U.S. patent application number 11/548824 was filed with the patent office on 2007-03-22 for uses of human zven proteins and polynucleotides.
This patent application is currently assigned to ZymoGenetics, Inc.. Invention is credited to Chung Chan, Richard M. Garcia, Susan D. Holderman, Stephen R. Jaspers, Katherine E. Lewis, Paul O. Sheppard, Penny J. Thompson, Robert R. West, Anitra Wolf.
Application Number | 20070065405 11/548824 |
Document ID | / |
Family ID | 32097184 |
Filed Date | 2007-03-22 |
United States Patent
Application |
20070065405 |
Kind Code |
A1 |
Thompson; Penny J. ; et
al. |
March 22, 2007 |
Uses of human Zven proteins and polynucleotides
Abstract
The present invention provides methods of using Zven1 and Zven2
polypeptides to increase chemokine production. The present
invention also provides methods for treating intestinal motility
disorders and improving gastrointestinal function with Zven1 and
Zven2 polypeptides.
Inventors: |
Thompson; Penny J.;
(Snohomish, WA) ; Lewis; Katherine E.; (Lake
Forest Park, WA) ; Jaspers; Stephen R.; (Edmonds,
WA) ; Garcia; Richard M.; (Kirkland, WA) ;
West; Robert R.; (Seattle, WA) ; Holderman; Susan
D.; (Seattle, WA) ; Chan; Chung; (Issaquah,
WA) ; Sheppard; Paul O.; (Fall City, WA) ;
Wolf; Anitra; (Seattle, WA) |
Correspondence
Address: |
ZYMOGENETICS, INC.;INTELLECTUAL PROPERTY DEPARTMENT
1201 EASTLAKE AVENUE EAST
SEATTLE
WA
98102-3702
US
|
Assignee: |
ZymoGenetics, Inc.
|
Family ID: |
32097184 |
Appl. No.: |
11/548824 |
Filed: |
October 12, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10680800 |
Oct 7, 2003 |
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11548824 |
Oct 12, 2006 |
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60416719 |
Oct 7, 2002 |
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60416718 |
Oct 7, 2002 |
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60434116 |
Dec 16, 2002 |
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60433918 |
Dec 16, 2002 |
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60508614 |
Oct 3, 2003 |
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60508603 |
Oct 3, 2003 |
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Current U.S.
Class: |
424/85.2 ;
424/94.2 |
Current CPC
Class: |
G01N 2800/06 20130101;
A61P 29/00 20180101; C07K 14/4702 20130101; A61K 38/1709 20130101;
A61P 1/00 20180101; G01N 33/6893 20130101; C07K 14/47 20130101;
G01N 2800/065 20130101; A61P 1/04 20180101; A61P 37/00
20180101 |
Class at
Publication: |
424/085.2 ;
424/094.2 |
International
Class: |
A61K 38/54 20060101
A61K038/54 |
Claims
1. A method of modulating gastrointestinal contractility, gastric
emptying or intestinal transit in a mammal in need thereof,
comprising administering to the mammal a polypeptide, wherein the
polypeptide comprises amino acid residues 28 to 129 of SEQ ID
NO:29.
2. The method according to claim 1, wherein the modulation is
inhibition.
3. The method according to claim 1, wherein the polypeptide is
administered in one or more administrations.
4. The method according to claim 3, wherein one or more of the
administrations of the polypeptide stimulates gastrointestinal
contractility, and wherein one or more of the administrations of
the polypeptide inhibits gastrointestinal contractility.
5. The method according to claim 4, wherein the one or more
administrations that stimulate gastrointestinal contractility are
administered before the one or more administrations that inhibit
gastrointestinal contractility.
6. The method according to claim 4, wherein the one or more
administrations that inhibit gastrointestinal contractility are
administered before the one or more administrations that stimulate
gastrointestinal contractility.
7. The method according to claim 4, wherein therapeutic control of
gastric contractility is achieved.
8. The method according to claim 1, wherein the polypeptide is
administered continually for a period of time.
9. A method of modulating gastric emptying in a mammal in need
thereof comprising administering to the mammal a polypeptide,
wherein the polypeptide comprises the amino acid sequence of amino
acid residues 28 to 129 of SEQ ID NO:29.
10. The method according to claim 9, wherein the modulation is
inhibition.
11. The method according to claim 10, wherein the polypeptide is
administered in one or more administration.
12. The method according to claim 11, wherein one or more of the
administrations of the polypeptide stimulates gastric emptying, and
wherein one or more of the administrations of the polypeptide
inhibits gastric emptying.
13. The method according to claim 12, wherein the one or more
administrations that stimulate gastric emptying are administered
before the one or more administrations that inhibit gastric
emptying.
14. The method according to claim 13, wherein the one or more
administrations that inhibit gastric emptying are administered
before the one or more administrations that stimulate gastric
emptying.
15. The method according to claim 12, wherein therapeutic control
of gastric emptying is achieved.
16. The method according to claim 9, wherein the polypeptide is
administered continually for a period of time.
17. A method of modulating intestinal transit in a mammal in need
thereof comprising administering to the mammal a polypeptide,
wherein the polypeptide comprises the amino acid sequence of amino
acid residues 28 to 129 of SEQ ID NO:29.
18. The method according to claim 17, wherein the modulation is
inhibition.
19. The method according to claim 18, wherein the polypeptide is
administered in one or more administration.
20. The method according to claim 19, wherein one or more of the
administrations of the polypeptide stimulates intestinal transit,
and wherein one or more of the administrations of the polypeptide
are effective in inhibiting intestinal transit.
21. The method according to claim 20, wherein the one or more
administrations that stimulate intestinal transit are administered
before the one or more administrations that inhibit intestinal
transit.
22. The method according to claim 20, wherein the one or more
administrations that inhibit intestinal transit are administered
before the one or more administrations that stimulate intestinal
transit.
23. The method according to claim 20, wherein therapeutic control
of intestinal transit is achieved.
24. The method according to claim 17, wherein the polypeptide is
administered continually for a period of time.
25. A method of treating gastroparesis in a mammal in thereof
comprising, administering to the mammal a polypeptide, wherein the
polypeptide comprises the amino acid sequence of amino acid
residues 28 to 129 of SEQ ID NO:29, and wherein gastrointestinal
contractility, gastric emptying, or intestinal transit is
improved.
26. The method according to claim 24, wherein the gastroparesis is
related to surgery.
27. The method according to claim 25, wherein the polypeptide is
administered to the mammal before or after the surgery.
28. The method according to claim 26, wherein the polypeptide is
administered to the mammal before or after the mammal is fed a
post-surgery meal.
29. The method according to claim 25, wherein the treatment is
characterized by an increase in contractility in the ileus.
30. The method according to claim 25, wherein the gastroparesis is
post-operative ileus, or paralytic ileus.
31. The method according to claim 24, wherein the gastroparesis is
not related to surgery.
32. The method according to claim 25, wherein the gastroparesis is
related to diabetes, intestinal pseudo-obstruction, chronic
constipation, dyspepsia, gastroesophageal reflux, emesis, paralytic
gastroparesis, sepsis, or consumption of medications.
33. The method according to claim 1, wherein the polypeptide is
administered orally, intraperitoneally, intravenously,
intramuscularly, or sub cutaneously.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 10/680,800, filed Oct. 7, 2003, which claims the benefit of
U.S. Provisional Application Ser. No. 60/416,719, filed Oct. 7,
2002; U.S. Provisional Application Ser. No. 60/416,718, filed Oct.
7, 2002; U.S. Provisional Application Ser. No. 60/434,116, filed
Dec. 16, 2002; U.S. Provisional Application Ser. No. 60/433,918,
filed Dec. 16, 2002; U.S. Provisional Application Ser. No.
60/508,614, filed Oct. 3, 2003; and U.S. Provisional Application
Ser. No. 60/508,603, filed Oct. 3, 2003, all of which are herein
incorporated by reference.
BACKGROUND OF THE INVENTION
[0002] Optimal gastrointestinal function includes mixing and
forward propulsion of contents in the stomach and intestine.
Gastric emptying is frequently abnormal in patients with critical
illness or who are recovering from surgery. Recovery of
gastrointestinal function and resumption of oral intake are
important determinants in recovery from an event that compromises
gastrointestinal function. Several events can lead to dysfunction
in the gastrointestinal system, including, for example, ileus
(post-operative and paralytic), chronic constipation, gastroparesis
(including diabetic gastroparesis), intestinal pseudo-obstruction,
dyspepsia, gastroesophageal reflux, and emesis.
[0003] Diseases and disorders of impaired or compromised
gastrointestinal function include ileus and gastroparesis.
Post-operative ileus (POI) is a condition of reduced intestinal
tract motility, including delayed gastric emptying, that occurs as
a result of disrupted muscle tone following surgery. It is
especially problematic following abdominal surgery. The problem may
arise from the surgery itself, from the residual effects of
anesthetic agents, and particularly, from pain-relieving narcotic
and opiate drugs used during and after surgery. Post-operative
ileus can be categorized as "uncomplicated", lasting two to three
days after surgery, or as "paralytic", lasting more than three days
after surgery. Thus, patients undergoing abdominal surgery who have
a delay in recovery of gastrointestinal function have prolonged
hospital stays, which can lead to increased medical costs and
potentially to other complications. An estimated 750 million to one
billion dollars is spent each year in increased hospitalization due
to post-operative ileus. Currently there are no drugs that have
been approved for treatment of this disease.
[0004] In addition to the need for a better therapeutic for
post-operative ileus, there is a need for a better therapeutic for
diabetic gastroparesis. Diabetic gastroparesis is paralysis of the
stomach brought about by a motor abnormality in the stomach, as a
complication of both type I and type II diabetes. Diabetic
gastroparesis is characterized by delayed gastric emptying,
post-prandial distention, nausea and vomiting. In diabetes, it is
thought to be due to a neuropathy, though it is also associated
with loss of interstitial cells of Cajal (ICC), which are the
"pacemaker cells" of the gut.
[0005] In the U.S. alone, there are at least 16 million individuals
with diabetes, affecting approximately 7% of the population. The
prevalence is continuing to increase and is growing worldwide.
Since up to two-thirds of individuals with diabetes suffer from
some degree of gastroparesis, this problem is significant. Episodes
are often acute, though long-term treatment is often required.
Moreover, symptoms associated with diabetic gastroparesis, such as
delayed gastric emptying, and emesis can cause water and
electrolyte imbalances, poor glycemic control, and ensuing
complications. If severe enough, it may require hospitalization for
control of diabetes, and treatment with intravenous fluids and
nutrition.
[0006] The often-acute nature of the episodes provides an
opportunity to treat with a prokinetic. Currently there are very
few drugs that can effectively treat diabetic gastroparesis, and
those that are available have side effects and/or cannot be taken
with other medications. Oral drugs may not be tolerated during
severe episodes, and thus, would require intravenous administration
of a prokinetic. In the United States, only two agents,
erythromycin and metoclopramide, are available to treat
gastroparesis.
[0007] Thus, a need still exists for therapeutic approaches to
treatment of gastric function disorders.
BRIEF SUMMARY OF THE INVENTION
[0008] The present invention provides proteins useful for the
treatment in recovery of gastronintestinal function and gastric
emptying. Other uses of Zven1 and Zven2 polypeptides are described
in more detail below.
DESCRIPTION OF THE INVENTION
1. OVERVIEW
[0009] The present invention is directed to novel uses of
previously described proteins, Zven1 and Zven2. See U.S. patent
application Ser. No. 09/712,529, now issued as U.S. Pat. No.
6,485,938. Zven1 and Zven2 are also known in the industry as
Prokineticin2 and Prokineticin1, respectively. As discussed herein,
Zven1 and Zven2, as well as variants and fragments thereof, can be
used to regulate gastrointestinal function and gastric emptying.
Receptors for Zven1 (Prokineticin2) and Zven2 (Prokineticin1) have
been identified as G protein-coupled receptors, GPCR73a and
GPCR73b. See Lin, D. et al., J. Biol. Chem. 277: 19276-19280, 2002.
The GPCR73a and GPCR73b receptors are also known as PK-R1 and
PK-R2.
[0010] The present invention provides methods of using human Zven
polypeptides and nucleic acid molecules that encode human Zven
polypeptides. An illustrative nucleic acid molecule containing a
sequence that encodes the Zven1 polypeptide has the nucleotide
sequence of SEQ ID NO:1. The encoded polypeptide has the following
amino acid sequence: MRSLCCAPLL LLLLLPPLLL TPRAGDAAVI TGACDKDSQC
GGGMCCAVSI WVKSIRICTP MGKLGDSCHP LTRKVPFFGR RMHHTCPCLP GLACLRTSFN
RFICLAQK (SEQ ID NO:2). Thus, the Zven1 nucleotide sequence
described herein encodes a polypeptide of 108 amino acids. The
putative signal sequences of Zven1 polypeptide reside at amino acid
residues 1 to 20, 1 to 21, and 1 to 22 of SEQ ID NO:2. The mature
form of the polypeptide comprises the amino acid sequence from
amino acid 28 to 108 as shown in SEQ ID NO:2.
[0011] A longer form of the sequence as shown in SEQ ID NO:2 is
included in the invention described herein. The longer form has the
following amino acid sequence: MRSLCCAPLL LLLLLPPLLL TPRAGDAAVI
TGACDKDSQC GGGMCCAVSI WVKSIRICTP MGKLGDSCHP LTRKNNFGNG RQERRKRKRS
KRKKEVPFFG RRMHHTCPCL PGLACLRTSF NRFICLAQK (SEQ ID NO:29). The
putative signal sequence of the longer form has a mature form that
comprises the amino acid sequence from amino acid 28 to 129 as
shown in SEQ ID NO:29.
[0012] An illustrative nucleic acid molecule containing a sequence
that encodes the Zven2 polypeptide has the nucleotide sequence of
SEQ ID NO:4. The encoded polypeptide has the following amino acid
sequence: MRGATRVSIM LLLVTVSDCA VITGACERDV QCGAGTCCAI SLWLRGLRMC
TPLGREGEEC HPGSHKVPFF RKRKHHTCPC LPNLLCSRFP DGRYRCSMDL KNINF (SEQ
ID NO:5). Thus, the Zven2 nucleotide sequence described herein
encodes a polypeptide of 105 amino acids. The putative signal
sequences of Zven2 polypeptide reside at amino acid residues 1 to
17, and 1 to 19 of SEQ ID NO:5.
[0013] As described below, the present invention provides isolated
polypeptides comprising an amino acid sequence that is at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, or at
least 95% identical to amino acid residues 23 to 108 of SEQ ID
NO:2, to amino acid residues 28 to 108 of SEQ ID NO:2, or to amino
acid residues 28 to 129 if SEQ ID NO:29. Certain of such isolated
polypeptides can specifically bind with an antibody that
specifically binds with a polypeptide consisting of the amino acid
sequence of SEQ ID NO:2. Particular polypeptides can increase or
decrease gastric contractility, gastric emptying and/or intestinal
transit. An illustrative polypeptide is a polypeptide that
comprises the amino acid sequence of SEQ ID NO:2.
[0014] Similarly, the present invention includes provides isolated
polypeptides comprising an amino acid sequence that is at least
70%, at least 75%, at least 80%, at least 85%, at least 90%, or at
least 95% identical to amino acid residues 20 to 105 of SEQ ID
NO:5, wherein such isolated polypeptides can specifically bind with
an antibody that specifically binds with a polypeptide consisting
of the amino acid sequence of SEQ ID NO:5. An illustrative
polypeptide is a polypeptide that comprises the amino acid sequence
of SEQ ID NO:5.
[0015] The present invention also provides polypeptides comprising
an amino acid sequence selected from the group consisting of: (1)
amino acid residues 21 to 108 of SEQ ID NO:2, (2) amino acid
residues 22 to 108 of SEQ ID NO:2, (3) amino acid residues 23 to
108 of SEQ ID NO:2, (4) amino acid residues 82 to 108 of SEQ ID
NO:2, (5) amino acid residues 1 to 78 (amide) of SEQ ID NO:2, (6)
amino acid residues 1 to 79 of SEQ ID NO:2, (7) amino acid residues
21 to 78 (amide) of SEQ ID NO:2, (8) amino acid residues 21 to 79
of SEQ ID NO:2, (9) amino acid residues 22 to 78 (amide) of SEQ ID
NO:2, (10) amino acid residues 22 to 79 of SEQ ID NO:2, (11) amino
acid residues 23 to 78 (amide) of SEQ ID NO:2, (12) amino acid
residues 23 to 79 of SEQ ID NO:2, (13) amino acid residues 20 to
108 of SEQ ID NO:2, (14) amino acid residues 20 to 72 of SEQ ID
NO:2, (15) amino acid residues 20 to 79 of SEQ ID NO:2, (16) amino
acid residues 20 to 79 (amide) of SEQ ID NO:2, (17) amino acid
residues 21 to 72 of SEQ ID NO:2, (18) amino acid residues 21 to 79
(amide) of SEQ ID NO:2, (19) amino acid residues 22 to 72 of SEQ ID
NO:2, (20) amino acid residues 22 to 79 (amide) of SEQ ID NO:2,
(21) amino acid residues 23 to 72 of SEQ ID NO:2, (22) amino acid
residues 23 to 79 (amide) of SEQ ID NO:2, (23) amino acid residues
28 to 108 of SEQ ID NO:2, (24) amino acid residues 28 to 72 of SEQ
ID NO:2, (25) amino acid residues 28 to 79 of SEQ ID NO:2, (26)
amino acid residues 28 to 79 (amide) of SEQ ID NO:2, (27) amino
acid residues 75 to 108 of SEQ ID NO:2, (28) amino acid residues 75
to 79 of SEQ ID NO:2, and (29) amino acid residues 75 to 78 (amide)
of SEQ ID NO:2. Illustrative polypeptides consist of amino acid
sequences (1) to (29). The present invention also included
polypeptide comprising an amino acid sequence comprising amino acid
28 to 129 as shown in SEQ ID NO:29, and/or fragments thereof.
[0016] The present invention further includes polypeptides
comprising an amino acid sequence selected from the group
consisting of: (a) amino acid residues 20 to 105 of SEQ ID NO:5,
(b) amino acid residues 18 to 105 of SEQ ID NO:5, (c) amino acid
residues 1 to 70 of SEQ ID NO:5, (d) amino acid residues 20 to 70
of SEQ ID NO:5, (e) amino acid residues 18 to 70 of SEQ ID NO:5,
(f) amino acid residues 76 to 105 of SEQ ID NO:5, (g) amino acid
residues 66 to 105 of SEQ ID NO:5, and (h) amino acid residues 82
to 105 of SEQ ID NO:5. Illustrative polypeptides consist of amino
acid sequences (a) to (h).
[0017] The present invention further provides antibodies and
antibody fragments that specifically bind with such polypeptides.
Exemplary antibodies include polyclonal antibodies, murine
monoclonal antibodies, humanized antibodies derived from murine
monoclonal antibodies, and human monoclonal antibodies.
Illustrative antibody fragments include F(ab').sub.2, F(ab).sub.2,
Fab', Fab, Fv, scFv, and minimal recognition units. The present
invention also includes anti-idiotype antibodies that specifically
bind with such antibodies or antibody fragments. The present
invention further includes compositions comprising a carrier and a
peptide, polypeptide, antibody, or anti-idiotype antibody described
herein.
[0018] The present invention also provides isolated nucleic acid
molecules that encode a Zven polypeptide, wherein the nucleic acid
molecule is selected from the group consisting of (a) a nucleic
acid molecule comprising the nucleotide sequence of SEQ ID NO:3,
(b) a nucleic acid molecule encoding the amino acid sequence of SEQ
ID NO:2, (c) a nucleic acid molecule that remains hybridized
following stringent wash conditions to a nucleic acid molecule
consisting of the nucleotide sequence of nucleotides 66 to 161 of
SEQ ID NO:1, the nucleotide sequence of nucleotides 288 to 389 of
SEQ ID NO:1, or to the complement of the nucleotide sequence of
either nucleotides 66 to 161 of SEQ ID NO:1 or nucleotides 288 to
389 of SEQ ID NO:1, (d) a nucleic acid molecule comprising the
nucleotide sequence of SEQ ID NO:6, (e) a nucleic acid molecule
encoding the amino acid sequence of SEQ ID NO:5, (f) a nucleic acid
molecule that remains hybridized following stringent wash
conditions to a nucleic acid molecule consisting of the nucleotide
sequence of nucleotides 334 to 405 of SEQ ID NO:4, or to the
complement of the nucleotide sequence of nucleotides 334 to 405 of
SEQ ID NO:4.
[0019] Illustrative nucleic acid molecules include those in which
any difference between the amino acid sequence encoded by the
nucleic acid molecule and the corresponding amino acid sequence of
either SEQ ID NO:2 or SEQ ID NO:5 is due to a conservative amino
acid substitution. The present invention further contemplates
isolated nucleic acid molecules that comprise a nucleotide sequence
of nucleotides 132 to 389 of SEQ ID NO:1, nucleotides 147 to 389 of
SEQ ID NO:1, and nucleotides 148 to 405 of SEQ ID NO:4.
[0020] The present invention also includes vectors and expression
vectors comprising such nucleic acid molecules. Such expression
vectors may comprise a transcription promoter, and a transcription
terminator, wherein the promoter is operably linked with the
nucleic acid molecule, and wherein the nucleic acid molecule is
operably linked with the transcription terminator. The present
invention further includes recombinant host cells comprising these
vectors and expression vectors. Illustrative host cells include
bacterial, yeast, avian, fungal, insect, mammalian, and plant
cells. Recombinant host cells comprising such expression vectors
can be used to prepare Zven polypeptides by culturing such
recombinant host cells that comprise the expression vector and that
produce the Zven protein, and, optionally, isolating the Zven
protein from the cultured recombinant host cells. The present
invention further includes products made by such processes.
[0021] In addition, the present invention provides pharmaceutical
compositions comprising a pharmaceutically acceptable carrier and
at least one of such an expression vector or recombinant virus
comprising such expression vectors.
[0022] The present invention also contemplates methods for
detecting the presence of Zven1 RNA in a biological sample,
comprising the steps of (a) contacting a Zven1 nucleic acid probe
under hybridizing conditions with either (i) test RNA molecules
isolated from the biological sample, or (ii) nucleic acid molecules
synthesized from the isolated RNA molecules, wherein the probe has
a nucleotide sequence comprising a portion of the nucleotide
sequence of SEQ ID NO:1, or its complement, and (b) detecting the
formation of hybrids of the nucleic acid probe and either the test
RNA molecules or the synthesized nucleic acid molecules, wherein
the presence of the hybrids indicates the presence of Zven1 RNA in
the biological sample. Analogous methods can be used to detect the
presence of Zven2 RNA in a biological sample, wherein the probe has
a nucleotide sequence comprising a portion of the nucleotide
sequence of SEQ ID NO:4, or its complement.
[0023] The present invention further provides methods for detecting
the presence of Zven polypeptide in a biological sample, comprising
the steps of: (a) contacting the biological sample with an antibody
or an antibody fragment that specifically binds with a polypeptide
either consisting of the amino acid sequence of SEQ ID NO:2 or
consisting of the amino acid sequence of SEQ ID NO:5, wherein the
contacting is performed under conditions that allow the binding of
the antibody or antibody fragment to the biological sample, and (b)
detecting any of the bound antibody or bound antibody fragment.
Such an antibody or antibody fragment may further comprise a
detectable label selected from the group consisting of
radioisotope, fluorescent label, chemiluminescent label, enzyme
label, bioluminescent label, and colloidal gold.
[0024] Illustrative biological samples include human tissue, such
as an autopsy sample, a biopsy sample, body fluids and digestive
components, and the like.
[0025] The present invention also provides kits for performing
these detection methods. For example, a kit for detection of Zven1
gene expression may comprise a container that comprises a nucleic
acid molecule, wherein the nucleic acid molecule is selected from
the group consisting of (a) a nucleic acid molecule comprising the
nucleotide sequence of nucleotides 66 to 161 of SEQ ID NO:1, (b) a
nucleic acid molecule comprising the nucleotide sequence of
nucleotides 288 to 389 of SEQ ID NO:1, (c) a nucleic acid molecule
comprising the complement of the nucleotide sequence of nucleic
acid molecules (a) or (b), (d) a nucleic acid molecule that is a
fragment of (a) consisting of at least eight nucleotides, (e) a
nucleic acid molecule that is a fragment of (b) consisting of at
least eight nucleotides, (f) a nucleic acid molecule that is a
fragment of (c) consisting of at least eight nucleotides, and (g) a
nucleic acid molecule that is a fragment of or consists of the
nucleic acid sequence as shown in SEQ ID NO: 12, 13, 15, 16, 17,
18, 19, 20, 23, or 24. A kit for detection of Zven2 gene expression
may comprise a container that comprises a nucleic acid molecule,
wherein the nucleic acid molecule is selected from the group
consisting of (a) a nucleic acid molecule comprising the nucleotide
sequence of nucleotides 334 to 405 of SEQ ID NO:4, (b) a nucleic
acid molecule comprising the complement of the nucleotide sequence
of (a), (c) a nucleic acid molecule that is a fragment of (a)
consisting of at least eight nucleotides, and (d) a nucleic acid
molecule that is a fragment of (b) consisting of at least eight
nucleotides. Such kits may also comprise a second container that
comprises one or more reagents capable of indicating the presence
of the nucleic acid molecule.
[0026] On the other hand, a kit for detection of Zven protein may
comprise a container that comprises an antibody, or an antibody
fragment, that specifically binds with a polypeptide consisting of
the amino acid sequence of SEQ ID NO:2 or consisting of the amino
acid sequence of SEQ ID NO:5.
[0027] The present invention also contemplates anti-idiotype
antibodies, or anti-idiotype antibody fragments, that specifically
bind an antibody or antibody fragment that specifically binds a
polypeptide consisting of the amino acid sequence of SEQ ID NO:2 or
the amino acid sequence of SEQ ID NO:5.
[0028] The present invention further provides variant Zven1
polypeptides, which comprise an amino acid sequence that shares an
identity with the amino acid sequence of SEQ ID NO:2 selected from
the group consisting of at least 70% identity, at least 80%
identity, at least 90% identity, at least 95% identity, or greater
than 95% identity, and wherein any difference between the amino
acid sequence of the variant polypeptide and the amino acid
sequence of SEQ ID NO:2 is due to one or more conservative amino
acid substitutions. Illustrative variant Zven2 polypeptides, which
comprise an amino acid sequence that shares an identity with the
amino acid sequence of SEQ ID NO:5 selected from the group
consisting of at least 70% identity, at least 80% identity, at
least 90% identity, at least 95% identity, or greater than 95%
identity, and wherein any difference between the amino acid
sequence of the variant polypeptide and the amino acid sequence of
SEQ ID NO:5 is due to one or more conservative amino acid
substitutions.
[0029] The present invention also provides fusion proteins
comprising a Zven1 polypeptide moiety or a Zven2 polypeptide
moiety. Such fusion proteins can further comprise an immunoglobulin
moiety. A suitable immunoglobulin moiety is an immunoglobulin heavy
chain constant region, such as a human Fc fragment. The present
invention also includes isolated nucleic acid molecules that encode
such fusion proteins.
[0030] The present invention further provides a method of treating
defective ileal contractility disease in a mammalian subject in
need of such treatment, comprising: administering to the mammalian
subject a Zven1 polypeptide, wherein the Zven1 polypeptide
comprises the amino acid sequence of amino acid residues 23 to 108
of SEQ ID NO:2. In one embodiment, the disease is diabetes
mellitus. In another method, the disease is post-operative ileus.
In another embodiment, the disease is sepsis-related
gastrointestinal stasis or ileus.
[0031] The present invention further provides a method of treating
defective ileal contractility disease in a mammalian subject in
need of such treatment, comprising: administering to the mammalian
subject a Zven1 polypeptide, wherein the Zven1 polypeptide
comprises the amino acid sequence of amino acid residues 28 to 108
of SEQ ID NO:2, the amino acid sequence of amino acid residues 20
to 105 of SEQ ID NO:5, or the amino acid sequence of amino acid
residues 28 to 129 of SEQ ID NO:29. In one embodiment, the disease
is diabetes mellitus. In another embodiment, the disease is
post-operative ileus. In another embodiment, the disease is
sepsis-related gastrointestinal stasis or ileus.
[0032] The invention further provides a method of modulating
gastrointestinal contractility in a mammal in need thereof
comprising administering to the mammal a polypeptide, wherein the
polypeptide comprises the amino acid sequence of amino acid
residues 28 to 108 of SEQ ID NO:2. In an embodiment, the modulation
is inhibition. In another embodiment, the polypeptide is
administered in one or more administrations. In a further
embodiment, one or more of the administrations of the polypeptide
stimulates gastrointestinal contractility, and wherein one or more
of the administrations of the polypeptide inhibits gastrointestinal
contractility. In another embodiment, the one or more
administrations that stimulate gastrointestinal contractility are
administered before the one or more administrations that inhibit
gastrointestinal contractility. In another embodiment, the one or
more administrations that inhibit gastrointestinal contractility
are administered before the one or more administrations that
stimulate gastrointestinal contractility. In a further embodiment,
therapeutic control of gastric contractility is achieved. In
another embodiment, the polypeptide is administered continually for
a period of time.
[0033] The invention also provides a method of stimulating
gastrointestinal contractility comprising administering to a mammal
in need thereof a polypeptide comprising the amino acid sequence of
amino acid residues acids 20 to 105 as shown in SEQ ID NO:5,
followed by administering a polypeptide comprising the amino acid
sequence of amino acids 28 to 108 of SEQ ID NO:2.
[0034] The invention also provides a method of modulating gastric
emptying in a mammal in need thereof comprising administering to
the mammal a polypeptide, wherein the polypeptide comprises the
amino acid sequence of amino acid residues 28 to 108 of SEQ ID
NO:2. In an embodiment, the modulation is inhibition. In another
embodiment, the polypeptide is administered in one or more
administration. In another embodiment, one or more of the
administrations of the polypeptide stimulates gastric emptying, and
wherein one or more of the administrations of the polypeptide
inhibits gastric emptying. In a further embodiment, the one or more
administrations that stimulate gastric emptying are administered
before the one or more administrations that inhibit gastric
emptying. In another further embodiment, the one or more
administrations that inhibit gastric emptying are administered
before the one or more administrations that stimulate gastric
emptying. In another embodiment, therapeutic control of gastric
emptying is achieved. In another embodiment the polypeptide is
administered continually for a period of time.
[0035] The invention also provides a method of modulating
intestinal transit in a mammal in need thereof comprising
administering to the mammal a polypeptide, wherein the polypeptide
comprises the amino acid sequence of amino acid residues 28 to 108
of SEQ ID NO:2. In an embodiment, the modulation is inhibition. In
another embodiment, the polypeptide is administered in one or more
administration. In a further embodiment, one or more of the
administrations of the polypeptide stimulates intestinal transit,
and wherein one or more of the administrations of the polypeptide
are effective in inhibiting intestinal transit. In a further
embodiment, the one or more administrations that stimulate
intestinal transit are administered before the one or more
administrations that inhibit intestinal transit. In another further
embodiment, the one or more administrations that inhibit intestinal
transit are administered before the one or more administrations
that stimulate intestinal transit. In another embodiment,
therapeutic control of intestinal transit is achieved. In another
embodiment, the polypeptide is administered continually for a
period of time.
[0036] The invention also provides a method of treating
gastroparesis in a mammal in thereof comprising, administering to
the mammal a polypeptide, wherein the polypeptide comprises the
amino acid sequence of amino acid residues 28 to 108 of SEQ ID
NO:2, and wherein gastrointestinal contractility, gastric emptying,
or intestinal transit is improved. In an embodiment, the
gastroparesis is related to surgery. In another embodiment, the
polypeptide is administered to the mammal before or after the
surgery. In another embodiment, the polypeptide is administered to
the mammal before or after the mammal is fed a post-surgery meal.
In another embodiment, the treatment is characterized by an
increase in contractility in the ileus. In another embodiment, the
gastroparesis is post-operative ileus, or paralytic ileus. In
another embodiment, the gastroparesis is not related to surgery. In
another embodiment, the gastroparesis is related to diabetes,
intestinal pseudo-obstruction, chronic constipation, dyspepsia,
gastroesophageal reflux, emesis, paralytic gastroparesis, sepsis,
or consumption of medications.
[0037] The invention also provides a method of stimulating
chemokine release in a mammal in need thereof, comprising
administering to the mammal a polypeptide, wherein the polypeptide
comprises the amino acid sequence of amino acid residues 28 to 108
of SEQ ID NO:2 or the amino acid sequence of amino acid residues 20
to 105 of SEQ ID NO:5.
[0038] The invention also provides a method of stimulating
chemokine release in a mammal in need thereof, comprising
administering to the mammal a polypeptide, wherein the polypeptide
comprises the amino acid sequence of amino acid residues 28 to 108
of SEQ ID NO:2 or the amino acid sequence of amino acid residues 20
to 105 of SEQ ID NO:5.
[0039] The invention also provides a method of stimulating
neutrophil infiltration in a mammal in need thereof, comprising
administering to the mammal a polypeptide, wherein the polypeptide
comprises the amino acid sequence of amino acid residues 28 to 108
of SEQ ID NO:2 or the amino acid sequence of amino acid residues 20
to 105 of SEQ ID NO:5.
[0040] The invention also provides a method of inducing or
increasing appetite or weight gain in a mammal in need thereof
comprising administering to the mammal a polypeptide, wherein the
polypeptide comprises the amino acid sequence of amino acid
residues 28 to 108 of SEQ ID NO:2 or the amino acid sequence of
amino acid residues 20 to 105 of SEQ ID NO:5.
[0041] The invention also provides a method of increasing
sensitization to a thermal, mechanical or painful stimulus in a
mammal in need thereof, comprising administering to the mammal a
polypeptide, wherein the polypeptide comprises the amino acid
sequence of amino acid residues 28 to 108 of SEQ ID NO:2 or the
amino acid sequence of amino acid residues 20 to 105 of SEQ ID
NO:5.
[0042] The invention also provides a method of decreasing
sensitization to a thermal, mechanical or painful stimulus in a
mammal in need thereof, comprising administering to the mammal an
antagonist to a polypeptide, wherein the polypeptide comprises the
amino acid sequence of amino acid residues 28 to 108 of SEQ ID NO:2
or the amino acid sequence of amino acid residues 20 to 105 of SEQ
ID NO:5. Within an embodiment, the antagonist is an antibody that
specifically binds to the polypeptide.
[0043] The invention also provides a method for inducing
vasculogenesis in cardiac stem cells, comprising administering a
polypeptide, wherein the polypeptide comprises the amino acid
sequence of amino acid residues 28 to 108 of SEQ ID NO:2. Within an
embodiment, the vasculogenesis is induced ex vivo or in vitro.
[0044] The invention also provides a for inducing angiogenesis in
cardiac stem cells, comprising administering a polypeptide, wherein
the polypeptide comprises the amino acid sequence of amino acid
residues 28 to 108 of SEQ ID NO:2. Within an embodiment, the
neogenesis is induced ex vivo or in vitro.
[0045] The invention also provides a method of modulating
gastrointestinal contractility, gastric emptying or intestinal
transit in a mammal in need thereof, comprising administering to
the mammal a polypeptide, wherein the polypeptide comprises the
amino acid sequence selected from: amino acid residues 28 to 108 of
SEQ ID NO:2; amino acid residues 20 to 105 of SEQ ID NO:5; and
amino acid residues 28 to 129 of SEQ ID NO:29. Within an
embodiment, the polypeptide is administered orally,
intraperitoneally, intravenously, intramuscularly, or
subcutaneously.
[0046] The invention also provides an isolated nucleic acid
comprising the nucleic acid sequence as shown in SEQ ID NO:14.
[0047] The invention also provides a method of producing a
polypeptide, comprising the step of culturing recombinant host
cells that comprise an expression vector, wherein the expression
vector comprises the isolated nucleic acid of as shown in SEQ ID
NO:14, a transcription promoter, and a transcription terminator,
wherein the promoter is operably linked with the nucleic acid, and
wherein the nucleic acid is operably linked with the transcription
terminator, and wherein the protein encoded by the nucleic acid is
produced by the recombinant cell. The invention also provides the
polypeptide produced by the method.
[0048] These and other aspects of the invention will become evident
upon reference to the following detailed description. In addition,
various references are identified below and are incorporated by
reference in their entirety.
2. DEFINITIONS
[0049] In the description that follows, a number of terms are used
extensively. The following definitions are provided to facilitate
understanding of the invention.
[0050] As used herein, "nucleic acid" or "nucleic acid molecule"
refers to polynucleotides, such as deoxyribonucleic acid (DNA) or
ribonucleic acid (RNA), oligonucleotides, fragments generated by
the polymerase chain reaction (PCR), and fragments generated by any
of ligation, scission, endonuclease action, and exonuclease action.
Nucleic acid molecules can be composed of monomers that are
naturally-occurring nucleotides (such as DNA and RNA), or analogs
of naturally-occurring nucleotides (e.g., .alpha.-enantiomeric
forms of naturally-occurring nucleotides), or a combination of
both. Modified nucleotides can have alterations in sugar moieties
and/or in pyrimidine or purine base moieties. Sugar modifications
include, for example, replacement of one or more hydroxyl groups
with halogens, alkyl groups, amines, and azido groups, or sugars
can be functionalized as ethers or esters. Moreover, the entire
sugar moiety can be replaced with sterically and electronically
similar structures, such as aza-sugars and carbocyclic sugar
analogs. Examples of modifications in a base moiety include
alkylated purines and pyrimidines, acylated purines or pyrimidines,
or other well-known heterocyclic substitutes. Nucleic acid monomers
can be linked by phosphodiester bonds or analogs of such linkages.
Analogs of phosphodiester linkages include phosphorothioate,
phosphorodithioate, phosphoroselenoate, phosphorodiselenoate,
phosphoroanilothioate, phosphoranilidate, phosphoramidate, and the
like. The term "nucleic acid molecule" also includes so-called
"peptide nucleic acids," which comprise naturally-occurring or
modified nucleic acid bases attached to a polyamide backbone.
Nucleic acids can be either single stranded or double stranded.
[0051] The term "complement of a nucleic acid molecule" refers to a
nucleic acid molecule having a complementary nucleotide sequence
and reverse orientation as compared to a reference nucleotide
sequence. For example, the sequence 5' ATGCACGGG 3' is
complementary to 5' CCCGTGCAT 3'.
[0052] The term "contig" denotes a nucleic acid molecule that has a
contiguous stretch of identical or complementary sequence to
another nucleic acid molecule. Contiguous sequences are said to
"overlap" a given stretch of a nucleic acid molecule either in
their entirety or along a partial stretch of the nucleic acid
molecule.
[0053] The term "degenerate nucleotide sequence" denotes a sequence
of nucleotides that includes one or more degenerate codons as
compared to a reference nucleic acid 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).
[0054] The term "structural gene" refers to a nucleic acid molecule
that is transcribed into messenger RNA (mRNA), which is then
translated into a sequence of amino acids characteristic of a
specific polypeptide.
[0055] An "isolated nucleic acid molecule" is a nucleic acid
molecule that is not integrated in the genomic DNA of an organism.
For example, a DNA molecule that encodes a growth factor that has
been separated from the genomic DNA of a cell is an isolated DNA
molecule. Another example of an isolated nucleic acid molecule is a
chemically-synthesized nucleic acid molecule that is not integrated
in the genome of an organism. A nucleic acid molecule that has been
isolated from a particular species is smaller than the complete DNA
molecule of a chromosome from that species.
[0056] A "nucleic acid molecule construct" is a nucleic acid
molecule, either single- or double-stranded, that has been modified
through human intervention to contain segments of nucleic acid
combined and juxtaposed in an arrangement not existing in
nature.
[0057] "Linear DNA" denotes non-circular DNA molecules having free
5' and 3' ends. Linear DNA can be prepared from closed circular DNA
molecules, such as plasmids, by enzymatic digestion or physical
disruption.
[0058] "Complementary DNA (cDNA)" is a single-stranded DNA molecule
that is formed from an mRNA template by the enzyme reverse
transcriptase. Typically, a primer complementary to portions of
mRNA is employed for the initiation of reverse transcription. Those
skilled in the art also use the term "cDNA" to refer to a
double-stranded DNA molecule consisting of such a single-stranded
DNA molecule and its complementary DNA strand. The term "cDNA" also
refers to a clone of a cDNA molecule synthesized from an RNA
template.
[0059] A "promoter" is a nucleotide sequence that directs the
transcription of a structural gene. Typically, a promoter is
located in the 5' non-coding region of a gene, proximal to the
transcriptional start site of a structural gene. Sequence elements
within promoters that function in the initiation of transcription
are often characterized by consensus nucleotide sequences. These
promoter elements include RNA polymerase binding sites, TATA
sequences, CAAT sequences, differentiation-specific elements (DSEs;
McGehee et al., Mol. Endocrinol. 7:551 (1993)), cyclic AMP response
elements (CREs), serum response elements (SREs; Treisman, Seminars
in Cancer Biol. 1:47 (1990)), glucocorticoid response elements
(GREs), and binding sites for other transcription factors, such as
CRE/ATF (O'Reilly et al., J. Biol. Chem. 267:19938 (1992)), AP2 (Ye
et al., J. Biol. Chem. 269:25728 (1994)), SP1, cAMP response
element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and
octamer factors (see, in general, Watson et al., eds., Molecular
Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing
Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J. 303:1
(1994)). If a promoter is an inducible promoter, then the rate of
transcription increases in response to an inducing agent. In
contrast, the rate of transcription is not regulated by an inducing
agent if the promoter is a constitutive promoter. Repressible
promoters are also known.
[0060] A "core promoter" contains essential nucleotide sequences
for promoter function, including the TATA box and start of
transcription. By this definition, a core promoter may or may not
have detectable activity in the absence of specific sequences that
may enhance the activity or confer tissue specific activity.
[0061] A "regulatory element" is a nucleotide sequence that
modulates the activity of a core promoter. For example, a
regulatory element may contain a nucleotide sequence that binds
with cellular factors enabling transcription exclusively or
preferentially in particular cells, tissues, or organelles. These
types of regulatory elements are normally associated with genes
that are expressed in a "cell-specific," "tissue-specific," or
"organelle-specific" manner.
[0062] An "enhancer" is a type of regulatory element that can
increase the efficiency of transcription, regardless of the
distance or orientation of the enhancer relative to the start site
of transcription.
[0063] "Heterologous DNA" refers to a DNA molecule, or a population
of DNA molecules, that does not exist naturally within a given host
cell. DNA molecules heterologous to a particular host cell may
contain DNA derived from the host cell species (i.e., endogenous
DNA) so long as that host DNA is combined with non-host DNA (i.e.,
exogenous DNA). For example, a DNA molecule containing a non-host
DNA segment encoding a polypeptide operably linked to a host DNA
segment comprising a transcription promoter is considered to be a
heterologous DNA molecule. Conversely, a heterologous DNA molecule
can comprise an endogenous gene operably linked with an exogenous
promoter. As another illustration, a DNA molecule comprising a gene
derived from a wild-type cell is considered to be heterologous DNA
if that DNA molecule is introduced into a mutant cell that lacks
the wild-type gene.
[0064] 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."
[0065] 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.
[0066] A peptide or polypeptide encoded by a non-host DNA molecule
is a "heterologous" peptide or polypeptide.
[0067] An "integrated genetic element" is a segment of DNA that has
been incorporated into a chromosome of a host cell after that
element is introduced into the cell through human manipulation.
Within the present invention, integrated genetic elements are most
commonly derived from linearized plasmids that are introduced into
the cells by electroporation or other techniques. Integrated
genetic elements are passed from the original host cell to its
progeny.
[0068] A "cloning vector" is a nucleic acid molecule, such as a
plasmid, cosmid, or bacteriophage, that has the capability of
replicating autonomously in a host cell. Cloning vectors typically
contain one or a small number of restriction endonuclease
recognition sites that allow insertion of a nucleic acid molecule
in a determinable fashion without loss of an essential biological
function of the vector, as well as nucleotide sequences encoding a
marker gene that is suitable for use in the identification and
selection of cells transformed with the cloning vector. Marker
genes typically include genes that provide tetracycline resistance
or ampicillin resistance.
[0069] An "expression vector" is a nucleic acid molecule encoding a
gene that is expressed in a host cell. Typically, an expression
vector comprises a transcription promoter, a gene, and a
transcription terminator. Gene expression is usually placed under
the control of a promoter, and such a gene is said to be "operably
linked to" the promoter. Similarly, a regulatory element and a core
promoter are operably linked if the regulatory element modulates
the activity of the core promoter.
[0070] A "recombinant host" is a cell that contains a heterologous
nucleic acid molecule, such as a cloning vector or expression
vector. In the present context, an example of a recombinant host is
a cell that produces a Zven1 or Zven2 peptide or polypeptide from
an expression vector. In contrast, such polypeptides can be
produced by a cell that is a "natural source" of Zven1 or Zven2,
and that lacks an expression vector.
[0071] "Integrative transformants" are recombinant host cells, in
which heterologous DNA has become integrated into the genomic DNA
of the cells.
[0072] A "fusion protein" is a hybrid protein expressed by a
nucleic acid molecule comprising nucleotide sequences of at least
two genes. For example, a fusion protein can comprise at least part
of a Zven1 or Zven2 polypeptide fused with a polypeptide that binds
an affinity matrix. Such a fusion protein provides a means to
isolate large quantities of Zven1 or Zven2 using affinity
chromatography.
[0073] The term "receptor" denotes a cell-associated protein that
binds to a bioactive molecule termed a "ligand." This interaction
mediates the effect of the ligand on the cell. Receptors can be
membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid
stimulating hormone receptor, beta-adrenergic receptor) or
multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3
receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor
and IL-6 receptor). Membrane-bound receptors are characterized by a
multi-domain structure comprising an extracellular ligand-binding
domain and an intracellular effector domain that is typically
involved in signal transduction. In certain membrane-bound
receptors, the extracellular ligand-binding domain and the
intracellular effector domain are located in separate polypeptides
that comprise the complete functional receptor.
[0074] In general, the binding of ligand to receptor results in a
conformational change in the receptor that causes an interaction
between the effector domain and other molecule(s) in the cell,
which in turn leads to an alteration in the metabolism of the cell.
Metabolic events that are often linked to receptor-ligand
interactions include gene transcription, phosphorylation,
dephosphorylation, increases in cyclic AMP production, mobilization
of cellular calcium, mobilization of membrane lipids, cell
adhesion, hydrolysis of inositol lipids and hydrolysis of
phospholipids.
[0075] The term "secretory signal sequence" denotes a DNA sequence
that encodes a peptide (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.
[0076] An "isolated polypeptide" is a polypeptide that is
essentially free from contaminating cellular components, such as
carbohydrate, lipid, or other proteinaceous impurities associated
with the polypeptide in nature. Typically, a preparation of
isolated polypeptide contains the polypeptide in a highly purified
form, i.e., at least about 80% pure, at least about 90% pure, at
least about 95% pure, greater than 95% pure, or greater than 99%
pure. One way to show that a particular protein preparation
contains an isolated polypeptide is by the appearance of a single
band following sodium dodecyl sulfate (SDS)-polyacrylamide gel
electrophoresis of the protein preparation and Coomassie Brilliant
Blue staining of the gel. However, 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.
[0077] 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.
[0078] The term "expression" refers to the biosynthesis of a gene
product. For example, in the case of a structural gene, expression
involves transcription of the structural gene into mRNA and the
translation of mRNA into one or more polypeptides.
[0079] 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 polypeptide encoded by a
splice variant of an mRNA transcribed from a gene.
[0080] As used herein, the term "immunomodulator" includes
cytokines, stem cell growth factors, lymphotoxins, co-stimulatory
molecules, hematopoietic factors, and synthetic analogs of these
molecules.
[0081] The term "complement/anti-complement pair" denotes
non-identical moieties that form a non-covalently associated,
stable pair under appropriate conditions. For instance, biotin and
avidin (or streptavidin) are prototypical members of a
complement/anti-complement pair. Other exemplary
complement/anti-complement pairs include receptor/ligand pairs,
antibody/antigen (or hapten or epitope) pairs, sense/antisense
polynucleotide pairs, and the like. Where subsequent dissociation
of the complement/anti-complement pair is desirable, the
complement/anti-complement pair preferably has a binding affinity
of less than 10.sup.9 M.sup.-1.
[0082] An "anti-idiotype antibody" is an antibody that binds with
the variable region domain of an immunoglobulin. In the present
context, an anti-idiotype antibody binds with the variable region
of an anti-Zven1 or anti-Zven2 antibody, and thus, an anti-idiotype
antibody mimics an epitope of Zven1 or Zven2.
[0083] An "antibody fragment" is a portion of an antibody such as
F(ab').sub.2, F(ab).sub.2, Fab', Fab, and the like. Regardless of
structure, an antibody fragment binds with the same antigen that is
recognized by the intact antibody. For example, an anti-Zven1
monoclonal antibody fragment binds with an epitope of Zven1.
[0084] The term "antibody fragment" also includes a synthetic or a
genetically engineered polypeptide that binds to a specific
antigen, such as polypeptides consisting of the light chain
variable region, "Fv" fragments consisting of the variable regions
of the heavy and light chains, recombinant single chain polypeptide
molecules in which light and heavy variable regions are connected
by a peptide linker ("scFv proteins"), and minimal recognition
units consisting of the amino acid residues that mimic the
hypervariable region.
[0085] A "chimeric antibody" is a recombinant protein that contains
the variable domains and complementary determining regions derived
from a rodent antibody, while the remainder of the antibody
molecule is derived from a human antibody.
[0086] "Humanized antibodies" are recombinant proteins in which
murine complementarity determining regions of a monoclonal antibody
have been transferred from heavy and light variable chains of the
murine immunoglobulin into a human variable domain.
[0087] A "detectable label" is a molecule or atom which can be
conjugated to an antibody moiety to produce a molecule useful for
diagnosis. Examples of detectable labels include chelators,
photoactive agents, radioisotopes, fluorescent agents, paramagnetic
ions, or other marker moieties.
[0088] The term "affinity tag" is used herein to denote a
polypeptide segment that can be attached to a second polypeptide to
provide for purification or detection 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 (1985)), substance P,
FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)),
streptavidin binding peptide, or other antigenic epitope or binding
domain. See, in general, Ford et al., Protein Expression and
Purification 2:95 (1991). DNAs encoding affinity tags are available
from commercial suppliers (e.g., Pharmacia Biotech, Piscataway,
N.J.).
[0089] A "naked antibody" is an entire antibody, as opposed to an
antibody fragment, which is not conjugated with a therapeutic
agent. Naked antibodies include both polyclonal and monoclonal
antibodies, as well as certain recombinant antibodies, such as
chimeric and humanized antibodies.
[0090] As used herein, the term "antibody component" includes both
an entire antibody and an antibody fragment.
[0091] A "target polypeptide" or a "target peptide" is an amino
acid sequence that comprises at least one epitope, and that is
expressed on a target cell, such as a tumor cell, or a cell that
carries an infectious agent antigen. T cells recognize peptide
epitopes presented by a major histocompatibility complex molecule
to a target polypeptide or target peptide and typically lyse the
target cell or recruit other immune cells to the site of the target
cell, thereby killing the target cell.
[0092] An "antigenic peptide" is a peptide, which will bind a major
histocompatibility complex molecule to form an MHC-peptide complex
which is recognized by a T cell, thereby inducing a cytotoxic
lymphocyte response upon presentation to the T cell. Thus,
antigenic peptides are capable of binding to an appropriate major
histocompatibility complex molecule and inducing a cytotoxic T
cells response, such as cell lysis or specific cytokine release
against the target cell which binds or expresses the antigen. The
antigenic peptide can be bound in the context of a class I or class
II major histocompatibility complex molecule, on an antigen
presenting cell or on a target cell.
[0093] In eukaryotes, RNA polymerase II catalyzes the transcription
of a structural gene to produce mRNA. A nucleic acid molecule can
be designed to contain an RNA polymerase II template in which the
RNA transcript has a sequence that is complementary to that of a
specific mRNA. The RNA transcript is termed an "anti-sense RNA" and
a nucleic acid molecule that encodes the anti-sense RNA is termed
an "anti-sense gene." Anti-sense RNA molecules are capable of
binding to mRNA molecules, resulting in an inhibition of mRNA
translation.
[0094] An "anti-sense oligonucleotide specific for Zven1" or a
"Zven1 anti-sense oligonucleotide" is an oligonucleotide having a
sequence (a) capable of forming a stable triplex with a portion of
the Zven1 gene, or (b) capable of forming a stable duplex with a
portion of an mRNA transcript of the Zven1 gene. Similarly, an
"anti-sense oligonucleotide specific for Zven2" or a "Zven2
anti-sense oligonucleotide" is an oligonucleotide having a sequence
(a) capable of forming a stable triplex with a portion of the Zven2
gene, or (b) capable of forming a stable duplex with a portion of
an mRNA transcript of the Zven2 gene.
[0095] A "ribozyme" is a nucleic acid molecule that contains a
catalytic center. The term includes RNA enzymes, self-splicing
RNAs, self-cleaving RNAs, and nucleic acid molecules that perform
these catalytic functions. A nucleic acid molecule that encodes a
ribozyme is termed a "ribozyme gene."
[0096] An "external guide sequence" is a nucleic acid molecule that
directs the endogenous ribozyme, RNase P, to a particular species
of intracellular mRNA, resulting in the cleavage of the mRNA by
RNase P. A nucleic acid molecule that encodes an external guide
sequence is termed an "external guide sequence gene."
[0097] The term "variant Zven1 gene" refers to nucleic acid
molecules that encode a polypeptide having an amino acid sequence
that is a modification of SEQ ID NO:2. Such variants include
naturally-occurring polymorphisms of Zven1 genes, as well as
synthetic genes that contain conservative amino acid substitutions
of the amino acid sequence of SEQ ID NO:2. Additional variant forms
of Zven1 genes are nucleic acid molecules that contain insertions
or deletions of the nucleotide sequences described herein. A
variant Zven1 gene can be identified by determining whether the
gene hybridizes with a nucleic acid molecule having the nucleotide
sequence of SEQ ID NO:1, or its complement, under stringent
conditions. Similarly, a variant Zven2 gene and a variant Zven2
polypeptide can be identified with reference to SEQ ID NO:4 and SEQ
ID NO:5, respectively.
[0098] Alternatively, variant Zven genes can be identified by
sequence comparison. Two amino acid sequences have "100% amino acid
sequence identity" if the amino acid residues of the two amino acid
sequences are the same when aligned for maximal correspondence.
Similarly, two nucleotide sequences have "100% nucleotide sequence
identity" if the nucleotide residues of the two nucleotide
sequences are the same when aligned for maximal correspondence.
Sequence comparisons can be performed using standard software
programs such as those included in the LASERGENE bioinformatics
computing suite, which is produced by DNASTAR (Madison, Wis.).
Other methods for comparing two nucleotide or amino acid sequences
by determining optimal alignment are well-known to those of skill
in the art (see, for example, Peruski and Peruski, The Internet and
the New Biology: Tools for Genomic and Molecular Research (ASM
Press, Inc. 1997), Wu et al. (eds.), "Information Superhighway and
Computer Databases of Nucleic Acids and Proteins," in Methods in
Gene Biotechnology, pages 123-151 (CRC Press, Inc. 1997), and
Bishop (ed.), Guide to Human Genome Computing, 2nd Edition
(Academic Press, Inc. 1998)). Particular methods for determining
sequence identity are described below.
[0099] Regardless of the particular method used to identify a
variant Zven1 gene or variant Zven1 polypeptide, a variant gene or
polypeptide encoded by a variant gene may be characterized by its
ability to bind specifically to an anti-Zven1 antibody. Similarly,
a variant Zven2 gene product or variant Zven2 polypeptide may be
characterized by its ability to bind specifically to an anti-Zven2
antibody.
[0100] 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.
[0101] 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.
[0102] "Paralogs" are distinct but structurally related proteins
made by an organism. Paralogs are believed to arise through gene
duplication. For example, .alpha.-globin, .beta.-globin, and
myoglobin are paralogs of each other.
[0103] The present invention includes functional fragments of Zven1
and Zven2 genes. Within the context of this invention, a
"functional fragment" of a Zven1 (or Zven2) gene refers to a
nucleic acid molecule that encodes a portion of a Zven1 (or Zven2)
polypeptide, which specifically binds with an anti-Zven1
(anti-Zven2) antibody.
[0104] Due to the imprecision of standard analytical methods,
molecular weights and lengths of polymers are understood to be
approximate values. When such a value is expressed as "about" X or
"approximately" X, the stated value of X will be understood to be
accurate to 110%.
3. PRODUCTION OF HUMAN ZVEN1 AND ZVEN2 GENES
[0105] Nucleic acid molecules encoding a human Zven1 gene can be
obtained by screening a human cDNA or genomic library using
polynucleotide probes based upon SEQ ID NO:1. Similarly, nucleic
acid molecules encoding a human Zven2 gene can be obtained by
screening a human cDNA or genomic library using polynucleotide
probes based upon SEQ ID NO:4. These techniques are standard and
well-established.
[0106] As an illustration, a nucleic acid molecule that encodes a
human Zven1 gene can be isolated from a human cDNA library. In this
case, the first step would be to prepare the cDNA library by
isolating RNA from tissues, such as testis or peripheral blood
lymphocytes, using methods well-known to those of skill in the art.
In general, RNA isolation techniques must provide a method for
breaking cells, a means of inhibiting RNase-directed degradation of
RNA, and a method of separating RNA from DNA, protein, and
polysaccharide contaminants. For example, total RNA can be isolated
by freezing tissue in liquid nitrogen, grinding the frozen tissue
with a mortar and pestle to lyse the cells, extracting the ground
tissue with a solution of phenol/chloroform to remove proteins, and
separating RNA from the remaining impurities by selective
precipitation with lithium chloride (see, for example, Ausubel et
al. (eds.), Short Protocols in Molecular Biology, 3.sup.rd Edition,
pages 4-1 to 4-6 (John Wiley & Sons 1995) ["Ausubel (1995)"];
Wu et al., Methods in Gene Biotechnology, pages 33-41 (CRC Press,
Inc. 1997) ["Wu (1997)"]). Alternatively, total RNA can be isolated
from tissue by extracting ground tissue with guanidinium
isothiocyanate, extracting with organic solvents, and separating
RNA from contaminants using differential centrifugation (see, for
example, Chirgwin et al., Biochemistry 18:52 (1979); Ausubel (1995)
at pages 4-1 to 4-6; Wu (1997) at pages 33-41).
[0107] In order to construct a cDNA library, poly(A).sup.+RNA must
be isolated from a total RNA preparation. Poly(A).sup.+ RNA can be
isolated from total RNA using the standard technique of
oligo(dT)-cellulose chromatography (see, for example, Aviv and
Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972); Ausubel (1995) at
pages 4-11 to 4-12).
[0108] Double-stranded cDNA molecules are synthesized from poly(A)+
RNA using techniques well-known to those in the art. (see, for
example, Wu (1997) at pages 41-46). Moreover, commercially
available kits can be used to synthesize double-stranded cDNA
molecules. For example, such kits are available from Life
Technologies, Inc. (Gaithersburg, Md.), CLONTECH Laboratories, Inc.
(Palo Alto, Calif.), Promega Corporation (Madison, Wis.) and
STRATAGENE (La Jolla, Calif.).
[0109] Various cloning vectors are appropriate for the construction
of a cDNA library. For example, a cDNA library can be prepared in a
vector derived from bacteriophage, such as a .lamda.gt10 vector.
See, for example, Huynh et al., "Constructing and Screening cDNA
Libraries in .lamda.gt10 and .lamda. gt11," in DNA Cloning: A
Practical Approach Vol. 1, Glover (ed.), page 49 (IRL Press, 1985);
Wu (1997) at pages 47-52.
[0110] Alternatively, double-stranded cDNA molecules can be
inserted into a plasmid vector, such as a PBLUESCRIPT vector
(STRATAGENE; La Jolla, Calif.), a LAMDAGEM-4 (Promega Corp.) or
other commercially available vectors. Suitable cloning vectors also
can be obtained from the American Type Culture Collection
(Manassas, Va.).
[0111] To amplify the cloned cDNA molecules, the cDNA library is
inserted into a prokaryotic host, using standard techniques. For
example, a cDNA library can be introduced into competent E. coli
DH5 cells, which can be obtained, for example, from Life
Technologies, Inc. (Gaithersburg, Md.).
[0112] A human genomic library can be prepared by means well-known
in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6;
Wu (1997) at pages 307-327). Genomic DNA can be isolated by lysing
tissue with the detergent Sarkosyl, digesting the lysate with
proteinase K, clearing insoluble debris from the lysate by
centrifugation, precipitating nucleic acid from the lysate using
isopropanol, and purifying resuspended DNA on a cesium chloride
density gradient.
[0113] DNA fragments that are suitable for the production of a
genomic library can be obtained by the random shearing of genomic
DNA or by the partial digestion of genomic DNA with restriction
endonucleases. Genomic DNA fragments can be inserted into a vector,
such as a bacteriophage or cosmid vector, in accordance with
conventional techniques, such as the use of restriction enzyme
digestion to provide appropriate termini, the use of alkaline
phosphatase treatment to avoid undesirable joining of DNA
molecules, and ligation with appropriate ligases. Techniques for
such manipulation are well-known in the art (see, for example,
Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages
307-327).
[0114] Nucleic acid molecules that encode a human Zven1 or Zven2
gene can also be obtained using the polymerase chain reaction (PCR)
with oligonucleotide primers having nucleotide sequences that are
based upon the nucleotide sequences described herein. General
methods for screening libraries with PCR are provided by, for
example, Yu et al., "Use of the Polymerase Chain Reaction to Screen
Phage Libraries," in Methods in Molecular Biology, Vol. 15: PCR
Protocols: Current Methods and Applications, White (ed.), pages
211-215 (Humana Press, Inc. 1993). Moreover, techniques for using
PCR to isolate related genes are described by, for example,
Preston, "Use of Degenerate Oligonucleotide Primers and the
Polymerase Chain Reaction to Clone Gene Family Members," in Methods
in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and
Applications, White (ed.), pages 317-337 (Humana Press, Inc.
1993).
[0115] Alternatively, human genomic libraries can be obtained from
commercial sources such as Research Genetics (Huntsville, Ala.) and
the American Type Culture Collection (Manassas, Va.).
[0116] A library containing cDNA or genomic clones can be screened
with one or more polynucleotide probes based upon SEQ ID NO:1,
using standard methods (see, for example, Ausubel (1995) at pages
6-1 to 6-11).
[0117] Anti-Zven antibodies, produced as described below, can also
be used to isolate DNA sequences that encode human Zven genes from
cDNA libraries. For example, the antibodies can be used to screen
.lamda.gt1 expression libraries, or the antibodies can be used for
immunoscreening following hybrid selection and translation (see,
for example, Ausubel (1995) at pages 6-12 to 6-16; Margolis et al.,
"Screening X expression libraries with antibody and protein
probes," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), pages 1-14 (Oxford University Press 1995)).
[0118] As an alternative, a Zven gene can be obtained by
synthesizing nucleic acid molecules using mutually priming long
oligonucleotides and the nucleotide sequences described herein
(see, for example, Ausubel (1995) at pages 8-8 to 8-9). Established
techniques using the polymerase chain reaction provide the ability
to synthesize DNA molecules at least two kilobases in length (Adang
et al., Plant Molec. Biol. 21:1131 (1993), Bambot et al., PCR
Methods and Applications 2:266 (1993), Dillon et al., "Use of the
Polymerase Chain Reaction for the Rapid Construction of Synthetic
Genes," in Methods in Molecular Biology, Vol. 15: PCR Protocols:
Current Methods and Applications, White (ed.), pages 263-268,
(Humana Press, Inc. 1993), and Holowachuk et al., PCR Methods Appl.
4:299 (1995)).
[0119] The nucleic acid molecules of the present invention can also
be synthesized with "gene machines" using protocols such as the
phosphoramidite method. If chemically-synthesized double stranded
DNA is required for an application such as the synthesis of a gene
or a gene fragment, then each complementary strand is made
separately. The production of short genes (60 to 80 base pairs) is
technically straightforward and can be accomplished by synthesizing
the complementary strands and then annealing them. For the
production of longer genes (>300 base pairs), however, special
strategies may be required, because the coupling efficiency of each
cycle during chemical DNA synthesis is seldom 100%. To overcome
this problem, synthetic genes (double-stranded) are assembled in
modular form from single-stranded fragments that are from 20 to 100
nucleotides in length. For reviews on polynucleotide synthesis,
see, for example, Glick and Pasternak, Molecular Biotechnology,
Principles and Applications of Recombinant DNA (ASM Press 1994),
Itakura et al., Ann. Rev. Biochem. 53:323 (1984), and Climie et
al., Proc. Nat'l Acad. Sci. USA 87:633 (1990).
[0120] The sequence of a Zven cDNA or Zven genomic fragment can be
determined using standard methods. Zven polynucleotide sequences
disclosed herein can also be used as probes or primers to clone 5'
non-coding regions of a Zven gene. Promoter elements from a Zven
gene can be used to direct the expression of heterologous genes in
tissues of, for example, transgenic animals or patients treated
with gene therapy. The identification of genomic fragments
containing a Zven promoter or regulatory element can be achieved
using well-established techniques, such as deletion analysis (see,
generally, Ausubel (1995)).
[0121] Cloning of 5' flanking sequences also facilitates production
of Zven proteins by "gene activation," as disclosed in U.S. Pat.
No. 5,641,670. Briefly, expression of an endogenous Zven gene in a
cell is altered by introducing into the Zven locus a DNA construct
comprising at least a targeting sequence, a regulatory sequence, an
exon, and an unpaired splice donor site. The targeting sequence is
a Zven 5' non-coding sequence that permits homologous recombination
of the construct with the endogenous Zven locus, whereby the
sequences within the construct become operably linked with the
endogenous Zven coding sequence. In this way, an endogenous Zven
promoter can be replaced or supplemented with other regulatory
sequences to provide enhanced, tissue-specific, or otherwise
regulated expression.
4. PRODUCTION OF ZVEN GENE VARIANTS
[0122] The present invention provides a variety of nucleic acid
molecules, including DNA and RNA molecules, which encode the Zven
polypeptides disclosed herein. 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 NOs:3 and 6 are a degenerate
nucleotide sequences that encompasses all nucleic acid molecules
that encode the Zven polypeptides of SEQ ID NOs:2 and 5,
respectively. Those skilled in the art will recognize that the
degenerate sequence of SEQ ID NO:3 also provides all RNA sequences
encoding SEQ ID NO:2, by substituting U for T, while the degenerate
sequence of SEQ ID NO:6 also provides all RNA sequences encoding
SEQ ID NO:5, by substituting U for T. Thus, the present invention
contemplates Zven1 polypeptide-encoding nucleic acid molecules
comprising nucleotide 66 to nucleotide 389 of SEQ ID NO:1, and
their RNA equivalents, as well as Zven2 polypeptide-encoding
nucleic acid molecules comprising nucleotide 91 to nucleotide 405
of SEQ ID NO:4, and their RNA equivalents.
[0123] Table 1 sets forth the one-letter codes used within SEQ ID
NOs:3 and 6 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. TABLE-US-00001 TABLE 1 Nucleotide
Resolution Complement Resolution A A T T C C G G G G C C T T A A R
A|G Y C|T Y C|T R A|G M A|C K G|T K G|T M A|C S C|G S C|G W A|T W
A|T H A|C|T D A|G|T B C|G|T V A|C|G V A|C|G B C|G|T D A|G|T H A|C|T
N A|C|G|T N A|C|G|T
[0124] The degenerate codons used in SEQ ID NOs:3 and 6,
encompassing all possible codons for a given amino acid, are set
forth in Table 2. TABLE-US-00002 TABLE 2 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 E 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 .cndot. TAA TAG TGA TRR Asn|Asp B RAY Glu|Gln Z SAR Any
X NNN
[0125] 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 an 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 sequence of SEQ ID NOs:2
and 5. Variant sequences can be readily tested for functionality as
described herein.
[0126] Different species can exhibit "preferential codon usage." In
general, see, Grantham et al., Nuc. Acids Res. 8:1893 (1980), Haas
et al. Curr. Biol. 6:315 (1996), Wain-Hobson et al., Gene 13:355
(1981), Grosjean and Fiers, Gene 18:199 (1982), Holm, Nuc. Acids
Res. 14:3075 (1986), Ikemura, J. Mol. Biol. 158:573 (1982), Sharp
and Matassi, Curr. Opin. Genet. Dev. 4:851 (1994), Kane, Curr.
Opin. Biotechnol. 6:494 (1995), and Makrides, Microbiol. Rev.
60:512 (1996). As used herein, the term "preferential codon usage"
or "preferential codons" is a term of art referring to protein
translation codons that are most frequently used in cells of a
certain species, thus favoring one or a few representatives of the
possible codons encoding each amino acid (See Table 2). For
example, the amino acid threonine (Thr) may be encoded by ACA, ACC,
ACG, or ACT, but in mammalian cells ACC is the most commonly used
codon; in other species, for example, insect cells, yeast, viruses
or bacteria, different Thr codons may be preferential. Preferential
codons for a particular species can be introduced into the
polynucleotides of the present invention by a variety of methods
known in the art. Introduction of preferential codon sequences into
recombinant DNA can, for example, enhance production of the protein
by making protein translation more efficient within a particular
cell type or species. Therefore, the degenerate codon sequences
disclosed in SEQ ID NOs:3 and 6 serve as templates for optimizing
expression of polynucleotides in various cell types and species
commonly used in the art and disclosed herein. Sequences containing
preferential codons can be tested and optimized for expression in
various species, and tested for functionality as disclosed
herein.
[0127] The present invention further provides variant polypeptides
and nucleic acid molecules that represent counterparts from other
species (orthologs). These species include, but are not limited to
mammalian, avian, amphibian, reptile, fish, insect and other
vertebrate and invertebrate species. Of particular interest are
Zven polypeptides from other mammalian species, including porcine,
ovine, bovine, canine, feline, equine, and other primate
polypeptides. Orthologs of human Zven 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 Zven. 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.
[0128] A Zven-encoding cDNA molecule 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 with primers designed from the
representative human Zven sequences 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 Zven polypeptide. Similar techniques
can also be applied to the isolation of genomic clones.
[0129] Those skilled in the art will recognize that the sequences
disclosed in SEQ ID NOs:1 and 4 represent single alleles of human
Zven1 and Zven2, respectively, and that allelic variation and
alternative splicing are 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 nucleotide sequences shown in SEQ ID NOs:1
and 4, 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 NOs:2 and 5. cDNA molecules generated
from alternatively spliced mRNAs, which retain the properties of
the Zven polypeptide 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.
[0130] Within certain embodiments of the invention, the isolated
nucleic acid molecules can hybridize under stringent conditions to
nucleic acid molecules comprising nucleotide sequences disclosed
herein. For example, such nucleic acid molecules can hybridize
under stringent conditions to nucleic acid molecules consisting of
the nucleotide sequence of SEQ ID NO:1, to nucleic acid molecules
consisting of the nucleotide sequence of nucleotides 66 to 161 of
SEQ ID NO:1, to nucleic acid molecules consisting of the nucleotide
sequence of nucleotides 288 to 389 of SEQ ID NO:1, to nucleic acid
molecules consisting of the nucleotide sequence of SEQ ID NO:4, to
nucleic acid molecules consisting of the nucleotide sequence of
nucleotides 334 to 405 of SEQ ID NO:4, or to nucleic acid molecules
consisting of nucleotide sequences that are the complements of such
sequences. 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.
[0131] A pair of nucleic acid molecules, such as DNA-DNA, RNA-RNA
and DNA-RNA, can hybridize if the nucleotide sequences have some
degree of complementarity. Hybrids can tolerate mismatched base
pairs in the double helix, but the stability of the hybrid is
influenced by the degree of mismatch. The T.sub.m of the mismatched
hybrid decreases by 1.degree. C. for every 1-1.5% base pair
mismatch. Varying the stringency of the hybridization conditions
allows control over the degree of mismatch that will be present in
the hybrid. The degree of stringency increases as the hybridization
temperature increases and the ionic strength of the hybridization
buffer decreases. Stringent hybridization conditions encompass
temperatures of about 5-25.degree. C. below the T.sub.m of the
hybrid and a hybridization buffer having up to 1 M Na.sup.+. Higher
degrees of stringency at lower temperatures can be achieved with
the addition of formamide which reduces the T.sub.m of the hybrid
about 1.degree. C. for each 1% formamide in the buffer solution.
Generally, such stringent conditions include temperatures of
20-70.degree. C. and a hybridization buffer containing up to
6.times.SSC and O-50% formamide. A higher degree of stringency can
be achieved at temperatures of from 40-70.degree. C. with a
hybridization buffer having up to 4.times.SSC and from 0-50%
formamide. Highly stringent conditions typically encompass
temperatures of 42-70.degree. C. with a hybridization buffer having
up to 1.times.SSC and O-50% formamide. Different degrees of
stringency can be used during hybridization and washing to achieve
maximum specific binding to the target sequence. Typically, the
washes following hybridization are performed at increasing degrees
of stringency to remove non-hybridized polynucleotide probes from
hybridized complexes.
[0132] The above conditions are meant to serve as a guide and it is
well within the abilities of one skilled in the art to adapt these
conditions for use with a particular polypeptide hybrid. The
T.sub.m for a specific target sequence is the temperature (under
defined conditions) at which 50% of the target sequence will
hybridize to a perfectly matched probe sequence. Those conditions
that influence the T.sub.m include, the size and base pair content
of the polynucleotide probe, the ionic strength of the
hybridization solution, and the presence of destabilizing agents in
the hybridization solution. Numerous equations for calculating
T.sub.m are known in the art, and are specific for DNA, RNA and
DNA-RNA hybrids and polynucleotide probe sequences of varying
length (see, for example, Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition (Cold Spring Harbor Press 1989);
Ausubel et al., (eds.), Current Protocols in Molecular Biology
(John Wiley and Sons, Inc. 1987); Berger and Kimmel (eds.), Guide
to Molecular Cloning Techniques, (Academic Press, Inc. 1987); and
Wetmur, Crit. Rev. Biochem. Mol. Biol. 26:227 (1990)). Sequence
analysis software such as OLIGO 6.0 (LSR; Long Lake, Minn.) and
Primer Premier 4.0 (Premier Biosoft International; Palo Alto,
Calif.), as well as sites on the Internet, are available tools for
analyzing a given sequence and calculating T.sub.m based on user
defined criteria. Such programs can also analyze a given sequence
under defined conditions and identify suitable probe sequences.
Typically, hybridization of longer polynucleotide sequences, >50
base pairs, is performed at temperatures of about 20-25.degree. C.
below the calculated T.sub.m. For smaller probes, <50 base
pairs, hybridization is typically carried out at the T.sub.m or
5-10.degree. C. below. This allows for the maximum rate of
hybridization for DNA-DNA and DNA-RNA hybrids.
[0133] The length of the polynucleotide sequence influences the
rate and stability of hybrid formation. Smaller probe sequences,
<50 base pairs, reach equilibrium with complementary sequences
rapidly, but may form less stable hybrids. Incubation times of
anywhere from minutes to hours can be used to achieve hybrid
formation. Longer probe sequences come to equilibrium more slowly,
but form more stable complexes even at lower temperatures.
Incubations are allowed to proceed overnight or longer. Generally,
incubations are carried out for a period equal to three times the
calculated Cot time. Cot time, the time it takes for the
polynucleotide sequences to reassociate, can be calculated for a
particular sequence by methods known in the art.
[0134] The base pair composition of polynucleotide sequence will
effect the thermal stability of the hybrid complex, thereby
influencing the choice of hybridization temperature and the ionic
strength of the hybridization buffer. A-T pairs are less stable
than G-C pairs in aqueous solutions containing sodium chloride.
Therefore, the higher the G-C content, the more stable the hybrid.
Even distribution of G and C residues within the sequence also
contribute positively to hybrid stability. In addition, the base
pair composition can be manipulated to alter the T.sub.m of a given
sequence. For example, 5-methyldeoxycytidine can be substituted for
deoxycytidine and 5-bromodeoxuridine can be substituted for
thymidine to increase the T.sub.m, whereas
7-deazz-2'-deoxyguanosine can be substituted for guanosine to
reduce dependence on T.sub.m.
[0135] The ionic concentration of the hybridization buffer also
affects the stability of the hybrid. Hybridization buffers
generally contain blocking agents such as Denhardt's solution
(Sigma Chemical Co., St. Louis, Mo.), denatured salmon sperm DNA,
tRNA, milk powders (BLOTTO), heparin or SDS, and a Na.sup.+ source,
such as SSC (1.times.SSC: 0.15 M sodium chloride, 15 mM sodium
citrate) or SSPE (1.times.SSPE: 1.8 M NaCl, 10 mM
NaH.sub.2PO.sub.4, 1 mM EDTA, pH 7.7). By decreasing the ionic
concentration of the buffer, the stability of the hybrid is
increased. Typically, hybridization buffers contain from between 10
mM-1 M Na.sup.+. The addition of destabilizing or denaturing agents
such as formamide, tetralkylammonium salts, guanidinium cations or
thiocyanate cations to the hybridization solution will alter the
T.sub.m of a hybrid. Typically, formamide is used at a
concentration of up to 50% to allow incubations to be carried out
at more convenient and lower temperatures. Formamide also acts to
reduce non-specific background when using RNA probes.
[0136] As an illustration, a nucleic acid molecule encoding a
variant Zven1 polypeptide can be hybridized with a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1 (or its
complement) at 42.degree. C. overnight in a solution comprising 50%
formamide, 5.times.SSC (1.times.SSC: 0.15 M sodium chloride and 15
mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5.times.
Denhardt's solution (100.times. Denhardt's solution: 2% (w/v)
Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine
serum albumin), 10% dextran sulfate, and 20 .mu.g/ml denatured,
sheared salmon sperm DNA. One of skill in the art can devise
variations of these hybridization conditions. For example, the
hybridization mixture can be incubated at a higher temperature,
such as about 65.degree. C., in a solution that does not contain
formamide. Moreover, premixed hybridization solutions are available
(e.g., EXPRESSHYB Hybridization Solution from CLONTECH
Laboratories, Inc.), and hybridization can be performed according
to the manufacturer's instructions.
[0137] Following hybridization, the nucleic acid molecules can be
washed to remove non-hybridized nucleic acid molecules under
stringent conditions, or under highly stringent conditions. Typical
stringent washing conditions include washing in a solution of
0.5.times.-2.times.SSC with 0.1% sodium dodecyl sulfate (SDS) at
55-65.degree. C. For example, nucleic acid molecules encoding
particular variant Zven1 polypeptides can remain hybridized with a
nucleic acid molecule consisting of the nucleotide sequence of
nucleotides 66 to 161 of SEQ ID NO:1, the nucleotide sequence of
nucleotides 288 to 389 of SEQ ID NO:1, or their complements,
following washing under stringent washing conditions, in which the
wash stringency is equivalent to 0.5.times.-2.times.SSC with 0.1%
SDS at 55-65.degree. C., including 0.5.times.SSC with 0.1% SDS at
55.degree. C., or 2.times.SSC with 0.1% SDS at 65.degree. C. In a
similar manner, nucleic acid molecules encoding particular Zven2
variants can remain hybridized with a nucleic acid molecule
consisting of the nucleotide sequence of nucleotides 334 to 405 of
SEQ ID NO:4, or its complement, following washing under stringent
washing conditions, in which the wash stringency is equivalent to
0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree. C., including
0.5.times.SSC with 0.1% SDS at 55.degree. C., or 2.times.SSC with
0.1% SDS at 65.degree. C. One of skill in the art can readily
devise equivalent conditions, for example, by substituting SSPE for
SSC in the wash solution.
[0138] Typical highly stringent washing conditions include washing
in a solution of 0.1.times.-0.2.times.SSC with 0.1% sodium dodecyl
sulfate (SDS) at 50-65.degree. C. As an illustration, nucleic acid
molecules encoding particular variant Zven1 polypeptides can remain
hybridized with a nucleic acid molecule consisting of the
nucleotide sequence of nucleotides 66 to 161 of SEQ ID NO:1, the
nucleotide sequence of nucleotides 288 to 389 of SEQ ID NO:1, or
their complements, following washing under highly stringent washing
conditions, in which the wash stringency is equivalent to
0.1.times.-0.2.times.SSC with 0.1% SDS at 50-65.degree. C.,
including 0.1.times.SSC with 0.1% SDS at 50.degree. C., or
0.2.times.SSC with 0.1% SDS at 65.degree. C. Similarly, nucleic
acid molecules encoding particular Zven2 variants remain hybridized
with a nucleic acid molecule consisting of the nucleotide sequence
of nucleotides 334 to 405 of SEQ ID NO:4, or its complement,
following washing under highly stringent washing conditions, in
which the wash stringency is equivalent to 0.1.times.-0.2.times.SSC
with 0.1% SDS at 50-65.degree. C., including 0.1.times.SSC with
0.1% SDS at 50.degree. C., or 0.2.times.SSC with 0.1% SDS at
65.degree. C.
[0139] The present invention also provides isolated Zven1
polypeptides that have a substantially similar sequence identity to
the polypeptides of SEQ ID NO:2, or their orthologs. The term
"substantially similar sequence identity" is used herein to denote
polypeptides having 85%, 90%, 95% or greater than 95% sequence
identity to the sequences shown in SEQ ID NO:2, or their orthologs.
Similarly, the present invention provides isolated Zven2
polypeptides having 85%, 90%, 95% or greater than 95% sequence
identity to the sequences shown in SEQ ID NO:5, or their
orthologs.
[0140] The present invention also contemplates Zven variant nucleic
acid molecules that can be identified using two criteria: a
determination of the similarity between the encoded polypeptide
with the amino acid sequence of SEQ ID NOs:2 or 5, and a
hybridization assay, as described above. For example, certain Zven1
gene variants include nucleic acid molecules (1) that remain
hybridized with a nucleic acid molecule consisting of the
nucleotide sequence of nucleotides 66 to 161 of SEQ ID NO:1, the
nucleotide sequence of nucleotides 288 to 389 of SEQ ID NO:1, or
their complements, following washing under stringent washing
conditions, in which the wash stringency is equivalent to
0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree. C., and (2)
that encode a polypeptide having 85%, 90%, 95% or greater than 95%
sequence identity to the amino acid sequence of SEQ ID NO:2.
Alternatively, certain Zven1 variant genes can be characterized as
nucleic acid molecules (1) that remain hybridized with a nucleic
acid molecule consisting of the nucleotide sequence of nucleotides
66 to 161 of SEQ ID NO:1, the nucleotide sequence of nucleotides
288 to 389 of SEQ ID NO:1, or their complements, following washing
under highly stringent washing conditions, in which the wash
stringency is equivalent to 0.1.times.-0.2.times.SSC with 0.1% SDS
at 50-65.degree. C., and (2) that encode a polypeptide having 85%,
90%, 95% or greater than 95% sequence identity to the amino acid
sequence of SEQ ID NO:2.
[0141] Moreover, certain Zven2 gene variants include nucleic acid
molecules (1) that remain hybridized with a nucleic acid molecule
consisting of the nucleotide sequence of nucleotides 334 to 405 of
SEQ ID NO:4, or its complement, following washing under stringent
washing conditions, in which the wash stringency is equivalent to
0.5.times.-2.times.SSC with 0.1% SDS at 55-65.degree. C., and (2)
that encode a polypeptide having 85%, 90%, 95% or greater than 95%
sequence identity to the amino acid sequence of SEQ ID NO:5.
Alternatively, certain Zven2 variant genes can be characterized as
nucleic acid molecules (1) that remain hybridized with a nucleic
acid molecule consisting of the nucleotide sequence of nucleotides
334 to 405 of SEQ ID NO:4, or its complement, following washing
under highly stringent washing conditions, in which the wash
stringency is equivalent to 0.1.times.-0.2.times.SSC with 0.1% SDS
at 50-65.degree. C., and (2) that encode a polypeptide having 85%,
90%, 95% or greater than 95% sequence identity to the amino acid
sequence of SEQ ID NO:5.
[0142] Percent sequence identity is determined by conventional
methods. See, for example, Altschul et al., Bull. Math. Bio. 48:603
(1986), and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA
89:10915 (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 3 (amino acids are
indicated by the standard one-letter codes). The percent identity
is then calculated as: ([Total number of identical matches]/[length
of the longer sequence plus the number of gaps introduced into the
longer sequence in order to align the two sequences])(100).
TABLE-US-00003 TABLE 3 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
[0143] Those skilled in the art appreciate that there are many
established algorithms available to align two amino acid sequences.
The "FASTA" similarity search algorithm of Pearson and Lipman is a
suitable protein alignment method for examining the level of
identity shared by an amino acid sequence disclosed herein and the
amino acid sequence of a putative Zven1 or Zven2 variant. The FASTA
algorithm is described by Pearson and Lipman, Proc. Nat'l Acad.
Sci. USA 85:2444 (1988), and by Pearson, Meth. Enzymol. 183:63
(1990).
[0144] 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. Preferred
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, Meth. Enzymol. 183:63 (1990).
[0145] 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 three to six, and most preferably,
three. The other parameters can be set as: gap opening penalty=10,
and gap extension penalty=1.
[0146] The present invention includes nucleic acid molecules that
encode a polypeptide having a conservative amino acid change,
compared with the amino acid sequence of SEQ ID NOs:2 or 5. That
is, variants can be obtained that contain one or more amino acid
substitutions of SEQ ID NOs:2 or 5, in which an alkyl amino acid is
substituted for an alkyl amino acid in a Zven1 or Zven2 amino acid
sequence, an aromatic amino acid is substituted for an aromatic
amino acid in a Zven1 or Zven2 amino acid sequence, a
sulfur-containing amino acid is substituted for a sulfur-containing
amino acid in a Zven1 or Zven2 amino acid sequence, a
hydroxy-containing amino acid is substituted for a
hydroxy-containing amino acid in a Zven1 or Zven2 amino acid
sequence, an acidic amino acid is substituted for an acidic amino
acid in a Zven1 or Zven2 amino acid sequence, a basic amino acid is
substituted for a basic amino acid in a Zven1 or Zven2 amino acid
sequence, or a dibasic monocarboxylic amino acid is substituted for
a dibasic monocarboxylic amino acid in a Zven1 or Zven2 amino acid
sequence.
[0147] Among the common amino acids, for example, a "conservative
amino acid substitution" is illustrated by a substitution among
amino acids within each of the following groups: (1) glycine,
alanine, valine, leucine, and isoleucine, (2) phenylalanine,
tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate
and glutamate, (5) glutamine and asparagine, and (6) lysine,
arginine and histidine.
[0148] The BLOSUM62 table is an amino acid substitution matrix
derived from about 2,000 local multiple alignments of protein
sequence segments, representing highly conserved regions of more
than 500 groups of related proteins (Henikoff and Henikoff, Proc.
Nat'l Acad. Sci. USA 89:10915 (1992)). Accordingly, the BLOSUM62
substitution frequencies can be used to define conservative amino
acid substitutions that may be introduced into the amino acid
sequences of the present invention. Although it is possible to
design amino acid substitutions based solely upon chemical
properties (as discussed above), the language "conservative amino
acid substitution" preferably refers to a substitution represented
by a BLOSUM62 value of greater than -1. For example, an amino acid
substitution is conservative if the substitution is characterized
by a BLOSUM62 value of 0, 1, 2, or 3. According to this system,
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
[0149] Particular variants of Zven1 or Zven2 are characterized by
having at least 70%, at least 80%, at least 85%, at least 90%, at
least 95% or greater than 95% sequence identity to a corresponding
amino acid sequence disclosed herein (i.e., SEQ ID NO:2 or SEQ ID
NO:5), wherein the variation in amino acid sequence is due to one
or more conservative amino acid substitutions.
[0150] Conservative amino acid changes in a Zven1 gene and a Zven2
gene can be introduced by substituting nucleotides for the
nucleotides recited in SEQ ID NO:1 and SEQ ID NO:4, respectively.
Such "conservative amino acid" variants can be obtained, for
example, by oligonucleotide-directed mutagenesis, linker-scanning
mutagenesis, mutagenesis using the polymerase chain reaction, and
the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson
(ed.), Directed Mutagenesis: A Practical Approach (IRL Press
1991)).
[0151] 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 vitro 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 typically 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 (1993), and Chung et al.,
Proc. Nat'l Acad. Sci. USA 90:10145 (1993).
[0152] 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 (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 (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 (1993)).
[0153] A limited number of non-conservative amino acids, amino
acids that are not encoded by the genetic code, non-naturally
occurring amino acids, and unnatural amino acids may be substituted
for Zven amino acid residues.
[0154] Amino acid sequence analysis indicates that Zven1 and Zven2
share several motifs. For example, one motif is "AVITGAC[DE][KR]D"
(SEQ ID NO:8), wherein acceptable amino acids for a given position
are indicated within square brackets. This motif occurs in Zven1 at
amino acid residues 28 to 37 of SEQ ID NO:2, and in Zven2 at amino
acid residues 20 to 29 of SEQ ID NO:5. Another motif is
"CHP[GL][ST][HR]KVPFFX[KR]RXHHTCPCLP" (SEQ ID NO:9), wherein
acceptable amino acids for a given position are indicated within
square brackets, and "X" can be any amino acid residue. This motif
occurs in Zven1 at amino acid residues 68 to 90 in SEQ ID NO:2, and
in Zven2 at amino acid residues 60 to 82 of SEQ ID NO:5. The
present invention includes peptides and polypeptides comprising
these motifs.
[0155] Sequence analysis also indicated that Zven1 and Zven2
include various conservative amino acid substitutions with respect
to each other. Accordingly, particular Zven1 variants can be
designed by modifying its sequence to include one or more amino
acid substitutions corresponding with the Zven2 sequence, while
particular Zven2 variants can be designed by modifying its sequence
to include one or more amino acid substitutions corresponding with
the Zven1 sequence. Such variants can be constructed using Table 4,
which presents exemplary conservative amino acid substitutions
found in Zven1 and Zven2. Although Zven1 and Zven2 variants can be
designed with any number of amino acid substitutions, certain
variants will include at least about X amino acid substitutions,
wherein X is selected from the group consisting of 2, 5, 7, 10, 12,
14, 16, 18, and 20. TABLE-US-00004 TABLE 4 Zven1 Zven2 Amino acid
Position Amino acid Position (SEQ ID NO: 2) Amino acid (SEQ ID NO:
5) Amino acid 4 Leu 4 Ala 7 Ala 7 Val 9 Leu 9 Ile 14 Leu 14 Val 35
Asp 27 Glu 36 Lys 28 Arg 42 Gly 34 Ala 48 Val 40 Ile 50 Ile 42 Leu
52 Val 44 Leu 53 Lys 45 Arg 55 Ile 47 Leu 63 Lys 55 Arg 66 Asp 58
Glu 71 Leu 63 Gly 72 Thr 64 Ser 73 Arg 65 His 80 Arg 72 Lys 93 Ala
85 Leu 102 Phe 94 Tyr
[0156] Essential amino acids in the polypeptides of the present
invention can be identified according to procedures known in the
art, such as site-directed mutagenesis or alanine-scanning
mutagenesis (Cunningham and Wells, Science 244:1081 (1989), Bass et
al., Proc. Nat'l Acad. Sci. USA 88:4498 (1991), Coombs and Corey,
"Site-Directed Mutagenesis and Protein Engineering," in Proteins:
Analysis and Design, Angeletti (ed.), pages 259-311 (Academic
Press, Inc. 1998)). In the latter technique, single alanine
mutations are introduced at every residue in the molecule, and the
resultant mutant molecules are tested for biological activity, such
as the ability to bind to an antibody, to identify amino acid
residues that are critical to the activity of the molecule. See
also, Hilton et al., J. Biol. Chem. 271:4699 (1996).
[0157] The location of Zven1 or Zven2 receptor binding domains can
also be determined by physical analysis of structure, as determined
by such techniques as nuclear magnetic resonance, crystallography,
electron diffraction or photoaffinity labeling, in conjunction with
mutation of putative contact site amino acids. See, for example, de
Vos et al., Science 255:306 (1992), Smith et al., J. Mol. Biol.
224:899 (1992), and Wlodaver et al., FEBS Lett. 309:59 (1992).
Moreover, Zven1 or Zven2 labeled with biotin or FITC can be used
for expression cloning of Zven1 or Zven2 receptors.
[0158] Multiple amino acid substitutions can be made and tested
using known methods of mutagenesis and screening, such as those
disclosed by Reidhaar-Olson and Sauer (Science 241:53 (1988)) or
Bowie and Sauer (Proc. Nat'l Acad. Sci. USA 86:2152 (1989)).
Briefly, these authors disclose methods for simultaneously
randomizing two or more positions in a polypeptide, selecting for
functional polypeptide, and then sequencing the mutagenized
polypeptides to determine the spectrum of allowable substitutions
at each position. Other methods that can be used include phage
display (e.g., Lowman et al., Biochem. 30:10832 (1991), Ladner et
al., U.S. Pat. No. 5,223,409, Huse, international publication No.
WO 92/06204, and region-directed mutagenesis (Derbyshire et al.,
Gene 46:145 (1986), and Ner et al., DNA 7:127, (1988)).
[0159] Variants of the disclosed Zven1 or Zven2 nucleotide and
polypeptide sequences can also be generated through DNA shuffling
as disclosed by Stemmer, Nature 370:389 (1994), Stemmer, Proc.
Nat'l Acad. Sci. USA 91:10747 (1994), and international publication
No. WO 97/20078. Briefly, variant DNA molecules are generated by in
vitro homologous recombination by random fragmentation of a parent
DNA followed by reassembly using PCR, resulting in randomly
introduced point mutations. This technique can be modified by using
a family of parent DNA molecules, such as allelic variants or DNA
molecules from different species, to introduce additional
variability into the process. Selection or screening for the
desired activity, followed by additional iterations of mutagenesis
and assay provides for rapid "evolution" of sequences by selecting
for desirable mutations while simultaneously selecting against
detrimental changes.
[0160] Mutagenesis methods as disclosed herein can be combined with
high-throughput, automated screening methods to detect activity of
cloned, mutagenized polypeptides in host cells. Mutagenized DNA
molecules that encode biologically active polypeptides, or
polypeptides that bind with anti-Zven1 or anti-Zven2 antibodies,
can be recovered from the host cells and rapidly sequenced using
modern equipment. These methods allow the rapid determination of
the importance of individual amino acid residues in a polypeptide
of interest, and can be applied to polypeptides of unknown
structure.
[0161] The present invention also includes "functional fragments"
of Zven1 or Zven2 polypeptides and nucleic acid molecules encoding
such functional fragments. Routine deletion analyses of nucleic
acid molecules can be performed to obtain functional fragments of a
nucleic acid molecule that encodes a Zven1 or Zven2 polypeptide. As
an illustration, DNA molecules having the nucleotide sequence of
SEQ ID NO:1 can be digested with Bal31 nuclease to obtain a series
of nested deletions. The fragments are then inserted into
expression vectors in proper reading frame, and the expressed
polypeptides are isolated and tested for the ability to bind
anti-Zven antibodies. One alternative to exonuclease digestion is
to use oligonucleotide-directed mutagenesis to introduce deletions
or stop codons to specify production of a desired fragment.
Alternatively, particular fragments of a Zven gene can be
synthesized using the polymerase chain reaction.
[0162] Methods for identifying functional domains are well-known to
those of skill in the art. For example, studies on the truncation
at either or both termini of interferons have been summarized by
Horisberger and Di Marco, Pharmac. Ther. 66:507 (1995). Moreover,
standard techniques for functional analysis of proteins are
described by, for example, Treuter et al., Molec. Gen. Genet.
240:113 (1993), Content et al., "Expression and preliminary
deletion analysis of the 42 kDa 2-5 A synthetase induced by human
interferon," in Biological Interferon Systems, Proceedings of
ISIR-TNO Meeting on Interferon Systems, Cantell (ed.), pages 65-72
(Nijhoff 1987), Herschman, "The EGF Receptor," in Control of Animal
Cell Proliferation, Vol. 1, Boynton et al., (eds.) pages 169-199
(Academic Press 1985), Coumailleau et al., J. Biol. Chem. 270:29270
(1995); Fukunaga et al., J. Biol. Chem. 270:25291 (1995); Yamaguchi
et al., Biochem. Pharmacol. 50:1295 (1995), and Meisel et al.,
Plant Molec. Biol. 30:1 (1996).
[0163] The present invention also contemplates functional fragments
of a Zven1 or Zven2 gene that have amino acid changes, compared
with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:5. A
variant Zven gene can be identified on the basis of structure by
determining the level of identity with the particular nucleotide
and amino acid sequences disclosed herein. An alternative approach
to identifying a variant gene on the basis of structure is to
determine whether a nucleic acid molecule encoding a potential
variant Zven1 or Zven2 gene can hybridize to a nucleic acid
molecule having the nucleotide sequence of SEQ ID NO:1 or SEQ ID
NO:4, as discussed above.
[0164] The present invention also provides polypeptide fragments or
peptides comprising an epitope-bearing portion of a Zven1 or Zven2
polypeptide described herein. Such fragments or peptides may
comprise an "immunogenic epitope," which is a part of a protein
that elicits an antibody response when the entire protein is used
as an immunogen. Immunogenic epitope-bearing peptides can be
identified using standard methods (see, for example, Geysen et al.,
Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
[0165] In contrast, polypeptide fragments or peptides may comprise
an "antigenic epitope," which is a region of a protein molecule to
which an antibody can specifically bind. Certain epitopes consist
of a linear or contiguous stretch of amino acids, and the
antigenicity of such an epitope is not disrupted by denaturing
agents. It is known in the art that relatively short synthetic
peptides that can mimic epitopes of a protein can be used to
stimulate the production of antibodies against the protein (see,
for example, Sutcliffe et al., Science 219:660 (1983)).
Accordingly, antigenic epitope-bearing peptides and polypeptides of
the present invention are useful to raise antibodies that bind with
the polypeptides described herein.
[0166] Antigenic epitope-bearing peptides and polypeptides can
contain at least four to ten amino acids, at least ten to fifteen
amino acids, or about 15 to about 30 amino acids of SEQ ID NOs:2 or
5. Such epitope-bearing peptides and polypeptides can be produced
by fragmenting a Zven1 or Zven2 polypeptide, or by chemical peptide
synthesis, as described herein. Moreover, epitopes can be selected
by phage display of random peptide libraries (see, for example,
Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et
al., Curr. Opin. Biotechnol. 7:616 (1996)). Standard methods for
identifying epitopes and producing antibodies from small peptides
that comprise an epitope are described, for example, by Mole,
"Epitope Mapping," in Methods in Molecular Biology, Vol. 10, Manson
(ed.), pages 105-116 (The Humana Press, Inc. 1992), Price,
"Production and Characterization of Synthetic Peptide-Derived
Antibodies," in Monoclonal Antibodies: Production, Engineering, and
Clinical Application, Ritter and Ladyman (eds.), pages 60-84
(Cambridge University Press 1995), and Coligan et al. (eds.),
Current Protocols in Immunology, pages 9.3.1-9.3.5 and pages
9.4.1-9.4.11 (John Wiley & Sons 1997).
[0167] Regardless of the particular nucleotide sequence of a
variant Zven1 or Zven2 gene, the gene encodes a polypeptide that
may be characterized by its ability to bind specifically to an
anti-Zven1 or anti-Zven2 antibody.
[0168] In addition to the uses described above, polynucleotides and
polypeptides of the present invention are useful as educational
tools in laboratory practicum kits for courses related to genetics
and molecular biology, protein chemistry, and antibody production
and analysis. Due to its unique polynucleotide and polypeptide
sequences, molecules of Zven1 or Zven2 can be used as standards or
as "unknowns" for testing purposes. For example, Zven1 or Zven2
polynucleotides can be used as an aid, such as, for example, to
teach a student how to prepare expression constructs for bacterial,
viral, or mammalian expression, including fusion constructs,
wherein Zven1 or Zven2 is the gene to be expressed; for determining
the restriction endonuclease cleavage sites of the polynucleotides;
determining mRNA and DNA localization of Zven1 or Zven2
polynucleotides in tissues (i.e., by northern and Southern blotting
as well as polymerase chain reaction); and for identifying related
polynucleotides and polypeptides by nucleic acid hybridization. As
an illustration, students will find that PvuII digestion of a
nucleic acid molecule consisting of the nucleotide sequence of
nucleotides 66 to 389 of SEQ ID NO:1 provides two fragments of
about 123 base pairs, and 201 base pairs, whereas HaeIII digestion
yields fragments of about 46 base pairs, and 278 base pairs.
[0169] Zven1 or Zven2 polypeptides can be used as an aid to teach
preparation of antibodies; identifying proteins by western
blotting; protein purification; determining the weight of expressed
Zven1 or Zven2 polypeptides as a ratio to total protein expressed;
identifying peptide cleavage sites; coupling amino and carboxyl
terminal tags; amino acid sequence analysis, as well as, but not
limited to monitoring biological activities of both the native and
tagged protein (i.e., protease inhibition) in vitro and in vivo.
For example, students will find that digestion of unglycosylated
Zven1 with cyanogen bromide yields four fragments having
approximate molecular weights of 148, 4337, 1909, 2402, and 2939,
whereas digestion of unglycosylated Zven1 with BNPS or NCS/urea
yields fragments having approximate molecular weights of 5231, and
6444.
[0170] Zven1 or Zven2 polypeptides can also be used to teach
analytical skills such as mass spectrometry, circular dichroism, to
determine conformation, especially of the four alpha helices, x-ray
crystallography to determine the three-dimensional structure in
atomic detail, nuclear magnetic resonance spectroscopy to reveal
the structure of proteins in solution. For example, a kit
containing Zven1 or Zven2 can be given to the student to analyze.
Since the amino acid sequence would be known by the instructor, the
protein can be given to the student as a test to determine the
skills or develop the skills of the student, the instructor would
then know whether or not the student has correctly analyzed the
polypeptide. Since every polypeptide is unique, the educational
utility of Zven1 or Zven2 would be unique unto itself.
[0171] The antibodies which bind specifically to Zven1 or Zven2 can
be used as a teaching aid to instruct students how to prepare
affinity chromatography columns to purify Zven1 or Zven2, cloning
and sequencing the polynucleotide that encodes an antibody and thus
as a practicum for teaching a student how to design humanized
antibodies. The Zven1 or Zven2 gene, polypeptide, or antibody would
then be packaged by reagent companies and sold to educational
institutions so that the students gain skill in art of molecular
biology. Because each gene and protein is unique, each gene and
protein creates unique challenges and learning experiences for
students in a lab practicum. Such educational kits containing the
Zven1 or Zven2 gene, polypeptide, or antibody are considered within
the scope of the present invention.
[0172] For any Zven polypeptide, including variants and fusion
proteins, one of ordinary skill in the art can readily generate a
fully degenerate polynucleotide sequence encoding that variant
using the information set forth in Tables 1 and 2 above. Moreover,
those of skill in the art can use standard software to devise Zven1
or Zven2 variants based upon the nucleotide and amino acid
sequences described herein. Accordingly, the present invention
includes a computer-readable medium encoded with a data structure
that provides at least one of the following sequences: SEQ ID NO:1,
SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID
NO:6. Suitable forms of computer-readable media include magnetic
media and optically-readable media. Examples of magnetic media
include a hard or fixed drive, a random access memory (RAM) chip, a
floppy disk, digital linear tape (DLT), a disk cache, and a ZIP
disk. Optically readable media are exemplified by compact discs
(e.g., CD-read only memory (ROM), CD-rewritable (RW), and
CD-recordable), and digital versatile/video discs (DVD) (e.g.,
DVD-ROM, DVD-RAM, and DVD+RW).
5. PRODUCTION OF ZVEN FUSION PROTEINS
[0173] Fusion proteins of Zven can be used to express a Zven
polypeptide or peptide in a recombinant host, and to isolate
expressed Zven polypeptides and peptides. One type of fusion
protein comprises a peptide that guides a Zven polypeptide from a
recombinant host cell. To direct a Zven polypeptide into the
secretory pathway of a eukaryotic host cell, a secretory signal
sequence (also known as a signal peptide, a leader sequence, prepro
sequence or pre sequence) is provided in the Zven expression
vector. While the secretory signal sequence may be derived from
Zven1 or Zven2, a suitable signal sequence may also be derived from
another secreted protein or synthesized de novo. The secretory
signal sequence is operably linked to a Zven1- or Zven2-encoding
sequence such that the two sequences are joined in the correct
reading frame and positioned to direct the newly synthesized
polypeptide into the secretory pathway of the host cell. Secretory
signal sequences are commonly positioned 5' to the nucleotide
sequence encoding the polypeptide of interest, although certain
secretory signal sequences may be positioned elsewhere in the
nucleotide 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).
[0174] Although the secretory signal sequence of Zven1, Zven2, or
another protein produced by mammalian cells (e.g., tissue-type
plasminogen activator signal sequence, as described, for example,
in U.S. Pat. No. 5,641,655) is useful for expression of Zven1 or
Zven2 in recombinant mammalian hosts, a yeast signal sequence is
preferred for expression in yeast cells. Examples of suitable yeast
signal sequences are those derived from yeast mating phermone
.alpha.-factor (encoded by the MF.alpha.1 gene), invertase (encoded
by the SUC2 gene), or acid phosphatase (encoded by the PHO5 gene).
See, for example, Romanos et al., "Expression of Cloned Genes in
Yeast," in DNA Cloning 2: A Practical Approach, 2.sup.nd Edition,
Glover and Hames (eds.), pages 123-167 (Oxford University Press
1995).
[0175] In bacterial cells, it is often desirable to express a
heterologous protein as a fusion protein to decrease toxicity,
increase stability, and to enhance recovery of the expressed
protein. For example, Zven1 or Zven2 can be expressed as a fusion
protein comprising a glutathione S-transferase polypeptide.
Glutathione S-transferease fusion proteins are typically soluble,
and easily purifiable from E. coli lysates on immobilized
glutathione columns. In similar approaches, a Zven1 or Zven2 fusion
protein comprising a maltose binding protein polypeptide can be
isolated with an amylose resin column, while a fusion protein
comprising the C-terminal end of a truncated Protein A gene can be
purified using IgG-Sepharose. Established techniques for expressing
a heterologous polypeptide as a fusion protein in a bacterial cell
are described, for example, by Williams et al., "Expression of
Foreign Proteins in E. coli Using Plasmid Vectors and Purification
of Specific Polyclonal Antibodies," in DNA Cloning 2: A Practical
Approach, 2.sup.nd Edition, Glover and Hames (Eds.), pages 15-58
(Oxford University Press 1995). In addition, commercially available
expression systems are available. For example, the PINPOINT Xa
protein purification system (Promega Corporation; Madison, Wis.)
provides a method for isolating a fusion protein comprising a
polypeptide that becomes biotinylated during expression with a
resin that comprises avidin.
[0176] Peptide tags that are useful for isolating heterologous
polypeptides expressed by either prokaryotic or eukaryotic cells
include polyHistidine tags (which have an affinity for
nickel-chelating resin), c-myc tags, calmodulin binding protein
(isolated with calmodulin affinity chromatography), substance P,
the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu
tag, and the FLAG tag (which binds with anti-FLAG antibodies). See,
for example, Luo et al., Arch. Biochem. Biophys. 329:215 (1996),
Morganti et al., Biotechnol. Appl. Biochem. 23:67 (1996), and Zheng
et al., Gene 186:55 (1997). Nucleic acid molecules encoding such
peptide tags are available, for example, from Sigma-Aldrich
Corporation (St. Louis, Mo.).
[0177] Another form of fusion protein comprises a Zven1 or Zven2
polypeptide and an immunoglobulin heavy chain constant region,
typically an Fc fragment, which contains two constant region
domains and a hinge region but lacks the variable region. As an
illustration, Chang et al., U.S. Pat. No. 5,723,125, describe a
fusion protein comprising a human interferon and a human
immunoglobulin Fc fragment. The C-terminal of the interferon is
linked to the N-terminal of the Fc fragment by a peptide linker
moiety. An example of a peptide linker is a peptide comprising
primarily a T cell inert sequence, which is immunologically inert.
An exemplary peptide linker has the amino acid sequence: GGSGG
SGGGG SGGGG S (SEQ ID NO:7). In this fusion protein, a preferred Fc
moiety is a human .gamma.4 chain, which is stable in solution and
has little or no complement activating activity. Accordingly, the
present invention contemplates a Zven fusion protein that comprises
a Zven1 or Zven2 polypeptide moiety and a human Fc fragment,
wherein the C-terminus of the Zven polypeptide moiety is attached
to the N-terminus of the Fc fragment via a peptide linker, such as
a peptide consisting of the amino acid sequence of SEQ ID NO:7.
[0178] In another variation, a Zven1 or Zven2 fusion protein
comprises an IgG sequence, a Zven polypeptide moiety covalently
joined to the amino terminal end of the IgG sequence, and a signal
peptide that is covalently joined to the amino terminal of the Zven
polypeptide moiety, wherein the IgG sequence consists of the
following elements in the following order: a hinge region, a
CH.sub.2 domain, and a CH.sub.3 domain. Accordingly, the IgG
sequence lacks a CH.sub.1 domain. The Zven polypeptide moiety
displays a Zven1 or Zven2 activity, such as the ability to bind
with a Zven1 or Zven2 receptor. This general approach to producing
fusion proteins that comprise both antibody and nonantibody
portions has been described by LaRochelle et al., EP 742830 (WO
95/21258).
[0179] Fusion proteins comprising a Zven1 or Zven2 polypeptide
moiety and an Fc moiety can be used, for example, as an in vitro
assay tool. For example, the presence of a Zven1 or Zven2 receptor
in a biological sample can be detected using these Zven1 or
Zven2-antibody fusion proteins, in which the Zven moiety is used to
target the cognate receptor, and a macromolecule, such as Protein A
or anti-Fc antibody, is used to detect the bound fusion
protein-ligand complex. In addition, antibody-Zven fusion proteins,
comprising antibody variable domains, are useful as therapeutic
proteins, in which the antibody moiety binds with a target antigen,
such as a tumor associated antigen.
[0180] Fusion proteins can be prepared by methods known to those
skilled in the art by preparing each component of the fusion
protein and chemically conjugating them. Alternatively, a
polynucleotide encoding both components of the fusion protein in
the proper reading frame can be generated using known techniques
and expressed by the methods described herein. General methods for
enzymatic and chemical cleavage of fusion proteins are described,
for example, by Ausubel (1995) at pages 16-19 to 16-25.
6. PRODUCTION OF ZVEN POLYPEPTIDES
[0181] The polypeptides of the present invention, including
full-length polypeptides, functional fragments, and fusion
proteins, can be produced in recombinant host cells following
conventional techniques. To express a Zven1 or Zven2 gene, a
nucleic acid molecule encoding the polypeptide must be operably
linked to regulatory sequences that control transcriptional
expression in an expression vector and then, introduced into a host
cell. In addition to transcriptional regulatory sequences, such as
promoters and enhancers, expression vectors can include
translational regulatory sequences and a marker gene, which is
suitable for selection of cells that carry the expression
vector.
[0182] Expression vectors that are suitable for production of a
foreign protein in eukaryotic cells typically contain (1)
prokaryotic DNA elements coding for a bacterial replication origin
and an antibiotic resistance marker to provide for the growth and
selection of the expression vector in a bacterial host; (2)
eukaryotic DNA elements that control initiation of transcription,
such as a promoter; and (3) DNA elements that control the
processing of transcripts, such as a transcription
termination/polyadenylation sequence. As discussed above,
expression vectors can also include nucleotide sequences encoding a
secretory sequence that directs the heterologous polypeptide into
the secretory pathway of a host cell. For example, a Zven1
expression vector may comprise a Zven1 gene and a secretory
sequence derived from a Zven1 gene or another secreted gene.
[0183] Zven1 or Zven2 proteins of the present invention may be
expressed in mammalian cells. Examples of suitable mammalian host
cells include African green monkey kidney cells (Vero; ATCC CRL
1587), human embryonic kidney cells (293-HEK; ATCC CRL 1573), baby
hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL
10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster
ovary cells (CHO-K1; ATCC CCL61; CHO DG44 [Chasin et al., Som.
Cell. Molec. Genet. 12:555 1986]), rat pituitary cells (GH1; ATCC
CCL82), HeLa S3 cells (ATCC CCL2.2), rat hepatoma cells (H-4-II-E;
ATCC CRL 1548) SV40-transformed monkey kidney cells (COS-1; ATCC
CRL 1650) and murine embryonic cells (NIH-3T3; ATCC CRL 1658).
[0184] For a mammalian host, the transcriptional and translational
regulatory signals may be derived from viral sources, such as
adenovirus, bovine papilloma virus, simian virus, or the like, in
which the regulatory signals are associated with a particular gene
which has a high level of expression. Suitable transcriptional and
translational regulatory sequences also can be obtained from
mammalian genes, such as actin, collagen, myosin, and
metallothionein genes.
[0185] Transcriptional regulatory sequences include a promoter
region sufficient to direct the initiation of RNA synthesis.
Suitable eukaryotic promoters include the promoter of the mouse
metallothionein I gene (Hamer et al., J. Molec. Appl. Genet. 1:273
(1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355
(1982)), the SV40 early promoter (Benoist et al., Nature 290:304
(1981)), the Rous sarcoma virus promoter (Gorman et al., Proc.
Natl. Acad. Sci. USA 79:6777 (1982)), the cytomegalovirus promoter
(Foecking et al., Gene 45:101 (1980)), and the mouse mammary tumor
virus promoter (see, generally, Etcheverry, "Expression of
Engineered Proteins in Mammalian Cell Culture," in Protein
Engineering: Principles and Practice, Cleland et al. (eds.), pages
163-181 (John Wiley & Sons, Inc. 1996)).
[0186] Alternatively, a prokaryotic promoter, such as the
bacteriophage T3 RNA polymerase promoter, can be used to control
Zven1 or Zven2 gene expression in mammalian cells if the
prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et
al., Mol. Cell. Biol. 10:4529 (1990), and Kaufman et al., Nucl.
Acids Res. 19:4485 (1991)).
[0187] An expression vector can be introduced into host cells using
a variety of standard techniques including calcium phosphate
transfection, liposome-mediated transfection,
microprojectile-mediated delivery, electroporation, and the like.
The transfected cells can be selected and propagated to provide
recombinant host cells that comprise the expression vector stably
integrated in the host cell genome. Techniques for introducing
vectors into eukaryotic cells and techniques for selecting such
stable transformants using a dominant selectable marker are
described, for example, by Ausubel (1995) and by Murray (ed.), Gene
Transfer and Expression Protocols (Humana Press 1991).
[0188] For example, one suitable selectable marker is a gene that
provides resistance to the antibiotic neomycin. In this case,
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 suitable 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. Alternatively, markers that introduce an altered phenotype,
such as green fluorescent protein, or cell surface proteins such as
CD4, CD8, Class I MHC, placental alkaline phosphatase may be used
to sort transfected cells from untransfected cells by such means as
FACS sorting or magnetic bead separation technology.
[0189] Zven1 or Zven2 polypeptides can also be produced by cultured
mammalian cells using a viral delivery system. Exemplary viruses
for this purpose 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 (1994), and Douglas and Curiel,
Science & Medicine 4:44 (1997)). Advantages of the adenovirus
system include the accommodation of relatively large DNA inserts,
the ability to grow to high-titer, the ability to infect a broad
range of mammalian cell types, and flexibility that allows use with
a large number of available vectors containing different
promoters.
[0190] 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. An
option is to delete the essential E1 gene from the viral vector,
which results in the inability to replicate unless the E1 gene is
provided by the host cell. Adenovirus vector-infected human 293
cells (ATCC Nos. CRL-1573, 45504, 45505), for example, can be grown
as adherent cells or in suspension culture at relatively high cell
density to produce significant amounts of protein (see Garnier et
al., Cytotechnol. 15:145 (1994)).
[0191] Zven1 or Zven2 genes may also be expressed in other higher
eukaryotic cells, such as avian, fungal, insect, yeast, or plant
cells. The baculovirus system provides an efficient means to
introduce cloned Zven1 or Zven2 genes into insect cells. Suitable
expression vectors are based upon the Autographa californica
multiple nuclear polyhedrosis virus (AcMNPV), and contain
well-known promoters such as Drosophila heat shockprotein (hsp) 70
promoter, Autographa californica nuclear polyhedrosis virus
immediate-early gene promoter (ie-1) and the delayed early 39K
promoter, baculovirus p10 promoter, and the Drosophila
metallothionein promoter. A second method of making recombinant
baculovirus utilizes a transposon-based system described by Luckow
(Luckow, et al., J. Virol. 67:4566 (1993)). This system, which
utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life
Technologies, Rockville, Md.). This system utilizes a transfer
vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to
move the DNA encoding the Zven polypeptide 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 (1990),
Bonning, et al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk, and
Rapoport, J. Biol. Chem. 270:1543 (1995). In addition, transfer
vectors can include an in-frame fusion with DNA encoding an epitope
tag at the C- or N-terminus of the expressed Zven polypeptide, for
example, a Glu-Glu epitope tag (Grussenmeyer et al., Proc. Nat'l
Acad. Sci. 82:7952 (1985)). Using a technique known in the art, a
transfer vector containing a Zven1 or Zven2 gene is transformed
into E. coli, and screened for bacmids, which contain an
interrupted lacZ gene indicative of recombinant baculovirus. The
bacmid DNA containing the recombinant baculovirus genome is then
isolated using common techniques.
[0192] The illustrative PFASTBAC vector can be modified to a
considerable degree. For example, the polyhedrin promoter can be
removed and substituted with the baculovirus basic protein promoter
(also known as Pcor, p6.9 or MP promoter) which is expressed
earlier in the baculovirus infection, and has been shown to be
advantageous for expressing secreted proteins (see, for example,
Hill-Perkins and Possee, J. Gen. Virol. 71:971 (1990), Bonning, et
al., J. Gen. Virol. 75:1551 (1994), and Chazenbalk and Rapoport, J.
Biol. Chem. 270:1543 (1995). In such transfer vector constructs, a
short or long version of the basic protein promoter can be used.
Moreover, transfer vectors can be constructed which replace the
native Zven1/Zven2 secretory signal sequences with secretory signal
sequences derived from insect proteins. For example, a secretory
signal sequence from Ecdysteroid Glucosyltransferase (EGT), honey
bee Melittin (Invitrogen Corporation; Carlsbad, Calif.), or
baculovirus gp67 (PharMingen: San Diego, Calif.) can be used in
constructs to replace the native Zven1/Zven2 secretory signal
sequence.
[0193] The recombinant virus or bacmid is used to transfect host
cells. Suitable insect host cells include cell lines derived from
IPLB-Sf-21, a Spodoptera frugiperda pupal ovarian cell line, such
as Sf9 (ATCC CRL 1711), Sf21AE, and Sf21 (Invitrogen Corporation;
San Diego, Calif.), as well as Drosophila Schneider-2 cells, and
the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni
(U.S. Pat. No. 5,300,435). Commercially available serum-free media
can be used to grow and to maintain the cells. Suitable media are
Sf90 II.TM. (Life Technologies) or ESF 921.TM. (Expression Systems)
for the Sf9 cells; and Ex-cellO405.TM. (JRH Biosciences, Lenexa,
Kans.) or Express FiveO.TM. (Life Technologies) for the T. ni
cells. When recombinant virus is used, the cells are typically
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.
[0194] Established techniques for producing recombinant proteins in
baculovirus systems are provided by Bailey et al., "Manipulation of
Baculovirus Vectors," in Methods in Molecular Biology, Volume 7:
Gene Transfer and Expression Protocols, Murray (ed.), pages 147-168
(The Humana Press, Inc. 1991), by Patel et al., "The baculovirus
expression system," in DNA Cloning 2: Expression Systems, 2nd
Edition, Glover et al. (eds.), pages 205-244 (Oxford University
Press 1995), by Ausubel (1995) at pages 16-37 to 16-57, by
Richardson (ed.), Baculovirus Expression Protocols (The Humana
Press, Inc. 1995), and by Lucknow, "Insect Cell Expression
Technology," in Protein Engineering: Principles and Practice,
Cleland et al. (eds.), pages 183-218 (John Wiley & Sons, Inc.
1996).
[0195] Fungal cells, including yeast cells, can also be used to
express the genes described herein. Yeast species of particular
interest in this regard include Saccharomyces cerevisiae, Pichia
pastoris, and Pichia methanolica. Suitable promoters for expression
in yeast include promoters from GAL1 (galactose), PGK
(phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1
(alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
Many yeast cloning vectors have been designed and are readily
available. These vectors include YIp-based vectors, such as YIp5,
YRp vectors, such as YRp17, YEp vectors such as YEp13 and YCp
vectors, such as YCp19. 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 suitable 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. Additional 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.
[0196] Transformation systems for other yeasts, including Hansenula
polymorpha, Schizosaccharomyces pombe, Kluyveromyces lactis,
Kluyveromyces fragilis, Ustilago maydis, Pichia pastoris, Pichia
methanolica, Pichia guillermondii and Candida maltosa are known in
the art. See, for example, Gleeson et al., J. Gen. Microbiol.
132:3459 (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.
[0197] For example, the use of Pichia methanolica as host for the
production of recombinant proteins is disclosed by Raymond, U.S.
Pat. No. 5,716,808, Raymond, U.S. Pat. No. 5,736,383, Raymond et
al., Yeast 14:11-23 (1998), and in international publication Nos.
WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565. DNA
molecules for use in transforming P. methanolica will commonly be
prepared as double-stranded, circular plasmids, which can be
linearized prior to transformation. For polypeptide production in
P. methanolica, the promoter and terminator in the plasmid can be
that of a P. methanolica gene, such as a P. methanolica alcohol
utilization gene (AUG1 or AUG2). Other useful promoters include
those of the dihydroxyacetone synthase (DHAS), formate
dehydrogenase (FMD), and catalase (CAT) genes. To facilitate
integration of the DNA into the host chromosome, it is preferred to
have the entire expression segment of the plasmid flanked at both
ends by host DNA sequences. A suitable selectable marker for use in
Pichia methanolica is a P. methanolica ADE2 gene, which encodes
phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21),
and which allows ade2 host cells to grow in the absence of adenine.
For large-scale, industrial processes where it is desirable to
minimize the use of methanol, it is possible to use host cells in
which both methanol utilization genes (AUG1 and AUG2) are deleted.
For production of secreted proteins, host cells can be used that
are deficient in vacuolar protease genes (PEP4 and PRB1).
Electroporation is used to facilitate the introduction of a plasmid
containing DNA encoding a polypeptide of interest into P.
methanolica cells. P. methanolica cells can be transformed by
electroporation using an exponentially decaying, pulsed electric
field having a field strength of from 2.5 to 4.5 kV/cm, preferably
about 3.75 kV/cm, and a time constant (t) of from 1 to 40
milliseconds, most preferably about 20 milliseconds.
[0198] Expression vectors can also be introduced into plant
protoplasts, intact plant tissues, or isolated plant cells. Methods
for introducing expression vectors into plant tissue include the
direct infection or co-cultivation of plant tissue with
Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA
injection, electroporation, and the like. See, for example, Horsch
et al., Science 227:1229 (1985), Klein et al., Biotechnology 10:268
(1992), and Miki et al., "Procedures for Introducing Foreign DNA
into Plants," in Methods in Plant Molecular Biology and
Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press,
1993).
[0199] Alternatively, Zven genes can be expressed in prokaryotic
host cells. Suitable promoters that can be used to express Zven1 or
Zven2 polypeptides in a prokaryotic host are well-known to those of
skill in the art and include promoters capable of recognizing the
T4, T3, Sp6 and T7 polymerases, the P.sub.R and P.sub.L promoters
of bacteriophage lambda, the trp, recA, heat shock, lacUV5, tac,
Ipp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.
subtilis, the promoters of the bacteriophages of Bacillus,
Streptomyces promoters, the int promoter of bacteriophage lambda,
the bla promoter of pBR322, and the CAT promoter of the
chloramphenicol acetyl transferase gene. Prokaryotic promoters have
been reviewed by Glick, J. Ind. Microbiol. 1:277 (1987), Watson et
al., Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins
1987), and by Ausubel et al. (1995).
[0200] Suitable prokaryotic hosts include E. coli and Bacillus
subtilus. Suitable strains of E. coli include BL21(DE3),
BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF',
DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109,
JM10, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for
example, Brown (ed.), Molecular Biology Labfax (Academic Press
1991)). Suitable strains of Bacillus subtilus include BR151, YB886,
MI119, MI120, and B170 (see, for example, Hardy, "Bacillus Cloning
Methods," in DNA Cloning: A Practical Approach, Glover (ed.) (IRL
Press 1985)).
[0201] When expressing a Zven 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.
[0202] Methods for expressing proteins in prokaryotic hosts are
well-known to those of skill in the art (see, for example, Williams
et al., "Expression of foreign proteins in E. coli using plasmid
vectors and purification of specific polyclonal antibodies," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
page 15 (Oxford University Press 1995), Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, page 137 (Wiley-Liss, Inc.
1995), and Georgiou, "Expression of Proteins in Bacteria," in
Protein Engineering: Principles and Practice, Cleland et al.
(eds.), Chapter 4, starting at page 101 (John Wiley & Sons,
Inc. 1996), and Rudolph, "Successful Refolding on an Industrial
Scale", Chapter 10).
[0203] Standard methods for introducing expression vectors into
bacterial, yeast, insect, and plant cells are provided, for
example, by Ausubel (1995).
[0204] General methods for expressing and recovering foreign
protein produced by a mammalian cell system are provided by, for
example, Etcheverry, "Expression of Engineered Proteins in
Mammalian Cell Culture," in Protein Engineering: Principles and
Practice, Cleland et al. (eds.), pages 163 (Wiley-Liss, Inc. 1996).
Standard techniques for recovering protein produced by a bacterial
system is provided by, for example, Grisshammer et al.,
"Purification of over-produced proteins from E. coli cells," in DNA
Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.),
pages 59-92 (Oxford University Press 1995). Established methods for
isolating recombinant proteins from a baculovirus system are
described by Richardson (ed.), Baculovirus Expression Protocols
(The Humana Press, Inc. 1995).
[0205] As an alternative, polypeptides of the present invention can
be synthesized by exclusive solid phase synthesis, partial solid
phase methods, fragment condensation or classical solution
synthesis. These synthesis methods are well-known to those of skill
in the art (see, for example, Merrifield, J. Am. Chem. Soc. 85:2149
(1963), Stewart et al., "Solid Phase Peptide Synthesis" (2nd
Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept.
Prot. 3:3 (1986), Atherton et al., Solid Phase Peptide Synthesis: A
Practical Approach (IRL Press 1989), Fields and Colowick,
"Solid-Phase Peptide Synthesis," Methods in Enzymology Volume 289
(Academic Press 1997), and Lloyd-Williams et al., Chemical
Approaches to the Synthesis of Peptides and Proteins (CRC Press,
Inc. 1997)). Variations in total chemical synthesis strategies,
such as "native chemical ligation" and "expressed protein ligation"
are also standard (see, for example, Dawson et al., Science 266:776
(1994), Hackeng et al., Proc. Nat'l Acad. Sci. USA 94:7845 (1997),
Dawson, Methods Enzymol. 287: 34 (1997), Muir et al, Proc. Nat'l
Acad. Sci. USA 95:6705 (1998), and Severinov and Muir, J. Biol.
Chem. 273:16205 (1998)).
[0206] Peptides and polypeptides of the present invention comprise
at least six, at least nine, or at least 15 contiguous amino acid
residues of SEQ ID NOs:2 and 5. Illustrative polypeptides of Zven2,
for example, include 15 contiguous amino acid residues of amino
acids 82 to 105 of SEQ ID NO:5. Exemplary polypeptides of Zven1
include 15 contiguous amino acid residues of amino acids 1 to 32 or
amino acids 75 to 108 of SEQ ID NO:2, whereas exemplary Zven2
polypeptides include amino acids 82 to 105 of SEQ ID NO:5. Within
certain embodiments of the invention, the polypeptides comprise 20,
30, 40, 50, 75, or more contiguous residues of SEQ ID NOs:2 or 5.
Nucleic acid molecules encoding such peptides and polypeptides are
useful as polymerase chain reaction primers and probes.
[0207] Examples for the production of Zven1 are shown in Examples
9, 10, 11, and 12.
[0208] The present invention contemplates compositions comprising a
peptide or polypeptide described herein. Such compositions can
further comprise a carrier. The carrier can be a conventional
organic or inorganic carrier. Examples of carriers include water,
buffer solution, alcohol, propylene glycol, macrogol, sesame oil,
corn oil, and the like.
7. ISOLATION OF ZVEN POLYPEPTIDES
[0209] The polypeptides of the present invention can be purified to
at least about 80% purity, to at least about 90% purity, to at
least about 95% purity, or even greater than 95% purity with
respect to contaminating macromolecules, particularly other
proteins and nucleic acids, and free of infectious and pyrogenic
agents. The polypeptides of the present invention can also be
purified to a pharmaceutically pure state, which is greater than
99.9% pure. In certain preparations, a purified polypeptide is
substantially free of other polypeptides, particularly other
polypeptides of animal origin.
[0210] Fractionation and/or conventional purification methods can
be used to obtain preparations of Zven1 or Zven2 purified from
natural sources, and recombinant Zven polypeptides and fusion Zven
polypeptides purified from recombinant host cells. In general,
ammonium sulfate precipitation and acid or chaotrope extraction may
be used for fractionation of samples. Exemplary purification steps
may include hydroxyapatite, size exclusion, FPLC and reverse-phase
high performance liquid chromatography. Suitable chromatographic
media include derivatized dextrans, agarose, cellulose,
polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and
Q derivatives are preferred. Exemplary chromatographic media
include those media derivatized with phenyl, butyl, or octyl
groups, such as Phenyl-Sepharose FF (Pharmacia), Toyopearl butyl
650 (Toso Haas, Montgomeryville, Pa.), Octyl-Sepharose (Pharmacia)
and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso
Haas) and the like. Suitable solid supports include glass beads,
silica-based resins, cellulosic resins, agarose beads, cross-linked
agarose beads, polystyrene beads, cross-linked polyacrylamide
resins and the like that are insoluble under the conditions in
which they are to be used. These supports may be modified with
reactive groups that allow attachment of proteins by amino groups,
carboxyl groups, sulfhydryl groups, hydroxyl groups and/or
carbohydrate moieties.
[0211] Examples of coupling chemistries include cyanogen bromide
activation, N-hydroxysuccinimide activation, epoxide activation,
sulfhydryl activation, hydrazide activation, and carboxyl and amino
derivatives for carbodiimide coupling chemistries. These and other
solid media are well known and widely used in the art, and are
available from commercial suppliers. Selection of a particular
method for polypeptide isolation and purification is a matter of
routine design and is determined in part by the properties of the
chosen support. See, for example, Affinity Chromatography:
Principles & Methods (Pharmacia LKB Biotechnology 1988), and
Doonan, Protein Purification Protocols (The Humana Press 1996).
[0212] Additional variations in Zven isolation and purification can
be devised by those of skill in the art. For example, anti-Zven
antibodies, obtained as described below, can be used to isolate
large quantities of protein by immunoaffinity purification.
Moreover, methods for binding receptors to ligand polypeptides,
such as Zven1 or Zven2, bound to support media are well known in
the art.
[0213] The polypeptides of the present invention can also be
isolated by exploitation of particular properties. For example,
immobilized metal ion adsorption (IMAC) chromatography can be used
to purify histidine-rich proteins, including those comprising
polyhistidine tags. Briefly, a gel is first charged with divalent
metal ions to form a chelate (Sulkowski, Trends in Biochem. 3:1
(1985)). Histidine-rich proteins will be adsorbed to this matrix
with differing affinities, depending upon the metal ion used, and
will be eluted by competitive elution, lowering the pH, or use of
strong chelating agents. Other methods of purification include
purification of glycosylated proteins by lectin affinity
chromatography and ion exchange chromatography (M. Deutscher,
(ed.), Meth. Enzymol. 182:529 (1990)). Within additional
embodiments of the invention, a fusion of the polypeptide of
interest and an affinity tag (e.g., maltose-binding protein, an
immunoglobulin domain) may be constructed to facilitate
purification.
[0214] Zven polypeptides or fragments thereof may also be prepared
through chemical synthesis, as described above. Zven polypeptides
may be monomers or multimers; glycosylated or non-glycosylated;
pegylated or non-pegylated; and may or may not include an initial
methionine amino acid residue.
8. ZVEN ANALOGS
[0215] As described above, the disclosed polypeptides can be used
to construct Zven variants. These polypeptides can be used to
identify Zven1 or Zven2 analogs. One type of Zven analog mimics
Zven by binding with a Zven receptor. Such an analog is considered
to be a Zven agonist if the binding of the analog with a Zven
receptor stimulates a response by a cell that expresses the
receptor. On the other hand, a Zven analog that binds with a Zven
receptor, but does not stimulate a cellular response, may be a Zven
antagonist. Such an antagonist may diminish Zven or Zven agonist
activity, for example, by a competitive or non-competitive binding
of the antagonist to the Zven receptor.
[0216] One general class of Zven analogs are agonists or
antagonists having an amino acid sequence that has at least one
mutation, deletion (amino- or carboxyl-terminus), or substitution
of the amino acid sequences disclosed herein. Another general class
of Zven analogs is provided by anti-idiotype antibodies, and
fragments thereof, as described below. Moreover, recombinant
antibodies comprising anti-idiotype variable domains can be used as
analogs (see, for example, Monfardini et al., Proc. Assoc. Am.
Physicians 108:420 (1996)). Since the variable domains of
anti-idiotype Zven antibodies mimic Zven, these domains can provide
either Zven agonist or antagonist activity. As an illustration, Lim
and Langer, J. Interferon Res. 13:295 (1993), describe
anti-idiotypic interferon-.alpha. antibodies that have the
properties of either interferon-.alpha. agonists or
antagonists.
[0217] A third approach to identifying Zven1 or Zven2 analogs is
provided by the use of combinatorial libraries. Methods for
constructing and screening phage display and other combinatorial
libraries are provided, for example, by Kay et al., Phage Display
of Peptides and Proteins (Academic Press 1996), Verdine, U.S. Pat.
No. 5,783,384, Kay, et. al., U.S. Pat. No. 5,747,334, and Kauffman
et al., U.S. Pat. No. 5,723,323.
[0218] The activity of a Zven polypeptide, agonist, or antagonist
can be determined using a standard cell proliferation or
differentiation assay. For example, assays measuring proliferation
include such assays as chemosensitivity to neutral red dye,
incorporation of radiolabeled nucleotides, incorporation of
5-bromo-2'-deoxyuridine in the DNA of proliferating cells, and use
of tetrazolium salts (Mosmann, J. Immunol. Methods 65:55 (1983);
Porstmann et al., J. Immunol. Methods 82:169 (1985); Alley et al.,
Cancer Res. 48:589 (1988); Cook et al., Analytical Biochem. 179:1
(1989); Marshall et al., Growth Reg. 5:69 (1995); Scudiero et al.,
Cancer Res. 48:4827 (1988); Cavanaugh et al., Investigational New
Drugs 8:347 (1990)). Assays measuring differentiation include, for
example, measuring cell-surface markers associated with
stage-specific expression of a tissue, enzymatic activity,
functional activity or morphological changes (Raes, Adv. Anim. Cell
Biol. Technol. Bioprocesses, pages 161-171 (1989; Watt, FASEB,
5:281 (1991); Francis, Differentiation 57:63 (1994)). Assays can be
used to measure other cellular responses, that include, chemotaxis,
adhesion, changes in ion channel influx, regulation of second
messenger levels and neurotransmitter release. Such assays are well
known in the art (see, for example, Chayen and Bitensky,
Cytochemical Bioassays: Techniques & Applications (Marcel
Dekker 1983)).
[0219] The effect of a variant Zven polypeptide, such as Zven1,
Zven2, as well as agonists, fragments, variants and/or chimeras
thereof, can also be determined by observing contractility of
tissues, including gastrointestinal tissues, with a tensiometer
that measures contractility and relaxation in tissues (see, for
example, Dainty et al., J. Pharmacol. 100:767 (1990); Rhee et al.,
Neurotox. 16:179 (1995); Anderson, Endocrinol. 114:364 (1984);
Downing, and Sherwood, Endocrinol. 116:1206 (1985)). For example,
methods for measuring vasodilatation of aortic rings are well known
in the art. As an illustration, aortic rings are removed from
four-month old Sprague Dawley rats and placed in a buffer solution,
such as modified Krebs solution (118.5 mM NaCl, 4.6 mM KCl, 1.2 mM
MgSO.sub.4.7H.sub.2O, 1.2 mM KH.sub.2PO.sub.4, 2.5 mM
CaCl.sub.2.2H.sub.2O, 24.8 mM NaHCO.sub.3 and 10 mM glucose). One
of skill in the art would recognize that this method can be used
with other animals, such as rabbits, other rat strains, Guinea
pigs, and the like. The rings are then attached to an isometric
force transducer (Radnoti Inc., Monrovia, Calif.) and the data are
recorded with a Ponemah physiology platform (Gould Instrument
systems, Inc., Valley View, Ohio) and placed in an oxygenated (95%
O.sub.2, 5% CO.sub.2) tissue bath containing the buffer solution.
The tissues are adjusted to one gram resting tension and allowed to
stabilize for about one hour before testing. The integrity of the
rings can be tested with norepinepherin (Sigma Co.; St. Louis, Mo.)
and carbachol, a muscarinic acetylcholine agonist (Sigma Co.).
After integrity is checked, the rings are washed three times with
fresh buffer and allowed to rest for about one hour. To test a
sample for vasodilatation, or relaxation of the aortic ring tissue,
the rings are contracted to two grams tension and allowed to
stabilize for fifteen minutes. A Zven polypeptide sample is then
added to one, two, or three of the four baths, without flushing,
and tension on the rings recorded and compared to the control rings
containing buffer only. Enhancement or relaxation of contractility
by Zven polypeptides, their agonists and antagonists is directly
measured by this method, and it can be applied to other contractile
tissues such as gastrointestinal tissues.
[0220] As another example, the effects of Zven1 were tested in a
standard guinea pig ileum organ bath. The organ bath system is a
standard method used to measure contractility in isolated tissue,
and the guinea pig ileum is routinely used for recording
contractile responses in the intestine ex vivo (Thomas E., et al.,
Mol Pharmacol 44:102-10, 1993). Because the components of the
enteric nervous system are located entirely within the gut, it may
be removed from the brain and the spinal cord and its reflex
behaviors studied. The classical response observed in
gastrointestinal tissue from guinea pig intestinal ileum is
longitudinal contraction by smooth muscle fibers orientated along
the long axis of the gut. As shown in Example 6, Zven1 treatment
stimulated smooth muscle contraction in the ileum at picomolar
concentrations as low as 0.75 ng/ml, which is equivalent to 75 pM.
Additionally, the highest response was observed at the 20 ng/ml
zven1 dose. Additional examples showing the effects of the Zven
molecules of the present invention are shown in Examples 7, 14, and
15.
[0221] The effect of a variant Zven polypeptide, such as Zven1,
Zven2, as well as agonists, fragments, variants and/or chimeras
thereof, on gastric motility would typically be measured in the
clinical setting as the time required for gastric emptying and
subsequent transit time through the gastrointestinal tract. Gastric
emptying scans are well known to those skilled in the art, and
briefly, comprise use of an oral contrast agent, such as barium, or
a radiolabeled meal. Solids and liquids can be measured
independently. Generally, a test food or liquid is radiolabeled
with an isotope (e.g., .sup.99mTc), and after ingestion or
administration, transit time through the gastrointestinal tract and
gastric emptying are measured by visualization using gamma cameras
(Meyer et al., Am. J. Dig. Dis. 21:296 (1976); Collins et al., Gut
24:1117 (1983); Maughan et al., Diabet. Med. 13:S6 (1996), and
Horowitz et al., Arch. Intern. Med. 145:1467 (1985)). The oral
administration of phenol red (test meal) to measure gastric
emptying and intestinal transit in rodents is a well-documented
model (Martinez V, Cuttitta F, Tache Y 1997 Endocrinology
138:3749-3755). Briefly, animals are deprived of food for 18 hours
but allowed free access to water. Animals receive oral
administration of 0.15 ml of test meal, consisting of a 1.5%
aqueous methylcellulose solution containing a non-absorbable dye,
0.05% phenol red (50 mg/100 ml Sigma Chemical Company Catalogue #
P4758). The effects of Zven1 on gastric emptying in an in vivo
mouse model are shown in Examples 4, 8, 21, and 22. Additional
studies can be performed before and after the administration of a
promotility agent to quantify the efficacy of the Zven
polypeptide.
[0222] Radiolabeled or affinity labeled Zven polypeptides can also
be used to identify or to localize Zven receptors in a biological
sample (see, for example, Deutscher (ed.), Methods in Enzymol.,
vol. 182, pages 721-37 (Academic Press 1990); Brunner et al., Ann.
Rev. Biochem. 62:483 (1993); Fedan et al., Biochem. Pharmacol.
33:1167 (1984)). Also see, Varthakavi and Minocha, J. Gen. Virol.
77:1875 (1996), who describe the use of anti-idiotype antibodies
for receptor identification.
9. PRODUCTION OF ANTIBODIES TO ZVEN PROTEINS
[0223] Antibodies to a Zven polypeptide can be obtained, for
example, using the product of a Zven expression vector or Zven
isolated from a natural source as an antigen. Particularly useful
anti-Zven1 and anti-Zven2 antibodies "bind specifically" with Zven1
and Zven2, respectively. Antibodies are considered to be
specifically binding if the antibodies exhibit at least one of the
following two properties: (1) antibodies bind to Zven1 or Zven2
with a threshold level of binding activity, and (2) antibodies do
not significantly cross-react with polypeptides related to Zven1 or
Zven2.
[0224] With regard to the first characteristic, antibodies
specifically bind if they bind to a Zven polypeptide, peptide or
epitope with 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 binding affinity of an antibody can be readily
determined by one of ordinary skill in the art, for example, by
Scatchard analysis (Scatchard, Ann. NY Acad. Sci. 51:660 (1949)).
With regard to the second characteristic, antibodies do not
significantly cross-react with related polypeptide molecules, for
example, if they detect Zven, but not known polypeptides (e.g.,
known Wnt inhibitors) using a standard Western blot analysis.
Particular anti-Zven1 antibodies bind Zven1, but not Zven2, while
certain anti-Zven2 antibodies bind Zven2, but not Zven1.
[0225] Anti-Zven1 and anti-Zven2 antibodies can be produced using
antigenic Zven1 or Zven2 epitope-bearing peptides and polypeptides.
Antigenic epitope-bearing peptides and polypeptides of the present
invention contain a sequence of at least four, or between 15 to
about 30 amino acids contained within SEQ ID NOs:2 or 5. However,
peptides or polypeptides comprising a larger portion of an amino
acid sequence of the invention, containing from 30 to 50 amino
acids, or any length up to and including the entire amino acid
sequence of a polypeptide of the invention, also are useful for
inducing antibodies that bind with Zven1 or Zven2. It is desirable
that the amino acid sequence of the epitope-bearing peptide is
selected to provide substantial solubility in aqueous solvents
(i.e., the sequence includes relatively hydrophilic residues, while
hydrophobic residues are preferably avoided). Moreover, amino acid
sequences containing proline residues may be also be desirable for
antibody production.
[0226] As an illustration, potential antigenic sites in Zven1 or
Zven2 were identified using the Jameson-Wolf method, Jameson and
Wolf, CABIOS 4:181, (1988), as implemented by the PROTEAN program
(version 3.14) of LASERGENE (DNASTAR; Madison, Wis.). Default
parameters were used in this analysis.
[0227] The Jameson-Wolf method predicts potential antigenic
determinants by combining six major subroutines for protein
structural prediction. Briefly, the Hopp-Woods method, Hopp et al.,
Proc. Nat'l Acad. Sci. USA 78:3824 (1981), was first used to
identify amino acid sequences representing areas of greatest local
hydrophilicity (parameter: seven residues averaged). In the second
step, Emini's method, Emini et al., J. Virology 55:836 (1985), was
used to calculate surface probabilities (parameter: surface
decision threshold (0.6)=1). Third, the Karplus-Schultz method,
Karplus and Schultz, Naturwissenschaften 72:212 (1985), was used to
predict backbone chain flexibility (parameter: flexibility
threshold (0.2)=1). In the fourth and fifth steps of the analysis,
secondary structure predictions were applied to the data using the
methods of Chou-Fasman, Chou, "Prediction of Protein Structural
Classes from Amino Acid Composition," in Prediction of Protein
Structure and the Principles of Protein Conformation, Fasman (ed.),
pages 549-586 (Plenum Press 1990), and Garnier-Robson, Garnier et
al., J. Mol. Biol. 120:97 (1978) (Chou-Fasman parameters:
conformation table=64 proteins; .alpha. region threshold=103;
.beta. region threshold=105; Garnier-Robson parameters: .alpha. and
.beta. decision constants=0). In the sixth subroutine, flexibility
parameters and hydropathy/solvent accessibility factors were
combined to determine a surface contour value, designated as the
"antigenic index." Finally, a peak broadening function was applied
to the antigenic index, which broadens major surface peaks by
adding 20, 40, 60, or 80% of the respective peak value to account
for additional free energy derived from the mobility of surface
regions relative to interior regions. This calculation was not
applied, however, to any major peak that resides in a helical
region, since helical regions tend to be less flexible.
[0228] The results of this analysis indicated that suitable
antigenic peptides of Zven1 include the following segments of the
amino acid sequence of SEQ ID NO:2: amino acids 22 to 27
("antigenic peptide 1"), amino acids 33 to 41 ("antigenic peptide
2"), amino acids 61 to 68 ("antigenic peptide 3"), amino acids 80
to 85 ("antigenic peptide 4"), amino acids 97 to 102 ("antigenic
peptide 5"), and amino acids 61 to 85 ("antigenic peptide 6"). The
present invention contemplates the use of any one of antigenic
peptides 1 to 6 to generate antibodies to Zven1. The present
invention also contemplates polypeptides comprising at least one of
antigenic peptides 1 to 6.
[0229] Similarly, analysis of the Zven2 amino acid sequence
indicated that suitable antigenic peptides of Zven2 include the
following segments of the amino acid sequence of SEQ ID NO:5: amino
acids 25 to 33 ("antigenic peptide 7"), amino acids 53 to 66
("antigenic peptide 8"), amino acids 88 to 95 ("antigenic peptide
9"), amino acids 98 to 103 ("antigenic peptide 10"), and amino
acids 88 to 103 ("antigenic peptide 11"). The present invention
contemplates the use of any one of antigenic peptides 7 to 11 to
generate antibodies to Zven2. The present invention also
contemplates polypeptides comprising at least one of antigenic
peptides 7 to 11.
[0230] Polyclonal antibodies to recombinant Zven protein or to Zven
isolated from natural sources can be prepared using methods
well-known to those of skill in the art. See, for example, Green et
al., "Production of Polyclonal Antisera," in Immunochemical
Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and
Williams et al., "Expression of foreign proteins in E. coli using
plasmid vectors and purification of specific polyclonal
antibodies," in DNA Cloning 2: Expression Systems, 2nd Edition,
Glover et al. (eds.), page 15 (Oxford University Press 1995). The
immunogenicity of a Zven polypeptide can 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
Zven 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.
[0231] Although polyclonal antibodies are typically raised in
animals such as horses, cows, dogs, chicken, rats, mice, rabbits,
guinea pigs, goats, or sheep, an anti-Zven antibody of the present
invention may also be derived from a subhuman primate antibody.
General techniques for raising diagnostically and therapeutically
useful antibodies in baboons may be found, for example, in
Goldenberg et al., international patent publication No. WO
91/11465, and in Losman et al., Int. J. Cancer 46:310 (1990).
[0232] Alternatively, monoclonal anti-Zven antibodies can be
generated. Rodent mono-clonal antibodies to specific antigens may
be obtained by methods known to those skilled in the art (see, for
example, Kohler et al., Nature 256:495 (1975), Coligan et al.
(eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7
(John Wiley & Sons 1991) ["Coligan"], Picksley et al.,
"Production of monoclonal antibodies against proteins expressed in
E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover
et al. (eds.), page 93 (Oxford University Press 1995)).
[0233] Briefly, monoclonal antibodies can be obtained by injecting
mice with a composition comprising a Zven gene product, verifying
the presence of antibody production by removing a serum sample,
removing the spleen to obtain B-lymphocytes, fusing the
B-lymphocytes with myeloma cells to produce hybridomas, cloning the
hybridomas, selecting positive clones which produce antibodies to
the antigen, culturing the clones that produce antibodies to the
antigen, and isolating the antibodies from the hybridoma
cultures.
[0234] In addition, an anti-Zven antibody of the present invention
may be derived from a human monoclonal antibody. Human monoclonal
antibodies are obtained from transgenic mice that have been
engineered to produce specific human antibodies in response to
antigenic challenge. In this technique, elements of the human heavy
and light chain locus are introduced into strains of mice derived
from embryonic stem cell lines that contain targeted disruptions of
the endogenous heavy chain and light chain loci. The transgenic
mice can synthesize human antibodies specific for human antigens,
and the mice can be used to produce human antibody-secreting
hybridomas. Methods for obtaining human antibodies from transgenic
mice are described, for example, by Green et al., Nature Genet.
7:13 (1994), Lonberg et al., Nature 368:856 (1994), and Taylor et
al., Int. Immun. 6:579 (1994).
[0235] Monoclonal antibodies can be isolated and purified from
hybridoma cultures by a variety of well-established techniques.
Such isolation techniques include affinity chromatography with
Protein-A Sepharose, size-exclusion chromatography, and
ion-exchange chromatography (see, for example, Coligan at pages
2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al., "Purification of
Immunoglobulin G (IgG)," in Methods in Molecular Biology, Vol. 10,
pages 79-104 (The Humana Press, Inc. 1992)).
[0236] For particular uses, it may be desirable to prepare
fragments of anti-Zven antibodies. Such antibody fragments can be
obtained, for example, by proteolytic hydrolysis of the antibody.
Antibody fragments can be obtained by pepsin or papain digestion of
whole antibodies by conventional methods. As an illustration,
antibody fragments can be produced by enzymatic cleavage of
antibodies with pepsin to provide a 5S fragment denoted
F(ab').sub.2. This fragment can be further cleaved using a thiol
reducing agent to produce 3.5S Fab' monovalent fragments.
Optionally, the cleavage reaction can be performed using a blocking
group for the sulfhydryl groups that result from cleavage of
disulfide linkages. As an alternative, an enzymatic cleavage using
pepsin produces two monovalent Fab fragments and an Fc fragment
directly. These methods are described, for example, by Goldenberg,
U.S. Pat. No. 4,331,647, Nisonoff et al., Arch Biochem. Biophys.
89:230 (1960), Porter, Biochem. J. 73:119 (1959), Edelman et al.,
in Methods in Enzymology Vol. 1, page 422 (Academic Press 1967),
and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
[0237] Other methods of cleaving antibodies, such as separation of
heavy chains to form monovalent light-heavy chain fragments,
further cleavage of fragments, or other enzymatic, chemical or
genetic techniques may also be used, so long as the fragments bind
to the antigen that is recognized by the intact antibody.
[0238] For example, Fv fragments comprise an association of V.sub.H
and V.sub.L chains. This association can be noncovalent, as
described by Inbar et al., Proc. Nat'l Acad. Sci. USA 69:2659
(1972). Alternatively, the variable chains can be linked by an
intermolecular disulfide bond or cross-linked by chemicals such as
glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech.
12:437 (1992)).
[0239] The Fv fragments may comprise V.sub.H and V.sub.L chains,
which are connected by a peptide linker. These single-chain antigen
binding proteins (scFv) are prepared by constructing a structural
gene comprising DNA sequences encoding the V.sub.H and V.sub.L
domains which are connected by an oligonucleotide. The structural
gene is inserted into an expression vector, which is subsequently
introduced into a host cell, such as E. coli. The recombinant host
cells synthesize a single polypeptide chain with a linker peptide
bridging the two V domains. Methods for producing scFvs are
described, for example, by Whitlow et al., Methods: A Companion to
Methods in Enzymology 2:97 (1991) (also see, Bird et al., Science
242:423 (1988), Ladner et al., U.S. Pat. No. 4,946,778, Pack et
al., Bio/Technology 11:1271 (1993), and Sandhu, supra).
[0240] As an illustration, a scFV can be obtained by exposing
lymphocytes to Zven polypeptide in vitro, and selecting antibody
display libraries in phage or similar vectors (for instance,
through use of immobilized or labeled Zven protein or peptide).
Genes encoding polypeptides having potential Zven polypeptide
binding domains can be obtained by screening random peptide
libraries displayed on phage (phage display) or on bacteria, such
as E. coli. Nucleotide sequences encoding the polypeptides can be
obtained in a number of ways, such as through random mutagenesis
and random polynucleotide synthesis. These random peptide display
libraries can be used to screen for peptides, which interact with a
known target that can be a protein or polypeptide, such as a ligand
or receptor, a biological or synthetic macromolecule, or organic or
inorganic substances. Techniques for creating and screening such
random peptide display libraries are known in the art (Ladner et
al., U.S. Pat. No. 5,223,409, Ladner et al., U.S. Pat. No.
4,946,778, Ladner et al., U.S. Pat. No. 5,403,484, Ladner et al.,
U.S. Pat. No. 5,571,698, and Kay et al., Phage Display of Peptides
and Proteins (Academic Press, Inc. 1996)) and random peptide
display libraries and kits for screening such libraries are
available commercially, for instance from CLONTECH Laboratories,
Inc. (Palo Alto, Calif.), Invitrogen Inc. (San Diego, Calif.), New
England Biolabs, Inc. (Beverly, Mass.), and Pharmacia LKB
Biotechnology Inc. (Piscataway, N.J.). Random peptide display
libraries can be screened using the Zven sequences disclosed herein
to identify proteins which bind to Zven.
[0241] Another form of an antibody fragment is a peptide coding for
a single complementarity-determining region (CDR). CDR peptides
("minimal recognition units") can be obtained by constructing genes
encoding the CDR of an antibody of interest. Such genes are
prepared, for example, by using the polymerase chain reaction to
synthesize the variable region from RNA of antibody-producing cells
(see, for example, Larrick et al., Methods: A Companion to Methods
in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation
of Monoclonal Antibodies," in Monoclonal Antibodies: Production,
Engineering and Clinical Application, Ritter et al. (eds.), page
166 (Cambridge University Press 1995), and Ward et al., "Genetic
Manipulation and Expression of Antibodies," in Monoclonal
Antibodies: Principles and Applications, Birch et al., (eds.), page
137 (Wiley-Liss, Inc. 1995)).
[0242] Alternatively, an anti-Zven antibody may be derived from a
"humanized" monoclonal antibody. Humanized monoclonal antibodies
are produced by transferring mouse complementary determining
regions from heavy and light variable chains of the mouse
immunoglobulin into a human variable domain. Typical residues of
human antibodies are then substituted in the framework regions of
the murine counterparts. The use of antibody components derived
from humanized monoclonal antibodies obviates potential problems
associated with the immunogenicity of murine constant regions.
General techniques for cloning murine immunoglobulin variable
domains are described, for example, by Orlandi et al., Proc. Nat'l
Acad. Sci. USA 86:3833 (1989). Techniques for producing humanized
monoclonal antibodies are described, for example, by Jones et al.,
Nature 321:522 (1986), Carter et al., Proc. Nat'l Acad. Sci. USA
89:4285 (1992), Sandhu, Crit. Rev. Biotech. 12:437 (1992), Singer
et al., J. Immun. 150:2844 (1993), Sudhir (ed.), Antibody
Engineering Protocols (Humana Press, Inc. 1995), Kelley,
"Engineering Therapeutic Antibodies," in Protein Engineering:
Principles and Practice, Cleland et al. (eds.), pages 399-434 (John
Wiley & Sons, Inc. 1996), and by Queen et al., U.S. Pat. No.
5,693,762 (1997).
[0243] Polyclonal anti-idiotype antibodies can be prepared by
immunizing animals with anti-Zven antibodies or antibody fragments,
using standard techniques. See, for example, Green et al.,
"Production of Polyclonal Antisera," in Methods In Molecular
Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana
Press 1992). Also, see Coligan at pages 2.4.1-2.4.7. Alternatively,
monoclonal anti-idiotype antibodies can be prepared using anti-Zven
antibodies or antibody fragments as immunogens with the techniques,
described above. As another alternative, humanized anti-idiotype
antibodies or subhuman primate anti-idiotype antibodies can be
prepared using the above-described techniques. Methods for
producing anti-idiotype antibodies are described, for example, by
Irie, U.S. Pat. No. 5,208,146, Greene, et. al., U.S. Pat. No.
5,637,677, and Varthakavi and Minocha, J. Gen. Virol. 77:1875
(1996).
10. THERAPEUTIC USES OF ZVEN POLYPEPTIDES
[0244] The present invention includes the use of proteins,
polypeptides, and peptides having Zven activity (such as Zven
polypeptides, Zven analogs, active Zven anti-idiotype antibodies,
and Zven fusion proteins) to a subject, which lacks an adequate
amount of this polypeptide. The present invention contemplates both
veterinary and human therapeutic uses. Illustrative subjects
include mammalian subjects, such as farm animals, domestic animals,
and human patients.
[0245] Zven polypeptides, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof, and
antagonists thereof are useful in diseases characterized by
dysfunction of the gastrointestinal tract due to limited
contractility, gastric emptying, and/or increased contractility, as
well as disorders associated with inflammation of the
intestine.
[0246] Dysfunction of the gastrointestinal tract due to limited
contractility and/or gastric emptying is a characteristic of
diseases and disorders including, but not limited to,
post-operative ileus, post-partum ileus, chronic constipation,
dyspepsia, intestinal pseudo-obstruction, gastroparesis, diabetic
gastroparesis, gastroesophageal reflux, emesis, use or consumption
of opiods and/or narcotics, muscular dystrophy, prgressive systemic
sclerosis, infectious diarrhea, and paralytic gastroparesis.
[0247] Postoperative inhibition of gastrointestinal motility
(postoperative ileus) is induced by laparotomy and intra-abdominal
procedures. The transient inhibition of gastrointestinal motility
occurring in humans mainly in the stomach and the colon may last
for several days and can considerably contribute to a patient's
postoperative discomfort. Oral food intake may be delayed until
post operative ileus has been resolved, and prolonged nasogastric
suction or, in rare cases, even relaparatomy becomes necessary
(Livingston, E. et al., Post operataive ileu. Dig. Dis Sci
35:121-132, 1990). Zven1 is a potent stimulator of gastrointestinal
contractility as shown in the following examples. As such, Zven
polypeptides, such as Zven1, Zven2, as well as agonists, fragments,
variants and/or chimeras thereof, can be used to stimulate
gastrointestinal contractility in patients after surgery. For
example, patients who have disorders such as, post-surgical
gastroparesis, including post-operative ileus, are good candidates
for administration of Zven polypeptides, such as Zven1, Zven2, as
well as agonists, fragments, variants and/or chimeras thereof.
[0248] Post-operative ileus (POI) is a condition of reduced
intestinal tract motility, including delayed gastric emptying, that
occurs as a result of disrupted muscle tone following surgery. It
is especially problematic following abdominal surgery. The problem
may arise from the surgery itself, from the residual effects of
anesthetic agents, and particularly, from pain-relieving narcotic
and opiate drugs used during and after surgery.
[0249] Post-operative ileus reduces gastrointestinal motility,
which also may delay the absorption of drugs administered orally.
Reduced intestinal motility following surgery is a major cause of
extended hospital stays, which are extremely expensive and result
in an increased chance of developing other complications. Extended
durations of POI may require the use of parenteral nutrition, which
also is expensive. With the increasing cost of medical care, the
expenses associated with hospital stays and parenteral feeding are
expected to increase even further.
[0250] The acute nature of this condition provides an opportunity
to treat with Zven polypeptides, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof. Moreover,
since oral drugs would be counter-indicated during POI, a drug
administered subcutaneously, intramuscularly, or intravenously,
such as Zven1 would be beneficial. "Prokinetics" have been found to
alleviate the symptoms associated with POI, and since Zven1 has
prokinetic properties, can be effective in treating POI. Currently
there are very few drugs that can effectively treat POI, and those
that are available have side effects, cannot be taken with other
medications, and/or are administered orally.
[0251] The effect of a Zven polypeptide, such as Zven1, Zven2, as
well as agonists, fragments, variants and/or chimeras thereof, on
POI can be measured in an in vivo model by administering it orally
(p.o.), intraperitoneally (i.p.), intraveneously (i.v.),
subcutaneously (s.c.), or intramuscularly (i.m.) to fasted animals
at an appropriate time prior to or following a laparotomy and cecal
manipulation performed under anesthesia. One of the Major Models
listed below may then be used to assess extent of gastric emptying
and/or intestinal transit at times ranging from 10 to 180 minutes
after removal of the anesthetic. In dog models, this time may be
greater (up to 50 h). Using several post-surgical time points
allows an estimate of the effects of surgery on gastric emptying
and transit along much of the gastrointestinal tract. This model
has been used extensively to evaluate the efficacy of prokinetic
drugs on gastric emptying and/or intestinal transit as a result of
POI (e.g. Martinez, Rivier, and Tache. J. Pharmacol. Experimental.
Therap. 290:629 (1999) and Furuta et al. Biol. Pharm. Bull.
25:103-1071 (2002)).
[0252] Additionally, Zven polypeptides, such as Zven1, Zven2, as
well as agonists, fragments, variants and/or chimeras thereof, can
be used to prevent POI. In this scenario, the Zven polypeptides,
such as Zven1, Zven2, as well as agonists, fragments, variants
and/or chimeras thereof, are administered to the patient
pre-operatively.
[0253] Diabetic gastroparesis is paralysis of the stomach brought
about by a motor abnormality in the stomach, as a complication of
both type I and type II diabetes. It is characterized by delayed
gastric emptying, post-prandial distention, nausea and vomiting. In
diabetes, it is thought to be due to a neuropathy, though it is
also associated with loss of interstitial cells of Cajal (ICC),
which are the "pacemaker cells" of the gut.
[0254] Patients who have diabetes mellitus may also have disorders
related to gastric emptying. For example, a patient who has had
diabetes mellitus for at least five years may have a prevalence of
significant delay in gastric emptying of >50% (Horowitz, M, et
al., J. Gastoenterology Hepatology: 1:97-113, 1986). Gastric
neuromuscular dysfunction occurs in up to 30-50% of patients after
10 years of type 1 or type 2 diabetes (Koch K., et al., Dig Dis Sci
44:1061-1075, 1999). Zven1, Zven2, and/or their agonists may also
be used as treatment for diabetic patients.
[0255] The often-acute nature of the episodes of diabetic
gastroparesis provides an opportunity to treat with Zven
polypeptides, such as Zven1, Zven2, as well as agonists, fragments,
variants and/or chimeras thereof. "Prokinetics" have been found to
alleviate the delayed gastric emptying associated with diabetic
gastroparesis. Currently there are very few drugs that can
effectively treat diabetic gastroparesis, and those that are
available have side effects and/or cannot be taken with other
medications. Oral drugs may not be tolerated during severe
episodes, and thus, would require intravenous administration of a
prokinetic.
[0256] The spontaneously diabetic NOD/LtJ mouse (available from
Jackson Laboratories) develops delayed gastric emptying, impaired
electrical pacemaking, and reduced motor neurotransmission. This is
described in Watkins et al. J. Clin. Invest. 106:373-384 (2000).
This strain also appears to have defects in interstitial cells of
Cajal (ICC) networks that are associated with impaired motility.
Streptozotocin treatment of rats and mice is a well-recognized and
acceptable method to induce diabetes; these animals are
characterized by impaired gastric emptying and intestinal transit,
and thus, show symptoms of diabetic gastroparesis (Yamano et al.
Naunyn-Schmiedeberg's Arch. Pharmacol. 356: 145-150 (1997) and
Watkins et al. J. Clin. Invest. 106:373-384 (2000)). The ability of
Zven polypeptide, such as Zven1, Zven2, as well as agonists,
fragments, variants and/or chimeras thereof, (administered via
p.o., i.v., i.p., s.c., or i.m.) to improve the impaired gastric
emptying and intestinal transit associated with the diabetes, can
also be measured by one of the Major Models described below.
[0257] Inflammatory reactions cause various clinical manifestations
frequently associated with abnormal motility of the
gastrointestinal tract, such as nausea, vomiting, ileus or
diarrhea. Bacterial lipopolysaccharide (LPS) exposure, for example,
induces such an inflammatory condition, which is observed in both
humans and experimental animals, and is characterized by biphasic
changes in gastrointestinal motility: increased transit in earlier
phases and delayed transit in later phases. Since Zven1 plays a
role in inflammation, and has biphasic activities at low
(prokinetic) and high (inhibitory) doses, it will be beneficial in
these inflammatory conditions.
[0258] Zven polypeptides, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof, can also be
used to treat gastrointestinal related sepsis. Experimental
"sepsis"/endotoxemia is produced in rodents using methods described
in Ceregrzyn et al. Neurogastroenterol. Mot. 13:605-613 (2001).
These animals develop biphasic alterations in gastrointestinal
transit. A Zven polypeptide, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof, can be
administered (via p.o., i.v., i.p., s.c., or i.m.) at either low
(prokinetic) or high (inhibitory) concentrations, depending on the
phase of the disease. Gastric emptying and/or intestinal transit
would then be measured using one of the Major Models described
below.
[0259] Morphine and other opioid analgesics are some of the most
common pain relievers used, especially following surgery. Because
they inhibit the release of acetylcholine from the mesenteric
plexus and thereby reduce the propulsive activity in the
gastrointestinal tract, individuals taking opioid analgesics often
suffer from reduced gastric emptying and intestinal transit. Since
Zven1 simulates intestinal contractility and has gastrointestinal
prokinetic activity, Zven1 may be beneficial in the treatment of
opioid-induced motility disorder(s).
[0260] The effect of Zven1 on opioid-induced gastroparesis in
experimental rodents can be measured by a well-known model. See
Suchitra et al. World J. Gastroenterol. 9:779-783 (2003) and Asai,
Arzneim.-Forsch./Drug Res. 48:802-805 (1998). Mice or rats are
administered a drug from the opioid class (e.g. morphine) via the
appropriate route of administration (p.o., i.p., i.v., s.c., i.m.)
at a dose known to inhibit gastric emptying and intestinal transit
(e.g. 1-5 mg/kg BW) at a set time prior to administration of the
test agent or method used to monitor gastric emptying and
intestinal transit (by use of one of the Major Models listed
below). The Zven polypeptide, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof, is
administered at the appropriate time point (via p.o., i.v., i.p.,
s.c., or i.m.) in relation to the opioid and test meal or method to
assess its efficacy in relieving opioid-induced
gastroparesis/ileus.
[0261] Individuals with neuropathies (e.g. as seen with diabetes)
often suffer from gastroparesis and reduced intestinal motility, as
a result of a malfunctioning nervous system. Since Zven1 appears to
induce intestinal contractility independently from nervous input,
this would suggest that Zven1 would be beneficial in individuals
suffering from neuropathy-induced gastrointestinal disorders.
[0262] The effect of Zven1 on vagotomy-induced gastroparesis in
experimental mammals can be measured in an animal model. Thoracic
vagotomy is performed in experimental mammals as described, for
example, in Takeda et al. Jpn. J. Pharmacol. 81:292-297 (1999) and
Hatanaka et al Neurogastroenterol. Motil. 8:227-233 (1996). These
animals are characterized by reduced gastric emptying and
intestinal transit. The Zven polypeptide, such as Zven1, Zven2, as
well as agonists, fragments, variants and/or chimeras thereof, is
administered (via p.o., i.p., i.v., s.c., or i.m.) as a means to
alleviate this vagotomy-induced gastroparesis/ileus, which is
monitored using one of the Major Models listed below.
[0263] Since Zven1 has contractile activity in the intestinal organ
bath system in the presence of atropine, Zven1 can also have
intestinal activity in vivo in the presence of atropine. Atropine
is a compound that blocks cholinergic nerve potential. Therefore,
the ability of Zven1 to be active in the presence of atropine
suggests that Zven1 can act independent of the cholinergic nervous
system, and thus, can be a beneficial treatment to those suffering
from neuropathy-associated gastroparesis and/or ileus.
[0264] Another indication where Zven1 and/or Zven2 can be used to
treat gastric dysfunction is gastreoesophageal reflux disease,
which is characterized by the backward flow of the stomach contents
into the esophagus, often as a result of a reduction in the
pressure barrier due to the failure of the lower esophageal
sphincter. Prokinetics, such as bethanechol (Urecholine) and
metoclopramide (Reglan) have been shown to help strengthen the
sphincter and make the stomach empty faster. Metoclopramide also
improves muscle action in the digestive tract, but these drugs have
frequent side effects that limit their usefulness. Thus a biologic
prokinetic, such as a Zven polypeptide, including Zven1, Zven2, as
well as agonists, fragments, variants and/or chimeras thereof, that
improves contraction in the stomach and gastrointestinal tract,
with or without improved stability of the esophageal sphincter will
be useful to treat gastroesophageal reflux disease.
[0265] Methods to investigate effects of atropine in vivo in
experimental rodents are well known in the art. See Chadhuri et al.
Life Sciences 66:847-854 (2000) and Kaneko et al. Digest. Dis. Sci.
40:2043-2051 (1995). Mice or rats are administered atropine via the
appropriate route of administration (p.o., i.p., i.v., s.c., i.m.)
at a dose known to inhibit gastric emptying and intestinal transit
(e.g. 0.1-2.0 mg/kg BW) at a set time prior to administration of
the test agent or method used to monitor gastric emptying and
intestinal transit (by use of one of the Major Models listed
below). Zven polypeptides, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof, is
administered at the appropriate time point (via p.o., i.v., i.p.,
s.c., or i.m.) in relation to the atropine and test meal or method
to assess its efficacy in the presence of atropine.
[0266] Additional indications where Zven polypeptides, such as
Zven1, Zven2, as well as agonists, fragments, variants and/or
chimeras thereof, can be used to treat gastric dysfunction are
gastroparesis, a paralysis of the stomach brought about by a motor
abnormality in the stomach or as a complication of diseases such as
diabetes, progressive systemic sclerosis, anorexia nervosa or
myotonic dystrophy. Diabetic gastroparesis results in delayed
gastric emptying, followed by post-prandial distention and
vomiting, which can result in poor glycemic control. It is often
associated with loss of interstitial cells of Cajal (ICC). Zven
polypeptides, such as Zven1, Zven2, as well as agonists, fragments,
variants and/or chimeras thereof, can also be used to treat gastric
dysfunction observed or associated with chronic constipation which
can be characterized by intestinal hypomotility, often due to lack
of intestinal muscle tone or intestinal spasticity. Another
indication where Zven polypeptides, such as Zven1, Zven2, as well
as agonists, fragments, variants and/or chimeras thereof, can be
used to treat gastric dysfunction is dyspepsia, which is defined as
an impairment of the power or function of digestion. It can be a
symptom of a primary gastrointestinal dysfunction, or a
complication of appendicitis, gall bladder disease or malnutrition.
Zven polypeptides, such as Zven1, Zven2, as well as agonists,
fragments, variants and/or chimeras thereof, can also be used to
treat gastric dysfunction from emesis which is characterized by
symptoms of nausea and vomiting, induced spontaneously, as a result
of delayed gastric emptying, or associated with emetogenic cancer
chemotherapy or irradiation therapy. In still another indication
where Zven polypeptides, such as Zven1, Zven2, as well as agonists,
fragments, variants and/or chimeras thereof, can be used to treat
gastric dysfunction associated with paralytic gastroparesis. This
is a paralysis of the stomach brought about by a motor abnormality
in the stomach or as a complication of diseases (other than
diabetes) such as progressive systemic sclerosis, anorexia nervosa
or myotonic dystrophy. It results in delayed gastric emptying,
followed by post-prandial distention and vomiting.
[0267] For disorders related to deficient gastrointestinal
function, clinical signs of improved function include, but are not
limited to, increased intestinal transit, increased gastric
emptying, flatus, and borborygmi, ability to consume liquids and
solids, and/or a reduction in nausea and/or emesis.
[0268] Additionally, since Zven1 and Zven2 reduce contractility
when administered at high doses, Zven polypeptides, such as Zven1,
Zven2, as well as agonists, fragments, variants and/or chimeras
thereof, can be used to reduce or inhibit contractility when such
effect is desired. This effect may be desired as a solo therapy to
treat, for example, diarrhea, including chronic diarrhea and
traveler's diarrhea.
[0269] For disorders related to hyperactive gastrointestinal
contractility, clinincal signs of improved gastrointestinal
function include, but are not limited to, slowed gastric emptying,
slowed intestinal transit, and/or a reduction in cramps associated
with diarrhea.
[0270] Zven polypeptides, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof, can be used
to stimulate chemokine production. Chemokines are small
pro-inflammatory proteins that have a broad range of activities
involved in the recruitment and function of leukocytes. Rat CINC-1,
murine KC, and human GRO.alpha. are members of the CXC subfamily of
chemokines. Chemokines, in general, can be divided into groups that
are chemotactic predominatly for neutrophils, and also have
angiogenic activity, and those that primarily attract T lymphocytes
and monocytes. See Banks, C. et al, J. Pathology 199: 28-35, 2002.
Chemokines in the first group display an ELR (Glu-Leu-Arg) amino
acid motif at the NH.sub.2 terminus. GRO.alpha., for example,
contains this motif. GRO.alpha. also has mitogenic and angiogenic
properties and is involved in wound healing and blood vessel
formation. (See, for example, Li and Thomhill, Cytokine 12:1409
(2000)). As illustrated by Examples 2, 3, and 17, Zven1 and Zven2
stimulated the release of chemokine CINC-1 (Cytokine Induced
Neutrophil Chemoattractant factor 1) in cell lines derived from the
thoracic aorta of rats, Zven1 stimulated the release of chemokine
KC from mice, and chemokine MIP-2 (mouse Macrophage Inflammatory
Protein-2) is up-regulated in response to a low dose
(intraperitoneal injection) of Zven1. Therefore, Zven polypeptides,
such as Zven1, Zven2, as well as agonists, fragments, variants
and/or chimeras thereof, can be used to stimulate the production
chemokines in vivo. The chemokines can be purified from culture
media and used in research or clinical settings. Zven variants can
also be identified by the ability to stimulate production of
chemokines in vitro or in vivo.
[0271] Upregulated chemokine expression correlates with increasing
activity of IBD. See Banks, C. et al, J. Pathology 199: 28-35,
2002. Chemokines are able to attract inflammatory cells and are
involved in their activation. Similarly, MIP-2 expression has been
found to be associated with neutrophil influx in various
inflammatory conditions. As polypeptides that stimulate the
production of chemokines, Zven polypeptides, such as Zven1, Zven2,
as well as agonists, fragments, variants and/or chimeras thereof,
may be useful in treating Inflammatory Bowel Disease by reducing,
inhibiting or preventing chemokine influx in the intestinal
tract.
[0272] Similarly, as shown in Example 3, Zven1 administration can
cause neutrophil infiltration. There are many aspects involved in
the immune response of a mammal to an injury or infection where
neutrophil infiltration would be desirable. As such, Zven
polypeptides, such as Zven1, Zven2, as well as agonists, fragments,
variants and/or chimeras thereof, will be useful as an agent to
induce neutrophil infiltration.
[0273] As a protein that can stimulate the production of
chemokines, Zven polypeptides, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof, may be
useful in treating infections, including fungal, bacterial, viral
and parasitic infections. Thus, the administration of a Zven
polypeptide, such as Zven1, Zven2, as well as an agonist, fragment,
variant and/or a chimera thereof, may be used as an immune booster
to a specific tissue site. For example, Zven1 administered to
gastrointestinal tissue, or to lung tissue, may be useful alone, or
in combination therapy to treat infections.
[0274] Example 5 demonstrates that Zven1 and Zven2 can stimulate
angiogenesis. Accordingly, Zven1, Zven2, Zven1 agonists, and Zven2
agonists can be used to stimulate proliferation of cardiac stem
cells. These molecules can be administered alone or in combination
with other angiogenic factors, such as vascular endothelial growth
factor.
11. MAJOR MODELS USED TO MEASURE GASTRIC EMPTYING AND INTESTINAL
TRANSIT
[0275] As described above, there are a number of in vivo models to
measure gastric function. A few of these models are represented
below.
[0276] Model 1: Method to Measure Rate and Extent of Gastric
Emptying and Intestinal Transit Using Phenol Red/Methyl Cellulose
in Experimental Mammals
[0277] Fasted animals are given Zven1 (or other Zven agent, Zven
polypeptides, such as Zven1, Zven2, as well as agonists, fragments,
variants and/or chimeras thereof) by the appropriate route (p.o.,
i.p., i.v., s.c., i.m.). At the appropriate time point, a
non-nutritive semi-solid meal consisting of methylcellulose and
phenol red is administered by gavage, and animals are sacrificed at
a set time following this meal administration. Transit is assessed
by the recovery and spectrophotometric determination of phenol red
from designated regions along the gastrointestinal tract. The
period of dye recovery in the gastrointestinal tract may be from 10
to 180 minutes, depending on the indication and intestinal site of
interest. This model has been used extensively to evaluate the
efficacy of other prokinetic drugs on gastric emptying and/or
intestinal transit.
[0278] Model 2: Method to Measure Rate and Extent of Intestinal
Transit Using Arabic Gum/Charcoal Meal in Experimental Mammals
[0279] Fasted animals would be given Zven1 (or other Zven agent,
Zven polypeptides, such as Zven1, Zven2, as well as agonists,
fragments, variants and/or chimeras thereof) by the appropriate
route (i.p., i.v., s.c., i.m., p.o.). At the appropriate time
point, a semi-solid meal consisting of gum arabic and charcoal is
administered by gavage, and animals are sacrificed at a set time
following this meal administration (Puig and Pol. J. Pharmacol.
Experiment. Therap. 287:1068 (1998)). Transit is assessed by the
distance that the charcoal meal traveled as a fraction of the total
distance of the intestine. The period of transit measurement in the
gastrointestinal tract may be from 10 to 180 minutes, depending on
the indication and intestinal site of interest. This model has been
used extensively to evaluate the efficacy of prokinetic drugs on
intestinal transit.
[0280] Model 3: Method to Measure Rate and Extent of Gastric
Emptying Using Polystyrene Beads (Undigestible Solids) in
Experimental Rodents
[0281] Gastric emptying is evaluated by determining the emptying of
polystyrene beads of a specific diameter (e.g. 1 mm for rats) from
the stomach of fasted (24 h) male or female experimental rodents in
response to Zven1 (or another Zven agent, Zven polypeptides, such
as Zven1, Zven2, as well as agonists, fragments, variants and/or
chimeras thereof) via p.o., i.p., s.c., i.v. or i.m. route of
administration. Polystyrene beads are administered by gavage and
assessed for emptying as previously described (Takeuchi et al.
Digest. Dis. Sci. 42; 251-258 (1997)). Animals are sacrificed at a
specified time after pellet administration (e.g. 20-180 min), and
the stomachs are removed. The number of the pellets remaining in
the stomach are counted. In control studies, 90% of pellets would
be expected to remain in the stomach after 30 min, and fewer than
10% in the stomach after 3 h. This model has been used extensively
to evaluate the gastric emptying efficacy of prokinetic drugs in
experimental rodents.
[0282] Model 4: Method to Measure Rate and Extent of Gastric
Emptying of a Liquid or Solid Test Meal in Experimental Mammals
Using Acetaminophen as the Tracer
[0283] Fasted animals are given a liquid or solid test meal
containing acetominophen as the tracer. The test compound (e.g.
Zven1) is administered p.o., i.v., i.p., s.c., or i.m. either
before or after test meal administration. Blood samples are
obtained at intervals between 0 and 120 min, and the plasma
concentration of acetaminophen (which is a measure of gastric
emptying) is measured by HPLC. This is described, for example, in
Trudel et al Peptides 24:531-534 (2003).
[0284] Model 5: Method to Measure Gastric Emptying of a Solid Meal
in Experimental Rodents
[0285] Mice or rats ("rodents") are separated into four groups
(Zven1-; positive control-[erythromycin, metoclopramide, or
cisapride]; negative control-[caerulein]; and vehicle-treated
groups). Each group contains approximately 10 animals. They are
deprived of food for 24 hours, but have free access to water during
fast period. Animals are housed one per cage, with floor grids
placed in the cages to prevent contact with the bedding or feces.
The fasted animals are treated with one of the above agents via one
of the following routes of administrations: oral; i.p., i.v., s.c.,
or i.m.). Animals are introduced to pre-weighed Purina chow
individually for a set period of time (e.g. 1 hr) in their home
cages (with bedding removed) at the appropriate time point
following or prior to administration of test agent. At the end of
the feeding period, animals are housed in their home cages without
food and water for an additional set period of time. They are then
euthanized, the abdominal cavity opened, and stomach removed after
clamping the pylorus and cardia. The stomach is weighed, opened,
and washed of the gastric content by tap water. The gastric wall is
wiped dry, and the empty stomach is weighed again. Gastric contents
are collected, dried, and weighed. The amount of food contained in
the stomach (as measured in grams) is calculated as the difference
between the total weight of the stomach with content and the weight
of the stomach wall after the contents are removed. The weight of
the pellet and spill in the cage is also measured at the end of the
feeding period. The solid food ingested by the animals is
determined by the difference between the weight of the Purina chow
before feeding and the weight of the pellet and spill at the end of
the feeding period. The gastric emptying for the designated period
is calculated according to the equation: % of gastric
emptying=(1-gastric content/food intake).times.100. This model has
been used extensively in the literature to assess gastric emptying
of a solid meal (Martinez et al. J. Pharmacol. Experiment. Ther.
301: 611-617 (2002)).
[0286] Model 6: Method to Measure Rate and Extent of Gastric
Emptying of a Solid Test Meal in Experimental Mammals
[0287] Fasted animals receive barium sulfate spheroids with a
standard meal, followed by administration of the test compound such
as Zven polypeptides, such as Zven1, Zven2, as well as agonists,
fragments, variants and/or chimeras thereof (via p.o., i.v., i.p.,
s.c., or i.m) either before or after the meal. Gastric emptying is
measured by means of X-ray location, with passage being monitored
at least every 15 min-2 h. This method is described, for example,
in Takeda et al. Jpn. J. Pharmacol. 81:292-297 (1999).
[0288] Model 7: Method to Measure Colonic Propulsive Motility in
Experimental Rodents.
[0289] This is used to demonstrate and characterize the
pharmacological effects of compounds on colonic propulsive motility
in experimental rodents as described (Martinez et al. J. Pharmacol.
Experiment. Ther. 301: 611-617 (2002)). The test is based on the
reflex expulsion of a glass bead from the distal colon, which is
indicative of drug effects on the reflex arc. This test is useful
in evaluating whether diarrhea is a side effect. Mice or rats are
fasted for one hour prior to administrations of the test compound,
such Zven polypeptides, such as Zven1, Zven2, as well as agonists,
fragments, variants and/or chimeras thereof, or vehicle by the
appropriate route (i.p., s.c., i.m., p.o., i.v.), followed 30
minutes (or other appropriate time) later by the insertion of a
glass bead into the distal colon. Rodents are marked for
identification and placed in large glass beakers (or other) for
observation. The time required for expulsion of the bead is noted
for each rodent.
[0290] Model 8: Model to Measure Gastrointestinal Motor Activity in
Dogs.
[0291] Dogs are anesthetized and the abdominal cavity opened.
Extraluminal force transducers (sensor to measure contraction) are
sutured onto five (5) sites, i.e., the gastric antrum, 3 cm
proximal to the pyloric ring, the duodenum, 5 cm distal to the
pyloric ring, the jejunum, 70 cm distal to the pyloric ring, the
ileum, 5 cm proximal to the ileum-colon junction, and the colon, 5
cm distal to the ileum-colon junction. The lead wires of these
force transducers are taken out of the abdominal cavity and then
brought out through a skin incision made between the scapulae, at
which a connector is connected. After the operation, a jacket
protector is placed on the dog to protect the connector.
Measurement of the gastrointestinal motor activity is started two
weeks after the operation. For ad libitum measurement, a telemeter
(electrowave data transmitter) is connected with the connector to
determine the contractive motility at each site of the
gastrointestinal tract. The data is stored in a computer via a
telemeter for analysis. A test compound, such as Zven polypeptides,
such as Zven1, Zven2, as well as agonists, fragments, variants
and/or chimeras thereof, is administered via the appropriate route
(p.o., i.v., i.p., s.c., i.m.) at the appropriate time point to
assess its ability to affect gastrointestinal motor activity. This
can be performed in normal dogs or dogs in which
gastroparesis/ileus has been induced. The above method is a
modification of those in Yoshida. and Ito. J. Pharmacol.
Experiment. Therap. 257, 781-787 (1991) and Furuta et al. Biol.
Pharm. Bull. 25:103-1071 (2002).
12. ADDITIONAL METHODS TO MEASURE GASTRIC FUNCTION
[0292] Model of Pain Assessment Associated with Gut Distention (in
Rats; Rabbits; Dogs)
[0293] Indication: Inflammatory Bowel Disease (IBD), Irritable Bowl
Syndrome (IBS), gastroparesis, ileus, dyspepsia.
[0294] Animals are surgically prepared with electrodes implanted on
the proximal colon and striated muscles, and catheters implanted in
lateral ventricles of the brain. Rectal distension is performed by
inflation of a balloon rectally inserted, and the pressure
eliciting a characteristic visceromotor response is measured. A
test compound, such as Zven polypeptides, such as Zven1, Zven2, as
well as agonists, fragments, variants and/or chimeras thereof, is
administered via the appropriate route (p.o., i.p., s.c., i.v., or
i.m.) and at the appropriate time (i.e. 20 min, if i.p. or i.c.v.)
prior to distention. Test compound is evaluated for its ability to
affect colonic motility, abdominal contractions, and visceral
pain.
[0295] Model to Assess Emesis (in Ferrets).
[0296] Indication: emesis (primary or as a result of
gastroparesis)
[0297] The anti-emetic activity of a test compound is tested by its
ability to inhibit cisplatin- or syrup of ipecac-induced emesis in
the ferret (since mice and rats can not vomit). In this model the
onset of retching and vomiting occurs approximately 1 h after the
administration of cisplatin (200 mg/m.sup.2 i.p.). At the first
retch in response to cisplatin, the test compound, Zven
polypeptides, such as Zven1, Zven2, as well as agonists, fragments,
variants and/or chimeras thereof, is administered (e.g. i.p., p.o.,
i.v., s.c., i.c.v.) and its effect on emesis determined by
comparison with appropriate controls (e.g. water). If using ipecac
to induce emesis, the test compound may be given at appropriate
time points prior to the ipecac. Latency to the first retch, the
first vomit and the number of retching and vomiting episodes are
recorded over 60 min. Data are expressed as the mean latency (in
min) to first retch or vomit; the mean number of emetic episodes
per ferret based on animals that did not exhibit emesis as well as
those that did, and the mean number of retches/vomits exhibited by
animals that remained responsive to ipecac ("responders"). Ferrets
that fail to exhibit emesis are omitted from the latter
calculation.
[0298] [i.e. (.+-.) cis-3-(2-methoxybenzylamino)-2-phenyl
piperidine exhibited anti-emetic activity when administered at a
dose of 3 mg/kg i.p.]
[0299] Models of (Interstitial Cells of Cajal) ICC Loss.
[0300] Loss of ICC results in serious gastrointestinal motor
dysfunction, and is seen in many diseases associated with altered
gastrointestinal function. Antibodies to Kit provide the
opportunity to evaluate ICC networks in gastrointestinal muscles in
motility disorders.
[0301] Indications: diabetic gastroparesis; IBD;
pseudo-obstruction, chronic constipation.
[0302] Inducible Example: BALB/c mouse pups are treated with a
monoclonal antibody (ACK2) to Kit for 4 d postnatally This
suppresses the development of c-kit, resulting in a severe disorder
of gut motility. A test compound, Zven polypeptides, such as Zven1,
Zven2, as well as agonists, fragments, variants and/or chimeras
thereof, is administered to assess its affect on gut motility.
Isolated segments of the intestine from the Kit-treated mice may
also be tested for rhythmic contraction and relaxation in vitro, in
response to the test compound.
[0303] Spontaneous Example: The spontaneously diabetic NOD/LtJ
mouse (Jackson Labs) develop delayed gastric emptying, impaired
electrical pacemaking, and reduced motor neurotransmission. A test
compound, Zven polypeptides, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof, administered
to assess its affect on gastric emptying (i.e. via phenol
red/methyl cellulose) and gut motility. Isolated segments of the
intestine from these mice may also be tested for rhythmic
contraction and relaxation in vitro, in response to the test
compound administration.
[0304] Zven polypeptides, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof, can also be
used to increase sensitization in mammals. For example, an ortholog
of Zven1 and Zven2, Bv8, was used to stimulate the PK-R1 and PK-R2
receptors in rats resulting in sensitization of peripheral
nociceptors to thermal and mechanic stimuli. See Negri, L. et al.,
Brit. J. Pharm. 137: 1147-1154, 2002. Thus, the Zven1 and Zven2
polypeptides of the present invention, including agonists, can be
used to increase sensitization (pain, heat, or mechanical) when
delivered locally or topically, systemically, or centrally. Also,
the polypeptides of the present invention can be administered to
enhance the sensitivity of brain cells in order to improve the
function out of the surviving neurons to neurotransmitters and
therefore might be effective in Parkinson's or Alzheimers disease.
Zven1 polypeptides, and other Zven1 agonists, can also be used to
alleviate pain, such as visceral pain or severe headache (e.g.,
migraine).
[0305] Similarly, where a patient has an increased sensitization to
pain, antagonists to Zven1 and Zven2 can be used to decrease the
sensation of pain in a patient with neuropathy. For example a
patients with diabetic neuropathy have chronic, enhanced pain, the
antagonist to zven1 may be useful to limit, prevent or decrease the
pain.
[0306] As shown in Example 5, Zven1 and Zven2 can stimulate
angiogenesis. Accordingly, Zven1, Zven2, Zven1 agonists, and Zven2
agonists can be used to induce growth of new blood vessels. These
molecules can be administered to a mammalian subject alone or in
combination with other angiogenic factors, such as vascular
endothelial growth factor.
[0307] Furthermore, prolonged gastrointestinal stasis often
complicates the course of patients with sepsis (Hemann G, et al.,
Am. J. Phys. Regul. Integr. Compr. Physiol. 276:R59-R68, 1999).
Activation of a systemic immune response by injury, infection,
radiation, or chemotherapy, is often accompanied by gastric stasis
which is perceived as nausea, loss of appetite and vomiting (Emch
G., et al., Am. J. Physiol. Gastrointest. Liver Physiol. 279:
G5582-G586, 2000). Thus, zven1 and it agonists may be useful in
treating sepsis related to gastrointestinal stasis or ileus.
[0308] Additionally, such as Zven1, Zven2, as well as agonists,
fragments, variants and/or chimeras thereof, may be useful in
treating patients with nausea and vomiting, especially where the
nausea and vomiting are related to, or a result of ileus or other
gastrointestinal motility disorders. These include when the
vomiting is related to treatment for cancer, such as a prophylaxis,
or post-administered, for chemotherapy.
[0309] Using telemetry in conscious male Sprague-Dawley rats, there
were no significant changes in blood pressure or heart rate in
response to an i.v. dose of 200 .mu.g/kg Zven1. Stool consistency
from these rats did not appear to be different during the 24 h
period following Zven1 administration. Rats do not appear to be
affected by these doses of Zven1. There were no reported outward
affects when mice were administered 10,000 .mu.g/kg Zven1 via an
i.p. injection.
[0310] The Zven polypeptides of the present invention, such as
Zven1, Zven2, as well as agonists, fragments, variants and/or
chimeras thereof, can also be used as a supplement to food. Zven2
polypeptides have been purified from bovine milk. See Masuda Y. et
al., Bioc. and Biophys. Res. Comm. 293:396-402, 2002. Additionally,
increased gastrointestinal contractility can be conducive to
improved metabolism and weight gain. As a protein that can be
administered orally, Zven1 or Zven2, or a combination of agonists,
variants, and/or fragments, can be useful as a supplement or
adjuvant to a feeding program wherein the mammalian subject suffers
from a lack of appetite and/or weight gain. Such conditions are
known, for example, as failure to thrive, cachexia, and wasting
syndromes. The polypeptides of the present invention may also be
useful adapting an infant mammal to digesting more conventional
types of food.
[0311] Generally, the dosage of administered polypeptide, protein
or peptide will vary depending upon such factors as the patient's
age, weight, height, sex, general medical condition and previous
medical history. Typically, it is desirable to provide the
recipient with a dosage of a molecule having Zven activity, which
is in the range of from about 1 pg/kg to 10 mg/kg (amount of
agent/body weight of patient), although a lower or higher dosage
also may be administered as circumstances dictate.
[0312] Zven1 polypeptides that were heated to 56 degrees C. for 30
minutes maintained some activity, when measured by a reporter
assay. See Example 13. Thus, the polypeptides of the present
invention may be effectively delivered orally.
[0313] Administration of a molecule having Zven activity to a
subject can be intravenous, intraarterial, intraperitoneal,
intramuscular, subcutaneous, intrapleural, intrathecal, by
perfusion through a regional catheter, inhalation, as a
suppository, or by direct intralesional injection. When
administering therapeutic proteins by injection, the administration
may be by continuous infusion or by single or multiple boluses.
Alternatively, Zven polypeptides, such as Zven1, Zven2, as well as
agonists, fragments, variants and/or chimeras thereof, can be
administered as a controlled release formulation.
[0314] Additional routes of administration include oral, dermal,
mucosal-membrane, pulmonary, and transcutaneous. Oral delivery is
suitable for polyester microspheres, zein microspheres, proteinoid
microspheres, polycyanoacrylate microspheres, and lipid-based
systems (see, for example, DiBase and Morrel, "Oral Delivery of
Microencapsulated Proteins," in Protein Delivery: Physical Systems,
Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)). The
feasibility of an intranasal delivery is exemplified by such a mode
of insulin administration (see, for example, Hinchcliffe and Illum,
Adv. Drug Deliv. Rev. 35:199 (1999)). Dry or liquid particles
comprising such as Zven1, Zven2, as well as agonists, fragments,
variants and/or chimeras thereof, can be prepared and inhaled with
the aid of dry-powder dispersers, liquid aerosol generators, or
nebulizers (e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton
et al., Adv. Drug Deliv. Rev. 35:235 (1999)). This approach is
illustrated by the AERX diabetes management system, which is a
hand-held electronic inhaler that delivers aerosolized insulin into
the lungs. Studies have shown that proteins as large as 48,000 kDa
have been delivered across skin at therapeutic concentrations with
the aid of low-frequency ultrasound, which illustrates the
feasibility of trascutaneous administration (Mitragotri et al.,
Science 269:850 (1995)). Transdermal delivery using electroporation
provides another means to administer such as Zven1, Zven2, as well
as agonists, fragments, variants and/or chimeras thereof, (Potts et
al., Pharm. Biotechnol. 10:213 (1997)).
[0315] Zven proteins can also be applied topically as, for example,
liposomal preparations, gels, salves, as a component of a glue,
prosthesis, or bandage, and the like.
[0316] Since chemokines can promote and accelerate tissue repair,
such as Zven1, Zven2, as well as agonists, fragments, variants
and/or chimeras thereof, can have a beneficial role in resolving
disease. For example, topical administration is useful for wound
healing applications, including the prevention of excess scaring
and granulation tissue, prevention of keyloids, and prevention of
adhesions following surgery.
[0317] A pharmaceutical composition comprising molecules having
Zven1 or Zven2 activity can be furnished in liquid form, in an
aerosol, or in solid form. Proteins having Zven1 or Zven2 activity
can be administered as a conjugate with a pharmaceutically
acceptable water-soluble polymer moiety. As an illustration, a
Zven1-polyethylene glycol conjugate is useful to increase the
circulating half-life of the interferon, and to reduce the
immunogenicity of the polypeptide. Liquid forms, including
liposome-encapsulated formulations, are illustrated by injectable
solutions and oral suspensions. Exemplary solid forms include
capsules, tablets, and controlled-release forms, such as a
miniosmotic pump or an implant. Other dosage forms can be devised
by those skilled in the art, as shown, for example, by Ansel and
Popovich, Pharmaceutical Dosage Forms and Drug Delivery Systems,
5.sup.th Edition (Lea & Febiger 1990), Gennaro (ed.),
Remington's Pharmaceutical Sciences, 19.sup.th Edition (Mack
Publishing Company 1995), and by Ranade and Hollinger, Drug
Delivery Systems (CRC Press 1996).
[0318] A pharmaceutical composition comprising a protein,
polypeptide, or peptide having Zven1 or Zven2 activity can be
formulated according to known methods to prepare pharmaceutically
useful compositions, whereby the therapeutic proteins are combined
in a mixture with a pharmaceutically acceptable carrier. A
composition is said to be a "pharmaceutically acceptable carrier"
if its administration can be tolerated by a recipient patient.
Sterile phosphate-buffered saline is one example of a
pharmaceutically acceptable carrier. Other suitable carriers are
well-known to those in the art. See, for example, Gennaro (ed.),
Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing
Company 1995).
[0319] For purposes of therapy, molecules having Zven1 or Zven2
activity and a pharmaceutically acceptable carrier are administered
to a patient in a therapeutically effective amount. A combination
of a protein, polypeptide, or peptide having Zven activity and a
pharmaceutically acceptable carrier is said to be administered in a
"therapeutically effective amount" if the amount administered is
physiologically significant. An agent is physiologically
significant if its presence results in a detectable change in the
physiology of a recipient patient.
[0320] For example, the present invention includes methods of
increasing or decreasing gastrointestinal contractility, gastric
emptyint, and/or intestinal transt, comprising the step of
administering a composition comprising a Zven2polypeptide, such as
Zven1, Zven2, as well as agonists, fragments, variants and/or
chimeras thereof, to the patient. In an in vivo approach, the
composition is a pharmaceutical composition, administered in a
therapeutically effective amount to a mammalian subject.
[0321] A pharmaceutical composition comprising molecules having
Zven activity can be furnished in liquid form, or in solid form.
Liquid forms, including liposome-encapsulated formulations, are
illustrated by injectable solutions and oral suspensions. Exemplary
solid forms include capsules, tablets, and controlled-release
forms, such as a miniosmotic pump or an implant. Other dosage forms
can be devised by those skilled in the art, as shown, for example,
by Ansel and Popovich, Pharmaceutical Dosage Forms and Drug
Delivery Systems, 5.sup.th Edition (Lea & Febiger 1990),
Gennaro (ed.), Remington's Pharmaceutical Sciences, 19.sup.th
Edition (Mack Publishing Company 1995), and by Ranade and
Hollinger, Drug Delivery Systems (CRC Press 1996).
[0322] Zven1 or Zven2 pharmaceutical compositions may be supplied
as a kit comprising a container that comprises Zven1 or Zven2, a
Zven1 or Zven2 agonist, or a Zven1 or Zven2 antagonist (e.g., an
anti-Zven1 or Zven2 antibody or antibody fragment). For example,
Zven1 or Zven2 can be provided in the form of an injectable
solution for single or multiple doses, or as a sterile powder that
will be reconstituted before injection. Alternatively, such a kit
can include a dry-powder disperser, liquid aerosol generator, or
nebulizer for administration of a therapeutic polypeptide. Such a
kit may further comprise written information on indications and
usage of the pharmaceutical composition. Moreover, such information
may include a statement that the Zven1 or Zven2 composition is
contraindicated in patients with known hypersensitivity to Zven1 or
Zven2.
13. THERAPEUTIC USES OF ZVEN NUCLEOTIDE SEQUENCES
[0323] The present invention includes the use of Zven nucleotide
sequences to provide Zven amino acid sequences to a subject in need
of proteins, polypeptides, or peptides having Zven activity, as
discussed in the previous section. In addition, a therapeutic
expression vector can be provided that inhibits Zven gene
expression, such as an anti-sense molecule, a ribozyme, or an
external guide sequence molecule.
[0324] There are numerous approaches to introduce a Zven gene to a
subject, including the use of recombinant host cells that express
Zven, delivery of naked nucleic acid encoding Zven, use of a
cationic lipid carrier with a nucleic acid molecule that encodes
Zven, and the use of viruses that express Zven, such as recombinant
retroviruses, recombinant adeno-associated viruses, recombinant
adenoviruses, and recombinant Herpes simplex viruses [HSV] (see,
for example, Mulligan, Science 260:926 (1993), Rosenberg et al.,
Science 242:1575 (1988), LaSalle et al., Science 259:988 (1993),
Wolff et al., Science 247:1465 (1990), Breakfield and Deluca, The
New Biologist 3:203 (1991)). In an ex vivo approach, for example,
cells are isolated from a subject, transfected with a vector that
expresses a Zven gene, and then transplanted into the subject.
[0325] In order to effect expression of a Zven gene, an expression
vector is constructed in which a nucleotide sequence encoding a
Zven gene is operably linked to a core promoter, and optionally a
regulatory element, to control gene transcription. The general
requirements of an expression vector are described above.
[0326] Alternatively, a Zven gene can be delivered using
recombinant viral vectors, including for example, adenoviral
vectors (e.g., Kass-Eisler et al., Proc. Nat'l Acad. Sci. USA
90:11498 (1993), Kolls et al., Proc. Nat'l Acad. Sci. USA 91:215
(1994), Li et al., Hum. Gene Ther. 4:403 (1993), Vincent et al.,
Nat. Genet. 5:130 (1993), and Zabner et al., Cell 75:207 (1993)),
adenovirus-associated viral vectors (Flotte et al., Proc. Nat'l
Acad. Sci. USA 90:10613 (1993)), alphaviruses such as Semliki
Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:857
(1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al.,
Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Pat.
Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus
vectors (Koering et al., Hum. Gene Therap. 5:457 (1994)), pox virus
vectors (Ozaki et al., Biochem. Biophys. Res. Comm. 193:653 (1993),
Panicali and Paoletti, Proc. Nat'l Acad. Sci. USA 79:4927 (1982)),
pox viruses, such as canary pox virus or vaccinia virus
(Fisher-Hoch et al., Proc. Nat'l Acad. Sci. USA 86:317 (1989), and
Flexner et al., Ann. N.Y. Acad. Sci. 569:86 (1989)), and
retroviruses (e.g., Baba et al., J. Neurosurg 79:729 (1993), Ram et
al., Cancer Res. 53:83 (1993), Takamiya et al., J. Neurosci. Res
33:493 (1992), Vile and Hart, Cancer Res. 53:962 (1993), Vile and
Hart, Cancer Res. 53:3860 (1993), and Anderson et al., U.S. Pat.
No. 5,399,346). Within various embodiments, either the viral vector
itself, or a viral particle which contains the viral vector may be
utilized in the methods and compositions described below.
[0327] As an illustration of one system, adenovirus, a
double-stranded DNA virus, is a well-characterized gene transfer
vector for delivery of a heterologous nucleic acid molecule (for a
review, see Becker et al., Meth. Cell Biol. 43:161 (1994); Douglas
and Curiel, Science & Medicine 4:44 (1997)). The adenovirus
system offers several advantages including: (i) the ability to
accommodate relatively large DNA inserts, (ii) the ability to be
grown to high-titer, (iii) the ability to infect a broad range of
mammalian cell types, and (iv) the ability to be used with many
different promoters including ubiquitous, tissue specific, and
regulatable promoters. In addition, adenoviruses can be
administered by intravenous injection, because the viruses are
stable in the bloodstream.
[0328] Using adenovirus vectors where portions of the adenovirus
genome are deleted, inserts are incorporated into the viral DNA by
direct ligation or by homologous recombination with a
co-transfected plasmid. In an exemplary system, the essential E1
gene is deleted from the viral vector, and the virus will not
replicate unless the E1 gene is provided by the host cell. When
intravenously administered to intact animals, adenovirus primarily
targets the liver. Although an adenoviral delivery system with an
E1 gene deletion cannot replicate in the host cells, the host's
tissue will express and process an encoded heterologous protein.
Host cells will also secrete the heterologous protein if the
corresponding gene includes a secretory signal sequence. Secreted
proteins will enter the circulation from tissue that expresses the
heterologous gene (e.g., the highly vascularized liver).
[0329] Moreover, adenoviral vectors containing various deletions of
viral genes can be used to reduce or eliminate immune responses to
the vector. Such adenoviruses are E1-deleted, and in addition,
contain deletions of E2A or E4 (Lusky et al., J. Virol. 72:2022
(1998); Raper et al., Human Gene Therapy 9:671 (1998)). The
deletion of E2b has also been reported to reduce immune responses
(Amalfitano et al., J. Virol. 72:926 (1998)). By deleting the
entire adenovirus genome, very large inserts of heterologous DNA
can be accommodated. Generation of so called "gutless"
adenoviruses, where all viral genes are deleted, are particularly
advantageous for insertion of large inserts of heterologous DNA
(for a review, see Yeh. and Perricaudet, FASEB J. 11:615
(1997)).
[0330] High titer stocks of recombinant viruses capable of
expressing a therapeutic gene can be obtained from infected
mammalian cells using standard methods. For example, recombinant
HSV can be prepared in Vero cells, as described by Brandt et al.,
J. Gen. Virol. 72:2043 (1991), Herold et al., J. Gen. Virol.
75:1211 (1994), Visalli and Brandt, Virology 185:419 (1991), Grau
et al., Invest. Ophthalmol. Vis. Sci. 30:2474 (1989), Brandt et
al., J. Virol. Meth. 36:209 (1992), and by Brown and MacLean
(eds.), HSV Virus Protocols (Humana Press 1997).
[0331] Alternatively, an expression vector comprising a Zven gene
can be introduced into a subject's cells 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. Nat'l Acad. Sci. USA 84:7413 (1987); Mackey
et al., Proc. Nat'l Acad. Sci. USA 85:8027 (1988)). The use of
lipofection to introduce exogenous genes into specific organs in
vivo has certain practical advantages. Liposomes can be used to
direct transfection to particular cell types, which is particularly
advantageous in a tissue with cellular heterogeneity, such as the
pancreas, liver, kidney, and brain. Lipids may be chemically
coupled to other molecules for the purpose of targeting. Targeted
peptides (e.g., hormones or neurotransmitters), proteins such as
antibodies, or non-peptide molecules can be coupled to liposomes
chemically.
[0332] Electroporation is another alternative mode of
administration of Zven nucleic acid molecules. For example, Aihara
and Miyazaki, Nature Biotechnology 16:867 (1998), have demonstrated
the use of in vivo electroporation for gene transfer into
muscle.
[0333] In an alternative approach to gene therapy, a therapeutic
gene may encode a Zven anti-sense RNA that inhibits the expression
of Zven. Suitable sequences for Zven anti-sense molecules can be
derived from the nucleotide sequences of Zven disclosed herein.
[0334] Alternatively, an expression vector can be constructed in
which a regulatory element is operably linked to a nucleotide
sequence that encodes a ribozyme. Ribozymes can be designed to
express endonuclease activity that is directed to a certain target
sequence in a mRNA molecule (see, for example, Draper and Macejak,
U.S. Pat. No. 5,496,698, McSwiggen, U.S. Pat. No. 5,525,468,
Chowrira and McSwiggen, U.S. Pat. No. 5,631,359, and Robertson and
Goldberg, U.S. Pat. No. 5,225,337). In the context of the present
invention, ribozymes include nucleotide sequences that bind with
Zven mRNA.
[0335] In another approach, expression vectors can be constructed
in which a regulatory element directs the production of RNA
transcripts capable of promoting RNase P-mediated cleavage of mRNA
molecules that encode a Zven gene. According to this approach, an
external guide sequence can be constructed for directing the
endogenous ribozyme, RNase P, to a particular species of
intracellular mRNA, which is subsequently cleaved by the cellular
ribozyme (see, for example, Altman et al., U.S. Pat. No. 5,168,053,
Yuan et al., Science 263:1269 (1994), Pace et al., international
publication No. WO 96/18733, George et al., international
publication No. WO 96/21731, and Werner et al., international
publication No. WO 97/33991). Preferably, the external guide
sequence comprises a ten to fifteen nucleotide sequence
complementary to Zven mRNA, and a 3'-NCCA nucleotide sequence,
wherein N is preferably a purine. The external guide sequence
transcripts bind to the targeted mRNA species by the formation of
base pairs between the mRNA and the complementary external guide
sequences, thus promoting cleavage of mRNA by RNase P at the
nucleotide located at the 5'-side of the base-paired region.
[0336] In general, the dosage of a composition comprising a
therapeutic vector having a Zven nucleotide acid sequence, such as
a recombinant virus, will vary depending upon such factors as the
subject's age, weight, height, sex, general medical condition and
previous medical history. Suitable routes of administration of
therapeutic vectors include intravenous injection, intraarterial
injection, intraperitoneal injection, intramuscular injection,
intratumoral injection, and injection into a cavity that contains a
tumor.
[0337] A composition comprising viral vectors, non-viral vectors,
or a combination of viral and non-viral vectors of the present
invention can be formulated according to known methods to prepare
pharmaceutically useful compositions, whereby vectors or viruses
are combined in a mixture with a pharmaceutically acceptable
carrier. As noted above, a composition, such as phosphate-buffered
saline is said to be a "pharmaceutically acceptable carrier" if its
administration can be tolerated by a recipient subject. Other
suitable carriers are well-known to those in the art (see, for
example, Remington's Pharmaceutical Sciences, 19th Ed. (Mack
Publishing Co. 1995), and Gilman's the Pharmacological Basis of
Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985)).
[0338] For purposes of therapy, a therapeutic gene expression
vector, or a recombinant virus comprising such a vector, and a
pharmaceutically acceptable carrier are administered to a subject
in a therapeutically effective amount. A combination of an
expression vector (or virus) and a pharmaceutically acceptable
carrier is said to be administered in a "therapeutically effective
amount" if the amount administered is physiologically significant.
An agent is physiologically significant if its presence results in
a detectable change in the physiology of a recipient subject.
[0339] When the subject treated with a therapeutic gene expression
vector or a recombinant virus is a human, then the therapy is
preferably somatic cell gene therapy. That is, the preferred
treatment of a human with a therapeutic gene expression vector or a
recombinant virus does not entail introducing into cells a nucleic
acid molecule that can form part of a human germ line and be passed
onto successive generations (i.e., human germ line gene
therapy).
14. DETECTION OF ZVEN1 GENE EXPRESSION WITH NUCLEIC ACID PROBES
[0340] Nucleic acid molecules can be used to detect the expression
of a Zven1 or Zven2 gene in a biological sample. Such probe
molecules include double-stranded nucleic acid molecules comprising
the nucleotide sequence of SEQ ID NO:1, or a fragment thereof, as
well as single-stranded nucleic acid molecules having the
complement of the nucleotide sequence of SEQ ID NO:1, or a fragment
thereof. Probe molecules may be DNA, RNA, oligonucleotides, and the
like.
[0341] Illustrative probes comprise a portion of the nucleotide
sequence of nucleotides 66 to 161 of SEQ ID NO:1, the nucleotide
sequence of nucleotides 288 to 389 of SEQ ID NO:1, or the
complement of such nucleotide sequences. An additional example of a
suitable probe is a probe consisting of nucleotides 354 to 382 of
SEQ ID NO:1, or a portion thereof. As used herein, the term
"portion" refers to at least eight nucleotides to at least 20 or
more nucleotides.
[0342] For example, nucleic acid molecules comprising a portion of
the nucleotide sequence of SEQ ID NO:1 can be used to detect
activated neutrophils. Such molecules can also be used to identity
therapeutic or prophylactic agents that modulate the response of a
neutrophil to a pathogen.
[0343] In a basic detection assay, a single-stranded probe molecule
is incubated with RNA, isolated from a biological sample, under
conditions of temperature and ionic strength that promote base
pairing between the probe and target Zven1 RNA species. After
separating unbound probe from hybridized molecules, the amount of
hybrids is detected.
[0344] Well-established hybridization methods of RNA detection
include northern analysis and dot/slot blot hybridization (see, for
example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et al. (eds.),
"Analysis of Gene Expression at the RNA Level," in Methods in Gene
Biotechnology, pages 225-239 (CRC Press, Inc. 1997)). Nucleic acid
probes can be detectably labeled with radioisotopes such as
.sup.32P or .sup.35 S. Alternatively, Zven RNA can be detected with
a nonradioactive hybridization method (see, for example, Isaac
(ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes
(Humana Press, Inc. 1993)). Typically, nonradioactive detection is
achieved by enzymatic conversion of chromogenic or chemiluminescent
substrates. Illustrative nonradioactive moieties include biotin,
fluorescein, and digoxigenin.
[0345] Zven1 oligonucleotide probes are also useful for in vivo
diagnosis. As an illustration, .sup.18F-labeled oligonucleotides
can be administered to a subject and visualized by positron
emission tomography (Tavitian et al., Nature Medicine 4:467
(1998)).
[0346] Numerous diagnostic procedures take advantage of the
polymerase chain reaction (PCR) to increase sensitivity of
detection methods. Standard techniques for performing PCR are
well-known (see, generally, Mathew (ed.), Protocols in Human
Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR
Protocols: Current Methods and Applications (Humana Press, Inc.
1993), Cotter (ed.), Molecular Diagnosis of Cancer (Humana Press,
Inc. 1996), Hanausek and Walaszek (eds.), Tumor Marker Protocols
(Humana Press, Inc. 1998), Lo (ed.), Clinical Applications of PCR
(Humana Press, Inc. 1998), and Meltzer (ed.), PCR in Bioanalysis
(Humana Press, Inc. 1998)).
[0347] One variation of PCR for diagnostic assays is reverse
transcriptase-PCR (RT-PCR). In the RT-PCR technique, RNA is
isolated from a biological sample, reverse transcribed to cDNA, and
the cDNA is incubated with Zven1 primers (see, for example, Wu et
al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR," in
Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)).
PCR is then performed and the products are analyzed using standard
techniques.
[0348] As an illustration, RNA is isolated from biological sample
using, for example, the guanidinium-thiocyanate cell lysis
procedure described above. Alternatively, a solid-phase technique
can be used to isolate mRNA from a cell lysate. A reverse
transcription reaction can be primed with the isolated RNA using
random oligonucleotides, short homopolymers of dT, or Zven1
anti-sense oligomers. Oligo-dT primers offer the advantage that
various mRNA nucleotide sequences are amplified that can provide
control target sequences. Zven1 sequences are amplified by the
polymerase chain reaction using two flanking oligonucleotide
primers that are typically 20 bases in length.
[0349] PCR amplification products can be detected using a variety
of approaches. For example, PCR products can be fractionated by gel
electrophoresis, and visualized by ethidium bromide staining.
Alternatively, fractionated PCR products can be transferred to a
membrane, hybridized with a detectably-labeled Zven1 probe, and
examined by autoradiography. Additional alternative approaches
include the use of digoxigenin-labeled deoxyribonucleic acid
triphosphates to provide chemiluminescence detection, and the
C-TRAK colorimetric assay.
[0350] Another approach for detection of Zven1 expression is
cycling probe technology (CPT), in which a single-stranded DNA
target binds with an excess of DNA-RNA-DNA chimeric probe to form a
complex, the RNA portion is cleaved with RNAase H, and the presence
of cleaved chimeric probe is detected (see, for example, Beggs et
al., J. Clin. Microbiol. 34:2985 (1996), Bekkaoui et al.,
Biotechniques 20:240 (1996)). Alternative methods for detection of
Zven1 sequences can utilize approaches such as nucleic acid
sequence-based amplification (NASBA), cooperative amplification of
templates by cross-hybridization (CATCH), and the ligase chain
reaction (LCR) (see, for example, Marshall et al., U.S. Pat. No.
5,686,272 (1997), Dyer et al., J. Virol. Methods 60:161 (1996),
Ehricht et al., Eur. J. Biochem. 243:358 (1997), and Chadwick et
al., J. Virol. Methods 70:59 (1998)). Other standard methods are
known to those of skill in the art.
[0351] Zven1 probes and primers can also be used to detect and to
localize Zven1 gene expression in tissue samples. Methods for such
in situ hybridization are well-known to those of skill in the art
(see, for example, Choo (ed.), In Situ Hybridization Protocols
(Humana Press, Inc. 1994), Wu et al. (eds.), "Analysis of Cellular
DNA or Abundance of mRNA by Radioactive In Situ Hybridization
(RISH)," in Methods in Gene Biotechnology, pages 259-278 (CRC
Press, Inc. 1997), and Wu et al. (eds.), "Localization of DNA or
Abundance of mRNA by Fluorescence In Situ Hybridization (RISH)," in
Methods in Gene Biotechnology, pages 279-289 (CRC Press, Inc.
1997)). Various additional diagnostic approaches are well-known to
those of skill in the art (see, for example, Mathew (ed.),
Protocols in Human Molecular Genetics (Humana Press, Inc. 1991),
Coleman and Tsongalis, Molecular Diagnostics (Humana Press, Inc.
1996), and Elles, Molecular Diagnosis of Genetic Diseases (Humana
Press, Inc., 1996)).
15. DETECTION OF ZVEN1 PROTEIN WITH ANTI-ZVEN1 ANTIBODIES
[0352] The present invention contemplates the use of anti-Zven1
antibodies to screen biological samples in vitro for the presence
of Zven1, and particularly for the upregulation of Zven1. In one
type of in vitro assay, anti-Zven1 antibodies are used in liquid
phase. For example, the presence of Zven1 in a biological sample
can be tested by mixing the biological sample with a trace amount
of labeled Zven1 and an anti-Zven1 antibody under conditions that
promote binding between Zven1 and its antibody. Complexes of Zven1
and anti-Zven1 in the sample can be separated from the reaction
mixture by contacting the complex with an immobilized protein which
binds with the antibody, such as an Fc antibody or Staphylococcus
protein A. The concentration of Zven1 in the biological sample will
be inversely proportional to the amount of labeled Zven1 bound to
the antibody and directly related to the amount of free-labeled
Zven1. Anti-Zven2 antibodies can be used in the same or a similar
fashion.
[0353] Alternatively, in vitro assays can be performed in which
anti-Zven1 antibody is bound to a solid-phase carrier. For example,
antibody can be attached to a polymer, such as aminodextran, in
order to link the antibody to an insoluble support such as a
polymer-coated bead, a plate or a tube. Other suitable in vitro
assays will be readily apparent to those of skill in the art.
[0354] In another approach, anti-Zven1 antibodies can be used to
detect Zven1 in tissue sections prepared from a biopsy specimen.
Such immunochemical detection can be used to determine the relative
abundance of Zven1 and to determine the distribution of Zven1 in
the examined tissue. General immunochemistry techniques are well
established (see, for example, Ponder, "Cell Marking Techniques and
Their Application," in Mammalian Development: A Practical Approach,
Monk (ed.), pages 115-38 (IRL Press 1987), Coligan at pages
5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley
Interscience 1990), and Manson (ed.), Methods In Molecular Biology,
Vol. 10: Immunochemical Protocols (The Humana Press, Inc.
1992)).
[0355] Immunochemical detection can be performed by contacting a
biological sample with an anti-Zven1 antibody, and then contacting
the biological sample with a detectably labeled molecule that binds
to the antibody. For example, the detectably labeled molecule can
comprise an antibody moiety that binds to anti-Zven1 antibody.
Alternatively, the anti-Zven1 antibody can be conjugated with
avidin/streptavidin (or biotin) and the detectably labeled molecule
can comprise biotin (or avidin/streptavidin). Numerous variations
of this basic technique are well-known to those of skill in the
art.
[0356] Alternatively, an anti-Zven1 antibody can be conjugated with
a detectable label to form an anti-Zven1 immunoconjugate. Suitable
detectable labels include, for example, a radioisotope, a
fluorescent label, a chemiluminescent label, an enzyme label, a
bioluminescent label or colloidal gold. Methods of making and
detecting such detectably-labeled immunoconjugates are well-known
to those of ordinary skill in the art, and are described in more
detail below.
[0357] The detectable label can be a radioisotope that is detected
by autoradiography. Isotopes that are particularly useful for the
purpose of the present invention are .sup.3H, .sup.125I, .sup.131I,
.sup.35S and .sup.14C.
[0358] Anti-Zven1 immunoconjugates can also be labeled with a
fluorescent compound. The presence of a fluorescently-labeled
antibody is determined by exposing the immunoconjugate to light of
the proper wavelength and detecting the resultant fluorescence.
Fluorescent labeling compounds include fluorescein isothiocyanate,
rhodamine, phycoerytherin, phycocyanin, allophycocyanin,
o-phthaldehyde and fluorescamine.
[0359] Alternatively, anti-Zven1 immunoconjugates can be detectably
labeled by coupling an antibody component to a chemiluminescent
compound. The presence of the chemiluminescent-tagged
immunoconjugate is determined by detecting the presence of
luminescence that arises during the course of a chemical reaction.
Examples of chemiluminescent labeling compounds include luminol,
isoluminol, an aromatic acridinium ester, an imidazole, an
acridinium salt and an oxalate ester.
[0360] Similarly, a bioluminescent compound can be used to label
anti-Zven1 immunoconjugates of the present invention.
Bioluminescence is a type of chemiluminescence found in biological
systems in which a catalytic protein increases the efficiency of
the chemiluminescent reaction. The presence of a bioluminescent
protein is determined by detecting the presence of luminescence.
Bioluminescent compounds that are useful for labeling include
luciferin, luciferase and aequorin.
[0361] Alternatively, anti-Zven1 immunoconjugates can be detectably
labeled by linking an anti-Zven1 antibody component to an enzyme.
When the anti-Zven1-enzyme conjugate is incubated in the presence
of the appropriate substrate, the enzyme moiety reacts with the
substrate to produce a chemical moiety, which can be detected, for
example, by spectrophotometric, fluorometric or visual means.
Examples of enzymes that can be used to detectably label
polyspecific immunoconjugates include .beta.-galactosidase, glucose
oxidase, peroxidase and alkaline phosphatase.
[0362] Those of skill in the art will know of other suitable
labels, which can be employed in accordance with the present
invention. The binding of marker moieties to anti-Zven1 antibodies
can be accomplished using standard techniques known to the art.
Typical methodology in this regard is described by Kennedy et al.,
Clin. Chim. Acta 70:1 (1976), Schurs et al., Clin. Chim. Acta 81:1
(1977), Shih et al., Int'l Cancer 46:1101 (1990), Stein et al.,
Cancer Res. 50:1330 (1990), and Coligan, supra.
[0363] Moreover, the convenience and versatility of immunochemical
detection can be enhanced by using anti-Zven1 antibodies that have
been conjugated with avidin, streptavidin, and biotin (see, for
example, Wilchek et al. (eds.), "Avidin-Biotin Technology," Methods
In Enzymology, Vol. 184 (Academic Press 1990), and Bayer et al.,
"Immunochemical Applications of Avidin-Biotin Technology," in
Methods In Molecular Biology, Vol. 10, Manson (ed.), pages 149-162
(The Humana Press, Inc. 1992).
[0364] Methods for performing immunoassays are well-established.
See, for example, Cook and Self, "Monoclonal Antibodies in
Diagnostic Immunoassays," in Monoclonal Antibodies: Production,
Engineering, and Clinical Application, Ritter and Ladyman (eds.),
pages 180-208, (Cambridge University Press, 1995), Perry, "The Role
of Monoclonal Antibodies in the Advancement of Immunoassay
Technology," in Monoclonal Antibodies: Principles and Applications,
Birch and Lennox (eds.), pages 107-120 (Wiley-Liss, Inc. 1995), and
Diamandis, Immunoassay (Academic Press, Inc. 1996).
[0365] In a related approach, biotin- or FITC-labeled Zven1 can be
used to identify cells that bind Zven1. Such can binding can be
detected, for example, using flow cytometry.
[0366] The present invention also contemplates kits for performing
an immunological diagnostic assay for Zven1 gene expression. Such
kits comprise at least one container comprising an anti-Zven1
antibody, or antibody fragment. A kit may also comprise a second
container comprising one or more reagents capable of indicating the
presence of Zven1 antibody or antibody fragments. Examples of such
indicator reagents include detectable labels such as a radioactive
label, a fluorescent label, a chemiluminescent label, an enzyme
label, a bioluminescent label, colloidal gold, and the like. A kit
may also comprise a means for conveying to the user that Zven1
antibodies or antibody fragments are used to detect Zven1 protein.
For example, written instructions may state that the enclosed
antibody or antibody fragment can be used to detect Zven1. The
written material can be applied directly to a container, or the
written material can be provided in the form of a packaging
insert.
[0367] The present invention, thus generally described, will be
understood more readily by reference to the following examples,
which are provided by way of illustration and are not intended to
be limiting of the present invention. The examples describe studies
using Zven1 protein produced in baculovirus with a C-terminal
Glu-Glu tag, following the methods generally described above. Zven2
("endocrine-gland-derived vascular endothelial growth factor")
protein was purchased from Peprotech, Inc. (Rocky Hill, N.J.).
16. EXAMPLES
Example 1
Stimulation of Responses in Wky12-22 Cells
[0368] Wky12-22 cells were derived from the medial layer of the
thoracic aorta of Wistar-Kyoto rat pups, as described by Lemire et
al., American Journal of Pathology 144:1068 (1994). These cells
respond to both Zven1 and Zven2 in a reporter luciferase assay
following transfection with NFkB/Ap-1 reporter construct. A control
cell line, Wky3M-22, derived from the same tissue in adult rat did
not signal. Activity was detected at concentrations ranging from
1-100 ng/ml of Zven1 or Zven2 (approximately 0.1 nM-10 nM). These
data suggest that Wky12-22 cells carry the Zven1 receptor, and that
Zven1 and Zven2 activate the NfKb/Ap-1 transcription factor.
[0369] In one experiment, Wky12-22 cells were loaded with the
fluorescent dye Fura. The emission peak of Fura shifts when bound
to calcium. Intracellular calcium release is detected by monitoring
the wavelength shift. Zven1 induced intracellular calcium release
at concentrations of 1-1000 ng/ml. Zven2 induced a similar
response.
[0370] Extracellular signal-regulated kinase/mitogen-activated
protein kinase (ERK-Map kinase) activity was measured in Wky12-22
cells in response to Zven1 treatment. Cells were incubated in Zven1
at concentrations ranging from 1 to 1,000 ng/ml for thirty minutes.
Cells were fixed and stained for phosphorylated ERK-Map kinase
using the Arrayscan, which measures the fluorescent intensities in
the cytosol and the nucleus of the treated cell. The difference in
fluorescence of the nucleus and the cytosol were quantified and
plotted. Zven1 induced ERK-Map kinase activity with an EC.sub.50 of
0.50 nM (approximately 5 ng/ml).
[0371] The binding of Zven1 to Wky12-22 cells was assessed using
I.sup.125-radiolabeled Zven1. Wky12-22 cells were seeded at low
cell density and cultured for three to four days until they reached
about 70% confluency. The cells were placed on ice, the medium was
removed, and the monolayers were washed. The cells were incubated
with increasing amounts of I.sup.125-Zven1 in the absence (total
binding) and presence (nonspecific binding) of a large excess of
unlabeled Zven1. After various times at 4.degree. C., the binding
media were removed, the monolayers were washed, and the cells were
solubilized with a small volume of 1.0 N NaOH. Cell associated
radioactivity was determined in a gamma counter. The specific
binding of I.sup.125-Zven1 was calculated as the difference between
the total and nonspecific values. The measured radioacitivity was
normalized to cell number that was determined on a set of parallel
cultures. Nonlinear regression using a two-site model was used to
fit the binding data for determination of Kd and Bmax. The high
affinity site exhibited a Kd of 1.5 nM and a Bmax of 350 fmol
bound/10.sup.6 cells whereas the low affinity site showed a Kd of
31 nM with a Bmax of 1025 fmol bound/10.sup.6 cells.
[0372] The results of these studies show that a neonatal rat aortic
cell expresses the Zven1 receptor while equivalent adult rat cells
do not. This suggests that Zven1 is involved with heart development
and vasculogenesis. Zven1 signals through NFkB/Ap1 and induces
chemokine release only in the neonatal cells, suggesting that it
may trigger a mitogenic response in fetal or neonatal heart. Zven1
may be a required factor necessary for the induction of
vasculogenesis/angiogenesis in cardiac stem cells. Zven1 induces
intracellular calcium release in the Wky12-22 cell line, an effect
consistent with chemokine activity. Consistent with its mitogenic
activity, Zven1 activates a mitogen activated protein kinase.
Example 2
Zven1 and Zven2 Stimulate Chemokine Release In Vitro
[0373] Confluent Wky12-22 or Wky3M22 cells were incubated with
varying concentrations of Zven1 for twenty-four hours. Conditioned
media were collected and assayed for the chemokine CINC-1 using a
commercially-available rat cytokine multiplex kit (Linco Research,
Inc.; St. Charles, Mo.). CINC-1, thought to be equivalent to human
growth-related oncogene-.alpha. (GRO-.alpha.), was detected at
levels ranging from 1.8-5 ng/ml in cells treated with 0.1 to 100
ng/ml of Zven1 respectively. Zven2 induced an equivalent level of
CINC-1 release from Wky12-22 cells. CINC-1 was not detected in
either the control Wky3M-22 cell line derived from adult rat aorta,
or non-treated controls.
Example 3
Zven1 Induces a Chemotactic Response and Stimulates Chemokine
Release and Neutrophil Infiltration In Vivo
[0374] Four groups of ten mice (BALB57/BL6 females at eight weeks
of age) were either not treated, or injected with vehicle buffer
control, 0.1 .mu.g of Zven1 or 1 .mu.g of Zven1. Four hours later,
peritoneal lavage fluid was collected, concentrated, and the cell
pellets were resuspended. The relative cell populations were
enumerated using the Cell Dyne, and cytospins were prepared for
CBC/diff counts. The non-treated and buffer control animals had
approximately 2% neutrophils in their lavage fluid, while the 0.1
.mu.g treated animals had approximately 30% neutrophils, indicating
an approximate 15-fold increase in neutrophils in the peritoneum of
the Zven1-treated animals. The 1 .mu.g Zven1-treated animals had
neutrophil levels consistent with the non-treated controls,
suggesting a biphasic Zven1 response. In sum, Zven1 induced
neutrophil infiltration into the peritoneum following
intraperitoneal injection.
[0375] Murine KC, the ortholog of GRO.alpha. in mice, was measured
in serum and lavage fluids obtained from the four groups of mice
using an ELISA kit (R&D Systems Inc.; MN). The 0.1.mu.g
Zven1-treated (low dose) mice had approximately 45 picograms/ml KC
in their peritoneal fluid, which was significantly higher than the
non-treated controls, the vehicle controls, and the 1.0 .mu.g
Zven1-treated (high dose) mice.
[0376] Serum levels of KC in the 0.1 .mu.g Zven1-treated mice were
considerably higher than the non-treated, the 1.0 .mu.g
Zven1-treated, and the vehicle-treated mice. The 0.1 .mu.g
Zven1-treated mice had KC levels of approximately 185 picograms/ml,
which is a six-fold increase. TABLE-US-00005 TABLE 5 Murine KC in
Zven1-treated mice following IP injection Concentration of Murine
KC (picogram/ml) Non-treated Vehicle 0.1 .mu.g 1.0 .mu.g animals
Control Zven1/animal Zven1/animal Lavage Fluid 10 21 45 8 Serum 30
38 185 50
[0377] These results are consistent with the stimulation of
chemokine release in vitro shown in Example 2. Furthermore these
results correlate with the observed neutriphil infiltration in the
peritoneum in the 0.1 .mu.g Zven1-treated (low dose) mice.
Example 4
Zven1 Effect on Gastric Emptying
[0378] Seven mice received an intraperitoneal injection of
approximately 200 .mu.g of Zven1 (10 .mu.g/g body weight) or
vehicle control followed by 7.5 mg phenol red. Gastric function was
measured by monitoring phenol red transport through the gut after
twenty minutes. The general behavior of Zven1 treated animals was
observed and was consistent with the behavior of the control
animals. In the Zven1-treated mice, gastric transit time was
reduced by approximately 50%.
[0379] These results show that, at high doses following
intraperitoneal injection, Zven1 reduces gastric transit. Zven1
administration did not appear to have any immediate toxic effects.
This reduction in transit may be the result of a massive muscle
contraction at such high doses. Zven1 may well increase motility in
vivo at low doses, and inhibit motility at high doses.
Example 5
Stimulation of Angiogenesis by Zven1 and Zven2
[0380] Thoracic aortas were removed from twelve-day, five-week, and
three-month old Wistar rats. The tissues were flushed with Hanks
basic salt solution to remove any blood cells and adventitial
tissues were removed. Aortic rings were prepared and plated on
Matrigel coated plates in serum free modified MCDB media from
Clonetics plus antibiotics, penicillin-streptomycin. Varying
concentrations of Zven1 and Zven2 were added to culture dish
approximately thirty minutes after plating. Proliferation was
measured visually and individual rings were photographed to record
results. Both Zven1 and Zven2 induced a proliferative response at
concentrations ranging from 1 to 100 ng/ml. This mitogenic effect
was observed in aortas from the animals at all three ages. Zven1
was also tested in the rat corneal model of anigiogenesis where no
effect was noted. The observed angiogenic effect in the aortic ring
cultures may be due to the mitogenic effects of the GRO.alpha.
homologue.
Example 6
Stimulation of Contractility in Guinea Pig Gastrointestinial Organ
Bath Assay
[0381] Male Hartley Guinea pigs at six weeks of age weighing
approximately 0.5 kg were euthanized by carbon monoxide. Intestinal
tissue was harvested as follows: 2-3 cm longitudinal sections of
ileum 10 cm rostral of the cecum, and 2-3 cm longitudinal sections
of duodenum, jejunum, and proximal and distal colon.
[0382] Tissue was washed in Krebs Ringer's Bicarbonate buffer
containing 118.2 mM NaCl, 4.6 mM KCl, 1.2 mm MgS0.sub.4, 24.8 mM
NaHC0.sub.3, 1.2 mM KH.sub.2P0.sub.4, 2.5 mM CaCl.sub.2 and 10 mM
glucose. Following a thorough wash, the tissue was mounted
longitudinally in a Radnoti organ bath perfusion system (SDR
Clinical Technology, Sydney Australia) containing oxygenated Krebs
buffer warmed and maintained at 37.degree. C. A one gram pre-load
was applied and the tissue strips were allowed to incubate for
approximately 30 minutes. Baseline contractions were then obtained.
Isometric contractions were measured with a force displacement
transducer and recorded on a chart recorder using Po-ne-mah
Physiology Platform Software. The neurotransmitter 5
Hydroxytryptophane (5HT) (Sigma) at 130 .mu.m, and atropine at 5-10
mM were used as controls. Atropine blocks the muscarinic effect of
acetylcholine.
[0383] Varying doses of Zven1 from 1-400 ng/ml were tested for
activity on strips of ileum. Muscle contractions were detected
immediately after adding zven1 protein and were recorded at
concentrations as low as 1 ng/ml or 100 picomolar. The EC 50 of
this response was approximately 10 ng/ml or 1 nM. Zven1 was tested
for activity in the presence of 5HT, and a secondary contraction
was observed. Zven1 was tested for activity in the presence of 0.1
.mu.M tetrodotoxin (TTX), the nerve action potential antagonist and
no reduction in the zven1 effect was observed. Zven1 was also
tested for activity in the presence of 100 nM Verapamil, the L-type
calcium channel blocker. A significant reduction in the amplitude
of the contractile response was observed.
[0384] Results of the effect of zven1 on contractions in the ileum
are shown in Table 6. TABLE-US-00006 TABLE 6 Summary of Ileum Organ
Bath Test Results Treatment Ileum 40 ng/ml Zven1 +C 40 ng/ml +C
Zven1 + 130 .mu.M 5HT 40 ng/ml Zven1 + 5 mM +C Atropine 40 ng/ml
Zven1 + 1 .mu.M - Verapamil 40 ng/nL Zven1 + 0.1 .mu.M +C TTX +C =
Contraction Observed - = No zven1 effect observed
[0385] Results of the effect of zven1 on contractions in duodenum,
jejunum, proximal colon, and distal colon were performed at a
concentration of 40 ng/ml did not produce contractions in duodenum,
jejunum, or distal colon. However, relaxation of the tissue of the
proximal colon was observed when the same concentration of zven1
was added.
Example 7
Effect of Dose on Contractility in Guinea Pig Ileal Organ Bath
Assay
[0386] All intestinal sections from the guinea pig ileum were
obtained and tested using the same protocol and reagents as
described in Example 6. Longitudinal strips of guinea pig ileum
were mounted in the organ bath and allowed to stabilize for
approximately 20 minutes. Acetylcholine (ACH) at a concentration of
10 .mu.g/ml was added to tissue to confirm contractile activity.
Two flush and fill cycles were run to wash ACH from the intestinal
tissue. Baseline activity was confirmed for approximately 25
minutes. Zven1 was added to the organ bath at a final concentration
of 1.0 ng/ml and an approximate 0.5 gram of deflection was
recorded. The 1.0 ng/ml zven1 dose was left on the tissue for 5
minutes to allow the tissue to return to baseline levels, and then
a 10 ng/ml dose was added. Another contractile response was noted
that resulted in a 2.0 gram deflection. The 10 ng/ml dose was left
on for another 5 minutes before dosing the tissue with a 20 ng/ml
dose of zven1. Another contractile response was observed, yielding
an approximate 2.2 gram deflection. Following a 5 minute
incubation, the tissue was treated with a 40 ng/ml dose of zven1.
The tissue contracted again, with an approximate 2.0 gram
deflection. The highest response was observed at the 20 ng/mL zven1
dose.
Example 8
Effect of Zven1 on Gastric Emptying and Intestinal Transit
[0387] Eight-week old female C57B1/6 mice were fed a test meal
consisting of a methylcellulose solution or a control, and both
gastric emptying and intestinal transit was measured by determining
the amount of phenol red recovered in different sections of the
intestine. The test meal consists of a 1.5% aqueous methylcellulose
solution containing a non-absorbable dye, 0.05% phenol red (50
mg/100 ml Sigma Chemical Company Catalogue # P4758). Medium
viscosity carboxy methylcellulose from Sigma (Catalogue #C4888)
with a final viscosity of 400-800 centipoises was used. One group
of animals was sacrificed immediately following administration of
test meal. These animals represent the standard group, 100% phenol
red in stomach or Group VIII. The remaining animals were sacrificed
20 minutes post administration of test meal. Following sacrifice,
the stomach was removed and the small intestine was sectioned into
proximal, mid and distal gut sections. The proximal gut consisted
approximately of duodenum, the mid gut consisted approximately of
duodenum and jejunum, and the distal gut consisted approximately of
ileum. All tissues were solubilized in 10 mls of 0.1 N NaOH using a
tissue homogenizer. Spectrophotometric analysis was used to
determine the OD and hence the level of gastric emptying and gut
transit.
[0388] Each treatment group consisted of 10 animals, except for the
animals being used as a standard group and the caerulein control
group where the n=5. The study was broken down into two days, such
that one half of all treatment groups are done on two consecutive
days. The animals were fasted for 18 hrs in elevated cages,
allowing access to water. The average weight of the mice was 16
grams.
[0389] Baculovirus-expressed Zven1 protein with a C-terminal
Glu-Glu tag formulated in 20 mM MES buffer, 20 mM NaCl, pH 6.5 was
diluted into 0.9% NaCl+0.1% BSA using siliconized tubes. (Sigma
sodium chloride solution 0.9%, and Sigma BSA 30% sterile TC tested
solution, Sigma Chemical Co, St Louis, Mo.). The protein
concentration was adjusted so as to be contained in a 0.2 ml volume
per mouse. Vehicle animals received an equivalent dose of zven1
formulation buffer based on the highest (775 ng/g) treatment
group.
[0390] Treatments were administered in a 0.2 ml volume via IP
(intraperitoneal) injection two minutes prior to receiving 0.15 ml
phenol red test meal as an oral gavage. Twenty minutes post
administration of phenol red, animals were euthanized and stomach
and intestinal segments removed. The intestine was measured and
divided into three equal segments: proximal, mid and distal gut.
The amount of phenol red in each sample was determined by
spectrophotometric analysis and expressed as the percent of total
phenol red in the stomach (Group VIII). These values were used to
determine the amount of gastric emptying and gut transit per tissue
collected. The CCK analogue caerulein at 40 ng/gram was used as a
positive control and was administered five minutes prior to gavage,
at which concentration it inhibits gastric emptying. Colormetric
analysis of phenol red recovered from each gut segment and stomach
was performed as follows. After euthanization, the stomach and
intestinal segments were placed into 10 mls of 0.1 N NaOH and
homogenized using a polytron tissue homogenizer. The homogenate was
incubated for 1 hour at room temperature then pelleted by
centrifugation on a table top centrifuge at 150.times.g for 20
minutes at 4 degrees C. Proteins were precipitated from 5.0 mls of
the homogenate by the addition of 0.5 ml of 20% trichloracetic
acid. Following centrifugation, 4 mls of supernatant was added to 4
mls of 0.5 N NaOH. A 200 .mu.l sample was read at 560 nm using
Molecular Devices Spectra Max 190 spectrophotometer. The amount of
gastric emptying was calculated using the following formula:
percent gastric emptying=(1-amount phenol red recovered from test
stomach/average amount of phenol red recovered from Group VII
stomach).times.100. The amount of gastric transit was expressed as
the percent of total phenol red recovered.
[0391] Results are shown in Table 7, below. Since test meal was not
detected in the distal gut under any conditions, these data are not
included. As expected, caerulein at 40 ng/ml inhibited gastric
emptying (93.8% of test meal in stomach after 20 minutes compared
to 63.8% with vehicle). Consistent with inhibited gastric emptying,
in the caerulein treated group only 2.6% of meal was measured in
the proximal gut and 1.2% in the mid gut.
[0392] At the lowest zven1 concentration, 0.78 ug/kg body weight, a
slight increase in gastric emptying compared to vehicle was
observed (56.3% of meal remaining versus 63.8% with vehicle).
Consistent with an increase in gastric emptying, increased meal was
detected in the proximal gut of the zven1 treated animals compared
to vehicle control, 25.5% and 18.4% respectively. At the 7.8 ug/kg
dose, zven1 treated animals had 20% less test meal in the stomach
(p=0.001), 16.6% more meal in the proximal gut (p=0.004) and 3.5%
more meal in the mid gut. The largest effect was observed with the
77.5 ug/kg animals where gastric emptying was increased
approximately 2 fold (37.8% test meal in zven1 treated animals and
63.8% in vehicle treated animals p=0.0002). Intestinal transit was
also increased significantly as a greater than 2 fold increase in
test meal in the mid gut was measured in the zven1 treated animals
over vehicle control (37.1% compared to 15% (p=0.004). At the
final, 775 ug/kg dose, increased gastric emptying was detected over
control 46.6% compared to 63.8%, but the effect was not as great as
the 77.5 .mu.g/kg dose. Increased intestinal transit was detected
in the mid gut (26% versus 15%), but the effect was not as
significant as that observed with the lower 77.5 ug/kg dose. These
data suggest that at higher concentrations, zven1 can inhibit
gastric emptying and intestinal transport. TABLE-US-00007 TABLE 7
Description of treatment groups and results Number of % Test Meal %
Test Meal in % Test Meal Treatment Groups Animals in Stomach
Proximal Gut in Mid Gut Group I Vehicle (Buffer N = 10 63.8% .+-.
3.8% 18.4% .+-. 2.4% 15% .+-. 3.3% SE for zven1) SE SE Group II
zven1 0.78 .mu.g/kg N = 10 56.3% .+-. 5.2% 25.5% .+-. 4.1% 14.6%
.+-. 4% SE body weight SE SE *Group III zven1 7.8 .mu.g/kg N = 10
43.7% .+-. 3.2% 35.0% .+-. 5.4% 18.5% .+-. 5.1% body weight SE SE
SE *p = .001 *p = .004 *Group IV zven1 N-10 37.8% .+-. 4.5% 26.6%
.+-. 5.1% 37.1% .+-. 7.1% 77.5 .mu.g/kg body weight SE SE SE *p =
.0002 *p = .004 *Group V zven1 775 .mu.g/kg N = 10 46.6% .+-. 4.5%
24.0% .+-. 5.9% 26% .+-. 4.3% SE body weight SE SE *p = .05 *p =
.009 Group VI Caerulein (CCK N = 10 93.8% .+-. 1.0% 2.6% .+-. 0.9%
1.2% .+-. 0.3% analogue positive control) SE SE SE 40 ng/g body
weight Group VII Sham non- N = 5 100% NA NA treated
Example 9
Baculovirus Expression of Zven
[0393] An expression vector containing a GLU-GLU tag,
pzBV32L:zven1cee, was designed and prepared to express zven1cee
polypeptides in insect cells.
[0394] A. Expression Vector:
[0395] An expression vector, pzBV32L:zven1cee, was prepared to
express human zven1 polypeptides having a carboxy-terminal Glu-Glu
tag, in insect cells as follows.
[0396] A 371 bp fragment containing sequence for zven1 and a
polynucleotide sequence encoding EcoR1 and Xba1 restriction sites
on the 5' and 3' ends, respectively, was generated by PCR
amplification using PCR SuperMix (Gibco BRL, Life Technologies) and
appropriate buffer from a plasmid containing zven1 cDNA
(zven1-zyt-1.contig) using primers ZC29463 (SEQ ID NO:23) and
ZC29462 (SEQ ID NO:24). (Note: the zven1 sequence and the Xba1 site
was out of frame. An additional 2 bases, CC--antisense, were added
to put in frame, which coded for an additional Gly between the
zven1 sequence and the CEE tag.) The PCR reaction conditions were
as follows: 1 cycle of 94.degree. C. for 3 minutes, followed by 25
cycles of 94.degree. C. for 30 seconds, 50.degree. C. for 30
seconds, and 68.degree. C. for 30 seconds; followed by a 4.degree.
C. hold. The fragment was visualized by gel electrophoresis (1%
Agarose-1 .mu.l of 10 mg/ml EtBr per 10 ml of agarose). A portion
of the PCR product was digested with EcoR1 and Xba1 restriction
enzymes in appropriate buffer, then run on an agarose gel. DNA
corresponding to the EcoR1/Xba1 digested zven1 coding sequence was
excised, purified using Qiagen Gel Extraction kit (#28704), and
ligated into an EcoR1/XbaI digested baculovirus expression donor
vector, pZBV32L. The pZBV32L vector is a modification of the
pFastBac1.TM. (Life Technologies) expression vector, where the
polyhedron promoter has been removed and replaced with the late
activating Basic Protein Promoter. In addition, the coding sequence
for the Glu-Glu tag (SEQ ID NO:10) as well as a stop signal is
inserted at the 3' end of the multiple cloning region. About 216
nanograms of the restriction digested zven1 insert and about 300 ng
of the corresponding vector were ligated overnight at 15.degree. C.
One .mu.l of ligation mix was electroporated into 35 .mu.l DH10B
cells (Life Technologies) at 2.1 kV. The electroporated DNA and
cells were diluted in 1 ml of LB media, grown for 1 hr at
37.degree. C., and plated onto LB plates containing 100 .mu.g/ml
ampicillin. Clones were analyzed by restriction digests and one
positive clone was selected and streaked on AMP+ plates to get
single colonies for confirmation by sequencing.
[0397] Sequencing revealed the presence of a initiation codon
upstream of the actual start codon which would possibly interfere
with proper translation. Therefore, the upstream codon was removed
using a Quick-change mutagenesis kit from Stratagene (La Jolla,
Calif.). This was accomplished by designing forward and reverse
primers that changed the upstream initiation ATG to a ATC, thereby
also eliminating a Nco restriction digest site and creating a Sma1
site instead. The new mutagenized plasmid containing the Sma1 and
Xba1 cleavage sites at the 5' and 3' ends of the zven1 sequence was
then electroporated into DH10B cells as before, analyzed by
restriction digests, this time with Sma1 and Xba1, and a positive
clone was selected and streaked on AMP+ plates to get a single
colony for confirmation by sequencing as before. A clone for the
Zven1 polynucleotide sequence could also be cloned without the
upstream initiation codon.
[0398] One to 5 ng of the positive clone donor vector was
transformed into 100 .mu.l DH10Bac Max Efficiency competent cells
(GIBCO-BRL, Gaithersburg, Md.) according to manufacturer's
instruction, by heat shock for 45 seconds in a 42.degree. C.
waterbath. The transformed cells were then diluted in 980 .mu.l SOC
media (2% Bacto Tryptone, 0.5% Bacto Yeast Extract, 10 ml 1M NaCl,
1.5 mM KCl, 10 mM MgCl.sub.2, 10 mM MgSO.sub.4 and 20 mM glucose)
out-grown in shaking incubator at 37.degree. C. for four hours and
plated onto Luria Agar plates containing 50 .mu.g/ml kanamycin, 7
.mu.g/ml gentamicin, 10 .mu.g/ml tetracycline, IPTG and Blue Gal.
The plated cells were incubated for 48 hours at 37.degree. C. A
color selection was used to identify those cells having zVen1cee
encoding donor insert that had incorporated into the plasmid
(referred to as a "bacmid"). Those colonies, which were white in
color, were picked for analysis. Bacmid DNA was isolated from
positive colonies using standard isolation technique according to
Life Technologies directions. Clones were screened for the correct
insert by amplifying DNA using primers to the transposable element
in the bacmid via PCR. The PCR reaction conditions were as follows:
35 cycles of 94.degree. C. for 45 seconds, 50.degree. C. for 45
seconds, and 72.degree. C. for 5 minutes; 1 cycle at 72.degree. C.
for 10 min.; followed by 4.degree. C. soak. The PCR product was run
on a 1% agarose gel to check the insert size. Those having the
correct insert size were used to transfect Spodoptera frugiperda
(Sf9) cells. The polynucleotide sequence is shown in SEQ ID NO:25.
The corresponding amino acid sequence is shown in SEQ ID NO:26.
[0399] B. Transfection in Insect Cells:
[0400] Sf9 cells were seeded at 1.times.10.sup.6 cells per 35 mm
plate and allowed to attach for 1 hour at 27.degree. C. Five
micrograms of bacmid DNA was diluted with 100 PI Sf-900 II SFM
medium (Life Technologies, Rockville, Md.). Fifteen .mu.l of
lipofectamine Reagent (Life Technologies) was diluted with 100
.mu.l Sf-900 II SFM. The bacmid DNA and lipid solutions were gently
mixed and incubated 30-45 minutes at room temperature. The media
from one plate of cells was aspirated. Eight hundred microliters of
Sf-900 II SFM was added to the lipid-DNA mixture. The DNA-lipid mix
was added to the cells. The cells were incubated at 27.degree. C.
overnight. The DNA-lipid mix was aspirated the following morning
and 2 ml of Sf-900 II media was added to each plate. The plates
were incubated at 27.degree. C., 90% humidity, for 168 hours after
which the virus was harvested.
[0401] C. Primary Amplification
[0402] Sf9 cells were seeded at 1.times.10.sup.6 cells per 35 mm
plate and allowed to attach for 1 hour at 27.degree. C. They were
then infected with 500 .mu.l of the viral stock from above and
incubated at 27.degree. C. for 4 days after which time the virus
was harvested according to standard methods known in the art.
[0403] D. Secondary Amplification
[0404] Sf9 cells were seeded at 1.times.10.sup.6 cells per 35 mm
plate and allowed to attach for 1 hour at 27.degree. C. They were
then infected with 20 .mu.l of the viral stock from above and
incubated at 27.degree. C. for 4 days after which time the virus
was harvested according to standard methods known in the art.
[0405] E. Tertiary Amplification
[0406] Sf9 cells were grown in 80 ml Sf-900 II SFM in 250 ml shake
flask to an approximate density of 1.times.10.sup.6 cells/ml. They
were then infected with 200 .mu.l of the viral stock from above and
incubated at 27.degree. C. for 4 days after which time the virus
was harvested according to standard methods known in the art.
[0407] F. Expression of Zven1Cee
[0408] Third round viral stock was titered by a growth inhibition
curve and the culture showing an MOI of "1" was allowed to proceed
for 48 hrs. The supernatant was analyzed via Western blot using a
primary monoclonal antibody specific for the n-terminal Glu Glu
epitope and a HRP conjugated Gt anti Mu secondary antibody. Results
indicated a band of the predicted molecular weight.
[0409] A large viral stock was then generated by the following
method: Sf9 cells were grown in IL Sf-900 II SFM in a 2800 ml shake
flask to an approximate density of 1.times.10.sup.6 cells/ml. They
were then infected with viral stock from the 3.sup.rd round amp.
and incubated at 27.degree. C. for 72 hrs after which time the
virus was harvested. Larger scale infections were completed to
provide material for downstream purification.
Example 10
Expression in E. coli
[0410] A. Generation of the Native Zven1 Expression Construct
[0411] A DNA fragment of native Zven1 (SEQ ID NO:11) was isolated
using PCR. Primer zc #40,821 (SEQ ID NO:12) containing 41 bp of
vector flanking sequence and 24 bp corresponding to the amino
terminus of Zven1, and primer zc#40,813 (SEQ ID NO:13) contained 38
bp corresponding to the 3' end of the vector which contained the
zven1 insert. Template was pZBV32L:zven1cee. The PCR conditions
were as follows: 25 cycles of 94.degree. C. for 30 seconds,
50.degree. C. for 30 seconds, and 72.degree. C. for 1 minute;
followed by a 4.degree. C. soak. A small sample (2-4 .mu.L) of the
PCR sample was run on a 1% agarose gel with 1.times.TBE buffer for
analysis, and the expected band of approximately 500 bp fragment
was seen. The remaining volume of the 100 .mu.L reaction was
precipitated with 200 .mu.L absolute ethanol. Pellet was
resuspended in 10 .mu.L water to be used for recombining into Sma1
cut recipient vector pTAP238 to produce the construct encoding the
zven1 as disclosed above. The clone with correct sequence was
designated as pTAP432. It was digested with Not1/Nco1 (10 .mu.l
DNA, 5 .mu.l buffer 3 New England BioLabs, 2 .mu.L Not 1, 2 .mu.L
Nco 1, 31 .mu.L water for 1 hour at 37.degree. C.) and religated
with T4 DNA ligase buffer (7 .mu.L of the previous digest, 2 .mu.L
of 5.times. buffer, 1 .mu.L of T4 DNA ligase). This step removed
the yeast sequence, CEN-ARS, to streamline the vector. The DNA was
diagnostically digested with Pvu 2 and Pst 1 to confirm the absence
of the yeast sequence. DNA was transformed into E. coli strain
W3110/pRARE.
[0412] B. Expression of the Native Zven1 in E. coli
[0413] E. coli was inoculated into 100 ml Superbroth II medium
(Becton Dickinson, Franklin Lakes, N.J.) with 0.01% Antifoam 289
(Sigma), 30 .mu.g/ml kanamycin, 35 .mu.g/ml chloramphenicol and
cultured overnight at 37.degree. C. A 5 ml inoculum was added to
500 ml of the same medium in a 2 L culture flask which was shaken
at 250 rpm at 37.degree. C. until the culture attained an
OD.sub.600 of 4. IPTG was then added to a final concentration of 1
mM and shaking was continued for another 2.5 hours. The cells were
centrifuged at 4,000.times.g for 10 min at 4.degree. C. The cell
pellets were frozen at -80.degree. C.
Example 11
Codon Optimization
[0414] A. Generation of the Codon Optimized Zven1 Expression
Construct
[0415] Native human Zven1 gene sequence could not be expressed in
E. coli strain W3110. Examination of the codons used in the Zven1
coding sequence indicated that it contained an excess of the least
frequently used codons in E. coli with a CAI value equal to 0.211.
The CAI is a statistical measure of synonymous codon bias and can
be used to predict the level of protein production (Sharp et al.,
Nucleic Acids Res. 15(3):1281-95, 1987). Genes coding for highly
expressed proteins tend to have high CAI values (>0.6), while
proteins encoded by genes with low CAI values (<0.2) are
generally inefficiently expressed. This suggested a reason for the
poor production of Zven1 in E. coli. Additionally, the rare codons
are clustered in the second half of the message leading to higher
probability of translational stalling, premature termination of
translation, and amino acid misincorporation (Kane J F. Curr. Opin.
Biotechnol. 6(5):494-500, 1995).
[0416] It has been shown that the expression level of proteins
whose genes contain rare codons can be dramatically improved when
the level of certain rare tRNAs is increased within the host
(Zdanovsky et al., ibid., 2000; Calderone et al., ibid., 1996;
Kleber-Janke et al., ibid., 2000; You et al., ibid., 1999). The
pRARE plasmid carries genes encoding the tRNAs for several codons
that are rarely used E. coli (argU, argW, leuW, proL, ileX and
glyT). The genes are under the control of their native promoters.
Co-expression with pRARE enhanced Zven1 production in E. coli and
yielded approximately 100 mg/L. Co-expression with pRARE also
decreased the level of truncated zven1 in E. coli lysate. These
data suggest that re-resynthesizing the gene coding for zven1 with
more appropriate codon usage provides an improved vector for
expression of large amounts of zven1.
[0417] The codon optimized zven1 coding sequence (SEQ ID NO:14) was
constructed from six overlaping oligonucleotides: zc45,048 (SEQ ID
NO:15), zc45,049 (SEQ ID NO:16), zc45,050 (SEQ ID NO:17), zc45,051
(SEQ ID NO:18), zc45,052 (SEQ ID NO:19) and zc45,053 (SEQ ID
NO:20). Primer extension of these overlapping oligonucleotides
followed by PCR amplification produced a full length zven1 gene
with codons optimized for expression in E. coli. The final PCR
product was inserted into expression vector pTAP237 by yeast
homologous recombination. The expression construct was extracted
from yeast and transformed into competent E. coli DH10B. Clones
resistance to kanamycin were identified by colony PCR. A positive
clone was verified by sequencing and subsequently transformed into
production host strain W3110. The expression vector with the
optimized zven1 sequence was named pSDH187. The resulting gene was
expressed very well in E. coli. Expression levels with the new
construct increased to around 150 mg/L.
[0418] B. Expression of the Codon Optimized Zven1 in E. coli
[0419] E. coli was inoculated into 100 ml Superbroth II medium
(Becton Dickinson) with 0.01% Antifoam 289 (Sigma), 30 .mu.g/ml
kanamycin and cultured overnight at 37.degree. C. A 5 ml inoculum
was added to 500 ml of same medium in a 2 L culture flask which was
shaken at 250 rpm at 37.degree. C. until the culture attained an
OD.sub.600 of 4. IPTG was then added to a final concentration of 1
mM and shaking was continued for another 2.5 hours. The cells were
centrifuged at 4,000.times.g for 10 min at 4.degree. C. The cell
pellets were frozen at -80.degree. C. until use at a later
time.
Example 12
Purification and Refolding of Zven1 Produced in E. coli
[0420] A. Inclusion Body Isolation:
[0421] Following induction of protein expression in either batch
ferment or shaker flask culture, the E. coli broth was centrifuged
in 1 liter bottles at 3000 RPM in a Sorvall swinging bucket rotor.
Additional washing of the cell paste to remove any broth
contaminants was performed with 50 mM Tris pH 8.0 containing 200 mM
NaCl and 5 mM EDTA until the supernate was clear.
[0422] The cell pellets were then suspended in ice cold lysis
buffer (50 mM Tris pH 8.0; 5 mM EDTA; 200 mM NaCl, 10% sucrose
(w/v); 5 mM DTT; 5 mM Benzamidine;) to 10-20 Optical Density units
at 600 nm. This slurry was then subjected to 2-3 passes at
8500-9000 psi in a chilled APV 2000 Lab Homogenizer producing a
disrupted cell lysate. The insoluble fraction (inclusion bodies)
was recovered by centrifugation of the cell lysate at
20,000.times.G for 1 hour at 4.degree. C.
[0423] The inclusion body pellet (resulting from the 20,000.times.G
spin) was re-suspended in wash buffer (50 mM Tris pH 8 containing
200 mM NaCl, 5 mM EDTA, 5 mM DTT, 5 mM Benzamidine) at 10 ml wash
buffer per gram inclusion bodies, and was completely dispersed
utilizing an OMNI international rotor stator generator. This
suspension was centrifuged at 20,000.times.G for 30 minutes at
4.degree. C. The wash cycle was repeated 3-5 times until the
supernatant was clear.
[0424] The final washed pellet was solubilized in 8M Urea, 50 mM
Borate buffer at pH 8.6 containing 0.1M Sodium Sulfite and 0.05 M
Sodium Tetrathionate at pH 8.2. The solubilization and sulfitolysis
reaction was allowed to proceed at 4.degree. C. overnight with
gentle shaking. The resulting pinkish colored solution was
centrifuged at 35,000.times.g for 1 hour at 4.degree. C. and the
clarified supernate, containing the soluble Zven1, was 0.45 um
filtered.
[0425] B. Zven1 Refolding:
[0426] The solubilized Zven1 was refolded by drop-wise dilution
into ice cold refolding buffer containing 55 mM Borate pH 8.6, 1.0
M Arginine, 0.55 M Guanidine HCL, 10.56 mM NaCl, 0.44 mM KCl,
0.055% PEG, 10 mM reduced Glutathione and 1.0 mM oxidized
Glutathione at a final zven1 concentration of 100-150 ug/ml. Once
diluted, the mixture was allowed to stir slowly in the cold room
for 48-72 hours.
[0427] C. Product Recovery & Purification:
[0428] After refolding, the solution was clarified by
centrifugation at 22,000.times.G, 1 hour, 4.degree. C. and/or by
filtration using a 0.45 micron membrane. The clarified supernate,
containing refolded zven1, was adjusted to 50 mM acetate and the pH
adjusted to 4.5 with addition of HCl. The pH adjusted material was
captured by cation exchange chromatography on a Pharmacia
Streamline SP column (33 mm ID.times.65 mm length) equilibrated in
50 mM acetate pH 4.5 buffer. The load flow rate was 10 ml/min with
inline dilution proportioning 1:5 in 50 mM acetate buffer at pH
4.5. This dilution lowers the ionic strength enabling efficient
binding of the target to this matrix. After sample loading was
complete, the column was washed to baseline absorbance with
equilibration buffer prior to step elution with 50 mM acetate pH
4.5 buffer containing 1 M NaCl.
[0429] The eluate pool from the cation exchange step was brought to
1% Acetic acid, pH 3.0 and Loaded to a column (22 mm.times.130 mm)
containing Toso Hass Amberchrom CG71m reverse phase media
equilibrated in 1% acetic acid, pH 3.0 at a flow rate of 10 ml/min.
Upon washing to baseline absorbance, the column was eluted with a
20 column volume gradient formed between equilibration buffer and
99% (V/V) acetonitrile, 1% (V/V) acetic acid.
[0430] The eluate pool from the reverse phase step was subjected to
another round of cation exchange chromatography. The pool was
directly loaded on to a Toso Haas SP 650 S column (10 mm.times.50
mm) equilibrated in 50 mM acetete pH 4.5 buffer at a flow rate of 3
ml/min. Upon completing the sample load, and washing to baseline
absorbance, the column was step eluted with 50 mM acetate pH 3.0
buffer containing 1.0 M NaCl. The protein eluate pool was
concentrated against a 3 k Da cutoff ultrafiltration membrane using
an Amicon concentration unit in preparation for the final
purification and buffer exchange size exclusion step.
[0431] D. Size Exclusion Buffer Exchange and Formulation:
[0432] The concentrated cation pool was injected onto a Pharmacia
Superdex Peptide size exclusion column (Pharmacia, now Pfizer, La
Jolla, Calif.) equilibrated in 25 mM Histidine; 120 mM NaCl at pH
6.5. The symetric eluate peak containing the product was pooled,
0.2 micron sterile-filtered, aliquoted and stored at -80.degree.
C.
Example 13
Activity of Zven1 and Zven2 in a Reporter Assay
[0433] A. Cell Lines
[0434] Rat2 fibroblast cells (ATCC #CRL-1764, American Type Culture
Collection, Manassass, Va.) were transfected with a SRE luciferase
reporter construct and selected for stable clones. These were then
transfected with constructs for either GPCR73a receptor (SEQ ID
NO:21) or GPCR73b receptor (SEQ ID NO:22).
[0435] B. Assay Procedure
[0436] Cells were trypsinized and seeded in Corning 96-well white
plates at 3,000 cells/well in media containing 1% serum and
incubated overnight at 37.degree. C. and 5% CO.sub.2. Media was
removed and samples were added in triplicate to cells in media
containing 0.5% BSA and incubated for four hours at 37.degree. C.
and 5% CO.sub.2. After media was removed the cells were lysed and
luciferase substrate was added according to the Promega luciferase
assay system (Promega Corp., Madison, Wis.)
[0437] C. Data and Conclusions
[0438] All data were reported as fold-induction of the RLU
(relative light units) from the luminometer divided by the basal
signal (media only). Zven1 was prepared in house. Zven2 used in the
assay was purchased from PeproTech Inc. (Rocky Hill, N.J.).
[0439] Tables 8 and 9 show that Zven1 was more active than Zven2 in
a dose-dependent manner with cells expressing the GPCR73a receptor.
TABLE-US-00008 TABLE 8 GPCR73a Fold-induction conc. (ng/ml) Zven1
(E. coli produced) Zven2 1000 17.8 20 320 20.7 24.4 100 19 11.4 32
15 5.8 10 8.4 2.5 3.2 4 1.6 1 1.9 1.2
[0440] TABLE-US-00009 TABLE 9 GPCR73a Fold-induction conc. (ng/ml)
Zven1 (E. coli produced) Zven2 1000 13.9 15 320 22 20.5 100 17.6
11.4 32 14.1 7.2 10 10.2 2.6 3.2 7.6 1.3 1 4.1 0.95
[0441] Table 10 and 11 show that Zven1 and Zven2 were similar in
activity with the cells expressing the GPCR73b receptor. Activity
of both molecules was lower in the cells expressing the GPCR73b
receptor. It is not known if the GPCR73b receptor numbers were
equivalent in both cell lines. TABLE-US-00010 TABLE 10 GPCR73b
Fold-induction conc. (ng/ml) Zven1 (E. coli produced) Zven2 1000
7.1 8.4 320 6.3 8.3 100 4.7 5.6 32 3 2.8 10 1.9 1.8 3.2 1.3 1.3 1
0.7 1.1
[0442] TABLE-US-00011 TABLE 11 GPCR73b Fold-induction conc. (ng/ml)
Zven1 (E. coli produced) Zven2 1000 4.8 6.1 320 5.2 5.8 100 4.4 4.1
32 2.6 2.7 10 1.7 1.8 3.2 1.2 1.4 1 1 1.1
[0443] Table 12 shows that Baculovirus-expressed Zven1 that has
been heated at 56.degree. C. for 30 minutes may have reduced
activity than fresh Zven1. TABLE-US-00012 TABLE 12 GPCR73a
Fold-induction conc. (ng/ml) Fresh Zven1 Heated Zven1 100 20.5 18.6
32 18.7 14.8 10 13.1 10 3.2 7.1 3.7 1 2.5 1.8
Example 14
Zven1 Activity in Organ Bath
[0444] Organ bath testing was also performed with Zven1 using at a
variety of tissues obtained from guinea pigs. A force transducer
was used to record the mechanical contraction using IOX software
(EMKa technologies, Falls Church, Va.) and Datanalyst software
(EMKa technologies, Falls Church, Va.). Tissues analyzed included:
duodenum, jejunum, ileum, trachea, esophagus, aorta, stomach, gall
bladder, bladder and uterus.
[0445] A. Organ Bath Methods
[0446] Two month old male guinea pigs (Hartley, Charles River Labs)
weighing 250 to 300 g were fasted with access to drinking water for
18 hours then euthanized by CO.sub.2 asphyxiation. All tissues were
rinsed with Krebs buffer (1.2 mM MgSO.sub.4, 115 mM NaCl, 11.5 mM
glucose, 23.4 mM NaHCO.sub.3, 4.7 mM KCl, 1.2 mM NaH.sub.2PO.sub.4,
and 2.4 mM CaCl.sub.2, oxygenated with 95% O.sub.2-5% CO.sub.2, pH
7.4, temperature 37.degree. C.) then suspended in the 5 ml organ
bath and pre-tensioned. All tissues were tested with positive
controls to establish their viability prior to running. Positive
controls used were CCK-8, acetylcholine (ACH), histamine, or 5HT,
and were purchased from Sigma (Saint Louis, Mo.). All tissues were
treated with a vehicle control, phosphate buffered saline (PBS), to
rule out the possibility of vehicle effects.
[0447] 1) Tissues that Did not Give a Response to Zven1 in the
Organ Bath: [0448] Tracheal ring: 3 mm wide tracheal ring (3 cm
away from brachial branches) was collected and allowed to
equilibrate at 5 gram tension prior to any treatments. The positive
control was 20 ug/ml ACH, which gave an approximate 1 gram
deflection. No effect seen with Zven1 at 80 ng/ml. [0449] Aortic
ring: 3 mm wide aortic ring (immediately adjacent to aortic arch)
was collected and allowed to equilibrate at 4 gram tension prior to
any treatments. The positive control was 2 mg/ml KCl, which gave an
average one gram deflection. Zven1 at 80 ng/ml did not cause a
visible effect. [0450] Esophagus: 2 cm in length esophagus (2 cm
away from cardia) was suspended and allowed to equilibrate at 1
gram tension prior to any treatments. Two mg/ml 5HT gave an
approximate 1.4 grams deflection. Zven1 at 20 ng/ml had no visible
effect. [0451] Gall bladder: Lumenal fluid was aspirated out with 1
ml syringe then longitudinally suspended and allowed to equilibrate
at 1 gram tension prior to any treatments. Five ng/ml of ACH gave a
0.4 gram deflection response. No effect was seen with 20 ng/ml
zven1. [0452] Bladder: 1.5 cm.times.0.3 cm longitudinal strip was
suspended and allowed to equilibrate to 0.5 gram tension prior to
any treatments. Positive controls induced a contractile response,
but no activity was seen at a 80 ng/ml Zven1 dose.
[0453] 2) Tissues that Responded to Zven1: [0454] Stomach/antrum:
1.5 cm.times.0.3 cm longitudinal strip was suspended and allowed to
equilibrate to 0.5 gram tension prior to any treatments. Treatment
with either 5 ng/ml ACH or 80 ng/ml CCK 8 resulted in an
approximate one gram deflection. Eighty ng/ml Zven1 also produced a
contractile response of approximately 0.5 gm deflection. [0455]
Duodenum: 2 cm in length duodenum (2 cm away from pylorus) was
suspended and allowed to equilibrate at 1 gram tension prior to any
treatments. ACH gave an approximate 0.75 gm deflection. Twenty
ng/ml Zven1 also gave a contractile response of approximately 0.5
grams deflection. [0456] Jejunum: 2 cm in length jejunum (midpoint
between pylorus and ileal-cecal junction) was suspended and allowed
to equilibrate at 1 gram tension prior to any treatments. ACH gave
an approximate 1.0 gram deflection and 20 ng/ml Zven1 gave an
approximate 0.5 gram deflection contractile response. [0457] Ileum:
8 cm in length ileum (2 cm away from ileal-cecal junction) was
collected and flushed with Krebs buffer to remove any fecal debris
if present then cut into four equal pieces. All tissues were
suspended and allowed to equilibrate at 1 gram tension prior to any
treatments. The ileum was run at the same time to compare Zven1
effects on the small intestine. ACH gave an approximate 1.5 gram
deflection, and 20 ng/ml Zven1 also gave a 1.5 gram deflection.
[0458] Proximal Colon: 2 cm in length colon (2 cm away from cecum)
was suspended and allowed to equilibrate at 0.5 gram tension prior
to any treatments. Zven1 at 20 ng/ml induced a relaxation effect
with a decrease in muscle tone and a decrease in the amplitude of
the contractions.
[0459] Zven1's contractile effects are specific to the
gastrointestinal tract. The greatest contractile response is seen
in the ileum, with lesser contraction seen in the duodenum,
jejunum, and antrum. The relaxation effect in the proximal colon is
suggestive of a coordinated effect on gut motility. As the smooth
muscle contraction is enhanced in the antrum and the small
intestine, the large intestine is preparing to accommodate the
approaching meal by relaxing. Coordinated contractile activity
between different parts of the gut will result in improved
gastrointestinal function.
Example 15
Comparative Activity of Zven1 and Zven2 in the Organ Bath
[0460] Both Zven1 and Zven2 have contractile effects on intestinal
tissue in the organ bath. Side by side comparisons were made to
compare activity in tissue derived from the same animal.
[0461] Ileal strips from guinea pig were tested for contractility
using methods described above. Zven2 was purchased from PeproTech
Inc. (Rocky Hill, N.J.). Activity was compared at 40, 12, and 3
ng/ml concentrations. ACH at 5 ng/ml was used as a positive
control. Contractile responses were normalized to the ACH response
in each tissue. All three doses were run on separate ileal
longitudinal tissue strips obtained from the same animal.
[0462] Results: Contractile effects were normalized to the ACH
positive control and are expressed as the ratio of Zven1 or Zven2
to ACH in the table below. TABLE-US-00013 TABLE 13 Zven1 Zven2 Conc
(ng/ml) ACH Zven1 Zven1:ACH ACH Zven2 Zven2:ACH 40 1.26 1.28 1.02
1.25 0.58 0.46 12 2.5 2.51 1.00 2.26 0.61 .027 3 1.38 .047 .034
1.73 .027 .016
[0463] Conclusions: Zven1 is approximately twice as active as Zven2
when comparing contractility in the ileum.
Example 16
Synergistic Effects of Zven1 and Zven2
[0464] In order to determine the combined effects of Zven1 and
zven2 on contractile activity, ileal tissues were pre-treated with
varying doses of zven2, followed by increasing doses of zven1.
[0465] All tissues are stabilized, treated with ACH, and again
stabilized prior to pre-treatment with zven2 at concentrations of
0.8, 3.0 or 12 ng/ml. Zven2 was left on tissue for approximately 20
minutes prior to dosing with 20 ng/ml zven1.
[0466] Results: Large 3 gram deflection contractions with Zven1
were observed when the tissue was pre-treated with 0.8 ng/ml zven2.
These contractions were larger than what is normally observed with
a 20 ng/ml dose of zven1, where contractile effects of
approximately 1.5 to 2.0 grams deflection are normally observed.
Zven2 alone at 0.8 ng/ml has a negligible contractile effect.
[0467] Conclusions: These data suggest that by pre-treating with a
low dose of zven2, and then treating with zven1, increased motility
effects may be obtained.
Example 17
MIP-2 Detection in Lavage Fluids and Serum of Mice Following IP
(Intraperitoneal) Injection of Zven1
[0468] As discussed in Example 3, above, mouse KC is the mouse
homolog of human GRO.alpha., and CINC-1 is the rat homolog.
Similarly, increased MIP-2 expression has been found to be
associated with neutrophil influx in various inflammatory
conditions. See Banks, C. et al, J. Path. 199: 28-35, 2003.
[0469] Similar to the methods used in Example 3, four groups of ten
mice were injected with Zven1 at 5 and 50 ug/kg, a vehicle control,
or no treatment. These mice weighed approximately 20 grams, so the
dose was 5 .mu.g/kg. MIP-2 levels were measured in both peritoneal
lavage fluid and serum using a Quantikine M Murine mouse MIP-2
ELISA kit (R and D Systems, Minneapolis, Minn.). Test results are
shown in Table 14. TABLE-US-00014 TABLE 14 MIP-2 picograms/ml Serum
Lavage Fluid Non-treated control 6.2 +/- 1.3 5.9 +/- 0.7 Vehicle
6.7 +/- 1.3 16.7 +/- 2.2 5 ug/kg Zven1 14.3 +/- 2.7 21.5 +/- 3.7 50
ug/kg Zven1 7.7 +/- 1.8 8.7 +/- 1.2 Data = mean +/- SEM
[0470] Conclusions: MIP-2 is up-regulated in serum and lavage fluid
in response to a low, (5 ug/kg), IP injection of zven1.
Concentrations in serum are approximately 2-fold higher in the
zven1 treated animals. There is a lesser effect in lavage fluid,
but that is due to the fact that some activation took place in the
vehicle treated animals over non-treated control animals. At the
higher (50 ug/kg dose) no effect was observed suggesting that at
elevated doses there is no chemotactic effect. These results
correlate with the neutrophil numbers, where in, neutrophil
infiltration was observed only in the animals administered the
lower (5 ug/kg) dose of Zven1.
Example 18
Production of Zven1 Polyclonal Antibodies
[0471] Polyclonal antibodies were prepared by immunizing 2 female
New Zealand white rabbits with the purified recombinant protein
huzven1-CEE-Bv (SEQ ID NO:24) The rabbits were each given an
initial intraperitoneal (ip) injection of 200 .mu.g of purified
protein in Complete Freund's Adjuvant followed by booster ip
injections of 100 .mu.g peptide in Incomplete Freund's Adjuvant
every three weeks. Seven to ten days after the administration of
the second booster injection (3 total injections), the animals were
bled and the serum was collected. The animals were then boosted and
bled every three weeks.
[0472] Polyclonal antibodies were purified from the immunized
rabbit serum using a 5 ml Protein A sepharose column (Pharmacia
LKB). Following purification, the polyclonal antibodies were
dialyzed with 4 changes of 20 times the antibody volume of PBS over
a time period of at least 8 hours. Huzven1-specific antibodies were
characterized by ELISA using 500 ng/ml of the purified recombinant
protein huzven1-CEE-Bv (SEQ ID NO:24) as the antibody target. The
lower limit of detection (LLD) of the rabbit anti-huzven1 purified
antibody was 1 ng/ml on its specific purified recombinant antigen
huzven1-CEE-Bv.
Example 19
Detection of Zven1 Protein
[0473] The purified polyclonal huzven1 antibodies were
characterized for their ability to bind recombinant human Zven1
polypeptides using the ORIGEN.RTM. Immunoassay System (IGEN Inc,
Gaithersburg, Md.). In this assay, the antibodies were used to
quantitatively determine the level of recombinant huzven1 in rat
serum samples. An immunoassay format was designed that consisted of
a biotinylated capture antibody and a detector antibody, which was
labeled with ruthenium (II) tris-bipyridal chelate, thereby
sandwiching the antigen in solution and forming an immunocomplex.
Streptavidin-coated paramagnetic beads were then bound to the
immunocomplex. In the presence of tripropylamine, the ruthenylated
Ab gave off light, which was measured by the ORIGEN analyzer.
Concentration curves of 0.1-50 ng/ml huzven1 made quantitation
possible using 50 microliters of sample. The resulting assay
exhibited a lower limit of detection of 200 pg/ml huzven1 in 5%
normal rat serum.
Example 20
Effect of Zven1 in Post-Operative Ileus In Vivo
[0474] Five to 25 male Sprague-Dawley rats (.about.240 g) per
treatment group were used for these POI studies. Animals were
fasted for 22-23 h (with 2 floor grids placed in their cages to
prevent them from having access to their bedding) with free access
to water. While under gas isoflurane anesthesia, the rat's abdomen
was shaved and wiped with betadine/70% ethanol. A midline incision
was then made through the skin and linea alba of the abdomen (3-4
cm long), such that intestines were visible and accessible. The
cecum was manipulated for 1 min with sterile saline-soaked gauze,
using a gentle, pulsatile-like pressure. This procedure was
consistent from animal to animal in order to reduce inter-animal
ileus variability. The linea alba was sutured with silk suture and
the skin closed with wound clips. Animals were kept on
water-jacketed heating pads during recovery from surgery and placed
back into their cages once they regained full consciousness.
[0475] When fully conscious, rats were administered 1.0 ml of the
test meal 15 minutes following completion of cecal manipulation
(CM); one minute or 20 minutes later, rats were administered 0.8 or
5 ug/kg BW E. coli-produced Zven1, or saline/0.1% w/v/BSA via
indwelling jugular venous catheter. Zven1 was diluted with
saline/0.1% BSA to the desired concentration (based on average BW
of rat [.about.240 g] and a 0.1 ml injection volume for i.v.)
immediately prior to study, using siliconized microfuge tubes.
[0476] The test meal consisted of 1.5% (w/v) aqueous
methylcellulose solution (medium viscosity methylcellulose from
Sigma 400 centipoises; catalog # M-0262) along with a
non-absorbable dye, 0.05% (50 mg/100 ml) phenol red (Sigma catalog
# P-4758; lot #120K3660). Twenty minutes following administration
of the test meal, animals were anesthetized under isoflurane and
sacrificed by cervical dislocation. The stomach and intestinal
segments were removed, and the amount of phenol red in each segment
was determined by spectrophotometric analysis (see below) and
expressed as the percent of total phenol red recovered per rat.
These values are used to determine the amount of gastric emptying
and gut transit per tissue collected.
[0477] Colorimetric analysis of phenol red recovered from each gut
segment and stomach were performed according to a modification of
the procedure outlined by Scarpinato and Bertaccini (1980) and
Izbeki et al (2002). Briefly, following euthanization, the stomach
and intestinal segments were placed into 20 ml of 0.1 N NaOH and
homogenized using a Polytron tissue homogenizer. The Polytron was
then rinsed with 5 ml of 0.1 N NaOH and added to the previous 20
ml, along with another 15 ml of 0.1 N NaOH. Homogenate was allowed
to settle for at least 1 hour at room temperature. Proteins were
precipitated from 5 ml of the supernate by the addition of 0.5 ml
of 20% trichloracetic acid. Following centrifugation (3000 rpm for
15 min), 1 ml of supernatant was added to 1 ml of 0.5 N NaOH. A 0.2
ml sample (in a 96-well plate) was read at 560 nm using Molecular
Devices Spectra Max 190 spectrophotometer. The extent of gastric
emptying and intestinal transit were expressed as percent of total
phenol red recovered per rat.
[0478] Data indicated that Zven1 (0.8 and 5.0 ug/kg, i.v.)
significantly increased gastric emptying and upper intestinal
transit of this semi-solid, non-nutritive meal by approximately 1.6
to 2.0-fold compared to emptying and transit observed in
vehicle-treated rats. Efficacy in this model was observed when
these doses of Zven1 are administered at either 1 min or 20 min
following meal administration.
Example 21
Effect of i.v. and ip. BV- and E. Coli-Produced Zven1 on Gastric
Emptying and Intestinal Transit of a Phenol Red Semi-Solid Meal in
Rats
[0479] Male Sprague-Dawley rats (.about.240 g) were used for this
study, with 6-12 animals per treatment group. Animals were fasted
for 24 h (with 2 floor grids placed in their cages to prevent them
from having access to their bedding) with free access to water. One
minute following the administration of 1.0 ml of test meal, rats
were administered varying doses of Zven1 (0.01 to 30 ug/kg BW) or
saline/0.1% w/v BSA via indwelling jugular venous catheter. For
i.p. dosing, Zven1 (0.1 to 100 ug/kg BW) or saline/0.1% BSA was
administered either 1 or 10 min prior to or 1 min after the meal.
Zven1 was diluted with saline/0.1% BSA to the desired concentration
(based on average BW of rat [.about.240 g] and a 0.1 ml injection
volume for i.v. or 0.5 ml injection volume for i.p.) immediately
prior to study, using siliconized microfuge tubes. The test meal
consisted of 1.5% (w/v) aqueous methylcellulose solution (medium
viscosity methylcellulose from Sigma 400 centipoises; catalog #
M-0262) along with a non-absorbable dye, 0.05% (50 mg/100 ml)
phenol red (Sigma catalog # P-4758; lot #120K3660). Fifteen or 20
min following administration of the test meal, rats were
anesthetized under isoflurane and sacrificed by cervical
dislocation.
[0480] The stomach and intestinal segments were removed, and the
amount of phenol red in each sample was determined by
spectrophotometric analysis (see below) and expressed as the
percent of total phenol red recovered per rat. These values were
used to determine the amount of gastric emptying and gut transit
per tissue collected.
[0481] Colorimetric analysis of phenol red recovered from each gut
segment and stomach were performed according to a modification of
the procedure outlined by Scarpinato et al Arch Int. Pharmacodyn.
246:286-294 (1980) and Piccinelli et al. Naunyn-Schmiedeberg's
Arch. Pharmacol 279: 75-82 (1973). Briefly, following
euthanization, the stomach and intestinal segments were placed into
20 ml of 0.1 N NaOH and homogenized using a Polytron tissue
homogenizer. The Polytron was then rinsed with 5 ml of 0.1 N NaOH
and added to the previous 20 ml, along with another 15 ml of 0.1 N
NaOH. Homogenate was allowed to settle for at least 1 hour at room
temperature. Proteins were precipitated from 5 ml of the supernate
by the addition of 0.5 ml of 20% trichloracetic acid. Following
centrifugation (3000 rpm for 15 min), 1 ml of supernatant was added
to 1 ml of 0.5 N NaOH. A 0.2 ml sample (in a 96-well plate) was
read at 560 nm using Molecular Devices Spectra Max 190
spectrophotometer. The extent of gastric emptying and intestinal
transit were expressed as percent of total phenol red recovered per
rat.
[0482] Gastric emptying and intestinal transit of this semi-solid
meal were increased by approximately two-fold following i.v.
administration of 0.1-1.0 .mu.g/kg BW BV- or E. coli-produced
Zven1. Inhibitory effects of gastric emptying and intestinal
transit were observed using higher doses (10-100 ug/kg BW for i.p.
dosing; 30 ug/kg BW for i.v. dosing) of BV- and E. coli-Zven1. The
inhibitory observations were especially evident when these higher
doses of Zven1 were administered i.v. at 1 minute following test
meal administration, or when administered i.p. at 10 minutes prior
to test meal administration. Similar results were observed when
Zven2 was administered i.v. at 30 .mu.g/kg.
Example 22
Effect of i.v. BV- and E. Coli-Produced Zven1 on Gastric Emptying
and Intestinal Transit of a Phenol Red Semi-Solid Meal in Mice
[0483] Female C57B1/6 mice, 8 to 10 weeks old, were used for the
study, which consisted of eight treatment groups and .about.9 mice
per group. The animals were fasted for 20 hrs in cages containing
floor screens, and allowed access to water. Animals were weighed to
determine proper dose, and their average weight was used to adjust
the protein concentration. Zven1 protein (in stock solutions of
either 20 mM Mes buffer/20 mM NaCl pH 6.5; or in PBS, pH 7.2)
dilutions were prepared in siliconized tubes just prior to
injections. Doses were based on the average weight of the study
animals (approximately 20 g) and adjusted with saline 0.1% w/v BSA
to 0.1 ml injection volumes per mouse. Zven1 and vehicle treatments
were administered via i.v. tail vein injection 1-2 minutes prior to
receiving 0.15 ml phenol red test meal as an oral gavage. The test
meal consisted of 1.5% w/v aqueous methylcellulose solution (medium
viscosity carboxy methylcellulose from Sigma with a final viscosity
of 400-800 centipoises; catalog # C-4888; lot #108H0052) containing
a non-absorbable dye, 0.05% phenol red (Sigma catalog # P-4758; lot
#120K3660). Twenty minutes post-administration of the test meal,
animals were euthanized and stomach and intestinal segments
removed. The small intestine was measured and divided into three
equal segments: proximal, mid and distal gut. The amount of phenol
red in each sample was determined by spectrophotometric analysis
(as described above for in Examples 20 and 21) and expressed as the
percent of total phenol red recovered per mouse. These values were
used to determine the amount of gastric emptying and gut transit
per tissue collected.
[0484] Results indicated that there were increases in gastric
emptying and intestinal transit in mice treated with i.v. Zven1 at
doses .about.1-10 ug/kg BW. Trends toward inhibition of gastric
emptying and intestinal transit were observed using higher doses
(>50 ug/kg i.v. in mice) of Zven1.
Example 23
Effect of BV- and E. coli-Produced Zven1 on Gross Morphology of
Stomach and Intestines of Urethane-Anesthetized Rats
[0485] Studies were conducted in urethane-anesthetized male
Sprague-Dawley rats to determine whether i.v. administration of BV-
or E. coli Zven1 (doses up to and including 30 ug/kg BW; a dose
known to induce intestinal motility) affected the gross appearance
of the stomach and small intestine.
[0486] Rats were fasted (with access to water) on double floor
grates in clean cages for 19 h. Between 07:00 and 08:30 am, rats
received an i.p injection of urethane (0.5 ml/100 g BW of a 25%
solution) and had a jugular venous catheter inserted. Anesthetized
rats were returned to their cages and kept on warming pads
(maintained at 37.degree. C.) throughout the day, with additional
i.p. doses of urethane administered as needed. An appropriate level
of anesthesia was monitored using the toe-pinch reflex test.
[0487] At .about.5 minute intervals between animals saline was
administered via the jugular vein, followed by either vehicle (PBS)
or BV- or E. coli-produced Zven1 at increasing doses (3, 10 and 30
ug/kg BW; 0.1 ml injection volume) every hour for 3 hours (total of
43 ug/kg BW). Zven1 protein dilutions were prepared just prior to
injection. Dose was based on the weight of the study animal
(approximately 225 grams) and adjusted so that it was contained in
0.1 ml total volume of diluent (saline/0.1% BSA). Protein was
diluted using siliconized microfuge tubes. Rats also received
infusions of saline via Harvard pumps at a rate of 0.5 ml per hour.
Approximately 8-9 hours later following the initial dose of
urethane, rats were sacrificed by cervical dislocation (under
anesthesia) and their stomachs and small intestine removed for
inspection and morphological evaluation.
[0488] There was no evidence of gastric or intestinal lesions in
any of the rats. A vehicle-treated rat had some dark fluid within a
small segment of the intestinal lumen; there was not any dark fluid
observed in the Zven1-treated rats. There was a significant amount
of mucous within the intestinal lumen in all treatment groups, most
likely as a result of the urethane anesthesia and fasting
protocol.
Example 24
Effects of BV-Produced Zven1 on In Vivo Gastrointestinal
Contractility in Anesthetized Experimental Mammals
[0489] "Sonomicrometry" is a technique, which utilizes
piezoelectric crystals to measure gastrointestinal distensibility,
compliance, and tone in vivo (Sonometrics, Corp. Ontario, Canada).
Crystals can be placed anywhere along the gastrointestinal tract in
experimental mammals. Peristaltic and segmentation contractions in
the stomach and/or intestine can then be accurately quantified and
qualified with great detail in response to the administration of
Zven1. This system offers a great deal of detailed and
sophisticated outcome measures of intestinal
motility/contractility.
[0490] This method of digital ultrasonomicrometry was used to
investigate motility and/or contractility in the ileum, jejunum,
cecum and proximal colon as described by Adelson et al.
Gastroenterology 122, A-554. (2002) in ten rats (two groups of 5
male Sprague-Dawley rats) following an i.v. infusion of the vehicle
(saline/0.1% w/v BSA) and escalating doses of BV-produced Zven1.
For these experiments, piezoelectric crystals were attached using a
small drop of cyanoacrylate glue (Vetbond, 3M Animal Care, St.
Paul, Minn.) to the relevant intestinal locations. After laparatomy
the urethane anesthetized rats were maintained at 37.degree. C. via
a feedback-controlled heater. Sonometric distance signals were
acquired continuously at a rate of 50 samples/sec via a digital
sonomicrometer (TRX-13, Sonometrics Corp, London ONT) connected to
a Pentium III class computer running SonoLAB software (Sonometrics
Corp, London, Ontario, Canada). Digitally-acquired distance data
were simultaneously recorded as analog signals via an installed
4-channel DAC. These sonometric analog signals, along with all
analog physiological data (rectal temperature, blood pressure, EKG,
respiratory rate) were acquired using a Microl401 A/D interface
(Cambridge Electronic Design, Ltd, Cambridge) connected to a
Pentium II class computer running Spike 2 (Cambridge Electronic
Design, Ltd, Cambridge) data acquisition software to allow
real-time observation and analysis of experiment progress. This
method allows simultaneous observation of distance measurements for
4 crystal pairs. Baseline levels were obtained between each vehicle
and Zven1 infusion. Both circular and longitudinal motion were
monitored using triads of piezoelectric crystals 1 mm in diameter
(Sonometrics Corp.) affixed so that two of the three were oriented
parallel to the longitudinal axis and the third was oriented to the
perpendicular axis.
[0491] Motility responses to applied stimuli may comprise tonic
and/or phasic components. Tonic and phasic components of responses
were analyzed separately. The tonic component of the trace was
obtained by replacing each point in the trace with the median value
of the trace over the surrounding 10 s. The phasic component was
obtained by applying to the original trace the inverse operation of
a smoothing function with a 10 s window, i.e. by removing the `DC
component` with a time constant of 10 s. Tonic responses were
analyzed in terms of mean value during a response, 1-min maximum
excursion from baseline, duration of response, and integrated
response (mean normalized response times duration). Phasic activity
was analyzed in terms of its rate and amplitude. Changes in
relationships between motility in different gut regions measured
simultaneously were analyzed using cross-correlation of continuous
signals and event correlations of peak positions.
[0492] Strong contractility responses were observed in the ileum of
Zven1-treated rats at i.v. doses as low as 3 ug/kg BW; contractions
were also noted in the jejunum and duodenum, though not as strong
as those observed for the ileum. Responses associated with a
relaxation were observed in the proximal colon.
Example 25
Effects of ip. Administration of BV-Produced Zven1 on Distal
Colonic Transit in Conscious Mice
[0493] Adult male C57/BL6 mice (6-8 weeks of age; Harlan, San
Diego, Calif.) were used for this study with 6-10 mice per
treatment group. Mice were maintained on a 12:12-h light-dark cycle
with controlled temperature (21-23.degree. C.) and humidity
(30-35%), and were group housed in cages with free access to food
(Purina Chow) and tap water. Mice were deprived of food for 18-20
h, with free access to water before the experiments. BV-produced
Zven1 in stock solution of 20 mmol MES and 20 mmol NaCl at pH 6.5
was stored at -80.degree. C. On the day of the experiment, Zven1
was diluted to 0.9% NaCl with 0.1% BSA. The pH for both vehicle and
Zven1 at various doses was 6.5.
[0494] Distal colonic transits were measured as previously
described (Martinez V, et al. J Pharmacol Exp Ther 301:
611-617(2002.)). Fasted mice had free access to water and
pre-weighed Purina chow for a 1-h period, then were briefly
anesthetized with enflurane (1-2 min; Ethrane-Anaquest, Madison,
Wis.) and a single 2-mm glass bead was inserted into the distal
colon at 2 cm from the anus. Bead insertion was performed with a
glass rod with a fire-polished end to avoid tissue damage. After
bead insertion the mice were placed individually in their home
cages without food and water. Mice regained consciousness within a
1-2 min period and thereafter showed normal behavior. Distal
colonic transit was determined to the nearest 0.1 min by monitoring
the time required for the expulsion of the glass bead (bead
latency).
[0495] At the end of the 1 h feeding period, mice were briefly
anesthetized with enflurane for bead insertion into the colon
followed by the intraperitoneal injection of either vehicle, or
Zven1 (3, 10, 30, or 100 .mu.g/kg). Animals were returned to their
home cages without food or water and the bead expulsion time was
monitored. Results were expressed as Mean.+-.S.E. and analyzed
using one-way ANOVA.
[0496] In mice, fasted for 18-20 h, re-fed for 1 h, Zven1 injected
i.p. (3, 10, 30, and 100 .mu.g/kg) showed no significant changes in
bead expulsion time in response to the i.p. injection of BV-Zven1
(3, 10 and 30 .mu.g/kg): 32.7.+-.6.1, 23.1.+-.14.5 and 34.2.+-.15.6
min respectively compared with 21.1.+-.13.9 min in i.p. vehicle
injected group. In a second group of mice, treated similarly except
administered higher doses of BV-Zven1, the measurement of distal
colonic transit showed a dose-related tendency to increase the time
at which the bead is expelled in response to the i.p. injection of
BV-Zven1 (30 and 100 .mu.g/kg) (29.8.+-.7.8 and 35.1.+-.3.7 min
respectively compared with 22.3.+-.5.7 min after i.p. injection of
vehicle) although changes did not reach statistical
significance.
Example 26
Expression of GPR73a and GPR73b in Rat Gastrointestinal Tract
[0497] Rats were fasted overnight and sacrificed. Intestines and
stomachs were isolated and four-centimeter tissue sections from the
stomach through the end of the colon were immediately flash frozen
in liquid nitrogen. Acid-Phenol extraction method was used for RNA
isolation. Briefly, tissue sections were grinded in liquid nitrogen
then lysed/homogenized in acid guanidium based lysis buffer (4M
Guanidine isothyocyanate, 25 mM sodium citrate (pH 7), 0.5%
sarcosyl), NaOAc (0.1M final concentration)+.beta.ME (1:100).
Lysates were spun down; supernatants were mixed with equal volume
of acid phenol and 1/10 volume chloroform. After spinning down,
equal volume of Isopropanol was added to the aqueous layer. Samples
were incubated at -20.degree. C. then pelleted down by spinning.
Pellets were washed with 70% EtOH and then resuspended in DEPC
treated water.
[0498] Taqman EZ RT-PCR Core Reagent Kit (Applied biosystems,
Foster City, Calif.) was used to determine GPR73a and GPR73b
receptor expression levels. Following manufacturer's instructions,
a standard curve was prepared using one of the RNA isolates which
had a high quality RNA and which showed expression of both
receptors at the same level. Standard curve dilutions of this RNA
sample were prepared at the following concentrations: 500 ng/.mu.l,
250 ng/.mu.l, 100 ng/.mu.l and 12.5 ng/.mu.l. These standard curve
dilutions were first used to test the primers designed for GPR73a
and GPR73b genes and for a housekeeping gene, rodent
glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Once the working
conditions of primer and standard curve were established, RNA
samples isolated from rat were tested.
[0499] The RNA samples were thawed on ice and diluted to 100
ng/.mu.l in RNase-free water (Invitrogen, Cat #750023). Diluted
samples were kept on ice during the experiment. Using the TaqMan EZ
RT-PCR Core Reagent Kit (Applied Biosystems, Cat# N808-0236),
master mix was prepared for GPR73a, GPR73b receptors and for the
house keeping gene. To assay samples in triplicate, 3.5 .mu.l of
each RNA samples were aliquoted. For positive controls, 3.5 .mu.l
of each standard curve dilutions were used in place of sample RNA.
For the negative control, 3.5 .mu.l RNase-free water was used for
the no template control. For endogenous controls (rodent GAPDH
message), 3.5 .mu.l of both standard curve dilutions and the sample
RNAs were aliquoted. Then 84 .mu.l of PCR master mix was added and
mixed well by pipetting.
[0500] A MicroAmp Optical 96-well Reaction Plate (Applied
Biosystems Cat# N801-0560) was placed on ice and 25 .mu.l of
RNA/master mix was added in triplicates to the appropriate wells.
Then MicroAmp 12-Cap Strips (Applied Biosystems Cat# N801-0534)
were used to cover entire plate. Then the plate was spun for two
minutes at 3000 RPM in the Qiagen Sigma 4-15 centrifuge.
[0501] The samples were run on a PE-ABI 7700 (Perkin Elmer, now
EG&G, Inc. Wellesley, Mass.). Sequence Detector was launched
and the default was set to Real Time PCR. Fluorochrome was set to
FAM. Plate template was set to indicate where standards and where
unknown test samples were.
[0502] Expression for each sample is reported as a Ct value. The Ct
value is the point at which the fluorochrome level or RT-PCR
product (a direct reflection of RNA abundance) is amplified to a
level, which exceeds the threshold or background level. The lower
the Ct value, the higher the expression level, since RT-PCR of a
highly expressing sample results in a greater accumulation of
fluorochrome/product which crosses the threshold sooner. A Ct value
of 40 means that there was no product measured and should result in
a mean expression value of zero. The Ct is converted to relative
expression value based on comparison to the standard curve. For
each sample tested, the amount of GPR73a, GPR73b and GAPDH
expression level was determined from the appropriate standard
curve. Then these calculated expression values of GPR73a and GPR73b
were divided by the GAPDH expression value of each sample in order
to obtain a normalized expression for each sample. Each normalized
expression value was divided by the normalized-calibrator value to
get the relative expression levels. Using GraphPad Prism software,
these normalized values were converted to fractions in which the
highest expression level was indicated as 1. TABLE-US-00015 TABLE
15 Normalized values (represented in fractions) for GPR73a and
GPR73b expressions in rat. GPR73a GPR73b normalized normalized
Samples value StDev N Samples value StDev N Forestomach 0.067 0.057
3 Forestomach 0.063 0.013 3 Fundus 0.003 0.023 3 Fundus 0.106 0.033
3 Antrum 0.000 0.016 3 Antrum 0.000 0.004 3 Pylorus/Antrum 0.041
0.016 3 Pylorus/Antrum 0.104 0.005 3 Duodenum 0.107 0.035 3
Duodenum 0.205 0.037 3 Jejunum-1 0.102 0.035 3 Jejunum-1 0.100
0.058 3 2 0.087 0.020 3 2 0.021 0.008 3 3 0.126 0.037 3 3 0.097
0.016 3 4 0.250 0.054 3 4 0.150 0.042 3 5 0.268 0.030 3 5 0.123
0.022 3 6 0.240 0.024 3 6 0.177 0.037 3 7 0.339 0.039 3 7 0.173
0.031 3 8 0.329 0.107 3 8 0.129 0.031 3 9 0.327 0.101 3 9 0.286
0.078 3 10 0.425 0.071 3 10 0.235 0.011 3 11 0.379 0.011 3 11 0.147
0.016 3 12 0.577 0.076 3 12 0.253 0.068 3 13 0.570 0.043 3 13 0.315
0.053 3 14 0.250 0.011 3 14 0.171 0.017 3 15 0.492 0.027 3 15 0.397
0.034 3 16 0.989 0.089 3 16 0.494 0.048 3 17 0.977 0.313 3 17 0.420
0.045 3 18 1.000 0.061 3 18 0.523 0.146 3 Ileum-1 0.797 0.080 3
Ileum-1 0.630 0.141 3 2 0.636 0.014 3 2 0.434 0.080 3 3 0.614 0.015
3 3 0.441 0.115 3 4 0.923 0.085 3 4 0.871 0.288 3 5 0.807 0.142 3 5
0.739 0.017 3 6 0.755 0.080 3 6 1.000 0.246 3 Cecum 0.088 0.020 3
Cecum 0.369 0.036 3 Proximal 0.171 0.060 3 Proximal 0.887 0.021 3
Middle 0.088 0.051 3 Middle 0.209 0.047 3 Distal 0.047 0.019 3
Distal 0.012 0.002 3
Example 27
Zven1 and Monoclonal Antibodies
[0503] Rat monoclonal antibodies are prepared by immunizing 4
female Sprague-Dawley Rats (Charles River Laboratories, Wilmington,
Mass.), with the purified recombinant protein from Example 6 or
Example 7, above. The rats are each given an initial
intraperitoneal (IP) injection of 25 .quadrature.g of the purified
recombinant protein in Complete Freund's Adjuvant (Pierce,
Rockford, Ill.) followed by booster IP injections of 10
.quadrature.g of the purified recombinant protein in Incomplete
Freund's Adjuvant every two weeks. Seven days after the
administration of the second booster injection, the animals are
bled and serum is collected.
[0504] The Zven1-specific rat sera samples are characterized by
ELISA using 1 ug/ml of the purified recombinant protein Zven1 as
the specific antibody target.
[0505] Splenocytes are harvested from a single high-titer rat and
fused to SP2/0 (mouse) myeloma cells using PEG 1500 in a single
fusion procedure (4:1 fusion ratio, splenocytes to myeloma cells,
"Antibodies: A Laboratory Manual, E. Harlow and D. Lane, Cold
Spring Harbor Press). Following 9 days growth post-fusion, specific
antibody-producing hybridoma pools are identified by
radioimmunoprecipitation (RIP) using the Iodine-125 labeled
recombinant protein Zven1 as the specific antibody target and by
ELISA using 500 ng/ml of the recombinant protein Zven1 as specific
antibody target. Hybridoma pools positive in either assay protocol
are analyzed further for their ability to block the
cell-proliferative activity ("neutralization assay") of purified
recombinant protein Zven1 on Baf3 cells expressing the receptor
sequence of GPR73a (SEQ ID NO:27) and/or GPR73b (SEQ ID NO:28).
[0506] Hybridoma pools yielding positive results by RIP only or RIP
and the "neutralization assay" are cloned at least two times by
limiting dilution.
[0507] Monoclonal antibodies purified from tissue culture media are
characterized for their ability to block the cell-proliferative
activity ("neutralization assay") of purified recombinant Zven1 on
Baf3 cells expressing the receptor sequences. "Neutralizing"
monoclonal antibodies are identified in this manner.
[0508] A similar procedure is followed to identify monoclonal
antibodies to Zven2 using the amino acid sequence in SEQ ID
NO:5.
[0509] 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 1
1
29 1 1496 DNA Homo sapiens CDS (66)...(389) 1 cgcccttact cactataggg
ctcgagcggc cgcccgggca ggtgccgccc agtcccgagg 60 gcgcc atg agg agc
ctg tgc tgc gcc cca ctc ctg ctc ctc ttg ctg ctg 110 Met Arg Ser Leu
Cys Cys Ala Pro Leu Leu Leu Leu Leu Leu Leu 1 5 10 15 ccg ccg ctg
ctg ctc acg ccc cgc gct ggg gac gcc gcc gtg atc acc 158 Pro Pro Leu
Leu Leu Thr Pro Arg Ala Gly Asp Ala Ala Val Ile Thr 20 25 30 ggg
gct tgt gac aag gac tcc caa tgt ggt gga ggc atg tgc tgt gct 206 Gly
Ala Cys Asp Lys Asp Ser Gln Cys Gly Gly Gly Met Cys Cys Ala 35 40
45 gtc agt atc tgg gtc aag agc ata agg att tgc aca cct atg ggc aaa
254 Val Ser Ile Trp Val Lys Ser Ile Arg Ile Cys Thr Pro Met Gly Lys
50 55 60 ctg gga gac agc tgc cat cca ctg act cgt aaa gtt cca ttt
ttt ggg 302 Leu Gly Asp Ser Cys His Pro Leu Thr Arg Lys Val Pro Phe
Phe Gly 65 70 75 cgg agg atg cat cac act tgc cca tgt ctg cca ggc
ttg gcc tgt tta 350 Arg Arg Met His His Thr Cys Pro Cys Leu Pro Gly
Leu Ala Cys Leu 80 85 90 95 cgg act tca ttt aac cga ttt att tgt tta
gcc caa aag taatcgctct 399 Arg Thr Ser Phe Asn Arg Phe Ile Cys Leu
Ala Gln Lys 100 105 ggagtagaaa ccaaatgtga atagccacat cttacctgta
aagtcttact tgtgattgtg 459 ccaaacaaaa aatgtgccag aaagaaatgc
tcttgcttcc tcaactttcc aagtaacatt 519 tttatctttg atttgtaaat
gatttttttt ttttttttta tcgaaagaga attttacttt 579 tggatagaaa
tatgaagtgt aaggcattat ggaactggtt cttatttccc tgtttgtgtt 639
ttggtttgat ttggcttttt tcttaaatgt caaaaacgta cccattttca caaaaatgag
699 gaaaataaga atttgatatt ttgttagaaa aacttttttt tttttttctc
accaccccaa 759 gccccatttg tgccctgccg cacaaataca cctacagctt
ttggtccctt gcctcttcca 819 cctcaaagaa tttcaaggct cttaccttac
tttatttttg tccatttctc ttccctcctc 879 ttgcatttta aagtggaggg
tttgtctctt tgagtttgat ggcagaatca ctgatgggaa 939 tccagctttt
tgctggcatt taaatagtga aaagagtgta tatgtgaact tgacactcca 999
aactcctgtc atggcacgga agctaggagt gctgctggac ccttcctaaa cctgtcactc
1059 aagaggactt cagctctgct gttgggctgg tgtgtggaca gaaggaatgg
aaagccaaat 1119 taatttagtc cagatttcta ggtttgggtt tttctaaaaa
taaaagatta catttacttc 1179 ttttactttt tataaagttt tttttcctta
gtctcctact tagagatatt ctagaaaatg 1239 tcacttgaag aggaagtatt
tattttaatc tggcacaaca ctaattacca tttttaaagc 1299 ggtattaagt
tgtaatttaa accttgtttg taactgaaag gtcgattgta atggattgcc 1359
gtttgtacct gtatcagtat tgctgtgtaa aaattctgta tcagaataat aacagtactg
1419 tatatcattt gatttatttt aatattatat ccttattttt gtcaaaaaaa
aaaaaaaaaa 1479 aaaaatatgc ggccgcg 1496 2 108 PRT Homo sapiens 2
Met Arg Ser Leu Cys Cys Ala Pro Leu Leu Leu Leu Leu Leu Leu Pro 1 5
10 15 Pro Leu Leu Leu Thr Pro Arg Ala Gly Asp Ala Ala Val Ile Thr
Gly 20 25 30 Ala Cys Asp Lys Asp Ser Gln Cys Gly Gly Gly Met Cys
Cys Ala Val 35 40 45 Ser Ile Trp Val Lys Ser Ile Arg Ile Cys Thr
Pro Met Gly Lys Leu 50 55 60 Gly Asp Ser Cys His Pro Leu Thr Arg
Lys Val Pro Phe Phe Gly Arg 65 70 75 80 Arg Met His His Thr Cys Pro
Cys Leu Pro Gly Leu Ala Cys Leu Arg 85 90 95 Thr Ser Phe Asn Arg
Phe Ile Cys Leu Ala Gln Lys 100 105 3 324 DNA Artificial Sequence
This degenerate sequence encodes the amino acid sequence of SEQ ID
NO2. misc_feature (1)...(324) n = A,T,C or G 3 atgmgnwsny
tntgytgygc nccnytnytn ytnytnytny tnytnccncc nytnytnytn 60
acnccnmgng cnggngaygc ngcngtnath acnggngcnt gygayaarga ywsncartgy
120 ggnggnggna tgtgytgygc ngtnwsnath tgggtnaarw snathmgnat
htgyacnccn 180 atgggnaary tnggngayws ntgycayccn ytnacnmgna
argtnccntt yttyggnmgn 240 mgnatgcayc ayacntgycc ntgyytnccn
ggnytngcnt gyytnmgnac nwsnttyaay 300 mgnttyatht gyytngcnca raar 324
4 1409 DNA Homo sapiens CDS (91)...(405) 4 tggcctcccc agcttgccag
gcacaaggct gagcgggagg aagcgagagg catctaagca 60 ggcagtgttt
tgccttcacc ccaagtgacc atg aga ggt gcc acg cga gtc tca 114 Met Arg
Gly Ala Thr Arg Val Ser 1 5 atc atg ctc ctc cta gta act gtg tct gac
tgt gct gtg atc aca ggg 162 Ile Met Leu Leu Leu Val Thr Val Ser Asp
Cys Ala Val Ile Thr Gly 10 15 20 gcc tgt gag cgg gat gtc cag tgt
ggg gca ggc acc tgc tgt gcc atc 210 Ala Cys Glu Arg Asp Val Gln Cys
Gly Ala Gly Thr Cys Cys Ala Ile 25 30 35 40 agc ctg tgg ctt cga ggg
ctg cgg atg tgc acc ccg ctg ggg cgg gaa 258 Ser Leu Trp Leu Arg Gly
Leu Arg Met Cys Thr Pro Leu Gly Arg Glu 45 50 55 ggc gag gag tgc
cac ccc ggc agc cac aag gtc ccc ttc ttc agg aaa 306 Gly Glu Glu Cys
His Pro Gly Ser His Lys Val Pro Phe Phe Arg Lys 60 65 70 cgc aag
cac cac acc tgt cct tgc ttg ccc aac ctg ctg tgc tcc agg 354 Arg Lys
His His Thr Cys Pro Cys Leu Pro Asn Leu Leu Cys Ser Arg 75 80 85
ttc ccg gac ggc agg tac cgc tgc tcc atg gac ttg aag aac atc aat 402
Phe Pro Asp Gly Arg Tyr Arg Cys Ser Met Asp Leu Lys Asn Ile Asn 90
95 100 ttt taggcgcttg cctggtctca ggatacccac catccttttc ctgagcacag
455 Phe 105 cctggatttt tatttctgcc atgaaaccca gctcccatga ctctcccagt
ccctacactg 515 actaccctga tctctcttgt ctagtacgca catatgcaca
caggcagaca tacctcccat 575 catgacatgg tccccaggct ggcctgagga
tgtcacagct tgaggctgtg gtgtgaaagg 635 tggccagcct ggttctcttc
cctgctcagg ctgccagaga ggtggtaaat ggcagaaagg 695 acattccccc
tcccctcccc aggtgacctg ctctctttcc tgggccctgc ccctctcccc 755
acatgtatcc ctcggtctga attagacatt cctgggcaca ggctcttggg tgcattgctc
815 agagtcccag gtcctggcct gaccctcagg cccttcacgt gaggtctgtg
aggaccaatt 875 tgtgggtagt tcatcttccc tcgattggtt aactccttag
tttcagacca cagactcaag 935 attggctctt cccagagggc agcagacagt
caccccaagg caggtgtagg gagcccaggg 995 aggccaatca gccccctgaa
gactctggtc ccagtcagcc tgtggcttgt ggcctgtgac 1055 ctgtgacctt
ctgccagaat tgtcatgcct ctgaggcccc ctcttaccac actttaccag 1115
ttaaccactg aagcccccaa ttcccacagc ttttccatta aaatgcaaat ggtggtggtt
1175 caatctaatc tgatattgac atattagaag gcaattaggg tgtttcctta
aacaactcct 1235 ttccaaggat cagccctgag agcaggttgg tgactttgag
gagggcagtc ctctgtccag 1295 attggggtgg gagcaaggga cagggagcag
ggcaggggct gaaaggggca ctgattcaga 1355 ccagggaggc aactacacac
caacctgctg gctttagaat aaaagcacca actg 1409 5 105 PRT Homo sapiens 5
Met Arg Gly Ala Thr Arg Val Ser Ile Met Leu Leu Leu Val Thr Val 1 5
10 15 Ser Asp Cys Ala Val Ile Thr Gly Ala Cys Glu Arg Asp Val Gln
Cys 20 25 30 Gly Ala Gly Thr Cys Cys Ala Ile Ser Leu Trp Leu Arg
Gly Leu Arg 35 40 45 Met Cys Thr Pro Leu Gly Arg Glu Gly Glu Glu
Cys His Pro Gly Ser 50 55 60 His Lys Val Pro Phe Phe Arg Lys Arg
Lys His His Thr Cys Pro Cys 65 70 75 80 Leu Pro Asn Leu Leu Cys Ser
Arg Phe Pro Asp Gly Arg Tyr Arg Cys 85 90 95 Ser Met Asp Leu Lys
Asn Ile Asn Phe 100 105 6 315 DNA Artificial Sequence This
degenerate sequence encodes the amino acid sequence of SEQ ID NO5.
misc_feature (1)...(315) n = A,T,C or G 6 atgmgnggng cnacnmgngt
nwsnathatg ytnytnytng tnacngtnws ngaytgygcn 60 gtnathacng
gngcntgyga rmgngaygtn cartgyggng cnggnacntg ytgygcnath 120
wsnytntggy tnmgnggnyt nmgnatgtgy acnccnytng gnmgngargg ngargartgy
180 cayccnggnw sncayaargt nccnttytty mgnaarmgna arcaycayac
ntgyccntgy 240 ytnccnaayy tnytntgyws nmgnttyccn gayggnmgnt
aymgntgyws natggayytn 300 aaraayatha aytty 315 7 16 PRT Artificial
Sequence Peptide linker. 7 Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly
Ser Gly Gly Gly Gly Ser 1 5 10 15 8 10 PRT Artificial Sequence
Motif. VARIANT (8)...(8) Xaa is Asp or Glu. VARIANT (9)...(9) Xaa
is Lys or Arg. VARIANT (1)...(10) Xaa = Any Amino Acid VARIANT
(1)...(10) Xaa = Any Amino Acid VARIANT (1)...(10) Xaa = Any Amino
Acid 8 Ala Val Ile Thr Gly Ala Cys Xaa Xaa Asp 1 5 10 9 23 PRT
Artificial Sequence Motif. VARIANT (4)...(4) Xaa is Gly or Leu.
VARIANT (5)...(5) Xaa is Ser or Thr. VARIANT (6)...(6) Xaa is His
or Arg. VARIANT (12)...(12) Xaa is any amino acid. VARIANT
(13)...(13) Xaa is Lys or Arg. VARIANT (15)...(15) Xaa is any amino
acid. 9 Cys His Pro Xaa Xaa Xaa Lys Val Pro Phe Phe Xaa Xaa Arg Xaa
His 1 5 10 15 His Thr Cys Pro Cys Leu Pro 20 10 6 PRT Artificial
Sequence Glu-Glu tag 10 Glu Tyr Met Pro Met Glu 1 5 11 249 DNA Homo
sapiens 11 atggccgtga tcaccggggc ttgtgacaag gactcccaat gtggtggagg
catgtgctgt 60 gctgtcagta tctgggtcaa gagcataagg atttgcacac
ctatgggcaa actgggagac 120 agctgccatc cactgactcg taaagttcca
ttttttgggc ggaggatgca tcacacttgc 180 ccgtgtctgc caggcttggc
ctgtttacgg acttcattta accgatttat ttgtttagcc 240 caaaagtaa 249 12 68
DNA Artificial Sequence oligonucleotide primer ZC40821 12
ctagaaataa ttttgtttaa ctttaagaag gagatatata tatggccgtg atcaccgggg
60 cttgtgac 68 13 67 DNA Artificial Sequence oligonucleotide primer
ZC40813 13 tctgtatcag gctgaaaatc ttatctcatc cgccaaaaca ttacttttgg
gctaaacaaa 60 taaatcg 67 14 249 DNA Artificial Sequence Codon
optimized polynucleotide sequence for Zven1 14 atggctgtta
ttaccggtgc ttgcgacaaa gactctcagt gtggtggtgg tatgtgctgc 60
gctgtttcta tctgggttaa atctatccgt atctgcactc ctatgggtaa actgggtgac
120 tcttgccatc cgctgactcg taaagttccg ttcttcggtc gtcgtatgca
tcacacctgt 180 ccgtgcctgc cgggtctggc ttgcctgcgt acctctttca
accgtttcat ttgcctggct 240 cagaagtaa 249 15 79 DNA Artificial
Sequence Oligonucleotide primer ZC45,048 15 agtcaatgga tgacaagaat
cacccaactt acccatagga gtacaaattc tgatagactt 60 aacccaaata gaaacagca
79 16 77 DNA Artificial Sequence Oligonucleotide primer ZC45049 16
ttcttgtcat ccattgacta gaaaggttcc attctttggt agaaggatgc atcacacttg
60 tccatgtttg ccaggtt 77 17 70 DNA Artificial Sequence
Oligonucleotide primer ZC45050 17 ttacttttga gccaaacaaa tgaatctgtt
gaaagaagtt ctcaaacaag ccaaacctgg 60 caaacatgga 70 18 68 DNA
Artificial Sequence Oligonucleotide primer ZC45051 18 attactggtg
cttgtgataa ggattctcaa tgtggtggtg gtatgtgttg tgctgtttct 60 atttgggt
68 19 65 DNA Artificial Sequence Oligonucleotide primer ZC45052 19
ttatcacaag caccagtaat aacagcagca tcaccggctc ttggagtcaa caacaatggt
60 ggcaa 65 20 59 DNA Artificial Sequence Oligonucleotide primer
ZC45053 20 atgagatctt tgtgttgtgc tccattgttg ttgttgttgt tgttgccacc
attgttgtt 59 21 1182 DNA Homo sapiens 21 atggagacca ccatggggtt
catggatgac aatgccacca acacttccac cagcttcctt 60 tctgtgctca
accctcatgg agcccatgcc acttccttcc cattcaactt cagctacagc 120
gactatgata tgcctttgga tgaagatgag gatgtgacca attccaggac gttctttgct
180 gccaagattg tcattgggat ggccctggtg ggcatcatgc tggtctgcgg
cattggaaac 240 ttcatcttta tcgctgccct ggtccgctac aagaaactgc
gcaacctcac caacctgctc 300 atcgccaacc tggccatctc tgacttcctg
gtggccattg tctgctgccc ctttgagatg 360 gactactatg tggtgcgcca
gctctcctgg gagcacggcc acgtcctgtg cacctctgtc 420 aactacctgc
gcactgtctc tctctatgtc tccaccaatg ccctgctggc catcgccatt 480
gacaggtatc tggctattgt ccatccgctg agaccacgga tgaagtgcca aacagccact
540 ggcctgattg ccttggtgtg gacggtgtcc atcctgatcg ccatcccttc
cgcctacttc 600 accaccgaga cggtcctcgt cattgtcaag agccaggaaa
agatcttctg cggccagatc 660 tggcctgtgg accagcagct ctactacaag
tcctacttcc tctttatctt tggcatagaa 720 ttcgtgggcc ccgtggtcac
catgaccctg tgctatgcca ggatctcccg ggagctctgg 780 ttcaaggcgg
tccctggatt ccagacagag cagatccgca agaggctgcg ctgccgcagg 840
aagacggtcc tggtgctcat gtgcatcctc accgcctacg tgctatgctg ggcgcccttc
900 tacggcttca ccatcgtgcg cgacttcttc cccaccgtgt ttgtgaagga
gaagcactac 960 ctcactgcct tctacatcgt cgagtgcatc gccatgagca
acagcatgat caacactctg 1020 tgcttcgtga ccgtcaagaa cgacaccgtc
aagtacttca aaaagatcat gttgctccac 1080 tggaaggctt cttacaatgg
cggtaagtcc agtgcagacc tggacctcaa gacaattggg 1140 atgcctgcca
ccgaagaggt ggactgcatc agactaaaat aa 1182 22 1155 DNA Homo sapiens
22 atggcagccc agaatggaaa caccagtttc acacccaact ttaatccacc
ccaagaccat 60 gcctcctccc tctcctttaa cttcagttat ggtgattatg
acctccctat ggatgaggat 120 gaggacatga ccaagacccg gaccttcttc
gcagccaaga tcgtcattgg cattgcactg 180 gcaggcatca tgctggtctg
cggcatcggt aactttgtct ttatcgctgc cctcacccgc 240 tataagaagt
tgcgcaacct caccaatctg ctcattgcca acctggccat ctccgacttc 300
ctggtggcca tcatctgctg ccccttcgag atggactact acgtggtacg gcagctctcc
360 tgggagcatg gccacgtgct ctgtgcctcc gtcaactacc tgcgcaccgt
ctccctctac 420 gtctccacca atgccttgct ggccattgcc attgacagat
atctcgccat cgttcacccc 480 ttgaaaccac ggatgaatta tcaaacggcc
tccttcctga tcgccttggt ctggatggtg 540 tccattctca ttgccatccc
atcggcttac tttgcaacag aaacggtcct ctttattgtc 600 aagagccagg
agaagatctt ctgtggccag atctggcctg tggatcagca gctctactac 660
aagtcctact tcctcttcat ctttggtgtc gagttcgtgg gccctgtggt caccatgacc
720 ctgtgctatg ccaggatctc ccgggagctc tggttcaagg cagtccctgg
gttccagacg 780 gagcagattc gcaagcggct gcgctgccgc aggaagacgg
tcctggtgct catgtgcatt 840 ctcacggcct atgtgctgtg ctgggcaccc
ttctacggtt tcaccatcgt tcgtgacttc 900 ttccccactg tgttcgtgaa
ggaaaagcac tacctcactg ccttctacgt ggtcgagtgc 960 atcgccatga
gcaacagcat gatcaacacc gtgtgcttcg tgacggtcaa gaacaacacc 1020
atgaagtact tcaagaagat gatgctgctg cactggcgtc cctcccagcg ggggagcaag
1080 tccagtgctg accttgacct cagaaccaac ggggtgccca ccacagaaga
ggtggactgt 1140 atcaggctga agtga 1155 23 28 DNA Artificial Sequence
Oligonucleotide primer ZC29463 23 ggaattcatg aggagcctgt gctgcgcc 28
24 31 DNA Artificial Sequence Oligonucleotide primer ZC29462 24
gctctagacc cttttgggct aaacaaataa a 31 25 348 DNA Artificial
Sequence Expression sequence 25 atgaggagcc tgtgctgcgc cccactcctg
ctcctcttgc tgctgccgcc gctgctgctc 60 acgccccgcg ctggggacgc
cgccgtgatc accggggctt gtgacaagga ctcccaatgt 120 ggtggaggca
tgtgctgtgc tgtcagtatc tgggtcaaga gcataaggat ttgcacacct 180
atgggcaaac tgggagacag ctgccatcca ctgactcgta aagttccatt ttttgggcgg
240 aggatgcatc acacttgccc gtgtctgcca ggcttggcct gtttacggac
ttcatttaac 300 cgatttattt gtttagccca aaagggtcta gaatacatgc cgatggac
348 26 116 PRT Artificial Sequence Expression sequence with Gly
linker and Glu-Glu-tag 26 Met Arg Ser Leu Cys Cys Ala Pro Leu Leu
Leu Leu Leu Leu Leu Pro 1 5 10 15 Pro Leu Leu Leu Thr Pro Arg Ala
Gly Asp Ala Ala Val Ile Thr Gly 20 25 30 Ala Cys Asp Lys Asp Ser
Gln Cys Gly Gly Gly Met Cys Cys Ala Val 35 40 45 Ser Ile Trp Val
Lys Ser Ile Arg Ile Cys Thr Pro Met Gly Lys Leu 50 55 60 Gly Asp
Ser Cys His Pro Leu Thr Arg Lys Val Pro Phe Phe Gly Arg 65 70 75 80
Arg Met His His Thr Cys Pro Cys Leu Pro Gly Leu Ala Cys Leu Arg 85
90 95 Thr Ser Phe Asn Arg Phe Ile Cys Leu Ala Gln Lys Gly Leu Glu
Tyr 100 105 110 Met Pro Met Asp 115 27 393 PRT Homo sapiens 27 Met
Glu Thr Thr Met Gly Phe Met Asp Asp Asn Ala Thr Asn Thr Ser 1 5 10
15 Thr Ser Phe Leu Ser Val Leu Asn Pro His Gly Ala His Ala Thr Ser
20 25 30 Phe Pro Phe Asn Phe Ser Tyr Ser Asp Tyr Asp Met Pro Leu
Asp Glu 35 40 45 Asp Glu Asp Val Thr Asn Ser Arg Thr Phe Phe Ala
Ala Lys Ile Val 50 55 60 Ile Gly Met Ala Leu Val Gly Ile Met Leu
Val Cys Gly Ile Gly Asn 65 70 75 80 Phe Ile Phe Ile Ala Ala Leu Val
Arg Tyr Lys Lys Leu Arg Asn Leu 85 90 95 Thr Asn Leu Leu Ile Ala
Asn Leu Ala Ile Ser Asp Phe Leu Val Ala 100 105 110 Ile Val Cys Cys
Pro Phe Glu Met Asp Tyr Tyr Val Val Arg Gln Leu 115 120 125 Ser Trp
Glu His Gly His Val Leu Cys Thr Ser Val Asn Tyr Leu Arg 130 135 140
Thr Val Ser Leu Tyr Val Ser Thr Asn Ala Leu Leu Ala Ile Ala Ile 145
150 155 160 Asp Arg Tyr Leu Ala Ile Val His Pro Leu Arg Pro Arg Met
Lys Cys 165 170 175 Gln Thr Ala Thr Gly Leu Ile Ala Leu Val Trp Thr
Val Ser Ile Leu 180 185 190 Ile Ala Ile Pro Ser Ala Tyr Phe Thr Thr
Glu Thr Val Leu Val Ile 195 200 205 Val Lys Ser Gln Glu Lys Ile Phe
Cys Gly Gln Ile Trp Pro
Val Asp 210 215 220 Gln Gln Leu Tyr Tyr Lys Ser Tyr Phe Leu Phe Ile
Phe Gly Ile Glu 225 230 235 240 Phe Val Gly Pro Val Val Thr Met Thr
Leu Cys Tyr Ala Arg Ile Ser 245 250 255 Arg Glu Leu Trp Phe Lys Ala
Val Pro Gly Phe Gln Thr Glu Gln Ile 260 265 270 Arg Lys Arg Leu Arg
Cys Arg Arg Lys Thr Val Leu Val Leu Met Cys 275 280 285 Ile Leu Thr
Ala Tyr Val Leu Cys Trp Ala Pro Phe Tyr Gly Phe Thr 290 295 300 Ile
Val Arg Asp Phe Phe Pro Thr Val Phe Val Lys Glu Lys His Tyr 305 310
315 320 Leu Thr Ala Phe Tyr Ile Val Glu Cys Ile Ala Met Ser Asn Ser
Met 325 330 335 Ile Asn Thr Leu Cys Phe Val Thr Val Lys Asn Asp Thr
Val Lys Tyr 340 345 350 Phe Lys Lys Ile Met Leu Leu His Trp Lys Ala
Ser Tyr Asn Gly Gly 355 360 365 Lys Ser Ser Ala Asp Leu Asp Leu Lys
Thr Ile Gly Met Pro Ala Thr 370 375 380 Glu Glu Val Asp Cys Ile Arg
Leu Lys 385 390 28 384 PRT Homo sapiens 28 Met Ala Ala Gln Asn Gly
Asn Thr Ser Phe Thr Pro Asn Phe Asn Pro 1 5 10 15 Pro Gln Asp His
Ala Ser Ser Leu Ser Phe Asn Phe Ser Tyr Gly Asp 20 25 30 Tyr Asp
Leu Pro Met Asp Glu Asp Glu Asp Met Thr Lys Thr Arg Thr 35 40 45
Phe Phe Ala Ala Lys Ile Val Ile Gly Ile Ala Leu Ala Gly Ile Met 50
55 60 Leu Val Cys Gly Ile Gly Asn Phe Val Phe Ile Ala Ala Leu Thr
Arg 65 70 75 80 Tyr Lys Lys Leu Arg Asn Leu Thr Asn Leu Leu Ile Ala
Asn Leu Ala 85 90 95 Ile Ser Asp Phe Leu Val Ala Ile Ile Cys Cys
Pro Phe Glu Met Asp 100 105 110 Tyr Tyr Val Val Arg Gln Leu Ser Trp
Glu His Gly His Val Leu Cys 115 120 125 Ala Ser Val Asn Tyr Leu Arg
Thr Val Ser Leu Tyr Val Ser Thr Asn 130 135 140 Ala Leu Leu Ala Ile
Ala Ile Asp Arg Tyr Leu Ala Ile Val His Pro 145 150 155 160 Leu Lys
Pro Arg Met Asn Tyr Gln Thr Ala Ser Phe Leu Ile Ala Leu 165 170 175
Val Trp Met Val Ser Ile Leu Ile Ala Ile Pro Ser Ala Tyr Phe Ala 180
185 190 Thr Glu Thr Val Leu Phe Ile Val Lys Ser Gln Glu Lys Ile Phe
Cys 195 200 205 Gly Gln Ile Trp Pro Val Asp Gln Gln Leu Tyr Tyr Lys
Ser Tyr Phe 210 215 220 Leu Phe Ile Phe Gly Val Glu Phe Val Gly Pro
Val Val Thr Met Thr 225 230 235 240 Leu Cys Tyr Ala Arg Ile Ser Arg
Glu Leu Trp Phe Lys Ala Val Pro 245 250 255 Gly Phe Gln Thr Glu Gln
Ile Arg Lys Arg Leu Arg Cys Arg Arg Lys 260 265 270 Thr Val Leu Val
Leu Met Cys Ile Leu Thr Ala Tyr Val Leu Cys Trp 275 280 285 Ala Pro
Phe Tyr Gly Phe Thr Ile Val Arg Asp Phe Phe Pro Thr Val 290 295 300
Phe Val Lys Glu Lys His Tyr Leu Thr Ala Phe Tyr Val Val Glu Cys 305
310 315 320 Ile Ala Met Ser Asn Ser Met Ile Asn Thr Val Cys Phe Val
Thr Val 325 330 335 Lys Asn Asn Thr Met Lys Tyr Phe Lys Lys Met Met
Leu Leu His Trp 340 345 350 Arg Pro Ser Gln Arg Gly Ser Lys Ser Ser
Ala Asp Leu Asp Leu Arg 355 360 365 Thr Asn Gly Val Pro Thr Thr Glu
Glu Val Asp Cys Ile Arg Leu Lys 370 375 380 29 129 PRT Homo sapiens
29 Met Arg Ser Leu Cys Cys Ala Pro Leu Leu Leu Leu Leu Leu Leu Pro
1 5 10 15 Pro Leu Leu Leu Thr Pro Arg Ala Gly Asp Ala Ala Val Ile
Thr Gly 20 25 30 Ala Cys Asp Lys Asp Ser Gln Cys Gly Gly Gly Met
Cys Cys Ala Val 35 40 45 Ser Ile Trp Val Lys Ser Ile Arg Ile Cys
Thr Pro Met Gly Lys Leu 50 55 60 Gly Asp Ser Cys His Pro Leu Thr
Arg Lys Asn Asn Phe Gly Asn Gly 65 70 75 80 Arg Gln Glu Arg Arg Lys
Arg Lys Arg Ser Lys Arg Lys Lys Glu Val 85 90 95 Pro Phe Phe Gly
Arg Arg Met His His Thr Cys Pro Cys Leu Pro Gly 100 105 110 Leu Ala
Cys Leu Arg Thr Ser Phe Asn Arg Phe Ile Cys Leu Ala Gln 115 120 125
Lys
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