U.S. patent application number 10/124591 was filed with the patent office on 2002-11-28 for human phospholipase a2 protein.
This patent application is currently assigned to Incyte Genomics, Inc.. Invention is credited to Bandman, Olga, Corley, Neil C., Guegler, Karl J., Hawkins, Phillip R., Shah, Purvi.
Application Number | 20020177208 10/124591 |
Document ID | / |
Family ID | 25511220 |
Filed Date | 2002-11-28 |
United States Patent
Application |
20020177208 |
Kind Code |
A1 |
Hawkins, Phillip R. ; et
al. |
November 28, 2002 |
Human phospholipase A2 protein
Abstract
The invention provides a human phospholipase A2 protein PHPLA2
and polynucleotides which identify and encode PHPLA2. The invention
also provides expression vectors, host cells, agonists, antibodies
and antagonists. The invention also provides methods for treating
disorders associated with expression of PHPLA2.
Inventors: |
Hawkins, Phillip R.;
(Mountain View, CA) ; Bandman, Olga; (Mountain
View, CA) ; Guegler, Karl J.; (Menlo Park, CA)
; Shah, Purvi; (Sunnyvale, CA) ; Corley, Neil
C.; (Mountain View, CA) |
Correspondence
Address: |
INCYTE GENOMICS, INC.
PATENT DEPARTMENT
3160 Porter Drive
Palo Alto
CA
94304
US
|
Assignee: |
Incyte Genomics, Inc.
|
Family ID: |
25511220 |
Appl. No.: |
10/124591 |
Filed: |
April 16, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10124591 |
Apr 16, 2002 |
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09489770 |
Jan 21, 2000 |
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6399301 |
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09489770 |
Jan 21, 2000 |
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08966317 |
Nov 7, 1997 |
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6103469 |
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Current U.S.
Class: |
435/196 ;
435/198; 435/320.1; 435/325; 435/6.11; 435/69.1; 536/23.2 |
Current CPC
Class: |
A61K 38/00 20130101;
C12Y 301/01004 20130101; C12N 9/20 20130101 |
Class at
Publication: |
435/196 ;
435/320.1; 435/6; 435/325; 435/69.1; 435/198; 536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; C12N 009/16; C12N 009/20; C12P 021/02; C12N 005/06 |
Claims
What is claimed is:
1. An isolated polypeptide selected from the group consisting of:
a) a polypeptide comprising an amino acid sequence of SEQ ID NO: 1,
b) a polypeptide comprising a naturally occurring amino acid
sequence at least 90% identical to an amino acid sequence of SEQ ID
NO: 1, c) a biologically active fragment of a polypeptide having an
amino acid sequence of SEQ ID NO:1, and d) an immunogenic fragment
of a polypeptide having an amino acid sequence of SEQ ID NO:1.
2. A isolated polypeptide of claim 1, comprising an amino acid
sequence of SEQ ID NO: 1.
3. An isolated polynucleotide encoding a polypeptide of claim
1.
4. An isolated polynucleotide encoding a polypeptide of claim
2.
5. An isolated polynucleotide of claim 4, comprising a
polynucleotide sequence of SEQ ID NO:2.
6. A recombinant polynucleotide comprising a promoter sequence
operably linked to a polynucleotide of claim 3.
7. A cell transformed with a recombinant polynucleotide of claim
6.
8. A transgenic organism comprising a recombinant polynucleotide of
claim 6.
9. A method for producing a polypeptide of claim 1, the method
comprising: a) culturing a cell under conditions suitable for
expression of the polypeptide, wherein said cell is transformed
with a recombinant polynucleotide, and said recombinant
polynucleotide comprises a promoter sequence operably linked to a
polynucleotide encoding the polypeptide of claim 1, and b)
recovering the polypeptide so expressed.
10. A method of claim 9, wherein the polypeptide comprises an amino
acid sequence of SEQ ID NO: 1.
11. An isolated antibody which specifically binds to a polypeptide
of claim 1.
12. An isolated polynucleotide selected from the group consisting
of: a) a polynucleotide comprising a polynucleotide sequence of SEQ
ID NO:2, b) a polynucleotide comprising a naturally occurring
polynucleotide sequence at least 90% identical to a polynucleotide
sequence of SEQ ID NO:2, c) a polynucleotide complementary to a
polynucleotide of a), d) a polynucleotide complementary to a
polynucleotide of b), and e) an RNA equivalent of a)-d).
13. An isolated polynucleotide comprising at least 60 contiguous
nucleotides of a polynucleotide of claim 12.
14. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) hybridizing the sample with a
probe comprising at least 20 contiguous nucleotides comprising a
sequence complementary to said target polynucleotide in the sample,
and which probe specifically hybridizes to said target
polynucleotide, under conditions whereby a hybridization complex is
formed between said probe and said target polynucleotide or
fragments thereof, and b) detecting the presence or absence of said
hybridization complex, and, optionally, if present, the amount
thereof.
15. A method of claim 14, wherein the probe comprises at least 60
contiguous nucleotides.
16. A method for detecting a target polynucleotide in a sample,
said target polynucleotide having a sequence of a polynucleotide of
claim 12, the method comprising: a) amplifying said target
polynucleotide or fragment thereof using polymerase chain reaction
amplification, and b) detecting the presence or absence of said
amplified target polynucleotide or fragment thereof, and,
optionally, if present, the amount thereof.
17. A composition comprising a polypepide of claim 1 and a
pharmaceutically acceptable excipient.
18. A composition of claim 17, wherein the polypeptide comprises an
amino acid sequence of SEQ ID NO: 1.
19. A method for treating a disease or condition associated with
decreased expression of functional PHPLA2, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method for screening a compound for effectiveness as an
agonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting agonist activity in the sample.
21. A composition comprising an agonist compound identified by a
method of claim 20 and a pharmaceutically acceptable excipient.
22. A method for treating a disease or condition associated with
decreased expression of functional PHPLA2, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method for screening a compound for effectiveness as an
antagonist of a polypeptide of claim 1, the method comprising: a)
exposing a sample comprising a polypeptide of claim 1 to a
compound, and b) detecting antagonist activity in the sample.
24. A composition comprising an antagonist compound identified by a
method of claim 23 and a pharmaceutically acceptable excipient.
25. A method for treating a disease or condition associated with
overexpression of functional PHPLA2, comprising administering to a
patient in need of such treatment a composition of claim 24.
26. A method of screening for a compound that specifically binds to
the polypeptide of claim 1, the method comprising: a) combining the
polypeptide of claim 1 with at least one test compound under
suitable conditions, and b) detecting binding of the polypeptide of
claim I to the test compound, thereby identifying a compound that
specifically binds to the polypeptide of claim 1.
27. A method of screening for a compound that modulates the
activity of the polypeptide of claim 1, said method comprising: a)
combining the polypeptide of claim 1 with at least one test
compound under conditions permissive for the activity of the
polypeptide of claim 1, b) assessing the activity of the
polypeptide of claim 1 in the presence of the test compound, and c)
comparing the activity of the polypeptide of claim 1 in the
presence of the test compound with the activity of the polypeptide
of claim 1 in the absence of the test compound, wherein a change in
the activity of the polypeptide of claim 1 in the presence of the
test compound is indicative of a compound that modulates the
activity of the polypeptide of claim 1.
28. A method for screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a polynucleotide sequence of claim 5, the
method comprising: a) exposing a sample comprising the target
polynucleotide to a compound, under conditions suitable for the
expression of the target polynucleotide, b) detecting altered
expression of the target polynucleotide, and c) comparing the
expression of the target polynucleotide in the presence of varying
amounts of the compound and in the absence of the compound.
29. A method for assessing toxicity of a test compound, the method
comprising: a) treating a biological sample containing nucleic
acids with the test compound, b) hybridizing the nucleic acids of
the treated biological sample with a probe comprising at least 20
contiguous nucleotides of a polynucleotide of claim 12 under
conditions whereby a specific hybridization complex is formed
between said probe and a target polynucleotide in the biological
sample, said target polynucleotide comprising a polynucleotide
sequence of a polynucleotide of claim 12 or fragment thereof, c)
quantifying the amount of hybridization complex, and d) comparing
the amount of hybridization complex in the treated biological
sample with the amount of hybridization complex in an untreated
biological sample, wherein a difference in the amount of
hybridization complex in the treated biological sample is
indicative of toxicity of the test compound.
30. A diagnostic test for a condition or disease associated with
the expression of PHPLA2 in a biological sample, the method
comprising: a) combining the biological sample with an antibody of
claim 11, under conditions suitable for the antibody to bind the
polypeptide and form an antibody:polypeptide complex, and b)
detecting the complex, wherein the presence of the complex
correlates with the presence of the polypeptide in the biological
sample.
31. The antibody of claim 11, wherein the antibody is: a) a
chimeric antibody, b) a single chain antibody, c) a Fab fragment,
d) a F(ab').sub.2 fragment, or e) a humanized antibody.
32. A composition comprising an antibody of claim II and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of PHPLA2 in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, wherein the antibody is labeled.
35. A method of diagnosing a condition or disease associated with
the expression of PHPLA2 in a subject, comprising administering to
said subject an effective amount of the composition of claim
34.
36. A method of preparing a polyclonal antibody with the
specificity of the antibody of claim 11, the method comprising: a)
immunizing an animal with a polypeptide having an amino acid
sequence of SEQ ID NO: 1, or an immunogenic fragment thereof, under
conditions to elicit an antibody response, b) isolating antibodies
from said animal, and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which binds
specifically to a polypeptide having an amino acid sequence of SEQ
ID NO: 1.
37. An antibody produced by a method of claim 36.
38. A composition comprising the antibody of claim 37 and a
suitable carrier.
39. A method of making a monoclonal antibody with the specificity
of the antibody of claim 11, the method comprising: a) immunizing
an animal with a polypeptide having an amino acid sequence of SEQ
ID NO:1, or an immunogenic fragment thereof, under conditions to
elicit an antibody response, b) isolating antibody producing cells
from the animal, c) fusing the antibody producing cells with
immortalized cells to form monoclonal antibody-producing hybridoma
cells, d) culturing the hybridoma cells, and e) isolating from the
culture monoclonal antibody which binds specifically to a
polypeptide having an amino acid sequence of SEQ ID NO: 1.
40. A monoclonal antibody produced by a method of claim 39.
41. A composition comprising the antibody of claim 40 and a
suitable carrier.
42. The antibody of claim 11, wherein the antibody is produced by
screening a Fab expression library.
43. The antibody of claim 11, wherein the antibody is produced by
screening a recombinant immunoglobulin library.
44. A method of detecting a polypeptide having an amino acid
sequence of SEQ ID NO: 1 in a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) detecting specific binding, wherein specific binding indicates
the presence of a polypeptide having an amino acid sequence of SEQ
ID NO:1 in the sample.
45. A method of purifying a polypeptide having an amino acid
sequence of SEQ ID NO: 1 from a sample, the method comprising: a)
incubating the antibody of claim 11 with a sample under conditions
to allow specific binding of the antibody and the polypeptide, and
b) separating the antibody from the sample and obtaining the
purified polypeptide having an amino acid sequence of SEQ ID NO:
1.
46. A microarray wherein at least one element of the microarray is
a polynucleotide of claim 13.
47. A method of generating an expression profile of a sample which
contains polynucleotides, the method comprising: a) labeling the
polynucleotides of the sample, b) contacting the elements of the
microarray of claim 46 with the labeled polynucleotides of the
sample under conditions suitable for the formation of a
hybridization complex, and c) quantifying the expression of the
polynucleotides in the sample.
48. An array comprising different nucleotide molecules affixed in
distinct physical locations on a solid substrate, wherein at least
one of said nucleotide molecules comprises a first oligonucleotide
or polynucleotide sequence specifically hybridizable with at least
30 contiguous nucleotides of a target polynucleotide, and wherein
said target polynucleotide is a polynucleotide of claim 12.
49. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 30
contiguous nucleotides of said target polynucleotide.
50. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to at least 60
contiguous nucleotides of said target polynucleotide.
51. An array of claim 48, wherein said first oligonucleotide or
polynucleotide sequence is completely complementary to said target
polynucleotide.
52. An array of claim 48, which is a microarray.
53. An array of claim 48, further comprising said target
polynucleotide hybridized to a nucleotide molecule comprising said
first oligonucleotide or polynucleotide sequence.
54. An array of claim 48, wherein a linker joins at least one of
said nucleotide molecules to said solid substrate.
55. An array of claim 48, wherein each distinct physical location
on the substrate contains multiple nucleotide molecules, and the
multiple nucleotide molecules at any single distinct physical
location have the same sequence, and each distinct physical
location on the substrate contains nucleotide molecules having a
sequence which differs from the sequence of nucleotide molecules at
another distinct physical location on the substrate.
56. A polypeptide of claim 1, comprising the amino acid sequence of
SEQ ID NO: 1.
57. A polynucleotide of claim 12, comprising the polynucleotide
sequence of SEQ ID NO:2.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/489,770, filed Jan. 21, 2000, which is a
divisional application of U.S. application Ser. No. 08/966,317,
filed Nov. 7, 1997, now U.S. Pat. No. 6,103,469, issued Aug. 15,
2000, both entitled HUMAN PHOSPHOLIPASE A2 PROTEIN, all of which
applications and patents are hereby incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] This invention relates to nucleic acid and amino acid
sequences of a human phospholipase A2 protein and to the use of
these sequences in the diagnosis, prevention, and treatment of
cancer and inflammation.
BACKGROUND OF THE INVENTION
[0003] The secretory phospholipase A2 (PLA2) superfamily comprises
a number of heterogeneous enzymes whose common feature is to
hydrolyze the sn-2 fatty acid acyl ester bond of phosphoglycerides.
Hydrolysis of the glycerophospholipids releases free fatty acids
and lysophospholipids. PLA2 activity generates precursors for the
biosynthesis of biologically active lipids, hydroxy fatty acids,
and platelet-activating factor.
[0004] PLA2 hydrolysis of the sn-2 ester bond in phospholipids
generates free fatty acids, such as arachidonic acid and
lysophospholipids. Arachidonic acid is processed into bioactive
lipid mediators of inflammation such as lyso-platelet-activating
factor and eicosanoids. The synthesis of arachidonic acid from
membrane phospholipids is the rate-limiting step in the
biosynthesis of the four major classes of eicosanoids
(prostaglandins, prostacyclins, thromboxanes and leukotrienes)
involved in pain, fever, and inflammation. Furthermore,
leukotriene-B4 is known to function in a feedback loop which
further increases PLA2 activity (Wijkander, J. et al. (1995) J.
Biol. Chem. 270:26543-26549).
[0005] PLA2s, first described as components of snake venoms and
pancreatic juice and later defined in mammals, have traditionally
been classified into several major groups and subgroups based on
their amino acid sequence, disulfide bond location, and divalent
cation requirements. The PLA2s of groups I, II, and III consist of
low molecular weight, secreted, Ca.sup.2+-dependent proteins, Group
IV PLA2s are primarily 85-kDa, Ca.sup.2+-dependent cytosolic
phospholipases, and an additional group comprises the
Ca.sup.2+-independent cytosolic phospholipases (Davidson, F. F. and
Dennis, E. A., (1990) J. Mol. Evol. 31: 228-238; and Dennis, E. F.
(1994) J. Biol Chem. 269:13057-13060).
[0006] The first PLA2s to be extensively characterized were the
Group I, II, and III PLA2s found in snake and bee venoms. These
venom PLA2s share many features with mammalian PLA2s including a
common catalytic mechanism, the same Ca+ requirement, and conserved
primary and tertiary structures. In addition to their role in the
digestion of prey, the venom PLA2s display neurotoxic, myotoxic,
anticoagulant, and proinflammatory effects in mammalian tissues.
This diversity of pathophysiological effects is due to the presence
of specific, high affinity receptors for these enzymes on various
cells and tissues (Lambeau, G. et al. (1995) J. Biol. Chem.
270:5534-5540).
[0007] At least four different groups of PLA2s have been
characterized in mammalian cells, including Group I (pancreatic),
Groups IIA, and IIC (inflammatory), and Group V (expressed in the
heart). The pancreatic PLA2s function in the digestion of dietary
lipids and have been proposed to play a role in cell proliferation,
smooth muscle contraction, and acute lung injury. The inflammatory
PLA2s are potent mediators of inflammatory processes and are highly
expressed in serum and synovial fluids of patients with
inflammatory disorders. These Group II PLA2s are found in most
human cell types assayed and are expressed in diverse pathological
processes such as septic shock, intestinal cancers, rheumatoid
arthritis, and epidermal hyperplasia. A Group V PLA2 has been
cloned from brain tissue and is strongly expressed in heart tissue.
Other PLA2s have been cloned from various human tissues and cell
lines, suggesting a large diversity of PLA2s. A human PLA2 was
recently cloned from fetal lung, and based on its structural
properties, appears to be the first member of a new group of
mammalian PLAs, referred to as Group X. (Chen J. et al. (1994) J.
Biol. Chem. 269:2365-2368; Kennedy, B. P., et al. (1995) J. Biol.
Chem. 270: 22378-22385; Komada, M., et al. (1990) Biochem. Biophys.
Res. Commun. 168: 1059-1065; and Cupillard, L. et al. (1997) J.
Biol. Chem. 272:15745-15752).
[0008] The discovery of a new human phospholipase A2 protein and
the polynucleotides encoding it satisfies a need in the art by
providing new compositions which are useful in the diagnosis,
prevention, and treatment of cancer and inflammation.
SUMMARY OF THE INVENTION
[0009] The invention features a substantially purified polypeptide,
human phospholipase A2 protein (PHPLA2), having the amino acid
sequence shown in SEQ ID NO: 1, or fragments thereof.
[0010] The invention further provides an isolated and substantially
purified polynucleotide sequence encoding the polypeptide
comprising the amino acid sequence of SEQ ID NO: 1 or fragments
thereof and a composition comprising said polynucleotide sequence.
The invention also provides a polynucleotide sequence which
hybridizes under stringent conditions to the polynucleotide
sequence encoding the amino acid sequence SEQ ID NO: 1, or
fragments of said polynucleotide sequence.
[0011] The invention further provides a polynucleotide sequence
comprising the complement of the polynucleotide sequence encoding
the amino acid sequence of SEQ ID NO: 1, or fragments or variants
of said polynucleotide sequence.
[0012] The invention also provides an isolated and purified
sequence comprising SEQ ID NO:2 or variants thereof. In addition,
the invention provides a polynucleotide sequence which hybridizes
under stringent conditions to the polynucleotide sequence of SEQ ID
NO:2. The invention also provides a polynucleotide sequence
comprising the complement of SEQ ID NO:2, or fragments or variants
thereof.
[0013] The present invention further provides an expression vector
containing at least a fragment of any of the claimed polynucleotide
sequences. In yet another aspect, the expression vector containing
the polynucleotide sequence is contained within a host cell.
[0014] The invention also provides a method for producing a
polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or a
fragment thereof, the method comprising the steps of: a) culturing
the host cell containing an expression vector containing at least a
fragment of the polynucleotide sequence encoding PHPLA2 under
conditions suitable for the expression of the polypeptide; and b)
recovering the polypeptide from the host cell culture.
[0015] The invention also provides a pharmaceutical composition
comprising a substantially purified PHPLA2 having the amino acid
sequence of SEQ ID NO: 1 in conjunction with a suitable
pharmaceutical carrier.
[0016] The invention also provides a purified antagonist of the
polypeptide of SEQ ID NO: 1. In one aspect the invention provides a
purified antibody which binds to a polypeptide comprising the amino
acid sequence of SEQ ID NO: 1.
[0017] Still further, the invention provides a purified agonist of
the polypeptide of SEQ ID NO: 1.
[0018] The invention also provides a method for treating or
preventing a cancer comprising administering to a subject in need
of such treatment an effective amount of an antagonist to
PHPLA2.
[0019] The invention also provides a method for treating or
preventing inflammation comprising administering to a subject in
need of such treatment an effective amount of an antagonist to
PHPLA2.
[0020] The invention also provides a method for detecting a
polynucleotide which encodes PHPLA2 in a biological sample
comprising the steps of: a) hybridizing the complement of the
polynucleotide sequence which encodes SEQ ID NO: 1 to nucleic acid
of a biological sample, thereby forming a hybridization complex;
and b) detecting the hybridization complex, wherein the presence of
the complex correlates with the presence of a polynucleotide
encoding PHPLA2 in the biological sample. In one aspect the nucleic
acid of the biological sample is amplified by the polymerase chain
reaction prior to hybridization.
BRIEF DESCRIPTION OF THE FIGURES
[0021] FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO:1)
and nucleic acid sequence (SEQ ID NO:2) of PHPLA2. The alignment
was produced using MacDNASIS PRO.TM. software (Hitachi Software
Engineering Co. Ltd. San Bruno, Calif.).
[0022] FIG. 2 shows the amino acid sequence alignments among PHPLA2
(816403; SEQ ID NO:1), mouse PLA2 (GI 984837; SEQ ID NO:3) and rat
PLA2 (GI 204319; SEQ ID NO:4), produced using the multisequence
alignment program of DNASTAR.TM. software (DNASTAR Inc, Madison
Wis.).
DESCRIPTION OF THE INVENTION
[0023] Before the present proteins, nucleotide sequences, and
methods are described, it is understood that this invention is not
limited to the particular methodology, protocols, cell lines,
vectors, and reagents described, as these may vary. It is also to
be understood that the terminology used herein is for the purpose
of describing particular embodiments only, and is not intended to
limit the scope of the present invention which will be limited only
by the appended claims.
[0024] It must be noted that as used herein and in the appended
claims, the singular forms "a", "an", and "the" include plural
reference unless the context clearly dictates otherwise. Thus, for
example, reference to "a host cell" includes a plurality of such
host cells, reference to the "antibody" is a reference to one or
more antibodies and equivalents thereof known to those skilled in
the art, and so forth.
[0025] Unless defined otherwise, all technical and scientific terms
used herein have the same meanings as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
any methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, the preferred methods, devices, and materials are now
described. All publications mentioned herein are incorporated
herein by reference for the purpose of describing and disclosing
the cell lines, vectors, and methodologies which are reported in
the publications which might be used in connection with the
invention. Nothing herein is to be construed as an admission that
the invention is not entitled to antedate such disclosure by virtue
of prior invention.
DEFINITIONS
[0026] PHPLA2, as used herein, refers to the amino acid sequences
of substantially purified PHPLA2 obtained from any species,
particularly mammalian, including bovine, ovine, porcine, murine,
equine, and preferably human, from any source whether natural,
synthetic, semi-synthetic, or recombinant.
[0027] The term "agonist", as used herein, refers to a molecule
which, when bound to PHPLA2, increases or prolongs the duration of
the effect of PHPLA2. Agonists may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to and modulate
the effect of PHPLA2.
[0028] An "allele" or "allelic sequence", as used herein, is an
alternative form of the gene encoding PHPLA2. Alleles may result
from at least one mutation in the nucleic acid sequence and may
result in altered mRNAs or polypeptides whose structure or function
may or may not be altered. Any given natural or recombinant gene
may have none, one, or many allelic forms. Common mutational
changes which give rise to alleles are generally ascribed to
natural deletions, additions, or substitutions of nucleotides. Each
of these types of changes may occur alone, or in combination with
the others, one or more times ill a given sequence.
[0029] "Altered" nucleic acid sequences encoding PHPLA2 as used
herein include those with deletions, insertions, or substitutions
of different nucleotides resulting in a polynucleotide that encodes
the same or a functionally equivalent PHPLA2. Included within this
definition are polymorphisms which may or may not be readily
detectable using a particular oligonucleotide probe of the
polynucleotide encoding PHPLA2, and improper or unexpected
hybridization to alleles, with a locus other than the normal
chromosomal locus for the polynucleotide sequence encoding PHPLA2.
The encoded protein may also be "altered" and contain deletions,
insertions, or substitutions of amino acid residues which produce a
silent change and result in a functionally equivalent PHPLA2.
Deliberate amino acid substitutions may be made on the basis of
similarity in polarity, charge, solubility, hydrophobicity,
hydrophilicity, and/or the amphipathic nature of the residues as
long as the biological or immunological activity of PHPLA2 is
retained. For example, negatively charged amino acids may include
aspartic acid and glutamic acid; positively charged amino acids may
include lysine and arginine; and amino acids with uncharged polar
head groups having similar hydrophilicity values may include
leucine, isoleucine, and valine, glycine and alanine, asparagine
and glutamine, serine and threonine, and phenylalanine and
tyrosine.
[0030] "Amino acid sequence" as used herein refers to an
oligopeptide, peptide, polypeptide, or protein sequence, and
fragment thereof, and to naturally occurring or synthetic
molecules. Fragments of PHPLA2 are preferably about 5 to about 15
amino acids in length and retain the biological activity or the
immunological activity of PHPLA2. Where "amino acid sequence" is
recited herein to refer to an amino acid sequence of a naturally
occurring protein molecule, amino acid sequence, and like terms,
are not meant to limit the amino acid sequence to the complete,
native amino acid sequence associated with the recited protein
molecule.
[0031] "Amplification" as used herein refers to the production of
additional copies of a nucleic acid sequence and is generally
carried out using polymerase chain reaction (PCR) technologies well
known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR
Primer, a Laboratory Manual, Cold Spring Harbor Press, Plainview,
N.Y.).
[0032] The term "antagonist" as used herein, refers to a molecule
which, when bound to PHPLA2, decreases the amount or the duration
of the effect of the biological or immunological activity of
PHPLA2. Antagonists may include proteins, nucleic acids,
carbohydrates, antibodies or any other molecules which decrease the
effect of PHPLA2.
[0033] As used herein, the term "antibody" refers to intact
molecules as well as fragments thereof, such as Fab, F(ab').sub.2,
and Fv, which are capable of binding the epitopic determinant.
Antibodies that bind PHPLA2 polypeptides can be prepared using
intact polypeptides or fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or oligopeptide
used to immunize an animal can be derived from the translation of
RNA or synthesized chemically and can be conjugated to a carrier
protein, if desired. Commonly used carriers that are chemically
coupled to peptides include bovine serum albumin and thyroglobulin,
keyhole limpet hemocyanin. The coupled peptide is then used to
immunize the animal (e.g., a mouse, a rat, or a rabbit).
[0034] The term "antigenic determinant", as used herein, refers to
that fragment of a molecule (i.e., an epitope) that makes contact
with a particular antibody. When a protein or fragment of a protein
is used to immunize a host animal, numerous regions of the protein
may induce the production of antibodies which bind specifically to
a given region or three-dimensional structure on the protein;
[0035] these regions or structures are referred to as antigenic
determinants. An antigenic determinant may compete with the intact
antigen (i.e., the immunogen used to elicit the immune response)
for binding to an antibody.
[0036] The term "antisense", as used herein, refers to any
composition containing nucleotide sequences which are complementary
to a specific DNA or RNA sequence. The term "antisense strand" is
used in reference to a nucleic acid strand that is complementary to
the "sense" strand. Antisense molecules include peptide nucleic
acids and may be produced by any method including synthesis or
transcription. Once introduced into a cell, the complementary
nucleotides combine with natural sequences produced by the cell to
form duplexes and block either transcription or translation. The
designation "negative" is sometimes used in reference to the
antisense strand, and "positive" is sometimes used in reference to
the sense strand.
[0037] The term "biologically active", as used herein, refers to a
protein having structural, regulatory, or biochemical functions of
a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic
PHPLA2, or any oligopeptide thereof, to induce a specific immune
response in appropriate animals or cells and to bind with specific
antibodies.
[0038] The terms "complementary" or "complementarity", as used
herein, refer to the natural binding of polynucleotides under
permissive salt and temperature conditions by base-pairing. For
example, the sequence "A-G-T" binds to the complementary sequence
"T-C-A". Complementarity between two single-stranded molecules may
be "partial", in which only some of the nucleic acids bind, or it
may be complete when total complementarity exists between the
single stranded molecules. The degree of complementarity between
nucleic acid strands has significant effects on the efficiency and
strength of hybridization between nucleic acid strands. This is of
particular portarice in amplification reactions, which depend upon
binding between nucleic acids strands and in the design and use of
PNA molecules.
[0039] A "composition comprising a given polynucleotide sequence"
as used herein refers broadly to any composition containing the
given polynucleotide sequence. The composition may comprise a dry
formulation or an aqueous solution. Compositions comprising
polynucleotide sequences encoding PHPLA2 (SEQ ID NO:1) or fragments
thereof (e.g., SEQ ID NO:2 and fragments thereof) may be employed
as hybridization probes. The probes may be stored in freeze-dried
form and may be associated with a stabilizing agent such as a
carbohydrate. In hybridizations, the probe may be deployed in an
aqueous solution containing salts (e.g., NaCl), detergents (e.g.,
SDS) and other components (e.g., Denhardt's solution, dry milk,
salmon sperm DNA, etc.).
[0040] "Consensus", as used herein, refers to a nucleic acid
sequence which has been resequenced to resolve uncalled bases, has
been extended using XL-PCR.TM. (Perkin Elmer, Norwalk, Conn.) in
the 5' and/or the 3' direction and resequenced, or has been
assembled from the overlapping sequences of more than one Incyte
Clone using a computer program for fragment assembly (e.g.,
GELVIEW.TM. Fragment Assembly system, GCG, Madison, Wis.). Some
sequences have been both extended and assembled to produce the
consensus sequence.
[0041] The term "correlates with expression of a polynucleotide",
as used herein, indicates that the detection of the presence of
ribonucleic acid that is similar to SEQ ID NO:2 by northern
analysis is indicative of the presence of mRNA encoding PHPLA2 in a
sample and thereby correlates with expression of the transcript
from the polynucleotide encoding the protein.
[0042] A "deletion", as used herein, refers to a change in the
amino acid or nucleotide sequence and results in the absence of one
or more amino acid residues or nucleotides.
[0043] The term "derivative", as used herein, refers to the
chemical modification of a nucleic acid encoding or complementary
to PHPLA2 or the encoded PHPLA2. Such modifications include, for
example, replacement of hydrogen by an alkyl, acyl, or amino group.
A nucleic acid derivative encodes a polypeptide which retains the
biological or immunological function of the natural molecule. A
derivative polypeptide is one which is modified by glycosylation,
pegylation, or any similar process which retains the biological or
immunological function of the polypeptide from which it was
derived.
[0044] The term "homology", as used herein, refers to a degree of
complementarity. There may be partial homology or complete homology
(i.e., identity). A partially complementary sequence that at least
partially inhibits an identical sequence from hybridizing to a
target nucleic acid is referred to using the functional term
"substantially homologous." The inhibition of hybridization of the
completely complementary sequence to the target sequence may be
examined using a hybridization assay (Southern or northern blot,
solution hybridization and the like) under conditions of low
stringency. A substantially homologous sequence or hybridization
probe will compete for and inhibit the binding of a completely
homologous sequence to the target sequence under conditions of low
stringency. This is not to say that conditions of low stringency
are such that non-specific binding is permitted; low stringency
conditions require that the binding of two sequences to one another
be a specific (i.e., selective) interaction. The absence of
non-specific binding may be tested by the use of a second target
sequence which lacks even a partial degree of complementarity
(e.g., less than about 30% identity). In the absence of
non-specific binding, the probe will not hybridize to the second
non-complementary target sequence.
[0045] Human artificial chromosomes (HACs) are linear
microchromosomes which may contain DNA sequences of 10K to 10M in
size and contain all of the elements required for stable mitotic
chromosome segregation and maintenance (Harrington, J. J. et al.
(1997) Nat. Genet. 15:345-355).
[0046] The term "humanized antibody", as used herein, refers to
antibody molecules in which amino acids have been replaced in the
non-antigen binding regions in order to more closely resemble a
human antibody, while still retaining the original binding
ability.
[0047] The term "hybridization", as used herein, refers to any
process by which a strand of nucleic acid binds with a
complementary strand through base pairing.
[0048] The term "hybridization complex", as used herein, refers to
a complex formed between two nucleic acid sequences by virtue of
the formation of hydrogen bonds between complementary G and C bases
and between complementary A and T bases; these hydrogen bonds may
be further stabilized by base stacking interactions. The two
complementary nucleic acid sequences hydrogen bond in an
antiparallel configuration. A hybridization complex may be formed
in solution (e.g., Cot or Rot analysis) or between one nucleic acid
sequence present in solution and another nucleic acid sequence
immobilized on a solid support (e.g., paper, membranes, filters,
chips, pins or glass slides, or any other appropriate substrate to
which cells or their nucleic acids have been fixed).
[0049] An "insertion" or "addition", as used herein, refers to a
change in an amino acid or nucleotide sequence resulting in the
addition of one or more amino acid residues or nucleotides,
respectively, as compared to the naturally occurring molecule.
[0050] "Microarray" refers to an array of distinct polynucleotides
or oligonucleotides arraigned or synthesized on a substrate, such
as paper, nylon or other type of membrane, filter, chip, glass
slide, or any other suitable solid support.
[0051] The term "modulate", as used herein, refers to a change in
the activity of PHPLA2. For example, modulation may cause an
increase or a decrease in protein activity, binding
characteristics, or any other biological, functional or
immunological properties of PHPLA2. "Nucleic acid sequence" as used
herein refers to an oligonucleotide, nucleotide, or polynucleotide,
and fragments thereof, and to DNA or RNA of genomic or synthetic
origin which may be single- or double-stranded, and represent the
sense or antisense strand. "Fragments" are those nucleic acid
sequences which are greater than 60 nucleotides than in length, and
most preferably includes fragments that are at least 100
nucleotides or at least 1000 nucleotides, and at least 10,000
nucleotides in length.
[0052] The term "oligonucleotide" refers to a nucleic acid sequence
of at least about 6 nucleotides to about 60 nucleotides, preferably
about 15 to 30 nucleotides, and more preferably about 20 to 25
nucleotides, which can be used in PCR amplification or a
hybridization assay, or a microarray. As used herein,
oligonucleotide is substantially equivalent to the terms
"amplimers","primers", "oligomers", and "probes", as commonly
defined in the art.
[0053] "Peptide nucleic acid", PNA as used herein, refers to an
antisense molecule or anti-gene agent which comprises an
oligonucleotide of at least five nucleotides in length linked to a
peptide backbone of amino acid residues which ends in lysine. The
terminal lysine confers solubility to the composition. PNAs may be
pegylated to extend their lifespan in the cell where they
preferentially bind complementary single stranded DNA and RNA and
stop transcript elongation (Nielsen, P. E. et al. (1993) Anticancer
Drug Des. 8:53-63).
[0054] The term "portion", as used herein, with regard to a protein
(as in "a portion of a given protein") refers to fragments of that
protein. The fragments may range in size from five amino acid
residues to the entire amino acid sequence minus one amino acid.
Thus, a protein "comprising at least a portion of the amino acid
sequence of SEQ ID NO:1" encompasses the full-length PHPLA2 and
fragments thereof.
[0055] The term "sample", as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acid
encoding PHPLA2, or fragments thereof, or PHPLA2 itself may
comprise a bodily fluid, extract from a cell, chromosome,
organelle, or membrane isolated from a cell, a cell, genomic DNA,
RNA, or cDNA in solution or bound to a solid support, a tissue, a
tissue print, and the like.
[0056] The terms "specific binding" or "specifically binding", as
used herein, refers to that interaction between a protein or
peptide and an agonist, an antibody and an antagonist. The
interaction is dependent upon the presence of a particular
structure (i.e., the antigenic determinant or epitope) of the
protein recognized by the binding molecule. For example, if an
antibody is specific for epitope "A", the presence of a protein
containing epitope A (or free, unlabeled A) in a reaction
containing labeled "A" and the antibody will reduce the amount of
labeled A bound to the antibody.
[0057] The terms "stringent conditions" or "stringency", as used
herein, refer to the conditions for hybridization as defined by the
nucleic acid, salt, and temperature. These conditions are well
known in the art and may be altered in order to identify or detect
identical or related polynucleotide sequences. Numerous equivalent
conditions comprising either low or high stringency depend on
factors such as the length and nature of the sequence (DNA, RNA,
base composition), nature of the target (DNA, RNA, base
composition), milieu (in solution or immobilized on a solid
substrate), concentration of salts and other components (e.g.,
formamide, dextran sulfate and/or polyethylene glycol), and
temperature of the reactions (within a range from about 5.degree.
C. below the melting temperature of the probe to about 20.degree.
C. to 25.degree. C. below the melting temperature). One or more
factors be may be varied to generate conditions of either low or
high stringency different from, but equivalent to, the above listed
conditions.
[0058] The term "substantially purified", as used herein, refers to
nucleic or amino acid sequences that are removed from their natural
environment, isolated or separated, and are at least 60% free,
preferably 75% free, and most preferably 90% free from other
components with which they are naturally associated.
[0059] A "substitution", as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0060] "Transformation", as defined herein, describes a process by
which exogenous DNA enters and changes a recipient cell. It may
occur under natural or artificial conditions using various methods
well known in the art. Transformation may rely on any known method
for the insertion of foreign nucleic acid sequences into a
prokaryotic or eukaryotic host cell. The method is selected based
on the type of host cell being transformed and may include, but is
not limited to, viral infection, electroporation, heat shock,
lipofection, and particle bombardment. Such "transformed" cells
include stably transformed cells in which the inserted DNA is
capable of replication either as an autonomously replicating
plasmid or as part of the host chromosome. They also include cells
which transiently express the inserted DNA or RNA for limited
periods of time.
[0061] A "variant" of PHPLA2, as used herein, refers to an amino
acid sequence that is altered by one or more amino acids. The
variant may have "conservative" changes, wherein a substituted
amino acid has similar structural or chemical properties, e.g.,
replacement of leucine with isoleucine. More rarely, a variant may
have "nonconservative" changes, e.g., replacement of a glycine with
a tryptophan. Analogous minor variations may also include amino
acid deletions or insertions, or both. Guidance in determining
which amino acid residues may be substituted, inserted, or deleted
without abolishing biological or immunological activity may be
found using computer programs well known in the art, for example,
DNASTAR software.
THE INVENTION
[0062] The invention is based on the discovery of a new human
phospholipase A2 protein (hereinafter referred to as "PHPLA2"), the
polynucleotides encoding PHPLA2, and the use of these compositions
for the diagnosis, prevention, or treatment of cancer and
inflammation.
[0063] Nucleic acids encoding the PHPLA2 of the present invention
were first identified in Incyte Clone 816403 from the ovarian tumor
cDNA library (OVARTUT01) using a computer search for amino acid
sequence alignments. SEQ ID NO:2 was derived from the extension of
Incyte Clone 816403 (OVARTUT01).
[0064] In one embodiment, the invention encompasses a polypeptide
comprising the amino acid sequence of SEQ ID NO:1, as shown in
FIGS. 1A and 1B. PHPLA2 is 165 amino acids in length and has a
phospholipase A2 active site signature sequence from residue
L.sub.129 to residue C.sub.139. PHPLA2 also has a potential
N-glycosylation site at residue N.sub.113, a potential casein
kinase II phosphorylation site at T.sub.93 and three potential
protein kinase C phosphorylation sites at residues S.sub.101,
T.sub.104, and S.sub.161. As shown in FIG. 2, PHPLA2 has chemical
and structural homology with mouse PHPLA2 (GI 984837; SEQ ID NO:3)
and rat PLA2 (GI 204319; SEQ ID NO:4). In particular, PHPLA2 and
mouse PLA2 share 36% identity, and PHPLA2 and rat PLA2 share 35%
identity. Northern analysis shows the expression of this sequence
in various libraries, at least 43% of which are immortalized or
cancerous and at least 43% of which involve an inflammatory
response.
[0065] The invention also encompasses PHPLA2 variants. A preferred
PHPLA2 variant is one having at least 80%, and more preferably at
least 90%, amino acid sequence identity to the PHPLA2 amino acid
sequence (SEQ ID NO:1) and which retains at least one biological,
immunological or other functional characteristic or activity of
PHPLA2. A most preferred PHPLA2 variant is one having at least 95%
amino acid sequence identity to SEQ ID NO:1.
[0066] The invention also encompasses polynucleotides which encode
PHPLA2. Accordingly, any nucleic acid sequence which encodes the
amino acid sequence of PHPLA2 can be used to produce recombinant
molecules which express PHPLA2. In a particular embodiment, the
invention encompasses the polynucleotide comprising the nucleic
acid sequence of SEQ ID NO:2 as shown in FIGS. 1A and 1B.
[0067] It will be appreciated by those skilled in the art that as a
result of the degeneracy of the genetic code, a multitude of
nucleotide sequences encoding PHPLA2, some bearing minimal homology
to the nucleotide sequences of any known and naturally occurring
gene, may be produced. Thus, the invention contemplates each and
every possible variation of nucleotide sequence that could be made
by selecting combinations based on possible codon choices. These
combinations are made in accordance with the standard triplet
genetic code as applied to the nucleotide sequence of naturally
occurring PHPLA2, and all such variations are to be considered as
being specifically disclosed.
[0068] Although nucleotide sequences which encode PHPLA2 and its
variants are preferably capable of hybridizing to the nucleotide
sequence of the naturally occurring PHPLA2 under appropriately
selected conditions of stringency, it may be advantageous to
produce nucleotide sequences encoding PHPLA2 or its derivatives
possessing a substantially different codon usage. Codons may be
selected to increase the rate at which expression of the peptide
occurs in a particular prokaryotic or eukaryotic host in accordance
with the frequency with which particular codons are utilized by the
host. Other reasons for substantially altering the nucleotide
sequence encoding PHPLA2 and its derivatives without altering the
encoded amino acid sequences include the production of RNA
transcripts having more desirable properties, such as a greater
half-life, than transcripts produced from the naturally occurring
sequence.
[0069] The invention also encompasses production of DNA sequences,
or fragments thereof, which encode PHPLA2 and its derivatives,
entirely by synthetic chemistry. After production, the synthetic
sequence may be inserted into any of the many available expression
vectors and cell systems using reagents that are well known in the
art. Moreover, synthetic chemistry may be used to introduce
mutations into a sequence encoding PHPLA2 or any fragment
thereof.
[0070] Also encompassed by the invention are polynucleotide
sequences that are capable of hybridizing to the claimed nucleotide
sequences, and in particular, those shown in SEQ ID NO:2, under
various conditions of stringency as taught in Wahl, G. M. and S. L.
Berger (1987; Methods Enzymol. 152:399-407) and Kimmel, A. R.
(1987; Methods Enzymol. 152:507-511).
[0071] Methods for DNA sequencing which are well known and
generally available in the art and may be used to practice any of
the embodiments of the invention. The methods may employ such
enzymes as the Klenow fragment of DNA polymerase I, Sequenase.RTM.
(US Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin
Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of polymerases and proofreading exonucleases such as
those found in the ELONGASE Amplification System marketed by
Gibco/BRL (Gaithersburg, Md.). Preferably, the process is automated
with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno,
Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown,
Mass.) and the ABI Catalyst and 373 and 377 DNA Sequencers (Perkin
Elmer).
[0072] The nucleic acid sequences encoding PHPLA2 may be extended
utilizing a partial nucleotide sequence and employing various
methods known in the art to detect upstream sequences such as
promoters and regulatory elements. For example, one method which
may be employed, "restriction-site" PCR, uses universal primers to
retrieve unknown sequence adjacent to a known locus (Sarkar, G.
(1993) PCR Methods Applic. 2:318-322). In particular, genomic DNA
is first amplified in the presence of primer to a linker sequence
and a primer specific to the known region. The amplified sequences
are then subjected to a second round of PCR with the same linker
primer and another specific primer internal to the first one.
Products of each round of PCR are transcribed with an appropriate
RNA polymerase and sequenced using reverse transcriptase.
[0073] Inverse PCR may also be used to amplify or extend sequences
using divergent primers based on a known region (Triglia, T. et al.
(1988) Nucleic Acids Res. 16:8186). The primers may be designed
using commercially available software such as OLIGO 4.06 Primer
Analysis software (National Biosciences Inc., Plymouth, Minn.), or
another appropriate program, to be 22-30 nucleotides in length, to
have a GC content of 50% or more, and to anneal to the target
sequence at temperatures about 68.degree.-72.degree. C. The method
uses several restriction enzymes to generate a suitable fragment in
the known region of a gene. The fragment is then circularized by
intramolecular ligation and used as a PCR template.
[0074] Another method which may be used is capture PCR which
involves PCR amplification of DNA fragments adjacent to a known
sequence in human and yeast artificial chromosome DNA (Lagerstrom,
M. et al. (1991) PCR Methods Applic. 1:111-119). In this method,
multiple restriction enzyme digestions and ligations may also be
used to place an engineered double-stranded sequence into an
unknown fragment of the DNA molecule before performing PCR.
[0075] Another method which may be used to retrieve unknown
sequences is that of Parker, J. D. et al. (1991; Nucleic Acids Res.
19:3055-3060). Additionally, one may use PCR, nested primers, and
PromoterFinder.TM. libraries to walk genomic DNA (Clontech, Palo
Alto, Calif.). This process avoids the need to screen libraries and
is useful in finding intron/exon junction screening for full-length
cDNAs, it is preferable to use libraries that have been
size-selected to include larger cDNAs. Also, random-primed
libraries are preferable, in that they will contain more sequences
which contain the 5' regions of genes. Use of a randomly primed
library may be especially preferable for situations in which an
oligo d(T) library does not yield a full-length cDNA. Genomic
libraries may be useful for extension of sequence into 5'
non-transcribed regulatory regions.
[0076] Capillary electrophoresis systems which are commercially
available may be used to analyze the size or confirm the nucleotide
sequence of sequencing or PCR products. In particular, capillary
sequencing may employ flowable polymers for electrophoretic
separation, four different fluorescent dyes (one for each
nucleotide) which are laser activated, and detection of the emitted
wavelengths by a charge coupled devise camera. Output/light
intensity may be converted to electrical signal using appropriate
software (e.g. Genotyper.TM. and Sequence Navigator.TM., Perkin
Elmer) and the entire process from loading of samples to computer
analysis and electronic data display may be computer controlled.
Capillary electrophoresis is especially preferable for the
sequencing of small pieces of DNA which might be present in limited
amounts in a particular sample.
[0077] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode PBPLA2 may be used in
recombinant DNA molecules to direct expression of PHPLA2, fragments
or functional equivalents thereof, in appropriate host cells. Due
to the inherent degeneracy of the genetic code, other DNA sequences
which encode substantially the same or a functionally equivalent
amino acid sequence may be produced, and these sequences may be
used to clone and express PHPLA2.
[0078] As will be understood by those of skill in the art, it may
be advantageous to produce PHPLA2-encoding nucleotide sequences
possessing non-naturally occurring codons. For example, codons
preferred by a particular prokaryotic or eukaryotic host can be
selected to increase the rate of protein expression or to produce
an RNA transcript having desirable properties, such as a half-life
which is longer than that of a transcript generated from the
naturally occurring sequence.
[0079] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter PHPLA2 encoding sequences for a variety of reasons, including
but not limited to, alterations which modify the cloning,
processing, and/or expression of the gene product. DNA shuffling by
random fragmentation and PCR reassembly of gene fragments and
synthetic oligonucleotides may be used to engineer the nucleotide
sequences. For example, site-directed mutagenesis may be used to
insert new restriction sites, alter glycosylation patterns, change
codon preference, produce splice variants, introduce mutations, and
so forth.
[0080] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding PHPLA2 may be
ligated to a heterologous sequence to encode a fusion protein. For
example, to screen peptide libraries for inhibitors of PHPLA2
activity, it may be useful to encode a chimeric PHPLA2 protein that
can be recognized by a commercially available antibody. A fusion
protein may also be engineered to contain a cleavage site located
between the PHPLA2 encoding sequence and the heterologous protein
sequence, so that PHPLA2 may be cleaved and purified away from the
heterologous moiety.
[0081] In another embodiment, sequences encoding PHPLA2 may be
synthesized, in whole or in part, using chemical methods well known
in the art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res.
Symp. Ser. 215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp.
Ser. 225-232). Alternatively, the protein itself may be produced
using chemical methods to synthesize the amino acid sequence of
PHPLA2, or a fragment thereof. For example, peptide synthesis can
be performed using various solid-phase techniques (Roberge, J. Y.
et al. (1995) Science 269:202-204) and automated synthesis may be
achieved, for example, using the ABI 431A Peptide Synthesizer
(Perkin Elmer).
[0082] The newly synthesized peptide may be substantially purified
by preparative high performance liquid chromatography (e.g.,
Creighton, T. (1983) Proteins, Structures and Molecular Principles,
WH Freeman and Co., New York, N.Y.). The composition of the
synthetic peptides may be confirmed by amino acid analysis or
sequencing (e.g., the Edman degradation procedure;
[0083] Creighton, supra). Additionally, the amino acid sequence of
PHPLA2, or any part thereof, may be altered during direct synthesis
and/or combined using chemical methods with sequences from other
proteins, or any part thereof, to produce a variant
polypeptide.
[0084] In order to express a biologically active PHPLA2, the
nucleotide sequences encoding PHPLA2 or functional equivalents, may
be inserted into appropriate expression vector, i.e., a vector
which contains the necessary elements for the transcription and
translation of the inserted coding sequence.
[0085] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding PHPLA2 and appropriate transcriptional and translational
control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. Such techniques are described in Sambrook, J. et al.
(1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor
Press, Plainview, N.Y., and Ausubel, F. M. et al. (1989) Current
Protocols in Molecular Biology, John Wiley & Sons, New York,
N.Y.
[0086] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding PHPLA2. These include,
but are not limited to, microorganisms such as bacteria transformed
with recombinant bacteriophage, plasmid, or cosmid DNA expression
vectors; yeast transformed with yeast expression vectors; insect
cell systems infected with virus expression vectors (e.g.
baculovirus); plant cell systems transformed with virus expression
vectors cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV)
or with bacterial expression vectors (e.g., Ti or pBR322 plasmids);
or animal cell systems. The invention is not limited by the host
cell employed.
[0087] The "control elements" or "regulatory sequences" are those
non-translated regions of the vector--enhancers, promoters, 5' and
3' untranslated regions--which interact with host cellular proteins
to carry out transcription and translation. Such elements may vary
in their strength and specificity. Depending on the vector system
and host utilized, any number of suitable transcription and
translation elements, including constitutive and inducible
promoters, may be used. For example, when cloning in bacterial
systems, inducible promoters such as the hybrid lacZ promoter of
the Bluescript.RTM. phagemid (Stratagene, La Jolla, Calif.) or
PSPORT1 plasmid (Gibco BRL) and the like may be used. The
baculovirus polyhedrin promoter may be used in insect cells.
Promoters or enhancers derived from the genomes of plant cells
(e.g., heat shock, RUBISCO; and storage protein genes) or from
plant viruses (e.g., viral promoters or leader sequences) may be
cloned into the vector. In mammalian cell systems, promoters from
mammalian genes or from mammalian viruses are preferable. If it is
necessary to generate a cell line that contains multiple copies of
the sequence encoding PHPLA2, vectors based on SV40 or EBV may be
used with an appropriate selectable marker.
[0088] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for PHPLA2. For example,
when large quantities of PHPLA2 are needed for the induction of
antibodies, vectors which direct high level expression of fusion
proteins that are readily purified may be used. Such vectors
include, but are not limited to, the multifunctional E. coli
cloning and expression vectors such as Bluescript.RTM.
(Stratagene), in which the sequence encoding PHPLA2 may be ligated
into the vector in frame with sequences for the amino-terminal Met
and the subsequent 7 residues of .beta.-galactosidase so that a
hybrid protein is produced; pIN vectors (Van Heeke, G. and S. M.
Schuster (1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX
vectors (Promega, Madison, Wis.) may also be used to express
foreign polypeptides as fusion proteins with glutathione
S-transferase (GST). In general, such fusion proteins are soluble
and can easily be purified from lysed cells by adsorption to
glutathione-agarose beads followed by elution in the presence of
free glutathione. Proteins made in such systems may be designed to
include heparin, thrombin, or factor XA protease cleavage sites so
that the cloned polypeptide of interest can be released from the
GST moiety at will.
[0089] In the yeast, Saccharomyces cerevisiae, a number of vectors
containing constitutive or inducible promoters such as alpha
factor, alcohol oxidase, and PGH may be used. For reviews, see
Ausubel et al. (supra) and Grant et al. (1987) Methods Enzymol.
153:516-544.
[0090] In cases where plant expression vectors are used, the
expression of sequences encoding PHPLA2 may be driven by any of a
number of promoters. For example, viral promoters such as the 35S
and 19S promoters of CaMV may be used alone or in combination with
the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J.
6:307-311). Alternatively, plant promoters such as the small
subunit of RUBISCO or heat shock promoters may be used (Coruzzi, G.
et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984)
Science 224:838-843; and Winter, J. et al. (1991) Results Probl.
Cell Differ. 17:85-105). These constructs can be introduced into
plant cells by direct DNA transformation or pathogen-mediated
transfection. Such techniques are described in a number of
generally available reviews (see, for example, Hobbs, S. or Murry,
L. E. in McGraw Hill Yearbook of Science and Technology (1992)
McGraw Hill, New York, N.Y.; pp. 191-196.
[0091] An insect system may also be used to express PHPLA2. For
example, in one such system, Autographa californica nuclear
polyhedrosis virus (AcNPV) is used as a vector to express foreign
genes in Spodoptera frugiperda cells or in Trichoplusia larvae. The
sequences encoding PHPLA2 may be cloned into a non-essential region
of the virus, such as the polyhedrin gene, and placed under control
of the polyhedrin promoter. Successful insertion of PHPLA2 will
render the polyhedrin gene inactive and produce recombinant virus
lacking coat protein. The recombinant viruses may then be used to
infect, for example, S. frugiperda cells or Trichoplusia larvae in
which PHPLA2 may be expressed (Engelhard, E. K. et al. (1994) Proc.
Nat. Acad. Sci. 91:3224-3227).
[0092] In mammalian host cells, a number of viral-based expression
systems may be utilized. In cases where an adenovirus is used as an
expression vector, sequences encoding PHPLA2 may be ligated into an
adenovirus transcription/translation complex consisting of the late
promoter and tripartite leader sequence. Insertion in a
non-essential E1 or E3 region of the viral-genome may be used to
obtain a viable virus which is capable of expressing PHPLA2 in
infected host cells (Logan, J. and Shenk, T. (1984) Proc. Natl.
Acad. Sci. 81:3655-3659). In addition, transcription enhancers,
such as the Rous sarcoma virus (RSV) enhancer, may be used to
increase expression in mammalian host cells.
[0093] Human artificial chromosomes (HACs) may also be employed to
deliver larger fragments of DNA than can be contained and expressed
in a plasmid. HACs of 6 to 10M are constructed and delivered via
conventional delivery methods (liposomes, polycationic amino
polymers, or vesicles) for therapeutic purposes.
[0094] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding PHPLA2. Such signals
include the ATG initiation codon and adjacent sequences. In cases
where sequences encoding PHPLA2, its initiation codon, and upstream
sequences are inserted into the appropriate expression vector, no
additional transcriptional or translational control signals may be
needed. However, in cases where only coding sequence, or a fragment
thereof, is inserted, exogenous translational control signals
including the ATG initiation codon should be provided. Furthermore,
the initiation codon should be in the correct reading frame to
ensure translation of the entire insert. Exogenous translational
elements and initiation codons may be of various origins, both
natural and synthetic. The efficiency of expression may be enhanced
by the inclusion of enhancers which are appropriate for the
particular cell system which is used, such as those described in
the literature (Scharf, D. et al. (1994) Results Probl. Cell
Differ. 20:125-162).
[0095] In addition, a host cell strain may be chosen for its
ability to modulate the expression of the inserted sequences or to
process the expressed protein in the desired fashion. Such
modifications of the polypeptide include, but are not limited to,
acetylation, carboxylation, glycosylation, phosphorylation,
lipidation, and acylation. Post-translational processing which
cleaves a "prepro" form of the protein may also be used to
facilitate correct insertion, folding and/or function. Different
host cells which have specific cellular machinery and
characteristic mechanisms for post-translational activities (e.g.,
CHO, HeLa, MDCK, HEK293, and W138), are available from the American
Type Culture Collection (ATCC; Bethesda, Md.) and may be chosen to
ensure the correct modification and processing of the foreign
protein.
[0096] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express PHPLA2 may be transformed using expression
vectors which may contain viral origins of replication and/or
endogenous expression elements and a selectable marker gene on the
same or on a separate vector. Following the Introduction of the
vector, cells may be allowed to grow for 1-2 days in an enriched
media before they are switched to selective media. The purpose of
the selectable marker is to confer resistance to selection, and its
presence allows growth and recovery of cells which successfully
express the introduced sequences. Resistant clones of stably
transformed cells may be proliferated using tissue culture
techniques appropriate to the cell type.
[0097] Any number of selection systems may be used to recover
transformed cell lines. These include, but are not limited to, the
herpes simplex virus thymidine kinase (Wigler, M. et al (1977) Cell
11:223-32) and adenine phosphoribosyltransferase (Lowy, I. et al.
(1980) Cell 22:817-23) genes which can be employed in tk.sup.- or
aprt.sup.- cells, respectively. Also, antimetabolite, antibiotic or
herbicide resistance can be used as the basis for selection; for
example, dhfr which confers resistance to methotrexate (Wigler, M.
et al. (1980) Proc. Natl. Acad. Sci. 77:3567-70); npt, which
confers resistance to the aminoglycosides neomycin and G-418
(Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14) and als
or pat, which confer resistance to chlorsulfuron and
phosphinotricin acetyltransferase, respectively (Murry, supra).
Additional selectable genes have been described, for example, trpB,
which allows cells to utilize indole in place of tryptophan, or
hisD, which allows cells to utilize histinol in place of histidine
(Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.
85:8047-51). Recently, the use of visible markers has gained
popularity with such markers as anthocyanins, .beta. glucuronidase
and its substrate GUS, and luciferase and its substrate luciferin,
being widely used not only to identify transformants, but also to
quantify the amount of transient or stable protein expression
attributable to a specific vector system (Rhodes, C. A. et al.
(1995) Methods Mol. Biol. 55:121-131).
[0098] Although the presence/absence of marker gene expression
suggests that the gene of interest is also present, its presence
and expression may need to be confirmed. For example, if the
sequence encoding PHPLA2 is inserted within a marker gene sequence,
transformed cells containing sequences encoding PHPLA2 can be
identified by the absence of marker gene function. Alternatively, a
marker gene can be placed in tandem with a sequence encoding PHPLA2
under the control of a single promoter. Expression of the marker
gene in response to induction or selection usually indicates
expression of the tandem gene as well.
[0099] Alternatively, host cells which contain the nucleic acid
sequence encoding PHPLA2 and express PHPLA2 may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridizations and protein bioassay or immunoassay techniques which
include membrane, solution, to chip based technologies for the
detection and/or quantification of nucleic acid or protein.
[0100] The presence of polynucleotide sequences encoding PHPLA2 can
be detected by DNA-DNA or DNA-RNA hybridization or amplification
using probes or fragments or fragments of polynucleotides encoding
PHPLA2. Nucleic acid amplification based assays involve the use of
oligonucleotides or oligomers based on the sequences encoding
PHPLA2 to detect transformants containing DNA or RNA encoding
PHPLA2.
[0101] A variety of protocols for detecting and measuring the
expression of PHPLA2, using either polyclonal or monoclonal
antibodies specific for the protein are known in the art. Examples
include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay
(RIA), and fluorescence activated cell sorting (FACS). A two-site,
monoclonal-based immunoassay utilizing monoclonal antibodies
reactive to two non-interfering epitopes on PHPLA2 is preferred,
but a competitive binding assay may be employed. These and other
assays are described, among other places, in Hampton, R. et al.
(1990; Serological Methods, a Laboratory Manual, APS Press, St
Paul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.
158:1211-1216).
[0102] A wide variety of labels and conjugation techniques are
known by those skilled in the art and may be used in various
nucleic acid and amino acid assays. Means for producing labeled
hybridization or PCR probes for detecting sequences related to
polynucleotides encoding PHPLA2 include oligolabeling, nick
translation, end-labeling or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding PHPLA2, or any
fragments thereof may be cloned into a vector for the production of
an mRNA probe. Such vectors are known in the art, are commercially
available, and may be used to synthesize RNA probes in vitro by
addition of an appropriate RNA polymerase such as T7, T3, or SP6
and labeled nucleotides. These procedures may be conducted using a
variety of commercially available kits (Pharmacia & Upjohn,
(Kalamazoo, Mich.); Promega (Madison Wis.); and U.S. Biochemical
Corp., Cleveland, Ohio). Suitable reporter molecules or labels,
which may be used for ease of detection, include radionuclides,
enzymes, fluorescent, chemiluminescent, or chromogenic agents as
well as substrates, cofactors, inhibitors, magnetic particles, and
the like.
[0103] Host cells transformed with nucleotide sequences encoding
PHPLA2 may be cultured under conditions suitable for the expression
and recovery of the protein from cell culture. The protein produced
by a transformed cell may be secreted or contained intracellularly
depending on the sequence and/or the vector used. As will be
understood by those of skill in the art, expression vectors
containing polynucleotides which encode PHPLA2 may be designed to
contain signal sequences which direct secretion of PHPLA2 through a
prokaryotic or eukaryotic cell membrane. Other constructions may be
used to join sequences encoding PHPLA2 to nucleotide sequence
encoding a polypeptide domain which will facilitate purification of
soluble proteins. Such purification facilitating domains include,
but are not limited to, metal chelating peptides such as
histidine-tryptophan modules that allow purification on immobilized
metals, protein A domains that allow purification on immobilized
immunoglobulin, and the domain utilized in the FLAGS
extension/affinity purification system (Immunex Corp., Seattle,
Wash.). The inclusion of cleavable linker sequences such as those
specific for Factor XA or enterokinase (Invitrogen, San Diego,
Calif.) between the purification domain and PHPLA2 may be used to
facilitate purification. One such expression vector provides for
expression of a fusion protein containing PHPLA2 and a nucleic acid
encoding 6 histidine residues preceding a thioredoxin or an
enterokinase cleavage site. The histidine residues facilitate
purification on IMAC (immobilized metal ion affinity chromatography
as described in Porath, J. et al. (1992, Prot. Exp. Purif. 3:
263-281) while the enterokinase cleavage site provides a means for
purifying PHPLA2 from the fusion protein. A discussion of vectors
which contain fusion proteins is provided in Kroll, D. J. et al.
(1993; DNA Cell Biol. 12:441-453).
[0104] In addition to recombinant production, fragments of PHPLA2
may be produced by direct peptide synthesis using solid-phase
techniques Merrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154).
Protein synthesis may be performed using manual techniques or by
automation. Automated synthesis may be achieved, for example, using
Applied Biosystems 431A Peptide Synthesizer Perkin Elmer). Various
fragments of PHPLA2 may be chemically synthesized separately and
combined using chemical methods to produce the full length
molecule.
THERAPEUTICS
[0105] Chemical and structural homology exists among PHPLA2
(816403; SEQ ID NO:1). mouse PLA2 (GI 984837; SEQ ID NO:3), and rat
PLA2 (GI 204319; SEQ ID NO:4). In addition, PHPLA2 contains a
phospholipase A2 active site signature sequence and is expressed in
cancerous and inflamed tissues. Therefore, PHPLA2 appears to play a
role in cancer and inflammation.
[0106] In one embodiment, an antagonist of PHPLA2 may be
administered to a subject to prevent or treat a cancer. Such
cancers may include, but are not limited to, adenocarcinoma,
leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma
and particularly cancers of the adrenal gland, bladder, bone, bone
marrow, brain, breast, cervix, gall bladder, ganglia,
gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary,
pancreas, parathyroid, penis, prostate, salivary glands, skin,
spleen, testis, thymus, thyroid, and uterus. In one aspect, an
antibody which specifically binds PHPLA2 may be used directly as an
antagonist or indirectly as a targeting or delivery mechanism for
bringing a pharmaceutical agent to cells or tissue which express
PHPLA2.
[0107] In another embodiment, a vector expressing the complement of
the polynucleotide encoding PHPLA2 may be administered to a subject
to treat or prevent the disorders including, but not limited to,
those cancers described above.
[0108] In another embodiment, an antagonist of PHPLA2 may be
administered to a subject to prevent or treat an inflammation. In
particular, inflammation may be associated with, but not limited
to, disorders such as AIDS, Addison's disease, adult respiratory
distress syndrome, allergies, anemia, asthma, atherosclerosis,
bronchitis, cholecystitis, Crohn's disease, ulcerative colitis,
atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema,
epidermal hyperplasia, erythema nodosum, atrophic gastritis,
glomerulonephritis, gout, Graves' disease, hypereosinophilia,
irritable bowel syndrome, lupus erythematosus, multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis, osteoporosis, pancreatitis, polymyostis, rheumatoid
arthritis, scleroderma, Sjogren's syndrome, toxic shock syndrome,
and autoimmune thyroiditis; complications of cancer, hemodialysis,
and extracorporeal circulation; viral, bacterial, fungal,
parasitic, protozoal, and helminthic infections; and trauma. In one
aspect, an antibody which specifically binds PHPLA2 may be used
directly as an antagonist or indirectly as a targeting or delivery
mechanism for bringing a pharmaceutical agent to cells or tissue
which express PHPLA.
[0109] In another embodiment, a vector expressing the complement of
the polynucleotide encoding PHPLA2 may be administered to a subject
to treat or prevent inflammation, and in particular, the disorders
provided above with which inflammation is associated.
[0110] In other embodiments, any of the proteins, antagonists,
antibodies, agonists, complementary sequences or vectors of the
invention may be administered in combination with other appropriate
therapeutic agents. Selection of the appropriate agents for use in
combination therapy may be made by one of ordinary skill in the
art, according to conventional pharmaceutical principles. The
combination of therapeutic agents may act synergistically to effect
the treatment or prevention of the various disorders described
above. Using this approach, one may be able to achieve therapeutic
efficacy with lower dosages of each agent, thus reducing the
potential for adverse side effects.
[0111] An antagonist of PHPLA2 may be produced using methods which
are generally known in the art. In particular, purified PHPLA2 may
be used to produce antibodies or to screen libraries of
pharmaceutical agents to identify those which specifically bind
PHPLA2.
[0112] Antibodies to PHPLA2 may be generated using methods that are
well known in the art. Such antibodies may include, but are not
limited to, polyclonal, monoclonal, chimeric, single chain, Fab
fragments, and fragments produced by a Fab expression library.
Neutralizing antibodies, (i.e., those which inhibit dimer
formation) are especially preferred for therapeutic use.
[0113] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others, may be immunized by
injection with PHPLA2 or any fragment or oligopeptide thereof which
has immunogenic properties. Depending on the host species, various
adjuvants may be used to increase immunological response. Such
adjuvants include, but are not limited to, Freund's, mineral gels
such as aluminum hydroxide, and surface active substances such as
lysolecithin, pluronic polyols, polyanions, peptides, oil
emulsions, keyhole limpet hemocyanin, and dinitrophenol. Among
adjuvants used in humans, BCG (bacilli Calmette-Guerin) and
Corynebacterium parvum are especially preferable.
[0114] It is preferred that the oligopeptides, peptides, or
fragments used to induce antibodies to PHPLA2 have an amino acid
sequence consisting of at least five amino acids and more
preferably at least 10 amino acids. It is also preferable that they
are identical to a portion of the amino acid sequence of the
natural protein, and they may contain the entire amino acid
sequence of a small, naturally occurring molecule. Short stretches
of PHPLA2 amino acids nay be fused with those of another protein
such as keyhole limpet hemocyanin and antibody produced against the
chimeric molecule.
[0115] Monoclonal antibodies to PHPLA2 may be prepared using any
technique which provides for the production of antibody molecules
by continuous cell lines in culture. These include, but are not
limited to, the hybridoma technique, the human B-cell hybridoma
technique, and the EBV-hybridoma technique (Kohler, G. et al.
(1975) Nature 256:495-497; Kozbor, D. et al. (1985) J. Immunol.
Methods 81:31-42; Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci.
80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.
62:109-120).
[0116] In addition, techniques developed for the production of
"chimeric antibodies", the splicing of mouse antibody genes to
human antibody genes to obtain a molecule with appropriate antigen
specificity and biological activity can be used (Morrison, S. L. et
al. (1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et
al. (1984) Nature 312:604-608; Takeda, S. et al. (1985) Nature
314:452-454). Alternatively, techniques described for the
production of single chain antibodies may be adapted, using methods
known in the art, to produce PHPLA2-specific single chain
antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be generated by chain shuffling from
random combinatorial immunoglobulin libraries (Kang, A. S. et al.
(1991) Proc. Natl. Acad. Sci. 88:11120-3).
[0117] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening
immunoglobulin libraries or panels of highly specific binding
reagents as disclosed in the literature (Orlandi, R. et al. (1989)
Proc. Natl. Acad. Sci. 86: 3833-3837; Winter, G. et al. (1991)
Nature 349:293-299).
[0118] Antibody fragments which contain specific binding sites for
PHPLA2 may also be generated. For example, such fragments include,
but are not limited to, the F(ab')2 fragments which can be produced
by pepsin digestion of the antibody molecule and the Fab fragments
which can be generated by reducing the disulfide bridges of the
F(ab)2 fragments. Alternatively, Fab expression libraries may be
constructed to allow rapid and easy identification of monoclonal
Fab fragments with the desired specificity (Huse, W. D. et al.
(1989) Science 254:1275-1281).
[0119] Various immunoassays may be used for screening to identify
antibodies having the desired specificity. Numerous protocols for
competitive binding or immunoradiometric assays using either
polyclonal or monoclonal antibodies with established specificities
are well known in the art. Such immunoassays typically involve the
measurement of complex formation between PHPLA2 and its specific
antibody. A two-site, monoclonal-based immunoassay utilizing
monoclonal antibodies reactive to two non-interfering PHPLA2
epitopes is preferred, but a competitive binding assay may also be
employed (Maddox, supra).
[0120] In another embodiment of the invention, the polynucleotides
encoding PHPLA2, or any fragment or complement thereof, may be used
for therapeutic purposes. In one aspect, the complement of the
polynucleotide encoding PHPLA2 may be used in situations in which
it would be desirable to block the transcription of the mRNA. In
particular, cells may be transformed with sequences complementary
to polynucleotides encoding PHPLA2. Thus, complementary molecules
or fragments may be used to modulate PHPLA2 activity, or to achieve
regulation of gene junction. Such technology is now well known in
the art, and sense or antisense oligonucleotides or larger
fragments, can be designed from various locations along the coding
or control regions of sequences encoding PHPLA2.
[0121] Expression vectors derived from retro viruses, adenovirus,
herpes or vaccinia viruses, or from various bacterial plasmids may
be used for delivery of nucleotide sequences to the targeted organ,
tissue or cell population. Methods which are well known to those
skilled in the art can be used to construct vectors which will
express nucleic acid sequence which is complementary to the
polynucleotides of the gene encoding PHPLA2. These techniques are
described both in Sambrook et al. (supra) and in Ausubel et al.
(supra).
[0122] Genes encoding PHPLA2 can be turned off by transforming a
cell or tissue with expression vectors which express high levels of
a polynucleotide or fragment thereof which encodes PHPLA2. Such
constructs may be used to introduce untranslatable sense or
antisense sequences into a cell. Even in the absence of integration
into the DNA, such vectors may continue to transcribe RNA molecules
until they are disabled by endogenous nucleases. Transient
expression may last for a month or more with a non-replicating
vector and even longer if appropriate replication elements are part
of the vector system.
[0123] As mentioned above, modifications of gene expression can be
obtained by designing complementary sequences or antisense
molecules (DNA, RNA, or PNA) to the control, 5' or regulatory
regions of the gene encoding PHPLA2 (signal sequence, promoters,
enhancers, and introns). Oligonucleotides derived from the
transcription initiation site, e.g., between positions -10 and +10
from the start site, are preferred. Similarly, inhibition can be
achieved using "triple helix" base-pairing methodology. Triple
helix pairing is useful because it causes inhibition of the ability
of the double helix to open sufficiently for the binding of
polymerases, transcription factors, or regulatory molecules. Recent
therapeutic advances using triplex DNA have been described in the
literature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I.
Carr, Molecular and Immunologic Approaches, Futura Publishing Co.,
Mt. Kisco, N.Y.). The complementary sequence or antisense molecule
may also be designed to block translation of mRNA by preventing the
transcript from binding to ribosomes.
[0124] Ribozymes, enzymatic RNA molecules, may also be used to
catalyze the specific cleavage of RNA. The mechanism of ribozyme
action involves sequence-specific hybridization of the ribozyme
molecule to complementary target RNA, followed by endonucleolytic
cleavage. Examples which may be used include engineered hammerhead
motif ribozyme molecules that can specifically and efficiently
catalyze endonucleolytic cleavage of sequences encoding PHPLA2.
[0125] Specific ribozyme cleavage sites within any potential RNA
target are initially identified by scanning the target molecule for
ribozyme cleavage sites which include the following sequences: GUA,
GUU, and GUC. Once identified, short RNA sequences of between 15
and 20 ribonucleotides corresponding to the region of the target
gene containing the cleavage site may be evaluated for secondary
structural features which may render the oligonucleotide
inoperable. The suitability of candidate targets may also be
evaluated by testing accessibility to hybridization with
complementary oligonucleotides using ribonuclease protection
assays.
[0126] Complementary ribonucleic acid molecules and ribozymes of
the invention may be prepared by any method known in the art for
the synthesis of nucleic acid molecules. These include techniques
for chemically synthesizing oligonucleotides such as solid phase
phosphoramidite chemical synthesis. Alternatively, RNA molecules
may be generated by in vitro and in vivo transcription of DNA
sequences encoding PHPLA2. Such DNA sequences may be incorporated
into a wide variety of vectors with suitable RNA polymerase
promoters such as T7 or SP6. Alternatively, these cDNA constructs
that synthesize complementary RNA constitutively or inducibly can
be introduced into cell lines, cells, or tissues.
[0127] RNA molecules may be modified to increase intracellular
stability and half-life. Possible modifications include, but are
not limited to, the addition of flanking sequences at the 5' and/or
3' ends of the molecule or the use of phosphorothioate or 2'
O-methyl rather than phosphodiesterase linkages within the backbone
of the molecule. This concept is inherent in the production of PNAs
and can be extended in all of these molecules by the inclusion of
nontraditional bases such as inosine, queosine, and wybutosine, as
well as acetyl-, methyl-, thio-, and similarly modified forms of
adenine, cytidine, guanine, thymine, and uridine which are not as
easily recognized by endogenous endonucleases.
[0128] Many methods for introducing vectors into cells or tissues
are available and equally suitable for use in vivo, in vitro, and
ex vivo. For ex vivo therapy, vectors may be introduced into stem
cells taken from the patient and clonally propagated for autologous
transplant back into that same patient. Delivery by transfection,
by liposome injections or polycationic amino polymers (Goldman, C.
K. et al. (1997) Nature Biotechnology 15:462-66; incorporated
herein by reference) may be achieved using methods which are well
known in the art.
[0129] Any of the therapeutic methods described above may be
applied to any subject in need of such therapy, including, for
example, mammals such as dogs, cats, cows, horses, rabbits,
monkeys, and most preferably, humans.
[0130] An additional embodiment of the invention relates to the
administration of a pharmaceutical composition, in conjunction with
a pharmaceutically acceptable carrier, for any of the therapeutic
effects discussed above. Such pharmaceutical compositions may
consist of PHPLA2, antibodies to PHPLA2, mimetics, agonists,
antagonists, or inhibitors of PHPLA2. The compositions may be
administered alone or in combination with at least one other agent,
such as stabilizing compound, which may be administered in any
sterile, biocompatible pharmaceutical carrier, including, but not
limited to, saline, buffered saline, dextrose, and water. The
compositions may be administered to a patient alone, or in
combination with other agents, drugs or hormones.
[0131] The pharmaceutical compositions utilized in this invention
may be administered by any number of routes including, but not
limited to, oral, intravenous, intramuscular, intra-arterial,
intramedullary, intrathecal, intraventricular, transdermal,
subcutaneous, intraperitoneal, intranasal, enteral, topical,
sublingual, or rectal means.
[0132] In addition to the active ingredients, these pharmaceutical
compositions may contain suitable pharmaceutically-acceptable
carriers comprising excipients and auxiliaries which facilitate
processing of the active compounds into preparations which can be
used pharmaceutically. Further details or techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0133] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral a dministration. Such carriers
enable the pharmaceutical compositions to be formulated as tablets,
pills, dragees, capsules, liquids, gels, syrups, slurries,
suspensions, and the like, for ingestion by the patient.
[0134] Pharmaceutical preparations for oral use can be obtained
through combination of active compounds with solid excipient,
optionally grinding a resulting mixture, and processing the mixture
of granules, after adding suitable auxiliaries, if desired, to
obtain tablets or dragee cores. Suitable excipients are
carbohydrate or protein fillers, such as sugars, including lactose,
sucrose, mannitol, or sorbitol; starch from corn, wheat, rice,
potato, or other plants; cellulose, such as methyl cellulose,
hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose;
gums including arabic and tragacanth; and proteins such as gelatin
and collagen. If desired, disintegrating or solubilizing agents may
be added, such as the cross-linked polyvinyl pyrrolidone, agar,
alginic acid, or a salt thereof, such as sodium alginate.
[0135] Dragee cores may be used in conjunction with suitable
coatings, such as concentrated sugar solutions, which may also
contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel,
polyethylene glycol, and/or titanium dioxide, lacquer solutions,
and suitable organic solvents or solvent mixtures. Dyestuffs or
pigments may be added to the tablets or dragee coatings for product
identification or to characterize the quantity of active compound,
i.e., dosage.
[0136] Pharmaceutical preparations which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with a
filler or binders, such as lactose or starches, lubricants, such as
talc or magnesium stearate, and, optionally, stabilizers. In soft
capsules, the active compounds may be dissolved or suspended in
suitable liquids, such as fatty oils, liquid, or liquid
polyethylene glycol with or without stabilizers.
[0137] Pharmaceutical formulations suitable for parenteral
administration may be formulated in aqueous solutions, preferably
in physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiologically buffered saline. Aqueous
injection suspensions may contain substances which increase the
viscosity of the suspension, such as sodium carboxymethyl
cellulose, sorbitol, or dextran. Additionally, suspensions of the
active compounds may be prepared as appropriate oily injection
suspensions. Suitable lipophilic solvents or vehicles include fatty
oils such as sesame oil, or synthetic fatty acid esters, such as
ethyl oleate or triglycerides, or liposomes. Non-lipid
polycatioriic amino polymers may also be used for delivery.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0138] For topical or nasal administration, penetrants appropriate
to the particular barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art.
[0139] The pharmaceutical compositions of the present invention may
be manufactured in a manner that is known in the art, e.g., by
means of conventional mixing, dissolving, granulating,
dragee-making, levigating, emulsifying, encapsulating, entrapping,
or lyophilizing processes.
[0140] The pharmaceutical composition may be provided as a salt and
can be formed with many acids, including but not limited to,
hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic,
etc. Salts tend to be more soluble in aqueous or other protonic
solvents than are the corresponding free base forms. In other
cases, the preferred preparation may be a lyophilized powder which
may contain any or all of the following: 1-50 mM histidine, 0.1%-2%
sucrose, and 2-7% mannitol, at a pH range of 4.5 to 5.5, that is
combined with buffer prior to use.
[0141] After pharmaceutical compositions have been prepared, they
can be placed in an appropriate container and labeled for treatment
of an indicated condition. For administration of PHPLA2, such
labeling would include amount, frequency, and method of
administration.
[0142] Pharmaceutical compositions suitable for use in the
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve the intended purpose
The determination of an effective dose is well within the
capability of those skilled in the art.
[0143] For any compound, the therapeutically effective dose can be
estimated initially either in cell culture assays, e.g., of
neoplastic cells, or in animal models, usually mice, rabbits, dogs,
or pigs. The animal model may also be used to determine the
appropriate concentration range and route of administration. Such
information can then be used to determine useful doses and routes
for administration in humans.
[0144] A therapeutically effective dose refers to that amount of
active ingredient, for example PHPLA2 or fragments thereof,
antibodies of PHPLA2, agonists, antagonists or inhibitors of
PHPLA2, which ameliorates the symptoms or condition. Therapeutic
efficacy and toxicity may be determined by standard pharmaceutical
procedures in cell cultures or experimental animals, e.g., ED50
(the dose therapeutically effective in 50% of the population) and
LD50 (the dose lethal to 50% of the population). The dose ratio of
toxic to therapeutic effects is the therapeutic index, which can be
expressed as the LD.sub.50/ED.sub.50 ratio. Pharmaceutical
compositions which exhibit large therapeutic indices are preferred.
The data obtained from cell culture assays and animal studies is
used in formulating a range of dosage for human use. The dosage
contained in such compositions is preferably within a range of
circulating concentrations that include the ED50 with little or no
toxicity. The dosage varies within this range depending upon the
dosage form employed, sensitivity of the patient, and the route of
administration.
[0145] The exact dosage will be determined by the practitioner, in
light of factors related to the subject that requires treatment.
Dosage and administration are adjusted to provide sufficient levels
of the active moiety or to maintain the desired effect. Factors
which may be taken into account include the severity of the disease
state, general health of the subject, age, weight, and gender of
the subject, diet, time and frequency of administration, drug
combination(s), reaction sensitivities, and tolerance/response to
therapy. Long-acting pharmaceutical compositions may be
administered every 3 to 4 days, every week, or once every two weeks
depending on half-life and clearance rate of the particular
formulation.
[0146] Normal dosage amounts may vary from 0.1 to 100,000
micrograms, up to a total dose of about 1 g, depending upon the
route of administration. Guidance as to particular dosages and
methods of delivery is provided in the literature and generally
available to practitioners in the art. Those skilled in the art
will employ different formulations for nucleotides than for
proteins or their inhibitors. Similarly, delivery of
polynucleotides or polypeptides will be specific to particular
cells, conditions, locations, etc.
DIAGNOSTICS
[0147] In another embodiment, antibodies which specifically bind
PHPLA2 may be used for the diagnosis of conditions or diseases
characterized by expression of PHPLA2, or in assays to monitor
patients being treated with PHPLA2, agonists, antagonists or
inhibitors The antibodies useful for diagnostic purposes may be
prepared in the same manner as those described above for
therapeutics. Diagnostic assays for PHPLA2 include methods which
utilize the antibody and a label to detect PHPLA2 in human body
fluids or extracts of cells or tissues. The antibodies may be used
with or without modification, and may be labeled by joining them,
either covalently or non-covalently, with a reporter molecule. A
wide variety of reporter molecules which are known in the art may
be used, several of which are described above.
[0148] A variety of protocols including ELISA, RIA, and FACS for
measuring PHPLA2 are known in the art and provide a basis for
diagnosing altered or abnormal levels of PHPLA2 expression. Normal
or standard values for PHPLA2 expression are established by
combining body fluids or cell extracts taken from normal mammalian
subjects, preferably human, with antibody to PHPLA2 under
conditions suitable for complex formation. The amount of standard
complex formation may be quantified by various methods, but
preferably by photometric, means. Quantities of PHPLA2 expressed in
subject, control and disease, samples from biopsied tissues are
compared with the standard values. Deviation between standard and
subject values establishes the parameters for diagnosing
disease.
[0149] In another embodiment of the invention, the polynucleotides
encoding PHPLA2 may be used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, complementary RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of PHPLA2 may be
correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of
PHPLA2, and to monitor regulation of PHPLA2 levels during
therapeutic intervention.
[0150] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding PHPLA2 or closely related molecules, may be
used to identify nucleic acid sequences which encode PHPLA2. The
specificity of the probe, whether it is made from a highly specific
region, e.g., 10 unique nucleotides in the 5' regulatory region, or
a less specific region, e.g., especially in the 3' coding region,
and the stringency of the hybridization or amplification (maximal,
high, intermediate, or low) will determine whether the probe
identifies only naturally occurring sequences encoding PHPLA2,
alleles, or related Probes may also be used for the detection of
related sequences, and should preferably contain at least 50% of
the nucleotides from any of the PHPLA2 encoding sequences. The
hybridization probes of the subject invention may be DNA or RNA and
derived from the nucleotide sequence of SEQ ID NO:2 or from genomic
sequence including promoter, enhancer elements, and introns of the
naturally occurring PHPLA2.
[0151] Means for producing specific hybridization probes for DNAs
encoding PHPLA2 include the cloning of nucleic acid sequences
encoding PHPLA2 or PHPLA2 derivatives into vectors for the
production of mRNA probes. Such vectors are known in the art,
commercially available, and may be used to synthesize RNA probes in
vitro by means of the addition of the appropriate RNA polymerases
and the appropriate labeled nucleotides. Hybridization probes may
be labeled by a variety of reporter groups, for example,
radionuclides such as 32P or 35S, or enzymatic labels, such as
alkaline phosphatase coupled to the probe via avidin/biotin
coupling systems, and the like.
[0152] Polynucleotide sequences encoding PHPLA2 may be used for the
diagnosis of conditions or disorders which are associated with
expression of PHPLA2. Examples of such conditions or disorders
include adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,
sarcoma, and teratocarcinoma and particularly cancers of the
adrenal gland, bladder, bone, bone marrow, brain, breast, cervix,
gall bladder, ganglia, gastrointestinal tract, heart, kidney,
liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate,
salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
and inflammation associated with conditions and disorders including
AIDS, Addison's disease, adult respiratory distress syndrome,
allergies, anemia, asthma, atherosclerosis, bronchitis,
cholecystitis, Crohn's disease, ulcerative colitis, atopic
dermatitis, dermatomyositis, diabetes mellitus, emphysema,
epidermal hyperplasia, erythema nodosum, atrophic gastritis,
glomerulonephritis, gout, Graves' disease, hypereosinophilia,
irritable bowel syndrome, lupus erythematosus, multiple sclerosis,
myasthenia gravis, myocardial or pericardial inflammation,
osteoarthritis, osteoporosis, pancreatitis, polymyositis,
rheumatoid arthritis, scleroderma, Sjogren's syndrome, toxic shock
syndrome, and autoimmune thyroiditis; complications of cancer,
hemodialysis, and extracorporeal circulation; viral, bacterial,
fungal, parasitic, protozoal, and helminthic infections; and
trauma. The polynucleotide sequences encoding PHPLA2 may be used in
Southern or northern analysis, dot blot, or other membrane-based
technologies; in PCR technologies; or in dipstick, pin, ELISA
assays or microarrays utilizing fluids or tissues from patient
biopsies to detect altered PHPLA2 expression. Such qualitative or
quantitative methods are well known in the art.
[0153] In a particular aspect, the nucleotide sequences encoding
PHPLA2 may be useful in assays that detect activation or induction
of various cancers, particularly those mentioned above. The
nucleotide sequences encoding PHPLA2 may be labeled by standard
methods, and added to a fluid or tissue sample from a patient under
conditions suitable for the formation of hybridization complexes.
After a suitable incubation period, the sample is washed and the
signal is quantitated and compared with a standard value. If the
amount of signal in the biopsied or extracted sample is
significantly altered from that of a comparable control sample, the
nucleotide sequences have hybridized with nucleotide sequences in
the sample, and the presence of altered levels of nucleotide
sequences encoding PHPLA2 in the sample indicates the presence of
the associated disease. Such assays may also be used to evaluate
the efficacy of a particular therapeutic treatment regimen in
animal studies, in clinical trials, or in monitoring the treatment
of an individual patient.
[0154] In order to provide a basis for the diagnosis of disease
associated with expression of PHPLA2, a normal or standard profile
for expression is established. This may be accomplished by
combining body fluids or cell extracts taken from normal subjects,
either animal or human, with a sequence, or a fragment thereof,
which encodes PHPLA2, under conditions suitable for hybridization
or amplification. Standard hybridization may be quantified by
comparing the values obtained from normal subjects with those from
an experiment where a known amount of a substantially purified
polynucleotide is used. Standard values obtained from normal
samples may be compared with values obtained from samples from
patients who are symptomatic for disease. Deviation between
standard and subject values is used to establish the presence of
disease.
[0155] Once disease is established and a treatment protocol is
initiated, hybridization assays may be repeated on a regular basis
to evaluate whether the level of expression in the patient begins
to approximate that which is observed in the normal patient. The
results obtained from successive assays may be used to show the
efficacy of treatment over a period ranging from several days to
months.
[0156] With respect to cancer, the presence of a relatively high
amount of transcript in biopsied tissue from an individual may
indicate a predisposition for the development of the disease, or
may provide a means for detecting the disease prior to the
appearance of actual clinical symptoms. A more definitive diagnosis
of this type may allow health professionals to employ preventative
measures or aggressive treatment earlier thereby preventing the
development or further progression of the cancer.
[0157] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding PHPLA2 may involve the use of PCR. Such
oligomers may be chemically synthesized, generated enzymatically,
or produced in vitro. Oligomers will preferably consist of two
nucleotide sequences, one with sense orientation (5'.fwdarw.3') and
another with antisense (3'.rarw.5'), employed under optimized
conditions for identification of a specific gene or condition. The
same two oligomers, nested sets of oligomers, or even a degenerate
pool of oligomers may be employed under less stringent conditions
for detection and/or quantitation of closely related DNA or RNA
sequences.
[0158] Methods which may also be used to quantitate the expression
of PHPLA2 include radiolabeling or biotinylating nucleotides,
coamplification of a control nucleic acid, and standard curves onto
which the experimental results are interpolated (Melby, P. C. et
al. (1993) J. Immunol. Methods, 159:235-244; Duplaa, C. et al.
(1993) Anal. Biochem. 212: 229-236). The speed of quantitation of
multiple samples may be accelerated by running the assay in an
ELISA format where the oligomer of interest is presented in various
dilutions and a spectrophotometric or calorimetric response gives
rapid quantitation.
[0159] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously (to produce a transcript image), and to identify
genetic variants, mutations and polymorphisms. This information may
be used to determine gene function, understanding the genetic basis
of disease, diagnosing disease, and in developing and in monitoring
the activities of therapeutic agents.
[0160] In further embodiments, oligonucleotides or longer fragments
derived from any of the polynucleotide sequences described herein
may be used as targets in a microarray. The microarray can be used
to monitor the expression level of large numbers of genes
simultaneously (to produce a transcript image), and to identify
genetic variants, mutations and polymorphisms. This information may
be used to determine gene function, understanding the genetic basis
of disease, diagnosing disease, and in developing and in monitoring
the activities of therapeutic agents.
[0161] In one embodiment, the microarray is prepared and used
according to the methods described in PCT application WO95/11995
(Chee et al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14:
1675-1680) and Schena, M. et al. (1996; Proc. Natl. Acad. Sci. 93:
10614-10619), all of which are incorporated herein in their
entirety by reference.
[0162] The microarray is preferably composed of a large number of
unique, single-stranded nucleic acid sequences, usually either
synthetic antisense oligonucleotides or fragments of cDNAs, fixed
to a solid support. The oligonucleotides are preferably about 6-60
nucleotides in length, more preferably 15-30 nucleotides in length,
and most preferably about 20-25 nucleotides in length. For a
certain type of microarray, it may be preferable to use
oligonucleotides which are only 7-10 nucleotides in length. The
microarray may contain oligonucleotides which cover the known 5',
or 3', sequence, sequential oligonucleotides which cover the full
length sequence; or unique oligonucleotides selected from
particular areas along the length of the sequence. Polynucleotides
used in the microarray may be oligonucleotides that are specific to
a gene or genes of interest in which at least a fragment of the
sequence is known or that are specific to one or more unidentified
cDNAs which are common to a particular cell type, developmental or
disease state.
[0163] In order to produce oligonucleotides to a known sequence for
a microarray, the gene of interest is examined using a computer
algorithm which starts at the 5' or more preferably at the 3' end
of the nucleotide sequence. The algorithm identifies oligomers of
defined length that are unique to the gene, have a GC content
within a range suitable for hybridization, and lack predicted
secondary structure that may interfere with hybridization. In
certain situations it may be appropriate to use pairs of
oligonucleotides on a microarray. The "pairs" will be identical,
except for one nucleotide which preferably is located in the center
of the sequence. The second oligonucleotide in the pair (mismatched
by one) serves as a control. The number of oligonucleotide pairs
may range from two to one million. The oligomers are synthesized at
designated areas on a substrate using a light-directed chemical
process. The substrate may be paper, nylon or other type of
membrane, filter, chip, glass slide or any other suitable solid
support.
[0164] In another aspect, an oligonucleotide may be synthesized on
the surface of the substrate by using a chemical coupling procedure
and an ink jet application apparatus, as described in PCT
application WO95/251116 (Baldeschweiler et al.) which is
incorporated herein in its entirety by reference. In another
aspect, a "gridded" array analogous to a dot (or slot) blot may be
used to arrange and link cDNA fragments or oligonucleotides to the
surface of a substrate using a vacuum system, thermal, UV,
mechanical or chemical bonding procedures. An array, such as those
described above, may be produced by hand or by using available
devices (slot blot or dot blot apparatus), materials (any suitable
solid support), and machines (including robotic instruments), and
may contain 8, 24, 96, 384, 1536 or 6144 oligonucleotides, or any
other number between two and one million which lends itself to the
efficient use of commercially available instrumentation.
[0165] In order to conduct sample analysis using a microarray, the
RNA or DNA from a biological sample is made into hybridization
probes. The mRNA is isolated, and cDNA is produced and used as a
template to make antisense RNA (aRNA). The aRNA is amplified in the
presence of fluorescent nucleotides, and labeled probes are
incubated with the microarray so that the probe sequences hybridize
to complementary oligonucleotides of the microarray. Incubation
conditions are adjusted so that hybridization occurs with precise
complementary matches or with various degrees of less
complementarity. After removal of nonhybridized probes, a scanner
is used to determine the levels and patterns of fluorescence. The
scanned images are examined to determine degree of complementarity
and the relative abundance of each oligonucleotide sequence on the
microarray. The biological samples may be obtained from any bodily
fluids (such as blood, urine, saliva, phlegm, gastric juices,
etc.), cultured cells, biopsies, or other tissue preparations. A
detection system may be used to measure the absence, presence, and
amount of hybridization for all of the distinct sequences
simultaneously. This data may be used for large scale correlation
studies on the sequences, mutations, variants, or polymorphisms
among samples (Heller, R. A. et al., (1997) Proc. Natl. Acad. Sci.
94:2150-55).
[0166] In another embodiment of the invention, the nucleic acid
sequences which encode PHPLA2 may also be used to generate
hybridization probes which are useful for mapping the naturally
occurring genomic sequence. The sequences may be mapped to a
particular chromosome, to a specific region of a chromosome or to
artificial chromosome constructions, such as human artificial
chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial
artificial chromosomes (BACs), bacterial P1 constructions or single
chromosome cDNA libraries as reviewed in Price, C. M. (1993) Blood
Rev. 7:127-134, and Trask, B. J. (1991) Trends Genet.
7:149-154.
[0167] Fluorescent in situ hybridization (FISH as described in
Veima et al. (1988) Human Chromosomes: A Manual of Basic
Techniques, Pergamon Press, New York, N.Y.) may be correlated with
other physical chromosome mapping techniques and genetic map data.
Examples of genetic map data can be found in various scientific
journals or at Online Mendelian Inheritance in Man (OMIM).
Correlation between the location of the gene encoding PHPLA2 on a
physical chromosomal map and a specific disease, or predisposition
to a specific disease, may help delimit the region of DNA
associated with that genetic disease. The nucleotide sequences of
the subject invention may be used to detect differences in gene
sequences between normal, carrier, or affected individuals.
[0168] In situ hybridization of chromosomal preparations and
physical mapping techniques such as linkage analysis using
established chromosomal markers may be used for extending genetic
maps. Often the placement of a gene on the chromosome of another
mammalian species, such as mouse, may reveal associated markers
even if the number or arm of a particular human chromosome is not
known. New sequences can be assigned to chromosomal arms, or parts
thereof, by physical mapping. This provides valuable information to
investigators searching for disease genes using positional cloning
or other gene discovery techniques. Once the disease or syndrome
has been crudely localized by genetic linkage to a particular
genomic region, for example, AT to 11q22-23 (Gatti, R. A. et al.
(1988) Nature 336:577-580), any sequences mapping to that area may
represent associated or regulatory genes for further investigation.
The nucleotide sequence of the subject invention may also be used
to detect differences in the chromosomal location due to
translocation, inversion, etc. among normal, carrier, or affected
individuals.
[0169] In another embodiment of the invention, PHPLA2, its
catalytic or immunogenic fragments or oligopeptides thereof, can be
used for screening libraries of compounds in any of a variety of
drug screening techniques. The fragment employed in such screening
may be free in solution, affixed to a solid support, borne on a
cell surface, or located intracellularly. The formation of binding
complexes, between PHPLA2 and the agent being tested, may be
measured.
[0170] Another technique for drug screening which may be used
provides for high throughput screening of compounds having suitable
binding affinity to the protein of interest as described in
published PCT application WO84/03564. In this method, as applied to
PHPLA2 large numbers of different small test compounds are
synthesized on a solid substrate, such as plastic pins or some
other surface. The test compounds are reacted with PHPLA2, or
fragments thereof, and washed. Bound PHPLA2 is then detected by
methods well known in the art. Purified PHPLA2 can also be coated
directly onto plates for use in the aforementioned drug screening
techniques. Alternatively, non-neutralizing antibodies can be used
to capture the peptide and immobilize it on a solid support.
[0171] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding PHPLA2 specifically compete with a test compound for
binding PHPLA2. In this manner, the antibodies can be used to
detect the presence of any peptide which shares one or more
antigenic determinants with PHPLA2.
[0172] In additional embodiments, the nucleotide sequences which
encode PHPLA2 may be used in any molecular biology techniques that
have yet to be developed, provided the new techniques rely on
properties of nucleotide sequences that are currently known,
including, but not limited to, such properties as the triplet
genetic code and specific base pair interactions.
[0173] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
I OVARTUT01 cDNA Library Construction
[0174] The OVARTUT01 cDNA library was constructed from tumorous
ovary tissue obtained from a 43 year old Caucasian female with a
malignant neoplasm (specimen #0123A; Mayo Clinic, Rochester Minn.).
The patient history indicated a previous normal delivery and a
vaginal hysterectomy. Also reported in the patient history were
previous diagnoses of hepatitis, cerebrovascular disease,
atherosclerosis and mitral valve disorder; however, the patient was
not taking medication for any of these conditions at the time of
surgery.
[0175] The frozen tissue was homogenized and lysed using a
Brinkniann Homogenizer Polytron PT-3000 (Brinkmann Instruments,
Westbury, N.Y.) in guanidinium isothiocyanate solution. The lysate
was centrifuged over a 5.7 M CsCl cushion using a Beckman SW28
rotor in a Beckman L8-70M Ultracentrifuge (Beckman Instruments) for
18 hours at 25,000 rpm at ambient temperature. The RNA was
extracted with acid phenol pH 4.7, precipitated using 0.3 M sodium
acetate and 2.5 volumes of ethanol, resuspended in RNAse-free
water, and DNase treated at 37.degree. C. The RNA extraction was
repeated with acid phenol pH 4.7 and precipitated With sodium
acetate and ethanol as before. The mRNA was then isolated using the
Qiagen Oligotex kit (QIAGEN, Inc., Chatsworth, Calif.) and used to
construct the cDNA library.
[0176] The mRNA was handled according to the recommended protocols
in the SuperScript Plasmid System for cDNA Synthesis and Plasmid
Cloning (Catalog #18248-013, Gibco/BRL). cDNAs were fractionated on
a Sepharose CL4B column (Catalog #275105-01, Pharmacia), and those
cDNAs exceeding 400 bp were ligated into PSPORT1. The plasmid
PSPORT1 was subsequently transformed into DH5.alpha. competent
cells (Catalog #18258-012, Gibco/BRL).
I Isolation and Sequencing of cDNA Clones
[0177] Plasmid DNA was released from the cells and purified using
the REAL Prep 96 Plasmid Kit (Catalog #26173; Qiagen, Inc). The
recommended protocol was employed except for the following changes:
1) the bacteria were cultured in 1 ml of sterile Terrific Broth
(Catalog #22711, Gibco/BRL) with carbenicillin at 25 mg/L and
glycerol at 0.4%; 2) the cultures were incubated for 19 hours after
the wells were inoculated and then lysed with 0.3 ml of lysis
buffer; 3) following isopropanol precipitation, the plasmid DNA
pellet was resuspended in 0.1 ml of distilled water. After the last
step in the protocol, samples were transferred to a Beckman 96-well
block for storage.
[0178] The cDNAs were sequenced by the method of Sanger, F. et al.
(1975); J. Mol. Biol. 94:441f), using a Hamilton Micro Lab 2200
(Hamilton, Reno Nev.) in combination with Peltier Thermal Cyclers
(PTC200 from MJ Research, Watertown Mass.) and Applied Biosystems
377 or 373 DNA Sequencing Systems; and the reading frame was
determined.
III Homology Searching of cDNA Clones and Their Deduced
Proteins
[0179] The nucleotide sequences of the Sequence Listing or amino
acid sequences deduced from them were used as query sequences
against databases such as GenBank, SwissProt, BLOCKS, and Pima II.
These databases which contain previously identified and annotated
sequences were searched for regions of homology (similarity) using
BLAST, which stands for Basic Local Alignment Search Tool (Altschul
(1993) supra, Altschul (1990) supra).
[0180] BLAST produces alignments of both nucleotide and amino acid
sequences to determine sequence similarity. Because of the local
nature of the alignments, BLAST is especially useful in determining
exact matches or in identifying homologs which may be of
prokaryotic (bacterial) or eukaryotic (animal, fungal or plant)
origin. Other algorithms such as the one described in Smith R F and
T F Smith (1992; Protein Engineering 5:35-51), incorporated herein
by reference, can be used when dealing with primary sequence
patterns and secondary structure gap penalties. As disclosed in
this application, the sequences have lengths of at least 49
nucleotides, and no more than 12% uncalled bases (where N is
recorded rather than A, C, G, or T).
[0181] The BLAST approach, as detailed in Karlin and Altschul
(supra) and incorporated herein by reference, searches for matches
between a query sequence and a database sequence, to evaluate the
statistical significance of any matches found, and to report only
those matches which satisfy the user-selected threshold of
significance. In this application, threshold was set at 10.sup.-25
for nucleotides and 10.sup.-14 for peptides.
[0182] Incyte nucleotide sequences were searched against the
GenBank databases for primate (pri), rodent (rod), and mammalian
sequences (mam), and deduced amino acid sequences from the same
clones are searched against GenBank functional protein databases,
mammalian (mamp), vertebrate (vrtp) and eukaryote (eukp), for
homology.
IV Northern Analysis
[0183] Northern analysis is a laboratory technique used to detect
the presence of a transcript of a gene and involves the
hybridization of a labeled nucleotide sequence to a membrane on
which RNAs from a particular cell type or tissue have been bound
(Sambrook et al., supra).
[0184] Analogous computer techniques using BLAST (Altschul, S. F.
(1993) J.Mol.Evol. 36:290-300; Altschul, S. F. et al. (1990)
J.Mol.Evol. 215:403-410) are used to search for identical or
related molecules in nucleotide databases such as GenBank or the
LIFESEQ.TM. database (Incyte Pharmaceuticals). This analysis is
much faster than multiple, membrane-based hybridizations. In
addition, the sensitivity of the computer search can be modified to
determine whether any particular match is categorized as exact or
homologous.
[0185] The basis of the search is the product score which is
defined as:
% sequence identity.times.% maximum BLAST score/100
[0186] The product score takes into account both the degree of
similarity between two sequences and the length of the sequence
match. For example, with a product score of 40, the match will be
exact within a 1-2% error; and at 70, the match will be exact.
Homologous molecules-are usually identified by selecting those
which show product scores between 15 and 40, although lower scores
may identify related molecules.
[0187] The results of northern analysis are reported as a list of
libraries in which the transcript encoding PHPLA2 occurs. Abundance
and percent abundance are also reported. Abundance directly
reflects the number of times a particular transcript is represented
in a cDNA library, and percent abundance is abundance divided by
the total number of sequences examined in the cDNA library.
V Extension of PHPLA2 Encoding Polynucleotides
[0188] The nucleic acid sequence of the Incyte Clone 816403 was
used to design oligonucleotide primers for extending a partial
nucleotide sequence to full length. One primer was synthesized to
initiate extension in the antisense direction, and the other was
synthesized to extend sequence in the sense direction. Primers were
used to facilitate the extension of the known sequence "outward"
generating amplicons containing new, unknown nucleotide sequence
for the region of interest. The initial primers were designed from
the cDNA using OLIGO 4.06 software (National Biosciences), or
another appropriate program, to be about 22 to about 30 nucleotides
in length, to have a GC content of 50% or more, and to anneal to
the target sequence at temperatures of about 68.degree. to about
72.degree. C. Any stretch of nucleotides which would result in
hairpin structures and primer-primer dimerizations was avoided.
[0189] Selected human cDNA libraries (Gibco/BRL) were used to
extend the sequence If more than one extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0190] High fidelity amplification was obtained by following the
instructions for the XL-PCR kit (Perkin Elmer) and thoroughly
mixing the enzyme and reaction mix. Beginning with 40 pmol of each
primer and the recommended concentrations of all other components
of the kit, PCR was performed using the Peltier Thermal Cycler
(PTC200; M.J. Research, Watertown, Mass.) and the following
parameters:
1 Step 1 94.degree. C. for 1 min (initial denaturation) Step 2
65.degree. C. for 1 min Step 3 68.degree. C. for 6 min Step 4
94.degree. C. for 15 sec Step 5 65.degree. C. for 1 min Step 6
68.degree. C. for 7 min Step 7 Repeat step 4-6 for 15 additional
cycles Step 8 94.degree. C. for 15 sec Step 9 65.degree. C. for 1
min Step 10 68.degree. C. for 7:15 min Step 11 Repeat step 8-10 for
12 cycles Step 12 72.degree. C. for 8 min Step 13 4.degree. C. (and
holding)
[0191] A 5-10 .mu.l aliquot of the reaction mixture was analyzed by
electrophoresis on a low 30 concentration (about 0.6-0.8%) agarose
mini-gel to determine which reactions were successful in extending
the sequence. Bands thought to contain the largest products were
excised from the gel, purified using QIAQuick.TM. (QIAGEN Inc.,
Chatsworth, Calif.), and trimmed of overhangs using Klenow enzyme
to facilitate religation and cloning.
[0192] After ethanol precipitation, the products were redissolved
in 13 .mu.l of ligation buffer, 1 .mu.l T4-DNA ligase (15 units)
and 1 .mu.T4 polynucleotide kinase were added, and the mixture was
incubated at room temperature for 2-3 hours or overnight at
16.degree. C. Competent E. coli cells (in 40 .mu.l of appropriate
media) were transformed with 3 .mu.l of ligation mixture and
cultured in 80 .mu.l of SOC medium (Sambrook et al., supra). After
incubation for one hour at 37.degree. C., the E. coli mixture was
plated on Luria Bertani (LB)-agar (Sambrook et al., supra)
containing 2.times. Carb. The following day, several colonies were
randomly picked from each plate and cultured in 150 ml of liquid
LB/2.times. Carb medium placed in an individual well of an
appropriate, commercially-available, sterile 96-well microtiter
plate. The following day, 5 .mu.l of each overnight culture was
transferred into a non-sterile 96-well plate and after dilution
1:10 with water, 5 .mu.l of each sample was transferred into a PCR
array.
[0193] For PCR amplification, 18 .mu.l of concentrated PCR reaction
mix (3.3.times.) containing 4 units of rTth DNA polymerase, a
vector primer, and one or both of the gene specific primers used
for the extension reaction were added to each well. Amplification
was performed using the following conditions:
2 Step 1 94.degree. C. for 60 sec Step 2 94.degree. C. for 20 sec
Step 3 55.degree. C. for 30 sec Step 4 72.degree. C. for 90 sec
Step 5 Repeat steps 2-4 for an additional 29 cycles Step 6
72.degree. C. for 180 sec Step 7 4.degree. C. (and holding)
[0194] Aliquots of the PCR reactions were run on agarose gels
together with molecular weight markers. The sizes of the PCR
products were compared to the original partial cDNAs, and
appropriate clones were selected, ligated into plasmid, and
sequenced.
[0195] In like manner, the nucleotide sequence of SEQ ID NO:2 is
used to obtain 5' regulatory sequences using the procedure above,
oligonucleotides designed for 5' extension, and an appropriate
genomic library.
VI Labeling and Use of Individual Hybridization Probes
[0196] Hybridization probes derived from SEQ ID NO:2 are employed
to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of
oligonucleotides, consisting of about 20 base-pairs, is
specifically described, essentially the same procedure is used with
larger nucleotide fragments. Oligonucleotides are designed using
state-of-the-art software such as OLIGO 4.06 software (National
Biosciences), labeled by combining 50 pmol of each oligomer and 250
.mu.Ci of [.gamma.-.sup.32P] adenosine triphosphate (Amersham) and
T4 polynucleotide kinase (DuPont NEN.RTM., Boston, Mass.). The
labeled oligonucleotides are substantially purified with Sephadex
G-25 superfine resin column (Pharmacia & Upjohn). A aliquot
containing 10.sup.7 counts per minute of the labeled probe is used
in a typical membrane-based hybridization analysis of human genomic
DNA digested with one of the following endonucleases (Ase I, Bgl
II, Eco RI, Pst I, Xba 1, or Pvu II; DuPont NEN.RTM.).
[0197] The DNA from each digest is fractionated on a 0.7 percent
agarose gel and transferred to nylon membranes (Nytran Plus,
Schleicher & Schuell, Durham, N.H.). Hybridization is carried
out for 16 hours at 40.degree. C. To remove nonspecific signals,
blots are sequentially washed at room temperature under
increasingly stringent conditions up to 0.1.times. saline sodium
citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR.TM. film
(Kodak, Rochester, N.Y.) is exposed to the blots, or the blots are
exposed in a PhosphorImager cassette (Molecular Dynamics,
Sunnyvale, Calif.), hybridization patterns are compared
visually.
VII Microarrays
[0198] To produce oligonucleotides for a microarray, SEQ ID NO:2 is
examined using a computer algorithm which starts at the 3' end of
the nucleotide sequence. The algorithm identifies oligomers of
defined length that are unique to the gene, have a GC content
within a range suitable for hybridization, and lack predicted
secondary structure that would interfere with hybridization. The
algorithm identifies-specific oligonucleotides of 20 nucleotides in
length (20-mers). A matched set of oligonucleotides is created in
which one nucleotide in the center of each sequence is altered.
This process is repeated for each gene in the microarray, and
double sets of twenty 20 mers are synthesized in the presence of
fluorescent or radioactive nucleotides and arranged on the surface
of the substrate. When the substrate is a silicon chip, a
light-directed chemical process is used for deposition (Chee, M. et
al., PCT/WO95/11995, incorporated herein by reference).
[0199] In the alternative, a chemical coupling procedure and an ink
jet device is used to synthesize oligomers on the surface of a
substrate (Baldeschweiler, J. D. et al., PCT/WO95/25116,
incorporated herein by reference). In another alternative, a
"gridded" array analogous to a dot (or slot) blot is used to
arrange and link cDNA fragments or oligonucleotides to the surface
of a substrate using a vacuum system, thermal, UV, mechanical or
chemical bonding procedures. A typical array may be produced by
hand or using available materials and machines and contain grids of
8 dots, 24 dots, 96 dots, 384 dots, 1536 dots or 6144 dots. After
hybridization, the microarray is washed to remove nonhybridized
probes, and a detection device as simple as X-ray film or
complicated as a light scanner is used to determine the levels and
patterns of radioactivity or fluorescence. Scanned fluorescent
images are examined to determine degree of complementarity and the
relative abundance/expression level of each oligonucleotide
sequence in the microarray.
VIII Complementary Polynucleotides
[0200] Sequence complementary to the PHPLA2-encoding sequence, or
any part thereof, is used to decrease or inhibit expression of
naturally occurring PHPLA2. Although use of oligonucleotides
comprising from about 15 to about 30 base-pairs is described,
essentially the same procedure is used with smaller or larger
sequence fragments. Appropriate oligonucleotides are designed using
Oligo 4.06 software and the coding sequence of PHPLA2, SEQ ID NO:
1. To inhibit transcription, a complementary oligonucleotide is
designed from the most unique 5' sequence and used to prevent
promoter binding to the coding sequence. To inhibit translation, a
complementary oligonucleotide is designed to prevent ribosomal
binding to the PHPLA2-encoding transcript.
IX Expression of PHPLA2
[0201] Expression of PHPLA2 is accomplished by subcloning the cDNAs
into appropriate vectors and transforming the vectors into host
cells. In this case, the cloning vector is also used to express
PHPLA2 in E. coli. Upstream of the cloning site, this vector
contains a promoter for B-galactosidase, followed by sequence
containing the amino-terminal Met, and the subsequent seven
residues of .beta.-galactosidase. Immediately following these eight
residues is a bacteriophage promoter useful for transcription and a
linker containing a number of unique restriction sites.
[0202] Induction of an isolated, transformed bacterial strain with
IPTG using standard methods produces a fusion protein which
consists of the first eight residues of .beta.-galactosidase, about
5 to 15 residues of linker, and the full length protein. The signal
residues direct the secretion of PHPLA2 into the bacterial growth
media which can be used directly in the following assay for
activity.
X Demonstration of PHPLA2 Activity
[0203] PHPLA2 activity can be demonstrated by an in vitro
hydrolysis assay with vesicles containing
1-palmitoyl-2-[1-14C]oleoyl phosphatidylcholine (Sigma Chemical
Co.). PHPLA2 phospholipase A2 activity is demonstrates by analysis
of the cleavage products isolated from the hydrolysis reaction
mixture.
[0204] Vesicles containing 1-palmitoyl-2-[1-.sup.14C]oleoyl
phosphatidylcholine (Amersham Corp.) are prepared by mixing 2.0
.mu.Ci of the radiolabeled phospholipid with 12.5 mg of unlabeled
1-palmitoyl-2-oleoyl phosphatidylcholine and drying the mixture
under N2. 2.5 ml of 150 mM Tris-HCl, pH 7.5, is added, and the
mixture is sonicated and centrifuged. The supernatant may be stored
at 4.degree. C. The final reaction mixtures contains 0.25 ml of
Hanks' buffered salt solution supplemented with 2.0 mM
taurochenodeoxycholate, 1.0% bovine serum albumin, 1.0 mM
CaCl.sub.2, pH 7.4, 150 .mu.g of 1-palmitoyl-2-[.sup.14C]- oleoyl
phosphatidylcholine vesicles, and various amount of PHPLA2 diluted
in PBS. After incubation for 30 min at 37.degree. C., 20 .mu.g each
of lyso-phosphatidylcholine and oleic acid are added as carriers
and each sample is extracted for total lipids. The lipids are
separated by thin layer chromatography, using a two solvent system
of chloroform:methanol:acetic acid:water (65:35:8:4) until the
solvent front is halfway up the plate, the process is then
continued with hexane:ether:acetic acid (86:16:1) until the solvent
front is at the top of the plate. The lipid-containing areas are
visualized with I2 vapor; the spots are scraped, and their
radioactivity is determined by scintillation counting. The amount
of radioactivity released as fatty acids will increase as a greater
amount of PHPLA2 is added to the assay mixture while the amount of
radioactivity released as lyso-phosphatidylchohne will remain low.
This demonstrates that PHPLA2 cleaves at the sn-2 and not the sn-1
position, exhibiting phospholipase A2 activity.
XI Production of PHPLA2 Specific Antibodies
[0205] PHPLA2 that is substantially purified using PAGE
electrophoresis (Sambrook, supra), or other purification
techniques, is used to immunize rabbits and to produce antibodies
using standard protocols. The amino acid sequence deduced from SEQ
ID NO:2 is analyzed using DNASTAR software (DNASTAR Inc) to
determine regions of high immunogenicity and a corresponding
oligopeptide is synthesized and used to raise antibodies by means
known to those of skill in the art. Selection of appropriate
epitopes, such as those near the C-terminus or in hydrophilic
regions, is described by Ausubel et al. (supra), and others.
[0206] Typically, the oligopeptides are 15 residues in length,
synthesized using an Applied Biosystems Peptide Synthesizer Model
431A using fmoc-chemistry, and coupled to keyhole limpet hemocyanin
(KLH, Sigma, St. Louis, Mo.) by reaction with
N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,
supra). Rabbits are immunized with the oligopeptide-KLH complex in
complete Freund's adjuvant. The resulting antisera are tested for
antipeptide activity, for example, by binding the peptide to
plastic, blocking with 1% BSA, reacting with rabbit antisera,
washing, and reacting with radio iodinated, goat anti-rabbit
IgG.
XII Purification of Naturally Occurring PHPLA2 Using Specific
Antibodies
[0207] Naturally occurring or recombinant PHPLA2 is substantially
purified by immunoaffinity chromatography using antibodies specific
for PHPLA2. An immunoaffinity column is constructed by covalently
coupling PHPLA2 antibody to an activated chromatographic resin,
such as CNBr-activated Sepharose (Pharmacia & Upjohn). After
the coupling, the resin is blocked and washed according to the
manufacturer's instructions.
[0208] Media containing PHPLA2 is passed over the immunoaffinity
column, and the column is washed under conditions that allow the
preferential absorbance of PHPLA2 (e.g., high ionic strength
buffers in the presence of detergent). The column is eluted under
conditions that disrupt antibody/PHPLA2 binding (e.g., a buffer of
pH 2-3 or a high concentration of a chaotrope, such as urea or
thiocyanate ion), and PHPLA2 is collected.
XIII Identification of Molecules Which Interact with PHPLA2
[0209] PHPLA2 or biologically active fragments thereof are labeled
with .sup.125I Bolton-Hunter reagent (Bolton et al. (1973) Biochem.
J. 133: 529). Candidate molecules previously arrayed in the wells
of a multi-well plate are incubated with the labeled PHPLA2, washed
and any wells with labeled PHPLA2 complex are assayed. Data
obtained using different concentrations of PHPLA2 are used to
calculate values for the number, affinity, and association of
PHPLA2 with the candidate molecules.
[0210] All publications and patents mentioned in the above
specification are herein incorporated by reference. Various
modifications and variations of the described method and system of
the invention will be apparent to those skilled in the art without
departing from the scope and spirit of the invention. Although the
invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed
should not be unduly limited to such specific embodiments. Indeed,
various modifications of the described modes for carrying out the
invention which are obvious to those skilled in molecular biology
or related fields are intended to be within the scope of the
following claims.
Sequence CWU 1
1
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