U.S. patent application number 10/485924 was filed with the patent office on 2004-12-23 for anti-tumour agents and methods of identifying anti-tumour agents.
Invention is credited to Chiquet-Ehrismann, Ruth, Orend, Gertraud.
Application Number | 20040259781 10/485924 |
Document ID | / |
Family ID | 9920128 |
Filed Date | 2004-12-23 |
United States Patent
Application |
20040259781 |
Kind Code |
A1 |
Chiquet-Ehrismann, Ruth ; et
al. |
December 23, 2004 |
Anti-tumour agents and methods of identifying anti-tumour
agents
Abstract
The FNIII13 domain of fibronectin and smaller fragments thereof
have a tumour cell proliferation inhibitory effect. Compositions
are provided comprising fragments of fibronectin having the FNIII
13 domain and fragments thereof. A system comprising cells exposed
to fibronectin and caused to proliferate by the presence of
tenascin are used as an in vitro method for screening possible
anti-tumour agents. Cell-free systems comprising a fibronectin
ligand and tenascin are also employed for screening potential
anti-tumour or anti-cancer agents. Test compounds are assayed for
the ability to disrupt binding of the fibronectin ligand to
tenascin. A further cell-free system additionally includes a
syndecan molecule.
Inventors: |
Chiquet-Ehrismann, Ruth;
(Reinach, CH) ; Orend, Gertraud; (Pratteln,
CH) |
Correspondence
Address: |
NOVARTIS
CORPORATE INTELLECTUAL PROPERTY
ONE HEALTH PLAZA 430/2
EAST HANOVER
NJ
07936-1080
US
|
Family ID: |
9920128 |
Appl. No.: |
10/485924 |
Filed: |
February 3, 2004 |
PCT Filed: |
August 8, 2002 |
PCT NO: |
PCT/EP02/08881 |
Current U.S.
Class: |
514/1.7 ;
435/7.23; 514/1.9; 514/14.9; 514/16.6; 514/19.3; 514/19.4; 514/9.3;
514/9.4 |
Current CPC
Class: |
A61K 38/39 20130101;
A61P 37/08 20180101; A61P 29/00 20180101; A61P 9/10 20180101; G01N
33/57484 20130101; G01N 33/5011 20130101; A61P 35/00 20180101; A61P
11/06 20180101; A61K 2039/505 20130101; A61P 7/02 20180101; A61P
17/02 20180101; A61P 37/06 20180101 |
Class at
Publication: |
514/012 ;
435/007.23 |
International
Class: |
A61K 038/18; G01N
033/574 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 9, 2001 |
GB |
0119476.0 |
Claims
1. A composition comprising a polypeptide as set forth in SEQ ID
NO:1 or a portion or variant thereof, wherein said portion
interferes with tenascin binding to fibronectin, for use as a
pharmaceutical.
2. A composition as claimed in claim 1 for the treatment or
prophylactic treatment of tumourigenesis or cancer.
3. A composition as claimed in claim 1 for the treatment or
prophylactic treatment of an immune related diseases, e.g.
rheumatism, asthma, allergic diseases, autoimmune diseases, and/or
transplant rejection.
4. A composition as claimed in claim 1 for the treatment or
prophylactic treatment of thrombosis.
5. A composition as claimed in claim 1 for the treatment or
prophylactic treatment of atherosclerosis.
6. A composition as claimed in claim 1 for use in wound
healing.
7. A composition as claimed in claim 1 for the treatment or
prophylactic treatment of any disease or condition dependent on the
interaction of tenascin with fibronectin.
8. A composition as claimed in claim 1, comprising said portion,
said portion comprising the first five amino acids of the amino
acid sequence set forth in SEQ ID NO:3, wherein Xaa is any amino
acid.
9. A composition as claimed in claim 8, wherein said portion
comprises the amino acid sequence set forth in SEQ ID NO:3 or a
fragment or variant thereof, wherein Xaa is any amino acid.
10. A composition as claimed in claim 8, wherein said portion is a
polypeptide having the amino acid sequence set forth in SEQ ID NO:4
or a fragment or variant thereof.
11. A composition as claimed in claim 8, wherein said portion is a
polypeptide having the amino acid sequence set forth in SEQ ID NO:2
or a fragment or variant thereof.
12. A composition as claimed in claim 8, wherein said portion is
free of any other fibronectin domain or module.
13. A composition as claimed in claim 1, further comprising a
pharmaceutically acceptable excipient, diluent or carrier.
14. A composition as claimed in claim 1, wherein said portion is
capable of restoring syndecan signalling.
15. A composition as claimed in claim 14, wherein the syndecan
signalling is mediated by syndecan-4.
16. A composition as claimed in claim 1, wherein said portion binds
to tenascin.
17. A composition as claimed in claim 1, wherein said portion binds
to tenascin C.
18. A composition as claimed in claim 1 wherein said portion
further binds to syndecan, preferably syndecan 4.
19. A composition as claimed in claim 1, wherein said portion
competes with the 13th fibronectin type III repeat of the native
fibronectin protein for tenascin binding.
20. A composition as claimed in claim 1, wherein said portion is
recombinant.
21. A polypeptide as set forth in SEQ ID NO:1 or a portion or
variant thereof, wherein said portion interferes with tenascin
binding to fibronectin, for use as a pharmaceutical.
22. A portion of the polypeptide as set forth in SEQ ID NO: 1, said
portion having any one or more of the features of said portions
referred to claim 8, for use as a pharmaceutical.
23. A use of the polypeptide of claim 21 or a portion or variant
thereof for the manufacture of a medicament for the treatment or
prophylactic treatment of tumourigenesis or cancer.
24. The use of the polypeptide of claim 21 or a portion or variant
thereof, for the manufacture of a medicament for the treatment or
prophylactic treatment of an immune related disease, e.g.
rheumatism, asthma, allergic diseases, autoimmune diseases, or
transplant rejection.
25. The use of the polypeptide of claim 21 or a portion or variant
thereof, for the manufacture of a medicament for the treatment or
prophylactic treatment of thrombosis.
26. The use of the polypeptide of claim 21 or a portion or variant
thereof, for the manufacture of a medicament for the treatment or
prophylactic treatment of atherosclerosis.
27. The use of the polypeptide of claim 21 or a portion or variant
thereof, for the manufacture of a medicament for use in wound
healing.
28. The use of the polypeptide of claim 21 or a portion or variant
thereof, for the manufacture of a medicament for the treatment or
prophylactic treatment of any disease or condition dependent on the
interaction of tenascin with fibronectin.
29. The use as claimed in claim 23, wherein the tumour or tumour
cells express tenascin, preferably tenascin C.
30. The use as claimed in claim 29, wherein the expression of
tenascin is at least two-fold greater than that in non-tumour
tissue or cells.
31. The use as claimed in claim 23, wherein the tumour is a solid
tumour.
32. The use as claimed in claim 23, wherein the tumour is
mesenchymal or epithelial cancer.
33. The use as claimed in claim 23, wherein the tumour is a
glioblastoma or breast carcinoma.
34. A method for the treatment or prophylactic treatment of
tumourigenesis or cancer, comprising administering an effective
amount of a polypeptide as set forth in SEQ ID NO:1 or a portion or
variant thereof, wherein said portion interferes with tenascin
binding to fibronectin.
35. The method as claimed in claim 34 for the treatment or
prophylactic treatment of an immune related disease, e.g.
rheumatism, asthma, allergic diseases, autoimmune diseases, or
transplant rejection.
36. The method as claimed in claim 34 for the treatment or
prophylactic treatment of thrombosis.
37. The method as claimed in claim 34 for the treatment or
prophylactic treatment of atherosclerosis.
38. The method as claimed in claim 34 for use in wound healing.
39. The method as claimed in claim 34 for the treatment or
prophylactic treatment of any disease or condition dependent on the
interaction of tenascin with fibronectin.
40. The method as claimed in claim 35, wherein said portion has the
amino acid sequence set out in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID
NO:4.
41. The method as claimed in claim 34, wherein said cancer
expresses tenascin, preferably tenascin C.
42. The method as claimed in claim 41, wherein the expression of
tenascin is at least two-fold greater than in non-tumour tissue or
cells.
43. A method as claimed in claim 41, wherein the cancer is a solid
tumour.
44. A method as claimed in claim 41, wherein the cancer is
mesenchymal or epithelial cancer.
45. A method as claimed in claim 41, wherein the cancer is a
glioblastoma or breast carcinoma.
46. An antibody specifically reactive against the polypeptide as
set forth in SEQ ID NO:2 or SEQ ID NO:4.
47. An antibody as claimed in claim 46 for use as a
pharmaceutical.
48. A method for identifying agents for the treatment or
prophylactic treatment of tumourigenesis or cancer, comprising
contacting a test compound with cells exposed to a polypeptide as
set forth in SEQ ID NO:1 or a portion or variant thereof, wherein
said portion interferes with tenascin binding to fibronectin, and
tenascin or a portion thereof and then measuring one or more of: a)
cell proliferation; b) DNA synthesis; c) cell adhesion; d) cell
spreading; e) focal adhesion and actin stress fiber formation on
fibronectin; f) the proportion of cells entering S-phase of the
cell cycle; g) the binding of cells to extracellular matrix (ECM),
preferably wherein the ECM is or comprises fibronectin; or h) any
other output from the syndecan signalling pathway.
49. A method as claimed in claim 48, wherein the cells express
syndecan, preferably syndecan-4.
50. A method as claimed in claim 48, further comprising measuring
(a), (b), (c), (d), (e), (f), (g) and/or (h) in control of cells
grown in the absence of test compound.
51. A method as claimed in claim 48, wherein the cells are cultured
on a solid substrate.
52. A method for identifying agents effective against any disease
or condition dependent on the interaction of tenascin with
fibronectin, comprising contacting a test compound with a system
comprising: i) a polypeptide as set forth in SEQ ID NO:1 or a
portion or variant thereof, wherein said portion interferes with
tenascin binding to fibronectin, ii) tenascin or a portion thereof
capable of binding (i), and/or iii) a syndecan molecule capable of
binding (i); and then measuring the binding of (i) and (ii) and/or
the binding of (i) and (iii).
53. A method for identifying agents as claimed in claim 52, further
comprising correlating a decrease in the binding of (i) and (ii)
with an anti-proliferative or anti-tumour agent.
54. A method for identifying agents as claimed in claim 52, further
comprising correlating an increase in the binding of (i) and (iii)
with an anti-proliferative or anti-tumour agent.
55. A method as claimed in claim 52, wherein the system further
comprises measuring the binding of (i) and (ii) and/or the binding
of (i) and (iii) in the absence of a test compound.
56. A method as claimed in claim 52, wherein said system comprises
said portion, said portion comprising the amino acid sequence as
set forth in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4.
57. A method as claimed in claim 52, wherein tenascin is intact
tenascin, preferably tenascin C.
58. A method as claimed in claim 54, wherein the syndecan molecule
is the ectodomain of syndecan, preferably that of syndecan 4.
59. A method as claimed in claim 54, wherein said polypeptide,
portion or variant thereof is attached to a solid phase.
60. A method as claimed in claim 54, wherein tenascin bound to said
polypeptide, portion or variant thereof is measured using an
antibody reactive against tenascin.
61. A method as claimed in claim 54, wherein tenascin is attached
to a solid phase.
62. A method as claimed in claim 54, wherein syndecan is attached
to a solid phase.
63. A method as claimed in claim 54, wherein one or more of the
components of fibronectin, tenascin, syndecan, or a variant or
fragment thereof is labeled.
64. A method as claim 63, wherein said label comprises radiolabels,
fluorescent dyes, electron dense reagents, enzymes, biotin/avidin,
dioxigenin, or haptens.
65. An agent identified by claim 48.
66. A method of diagnosing or prognosing cancer comprising: i)
obtaining a sample from an individual; j) analysing said sample for
the presence of accessible FNIII13 or a portion or variant thereof;
and k) correlating the presence of accessible FNIII13 or a portion
or variant thereof with a favourable prognosis or diagnosis.
67. A method of diagnosing or prognosing cancer as claimed in claim
66 wherein said accessible FNIII13 or a portion or variant thereof
is detected using an antibody specific for FNIII13 or a portion or
variant thereof, optionally wherein a control antibody specific to
a part of fibronectin other than FNIII13 is used in a control
reaction.
68. A method of diagnosing or prognosing cancer as claimed in claim
66 or in claim 67 wherein said sample comprises cells grown in
culture.
Description
[0001] The present invention relates to active agents having
anti-tumour and/or anti-tumourigenic activity, pharmaceutical
compositions of these agents and the pharmaceutical uses of such
agents and compositions. The invention also relates to in vitro
methods of screening agents for anti-tumour and/or
anti-tumourigenic activity.
[0002] Tenascin-C is an adhesion-modulatory extracellular matrix
(ECM) molecule for a variety of cell types (reviewed in Vollmer, G.
(1997) Crit Rev Oncol Hematol 25: 187-210). Tenascin-C is
prominently expressed in the stroma of most solid tumours
(Chiquet-Ehrismann, R. et al (1986) Cell 47: 131-139) and is found
around newly formed blood vessels (Schnyder, B. et al (1997) Int J
Cancer 72: 217-224). Tenascin-C expression precedes the
manifestation of mammary neoplasia in stromelysin/MMP3 transgenic
mice suggesting that tenascin-C might be involved in early steps of
tumourigenesis (Thomasset, N. et al (1998) Am J Pathol 153:
457-467). Despite intriguing expression patterns the roles of
tenascin-C in tumourigenesis and tumour progression are not
known.
[0003] The ECM has an important regulatory function in tissue
homeostasis and, together with oncogenes and tumour suppressor
genes is critically involved in tumourigenesis (reviewed in
Boudreau, N. & Bissell, M. J. (1998) Curr Opin Cell Biol 10:
640-646 and Ruoslahti, E. (1999) Adv Cancer Res 76: 1-20). Enforced
interaction of tumour cells with fibronectin can block
proliferation in cell culture and can decrease tumour growth in
nude mice (Akamatsu H. et al (1996) Cancer Res 56: 4541-4546 and
Giancotti, F. G & Ruoslahti, E. (1990) Cell 60: 849-859).
Tenascin-C was shown to disrupt the interaction of cells with
fibronectin and by that may enhance tumour cell proliferation.
Chiquet-Ehrismann, R. et al (1988) Cell 53: 383-390 were the first
to show that tenascin-C binds to fibronectin, blocks cell
attachment to fibronectin and increases proliferation of rat breast
adenocarcinoma cells (Chiquet-Ehrismann, R. et al (1986) Cell 47:
131-139). Tenascin-C binds fibronectin in an RGD-independent manner
indicating that tenascin-C does not block the RGD cell binding site
in fibronectin (Chiquet-Ehrismann, R. et al (1988) Cell 53:
383-390). The mechanism of how tenascin-C blocks cell attachment to
fibronectin is not known.
[0004] Upon cell adhesion, signals from the ECM are coupled to the
cytoskeleton through specific cell surface receptors (see the
review by Hynes, R. O. (1999) Trends Cell Biol 9: M33-37). In
particular, cell adhesion to fibronectin involving integrins and
proteoglycans orchestrates an assembly of adhesion complexes and a
rearrangement of the actin cytoskeleton, thereby triggering
cytoplasmic signalling that determines cell behaviour, e.g.
survival and proliferation (see the review by Giancotti, F. G.
& Ruoslahti, E. (1990) Cell 60: 849-859).
[0005] Fibroblasts can attach on the cell binding site of
fibronectin (RGD and synergy site) but, for full spreading
including focal contact and actin stress fibre formation,
additional activation of syndecan-4 is required (Woods, A. &
Couchman, J. R. (1994) Mol Biol Cell 5: 183-192). Syndecan-4 is
documented to be required for full cell spreading through
interaction with the HepII site in fibronectin (Tumova, S. et al
(2000) J Biol Chem 275: 9410-9417 and Saoncella, S. et al (1999)
P.N.A.S. 95: 2805-2810). Cell binding of syndecan-4 was shown to be
mediated through the heparin binding site II (HepII site) in
fibronectin (Woods, A. et al (2000) Arch Biochem Biophys 374:
66-72) and upon clustering, syndecan-4 initiates cytoplasmic
signalling involving integrins (see review by Rapraeger, A. C.
(2000) J Cell Biol 149: 995-998).
[0006] Zvibel, I. et al (2001) Int J Cancer 91: 316-321 showed that
the expression of growth promoting erb-B2 and erb-B3 is increased
upon addition of soluble syndecan-4 to colon cancer cells.
[0007] Several integrins have been characterised as cell surface
receptors for tenascin-C (Sriramarao, P. et al (1993) J Cell Sci
105: 1001-1012; Yokosaki, Y. et al (1994) J Biol Chem 269:
26691-26696; Schnapp, L. M. et al (1995) J Biol Chem 270:
23196-23202; and Yokoyama, K. et al (2000) J Biol Chem 275:
16891-16898). Tenascin-C has also been shown to bind to syndecan
(Salmivirta, M. et al (1991) J Biol Chem 266: 7733-7739) and other
sulphated glycosaminoglycans (Chiquet, M. & Fambrough, D. M.
(1984) J Cell Biol 98: 1937-1946; Vaughan, L. et al (1987) EMBO J
6: 349-353; Barnea, G. et al (1994) J Biol Chem 269: 14349-14352;
Grumet, M. et al (1994) J Biol Chem 269: 12142-12146; Milev, P. et
al (1994) J Cell Biol 127: 1703-1715; Vaughan, L. et al (1994)
Perspect Dev Neurobiol 2: 43-52; and Chung, C. Y. & Erikson, H.
P. (1987) J Cell Sci 110: 1413-1419). Whether binding of tenascin-C
to any of these receptors plays a role in tenascin-C-induced
adhesion modulation on fibronectin affecting tumour cell
proliferation is not known.
[0008] Blocking integrin function by competition mechanisms is an
emerging topic in cell adhesion modulation. Examples are high
molecular kininogen masking the .alpha.v.beta.3 integrin binding
site in vitronectin (Asakura, S. et al (1998) J Biochem (Tokyo)
124: 473-484) and the melanoma inhibitory activity (MIA) that
appears to compete with .alpha.4.beta.1 integrin binding to the
14.sup.th fibronectin type III repeat in fibronectin through its
SH3 like domain (Stoll, R. et al (2001) EMBO J 20: 340-349).
[0009] Fibronectin is a large multidomain glycoprotein found in
connective tissue, on cell surfaces, and in plasma and other body
fluids. Fibronectin interacts with a variety of macromolecules
including components of the cytoskeleton and the extracellular
matrix, circulating components of the blood clotting, fibrinolytic,
acute phase and complement systems, and with cell-surface receptors
on a variety of cells including fibroblasts, neurons, phagocytes
and bacteria. Fibronectin also interacts with itself, forming
fibrillar entities whose structure is poorly understood.
[0010] The amino acid sequence of FN reveals three types of
internally homologous repeats or modules, usually separated by
short connecting sequences. There are twelve type I, two type II
and fifteen type III modules, also referred to as FNI, FNII and
FNIII. Each module constitutes an independently folded unit, often
referred to as a domain, but not to be confused with "functional
domains" that frequently contain more than one module. Modules
homologous to those in fibronectin are also found in other
proteins, especially the type III which is one of the most
ubiquitous of all modules, being found in about 2% of animal
proteins. Amino acid sequences of fibronectin modules are highly
conserved. All three fibronectin modules contain several conserved
core residues.
[0011] There are four sites of alternate splicing of fibronectin
mRNA. Of these, the first two result in the insertion of extra type
III domains (EDA and EDB) after modules III-11 and III-7
respectively. These modules are virtually absent from adult tissue
but are differentially expressed during embryonic development and
again in malignant or injured tissue and during angiogenesis. The
extra EDA module renders fibronectin a better substrate for cell
spreading and migration and has been used as a marker for certain
types of cancer. No specific ligands have been identified for
either of these domains.
[0012] Cell adhesion to fibronectin plays an important role in
tumourigenesis and angiogenesis with an inverse correlation of
tumourigenesis and adhesion of tumour cells to fibronectin
(Akiyama, S. K. et al (1995) Cancer Metastasis Rev, 14: 173-189;
and Ruoslahti, E. (1997) Kidney Int. 51: 1413-1417). In particular,
blocking the .alpha.5.beta.1 integrin enhances DNA replication
(Gong, J. et al (1998) J Biol Chem 273: 1662-1669) and
overexpression of integrin .alpha.5.beta.1 decreases proliferation
and tumourigenesis of CHO cells in nude mice (Giancotti, F. G.
& Ruoslahti, E. (1990) Cell 60: 849-859; Gong, J. et al (1997)
Cell Growth Differ 8: 83-90). Although the single animal model
analysed does not support a tumourigenesis-enhancing effect of
tenascin-C (Talts, J. F. et al (1999) J Cell Sci 112: 1855-1864), a
wealth of immunohistochemical studies (Tan, M. I. et al (1999)
Cancer Lett 140: 145-152; and Jahkola, T. et al (1998) Eur J Cancer
34: 1687-1692) and cell culture experiments (Chiquet-Ehrismann, R.
et al (1986) Cell 47: 131-139) suggest a role of tenascin-C in
tumourigenesis especially by enhancing proliferation of cancer
cells in situ.
[0013] U.S. Pat. No. 5,641,483 (Beaulieu) discloses topical gel and
cream formulations containing human plasma fibronectin for the
healing of cutaneous wounds. The formulations provide slow release
and increased contact time of fibronectin to the wound site leading
to effective absorption of an effective wound healing amount of
fibronectin in the skin.
[0014] U.S. Pat. No. 5,958,874 (Clark et al) provides an
extracellular matrix for wound healing comprising a recombinant
fibronectin protein and a backbone matrix. The recombinant
fibronectin protein comprises peptides from at least three
fibronectin domains; the three fibronectin domains being the cell
binding domain, the IIICS domain, and the heparin II binding
domain.
[0015] U.S. Pat. No. 5,750,378 (Goodheart et al) teaches a method
of producing cellular fibronectin in cell culture and then
harvesting the fibronectin. One of the suggested uses of the
cellular fibronectin is the treatment of cancer resections in human
or veterinary medicine.
[0016] U.S. Pat. No. 6,060,317 (Malech) teaches a method of
transducing mammalian cells, and products related thereto including
contacting cells with a viral-vector in the presence of a
multi-functional chemical moiety. A multifunctional chemical moiety
has at least one cell-surface binding domain of e.g. fibronectin or
tenascin linked to at least one virus binding domain, e.g. heparin
II binding domain of fibronectin. One of the suggested uses of this
method treatment of genetic deficiencies such as neoplasias.
[0017] U.S. Pat. No. 6,180,610 (Chen et al) concerns osteogenic
compositions comprising a matrix, an osteoinductive factor (e.g.
mineralized bone) and an extracellular matrix protein. In one
claimed embodiment the extracellular matrix protein is
fibronectin.
[0018] U.S. Pat. No. 6,025,150 (Livant) discloses a wound healing
composition comprising a fibronectin-derived peptide containing the
amino acid sequence PHSRN.
[0019] U.S. Pat. No. 6,194,378 (Clark et al) discloses fibronectin
peptides-based extracellular matrix for wound healing containing
peptides from two or more fibronectin domains in a backbone matrix,
e.g. containing cell-binding domain and heparin II binding
domain.
[0020] WO 94 13692 A1 (Regents of the University of Minnesota et
al) teaches a method for treating acute or chronic inflammatory or
autoimmune disorders comprising administering a polypeptide having
at least three amino acids corresponding to an amino acid sequence
within the heparin-binding region of fibronectin or an
RGD-containing amino acid sequence.
[0021] WO 00 55181 A1 (The General Hospital Corp.) concerns a
method of modulating cell attachment and migration which includes
administering an agent which modulates the interaction (binding) of
the syndecan-4 ectodomain with a counterligand, e.g. the
heparin-binding domain of an ECM. Examples for such agents are
syndecan binding or cell binding domain of fibronectin,
vitronectin, laminin, collagen, or a syndecan-4 binding portion of
such an ECM molecule or any other peptide which binds to the
ectodomain of syndecan-4.
[0022] WO 01 72776 A2 (Wisconsin Alumni Research Foundation)
concerns a method for reducing cell contacts and matrix
organization in trabecular meshwork of a human or nonhuman eye is
disclosed which includes the step of administering a suitable
peptide having a sequence found in the Hep II domain of fibronectin
where the peptide has an ability to disrupt cell contacts and
matrix formation. In particular, a pentapeptide (PRARI) of the
FNIII 14 is disclosed which binds syndecan. FNIII14 is disclosed to
be the most active region of the Hep II domain.
[0023] EP 0399 806 (Takara Shuzo Co Ltd) discloses a functional
polypeptide which has the cell binding domain of human fibronectin
bound directly or by means of a linker amino acid or peptide with
the heparin-binding domain of human fibronectin which has not been
identified in detail but is believed to consist of three type III
repeating sequences which are made of about 90 amino acids each.
Disclosed is also a method for its preparation and the use of such
functional polypeptide to inhibit angiogenesis
[0024] EP 0 837 074 A2 (Hisamitsu Pharmaceutical Co Inc) teaches
fibronectin peptides of 30 amino acids or less comprising the amino
acid sequence YTIYVIAL and having cell adhesion inhibition
activity. Such peptides fall within the FNIII-14 domain of
fibronectin and are suggested for use in the treatment of a variety
of diseases or conditions including inter alia cancer, rheumatism,
asthma, allergic disease, thrombosis, transplant rejection, wound
healing, inflammation, immunological inflammation containing
enteritis nephrocalcinosis (such as colitis ulcerosa), and
autoimmune disease.
[0025] The present inventors have investigated cell adhesion and
proliferation of tumour cells and discovered that tenascin-C blocks
cell attachment and spreading on fibronectin by specific binding to
the 13th fibronectin type III repeat (FNIII13) (SEQ ID NO:1) of the
HepII site, thereby interfering with syndecan-4 binding to
fibronectin. This has been found to correlate with an enhanced
proliferation of tumour cells that can be neutralised by
recombinant FNIII13. The inventors have also discovered that
tenascin-C competes with binding of syndecan-4 to the HepII site in
fibronectin, thereby blocking or altering syndecan-4 function. This
prevents fibronectin adhesion signalling (observed as compromised
cell attachment and spreading, lack of fibronectin specific cell
adhesion structures, lack of actin stress fibres and increased
proliferation of tumour cells on fibronectin). Overexpression of
syndecan-4 as well as addition of FNIII13 or of a smaller peptide
of FNIII 13 has been discovered to restore tenascin-C-compromised
cell spreading. The inventors conclude that FNIII13 in fibronectin
serves as a ligand for syndecan-4.
[0026] In summary, the inventors have discovered that tenascin-C
blocks fibronectin specific adhesion signalling by masking the
syndecan-4 binding site in fibronectin through direct interaction
with FNIII13 in fibronectin. In consequence, interference with
syndecan-4 function triggered enhanced proliferation of tumour
cells by tenascin-C. The inventors have therefore discovered that
tenascin-C impairs the adhesive function of fibronectin through
blocking the co-receptor function of syndecan-4 in integrin
signalling, thereby triggering tumour cell proliferation.
[0027] Accordingly, the present invention provides a composition
for the prevention or prophylactic treatment of tumourigenesis or
the treatment or prophylactic treatment of tumours or of any one or
more of rheumatism, asthma, allergic diseases, autoimmune diseases,
prevention of transplant rejection or the treatment or prophylactic
treatment of any disease or condition involving
tenascin/fibronectin interactions, e.g., thrombosis and
atherosclerosis comprising a fragment of fibronectin having the
heparin binding site II (HepII site).
[0028] The present invention also provides a composition for the
prevention or prophylactic treatment of tumourigenesis or the
treatment or prophylactic treatment of tumours or cancer or of any
one or more of rheumatism, asthma, allergic diseases, autoimmune
diseases, prevention of transplant rejection, thrombosis,
atherosclerosis, or the treatment or prophylactic treatment of any
disease or condition dependent on the interaction of tenascin with
fibronectin comprising a fragment of fibronectin having all or a
portion of the 13.sup.th type III repeat (FNIII13) (SEQ ID NO:1) of
fibronectin, wherein said portion interferes with tenascin binding
to fibronectin. In a preferred embodiment the invention provides a
composition comprising a portion of the FNIII13 fibronectin
fragment, wherein said portion comprises the first five amino acids
of the amino acid sequence set forth in SEQ ID NO: 3, wherein Xaa
is any amino acid. Preferably, Xaa is a hydrophibic amino acid. In
a more preferred embodiment the invention provides a composition
comprising a portion of the FNIII13 fibronectin fragment, wherein
the portion comprises the amino acid sequence set forth in SEQ ID
NO: 3. Preferably, Xaa of the amino acid number 3, 5, 8, 13, 15,
and 17 of SEQ ID NO:3 is a hydrophobic amino acid, Xaa of the amino
acid number 7 and 10 of SEQ ID NO:3 is a charged amino acid, and
Xaa of the amino acid number 6, 9,11, 12, 14, 16, and 19 of SEQ ID
NO:3 is a neutral amino acid (most preferably with a hydroxy
group). In a most preferred embodiment the invention provides a
composition comprising a portion of the FNIII13 fibronectin
fragment, wherein said portion is a polypeptide having the amino
acid sequence set forth in SEQ ID NO:4 or avariant thereof.
Preferably, the invention provides a fragment comprising the first
ten amino acids of the amino acid sequence set forth in SEQ ID
NO:4. In another embodiment the invention provides a composition
comprising a portion of the FNIII13 fibronectin fragment, wherein
said portion is a polypeptide having the amino acid sequence set
forth in SEQ ID NO:2 or a fragment or variant thereof. Preferably,
the invention provides a composition comprising a portion of a
FNIII13 fibronectin fragment as hereinabove described, wherein said
portion is free of any other fibronectin domain or module. All of
the hereinbefore described portions are portions which are capable
of interfering with tenascin binding to fibronectin and which are
capable of restoring a cell spreading defect imposed by
tenascin.
[0029] The compositions of the invention are also useful for the
treatment in wound healing. In particular, the invention provides a
composition for wound healing comprising a fragment of fibronectin
having all or a portion of the 13.sup.th fibronectin type III
repeat (FNIII13) free of any other fibronectin modules or domains.
In another embodiment the invention provides a composition for
wound healing comprising a portion of a fibronectin fragment,
wherein said portion comprises the first five amino acids of the
amino acid sequence set forth in SEQ ID NO: 3, wherein Xaa is any
amino acid, free of any other fibronectin modules or domains. In
yet another embodiment the invention provides a composition for
wound healing comprising a portion of a fibronectin fragment,
wherein said portion comprises the amino acid sequence set forth in
SEQ ID NO: 3 or a fragment or variant thereof, wherein Xaa is any
amino acid, free of any other fibronectin modules or domains. In
yet another embodiment the invention provides a composition for
wound healing comprising a portion of a fibronectin fragment,
wherein said portion is a polypeptide having the amino acid
sequence set forth in SEQ ID NO:4 or in SEQ ID NO: 2 or a fragment
or variant thereof, free of any other fibronectin modules or
domains.
[0030] The fibronectin fragments of the compositions of the
invention may be the same or different, that is to say the
compositions may comprise one or more species of fragment. Although
the HepII site of fibronectin comprises FNIII12, FNIII13 and
FNIII14, preferred fragments comprise just all or a portion of the
13th fibronectin type III repeat (FNIII13). Thus, a fibronectin
fragment includes any polypeptide (i.e. protein) species of
fibronectin that has fewer amino acids than the native whole
fibronectin. In other words, fibronectin fragments may fall within
the range 5 to n-1 amino acid residues, wherein n is the full
length, native fibronectin. In preferred embodiments, a fibronectin
fragment is FNIII13 (SEQ ID NO:1), or a portion or a variant
thereof, preferably free of any other fibronectin domains.
[0031] "Fragments" and "portion" of fragments as small as 5 amino
acid residues are within the scope of the invention. In a preferred
embodiment the fibronectin fragments of the compositions of the
invention comprise the amino acid sequence Arg 98 to Arg 146
according to the numbering used in Sharma et al EMBO J
18:1468-1479. This fragment comprising amino acids 98 to 146 can
restore compromised fibronectin adhesion signalling induced by
tenascin-C. The peptide is shown in SEQ ID NO 2.
[0032] A particularly preferred fragment is the fragment of SEQ ID
NO:4 although larger or smaller fragments may be used. The
fragments and portion of fragments may fall in the range 5 to 100
amino acid residues, preferably having 89, more preferably having
49, even more preferably having at least 5 or 6, even more
preferably having at least 10, most preferably having at least 20
amino acid residues. Other properties of the peptide (such as
solubility) may govern the actual length of peptide used. In one
embodiment the invention provides a fragment of fibronectin having
all or a portion of the 89 amino acids of the sequence of the FNIII
13 fragment (SEQ ID NO:1). In another embodiment the invention
provides a fragment of fibronectin having all or a portion of the
49 amino acids of the sequence as set forth in SEQ ID NO:2, or
having all or a portion of the 20 amino acids of the sequence as
set forth in SEQ ID NO:3 or SEQ ID NO:4. In an alternative
embodiment the invention provides a portion of fragment having the
first five amino acids of SEQ ID NO:3 or SEQ ID NO:4. In another
embodiment the invention provides a portion of fragment having the
first six amino acids of SEQ ID NO:3 or SEQ ID NO:4. Preferably,
the invention provides a portion of fragment having the first ten
amino acids of the amino acid sequence set forth in SEQ ID NO:3 or
SEQ ID NO:4. Xaa in SEQ ID NO:3 may be any amino acid, preferably
an amino acid as hereinbefore described.
[0033] The compositions of the invention may also include other
individual fibronectin fragments in addition to those that are or
comprise the HepII site or the FNIII13 domain or portion thereof.
These other fragments may be selected from FNIII1to FNIII12,
FNIII14 and FNIII15, CS or the HepI site, or fragments thereof, for
example. The other preferred fragments also include FNIII4, FNIII5
and FNIII6, including contiguous fragments comprising two or more
of these domains, or fragments thereof. In preferred embodiments,
the fibronectin fragments of the compositions of the invention are
free of other fibronectin sequences or domains. In these
circumstances, the compositions of the invention can include other
proteins, peptides or fragments from proteins other than
fibronectin.
[0034] The invention therefore provides pharmaceutical compositions
for humans or veterinary compositions for animal use that comprise
one or more of the aforementioned active fragments of fibronectin.
The compositions may also include other active or non-active
agents. Non-active agents may include a pharmaceutically acceptable
excipient, diluent or carrier, but not limited to saline, buffered
saline, dextrose and water.
[0035] The fibronectin fragment is preferably one which is capable
of restoring a cell spreading defect imposed by tenascin such as
restoring syndecan signalling although there may be other potential
targets. Syndecan signalling manifests itself in the way in which
cells spread when cultured in vitro. Instead of a rounded
morphology the cells adopt a flattened, and stretched appearance
visible under the light microscope. The quality of spreading which
is restored can be determined by observation or measured by
morphometric analysis (determining the surface area of the cell
attached to the substrate). Other manifestations of syndecan
signalling include the restored adhesion of cells to fibronectin in
vitro, the presence of specific cell adhesion structures and actin
stress fibres.
[0036] Various approaches and practical methods of measuring
syndecan signalling are described hereinafter.
[0037] There are a number of syndecan molecules, including syndecan
1, syndecan 2, syndecan 3 and syndecan 4. Preferred compositions of
the invention restore syndecan 4-mediated signalling.
[0038] In preferred embodiments the fibronectin fragment binds to
tenascin; preferably the tenascin binds to the HepII site of the
fibronectin fragment, more preferably the tenascin binds to the
FNIII13 site or portion thereof of the fibronectin fragment, or
even more preferably the tenascin binds to the peptide (6-25) (SEQ
ID NO:4) of the FNIII 13 site, or to any portion of a fibronectin
fragment as hereinbefore described, preferably to the portion of
fragment having the first ten amino acids of the amino acid
sequence set forth in SEQ ID NO:4. In particularly preferred
embodiments the tenascin is tenascin C, although in other
embodiments the tenascin may be tenascin X or tenascin Y, R or
W.
[0039] In particularly preferred embodiments heparin competes with
syndecan for binding to the HepII site of the fibronectin fragment,
or for binding to the FNIII13 site or portion thereof of the
fibronectin fragment. In a more particularly preferred embodiments
the portion of a fibronectin fragment of the invention competes
with the FNIII13 site or portion thereof of the native fibronectin
protein for tenascin binding.
[0040] In a particularly preferred embodiment tenascin binds to the
heparin binding site(s) of FNIII13 or FNIII13 fragments of the
invention.
[0041] The invention also provides compositions wherein the
fibronectin fragment(s) are produced by recombinant means. In so
doing, the fragment(s) may be engineered so that the HepII site or
FNIII13 site or portion thereof is present but that the flanking
sequences of the remainder of the fibronectin fragment can comprise
amino acid sequence that the skilled person may find beneficial in
other practical respects, e.g. stability, solubility, or the
possession of some additional biological function, e.g. cell
binding activity via integrin binding sites. Such variant fragments
made by recombinant means familiar to one of skill in the art
comprise a fibronectin fragment having the 13th fibronectin type
III repeat (FNIII13), or a portion or variant of the invention as
hereinabove described, and any one or more of the other fibronectin
domains or portions thereof, or other proteins, peptides or
fragments from proteins other than fibronectin.
[0042] The complete nucleotide sequence of fibronectin is provided
in U.S. Pat. No. 5,679,230. The amino acid sequence is provided in
Kornblihtt et al EMBO J 4: 1755-1759.
[0043] Also included within the invention are variants and
derivatives of the fibronectin fragment, whether produced by
recombinant means or synthetic means or isolated from naturally
occurring sources. For example, peptides having modified amino
acids/peptide linkages, and peptides containing non-naturally
occurring amino acids and/or cyclic peptides, which may have
improved properties such as stability or activity are included. In
addition the peptides of the invention may be in the form of a
fusion with another protein, for example, tags for the targeted
delivery or detection of the HepII site or the FNIII13 fragments or
portions or variants thereof.
[0044] A "variant" in terms of amino acid sequence defines an amino
acid sequence that differs by one or more amino acids from another,
usually related amino acid sequence. The variant may have
"conservative" changes, wherein a substituted amino acid has
similar structural or chemical properties (e.g. replacement of
leucine with isoleucine). Less likely, a variant may have
"non-conservative" changes, e.g. replacement of a glycine with a
tryptophan. Similar minor variations may also include amino acid
deletions or insertions (i.e. additions), or both. Guidance in
determining which and how many amino acid residues may be
substituted, inserted or deleted without abolishing activity may be
found using computer programs well known in the art.
[0045] The present invention therefore provides a fragment of
fibronectin as hereinbefore described having the heparin II binding
site (HepII site) for use as a pharmaceutical. The present
invention also provides a fragment of fibronectin as hereinbefore
described having the 13.sup.th fibronectin type III repeat
(FNIII13) or a portion or variant thereof, wherein said portion
interferes with tenascin binding to fibronectin, for use as a
pharmaceutical. The present invention also provides a portion of
fragment of fibronectin for use as a pharmaceutical, wherein the
portion has any one or more of the features as hereinbefore
described.
[0046] The present invention further provides the use of a fragment
of fibronectin as hereinbefore described having the heparin binding
site II (HepII site), or having all or a portion of the 13.sup.th
type III repeat (FNIII13), or having all or a portion of the
peptide having the amino acid sequence as set forth in any one or
more of the sequences of SEQ ID NOS:2 to 4 or fragments or variants
thereof as hereinbefore described, for the manufacture of a
medicament for the prevention or prophylactic treatment of
tumourigenesis or the treatment or prophylactic treatment of
tumours or cancer. The invention also includes the use of the
fibronectin fragments of the invention for the manufacture of a
medicament for the treatment or prophylactic treatment of any one
or more of an immune related disease, including without limitation
rheumatism, asthma, allergic diseases, autoimmune diseases,
transplant rejection. The invention further includes the use of the
fibronectin fragments of the invention for the manufacture of a
medicament for the treatment or prophylactic treatment of any one
or more of thrombosis, artherosclerosis, wound healing and any
other disease or condition dependent on the interaction of tenascin
with fibronectin.
[0047] The compositions and medicaments of the invention may
therefore be used prophylactically in order to prevent tumours from
forming, or they may be used in a curative or partly curative way
to treat or contain a pre-existing tumourous condition. As well as
tumours, cancerous or malignant conditions may be prevented or
treated with compositions or medicaments of the invention.
[0048] The tumours or tumour cells are preferably those which
express tenascin in the stroma, more preferably tenascin C. The
expression of tenascin is preferably at least two-fold greater than
non-tumour tissue or cells. In particularly preferred embodiments
the tumours are solid tumours, e.g. mesenchymal tumours such as
glioblastoma or epithelial cancers such as glioblastoma or breast
carcinoma.
[0049] The invention also provides a method of preventing or
prophylactic treatment of tumourigenesis or of treatment or
prophylactic treatment of tumours or cancer or of any one or more
of rheumatism, asthma, allergic diseases, autoimmune diseases,
prevention of transplant rejection, thrombosis, atherosclerosis or
the treatment or prophylactic treatment of any disease or condition
dependent on the interaction of tenascin with fibronectin in an
individual comprising administering an effective amount of a
fibronectin fragment having all or a portion of the 13.sup.th type
III repeat (FNIII13), or having all or a portion of the peptide
having the amino acid sequence as set forth in any one or more of
the sequences of SEQ ID NOS:2 to 4 or a portion or variant of the
invention.
[0050] The invention further provides a method of wound healing
comprising administering an effective amount of the fibronectin
fragments of the invention.
[0051] The determination of an effective dose is well within the
capability of those skilled in the art. For any compound, the
therapeutically effective dose can be estimated initially either in
cell culture assays or in an appropriate animal model. The animal
model is also used to achieve a desirable concentration range and
route of administration. Such information can then be used to
determine useful doses and routes for administration in humans.
[0052] A therapeutically effective dose refers to that amount of
active agent which ameliorates the symptoms or condition.
Therapeutic efficacy and toxicity of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals (e.g., ED.sub.50, the dose therapeutically
effective in 50% of the population; and LD.sub.50, the dose lethal
to 50% of the population). The dose ratio between therapeutic and
toxic effects is the therapeutic index, and it can be expressed as
the ratio, LD.sub.50/ED.sub.50. Pharmaceutical compositions which
exhibit large therapeutic indices are preferred. The data obtained
from cell culture assays and animal studies is used in formulating
a range of dosage for human use. The dosage of such compounds lies
preferably within a range of circulating concentrations that
include the ED.sub.50 with little or no toxicity. The dosage varies
within this range depending upon the dosage form employed,
sensitivity of the patient, and the route of administration.
[0053] The exact dosage may be chosen by the individual physician
in view of the patient to be treated. Dosage and administration can
be adjusted to provide sufficient levels of the active moiety or to
maintain the desired effect. Additional factors which may be taken
into account include the severity of the disease state (e.g. tumour
size and location); age, weight and gender of the patient; diet;
time and frequency of administration; drug combination(s); reaction
sensitivities; and tolerance/response to therapy. Long acting
pharmaceutical compositions can be administered on a daily basis,
every 3 to 4 days, every week, or once every two weeks, depending
on half-life and clearance rate of the particular formulation.
[0054] The invention further provides an antibody specifically
reactive against a fragment of fibronectin as hereinbefore
described having all or a portion of the 13.sup.th type III repeat
(FNIII13). The antibody may be monoclonal or polyclonal. Methods of
producing polyclonal and monoclonal antibodies are known to those
of skill in the art and described in the scientific and patent
literature, see, e.g., Coligan, Current Protocols in Immunology,
Wiley/Green, N.Y. (1991); Stites (eds.) Basic and Clinical
Immunology (7th ed.) Lange Medical Publications, Los Altos, Calif.,
and references cited therein (Stites); Goding, Monoclonal
Antibodies: Principles and Practice (2nd ed.) Academic Press, New
York, N.Y. (1986); and Kohler (1975) Nature 256: 495. Such
techniques include selection of antibodies from libraries of
recombinant antibodies displayed in phage or similar on cells. See,
Huse (1989) Science 246: 1275 and Ward (1989) Nature 341: 544.
Recombinant antibodies can be expressed by transient or stable
expression vectors in mammalian cells, as in Norderhaug (1997) J.
Immunol. Methods 204: 77-87.
[0055] The invention also provides other useful aspects including a
method for identifying agents for the treatment or prophylactic
treatment of tumourigenesis or cancer comprising contacting a test
compound with cells exposed to ECM, fibronectin, FNIII13 fragment
or a portion thereof and caused to proliferate by the presence of
tenascin or a fragment thereof and then measuring one or more
of:
[0056] a) cell proliferation;
[0057] b) DNA synthesis;
[0058] c) cell adhesion;
[0059] d) cell spreading;
[0060] e) focal adhesion and actin stress fibre formation on
fibronectin;
[0061] f) the proportion of cells entering S-phase of the cell
cycle; and
[0062] g) the binding of cells to extracellular matrix (ECM),
preferably wherein the ECM is or comprises fibronectin; or
[0063] h) any other output from the syndecan signalling
pathway.
[0064] Although the hereinabove described method teaches a method
for identifying agents for the treatment or prophylactic treatment
of tumourigenesis or cancer, the method may also include
identifying agents for the treatment or prophylactic treatment of
any disease or condition dependent on the interaction of tenascin
with fibronectin, which include without limitation rheumatism,
asthma, allergic diseases, autoimmune diseases, transplant
rejection, thrombosis, atherosclerosis, and/or wound healing.
[0065] The proliferating cells may be encouraged to proliferate by
the addition of tenascin to the cell culture, preferably by coating
the solid substrate therewith. The cell cultures are preferably
grown on a solid substrate or in a liquid medium. A first
measurement of one or more of (a) to (h) may be made prior to
contacting the cells with a test compound. A second measurement may
be made thereafter. A multiplicity of further measurements may be
made over a period of hours or days after contact of the cells with
the test compound. In this way a time course of the cellular
response(s) may be obtained and analysed.
[0066] In preferred aspects one or more of the following arising
after contacting cells with a test compound may indicate an
anti-proliferative or anti-tumour agent:
[0067] (a) a reduction in cell proliferation;
[0068] (b) a reduction in DNA synthesis;
[0069] (c) an increase in cell adhesion, preferably to
fibronectin;
[0070] (d) an increase in cell spreading;
[0071] (e) an increase in focal adhesion and actin stress fibre
formation on fibronectin;
[0072] (f) a decrease in the proportion of cells entering S-phase
of the cell cycle;
[0073] (g) an increase in the binding of cells to ECM, preferably
fibronectin, FNII13 or a portion thereof; and
[0074] (h) an increase in any other output from the syndecan
signalling pathway.
[0075] Any adhesive cell may be used in performing the method,
including both normal and transformed cells. Such cells may be
fibroblasts, epithelial cells, neuronal cells, endothelial cells,
smooth muscle cells and astrocytes, for example.
[0076] Actin stress fibre formation may be assayed according to the
Actin Assembly Assay described in Bloom, L et al(1999) Mol Biol
Cell 10: 1521-1536.
[0077] Adhesion assays may be performed according to the method
described in Bloom, L et al (1999) or as described in the Examples
below.
[0078] In preferred methods the cells express syndecan, in
particular syndecan 4 and are grown in the presence of fibronectin
and tenascin whether before, during or after contact with the test
compound.
[0079] In other embodiments, the method of the invention may
further comprise control cells grown in the absence of test
compound and (a), (b), (c), (d), (e), (f), (g) and/or (h) are
measured in both control and test cultures. The test measurements
can thereby be normalised with respect to the control.
[0080] In another aspect the invention provides a method for the
identification of potential anti-tumour or anti-tumourigenic
compounds or compounds for the treatment or prophylactic treatment
of any one or more of rheumatism, asthma, allergic diseases,
autoimmune diseases, prevention of transplant rejection or the
treatment or prophylactic treatment of any disease or condition
dependent on the interaction of tenascin with fibronectins, e.g.,
thrombosis, wound healing and atherosclerosis in accordance with
the invention comprising contacting a test compound with a system
comprising:
[0081] (i) a fibronectin molecule, FNII13, or a portion or variant
thereof, and
[0082] (ii) tenascin or a portion thereof capable of binding
(i);
[0083] and then measuring the binding of (i) and (ii).
[0084] This method as hereinabove described provides an essentially
cell-free system for the identification of potential anti-tumour or
tumour preventing agents. The fibronectin molecule, portion,
variant or fragment thereof is preferably FNII13, or a portion,
variant or fragment thereof that is sufficient to permit measurable
binding of tenascin thereto. This method relies on the ability of a
potential anti-tumour agent to prevent, inhibit or disrupt the
binding between fibronectin and tenascin. The nature of any
disruption of the fibronectin and tenascin binding may be
determined by the person of ordinary skill by performing a binding
assay for fibronectin and tenascin. The relative amounts or
concentrations of reagents and test compound may be varied, thereby
permitting calculation of inhibition constants and other
parameters, e.g. binding affinities. The optimisation of assay
conditions will be well within the realm of one of ordinary skill
in the art.
[0085] The system may further comprise a control without test
compound and the binding of (i) and (ii) is measured in that
control, thereby permitting corresponding measurements in the test
system to be normalised relative to the control.
[0086] Another screening method for identifying agents effective
against any disease or condition dependent on the interaction of
tenascin with fibronectin comprises contacting a test compound with
a system comprising:
[0087] (i) a fibronectin molecule, in particular FNII13, a portion
or variant thereof,
[0088] (ii) a tenascin molecule or portion thereof capable of
binding (i), and/or
[0089] (iii) a syndecan molecule capable of binding (i);
[0090] and then measuring the binding of (i) and (ii) and/or the
binding of (i) and (iii).
[0091] In this way the potential of a test compound to be able to
disrupt, inhibit or prevent tenascin binding to fibronectin may be
correlated with its potential to restore syndecan binding to
fibronectin. A decrease in the binding of (i) and (ii) correlates
with an anti-proliferative or anti-tumour agent and an increase in
the binding of (i) and (iii) correlates with an anti-proliferative
or anti-tumour agent
[0092] As with the previous screening aspect of the invention, this
system may further comprise a control without test compound and the
binding of (i) and (ii) and/or the binding of (i) and (iii) is
measured.
[0093] A disease or condition dependent on the interaction of
tenascin with fibronectin may include without limiation
tumourigenesis or cancer, rheumatism, asthma, allergic diseases,
autoimmune diseases, transplant rejection, thrombosis,
atherosclerosis, and/or wound healing. In a preferred embodiment,
the present invention provides agents which are effective against
tumourigenesis or cancer, preferably anti-tumour or
anti-proliferative agents.
[0094] In all of the aforementioned screening aspects of the
invention the fibronectin molecule, portion or variant thereof may
be selected from:
[0095] (a) intact fibronectin,
[0096] (b) FNIII13,
[0097] (c) a fragment of FNIII13 of at least 5 amino acid residues,
or
[0098] (d) a fragment of fibronectin comprising the amino acid
sequence as set forth in SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 or
a portion or variant thereof.
[0099] In any of aforementioned methods of the invention the
tenascin is preferably intact tenascin, more preferably tenascin C.
Also, the syndecan molecule preferably comprises the ectodomain of
a syndecan, more preferably that of syndecan 4 (see McFall and
Rapraeger (1998) J. Biol. Chem. 273:28270-28276). However, active
fragments or portion of fragments of these molecules may be
employed.
[0100] One of the fibronectin molecule, portion or variant thereof,
tenascin or syndecan or active fragments or portions thereof may be
attached to a solid phase. The solid phase may comprise particles
or be a surface. For example, particles such as latex beads or
cellulose may be used. Examples of solid substrates include
membrane filters, e.g. nitrocellulose, or polystyrene, e.g. in the
form of a microtiter plate.
[0101] Where one component of the assay (screening) systems of the
invention is coupled to a solid particle or substrate, then one or
more of the other components not so coupled may be labelled.
Examples of labels include radiolabels e.g. .sup.14C or .sup.3H,
dyes, metal sols, enzymes or biotin/avidin. By attaching such
labels to "free" components in the system any binding assay may be
carried out in solution in accordance with procedures well known in
the art. After allowing the components to react solid phase
particles can be separated from solution, e.g. by filtration or
sedimentation (including centrifugation). In some embodiments
immunoprecipitation may be used to separate bound and free labelled
components. Usually, an antibody may be employed to bring an
unlabelled component out of solution (whether or not this component
has bound to another labelled component or not). After separation,
the label present in solution (free) and the label present in or on
the solid phase (bound) may be measured. Standard analyses of such
bound and free data, e.g. Scatchard plots and the determination of
affinity and inhibition constants for binding are well known to the
person of ordinary skill in the art.
[0102] Where the solid phase is not particulate, e.g. in the form
of a surface, such as a microtiter plate well, then binding assays
measuring bound and free label may be performed but this will
normally involve the removal of liquid phase from the wells after
binding reactions have occurred. Advantageously, this assay format
may dispense with the need for providing specifically labelled
reaction components. Instead, labelled antibodies may be used to
measure the binding of previously free reaction components to solid
phase components.
[0103] In some embodiments the fibronectin molecule, variant or
fragment thereof may be attached directly to a solid phase. In
preferred immunoassay embodiments of this type, tenascin bound to
fibronectin is measured using an antibody reactive against
tenascin. The assays may also be performed in the presence of
syndecan.
[0104] In other embodiments tenascin may be attached to a solid
phase. In preferred immunoassay embodiments of this type,
fibronectin molecule, variant or fragment thereof bound to tenascin
is measured using an antibody reactive against fibronectin. The
assays may also be performed in the presence of syndecan.
[0105] In further embodiments, syndecan may be attached to a solid
phase and the binding assay with fibronectin molecule, variant or
fragment thereof performed in the presence of tenascin. In
preferred immunoassay embodiments of this type, fibronectin
molecule, variant or fragment thereof bound to syndecan is measured
using an antibody reactive against fibronectin.
[0106] Immunological binding assays are known in the art. For a
review, see Methods in Cell Biology Vol. 37: Antibodies in Cell
Biology, Asai, (Ed.) Academic Press, Inc. New York (1993).
[0107] A label may be any detectable composition whereby the
detection can be spectroscopic, photochemical, biochemical,
immunochemical, physical or chemical. For example, useful labels
can include .sup.32p, .sup.35S, .sup.3H, .sup.14C, .sup.125I,
.sup.131I, fluorescent dyes (e.g. FITC, rhodamine and lanthanide
phosphors), electron-dense reagents, enzymes, e.g. as commonly used
in ELISA (e.g. horseradish peroxidase, beta-galactosidase,
luciferase and alkaline phosphatase), biotin, dioxigenin, or
haptens and proteins for which antisera or monoclonal antibodies
are available. The label may be directly incorporated into a target
compound to be detected, or it may be attached to a probe or
antibody which binds to the target.
[0108] Throughout the assays of the invention, incubation and/or
washing steps may be required after each application of reagent or
incubation of combinations of reagents. Incubation steps may vary
from about 5 minutes to several hours, perhaps from about 30
minutes to about 6 hours. However, the incubation time usually
depends upon the assay format, analyte, volume of solution,
concentrations, and so forth. Usually, the assays should be carried
out at ambient temperature, although they may be conducted at
temperatures; in the range 10.degree. C. to 40.degree. C., for
example.
[0109] A particularly preferred assay format is an enzyme-linked
immunosorbent assay (ELISA).
[0110] All of the aforementioned methods of screening of the
invention are equally applicable to the screening of substances for
biological activity and potential agents for wound healing or
treatment of artherosclerosis, or for the treatment or prophylactic
treatment of any disease or condition dependent on the interaction
of tenascin with fibronectin.
[0111] Also included within the scope of the present invention are
anti-tumourigenic, anti-tumour, anti-metastastic, wound healing or
anti-artherosclerosis substances or substances for the treatment or
prophylactic treatment of any one or more of rheumatism, asthma,
allergic disease, autoimmune disease, prevention of transplant
rejection or for the treatment or prophylactic treatment of any
disease or condition dependent on the interaction of tenascin with
fibronectin, e.g thrombosis, identified by any of the screening
methods of the invention. These substances may be proteins,
polypeptides or small organic molecules (drugs). The invention
therefore includes pharmaceutical compositions for preventing or
treating tumours, metastasis, wound healing or artherosclerosis and
comprising one or more of the substances identified by a method of
the invention.
[0112] The present invention also provides a method of diagnosing
or prognosing cancer comprising:
[0113] a) obtaining a sample from an individual;
[0114] b) analysing said sample for the presence of accessible
FNIII 13 or a portion or variant thereof; and
[0115] c) correlating the presence of accessible FNII13 or a
portion or variant thereof with a favourable prognosis or
diagnosis.
[0116] Accessible FNIII13 or a portion or variant thereof may be
detected using an antibody specific for FNIII13 or a fragment
thereof, and a control assay can be carried out using an antibody
specific for a different area of fibronectin. The sample is
preferably a tissue sample mounted onto a solid surface for
histochemical analysis. The presence of detectable, accessible
FNIII13 or a portion or variant thereof indicates that FNIII13 is
accessible to cells for binding. In particular, if an
FNIII13-specific antibody reacts with FNIII13 in a tissue section,
then there is an expectation that cells will also be able to
interact with FNIII13 in that sample. This leads to a favourable
diagnosis or prognosis. If, on the other hand, the antibody does
not react with FNIII13 in the tissue section, e.g. because tenascin
is masking the FNIII13 site, then there is an expectation that
cells cannot interact. This leads to an unfavourable diagnosis or
prognosis.
[0117] In a preferred embodiment, the invention provides kits
suitable for use in the diagnostic or prognostic methods of the
invention. Such kits comprise reagents useful for carrying out
these methods, for example, antibodies from one or more species
specific for FNIII13. Additionally, kits can contain antibodies
from one or more species specific for other parts of the
fibronectin molecule, for use as a control. Secondary antibodies
that recognise either or both such primary anti-fibronectin
antibodies can also be included for the purpose of recognition and
detection of primary antibody binding to a sample. Such secondary
antibodies can be labelled for detection e.g. with fluorophores,
enzymes, radioactive labels or otherwise. Other detection labels
will occur to those skilled in the art. Alternatively, the primary
anti-fibronectin antibodies can be labelled for direction
detection.
[0118] Methods for the production of polyclonal and monoclonal
antibodies are known to those of skill in the art. In brief, an
immunogen is mixed with a suitable adjuvant and animals are
immunised. The immune response to the immunogen preparation is
monitored by taking test bleeds and determining the titer of
reactivity to the immunogen. When appropriately high titers of
antibody to the immunogen are obtained, blood is collected from the
animal and antisera are prepared. The antisera may be further
fractionated to enrich for the reactive species of antibodies.
[0119] Monoclonal antibodies to fibronectin molecule, variant or
fragment thereof, tenascin or syndecan may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include but are not
limited to the hybridoma technique originally described by Koehler
and Milstein (Koehler and Milstein (1975) Nature 256: 495-497), the
human B-cell hybridoma technique (Kosbor et al (1983) Immunol Today
4: 72; Cote et al (1983) Proc Natl Acad Sci 80: 2026-2030) and the
EBV-hybridoma technique (Cole et al (1985) Monoclonal Antibodies
and Cancer Therapy, Alan R Liss Inc, New York N.Y., pp 77-96).
Large amounts of monoclonal antibodies for use in immunoassays may
be obtained by various techniques familiar to those skilled in the
art. Briefly, spleen cells from an animal immunised with the
desired protein are immortalised, commonly by fusion with a myeloma
cell. Alternative methods of immortalisation include transformation
with Epstein Barr Virus, oncogenes, or retroviruses, or other
methods well known in the art. Colonies arising from single
immortalised cells are screened for production of antibodies of the
desired specificity and affinity for fibronectin molecule, variant
or fragment thereof, tenascin or syndecan. The yield of the
monoclonal antibodies produced by such cells may be enhanced by
various techniques, including injection into the peritoneal cavity
of a vertebrate host. Alternatively, one may isolate DNA sequences
which encode a monoclonal antibody or a binding fragment thereof by
screening a DNA library from appropriate human B cells, i.e.
immunised according to a general protocol.
[0120] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, etc may be immunised by injection with
fibronectin molecule, variant or fragment thereof, tenascin or
syndecan or any portion or fragment that retains immunogenic
properties. Depending on the host species, various adjuvants may be
used to Increase immunological response. Such adjuvants are
commercially available, and include but are not limited to
Freund's, mineral gels such as aluminium hydroxide, and surface
active substances such as lysolecithin, pluronic polyols,
polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and
dinitrophenol. BCG (Bacillus Calmette-Guerin) and Corynebacterium
parvum are potentially useful adjuvants.
[0121] Animals (e.g. inbred strain of mice or rabbits) can be
immunised with immunogen using a standard adjuvant, such as
Freund's adjuvant, and a standard immunisation protocol.
Alternatively, a synthetic peptide conjugated to a carrier protein
can be used an immunogen. Polyclonal sera are collected and titered
against the immunogen in an immunoassay, for example, a solid phase
immunoassay with the immunogen immobilised on a solid support.
Polyclonal antisera with a titer of, for example, 10.sup.4 or
greater are selected and tested for their cross-reactivity against
homologous proteins from other organisms and/or non-immunogen
protein, using, for example, a competitive binding immunoassay.
Specific monoclonal and polyclonal antibodies and antisera will
usually bind with a K.sub.D of at least about 1 .mu.M, preferably
at least about 0.1 .mu.M or better, and most preferably, 0.01 .mu.M
or better.
[0122] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening recombinant
immunoglobulin libraries or panels of highly specific binding
reagents (Orlandi et al (1989) Proc Natl Acad Sci 86: 3833; and
Winter and Milstein (1991) Nature 349: 293.
[0123] Antibody fragments which contain specific binding sites for
the immunogens may also be generated. For example, such fragments
include, but are not limited to, the F(ab').sub.2 fragments which
can be produced by pepsin digestion of the antibody molecule and
the Fab fragments which can be generated by reducing the disulfide
bridges of the F(ab').sub.2 fragments. Alternatively, Fab
expression libraries may be constructed to allow rapid and easy
identification of monoclonal Fab fragments with the desired
specificity (Huse et al (1989) Science 256:1275.
[0124] Administration of pharmaceutical compositions of the
invention may be accomplished orally or parenterally. Methods of
parenteral delivery include topical, intra-arterial (e.g. directly
to the tumour), intramuscular, subcutaneous, intramedullary,
intrathecal, intraventricular, intravenous, intraperitoneal, or
intranasal administration. In addition to the active ingredients,
these pharmaceutical compositions can contain suitable
pharmaceutically acceptable carriers comprising excipients and
other compounds that facilitate processing of the active compounds
into preparations which can be used pharmaceutically. Further
details on techniques for formulation and administration can be
found in the latest edition of Remington's Pharmaceutical Sciences
(Maack Publishing Co, Easton Pa.).
[0125] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, etc, suitable for ingestion by the patient.
[0126] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable additional compounds, if
desired, to obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers include, but are not limited to
sugars, including lactose, sucrose, mannitol, or sorbitol; starch
from corn, wheat, rice, potato, or other plants; cellulose such as
methyl cellulose, hydroxypropylmethyl-cellulose, or sodium
carboxymethylcellulose; and gums including arabic and tragacanth;
as well as proteins such as gelatin and collagen. If desired,
disintegrating or solubilizing agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt
thereof, such as sodium alginate.
[0127] Dragee cores can be provided with suitable coatings such as
concentrated sugar solutions, which may also contain gum arabic,
talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol,
and/or titanium dioxide, lacquer solutions, and suitable organic
solvents or solvent mixtures. Dyestuffs or pigments may be added to
the tablets or dragee coatings for product identification or to
characterise the quantity of active compound (i.e. dosage).
[0128] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders such as lactose or starches, lubricants such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds can be dissolved or suspended in
suitable liquids, such as fatty oils, liquid paraffin, or liquid
polyethylene glycol with or without stabilizers.
[0129] Pharmaceutical formulations for parenteral administration
include aqueous solutions of active compounds. For injection, the
pharmaceutical compositions of the invention may be formulated in
aqueous solutions, preferably in physiologically compatible buffers
such as Hanks's solution, Ringer's solution, or physiologically
buffered saline. Aqueous injection suspensions can contain
substances which increase the viscosity of the suspension, such as
sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,
suspensions of the active compounds can be prepared as appropriate
oily injection suspensions. Suitable lipophilic solvents or
vehicles include fatty oils such as sesame oil, or synthetic fatty
acid esters, such as ethyl oleate or triglycerides, or liposomes.
Optionally, the suspension can also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0130] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0131] The pharmaceutical compositions of the present invention can
be manufactured in substantial accordance with standard
manufacturing procedures known in the art (e.g. by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilising
processes).
[0132] The invention is further described below, for the purpose of
illustration only, in the following examples:
EXAMPLE 1
Preparation of Tenascin-C, Fibronectin and Recombinant Fibronectin
Proteins, and Syndecan
[0133] Full length chicken tenascin-C TN260 was cloned by insertion
of all known extra fibronectin type III repeats of tenascin-C into
construct pCDNA/TN 190 (Fischer, D. et al (1995) J Biol Chem 270:
3378-3384) subcloned into the pCEP-Pu vector (Kohfeldt, E. et al
(1997) FEBS Lett 414: 557-561), transfected into human embryonic
HEK-293 cells. Stable expressor cells were selected with puromycin.
Recombinant tenascin-C was made by growing cells to 2/3 confuence
in 10% FCS-containing medium. The medium was replaced by serum-free
Dulbeccos's Modified Eagle Medium (DMEM) and conditioned medium was
collected after 2 days. Conditioned medium was collected up to
6-times interrupted by 18 h of keeping cells in serum-containing
medium between cycles. Recombinant tenascin-C was purified by
immunoaffinity chromatography as described (Fischer, D et al (1995)
J Biol Chem 270: 3378-3384). Conditioned medium was sequentially
passed over columns of gelatin agarose, and Sepharose 4B
(Pharmacia, Uppsala, Sweden) coupled with anti-tenascin-C (mAbM1)
and (mAb60) (for antibodies see Fischer, D. et al (1997) Mol Biol
Cell 8: 2055-2075). Bound tenascin-C was washed with 0.05% Triton
X-100, 1M NaCl, 0.01M Tris-HCl pH8.3, eluted with 50 mM
diethylamine in PBS containing 0.01%Tween-20, and dialysed against
0.01%Tween-20 in PBS.
[0134] Fibronectin was prepared by gelatin agarose chromatography
as described (Fischer, D. et al (1997) J Cell Sci 110: 1513-1522).
Horse serum was passed over a gelatin agarose column, washed with
0.05%Triton X-100, 1M NaCl, 0.01M Tris-HCl pH8.3, eluted with 4M
urea and dialysed against PBS. Recombinant fibronectin proteins
were isolated on Ni-NTA resins according to the manufacturer's
instructions (Quiagen).
[0135] The following recombinant proteins were obtained as
indicated: FNIII13 (Bloom, L. et al(1999) Mol Biol Cell 10:
1521-1536), FNIII17-10 (Redick, S. D. et al(2000) J Cell Biol 149:
521-527), FNIII4-6 and FNIII12-15+CS and other fibronectin
constructs (prepared according to the methods of Bloom, L. et al
(1999), cDNAs for mouse syndecan-1 (Liu, W. et al (1988) J Biol
Chem 273: 22825-22832), syndecan-2 (Klass, C. M. et al (2000) J
Cell Sci 113: 493-506) and syndecan-4 (Oh, E. S. et al (1997) J
Biol Chem 272: 11805-11811).
[0136] Fragments of FNIII13 having the amino acid sequence set
forth in SEQ ID NO: 4 or the first ten amino acids thereof, were
synthesized according to the common solid phase peptide synthesis
method using t-Boc and Fmoc chemistry (see for example Chan, W. C.
and White, P. D. (2000), Fmoc Solid Phase Peptide Synthesis: A
Practical Approach, Oxford University Press, 368 pp., or Rovero, P.
et. al. (1991) Int. J. Peptide Protein Res., 37, 140-4). The
peptides were purified by HPLC (High pressure liquid
chromatography) and veryfied using mass spectrometry.
EXAMPLE 2
Cell Lines, Cell Culture and Transfection
[0137] All cell lines were originally derived from ATCC if not
mentioned otherwise; human KRIB osteosarcoma, MDA-MB435 breast
carcinoma, T98G glioblastoma and Chinese hamster CHO-K1 and
derivatives (Bauer, J. S et al (1992) J Cell Biol 116: 447-487 and
Schreiner, C. L. et al (1989) J Cell Biol 109: 3157-3167). Cells
were cultured in DMEM or .alpha.MEM with 10% FCS and antibiotics
(0.36 mg/ml penicillin, 1 mg/ml streptomycin). Transfections were
done with Fugene6 according to the manufacturer's protocol. For
selection of stable syndecan-4 overexpressors T98G cells were grown
with G418 and expression was analysed by immunofluorescence. Clonal
lines were derived by limited dilution.
EXAMPLE 3
Adhesion Assay
[0138] 60-well microtiter plates (Nunc) were coated with 10
.mu.g/ml ECM molecules giving rise to 1 .mu.g/cm.sup.2 (fibronectin
and tenascin-C) and 4 .mu.g/cm.sup.2 (FNIII13) for 1 h at
37.degree. C. ECM proteins were coated separately, starting with
fibronectin, followed by tenascin-C, FNIII13 and BSA. The
non-coated plastic surface was blocked with 1% heat-inactivated BSA
in PBS giving rise to 10 .mu.g/cm.sup.2 protein. Similarly, mixed
substrata of collagen I and laminin 1, respectively with tenascin-C
were prepared and tenascin-C was detected by ELISA.
[0139] Efficient fibronectin and tenascin-C coating was determined
by ELISA with an anti-fibronectin and anti-tenascin-C antibody
(Fischer, D. et al (1995) J Biol Chem 270: 3378-3384), respectively
and by PAGE analysis of lysed surface-bound ECM material combined
with Coomassie Brilliant Blue staining. Before plating, cells were
serum starved for 18 h in DMEM containing 1.times.ITS (insulin,
transferrin, selene) supplement, trypsinised, trypsin was blocked
with 100 .mu.g/ml soybean trypsin inhibitor in PBS and, cells were
resuspended in serum-free medium and counted. 200-500 cells per
well were plated for the indicated time points, fixed by addition
of glutaraldehyde (2% final concentration) for 15 minutes and
stained with 0.1% crystalviolet in 20% methanol for 30 minutes.
Cells were observed under a Nikon microscope (Nikon diaphot) and
pictures were taken with a Nikon camera.
EXAMPLE 4
DNA Replication and Proliferation Assay
[0140] 96-well plates (Falcon) were coated as described above.
Cells were serum starved overnight and trypsinised as described.
10.sup.4 cells were transferred onto the coated plates in the
presence of the indicated mitogens. 14 h later cells were labelled
with radioactive .sup.3H-thymidine (0.5 .mu.Ci/well) for 4 h at
37.degree. C., incorporated .sup.3H-thymidine was precipitated with
10% TCA and determined with a Beckman scintillation counter after
cell lysis in 0.3N NaOH, 2% SDS. For long-term cell proliferation
assays 2.times.10.sup.3 MDA-MB435 cells were plated into ECM-coated
96-well plates in the presence of 100 ng/ml insulin and incubated
for the indicated time points in a humidified chamber at 37.degree.
C. in a CO.sup.2-incubator. 50% fresh medium with growth factor was
added every 24 h. At different time points cells were trypsinised
and counted.
EXAMPLE 5
In Vitro Binding Assay (ELISA)
[0141] 96-well ELISA plates were coated with the indicated ECM
proteins for 1 h at 37.degree. C., blocked with 1% milkpowder,
0.05% Tween-20 in PBS. ECM proteins were added at the indicated
concentrations in blocking solution for 1 h, washed with blocking
solution and anti-fibronectin or anti-tenascin-C antibodies were
added. Bound proteins were detected by immune reaction with a
peroxidase-coupled secondary antibody specifically recognising
anti-fibronectin and anti-tenascin-C antibodies, respectively,
followed by colour reaction with 21 mg/ml citric acid 1-hydrate, 34
mg/ml Na.sub.2HPO.sub.4.2H.sub.2O, 0.4 mg/ml phenylenediamine, 1
.mu.l H.sub.2O.sub.2 that was stopped with 4M sulphuric acid. The
absorbance was read at 590 nm.
EXAMPLE 6
Immunofluorescence Microscopy
[0142] 10.sup.4 cells were transferred onto 4-well Cellstar plastic
plates (Greiner) that were coated with ECM proteins as described.
Cells were fixed with 4% paraformaldehyde, 50 mM phosphate buffer,
5 mM EDTA in PBS for 15 minutes, blocked with 3% BSA, 0.5% Tween-20
in PBS and incubated with primary and secondary antibodies in
blocking solution. Slides were embedded in 10.5% Mowiol containing
2.5% DABCO as antifade agent. Expression of syndecans was
determined by immunofluorescence.
[0143] Cells were fixed in methanol and incubated with
anti-syndecan-1, anti-syndecan-2 and anti-syndecan-4 antibodies at
a dilution of 1:50 each, respectively. Cells were analysed by
microscopy.
EXAMPLE 7
Tenascin-C Blocks Cell Attachment and Compromises Cell Spreading on
Fibronectin
[0144] Ten different tumour cell lines were plated on adhesive
substrata such as fibronectin, collagen I and laminin 1, or on the
large tumour-expressed form of tenascin-C (Borsi, L. et al (1992)
Int J Cancer 52: 688-692) (see Table 1 below):
1TABLE 1 Cell Line Time TN FN/TN BSA T98G 1 h 9.3 +/-0.9 31.9
+/-5.2 8.1 +/-0.8 MDA MB 435 8.2 +/-3.9 24.5 +/-2.8 7.4 +/-4.1
T98G.sup.a 20 h 14.6 +/-0.4 50.4 +/-2.7 11.1 +/-2.7 MDA MB
435.sup.b 4.0 +/-0.2 62.2 +/-5.1 4.7 +/-2.1 CHO-K1.sup.c 4.7 +/-0.2
37.9 +/-2.1 7.4 +/-1.4 CHO-B2.sup.c 8.0 +/-2.9 31.5 +/-9.6 7.4
+/-2.4 CHO-B2.alpha.27.sup.c 4.3 +/-0.7 45.1 +/-3.7 8.1 +/-2.1
[0145] Table 1 shows compromised cell adhesion by tenascin-C. The
results of 1 h-attachment and 20 h-adhesion assays are summarized
for the indicated tumour cell lines. Cells were plated in serum
free medium or in medium supplemented with 40 ng/ml
platelet-derived growth factor BB (PDGF-BB) (a), 100 ng/ml insulin
(b) and ITS (c). The numbers of attached cells are described as %
of cells attached on fibronectin.
[0146] Whereas most cells adhered to fibronectin, collagen I and
laminin 1; less than 10% of cells attached on tenascin-C and
remained rounded 1 h after plating showing that tenascin-C was not
adhesive for all cell lines tested. Tenascin-C remained
anti-adhesive for a long time, since all cells still failed to
attach 20 h after plating in serum-free medium (see Table 1).
Mitogen addition of PDGF-BB, insulin, endothelial growth factor
(EGF), lysophosphatidic acid (LPA) and transforming growth
factor-beta (TGF.beta.) did not reduce the anti-adhesiveness of
tenascin-C (see Table 1).
[0147] In the tissue context cells are encountering tenascin-C in
combination with other ECM molecules. An experiment was performed
to establish whether tenascin-C would be anti-adhesive in a
situation where cells were allowed to attach to an adhesive ECM
such as fibronectin, laminin 1 or collagen I. Tenascin-C was found
to compromise cell attachment when offered together with
fibronectin both 1 h as well as 20 h after plating cells on a
substratum containing equimolar amounts of tenascin-C and
fibronectin (see Table 1). Even in a molar ratio of tenascin-C to
fibronectin of 1:8 (that does not affect efficient coating of
tenascin-C; see Example 9), tenascin-C blocked cell attachment on
fibronectin as efficiently as in an equimolar ratio. In contrast to
a mixed fibronectin/tenascin-C substratum, other substrata-of
collagen I and laminin 1 that contained equimolar amounts of
tenascin-C were equally adhesive as without tenascin-C. Similar
numbers of T98G cells and MRC-5 fibroblasts attached and spread on
these mixed substrata as on the single ECM molecules. But on the
mixed fibronectin/tenascin-C substratum only 45% of T98G cells were
partially spread after 20 h. Tenascin-C blocks cell attachment and
interferes with proper cell spreading on fibronectin, but not on
collagen I nor laminin 1 in all cell lines tested. Tenascin-C
enhances proliferation of a variety of tumour cell lines tested
including glioblastoma and breast carcinoma cells. Thus in the
tissue context tenascin-C appears to increase tumour mass by
elevating the number of tumour cells. There was no evidence for an
altered apoptosis rate but a higher proportion of cells on the
fibronectin/tenascin-C mixture entered the DNA synthesis phase of
the cell cycle than counterparts that were grown attached on
fibronectin. Apparently, cells that were not yet determined to
enter S-phase on fibronectin were triggered to start DNA synthesis
upon tenascin-C compromised adhesion to fibronectin.
EXAMPLE 8
Increased Integrin Expression Does Not Overcome Compromised Cell
Attachment by Tenascin-C
[0148] The inventors tested whether expression of integrins capable
of binding to fibronectin influences tenascin-C-induced compromised
attachment on fibronectin. CHO-K1 cells (which express moderate
levels of .alpha.5.beta.1 (Bauer, J. S. et al (1992) J Cell Biol
116: 477-487)) were compared for levels of adhesion on fibronectin
and other ECM substrates with that of cells that either express
essentially no fibronectin binding integrins (CHO-B2), or
overexpress .alpha.5.beta.1 (CHO-B2.alpha.27) and .alpha.v.beta.1
(CHO-B2v7), respectively (Schreiner, C. et al (1991) Cancer Res 51:
1738-1740). Experiments showed that cell adhesion on the mixed
substratum was blocked similarly in all cell lines irrespective of
the nature of integrin expression (see Table 1). Also,
overexpression of the .alpha.5.beta.1 integrin in human HT29 colon
carcinoma cells did not support cell attachment and spreading on
the mixed fibronectin/tenascin-C substratum. .beta.1 integrins are
probably not the primary target of tenascin-C action.
EXAMPLE 9
Increased Tumour Cell Proliferation on a Mixed
Fibronectin/Tenascin-C Substratum
[0149] Cell proliferation and DNA synthesis of MDA-MB435 breast
carcinoma, T98G glioblastoma, and Chinese hamster ovary carcinoma
cells (CHO) grown on fibronectin, tenascin-C and on mixed
fibronectin/tenascin-C substrata was determined. MDA-MB435 breast
carcinoma cells were grown on fibronectin or on an equimolar
mixture of fibronectin and tenascin-C in the presence of 100 ng/ml
insulin. After 27 h, 51 h, and 75 h cells were counted. 24% and 33%
more cells were counted on fibronectin/tenascin-C than on
fibronectin after 51 h and 75 h of culturing, respectively. T98G
glioblastoma cells were cultured on fibronectin, collagen I,
laminin 1 and on mixtures of these ECM molecules with tenascin-C in
the presence of 40 ng/ml PDGF-BB. .sup.3H-thymidine incorporation
per attached cell was measured as fold over cpm on fibronectin. The
DNA replication indices (cpm/cell) of MDA-MB435 and T98G cells were
about 2- and 3-fold increased on the mixed fibronectin/tenascin-C
substratum in comparison to fibronectin alone (see Table 2
below).
2 TABLE 2 Cell Line TN FN/TN T98G.sup.a 3.0 +/-0.9 3.0 +/-0.2 MDA
MB 435.sup.b 2.7 +/-0.6 1.8 +/-0.6 CHO-K1.sup.c 5.9 +/-1.7 2.3
+/-0.2 CHO-B2.sup.c 7.1 +/-0.2 2.6 +/-0.3 CHO-B2.alpha.27.sup.c 11
+/-3.9 2.5 +/-0.6
[0150] Table 2 shows enhanced DNA synthesis by tenascin-C. DNA
replication was determined by .sup.3H-thymidine incorporation and
is described as relative increase in cpm over cells plated on
fibronectin. Serum starved cells were triggered to enter S-phase by
40 ng/ml PDGF-BB (a), 100 ng/ml insulin (b) and ITS (c).
[0151] The possibility that rounded cells might have taken up more
.sup.3H-thymidine than attached cells could be ruled out as similar
total cellular cpm were counted from cells plated on the different
substrata after 4 h when T98G cells would not have entered S-phase.
Increased DNA synthesis levels were also observed for the other
tumour cell lines tested (see Table 2). This demonstrated that a
mixed fibronectin/tenascin-C substratum triggered more cells to
enter S-phase of the cell cycle than fibronectin alone. Since all
tumour cell lines tested were equally compromised in cell adhesion
on a mixed fibronectin/tenascin-C substratum two representative
cell lines, human MDA-MB435 breast carcinoma and T98G glioblastoma
cells were chosen for further study.
[0152] In order to address whether tenascin-C alone and in ECM
contexts other than fibronectin might also enhance proliferation,
an investigation was carried out into DNA synthesis on a pure
tenascin-C substratum where cells remained rounded and, on mixed
substrata of collagen I and laminin 1 that contained tenascin-C
where cells spread normally. These experiments revealed that the
DNA replication indices of all cell lines tested were increased
with levels as high as 11-fold on pure tenascin-C (see Table 2).
However, on adhesive substrata containing tenascin-C (mixtures with
collagen I and laminin 1, respectively), DNA synthesis rates were
identical to those on the single ECM molecules. To rule out that
tenascin-C may shorten S-phase of the cell cycle in tumour cells,
DNA synthesis of T98G cells was determined by assay every two hours
starting 11 h after plating. Therefore, T98G glioblastoma cells
were grown on fibronectin or on a mixture of fibronectin and
tenascin-C in the presence of 40 ng/ml PDGF-BB, labelled with
.sup.3H-thymidine for 1 h and harvested and counted every two hours
starting 11 h after plating. This experiment revealed that on the
mixed substratum DNA replication followed similar kinetics as on
fibronectin. These observations show that tenascin-C stimulates a
subset of cells to enter S-phase of the cell cycle by interfering
with fibronectin-specific cell adhesion signalling.
EXAMPLE 10
Tenascin-C Binds to the 13.sup.th Fibronectin Type III Repeat of
the HepII Site in Fibronectin
[0153] The anti-adhesive and proliferation-stimulatory effect of
tenascin-C was found to be specific for cells on a mixed
fibronectin/tenascin-C substratum. An ELISA assay was performed
(see Example 5), and it was shown that fibronectin binds to
substratum-immobilised tenascin-C in a dose-dependent manner.
Further ELISA assays showed that recombinant fragments FNIII 4-6,
FN12-15+CS and FNIII13 that are part of the heparin and cell
binding sites HepIII and HepII in fibronectin respectively bound to
tenascin-C in a dose-dependent manner reaching saturation (e.g.
FNIII13). Bound fragments were detected with a polyclonal
anti-fibronectin antibody, bound tenascin-C was detected with a
monoclonal anti-tenascin-C antibody.
[0154] Tenascin-C and FNIII13 were also found to form complexes in
co-immunoprecipitation experiments. Moreover, binding of tenascin-C
to surface-immobilised intact fibronectin could be competed for by
FNIII13 in a concentration-dependent fashion. No binding of the
recombinant fragment FNIII7-10, including the RGD and synergy
sites, to tenascin-C was detected in an ELISA assay. This shows
that binding of tenascin-C to FNIII13 in the HepII site of
fibronectin was specific.
EXAMPLE 11
The 13.sup.th Fibronectin Type III Repeat Restores the
Tenascin-C-Induced Cell Spreading Defect on Fibronectin
[0155] Attachment assays showed that the majority of T98G cells,
MDA-MB435 cells and rat embryo fibroblasts REF52 spread on a triple
matrix of fibronectin/tenascin-C/FNIII13. Cells were plated on
substrata (FN, FN/TN, FN/TN/III13, FN/TN+III13, III13, or BSA) for
1 h and 2 h, fixed and stained with crystal violet or vinculin and
TRITC-labelled phalloidin. Adherent cells were photographed and
spread cells were counted. Six-fold molar excess of FNIII13 (4
.mu.g/cm.sup.2) was either bound to a mixed fibronectin/tenascin-C
substratum prior to addition of the cells (FN/TN/III13) or, was
added in solution together with the cells (FN/TN+FNIII13).
Proliferation of MDA-MB435 cells and DNA replication of T98G
glioblastoma cells were determined as described in Examples 4 and
5. This revealed that FNIII13 could restore the cell spreading
defect caused by tenascin-C in all cell lines tested. The FNIII13
fragment did not provide any spreading signal by itself because
cells poorly attached and remained rounded on a FNIII13 substratum.
Cell spreading was also restored when cells were added to a
fibronectin/tenascin-C substratum together with soluble FNIII13. In
contrast to FNIII13, FNIII4-6 did not restore cell spreading on
fibronectin/tenascin-C, even when offered immobilised or in
solution. These results were confirmed by analysing the
cytoskeleton and adhesion structures of cells plated on the
different substrata. T98G cells plated for 2 h on fibronectin or on
the mixed substratum were stained for vinculin. Focal contacts were
found to have formed on fibronectin but not on
fibronectin/tenascin-C. In addition, polymerisation of actin into
stress fibres was blocked by tenascin-C. No polymerised actin was
found.
[0156] Since the FNIII13 fragment restored cell spreading on
fibronectin/tenascin-C an experiment was performed to see whether
actin stress fibres and focal contacts were restored by FNIII13.
Immunofluorescence experiments revealed that FNIII13 largely
restored actin stress fibres and focal contacts in T98G and REF52
cells upon addition of the FNIII13 fragment to a
fibronectin/tenascin-C substratum.
[0157] Tenascin-C specifically interferes with cell attachment and
spreading on fibronectin. Tenascin-C compromised focal contact and
actin stress fibre formation, both of which are hallmarks of
integrin mediated cell adhesion. In addition, .beta.1 integrins did
not localize to focal contacts and, neither integrin activation by
MnCl.sub.2 nor overexpression of the .alpha.5.beta.1 integrin
caused reversion of the tenascin-C phenotype. Tenascin-C affects
integrin function by an indirect mechanism.
[0158] Tenascin-C binds to fibronectin specifically to the HepII
and HepIII cell binding sites in fibronectin. Recombinant fragments
including the HepIII (FNIII4-6) and HepII (FNIII12-15+CS, FNIII13)
sites of fibronectin bind tenascin-C in a dose-dependent and
competable (FNIII13) manner. In contrast, a fragment encompassing
the RGD and synergy cell binding site (FNIII17-10) does not bind
tenascin-C. Whereas FNIII4-6 does not affect cell spreading of T98G
cells, FNIII13 neutralizes the spreading defect and restores focal
contacts and actin stress fibre formation on the mixed substratum
of fibronectin and tenascin-C. Tenascin-C efficiently blocks cell
access to fibronectin by directly binding to the cell binding site
located in FNIII13, thereby inhibiting full cell spreading.
[0159] In summary, tenascin-C interferes with cell spreading, focal
contact and actin stress fibre formation on fibronectin and this
effect can be neutralised by the addition of FNIII13.
EXAMPLE 12
Fragments of the 13.sup.th Fibronectin Type III Repeat Restore the
Tenascin-C-Induced Cell Spreading Defect on Fibronectin
[0160] As described in Example 11, it could also be shown that the
recombinant fragment of FNIII13 having the amino acid sequence set
forth in SEQ ID NO:4 restored cell spreading on
fibronectin/tenascin-C. Immunofluorescence experiments revealed
that the recombinant fragment of FNIII13 having the amino acid
sequence set forth in SEQ ID NO:4 also largely restored actin
stress fibres and focal contacts in T98G and REF52 cells upon
addition of the recombinant fragment to a fibronectin/tenascin-C
substratum.
[0161] Therefore, tenascin-C efficiently blocks cell access to
fibronectin by directly binding to the binding site located in the
recombinant fragment of FNIII13 having the amino acid sequence set
forth in SEQ ID NO:4, thereby inhibiting full cell spreading.
Tenascin-C interferes with cell spreading, focal contact and actin
stress fibre formation on fibronectin and this effect can be
neutralised by the addition of the recombinant fragment of FNIII13
having the amino acid sequence set forth in SEQ ID NO:4.
[0162] The same experiments were performed using the recombinant
fragment of FNIII13 having the first ten amino acids of the amino
acid sequence set forth in SEQ ID NO:4 and similar results were
obtained but to a lesser effect.
EXAMPLE 13
The 13.sup.th Fibronectin Type III Repeat Neutralises the Cell
Proliferation Stimulatory Effect of Tenascin-C
[0163] Since detachment by tenascin-C correlated with enhanced
tumour cell proliferation, and FNIII13 restored cell spreading, an
experiment was performed to see whether FNIII13 also reduced
proliferation of cells on the mixed substratum to levels as on
fibronectin. This was confirmed. Similar numbers of MDA-MB435
breast carcinoma cells were counted on the triple matrix containing
FNIII13 as on fibronectin alone 75 h after plating and for T98G
cells similar DNA synthesis levels were determined on
fibronectin/tenascin-C/FNIII13 as on fibronectin.
[0164] The same experiment was performed to see whether the peptide
of the sequence set forth in SEQ ID NO:4 also reduced proliferation
of cells on the mixed substratum to levels as on fibronectin which
was confirmed. Similar numbers of MDA-MB435 breast carcinoma cells
were counted on the triple matrix containing the recombinant
fragment of FNIII13 having the amino acid sequence set forth in SEQ
ID NO:4 as on fibronectin alone 75 h after plating and for T98G
cells similar DNA synthesis levels were determined on
fibronectin/tenascin-C/fragment of FNIII13 as on fibronectin.
[0165] The same experiment was performed with the recombinant
fragment of FNIII13 having the first ten amino acids of the amino
acid sequence set forth in SEQ ID NO:4.
EXAMPLE 14
Overexpression of Syndecan-4 Restores Cell Spreading on a Mixed
Fibronectin/Tenascin-C Substrate
[0166] An experiment was performed to check for the possibility
that tenascin-C might compete with syndecan-4 for binding to the
HepII site in fibronectin. The experiment tested whether activation
of syndecan-4 through overexpression could rescue the
tenascin-C-induced spreading defect on fibronectin. Parent T98G
cells, pools (T98G:S4) or a selected clone (T98G:S4*) of
glioblastoma cells stably overexpressing syndecan-4 (T98G:S4) were
plated on the indicated substrata for 30 minutes, 2 h, 18 h or
fixed, stained with crystal violet, photographed, and counted or
stained with TRITC-labelled phalloidin and vinculin. Syndecan-4 was
detected by immunofluorescence with an anti-syndecan-4 antibody.
There was no staining with an unspecific anti-His monoclonal
antibody (control), nor with the secondary antibody alone. FAK
(focal adhesion kinase) autophosphorylation at Y397 and total FAK
expression levels were assessed by immunoblotting with specific
antibodies. DNA replication was determined as described in Example
4 and 5. As a result, pools of T98G cells (T98G:S4) that
overexpressed syndecan-4 thereby activating syndecan-4 attached and
spread on the mixed fibronectin/tenascin-C substratum. The majority
of the 64.7% of T98G:S4 cells that attached also spread on the
fibronectin/tenascin-C substratum. This was in contrast to T98G
cells with low endogenous syndecan-4 expression levels, that only
spread to a minimal extent (3.5%) on a mixed substratum. Spreading
of the syndecan-4-overexpressing clone T98G:S4* was completely
restored on the mixed fibronectin/tenascin-C substratum 2 h after
plating.
[0167] In addition to stimulating cell attachment and spreading on
the mixed fibronectin/tenascin-C substratum, overexpression of
syndecan-4 in clonal T98G:S4* also completely restored focal
contact formation and actin polymerisation into stress fibres. This
observation supports a function of syndecan-4 in cell spreading
linked to Rho-mediated actin stress fibre formation as recently has
been shown by Saoncella, S. et al (1999) P.N.A.S. 96: 2805-2810.
Activation of focal adhesion kinase (FAK) by autophosphorylation at
Y397 is an early step in cell adhesion signalling. This is
compromised in T98G parental cells and in T98G:S4* cells on a
fibronectin/tenascin-C substratum. In contrast, plating on a
substratum of fibronectin/tenascin-C that contains FNIII13 largely
restored FAK autophosphorylation indicating that activation of
syndecan-4 by FNIII13 is linked to restoration of cell adhesion
signalling by FNIII13. In addition to rescuing cell spreading on a
mixed fibronectin/tenascin-C substratum overexpression of
syndecan-4 also reduced DNA replication levels to that on
fibronectin.
[0168] Overexpression of syndecan-4 in T98G cells rescued the
spreading defect and the lack of actin stress fibre formation on
the mixture of fibronectin and tenascin-C. This was specific for
syndecan-4 since overexpression of syndecans-1 and -2 did not
neutralize the tenascin-C-induced phenotype. We showed that both
the addition of FNIII13 and the overexpression of syndecan-4
restored tenascin-C-compromised cell spreading on fibronectin.
Therefore, FNIII13 neutralization of the tenascin-C effect is
mediated through syndecan-4 and that within the characterized
syndecan-4 recognition sequence in fibronectin (FNIII12-15) (Woods,
A. et al(2000) Arch Biochem Biophys 374: 66-72) binding of
syndecan-4 occurs through FNIII13. Also, FNIII13 binds to
syndecan-4 overexpressing T98G cells in a dose-dependent and
heparin-competable manner. Syndecan-4 binds to the same site in
fibronectin as tenascin-C (FNIII13) and the interaction of
tenascin-C with FNIII13 competes with syndecan-4 binding. FNIII14
is not relevant for the tenascin-C induced cell spreading defect
since addition of FNIII13 alone was sufficient to restore cell
spreading.
[0169] The blocking of syndecan-4 function by tenascin-C enhances
tumour cell proliferation. The inventors conclude that higher
levels of syndecan-4 in glioblastoma and breast cancer cells can
attenuate tumour cell proliferation in the context of fibronectin
and tenascin-C.
EXAMPLE 15
The 13.sup.th Fibronectin Type III Repeat (FNIII13) of Fibronectin
Binds to Syndecan-4
[0170] To investigate FNIII13 as a potential ligand of syndecan-4,
the interaction of syndecan-4 with FNIII13 was tested. Upon
addition of FNIII13 to T98G:S4* cells, FNIII13 was detected in a
syndecan-4 immunoprecipitation followed by western blotting with a
GST specific antibody. This interaction was blocked upon addition
of heparin. Parental and syndecan-4 overexpressing T98G:S4* cells
were incubated with (100 .mu.g/ml) FNIII13 for 1 h in the presence
or absence of 0.5 mg/ml heparin. Syndecan-4-bound FNIII13 was
detected by immunoprecipitation of syndecan-4 and immunoblotting
for FNIII13. Recombinant FNIII13 was added to the cells in the
absence or presence of (0.5 mg/ml) heparin. This shows that FNIII13
is a ligand for syndecan-4. To further test whether FNIII13 binds
to syndecan-4, FNIII13 was added to T98G cells and cell
surface-bound FNIII13 was detected by pull down of the GST-tagged
FNIII13 with glutathione Sepharose followed by western blotting
with an anti-His antibody or by immunofluorescence with an anti-GST
antibody. FNIII13 bound to the cell surface of T98G:S4* with
several fold increased levels compared to parental T98G, but not to
T98G:S1 and T98G:S2 cells (which overexpress syndecan-1 and
syndecan-2 respectively, see below). This interaction could be
blocked by heparin showing that syndecan-4 bound FNIII13 in a
glycosaminoglycan-dependent manner. In addition, cell surface-bound
FNIII13 was detected by immunofluorescence with an anti-His and
anti-GST antibody. To test whether overexpression of syndecans-1
and -2 could also rescue the tenascin-C-specific cell spreading
defect. T98G cells that stably overexpress syndecan-1 (T98G:S1) and
-2 (T98G:S2) were generated and were plated for 2h on
fibronectin/tenascin-C. In contrast to syndecan-4, neither
syndecan-1 nor syndecan-2 overexpression allowed cells to spread on
the mixed substratum. Like T98G:S4*, syndecan-1 and -2
overexpressors did not bind to FNIII13, nor to tenascin-C. Syndecan
overexpression in T98G:S1 and T98G:S2 was demonstrated by
immunofluorescence with syndecan-1 and -2 specific antibodies.
Syndecan-2 expression was determined by incubation with chicken
anti-syndecan-2, followed by rabbit anti-chicken and FITC coupled
goat anti-rabbit immunostaining.
[0171] Rescue of cell spreading was only accomplished by activation
of syndecan-4 and not by syndecans-1 or -2. FNIII13 is therefore a
ligand for syndecan-4 and tenascin-C specifically competes with
binding of syndecan-4 to FNIII13 and thereby prevents cell
spreading on fibronectin.
EXAMPLE 16
Fragments of the 13.sup.th Fibronectin Type III Repeat (FNIII13) of
Fibronectin Bind to Syndecan-4
[0172] The same experiment as described in Example 15 is performed
to investigate the recombinant fragment of FNIII13 having the amino
acid sequence set forth in SEQ ID NO:4, and the fragment having the
first ten amino acids of the sequence set forth in SEQ ID NO:4, as
a potential ligand of syndecan-4.
Sequence CWU 1
1
4 1 89 PRT Homo sapiens DOMAIN (1)...(89) 13th fibronectin type III
repeat (FNIII13) 1 Asn Val Ser Pro Pro Arg Arg Ala Arg Val Thr Asp
Ala Thr Glu Thr 1 5 10 15 Thr Ile Thr Ile Ser Trp Arg Thr Lys Thr
Glu Thr Ile Thr Gly Phe 20 25 30 Gln Val Asp Ala Val Pro Ala Asn
Gly Gln Thr Pro Ile Gln Arg Thr 35 40 45 Ile Lys Pro Asp Val Arg
Ser Tyr Thr Ile Thr Gly Leu Gln Pro Gly 50 55 60 Thr Asp Tyr Lys
Ile Tyr Leu Tyr Thr Leu Asn Asp Asn Ala Arg Ser 65 70 75 80 Ser Pro
Val Val Ile Asp Ala Ser Thr 85 2 49 PRT Homo sapiens PEPTIDE
(1)...(49) portion of the FNIII13 fibronectin fragment 2 Arg Arg
Ala Arg Val Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1 5 10 15
Trp Arg Thr Lys Thr Glu Thr Ile Thr Gly Phe Gln Val Asp Ala Val 20
25 30 Pro Ala Asn Gly Gln Thr Pro Ile Gln Arg Thr Ile Lys Pro Asp
Val 35 40 45 Arg 3 20 PRT Homo sapiens PEPTIDE (1)...(20) a portion
of the FNIII13 fibronectin fragment, wherein said portion comprises
the first five amino acids of the amino acid sequence set forth 3
Arg Arg Xaa Arg Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5
10 15 Xaa Arg Xaa Lys 20 4 20 PRT Homo sapiens PEPTIDE (1)...(20) a
portion of the FNIII13 fibronectin fragment 4 Arg Arg Ala Arg Val
Thr Asp Ala Thr Glu Thr Thr Ile Thr Ile Ser 1 5 10 15 Trp Arg Thr
Lys 20
* * * * *