U.S. patent application number 11/806648 was filed with the patent office on 2008-02-14 for inhibition of secretion from non-neuronal cells.
Invention is credited to John Andrew Chaddock, Keith Alan Foster, John Robert Purkiss, Conrad Padraig Quinn.
Application Number | 20080038274 11/806648 |
Document ID | / |
Family ID | 46328821 |
Filed Date | 2008-02-14 |
United States Patent
Application |
20080038274 |
Kind Code |
A1 |
Foster; Keith Alan ; et
al. |
February 14, 2008 |
Inhibition of secretion from non-neuronal cells
Abstract
The present invention relates to treatment of disease by
inhibition of cellular secretory processes, to agents and
compositions therefor, and to manufacture of those agents and
compositions. The present invention relates particularly, to
treatment of disease dependent upon the exocytotic activity of
endocrine cells, exocrine cells, inflammatory cells, cells of the
immune system, cells of the cardiovascular system and bone
cells.
Inventors: |
Foster; Keith Alan;
(Salisbury, GB) ; Chaddock; John Andrew;
(Salisbury, GB) ; Quinn; Conrad Padraig; (Lilburn,
GA) ; Purkiss; John Robert; (Salisbury, GB) |
Correspondence
Address: |
MERCHANT & GOULD PC
P.O. BOX 2903
MINNEAPOLIS
MN
55402-0903
US
|
Family ID: |
46328821 |
Appl. No.: |
11/806648 |
Filed: |
June 1, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11327855 |
Jan 9, 2006 |
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11806648 |
Jun 1, 2007 |
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10088665 |
Aug 14, 2002 |
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PCT/GB00/03681 |
Sep 25, 2000 |
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11327855 |
Jan 9, 2006 |
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Current U.S.
Class: |
424/145.1 ;
424/130.1; 435/375; 514/21.5; 514/21.6; 514/21.8; 514/21.9;
514/7.6; 514/8.5 |
Current CPC
Class: |
A61K 38/1808 20130101;
A61P 11/06 20180101; A61P 5/00 20180101; A61K 38/4886 20130101;
A61P 37/08 20180101; C12Y 304/24069 20130101; C07K 2319/00
20130101; C12N 9/52 20130101; A61K 47/64 20170801; A61P 43/00
20180101; A61P 3/00 20180101; A61P 35/00 20180101; A61P 19/08
20180101; C07K 16/1282 20130101; A61P 29/00 20180101 |
Class at
Publication: |
424/145.1 ;
424/130.1; 435/375; 514/014; 514/015; 514/017; 514/018;
514/002 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61K 39/00 20060101 A61K039/00; A61K 39/395 20060101
A61K039/395; A61P 43/00 20060101 A61P043/00; C12N 5/06 20060101
C12N005/06 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 23, 1999 |
GB |
9922558.3 |
Claims
1. A method for inhibiting secretion from a non-neuronal
inflammatory cell, said method comprising administering an agent
comprising at least first and second domains, wherein the first
domain cleaves one or more proteins essential to exocytosis and the
second domain translocates the first domain into the inflammatory
cell.
2. The method according to claim 1, for treatment of disease
caused, exacerbated or maintained by secretion from said
non-neuronal inflammatory cell.
3. The method according to claim 1 or 2, wherein the agent further
comprises a third domain for targeting the agent to said
non-neuronal inflammatory cell.
4. The method according to claim 3 wherein the third domain
comprises or consists of a growth factor or an integrin-binding
protein; or a ligand selected from (i) for mast cells, complement
receptors in general, including C4 domain of the Fc IgE, and
antibodies/ligands to the C3a/C4a-R complement receptor; (ii) for
eosinophils, antibodies/ligands to the C3a/C4a-R complement
receptor, anti VLA-4 monoclonal antibody, anti-IL5 receptor,
antigens or antibodies reactive toward CR4 complement receptor;
(iii) for macrophages and monocytes, macrophage stimulating factor,
(iv) for macrophages, monocytes and neutrophils, bacterial IPS and
yeast B-glucans which bind to CR3, (v) for neutrophils, antibody to
0X42, an antigen associated with the iC3b complement receptor, or
IL8; (vi) for fibroblasts, mannose 6-phosphate/insulin-like growth
factor-beta (M6P/IGFII) receptor and PA2.26, antibody to a
cell-surface receptor for active fibroblasts in mice.
5. The method according to claim 1 for the treatment of a disease
selected from the group consisting of allergies (seasonal allergic
rhinitis (hay fever), allergic conjunctivitis, vasomotor rhinitis
and food allergy), eosinophilia, asthma, rheumatoid arthritis,
systemic lupus erythematosus, discoid lupus erythematosus,
ulcerative colitis, Crohn's disease, hemorrhoids, pruritus,
glomerulonephritis, hepatitis, pancreatitis, gastritis, vasculitis,
myocarditis, psoriasis, eczema, chronic radiation-induced fibrosis,
lung scarring and other fibrotic disorders.
6. The method according to claim 1, wherein the agent comprises a
first domain that cleaves a protein selected from SNAP-25,
synaptobrevin and syntaxin.
7. The method according to claim 1 wherein the first domain
comprises a light chain of a clostridial neurotoxin, or a fragment,
variant or derivative thereof which inhibits exocytosis.
8. The method according to claim 1, wherein the second domain
comprises a HN region of a clostridial polypeptide, or a fragment,
variant or derivative thereof that translocates the exocytosis
inhibiting activity of the first domain into the cell.
9. The method according to claim 1 for inhibition of constitutive
and regulated release from non-neuronal inflammatory cells.
10. The method according to claim 1, wherein the agent is in the
form of a pharmaceutical composition comprising a pharmaceutically
acceptable carrier.
11. The method according to claim 3, wherein the third domain is
epidermal growth factor.
12. The method according to claim 3, wherein the third domain is an
integrin-binding protein.
13. The method according to claim 12, wherein the third domain
comprises the tri-peptide amino acid sequence Arg-Gly-Asp.
14. The method according to claim 12, wherein the third domain
comprises a sequence selected from Arg-Gly-Asp-Phe-Val (SEQ ID NO:
23); Arg-Gly-Asp-{D-Phe}-{N-methyl-Val} (SEQ ID NO: 23); RGDFV (SEQ
ID NO: 23); RGDfNMeV (SEQ ID NO: 23); GGRGDMFGA (SEQ ID NO: 21);
GGCRGDMFGCA (SEQ ID NO: 22); GRGDSP (SEQ ID NO: 26); GRGESP (SEQ ID
NO: 27); PLAEIDGIEL (SEQ ID NO: 24) and CPLAEIDGIELC (SEQ ID NO:
25), or a sequence having at least 80% identity therewith.
15. The method according to claim 2, wherein the agent further
comprises a third domain for targeting the agent to said
non-neuronal inflammatory cell.
Description
[0001] This application is a continuation-in-part of U.S. patent
application Ser. No. 11/327,855, filed on Jan. 9, 2006, which is a
continuation of U.S. patent application Ser. No. 10/088,665, filed
Aug. 14, 2002, which is a national phase entry of PCT/GB00/03681,
filed Sep. 25, 2000, which claims the benefit of priority of GB
9922558.3, filed Sep. 23, 1999. Each of these applications is
hereby incorporated by reference in their entirety.
[0002] The present invention relates to treatment of disease by
inhibition of cellular secretory processes, to agents and
compositions therefor, and to manufacture of those agents and
compositions. The present invention relates particularly, to
treatment of diseases dependent upon the exocytotic activity of
endocrine cells, exocrine cells, inflammatory cells, cells of the
immune system, cells of the cardiovascular system and bone
cells.
[0003] Exocytosis is the fusion of secretory vesicles with the
plasma membrane and results in the discharge of vesicle content--a
process also known as cell secretion. Exocytosis can be
constitutive or regulated. Constitutive exocytosis is thought to
occur in every cell type whereas regulated exocytosis occurs from
specialised cells.
[0004] The understanding of the mechanisms involved in exocytosis
has increased rapidly, following the proposal of the SNARE
hypothesis (Rothman, 1994, Nature 372, 55-63). This hypothesis
describes protein markers on vesicles, which recognise target
membrane markers. These so-called cognate SNARES (denoted v-SNARE
for vesicle and t-SNARE for target) facilitate docking and fusion
of vesicles with the correct membranes, thus directing discharge of
the vesicular contents into the appropriate compartment. Key to the
understanding of this process has been the identification of the
proteins involved. Three SNARE protein families have been
identified for exocytosis: SNAP-25 and SNAP-23, and syntaxins are
the t-SNARE families in the membrane; and VAMPs (vesicle-associated
membrane protein), including synaptobrevin and cellubrevin, are the
v-SNARE family on secretory vesicles. Key components of the fusion
machinery including SNARES are involved in both regulated and
constitutive exocytosis (De Camilli, 1993, Nature, 364,
387-388).
[0005] The clostridial neurotoxins are proteins with molecular
masses of the order of 150 kDa. They are produced by various
species of the genus Clostridium, most importantly C. tetani and
several strains of C. botulinum. There are at present eight
different classes of the neurotoxins known: tetanus toxin and
botulinum neurotoxin in its serotypes A, B, C.sub.1, D, E, F and G,
and they all share similar structures and modes of action. The
clostridial neurotoxins are synthesized by the bacterium as a
single polypeptide that is modified post-translationally to form
two polypeptide chains joined together by a disulphide bond. The
two chains are termed the heavy chain (H) which has a molecular
mass of approximately 100 kDa and the light chain (LC) which has a
molecular mass of approximately 50 kDa. The clostridial neurotoxins
are highly selective for neuronal cells, and bind with high
affinity thereto [see Black, J. D. and Dolly, J. O. (1987)
Selective location of acceptors for BoNT/A in the central and
peripheral nervous systems. Neuroscience, 23, pp. 767-779;
Habermann, E. and Dreyer, F. (1986) Clostridial
neurotoxins:handling and action at the cellular and molecular
level. Curr. Top. Microbiol. Immunol. 129, pp. 93-179; and
Sugiyama, H. (1980) Clostridium botulinum neurotoxin. Microbiol.
Rev., 44, pp. 419-448 (and internally cited references)].
[0006] The functional requirements of neurointoxication by the
clostridial neurotoxins can be assigned to specific domains within
the neurotoxin structure. The clostridial neurotoxins bind to an
acceptor site on the cell membrane of the motor neuron at the
neuromuscular junction and, following binding to the highly
specific receptor, are internalised by an endocytotic mechanism.
The specific neuromuscular junction binding activity of clostridial
neurotoxins is known to reside in the carboxy-terminal portion of
the heavy chain component of the dichain neurotoxin molecule, a
region known as H.sub.C. The internalised clostridial neurotoxins
possess a highly specific zinc-dependent endopeptidase activity
that hydrolyses a specific peptide bond in at least one of three
protein families, synaptobrevin, syntaxin or SNAP-25, which are
crucial components of the neurosecretory machinery. The
zinc-dependent endopeptidase activity of clostridial neurotoxins is
found to reside in the L-chain (LC). The amino-terminal portion of
the heavy chain component of the dichain neurotoxin molecule, a
region known as H.sub.N, is responsible for translocation of the
neurotoxin, or a portion of it containing the endopeptidase
activity, across the endosomal membrane following internalisation,
thus allowing access of the endopeptidase to the neuronal cytosol
and its substrate protein(s). The result of neurointoxication is
inhibition of neurotransmitter release from the target neuron due
to prevention of release of synaptic vesicle contents.
[0007] The mechanism by which the H.sub.N domain effects
translocation of the endopeptidase into the neuronal cytosol is not
fully characterised but is believed to involve a conformational
change, insertion into the endosomal membrane and formation of some
form of channel or pore through which the endopeptidase can gain
access to the neuronal cytosol. Following binding to its specific
receptor at the neuronal surface pharmacological and morphologic
evidence indicate that the clostridial neurotoxins enter the cell
by endocytosis [Black & Dolly (1986) J. Cell Biol. 103, 535-44]
and then have to pass through a low pH step for neuron intoxication
to occur [Simpson et al (1994) J. Pharmacol Exp. Ther., 269,
256-62]. Acidic pH does not activate the toxin directly via a
structural change, but is believed to trigger the process of LC
membrane translocation from the neuronal endosomal vesicle lumen to
the neuronal cytosol [Montecucco et al (1994) FEBS Lett. 346,
92-98]. There is a general consensus that toxin-determined channels
are related to the translocation process into the cytosol [Schiavo
& Montecucco (1997) in Bacterial Toxins (ed. K. Aktories)].
This model requires that the H.sub.N domain forms a transmembrane
hydrophobic pore across the acidic vesicle membrane that allows the
partially unfolded LC passage through to the cytosol. The requisite
conformational change is believed to be triggered by environmental
factors in the neuronal endosomal compartment into which the
neurotoxin is internalised, and a necessary feature of the binding
domain of the H.sub.C is to target binding sites which enable
internalisation into the appropriate endosomal compartment.
Therefore clostridial neurotoxins have evolved to target cell
surface moieties that fulfil this requirement.
[0008] Hormones are chemical messengers that are secreted by the
endocrine glands of the body. They exercise specific physiological
actions on other organs to which they are carried by the blood. The
range of processes regulated by hormones includes various aspects
of homeostasis (e.g. insulin regulates the concentration of glucose
in the blood), growth (e.g. growth hormone promotes growth and
regulates fat, carbohydrate and protein metabolism), and maturation
(e.g. sex hormones promote sexual maturation and reproduction).
Endocrine hyperfunction results in disease conditions which are
caused by excessive amounts of a hormone or hormones in the
bloodstream. The causes of hyperfunction are classified as
neoplastic, autoimmune, iatrogenic and inflammatory. The endocrine
hyperfunction disorders are a complex group of diseases, not only
because there is a large number of glands that can cause a
pathology (e.g. anterior pituitary, posterior pituitary, thyroid,
parathyroid, adrenal cortex, adrenal medulla, pancreas, ovaries,
testis) but because many of the glands produce more than one
hormone (e.g. the anterior pituitary produces corticotrophin,
prolactin, luteinizing hormone, follicle stimulating hormone,
thyroid stimulating hormone and gonadotrophins). The majority of
disorders that cause hormone excess are due to neoplastic growth of
hormone producing cells. However, certain tumours of non-endocrine
origin can synthesise hormones causing endocrine hyperfunction
disease symptoms. The hormone production under these conditions is
termed "ectopic". Surgical removal or radiation induced destruction
of part or all of the hypersecreting tissue is frequently the
treatment of choice. However, these approaches are not always
applicable, result in complete loss of hormone production or have
to be repeated due to re-growth of the secreting tissue.
[0009] A further level of complexity in endocrine hyperfunction
disorders arises in a group of conditions termed multiple endocrine
neoplasia (MEN) where two or more endocrine glands are involved.
The multiple endocrine neoplasia syndromes (MEN1 and MEN2) are
familial conditions with an autosomal dominant pattern of
inheritance. MEN1 is characterised by the association of
parathyroid hyperplasia, pancreatic endocrine tumours, and
pituitary adenomas, and has a prevalence of about 1 in 10000. MEN2
is the association of medullary cell carcinoma of the thyroid and
phaeochromocytoma, though parathyroid hyperplasia may also occur in
some sufferers.
[0010] Most of the morbidity associated with MEN1 is due to the
effects of pancreatic endocrine tumours. Often surgery is not
possible and the therapeutic aim is to reduce hormone excess. Aside
from reducing tumour bulk, which is often precluded, inhibition of
hormone secretion is the preferred course of action. Current
procedures include subcutaneous application of the somatostatin
analogue, octreotide. However, this approach is only temporarily
effective, and the success diminishes over a period of months.
[0011] Many further disease states are known that involve secretion
from other non-endocrine, non-neuronal cells. It would accordingly
be desirable to treat, reduce or prevent secretion by non-neuronal
cells, such as hyperfunction of the endocrine cells that causes or
leads to these disease conditions.
[0012] The activity of the botulinum neurotoxins is exclusively
restricted to inhibition of neurotransmitter release from neurons.
This is due to the exclusive expression of high affinity binding
sites for clostridial neurotoxins on neuronal cells [see
Daniels-Holgate, P. U. and Dolly, J. O. (1996) Productive and
non-productive binding of botulinum neurotoxin to motor nerve
endings are distinguished by its heavy chain. J. Neurosci. Res. 44,
263-271].
[0013] Non-neuronal cells do not possess the high affinity binding
sites for clostridial neurotoxins, and are therefore refractory to
the inhibitory effects of exogenously applied neurotoxin. Simple
application of clostridial neurotoxins to the surface of
non-neuronal cells does not therefore lead to inhibition of
secretory vesicle exocytosis.
[0014] The productive binding or lack of productive binding of
clostridial neurotoxins thereby defines neuronal and non-neuronal
cells respectively.
[0015] In addition to lacking high affinity binding sites for
clostridial neurotoxins, absence of the correct internalisation and
intracellular routing mechanism, or additional factors that are not
yet understood, would prevent clostridial neurotoxin action in
non-neuronal cells.
[0016] It is known from WO96/33273 that hybrid clostridial
neurotoxins endopeptidases can be prepared and that these hybrids
effectively inhibit release of neurotransmitters from neuronal
cells to which they are targeted, such as pain transmitting
neurons. WO96/33273 describes the activity of hybrids only in
neuronal systems where neuronal mechanisms of internalisation and
vesicular routing are operational.
[0017] Non-neuronal cells are, however, refractory to the effects
of clostridial neurotoxins, since simple application of clostridial
neurotoxins to the surface of non-neuronal cells does not lead to
inhibition of secretory vesicle exocytosis. This insensitivity of
non-neuronal cells to clostridial neurotoxins may be due to absence
of the requisite receptor, absence of the correct internalisation
& intracellular routing mechanism, or additional factors that
are not yet understood.
[0018] WO95/17904 describes the use of C. botulinum holotoxin in
the treatment of various disorders such as excessive sweating,
lacrimation and mucus secretion, and pain. WO95/17904 describes
treatment by targeting neuronal cells
[0019] It is an object of the present invention to provide methods
and compositions for inhibition of secretion from non-neuronal
cells.
[0020] Accordingly, the present invention is based upon the use of
a composition which inhibits the exocytotic machinery in neuronal
cells and which surprisingly has been found to be effective at
inhibiting exocytotic processes in non-neuronal cells.
[0021] A first aspect of the invention thus provides a method of
inhibiting secretion from a non-neuronal cell comprising
administering an agent comprising at least first and second
domains, wherein the first domain cleaves one or more proteins
essential to exocytosis and the second domain translocates the
first domain into the cell.
[0022] Advantageously, the invention provides for inhibition of
non-neuronal secretion and enables treatment of disease caused,
exacerbated or maintained by such secretion.
[0023] An agent for use in the invention is suitably prepared by
replacement of the cell-binding H.sub.C domain of a clostridial
neurotoxin with a ligand capable of binding to the surface of
non-neuronal cells. Surprisingly, this agent is capable of
inhibiting the exocytosis of a variety of secreted substances from
non-neuronal cells. By covalently linking a clostridial neurotoxin,
or a hybrid of two clostridial neurotoxins, in which the H.sub.C
region of the H-chain has been removed or modified, to a new
molecule or moiety, the Targeting Moiety (TM), an agent is produced
that binds to a binding site (BS) on the surface of the relevant
non-neuronal secretory cells. A further surprising aspect of the
present invention is that if the L-chain of a clostridial
neurotoxin, or a fragment, variant or derivative of the L-chain
containing the endopeptidase activity, is covalently linked to a TM
which can also effect internalisation of the L-chain, or a fragment
of the endopeptidase activity, into the cytoplasm of a non-neuronal
secretory cell, this also produces an agent capable of inhibiting
secretion. Thus, the present invention overcomes the
insusceptibility of non-neuronal cells to the inhibitory effects of
clostridial neurotoxins.
[0024] An example of an agent of the invention is a polypeptide
comprising first and second domains, wherein said first domain
cleaves one or more vesicle or plasma-membrane associated proteins
essential to neuronal exocytosis and wherein said second domain
translocates the polypeptide into the cell or translocates at least
that portion responsible for the inhibition of exocytosis into the
non-neuronal cell. The polypeptide can be derived from a neurotoxin
in which case the polypeptide is typically free of clostridial
neurotoxin and free of any clostridial neurotoxin precursor that
can be converted into toxin by proteolytic action, being
accordingly substantially non-toxic and suitable for therapeutic
use. Accordingly, the invention may thus use polypeptides
containing a domain equivalent to a clostridial toxin light chain
and a domain providing the translocation function of the H.sub.N of
a clostridial toxin heavy chain, whilst lacking the functional
aspects of a clostridial toxin H.sub.C domain.
[0025] In use of the invention, the polypeptide is administered in
vivo to a patient, the first domain is translocated into a
non-neuronal cell by action of the second domain and cleaves one or
more vesicle or plasma-membrane associated proteins essential to
the specific cellular process of exocytosis, and cleavage of these
proteins results in inhibition of exocytosis, thereby resulting in
inhibition of secretion, typically in a non-cytotoxic manner.
[0026] The polypeptide of the invention may be obtained by
expression of a recombinant nucleic acid, preferably a DNA, and can
be a single polypeptide, that is to say not cleaved into separate
light and heavy chain domains or two polypeptides linked for
example by a disulphide bond.
[0027] The first domain preferably comprises a clostridial toxin
light chain or a functional fragment or variant of a clostridial
toxin light chain. The fragment is optionally an N-terminal, or
C-terminal fragment of the light chain, or is an internal fragment,
so long as it substantially retains the ability to cleave the
vesicle or plasma-membrane associated protein essential to
exocytosis. The minimal domains necessary for the activity of the
light chain of clostridial toxins are described in J. Biol. Chem.,
Vol. 267, No. 21, July 1992, pages 14721-14729. The variant has a
different peptide sequence from the light chain or from the
fragment, though it too is capable of cleaving the vesicle or
plasma-membrane associated protein. It is conveniently obtained by
insertion, deletion and/or substitution of a light chain or
fragment thereof. A variety of variants are possible, including (i)
an N-terminal extension to a clostridial toxin light chain or
fragment (ii) a clostridial toxin light chain or fragment modified
by alteration of at least one amino acid (iii) a C-terminal
extension to a clostridial toxin light chain or fragment, or (iv)
combinations of 2 or more of (i)-(iii). In further embodiments of
the invention, the variant contains an amino acid sequence modified
so that (a) there is no protease sensitive region between the LC
and H.sub.N components of the polypeptide, or (b) the protease
sensitive region is specific for a particular protease. This latter
embodiment is of use if it is desired to activate the endopeptidase
activity of the light chain in a particular environment or cell,
though, in general, the polypeptides of the invention are in an
active form prior to administration.
[0028] The first domain preferably exhibits endopeptidase activity
specific for a substrate selected from one or more of SNAP-25,
synaptobrevin/VAMP and syntaxin. The clostridial toxin from which
this domain can be obtained or derived is preferably botulinum
toxin or tetanus toxin. The polypeptide can further comprise a
light chain or fragment or variant of one toxin type and a heavy
chain or fragment or variant of another toxin type.
[0029] The second domain preferably comprises a clostridial toxin
heavy chain H.sub.N portion or a fragment or variant of a
clostridial toxin heavy chain H.sub.N portion. The fragment is
optionally an N-terminal or C-terminal or internal fragment, so
long as it retains the function of the H.sub.N domain. Teachings of
regions within the H.sub.N responsible for its function are
provided for example in Biochemistry 1995, 34, pages 15175-15181
and Eur. J. Biochem, 1989, 185, pages 197-203. The variant has a
different sequence from the H.sub.N domain or fragment, though it
too retains the function of the H.sub.N domain. It is conveniently
obtained by insertion, deletion and/or substitution of a H.sub.N
domain or fragment thereof, and examples of variants include (i) an
N-terminal extension to a H.sub.N domain or fragment, (ii) a
C-terminal extension to a H.sub.N domain or fragment, (iii) a
modification to a H.sub.N domain or fragment by alteration of at
least one amino acid, or (iv) combinations of 2 or more of
(i)-(iii). The clostridial toxin is preferably botulinum toxin or
tetanus toxin.
[0030] In preparation of the polypeptides by recombinant means,
methods employing fusion proteins can be employed, for example a
fusion protein comprising a fusion of (a) a polypeptide of the
invention as described above with (b) a second polypeptide adapted
for binding to a chromatography matrix so as to enable purification
of the fusion protein using said chromatography matrix. It is
convenient for the second polypeptide to be adapted to bind to an
affinity matrix, such as glutathione Sepharose, enabling rapid
separation and purification of the fusion protein from an impure
source, such as a cell extract or supernatant.
[0031] One second purification polypeptide is
glutathione-S-transferase (GST), and others may be chosen so as to
enable purification on a chromatography column according to
conventional techniques.
[0032] In a second aspect of the invention there is provided a
method of inhibiting secretion from selected non-neuronal cells
responsible for regulated secretion by administering an agent of
the invention.
[0033] In a third aspect of the invention there is provided a
method of treatment of disease resulting, or caused or maintained
by secretions from non-neuronal cells, comprising administering an
agent of the invention.
[0034] In further aspects of the invention there are provided
agents of the invention targeted to non-neuronal cells responsible
for secretion.
[0035] In one embodiment of the invention, an agent is provided for
the treatment of conditions resulting from hyperfunction of
endocrine cells, for example endocrine neoplasia.
[0036] Accordingly, an agent of the invention is used in the
treatment of endocrine hyperfunction, to inhibit secretion of
endocrine cell-derived chemical messengers. An advantage of the
invention is that effective treatment of endocrine hyperfunction
and associated disease states is now provided, offering relief to
sufferers where hitherto there was none and no such agent
available.
[0037] A further advantage of the invention is that agents are made
available which, in use, result in the inhibition of or decrease in
hypersecretion of multiple hormones from a single endocrine gland.
Thus, the multitude of disorders that result from hyperfunction of
one gland (e.g. the anterior pituitary) will be simultaneously
treated by a reduction in the function of the hypersecreting
gland.
[0038] The agent preferably comprises a ligand or targeting domain
which binds to an endocrine cell, and is thus rendered specific for
these cell types. Examples of suitable ligands include iodine;
thyroid stimulating hormone (TSH); TSH receptor antibodies;
antibodies to the islet-specific monosialo-ganglioside GM2-1;
insulin, insulin-like growth factor and antibodies to the receptors
of both; TSH releasing hormone (protirelin) and antibodies to its
receptor; FSH/LH releasing hormone (gonadorelin) and antibodies to
its receptor; corticotrophin releasing hormone (CRH) and antibodies
to its receptor; and ACTH and antibodies to its receptor. According
to the invention, an endocrine targeted agent may thus be suitable
for the treatment of a disease selected from: endocrine neoplasia
including MEN; thyrotoxicosis and other diseases dependent on
hypersecretions from the thyroid; acromegaly, hyperprolactinaemia,
Cushings disease and other diseases dependent on anterior pituitary
hypersecretion; hyperandrogenism, chronic anovulation and other
diseases associated with polycystic ovarian syndrome.
[0039] In a further embodiment, an agent of the invention is used
for the treatment of conditions resulting from secretions of
inflammatory cells, for example allergies. Ligands suitable to
target agent to these cells include (i) for mast cells, complement
receptors in general, including C4 domain of the Fc IgE, and
antibodies/ligands to the C3a/C4a-R complement receptor; (ii) for
eosinophils, antibodies/ligands to the C3a/C4a-R complement
receptor, anti VLA-4 monoclonal antibody, anti-IL5 receptor,
antigens or antibodies reactive toward CR4 complement receptor;
(iii) for macrophages and monocytes, macrophage stimulating factor,
(iv) for macrophages, monocytes and neutrophils, bacterial LPS and
yeast B-glucans which bind to CR3, (v) for neutrophils, antibody to
OX42, an antigen associated with the iC3b complement receptor, or
IL8; (vi) for fibroblasts, mannose 6-phosphate/insulin-like growth
factor-beta (M6P/IGF-II) receptor and PA2.26, antibody to a
cell-surface receptor for active fibroblasts in mice.
[0040] According to a preferred embodiment of the present
invention, the TM is a growth factor, preferably an epidermal
growth factor (EGF), vascular endothelial growth factor,
platelet-derived growth factor, keratinocyte growth factor,
hepatocyte growth factor, transforming growth factor alpha,
transforming growth factor beta.
[0041] According to another preferred embodiment of the present
invention, the TM is a peptide or protein that binds to an
inflammatory cell. A preferred example of such a TM is an
integrin-binding protein.
[0042] Integrins are obligate heterodimer transmembrane proteins
containing two distinct chains a (alpha) and D (beta) subunits. In
mammals, 19 alpha and 8 beta subunits have been characterised--see
Humphries, M. J. (2000), Integrin structure. Biochem Soc Trans. 28:
311-339, which is herein incorporated by reference thereto.
Integrin subunits span through the plasma membrane, and in general
have very short cytoplasmic domains of about 40-70 amino acids.
Outside the cell plasma membrane, the alpha and beta chains lie
close together along a length of about 23 nm, the final 5 nm
NH.sub.2-termini of each chain forming a ligand-binding region to
which an agent of the present invention binds.
[0043] Preferred integrin-binding proteins of the present invention
comprise the amino sequence Arg-Gly-Asp ("RGD"), which binds to the
above-described ligand-binding region--see Craig. D et al. (2004),
Structural insights into how the MIDAS ion stabilizes integrin
binding to an RGD peptide under force. Structure, vol. 12, pp
2049-2058, which is herein incorporated by reference thereto.
[0044] In one embodiment, the integrin-binding protein TMs of the
present invention have an amino acid length of between 3 and 100,
preferably between 3 and 50, more preferably between 5 and 25, and
particularly preferably between 5 and 15 amino acid residues.
[0045] The TMs of the present invention may form linear or cyclic
structures.
[0046] Preferred integrin-binding TMs of the present invention
include actin, alpha-actinin, focal contact adhesion kinase,
paxillin, talin, RACK1, collagen, laminin, fibrinogen, heparin,
phytohaemagglutinin, fibronectin, vitronectin, VCAM-1, ICAM-1,
ICAM-2 and serum protein. Many integrins recognise the triple
Arg-Gly-Asp (RGD) peptide sequence (Ruoslahti, 1996). The RGD motif
is found in over 100 proteins including fibronectin, tenascin,
fibrinogen and vitronectin. The RGD-integrin interaction is
exploited as a conserved mechanism of cell entry by many pathogens
including coxsackievirus (Roivaninen et al., 1991) and adenovirus
(Mathias et al., 1994).
[0047] Additionally preferred integrin-binding TMs of the present
invention include proteins selected from the following sequences:
Arg-Gly-Asp-Phe-Val (SEQ ID NO:23);
Arg-Gly-Asp-{D-Phe}-{N-methyl-Val} (SEQ ID NO:23); RGDFV (SEQ ID
NO:23); RGDfNMeV (SEQ ID NO:23); GGRGDMFGA (SEQ ID NO:21);
GGCRGDMFGCA (SEQ ID NO:22); GRGDSP (SEQ ID NO:26); GRGESP (SEQ ID
NO:27); PLAEIDGIEL (SEQ ID NO:24 and CPLAEIDGIELC (SEQ ID NO:25).
Reference to the above sequences embraces linear and cyclic forms,
together with peptides exhibiting at least 80%, 85%, 90%, 95%, 98%,
99% sequence identity with said sequences. All of said TMs
preferably retain the "RGD" tri-peptide sequence.
[0048] Diseases thus treatable according to the invention include
diseases selected from allergies (seasonal allergic rhinitis (hay
fever), allergic conjunctivitis, vasomotor rhinitis and food
allergy), eosinophilia, asthma, rheumatoid arthritis, systemic
lupus erythematosus, discoid lupus erythematosus, ulcerative
colitis, Crohn's disease, hemorrhoids, pruritus,
glomerulonephritis, hepatitis, pancreatitis, gastritis, vasculitis,
myocarditis, psoriasis, eczema, chronic radiation-induced fibrosis,
lung scarring and other fibrotic disorders.
[0049] VAMP expression has been demonstrated in B-lymphocytes [see
Olken, S. K. and Corley, R. B. 1998, Mol. Biol. Cell. 9, 207a].
Thus, an agent according to the present invention, when targeted to
a B-lymphocyte and following internalisation and retrograde
transport, may exert its inhibitory effect on such target
cells.
[0050] In a further embodiment, an agent of the invention is
provided for the treatment of conditions resulting from secretions
of the exocrine cells, for example acute pancreatitis (Hansen et
al, 1999, J. Biol. Chem. 274, 22871-22876). Ligands suitable to
target agent to these cells include pituitary adenyl cyclase
activating peptide (PACAP-38) or an antibody to its receptor. The
present invention also concerns treatment of mucus hypersecretion
from mucus-secreting cells located in the alimentary tract, in
particular located in the colon.
[0051] Gaisano, H. Y. et al. (1994) J. Biol. Chem. 269, pp.
17062-17066 has demonstrated that, following in vitro membrane
permeabilisation to permit cellular entry, tetanus toxin light
chain cleaves a vesicle-associated membrane protein (VAMP) isoform
2 in rat pancreatic zymogen granules, and inhibits enzyme
secretion. Thus, an agent according to the present invention, when
targeted to a pancreatic cell and following internalisation and
retrograde transport, may exert its inhibitory effect on such
target cells.
[0052] In a further embodiment, an agent of the invention is used
for the treatment of conditions resulting from secretions of
immunological cells, for example autoimmune disorders where B
lymphocytes are to be targeted (immunosuppression). Ligands
suitable to target agent to these cells include Epstein Barr virus
fragment/surface feature or idiotypic antibody (binds to CR2
receptor on B-lymphocytes and lymph node follicular dendritic
cells). Diseases treatable include myasthenia gravis, rheumatoid
arthritis, systemic lupus erythematosus, discoid lupus
erythematosus, organ transplant, tissue transplant, fluid
transplant, Graves disease, thyrotoxicosis, autoimmune diabetes,
hemolytic anaemia, thrombocytopenic purpura, neutropenia, chronic
autoimmune hepatitis, autoimmune gastritis, pernicious anaemia,
Hashimoto's thyroiditis, Addison's disease, Sjogren's syndrome,
primary biliary cirrhosis, polymyositis, scleroderma, systemic
sclerosis, pemphigus vulgaris, bullous pemphigoid, myocarditis,
rheumatic carditis, glomerulonephritis (Goodpasture type), uveitis,
orchitis, ulcerative colitis, vasculitis, atrophic gastritis,
pernicious anaemia, type 1 diabetes mellitus.
[0053] By using cell permeabilisation techniques it has been
possible to internalise BoNT/C into eosinophils [see Pinxteren J A,
et al (2000) Biochimie, April; 82(4):385-93 Thirty years of
stimulus-secretion coupling: from Ca(2.sup.+) to GTP in the
regulation of exocytosis]. Following internalisation, BoNT/C
exerted an inhibitory effect on exocytosis in eosinophils. Thus, an
agent according to the present invention, when targeted to an
eosinophil and following internalisation and retrograde transport,
may exert its inhibitory effect on such target cells.
[0054] In a further embodiment of the invention, an agent is
provided for the treatment of conditions resulting from secretions
of cells of the cardiovascular system. Suitable ligands for
targeting platelets for the treatment of disease states involving
inappropriate platelet activation and thrombus formation include
thrombin and TRAP (thrombin receptor agonist peptide) or antibodies
to CD31/PECAM-1, CD24 or CD106NCAM-1, and ligands for targeting
cardiovascular endothelial cells for the treatment of hypertension
include GP1b surface antigen recognising antibodies.
[0055] In a further embodiment of the invention, an agent is
provided for the treatment of bone disorders. Suitable ligands for
targeting osteoblasts for the treatment of a disease selected from
osteopetrosis and osteoporosis include calcitonin, and for
targeting an agent to osteoclasts include osteoclast
differentiation factors (eg. TRANCE, or RANKL or OPGL), and an
antibody to the receptor RANK.
[0056] In use of the invention, a Targeting moiety (TM) provides
specificity for the BS on the relevant non-neuronal secretory
cells. The TM component of the agent can comprise one of many cell
binding molecules, including, but not limited to, antibodies,
monoclonal antibodies, antibody fragments (Fab, F(ab)'.sub.2, Fv,
ScFv, etc.), lectins, hormones, cytokines, growth factors,
peptides, carbohydrates, lipids, glycons, nucleic acids or
complement components.
[0057] The TM is selected in accordance with the desired cell-type
to which the agent of the present invention is to be targeted, and
preferably has a high specificity and/or affinity for non-neuronal
target cells. Preferably, the TM does not substantially bind to
neuronal cells of the presynaptic muscular junction, and thus the
agent is substantially non-toxic in that it is not capable of
effecting muscular paralysis. This is in contrast to clostridial
holotoxin which targets the presynaptic muscular junction and
effects muscular paralysis. In addition, preferably the TM does not
substantially bind to neuronal peripheral sensory cells, and thus
the agent does not exert any substantial analgesic effect.
Preferably, the TM does not substantially bind to neuronal cells,
and does not therefore permit the agent to exert an inhibitory
effect on secretion in a neuronal cell.
[0058] It is known in the art that the H.sub.C portion of the
neurotoxin molecule can be removed from the other portion of the
H-chain, known as H.sub.N, such that the H.sub.N fragment remains
disulphide linked to the L-chain of the neurotoxin providing a
fragment known as LH.sub.N. Thus, in one embodiment of the present
invention the LH.sub.N fragment of a clostridial neurotoxin is
covalently linked, using linkages which may include one or more
spacer regions, to a TM.
[0059] In another embodiment of the invention, the H.sub.C domain
of a clostridial neurotoxin is mutated, blocked or modified, e.g.
by chemical modification, to reduce or preferably incapacitate its
ability to bind the neurotoxin to receptors at the neuromuscular
junction. This modified clostridial neurotoxin is then covalently
linked, using linkages which may include one or more spacer
regions, to a TM.
[0060] In another embodiment of the invention, the heavy chain of a
clostridial neurotoxin, in which the H.sub.C domain is mutated,
blocked or modified, e.g. by chemical modification, to reduce or
preferably incapacitate its ability to bind the neurotoxin to
receptors at the neuromuscular junction, is combined with the
L-chain of a different clostridial neurotoxin. This hybrid,
modified clostridial neurotoxin is then covalently linked, using
linkages which may include one or more spacer regions, to a TM.
[0061] In another embodiment of the invention, the H.sub.N domain
of a clostridial neurotoxin is combined with the L-chain of a
different clostridial neurotoxin. This hybrid LH.sub.N is then
covalently linked, using linkages which may include one or more
spacer regions, to a TM.
[0062] In another embodiment of the invention, the light chain of a
clostridial neurotoxin, or a fragment of the light chain containing
the endopeptidase activity, is covalently linked, using linkages
which may include one or more spacer regions, to a TM which can
also effect the internalisation of the L-chain, or a fragment of
the L-chain containing the endopeptidase activity, into the
cytoplasm of the relevant non-neuronal cells responsible for
secretion.
[0063] In another embodiment of the invention, the light chain of a
clostridial neurotoxin, or a fragment of the light chain containing
the endopeptidase activity, is covalently linked, using linkages
which may include one or more spacer regions, to a translocation
domain to effect transport of the endopeptidase fragment into the
cytosol. Examples of translocation domains derived from bacterial
neurotoxins are as follows:
[0064] Botulinum type A neurotoxin--amino acid residues
(449-871)
[0065] Botulinum type B neurotoxin--amino acid residues
(441-858)
[0066] Botulinum type C neurotoxin--amino acid residues
(442-866)
[0067] Botulinum type D neurotoxin--amino acid residues
(446-862)
[0068] Botulinum type E neurotoxin--amino acid residues
(423-845)
[0069] Botulinum type F neurotoxin--amino acid residues
(440-864)
[0070] Botulinum type G neurotoxin--amino acid residues
(442-863)
[0071] Tetanus neurotoxin--amino acid residues (458-879)
[0072] other clostridial sources include--C. butyricum, and C.
argentinense.
[0073] [for the genetic basis of toxin production in Clostridium
botulinum and C. tetani, see Henderson et al (1997) in The
Clostridia: Molecular Biology and Pathogenesis, Academic
press].
[0074] In addition to the above translocation domains derived from
clostridial sources, other non-clostridial sources may be employed
in an agent according to the present invention. These include, for
example, diphtheria toxin [London, E. (1992) Biochem. Biophys.
Acta., 1112, pp. 25-51], Pseudomonas exotoxin A [Prior et al (1992)
Biochem., 31, pp. 3555-3559], influenza virus haemagglutinin
fusogenic peptides [Wagner et al (1992) PNAS, 89, pp. 7934-7938],
and amphiphilic peptides [Murata et al (1992) Biochem., 31, pp.
1986-1992].
[0075] In use, the domains of an agent according to the present
invention are associated with each other. In one embodiment, two or
more of the Domains may be joined together either directly (e.g. by
a covalent linkage), or via a linker molecule. Conjugation
techniques suitable for use in the present invention have been well
documented:--Chemistry of protein conjugation and cross-linking.
Edited by Wong, S. S. 1993, CRC Press Inc., Florida; and
Bioconjugate techniques, Edited by Hermanson, G. T. 1996, Academic
Press, London, UK.
[0076] Direct linkage of two or more of Domains is now described
with reference to clostridial neurotoxins and to the present
Applicant's nomenclature of clostridial neurotoxin domains, namely
Domain B (contains the binding domain), Domain T (contains the
translocation domain) and Domain E (contains the protease domain),
although no limitation thereto is intended.
[0077] In one embodiment of the present invention, Domains E and T
may be mixed together in equimolar quantities under reducing
conditions and covalently coupled by repeated dialysis (e.g. at
4.degree. C., with agitation), into physiological salt solution in
the absence of reducing agents. At this stage, in contrast to
Example 6 of WO94/21300, the E-T complex is not blocked by
iodoacetamide, therefore any remaining free --SH groups are
retained.
[0078] Domain B is then modified, for example, by derivatisation
with SPDP followed by subsequent reduction. In this reaction, SPDP
does not remain attached as a spacer molecule to Domain B, but
simply increases the efficiency of this reduction reaction.
[0079] Reduced domain B and the E-T complex may then be mixed under
non-reducing conditions (e.g. at 4.degree. C.) to form a
disulphide-linked E-T-B "agent".
[0080] In another embodiment, a coupled E-T complex may be prepared
according to Example 6 of WO94/21300, including the addition of
iodoacetamide to block free sulphydryl groups. However, the E-T
complex is not further derivatised, and the remaining chemistry
makes use of the free amino (--NH.sub.2) groups on amino acid side
chains (e.g. lysine, and arginine amino acids).
[0081] Domain B may be derivatised using carbodiimide chemistry
(e.g. using EDC) to activate carboxyl groups on amino acid side
chains (e.g. glutamate, and aspartate amino acids), and the E-T
complex mixed with the derivatised Domain B to result in a
covalently coupled (amide bond) E-T-B complex.
[0082] Suitable methodology for the creation of such an agent is,
for example, as follows:--
[0083] Domain B was dialysed into MES buffer (0.1 M MES, 0.1 M
sodium chloride, pH 5.0) to a final concentration of 0.5 mg/ml.
EDAC (1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride)
was added to final concentrations of 0.2 mg/ml and reacted for 30
min at room temperature. Excess EDAC was removed by desalting over
a MES buffer equilibrated PD-10 column (Pharmacia). The derivatised
domain B was concentrated (to >2 mg/ml) using Millipore Biomax
10 concentrators. The E-T complex (1 mg/ml) was mixed for 16 hours
at 4.degree. C., and the E-T-B complex purified by size-exclusion
chromatography over a Superose 12 HR10/30 column (Pharmacia) to
remove unreacted Domain B (column buffer: 50 mM sodium phosphate
pH6.5+20 mM NaCl).
[0084] As an alternative to direct covalent linkage of the various
Domains of an agent according to the present invention, suitable
spacer molecules may be employed. The term linker molecule is used
synonymously with spacer molecule. Spacer technology was readily
available prior to the present application.
[0085] For example, one particular coupling agent (SPDP) is
described in Example 6 of WO94/21300 (see lines 3-5 on page 16). In
Example 6, SPDP is linked to an E-T complex, thereby providing an
E-T complex including a linker molecule. This complex is then
reacted a Domain B, which becomes attached to the E-T complex via
the linker molecule. In this method, SPDP results in a spacing
region of approximately 6.8 Angstroms between different Domains of
the "agent" of the present invention.
[0086] A variant of SPDP known as LC-SPDP is identical in all
respects to SPDP but for an increased chain length. LC-SPDP may be
used to covalently link two Domains of the "agent" of the present
invention resulting in a 15.6 Angstrom spacing between these
Domains.
[0087] Examples of spacer molecules include, but are not limited
to:--
[0088] (GGGGS).sub.2 (SEQ ID NO:28), elbow regions of Fab--[see
Anand et al., (1991) J. Biol. Chem. 266, 21874-9];
[0089] (GGGGS).sub.3 (SEQ ID NO:28)-[see Brinkmann et al. (1991)
Proc. Natl. Acad. Sci. 88, 8616-20];
[0090] the interdomain linker of cellulose--[see Takkinen et al.
(1991) Protein Eng, 4, 837-841];
[0091] PPPIEGR (SEQ ID NO:29)-[see Kim (1993) Protein Science, 2,
348-356];
[0092] Collagen-like spacer--[see Rock (1992) Protein Engineering,
vol 5, No 6, pp 583-591]; and
[0093] Trypsin-sensitive diphtheria toxin peptide-[see O'Hare
(1990) FEBS, vol 273, No 1, 2, pp 200-204].
[0094] In a further embodiment of the present invention, an agent
having the structure E-X-T-X-B, where "X" is a spacer molecule
between each domain, may be prepared, for example, as
follows:--
[0095] Domain E is derivatised with SPDP, but not subsequently
reduced. This results in an SPDP-derivatised Domain E.
[0096] Domain T is similarly prepared, but subsequently reduced
with 10 mM dithiothreitol (DTT). The 10 mM DTT present in the
Domain T preparation, following elution from the QAE column (see
Example 6 in WO94/21300), is removed by passage of Domain T through
a sephadex G-25 column equilibrated in PBS.
[0097] Domain T free of reducing agent is then mixed with the
SPDP-derivatised Domain E, with agitation at 4.degree. C. for 16
hours. E-T complex is isolated from free Domain E and from free
Domain T by size-exclusion chromatography (Sephadex G-150).
Whereafter, the same procedure can be followed as described in
Example 6 of WO94/21300 for rederivatisation of the E-T complex
with SPDP, and subsequent coupling thereof to the free sulphydryl
on Domain B.
[0098] The agents according to the present invention may be
prepared recombinantly.
[0099] In one embodiment, the preparation of a recombinant agent
may involve arrangement of the coding sequences of the selected TM
and clostridial neurotoxin component in a single genetic construct.
These coding sequences may be arranged in-frame so that subsequent
transcription and translation is continuous through both coding
sequences and results in a fusion protein. All constructs would
have a 5' ATG codon to encode an N-terminal methionine, and a
C-terminal translational stop codon.
[0100] Thus, a the light chain of a clostridial neurotoxin (or a
fragment of the light chain containing the endopeptidase activity)
may be expressed recombinantly as a fusion protein with a TM which
can also effect the internalisation of the L-chain (or a fragment
thereof) into the cytoplasm of the relevant non-neuronal cells
responsible for secretion. The expressed fusion protein may also
include one or more spacer regions.
[0101] In the case of an agent based on clostridial neurotoxin, the
following information would be required to produce said agent
recombinantly:--(i) DNA sequence data relating to a selected TM;
(ii) DNA sequence data relating to the clostridial neurotoxin
component; and (iii) a protocol to permit construction and
expression of the construct comprising (i) and (ii).
[0102] All of the above basic information (i)-(iii) are either
readily available, or are readily determinable by conventional
methods. For example, both WO98/07864 and WO99/17806 exemplify
clostridial neurotoxin recombinant technology suitable for use in
the present application.
[0103] In addition, methods for the construction and expression of
the constructs of the present invention may employ information from
the following references and others:
[0104] Lorberboum-Galski, H., FitzGerald, D., Chaudhary, V., Adhya,
S., Pastan, I. (1988). Cytotoxic activity of an interleukin
2-Pseudomonas exotoxin chimeric protein produced in Escherichia
coli. Proc Natl Acad Sci USA 85(6):1922-6;
[0105] Murphy, J. R. (1988) Diphtheria-related peptide hormone gene
fusions: a molecular genetic approach to chimeric toxin
development. Cancer Treat Res; 37:123-40;
[0106] Williams, D. P., Parker, K., Bacha, P., Bishai, W.,
Borowski, M., Genbauffe, F., Strom, T. B., Murphy, J. R. (1987).
Diphtheria toxin receptor binding domain substitution with
interleukin-2: genetic construction and properties of a diphtheria
toxin-related interleukin-2 fusion protein. Protein Eng;
1(6):493-8;
[0107] Arora, N., Williamson, L. C., Leppla, S. H., Halpern, J. L.
(1994). Cytotoxic effects of a chimeric protein consisting of
tetanus toxin light chain and anthrax toxin lethal factor in
non-neuronal cells J Biol Chem, 269(42):26165-71;
[0108] Brinkmann, U., Reiter, Y., Jung, S. H., Lee, B., Pastan, I.
(1993). A recombinant immunotoxin containing a
disulphide-stabilized Fv fragment. Proc Natl Acad Sci USA;
90(16):7538-42; and
[0109] O'Hare, M., Brown, A. N., Hussain, K., Gebhardt, A., Watson,
G., Roberts, L. M., Vitetta, E. S., Thorpe, P. E., Lord, J. M.
(1990). Cytotoxicity of a recombinant ricin-A-chain fusion protein
containing a proteolytically-cleavable spacer sequence. FEBS Lett
October 29; 273(1-2):200-4.
[0110] Suitable clostridial neurotoxin sequence information
relating to L- and LH.sub.N-chains may be obtained from, for
example, Kurazono, H. (1992) J. Biol. Chem., vol. 267, No. 21, pp.
14721-14729; and Popoff, M. R., and Marvaud, J.-C. (1999) The
Comprehensive Sourcebook of Bacterial Protein Toxins, 2nd edition
(ed. Alouf, J. E., and Freer, J. H.), Academic Press, pp.
174-201.
[0111] Similarly, suitable TM sequence data are widely available in
the art. Alternatively, any necessary sequence data may be obtained
by techniques which were well-known to the skilled person.
[0112] For example, DNA encoding the TM component may be cloned
from a source organism by screening a cDNA library for the correct
coding region (for example by using specific oligonucleotides based
on the known sequence information to probe the library), isolating
the TM DNA, sequencing this DNA for confirmation purposes, and then
placing the isolated DNA in an appropriate expression vector for
expression in the chosen host.
[0113] As an alternative to isolation of the sequence from a
library, the available sequence information may be employed to
prepare specific primers for use in PCR, whereby the coding
sequence is then amplified directly from the source material and,
by suitable use of primers, may be cloned directly into an
expression vector.
[0114] Another alternative method for isolation of the coding
sequence is to use the existing sequence information and synthesise
a copy, possibly incorporating alterations, using DNA synthesis
technology. For example, DNA sequence data may be generated from
existing protein and/or RNA sequence information. Using DNA
synthesis technology to do this (and the alternative described
above) enables the codon bias of the coding sequence to be modified
to be optimal for the chosen expression host. This may give rise to
superior expression levels of the fusion protein.
[0115] Optimisation of the codon bias for the expression host may
be applied to the DNA sequences encoding the TM and clostridial
components of the construct. Optimisation of the codon bias is
possible by application of the protein sequence into freely
available DNA/protein database software, e.g. programs available
from Genetics Computer Group, Inc.
[0116] According to a further aspect of the present invention,
nucleic acid encoding the light chain of a clostridial neurotoxin
(or a fragment of the light chain containing the endopeptidase
activity), may be associated with a TM which can also effect the
internalisation of the nucleic acid encoding the L-chain (or a
fragment thereof) into the cytoplasm of the relevant non-neuronal
cells responsible for secretion. The nucleic acid sequence may be
coupled to a translocation domain, and optionally to a targeting
moiety, by for example direct covalent linkage or via spacer
molecule technology. Ideally, the coding sequence will be expressed
in the target cell.
[0117] Thus, the agent of the present invention may be the
expression product of a recombinant gene delivered independently to
the preferred site of action of the agent. Gene delivery
technologies are widely reported in the literature [reviewed in
"Advanced Drug Delivery Reviews" Vol. 27, (1997), Elsevier Science
Ireland Ltd].
[0118] According to another aspect, the present invention therefore
provides a method of treating a condition or disease which is
susceptible of treatment with a nucleic acid in a mammal e.g. a
human which comprises administering to the sufferer an effective,
non-toxic amount of a compound of the invention. A condition or
disease which is susceptible of treatment with a nucleic acid may
be for example a condition or disease which may be treated by or
requiring gene therapy. The preferred conditions or diseases
susceptible to treatment according to the present invention,
together with the preferred TMs, have been described previously in
this specification. Similarly, the preferred first domains which
cleave one or more proteins (eg. SNAP-25, synaptobrevin and
syntaxin) essential to exocytosis have been described previously in
this specification. The various domains of an agent for use in gene
therapy may be directly linked (e.g. via a covalent bond) or
indirectly linked (e.g. via a spacer molecule), as for example
previously described in this specification.
[0119] The invention further provides a compound of the invention
for use as an active therapeutic substance, in particular for use
in treating a condition or disease as set forth in the present
claims.
[0120] The invention further provides pharmaceutical compositions
comprising an agent or a conjugate of the invention and a
pharmaceutically acceptable carrier.
[0121] In use the agent or conjugate will normally be employed in
the form of a pharmaceutical composition in association with a
human pharmaceutical carrier, diluent and/or excipient, although
the exact form of the composition will depend on the mode of
administration.
[0122] The conjugate may, for example, be employed in the form of
an aerosol or nebulisable solution for inhalation or a sterile
solution for parenteral administration, intra-articular
administration or intra-cranial administration.
[0123] For treating endocrine targets, i.v. injection, direct
injection into gland, or aerosolisation for lung delivery are
preferred; for treating inflammatory cell targets, i.v. injection,
sub-cutaneous injection, or surface patch administration are
preferred; for treating exocrine targets, i.v. injection, or direct
injection into the gland are preferred; for treating immunological
targets, i.v. injection, or injection into specific tissues e.g.
thymus, bone marrow, or lymph tissue are preferred; for treatment
of cardiovascular targets, i.v. injection is preferred; and for
treatment of bone targets, i.v. injection, or direct injection is
preferred. In cases of i.v. injection, this should also include the
use of pump systems.
[0124] The dosage ranges for administration of the compounds of the
present invention are those to produce the desired therapeutic
effect. It will be appreciated that the dosage range required
depends on the precise nature of the conjugate, the route of
administration, the nature of the formulation, the age of the
patient, the nature, extent or severity of the patient's condition,
contraindications, if any, and the judgement of the attending
physician.
[0125] Suitable daily dosages are in the range 0.0001-1 mg/kg,
preferably 0.0001-0.5 mg/kg, more preferably 0.002-0.5 mg/kg, and
particularly preferably 0.004-0.5 mg/kg. The unit dosage can vary
from less that 1 microgram to 30 mg, but typically will be in the
region of 0.01 to 1 mg per dose, which may be administered daily or
less frequently, such as weekly or six monthly.
[0126] Wide variations in the required dosage, however, are to be
expected depending on the precise nature of the conjugate, and the
differing efficiencies of various routes of administration. For
example, oral administration would be expected to require higher
dosages than administration by intravenous injection.
[0127] Variations in these dosage levels can be adjusted using
standard empirical routines for optimisation, as is well understood
in the art.
[0128] Compositions suitable for injection may be in the form of
solutions, suspensions or emulsions, or dry powders which are
dissolved or suspended in a suitable vehicle prior to use.
[0129] Fluid unit dosage forms are typically prepared utilising a
pyrogen-free sterile vehicle. The active ingredients, depending on
the vehicle and concentration used, can be either dissolved or
suspended in the vehicle.
[0130] Solutions may be used for all forms of parenteral
administration, and are particularly used for intravenous
injection. In preparing solutions the compound can be dissolved in
the vehicle, the solution being made isotonic if necessary by
addition of sodium chloride and sterilised by filtration through a
sterile filter using aseptic techniques before filling into
suitable sterile vials or ampoules and sealing. Alternatively, if
solution stability is adequate, the solution in its sealed
containers may be sterilised by autoclaving.
[0131] Advantageously additives such as buffering, solubilising,
stabilising, preservative or bactericidal, suspending or
emulsifying agents and/or local anaesthetic agents may be dissolved
in the vehicle.
[0132] Dry powders which are dissolved or suspended in a suitable
vehicle prior to use may be prepared by filling pre-sterilised drug
substance and other ingredients into a sterile container using
aseptic technique in a sterile area.
[0133] Alternatively the agent and other ingredients may be
dissolved in an aqueous vehicle, the solution is sterilized by
filtration and distributed into suitable containers using aseptic
technique in a sterile area. The product is then freeze dried and
the containers are sealed aseptically.
[0134] Parenteral suspensions, suitable for intramuscular,
subcutaneous or intradermal injection, are prepared in
substantially the same manner, except that the sterile compound is
suspended in the sterile vehicle, instead of being dissolved and
sterilisation cannot be accomplished by filtration. The compound
may be isolated in a sterile state or alternatively it may be
sterilised after isolation, e.g. by gamma irradiation.
[0135] Advantageously, a suspending agent for example
polyvinylpyrrolidone is included in the composition to facilitate
uniform distribution of the compound.
[0136] Compositions suitable for administration via the respiratory
tract include aerosols, nebulisable solutions or microfine powders
for insufflation. In the latter case, particle size of less than 50
microns, especially less than 10 microns, is preferred. Such
compositions may be made up in a conventional manner and employed
in conjunction with conventional administration devices.
[0137] The agent described in this invention can be used in vivo,
either directly or as a pharmaceutically acceptable salt, for the
treatment of conditions involving secretion from non-neuronal
cells, such as hypersecretion of endocrine cell derived chemical
messengers, hypersecretion from exocrine cells, secretions from the
cells of the immune system, the cardiovascular system and from bone
cells.
[0138] The present invention will now be described by reference to
the following examples illustrated by the accompanying drawings in
which:--
[0139] FIG. 1 shows SDS-PAGE analysis of WGA-LH.sub.N/A
purification scheme;
[0140] FIG. 2 shows activity of WGA-LH.sub.N/A on release of
transmitter from HIT-T15 cells;
[0141] FIG. 3 shows correlation of SNAP-25 cleavage with inhibition
of neurotransmitter release following application of WGA-LH.sub.N/A
to HIT-T15 cells;
[0142] FIG. 4 shows activity of WGA-LH.sub.N/A on release of
[.sup.3H]-noradrenaline from undifferentiated PC12 cells;
[0143] FIG. 5 shows a Western blot indicating expression of
recLH.sub.N/B in E. coli;
[0144] FIG. 6 shows in vitro cleavage of synthetic VAMP peptide by
recLH.sub.N/B;
[0145] FIG. 7 shows the effect of low pH and BoNT/B treatment on
stimulated von Willebrands Factor (vWF) release from human
umbilical vein endothelial cells;
[0146] FIG. 8 shows release of [.sup.3H]-glucosamine labelled high
molecular weight material from LS180 cells;
[0147] FIG. 9 shows the effect of low pH and BoNT/B treatment on
stimulated .beta.-glucuronidase release from differentiated HL60
cells;
[0148] FIG. 10 shows purification of a LHN/C-EGF fusion
protein;
[0149] FIG. 11 shows purification of a LHN/B-EGF fusion
protein;
[0150] FIG. 12 shows purification of a LHN/C-RGD fusion
protein;
[0151] FIG. 13 shows purification of a LHN/C-cyclic RGD fusion
protein;
[0152] FIG. 14 shows purification of a LC/C-RGD-HN/C fusion
protein;
[0153] FIG. 15 shows VAMP cleavage activity of LHN/B-EGF;
[0154] FIG. 16 shows effect of 10 nm Syntaxin compounds con
LPS-mediated IL-8 secretion by THP-1 cells;
[0155] FIG. 17 shows effect of 10 nm Syntaxin compounds con
LPS-mediated IL-10 secretion by RPMI-8226 cells;
[0156] FIG. 18 shows effect of EGF and fusions on IL-8 production
and on LPS-stimulated IL-8 secretion; and
[0157] FIG. 19 shows effect of EGF and fusions on IP-10 production
and on PHA-stimulated IP-10 secretion.
[0158] FIGS. 5-19 are now described in more detail.
[0159] Referring to FIG. 5, MBP-LH.sub.N/B was expressed in E. coli
as described in Example 4. Lane 1 represents the profile of the
expressed fusion protein in E. coli. Lane 2 represents the profile
of fusion protein expression in the crude E. coli lysate. Lane 3
represents the profile of the MBP-LH.sub.N/B following purification
by immobilised amylose. Molecular weights in kDa are indicated to
the right side of the Figure.
[0160] Referring to FIG. 6, dilutions of recLH.sub.N/B (prepared as
described in Example 4) and BoNT/B were compared in an in vitro
peptide cleavage assay. Data indicate that the recombinant product
has similar catalytic activity to that of the native neurotoxin,
indicating that the recombinant product has folded correctly into
an active conformation.
[0161] Referring to FIG. 7, cells were exposed to pH 4.7 media with
or without 500 nM BoNT/B (control cells received pH7.4 medium) for
2.5 hours then washed. 24 hours later release of vWF was stimulated
using 1 mM histamine and the presented results are the net
stimulated release with basal subtracted. Results are presented in
mIU of vWF/ml and are the mean +/-SEM of three determinations apart
from pH 4.7 alone which is two determinations. pH 4.7+BoNT/B has
reduced vWF release by 27.4% compared to pH 4.7 controls.
[0162] Referring to FIG. 8, high molecular weight mucin
synthesising colon carcinoma LS180 cells were treated with pH 4.7
medium and pH 4.7 medium containing 500 nM botulinum neurotoxin
type B (BoNT/B) for four hours then labelled with
[.sup.3H]-glucosamine for 18 hours. Release of high molecular
weight material was stimulated with 10 .mu.M ionomycin and
[.sup.3H]-glucosamine labelled material recovered by
ultracentrifugation and centrifugal molecular weight sieving.
Radiolabel of release of labelled high molecular weight material
was determined by scintillation counting and net stimulated release
calculated by subtracting non-stimulated basal values. Data are
expressed as disintegrations per minute (dpm)+/-SEM of three
determinations. BoNT/B co-treatment clearly inhibits the release of
high molecular weight material from these mucin synthesising cells
and in this experiment a 74.5% reduction was seen.
[0163] Referring to FIG. 9, cells were exposed to pH 4.8 media with
or without 500 nM BoNT/B (control cells received pH 7.4 medium) for
2.5 hours then washed and differentiated for 40 hours by the
addition of 300 .mu.M dibutyryl cyclic AMP (dbcAMP). Cells were
stimulated with fMet-Leu-Phe (1 .mu.M)+ATP (100 .mu.M) in the
presence of cytochalasin B (5 .mu.M) for 10 minutes and released
.beta.-glucuronidase determined by colourimetric assay. Net
stimulated release was calculated by subtraction of unstimulated
basal release values from stimulated values and released activity
is expressed as a percentage of the total activity present in the
cells. Data are the mean +/-SEM of three determinations. BoNT/B
treatment in low pH medium significantly inhibited stimulated
release of .beta.-glucuronidase compared to cells treated with low
pH alone (p=0.0315 when subjected to a 2 tailed Student T test with
groups of unequal variance).
[0164] Referring to FIG. 10, using the methodology outlined in
Example 11, a LHN/C-EGF fusion protein was purified from E. coli
BL21 cells. Briefly, the soluble products obtained following cell
disruption were applied to a nickel-charged affinity capture
column. Bound proteins were eluted with 100 mM imidazole, treated
with Factor Xa to activate the fusion protein and remove the
maltose-binding protein (MBP) tag, then re-applied to a second
nickel-charged affinity capture column. Samples from the
purification procedure were assessed by SDS-PAGE. Lane 1 & 6:
Molecular mass markers (kDa), lane 2: Clarified crude cell lysate,
lane 3: First nickel chelating Sepharose column eluant, lane 4:
Factor Xa digested protein, lane 5: Purified LHN/C-EGF under
non-reducing conditions, lane 7: Purified LHN/C-EGF under reduced
conditions.
[0165] Referring to FIG. 11, using the methodology outlined in
Example 12, a LHN/B-EGF fusion protein was purified from E. coli
BL21 cells. Briefly, the soluble products obtained following cell
disruption were applied to a nickel-charged affinity capture
column. Bound proteins were eluted with 100 mM imidazole, treated
with Factor Xa and enterokinase to activate the fusion protein and
remove the maltose-binding protein (MBP) tag, then re-applied to a
second nickel-charged affinity capture column. Samples from the
purification procedure were assessed by SDS-PAGE. The final
purified material in the absence and presence of reducing agent is
identified in the lanes marked [-] and [+] respectively.
[0166] Referring to FIG. 12, using the methodology outlined in
Example 13, a LHN/C-RGD fusion protein was purified from E. coli
BL21 cells. Briefly, the soluble products obtained following cell
disruption were applied to a nickel-charged affinity capture
column. Bound proteins were eluted with 100 mM imidazole, treated
with Factor Xa to activate the fusion protein and remove the
maltose-binding protein (MBP) tag, then re-applied to a second
nickel-charged affinity capture column. Samples from the
purification procedure were assessed by SDS-PAGE. The final
purified material in the absence and presence of reducing agent is
identified in the lanes marked [-] and [+] respectively.
[0167] Referring to FIG. 13, using the methodology outlined in
Example 14, a LHN/C-cyclic RGD fusion protein was purified from E.
coli BL21 cells. Briefly, the soluble products obtained following
cell disruption were applied to a nickel-charged affinity capture
column. Bound proteins were eluted with 100 mM imidazole, treated
with Factor Xa to activate the fusion protein and remove the
maltose-binding protein (MBP) tag, then re-applied to a second
nickel-charged affinity capture column. Samples from the
purification procedure were assessed by SDS-PAGE. The final
purified material in the absence and presence of reducing agent is
identified in the lanes marked [-] and [+] respectively.
[0168] Referring to FIG. 14, using the methodology outlined in
Example 15, a LC/C-RGD-HN/C fusion protein was purified from E.
coli BL21 cells. Briefly, the soluble products obtained following
cell disruption were applied to a nickel-charged affinity capture
column. Bound proteins were eluted with 100 mM imidazole, treated
with Factor Xa to activate the fusion protein and remove the
maltose-binding protein (MBP) tag, then re-applied to a second
nickel-charged affinity capture column. Samples from the
purification procedure were assessed by SDS-PAGE. The final
purified material in the absence and presence of reducing agent is
identified in the lanes marked [-] and [+] respectively.
[0169] Referring to FIG. 15, using the methodology outlined in
example 16, BoNT/B (.circle-solid.), LHN/B (.box-solid.) and
LHN/B-EGF (.tangle-solidup.) were assayed for VAMP cleavage
activity.
[0170] Referring to FIG. 16, using the methodology outlined in
Example 17, the activity of EGF-LHN/C(SXN100501) and EGF-LHN/B
(SXN100328) was assessed in THP-1 immune cells. The quantity of
secreted IL-8 was determined by Luminex-based technology. Data are
presented as % of LPS control.
[0171] Referring to FIG. 17, using the methodology outlined in
Example 18, the activity of EGF-LHN/C (SXN100501) and EGF-LHN/B
(SXN100328) was assessed in RPMI-8226 immune cells. The quantity of
secreted IL-10 was determined by Luminex-based technology. Data are
presented as % of LPS control.
[0172] Referring to FIG. 18, using the methodology outlined in
Example 19, the activity of EGF-LHN/C (SXN100501) and EGF-LHN/B
(SXN100328) and CP-RGD-LHN/C (SXN100221) was assessed in PBMC
immune cells. The quantity of secreted IL-8 was determined by
Luminex-based technology. Data are presented as % of LPS
control.
[0173] Referring to FIG. 19, using the methodology outlined in
Example 20, the activity of EGF-LHN/C (SXN100501) and EGF-LHN/B
(SXN100328) and CP-RGD-LHN/C (SXN100221) was assessed in PBMC
immune cells. The quantity of secreted IP-10 was determined by
Luminex-based technology. Data are presented as % of PHA
control.
EXAMPLES
Example 1
Production of a Conjugate of a Lectin from Triticum vulgaris and
LH.sub.N/A
Materials
[0174] Lectin from Triticum vulgaris (Wheat Germ Agglutinin-WGA)
was obtained from Sigma Ltd.
[0175] SPDP was from Pierce Chemical Co.
[0176] PD-10 desalting columns were from Pharmacia.
[0177] Dimethylsulphoxide (DMSO) was kept anhydrous by storage over
a molecular sieve.
[0178] Denaturing sodium dodecylsulphate polyacrylamide gel
electrophoresis (SDS-PAGE) and non-denaturing polyacrylamide gel
electrophoresis was performed using gels and reagents from
Novex.
[0179] Additional reagents were obtained from Sigma Ltd.
[0180] LH.sub.N/A was prepared according to a previous method
(Shone, C. C. and Tranter, H. S. (1995) in "Clostridial
Neurotoxins--The molecular pathogenesis of tetanus and botulism",
(Montecucco, C., Ed.), pp. 152-160, Springer). FPLC chromatography
media and columns were obtained from Amersham Pharmacia Biotech,
UK. Affi-gel.RTM. Hz matrix and materials were from BioRad, UK.
Preparation of an Anti-BoNT/A Antibody-Affinity Column
[0181] An antibody-affinity column was prepared with specific
monoclonal antibodies essentially as suggested by the
manufacturers.quadrature. protocol. Briefly, monoclonal antibodies
5BA2.3 & 5BA9.3 which have different epitope recognition in the
H.sub.C domain (Hallis, B., Fooks, S., Shone, C. and Hambleton, P.
(1993) in "Botulinum and Tetanus Neurotoxins", (DasGupta, B. R.,
Ed.), pp. 433-436, Plenum Press, New York) were purified from mouse
hybridoma tissue culture supernatant by Protein G (Amersham
Pharmacia Biotech) chromatography. These antibodies represent a
source of BoNT/A H.sub.C-specific binding molecules and can be
immobilised to a matrix or used free in solution to bind BoNT/A. In
the presence of partially purified LH.sub.N/A (which has no H.sub.C
domain) these antibodies will only bind to BoNT/A. The antibodies
5BA2.3 & 5BA9.3 were pooled in a 3:1 ratio and two mg of the
pooled antibody was oxidised by the addition of sodium periodate
(final concentration of 0.2%) prior coupling to 1 ml Affi-Gel
Hz.TM. gel (16 hours at room temperature). Coupling efficiencies
were routinely greater than 65%. The matrix was stored at 4.degree.
C. in the presence of 0.02% sodium azide.
Purification Strategy for the Preparation of Pure LH.sub.N/A
[0182] BoNT/A was treated with 17 .mu.g trypsin per mg BoNT/A for a
period of 72-120 hours. After this time no material of 150 kDa was
observed by SDS-PAGE and Coomassie blue staining. The trypsin
digested sample was chromatographed (FPLC system, Amersham
Pharmacia Biotech) on a Mono Q column (HR5/5) to remove trypsin and
separate the majority of BoNT/A from LH.sub.N/A. The crude sample
was loaded onto the column at pH 7 in 20 mM HEPES, 50 mM NaCl and 2
ml LH.sub.N/A fractions eluted in a NaCl gradient from 50 mM to 150
mM. The slightly greater pl of BoNT/A (6.3) relative to LH.sub.N/A
(5.2) encouraged any BoNT/A remaining after trypsinisation to elute
from the anion exchange column at a lower salt concentration than
LH.sub.N/A. LH.sub.N/A containing fractions (as identified by
SDS-PAGE) were pooled for application to the antibody column.
[0183] The semi-purified LH.sub.N/A mixture was applied and
reapplied at least 3 times to a 1-2 ml immobilised monoclonal
antibody matrix at 20.degree. C. After a total of 3 hours in
contact with the immobilised antibodies, the LH.sub.N/A-enriched
supernatant was removed. Entrapment of the BoNT/A contaminant,
rather than specifically binding the LH.sub.N/A, enables the
elution conditions to be maintained at the optimum for LH.sub.N
stability. The use of harsh elution conditions e.g. low pH, high
salt, chaotropic ions, which may have detrimental effects on
LH.sub.N polypeptide folding and enzymatic activity, are therefore
avoided. Treatment of the immobilised antibody column with 0.2M
glycine/HCl pH2.5 resulted in regeneration of the column and
elution of BoNT/A-reactive proteins of 150 kDa.
[0184] The LH.sub.N/A enriched sample was then applied 2 times to a
1 ml HiTrap.TM. Protein G column (Amersham Pharmacia Biotech) at
20.degree. C. Protein G was selected since it has a high affinity
for mouse monoclonal antibodies. This step was included to remove
BoNT/A-antibody complexes that may leach from the immunocolumn.
Antibody species bind to the Protein G matrix allowing purified
LH.sub.N/A to elute, essentially by the method of Shone C. C.,
Hambleton, P., and Melling, J. 1987, Eur. J. Biochem. 167, 175-180,
and as described in PCT/GB00/03519.
Methods
[0185] The lyophilised lectin was rehydrated in phosphate buffered
saline (PBS) to a final concentration of 10 mg/ml. Aliquots of this
solution were stored at -20.degree. C. until use.
[0186] The WGA was reacted with an equal concentration of SPDP by
the addition of a 10 mM stock solution of SPDP in DMSO with mixing.
After one hour at room temperature the reaction was terminated by
desalting into PBS over a PD-10 column.
[0187] The thiopyridone leaving group was removed from the product
to release a free --SH group by reduction with dithiothreitol (DTT;
5 mM; 30 min). The thiopyridone and DTT were removed by once again
desalting into PBS over a PD-10 column.
[0188] The LH.sub.N/A was desalted into PBSE (PBS containing 1 mM
EDTA). The resulting solution (0.5-1.0 mg/ml) was reacted with a
four-fold molar excess of SPDP by addition of a 10 mM stock
solution of SPDP in DMSO. After 3 h at room temperature the
reaction was terminated by desalting over a PD-10 column into
PBSE.
[0189] A portion of the derivatized LH.sub.N/A was removed from the
solution and reduced with DTT (5 mM, 30 min). This sample was
analyzed spectrophotometrically at 280 nm and 343 nm to determine
the degree of derivatisation. The degree of derivatisation achieved
was 3.53+/-0.59 mol/mol.
[0190] The bulk of the derivatized LH.sub.N/A and the derivatized
WGA were mixed in proportions such that the WGA was in greater than
three-fold molar excess. The conjugation reaction was allowed to
proceed for >16 h at 4.degree. C.
[0191] The product mixture was centrifuged to clear any precipitate
that had developed. The supernatant was concentrated by
centrifugation through concentrators (with 10000 molecular weight
exclusion limit) before application to a Superose 12 column on an
FPLC chromatography system (Pharmacia). The column was eluted with
PBS and the elution profile followed at 280 nm.
[0192] Fractions were analyzed by SDS-PAGE on 4-20% polyacrylamide
gradient gels, followed by staining with Coomassie Blue. The major
conjugate products have an apparent molecular mass of between
106-150 kDa, these are separated from the bulk of the remaining
unconjugated LH.sub.N/A and more completely from the unconjugated
WGA. Fractions containing conjugate were pooled prior to addition
to PBS-washed N-acetylglucosamine-agarose. Lectin-containing
proteins (i.e. WGA-LH.sub.N/A conjugate) remained bound to the
agarose during washing with PBS to remove contaminants
(predominantly unconjugated LH.sub.N/A). WGA-LH.sub.N/A conjugate
was eluted from the column by the addition of 0.3M
N-acetylglucosamine (in PBS) and the elution profile followed at
280 nm. See FIG. 1 for SDS-PAGE profile of the whole purification
scheme.
[0193] The fractions containing conjugate were pooled, dialysed
against PBS, and stored at 4.degree. C. until use.
Example 2
Activity of WGA-LH.sub.N/A in Cultured Endocrine Cells
(HIT-T15)
[0194] The hamster pancreatic B cell line HIT-T15 is an example of
a cell line of endocrine origin. It thus represents a model cell
line for the investigation of inhibition of release effects of the
agents. HIT-T15 cells possess surface moieties that allow for the
binding, and internalisation, of WGA-LH.sub.N/A.
[0195] In contrast, HIT-T15 cells lack suitable receptors for
clostridial neurotoxins and are therefore not susceptible to
botulinum neurotoxins (BoNTs).
[0196] FIG. 2 illustrates the inhibition of release of insulin from
HIT-T15 cells after prior incubation with WGA-LH.sub.N/A. It is
clear that dose-dependent inhibition is observed, indicating that
WGA-LH.sub.N/A can inhibit the release of insulin from an endocrine
cell model.
[0197] Inhibition of insulin release was demonstrated to correlate
with cleavage of the SNARE protein, SNAP-25 (FIG. 3). Thus,
inhibition of release of chemical messenger is due to a clostridial
endopeptidase-mediated effects of SNARE-protein cleavage.
Materials
[0198] Insulin radioimmunoassay kits were obtained from Linco
Research Inc., USA. Western blotting reagents were obtained from
Novex.
Methods
[0199] HIT-T15 cells were seeded onto 12 well plates and cultured
in RPMI-1640 medium containing 5% foetal bovine serum, 2 mM
L-glutamine for 5 days prior to use. WGA-LH.sub.N/A was applied for
4 hours on ice, the cells were washed to remove unbound
WGA-LH.sub.N/A, and the release of insulin assayed 16 hours later.
The release of insulin from HIT-T15 cells was assessed by
radioimmunoassay exactly as indicated by the manufacturer's
instructions.
[0200] Cells were lysed in 2M acetic acid/0.1% TFA. Lysates were
dried then resuspended in 0.1 M Hepes, pH 7.0. To extract the
membrane proteins Triton-X-114 (10%, v/v) was added and incubated
at 4.degree. C. for 60 min. The insoluble material was removed by
centrifugation and the supernatants were warmed to 37.degree. C.
for 30 min. The resulting two phases were separated by
centrifugation and the upper phase discarded. The proteins in the
lower phase were precipitated with chloroform/methanol for analysis
by Western blotting.
[0201] The samples were separated by SDS-PAGE and transferred to
nitrocellulose. Proteolysis of SNAP-25, a crucial component of the
neurosecretory process and the substrate for the zinc-dependent
endopeptidase activity of BoNT/A, was then detected by probing with
an antibody (SMI-81) that recognises both the intact and cleaved
forms of SNAP-25.
Example 3
Activity of WGA-LH.sub.N/A in Cultured Neuroendocrine Cells
(PC12)
[0202] The rat pheochromocytoma PC12 cell line is an example of a
cell line of neuroendocrine origin. In its undifferentiated form it
has properties associated with the adrenal chromaffin cell [Greene
and Tischler, in "Advances in Cellular Neurobiology" (Federoff and
Hertz, eds), Vol. 3, p 373-414. Academic Press, New York, [982]. It
thus represents a model cell line for the investigation of
inhibition of release effects of the agents. PC12 cells possess
surface moieties that allow for the binding, and internalisation,
of WGA-LH.sub.N/A. FIG. 4 illustrates the inhibition of release of
noradrenaline from PC12 cells after prior incubation with
WGA-LH.sub.N/A. It is clear that dose-dependent inhibition is
observed, indicating that WGA-LH.sub.N/A can inhibit the release of
hormone from a neuroendocrine cell model. Comparison of the
inhibition effects observed with conjugate and the untargeted
LH.sub.N/A demonstrate the requirement for a targeting moiety (TM)
for efficient inhibition of transmitter release.
Methods
[0203] PC12 cells were cultured on 24 well plates in RPMI-1640
medium containing 10% horse serum, 5% foetal bovine serum, 1%
L-glutamine. Cells were treated with a range of concentrations of
WGA-LH.sub.N/A for three days. Secretion of noradrenaline was
measured by labelling cells with [.sup.3\H]-noradrenaline (2
.mu.Ci/ml, 0.5 ml/well) for 60 min. Cells were washed every 15 min
for 1 hour then basal release determined by incubation with a
balanced salt solution containing 5 mM KCl for 5 min. Secretion was
stimulated by elevating the concentration of extracellular
potassium (100 mM KCl) for 5 min. Radioactivity in basal and
stimulated superfusates was determined by scintillation counting.
Secretion was expressed as a percentage of the total uptake and
stimulated secretion was calculated by subtracting basal.
Inhibition of secretion was dose-dependent with an observed
IC.sub.50 of 0.63+/-0.15 .mu.g/ml (n=3). Inhibition was
significantly more potent when compared to untargeted endopeptidase
(LH.sub.N/A in FIG. 4). Thus WGA-LH.sub.N/A inhibits release of
neurotransmitter from a model neuroendocrine cell type.
Example 4
Expression and Purification of Catalytically Active Recombinant
LH.sub.N/B
[0204] The coding region for LH.sub.N/B was inserted in-frame to
the 3' of the gene encoding maltose binding protein (MBP) in the
expression vector pMAL (New England Biolabs). In this construct,
the expressed MBP and LH.sub.N/B polypeptides are separated by a
Factor Xa cleavage site.
[0205] Expression of the MBP-LH.sub.N/B in E. coli TG1 was induced
by addition of IPTG to the growing culture at an approximate OD600
nm of 0.8. Expression was maintained for a further 3 hours in the
presence of inducing agent prior to harvest by centrifugation. The
recovered cell paste was stored at -20.degree. C. until
required.
[0206] The cell paste was resuspended in resuspension buffer (50 mM
Hepes pH7.5+150 mM NaCl.sup.+ a variety of protease inhibitors) at
6 ml buffer per gram paste. To this suspension was added lysozyme
to a final concentration of 1 mg/ml. After 10 min at 0.degree. C.,
the suspension was sonicated for 6.times.30 seconds at 24.mu. at
0.degree. C. The broken cell paste was then centrifuged to remove
cell debris and the supernatant recovered for chromatography.
[0207] In some situations, the cell paste was disrupted by using
proprietary disruption agents such as BugBuster.TM. (Novagen) as
per the manufacturers protocol. These agents were satisfactory for
disruption of the cells to provide supernatant material for
affinity chromatography.
[0208] The supernatant was applied to an immobilised amylose matrix
at 0.4 ml/min to facilitate binding of the fusion protein. After
binding, the column was washed extensively with resuspension buffer
to remove contaminating proteins. Bound proteins were eluted by the
addition of elution buffer (resuspension buffer+10 mM maltose) and
fractions collected. Eluted fractions containing protein were
pooled for treatment with Factor Xa.
[0209] On some occasions a further purification step was
incorporated into the scheme, prior to the addition of Factor Xa.
In these instances, the eluted fractions were made to 5 mM DTT and
applied to a Pharmacia Mono-Q HR5/5 column (equilibrated in
resuspension buffer) as part of an FPLC system. Proteins were bound
to the column at 150 mM NaCl, before increased to 500 mM NaCl over
a gradient. Fractions were collected and analysed for the presence
of MBP-LH.sub.N/B by Western blotting (probe antibody=guinea pig
anti-BoNT/B or commercially obtained anti-MBP).
[0210] Cleavage of the fusion protein by Factor Xa was as described
in the protocol supplied by the manufacturer (New England Biolabs).
Cleavage of the fusion protein resulted in removal of the MBP
fusion tag and separation of the LC and H.sub.N domains of
LH.sub.N/B. Passage of the cleaved mixture through a second
immobilised maltose column removed free MBP from the mixture to
leave purified disulphide-linked LH.sub.N/B. This material was used
for conjugation.
[0211] See FIG. 5 for an illustration of the purification of
LH.sub.N/B.
[0212] See FIG. 6 for an illustration of the in vitro catalytic
activity of LH.sub.N/B.
Example 5
Production of a Conjugate of a Lectin from Triticum vulgaris and
LH.sub.N/B
Materials
[0213] Lectin from Triticum vulgaris (WGA) was obtained from Sigma
Ltd.
[0214] LH.sub.N/B was prepared as described in Example 4.
[0215] SPDP was from Pierce Chemical Co.
[0216] PD-10 desalting columns were from Pharmacia.
[0217] Dimethylsulphoxide (DMSO) was kept anhydrous by storage over
a molecular sieve.
[0218] Polyacrylamide gel electrophoresis was performed using gels
and reagents from Novex.
[0219] Additional reagents were obtained from Sigma Ltd.
Methods
[0220] The lyophilised lectin was rehydrated in phosphate buffered
saline (PBS) to a final concentration of 10 mg/ml. Aliquots of this
solution were stored at -20.degree. C. until use.
[0221] The WGA was reacted with an equal concentration of SPDP by
the addition of a 10 mM stock solution of SPDP in DMSO with mixing.
After one hour at room temperature the reaction was terminated by
desalting into PBS over a PD-10 column.
[0222] The thiopyridone leaving group was removed from the product
to release a free --SH group by reduction with dithiothreitol (DTT;
5 mM; 30 min). The thiopyridone and DTT were removed by once again
desalting into PBS over a PD-10 column.
[0223] The recLH.sub.N/B was desalted into PBS. The resulting
solution (0.5-1.0 mg/ml) was reacted with a four-fold molar excess
of SPDP by addition of a 10 mM stock solution of SPDP in DMSO.
After 3 h at room temperature the reaction was terminated by
desalting over a PD-10 column into PBS.
[0224] A portion of the derivatized recLH.sub.N/B was removed from
the solution and reduced with DTT (5 mM, 30 min). This sample was
analysed spectrophotometrically at 280 nm and 343 nm to determine
the degree of derivatisation.
[0225] The bulk of the derivatized recLH.sub.N/B and the
derivatized WGA were mixed in proportions such that the WGA was in
greater than three-fold molar excess. The conjugation reaction was
allowed to proceed for >16 h at 4.degree. C.
[0226] The product mixture was centrifuged to clear any precipitate
that had developed. The supernatant was concentrated by
centrifugation through concentrators (with 10000 molecular weight
exclusion limit) before application to a Superdex G-200 column on
an FPLC chromatography system (Pharmacia). The column was eluted
with PBS and the elution profile followed at 280 nm.
[0227] Fractions were analysed by SDS-PAGE on 4-20% polyacrylamide
gradient gels, followed by staining with Coomassie Blue. The major
conjugate products have an apparent molecular mass of between
106-150 kDa, these are separated from the bulk of the remaining
unconjugated recLH.sub.N/B and more completely from the
unconjugated WGA. Fractions containing conjugate were pooled prior
to addition to PBS-washed N-acetylglucosamine-agarose.
Lectin-containing proteins (i.e. WGA-recLH.sub.N/B conjugate)
remained bound to the agarose during washing with PBS to remove
contaminants (predominantly unconjugated recLH.sub.N/B).
WGA-recLH.sub.N/B conjugate was eluted from the column by the
addition of 0.3M N-acetylglucosamine (in PBS) and the elution
profile followed at 280 nm.
[0228] The fractions containing conjugate were pooled, dialysed
against PBS, and stored at 4.degree. C. until use.
Example 6
Activity of BoNT/B in Vascular Endothelial Cells
[0229] Human umbilical vein endothelial cells (HUVEC) secrete von
Willebrands Factor (vWF) when stimulated with a variety of cell
surface receptor agonists including histamine. These cells maintain
this property when prepared from full term umbilical cords and
grown in culture (Loesberg et al 1983, Biochim. Biophys. Acta. 763,
160-168). The release of vWF by HUVEC thus represents a secretory
activity of a non-neuronal cell type derived from the
cardiovascular system. FIG. 7 illustrates the inhibition of the
histamine stimulated release of vWF by HUVEC when previously
treated with BoNT/B in low pH medium. Treatment of cells with
toxins in low pH can be used as a technique for facilitating toxin
penetration of the plasmalemma of cells refractory to exogenously
applied clostridial neurotoxins.
[0230] This result clearly shows the ability of botulinum
neurotoxins to inhibit secretory activity of non-neuronal cells in
the cardiovascular system (see FIG. 7).
Methods
[0231] HUVEC were prepared by the method of Jaffe et al 1973, J.
Clin. Invest. 52, 2745-2756. Cells were passaged once onto 24 well
plates in medium 199 supplemented with 10% foetal calf serum, 10%
newborn calf serum, 5 mM L-glutamine, 100 units/ml penicillin, 100
units/ml streptomycin, 20 .mu.g/ml endothelial cell growth factor
(Sigma). Cells were treated with DMEM pH 7.4, DMEM pH 4.7 (pH
lowered with HCl) or DMEM, pH 4.7 with 500 nM BoNT/B for 2.5 hours
then washed three times with HUVEC medium. 24 hours later cells
were washed with a balanced salt solution, pH 7.4 and exposed to
this solution for 30 minutes for the establishment of basal
release. This was removed and BSS containing 1 mM histamine applied
for a further 30 minutes. Superfusates were centrifuged to remove
any detached cells and the quantity of vWF determined using an
ELISA assay as described by Paleolog et al 1990, Blood. 75,
688-695. Stimulated secretion was then calculated by subtracting
basal from the histamine stimulated release. Inhibition by BoNT/B
treatment at pH 4.7 was calculated at 27.4% when compared to pH 4.7
treatment alone.
Example 7
Activity of BoNT/B in Mucus Secreting Cells
[0232] The LS180 colon carcinoma cell line is recognised as a model
of mucin secreting cells (McCool, D. J., Forstner, J. F. and
Forstner, G. G. 1994 Biochem. J. 302, 111-118). These cells have
been shown to adopt goblet cell morphology and release high
molecular weight mucin when stimulated with muscarinic agonists (eg
carbachol), phorbol esters (PMA) and Ca.sup.2+ ionophores (eg
A23187) (McCool, D. J., Forstner, J. F. and Forstner, G. G. 1995
Biochem. J. 312, 125-133). These cells thus represent a
non-neuronal cell type derived from the colon which can undergo
regulated mucin secretion. FIG. 8 illustrates the inhibition of the
ionomycin stimulated release of high molecular weight,
[.sup.3H]-glucosamine labelled material from LS180 cells by
pretreatment with BoNT/B in low pH medium. Ionomycin is a Ca.sup.2+
ionophore and treatment of cells with low pH medium has been
previously shown to facilitate toxin entry into cells.
[0233] This result clearly shows the ability of botulinum
neurotoxins to inhibit secretory activity of non-neuronal cells
able to release mucin when stimulated with a secretagogue (see FIG.
8).
Methods
[0234] Mucin synthesising colon carcinoma LS180 cells were grown on
Matrigel coated 24 well plates in minimum essential medium
supplemented with 10% foetal calf serum, 2 mM L-glutamine and 1%
non-essential amino acids (Sigma) Cells were treated with pH 7.4
medium, pH 4.7 medium and pH 4.7 medium containing 500 nM botulinum
neurotoxin type B (BoNT/B) for four hours then labelled with
[.sup.3H]-glucosamine (1 .mu.Ci/ml, 0.5 ml/well) for 18 hours in
L15 glucose free medium. Cells were then washed twice with a
balanced salt solution (BSS) pH 7.4 and then 0.5 ml of BSS was
applied for 30 minutes. This material was removed and 0.5 ml of BSS
containing 10 .mu.M ionomycin applied to stimulate mucin release.
The stimulating solution was removed and all superfusates
centrifuged to remove any detached cells. Supernatants were then
centrifuged at 100,000.times.g for 1 hour. Supernatants were
applied to Centricon centrifugal concentrators with a molecular
weight cut-off of 100 kDa and centrifuged (2,500.times.g) until all
liquid had passed through the membrane. Membranes were washed with
BSS by centrifugation three times and then the membrane
scintillation counted for retained, [.sup.3H]-glucosamine labelled
high molecular weight material.
Example 8
Activity of BoNT/B in Inflammatory Cells
[0235] The promyelocytic cell line HL60 can be differentiated into
neutrophil like cells by the addition of dibutyryl cyclic AMP to
the culture medium. Upon differentiation these cells increase their
expression of characteristic enzymes such as .beta.-glucuronidase.
In this condition these cells therefore represent a model of a
phagocytic cell type which contributes to the inflammatory response
of certain disease states (eg rheumatoid arthritis). FIG. 9
illustrates the significant (p>0.05) inhibition of stimulated
release of .beta.-glucuronidase from dbcAMP differentiated HL60
cells by pre-treatment with BoNT/B in low pH medium.
[0236] This result clearly shows the ability of botulinum
neurotoxins to inhibit the secretory activity of a non-neuronal
cell type which is a model of the neutrophil a cell which
participates in inflammation.
Methods
[0237] HL60 cells were cultured in RPMI 1640 medium containing 10%
foetal calf serum and 2 mM glutamine. Cells were exposed to low pH
and toxin for 2.5 hours then washed 3 times and differentiated by
the addition of dibutyryl cyclic AMP (dbcAMP) to a final
concentration of 300 .mu.M. Cells were differentiated for 40 hours
and then stimulated release of .beta.-glucuronidase activity was
determined. Cells were treated with cytochalasin B (5 .mu.M) 5
minutes before stimulation. Cells were stimulated with 1 .mu.M
N-formyl-Met-Leu-Phe with 100 .mu.M ATP for 10 minutes then
centrifuged and the supernatant taken for assay of
.beta.-glucuronidase activity. Activity was measured in cell
lysates and the amount released expressed as a percentage of the
total cellular content of enzyme.
[0238] .beta.-glucuronidase activity was determined according to
the method of Absolom D. R. 1986, (Methods in Enzymology, 132, 160)
using p-Nitrophenyl-.beta.-D-glucuronide as the substrate.
Example 9
Preparation of a LHN/B Backbone Construct
[0239] The following procedure creates a clone for use as an
expression backbone for multidomain fusion expression. This example
is based on preparation of a serotype B based clone (SEQ ID
NO:1).
Preparation of Cloning and Expression Vectors
[0240] pCR 4 (Invitrogen) is the chosen standard cloning vector
chosen due to the lack of restriction sequences within the vector
and adjacent sequencing primer sites for easy construct
confirmation. The expression vector is based on the pMAL (NEB)
expression vector which has the desired restriction sequences
within the multiple cloning site in the correct orientation for
construct insertion (BamHI-SalI-PstI-HindIII). A fragment of the
expression vector has been removed to create a non-mobilisable
plasmid and a variety of different fusion tags have been inserted
to increase purification options.
Preparation of LC/B
The LC/B is created by one of two ways:
[0241] The DNA sequence is designed by back translation of the LC/B
amino acid sequence (obtained from freely available database
sources such as GenBank (accession number P10844) or Swissprot
(accession locus BXB_CLOBO) using one of a variety of reverse
translation software tools (for example EditSeq best E. coli
reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0
(Entelechon)). BamHI/SalI recognition sequences are incorporated at
the 5' and 3' ends respectively of the sequence maintaining the
correct reading frame. The DNA sequence is screened (using software
such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage
sequences incorporated during the back translation. Any cleavage
sequences that are found to be common to those required by the
cloning system are removed manually from the proposed coding
sequence ensuring common E. coli codon usage is maintained. E. coli
codon usage is assessed by reference to software programs such as
Graphical Codon Usage Analyser (Geneart), and the % GC content and
codon usage ratio assessed by reference to published codon usage
tables (for example GenBank Release 143, Sep. 13, 2004). This
optimised DNA sequence containing the LC/B open reading frame (ORF)
is then commercially synthesized (for example by Entelechon,
Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
[0242] The alternative method is to use PCR amplification from an
existing DNA sequence with BamHI and SalI restriction enzyme
sequences incorporated into the 5' and 3' PCR primers respectively.
Complementary oligonucleotide primers are chemically synthesised by
a Supplier (for example MWG or Sigma-Genosys) so that each pair has
the ability to hybridize to the opposite strands (3' ends pointing
"towards" each other) flanking the stretch of Clostridium target
DNA, one oligonucleotide for each of the two DNA strands. To
generate a PCR product the pair of short oligonucleotide primers
specific for the Clostridium DNA sequence are mixed with the
Clostridium DNA template and other reaction components and placed
in a machine (the `PCR machine`) that can change the incubation
temperature of the reaction tube automatically, cycling between
approximately 94.degree. C. (for denaturation), 55.degree. C. (for
oligonucleotide annealing), and 72.degree. C. (for synthesis).
Other reagents required for amplification of a PCR product include
a DNA polymerase (such as Taq or Pfu polymerase), each of the four
nucleotide dNTP building blocks of DNA in equimolar amounts (50-200
.mu.M) and a buffer appropriate for the enzyme optimised for Mg2+
concentration (0.5-5 mM).
[0243] The amplification product is cloned into pCR 4 using either,
TOPO TA cloning for Taq PCR products or Zero Blunt TOPO cloning for
Pfu PCR products (both kits commercially available from
Invitrogen). The resultant clone is checked by sequencing. Any
additional restriction sequences which are not compatible with the
cloning system are then removed using site directed mutagenesis
(for example using Quickchange (Stratagene Inc.)).
Preparation of HN/B Insert
The HN is created by one of two ways:
[0244] The DNA sequence is designed by back translation of the HN/B
amino acid sequence (obtained from freely available database
sources such as GenBank (accession number P10844) or Swissprot
(accession locus BXB_CLOBO)) using one of a variety of reverse
translation software tools (for example EditSeq best E. coli
reverse translation (DNASTAR Inc.), or Back translation tool v2.0
(Entelechon)). A PstI restriction sequence added to the N-terminus
and XbaI-stop codon-HindIII to the C-terminus ensuring the correct
reading frame in maintained. The DNA sequence is screened (using
software such as MapDraw, DNASTAR Inc.) for restriction enzyme
cleavage sequences incorporated during the back translation. Any
sequences that are found to be common to those required by the
cloning system are removed manually from the proposed coding
sequence ensuring common E. coli codon usage is maintained. E. coli
codon usage is assessed by reference to software programs such as
Graphical Codon Usage Analyser (Geneart), and the % GC content and
codon usage ratio assessed by reference to published codon usage
tables (for example GenBank Release 143, Sep. 13, 2004). This
optimised DNA sequence is then commercially synthesized (for
example by Entelechon, Geneart or Sigma-Genosys) and is provided in
the pCR 4 vector.
[0245] The alternative method is to use PCR amplification from an
existing DNA sequence with PstI and XbaI-stop codon-HindIII
restriction enzyme sequences incorporated into the 5' and 3' PCR
primers respectively. The PCR amplification is performed as
described above. The PCR product is inserted into pCR 4 vector and
checked by sequencing. Any additional restriction sequences which
are not compatible with the cloning system are then removed using
site directed mutagenesis (for example using Quickchange
(Stratagene Inc.)).
Preparation of the Spacer (LC-HN Linker)
[0246] The LC-HN linker can be designed from first principle, using
the existing sequence information for the linker as the template.
For example, the serotype B linker (in this case defined as the
inter-domain polypeptide region that exists between the cysteines
of the disulphide bridge between LC and HN) has the sequence
KSVKAPG (SEQ ID NO:30). This sequence information is freely
available from available database sources such as GenBank
(accession number P10844) or Swissprot (accession locus BXB_CLOBO).
For generation of a specific protease cleavage site, the
recognition sequence for enterokinase is inserted into the
activation loop to generate the sequence VDEEKLYDDDDKDRWGSSLQ (SEQ
ID NO:31). Using one of a variety of reverse translation software
tools (for example EditSeq best E. coli reverse translation
(DNASTAR Inc.), or Backtranslation tool v2.0 (Entelechon)), the DNA
sequence encoding the linker region is determined. BamHI/SalI and
PstI/XbaI/stop codon/HindIII restriction enzyme sequences are
incorporated at either end, in the correct reading frames. The DNA
sequence is screened (using software such as MapDraw, DNASTAR Inc.)
for restriction enzyme cleavage sequences incorporated during the
back translation. Any sequences that are found to be common to
those required by the cloning system are removed manually from the
proposed coding sequence ensuring common E. coli codon usage is
maintained. E. coli codon usage is assessed by reference to
software programs such as Graphical Codon Usage Analyser (Geneart),
and the % GC content and codon usage ratio assessed by reference to
published codon usage tables (for example GenBank Release 143, Sep.
13, 2004). This optimised DNA sequence is then commercially
synthesized (for example by Entelechon, Geneart or Sigma-Genosys)
and is provided in the pCR 4 vector. If it is desired to clone the
linker out of pCR 4 vector, the vector (encoding the linker) is
cleaved with either BamHI+SalI or PstI+XbaI combination restriction
enzymes. This cleaved vector then serves as the recipient vector
for insertion and ligation of either the LC DNA (cleaved with
BamHI/SalI) or HN DNA (cleaved with PstI/XbaI). Once the LC or the
HN encoding DNA is inserted upstream or downstream of the linker
DNA, the entire LC-linker or linker-HN DNA fragment can the be
isolated and transferred to the backbone clone.
[0247] As an alternative to independent gene synthesis of the
linker, the linker-encoding DNA can be included during the
synthesis or PCR amplification of either the LC or HN.
Assembly and Confirmation of the Backbone Clone
[0248] The LC or the LC-linker is cut out from the pCR 4 cloning
vector using BamHI/SalI or BamHI/PstI restriction enzymes digests.
The pMAL expression vector is digested with the same enzymes but is
also treated with calf intestinal protease (CIP) as an extra
precaution to prevent re-circularisation. Both the LC or LC-linker
region and the pMAL vector backbone are gel purified. The purified
insert and vector backbone are ligated together using T4 DNA ligase
and the product is transformed with TOP10 cells which are then
screened for LC insertion using BamHI/SalI or BamHI/PstI
restriction digestion. The process is then repeated for the HN or
linker-HN insertion into the PstI/HindIII or SalI/HindIII sequences
of the pMAL-LC construct.
[0249] Screening with restriction enzymes is sufficient to ensure
the final backbone is correct as all components are already
sequenced confirmed, either during synthesis or following PCR
amplification. However, during the sub-cloning of some components
into the backbone, where similar size fragments are being removed
and inserted, sequencing of a small region to confirm correct
insertion is required.
Example 10
Preparation of a LHN/C Backbone Construct
[0250] The following procedure creates a clone for use as an
expression backbone for multidomain fusion expression. This example
is based on preparation of a serotype C based clone (SEQ ID
NO:2).
Preparation of Cloning and Expression Vectors
[0251] pCR 4 (Invitrogen) is the chosen standard cloning vector
chosen due to the lack of restriction sequences within the vector
and adjacent sequencing primer sites for easy construct
confirmation. The expression vector is based on the pMAL (NEB)
expression vector which has the desired restriction sequences
within the multiple cloning site in the correct orientation for
construct insertion (BamHI-SalI-PstI-HindIII). A fragment of the
expression vector has been removed to create a non-mobilisable
plasmid and a variety of different fusion tags have been inserted
to increase purification options.
Preparation of LC/C
The LC/C is created by one of two ways:
[0252] The DNA sequence is designed by back translation of the LC/C
amino acid sequence (obtained from freely available database
sources such as GenBank (accession number P18640) or Swissprot
(accession locus BXC1_CLOBO) using one of a variety of reverse
translation software tools (for example EditSeq best E. coli
reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0
(Entelechon)). BamHI/SalI recognition sequences are incorporated at
the 5' and 3' ends respectively of the sequence maintaining the
correct reading frame. The DNA sequence is screened (using software
such as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage
sequences incorporated during the back translation. Any cleavage
sequences that are found to be common to those required by the
cloning system are removed manually from the proposed coding
sequence ensuring common E. coli codon usage is maintained. E. coli
codon usage is assessed by reference to software programs such as
Graphical Codon Usage Analyser (Geneart), and the % GC content and
codon usage ratio assessed by reference to published codon usage
tables (for example GenBank Release 143, Sep. 13, 2004). This
optimised DNA sequence containing the LC/C open reading frame (ORF)
is then commercially synthesized (for example by Entelechon,
Geneart or Sigma-Genosys) and is provided in the pCR 4 vector.
[0253] The alternative method is to use PCR amplification from an
existing DNA sequence with BamHI and SalI restriction enzyme
sequences incorporated into the 5' and 3' PCR primers respectively.
Complementary oligonucleotide primers are chemically synthesised by
a Supplier (for example MWG or Sigma-Genosys) so that each pair has
the ability to hybridize to the opposite strands (3' ends pointing
"towards" each other) flanking the stretch of Clostridium target
DNA, one oligonucleotide for each of the two DNA strands. To
generate a PCR product the pair of short oligonucleotide primers
specific for the Clostridium DNA sequence are mixed with the
Clostridium DNA template and other reaction components and placed
in a machine (the `PCR machine`) that can change the incubation
temperature of the reaction tube automatically, cycling between
approximately 94.degree. C. (for denaturation), 55.degree. C. (for
oligonucleotide annealing), and 72.degree. C. (for synthesis).
Other reagents required for amplification of a PCR product include
a DNA polymerase (such as Taq or Pfu polymerase), each of the four
nucleotide dNTP building blocks of DNA in equimolar amounts (50-200
.mu.M) and a buffer appropriate for the enzyme optimised for Mg2+
concentration (0.5-5 mM).
[0254] The amplification product is cloned into pCR 4 using either,
TOPO TA cloning for Taq PCR products or Zero Blunt TOPO cloning for
Pfu PCR products (both kits commercially available from
Invitrogen). The resultant clone is checked by sequencing. Any
additional restriction sequences which are not compatible with the
cloning system are then removed using site directed mutagenesis
(for example using Quickchange (Stratagene Inc.)).
Preparation of HN/C Insert
The HN is created by one of two ways:
[0255] The DNA sequence is designed by back translation of the HN/C
amino acid sequence (obtained from freely available database
sources such as GenBank (accession number P18640) or Swissprot
(accession locus BXC1_CLOBO)) using one of a variety of reverse
translation software tools (for example EditSeq best E. coli
reverse translation (DNASTAR Inc.), or Back translation tool v2.0
(Entelechon)). A PstI restriction sequence added to the N-terminus
and XbaI-stop codon-HindIII to the C-terminus ensuring the correct
reading frame in maintained. The DNA sequence is screened (using
software such as MapDraw, DNASTAR Inc.) for restriction enzyme
cleavage sequences incorporated during the back translation. Any
sequences that are found to be common to those required by the
cloning system are removed manually from the proposed coding
sequence ensuring common E. coli codon usage is maintained. E. coli
codon usage is assessed by reference to software programs such as
Graphical Codon Usage Analyser (Geneart), and the % GC content and
codon usage ratio assessed by reference to published codon usage
tables (for example GenBank Release 143, Sep. 13, 2004). This
optimised DNA sequence is then commercially synthesized (for
example by Entelechon, Geneart or Sigma-Genosys) and is provided in
the pCR 4 vector.
[0256] The alternative method is to use PCR amplification from an
existing DNA sequence with PstI and XbaI-stop codon-HindIII
restriction enzyme sequences incorporated into the 5' and 3' PCR
primers respectively. The PCR amplification is performed as
described above. The PCR product is inserted into pCR 4 vector and
checked by sequencing. Any additional restriction sequences which
are not compatible with the cloning system are then removed using
site directed mutagenesis (for example using Quickchange
(Stratagene Inc.)).
Preparation of the Spacer (LC-HN Linker)
[0257] The LC-HN linker can be designed from first principle, using
the existing sequence information for the linker as the template.
For example, the serotype C linker (in this case defined as the
inter-domain polypeptide region that exists between the cysteines
of the disulphide bridge between LC and HN) has the sequence
HKAIDGRSLYNKTLD (SEQ ID NO:32). This sequence information is freely
available from available database sources such as GenBank
(accession number P18640) or Swissprot (accession locus
BXC1_CLOBO). For generation of a specific protease cleavage site,
the recognition sequence for enterokinase is inserted into the
activation loop to generate the sequence VDGIITSKTKSDDDDKNKALNLQ
(SEQ ID NO:33). Using one of a variety of reverse translation
software tools (for example EditSeq best E. coli reverse
translation (DNASTAR Inc.), or Backtranslation tool v2.0
(Entelechon)), the DNA sequence encoding the linker region is
determined. BamHI/SalI and PstI/XbaI/stop codon/HindIII restriction
enzyme sequences are incorporated at either end, in the correct
reading frames. The DNA sequence is screened (using software such
as MapDraw, DNASTAR Inc.) for restriction enzyme cleavage sequences
incorporated during the back translation. Any sequences that are
found to be common to those required by the cloning system are
removed manually from the proposed coding sequence ensuring common
E. coli codon usage is maintained. E. coli codon usage is assessed
by reference to software programs such as Graphical Codon Usage
Analyser (Geneart), and the % GC content and codon usage ratio
assessed by reference to published codon usage tables (for example
GenBank Release 143, Sep. 13, 2004). This optimised DNA sequence is
then commercially synthesized (for example by Entelechon, Geneart
or Sigma-Genosys) and is provided in the pCR 4 vector. If it is
desired to clone the linker out of pCR 4 vector, the vector
(encoding the linker) is cleaved with either BamHI+SalI or
PstI+XbaI combination restriction enzymes. This cleaved vector then
serves as the recipient vector for insertion and ligation of either
the LC DNA (cleaved with BamHI/SalI) or HN DNA (cleaved with
PstI/XbaI). Once the LC or the HN encoding DNA is inserted upstream
or downstream of the linker DNA, the entire LC-linker or linker-HN
DNA fragment can the be isolated and transferred to the backbone
clone.
[0258] As an alternative to independent gene synthesis of the
linker, the linker-encoding DNA can be included during the
synthesis or PCR amplification of either the LC or HN.
Assembly and Confirmation of the Backbone Clone
[0259] The LC or the LC-linker is cut out from the pCR 4 cloning
vector using BamHI/SalI or BamHI/PstI restriction enzymes digests.
The pMAL expression vector is digested with the same enzymes but is
also treated with calf intestinal protease (CIP) as an extra
precaution to prevent re-circularisation. Both the LC or LC-linker
region and the pMAL vector backbone are gel purified. The purified
insert and vector backbone are ligated together using T4 DNA ligase
and the product is transformed with TOP10 cells which are then
screened for LC insertion using BamHI/SalI or BamHI/PstI
restriction digestion. The process is then repeated for the HN or
linker-HN insertion into the PstI/HindIII or SalI/HindIII sequences
of the pMAL-LC construct.
[0260] Screening with restriction enzymes is sufficient to ensure
the final backbone is correct as all components are already
sequenced confirmed, either during synthesis or following PCR
amplification. However, during the sub-cloning of some components
into the backbone, where similar size fragments are being removed
and inserted, sequencing of a small region to confirm correct
insertion is required.
Example 11
Construction, Expression, and Purification of a LHN/C-EGF Fusion
Protein
Preparation of Spacer-EGF Insert
[0261] For presentation of an EGF sequence at the C-terminus of the
HN domain, a DNA sequence is designed to flank the spacer and
targeting moiety (TM) regions allowing incorporation into the
backbone clone (SEQ ID NO:2). The DNA sequence can be arranged as
BamHI-SalI-PstI-XbaI-spacer-EGF-stop codon-HindIII (SEQ ID NO:3).
The DNA sequence can be designed using one of a variety of reverse
translation software tools (for example EditSeq best E. coli
reverse translation (DNASTAR Inc.), or Backtranslation tool v2.0
(Entelechon)). Once the TM DNA is designed, the additional DNA
required to encode the preferred spacer is created in silico. It is
important to ensure the correct reading frame is maintained for the
spacer, EGF and restriction sequences and that the XbaI sequence is
not preceded by the bases, TC which would result on DAM
methylation. The DNA sequence is screened for restriction sequence
incorporated and any additional sequences are removed manually from
the remaining sequence ensuring common E. coli codon usage is
maintained. E. coli codon usage is assessed by reference to
software programs such as Graphical Codon Usage Analyser (Geneart),
and the % GC content and codon usage ratio assessed by reference to
published codon usage tables (for example GenBank Release 143, Sep.
13, 2004). This optimised DNA sequence is then commercially
synthesized (for example by Entelechon, Geneart or Sigma-Genosys)
and is provided in the pCR 4 vector.
Insertion of Spacer-EGF into Backbone
[0262] In order to create a LC-linker-HN-spacer-EGF construct (SEQ
ID NO:4) using the backbone construct (SEQ ID NO:2) and the newly
synthesised pCR 4-spacer-TM vector encoding the EGF TM (SEQ ID
NO:3), the following two-step method is employed. Firstly, the HN
domain is excised from the backbone clone using restriction enzymes
PstI and XbaI and ligated into similarly digested pCR 4-spacer-EGF
vector. This creates an HN-spacer-EGF ORF in pCR 4 that can be
excised from the vector using restriction enzymes PstI and HindIII
for subsequent ligation into similarly cleaved backbone or
expression construct. The final construct contains the
LC-linker-HN-spacer-EGF ORF (SEQ ID NO:4) for transfer into
expression vectors for expression to result in a fusion protein of
the sequence illustrated in SEQ ID NO:5.
[0263] Screening with restriction enzymes is sufficient to ensure
the final backbone is correct as all components are already
sequenced confirmed, either during synthesis or following PCR
amplification. However, during the sub-cloning of some components
into the backbone, where similar size fragments are being removed
and inserted, sequencing of a small region to confirm correct
insertion is required.
Alternative Construction Approach
[0264] As an alternative to the methodologies described above for
the construction of LHN/C-EGF, complete gene synthesis has been
used to create a single DNA insert that encodes the LC, the HN,
linkers, spacers and a protease activation site. The synthetic DNA
is designed to have a NdeI restriction site at the 5' end and a
HindIII restriction site at the 3' end to facilitate direct cloning
into expression vectors. The sequence of the engineered coding
region is subject to the same codon utilisation analysis as
described above. The sequence of the synthetic DNA is illustrated
in SEQ ID NO:19, and the protein that it encodes is illustrated in
SEQ ID NO:20.
Expression of LHN/C-EGF Fusion Protein
[0265] Expression of the LHN/C-EGF fusion protein is achieved using
the following protocol. Inoculate 100 ml of modified TB containing
0.2% glucose and 100 mg/ml ampicillin in a 250 ml flask with a
single colony from the LHN/C-EGF expression strain. Grow the
culture at 37.degree. C., 225 rpm for 16 hours. Inoculate 1 L of
modified TB containing 0.2% glucose and 100 .quadrature.g/ml
ampicillin in a 2 L flask with 10 ml of overnight culture. Grow
cultures at 37.degree. C. until an approximate OD600 nm of 0.5 is
reached at which point reduce the temperature to 16.degree. C.
After 1 hour induce the cultures with 1 mM IPTG and grow at
16.degree. C. for a further 16 hours.
Purification of LHN/C-EGF Fusion Protein
[0266] Defrost falcon tube containing 25 ml 50 mM HEPES pH 7.2 200
mM NaCl and approximately 10 g of E. coli BL21 cell paste. Sonicate
the cell paste on ice 30 seconds on, 30 seconds off for 10 cycles
at a power of 22 microns ensuring the sample remains cool. Spin the
lysed cells at 18 000 rpm, 4.degree. C. for 30 minutes. Load the
supernatant onto a 0.1 M NiSO4 charged Chelating column (20-30 ml
column is sufficient) equilibrated with 50 mM HEPES pH 7.2 200 mM
NaCl. Using a step gradient of 10 and 40 mM imidazole, wash away
the non-specific bound protein and elute the fusion protein with
100 mM imidazole. Dialyse the eluted fusion protein against 5 L of
50 mM HEPES pH 7.2 200 mM NaCl at 4.degree. C. overnight and
measure the OD of the dialysed fusion protein. Add 1 unit of factor
Xa per 100 .quadrature.g fusion protein and incubate at 25.degree.
C. static overnight. Load onto a 0.1 M NiSO4 charged Chelating
column (20-30 ml column is sufficient) equilibrated with 50 mM
HEPES pH 7.2 200 mM NaCl. Wash column to baseline with 50 mM HEPES
pH 7.2 200 mM NaCl. Using a step gradient of 10 and 40 mM
imidazole, wash away the non-specific bound protein and elute the
fusion protein with 100 mM imidazole. Dialyse the eluted fusion
protein against 5 L of 50 mM HEPES pH 7.2 200 mM NaCl at 4.degree.
C. overnight and concentrate the fusion to about 2 mg/ml, aliquot
sample and freeze at -20.degree. C. Test purified protein using OD,
BCA and purity analysis. FIG. 8 demonstrates the purified protein
as analysed be SDS-PAGE.
Example 12
Construction, Expression and Purification of a LHN/B-EGF Fusion
Protein
[0267] The LC-HN linker is designed using the methods described in
example 11 using the B serotype linker arranged as
BamHI-SalI-PstI-XbaI-spacer-EGF-stop codon-HindIII (SEQ ID NO:3).
The LHN/B-EGF fusion is then assembled using the LHN/B backbone
clone (SEQ ID NO:1) made using the methods described in example 9
and constructed using methods described in example 11. The final
construct contains the LC-linker-HN-spacer-EGF ORF (SEQ ID NO:6)
for transfer into expression vectors for expression to result in a
fusion protein of the sequence illustrated in SEQ ID NO:7. The
resultant expression plasmid, pMAL LHN/B-EGF is transformed into E.
coli BL21 for recombinant protein expression. Expression and
purification of the fusion protein was carried out as described in
example 6 except that enterokinase replaced factor Xa in the
activation of the fusion protein. FIG. 9 demonstrates the purified
protein as analysed by SDS-PAGE.
Example 13
Preparation and Purification of a LHN/C-RGD Fusion Protein
Preparation of Spacer-RGD Insert
[0268] For presentation of an RGD sequence at the C-terminus of the
HN domain, a DNA sequence is designed to flank the spacer and TM
regions allowing incorporation into the backbone clone (SEQ ID
NO:2). The DNA sequence can be arranged as
BamHI-SalI-PstI-XbaI-spacer-SpeI-RGD-stop codon-HindIII (SEQ ID
NO:8). The DNA sequence can be designed using one of a variety of
reverse translation software tools (for example EditSeq best E.
coli reverse translation (DNASTAR Inc.), or Backtranslation tool
v2.0 (Entelechon)). Once the TM DNA is designed, the additional DNA
required to encode the preferred spacer is created in silico. It is
important to ensure the correct reading frame is maintained for the
spacer, RGD and restriction sequences and that the XbaI sequence is
not preceded by the bases, TC which would result on DAM
methylation. The DNA sequence is screened for restriction sequence
incorporated and any additional sequences are removed manually from
the remaining sequence ensuring common E. coli codon usage is
maintained. E. coli codon usage is assessed by reference to
software programs such as Graphical Codon Usage Analyser (Geneart),
and the % GC content and codon usage ratio assessed by reference to
published codon usage tables (for example GenBank Release 143, Sep.
13, 2004). This optimised DNA sequence is then commercially
synthesized (for example by Entelechon, Geneart or Sigma-Genosys)
and is provided in the pCR 4 vector.
Insertion of Spacer-RGD into Backbone
[0269] In order to create a LC-linker-HN-spacer-RGD construct (SEQ
ID NO:9) using the backbone construct (SEQ ID NO:2) and the newly
synthesised pCR 4-spacer-TM vector encoding the RGD TM (SEQ ID
NO:8), the following two-step method is employed. Firstly, the HN
domain is excised from the backbone clone using restriction enzymes
PstI and XbaI and ligated into similarly digested pCR 4-spacer-RGD
vector. This creates an HN-spacer-RGD ORF in pCR 4 that can be
excised from the vector using restriction enzymes PstI and HindIII
for subsequent ligation into similarly cleaved backbone or
expression construct. The final construct contains the
LC-linker-HN-spacer-RGD ORF (SEQ ID NO:9) for transfer into
expression vectors for expression to result in a fusion protein of
the sequence illustrated in SEQ ID NO:10.
[0270] Screening with restriction enzymes is sufficient to ensure
the final backbone is correct as all components are already
sequenced confirmed, either during synthesis or following PCR
amplification. However, during the sub-cloning of some components
into the backbone, where similar size fragments are being removed
and inserted, sequencing of a small region to confirm correct
insertion is required.
[0271] Expression and purification of the fusion protein was
carried out as described in example 11. FIG. 10 demonstrates the
purified protein as analysed by SDS-PAGE.
Example 14
Preparation and Purification of a LHN/C-cyclic RGD Fusion
Protein
[0272] The LC-HN linker can be designed using the methods described
in example 13 using the C serotype linker arranged as
BamHI-SalI-PstI-XbaI-spacer-SpeI-cyclic RGD-stop codon-HindIII (SEQ
ID NO:11). The LHN/C-cyclic RGD fusion is then assembled using the
LHN/C backbone clone (SEQ ID NO:2) made using the methods described
in example 10 and constructed using methods described in example
13. The final construct contains the LC-linker-HN-spacer-cyclic RGD
ORF (SEQ ID NO:11) for transfer into expression vectors for
expression to result in a fusion protein of the sequence
illustrated in SEQ ID NO:13. The resultant expression plasmid, pMAL
LHN/C-cyclic RGD was transformed into E. coli BL21 for recombinant
protein expression. Expression and purification of the fusion
protein was carried out as described in example 11. FIG. 11
demonstrates the purified protein as analysed by SDS-PAGE.
Example 15
Preparation and Purification of a LC/C-RGD-HN/C Fusion Protein
[0273] In order to create the LC-linker-RGD-spacer-HN construct
(SEQ ID NO:15), the pCR 4 vector encoding the linker (SEQ ID NO:14)
is cleaved with BamHI+SalI restriction enzymes. This cleaved vector
then serves as the recipient vector for insertion and ligation of
the LC/C DNA (SEQ ID NO:2) cleaved with BamHI+SalI. The resulting
plasmid DNA is then cleaved with PstI+XbaI restriction enzymes and
serves as the recipient vector for the insertion and ligation of
the HN/C DNA (SEQ ID NO:2) cleaved with PstI+XbaI. The final
construct contains the LC-linker-RGD-spacer-HN ORF (SEQ ID NO:15)
for transfer into expression vectors for expression to result in a
fusion protein of the sequence illustrated in SEQ ID NO:16. The
resultant expression plasmid, pMAL LC/C-RGD-HN/C was transformed
into E. coli BL21 for recombinant protein expression. Expression
and purification of the fusion protein was carried out as described
in example 11. FIG. 12 demonstrates the purified protein as
analysed by SDS-PAGE.
Alternative Construction Approach
[0274] As an alternative to the methodologies described above for
the construction of LC-linker-RGD-spacer-HN, complete gene
synthesis has been used to create a single DNA insert that encodes
the LC, the HN, linkers, spacers and a protease activation site.
The synthetic DNA is designed to have a NdeI restriction site at
the 5' end and a HindIII restriction site at the 3' end to
facilitate direct cloning into expression vectors. The sequence of
the engineered coding region is subject to the same codon
utilisation analysis as described above. The sequence of the
synthetic DNA is illustrated in SEQ ID NO:17, and the protein that
it encodes is illustrated in SEQ ID NO:18.
Example 16
VAMP Cleavage Activity Assay
[0275] A range of concentrations of LHN/B-EGF in cleavage buffer
(50 mM HEPES pH7.4, 10 mM DTT, 20 .quadrature.M ZnCl2, 1% FBS) are
incubated with biotinylated VAMP substrate (1 mg/ml) for two hours
at 37.degree. C. in a shaking incubator. The cleavage reaction is
transferred to a washed 96-well streptavidin coated plate and
incubated at 37.degree. C. in a shaking incubator for 5 minutes.
The plate is washed three times with PBS-0.1% tween-20 (PBS-T). The
wells are blocked with blocking buffer (5% FCS in PBS-T) for 1 hour
at 37.degree. C. The primary antibody (anti-FESS) is added at a
dilution of 1 in 500 in blocking buffer and the plate is incubated
at 37.degree. C. for 1 hour. The plate is washed three times with
PBS-T and the secondary antibody (anti guinea pig HRP conjugate)
diluted 1 in 1000 in blocking buffer is applied. Following 1 hour
incubation at 37.degree. C. the plate is developed with bioFX TMB
substrate. Colour development is allowed to proceed for 1-5 minutes
and then stopped with stop solution. The absorbance is measured at
450 nm. FIG. 13 shows the VAMP cleavage activity of LHN/B-EGF
fusion protein.
Example 17
Activity of EGF-LHN/C and EGF-LHN/B in THP-1 Immune Cells
[0276] The THP-1 cell line is a human-derived suspension
(non-adherent) culture that is used frequently to provide a model
system for primary monocytes. It is a well characterized model and
over 2000 reviewed publications have utilized the THP-1 line to
investigate molecular and cellular processes. Recent studies have
demonstrated the utility of the THP-1 cell line as a model to
assess the secretion of anti- and pro-inflammatory cytokines (Qiu
et. al. 2007 J. Lipid Res. 48(2) 385-394, Prunet et. al. 2006
Cytometry A. 69, 359-373 and Segura et al 2002 Clin. Exp. Immunol.
127(2) 243-254).
[0277] FIG. 14 illustrates the significant inhibition of
LPS-stimulated release of IL-8 from THP-1 cells in culture by
pretreatment with either EGF-LHN/C (SXN 100501) or with EGF-LHN/B
(SXN 100328).
[0278] This result shows clearly the ability of fusion proteins to
inhibit the pro-inflammatory cytokine secretory activity of a
non-neuronal immune cell type that is a model for the monocyte cell
which participates in inflammation.
Methods
[0279] THP-1, cells were pre-incubated with 10 nM compound or
vehicle control for 48 hours at 37.degree. C./5% CO2. After the
pre-incubation, LPS was added at a final concentration of 1 mg/ml
and the cells incubated for a further 16 hours (overnight). For
inhibitory controls; cells were treated with Staurosporine (1
.mu.M) or Dexamethasone (1 .mu.M) for 30 minutes prior to adding
the LPS, and then incubated for 16 hours (overnight). Culture
supernatant from each well was harvested and analyzed for cytokine
by Luminex-based technology (BioSource). All estimations were
performed in triplicate.
Example 18
Activity of EGF-LHN/C and EGF-LHN/B in RPMI Immune Cells
[0280] The RPMI-8226 cell line is a human-derived culture that is
used frequently to provide a model system for primary
B-lymphocytes. It is a well characterized model and over 250
reviewed publications have utilized the RPMI-8226 line to
investigate molecular and cellular processes. Recent studies have
demonstrated the utility of the RPMI-8226 cell line as a model to
assess the secretion of cytokines (Xu et. al. J. Leukoc. Biol.
2002, 72(2) 410-416 and Gupta et. al. 2001, 15(12) 1950-1961).
[0281] FIG. 15 illustrates the significant inhibition of
LPS-stimulated release of IL-10 from RPMI-8226 cells in culture by
pretreatment with either EGF-LHN/C (SXN 100501) or with EGF-LHN/B
(SXN 100328).
[0282] This result shows clearly the ability of fusion proteins to
inhibit the cytokine secretory activity of a non-neuronal immune
cell type that is a model for the B-lymphocyte cell which
participates in immune responses.
[0283] Methods
[0284] RPMI-8226 cells were pre-incubated with 10 nM compound or
vehicle control for 48 hours at 37oC/5% CO2. After the
pre-incubation, LPS was added at a final concentration of 1 mg/ml
and the cells incubated for a further 16 hours (overnight). For
inhibitory controls; cells were treated with Staurosporine (1
.mu.M) or Dexamethasone (1 .mu.M) for 30 minutes prior to adding
the LPS, and then incubated for 16 hours (overnight). Culture
supernatant from each well was harvested and analyzed for cytokine
by Luminex-based technology (BioSource). All estimations were
performed in triplicate.
Example 19
Activity of EGF-LHN/C, CP-RGD-LHN/C and EGF-LHN/B in Human PBMC
Immune Cells
[0285] PBMC are peripheral blood mononuclear cells providing a
primary culture that is highly diverse in constituent cell
phenotype. It is a well characterized model and over 3000 reviewed
publications have utilized primary human PBMC to investigate
molecular and cellular processes. Recent studies have demonstrated
the utility of human PBMC as a model to assess the secretion of
cytokines (Bachmann et. al. Cell Microbiol. 2006, 8(2) 289-300,
Siejka et. al. Endocr. Regul. 2005, 39(1) 7-11, Reddy et. al. 2004,
293(1-2) 127-142).
[0286] FIG. 16 illustrates the significant inhibition of
LPS-stimulated release of IL-8 from human PBMC cells in culture by
pretreatment with CP-RGD-LHN/C (SXN 100221), EGF-LHN/C (SXN 100501)
or with EGF-LHN/B (SXN 100328).
[0287] This result shows clearly the ability of fusion proteins to
inhibit the cytokine secretory activity of non-neuronal human
immune cells which participates in immune responses.
Methods
[0288] PBMC cells were pre-incubated with 10 nM compound or vehicle
control for 24 hours at 37.degree. C./5% CO2. After the
pre-incubation, LPS was added at a final concentration of 1 mg/ml
and the cells incubated for a further 16 hours (overnight). For
inhibitory controls; cells were treated with Staurosporine (1
.mu.M) or Dexamethasone (1 .mu.M) for 30 minutes prior to adding
the LPS, and then incubated for 16 hours (overnight). Culture
supernatant from each well was harvested and analyzed for cytokine
by Luminex-based technology (BioSource). All estimations were
performed in triplicate.
Example 20
Activity of EGF-LHN/C, CP-RGD-LHN/C and EGF-LHN/B in Human PBMC
Immune Cells
[0289] PBMC are peripheral blood mononuclear cells providing a
primary culture that is highly diverse in constituent cell
phenotype. It is a well characterized model and over 3000 reviewed
publications have utilized primary human PBMC to investigate
molecular and cellular processes. Recent studies have demonstrated
the utility of human PBMC as a model to assess the secretion of
cytokines (Bachmann et. al. Cell Microbiol. 2006, 8(2) 289-300,
Siejka et. al. Endocr. Regul. 2005, 39(1) 7-11, Reddy et. al. 2004,
293(1-2) 127-142).
[0290] FIG. 17 illustrates the significant inhibition of
PHA-stimulated release of IP-10 from human PBMC cells in culture by
pretreatment with CP-RGD-LHN/C (SXN 100221), EGF-LHN/C (SXN 100501)
or with EGF-LHN/B (SXN 100328).
[0291] This result shows clearly the ability of fusion proteins to
inhibit the cytokine secretory activity of non-neuronal human
immune cells which participates in immune responses.
Methods
[0292] PBMC cells were pre-incubated with 10 nM compound or vehicle
control for 24 hours at 37.degree. C./5% CO2. After the
pre-incubation, PHA was added at a final concentration of 2 mg/ml
and the cells incubated for a further 16 hours (overnight). For
inhibitory controls; cells were treated with Staurosporine (1
.mu.M) or Dexamethasone (1 .mu.M) for 30 minutes prior to adding
the PHA, and then incubated for 16 hours (overnight). Culture
supernatant from each well was harvested and analyzed for cytokine
by Luminex-based technology (BioSource). All estimations were
performed in triplicate.
Example 21
Clinical Example
[0293] A 54 year old male suffering from asthma presents at his GP.
Despite daily treatment with his preventer inhaler, the use of his
reliever inhaler has increased significantly. The patient presents
with difficulty in performing everyday tasks due continued
shortness of breath and frequent asthma attacks. The GP prescribes
a 6-month course of SXN100501 (as prepared in previous examples) in
nebuliser form, 80 .mu.g to be taken monthly. Following discussion
with the physician, the patient selects the most appropriate
nebuliser for their personal situation from a range of suitable
devices. After a single dose of SXN100501 the patient experiences a
reduced frequency of attacks and a general improvement in FEV1.
Further treatment enhances these parameters further and improves
quality of life.
Example 22
Clinical Example
[0294] A 26 year old female suffering from seasonal allergic
rhinitis (hay fever) presents at her GP. Despite completion of a
course of preventer treatment (consisting of daily treatment with
flixonase for a period of 3 weeks) and subsequent treatment with
OTC anti-histamines, the frequency and severity of rhinitis
increases. The GP prescribes a 4-month course of SXN100328 (as
prepared in previous examples), 80 .mu.g to be taken monthly in the
form of a nasal spray. After a single dose of SXN100328 the patient
experiences a reduced frequency of rhinitis and generally improved
quality of life. Further treatments continue to decrease the
severity of the rhinitis.
SEQ ID List
[0295] SEQ ID NO:1 DNA sequence of LHN/B [0296] SEQ ID NO:2 DNA
sequence of LHN/C [0297] SEQ ID NO:3 DNA sequence of the EGF linker
[0298] SEQ ID NO:4 DNA sequence of the EGF-C fusion [0299] SEQ ID
NO:5 Protein sequence of the EGF-C fusion [0300] SEQ ID NO:6 DNA
sequence of the EGF-B fusion [0301] SEQ ID NO:7 Protein sequence of
the EGF-B fusion [0302] SEQ ID NO:8 DNA sequence of the RGD linker
[0303] SEQ ID NO:9 DNA sequence of the RGD-C fusion [0304] SEQ ID
NO:10 Protein sequence of the RGD-C fusion [0305] SEQ ID NO:11 DNA
sequence of the cyclic RGD linker [0306] SEQ ID NO:12 DNA sequence
of the cyclic RGD-C fusion [0307] SEQ ID NO:13 Protein sequence of
the cyclic RGD-C fusion [0308] SEQ ID NO:14 DNA sequence of the
LC/C-RGD-HN/C linker [0309] SEQ ID NO:15 DNA sequence of the
LC/C-RGD-HN/C fusion [0310] SEQ ID NO:16 Protein sequence of the
LC/C-RGD-HN/C fusion [0311] SEQ ID NO:17 DNA sequence of the fully
synthesised LC/C-RGD-HN/C fusion [0312] SEQ ID NO:18 Protein
sequence of the fully synthesised LC/C-RGD-HN/C fusion [0313] SEQ
ID NO:19 DNA sequence of the fully synthesised EGF-LHN/C fusion
[0314] SEQ ID NO:20 Protein sequence of the fully synthesised
EGF-LHN/C fusion [0315] SEQ ID NO:21 Integrin binding peptide
sequence [0316] SEQ ID NO:22 Integrin binding peptide sequence
[0317] SEQ ID NO:23 Cyclic RGD peptide [0318] SEQ ID NO:24 Linear
integrin binding sequence [0319] SEQ ID NO:25 Cyclic integrin
binding sequence
Sequence CWU 1
1
25 1 2631 DNA Unknown DNA sequence of LHN/B 1 ggatccatgc cggttaccat
caacaacttc aactacaacg acccgatcga caacaacaac 60 atcattatga
tggaaccgcc gttcgcacgt ggtaccggac gttactacaa ggcttttaag 120
atcaccgacc gtatctggat catcccggaa cgttacacct tcggttacaa acctgaggac
180 ttcaacaaga gtagcgggat tttcaatcgt gacgtctgcg agtactatga
tccagattat 240 ctgaatacca acgataagaa gaacatattc cttcagacta
tgattaaact cttcaaccgt 300 atcaaaagca aaccgctcgg tgaaaaactc
ctcgaaatga ttatcaacgg tatcccgtac 360 ctcggtgacc gtcgtgtccc
gcttgaagag ttcaacacca acatcgcaag cgtcaccgtc 420 aacaaactca
tcagcaaccc aggtgaagtc gaacgtaaaa aaggtatctt cgcaaacctc 480
atcatcttcg gtccgggtcc ggtcctcaac gaaaacgaaa ccatcgacat cggtatccag
540 aaccacttcg caagccgtga aggtttcggt ggtatcatgc agatgaaatt
ctgcccggaa 600 tacgtcagtg tcttcaacaa cgtccaggaa aacaaaggtg
caagcatctt caaccgtcgt 660 ggttacttca gcgacccggc actcatcctc
atgcatgaac tcatccacgt cctccacggt 720 ctctacggta tcaaagttga
cgacctcccg atcgtcccga acgagaagaa attcttcatg 780 cagagcaccg
acgcaatcca ggctgaggaa ctctacacct tcggtggcca agacccaagt 840
atcataaccc cgtccaccga caaaagcatc tacgacaaag tcctccagaa cttcaggggt
900 atcgtggaca gactcaacaa agtcctcgtc tgcatcagcg acccgaacat
caatatcaac 960 atatacaaga acaagttcaa agacaagtac aaattcgtcg
aggacagcga aggcaaatac 1020 agcatcgacg tagaaagttt cgacaagctc
tacaaaagcc tcatgttcgg tttcaccgaa 1080 accaacatcg ccgagaacta
caagatcaag acaagggcaa gttacttcag cgacagcctc 1140 ccgcctgtca
aaatcaagaa cctcttagac aacgagattt acacaattga agagggcttc 1200
aacatcagtg acaaagacat ggagaaggaa tacagaggtc agaacaaggc tatcaacaaa
1260 caggcatacg aggagatcag caaagaacac ctcgcagtct acaagatcca
gatgtgcgtc 1320 gacgaagaaa agctgtacga cgacgacgac aaagaccgtt
ggggttcttc gctgcagtgc 1380 atcgacgttg acaacgaaga cctgttcttc
atcgctgaca aaaacagctt cagtgacgac 1440 ctgagcaaaa acgaacgtat
cgaatacaac acccagagca actacatcga aaacgacttc 1500 ccgatcaacg
aactgatcct ggacaccgac ctgataagta aaatcgaact gccgagcgaa 1560
aacaccgaaa gtctgaccga cttcaacgtt gacgttccgg tttacgaaaa acagccggct
1620 atcaagaaaa tcttcaccga cgaaaacacc atcttccagt acctgtacag
ccagaccttc 1680 ccgctggaca tccgtgacat cagtctgacc agcagtttcg
acgacgctct gctgttcagc 1740 aacaaagttt acagtttctt cagcatggac
tacatcaaaa ccgctaacaa agttgttgaa 1800 gcagggctgt tcgctggttg
ggttaaacag atcgttaacg acttcgttat cgaagctaac 1860 aaaagcaaca
ctatggacaa aatcgctgac atcagtctga tcgttccgta catcggtctg 1920
gctctgaacg ttggtaacga aaccgctaaa ggtaactttg aaaacgcttt cgagatcgct
1980 ggtgcaagca tcctgctgga gttcatcccg gaactgctga tcccggttgt
tggtgctttc 2040 ctgctggaaa gttacatcga caacaaaaac aagatcatca
aaaccatcga caacgctctg 2100 accaaacgta acgaaaaatg gagtgatatg
tacggtctga tcgttgctca gtggctgagc 2160 accgtcaaca cccagttcta
caccatcaaa gaaggtatgt acaaagctct gaactaccag 2220 gctcaggctc
tggaagagat catcaaatac cgttacaaca tctacagtga gaaggaaaag 2280
agtaacatca acatcgactt caacgacatc aacagcaaac tgaacgaagg tatcaaccag
2340 gctatcgaca acatcaacaa cttcatcaac ggttgcagtg ttagctacct
gatgaagaag 2400 atgatcccgc tggctgttga aaaactgctg gacttcgaca
acaccctgaa aaagaacctg 2460 ctgaactaca tcgacgaaaa caagctgtac
ctgatcggta gtgctgaata cgaaaaaagt 2520 aaagtgaaca aatacctgaa
gaccatcatg ccgttcgacc tgagtatcta caccaacgac 2580 accatcctga
tcgaaatgtt caacaaatac aactctctag actagaagct t 2631 2 2620 DNA
Unknown DNA sequence of LHN/C 2 ggatccatgc cgatcaccat caacaacttc
aactacagcg atccggtgga taacaaaaac 60 atcctgtacc tggataccca
tctgaatacc ctggcgaacg aaccggaaaa agcgtttcgt 120 atcaccggca
acatttgggt tattccggat cgttttagcc gtaacagcaa cccgaatctg 180
aataaaccgc cgcgtgttac cagcccgaaa agcggttatt acgatccgaa ctatctgagc
240 accgatagcg ataaagatac cttcctgaaa gaaatcatca aactgttcaa
acgcatcaac 300 agccgtgaaa ttggcgaaga actgatctat cgcctgagca
ccgatattcc gtttccgggc 360 aacaacaaca ccccgatcaa cacctttgat
ttcgatgtgg atttcaacag cgttgatgtt 420 aaaacccgcc agggtaacaa
ttgggtgaaa accggcagca ttaacccaag cgtgattatt 480 accggtccgc
gcgaaaacat tattgatccg gaaaccagca cctttaaact gaccaacaac 540
acctttgcgg cgcaggaagg ttttggcgcg ctgagcatta ttagcattag cccgcgcttt
600 atgctgacct atagcaacgc gaccaacgat gttggtgaag gccgtttcag
caaaagcgaa 660 ttttgcatgg acccgatcct gatcctgatg catgaactga
accatgcgat gcataacctg 720 tatggcatcg cgattccgaa cgatcagacc
attagcagcg tgaccagcaa catcttttac 780 agccagtaca acgtgaaact
ggaatatgcg gaaatctatg cgtttggcgg tccgaccatt 840 gatctgattc
cgaaaagcgc gcgcaaatac ttcgaagaaa aagcgctgga ttactatcgc 900
agcattgcga aacgtctgaa cagcattacc accgcgaatc cgagcagctt caacaaatat
960 atcggcgaat ataaacagaa actgatccgc aaatatcgct ttgtggtgga
aagcagcggc 1020 gaagttaccg ttaaccgcaa taaattcgtg gaactgtaca
acgaactgac ccagatcttc 1080 accgaattta actatgcgaa aatctataac
gtgcagaacc gtaaaatcta cctgagcaac 1140 gtgtataccc cggtgaccgc
gaatattctg gatgataacg tgtacgatat ccagaacggc 1200 tttaacatcc
cgaaaagcaa cctgaacgtt ctgtttatgg gccagaacct gagccgtaat 1260
ccggcgctgc gtaaagtgaa cccggaaaac atgctgtacc tgttcaccaa attttgcgtc
1320 gacgcgattg atggtcgtag cctgtacaac aaaaccctgc agtgtcgtga
actgctggtg 1380 aaaaacaccg atctgccgtt tattggcgat atcagcgatg
tgaaaaccga tatcttcctg 1440 cgcaaagata tcaacgaaga aaccgaagtg
atctactacc cggataacgt gagcgttgat 1500 caggtgatcc tgagcaaaaa
caccagcgaa catggtcagc tggatctgct gtatccgagc 1560 attgatagcg
aaagcgaaat tctgccgggc gaaaaccagg tgttttacga taaccgtacc 1620
cagaacgtgg attacctgaa cagctattac tacctggaaa gccagaaact gagcgataac
1680 gtggaagatt ttacctttac ccgcagcatt gaagaagcgc tggataacag
cgcgaaagtt 1740 tacacctatt ttccgaccct ggcgaacaaa gttaatgcgg
gtgttcaggg cggtctgttt 1800 ctgatgtggg cgaacgatgt ggtggaagat
ttcaccacca acatcctgcg taaagatacc 1860 ctggataaaa tcagcgatgt
tagcgcgatt attccgtata ttggtccggc gctgaacatt 1920 agcaatagcg
tgcgtcgtgg caattttacc gaagcgtttg cggttaccgg tgtgaccatt 1980
ctgctggaag cgtttccgga atttaccatt ccggcgctgg gtgcgtttgt gatctatagc
2040 aaagtgcagg aacgcaacga aatcatcaaa accatcgata actgcctgga
acagcgtatt 2100 aaacgctgga aagatagcta tgaatggatg atgggcacct
ggctgagccg tattatcacc 2160 cagttcaaca acatcagcta ccagatgtac
gatagcctga actatcaggc gggtgcgatt 2220 aaagcgaaaa tcgatctgga
atacaaaaaa tacagcggca gcgataaaga aaacatcaaa 2280 agccaggttg
aaaacctgaa aaacagcctg gatgtgaaaa ttagcgaagc gatgaataac 2340
atcaacaaat tcatccgcga atgcagcgtg acctacctgt tcaaaaacat gctgccgaaa
2400 gtgatcgatg aactgaacga atttgatcgc aacaccaaag cgaaactgat
caacctgatc 2460 gatagccaca acattattct ggtgggcgaa gtggataaac
tgaaagcgaa agttaacaac 2520 agcttccaga acaccatccc gtttaacatc
ttcagctata ccaacaacag cctgctgaaa 2580 gatatcatca acgaatactt
caatctagac taataagctt 2620 3 249 DNA Unknown DNA sequence of EGF
linker 3 ggatccgtcg acctgcaggg tctagaaggc ggtggcggta gcggcggtgg
cggtagcggc 60 ggtggcggta gcgcactaga caactctgac tctgaatgcc
cgctgtctca cgacggttac 120 tgcctgcacg acggtgtttg catgtacatc
gaagctctgg acaaatacgc ttgcaactgc 180 gttgttggtt acatcggtga
acgttgccag taccgtgacc tgaaatggtg ggaactgcgt 240 tgaaagctt 249 4
2838 DNA Unknown DNA sequence of EGF-C fusion 4 ggatccgaat
tcatgccgat caccatcaac aacttcaact acagcgatcc ggtggataac 60
aaaaacatcc tgtacctgga tacccatctg aataccctgg cgaacgaacc ggaaaaagcg
120 tttcgtatca ccggcaacat ttgggttatt ccggatcgtt ttagccgtaa
cagcaacccg 180 aatctgaata aaccgccgcg tgttaccagc ccgaaaagcg
gttattacga tccgaactat 240 ctgagcaccg atagcgataa agataccttc
ctgaaagaaa tcatcaaact gttcaaacgc 300 atcaacagcc gtgaaattgg
cgaagaactg atctatcgcc tgagcaccga tattccgttt 360 ccgggcaaca
acaacacccc gatcaacacc tttgatttcg atgtggattt caacagcgtt 420
gatgttaaaa cccgccaggg taacaattgg gtgaaaaccg gcagcattaa cccgagcgtg
480 attattaccg gtccgcgcga aaacattatt gatccggaaa ccagcacctt
taaactgacc 540 aacaacacct ttgcggcgca ggaaggtttt ggcgcgctga
gcattattag cattagcccg 600 cgctttatgc tgacctatag caacgcgacc
aacgatgttg gtgaaggccg tttcagcaaa 660 agcgaatttt gcatggaccc
gatcctgatc ctgatgcatg aactgaacca tgcgatgcat 720 aacctgtatg
gcatcgcgat tccgaacgat cagaccatta gcagcgtgac cagcaacatc 780
ttttacagcc agtacaacgt gaaactggaa tatgcggaaa tctatgcgtt tggcggtccg
840 accattgatc tgattccgaa aagcgcgcgc aaatacttcg aagaaaaagc
gctggattac 900 tatcgcagca ttgcgaaacg tctgaacagc attaccaccg
cgaatccgag cagcttcaac 960 aaatatatcg gcgaatataa acagaaactg
atccgcaaat atcgctttgt ggtggaaagc 1020 agcggcgaag ttaccgttaa
ccgcaataaa ttcgtggaac tgtacaacga actgacccag 1080 atcttcaccg
aatttaacta tgcgaaaatc tataacgtgc agaaccgtaa aatctacctg 1140
agcaacgtgt ataccccggt gaccgcgaat attctggatg ataacgtgta cgatatccag
1200 aacggcttta acatcccgaa aagcaacctg aacgttctgt ttatgggcca
gaacctgagc 1260 cgtaatccgg cgctgcgtaa agtgaacccg gaaaacatgc
tgtacctgtt caccaaattt 1320 tgcgtcgacg cgattgatgg tcgtagcctg
tacaacaaaa ccctgcagtg tcgtgaactg 1380 ctggtgaaaa acaccgatct
gccgtttatt ggcgatatca gcgatgtgaa aaccgatatc 1440 ttcctgcgca
aagatatcaa cgaagaaacc gaagtgatct actacccgga taacgtgagc 1500
gttgatcagg tgatcctgag caaaaacacc agcgaacatg gtcagctgga tctgctgtat
1560 ccgagcattg atagcgaaag cgaaattctg ccgggcgaaa accaggtgtt
ttacgataac 1620 cgtacccaga acgtggatta cctgaacagc tattactacc
tggaaagcca gaaactgagc 1680 gataacgtgg aagattttac ctttacccgc
agcattgaag aagcgctgga taacagcgcg 1740 aaagtttaca cctattttcc
gaccctggcg aacaaagtta atgcgggtgt tcagggcggt 1800 ctgtttctga
tgtgggcgaa cgatgtggtg gaagatttca ccaccaacat cctgcgtaaa 1860
gataccctgg ataaaatcag cgatgttagc gcgattattc cgtatattgg tccggcgctg
1920 aacattagca atagcgtgcg tcgtggcaat tttaccgaag cgtttgcggt
taccggtgtg 1980 accattctgc tggaagcgtt tccggaattt accattccgg
cgctgggtgc gtttgtgatc 2040 tatagcaaag tgcaggaacg caacgaaatc
atcaaaacca tcgataactg cctggaacag 2100 cgtattaaac gctggaaaga
tagctatgaa tggatgatgg gcacctggct gagccgtatt 2160 atcacccagt
tcaacaacat cagctaccag atgtacgata gcctgaacta tcaggcgggt 2220
gcgattaaag cgaaaatcga tctggaatac aaaaaataca gcggcagcga taaagaaaac
2280 atcaaaagcc aggttgaaaa cctgaaaaac agcctggatg tgaaaattag
cgaagcgatg 2340 aataacatca acaaattcat ccgcgaatgc agcgtgacct
acctgttcaa aaacatgctg 2400 ccgaaagtga tcgatgaact gaacgaattt
gatcgcaaca ccaaagcgaa actgatcaac 2460 ctgatcgata gccacaacat
tattctggtg ggcgaagtgg ataaactgaa agcgaaagtt 2520 aacaacagct
tccagaacac catcccgttt aacatcttca gctataccaa caacagcctg 2580
ctgaaagata tcatcaacga atacttcaat ctagaaggtg gcggtgggtc cggtggcggt
2640 ggctcaggcg ggggcggtag cgcactagac aactctgact ctgaatgccc
gctgtctcac 2700 gacggttact gcctgcacga cggtgtttgc atgtacatcg
aagctctgga caaatacgct 2760 tgcaactgcg ttgttggtta catcggtgaa
cgttgccagt accgtgacct gaaatggtgg 2820 gaactgcgtt gaaagctt 2838 5
945 PRT Unknown Protein sequence of the EGF-C fusion 5 Gly Ser Glu
Phe Met Pro Ile Thr Ile Asn Asn Phe Asn Tyr Ser Asp 1 5 10 15 Pro
Val Asp Asn Lys Asn Ile Leu Tyr Leu Asp Thr His Leu Asn Thr 20 25
30 Leu Ala Asn Glu Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp
35 40 45 Val Ile Pro Asp Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu
Asn Lys 50 55 60 Pro Pro Arg Val Thr Ser Pro Lys Ser Gly Tyr Tyr
Asp Pro Asn Tyr 65 70 75 80 Leu Ser Thr Asp Ser Asp Lys Asp Thr Phe
Leu Lys Glu Ile Ile Lys 85 90 95 Leu Phe Lys Arg Ile Asn Ser Arg
Glu Ile Gly Glu Glu Leu Ile Tyr 100 105 110 Arg Leu Ser Thr Asp Ile
Pro Phe Pro Gly Asn Asn Asn Thr Pro Ile 115 120 125 Asn Thr Phe Asp
Phe Asp Val Asp Phe Asn Ser Val Asp Val Lys Thr 130 135 140 Arg Gln
Gly Asn Asn Trp Val Lys Thr Gly Ser Ile Asn Pro Ser Val 145 150 155
160 Ile Ile Thr Gly Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr
165 170 175 Phe Lys Leu Thr Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe
Gly Ala 180 185 190 Leu Ser Ile Ile Ser Ile Ser Pro Arg Phe Met Leu
Thr Tyr Ser Asn 195 200 205 Ala Thr Asn Asp Val Gly Glu Gly Arg Phe
Ser Lys Ser Glu Phe Cys 210 215 220 Met Asp Pro Ile Leu Ile Leu Met
His Glu Leu Asn His Ala Met His 225 230 235 240 Asn Leu Tyr Gly Ile
Ala Ile Pro Asn Asp Gln Thr Ile Ser Ser Val 245 250 255 Thr Ser Asn
Ile Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala 260 265 270 Glu
Ile Tyr Ala Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser 275 280
285 Ala Arg Lys Tyr Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile
290 295 300 Ala Lys Arg Leu Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser
Phe Asn 305 310 315 320 Lys Tyr Ile Gly Glu Tyr Lys Gln Lys Leu Ile
Arg Lys Tyr Arg Phe 325 330 335 Val Val Glu Ser Ser Gly Glu Val Thr
Val Asn Arg Asn Lys Phe Val 340 345 350 Glu Leu Tyr Asn Glu Leu Thr
Gln Ile Phe Thr Glu Phe Asn Tyr Ala 355 360 365 Lys Ile Tyr Asn Val
Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr 370 375 380 Thr Pro Val
Thr Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln 385 390 395 400
Asn Gly Phe Asn Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly 405
410 415 Gln Asn Leu Ser Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu
Asn 420 425 430 Met Leu Tyr Leu Phe Thr Lys Phe Cys Val Asp Ala Ile
Asp Gly Arg 435 440 445 Ser Leu Tyr Asn Lys Thr Leu Gln Cys Arg Glu
Leu Leu Val Lys Asn 450 455 460 Thr Asp Leu Pro Phe Ile Gly Asp Ile
Ser Asp Val Lys Thr Asp Ile 465 470 475 480 Phe Leu Arg Lys Asp Ile
Asn Glu Glu Thr Glu Val Ile Tyr Tyr Pro 485 490 495 Asp Asn Val Ser
Val Asp Gln Val Ile Leu Ser Lys Asn Thr Ser Glu 500 505 510 His Gly
Gln Leu Asp Leu Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu 515 520 525
Ile Leu Pro Gly Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn 530
535 540 Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu
Ser 545 550 555 560 Asp Asn Val Glu Asp Phe Thr Phe Thr Arg Ser Ile
Glu Glu Ala Leu 565 570 575 Asp Asn Ser Ala Lys Val Tyr Thr Tyr Phe
Pro Thr Leu Ala Asn Lys 580 585 590 Val Asn Ala Gly Val Gln Gly Gly
Leu Phe Leu Met Trp Ala Asn Asp 595 600 605 Val Val Glu Asp Phe Thr
Thr Asn Ile Leu Arg Lys Asp Thr Leu Asp 610 615 620 Lys Ile Ser Asp
Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu 625 630 635 640 Asn
Ile Ser Asn Ser Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala 645 650
655 Val Thr Gly Val Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile
660 665 670 Pro Ala Leu Gly Ala Phe Val Ile Tyr Ser Lys Val Gln Glu
Arg Asn 675 680 685 Glu Ile Ile Lys Thr Ile Asp Asn Cys Leu Glu Gln
Arg Ile Lys Arg 690 695 700 Trp Lys Asp Ser Tyr Glu Trp Met Met Gly
Thr Trp Leu Ser Arg Ile 705 710 715 720 Ile Thr Gln Phe Asn Asn Ile
Ser Tyr Gln Met Tyr Asp Ser Leu Asn 725 730 735 Tyr Gln Ala Gly Ala
Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys 740 745 750 Tyr Ser Gly
Ser Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn Leu 755 760 765 Lys
Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn 770 775
780 Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu
785 790 795 800 Pro Lys Val Ile Asp Glu Leu Asn Glu Phe Asp Arg Asn
Thr Lys Ala 805 810 815 Lys Leu Ile Asn Leu Ile Asp Ser His Asn Ile
Ile Leu Val Gly Glu 820 825 830 Val Asp Lys Leu Lys Ala Lys Val Asn
Asn Ser Phe Gln Asn Thr Ile 835 840 845 Pro Phe Asn Ile Phe Ser Tyr
Thr Asn Asn Ser Leu Leu Lys Asp Ile 850 855 860 Ile Asn Glu Tyr Phe
Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly 865 870 875 880 Gly Ser
Gly Gly Gly Gly Ser Ala Leu Asp Asn Ser Asp Ser Glu Cys 885 890 895
Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val Cys Met Tyr 900
905 910 Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val Gly Tyr
Ile 915 920 925 Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu
Leu Arg Lys 930 935 940 Leu 945 6 2850 DNA Unknown DNA sequence of
the EGF-B fusion 6 ggatccatgc cggttaccat caacaacttc aactacaacg
acccgatcga caacaacaac 60 atcattatga tggaaccgcc gttcgcacgt
ggtaccggac gttactacaa ggcttttaag 120 atcaccgacc gtatctggat
catcccggaa cgttacacct tcggttacaa acctgaggac 180 ttcaacaaga
gtagcgggat tttcaatcgt gacgtctgcg agtactatga tccagattat 240
ctgaatacca acgataagaa gaacatattc cttcagacta tgattaaact cttcaaccgt
300 atcaaaagca aaccgctcgg tgaaaaactc ctcgaaatga ttatcaacgg
tatcccgtac 360 ctcggtgacc gtcgtgtccc gcttgaagag ttcaacacca
acatcgcaag cgtcaccgtc 420 aacaaactca tcagcaaccc aggtgaagtc
gaacgtaaaa aaggtatctt cgcaaacctc 480 atcatcttcg gtccgggtcc
ggtcctcaac gaaaacgaaa ccatcgacat
cggtatccag 540 aaccacttcg caagccgtga aggtttcggt ggtatcatgc
agatgaaatt ctgcccggaa 600 tacgtcagtg tcttcaacaa cgtccaggaa
aacaaaggtg caagcatctt caaccgtcgt 660 ggttacttca gcgacccggc
actcatcctc atgcatgaac tcatccacgt cctccacggt 720 ctctacggta
tcaaagttga cgacctcccg atcgtcccga acgagaagaa attcttcatg 780
cagagcaccg acgcaatcca ggctgaggaa ctctacacct tcggtggcca agacccaagt
840 atcataaccc cgtccaccga caaaagcatc tacgacaaag tcctccagaa
cttcaggggt 900 atcgtggaca gactcaacaa agtcctcgtc tgcatcagcg
acccgaacat caatatcaac 960 atatacaaga acaagttcaa agacaagtac
aaattcgtcg aggacagcga aggcaaatac 1020 agcatcgacg tagaaagttt
cgacaagctc tacaaaagcc tcatgttcgg tttcaccgaa 1080 accaacatcg
ccgagaacta caagatcaag acaagggcaa gttacttcag cgacagcctc 1140
ccgcctgtca aaatcaagaa cctcttagac aacgagattt acacaattga agagggcttc
1200 aacatcagtg acaaagacat ggagaaggaa tacagaggtc agaacaaggc
tatcaacaaa 1260 caggcatacg aggagatcag caaagaacac ctcgcagtct
acaagatcca gatgtgcgtc 1320 gacgaagaaa agctgtacga cgacgacgac
aaagaccgtt ggggttcttc gctgcagtgc 1380 atcgacgttg acaacgaaga
cctgttcttc atcgctgaca aaaacagctt cagtgacgac 1440 ctgagcaaaa
acgaacgtat cgaatacaac acccagagca actacatcga aaacgacttc 1500
ccgatcaacg aactgatcct ggacaccgac ctgataagta aaatcgaact gccgagcgaa
1560 aacaccgaaa gtctgaccga cttcaacgtt gacgttccgg tttacgaaaa
acagccggct 1620 atcaagaaaa tcttcaccga cgaaaacacc atcttccagt
acctgtacag ccagaccttc 1680 ccgctggaca tccgtgacat cagtctgacc
agcagtttcg acgacgctct gctgttcagc 1740 aacaaagttt acagtttctt
cagcatggac tacatcaaaa ccgctaacaa agttgttgaa 1800 gcagggctgt
tcgctggttg ggttaaacag atcgttaacg acttcgttat cgaagctaac 1860
aaaagcaaca ctatggacaa aatcgctgac atcagtctga tcgttccgta catcggtctg
1920 gctctgaacg ttggtaacga aaccgctaaa ggtaactttg aaaacgcttt
cgagatcgct 1980 ggtgcaagca tcctgctgga gttcatcccg gaactgctga
tcccggttgt tggtgctttc 2040 ctgctggaaa gttacatcga caacaaaaac
aagatcatca aaaccatcga caacgctctg 2100 accaaacgta acgaaaaatg
gagtgatatg tacggtctga tcgttgctca gtggctgagc 2160 accgtcaaca
cccagttcta caccatcaaa gaaggtatgt acaaagctct gaactaccag 2220
gctcaggctc tggaagagat catcaaatac cgttacaaca tctacagtga gaaggaaaag
2280 agtaacatca acatcgactt caacgacatc aacagcaaac tgaacgaagg
tatcaaccag 2340 gctatcgaca acatcaacaa cttcatcaac ggttgcagtg
ttagctacct gatgaagaag 2400 atgatcccgc tggctgttga aaaactgctg
gacttcgaca acaccctgaa aaagaacctg 2460 ctgaactaca tcgacgaaaa
caagctgtac ctgatcggta gtgctgaata cgaaaaaagt 2520 aaagtgaaca
aatacctgaa gaccatcatg ccgttcgacc tgagtatcta caccaacgac 2580
accatcctga tcgaaatgtt caacaaatac aactctctag atctagaagg tggcggtggg
2640 tccggtggcg gtggctcagg cgggggcggt agcgcactag acaactctga
ctctgaatgc 2700 ccgctgtctc acgacggtta ctgcctgcac gacggtgttt
gcatgtacat cgaagctctg 2760 gacaaatacg cttgcaactg cgttgttggt
tacatcggtg aacgttgcca gtaccgtgac 2820 ctgaaatggt gggaactgcg
ttgaaagctt 2850 7 949 PRT Unknown Protein sequence of the EGF-B
fusion 7 Gly Ser Met Pro Val Thr Ile Asn Asn Phe Asn Tyr Asn Asp
Pro Ile 1 5 10 15 Asp Asn Asn Asn Ile Ile Met Met Glu Pro Pro Phe
Ala Arg Gly Thr 20 25 30 Gly Arg Tyr Tyr Lys Ala Phe Lys Ile Thr
Asp Arg Ile Trp Ile Ile 35 40 45 Pro Glu Arg Tyr Thr Phe Gly Tyr
Lys Pro Glu Asp Phe Asn Lys Ser 50 55 60 Ser Gly Ile Phe Asn Arg
Asp Val Cys Glu Tyr Tyr Asp Pro Asp Tyr 65 70 75 80 Leu Asn Thr Asn
Asp Lys Lys Asn Ile Phe Leu Gln Thr Met Ile Lys 85 90 95 Leu Phe
Asn Arg Ile Lys Ser Lys Pro Leu Gly Glu Lys Leu Leu Glu 100 105 110
Met Ile Ile Asn Gly Ile Pro Tyr Leu Gly Asp Arg Arg Val Pro Leu 115
120 125 Glu Glu Phe Asn Thr Asn Ile Ala Ser Val Thr Val Asn Lys Leu
Ile 130 135 140 Ser Asn Pro Gly Glu Val Glu Arg Lys Lys Gly Ile Phe
Ala Asn Leu 145 150 155 160 Ile Ile Phe Gly Pro Gly Pro Val Leu Asn
Glu Asn Glu Thr Ile Asp 165 170 175 Ile Gly Ile Gln Asn His Phe Ala
Ser Arg Glu Gly Phe Gly Gly Ile 180 185 190 Met Gln Met Lys Phe Cys
Pro Glu Tyr Val Ser Val Phe Asn Asn Val 195 200 205 Gln Glu Asn Lys
Gly Ala Ser Ile Phe Asn Arg Arg Gly Tyr Phe Ser 210 215 220 Asp Pro
Ala Leu Ile Leu Met His Glu Leu Ile His Val Leu His Gly 225 230 235
240 Leu Tyr Gly Ile Lys Val Asp Asp Leu Pro Ile Val Pro Asn Glu Lys
245 250 255 Lys Phe Phe Met Gln Ser Thr Asp Ala Ile Gln Ala Glu Glu
Leu Tyr 260 265 270 Thr Phe Gly Gly Gln Asp Pro Ser Ile Ile Thr Pro
Ser Thr Asp Lys 275 280 285 Ser Ile Tyr Asp Lys Val Leu Gln Asn Phe
Arg Gly Ile Val Asp Arg 290 295 300 Leu Asn Lys Val Leu Val Cys Ile
Ser Asp Pro Asn Ile Asn Ile Asn 305 310 315 320 Ile Tyr Lys Asn Lys
Phe Lys Asp Lys Tyr Lys Phe Val Glu Asp Ser 325 330 335 Glu Gly Lys
Tyr Ser Ile Asp Val Glu Ser Phe Asp Lys Leu Tyr Lys 340 345 350 Ser
Leu Met Phe Gly Phe Thr Glu Thr Asn Ile Ala Glu Asn Tyr Lys 355 360
365 Ile Lys Thr Arg Ala Ser Tyr Phe Ser Asp Ser Leu Pro Pro Val Lys
370 375 380 Ile Lys Asn Leu Leu Asp Asn Glu Ile Tyr Thr Ile Glu Glu
Gly Phe 385 390 395 400 Asn Ile Ser Asp Lys Asp Met Glu Lys Glu Tyr
Arg Gly Gln Asn Lys 405 410 415 Ala Ile Asn Lys Gln Ala Tyr Glu Glu
Ile Ser Lys Glu His Leu Ala 420 425 430 Val Tyr Lys Ile Gln Met Cys
Val Asp Glu Glu Lys Leu Tyr Asp Asp 435 440 445 Asp Asp Lys Asp Arg
Trp Gly Ser Ser Leu Gln Cys Ile Asp Val Asp 450 455 460 Asn Glu Asp
Leu Phe Phe Ile Ala Asp Lys Asn Ser Phe Ser Asp Asp 465 470 475 480
Leu Ser Lys Asn Glu Arg Ile Glu Tyr Asn Thr Gln Ser Asn Tyr Ile 485
490 495 Glu Asn Asp Phe Pro Ile Asn Glu Leu Ile Leu Asp Thr Asp Leu
Ile 500 505 510 Ser Lys Ile Glu Leu Pro Ser Glu Asn Thr Glu Ser Leu
Thr Asp Phe 515 520 525 Asn Val Asp Val Pro Val Tyr Glu Lys Gln Pro
Ala Ile Lys Lys Ile 530 535 540 Phe Thr Asp Glu Asn Thr Ile Phe Gln
Tyr Leu Tyr Ser Gln Thr Phe 545 550 555 560 Pro Leu Asp Ile Arg Asp
Ile Ser Leu Thr Ser Ser Phe Asp Asp Ala 565 570 575 Leu Leu Phe Ser
Asn Lys Val Tyr Ser Phe Phe Ser Met Asp Tyr Ile 580 585 590 Lys Thr
Ala Asn Lys Val Val Glu Ala Gly Leu Phe Ala Gly Trp Val 595 600 605
Lys Gln Ile Val Asn Asp Phe Val Ile Glu Ala Asn Lys Ser Asn Thr 610
615 620 Met Asp Lys Ile Ala Asp Ile Ser Leu Ile Val Pro Tyr Ile Gly
Leu 625 630 635 640 Ala Leu Asn Val Gly Asn Glu Thr Ala Lys Gly Asn
Phe Glu Asn Ala 645 650 655 Phe Glu Ile Ala Gly Ala Ser Ile Leu Leu
Glu Phe Ile Pro Glu Leu 660 665 670 Leu Ile Pro Val Val Gly Ala Phe
Leu Leu Glu Ser Tyr Ile Asp Asn 675 680 685 Lys Asn Lys Ile Ile Lys
Thr Ile Asp Asn Ala Leu Thr Lys Arg Asn 690 695 700 Glu Lys Trp Ser
Asp Met Tyr Gly Leu Ile Val Ala Gln Trp Leu Ser 705 710 715 720 Thr
Val Asn Thr Gln Phe Tyr Thr Ile Lys Glu Gly Met Tyr Lys Ala 725 730
735 Leu Asn Tyr Gln Ala Gln Ala Leu Glu Glu Ile Ile Lys Tyr Arg Tyr
740 745 750 Asn Ile Tyr Ser Glu Lys Glu Lys Ser Asn Ile Asn Ile Asp
Phe Asn 755 760 765 Asp Ile Asn Ser Lys Leu Asn Glu Gly Ile Asn Gln
Ala Ile Asp Asn 770 775 780 Ile Asn Asn Phe Ile Asn Gly Cys Ser Val
Ser Tyr Leu Met Lys Lys 785 790 795 800 Met Ile Pro Leu Ala Val Glu
Lys Leu Leu Asp Phe Asp Asn Thr Leu 805 810 815 Lys Lys Asn Leu Leu
Asn Tyr Ile Asp Glu Asn Lys Leu Tyr Leu Ile 820 825 830 Gly Ser Ala
Glu Tyr Glu Lys Ser Lys Val Asn Lys Tyr Leu Lys Thr 835 840 845 Ile
Met Pro Phe Asp Leu Ser Ile Tyr Thr Asn Asp Thr Ile Leu Ile 850 855
860 Glu Met Phe Asn Lys Tyr Asn Ser Leu Asp Leu Glu Gly Gly Gly Gly
865 870 875 880 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala Leu
Asp Asn Ser 885 890 895 Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr
Cys Leu His Asp Gly 900 905 910 Val Cys Met Tyr Ile Glu Ala Leu Asp
Lys Tyr Ala Cys Asn Cys Val 915 920 925 Val Gly Tyr Ile Gly Glu Arg
Cys Gln Tyr Arg Asp Leu Lys Trp Trp 930 935 940 Glu Leu Arg Lys Leu
945 8 120 DNA Unknown DNA sequence of the RGD linker 8 ggatccgtcg
acctgcaggg tctagaaggc ggtggcggta gcggcggtgg cggtagcggc 60
ggtggcggta gcgcactagt gggtggtcgt ggtgacatgt tcggtgcttg ataaaagctt
120 9 2709 DNA Unknown DNA sequence of the RGD-C fusion 9
ggatccgaat tcatgccgat caccatcaac aacttcaact acagcgatcc ggtggataac
60 aaaaacatcc tgtacctgga tacccatctg aataccctgg cgaacgaacc
ggaaaaagcg 120 tttcgtatca ccggcaacat ttgggttatt ccggatcgtt
ttagccgtaa cagcaacccg 180 aatctgaata aaccgccgcg tgttaccagc
ccgaaaagcg gttattacga tccgaactat 240 ctgagcaccg atagcgataa
agataccttc ctgaaagaaa tcatcaaact gttcaaacgc 300 atcaacagcc
gtgaaattgg cgaagaactg atctatcgcc tgagcaccga tattccgttt 360
ccgggcaaca acaacacccc gatcaacacc tttgatttcg atgtggattt caacagcgtt
420 gatgttaaaa cccgccaggg taacaattgg gtgaaaaccg gcagcattaa
cccgagcgtg 480 attattaccg gtccgcgcga aaacattatt gatccggaaa
ccagcacctt taaactgacc 540 aacaacacct ttgcggcgca ggaaggtttt
ggcgcgctga gcattattag cattagcccg 600 cgctttatgc tgacctatag
caacgcgacc aacgatgttg gtgaaggccg tttcagcaaa 660 agcgaatttt
gcatggaccc gatcctgatc ctgatgcatg aactgaacca tgcgatgcat 720
aacctgtatg gcatcgcgat tccgaacgat cagaccatta gcagcgtgac cagcaacatc
780 ttttacagcc agtacaacgt gaaactggaa tatgcggaaa tctatgcgtt
tggcggtccg 840 accattgatc tgattccgaa aagcgcgcgc aaatacttcg
aagaaaaagc gctggattac 900 tatcgcagca ttgcgaaacg tctgaacagc
attaccaccg cgaatccgag cagcttcaac 960 aaatatatcg gcgaatataa
acagaaactg atccgcaaat atcgctttgt ggtggaaagc 1020 agcggcgaag
ttaccgttaa ccgcaataaa ttcgtggaac tgtacaacga actgacccag 1080
atcttcaccg aatttaacta tgcgaaaatc tataacgtgc agaaccgtaa aatctacctg
1140 agcaacgtgt ataccccggt gaccgcgaat attctggatg ataacgtgta
cgatatccag 1200 aacggcttta acatcccgaa aagcaacctg aacgttctgt
ttatgggcca gaacctgagc 1260 cgtaatccgg cgctgcgtaa agtgaacccg
gaaaacatgc tgtacctgtt caccaaattt 1320 tgcgtcgacg cgattgatgg
tcgtagcctg tacaacaaaa ccctgcagtg tcgtgaactg 1380 ctggtgaaaa
acaccgatct gccgtttatt ggcgatatca gcgatgtgaa aaccgatatc 1440
ttcctgcgca aagatatcaa cgaagaaacc gaagtgatct actacccgga taacgtgagc
1500 gttgatcagg tgatcctgag caaaaacacc agcgaacatg gtcagctgga
tctgctgtat 1560 ccgagcattg atagcgaaag cgaaattctg ccgggcgaaa
accaggtgtt ttacgataac 1620 cgtacccaga acgtggatta cctgaacagc
tattactacc tggaaagcca gaaactgagc 1680 gataacgtgg aagattttac
ctttacccgc agcattgaag aagcgctgga taacagcgcg 1740 aaagtttaca
cctattttcc gaccctggcg aacaaagtta atgcgggtgt tcagggcggt 1800
ctgtttctga tgtgggcgaa cgatgtggtg gaagatttca ccaccaacat cctgcgtaaa
1860 gataccctgg ataaaatcag cgatgttagc gcgattattc cgtatattgg
tccggcgctg 1920 aacattagca atagcgtgcg tcgtggcaat tttaccgaag
cgtttgcggt taccggtgtg 1980 accattctgc tggaagcgtt tccggaattt
accattccgg cgctgggtgc gtttgtgatc 2040 tatagcaaag tgcaggaacg
caacgaaatc atcaaaacca tcgataactg cctggaacag 2100 cgtattaaac
gctggaaaga tagctatgaa tggatgatgg gcacctggct gagccgtatt 2160
atcacccagt tcaacaacat cagctaccag atgtacgata gcctgaacta tcaggcgggt
2220 gcgattaaag cgaaaatcga tctggaatac aaaaaataca gcggcagcga
taaagaaaac 2280 atcaaaagcc aggttgaaaa cctgaaaaac agcctggatg
tgaaaattag cgaagcgatg 2340 aataacatca acaaattcat ccgcgaatgc
agcgtgacct acctgttcaa aaacatgctg 2400 ccgaaagtga tcgatgaact
gaacgaattt gatcgcaaca ccaaagcgaa actgatcaac 2460 ctgatcgata
gccacaacat tattctggtg ggcgaagtgg ataaactgaa agcgaaagtt 2520
aacaacagct tccagaacac catcccgttt aacatcttca gctataccaa caacagcctg
2580 ctgaaagata tcatcaacga atacttcaat ctagaaggcg gtggcggtag
cggcggtggc 2640 ggtagcggcg gtggcggtag cgcactagtg ggtggtcgtg
gtgacatgtt cggtgcttga 2700 taaaagctt 2709 10 901 PRT Unknown
Protein sequenc eof RGD-C fusion 10 Gly Ser Glu Phe Met Pro Ile Thr
Ile Asn Asn Phe Asn Tyr Ser Asp 1 5 10 15 Pro Val Asp Asn Lys Asn
Ile Leu Tyr Leu Asp Thr His Leu Asn Thr 20 25 30 Leu Ala Asn Glu
Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp 35 40 45 Val Ile
Pro Asp Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys 50 55 60
Pro Pro Arg Val Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr 65
70 75 80 Leu Ser Thr Asp Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile
Ile Lys 85 90 95 Leu Phe Lys Arg Ile Asn Ser Arg Glu Ile Gly Glu
Glu Leu Ile Tyr 100 105 110 Arg Leu Ser Thr Asp Ile Pro Phe Pro Gly
Asn Asn Asn Thr Pro Ile 115 120 125 Asn Thr Phe Asp Phe Asp Val Asp
Phe Asn Ser Val Asp Val Lys Thr 130 135 140 Arg Gln Gly Asn Asn Trp
Val Lys Thr Gly Ser Ile Asn Pro Ser Val 145 150 155 160 Ile Ile Thr
Gly Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr 165 170 175 Phe
Lys Leu Thr Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala 180 185
190 Leu Ser Ile Ile Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn
195 200 205 Ala Thr Asn Asp Val Gly Glu Gly Arg Phe Ser Lys Ser Glu
Phe Cys 210 215 220 Met Asp Pro Ile Leu Ile Leu Met His Glu Leu Asn
His Ala Met His 225 230 235 240 Asn Leu Tyr Gly Ile Ala Ile Pro Asn
Asp Gln Thr Ile Ser Ser Val 245 250 255 Thr Ser Asn Ile Phe Tyr Ser
Gln Tyr Asn Val Lys Leu Glu Tyr Ala 260 265 270 Glu Ile Tyr Ala Phe
Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser 275 280 285 Ala Arg Lys
Tyr Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile 290 295 300 Ala
Lys Arg Leu Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn 305 310
315 320 Lys Tyr Ile Gly Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg
Phe 325 330 335 Val Val Glu Ser Ser Gly Glu Val Thr Val Asn Arg Asn
Lys Phe Val 340 345 350 Glu Leu Tyr Asn Glu Leu Thr Gln Ile Phe Thr
Glu Phe Asn Tyr Ala 355 360 365 Lys Ile Tyr Asn Val Gln Asn Arg Lys
Ile Tyr Leu Ser Asn Val Tyr 370 375 380 Thr Pro Val Thr Ala Asn Ile
Leu Asp Asp Asn Val Tyr Asp Ile Gln 385 390 395 400 Asn Gly Phe Asn
Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly 405 410 415 Gln Asn
Leu Ser Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn 420 425 430
Met Leu Tyr Leu Phe Thr Lys Phe Cys Val Asp Ala Ile Asp Gly Arg 435
440 445 Ser Leu Tyr Asn Lys Thr Leu Gln Cys Arg Glu Leu Leu Val Lys
Asn 450 455 460 Thr Asp Leu Pro Phe Ile Gly Asp Ile Ser Asp Val Lys
Thr Asp Ile 465 470 475 480 Phe Leu Arg Lys Asp Ile Asn Glu Glu Thr
Glu Val Ile Tyr Tyr Pro 485 490 495 Asp Asn Val Ser Val Asp Gln Val
Ile Leu Ser Lys Asn Thr Ser Glu 500 505 510 His Gly Gln Leu Asp Leu
Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu 515 520 525 Ile Leu Pro Gly
Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn 530 535 540 Val Asp
Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser 545 550 555
560 Asp Asn Val Glu Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu
565 570 575 Asp Asn Ser Ala Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala
Asn Lys 580 585 590 Val Asn Ala Gly Val Gln Gly Gly Leu Phe Leu Met
Trp Ala Asn Asp 595 600 605 Val Val Glu Asp Phe Thr Thr Asn Ile Leu
Arg Lys Asp Thr Leu Asp 610 615
620 Lys Ile Ser Asp Val Ser Ala Ile Ile Pro Tyr Ile Gly Pro Ala Leu
625 630 635 640 Asn Ile Ser Asn Ser Val Arg Arg Gly Asn Phe Thr Glu
Ala Phe Ala 645 650 655 Val Thr Gly Val Thr Ile Leu Leu Glu Ala Phe
Pro Glu Phe Thr Ile 660 665 670 Pro Ala Leu Gly Ala Phe Val Ile Tyr
Ser Lys Val Gln Glu Arg Asn 675 680 685 Glu Ile Ile Lys Thr Ile Asp
Asn Cys Leu Glu Gln Arg Ile Lys Arg 690 695 700 Trp Lys Asp Ser Tyr
Glu Trp Met Met Gly Thr Trp Leu Ser Arg Ile 705 710 715 720 Ile Thr
Gln Phe Asn Asn Ile Ser Tyr Gln Met Tyr Asp Ser Leu Asn 725 730 735
Tyr Gln Ala Gly Ala Ile Lys Ala Lys Ile Asp Leu Glu Tyr Lys Lys 740
745 750 Tyr Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser Gln Val Glu Asn
Leu 755 760 765 Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala Met Asn
Asn Ile Asn 770 775 780 Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu
Phe Lys Asn Met Leu 785 790 795 800 Pro Lys Val Ile Asp Glu Leu Asn
Glu Phe Asp Arg Asn Thr Lys Ala 805 810 815 Lys Leu Ile Asn Leu Ile
Asp Ser His Asn Ile Ile Leu Val Gly Glu 820 825 830 Val Asp Lys Leu
Lys Ala Lys Val Asn Asn Ser Phe Gln Asn Thr Ile 835 840 845 Pro Phe
Asn Ile Phe Ser Tyr Thr Asn Asn Ser Leu Leu Lys Asp Ile 850 855 860
Ile Asn Glu Tyr Phe Asn Leu Glu Gly Gly Gly Gly Ser Gly Gly Gly 865
870 875 880 Gly Ser Gly Gly Gly Gly Ser Ala Leu Val Gly Gly Arg Gly
Asp Met 885 890 895 Phe Gly Ala Lys Leu 900 11 126 DNA Unknown DNA
sequence of cyclic RGD linker 11 ggatccgtcg acctgcaggg tctagaaggc
ggtggcggta gcggcggtgg cggtagcggc 60 ggtggcggta gcgcactagt
gggtggttgc cgtggtgaca tgttcggttg cgcttgataa 120 aagctt 126 12 2715
DNA Unknown DNA sequence of cyclic RGD-C fusion 12 ggatccgaat
tcatgccgat caccatcaac aacttcaact acagcgatcc ggtggataac 60
aaaaacatcc tgtacctgga tacccatctg aataccctgg cgaacgaacc ggaaaaagcg
120 tttcgtatca ccggcaacat ttgggttatt ccggatcgtt ttagccgtaa
cagcaacccg 180 aatctgaata aaccgccgcg tgttaccagc ccgaaaagcg
gttattacga tccgaactat 240 ctgagcaccg atagcgataa agataccttc
ctgaaagaaa tcatcaaact gttcaaacgc 300 atcaacagcc gtgaaattgg
cgaagaactg atctatcgcc tgagcaccga tattccgttt 360 ccgggcaaca
acaacacccc gatcaacacc tttgatttcg atgtggattt caacagcgtt 420
gatgttaaaa cccgccaggg taacaattgg gtgaaaaccg gcagcattaa cccgagcgtg
480 attattaccg gtccgcgcga aaacattatt gatccggaaa ccagcacctt
taaactgacc 540 aacaacacct ttgcggcgca ggaaggtttt ggcgcgctga
gcattattag cattagcccg 600 cgctttatgc tgacctatag caacgcgacc
aacgatgttg gtgaaggccg tttcagcaaa 660 agcgaatttt gcatggaccc
gatcctgatc ctgatgcatg aactgaacca tgcgatgcat 720 aacctgtatg
gcatcgcgat tccgaacgat cagaccatta gcagcgtgac cagcaacatc 780
ttttacagcc agtacaacgt gaaactggaa tatgcggaaa tctatgcgtt tggcggtccg
840 accattgatc tgattccgaa aagcgcgcgc aaatacttcg aagaaaaagc
gctggattac 900 tatcgcagca ttgcgaaacg tctgaacagc attaccaccg
cgaatccgag cagcttcaac 960 aaatatatcg gcgaatataa acagaaactg
atccgcaaat atcgctttgt ggtggaaagc 1020 agcggcgaag ttaccgttaa
ccgcaataaa ttcgtggaac tgtacaacga actgacccag 1080 atcttcaccg
aatttaacta tgcgaaaatc tataacgtgc agaaccgtaa aatctacctg 1140
agcaacgtgt ataccccggt gaccgcgaat attctggatg ataacgtgta cgatatccag
1200 aacggcttta acatcccgaa aagcaacctg aacgttctgt ttatgggcca
gaacctgagc 1260 cgtaatccgg cgctgcgtaa agtgaacccg gaaaacatgc
tgtacctgtt caccaaattt 1320 tgcgtcgacg cgattgatgg tcgtagcctg
tacaacaaaa ccctgcagtg tcgtgaactg 1380 ctggtgaaaa acaccgatct
gccgtttatt ggcgatatca gcgatgtgaa aaccgatatc 1440 ttcctgcgca
aagatatcaa cgaagaaacc gaagtgatct actacccgga taacgtgagc 1500
gttgatcagg tgatcctgag caaaaacacc agcgaacatg gtcagctgga tctgctgtat
1560 ccgagcattg atagcgaaag cgaaattctg ccgggcgaaa accaggtgtt
ttacgataac 1620 cgtacccaga acgtggatta cctgaacagc tattactacc
tggaaagcca gaaactgagc 1680 gataacgtgg aagattttac ctttacccgc
agcattgaag aagcgctgga taacagcgcg 1740 aaagtttaca cctattttcc
gaccctggcg aacaaagtta atgcgggtgt tcagggcggt 1800 ctgtttctga
tgtgggcgaa cgatgtggtg gaagatttca ccaccaacat cctgcgtaaa 1860
gataccctgg ataaaatcag cgatgttagc gcgattattc cgtatattgg tccggcgctg
1920 aacattagca atagcgtgcg tcgtggcaat tttaccgaag cgtttgcggt
taccggtgtg 1980 accattctgc tggaagcgtt tccggaattt accattccgg
cgctgggtgc gtttgtgatc 2040 tatagcaaag tgcaggaacg caacgaaatc
atcaaaacca tcgataactg cctggaacag 2100 cgtattaaac gctggaaaga
tagctatgaa tggatgatgg gcacctggct gagccgtatt 2160 atcacccagt
tcaacaacat cagctaccag atgtacgata gcctgaacta tcaggcgggt 2220
gcgattaaag cgaaaatcga tctggaatac aaaaaataca gcggcagcga taaagaaaac
2280 atcaaaagcc aggttgaaaa cctgaaaaac agcctggatg tgaaaattag
cgaagcgatg 2340 aataacatca acaaattcat ccgcgaatgc agcgtgacct
acctgttcaa aaacatgctg 2400 ccgaaagtga tcgatgaact gaacgaattt
gatcgcaaca ccaaagcgaa actgatcaac 2460 ctgatcgata gccacaacat
tattctggtg ggcgaagtgg ataaactgaa agcgaaagtt 2520 aacaacagct
tccagaacac catcccgttt aacatcttca gctataccaa caacagcctg 2580
ctgaaagata tcatcaacga atacttcaat ctagaaggcg gtggcggtag cggcggtggc
2640 ggtagcggcg gtggcggtag cgcactagtg ggtggttgcc gtggtgacat
gttcggttgc 2700 gcttgataaa agctt 2715 13 903 PRT Unknown Protein
sequence of cyclic RGD-C fusion 13 Gly Ser Glu Phe Met Pro Ile Thr
Ile Asn Asn Phe Asn Tyr Ser Asp 1 5 10 15 Pro Val Asp Asn Lys Asn
Ile Leu Tyr Leu Asp Thr His Leu Asn Thr 20 25 30 Leu Ala Asn Glu
Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp 35 40 45 Val Ile
Pro Asp Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys 50 55 60
Pro Pro Arg Val Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr 65
70 75 80 Leu Ser Thr Asp Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile
Ile Lys 85 90 95 Leu Phe Lys Arg Ile Asn Ser Arg Glu Ile Gly Glu
Glu Leu Ile Tyr 100 105 110 Arg Leu Ser Thr Asp Ile Pro Phe Pro Gly
Asn Asn Asn Thr Pro Ile 115 120 125 Asn Thr Phe Asp Phe Asp Val Asp
Phe Asn Ser Val Asp Val Lys Thr 130 135 140 Arg Gln Gly Asn Asn Trp
Val Lys Thr Gly Ser Ile Asn Pro Ser Val 145 150 155 160 Ile Ile Thr
Gly Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr 165 170 175 Phe
Lys Leu Thr Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala 180 185
190 Leu Ser Ile Ile Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn
195 200 205 Ala Thr Asn Asp Val Gly Glu Gly Arg Phe Ser Lys Ser Glu
Phe Cys 210 215 220 Met Asp Pro Ile Leu Ile Leu Met His Glu Leu Asn
His Ala Met His 225 230 235 240 Asn Leu Tyr Gly Ile Ala Ile Pro Asn
Asp Gln Thr Ile Ser Ser Val 245 250 255 Thr Ser Asn Ile Phe Tyr Ser
Gln Tyr Asn Val Lys Leu Glu Tyr Ala 260 265 270 Glu Ile Tyr Ala Phe
Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser 275 280 285 Ala Arg Lys
Tyr Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser Ile 290 295 300 Ala
Lys Arg Leu Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe Asn 305 310
315 320 Lys Tyr Ile Gly Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr Arg
Phe 325 330 335 Val Val Glu Ser Ser Gly Glu Val Thr Val Asn Arg Asn
Lys Phe Val 340 345 350 Glu Leu Tyr Asn Glu Leu Thr Gln Ile Phe Thr
Glu Phe Asn Tyr Ala 355 360 365 Lys Ile Tyr Asn Val Gln Asn Arg Lys
Ile Tyr Leu Ser Asn Val Tyr 370 375 380 Thr Pro Val Thr Ala Asn Ile
Leu Asp Asp Asn Val Tyr Asp Ile Gln 385 390 395 400 Asn Gly Phe Asn
Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met Gly 405 410 415 Gln Asn
Leu Ser Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu Asn 420 425 430
Met Leu Tyr Leu Phe Thr Lys Phe Cys Val Asp Ala Ile Asp Gly Arg 435
440 445 Ser Leu Tyr Asn Lys Thr Leu Gln Cys Arg Glu Leu Leu Val Lys
Asn 450 455 460 Thr Asp Leu Pro Phe Ile Gly Asp Ile Ser Asp Val Lys
Thr Asp Ile 465 470 475 480 Phe Leu Arg Lys Asp Ile Asn Glu Glu Thr
Glu Val Ile Tyr Tyr Pro 485 490 495 Asp Asn Val Ser Val Asp Gln Val
Ile Leu Ser Lys Asn Thr Ser Glu 500 505 510 His Gly Gln Leu Asp Leu
Leu Tyr Pro Ser Ile Asp Ser Glu Ser Glu 515 520 525 Ile Leu Pro Gly
Glu Asn Gln Val Phe Tyr Asp Asn Arg Thr Gln Asn 530 535 540 Val Asp
Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu Ser Gln Lys Leu Ser 545 550 555
560 Asp Asn Val Glu Asp Phe Thr Phe Thr Arg Ser Ile Glu Glu Ala Leu
565 570 575 Asp Asn Ser Ala Lys Val Tyr Thr Tyr Phe Pro Thr Leu Ala
Asn Lys 580 585 590 Val Asn Ala Gly Val Gln Gly Gly Leu Phe Leu Met
Trp Ala Asn Asp 595 600 605 Val Val Glu Asp Phe Thr Thr Asn Ile Leu
Arg Lys Asp Thr Leu Asp 610 615 620 Lys Ile Ser Asp Val Ser Ala Ile
Ile Pro Tyr Ile Gly Pro Ala Leu 625 630 635 640 Asn Ile Ser Asn Ser
Val Arg Arg Gly Asn Phe Thr Glu Ala Phe Ala 645 650 655 Val Thr Gly
Val Thr Ile Leu Leu Glu Ala Phe Pro Glu Phe Thr Ile 660 665 670 Pro
Ala Leu Gly Ala Phe Val Ile Tyr Ser Lys Val Gln Glu Arg Asn 675 680
685 Glu Ile Ile Lys Thr Ile Asp Asn Cys Leu Glu Gln Arg Ile Lys Arg
690 695 700 Trp Lys Asp Ser Tyr Glu Trp Met Met Gly Thr Trp Leu Ser
Arg Ile 705 710 715 720 Ile Thr Gln Phe Asn Asn Ile Ser Tyr Gln Met
Tyr Asp Ser Leu Asn 725 730 735 Tyr Gln Ala Gly Ala Ile Lys Ala Lys
Ile Asp Leu Glu Tyr Lys Lys 740 745 750 Tyr Ser Gly Ser Asp Lys Glu
Asn Ile Lys Ser Gln Val Glu Asn Leu 755 760 765 Lys Asn Ser Leu Asp
Val Lys Ile Ser Glu Ala Met Asn Asn Ile Asn 770 775 780 Lys Phe Ile
Arg Glu Cys Ser Val Thr Tyr Leu Phe Lys Asn Met Leu 785 790 795 800
Pro Lys Val Ile Asp Glu Leu Asn Glu Phe Asp Arg Asn Thr Lys Ala 805
810 815 Lys Leu Ile Asn Leu Ile Asp Ser His Asn Ile Ile Leu Val Gly
Glu 820 825 830 Val Asp Lys Leu Lys Ala Lys Val Asn Asn Ser Phe Gln
Asn Thr Ile 835 840 845 Pro Phe Asn Ile Phe Ser Tyr Thr Asn Asn Ser
Leu Leu Lys Asp Ile 850 855 860 Ile Asn Glu Tyr Phe Asn Leu Glu Gly
Gly Gly Gly Ser Gly Gly Gly 865 870 875 880 Gly Ser Gly Gly Gly Gly
Ser Ala Leu Val Gly Gly Cys Arg Gly Asp 885 890 895 Met Phe Gly Cys
Ala Lys Leu 900 14 156 DNA Unknown DNA sequence of the
LC/C-RGD-HN/C LINKER 14 ggatccacgc acgtcgacgc gattgatggt cgtggtggtc
gtggtgacat gttcggtgct 60 gcgctagcgg gcggtggcgg tagcggcggt
ggcggtagcg gcggtggcgg tagcgcacta 120 gtgctgcaga cgcacggtct
agaatgataa aagctt 156 15 2733 DNA Unknown DNA sequence of the
LC/C-RGD-HN/C fusion 15 ggatccgaat tcatgccgat caccatcaac aacttcaact
acagcgatcc ggtggataac 60 aaaaacatcc tgtacctgga tacccatctg
aataccctgg cgaacgaacc ggaaaaagcg 120 tttcgtatca ccggcaacat
ttgggttatt ccggatcgtt ttagccgtaa cagcaacccg 180 aatctgaata
aaccgccgcg tgttaccagc ccgaaaagcg gttattacga tccgaactat 240
ctgagcaccg atagcgataa agataccttc ctgaaagaaa tcatcaaact gttcaaacgc
300 atcaacagcc gtgaaattgg cgaagaactg atctatcgcc tgagcaccga
tattccgttt 360 ccgggcaaca acaacacccc gatcaacacc tttgatttcg
atgtggattt caacagcgtt 420 gatgttaaaa cccgccaggg taacaattgg
gtgaaaaccg gcagcattaa cccgagcgtg 480 attattaccg gtccgcgcga
aaacattatt gatccggaaa ccagcacctt taaactgacc 540 aacaacacct
ttgcggcgca ggaaggtttt ggcgcgctga gcattattag cattagcccg 600
cgctttatgc tgacctatag caacgcgacc aacgatgttg gtgaaggccg tttcagcaaa
660 agcgaatttt gcatggaccc gatcctgatc ctgatgcatg aactgaacca
tgcgatgcat 720 aacctgtatg gcatcgcgat tccgaacgat cagaccatta
gcagcgtgac cagcaacatc 780 ttttacagcc agtacaacgt gaaactggaa
tatgcggaaa tctatgcgtt tggcggtccg 840 accattgatc tgattccgaa
aagcgcgcgc aaatacttcg aagaaaaagc gctggattac 900 tatcgcagca
ttgcgaaacg tctgaacagc attaccaccg cgaatccgag cagcttcaac 960
aaatatatcg gcgaatataa acagaaactg atccgcaaat atcgctttgt ggtggaaagc
1020 agcggcgaag ttaccgttaa ccgcaataaa ttcgtggaac tgtacaacga
actgacccag 1080 atcttcaccg aatttaacta tgcgaaaatc tataacgtgc
agaaccgtaa aatctacctg 1140 agcaacgtgt ataccccggt gaccgcgaat
attctggatg ataacgtgta cgatatccag 1200 aacggcttta acatcccgaa
aagcaacctg aacgttctgt ttatgggcca gaacctgagc 1260 cgtaatccgg
cgctgcgtaa agtgaacccg gaaaacatgc tgtacctgtt caccaaattt 1320
tgcgtcgacg cgattgatgg tcgtggtggt cgtggtgaca tgttcggtgc tgcgctagcg
1380 ggcggtggcg gtagcggcgg tggcggtagc ggcggtggcg gtagcgcact
agtgctgcag 1440 tgtcgtgaac tgctggtgaa aaacaccgat ctgccgttta
ttggcgatat cagcgatgtg 1500 aaaaccgata tcttcctgcg caaagatatc
aacgaagaaa ccgaagtgat ctactacccg 1560 gataacgtga gcgttgatca
ggtgatcctg agcaaaaaca ccagcgaaca tggtcagctg 1620 gatctgctgt
atccgagcat tgatagcgaa agcgaaattc tgccgggcga aaaccaggtg 1680
ttttacgata accgtaccca gaacgtggat tacctgaaca gctattacta cctggaaagc
1740 cagaaactga gcgataacgt ggaagatttt acctttaccc gcagcattga
agaagcgctg 1800 gataacagcg cgaaagttta cacctatttt ccgaccctgg
cgaacaaagt taatgcgggt 1860 gttcagggcg gtctgtttct gatgtgggcg
aacgatgtgg tggaagattt caccaccaac 1920 atcctgcgta aagataccct
ggataaaatc agcgatgtta gcgcgattat tccgtatatt 1980 ggtccggcgc
tgaacattag caatagcgtg cgtcgtggca attttaccga agcgtttgcg 2040
gttaccggtg tgaccattct gctggaagcg tttccggaat ttaccattcc ggcgctgggt
2100 gcgtttgtga tctatagcaa agtgcaggaa cgcaacgaaa tcatcaaaac
catcgataac 2160 tgcctggaac agcgtattaa acgctggaaa gatagctatg
aatggatgat gggcacctgg 2220 ctgagccgta ttatcaccca gttcaacaac
atcagctacc agatgtacga tagcctgaac 2280 tatcaggcgg gtgcgattaa
agcgaaaatc gatctggaat acaaaaaata cagcggcagc 2340 gataaagaaa
acatcaaaag ccaggttgaa aacctgaaaa acagcctgga tgtgaaaatt 2400
agcgaagcga tgaataacat caacaaattc atccgcgaat gcagcgtgac ctacctgttc
2460 aaaaacatgc tgccgaaagt gatcgatgaa ctgaacgaat ttgatcgcaa
caccaaagcg 2520 aaactgatca acctgatcga tagccacaac attattctgg
tgggcgaagt ggataaactg 2580 aaagcgaaag ttaacaacag cttccagaac
accatcccgt ttaacatctt cagctatacc 2640 aacaacagcc tgctgaaaga
tatcatcaac gaatacttca atctagaagc actagcgagt 2700 gggcaccatc
accatcacca ttaatgaaag ctt 2733 16 909 PRT Unknown Protein sequence
of the LC/C-RGD-HN/C fusion 16 Gly Ser Glu Phe Met Pro Ile Thr Ile
Asn Asn Phe Asn Tyr Ser Asp 1 5 10 15 Pro Val Asp Asn Lys Asn Ile
Leu Tyr Leu Asp Thr His Leu Asn Thr 20 25 30 Leu Ala Asn Glu Pro
Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile Trp 35 40 45 Val Ile Pro
Asp Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn Lys 50 55 60 Pro
Pro Arg Val Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn Tyr 65 70
75 80 Leu Ser Thr Asp Ser Asp Lys Asp Thr Phe Leu Lys Glu Ile Ile
Lys 85 90 95 Leu Phe Lys Arg Ile Asn Ser Arg Glu Ile Gly Glu Glu
Leu Ile Tyr 100 105 110 Arg Leu Ser Thr Asp Ile Pro Phe Pro Gly Asn
Asn Asn Thr Pro Ile 115 120 125 Asn Thr Phe Asp Phe Asp Val Asp Phe
Asn Ser Val Asp Val Lys Thr 130 135 140 Arg Gln Gly Asn Asn Trp Val
Lys Thr Gly Ser Ile Asn Pro Ser Val 145 150 155 160 Ile Ile Thr Gly
Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser Thr 165 170 175 Phe Lys
Leu Thr Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly Ala 180 185 190
Leu Ser Ile Ile Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser Asn 195
200 205 Ala Thr Asn Asp Val Gly Glu Gly Arg Phe Ser Lys Ser Glu Phe
Cys 210 215 220 Met Asp Pro Ile Leu Ile Leu Met His Glu Leu Asn His
Ala Met His 225 230 235 240 Asn Leu Tyr Gly Ile Ala Ile Pro Asn Asp
Gln Thr Ile Ser Ser Val 245 250 255 Thr
Ser Asn Ile Phe Tyr Ser Gln Tyr Asn Val Lys Leu Glu Tyr Ala 260 265
270 Glu Ile Tyr Ala Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys Ser
275 280 285 Ala Arg Lys Tyr Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg
Ser Ile 290 295 300 Ala Lys Arg Leu Asn Ser Ile Thr Thr Ala Asn Pro
Ser Ser Phe Asn 305 310 315 320 Lys Tyr Ile Gly Glu Tyr Lys Gln Lys
Leu Ile Arg Lys Tyr Arg Phe 325 330 335 Val Val Glu Ser Ser Gly Glu
Val Thr Val Asn Arg Asn Lys Phe Val 340 345 350 Glu Leu Tyr Asn Glu
Leu Thr Gln Ile Phe Thr Glu Phe Asn Tyr Ala 355 360 365 Lys Ile Tyr
Asn Val Gln Asn Arg Lys Ile Tyr Leu Ser Asn Val Tyr 370 375 380 Thr
Pro Val Thr Ala Asn Ile Leu Asp Asp Asn Val Tyr Asp Ile Gln 385 390
395 400 Asn Gly Phe Asn Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met
Gly 405 410 415 Gln Asn Leu Ser Arg Asn Pro Ala Leu Arg Lys Val Asn
Pro Glu Asn 420 425 430 Met Leu Tyr Leu Phe Thr Lys Phe Cys Val Asp
Ala Ile Asp Gly Arg 435 440 445 Gly Gly Arg Gly Asp Met Phe Gly Ala
Ala Leu Ala Gly Gly Gly Gly 450 455 460 Ser Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Ala Leu Val Leu Gln 465 470 475 480 Cys Arg Glu Leu
Leu Val Lys Asn Thr Asp Leu Pro Phe Ile Gly Asp 485 490 495 Ile Ser
Asp Val Lys Thr Asp Ile Phe Leu Arg Lys Asp Ile Asn Glu 500 505 510
Glu Thr Glu Val Ile Tyr Tyr Pro Asp Asn Val Ser Val Asp Gln Val 515
520 525 Ile Leu Ser Lys Asn Thr Ser Glu His Gly Gln Leu Asp Leu Leu
Tyr 530 535 540 Pro Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly Glu
Asn Gln Val 545 550 555 560 Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp
Tyr Leu Asn Ser Tyr Tyr 565 570 575 Tyr Leu Glu Ser Gln Lys Leu Ser
Asp Asn Val Glu Asp Phe Thr Phe 580 585 590 Thr Arg Ser Ile Glu Glu
Ala Leu Asp Asn Ser Ala Lys Val Tyr Thr 595 600 605 Tyr Phe Pro Thr
Leu Ala Asn Lys Val Asn Ala Gly Val Gln Gly Gly 610 615 620 Leu Phe
Leu Met Trp Ala Asn Asp Val Val Glu Asp Phe Thr Thr Asn 625 630 635
640 Ile Leu Arg Lys Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Ala Ile
645 650 655 Ile Pro Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn Ser Val
Arg Arg 660 665 670 Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val
Thr Ile Leu Leu 675 680 685 Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala
Leu Gly Ala Phe Val Ile 690 695 700 Tyr Ser Lys Val Gln Glu Arg Asn
Glu Ile Ile Lys Thr Ile Asp Asn 705 710 715 720 Cys Leu Glu Gln Arg
Ile Lys Arg Trp Lys Asp Ser Tyr Glu Trp Met 725 730 735 Met Gly Thr
Trp Leu Ser Arg Ile Ile Thr Gln Phe Asn Asn Ile Ser 740 745 750 Tyr
Gln Met Tyr Asp Ser Leu Asn Tyr Gln Ala Gly Ala Ile Lys Ala 755 760
765 Lys Ile Asp Leu Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu Asn
770 775 780 Ile Lys Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val
Lys Ile 785 790 795 800 Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile
Arg Glu Cys Ser Val 805 810 815 Thr Tyr Leu Phe Lys Asn Met Leu Pro
Lys Val Ile Asp Glu Leu Asn 820 825 830 Glu Phe Asp Arg Asn Thr Lys
Ala Lys Leu Ile Asn Leu Ile Asp Ser 835 840 845 His Asn Ile Ile Leu
Val Gly Glu Val Asp Lys Leu Lys Ala Lys Val 850 855 860 Asn Asn Ser
Phe Gln Asn Thr Ile Pro Phe Asn Ile Phe Ser Tyr Thr 865 870 875 880
Asn Asn Ser Leu Leu Lys Asp Ile Ile Asn Glu Tyr Phe Asn Leu Glu 885
890 895 Ala Leu Ala Ser Gly His His His His His His Lys Leu 900 905
17 2721 DNA Unknown DNA sequence of the fully synthesised
LC/C-RGD-HN/C fusion 17 catatgggct ccgaatttat gccgataaca attaacaatt
tcaattactc ggatccggtg 60 gacaacaaaa acattctgta tctggataca
catttaaata ctcttgcgaa tgaaccagaa 120 aaagcgttca gaattacggg
aaatatctgg gtcatcccgg atcgcttttc gagaaactca 180 aaccccaacc
tgaacaaacc gccccgtgtt acaagtccga aaagcggcta ttacgatcca 240
aactaccttt cgaccgactc ggacaaagat acgtttctta aagagataat taaactgttt
300 aaacgtatca attcacgcga aattggggaa gagttaattt accgcctctc
caccgacatt 360 ccgtttccag gcaataacaa tacaccgatt aacacctttg
atttcgacgt ggacttcaac 420 agcgtggatg ttaaaacgcg ccagggtaat
aactgggtaa agacgggatc gattaacccg 480 agtgttatta tcaccggtcc
tcgcgaaaat atcatagacc cggaaactag cacgtttaaa 540 cttactaata
acacattcgc ggcccaagaa gggttcggcg ccctgtcaat tataagcatc 600
agtccgcgct ttatgctgac ttacagtaat gctactaatg acgtgggtga gggccggttc
660 tctaaatcag aattttgcat ggatccaatc ctgattctga tgcatgagct
gaatcacgct 720 atgcacaatc tgtatggtat tgctattccg aacgatcaga
caattagttc agtgacgtct 780 aacatattct attctcaata taatgtgaaa
ttggagtatg cggaaattta tgcatttggt 840 ggcccaacca tcgatcttat
cccaaaatcc gcgcgcaagt atttcgaaga gaaagcatta 900 gattattacc
ggtctatcgc aaagcgtctg aatagcataa ctacggctaa tccgagttcg 960
tttaacaaat atattggcga atataaacag aaactgatcc gtaaatatcg tttcgtagtg
1020 gaatcatccg gtgaagttac agtcaatcgt aataaatttg tggagttata
caatgagctg 1080 acccaaatct tcaccgaatt caactatgct aaaatttata
atgttcagaa ccgcaaaatc 1140 tacctgagta acgtgtatac gcctgtaaca
gccaatattc tggatgacaa cgtgtatgat 1200 atccagaatg gctttaacat
acctaaaagt aacttgaatg ttctctttat gggtcaaaat 1260 ctttcccgca
atccggctct ccgaaaggta aatccggaaa acatgctcta tcttttcacc 1320
aaattttgcg tcgacgcaat cgatggacgt ggtgggagag gtgatatgtt tggggccgca
1380 ttagcgggtg gcgggggatc cggcggtggc ggtagtggcg ggggcggaag
cgcgctggta 1440 ctgcagtgtc gcgaactttt agttaagaat actgatctgc
cattcattgg tgatatctca 1500 gatgtcaaga ccgatatttt cctccgtaaa
gatatcaatg aggaaacaga ggtaatttac 1560 tatccggata atgtatctgt
cgatcaggtc attctgtcca aaaatacctc tgaacacggt 1620 caactggatc
tgctctaccc ctcgattgac tccgaatctg aaatcctccc tggagaaaac 1680
caggtctttt atgacaatcg tacccagaac gtggactact taaactctta ttactatttg
1740 gagagccaaa agttgtccga taacgttgaa gactttactt ttacccgatc
tatagaagag 1800 gcattagaca actcggcgaa ggtttacacc tatttcccta
ccttagccaa taaagtgaac 1860 gcaggtgtgc agggagggct gtttttgatg
tgggccaatg atgtcgttga ggatttcaca 1920 accaacattc tgcgcaaaga
cactttagat aaaatctcag atgtatcggc gatcattccc 1980 tacattggcc
ctgcccttaa catttctaat tccgttcgtc gcggcaattt tactgaggcg 2040
tttgctgtca ccggtgtgac gatcttgctg gaggcttttc ctgaatttac cattcccgca
2100 ctgggggcat tcgttatcta cagtaaggtt caggaacgga acgaaattat
aaaaacaatc 2160 gataattgcc tggaacagcg tatcaaacgg tggaaagata
gctacgaatg gatgatgggc 2220 acgtggttga gccgcataat tacgcagttt
aataacatct catatcaaat gtatgactcc 2280 ctgaattacc aggcgggcgc
gattaaagcc aaaatcgatc tggagtacaa aaagtattca 2340 ggcagcgaca
aagagaacat taaaagtcag gttgaaaacc tgaagaattc actggatgtg 2400
aaaatcagcg aagccatgaa taacattaat aaattcatcc gtgaatgtag tgtgacctat
2460 ctctttaaga atatgttgcc gaaagttatc gatgagctga acgagtttga
tcgaaatacc 2520 aaagcaaagc tgattaattt aattgacagc cataatatta
tactggtcgg cgaagtggat 2580 aaactgaagg ccaaggtaaa caattctttt
caaaacacga taccattcaa catcttttct 2640 tatacgaata acagccttct
gaaggacatt attaacgaat attttaattt ggaagccttg 2700 gctagcggat
aatgaaagct t 2721 18 902 PRT Unknown Protein sequence of the fully
synthesised LC/C-RGD-HN/C fusion 18 Met Gly Ser Glu Phe Met Pro Ile
Thr Ile Asn Asn Phe Asn Tyr Ser 1 5 10 15 Asp Pro Val Asp Asn Lys
Asn Ile Leu Tyr Leu Asp Thr His Leu Asn 20 25 30 Thr Leu Ala Asn
Glu Pro Glu Lys Ala Phe Arg Ile Thr Gly Asn Ile 35 40 45 Trp Val
Ile Pro Asp Arg Phe Ser Arg Asn Ser Asn Pro Asn Leu Asn 50 55 60
Lys Pro Pro Arg Val Thr Ser Pro Lys Ser Gly Tyr Tyr Asp Pro Asn 65
70 75 80 Tyr Leu Ser Thr Asp Ser Asp Lys Asp Thr Phe Leu Lys Glu
Ile Ile 85 90 95 Lys Leu Phe Lys Arg Ile Asn Ser Arg Glu Ile Gly
Glu Glu Leu Ile 100 105 110 Tyr Arg Leu Ser Thr Asp Ile Pro Phe Pro
Gly Asn Asn Asn Thr Pro 115 120 125 Ile Asn Thr Phe Asp Phe Asp Val
Asp Phe Asn Ser Val Asp Val Lys 130 135 140 Thr Arg Gln Gly Asn Asn
Trp Val Lys Thr Gly Ser Ile Asn Pro Ser 145 150 155 160 Val Ile Ile
Thr Gly Pro Arg Glu Asn Ile Ile Asp Pro Glu Thr Ser 165 170 175 Thr
Phe Lys Leu Thr Asn Asn Thr Phe Ala Ala Gln Glu Gly Phe Gly 180 185
190 Ala Leu Ser Ile Ile Ser Ile Ser Pro Arg Phe Met Leu Thr Tyr Ser
195 200 205 Asn Ala Thr Asn Asp Val Gly Glu Gly Arg Phe Ser Lys Ser
Glu Phe 210 215 220 Cys Met Asp Pro Ile Leu Ile Leu Met His Glu Leu
Asn His Ala Met 225 230 235 240 His Asn Leu Tyr Gly Ile Ala Ile Pro
Asn Asp Gln Thr Ile Ser Ser 245 250 255 Val Thr Ser Asn Ile Phe Tyr
Ser Gln Tyr Asn Val Lys Leu Glu Tyr 260 265 270 Ala Glu Ile Tyr Ala
Phe Gly Gly Pro Thr Ile Asp Leu Ile Pro Lys 275 280 285 Ser Ala Arg
Lys Tyr Phe Glu Glu Lys Ala Leu Asp Tyr Tyr Arg Ser 290 295 300 Ile
Ala Lys Arg Leu Asn Ser Ile Thr Thr Ala Asn Pro Ser Ser Phe 305 310
315 320 Asn Lys Tyr Ile Gly Glu Tyr Lys Gln Lys Leu Ile Arg Lys Tyr
Arg 325 330 335 Phe Val Val Glu Ser Ser Gly Glu Val Thr Val Asn Arg
Asn Lys Phe 340 345 350 Val Glu Leu Tyr Asn Glu Leu Thr Gln Ile Phe
Thr Glu Phe Asn Tyr 355 360 365 Ala Lys Ile Tyr Asn Val Gln Asn Arg
Lys Ile Tyr Leu Ser Asn Val 370 375 380 Tyr Thr Pro Val Thr Ala Asn
Ile Leu Asp Asp Asn Val Tyr Asp Ile 385 390 395 400 Gln Asn Gly Phe
Asn Ile Pro Lys Ser Asn Leu Asn Val Leu Phe Met 405 410 415 Gly Gln
Asn Leu Ser Arg Asn Pro Ala Leu Arg Lys Val Asn Pro Glu 420 425 430
Asn Met Leu Tyr Leu Phe Thr Lys Phe Cys Val Asp Ala Ile Asp Gly 435
440 445 Arg Gly Gly Arg Gly Asp Met Phe Gly Ala Ala Leu Ala Gly Gly
Gly 450 455 460 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ala
Leu Val Leu 465 470 475 480 Gln Cys Arg Glu Leu Leu Val Lys Asn Thr
Asp Leu Pro Phe Ile Gly 485 490 495 Asp Ile Ser Asp Val Lys Thr Asp
Ile Phe Leu Arg Lys Asp Ile Asn 500 505 510 Glu Glu Thr Glu Val Ile
Tyr Tyr Pro Asp Asn Val Ser Val Asp Gln 515 520 525 Val Ile Leu Ser
Lys Asn Thr Ser Glu His Gly Gln Leu Asp Leu Leu 530 535 540 Tyr Pro
Ser Ile Asp Ser Glu Ser Glu Ile Leu Pro Gly Glu Asn Gln 545 550 555
560 Val Phe Tyr Asp Asn Arg Thr Gln Asn Val Asp Tyr Leu Asn Ser Tyr
565 570 575 Tyr Tyr Leu Glu Ser Gln Lys Leu Ser Asp Asn Val Glu Asp
Phe Thr 580 585 590 Phe Thr Arg Ser Ile Glu Glu Ala Leu Asp Asn Ser
Ala Lys Val Tyr 595 600 605 Thr Tyr Phe Pro Thr Leu Ala Asn Lys Val
Asn Ala Gly Val Gln Gly 610 615 620 Gly Leu Phe Leu Met Trp Ala Asn
Asp Val Val Glu Asp Phe Thr Thr 625 630 635 640 Asn Ile Leu Arg Lys
Asp Thr Leu Asp Lys Ile Ser Asp Val Ser Ala 645 650 655 Ile Ile Pro
Tyr Ile Gly Pro Ala Leu Asn Ile Ser Asn Ser Val Arg 660 665 670 Arg
Gly Asn Phe Thr Glu Ala Phe Ala Val Thr Gly Val Thr Ile Leu 675 680
685 Leu Glu Ala Phe Pro Glu Phe Thr Ile Pro Ala Leu Gly Ala Phe Val
690 695 700 Ile Tyr Ser Lys Val Gln Glu Arg Asn Glu Ile Ile Lys Thr
Ile Asp 705 710 715 720 Asn Cys Leu Glu Gln Arg Ile Lys Arg Trp Lys
Asp Ser Tyr Glu Trp 725 730 735 Met Met Gly Thr Trp Leu Ser Arg Ile
Ile Thr Gln Phe Asn Asn Ile 740 745 750 Ser Tyr Gln Met Tyr Asp Ser
Leu Asn Tyr Gln Ala Gly Ala Ile Lys 755 760 765 Ala Lys Ile Asp Leu
Glu Tyr Lys Lys Tyr Ser Gly Ser Asp Lys Glu 770 775 780 Asn Ile Lys
Ser Gln Val Glu Asn Leu Lys Asn Ser Leu Asp Val Lys 785 790 795 800
Ile Ser Glu Ala Met Asn Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser 805
810 815 Val Thr Tyr Leu Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu
Leu 820 825 830 Asn Glu Phe Asp Arg Asn Thr Lys Ala Lys Leu Ile Asn
Leu Ile Asp 835 840 845 Ser His Asn Ile Ile Leu Val Gly Glu Val Asp
Lys Leu Lys Ala Lys 850 855 860 Val Asn Asn Ser Phe Gln Asn Thr Ile
Pro Phe Asn Ile Phe Ser Tyr 865 870 875 880 Thr Asn Asn Ser Leu Leu
Lys Asp Ile Ile Asn Glu Tyr Phe Asn Leu 885 890 895 Glu Ala Leu Ala
Ser Gly 900 19 2859 DNA Unknown DNA sequence of the fully
synthesised EGF-LHN/C fusion 19 catatgattt ccgaatttgg ctcggagttc
atgccaatta cgattaacaa ttttaactat 60 agtgatccgg tggataataa
aaacatttta tacctggata cccacttgaa tactcttgcc 120 aatgagcctg
aaaaagcctt tcgcataacg ggtaacattt gggtcattcc ggaccgtttt 180
agccggaact ctaaccctaa tctgaataaa cctccgcgtg tcacgtctcc gaaaagtggg
240 tattacgatc caaattatct gagtaccgat tcagacaagg atacgtttct
gaaagaaatc 300 ataaaacttt tcaaaagaat caactcccgt gaaatcggtg
aagagctgat ctaccgtctg 360 tcgacggaca ttccttttcc gggaaacaat
aacactccca ttaatacctt cgactttgat 420 gtcgatttca actcagtcga
tgtgaaaacc cgccagggta ataactgggt taaaactgga 480 tccattaacc
cgtccgttat tatcacaggt cctcgtgaaa atattataga tcctgagacc 540
tccacgttca agctgacgaa taacactttt gcggcacagg aagggtttgg tgccctttca
600 attatctcta tctctccgcg cttcatgtta acgtattcta acgcaaccaa
cgatgttggc 660 gagggccgct tcagcaaaag tgaattctgt atggatccca
ttctgatctt gatgcatgag 720 cttaaccacg ctatgcataa tctttatggt
attgcaatcc caaacgatca gacgatctcc 780 agcgttacat ctaacatatt
ctacagccaa tataatgtga agctcgaata tgcagagatt 840 tacgccttcg
gtgggccgac cattgacctc attccaaagt ctgcccgtaa gtactttgag 900
gaaaaagcgt tggattacta tcgtagcatc gcgaaacgcc tgaattcaat tacaactgca
960 aacccatcta gcttcaacaa atacatcgga gaatataaac aaaagctgat
acgcaaatat 1020 cgctttgtgg tcgaatcgtc cggggaagtg acagttaatc
gaaataaatt tgttgaactc 1080 tataatgaat taacgcagat cttcacagaa
tttaattatg ctaaaatcta taatgtacag 1140 aaccggaaaa tttatctcag
taatgtatac acaccggtga ctgctaacat tctggacgat 1200 aacgtctacg
atattcaaaa tggctttaat atcccgaaga gcaacttgaa tgtcctcttc 1260
atggggcaga acttgtcacg taacccagcg ctgcgaaaag ttaacccaga aaatatgttg
1320 tacctcttta caaaattctg tgtagacgcc attgacggac gctcactgta
caacaaaacc 1380 ctgcaatgcc gtgaacttct ggttaagaac accgacctgc
cgttcattgg ggacatcagt 1440 gatgtcaaaa cggatatttt tcttcggaag
gatattaatg aggaaaccga agtgatatac 1500 tatcctgaca atgtgtcggt
agatcaggta atcctgagta agaacaccag cgagcatggg 1560 cagctggatc
tgttgtatcc gagcattgac agcgagtcgg aaatactgcc cggcgaaaat 1620
caagtttttt atgacaatcg gacccagaat gttgattatc tgaatagtta ctattacttg
1680 gagagccaaa aattatcaga taatgtggaa gactttacct ttacccggtc
tatcgaagag 1740 gcgctggata acagcgcgaa agtttacact tattttccca
cgctcgcaaa caaagttaat 1800 gctggcgtac agggtggatt atttcttatg
tgggcgaatg atgtggtaga ggactttaca 1860 accaacatcc tgcgcaaaga
cactttagac aaaatttctg acgtctcggc cattatcccg 1920 tatataggtc
cggccttaaa cataagcaat tcggttcgcc gtggcaactt cacagaagcc 1980
ttcgctgtga ctggtgtgac cattctgttg gaagcatttc ctgagtttac gatcccggct
2040 ctgggcgcat ttgtaattta ctctaaagtt caggaacgaa atgaaattat
aaaaactatc 2100 gataattgcc tggaacagcg tatcaagaga tggaaggatt
cctatgagtg gatgatgggg 2160 acctggctgt caagaattat cacacagttt
aataacatat cctatcaaat gtatgatagc 2220 ttaaactatc aagcaggagc
gataaaggcg aaaattgacc tggaatacaa gaaatattct 2280 ggttcggata
aagagaatat taaaagtcag gtggaaaatc tgaaaaatag tttagatgtc 2340
aaaatttctg aggcgatgaa taacattaac aaattcatcc gcgagtgcag tgtaacttat
2400 ttgtttaaga atatgttacc caaagttatc gacgaactga atgaatttga
tcgtaatacc 2460 aaagccaaat tgatcaacct catcgactct cataacatca
ttctggtggg agaagtcgac 2520 aaactgaaag
ctaaggtgaa taacagcttc cagaatacaa ttccgtttaa tattttctca 2580
tacaccaata actcgctgct taaagatatt atcaacgaat attttaatct ggagggtggc
2640 ggtggcagtg gcggtggcgg atccggcggt ggcggtagcg cactggataa
ttcagattcc 2700 gaatgtccac tgtcacacga tggttattgt cttcatgatg
gcgtgtgcat gtatatagaa 2760 gcgttagata aatacgcttg caactgcgtg
gttggctata tcggcgaacg ttgtcagtat 2820 cgtgatttaa agtggtggga
attacgctaa tgaaagctt 2859 20 948 PRT Unknown Protein sequence of
the fully synthesised EGF-LHN/C fusion 20 Met Ile Ser Glu Phe Gly
Ser Glu Phe Met Pro Ile Thr Ile Asn Asn 1 5 10 15 Phe Asn Tyr Ser
Asp Pro Val Asp Asn Lys Asn Ile Leu Tyr Leu Asp 20 25 30 Thr His
Leu Asn Thr Leu Ala Asn Glu Pro Glu Lys Ala Phe Arg Ile 35 40 45
Thr Gly Asn Ile Trp Val Ile Pro Asp Arg Phe Ser Arg Asn Ser Asn 50
55 60 Pro Asn Leu Asn Lys Pro Pro Arg Val Thr Ser Pro Lys Ser Gly
Tyr 65 70 75 80 Tyr Asp Pro Asn Tyr Leu Ser Thr Asp Ser Asp Lys Asp
Thr Phe Leu 85 90 95 Lys Glu Ile Ile Lys Leu Phe Lys Arg Ile Asn
Ser Arg Glu Ile Gly 100 105 110 Glu Glu Leu Ile Tyr Arg Leu Ser Thr
Asp Ile Pro Phe Pro Gly Asn 115 120 125 Asn Asn Thr Pro Ile Asn Thr
Phe Asp Phe Asp Val Asp Phe Asn Ser 130 135 140 Val Asp Val Lys Thr
Arg Gln Gly Asn Asn Trp Val Lys Thr Gly Ser 145 150 155 160 Ile Asn
Pro Ser Val Ile Ile Thr Gly Pro Arg Glu Asn Ile Ile Asp 165 170 175
Pro Glu Thr Ser Thr Phe Lys Leu Thr Asn Asn Thr Phe Ala Ala Gln 180
185 190 Glu Gly Phe Gly Ala Leu Ser Ile Ile Ser Ile Ser Pro Arg Phe
Met 195 200 205 Leu Thr Tyr Ser Asn Ala Thr Asn Asp Val Gly Glu Gly
Arg Phe Ser 210 215 220 Lys Ser Glu Phe Cys Met Asp Pro Ile Leu Ile
Leu Met His Glu Leu 225 230 235 240 Asn His Ala Met His Asn Leu Tyr
Gly Ile Ala Ile Pro Asn Asp Gln 245 250 255 Thr Ile Ser Ser Val Thr
Ser Asn Ile Phe Tyr Ser Gln Tyr Asn Val 260 265 270 Lys Leu Glu Tyr
Ala Glu Ile Tyr Ala Phe Gly Gly Pro Thr Ile Asp 275 280 285 Leu Ile
Pro Lys Ser Ala Arg Lys Tyr Phe Glu Glu Lys Ala Leu Asp 290 295 300
Tyr Tyr Arg Ser Ile Ala Lys Arg Leu Asn Ser Ile Thr Thr Ala Asn 305
310 315 320 Pro Ser Ser Phe Asn Lys Tyr Ile Gly Glu Tyr Lys Gln Lys
Leu Ile 325 330 335 Arg Lys Tyr Arg Phe Val Val Glu Ser Ser Gly Glu
Val Thr Val Asn 340 345 350 Arg Asn Lys Phe Val Glu Leu Tyr Asn Glu
Leu Thr Gln Ile Phe Thr 355 360 365 Glu Phe Asn Tyr Ala Lys Ile Tyr
Asn Val Gln Asn Arg Lys Ile Tyr 370 375 380 Leu Ser Asn Val Tyr Thr
Pro Val Thr Ala Asn Ile Leu Asp Asp Asn 385 390 395 400 Val Tyr Asp
Ile Gln Asn Gly Phe Asn Ile Pro Lys Ser Asn Leu Asn 405 410 415 Val
Leu Phe Met Gly Gln Asn Leu Ser Arg Asn Pro Ala Leu Arg Lys 420 425
430 Val Asn Pro Glu Asn Met Leu Tyr Leu Phe Thr Lys Phe Cys Val Asp
435 440 445 Ala Ile Asp Gly Arg Ser Leu Tyr Asn Lys Thr Leu Gln Cys
Arg Glu 450 455 460 Leu Leu Val Lys Asn Thr Asp Leu Pro Phe Ile Gly
Asp Ile Ser Asp 465 470 475 480 Val Lys Thr Asp Ile Phe Leu Arg Lys
Asp Ile Asn Glu Glu Thr Glu 485 490 495 Val Ile Tyr Tyr Pro Asp Asn
Val Ser Val Asp Gln Val Ile Leu Ser 500 505 510 Lys Asn Thr Ser Glu
His Gly Gln Leu Asp Leu Leu Tyr Pro Ser Ile 515 520 525 Asp Ser Glu
Ser Glu Ile Leu Pro Gly Glu Asn Gln Val Phe Tyr Asp 530 535 540 Asn
Arg Thr Gln Asn Val Asp Tyr Leu Asn Ser Tyr Tyr Tyr Leu Glu 545 550
555 560 Ser Gln Lys Leu Ser Asp Asn Val Glu Asp Phe Thr Phe Thr Arg
Ser 565 570 575 Ile Glu Glu Ala Leu Asp Asn Ser Ala Lys Val Tyr Thr
Tyr Phe Pro 580 585 590 Thr Leu Ala Asn Lys Val Asn Ala Gly Val Gln
Gly Gly Leu Phe Leu 595 600 605 Met Trp Ala Asn Asp Val Val Glu Asp
Phe Thr Thr Asn Ile Leu Arg 610 615 620 Lys Asp Thr Leu Asp Lys Ile
Ser Asp Val Ser Ala Ile Ile Pro Tyr 625 630 635 640 Ile Gly Pro Ala
Leu Asn Ile Ser Asn Ser Val Arg Arg Gly Asn Phe 645 650 655 Thr Glu
Ala Phe Ala Val Thr Gly Val Thr Ile Leu Leu Glu Ala Phe 660 665 670
Pro Glu Phe Thr Ile Pro Ala Leu Gly Ala Phe Val Ile Tyr Ser Lys 675
680 685 Val Gln Glu Arg Asn Glu Ile Ile Lys Thr Ile Asp Asn Cys Leu
Glu 690 695 700 Gln Arg Ile Lys Arg Trp Lys Asp Ser Tyr Glu Trp Met
Met Gly Thr 705 710 715 720 Trp Leu Ser Arg Ile Ile Thr Gln Phe Asn
Asn Ile Ser Tyr Gln Met 725 730 735 Tyr Asp Ser Leu Asn Tyr Gln Ala
Gly Ala Ile Lys Ala Lys Ile Asp 740 745 750 Leu Glu Tyr Lys Lys Tyr
Ser Gly Ser Asp Lys Glu Asn Ile Lys Ser 755 760 765 Gln Val Glu Asn
Leu Lys Asn Ser Leu Asp Val Lys Ile Ser Glu Ala 770 775 780 Met Asn
Asn Ile Asn Lys Phe Ile Arg Glu Cys Ser Val Thr Tyr Leu 785 790 795
800 Phe Lys Asn Met Leu Pro Lys Val Ile Asp Glu Leu Asn Glu Phe Asp
805 810 815 Arg Asn Thr Lys Ala Lys Leu Ile Asn Leu Ile Asp Ser His
Asn Ile 820 825 830 Ile Leu Val Gly Glu Val Asp Lys Leu Lys Ala Lys
Val Asn Asn Ser 835 840 845 Phe Gln Asn Thr Ile Pro Phe Asn Ile Phe
Ser Tyr Thr Asn Asn Ser 850 855 860 Leu Leu Lys Asp Ile Ile Asn Glu
Tyr Phe Asn Leu Glu Gly Gly Gly 865 870 875 880 Gly Ser Gly Gly Gly
Gly Ser Gly Gly Gly Gly Ser Ala Leu Asp Asn 885 890 895 Ser Asp Ser
Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp 900 905 910 Gly
Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys 915 920
925 Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp
930 935 940 Trp Glu Leu Arg 945 21 9 PRT Unknown Integrin binding
peptide sequence 21 Gly Gly Arg Gly Asp Met Phe Gly Ala 1 5 22 11
PRT Unknown Integrin binding peptide sequence 22 Gly Gly Cys Arg
Gly Asp Met Phe Gly Cys Ala 1 5 10 23 5 PRT Unknown cyclic RGD
peptide MISC_FEATURE (4)..(4) D-Phe MISC_FEATURE (5)..(5)
N-methyl-Val 23 Arg Gly Asp Phe Val 1 5 24 10 PRT Unknown linear
integrin binding sequence 24 Pro Leu Ala Glu Ile Asp Gly Ile Glu
Leu 1 5 10 25 12 PRT Unknown cyclic integrin binding sequence 25
Cys Pro Leu Ala Glu Ile Asp Gly Ile Glu Leu Cys 1 5 10
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