U.S. patent application number 12/543026 was filed with the patent office on 2010-04-01 for mn gene and protein.
Invention is credited to Jaromir Pastorek, Silvia Pastorekova, Jan Zavada.
Application Number | 20100080815 12/543026 |
Document ID | / |
Family ID | 27430081 |
Filed Date | 2010-04-01 |
United States Patent
Application |
20100080815 |
Kind Code |
A1 |
Zavada; Jan ; et
al. |
April 1, 2010 |
MN Gene and Protein
Abstract
A new gene--MN--and proteins/polypeptides encoded therefrom are
disclosed. Recombinant nucleic acid molecules for expressing MN
proteins/polypeptides and recombinant proteins are provided.
Expression of the MN gene is disclosed as being associated with
tumorigenicity, and the invention concerns methods and compositions
for detecting and/or quantitating MN antigen and/or MN-specific
antibodies in vertebrate samples that are diagnostic/prognostic for
neoplastic and pre-neoplastic disease. Test kits embodying the
immunoassays of this invention are provided. MN-specific antibodies
are disclosed that can be used diagnostically/prognostically,
therapeutically, for imaging, and/or for affinity purification of
MN proteins/polypeptides. Also provided are nucleic acid probes for
the MN gene as well as test kits comprising said probes. The
invention also concerns vaccines comprising MN
proteins/polypeptides which are effective to immunize a vertebrate
against neoplastic diseases associated with the expression of MN
proteins. The invention still further concerns antisense nucleic
acid sequences that can be used to inhibit MN gene expression.
Inventors: |
Zavada; Jan; (Prague,
CZ) ; Pastorekova; Silvia; (Bratislava, SK) ;
Pastorek; Jaromir; (Bratislava, SK) |
Correspondence
Address: |
LEONA L. LAUDER
235 MONTGOMERY STREET, SUITE 1026
SAN FRANCISCO
CA
94104-0332
US
|
Family ID: |
27430081 |
Appl. No.: |
12/543026 |
Filed: |
August 18, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10795933 |
Mar 8, 2004 |
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12543026 |
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08260190 |
Jun 15, 1994 |
6774117 |
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10795933 |
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08177093 |
Dec 30, 1993 |
6051226 |
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08260190 |
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07964589 |
Oct 21, 1992 |
5387676 |
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08177093 |
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Current U.S.
Class: |
424/178.1 ;
435/252.33; 435/320.1; 435/348; 435/69.1; 435/7.1; 530/350;
530/387.9; 530/388.1; 530/389.1; 530/391.3; 530/391.7;
536/23.5 |
Current CPC
Class: |
A61K 39/00 20130101;
A61P 35/00 20180101; C07K 2319/00 20130101; C12N 9/88 20130101;
C07K 16/08 20130101; C07K 16/32 20130101; A61K 38/00 20130101; C07K
2317/34 20130101; C07K 7/06 20130101; C07K 16/3069 20130101; C07K
14/82 20130101 |
Class at
Publication: |
424/178.1 ;
536/23.5; 435/320.1; 435/348; 435/252.33; 530/350; 435/69.1;
530/389.1; 530/388.1; 530/387.9; 530/391.7; 530/391.3; 435/7.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07H 21/04 20060101 C07H021/04; C12N 15/63 20060101
C12N015/63; C12N 5/10 20060101 C12N005/10; C12N 1/21 20060101
C12N001/21; C07K 14/00 20060101 C07K014/00; C12P 21/00 20060101
C12P021/00; C07K 16/18 20060101 C07K016/18; G01N 33/53 20060101
G01N033/53; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 11, 1992 |
CZ |
PV-709-92 |
Claims
1. An isolated nucleic acid containing at least fifty nucleotides
wherein the nucleotide sequence for said nucleic acid is selected
from the group consisting of: (a) SEQ. ID. NOS.: 1, 5, 23 and
nucleotide sequences complementary to SEQ. ID. NOS.: 1, 5 or 23;
(b) nucleotide sequences that hybridize under standard stringent
hybridization conditions to one or more of the following nucleotide
sequences: SEQ. ID. NOS.: 1, 5, 23 and the respective complements
of SEQ. ID. NOS.: 1, 5 and 23; and (c) nucleotide sequences that
but for the degeneracy of the genetic code would hybridize under
standard stringent hybridization conditions to one or more of the
following nucleotide sequences: SEQ. ID. NOS.: 1, 5, 23 and the
respective complements of SEQ. ID. NOS.: 1, 5 and 23.
2. An isolated nucleic acid according to claim 1 which encodes an
MN protein or polypeptide.
3. An isolated nucleic acid according to claim 1 wherein the
nucleotide sequence for said nucleic acid is selected from the
group consisting of: (a) SEQ. ID. NO.: 23 and its complement; (b)
nucleotide sequences that hybridize under standard stringent
hybridization conditions to SEQ. ID. NO.: 23 or to its complement;
and (c) nucleotide sequences that but for the degeneracy of the
genetic code would hybridize under standard stringent hybridization
conditions to SEQ. ID. NO.: 23 or to its complement; wherein the
nucleic acids represented by the nucleotide sequences of (b) and
(c) function to identify MN nucleic acid sequences.
4-5. (canceled)
6. An isolated nucleic acid according to claim 1 operatively linked
to an expression control sequence within a vector.
7. A unicellular host which is either prokaryotic or eukaryotic
that is transformed or transfected with the isolated nucleic acid
operatively linked to an expression control sequence in a vector
according to claim 6.
8. A unicellular host according to claim 7 which is an insect cell
or an E. coli cell.
9. An MN protein or polypeptide encoded by the isolated nucleic
acid according to claim 2.
10. A fusion protein comprising at least a first and a second amino
acid wherein the first amino acid is encoded by an isolated nucleic
acid according to claim 2, and wherein the second amino acid is a
non-MN protein or polypeptide.
11. (canceled)
12. A method of recombinantly producing an MN protein or
polypeptide wherein a baculovirus expression system is used
comprising the steps of: (a) transforming an appropriate
unicellular host with an isolated nucleic acid operatively linked
to an expression control sequence in a vector according to claim 6;
(b) culturing said unicellular host so that said MN protein or
polypeptide is expressed; and (c) extracting and isolating said MN
protein or polypeptide.
13. An antibody which specifically binds to an epitope of an MN
protein or polypeptide according to claim 9.
14. An antibody according to claim 13 which is monoclonal.
15-16. (canceled)
17. An antibody according to claim 13 which specifically binds to
an MN antigen epitope selected from the group of epitopes
represented by the following amino acid sequences: SEQ. ID. NOS.
10-16.
18-21. (canceled)
22. An antibody according to claim 13 which is linked to a
chemotherapeutic agent or a toxic agent.
23. A method of delivering a chemotherapeutic agent or toxic agent
to a cancer cell which comprises contacting said cell with an
antibody according to claim 22.
24. An antibody according to claim 13 which is linked to an imaging
agent.
25. A method of imaging pre-neoplastic or neoplastic disease in a
patient comprising: (a) injecting said patient with antibody
according to claim 24; and (b) detecting the binding of said
antibody.
26. A method of detecting and/or quantitating in a vertebrate
sample MN antigen comprising the steps of: (a) contacting said
sample with an antibody according to claim 13; and (b) detecting
and/or quantitating binding of said antibody in said sample.
27. The method according to claim 26 wherein said detecting and/or
quantitating is by immunohistochemical staining.
28. The method according to claim 26 which is diagnostic/prognostic
for pre-neoplastic/neoplastic disease.
29. A method of detecting and/or quantitating MN-specific
antibodies in a vertebrate sample comprising the steps of: (a)
contacting and incubating the vertebrate sample with a fusion
protein according to claim 10; and (b) detecting and/or
quantitating binding of said fusion protein to antibody in said
sample.
30. (canceled)
Description
[0001] This application is a continuation application of U.S. Ser.
No. 10/795,933 (filed Mar. 8, 2004) which is a continuation
application of U.S. Ser. No. 08/260,190 (filed Jun. 15, 1994) which
issued on Aug. 10, 2004 as U.S. Pat. No. 6,774,117 B1, which is a
continuation-in-part of U.S. Ser. No. 08/177,093 (filed Dec. 30,
1993) which issued on Apr. 18, 2000 as U.S. Pat. No. 6,051,226,
which is in turn a continuation-in-part of U.S. Ser. No. 07/964,589
(filed Oct. 21, 1992) which issued on Feb. 7, 1995 as U.S. Pat. No.
5,387,676. This application declares priority under 35 USC
.sctn.120 from those U.S. applications, and also under 35 USC
.sctn.119 from the now pending Czechoslovakian patent application
PV-709-92 (filed Mar. 11, 1992).
FIELD OF THE INVENTION
[0002] The present invention is in the general area of medical
genetics and in the fields of biochemical engineering and
immunochemistry. More specifically, it relates to the
identification of a new gene--the MN gene--a cellular gene coding
for the MN protein. The inventors hereof found MN proteins to be
associated with tumorigenicity. Evidence indicates that the MN
protein appears to represent a potentially novel type of
oncoprotein. Identification of MN antigen as well as antibodies
specific therefor in patient samples provides the basis for
diagnostic/prognostic assays for cancer.
BACKGROUND OF THE INVENTION
[0003] A novel quasi-viral agent having rather unusual properties
was detected by its capacity to complement mutants of vesicular
stomatitis virus (VSV) with heat-labile surface G protein in HeLa
cells (cell line derived from human cervical adenocarcinoma), which
had been cocultivated with human breast carcinoma cells. [Zavada et
al., Nature New Biol., 240: 124 (1972); Zavada et al., J. Gen.
Virol., 24: 327 (1974); Zavada, J., Arch. Virol., 50:1 (1976);
Zavada, J., J. Gen. Virol., 63: 15-24 (1982); Zavada and Zavadova,
Arch, Virol., 118: 189 (1991).] The quasi viral agent was called
MaTu as it was presumably derived from a human mammary tumor.
[0004] There was significant medical interest in studying and
characterizing MaTu as it appeared to be an entirely new type of
molecular parasite of living cells, and possibly originated from a
human tumor. Described herein is the elucidation of the biological
and molecular nature of MaTu which resulted in the discovery of the
MN gene and protein. MaTu was found by the inventors to be a
two-component system, having an exogenous transmissible component,
MX, and an endogenous cellular component, MN. As described herein,
the MN component was found to be a cellular gene, showing only very
little homology with known DNA sequences. The MN gene was found to
be present in the chromosomal DNA of all vertebrates tested, and
its expression was found to be strongly correlated with
tumorigenicity.
[0005] The exogenous MaTu-MX transmissible agent was identified as
lymphocytic choriomeningitis virus (LCMV) which persistently
infects HeLa cells. The inventors discovered that the MN expression
in HeLa cells is positively regulated by cell density, and also its
expression level is increased by persistent infection with
LCMV.
[0006] Research results provided herein show that cells transfected
with MN cDNA undergo changes indicative of malignant
transformation. Further research findings described herein indicate
that the disruption of cell cycle control is one of the mechanisms
by which MN may contribute to the complex process of tumor
development.
[0007] Described herein is the cloning and sequencing of the MN
gene and the recombinant production of MN proteins. Also described
are antibodies prepared against MN proteins/polypeptides. MN
proteins/polypeptides can be used in serological assays according
to this invention to detect MN-specific antibodies. Further, MN
proteins/polypeptides and/or antibodies reactive with MN antigen
can be used in immunoassays according to this invention to detect
and/or quantitate MN antigen. Such assays may be diagnostic and/or
prognostic for neoplastic/pre-neoplastic disease.
SUMMARY OF THE INVENTION
[0008] Herein disclosed is the MN gene, a cellular gene which is
the endogenous component of the MaTu agent. cDNA sequences for the
MN gene are shown in FIGS. 1A-B [SEQ. ID. NO.: 1] and FIG. 15 [SEQ.
ID. NO.: 5]. FIG. 25 provides the sequence of a MN genomic clone
containing a promoter region [SEQ. ID. NO.: 23].
[0009] This invention is directed to said MN gene, fragments
thereof and the related cDNA which are useful, for example, as
follows: 1) to produce MN proteins/polypeptides by biochemical
engineering; 2) to prepare nucleic acid probes to test for the
presence of the MN gene in cells of a subject; 3) to prepare
appropriate polymerase chain reaction (PCR) primers for use, for
example, in PCR-based assays or to produce nucleic acid probes; 4)
to identify MN proteins and polypeptides as well as homologs or
near homologs thereto; 5) to identify various mRNAs transcribed
from MN genes in various tissues and cell lines, preferably human;
and 6) to identify mutations in MN genes. The invention further
concerns purified and isolated DNA molecules comprising the MN gene
or fragments thereof, or the related cDNA or fragments thereof.
[0010] Thus, this invention in one aspect concerns isolated nucleic
acid sequences that encode MN proteins or polypeptides wherein the
nucleotide sequences for said nucleic acids are selected from the
group consisting of:
[0011] (a) SEQ. ID. NO.: 1;
[0012] (b) SEQ. ID. NO.: 5;
[0013] (c) nucleotide sequences that hybridize under stringent
conditions to SEQ. ID. NO.: 1 or to its complement;
[0014] (d) nucleotide sequences that hybridize under stringent
conditions to SEQ. ID. NO.: 5 or to its complement; and
[0015] (e) nucleotide sequences that differ from SEQ. ID. NO.: 1 or
SEQ. ID NO.: 5, or from the nucleotide sequences of (c) and (d) in
codon sequence because of the degeneracy of the genetic code, that
is, sequences that are degenerate variants of those sequences.
Further, such nucleic acid sequences are selected from nucleotide
sequences that but for the degeneracy of the genetic code would
hybridize to either SEQ. ID. NO.: 1 or SEQ. ID. NO.: 5 under
stringent hybridization conditions.
[0016] Further, such isolated nucleic acids that encode MN proteins
or polypeptides can also include the MN nucleic acid of the genomic
clone shown in FIG. 25a-b, that is, SEQ. ID. NO.: 23, as well as
sequences that hybridize to it or its complement under stringent
conditions, or would hybridize to SEQ. ID. NO.: 23 or its
complement under such conditions, but for the degeneracy of the
genetic code.
[0017] Further, this invention concerns nucleic acid probes which
are fragments of the isolated nucleic acids that encode MN proteins
or polypeptides as described above. Preferably said nucleic acid
probes are comprised of at least 50 nucleotides, more preferably at
least 100 nucleotides, and still more preferably at least 150
nucleotides.
[0018] Still further, this invention is directed to isolated
nucleic acids selected from the group consisting of:
[0019] (a) a nucleic acid having the nucleotide sequence shown in
FIG. 25 [SEQ. ID. NO.: 23] and its complement;
[0020] (b) nucleic acids that hybridize under standard stringent
hybridization conditions to the nucleic acid of (a) or to its
complement; and
[0021] (c) nucleic acids that differ from the nucleic acids of (a)
and (b) in codon sequence because of the degeneracy of the genetic
code. The invention also concerns nucleic acids that but for the
degeneracy of the genetic code would hybridize to the nucleic acid
of (a) or to its complement under standard stringent hybridization
conditions. The nucleic acids of (b) and (c) that hybridize to the
coding region of SEQ. ID. NO.: 23 preferably have a length of at
least 50 nucleotides, whereas the nucleic acids of (b) and (c) that
hybridize partially or wholly to the non-coding region of SEQ. ID.
NO.: 23 or its complement are those that function as nucleic acid
probes to identify MN nucleic acid sequences. Conventional
technology can be used to determine whether the nucleic acids of
(b) and (c) or of fragments of SEQ. ID. NO.: 23 are useful to
identify MN nucleic acid sequences, for example, as outlined in
Benton and Davis, Science, 196: 180 (1977) and Fuscoe et al.
Genomics, 5: 100 (1989). In general, the nucleic acids of (b) and
(c) are preferably at least 50 nucleotides, more preferably at
least 100 nucleotides, and still more preferably at least 150
nucleotides.
[0022] Test kits of this invention can comprise the nucleic acid
probes of the invention which are useful
diagnostically/prognostically for neoplastic and/or pre-neoplastic
disease. Preferred test kits comprise means for detecting or
measuring the hybridization of said probes to the MN gene or to the
mRNA product of the MN gene, such as a visualizing means.
[0023] Fragments of the isolated nucleic acids of the invention,
can also be used as PCR primers to amplify segments of MN genes,
and may be useful in identifying mutations in MN genes. Typically,
said PCR primers are olignucleotides, preferably at least 16
nucleotides, but they may be considerably longer. Exemplary primers
may be from about 16 nucleotides to about 50 nucleotides,
preferably from about 19 nucleotides to about 45 nucleotides.
[0024] This invention also concerns nucleic acids which encode MN
proteins or polypeptides that are specifically bound by monoclonal
antibodies designated M75 that are produced by the hybridoma VU-M75
deposited at the American Type Culture Collection (ATCC) 10801
University Blvd., Manassas, Va. 20110-2209 (USA) under ATCC No. HB
11128, and/or by monoclonal antibodies designated MN12 produced by
the hybridoma MN 12.2.2 deposited at the ATCC under ATCC No. HB
11647.
[0025] The invention further concerns the discovery of a hitherto
unknown protein--MN, encoded by the MN gene. The expresssion of MN
proteins is inducible by growing cells in dense cultures, and such
expression was discovered to be associated with tumorigenic
cells.
[0026] MN proteins were found to be produced by some human tumor
cell lines in vitro, for example, by HeLa (cervical carcinoma), T24
(bladder carcinoma) and T47D (mammary carcinoma) and SK-MeI 1477
(melanoma) cell lines, by tumorigenic hybrid cells and by cells of
some human cancers in vivo, for example, by cells of uterine
cervical, ovarian and endometrial carcinomas as well as cells of
some benign neoplasias such as mammary papillomas. MN proteins were
not found in non-tumorigenic hybrid cells, and are generally not
found in the cells of normal tissues, although they have been found
in a few normal tissues, most notably and abundantly in normal
stomach tissues. MN antigen was found by immunohistochemical
staining to be prevalent in tumor cells and to be present sometimes
in morphologically normal appearing areas of tissue specimens
exhibiting dysplasia and/or malignancy. Thus, the MN gene is
strongly correlated with tumorigenesis and is considered to be a
putative oncogene.
[0027] In HeLa and in tumorigenic HeLa.times.fibroblast hybrid
(H/F-T) cells, MN protein is manifested as a "twin" protein
p54/58N; it is glycosylated and forms disulfide-linked oligomers.
As determined by electrophoresis upon reducing gels, MN proteins
have molecular weights in the range of from about 40 kd to about 70
kd, preferably from about 45 kd to about 65 kd, more preferably
from about 48 kd to about 58 kd. Upon non-reducing gels, MN
proteins in the form of oligomers have molecular weights in the
range of from about 145 kd to about 160 kd, preferably from about
150 to about 155 kd, still more preferably from about 152 to about
154 kd. The predicted amino acid sequences for preferred MN
proteins of this invention are shown in FIG. 1A-1B [SEQ. ID. NO. 2]
and in FIG. 15 [SEQ. ID. NO.: 6].
[0028] The discovery of the MN gene and protein and thus, of
substantially complementary MN genes and proteins encoded thereby,
led to the finding that the expression of MN proteins was
associated with tumorigenicity. That finding resulted in the
creation of methods that are diagnostic/prognostic for cancer and
precancerous conditions. Methods and compositions are provided for
identifying the onset and presence of neoplastic disease by
detecting and/or quantitating MN antigen in patient samples,
including tissue sections and smears, cell and tissue extracts from
vertebrates, preferably mammals and more preferably humans. Such MN
antigen may also be found in body fluids.
[0029] MN proteins and genes are of use in research concerning the
molecular mechanisms of oncogenesis, in cancer
diagnostics/prognostics, and may be of use in cancer immunotherapy.
The present invention is useful for detecting a wide variety of
neoplastic and/or pre-neoplastic diseases. Exemplary neoplastic
diseases include carcinomas, such as mammary, bladder, ovarian,
uterine, cervical, endometrial, squamous cell and adenosquamous
carcinomas; and head and neck cancers; mesodermal tumors, such as
neuroblastomas and retinoblastomas; sarcomas, such as osteosarcomas
and Ewing's sarcoma; and melanomas. Of particular interest are head
and neck cancers, gynecologic cancers including ovarian, cervical,
vaginal, endometrial and vulval cancers; gastrointestinal cancer,
such as, stomach, colon and esophageal cancers; urinary tract
cancer, such as, bladder and kidney cancers; skin cancer; liver
cancer; prostate cancer; lung cancer; and breast cancer. Of still
further particular interest are gynecologic cancers; breast cancer;
urinary tract cancers, especially bladder cancer; lung cancer; and
liver cancer. Even further of particular interest are gynecologic
cancers and breast cancer. Gynecologic cancers of particular
interest are carcinomas of the uterine cervix, endometrium and
ovaries; more particularly such gynecologic cancers include
cervical squamous cell carcinomas, adenosquamous carcinomas,
adenocarcinomas as well as gynecologic precancerous conditions,
such as metaplastic cervical tissues and condylomas.
[0030] The invention further relates to the biochemical engineering
of the MN gene, fragments thereof or related cDNA. For example,
said gene or a fragment thereof or related cDNA can be inserted
into a suitable expression vector; host cells can be transformed
with such an expression vector; and an MN protein/polypeptide,
preferably an MN protein, is expressed therein. Such a recombinant
protein or polypeptide can be glycosylated or nonglycosylated,
preferably glycosylated, and can be purified to substantial purity.
The invention further concerns MN proteins/polypeptides which are
synthetically or otherwise biologically prepared.
[0031] Said MN proteins/polypeptides can be used in assays to
detect MN antigen in patient samples and in serological assays to
test for MN-specific antibodies. MN proteins/polypeptides of this
invention are serologically active, immunogenic and/or antigenic.
They can further be used as immunogens to produce MN-specific
antibodies, polyclonal and/or monoclonal, as well as an immune
T-cell response.
[0032] The invention further is directed to MN-specific antibodies,
which can be used diagnostically/prognostically and may be used
therapeutically. Preferred according to this invention are
MN-specific antibodies reactive with the epitopes represented
respectively by the amino acid sequences of the MN protein shown in
FIG. 15 as follows: from AA 62 to AA 67 [SEQ. ID. NO.: 10]; from AA
55 to AA 60 [SEQ. ID. NO.: 11]; from AA 127 to AA 147 [SEQ. ID.
NO.: 12]; from AA 36 to AA 51 [SEQ. ID. NO.: 13]; from AA 69 to AA
83 [SEQ. ID. NO.: 14]; from AA 279 to AA 291 [SEQ. ID. NO.: 15];
and from AA 450 to AA 462 [SEQ. ID. NO.: 16]. More preferred are
antibodies reactive with epitopes represented by SEQ. ID. NOS.: 10,
11 and 12. Still more preferred are antibodies reactive with the
epitopes represented by SEQ. ID NOS: 10 and 11, as for example,
respectively Mabs M75 and MN12. Most preferred are monoclonal
antibodies reactive with the epitope represented by SEQ. ID. NO.:
10.
[0033] Also preferred according to this invention are antibodies
prepared against recombinantly produced MN proteins as, for
example, GEX-3X-MN and MN 20-19. Also preferred are MN-specific
antibodies prepared against glycosylated MN proteins, such as, MN
20-19 expressed in baculovirus infected Sf9 cells.
[0034] A hybridoma that produces a representative MN-specific
antibody, the monoclonal antibody M75 (Mab M75), was deposited at
the ATCC under Number HB 11128 as indicated above. The M75 antibody
was used to discover and identify the MN protein and can be used to
identify readily MN antigen in Western blots, in radioimmunoassays
and immunohistochemically, for example, in tissue samples that are
fresh, frozen, or formalin-, alcohol-, acetone- or otherwise fixed
and/or paraffin-embedded and deparaffinized. Another representative
MN-specific antibody, Mab MN12, is secreted by the hybridoma MN
12.2.2, which was deposited at the ATCC under the designation HB
11647.
[0035] MN-specific antibodies can be used, for example, in
laboratory diagnostics, using immunofluorescence microscopy or
immunohistochemical staining; as a component in immunoassays for
detecting and/or quantitating MN antigen in, for example, clinical
samples; as probes for immunoblotting to detect MN antigen; in
immunoelectron microscopy with colloid gold beads for localization
of MN proteins and/or polypeptides in cells; and in genetic
engineering for cloning the MN gene or fragments thereof, or
related cDNA. Such MN-specific antibodies can be used as components
of diagnostic/prognostic kits, for example, for in vitro use on
histological sections; such antibodies can also and used for in
vivo diagnostics/prognostics, for example, such antibodies can be
labeled appropriately, as with a suitable radioactive isotope, and
used in vivo to locate metastases by scintigraphy. Further such
antibodies may be used in vivo therapeutically to treat cancer
patients with or without toxic and/or cytostatic agents attached
thereto. Further, such antibodies can be used in vivo to detect the
presence of neoplastic and/or pre-neoplastic disease. Still
further, such antibodies can be used to affinity purify MN proteins
and polypeptides.
[0036] This invention also concerns recombinant DNA molecules
comprising a DNA sequence that encodes for an MN protein or
polypeptide, and also recombinant DNA molecules that encode not
only for an MN protein or polypeptide but also for an amino acid
sequence of a non-MN protein or polypeptide. Said non-MN protein or
polypeptide may preferably be nonimmunogenic to humans and not
typically reactive to antibodies in human body fluids. Examples of
such a DNA sequence is the alpha-peptide coding region of
beta-galactosidase and a sequence coding for glutathione
S-transferase or a fragment thereof. However, in some instances, a
non-MN protein or polypeptide that is serologically active,
immunogenic and/or antigenic may be preferred as a fusion partner
to a MN antigen. Further, claimed herein are such recombinant
fusion proteins/polypeptides which are substantially pure and
non-naturally occurring. An exemplary fusion protein of this
invention is GEX-3X-MN.
[0037] This invention also concerns methods of treating neoplastic
disease and/or pre-neoplastic disease comprising inhibiting the
expression of MN genes by administering antisense nucleic acid
sequences that are substantially complementary to mRNA transcribed
from MN genes. Said antisense nucleic acid sequences are those that
hybridize to such mRNA under stringent hybridization conditions.
Preferred are antisense nucleic acid sequences that are
substantially complementary to sequences at the 5' end of the MN
cDNA sequences shown in FIG. 1A-1B and/or in FIG. 15. Preferably
said antisense nucleic acid sequences are oligonucleotides.
[0038] This invention also concerns vaccines comprising an
immunogenic amount of one or more substantially pure MN proteins
and/or polypeptides dispersed in a physiologically acceptable,
nontoxic vehicle, which amount is effective to immunize a
vertebrate, preferably a mammal, more preferably a human, against a
neoplastic disease associated with the expression of MN proteins.
Said proteins can be recombinantly, synthetically or otherwise
biologically produced. Recombinent MN proteins include GEX-3X-MN
and MN 20-19. A particular use of said vaccine would be to prevent
recidivism and/or metastasis. For example, it could be administered
to a patient who has had an MN-carrying tumor surgically removed,
to prevent recurrence of the tumor.
[0039] The immunoassays of this invention can be embodied in test
kits which comprise MN proteins/polypeptides and/or MN-specific
antibodies. Such test kits can be in solid phase formats, but are
not limited thereto, and can also be in liquid phase format, and
can be based on immunohistochemical assays, ELISAs, particle
assays, radiometric or fluorometric assays either unamplified or
amplified, using, for example, avidin/biotin technology.
ABBREVIATIONS
[0040] The following abbreviations are used herein: [0041]
AA--amino acid [0042] ATCC--American Type Culture Collection [0043]
by--base pairs [0044] BSA--bovine serum albumin [0045]
BRL--Bethesda Research Laboratories [0046] CA--carbonic anhydrase
[0047] Ci--curie [0048] cm--centimeter [0049] cpm--counts per
minute [0050] C-terminus--carboxyl-terminus [0051] .degree.
C.--degrees centigrade [0052] DAB--diaminobenzidine [0053]
dH.sub.2O--deionized water [0054] DMEM--Dulbecco modified Eagle
medium [0055] DTT--dithiothreitol [0056]
EDTA--ethylenediaminetetracetate [0057] EIA--enzyme immunoassay
[0058] ELISA--enzyme-linked immunosorbent assay [0059]
EtOH--ethanol [0060] F--fibroblasts [0061] FCS--fetal calf serum
[0062] FIBR--fibroblasts [0063] FITC--fluorescein isothiocyanate
[0064] GEX-3X-MN--fusion protein MN glutathione S-transferase
[0065] H--HeLa cells [0066] H.sub.2O.sub.2--hydrogen peroxide
[0067] HCA--Hydrophobic Cluster Analysis [0068] HEF--human embryo
fibroblasts [0069] HeLa K--standard type of HeLa cells [0070] HeLa
S--Stanbridge's mutant HeLa D98/AH.2 [0071] H/F-T--hybrid HeLa
fibroblast cells that are tumorigenic; derived from HeLa D98/AH.2
[0072] H/F-N--hybrid HeLa fibroblast cells that are nontumorigenic;
derived from HeLa D98/AH.2 [0073] HGPRT.sup.---hypoxanthine guanine
phosphoribosyl transferase-deficient [0074] HLH--helix-loop-helix
[0075] HRP--horseradish peroxidase [0076]
IPTG--isopropyl-beta-D-thiogalacto-pyranoside [0077] kb--kilobase
[0078] kbp--kilobase pairs [0079] kd--kilodaltons [0080]
KPL--Kirkegaard & Perry Laboratories, Inc. [0081]
LCMV--lymphocytic choriomeningitis virus [0082] LTR--long terminal
repeat [0083] M--molar [0084] mA--milliampere [0085]
MAb--monoclonal antibody [0086] ME--mercaptoethanol [0087]
MEM--minimal essential medium [0088] min.--minute(s) [0089]
mg--milligram [0090] ml--milliliter [0091] MM--millimolar [0092]
MMC--mitomycin C [0093] MTV--mammary tumor virus [0094] N--normal
concentration [0095] ng--nanogram [0096] NGS--normal goat serum
[0097] N-terminus--amino-terminus [0098] ODN--oligodeoxynucleotide
[0099] PAGE--polyacrylamide gel electrophoresis [0100]
PBS--phosphate buffered saline [0101] PCR--polymerase chain
reaction [0102] PEST--combination of one-letter abbreviations for
proline, glutamic acid, serine, threonine [0103] pI--isoelectric
point [0104] RIP--radioimmunoprecipitation [0105]
RIPA--radioimmunoprecipitation assay [0106] SAC--protein
A-Staphylococcus aureus cells [0107] SDRE--serum dose response
element [0108] SDS--sodium dodecyl sulfate [0109] SDS-PAGE--sodium
dodecyl sulfate-polyacrylamide gel electrophoresis [0110]
SP-RIA--solid-phase radioimmunoassay [0111] SSPE--NaCl (0.18 M),
sodium phosphate (0.01 M), EDTA (0.001 M) [0112]
TBE--Tris-borate/EDTA electrophoresis buffer [0113]
TCA--trichloroacetic acid [0114] TC media--tissue culture media
[0115] TMB--tetramethylbenzidine [0116]
Tris--tris(hydroxymethyl)aminomethane [0117] .mu.Ci--microcurie
[0118] .mu.g--microgram [0119] .mu.l--microliter [0120]
.mu.M--micromolar [0121] VSV--vesicular stomatitis virus [0122]
X-MLV--xenotropic murine leukemia virus
Cell Lines
[0123] The following cell lines were used in the experiments herein
described: [0124] HeLa K--standard type of HeLa cells; aneuploid,
epithelial-like cell line isolated from a human cervical
adenocarcinoma [Gey et al., Cancer Res., 12: 264 (1952); Jones et
al., Obstet. Gynecol., 38: 945-949 (1971)] obtained from Professor
B. Korych, [Institute of Medical Microbiology and Immunology,
Charles University; Prague, Czech Republic] [0125] HeLa
D98/AH.2--Mutant HeLa clone that is hypoxanthine [0126] (also HeLa
S) guanine phosphoribosyl transferase-deficient (HGPRT.sup.-)
kindly provided by Eric J. Stanbridge [Department of Microbiology,
College of Medicine, University of California, Irvine, Calif.
(USA)] and reported in Stanbridge et al., Science, 215: 252-259 (15
Jan. 1982); parent of hybrid cells H/F-N and H/F-T, also obtained
from E. J. Stanbridge. [0127] NIH-3T3--murine fibroblast cell line
reported in Aaronson, Science, 237: 178 (1987). [0128] T47D--cell
line derived from a human mammary carcinoma [Keydar et al., Eur. J.
Cancer, 15: 659-670 (1979)]; kindly provided by J. Keydar [Haddasah
Medical School; Jerusalem, Israel] [0129] T24--cell line from
urinary bladder carcinoma [Bubenik et al., Int. J. Cancer, 11:
765-773 (1973)] kindly provided by J. Bubenik [Institute of
Molecular Genetics, Czechoslovak Academy of Sciences; Prague, Czech
Republic] [0130] HMB2--cell line from melanoma [Svec et al.,
Neoplasma, 35: 665-681 (1988)] [0131] HEF--human embryo fibroblasts
[Zavada et al., Nature New Biology, 240: 124-125 (1972)] [0132]
SIRC--cell line from rabbit cornea (control and X-MLV-infected)
[Zavada et al., Virology, 82: 221-231 (1977)] [0133] Vero
cells--African green monkey cell line [Zavada et al. (1977)] [0134]
myeloma cell--myeloma cell line used as a fusion parent [0135] line
NS-0 in production of monoclonal antibodies [Galfre and Milstein,
Methods Enzymol., 73: 3-46 (1981)] [0136] SK-Mel 1477--human
melanoma cell line kindly provided by K. E. Hellstrom [Division of
Tumor Immunology, Fred Hutchins Cancer Research Center; Seattle,
Wash. (USA)] [0137] XC--cells derived from a rat rhabdomyosarcoma
induced with Rous sarcoma virus-induced rat sarcoma [Svoboda, J.,
Natl. Cancer Center Institute Monograph No. 17, IN: "International
Conference on Avian Tumor Viruses" (J. W. Beard ed.), pp. 277-298
(1964)], kindly provided by Jan Svoboda [Institute of Molecular
Genetics, Czechoslovak Academy of Sciences; Prague, Czech
Republic]; and [0138] Rat 2-Tk.sup.---a thymidine kinase deficient
cell line, kindly provided by L. Kutinova [Institute of Sera and
Vaccines; Prague, Czech Republic] [0139] CGL1--H/F-N hybrid cells
(HeLa D98/AH.2 derivative) [0140] CGL2--H/F-N hybrid cells (HeLa
D98/AH.2 derivative) [0141] CGL3--H/F-T hybrid cells (HeLa D98/AH.2
derivative) [0142] CGL4--H/F-T hybrid cells (HeLa D98/Ah.2
derivative)
Nucleotide and Amino Acid Sequence Symbols
[0143] The following symbols are used to represent nucleotides
herein:
TABLE-US-00001 Base Symbol Meaning A adenine C cytosine G guanine T
thymine U uracil I inosine M A or C R A or G W A or T/U S C or G Y
C or T/U K G or T/U V A or C or G H A or C or T/U D A or G or T/U B
C or G or T/U N/X A or C or G or T/U
[0144] There are twenty main amino acids, each of which is
specified by a different arrangement of three adjacent nucleotides
(triplet code or codon), and which are linked together in a
specific order to form a characteristic protein. A three-letter or
one-letter convention is used herein to identify said amino acids,
as, for example, in FIGS. 1A-B and FIG. 15, respectively, as
follows:
TABLE-US-00002 3 Ltr. 1 Ltr. Amino acid name Abbrev. Abbrev.
Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic Acid Asp D
Cysteine Cys C Glutamic Acid Glu E Glutamine Gln Q Glycine Gly G
Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K
Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S
Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V
Unknown or other X
BRIEF DESCRIPTION OF THE FIGURES
[0145] FIG. 1A-1B provides the nucleotide sequence for a MN cDNA
[SEQ. ID. NO.: 1] clone isolated as described herein and the
predicted amino acid sequence [SEQ. ID. NO.: 2] encoded by the
cDNA. That sequence data has been sent to the EMBL Data Library in
Heidelberg, Germany and is available under Accession No.
X66839.
[0146] FIG. 2 provides SDS-PAGE and immunoblotting analyses of
recombinant MN protein expressed from a pGEX-3X bacterial
expression vector. Two parallel samples of purified recombinant MN
protein (twenty .mu.g in each sample) were separated by SDS-PAGE on
a 10% gel. One sample (A in FIG. 2) was stained with Coomassie
brilliant blue; whereas the other sample (B) was blotted onto a
Hybond C membrane [Amersham; Aylesbury, Bucks, England]. The blot
was developed by autoradiography with .sup.125I-labeled Mab
M75.
[0147] FIG. 3 illustrates inhibition of p54/58 expression by
antisense oligodeoxynucleotides (ODNs). HeLa cells cultured in
overcrowded conditions were incubated with (A) 29-mer ODNI [SEQ.
ID. NO.: 3]; (B) 19-mer ODN2 [SEQ. ID. NO.: 4]; (C) both ODNI and
ODN2; and (D) without ODNS. Example 10 provides details of the
procedures used.
[0148] FIG. 4 shows the results of Northern blotting of MN mRNA in
human cell lines. Total RNA was prepared from the following cell
lines: HeLa cells growing in dense (A) and sparse (B) culture;
(C)H/F-N; (D) and (E) H/F-T; and (F) human embryo fibroblasts.
Example 11 details the procedure and results.
[0149] FIG. 5 illustrates the detection of the MN gene in genomic
DNAs by Southern blotting. Chromosomal DNA digested by PstI was as
follows: (A) chicken; (B) bat; (C) rat; (D) mouse; (E) feline; (F)
pig; (G) sheep; (H) bovine; (I) monkey; and (J) human HeLa cells.
The procedures used are detailed in Example 12.
[0150] FIG. 6 graphically illustrates the expression of MN- and
MX-specific proteins in human fibroblasts (F), in HeLa cells (H)
and in H/F-N and H/F-T hybrid cells and contrasts the expression in
MX-infected and MX-uninfected cells. Example 5 details the
procedures and results.
[0151] FIG. 7 (discussed in Example 5) provides immunoblots of MN
proteins in fibroblasts (FIBR) and in HeLa to K, HeLa S, H/F-N and
H/F-T hybrid cells.
[0152] FIG. 8 (discussed in Example 6) shows immunoblots of MN
proteins in cell culture extracts prepared from the following: (A)
MX-infected HeLa cells; (B) human fibroblasts; (C) T24; (D) T47D;
(E) SK-MeI 1477; and (F) HeLa cells uninfected with MX. The symbols
+ME and O ME indicate that the proteins were separated by PAGE
after heating in a sample buffer, with and without 3%
mercaptoethanol (ME), respectively.
[0153] FIG. 9 (discussed in Example 6) provides immunoblots of MN
proteins from human tissue extracts. The extracts were prepared
from the following: (A) MX-infected HeLa cells; (B) full-term
placenta; (C) corpus uteri; (D, M) adenocarcinoma endometrii; (E,
N) carcinoma ovarii; (F, G) trophoblasts; (H) normal ovary; (I)
myoma uteri; (J) mammary papilloma; (K) normal mammary gland; (L)
hyperplastic endometrium; (O) cervical carcinoma; and (P)
melanoma.
[0154] FIG. 10 (discussed in Example 7) provides immunoblots of MN
proteins from (A) MX-infected HeLa cells and from (B) Rat2-Tk.sup.-
cells. (+ME and 0 ME have the same meanings as explained in the
legend to FIG. 8.)
[0155] FIG. 11 (discussed in Example 8) graphically illustrates the
results from radioimmunoprecipitation experiments with
.sup.125I-GEX-3X-MN protein and different antibodies. The
radioactive protein (15.times.10.sup.3 cpm/tube) was precipitated
with ascitic fluid or sera and SAC as follows: (A) ascites with MAb
M75; (B) rabbit anti-MaTu serum; (C) normal rabbit serum; (D) human
serum L8; (E) human serum KH; and (F) human serum M7.
[0156] FIG. 12 (discussed in Example 8) shows the results from
radioimmunoassays for MN antigen. Ascitic fluid (dilution
precipitating 50% radioactivity) was allowed to react for 2 hours
with (A) "cold" (unlabeled) protein GEX-3X-MN, or with extracts
from cells as follows: (B) HeLa+MX; (C) Rat-2Tk.sup.-; (D) HeLa;
(E) rat XC; (F) T24; and (G) HEF. Subsequently .sup.125I-labeled
GEX-3X-MN protein (25.times.10.sup.3 cpm/tube) was added and
incubated for an additional 2 hours. Finally, the radioactivity to
MAb M75 was adsorbed to SAC and measured.
[0157] FIG. 13 (discussed in Example 9) provides results of
immunoelectron and scanning microscopy of MX-uninfected (control)
and MX-infected HeLa cells. Panels A-D show ultrathin sections of
cells stained with MAb M75 and immunogold; Panels E and F are
scanning electron micrographs of cells wherein no immunogold was
used. Panels E and F both show a terminal phase of cell division.
Panels A and E are of control HeLa cells; panels B, C, D and F are
of MX-infected HeLa cells. The cells shown in Panels A, B and C
were fixed and treated with M75 and immunogold before they were
embedded and sectioned. Such a procedure allows for immunogold
decoration only of cell surface antigens. The cells in Panel D were
treated with M75 and immunogold only once they had been embedded
and sectioned, and thus antigens inside the cells could also be
decorated.
[0158] FIG. 14 compares the results of immunizing baby rats to XC
tumor cells with rat serum prepared against the fusion protein MN
glutathione S-transferase (GEX-3X-MN) (the IM group) with the
results of immunizing baby rats with control rat sera (the C
group). Each point on the graph represents the tumor weight of a
tumor from one rat. Example 14 details those experiments.
[0159] FIG. 15A-C shows a complete nucleotide sequence of a MN cDNA
[SEQ. ID. NO.: 5]. Also shown is the deduced amino acid sequence
[SEQ. ID. NO.: 6]. The polyadenylation signal (AATAAA) and the mRNA
instability motif (ATTTA) are located at nucleotides (nts)
1507-1512 and at nts 1513-1518, respectively. The amino acid
residues of the putative signal peptide as well as the
membrane-spanning segment are are located at amino acids (aa) 1-37
and at aa 415-433, respectively. The N-glycosylation site is
located at aa 346. The S/TPXX elements are are located at amino
acids 7-10, 47-50, 71-74, 153-156, 162-165, 333-336, and
397-400.
[0160] FIG. 16 is a restriction map of the full-length MN cDNA. The
open reading frame is shown as an open box. The thick lines below
the restriction map illustrate the sizes and positions of two
overlapping cDNA clones. The horizontal arrows indicate the
positions of primers R1 [SEQ. ID. NO.: 7] and R2 [SEQ. ID. NO.: 8]
used for the 5' end RACE. Relevant restriction sites are BamHI (B),
EcoRV (V), EcoRI (E), PstI (Ps), PvuII (Pv).
[0161] FIG. 17 shows a restriction analysis of the MN gene. Genomic
DNA from HeLa cells was cleaved with the following restriction
enzymes: EcoRI (1), EcoRV (2), HindIII (3), KpnI (4), NcoI (5),
PstI (6), and PvuII (7), and then analyzed by Southern
hybridization under stringent conditions using MN cDNA as a
probe.
[0162] FIG. 18 provides a hydrophilicity profile of the MN protein
shown in FIG. 15. The profile was computed using an average group
length of 6 amino acids.
[0163] FIG. 19(a) shows an alignment of HCA plots derived from MN,
human CA VI (hCA) and CA II (CA2). A one-letter code is used for
all amino acids with exception of P (stars), G (diamond-shaped
symbol), T and S (open and dotted squares, respectively). Strands
D, E, F and G are essential for the structural core of CA.
Topologically conserved hydrophobic amino acids are shaded (in hCA
VI and MN). Ligands of the catalytic zinc ion (His residues) are
indicated by arrowheads.
[0164] FIG. 19(b) presents a stereoview of the CA II
three-dimensional structure illustrating a superposition of the
complete CA II structure (thin ribbon) with the structure which is
well conserved in MN (open thick ribbon).
[0165] FIG. 19(c) presents an HCA comparison of the
basic/helix-loop-helix/zipper domains of Max and Myf-3 with the
N-terminal part of MN. The 3D structure of Max is indicated above
its plot with delineation of the segments b: basic, h1: helix 1, L:
loop, h2: helix 2, z: zipper. The I(6X)Y motif is shaded within
helix 2.
[0166] FIG. 19(d) schematically represents the sequence elements
and structural domains predicted from the deduced amino acid
sequence of MN.
[0167] FIG. 20 schematically represents an MN promoter region. The
consensus sequences are as follows: CAT-CCAAT; TATA-ATAAATATA;
AP2-1-GSSWSCC; AP2-YSSCCMNSSS [SEQ. ID. NO.: 19]; SP1-KMGGCCKRRY
[SEQ. ID. NO.: 20]; p53-RRRCWWGYYY [SEQ. ID. NO.: 21]; and
SDRE--CACCSCAC.
[0168] FIG. 21 schematically represents the 5' MN genomic region of
an MN genomic clone.
[0169] FIG. 22(a) shows the zinc-binding activity of MN protein
extracted from HeLa cells persistently infected with LCMV. Samples
were concentrated by immunoprecipitation with Mab M75 before
loading (A), and after elution from ZnCl.sub.2-saturated (B) or
ZnCl.sub.2-free Fast-Flow chelating Sepharase column (c).
Immunoprecipitates were analyzed by Western blotting using
iodinated M75 antibody.
[0170] FIG. 22 (b) shows MN protein binding to DNA-cellulose.
Proteins extracted from LCMV-infected HeLa cells were incubated
with DNA-cellulose (A). Proteins that bound to DNA-cellulose in the
presence of ZnCl.sub.2 and absence of DTT (B), in the presence of
both ZnCl.sub.2 and DTT (C), and in the absence of both ZnCl.sub.2
and DTT (D) were eluted, and all samples were analyzed as
above.
[0171] FIG. 22(c) shows the results of endoglycosidase H and F
digestion. MN protein immunoprecipitated with Mab M75 was treated
with Endo F (F) and Endo H(H). Treated (+) and control samples (-)
were analyzed by Western blotting as above.
[0172] FIG. 23 shows the morphology and growth kinetics of control
(a, c, e and g) and MN-expressing (b, d, f and h) NIH 3T3 cells.
The micrographs are of methanol fixed and Giemsa stained cells at a
magnification X 100. Cells were grown to confluency (a, b), or as
individual colonies in Petri dishes (c, d) and in soft agar (e, f).
The (g) and (h) graphs provide growth curves of cells cultured in
DMEM medium containing respectively, 10% and 1% FCS. The mean
values of triplicate determinations were plotted against time.
[0173] FIG. 24 illustrates flow cytometric analyses of asynchromous
cell populations of control and MN cDNA-transfected NIH 3T3
cells.
[0174] FIG. 25a-b is the complete sequence of an MN genomic clone
of this invention [SEQ. ID. NO.: 23]. It is 5052 nucleotides long
with the transcription start site at position 3534 (starting with
ACAGTCA . . . ). The presumed promoter region is about 300 to 400
nucleotides upstream of the transcription start site.
DETAILED DESCRIPTION
[0175] As demonstrated herein MaTu was found to be a two-component
system. One part of the complex, exogenous MX, is transmissible,
and is manifested by a protein, p58X, which is a cytoplasmic
antigen which reacts with some natural sera, of humans and of
various animals. The other component, MN, is endogenous to human
cells. The level of MN expression has been found to be considerably
increased in the presence of the MaTu-MX transmissible agent, which
has been now identified as lymphocytic choriomeningitis virus
(LCMV) which persistently infects HeLa cells.
[0176] MN is a cellular gene, showing only very little homology
with known DNA sequences. It is rather conservative and is present
as a single copy gene in the chromosomal DNA of various
vertebrates. Described herein is the cloning and sequencing of the
MN cDNA, and the genetic engineering of MN proteins--such as the
GEX-3X-MN and MN 20-19 proteins. The recombinant MN proteins can be
conveniently purified by affinity chromatography.
[0177] MN is manifested in HeLa cells by a twin protein, p54/58N,
that is localized on the cell surface and in the nucleus.
Immunoblots using a monoclonal antibody reactive with p54/58N (MAb
M75) revealed two bands at 54 kd and 58 kd. Those two bands may
correspond to one type of protein that differs by glycosylation
pattern or by how it is processed. (Both p54N and p58N are
glycosylated with oligosaccharidic residues containing mannose, but
only p58N also contains glucosamine.) Herein, the phrase "twin
protein" indicates p54/58N.
[0178] MN is absent in rapidly growing, sparse cultures of HeLa,
but is inducible either by keeping the cells in dense cultures or,
more efficiently, by infecting them with MX (LCMV). Those inducing
factors are synergistic. p54/58N and not p58X is associated with
virions of vesicular stomatitis virus (VSV), reproduced in
MaTu-infected HeLa. Whereas the twin protein p54/58N is
glycosylated and forms oligomers linked by disulfidic bonds, p58X
is not glycosylated and does not form S--S-linked oligomers.
[0179] VSV assembles p54/58N into virions in HeLa cells, indicating
that the twin protein is responsible for complementation of VSV
G-protein mutants and for formation of VSV(MaTu) pseudotypes. As
only enveloped viruses provide surface glycoproteins for the
formation of infectious, functioning pseudotypes, which can perform
such specific functions as adsorption and penetration of virions
into cells [Zavada, J., J. Gen. Virol., 63: 15-24 (1982)], that
observation implies that the MN gene behaves as a quasi-viral
sequence.
[0180] The surface proteins of enveloped viruses, which participate
in the formation of VSV pseudotypes, are glycosylated as is the MN
twin protein, p54/58N. MN proteins also resemble viral
glycoproteins in the formation of oligomers (preferably tri- or
tetramers); such oligomerization, although not necessarily
involving S--S bonds (disulfidic bonds), is essential for the
assembly of virions [Kreis and Lodish, Cell, 46: 929-937 (1986)].
The disulfidic bonds can be disrupted by reduction with
2-mercaptoethanol.
[0181] As reported in Pastorekova et al., Virology, 187: 620-626
(1992), after reduction with mercaptoethanol, p54/58N from cell
extracts or from VSV looks very similar on immunoblot. Without
reduction, in cell extracts, it gives several bands around 150 kd,
suggesting that the cells may contain several different oligomers
(probably with a different p54:p58 ratio), but VSV selectively
assembles only one of them, with a molecular weight of about 153
kd. That oligomer might be a trimer, or rather a tetramer,
consisting of 54 kd and 58 kd proteins. The equimolar ratio of
p54:p58 in VSV virions is indicated by approximately the same
strength of 54 kd and 58 kd bands in a VSV sample analyzed under
reducing conditions.
[0182] The expression of MN proteins appears to be
diagnostic/prognostic for neoplastic disease. The MN twin protein,
p54/58N, was found to be expressed in HeLa cells and in
Stanbridge's tumorigenic (H/F-T) hybrid cells [Stanbridge et al.,
Somatic Cell Genet, 7: 699-712 (1981); and Stanbridge et al.,
Science, 215: 252-259 (1982)] but not in fibroblasts or in
non-tumorigenic (H/F-N) hybrid cells [Stanbridge et al., id.]. In
early studies, MN proteins were found in immunoblots prepared from
human ovarian, endometrial and uterine cervical carcinomas, and in
some benign neoplasias (as mammary papilloma) but not from normal
ovarian, endometrial, uterine or placental tissues. Example 13
details further research on MN gene expression wherein MN antigen,
as detected by immunohistochemical staining, was found to be
prevalent in tumor cells of a number of cancers, including
cervical, bladder, head and neck, and renal cell carcinomas among
others. Further, the immunohistochemical staining experiments of
Example 13 show that among normal tissues tested, only normal
stomach tissues showed routinely and extensively the presence of MN
antigen. MN antigen is further shown herein to be present sometimes
in morphologically normal-appearing areas of tissue specimens
exhibiting dysplasia and/or malignancy.
[0183] In HeLa cells infected with MX, observed were conspicuous
ultrastructural alterations, that is, the formation of abundant
filaments on cell surfaces and the amplification of mitochondria.
Using an immunogold technique, p54/58N was visualized on the
surface filaments and in the nucleus, particularly in the nucleoli.
Thus MN proteins appear to be strongly correlated with
tumorigenicity, and do not appear to be produced in general by
normal non-tumor cells.
[0184] Examples herein show that MX and MN are two different
entities, that can exist independently of each other. MX (LCMV) as
an exogenous, transmissible agent can multiply in fibroblasts and
in H/F-N hybrid cells which are not expressing MN-related proteins
(FIG. 6). In such cells, MX does not induce the production of MN
protein. MN protein can be produced in HeLa and other tumor cells
even in the absence of MX as shown in FIGS. 6-9. However, MX is a
potent inducer of MN-related protein in HeLa cells; it increases
its production thirty times over the concentration observed in
uninfected cells (FIGS. 7 and 12, Table 1 in Example 8, below).
MN Gene--Cloning and Sequencing
[0185] FIGS. 1A-1B and 15 provide the nucleotide sequences for MN
cDNA clones isolated as described below, respectively SEQ. ID.
NOS.: 1 and 5. FIG. 25a-b provides the sequence of a MN genomic
clone containing a promoter region [SEQ. ID. NO.: 23].
[0186] It is understood that because of the degeneracy of the
genetic code, that is, that more than one codon will code for one
amino acid [for example, the codons TTA, TTG, CTT, CTC, CTA and CTG
each code for the amino acid leucine (leu)], that variations of the
nucleotide sequences in, for example, SEQ. ID. NOS.: 1, 5, and 23
wherein one codon is substituted for another, would produce a
substantially equivalent protein or polypeptide according to this
invention. All such variations in the nucleotide sequences of the
MN cDNA and complementary nucleic acid sequences are included
within the scope of this invention.
[0187] It is further understood that the nucleotide sequences
herein described and shown in FIGS. 1A-1B, 15 and 25, represent
only the precise structures of the cDNA and genomic nucleotide
sequences isolated and described herein. It is expected that
slightly modified nucleotide sequences will be found or can be
modified by techniques known in the art to code for substantially
similar or homologous MN proteins and polypeptides, for example,
those having similar epitopes, and such nucleotide sequences and
proteins/polypeptides are considered to be equivalents for the
purpose of this invention. DNA or RNA having equivalent codons is
considered within the scope of the invention, as are synthetic
nucleic acid sequences that encode proteins/polypeptides homologous
or substantially homologous to MN proteins/polypeptides, as well as
those nucleic acid sequences that would hybridize to said exemplary
sequences [SEQ. ID. NOS. 1, 5 and 23] under stringent conditions or
that but for the degeneracy of the genetic code would hybridize to
said cDNA nucleotide sequences under stringent hybridization
conditions. Modifications and variations of nucleic acid sequences
as indicated herein are considered to result in sequences that are
substantially the same as the exemplary MN sequences and fragments
thereof.
Partial cDNA Clone
[0188] To find the MN gene, a lambda gt11 cDNA library from
MX-infected HeLa cells was prepared. Total RNA from MX-infected
HeLa cells was isolated by a guanidinium-thiocyanate-CsCl method
[Chirgwin et al., Biochemistry, 18: 5249 (1979)], and the mRNA was
affinity separated on oligo dT-cellulose [Ausubel et al., Short
Protocols in Molecular Biology, (Greene Publishing Assocs. and
Wiley-Interscience; NY, USA, 1989]. The synthesis of the cDNA and
its cloning into lambda gt11 was carried out using kits from
Amersham, except that the EcoRI-NotI adaptor was from Stratagene
[La Jolla, Calif. (USA)]. The library was subjected to
immunoscreening with Mab M75 in combination with goat anti-mouse
antibodies conjugated with alkaline phosphatase. That
immunoscreening method is described in Young and Davis, PNAS (USA),
80: 1194-1198 (1983). About 4.times.10.sup.5 primary plaques on E.
coli Y1090 cells, representing about one-half of the whole library,
were screened using Hybond N+ membrane [Amersham] saturated with 10
mM IPTG and blocked with 5% FCS. Fusion proteins were detected with
Mab M75 in combination with goat anti-mouse antibodies conjugated
with alkaline phosphatase. One positive clone was picked.
[0189] pBluescript-MN. The positive clone was subcloned into the
NotI site of pBluescript KS [Stratagene] thereby creating
pBluescript-MN. Two oppositely oriented nested deletions were made
using Erase-a-Base.TM. kit [Promega; Madison, Wis. (USA)] and
sequenced by dideoxy method with a T7 sequencing kit [Pharmacia;
Piscataway, N.J. (USA)]. The sequencing showed a partial cDNA
clone, the insert being 1397 by long. That sequence is shown in
FIG. 1A-1B. The sequence comprises a large 1290 by open reading
frame and 107 by 3' untranslated region containing a
polyadenylation signal (AATAAA). Another interesting feature of the
sequence is the presence of a region contributing to instability of
the mRNA (AUUUA at position 1389) which is characteristic for mRNAs
of some oncogenes and lymphokines [Shaw and Kamen, Cell, 46:
659-667 (1986)]. However, the sequence surrounding the first ATG
codon in the open reading frame (ORF) did not fit the definition of
a translational start site. In addition, as follows from a
comparison of the size of the MN clone with that of the
corresponding mRNA in a Northern blot (FIG. 4), the cDNA was
missing about 100 by from the 5' end of its sequence.
Full-Length cDNA Clone
[0190] Attempts to isolate a full-length clone from the original
cDNA library failed. Therefore, we performed a rapid amplification
of cDNA ends (RACE) using MN-specific primers, R1 and R2, derived
from the 5' region of the original cDNA clone. The RACE product was
inserted into pBluescript, and the entire population of recombinant
plasmids was sequenced with an MN-specific primer ODN1. In that
way, we obtained a reliable sequence at the very 5' end of the MN
cDNA as shown in FIG. 15 [SEQ. ID. NO.: 5].
[0191] Specifically, RACE was performed using 5' RACE System [GIBCO
BRL; Gaithersburg, Md. (USA)] as follows. 1 .mu.g of mRNA (the same
as above) was used as a template for the first strand cDNA
synthesis which was primed by the MN-specific antisense
oligonucleotide, R1 (5'-TGGGGTTCTTGAGGATCTCCAGGAG-3') [SEQ. ID.
NO.: 7]. The first strand product was precipitated twice in the
presence of ammonium acetate and a homopolymeric C tail was
attached to its 3' end by TdT. Tailed cDNA was then amplified by
PCR using a nested primer, R2 (5'-CTCTAACTTCAGGGAGCCCTCTTCTT-3')
[SEQ. ID. NO.: 8] and an anchor primer that anneals to the
homopolymeric tail
(5'-CUACUACUACUAGGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3') [SEQ. ID.
NO.: 9]. Amplified product was digested with BamHI and SalI
restriction enzymes and cloned into pBluescript II KS plasmid.
After transformation, plasmid DNA was purified from the whole
population of transformed cells and used as a template for the
sequencing with the MN-specific primer ODN1 [SEQ. ID. NO.: 3; a
29-mer, the sequence for which is shown in Example 10].
[0192] The full-length MN cDNA sequence is 1519 base pairs (bp)
long (FIG. 15). It contains a single ORF of 1400 bp, starting at
position 12, with an ATG codon that is in a good context (GCGCATGG)
with the rule proposed for translation initiation [Kozak, J. Cell.
Biol., 108: 229-241 (1989)]. The AT rich 3' untranslated region
contains, as indicated above, a polyadenylation signal (AATAAA)
preceding the end of the cDNA by 10 bp. Surprisingly, the sequence
from the original clone as well as from four additional clones
obtained from the same cDNA library did not reveal any poly(A)
tail. Moreover, also as indicated above, just downstream of the
poly(A) signal we found an ATTTA motif that is thought to
contribute to mRNA instability (Shaw and Kamen, supra). This fact
raised possibility that the poly (A) tail is missing due to the
specific degradation of the MN mRNA.
Genomic Clone
[0193] To study MN regulation, an MN genomic clone was isolated
from a human cosmid library prepared from fetal brain using both
the MN cDNA probe and the MN-specific primers derived from the 5'
end of the cDNA [SEQ. ID. NOS.: 3 and 4; ODN1 AND ODN2; see Example
10]. The sequence for that genomic clone is shown in FIG. 25 [SEQ.
ID. NO.: 23]. Sequence analysis revealed that the genomic clone
covers a region upstream from the MN transcription start site and
ending with the BamHI restriction site localized inside the MN
cDNA. Other MN genomic clones can be similarly isolated.
[0194] The promoter region is GC-rich and contains one putative
TATA-box 578 by upstream from the transcription start. The promoter
contains several consensus sequences for binding sites of
regulatory elements, including two p53 sites, two AP-2 sites, an
AP-1 site, a SP-1 site, and a SDRE site. FIG. 20 provides a
schematic of the MN promoter region, and FIG. 21 provides a
schematic of the 5' MN genomic region.
[0195] Interestingly, the 5' end region of the isolated genomic
clone is strongly homologous to the 5' long terminal repeat (LTR)
of human endogenous retroviruses HERV K. As shown in FIG. 21, there
is no coding sequence, only two Alu repeats, between the LTR-like
region and the promoter. That fact suggests that the LTR, although
not necessarily belonging to MN, may provide an enhancer for MN
transcription.
Deduced Amino Acid Sequences
[0196] The open reading frame of the MN cDNA clone shown in FIG.
1A-1B encodes a putative protein of 429 amino acids with a
calculated molecular weight of about 48 kd. The hydrophilicity
profile reveals a hydrophobic sequence of amino acids (at positions
371-395) probably representing the region spanning the plasma
membrane and containing also a potential cleavage signal. The
profile fits well with the observation that p54/58N proteins are
localized on the cell membrane. There are no PEST regions in the MN
amino acid sequence, suggesting that the product of the MN gene is
a stable long-lived protein [Rogers et al., Science, 234: 364-368
(1986)]. Such a feature explains our experience with inefficient
metabolic labeling of p54/58N. The deduced amino acid sequence
displays also other features namely, 10 potential phosphorylation
and 7 myristylation sites, and 3 antigenic determinants.
[0197] The deduced amino acid sequence from the partial cDNA
sequence shown in FIG. 1A-1B can be compared to that shown in FIG.
15 from the full-length cDNA. The partial sequence is missing the
N-terminal 37 amino acids--the putative signal peptide. The ORF of
the MN cDNA shown in FIG. 15 has the coding capacity for a 466
amino acid protein with a calculated molecular weight of 51.5 kd.
As assessed by amino acid sequence analysis, the deduced primary
structure of the MN protein can be divided into four distinct
regions. The initial hydrophobic region of 37 amino acids (AA)
corresponds to a signal peptide. The mature protein has an
N-terminal part of 377 AA, a hydrophobic transmembrane segment of
20 AA and a C-terminal region of 32 AA. The overall amino acid
composition is rather basic, with a predicted isoelectric point of
8.92. The MN protein is rich in leucine (11.16%), proline (10.3%),
alanine (9.44%), arginine (9.23%), and serine (9.01%).
[0198] More detailed insight into MN protein primary structure
disclosed the presence of several consensus sequences. One
potential N-glycosylation site was found at position 345 of FIG.
15, and a putative nuclear localization signal composed of a
stretch of basic amino acids RRARKK [Blank et al., EMBO J., 10:
4159-4167 (1991); Wang and Reed, Nature, 364: 121-126 (1993)] was
recognized in the middle of the protein, at position 279-284 of
FIG. 15. These features, together with the predicted
membrane-spanning region mentioned above, are consistent with the
results, in which MN was shown to be an N-glycosylated protein
localized both in the plasma membrane and in the nucleus. MN
protein sequence deduced from cDNA was also found to contain six
S/TPXX sequence elements [SEQ. ID. NOS.: 25 AND 26] (one of them is
in the signal peptide) defined by Suzuki, J. Mol. Biol., 207: 61-84
(1989) as motifs frequently found in gene regulatory proteins.
However, only two of them are composed of the suggested consensus
amino acids.
Sequence Similarities and HCA
[0199] Computer analysis of the MN cDNA sequence was carried out
using DNASIS and PROSID (Pharmacia Software packages). GenBank,
EMBL, Protein Identification Resource and SWISS-PROT databases were
searched for all possible sequence similarities. In addition, a
search for proteins sharing sequence similarities with MN was
performed in the MIPS databank with the FastA program [Pearson and
Lipman, PNAS (USA), 85: 2444 (1988)].
[0200] The MN gene was found to clearly be a novel sequence derived
from the human genome. Searches for amino acid sequence
similarities in protein databases revealed as the closest homology
a level of sequence identity (44% of 170 AAs) between the central
part of the MN protein [AAs 221-390 of FIG. 15 (SEQ. ID. NO.: 6)]
and carbonic anhydrases (CA). However, the overall sequence
homology between the cDNA MN sequence and cDNA sequences encoding
different CA isoenzymes is in a homology range of 48-50% which is
considered by ones in the art to be low. Therefore, the MN cDNA
sequence is not closely related to any CA cDNA sequences.
[0201] Only very closely related sequences having a homology of at
least 80-90% would hybridize to each other under stringent
conditions. A sequence comparison of the MN cDNA sequence shown in
FIG. 1A-1B and a corresponding cDNA of the human carbonic anhydrase
II (CA II) showed that there are no stretches of identity between
the two sequences that would be long enough to allow for a segment
of the CA II cDNA sequence having 50 or more nucleotides to
hybridize under stringent hybridization conditions to the MN cDNA
or vice versa.
[0202] Although MN deduced amino acid sequences show some homology
to known carbonic anhydrases, they differ from them in several
repects. Seven carbonic anhydrases are known [Dodgson et al.
(eds.), The Carbonic Anhydrases, (Plenum Press; New York/London
(1991)]. All of the known carbonic anhydrases are proteins of about
30 kd, smaller than the p54/58N-related products of the MN gene.
Further, the carbonic anhydrases do not form oligomers as do the
MN-related proteins.
[0203] HCA. Hydrophobic Cluster Analysis (HCA) was used as a very
sensitive method to detect similarities in the secondary and
tertiary folding of protein domains even if the sequence homology
is low. [Lemesle-Varloot et al., Biochimie, 72: 555 (1990); Thoreau
et al., FEBS Lett., 282: 26 (1991); Gaboriaud et al., FEBS Lett.,
224: 189 (1987).] Comparison of the HCA plots for MN, human CA VI
and CA II [FIG. 19(a)] showed that only the middle and C-terminal
part of CA are highly conserved in MN with the conserved zinc
binding site and the enzyme's active center. A 44% sequence
identity and 87% HCA score was calculated between MN and CA VI
isoenzyme. [Aldred et al., Biochemistry, 30: 569 (1991).] Those
values are within the range of those observed for closely related
structures [Lemesle-Varloot et al., supra].
[0204] HCA was also used in combination with the one-dimensional
analysis programs, to analyze the N-terminal part of MN. When
screened against the MIPS sequence databank using the FastA program
[Pearson and Lipman, supra], the N-terminal segment of MN (AAs
38-114 of SEQ. ID. NO.: 6) repeatedly matched helical protein
domains involved in the regulation of gene expression (i.e.,
transforming protein Myb, accession number 504897; myogenic
determination factor Myf-3, accession number S06947;
transcriptional activator YAB, accession number JE0416;
translational activator PET127, accession number S17029, etc.). HCA
suggested [FIG. 19(c)] that although those hits were of low level
sequence identity, they might be structurally valid.
[0205] The most significant is a predicted structural similarity of
MN to members of the HLH protein family--represented by Myf-3 and
Max protein. The conserved I(6X)Y motif is revealed in FIG. 19(c),
a motif that is a common characteristic of an HLH protein
dimerization domain [Ferre-D'Amare et al., Nature, 363: 38 (1993)].
The region between the CA-like domain and the putative HLH
(covering AAs 120-220 of SEQ. ID. NO.: 6) domain is rich in
imperfect repeats of Ser (15%), Pro (16%), Gly and acidic residues
with few hydrophobic amino acids, resembling, thus, an activation
region of transcription factors. [Lautenberger et al., Oncogene, 7:
1713 (1992).]
[0206] In experiments, the results for which are shown in FIG.
22(a), it was determined that MN protein is able to bind zinc
cations, as shown by affinity chromatography using Zn-charged
chelating sepharose. MN protein immunoprecipitated from HeLa cells
by Mab M75 was found to have weak catalytic activity of CA. The
CA-like domain of MN has a structural predisposition to serve as a
binding site for small soluble domains. Thus, MN protein could
mediate some kind of signal transduction.
[0207] MN protein from LCMV-infected HeLA cells was shown by using
DNA cellulose affinity chromatography [FIG. 22(b)] to bind to
immobilized double-stranded salmon sperm DNA. The binding activity
required both the presence of zinc cations and the absence of a
reducing agent in the binding buffer.
MN Twin Protein
[0208] The possibility that the 4 kd difference between the
molecular weights of the two MN proteins is caused by different
glycosylation was ruled out, since after in vitro treatment with
endoglycosidases H and F, respectively, both peptide portions lost
about 3 kd in weight. This result indicates, in addition, that the
molecular weight of the smaller 54 kd MN protein without its 3 kd
sugar moiety, roughly corresponds to the molecular weight of MN
calculated from the full-length cDNA. Western blot analysis of MN
proteins from cervical carcinoma and normal stomach shows that in
both tissues MN protein consists of two 54 and 58 kd peptide
portions.
[0209] To determine whether both p54/58N proteins were encoded by
one gene, antisense ODNs were used to inhibit specifically MN gene
expression. [Such use of antisense ODNs is reviewed in Stein and
Cohen, Cancer Res., 48: 2659-2668 (1988).] Those experiments are
detailed in Example 10. The findings indicated that cultivation of
HeLa cells with ODNs resulted in a considerable inhibition of
p54/58N synthesis, whereas the amount of different HeLa cell
proteins produced remained approximately the same. Further, and
importantly, on immunoblotting, the specific inhibition by ODNs
affected both of the p54/58N proteins (FIG. 3). Thus, it was
concluded that the MN gene that was cloned codes for both of the
p54/58N proteins in HeLa cells.
MN Proteins and/or Polypeptides
[0210] The phrase "MN proteins and/or polypeptides" (MN
proteins/polypeptides) is herein defined to mean proteins and/or
polypeptides encoded by an MN gene or fragments thereof. Exemplary
and preferred MN proteins according to this invention have the
deduced amino acid sequences shown in FIGS. 1A-1B and 15. Preferred
MN proteins/polypeptides are those proteins and/or polypeptides
that have substantial homology with the MN proteins shown in FIGS.
1A-1B and 15. For example, such substantially homologous MN
proteins/polypeptides are those that are reactive with the
MN-specific antibodies of this invention, preferably the Mabs M75,
MN12 and MN7 or their equivalents.
[0211] A "polypeptide" is a chain of amino acids covalently bound
by peptide linkages and is herein considered to be composed of 50
or less amino acids. A "protein" is herein defined to be a
polypeptide composed of more than 50 amino acids.
[0212] MN proteins exhibit several interesting features: cell
membrane, and at the same time, nuclear localization (similar to E6
protein of HPV16), cell density dependent expression in HeLa cells,
correlation with the tumorigenic phenotype of HeLa.times.fibroblast
somatic cell hybrids, and expression in several human carcinomas
among other tissues. As demonstrated herein, for example, in
Example 13, MN protein can be found directly in tumor tissue
sections but not in general in counterpart normal tissues
(exceptions noted infra in Example 13 as in normal stomach
tissues). MN is also expressed sometimes in morphologically normal
appearing areas of tissue specimens exhibiting dysplasia and/or
malignancy. Taken together, these features suggest a possible
involvement of MN in the regulation of cell proliferation,
differentiation and/or transformation.
[0213] It can be appreciated that a protein or polypeptide produced
by a neoplastic cell in vivo could be altered in sequence from that
produced by a tumor cell in cell culture or by a transformed cell.
Thus, MN proteins and/or polypeptides which have varying amino acid
sequences including without limitation, amino acid substitutions,
extensions, deletions, truncations and combinations thereof, fall
within the scope of this invention. It can also be appreciated that
a protein extant within body fluids is subject to degradative
processes, such as, proteolytic processes; thus, MN proteins that
are significantly truncated and MN polypeptides may be found in
body fluids, such as, sera. The phrase "MN antigen" is used herein
to encompass MN proteins and/or polypeptides.
[0214] It will further be appreciated that the amino acid sequence
of MN proteins and polypeptides can be modified by genetic
techniques. One or more amino acids can be deleted or substituted.
Such amino acid changes may not cause any measurable change in the
biological activity of the protein or polypeptide and result in
proteins or polypeptides which are within the scope of this
invention.
[0215] The MN proteins and polypeptides of this invention can be
prepared in a variety of ways according to this invention, for
example, recombinantly, synthetically or otherwise biologically,
that is, by cleaving longer proteins and polypeptides enzymatically
and/or chemically. A preferred method to prepare MN proteins is by
a recombinant means. Particularly preferred methods of
recombinantly producing MN proteins are described below for the
GEX-3X-MN and MN 20-19 proteins.
Recombinant Production of MN Proteins and Polypeptides
[0216] A representative method to prepare the MN proteins shown in
FIGS. 1A-1B and 15 or fragments thereof would be to insert the
appropriate fragment of MN cDNA into an appropriate expression
vector as exemplified below. The fusion protein GEX-3X-MN expressed
from XL1-Blue cells is nonglycosylated. Representative of a
glycosylated, recombinantly produced MN protein is the MN 20-19
protein expressed from insect cells. The MN 20-19 protein was also
expressed in a nonglycosylated form in E. coli using the expression
plasmid pEt-22b [Navagen].
[0217] Baculovirus Expression Systems. Recombinant baculovirus
express vectors have been developed for infection into several
types of insect cells. For example, recombinant baculoviruses have
been developed for among others: Aedes aegypti, Autographa
californica, Bombyx mor, Drosphila melanogaster, Heliothis zea,
Spodoptera frugiperda, and Trichoplusia ni [PCT Pub. No. WO
89/046699; Wright, Nature, 321: 718 (1986); Fraser et al., In Vitro
Cell Dev. Biol., 25: 225 (1989). Methods of introducing exogenous
DNA into insect hosts are well-known in the art. DNA transfection
and viral infection procedures usually vary with the insect genus
to be transformed. See, for example, Autographa [Carstens et al.,
Virology, 101: 311 (1980)]; Spodoptera [Kang, "Baculovirus Vectors
for Expression of Foreign Genes," in: Advances in Virus Research,
35 (1988)]; and Heliothis (virescens) [PCT Pub. No. WO
88/02030].
[0218] A wide variety of other host-cloning vector combinations may
be usefully employed in cloning the MN DNA isolated as described
herein. For example, useful cloning vehicles may include
chromosomal, nonchromosomal and synthetic DNA sequences such as
various known bacterial plasmids such as pBR322, other E. coli
plasmids and their derivatives and wider host range plasmids such
as RP4, phage DNA, such as, the numerous derivatives of phage
lambda, e.g., NB989 and vectors derived from combinations of
plasmids and phage DNAs such as plasmids which have been modified
to employ phage DNA expression control sequences.
[0219] Useful hosts may be eukaryotic or prokaryotic and include
bacterial hosts such as E. coli and other bacterial strains, yeasts
and other fungi, animal or plant hosts such as animal or plant
cells in culture, insect cells and other hosts. Of course, not all
hosts may be equally efficient. The particular selection of
host-cloning vehicle combination may be made by those of skill in
the art after due consideration of the principles set forth herein
without departing from the scope of this invention.
[0220] The particular site chosen for insertion of the selected DNA
fragment into the cloning vehicle to form a recombinant DNA
molecule is determined by a variety of factors. These include size
and structure of the protein or polypeptide to be expressed,
susceptibility of the desired protein or polypeptide to
endoenzymatic degradation by the host cell components and
contamination by its proteins, expression characteristics such as
the location of start and stop codons, and other factors recognized
by those of skill in the art.
[0221] The recombinant nucleic acid molecule containing the MN
gene, fragment thereof, or cDNA therefrom, may be employed to
transform a host so as to permit that host (transformant) to to
express the structural gene or fragment thereof and to produce the
protein or polypeptide for which the hybrid DNA encodes. The
recombinant nucleic acid molecule may also be employed to transform
a host so as to permit that host on replication to produce
additional recombinant nucleic acid molecules as a source of MN
nucleic acid and fragments thereof. The selection of an appropriate
host for either of those uses is controlled by a number of factors
recognized in the art. These include, for example, compatibility
with the chosen vector, toxicity of the co-products, ease of
recovery of the desired protein or polypeptide, expression
characteristics, biosafety and costs.
[0222] Where the host cell is a procaryote such as E. coli,
competent cells which are capable of DNA uptake are prepared from
cells harvested after exponential growth phase and subsequently
treated by the CaCl.sub.2 method by well known procedures.
Transformation can also be performed after forming a protoplast of
the host cell.
[0223] Where the host used is an eukaryote, transfection methods
such as the use of a calcium phosphate-precipitate,
electroporation, conventional mechanical procedures such as
microinjection, insertion of a plasmid encapsulated in red blood
cell ghosts or in liposomes, treatment of cells with agents such as
lysophosphatidyl-choline or use of virus vectors, or the like may
be used.
[0224] The level of production of a protein or polypeptide is
governed by three major factors: (1) the number of copies of the
gene or DNA sequence encoding for it within the cell; (2) the
efficiency with which those gene and sequence copies are
transcribed and translated; and (3) the stability of the mRNA.
Efficiencies of transcription and translation (which together
comprise expression) are in turn dependent upon nucleotide
sequences, normally situated ahead of the desired coding sequence.
Those nucleotide sequences or expression control sequences define,
inter alia, the location at which an RNA polymerase interacts to
initiate transcription (the promoter sequence) and at which
ribosomes bind and interact with the mRNA (the product of
transcription) to initiate translation. Not all such expression
control sequences function with equal efficiency. It is thus of
advantage to separate the specific coding sequences for the desired
protein from their adjacent nucleotide sequences and fuse them
instead to known expression control sequences so as to favor higher
levels of expression. This having been achieved, the newly
engineered DNA fragment may be inserted into a multicopy plasmid or
a bacteriophage derivative in order to increase the number of gene
or sequence copies within the cell and thereby further improve the
yield of expressed protein.
[0225] Several expression control sequences may be employed. These
include the operator, promoter and ribosome binding and interaction
sequences (including sequences such as the Shine-Dalgarno
sequences) of the lactose operon of E. coli ("the lac system"), the
corresponding sequences of the tryptophan synthetase system of E.
coli ("the trp system"), a fusion of the trp and lac promoter ("the
tac system"), the major operator and promoter regions of phage
lambda (O.sub.LP.sub.L and O.sub.RP.sub.R), and the control region
of the phage fd coat protein. DNA fragments containing these
sequences are excised by cleavage with restriction enzymes from the
DNA isolated from transducing phages that carry the lac or trp
operons, or from the DNA of phage lambda or fd. Those fragments are
then manipulated in order to obtain a limited population of
molecules such that the essential controlling sequences can be
joined very close to, or in juxtaposition with, the initiation
codon of the coding sequence.
[0226] The fusion product is then inserted into a cloning vehicle
for transformation or transfection of the appropriate hosts and the
level of antigen production is measured. Cells giving the most
efficient expression may be thus selected. Alternatively, cloning
vechicles carrying the lac, trp or lambda P.sub.L control system
attached to an initiation codon may be employed and fused to a
fragment containing a sequence coding for a MN protein or
polypeptide such that the gene or sequence is correctly translated
from the initiation codon of the cloning vehicle.
[0227] The phrase "recombinant nucleic acid molecule" is herein
defined to mean a hybrid nucleotide sequence comprising at least
two nucleotide sequences, the first sequence not normally being
found together in nature with the second.
[0228] The phrase "expression control sequence" is herein defined
to mean a sequence of nucleotides that controls and regulates
expression of structural genes when operatively linked to those
genes.
[0229] The following are representative examples of genetically
engineering MN proteins of this invention. The descriptions are
exemplary and not meant to limit the invention in any way.
Production of Fusion Protein GEX-3X-MN
[0230] To confirm whether the partial cDNA clone codes for the
p54/58N-specific protein, it was subcloned into the bacterial
expression vector pGEX-3X [Pharmacia; Upsala, Sweden], constructed
to express a fusion protein containing the C-terminus of
glutathione S-transferase. The partial cDNA insert from the
above-described pBluescript-MN was released by digesting the
plasmid DNA by NotI. It was then treated with S1 nuclease to obtain
blunt ends and then cloned into a dephosphorylated SmaI site of
pGEX-3X[Pharmacia]. After transformation of XL1-Blue cells [E. coli
strain; Stratagene] and induction with IPTG, a fusion protein was
obtained.
[0231] The fusion protein--MN glutathione S-transferase (GEX-3X-MN)
was purified by affinity chromatography on Glutathione-S-Sepharose
4B [Pharmacia]. Twenty micrograms of the purified recombinant
protein in each of two parallel samples were separated by SDS-PAGE
on a 10% gel. One of the samples (A) was stained with Coomassie
brilliant blue, whereas the other (B) was blotted onto a Hybond C
membrane [Amersham]. The blot was developed by autoradiography with
.sup.125I-labeled MAb M75. The results are shown in FIG. 2.
[0232] SDS-PAGE analysis provided an interesting result: a number
of protein bands with different molecular weights (FIG. 2A). A
similar SDS-PAGE pattern was obtained with another representative
fusion protein produced according to this invention,
beta-galactosidase-MN that was expressed from lambda gt11 lysogens.
It appears that those patterns are due to translation errors caused
by the presence of 9 AGGAGG codon tandems in the MN sequence. The
use of those codons is strongly avoided in bacterial genes because
of the shortage of corresponding tRNAs. Thus, during the
translation of AGGAGG tandems from foreign mRNA, +1 ribosomal
frameshifts arise with a high frequency (about 50%) [Spanjaard et
al., Nuc. Acid Res., 18: 5031-5036 (1990)].
[0233] By immunoblotting, a similar pattern was obtained: all the
bands seen on stained SDS-PAGE gel reacted with the MN-specific MAb
M75 (FIG. 2B), indicating that all the protein bands are
MN-specific. Also, that result indicates that the binding site for
MAb M75 is on the N-terminal part of the MN protein, which is not
affected by frameshifts.
[0234] As shown in Example 8 below, the fusion protein GEX-3X-MN
was used in radioimmunoassays for MN-specific antibodies and for MN
antigen.
Expression of MN 20-19 Protein
[0235] Another representative, recombinantly produced MN protein of
this invention is the MN 20-19 protein which, when produced in
baculovirus-infected Sf9 cells [Spodoptera frugiperda cells;
Clontech; Palo Alto, Calif. (USA)], is glycosylated. The MN 20-19
protein misses the putative signal peptide (AAs 1-37) of SEQ. ID.
NO.: 6 (FIG. 15), has a methionine (Met) at the N-terminus for
expression, and a Leu-Glu-His-His-His-His-His-His [SEQ. ID NO.: 22]
added to the C-terminus for purification. In order to insert the
portion of the MN coding sequence for the GEX-3X-MN fusion protein
into alternate expression systems, a set of primers for PCR was
designed. The primers were constructed to provide restriction sites
at each end of the coding sequence, as well as in-frame start and
stop codons. The sequences of the primers, indicating restriction
enzyme cleavage sites and expression landmarks, are shown
below.
TABLE-US-00003 Primer #20:N-terminus Translation start 5'
GTCGCTAGCTCCATGGGTCATATGCAGAGGTTGCCCCGGATGCAG 3' [SEQ. ID. NO. 17]
NheI NcoI NdeI MN cDNA #1 Primer #19:C-terminus Translation stop 5'
GAAGATCTCTTACTCGAGCATTCTCCAAGATCCAGCCTCTAGG 3' [SEQ. ID. NO. 18]
BglII XhoI MN cDNA
The SEQ. ID. NOS.: 17 and 18 primers were used to amplify the MN
coding sequence present in the pGEX-3X-MN vector using standard PCR
techniques. The resulting PCR product (termed MN 20-19) was
electrophoresed on a 0.5% agarose/1X TBE gel; the 1.3 kb band was
excised; and the DNA recovered using the Gene Clean II kit
according to the manufacturer's instructions [Bio101; LaJolla,
Calif. (USA)].
[0236] MN 20-19 and plasmid pET-22b [Novagen, Inc.; Madison, Wis.
(USA)] were cleaved with the restriction enzymes NdeI and XhoI,
phenol-chloroform extracted, and the appropriate bands recovered by
agarose gel electrophoresis as above. The isolated fragments were
ethanol co-precipitated at a vector:insert ratio of 1:4. After
resuspension, the fragments were ligated using T4 DNA ligase. The
resulting product was used to transform competent Novablue E. coli
cells [Novagen, Inc.]. Plasmid mini-preps [Magic Minipreps;
Promega] from the resultant ampicillin resistant colonies were
screened for the presence of the correct insert by restriction
mapping. Insertion of the gene fragment into the pET-22b plasmid
using the NdeI and XhoI sites added a 6-histidine tail to the
protein that could be used for affinity isolation.
[0237] To prepare MN 20-19 for insertion into the baculovirus
expression system, the MN 20-19 gene fragment was excised from
pET-22b using the restriction endonucleases XbaI and PvuI. The
baculovirus shuttle vector pBacPAK8 [Clontech] was cleaved with
XbaI and PacI. The desired fragments (1.3 kb for MN 20-19 and 5.5
kb for pBacPAK8) were isolated by agarose gel electrophoresis,
recovered using Gene Clean II, and co-precipitated at an
insert:vector ratio of 2.4:1.
[0238] After ligation with T4 DNA ligase, the DNA was used to
transform competent NM522 E. coli cells (Stratagene). Plasmid
mini-preps from resultant ampicillin resistant colonies were
screened for the presence of the correct insert by restriction
mapping. Plasmid DNA from an appropriate colony and linearized
BacPAK6 baculovirus DNA [Clontech] were used to transform Sf9 cells
by standard techniques. Recombination produced BacPAK viruses
carrying the MN 20-19 sequence. Those viruses were plated onto Sf9
cells and overlaid with agar.
[0239] Plaques were picked and plated onto Sf9 cells. The
conditioned media and cells were collected. A small aliquot of the
conditioned media was set aside for testing. The cells were
extracted with PBS with 1% Triton X100.
[0240] The conditioned media and the cell extracts were dot blotted
onto nitrocellulose paper. The blot was blocked with 5% non-fat
dried milk in PBS. Mab M75 were used to detect the MN 20-19 protein
in the dot blots. A rabbit anti-mouse Ig-HRP was used to detect
bound Mab M75. The blots were developed with TMB/H.sub.2O.sub.2
with a membrane enhancer [KPL; Gaithersburg, Md. (USA)]. Two clones
producing the strongest reaction on the dot blots were selected for
expansion. One was used to produce MN 20-19 protein in High Five
cells [Invitrogen Corp., San Diego, Calif. (USA); BTI-TN-5BI-4;
derived from Trichoplusia ni egg cell homogenate]. MN 20-19 protein
was purified from the conditioned media from the virus infected
High Five cells.
[0241] The MN 20-19 protein was purified from the conditioned media
by immunoaffinity chromatography. 6.5 mg of Mab M75 was coupled to
1 g of Tresyl activated Toyopearl.TM. [solid support in bead form;
Tosoh, Japan (#14471)]. Approximately 150 ml of the conditioned
media was run through the M75-Toyopearl column. The column was
washed with PBS, and the MN 20-19 protein was eluted with 1.5 M
MgCl. The eluted protein was then dialyzed against PBS.
Synthetic and Biologic Production of MN Proteins and
Polypeptides
[0242] MN proteins and polypeptides of this invention may be
prepared not only by recombinant means but also by synthetic and by
other biologic means. Synthetic formation of the polypeptide or
protein requires chemically synthesizing the desired chain of amino
acids by methods well known in the art. Exemplary of other biologic
means to prepare the desired polypeptide or protein is to subject
to selective proteolysis a longer MN polypeptide or protein
containing the desired amino acid sequence; for example, the longer
polypeptide or protein can be split with chemical reagents or with
enzymes.
[0243] Chemical synthesis of a peptide is conventional in the art
and can be accomplished, for example, by the Merrifield solid phase
synthesis technique [Merrifield, J., Am. Chem. Soc., 85: 2149-2154
(1963); Kent et al., Synthetic Peptides in Biology and Medicine, 29
f.f. eds. Alitalo et al., (Elsevier Science Publishers 1985); and
Haug, J. D., "Peptide Synthesis and Protecting Group Strategy",
American Biotechnology Laboratory, 5(1): 40-47 (January/February
1987)].
[0244] Techniques of chemical peptide synthesis include using
automatic peptide synthesizers employing commercially available
protected amino acids, for example, Biosearch [San Rafael, Calif.
(USA)] Models 9500 and 9600; Applied Biosystems, Inc. [Foster City,
Calif. (USA)] Model 430; Milligen [a division of Millipore Corp.;
Bedford, Mass. (USA)] Model 9050; and Du Pont's RAMP (Rapid
Automated Multiple Peptide Synthesis) [Du Pont Compass, Wilmington,
Del. (USA)].
Regulation of MN Expression and MN Promoter
[0245] MN appears to be a novel regulatory protein that is directly
involved in the control of cell proliferation and in cellular
transformation. In HeLa cells, the expression of MN is positively
regulated by cell density. Its level is increased by persistent
infection with LCMV. In hybrid cells between HeLa and normal
fibroblasts, MN expression correlates with tumorigenicity. The fact
that MN is not present in nontumorigenic hybrid cells (CGL1), but
is expressed in a tumorigenic segregant lacking chromosome 11,
indicates that MN is negatively regulated by a putative suppressor
in chromosome 11.
[0246] Evidence supporting the regulatory role of MN protein was
found in the generation of stable transfectants of NIH 3T3 cells
that constitutively express MN protein as described in Example 15.
As a consequence of MN expression, the NIH 3T3 cells acquired
features associated with a transformed phenotype: altered
morphology, increased saturation density, proliferative advantage
in serum-reduced media, enhanced DNA synthesis and capacity for
anchorage-independent growth. Further, as shown in Example 16, flow
cytometric analyses of asynchronous cell populations indicated that
the expression of MN protein leads to accelerated progression of
cells through G1 phase, reduction of cell size and the loss of
capacity for growth arrest under inappropriate conditions. Also,
Example 16 shows that MN expressing cells display a decreased
sensitivity to the DNA damaging drug mitomycin C.
[0247] Nontumorigenic human cells, CGL1 cells, were also
transfected with the full-length MN cDNA. The same pSG5C-MN
construct in combination with pSV2neo plasmid as used to transfect
the NIH 3T3 cells (Example 15) was used. Also the protocol was the
same except that the G418 concentration was increased to 1000
.mu.g/ml.
[0248] Out of 15 MN-positive clones (tested by SP-RIA and Western
blotting), 3 were chosen for further analysis. Two MN-negative
clones isolated from CGL1 cells transfected with empty plasmid were
added as controls. Initial analysis indicates that the morphology
and growth habits of MN-transfected CGL1 cells are not changed
dramatically, but their proliferation rate and plating efficiency
is increased.
[0249] MN cDNA and promoter. When the promoter region from the MN
genomic clone, isolated as described above, was linked to MN cDNA
and transfected into CGL1 hybrid cells, expression of MN protein
was detectable immediately after selection. However, then it
gradually ceased, indicating thus an action of a feedback
regulator. The putative regulatory element appeared to be acting
via the MN promoter, because when the full-length cDNA (not
containing the promoter) was used for transfection, no similar
effect was observed.
[0250] An "antisense" MN cDNA/MN promoter construct was used to
transfect CGL3 cells. The effect was the opposite of that of the
CGL1 cells transfected with the "sense" construct. Whereas the
transfected CGL1 cells formed colonies several times larger than
the control CGL1, the transfected CGL3 cells formed colonies much
smaller than the control CGL3 cells.
[0251] For those experiments, the part of the promoter region that
was linked to the MN cDNA through BamHI site was derived from
NcoI-BamHI fragment of the MN genomic clone and represents the
region 233 by upstream from the transcription initiation site.
After the ligation, the joint DNA was inserted into a pBK-CMV
expression vector [Stratagene]. The required orientation of the
inserted sequence was ensured by directional cloning and
subsequently verified by restriction analysis. The transfection
procedure was the same as used in transfecting the NIH 3T3 cells
(Example 15), but co-transfection with the pSV2neo plasmid was not
necessary since the neo selection marker was already included in
the pBK-CMV vector.
[0252] After two weeks of selection in a medium containing G418,
remarkable differences between the numbers and sizes of the
colonies grown were evident as noted above. Immediately following
the selection and cloning, the MN-transfected CGL1 and CGL3 cells
were tested by SP-RIA for expression and repression of MN,
respectively. The isolated transfected CGL1 clones were MN positive
(although the level was lower than obtained with the full-length
cDNA), whereas MN protein was almost absent from the transfected
CGL3 clones. However, in subsequent passages, the expression of MN
in transfected CGL1 cells started to cease, and was then blocked
perhaps evidencing a control feedback mechanism.
[0253] As a result of the very much lowered proliferation of the
transfected CGL3 cells, it was difficult to expand the majority of
cloned cells (according to SP-RIA, those with the lowest levels of
MN), and they were lost during passaging. However, some clones
overcame that problem and again expressed MN. It is possible that
once those cells reached a higher quantity, that the level of
endogenously produced MN mRNA increased over the amount of
ectopically expressed antisense mRNA.
Nucleic Acid Probes and Test Kits
[0254] Nucleic acid probes of this invention are those comprising
sequences that are complementary or substantially complementary to
the MN cDNA sequences shown in FIGS. 1A-1B and 15 or to other MN
gene sequences, such as, the genomic clone sequence of FIG. 25
[SEQ. ID. NO.: 23]. The phrase "substantially complementary" is
defined herein to have the meaning as it is well understood in the
art and, thus, used in the context of standard hybridization
conditions. The stringency of hybridization conditions can be
adjusted to control the precision of complementarity. Exemplary are
the stringent hybridization conditions used in Examples 11 and 12.
Two nucleic acids are, for example, substantially complementary to
each other, if they hybridize to each other under such stringent
hybridization conditions.
[0255] Stringent hybridization conditions are considered herein to
conform to standard hybridization conditions understood in the art
to be stringent. For example, it is generally understood that
stringent conditions encompass relatively low salt and/or high
temperature conditions, such as provided by 0.02 M to 0.15 M NaCl
at temperatures of 50.degree. C. to 70.degree. C. Less stringent
conditions, such as, 0.15 M to 0.9 M salt at temperatures ranging
from 20.degree. C. to 55.degree. C. can be made more stringent by
adding increasing amounts of formamide, which serves to destabilize
hybrid duplexes as does increased temperature.
[0256] Exemplary stringent hybridization conditions are described
in Examples 11 and 12, infra; the hybridizations therein were
carried out "in the presence of 50% formamide at 42.degree. C."
[See Sambrook et al., Molecular Cloning: A Laboratory Manual, pages
1.91 and 9.47-9.51 (Second Edition, Cold Spring Harbor Laboratory
Press; Cold Spring Harbor, N.Y.; 1989); Maniatis et al., Molecular
Cloning: A Laboratory Manual, pages 387-389 (Cold Spring Harbor
Laboratory; Cold Spring Harbor, N.Y.; 1982); Tsuchiya et al., Oral
Surgery, Oral Medicine, Oral Pathology, 71(6): 721-725 (June
1991).]
[0257] Preferred nucleic acid probes of this invention are
fragments of the isolated nucleic acid sequences that encode MN
proteins or polypeptides according to this invention. Preferably
those probes are composed of at least fifty nucleotides.
[0258] However, nucleic acid probes of this invention need not
hybridize to a coding region of MN. For example, nucleic acid
probes of this invention may hybridize partially or wholly to a
non-coding region of the genomic clone of FIG. 25a-b [SEQ. ID. NO.:
23]. Conventional technology can be used to determine whether
fragments of SEQ. ID. NO.: 23 or related nucleic acids are useful
to identify MN nucleic acid sequences. [See, for example, Benton
and Davis, supra and Fuscoe et al., supra.]
[0259] Nucleic acid probes of this invention can be used to detect
MN DNA and/or RNA, and thus can be used to test for the presence or
absence of MN genes, and amplification(s), mutation(s) or genetic
rearrangements of MN genes in the cells of a patient. For example,
overexpression of an MN gene may be detected by Northern blotting
using probes of this invention. Gene alterations, as
amplifications, translocations, inversions, and deletions among
others, can be detected by using probes of this invention for in
situ hybridization to chromosomes from a patient's cells, whether
in metaphase spreads or interphase nuclei. Southern blotting could
also be used with the probes of this invention to detect
amplifications or deletions of MN genes. Restriction Fragment
Length Polymorphism (RFLP) analysis using said probes is a
preferred method of detecting gene alterations, mutations and
deletions. Said probes can also be used to identify MN proteins
and/or polypeptides as well as homologs or near homologs thereto by
their hybridization to various mRNAs transcribed from MN genes in
different tissues.
[0260] Probes of this invention thus can be useful
diagnostically/prognostically. Said probes can be embodied in test
kits, preferably with appropriate means to enable said probes when
hybridized to an appropriate MN gene or MN mRNA target to be
visualized. Such samples include tissue specimens including smears,
body fluids and tissue and cell extracts.
[0261] PCR Assays. To detect relatively large genetic
rearrangements, hybridization tests can be used. To detect
relatively small genetic rearrangements, as, for example, small
deletions or amplifications, or point mutations, the polymerase
chain reaction (PCR) would preferably be used. [U.S. Pat. Nos.
4,800,159; 4,683,195; 4,683,202; and Chapter 14 of Sambrook et al.,
Molecular Cloning: A Laboratory Manual, supra]
[0262] An exemplary assay would use cellular DNA from normal and
cancerous cells, which DNA would be isolated and amplified
employing appropriate PCR primers. The PCR products would be
compared, preferably initially, on a sizing gel to detect size
changes indicative of certain genetic rearrangements. If no
differences in sizes are noted, further comparisons can be made,
preferably using, for example, PCR-single-strand conformation
polymorphism (PCR-SSCP) assay or a denaturing gradient gel
electrophoretic assay. [See, for example, Hayashi, K., "PCR-SSCP: A
Simple and Sensitive Method for Detection of Mutations in the
Genomic DNA," in PCR Methods and Applications, 1: 34-38 (1991); and
Meyers et al., "Detection and Localization of Single Base Changes
by Denaturing Gradient Gel Electrophoresis," Methods in Enzymology,
155: 501 (1987).]
Assays
[0263] Assays according to this invention are provided to detect
and/or quantitate MN antigen or MN-specific antibodies in
vertebrate samples, preferably mammalian samples, more preferably
human samples. Such samples include tissue specimens, body fluids,
tissue extracts and cell extracts. MN antigen may be detected by
immunoassay, immunohistochemical staining, immunoelectron and
scanning microscopy using immunogold among other techniques.
[0264] Preferred tissue specimens to assay by immunohistochemical
staining include cell smears, histological sections from biopsied
tissues or organs, and imprint preparations among other tissue
samples. Such tissue specimens can be variously maintained, for
example, they can be fresh, frozen, or formalin-, alcohol- or
acetone- or otherwise fixed and/or paraffin-embedded and
deparaffinized. Biopsied tissue samples can be, for example, those
samples removed by aspiration, bite, brush, cone, chorionic villus,
endoscopic, excisional, incisional, needle, percutaneous punch, and
surface biopsies, among other biopsy techniques.
[0265] Preferred cervical tissue specimens include cervical smears,
conization specimens, histologic sections from hysterectomy
specimens or other biopsied cervical tissue samples. Preferred
means of obtaining cervical smears include routine swab, scraping
or cytobrush techniques, among other means. More preferred are
cytobrush or swab techniques. Preferably, cell smears are made on
microscope slides, fixed, for example, with 55% EtOH or an alcohol
based spray fixative and air-dried.
[0266] Papanicolaou-stained cervical smears (Pap smears) can be
screened by the methods of this invention, for example, for
retrospective studies. Preferably, Pap smears would be decolorized
and re-stained with labeled antibodies against MN antigen. Also
archival specimens, for example, matched smears and biopsy and/or
tumor specimens, can be used for retrospective studies. Prospective
studies can also be done with matched specimens from patients that
have a higher than normal risk of exhibiting abnormal cervical
cytopathology.
[0267] Preferred samples in which to assay MN antigen by, for
example, Western blotting or radioimmunoassay, are tissue and/or
cell extracts. However, MN antigen may be detected in body fluids,
which can include among other fluids: blood, serum, plasma, semen,
breast exudate, saliva, tears, sputum, mucous, urine, lymph,
cytosols, ascites, pleural effusions, amniotic fluid, bladder
washes, bronchioalveolar lavages and cerebrospinal fluid. It is
preferred that the MN antigen be concentrated from a larger volume
of body fluid before testing. Preferred body fluids to assay would
depend on the type of cancer for which one was testing, but in
general preferred body fluids would be breast exudate, pleural
effusions and ascites.
[0268] MN-specific antibodies can be bound by serologically active
MN proteins/polypeptides in samples of such body fluids as blood,
plasma, serum, lymph, mucous, tears, urine, spinal fluid and
saliva; however, such antibodies are found most usually in blood,
plasma and serum, preferably in serum. A representative assay to
detect MN-specific antibodies is shown in Example 8 below wherein
the fusion protein GEX-3X-MN is used. Correlation of the results
from the assays to detect and/or quantitate MN antigen and
MN-specific antibodies reactive therewith, provides a preferred
profile of the disease condition of a patient.
[0269] The assays of this invention are both diagnostic and/or
prognostic, i.e., diagnostic/prognostic. The term
"diagnostic/prognostic" is herein defined to encompass the
following processes either individually or cumulatively depending
upon the clinical context: determining the presence of disease,
determining the nature of a disease, distinguishing one disease
from another, forecasting as to the probable outcome of a disease
state, determining the prospect as to recovery from a disease as
indicated by the nature and symptoms of a case, monitoring the
disease status of a patient, monitoring a patient for recurrence of
disease, and/or determining the preferred therapeutic regimen for a
patient. The diagnostic/prognostic methods of this invention are
useful, for example, for screening populations for the presence of
neoplastic or pre-neoplastic disease, determining the risk of
developing neoplastic disease, diagnosing the presence of
neoplastic and/or pre-neoplastic disease, monitoring the disease
status of patients with neoplastic disease, and/or determining the
prognosis for the course of neoplastic disease. For example, it
appears that the intensity of the immunostaining with MN-specific
antibodies may correlate with the severity of dysplasia present in
samples tested.
[0270] The present invention is useful for screening for the
presence of a wide variety of neoplastic diseases including
carcinomas, such as, mammary, urinary tract, ovarian, uterine,
cervical, endometrial, squamous cell and adenosquamous carcinomas;
head and neck cancers; mesodermal tumors, such as, neuroblastomas
and retinoblastomas; sarcomas, such as osteosarcomas and Ewing's
sarcoma; and melanomas. Of particular interest are gynecological
cancers including ovarian, uterine, cervical, vaginal, vulval and
endometrial cancers, particularly ovarian, uterine cervical and
endometrial cancers. Also of particular interest are cancers of the
breast, of the stomach including esophagus, of the colon, of the
kidney, of the prostate, of the liver, of the urinary tract
including bladder, of the lung, and of the head and neck.
[0271] The invention provides methods and compositions for
evaluating the probability of the presence of malignant or
pre-malignant cells, for example, in a group of cells freshly
removed from a host. Such an assay can be used to detect tumors,
quantitate their growth, and help in the diagnosis and prognosis of
disease. The assays can also be used to detect the presence of
cancer metastasis, as well as confirm the absence or removal of all
tumor tissue following surgery, cancer chemotherapy and/or
radiation therapy. It can further be used to monitor cancer
chemotherapy and tumor reappearance.
[0272] The presence of MN antigen or antibodies can be detected
and/or quantitated using a number of well-defined diagnostic
assays. Those in the art can adapt any of the conventional
immunoassay formats to detect and/or quantitate MN antigen and/or
antibodies. Example 8 details the format of a preferred diagnostic
method of this invention--a radioimmunoassay. Immunohistochemical
staining is another preferred assay format as exemplified in
Example 13.
[0273] Many other formats for detection of MN antigen and
MN-specific antibodies are, of course available. Those can be
Western blots, ELISAs (enzyme-linked immunosorbent assays), RIAs
(radioimmunoassay), competitive EIA or dual antibody sandwich
assays, among other assays all commonly used in the diagnostic
industry. In such immunoassays, the interpretation of the results
is based on the assumption that the antibody or antibody
combination will not cross-react with other proteins and protein
fragments present in the sample that are unrelated to MN.
[0274] Representative of one type of ELISA test for MN antigen is a
format wherein a microtiter plate is coated with antibodies made to
MN proteins/polypeptides or antibodies made to whole cells
expressing MN proteins, and to this is added a patient sample, for
example, a tissue or cell extract. After a period of incubation
permitting any antigen to bind to the antibodies, the plate is
washed and another set of anti-MN antibodies which are linked to an
enzyme is added, incubated to allow reaction to take place, and the
plate is then rewashed. Thereafter, enzyme substrate is added to
the microtiter plate and incubated for a period of time to allow
the enzyme to work on the substrate, and the absorbance of the
final preparation is measured. A large change in absorbance
indicates a positive result.
[0275] It is also apparent to one skilled in the art of
immunoassays that MN proteins and/or polypeptides can be used to
detect and/or quantitate the presence of MN antigen in the body
fluids, tissues and/or cells of patients. In one such embodiment, a
competition immunoassay is used, wherein the MN protein/polypeptide
is labeled and a body fluid is added to compete the binding of the
labeled MN protein/polypeptide to antibodies specific to MN
protein/polypeptide. Such an assay can be used to detect and/or
quantitate MN antigen as described in Example 8.
[0276] In another embodiment, an immunometric assay may be used
wherein a labeled antibody made to a MN protein or polypeptide is
used. In such an assay, the amount of labeled antibody which
complexes with the antigen-bound antibody is directly proportional
to the amount of MN antigen in the sample.
[0277] A representative assay to detect MN-specific antibodies is a
competition assay in which labeled MN protein/polypeptide is
precipitated by antibodies in a sample, for example, in combination
with monoclonal antibodies recognizing MN proteins/polypeptides.
One skilled in the art could adapt any of the conventional
immunoassay formats to detect and/or quantitate MN-specific
antibodies. Detection of the binding of said antibodies to said MN
protein/polypeptide could be by many ways known to those in the
art, e.g., in humans with the use of anti-human labeled IgG.
[0278] An exemplary immunoassay method of this invention to detect
and/or quantitate MN antigen in a vertebrate sample comprises the
steps of:
[0279] a) incubating said vertebrate sample with one or more sets
of antibodies (an antibody or antibodies) that bind to MN antigen
wherein one set is labeled or otherwise detectable;
[0280] b) examining the incubated sample for the presence of immune
complexes comprising MN antigen and said antibodies.
[0281] Another exemplary immunoassay method according to this
invention is that wherein a competition immunoassay is used to
detect and/or quantitate MN antigen in a vertebrate sample and
wherein said method comprises the steps of:
[0282] a) incubating a vertebrate sample with one or more sets of
MN-specific antibodies and a certain amount of a labeled or
otherwise detectable MN protein/polypeptide wherein said MN
protein/polypeptide competes for binding to said antibodies with MN
antigen present in the sample;
[0283] b) examining the incubated sample to determine the amount of
labeled/detectable MN protein/polypeptide bound to said antibodies;
and
[0284] c) determining from the results of the examination in step
b) whether MN antigen is present in said sample and/or the amount
of MN antigen present in said sample.
[0285] Once antibodies (including biologically active antibody
fragments) having suitable specificity have been prepared, a wide
variety of immunological assay methods are available for
determining the formation of specific antibody-antigen complexes.
Numerous competitive and non-competitive protein binding assays
have been described in the scientific and patent literature, and a
large number of such assays are commercially available. Exemplary
immunoassays which are suitable for detecting a serum antigen
include those described in U.S. Pat. Nos. 3,791,932; 3,817,837;
3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262;
3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and
4,098,876.
[0286] Antibodies employed in assays may be labeled or unlabeled.
Unlabeled antibodies may be employed in agglutination; labeled
antibodies may be employed in a wide variety of assays, employing a
wide variety of labels.
[0287] Suitable detection means include the use of labels such as
radionuclides, enzymes, coenzymes, fluorescers, chemiluminescers,
chromogens, enzyme substrates or co-factors, enzyme inhibitors,
free radicals, particles, dyes and the like. Such labeled reagents
may be used in a variety of well known assays, such as
radioimmunoassays, enzyme immunoassays, e.g., ELISA, fluorescent
immunoassays, and the like. See for example, U.S. Pat. Nos.
3,766,162; 3,791,932; 3,817,837; and 4,233,402.
[0288] Methods to prepare antibodies useful in the assays of the
invention are described below. The examples below detail
representative assays according to this invention.
Immunoassay Test Kits
[0289] The above outlined assays can be embodied in test kits to
detect and/or quantitate MN antigen and/or MN-specific antibodies
(including biologically active antibody fragments). Kits to detect
and/or quantitate MN antigen can comprise MN
protein(s)/polypeptides(s) and/or MN-specific antibodies,
polyclonal and/or monoclonal. Such diagnostic/prognostic test kits
can comprise one or more sets of antibodies, polyclonal and/or
monoclonal, for a sandwich format wherein antibodies recognize
epitopes on the MN antigen, and one set is appropriately labeled or
is otherwise detectable.
[0290] Test kits for an assay format wherein there is competition
between a labeled (or otherwise detectable) MN protein/polypeptide
and MN antigen in the sample, for binding to an antibody, can
comprise the combination of the labeled protein/polypeptide and the
antibody in amounts which provide for optimum sensitivity and
accuracy.
[0291] Test kits for MN-specific antibodies preferably comprise
labeled/detectable MN proteins(s) and/or polypeptides(s), and may
comprise other components as necessary, for example, to perform a
preferred assay as outlined in Example 8 below, such as, controls,
buffers, diluents and detergents. Such test kits can have other
appropriate formats for conventional assays.
[0292] A kit for use in an enzyme-immunoassay typically includes an
enzyme-labelled reagent and a substrate for the enzyme. The enzyme
can, for example, bind either an MN-specific antibody of this
invention or to an antibody to such an MN-specific antibody.
Preparation of MN-Specific Antibodies
[0293] The term "antibodies" is defined herein to include not only
whole antibodies but also biologically active fragments of
antibodies, preferably fragments containing the antigen binding
regions. Such antibodies may be prepared by conventional
methodology and/or by genetic engineering. Antibody fragments may
be genetically engineered, preferably from the variable regions of
the light and/or heavy chains (V.sub.H and V.sub.L), including the
hypervariable regions, and still more preferably from both the
V.sub.H and V.sub.L regions. For example, the term "antibodies" as
used herein comprehends polyclonal and monoclonal antibodies and
biologically active fragments thereof including among other
possibilities "univalent" antibodies [Glennie et al., Nature, 295:
712 (1982)]; Fab proteins including Fab' and F(ab').sub.2 fragments
whether covalently or non-covalently aggregated; light or heavy
chains alone, preferably variable heavy and light chain regions
(V.sub.H and V.sub.L regions), and more preferably including the
hypervariable regions [otherwise known as the complementarity
determining regions (CDRs) of said V.sub.H and V.sub.L regions];
F.sub.c proteins; "hybrid" antibodies capable of binding more than
one antigen; constant-variable region chimeras; "composite"
immunoglobulins with heavy and light chains of different origins;
"altered" antibodies with improved specificity and other
characteristics as prepared by standard recombinant techniques and
also by oligonucleotide-directed mutagenesis techniques
[Dalbadie-McFarland et al., PNAS (USA), 79: 6409 (1982)].
[0294] It may be preferred for therapeutic and/or imaging uses that
the antibodies be biologically active antibody fragments,
preferably genetically engineered fragments, more preferably
genetically engineered fragments from the V.sub.H and/or V.sub.L
regions, and still more preferably comprising the hypervariable
regions thereof.
[0295] There are conventional techniques for making polyclonal and
monoclonal antibodies well-known in the immunoassay art. Immunogens
to prepare MN-specific antibodies include MN proteins and/or
polypeptides, preferably purified, and MX-infected tumor line
cells, for example, MX-infected HeLa cells, among other
immunogens.
[0296] Anti-peptide antibodies are also made by conventional
methods in the art as described in European Patent Publication No.
44,710 (published Jan. 27, 1982). Briefly, such anti-peptide
antibodies are prepared by selecting a peptide from an MN amino
acid sequence as from FIGS. 1A-B or 15, chemically synthesizing it,
conjugating it to an appropriate immunogenic protein and injecting
it into an appropriate animal, usually a rabbit or a mouse; then,
either polyclonal or monoclonal antibodies are made, the latter by
a Kohler-Milstein procedure, for example.
[0297] Besides conventional hybridoma technology, newer
technologies can be used to produce antibodies according to this
invention. For example, the use of the PCR to clone and express
antibody V-genes and phage display technology to select antibody
genes encoding fragments with binding activities has resulted in
the isolation of antibody fragments from repertoires of PCR
amplified V-genes using immunized mice or humans. [Marks et al.,
BioTechnology, 10: 779 (July 1992) for references; Chiang et al.,
BioTechniques, 7(4): 360 (1989); Ward et al., Nature, 341: 544
(Oct. 12, 1989); Marks et al., J. Mol. Biol., 222: 581 (1991);
Clackson et al., Nature, 352: (15 Aug. 1991); and Mullinax et al.,
PNAS (USA), 87: 8095 (October 1990).]
[0298] Descriptions of preparing antibodies, which term is herein
defined to include biologically active antibody fragments, by
recombinant techniques can be found in U.S. Pat. No. 4,816,567
(issued Mar. 28, 1989); European Patent Application Publication
Number (EP) 338,745 (published Oct. 25, 1989); EP 368,684
(published Jun. 16, 1990); EP 239,400 (published Sep. 30, 1987); WO
90/14424 (published Nov. 29, 1990); WO 90/14430 (published May 16,
1990); Huse et al., Science, 246: 1275 (Dec. 8, 1989); Marks et
al., BioTechnology, 10: 779 (July 1992); La Sastry et al., PNAS
(USA), 86: 5728 (August 1989); Chiang et al., BioTechniques, 7(40):
360 (1989); Orlandi et al., PNAS (USA), 86: 3833 (May 1989); Ward
et al. Nature, 341: 544 (Oct. 12, 1989); Marks et al., J. Mol.
Biol., 222: 581 (1991); and Hoogenboom et al., Nucleic Acids Res.,
19(15): 4133 (1991).
Representative Mabs
[0299] Monoclonal antibodies for use in the assays of this
invention may be obtained by methods well known in the art for
example, Galfre and Milstein, "Preparation of Monoclonal
Antibodies: Strategies and Procedures," in Methods in Enzymology:
Immunochemical Techniques, 73: 1-46 [Langone and Vanatis (eds);
Academic Press (1981)]; and in the classic reference, Milstein and
Kohler, Nature, 256: 495-497 (1975).]
[0300] Although representative hybridomas of this invention are
formed by the fusion of murine cell lines, human/human hybridomas
[Olsson et al., PNAS (USA), 77: 5429 (1980)] and human/murine
hybridomas [Schlom et al., PNAS (USA), 77: 6841 (1980); Shearman et
al. J. Immunol., 146: 928-935 (1991); and Gorman et al., PNAS
(USA), 88: 4181-4185 (1991)] can also be prepared among other
possiblities. Such humanized monoclonal antibodies would be
preferred monoclonal antibodies for therapeutic and imaging
uses.
[0301] Monoclonal antibodies specific for this invention can be
prepared by immunizing appropriate mammals, preferably rodents,
more preferably rabbits or mice, with an appropriate immunogen, for
example, MaTu-infected HeLa cells, MN fusion proteins, or MN
proteins/polypeptides attached to a carrier protein if necessary.
Exemplary methods of producing antibodies of this invention are
described below.
[0302] The monoclonal antibodies useful according to this invention
to identify MN proteins/polypeptides can be labeled in any
conventional manner, for example, with enzymes such as horseradish
peroxidase (HRP), fluorescent compounds, or with radioactive
isotopes such as, .sup.125I, among other labels. A preferred label,
according to this invention is .sup.125I, and a preferred method of
labeling the antibodies is by using chloramine-T [Hunter, W. M.,
"Radioimmunoassay," In: Handbook of Experimental Immunology, pp.
14.1-14.40 (D. W. Weir ed.; Blackwell,
Oxford/London/Edinburgh/Melbourne; 1978)].
[0303] Representative mabs of this invention include Mabs M75, MN9,
MN12 and MN7 described below. Monoclonal antibodies of this
invention serve to identify MN proteins/polypeptides in various
laboratory diagnostic tests, for example, in tumor cell cultures or
in clinical samples.
Mabs Prepared Against HeLa Cells
[0304] MAb M75. Monoclonal antibody M75 (MAb M75) is produced by
mouse lymphocytic hybridoma VU-M75, which was initially deposited
in the Collection of Hybridomas at the Institute of Virology,
Slovak Academy of Sciences (Bratislava, Slovakia) and was deposited
under ATCC Designation HB 11128 on Sep. 17, 1992 at the American
Type Culture Collection (ATCC) in Manassas, Va. (USA).
[0305] Hybridoma VU-M75 was produced according to the procedure
described in Gerhard, W., "Fusion of cells in suspension and
outgrowth of hybrids in conditioned medium," In: Monoclonal
Antibodies. Hybridomas: A New Dimension in Biological Analysis,
page 370 [Kennet et al. (eds.); Plenum NY (USA)]. BALE/C mice were
immunized with MaTu-infected HeLa cells, and their spleen cells
were fused with myeloma cell line NS-0. Tissue culture media from
the hybridomas were screened for monoclonal antibodies, using as
antigen the p58 immunoprecipitated from cell extracts of
MaTu-infected HeLa with rabbit anti-MaTu serum and protein
A-Staphylococcus aureus cells (SAC) [Zavada and Zavadova, Arch.
Virol., 118 189-197 (1991)], and eluted from SDS-PAGE gels.
Monoclonal antibodies were purified from TC media by affinity
chromatography on protein A-Sepharose [Harlow and Lane,
"Antibodies: A Laboratory Manual," Cold Spring Harbor, Cold Spring
Harbor, N.Y. (USA); 1988].
[0306] Mab M75 recognizes both the nonglycosylated GEX-3X-MN fusion
protein and native MN protein as expressed in CGL3 cells equally
well. Mab M75 was shown by epitope mapping to be reactive with the
epitope represented by the amino acid sequence from AA 62 to AA 67
[SEQ. ID. NO.: 10] of the MN protein shown in FIG. 15.
[0307] Mabs M16 and M67. Also produced by the method described for
producing MAb M75 (isotype IgG2B) were MAbs M16 (isotype IgG2A) and
M67 (isotype IgG1). Mabs M16 and M67 recognize MX protein, as
described in the examples below.
[0308] MAb H460. Monoclonal antibody H460 (MAb H460) was prepared
in a manner similar to that for MAb M75 except that the mice were
immunized with HeLa cells uninfected with MaTu, and lymphocytes of
the mice rather than spleen cells were fused with cells from
myeloma cell line NS-0. MAb H460 reacts about equally with any
human cells.
Mabs Prepared Against Fusion Protein GEX-3X-MN
[0309] Monoclonal antibodies of this invention were also prepared
against the MN glutathione S-transferase fusion protein (GEX-3X-MN)
purified by affinity chromatography as described above. BALE/C mice
were immunized intraperitoneally according to standard procedures
with the GEX-3X-MN fusion protein in Freund's adjuvant. Spleen
cells of the mice were fused with SP/20 myeloma cells [Milstein and
Kohler, supra].
[0310] Tissue culture media from the hybridomas were screened
against CGL3 and CGL1 membrane extracts in an ELISA employing HRP
labelled-rabbit anti-mouse. The membrane extracts were coated onto
microtiter plates. Selected were antibodies reacted with the CGL3
membrane extract. Selected hybridomas were cloned twice by limiting
dilution.
[0311] The mabs prepared by the just described method were
characterized by Western blots of the GEX-3X-MN fusion protein, and
with membrane extracts from the CGL1 and CGL3 cells. Representative
of the mabs prepared are Mabs MN9, MN12 and MN7.
[0312] Mab MN9. Monoclonal antibody MN9 (Mab MN9) reacts to the
same epitope as Mab M75, represented by the sequence from AA 62 to
AA 67 [SEQ. ID. NO.: 10] of the FIG. 15 MN protein. As Mab M75, Mab
MN9 recognizes both the GEX-3X-MN fusion protein and native MN
protein equally well.
[0313] Mabs corresponding to Mab MN9 can be prepared reproducibly
by screening a series of mabs prepared against an MN
protein/polypeptide, such as, the GEX-3X-MN fusion protein, against
the peptide representing the epitope for Mabs M75 and MN9. That
peptide is Arg Arg Ile Cys Pro Val [SEQ. ID. NO.: 10].
Alternatively, the Novatope system [Novagen] or competition with
the deposited Mab M75 could be used to select mabs comparable to
Mabs M75 and MN9.
[0314] Mab MN12. Monoclonal antibody MN12 (Mab MN12) is produced by
the mouse lymphocytic hybridoma MN 12.2.2 which was deposited under
ATCC Designation HB 11647 on Jun. 9, 1994 at the American Type
Culture Collection (ATCC) at 10801 University Blvd., Manassas, Va.
20110-2209 (USA). Antibodies corresponding to Mab MN12 can also be
made, analogously to the method outlined above for Mab MN9, by
screening a series of antibodies prepared against an MN
protein/polypeptide, against the peptide representing the epitope
for Mab MN12. That peptide is Gly Lys Met Thr His Trp [SEQ. ID.
NO.: 11]. The Novatope system could also be used to find antibodies
specific for said epitope.
[0315] Mab MN7. Monoclonal antibody MN7 (Mab MN7) was selected from
mabs prepared against nonglycosylated GEX-3X-MN as described above.
It recognizes the epitope on MN represented by the amino acid
sequence from AA 127 to AA 147 [SEQ. ID. NO.: 12; Asn Asn Ala His
Arg Asp Lys Glu Gly Asp Asp Gln Ser His Trp Arg Tyr Gly Gly Asp
Pro] of the FIG. 15 MN protein. Analogously to methods described
above for Mabs MN9 and MN12, mabs corresponding to Mab MN7 can be
prepared by selecting mabs prepared against an MN
protein/polypeptide that are reactive with the peptide having SEQ.
ID. NO.: 12, or by the stated alternative means.
Epitope Mapping
[0316] Epitope mapping was performed by the Novatope system, a kit
for which is commercially available from Novagen, Inc. [See, for
analogous example, Li et al., Nature, 363: 85-88 (6 May 1993).] In
brief, the MN cDNA was cut into overlapping short fragments of
approximately 60 base pairs. The fragments were expressed in E.
coli, and the E. coli colonies were transferred onto nitrocellulose
paper, lysed and probed with the mab of interest. The MN cDNA of
clones reactive with the mab of interest was sequenced, and the
epitopes of the mabs were deduced from the overlapping polypeptides
found to be reactive with each mab.
Therapeutic Use of MN-Specific Antibodies
[0317] The MN-specific antibodies of this invention, monoclonal
and/or polyclonal, preferably monoclonal, and as outlined above,
may be used therapeutically in the treatment of neoplastic and/or
pre-neoplastic disease, either alone or in combination with
chemotherapeutic drugs or toxic agents, such as ricin A. Further
preferred for therapeutic use would be biologically active antibody
fragments as described herein. Also preferred MN-specific
antibodies for such therapeutic uses would be humanized monoclonal
antibodies.
[0318] The MN-specific antibodies can be administered in a
therapeutically effective amount, preferably dispersed in a
physiologically acceptable, nontoxic liquid vehicle.
Imaging Use of Antibodies
[0319] Further, the MN-specific antibodies of this invention when
linked to an imaging agent, such as a radionuclide, can be used for
imaging. Biologically active antibody fragments or humanized
monoclonal antibodies, may be preferred for imaging use.
[0320] A patient's neoplastic tissue can be identified as, for
example, sites of transformed stem cells, of tumors and locations
of any metastases. Antibodies, appropriately labeled or linked to
an imaging agent, can be injected in a physiologically acceptable
carrier into a patient, and the binding of the antibodies can be
detected by a method appropriate to the label or imaging agent, for
example, by scintigraphy.
Antisense MN Nucleic Acid Sequences
[0321] MN genes are herein considered putative oncogenes and the
encoded prcteins thereby are considered to be putative
oncoproteins. Antisense nucleic acid sequences substantially
complementary to mRNA transcribed from MN genes, as represented by
the antisense oligodeoxynucleotides (ODNs) of Example 10, infra,
can be used to reduce or prevent expression of the MN gene.
[Zamecnick, P.C., "Introduction: Oligonucleotide Base Hybridization
as a Modulator of Genetic Message Readout," pp. 1-6, Prospects for
Antisense Nucleic Acid Therapy of Cancer and AIDS, (Wiley-Liss,
Inc., New York, N.Y., USA; 1991); Wickstrom, E., "Antisense DNA
Treatment of HL-60 Promyelocytic Leukemia Cells: Terminal
Differentiation and Dependence on Target Sequence," pp. 7-24, id.;
Leserman et al., "Targeting and Intracellular Delivery of Antisense
Oligonucleotides Interfering with Oncogene Expression," pp. 25-34,
id.; Yokoyama, K., "Transcriptional Regulation of c-myc
Proto-oncogene by Antisense RNA," pp. 35-52, id.; van den Berg et
al., "Antisense fos Oligodeoxyribonucleotides Suppress the
Generation of Chromosomal Aberrations," pp. 63-70, id.; Mercola,
D., "Antisense fos and fun RNA," pp. 83-114, id.; Inouye, Gene, 72:
25-34 (1988); Miller and Ts'o, Ann. Reports Med. Chem., 23: 295-304
(1988); Stein and Cohen, Cancer Res., 48: 2659-2668 (1988);
Stevenson and Inversen, J. Gen. Virol., 70: 2673-2682 (1989);
Goodchild, "Inhibition of Gene Expression by Oligonucleotides," pp.
53-77, Oligodeoxynucleotides: Antisense Inhibitors of Gene
Expression (Cohen, J. S., ed; CRC Press, Boca Raton, Fla., USA;
1989); Dervan et al., "Oligonucleotide Recognition of
Double-helical DNA by Triple-helix Formation," pp. 197-210, id.;
Neckers, L. M., "Antisense Oligodeoxynucleotides as a Tool for
Studying Cell Regulation: Mechanisms of Uptake and Application to
the Study of Oncogene Function," pp. 211-232, id.; Leitner et al.,
PNAS (USA), 87: 3430-3434 (1990); Bevilacqua et al., PNAS (USA),
85: 831-835 (1988); Loke et al. Curr. Top. Microbiol. Immunol.,
141: 282-288 (1988); Sarin et al., PNAS (USA), 85: 7448-7451
(1988); Agrawal et al., "Antisense Oligonucleotides: A Possible
Approach for Chemotherapy and AIDS," International Union of
Biochemistry Conference on Nucleic Acid Therapeutics (Jan. 13-17,
1991; Clearwater Beach, Fla., USA); Armstrong, L., Ber. Week, pp.
88-89 (Mar. 5, 1990); and Weintraub et al., Trends, 1: 22-25
(1985).] Such antisense nucleic acid sequences, preferably
oligonucleotides, by hybridizing to the MN mRNA, particularly in
the vicinity of the ribosome binding site and translation
initiation point, inhibits translation of the mRNA. Thus, the use
of such antisense nucleic acid sequences may be considered to be a
form of cancer therapy.
[0322] Preferred antisense oligonucleotides according to this
invention are gene-specific ODNs or oligonucleotides complementary
to the 5' end of MN mRNA. Particularly preferred are the 29-mer
ODN1 and 19-mer ODN2 for which the sequences are provided in
Example 10, infra. Those antisense ODNs are representative of the
many antisense nucleic acid sequences that can function to inhibit
MN gene expression. Ones of ordinary skill in the art could
determine appropriate antisense nucleic acid sequences, preferably
antisense oligonucleotides, from the nucleic acid sequences of
FIGS. 1A-1B, 15 and 25a-b.
[0323] Also, as described above, CGL3 cells transfected with an
"antisense" MN cDNA/promoter construct formed colonies much smaller
than control CGL3 cells.
Vaccines
[0324] It will be readily appreciated that MN proteins and
polypeptides of this invention can be incorporated into vaccines
capable of inducing protective immunity against neoplastic disease
and a dampening effect upon tumorigenic activity. Efficacy of a
representative MN fusion protein GEX-3X-MN as a vaccine in a rat
model is shown in Example 14.
[0325] MN proteins and/or polypeptides may be synthesized or
prepared recombinantly or otherwise biologically, to comprise one
or more amino acid sequences corresponding to one or more epitopes
of the MN proteins either in monomeric or multimeric form. Those
proteins and/or polypeptides may then be incorporated into vaccines
capable of inducing protective immunity. Techniques for enhancing
the antigenicity of such polypeptides include incorporation into a
multimeric structure, binding to a highly immunogenic protein
carrier, for example, keyhole limpet hemocyanin (KLH), or
diphtheria toxoid, and administration in combination with adjuvants
or any other enhancers of immune response.
[0326] Preferred MN proteins/polypeptides to be used in a vaccine
according to this invention would be genetically engineered MN
proteins. Preferred recombinant MN protein are the GEX-3X-MN and MN
20-19 proteins.
[0327] A preferred exemplary use of such a vaccine of this
invention would be its administration to patients whose MN-carrying
primary cancer had been surgically removed. The vaccine may induce
active immunity in the patients and prevent recidivism or
metastasis.
[0328] It will further be appreciated that anti-idiotype antibodies
to antibodies to MN proteins/polypeptides are also useful as
vaccines and can be similarly formulated.
[0329] An amino acid sequence corresponding to an epitope of an MN
protein/polypeptide either in monomeric or multimeric form may also
be obtained by chemical synthetic means or by purification from
biological sources including genetically modified microorganisms or
their culture media. [See Lerner, "Synthetic Vaccines", Sci. Am.
248(2): 66-74 (1983).] The protein/polypeptide may be combined in
an amino acid sequence with other proteins/polypeptides including
fragments of other proteins, as for example, when synthesized as a
fusion protein, or linked to other antigenic or non-antigenic
polypeptides of synthetic or biological origin. In some instances,
it may be desirable to fuse a MN protein or polypeptide to an
immunogenic and/or antigenic protein or polypeptide, for example,
to stimulate efficacy of a MN-based vaccine.
[0330] The term "corresponding to an epitope of an MN
protein/polypeptide" will be understood to include the practical
possibility that, in some instances, amino acid sequence variations
of a naturally occurring protein or polypeptide may be antigenic
and confer protective immunity against neoplastic disease and/or
anti-tumorigenic effects. Possible sequence variations include,
without limitation, amino acid substitutions, extensions,
deletions, truncations, interpolations and combinations thereof.
Such variations fall within the contemplated scope of the invention
provided the protein or polypeptide containing them is immunogenic
and antibodies elicited by such a polypeptide or protein
cross-react with naturally occurring MN proteins and polypeptides
to a sufficient extent to provide protective immunity and/or
anti-tumorigenic activity when administered as a vaccine.
[0331] Such vaccine compositions will be combined with a
physiologically acceptable medium, including immunologically
acceptable diluents and carriers as well as commonly employed
adjuvants such as Freund's Complete Adjuvant, saponin, alum, and
the like. Administration would be in immunologically effective
amounts of the MN proteins or polypeptides, preferably in
quantities providing unit doses of from 0.01 to 10.0 micrograms of
immunologically active MN protein and/or polypeptide per kilogram
of the recipient's body weight. Total protective doses may range
from 0.1 to about 100 micrograms of antigen.
[0332] Routes of administration, antigen dose, number and frequency
of injections are all matters of optimization within the scope of
the ordinary skill in the art.
[0333] The following examples are for purposes of illustration only
and not meant to limit the invention in any way.
Materials and Methods
[0334] The following materials and methods were used in examples
below.
MaTu-Infected and Uninfected HeLa Cells
[0335] MaTu agent [Zavada et al., Nature New Biol., 240: 124-125
(1972); Zavada et al., J. Gen. Virol, 24: 327-337 (1974)] was from
original "MaTu" cells [Widmaier et al., Arch. Geschwulstforsch, 44:
1-10 (1974)] transferred into our stock of HeLa by cocultivation
with MaTu cells treated with mitomycin C, to ensure that control
and MaTu-infected cells were comparable. MaTu cells were incubated
for 3 hours at 37.degree. C. in media with 5 .mu.g/ml of mitomycin
C [Calbiochem; LaJolla, Calif. (USA)]. Mixed cultures were set to
2.times.10.sup.5 of mitomycin C-treated cells and 4.times.10.sup.5
of fresh recipient cells in 5 ml of medium. After 3 days they were
first subcultured and further passaged 1-2 times weekly.
[0336] Control HeLa cells were the same as those described in
Zavada et al. (1972), supra.
Sera
[0337] Human sera from cancer patients, from patients suffering
with various non-tumor complaints and from healthy women were
obtained from the Clinics of Obstetrics and Gynaecology at the
Postgraduate Medical School, Bratislava, Slovakia. Human serum KH
was from a fifty year old mammary carcinoma patient, fourteen
months after resection. That serum was one of two sera out of 401
serum samples that contained neutralizing antibodies to the
VSV(MaTU) pseudotype as described in Zavada et al. (1972), supra.
Serum L8 was from a patient with Paget's disease. Serum M7 was from
a healthy donor.
[0338] Rabbit anti-MaTu serum was prepared by immunizing a rabbit
three times at intervals of 30 days with 1-5.times.10.sup.7 viable
MaTu-infected HeLa cells.
RIP and PAGE
[0339] RIP and PAGE were performed essentially as described in
Zavada and Zavadova, supra, except that in the experiments
described herein [.sup.35S]methionine (NEN), 10 .mu.Ci/ml of
methionine-free MEM medium, supplemented with 2% FCS and 3%
complete MEM were used. Confluent petri dish cultures of cells were
incubated overnight in that media.
[0340] For RIP, the SAC procedure [Kessler, J. Immunol., 115:
1617-1624 (1975)] was used. All incubations and centrifugations
were performed at 0-4.degree. C. Cell monolayers were extracted
with RIPA buffer (0.14 M NaCl, 7.5 mM phosphate buffer, pH 7.2, 1%
Triton X-100, 0.1% sodium deoxycholate, 1 mM phenylmethylsulfonyl
fluoride and Trasylol). To reduce non-specific reactions, antisera
were preabsorbed with fetal calf serum [Barbacid et al., PNAS
(USA), 77: 1617-1621 (1980)] and antigenic extracts with SAC.
[0341] For PAGE (under reducing conditions) we used 10% gels with
SDS [Laemmli, Nature, 227: 680-685 (1970)]. As reference marker
proteins served the Sigma kit [product MW-SDS-200; St. Louis, Mo.
(USA)]. For fluorography we used salicylate [Heegaard et al.,
Electrophoresis, 5: 263-269 (1984)].
Immunoblots
[0342] Immunoblotting used as described herein follows the method
of Towbin et al., PNAS (USA), 76: 4350-4354 (1979). The proteins
were transferred from the gels onto nitrocellulose [Schleicher and
Schuell; Dassel Germany; 0.45 .mu.m porosity] in Laemmli electrode
buffer diluted 1:10 with distilled water, with no methanol or SDS.
The transfer was for 21/2 hours at 1.75 mA/cm.sup.2. The blots were
developed with .sup.125I-labeled MAbs and autoradiography was
performed using intensifying screens, with X-ray films exposed at
-70.degree. C.
[0343] In extracts from cell cultures containing only small amounts
of MN antigen, we concentrated the antigen from 0.5 or 1 ml of an
extract by adding 50 .mu.l of a 10% SAC suspension, pre-loaded with
MAb M75. This method allowed the concentration of MN antigen even
from clinical specimens, containing human IgG; preliminary control
experiments showed that such a method did not interfere with the
binding of the MN antigen to SAC-adsorbed M75. Tissue extracts were
made by grinding the tissue with a mortar and pestle and sand
(analytical grade). To the homogenates was added RIPA buffer, 10:1
(volume to weight) of original tissue. The extracts were clarified
for 3 minutes on an Eppendorf centrifuge.
Example 1
Immunofluorescence of MaTu-Specific Antigens
[0344] Immunofluorescence experiments were performed on control and
MaTu-infected HeLa cells with monoclonal antibodies, prepared as
described above, which are specific for MaTu-related antigens.
FITC-conjugated anti-mouse IgG was used to detect the presence of
the monoclonal antibodies. Staining of the cells with Giemsa
revealed no clear differences between control and MaTu-infected
HeLa cells.
[0345] MAbs, which in preliminary tests proved to be specific for
MaTu-related antigens, showed two different reactivities in
immunofluorescence. A representative of the first group, MAb M67,
gave a granular cytoplasmic fluorescence in MaTu-infected HeLa,
which was only seen in cells fixed with acetone; living cells
showed no fluorescence. MAb M16 gave the same type of fluorescence.
With either M67 or M16, only extremely weak "background"
fluorescence was seen in control HeLa cells.
[0346] Another MAb, M75, showed a granular membrane fluorescence on
living MaTu-infected cells and a granular nuclear fluorescence in
acetone-fixed cells. However, M75 sometimes showed a similar,
although much weaker, fluorescence on uninfected HeLa cells. A
relationship was observed based upon the conditions of growth: in
HeLa cells uninfected with MaTu, both types of fluorescence with
MAb M75 were observed only if the cells were grown for several
passages in dense cultures, but not in sparse ones.
[0347] The amount of M75-reactive cell surface antigen was analyzed
cytofluorometrically and was dependent on the density of the cell
cultures and on infection with MaTu. Control and MaTu infected HeLa
cells were grown for 12 days in dense or sparse cultures. The cells
were released with Versene (EDTA), and incubated with MAb M75 or
with no MAb, and subsequently incubated with FITC-conjugated
anti-mouse IgG. The intensity of fluorescence was measured.
[0348] It appeared that the antigen binding MAb M75 is inducible:
it was found to be absent in control HeLa grown in sparse culture,
and to be induced either by the growth of HeLa in dense culture or
by infection with MaTu. Those two factors were found to have an
additive or synergistic effect. Those observations indicated along
with other results described herein that there were two different
agents involved: exogenous, transmissible MX, reactive with M67,
and endogenous, inducible MN, detected by MAb M75.
Example 2
Immunoblot Analysis of Protein(s) Reactive with MAb M75
[0349] To determine whether MAb M75 reacts with the same protein in
both uninfected and MaTu-infected HeLa, and to determine the
molecular weight of the protein, extracts of those cells were
analyzed by PAGE and immunoblotting (as described above). HeLa
cells uninfected or MaTu-infected, that had been grown for 12 days
in dense or sparse cultures, were seeded in 5-cm petri dishes, all
variants at 5.times.10.sup.5 cells per dish. Two days later, the
cells were extracted with RIPA buffer (above described), 200
.mu.l/dish. The extracts were mixed with 2.times. concentrated
Laemmli sample buffer containing 6% mercaptoethanol and boiled for
five minutes. Proteins were separated by SDS-PAGE and blotted on
nitrocellulose. The blots were developed with .sup.125I-labeled MAb
M75 and autoradiography.
[0350] MAb M75 reacted with two MN-specific protein bands of 54 kd
and 58 kd, which were the same in uninfected HeLa grown at high
density and in MaTu-infected HeLa, evidencing that M75 recognizes
the same protein(s) in both uninfected and MaTu-infected HeLa
cells. Consistent with the cytofluorometric results, the amount of
the antigen depended both on cell density and on infection with
MaTu, the latter being a much more potent inducer of p54/58N.
Example 3
Radioimmunoassay of MaTu-Specific Antigens In Situ
[0351] In contrast to the results with M75, the other MAb,
[0352] M67, appeared to be specific for the exogenous,
transmissible agent MX. With M67 we observed no immunofluorescence
in control HeLa, regardless of whether the cells were grown in
dense or in sparse culture. That difference was clearly evidenced
in radioimmunoassay experiments wherein .sup.125I-labeled MAbs M67
and M75 were used.
[0353] For such experiments, parallel cultures of uninfected and
MaTu-infected cells were grown in dense or sparse cultures. The
cultures were either live (without fixation), or fixed (with
methanol for five minutes and air-dried). The cultures were
incubated for two hours in petri dishes with the .sup.125I-labeled
MAbs, 6.times.10.sup.4 cpm/dish. Afterward, the cultures were
rinsed four times with PBS and solubilized with 1 ml/dish of 2 N
NaOH, and the radioactivity was determined on a gamma counter.
[0354] The simple radioimmunoassay procedure of this example was
performed directly in petri dish cultures. Sixteen variants of the
radioimmunoassay enabled us to determine whether the MX and MN
antigens are located on the surface or in the interior of the cells
and how the expression of those two antigens depends on infection
with MaTu and on the density, in which the cells had been grown
before the petri dishes were seeded. In live, unfixed cells only
cell surface antigens can bind the MAbs. In those cells, M67 showed
no reaction with any variant of the cultures, whereas M75 reacted
in accord with the results of Examples 1 and 2 above.
[0355] Fixation of the cells with methanol made the cell membrane
permeable to the MAbs: M67 reacted with HeLa infected with MaTu,
independently of previous cell density, and it did not bind to
control HeLa. MAb M75 in methanol-fixed cells confirmed the absence
of corresponding antigen in uninfected HeLa from sparse cultures
and its induction both by growth in dense cultures and by infection
with MaTu.
Example 4
Identification of MaTu Components Reactive with Animal Sera or
Associated with VSV Virions
[0356] Immunoblot analyses of MaTu-specific proteins from RIPA
extracts from uninfected or MaTu-infected HeLa and from purified
VSV reproduced in control or in MaTu-infected HeLa, identified
which of the antigens, p58X or p54/58N, were
radioimmunoprecipitated with animal sera, and which of them was
responsible for complementation of VSV mutants and for the
formation of pseudotype virions. Details concerning the procedures
can be found in Pastorekova et al., Virology, 187: 620-626
(1992).
[0357] The serum of a rabbit immunized with MaTu-infected HeLa
immunoprecipitated both MAb M67- and MAb M75-reactive proteins
(both p58X and p54/58N), whereas the "spontaneously" immune sera of
normal rabbit, sheep or leukemic cow immunoprecipitated only the
M67-reactive protein (p58X). On the other hand, in VSV reproduced
in MaTu-infected HeLa cells and subsequently purified, only the
M75-reactive bands of p54/58N were present. Thus, it was concluded
that MX and MN are independent components of MaTu, and that it was
p54/58N that complemented VSV mutants and was assembled into
pseudotype virions.
[0358] As shown in FIG. 6 discussed below in Example 5, MX antigen
was found to be present in MaTu-infected fibroblasts. In Zavada and
Zavadova, supra, it was reported that a p58 band from MX-infected
fibroblasts could not be detected by RIP with rabbit anti-MaTu
serum. That serum contains more antibodies to MX than to MN
antigen. The discrepancy can be explained by the extremely slow
spread of MX in infected cultures. The results reported in Zavada
and Zavadova, supra were from fibroblasts tested 6 weeks after
infection, whereas the later testing was 4 months after infection.
We have found by immunoblots that MX can be first detected in both
H/F-N and H/F-T hybrids after 4 weeks, in HeLa cells after six
weeks and in fibroblasts only 10 weeks after infection.
Example 5
Expression of MN- and MX-Specific Proteins
[0359] FIG. 6 graphically illustrates the expression of MN- and
MX-specific proteins in human fibroblasts, in HeLa cells and in
H/F-N and H/F-T hybrid cells, and contrasts the expression in
MX-infected and uninfected cells. Cells were infected with MX by
co-cultivation with mitomycin C-treated MX-infected HeLa. The
infected and uninfected cells were grown for three passages in
dense cultures. About four months after infection, the infected
cells concurrently with uninfected cells were grown in petri dishes
to produce dense monolayers.
[0360] A radioimmunoassay was performed directly in confluent petri
dish (5 cm) culture of cells, fixed with methanol essentially as
described in Example 3, supra. The monolayers were fixed with
methanol and treated with .sup.125I-labeled MAbs M67 (specific for
exogenous MX antigen) or M75 (specific for endogenous MN antigen)
at 6.times.10.sup.4 cpm/dish. The bound radioactivity was measured;
the results are shown in FIG. 6.
[0361] FIG. 6 shows that MX was transmitted to all four cell lines
tested, that is, to human embryo fibroblasts, to HeLa and to both
H/F-N and H/F-T hybrids; at the same time, all four uninfected
counterpart cell lines were MX-negative (top graph of FIG. 6). MN
antigens are shown to be present in both MX-infected and uninfected
HeLa and H/F-T cells, but not in the fibroblasts (bottom graph of
FIG. 6). No MN antigen was found in the control H/F-N, and only a
minimum increase over background of MN antigen was found in MaTu
infected H/F-N. Thus, it was found that in the hybrids, expression
of MN antigen very strongly correlates with tumorigenicity.
[0362] Those results were consistent with the results obtained by
immunoblotting as shown in FIG. 7. The MN-specific twin protein
p54/58N was detected in HeLa cell lines (both our standard type,
that is, HeLa K, and in the Stanbridge mutant HeLa, that is,
D98/AH.2 shown as HeLa S) and in tumorigenic H/F-T; however,
p54/58N was not detected in the fibroblasts nor in the
non-tumorigenic H/F-N even upon deliberately long exposure of the
film used to detect radioactivity. Infection of the HeLa cells with
MX resulted in a strong increase in the concentration of the
p54/58N protein(s).
[0363] The hybrid cells H/F-N and H/F-T were constructed by Eric J.
Stanbridge [Stanbridge et al., Somatic Cell Genetics, 7: 699-712
(1981); and Stanbridge et al., Science, 215: 252-259 (1982)]. His
original hybrid, produced by the fusion of a HeLa cell and a human
fibroblast was not tumorigenic in nude mice, although it retained
some properties of transformed cells, for example, its growth on
soft agar. Rare segregants from the hybrid which have lost
chromosome 11 are tumorigenic. The most likely explanation for the
tumorigenicity of those segregants is that chromosome 11 contains a
suppressor gene (an antioncogene), which blocks the expression of a
as yet unknown oncogene. The oncoprotein encoded by that oncogene
is critical for the capacity of the H/F hybrids to produce tumors
in nude mice. Since the p54/58N protein shows a correlation with
the tumorigenicity of H/F hybrids, it is a candidate for that
putative oncoprotein.
Example 6
Immunoblots of MN Antigen from Human Tumor Cell Cultures and from
Clinical Specimens of Human Tissues
[0364] The association of MN antigen with tumorigenicity in the H/F
hybrid cells as illustrated by Example 5 prompted testing for the
presence of MN antigen in other human tumor cell cultures and in
clinical specimens. Preliminary experiments indicated that the
concentration of MN antigen in the extracts from other human tumor
cell cultures was lower than in HeLa; thus, it was realized that
long exposure of the autoradiographs would be required. Therefore,
the sensitivity of the method was increased by the method indicated
under Materials and Methods: Immunoblotting, supra, wherein the MN
antigen was concentrated by precipitation with MAb M75-loaded
SAC.
[0365] FIG. 8 shows the immunoblots wherein lane A, a cell culture
extract from MX-infected HeLa cells was analysed directly (10 .mu.l
per lane) whereas the antigens from the other extracts (lanes B-E)
were each concentrated from a 500 .mu.l extract by precipitation
with MAb M75 and SAC.
[0366] FIG. 8 indicates that two other human carcinoma cell lines
contain MN-related proteins--T24 (bladder carcinoma; lane C) and
T47D (mammary carcinoma; lane D). Those cells contain proteins
which react with MAb M75 that under reducing conditions have
molecular weights of 54 kd and 56 kd, and under non-reducing
conditions have a molecular weight of about 153 kd. The intensity
of those bands is at least ten times lower than that for the
p54/58N twin protein from HeLa cells.
[0367] An extremely weak band at approximately 52 kd could be seen
under reducing conditions from extracts from human melanoma cells
(SK-MeI 1477;lane E), but no bands for human fibroblast extracts
(lane B) could be seen either on the reducing or non-reducing
gels.
[0368] FIG. 9 shows immunoblots of human tissue extracts including
surgical specimens as compared to a cell extract from MX-infected
HeLa (lane A). The MN-related antigen from all the extracts but for
lane A (analysed directly at 10 .mu.l per lane) was first
concentrated from a 1 ml extract as explained above. MN proteins
were found in endometrial (lanes D and M), ovarian (lanes E and N)
and in uterine cervical (lane 0) carcinomas. In those extracts
MN-related proteins were found in three bands having molecular
weights between about 48 kd and about 58 kd. Another MN-related
protein was present in the tissue extract from a mammary papilloma;
that protein was seen as a single band at about 48 kd (lane J).
[0369] Clearly negative were the extracts from full-term placenta
(lane B), normal mammary gland (lane K), hyperplastic endometrium
(lane L), normal ovaries (lane H), and from uterine myoma (lane I).
Only extremely slightly MN-related bands were seen in extracts from
trophoblasts (lanes F and G) and from melanoma (lane P).
[0370] The observations that antigen related to p54/58N was
expressed in clinical specimens of several types of human
carcinomas but not in general in normal tissues of the
corresponding organs (exceptions delineated in Example 13) further
strengthened the association of MN antigen with tumorigenesis.
However, it should be noted that for human tumors, a normal tissue
is never really an adequate control in that tumors are believed not
to arise from mature, differentiated cells, but rather from some
stem cells, capable of division and of differentiation. In body
organs, such cells may be quite rare.
Example 7
MN Antigen in Animal Cell Lines
[0371] Since the MN gene is present in the chromosomal DNA of all
vertebrate species that were tested, MN-related antigen was
searched for also in cell lines derived from normal tissues and
from tumors of several animal species. MN-related protein was found
in two rat cell lines: one of them was the XC cell line derived
from rat rhabdomyosarcoma induced with Rous sarcoma virus; the
other was the Rat2-Tk.sup.- cell line. In extracts from both of
those rat cell lines, a single protein band was found on the blots:
its molecular weight on blots produced from a reducing gel and from
a non-reducing gel was respectively 53.5 kd and 153 kd. FIG. 10
shows the results with Rat2-Tk.sup.- cell extracts (lane B),
compared with extracts from MX-infected HeLa (lane A); the
concentration of MN antigen in those two cell lines is very
similar. The extracts were analysed directly (40 .mu.l per
lane).
[0372] MN-related protein from XC cells showed the same pattern as
for Rat2-Tk.sup.- cells both under reducing and non-reducing
conditions, except that its concentration was about 30.times.
lower. The finding of a MN-related protein--p53.5N--in two rat cell
lines (FIGS. 10 and 12) provides the basis for a model system.
[0373] None of the other animal cell lines tested contained
detectable amounts of MN antigen, even when the highly sensitive
immunoblot technique in which the MN antigens are concentrated was
used. The MN-negative cells were: Vero cells (African green
monkey); mouse L cells; mouse NIH-3T3 cells normal, infected with
Moloney leukemia virus, or transformed with Harvey sarcoma virus;
GR cells (mouse mammary tumor cells induced with MTV), and NMG
cells (normal mouse mammary gland).
Example 8
Radioimmunoassays in Liquid Phase Using Recombinant MN Protein for
MN-Specific Antibodies and for MN Antigen
[0374] The genetically engineered MN protein fused with glutathione
S-transferase--GEX-3X-MN--prepared and purified as described above
was labeled with .sup.125I by the chloramine T method [Hunter
(1978)]. The purified protein enabled the development of a
quantitative RIA for MN-specific antibodies as well as for MN
antigens. All dilutions of antibodies and of antigens were prepared
in RIPA buffer (1% TRITON X-100 and 0.1% sodium deoxycholate in
PBS--phosphate buffered saline, pH 7.2), to which was added 1% of
fetal calf serum (FCS). Tissue and cell extracts were prepared in
RIPA buffer containing 1 mM phenylmethylsulfonylfluoride and 200
trypsin inhibiting units of Trasylol (aprotinin) per ml, with no
FCS. .sup.125I-labeled GEX-3X-MN protein (2.27 .mu.Ci/.mu.g of
TCA-precipitable protein) was before use diluted with RIPA+1% FCS,
and non-specifically binding radioactivity was adsorbed with a
suspension of fixed protein A-Staphylococcus aureus cells
(SAC).
[0375] In an RIA for MN-specific antibodies, MAb-containing ascites
fluids or test sera were mixed with .sup.125I-labeled protein and
allowed to react in a total volume of 1 ml for 2 hours at room
temperature. Subsequently, 50 .mu.l of a 10% suspension of SAC
[Kessler, supra] was added and the mixture was incubated for 30
minutes. Finally, the SAC was pelleted, 3.times. washed with RIPA,
and the bound radioactivity was determined on a gamma counter.
[0376] Titration of antibodies to MN antigen is shown in FIG. 11.
Ascitic fluid from a mouse carrying M75 hybridoma cells (A) is
shown to have a 50% end-point at dilution 1:1.4.times.10.sup.-6. At
the same time, ascitic fluids with MAbs specific for MX protein
(M16 and M67) showed no precipitation of .sup.125I-labeled
GEX-3X-MN even at dilution 1:200 (result not shown). Normal rabbit
serum (C) did not significantly precipitate the MN antigen; rabbit
anti-MaTu serum (B), obtained after immunization with live
MX-infected HeLa cells, precipitated 7% of radioactive MN protein,
when diluted 1:200. The rabbit anti-MaTu serum is shown by
immunoblot in Example 4 (above) to precipitate both MX and MN
proteins.
[0377] Only one out of 180 human sera tested (90 control and 90
sera of patients with breast, ovarian or uterine cervical cancer)
showed a significant precipitation of the radioactively labeled MN
recombinant protein. That serum--L8--(D) was retested on immunoblot
(as in Example 4), but it did not precipitate any p54/58N from
MX-infected HeLa cells. Also, six other human sera, including KH
(E), were negative on immunoblot. Thus, the only positive human
serum in the RIA, L8, was reactive only with the genetically
engineered product, but not with native p54/58N expressed by HeLa
cells.
[0378] In an RIA for MN antigen, the dilution of MAb M75, which in
the previous test precipitated 50% of maximum precipitable
radioactivity (=dilution 1:1.4.times.10.sup.-6) was mixed with
dilutions of cell extracts and allowed to react for 2 hours. Then,
.sup.125I-labeled GEX-3X-MN (25.times.10.sup.3 cpm/tube) was added
for another 2 hours. Finally, the radioactivity bound to MAb M75
was precipitated with SAC and washed as above. One hundred percent
precipitation (=0 inhibition) was considered the maximum
radioactivity bound by the dilution of MAb used. The concentration
of the MN antigen in the tested cell extracts was calculated from
an inhibition curve obtained with "cold" GEX-3X-MN, used as the
standard (A in FIG. 12).
[0379] The reaction of radioactively labeled GEX-3X-MN protein with
MAb M75 enabled us to quantitate MN antigen directly in cell
extracts. FIG. 12 shows that 3 ng of "cold" GEX-3X-MN (A) caused a
50% inhibition of precipitation of "hot" GEX-3X-MN; an equivalent
amount of MN antigen is present in 3.times.10.sup.3 ng of proteins
extracted from MaTu-infected HeLa (B) or from Rat2-Tk.sup.- cells
(C). Concentrations of MN protein in cell extracts, determined by
this RIA, are presented in Table 1 below. It must be understood
that the calculated values are not absolute, since MN antigens in
cell extracts are of somewhat different sizes, and also since the
genetically engineered MN protein is a product containing molecules
of varying size.
TABLE-US-00004 TABLE 1 Concentration of MN Protein in Cell Extracts
ng MN/mg Cells total protein HeLa + MX 939.00 Rat2-Tk.sup.- 1065.00
HeLa 27.50 XC 16.40 T24 1.18 HEF 0.00
The data were calculated from the results shown in FIG. 12.
Example 9
Immunoelectron and Scanning Microscopy of Control and of
MX-infected HeLa Cells
[0380] As indicated above in Example 1, MN antigen, detected by
indirect immunofluorescence with MAb M75, is located on the surface
membranes and in the nuclei of MX-infected HeLa cells or in HeLa
cells grown in dense cultures. To elucidate more clearly the
location of the MN antigen, immunoelectron microscopy was used
wherein MAb M75 bound to MN antigen was visualized with immunogold
beads. [Herzog et al., "Colloidal gold labeling for determining
cell surface area," IN: Colloidal Gold, Vol. 3 (Hayat, M. A., ed.),
pp. 139-149 (Academic Press Inc.; San Diego, Calif.).]
[0381] Ultrathin sections of control and of MX-infected HeLa cells
are shown in FIG. 13 A-D. Those immuno-electron micrographs
demonstrate the location of MN antigen in the cells, and in
addition, the striking ultrastructural differences between control
and MX-infected HeLa. A control HeLa cell (FIG. 13A) is shown to
have on its surface very little MN antigen, as visualised with gold
beads. The cell surface is rather smooth, with only two little
protrusions. No mitochondria can be seen in the cytoplasm. In
contrast, MX-infected HeLa cells (FIGS. 13B and C) show the
formation of abundant, dense filamentous protrusions from their
surfaces. Most of the MN antigen is located on those filaments,
which are decorated with immunogold. The cytoplasm of MX-infected
HeLa contains numerous mitochondria (FIG. 13C). FIG. 13D
demonstrates the location of MN antigen in the nucleus: some of the
MN antigen is in nucleoplasm (possibly linked to chromatin), but a
higher concentration of the MN antigen is in the nucleoli. Again,
the surface of normal HeLa (panels A and E of FIG. 13) is rather
smooth whereas MX-infected HeLa cells have on their surface,
numerous filaments and "blebs". Some of the filaments appear to
form bridges connecting them to adjacent cells.
[0382] It has been noted that in some instances of in vitro
transformed cells compared to their normal parent cells that one of
the differences is that the surface of normal cells was smooth
whereas on the transformed cells were numerous hair-like
protrusions [Darnell et al. "Molecular Cell Biology," (2nd edition)
Sci. Am. Books; W.H. Freeman and Co., New York (1990)]. Under that
criteria MX-infected HeLa cells, as seen in FIG. 13F, has a
supertransformed appearance.
[0383] Further in some tumors, amplification of mitochondria has
been described [Bernhard, W., "Handbook of Molecular Cytology," pp.
687-715, Lima de Faria (ed.), North Holland Publishing Co.;
Amsterdam-London (1972)]. Such amplification was noted for
MX-infected HeLa cells which stained very intensely with Janus'
green, specific for mitochondria whereas control HeLa were only
weakly stained.
[0384] It should be noted that electron microscopists were unable
to find any structural characteristics specific for tumor
cells.
Example 10
Antisense ODNs Inhibit MN Gene Expression
[0385] To determine whether both of the p54/58N proteins were
encoded by one gene, the following experiments with antisense ODNs
were performed. Previously sparse-growing HeLa cells were seeded to
obtain an overcrowded culture and incubated for 130 hours either in
the absence or in the presence of two gene-specific ODNs
complementary to the 5' end of MN mRNA. HeLa cells were subcultured
at 8.times.10.sup.5 cells per ml of DMEM with 10% FCS.
Simultaneously, ODNs were added to the media as follows: (A) 29-mer
ODN1 (5' CGCCCAGTGGGTCATCTTCCCCAGAAGAG 3' [SEQ. ID. NO.: 3],
complementary to positions 44-72 of FIG. 1A-1B) in 4 .mu.M final
concentration, (B) 19-mer ODN2 (5' GGAATCCTCCTGCATCCGG 3' [SEQ. ID.
NO.: 4], complementary to positions 12-30) in 4 .mu.M final
concentration and (C) both ODN1 and ODN2 in 2 .mu.M final
concentration each. (D) Cells treated in the same way, but
incubated without ODNs, served as a control. After 130 hours,
extracts from the cells were prepared and analyzed by
immunoblotting using .sup.125I-labeled MAb M75. Protein extracts
from the cells were analyzed by immunoblotting and RIA using MAb
M75. FIG. 3 provides the immunoblot results of those
experiments.
[0386] It was found that cultivation of HeLa cells with the ODNs
resulted in considerable inhibition of p54/58N synthesis. The
19-mer ODN2 (FIG. 3B) in 4 .mu.M final concentration was very
effective; as determined by RIA, it caused 40% inhibition, whereas
the 29-mer ODN1 (4 .mu.M) (FIG. 3A) and a combination of the two
ODNs (FIG. 3C), each in 2 .mu.M final concentration, were less
effective in RIA showing a 25-35% decrease of the MN-related
proteins. At the same time, the amount of different HeLa cell
protein determined by RIA using specific MAb H460 was in all cell
variants approximately the same. Most importantly was that on
immunoblot it could be seen that specific inhibition by the ODNs
affected both of the p54/58N proteins. Thus, we concluded that the
MN gene we cloned coded for both p54/58N proteins in HeLa
cells.
[0387] The results indicated that the MN twin proteins arise by
translation of a single mRNA (consistent with the Northern blotting
data). Thus, the twin proteins may represent either differences in
post-translational modification (phosphorylation, protease
processing, etc.), or the use of alternative translational
initiation sites.
Example 11
Northern Blotting of MN mRNA in Tumorigenic and Non-Tumorigenic
Cell Lines
[0388] FIG. 4 shows the results of Northern blotting of MN mRNA in
human cell lines. Total RNA was prepared from the following cell
lines by the guanidinium thiocyanate-CsCl method: HeLa cells
growing in a dense (A) and sparse (B) culture; CGL1 (H/F-N) hybrid
cells (C); CGL3 (D) and CGL4 (E) segregants (both H/F-T); and human
embryo fibroblasts (F). Fifteen .mu.g of RNA were separated on a
1.2% formaldehyde gel and blotted onto a Hybond C Super membrane
[Amersham]. MN cDNA NotI probe was labeled by random priming
[Multiprime DNA labelling system; Amersham]. Hybridization was
carried out in the presence of 50% formamide at 42.degree. C., and
the final wash was in 0.1% SSPE and 0.1% SDS at 65.degree. C. An
RNA ladder (0.24-9.5 kb) [BRL; Bethesda, Md. (USA)] was used as a
size standard. Membranes were exposed to films at -70.degree. C.,
with intensifying screens.
[0389] Detected was a 1.5 kb MN-specific mRNA only in two
tumorigenic segregant clones--CGL3 and CGL4 (H/F-T), but not in the
non-tumorigenic hybrid clone CGL1 (H/F-N) or in normal human
fibroblasts. Further, the 1.5 kb mRNA was found in the HeLa cells
growing in dense (FIG. 4A) but not in sparse to (FIG. 4B)
culture.
[0390] Thus, the results of the Northern blotting were consistent
with other examples in regard to MN-related proteins being
associated with tumorigenicity.
Example 12
Southern Blotting of Genomic DNAs from Different Vertebrate Species
to Detect MN Gene and Restriction Analysis of Genomic DNA of HeLa
Cells
[0391] FIG. 5 illustrates the detection of MN genes in the genomic
DNAs of various vertebrates by Southern blotting. Chromosomal DNA
digested by PstI was as follows: (A) chicken; (B) bat; (C) rat; (D)
mouse; (E) feline; (F) pig; (G) sheep; (H) bovine; (I) monkey; and
(J) human HeLa cells. Restriction fragments were separated on a
0.7% agarose gel and alkali blotted onto a Hybond N membrane
[Amersham]. The MN cDNA probe labelling and hybridization
procedures were the same as for the Northern blotting analyses
shown in FIG. 4 and described in Example 11. The Southern blot of
FIG. 5 made with PstI indicates that the MN gene is conserved in a
single copy in all vertebrate genomes tested.
[0392] HeLa. Further, genomic DNA from HeLa cells was prepared as
described by Ausubel et al., Short Protocols in Molecular Biology
[Greene Publishing Associates and Wiley-Interscience; New York
(1989)], digested with different restriction enzymes, resolved on
an agarose gel and transferred to Hybond N+ membrane [Amersham].
The HeLa genomic DNA was cleaved with the following restriction
enzymes with the results shown in FIG. 17 (wherein the numbers in
parentheses after the enzymes indicate the respective lanes in FIG.
17): EcoRI (1), EcoRV (2), HindIII (3), KpnI (4), NcoI (5), PstI
(6), and PvuII (7), and then analyzed by Southern hybridization
under stringent conditions using MN cDNA as a probe.
[0393] The prehybridization and hybridization using an MN cDNA
probe labelled with .sup.32P-dCTP by random priming [Multi-prime
DNA labelling system; Amersham] as well as wash steps were carried
out according to Amersham's protocols at high stringency. A 1 kb
DNA Ladder [from BRL; Bethesda, Md. (USA)] was used as a size
standard. Membranes were exposed to films at -70.degree. C., with
intensifying screens.
[0394] The Southern blotting analysis of HeLa chromosomal DNA
showed that the gene coding for MN is present in the human genome
in a single copy (FIG. 17). The sizes and distribution of
MN-positive restriction fragments obtained using the restriction
enzymes KpnI, NcoI and HindIII indicate that the MN gene contains
introns, since those enzymes cut the MN genomic sequences despite
the absence of their restriction sites in MN cDNA.
Example 13
Immunohistochemical Staining of Tissue Specimens
[0395] To study and evaluate the tissue distribution range and
expression of MN proteins, the monoclonal antibody M75 was used to
stain immunohistochemically a variety of human tissue specimens.
The primary antibody used in these immunohistochemical staining
experiments was the M75 monoclonal antibody. A biotinylated second
antibody and streptavidin-peroxidase were used to detect the M75
reactivity in sections of formalin-fixed, paraffin-embedded tissue
samples. A commercially available amplification kit, specifically
the DAKO LSAB.TM. kit [DAKO Corp., Carpinteria, Calif. (USA)] which
provides matched, ready made blocking reagent, secondary antibody
and steptavidin-horseradish peroxidase was used in these
experiments.
[0396] M75 immunoreactivity was tested according to the methods of
this invention in multiple-tissue sections of breast, colon,
cervical, lung and normal tissues. Such multiple-tissue sections
were cut from paraffin blocks of tissues called "sausages" that
were purchased from the City of Hope [Duarte, Calif. (USA)].
Combined in such a multiple-tissue section were normal, benign and
malignant specimens of a given tissue; for example, about a score
of tissue samples of breast cancers from different patients, a
similar number of benign breast tissue samples, and normal breast
tissue samples would be combined in one such multiple-breast-tissue
section. The normal multiple-tissue sections contained only normal
tissues from various organs, for example, liver, spleen, lung,
kidney, adrenal gland, brain, prostate, pancreas, thyroid, ovary,
and testis.
[0397] Also screened for MN gene expression were multiple
individual specimens from cervical cancers, bladder cancers, renal
cell cancers, and head and neck cancers. Such specimens were
obtained from U.C. Davis Medical Center in Sacramento, Calif. and
from Dr. Shu Y. Liao [Department of Pathology; St. Joseph Hospital;
Orange, Calif. (USA)].
[0398] Controls used in these experiments were the cell lines CGL3
(H/F-T hybrid cells) and CGL1 (H/F-N hybrid cells) which are known
to stain respectively, positively and negatively with the M75
monoclonal antibody. The M75 monoclonal antibody was diluted to a
1:5000 dilution wherein the diluent was either PBS [0.05 M
phosphate buffered saline (0.15 M NaCl), pH 7.2-7.4] or PBS
containing 1% protease-free BSA as a protein stabilizer.
Immunohistochemical Staining Protocol
[0399] The immunohistochemical staining protocol was followed
according to the manufacturer's instructions for the DAKO LSAB.TM.
kit. In brief, the sections were dewaxed, rehydrated and blocked to
remove non-specific reactivity as well as endogenous peroxidase
activity. Each section was then incubated with dilutions of the M75
monoclonal antibody. After the unbound M75 was removed by rinsing
the section, the section was sequentially reacted with a
biotinylated antimouse IgG antibody and streptavidin conjugated to
horseradish peroxidase; a rinsing step was included between those
two reactions and after the second reaction. Following the last
rinse, the antibody-enzyme complexes were detected by reaction with
an insoluble chromogen (diaminobenzidine) and hydrogen peroxide. A
positive result was indicated by the formation of an insoluble
reddish-brown precipitate at the site of the primary antibody
reaction. The sections were then rinsed, counterstained with
hematoxylin, dehydrated and cover slipped. Then the sections were
examined using standard light microscopy. The following is an
outline of exemplary steps of the immunohistochemical staining
protocol.
TABLE-US-00005 1. Series of ETOH-baths 100, 100, 95, 2 min. .+-. 1
min. each 95, 70% 2. dH.sub.20 wash - 2x 2 min. .+-. 1 min. each 3.
3% H.sub.20.sub.2 as endogenous peroxidase 5 min. block 4. PBS wash
- 2x 2 min. .+-. 1 min. 5. normal serum block (1.5% NGS) 30 min. 6.
primary antibody (Mab M75) 60 min. .+-. 5 min. 7. PBS wash - 2x 2
min. .+-. 1 min. 8. biotinylated secondary antibody 20-30 min. .+-.
2 min. 9. PBS wash - 2x 2 min. .+-. 1 min. 10.
streptavidin-peroxidase reagent 20-30 min. .+-. 2 min. 11. PBS wash
- 2x 2 min. .+-. 1 min. 12. DAB (150 ml Tris, 90.PHI.l
H.sub.20.sub.2, 3 ml KPL 5-6 min. DAB) 13. PBS rinse, dH.sub.20
wash 1-2 min. 14. Hematoxylin counterstain 2 min. .+-. 1 min. 15.
wash with running tap water until clear 16. 0.05% ammonium
hydroxide 20 sec. .+-. 10 sec. 17. dH.sub.20 wash - 2x 3 min. .+-.
1 min. 18. dehydrate 70, 95, 95, 100, 100% 2 min. .+-. 1 min. each
EtOH 19. xylene 3x 3 min. .+-. 1 min. each 20. coverslip with
Permount .TM. [Fisher Scientific Pittsburgh, PA (USA)] 21. wait 10
min. before viewing results.
[0400] Interpretation. A deposit of a reddish brown precipitate
over the plasma membrane was taken as evidence that the M75
antibody had bound to a MN antigen in the tissue. The known
positive control (CGL3) had to be stained to validate the assay.
Section thickness was taken into consideration to compare staining
intensities, as thicker sections produce greater staining intensity
independently of other assay parameters.
[0401] The above-described protocol was optimized for
formalin-fixed tissues, but can be used to stain tissues prepared
with other fixatives.
Results
[0402] Preliminary examination of cervical specimens showed that 62
of 68 squamous cell carcinoma specimens (91.2%) stained positively
with M75. Additionally, 2 of 6 adenocarcinomas and 2 of 2
adenosquamous cancers of the cervix also stained positively. In
early studies, 55.6% (10 of 18) of cervical dysplasias stained
positively. A total of 9 specimens including both cervical
dysplasias and tumors, exhibited some MN expression in normal
appearing areas of the endocervical glandular epithelium, usually
at the basal layer. In some specimens, whereas morphologically
normal-looking areas showed expression of MN antigen, areas
exhibiting dysplasia and/or malignancy did not show MN
expression.
[0403] M75 positive immunoreactivity was most often localized to
the plasma membrane of cells, with the most apparent stain being
present at the junctions between adjacent cells. Cytoplasmic
staining was also evident in some cells; however, plasma membrane
staining was most often used as the main criterion of
positivity.
[0404] M75 positive cells tended to be near areas showing keratin
differentiation in cervical specimens. In some specimens, positive
staining cells were located in the center of nests of non-staining
cells. Often, there was very little, if any, obvious morphological
difference between staining cells and non-staining cells. In some
specimens, the positive staining cells were associated with
adjacent areas of necrosis.
[0405] In most of the squamous cell carcinomas of the cervix, the
M75 immunoreactivity was focal in distribution, i.e., only certain
areas of the specimen stained. Although the distribution of
positive reactivity within a given specimen was rather sporadic,
the intensity of the reactivity was usually very strong. In most of
the adenocarcinomas of the cervix, the staining pattern was more
homogeneous, with the majority of the specimen staining
positively.
[0406] Among the normal tissue samples, intense, positive and
specific M75 immunoreactivity was observed only in normal stomach
tissues, with diminishing reactivity in the small intestine,
appendix and colon. No other normal tissue stained extensively
positively for M75. Occasionally, however, foci of intensely
staining cells were observed in normal intestine samples (usually
at the base of the crypts) or were sometimes seen in
morphologically normal appearing areas of the epithelium of
cervical specimens exhibiting dysplasia and/or malignancy. In such,
normal appearing areas of cervical specimens, positive staining was
seen in focal areas of the basal layer of the ectocervical
epithelium or in the basal layer of endocervical glandular
epithelium. In one normal specimen of human skin, cytoplasmic MN
staining was observed in the basal layer. The basal layers of these
epithelia are usually areas of proliferation, suggesting the MN
expression may be involved in cellular growth. In a few cervical
biopsied specimens, MN positivity was observed in the
morphologically normal appearing stratified squamous epithelium,
sometimes associated with cells undergoing koilocytic changes.
[0407] Some colon adenomas (4 of 11) and adenocarcinomas (9 of 15)
were positively stained. One normal colon specimen was positive at
the base of the crypts. Of 15 colon cancer specimens, 4
adenocarcinomas and 5 metastatic lesions were MN positive. Fewer
malignant breast cancers (3 of 25) and ovarian cancer specimens (3
of 15) were positively stained. Of 4 head and neck cancers, 3
stained very intensely with M75.
[0408] Although normal stomach tissue was routinely positive, 4
adenocarcinomas of the stomach were MN negative. Of 3 bladder
cancer specimens (1 adenocarcinoma, 1 non-papillary transitional
cell carcinoma, and 1 squamous cell carcinoma), only the squamous
cell carcinoma was MN positive. Approximately 40% (12 of 30) of
lung cancer specimens were positive; 2 of 4 undifferentiated
carcinomas; 3 of 8 adenocarcinomas; 2 of 8 oat cell carcinomas;
and, 5 of 10 squamous cell carcinomas. One hundred percent (4 of 4)
of the renal cell carcinomas were MN positive.
[0409] In summary, MN antigen, as detected by M75 and
immunohistochemistry in the experiments described above, was shown
to be prevalent in tumor cells, most notably in tissues of cervical
cancers. MN antigen was also found in some cells of normal tissues,
and sometimes in morphologically normal appearing areas of
specimens exhibiting dysplasia and/or malignancy. However, MN is
not usually extensively expressed in most normal tissues, except
for stomach tissues where it is extensively expressed and in the
tissues of the lower gastrointestinal tract where it is less
extensively expressed. MN expression is most often localized to the
cellular plasma membrane of tumor cells and may play a role in
intercellular communication or cell adhesion. Representative
results of experiments performed as described above are tabulated
in Table 2.
TABLE-US-00006 TABLE 2 Immunoreactivity of M75 in Various Tissues
POS/NEG TISSUE TYPE (#pos/#tested) liver, spleen, lung, normal NEG
(all) kidney, adrenal gland, brain, prostate, pancreas, thyroid,
ovary, testis skin normal POS (in basal layer) (1/1) stomach normal
POS small intestine normal POS colon normal POS breast normal NEG
(0/10) cervix normal NEG (0/2) breast benign NEG (0/17) colon
benign POS (4/11) cervix benign POS (10/18) breast malignant POS
(3/25) colon malignant POS (9/15) ovarian malignant POS (3/15) lung
malignant POS (12/30) bladder malignant POS (1/3) head & neck
malignant POS (3/4) kidney malignant POS (4/4) stomach malignant
NEG (0/4) cervix malignant POS (62/68)
[0410] The results recorded in this example indicate that the
presence of MN proteins in a tissue sample from a patient may, in
general, depending upon the tissue involved, be a marker signaling
that a pre-neoplastic or neoplastic process is occurring. Thus, one
may conclude from these results that diagnostic/prognostic methods
that detect MN antigen may be particularly useful for screening
patient samples for a number of cancers which can thereby be
detected at a pre-neoplastic stage or at an early stage prior to
obvious morphologic changes associated with dysplasia and/or
malignancy being evident or being evident on a widespread
basis.
Example 14
Vaccine--Rat Model
[0411] As shown above in Example 7, in some rat tumors, for
example, the XC tumor cell line (cells from a rat
rhabdomyosarcoma), a rat MN protein, related to human MN, is
expressed. Thus a model was afforded to study antitumor immunity
induced by experimental MN-based vaccines. The following
representative experiments were performed.
[0412] Nine- to eleven-day-old Wistar rats from several families
were randomized, injected intraperitoneally with 0.1 ml of either
control rat sera (the C group) or with rat serum against the MN
fusion protein GEX-3X-MN (the IM group). Simultaneously both groups
were injected subcutaneously with 10.sup.6 XC tumor cells.
[0413] Four weeks later, the rats were sacrificed, and their tumors
weighed. The results are shown in FIG. 14. Each point on the graph
represents a tumor from one rat. The difference between the two
groups--C and IM--was significant by Mann-Whitney rank test (U=84,
.alpha.<0.025). The results indicate that the IM group of baby
rats developed tumors about one-half the size of the controls, and
5 of the 18 passively immunized rats developed no tumor at all,
compared to 1 of 18 controls.
Example 15
Expression of Full-Length MN cDNA in NIH 3T3 Cells
[0414] The role of MN in the regulation of cell proliferation was
studied by expressing the full-length cDNA in NIH 3T3 cells. That
cell line was chosen since it had been used successfully to
demonstrate the phenotypic effect of a number of proto-oncogenes
[Weinberg, R. A., Cancer Res., 49: 3713 (1989); Hunter, T., Cell,
64: 249 (1991)]. Also, NIH 3T3 cells express no endogenous
MN-related protein that is detectable by Mab M75.
[0415] The full length MN cDNA was obtained by ligation of the two
cDNA clones using the unique BamHI site and subcloned from
pBluescript into KpnI-SacI sites of the expression vector pSG5C.
pSG5C was kindly provided by Dr. Richard Kettman [Department of
Molecular Biology, Faculty of Agricultural Sciences, B-5030
Gembloux, Belgium]. pSG5C was derived from pSG5 [Stratagene] by
inserting a polylinker consisting of a sequence having several
neighboring sites for the following restriction enzymes: EcoRI,
XhoI, KpnI, BamHI, SacI, 3 times TAG stop codon and BglII.
[0416] The recombinant pSG5C-MN plasmid was co-transfected in a
10:1 ratio (10 .mu.g:1 .mu.g) with the pSV2neo plasmid [Southern
and Berg, J. Mol. Appl. Genet., 1: 327 (1982)] which contains the
neo gene as a selection marker. The co-transfection was carried out
by calcium phosphate precipitation method [Mammalian Transfection
Kit; Stratagene] into NIH 3T3 cells plated a day before at a
density of 1.times.10.sup.5 per 60 mm dish. As a control, pSV2neo
was co-transfected with empty pSG5C.
[0417] Transfected cells were cultured in DMEM medium supplemented
with 10% FCS and 600 .mu.ml.sup.-1 of G418 [Gibco BRL] for 14 days.
The G418-resistant cells were clonally selected, expanded and
analysed for expression of the transfected cDNA by Western blotting
using iodinated Mab M75.
[0418] For an estimation of cell proliferation, the clonal cell
lines were plated in triplicates (2.times.10.sup.4 cells/well) in
24-well plates and cultivated in DMEM with 10% FCS and 1% FCS,
respectively. The medium was changed each day, and the cell number
was counted using a hemacytometer.
[0419] To determine the DNA synthesis, the cells were plated in
triplicate in 96-well plate at a density of 10.sup.4/well in DMEM
with 10% FCS and allowed to attach overnight. Then the cells were
labeled with .sup.3H-thymidine for 24 hours, and the incorporated
radioactivity was counted.
[0420] For the anchorage-independent growth assay, cells
(2.times.10.sup.4) were suspended in a 0.3% agar in DMEM containing
10% FCS and overlaid onto 0.5% agar medium in 60 mm dish. Colonies
grown in soft agar were counted two weeks after plating.
[0421] Several clonal cell lines constitutively expressing both 54
and 58 kd forms of MN protein in levels comparable to those found
in LCMV-infected HeLa cells were obtained. Selected MN-positive
clones and negative control cells (mock-transfected with an empty
pSG5C plasmid) were subjected to further analyses directed to the
characterization of their phenotype and growth behavior.
[0422] The MN-expressing NIH 3T3 cells displayed spindle-shaped
morphology, and increased refractility; they were less adherent to
the solid support and smaller in size. The control (mock
transfected cells) had a flat morphology, similar to parental NIH
3T3 cells. In contrast to the control cells that were aligned and
formed a monolayer with an ordered pattern, the cells expressing MN
lost the capacity for growth arrest and grew chaotically on top of
one another (FIG. 23a-d). Correspondingly, the MN-expressing cells
were able to reach significantly higher (more than 2.times.)
saturation densities (Table 3) and were less dependent on growth
factors than the control cells (FIG. 23g-h).
[0423] MN transfectants also showed faster doubling times (by 15%)
and enhanced DNA synthesis (by 10%), as determined by the amount of
[.sup.3H]-thymidine incorporated in comparison to control cells.
Finally, NIH 3T3 cells expressing MN protein grew in soft agar. The
diameter of colonies grown for 14 days ranged from 0.1 to 0.5 mm
(FIG. 23f); however, the cloning efficiency of MN transfectants was
rather low (2.4%). Although that parameter of NIH 3T3 cells seems
to be less affected by MN than by conventional oncogenes, all other
data are consistent with the idea that MN plays a role in cell
growth control.
TABLE-US-00007 TABLE 3 Growth Properties of NIH 3T3 Cells
Expressing MN Protein Transfected pSG5C/ pSG5C-MN/ DNA pSV2neo
pSV2neo Doubling time.sup.a 27.9 .+-. 0.5 24.1 .+-. 1.3 (hours)
Saturation density.sup.b 4.9 .+-. 0.2 11.4 .+-. 0.4 (cells .times.
10.sup.4/cm.sup.2) Cloning <0.01 2.4 .+-. 0.2 efficiency
(%).sup.c .sup.aFor calculation of the doubling time, the
proliferation rate of exponentially growing cells was used.
.sup.bThe saturation cell density was derived from the cell number
4 days after reaching confluency. .sup.cColonies greater than 0.1
mm in diameter were scored at day 14. Cloning efficiency was
estimated as a percentage of colonies per number of cells plated,
with correction for cell viability.
Example 16
Acceleration of G1 Transit and Decrease in Mitomycin C Sensitivity
Caused by MN Protein
[0424] For the experiments described in this example, the stable MN
transfectants of NIH 3T3 cells generated as described in Example 15
were used. Four selected MN-positive clones and four control
mock-transfected clones were either used individually or in
pools.
[0425] Flow cytometric analyses of asynchronous cell populations.
For the results shown in FIG. 24(a), cells that had been grown in
dense culture were plated at 1.times.10.sup.6 cells per 60 mm dish.
Four days later, the cells were collected by trypsinization,
washed, resuspended in PBS, fixed by dropwise addition of 70%
ethanol and stained by propidium iodine solution containing RNase.
Analysis was performed by FACStar using DNA cell cycle analysis
software [Becton Dickinson; Franklin Lakes, N.J. (USA)].
[0426] For the analyses shown in FIG. 24(h) and (c), exponentially
growing cells were plated at 5.times.10.sup.5 cells per 60 mm dish
and analysed as above 2 days later. Forward light scatter was used
for the analysis of relative cell sizes. The data were evaluated
using Kolmogorov-Smirnov test [Young, J. Histochem. Cytochem., 25:
935 (1977)]. D is the maximum difference between summation curves
derived from histograms. D/s(n) is a value which indicates the
similarity of the compared curves (it is close to zero when curves
are similar).
[0427] The flow cytometric analyses revealed that clonal
populations constitutively expressing MN protein showed a decreased
percentage of cells in G1 phase and an increased percentage of
cells in G2-M phases. Those differences were more striking in cell
populations grown throughout three passages in high density
cultures [FIG. 24(a)], than in exponentially growing subconfluent
cells [FIG. 24(b)]. That observation supports the idea that MN
protein has the capacity to perturb contact inhibition.
[0428] Also observed was a decrease in the size of MN expressing
cells seen in both exponentially proliferating and high density
cultures. It is possible that the MN-mediated acceleration of G1
transit is related to the above-noted shorter doubling time (by
about 15%) of exponentially proliferating MN-expressing NIH 3T3
cells. Also, MN expressing cells displayed substantially higher
saturation density and lower serum requirements than the control
cells. Those facts suggest that MN-transfected cells had the
capacity to continue to proliferate despite space limitations and
diminished levels cf serum growth factors, whereas the control
cells were arrested in G1 phase.
[0429] Limiting conditions. The proliferation of MN-expressing and
control cells was studied both in optimal and limiting conditions.
Cells were plated at 2.times.10.sup.4 per well of 24-well plate in
DMEM with 10% FCS. The medium was changed at daily intervals until
day 4 when confluency was reached, and the medium was no longer
renewed. Viable cells were counted in a hemacytometer at
appropriate times using trypan blue dye exclusion. The numbers of
cells were plotted versus time wherein each plot point represents a
mean value of triplicate determination.
[0430] The results showed that the proliferation of MN expressing
and control cells was similar during the first phase when the
medium was renewed daily, but that a big difference in the number
of viable cells occurred after the medium was not renewed. More
than half of the control cells were not able to withstand the
unfavorable growth conditions. In contrast, the MN-expressing cells
continued to proliferate even when exposed to increasing
competition for nutrients and serum growth factors.
[0431] Those results were supported also by flow cytometric
analysis of serum starved cells grown for two days in medium
containing 1% FCS. While 83% of control cells accumulated in G0-G1
phase (S=5%, G2-M=12%), expression of MN protein partially reversed
the delay in G1 as indicated by cell cycle distribution of MN
transfectants (G0-G1=65%, S=10%, G2-M=26%). The results of the
above-described experiments suggest that MN protein might function
to release the G1/S checkpoint and allow cells to proliferate under
unfavorable conditions.
[0432] MMC. To test that assumption, unfavorable conditions were
simulated by treating cells with the DNA damaging drug mitomycin C
(MMC) and then following their proliferation and viability. The
mechanism of action of MMC is thought to result from its
intracellular activation and subsequent DNA alkylation and
crosslinking [Yier and Szybalski, Science, 145: 55 (1964)].
Normally, cells respond to DNA damage by arrest of their cell cycle
progression to repair defects and prevent acquisition of genomic
instability. Large damage is accompanied by marked cytotoxicity.
However, many studies [for example, Peters et al., Int. J. Cancer,
54: 450 (1993)] concern the emergence of drug resistant cells both
in tumor cell populations and after the introduction of oncogenes
into nontransformed cell lines.
[0433] The response of MN-transfected NIH 3T3 cells to increasing
concentrations of MMC was determined by continuous
[.sup.3H]-thymidine labeling. Cells were plated in 96-well
microtiter plate concentration of 10.sup.4 per well and incubated
overnight in DMEM with 10% FCS to attach. Then the growth medium
was replaced with 100 .mu.l of medium containing increasing
concentrations of MMC from 1 .mu.l/ml to 32 .mu.g/ml. All the drug
concentrations were tested in three replicate wells. After 5 hours
of treatment, the MMC was removed, cells were washed with PBS and
fresh growth medium without the drug was added. After overnight
recovery, the fractions of cells that were actively participating
in proliferation was determined by continuous 24-hr labeling with
[.sup.3H]-thymidine. The incorporation by the treated cells was
compared to that of the control, untreated cells, and the
proliferating fractions were considered as a percentage of the
control's incorporation.
[0434] The viability of the treated cells was estimated three days
later by a CellTiter 96 AQ Non-Radioactive Cell Proliferation Assay
[Promega] which is based on the bioreduction of methotrexate (MTX)
into a water soluble formazan that absorbs light at 490 nm. The
percentage of surviving cells was derived from the values of
absorbance obtained after subtraction of background.
[0435] The control and MN-expressing NIH 3T3 cells showed
remarkable differences in their responses to MMC. The sensitivity
of the MN-transfected cells appeared considerably lower than the
control's in both sections of the above-described experiments. The
results suggested that the MN-transfected cells were able to
override the negative growth signal mediated by MMC.
[0436] ATCC Deposits. The material listed below was deposited with
the American Type Culture Collection (ATCC) at 10801 University
Blvd., Manassas, Va. 20110-2209 (USA). The deposits were made under
the provisions of the Budapest Treaty on the International
Recognition of Deposited Microorganisms for the Purposes of Patent
Procedure and Regulations thereunder (Budapest Treaty). Maintenance
of a viable culture is assured for thirty years from the date of
deposit. The organism will be made available by the ATCC under the
terms of the Budapest Treaty, and subject to an agreement between
the Applicants and the ATCC which assures unrestricted availability
of the deposited hybridomas to the public upon the granting of
patent from the instant application. Availability of the deposited
strain is not to be construed as a license to practice the
invention in contravention of the rights granted under the
authority of any Government in accordance with its patent laws.
TABLE-US-00008 Hybridoma Deposit Date ATCC # VU-M75 Sep. 17, 1992
HB 11128 MN 12.2.2 Jun. 9, 1994 HB 11647
[0437] The description of the foregoing embodiments of the
invention have been presented for purposes of illustration and
description. They are not intended to be exhaustive or to limit the
invention to the precise form disclosed, and obviously many
modifications and variations are possible in light of the above
teachings. The embodiments were chosen and described in order to
explain the principles of the invention and its practical
application to enable thereby others skilled in the art to utilize
the invention in various embodiments and with various modifications
as are suited to the particular use contemplated.
[0438] All references cited herein are hereby incorporated by
reference.
Sequence CWU 1
1
2611399DNAHUMANCDS(1)..(1266) 1cag agg ttg ccc cgg atg cag gag gat
tcc ccc ttg gga gga ggc tct 48Gln Arg Leu Pro Arg Met Gln Glu Asp
Ser Pro Leu Gly Gly Gly Ser1 5 10 15tct ggg gaa gat gac cca ctg ggc
gag gag gat ctg ccc agt gaa gag 96Ser Gly Glu Asp Asp Pro Leu Gly
Glu Glu Asp Leu Pro Ser Glu Glu 20 25 30gat tca ccc aga gag gag gat
cca ccc gga gag gag gat cta cct gga 144Asp Ser Pro Arg Glu Glu Asp
Pro Pro Gly Glu Glu Asp Leu Pro Gly 35 40 45gag gag gat cta cct gga
gag gag gat cta cct gaa gtt aag cct aaa 192Glu Glu Asp Leu Pro Gly
Glu Glu Asp Leu Pro Glu Val Lys Pro Lys 50 55 60tca gaa gaa gag ggc
tcc ctg aag tta gag gat cta cct act gtt gag 240Ser Glu Glu Glu Gly
Ser Leu Lys Leu Glu Asp Leu Pro Thr Val Glu65 70 75 80gct cct gga
gat cct caa gaa ccc cag aat aat gcc cac agg gac aaa 288Ala Pro Gly
Asp Pro Gln Glu Pro Gln Asn Asn Ala His Arg Asp Lys 85 90 95gaa ggg
gat gac cag agt cat tgg cgc tat gga ggc gac ccg ccc tgg 336Glu Gly
Asp Asp Gln Ser His Trp Arg Tyr Gly Gly Asp Pro Pro Trp 100 105
110ccc cgg gtg tcc cca gcc tgc gcg ggc cgc ttc cag tcc ccg gtg gat
384Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Phe Gln Ser Pro Val Asp
115 120 125atc cgc ccc cag ctc gcc gcc ttc tgc ccg gcc ctg cgc ccc
ctg gaa 432Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala Leu Arg Pro
Leu Glu 130 135 140ctc ctg ggc ttc cag ctc ccg ccg ctc cca gaa ctg
cgc ctg cgc aac 480Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu Leu
Arg Leu Arg Asn145 150 155 160aat ggc cac agt gtg caa ctg acc ctg
cct cct ggg cta gag atg gct 528Asn Gly His Ser Val Gln Leu Thr Leu
Pro Pro Gly Leu Glu Met Ala 165 170 175ctg ggt ccc ggg cgg gag tac
cgg gct ctg cag ctg cat ctg cac tgg 576Leu Gly Pro Gly Arg Glu Tyr
Arg Ala Leu Gln Leu His Leu His Trp 180 185 190ggg gct gca ggt cgt
ccg ggc tcg gag cac act gtg gaa ggc cac cgt 624Gly Ala Ala Gly Arg
Pro Gly Ser Glu His Thr Val Glu Gly His Arg 195 200 205ttc cct gcc
gag atc cac gtg gtt cac ctc agc acc gcc ttt gcc aga 672Phe Pro Ala
Glu Ile His Val Val His Leu Ser Thr Ala Phe Ala Arg 210 215 220gtt
gac gag gcc ttg ggg cgc ccg gga ggc ctg gcc gtg ttg gcc gcc 720Val
Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu Ala Val Leu Ala Ala225 230
235 240ttt ctg gag gag ggc ccg gaa gaa aac agt gcc tat gag cag ttg
ctg 768Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala Tyr Glu Gln Leu
Leu 245 250 255tct cgc ttg gaa gaa atc gct gag gaa ggc tca gag act
cag gtc cca 816Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser Glu Thr
Gln Val Pro 260 265 270gga ctg gac ata tct gca ctc ctg ccc tct gac
ttc agc cgc tac ttc 864Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser Asp
Phe Ser Arg Tyr Phe 275 280 285caa tat gag ggg tct ctg act aca ccg
ccc tgt gcc cag ggt gtc atc 912Gln Tyr Glu Gly Ser Leu Thr Thr Pro
Pro Cys Ala Gln Gly Val Ile 290 295 300tgg act gtg ttt aac cag aca
gtg atg ctg agt gct aag cag ctc cac 960Trp Thr Val Phe Asn Gln Thr
Val Met Leu Ser Ala Lys Gln Leu His305 310 315 320acc ctc tct gac
acc ctg tgg gga cct ggt gac tct cgg cta cag ctg 1008Thr Leu Ser Asp
Thr Leu Trp Gly Pro Gly Asp Ser Arg Leu Gln Leu 325 330 335aac ttc
cga gcg acg cag cct ttg aat ggg cga gtg att gag gcc tcc 1056Asn Phe
Arg Ala Thr Gln Pro Leu Asn Gly Arg Val Ile Glu Ala Ser 340 345
350ttc cct gct gga gtg gac agc agt cct cgg gct gct gag cca gtc cag
1104Phe Pro Ala Gly Val Asp Ser Ser Pro Arg Ala Ala Glu Pro Val Gln
355 360 365ctg aat tcc tgc ctg gct gct ggt gac atc cta gcc ctg gtt
ttt ggc 1152Leu Asn Ser Cys Leu Ala Ala Gly Asp Ile Leu Ala Leu Val
Phe Gly 370 375 380ctc ctt ttt gct gtc acc agc gtc gcg ttc ctt gtg
cag atg aga agg 1200Leu Leu Phe Ala Val Thr Ser Val Ala Phe Leu Val
Gln Met Arg Arg385 390 395 400cag cac aga agg gga acc aaa ggg ggt
gtg agc tac cgc cca gca gag 1248Gln His Arg Arg Gly Thr Lys Gly Gly
Val Ser Tyr Arg Pro Ala Glu 405 410 415gta gcc gag act gga gcc
tagaggctgg atcttggaga atgtgagaag 1296Val Ala Glu Thr Gly Ala
420ccagccagag gcatctgagg gggagccggt aactgtcctg tcctgctcat
tatgccactt 1356ccttttaact gccaagaaat tttttaaaat aaatatttat aat
13992422PRTHUMAN 2Gln Arg Leu Pro Arg Met Gln Glu Asp Ser Pro Leu
Gly Gly Gly Ser1 5 10 15Ser Gly Glu Asp Asp Pro Leu Gly Glu Glu Asp
Leu Pro Ser Glu Glu 20 25 30Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly
Glu Glu Asp Leu Pro Gly 35 40 45Glu Glu Asp Leu Pro Gly Glu Glu Asp
Leu Pro Glu Val Lys Pro Lys 50 55 60Ser Glu Glu Glu Gly Ser Leu Lys
Leu Glu Asp Leu Pro Thr Val Glu 65 70 75 80Ala Pro Gly Asp Pro Gln
Glu Pro Gln Asn Asn Ala His Arg Asp Lys 85 90 95Glu Gly Asp Asp Gln
Ser His Trp Arg Tyr Gly Gly Asp Pro Pro Trp 100 105 110Pro Arg Val
Ser Pro Ala Cys Ala Gly Arg Phe Gln Ser Pro Val Asp 115 120 125Ile
Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala Leu Arg Pro Leu Glu 130 135
140Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro Glu Leu Arg Leu Arg
Asn145 150 155 160Asn Gly His Ser Val Gln Leu Thr Leu Pro Pro Gly
Leu Glu Met Ala 165 170 175Leu Gly Pro Gly Arg Glu Tyr Arg Ala Leu
Gln Leu His Leu His Trp 180 185 190Gly Ala Ala Gly Arg Pro Gly Ser
Glu His Thr Val Glu Gly His Arg 195 200 205Phe Pro Ala Glu Ile His
Val Val His Leu Ser Thr Ala Phe Ala Arg 210 215 220Val Asp Glu Ala
Leu Gly Arg Pro Gly Gly Leu Ala Val Leu Ala Ala225 230 235 240Phe
Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala Tyr Glu Gln Leu Leu 245 250
255Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser Glu Thr Gln Val Pro
260 265 270Gly Leu Asp Ile Ser Ala Leu Leu Pro Ser Asp Phe Ser Arg
Tyr Phe 275 280 285Gln Tyr Glu Gly Ser Leu Thr Thr Pro Pro Cys Ala
Gln Gly Val Ile 290 295 300Trp Thr Val Phe Asn Gln Thr Val Met Leu
Ser Ala Lys Gln Leu His305 310 315 320Thr Leu Ser Asp Thr Leu Trp
Gly Pro Gly Asp Ser Arg Leu Gln Leu 325 330 335Asn Phe Arg Ala Thr
Gln Pro Leu Asn Gly Arg Val Ile Glu Ala Ser 340 345 350Phe Pro Ala
Gly Val Asp Ser Ser Pro Arg Ala Ala Glu Pro Val Gln 355 360 365Leu
Asn Ser Cys Leu Ala Ala Gly Asp Ile Leu Ala Leu Val Phe Gly 370 375
380Leu Leu Phe Ala Val Thr Ser Val Ala Phe Leu Val Gln Met Arg
Arg385 390 395 400Gln His Arg Arg Gly Thr Lys Gly Gly Val Ser Tyr
Arg Pro Ala Glu 405 410 415Val Ala Glu Thr Gly Ala 420329DNAHUMAN
3cgcccagtgg gtcatcttcc ccagaagag 29419DNAHUMAN 4ggaatcctcc
tgcatccgg 1951522DNAHUMANCDS(13)..(1389)mat_peptide(124)..(1389)
5acagtcagcc gc atg gct ccc ctg tgc ccc agc ccc tgg ctc cct ctg ttg
51 Met Ala Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu -35 -30
-25atc ccg gcc cct gct cca ggc ctc act gtg caa ctg ctg ctg tca ctg
99Ile Pro Ala Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu Ser Leu
-20 -15 -10ctg ctt ctg atg cct gtc cat ccc cag agg ttg ccc cgg atg
cag gag 147Leu Leu Leu Met Pro Val His Pro Gln Arg Leu Pro Arg Met
Gln Glu -5 -1 1 5gat tcc ccc ttg gga gga ggc tct tct ggg gaa gat
gac cca ctg ggc 195Asp Ser Pro Leu Gly Gly Gly Ser Ser Gly Glu Asp
Asp Pro Leu Gly 10 15 20gag gag gat ctg ccc agt gaa gag gat tca ccc
aga gag gag gat cca 243Glu Glu Asp Leu Pro Ser Glu Glu Asp Ser Pro
Arg Glu Glu Asp Pro25 30 35 40ccc gga gag gag gat cta cct gga gag
gag gat cta cct gga gag gag 291Pro Gly Glu Glu Asp Leu Pro Gly Glu
Glu Asp Leu Pro Gly Glu Glu 45 50 55gat cta cct gaa gtt aag cct aaa
tca gaa gaa gag ggc tcc ctg aag 339Asp Leu Pro Glu Val Lys Pro Lys
Ser Glu Glu Glu Gly Ser Leu Lys 60 65 70tta gag gat cta cct act gtt
gag gct cct gga gat cct caa gaa ccc 387Leu Glu Asp Leu Pro Thr Val
Glu Ala Pro Gly Asp Pro Gln Glu Pro 75 80 85cag aat aat gcc cac agg
gac aaa gaa ggg gat gac cag agt cat tgg 435Gln Asn Asn Ala His Arg
Asp Lys Glu Gly Asp Asp Gln Ser His Trp 90 95 100cgc tat gga ggc
gac ccg ccc tgg ccc cgg gtg tcc cca gcc tgc gcg 483Arg Tyr Gly Gly
Asp Pro Pro Trp Pro Arg Val Ser Pro Ala Cys Ala105 110 115 120ggc
cgc ttc cag tcc ccg gtg gat atc cgc ccc cag ctc gcc gcc ttc 531Gly
Arg Phe Gln Ser Pro Val Asp Ile Arg Pro Gln Leu Ala Ala Phe 125 130
135tgc ccg gcc ctg cgc ccc ctg gaa ctc ctg ggc ttc cag ctc ccg ccg
579Cys Pro Ala Leu Arg Pro Leu Glu Leu Leu Gly Phe Gln Leu Pro Pro
140 145 150ctc cca gaa ctg cgc ctg cgc aac aat ggc cac agt gtg caa
ctg acc 627Leu Pro Glu Leu Arg Leu Arg Asn Asn Gly His Ser Val Gln
Leu Thr 155 160 165ctg cct cct ggg cta gag atg gct ctg ggt ccc ggg
cgg gag tac cgg 675Leu Pro Pro Gly Leu Glu Met Ala Leu Gly Pro Gly
Arg Glu Tyr Arg 170 175 180gct ctg cag ctg cat ctg cac tgg ggg gct
gca ggt cgt ccg ggc tcg 723Ala Leu Gln Leu His Leu His Trp Gly Ala
Ala Gly Arg Pro Gly Ser185 190 195 200gag cac act gtg gaa ggc cac
cgt ttc cct gcc gag atc cac gtg gtt 771Glu His Thr Val Glu Gly His
Arg Phe Pro Ala Glu Ile His Val Val 205 210 215cac ctc agc acc gcc
ttt gcc aga gtt gac gag gcc ttg ggg cgc ccg 819His Leu Ser Thr Ala
Phe Ala Arg Val Asp Glu Ala Leu Gly Arg Pro 220 225 230gga ggc ctg
gcc gtg ttg gcc gcc ttt ctg gag gag ggc ccg gaa gaa 867Gly Gly Leu
Ala Val Leu Ala Ala Phe Leu Glu Glu Gly Pro Glu Glu 235 240 245aac
agt gcc tat gag cag ttg ctg tct cgc ttg gaa gaa atc gct gag 915Asn
Ser Ala Tyr Glu Gln Leu Leu Ser Arg Leu Glu Glu Ile Ala Glu 250 255
260gaa ggc tca gag act cag gtc cca gga ctg gac ata tct gca ctc ctg
963Glu Gly Ser Glu Thr Gln Val Pro Gly Leu Asp Ile Ser Ala Leu
Leu265 270 275 280ccc tct gac ttc agc cgc tac ttc caa tat gag ggg
tct ctg act aca 1011Pro Ser Asp Phe Ser Arg Tyr Phe Gln Tyr Glu Gly
Ser Leu Thr Thr 285 290 295ccg ccc tgt gcc cag ggt gtc atc tgg act
gtg ttt aac cag aca gtg 1059Pro Pro Cys Ala Gln Gly Val Ile Trp Thr
Val Phe Asn Gln Thr Val 300 305 310atg ctg agt gct aag cag ctc cac
acc ctc tct gac acc ctg tgg gga 1107Met Leu Ser Ala Lys Gln Leu His
Thr Leu Ser Asp Thr Leu Trp Gly 315 320 325cct ggt gac tct cgg cta
cag ctg aac ttc cga gcg acg cag cct ttg 1155Pro Gly Asp Ser Arg Leu
Gln Leu Asn Phe Arg Ala Thr Gln Pro Leu 330 335 340aat ggg cga gtg
att gag gcc tcc ttc cct gct gga gtg gac agc agt 1203Asn Gly Arg Val
Ile Glu Ala Ser Phe Pro Ala Gly Val Asp Ser Ser345 350 355 360cct
cgg gct gct gag cca gtc cag ctg aat tcc tgc ctg gct gct ggt 1251Pro
Arg Ala Ala Glu Pro Val Gln Leu Asn Ser Cys Leu Ala Ala Gly 365 370
375gac atc cta gcc ctg gtt ttt ggc ctc ctt ttt gct gtc acc agc gtc
1299Asp Ile Leu Ala Leu Val Phe Gly Leu Leu Phe Ala Val Thr Ser Val
380 385 390gcg ttc ctt gtg cag atg aga agg cag cac aga agg gga acc
aaa ggg 1347Ala Phe Leu Val Gln Met Arg Arg Gln His Arg Arg Gly Thr
Lys Gly 395 400 405ggt gtg agc tac cgc cca gca gag gta gcc gag act
gga gcc 1389Gly Val Ser Tyr Arg Pro Ala Glu Val Ala Glu Thr Gly Ala
410 415 420tagaggctgg atcttggaga atgtgagaag ccagccagag gcatctgagg
gggagccggt 1449aactgtcctg tcctgctcat tatgccactt ccttttaact
gccaagaaat tttttaaaat 1509aaatatttat aat 15226459PRTHUMAN 6Met Ala
Pro Leu Cys Pro Ser Pro Trp Leu Pro Leu Leu Ile Pro Ala -35 -30
-25Pro Ala Pro Gly Leu Thr Val Gln Leu Leu Leu Ser Leu Leu Leu Leu
-20 -15 -10Met Pro Val His Pro Gln Arg Leu Pro Arg Met Gln Glu Asp
Ser Pro -5 -1 1 5 10Leu Gly Gly Gly Ser Ser Gly Glu Asp Asp Pro Leu
Gly Glu Glu Asp 15 20 25Leu Pro Ser Glu Glu Asp Ser Pro Arg Glu Glu
Asp Pro Pro Gly Glu 30 35 40Glu Asp Leu Pro Gly Glu Glu Asp Leu Pro
Gly Glu Glu Asp Leu Pro 45 50 55Glu Val Lys Pro Lys Ser Glu Glu Glu
Gly Ser Leu Lys Leu Glu Asp 60 65 70 75Leu Pro Thr Val Glu Ala Pro
Gly Asp Pro Gln Glu Pro Gln Asn Asn 80 85 90Ala His Arg Asp Lys Glu
Gly Asp Asp Gln Ser His Trp Arg Tyr Gly 95 100 105Gly Asp Pro Pro
Trp Pro Arg Val Ser Pro Ala Cys Ala Gly Arg Phe 110 115 120Gln Ser
Pro Val Asp Ile Arg Pro Gln Leu Ala Ala Phe Cys Pro Ala 125 130
135Leu Arg Pro Leu Glu Leu Leu Gly Phe Gln Leu Pro Pro Leu Pro
Glu140 145 150 155Leu Arg Leu Arg Asn Asn Gly His Ser Val Gln Leu
Thr Leu Pro Pro 160 165 170Gly Leu Glu Met Ala Leu Gly Pro Gly Arg
Glu Tyr Arg Ala Leu Gln 175 180 185Leu His Leu His Trp Gly Ala Ala
Gly Arg Pro Gly Ser Glu His Thr 190 195 200Val Glu Gly His Arg Phe
Pro Ala Glu Ile His Val Val His Leu Ser 205 210 215Thr Ala Phe Ala
Arg Val Asp Glu Ala Leu Gly Arg Pro Gly Gly Leu220 225 230 235Ala
Val Leu Ala Ala Phe Leu Glu Glu Gly Pro Glu Glu Asn Ser Ala 240 245
250Tyr Glu Gln Leu Leu Ser Arg Leu Glu Glu Ile Ala Glu Glu Gly Ser
255 260 265Glu Thr Gln Val Pro Gly Leu Asp Ile Ser Ala Leu Leu Pro
Ser Asp 270 275 280Phe Ser Arg Tyr Phe Gln Tyr Glu Gly Ser Leu Thr
Thr Pro Pro Cys 285 290 295Ala Gln Gly Val Ile Trp Thr Val Phe Asn
Gln Thr Val Met Leu Ser300 305 310 315Ala Lys Gln Leu His Thr Leu
Ser Asp Thr Leu Trp Gly Pro Gly Asp 320 325 330Ser Arg Leu Gln Leu
Asn Phe Arg Ala Thr Gln Pro Leu Asn Gly Arg 335 340 345Val Ile Glu
Ala Ser Phe Pro Ala Gly Val Asp Ser Ser Pro Arg Ala 350 355 360Ala
Glu Pro Val Gln Leu Asn Ser Cys Leu Ala Ala Gly Asp Ile Leu 365 370
375Ala Leu Val Phe Gly Leu Leu Phe Ala Val Thr Ser Val Ala Phe
Leu380 385 390 395Val Gln Met Arg Arg Gln His Arg Arg Gly Thr Lys
Gly Gly Val Ser 400 405 410Tyr Arg Pro Ala Glu Val Ala Glu Thr Gly
Ala 415 420725DNAHUMAN 7tggggttctt gaggatctcc aggag 25826DNAHUMAN
8ctctaacttc agggagccct cttctt 26948DNAHUMANprimer_bind(1)..(48)
9cuacuacuac uaggccacgc gtcgactagt acgggnnggg nngggnng 48106PRTHUMAN
10Glu Glu Asp Leu Pro Ser1 5116PRTHUMAN 11Gly Glu Asp Asp Pro Leu1
51221PRTHUMAN 12Asn Asn Ala His Arg Asp Lys Glu Gly Asp Asp Gln Ser
His Trp Arg1 5 10
15Tyr Gly Gly Asp Pro 201316PRTHUMAN 13His Pro Gln Arg Leu Pro Arg
Met Gln Glu Asp Ser Pro Leu Gly Gly1 5 10 151424PRTHUMAN 14Glu Glu
Asp Ser Pro Arg Glu Glu Asp Pro Pro Gly Glu Glu Asp Leu1 5 10 15Pro
Gly Glu Glu Asp Leu Pro Gly 201513PRTHUMAN 15Leu Glu Glu Gly Pro
Glu Glu Asn Ser Ala Tyr Glu Gln1 5 101616PRTHUMAN 16Met Arg Arg Gln
His Arg Arg Gly Thr Lys Gly Gly Val Ser Tyr Arg1 5 10
151745DNAHUMAN 17gtcgctagct ccatgggtca tatgcagagg ttgccccgga tgcag
451843DNAHUMAN 18gaagatctct tactcgagca ttctccaaga tccagcctct agg
431910DNAHUMANmisc_feature(1)..(10) 19yssccmnsss
102010DNAHUMANmisc_feature(1)..(10) 20kmggcckrry
102110DNAHUMANmisc_feature(1)..(10) 21rrrcwwgyyy 10228PRTHUMAN
22Leu Glu His His His His His His1
5235052DNAHUMANmisc_feature(1)..(5052) 23ggatcctgtt gactcgtgac
cttaccccca accctgtgct ctctgaaaca tgagctgtgt 60ccactcaggg ttaaatggat
taagggcggt gcaagatgtg ctttgttaaa cagatgcttg 120aaggcagcat
gctcgttaag agtcatcacc aatccctaat ctcaagtaat cagggacaca
180aacactgcgg aaggccgcag ggtcctctgc ctaggaaaac cagagacctt
tgttcacttg 240tttatctgac cttccctcca ctattgtcca tgaccctgcc
aaatccccct ctgtgagaaa 300cacccaagaa ttatcaataa aaaaataaat
ttaaaaaaaa aatacaaaaa aaaaaaaaaa 360aaaaaaaaaa gacttacgaa
tagttattga taaatgaata gctattggta aagccaagta 420aatgatcata
ttcaaaacca gacggccatc atcacagctc aagtctacct gatttgatct
480ctttatcatt gtcattcttt ggattcacta gattagtcat catcctcaaa
attctccccc 540aagttctaat tacgttccaa acatttaggg gttacatgaa
gcttgaacct actaccttct 600ttgcttttga gccatgagtt gtaggaatga
tgagtttaca ccttacatgc tggggattaa 660tttaaacttt acctctaagt
cagttgggta gcctttggct tatttttgta gctaattttg 720tagttaatgg
atgcactgtg aatcttgcta tgatagtttt cctccacact ttgccactag
780gggtaggtag gtactcagtt ttcagtaatt gcttacctaa gaccctaagc
cctatttctc 840ttgtactggc ctttatctgt aatatgggca tatttaatac
aatataattt ttggagtttt 900tttgtttgtt tgtttgtttg tttttttgag
acggagtctt gcatctgtca tgcccaggct 960ggagtagcag tggtgccatc
tcggctcact gcaagctcca cctcccgagt tcacgccatt 1020ttcctgcctc
agcctcccga gtagctggga ctacaggcgc ccgccaccat gcccggctaa
1080ttttttgtat ttttggtaga gacggggttt caccgtgtta gccagaatgg
tctcgatctc 1140ctgacttcgt gatccacccg cctcggcctc ccaaagttct
gggattacag gtgtgagcca 1200ccgcacctgg ccaatttttt gagtctttta
aagtaaaaat atgtcttgta agctggtaac 1260tatggtacat ttccttttat
taatgtggtg ctgacggtca tataggttct tttgagtttg 1320gcatgcatat
gctacttttt gcagtccttt cattacattt ttctctcttc atttgaagag
1380catgttatat cttttagctt cacttggctt aaaaggttct ctcattagcc
taacacagtg 1440tcattgttgg taccacttgg atcataagtg gaaaaacagt
caagaaattg cacagtaata 1500cttgtttgta agagggatga ttcaggtgaa
tctgacacta agaaactccc ctacctgagg 1560tctgagattc ctctgacatt
gctgtatata ggcttttcct ttgacagcct gtgactgcgg 1620actatttttc
ttaagcaaga tatgctaaag ttttgtgagc ctttttccag agagaggtct
1680catatctgca tcaagtgaga acatataatg tctgcatgtt tccatatttc
aggaatgttt 1740gcttgtgttt tatgctttta tatagacagg gaaacttgtt
cctcagtgac ccaaaagagg 1800tgggaattgt tattggatat catcattggc
ccacgctttc tgaccttgga aacaattaag 1860ggttcataat ctcaattctg
tcagaattgg tacaagaaat agctgctatg tttcttgaca 1920ttccacttgg
taggaaataa gaatgtgaaa ctcttcagtt ggtgtgtgtc cctngttttt
1980ttgcaatttc cttcttactg tgttaaaaaa aagtatgatc ttgctctgag
aggtgaggca 2040ttcttaatca tgatctttaa agatcaataa tataatcctt
tcaaggatta tgtctttatt 2100ataataaaga taatttgtct ttaacagaat
caataatata atcccttaaa ggattatatc 2160tttgctgggc gcagtggctc
acacctgtaa tcccagcact ttgggtggcc aaggtggaag 2220gatcaaattt
gcctacttct atattatctt ctaaagcaga attcatctct cttccctcaa
2280tatgatgata ttgacagggt ttgccctcac tcactagatt gtgagctcct
gctcagggca 2340ggtagngttt tttgtttttg tttttgtttt tcttttttga
gacagggtct tgctctgtca 2400cccaggccag agtgcaatgg tacagtctca
gctcactgca gcctcaacgc ctcggctcaa 2460accatcatcc catttcagcc
tcctgagtag ctgggactac aggcacatgc cattacacct 2520ggctaatttt
tttgtatttc tagtagagac agggtttggc catgttgccc gggctggtct
2580cgaactcctg gactcaagca atccacccac ctcagcctcc caaaatgagg
gaccgtgtct 2640tattcatttc catgtcccta gtccatagcc cagtgctgga
cctatggtag tactaaataa 2700atatttgttg aatgcaatag taaatagcat
ttcagggagc aagaactaga ttaacaaagg 2760tggtaaaagg tttggagaaa
aaaataatag tttaatttgg ctagagtatg agggagagta 2820gtaggagaca
agatggaaag gtctcttggg caaggttttg aaggaagttg gaagtcagaa
2880gtacacaatg tgatatcgtg gcaggcagtg gggagccaat gaaggctttt
gagcaggaga 2940gtaatgtgtt gaaaaataaa tataggttaa acctatcaga
gcccctctga cacatacact 3000tgcttttcat tcaagctcaa gtttgtctcc
cacataccca ttacttaact caccctcggg 3060ctcccctagc agcctgccct
acctctttac ctgcttcctg gtggagtcag ggatgtatac 3120atgagctgct
ttccctctca gccagagaca tggggggccc cagctcccct gcctttcccc
3180ttctgtgcct ggagctggga agcaggccag ggttagctga ggctggctgg
caagcagctg 3240ggtggtgcca gggagagcct gcatagtgcc aggtggtgcc
ttgggttcca agctagtcca 3300tggccccgat aaccttctgc ctgtgcacac
acctgcccct cactccaccc ccatcctagc 3360tttggtatgg gggagagggc
acagggccag acaaacctgt gagactttgg ctccatctct 3420gcaaaagggc
gctctgtgag tcagcctgct cccctccagg cttgctcctc ccccacccag
3480ctctcgtttc caatgcacgt acagcccgta cacaccgtgt gctgggacac
cccacagtca 3540gcgcatggct cccctgtgcc ccagcccctg gctccctctg
ttgatcccgg cccctgctcc 3600aggcctcact gtgcaactgc tgctgtcact
gctgcttctg atgcctgtcc atccccagag 3660gttgccccgg atgcaggagg
attccccctt ggaggaggct cttctgggga agatgaccca 3720ctgggcgagg
aggatctgcc cagtgaagag gattcaccca gagaggagga tccacccgga
3780gaggaggatc tacctggaga ggaggatcta cctggagagg aggatctacc
tgaagttaat 3840gcctaaatca gaagaagagg gctccctgaa gttagaggat
ctacctactg ttgaggctcc 3900tggagatcct caagaacccc agaataatgc
ccacagggac aaagaagggg atgaccagag 3960tcattggcgc tatggaggcg
acccgcctgg ccccgggtgt ccccagcctg cgcgggccgc 4020ttccagtccc
cggtggatat ccgcccccag ctcgccgcct tctgcccggc cctgcgcccc
4080ctggaactcc tgggcttcca gctcccgccg ctcccagaac tgcgcctgca
gacaatggcc 4140acagtgtgca actgaccctg cctcctgggc tagagatggc
tctgggtccc gggcgggagt 4200accggctctg cagctgcatc tgcactgggg
ggctgcaggt cgtccgggct cggagcacac 4260tgtggaaggc caccgtttcc
ctgccgagat ccacgtggtt cacctcagca ccgcctttgc 4320cagagttgac
gaggccttgg ggcgcccggg aggcctggcc gtgttggcgc ctttctggag
4380gagggcccgg aagaaaacag tgtcctatga gcagttgctg tctcgcttgg
aagaaatcgc 4440tgaggaaggc tcagagactc aggtcccagg actggacata
tctgcactcc tgccctctga 4500cttcagccgc tacttccaat atgaggggtc
tctgactaca ccgccctgtg cccagggtgt 4560catctggact gtgtttaacc
agacagtgat gctgagtgct aagcagctcc acaccctctc 4620tgacaccctg
tggggacctg gtgactctcg gctacagctg aacttccgag cgacgcagcc
4680tttgaatggg cgagtgattg aggcctcctt ccctgctgga gtggacagca
gtcctcgggc 4740tgctgagcca gtccagctga attcctgcct ggctgctggt
gacatcctag ccctggtttt 4800tggcctcctt tttgctgtca ccagcgtcgc
gttccttgtg cagatgagaa ggcagcacag 4860aaggggaacc aaagggggtg
tgagcgtacc gcccagcaga ggtagccgag actggagcct 4920agaggctgga
tcttggagaa tgtgagaagc cagccagagg catctgaggg ggagccggta
4980actgtcctgt cctgctcatt atgccacttc cttttaactg ccaagaaatt
ttttaaaata 5040aatatttata at 5052246PRTHUMAN 24Arg Arg Ala Arg Lys
Lys1 5254PRTHUMANSITE(1)..(4) 25Ser Pro Xaa
Xaa1264PRTHUMANSITE(1)..(4) 26Thr Pro Xaa Xaa1
* * * * *