U.S. patent application number 09/883375 was filed with the patent office on 2004-02-19 for enhanced affinity hyaluronan binding peptides.
Invention is credited to Turley, Eva.
Application Number | 20040034201 09/883375 |
Document ID | / |
Family ID | 22081606 |
Filed Date | 2004-02-19 |
United States Patent
Application |
20040034201 |
Kind Code |
A1 |
Turley, Eva |
February 19, 2004 |
Enhanced affinity hyaluronan binding peptides
Abstract
Novel hyaluronan-binding peptides are provided. The peptides are
useful in preventing and treating disorders associated with altered
tissue levels of hyaluronan or RHAMM, including cancer,
inflammatory and autoimmune disorders and fibrotic disorders
associated with tissue trauma.
Inventors: |
Turley, Eva; (Toronto,
CA) |
Correspondence
Address: |
Micheline Gravelle
Bereskin & Parr
Box 401
40 King Street West
Toronto
ON
M5H 3Y2
CA
|
Family ID: |
22081606 |
Appl. No.: |
09/883375 |
Filed: |
June 19, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09883375 |
Jun 19, 2001 |
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09210896 |
Dec 16, 1998 |
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6271344 |
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60068285 |
Dec 19, 1997 |
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Current U.S.
Class: |
530/388.22 ;
530/324 |
Current CPC
Class: |
C07K 14/47 20130101;
A61K 38/00 20130101; C07K 7/08 20130101 |
Class at
Publication: |
530/388.22 ;
530/324 |
International
Class: |
C07K 016/28; C07K
007/08 |
Claims
I claim:
1. A HA-binding peptide comprising: (a) a sequence of the formula
I:X.sub.1-X.sub.2-X.sub.1-X.sub.3-X.sub.4-X.sub.3-X.sub.4-X.sub.3-X.sub.3-
-X.sub.3-X.sub.5-X.sub.6-X.sub.6-X.sub.6-X.sub.1 wherein each
X.sub.1 is independently selected from a hydroxy amino acid
residue; each X.sub.2 is independently selected from a sulfur
containing amino acid residue; each X.sub.3 is independently
selected from a basic amino acid residue; each X.sub.4 is
independently selected from an imino or aromatic amino acid
residue; each X.sub.5 is independently selected from a dicarboxylic
acid amino acid residue; and each X.sub.6 is independently selected
from an aliphatic amino acid residue, and fragments, analogs or
derivatives of the peptide which can bind HA; (b) a sequence of the
formula
II:Y.sub.1-Y.sub.1-Y.sub.2-Y.sub.2-Y.sub.1-Y.sub.3-Y.sub.1-Y.sub.3-Y.sub.-
3-Y.sub.1-Y.sub.3-Y.sub.1-Y.sub.2-Y.sub.3-Y.sub.3 wherein each
Y.sub.1 is independently selected from a hydroxy amino acid
residue; each Y.sub.2 is independently selected from a sulfur
containing amino acid residue; and each Y.sub.3 is independently
selected from a basic amino acid residue, and fragments, analogs or
derivatives of the peptide which bind HA; or (c) a sequence of the
formula III:Z.sub.1-Z.sub.1-Z.sub.2-Z.sub.2-Z.sub.1-
-Z.sub.3-Z.sub.1-Z.sub.3-Z.sub.3-Z.sub.1-Z.sub.3-Z.sub.1-Z.sub.3-Z.sub.3
wherein each Z.sub.1 is independently selected from a hydroxy amino
acid residue; each Z.sub.2 is independently selected from a sulfur
containing amino acid residue; and each Z.sub.3 is independently
selected from a basic amino acid residue, and fragments, analogs or
derivatives of the peptide which bind HA.
2. A HA-binding peptide comprising a sequence of the formula I as
defined in claim 1 wherein each X.sub.1 is independently selected
from threonine or serine; each X.sub.2 is independently selected
from methionine or cysteine; each X.sub.3 is independently selected
from arginine, lysine or histidine; each X.sub.4 is independently
selected from proline, phenylalanine or tryptophan; each X.sub.5 is
independently selected from asparagine or glutamine; and each
X.sub.6 is independently selected from leucine, isoleucine, valine
or alanine, and fragments, analogs or derivatives of the peptide
which can bind HA.
3. A peptide according to claim 2 comprising the amino acid
sequence TMTRPHFHKRQLVLS.
4. A peptide according to claim 3 wherein the amino acids in the
peptide are the levorotatory (L) form.
5. A HA-binding peptide comprising a sequence of the Formula II as
defined in claim 1, wherein each Y.sub.1 is independently selected
from serine or threonine; each Y.sub.2 is independently selected
from methionine or cysteine; and each Y.sub.3 is independently
selected from arginine, lysine or histidine, and fragments, analogs
or derivatives of the peptide which bind HA.
6. A peptide according to claim 5 comprising the amino acid
sequence STMMSRSHKTRSCHH.
7. A peptide according to claim 6 wherein the amino acids in the
peptide are the levorotatory (L) form.
8. A HA-binding peptide comprising a sequence of the formula III as
defined in claim 1, wherein each Z.sub.1 is independently selected
from serine or threonine; each Z.sub.2 is independently selected
from methionine or cysteine; and each Z.sub.3 is independently
selected from arginine, lysine or histidine, and fragments, analogs
or derivatives of the peptide which bind HA.
9. A peptide according to claim 8 comprising the amino acid
sequence STMMSRSHKTRSHH.
10. A peptide according to claim 9 wherein the amino acids in the
peptide are the levorotatory (L) form.
11. A peptide according to claim 8 comprising the amino acid
sequence STMMSRSHKTRSHHV.
12. A peptide according to claim 11 wherein the amino acids in the
peptide are the levorotatory (L) form.
13. An isolated nucleic acid molecule encoding a HA binding peptide
according to claim 1.
14. An isolated nucleic acid molecule according to claim 13
encoding a HA binding peptide and comprising a nucleotide sequence
selected from the group consisting of: (a) the nucleotide sequence
shown in SEQ ID NO. 5; (b) the nucleotide sequence shown in SEQ ID
NO. 6; (c) the nucleotide sequence shown in SEQ ID NO. 7; (d) the
nucleotide sequence shown in SEQ ID NO. 8; (e) the nucleotide
sequence shown in SEQ ID NO. 9; (f) the nucleotide sequence shown
in SEQ ID NO. 10; (g) the nucleotide sequence shown in SEQ ID NO.
11; and (h) the nucleotide sequence shown in SEQ ID NO. 12.
15. An expression vector comprising an isolated nucleic acid
molecule according to claim 13 and regulatory sequences suitable
for expression of the nucleic acid molecule.
16. A method of modulating cell locomotion comprising administering
an effective amount of one or more hyaluronan-binding peptides
according to claim 1 to a cell or animal in need thereof.
17. A method of modulating cell locomotion comprising administering
an effective amount of hyaluronan-binding peptide according to
claim 3 to a cell or animal in need thereof.
18. A method of modulating cell locomotion comprising administering
an effective amount of hyaluronan-binding peptide according to
claim 6 to a cell or animal in need thereof.
19. A method of modulating cell locomotion comprising administering
an effective amount of hyaluronan-binding peptide according to
claim 9 to a cell or animal in need thereof.
20. A method of modulating cell locomotion comprising administering
an effective amount of hyaluronan-binding peptide according to
claim 11 to a cell or animal in need thereof.
21. A method of preventing or inhibiting tissue fibrosis comprising
administering an effective amount of one or more hyaluronan-binding
peptides according to claim 1 to an animal in need thereof.
22. A method of preventing or inhibiting tissue fibrosis comprising
administering an effective amount of hyaluronan-binding peptide
according to claim 3 to an animal in need thereof.
23. A method of preventing or inhibiting tissue fibrosis comprising
administering an effective amount of hyaluronan-binding peptide
according to claim 6 to an animal in need thereof.
24. A method of preventing or inhibiting tissue fibrosis comprising
administering an effective amount of hyaluronan-binding peptide
according to claim 9 to an animal in need thereof.
25. A method of preventing or inhibiting tissue fibrosis comprising
administering an effective amount of hyaluronan-binding peptide
according to claim 11 to an animal in need thereof.
26. A method according to claim 21 wherein the tissue fibrosis is
caused by a disorder selected from the group consisting of tissue
malfunction due to keloids, hypertrophic scars, anatomonic
strictures, intra-abdominal adhesions, cirrhosis of the liver,
neurological deficits following spinal cord injury, valvular heart
diseases, burn-injured joints, failure of anastomosis and adhesions
following surgery.
27. A method of treating or preventing cancer comprising
administering an effective amount of one or more hyaluronan-binding
peptides according to claim 1 to an animal in need thereof.
28. A method of preventing or reducing the metastasis of cancer
cells comprising administering an effective amount of one or more
hyaluronan-binding peptides according to claim 1 to an animal in
need thereof.
Description
[0001] This application claims the benefit under 35 USC.sctn.119(e)
of U.S. provisional application serial No. 60/068,285, filed on
Dec. 19, 1997.
FIELD OF THE INVENTION
[0002] The present invention relates to novel enhanced affinity
hyaluronan binding peptides, to methods for modulating cell
locomotion and for modulating physiological and pathological
conditions involving cell locomotion utilizing the peptides of the
invention and to pharmaceutical compositions containing the
peptides.
BACKGROUND OF THE INVENTION
[0003] Hyaluronic acid or hyaluronan (HA) is a large negatively
charged glucosaminoglycan consisting of repeating disaccharides of
N-acetylglucosamine and -glucuronic acid. This polymer is
ubiquitous in the extracellular matrix and is the major component
of skin, cartilage and brain tissue. In addition to its known
macrostructural functions, it also performs physicochemical
functions, for example, by acting as a lubricant in the synovial
fluid in joints.
[0004] Synthesis of HA has been associated with the morphogenesis
of many tissues, with wound repair, tumour invasion and cellular
immune function (Toole, B. P., Connect. Tissue Res. 10: 93-100,
1982; Pauli, B. V. et al., Cancer Met. Rev. 2: 129-152, 1983;
Toole, B. P. et al., Ciba Found. Symp. 143: 150-169, 1989; Turley,
E. A., Cancer Met. Rev. 3: 325-339, 1984; Iozzo, R. V., Lab.
Invest. 53: 373-396, 1985; Weigel, P. H. et al., J. Theol. Biol.
119: 219-234, 1986; Weigel, P. H. et al., Ciba Found. Symp. 143
248-264, 1989; Boudreax, N. et al., Dev. Biol. 143: 235-247, 1991;
Turley, E. A., Cancer Met. Rev. 11: 21-30, 1992). The underlying
mechanism of action at the cellular level is believed to involve
the ability of HA to elicit receptor-mediated alterations of cell
motility. High affinity HA receptors have been identified and
characterized on a variety of cell types and these are namely the
receptor for hyaluronan mediated mobility (or RHAMM) (Turley, E. A.
et al., J. Cell. Biol. 112: 1041-1047, 1991; Hardwick, C. et al.,
J. Cell. Biol. 117: 1343-1350, 1992; Yang, B. et al., J. Biol.
Chem. 268: 8617-8623, 1993), intercellular adhesion molecule-1 (or
ICAM-1) (McCourt, P. A. G. et al., J. Biol. Chem. 269: 30081-30084,
1994) and CD44 (Underhill, C. B. et al., J. Biol. Chem. 262:
13142-13146, 1987; Stamenkovic, I. et al., Cell 56: 1057-1062,
1989; Aruffo, A. et al., Cell 61: 1303-1313, 1990; Lesley, J. et
al., Exp. Cell Res. 187: 224-233, 1990; Miyake, K. et al., J. Exp.
Med. 172: 69-76, 1990). Both RHAMM and CD44 have been shown to be
associated with cell locomotion, cell proliferation and
differentiation. Other HA-binding proteins in the extracellular
milieu have also been identified and they are namely link protein,
aggrecan, versican, GHAP, collagen type VI and TSG-6.
[0005] In tissue trauma, an acute, transient increase in production
of HA is observed which is accompanied by an increase in expression
of HA receptors such as the receptor for hyaluronan mediated
mobility (RHAMM) and CD44. The in vivo physiological implications
of these molecular events has not been fully elucidated although an
increased production of HA and its accumulation has been shown to
regulate locomotion of fibroblasts, inflammatory cells and
epidermal cells depending upon the concentration of HA used.
Further, HA affects collagen fibril formation and white blood cell
phagocytic function, peroxide generation and cytokine expression.
Prior research studies have demonstrated that high concentrations
of HA act as a RHAMM antagonist to injured tissues including skin
burns, ulcers, ruptured tympanic membranes and abraded cornea by
reducing tissue fibrosis (Goz, K. L. and Benfield, P., Drugs 47:
536, 1994; King, S. R. et al., Surgery 109: 76, 1991;
Riquelme-Saquier, J. L., Dev. Ophthamol. 22: 50, 1991; Chung, J. H.
et al., Exp. Eye Res. 48: 569, 1989; Hellstrom, S. and Laurent, C.,
Acta Otolaryngol. 442 (Suppl.): 54, 1987; Retanda, G. G., Ital.
Dermatol. Venereol. 120: 71-75, 1985; Abatangelo, G. et al., J.
Surg. Res. 35: 410, 1983). Fibrosis of adult tissues after trauma
is a serious clinical problem that can result in pathologies
including malfunction of tissues due to keloids, hypertrophic
scars, anatomonic strictures, intra-abdominal adhesions, cirrhosis
of the liver, neurological deficits following spinal cord injury,
valvular heart diseases, burn-injured joints as well as failure of
anastomosis and adhesions following surgery (Bleacher, J. C. et
al., Dermatologic Clinics 11: 677-683, 1993; Clark, R. A. et al.,
Am. J. Med. Sci. 306: 42-48, 1993; Hebda, P. A. et al.,
Dermatologic Clinics 11: 685-696, 1993; Adzick, N. S. and Longaker,
M. T., Ann. Surg. 215: 3-7, 1992; Folkman, J., Ann. Surg. 215: 1-2,
1992).
[0006] Antagonism of RHAMM by antibodies and peptides has also been
demonstrated to inhibit HA-promoted cell locomotion (International
application PCT/CA93/00158 published as WO 93/21312). For instance,
HA-promoted fibroblast locomotion was inhibited by application of a
polyclonal or monoclonal antibody against the HA-receptor complex
(HARC).
[0007] Tumorigenesis is commonly manifested by an uncontrolled
proliferation of cells, and the metastatic spread of tumour tissue
is associated with the ability of these cells to locomote and
invade. Oncogenes and tumour suppressor genes are important factors
in the control of tumour cell growth, but extracellular matrix
(ECM) molecules such as HA and their receptors also play
significant roles. HA and RHAMM have been shown to regulate cell
proliferation and differentiation and are implicated in cell
transformation and tumour metastasis.
[0008] The transforming oncogene H-ras promotes cell locomotion by
enhancing the formation and release of autocrine motility factors,
growth factors and extracellular matrix (ECM) molecules such as HA
(Toole, B. P., Curr. Opin. Cell Biol. 2: 839-844, 1990; Stoker, M.
et al., Biochim. Biophys. Acta 1072: 81-102, 1991; Hardingham, T.
E. and Fosang, A. J., FASEB J. 6: 861-870, 1992; Laurent, T. C. and
Fraser, J. R. E., FASEB J. 6: 2397-2404, 1992; Pilarski, L. M. et
al., Leuk, Lymp. 14: 363-374, 1994). Enhancement of ras-transformed
cell locomotion by HA has been found to depend on the presence of a
HA-receptor complex termed HARC occurring at the cell surface or
released as soluble proteins (Hall, C. L. et al., Cell 82: 1-20,
1995; Hall, C. L. and Turley, E. A., J. Neuro-Oncol. 26: 221-229,
1995; Turley, E. A. et al., Blood 81: 446-453, 1993; Turley, E. A.
et al., Exp. Cell Res. 207: 277-282, 1993; Turley, E. A., Cancer
Metast. Rev. 11: 21-30, 1992; Turley, E. A. et al., J. Cell Biol.
112: 1041-1047, 1991). Turley et al. (Exp. Cell Res. 207: 277-282,
1993) reported that such HA-promoted cell locomotion was inhibited
by monoclonal antibodies specific to RHAMM thereby implicating
RHAMM as the major HA-binding component of HARC in tumorigenesis
and metastasis.
[0009] Under normal physiological conditions, RHAMM is not
detectable on B-lymphocytes found in the blood, spleen or lymph
node. Among B-cell malignancies, RHAMM is overexpressed on most
terminally differentiated B-cells from multiple myeloma bone
marrows, certain non-Hodgkin's lymphomas, and splenic hairy
leukemic cells (Turley, E. A. et al., Blood 81: 446-453, 1993;
Masellis-Smith, A. et al., Blood 87: 1891-1899, 1996). RHAMM is
also overexpressed in breast carcinoma cells (Turley, E. A. et al.,
Exp. Cell Res. 207: 277-282, 1993; Hall, C. L. & Turley, E. A.,
J. Neuro-oncol. 26: 221-229, 1995), and in combination with an
increased level of HA, are responsible for their enhanced motility
and metastasis. Administration of RHAMM-transfected cells into
animals results in spontaneous metastasis and formation of lung
tumour colonies.
[0010] RHAMM was one of the first HA receptors to be isolated and
characterized at the biochemical and molecular levels. It is an
N-linked glycoprotein that binds HA with high affinity (Kd: 1 nM)
and specificity. Several isoforms of RHAMM with different
subcellular distribution have been identified. Isoforms found
intracellularly and on the plasma membrane are designated iRHAMM
and pRHAMM, respectively, and the secreted isoform is designated
sRHAMM. The molecular structure of the various RHAMM isoforms may
be differentially regulated by phosphorylation and/or glycosylation
statuses. The precise roles of the RHAMM isoforms have not been
fully elucidated, but it is believed that pRHAMM and sRHAMM elicit
opposite activities and the net functional behaviour of a HA-RHAMM
interaction depends at least in part on the balance of pRHAMM
versus sRHAMM expressed by the cells involved.
[0011] Yang, B. et al. (J. Biol. Chem. 268: 8617-8623, 1993) have
identified two discrete HA binding domains in RHAMM that occur at
the carboxyl terminus of the protein. These domains are the only HA
binding regions in the receptor protein and they each contribute
approximately equally to the HA binding ability of RHAMM. Mutation
studies have revealed that Domain I contains two sets of two basic
amino acid residues spaced seven amino acids apart are important
for HA binding of the receptor. Similarly, Domain II contains a
lysine residue at position 423 and arginine at position 431 also
spaced seven amino acids apart which are critical for HA binding
activity. Collectively, these data predicted a generic binding
motif with a structure of B.sup.1-A.sub.n-B.sup.2 representing a
minimal binding requirement for HA and RHAMM. B.sup.1 and B.sup.2
are the same or different basic amino acid residues and A.sub.n is
a peptide sequence containing seven or eight amino acid residues
which are the same or different and are neutral or basic amino
acids. This generic binding motif was also found to be present in
CD44, link protein and all other HA binding proteins discovered to
date.
[0012] A full-length murine RHAMM cDNA has been cloned successfully
from a GT11 3T3 cDNA expression library (Hardwick, C. et al., J.
Cell Biol. 117: 1343-1350, 1992). Immunoblot analyses of cell
lysates using antibodies to peptides encoded in the cDNA reacted
specifically reacted with RHAMM protein. Using a fragment of the
clone DNA sequence, a mouse fibroblast genomic library was screened
to clone the genomic RHAMM gene which spans at least 20 kb and
comprises 14 exons ranging in size from 75 to 1099 bp (Entwistle,
J. et al., Gene 163: 233-238, 1995).
[0013] Similarly, a human RHAMM cDNA clone was also isolated
successfully by a combination of screening a human breast cDNA
expression library with the murine RHAMM cDNA as well as 5' RACE
and reverse transcription-polymerase chain reaction using messenger
RNA from the human breast cell line MCF-10A (Wang, C. et al., Gene
174: 299-306, 1996). The full-length human RHAMM cDNA encodes for a
725 amino acid protein and shares a 85% homology with the murine
transcripts, RHAMM v4. More importantly, the HA binding motif
B.sup.1-A.sub.n-B.sup.2 which is shown to be critical for the
signalling capability of RHAMM is 100% conserved between human and
mouse.
[0014] PCT published patent application no. WO 93/21312 to the
present inventor describes short peptides of nine or ten amino acid
residues which mimic the HA binding motif of RHAMM. These RHAMM
peptides possess the ability to bind HA and share a common generic
peptide sequence represented by B.sup.1-A.sub.n-B.sup.2 as
described above.
[0015] However, the published application discloses key
restrictions associated with the sequence of these RHAMM peptides
with respect to their HA binding affinity. For example, it is clear
from the patent publication that if A.sub.n is a peptide sequence
containing less than seven or greater than eight amino acid
residues, HA binding affinity is lost. Acidic amino acids are
incompatible with HA binding as substitution of neutral or basic
amino acid residues by acidic amino acid residues also abolishes HA
binding affinity. Moreover, the basic amino acid histidine at the
carboxy-terminal end is not compatible with HA binding which is
indicated by the fact that replacement of the carboxy-terminal
lysine or arginine by histidine completely destroyed HA-binding
ability of the RHAMM peptides. In particular, a peptide with the
sequence KLRSQLVHHH was unable to bind to HA.
[0016] PCT published patent application no. WO 97/24111 to the
present inventor also describes HA-binding peptides consisting of
dextrorotatory, D-amino acids and their ability to bind naturally
occuring hyaluronic acid in the body which prevents hyaluronic acid
from stimulating its receptors. Correspondingly, through the
inhibition of hyaluronic acid receptor activation, said D-forms of
HA-binding peptides were speculated to be useful when combined with
a second medicine or therapeutic agent such as a surfactant for the
treatment of herpes infection, an anti-microbial agent for the
treatment of mononucleosis, dimethyl sulphoxide for AIDS therapy,
insulin for the treatment of diabetes and a calcium channel blocker
for the treatment of hypertension.
[0017] The invention of WO 97/24111 is distinct from the peptides
and uses of the present invention in two major respects. First,
based on past empirical evidence, it is well known that many
physiologically and therapeutically important peptides of similar
sizes to the peptides of the present invention exhibit significant
stereospecificity in their biological actions. In many instances,
subsitution of L-amino acid(s) by D-amino acid(s) or vice versa
resulted in peptides that produce physiological effects that are
opposite to those originally observed. Such a seemingly
straightforward change of one or more amino acid residue(s) by its
enantiomeric counterpart(s) can therefore create peptides that have
distinctly different therapeutic uses.
[0018] For example, vasopressin is a nonapeptide and substitution
of L-amino acid residue(s) to D-amino acid residue(s) in the
SK&F vasopressin analogs dramatically reversed their
bioactivity as vasopressin agonists to vasopressin antagonists
(Albrightson-Winslow, C. et al., J. Pharmacol. Exp. Ther. 256:
335-340, 1991; Brooks, D. P. et al., Eur. J. Pharmacol. 160:
159-152, 1989). Similarly, enantiomeric substitutions of L- and
D-amino acids in substance P, an undecapeptide, also dramatically
changed its agonistic and antagonistic activities as well as the
peptide's specificity to stimulate different receptor subtypes and
to elicit totally different physiological responses (Cross, M. et
al., Eur. J. Pharmacol. 291: 291-300, 1995; Dutta, A. S. et al., J.
Med. Chem. 29:1163-1171, 1986; Dutta, A. S. et al., J. Med. Chem.
29:1171-1178, 1986). To further illustrate this phenomen, substance
P with an L-proline at position 9 induced a marked scratching
response in the rat but does not produce any response in colon
smooth muscle. Conversely, substitution of this single amino acid
by a D-proline resulted in a peptide that elicited no scratching
response but produced sigificant contraction of colon smooth muscle
(Piercey, M. F. et al., Life Sci. 36: 777-780, 1985).
[0019] Similarly, Casteels, P. and Tempst, P. reported the
stereospecific requirements of apidaecin-type antibacterial
peptides in which the L-enantiomer of apidaecin is a lethal
non-poreforming bacteriotoxin but the D-enantiomer is completely
devoid of antibacterial activity (Casteels, P. and Tempst, P.,
Biochem. Biophys. Res. Commun. 199: 339-345, 1994). Oren, Z. et al.
also reported the detrimental effects of D-amino acid substitution
on the alpha-helical structure of diastereomeric dodecapeptides and
their antibacterial effects (Oren, Z. et al., J. Biol. Chem. 272:
14643-14649, 1997).
[0020] Furthermore, peptidyl dipeptide hydrolase catalyzes the
hydrolysis of the decapeptide angiotensin I to the physiologically
active octapeptide angiotensin II. This enzyme has been shown to
exhibit an absolute stereospecific requirement for L-amino acid
residues at the C-terminus of the angiotensin I peptide in order to
achieve activation (Oshima, G. and Nagasawa, K., J. Biochem. 86:
1719-1724, 1979; Oparil, S. et al., Circ. Res. 29: 682-690,
1971).
[0021] In view of the above, the biological activities of one
enantiomer of a given peptide are quite distinct from those of
another enantiomer of the same peptide. The speculation of certain
activities for the D-forms of the HA-binding peptides in WO
97/24111 therefore cannot be used to predict the clinical utilities
of the L-forms of said peptides. This is supported by the fact that
the L-peptides of the present invention, when given alone, are
useful in the prevention of skin tissue fibrosis which is clearly
in contrast to the combinational therapeutic regimens and the
therapeutic areas described in WO 97/24111.
[0022] Second, notwithstanding the terminology "HA-binding
peptides" and that the actions of the D-peptides in WO 97/24111
depend on their ability to inhibit HA interaction with its
receptors, the L-forms of the peptides of the present invention do
not elicit its biological actions by inhibiting HA interaction with
its receptors (RHAMM and CD44). This aspect of the invention is
demonstrated in greater detail below.
SUMMARY OF THE INVENTION
[0023] The present inventor has isolated novel peptides which can
bind hyaluronic acid (HA) or hyaluronan with enhanced affinity. The
peptides of the present invention differ from the previous
HA-binding peptides and do not fall within the previously described
formula.
[0024] In one embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the formula I:
X.sub.1-X.sub.2-X.sub.1-X.sub.3-X.sub.4-X.sub.3-X.sub.4-X.sub.3-X.sub.3-X.-
sub.3-X.sub.5-X.sub.6-X.sub.6-X.sub.6-X.sub.1
[0025] wherein
[0026] each X.sub.1 is independently selected from a hydroxy amino
acid residue;
[0027] each X.sub.2 is independently selected from a sulfur
containing amino acid residue;
[0028] each X.sub.3 is independently selected from a basic amino
acid residue;
[0029] each X.sub.4 is independently selected from an imino or
aromatic amino acid residue;
[0030] each X.sub.5 is independently selected from a dicarboxylic
acid amino acid residue; and
[0031] each X.sub.6 is independently selected from an aliphatic
amino acid residue, and fragments, analogs or derivatives of the
peptide which can bind HA.
[0032] In a preferred embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the formula I:
X.sub.1-X.sub.2-X.sub.1-X.sub.3-X.sub.4-X.sub.3-X.sub.4-X.sub.3-X.sub.3-X.-
sub.3-X.sub.5-X.sub.6-X.sub.6-X.sub.6-X.sub.1
[0033] wherein
[0034] each X.sub.1 is independently selected from threonine or
serine;
[0035] each X.sub.2 is independently selected from methionine or
cysteine;
[0036] each X.sub.3 is independently selected from arginine, lysine
or histidine;
[0037] each X.sub.4 is independently selected from proline,
phenylalanine or tryptophan;
[0038] each X.sub.5 is independently selected from asparagine or
glutamine; and
[0039] each X.sub.6 is independently selected from leucine,
isoleucine, valine or alanine, and fragments, analogs or
derivatives of the peptide which can bind HA.
[0040] A preferred peptide of Formula I is TMTRPHFHKRQLVLS
(SEQ.ID.NO.:1).
[0041] In another embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the Formula II:
Y.sub.1-Y.sub.1-Y.sub.2-Y.sub.2-Y.sub.1-Y.sub.3-Y.sub.1-Y.sub.3-Y.sub.3-Y.-
sub.1-Y.sub.3-Y.sub.1-Y.sub.2-Y.sub.3-Y.sub.3
[0042] wherein
[0043] each Y.sub.1 is independently selected from a hydroxy amino
acid residue;
[0044] each Y.sub.2 is independently selected from a sulfur
containing amino acid residue; and
[0045] each Y.sub.3 is independently selected from a basic amino
acid residue, and fragments, analogs or derivatives of the peptide
which bind HA.
[0046] In a preferred embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the Formula II:
Y.sub.1-Y.sub.1-Y.sub.2-Y.sub.2-Y.sub.1-Y.sub.3-Y.sub.1-Y.sub.3-Y.sub.3-Y.-
sub.1-Y.sub.3-Y.sub.1-Y.sub.2-Y.sub.3-Y.sub.3
[0047] wherein
[0048] each Y.sub.1 is independently selected from serine or
threonine;
[0049] each Y.sub.2 is independently selected from methionine or
cysteine; and
[0050] each Y.sub.3 is independently selected from arginine, lysine
or histidine, and fragments, analogs or derivatives of the peptide
which bind HA.
[0051] A preferred peptide of the Formula II is STMMSRSHKTRSCHH
(SEQ.ID.NO.:2).
[0052] In another embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the Formula III:
Z.sub.1-Z.sub.1-Z.sub.2-Z.sub.2-Z.sub.1-Z.sub.3-Z.sub.1-Z.sub.3-Z.sub.1-Z.-
sub.3-Z.sub.1-Z.sub.3-Z.sub.3
[0053] wherein
[0054] each Z.sub.1 is independently selected from a hydroxy amino
acid residue;
[0055] each Z.sub.2 is independently selected from a sulfur
containing amino acid residue; and
[0056] each Z.sub.3 is independently selected from a basic amino
acid residue, and fragments, analogs or derivatives of the peptide
which bind HA.
[0057] In a preferred embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the Formula III:
Z.sub.1-Z.sub.1-Z.sub.2-Z.sub.2-Z.sub.1-Z.sub.3-Z.sub.1-Z.sub.3-Z.sub.3-Z.-
sub.1-Z.sub.3-Z.sub.1-Z.sub.3-Z.sub.3
[0058] wherein
[0059] each Z.sub.1 is independently selected from serine or
threonine;
[0060] each Z.sub.2 is independently selected from methionine or
cysteine; and
[0061] each Z.sub.3 is independently selected from arginine, lysine
or histidine, and fragments, analogs or derivatives of the peptide
which bind HA.
[0062] A preferred peptide of the Formula III is STMMSRSHKTRSHH
(SEQ.ID.NO.:3). A peptide of the Formula III may optionally contain
a valine residue at the C-terminal and have the following sequence:
STMMSRSHKTRSHHV (SEQ.ID.NO.:4).
[0063] The term "HA-binding peptide" as used herein means a peptide
that can bind hyaluronic acid (or hyaluronan) and is of the Formula
I, II, or III as defined above. The term includes fragments,
analogs and derivatives of the peptides which maintain the ability
to bind HA. Collectively, the HA binding peptides defined herein
are referred to as "the peptides of the invention". Preferably, the
peptides of the invention consist of amino acids in the
levorotatory (L) form which corresponds to how amino acids occur in
nature.
[0064] The present invention also provides a purified and isolated
nucleic acid having a nucleotide sequence encoding an HA-binding
peptide comprising a sequence of the formula I, II or III described
above. The present invention also provides an expression vector
comprising a DNA molecule encoding the HA-binding peptide adapted
for transfection or transformation of a host cell.
[0065] In a further aspect, the present invention provides a
pharmaceutical composition containing one or more HA-binding
peptides or nucleic acid molecules encoding a HA-binding peptide of
the invention in admixture with a pharmaceutically acceptable
carrier, diluent or excipient for modulating cell locomotion and
for the prophylaxis or treatment of cancer, inflammatory and
autoimmune disorders and fibrotic disorders associated with tissue
trauma and its recovery.
[0066] The invention also contemplates a method for preventing or
treating cancer, inflammatory and autoimmune disorders and fibrotic
disorders associated with tissue trauma and its recovery by
administering an effective amount of one or more HA-binding
peptides or nucleic acid molecules encoding a HA-binding peptide to
an animal in need thereof.
[0067] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description and attached drawings. It should be understood,
however, that the detailed description and associated examples are
given by way of illustration only, and various changes and
modifications thereto falling within the scope of the invention
will become apparent to those skilled in the art. In addition,
reference is made herein to various publications, patents and
patent applications which are hereby incorporated by reference in
their entirety.
BRIEF DESCRIPTION OF THE DRAWINGS
[0068] The invention will be better understood with reference to
the drawings in which:
[0069] FIG. 1 is a schematic diagram illustrating the isolation of
enhanced affinity hyaluronan binding peptides from a 15-mer phage
display library.
[0070] FIGS. 2A-F illustrates the stimulation of focal contact
formation by enhanced affinity hyaluronan binding peptides.
[0071] FIG. 2G is a graph illustrating the formation of
vinculin-positive focal contacts in ras-transformed 10T 1/2
fibroblast versus the concentration of hyaluronidase.
[0072] FIG. 3A is a Western blot of the data from image analysis on
the inhibition of cell motility fluctuations on fibronectin
substrata by enhanced affinity hyaluronan binding peptides.
[0073] FIG. 3B is an SDS-PAGE 1f the data from image analysis on
the inhibition of cell motility fluctuations on fibronectin
substrata by enhanced affinity hyaluronan binding peptides.
[0074] FIGS. 3C-3E shows the densitometry analysis of the data in
the Western blot in FIG. 3A.
[0075] FIG. 3F is a Western blot of the data illustrating that
fluctuations in cell motility is correlated with fluctuations in
protein tyrosine phosphorylation and that addition of enhanced
affinity hyaluronan binding peptides reduced protein
phosphorylation/dephosphoryla- tion cycles;
[0076] FIG. 3G shows Coomassie blue staining of lysate proteins
separated by SDS-PAGE;
[0077] FIG. 3H shows densitometry data of Western blots in FIG.
3F;
[0078] FIG. 3I shows inhibition of cell motility fluctuations by
enhanced affinity hyaluronan binding peptides for over 18 h as
compared to null effect by the scrambled peptides (controls).
[0079] FIG. 4 presents data from RT-PCR analysis showing increases
in RHAMM and CD44 mRNA at 24 h after injury.
[0080] FIG. 5 shows data from immunohistochemical analysis showing
expression of RHAMM (A) immediately after injury (B) 24 h after
injury (C) 48-72 h after injury (D) represents a biopsy site
stained with IgG at 24 h after injury.
[0081] FIG. 6 presents (A) Western analysis data and (B)
densitometry analysis data showing the presence of 3 major RHAMM
isoforms (80 kDa, 68-70 kDa and 60 kDa proteins) in uninjured skin
(1).
[0082] FIG. 7 shows (A) RT-PCR analysis data and (B) densitometry
analysis data illustrating increases in mRNA for total RHAMM
isoforms (designated RHAMM) and for RHAMM v4 (an isoform implicated
in regulation of cell motility and growth) 6 h after injury
(3).
[0083] FIG. 8 is a graph illustrating the concentrating of HA
versus time after injury.
[0084] FIGS. 9A-D illustrates immunohistochemical data on
distribution of HA in wound tissue.
[0085] FIGS. 10A and B shows results of (A) RT-PCR and (B)
densitometry analyses of collagen type I mRNA. The times post
injury are: (3) 48 h (4) 72 h, (5) 96 h, (6) 120 h and (7) 2
weeks.
[0086] FIGS. 10C and D shows results of (C) RT-PCR and (D)
densitometry analyses of collagen type III mRNA. Collagen type III
mRNA was present within uninjured skin at low levels (1) and
increased at 6 h after injury (3) which reached a maximum at 12-24
h (4, 5) and declined thereafter. By 407 days post injury (7,8),
collagen type III mRNA returned to background levels.
[0087] FIG. 11 is a graph showing glucosaminidase levels in wounds
versus time after injury.
[0088] FIG. 12 shows data from histological analysis of wound site
at 24 h after injury treated with (I) Vehicle or scrambled peptide
(control); (II) HA binding peptides; (III) IgG antibodies and (IV)
anti-RHAMM antibodies.
[0089] FIG. 13 is a graph showing the number of macrophages at the
wound site in the presence of a HA binding peptides, scrambled
peptides and untreated.
[0090] FIG. 14 is a graph showing that the HA binding peptides
reduced glucosaminidase activity at the wound site in a
dose-dependent manner as compared to the null effect of the
scrambled peptide.
[0091] FIG. 15 shows the effect of the HA binding peptides on
collagen type I mRNA levels as detected by RT-PCR analysis.
Collagen type I mRNA levels increased by 72 h after injury (2, 3)
above control levels with vehicle (1) or scrambled peptide (3).
Wounds treated with enhanced affinity HA binding peptide exhibited
low collagen type I mRNA levels (5).
[0092] FIGS. 16A and B shows the results of (A) RT-PCR and (B)
densitometry analyses of the effect of the HA binding peptides on
collagen type III mRNA levels as detected by RT-PCR analysis.
Collagen type III mRNA levels was low in uninjured skin but were
increased at 24 h after injury in wounds treated with vehicle and
scrambled peptide (2). Wounds treated with the HA binding peptide
exhibited low collagen type III mRNA levels (3).
[0093] FIG. 17 shows photographs of paraffin sections of wound
biopsies 2 weeks after injury which were stained with hematoxylin
(A, B) or for RHAMM (C, D).
[0094] FIG. 18 shows a graph illustrating the inhibition of human
foreskin fibroblasts contraction by 1 ng/mL and 500 mg/mL of
HA-binding peptides as compared to the null effect of the negative
control vehicle.
[0095] FIG. 19 shows a graph illustrating the inhibition of human
foreskin fibroblasts contraction of HA-binding peptides in a
dose-dependent manner as compared to the null effect of the
negative control vehicle.
[0096] FIG. 20 shows a graph illustrating the null effect of
RHAMM-motif peptide (1 ng/mL and 500 mg/mL) on human foreskin
fibroblasts contraction.
[0097] FIG. 21 shows a graph illustrating the null effect of
anti-RHAMM (exon 5) antibodies on human foreskin fibroblasts
contraction.
[0098] FIG. 22 shows a graph illustrating the null effect of
anti-RHAMM (exon 9) antibodies on human foreskin fibroblasts
contraction.
[0099] FIG. 23 shows a graph illustrating the null effect of
anti-CD44 antibodies on human foreskin fibroblasts contraction.
DETAILED DESCRIPTION OF THE INVENTION
[0100] The following standard one letter and three letter
abbreviations for the amino acid residues may be used throughout
the specification: A, Ala-alanine; R, Arg-Arginine; N,
Asn-Asparagine; D, Asp-Aspartic acid; C, Cys-Cysteine; Q,
Gln-Glutamine; E, Glu-Glutamic acid; G, Gly-Glycine; H,
His-Histidine; I, Ile-Isoleucine; L, Leu-Leucine; K, Lys-Lysine; M,
Met-Methionine; F, Phe-Phenyalanine; P, Pro-Proline; S, Ser-Serine;
T, Thr-Threonine; W, Trp-Tryptophan; Y, Tyr-Tyrosine; and V,
Val-Valine;
[0101] HA Binding Peptides
[0102] The present inventor has isolated, sequenced and
characterized novel peptides of about 14 or 15 amino acid residues
which bind hyaluronic acid or hyaluronan with enhanced
affinity.
[0103] In one embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the formula I:
X.sub.1-X.sub.2-X.sub.1-X.sub.3-X.sub.4-X.sub.3-X.sub.4-X.sub.3-X.sub.3-X.-
sub.3-X.sub.5-X.sub.6-X.sub.6-X.sub.6-X.sub.1
[0104] wherein
[0105] each X.sub.1 is independently selected from a hydroxy amino
acid residue;
[0106] each X.sub.2 is independently selected from a sulfur
containing amino acid residue;
[0107] each X.sub.3 is independently selected from a basic amino
acid residue;
[0108] each X.sub.4 is independently selected from an imino or
aromatic amino acid residue;
[0109] each X.sub.5 is independently selected from a dicarboxylic
acid amino acid residue; and
[0110] each X.sub.6 is independently selected from an aliphatic
amino acid residue, and fragments, analogs or derivatives of the
peptide which can bind HA.
[0111] In a preferred embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the formula I:
X.sub.1-X.sub.2-X.sub.1-X.sub.3-X.sub.4-X.sub.3-X.sub.4-X.sub.3-X.sub.3-X.-
sub.3-X.sub.5-X.sub.6-X.sub.6-X.sub.6-X.sub.1
[0112] wherein
[0113] each X.sub.1 is independently selected from threonine or
serine;
[0114] each X.sub.2 is independently selected from methionine or
cysteine;
[0115] each X.sub.3 is independently selected from arginine, lysine
or histidine;
[0116] each X.sub.4 is independently selected from proline,
phenylalanine or tryptophan;
[0117] each X.sub.5 is independently selected from asparagine or
glutamine; and
[0118] each X.sub.6 is independently selected from leucine,
isoleucine, valine or alanine, and fragments, analogs or
derivatives of the peptide which can bind HA.
[0119] A preferred peptide of Formula I is TMTRPHFHKRQLVLS
(SEQ.ID.NO.:1).
[0120] In another embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the Formula II:
Y.sub.1-Y.sub.1-Y.sub.2-Y.sub.2-Y.sub.1-Y.sub.3-Y.sub.1-Y.sub.3-Y.sub.3-Y.-
sub.1-Y.sub.3-Y.sub.1-Y.sub.2-Y.sub.3-Y.sub.3
[0121] wherein
[0122] each Y.sub.1 is independently selected from a hydroxy amino
acid residue;
[0123] each Y.sub.2 is independently selected from a sulfur
containing amino acid residue; and
[0124] each Y.sub.3 is independently selected from a basic amino
acid residue, and fragments, analogs or derivatives of the peptide
which bind HA.
[0125] In a preferred embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the Formula II:
Y.sub.1-Y.sub.1-Y.sub.2-Y.sub.2-Y.sub.1-Y.sub.3-Y.sub.1-Y.sub.3-Y.sub.3-Y.-
sub.1-Y.sub.3-Y.sub.1-Y.sub.2-Y.sub.3-Y.sub.3
[0126] wherein
[0127] each Y.sub.1 is independently selected from serine or
threonine;
[0128] each Y.sub.2 is independently selected from methionine or
cysteine; and
[0129] each Y.sub.3 is independently selected from arginine, lysine
or histidine, and fragments, analogs or derivatives of the peptide
which bind HA.
[0130] A preferred peptide of the Formula II is STMMSRSHKTRSCHH
(SEQ.ID.NO.:2).
[0131] In another embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the Formula III:
Z.sub.1-Z.sub.1-Z.sub.2-Z.sub.2-Z.sub.1-Z.sub.3-Z.sub.1-Z.sub.3-Z.sub.3-Z.-
sub.1-Z.sub.3-Z.sub.1-Z.sub.3-Z.sub.3
[0132] wherein
[0133] each Z.sub.1 is independently selected from a hydroxy amino
acid residue;
[0134] each Z.sub.2 is independently selected from a sulfur
containing amino acid residue; and
[0135] each Z.sub.3 is independently selected from a basic amino
acid residue, and fragments, analogs or derivatives of the peptide
which bind HA.
[0136] In a preferred embodiment, the present invention provides a
HA-binding peptide comprising a sequence of the Formula III:
Z.sub.1-Z.sub.1-Z.sub.2-Z.sub.2-Z.sub.1-Z.sub.3-Z.sub.1-Z.sub.3-Z.sub.3-Z.-
sub.1-Z.sub.3-Z.sub.1-Z.sub.3-Z.sub.3
[0137] wherein
[0138] each Z.sub.1 is independently selected from serine or
threonine;
[0139] each Z.sub.2 is independently selected from methionine or
cysteine; and
[0140] each Z.sub.3 is independently selected from arginine, lysine
or histidine, and fragments, analogs or derivatives of the peptide
which bind HA.
[0141] A preferred peptide of the Formula III is STMMSRSHKTRSHH
(SEQ.ID.NO.:3). A peptide of the Formula III may optionally contain
a valine residue at the C-terminal and have the following sequence:
STMMSRSHKTRSHHV (SEQ.ID.NO.:4).
[0142] The term "HA-binding peptide" as used herein means peptide
that can bind hyaluronic acid (or hyaluronan) and is of the Formula
I, II, or III as defined above. The term includes fragments,
analogs and derivatives of the peptides which maintain the ability
to bind HA. Collectively, the HA binding peptides defined herein
are referred to as the peptides of the invention. Preferably, the
peptides of the invention consist of amino acids in the
levorotatory (L) form which corresponds to how amino acids occur in
nature.
[0143] The term "analog" includes any peptide having an amino acid
residue sequence substantially identical to the sequence of the HA
binding peptides shown in Formula I, II, or III shown herein in
which one or more residues have been conservatively substituted
with a functionally similar residue and which displays the ability
to mimic a HA binding peptide. Examples of conservative
substitutions include the substitution of one non-polar
(hydrophobic) residue such as alanine, isoleucine, valine, leucine
or methionine for another, the substitution of one polar
(hydrophilic) residue for another such as between arginine and
lysine, between glutamine and asparagine, between glycine and
serine, the substitution of one basic residue such as lysine,
arginine or histidine for another, or the substitution of one
acidic residue, such as aspartic acid or glutamic acid for another.
The phrase "conservative substitution" also includes the use of a
chemically derivatized residue in place of a non-derivatized
residue provided that such polypeptide displays the requisite
activity.
[0144] "Derivative" refers to a peptide having one or more residues
chemically derivatized by reaction of a functional side group. Such
derivatized molecules include for example, those molecules in which
free amino groups have been derivatized to form amine
hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups,
t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
Free carboxyl groups may be derivatized to form salts, methyl and
ethyl esters or other types of esters or hydrazides. Free hydroxyl
groups may be derivatized to form O-acyl or O-alkyl derivatives.
The imidazole nitrogen of histidine may be derivatized to form
N-im-benzylhistidine. Also included as derivatives are those
peptides which contain one or more naturally occurring amino acid
derivatives of the twenty standard amino acids. For examples:
4-hydroxyproline may be substituted for proline; 5-hydroxylysine
may be substituted for lysine; 3-methylhistidine may be substituted
for histidine; homoserine may be substituted for serine; and
ornithine may be substituted for lysine. HA-binding peptides of the
present invention also include any peptide having one or more
additions and/or deletions or residues relative to the sequence of
a polypeptide whose sequence is shown herein, so long as the
requisite activity is maintained or increased.
[0145] The term "fragment" refers to any subject peptide having an
amino acid residue sequence shorter than that of a peptide whose
amino acid residue sequence is shown herein.
[0146] The invention includes cyclic derivatives of the HA binding
peptides of the invention. Cyclization allows the peptide to assume
a more favourable conformation. Cyclization of the peptides may be
achieved using techniques known in the art. In particular,
disulphide bonds may be formed between two appropriately spaced
components having free sulfhydryl groups. The bonds may be formed
between side chains of amino acids, non-amino acid components or a
combination of the two.
[0147] Peptides of the present invention may be converted into
pharmaceutical salts by reacting with inorganic acids including
hydrochloric acid, sulphuric acid, hydrobromic acid, phosphoric
acid, etc., or organic acids including formic acid, acetic acid,
propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic
acid, succinic acid, malic acid, tartaric acid, citric acid,
benzoic acid, salicylic acid, benzenesulphonic acid, and
tolunesulphonic acids.
[0148] The peptides of the invention may be prepared as N-terminal
or C-terminal fusion proteins. The fusion proteins may be prepared
by fusing, through recombinant techniques or by chemical
crosslinking, the N-terminal or C-terminal of the peptide, and the
sequence of a selected protein or selectable marker with a desired
biological function. The resultant fusion proteins contain the
peptide fused to the selected protein or marker protein as
described herein. Examples of proteins which may be used to prepare
fusion proteins include immunoglobulins, glutathione-S-transferase,
hemagglutinin, and truncated myc.
[0149] The peptides of the invention may be used to prepare
monoclonal or polyclonal antibodies. Conventional methods can be
used to prepare the antibodies. As to the details relating to the
preparation of monoclonal antibodies reference can be made to
Goding, J. W., Monoclonal Antibodies: Principles and Practice, 2nd
Ed., Academic Press, London, 1986. As discussed below, the
antibodies may be used to identify proteins that bind HA.
[0150] The peptides and antibodies specific for the peptides of the
invention may be labelled using conventional methods with various
enzymes, fluorescent materials, luminescent materials and
radioactive material. Suitable enzymes, fluorescent materials,
luminescent materials, and radioactive material are well known to
the skilled artisan. Labelled antibodies specific for the peptides
of the invention may be used to screen for proteins with HA binding
sites as discussed in further detail below.
[0151] Preparation of the Peptides
[0152] The peptides of the invention may be prepared by chemical
synthesis using techniques well known in the chemistry of proteins
such as solid phase synthesis (Merrifield, J. Am. Chem. Assoc.
85:2149-2154 (1964)) or synthesis in homogenous solution
(Houbenweyl, Methods of Organic Chemistry, ed. E. Wansch, Vol. 15 I
and II, Thieme, Stuttgart (1987)).
[0153] The peptides of the invention may also be produced by
recombinant DNA technology. To prepare the peptides of the
invention by recombinant DNA techniques, a DNA sequence encoding
the HA-binding peptide must be prepared. Consequently, the present
invention also provides purified and isolated nucleic acid having a
nucleotide sequence encoding a HA-binding peptide comprising an
amino acid sequence of the formula I, II or III wherein the
substituents are described above. In one embodiment, the DNA
sequence encoding a HA-binding peptide of the formula I comprises a
nucleotide sequence of ACC ATG ACC CGT CCG CAC TTC CAC AAA CGT CAG
CTG GTT CTG TCT (SEQ.ID.NO.:5) or ACS ATG ACS CGS CCS CAC TTC CAC
AAG CGS CAG CTS GTS CTS WSS wherein S is C or G and W is A or T
(SEQ.ID.NO.: 6). In a second embodiment, the DNA sequence encoding
a HA-binding peptide of the formula II comprises a nucleotide
sequence of TCT ACC ATG ATG TCT CGT TCT CAC AAA ACC CGT TCT TGT CAC
CAC (SEQ.ID.NO.: 7) or WSS ACS ATG ATG WSS CGS WSS CAC AAG ACS CGS
WSS TGC CAC CAC wherein S is C or G and W is A or T (SEQ.ID.NO.:8).
In a third embodiment, the DNA sequence encoding a HA-binding
peptide of the formula III comprises a nucleotide sequence: TCT ACC
ATG ATG TCT CGT TCT CAC AAA ACC CGT TCT CAC CAC (SEQ.ID.NO.:9) or
WSS ACS ATG ATG WSS CGS WSS CAC AAG ACS CGS WSS CAC CAC wherein S
is C or G and W is A or T (SEQ.ID.NO.:10) or TCT ACC ATG ATG TCT
CGT TCT CAC AAA ACC CGT TCT CAC CAC GTG (SEQ.ID.NO.:11) or WSS ACS
ATG ATG WSS CGS WSS CAC AAG ACS CGS WSS CAC CAC GTC wherein S is C
or G and W is A or T (SEQ.ID.NO.:12).
[0154] The present invention also provides an expression vector
comprising a DNA molecule encoding a HA-binding peptide adapted for
transfection or transformation of a host cell. The nucleic acid
molecules of the present invention may be incorporated in a known
manner into an appropriate expression vector which ensures
expression of the protein. Possible expression vectors include but
are not limited to cosmids, plasmids, or modified viruses (e.g.
replication defective retroviruses, adenoviruses and
adeno-associated viruses). The vector should be compatible with the
host cell used. The expression vectors are "suitable for
transformation of a host cell", which means that the expression
vectors contain a nucleic acid molecule of the invention and
regulatory sequences selected on the basis of the host cells to be
used for expression, which is operatively linked to the nucleic
acid molecule. Operatively linked is intended to mean that the
nucleic acid is linked to regulatory sequences in a manner which
allows expression of the nucleic acid.
[0155] The invention therefore contemplates a recombinant
expression vector containing a nucleic acid molecule of the
invention, or a fragment thereof, and the necessary regulatory
sequences for the transcription and translation of the inserted
protein-sequence.
[0156] Suitable regulatory sequences may be derived from a variety
of sources, including bacterial, fungal, viral, mammalian, or
insect genes (For example, see the regulatory sequences described
in Goeddel, Gene Expression Technology: Methods in Enzymology 185,
Academic Press, San Diego, Calif. 1990). Selection of appropriate
regulatory sequences is dependent on the host cell chosen as
discussed below, and may be readily accomplished by one of ordinary
skill in the art. Examples of such regulatory sequences include: a
transcriptional promoter and enhancer or RNA polymerase binding
sequence, a ribosomal binding sequence, including a translation
initiation signal. Additionally, depending on the host cell chosen
and the vector employed, other sequences, such as an origin of
replication, additional DNA restriction sites, enhancers, and
sequences conferring inducibility of transcription may be
incorporated into the expression vector. It will also be
appreciated that the necessary regulatory sequences may be supplied
by the native A and B chains and/or its flanking regions.
[0157] The recombinant expression vectors of the invention may also
contain a selectable marker gene which facilitates the selection of
host cells transformed or transfected with a recombinant molecule
of the invention. Examples of selectable marker genes are genes
encoding a protein such as G418 and hygromycin which confer
resistance to certain drugs, .beta.-galactosidase, chloramphenicol
acetyltransferase, firefly luciferase, or an immunoglobulin or
portion thereof such as the Fc portion of an immunoglobulin
preferably IgG. Transcription of the selectable marker gene is
monitored by changes in the concentration of the selectable marker
protein such as .beta.-galactosidase, chloramphenicol
acetyltransferase, or firefly luciferase. If the selectable marker
gene encodes a protein conferring antibiotic resistance such as
neomycin resistance transformant cells can be selected with G418.
Cells that have incorporated the selectable marker gene will
survive, while the other cells die. This makes it possible to
visualize and assay for expression of recombinant expression
vectors of the invention and in particular to determine the effect
of a mutation on expression and phenotype. It will be appreciated
that selectable markers can be introduced on a separate vector from
the nucleic acid of interest.
[0158] Recombinant expression vectors can be introduced into host
cells to produce a transformant host cell. The term "transformant
host cell" is intended to include prokaryotic and eukaryotic cells
which have been transformed or transfected with a recombinant
expression vector of the invention. The terms "transformed with",
"transfected with", "transformation" and "transfection" are
intended to encompass introduction of nucleic acid (e.g. a vector)
into a cell by one of many possible techniques known in the art.
Prokaryotic cells can be transformed with nucleic acid by, for
example, electroporation or calcium-chloride mediated
transformation. Nucleic acid can be introduced into mammalian cells
via conventional techniques such as calcium phosphate or calcium
chloride co-precipitation, DEAE-dextran mediated transfection,
lipofectin, electroporation or microinjection. Suitable methods for
transforming and transfecting host cells can be found in Sambrook
et al. (Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold
Spring Harbor Laboratory press (1989)), and other laboratory
textbooks.
[0159] Suitable host cells include a wide variety of prokaryotic
and eukaryotic host cells. For example, the proteins of the
invention may be expressed in bacterial cells such as E. coli,
insect cells (using baculovirus), yeast cells or mammalian cells.
Other suitable host cells can be found in Goeddel, Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego,
Calif. (1991).
[0160] More particularly, bacterial host cells suitable for
carrying out the present invention include E. coli, B. subtilis,
Salmonella typhimurium, and various species within the genus'
Pseudomonas, Streptomyces, and Staphylococcus, as well as many
other bacterial species well known to one of ordinary skill in the
art. Suitable bacterial expression vectors preferably comprise a
promoter which functions in the host cell, one or more selectable
phenotypic markers, and a bacterial origin of replication.
Representative promoters include the .beta.-lactamase
(penicillinase) and lactose promoter system (see Chang et al.,
Nature 275:615 (1978)), the trp promoter (Nichols and Yanofsky,
Meth. in Enzymology 101:155, 1983) and the tac promoter (Russell et
al., Gene 20: 231, 1982). Representative selectable markers include
various antibiotic resistance markers such as the kanamycin or
ampicillin resistance genes. Suitable expression vectors include
but are not limited to bacteriophages such as lambda derivatives or
plasmids such as pBR322 (see Bolivar et al., Gene 2:9S, (1977)),
the pUC plasmids pUC18, pUC19, pUC118, pUC119 (see Messing, Meth in
Enzymology 101:20-77, 1983 and Vieira and Messing, Gene 19:259-268
(1982)), and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene,
La Jolla, Calif.). Typical fusion expression vectors which may be
used are discussed above, e.g. pGEX (Amrad Corp., Melbourne,
Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5
(Pharmacia, Piscataway, N.J.). Examples of inducible non-fusion
expression vectors include pTrc (Amann et al., Gene 69:301-315
(1988)) and pET 11d (Studier et al., Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif., 60-89
(1990)).
[0161] Yeast and fungi host cells suitable for carrying out the
present invention include, but are not limited to Saccharomyces
cerevisae, the genera Pichia or Kluyveromyces and various species
of the genus Aspergillus. Examples of vectors for expression in
yeast S. cerivisae include pYepSec1 (Baldari. et al., Embo J.
6:229-234 (1987)), pMFa (Kurjan and Herskowitz, Cell 30:933-943
(1982)), pJRY88 (Schultz et al., Gene 54:113-123 (1987)), and pYES2
(Invitrogen Corporation, San Diego, Calif.). Protocols for the
transformation of yeast and fungi are well known to those of
ordinary skill in the art.(see Hinnen et al., Proc. Natl. Acad.
Sci. USA 75:1929 (1978); Itoh et al., J. Bacteriology 153:163
(1983), and Cullen et al. (Bio/Technology 5:369 (1987)).
[0162] Mammalian cells suitable for carrying out the present
invention include, among others: COS (e.g., ATCC No. CRL 1650 or
1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa
(e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1 cells.
Suitable expression vectors for directing expression in mammalian
cells generally include a promoter (e.g., derived from viral
material such as polyoma, Adenovirus 2, cytomegalovirus and Simian
Virus 40), as well as other transcriptional and translational
control sequences. Examples of mammalian expression vectors include
pCDM8 (Seed, B., Nature 329:840 (1987)) and pMT2PC (Kaufman et al.,
EMBO J. 6:187-195 (1987)).
[0163] Given the teachings provided herein, promoters, terminators,
and methods for introducing expression vectors of an appropriate
type into plant, avian, and insect cells may also be readily
accomplished. For example, within one embodiment, the proteins of
the invention may be expressed from plant cells (see Sinkar et al.,
J. Biosci (Bangalore) 11:47-58 (1987), which reviews the use of
Agrobacterium rhizogenes vectors; see also Zambryski et al.,
Genetic Engineering, Principles and Methods, Hollaender and Setlow
(eds.), Vol. VI, pp. 253-278, Plenum Press, New York (1984), which
describes the use of expression vectors for plant cells, including,
among others, pAS2022, pAS2023, and pAS2034).
[0164] Insect cells suitable for carrying out the present invention
include cells and cell lines from Bombyx or Spodotera species.
Baculovirus vectors available for expression of proteins in
cultured insect cells (SF 9 cells) include the pAc series (Smith et
al., Mol. Cell Biol. 3:2156-2165 (1983)) and the pVL series
(Lucklow, V. A., and Summers, M. D., Virology 170:31-39 (1989)).
Some baculovirus-insect cell expression systems suitable for
expression of the recombinant proteins of the invention are
described in PCT/US/02442.
[0165] The recombinant expression vectors containing the nucleotide
sequences encoding the HA-binding peptides may also contain genes
which encode a fusion moiety which provides increased expression of
the recombinant peptide; increased solubility of the recombinant
peptide; and aid in the purification of the target recombinant
peptide by acting as a ligand in affinity purification. For
example, a proteolytic cleavage site may be added to the target
recombinant protein to allow separation of the recombinant protein
from the fusion moiety subsequent to purification of the fusion
protein. Typical fusion expression vectors include pGEX (Amrad
Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly,
Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse
glutathione S-transferase (GST), maltose E binding protein, or
protein A, respectively, to the recombinant protein. By way of
illustration, the DNA sequence encoding an enhanced affinity HA
binding peptide may be cloned into a pGEX-type plasmid for
co-expression with a 26 kD protein glutathione-S-transferase (GST):
pGEX-2T, pGEX-2TK, pGEX-3X, pGEX-1T, pGEX-4T, pGEX-5X. Said
plasmids are transformed into Escherichia coli HB101 cells, and
positive clones can then be selected by standard hybridization
techniques identifying GST-HA binding peptide fusion protein.
Colonies with the pGEX-2T plasmids containing DNA encoding an
enhanced affinity HA binding peptide may be grown in 5 mL of LB/amp
medium at 37.degree. C. for overnight. Isopropylthio-D-galactoside
is added to the culture to a final concentration of 0.1 mM to
induce the biosynthesis of the fusion protein. The cultures are
grown for 90 min post induction and the cells are harvested by
centrifugation and lysed in 1 mL of 50 mM Tris-HCl, pH 8.0,
containing 2 M urea and 1% Triton X-100. Cells can then be further
disrupted by sonication and centrifuged at 15,000.times.g for 20
min to separate soluble from insoluble fractions. The supernatant
containing the GST-HA binding peptide fusion protein is then used
for extraction of the fusion protein by standard procedures (GST
Purification Module: Pharmacia Cat. #27-4570-01, -02).
Subsequently, the fusion protein may be treated with an enzyme to
release the enhanced affinity HA-binding peptide (thrombin for
pGEX-2T, pGEX-2TK, pGEX-1T, pGEX-4T; factor Xa for pGEX-3X and
pGEX-5X).
[0166] Another aspect of the invention provides a nucleotide
sequence which hybridizes under high stringency conditions to a
nucleotide sequence encoding a HA-binding peptide. Appropriate
stringency conditions which promote DNA hybridization are known to
those skilled in the art, or can be found in Current Protocols in
Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1 6.3.6.
For example, 6.0.times.sodium chloride/sodium citrate (SSC) at
about 45.degree. C., followed by a wash of 2.0.times.SSC at
50.degree. C. may be employed. The stringency may be selected based
on the conditions used in the wash step. By way of example, the
salt concentration in the wash step can be selected from a high
stringency of about 0.2.times.SSC at 50.degree. C. In addition, the
temperature in the wash step can be at high stringency conditions,
at about 65.degree. C.
[0167] Isolation of HA-Binding Peptides
[0168] HA-binding peptides may be isolated by assaying a sample for
peptides that bind to HA. Any assay system or testing method that
detects protein-protein interactions may be used including
co-immunoprecipitation, crosslinking and co-purification through
gradients or chromatographic columns may be used. Biological
samples and commercially available libraries may be tested for
HA-binding peptides. For example, labelled HA may be used to probe
phage display libraries as is described in greater detail in
Example 1. In addition, antibodies prepared to the peptides of the
invention may be used to isolate other peptides with HA binding
affinity. For example, labelled antibodies may be used to probe
phage display libraries or biological samples.
[0169] Additionally, a DNA sequence encoding a HA protein may be
used to probe biological samples or libraries for nucleic acids
that encode HA-binding proteins.
[0170] Applications of the Peptides
[0171] The inventor has demonstrated that the HA binding peptides
of the present invention are able to influence and inhibit cell
motility and locomotion in vivo and in vitro. Consequently, the
present invention includes the use of one or more HA-binding
peptides of the invention to modulate cell locomotion. Accordingly,
the present invention provides a method of modulating cell
locomotion comprising administering an effective amount of a HA
binding peptide or a nucleic acid molecule encoding a HA binding
peptide of the invention to a cell or animal in need thereof.
[0172] The term "effective amount" as used herein means an amount
effective, at dosages and for periods of time necessary to achieve
desired results.
[0173] More specifically, the peptides of the invention may be used
in the prophylaxis or treatment of pathological conditions
involving cell locomotion such as cancer, inflammatory and
autoimmune disorders, and fibrotic disorders associated with tissue
trauma and its recovery in a mammal.
[0174] In one embodiment, the HA binding peptides of the present
invention are useful for prevention of fibrosis of adult human
tissues thereby eliminating clinical pathologies resulting from the
malfunction of tissues due to keloids, hypertrophic scars,
anatomonic strictures, intra-abdominal adhesions, cirrhosis of the
liver, neurological deficits following spinal cord injury, valvular
heart diseases, burn-injured joints as well as failure of
anastomosis and adhesions following surgery. Accordingly, the
present invention provides a method of preventing or inhibiting
tissue fibrosis comprising administering an effective amount of a
HA binding peptide of the invention to an animal in need
thereof.
[0175] Pretreatment of skin wounds with enhanced affinity HA
binding peptides have resulted in significant reduction in
fibroblast activity and deposition of collagen at the wound site
thereby preventing wound contraction and tissue fibrosis. As
mentioned above, fibrosis of adult human tissues is a serious
clinical pathology which can result in malfunction of tissues due
to keloids, hypertrophic scars, anatomonic strictures,
intra-abdominal adhesions, cirrhosis of the liver, neurological
deficits following spinal cord injury, valvular heart diseases,
burn-injured joints as well as failure of anastomosis and adhesions
following surgery. The application of enhanced affinity HA binding
peptides therapeutically to skin injuries can reduce or eliminate
the adversities associated with tissue fibrosis during wound
healing and these peptides possess important clinical utilities,
both for therapeutic and for aesthetic purposes. Notable examples
of surgical procedures which may benefit from the treatment with
RHAMM-peptides include coronary balloon angioplasty (prevention of
restenosis), small intestinal resections (e.g. in Crohn's Disease),
surgery of the renal system (e.g. ureteral connection in renal
transplants), and vascular surgery. Similarly, the application of
RHAMM-peptides in plastic and cosmetic surgery can minimize the
aesthetic consequences of hypertrophic scars and skin
disfiguration.
[0176] Accordingly, the present invention provides a method of
preventing or reducing tissue scarring comprising administering an
effective amount of a HA binding peptide or a nucleic acid molecule
encoding a HA binding peptide of the invention to an animal in need
thereof.
[0177] In a further embodiment, the peptides of the invention are
useful in treating cancers that are associated with the activation
of the ras-oncogene including cancer of the lung, gastrointestinal,
breast, bladder, skin cancer (melanoma and non-melanoma), brain,
cervix, and leukemia. Accordingly, the present invention provides a
method of preventing or treating cancer comprising administering an
effective amount of a HA binding peptide or a nucleic acid molecule
encoding a HA binding peptide of the invention to an animal in need
thereof.
[0178] Locomotion and motility of tumour cells are fundamental to
their ability to invade other tissues and metastasize and prior
studies have shown that highly metastatic cancer cells locomote
more rapidly than slow- or non-metastatic cells (Mohler, J. L. et
al., J. Urol. 138: 168-170, 1987; Raz, A. et al., Cancer Metas.
Rev. 6: 3021, 1987; Hosaka, S. et al., Gann 69: 273-276, 1978).
Furthermore, enhanced affinity HA binding peptides of this
invention may also elicit beneficial impact on activation of the
ras oncogene and carcinogenesis. Initial studies have shown that
administration of enhanced affinity HA binding peptides terminated
replication of malignant cells at the G.sub.2/M phase of the cell
cycle and induced apoptosis.
[0179] The ability of the present enhanced affinity HA binding
peptides to inhibit the motility ras-transformed cells implicates
their effectiveness in preventing tumour metastasis and their
utility as cancer chemotherapeutic agents. Accordingly, the present
invention provides a method of preventing or reducing tumour
metastasis comprising administering an effective amount of a HA
binding peptide or a nucleic acid molecule encoding a HA binding
peptide of the invention to an animal in need thereof.
[0180] The peptides and nucleic acid molecules of the invention may
be formulated into pharmaceutical compositions for adminstration to
subjects in a biologically compatible form suitable for
administration. By biologically compatible form suitable for
administration is meant a form of the substance to be administered
in which any toxic effects are outweighed by the therapeutic
effects. The substances may be administered to living organisms
including humans, and animals in a therapeutically effective
amount. Administration of an effective amount of the pharmaceutical
compositions of the present invention is defined as an amount
effective, at dosages and for periods of time necessary to achieve
the desired result. For example, a therapeutically active amount of
a substance may vary according to factors such as the disease
state, age, sex, and weight of the individual, and the ability of
peptide to elicit a desired response in the individual. Dosage
regime may be adjusted to provide the optimum therapeutic response.
For example, several divided doses may be administered daily or the
dose may be proportionally reduced as indicated by the exigencies
of the therapeutic situation.
[0181] The active substance may be administered in a convenient
manner such as by topical or transdermal application, injection
(subcutaneous, intravenous, etc.), oral administration, inhalation,
or rectal administration. Depending on the route of administration,
the active substance may be coated in a material to protect the
compound from the action of enzymes, acids and other natural
conditions which may inactivate the compound.
[0182] Several modes of administration are available when using a
composition containing a nucleic acid molecule encoding a HA
binding peptide of the invention. Recombinant molecules comprising
an nucleic acid sequence encoding a HA binding protein (as
described above), or fragment thereof, may be directly introduced
into cells or tissues in vivo using delivery vehicles such as
retroviral vectors, adenoviral vectors and DNA virus vectors. They
may also be introduced into cells in vivo using physical techniques
such as microinjection and electroporation or chemical methods such
as coprecipitation and incorporation of DNA into liposomes.
Recombinant molecules may also be delivered in the form of an
aerosol or by lavage. The nucleic acid molecules of the invention
may also be applied extracellularly such as by direct injection
into cells.
[0183] The compositions described herein can be prepared by per se
known methods for the preparation of pharmaceutically acceptable
compositions which can be administered to subjects, such that an
effective quantity of the active substance is combined in a mixture
with a pharmaceutically acceptable vehicle. Suitable vehicles are
described, for example, in Remington's Pharmaceutical Sciences
(Remington's Pharmaceutical Sciences, Mack Publishing Company,
Easton, Pa., USA 1985). On this basis, the compositions include,
albeit not exclusively, solutions of the substances in association
with one or more pharmaceutically acceptable vehicles or diluents,
and contained in buffered solutions with a suitable pH and
iso-osmotic with the physiological fluids.
[0184] Compositions for injection include, albeit not exclusively,
the peptides or nucleic acids in association with one or more
pharmaceutically acceptable vehicles or diluents, and contained in
buffered solutions with a suitable pH and iso-osmotic with the
physiological fluids. Any pharmaceutically suitable diluent can be
used in the composition for injections: distilled water,
physiological or a salt solution, and/or a buffer solution. The
composition for injections may be prepared by conventional
volume-weight procedures. A certain amount of the peptide is
diluted to the necessary volume with a diluent or solvent. The
solution is then filtered through sterilized filters, bottled or
ampouled. The resultant solution is a stable transparent liquid,
and does not contain any chemical or other impurities.
[0185] Solid form preparations for oral administration can be made
in the form of tablets, powders, or capsules. It may contain a
medium for the active substance and other additives, including
dyes, aromas, etc.
[0186] The compositions and treatments are indicated as therapeutic
agents or treatments either alone or in conjunction with other
therapeutic agents or other forms of treatment.
EXAMPLES
Example 1
[0187] Isolation of HA-Binding Peptides Using Phage Display
Technology
[0188] A 15-mer phage display library was obtained from Chiron Inc.
(CA). Hyaluronan (HA) was biotinylated and mixed with
streptavidin-sepharose (Sigma) and washed. The Biotinylated HA was
incubated with the phage library, then recovered with
streptavidin-sepharose. The phage biotinylated HA-streptavidin
-sepharose mixture was washed 4 times with phosphate buffered
saline (PBS) and recovered phage were plated on a bacteria lawn in
petri dishes. Clones were isolated and assessed for their ability
to bind to HA. For the first screen, a total 75 clones were
obtained after 4 cycles of screening. To assess the specificity of
binding of these clones to HA, two approaches were taken. In the
first, the ability of the clones to bind to biotin-sepharose or to
streptavidin-sepharose were assessed. Clones were recovered from
the bacteria lawn and mixed with biotin-sepharose and
streptavidin-sepharose. Sepharose beads were washed 4 times with
PBS and plated onto a bacteria lawn. In the second assay, clones
were mixed with excess (100 ug/mL) HA (mean molecular weight:
700,000 daltons (Hyal, Ontario, Canada) together with biotinylated
HA. After mixing the biotinylated HA was recovered with
streptavidin-sepharose, washed as above and plated onto a bacteria
lawn. Clones were sequenced in an automatic sequencer. A schematic
diagram illustrating the isolation of enhanced affinity hyaluronan
binding peptides from the 15-mer phage display library is shown in
FIG. 1.
[0189] Assessment of HA-Binding Properties of Peptides
[0190] Enhanced affinity HA binding peptides mimicking sequences
obtained from the phage display library and scrambled peptides
prepared from these sequences were tested for their HA binding
properties. HA was derivatized to NUNC ELISA plates and
biotinylated peptides were added to the plates and incubated for 2
h at room temperature. Plates were washed 4 times with PBS and then
incubated with streptavidin-peroxidase for 1 h, washed, developed
with hydrogen peroxide and their optical densities were determined.
Controls were conducted using BSA-coated ELISA wells.
[0191] Enhanced affinity binding peptides were determined and found
to possess the two following sequences. The first sequence (45
clones) encoded STMMSRSHKTRSHHV while the second sequence (30
clones) encoded TMTRPHFHKRQLVLS. The inventor has shown that a
peptide of the first sequence that has the C-terminal valine
removed (i.e., STMMSRSHKTRSHH) or has a cysteine residue at
position 13 inserted and the C-terminal valine deleted (i.e.,
STMMSRSHKTRSCHH) also can bind to HA.
[0192] The peptide sequences of the present invention are different
from the HA binding motif disclosed by Yang et al. and represented
by the generic formula B.sup.1-A.sub.n-B.sup.2 (wherein B.sup.1 and
B.sup.2 are the same or different basic amino acid residues and
A.sub.n is a peptide sequence containing seven or eight amino acid
residues which are the same or different and are neutral or basic
amino acids (Yang, B. et al., J. Biol. Chem. 268: 8617-8623,
1993)). In particular, Yang et al. state that when B.sup.2 is a
histidine residue the HA-binding ability of the RHAMM peptides is
abolished. Consequently, the peptides STMMSRSHKTRSHHV,
STMMSRSHKTRSHH and STMMSRSHKTRSCHH would not be expected to bind HA
according to the Yang et al. formula. In addition, the second
sequence TMTRPHFHKRQLVLS does not contain a B.sup.1-A.sub.n-B.sup.2
motif as n can not be a peptide sequence containing five amino acid
residues. Yang et al. demonstrated a lost in binding activity if
A.sub.n is less than seven or greater than eight amino acid
residues. Therefore, the peptides of the present invention are
distinct to those described in this reference based on the
exclusion criteria clearly defined as critical by Yang et al.
Moreover, the novel enhanced affinity hyaluronan binding peptides
of the present invention exhibit higher HA binding affinity
(nanomolar range) than the nonomeric and decameric peptides
previously described.
Example 2
[0193] Effect of Enhanced affinity HA-Binding Peptide on Cell
Locomotion In Vitro
[0194] Cell Culture
[0195] Ras-transformed 10T1/2 or parental 10T1/2 fibroblasts (Egan,
S. E. et al., Science 238: 202-205, 1987; Hardwick, C. et al., J.
Biol. Chem. 117: 1343-1350, 1992) were cultured in DMEM containing
10% fetal bovine serum (FBS) and maintained in 5% CO.sub.2
humidified atmospheric condition. Every second passage, cells were
maintained in 600 ug/mL G418 for one passage to select for
maintenance of the mutant active ras-geneticin construct in cells.
Cells were passaged at confluence and utilized under 20 passages.
Macrophages were obtained from lung lavage 1 h after bleomycin
insertion (Gelzleichter, T. R. et al., Fund. Appl. Toxicol. 30:
39-46, 1996) and used immediately in assays. Neutrophils were
obtained from human blood as described and used immediately.
[0196] Immunofluorescence
[0197] Ras-transformed or parental 10T1/2 fibroblasts (Egan, S. E.
et al., Science 238: 202-205, 1987) were subcultured onto
fibronectin coated (20 ug/mL) glass coverslips and cultured for
24-48 h. The enhanced affinity hyaluronan binding peptide (shown in
SEQ.ID.NO.:3) or scrambled peptides were added at 100 ng/mL to
cultures and cells were fixed at 10 min, 20 min, 1 h and 2 h after
the addition of peptides. Cultures were washed carefully in
phosphate buffered saline (PBS) and then fixed in freshly prepared
3.5% paraformaldehyde for 20 min at 4.degree. C. Fixed monolayers
were washed in PBS and then incubated in 1.0 M glycine to reduce
autofluorescence. Cultures were washed again and then incubated
with anti-vinculin antibody (1:100, Pharmingen) for 2 h. Cultures
were mounted and observed with an epifluorescence microscope
(Zeiss).
[0198] For routine analysis of random locomotion, 10T1/2
fibroblasts or leukocytes were subcultured into culture flasks
coated with 20 ug/mL fibronectin and maintained in DMEM plus 10%
FBS for 24-48 h. The enhanced affinity hyaluronan binding peptide
(shown in SEQ.ID.NO.:3) or scrambled peptides were incubated with
the cultures at concentrations of 50 ng to 1 ug per mL.
Concentrations of 50-100 ng/mL were found to be optimal for
inhibiting cell motility. Cells were observed with Hoffman optics
and the image analysis program automatically follows the outline of
the cells. Filming was conducted for up to 24 h with images
recorded every 20 min. A total of 100 cells were observed for each
experimental condition.
[0199] For serum stimulation of cell motility, cells were plated
onto fibronectin-coated culture dishes (30 ug/mL) and grown in 10%
FBS to semi-confluence. Cells were then maintained in defined
medium for 24 h. Medium was removed and replaced with medium
containing 10% FBS and cell motility was monitored as above.
[0200] To assess the effects of enhanced affinity hyaluronan
binding peptides and scrambled peptides on chemotaxis, a modified
multi-well boyden chamber was utilized as described by Shi, Y. et
al. (J. Immunol. Methods 164: 149-154, 1993).
[0201] Protein Tyrosine Phosphorylation
[0202] Cultures were grown to 70-80% confluence and were then
serum-starved for 24 h. DMEM medium containing 10% FBS was added to
cells in the presence or absence of the enhanced affinity
hyaluronan binding peptide (shown in SEQ.ID.NO.:3) or scrambled
peptides, and the cells were monitored for their motility for
periods of up to 3 h. At the end of each filming period, cell
monolayers were extracted with RIPA and 10 ug of protein was
electrophoresed on SDS-PAGE. Separated protein was transferred to
nitrocellulose blots and was then processed for protein tyrosine
phosphorylation using an anti-phosphotyrosine antibody (Shi, Y. et
al., J. Cell. Biol. 126: 575-588, 1994). An equal amount of sample
was also electrophoresed on SDS-PAGE in an identical manner and was
stained with Coomassie Blue to assess protein loading.
[0203] FAK Immunoprecipitation
[0204] Cultures were tested with peptides as described above then
monolayers were extracted with RIPA buffer. The extract was
microcentrifuged for 3 min. to remove particulate material.
Anti-FAK antibody was incubated with the extract for 2 h then the
antibody was captured using anti-mouse IgG-sepharose. The complex
was washed 3 times then FAK was removed from the complex by boiling
in SDS-PAGE loading buffer and electrophoresed in SDS-PAGE. Protein
was then transferred to a nitrocellulose blot and the blot was
stained for protein tyrosine phosphorylation using an anti-tyrosine
antibody. Blots were then stripped and reincubated with the
anti-FAK antibody and processed as above to detect loading of
immunoprecipitated FAK.
[0205] Cell Proliferation
[0206] 10T1/2 fibroblasts were grown to semi-confluence and were
incubated with enhanced affinity hyaluronan binding peptide (shown
in SEQ.ID.NO.:3) at a concentration of 100 ug/mL for 48 h. The
cells were then trypsinized in 0.12% trypsin and counted using a
hematocytometer. Released cells were stained with trypan blue and
only viable cells were counted.
[0207] Apoptosis
[0208] Ras-transformed 10T1/2 fibroblasts were grown to
sub-confluence and were incubated with enhanced affinity hyaluronan
binding peptide (shown in SEQ.ID.NO.:3) at a concentration of 100
ug/mL for 48 h. The cells were then stained with Hoechst dye using
standard procedures and the percentage of apoptotic cells were
determined by detection of positive stain in the nucleus of
randomly selected cells.
[0209] Results on Cell Locomotion and Protein Tyrosine
Phosphorylation
[0210] HA/RHAMM interactions have been shown to control focal
adhesion turnover during cell locomotion and levels of protein
tyrosine phosphorylation. The effects of enhanced affinity
hyaluronan binding peptides on focal adhesion formation and protein
tyrosine phosphorylation were examined.
[0211] Enhanced affinity hyaluronan binding peptides were shown to
strongly inhibit fibroblast random motility (TABLES II and III).
The formation of focal adhesions was observed when the cells were
plated on fibronectin substrata (FIGS. 2A-F). Focal adhesion
assembly was enhanced by 20 min following the addition of enhanced
affinity hyaluronan binding peptides and was maintained at high
levels for several hours. This effect appeared to be due to the
ability of the peptides to sequester HA as treatment of cells with
Streptomyces with hyaluronidase produced a similar effect on focal
adhesion formation (FIG. 2G).
[0212] Serum-starved cells exhibited slowed cell motility, and
re-introduction of serum enhanced cell locomotion (FIGS. 3F-H).
When cell motility was monitored over short time periods, regular
fluctuations in the rate of locomotion was apparent. Such
fluctuations in motility coincided with cycles of protein tyrosine
phosphorylation/dephosphorylati- on of various proteins. The rate
of protein tyrosine phosphorylation was reciprocally correlated
with motility rate. Addition of enhanced affinity hyaluronan
binding peptides inhibited cycling of protein tyrosine
dephosphorylation (most notably of proteins of MW 125, 85 and 70
kD) and inhibited fluctuations in cell motility (FIGS. 3A-H). The
duration of inhibitory effect was over 18 h following
administration (FIG. 3I).
Example 3
[0213] Effect of Enhanced affinity HA-Binding Peptide on Wound
Repair In Vivo
[0214] Punch Biopsy Model
[0215] Response to injury of skin involves extensive extracellular
remodelling initiated by infiltration of fibroblasts and
leucocytes. The adult skin healing process involves an acute and
transient increase in HA production followed by deposition of
extracellular matrix glycoproteins such as collagen. Healing is
achieved with extensive leucocyte infiltration and the consequent
release of growth factors that attract and stimulate and enhance
proliferation of fibroblasts. This fibroproliferative response is
accompanied by wound contraction and fibrosis due to the presence
of myobroblasts and to the enhanced production of collagen.
[0216] The early deposition of HA following skin injury has been
shown to regulate leucocyte chemotaxis and function. Neutral
hyaluronidases are released by macrophages and the production of
oligosaccharides and from polymeric HA may in turn stimulate the
release of growth factors such as TGF.beta.-1 that alter macrophage
and fibroblast function. However, as mentioned above, the effects
of HA is governed by the level of accumulation wherein high HA
concentrations paradoxically inhibits fibroblast migration,
angiogenesis, and the ability of fibroblasts to contract collagen
gels.
[0217] As also aforementioned, the major biological responses to HA
in tissue remodelling are mediated through at least two HA
receptors, CD44 and RHAMM. CD44 has been implicated in to be
involved in promoting the motility and proliferation within
epidermal structures and to be required for the efficient repair of
skin. Similarly, RHAMM has been shown to be overexpressed in
activated white cells and fibroblasts migrating in response to
growth factors.
[0218] In order to further delineate the significance of HA/RHAMM
interaction in the wound healing process and to demonstrate the
clinical utility of enhanced affinity HA-binding peptides, a rat
punch biopsy model of skin repair was used to investigate the
expression of RHAMM and the in vivo effects of the HA binding
peptides of the present invention with respect to the severity of
fibrotic response following tissue trauma. Collagen was used as a
vehicle to stimulate inflammation and fibrosis as rat skin normally
exhibits minimal fibrosis.
[0219] Three-month-old female Fischer rats were anesthesized and
subjected to 47 mm full thickness punch biopsies. Wounds were
filled with a sterile solution of 1% collagen type 1 gel containing
either PBS alone, HA binding peptide (shown in SEQ.ID.NO.:3) (20
mg/mL) or scrambled peptide (20 mg/mL) that contained the same
amino acids as the HA binding peptide but in a random order. 100 uL
of the collagen solution was applied to the punch biopsy wound and
allowed to gel over 20 min. The wounds were then covered with a
protective plastic shield that was sewn into the skin. Wounds were
recovered from euthenized rats with a 9 mm punch biopsy.
[0220] Western Analyses
[0221] Recovered punch biopsies were extracted with RIPA buffer and
total protein was determined with a Biorad protein assay. Protein
(50 ug) was loaded onto each lane and proteins were separated by
electrophoresis on SDS-PAGE. Separated proteins were transferred to
a nitrocellulose blot and the blot was processed for reactivity to
anti-RHAMM antisera or anti-CD44 antibody (Dillon, P. W. et al., J.
Surg. Res. 57: 170-173, 1994).
[0222] Isolation of mRNA and RT-PCR
[0223] Messenger RNA was isolated by Micro-FastTrack Kit
(Invitrogen, San Diego, Calif.). The quality of RNA samples was
confirmed by denaturing gel electrophoresis. Reverse transcription
was performed by using First Strand cDNA Synthesis kit (Clontech,
Polo Alco, Calif.). Briefly, 0.2 ug of mRNA was used to generate
cDNA using oligo(dT) 18 primer. Samples containing 0.2 ug of mRNA
per time point was heat-denatured in DEPC-treated water for 2 min
at 70.degree. C. and incubated at 42.degree. C. for 1 h in a total
volume of 20 uL of 20 pmol primers, 0.5 mM of each dNTP, 1 unit/uL
of RNase inhibitor and 200 units/ug RNA of MMLV. The reaction was
stopped by heating at 94.degree. C. for 5 min and to destroy any
DNase activity. The reaction mixture was diluted to a final volume
of 100 uL and aliquots were stored at -80.degree. C. until further
use.
[0224] To distinguish between amplication of genomic DNA and
complementary DNA, the primer pairs were designed to enclose at
least one intron on the genomic sequences of RHAMM and beta-actin.
The primers were synthesized by GIBCO and the sequences of the
primers were as follows: Beta-actin A: 5' GTA ACC AAC TGG GAC GAT
AT 3' (1554-1574) SEQ.ID.NO.:13; B: GAT CTT GAT CTT CAT GGT GCT 3'
(2991-2841) SEQ.ID.NO.:14. RHAMM sense: 5' GGG TTA GTT ATG TTG GTT
GGT 3' (SEQ.ID.NO.:15) (10-31 (Entwistle, J. et al., J. Cell.
Biochem. 61: 569-577, 1996)), and antisense: 5' CTG AAG ATG AGC AGA
CAG TTC (426-406) SEQ.ID.NO.:16. CD44 sense: 5' AAT GGC CGC TAC AGT
ATC TCC AGG ACT GAA (263-266) SEQ.ID.NO.:17, CD44 antisense: 5' GTT
GAT CAC CAG CTT CTT CTT CTG CCC ACA (1451-1421 (Gunthert, U. et
al., Cell 65: 13-24, 1991)) SEQ.ID.NO.:18. Collagen type I alpha 2
sense: 5' CCC ACG TAG GTG TCC TAA AGT SEQ.ID.NO.:19, and collagen
type I alpha 2 antisense: 5' CCG TGG TGC TAA AAT AAT AAA
SEQ.ID.NO.:20. Collagen type III sense: 5' CGA GGT AAC AGA GGT GAA
AGA SEQ.ID.NO.:21, and collagen type III antisense: 5' AAC CCA GTA
TTC TCC GCT CTT (Power, W. J. et al., Curr. Eye Res. 14: 879-886,
1995) SEQ.ID.NO.:22. The PCR amplification of cDNA was carried out
by using Taq polymerase. Briefly, an aliquot of cDNA was added to a
100 uL reaction mixture containing 0.2 uM primers and 2.5 units of
Taq polymerase. Amplification was performed for 30 cycles at
94.degree. C. for 1 min of denaturation, 60.degree. C. for 1 min of
annealing and at 72.degree. C. for 2 min of extension. The PCR
products were electrophoresed in a 1% agarose gel and transferred
to nylon transfer membranes (Amersham) and checked by Southern blot
using an inter-nest probe.
[0225] Immunocytochemistry
[0226] Punch biopsies collected using an 11 mm punch were fixed in
freshly prepared 3.5% paraformaldehyde and processed for paraffin
sections. Sections of thickness of 15 um were obtained and
deparaffinized in a series of alcohol. Sections were stained for
RHAMM (Gunthert, U. et al., Cell 65: 13-24, 1991; Power, W. J. et
al., Curr. Eye Res. 14: 879-886, 1995), ED-1 (Chandler, D. B. et
al., Am. J. Pathol. 112: 170-177, 1983) and mac-3 (Pharmingen)
using either anti-rabbit or anti-mouse IgG conjugated with
horseradish peroxidase. Sections were developed as described in
Gunthert and Power as described above.
[0227] Detection of HA
[0228] HA was detected using biotinylated aggrecan in either an
ELISA or by histochemical staining (Hendin, B. N. et al., Am. J.
Card. Pathol. 3: 209-215, 1990). Aggrecan was purified from calf's
noses and biotinylated.
[0229] Enzyme Assays
[0230] To determine the specificity of biotinylated aggrecan for
HA, tissues sections were first exposed to 0.1 TRU of Streptomyces
hyaluronidase (Sigma) for 3 h. Sections were washed then incubated
with biotinylated aggrecan as described above. To assess the effect
of hyaluronidase on focal contraction formation of ras-transformed
cells, varying concentrations of the enzyme were added to adherent
cells for 1 h in buffer. Cells were then washed, fixed and
processed for vinculin staining.
[0231] To quantify leucocytes present within wounds, tissue
biopsies were extracted with RIPA buffer then processed for
detection of glucosaminidase (Podhajsky, R. J. et al., Exp. Neurol.
143: 153-161, 1997; Gelzleichter, T. R. et al., Fund. Appl.
Toxicol. 30: 39-46, 1996).
[0232] Staining disappeared when tissue sections were pretreated
with Streptomyces hyaluronidase indicating that the biotinylated
aggrecan used to detect HA was specific for this
glycosaminoglycan.
[0233] Control Wound Repair Results
[0234] Western analysis and RT-PCR analysis of mRNA extracted from
the wound site showed that RHAMM and CD44 expression was
significantly increased after injury. As shown in FIGS. 4, 5A-D,
6A-B and 7A-B, CD44 and RHAMM expression was low in uninjured adult
skin which increased after injury. This is confirmed by
immunohistochemical data showing the virtual absence of RHAMM in
uninjured skin.
[0235] Several RHAMM isoforms with molecular weight ranging from
60-80 kDa were detected by Western analysis and densitometry
analysis showed that these isoforms increased above control levels
by 24 h following injury. Histochernical analysis revealed that
RHAMM expression was strongly upregulated in keratinocytes which
was maintained at high levels for two weeks (FIGS. 5A-D and 17). At
24 h, RHAMM staining appeared to be at the cell surface and was
localized primarily within the suprabasal epidermal layer. By 72 h,
the entire epidermal layer was positive for RHAMM and staining then
appeared to be primarily intracellular. RHAMM expression in
infiltrated fibroblasts also showed marked increases in RHAMM
expression and was observed to a lesser extent in infiltrated white
blood cells. At 2 weeks after injury, RHAMM levels had diminished
in both the dermal and epidermal layers of the skin but remained
higher than uninjured levels.
[0236] RT-PCR analysis of mRNA isolated from the wound site
confirmed the above findings by showing parallel increases in RHAMM
expression (FIGS. 7A-B). The use of oligonucleotide primers that
detected all RHAMM isoforms showed an increase in RHAMM mRNA at 12
h after injury and levels were sustained above background for a
week after injury. RHAMM expression was maximal at 48 h after
injury. RHAMM 4 was a minor fraction of total RHAMM assessed with
RT-PCR and an increase in the level of this isoform was evident at
6 h after injury and declined towards baseline levels
thereafter.
[0237] Injury to the skin was also accompanied by additional
changes in extracellular matrix remodelling. HA content of control
wounds detected by biotinylated aggrecan using an ELISA was
increased by 8 h, and glucosaminidase activity indicating
macrophage content was also increased by 24 h after injury (FIGS. 8
and 9A-D). The cell type responsible for the enhanced HA production
was not ascertained but histochemical analysis indicated that
accumulation occurred within the regenerating dermis and subdermis
of the wound site (FIGS. 9A-D). HA levels returned to background
levels at 36 h after injury. Such changes to skin HA levels were
confirmed by enzyme assays.
[0238] Collagen production as determined by histochemical and
PT-PCR analyses was also found to be increased following skin
injury. Increase in collagen type I production paralleled the
infiltration of fibroblasts into the wound site. Level of collagen
type I mRNA increased to a maximum at 3 to 4 days and declined
towards basal levels over the subsequent 2 weeks (FIGS. 10A-B). The
level of collagen type III which is predominantly produced by
fibroblasts and white blood cells increased more rapidly (FIGS.
10C-D). Collagen type III mRNA level was notably increased by 6 h
following injury, peaked at 24 h, and declined towards baseline
values by 4 days (FIG. 10D). The timing of the acute increases in
HA (FIG. 8) and collagen type III (FIG. 10D) coincided with the
infiltration of leukocytes into the wound site and the resurfacing
of the wound by the epidermal layer. Similarly, the timing of
collagen type I elevation coincided with the infiltration of
fibroblasts into the wound site (FIGS. 10B and 13).
[0239] Effects of HA-Binding Peptides on Wound Repair
[0240] Enhanced affinity HA binding peptides obtained from the
phage display library were assessed for their effect on the course
of wound repair. Administration of enhanced affinity HA binding
peptide (shown in SEQ.ID.NO.:3) inhibited keratinocyte resurfacing
of the wound (FIGS. 12 II & IV) and reduced the number of
fibroblasts at the wound site (FIG. 17B). Leukocyte infiltration
into the wound, as assessed by morphological examination, mac-3 and
ED-1 staining and glucosaminidase measurement, was also reduced by
enhanced affinity HA binding peptides relative to the scrambled
peptide controls (FIG. 12(IV)). Leukocytes appeared to accumulate
at the wound periphery but did not infiltrate into the wound
site.
[0241] The effect of enhanced affinity HA binding peptides on HA
accumulation and collagen production were also assessed. As shown
in FIGS. 9A-D, treatment of wound sites with enhanced affinity HA
binding peptide (shown in SEQ.ID.NO.:3) resulted in an increase in
HA accumulation within the remodelling dermal layer at 24 h.
Staining disappeared when tissue sections were pretreated with
Streptomyces hyaluronidase indicating that the biotinylated
aggrecan used to detect HA was specific for this glycosaminoglycan
(FIGS. 9A-D). These results are consistent with the ELISA analysis
(FIG. 8) of HA levels in injured skin that showed a peak
accumulation of HA at 8 h and a reduction to background levels by
24-36 h following injury. Enhanced affinity HA binding peptides
also reduced levels of collagen types I and III at 24 h as
confirmed by mRNA determination by RT-PCR (FIGS. 15 and 16A-B).
[0242] Treatment of wound sites with enhanced affinity HA binding
peptides resulted in down-regulation of RHAMM and CD44 receptors as
analyzed by RT-PCR (FIG. 4). Immunohistochemical analysis indicated
that these receptors were depleted in keratinocytes, leucocytes and
fibroblasts. The results indicate that RHAMM display was reduced
per cell present within the wound and did not reflect an artifact
associated with the reduced number of cells such as leucocytes and
fibroblasts within the wound site. Similarly, addition of a
monoclonal antibody specific for RHAMM (3T3-5 (Turley, E. A. et
al., J. Cell. Biol. 112: 1041-1047, 1991)), previously shown to
block cell motility (Turley, E. A. et al., J. Cell. Biol. 112:
1041-1047, 1991; Hall, C. L. et al., Cell 82: 19-28, 1995),
inhibited re-epithelialization and leukocyte infiltration (FIG. 12
IV) to a similar extent to the enhanced affinity HA binding
peptides (FIG. 12 II). These data confirm that RHAMM, in addition
to CD44, is involved in cell migration and wound repair and that
the effect of enhanced affinity HA binding peptides is mediated
through RHAMM antagonism.
[0243] These changes in remodelling of the extracellular matrix
coincided with a change in the morphology of the wound site at 2
weeks post injury (FIG. 17). The epidermal layer overlying the
wound site was thicker in HA-binding peptide-treated skin as
compared to control skin. In control skin, the injured site is
contracted while fibroblasts and collagen fibrils were aligned
parallel to each other as determined by trichrome staining (FIGS.
17A-B). In contrast, skin treated with HA-binding peptides did not
show contraction and the skin resembles the surrounding uninjured
skin. Tensometry analysis indicated that the skin strength of
control versus treated wounds was not different and wound breaking
was not observed.
Example 4
[0244] Mechanism of Action for HA-Binding Peptides in Tissue
Fibrosis
[0245] Collagen Fibrillar Matrix (CFM) Assay
[0246] Tissue repair primarily consists of three overlapping phases
consisting local inflammation, granulation tissue formation, and
scar formation. The fibroproliferative response in wound repair is
characterized by the concentric alignment of fibroblasts around the
wound and production of collagen type I to form the major
extracellular matrix or scar. Fibrotic wound repair is a pathology
where excesssive formation of scar tissue is formed at the wound
site as a result of excessive wound contraction and collagen
deposition by fibroblasts.
[0247] In order to examine the ability of HA-binding peptides to
reduce tissue fibrosis, a floating collagen fibrillar matrix (CFM)
assay was established as a measure of fibroblast contraction using
Vitrogen 100 (Collagen Corporation, Palo Alto, Calif.).
Furthermore, the effects of 3 anti-RHAMM monoclonal antibodies
(against exons 4, 5 and 9 of RHAMM protein), an anti-CD44
monoclonal antibody, as well as a RHAMM-motif peptide as described
in PCT published application no. WO 93/21312, were also examined
for elucidation of the underlying mechanism(s) of action. These
monoclonal antibodies and RHAMM-motif peptide have been previously
shown to inhibit HA binding to its receptors, RHAMM and CD44.
[0248] In the assay, CFM was formed in 24-well non-stick
cultureware and were pre-equilibrated with 2 changes of
alpha-modified Eagle's medium (alpha-MEM). The concentrations of
HA-binding peptide studied were 1 ng/mL to 500 ug/mL. Each
concentration of HA-binding peptide was resuspended in 10% fetal
bovine serum (FBS) in alpha-MEM medium and 0.9 mL of such
HA-binding peptide dilution was added to each well. Gels and media
with or without HA-binding peptide were allowed to equilibrate
overnight at 37.degree. C. with 5% CO.sub.2. On the following day,
100 uL of human foreskin fibroblasts were added to each CFM well in
10% FBS and alpha-MEM at a concentration of 0.1.times.10.sup.6
cells/mL. Cells were evenly distributed by rotating the cultureware
for 15 seconds. Final culture media composed of 1 ng/mL, 10 ng/mL,
100 ng/mL. 100 ug/mL or 500 ug/mL of HA-binding peptide in 1 mL
alpha-MEM containing 10% FBS. Each experimental CFM with
fibroblasts with or without HA-binding peptide were incubated at
37.degree. C. with 5% CO.sub.2. After plating the fibroblasts for 2
h, CFMs were detached from the bottoms of the wells. Photographs
were taken at different times, up to 48 h. Areas of CFMs were
measured by using NIH Image 1.6267.
[0249] CFM Results
[0250] The current CFM assay showed a dose-dependent inhibition of
human foreskin fibroblasts contraction by enhanced affinity
HA-binding peptides of the present invention as compared to control
(FIGS. 18 and 19) thereby indicating its efficacy in reducing wound
contraction in fibrotic wound healing. The effective concentration
range of said HA-binding peptides is 1 ng/mL to 500 ug/mL.
[0251] In order to further distinguish the HA-binding peptides of
the present invention from the RHAMM-motif peptides as described in
PCT published application no. WO 93/21312, the present
experimentation has shown that the latter RHAMM-motif peptides,
over the same concentration range, are not effective in inhibiting
human foreskin fibroblasts contraction (FIG. 20). Since the
RHAMM-motif peptides resemble the receptor domains in RHAMM
responsible for HA binding and are known to inhibit HA binding to
RHAMM, the failure of RHAMM-motif peptides to reduce tissue
contraction indicates that the enhanced affinity HA-binding
peptides of the present invention do not inhibit tissue contraction
by interfering with HA binding to RHAMM. This observation is
further supported by the fact that addition of 2 anti-RHAMM
(anti-exon-5 and anti-exon-9) monoclonal antibodies did not affect
human fibroblasts contraction as determined by the CFM assay (FIGS.
21 and 22).
[0252] Furthermore, addition of an anti-CD44 monoclonal antibody
also did not inhibit fibroblasts contraction in the CFM assay
suggesting that the inhibitory effects of the HA-binding peptides
of the present invention are not mediated by interference of HA
binding to CD44 (FIG. 23).
[0253] Having illustrated and described the principles of the
invention in a preferred embodiment, it should be appreciated by
those skilled in the art that invention can be modified in
arrangement and detail without departure from such principles. I
claim all modifications coming within the scope of the following
claims.
1TABLE I DNA Sequences for An Enhanced Affinity HA-Binding Peptides
SEQ ID NO.5 For expression in Escherichia coli A C C A T G A C C C
G T C C G C A C T T C C A C A A A C G T C A G C T G G T T C T G T C
T SEQ ID NO.6 For expression in Streptomyces lividans A C S A T G A
C S C G S C C S C A C T T C C A C A A G C G S C A G C T S G T S C T
S W S S wherein S is C or G and W is A or T. SEQ ID NO.7 For
expression in Escherichia coli T C T A C C A T G A T G T C T C G T
T C T C A C A A A A C C C G T T C T C A C C A C T G T SEQ ID NO.8
For expression in Streptomyces lividans W S S A C S A T G A T G W S
S C G S W S S C A C A A G A C S C G S W S S C A C C A C T G C
wherein S is C or G and W is A or T. SEQ.ID.NO.9 For expression in
Escherichia coli TCT ACC ATG ATG TCT CGT TCT CAC AAA ACC CGT TCT
CAC CAC wherein S is C or G and W is A or T. SEQ.ID.NO.10 For
expression in Streptomyces lividans WSS ACS ATG ATG WSS CGS WSS CAC
AAG ACS CGS WSS CAC CAC wherein S is C or G and W is A or T.
SEQ.ID.NO.:11 For expression in Escherichia coli TCT ACC ATG ATG
TCT CGT TCT CAC AAA ACC CGT TCT CAC CAC GTG wherein S is C or G and
W is A or T. SEQ.ID.NO.:12 For expression in Streptomyces lividans
WSS ACS ATG ATG WSS CGS WSS CAC AAG ACS CGS WSS CAC CAC GTC wherein
S is C or G and W is A or T.
[0254]
2TABLE II Effect of Peptide on Cell Locomotion Rate of Locomotion
HA-Binding Peptide Scrambled (STMMSRSHKTRSHH) A: Cell Type Peptide
(um/h) (47) (um/h) a) Random locomotion* Fibroblast (ras
transformed) 34.04 .+-. 3.57 10.83 .+-. 1.07 Alveolar macrophages
8.67 .+-. 1.13 3.24 .+-. 0.53 b) Chemotaxist.sup.+ Human
neutrophils (to IL-8) 0.287 .+-. 0.013 0.088 .+-. 0.009 Rat
alveolar macrophages 0.768 .+-. 0.063 0.425 .+-. 0.028 (to C5a) S1
macrophage cell line 0.676 .+-. 0.046 0.238 .+-. 0.014 (to C5a) n =
100 cells for random locomotion and n = 3 replicates .+-. SD of
mean for chemotaxis assays. *Random locomotion was assayed by
quantifying nuclear displacement using image analysis.
.sup.+Chemotaxis was measured in a standard Boyden chamber assay
using MTT blue to detect live cells. Reselts are means .+-.
SEM.
[0255]
3TABLE III Effect of HA-Binding Peptides on Cell Proliferation in
Response to Serum and Apoptosis Number of Cells % Treatment*
(.times.10.sup.6) Hoechst Positive Nuclei Control 2.5 .+-. 0.2 2.0
.+-. 0.8 Positive Control 0.5 .+-. 0.1 40 .+-. 2.5 (PD98059, MEK
inhibitor) HA-Binding Peptide 2.4 .+-. 0.2 4.9 .+-. 0.9
(STMMSRSHKTRSHH) *Cells were treated with 100 ug/mL peptide or a
MEK inhibitor PD98059 (50 ug/mL). Cell number was determined with
hemocytometer counts and apoptosis by the number of Hoechst dye
positive nuclei.
[0256]
Sequence CWU 1
1
* * * * *