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Patent 2376641 Summary

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(12) Patent Application: (11) CA 2376641
(54) English Title: CHIMERIC POLYPEPTIDES OF SERUM ALBUMIN AND USES RELATED THERETO
(54) French Title: POLYPEPTIDES CHIMERES D'ALBUMINE SERIQUE ET UTILISATIONS ASSOCIEES
Status: Dead
Bibliographic Data
(51) International Patent Classification (IPC):
  • C12N 15/62 (2006.01)
  • A61K 38/17 (2006.01)
  • C07K 14/705 (2006.01)
  • C07K 14/71 (2006.01)
  • C07K 14/715 (2006.01)
  • C07K 14/72 (2006.01)
  • C07K 14/76 (2006.01)
  • C07K 14/765 (2006.01)
  • C07K 14/78 (2006.01)
  • C12N 9/12 (2006.01)
  • C12N 9/68 (2006.01)
  • A61K 38/00 (2006.01)
(72) Inventors :
  • MORRIS, AARON (United States of America)
  • GYURIS, JENO (United States of America)
  • LAMPHERE, LOU (United States of America)
(73) Owners :
  • GPC BIOTECH, INC. (United States of America)
(71) Applicants :
  • GPC BIOTECH, INC. (United States of America)
(74) Agent: BORDEN LADNER GERVAIS LLP
(74) Associate agent:
(45) Issued:
(86) PCT Filing Date: 2000-07-19
(87) Open to Public Inspection: 2001-01-25
Availability of licence: N/A
(25) Language of filing: English

Patent Cooperation Treaty (PCT): Yes
(86) PCT Filing Number: PCT/US2000/019689
(87) International Publication Number: WO2001/005826
(85) National Entry: 2002-01-18

(30) Application Priority Data:
Application No. Country/Territory Date
60/144,534 United States of America 1999-07-19

Abstracts

English Abstract




The present invention relates to chimeric polypeptides in which a serum
albumin protein has been altered to include one or more biologically active
heterologous peptide sequences. The chimeric polypeptides may exhibit
therapeutic activity related to the heterologous peptide sequences coupled
with the improved serum half-lives derived from the serum albumin protein
fragments. Heterologous peptide sequences may be chosen to promote any
biological effect, including angiogenesis inhibition, antitumor activity, and
induction of apoptosis. The therapeutic effect may be achieved by direct
administration of the chimeric polypeptide, or by transfecting cells with a
vector including a nucleic acid encoding such a chimeric polypeptide.


French Abstract

La présente invention concerne des polypeptides chimères constitués d'une protéine d'albumine sérique altérée par l'inclusion d'une ou de plusieurs séquences de peptides hétérologues biologiquement actifs. Les polypeptides chimères peuvent montrer une activité thérapeutique se rapportant aux séquences peptidiques hétérologues couplées avec les demi-vies sériques améliorées dérivées des fragments de protéine d'albumine sérique. On peut choisir des séquences de peptide hétérologue afin de promouvoir un quelconque effet biologique, y compris une inhibition de l'angiogénèse, une activité antitumorale et une induction d'apoptose. Il est possible d'obtenir l'effet thérapeutique par administration directe du polypeptide chimère, ou par transfection de cellules au moyen d'un vecteur contenant un acide nucléique codant pour un tel polypeptide chimère.

Claims

Note: Claims are shown in the official language in which they were submitted.


We claim:
1. A chimeric polypeptide comprising serum albumin protein (SA) having a
biologically active
heterologous peptide sequence inserted therein.
2. A chimeric polypeptide having the structure A-B-C, wherein:
A represents a first fragment of serum albumin (SA);
B represents a biologically active heterologous peptide sequence; and
C represents a second peptide fragment of SA.
3. A chimeric polypeptide comprising:
a first peptide fragment, comprising an N-terminal fragment of serum albumin
(SA)
protein;
a second peptide fragment, comprising a biologically active heterologous
peptide
sequence, and
a third peptide fragment, comprising a C-terminal fragment of SA.
4. The chimeric polypeptide of claim 1, 2, or 3, wherein the heterologous
peptide sequence
comprises a fragment of an angiogenesis-inhibiting protein or polypeptide.
5. The chimeric polypeptide of claim 4, wherein said angiogenesis-inhibiting
protein or
polypeptide is selected from the group consisting of angiostatin, endostatin,
and peptide
fragments thereof.
6. The chimeric polypeptide of claim 1, 2, or 3, wherein the heterologous
peptide sequence binds
to a cell surface receptor protein.
7. The chimeric polypeptide of claim 6, wherein the receptor protein is a G-
protein coupled
receptor.
8. The chimeric polypeptide of claim 6, wherein the receptor protein is a
tyrosine kinase receptor.
9. The chimeric polypeptide of claim 6, wherein the receptor protein is a
cytokine receptor.
-36-


10. The chimeric polypeptide of claim 6, wherein the receptor protein is an
MIRR receptor.
11. The chimeric polypeptide of claim 6, wherein the receptor protein is an
orphan receptor.
12. The chimeric polypeptide of claim 1, 2, or 3, wherein the chimeric
polypeptide binds to an
extracellular receptor or an ion channel.
13. The chimeric polypeptide of claim 12, wherein the chimeric polypeptide is
an agonist of said
receptor or ion channel.
14. The chimeric polypeptide of claim 12, wherein the chimeric polypeptide is
an antagonist of
said receptor or ion channel.
15. The chimeric polypeptide of claim 1, 2, or 3, wherein the chimeric
polypeptide induces
apoptosis.
16. The chimeric polypeptide of claim 1, 2, or 3, wherein the chimeric
polypeptide modulates
cell proliferation.
17. The chimeric polypeptide of claim 1, 2, or 3, wherein the chimeric
polypeptide modulates
differentiation of cell types.
18. The chimeric polypeptide of claim 1, 2, or 3, wherein the heterologous
peptide sequence
comprises between 4 and 400 residues.
19. The chimeric polypeptide of claim 1, 2, or 3, wherein the heterologous
peptide sequence
comprises between 4 and 200 residues.
20. The chimeric polypeptide of claim 1, 2, or 3, wherein the heterologous
peptide sequence
comprises between 4 and 100 residues.
-37-


21. The chimeric polypeptide of claim 1, 2, or 3, wherein the heterologous
peptide sequence
comprises between 4 and 20 residues.
22. The chimeric polypeptide of claim 1, 2, or 3, wherein the tertiary
structure of the chimeric
polypeptide is similar to the tertiary structure of native SA.
23. The chimeric polypeptide of claim 1, wherein the inserted peptide sequence
replaces a
portion of native SA sequence.
24. The chimeric polypeptide of claim 23, wherein the inserted peptide
sequence and the replaced
portion of native SA sequence are of unequal length.
25. The chimeric polypeptide of claim 1, 2, or 3, wherein the half-life of the
polypeptide in the
blood is no less than 14 days.
26. The chimeric polypeptide of claim 1, 2, or 3, wherein the half-life of the
polypeptide in the
blood is no less than 10 days.
27. The chimeric polypeptide of claim 1, 2, or 3, wherein in the half-life of
the polypeptide in
the blood is no less than 50% of the half-life of native SA.
28. A nucleic acid encoding the chimeric polypeptide of claim 1, 2, or 3.
29. A delivery vector comprising the nucleic acid of claim 28.
30. The delivery vector of claim 29, wherein said delivery vector comprises a
virus or retrovirus.
31. The delivery vector of claim 30, wherein said virus or retrovirus is
selected from the group
consisting of adenoviruses, adeno-associated viruses, herpes simplex viruses,
human
immunodeficiency viruses, or vaccinia viruses.
32. Transfected cells comprising target cells which have been exposed to the
delivery vector of
claim 29.
-38-


33. The transfected cells of claim 32, wherein the cells are selected from the
group consisting of
blood cells, skeletal muscle cells, stem cells, skin cells, liver cells,
secretory gland cells,
hematopoietic cells, and marrow cells.
34. A pharmaceutical preparation comprising a pharmaceutically acceptable
excipient and the
chimeric polypeptide of claim 1, 2, or 3
35. A method for treating disease in an organism, comprising administering as
a pharmaceutical
preparation to the organism the chimeric polypeptide of claim 1, 2, or 3.
36. A method for treating disease in an organism, said method comprising:
providing a delivery vector comprising genetic material which encodes the
chimeric
polypeptide of claim 1, 2, or 3; and
introducing said vector into target cells in vivo, under conditions sufficient
to induce said
target cells to express said polypeptide.
37. A method for treating a disease in an organism comprising:
providing a delivery vector comprising genetic material which encodes the
chimeric
polypeptide of claim 1, 2, or 3;
introducing said vector into target cells ex vivo; and
introducing said target cells containing the introduced vector into the
organism under
conditions sufficient to induce said target cells to express said polypeptide.
38. The method of claim 36 or 37, wherein the target cells are selected from
the group consisting
of blood cells, skeletal muscle cells, stem cells, skin cells, liver cells,
secretory gland cells,
hematopoietic cells, and marrow cells.
39. A chimeric polypeptide having the structure (A-B-C)n, wherein:
A, independently for each occurrence, represents a fragment of serum albumin
(SA);
B, independently for each occurrence, represents a biologically active
heterologous
peptide sequence;
-39-


C, independently for each occurrence, represents a second biologically active
heterologous peptide sequence or a fragment of serum albumin (SA); and
n is an integer greater than 0.
40. The polypeptide of claim 39, wherein B and C comprise identical sequences.
41. The polypeptide of claim 39, wherein B and C comprise fragments of a
single protein.
42. The polypeptide of claim 39, wherein B and C comprise fragments two
different proteins.
43. A chimeric polypeptide comprising serum albumin protein (SA) having at
least two
biologically active heterologous peptide sequences inserted therein.
44. The polypeptide of claim 43, wherein the heterologous peptide sequences
are identical.
45. The polypeptide of claim 43, wherein the heterologous peptide sequences
comprise distinct
sequences of a protein.
46. The polypeptide of claim 44, wherein the heterologous peptide sequences
comprise
sequences from at least two different proteins.
47. A method for modulating one or more of cell proliferation, cell
differentiation, and cell
death in an organism, comprising administering as a pharmaceutical preparation
to the organism
the chimeric polypeptide of claim 1, 2, or 3.
47. A method for modulating one or more of cell proliferation, cell
differentiation, and cell
death in an organism, comprising:
providing a delivery vector comprising genetic material which encodes the
chimeric
polypeptide of claim 1, 2, or 3; and
-40-


introducing said vector into target cells in vivo, under conditions sufficient
to induce said
target cells to express said polypeptide.
-41-

Description

Note: Descriptions are shown in the official language in which they were submitted.



CA 02376641 2002-O1-18
WO 01/05826 PCT/US00/19689
CHIMERIC POLYPEPTIDES OF SERUM ALBUMIN AND USES RELATED THERETO
This application is based on U.S. Provisional Application No. 60/144,534,
filed July
19, 1999, the specification of which is hereby incorporated by reference in
its entirety.
Background of the Invention
Recent advances in recombinant DNA technology have made available a wide range
of biologically active peptides. Although in some instances molecular
remodeling, for
instance by ligated gene fusion or by site directed mutagenesis, has endowed
such proteins
with properties compatible with optimal activity, it is generally the case
that effective use of
these products can only be achieved through delivery systems.
Polypeptide therapeutic agents, despite their promise in a number of disease
treatments, are readily decomposed by gastric juices and by intestinal
proteinases such as
pepsin and trypsin. As a result, when these polypeptides are orally
administered, they are
barely absorbed and produce no effective pharmacological action. In order to
obtain the
desired biological activity, the polypeptides are at present usually dispensed
in injectable
dosage forms. However, the injectable route is inconvenient and painful to the
patient,
particularly when administration must occur on a regular and frequent basis.
Consequently,
efforts have focused recently on alternative methods for administration of
such polypeptides.
Such agents usually exhibit a short half life in the circulation, being
rapidly excreted
through the kidneys or taken up by the reticuloendothelial system (RES) and
other tissues.
To compensate for such premature drug loss, larger doses are required so that
sufficient
amounts of drug can concentrate in areas in need of treatment. However, this
is not only
costly; it can also lead to toxicity and an immune response to the foreign
protein. Sustained-
release formulations (Putney, S.D. et al. Nature Biotechnology 1998, 16, 153-
157) generally
reduce the necessary dosage, but still depend on injection or more
objectionable forms of
delivery. A therapeutic protein with a longer half life in the body would
maintain a more
stable blood level in much the same way as a sustained-release formulation,
but would not
entail the difficulties of preparing a sustained-release formulation and would
require an even
lower dosage because it is destroyed less quickly. For instance, cytokines
such as interferon
(IFN-gamma) and interleukin-2 (IL-2) would be more effective, less toxic and
could be used
in smaller quantities, if their presence in the circulation could be extended.
-1-


CA 02376641 2002-O1-18
WO 01/05826 PCT/US00/19689
One aspect of the present invention provides a chimeric polypeptide comprising
a
biologically active heterologous peptide fragment inserted into a serum
albumin protein or a
homolog thereof. The heterologous peptide fragment may optionally replace a
portion of the
serum albumin protein sequence. A peptide fragment which replaces a portion of
the serum
albumin protein sequence need not be of the same length as the fragment it
replaces. A
chimeric polypeptide according to this aspect may include more than one
heterologous
peptide fragment which replaces a portion of the serum albumin protein
sequence. The
included fragments may be identical, may be distinct sequences from a protein
unrelated to
serum albumin protein, or may be distinct sequences of unrelated origin.
A chimeric polypeptide of this aspect, for example, may comprise the structure
A-B-
C, wherein A represents a first fragment of a serum albumin protein or homolog
thereof, B
represents a biologically active heterologous peptide sequence, and C
represents another
fragment of a serum albumin protein or a homolog thereof. Similarly, a
chimeric polypeptide
may comprise the structure A-B-C-D-E, wherein A, C, and E represent fragments
of a serum
albumin protein and B and D represent identical biologically active
heterologous peptide
sequences, two different biologically active sequences of a protein unrelated
to serum
albumin protein, or two different biologically active sequences of two
different proteins
unrelated to serum albumin protein. Analogously, a chimeric polypeptide may
comprise the
structure A-B-C-D-E-F-G, wherein A, C, E, and G represent fragments of a serum
albumin
protein and B, D, and F represent identical biologically active heterologous
peptide
sequences, at least two different biologically active sequences of a protein
unrelated to serum
albumin protein, or at least two different biologically active sequences of
two different
proteins unrelated to serum albumin protein. In certain embodiments, a peptide
fragment of
serum albumin or a heterologous peptide sequence includes at least 6 amino
acids, at least 12
amino acids, or at least 18 amino acids.
A chimeric polypeptide may comprise the structure (A-B-C)n, e.g., -HN-(A-B-C)~-

CO- or HzN-(A-B-C)n-COzH, wherein A, independently for each occurrence,
represents a
fragment of serum albumin (SA), B, independently for each occurrence,
represents a
biologically active heterologous peptide sequence, C, independently for each
occurrence,
represents a second biologically active heterologous peptide sequence or a
fragment of serum
albumin (SA), and n is an integer greater than 0. In certain embodiments, a
peptide fragment
-2-


CA 02376641 2002-O1-18
WO 01/05826 PCT/US00/19689
of serum albumin or a heterologous peptide sequence includes at least 6 amino
acids, at least
12 amino acids, or at least 18 amino acids.
Alternatively, such a chimeric polypeptide may comprise an N-terminal fragment
of a
serum albumin protein or a homolog thereof, a biologically active heterologous
peptide
sequence, and a C-terminal fragment of a serum albumin protein or a homolog
thereof. The
heterologous peptide sequence may be between about 3 and about 500 or between
about 4
and about 400 residues in length, preferably between about 4 and about 200
residues, more
preferably between about 4 and 100 residues, and most preferably between about
4 and about
20 residues.
In one embodiment, the chimeric polypeptide has a half life in the blood no
less than
days, preferably no less than about 14 days, and most preferably no less than
50% of the
half life of the native serum albumin protein or homolog thereof.
In another embodiment, the heterologous peptide sequence is capable of binding
to a
cell surface receptor protein. Examples of such a receptor protein include a G
protein-coupled
receptor, a tyrosine kinase receptor, a cytokine receptor, an MIRR receptor,
and an orphan
receptor.
In another embodiment, the chimeric polypeptide is capable of binding to an
extracellular receptor or ion channel. The chimeric polypeptide may be an
agonist or an
antagonist of an extracellular receptor or ion channel. The chimeric
polypeptide of this
embodiment may, for example, induce apoptosis, modulate cell proliferation, or
modulate
differentiation of cell types.
The invention also comprises a nucleic acid sequence which encodes a chimeric
polypeptide as described above.
The invention further comprises a delivery vector, such as a viral or
retroviral vector
comprising a nucleic acid sequence encoding the chimeric polypeptide. Suitable
vectors may
include, for example, an adenovirus, an adeno-associated virus, a herpes
simplex virus, a
human immunodeficiency viruses, or a vaccinia virus.
The invention also comprises a pharmaceutical composition comprising a
chimeric
polypeptide as described above, and methods for treating a disease in an
organism by
administering an effective dose of such a pharmaceutical composition to the
organism. In a
currently preferred embodiment, a chimeric polypeptide according to the
invention comprises
a fragment of an angiogenesis-inhibiting protein, such as angiostatin or
endostatin, as the
heterologous peptide sequence and is capable of inhibiting angiogenesis. For
example, a
-3-


CA 02376641 2002-O1-18
WO 01/05826 PCT/US00/19689
peptide fragment that inhibits angiogenesis and which may be incorporated into
a subject
polypeptide is RGD (Arg-Gly-Asp), or a sequence which includes the sequence
RGD (e.g.,
VRGDF). Analogous methods may be used to modulate conditions such as cell
proliferation,
cell differentiation, and cell death.
In a currently preferred embodiment, the present invention provides a method
of
treating a disease in an organism by introducing into cells of the organism
genetic material
encoding a chimeric polypeptide protein comprising serum albumin protein or
segments
thereof and one or more therapeutic proteins or polypeptides or fragments
thereof, such that
the introduced genetic material is expressed by the transfected cells of the
organism.
Analogous methods may be used to modulate conditions such as cell
proliferation, cell
differentiation, and cell death.
In another aspect, the present invention provides a method for treating a
disease in an
organism by introducing genetic material encoding a chimeric polypeptide
comprising serum
albumin protein or segments thereof and one or more therapeutic proteins or
polypeptides or
fragments thereof into target cells ex vivo under conditions sufficient to
cause the genetic
material to be incorporated into the cell, thereby causing the cell to express
the genetic
material encoding said proteins or polypeptides. The target cells are then
introduced into the
host organism such that the introduced genetic material encoding said proteins
or
polypeptides is expressed by the target cells in the organism. The target
cells may be selected
from the group consisting of blood cells, skeletal muscle cells, smooth muscle
cells, stem
cells, skin cells, liver cells, secretory gland cells, hematopoietic cells,
and marrow cells.
Another aspect of the present invention provides transfected cells comprising
target
cells which have been exposed to a delivery vector comprising a nucleic acid
encoding the
chimeric protein or polypeptide of this invention. These cells are preferably
selected from the
group consisting of blood cells, skeletal muscle cells, smooth muscle cells,
stem cells, skin
cells, liver cells, secretory gland cells, hematopoietic cells, and marrow
cells.
Figure 1 shows the tertiary structure of human serum albumin (HSA).
Figure 2 illustrates the transfection of cells with mouse serum albumin (MSA)-
Myc
fusion constructs and successful expression of the fusion protein, as well as
binding of MSA
and Myc antibodies to MSA-Myc fusion proteins depending on the location of the
heterologous sequence in the MSA protein.
-4-


CA 02376641 2002-O1-18
WO 01/05826 PCT/US00/19689
Figure 3 depicts inhibition of FGF-induced proliferation of bovine capillary
endothelial cells by RGD peptide and by MSA-myc-RGD fusion proteins.
Detailed Description of the Invention
The systems and methods disclosed herein are directed towards increasing the
lifetime
of therapeutic polypeptides in the bloodstream by creating chimeric
polypeptides containing
segments of serum albumin (SA) and segments of biologically active
heterologous peptide
sequences. SA is the major protein constituent of the circulatory system, has
a half life in the
blood of about three weeks (Rothschild, M.A. et al. Hepatology 1988, 8, 385-
401), and is
present in quantity (40 g/L in the serum). It is also known that the normal
adult human liver
produces approximately 15 grams of human serum albumin (HSA) per day, or about
200 mg
per kilogram of body weight. Serum albumin has no immunological activity or
enzymatic
function, and is a natural carrier protein used to transport many natural and
therapeutic
molecules. Fusion proteins wherein a therapeutic polypeptide has been
covalently linked to
serum albumin have been shown to have serum half lives many times longer than
the half life
of the therapeutic peptide itself (Syed, S. et al. Blood 1997, 89, 3243-3252;
Yeh, P. et al.
Proc. Natl. Acad. Sci. USA 1992, 89, 1904-1908). In both cited publications,
the half life of
the fusion protein was more than 140 times greater than that of the
therapeutic polypeptide
itself, and approached the half life of unfused serum albumin. Furthermore,
the amino-
terminal portion of serum albumin has been found to favor particularly
efficient translocation
and export of the fusion proteins in eukaryotic cells (PCT publication WO
90/13653).
Generally, this means that such proteins are more efficiently secreted by a
cell manufacturing
such proteins than are the free therapeutic polypeptides themselves.
From a drug delivery standpoint, chimeric polypeptides of serum albumin
proteins
offer substantial promise because serum albumins are found in tissues and
secretions
throughout the body. It is known, for example, that serum albumin is
responsible for the
transport of compounds across organ-circulatory interfaces into such organs as
the liver,
intestine, kidney, and brain. Chimeric proteins of serum albumin may thus
manifest their
biological activity anywhere in the body, crossing even the daunting blood-
brain barrier.
The three-dimensional structure and the chemistry of SA have been well studied
(Carter, D.C. et al. Eur. J. Biochem. 1994, 226, 1049-1052; He, X.M. et al.
Nature 1992, 358,
209-215; Carter, D.C. et al. Science 1989, 244, 1195-1198). Thus, rather than
relying on
-5-


CA 02376641 2002-O1-18
WO 01/05826 PCT/US00/19689
simple, binary fusion proteins as discussed above, portions of the SA protein
may be
strategically or combinatorially replaced by therapeutic polypeptides.
Techniques of combinatorial mutagenesis combined with structurally motivated
grafting procedures allow the random preparation of a library of many related
polypeptides
which carry a biologically active peptide fragment and are substantially
similar to serum
albumin in tertiary structure. For example, a chimeric polypeptide of the
present invention
may include a biologically active heterologous peptide sequence inserted into
the peptide
sequence of a serum albumin protein. The inserted sequence may optionally
replace a portion
of the serum albumin sequence, whether that portion is of similar or
dissimilar length. In
some cases, more than one insertion may be required to obtain the desired
biological activity.
Alternatively, a biologically active heterologous peptide sequence may be
placed between
two fragments of a serum albumin sequence to create such a chimeric
polypeptide.
Optionally, one or more additional biologically active peptide sequences may
be placed
between fragments of serum albumin protein. Chimeric polypeptides of the
present invention
may also be described as a biologically active heterologous peptide sequence
flanked on one
side by an N-terminal fragment of serum albumin protein and on the other side
by a C-
terminal fragment of serum albumin protein.
The advantage of such chimeric polypeptides is that the similarity to serum
albumin
protein in structure may camouflage these polypeptides to biological
mechanisms which
degrade foreign peptides even more effectively than known fusion proteins,
because the
foreign polypeptide fragments are carried on a protein that is substantially
similar to a protein
that is pervasive within the organism. Such proteins may retain the beneficial
characteristics
of serum albumin (non-immunogenicity, high level of expression, efficient
secretion, and
long half life), while supporting the additional desired biological function.
Many therapeutic applications of such chimeric polypeptides will be obvious to
those
skilled in the art. For example, inclusion of a peptide fragment which
inhibits cell
proliferation might serve as a treatment for cancer and other diseases
characterized by cell
proliferation known to those in the art. Inclusion of a peptide fragment which
modulates the
differentiation of immature cells into particular cell types may create a
chimeric polypeptide
which may be effective in the treatment of neurological conditions, e.g.,
nerve damage and
neurodegenerative diseases, hyperplastic and neoplastic disorders of
pancreatic tissue, and
other conditions characterized by undesirable proliferation and
differentiation of tissue.
Inclusion of a peptide fragment which induces apoptosis may provide a
polypeptide effective
-6-


CA 02376641 2002-O1-18
WO 01/05826 PCT/US00/19689
in treating diseases marked by unwanted cell proliferation, such as cancer,
and other
conditions known to those in the art as amenable to apoptotic therapy.
Inclusion of an anti-
angiogenic peptide fragment, e.g., a fragment of angiostatin or endostatin,
may yield a
chimeric polypeptide useful in the treatment of cancer and other conditions
resulting from or
enabled by angiogenesis.
The term 'peptide' refers to an oligomer in which the monomers are amino acids
(usually
alpha-amino acids) joined together through amide bonds. Peptides are two or
more amino acid
monomers long, but more often are between 5 to 10 amino acid monomers long and
can be even
longer, i.e., up to 20 amino acids or more, although peptides longer than 20
amino acids are more
likely to be called 'polypeptides'. The term 'protein' is well known in the
art and usually refers
to a very large polypeptide, or set of associated homologous or heterologous
polypeptides, that
has some biological function. For purposes of the present invention the terms
'peptide',
'polypeptide', and 'protein' are largely interchangeable as all three types
are collectively referred
to as peptides.
The interchangeable terms 'fusion' and 'chimeric', as used herein to describe
proteins and
polypeptides, relate to polypeptides or proteins wherein two individual
polypeptides or portions
thereof are fused to form a single amino acid chain. Such fusion may arise
from the expression
of a single continuous coding sequence formed by recombinant DNA techniques.
Thus, 'fusion'
polypeptides and 'chimeric' polypeptides include contiguous polypeptides
comprising a first
polypeptide covalently linked via an amide bond to one or more amino acid
sequences which
define polypeptide domains that are foreign to and not substantially
homologous with any
domain of the first polypeptide.
Gene constructs encoding fusion proteins are likewise referred to a 'chimeric
genes' or
'fusion genes'.
'Homology' and 'identity' each refer to sequence similarity between two
polypeptide
sequences, with identity being a more strict comparison. Homology and identity
can each be
determined by comparing a position in each sequence which may be aligned for
purposes of
comparison. When a position in the compared sequence is occupied by the same
amino acid
residue, then the polypeptides can be referred to as identical at that
position; when the equivalent
site is occupied by the same amino acid (e.g., identical) or a similar amino
acid (e.g., similar in
steric and/or electronic nature), then the molecules can be referred to as
homologous at that


CA 02376641 2002-O1-18
WO 01/05826 PCT/US00/19689
position. A percentage of homology or identity between sequences is a function
of the number
of matching or homologous positions shared by the sequences. An 'unrelated',
'heterologous',
or 'non-homologous' sequence shares less than 40 percent identity, though
preferably less than
25 percent identity, with a sequence to which it is compared. Thus, a
'heterologous peptide
sequence' is a peptide sequence substantially dissimilar to a sequence to
which it is compared.
The term 'serum albumin' (SA) is intended to include (but not necessarily to
be restricted
to) serum albumin proteins of living organisms, preferably mammalian serum
albumins, even
more preferably known or yet-to-be-discovered polymorphic forms of human serum
albumin
(HSA), and variants thereof. For example, the human serum albumin Naskapi has
Lys-372 in
place of Glu-372, and albumin Christchurch has an altered pro-sequence. The
term 'variants' is
intended to include (but not necessarily be restricted to) homologs of SA
proteins with minor
artificial variations in sequence (such as molecules lacking one or a few
residues, having
conservative substitutions or minor insertions of residues, or having minor
variations of amino
acid structure). Thus, polypeptides which have 80%, 85%, 90%, or 99% homology
with a native
SA are deemed to be 'variants'. It is also preferred for such variants to
share at least one
pharmacological utility with a native SA. Any putative variant which is to be
used
pharmacologically should be non-immunogenic in the animal (especially human)
being treated.
Sequences of a number of contemplated serum albumin proteins can be obtained
from GenBank
(National Center for Biotechnology Information), including human, bovine,
mouse, pig, horse,
sheep, and chick serum albumins.
The term 'native' is used to describe a protein which occurs naturally in a
living organism.
Wild-type proteins are thus native proteins. Proteins which are non-native are
those which have
been generated by artificial mutation, recombinant design, or other laboratory
modification and
are not known in natural populations.
'Conservative substitutions' are those where one or more amino acids are
substituted for
others having similar properties such that one skilled in the art of
polypeptide chemistry would
expect at least the secondary structure, and preferably the tertiary
structure, of the polypeptide
to be substantially unchanged. For example, typical such substitutions include
asparagine for
glutamine, serine for asparagine, and arginine for lysine. The term
'physiologically functional
equivalents' also encompasses larger molecules comprising the native sequence
plus a further
sequence at the N-terminus (for example, pro-HSA, pre-pre-HSA, and met-HSA).
'Tertiary structure' refers to the three-dimensional structure of a protein.
Proteins which
have similar tertiary structures will have similar shapes and surfaces, even
if the amino acid
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sequences (the 'secondary structure') is not identical. Tertiary structure is
a consequence of the
folding and twisting of an amino acid chain upon itself and can be disrupted
by chemical means,
e.g., strong acid or base, or by physical means, e.g., heating.
The term 'biologically active' refers to an entity which interacts in some way
with a living
organism on a molecular level. Entities which are biologically active may
activate a receptor,
provoke an immune reaction, interact with a membrane or ion channel, or
otherwise induce a
change in a biological function of an organism or any part of an organism.
The term 'ligand' refers to a molecule that is recognized by a particular
protein, e.g., a
receptor. Any agent bound by or reacting with a protein is called a 'ligand',
so the term
encompasses the substrate of an enzyme and the reactants of a catalyzed
reaction. The term
'ligand' does not imply any particular molecular size or other structural or
compositional feature
other than that the substance in question is capable of binding or otherwise
interacting with a
protein. A 'ligand' may serve either as the natural ligand to which the
protein binds or as a
functional analogue that may act as an agonist or antagonist.
The term 'vector' refers to a DNA molecule, capable of replication in a host
cell, into
which a gene can be inserted to construct a recombinant DNA molecule. Examples
of vectors
include plasmids and infective microorganisms such as viruses, or non-viral
vectors such as
ligand-DNA conjugates, liposomes, or lipid-DNA complexes.
As used herein, 'cell surface receptor' refers to molecules that occur on the
surface of
cells, interact with the extracellular environment, and (directly or
indirectly) transmit or
transduce the information regarding the environment intracellularly in a
manner that may
modulate intracellular second messenger activities or transcription of
specific promoters,
resulting in transcription of specific genes.
As used herein, 'extracellular signals' include a molecule or other change in
the
extracellular environment that is transduced intracellularly via cell surface
proteins that interact,
directly or indirectly, with the signal. An extracellular signal or effector
molecule includes any
compound or substance that in some manner alters the activity of a cell
surface protein.
Examples of such signals include, but are not limited to, molecules such as
acetylcholine, growth
factors and hormones, lipids, sugars and nucleotides that bind to cell surface
and/or intracellular
receptors and ion channels and modulate the activity of such receptors and
channels.
As used herein, 'extracellular signals' also include as yet unidentified
substances that
modulate the activity of a cellular receptor, and thereby influence
intracellular functions. Such
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extracellular signals are potential pharmacological agents that may be used to
treat specific
diseases by modulating the activity of specific cell surface receptors.
'Orphan receptors' is a designation given to receptors for which no specific
natural ligand
has been described and/or for which no function has been determined.
The term 'target cells' as used herein means cells, either in vivo or ex vivo,
into which it
is desired to introduce exogenous genetic material. Target cells may be any
type of cell,
including blood cells, skeletal muscle cells, stem cells, skin cells, liver
cells, secretory gland
cells, hematopoietic cells, and marrow cells.
An 'effective amount' of a fusion polypeptide, with respect to the subject
method of
treatment, refers to an amount of the polypeptide in a preparation which, when
applied as part
of a desired dosage regimen, provides inhibition of angiogenesis so as to
reduce or cure a
disorder according to clinically acceptable standards.
'Serum half life' as used herein refers to the time required for half of a
quantity of a
peptide in the bloodstream to be degraded.
F.xP~~l~cation
As set out above, the chimeric polypeptide of the present invention can be
constructed
as a chimeric polypeptide containing a sequence homologous to at least a
portion of a serum
albumin and at least a portion of one or more heterologous proteins, expressed
as one contiguous
polypeptide chain. In preparing the chimeric polypeptide, a fusion gene is
constructed
comprising DNA encoding at least one sequence each of a serum albumin, a
heterologous
protein, and, optionally, a peptide linker sequence to span the fragments. If
more than one
heterologous sequences are included in the chimeric polypeptide, they may be
identical, related,
or unrelated sequences. Identical sequences may be included to increase the
effective
concentration of the sequence. Related sequences may be included to more
accurately mimic the
native protein from which they are derived. Unrelated sequences may be useful
for activating two
or more distinct receptors that stimulate the same response, or for imparting
two or more distinct
activities to the chimeric polypeptide. For example, the chimeric polypeptide
might include a
sequence that has antiangiogenic activity and a sequence which induces
apoptosis of tumor cells.
To make this chimeric polypeptide, an entire protein can be cloned and
expressed as part
of the protein, or alternatively, a suitable fragment thereof containing a
biologically active
moiety can be used. The use of recombinant DNA techniques to create a fusion
gene, with the
translational product being the desired chimeric polypeptide, is well known in
the art. Both the
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coding sequence of a gene and its regulatory regions can be redesigned to
change the functional
properties of the protein product, the amount of protein made, or the cell
type in which the
protein is produced. The coding sequence of a gene can be extensively altered,
for example, by
fusing part of it to the coding sequence of a different gene to produce a
novel hybrid gene that
encodes a fusion protein. Examples of methods for producing fusion proteins
are described in
PCT applications PCT/LTS87/02968, PCT/US89/03587 and PCT/LTS90/07335, as well
as
Traunecker et al. (1989) Nature 339:68, all of which are incorporated by
reference herein.
Techniques for making fusion genes are well known. Essentially, the joining of
various
DNA fragments coding for different polypeptide sequences is performed in
accordance with
conventional techniques, employing blunt-ended or stagger-ended termini for
ligation, restriction
enzyme digestion to provide for appropriate termini, filling in of cohesive
ends as appropriate,
alkaline phosphatase treatment to avoid undesirable joining, and enzymatic
ligation.
Alternatively, the fusion gene can be synthesized by conventional techniques
including
automated DNA synthesizers. In another method, PCR amplification of gene
fragments can be
carried out using anchor primers which give rise to complementary overhangs
between two
consecutive gene fragments which can subsequently be annealed to generate a
chimeric gene
sequence (see, for example, Current Protocols in Molecular Biology, Eds.
Ausubel et al. John
Wiley & Sons: 1992).
This invention also provides expression vectors comprising a nucleotide
sequence
encoding a subject chimeric polypeptide operably linked to at least one
regulatory sequence.
'Operably linked' is intended to mean that the nucleotide sequence is linked
to a regulatory
sequence in a manner which allows expression of the nucleotide sequence.
Regulatory
sequences are art-recognized and are selected to direct expression of the
encoded polypeptide.
Accordingly, the term regulatory sequence includes promoters, enhancers and
other expression
control elements. Exemplary regulatory sequences are described in Goeddel;
Gene Expression
Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
For instance,
any of a wide variety of expression control sequences-sequences that control
the expression of
a DNA sequence when operatively linked to it may be used in these vectors to
express DNA
sequences encoding the chimeric polypeptides of this invention. Such useful
expression control
sequences, include, for example, the early and late promoters of SV40,
adenovirus or
cytomegalovirus immediate early promoter, the lac system, the trp system, the
TAC or TRC
system, T7 promoter whose expression is directed by T7 RNA polymerase, the
major operator
and promoter regions of phage lambda, the control regions for fd coat protein,
the promoter for
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3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid
phosphatase, e.g.,
PhoS, the promoters of the yeast a-mating factors, the polyhedron promoter of
the baculovirus
system and other sequences known to control the expression of genes of
prokaryotic or
eukaryotic cells or their viruses, and various combinations thereof. It should
be understood that
the design of the expression vector may depend on such factors as the choice
of the host cell to
be transformed and/or the type of protein desired to be expressed. Moreover,
the vector's copy
number, the ability to control that copy number and the expression of any
other proteins encoded
by the vector, such as antibiotic markers, should also be considered.
As will be apparent, the subject gene constructs can be used to cause
expression of the
subject chimeric polypeptides in cells propagated in culture, e.g., to produce
chimeric
polypeptides, for purification. This represents a method for preparing
substantial quantities of
the polypeptide, e.g., for research, clinical, and pharmaceutical uses.
In certain therapeutic applications, the ex vivo-derived chimeric polypeptides
are utilized
in a manner appropriate for therapy in general. For such therapy, the
polypeptides of the
invention can be formulated for a variety of modes of administration,
including systemic and
topical or localized administration. In such embodiments, the polypeptide may
by combined with
a pharmaceutically acceptable excipient, e.g., a non-pyrogenic excipient.
Techniques and
formulations generally may be found in Remmington's Pharmaceutical Sciences,
Meade
Publishing Co., Easton, PA. For systemic administration, injection being
preferred, including
intramuscular, intravenous, intraperitoneal, and subcutaneous injection, the
polypeptides of the
invention can be formulated in liquid solutions, preferably in physiologically
compatible buffers
such as Hank's solution or Ringer's solution. In addition, the polypeptides
may be formulated
in solid form and redissolved or suspended immediately prior to use.
Lyophilized forms are also
included.
Systemic administration can also be by transmucosal or transdermal means, or
the
compounds can be administered orally. For transmucosal or transdermal
administration,
penetrants appropriate to the barner to be permeated are used in the
formulation. Such
penetrants are generally known in the art, and include, for example, for
transmucosal
administration bile salts and fusidic acid derivatives. In addition,
detergents may be used to
facilitate permeation. Transmucosal administration may be through nasal sprays
or using
suppositories. For oral administration, the peptides are formulated into
conventional oral
administration forms such as capsules, tablets, and tonics. For topical
administration,
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particularly cosmetic formulations, the oligomers of the invention are
formulated into ointments,
salves, gels, or creams as generally known in the art.
Alternative means of administration of peptides have been developed. Sustained-
release
formulations (Putney, et al. Nature Biotechnology 1998, 16, 153-157) are
advantageous,
requiring fewer administrations and, often, lower dosages. Techniques for oral
delivery of
peptides have been reviewed (Fasano, A. Trends in Biotechnology 1998, 16, 152-
157), as have
several site-specific means of peptide delivery (Pettit, D.K. et al. Trends in
Biotechnology 1998,
16, 343-349). Additional techniques for therapeutic administration of peptides
are known to
those of skill in the art.
Genetic material of the present invention can be delivered, for example, as an
expression
plasmid which, when transcribed in the cell, produces the desired chimeric
polypeptide.
In another embodiment, the genetic material is provided by use of an
"expression"
construct, which can be transcribed in a cell to produce the chimeric
polypeptide. Such
expression constructs may be administered in any biologically effective
carrier, e.g., any
formulation or composition capable of effectively transfecting cells either ex
vivo or in vivo with
genetic material encoding a chimeric polypeptide. Approaches include insertion
of the antisense
nucleic acid in viral vectors including recombinant retroviruses,
adenoviruses, adeno-associated
viruses, human immunodeficiency viruses, and herpes simplex viruses-l, or
recombinant
bacterial or eukaryotic plasmids. Viral vectors can be used to transfect cells
directly; plasmid
DNA can be delivered with the help of, for example, cationic liposomes
(lipofectin) or
derivatized (e.g., antibody conjugated), polylysine conjugates, gramicidin S,
artificial viral
envelopes or other such intracellular carriers, as well as direct injection of
the gene construct or
calcium phosphate precipitation carried out in vivo. It will be appreciated
that because
transduction of appropriate target cells represents the critical first step in
gene therapy, choice
of the particular gene delivery system will depend on such factors as the
phenotype of the
intended target and the route of administration, e.g., locally or
systemically.
A preferred approach for in vivo introduction of genetic material encoding one
of the
subject proteins into a cell is by use of a viral vector containing said
genetic material. Infection
of cells with a viral vector has the advantage that a large proportion of the
targeted cells can
receive the nucleic acid. Additionally, chimeric polypeptides encoded by
genetic material in the
viral vector, e.g., by a nucleic acid contained in the viral vector, are
expressed efficiently in cells
which have taken up viral vector nucleic acid. Such a strategy may be
particularly effective when
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skeletal muscle cells are the targets of the vector (Fisher, K.J. et al.
Nature Medicine 1997, 3,
306-312).
Retrovirus vectors and adeno-associated virus vectors are generally understood
to be the
recombinant gene delivery system of choice for the transfer of exogenous genes
in vivo,
particularly into humans. These vectors provide efficient delivery of genes
into cells, and the
transferred nucleic acids are stably integrated into the chromosomal DNA of
the host. A major
prerequisite for the use of retroviruses is to ensure the safety of their use,
particularly with regard
to the possibility of the spread of wild-type virus in the cell population.
The development of
specialized cell lines (termed "packaging cells") which produce only
replication-defective
retroviruses has increased the utility of retroviruses for gene therapy, and
defective retroviruses
are well characterized for use in gene transfer for gene therapy purposes (for
a review see Miller,
A.D. (1990) Blood 76:271). Thus, recombinant retrovirus can be constructed in
which part of
the retroviral coding sequence (gag, pol, env) has been replaced by nucleic
acid encoding one
of the antisense E6AP constructs, rendering the retrovirus replication
defective. The replication
defective retrovirus is then packaged into virions which can be used to infect
a target cell through
the use of a helper virus by standard techniques. Protocols for producing
recombinant
retroviruses and for infecting cells in vitro or in vivo with such viruses can
be found in Current
Protocols in Molecular Biolo~v, Ausubel, F.M. et al. (eds.) Greene Publishing
Associates,
(1989), Sections 9.10-9.14, and other standard laboratory manuals. Examples of
suitable
retroviruses include pLJ, pZIP, pWE and pEM which are well known to those
skilled in the art.
Examples of suitable packaging virus lines for preparing both ecotropic and
amphotropic
retroviral systems include yrCrip, yrCre, y~2 and yrAm. Retroviruses have been
used to introduce
a variety of genes into many different cell types, including neural cells,
epithelial cells,
endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in
vitro and/or in vivo
(see for example Eglitis, et al. (1985) Science 230:1395-1398; Danos and
Mulligan (1988) Proc.
Natl. Acad. Sci. USA 85:6460-6464; Wilson et al. (1988) Proc. Natl. Acad. Sci.
USA 85:3014-
3018; Armentano et al. (1990) Proc. Natl. Acad. Sci. USA 87:6141-6145; Huber
et al. (1991)
Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al. (1991) Proc. Natl. Acad.
Sci. USA
88:8377-8381; Chowdhury et al. (1991) Science 254:1802-1805; van Beusechem et
al. (1992)
Proc. Natl. Acad. Sci. USA 89:7640-7644; Kay et al. (1992) Human Gene Therapy
3:641-647;
Dai et al. (1992) Proc. Natl. Acad. Sci. USA 89:10892-10895; Hwu et al. (1993)
J. Immunol.
150:4104-4115; U.S. Patent No. 4,868,116; U.S. Patent No. 4,980,286; PCT
Application WO
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CA 02376641 2002-O1-18
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89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345; and PCT
Application WO 92/07573).
In choosing retroviral vectors as a gene delivery system for genetic material
encoding the
subject chimeric polypeptides, it is important to note that a prerequisite for
the successful
infection of target cells by most retroviruses, and therefore of stable
introduction of the genetic
material, is that the target cells must be dividing. In general, this
requirement will not be a
hindrance to use of retroviral vectors. In fact, such limitation on infection
can be beneficial in
circumstances wherein the tissue (e.g., nontransformed cells) surrounding the
target cells does
not undergo extensive cell division and is therefore refractory to infection
with retroviral vectors.
Furthermore, it has been shown that it is possible to limit the infection
spectrum of
retroviruses and consequently of retroviral-based vectors, by modifying the
viral packaging
proteins on the surface of the viral particle (see, for example, PCT
publications W093/25234,
W094/06920, and W094/11524). For instance, strategies for the modification of
the infection
spectrum of retroviral vectors include: coupling antibodies specific for cell
surface antigens to
the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Julan et al.
(1992) J. Gen Virol
73:3251-3255; and Goud et al. (1983) Virology 163:251-254); or coupling cell
surface ligands
to the viral env proteins (Neda et al. (1991) JBiol Chem 266:14143-14146).
Coupling can be
in the form of the chemical cross-linking with a protein or other variety
(e.g., lactose to convert
the env protein to an asialoglycoprotein), as well as by generating chimeric
proteins (e.g., single-
chain antibody/env chimeric proteins). This technique, while useful to limit
or otherwise direct
the infection to certain tissue types, and can also be used to convert an
ecotropic vector in to an
amphotropic vector.
Moreover, use of retroviral gene delivery can be further enhanced by the use
of tissue-
or cell-specific transcriptional regulatory sequences which control expression
of the genetic
material of the retroviral vector.
Another viral gene delivery system useful in the present invention utilizes
adenovirus-
derived vectors. The genome of an adenovirus can be manipulated such that it
encodes a gene
product of interest, but is inactive in terms of its ability to replicate in a
normal lytic viral life
cycle (see, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld
et al. (1991)
Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155). Suitable
adenoviral vectors
derived from the adenovirus strain Ad type 5 d1324 or other strains of
adenovirus (e.g., Ad2,
Ad3, Ad7, etc.) are well known to those skilled in the art. Recombinant
adenoviruses can be
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advantageous in certain circumstances in that they are capable of infecting
non-dividing cells and
can be used to infect a wide variety of cell types, including airway
epithelium (Rosenfeld et al.
(1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl.
Acad. Sci. USA
89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc. Natl. Acad. Sci. USA
90:2812-2816)
and muscle cells (Quantin et al. (1992) Proc. Natl. Acad. Sci. USA 89:2581-
2584). Furthermore,
the virus particle is relatively stable and amenable to purification and
concentration, and, as
above, can be modified so as to affect the spectrum of infectivity.
Additionally, introduced
adenoviral DNA (and foreign DNA contained therein) is not integrated into the
genome of a host
cell but remains episomal, thereby avoiding potential problems that can occur
as a result of
insertional mutagenesis in situations where introduced DNA becomes integrated
into the host
genome (e.g., retroviral DNA). Moreover, the carrying capacity of the
adenoviral genome for
foreign DNA is large (up to 8 kilobases) relative to other gene delivery
vectors (Berkner et al.,
supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267). Most replication-
defective adenoviral
vectors currently in use and therefore favored by the present invention are
deleted for all or parts
of the viral El and E3 genes but retain as much as 80% of the adenoviral
genetic material (see,
for example, Jones et al. (1979) Cell 16:683; Berkner et al., supra; and
Graham et al. in
in Molecular Biolo~v, E.J. Murray, Ed. (Humana, Clifton, NJ, 1991) vol. 7. pp.
109-127).
Expression of the inserted genetic material can be under control of, for
example, the ElA
promoter, the major late promoter (MLP) and associated leader sequences, the
E3 promoter, or
exogenously added promoter sequences.
Yet another viral vector system useful for delivery of genetic material
encoding the
subject chimeric polypeptides is the adeno-associated virus (AAV). Adeno-
associated virus is
a naturally occurring defective virus that requires another virus, such as an
adenovirus or a
herpes virus, as a helper virus for efficient replication and a productive
life cycle. (For a review
see Muzyczka et al. Curr. Topics in Micro. and Immunol. (1992) 158:97-129). It
is also one of
the few viruses that may integrate its DNA into non-dividing cells, and
exhibits a high frequency
of stable integration (see for example Flotte et al. (1992) Ana. J. Respir.
Cell. Mol. Biol. 7:349-
356; Samulski et al. (1989) J. Virol. 63:3822-3828; and McLaughlin et al.
(1989) J. Virol.
62:1963-1973). Vectors containing as little as 300 base pairs of AAV can be
packaged and can
integrate. Space for exogenous DNA is limited to about 4.5 kb. An AAV vector
such as that
described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used
to introduce DNA
into cells. A variety of nucleic acids have been introduced into different
cell types using AAV
vectors (see for example Hermonat et al. ( 1984) Proc. Natl. Acad. Sci. USA
81:6466-6470;
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Tratschin et al. (1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al. (1988)
Mol. Endocrinol.
2:32-39; Tratschin et al. (1984) J. Virol. 51:611-619; and Flotte et al.
(1993) J. Biol. Chem.
268:3781-3790).
Other viral vector systems that may have application in gene therapy have been
derived
from herpes virus, vaccinia virus, and several RNA viruses.
In addition to viral transfer methods, such as those illustrated above, non-
viral methods
can also be employed to cause expression of genetic material encoding the
subject chimeric
polypeptides in the tissue of an animal. Most nonviral methods of gene
transfer rely on normal
mechanisms used by mammalian cells for the uptake and intracellular transport
of
macromolecules. In preferred embodiments, non-viral gene delivery systems of
the present
invention rely on endocytic pathways for the uptake of genetic material by the
targeted cell.
Exemplary gene delivery systems of this type include liposomal derived
systems, polylysine
conjugates, and artificial viral envelopes.
In a representative embodiment, genetic material can be entrapped in liposomes
bearing
positive charges on their surface (e.g., lipofectins) and, optionally, which
are tagged with
antibodies against cell surface antigens of the target tissue (Mizuno et al.
(1992) No Shinkei Geka
20:547-551; PCT publication W091/06309; Japanese patent application 1047381;
and European
patent publication EP-A-43075). For example, lipofection of papilloma-infected
cells can be
carned out using liposomes tagged with monoclonal antibodies against PV-
associated antigen
(see Viac et al. (1978) Jlnvest Dermatol 70:263-266; see also Mizuno et al.
(1992) Neurol. Med.
Chir. 32:873-876).
In yet another illustrative embodiment, the gene delivery system comprises an
antibody
or cell surface ligand which is cross-linked with a gene binding agent such as
polylysine (see,
for example, PCT publications W093/04701, W092/22635, W092/20316, W092/19749,
and
W092/06180). For example, genetic material encoding the subject chimeric
polypeptides can
be used to transfect hepatocytic cells in vivo using a soluble polynucleotide
carrier comprising
an asialoglycoprotein conjugated to a polycation, e.g., polylysine (see U.S.
Patent 5,166,320).
It will also be appreciated that effective delivery of the subject nucleic
acid constructs via
mediated endocytosis can be improved using agents which enhance escape of the
gene from the
endosomal structures. For instance, whole adenovirus or fusogenic peptides of
the influenza HA
gene product can be used as part of the delivery system to induce efficient
disruption of DNA-
containing endosomes (Mulligan et al. (1993) Science 260-926; Wagner et al.
(1992) PNAS
89:7934; and Christiano et al. (1993) PNAS 90:2122).
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In clinical settings, the gene delivery systems can be introduced into a
patient by any of
a number of methods, each of which is familiar in the art. For instance, a
pharmaceutical
preparation of the gene delivery system can be introduced systemically, e.g.,
by intravenous
injection, and specific transduction of the target cells occurs predominantly
from specificity of
transfection provided by the gene delivery vehicle, cell-type or tissue-type
expression due to the
transcriptional regulatory sequences controlling expression of the gene, or a
combination thereof.
In other embodiments, initial delivery of the recombinant gene is more limited
with introduction
into the animal being quite localized. For example, the gene delivery vehicle
can be introduced
by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g.,
Chen et al. (1994) PNAS
91: 3054-3057).
Moreover, the pharmaceutical preparation can consist essentially of the gene
delivery
system in an acceptable diluent, or can comprise a slow release matrix in
which the gene delivery
vehicle is imbedded. Alternatively, where the complete gene delivery system
can be produced
intact from recombinant cells, e.g., retroviral packages, the pharmaceutical
preparation can
comprise one or more cells which produce the gene delivery system. In the
latter case, methods
of introducing the viral packaging cells may be provided by, for example,
rechargeable or
biodegradable devices. Various slow release polymeric devices have been
developed and tested
in vivo in recent years for the controlled delivery of drugs, including
proteinaceous
biopharmaceuticals, and can be adapted for release of viral particles through
the manipulation
of the polymer composition and form. A variety of biocompatible polymers
(including
hydrogels), including both biodegradable and non-degradable polymers, can be
used to form an
implant for the sustained release of an the viral particles by cells implanted
at a particular target
site. Such embodiments of the present invention can be used for the delivery
of an exogenously
purified virus, which has been incorporated in the polymeric device, or for
the delivery of viral
particles produced by a cell encapsulated in the polymeric device.
By choice of monomer composition or polymerization technique, the amount of
water,
porosity and consequent permeability characteristics can be controlled. The
selection of the
shape, size, polymer, and method for implantation can be determined on an
individual basis
according to the disorder to be treated and the individual patient response.
The generation of such
implants is generally known in the art. See, for example, Concise Encyclopedia
of Medical &
Dental Materials, ed. by David Williams (MIT Press: Cambridge, MA, 1990); and
the Sabel et
al. U.S. Patent No. 4,883,666. In another embodiment of an implant, a source
of cells producing
a the recombinant virus is encapsulated in implantable hollow fibers. Such
fibers can be pre-
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CA 02376641 2002-O1-18
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spun and subsequently loaded with the viral source (Aebischer et al. U.S.
Patent No. 4,892,538;
Aebischer et al. U.S. Patent No. 5,106,627; Hoffinan et al. (1990) Expt.
Neurobiol. 110:39-44;
Jaeger et al. (1990) Prog. Brain Res. 82:41-46; and Aebischer et al. (1991) J.
Biomech. Eng.
113:178-183), or can be co-extruded with a polymer which acts to form a
polymeric coat about
the viral packaging cells (Lim U.S. Patent No. 4,391,909; Sefton U.S. Patent
No. 4,353,888;
Sugamori et al. (1989) Trans. Am. Artif. Intern. Organs 35:791-799; Sefton et
al. (1987)
Biotechnol. Bioeng. 29:1135-1143; and Aebischer et al. (1991) Biomaterials
12:50-55). Again,
manipulation of the polymer can be carried out to provide for optimal release
of viral particles.
Chimeric polypeptides of the present invention can be designed by using
molecular
modeling. A computer model of serum albumin may be altered to include a
selected heterologous
sequence and the resulting structure may be submitted to calculations designed
to determine how
the resulting peptide will change in shape, how much strain the alteration
introduces into the
polypeptide, how the heterologous sequence is displayed in three dimensions,
and other data
relevant to the resulting structure of the chimeric polypeptide.
Alternatively, the nature of the
sequence to be included might be determined by the calculation, based on
knowledge of a
receptor or binding pocket. In another embodiment, the calculations might best
determine how
to insert a desired sequence to maintain the tertiary structure of the serum
albumin backbone, or
to display the insertion in the proper orientation. Other calculational
strategies will be known to
those skilled in the art. Calculations such as these can be useful for
directing the synthesis of
chimeric polypeptides of the present invention in a time- and material-
efficient manner, before
actual synthesis and screening techniques begin.
Methods for screening chimeric polypeptides of the present invention are well
known in
the art, independent of the use of computer modeling. The use of peptide
libraries is one way of
screening large numbers of polypeptides at once. In one screening assay, the
candidate peptides
are displayed on the surface of a cell or viral particle, and the ability of
particular cells or viral
particles to bind a target molecule, such as a receptor protein via this gene
product is detected in
a "panning assay". For instance, the gene library can be cloned into the gene
for a surface
membrane protein of a bacterial cell, and the resulting chimeric polypeptide
detected by panning
(Ladner et al., WO 88/06630; Fuchs et al. (1991) BiolTechnology 9:1370-1371;
and Goward et
al. (1992) TIBS 18:136-140).
In an alternate embodiment, the peptide library is expressed as chimeric
polypeptides on
the surface of a viral particle. For instance, in the filamentous phage
system, foreign peptide
sequences can be expressed on the surface of infectious phage, thereby
conferring two significant
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benefits. First, since these phage can be applied to affinity matrices at very
high concentrations,
a large number of phage can be screened at one time. Second, since each
infectious phage
displays the combinatorial gene product on its surface, if a particular phage
is recovered from an
affinity matrix in low yield, the phage can be amplified by another round of
infection. The group
of almost identical E. coli filamentous phages M13, fd, and fl are most often
used in phage
display libraries, as either of the phage gIII or gVIII coat proteins can be
used to generate
chimeric polypeptides without disrupting the ultimate packaging of the viral
particle (Ladner et
al. PCT publication WO 90/02809; Garrard et al., PCT publication WO 92/09690;
Marks et al.
(1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-
734; Clackson
et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).
The field of combinatorial peptide libraries has been reviewed (Gallop et al.
J. Med.
Chem. 1994, 37, 1233-1251), and additional techniques are known in the art
(Gustin, K. Virology
1993, 193, 653-660; Goeddel et al. U.S. Patent 5,223,408; Markland et al. PCT
publication
W092/15679; Bass et al. Proteins: Structure, Function and Genetics 1990, 8,
309-
314;Cunningham, B.C. Science 1990, 247, 1461-1465; Lowman, H.B. Biochemistry
1991, 30,
10832-10838; Fowlkes et al. U.S. Patent No. 5,789,184; Houghton, Proc. Natl.
Acad. Sci. U.S.A.
1985, 82, 5131-5135) for generating and screening peptide libraries.
U.S. patent application 09/174,943, filed October 19, 1998, discloses a method
for
isolating biologically active peptides. Using the techniques disclosed
therein, a chimeric
polypeptide of the present invention may be developed which interacts with a
chosen receptor.
In a representative example, this method is utilized to identify polypeptides
which have
antiproliferative activity with respect to one or more types of cells. One of
skill in the art will
readily be able to modify the procedures outlined below to find polypeptides
with any desired
activity. In the example, in the display mode, the chimeric polypeptide
library can be panned
with the target cells for which an antiproliferative is desired in order to
enrich for polypeptides
which bind to that cell. At that stage, the polypeptide library can also be
panned against one or
more control cell lines in order to remove polypeptides which bind the control
cells. In this
manner, the polypeptide library which is then tested in the secretion mode can
be enriched for
polypeptides which selectively bind target cells (relative to the control
cells). Thus, for example,
the display mode can produce a polypeptide library enriched for polypeptides
which
preferentially bind tumor cells relative to normal cells, which preferentially
bind p53- cells
relative to p53+ cells, which preferentially bind hair follicle cells relative
to other epithelial cells,
or any other differential binding characteristic.
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In the secretion mode, the polypeptides are tested for antiproliferative
activity against the
target cell using any of a number of techniques known in the art. For
instance, BrdU or other
nucleotide uptake can be measured as an indicator of proliferation. As above,
the secretion mode
can include negative controls in order to select for polypeptides with
specific antiproliferative
activity.
In similar fashion, polypeptides can be isolated from the library based on
their ability to
induce apoptosis or cell lysis, for example, in a cell-selective manner.
Also, this method can be used to identify polypeptides with angiogenic or
antiangiogenic
activity. For instance, the polypeptide library can be enriched for
polypeptides that bind to
endothelial cells but which do not bind to fibroblasts. The resulting sub-
library can be screened
for polypeptides which inhibit capillary endothelial cell proliferation and/or
endothelial cell
migration. Polypeptides scoring positive for one or both of these activities
can also be tested for
activity against other cell types, such as smooth muscle cells or fibroblasts,
in order to select
polypeptides active only against endothelial cells.
Furthermore, this method can be used to identify anti-infective polypeptides,
for example,
which are active as anti-fungal or antibacterial agents.
In addition, this assay can be used for identifying effectors of a receptor
protein or
complex thereof. In general, the assay is characterized by the use of a test
cell which includes
a target receptor or ion channel protein whose signal transduction activity
can be modulated by
interaction with an extracellular signal, the transduction activity being able
to generate a
detectable signal.
In general, such assays are characterized by the use of a mixture of cells
expressing a
target receptor protein or ion channel capable of transducing a detectable
signal in the reagent
cell. The receptor/channel protein can be either endogenous or heterologous.
In combination
with the disclosed detection means, a culture of the instant reagent cells
will provide means for
detecting agonists or antagonists of receptor function.
The ability of particular polypeptides to modulate a signal transduction
activity of the
target receptor or channel can be scored for by detecting up or down-
regulation of the detection
signal. For example, second messenger generation (e.g., GTPase activity,
phospholipid
hydrolysis, or protein phosphorylation patterns as examples) can be measured
directly.
Alternatively, the use of an indicator gene can provide a convenient readout.
In other
embodiments a detection means consists of an indicator gene. In any event, a
statistically
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significant change in the detection signal can be used to facilitate
identification of compounds
which modulate receptor or ion channel activities.
By this method, polypeptides which induce a signal pathway from a particular
receptor
or channel can be identified. If a test polypeptide does not appear to induce
the activity of the
receptor/channel protein, the assay may be repeated as described above, and
modified by the
introduction of a step in which the reagent cell is first contacted with a
known activator of the
target receptor/channel to induce signal transduction, and the test peptide
can be assayed for its
ability to inhibit the activated receptor/channel, for example, to identify
antagonists. In yet other
embodiments, peptides can be screened for those which potentiate the response
to a known
activator of the receptor.
In particular, the assays can be used to test functional ligand-receptor or
ligand-ion
channel interactions for cell surface-localized receptors and channels. As
described in more
detail below, the subject assay can be used to identify effectors of, for
example, G protein-
coupled receptors, receptor tyrosine kinases, cytokine receptors, and ion
channels. In certain
embodiments the method described herein is used for identifying ligands for
"orphan receptors"
for which no ligand is known.
In some examples, the receptor is a cell surface receptor, such as: a receptor
tyrosine
kinase, for example, an EPH receptor; an ion channel; a cytokine receptor; an
multisubunit
immune recognition receptor, a chemokine receptor; a growth factor receptor,
or a G-protein
coupled receptor, such as a chemoattracttractant peptide receptor, a
neuropeptide receptor, a light
receptor, a neurotransmitter receptor, or a polypeptide hormone receptor.
Preferred G protein-coupled receptors include alA-adrenergic receptor, alB-
adrenergic
receptor, a2-adrenergic receptor, a2B-adrenergic receptor, 1-adrenergic
receptor, X32-adrenergic
receptor, ~3-adrenergic receptor, ml acetylcholine receptor (AChR), m2 AChR,
m3 AChR, m4
AChR, m5 AChR, D1 dopamine receptor, D2 dopamine receptor, D3 dopamine
receptor, D4
dopamine receptor, DS dopamine receptor, Al adenosine receptor, A2b adenosine
receptor, 5-
HT 1 a receptor, 5-HT 1 b receptor, SHT 1-like receptor, 5-HT 1 d receptor,
SHT 1 d-like receptor,
SHTId beta receptor, substance K (neurokinin A) receptor, fMLP receptor, fMLP-
like receptor,
angiotensin II type 1 receptor, endothelin ETA receptor, endothelin ETB
receptor, thrombin
receptor, growth hormone-releasing hormone (GHRH) receptor, vasoactive
intestinal peptide
receptor, oxytocin receptor, somatostatin SSTR1 and SSTR2, SSTR3, cannabinoid
receptor,
follicle stimulating hormone (FSH) receptor, leutropin (LH/HCG) receptor,
thyroid stimulating
hormone (TSH) receptor, thromboxane A2 receptor, platelet-activating factor
(PAF) receptor,
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CSa anaphylatoxin receptor, Interleukin 8 (IL-8) IL-BRA, IL-8RB, Delta Opioid
receptor, Kappa
Opioid receptor, mip-1/RANTES receptor, Rhodopsin, Red opsin, Green opsin,
Blue opsin,
metabotropic glutamate mGluR1-6, histamine H2 receptor, ATP receptor,
neuropeptide Y
receptor, amyloid protein precursor receptor, insulin-like growth factor II
receptor, bradykinin
receptor, gonadotropin-releasing hormone receptor, cholecystokinin receptor,
melanocyte
stimulating hormone receptor receptor, antidiuretic hormone receptor, glucagon
receptor, and
adrenocorticotropic hormone II receptor.
Preferred EPH receptors inlcude eph, elk, eck, sek, mek4, hek, hek2, eek, erk,
tyrol , tyro4,
tyros, tyro6, tyroll, cek4, cek5, cek6, cek7, cek8, cek9, cekl0, bsk, rtkl,
rtk2, rtk3, mykl, myk2,
ehkl, ehk2, pagliaccio, htk, erk and nuk receptors.
A. ~;vtoki~Ze Receptors
In one example the target receptor is a cytokine receptor. Cytokines are a
family of
soluble mediators of cell-to-cell communication that includes interleukins,
interferons, and
colony-stimulating factors. The characteristic features of cytokines lie in
their functional
redundancy and pleiotropy. Most of the cytokine receptors that constitute
distinct superfamilies
do not possess intrinsic protein tyrosine kinase domains, yet receptor
stimulation usually invokes
rapid tyrosine phosphorylation of intracellular proteins, including the
receptors themselves.
Many members of the cytokine receptor superfamily activate the Jak protein
tyrosine kinase
family, with resultant phosphorylation of the STAT transcriptional activator
factors. IL-2, IL-7,
IL-2 and Interferon y have all been shown to activate Jak kinases (Frank et al
(1995) Proc Natl
Acad Sci USA 92:7779-7783); Scharfe et al. (1995) Blood 86:2077-2085); (Bacon
et al. (1995)
Proc Natl Acad Sci USA 92:7307-7311); and (Sakatsume et al (1995) J. Biol Chem
270:17528-
17534). Events downstream of Jak phosphorylation have also been elucidated.
For example,
exposure of T lymphocytes to IL-2 has been shown to lead to the
phosphorylation of signal
transducers and activators of transcription (STAT) proteins STATla,, STAT2(3,
and STAT3, as
well as of two STAT-related proteins, p94 and p95. The STAT proteins were
found to
translocate to the nucleus and to bind to a specific DNA sequence, thus
suggesting a mechanism
by which IL-2 may activate specific genes involved in immune cell function
(Frank et al. supra).
Jak3 is associated with the gamma chain of the IL-2, IL-4, and IL-7 cytokine
receptors (Fujii et
al. (1995) Proc Natl Acad Sci 92:5482-5486) and (Musso et al (1995) J Exp Med.
181:1425-
1431 ). The Jak kinases have also been shown to be activated by numerous
ligands that signal
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via cytokine receptors such as, growth hormone and erythropoietin and IL-6
(Kishimoto ( 1994)
Stem cells Suppl 12:37-44).
Detection means which may be scored for in the present assay, in addition to
direct
detection of second messengers, such as by changes in phosphorylation,
includes reporter
constructs or indicator genes which include transcriptional regulatory
elements responsive to the
STAT proteins. Described infra.
B Multicz~bunit Immune Recognition RecentOY (M
In another example the receptor is a multisubunit receptor. Receptors can be
comprised
of multiple proteins referred to as subunits, one category of which is
referred to as a multisubunit
receptor is a multisubunit immune recognition receptor (MIRR). MIRRs include
receptors having
multiple noncovalently associated subunits and are capable of interacting with
src-family
tyrosine kinases. MIRRs can include, but are not limited to, B cell antigen
receptors, T cell
antigen receptors, Fc receptors and CD22. One example of an MIRR is an antigen
receptor on
the surface of a B cell. To further illustrate, the MIRR on the surface of a B
cell comprises
membrane-bound immunoglobulin (mIg) associated with the subunits Ig-a and Ig-
or Ig-y,
which forms a complex capable of regulating B cell function when bound by
antigen. An antigen
receptor can be functionally linked to an amplifier molecule in a manner such
that the amplifier
molecule is capable of regulating gene transcription.
Src-family tyrosine kinases are enzymes capable of phosphorylating tyrosine
residues of
a target molecule. Typically, a src-family tyrosine kinase contains one or
more binding domains
and a kinase domain. A binding domain of a src-family tyrosine kinase is
capable of binding to
a target molecule and a kinase domain is capable of phosphorylating a target
molecule bound to
the kinase. Members of the src family of tyrosine kinases are characterized by
an N-terminal
unique region followed by three regions that contain different degrees of
homology among all
the members of the family. These three regions are referred to as src homology
region 1 (SHl),
src homology region 2 (SH2) and src homology region 3 (SH3). Both the SH2 and
SH3 domains
are believed to have protein association functions important for the formation
of signal
transduction complexes. The amino acid sequence of an N-terminal unique
region, varies
between each src-family tyrosine kinase. An N-terminal unique region can be at
least about the
first 40 amino acid residues of the N-terminal of a src-family tyrosine
kinase.
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Syk-family kinases are enzymes capable of phosphorylating tyrosine residues of
a target
molecule. Typically, a syk-family kinase contains one or more binding domains
and a kinase
domain. A binding domain of a syk-family tyrosine kinase is capable of binding
to a target
molecule and a kinase domain is capable of phosphorylating a target molecule
bound to the
kinase. Members of the syk family of tyrosine kinases are characterized by two
SH2 domains
for protein association function and a tyrosine kinase domain.
A primary target molecule is capable of further extending a signal
transduction pathway
by modifying a second messenger molecule. Primary target molecules can
include, but are not
limited to, phosphatidylinositol 3-kinase (PI-3K), P2lrasGAPase-activating
protein and
associated P190 and P62 protein, phospholipases such as PLCyI and PLC 2, MAP
kinase, Shc
and VAV. A primary target molecule is capable of producing second messenger
molecule which
is capable of further amplifying a transduced signal. Second messenger
molecules include, but
are not limited to diacylglycerol and inositol 1,4,5-triphosphate (IP3).
Second messenger
molecules are capable of initiating physiological events which can lead to
alterations in gene
transcription. For example, production of IP3 can result in release of
intracellular calcium, which
can then lead to activation of calmodulin kinase II, which can then lead to
serine phosphorylation
of a DNA binding protein referred to as ets-1 proto-onco-protein.
Diacylglycerol is capable of
activating the signal transduction protein, protein kinase C which affects the
activity of the AP 1
DNA binding protein complex. Signal transduction pathways can lead to
transcriptional
activation of genes such as c-fos, egr-1, and c-myc.
Shc can be thought of as an adaptor molecule. An adaptor molecule comprises a
protein
that enables two other proteins to form a complex (e.g., a three molecule
complex). Shc protein
enables a complex to form which includes Grb2 and SOS. Shc comprises an SH2
domain that
is capable of associating with the SH2 domain of Grb2.
Molecules of a signal transduction pathway can associate with one another
using
recognition sequences. Recognition sequences enable specific binding between
two molecules.
Recognition sequences can vary depending upon the structure of the molecules
that are
associating with one another. A molecule can have one or more recognition
sequences, and as
such can associate with one or more different molecules.
Signal transduction pathways for MIRR complexes are capable of regulating the
biological functions of a cell. Such functions can include, but are not
limited to the ability of a
cell to grow, to differentiate and to secrete cellular products. MIRR-induced
signal transduction
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pathways can regulate the biological functions of specific types of cells
involved in particular
responses by an animal, such as immune responses, inflammatory responses and
allergic
responses. Cells involved in an immune response can include, for example, B
cells, T cells,
macrophages, dendritic cells, natural killer cells and plasma cells. Cells
involved in
inflammatory responses can include, for example, basophils, mast cells,
eosinophils, neutrophils
and macrophages. Cells involved in allergic responses can include, for example
mast cells,
basophils, B cells, T cells and macrophages.
In certain examples, the detection signal is a second messenger, such as a
phosphorylated
src-like protein, including reporter constructs or indicator genes which
include transcriptional
regulatory elements such as serum response element (SRE), 12-O-tetradecanoyl-
phorbol-13-
acetate response element, cyclic AMP response element, c- fos promoter, or a
CREB-responsive
element.
C. Rece for >rosine kinases.
In still another example, the target receptor is a receptor tyrosine kinase.
The receptor
tyrosine kinases can be divided into five subgroups on the basis of structural
similarities in their
extracellular domains and the organization of the tyrosine kinase catalytic
region in their
cytoplasmic domains. Sub-groups I (epidermal growth factor (EGF) receptor-
like), II (insulin
receptor-like) and the eph/eck family contain cysteine-rich sequences (Hirai
et al., ( 1987) Science
238:1717-1720 and Lindberg and Hunter, (1990) Mol. Cell. Biol. 10:6316-6324).
The functional
domains of the kinase region of these three classes of receptor tyrosine
kinases are encoded as
a contiguous sequence ( Hanks et al. (1988) Science 241:42-52). Subgroups III
(platelet-derived
growth factor (PDGF) receptor-like) and IV (the fibro-blast growth factor
(FGF) receptors) are
characterized as having immunoglobulin (Ig)-like folds in their extracellular
domains, as well
as having their kinase domains divided in two parts by a variable stretch of
unrelated amino acids
(Yanden and Ullrich (1988) supra and Hanks et al. (1988) supra).
The family with by far the largest number of known members is the EPH family.
Since
the description of the prototype, the EPH receptor (Hirai et al. (1987)
Science 238:1717-1720),
sequences have been reported for at least ten members of this family, not
counting apparently
orthologous receptors found in more than one species. Additional partial
sequences, and the rate
at which new members are still being reported, suggest the family is even
larger (Maisonpierre
et al. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene 9:1461-1467;
Henkemeyer
et al. (1994) Oncogene 9:1001-1014; Ruiz et al. (1994) Mech Dev 46:87-100; Xu
et al. (1994)
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Development 120:287-299; Zhou et al. (1994) JNeurosci Res 37:129-143; and
references in Tuzi
and Gullick (1994) BrJCancer 69:417-421). Remarkably, despite the large number
of members
in the EPH family, all of these molecules were identified as orphan receptors
without known
ligands.
The expression patterns determined for some of the EPH family receptors have
implied
important roles for these molecules in early vertebrate development. In
particular, the timing and
pattern of expression of sek, mek4 and some of the other receptors during the
phase of
gastrulation and early organogenesis has suggested functions for these
receptors in the important
cellular interactions involved in patterning the embryo at this stage (Gilardi-
Hebenstreit et al.
(1992) Oncogene 7:2499-2506; Nieto et al. (1992) Development 116:1137-1150;
Henkemeyer
et al., supra; Ruiz et al., supra; and Xu et al., supra). Sek, for example,
shows a notable early
expression in the two areas of the mouse embryo that show obvious
segmentation, namely the
somites in the mesoderm and the rhombomeres of the hindbrain; hence the name
sek, for
segmentally expressed kinase (Gilardi-Hebenstreit et al., supra; Nieto et al.,
supra). As in
Drosophila, these segmental structures of the mammalian embryo are implicated
as important
elements in establishing the body plan. The observation that Sek expression
precedes the
appearance of morphological segmentation suggests a role for sek in forming
these segmental
structures, or in determining segment-specific cell properties such as lineage
compartmentation
(Nieto et al., supra). Moreover, EPH receptors have been implicated, by their
pattern of
expression, in the development and maintenance of nearly every tissue in the
embryonic and
adult body. For instance, EPH receptors have been detected throughout the
nervous system, the
testes, the cartilaginous model of the skeleton, tooth primordia, the
infundibular component of
the pituitary, various epithelial tissues, lung, pancreas, liver and kidney
tissues. Observations
such as this have been indicative of important and unique roles for EPH family
kinases in
development and physiology, but further progress in understanding their action
has been severely
limited by the lack of information on their ligands.
As used herein, the terms "EPH receptor" or "EPH-type receptor" refer to a
class of
receptor tyrosine kinases, comprising at least eleven paralogous genes, though
many more
orthologs exist within this class, e.g., homologs from different species. EPH
receptors, in
general, are a discrete group of receptors related by homology and easily
recognizable, for
example, they are typically characterized by an extracellular domain
containing a characteristic
spacing of cysteine residues near the N-terminus and two fibronectin type III
repeats (Hirai et
al. (1987) Science 238:1717-1720; Lindberg et al. (1990) Mol Cell Biol 10:6316-
6324; Chan et
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al. (1991) Oncogene 6:1057-1061; Maisonpierre et al. (1993) Oncogene 8:3277-
3288; Andres
et al. (1994) Oncogene 9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-
1014; Ruiz
et al. (1994) Mech Dev 46:87-100; Xu et al. (1994) Development 120:287-299;
Zhou et al. (1994)
JNeurosci Res 37:129-143; and references in Tuzi and Gullick (1994) BrJCancer
69:417-421).
Exemplary EPH receptors include the eph, elk, eck, sek, mek4, hek, hek2, eek,
erk, tyrol , tyro4,
tyros, tyro6, tyrol I , cek4, cek5, cek6, cek7, cek8, cek9, cekl0, bsk, rtkl ,
rtk2, rtk3, mykl , myk2,
ehkl, ehk2, pagliaccio, htk, erk and nuk receptors. The term "EPH receptor"
refers to the
membrane form of the receptor protein, as well as soluble extracellular
fragments which retain
the ability to bind the ligand of the present invention.
In certain examples, the detection signal is provided by detecting
phosphorylation of
intracellular proteins, e.g., MEKKs, MEKs, or Map kinases, or by the use of
reporter constructs
or indicator genes which include transcriptional regulatory elements
responsive to c-fos and/or
c jun. Described infra.
D. (T Protein-Coupled Rece t
One family of signal transduction cascades found in eukaryotic cells utilizes
heterotrimeric "G proteins." Many different G proteins are known to interact
with receptors. G
protein signaling systems include three components: the receptor itself, a GTP-
binding protein
(G protein), and an intracellular target protein.
The cell membrane acts as a switchboard. Messages arriving through different
receptors
can produce a single effect if the receptors act on the same type of G
protein. On the other hand,
signals activating a single receptor can produce more than one effect if the
receptor acts on
different kinds of G proteins, or if the G proteins can act on different
effectors.
In their resting state, the G proteins, which consist of alpha (a), beta ((3)
and gamma (y)
subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and
are in contact
with receptors. When a hormone or other first messenger binds to receptor, the
receptor changes
conformation and this alters its interaction with the G protein. This spurs
the a subunit to release
GDP, and the more abundant nucleotide guanosine triphosphate (GTP), replaces
it, activating
the G protein. The G protein then dissociates to separate the a subunit from
the still complexed
beta and gamma subunits. Either the Ga subunit, or the G(3y complex, depending
on the
pathway, interacts with an effector. The effector (which is often an enzyme)
in turn converts an
inactive precursor molecule into an active "second messenger," which may
diffuse through the
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CA 02376641 2002-O1-18
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cytoplasm, triggering a metabolic cascade. After a few seconds, the Goc
converts the GTP to
GDP, thereby inactivating itself. The inactivated Ga, may then reassociate
with the G(3y complex.
Hundreds, if not thousands, of receptors convey messages through
heterotrimeric G
proteins, of which at least 17 distinct forms have been isolated. Although the
greatest variability
has been seen in the a, subunit, several different (3 and y structures have
been reported. There are,
additionally, several different G protein-dependent effectors.
Most G protein-coupled receptors are comprised of a single protein chain that
is threaded
through the plasma membrane seven times. Such receptors are often referred to
as seven-
transmembrane receptors (STRs). More than a hundred different STRs have been
found,
including many distinct receptors that bind the same ligand, and there are
likely many more
STRs awaiting discovery.
In addition, STRs have been identified for which the natural ligands are
unknown; these
receptors are termed "orphan" G protein-coupled receptors, as described above.
Examples
include receptors cloned by Neote et al. (1993) Cell 72, 415; Kouba et al.
FEBSLett. (1993) 321,
173; Birkenbach et a1.(1993) J. Virol. 67, 2209.
The 'exogenous receptors' of this example may be any G protein-coupled
receptor which
is exogenous to the cell which is to be genetically engineered for the purpose
of the present
invention. This receptor may be a plant or animal cell receptor. Screening for
binding to plant
cell receptors may be useful in the development of, for example, herbicides.
In the case of an
animal receptor, it may be of invertebrate or vertebrate origin. If an
invertebrate receptor, an
insect receptor is preferred, and would facilitate development of
insecticides. The receptor may
also be a vertebrate, more preferably a mammalian, still more preferably a
human, receptor. The
exogenous receptor is also preferably a seven transmembrane segment receptor.
Known ligands for G protein coupled receptors include: purines and
nucleotides, such
as adenosine, cAMP, ATP, UTP, ADP, melatonin and the like; biogenic amines
(and related
natural ligands), such as S-hydroxytryptamine, acetylcholine, dopamine,
adrenaline, adrenaline,
adrenaline., histamine, noradrenaline, noradrenaline, noradrenaline.,
tyramine/octopamine and
other related compounds; peptides such as adrenocorticotrophic hormone (acth),
melanocyte
stimulating hormone (msh), melanocortins, neurotensin (nt), bombesin and
related peptides,
endothelins, cholecystokinin, gastrin, neurokinin b (nk3), invertebrate
tachykinin-like peptides,
substance k (nk2), substance p (nkl), neuropeptide y (npy), thyrotropin
releasing-factor (trf),
bradykinin, angiotensin ii, beta-endorphin, c5a anaphalatoxin, calcitonin,
chemokines (also
called intercrines), corticotrophic releasing factor (crf), dynorphin,
endorphin, fmlp and other
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formylated peptides, follitropin (fsh), fungal mating pheremones, galanin,
gastric inhibitory
polypeptide receptor (gip), glucagon-like peptides (glps), glucagon,
gonadotropin releasing
hormone (gnrh), growth hormone releasing hormone(ghrh), insect diuretic
hormone, interleukin-
8, leutropin (lh/hcg), met-enkephalin, opioid peptides, oxytocin, parathyroid
hormone (pth) and
pthrp, pituitary adenylyl cyclase activiating peptide (pacap), secretin,
somatostatin, thrombin,
thyrotropin (tsh), vasoactive intestinal peptide (vip), vasopressin,
vasotocin; eicosanoids such
as ip-prostacyclin, pg-prostaglandins, tx-thromboxanes; retinal based
compounds such as
vertebrate 11-cis retinal, invertebrate 11-cis retinal and other related
compounds; lipids and lipid-
based compounds such as cannabinoids, anandamide, lysophosphatidic acid,
platelet activating
factor, leukotrienes and the like; excitatory amino acids and ions such as
calcium ions and
glutamate.
Suitable examples of G-protein coupled receptors include, but are not limited
to,
dopaminergic, muscarinic cholinergic, a-adrenergic, b-adrenergic, opioid
(including delta and
mu), cannabinoid, serotoninergic, and GABAergic receptors. Preferred receptors
include the
SHT family of receptors, dopamine receptors, CSa receptor and FPRL-1 receptor,
cyclo-histidyl-
proline-diketoplperazine receptors, melanocyte stimulating hormone release
inhibiting factor
receptor, and receptors for neurotensin, thyrotropin releasing hormone,
calcitonin,
cholecytokinin-A, neurokinin-2, histamine-3, cannabinoid, melanocortin, or
adrenomodulin,
neuropeptide-Y1 or galanin. Other suitable receptors are listed in the art.
The term 'receptor,' as
used herein, encompasses both naturally occurnng and mutant receptors.
Many of these G protein-coupled receptors, like the yeast a- and a,-factor
receptors,
contain seven hydrophobic amino acid-rich regions which are assumed to lie
within the plasma
membrane. Specific human G protein-coupled STRs for which genes have been
isolated and for
which expression vectors could be constructed include those listed herein and
others known in
the art. Thus, the gene would be operably linked to a promoter functional in
the cell to be
engineered and to a signal sequence that also functions in the cell. For
example in the case of
yeast, suitable promoters include S~e2, and gallQ. Suitable signal sequences
include those
of ~2, 5~.~ and of other genes which encode proteins secreted by yeast cells.
Preferably, when
a yeast cell is used, the codons of the gene would be optimized for expression
in yeast. See
Hoekema et a1.,(1987) Mol. Cell. Biol., 7:2914-24; Sharp, et al.,
(1986)14:5125-43.
The homology of STRs is discussed in Dohlman et al., Ann. Rev. Biochem.,
(1991)
60:653-88. When STRs are compared, a distinct spatial pattern of homology is
discernible. The
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CA 02376641 2002-O1-18
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transmembrane domains are often the most similar, whereas the N- and C-
terminal regions, and
the cytoplasmic loop connecting transmembrane segments V and VI are more
divergent.
The functional significance of different STR regions has been studied by
introducing
point mutations (both substitutions and deletions) and by constructing
chimeras of different but
related STRs. Synthetic peptides corresponding to individual segments have
also been tested for
activity. Affinity labeling has been used to identify ligand binding sites.
It is conceivable that when the host cell is a yeast cell, a foreign receptor
will fail to
functionally integrate into the yeast membrane, and there interact with the
endogenous yeast G
protein. More likely, either the receptor will need to be modified (e.g., by
replacing its V-VI loop
with that of the yeast STE2 or STE3 receptor), or a compatible G protein
should be provided.
If the wild-type exogenous G protein-coupled receptor cannot be made
functional in
yeast, it may be mutated for this purpose. A comparison would be made of the
amino acid
sequences of the exogenous receptor and of the yeast receptors, and regions of
high and low
homology identified. Trial mutations would then be made to distinguish regions
involved in
ligand or G protein binding, from those necessary for functional integration
in the membrane.
The exogenous receptor would then be mutated in the latter region to more
closely resemble the
yeast receptor, until functional integration was achieved. If this were
insufficient to achieve
functionality, mutations would next be made in the regions involved in G
protein binding.
Mutations would be made in regions involved in ligand binding only as a last
resort, and then
an effort would be made to preserve ligand binding by making conservative
substitutions
whenever possible.
Preferably, the yeast genome is modified so that it is unable to produce the
yeast
receptors which are homologous to the exogenous receptors in functional form.
Otherwise, a
positive assay score might reflect the ability of a peptide to activate the
endogenous G protein-
coupled receptor, and not the receptor of interest.
(i). Chemoattractant receptors
The N-formyl peptide receptor is a classic example of a calcium mobilizing G
protein-
coupled receptor expressed by neutrophils and other phagocytic cells of the
mammalian immune
system (Snyderman et al. (1988) In Inflammation: Basic Principles and Clinical
Correlates, pp.
309-323). N-Formyl peptides of bacterial origin bind to the receptor and
engage a complex
activation program that results in directed cell movement, release of
inflammatory granule
contents, and activation of a latent NADPH oxidase which is important for the
production of
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CA 02376641 2002-O1-18
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metabolites of molecular oxygen. This pathway initiated by receptor-ligand
interaction is critical
in host protection from pyogenic infections. Similar signal transduction
occurs in response to
the inflammatory peptides CSa and IL-8.
Two other formyl peptide receptor like (FPRL) genes have been cloned based on
their
ability to hybridize to a fragment of the NFPR cDNA coding sequence. These
have been named
FPRL1 (Murphy et al. (1992) J. Biol Chem. 267:7637-7643) and FPRL2 (Ye et al.
(1992)
Biochem Biophys Res. Comm. 184:582-589). FPRL2 was found to mediate calcium
mobilization
in mouse fibroblasts transfected with the gene and exposed to formyl peptide.
In contrast,
although FPRL1 was found to be 69% identical in amino acid sequence to NFPR,
it did not bind
prototype N-formyl peptides ligands when expressed in heterologous cell types.
This lead to the
hypothesis of the existence of an as yet unidentified ligand for the FPRLl
orphan receptor
(Murphy et al. supra).
(ii.) G proteins
In the case of an exogenous Gprotein-coupled receptor, the yeast cell must be
able to
produce a G protein which is activated by the exogenous receptor, and which
can in turn activate
the yeast effector(s). The art suggests that the endogenous yeast Ga subunit
(e.g., GPA) will be
often be sufficiently homologous to the "cognate" Ga subunit which is natively
associated with
the exogenous receptor for coupling to occur. More likely, it will be
necessary to genetically
engineer the yeast cell to produce a foreign Ga subunit which can properly
interact with the
exogenous receptor. For example, the Ga subunit of the yeast G protein may be
replaced by the
Ga subunit natively associated with the exogenous receptor.
Dietzel and Kurjan, (1987) Cell, 50:1001) demonstrated that rat Gas
functionally coupled
to the yeast G(3y complex. However, rat Gai2 complemented only when
substantially
overexpressed, while Ga0 did not complement at all. Kang, et al., Mol. Cell.
Biol.,
( 1990) 10:2582). Consequently, with some foreign Ga subunits, it is not
feasible to simply
replace the yeast Ga.
If the exogenous G protein coupled receptor is not adequately coupled to yeast
G(3y by
the Ga subunit natively associated with the receptor, the Ga subunit may be
modified to
improve coupling. These modifications often will take the form of mutations
which increase the
resemblance of the Ga subunit to the yeast Ga while decreasing its resemblance
to the receptor-
associated Ga. For example, a residue may be changed so as to become identical
to the
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CA 02376641 2002-O1-18
WO 01/05826 PCT/US00/19689
corresponding yeast Ga residue, or to at least belong to the same exchange
group of that residue.
After modification, the modified Ga subunit might or might not be
"substantially homologous"
to the foreign and/or the yeast Ga subunit.
The modifications are preferably concentrated in regions of the Ga which are
likely to
be involved in G(3y binding. In some examples, the modifications will take the
form of replacing
one or more segments of the receptor-associated Ga with the corresponding
yeast Ga
segment(s), thereby forming a chimeric Ga subunit. (For the purpose of the
appended claims,
the term "segment" refers to three or more consecutive amino acids.) In other
examples, point
mutations may be sufficient.
This chimeric Ga subunit will interact with the exogenous receptor and the
yeast G(3y
complex, thereby permitting signal transduction. While use of the endogenous
yeast G(3y is
preferred, if a foreign or chimeric G(3y is capable of transducing the signal
to the yeast effector,
it may be used instead.
Although many of the techniques presented above require specific knowledge of
a
receptor active in a particular biological pathway, it will be recognized by
those skilled in the art
that such knowledge is not required for the screening of a library of chimeric
polypeptides of the
present invention. Rather, cell-based assays are well known in the art in
which cells of a selected
phenotype can be used to screen chimeric polypeptides for those which induce a
particular
alteration in the phenotype. In this way, chimeric polypeptides can be found
that have a desired
biological function that is not understood on a molecular level.
The invention now being generally described, it will be more readily
understood by
reference to the following examples which are included merely for purposes of
illustration of
certain aspects and embodiments of the present invention, and are not intended
to limit the
invention.
Serum albumin loop regions. A space-filling model of human serum albumin (HSA)
is shown in Figure 1. The tertiary structure of HSA reveals the presence of
ten approximate
helical regions or loops, each constrained by disulfide bonded cysteine pairs.
The space-filling
model was used to predict loop regions that are exposed on the surface of the
protein. Two amino
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CA 02376641 2002-O1-18
WO 01/05826 PCT/US00/19689
acid segments were chosen to represent surface exposed regions (loop 53-62 and
loop 360-369)
and a third to represent a region assumed to be buried within the protein
(loop 450-463).
Myc epitome display in MSA loop regions. In order to determine whether the
predicted
loops were indeed exposed on the surface of the albumin molecule, mouse serum
albumin
(MSA) was modified to include the myc epitope, EQKLISEEDL. The myc epitope was
inserted
in the middle of each of three amino acid segments: between amino acids 57-58
for loop 53-62,
amino acids 364-365 for loop 360-369 and amino acids 467-468 for loop 450-467.
Cos7 cells
were transfected with either wild type MSA or the various myc containing MSA
constructs. The
presence of the proteins in the medium was first determined by Western blot
analysis using
antibodies specific for MSA and the myc epitope. As can be seen in the left
half of Figure 2, only
samples from media from cells transfected with MSA or MSA-Myc reveal the
presence of the
albumin protein. Additionally, only the samples from cells transfected with
MSA-Myc are
positive for the myc epitope. As the samples are all denatured by virtue of
the SDS-PAGE
system, this analysis does not allow for the differentiation of myc epitopes
that would be exposed
on the surface versus one that was buried within the protein. For this
analysis
immunoprecipitation with the myc-specific antibody was utilized. In this
experiment, the
conditioned media was either mixed directly with the antibody (N, native) or
first denatured in
the presence of 0.1% SDS, 1 mM 13-mercapthoethanol and heat (100 °C for
10 min) and then
antibody added (D, denatured). Following immunoprecipitation the presence of
the proteins that
could be precipitated by the myc antibody were revealed by Western blot
analysis using the
MSA specific antibody. The right panel of Figure 2 shows that, as predicted,
the albumin
proteins with myc inserted in loops 53-62 and 360-369 were bound by the myc
antibody
regardless of whether the protein was in its native or denatured form. On the
other hand, when
myc was inserted in the predicted buried region, loop 450-463, the protein
only bound the
antibody when it was first denatured. This experiment clearly demonstrates
that loops 53-62 and
360-369 are exposed on the surface of the MSA protein and therefore good for
display.
Additionally, the 450-463 loop is buried.
Inhibition of bovine capillary endothelial cells (BCE) MSA-RGD. The goal of
this
experiment was determine the function of MSA with the RGD peptide (VRGDF) was
displayed
on the surface of the protein in the loop 53-58 region (MSA-myc-RGD). RGD was
chosen as this
peptide can efficiently bind to av(33 integrin receptors on endothelial cells
and inhibit their
proliferation. Triplicate wells of Cos7 cells were transfected with the
following constructs:
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CA 02376641 2002-O1-18
WO 01/05826 PCT/LTS00/19689
MSA-myc (the myc epitope was added to the C-terminal tail of MSA in this
iteration);
MSA-myc-RGD; or pAM7-stuffer. These Cos7 cells were grown in the lower chamber
of a
Transwell~ tissue culture plate with BCE cells in the upper chamber. To
stimulate growth of the
BCE cells, FGF was added to the lower chamber or not in the case of no FGF
control and the
cells allowed to grow for 72 hours. To one set of wells, those with pAM7-
stuffer, 6.25 ~M
c-RGD peptide was also added. Cell growth was determined by a Calcein-binding
fluorescence
assay. The left panel of Figure 3 is a graph of the optical density (OD) for
each. The data reveals
the addition of FGF results in a 2-fold stimulation of growth of the BCE
cells. This growth was
inhibited by the c-RGD peptide and also by the secreted MSA-myc-RGD protein.
The right panel
is a different way of looking at the same data. In this instance the degree of
inhibition of growth
is graphed for each. The data shows that the MSA-Myc-RGD protein inhibited the
growth of the
BCE cell by 53% and the degree of inhibition was equivalent to that of the
added RGD peptide.
The RGD peptide displayed on the surface of the MSA molecule inhibited BCE
cell growth as
efficiently as the endogenously added free RGD peptide demonstrating that the
peptide retains
its activity in the looped orientation.
The skilled arx'san will recognize many equivalents to the disclosed
invention, all of
which are intended to be within the scope of the present invention. All
articles, patents, and
applications cited above are incorporated herein by reference.
-35-

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A single figure which represents the drawing illustrating the invention.
Administrative Status

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Administrative Status

Title Date
Forecasted Issue Date Unavailable
(86) PCT Filing Date 2000-07-19
(87) PCT Publication Date 2001-01-25
(85) National Entry 2002-01-18
Dead Application 2005-07-19

Abandonment History

Abandonment Date Reason Reinstatement Date
2004-07-19 FAILURE TO PAY APPLICATION MAINTENANCE FEE

Payment History

Fee Type Anniversary Year Due Date Amount Paid Paid Date
Application Fee $300.00 2002-01-18
Maintenance Fee - Application - New Act 2 2002-07-19 $100.00 2002-07-03
Registration of a document - section 124 $100.00 2002-07-05
Registration of a document - section 124 $100.00 2002-07-05
Maintenance Fee - Application - New Act 3 2003-07-21 $100.00 2003-07-04
Owners on Record

Note: Records showing the ownership history in alphabetical order.

Current Owners on Record
GPC BIOTECH, INC.
Past Owners on Record
GYURIS, JENO
LAMPHERE, LOU
MITOTIX, INC.
MORRIS, AARON
Past Owners that do not appear in the "Owners on Record" listing will appear in other documentation within the application.
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Representative Drawing 2002-07-18 1 21
Description 2002-01-18 35 2,184
Cover Page 2002-07-19 1 57
Abstract 2002-01-18 1 70
Claims 2002-01-18 6 177
Drawings 2002-01-18 3 93
PCT 2002-01-18 5 481
Assignment 2002-01-18 3 90
Correspondence 2002-07-16 1 25
Assignment 2002-07-05 9 326
PCT 2002-01-19 6 264