U.S. patent application number 09/778885 was filed with the patent office on 2002-04-04 for antibodies to kunitz domain polypeptides.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Conklin, Darrell C., Foster, Donald C., Gao, Zeren.
Application Number | 20020039748 09/778885 |
Document ID | / |
Family ID | 23244869 |
Filed Date | 2002-04-04 |
United States Patent
Application |
20020039748 |
Kind Code |
A1 |
Conklin, Darrell C. ; et
al. |
April 4, 2002 |
Antibodies to kunitz domain polypeptides
Abstract
Proteinase inhibitors comprising a Kunitz domain are disclosed.
The Kunitz domain comprises a sequence of amino acid residues as
shown in SEQ ID NO: 5, wherein the sequence is at least 90%
identical to SEQ ID NO: 2. Also disclosed are methods for making
the proteinase inhibitors, and expression vectors and cultured
cells that are useful within the methods. The proteinase
inhibitors. may be used as components of cell culture media, in
protein purification, and in certain therapeutic and diagnostic
applications.
Inventors: |
Conklin, Darrell C.;
(Seattle, WA) ; Foster, Donald C.; (Lake Forest
Park, WA) ; Gao, Zeren; (Redmond, WA) |
Correspondence
Address: |
Gary E. Parker
Patent Department
ZymoGenetics, Inc.
1201 Eastlake Avenue East
Seattle
WA
98102
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
23244869 |
Appl. No.: |
09/778885 |
Filed: |
February 6, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09778885 |
Feb 6, 2001 |
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09523487 |
Mar 10, 2000 |
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6232098 |
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09523487 |
Mar 10, 2000 |
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09320095 |
May 26, 1999 |
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6087473 |
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60087032 |
May 28, 1998 |
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Current U.S.
Class: |
435/7.1 ;
435/184; 435/189; 435/320.1; 435/325; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C07K 14/8114 20130101 |
Class at
Publication: |
435/7.1 ;
435/189; 435/184; 435/325; 435/320.1; 536/23.2 |
International
Class: |
G01N 033/53; C07H
021/04; C12N 009/99; C12N 009/02 |
Claims
We claim:
1. An isolated protein comprising a sequence of amino acid residues
as shown in SEQ ID NO: 5, wherein said sequence is at least 90%
identical to residues 9 through 59 of SEQ ID NO: 2 and wherein said
protein has proteinase inhibiting activity.
2. The isolated protein of claim 1 wherein said protein is from 51
to 81 amino acid residues in length.
3. The isolated protein of claim 1 wherein said protein is from 51
to 67 residues in length.
4. The isolated protein of claim 1 wherein said sequence is
selected from the group consisting of: (a) residues 9 through 59 of
SEQ ID NO: 2; and (b) residues 9 through 59 of SEQ ID NO: 4.
5. The isolated protein of claim 4 wherein said protein is from 51
to 67 residues in length.
6. The isolated protein of claim 1 consisting of from 51-557
contiguous amino acid residues of SEQ ID NO: 10.
7. The isolated protein of claim 1 further comprising an affinity
tag.
8. The isolated protein of claim 7 wherein said affinity tag is
maltose binding protein, polyhistidine, or Glu-Tyr-Met-Pro-Met-Glu
(SEQ ID NO: 18).
9. An expression vector comprising the following operably linked
elements: (a) a transcription promoter; (b) a DNA segment encoding
a protein comprising a sequence of amino acid residues as shown in
SEQ ID NO: 5, wherein said sequence of amino acid residues is at
least 90% identical to residues 9 through 59 of SEQ ID NO: 2 and
wherein said protein has proteinase inhibiting activity; and (c) a
transcription terminator.
10. The expression vector of claim 9 wherein said protein is from
51 to 81 amino acid residues in length.
11. The expression vector of claim 9 further comprising a secretory
signal sequence operably linked to the DNA segment.
12. The expression vector of claim 9 wherein said sequence of amino
acid residues is selected from the group consisting of: (a)
residues 9 through 59 of SEQ ID NO: 2; and (b) residues 9 through
59 of SEQ ID NO: 4.
13. The expression vector of claim 12 wherein said protein is from
51 to 67 residues in length.
14. The expression vector of claim 9 wherein said protein is from
51 to 67 residues in length.
15. The expression vector of claim 9 wherein said DNA segment
further encodes an affinity tag.
16. The expression vector of claim 15 wherein said affinity tag is
maltose binding protein, polyhistidine, or Glu-Tyr-Met-Pro-Met-Glu
(SEQ ID NO: 18).
17. A cultured cell containing an expression vector according to
claim 9, wherein said cell expresses the DNA segment.
18. A method of making a protein having proteinase inhibiting
activity comprising: culturing the cell of claim 17 under
conditions whereby said DNA segment is expressed; and recovering
the protein encoded by the DNA segment.
19. An antibody that specifically binds to a protein as shown in
SEQ ID NO: 2 or SEQ ID NO: 4.
Description
BACKGROUND OF THE INVENTION
[0001] In animals, proteinases are important in wound healing,
extracellular matrix destruction, tissue reorganization, and in
cascades leading to blood coagulation, fibrinolysis, and complement
activation. Proteinases are released by inflammatory cells for
destruction of pathogens or foreign materials, and by normal and
cancerous cells as they move through their surroundings.
[0002] The activity of proteinases is regulated by inhibitors; 10%
of the proteins in blood serum are proteinase inhibitors (Roberts
et al., Critical Reviews in Eukaryotic Gene Expression 5:385-436,
1995). One family of proteinase inhibitors, the Kunitz inhibitors,
includes inhibitors of trypsin, chymotrypsin, elastase, kallikrein,
plasmin, coagulation factors XIa and IXa, and cathepsin G. These
inhibitors thus regulate a variety of physiological processes,
including blood coagulation, fibrinolysis, and inflammation.
[0003] Proteinase inhibitors regulate the proteolytic activity of
target proteinases by occupying the active site and thereby
preventing occupation by normal substrates. Although proteinase
inhibitors fall into several unrelated structural classes, they all
possess an exposed loop (variously termed an "inhibitor loop", a
"reactive core", a "reactive site", or a "binding loop") which is
stabilized by intermolecular interactions between residues flanking
the binding loop and the protein core (Bode and Huber, Eur. J.
Biochem. 204:433-451, 1992). Interaction between inhibitor and
enzyme produces a stable complex which disassociates very slowly,
releasing either virgin (uncleaved) inhibitor, or a modified
inhibitor that is cleaved at the scissile bond of the binding
loop.
[0004] One class of proteinase inhibitors, the Kunitz inhibitors,
are generally basic, low molecular weight proteins comprising one
or more inhibitory domains ("Kunitz domains"). The Kunitz domain is
a folding domain of approximately 50-60 residues which forms a
central anti-parallel beta sheet and a short C-terminal helix. This
characteristic domain comprises six cysteine residues that form
three disulfide bonds, resulting in a double-loop structure.
Between the N-terminal region and the first beta strand resides the
active inhibitory binding loop. This binding loop is disulfide
bonded through the P2 Cys residue to the hairpin loop formed
between the last two beta strands. Isolated Kunitz domains from a
variety of proteinase inhibitors have been shown to have inhibitory
activity (e.g., Petersen et al., Eur. J. Biochem. 125:310-316,
1996; Wagner et al., Biochem. Biophys. Res. Comm. 186:1138-1145,
1992; Dennis et al., J. Biol. Chem. 270:25411-25417, 1995).
[0005] Proteinase inhibitors comprising one or more Kunitz domains
include tissue factor pathway inhibitor (TFPI), tissue factor
pathway inhibitor 2 (TFPI-2), amyloid .beta.-protein precursor
(A.beta.PP), aprotinin, and placental bikunin. TFPI, an extrinsic
pathway inhibitor and a natural anticoagulant, contains three
tandemly linked Kunitz inhibitor domains. The amino-terminal Kunitz
domain inhibits factor VIIa, plasmin, and cathepsin G; the second
domain inhibits factor Xa, trypsin, and chymotrypsin; and the third
domain has no known activity (Petersen et al., ibid.) . TFPI-2 has
been shown to be an inhibitor of the amidolytic and proteolytic
activities of human factor VIIa-tissue factor complex, factor XIa,
plasma kallikrein, and plasmin (Sprecher et al., Proc. Natl. Acad.
Sci. USA 91:3353-3357, 1994; Petersen et al., Biochem. 35:266-272,
1996). The ability of TFPI-2 to inhibit the factor VIIa-tissue
factor complex and its relatively high levels of transcription in
umbilical vein endothelial cells, placenta and liver suggests a
specialized role for this protein in hemostasis (Sprecher et al.,
ibid.). Aprotinin (bovine pancreatic trypsin inhibitor) is a broad
spectrum Kunitz-type serine proteinase inhibitor that has been
shown to prevent activation of the clotting cascade. Aprotinin is a
moderate inhibitor of plasma kallikrein and plamin, and blockage of
fibrinolysis and extracorporeal coagulation have been detected in
patients given aprotinin during open heart surgery (Davis and
Whittington, Drugs 49:954-983, 1995; Dietrich et al., Thorac.
Cardiovasc. Surg. 37:92-98, 1989). Aprotinin has also been used in
the treatment of septic shock, adult respiratory distress syndrome,
acute pancreatitis, hemorrhagic shock, and other conditions
(Westaby, Ann. Thorac. Surg. 55:1033-1041, 1993; Wachtfogel et al.,
J. Thorac. Cardiovasc. Surg. 106:1-10, 1993). The clinical utility
of aprotinin is believed to arise from its inhibitory activity
towards plasma kallikrein or plasmin (Dennis et al., ibid.).
Placental bikunin is a serine proteinase inhibitor containing two
Kunitz domains (Delaria et al., J. Biol. Chem. 272:12209-12214,
1997). Individual Kunitz domains of bikunin have been expressed and
shown to be potent inhibitors of trypsin, chymotrypsin, plasmin,
factor XIa, and tissue and plasma kallikrein (Delaria et al.,
ibid.).
[0006] Known Kunitz-type inhibitors lack specificity and may have
low potency. Lack of specificity can result in undesirable side
effects, such as nephrotoxicity that occurs after repeated
injections of high doses of aprotinin. These limitations may be
overcome by preparing isolated Kunitz domains, which may have fewer
side effects than traditional anticoagulants. Hence, there is a
need in the art for additional Kunitz-type proteinase
inhibitors.
SUMMARY OF THE INVENTION
[0007] It is an object of the present invention to provide novel
Kunitz inhibitor proteins and compositions comprising the proteins.
It is another object of the invention to provide materials and
methods for making the Kunitz inhibitor proteins. It is a further
object of the invention to provide antibodies that specifically
bind to the Kunitz inhibitor proteins.
[0008] Within one aspect, the invention provides an isolated
protein comprising a sequence of amino acid residues as shown in
SEQ ID NO: 5, wherein the sequence is at least 90% identical to
residues 9 through 59 of SEQ ID NO: 2 and wherein the protein has
proteinase inhibiting activity. within one embodiment, the protein
is from 51 to 81 amino acid residues in length. Within other
embodiments, the protein is from 51 to 67 residues in length,
preferably from 55 to 62 residues in length. Within another
embodiment, the sequence is selected from the group consisting of
residues 9 through 59 of SEQ ID NO: 2 and residues 9 through 59 of
SEQ ID NO: 4. Within a further embodiment, the protein consists of
from 51-557 contiguous amino acid residues of SEQ ID NO: 10. Within
an additional embodiment, the protein further comprises an affinity
tag. Suitable affinity tags include maltose binding protein,
polyhistidine, and Glu-Tyr-Met-Pro-Met-Glu (SEQ ID NO: 18).
[0009] Within a second aspect, the invention provides an expression
vector comprising the following operably linked elements: (a) a
transcription promoter; (b) a DNA segment encoding a protein as
disclosed above; and (c) a transcription terminator. Within one
embodiment, the expression vector further comprises a secretory
signal sequence operably linked to the DNA segment.
[0010] Within a third aspect, the invention provides a cultured
cell containing an expression vector as disclosed above, wherein
the cell expresses the DNA segment.
[0011] Within a fourth aspect of the invention there is provided a
method of making a protein having proteinase inhibiting activity
comprising culturing a cell as disclosed above under conditions
whereby the DNA segment is expressed, and recovering the protein
encoded by the DNA segment.
[0012] Within a fifth aspect of the invention there is provided an
antibody that specifically binds to a protein as shown in SEQ ID
NO: 2 or SEQ ID NO: 4.
[0013] These and other aspects of the invention will become evident
upon reference to the following detailed description and the
attached drawing.
BRIEF DESCRIPTION OF THE DRAWING
[0014] The Figure shows an amino acid sequence alignment of a
representative polypeptide of the present invention (SEQ ID NO: 2),
designated "ZKUN5", with the sequence of the Kunitz domain of human
alpha 3 type VI collagen (SEQ ID NO: 8), designated "1KNT".
DETAILED DESCRIPTION OF THE INVENTION
[0015] Prior to setting forth the invention in detail, it may be
helpful to the understanding thereof to define the following
terms:
[0016] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification of the second polypeptide or provide sites
for attachment of the second polypeptide to a substrate. In
principal, any peptide or protein for which an antibody or other
specific binding agent is available can be used as an affinity tag.
Affinity tags include a poly-histidine tract, protein A (Nilsson et
al., EMBO J. 4:1075, 1985; Nilsson et al., Methods Enzymol. 198:3,
1991), glutathione S transferase (Smith and Johnson, Gene 67:31,
1988), Glu-Glu affinity tag (Grussenmeyer et al., Proc. Natl. Acad.
Sci. USA 82:7952-4, 1985) (SEQ ID NO: 18), substance P, Flag.TM.
peptide (Hopp et al., Biotechnology 6:1204-1210, 1988),
streptavidin binding peptide, maltose binding protein (Guan et al.,
Gene 67:21-30, 1987), cellulose binding protein, thioredoxin,
ubiquitin, T7 polymerase, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2:95-107, 1991. DNAs encoding affinity tags and other
reagents are available from commercial suppliers (e.g., Pharmacia
Biotech, Piscataway, N.J.; New England Biolabs, Beverly, Mass.;
Eastman Kodak, New Haven, Conn.).
[0017] The term "allelic variant" is used herein to denote any of
two or more alternative forms of a gene occupying the same
chromosomal locus. Allelic variation arises naturally through
mutation, and may result in phenotypic polymorphism within
populations. Gene mutations can be silent (no change in the encoded
polypeptide) or may encode polypeptides having altered amino acid
sequence. The term allelic variant is also used herein to denote a
protein encoded by an allelic variant of a gene.
[0018] The terms "amino-terminal" and "carboxyl-terminal" are used
herein to denote positions within polypeptides. Where the context
allows, these terms are used with reference to a particular
sequence or portion of a polypeptide to denote proximity or
relative position. For example, a certain sequence positioned
carboxyl-terminal to a reference sequence within a polypeptide is
located proximal to the carboxyl terminus of the reference
sequence, but is not necessarily at the carboxyl terminus of the
complete polypeptide.
[0019] A "complement" of a polynucleotide molecule is a
polynucleotide molecule having a complementary base sequence and
reverse orientation as compared to a reference sequence. For
example, the sequence 5' ATGCACGGG 3' is complementary to 5.degree.
CCCGTGCAT 3'.
[0020] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons (as
compared to a reference polynucleotide molecule that encodes a
polypeptide). Degenerate codons contain different triplets of
nucleotides, but encode the same amino acid residue (i.e., GAU and
GAC triplets each encode Asp).
[0021] A "DNA segment" is a portion of a larger DNA molecule having
specified attributes. For example, a DNA segment encoding a
specified polypeptide is a portion of a longer DNA molecule, such
as a plasmid or plasmid fragment, that, when read from the 5' to
the 3' direction, encodes the sequence of amino acids of the
specified polypeptide.
[0022] The term "expression vector" is used to denote a DNA
molecule, linear or circular, that comprises a segment encoding a
polypeptide of interest operably linked to additional segments that
provide for its transcription. Such additional segments include
promoter and terminator sequences, and may also include one or more
origins of replication, one or more selectable markers, an
enhancer, a polyadenylation signal, etc. Expression vectors are
generally derived from plasmid or viral DNA, or may contain
elements of both.
[0023] The term "isolated", when applied to a polynucleotide,
denotes that the polynucleotide has been removed from its natural
genetic milieu and is thus free of other extraneous or unwanted
coding sequences, and is in a form suitable for use within
genetically engineered protein production systems. Such isolated
molecules are those that are separated from their natural
environment and include cDNA and genomic clones. Isolated DNA
molecules of the present invention are free of other genes with
which they are ordinarily associated, but may include naturally
occurring 5' and 3' untranslated regions such as promoters and
terminators. The identification of associated regions will be
evident to one of ordinary skill in the art (see for example, Dynan
and Tijan, Nature 316:774-78, 1985).
[0024] An "isolated" polypeptide or protein is a polypeptide or
protein that is found in a condition other than its native
environment, such as apart from blood and animal tissue. In a
preferred form, the isolated polypeptide is substantially free of
other polypeptides, particularly other polypeptides of animal
origin. It is preferred to provide the polypeptides in a highly
purified form, i.e. greater than 95% pure, more preferably greater
than 99% pure. When used in this context, the term "isolated" does
not exclude the presence of the same polypeptide in alternative
physical forms, such as dimers or alternatively glycosylated or
derivatized forms.
[0025] The term "operably linked", when referring to DNA segments,
indicates that the segments are arranged so that they function in
concert for their intended purposes, e.g., transcription initiates
in the promoter and proceeds through the coding segment to the
terminator.
[0026] The term "ortholog" denotes a polypeptide or protein
obtained from one species that is the functional counterpart of a
polypeptide or protein from a different species. Sequence
differences among orthologs are the result of speciation.
[0027] A "polynucleotide" is a single-or double-stranded polymer of
deoxyribonucleotide or ribonucleotide bases read from the 5' to the
3' end. Polynucleotides include RNA and DNA, and may be isolated
from natural sources, synthesized in vitro, or prepared from a
combination of natural and synthetic molecules. Sizes of
polynucleotides are expressed as base pairs (abbreviated "bp"),
nucleotides ("nt"), or kilobases ("kb"). Where the context allows,
the latter two terms may describe polynucleotides that are
single-stranded or double-stranded. When these terms are applied to
double-stranded molecules they are used to denote overall length
and will be understood to be equivalent to the term "base pairs".
It will be recognized by those skilled in the art that the two
strands of a double-stranded polynucleotide may differ slightly in
length and that the ends thereof may be staggered as a result of
enzymatic cleavage; thus all nucleotides within a double-stranded
polynucleotide molecule may not be paired. Such unpaired ends will
in general not exceed 20 nt in length.
[0028] A "polypeptide" is a polymer of amino acid residues joined
by peptide bonds, whether produced naturally or synthetically.
Polypeptides of less than about 10 amino acid residues are commonly
referred to as "peptides".
[0029] The term "promoter" is used herein for its art-recognized
meaning to denote a portion of a gene containing DNA sequences that
provide for the binding of RNA polymerase and initiation of
transcription. Promoter sequences are commonly, but not always,
found in the 5' non-coding regions of genes.
[0030] A "protein" is a macromolecule comprising one or more
polypeptide chains. A protein may also comprise non-peptidic
components, such as carbohydrate groups. Carbohydrates and other
non-peptidic substituents may be added to a protein by the cell in
which the protein is produced, and will vary with the type of cell.
Proteins are defined herein in terms of their amino acid backbone
structures; substituents such as carbohydrate groups are generally
not specified, but may be present nonetheless.
[0031] The term "secretory signal sequence" denotes a DNA sequence
that encodes a polypeptide (a "secretory peptide") that, as a
component of a larger polypeptide, directs the larger polypeptide
through a secretory pathway of a cell in which it is synthesized.
The larger polypeptide is commonly cleaved to remove the secretory
peptide during transit through the secretory pathway.
[0032] The term "splice variant" is used herein to denote
alternative forms of RNA transcribed from a gene. Splice variation
arises naturally through use of alternative splicing sites within a
transcribed RNA molecule, or less commonly between separately
transcribed RNA molecules, and may result in several mRNAs
transcribed from the same gene. Splice variants may encode
polypeptides having altered amino acid sequence. The term splice
variant is also used herein to denote a protein encoded by a splice
variant of an mRNA transcribed from a gene.
[0033] Molecular weights and lengths of polymers determined by
imprecise analytical methods (e.g., gel electrophoresis) will be
understood to be approximate values. When such a value is expressed
as "about" X or l"approximately" X, the stated value of X will be
understood to be accurate to .+-.10%.
[0034] All references cited herein are incorporated by reference in
their entirety.
[0035] The present invention provides, in part, novel serine
proteinases comprising a Kunitz domain. This Kunitz domain,
including sequence variants thereof and proteins containing it, is
referred to herein as "zkun5". The zkun5 polypeptide sequence shown
in SEQ ID NO: 2 comprises this Kunitz domain, which is bounded at
the amino and carboxyl termini by cysteine residues at positions 9
and 59, respectively.
[0036] Zkun5 has 50% residue identity with the kunitz domain in
human alpha 3 type VI collagen (shown in SEQ ID NO: 8). The
structure of the latter domain has been solved by X-ray
crystallography and by NMR (Arnoux et al., J. Mol. Biol.
246:609-617, 1995; Sorensen et al., Biochemistry 36:10439-10450,
1997). An alignment of zkun5 and the collagen Kunitz domain (see
Figure) can be combined with a homology model of zkun5 based on the
X-ray structure to predict the function of certain residues in
zkun5. Referring to SEQ ID NO: 2, disulfide bonds are predicted to
be formed by paired cysteine residues Cys9-Cys59; Cys18-Cys42; and
Cys34-Cys55. The protease binding loop (P3-P4') is expected to
comprise residues 17-23 of SEQ ID NO: 2
(Asn-Cys-Gly-Glu-Tyr-Val-Val), with the P1 residue being Gly19, and
the P1' residue being Glu20. Zkun5 further comprises a potential
glycosylation site at position Asn43 of SEQ ID NO: 2.
[0037] The kunitz domain of human alpha 3 type VI collagen is not
known to have antiproteinase activity. Suspected reasons for this
include unfavorable steric hindrances with trypsin involving the
residue Aspl15 of collagen (corresponding to Glu20 in zkun5). From
these data it is predicted that a Glu20Ala mutant of zkun5 may show
increased antiproteinase activity. The present invention thus
contemplates zkun5 proteins wherein residue 20 of SEQ ID NO: 2 is
replaced with Ala as shown in SEQ ID NO: 4.
[0038] Additional amino acid substitions can be made within the
zkun5 sequence so long as the conserved cysteine residues are
retained and the higher order structure is not disrupted. It is
preferred to make substitutions within the zkun5 Kunitz domain by
reference to the sequences of other Kunitz domains. SEQ ID NO: 5 is
a generalized Kunitz domain sequence that shows allowable amino
acid substitutions based on such an alignment. The 51-residue
sequence shown in SEQ ID NO: 5 conforms to the pattern:
C-X(8)-C-X(15)-C-X(7)-C-X(12)-C-X(3)-C
[0039] wherein C denotes cysteine; X is any naturally occuring
amino acid residue, subject to the limitations set forth in the
attached Sequence Listing for SEQ ID NO: 5; and the numerals
indicate the number of such variable residues. The second cysteine
residue is in the P2 position.
[0040] Within the present invention up to 10% of the amino acid
residues in the zkun5 Kunitz domain (residues 9 through 59 of SEQ
ID NO: 2) can be replaced with other amino acid residues, subject
to the limitation that the resulting substituted sequence is one of
the sequences disclosed in SEQ ID NO: 5. The present invention thus
provides a family of proteins comprising a sequence of amino acid
residues as shown in SEQ ID NO: 5, wherein the sequence is at least
90% identical to residues 9 through 59 of SEQ ID NO: 2. It is
preferred that the proteins of the present invention comprise such
a sequence that is at least 95% identical to residues 9 through 59
of SEQ ID NO: 2.
[0041] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio.
48:603-616, 1986, and Henikoff and Henikoff, Proc. Natl. Acad. Sci.
USA 89:10915-10919, 1992. Briefly, two amino acid sequences are
aligned to optimize the alignment scores using a gap opening
penalty of 10, a gap extension penalty of 1, and the "BLOSUM62"
scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 2
(amino acids are indicated by the standard one-letter codes). The
percent identity is then calculated as: 1 Total number of identical
matches [length of the longer sequence plus the number of gaps
introduced into the longersequence in order to align the
twosequences] .times. 100
1 TABLE 2 A R N D C Q E G H I L K M F P S T W Y V A 4 R -1 5 N -2 0
6 D -2 -2 1 6 C 0 -3 -3 -3 9 Q -1 1 0 0 -3 5 E -1 0 0 2 -4 2 5 G 0
-2 0 -1 -3 -2 -2 6 H -2 0 1 -1 -3 0 0 -2 8 I -1 -3 -3 -3 -1 -3 -3
-4 -3 4 L -1 -2 -3 -4 -1 -2 -3 -4 -3 2 4 K -1 2 0 -1 -3 1 1 -2 -1
-3 -2 5 M -1 -1 -2 -3 -1 0 -2 -3 -2 1 2 -1 5 F -2 -3 -3 -3 -2 -3 -3
-3 -1 0 0 -3 0 6 P -1 -2 -2 -1 -3 -1 -1 -2 -2 -3 -3 -1 -2 -4 7 S 1
-1 1 0 -1 0 0 0 -1 -2 -2 0 -1 -2 -1 4 T 0 -1 0 -1 -1 -1 -1 -2 -2 -1
-1 -1 -1 -2 -1 1 5 W -3 -3 -4 -4 -2 -2 -3 -2 -2 -3 -2 -3 -1 1 -4 -3
-2 11 Y -2 -2 -2 -3 -2 -1 -2 -3 2 -1 -1 -2 -1 3 -3 -2 -2 2 7 V 0 -3
-3 -3 -1 -2 -2 -3 -3 3 1 -2 1 -1 -2 -2 0 -3 -1 4
[0042] The level of identity between amino acid sequences can be
determined using the "FASTA" similarity search algorithm of Pearson
and Lipman (Proc. Natl. Acad. Sci. USA 85:2444, 1988) and Pearson
(Meth. Enzymol. 183:63, 1990). Briefly, FASTA first characterizes
sequence similarity by identifying regions shared by the query
sequence (e.g., SEQ ID NO: 2) and a test sequence that have either
the highest density of identities (if the ktup variable is 1) or
pairs of identities (if ktup=2), without considering conservative
amino acid substitutions, insertions, or deletions. The ten regions
with the highest density of identities are then rescored by
comparing the similarity of all paired amino acids using an amino
acid substitution matrix, and the ends of the regions are "trimmed"
to include only those residues that contribute to the highest
score. If there are several regions with scores greater than the
"cutoff" value (calculated by a predetermined formula based upon
the length of the sequence and the ktup value), then the trimmed
initial regions are examined to determine whether the regions can
be joined to form an approximate alignment with gaps. Finally, the
highest scoring regions of the two amino acid sequences are aligned
using a modification of the Needleman-Wunsch-Sellers algorithm
(Needleman and Wunsch, J. Mol. Biol. 48:444, 1970; Sellers, SIAM J.
Appl. Math. 26:787, 1974), which allows for amino acid insertions
and deletions. Illustrative parameters for FASTA analysis are:
ktup=1, gap opening penalty=10, gap extension penalty=1, and
substitution matrix=BLOSUM62. These parameters can be introduced
into a FASTA program by modifying the scoring matrix file
("SMATRIX"), as explained in Appendix 2 of Pearson, 1990
(ibid.).
[0043] FASTA can also be used to determine the sequence identity of
nucleic acid molecules using a ratio as disclosed above. For
nucleotide sequence comparisons, the ktup value can range between
one to six, preferably from four to six.
[0044] The proteins of the present invention can also comprise
non-naturally occurring amino acid residues. Non-naturally
occurring amino acids include, without limitation,
trans-3-methylproline, 2,4-methanoproline, cis-4-hydroxyproline,
trans-4-hydroxyproline, N-methylglycine, allo-threonine,
methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine,
nitroglutamine, homoglutamine, pipecolic acid, thiazolidine
carboxylic acid, dehydroproline, 3-and 4-methylproline,
3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine,
3-azaphenylalanine, 4-azaphenylalanine, and 4-fluorophenylalanine.
Several methods are known in the art for incorporating
non-naturally occurring amino acid residues into proteins. For
example, an in vi tro system can be employed wherein nonsense
mutations are suppressed using chemically aminoacylated suppressor
tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA
are-known in the art. Transcription and translation of plasmids
containing nonsense mutations is carried out in a cell-free system
comprising an E. coli S30 extract and commercially available
enzymes and other reagents. Proteins are purified by
chromatography. See, for example, Robertson et al., J. Am. Chem.
Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991;
Chung et al., Science 259:806-9, 1993; and Chung et al., Proc.
Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method,
translation is carried out in Xenopus oocytes by microinjection of
mutated MRNA and chemically aminoacylated suppressor tRNAs
(Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third
method, E. coli cells are cultured in the absence of a natural
amino acid that is to be replaced (e.g., phenylalanine) and in the
presence of the desired non-naturally occurring amino acid(s)
(e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine,
or 4-fluorophenylalanine) . The non-naturally occurring amino acid
is incorporated into the protein in place of its natural
counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally
occurring amino acid residues can be converted to non-naturally
occurring species by in vitro chemical modification. Chemical
modification can be combined with site-directed mutagenesis to
further expand the range of substitutions (Wynn and Richards,
Protein Sci. 2:395-403, 1993).
[0045] Additional polypeptides may be joined to the amino and/or
carboxyl termini of the zkun5 Kunitz domain (residues 9-59 of SEQ
ID NO: 2) or a derivative of the zkun5 Kunitz domain as disclosed
above. Within one embodiment, the extensions are those shown in SEQ
ID NO: 10, wherein the zkun5 Kunitz domain is located at residues
504-554. Particularly preferred proteins in this regard include
residues 1-62 of SEQ ID NO: 2 or SEQ ID NO: 4. Amino and carboxyl
extensions of the zkun5 Kunitz domain will be selected so as not to
destroy or mask the proteinase-inhibiting activity of the protein
by, for example, burying the Kunitz domain within the interior of
the protein. There is a consequent preference for shorter
extensions, typically 10-15 residues in length, preferably not
exceeding 8 residues in length. There is considerable latitude in
the permissible sequence of these extensions, although it is
preferred to avoid the addition of cysteine residues in close
proximity to the the Kunitz domain itself. For example, a zkun5
protein can comprise residues 9-59 of SEQ ID NO: 2 or SEQ ID NO: 4
with amino-and carboxyl-terminal dipeptides, wherein the individual
amino acid residues of the dipeptides are any amino acid residue
except cysteine.
[0046] Other amino-and carboxyl-terminal extensions that can be
included in the proteins of the present invention include, for
example, an amino-terminal methionine residue, a small linker
peptide of up to about 20-25 residues, or an affinity tag as
disclosed above. A protein comprising such an extension may further
comprise a polypeptide linker and/or a proteolytic cleavage site
between the zkun5 portion and the affinity tag. Preferred cleavage
sites include thrombin cleavage sites and factor Xa cleavage sites.
For example, the zkun5 protein shown in SEQ ID NO: 2 may be
expressed as a fusion comprising, from amino terminus to carboxyl
terminus: maltose binding protein-polyhistidine-thrombin cleavage
site (Leu-Val-Pro-Arg; SEQ ID NO: 11)-SEQ ID NO: 2. Linker peptides
and affinity tags provide for additional functions, such as binding
to substrates, antibodies, binding proteins, and the like, and
facilitate purification, detection, and delivery of zkun5 proteins.
In another example, a zkun5 Kunitz domain can be expressed as a
secreted protein comprising a carboxyl-terminal receptor
transmembrane domain, permitting the Kunitz domain to be displayed
on the surface of a cell. To span the lipid bilayer of the cell
membrane, a minimum of about 20 amino acids are required in the
transmembrane domain; these should predominantly be hydrophobic
amino acids. The Kunitz domain can be separated from the
transmembrane domain by a spacer polypeptide, and can be contained
within an extended polypeptide comprising a carboxyl-terminal
transmembrane domain--spacer polypeptide--Kunitz
domain--amino-terminal polypeptide. Many receptor transmembrane
domains and polynucleotides encoding them are known in the art. The
spacer polypeptide will generally be at least about 50 amino acid
residues in length, up to 200-300 or more residues. The amino
terminal polypeptide may be up to 300 or more residues in
length.
[0047] The present invention further provides polynucleotide
molecules, including DNA and RNA molecules, encoding zkun5
proteins. The polynucleotides of the present invention include the
sense strand; the anti-sense strand; and the DNA as
double-stranded, having both the sense and anti-sense strand
annealed together by their respective hydrogen bonds.
Representative DNA sequences encoding zkun5 proteins are set forth
in SEQ IDNO: 1, SEQ ID NO: 3, and SEQ ID NO: 9. DNA sequences
encoding other zkun5 proteins can be readily generated by those of
ordinary skill in the art based on the genetic code. Counterpart
RNA sequences can be generated by substitution of U for T.
[0048] Those skilled in the art will readily recognize that, in
view of the degeneracy of the genetic code, considerable sequence
variation is possible among these polynucleotide molecules. SEQ ID
NO: 6 is a degenerate DNA sequence that encompasses all DNAs that
encode the zkun5 polypeptide of SEQ ID NO: 2. SEQ ID NO: 7 is a
degenerate DNA sequence that encompasses all DNAs that encode the
zkun5 polypeptide of SEQ ID NO: 4. Those skilled in the art will
recognize that the degenerate sequences of SEQ ID NO: 6 and SEQ ID
NO: 7 also provide all RNA sequences encoding SEQ ID NO: 2 and SEQ
ID NO: 4, respectively, by substituting U for T. Thus, zkun5
polypeptide-encoding polynucleotides comprising nucleotide 1 to
nucleotide 186 of SEQ ID NO: 6, nucleotide 1 to nucleotide 186 of
SEQ ID NO: 7, and their respective RNA equivalents are contemplated
by the present invention. Table 2 sets forth the one-letter codes
used within SEQ ID NOS: 6 and 7 to denote degenerate nucleotide
positions. "Resolutions" are the nucleotides denoted by a code
letter. "Complement" indicates the code for the complementary
nucleotide(s). For example, the code Y denotes either C or T, and
its complement R denotes A or G, A being complementary to T, and G
being complementary to C.
2 TABLE 2 Nucleotide Resolution Nucleotide Complement A A T T C C G
G G G C C T T A A R A.vertline.G Y C.vertline.T Y C.vertline.T R
A.vertline.G M A.vertline.C K G.vertline.T K G.vertline.T M
A.vertline.C S C.vertline.G S C.vertline.G W A.vertline.T W
A.vertline.T H A.vertline.C.vertline.T D A.vertline.G.vertline.T B
C.vertline.G.vertline.T V A.vertline.C.vertline.G V
A.vertline.C.vertline.G B C.vertline.G.vertline.T D
A.vertline.G.vertline.T H A.vertline.C.vertline.T N
A.vertline.C.vertline.G.vertline.T N
A.vertline.C.vertline.G.vertline.T
[0049] The degenerate codons used in SEQ ID NOS: 6 and 5 7,
encompassing all possible codons for a given amino acid, are set
forth in Table 3.
3TABLE 3 One Amino Letter Degenerate Acid Code Codons Codon Cys C
TGC TGT TGY Ser S AGC AGT TCA TCC TCG TCT WSN Thr T ACA ACC ACG ACT
ACN Pro P CCA CCC CCG CCT CCN Ala A GCA GCC GCG GCT GCN Gly G GGA
GGC GGG GGT GGN Asn N AAC AAT AAY Asp D GAC GAT GAY Glu F GAA GAG
GAR Gln Q CAA CAG CAR His H CAC CAT CAY Arg R AGA AGG CGA CGC CGG
CGT MGN Lys K AAA AAG AAR Met M ATG ATG Ile I ATA ATC ATT ATH Leu L
CTA CTC CTG CTT TTA TTG YTN Val V GTA GTC GTG GTT GTN Phe F TTC TTT
TTY Tyr Y TAC TAT TAY Trp W TGG TGG Ter . TAA TAG TGA TRR
Asn.vertline.Asp B RAY Glu.vertline.Gln Z SAR Any X NNN
[0050] One of ordinary skill in the art will appreciate that some
ambiguity is introduced in determining a degenerate codon,
representative of all possible codons encoding each amino acid. For
example, the degenerate codon for serine (WSN) can, in some
circumstances, encode arginine (AGR), and the degenerate codon for
arginine (MGN) can, in some circumstances, encode serine (AGY). A
similar relationship exists between codons encoding phenylalanine
and leucine. Thus, some polynucleotides encompassed by the
degenerate sequence may encode variant amino acid sequences, but
one of ordinary skill in the art can easily identify such variant
sequences by reference to the amino acid sequences shown in SEQ ID
NOS: 2 and 4. Variant sequences can be readily tested for
functionality as described herein.
[0051] One of ordinary skill in the art will also appreciate that
different species can exhibit preferential codon usage, that is, a
bias in codon usage within the genome of a species. See, in
general, Grantham et al., Nuc. Acids Res. 8:1893-912, 1980; Haas et
al. Curr. Biol. 6:315-24, 1996; Wain-Hobson et al., Gene 13:355-64,
1981; Grosjean and Fiers, Gene 18:199-209, 1982; Holm, Nuc. Acids
Res. 14:3075-87, 1986; and Ikemura, J. Mol. Biol. 158:573-97, 1982.
Preferred codons for a particular species can be introduced into
the polynucleotides of the present invention by a variety of
methods known in the art, thereby increasing translation efficiency
within a particular cell type or species. The degenerate codon
sequences disclosed in SEQ ID NOS:6 and 7 serve as templates for
optimizing expression of polynucleotides in various cell types and
species commonly used in the art and disclosed herein. Sequences
containing preferred codons can be tested and optimized for
expression in various host cell species, and tested for
functionality as disclosed herein.
[0052] It is preferred that zkun5 polynucleotides hybridize to
similar sized regions of SEQ IDNO: 1, or a sequence complementary
thereto, under stringent conditions. In general, stringent
conditions are selected to be about 5.degree. C. lower than the
thermal melting point (T.sub.m) for the specific sequence at a
defined ionic strength and pH. The T.sub.m is the temperature
(under defined ionic strength and pH) at which 50% of the target
sequence hybridizes to a perfectly matched probe. Typical stringent
conditions are those in which the salt concentration is up to about
0.03 M at pH 7 and the temperature is at least about 60.degree.
C.
[0053] As previously noted, zkun5 polynucleotides provided by the
present invention include DNA and RNA. Methods for preparing DNA
and RNA are well known in the art. In general, RNA is isolated from
a tissue or cell that produces large amounts of zkun5 RNA. Such
tissues and cells are identified by Northern blotting (Thomas,
Proc. Natl. Acad. Sci. USA 77:5201, 1980), and include spinal cord,
trachea, heart, colon, small intestine, and stomach. Total RNA can
be prepared using guanidine-HCl extraction followed by isolation by
centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry
18:52-94, 1979). Poly (A)+RNA is prepared from total RNA using the
method of Aviv and Leder (Proc. Natl. Acad. Sci. USA 69:1408-12,
1972). Complementary DNA (cDNA) is prepared from poly(A)+RNA using
known methods. In the alternative, genomic DNA can be isolated.
Polynucleotides encoding zkun5 polypeptides are then identified and
isolated by, for example, hybridization or PCR.
[0054] A full-length clone encoding zkun5 can be obtained by
conventional cloning procedures. Complementary DNA (cDNA) clones
are preferred, although for some applications (e.g., expression in
transgenic animals) it may be preferable to use a genomic clone, or
to modify a cDNA clone to include at least one genomic intron.
Methods for preparing cDNA and genomic clones are well known and
within the level of ordinary skill in the art, and include the use
of the sequence disclosed herein, or parts thereof, for probing or
priming a library. Expression libraries can be probed with
antibodies to zkun5, receptor fragments, or other specific binding
partners.
[0055] The polynucleotides of the present invention can also be
synthesized using automated equipment ("gene machines"). Gene
synthesis methods are well known in the art. See, for example,
Glick and Pasternak, Molecular Biotechnology. Principles &
Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994;
Itakura et al., Annu. Rev. 1iochem. 53:323-3S6, 1984; and Climie et
al., Proc. Natl. Acad. Sci. USA 87:633-637, 1990.
[0056] The zkun5 polynucleotide sequences disclosed herein can be
used to isolate counterpart polynucleotides from other species
(orthologs). These orthologous polynucleotides can be used, inter
alia, to prepare the respective orthologous proteins. These other
species include, but are not limited to mammalian, avian,
amphibian, reptile, fish, insect and other vertebrate and
invertebrate species. Of particular interest are zkun5
polynucleotides abd polypeptides from other mammalian species,
including murine, porcine, ovine, bovine, canine, feline, equine,
and other primate polypeptides. Orthologs of human zkun5 can be
cloned using information and compositions provided by the present
invention in combination with conventional cloning techniques. For
example, a cDNA can be cloned using mRNA obtained from a tissue or
cell type that expresses zkun5 as disclosed herein. Suitable
sources of mRNA can be identified by probing Northern blots with
probes designed from the sequences disclosed herein. A library is
then prepared from mRNA of a positive tissue or cell line. A
zkun5-encoding cDNA can then be isolated by a variety of methods,
such as by probing with a complete or partial human cDNA or with
one or more sets of degenerate probes based on the disclosed
sequences. A cDNA can also be cloned using the polymerase chain
reaction, or PCR (Mullis, U.S. Pat. No. 4,683,202), using primers
designed from the representative human zkun5 sequence disclosed
herein. Within an additional method, the cDNA library can be used
to transform or transfect host cells, and expression of the cDNA of
interest can be detected with an antibody to zkun5 polypeptide.
Similar techniques can also be applied to the isolation of genomic
clones.
[0057] Those skilled in the art will recognize that the sequence
disclosed in SEQ IDNO: 1 represents a single allele of human zkun5
and that natural variation, including allelic variation and
alternative splicing, is expected to occur. Allelic variants of
this sequence can be cloned by probing cDNA or genomic libraries
from different individuals according to standard procedures.
Allelic variants of the DNA sequence shown in SEQ ID NO: 1,
including those containing silent mutations and those in which
mutations result in amino acid sequence changes, are within the
scope of the present invention, as are proteins which are allelic
variants of SEQ ID NO: 2. cDNAs generated from alternatively
spliced mRNAs, which retain the proteinase inhibiting activity of
zkun5 are included within the scope of the present invention, as
are polypeptides encoded by such cDNAs and mRNAs. Allelic variants
and splice variants of these sequences can be cloned by probing
cDNA or genomic libraries from different individuals or tissues
according to standard procedures known in the art.
[0058] Zkun5 proteins, including variants of wild-type zkun5, are
tested for activity in protease inhibition assays, a variety of
which are known in the art. Preferred assays include those
measuring inhibition of trypsin, chymotrypsin, plasmin, cathepsin
G, and human leukocyte elastase. See, for example, Petersen et al.,
Eur. J. Biochem. 235:310-316, 1996. In a typical procedure, the
inhibitory activity of a test compound is measured by incubating
the test compound with the proteinase, then adding an appropriate
substrate, typically a chromogenic peptide substrate. See, for
example, Norris et al. (Biol. Chem. Hoppe-Seyler 371:37-42, 1990).
Briefly, various concentrations of the inhibitor are incubated in
the presence of trypsin, plasmin, and plasma kallikrein in a
low-salt buffer at. pH 7.4, 25.degree. C. After 30 minutes, the
residual enzymatic activity is measured by the addition of a
chromogenic substrate (e.g., S2251 (D-Val-Leu-Lys-Nan) or S2302
(D-Pro-Phe-Arg-Nan), available from Kabi, Stockholm, Sweden) and a
30-minute incubation. Inhibition of enzyme activity is indicated by
a decrease in absorbance at 405 nm or fluorescence Em at 460 nm.
From the results, the apparent inhibition constant K.sub.i is
calculated. The inhibition of coagulation factors (e.g., factor
VIIa, factor Xa) can be measured using chromogenic substrates or in
conventional coagulation assays (e.g., clotting time of normal
human plasma; Dennis et al., ibid.).
[0059] Zkun5 proteins can be tested in animal models of disease,
particularly tumor models, models of fibrinolysis, and models of
imbalance of hemostasis. Suitable models are known in the art. For
example, inhibition of tumor metastasis can be assessed in mice
into which cancerous cells or tumor tissue have been introduced by
implantation or injection (e.g., Brown, Advan. Enzyme Regul.
35:293-301, 1995; Conway et al., Clin. Exp. Metastasis 14:115-124,
1996). Effects on fibrinolysis can be measured in a rat model
wherein the enzyme batroxobin and radiolabeled fibrinogen are
administered to test animals. Inhibition of fibrinogen activation
by a test compound is seen as a reduction in the circulating level
of the label as compared to animals not receiving the test
compound. See, Lenfors and Gustafsson, Semin. Thromb. Hemost.
22:335-342, 1996. Zkun5 proteins can be delivered to test animals
by injection or infusion, or can be produced in vivo by way of, for
example, viral or naked DNA delivery systems or transgenic
expression.
[0060] Exemplary viral delivery systems include adenovirus,
herpesvirus, vaccinia virus and adeno-associated virus (AAV).
Adenovirus, a double-stranded DNA virus, is currently the best
studied gene transfer vector for delivery of heterologous nucleic
acid (for a review, see Becker et al., Meth. Cell Biol. 43:161-189,
1994; and Douglas and Curiel, Science & Medicine 4:44-53,
1997). The adenovirus system offers several advantages: adenovirus
can (i) accommodate relatively large DNA inserts; (ii) be grown to
high titer; (iii) infect a broad range of mammalian cell types; and
(iv) be used with a large number of available vectors containing
different promoters. Also, because adenoviruses are stable in the
bloodstream, they can be administered by intravenous injection. By
deleting portions of the adenovirus genome, larger inserts (up to 7
kb) of heterologous DNA can be accommodated. These inserts can be
incorporated into the viral DNA by direct ligation or by homologous
recombination with a co-transfected plasmid. In an exemplary
system, the essential El gene is deleted from the viral vector, and
the virus will not replicate unless the El gene is provided by the
host cell (e.g., the human 293 cell line). When intravenously
administered to intact animals, adenovirus primarily targets the
liver. If the adenoviral delivery system has an El gene deletion,
the virus cannot replicate in the host cells. However, the host's
tissue (e.g., liver) will express and process (and, if a signal
sequence is present, secrete) the heterologous protein. Secreted
proteins will enter the circulation in the highly vascularized
liver, and effects on the infected animal can be determined.
[0061] An alternative method of gene delivery comprises removing
cells from the body and introducing a vector into the cells as a
naked DNA plasmid. The transformed cells are then re-implanted in
the body. Naked DNA vectors are introduced into host cells by
methods known in the art, including transfection, electroporation,
microinjection, transduction, cell fusion, DEAE dextran, calcium
phosphate precipitation, use of a gene gun, or use of a DNA vector
transporter. See, Wu et al., J. Biol. Chem. 263:14621-14624, 1988;
Wu et al., J. Biol. Chem. 267:963-967, 1992; and Johnston and Tang,
Meth. Cell Biol. 43:353-365, 1994.
[0062] Transgenic mice, engineered to express a zkun5 gene, and
mice that exhibit a complete absence of zkun5 gene function,
referred to as "knockout mice" (Snouwaert et al., Science 257:1083,
1992), can also be generated (Lowell et al., Nature 366:740-742,
1993). These mice are employed to study the zkun5 gene and the
encoded protein in an in vivo system. Transgenic mice are
particularly useful for investigating the role of zkun5 proteins in
early development because they allow the identification of
developmental abnormalities or blocks resulting from the over-or
underexpression of a specific factor.
[0063] The zkun5 polypeptides of the present invention, including
full-length polypeptides, biologically active fragments, and fusion
polypeptides can be produced in genetically engineered host cells
according to conventional techniques. Suitable host cells are those
cell types that can be transformed or transfected with exogenous
DNA and grown in culture, and include bacteria, fungal cells, and
cultured higher eukaryotic cells. Techniques for manipulating
cloned DNA molecules and introducing exogenous DNA into a variety
of host cells are disclosed by Sambrook et al., Molecular Cloning:
A Laboratory Manual, 2 nd ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y., 1989, and Ausubel et al., eds., Current
Protocols in Molecular Biology, John Wiley and Sons, Inc., N.Y.,
1987.
[0064] In general, a DNA sequence encoding a zkun5 polypeptide is
operably linked to other genetic elements required for its
expression, generally including a transcription promoter and
terminator, within an expression vector. The vector will also
commonly contain one or more selectable markers and one or more
origins of replication, although those skilled in the art will
recognize that within certain systems selectable markers may be
provided on separate vectors, and replication of the exogenous DNA
may be provided by integration into the host cell genome. Selection
of promoters, terminators, selectable markers, vectors and other
elements is a matter of routine design within the level of ordinary
skill in the art. Many such elements are described in the
literature and are available through commercial suppliers.
[0065] To direct a zkun5 polypeptide into the secretory pathway of
a host cell, a secretory signal sequence (also known as a leader
sequence, prepro sequence or pre sequence) is provided in the
expression vector. The secretory signal sequence may be that of
zkun5, or may be derived from another secreted protein (e.g., t-PA;
see, U.S. Pat. No. 5,641,655) or synthesized de novo. The secretory
signal sequence is operably linked to the zkun5 DNA sequence, i.e.,
the two sequences are joined in the correct reading frame and
positioned to direct the newly sythesized polypeptide into the
secretory pathway of the host cell. Secretory signal sequences are
commonly positioned 5' to the DNA sequence encoding the polypeptide
of interest, although certain signal sequences may be positioned
elsewhere in the DNA sequence of interest (see, e.g., Welch et al.,
U.S. Pat. No. 5,037,743; Holland et al., U.S. Pat. No.
5,143,830).
[0066] Cultured mammalian cells are suitable hosts for use within
the present invention. Methods for introducing exogenous DNA into
mammalian host cells include calcium phosphate-mediated
transfection (Wigler et al., Cell 14:725, 1978; Corsaro and
Pearson, Somatic Cell Genetics 7:603, 1981: Graham and Van der Eb,
Virology 52:456, 1973), electroporation (Neumann et al., EMBO J.
1:841-845, 1982), DEAE-dextran mediated transfection (Ausubel et
al., ibid.), and liposome-mediated transfection (Hawley-Nelson et
al., Focus 15:73, 1993; Ciccarone et al., Focus 15:80, 1993). The
production of recombinant polypeptides in cultured mammalian cells
is disclosed, for example, by Levinson et al., U.S. Pat. No.
4,713,339; Hagen et al., U.S. Pat. No. 4,784,950; Palmiter et al.,
U.S. Pat. No. 4,579,821; and Ringold, U.S. Pat. No. 4,656,134.
Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL
1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570
(ATCC No. CRL 10314), 293 (ATCC No. CRL 1573; Graham et al., J.
Gen. Virol. 36:59-72, 1977) and Chinese hamster ovary (e.g. CHO-K1;
ATCC No. CCL 61) cell lines. Additional suitable cell lines are
known in the art and available from public depositories such as the
American Type Culture Collection, Rockville, Md. In general, strong
transcription promoters are preferred, such as promoters from SV-40
or cytomegalovirus. See, e.g., U.S. Pat. No. 4,956,288. Other
suitable promoters include those from metallothionein genes (U.S.
Pat. Nos. 4,579,821 and 4,601,978) and the adenovirus major late
promoter. Expression vectors for use in mammalian cells include
pZP-1 and pZP-9, which have been deposited with the American Type
Culture Collection, Rockville, Md. USA under accession numbers
98669 and 98668, respectively.
[0067] Drug selection is generally used to select for cultured
mammalian cells into which foreign DNA has been inserted. Such
cells are commonly referred to as "transfectants". Cells that have
been cultured in the presence of the selective agent and are able
to pass the gene of interest to their progeny are referred to as
"stable transfectants." A preferred selectable marker is a gene
encoding resistance to the antibiotic neomycin. Selection is
carried out in the presence of a neomycin-type drug, such as G-418
or the like. Selection systems can also be used to increase the
expression level of the gene of interest, a process referred to as
"amplification." Amplification is carried out by culturing
transfectants in the presence of a low level of the selective agent
and then increasing the amount of selective agent to select for
cells that produce high levels of the products of the introduced
genes. A preferred amplifiable selectable marker is dihydrofolate
reductase, which confers resistance to methotrexate. Other drug
resistance genes (e.g. hygromycin resistance, multi-drug
resistance, puromycin acetyltransferase) can also be used.
[0068] Other higher eukaryotic cells can also be used as hosts,
including insect cells, plant cells and avian cells. The use of
Agrobacterium rhizogenes as a vector for expressing genes in plant
cells has been reviewed by Sinkar et al., J. Biosci. (Bangalore)
11:47-58, 1987. Transformation of insect cells and production of
foreign polypeptides therein is disclosed by Guarino et al., U.S.
Pat. No. 5,162,222 and WIPO publication WO 94/06463.
[0069] Insect cells can be infected with recombinant baculovirus,
commonly derived from Autographa californica nuclear polyhedrosis
virus (AcNPV). See, King and Possee, The Baculovirus Expression
System: A Laboratory Guide, London, Chapman & Hall; O'Reilly et
al., Baculovirus Expression Vectors: A Laboratory Manual, New York,
Oxford University Press., 1994; and Richardson, Ed., Baculovirus
Expression Protocols. Methods in Molecular Biology, Humana Press,
Totowa, N.J., 1995. Recombinant baculovirus can also be produced
through the use of a transposon-based system described by Luckow et
al. (J. Virol. 67:4566-4579, 1993). This system, which utilizes
transfer vectors, is commercially available in kit form
(Bac-to-Bac.TM. kit; Life Technologies, Rockville, Md.). The
transfer vector (e.g., pFastBac1.TM.; Life Technologies) contains a
Tn7 transposon to move the DNA encoding the protein of interest
into a baculovirus genome maintained in E. coli as a large plasmid
called a "bacmid." See, Hill-Perkins and Possee, J. Gen. Virol.
71:971-976, 1990; Bonning et al., J. Gen. Virol. 75:1551-1556,
1994; and Chazenbalk and Rapoport, J. Biol. Chem. 270:1543-1549,
1995. In addition, transfer vectors can include an in-frame fusion
with DNA encoding a polypeptide extension or affinity tag as
disclosed above. Using techniques known in the art, a transfer
vector containing a zkun5 -encoding sequence is transformed into E.
coli host cells, and the cells are screened for bacmids which
contain an interrupted lacZ gene indicative of recombinant
baculovirus. The bacmid DNA containing the recombinant baculovirus
genome is isolated, using common techniques, and used to transfect
Spodoptera frugiperda cells, such as Sf9 cells. Recombinant virus
that expresses zkun5 protein is subsequently produced. Recombinant
viral stocks are made by methods commonly used the art.
[0070] For protein production, the recombinant virus is used to
infect host cells, typically a cell line derived from the fall
armyworm, Spodoptera frugiperda (e.g., Sf9 or Sf21 cells) or
Trichoplusia ni (e.g., High Five.TM. cells; Invitrogen, Carlsbad,
Calif.). See, in general, Glick and Pasternak, Molecular
Biotechnology: Principles and Applications of Recombinant DNA, ASM
Press, Washington, D.C., 1994. See also, U.S. Pat. No. 5,300,435.
Serum-free media are used to grow and maintain the cells. Suitable
media formulations are known in the art and can be obtained from
commercial suppliers. The cells are grown up from an inoculation
density of approximately 2-5.times.10.sup.5 cells to a density of
1-2.times.10.sup.6 cells, at which time a recombinant viral stock
is added at a multiplicity of infection (MOI) of 0.1 to 10, more
typically near 3. Procedures used are generally described in
available laboratory manuals (e.g., King and Possee, ibid.;
O'Reilly et al., ibid.; Richardson, ibid.).
[0071] Fungal cells, including yeast cells, can also be used within
the present invention. Yeast species of particular interest in this
regard include Saccharomyces cerevisiae, Pichia pastoris, and
Pichia methanolica. Methods for transforming S. cerevisiae cells
with exogenous DNA and producing recombinant polypeptides therefrom
are disclosed by, for example, Kawasaki, U.S. Pat. No. 4,599,311;
Kawasaki et al., U.S. Pat. No. 4,931,373; Brake, U.S. Pat. No.
4,870,008; Welch et al., U.S. Pat. No. 5,037,743; and Murray et
al., U.S. Pat. No. 4,845,075. Transformed cells are selected by
phenotype determined by the selectable marker, commonly drug
resistance or the ability to grow in the absence of a particular
nutrient (e.g., leucine). A preferred vector system for use in
Saccharomyces cerevisiae is the POT1 vector system disclosed by
Kawasaki et al. (U.S. Pat. No. 4,931,373), which allows transformed
cells to be selected by growth in glucose-containing media.
Suitable promoters and terminators for use in yeast include those
from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Pat. No.
4,599,311; Kingsman et al., U.S. Pat. No. 4,615,974; and Bitter,
U.S. Pat. No. 4,977,092) and alcohol dehydrogenase genes. See also
U.S. Pat. Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454.
Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragills, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondil and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459-3465, 1986 and Cregg, U.S. Pat. No. 4,882,279. Aspergillus
cells may be utilized according to the methods of McKnight et al.,
U.S. Pat. No. 4,935,349. Methods for transforming Acremonium
chrysogenum are disclosed by Sumino et al., U.S. Pat. No.
5,162,228. Methods for transforming Neurospora are disclosed by
Lambowitz, U.S. Pat. No. 4,486,533. The use of Pichia methanolica
as host for the production of recombinant proteins is disclosed in
U.S. Pat. No. 5,716,808, 5,736,383, 5,854,039, and 5,888,768; and
WIPO Publications WO 97/17450 and WO97/17451.
[0072] Prokaryotic host cells, including strains of the bacteria
Escherichia coll, Bacillus and other genera are also useful host
cells within the present invention. Techniques for transforming
these hosts and expressing foreign DNA sequences cloned therein are
well known in the art (see, e.g., Sambrook et al., ibid.). When
expressing a zkun5 polypeptide in bacteria such as E. coli, the
polypeptide may be retained in the cytoplasm, typically as
insoluble granules, or may be directed to the periplasmic space by
a bacterial secretion sequence. In the former case, the cells are
lysed, and the granules are recovered and denatured using, for
example, guanidine isothiocyanate or urea. The denatured
polypeptide can then be refolded and dimerized by diluting the
denaturant, such as by dialysis against a solution of urea and a
combination of reduced and oxidized glutathione, followed by
dialysis against a buffered saline solution. In the latter case,
the polypeptide can be recovered from the periplasmic space in a
soluble and functional form by disrupting the cells (by, for
example, sonication or osmotic shock) to release the contents of
the periplasmic space and recovering the protein, thereby obviating
the need for denaturation and refolding.
[0073] Transformed or transfected host cells are cultured according
to conventional procedures in a culture medium containing nutrients
and other components required for the growth of the chosen host
cells. A variety of suitable media, including defined media and
complex media, are known in the art and generally include a carbon
source, a nitrogen source, essential amino acids, vitamins and
minerals. Media may also contain such components as growth factors
or serum, as required. The growth medium will generally select for
cells containing the exogenously added DNA by, for example, drug
selection or deficiency in an essential nutrient which is
complemented by the selectable marker carried on the expression
vector or co-transfected into the host cell.
[0074] Zkun5 polypeptides, particularly shorter polypeptides, can
also be prepared through chemical synthesis according to methods
known in the art, including exclusive solid phase synthesis,
partial solid phase methods, fragment condensation or classical
solution synthesis. See, for example, Merrifield, J. Am. Chem. Soc.
85:2149, 1963; Stewart et al., Solid Phase Peptide Synthesis (2 nd
edition), Pierce Chemical Co., Rockford, IL, 1984; Bayer and Rapp,
Chem. Pept. Prot. 3:3, 1986; and Atherton et al., Solid Phase
Peptide Synthesis: A Practical Ap-roach, IRL Press, Oxford,
1989.
[0075] It is preferred to purify the proteins of the present
invention to .gtoreq.80% purity, more preferably to .gtoreq.90%
purity, even more preferably .gtoreq.95% purity, and particularly
preferred is a pharmaceutically pure state, that is greater than
99.9% pure with respect to contaminating macromolecules,
particularly other proteins and nucleic acids, and free of
infectious and pyrogenic agents. Preferably, a purified protein is
substantially free of other proteins, particularly other proteins
of animal origin.
[0076] Zkun5 proteins are purified by conventional protein
purification methods, typically by a combination of chromatographic
techniques. Polypeptides comprising a polyhistidine affinity tag
(typically about 6 histidine residues) are purified by affinity
chromatography on a nickel chelate resin. See, for example,
Houchuli et al., Bio/Technol. 6:1321-1325, 1988.
[0077] Using methods known in the art, zkun5 proteins can be
produced glycosylated or non-glycosylated; pegylated or
non-pegylated; and may or may not include an initial methionine
amino acid residue.
[0078] The zkun5 proteins are contemplated for use in the treatment
or prevention of conditions associated with excessive proteinase
activity, in particular an excess of trypsin, plasmin, kallikrein,
elastase, cathepsin G, proteinase-3, thrombin, factor VIIa, factor
IXa, factor Xa, factor XIa, factor XIIa, or matrix
metalloproteinases. Such conditions include, but are not limited
to, acute pancreatitis, cardiopulmonary bypass (CPB)-induced
pulmonary injury, allergy-induced protease release, deep vein
thrombosis, myocardial infarction, shock (including septic shock),
hyperfibrinolytic hemorrhage, emphysema, rheumatoid arthritis,
adult respiratory distress syndrome, chronic inflammatory bowel
disease, psoriasis, and other inflammatory conditions. Zkun5
proteins are also contemplated for use in preservation of platelet
function, organ preservation, and wound healing.
[0079] Zkun5 proteins may be useful in the treatment of conditions
arising from an imbalance in hemostasis, including acquired
coagulopathies, primary fibrinolysis and fibrinolysis due to
cirrhosis, and complications from high-dose thrombolytic therapy.
Acquired coagulopathies can result from liver disease, uremia,
acute disseminated intravascular coagulation, post-cardiopulmonary
bypass, massive transfusion, or Warfarin overdose (Humphries,
Transfusion Medicine 1:1181-1201, 1994). A deficiency or
dysfunction in any of the procoagulant mechanisms predisposes the
patient to either spontaneous hemorrhage or excess blood loss
associated with trauma or surgery. Acquired coagulopathies usually
involve a combination of deficiencies, such as deficiencies of a
plurality of coagulation factors, and/or platelet dysfunction. In
addition, patients with liver disease commonly experience increased
fibrinolysis due to an inability to maintain normal levels of
a2-antiplasmin and/or decreased hepatic clearance of plasminogen
activators (Shuman, Hemorrhagic Disorders, in Bennet and Plum, eds.
Cecil Textbook of Medicine, 20 th ed., W. B. Saunders Co., 1996).
Primary fibrinolysis results from a massive release of plasminogen
activator. Conditions associated with primary fibrinolysis include
carcinoma of the prostate, acute promyelocytic leukemia,
hemangiomas, and sustained release of plasminogen activator by
endothelial cells due to injection of venoms. The condition becomes
critical when enough plasmin is activated to deplete the
circulating level of .alpha..sub.2-antiplasmin (Shuman, ibid.).
Data suggest that plasmin on endothelial cells may be related to
the pathophysiology of bleeding or rethrombosis observed in
patients undergoing high-dose thrombolytic therapy for thrombosis.
Plasmin may cause further damage to the thrombogenic surface of
blood vessels after thrombolysis, which may result in rethrombosis
(Okajima, J. Lab. Clin. Med. 126:1377-1384, 1995).
[0080] Additional antithrombotic uses of zkun5 proteins include
treatment or prevention of deep vein thrombosis, pulmonary
embolism, and post-surgical thrombosis.
[0081] Zkun5 proteins may also be used within methods for
inhibiting blood coagulation in mammals, such as in the treatment
of disseminated intravascular coagulation. Zkun5 proteins may thus
be used in place of known anticoagulants such as heparin, coumarin,
and anti-thrombin III. Such methods will generally include
administration of the protein in an amount sufficient to produce a
clinically significant inhibition of blood coagulation. Such
amounts will vary with the nature of the condition to be treated,
but can be predicted on the basis of known assays and experimental
animal models, and will in general be within the ranges disclosed
below.
[0082] Zkun5 proteins may also find therapeutic use in the blockage
of proteolytic tissue degradation. Proteolysis of extracellular
matrix, connective tissue, and other tissues and organs is an
element of many diseases. This tissue destruction is beleived to be
initiated when plasmin activates one or more matrix
metalloproteinases (e.g., collagenase and metallo-elastases).
Inhibition of plasmin by zkun5 proteins may thus be beneficial in
the treatment of these conditions.
[0083] Matrix metalloproteinases (MMPs) are believed to play a role
in metastases of cancers, abdominal aortic aneurysm, multiple
sclerosis, rheumatoid arthritis, osteoarthritis, trauma and
hemorrhagic shock, and cornial ulcers. MMPs produced by tumor cells
break down and remodel tissue matrices during the process of
metastatic spread. There is evidence to suggest that MMP inhibitors
may block this activity (Brown, Advan. Enzyme Regul. 35:293-301,
1995). Abdominal aortic aneurysm is characterized by the
degradation of extracellular matrix and loss of structural
integrity of the aortic wall. Data suggest that plasmin may be
important in the sequence of events leading to this destruction of
aortic matrix (Jean-Claude et al., Surgery 116:472-478, 1994).
Proteolytic enzymes are also believed to contribute to the
inflammatory tissue damage of multiple sclerosis (Gijbels, J. Clin.
Invest. 94:2177-2182, 1994). Rheumatoid arthritis is a chronic,
systemic inflammatory disease predominantly affecting joints and
other connective tissues, wherein proliferating inflammatory tissue
(panus) may cause joint deformities and dysfunction (see, Arnett,
in Cecil Textbook of Medicine, ibid.). Osteoarthritis is a chronic
disease causing deterioration of the joint cartilage and other
joint tissues and the formation of new bone (bone spurs) at the
margins of the joints. There is evidence that MMPs participate in
the degradation of collagen in the matrix of osteoarthritic
articular cartilage. Inhition of MMPs results in the inhibition of
the removal of collagen from cartilage matrix (Spirito, Inflam.
Res. 44 (supp. 2):S131-S132, 1995; O'Byrne, Inflam. Res. 44 (supp.
2):S117-S118, 1995; Karran, Ann. Rheumatic Disease 54:662-669,
1995). Zkun5 proteins may also be useful in the treatment of trauma
and hemorrhagic shock. Data suggest that administration of an MMP
inhibitor after hemorrhage improves cardiovascular response,
hepatocellular function, and microvascular blood flow in various
organs (Wang, Shock 6:377-382, 1996). Corneal ulcers, which can
result in blindness, manifest as a breakdown of the collagenous
stromal tissue. Damage due to thermal or chemical injury to corneal
surfaces often results in a chronic wound-healing situation. There
is direct evidence for the role of MMPs in basement membrane
defects associated with failure to re-epithelialize in cornea or
skin (Fini, Am. J. Pathol. 149:1287-1302, 1996).
[0084] The zkun5 proteins of the present invention may be combined
with other therapeutic agents to augment the activity (e.g.,
antithrombotic or anticoagulant activity) of such agents. For
example, a zkun5 protein may be used in combination with tissue
plasminogen activator in thrombolytic therapy.
[0085] Doses of zkun5 proteins will vary according to the severity
of the condition being treated and may range from approximately 10
.mu.g/kg to 10 mg/kg body weight, preferably 100 .mu.g/kg to 5
mg/kg, more preferably 100 .mu.g/kg to 1 mg/kg. The proteins
formulated in a pharmaceutically acceptable carrier or vehicle. It
is preferred to prepare them in a form suitable for injection or
infusion, such as by dilution with with sterile water, an isotonic
saline or glucose solution, or similar vehicle. In the alternative,
the protein may be packaged as a lyophilized powder, optionally in
combination with a pre-measured diluent, and resuspended
immediately prior to use. Pharmaceutical compositions may further
include one or more excipients, preservatives, solubilizers,
buffering agents, albumin to prevent protein loss on vial surfaces,
etc. Formulation methods are within the level of ordinary skill in
the art. See, Remington: The Science and Practice of Pharmacy,
Gennaro, ed., Mack Publishing Co., Easton, Pa., 19 th ed.,
1995.
[0086] Gene therapy provides an alternative therapeutic approach
for delivery of zkun5 proteins. If a mammal has a mutated or absent
zkun5 gene, a polynucleotide encoding a zkun5 protein can be
introduced into the cells of the mammal. In one embodiment, a gene
encoding a zkun5 protein is introduced in vivo in a viral vector.
Such vectors include an attenuated or defective DNA virus, such as
herpes simplex virus (HSV), papillomavirus, Epstein Barr virus
(EBV), adenovirus, adeno-associated virus (AAV), and the like.
Defective viruses, which entirely or almost entirely lack viral
genes, are preferred. A defective virus is not infective after
introduction into a cell. Use of defective viral vectors allows for
administration to cells in a specific, localized area, without
concern that the vector can infect other cells. Examples of
particular vectors include, without limitation, a defective herpes
simplex virus 1 (HSV1) vector (Kaplitt et al., Molec. Cell.
Neurosci. 2:320-30, 1991); an attenuated adenovirus vector, such as
the vector described by Stratford-Perricaudet et al., J. Clin.
Invest. 90:626-30, 1992; and a defective adeno-associated virus
vector (Samulski et al., J. Virol. 61:3096-101, 1987; Samulski et
al., J. Virol. 63:3822-8, 1989).
[0087] Within another embodiment, a zkun5 polynucleotide can be
introduced in a retroviral vector, as described, for example, by
Anderson et al., U.S. Pat. No. 5,399,346; Mann et al. Cell 33:153,
1983; Temin et al., U.S. Pat. No. 4,650,764; Temin et al., U.S.
Pat. No. 4,980,289; Markowitz et al., J. Virol. 62:1120, 1988;
Temin et al., U.S. Pat. No. 5,124,263; Dougherty et al., WIPO
Publication No. WO 95/07358; and Kuo et al., Blood 82:845, 1993.
Alternatively, the vector can be introduced by lipofection in vivo
using liposomes. Synthetic cationic lipids can be used to prepare
liposomes for in vivo transfection of a gene encoding a marker
(Felgner et al., Proc. Nati. Acad. Sci. USA 84:7413-7, 1987; Mackey
et al., Proc. Natl. Acad. Sci. USA 85:8027-31, 1988).
[0088] Within a further embodiment, target cells are removed from
the body, and a vector is introduced into the cells as a naked DNA
plasmid. The transformed cells are then re-implanted into the body.
Naked DNA vectors for gene therapy can be introduced into the
desired host cells by methods known in the art, e.g., transfection,
electroporation, microinjection, transduction, cell fusion, DEAE
dextran, calcium phosphate precipitation, use of a gene gun or use
of a DNA vector transporter. See, for example, Wu et al., J. Biol.
Chem. 267:963-7, 1992; Wu et al., J. Biol. Chem. 263:14621-4,
1988.
[0089] Zkun5 proteins can also be used to prepare antibodies that
specifically bind to zkun5 proteins. As used herein, the term
"antibodies" includes polyclonal antibodies, monoclonal antibodies,
antigen-binding fragments thereof such as F(ab').sub.2 and Fab
fragments, single chain antibodies, and the like, including
genetically engineered antibodies. Non-human antibodies can be
humanized by grafting only non-human CDRs onto human framework and
constant regions, or by incorporating the entire non-human variable
domains (optionally "cloaking" them with a human-like surface by
replacement of exposed residues, wherein the result is a "veneered"
antibody). In some instances, humanized antibodies may retain
non-human residues within the human variable region framework
domains to enhance proper binding characteristics. Through
humanizing antibodies, biological half-life may be increased, and
the potential for adverse immune reactions upon administration to
humans is reduced. One skilled in the art can generate humanized
antibodies with specific and different constant domains (i.e.,
different Ig subclasses) to facilitate or inhibit various immune
functions associated with particular antibody constant domains.
Alternative techniques for generating or selecting antibodies
useful herein include in vitro exposure of lymphocytes to a zkun5
protein, and selection of antibody display libraries in phage or
similar vectors (for instance, through use of immobilized or
labeled zkun5 polypeptide). Antibodies are defined to be
specifically binding if they bind to a zkun5 protein with an
affinity at least 10-fold greater than the binding affinity to
control (non-zkun5) polypeptide. It is preferred that the
antibodies exhibit a binding affinity (K.sub.a) of 10.sup.6
M.sup.-1 or greater, preferably 10.sup.7 M.sup.-1 or greater, more
preferably 10.sup.8 M.sup.-1 or greater, and most preferably
10.sup.9 M.sup.-1 or greater. The affinity of a monoclonal antibody
can be readily determined by one of ordinary skill in the art (see,
for example, Scatchard, Ann. NY Acad. Sci. 51: 660-672, 1949).
[0090] Methods for preparing polyclonal and monoclonal antibodies
are well known in the art (see for example, Hurrell, J. G. R., Ed.,
Monoclonal Hybridoma Antibodies: Techniques and Applications, CRC
Press, Inc., Boca Raton, Fla., 1982). As would be evident to one of
ordinary skill in the art, polyclonal antibodies can be generated
from a variety of warm-blooded animals such as horses, cows, goats,
sheep, dogs, chickens, rabbits, mice, and rats. The immunogenicity
of a zkun5 protein may be increased through the use of an adjuvant
such as alum (aluminum hydroxide) or Freund's complete or
incomplete adjuvant. Polypeptides useful for immunization also
include fusion polypeptides, such as fusions of a zkun5 protein or
a portion thereof with an immunoglobulin polypeptide or with
maltose binding protein. The polypeptide immunogen may be a
full-length molecule or a portion thereof. If the polypeptide
portion is "hapten-like", such portion may be advantageously joined
or linked to a macromolecular carrier (such as keyhole limpet
hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for
immunization.
[0091] Immunogenic zkun5 polypeptides may be as small as 5
residues. It is preferred to use polypeptides that are hydrophilic
or comprise a hydrophilic region. A preferred such region of SEQ ID
NO: 2 includes residues 43-59.
[0092] A variety of assays known to those skilled in the art can be
utilized to detect antibodies that specifically bind to a zkun5
protein. Exemplary assays are described in detail in Antibodies: A
Laboratory Manual, Harlow and Lane (Eds.), Cold Spring Harbor
Laboratory Press, 1988. Representative examples of such assays
include concurrent immunoelectrophoresis, radio-immunoassays,
radio-immunoprecipitations, enzyme-linked immunosorbent assays
(ELISA), dot blot assays, Western blot assays, inhibition or
competition assays, and sandwich assays.
[0093] Antibodies to zkun5 may be used for affinity purification of
zkun5 proteins; within diagnostic assays for determining
circulating levels of zkun5 proteins; for detecting or quantitating
soluble zkun5 protein as a marker of underlying pathology or
disease; for immunolocalization within whole animals or tissue
sections, including immunodiagnostic applications; for
immunohistochemistry; for screening expression libraries; and for
other uses that will be evident to those skilled in the art. For
certain applications, including in vitro and in vivo diagnostic
uses, it is advantageous to employ labeled antibodies. Suitable
direct tags or labels include radionuclides, enzymes, substrates,
cofactors, inhibitors, fluorescent markers, chemiluminescent
markers, magnetic particles and the like; indirect tags or labels
may feature use of biotin-avidin or other
complement/anti-complement pairs as intermediates.
[0094] Zkun5 proteins may be used in the laboratory or commercial
preparation of proteins from cultured cells. The proteins can be
used alone to inhibit specific proteolysis or can be combined with
other proteinase inhibitors to provide a "cocktail" with a broad
spectrum of activity. Of particular interest is the inhibition of
cellular proteases, which can be release during cell lysis. The
proteins can also be used in the laboratory as a tissue culture
additive to prevent cell detachment.
[0095] The present invention also provides reagents for use in
diagnostic applications. For example, the zkun5 gene, a probe
comprising zkun5 DNA or RNA or a subsequence thereof can be used to
determine if the zkun5 gene is present on chromosome 7 or if a
mutation has occurred. Detectable chromosomal aberrations at the
zkun5 gene locus include, but are not limited to, aneuploidy, gene
copy number changes, insertions, deletions, restriction site
changes and rearrangements. Such aberrations can be detected using
polynucleotides of the present invention by employing molecular
genetic techniques, such as restriction fragment length
polymorphism (RFLP) analysis, short tandem repeat (STR) analysis
employing PCR techniques, and other genetic linkage analysis
techniques known in the art (Sambrook et al., ibid.; Ausubel et.
al., ibid.; Marian, Chest 108:255-65, 1995).
[0096] Zkun5 polynucleotides can also be used for chromosomal
mapping. The human zkun5 gene has be localized to 7p22.2-p22.1.
Localization of the zkun5 gene facilitates the establishment of
directly proportional physical distances between newly discovered
genes of interest and previously mapped markers, including zkun5.
The precise knowledge of a gene's position can be useful for a
number of purposes, including: 1) determining if a newly identified
sequence is part of an previously identified gene or gene segment
and obtaining additional surrounding genetic sequences in various
forms, such as YACs, BACs or cDNA clones; 2) providing a possible
candidate gene for an inheritable disease which shows linkage to
the same chromosomal region; and 3) cross-referencing model
organisms, such as mouse, which may aid in determining what
function a particular gene might have. A useful technique in this
regard is radiation hybrid mapping, a somatic cell genetic
technique developed for constructing high-resolution, contiguous
maps of mammalian chromosomes (Cox et al., Science 250:245-50,
1990). Partial or full knowledge of a gene's sequence allows one to
design PCR primers suitable for use with chromosomal radiation
hybrid mapping panels. Radiation hybrid mapping panels, which are
commercially available (e.g., the Stanford G3 RH Panel and the
GeneBridge 4 RH Panel, available from Research Genetics, Inc.,
Huntsville, Ala.), cover the entire human genome. These panels
enable rapid, PCR-based chromosomal localizations and ordering of
genes, sequence-tagged sites (STSs), and other nonpolymorphic and
polymorphic markers within a region of interest.
[0097] Sequence tagged sites (STSs) can also be used independently
for chromosomal localization. An STS is a DNA sequence that is
unique in the human genome and can be used as a reference point for
a particular chromosome or region of a chromosome. An STS is
defined by a pair of oligonucleotide primers that are used in a
polymerase chain reaction to specifically detect this site in the
presence of all other genomic sequences. Since STSs are based
solely on DNA sequence they can be completely described within an
electronic database (for example, Database of Sequence Tagged Sites
(dbSTS), GenBank; National Center for Biological Information,
National Institutes of Health, Bethesda, Md.;
http://www.ncbi.nlm.nih.gov) and can be searched with a gene
sequence of interest for the mapping data contained within these
short genomic landmark STS sequences.
[0098] The invention is further illustrated by the following
non-limiting examples.
EXAMPLES
Example 1
[0099] The 139-nucleotide sequence of an expressed sequence tag
(EST) was analyzed and found to encode a C-terminal portion of a
Kunitz domain. A clone corresponding to this EST was obtained and
sequenced. The clone contained the sequence shown in SEQ ID NO: 1.
This Kunitz domain sequence was designatied "zkun5".
[0100] Analysis of tissue distribution of zkun5 was performed by
Northern blotting (using Human Multiple Tissue Blots I, II, and
III, and Human RNA Master blot from Clontech Laboratories, Inc.,
Palo Alto, Calif.). A probe was made from a gel-purified EcoRI-XhoI
fragment of the origninal zkun5 clone, and was radioactively
labeled using a commercially available labeling kit (Rediprime.TM.
DNA labeling system, Amersham Corp., Arlington Heights, Ill.)
according to the manufacturer's specifications. The probe was
purified using a commercially available push column (NucTrap.RTM.
column; Stratagene, La Jolla, Calif.; see U.S. Pat. No. 5,336,412).
A commercially available hybridization solution (ExpressHyb.TM.
Hybridization Solution; Clontech Laboratories, Inc., Palo Alto,
Calif.) was used for prehybridization and as a hybridization
solution for the blots. Hybridization took place overnight at
65.degree. C., and the blots were then washed in 2.times.SSC and
0.05% SDS at room temperature, followed by a wash in 0.1.times.SSC
and 0.1% SDS at 55.degree. C. Two major transcript were observed at
sizes of 7.5 kb and 5.0 kb. Signals were present in many tissues,
including spinal cord, trachea, heart, colon, small intestine,
stomach, placenta, skeletal muscle, kidney, pancreas, prostate,
testis, thyroid, and adrenal gland. The RNA appeared to be subject
to a tissue specific splicing event since trachea, testis and
placenta give different size bands from the others.
Example 2
[0101] Based on the tissue distribution from the Northern blotting
experiments (see Example 1), 5' RACE was performed on cDNAs made
from several tissues including pancreas, heart, stomach and testis.
The cDNAs were prepared using a a commercially available kit
(Marathon.TM. cDNA Amplification Kit from Clontech Laboratories,
Inc., Palo Alto, Calif.) and an oligo(dT) primer.
[0102] To amplify the zkun5 DNA, 5 .mu.l each of 1/100 diluted
cDNAs, 20 pmoles each of oligonucleotide primers ZC9739 (SEQ ID NO:
12) and ZC15,999 (SEQ ID NO: 13), and 1 U of a 2:1 mixture of
ExTag.TM. DNA polymerase (TaKaRa Biomedicals) and Pfu DNA
polymerasse (Stratagene, La Jolla, Calif.) (ExTaq/Pfu) were used in
25.mu.l reaction mixtures. The reaction mixtures were incubated at
94.degree. C. for 2 minutes; 25 cycles of 94.degree. C. for 15
seconds, 66.degree. C. for 20 seconds, and 72.degree. C. for 30
seconds; and a 1-minute incubation at 72.degree. C. 1 .mu.l each of
1/100 diluted first PCR product was used as template for a nested
PCR. 20 pmoles each of oligonucleotide primers ZC9719 (SEQ ID NO:
14) and ZC15,998 (SEQ ID NO: 15), and 1 U of ExTaq/Pfu were used in
25-.mu.l reaction mixtures. The mixtures were incubated at
94.degree. C. for 2 minutes; 2 cycles of 94.degree. C. for 15
seconds, 66.degree. C. for 20 seconds, 72.degree. C. for 30
seconds; 25 cycles of 94.degree. C. for 15 seconds, 64.degree. C.
for 20 seconds, 72.degree. C. for 30 seconds; and a 1-minute
incubation at 72.degree. C. The PCR products were gel purified and
sequenced. Sequencing results indicated that the PCR products
extended the original EST clone to include an intact Kunitz domain.
The sequence of the PCR-generated clone is shown in SEQ ID NO:
9.
[0103] To construct an expression vector for the zkun5 Kunitz
domain, PCR was performed on cDNA prepared from pancreas as
disclosed above. Based on the domain comparison with other known
Kunitz domains, primers were designed such that the PCR product
would encode an intact Kunitz domain with restriction sites Bam HI
in sense primer ZC17,238 (SEQ ID NO: 16) and Xho I in antisense
primer ZC17,240 (SEQ ID NO: 17) to facilitate subcloning into an
expression vector. A silent mutation (nucleotide T to C) was
introduced in the sense primer ZC17,238 (SEQ ID NO: 16) to remove
an internal Bam HI site within the Kunitz domain sequence. 5 .mu.l
of 1/100 diluted cDNA, 20 pmoles each of oligonucleotide primers
ZC17,238 (SEQ ID NO: 16) and ZC17,240 (SEQ ID NO: 17), and 1 U of
ExTaq/Pfu were used in 25-.mu.l reaction mixtures. The mixtures
were incubated at 94.degree. C. for 2 minutes; 3 cycles of
94.degree. C. for 30 seconds, 50.degree. C. for 30 seconds,
72.degree. C. for 30 seconds; 35 cycles of 94.degree. C. for 30
seconds, 68.degree. C. for 30 seconds; and a 7-minute incubation at
72.degree. C. The PCR product was gel purified and restriction
digested with Bam HI and Xho I overnight.
[0104] A mammalian expression vector was constructed with the
dihyrofolate reductase gene selectable marker under control of the
SV40 early promoter, SV40 polyadenylation site, a cloning site to
insert the gene of interest under control of the mouse
metallothionein 1 (MT-1) promoter and the hGH polyadenylation site.
The expression vector was designated pZP-9 and has been deposited
at the American Type Culture Collection, 12301 Parklawn Drive,
Rockville, Md. under accession no 98668. To facilitate protein
purification, the pZP9 vector was modified by addition of a tissue
plasminogen activator (t-PA) secretory signal sequence (see U.S.
Pat. No. 5,641,655) and a GluGlu tag sequence (SEQ ID NO: 18)
between the MT-1 promoter and hGH terminator. The t-PA secretory
signal sequence replaces the native secretory signal sequence for
DNAs encoding polypeptides of interest that are inserted into this
vector, and expression results in an N-terminally tagged protein.
The N-terminally tagged vector was designated pZP9NEE. The vector
was digested with Bam HI and Xho I, and the zkun5 fragment was
inserted. The resulting construct was confirmed by sequencing.
Example 3
[0105] The human zkun5 gene was mapped to chromosome 7 using the
commercially available GeneBridge 4 Radiation Hybrid Panel
(Research Genetics, Inc., Huntsville, Ala.) The GeneBridge 4
Radiation Hybrid Panel contains PCRable DNAs from each of 93
radiation hybrid clones, plus two control DNAs (the HFL donor and
the A23 recipient). A publicly available world-wide web server
(http://www-genome.wi.mit.edu/cgi-bin/contig/rhmapp- er.pl) allows
mapping relative to the Whitehead Institute/MIT Center for Genome
Research's radiation hybrid map of the human genome (the "WICGR"
radiation hybrid map), which was constructed with the GeneBridge 4
Radiation Hybrid Panel.
[0106] For the mapping of the zkun5 gene, 20-.mu.l reaction
mixtures were set up in a PCRable 96-well microtiter plate
(Stratagene, La Jolla, Calif.) and used in a thermal cycler
(RoboCycler.RTM. Gradient 96; Stratagene). Each of the 95 PCR
reaction mixtures contained 2 .mu.l buffer (10.times. KlenTaq PCR
reaction buffer; Clontech Laboratories, Inc., Palo Alto, Calif.),
1.6 .mu.l dNTPs mix (2.5 mM each, PERKIN-ELMER, Foster City,
Calif.), 1 .mu.l sense primer (ZC16,523; SEQ ID NO: 19), 1 .mu.l
antisense primer (ZC16,522; SEQ ID NO: 20), 2 .mu.l of a density
increasing agent and tracking dye (RediLoad, Research Genetics,
Inc., Huntsville, Ala.), 0.4 .mu.l of a commercially available DNA
polymerase/antibody mix (50.times. Advantage.TM. KlenTaq Polymerase
Mix; Clontech Laboratories, Inc.), 25 ng of DNA from an individual
hybrid clone or control and .times..mu.l ddH.sub.2O, for a total
volume of 20 .mu.l. The mixtures were overlaid with an equal amount
of mineral oil and sealed. The PCR cycler conditions were as
follows: an initial 5 minute denaturation at 95.degree. C.; 35
cycles of a 1 minute denaturation at 95.degree. C., 1 minute
annealing at 64.degree. C., and 1.5 minute extension at 72.degree.
C.; followed by a final extension of 7 minutes at 72.degree. C. The
reaction products were separated by electrophoresis on a 2% agarose
gel (Life Technologies, Gaithersburg, Md.).
[0107] The results showed that the zkun5 gene maps 2.74
cR.sub.--3000 from the framework marker D7S481 on the chromosome 7
WICGR radiation hybrid map. Proximal and distal framework markers
were D7S481 and CHLC.GATA84AO8, respectively. The use of
surrounding markers positions the zkun5 gene in the 7p22.2-p22.1
region on the integrated LDB chromosome 7 map (The Genetic Location
Database, University of Southhampton, WWW server:
http://cedar.genetics. soton.ac.uk/public_html/- ).
[0108] From the foregoing, it will be appreciated that, although
specific embodiments of the invention have been described herein
for purposes of illustration, various modifications may be made
without deviating from the spirit and scope of the invention.
Accordingly, the invention is not limited except as by the appended
claims.
Sequence CWU 0
0
* * * * *
References