U.S. patent application number 12/836480 was filed with the patent office on 2011-03-31 for peptides comprising an isodgr motif.
This patent application is currently assigned to MOLMED SPA. Invention is credited to ANGELO CORTI, FLAVIO CURNIS.
Application Number | 20110076234 12/836480 |
Document ID | / |
Family ID | 36602131 |
Filed Date | 2011-03-31 |
United States Patent
Application |
20110076234 |
Kind Code |
A1 |
CORTI; ANGELO ; et
al. |
March 31, 2011 |
PEPTIDES COMPRISING AN ISODGR MOTIF
Abstract
Disclosed herein are peptides which include an isoDGR motif and
which selectively inhibit .alpha.v.beta.3 integrin. In some
embodiments, the isoDGR motif results from the deamidation of an
NGR motif.
Inventors: |
CORTI; ANGELO; (AZZANO SAN
PAOLO (BG), IT) ; CURNIS; FLAVIO; (ALZANO LOMBARDO
(BG), IT) |
Assignee: |
MOLMED SPA
FONDAZIONE CENTRO SAN RAFFAELE DEL MONTE TABOR
|
Family ID: |
36602131 |
Appl. No.: |
12/836480 |
Filed: |
July 14, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11793417 |
Nov 8, 2007 |
7795386 |
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PCT/IB2005/004058 |
Dec 21, 2005 |
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12836480 |
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60638158 |
Dec 23, 2004 |
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Current U.S.
Class: |
424/9.1 ;
424/450; 424/649; 424/85.5; 514/1.1; 514/19.3; 530/317; 530/322;
530/326; 530/327; 530/328; 530/329; 530/330; 530/350; 530/351;
530/391.7; 530/391.9 |
Current CPC
Class: |
A61P 27/02 20180101;
A61P 43/00 20180101; C07K 14/78 20130101; A61P 35/00 20180101; A61P
19/10 20180101; A61P 3/10 20180101; A61K 47/64 20170801; A61P 31/00
20180101; A61P 13/12 20180101; A61P 19/02 20180101; A61P 9/00
20180101 |
Class at
Publication: |
424/9.1 ;
530/330; 530/329; 530/328; 530/326; 530/327; 530/317; 514/19.3;
514/1.1; 530/350; 530/351; 424/85.5; 424/450; 424/649; 530/322;
530/391.7; 530/391.9 |
International
Class: |
A61K 49/00 20060101
A61K049/00; C07K 7/06 20060101 C07K007/06; C07K 7/08 20060101
C07K007/08; C07K 7/64 20060101 C07K007/64; A61K 38/00 20060101
A61K038/00; A61K 38/19 20060101 A61K038/19; C07K 14/00 20060101
C07K014/00; C07K 14/57 20060101 C07K014/57; C07K 14/54 20060101
C07K014/54; A61K 38/21 20060101 A61K038/21; A61K 9/127 20060101
A61K009/127; A61K 33/24 20060101 A61K033/24; A61P 3/10 20060101
A61P003/10; A61P 13/12 20060101 A61P013/12; A61P 19/02 20060101
A61P019/02; A61P 19/10 20060101 A61P019/10; A61P 35/00 20060101
A61P035/00 |
Claims
1-111. (canceled)
112. A peptide comprising a .sub.DDGR motif.
113. A peptide according to claim 112 comprising the sequence
X.sub.DDGRX', wherein: (a) X is selected from the group consisting
of L, V, A, C, G, Y, P, H, K, Q and I; and (b) X' is selected from
the group consisting of C, G, H, L, E, T, Q, R, S and P.
114. A peptide according to claim 112, wherein the peptide
comprises a sequence selected from the group consisting of
C.sub.DDGRCVSGCAGRC, .sub.DDGRAHA, G.sub.DDGRG, CVL.sub.DDGRMEC,
C.sub.DDGRC, C.sub.DDGRCG, L.sub.DDGRE, Y.sub.DDGRT,
LQCICTG.sub.DDGRGEWKCE, LQCISTG.sub.DDGRGEWKCE,
CICTG.sub.DDGRGEWKC, CISTG.sub.DDGRGEWKC, MRCTCVG.sub.DDGRGEWTCY,
MRCTSVG.sub.DDGRGEWTCY, CTCVG.sub.DDGRGEWTC and
CTSVG.sub.DDGRGEWTC.
115. A peptide according to claim 114, wherein the peptide
comprises a sequence selected from the group consisting of
cycloCVL.sub.DDGRMEC, linear C.sub.DDGRC, cyclic C.sub.DDGRC,
linear C.sub.DDGRCG and cyclic C.sub.DDGRCG.
116. A peptide according to claim 112, wherein the peptide
comprises a deamidation product of an extracellular matrix protein
or a fragment or derivative thereof.
117. A peptide according to claim 116, wherein the peptide
comprises a deamidation product of fibronectin, vitronectin,
collagen, laminin, or a fragment or derivative thereof.
118. A peptide according to claim 117, wherein the peptide
comprises a deamidation product of the FN-I.sub.5, FN-I.sub.7,
FN-II.sub.1 or FN-III.sub.9 module of fibronectin or a fragment or
derivative thereof.
119. A peptide according to claim 112, wherein the peptide inhibits
tumor growth.
120. A peptide according to claim 112, wherein the peptide inhibits
angiogenisis.
121. A peptide according to claim 112, wherein the peptide does not
comprise an additional therapeutic agent.
122. A peptide according to claim 121, wherein the additional
therapeutic agent is an anti-cancer peptide.
123. A peptide according to claim 121, wherein the additional
therapeutic agent is a cytokine.
124. A peptide according to claim 112, wherein the peptide
comprises a turn involving the G and R residues of the DGR
motif.
125. A peptide according to claim 112, wherein the peptide
comprises up to 350 amino acids.
126. A peptide according to claim 112, other than when formed by
metabolism of a peptide comprising the NGR motif.
127. A conjugation product comprising a peptide as defined in claim
112, and a drug, cytokine, cytokine fragment, toxin, apoptotic
peptide, biological response modifier, radionuclide, viral
particle, gene or an imaging compound.
128. A pharmaceutical composition comprising an effective amount of
a peptide as defined in claim 112.
129. A composition according to claim 128 together with a
pharmaceutically acceptable carrier, diluent or excipient.
130. A composition according to claim 128 comprising essentially no
peptide having a corresponding NGR motif.
131. A conjugation product between a peptide as defined claim 112,
and a cytokine.
132. A conjugation product between a cytokine and a targeting
moiety comprising the DGR motif, wherein the DGR is a
.sub.DDGR.
133. A conjugation product according to claim 132, wherein the
targeting moiety comprises the sequence X.sub.DDGRX' wherein: (a) X
is selected from the group consisting of L, V, A, C, G, Y, P, H, K,
Q and I; and (b) X' is selected from the group consisting of C, G,
H, L, E, T, Q, R, S and P.
134. A conjugation product according to claim 132, wherein the
targeting moiety is selected from the group consisting of
C.sub.DDGRCVSGCAGRC, .sub.DDGRAHA, G.sub.DDGRG, CVL.sub.DDGRMEC,
C.sub.DDGRC, C.sub.DDGRCG, L.sub.DDGRE, Y.sub.DDGRT,
LQCICTG.sub.DDGRGEWKCE, LQCISTG.sub.DDGRGEWKCE,
CICTG.sub.DDGRGEWKC, CISTG.sub.DDGRGEWKC, MRCTCVG.sub.DDGRGEWTCY,
MRCTSVG.sub.DDGRGEWTCY, CTCVG.sub.DDGRGEWTC and
CTSVG.sub.DDGRGEWTC.
135. A conjugation product according to claim 134, wherein the
targeting moiety is selected from the group consisting of
cycloCVL.sub.DDGRMEC, linear C.sub.DDGRC, cyclic C.sub.DDGRC,
linear C.sub.DDGRCG and cyclic C.sub.DDGRCG.
136. A conjugation product according to claim 131, wherein the
cytokine is derivatized with polyethylene glycol or an acyl
residue.
137. A conjugation product according to claim 131, wherein the
cytokine is further conjugated with a compound selected from the
group consisting of an antibody, an antibody fragment, and biotin,
wherein said antibody or fragment thereof is directed to a compound
selected from the group consisting of a tumoral antigen, a tumoral
angiogenic marker, and a component of the extracellular matrix.
138. A conjugation product according to claim 137, wherein the
cytokine is TNF and is conjugated to both the targeting moiety and
a compound selected from the group consisting of an antibody, and
antibody fragment, and biotin.
139. A conjugation product according to claim 131, other than when
formed in vivo by metabolism of a peptide comprising the NGR
motif.
140. A conjugation product according to claim 131, wherein the
cytokine is linked to the peptide or targeting moiety through a
spacer.
141. A conjugation product according to claim 140, wherein the
spacer is a glycine (G).
142. A conjugation product according to claim 131, wherein the
cytokine is selected from the group consisting of TNF, IFN-.gamma.,
IL-12, IP-10, IL-7 and EMAP II.
143. A conjugation product according to claim 131, wherein the
cytokine is TNF or IFN.gamma..
144. A conjugation product according to claim 143, wherein the
cytokine is TNF.
145. A conjugation product according to claim 144, wherein the
cytokine is TNF.alpha. or TNF.beta..
146. A pharmaceutical composition comprising an effective amount of
a conjugation product as claimed in claim 131, together with a
pharmaceutically acceptable carrier, diluent or excipient.
147. A pharmaceutical composition comprising an effective amount of
a conjugation product of TNF and a peptide as defined in claim 112,
and an effective amount of IFN.gamma. or a polynucleotide encoding
therefor.
148. A composition according to claim 128, in the form of an
injectable solution or suspension or a liquid for infusions.
149. A composition according to claim 128, in the form of
liposomes.
150. A composition according to claim 128, further comprising
another antitumor agent or a diagnostic tumor-imaging compound.
151. A composition according to claim 150, wherein the other
antitumor agent is doxorubicin, melphalan, cis-platin, gemcitabine
or taxol.
152. A method of treating or diagnosing a patient suffering from
disorders involving .alpha.v.beta.3 comprising administering the
peptide of claim 112.
153. A method of treating or diagnosing a patient suffering from
osteoporosis, arthritis, diabetic retinopathy, macular
degeneration, restenosis or hemangioma comprising administering the
peptide claim 112.
154. A method of treating or diagnosing a patient suffering from
cancer comprising administering peptide claim 112.
155. A method according to claim 154, wherein cancer comprises a
solid tumor.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/793,417, filed Nov. 8, 2007, which is the
National Phase of PCT/IB2005/004058, filed Dec. 21, 2005, which
claims the benefit of priority from U.S. Provisional Patent
Application No. 60/638,158, filed Dec. 23, 2004. The contents of
these applications are incorporated herein by reference in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to novel peptides which may be
used in the treatment of diseases associated with angiogenesis and
cancer. More particularly, the invention relates to extracellular
matrix peptides and derivatives thereof, and, in particular to
peptides comprising a DGR motif and isomers thereof. The present
invention also relates to modified cytokines, in particular to
cytokine derivatives capable of "homing" angiogenic vessels.
BACKGROUND TO THE INVENTION
[0003] The extracellular matrix is an important regulator of cell
and tissue behaviour. Proteins of the extracellular matrix, such as
fibronectin, vitronectin, collagens and laminin bind to a number of
cell receptors that control many medically important biological
phenomena, such as angiogenisis, cell migration, tissue repair,
cancer cell differentiation, platelet aggregation and homing of
immune system cells and neuronal processes to target sites. An
important family of receptors that bind to the extracellular matrix
are the integrins.
[0004] Fibronectins are large extracellular matrix glycoproteins
(.about.450 KDa) that have been implicated in integrin binding.
They are composed of two nearly identical disulfide bonded
subunits, each subunit consisting of three types of repeating
homologous modules termed FN-I, FN-II and FN-III repeats. In
addition, alternatively spliced modules, called EDA, EDB and IIICS,
can also be present (Mohri, 1997; Pankov and Yamada, 2002). Single
modules or groups of modules may contain binding sites for
different molecules, including sulfated glycosaminoglycans, DNA,
gelatin, heparin and fibrin (Mohri, 1997; Pankov and Yamada, 2002;
Yamada, 1989). Furthermore, fibronectins contains binding sites for
about half of the known cell surface integrin receptors (Johansson
et al., 1997; Plow et al., 2000). In particular, the FM-III.sub.10
repeat contains an RGD site that can bind
.alpha..sub.3.beta..sub.1, .alpha..sub.5.beta..sub.1,
.alpha..sub.v.beta..sub.1, .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.v.beta..sub.6,
.alpha..sub.8.beta..sub.1, .alpha.II.sub.b.beta..sub.3 integrins,
while the FN-III.sub.9 repeat contains the so-called "synergy site"
PHSRN (SEQ ID No: 51) that cooperates with RGD in the binding of
.alpha..sub.5.beta..sub.1 and .alpha.II.sub.b.beta..sub.3 (Busk et
al., 1992; Dedhar and Gray, 1990; Gardner and Hynes, 1985;
Johansson et al., 1997; Pankov and Yamada, 2002; Plow et al., 1985;
Pytela et al., 1985; Smith et al., 1990; Takada et al., 1987; Vogel
et at., 1990; Weinacker et at., 1994). The FN-III.sub.14-IIICS
region contains three sites (called CS.sub.1, CS.sub.5 and H.sub.1)
characterised by the presence of LDV motif and accessory REDV (SEQ
ID No: 52) and IDA sites involved in .alpha..sub.4.beta..sub.1 and
.alpha..sub.4.beta..sub.7 recognition (Johansson et at., 1997;
Pankov and Yamada, 2002). Moreover, the FN-III.sub.5 repeat
contains the KLDAPT (SEQ ID No: 53) sequence that bind
.alpha..sub.4.beta..sub.1 and .alpha..sub.4.beta..sub.7 (Moyano et
al., 1997) whereas FN-I.sub.1-9 and FN-II.sub.1-2 contain sites,
not yet identified, that can interact with
.alpha..sub.5.beta..sub.1 (Hocking et al., 1998).
[0005] Primary and tertiary structure analysis of human
fibronectin, showed that this protein contains two GNGRG (SEQ ID
No: 4) loops, located in FN-I.sub.5 and FN-I.sub.7 modules, that
are conserved in bovine, murine, rat, amphibian and fish (Di Matteo
et al.). Two additional NGR sites, less conserved, are also present
in human FN-II.sub.1 and FN-III.sub.9. Recent experimental work
showed that peptides containing the NGR motif can inhibit
.alpha..sub.5.beta..sub.1 and .alpha..sub.v.beta..sub.1-mediated
cell adhesion to fibronectin (Koivunen et al., 1993).
[0006] The interaction between cell surface anchored integrins and
extracellular matrix components have been implicated in
angiogenesis, an important process in neonatal growth and in the
pathogenesis of a large variety of clinical diseases including
tissue inflammation, arthritis, tumor growth, diabetic retinopathy,
macular degeneration by neovascularization of retina and the like
conditions. It is known that .alpha..sub.v.beta..sub.3 integrin,
the vitronectin receptor, plays a critical role in angiogenesis
(Hynes, 2002). Compounds able to inhibit the interaction of this
integrin with extracellular matrix proteins are known to inhibit
angiogenesis and tumor growth (Brooks et al., 1994; Brooks et al.,
1995; Friedlander et al., 1995; Friedlander et al., 1996; Hammes et
al., 1996). However, a problem associated with these inhibitors is
their cross-reactivity with many integrin species.
[0007] In the light of increasing awareness of the role of the
extracellular matrix in biological processes and disease
development, there remains a need for novel therapeutics that
target this area. The present invention addresses this need.
[0008] The antitumoral activity of some cytokines is well known and
described. Some cytokines have already been used therapeutically
also in humans (Fiers et al., 1995). For example, such cytokines as
interleukine-2 (IL-2) and interferon .alpha. (IFN.alpha.) have
shown positive antitumoral activity in patients with different
types of tumors, such as kidney metastatic carcinoma, hairy cell
leukemia, Kaposi sarcoma, melanoma, multiple mieloma, and the like.
Other cytokines like IFN.beta., the Tumor Necrosis Factor (TNF)
.alpha., TNF.beta., IL-1, 4, 6, 12, 15 and the Colony Stimulating
Factors (CFSs) have shown a certain antitumoral activity on some
types of tumors and therefore are the object of further
studies.
[0009] In general, the therapeutic use of cytokines is strongly
limited by their systemic toxicity. TNF, for example, was
originally discovered for its capacity of inducing the hemorrhagic
necrosis of some tumors Carswell et al., 1975), and for its in
vitro cytotoxic effect on different tumoral lines Helson et al.,
1975), but it subsequently proved to have strong pro-inflammatory
activity, which can, in case of overproduction conditions,
dangerously affect the human body (Tracey et al., 1993).
[0010] As the systemic toxicity is a fundamental problem with the
use of pharmacologically active amounts of cytokines in humans,
novel derivatives and therapeutic strategies are now under
evaluation, aimed at reducing the toxic effects of this class of
biological effectors while keeping their therapeutic efficacy. For
example, WO01/61017 discloses a conjugation product between TNF or
IFN.gamma. and a ligand of the CD13 receptor; WO03/093478 discloses
a pharmaceutical composition comprising a conjugate of a cytokine
and a tumor targeting moiety wherein the cytokine is present in an
amount which does not induce a negative feedback mechanism; and
WO03/092737 discloses conjugates of various cytokines and tumor
targeting moieties. The present invention provides novel cytokine
conjugates that have therapeutic potential.
SUMMARY OF THE INVENTION
[0011] Asparagine (N) deamidation, a non-enzymatic protein
post-translational modification, is generally viewed as a
deleterious event associated with protein aging. In the present
application we show that an Asparagine-Glycine-Arginine (NGR)
sequence of the extracellular matrix can promote endothelial cell
adhesion via an unusual mechanism based on non-enzymatic
deamidation of asparagine to aspartic acid and isoaspartic acid,
generating a new cell adhesion motif. In particular, we show that
this deamidation is associated with a "gain of function;"
deamidated fragments being able to inhibit endothelial cell
adhesion to vitronectin, inhibit (.alpha..sub.v.beta..sub.3
integrin and inhibit tumor growth.
[0012] In particular, we have shown that the .sub.LisoDGR and
.sub.DDGR isomers show enhanced cell adhesion binding and
anti-cancer properties over other isomeric forms. This is
surprising given that the vast majority of peptides will comprise
the L enantiomer of aspartic acid. Peptide produced by gene
expression will all comprise the L enantiomer.
[0013] We have also found that cytokines conjugated to deamidated
NGR peptides have surprising activity. We have previously found
that the therapeutic index of certain cytokines can be remarkably
improved and their immunotherapeutic properties can be enhanced by
coupling with a ligand comprising the NGR motif, such as a modified
ligand of aminopeptidase-N receptor (CD13). CD13 is a
trans-membrane glycoprotein of 150 kDa highly conserved in various
species. It is expressed on normal cells as well as in myeloid
tumor lines, in the angiogenic endothelium and is some epithelia.
CD13 receptor is usually identified as "NOR" receptor, in that its
peptide ligands share the amino acidic "NGR" motif. We have now
surprisingly found that similar or indeed improved results may be
achieved using a modified ligand in which the asparaginyl (Asn or
N) residue in the NGR motif is modified to aspartyl (Asp or D),
i.e. a "DGR" motif, by deamidation. TNF coupled with such a
modified ligand of CD13 receptor and IFN.gamma. may also act
synergistically so that effective anti-tumor activity may be seen
upon co-administration at dosages which are below the effective
doses individually. In addition, we have found that the anti-tumor
activity of a combination of the modified TNF and another
anti-tumor agent, such as mephalan, or doxorubicin or cis-platin or
gemcitabine or taxol, is increased by administration of
IFN.gamma..
STATEMENTS OF THE INVENTION
[0014] According to a first aspect of the invention there is
provided a peptide which selectively inhibits
.alpha..sub.v.beta..sub.3 integrin which comprises the deamidation
product of a peptide comprising the NGR motif.
[0015] According to another aspect of the invention there is
provided a peptide which inhibits endothelial cell adhesion to
vitronectin which comprises the deamidation product of a peptide
comprising the NGR motif.
[0016] According to another aspect of the invention there is
provided a peptide comprising the deamidation product of an
extracellular matrix protein such as, but not limited to,
fibronectin, vitronectin, collagen or laminin or a fragment or
derivative thereof. Preferably the fragment comprises at least 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, or 100 consecutive amino acids
of the deamidated matrix protein and preferably comprises a
deamidated NOR motif of said protein.
[0017] Preferably the peptide comprises the deamidation product of
fibronectin, more preferably the deamidation product of the
FN-I.sub.5, FN-I.sub.7, FN-II.sub.1 or FN-III.sub.9 module of
fibronectin, more preferably the deamidation product of the
FN-I.sub.5 module of fibronectin, or a fragment or derivative
thereof or the deamidation product of the FN-I.sub.7 module of
fibronectin, or a fragment or derivative thereof. Preferably the
fragment comprises at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50,
or 100 consecutive amino acids of fibronectin and preferably
comprises a deamidated NGR motif of said protein.
[0018] In one embodiment the peptide consists of the deamidation
product of the extracellular matrix protein or fragment or
derivative thereof.
[0019] According to another aspect of the invention there is
provided the deamidation product of a peptide comprising the
sequence XNGRX' (SEQ ID No: 1) wherein X is selected from the group
consisting of L, V, A, C, G, Y, P, H, K, Q and I and X' is selected
from the group consisting of C, G, H, L, E, T, Q, R, S and P.
[0020] Preferably the peptide comprises the deamidation product of
a peptide comprising a sequence selected from CNGRCVSGCAGRC (SEQ ID
No: 2), NGRAHA (SEQ ID No: 3), GNGRG (SEQ ID No: 4), CVLNGRMEC (SEQ
ID No: 5), CNGRC (SEQ ID No: 6), CNGRCG (SEQ ID No: 7), LNGRE (SEQ
ID No: 8), YNGRT (SEQ ID No: 9), LQCICTGNGRGEWKCE (SEQ ID No: 10),
LQCISTGNGRGEWKCE (SEQ ID No: 11), CICTGNGRGEWKC (SEQ ID No: 12),
CISTGNGRGEWKC (SEQ ID No: 13), MRCTCVGNGRGEWTCY (SEQ ID No: 14),
MRCTSVGNGRGEWTCY (SEQ ID No: 15), CTCVGNGRGEWTC (SEQ ID No: 16),
and CTSVGNGRGEWTC (SEQ ID No: 17).
[0021] In one embodiment the peptide consists of the deamidation
product of a peptide having a sequence selected from CNGRCVSOCAGRC
(SEQ ID No: 2), NGRAHA (SEQ ID No: 3), GNGRG (SEQ ID No: 4),
CVLNGRMEC (SEQ ID No: 5), CNGRC (SEQ ID No: 6), LNGRE (SEQ ID No:
8), YNGRT (SEQ ID No: 9), LQCICTGNGRGEWKCE (SEQ ID No: 10),
LQCISTGNGRGEWKCE (SEQ ID No: 11), CICTGNGRGEWKC (SEQ ID No: 12),
CISTGNGRGEWKC (SEQ ID No: 13), MRCTCVGNGRGEWTCY(SEQ ID No: 14),
MRCTSVGNGRGEWTCY (SEQ ID No: 15), CTCVGNGRGEWTC (SEQ ID No: 16),
and CTSVGNGRGEWTC (SEQ ID No: 17).
[0022] Preferably the peptide comprises the deamidation product of
a peptide comprising a sequence selected from cycloCVLNGRMEC (SEQ
ID No: 5), linear CNGRC (SEQ ID No: 6), cyclic CNGRC (SEQ ID No:
6), linear CNGRCG (SEQ ID No: 7), and cyclic CNGRCG (SEQ ID No:
7).
[0023] In one embodiment the peptide consists of the deamidation
product of a peptide having a sequence selected from cycloCVLNGRMEC
(SEQ ID No: 5), linear CNGRC (SEQ ID No: 6), cyclic CNGRC (SEQ ID
No: 6), linear CNGRCG (SEQ ID No: 7), and cyclic CNGRCG (SEQ ID No:
7).
[0024] According to another aspect of the present invention there
is provided a peptide comprising the DGR motif wherein said peptide
selectively inhibits .alpha..sub.v.beta..sub.3 integrin.
[0025] According to another aspect of the present invention there
is provided a peptide comprising the DGR motif wherein said peptide
inhibits endothelial cell adhesion to vitronectin.
[0026] Preferably the peptide comprises the sequence XDGRX' (SEQ ID
No: 18) wherein X is selected from the group consisting of L, V, A,
C, G, Y, P, H, K, Q and I and X' is selected from the group
consisting of C, G, H, L, E, T, Q, R, S and P, and wherein the "D"
of the SEQ ID NO: 18 DGR motif is the .sub.DD entantiomer.
[0027] Preferably the peptide comprises a sequence selected from
CDGRCVSGCAGRC (SEQ ID No: 19), DGRAHA (SEQ ID No: 20), GDGRG (SEQ
ID No: 21), CVLDGRMEC (SEQ ID No: 22), CDGRC (SEQ ID No: 23),
CDGRCG (SEQ ID No: 24), LDGRE (SEQ ID No: 25), YDGRT (SEC) ID No:
26), LQCICTGDGRGEWKCE (SEQ ID No: 27), LQCISTGDGRGEWKCE (SEQ ID No:
28), CICTGDGRGEWKC (SEQ ID No: 29), CISTGDGRGEWKC (SEQ ID No: 30),
MRCTCVGDGRGEWTCY (SEQ ID No: 31), MRCTSVGDGRGEWTCY (SEQ ID No: 32),
CTCVGDGRGEWTC (SEQ ID No: 33), or CTSVGDGRGEWTC (SEQ ID No: 34),
more preferably a sequence selected from cycloCVLDGRMEC (SEQ ID No:
22.times.), linear CDGRC (SEQ ID No: 23), cyclic CDGRC (SEQ ID No:
23), linear CDGRCG (SEQ ID No: 24), and cyclic CDGRCG (SEQ ID NO:
24), and wherein the "D" of the DGR motif of each of the SEQ ID
NOs: is the .sub.DD entantiomer.
[0028] In one embodiment the peptide consists of a sequence
selected from CDGRCVSGCAGRC (SEQ ID No: 19), DGRAHA (SEQ ID No:
20), GDGRG (SEQ ID No: 21), CVLDGRMEC (SEQ ID No: 22), CDGRC (SEQ
ID No: 23), CDGRCG (SEQ ID No: 24), LDGRE (SEQ ID No: 25), YDGRT
(SEQ ID No: 26), LQCICTGDGRGEWKCE (SEQ ID No: 27), LQCISTGDGRGEWKCE
(SEQ ID No: 28), CICTGDGRGEWKC (SEQ ID No: 29), CISTGDGRGEWKC (SEQ
ID No: 30), MRCTCVGDGRGEWTCY (SEQ ID No: 31), MRCTSVGDGRGEWTCY (SEQ
ID No: 32), CTCVGDGRGEWTC (SEQ ID No: 33), or CTSVGDGRGEWTC (SEQ ID
No: 34), more preferably a sequence selected from cycloCVLDGRMEC
(SEQ ID No: 22X), linear CDGRC (SEQ ID No: 23), cyclic CDGRC (SEQ
ID No: 23), linear CDGRCG (SEQ ID No: 24), and cyclic CDGRCG (SEQ
ID NO: 24), and wherein the "D" of the DGR motif of each of the SEQ
ID NOs: is the .sub.DD entantiomer.
[0029] According to another aspect of the present invention there
is provided a peptide comprising an isoDGR motif.
[0030] Preferably the peptide comprises the sequence XisoDGRX' (SEQ
ID No: 54) wherein X is selected from the group consisting of L, V,
A, C, G, Y, P, H, K, Q and I and X' is selected from the group
consisting of C, G, H, L, E, T, Q, R, S and P.
[0031] Preferably the peptide comprises a sequence selected from
CisoDGRCVSGCAGRC (SEQ ID No: 55), isoDGRAHA (SEQ ID No: 56),
GisoDGRG (SEQ ID No: 57), CVLisoDGRMEC (SEQ ID No: 58), CisoDGRC
(SEQ ID No: 59), CisoDGRCG (SEQ ID No: 60), LisoDGRE (SEQ ID No:
61), YisoDGRT (SEQ ID No: 62), LQCICTGisoDGRGEWKCE (SEQ ID No: 63),
LQCISTGisoDGRGEWKCE (SEQ ID No: 64), CICTGisoDGRGEWKC (SEQ ID No:
65), CICTGisoDGRGEWKC (SEQ ID No: 66), MRCTCVGisoDGRGEWTCY (SEQ ID
No: 67), MRCTSVGisoDGRGEWTCY (SEQ ID No: 68), CTCVGisoDGRGEWTC (SEQ
ID No: 69), or CTSVGisoDGRGEWTC (SEQ ID No: 70), more preferably a
sequence selected from cycloCVLisoDGRMEC (SEQ ID No: 58), linear
CisoDGRC (SEQ ID No: 59), cyclic CisoDGRC (SEQ ID No: 59), linear
CisoDGRCG (SEQ ID No: 60), and cyclic CisoDGRCG (SEQ ID No:
60).
[0032] In one embodiment the peptide consists of a sequence
selected from CisoDGRCVSGCAGRC (SEQ ID No: 55), isoDGRAHA (SEQ ID
No: 56), GisoDGRG (SEQ ID No: 57), CVLisoDGRMEC (SEQ ID No: 58),
CisoDGRC (SEQ ID No: 59), CisoDGRCG (SEQ ID No: 60), LisoDGRE (SEQ
ID No: 61), YisoDGRT (SEQ ID No: 62), LQCICTGisoDGRGEWKCE (SEQ ID
No: 63), LQCISTGisoDGRGEWKCE (SEQ ID No: 64), CICTGisoDGRGEWKC (SEQ
ID No: 65), CICTGisoDGRGEWKC (SEQ ID No: 66), MRCTCVGisoDGRGEWTCY
(SEQ ID No: 67), MRCTSVGisoDGRGEWTCY (SEQ ID No: 68),
CTCVGisoDGRGEWTC (SEQ ID No: 69), and CTSVGisoDGRGEWTC (SEQ ID No:
70), more preferably a sequence selected from cycloCVLisoDGRMEC
(SEQ ID No: 58), linear CisoDGRC (SEQ ID No: 59), cyclic CisoDGRC
(SEQ ID No: 59), linear CisoDGRCG (SEQ ID No: 60), and cyclic
CisoDGRCG (SEQ ID No: 60),
[0033] According to another aspect of the present invention there
is provided a peptide comprising an .sub.LisoDGR motif
[0034] Preferably the peptide comprises the sequence
X.sub.LisoDGRX' (SEQ ID No: 54) sequence wherein X is selected from
the group consisting of L, V, A, C, G, Y, P, H, K, Q or I and X' is
selected from the group consisting of C, G, H, L, E, T, Q, R, S or
P.
[0035] Preferably the peptide comprises a sequence selected from
C.sub.LisoDGRCVSGCAGRC (SEQ ID No: 55), .sub.LisoDGRAHA (SEQ ID No:
56), G.sub.LisoDGRG (SEQ ID No: 57), CVL.sub.LisoDGRMEC (SEQ ID No:
58), C.sub.LisoDGRC (SEQ ID No: 59), C.sub.LisoDGRCG (SEQ ID No:
60), L.sub.LisoDGRE (SEQ ID No: 61), Y.sub.LisoDGRT (SEQ ID No:
62), LQCICTG.sub.LisoDGRGEWKCE (SEQ ID No: 63),
LQCISTG.sub.LisoDGRGEWKCE (SEQ ID No: 64), CICTG.sub.LisoDGRGEWKC
(SEQ ID No: 65), CISTG.sub.LisoDGRGEWKC (SEQ ID No: 66),
MRCTCVG.sub.LisoDGRGEWTCY (SEQ ID No: 67),
MRCTSVG.sub.LisoDGRGEWTCY (SEQ ID No: 68), CTCVG.sub.LisoDGRGEWTC
(SEQ ID No: 69), and CTSVG.sub.LisoDGRGEWTC (SEQ ID No: 70), more
preferably a sequence selected from cycloCVL.sub.LisoDGRMEC (SEQ ID
No: 58), linear C.sub.LisoDGRC (SEQ ID No: 59), cyclic
C.sub.LisoDGRC (SEQ ID No: 59), linear C.sub.LisoDGRCG (SEQ ID No:
60), and cyclic C.sub.LisoDGRCG (SEQ ID No: 60).
[0036] In one embodiment the peptide consists of a sequence
selected from C.sub.LisoDGRCVSGCAGRC (SEQ ID No: 55),
.sub.LisoDGRAHA (SEQ ID No: 56), G.sub.LisoDGRG (SEQ ID No: 57),
CVL.sub.LisoDGRMEC (SEQ ID No: 58), C.sub.LisoDGRC (SEQ ID No: 59),
C.sub.LisoDGRCG (SEQ ID No: 60), L.sub.LisoDGRE (SEQ ID No: 61),
Y.sub.LisoDGRT (SEQ ID No: 62), LQCICTG.sub.LisoDGRGEWKCE (SEQ ID
No: 63), LQCISTG.sub.LisoDGRGEWKCE (SEQ ID No: 64),
CICTG.sub.LisoDGRGEWKC (SEQ ID No: 65), CISTG.sub.LisoDGRGEWKC (SEQ
ID No: 66), MRCTCVG.sub.LisoDGRGEWTCY (SEQ ID No: 67),
MRCTSVG.sub.LisoDGRGEWTCY (SEQ ID No: 68), CTCVG.sub.LisoDGRGEWTC
(SEQ ID No: 69), and CTSVG.sub.LisoDGRGEWTC (SEQ ID No: 70), more
preferably a sequence selected from cycloCVL.sub.LisoDGRMEC (SEQ ID
No: 58), linear C.sub.LisoDGRC (SEQ ID No: 59), cyclic
C.sub.LisoDGRC (SEQ ID No: 59), linear C.sub.LisoDGRCG (SEQ ID No:
60), and cyclic C.sub.LisoDGRCG (SEQ ID No: 60).
[0037] According to another aspect of the present invention there
is provided a peptide comprising a .sub.DDGR motif
[0038] Preferably the peptide comprises the sequence X.sub.DDGRX'
(SEQ ID No: 54) wherein X is selected from L, V, A, C, G, Y, P, H,
K, Q or I and X' is selected from C, G, H, L, E, T, Q, R, S or P,
and wherein the isoD of SEQ ID NO: 54 is the .sub.LisoD
entantiomer.
[0039] Preferably the peptide comprises a sequence selected from
C.sub.DDGRCVSGCAGRC (SEQ ID No: 55), .sub.DDGRAHA (SEQ ID No: 56),
G.sub.DDGRG (SEQ ID No: 57), CVL.sub.DDGRMEC (SEQ ID No: 58),
C.sub.DDGRC (SEQ ID No: 59), C.sub.DDGRCG (SEQ ID No: 60),
L.sub.DDGRE (SEQ ID No: 61), Y.sub.DDGRT (SEQ ID No: 62),
LQCICTG.sub.DDGRGEWKCE (SEQ ID No: 63), LQCISTG.sub.DDGRGEWKCE (SEQ
ID No: 64), CICTG.sub.DDGRGEWKC (SEQ ID No: 65),
CISTG.sub.DDGRGEWKC (SEQ ID No: 66), MRCTCVG.sub.DDGRGEWTCY (SEQ ID
No: 67), MRCTSVG.sub.DDGRGEWTCY (SEQ ID No: 68),
CTCVG.sub.DDGRGEWTC (SEQ ID No: 69), and CTSVG.sub.DDGRGEWTC (SEQ
ID No: 70), more preferably a sequence selected from
cycloCVL.sub.DDGRMEC (SEQ ID No: 58), linear C.sub.DDGRC (SEQ ID
No: 59), cyclic C.sub.DDGRC (SEQ ID No: 59), linear C.sub.DDGRCG
(SEQ ID No: 60), and cyclic C.sub.DDGRCG (SEQ ID NO: 60), and
wherein the isoD of each of the SEQ ID NOs: is the .sub.LisoD
enantiomer.
[0040] In one embodiment the peptide consists of a sequence
selected from C.sub.DDGRCVSGCAGRC (SEQ ID No: 55), .sub.DDGRAHA
(SEQ ID No: 56), G.sub.DDGRG (SEQ ID No: 57), CVL.sub.DDGRMEC (SEQ
ID No: 58), C.sub.DDGRC (SEQ ID No: 59), C.sub.DDGRCG (SEQ ID No:
60), L.sub.DDGRE (SEQ ID No: 61), Y.sub.DDGRT (SEQ ID No: 62),
LQCICTG.sub.DDGRGEWKCE (SEQ ID No: 63), LQCISTG.sub.DDGRGEWKCE (SEQ
ID No: 64), CICTG.sub.DDGRGEWKC (SEQ ID No: 65),
CISTG.sub.DDGRGEWKC (SEQ ID No: 66), MRCTCVG.sub.DDGRGEWTCY (SEQ ID
No: 67), MRCTCVG.sub.DDGRGEWTCY (SEQ ID No: 68),
CTCVG.sub.DDGRGEWTC (SEQ ID No: 69), and CTCVG.sub.DDGRGEWTC (SEQ
ID No: 70), more preferably a sequence selected from
cycloCVL.sub.DDGRMEC (SEQ ID No: 58), linear C.sub.DDGRC (SEQ ID
No: 59), cyclic C.sub.DDGRC (SEQ ID No: 59), linear C.sub.DDGRCG
(SEQ ID No: 60), and cyclic C.sub.DDGRCG (SEQ ID NO: 60), and
wherein the isoD of each of the SEQ ID NOs: is the .sub.LisoD
enantiomer.
[0041] In one embodiment the peptide comprising the isoDGR motif,
.sub.LisoDGR motif or .sub.DDGR motif comprises a deamidation
product of an extracellular matrix protein, such as, but not
limited to fibronectin, vitronectin, collagen or laminin, more
preferably a deamidation product fibronectin, more preferably a
deamidation product of FN-I.sub.5, FN-II.sub.1 or FN-III.sub.9
module of fibronectin, or a fragment or derivative thereof.
[0042] Preferably the fragment comprises at least 5, 10, 15, 20,
25, 30, 35, 40, 45, 50, or 100 consecutive amino acids of the
extracellular matrix protein or module.
[0043] Preferably the peptide of the present invention selectively
inhibits .alpha..sub.v.beta..sub.3 integrin and/or inhibits
endothelial cell adhesion to vitronectin. Preferably the peptide
inhibits .alpha..sub.v.beta..sub.3 but does not inhibit
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1 and
.alpha..sub.1.beta..sub.1.
[0044] Preferably the IC.sub.50 of the peptide in respect of
.alpha..sub.v.beta..sub.3 inhibition is <0.5 .mu.M, preferably
<0.2 .mu.M. Preferably the assay used to determine the IC.sub.50
is determined using streptavidin-peroxidase conjugate complexes as
described in the materials and methods. Preferably the IC.sub.50 of
the peptide in respect of .alpha..sub.v.beta..sub.5 and/or
.alpha..sub.5.beta..sub.1 and/or .alpha..sub.1.beta..sub.1 is
>1.0 .mu.M, more preferably >2.0 .mu.M, >3.0 .mu.M,
>4.0 .mu.M.
[0045] Preferably the peptide of the present invention inhibits
angiogenisis.
[0046] In one embodiment the peptide of the present invention does
not comprise an additional therapeutic agent, including an
anticancer agent or cytokine.
[0047] Preferably the peptide of the present invention comprises a
turn involving the G and R residues of the DGR motif.
[0048] In one embodiment the peptide of the present invention
comprises up to 350, 100, 50, 25, 15, 10 or 5 amino acids.
[0049] According to another aspect of the present invention there
is provided a conjugation product comprising a peptide of the
present invention. Preferably the conjugation product is between a
peptide of the present invention and a drug, cytokine, cytokine
fragment toxin, apoptotic peptide, biological response modifier
radionuclide, viral particle, gene or an imaging compound. In one
embodiment the drug is an anticancer agent such as doxorubicin,
melphalan, cis-platin, gemcitabine or taxol.
[0050] According to another aspect of the present invention there
is provided a pharmaceutical composition comprising a
pharmaceutically effective amount of a peptide or conjugation
product of the present invention, preferably comprising a
pharmaceutically acceptable carrier, diluent or excipient.
[0051] Preferably the composition comprises essentially no peptide
or conjugate having an NGR motif. In particular, when the
composition comprises a deamidated peptide, it preferably comprises
essentially no precursor peptide i.e. essentially no starting
material. Preferably the peptide or conjugate of the composition
comprises less than 40, 30, 20, 15, 10, 5, 3 or 1% starting
material.
[0052] Preferably compositions of the present invention comprising
peptides and conjugates which contain DGR motifs are substantially
free of the corresponding peptides and conjugates which contain the
corresponding NGR motif. Preferably the proportion of DGR
containing peptide relative to the total peptide (i.e., DGR and NGR
containing peptide) is more than 60%, more preferably more than
70%, more preferably more than 80%, more preferably more than 85%,
more preferably more than 90%, more preferably more than 95%, more
preferably more than 97%, more preferably more than 99% w/w.
[0053] The composition of the present invention may be in the form
of an injectable solution or suspension or a liquid for
infusions.
[0054] The composition of the present invention may be in the form
of liposomes.
[0055] According to another aspect of the present invention there
is provided a method of treating or diagnosing a patient suffering
from disorders involving .alpha..sub.v.beta..sub.3 such as but not
limited to osteoporosis, arthritis, diabetic retinopathy, macular
degeneration, restenosis or hemangioma comprising administering the
peptide, conjugation product or pharmaceutical composition of the
invention.
[0056] According to another aspect of the present invention there
is provided a method of treating or diagnosing a patient suffering
from osteoporosis, arthritis, diabetic retinopathy, macular
degeneration, restenosis or hemangioma comprising administering the
peptide, conjugation product or pharmaceutical composition of the
invention.
[0057] According to another aspect of the present invention there
is provided a method of treating or diagnosing a patient suffering
from a cancer, such as but not limited to lung, pancreas, breast,
colon, larynx or ovary cancer, comprising administering the
peptide, conjugation product or pharmaceutical composition of the
invention.
[0058] According to another aspect of the present invention there
is provided a conjugation product between a peptide of the present
invention and a cytokine.
[0059] According to another aspect of the present invention there
is provided a conjugation product between a cytokine and a
targeting moiety comprising the deamidation product of the NGR
motif.
[0060] According to another aspect of the present invention there
is provided a conjugation product between a cytokine and a
targeting moiety comprising the DGR motif, including but not
limited to CDGRCVSGCAGRC (SEQ ID No: 19), DGRAHA (SEQ ID No: 20),
GDGRG (SEQ ID No: 21), CVLDGRMEC (SEQ ID No: 22), CDGRC (SEQ ID No:
23), CDGRCG (SEQ ID No: 24), LDGRE (SEQ ID No: 25), YDGRT (SEQ ID
No: 26), LQCICTGDGRGEWKCE (SEQ ID No: 27), LQCISTGDGRGEWKCE (SEQ ID
No: 28), CICTGDGRGEWKC (SEQ ID No: 29), CISTGDGRGEWKC (SEQ ID No:
30), MRCTCVGDGRGEWTCY (SEQ ID No: 31), MRCTSVGDGRGEWTCY (SEQ ID No:
32), CTCVGDGRGEWTC (SEQ ID No: 33), or CTSVGDGRGEWTC (SEQ ID No:
34), more preferably cycloCVLDGRMEC (SEQ ID No: 22), linear CDGRC
(SEQ ID No: 23), cyclic CDGRC (SEQ ID No: 23), linear CDGRCG (SEQ
ID No: 24), or cyclic CDGRCG (SEQ ID No: 24). Preferably the DGR
motif is isoDGR motif, more preferably .sub.LisoDGR. In one
embodiment the DGR motif is .sub.DDGR.
[0061] A non-limiting list of cytokines used in the conjugate of
the present invention is TNF.alpha., TNF.beta., IFN.beta.,
IFN.beta., IFN.gamma., IL-1, 2, 4, 6, 7, 12, 15, EMAP II, vascular
endothelial growth factor (VEGF), PDGF, PD-ECGF or a chemokine.
Preferably the cytokine of the conjugate is selected from TNF,
IFN.gamma., IL-12 IP-10, IL-7 or EMAP II. More preferably the
cytokine is selected from TNF, IFN.gamma. or IL-12
[0062] Preferably the TNF is TNF.alpha. or TNF.beta..
[0063] In one embodiment the cytokine is derivatized with
polyethylene glycol or an acyl residue.
[0064] In another embodiment the cytokine is further conjugated
with a compound selected from the group consisting of an antibody,
an antibody fragment, and biotin, wherein said antibody or fragment
thereof is directed to a compound selected from the group
consisting of a tumoral antigen, a tumoral angiogenic marker or a
component of the extracellular matrix.
[0065] In another embodiment the cytokine is TNF and is conjugated
to both the targeting moiety and a compound selected from the group
consisting of an antibody, and antibody fragment, and biotin.
[0066] According to another aspect of the present invention there
is provided a pharmaceutical composition comprising an effective
amount of a conjugation product of TNF of the present invention,
and an effective amount of IFN.gamma. or a polynucleotide encoding
therefor.
[0067] Preferably the peptide or conjugate of the present invention
is other than when formed in vivo by metabolism of a peptide or
conjugate comprising an NGR motif.
[0068] According to another aspect of the present invention there
is provided a peptide or conjugate of the present invention
obtainable by heat treatment.
[0069] The composition of the present invention may further
comprise another antitumor agent, such as, but not limited to
doxorubicin or melphalan, or cis-platin or gemcitabine or taxol or
a diagnostic tumor-imaging compound.
DETAILED DESCRIPTION
[0070] Various preferred features and embodiment of the present
invention will now be described by way of non-limiting example.
[0071] The practice of the present invention will employ, unless
otherwise indicated, conventional techniques of chemistry,
molecular biology, microbiology, recombinant DNA and immunology,
which are within the capabilities of a person of ordinary skill in
the art. Such techniques are explained in the literature. See, for
example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989,
Molecular Cloning: A Laboratory Manual, Second Edition, Books 1-3,
Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995
and periodic supplements; Current Protocols in Molecular Biology,
ch. 9, 13, and 16, John Wiley & Sons, New York, N.Y.); B. Roe,
J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing:
Essential Techniques, John Wiley & Sons; J. M. Polak and James
O'D. McGee, 1990, In Situ Hybridization: Principles and Practice;
Oxford University Press; M. J. Gait (Editor), 1984, Oligonucleotide
Synthesis: A Practical Approach, Ir1 Press; and, D. M. J. Lilley
and J. E. Dahlberg, 1992, Methods of Enzymology: DNA Structure Part
A: Synthesis and Physical Analysis of DNA Methods in Enzymology,
Academic Press. Each of these general texts is herein incorporated
by reference.
DGR Motif
[0072] It is well known that aspartic acid may exist in a different
structural isomeric form, namely isoaspartic acid (see FIG.
14).
[0073] Aspartic acid and isoaspartic acid are each chiral
molecules, and the different isomers can be referred to as
.sub.L-Asp (.sub.LD), .sub.LisoAsP (.sub.LisoD), .sub.DAsp
(.sub.DD) and .sub.DisoAsp (.sub.DisoD) where .sub.LisoD and
.sub.DisoD represent the enantiomers of isoaspartic acid and
.sub.LD and .sub.DD represent the enantiomers of aspartic acid.
[0074] When the prior art refers to DGR, it in essence refers to
.sub.LDGR. As used herein, the term DGR refers to a DGR motif that
comprises .sub.DD and/or .sub.LisoD or mixtures thereof and which
may further comprise .sub.LD and .sub.DisoD. Preferably, the DGR is
generated by deamidation of the corresponding NGR motif. In one
embodiment the DGR motif comprises at least 10 w/w % .sub.LisoDGR.
In another embodiment the DGR motif comprises at least 10 w/w %
.sub.DDGR
[0075] By a peptide or targeting moiety comprising an isoDGR motif
it is meant a peptide or targeting moiety wherein the DGR motif is
substantially in the form of isoDGR. By substantially, it is meant
the w/w % of peptide or targeting moiety comprising the isoDGR
motif relative to total DGR containing peptide or targeting moiety
is greater than 55%, more preferably greater than 60% more
preferably greater than 65%, more preferably greater than 70%, more
preferably greater than 75%, more preferably greater than 80%, more
preferably greater than 85%, more preferably greater than 90%, more
preferably greater than 95%, more preferably greater than 97%, more
preferably greater than 99%. The isoDGR may comprise both
enantiomers of .sub.L/DisoD, but preferably comprises at least 5,
more preferably at least 10, more preferably at least 30, more
preferably at least 40, more preferably at least 50 wt % of
.sub.LisoD.
[0076] By a peptide or targeting moiety comprising an .sub.LisoDGR
motif it is meant a peptide or targeting moiety wherein the DGR
motif is substantially in the form of .sub.LisoDGR. By
substantially it is meant the w/w % of peptide or targeting moiety
comprising the .sub.LisoDGR motif relative to total DGR containing
peptide or targeting moiety is greater than 55%, more preferably
greater than. 60% more preferably greater than 65%, more preferably
greater than 70%, more preferably greater than 75%, more preferably
greater than 80%, more preferably greater than 85%, more preferably
greater than 90%, more preferably greater than 95%, more preferably
greater than 97%, more preferably greater than 99%.
[0077] By a peptide or targeting moiety comprising a .sub.DDGR
motif it is meant a peptide or targeting moiety wherein the DGR
motif is substantially in the form of .sub.DDGR. By substantially
it is meant the w/w % of peptide or targeting moiety comprising the
.sub.DDGR motif relative to total DGR containing peptide or
targeting moiety is greater than 55%, more preferably greater than
60% more preferably greater than 65%, more preferably greater than
70%, more preferably greater than 75%, more preferably greater than
80%, more preferably greater than 85%, more preferably greater than
90%, more preferably greater than 95%, more preferably greater than
97%, more preferably greater than 99%.
[0078] The DGR motif of the present invention preferably comprises
a turn involving the G and R residues. When the DGR motif is
arrived at by deamidation of the corresponding NGR motif, it is
preferable that the NGR motif comprises a turn involving the G and
R residues. The structure-activity relationship of linear and
cyclic peptides containing the NGR motif and their ability to
target tumors is discussed in Colombo et al., J. Biol. Chem., 2002,
49, 47891-47897. The Experiments carried out in animal models
showed that both GNGRG (SEQ ID No: 4), and CNGRC (SEQ ID No: 6),
can target TNF to tumors. Molecular dynamic simulation of cyclic
CNGRC (SEQ ID No: 6), showed the presence of a bend geometry
involving residues Gly.sup.3-Argo.sup.4, stabilised by the
formation of a disulphide bridge. Molecular dynamic simulation of
the same peptide without disulfide constraints showed that the most
populated and thermodynamically favoured configuration is
characterised by the presence of a .beta.-turn involving residues
Gly.sup.3-Arg.sup.4. These results suggest that the NGR motif has a
strong propensity to form .beta.-turn in linear peptides and may
explain the finding that GNGRG (SEQ ID No: 4), peptide can target
TNF to tumors, albeit to a lower extent than CNGRC (SEQ ID No:
6),
[0079] Three-dimensional structure analysis of the fibronectin
domain FN-I.sub.5 showed that the GNGRG (SEQ ID No: 4), motif forms
an exposed loop, likely accessible to water and to receptors (see
FIG. 1B). The molecular dynamic simulation of an NGR-peptide with
flanking cysteines (CNGRC (SEQ ID No: 6)), used in the present
application as a molecular surrogate of the fibronectin NGR motif,
showed that its structure is superimposable to that of the FN loop
(Di Matteo et al., 2002).
Deamidation
[0080] Deamidation of asparagine residues results in the conversion
to isoaspartate and aspartate. The process is shown in FIG. 14.
[0081] The deamidation referred to herein may be carried under any
suitable conditions readily identified by a person of skill in the
art. The deamidation of the present invention is carried out in
vitro.
[0082] In one embodiment, the deamidation is carried out by heating
the NGR comprising peptide. The temperature required may vary
depending on the exact nature of the peptide and are readily
determinable by one skilled in the art. Preferably the temperature
is sufficient to induce deamidation of the asparagine residue, but
is not so high as to denature or damage the peptide. A suitable
temperatures may be, for example, between 25-50, 30-40 or
35-40.degree. C.
[0083] The deamidation may also be performed by incubating the
peptide in basic conditions, usually about pH 8 e.g., by incubating
in an ammonium bicarbonate solution. The exact nature of the basic
conditions may vary depending on the exact nature of the peptide
but are readily determinable by one skilled in the art. Preferably
the basic conditions are sufficient to induce deamidation of the
asparagine residue, but are not such that the remaining peptide is
denatured or damaged. Suitable basic conditions may be, for
example, between pH 7.5-9.0, 8.0-9.0 or 8.0-8.5.
[0084] In a preferred embodiment the deamidation is carried out
under heating in a basic solvent.
Peptide
[0085] The term "peptide" as used herein includes polypeptides and
proteins. The term "polypeptide" includes single-chain polypeptide
molecules as well as multiple-polypeptide complexes where
individual constituent polypeptides are linked by covalent or
non-covalent means. The term "polypeptide" includes peptides of two
or more amino acids in length, typically having more than 5, 10,
20, 30, 40, 50 or 100, amino acids.
[0086] Peptides of the present invention may be administered
therapeutically to patients. It is preferred to use peptides that
do not consisting solely of naturally-occurring amino acids but
which have been modified, for example to reduce immunogenicity, to
increase circulatory half-life in the body of the patient, to
enhance bioavailability and/or to enhance efficacy and/or
specificity.
[0087] A number of approaches have been used to modify peptides for
therapeutic application. One approach is to link the peptides or
proteins to a variety of polymers, such as polyethylene glycol
(PEG) and polypropylene glycol (PPG)--see for example U.S. Pat.
Nos. 5,091,176, 5,214,131 and U.S. Pat. No. 5,264,209.
[0088] Replacement of naturally-occurring amino acids with a
variety of uncoded or modified amino acids such as D-amino acids
and N-methyl amino acids may also be used to modify peptides
[0089] Another approach is to use bifunctional crosslinkers, such
as N-succinimidyl 3-(2 pyridyldithio) propionate, succinimidyl
6-[3-(2 pyridyldithio) propionamido]hexanoate, and
sulfosuccinimidyl 6-[3-(2 pyridyldithio) propionamido]hexanoate
(see U.S. Pat. No. 5,580,853).
[0090] It may be desirable to use derivatives of the peptides of
the invention which are conformationally constrained.
Conformational constraint refers to the stability and preferred
conformation of the three-dimensional shape assumed by a peptide.
Conformational constraints include local constraints, involving
restricting the conformational mobility of a single residue in a
peptide; regional constraints, involving restricting the
conformational mobility of a group of residues, which residues may
form some secondary structural unit; and global constraints,
involving the entire peptide structure.
[0091] The active conformation of the peptide may be stabilised by
a covalent modification, such as cyclization or by incorporation of
gamma-lactam or other types of bridges. For example, side chains
can be cyclized to the backbone so as create a L-gamma-lactam
moiety on each side of the interaction site. See, generally, Hruby
et al., "Applications of Synthetic Peptides," in Synthetic
Peptides: A User's Guide: 259-345 (W.H. Freeman & Co. 1992).
Cyclization also can be achieved, for example, by formation of
cysteine bridges, coupling of amino and carboxy terminal groups of
respective terminal amino acids, or coupling of the amino group of
a Lys residue or a related homolog with a carboxy group of Asp, Glu
or a related homolog. Coupling of the .alpha-amino group of a
polypeptide with the epsilon-amino group of a lysine residue, using
iodoacetic anhydride, can be also undertaken. See Wood and Wetzel,
1992, Int'l J. Peptide Protein Res. 39: 533-39.
[0092] Another approach described in U.S. Pat. No. 5,891,418 is to
include a metal-ion complexing backbone in the peptide structure.
Typically, the preferred metal-peptide backbone is based on the
requisite number of particular coordinating groups required by the
coordination sphere of a given complexing metal ion. In general,
most of the metal ions that may prove useful have a coordination
number of four to six. The nature of the coordinating groups in the
peptide chain includes nitrogen atoms with amine, amide, imidazole,
or guanidino functionalities; sulfur atoms of thiols or disulfides;
and oxygen atoms of hydroxy, phenolic, carbonyl, or carboxyl
functionalities. In addition, the peptide chain or individual amino
acids can be chemically altered to include a coordinating group,
such as for example oxime, hydrazino, sulfhydryl, phosphate, cyano,
pyridino, piperidino, or morpholino. The peptide construct can be
either linear or cyclic, however a linear construct is typically
preferred. One example of a small linear peptide is Gly-Gly-Gly-Gly
(SEQ ID No: 35) which has four nitrogens (an N.sub.4 complexation
system) in the back bone that can complex to a metal ion with a
coordination number of four.
[0093] A further technique for improving the properties of
therapeutic peptides is to use non-peptide peptidomimetics. A wide
variety of useful techniques may be used to elucidating the precise
structure of a peptide. These techniques include amino acid
sequencing, x-ray crystallography, mass spectroscopy, nuclear
magnetic resonance spectroscopy, computer-assisted molecular
modelling, peptide mapping, and combinations thereof. Structural
analysis of a peptide generally provides a large body of data which
comprise the amino acid sequence of the peptide as well as the
three-dimensional positioning of its atomic components. From this
information, non-peptide peptidomimetics may be designed that have
the required chemical functionalities for therapeutic activity but
are more stable, for example less susceptible to biological
degradation. An example of this approach is provided in U.S. Pat.
No. 5,811,512.
[0094] Techniques for chemically synthesising therapeutic peptides
of the invention are described in the above references and also
reviewed by Borgia and Fields, 2000, TibTech 18: 243-251 and
described in detail in the references contained therein.
Peptide Variants, Derivatives and Fragments
[0095] The terms "variant" or "derivative" in relation to the amino
acid sequences of the present invention includes any substitution
of, variation of, modification oi; replacement of, deletion of or
addition of one (or more) amino acids from or to the sequence
providing the resultant amino acid sequence preferably has
targeting activity, preferably having at least 25 to 50% of the
activity as the polypeptides presented in the sequence listings,
more preferably at least substantially the same activity.
[0096] Thus, sequences may be modified for use in the present
invention. Typically, modifications are made that maintain the
activity of the sequence. Thus, in one embodiment, amino acid
substitutions may be made, for example from 1, 2 or 3 to 10, 20 or
30 substitutions provided that the modified sequence retains at
least about 25 to 50% of, or substantially the same activity.
However, in an alternative embodiment, modifications to the amino
acid sequences of a polypeptide of the invention may be made
intentionally to reduce the biological activity of the polypeptide.
For example truncated polypeptides that remain capable of binding
to target molecule but lack functional effector domains may be
useful.
[0097] In general, preferably less than 20%, 10% or 5% of the amino
acid residues of a variant or derivative are altered as compared
with the corresponding region depicted in the sequence
listings.
[0098] Amino acid substitutions may include the use of
non-naturally occurring analogues, for example to increase blood
plasma half-life of a therapeutically administered polypeptide (see
below for further details on the production of peptide derivatives
for use in therapy).
[0099] Conservative substitutions may be made, for example
according to the Table below. Amino acids in the same block in the
second column and preferably in the same line in the third column
may be substituted for each other:
TABLE-US-00001 ALIPHATIC Non-polar GAP ILV Polar - uncharged CSTM
NQ Polar - charged DE KRH AROMATIC FWY
[0100] Polypeptides of the invention also include fragments of the
above mentioned polypeptides and variants thereof; including
fragments of the sequences. Preferred fragments include those which
include an epitope or binding domain. Suitable fragments will be at
least about 5, e.g. 10, 12, 15 or 20 amino acids in length. They
may also be less than 200, 100 or 50 amino acids in length.
Polypeptide fragments of the proteins and allelic and species
variants thereof may contain one or more (e.g. 2, 3, 5, or 10)
substitutions, deletions or insertions, including conserved
substitutions. Where substitutions, deletion and/or insertions have
been made, for example by means of recombinant technology,
preferably less than 20%, 10% or 5% of the amino acid residues
depicted in the sequence listings are altered.
[0101] Polypeptides and conjugates of the invention are typically
made by recombinant means, for example as described below. However
they may also be made by synthetic means using techniques well
known to skilled persons such as solid phase synthesis. Various
techniques for chemical synthesising peptides are reviewed by
Borgia and Fields, 2000, TibTech 18: 243-251 and described in
detail in the references contained therein.
Polynucleotides
[0102] Polynucleotides for use in the invention comprise nucleic
acid sequences encoding peptides and conjugates of the invention.
In particular, the polynucleotides may encode precursor peptides or
conjugates comprising the NGR motif which, upon deamidmion, renders
peptides and conjugates, comprising the corresponding DGR motif
[0103] It will be understood by a skilled person that numerous
different polynucleotides can encode the same polypeptide as a
result of the degeneracy of the genetic code. In addition, it is to
be understood that skilled persons may, using routine techniques,
make nucleotide substitutions that do not affect the polypeptide
sequence encoded by the polynucleotides of the invention to reflect
the codon usage of any particular host organism in which the
polypeptides of the invention are to be expressed.
[0104] Polynucleotides of the invention may comprise DNA or RNA.
They may be single-stranded or double-stranded. They may also be
polynucleotides which include within them synthetic or modified
nucleotides. A number of different types of modification to
oligonucleotides are known in the art. These include
methylphosphonate and phosphorothioate backbones, addition of
acridine or polylysine chains at the 3' and/or 5' ends of the
molecule. For the purposes of the present invention, it is to be
understood that the polynucleotides described herein may be
modified by any method available in the field of the invention.
[0105] Such modifications may be carried out in order to enhance
the in vivo activity or life span of polynucleotides.
[0106] Polynucleotides of the invention can be incorporated into a
recombinant replicable vector. The vector may be used to replicate
the nucleic acid in a compatible host cell. Thus in a further
embodiment, the invention provides a method of making
polynucleotides of the invention by introducing a polynucleotide of
the invention into a replicable vector, introducing the vector into
a compatible host cell, and growing the host cell under conditions
which bring about replication of the vector. The vector may be
recovered from the host cell. Suitable host cells include bacteria
such as E. coli, yeast, mammalian cell lines and other eukaryotic
cell lines, for example insect Sf9 cells.
[0107] Preferably, a polynucleotide of the invention in a vector is
operably linked to a control sequence that is capable of providing
for the expression of the coding sequence by the host cell, i.e.
the vector is an expression vector. The term "operably linked"
means that the components described are in a relationship
permitting them to function in their intended manner. A regulatory
sequence "operably linked" to a coding sequence is ligated in such
a way that expression of the coding sequence is achieved under
condition compatible with the control sequences.
[0108] The control sequences may be modified, for example by the
addition of further transcriptional regulatory elements to make the
level of transcription directed by the control sequences more
responsive to transcriptional modulators.
[0109] Vectors of the invention may be transformed or transfected
into a suitable host cell as described below to provide for
expression of a protein of the invention. This process may comprise
culturing a host cell transformed with an expression vector as
described above under conditions to provide for expression by the
vector of a coding sequence encoding the protein, and optionally
recovering the expressed protein.
[0110] The vectors may be for example, plasmid or virus vectors
provided with an origin of replication, optionally a promoter for
the expression of the said polynucleotide and optionally a
regulator of the promoter. The vectors may contain one or more
selectable marker genes, for example an ampicillin resistance gene
in the case of a bacterial plasmid or a neomycin resistance gene
for a mammalian vector. Vectors may be used, for example, to
transfect or transform a host cell.
[0111] Control sequences operably linked to sequences encoding the
protein of the invention include promoters/enhancers and other
expression regulation signals. These control sequences may be
selected to be compatible with the host cell for which the
expression vector is designed to be used in. The term "promoter" is
well-known in the art and encompasses nucleic acid regions ranging
in size and complexity from minimal promoters to promoters
including upstream elements and enhancers.
[0112] The promoter is typically selected from promoters which are
functional in mammalian cells, although prokaryotic promoters and
promoters functional in other eukaryotic cells may be used. The
promoter is typically derived from promoter sequences of viral or
eukaryotic genes. For example, it may be a promoter derived from
the genome of a cell in which expression is to occur. With respect
to eukaryotic promoters, they may be promoters that function in a
ubiquitous manner (such as promoters of a-actin, b-actin, tubulin)
or, alternatively, a tissue-specific manner (such as promoters of
the genes for pyruvate kinase). Tissue-specific promoters specific
for certain cells may also be used. They may also be promoters that
respond to specific stimuli, for example promoters that bind
steroid hormone receptors. Viral promoters may also be used, for
example the Moloney murine leukaemia virus long terminal repeat
(MMLV LTR) promoter, the rows sarcoma virus (RSV) LTR promoter or
the human cytomegalovirus (CMV) IE promoter.
[0113] It may also be advantageous for the promoters to be
inducible so that the levels of expression of the heterologous gene
can be regulated during the life-time of the cell. Inducible means
that the levels of expression obtained using the promoter can be
regulated.
[0114] In addition, any of these promoters may be modified by the
addition of further regulatory sequences, for example enhancer
sequences. Chimeric promoters may also be used comprising sequence
elements from two or more different promoters described above.
[0115] Vectors and polynucleotides of the invention may be
introduced into host cells for the purpose of replicating the
vectors/polynucleotides and/or expressing the proteins of the
invention encoded by the polynucleotides of the invention. Although
the proteins of the invention may be produced using prokaryotic
cells as host cells, it is preferred to use eukaryotic cells, for
example yeast, insect or mammalian cells, in particular mammalian
cells.
[0116] Vectors/polynucleotides of the invention may introduced into
suitable host cells using a variety of techniques known in the art,
such as transfection, transformation and electroporation. Where
vectors/polynucleotides of the invention are to be administered to
animals, several techniques are known in the art, for example
infection with recombinant viral vectors such as retroviruses,
herpes simplex viruses and adenoviruses, direct injection of
nucleic acids and biolistic transformation. Host cells comprising
polynucleotides of the invention may be used to express conjugates
of the invention. Host cells may be cultured under suitable
conditions which allow expression of the polypeptides and
conjugates of the invention. Expression of the products of the
invention may be constitutive such that they are continually
produced, or inducible, requiring a stimulus to initiate
expression. In the case of inducible expression, protein production
can be initiated when required by, for example, addition of an
inducer substance to the culture medium, for example dexamethasone
or IPTG.
Conjugates
[0117] The present invention relates to a conjugate which is a
molecule comprising a peptide or targeting moiety of the invention
linked to at least one other agent, including, but not limited to,
a drug, a cytokine, a toxin, an apoptotic peptide, a radionuclide,
a viral particle, a gene or an imaging compound, formed through
genetic fusion or chemical coupling. A non-limiting list of
cytokines used in the conjugate of the present invention is
TNF.alpha., TNF.beta., IFN.alpha., IFN.beta., IFN.gamma., IL-1, 2,
4, 6, 7, 12, 15, EMAP II, vascular endothelial growth factor
(VEGF), PDGF, PD-ECGF or a chemokine.
[0118] Conjugates include fusion proteins in which the peptide or
targeting moiety is linked to an agent via its polypeptide backbone
through genetic expression of a DNA molecule encoding these
proteins, directly synthesised proteins and coupled proteins in
which pre-formed sequences are associated by a cross-linking agent.
The term is also used herein to include associations, such as
aggregates.
[0119] The peptide or targeting moiety can be coupled directly to
the agent or indirectly through a spacer, which can be a single
amino acid (e.g., G (glycine)), an amino acid sequence or an
organic residue, such as 6-aminocapryl-N-hydroxysuccinimide.
[0120] In one embodiment the peptide or targeting moiety is linked
to the cytokine N-terminus or C-terminus thus minimising any
interference in the binding of the modified cytokine to its
receptor. Alternatively, the peptide or targeting moiety can be
linked to amino acid residues which are amido- or carboxylic-bond
acceptors, which may be naturally occurring on the molecule or
artificially inserted using genetic engineering techniques.
[0121] In one embodiment, the peptide ligand preferably is linked
to the cytokine N-terminus or C-terminus thus minimizing any
interference in the binding of the modified cytokine to its
receptor. Alternatively, the peptide can be linked to amino acid
residues which are amido- or carboxylic-bonds acceptors, naturally
occurring on the molecule or artificially inserted with genetic
engineering techniques. The modified cytokine is preferably
prepared by use of a cDNA comprising a 5'-contiguous sequence
encoding the peptide.
[0122] According to a preferred embodiment, there is provided a
conjugation product between TNF and the CDGRC (SEQ ID No: 23),
CisoDGRC (SEQ ID No: 59), C.sub.LisoDGRC (SEQ ID No: 59), or
C.sub.DDGRC peptide. Preferably the amino-terminal of TNF is linked
to the peptide, preferably through a spacer. Preferably the spacer
is G (glycine).
[0123] According to another preferred embodiment, there is provided
a conjugation product between IFN.gamma. and the CDGRC (SEQ ID No:
23), CisoDGRC (SEQ ID No: 59), C.sub.LisoDGRC (SEQ ID No: 59), or
C.sub.DDGRC peptide. Preferably the amino-terminal of TNF is linked
to the peptide, preferably through a spacer. Preferably the spacer
is G (glycine).
[0124] According to another preferred embodiment, there is provided
a conjugation product between IL-12 and the CDGRC (SEQ ID No: 23),
CisoDGRC (SEQ ID No: 59), C.sub.LisoDGRC (SEQ ID No: 59), or
C.sub.DDGRC peptide.
[0125] Preferably the amino-terminal of TNF is linked to the
peptide, preferably through a spacer. Preferably the spacer is G
(glycine).
[0126] The therapeutic index of the TNF/DGR conjugates can be
further improved by using a mutant form of TNF capable of
selectively binding to one of the two TNF receptors, p75TNFR and
p55TNFR. Said TNF mutant can be obtained by site-directed
mutagenesis (Loetscher et al., 1993, Van Ostade et al., 1993).
[0127] The pharmacokinetic of the modified cytokines according to
the invention can be improved by preparing polyethylene glycol
derivatives, which allow to extend the plasmatic half-life of the
cytokines themselves.
Bifunctional Derivatives
[0128] A further embodiment of the invention is provided by
bifunctional derivatives in which peptides or conjugates of the
invention are conjugated with antibodies, or their fragments,
against tumoral antigens or other tumor angiogenic markers, e.g.
.alpha.v integrins, metalloproteases or the vascular growth factor,
or antibodies or fragments thereof directed against components of
the extracellular matrix, such as anti-tenascin antibodies or
anti-fibronectin EDB domain. The preparation of a fusion product
between TNF and the hinge region of a mAb against the
tumor-associated TAG72 antigen expressed by gastric and ovarian
adenocarcinoma has recently been reported (Yang et al., 1995).
[0129] A further embodiment of the invention is provided by the
tumoral pre-targeting with the biotin/avidin system. According to
this approach, a ternary complex is obtained on the tumoral
antigenic site, at different stages, which is formed by 1)
biotinylated mAb, 2) avidin (or streptavidin) and 3) a peptide or
conjugate of the invention and biotin. A number of papers proved
that the pre-targeting approach, compared with conventional
targeting with immunoconjugates, can actually increase the ratio of
active molecule homed at the target to free active molecule, thus
reducing the treatment toxicity (Colombo et al., 1993; Modorati et
al., 1994; Paganelli et al., 1995, 1991). This approach produced
favorable results with biotinylated TNF, which was capable of
inducing cytotoxicity in vitro and decreasing the tumor cells
growth under conditions in which normal TNF was inactive (Moro et
al., 1997; Gasparri et al., 1999). The pre-targeting approach can
also be carried out with a two-phase procedure by using a
bispecific antibody which at the same time binds the tumoral
antigen and the modified cytokine. The use of a bispecific antibody
directed against a carcinoembryonic antigen and TNF has recently
been described as a means for TNF tumoral pre-targeting (Robert et
al., 1996).
[0130] According to a further embodiment, the invention comprises a
TNF molecule conjugated to both a peptide of the invention (e.g.,
peptide comprising DGR) and an antibody, or a fragment thereof
(directly or indirectly via a biotin-avidin bridge), on different
TNF subunits, where the antibody or its fragments are directed
against an antigen expressed on tumor cells or other components of
the tumor stroma, e.g. tenascin and fibronectin EDB domain. This
results in a further improvement of the tumor homing properties of
the modified cytokine and in the slow release of the latter in the
tumor microenvironment through trimer-monomer-trimer transitions.
As shown in previous works, in fact, the modified subunits of TNF
conjugates can dissociate from the targeting complexes and
reassociate so as to form unmodified trimeric TNF molecules, which
then diffuse in the tumor microenvironment. The release of
bioactive TNF has been shown to occur within 24-48 hours after
targeting (Corti et al., 1998).
Extracellular Matrix
[0131] The term "extracellular matrix component" (ECM), as used
herein, means a molecule that occupies the extracellular spaces of
tissues.
[0132] In one embodiment the ECM component is selected from
fibronectin, vitronectin, collagen or laminin. Preferred ECM
components are the FN-I.sub.5, FN-I.sub.7, FN-II.sub.1 or
FN-III.sub.9 modules of fibronectin, or fragments thereof.
Preferably the fragments comprise the NGR motif, or, following
deamidation, the DGR motif.
[0133] Preferred components of the ECM according to the present
invention are those which show modified properties, such as
increased endothelial cell adhesion and/or increased integrin
(e.g., .alpha..sub.v.beta..sub.3) binding following deamidation of
asparagine residues. Particularly preferred ECM components are
those that comprise an NGR motif that undergoes deamidation to DGR,
preferably .sub.LisoDGR or .sub.DDGR.
Cytokines
[0134] Drug penetration into neoplastic cells is critical for the
effectiveness of solid-tumor chemotherapy. To reach cancer cells in
solid tumors, chemotherapeutic drugs must enter the drug blood
vessels, cross the vessel wall and finally migrate through the
interstitium. Heterogeneous tumor perfusion, vascular permeability
and cell density, and increased interstitial pressure may represent
critical barriers that may limit the penetration of drugs into
neoplastic cells and, consequently, the effectiveness of
chemotherapy. Cytokines which have the effect of affecting these
factors are therefore useful in the present invention. A
non-limiting list of cytokines which may be used in the present
invention is: TNF.alpha., TNF.beta., IFN.alpha., IFN.beta.,
IFN.gamma., IL-1, 2, 4, 6, 7, 12, 15, IP-10, EMAP II, vascular
endothelial growth factor (VEGF), PDGF, PD-ECGF or a chemokine.
TNF
[0135] TNF acts as an inflammatory cytokine and has the effect of
inducing alteration of the endothelial barrier function, reducing
of tumor interstitial pressure, and increasing chemotherapeutic
drug penetration and tumor vessel damage.
[0136] The first suggestion that a tumor necrotizing molecule
existed was made when it was observed that cancer patients
occasionally showed spontaneous regression of their tumors
following bacterial infections. Subsequent studies in the 1960s
indicated that host-associated (or endogenous) mediators,
manufactured in response to bacterial products, were likely
responsible for the observed effects. In 1975 it was shown that a
bacterially-induced circulating factor had strong anti-tumor
activity against tumors implanted in the skin in mice. This factor,
designated tumor necrosis factor (TNF), was subsequently isolated,
cloned, and found to be the prototype of a family of molecules that
are involved with immune regulation and inflammation. The receptors
for TNF and the other members of the TNF superfamily also
constitute a superfamily of related proteins.
[0137] TNF-related ligands usually share a number of common
features. These features do not include a high degree of overall
amino acid (aa) sequence homology. With the exception of nerve
growth factor (NGF) and TNF-beta, all ligands are synthesised as
type II transmembrane proteins (extracellular C-terminus) that
contain a short cytoplasmic segment (10-80 as residues) and a
relatively long extracellular region (140-215 as residues). NGF,
which is structurally unrelated to TNF, is included in this
superfamily only because of its ability to bind to the TNFRSF low
affinity NGF receptor (LNGFR). NGF has a classic signal sequence
peptide and is secreted. TNF-.beta., in contrast, although also
fully secreted, has a primary structure much more related to type
II transmembrane proteins. TNF-.beta. might be considered as a type
H protein with a non-functional, or inefficient, transmembrane
segment. In general, TNFSF members form trimeric structures, and
their monomers are composed of beta strands that orient themselves
into a two sheet structure. As a consequence of the trimeric
structure of these molecules, it is suggested that the ligands and
receptors of the TNSF and TNFRSF superfamilies undergo "clustering"
during signal transduction.
[0138] TNF-.alpha.: Human TNF-.alpha. is a 233 aa residue,
nonglycosylated polypeptide that exists as either a transmembrane
or soluble protein. When expressed as a 26 kDa membrane bound
protein, TNF-.alpha. consists of a 29 aa residue cytoplasmic
domain, a 28 aa residue transmembrane segment, and a 176 aa residue
extracellular region. The soluble protein is created by a
proteolytic cleavage event via an 85 kDa TNF-alpha converting
enzyme (TACE), which generates a 17 kDa, 157 as residue molecule
that normally circulates as a homotrimer.
[0139] TNF-.beta./LT-.alpha.: TNF-.beta., otherwise known as
lymphotoxin-.alpha. (LT-.alpha.) is a molecule whose cloning was
contemporary with that of TNF-.alpha.. Although TNF-.beta.
circulates as a 171 aa residue, 25 kDa glycosylated polypeptide, a
larger form has been found that is 194 aa residues long. The human
TNF-.beta. cDNA codes for an open reading frame of 205 aa residues
(202 in the mouse), and presumably some type of proteolytic
processing occurs during secretion. As with TNF-.alpha.,
circulating TNF-.beta. exists as a non-covalently linked trimer and
is known to bind to the same receptors as TNF-.alpha..
[0140] In one embodiment the TNF is a mutant form of TNF capable of
selectively binding to one of the TNF receptors (Loetscher H et al
(1993) J Biol Chem 268:26350-7; Van Ostade X et al (1993) Nature
361:266-9).
[0141] The maximum tolerated dose of bolus TNF in humans is 218-410
.mu.g/m.sup.2 (Fraker et al., 1995) about 10-fold lower than the
effective dose in animals. Based on data from murine models it is
believed that an at least 10 times higher dose is necessary to
achieve anti-tumor effects in humans (Schraffordt Koops et al.,
1998). In the first clinical study on hyperthermic isolated-limb
perfusion, high response rates were obtained with' the unique dose
of 4 mg of TNF in combination with melphalan and interferon .gamma.
(Lienard et al., 1992). Other works showed that interferon .gamma.
can be omitted and that even lower doses of TNF can be sufficient
to induce a therapeutic response (Hill et al., 1993; Eggermont et
al., 1996). As the two cytokines exert synergistic effects on
endothelial cells, their combined, selective targeting thereon is
likely to result in stronger anti-tumor activity thus allowing to
overcome the problems of systemic toxicity usually encountered in
cancer therapy with the same cytokines used in combination.
Furthermore, it is known that TNF can decrease the barrier function
of the endothelial lining vessels, thus increasing their
permeability to macromolecules. Taking advantage. of the lower
toxicity of treatment with the modified TNF molecules according to
the invention, and of their tumor vessels homing properties, an
alternative application is their use to increase the permeability
of tumor vessels to other compounds, either for therapeutic or
diagnostic purposes. For instance the modified TNF can be used to
increase the tumor uptake of radiolabelled antibodies or hormones
(tumor-imaging compounds) in radioimmunoscintigraphy or
radioimmunotherapy of tumors. Alternatively, the uptake of
chemotherapeutic drugs, immunotoxins, liposomes carrying drugs or
genes, or other anticancer drugs could also be increased, so that
their antitumor effects are enhanced.
[0142] Many other inflammatory cytokines also have the property of
increasing endothelial vessel permeability, and it will be
appreciated that the invention can be applied to such cytokines,
together with agents which increase expression of such cytokines.
Inflammatory cytokines, also known as pro-inflammatory cytokines,
are a number of polypeptides and glycoproteins with molecular
weights between SkDa and 70 kDa. They have a stimulating effect on
the inflammatory response. The most important inflammatory
cytokines are TNF, IL-1, IL-6 and IL-8.
[0143] A Table of some cytokines classified as inflammatory
cytokines is shown below:
TABLE-US-00002 Inflammatory Cytokines Group Individual cytokines
Endogenous cytokines IL-1, TNF-.alpha., IL-6 Up-regulation IL-1,
TNF-.alpha., IL-6, IFN-.alpha., INF-.beta., chemokines Stimulation
of the production IL-1, IL-6, IL-11, TNF-.alpha., INF-.gamma., of
acute phase reactants TGF-.beta., LIF, OSM, CNTF Chemoattractant
cytokines CXC chemokines IL-8, PF-4, PBP, NAP-2, .beta.-TG CC
chemokines MIP-1.alpha., MIP-1.beta., MCP-1, MCP-2, MCP-3, RANTES C
chemokines Lymphotactin Stimulation of inflammatory IL-12
cytokines
[0144] TGF-.beta.: transforming growth factor, LIF: leukemia
inhibitory factor, OSM: oncostatin M; CNTF: ciliary neurotrophic
factor; PF-4: platelet factor 4; PBP: platelet basic protein;
NAP-2: neutrophil activating protein 2; .beta.-TG:
.beta.-thromboglobulin; MIP: macrophage inflammatory protein; MCP:
monocyte chemoattractant protein.
[0145] The up-regulation of inflammatory response is also performed
by IL-11, IFN-.alpha., IFN-.beta., and especially by the members of
the chemokine superfamily. TGF-.beta. in some situations has a
number of inflammatory activities including chemoattractant effects
on neutrophils, T lymphocytes and inactivated monocytes.
IFN-.gamma.
[0146] A large body of evidence suggests that interferon-.gamma.
(IFN.gamma.), a pleiotropic cytokine mainly produced by
T-lymphocytes and natural killer cells (Farrar, et al., 1993; Boehm
et al., 1997) can promote anti-tumor responses (Curnis et al.,
2005). For instance, IFN.gamma. can induce anti-proliferative and
pro-apoptotic effects on many tumor cell types, can inhibit tumor
angiogenesis and activate natural killer cells and macrophages to
kill a variety of tumor cell targets. IFN.gamma. is also an
important regulator of CD4.sup.+T helper cells, is the major
physiological macrophage-activating factor and can augment the
expression of MMC-I and I1 on cancer and endothelial cells. Within
tumor stroma IFN.gamma. can induce cytokine and chemokine
secretion, including IP-10 (IFN-inducible Protein 10), an
angiostatic protein and a chemoattractant factor for lymphocytes
and monocytes. Evidence has been obtained to suggest that
IFN.gamma. produced by tumor-infiltrating macrophages plays a role
in tumor blood vessel destruction. Combined treatment of
endothelial cells with IFN.gamma. and tumor necrosis factor-.alpha.
(TNF) results in synergistic cytotoxic effects, likely important
for tumor vasculature destruction. IFN.gamma. can also increase the
production of TNF by activated macrophages, as well as the
expression of TNF-receptors in various cell types. As a consequence
of these effects on tumor vasculature and on cells of the immune
system IFN.gamma. can activate inflammatory/immune responses
against established tumors and inhibit tumor growth.
[0147] IFN.gamma. exists as a homodimer of two noncovalently bound
polypeptide subunits. The primary sequence of wildtype human
IFN.gamma. (huIFNG) was reported by Gray et al., 1982; Taya et al.,
1982; Devos et al., 1982; and Rinderknecht et at., 1984, and in EP
77670, EP 89676 and EP 110044. The 3D structure of huIFNG was
reported by Ealick et al., 1991.
IL-12
[0148] Interleukin 12 (IL-12), also referred to as natural killer
cell stimulatory factor ("NKSF") or cytotoxic lymphocyte maturation
factor ("CLMF"), is a potent immunoregulatory molecule that plays a
role in a wide range of diseases. Human IL-12 has been
characterized as a cytokine with a unique structure and pleiotropic
effects (Kobayashi, et al., 1989; Seder, et al., 1993; Ling, et
al., 1995; Podlaski, et al., 1992). IL-12 plays a critical role in
the pathology associated with several diseases involving immune and
inflammatory responses. A review of IL-12, its biological
activities, and its role in disease can be found in Gately et al.,
1998. An important role of IL-12 in vivo is its ability to induce
IFN.gamma. production by both natural killer (NK) and T cells.
[0149] Structurally, IL-12 is a heterodimeric protein comprising a
35 kDa subunit (p35) and a 40 kDa subunit (p40) which are both
linked together by a disulfide bridge (referred to as the "p70
subunit"). The heterodimeric protein is produced primarily by
antigen-presenting cells such as monocytes, macrophages and
dendritic cells. These cell types also secrete an excess of the p40
subunit relative to p70 subunit.
IL-2
[0150] Because of the central role of the IL-2/IL-2R system in
mediation of the immune and inflammatory responses, it is obvious
that monitoring and manipulation of this system has important
diagnostic and therapeutic implications. IL-2 has shown promise as
an anti-cancer drug by virtue of its ability to stimulate the
proliferation and activities of tumor-attacking LAK and TIL
(tumor-infiltrating lymphocytes) cells. However, problems with IL-2
toxicity are still of concern and merit investigation. The present
invention addresses this problem.
IL-15
[0151] Interleukin 15 (IL-15) is a novel cytokine that shares many
biological properties with, but lacks amino acid sequence homology
to, IL-2. IL-15 was originally identified in media conditioned by a
monkey kidney epithelial cell line (CVI/EBNA) based on its
mitogenic activity on the murine T cell line, CTLL-2. IL-15 was
also independently discovered as a cytokine produced by a human
adult T cell leukemia cell line (HuT-102) that stimulated T cell
proliferation and was designated IL-T. By virtue of its activity as
a stimulator of T cells, NK cells, LAK cells, and TILs, IL-2 is
currently in clinical trials testing its potential use in
treatments for cancer and for viral infections. Because of its
similar biological activities, IL-15 should have similar
therapeutic potential.
Chemokines
[0152] Chemokines are a superfamily of mostly small, secreted
proteins that function in leukocyte trafficking, recruiting, and
recirculation. They also play a critical role in many
pathophysiological processes such as allergic responses, infectious
and autoimmune diseases, angiogenesis, inflammation, tumor growth,
and hematopoietic development. Approximately 80 percent of these
proteins have from 66 to 78 amino acids (aa) in their mature foim.
The remainder are larger with additional as occurring upstream of
the protein core or as part of an extended C-terminal segment. All
chemokines signal through seven transmembrane domain G-protein
coupled receptors. There are at least seventeen known chemokine
receptors, and many of these receptors exhibit promiscuous binding
properties whereby several different chemokines can signal through
the same receptor.
[0153] Chemokines are divided into subfamilies based on conserved
aa sequence motifs. Most family members have at least four
conserved cysteine residues that form two intramolecular disulfide
bonds. The subfamilies are defined by the position of the first two
cysteine residues: [0154] The alpha subfamily, also called the CXC
chemokines, have one aa separating the first two cysteine residues.
This group can be further subdivided based on the presence or
absence of a glu-leu-arg (ELR) aa motif immediately preceding the
first cysteine residue. There are currently five CXC-specific
receptors and they are designated CXCR1 to CXCR5. The ELR
chemokines bind to CXCR2 and generally act as neutrophil
chemoattractants and activators. The ELR chemokines bind CXCR3 to
-5 and act primarily on lymphocytes. At the time of this writing,
14 different human genes encoding CXC chemokines have been reported
in the scientific literature with some additional diversity
contributed by alternative splicing. [0155] In the beta subfamily,
also called the CC chemokines, the first two cysteines are adjacent
to one another with no intervening aa. There are currently 24
distinct human beta subfamily members. The receptors for this group
are designated CCR1 to CCR11. Target cells for different CC family
members include most types of leukocytes. [0156] There are two
known proteins with chemokine homology that fall outside of the
alpha and beta subfamilies. Lymphotactin is the lone member of the
gamma class (C chemokine) which has lost the first and third
cysteines. The lymphotactin receptor is designated XCR1.
Fractalkine, the only known member of the delta class (CX.sub.3C
chemokine), has three intervening as between the first two cysteine
residues. This molecule is unique among chemokines in that it is a
transmembrane protein with the N-terminal chemokine domain fused to
a long mucin-like stalk. The fractalkine receptor is known as
CX.sub.3CR1.
VEGF
[0157] The present invention is also applicable to Vasculature
Endothelial Growth Factor (VEGF). Angiogenesis is a process of new
blood vessel development from preexisting vasculature. It plays an
essential role in embryonic development, normal growth of tissues,
wound healing, the female reproductive cycle (i.e., ovulation,
menstruation and placental development), as well as a major role in
many diseases. Particular interest has focused on cancer, since
tumors cannot grow beyond a few millimeters in size without
developing a new blood supply. Angiogenesis is also necessary for
the spread and growth of tumor cell metastases.
[0158] One of the most important growth and survival factors for
endothelium is VEGF. VEGF induces angiogenesis and endothelial cell
proliferation and it plays an important role in regulating
vasculogenesis. VEGF is a heparin-binding glycoprotein that is
secreted as a homodimer of 45 kDa. Most types of cells, but usually
not endothelial cells themselves, secrete VEGF. Since the initially
discovered VEGF, VEGF-A, increases vascular permeability, it was
known as vascular permeability factor. In addition, VEGF causes
vasodilatation, partly through stimulation of nitric oxide synthase
in endothelial cells. VEGF can also stimulate cell migration and
inhibit apoptosis. There are several splice variants of VEGF-A. The
major ones include: 121, 165, 189 and 206 amino acids (aa), each
one comprising a specific exon addition.
EMAP II
[0159] Endothelial-Monocyte Activating Polypeptide-II (EMAP-II) is
a cytokine that is an antiangiogenic factor in tumor vascular
development, and strongly inhibits tumor growth. Recombinant human
EMAP-II is an 18.3 kDa protein containing 166 amino acid residues.
EMAP II has also been found to increase endothelial vessel
permeability.
PDGF
[0160] It has also been proposed that platelet-derived growth
factor (PDGF) antagonists might increase drug-uptake and
therapeutic effects of a broad range of anti-tumor agents in common
solid tumors. PDGF is a cytokine of 30 kDA and is released by
platelets on wounding and stimulates nearby cells to grow and
repair the wound.
PD-ECGF
[0161] As its name suggests, platelet-derived endothelial cell
growth factor (PD-ECGF) was originally isolated from platelets
based on its ability to induce mitosis in endothelial cells. Its
related protein is gliostatin.
Antibodies
[0162] As used herein, an "antibody" refers to a protein consisting
of one or more polypeptides substantially encoded by immunoglobulin
genes or fragments of immunoglobulin genes. Antibodies may exist as
intact immunoglobulins or as a number of fragments, including those
well-characterised fragments produced by digestion with various
peptidases. While various antibody fragments are defined in terms
of the digestion of an intact antibody, one of skill will
appreciate that antibody fragments may be synthesised de novo
either chemically or by utilising recombinant DNA methodology.
Thus, the term antibody, as used herein also includes antibody
fragments either produced by the modification of whole antibodies
or synthesised de novo using recombinant DNA methodologies.
Antibody fragments encompassed by the use of the term "antibodies"
include, but are not limited to, Fab, Fab', F (ab') 2, scFv, Fv,
dsFv diabody, and Fd fragments.
[0163] The invention also provides monoclonal or polyclonal
antibodies to the surface proteins. Thus, the present invention
further provides a process for the production of monoclonal or
polyclonal antibodies to polypeptides of the invention.
[0164] If polyclonal antibodies are desired, a selected mammal
(e.g., mouse, rabbit, goat, horse, etc.) is immunised with an
immunogenic polypeptide bearing an epitope(s). Serum from the
immunised animal is collected and treated according to known
procedures. If serum containing polyclonal antibodies to an epitope
contains antibodies to other antigens, the polyclonal antibodies
can be purified by immunoaffinity chromatography. Techniques for
producing and processing polyclonal antisera are known in the art.
In order that such antibodies may be made, the invention also
provides polypeptides of the invention or fragments thereof
haptenised to another polypeptide for use as immunogens in animals
or humans.
[0165] Monoclonal antibodies directed against binding cell surface
epitopes in the polypeptides can also be readily produced by one
skilled in the art The general methodology for making monoclonal
antibodies by hybridomas is well known. Immortal antibody-producing
cell lines can be created by cell fusion, and also by other
techniques such as direct transformation of B lymphocytes with
oncogenic DNA, or transfection with Epstein-Barr virus. Panels of
monoclonal antibodies produced against epitopes can be screened for
various properties; i.e., for isotype and epitope affinity.
[0166] An alternative technique involves screening phage display
libraries where, for example the phage express scFv fragments on
the surface of their coat with a large variety of complementarity
determining regions (CDRs). This technique is well known in the
art.
[0167] For the purposes of this invention, the term "antibody,"
unless specified to the contrary, includes fragments of whole
antibodies which retain their binding activity for a target
antigen. As mentioned above such fragments include Fv, F(ab') and
F(ab').sub.2 fragments, as well as single chain antibodies (scFv).
Furthermore, the antibodies and fragments thereof may be humanised
antibodies, for example as described in EP-A-239400.
Agent
[0168] As used herein, the term "agent" includes, but is not
limited to, a compound, such as a test compound, which may be
obtainable from or produced by any suitable source, whether natural
or not. The agent may be designed or obtained from a library of
compounds which may comprise peptides, as well as other compounds,
such as small organic molecules and particularly new lead
compounds. By way of example, the agent may be a natural substance,
a biological macromolecule, or an extract made from biological
materials such as bacteria, fungi, or animal (particularly
mammalian) cells or tissues, an organic or an inorganic molecule, a
synthetic test compound, a semi-synthetic test compound, a
structural or functional mimetic, a peptide, a peptidomimetics, a
derivatised test compound, a peptide cleaved from a whole protein,
or a peptides synthesised synthetically (such as, by way of
example, either using a peptide synthesizer) or by recombinant
techniques or combinations thereof, a recombinant test compound, a
natural or a non-natural test compound, a fusion protein or
equivalent thereof and mutants, derivatives or combinations
thereof.
[0169] The agent can be an amino acid sequence or a chemical
derivative thereof. The substance may even be an organic compound
or other chemical. The agent may even be a nucleotide
sequence--which may be a sense sequence or an anti-sense
sequence.
Pharmaceutical Formulations
[0170] The present invention also provides a pharmaceutical
composition for treating an individual wherein the composition
comprises a therapeutically effective amount of a peptide,
polynucleotide, conjugate and drug combinations of the present
invention.
[0171] A composition of the present invention comprising a peptide
or conjugate comprising an isoDGR motif is substantially free of
peptides or conjugates comprising other forms of DGR By
substantially free, it is meant the w/w % of isoDGR containing
peptide or conjugate (i.e., peptide or conjugate wherein the DGR
motif is all in the isoDGR form) present in the composition
relative to total DGR containing peptide or conjugate (i.e. in all
isomeric forms) present in the composition is greater than 50%,
more preferably greater than 55% more preferably greater than 60%,
more preferably greater than 65%, more preferably greater than 70%,
more preferably greater than 75%, more preferably greater than 80%,
more preferably greater than 85%, more preferably greater than 90%,
more preferably greater than 95%, more preferably greater than 97%,
more preferably greater than 99%.
[0172] A composition of the present invention comprising a peptide
or conjugate comprising an .sub.LisoDGR motif is substantially free
of peptides or conjugates comprising other forms of DGR. By
substantially free, it is meant the w/w % of the .sub.LisoDGR
containing peptide or conjugate (i.e., peptide or conjugate wherein
the DGR motif is all in the .sub.LisoDGR form) present in the
composition relative to total DGR containing peptide or conjugate
(i.e. in all isomeric forms) present in the composition is greater
than 50%, more preferably greater than 55%, more preferably greater
than 60% more preferably greater than 65%, more preferably greater
than 70%, more preferably greater than 75%, more preferably greater
than 80%, more preferably greater than 85%, more preferably greater
than 90%, more preferably greater than 95%, more preferably greater
than 97%, more preferably greater than 99%.
[0173] A composition of the present invention comprising a peptide
or conjugate comprising a .sub.DDGR motif is substantially free of
peptides or conjugates comprising other forms of DGR. By
substantially free, it is meant the w/w % of the .sub.DDGR
containing peptide or conjugate (i.e., peptide or conjugate wherein
the DGR motif is all in the .sub.DDGR form) present in the
composition relative to total DGR containing peptide or conjugate
(i.e. in all isomeric forms) present in the composition is greater
than 50%, more preferably greater than 55%, more preferably greater
than 60% more preferably greater than 65%, more preferably greater
than 70%, more preferably greater than 75%, more preferably greater
than 80%, more preferably greater than 85%, more preferably greater
than 90%, more preferably greater than 95%, more preferably greater
than 97%, more preferably greater than 99%.
[0174] The pharmaceutical composition may be for human or animal
usage. Typically, a physician will determine the actual dosage
which will be most suitable for an individual subject and it will
vary with the age, weight and response of the particular
individual.
[0175] The composition may optionally comprise a pharmaceutically
acceptable carrier, diluent, excipient or adjuvant. The choice of
pharmaceutical carrier, excipient or diluent can be selected with
regard to the intended route of administration and standard
pharmaceutical practice. The pharmaceutical compositions may
comprise as--or in addition to--the carrier, excipient or diluent
any suitable binder(s), lubricant(s), suspending agent(s), coating
agent(s), solubilising agent(s), and other carrier agents that may
aid or increase the viral entry into the target site (such as for
example a lipid delivery system). Suitable carriers and diluents
include isotonic saline solutions, for example phosphate-buffered
saline. Details of excipients may be found in The Handbook of
Pharmaceutical Excipients, 2nd Edn, Eds Wade & Weller, American
Pharmaceutical Association.
[0176] Where appropriate, the pharmaceutical compositions can be
administered by any one or more of: inhalation, in the form of a
suppository or pessary, topically in the form of a lotion,
solution, cream, ointment or dusting powder, by use of a skin
patch, orally in the form of tablets containing excipients such as
starch or lactose, or in capsules or ovules either alone or in
admixture with excipients, or in the form of elixirs, solutions or
suspensions containing flavouring or colouring agents, or they can
be injected parenterally, for example intracavernosally,
intravenously, intramuscularly or subcutaneously. For parenteral
administration, the compositions may be best used in the form of a
sterile aqueous solution which may contain other substances, for
example enough salts or monosaccharides to make the solution
isotonic with blood. For buccal or sublingual administration the
compositions may be administered in the form of tablets or lozenges
which can be formulated in a conventional manner.
[0177] Formulations for oral or parenteral administration are
preferred. Formualtions for parenteral administration comprise
injectable solutions or suspensions and liquids for infusions. For
the preparation of the parenteral forms, an effective amount of the
active ingredient will be dissolved or suspended in a sterile
carrier, optionally adding excipients such as solubilizers,
isotonicity agents, preservatives, stabilizers, emulsifiers or
dispersing agents, and it will be subsequently distributed in
sealed vials or ampoules.
[0178] The composition may be formulated such that administration
daily, weekly or monthly will provide the desired daily dosage. It
will be appreciated that the composition may be conveniently
formulated for administrated less frequently, such as every 2, 4,
6, 8, 10 or 12 hours.
[0179] Polynucleotides/vectors encoding polypeptide components may
be administered directly as a naked nucleic acid construct,
preferably further comprising flanking sequences homologous to the
host cell genome.
[0180] Uptake of naked nucleic acid constructs by mammalian cells
is enhanced by several known transfection techniques for example
those including the use of transfection agents. Example of these
agents include cationic agents (for example calcium phosphate and
DEAE-dextran) and lipofectants (for example Lipofectam.TM. and
Transfectam.TM.). Typically, nucleic acid constructs are mixed with
the transfection agent to produce a composition.
[0181] Preferably the polynucleotide or vector of the invention is
combined with a pharmaceutically acceptable carrier or diluent to
produce a pharmaceutical composition. Suitable carriers and
diluents include isotonic saline solutions, for example
phosphate-buffered saline. The composition may be formulated for
parenteral, intramuscular, intravenous, subcutaneous, intraocular
or transdermal administration.
[0182] The routes of administration and dosage regimens described
are intended only as a guide since a skilled practitioner will be
able to determine readily the optimum route of administration and
dosage regimens for any particular patient and condition.
[0183] The preparation of peptides, and in particular cytokines, in
form of liposomes can improve the biological activity thereof. It
has, in fact, been observed that acylation of the TNF amino groups
induces an increase in its hydrophobicity without loss of
biological activity in vitro. Furthermore, it has been reported
that TNF bound to lipids has unaffected cytotoxicity in vitro,
immunomodulating effects and reduced toxicity in vivo (Deb et al.,
1989, 1990).
[0184] Preferably compositions of the present invention comprising
peptides and conjugates which contain DGR motifs according to the
present invention are substantially free of the corresponding
peptides and conjugates which contain the corresponding NGR motif
Preferably the proportion of DGR containing peptide relative to the
total peptide (i.e., DGR and NGR containing peptide) is more than
60%, more preferably 70%, more preferably 80%, more preferably 85%,
more preferably 90%, more preferably 95%, more preferably 97%, more
preferably 99% w/w.
Treatment
[0185] The peptides, conjugates and compositions of the invention
may be used in therapeutic treatment.
[0186] It is to be appreciated that all references herein to
treatment include curative, palliative and prophylactic
treatment.
[0187] The patient treated in the present invention in its many
embodiments is desirably a human patient, although it is to be
understood that the principles of the invention indicate that the
invention is effective with respect to all mammals, which are
intended to be included in the term "patient." In this context, a
mammal is understood to include any mammalian species in which
treatment of diseases associated with angiogenesis is desirable,
particularly agricultural and domestic mammalian species.
[0188] In one embodiment the peptides, conjugates or pharmaceutical
compositions may be used to treat or prevent cancer including but
not limited to cancer of the lung, pancreas, breast, colon, larynx
or ovary. Preferably the cancer comprises a solid tumor.
[0189] In another embodiment, the peptides and/or conjugates may be
used to treat or prevent diseases involving angiogenisis, such as
diseases associated with .alpha..sub.v.beta..sub.3 expression.
[0190] Angiogenesis is a process of tissue vascularization that
involves the growth of new developing blood vessels into a tissue,
and is also referred to as neo-vascularization. The process is
mediated by the infiltration of endothelial cells and smooth muscle
cells. The process is believed to proceed in any one of three ways:
the vessels can sprout from pre-existing vessels, de-novo
development of vessels can arise from precursor cells
(vasculogenesis), or existing small vessels can enlarge in diameter
(Blood et al., 1990).
[0191] There are a variety of diseases in which angiogenesis is
believed to be important, referred to as angiogenic diseases,
including but not limited to, inflammatory disorders such as immune
and non-immune inflammation, arthritis, disorders associated with
inappropriate or inopportune invasion of vessels such as diabetic
retinopathy, macular degeneration, neovascular glaucoma,
restenosis, capillary proliferation in atherosclerotic plaques and
osteoporosis, and cancer associated disorders, such as solid
tumors, solid tumor metastases, angiofibromas, retrolental
fibroplasia, hemangiomas, Kaposi sarcoma and the like which require
neovascularization to support tumor growth.
[0192] Thus, methods which inhibit angiogenesis in a diseased
tissue ameliorates symptoms of the disease and, depending upon the
disease, can contribute to cure of the disease.
[0193] In another related embodiment, a tissue to be treated is a
retinal tissue of a patient with diabetic retinopathy, macular
degeneration or neovascular glaucoma and the angiogenesis to be
inhibited is retinal tissue angiogenesis where there is
neovascularization of retinal tissue.
[0194] In an additional related embodiment, a tissue to be treated
is a tumor tissue of a patient with a solid tumor, a metastases, a
skin cancer, a breast cancer, a hemangioma or angiofibroma and the
like cancer, and the angiogenesis to be inhibited is tumor tissue
angiogenesis where there is neovascularization of a tumor tissue.
Typical solid tumor tissues treatable by the present methods
include lung, pancreas, breast, colon, laryngeal, ovarian, and the
like tissues. Exemplary tumor tissue angiogenesis, and inhibition
thereof, is described in the Examples.
[0195] Inhibition of tumor tissue angiogenesis is a particularly
preferred embodiment because of the important role
neovascularization plays in tumor growth. In the absence of
neovascularization of tumor tissue, the tumor tissue does not
obtain the required nutrients, slows in growth, ceases additional
growth, regresses and ultimately becomes necrotic resulting in
killing of the tumor.
[0196] The methods are also particularly effective against the
formation of metastases because (1) their formation requires
vascularization of a primary tumor so that the metastatic cancer
cells can exit the primary tumor and (2) their establishment in a
secondary site requires neovascularization to support growth of the
metastases.
[0197] In a related embodiment, the invention contemplates the
practice of the method in conjunction with other therapies such as
conventional chemotherapy directed against solid tumors and for
control of establishment of metastases. The administration of
angiogenesis inhibitor is typically conducted during or after
chemotherapy, although it is preferably to inhibit angiogenesis
after a regimen of chemotherapy at times where the tumor tissue
will be responding to the toxic assault by inducing angiogenesis to
recover by the provision of a blood supply and nutrients to the
tumor tissue. In addition, it is preferred to administer the
angiogenesis inhibition methods after surgery where solid tumors
have been removed as a prophylaxis against metastases.
[0198] Insofar as the present methods apply to inhibition of tumor
neovascularization, the methods can also apply to inhibition of
tumor tissue growth, to inhibition of tumor metastases formation,
and to regression of established tumors.
[0199] Restenosis is a process of smooth muscle cell (SMC)
migration and proliferation at the site of percutaneous
transluminal coronary angioplasty which hampers the success of
angioplasty. The migration and proliferation of SMC's during
restenosis can be considered a process of angiogenesis which is
inhibited by the present methods. Therefore, the invention also
contemplates inhibition of restenosis by inhibiting angiogenesis
according to the present methods in a patient following angioplasty
procedures.
[0200] The peptides, conjugates and pharmaceutical compositions of
the invention can be used in combined, separated or sequential
preparations, also with other diagnostic or therapeutic
substances.
TNF/IFN Combinations
[0201] Another aspect of the present invention relates to the use
of a combination of the modified TNF, and IFN.gamma.. This
combination can be used in combined, separated or sequential
preparations. Advantageously the combination is also with other
diagnostic or therapeutic substances, in the treatment or in the
diagnosis of cancer, such as doxorubicin and mephalan. Thus the
present invention provides a pharmaceutical composition comprising
a combination of the modified TNF and IFN-.gamma., and optionally
another tumor-diagnostic or anti-tumor therapeutic substance.
Again, this combination can be used in combined, separated or
sequential preparations.
[0202] In our International patent publication number WO03/093478,
we found targeted delivery of picogram doses of cytokines enhances
the penetration of chemotherapeutic drugs, providing a novel and
surprising strategy for increasing the therapeutic index of
chemotherapeutic drugs. International patent publication number
WO03/093478 is hereby incorporated by reference in its entirety. In
more detail, we have found that delivery of very low doses of
cytokines to tumors and the tumor-associated environment including
tumor vasculature represents a new approach to avoiding negative
feedback mechanisms and to preserve its ability to alter
drug-penetration barriers.
[0203] In one embodiment of this aspect of the present invention, a
conjugate of the present invention may be administered at a dose of
from in the range of 0.5 to 500 ng/kg, preferably in the range of 1
to 50 ng/kg, more preferably in the range of 5 to 15 ng/kg.
[0204] In an alternative embodiment of this aspect of the invention
there is provided a pharmaceutical composition comprising a
conjugate of the present invention in combination with IFN.gamma.,
wherein the conjugate is present in an amount such that the
conjugate or a metabolite thereof is provided to the blood plasma
of the subject to be treated in an amount of no greater than about
35,000 ng/day, preferably about 3,500 ng/day, more preferably about
1,000 ng/day.
[0205] The above dosage relate to a dosage for a 70 kg subject. A
person skilled in the art would readily be able to modify the
recited dosage for a subject having as mass other than 70 kg.
[0206] The routes of administration and dosage regimens described
are intended only as a guide since a skilled practitioner will be
able to determine readily the optimum route of administration and
dosage regimens for any particular patient and condition.
DESCRIPTION OF THE FIGURES
[0207] FIGS. 1A-1C. Schematic representation, three-dimensional
structure and primary sequence of fibronectin fragments.
[0208] (A) The repeats in each fragments and in fibronectin subunit
are schematically represented: type I repeats (rectangles); type II
repeats (ovals); type III repeats (squares). Modules containing NGR
and RGD sequences are indicated (SEQ ID NOS 4, 9, 4 and 8
disclosed, respectively, in order of appearance). Natural fragments
obtained by proteolytic digestion with cathepsin (FN-70 KDa) or
cathepsin-trypsin (FN-45 and FN-30 KDa) are schematically shown.
Retronectin, recombinant FN-I.sub.4-5 fragment, and synthetic
FN-I.sub.5 are also represented.
[0209] (B) Three dimensional PDB-structure of FN-I.sub.4-5 (Protein
Data Bank code: 1FBR.pdb). The side-chains of the NGR motif
(Asn.sup.263, Gly.sup.264 and Arg.sup.265) are shown.
[0210] (C) Primary sequence of synthetic FN-I.sub.5 and Loop-GNGRG
(SEQ ID No: 4), and Loop-RGGNG (SEQ ID No: 50) peptides, and
FN-I.sub.7 and Loop-GNGRG (SEQ ID No: 4) peptide (see Methods) (SEQ
ID NOS 45-49 disclosed, respectively, in order of appearance). The
sequences presented in FIG. 1C are, from top to bottom and left to
right, SEQ ID NO: 45, SEQ ID NO: 4, SEQ ID NO: 46, SEQ ID NO: 50,
SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 4 and SEQ ID NO: 49.
[0211] FIG. 2. Cell adhesion promoting properties of natural
fibronectin fragments.
[0212] Microtiter plates coated with natural proteolytic fragments
(FN-70 KDa, FN-45 KDa and FN-30 KDa) promote the adhesion of
endothelial EA.hy926 cells.
[0213] FIGS. 3A-3D. Accelerated aging of FN fragments increase
their pro-adhesive properties.
[0214] Adhesion of EA.hy926 cells to microtiter plates coated with
various peptides and proteins before and after accelerated aging.
Accelerated aging was performed by peptide incubation in 0.1 M
ammonium bicarbonate buffer, pH 8.5, for 16 h at 37.degree. C.
("heat-treatment"). Adhesion to: recombinant FN-I.sub.4-5 or
control protein (FN-I.sub.4-5 (SGS)) (A), synthetic FN-I.sub.5 (B),
recombinant CNGRC (SEQ ID No: 7)-TNF, CNGRC-TNF.sub.1-11 and
control CARAC (SEQ ID No: 71)-TNF.sub.1-11 peptide (C), CDGRC-TNF
(D). Heat-treated (37.degree. C.) and untreated (-20.degree. C.)
products were adsorbed onto the plastic surface of microtiter wells
(overnight at 4.degree. C.). Microphotograph of wells coated with
30 .mu.g/ml of heat-treated FN-I.sub.5 or BSA (B, right) or with 10
.mu.g/ml of recombinant CNGRC-TNF or CDGRC-TNF (C, right),
.times.200. Cell adhesion assay was carried out as described in
Materials and Methods. [CNGRC (SEQ ID NO: 6); CDGRC (SEQ ID NO:
23); CARAC (SEQ ID NO: 36)] The TNF fusion constructs, including
the TNF.sub.1-11 fusion constructs, were made as described
Materials and Methods, and include a "G" linker.
[0215] FIG. 4. The pro-adhesive properties of EN fragments are
inhibited by PIMT.
[0216] Microtiterplates were coated with heat-treated FN-I.sub.5 or
NGR-TNF (A), or with natural FN-30 KDa, FN-45 KDa or retronectin
(RN) (CNGRC disclosed as SEQ ID NO: 6) (B). The adsorbed proteins
were then treated with PIMT enzyme for 16 h at 37.degree. C. After
incubation the enzyme solution was removed by washing. EA.hy926
cell adhesion assay was then performed as described in Materials
and Methods.
[0217] FIG. 5. Stability of NGR, DGR and isoDGR motives.
[0218] RP-HPLC analysis of NGR-pep and its synthetic deamidation
products (DGR-pep and isoDGR-pep) before and after incubation for 4
h at 37.degree. C. in 0.1 M ammonium bicarbonate buffer, pH 8.5 (A
and B) and in DMEM, pH 7.3 (A, inset). RP-HPLC was performed on a
C-18 column (PepMap C18, PerSeptive Biosystem) as follows: buffer
A, 0.1% trifluoroacetic acid (TFA) in water; buffer B, 95%
acetonitrile, 0.1% TFA; 0% B for 10 min, linear gradient 20-40% B
in 30 min, 100% B for 10 min; 0% B for 15 min (flow rate, 0.5
ml/min). Peak 1, 2 and 3 were identified by spiking the NGR-pep,
after 2 h of incubation, with equal amount of the indicated peptide
(C). NGR-pep (peak 1) rapidly converts to compounds corresponding
to DGR-pep (peak 2) and isoDGR-pep (peak 3) (half-life, 2-4 h).
[0219] FIG. 6. EN-I.sub.5 and peptides containing the NGR motif
bind to .alpha..sub.v.beta..sub.3 integrin after accelerated
aging.
[0220] (A) Binding of heat-treated CNGRC (SEQ ID NO: 6)-TNF and TNF
to solid phases coated with purified human integrins. Single
concentration (5 .mu.g/ml) or various concentrations (inset) of
CNGRC (SEQ ID NO: 6)-TNF and TNF were used. Binding was detected
with anti-TNF antibodies as described in Materials and Methods.
[0221] (B) Binding of biotinylated CNGRC (SEQ ID NO:
6)-TNF.sub.1-11, CARAC (SEQ ID NO: 36)-TNF.sub.1-11 and FN-I.sub.5
(complexed with streptavidin-peroxidase) to purified human
integrins. Binding was detected by chromogenic reaction with
o-phenylendiammine.
[0222] (C) Competitive binding of biotinylated
FN-I5/streptavidin-peroxidase complexes to
.alpha..sub.v.beta..sub.3-coated plates with various amounts of
FN-I.sub.4-5 or FN-I.sub.4-5SGS (upper panel) or peptides
corresponding to the NOR loop (residues 258-271) of FN-I.sub.5
before and after heat treatment (lower panel). [RGGNG (SEQ ID NO:
50); GNGRG (SEQ ID NO:4)]
[0223] FIG. 7 Competitive binding of NGR-TNF to
.alpha..sub.v.beta..sub.3 with NGR-pep and its deamidated products
to .alpha..sub.v.beta..sub.3.
[0224] Biotinylated NGR-TNF (4 .mu.g/ml) was mixed with different
amount of the indicated peptides and added to a.sub.v.beta..sub.3
coated plates. Binding was detected as described in Materials and
Methods. IC.sub.50 was determined by nonlinear regression analysis
using the GraphPad Prism software. The kinetics of deamidation of
FN-I.sub.5 was determined by plotting the inhibitory concentration
(IC.sub.50) versus the incubation times. Bars represent the
confidence intervals for each determined IC.sub.50.
[0225] FIG. 8. Deamidated FN-I.sub.5 fragment inhibits in vitro
cell adhesion to vitronectin and tumor growth in a mouse model.
[0226] (A) Inhibition of EA.hy926 cell adhesion to vitronectin by
heat-treated FN-I.sub.5. Vitronectin (3 .mu.g/ml) was adsorbed to
microtiter plate and cell adhesion assay was performed as described
in Materials and Methods.
[0227] (B) Anti-tumor effects of repeated administration of
"heat-treated" FN-I.sub.5 to RMA-tumor bearing mice. Animals
(5/group) were treated with 200 .mu.g of "heat treated" FN-I.sub.5
(i.p.) at the indicated times (arrows) starting 4 days after tumor
implantation. *p=0.0003 at day 14, statistical analysis by two
tailed t-test.
[0228] FIG. 9. RP-HPLC analysis of NGR-TNF.
[0229] RP-HPLC of NGR-TNF revealed two major peaks, F2 and F4, and
two minor peaks F1 and F3. The presence of deamidated forms in
these peaks was identified by measuring the amount of isoAsp in
these fraction. The results showed that F2 contained much more
isoAsp then F4 (Table 1).
[0230] FIG. 10. RP-HPLC and SDS-PAGE of F2 and F4.
[0231] A) Re-chromatography of F2 and F4 by RP-HPLC showed that the
retention time did not change after separation. B) Coomassie
staining of non-reducing SDS-PAGE revealed a major band of 17-18
kDa in both F2 and F4 products, as expected for TNF monomers.
[0232] FIG. 11. Anti-tumor activity of F2 in the RMA lymphoma
model. Two experiments were performed (Exp.1 and Exp.2). C57BL/6
mice (Charles River Laboratories, Calco, Italy) weighing 16-18 g
were challenged with subcutaneous injection in the left flank of
7.times.10.sup.4 RMA living cells; 10 days later, mice were treated
with 0, 25 or 100 pg of F2 (100 .mu.l) followed 2 h later by
administration of melphalan (50 .mu.g in 100 .mu.l. Tumor sizes are
shown as mean.+-.SE (5 animals/group).
[0233] FIG. 12. RP-HPLC of NGR-TNF after treatment at 37.degree. C.
in ammonium bicarbonate pH 8.5 for various times.
[0234] NGR-TNF was diluted in 0.1 M ammonium bicarbonate buffer, pH
8.5 and incubated at 37.degree. C. for various time. The heat
treated NGR-TNF was analyzed by RP-HPLC.
[0235] FIG. 13. RP-HPLC (A) and SDS-PAGE (B) of recombinant DGR-TNF
and NGR-TNF.
[0236] The following examples further illustrate the invention.
[0237] FIG. 14 shows a different structural isomeric form of
aspartic acid, namely isoaspartic acid.
Example 1
Materials and Methods for Examples 1 to 7
Cell Lines and Reagents
[0238] Mouse RMA lymphoma cells and EA.hy926 cells (human
endothelial cells fused with human lung carcinoma A549 cells) were
cultured as described previously (Curnis et al., 2005; Ljunggren
and Kane, 1985). Crystal violet (Fluka Chemie); bovine serum
albumin (BSA), goat anti-rabbit IgG horseradish peroxidase
conjugate, FN-70 KDa, FN-45 KDa and FN-30 KDa fragments (Sigma);
RetroNectin (Takara Biomedicals). Human .alpha..sub.v.beta..sub.3,
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1 and
.alpha..sub.1.beta..sub.1 integrins (Immunological Sciences);
streptavidin-peroxidase (Societa Prodotti Antibiotici).
Preparation and Characterization of Recombinant FN-.sub.4-5 and
FN-I.sub.4-5SGS
[0239] The cDNA coding for human fibronectin 4-5.sup.th type I
repeats (FN-I.sub.4-5; residue 184-273 of fibronectin) was prepared
by RT-PCR on MSR-3-mel cells total RNA (Tanzarella et al., 1999)
using the following primers: 5'-CTGGATCCGAGAAGTGTTTTGATCATGCTGCTGGG
(SEQ ID NO: 37) (forward) and 5' TATATTAAGCTTTCAGTGCCTCTCACACTTCC
(SEQ ID NO: 38) (reverse). A control fragment with NGR replaced
with SGS (FN-I.sub.4-5SGS), was generated by PCR on FN-I.sub.4-5
plasmid using the above forward primer and the following reverse
primer: 5'-TATATTAAGCTTTCAGTGCCTCTCACACTTCCACTCTCCACTGCCGCTG (SEQ
ID NO: 39). Amplified fragments were cloned into a pRSET-A plasmid
(Invitrogen), expressed in BL21(DE3)pLysS E. coli cells as soluble
proteins (with an His-tag at the N-terminus) and purified from cell
extracts by metal-chelate affinity chromatography.
Preparation and Characterization of Synthetic Peptides
[0240] Various peptides (biotinylated and non biotinylated) were
prepared by chemical synthesis using an Applied Biosystem model
433A peptide synthesizer. Aminoacids in L-configuration were used
except when indicated. A synthetic peptide corresponding to the
5.sup.th type I repeat of human fibronectin (FN-I.sub.5), residues
230-274 of mature protein (accession number P02751, Swiss-Prot),
was also synthesized with an additional N-terminal acetyl-glycine
and with Glu.sup.250 in place of Asp.sup.250. To promote disulfide
formation between Cys.sup.258-Cys.sup.270 and
Cys.sup.231-Cys.sup.260 in FN-I.sub.5 (see FIG. 1), Cys.sup.258 and
Cys.sup.270 were protected with S-trityl groups (removable by
trifluoroacetic acid after side-chain deprotection and cleavage
from the resin), whereas Cys.sup.231 and Cys.sup.26.degree. were
protected with S-acetamidomethyl groups (removable by treatment
with iodine). After RP-HPLC purification of peptide, the
Cys.sup.258-Cys.sup.27.degree. disulfide bridge was formed by
incubating 20 .mu.moles of peptide in 300 ml of 8% DMSO at pH 7
(room temperature, overnight). The peptide was then purified by
RP-HPLC and lyophilized. To form the second
Cys.sup.231-Cys.sup.26.degree. disulfide bridge, 13.5 .mu.moles of
peptide was dissolved in 67 ml of 80% v/v acetic acid and mixed
with 0.135 ml of 1 N hydrochloric acid. Then, 21 mg of iodine,
dissolved in 2 ml of methanol, was added to the peptide solution
under stirring. After 90 min, 1 mmole of ascorbic acid was added,
to quench iodine. The peptide was then purified by RP-HPLC.
[0241] All peptides were dissolved in sterile water and stored in
aliquots at -20.degree. C. Peptide purity was analyzed by RP-HPLC.
Free sulfhydryl groups in all peptide preparations were <0.1% as
checked by titration with Ellman's reagent (Pierce, Rockford,
Ill.). Peptide identity was checked by MALDI-TOF or ESI-MS mass
spectrometry. The molecular mass of peptides used throughout this
work was similar to the expected value. ESI-MS analysis of
FN-I.sub.5 showed the presence of an additional component of +144
Da, corresponding to FN-I.sub.5 with unremoved acetamidomethyl
groups.
Preparation and Characterization of CNGRC (SEQ ID NO: 6)-TNF, CDGRC
(SEQ ID NO: 23)-TNF and CNGRC (SEQ ID NO: 6)-TNF.sub.1-11
Conjugates
[0242] Marine TNF and CNGRC (SEQ ID NO: 6)-TNF (consisting of
murine TNF fused with the C-terminus of CNGRCG (SEQ ID NO: 7)) were
prepared by recombinant DNA technology as described (Curnis et al.,
2000).
[0243] The cDNA coding for CDGRC (SEQ ID NO: 23)-TNF (murine TNF
fused with the C-terminus of CDGRCG (SEQ ID NO: 24)) was prepared
by PCR on CNGRC (SEQ ID NO: 6)-TNF plasmid (Curnis et al., 2000)
using the following primers: 5'-CACCATGGGCAACGGCCGTGGCGGCGTC (SEQ
ID NO: 40) (forward); 5'-TCAGGATCCTCACAGGGCAATGATCCCAAAGTAGAC (SEQ
ID NO: 41) (reverse). The amplified cDNA was cloned into the
pET101/D-TOPO plasmid (Invitrogen), expressed in BL21(DE3) E. coli
cells (Novagen) and purified from cell extracts by affinity
chromatography on soluble p75-TNF receptor-Sepharose essentially as
described previously for CNGRC (SEQ ID NO: 6)-TNF (Colombo et al.,
2002). Both CNGRC (SEQ ID NO: 6)-TNF and CDGRC (SEQ ID NO: 23)-TNF
were subjected to a refolding procedure as described previously
(Curnis et al., 2000).
[0244] Protein purity and identity were checked by SDS-PAGE,
electrospray mass spectrometry and gel-filtration chromatography.
The in vitro cytolytic activity of CNGRC (SEQ ID NO: 6)-TNF and
CDGRC (SEQ ID NO: 23)-TNF, measured by standard cytolytic assay
with L-M mouse fibroblasts (Corti et al., 1994), were 2.96
(.+-.0.56).times.10.sup.8 U/mg and 2.59 (.+-.0.69).times.10.sup.8
U/mg, respectively.
[0245] CNGRC (SEQ ID NO: 6)-TNF.sub.1-11, corresponding to the
N-teiininal sequence of NGR-TNF (lacking TNF activity) and CARAC
(SEQ ID NO: 36)-TNF.sub.1-11 peptide, were prepared by chemical
synthesis as described above.
Accelerated Aging (Heat Treatment) of Fibronectin Fragments and
Peptides
[0246] Fibronectin fragments, peptides and peptide-TNF conjugates
were diluted in 0.1 M ammonium bicarbonate buffer, pH 8.5,
incubated for 16 h at 37.degree. C., and stored at -20.degree. C.
until analysis. These products are hereinafter referred to as "heat
treated."
Isoaspartate (isoAsp) Quantification
[0247] IsoAsp content in untreated and "heat treated" CNGRC (SEQ ID
NO: 6)-TNF and peptides was quantified by using the ISOQUANT.TM.
Isoaspartate Detection Kit (Promega).
Cell Adhesion Assay and PIMT Treatment
[0248] Untreated and "heat treated" fibronectin fragments, peptides
and peptide-TNF conjugates were diluted at the desired
concentration with 150 mM sodium chloride, 50 mM sodium phosphate,
pH 7.3, and added to 96-well polyvinyl chloride microtiter plates
(Falcon, Becton Dickinson). After overnight incubation at 4.degree.
C., the plates were washed, seeded with EA.hy926 cell in DMEM
containing 0.1% BSA (40000 cells/well) and left to incubate for 2-3
h at 37.degree. C., 5% CO.sub.2. Adherent cells were fixed and
stained with crystal violet as described (Curnis et al., 2005).
[0249] The effect of proteinL-isoAsp/D-Asp/D-methyltransferase
(PIMT) on the pro-adhesive properties of different fragments was
investigated as follows: microtiter plates were coated with various
products as described above and washed with 0.9% sodium chloride.
Each well was then filled with 45 .mu.l of a solution containing
0.02 mM S-adenosyl-L-methionine in 150 mM sodium chloride, 50 mM
sodium phosphate buffer, pH 6.8, and 5 .mu.l of PIMT solution (from
the Isoquant Isoaspartate Detection kit, Promega) (final volume 50
.mu.l/well) and incubated at 37.degree. C. for 16 h. After
incubation the plates were washed with 0.9% sodium chloride and
cell adhesion assay was performed as described above.
Binding of Peptides and Proteins to Integrins
[0250] Human .alpha..sub.v.beta..sub.3, .alpha..sub.v.beta..sub.5,
.alpha..sub.5.beta..sub.1, and .alpha..sub.1.beta..sub.1 integrins
solutions, 0.5-2 .mu.g/ml in phosphate buffered saline with
Ca.sup.2+ and Mg.sup.2+ (DPBS, Cambrex), were added to 96-well
polyvinylchloride microtiter plates (50 .mu.l/well) and left to
incubate overnight at 4.degree. C. All subsequent steps were
carried out at room temperature. The plates were washed with DPBS
and further incubated with DPBS containing 3% BSA (200 .mu.l/well,
1 h). The plates were then washed and filled with CNGRC (SEQ ID NO:
6)-TNF or TNF solutions (5 .mu.g/ml, 50 .mu.l/well in 3% BSA-DPBS)
and left to incubate for 2 h. After washing with DPBS, each well
was incubated with purified rabbit anti-murine TNF IgGs in 3%
BSA-DPBS containing 1% normal goat serum (10 .mu.g/ml, 50
.mu.l/well, 1 h) followed by a goat anti-rabbit peroxidase
conjugate in the same buffer (50 .mu.l/well, 1 h). Bound peroxidase
was detected by adding o-phenylendiammine chromogenic
substrate.
[0251] Binding of biotinylated peptides (CNGRC (SEQ ID NO:
6)-TNF.sub.1-11, CARAC (SEQ ID NO: 36)-TNF.sub.1-11 and FN-I.sub.5)
to purified integrins was studied using streptavidin-peroxidase
conjugate complexes. Complexes were prepared by mixing various
quantities (0.5-1 .mu.g) of heat-treated biotinylated peptides in
DPBS containing 3% BSA with 0.03 units of streptavidinperoxidase
(binding capacity: 1 .mu.g of biotin/unit of
streptavidin-peroxidase) (final volume 15 .mu.l). Complexes were
diluted in 3% BSA-DPBS (1:500), added to microtiterplates coated
with integrins as described above, and incubated for 2 h at room
temperature. After washing with DPBS, bound peroxidase was detected
by chromogenic reaction as described above. Each assay was carried
out in triplicate.
In Vivo Studies
[0252] Studies on animal models were approved by the Ethical
Committee of the San Raffaele H Scientific Institute and performed
according to the prescribed guidelines. C57BL/6N mice (Charles
River Laboratories, Calco, Italy) weighing 16-18 g were challenged
with subcutaneous injection in the left flank of 7.times.10.sup.4
RMA living cells; 4 days later, the mice were treated daily with
200 .mu.g of heat-treated FN-I.sub.5 (100 .mu.l) in 0.9% sodium
chloride (i.p.). Tumor growth was monitored by measuring the tumors
with calipers as previously described (Gasparri et al., 1999).
Animals were sacrificed before the tumors reached 1.0-1.3 cm in
diameter. Tumor sizes are shown as mean.+-.SE (5
animals/group).
Example 2
Accelerated Aging of Fibronectin Fragments Generates NGR-Dependent
Adhesion Stes
Accelerated Aging of Fibronectin Fragments Generates NGR-Dependent
Adhesion Sites
[0253] The adhesion of EA.hy926 cells to natural, recombinant and
synthetic fibronectin fragments containing the NGR motif was
studied. First, the following proteolytic fragments of fibronectin
were studied: a) FN-70 KDa, containing the FN-I.sub.1-9 and
FN-II.sub.1-2 repeats; b) FN-30 KDa, containing the FN-I.sub.1-5
repeats; c) FN-45 KDa, containing the FN-I.sub.6-9 and
FN-II.sub.1-2 (see FIG. 1 for a schematic representation). All
fragments, after adsorption to microtiterplates, induced cell
adhesion and spreading (FIG. 2), suggesting the presence of
pro-adhesive sites in these regions.
[0254] To investigate the role of the NGR motif in the FN-I.sub.5
repeat, we then analyzed the pro-adhesive properties of FN-I.sub.5,
FN-I.sub.4-5 and FN-I.sub.4-5SGS fragments, the latter
corresponding to a mutant with SGS in place of NGR (residues
263-265). Recombinant FN-I.sub.4-5, but not the SGS mutant,
promoted cell adhesion (FIG. 3A). When we incubated these fragments
in 0.1 M ammonium bicarbonate buffer, pH 8.5, for 16 h at
37.degree. C. (from now on this treatment called "heat-treatment")
we observed an increase of cell adhesion to FN-I.sub.4-5, but not
to FN-I.sub.4-5SGS (FIG. 3A). This suggests that a cell adhesion
site, somehow related to the NGR sequence, was generated by
accelerated aging. Similar results were obtained with synthetic
FN-I.sub.5 (FIG. 3B).
[0255] To assess whether the NGR motif was sufficient for mediating
this phenomenon, we investigated the pro-adhesive properties of
short peptides containing the NGR-motif, before and after heat
treatment. Since the NGR tripeptide is unlikely to fold and to bind
to microtiterplates, we introduced two flanking cysteines. The
rationale for using flanking cysteines was based on the fact that
the predicted conformation of CNGRC (SEQ ID NO: 6) peptide, by
molecular dynamic simulation, is similar to that of the GNGRG (SEQ
ID NO: 4) loop of FN-I.sub.5 repeat (Colombo et al., 2002).
Furthermore, we fused this peptide to TNF (CNGRC (SEQ ID NO:
6)-TNF) or to the first eleven residues of TNF (CNGRC (SEQ ID NO:
6)-TNF.sub.1-11, devoid of TNF activity) to enable adsorption to
microtiterplates. As expected heat treatment of CNGRC (SEQ ID NO:
6)-TNF increased cell adhesion (FIG. 3C). No adhesion was observed
to TNF alone either before or after heat treatment (data not
shown). Similarly, heat treatment of CNGRC (SEQ ID NO:
6)-TNF.sub.1-11, but not of CARAC (SEQ ID NO: 36)-TNF.sub.1-11 (a
control peptide), increased cell adhesion (FIG. 3C, right).
[0256] These results support the hypothesis that the NGR motif is
sufficient for promoting cell adhesion after heat treatment.
Interestingly, a CDGRC (SEQ ID NO: 23)-TNF conjugate, prepared by
recombinant DNA technology, was completely inactive (FIG. 3D) even
after heat treatment (not shown), suggesting that Asn is a crucial
residue for the enhanced activity. In conclusion these results
suggest that structural changes somehow related to the Asn residue
of NGR motif in FN-I.sub.5 lead to generation of a pro-adhesive
site.
Example 3
Deamidation of the NGR is Associated with Increased Cell
Adhesion
[0257] It is well known that the Asn residues, particularly when
followed by Gly, can undergo non-enzymatic deamidation at
physiological pH (Robinson and Robinson, 2001; Robinson et al.,
2004; Stephenson and Clarke, 1989; Tyler-Cross and Schirch, 1991).
This reaction leads to formation of Asp or isoAsp, L-Asp (.sub.LD),
L-isoAsp, (.sub.LisoD), D-Asp (.sub.DD), and D-isoAsp (.sub.DisoD),
although the L-configuration predominates (Geiger and Clarke,
1987). Accordingly we found that the molecular mass of FN-I.sub.5,
CNGRC (SEQ ID NO: 6)-TNF and CNGRC (SEQ ID NO: 6) peptides was
increased by about 1 Da, by mass spectrometry analysis, after heat
treatment. Furthermore, isoAsp analysis of CNGRC (SEQ ID NO: 6)-TNF
showed the presence of >0.5 pmol isoAsp/mol of CNGRC (SEQ ID NO:
6)-TNF subunit after heat treatment.
[0258] To assess whether the enhanced adhesive properties of
FN-I.sub.5 after heat-treatment depended on NGR deamidation, we
incubated the "heat treated" FN-I.sub.5 and CNGRC (SEQ ID NO:
6)-TNF with the proteinL-isoAsp/D-Asp/D-methyltransferase (PIMT),
an enzyme that converts L-isoAsp and D-Asp residues to L-Asp
(Aswad, 1984; Galletti et al., 1988; Lowenson and Clarke, 1992;
McFadden and Clarke, 1987; Murray and Clarke, 1984). PIMT almost
completely inhibited the pro-adhesive activity of heat-treated
FN-I.sub.5 and CNGRC (SEQ ID NO: 6)-TNF (FIG. 4A). Moreover, this
enzymatic treatment partially inhibited the pro-adhesive properties
of natural FN-30 KDa (FIG. 4B) and FN-45 KDa fragment (not shown)
suggesting that Asn deamidation can occur also in natural
fibronectin fragments. To assess the specificity of this reaction
we evaluated also the effect of PIMT on retronectin, a FN fragment
that is known to promote cell adhesion via RGD (see FIG. 1). As
expected, in this case no inhibition was observed after enzymatic
treatment (FIG. 4B). Conversely, in this case we observed a modest
but significant increase. This was not totally unexpected as also
RGD can undergo isomerization of Asp residues, with formation of
isoAsp and loss of function (Geiger and Clarke, 1987; Lanthier and
Desrosiers, 2004; Reissner and Aswad, 2003; Stephenson and Clarke,
1989). Thus, in this case PIMT is expected to "repair" RGD and
increase cell adhesion. In conclusion, these results suggest that
NGR deamidation, in contrast to RGD isomerization, is associated
with a "gain of function" in cell adhesion assays.
Example 4
Kinetics of NGR Deamidation
[0259] The stability of NGR and the kinetics of .sub.LisoDGR or
.sub.DDGR formation at pH 7.3 (DMEM) and pH 8.5 (ammonium
bicarbonate), were then investigated. To this aim the CNGRCGVRY(SEQ
ID NO: 42) peptide (called NGR-pep) was synthesized and analyzed by
reverse-phase HPLC, before and after incubation at 37.degree. C.
Residues GVRY (SEQ ID NO: 72) were added to the C-terminus of CNGRC
(SEQ ID NO: 6) to enable column adsorption. In addition five
peptides called, DGR-pep, .sub.D-DGR-pep, isoDGR-pep, .sub.D-isoDGR
and SGR-pep, corresponding to the same sequence of NGR-pep except
for the presence of D, .sub.D-D, .sub.L-isoD, .sub.D-isoD and S,
respectively in place of N were prepared. The half-life of NGR-pep
at pH 7.3 and 8.5, estimated from the height of the main
chromatographic peak (peak 1), was about 4 and 2 h, respectively
(FIG. 5A, inset). Peptides corresponding to deamidation products
were stable under these conditions (FIG. 5B).
[0260] To identify peaks and to verify that the measured half-life
correspond to Asn deamidation, and not to other structural changes,
we analyzed heat-treated NGR-pep peptide spiked with fresh NGR-pep,
or DGR-pep or isoDGR-pep peptides. The results showed that peak 1,
2 and 3 observed after NGR-pep incubation correspond to NGR-pep,
DGR-pep and isoDGR-pep peptides, respectively. Of note the NGR-pep
was stable for more than one week at 37.degree. C. when stored in
water.
Example 5
.alpha..sub.v.beta..sub.3 is a Receptor of .sub.DisoDGR and
.sub.DDGR Motifs
[0261] The binding of heat-treated FN-I.sub.5, CNGRC (SEQ ID NO:
6)-TNF and short peptides to purified .alpha..sub.v.beta..sub.3
.alpha..sub.v.beta..sub.5, .alpha..sub.5.beta..sub.1 and
.alpha..sub.1.beta..sub.1 integrins was then investigated by direct
and competitive ELISA with integrins adsorbed on
microtiterplates.
[0262] We observed binding of all NGR-containing molecules
(heat-treated) to .alpha..sub.v.beta..sub.3, but little, or not at
all, to the other integrins (FIGS. 6A and B). No binding was
observed with heat-treated TNF or with a control CARAC (SEQ ID NO:
36)-TNF.sub.1-11 peptide (FIGS. 6A and B), suggesting that NGR was
critical. Of note, we observed binding of CNGRC (SEQ ID NO: 6)-TNF
to .alpha..sub.v.beta..sub.3 even before heat treatment (data not
shown), although to a lower extent, possibly due to deamidation
occurring during preparation and/or assay incubation.
[0263] To verify the importance of the NGR loop of FN-I.sub.5 for
integrin binding we performed competitive binding experiments with
recombinant FN-I.sub.4-5 and FN-I.sub.4-5SGS, the latter lacking
NGR. As expected only the fragment with NGR could compete the
binding of FN-I.sub.5 to .alpha..sub.v.beta..sub.3 (FIG. 6C).
Binding competition was observed also with a peptide corresponding
to the entire 256-271 loop of FN-I.sub.5 (see FIG. 1C), but not
with a control peptide with a scrambled sequence at the GNGRG (SEQ
ID NO: 4) site (FIG. 6 C).
[0264] Accordingly this binding was efficiently competed with
.sub.DDGR-pep and isoDGR-pep (EC.sub.50, 0.1 .mu.M) and
heat-treated FN-I.sub.5 (EC.sub.50, 0.4 .mu.M) and less efficiently
with .sub.DisoDGR-pep or DGR-pep, NGR-pep, SGR-pep (EC.sub.50,
>10 .mu.M) (FIG. 7).
[0265] Considering that the isoDGR-pep or .sub.DDGR-pep peptides
are stable under these assay conditions these results suggest that
isoDGR-pep or .sub.DDGR-pep motives new ligands of
.alpha..sub.v.beta..sub.3.
Example 6
Kinetics of Integrin Binding Site Formation in FN-I.sub.5
[0266] To investigate the kinetics of integrin binding site in
FN-I.sub.5 we have performed additional competitive binding studies
with various doses of FN-I.sub.5, incubated for different times at
37.degree. C. in DMEM. Interestingly, when we plotted the
inhibitory concentration (IC.sub.50) versus the incubation times we
observed that maximal competitive binding activity was reached
after 24-48 h of incubation (half life 3.4 h) (FIG. 7B). This value
corresponds very well to the half fife of NGR-pep deamidation
described above, and further support the concept that the integrin
binding proprieties of heat treated FN-I.sub.5 were indeed related
to the NGR-deamidation.
Example 7
Deamidated FN-I.sub.5 Fragments Inhibits In Vitro Cell Adhesion to
Vitronectin and Tumor Growth in a Mouse Model
[0267] It is well known that .alpha..sub.v.beta..sub.3 integrin,
the vitronectin receptor, plays a critical role in angiogenesis
(Hynes, 2002). Compounds able to inhibit the interaction of this
integrin with ECM proteins are known to inhibit angiogenesis and
tumor growth (Brooks et al., 1994; Brooks et al., 1995; Friedlander
et al., 1995; Friedlander et al., 1996; Hammes et al., 1996) We
have observed that deamidated FN-I.sub.5 fragments could inhibit
the adhesion of EA.hy926 cells to vitronectin (FIG. 8A).
Furthermore, this fragment, when was daily administered to RMA
lymphoma bearing mice, significantly inhibited tumor growth in vivo
(FIG. 8B). These results suggest that deamidated fragments of FN
could play a role in cell adhesion and tumor biology.
Example 8
Isolation of a Deamidated Form of NGR-TNF
[0268] To prepare murine Cys-Asp-Gly-Arg-Cys-Gly (SEQ ID NO:
24)-TNF (DGR TNF) and Cys-isoAsp-Gly-Arg-Cys-Gly (SEQ ID NO:
60)-TNF (isoDGR-TNF) we cloned and expressed
Cys-Asn-Gly-Arg-Cys-Gly (SEQ ID NO: 7)-TNF (NGR-TNF) in E. coli
cells and purified deamidated forms (spontaneously produced during
the production process) from this product.
[0269] The cDNA coding for NGR-TNF (consisting of TNF fused with
the C-terminus of CNGRCG (SEQ ID NO: 7)) was first prepared by
recombinant DNA technology using E. coli cells and purified as
described (Curnis et al., 2000). To isolate the deamidated forms
from this preparation we performed a further purification step
based on reversed phase chromatography (RP-HPLC) on a C-4 column
(Hi-pore Reversed Phase column, 250.times.4.6 mm, Biorad), as
follows: mobile phase A; 5 mM sodium phosphate buffer, pH 6.8,
containing 5% acetonitrile in water, mobile phase B, 5 mM sodium
phosphate buffer, pH 6.8, containing 70% acetonitrile in water; 0%
B for 10 30% B for 5 min, linear gradient 30-65% B in 35 min, 100%
B for 10 min, 0% B for 10 min, flow rate 2 ml/min. The elution was
monitored at 214 nm and 280 nm (HPLC, LC-126 nM, Beckman Coulter).
RP-HPLC of NGR-TNF revealed two major peaks, called F2 and F4, and
two minor peaks called F1 and F3 (FIG. 9). The presence of
deamidated forms in these peaks was identified by measuring the
amount of isoAsp in these fraction. The results showed that F2
contained much more isoAsp then F4 (Table 1).
[0270] Re-chromatography of F2 and F4 by RP-HPLC showed that the
retention time did not change after separation, suggesting that
these products were stable. Coomassie staining of non-reducing
SDS-PAGE revealed a major band of 17-18 kDa in both F2 and F4
products, as expected for TNF monomers (FIG. 10, inset)
[0271] The biochemical and biological properties of F2 and F4 were
further characterized (Table 1). Electrospray mass spectrometry
analysis showed that molecular mass of F2 was 1 Da greater than
that of F4. Moreover, the cytolytic activities of F2 and F4 against
L-M cell were similar. This result, together with the data on
isoAsp content, suggests that F2 corresponds to a deamidated form
of NGR-TNF.
[0272] To assess whether deamidation took place in the N-terminal
CNGRC (SEQ ID NO: 6) domain we performed sequence analysis of the
N-terminus by the Edman degradation method (not shown). The results
confirmed that deamidation took place at the N-terminus, as
Asp.sup.2 was detected in place of Asn.sup.2. This and the above
data suggest that F2 was a mixture of isoDGR-TNF (75%) and DGR-TNF,
with isoaspartyl and aspartyl residues in position 2.
[0273] The in vivo anti-tumor properties of F2 were then
investigated using the RMA lymphoma model. Studies on animals were
approved by the Ethical Committee of the San Raffaele H Scientific
Institute and performed according to the prescribed guidelines.
C57BL/6 mice (Charles River Laboratories, Calco, Italy) weighing
16-18 g were challenged with subcutaneous injection in the left
flank of 7.times.10.sup.4 RMA living cells; 10 days later, mice
were treated with 0, 25 or 100 pg of F2 (100 .mu.l) followed 2 h
later by administration of melphalan (50 .mu.g in 100 .mu.l) (Glaxo
Wellcome Operations, Dartford, Great Britain). All drugs, diluted
with 0.9% sodium chloride containing 100 .mu.g/ml endotoxin-free
human serum albumin (Farina-Biagini SpA, Lucca, Italy), were
administered intraperitoneally. Tumor growth was monitored daily by
measuring the tumors with calipers as previously described (34).
Animals were sacrificed before the tumors reached 1.0-1.3 cm in
diameter. Tumor sizes are shown as mean.+-.SE (5
animals/group).
[0274] The results showed that very low doses (25 pg) of F2 could
induce anti-tumor effects when administered in combination with
melphalan (FIG. 11).
Example 9
Preparation of isoDGR-TNF/DGR-TNF by Treatment of NGR-TNF with 0.1
M Ammonium Bicarbonate Buffer
[0275] Buffer composition, ionic strength, pH and temperature can
increased the deamidation/isomerization rate of the Asn residue.
Thus, to develop a rapid method for the preparation of F2
(iDGR-TNF/DGR-TNF) we have diluted NGR-TNF in 0.1 M ammonium
bicarbonate buffer, pH 8.5 and incubated at 37.degree. C. for
various times. The heat treated NGR-TNF was analyzed by RP-HPLC.
FIG. 12 shows that F4 was rapidly converted into F2 after this
treatment. Remarkably, no loss of cytolytic activity was observed
after 4 h of treatment, indicating that this process can be
exploited for producing isoDGR-TNF/DGR-TNF.
Example 10
Preparation of DGR-TNF by Recombinant DNA Technology
[0276] The cDNA coding for DGR-TNF (mouse TNF fused with the
C-terminus of CDGRCG (SEQ ID NO: 24)) was obtained by PCR on
NGR-TNF plasmid, using the following primers: 5'
CACCATGTGCGACGGCCGTTGCGGC 3' (SEP ID NO: 43) (5' primer); 5'
CTGGATCCTCACAGAGCAATGATCCCAAAG '3 (SEQ ID NO: 44) (3' primer). Both
primers were designed to allow the directional cloning into a
pET101/D-TOPO plasmid expression kit (Invitrogen). The underlined
sequence in 5' primer contains the sequence necessary to enable the
directional cloning and the initial translation codon whereas the
3' primer contains the stop codon (underlined). The PCR reaction
and the cloning procedure were performed as recommended by the
manufacturer's instruction. The DGR-TNF cDNA was expressed in
BL21(DE3) E. coli cells (Novagen) and purified from cell extracts
by affinity chromatography on soluble p75-TNF receptor-Sepharose as
follows: 10 mg of commercially available recombinant p75-TNF
receptor (Embrel) were coupled to 2.5 ml of activated-CH-Sepharose
(Amersham Biosciences Europe GmbH, Freiburg, Germany) according to
the manufacturer's instruction. The column was washed extensively
100 mM Tris-HCl buffer containing pH 8.0, loaded with DGR-TNF crude
extract, diluted in 20 mM Tris-HCl buffer containing 2 mM EDTA pH
8.0, and desorbed by elution with 7 M urea, 100 mM Tris-HCl, pH
8.0. DGR-TNF was then refolded as described (1). Briefly, the
denatured products were dialyzed against 2.33 M urea, 100 mM
Tris-HCl, pH 8.0 at 4.degree. C. (140 min), followed by 1.55 M
urea, 100 mM Tris-HCl, pH 8.0 (140 min), and 1 M urea, 100 mM
Tris-HCl, pH 8 (140 min). Finally the products were dialyzed
against 100 mM Tris-HCl, pH 8.0 (48 h). The products were
centrifuged at 13000.times.g (10 min, 4.degree. C.) and
concentrated by ultra-filtration through a 10 KDa cut-off filter.
All solutions used in the purification and refolding steps were
prepared with sterile and endotoxin-free water (S.A.L.F.
Laboratorio Farmacologico SpA, Bergamo, Italy). Protein
concentration was measured using the BCA Protein Assay Reagent
(Pierce, Rockford, Ill.). Protein purity and identity were checked
by SDS-PAGE, electrospray mass spectrometry (ESI-MS) and analytical
gel-filtration chromatography. The results confirmed the identity
of the product (data not shown). The in vitro cytolytic activity of
DGR-TNF (2.5.times.10.sup.8 U/mg) was measured by standard
cytolytic assay with L-M mouse fibroblasts.
[0277] RP-HPLC of DGR-TNF showed that its retention time was
similar to that of F2 obtained from NGR-TNF (FIG. 13).
TABLE-US-00003 TABLE 1 Molecular mass, cytolytic activity and
isoAsp content of F2 and F4. Molecular Cytolytic Activity isoAsp
Sample mass (Da) .sup.a) (U/mg) .sup.b) (%) .sup.c) NGR-TNF --
.sup. 2.9 .times. 10.sup.8 29.8 F2 17844.5 (3.5 .+-. 0.6) .times.
10.sup.8 75 F4 17843.5 (2.9 .+-. 0.7) .times. 10.sup.8 14 TNF --
.sup. 8 .times. 10.sup.8 2.6 .sup.a) By electrospray
mass-spectrometry .sup.b) By cytolytic assay using L-M murine
fibroblasts (1). .sup.c) Isoaspartyl residues were quantified by
enzymatic assay (ISOQUANT, protein deamidation detection kit,
Promega) and expressed as pmol of isoAsp/pmol of peptide (%).
[0278] All publications mentioned in the above specification are
herein incorporated by reference. Various modifications and
variations of the described methods and system of the present
invention will be apparent to those skilled in the art without
departing from the scope and spirit of the present invention.
Although the present invention has been described in connection
with specific preferred embodiments, it should be understood that
the invention as claimed should not be unduly limited to such
specific embodiments. Indeed, various modifications of the
described modes for carrying out the invention which are obvious to
those skilled in biochemistry and biotechnology or related fields
are intended to be within the scope of the following claims.
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Sequence CWU 1
1
7215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 1Xaa Asn Gly Arg Xaa1 5213PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 2Cys
Asn Gly Arg Cys Val Ser Gly Cys Ala Gly Arg Cys1 5
1036PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 3Asn Gly Arg Ala His Ala1 545PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 4Gly
Asn Gly Arg Gly1 559PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 5Cys Val Leu Asn Gly Arg Met Glu Cys1
565PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Cys Asn Gly Arg Cys1 576PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 7Cys
Asn Gly Arg Cys Gly1 585PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 8Leu Asn Gly Arg Glu1
595PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 9Tyr Asn Gly Arg Thr1 51016PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 10Leu
Gln Cys Ile Cys Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu1 5 10
151116PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 11Leu Gln Cys Ile Ser Thr Gly Asn Gly Arg Gly Glu
Trp Lys Cys Glu1 5 10 151213PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 12Cys Ile Cys Thr Gly Asn Gly
Arg Gly Glu Trp Lys Cys1 5 101313PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 13Cys Ile Ser Thr Gly Asn
Gly Arg Gly Glu Trp Lys Cys1 5 101416PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 14Met
Arg Cys Thr Cys Val Gly Asn Gly Arg Gly Glu Trp Thr Cys Tyr1 5 10
151516PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 15Met Arg Cys Thr Ser Val Gly Asn Gly Arg Gly Glu
Trp Thr Cys Tyr1 5 10 151613PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 16Cys Thr Cys Val Gly Asn Gly
Arg Gly Glu Trp Thr Cys1 5 101713PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 17Cys Thr Ser Val Gly Asn
Gly Arg Gly Glu Trp Thr Cys1 5 10185PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 18Xaa
Asp Gly Arg Xaa1 51913PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 19Cys Asp Gly Arg Cys Val Ser
Gly Cys Ala Gly Arg Cys1 5 10206PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 20Asp Gly Arg Ala His Ala1
5215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 21Gly Asp Gly Arg Gly1 5229PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 22Cys
Val Leu Asp Gly Arg Met Glu Cys1 5235PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 23Cys
Asp Gly Arg Cys1 5246PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 24Cys Asp Gly Arg Cys Gly1
5255PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 25Leu Asp Gly Arg Glu1 5265PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 26Tyr
Asp Gly Arg Thr1 52716PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 27Leu Gln Cys Ile Cys Thr Gly
Asp Gly Arg Gly Glu Trp Lys Cys Glu1 5 10 152816PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Leu
Gln Cys Ile Ser Thr Gly Asp Gly Arg Gly Glu Trp Lys Cys Glu1 5 10
152913PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 29Cys Ile Cys Thr Gly Asp Gly Arg Gly Glu Trp Lys
Cys1 5 103013PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 30Cys Ile Ser Thr Gly Asp Gly Arg Gly
Glu Trp Lys Cys1 5 103116PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 31Met Arg Cys Thr Cys Val Gly
Asp Gly Arg Gly Glu Trp Thr Cys Tyr1 5 10 153216PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 32Met
Arg Cys Thr Ser Val Gly Asp Gly Arg Gly Glu Trp Thr Cys Tyr1 5 10
153313PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 33Cys Thr Cys Val Gly Asp Gly Arg Gly Glu Trp Thr
Cys1 5 103413PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 34Cys Thr Ser Val Gly Asp Gly Arg Gly
Glu Trp Thr Cys1 5 10354PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 35Gly Gly Gly
Gly1365PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 36Cys Ala Arg Ala Cys1 53735DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
37ctggatccga gaagtgtttt gatcatgctg ctggg 353832DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
38tatattaagc tttcagtgcc tctcacactt cc 323949DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
39tatattaagc tttcagtgcc tctcacactt ccactctcca ctgccgctg
494028DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 40caccatgggc aacggccgtg gcggcgtc
284136DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 41tcaggatcct cacagggcaa tgatcccaaa gtagac
36429PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 42Cys Asn Gly Arg Cys Gly Val Arg Tyr1
54325DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 43caccatgtgc gacggccgtt gcggc 254430DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
44ctggatcctc acagagcaat gatcccaaag 304546PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Gly
Arg Cys Asn Asp Gln Asp Thr Arg Thr Ser Tyr Arg Ile Gly Asp1 5 10
15Thr Trp Ser Lys Lys Glu Asn Arg Gly Asn Leu Leu Gln Cys Ile Cys
20 25 30Thr Gly Asn Gly Arg Gly Glu Trp Lys Cys Glu Arg His Thr 35
40 454616PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Leu Gln Cys Ile Ser Thr Gly Asn Gly Arg Gly Glu
Trp Lys Cys Glu1 5 10 154716PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 47Leu Gln Cys Ile Ser Thr Arg
Gly Gly Asn Gly Glu Trp Lys Cys Glu1 5 10 154844PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 48Glu
Ile Cys Thr Thr Asn Glu Gly Val Met Tyr Arg Ile Gly Asp Gln1 5 10
15Trp Asp Lys Gln His Asp Met Gly His Met Met Arg Cys Thr Cys Val
20 25 30Gly Asn Gly Arg Gly Glu Trp Thr Cys Tyr Ala Tyr 35
404916PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Met Arg Cys Thr Cys Val Gly Asn Gly Arg Gly Glu
Trp Thr Cys Tyr1 5 10 15505PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 50Arg Gly Gly Asn Gly1
5515PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Pro His Ser Arg Asn1 5524PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 52Arg
Glu Asp Val1536PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 53Lys Leu Asp Ala Pro Thr1
5545PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 54Xaa Asp Gly Arg Xaa1 55513PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 55Cys
Asp Gly Arg Cys Val Ser Gly Cys Ala Gly Arg Cys1 5
10566PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 56Asp Gly Arg Ala His Ala1 5575PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Gly
Asp Gly Arg Gly1 5589PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 58Cys Val Leu Asp Gly Arg Met
Glu Cys1 5595PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 59Cys Asp Gly Arg Cys1 5606PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 60Cys
Asp Gly Arg Cys Gly1 5615PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 61Leu Asp Gly Arg Glu1
5625PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Tyr Asp Gly Arg Thr1 56316PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 63Leu
Gln Cys Ile Cys Thr Gly Asp Gly Arg Gly Glu Trp Lys Cys Glu1 5 10
156416PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Leu Gln Cys Ile Ser Thr Gly Asp Gly Arg Gly Glu
Trp Lys Cys Glu1 5 10 156513PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 65Cys Ile Cys Thr Gly Asp Gly
Arg Gly Glu Trp Lys Cys1 5 106613PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 66Cys Ile Ser Thr Gly Asp
Gly Arg Gly Glu Trp Lys Cys1 5 106716PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 67Met
Arg Cys Thr Cys Val Gly Asp Gly Arg Gly Glu Trp Thr Cys Tyr1 5 10
156816PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 68Met Arg Cys Thr Ser Val Gly Asp Gly Arg Gly Glu
Trp Thr Cys Tyr1 5 10 156913PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 69Cys Thr Cys Val Gly Asp Gly
Arg Gly Glu Trp Thr Cys1 5 107013PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 70Cys Thr Ser Val Gly Asp
Gly Arg Gly Glu Trp Thr Cys1 5 10716PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 71Cys
Ala Arg Ala Cys Gly1 5724PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 72Gly Val Arg Tyr1
* * * * *