the promotion of wound healing and inhibition of fibrosis.
SUMMARY OF THE INVENTION
The invention provides a method of stimulating TGF-B activity,
comprising contacting latent TGF-B with an amount of TSP or an activating
peptide
from TSP effective to convert latent TGF-B to active TGF-B. Also provided is a
method of inhibiting the stimulation of TGF-B activity, comprising contacting
latent
TGF-Li with a ligand specific for TSP effective to bind TSP and prevent
activation of
TGF-P or an amount of an inhibiting peptide having a sequence that corresponds
to a
sequence of four consecutive amino acids of TSP, effective to inhibit the
conversion of
latent TGF-P to active TGF-1i.
The invention also provides a method of enhancing wound healing,
comprising administering to a wound site an amount of TSP or an activating
peptide
effective to convert latent TGF-!3 to active TGF-0, the activation of TGF-B
resulting
in enhanced wound healing.
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A method of preventing fibrosis stimulated by TGF-B in pathology is
also provided. The method comprises administering to a site of potential
fibrosis an
amount of a ligand specific for TSP effective to bind TSP and inhibit
activation of
TGF-p or an inhibiting peptide from TSP effective to inhibit conversion of
latent TGF-
= 5~3 to active TGF- a, resulting in reduced fibrosis.
The invention also provides a method of blocking TGF-a-mediated
inhibition of endothelial cell proliferation comprising contacting the
endothelial cells
with a ligand specific for TSP effective to bind TSP and inhibit activation of
TGF-P or
an inhibiting peptide from TSP effective to inhibit conversion of latent TGF-R
to active
TGF-R, resulting in proliferation of endothelial cells.
DESCRIPTION OF THE PREFERRED EMBODIlVIENT
The present invention may be understood more readily by reference to
the following detailed description of specific embodiments and the Examples
included
herein.
In one embodiment, the present invention provides a method of
stimulating TGF-B activity, comprising contacting latent TGF-B with an amount
of
purified TSP or a purified activating peptide from TSP effective to convert
latent TGF-
B to active TGF-B. "Activated TGF-or "TGF-P activity" as used herein describes
the TGF-P protein present in a conformation whereby the TGF-(i protein exerts
an
effect on cells to which it is exposed, the effect being proliferation,
differentiation,
angiogenesis, etc. "Latent TGF-R" as used herein means the TGF-(3 protein
present
in a conformation whereby the active domain of the TGF- (i protein is
complexed to
LAP and therefore, does not exert an effect on cells to which it is exposed.
The term
"activating peptide" as used herein is defined as a peptide sequence or
peptide mimetic,
either synthetic or generated from a native protein or by recombinant methods,
comprising a minimum of three amino acids, which when exposed to latent TGF-R,
converts latent TGF-P to activated TGF-P. The peptides of the invention
correspond
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6
to sequences of TSP or they can be derived from the functional sequences of
TSP.
The term "purified" as used herein means separated from other proteins,
peptides and
contaminants.
In the method of stimulating TGF-P activity by contacting latent TGF- =
(3 with an amount of an activating peptide from TSP, the activating peptide
can be
from the first, second and third type 1 repeat regions of TSP. For example,
SEQ ID
NOS: 5, 9, 14, 15 and 16 are from the second type 1 repeat region. As used
herein,
"second type 1 repeat region" means the second type 1 repeating sequence unit,
as
measured from the amino tenminus of the three type 1 repeats and consisting of
amino
acids 412-473 of human TSP1. The activating.peptide can be selected from the
group
consisting of: KRFK (SEQ ID NO:5), ERFK (SEQ IDNO:6), RKPK (SEQ ID
NO:7), QRFK (SEQ ID NO:8), KRFKQDGG (SEQ ID NO:9), RWRPWTAWSE
(SEQ ID NO:10), TAYRWRLSHRPKTGIRV (SEQ ID NO: 11),
KRFKQDGGASHASPASS (SEQ ID NO: 12), KRFKQDGGASHASP (SEQ ID
NO:13), KRFKQDGGWSHWSP (SEQ ID NO: 14), KRFKQDGGWSHWSPWSS
(SEQ ID NO:15), KRFKQDGGWSHW (SEQ ID NO:16) and KRFKQDGGWWSP
(SEQ ID NO: 17) or can consist of the amino acid sequence RFK (SEQ ID NO: 18).
The activating peptide may contain partial or full retro-inverso modifications
of the
sequences or appropriate non-natural amino acids. Such sequences as well as
others
corresponding to or derived from TSP are determined to be activating sequences
by
screening for TGF-(3 activating function in a soft agar NRK colony formation
assay
and an endothelial cell proliferation assay as described in the Examples.
Also provided is a method of inhibiting the stimulation of TGF-B
activity by contacting cells producing TGF-B with an amount of a ligand
specific for
TSP effective to bind TSP and prevent activation of TGF-B. "Specific" as used
herein
describes an antibody or other ligand that does not cross react substantially
with any
moiety other than the one specified, in this case, TSP. As contemplated
herein, the
ligand includes any reagent which binds TSP, for example, an intact antibody,
a
fragment of an antibody or another reagent that has specific reactivity with
TSP.
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Ligands, such as antibodies can be those provided in the Examples or can be
made as
described in the art using routine procedures (61).
In another embodiment, the invention provides a method of inhibiting
the stimulation of TGF-B activity comprising contacting latent TGF-B with an
amount
of a purified inhibiting peptide from TSP effective to inhibit the conversion
of latent
TGF-P to activated TGF-p. As used herein, "inhibitory peptide" means a peptide
sequence comprising a minimum of four amino acids derived from the functional
regions of TSP which, when exposed to latent TGF-a, inhibits the conversion of
latent
TGF-(3 to active TGF-P. The purified inhibiting peptide can have a sequence
that
corresponds to a sequence of four consecutive amino acids of TSP, effective to
inhibit
the conversion of latent TGF-P to active transforming TGF-P .
In the method of inhibiting the stimulation of TGF-P activity, the
inhibiting peptide can be from the first, second and third type 1 repeat
region of TSP.
For example, SEQ ID NOS: 25, 26, 27 and 3 are from the second type 1 repeat.
The
inhibiting peptide derived from TSP can consist of the aniino acid sequence
GGWSHW (SEQ ID NO:3) or selected from the group consisting of: WNDWI (SEQ
ID NO:19), WSSWS (SEQ ID NO:20), LSKL (SEQ ID NO:21), AAWSHW (SEQ ID
NO:22), DGWSPW (SEQ ID NO:23), GGWGPW (SEQ ID NO:24), WSPWS (SEQ
ID NO:25), GWSHW (SEQ ID NO:26) and WSHWS (SEQ ID NO:27). The
activating peptide may contain partial or full retro-inverso modifications of
the
sequences or appropriate non-natural amino acids. These and other sequences
derived
from TSP are determined to be inhibiting sequences by screening for inhibition
of
TGF-p activating function in a soft agar NRK colony formation assay as
described in
the Examples.
TGF-P is known to regulate wound healing. Thus, the present
invention also provides a method of enhancing wound healing by administering
to a
30. wound site an amount of purified TSP or a purified activating peptide from
TSP
effective to convert latent TGF-P to active TGF-B, the activation of TGF-13
resulting in
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enhanced wound healing. The activating peptides can be those described herein.
As
used herein, "enhanced wound healing" is defined as a statistically
significant increase
in the rate of wound healing, as determined by histological analysis, tensile
strength
and total protein and collagen content of a wound treated with an amount of
TSP or an
activating peptide from TSP, as compared to a similar untreated wound or a
similar =
wound treated with a non-activating control. Histological analysis includes
examination for the presence of fibroblasts and capillary endothelial cells,
which are
early signs of wound healing. One example of this method, using the peptide
KRFK
(SEQ ID NO:5), is provided in the Examples.
In the method of enhancing wound healing, the activating peptide can
be selected from the group consisting of: KRFK (SEQ ID NO:5), HRFK (SEQ ID
NO:6), RKPK (SEQ ID NO:7), QRFK (SEQ ID NO:8), KRFKQDGG (SEQ ID
NO:9), RWRPWTAWSE (SEQ ID NO:10), TAYRWRLSHItPKTGIRV (SEQ ID
NO:11), KRFKQDGGASHASPASS (SEQ ID NO:12), KRFKQDGGASHASP (SEQ
ID NO:13), KRFKQDGGWSHWSP (SEQ ID NO:-14), KRFKQDGGWSHWSPWSS
(SEQ ID NO:15), KRFKQDGGWSHW (SEQ ID NO:16) and KRFKQDGGWWSP
(SEQ ID NO:17) or can consist of the amino acid sequence RFK (SEQ ID NO:18).
Such sequences are determined to be activating sequences which enhance wound
healing by screening for enhanced wound healing in rat models of wound healing
as
described in the Examples.
Because TGF-P plays a role in the development of fibrosis, the present
invention also provides a method of preventing fibrosis stimulated by TGF-B in
pathology by administering to a site of potential fibrosis an amount of a
purified
inhibiting peptide from TSP effective to inhibit conversion of latent TGF-P to
active
TGF-R, resulting in reduced fibrosis. The inhibiting peptides can include
those
described herein. As used herein, "fibrosis" means the abnormal formation of
fibrous
tissue (60,64). "Reduced fibrosis," as used herein, is defined as the
statistically
significant reduction in the level of abnormal formation of fibrous tissue as
determined
by histological analysis, tensile strength and total protein and collagen
content in a
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wound treated with an inhibitory peptide from TSP as compared to the level of
abnormal formation of fibrous tissue in a similar untreated wound or a similar
wound
. treated with a peptide having no activity under conditions such that
fibrosis is expected
to develop. One example of this method, using the TSP peptide GGWSHW (SEQ ID
= 5 NO:3), is provided in the Examples.
In the method of preventing fibrosis stimulated by TGF-B in pathology,
the inhibiting peptide can also be selected from the group consisting of:
WNDWI
(SEQ ID NO:19), WSSWS (SEQ ID NO:20), LSKL (SEQ ID NO:21), AAWSHW
(SEQ ID NO:22), DGWSPW (SEQ ID NO:23), GGWGPW (SEQ ID NO:24),
WSPWS (SEQ ID NO:25), GWSHW (SEQ ID NO:26) and WSHWS (SEQ ID
NO:27). Such sequences are determined to be inhibiting sequences which prevent
fibrosis by screening for prevention of fibrosis in rat models of fibrosis
formation as
described in the Examples.
A method is also provided for preventing excessive fibrosis stimulated
by TGF-B in certain pathologies by administering to a site of potential
fibrosis an
amount of a ligand specific for TSP, effective to bind TSP, thereby inhibiting
the
stimulation of TGF-B, resulting in reduced fibrosis.
Active TGF-P inhibits the proliferation of endothelial and epithelial
cells. Thus, in another embodiment, the present invention provides a method of
blocldng TGF-P -mediated inhibition of endothelial or epithelial cell
proliferation,
comprising contacting the cells with an amount of TSP or a purified inhibiting
peptide
from TSP effective to inhibit conversion of latent TGF-P to active TGF-P,
resulting in
proliferation of the cells. As used herein, "proliferation" means an increase
in the
number of cells.
Also provided is a method of blocking TGF-P -mediated inhibition of
endothelial or epithelial cell proliferation, comprising contacting the cells
with a ligand
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specific for TSP effective to bind TSP and inhibit activation of TGF-P,
resulting in
proliferation of the cells.
In the methods of blocking TGF-p -mediated inhibition of cell
5 proliferation, the cells can be arterial endothelial cells. Other cells that
can proliferate =
in response to these methods are capillary endothelial cells. The inhibiting
peptide can
be selected from the group consisting of WNDWI (SEQ II) NO:19), WSSWS (SEQ
ID NO:20), LSKL (SEQ ID NO:21), AAWSHW (SEQ ID NO:22), DGWSPW (SEQ
ID NO:23), GGWGPW (SEQ ID NO:24), WSPWS (SEQ ID NO:25), GWSHW
10 (SEQ ID NO:26) and WSHWS (SEQ ID NO:27) or can consist of the amino acid
sequence GGWSHW (SEQ ID NO:3). Such sequences are determined to be inhibiting
sequences by screening for TGF-P -mediated inhibition of cell proliferation,
as
described in the Examples.
The invention provides TGF-(3 activating and inhibiting peptides
derived from the functional sequences of TSP. The present invention also
provides a
purified peptide having 3 to 30 amino acids, wherein the peptide comprises a
subsequence RI-Xj-X2-X3-R2, wherein Xl is selected from the group consisting
of Arg
and Lys, X2 is selected from the group consisting of Pro and Phe, X3 is
selected from
the group consisting of Lys and Arg, Rl is H2, acyl, or a peptide from I to 26
amino
acids, R2 is H, NH2, or a peptide of from 1 to 26 amino acids, and wherein the
peptide
converts latent TGF-P to active TGF-P.
The purified peptide can be selected from the group consisting of: RFK
(SEQ ID NO:18) KRFK (SEQ ID NO:5), HRFK (SEQ ID NO:6), RKPK (SEQ ID
NO:7), QRFK (SEQ ID NO:8), KRFKQDGG (SEQ ID NO:9),
TAYRWRLSHRPKTGIRV (SEQ ID NO:11), and KRFKQDGGASHASPASS (SEQ
ID NO: 12) or can consist of the amino acid sequence RWRPWTAWSE (SEQ ID
NO: 10).
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The purified peptide can be conjugated to a water soluble polymer
using standard protein conjugation protocols (61). For example, suitable water
soluble
polymers include polysucrose, dextran, polyethylene glycol and polyvinyl
alcohol.
= 5 The purified peptide can also be selected from the group consisting of
partial and full retro-inverso peptide sequences. As used herein, "partial and
full retro-
inverso peptide sequences" means peptide sequences, determined to be either
activating or inhibiting, which comprise some D-amino acids (partial) or
consist
entirely of D-amino acids (full), gem-diaminoalkyl residues, and alkylmalohyl
residues.
These can have unmodified termini, or can include appropriate alkyl, acyl, or
amine
substitutions to modify the charge of the terminal amino acid residues.
The present invention further provides purified peptides consisting of
the amino acid sequences LSKL (SEQ ID NO:21) and acetyl- WHSWAA-NH2 (SEQ
ID NO:28), and their partial and full retro-inverso peptide sequences.
Due to the relatively short half-life of peptides in vivo, the effects of
modified peptides with longer half-lives can be examined. For'example, the
retro-
inverso amino acid sequences (i.e., composed of D-amino acids) of the peptides
described herein, such as the KRFKQDGGWSHWSPWSS (SEQ ID NO: 15) and
GGWSHW (SEQ ID NO:3) peptides, can be employed as described. These are
expected to have a longer half life, because D-amino acids cannot be
metabolized by
ceIls as can naturally occurring L-forms of amino acids in proteins (65). Such
Tetro-
inverso peptides can be synthesized by standard peptide synthesis methods
using
commercially available D-amino acids (74). Peptide mimetics may be employed as
substitutes for the natural peptide sequences based on established methods
(75).
The described peptides can be applied in in vivo models to verify their
modulation of TGF-P -mediated effects of wound healing and fibrosis formation.
For
example, rat models of wound healing can be used to evaluate the effectiveness
of
KRFK (SEQ ID NO:5) in stimulating wound healing in comparison to active TGF-P
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(62,63). An inactive peptide can be used as a negative control (e.g., TRIR
(SEQ ID
NO:30), KRAK (SEQ ID NO:35)).
The GGWSHW (SEQ ID NO:3) peptide or other inhibiting peptides
provided herein can also be examined for any effect on inhibiting wound
healing by
blocking TGF-a activation or for any effect in keloid formation. Inactive
analogues of
this peptide can be used as negative controls.
The in vivo protocol of Sporn et al. (62) can be used to determine the
relative effectiveness of the TSP or activating peptides described herein on
wound
healing. For example, 2 cm by 1 cm wire mesh wound chambers can be implanted
in
the backs of rats. After a wound healing response is initiated (day 4), rats
can be given
daily injections of either 1000 ng TGF-P, 100-1000nM of activating peptides,
100-1000nM TSP, 1000 ng albumin or vehicle control per injection site at the
wound
site. On day 9, the animals can be sacrificed and tissues in the wound
chambers can be
examined histologically, assayed for total protein and collagen content (by
measurement of hydroxy-proline content) and relative levels of TGF-R in the
wound
tissue can be examined by immunohistochemical techniques.
Alternately, a rat model of incisional wound healing as described by
Cromack et al. (63), can be used. In this system, a 6 cm linear incision can
be made on
the dorsal skin of a rat, the wound can be coapted with surgical clamps and
100-
1,000nM of TSP or 100-1000nM of activating peptides can be injected at the
wound
site in 3% methylcellulose as a vehicle. After 7-10 days, the wound strips can
be
harvested and evaluated for tensile strength using a tensiometer and for
histological
analysis as described herein.
The above-described protocols can be applied to humans, because
wound healing and fibrosis formation in rats, rabbits and pigs are commonly
used as =
models for the study of wound healing and fibrosis formation in humans. (66-
69).
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In a clinical application, 1 g to 100 mg of TSP or the activating
peptides from TSP can be used to impregnate bandages or as part of an ointment
to be
applied to wound areas for the purpose of enhancing wound healing or
preventing
fibrosis. A skilled clinician would be able to determine, more specificatly,
the amount
= 5 of peptides and length of treatment necessary to enhance wound healing or
inhibit
fibrosis.
The ligands, TSP or peptides from TSP may be administered
parenterally (e.g., intravenously), by intramuscular injection, by
intraperitoneal
injection, topically, transdermally, or the like, although topical
administration is
typically preferred. The exact amount of such compounds required will vary
from
subject to subject, depending on the species, age, weight and general
condition of the
subject, the severity of the wound or disease that is being treated, the
particular
compound used, its mode of administration, and the like. Thus, it is not
possible to
specify an exact amount. However, an appropriate amount may be determined by
one
of ordinary skill in the art using methods well known in the art. -
For topical administration, the compounds of the present invention can
be in pharmaceutical compositions in the forrn of solid, semi-solid or liquid
dosage
forms, such as, for example powders, liquids, suspension, lotions, creams,
gels or the
like, preferably in unit dosage form suitable for single administration of a
precise
dosage. The compositions will include, as noted above, an effective amount of
the
selected compound in combination with a pharmaceutically acceptable carrier
and, in
addition, may include other medicinal agents, pharmaceutical agents, carriers,
adjuvants, diluents, etc. By "phanmaceutically acceptable" is meant a material
that is
not biologically or otherwise undesirable, i.e., the material may be
administered to an
individual along with the selected compound without causing any undesirable
biological effects or interacting in a deleterious manner with any of the
other
components of the pharmaceutical composition in which it is contained.
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Parenteral administration, if used, is generally characterized by
injection. Injectables can be prepared in conventional forms, either as liquid
solutions
or suspensions, solid forms suitable for solution or suspension in liquid
prior to
injection, or as emulsions. Parenteral administration can also employ the use
of a slow
release or sustained release system, such that a constant level of dosage is
maintained =
(See, for example, U.S. Patent No. 3,710,795).
Utility
The present invention also provides a bioassay for screening substances
for their ability to modulate the activity of TSP. For example, the present
invention
provides a bioassay for screening substances for their ability to enhance the
activity of
TSP, for example, for use in therapies to promote wound healing. In another
example,
the present invention provides a bioassay for screening substances for their
ability to
inhibit the activity of TSP, for example, for use in therapies for prevention
of fibrosis.
Briefly, these bioassays can be performed in vitro by administering a
substance to NRK
cells with TSP or peptides from TSP and latent TGF-P and assaying for soft
agar
colony formation as described in the Examples. Alternatively, these bioassays
can be
performed in vitro by administering a substance to BAE cells and measuring
cell
proliferation as described in the Examples. The current use of such screening
methods
is set forth in the Examples, which show that the peptide KRFK (SEQ ID NO:5)
activates TGF-P and the peptide GGWSHW (SEQ ID NO:3) inhibits TSP-mediated
activation of latent TGF-P. In vivo, these bioassays can be performed by
administering
substances to the wound chambers and wound sites as described herein to screen
for
substances which play a role in wound healing and fibrosis.
The present invention further provides peptides which activate and
inhibit TGF-R, which can be used as controls in in vitro bioassays for
screening
substances for their ability to modulate the activity of TGF-P. For example,
the
substances and TGF-R can be administered to NRK cells which can then be
assayed
for soft agar colony formation as described in the Examples. Activating or
inhibiting
peptides can be administered to NRK cells which can then be assayed for soft
agar
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colony formation as positive controls for TGF-P activation and inhibition.
These
activating and inhibiting peptides can also be used as controls in in vivo
bioassays for
screening substances for their ability to modulate the activity of TGF-(3. For
example,
substances can be applied to the wounds and sites of potential fibrosis in the
assays
5 described in the Examples and evaluated for their ability to enhance wound
healing and
reduce fibrosis. The activating and inhibiting peptides can be used as
controls in the
described Examples.
The invention provides a method of generating a purified antibody
10 specifically reactive with TSP or a peptide of the invention. The
antibodies made can
be used to detect the presence of TSP. Antibodies can be made as described in
the
art (61). Briefly, purified TSP, purified peptide alone or peptide conjugated
to a
carrier protein can be injected into an animal in an amount and in intervals
sufficient to
elicit an immune response. Antibodies can either be purified directly, or, for
15 monoclonal antibodies, spleen cells can be obtained from the animal. The
cells are then
fused with an immortal cell line and screened for antibody secretion.
The following examples are intended to illustrate, but not limit, the
invention. While they are typical of those that might be used, other
procedures known
to those skilled in the art may be alternatively employed.
The present invention is more particularly described in the following
examples which are intended as illustrative. only since numerous modifications
and
variations therein will be apparent to those skilled in the art.
EXAMPLES
Thrombospondin Purification
TSP was purified as previously described (34). Briefly, 8-10 units of
fresh human platelets were purchased from the Birmingham American Red Cross
and
washed with Hepes wash buffer (10% ACD, 0.05M Hepes, 0. 15M NaC1, and 5mM
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16
dextrose), pH 7.6. The platelets were thrombin-stimulated and the platelet
releasate
was applied to a heparin-Sepharosem CL-6B (Pharmacia, Piscataway, New Jersey)
affinity column pre-equilibrated with TBS-C (0.O1M Tris-HCI, 0. 15M NaCI,
0.1mM
CaCI, pH 7.4). The bound TSP was eluted with 0.55M NaCI/ TBS with 1mM CaCI 5
and applied to an A0.5M gel filtration column (Bio-Rad, Richmond,California)
pre-equilibrated with TBS-C, pH 11, to remove associated TGF-P, yielding TSP
stripped of TGF-P activity (sTSP). Purity was assessed by sodium dodecyl
sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue or silver
staining. No contaminating TGF-0 activity was found associated with sTSP in
normal
rat kidney (NRK) soft agar colony formation assays.
Cells
Bovine aortic endothelial (BAE) cells were isolated from aortas
obtained at a local abattoir, and were characterized by the uptake of
acetylated low
density lipoproteins (Dil-AcLDL) and staining for Factor VIII antigen. Cells
were
cultured in Dulbecco's modified Eagle's medium (DMEM; Cell-Gro, Mediatech,
Herndon, VA) supplemented with 4.5 g/L glucose, 2mM glutamine, and 20% fetal
bovine serum (FBS; Hyclone Laboratories, Logan, UT) as previously described
(33).
NRK clone 49F cells (ATCC Accession No. CRL 1570) were cultured in DMEM
supplemented with 4.5 g/L glucose, 2mM glutamine, and 10% calf serum (Hyclone
Laboratories, Logan, UT) as described (1). Calf serum was tested and chosen
for low
levels of active TGF-P. M'uik lung epithelial cells (ATCC Accession No. CCL
64)
(Mv 1 Lu) were cultured in minimum Eagle's medium (MEM; Cell-Gro, Mediatech,
Herndon, VA) supplemented with 4.5g/L glucose, 2mM glutamine, and 10% FBS.
Stocks were cultured in F-12 (Cell-Gro, Mediatech, Herndon, VA) supplemented
with
10% FBS. All cells tested negative forMycoplasma contamination.
Antibodies
Mouse anti-TSP 133 was raised against sTSP and developed at the
Monoclonal Antibody Core facility at the University of Alabama at Birmingham.
This
antibody is a IgG2b which recognizes the 50kDa chymotryptic fragment of sTSP
by
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17
Western blotting. Mab TSP-B7 ascites was raised against human platelet
releasate and
is specific for TSP (11) (Sigma Chemical, St. Louis, Missouri).
A chicken anti-TGF-P antibody was purchased from Oncomembrane,
= 5 Seattle, Waslzington and a mouse monoclonal anti-TGF-R antibody was
purchased
from Genzyme, Cambridge, Masschusetts. Anti-vitronectin monoclonal and
polyclonal
antibodies were purchased from Telios, San Diego, California. A polyclonal
anti-platelet factor 4 antibody was purchased from Atlantic Antibodies,
Scarborough,
ME. Mouse monoclonal anti-basic fibroblast growth factor (bFGF) was obtained
from
Upstate Biotech. Inc., Lake Placid, New York.
Peptide Synthesis
Peptides corresponding to sequences of human TSP 1 as deduced from
a cDNA sequence for TSPI (70), were synthesized on a Mode19600 peptide
synthesizer (Biosearch, San Rafael, California) using standard Merifield solid
phase
synthesis protocols and t-Boc chemistry (56,57,71). Peptides were analyzed for
purity
by reverse-phase HPLC. Larger peptides were also characterized by amino acid
sequencing.
Production of the Second Type 1 Repeat Fusion Construct
The second type I repeat of TSP 1 corresponding to exon 9 was
produced by making PCR primers which correspond to the intron-exon boundaries
of
exons 8-9 and 9-10. The cDNA strands were expanded by polymerase chain
reaction
(PCR) and expressed in E. coli cells using glutathione-S-transferase. The
expressed
proteins were characterized by sequence analysis and gel electrophoresis.
NRK Colony Formation in Soft Agar
TGF-(3 activity was assayed by determining colony formation ofNRK
cells in soft agar assays as described (34), except assays were performed in
24 well
tissue culture plates. Briefly, 5% Noble agar (Difco, Detroit, IVfichigan) was
diluted
ten-fold in 10% calf serum/DMEM and 0.5 ml of this 0.5% agar dilution was
added
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per well to a 24 well tissue culture plate as a base layer and allowed to
harden. A 0.2
mi sample containing 5 ng epidermal growth factor (EGF) was combined with 0.6
ml
0.5% agar and 0.2 ml (2 x 103) of an NRK cell suspension in 10% calf
serum/DMEM.
A 0.5 ml aliquot of this 0.3% agar-sample solution was added to the cooled
agar base
layer and the plates were incubated for seven days at 37 C, 5% COZ. Colonies
greater =
than 62 m in diameter (>8-10 cells) were counted. Experiments were performed
in
triplicate.
BAE Cell Proliferation Assays
BAE cells were plated at 5000 cells per well in 1 ml of DMEM with 20% FBS
in 24 well tissue culture plates and incubated overnight at 37 C, 5% CO2. The
cells
were rinsed once in serum-free DMEM. Test samples in 0.5 m12.5% FBS DMEM
were added to each well in triplicate (day 0). On day 2, cells received fresh
aliquots of
test sample in 0.5 ml without removing the original medium (to give a final
volume of
1 ml). CeDs were grown for another two days, then culture medium was removed
and
cells were trypsinized with 0.5 ml trypsin-EDTA (Gibco, Grand Island, NY) and
harvested. The number of harvested cells was determined using a model ZM
Coulter
Counter(Coulter Electronics, H'ialeah, FL).
Preparation of BAE Conditioned Medium
BAEs were plated at a density of 100,000 cells in a 25 cm2 flask in 20%
or 0.2% FBS/DMEM and incubated overnight at 37 C, 5% COZ. This density was
determined by comparing the ability of sTSP to activate latent TGF-P in
sparse,
sub-confluent, and confluent BAE cultures. This cell density showed the
greatest
difference in levels of active TGF-P between control and TSP-treated medium.
Flasks
were rinsed once with 2 ml serum-free DMEM and then test samples were added in
2.5 ml of DMEM with either 0.2% or 20% FBS. The flasks were incubated for
additional times at 37 C, 5% CO2. Conditioned medium was collected,
centrifuged at
1200 rpm for five minutes to remove cellular debris, and stored at 4 C in
polypropylene tubes for no more than 3 days before testing in NRK soft agar
assay to
determine TGF-P activity.
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Activation of Purified Latent Recombinant TGF-P by sTSP or Peptides
Various concentrations of sTSP or the synthesized peptides were
incubated with 2nM (200 ng/ml) purified small latent recombinant TGF-P (LTGF-
R)
(Bristol-Myers Squibb, Seattle, WA) in a final volume of 0.5 ml phosphate
buffered
saline (PBS) for one hour at 37 C. As a positive control, 4mM HCL was used for
TGF-(3 activation. PBS was added to cells to establish a baseline for cell
proliferation.
Bovine serum albumin (BSA) (0.1%) was added to all of the samples to reduce
non-
specific binding of TGF-P to the tube. Samples were tested in soft agar NRK
colony
formation assays for TGF-R activity.
Effect of Protease Inhibitors on the Sensitivity of sTSP-Mediated Stimulation
of
TGF- P Activity
BAE cells were seeded at 1 x 105 cells/ 25 cm2 flask in DMEM with
20% FBS and incubated overnight. Cells were washed with DMEM, and one of the
following protease inhibitors: e-aminocaproic acid (EACA, 0.3mM), aprotinin
(6mM),
and alpha2-antiplasmin(0.6uM) was added, with 1 g sTSP (0.4 ug/ml), to each
flask
in DMEM with either 2.5 ml 0.2% or 20% FBS and then incubated with cells an
additional 48 hrs. Aliquots of conditioned medium were tested in NRK colony
forming
soft agar assays to determine TGF- P activity. Recombinant TGF- P (5 ng/ml)
was also
incubated with the inhibitors and assayed for colony forming activity.
Inhibitors alone
were also added to the conditioned medium.
Effect of sTSP on Mature rTGF-P Activity and Stimulation of TGF-P Activity in
FBS
The NRK colony forming assay was performed as desccribed herein.
All samples contained 5 ng/ml EGF in 10% calf serum/DMEM, except samples in
which EGF was in 0.2% FBS. Stripped TSP (1 g/ml) was preincubated with 1
ng/nil
rTGF-P for two hrs at 37 C and compared for relative activity versus I ng/ml
rTGF-
or 1 g/mi sTSP. Anti-sTSP antibody Mab133 (10 g/ml) was pre-incubated with
recombinant TGF-P (rTGF-P) (Bristol Myers Squibb, Seattle, WA) for two hrs at
37 C before addition to NRK cells in soft agar. sTSP (3 g/ml) was also
incubated
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with 0.2% FBS for two hrs at 37 C and tested for relative activity versus sTSP
(3
g/ml) or 0.2% FBS. All samples were tested in the NRK soft agar for seven days
at
37 C, 5% CO2.
5 TSP Stripped of TGF-(3 Activity Inhibits the Growth of BAE Cells To examine
whether TSP stripped of associated TGF-P (sTSP) activity
inhibited BAE growth, ceU proliferation assays were performed using sTSP. BAE
cells
were exposed to increasing concentrations of either native TSP (TGF-P activity
associated with TSP), or sTSP (no associated TGF-P activity) in medium
10 supplemented with 2.5% FBS for a period of four days, at which time cell
number was
determined. Native TSP and sTSP significantly inhibited the proliferation of
BAE cells
as compared to 2.5% FBS alone. Furthermore, the dose response curves were
nearly
identical for native and sTSPs. No significant ceU death was observed. The
inhibition
of BAE proliferation by TSP was concentration dependent with 1 g/mi sTSP
15 inhibiting 75% of growth. Cells grown in the presence of sTSP assumed a
more
elongated, fibroblastic shape and had prominent nucleoli as compared to the
polygonal
cells in the 2.5% FBS medium control. Similarly, TGF-P treated cells were
elongated
with numerous processes and prominent nucleoli.
20 A neutralizing antibody to TGF-p reversed the growth inhibitory effects
of sTSP by 42%. Addition of the neutralizing antibody against TGF- (3 to wells
containing sTSP also caused a partial reversion to a smaller, more polygonal
cell,
characteristic of normal BAE cells. Similar results were obtained with both
mouse and
chick anti-TGF-(3 antibodies. In contrast, a polyclonal antibody and various
monoclonal antibodies specific for native TSP were not able to neutralize
sTSP-mediated growth-inhibition. Antibodies alone did not affect cell growth.
Thus,
growth inhibition of BAE cells by TSP stripped of associated TGF-P activity
may be at
least partially due to a TGF-p -dependent component.
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Stripped TSP Activates TGF-P in BAE Conditioned Media (CM)
Since sTSP-mediated BAE growth inhibition is partially TGF-P
dependent, it is possible that sTSP incubation with BAE cells is causing
activation of
endogenous latent TGF-R. TGF-P is secreted from endothelial cells as an
inactive
molecule (18) and it is not entirely clear how endothelial cell latent TGF-(i
is activated.
In order to test the hypothesis that sTSP activates latent TGF-P secreted by
endothelial cells, sTSP was added to BAE cells in DMEM with 0.2% FBS for 0-48
hrs.
Aliquots of the conditioned medium were tested in NRK colony
forming soft agar assays for the presence of TGF-P activity. Stripped TSP at
0.4
g/ml (0.9nM) increased colony forming activity in the conditioned media by 2-3
fold
as compared to conditioned medium alone. Increases in TGF-P activity were
observed
as early as 15 minutes after addition of sTSP to cells and persisted above
control levels
for at least 48 hrs. Similar levels of activation were observed when cells
were
conditioned in media where serum levels were raised from 0.2% to 20%,
suggesting
that sTSP-mediated stimulation of TGF-P activity is independent of serum
factors.
Stimulation of TGF-P Activity is Dependent on sTSP Concentration.
To assess whether the stimulation of TGF-P activity in BAE
conditioned media was dependent on the concentration of sTSP present, varying
doses
of sTSP ranging from 10 ng to 10 g were added to BAE cells in 2.5 ml of
medium.
Concentrations of sTSP between 40-400 ng/ml (100-1000 ng added) were effective
at
stimulating NRK colony formation in soft agar. The maximal response was
repeatedly
observed with 1 g sTSP/25 cm2 flask (0.4 g/ml, or 0.9 nM). When compared
rTGF-P, the level of maximal NRK colony formation induced by sTSP correlated
to
approximately 0.1 ng/ml of TGF- a activity.
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Stripped TSP Does Not Affect the Activity of Mature rTGF-p or Stimulate TGF-
P Activity in FBS
To rule out that the increase in TGF-(3 activity in the NRK soft agar
assays was due to sTSP acting at the level of the NRK cells, experiments were
done to
determine whether sTSP affected mature rTGF-(3 activity and whether anti-sTSP
antibody 133, which inhibits sTSP stimulation of TGF-p activity in the
conditioned
medium, affected TGF-P activity in the NRK assay. There was no modulation of
rTGF-(3 activity by either sTSP or anti-TSP antibodies, nor did sTSP by itself
stimulate
colony formation. Stripped TSP also did not activate the latent TGF-P present
in
0.2% FBS.
Stimulation of TGF-P Activity in BAE Conditioned Media is Specific for sTSP
Other extracellular matrix proteins were tested for their ability to
activate endothelial cell secreted latent TGF-P. Equimolar amounts of
tenascin,
fibronectin, BSA, or laminin did not stimulate TGF-0 activation. Basic FGF, in
contrast to a previous report (13), did not stimulate increased TGF-P activity
in our
system. These results show that stimulation of TGF-P activity in BAE
conditioned
medium is not a general property of extracellular matrix molecules, including
TGF-P -binding molecules such as fibronectin, and, therefore, is a specific
property of
sTSP.
Antibodies to sTSP Inhibit Stimulation of TGF R Activity by sTSP.
To eliminate the possibility that the observed increase in TGF-P activity
was due to potential components associated with sTSP, an attempt was made to
block
stimulation with antibodies to TSP. Monoclonal antibody 133 ascites, which
recognizes an epitope in the 50kDa chymotryptic fragment of sTSP, completely
inhibited the stimulation of TGF-P activity by sTSP. Mab TSP-B7 ascites, which
is
specific for the 70kDa core of platelet TSP (11) also blocked this effect of
sTSP.
However, another monoclonal antibody which recognizes an epitope in the 70kDa
fragment of native TSP only inhibited sTSP activation of latent TGF-P by 32%.
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Antibodies alone had no effect on these assays and did not interfere with the
ability of
rTGF-0 to form colonies in soft agar.
Colony formation was also TGF-P dependent, since a polyclonal
chicken anti-TGF-0 antibody and a monoclonal mouse anti-TGF-P neutralizing
antibody completely inhibited colony formation. These results showed that the
factor
activated by sTSP in BAE conditioned medium is TGF-(3.
In contrast, antibodies to vitronectin (both monoclonal and polyclonal),
platelet factor-4, bFGF, and control ascites, did not inhibit the stimulation
by sTSP.
These data showed that increases in TGF- P activity observed in the NRK soft
agar
assays were not due to the presence of commonly associated matrix and platelet
proteins, but were dependent on sTSP and TGF-R.
Stripped TSP Stimulation of TGF-P Activity in BAE Conditioned Medium
Occurs Independently of Binding to the Cell Surface.
A proposed mechanism of latent TGF-P activation in vivo is through
binding to and internalization of latent TGF-P by mannose-6-phosphate
receptors and
subsequent processing in acidifying vesicles or processing by plasmin at the
cell surface
(12,20,21). Experiments were performed to determine whether sTSP requires
interactions with cell surface molecules in order to activate latent TGF- R.
After
incubating BAE cells in DMEM with 0.2% FBS overnight, the medium was removed
from the culture flasks and incubated in polypropylene tubes in the presence
or absence
of sTSP (0.4 pg/ml) for the indicated times. This was done in direct
comparison with
sTSP incubated in the presence of cells. Aliquots of the conditioned medium
were
then tested in NRK colony forming soft agar assays for sTSP-mediated
activation of
TGF-P. These data showed that sTSP was able to activate TGF-P in the absence
of
cells to a similar extent and with similar Icinetics to sTSP incubated in the
presence of
cells. Cell-conditioned medium incubated with sTSP in the absence of cells
demonstrated increased TGF-(3 activity as early as 15 min after addition of
sTSP.
Maximal levels were reached by two hrs and persisted above baseline for at
least 48
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hrs. Thus, in contrast to previously reported mechanisms of activation, TSP-
mediated
activation of latent TGF-(3 does not require interactions with cell surface
molecules.
Stripped TSP Mediated Stimulation of TGF-P Activity is Insensitive to Serine
Protease Inhibitors.
Previous studies have shown that plasmin can activate latent TGF-P in vitro
(22,23). In co-cultures of endothelial and smooth muscle cells, plasmin levels
have
been shown to be upregulated, thus, activating latent TGF-P (40,4 1). A common
motif is the involvement of a serine protease in the activation of latent TGF-
P.
Therefore, the effects of different serine protease inhibitors on the
activation of TGF-P
by sTSP in BAE conditioned medium were tested. BAE cells were incubated with
sTSP (0.4 g/ml) in addition to either e-aminocaproic acid (EACA, 0.3mM),
aprotinin
(6mM), or alpha2-antiplasmin (0.6uM). The concentrations of these inhibitors
were
chosen based on previous studies (40) and dose response assays. These
inhibitors did
not inhibit sTSP-mediated activation of TGF-P and had no effect on rTGF-P
activity
in soft agar assay. Inhibitors alone were also added to the conditioned medium
and
had no effect on the assay.
Due to evidence that TSP can interact with these serine proteases (7),
sTSP was also tested for associated plasmin and thrombin activity using enzyme
assays
measuring generation of chromogens from specific substrates (Boehringer-
1Vlannheim,
Indianapolis, IN). No associated plasmin or thrombin activity was detected in
sTSP
and there was no generation of plasmin activity in sTSP-conditioned medium as
compared to control conditioned medium.
These data showed that, in contrast to activation of endothelial
cell-derived latent TGF-R by bFGF or in co-culture systems, latent TGF-a
activation
by sTSP does not involve serine proteases.
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Stripped TSP Can Activate Purified Recombinant Latent TGF-P (LTGF-P)
To determine if sTSP was activating latent TGF-P without the
involvement of cell-secreted factors, sTSP was incubated with LTGF-P for two
hrs
and then assayed for TGF-p activity. Stripped TSP was able to activate LTGF-P
at
5 both 37 C and 4 C. Stripped TSP at a concentration of l3nM could activate
approximately half of the acid-activatable LTGF-P. These results showed that
sTSP
was able to activate LTGF-P directly, without the involvement of cell-secreted
factors
such as proteases.
10 A Unique Peptide in the Second Type 1 Repeat Activates Latent TGF-P
To determine if TGF-P activation was due to either of the type I
consensus sequences, CSVTCG (SEQ ID NO: 1) and WSXW (SEQ ID NO:2), the
peptides, VTCGGGVQKRSRL (SEQ ID NO:29) and KRFKQDGGWSHWSPWSS
(SEQ ID NO: 15), were constructed and analyzed to determine the effect of
these
15 sequences on activation ofTGF-P.
Latent TGF-P was incubated with equimolar concentrations of sTSP or
the above peptides and activation of TGF- j3 was assayed by soft agar NRK
colony
formation. As shown in Table I, the addition of TGF-P incubated with I InM
sTSP
20 increased NRK colony formation approximately twofold over the PBS baseline
control. The VTCGGGVQKRSRL (SEQ ID NO:29) peptide failed to activate latent
TGF-P when tested at concentrations up to 11 M. The KRFKQDGGWSHWSPWSS
(SEQ ID NO: 15) peptide increased colony formation to levels equal to those
observed
with sTSP. These data indicated that the mechanism by which TSP activates
latent
25 TGF-p is independent of the CSVTCG (SEQ ID NO:1) cell adhesion motif, and
is
associated with the KRFKQDGGWSHWSPWSS (SEQ ID NO: 15) peptide.
The amino acid sequence, arginine-phenylalanine-lysine activates latent TGF-P
To further localize which region within the
KRFKQDGGWSHWSPWSS (SEQ IDNOA) sequence activates latent TGF-(3, the
following peptides, containing deletions at the carboxy-terminal of the
peptide, were
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26
constructed: KRFKQDGGWSHWSP (SEQ ID NO: 14), KRFKQDGGWSHW (SEQ
ID NO:16), KRFKQDGGWWSP (SEQ ID NO:17), KRFKQDGG (SEQ ID NO:9),
KRFK (SEQ ID NO:5) and RFK (SEQ ID NO:18). Recombinant latent TGFP was
incubated with equimolar concentrations of the peptides, sTSP or the
KRFKQDGGWSHWSPWSS (SEQ ID NO:15) peptide and tested for TGF-p activity.
As shown in Table I, all peptides containing the amino-terminal basic residues
of
KRFKQDGGWSHWSPWSS (SEQ ID NO: 15) activated latent TGF-P to levels
comparable to sTSP. The tripeptide, RFK (SEQ ID NO:18), represented the
minimal
sequence required to activate TGF-P. Deletion of the WSHW (SEQ ID NO:2)
sequence from the peptide failed to diminish the TGF-P activating potential,
which
indicates that the consensus sequence WSXW may have no direct role in the
activation
of latent TGF-(3 by TSP.
These data were consistent with the results of experiments
demonstrating the inability of fusion constructs of the amino acids encoded by
exon 9
to activate latent TGF-(3. Exon 9 encodes amino acid residues Lys 415 through
Ile
473 and contains the entire second type I repeat sequence of TSPl. However,
the
fusion protein produced lacks the KRF sequence. These data provided fiuther
support
that the sequence (K)RFK is required for activation of latent TGF-P.
Certain Amino Acids Within the KRFK Sequence are Necessary for Activity
To determine which amino acids in the (K)RFK sequence are necessary
for activation of latent TGF-P, peptides containing anuno acid substitutions
were
synthesized and tested for their ability to activate latent TGF-(3. The
results of this
experiment are shown in Table H.
As shown in Table II, the peptide, RRFK (SEQ ID NO:5), activated
latent TGF-P twofold over the baseline control. The peptide, TRIR (SEQ ID
NO:30)
(the corresponding sequence in the second type 1 repeat in TSP2), did not
activate
latent TGF-P, indicating that the activation of TGF-R is a function specific
to TSP1.
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The substitution of Lys 412 with amine group-containing residues, Gin
(QRFK (SEQ ID NO:8)) or His (HRFK (SEQ ID NO:6)), did not diminish activity.
Substitution of Lys 412 with Ala (ARFK (SEQ ID NO:31)), which lacks an amine
group, abrogated activity (Table II).
Arg 413 is also important for activity. Arg was replaced with Lys
(KI,CFK (SEQ ID NO:32)) without diminishing activity, but substitution with a
Gln,
which lacks a positively charged amine group, (KQFK (SEQ II) NO:33)) abolished
activity. This suggests that a positively charged amine or guanidino group in
position
413 is necessary for activity. Similarly, substitution of Lys 415 with Gln
(KRFQ(SEQ
ID NO:34)) resulted in loss of activity.
As further demonstrated in Table II, Phe 414 was required for activity.
Substituting Ala (KRAK (SEQ ID NO:35)) for Phe 414 resulted in loss of
activity.
Other aromatic residues such as Tyr (KRYK (SEQ ID NO:36)) or Trp (KRWK (SEQ
ID NO:37)) cannot substitute for Phe 414, because these amino acid
substitutions
inactived the peptide. These experiments demonstrated a specific requirement
for Phe
in this position.
The tripeptide, RFK (SEQ ID NO:18) activated latent TGF-(3 2.5 fold
over baseline controls. However, substitution of Lys 415 with Arg (RFR (SEQ ID
NO:43)) abolished activity, as did replacement of both Phe 414 and Lys 415
with Trp
and Arg (RWR (SEQ ID NO:44)), respectively (Table II).
In addition, a peptide with an inverted sequence of
KRFKQDGGWSHWSPWSS (SEQ ID NO:15), composed of D-amino acids and
modified with an N-terminal acetyl and a C-ternunal amide, was synthesized to
obtain a
peptide with a longer physiologic half-life. This retro-inverso peptide
activated latent
TGF-P at equimolar concentrations of sTSP. These data indicated that this
peptide
might have more sustained TGF-P modulating activity under physiologic (in
vivo)
conditions than the standard peptide.
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The peptides listed in Table III either activate or inhibit activation of
latent TGF- P as determined by analysis using the experimental protocols
described in
the Examples herein.
Activation of latent TGF-P by (K)RFK is Independent of Heparin-binding
Activity
BBxB is a well known heparin-binding motif wherein B represents a
basic amino acid (K,R,H) and x is any amino acid (72). The
KRFKQDGGWSHWSPWSS (SEQ ID NO: 15) peptide of TSP has been shown to
bind heparin, but this activity is localized to the WSHW (SEQ ID NO:4) region
of the
peptide (56,7 1). To examine whether activation of latent TGF-P by TSP was
associated with the BBxB motif, TSP peptide Hep II,
ASLRQMKKTRGTLLALERKDHS (SEQ ID NO:38) (residues 74-95), which
contains the BBxB motif and binds heparin (73), and a second heparin-binding
TSP
peptide laclcing this consensus motif, Hep I, ELTGAARKGSGRRLVKGPD (amino
acids 17-35) (SEQ ID NO:39), were analyzed for TGF-R activating capability.
Neither Hep I nor Hep II activated latent TGF-P when assayed at concentrations
up to
11 M. These results demonstrated that activation of latent TGF-R is a
specific
function of the KRFK (SEQ ID NO:5) sequence of TSP and is not dependent upon
the
BBxB motif.
Because heparin is an anionic polysaccharide, experiments were
conducted to determine if heparin blocked TGF-R activation by either sTSP or
the
peptides. It was found to have no effect. Based on these results, it was
determined that
the binding/activation of latent TGF-R by sTSP or the peptides is not mediated
via
carbohydrate interactions.
The RFK Sequence Present in other Proteins does not have TGF-P Activating
Function
Calcineurin (Sigma Chemicals, St. Louis, Missouri) and BSA, both of
which contain the RFK (SEQ ID NO:5) sequence, were examined to determine if
CA 02590837 2007-06-18
WO 95/05191 PCT/US94/09193
29
activation of latent TGF-a by this sequence is a function of this peptide
sequence in
other proteins. Latent TGF-P was incubated with equimolar amounts of sTSP,
calcineurin, or BSA and assayed for activation. As determined by soft agar NRK
colony formation, only sTSP activated TGF-a, indicating that the RFK (SEQ ID
NO:5) sequence as it is presented within the calcineurin (KRFK (SEQ ID NO:5))
and
BSA (HRFK (SEQ ID NO:6)) proteins lacks the TGF-P activity function.
Modification of the Trp Residues in the Larger Peptides Results in a Loss of
Activity
The above results showed that the sequence (K)RFK is directly
responsible for the activation of latent TGF-P by sTSP. To determine whether
other
residues in the larger peptides are important for TGF-p activation, amino acid
residues
Trp 420, Trp 423 and Trp 426 were all substituted to Ala residues in the
KRFKQDGGWSHWSPWSS (SEQ ID NO:15) peptide and KRFKQDGGWSHWSP
(SEQ ID NO: 14) peptide to produce the peptides KRFKQDGGASHASPASS (SEQ
ID NO:12) and KRFKQDGGASHASP (SEQ ID NO:13) and each of these was tested
for TGF- R activating potential. Latent TGF- R was incubated with increasing
concentrations of each of the four peptides or TSP and assayed for NRK colony
forming activity. The substitution of Trp residues with Ala residues abolished
the
TGF- P activating function of the Trp-containing peptides at the nanomolar
concentrations previously shown to be effective for the unmodified peptides or
sTSP.
However, peptides lacking the Trp residues activated latent TGF-P when applied
at
concentrations greater than 1 M. These data showed that while the (K)RFK
sequence
alone is sufficient to activate latent TGF-(3, other amino acid specificities
appear to be
required to properly orient the (K)RFK sequence within larger peptides and,
similarly,
within intact TSP.
The sequence GGWSHW inhibits sTSP-mediated activation of latent TGF P
To determine whether the GGWSHW (SEQ ID NO:3) sequence
competitively blocks the activation of TGF-P by the TSP trimer, latent TGF-R
was
incubated with sTSP in the presence of increasing concentrations of the
peptide
CA 02590837 2007-06-18
WO 95/05191 PCT/US94/09193
GGWSHW (SEQ ID NO:3) and assayed for activation. In a soft agar NRK colony
formation assay, TGF-P, combined with only sTSP up to concentrations of 11W
increased colony formation by approximately twofold over PBS baseline
controls.
However, when latent TGF-~ was incubated with 11nM sTSP in the presence of a
5 100-fold molar excess (1.1 M) of the GGWSHW (SEQ ID NO:3) peptide, TGF-P
activity was completely inhibited. The inhibition of sTSP-mediated activation
of latent
TGF-P by the GGWSHW (SEQ ID NO:3) peptide was dependent on the peptide
concentration, with 100% inhibition observed after applying the peptide at a
concentration of 1.luM.
Members of the TGF-P receptor superfamily also contain the sequence
WSXW (SEQ ID NO:2) and it is possible that the decrease in activity observed
may be
due to competition of the peptide with active TGF-P for TGF-P receptor binding
sites
rather than a physical blocldng of TSP-TGF-R interactions. To examine this
possibility, human platelet TGF-P (R&D Systems, M'inneapolis, Mmnesota) was
activated according to the manufacturer's instructions with 4mM HCI and pre-
incubated with 1.1 M of the GGWSHW (SEQ ID NO:3) peptide for 30 minutes to
maximize the possible interactions between the peptide and TGF-P. The TGF-P
activity of this sample was assayed by soft agar NRK colony formation assay
and
20. compared with the TGF-(3 activity of activated human platelet TGF-P
incubated
without the peptide. The GGWSHW (SEQ ID NO:3) peptide had no effect on the
activation of TGF-P as compared to human platelet TGF-(3 alone. Thus, the
inhibitory
effect of the GGWSHW (SEQ ID NO:3) peptide does not appear to be at the level
of
competitive bloclcing of TGF-p receptor binding sites.
The Sequence GGWSHW Blocks TGF-p-mediated Inhibition of Endothelial Cell
Growth
BAE cells were seeded at 5 x 103 cells/well in a 24 well plate (Corning,
Corning, New York) and allowed to attach overnight at 37 C, 5% CO2. The wells
=
were washed once with DMEM containing no FBS. Samples of the described
peptides
or intact sTSP were added in 0.5 ml 2.5% FBS/DMEM and incubated for four days
at
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31
37 C, 5% COZ. Cells were fed after 48 hrs with additional sTSP or peptides and
incubated an additional 48 hrs in a total volume of 1 ml of medium. Cells were
then
trypsinized and counted on a Coulter Cell Counter model ZM (Coulter
Electronics,
Hialeah, Florida). Inhibiting peptides resulted in higher levels of cell
proliferation
compared to peptides having no activity.
In these experiments, GGWSHW (SEQ ID NO:3) in 1000-10,000-fold
molar excess blocked 36-47% of TSP-mediated BAE growth inhibition (Table M.
These experiments suggest that the inhibitory GGWSHW (SEQ ID NO:3) peptide can
be an effective reagent to block TSP activation of latent TGF-(3 in a cellular
environment. Other inhibiting peptides are expected to have the same effect on
cell
proliferation because of their similar action on TGF-P.
The Sequence KRFKQDGGWSHWSPWSSC (SEQ ID NO:45) Inhibits
Proliferation of Endothelial CeAs
Corneal bovine endothelial cells (BCE cells) were used at passages 2
through 8 (76). BCE cell cultures were maintained in DMEM (low glucose),
containing 10% FCS, 4mM glutamine, 2.5 g/mi amphotericin B, and 500 U/ml each
of penicillin G potassium and streptomycin sulfate (all media components were
from
Biofluids Inc., Rockville, Maryland). BCE cells were grown at 34 C in 5% CO2.
The
medium was changed every 2-3 days.
Endothelial cell proliferation was measured using the CELL TITER
96~ assay (Promega, Madison, Wisconsin). 5 x 103 cells were plated into each
well
of a 96-well culture plate in 0.5 or 5% FCS-containing medium together with
the
indicated concentrations of growth effectors. After 72 hrs, 15 l of dye
solution were
added to each well, and the plates were incubated for an additional four hrs.
Solubilization solution was added and absorbance at 570 nm was deten;nined
after 24
hrs, as described by the manufacturer.
CA 02590837 2007-06-18
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32
The activating peptide KRFKQDGGWSHWSPWSSC (SEQ ID
NO:45) conjugated to FICOLL was a potent inhibitor of endothelial cell
proliferation,
with an inhibitory concentration so (IC50) consistently less than 1 pM. The
FICOLL
carrier without peptide was inactive. The FICOLL conjugate of the activating
peptide
GGWSHWSPWSSC (SEQ ID NO:46), which lacks the amino terminal basic amino
acid sequence, was also strongly active for inhibiting proliferation of
endothelial cells.
Similar inhibitory activities were observed using cells grown in 0.5% or 5%
fetal calf
serum. Other peptides that activate TGF-P should have the same effect because
of
their action on TGF-P.
Enhancement of Wound Healing by Administration of the KRFK Peptide
A 2 cm by 1 cm wire mesh wound chamber is implanted into the backs
of rats. After a wound healing response is initiated (day 4), the rats are
given daily
injections of 100-1000nM KRFK (SEQ ID NO:5), 1000 ng TGF-P, 1000 ng albumin
or vehicle control per injection site at the wound site. On day 9, the animals
are
sacrificed and tissues in the wound chamber are examined histologically and
assayed
for total protein and collagen content (by measurement of hydroxy-proline
content).
Relative levels of TGF-P are examined in the wound tissue
by=immunohistochemical
techniques..
Alternatively, a six cm linear incision is made in the dorsal skin of a rat,
the wound is coapted with surgical clamps and 100-1000nM KRFK, 1000 ng TGF-(3,
1000 ng albumin (in 3% methylcellulose as a vehicle) or vehicle control per
injection
site is injected at the wound site. Other controls could include inactive
analogs of
KRFK (SEQ ID NO:5), such as KRAK (SEQ ID NO:35), TRIR (SEQ ID NO:30) or
KRWK (SEQ ID NO:37). After 7-10 days, wound strips are harvested and evaluated
for tensile strength using a tensiometer and for histological analysis as
described above.
Enhanced wound healing would be determined by histological evaluation of
cellularity
of the wound site (measurement of DNA content), the presence of collagen
Sbrils, and
of re-epithelialization of the wound surface. Clinicians familiar with this
condition
would be able to make a determination that a statistically significant
increase in the rate
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33
of wound healing has occurred. For example, one skilled in the art could
evaluate how
much change in the DNA content is indicative of enhanced wound healing in a
treated
wound relative to an untreated wound. Also, a skilled artisan could readily
determine
the amount of re-epithelialization required for enhanced wound healing.
Furthermore;
one skilled in the art could readily assess the tensile strength of a wound in
evaluating
enhanced wound healing.
Prevention of Fibrosis by Administration of the GGWSHW Peptide
A 2 cm by 1 cm wire mesh wound chamber is implanted into the backs
of rats. After a wound healing response is initiated (day 4), the rats are
given daily
injections of 100-1000nM GGWSHW (SEQ ID NO:3) peptides, 100-2000 ng TGF-
(3, 100-2000 ng albumin or vehicle control per injection site at the wound
site. On
day 9, the animals are sacrificed and tissues in the wound chamber are
examined
histologically and assayed for total protein and collagen content (by
measurement of =
15. hydroxy-proline content). Relative levels of TGF-R are examined in the
wound tissue
by immunohistochemical techniques.
Alternatively, a six cm linear incision is made in the dorsal skin of a rat,
the wound is coapted with surgical clamps and 100-1000nM GGWSHW. (SEQ ID
NO:3), 100-2000 ng TGF-P, 100-2000 ng albumin (in 3% methylcellulose as a
vehicle) or vehicle control per injection site is injected into the wound
site. Other
controls could include inactive analogs of GGWSHW (SEQ ID NO:3), such as
SHWWSS (SEQ ID NO:40), GGWSHY (SEQ ID NO:41) and GGWSKW (SEQ ID
NO:42). After 7-10 days, the wound strips are harvested and evaluated for
tensile
strength using a tensiometer and for histological analysis as described above.
The
primary measure of fibrosis would be an evaluation of the collagen content of
the
wound by histological analysis with a trichrome stain for connective tissue
and by
measurement of hydroxyproline content of a defined wound area from a punch
biopsy.
Clinicians familiar with this condition would be able to make a determination
that a
statistically significant reduction in fibrosis has occurred. For example, one
skilled in
the art could evaluate how much change in the collagen content of a treated
wound is
CA 02590837 2007-06-18
WO 95/05191 PCTIUS94/09193
34
indicative of reduced fibrosis relative to an untreated wound. Also, a skilled
artisan
could readily determine the amount of hydroxyproline which is indicative of
reduced
fibrosis.
Topical Treatment of a Wound with TSP or Peptides from TSP to Eahance
Wound Healing
In a clinical application, 1 g to 100 mg of purified TSP or activating
peptides from TSP are impregnated in a bandage which is applied directly to a
wound
or are incorporated into an ointment which is applied directly to the wound. A
skilled
clinician would be able to determine the amount of TSP or peptides from TSP
and
length of treatment necessary to enhance wound healing, depending on the
patient's
age, size and the site and condition of the wound.
Topical Treatment of a Site of Potential Fibrosis with TSP Peptides to Reduce
Fibrosis
In a clinical application, 1 g to 100 mg of purified inhibiting peptides
of TSP are impregnated in a bandage which is applied directly to a site of
potential
fibrosis or are incorporated into an ointment which is applied directly to a
site of
potential fibrosis. A skilled clinician would be able to determine the amount
of
peptides and length of treatment with the peptides necessary to reduce
fibrosis,
depending on the patient's age, size and the condition of the site of
potential fibrosis.
Throughout this application, various publications are referenced. The
disclosures of these publications in their entireties are hereby incorporated
by reference
into this application in order to more fully describe the state of the art to
which this
invention pertains.
Although the present process has been described with reference to
specific details of certain embodiments thereof, it is not intended that such
details
should be regarded as limitations upon the scope of the invention except as
and to the
extent that they are included in the accompanying claims.
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WO 95/05191 PCT/US94/09193
TABLE I. The minimal sequence required for activation of latent TGF-0 is the
RFK
peptide sequence of TSP.
Fold
Sequence Activation of
Latent TGF- P
over Baseline
5 sTSP 2x f 0
VTCGGGVQKRSRL (SEQ ID NO:29) none
KRFKQDGGWSHWSPWSS (SEQ ID NO:15) 2x f 0
KRFKQDGGWSHWSP (SEQ ID NO: 14) 2.3x 0.2
10 KRFKQDGGWSHW (SEQ ID NO:16) 1.9x f 0.2
KRFKQDGGWWSP (SEQ ID NO:17) 2.1x f 0.2
KRFKQDGG (SEQ ID NO:9) 2.lx f 0.1
KRFK (SEQ ID NO:5) 2x f 0.1
RFK(SEQID NO:18) 2.5xt0.5
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36
TABLE U. Certain amino acids within the KRFK sequence of TSP are necessary for
TGF-P stimulating activity.
Fold Activation of
Sequence Latent TGF-P over
Baseline
sTSP 2x t 0
KRFK (SEQID NO:5) 2xf0.1
TRIR (SEQ ID NO:30) none
QRFK (SEQ ID NO:8) 2X f 0
HRFK (SEQ ID NO:6) 2X f 0
KKFK(SEQIDNO:32) 2.2t0.15
KQFK (SEQ ID NO:33) none
KRFQ (SEQ ID NO:34) none
KRAK (SEQ ID NO:35) none
KRYK (SEQ ID NO:36) none
KRWK (SEQ ID NO:37) none
RFK (SEQ ID NO:18) 2.5x f 0.5
RFR (SEQ ID NO:43) none
RWR (SEQ ID NO:44) none
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37
TABLE III: Peptides which Activate or Inhibit Activation of Latent TGF-0
Activating Peptides Inhibiting Peptides
KRFK (SEQ ID NO:5) WNDWI (SEQ ID NO:19)
HRFK (SEQ ID NO:6) GGWSHW (SEQ ID
NO:3)
RKPK (SEQ ID NO:7) LSKL (SEQ ID NO:21)
QRFK (SEQ ID NO:8) DGWSPW (SEQ ID
NO:23)
RFK (SEQ ID NO:18) GGWGPW (SEQ ID
NO:24)
KRFKQDGG (SEQ ID NO:9) WSPWS (SEQ ID
NO:25)
RWRPWTAWSE (SEQ ID NO: 10) GWSHW (SEQ ID
NO:26)
TAYRWRLSHRPKTGIRV (SEQ ID NO:11) WSHWS (SEQ ID
NO:27)
KRFKQDGGASHASPASS (SEQ ID NO:12) WSSWS (SEQ ID
NO:20)
KRFKQDGGASHASP (SEQ ID NO: 13) retro-inverso acetyl
WHSWAA (SEQ ID
NO:28)-NH2
KRFKQDGGWSHWSPWSSC (SEQ ID NO:45) AAWSHW (SEQ ID
NO:22)
GGWSHW2SPWSSC (SEQ ID NO:46)
TABLE IV. The inhibitory peptide, GGWSHW, blocks TGF-p-mediated inhibition
of BAE cell growth.
Treatment of BAE Cells Cells/Well
onDay4
2.5% FBS 165,491 f 1530
TSP 1.0 }ig/ml 64,331 t 3841
TSP + GGWSHW ( SEQ ID NO: 3) (1.1 M) 100,522 f 2990
TSP + GGWSHW (SEQ ID NO:3) (11pM) 112,276 f 3730
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38
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44. Yamaguchi, Y., D.M. Mann, and E. Ruoslahti. 1990. Negative Regulation of
Transfonming Growth Factor-beta by the Proteoglycan Decorin. Nature
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45. ZentaUa, A and J. Massague. 1992. TGF-P Induces Myoblast Differentiation
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SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: MURPHY-ULLRICH, JOANNE E.
ROBERTS, DAVID D.
SCHULTZ-CHERRY, STACEY
KRUTZSCH, HENRY C.
(ii) TITLE OF INVENTION: METHODS AND COMPOSITIONS FOR
STIMULATING AND INHIBITING TGF-BETA ACTIVITY
(iii) NUMBER OF SEQUENCES: 46
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: NEEDLE & ROSENBERG, P.C.
(B) STREET: 127 Peachtree Street, NE
(C) CITY: Atlanta
(D) STATE: Georgia
(E) COUNTRY: USA
(F) ZIP: 30303-1811
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: SPRATT, GWENDOLYN D.
(B) REGISTRATION NUMBER: 36,016
(C) REFERENCE/DOCKET NUMBER: 2180.0181
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (404) 688-0770
(B) TELEFAX: (404) 688-9880
(2) INFORMATION FOR SEQ ID N0:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
Cys Ser Val Thr Cys Gly
1 5
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Trp Ser Xaa Trp
1
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
Gly Gly Trp Ser His Trp
1 5
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(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Trp Ser His Trp
1
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
Lys Arg Phe Lys
1
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
His Arg Phe Lys
1
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Arg Lys Pro Lys
1
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
Gln Arg Phe Lys
1
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Lys Arg Phe Lys Gln Asp Gly Gly
1 5
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Arg Trp Arg Pro Trp Thr Ala Trp Ser Glu
1 5 10
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Thr Ala Tyr Arg Trp Arg Leu Ser His Arg Pro Lys Thr Gly Ile
Arg
1 5 10
Val
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(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Lys Arg Phe Lys Gln Asp Gly Gly Ala Ser His Ala Ser Pro Ala
Ser
1 5 10 15
Ser
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Lys Arg Phe Lys Gln Asp Gly Gly Ala Ser His Ala Ser Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 14 amino acids
(B) TYPE: amino acid
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(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
Lys Arg Phe Lys Gln Asp Gly Gly Trp Ser His Trp Ser Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 amino acids
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Lys Arg Phe Lys Gln Asp Gly Gly Trp Ser His Trp Ser Pro Trp
Ser
1 5 .10 15
Ser
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
Lys Arg Phe Lys Gln Asp Gly Gly Trp Ser His Trp
1 5 10
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(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
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(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Lys Arg Phe Lys Gln Asp Gly Gly Trp Trp Ser Pro
1 5 10
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
Arg Phe Lys
1
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
Trp Asn Asp Trp Ile
1 5
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
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(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Trp Ser Ser Trp Ser
1 5
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
Leu Ser Lys Leu
1
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Ala Ala Trp Ser His Trp
1 5
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii)-MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
Asp Gly Trp Ser Pro Trp
1 5
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
Gly Gly Trp Gly Pro Trp
1 5
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Trp Ser Pro Trp Ser
1 5
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
Gly Trp Ser His Trp
1 5
(2) INFORMATION FOR SEQ ID NO:27:
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(A) LENGTH: 5 amino acids
(B) TYPE: amino acid
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Trp Ser His Trp Ser
1 5
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
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(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
Trp His Ser Trp Ala Ala
1 5
(2) INFORMATION FOR SEQ ID NO:29:
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(A) LENGTH: 13 amino acids
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Val Thr Cys Gly Gly Gly Val Gin Lys Arg Ser Arg Leu
1 5 10
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
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(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Thr Arg Ile Arg
1
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(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
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(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Ala Arg Phe Lys
1
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Lys Lys Phe Lys
1
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Lys Gln Phe Lys
1
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
Lys Arg Phe Gin
1
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
Lys Arg Ala Lys
1
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID N0:36:
Lys Arg Tyr Lys
1
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 4 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Lys Arg Trp Lys
= 1
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
Ala Ser Leu Arg Gln Met Lys Lys Thr Arg Gly Thr Leu Leu Ala
Leu
1 5 10 15
Glu Arg Lys Asp His Ser
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(2) INFORMATION FOR SEQ ID NO:39:
(i)'SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Glu Leu Thr Gly Ala Ala Arg Lys Gly Ser Gly Arg Arg Leu Val
Lys
1 5 10 15
Gly Pro Asp
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
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(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
Ser His Trp Trp Ser Ser
1 5
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
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(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
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(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
Gly Gly Trp Ser His Tyr
1 5
(2) INFORMATION FOR SEQ ID NO:42:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Gly Gly Trp Ser Lys Trp
1 . 5
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Arg Phe Arg
1
(2) INFORMATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3 amino acids =
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02590837 2007-06-18
WO 95/05191 PCT/US94/09193
61
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Arg Trp Arg
1
(2) INFORMATION FOR SEQ ID NO:45:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Lys Arg Phe Lys Gln Asp Gly Gly Trp Ser His Trp Ser Pro Trp
Ser
1 5 10 15
Ser Cys
(2) INFORMATION FOR SEQ ID NO:46:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:
Gly Gly Trp Ser His Trp Ser Pro Trp Ser Ser Cys
1 5 10
