U.S. patent application number 10/316761 was filed with the patent office on 2003-06-12 for novel human pancreatitis-associated protein.
This patent application is currently assigned to Incyte Genomics, Inc.. Invention is credited to Goli, Surya K., Hillman, Jennifer L..
Application Number | 20030109004 10/316761 |
Document ID | / |
Family ID | 25235597 |
Filed Date | 2003-06-12 |
United States Patent
Application |
20030109004 |
Kind Code |
A1 |
Hillman, Jennifer L. ; et
al. |
June 12, 2003 |
Novel human pancreatitis-associated protein
Abstract
The present invention provides a novel human C-type lectin
(human PAP-2) and polynucleotides which identify and encode human
PAP-2. The invention also provides expression vectors, host cells,
agonists, antibodies or antagonists. The invention also provides
methods for treating or preventing diseases associated with
expression of human PAP-2.
Inventors: |
Hillman, Jennifer L.; (Santa
Cruz, CA) ; Goli, Surya K.; (San Jose, CA) |
Correspondence
Address: |
INCYTE CORPORATION (formerly known as Incyte
Genomics, Inc.)
3160 PORTER DRIVE
PALO ALTO
CA
94304
US
|
Assignee: |
Incyte Genomics, Inc.
Palo Alto
CA
|
Family ID: |
25235597 |
Appl. No.: |
10/316761 |
Filed: |
December 10, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10316761 |
Dec 10, 2002 |
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09226852 |
Jan 7, 1999 |
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6492499 |
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09226852 |
Jan 7, 1999 |
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08822261 |
Mar 20, 1997 |
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5935813 |
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Current U.S.
Class: |
435/69.1 ;
435/320.1; 435/325; 435/6.14; 530/395; 536/23.5 |
Current CPC
Class: |
A61P 1/18 20180101; A61K
38/00 20130101; C07K 14/4733 20130101 |
Class at
Publication: |
435/69.1 ; 435/6;
435/320.1; 435/325; 530/395; 536/23.5 |
International
Class: |
C12Q 001/68; C07H
021/04; C07K 014/47; 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. An 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 of 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 of 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 of 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 polypeptide 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 PAP-2, comprising administering
to a patient in need of such treatment the composition of claim
17.
20. A method of 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 PAP-2, comprising administering
to a patient in need of such treatment a composition of claim
21.
23. A method of 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 PAP-2, 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 1 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, the 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 of screening a compound for effectiveness in altering
expression of a target polynucleotide, wherein said target
polynucleotide comprises a 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 of 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 method for a diagnostic test for a condition or disease
associated with the expression of PAP-2 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 11 and an
acceptable excipient.
33. A method of diagnosing a condition or disease associated with
the expression of PAP-2 in a subject, comprising administering to
said subject an effective amount of the composition of claim
32.
34. A composition of claim 32, further comprising a label.
35. A method of diagnosing a condition or disease associated with
the expression of PAP-2 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 consisting of 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 the animal, and c) screening the isolated antibodies with the
polypeptide, thereby identifying a polyclonal antibody which
specifically binds to a polypeptide comprising an amino acid
sequence of SEQ ID NO: 1.
37. A polyclonal antibody produced by a method of claim 36.
38. A composition comprising the polyclonal 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 consisting of 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 specifically binds to a
polypeptide comprising 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 monoclonal 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 comprising an amino acid
sequence of SEQ ID NO: 1 in a sample, the method comprising: a)
incubating the antibody of claim 11 with the 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 comprising an amino
acid sequence of SEQ ID NO: 1 in the sample.
45. A method of purifying a polypeptide comprising an amino acid
sequence of SEQ ID NO: 1 from a sample, the method comprising: a)
incubating the antibody of claim 11 with the 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 comprising 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.
Description
[0001] This application is a divisional application of U.S.
application Ser. No. 09/226,852, filed Jan. 7, 1999, which is a
divisional application of U.S. application Ser. No. 08/822,261,
filed Mar. 20, 1997, now U.S. Pat. No. 5,935,813, issued Aug. 10,
1999, all of which are entitled NOVEL HUMAN PANCREATITIS-ASSOCIATED
PROTEIN, and all of which are hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to nucleic acid and amino acid
sequences of a novel human pancreatitis-associated (PAP) protein,
which comprises a soluble C-type lectin. This novel human PAP
protein shares features with other proteins in the reg/PSP
multigene family which are involved in the regulation of cell
growth. The present invention relates to the use of these novel
sequences in the diagnosis, prevention, and treatment of
disease.
BACKGROUND ART
[0003] Lectins are proteins which are defined by their ability to
bind carbohydrates specifically and to agglutinate cells. Lectins
have been shown to be involved in a wide variety of cellular
functions, including cell-cell and cell-matrix interactions.
Lectins are widespread among plants, invertebrates, and
mammals.
[0004] Animal lectins have been grouped into four distinct
families: 1) C-type lectins, which include selectins; 2) P-type
lectins; 3) galectins (formerly termed S-type lectins or S-Lac
lectins); and 4) pentraxins [Barondes SH et al. (1994) J. Biol.
Chem. 269:20807-10]. The C-type lectins bind carbohydrate ligands
in a Ca.sup.2+-dependent manner and are structurally related to the
asialoglycoprotein receptor. Selectins, a subcategory of the C-type
lectins, are composite transmembrane molecules which are involved
in cell-cell interactions. The selectins include lymphocyte homing
receptors and platelet/endothelial cell surface receptors [Stoolnan
(1989) Cell 56:907-10].
[0005] C-type animal lectins contain Ca.sup.2+-dependent
carbohydrate-recognition domains (CRDs). The prototypical C-type
animal lectins are integral membrane proteins (e.g., the
asialoglycoprotein receptor). However, a number of soluble C-type
animal lectins have been identified. One group of soluble C-type
animal lectins, termed collectins or Group III C-type lectins,
comprises proteins having both lectin- (i.e., CRD) and
collagenous-like domains within a single polypeptide [Drickamer
(1993) Curr. Opin. Struct. Biol. 3:393]. Another group of soluble
C-type animal lectins, termed Group IV C-type lectins, comprises
free CRDs which are not joined to other polypeptide domains (other
than a signal peptide utilized in secretion) [Drickamer (1993),
supra]. The soluble C-type animal lectins comprising free CRDs
found in mammals are most closely related to proteins identified in
invertebrates and lower vertebrates (e.g., snakes).
[0006] Proteins recognized as members of the Group IV C-type
lectins appear to be members of a multigene family termed the
reg/PSP multigene family [Drickamer (1993), supra and Unno et al.
(1993) J. Biol. Chem. 268:15974]. The reg/PSP multigene family
comprises genes encoding secretory proteins which are expressed in
the pancreas. The ectopic expression (i.e., expression in a tissue
which does not normally express reg/PSP proteins) of some members
of the reg/PSP family is associated with disease states such as
tumors and Alzheimer's disease.
[0007] The first member of the reg/PSP multigene family was
identified in a cDNA library derived from rat regenerating
pancreatic islets [Terazono et al. (1988) J. Biol. Chem. 263:2111].
This gene was termed reg (regenerating gene) and is now known as
the regI.alpha. gene. The regI.alpha. gene product has been called
by different investigators reg protein, regI.alpha. protein,
lithostathine, islet cell regeneration factor (ICRF), pancreatic
stone protein (PSP) and pancreatic thread protein (PTP) [Terazono
et al. (1988), supra; Moriizumi et al. (1994) Biochem. Biophys.
Acta 1217:199; Dusetti et al. (1993) Biochem. Biophys. Acta
1174:99; Rouquier et al. (1991) J. Biol. Chem. 266:786; and de la
Monte et al. (1990) J. Clin. Invest. 86:1004]. The clear
association between reg gene expression and islet cell replication
in vitro has lead to the suggestion that the
regI.alpha./lithostathine protein has a growth-promoting activity
for islet .beta.-cells [Unno et al. (1993), supra]. Human
regI.alpha. mRNA is expressed in colon and rectal tumors although
it is not expressed in normal colon or rectal tissue. Thus, ectopic
expression of regI.alpha. protein is associated with tumorigenesis.
Elevated levels of regI.alpha. protein have been found in the
brains of patients suffering from Alzheimer's disease, as well as
in the brains of middle-aged individuals with Down's syndrome
[Ozturk et al. (1989) Proc. Natl. Acad. Sci. USA 86:419 and de la
Monte et al. (1990) J. Clin. Invest. 86:1004]. RegI.alpha. mRNA is
expressed in the developing human brain, but not in normal adult
brain, while expression of regI.alpha. is seen in adult brain which
undergoing regenerative sprouting. Given its pattern of expression
(e.g., expression in regenerating pancreatic islets and brain,
expression in tumors), it appears that regI.alpha. protein is
associated with cell growth.
[0008] Other members of the reg/PSP multigene family include genes
encoding pancreatitis-associated proteins (PAPs), which have been
identified in humans, nice and rats [Iovanna et al. (1991) J. Biol.
Chem. 266:24664; Orelle et al. (1992) J. Clin. Invest. 90:2284;
Itoh and Teraoka (1993) Biochem. Biophys. Acta 1172:184; and
Dusetti et al. (1994) Genomics 19:108]. The reg/lithostathine and
PAP proteins characterized to date share about 45-65% identity on
the amino acid level.
[0009] The PAP proteins are secretory proteins which are stored in
zymogen granules prior to secretion [Keim et al. (1991)
Gastroenterol. 100:775]. PAP is present at low levels in normal
pancreas but is rapidly overexpressed during the acute phase of
pancreatitis. PAP, like other members of the reg/PSP family, shares
sequence similarity with the carbohydrate-binding domain of C-type
lectins, which likely explains the ability of PAP to induce
aggregation of bacteria [Iovanna et al. (1991), supra]. The ability
to aggregate bacteria has lead to the suggestion that PAP is
involved in the control of bacterial proliferation, a frequent
complication of pancreatitis. PAP has been shown to be able to bind
lactose [Christa et al. (1994) FEBS Lett. 337:114].
[0010] Three PAP genes, PAP I-III, have been identified in rats.
All three PAP genes are expressed during the acute phase of
pancreatitis. Rat PAP I and PAP III are expressed constitutively in
the intestine, where their expression is induced by feeding. Rat
PAP II is not expressed in the intestine. Rat PAP I and PAP III
share 66% amino acid identity; rat PAP II and PAP III share 63%
amino acid identity; rat PAP I and PAP II share 58% amino acid
identity. A homolog of rat PAP I has been identified in cows [BPTP;
de la Monte et al. (1990), supra].
[0011] A human homolog of the rat PAP I gene, human PAP or human
PAP I, has been identified [Orelle et al. (1992) J. Clin. Invest.
90:2284]. The human PAP I protein is the same size as the rat PAP I
protein (175 amino acids), and the two proteins share 71% amino
acid identity, including conservation of 7 cystine residues. Both
the rat and the human PAP I proteins are synthesized as preproteins
having an N-terminal signal peptide of 26 amino acids. Expression
of the human PAP I mRNA is increased in necrohemorragic
pancreatitis. Serum levels of human PAP I were found to be near
background levels in normal individuals. In individuals suffering
from acute pancreatitis or acute exacerbations of chronic
pancreatitis, human PAP I levels increased 24-140 times the
background level [Orelle et al. (1992), supra]. Thus, human PAP I
appears to serve as a marker of acute pancreatitis.
[0012] The human PAP I gene is also referred to as the HIP gene
[Lasserre et al. (1992) Cancer Res. 52:5089]. The HIP gene was
identified by differential screening of a human primary liver
cancer (hepatocellular carcinoma) library. The human PAP I/HIP gene
is not expressed in normal adult or fetal liver as expression of
PAP I/HIP is limited to the pancreas and small intestine in normal
tissues. The ectopic expression of PAP I/HIP is associated with
tumorigenesis in the liver. In addition, PAP I/HIP mRNA is
expressed in human pancreatic cell hyperplasia (hyperinsulinism)
(Lasserre et al., supra).
[0013] Proteins expressed by the reg/PSP multigene family represent
an important family of proteins which are involved in the
maintenance of proper pancreatic function as well as in the
regulation of cell proliferation and/or differentiation. Discovery
of new molecules related to or in the mammalian reg/PSP multigene
family is useful for the development of new diagnostic or
therapeutic compositions.
DISCLOSURE OF THE INVENTION
[0014] The present invention features a novel Group IV C-type
lectin protein hereinafter designated human PAP-2 and characterized
as having similarity to the human PAP 1 protein. Human PAP-2 is a
member of the PAP branch of the reg/PSP multigene family.
[0015] Accordingly, the invention features a substantially purified
polypeptide having the amino acid sequence shown in SEQ ID NO: 1 or
fragments thereof. Preferred fragments of SEQ ID NO: 1 are
fragments which retain biological activity or immunological
activity (i.e., capable of eliciting anti-human PAP-2 antibodies).
Fragments of SEQ ID NO: 1 which are at least 15 amino acids, at
least 50 amino acids, at least 100 amino acids, at least 125 amino
acids and at least 200 amino acids in length are contemplated. The
invention specifically contemplates secretory (i.e., the signal
peptide is cleaved; E.sub.27-D.sub.175 of SEQ ID NO: 1) and
nonsecretory (i.e., signal peptide remains) forms of a
substantially purified human PAP-2 as well as any proteolytic
fragments thereof.
[0016] The present invention further provides isolated and
substantially purified polynucleotide sequences encoding the
polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or
fragments thereof. In a particular aspect, the polynucleotide is
the nucleotide sequence of SEQ ID NO: 2 or variants thereof. In
another embodiment, the present invention provides polynucleotides
comprising fragments of SEQ ID NO: 2 having a length of at least
seven nucleotides. The invention further contemplates fragments of
this polynucleotide sequence (i.e., SEQ ID NO: 2) that are at least
50 nucleotides, at least 100 nucleotides, at least 250 nucleotides,
at least 500 nucleotides and at least 750 nucleotides in
length.
[0017] In addition, the invention provides polynucleotide sequences
which hybridize under stringent conditions to the polynucleotide
sequence of SEQ ID NO: 2. In another embodiment the present
invention provides a composition comprising an isolated and
purified polynucleotide sequence encoding human PAP-2.
[0018] The invention provides polynucleotide sequences comprising
the complement of SEQ ID NO: 2 or variants thereof; these
complementary nucleic acid sequences may comprise the complement of
the entire nucleic acid sequence of SEQ ID NO: 2 or fragments
thereof. In another embodiment the present invention provides a
composition comprising an isolated and purified polynucleotide
sequence comprising the complement of SEQ ID NO: 2 or variants
thereof.
[0019] The invention additionally features nucleic acid sequences
encoding polypeptides, oligonucleotides, peptide nucleic acids
(PNA), fragments, portions or antisense molecules thereof, and
expression vectors and host cells comprising polynucleotides that
encode human PAP-2.
[0020] In another embodiment the present invention provides an
isolated polynucleotide comprising at least a portion of the
nucleic acid sequence of SEQ ID NO: 2 or variants thereof contained
on a recombinant expression vector. In yet another embodiment, the
expression vector containing the polynucleotide sequence is
contained within a host cell. The invention is not limited by the
nature of the host cell employed. For example, the host cell may be
an E. coli cell, a yeast cell, an insect cell, a mammalian cell,
etc.
[0021] The present invention also provides a method for producing a
polypeptide comprising the amino acid sequence of SEQ ID NO: 1 or
fragments thereof, the method comprising the steps of: a) culturing
the host cell containing an expression vector containing an
isolated polynucleotide encoding at least a fragment of the human
PAP-2 polypeptide under conditions suitable for the expression of
the polypeptide; and b) recovering the polypeptide from the host
cell culture.
[0022] In another embodiment, the invention provides a
pharmaceutical composition comprising a substantially purified
human PAP-2 protein having the amino acid sequence of SEQ ID NO: 1
in conjunction with a suitable pharmaceutical carrier.
[0023] The invention also provides a purified antibody which binds
specifically to a polypeptide comprising at least a portion of the
amino acid sequence of SEQ ID NO: 1.
[0024] Still further, the invention provides a purified agonist
which specifically binds to and modulates the activity of a
polypeptide comprising at least a portion of the amino acid
sequence of SEQ ID NO: 1. The present invention further provides a
pharmaceutical composition comprising a purified agonist which
specifically binds to and modulates the activity of a polypeptide
comprising at least a portion of the amino acid sequence of SEQ ID
NO: 1. In another embodiment, the invention provides a purified
antagonist which specifically binds to and modulates the activity
of a polypeptide comprising at least a portion of the amino acid
sequence of SEQ ID NO: 1. The present invention further provides a
pharmaceutical composition comprising a purified antagonist which
specifically binds to and modulates the activity of a polypeptide
comprising at least a portion of the amino acid sequence of SEQ ID
NO: 1.
[0025] The invention also provides a method for treating
pancreatitis (particularly for preventing or controlling bacterial
infections associated with pancreatitis) comprising administering
to a subject in need of such treatment an effective amount of a
pharmaceutical composition comprising a purified agonist which
specifically binds to and modulates the activity of a polypeptide
comprising at least a portion of the amino acid sequence of SEQ ID
NO: 1. The treatment of a variety of tumors, including but not
limited to tumors of the pancreas, esophagus lung and large
intestine (e.g., cecum and colon), using agonists as well as
antagonists of human PAP-2 is also contemplated by the present
invention.
[0026] The invention also provides a method for detection of
polynucleotides encoding human PAP-2 in a biological sample
comprising the steps of: a) hybridizing a polynucleotide sequence
encoding human PAP-2 (SEQ ID NO: 1) to nucleic acid material 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
human PAP-2 in the biological sample. In a preferred embodiment,
prior to hybridization, the nucleic acid material of the biological
sample is amplified by the polymerase chain reaction.
BRIEF DESCRIPTION OF DRAWINGS
[0027] FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO: 1)
and nucleic acid sequence (SEQ ID NO: 2) of human PAP-2. The
alignment was produced using MacDNASIS PRO software (Hitachi
Software Engineering Co., Ltd., San Bruno, Calif.).
[0028] FIG. 2 shows the amino acid sequence alignments among the
human PAP-2 protein (SEQ ID NO: 1) and human PAP 1 proteins (GI
262369, SEQ ID NO: 3; and GI 189601, SEQ ID NO: 4). The alignment
was produced using the multisequence alignment program of DNASTAR
software (DNASTAR Inc., Madison Wis.).
[0029] FIGS. 3A and 3B show the hydrophobicity plot (MacDNASIS PRO
software) for human PAP-2 (SEQ ID NO: 1) and human PAP I (SEQ ID
NO: 3), respectively. The positive X axis reflects amino acid
position, and the negative Y axis reflects hydrophobicity.
[0030] FIG. 4 shows the northern analysis for SEQ ID NO: 2. The
northern analysis was produced electronically using the LIFESEQ
database (Incyte Pharmaceuticals, Inc., Palo Alto, Calif.).
MODES FOR CARRYING OUT THE INVENTION
[0031] 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.
[0032] 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.
[0033] 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.
[0034] Definitions
[0035] "Nucleic acid sequence" as used herein refers to an
oligonucleotide, nucleotide, or polynucleotide, and fragments or
portions 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. Similarly, "amino acid sequence" as used herein
refers to an oligopeptide, peptide, polypeptide, or protein
sequence, and fragments or portions thereof, and to naturally
occurring or synthetic molecules.
[0036] A "composition comprising a given polynucleotide sequence"
as used herein refers broadly to any composition containing the
given polynucleotide sequence. The composition may comprise an
aqueous solution. Compositions comprising polynucleotide sequences
encoding human PAP-2 (SEQ ID NO: 1) or fragments thereof (e.g., SEQ
ID NO: 2 and fragments thereof) may be employed as hybridization
probes. In this case, the human PAP-2-encoding nucleotide sequences
are typically employed 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.).
[0037] 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, such as "polypeptide" or
"protein" are not meant to limit the amino acid sequence to the
complete, native amino acid sequence associated with the recited
protein molecule.
[0038] "Peptide nucleic acid", as used herein, refers to a molecule
which comprises an oligomer to which an amino acid residue, such as
lysine, and an amino group have been added. These small molecules,
also designated anti-gene agents, stop transcript elongation by
binding to their complementary strand of nucleic acid (Nielsen, P.
E. et al. (1993) Anticancer Drug Des. 8:53-63).
[0039] As used herein the "reg/PSP multigene family" refers to
genes encoding any of the following proteins: regenerating protein,
reg protein, regI.alpha. protein, regI.beta., lithostathine, islet
cell regeneration factor (ICRF), pancreatic stone protein (PSP),
pancreatic thread protein (PTP), HIP protein,
pancreatitis-associated protein (PAP) and the novel human PAP-2 of
the present invention, as well as other genes which encode proteins
sharing at least 21% identity with the listed proteins. Members of
the reg/PSP multigene family share a number of features including
expression in the pancreas and the presence of sequences conserved
among the CRD of C-type lectins. On the amino acid level, members
of the reg/PSP multigene family share about 30-87% identity.
Protein sequences comprising typical amino acid compositions (i.e.,
amino acids are present at their observed normal frequencies) which
share an identity of greater than 20% are defined as "homologous"
or related proteins; this assumes that only a limited number of
insertions and deletions are made to align the sequences being
compared [Creighton, Proteins, Structure and Molecular Properties,
2nd ed., W. H. Freeman, NY, pp. 108-109 (1993)].
[0040] Human PAP-2, as used herein, refers to the amino acid
sequences of substantially purified human PAP-2 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.
[0041] "Consensus", as used herein, refers to a nucleic acid
sequence which has been resequenced to resolve uncalled bases, or
which has been extended using XL-PCR (Perkin Elmer, Norwalk, Conn.)
in the 5' and/or the 3' direction and resequenced, or which has
been assembled from the overlapping sequences of more than one
Incyte clone using the GELVIEW fragment assembly system (GCG,
Madison, Wis.), or which has been both extended and assembled.
[0042] A "variant" of human PAP-2, 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. Similar 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.
[0043] A "deletion", as used herein, refers to a change in either
amino acid or nucleotide sequence in which one or more amino acid
or nucleotide residues, respectively, are absent.
[0044] 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 or nucleotide residues,
respectively, as compared to the naturally occurring molecule.
[0045] A "substitution", as used herein, refers to the replacement
of one or more amino acids or nucleotides by different amino acids
or nucleotides, respectively.
[0046] 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
human PAP-2, or any oligopeptide thereof, to induce a specific
immune response in appropriate animals or cells and to bind with
specific antibodies.
[0047] The term "agonist", as used herein, refers to a molecule
which, when bound to human PAP-2, causes a change in human PAP-2
which modulates the activity of human PAP-2. Agonists may include
proteins, nucleic acids, carbohydrates, or any other molecules
which bind to human PAP-2.
[0048] The terms "antagonist" or "inhibitor", as used herein, refer
to a molecule which, when bound to human PAP-2, blocks or modulates
the biological or immunological activity of human PAP-2.
Antagonists and inhibitors may include proteins, nucleic acids,
carbohydrates, or any other molecules which bind to human
PAP-2.
[0049] The term "modulate", as used herein, refers to a change or
an alteration in the biological activity of human PAP-2. Modulation
may be an increase or a decrease in protein activity, a change in
binding characteristics, or any other change in the biological,
functional, or immunological properties of human PAP-2.
[0050] The term "mimetic", as used herein, refers to a molecule,
the structure of which is developed from knowledge of the structure
of human PAP-2 or portions thereof and, as such, is able to effect
some or all of the actions of human PAP-2-like molecules.
[0051] The term "derivative", as used herein, refers to the
chemical modification of a nucleic acid encoding human PAP-2 or the
encoded human PAP-2. Illustrative of such modifications would be
replacement of hydrogen by an alkyl, acyl, or amino group. A
nucleic acid derivative would encode a polypeptide which retains
essential biological characteristics of the natural molecule.
[0052] 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.
[0053] "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.).
[0054] 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.
[0055] 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., C.sub.0t or R.sub.0t analysis) or between one
nucleic acid sequence present in solution and another nucleic acid
sequence immobilized on a solid support (e.g., membranes, filters,
chips, pins or glass slides to which cells have been fixed for in
situ hybridization).
[0056] 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, for 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 importance in
amplification reactions, which depend upon binding between nucleic
acids strands.
[0057] 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 is one that at
least partially inhibits an identical sequence from hybridizing to
a target nucleic acid; it 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 probe will
compete for and inhibit the binding (i.e., the hybridization) of a
completely homologous sequence or probe 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.
[0058] As known in the art, numerous equivalent conditions may be
employed to comprise either low or high stringency conditions.
Factors such as the length and nature (DNA, RNA, base composition)
of the sequence, nature of the target (DNA, RNA, base composition,
presence in solution or immobilization, etc.), and the
concentration of the salts and other components (e.g., the presence
or absence of formamide, dextran sulfate and/or polyethylene
glycol) are considered and the hybridization solution may be varied
to generate conditions of either low or high stringency different
from, but equivalent to, the above listed conditions.
[0059] The term "stringent conditions", as used herein, is the
"stringency" which occurs within a range from about Tm-5.degree. C.
(5.degree. C. below the melting temperature (Tm) of the probe) to
about 20.degree. C. to 25.degree. C. below Tm. As will be
understood by those of skill in the art, the stringency of
hybridization may be altered in order to identify or detect
identical or related polynucleotide sequences. Under "stringent
conditions" SEQ ID NO: 2 or fragments thereof will hybridize to its
exact complement and closely related sequences. The stringent
conditions are chosen such that SEQ ID NO: 2 or fragments thereof
will hybridize to sequences encoding human PAP-2 but not to
sequences encoding human PAP I proteins (i.e., SEQ ID NOs: 5 and 6
or their RNA equivalents). When fragments of SEQ ID NO: 2 are
employed in hybridization reactions, the stringent conditions
include the choice of fragments of SEQ ID NO: 2 to be used.
Fragments of SEQ ID NO: 2 which contain unique sequences (i.e.,
regions which are either non-homologous to or which contain less
than about 50% homology or complementarity with SEQ ID NOS: 4
and/or 6 are preferentially employed. SEQ ID NOS:5 and 6 represent
DNA sequences encoding human PAP I proteins; these DNA sequences
can be found in GenBank under GI 262368 (SEQ ID NO: 5) and GI
189600 (SEQ ID NO: 6), respectively.
[0060] The term "antisense", as used herein, refers to 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 may be produced by any method, including
synthesis by ligating the gene(s) of interest in a reverse
orientation to a viral promoter which permits the synthesis of a
complementary strand. Once introduced into a cell, this transcribed
strand combines with natural sequences produced by the cell to form
duplexes. These duplexes then block either the further
transcription or translation. In this manner, mutant phenotypes may
be generated. The designation "negative" is sometimes used in
reference to the antisense strand, and "positive" is sometimes used
in reference to the sense strand.
[0061] 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 four 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 human PAP-2
and fragments thereof.
[0062] "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 host cell being transformed and may include, but is not
limited to, viral infection, electroporation, 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.
[0063] The term "antigenic determinant", as used herein, refers to
that portion of a molecule that makes contact with a particular
antibody (i.e., an epitope). 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; 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.
[0064] The terms "specific binding" or "specifically binding", as
used herein, in reference to the interaction of an antibody and a
protein or peptide, mean that the interaction is dependent upon the
presence of a particular structure (i.e., the antigenic determinant
or epitope) on the protein; in other words, the antibody is
recognizing and binding to a specific protein structure rather than
to proteins in general. 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.
[0065] The term "sample", as used herein, is used in its broadest
sense. A biological sample suspected of containing nucleic acid
encoding human PAP-2 or fragments thereof may comprise a cell,
chromosomes isolated from a cell (e.g., a spread of metaphase
chromosomes), genomic DNA (in solution or bound to a solid support
such as for Southern analysis), RNA (in solution or bound to a
solid support such as for northern analysis), cDNA (in solution or
bound to a solid support), an extract from cells or a tissue, and
the like.
[0066] 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 human PAP-2
in a sample and thereby correlates with expression of the
transcript from the polynucleotide encoding the protein.
[0067] "Alterations" in the polynucleotide of SEQ ID NO: 2, as used
herein, comprise any alteration in the sequence of polynucleotides
encoding human PAP-2 including deletions, insertions, and point
mutations that may be detected using hybridization assays. Included
within this definition is the detection of alterations to the
genomic DNA sequence which encodes human PAP-2 (e.g., by
alterations in the pattern of restriction fragment length
polymorphisms capable of hybridizing to SEQ ID NO: 2), the
inability of a selected fragment of SEQ ID NO: 2 to hybridize to a
sample of genomic DNA (e.g., using allele-specific oligonucleotide
probes), and improper or unexpected hybridization, such as
hybridization to a locus other than the normal chromosomal locus
for the polynucleotide sequence encoding human PAP-2 (e.g., using
fluorescent in situ hybridization [FISH] to metaphase chromosomes
spreads).
[0068] 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 human PAP-2 polypeptides can be prepared using
intact polypeptides or fragments containing small peptides of
interest as the immunizing antigen. The polypeptide or peptide used
to immunize an animal can be derived from the transition 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. The
coupled peptide is then used to immunize the animal (e.g., a mouse,
a rat, or a rabbit).
[0069] 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.
[0070] The Invention
[0071] Given the role C-type lectins play in regulating cell growth
and development, the discovery of new molecules related to or in
the C-type lectin gene family, and in the human reg/PSP multigene
family in particular, is useful for developing diagnostic or
therapeutic compositions directed at detecting or preventing
neoplasia and/or metastasis. In addition, overexpression of PAP
proteins is seen in acute pancreatitis and thus, novel human PAP
genes are useful for developing diagnostic or therapeutic
compositions directed at detection and treatment of pancreatitis
and other disorders of the pancreas (e.g., pancreatic cell
hyperplasia or hyperinsulinism).
[0072] As aberrant (e.g., ectopic) expression of members within the
reg/PSP gene family is associated with tumorigenesis, the discovery
of new molecules related to or in the reg/PSP gene family is useful
for developing diagnostic or therapeutic compositions directed at a
variety of tumors. Furthermore, new molecules related to or in the
reg/PSP gene family are useful for developing diagnostic or
therapeutic compositions directed at correcting diseases associated
with the overexpression or underexpresssion of reg/PSP
proteins.
[0073] The invention is based on the discovery of a novel human
C-type lectin which is a member of the human reg/PSP multigene
family integral membrane protein (human PAP-2), the polynucleotides
encoding human PAP-2, and the use of these compositions for the
diagnosis, prevention, or treatment of diseases associated with
abnormal pancreatic tissue, including pancreatic tumors. In
addition, as mRNA encoding human PAP-2 is found in a number of
other tumors, human PAP-2 serves as a marker for cancerous cells,
particularly esophageal, lung and intestinal (cecal and colon)
tumor cells.
[0074] Nucleic acids encoding the human PAP-2 of the present
invention were first identified in Incyte Clone 2072483 from the
ISLTNOT01 cDNA library through a computer-generated search for
amino acid sequence alignments. A consensus sequence, SEQ ID NO: 2,
was derived from the following overlapping and/or extended nucleic
acid sequences: Incyte Clones 225534 (PANCNOT01), 227084
(PANCNOT01), 229613 (PANCNOT01), and 2072483 (ISLNOT01).
[0075] 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. Human PAP-2 contains 175 amino acids, a number of
which are residues shown to be conserved among mammalian PAP and
Reg proteins and among the CRD of C-type animal lectins. The
conserved sequence motif found in C-type CRDs is described by
Drickamer [Curr. Opin. Struc. Biol. (1993) 3:393]. Sequences
corresponding to this conserved motif within the human PAP-2 of the
present invention include G.sub.48, C.sub.68, G.sub.105, D.sub.108,
W.sub.133, G.sub.144, C.sub.146, W.sub.158, D.sub.160, C.sub.163,
and C.sub.171. A version of this motif is found in the PROSITE
database as the C-type lectin domain signature (CTL); residues
146-171 of SEQ ID NO: 1 comprise the CTL listed in the PROSITE
database.
[0076] As shown in FIG. 2, human PAP-2 contains sequences that
closely match the EGF-like domain cysteine pattern signature at
residues 40-51 of SEQ ID NO: 1. Human PAP-2 contains sequences that
closely match the glucagon/GIP/secretin/VIP family signature
consensus sequence (i.e., residues 82-104 of SEQ ID NO: 1).
Proteins within the glucagon/GIP/secretin/VIP family, like the PAP
proteins including PAP-2, are expressed in the intestine or the
pancreas.
[0077] The amino-terminal 26 residues of the human PAP-2 of the
present invention comprise a putative signal sequence, a feature
common to mammalian Reg and PAP proteins in general and to human
and rat PAP I proteins in particular (Orelle et al., supra). The
first amino acid of the mature or processed form of PAP-2 is
therefore E.sub.27 of SEQ ID NO: 1.
[0078] Human PAP-2 contains eight cysteine residues (i.e.,
C.sub.17, C.sub.22, C.sub.40, C.sub.51, C.sub.68, C.sub.146,
C.sub.163, and C.sub.171). In addition to providing sites for
disulfide bond formation, the cysteine residues provide potential
sites for palmitoylation. Seven of the eight cysteine residues
found in human PAP-2 are conserved in location with cysteine
residues found in the human PAP I (i.e., C.sub.17, C.sub.40,
C.sub.51, C.sub.68, C.sub.146, C.sub.163, and C.sub.171 of human
PAP-2). The human PAP-2 of the present invention contains numerous
potential O-linked glycosylation sites (i.e., serine and threonine
residues). Human PAP-2 has a single potential N-linked
glycoslyation site (i.e., Asn-X-Ser/Thr) (i.e., N.sub.136) which is
conserved in location with the single N-linked glycoslyation site
found in the human PAP I protein. In addition, the human PAP-2 of
the present invention contains numerous potential phosphorylation
sites (i.e., typically the hydroxyl groups of serine, threonine and
tyrosine residues although asparagine, histidine and lysine
residues may also be phosphorylated), including two potential sites
for phosphorylation by cAMP-dependent protein kinase (e.g.,
R-X-S/T) (i.e., S.sub.73 and T.sub.153). These sites are conserved
in location with the two potential cAMP-dependent protein kinase
phosphorylation sites found in the human PAP I protein.
[0079] Human PAP-2 has chemical and structural homology with the
human PAP I protein (GI 262369; SEQ ID NO: 3) (Orelle et al.,
supra). In particular, human PAP-2 and PAP I share 85% identity and
92% similarity. A pair of residues are said to be similar if they
represent conservative substitutions. FIG. 2 provides an alignment
between the amino acid sequences of SEQ ID NOS: 1 and 3.
[0080] The human PAP-2 protein of the present invention, like the
human PAP 1 protein (GI 262369), has a nearly neutral isoelectric
point (pI) (human PAP-2 has a pI of 6.84 and PAP I has a pI of
7.54). As illustrated by FIGS. 3A and 3B, human PAP-2 and PAP I (GI
262369) have similar hydrophobicity plots.
[0081] Northern analysis (FIG. 4) shows the expression of human
PAP-2-encoding sequences in various libraries, 25% of which are
cancerous and 20% of which are involved with the immune response,
including inflammatory and/or autoiimune disease (e.g., ulcerative
colitis, Crohn's disease). Of particular note is the expression of
human PAP-2 mRNA in pancreatic tumor ({fraction (1/20)}),
esophageal tumor ({fraction (1/20)}), cecal tumor ({fraction
(1/20)}) and colon tumor ({fraction (1/20)}) libraries. Human
PAP-2, like other members of the reg/PSP multigene family, is
expressed in the pancreas. Human PAP-2 is expressed at relatively
high levels in apparently normal pancreatic tissue (i.e., tissue
not identified as being tumorous or otherwise obviously diseased)
while the expression of other PAP mRNAs (e.g., human, mouse and rat
PAP 1) is reported to be low in normal pancreatic tissue and
elevated during the acute phase of pancreatitis (Itoh and Teraoka,
supra). Human PAP-2 is also expressed at relatively high levels in
the small intestine, a feature in common with human, mouse and rat
PAP I (Itoh and Teraoka, supra). Thus, human PAP-2 sequences serve
as a marker for the small intestine, especially the ileum
[0082] The invention also encompasses human PAP-2 variants. A
preferred human PAP-2 variant is one having at least 86%, and more
preferably 90%, amino acid sequence identity to the human PAP-2
amino acid sequence (SEQ ID NO: 1). A most preferred human PAP-2
variant is one having at least 95% amino acid sequence identity to
SEQ ID NO: 1.
[0083] The invention also encompasses polynucleotides which encode
human PAP-2. Accordingly, any nucleic acid sequence which encodes
the amino acid sequence of human PAP-2 can be used to generate
recombinant molecules which express human PAP-2. 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.
[0084] 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 human PAP-2, 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 human PAP-2, and all such variations are to be considered
as being specifically disclosed.
[0085] Although nucleotide sequences which encode human PAP-2 and
its variants are preferably capable of hybridizing to the
nucleotide sequence of the naturally occurring human PAP-2 under
appropriately selected conditions of stringency, it may be
advantageous to produce nucleotide sequences encoding human PAP-2
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 human PAP-2
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.
[0086] The invention also encompasses production of DNA sequences,
or portions thereof, which encode human PAP-2 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 at the time of the filing of this application. Moreover,
synthetic chemistry may be used to introduce mutations into a
sequence encoding IMP- or any portion thereof.
[0087] 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. Hybridization conditions are
based on the melting temperature (Tm) of the nucleic acid binding
complex or probe, 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), and may be used at a defined stringency.
[0088] Altered nucleic acid sequences encoding human PAP-2 which
are encompassed by the invention include deletions, insertions, or
substitutions of different nucleotides resulting in a
polynucleotide that encodes the same or a functionally equivalent
human PAP-2. The encoded protein may also contain deletions,
insertions, or substitutions of amino acid residues which produce a
silent change and result in a functionally equivalent human PAP-2.
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 activity of human PAP-2 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; phenylalanine and tyrosine.
[0089] Also included within the scope of the present invention are
alleles of the genes encoding human PAP-2. As used herein, an
"allele" or "allelic sequence" is an alternative form of the gene
which may result from at least one mutation in the nucleic acid
sequence. Alleles may result in altered mRNAs or polypeptides whose
structure or function may or may not be altered. Any given 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 in a given sequence.
[0090] Methods for DNA sequencing which are well known and
generally available in the art may be used to practice any
embodiments of the invention. The methods may employ such enzymes
as the Klenow fragment of DNA polymerase I, SEQUENASE (US
Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer),
thermostable T7 polymerase (Amersham, Chicago, Ill.), or
combinations of recombinant polymerases and proofreading
exonucleases such as the ELONGASE amplification system marketed by
Gibco BRL (Gaithersburg, Md.). Preferably, the process is automated
with machines such as the Hamilton MICROLAB 2200 (Hamilton, Reno,
Nev.), Peltier PTC200 thermal cycler (M J Research, Watertown,
Mass.), and ABI 377 DNA sequencers (Perkin Elmer).
[0091] The nucleic acid sequences encoding human PAP-2 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 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.
[0092] 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 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.
[0093] 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 portion of the DNA molecule before performing PCR.
[0094] 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 libraries to walk in genomic DNA (Clontech, Palo
Alto, Calif.). This process avoids the need to screen libraries and
is useful in finding intron/exon junctions.
[0095] When 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 the 5' and 3' non-transcribed regulatory regions.
[0096] 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 and SEQUENCE NAVIGATOR, 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.
[0097] In another embodiment of the invention, polynucleotide
sequences or fragments thereof which encode human PAP-2, or fusion
proteins or functional equivalents thereof, may be used in
recombinant DNA molecules to direct expression of human PAP-2 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 human
PAP-2.
[0098] As will be understood by those of skill in the art, it may
be advantageous to produce human PAP-2-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 a recombinant RNA transcript having desirable properties,
such as a half-life which is longer than that of a transcript
generated from the naturally occurring sequence.
[0099] The nucleotide sequences of the present invention can be
engineered using methods generally known in the art in order to
alter human PAP-2 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, or introduce mutations,
and so forth.
[0100] In another embodiment of the invention, natural, modified,
or recombinant nucleic acid sequences encoding human PAP-2 may be
ligated to a heterologous sequence to encode a fusion protein. For
example, to screen peptide libraries for inhibitors of human PAP-2
activity, it may be useful to encode a chimeric human PAP-2 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 human PAP-2 encoding sequence and the
heterologous protein sequence, so that human PAP-2 may be cleaved
and purified away from the heterologous moiety.
[0101] In another embodiment, sequences encoding human PAP-2 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
human PAP-2, or a portion 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).
[0102] The newly synthesized peptide may be substantially purified
by preparative high performance liquid chromatography (e.g.,
Creighton, T. (1983) Proteins, Structures and Molecular Principles,
W H 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; Creighton,
supra). Additionally, the amino acid sequence of human PAP-2, 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.
[0103] In order to express a biologically active human PAP-2, the
nucleotide sequences encoding human PAP-2 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.
[0104] Methods which are well known to those skilled in the art may
be used to construct expression vectors containing sequences
encoding human PAP-2 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.
[0105] A variety of expression vector/host systems may be utilized
to contain and express sequences encoding human PAP-2. 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 (e.g., cauliflower mosaic virus, CaMV; tobacco
mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti
or pBR322 plasmids); or animal cell systems.
[0106] 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 phagemid (Stratagene, LaJolla, 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 human PAP-2, vectors based on SV40 or EBV may be used with
an appropriate selectable marker.
[0107] In bacterial systems, a number of expression vectors may be
selected depending upon the use intended for human PAP-2. For
example, when large quantities of human PAP-2 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
(Stratagene), in which the sequence encoding human PAP-2 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.
[0108] 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. In cases where plant expression vectors are used, the
expression of sequences encoding human PAP-2 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.
[0109] An insect system may also be used to express human PAP-2.
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 human PAP-2 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 human
PAP-2 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 human PAP-2 may be expressed
(Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci.
91:3224-3227).
[0110] 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 human PAP-2 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 human PAP-2 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.
[0111] Specific initiation signals may also be used to achieve more
efficient translation of sequences encoding human PAP-2. Such
signals include the ATG initiation codon and adjacent sequences. In
cases where sequences encoding human PAP-2, 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 portion 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).
[0112] 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 such as CHO (ATCC CCL 61 and CRL 9618), HeLa (ATCC CCL
2), MDCK (ATCC CCL 34 and CRL 6253), HEK 293 (ATCC CRL 1573), and
WI-38 (ATCC CCL 75) (ATCC: American Type Culture Collection,
Rockville, Md.), which have specific cellular machinery and
characteristic mechanisms for such post-translational activities,
may be chosen to ensure the correct modification and processing of
the foreign protein.
[0113] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express human PAP-2 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.
[0114] 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. 159:1-14) 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).
[0115] 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 human PAP-2 is inserted within a marker gene
sequence, recombinant cells containing sequences encoding human
PAP-2 can be identified by the absence of marker gene function.
Alternatively, a marker gene can be placed in tandem with a
sequence encoding human PAP-2 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.
[0116] Alternatively, host cells which contain the nucleic acid
sequence encoding human PAP-2 and express human PAP-2 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, or chip based
technologies for the detection and/or quantification of nucleic
acid or protein.
[0117] The presence of polynucleotide sequences encoding human
PAP-2 can be detected by DNA-DNA or DNA-RNA hybridization or
amplification using probes or portions or fragments of
polynucleotides encoding human PAP-2. Nucleic acid amplification
based assays involve the use of oligonucleotides or oligomers based
on the sequences encoding human PAP-2 to detect transformants
containing DNA or RNA encoding human PAP-2. As used herein
"oligonucleotides" or "oligomers" refer to a nucleic acid sequence
of at least about 10 nucleotides and as many as about 60
nucleotides, preferably about 15 to 30 nucleotides, and more
preferably about 20-25 nucleotides, which can be used as a probe or
amplimer.
[0118] A variety of protocols for detecting and measuring the
expression of human PAP-2, 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 human PAP-2 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).
[0119] 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 human PAP-2 include oligolabeling, nick
translation, end-labeling or PCR amplification using a labeled
nucleotide. Alternatively, the sequences encoding human PAP-2, or
any portions 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, include radionuclides, enzymes,
fluorescent, chemiluminescent, or chromogenic agents as well as
substrates, cofactors, inhibitors, magnetic particles, and the
like.
[0120] Host cells transformed with nucleotide sequences encoding
human PAP-2 may be cultured under conditions suitable for the
expression and recovery of the protein from cell culture. The
protein produced by a recombinant 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 human PAP-2 may be
designed to contain signal sequences which direct secretion of
human PAP-2 through a prokaryotic or eukaryotic cell membrane. The
signal sequence may be encoded by the expression vector (i.e., a
heterologous signal sequence) or by a sequence encoding the
naturally occurring PAP-2 signal sequence (i.e., sequences encoding
amino acid residues 1-26 of SEQ ID NO: 1).
[0121] Other recombinant constructions may be used to join
sequences encoding human PAP-2 to nucleotide sequences 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 human PAP-2 may be used
to facilitate purification. One such expression vector provides for
expression of a fusion protein containing human PAP-2 and a nucleic
acid encoding 6 histidine residues preceding a thioredoxin or an
enterokinase cleavage site. The histidine residues facilitate
purification on IMIAC (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 human PAP-2 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).
[0122] In addition to recombinant production, fragments of human
PAP-2 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
an Applied Biosystems 431A peptide synthesizer (Perkin Elmer).
Various fragments of human PAP-2 may be chemically synthesized
separately and combined using chemical methods to produce the full
length molecule.
[0123] Therapeutics
[0124] Based on the chemical and structural homology among human
PAP-2 (SEQ ID NO: 1) and the human PAP I protein (SEQ ID NOs: 3 and
4), human PAP-2 appears to be a member of the reg/PSP multigene
family and in particular a member of the PAP branch of this family.
Human PAP I (and rat PAP I, mouse PAP I and rat PAP III) have been
shown to be expressed at high levels during the acute phase of
pancreatitis and in normal small intestine. Based on the homology
between human PAP-2 and human PAP I and the pattern of PAP-2 mRNA
expression, human PAP-2 protein and nucleotide sequences are
believed to serve as a marker of acute pancreatitis. Human PAP-2
also shares chemical and structural homology with rat PAP I; rat
PAP I has been shown to have the ability to aggregate bacteria.
Therefore, human PAP-2 may be used therapeutically to control
bacterial proliferation, a frequent complication of pancreatitis.
The ectopic expression of members of the reg/PSP multigene family,
including the PAP genes, is associated with a variety of disease
states [Watanabbe et al. (1990) J. Biol. Chem. 265:7432; Lasserre
et al., supra]. Expression of human PAP I in adult liver is
associated with liver cancer; human PAP I is not expressed in
normal adult or fetal liver (Lasserre et al., supra). As shown
herein, PAP-2 is expression in a variety of tumor types, including
pancreatic, esophageal, lung, cecal and colon tumors. Therefore,
inhibition of human PAP-2 expression (e.g., using antisense PAP-2
transcripts), particularly in tissues in which PAP-2 is normally
absent or expressed at low levels (i.e., ectopic expression), may
be therapeutic. The PAP-2 amino acid and nucleic acid sequences
provided herein provide a means of producing therapeutic compounds
for the treatment of disease states associated with altered PAP-2
expression.
[0125] Therefore, in one embodiment, human PAP-2 or a fragment or
derivative thereof may be administered to a subject to control the
bacterial proliferation associated with infections or conditions
such as pancreatitis.
[0126] In another embodiment, a vector capable of expressing human
PAP-2, or a fragment or a derivative thereof, may also be
administered to a subject to treat the bacterial proliferation
described above.
[0127] In another embodiment, human PAP-2 may be administered in
combination with other conventional chemotherapeutic agents
(including antimicrobial agents). The combination of therapeutic
agents having different mechanisms of action will have synergystic
effects allowing for the use of lower effective doses of each agent
and lessening side effects.
[0128] In one aspect, agonists of human PAP-2 may be used to
increase the activity of human PAP-2 in cells having reduced human
PAP-2 levels. Antibodies which are specific for human PAP-2 may be
used directly as an agonist, or indirectly as a targeting or
delivery mechanism for bringing a pharmaceutical agent to cells or
tissue which express human PAP-2.
[0129] In one embodiment, antagonists or inhibitors of human PAP-2
may be administered to a subject to treat or prevent tumors,
particularly pancreatic, esophageal, lung, cecal and colon tumors
as well as Crohn's disease and ulcerative colitis.
[0130] In another embodiment, a vector expressing antisense of the
polynucleotide encoding human PAP-2 may be administered to a
subject to treat or prevent tumors, particularly pancreatic,
esophageal, lung, cecal and colon tumors as well as Crohn's disease
and ulcerative colitis.
[0131] Antagonists or inhibitors of human PAP-2 may be produced
using methods which are generally known in the art. In particular,
purified human PAP-2 may be used to produce antibodies or to screen
libraries of pharmaceutical agents to identify those which
specifically bind human PAP-2.
[0132] Antibodies which are specific for human PAP-2 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 human PAP-2. The antibodies 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
reduce or abolish human PAP-2 activity) are especially preferred
for therapeutic use.
[0133] For the production of antibodies, various hosts including
goats, rabbits, rats, mice, humans, and others, may be immunized by
injection with human PAP-2 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.
[0134] It is preferred that the peptides, fragments, or
oligopeptides used to induce antibodies to human PAP-2 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 human PAP-2 amino acids may be fused with those of another
protein such as keyhole limpet hemocyanin and antibody produced
against the chimeric molecule.
[0135] Monoclonal antibodies to human PAP-2 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).
[0136] 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 human PAP-2-specific single chain
antibodies. Antibodies with related specificity, but of distinct
idiotypic composition, may be generated by chain shuffling from
random combinatorial immunoglobulin libraries (Burton D. R. (1991)
Proc. Natl. Acad. Sci. 88:11120-3).
[0137] Antibodies may also be produced by inducing in vivo
production in the lymphocyte population or by screening recombinant
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).
[0138] Antibody fragments which contain specific binding sites for
human PAP-2 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).
[0139] 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 human PAP-2 and its
specific antibody. A two-site, monoclonal-based immunoassay
utilizing monoclonal antibodies reactive to two non-interfering
human PAP-2 epitopes is preferred, but a competitive binding assay
may also be employed (Maddox, supra).
[0140] In another embodiment of the invention, the polynucleotides
encoding human PAP-2, or any fragment thereof, or antisense
molecules, may be used for therapeutic purposes. In one aspect,
antisense to the polynucleotide encoding human PAP-2 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 human
PAP-2. Thus, antisense molecules may be used to modulate human
PAP-2 activity, or to achieve regulation of gene function. Such
technology is now well known in the art, and sense or antisense
oligomers or larger fragments, can be designed from various
locations along the coding or control regions of sequences encoding
human PAP-2.
[0141] Expression vectors derived from retroviruses, 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 recombinant vectors
which will express antisense molecules complementary to the
polynucleotides of the gene encoding human PAP-2. These techniques
are described both in Sambrook et al. (supra) and in Ausubel et al.
(supra).
[0142] Genes encoding human PAP-2 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 human PAP-2.
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.
[0143] As mentioned above, modifications of gene expression can be
obtained by designing antisense molecules, DNA, RNA, or PNA, to the
control regions of the gene encoding human PAP-2, i.e., the
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 antisense molecules may also be designed to
block translation of mRNA by preventing the transcript from binding
to ribosomes.
[0144] 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 human
PAP-2.
[0145] 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.
[0146] Antisense 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
human PAP-2. 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
antisense RNA constitutively or inducibly can be introduced into
cell lines, cells, or tissues.
[0147] 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.
[0148] 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
and by liposome injections may be achieved using methods which are
well known in the art.
[0149] 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.
[0150] 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 human PAP-2, antibodies to human PAP-2, mimetics,
agonists, antagonists, or inhibitors of human PAP-2. 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.
[0151] 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.
[0152] 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 on techniques for
formulation and administration may be found in the latest edition
of Remington's Pharmaceutical Sciences (Maack Publishing Co.,
Easton, Pa.).
[0153] Pharmaceutical compositions for oral administration can be
formulated using pharmaceutically acceptable carriers well known in
the art in dosages suitable for oral administration. 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.
[0154] 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.
[0155] 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.
[0156] 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.
[0157] 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. 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.
[0158] 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.
[0159] 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.
[0160] 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.
[0161] 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 human PAP-2, such
labeling would include amount, frequency, and method of
administration.
[0162] 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.
[0163] 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.
[0164] A therapeutically effective dose refers to that amount of
active ingredient, for example human PAP-2 or fragments thereof,
antibodies of human PAP-2, agonists, antagonists or inhibitors of
human PAP-2, 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 ratio, LD50/ED50.
[0165] 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 includes 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.
[0166] 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.
[0167] 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.
[0168] Diagnostics
[0169] In another embodiment, antibodies which specifically bind
human PAP-2 may be used for the diagnosis of conditions or diseases
characterized by expression of human PAP-2, or in assays to monitor
patients being treated with human PAP-2, 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 human PAP-2 include methods
which utilize the antibody and a label to detect human PAP-2 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.
[0170] A variety of protocols including ELISA, RIA, and FACS for
measuring human PAP-2 are known in the art and provide a basis for
diagnosing altered or abnormal levels of human PAP-2 expression.
Normal or standard values for human PAP-2 expression are
established by combining body fluids or cell extracts taken from
normal mammalian subjects, preferably human, with antibody to human
PAP-2 under conditions suitable for complex formation. The amount
of standard complex formation may be quantified by various methods,
preferably by photometric means. Quantities of human PAP-2
expressed in subject samples from biopsied tissues are compared
with the standard values. Deviation between standard and subject
values establishes the parameters for diagnosing disease.
[0171] In another embodiment of the invention, the polynucleotides
encoding human PAP-2 are used for diagnostic purposes. The
polynucleotides which may be used include oligonucleotide
sequences, antisense RNA and DNA molecules, and PNAs. The
polynucleotides may be used to detect and quantitate gene
expression in biopsied tissues in which expression of human PAP-2
may be correlated with disease. The diagnostic assay may be used to
distinguish between absence, presence, and excess expression of
human PAP-2, and to monitor regulation of human PAP-2 levels during
therapeutic intervention.
[0172] In one aspect, hybridization with PCR probes which are
capable of detecting polynucleotide sequences, including genomic
sequences, encoding human PAP-2 or closely related molecules, may
be used to identify nucleic acid sequences which encode human
PAP-2. 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 human PAP-2, alleles, or related sequences.
[0173] 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 human PAP-2 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 human PAP-2.
[0174] Means for producing specific hybridization probes for DNAs
encoding human PAP-2 include the cloning of nucleic acid sequences
encoding human PAP-2 or human PAP-2 derivatives into vectors for
the production of mRNA probes. Such vectors are known in the art,
are 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.
[0175] Polynucleotide sequences encoding human PAP-2 may be used
for the diagnosis of conditions or diseases which are associated
with expression of human PAP-2. Examples of such conditions or
diseases include cancers of the pancreas, esophagus, lung, cecum
and colon and Crohn's disease and ulcerative colitis. The
polynucleotide sequences encoding human PAP-2 may be used in
Southern or northern analysis, dot blot, or other membrane-based
technologies; in PCR technologies; or in dip stick, pin, ELISA or
chip assays utilizing fluids or tissues from patient biopsies to
detect altered human PAP-2 expression. Such qualitative or
quantitative methods are well known in the art.
[0176] In a particular aspect, the nucleotide sequences encoding
human PAP-2 provide the basis for assays that detect activation or
induction of various cancers, particularly those mentioned above;
in addition the lack of expression of human PAP-2 may be detected
using the human PAP-2-encoding nucleotide sequences disclosed
herein. The nucleotide sequences encoding human PAP-2 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 human PAP-2 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.
[0177] In order to provide a basis for the diagnosis of disease
associated with expression of human PAP-2, 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 human PAP-2, 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.
[0178] 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.
[0179] With respect to cancer, the presence of a relatively low or
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.
[0180] Additional diagnostic uses for oligonucleotides designed
from the sequences encoding human PAP-2 may involve the use of PCR.
Such oligomers may be chemically synthesized, generated
enzymatically, or produced from a recombinant source. 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.
[0181] Methods which may also be used to quantitate the expression
of human PAP-2 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 colorimetric response gives
rapid quantitation.
[0182] In another embodiment of the invention, the nucleic acid
sequences which encode human PAP-2 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 or to a specific region of the chromosome
using well known techniques. Such techniques include FISH, FACS, or
artificial chromosome constructions, such as yeast artificial
chromosomes, bacterial artificial chromosomes, 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.
[0183] FISH (as described in Verma 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 the
1994 Genome Issue of Science (265: 1981f). Correlation between the
location of the gene encoding human PAP-2 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.
[0184] 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.
[0185] In another embodiment of the invention, human PAP-2, 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 human PAP-2 and the agent being tested, may be
measured.
[0186] 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
human PAP-2 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 human PAP-2, or
fragments thereof, and washed. Bound human PAP-2 is then detected
by methods well known in the art. Purified human PAP-2 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.
[0187] In another embodiment, one may use competitive drug
screening assays in which neutralizing antibodies capable of
binding human PAP-2 specifically compete with a test compound for
binding human PAP-2. In this manner, the antibodies can be used to
detect the presence of any peptide which shares one or more
antigenic determinants with human PAP-2.
[0188] In additional embodiments, the nucleotide sequences which
encode human PAP-2 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.
[0189] The examples below are provided to illustrate the subject
invention and are not included for the purpose of limiting the
invention.
EXAMPLES
[0190] I ISLTNOT01 cDNA Library Construction
[0191] The ISLTNOT01 cDNA library was constructed from total RNA
isolated from microscopically normal pancreatic islet cells
(specimen #A143, Pfizer, Inc., New York, N.Y.). The frozen tissue
was homogenized and lysed using a Polytron PT-3000 homogenizer
(Brinkmann Instruments, Westbury, N.J.) in guanidinium
isothiocyanate solution. The lysate was centrifuged over a 5.7 M
CsCl cushion using an SW28 rotor in an 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.
Extraction and precipitation were repeated. The mRNA was isolated
with the OLIGOTEX kit (QIAGEN, Inc. Chatsworth, Calif.) and used to
construct the cDNA library.
[0192] The mRNA was handled according to the recommended protocols
in the SUPERSCRIPT plasmid system for cDNA synthesis and plasmid
cloning (Cat. #18248-013, Gibco/BRL, Gaithersburg, Md.). The cDNAs
were fractionated on a SEPHAROSE CL4B column (Cat. #275105-01;
Pharmacia), and those cDNAs exceeding 400 bp were ligated into
pINCY1. The plasmid pINCY1 was subsequently transformed into
DH5.alpha. competent cells (Cat. #18258-012; Gibco/BRL).
[0193] II Isolation and Sequencing of cDNA clones
[0194] Plasmid DNA was released from the cells and purified using
the R.E.A.L. PREP 96 plasmid kit (Catalog #26173; QIAGEN, Inc.).
This kit enabled the simultaneous purification of 96 samples in a
96-well block using multi-channel reagent dispensers. 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) after inoculation, the cultures were incubated
for 19 hours and at the end of incubation, the cells were lysed
with 0.3 ml of lysis buffer; and 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 96-well block for storage at 4.degree. C.
[0195] The cDNAs were sequenced by the method of Sanger et al.
(1975, J. Mol. Biol. 94:441f), using a MICROLAB 2200 (Hamilton,
Reno, Nev.) in combination with Peltier PTC200 thermal cyclers (M J
Research, Watertown, Mass.) and Applied Biosystems 377 DNA
sequencing systems.
[0196] III Homology Searching of cDNA Clones and Their Deduced
Proteins
[0197] 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).
[0198] 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).
[0199] 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 10-25 for
nucleotides and 10.sup.-14 for peptides.
[0200] 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. The relevant database for a particular match were
reported as a GIxxx.+-.p (where xxx is pri, rod, etc and if
present, p=peptide) as shown in Table 1. In column 3 of Table 1,
the product score is calculated as follows: the % nucleotide or
amino acid identity [between the query and reference sequences] in
BLAST is multiplied by the % maximum possible BLAST score [based on
the lengths of query and reference sequences] and then divided by
100. Where an Incyte Clone was homologous to several sequences, up
to five matches were provided with their relevant scores. In an
analogy to the hybridization procedures used in the laboratory, the
electronic stringency for an exact match was set at 70, and the
conservative lower limit for an exact match was set at
approximately 40 (with 1-2% error due to uncalled bases). Column 4
provides the log-likelihood where the value reflects the log of
(probability divided by threshold); column 5, the relevant GenBank
release; and column 6, a GenBank description of the protein, or an
edited version thereof. Some of the GenBank descriptions presented
in the tables of this application were standardized with respect to
abbreviations and spelling.
[0201] A comparison of the full-length and partial cDNA sequences
and the deduced amino acid sequences corresponding to the human
PAP-2 gene and human PAP-2 protein with known nucleotide and
protein sequences in GenBank revealed that the full-length human
PAP-2 cDNA and protein sequences (i.e., SEQ ID NOS: 1 and 2) were
unique (i.e., not previously identified). This search revealed that
the human PAP-2 protein shared some homology with the human PAP I
protein (SEQ ID NOS: 3 and 4) as well as other mammalian PAP
proteins (e.g., the mouse PAP I protein, rat PAP I, II and III
proteins, and bovine PTP) as well as a number of mammalian
reg/litostathine proteins.
[0202] IV Northern Analysis
[0203] 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).
[0204] Analogous computer techniques using BLAST (Altschul, S. F.
1993 and 1990, supra) are used to search for identical or related
molecules in nucleotide databases such as GenBank or the LIFESEQ
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.
[0205] The basis of the search is the product score which is
defined as: 1 % sequence identity .times. % maximum BLAST score
100
[0206] 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.
[0207] The results of northern analysis are reported as a list of
libraries in which the transcript encoding human PAP-2 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.
[0208] Electronic northern analysis (FIG. 4) revealed that mRNA
encoding human PAP-2 (SEQ ID NO: 1) was present in libraries
generated primarily from pancreatic and intestinal tissues. Thus,
the expression pattern of PAP-2 is similar to that seen with other
mammalian PAP proteins. Human PAP-2 cDNA is expressed in a variety
of tumors, including pancreas, esophagus, lung, cecal and colon
tumors. Human PAP-2 cDNA is also expressed in a number of tissues
that are involved with inflammatory and/or autoimmune disease
(e.g., ulcerative colitis, Crohn's disease).
[0209] V Extension of PAP-2-Encoding Polynucleotide Sequences
[0210] Full length human PAP-2-encoding nucleic acid sequence (SEQ
ID NO: 2) is used to design oligonucleotide primers for extending a
partial nucleotide sequence to full length or for obtaining 5' or
3', intron or other control sequences from genomic libraries. One
primer is synthesized to initiate extension in the antisense
direction (XLR) and the other is synthesized to extend sequence in
the sense direction (XLF). Primers are 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 are designed from the cDNA using
OLIGO 4.06 software (National Biosciences), 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. Any stretch of nucleotides which
would result in hairpin structures and primer-primer dimerizations
is avoided.
[0211] The original, selected cDNA libraries, or a human genomic
library are used to extend the sequence; the latter is most useful
to obtain 5' upstream regions. If more extension is necessary or
desired, additional sets of primers are designed to further extend
the known region.
[0212] By following the instructions for the XL-PCR kit (Perkin
Elmer) and thoroughly mixing the enzyme and reaction mix, high
fidelity amplification is obtained. Beginning with 40 pmol of each
primer and the recommended concentrations of all other components
of the kit, PCR is performed using the Peltier thermal cycler
(PTC200; M. J. Research, Watertown, Mass.) and the following
parameters:
[0213] Step 1 94.degree. C. for 1 min (initial denaturation)
[0214] Step 2 65.degree. C. for 1 min
[0215] Step 3 68.degree. C. for 6 min
[0216] Step 4 94.degree. C. for 15 sec
[0217] Step 5 65.degree. C. for 1 min
[0218] Step 6 68.degree. C. for 7 min
[0219] Step 7 Repeat step 4-6 for 15 additional cycles
[0220] Step 8 94.degree. C. for 15 sec
[0221] Step 9 65.degree. C. for 1 min
[0222] Step 10 68.degree. C. for7:15 min
[0223] Step 11 Repeat step 8-10 for 12 cycles
[0224] Step 12 72.degree. C. for 8 min
[0225] Step 13 4.degree. C. (and holding)
[0226] A 5- 10 .mu.l aliquot of the reaction mixture is analyzed by
electrophoresis on a low concentration (about 0.6-0.8%) agarose
mini-gel to deteminne which reactions were successful in extending
the sequence. Bands thought to contain the largest products are
selected and removed from the gel. Further purification involves
using a commercial gel extraction method such as QIAQUICK (QIAGEN
Inc., Chatsworth, Calif.). After recovery of the DNA, Klenow enzyme
is used to trim single-stranded, nucleotide overhangs creating
blunt ends which facilitate religation and cloning.
[0227] After ethanol precipitation, the products are redissolved in
13 .mu.l of ligation buffer, 1.mu.l T4-DNA ligase (15 units) and 1
.mu.l T4 polynucleotide kinase are added, and the mixture is
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) are 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 whole transformation
mixture is plated on Luria Bertani (LB)-agar (Sambrook et al.,
supra) containing 2.times.Carb. The following day, several colonies
are randomly picked from each plate and cultured in 150 .mu.l 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 is
transferred into a non-sterile 96-well plate and after dilution
1:10 with water, 5 .mu.l of each sample is transferred into a PCR
array.
[0228] 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 are added to each well. Amplification is
performed using the following conditions:
[0229] Step 1 94.degree. C. for 60 sec
[0230] Step 2 94.degree. C. for 20 sec
[0231] Step 3 55.degree. C. for 30 sec
[0232] Step 4 72.degree. C. for 90 sec
[0233] Step 5 Repeat steps 2-4 for an additional 29 cycles
[0234] Step 6 72.degree. C. for 180 sec
[0235] Step 7 4.degree. C. (and holding)
[0236] Aliquots of the PCR reactions are run on agarose gels
together with molecular weight markers. The sizes of the PCR
products are compared to the original partial cDNAs, and
appropriate clones are selected, ligated into plasmid, and
sequenced.
[0237] VI Labeling and Use of Hybridization Probes
[0238] 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 cDNA 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, Boston, Mass.). The labeled
oligonucleotides are substantially purified with SEPHADEX G-25
superfine resin column (Pharmacia & Upjohn). A portion
containing 10.sup.7 counts per minute of each of the sense and
antisense oligonucleotides 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).
[0239] 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 film
(Kodak, Rochester, N.Y.) is exposed to the blots, or the blots are
exposed to a phosphoimager cassette (Molecular Dynamics, Sunnyvale,
Calif.), hybridization patterns are compared visually.
[0240] II Antisense Molecules
[0241] Antisense molecules or sequence complementary to the human
PAP-2-encoding sequence, or any part thereof, is used to inhibit in
vivo or in vitro expression of naturally occurring human PAP-2.
Although use of antisense oligonucleotides, comprising about 20
base-pairs, is specifically described, essentially the same
procedure is used with larger cDNA fragments. An oligonucleotide
based on the coding sequences of human PAP-2, as shown in FIGS. 1A
and 1B, is used to inhibit expression of naturally occurring human
PAP-2. The complementary oligonucleotide is designed from the most
unique 5' sequence as shown in FIGS. 1A and 1B and used either to
inhibit transcription by preventing promoter binding to the
upstream nontranslated sequence or translation of an human
PAP-2-encoding transcript by preventing the ribosome from binding.
Using an appropriate portion of the signal and 5' sequence of SEQ
ID NO: 2, an effective antisense oligonucleotide includes any 15-20
nucleotides spanning the region which translates into the signal or
5' coding sequence of the polypeptide as shown in FIGS. 1A and
1B.
[0242] VIII Expression of Human PAP-2
[0243] Expression of human PAP-2 is accomplished by subcloning the
cDNAs into appropriate vectors and transforming the vectors into
host cells. In this case, the vector, PSPORT (Gibco/BRL), is used
to express human PAP-2 in E. coli. Upstream of the cloning site,
this vector contains a promoter for .beta.-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.
[0244] 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 or
fragments thereof. Sequences encoding human PAP-2 fusion proteins
lacking the naturally occurring signal sequence at residues 1-26 of
SEQ ID NO: 1 are preferentially employed for the production of
recombinant human PAP-2. The signal residues present on the PSPORT
vector direct the secretion of human PAP-2 into the bacterial
growth media which can be used directly in the following assays for
activity.
[0245] IX Demonstration of Human PAP-2 Activity
[0246] Given the chemical and structural similarity between the
human PAP-2 and the human and rat PAP I proteins as well as other
members of the reg/PSP multigene family, human PAP-2 is presumed to
be a C-type lectin and therefore capable of binding to
carbohydrates. The ability of recombinant human PAP-2 to bind
carbohydrates may be demonstrated by examining the ability of human
PAP-2 to bind to affinity columns comprising carbohydrates (e.g.,
lactose, maltose, D-mannose, D-galactose, etc. which are available
from Sigma Chemical Corp., St. Louis, Mo.) or by using the assay
described by Christa et al. (1994), supra.
[0247] C-type lectins, including rat PAP I, are known to
agglutinate bacteria. The ability of human PAP-2 to agglutinate
bacteria is demonstrated using the assay described by Iovanna et
al. [(1991), supra]. Briefly, bacteria (e.g., E. coli strains KH802
or JM101) are grown at 37.degree. C. to stationary phase in
L-broth. The bacteria are then collected by centrifugation and
washed in PBS. The washed bacteria are resuspended in PBS
containing 0.5 mM CaCl.sub.2 (PBS/CaCl.sub.2) and are placed in the
wells of microtiter plates at a concentration of approximately
5.times.10.sup.7 bacteria/200 .mu.l PBS/CaCl.sub.2. Human PAP-2 is
then added at a variety of concentrations (e.g., 1 to 50 .mu.g/ml)
and the presence of macroscopic aggregation is monitored following
a 3 hour incubation at 25.degree. C. Concanavalin A and albumin at
50 .mu.g/ml may be employed as positive and negative controls,
respectively.
[0248] X Production of Human PAP-2 Specific Antibodies
[0249] Human PAP-2 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
oligopolypeptide 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.
[0250] Typically, the oligopeptides are 15 residues in length,
synthesized using an Applied Biosystems 431A peptide synthesizer
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 radioiodinated, goat anti-rabbit
IgG.
[0251] XI Purification of Naturally Occurring Human PAP-2 Using
Specific Antibodies
[0252] Naturally occurring or recombinant human PAP-2 is
substantially purified by immunoaffinity chromatography using
antibodies specific for human PAP-2. An immunoaffinity column is
constructed by covalently coupling human PAP-2 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.
[0253] Media containing human PAP-2 is passed over the
immunoaffinity column, and the column is washed under conditions
that allow the preferential absorbance of human PAP-2 (e.g., high
ionic strength buffers in the presence of detergent). The column is
eluted under conditions that disrupt antibody/human PAP-2 binding
(eg, a buffer of pH 2-3 or a high concentration of a chaotrope,
such as urea or thiocyanate ion), and human PAP-2 is collected.
[0254] XII Identification of Molecules Which Interact with Human
PAP-2
[0255] Human PAP-2 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 human
PAP-2, washed and any wells with labeled human PAP-2 complex are
assayed. Data obtained using different concentrations of human
PAP-2 are used to calculate values for the number, affinity, and
association of human PAP-2 with the candidate molecules.
[0256] 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
* * * * *