U.S. patent application number 12/021151 was filed with the patent office on 2009-11-12 for mesothelin, a differentiation antigen present on mesothelium, mesotheliomas, and ovarian cancers and methods and kits for targeting the antigen.
This patent application is currently assigned to The Govt. of the U.S.A. ad represented by the Secretary of the Dept. of Health & Human services. Invention is credited to Kai Chang, Ira Pastan.
Application Number | 20090280556 12/021151 |
Document ID | / |
Family ID | 39387568 |
Filed Date | 2009-11-12 |
United States Patent
Application |
20090280556 |
Kind Code |
A1 |
Pastan; Ira ; et
al. |
November 12, 2009 |
Mesothelin, A Differentiation Antigen Present On Mesothelium,
Mesotheliomas, and Ovarian Cancers and Methods and Kits for
Targeting the Antigen
Abstract
This invention relates to the discovery of a differentiation
antigen termed mesothelin which is associated with mesotheliomas
and ovarian cancers. Mesothelin is about 69 kD in its full-length
form. The invention includes uses for the amino acid and nucleic
acid sequences for mesothelin, recombinant cells expressing it,
methods for targeting and/or inhibiting the growth of cells bearing
mesothelin, methods for detecting the antigen and its expression
level as an indication of the presence of tumor cells, and kits for
such detection.
Inventors: |
Pastan; Ira; (Potomac,
MD) ; Chang; Kai; (Rockville, MD) |
Correspondence
Address: |
TOWNSEND AND TOWNSEND AND CREW, LLP
TWO EMBARCADERO CENTER, 8TH FLOOR
SAN FRANCISCO
CA
94111
US
|
Assignee: |
The Govt. of the U.S.A. ad
represented by the Secretary of the Dept. of Health & Human
services
Rockville
MD
|
Family ID: |
39387568 |
Appl. No.: |
12/021151 |
Filed: |
January 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09684599 |
Oct 5, 2000 |
7375183 |
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12021151 |
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09215035 |
Dec 17, 1998 |
6153430 |
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09684599 |
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08776271 |
Jan 12, 1998 |
6083502 |
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PCT/US97/00224 |
Jan 3, 1997 |
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09215035 |
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60010166 |
Jan 5, 1996 |
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Current U.S.
Class: |
435/252.33 ;
536/23.1 |
Current CPC
Class: |
C07K 14/4748
20130101 |
Class at
Publication: |
435/252.33 ;
536/23.1 |
International
Class: |
C12N 1/21 20060101
C12N001/21; C07H 21/04 20060101 C07H021/04 |
Claims
1. An isolated nucleic acid encoding a chimeric molecule, said
chimeric molecule comprising a targeting molecule and an effector
molecule, wherein said targeting molecule specifically binds to a
portion of mesothelin (SEQ ID NO.:2) that is not bound by
monoclonal antibody K1, a monoclonal antibody secreted by a
hybridoma deposited as ATCC Accession No. HB 10570.
2. The isolated nucleic acid of claim 1, wherein said effector
molecule is a cytotoxin or a drug.
3. The isolated nucleic acid of claim 1, wherein said effector
molecule is a cytotoxin.
4. The isolated nucleic acid of claim 3, wherein said cytotoxin is
selected from the group consisting of a modified Pseudomonas
exotoxin A, ricin, abrin and a modified Diphtheria toxin.
5. The isolated nucleic acid of claim 1, further wherein said
nucleic acid is operably linked to an expression control
sequence.
6. A transfected cell comprising a recombinant nucleic acid
encoding a chimeric molecule, said chimeric molecule comprising a
targeting molecule and an effector molecule, wherein said targeting
molecule specifically binds to a portion of mesothelin (SEQ ID
NO.:2) that is not bound by monoclonal antibody K1, a monoclonal
antibody secreted by a hybridoma deposited as ATCC Accession No. HB
10570.
7. The transfected cell of claim 6, wherein said effector molecule
is a cytotoxin or a drug.
8. The transfected cell of claim 7, wherein said effector molecule
is a cytotoxin.
9. The transfected cell of claim 7, wherein said cytotoxin is
selected from the group consisting of a modified Pseudomonas
exotoxin A, ricin, abrin and a modified Diphtheria toxin.
10. The transfected cell of claim 6, wherein said cell is an E.
coli cell.
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/684,599, filed Oct. 5, 2000, which is a
divisional of U.S. patent application Ser. No. 09/215,035, filed
Dec. 17, 1999, now U.S. Pat. No. 6,153,430, which was a divisional
of U.S. patent application Ser. No. 08/776,271, filed Jan. 12,
1998, now U.S. Pat. No. 6,083,502, which was a national stage
application under 35 U.S.C. .sctn. 371 of PCT/US97/00224, filed
Jan. 3, 1997, which claims priority from U.S. Provisional
Application No. 60/010,166, filed Jan. 5, 1996. The contents of
each of these applications is hereby incorporated by reference.
STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH AND DEVELOPMENT
[0002] Not applicable.
FIELD OF THE INVENTION
[0003] This invention relates to the identification of a specific
antigen found on tumor cells, particularly mesotheliomas and
ovarian tumor cells and, inter alia, methods and compositions for
targeting cells bearing the antigen.
BACKGROUND OF THE INVENTION
[0004] Monoclonal antibodies are currently being used to diagnose
and treat cancer (Mach, J., et al., Current Opinion Immunol. B,
685-693 (1991); Grossbard, M. L., et al., Blood 80 (4):863-878
(1992)). To be useful for therapy, the antibody should recognize an
antigen that is present in large amounts on the cancer cells and in
negligible amounts in normal cells. Alternatively the antigen can
be present in substantial amounts on normal cells, if the normal
cells are not components of an essential organ. This approach has
been useful in developing new treatments for leukemias and
lymphomas. Several differentiation antigens have been identified on
lymphomas and leukemias which are good targets for immunotherapy,
because they are not present on the stem cells which give rise to
differentiated lymphocytes (Grossbard, M. L., et al., Blood 80
(4):863-878 (1992)). Thus, normal lymphocytes that are killed by
immunotherapy can be regenerated. Some examples of lymphocyte
antigens of this type are CD19, CD22, CD25 and CD30 (Grossbard, M.
L., et al., Blood 80 (4):863-878 (1992); Engert, A., et al., Cancer
Research 50, 84-88 (1990)). Clearly, it would be very useful to
have antibodies that recognize differentiation antigens on solid
tumors, but only a small number of these are available. One reason
contributing to the paucity of such antibodies is that efforts to
identify differentiation antigens on various types of epithelial
cells have been relatively modest compared with the intense efforts
made to identify differentiation antigens on cells of the
hematopoietic system.
[0005] Ovarian cancer represents one of the diseases which could be
treated by immunotherapy, because the ovaries are always removed
during surgery for this disease and reactivity with normal ovarian
tissue is not a problem. Several antibodies that recognize
differentiation antigens on ovarian cancer cells have been
generated. One of these is OC125 that recognizes the CA125 antigen
(Bast, R., et al., N. Eng. J. Med. 309, 883-887 (1983)). CA125 is a
high molecular weight glycoprotein that is shed by ovarian cancer
cells and has been useful in the diagnosis of ovarian cancer.
However, antibodies to CA 125 are not useful for immunotherapy
because the CA 125 antigen is shed into the blood stream (Bast, R.,
et al., N. Eng. J. Med. 309, 883-887 (1983)). Another is MOV18
which recognizes the folate binding protein. This protein is
abundant in ovarian cancers as well as in some other tumors.
Unfortunately, this protein is also abundantly expressed in kidney
(Campbell, I. G., et al., Cancer Res. 51, 5329-5338 (1991)). An
antibody we previously isolated and termed MAb K1 reacts with many
ovarian cancers and many mesotheliomas. Like OC125, the antibody
also reacts with normal mesothelial cells, but it does not react
with other cell types except for weak reactivity with some cells in
the trachea (Chang, K., et al., Int. J. Cancer 50, 373-381 (1992);
Chang, K., et al., Cancer Res. 52, 181-186 (1992), see also U.S.
Pat. No. 5,320,956). The antigen recognized by MAb K1 appears to be
a differentiation antigen present on mesothelium and is expressed
on cancers derived from mesothelium such as epithelioid type
mesotheliomas as well as on most ovarian cancers. Thus
immunotherapy directed at the CAK1 antigen should take into account
the potential risk of damaging normal mesothelial cells and perhaps
cells of the trachea (Chang, K., et al., Int. J. Cancer 50, 373-381
(1992); Chang, K., et al., Cancer Res. 52, 181-186 (1992); Chang,
K., et al., Int. J. Cancer 51, 548-554 (1992); Chang, K., et al.,
Am. J. Surg. Pathol. 16, 259-268 (1992)).
[0006] Using the ovarian cancer cell line OVCAR-3 as well as HeLa
cells, the antigen has been shown to be an approximately 40 kD
glycoprotein that is attached to the cell surface by
phosphatidylinositol. The protein is released when cells are
treated with phosphotidylinositol specific phospholipase C (Chang,
K., et al., Cancer Res. 52, 181-186 (1992)). We had previously
attempted to clone a cDNA encoding the CAK1 antigen but instead
cloned cDNAs encoding two different intracellular proteins which
also react with MAb K1 (Chang, K., and Pastan, I., Int. J. Cancer
57, 90-97 (1994)). Neither of these is the cell surface membrane
antigen recognized by MAb K1.
SUMMARY OF THE INVENTION
[0007] The present invention provides uses for isolated
polypeptides comprising at least 10 contiguous amino acids from the
polypeptide sequence of SEQ ID NO:2, wherein the polypeptide binds
to antisera raised against the full-length polypeptide of SEQ ID
NO:2 as an immunogen, which has been fully immunoadsorbed with a 40
kD polypeptide attached to the cell surface of OVCAR-3 and HeLa
cells (the K1 antigen). Full-length polypeptides of the invention
are typically about 69 kD in size, although they are larger when
glycosylated or incorporated into a construct such as an eukaryotic
expression vector. The polypeptides of the present invention may be
present in several forms, including isolated naturally occurring
endoproteolytic polypeptides, recombinantly produced polypeptides,
and as portions of recombinant polypeptides such as fusion
proteins.
[0008] The present invention also provides uses for isolated
nucleic acids which encode the polypeptides described above.
Exemplary nucleic acids include those described in SEQ ID NO: 1. In
preferred embodiments, the nucleic acid is part of a recombinant
vector such as a plasmid or virus or may be used as a probe to
detect for the antigen. In preferred embodiments, the nucleic acid
selectively hybridizes to the nucleic acid of SEQ ID NO: 1. The
nucleic acid sequence may encode, e.g., a mesothelin polypeptide
with complete sequence identity to a naturally occurring mesothelin
protein. The nucleic acid may also encode a mesothelin polypeptide
which is not identical to a naturally occurring mesothelin
polypeptide, such as a fusion protein, or a genetically engineered
mutant mesothelin protein which retains the bases critical for
protein function or immunogenicity as described herein.
[0009] Recombinant cells which comprise a nucleic acid of the
present invention are also provided, including eukaryotic and
prokaryotic cells. The present invention also provides antibodies
which bind specifically to the polypeptides of the present
invention. The invention further provides methods for targeting
and/or inhibiting the growth of cells bearing mesothelin; methods
for detecting the antigen and its expression level as an indication
of the presence of tumor cells; and kits for such detection.
DEFINITIONS
[0010] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by those
of ordinary skill in the art to which this invention belongs.
Although any methods and materials similar or equivalent to those
described herein can be used in the practice or testing of the
present invention, the preferred methods and materials are
described. For purposes of the present invention, the following
terms are defined below.
[0011] The term "antibody" as used herein, includes various forms
of modified or altered antibodies, such as an intact
immunoglobulin, various fragments such as an Fv fragment, an Fv
fragment containing only the light and heavy chain variable
regions, an Fv fragment linked by a disulfide bond (Brinkmann, et
al. Proc. Natl. Acad. Sci. USA, 90: 547-551 (1993)), an Fab or
(Fab)'.sub.2 fragment containing the variable regions and parts of
the constant regions, a single-chain antibody and the like (Bird et
al., Science 242: 424-426 (1988); Huston et al., Proc. Nat. Acad.
Sci. USA 85: 5879-5883 (1988)). The antibody may be of animal
(especially mouse or rat) or human origin or may be chimeric
(Morrison et al., Proc Nat. Acad. Sci. USA 81: 6851-6855 (1984)) or
humanized (Jones et al., Nature 321: 522-525 (1986), and published
UK patent application #8707252).
[0012] The term "immunoassay" is an assay that utilizes an antibody
to specifically bind an analyte or antigen. The immunoassay is
characterized by the use of specific binding properties of a
particular antibody to isolate, target, and/or quantify the
analyte.
[0013] The terms "isolated," "purified," or "biologically pure"
refer to material which is substantially or essentially free from
components which normally accompany it as found in its native
state.
[0014] The term "nucleic acid" refers to a deoxyribonucleotide or
ribonucleotide polymer in either single- or double-stranded form,
and unless otherwise limited, encompasses known analogs of natural
nucleotides that can function in a similar manner as naturally
occurring nucleotides.
[0015] The term "nucleic acid probe" refers to a molecule which
binds to a specific sequence or subsequence of a nucleic acid. A
probe is preferably a nucleic acid which binds through
complementary base pairing to the full sequence or to a subsequence
of a target nucleic acid. It will be understood by one of skill in
the art that probes may bind target sequences lacking complete
complementarity with the probe sequence depending upon the
stringency of the hybridization conditions. The probes are
preferably directly labelled as with isotopes, chromophores,
lumiphores, chromogens, or indirectly labelled such as with biotin
to which a streptavidin complex may later bind. By assaying for the
presence or absence of the probe, one can detect the presence or
absence of the select sequence or subsequence.
[0016] The terms "polypeptide", "peptide" and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to amino acid polymers in which one or
more amino acid residue is an artificial chemical analogue of a
corresponding naturally occurring amino acid, as well as to
naturally occurring amino acid polymers.
[0017] The term "recombinant" when used with reference to a cell
indicates that the cell encodes a DNA whose origin is exogenous to
the cell-type. Thus, for example, recombinant cells express genes
that are not found within the native (non-recombinant) form of the
cell.
[0018] The term "identical" in the context of two nucleic acids or
polypeptide sequences refers to the residues in the two sequences
which are the same when aligned for maximum correspondence. Optimal
alignment of sequences for comparison can be conducted, e.g., by
the local homology algorithm of Smith and Waterman Adv. Appl. Math.
2: 482 (1981), by the homology alignment algorithm of Needleman and
Wunsch J. Mol. Biol. 48:443 (1970), by the search for similarity
method of Pearson and Lipman Proc. Natl. Acad. Sci. (U.S.A.) 85:
2444 (1988), by computerized implementations of these algorithms
(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software
Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.),
or by inspection.
[0019] The term "substantial identity" or "substantial similarity"
in the context of a polypeptide indicates that a polypeptide
comprises a sequence with at least 70% sequence identity to a
reference sequence, or preferably 80%, or more preferably 85%
sequence identity to the reference sequence, or most preferably 90%
identity over a comparison window of about 10-20 amino acid
residues. An indication that two polypeptide sequences are
substantially identical is that one peptide is immunologically
reactive with antibodies raised against the second peptide. Thus, a
polypeptide is substantially identical to a second polypeptide, for
example, where the two peptides differ only by a conservative
substitution.
[0020] An indication that two nucleic acid sequences are
substantially identical is that the polypeptide which the first
nucleic acid encodes is immunologically cross reactive with the
polypeptide encoded by the second nucleic acid.
[0021] Another indication that two nucleic acid sequences are
substantially identical is that the two molecules hybridize to each
other under stringent conditions. Stringent conditions are sequence
dependent and are different under different environmental
parameters. Generally, stringent conditions are selected to be
about 5.degree. C. to 20.degree. C. lower than the thermal melting
point (Tm) for the specific sequence at a defined ionic strength
and pH. The Tm is the temperature (under defined ionic strength and
pH) at which 50% of the target sequence hybridizes to a perfectly
matched probe. However, nucleic acids which do not hybridize to
each other under stringent conditions are still substantially
identical if the polypeptides which they encode are substantially
identical. This occurs, e.g., when a copy of a nucleic acid is
created using the maximum codon degeneracy permitted by the genetic
code.
[0022] The phrases "specifically binds to a protein" or
"specifically hybridizes to" or "specifically immunoreactive with",
when referring to an antibody refers to a binding reaction which is
determinative of the presence of the protein in the presence of a
heterogeneous population of proteins and other biologics. Thus,
under designated immunoassay conditions, the specified antibodies
bind preferentially to a particular protein and do not bind in a
significant amount to other proteins present in the sample.
Specific binding to a protein under such conditions requires an
antibody that is selected for its specificity for a particular
protein. A variety of immunoassay formats may be used to select
antibodies specifically immunoreactive with a particular protein.
For example, solid-phase ELISA immunoassays are routinely used to
select monoclonal antibodies specifically immunoreactive with a
protein. See Harlow and Lane (1988) Antibodies, A Laboratory
Manual, Cold Spring Harbor Publications, New York, for a
description of immunoassay formats and conditions that can be used
to determine specific immunoreactivity.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1: Nucleotide sequence (SEQ ID NO: 1) and deduced amino
acid sequence (SEQ ID NO:2) of the CAK1-9 cDNA. The nucleotide
sequence (upper line) and the deduced amino acid sequence (lower
line) of the CAK1 cDNA is listed with nucleotide numbers at left.
The translation of CAK1 starts at nucleotides 100-102 (ATG) and
terminates at 1986-88 (TGA). The putative signal peptide is
underlined and a typical hydrophobic sequence for GPI anchorage is
double-underlined. A likely furin cleavage site RPRFRR (SEQ ID
NO:8) is underlined and the cleavage site shown by an arrow. There
are four potential N-linked glycosylation sites (in bold letters).
A variant polyadenylation signal (AGTAAA) is present 22 base pairs
upstream from the polyadenylation tail. The original p6-1 cDNA
sequence spans nucleotides 721 to 2138.
[0024] FIG. 2: Different forms of the CAK1 tumor antigen.
S.P.=putative signal peptide; H.P.=GPI anchorage dependent
hydrophobic peptide; CHO=carbohydrates; M=membrane, AA=amino
acids.
DETAILED DESCRIPTION
[0025] This invention relates to the discovery of an antigen,
referred to herein as mesothelin, found on mesothelium,
mesotheliomas, ovarian cancer cells and some squamous cell
carcinomas. Previously, an antibody designated monoclonal antibody
K1 was described which reacts with an antigen found on OVCAR-3
cells (from a human ovarian tumor cell line) having a molecular
weight of 40 kD (kilodaltons). See, e.g. U.S. Pat. No. 5,320,956.
The antigen described and claimed here was unexpectedly obtained
during an attempt to clone and sequence the K1 antigen. Mesothelin
in its full-length form has an apparent molecular weight of about
69 kD and appears to be the precursor protein for the 40 kD K1
antigen. The K1 antigen itself proved difficult to clone and our
first attempts resulted in the cloning of two different
intracellular proteins as mentioned above (see Chang & Pastan,
Int. J. Cancer, supra). Though the existence of the K1 antigen was
known, its cDNA was not routine to clone. First, we were not able
to obtain sufficient amounts of it to clone. The methods used here
were more laborious, but successful because unbeknownst to us the
K1 antigen was derived from a larger molecule that we did not know
existed. The DNA sequence and corresponding amino acid sequence for
full-length mesothelin are set out in FIG. 1 and in SEQ ID NOS: 1
and 2, respectively.
[0026] Reference to mesothelin herein refers to both the isolated
full-length polypeptide and isolated polypeptide fragments of at
least 10 contiguous amino acids from the full-length sequence
wherein the fragment binds to antisera raised against the
full-length polypeptide, which has been fully immunosorbed with the
40 kD K1 antigen.
[0027] Mesothelin, as described here represents an antigen which is
found on mesothelium, mesotheliomas, ovarian cancers and some
squamous cell carcinomas. We have designated this antigen
mesothelin to reflect its presence on mesothelial cells. The
full-length cDNA for mesothelin is 2138 bp in length and contains
an open reading frame of 1884 bp. The protein it encodes contains
628 amino acids with a calculated molecular weight of about 69000
daltons in its full-length form.
[0028] The protein contains four potential N-linked glycosylation
sites N--X--S or
[0029] N--X-T that are shown in bold letters in FIG. 1. A typical
signal sequence is not present at the amino terminus. However, a
short hydrophobic segment is located 15 amino acids from the first
methionine (FIG. 1). This sequence might function as a signal
sequence for membrane insertion, because the protein is found on
the cell surface and is inserted into microsomes during cell free
translation. Also present is a putative proteolytic processing
site, RPRFRR (SEQ ID NO:8), beginning at amino acid 293 (FIG. 1).
This site is recognized by furin, a protease important in the
processing of several membrane proteins as well as in the
activation of Pseudomonas and diphtheria toxins (Chiron, M. F., et
al., J.B.C. 269(27):18169-18176 (1994)).
[0030] The 40 kD form ("K1") appears to be derived from a 69 kD
precursor by several processing steps. These are summarized in FIG.
2. Initially, mesothelin is made as a 69 kD polypeptide with a
hydrophobic tail which is probably removed and replaced by
phosphatidylinositol (Chang, K., et al., Cancer Res. 52, 181-186
(1992)). After glycosylation at one or more of its four putative
N-linked glycosylation sites, it is cleaved by a protease to yield
higher molecular weight forms, the 40 kD fragment (or doublet)
found on the surface of OVCAR-3 cells and a smaller (31 kD)
fragment. The latter could be released into the medium and/or
further degraded. We found that the amino terminal fragment was
detected in the medium of OVCAR-3 cells.
[0031] Mesothelin is one of many proteins and glycoproteins that
are attached to the cell surface by phosphatidylinositol. Several
functions have been ascribed to these molecules. Some are receptors
involved in cell signaling; others are involved in cellular
recognition and/or adhesion (Dustin, M. L., et al., Nature 329,
846-848 (1987); Stiemberg, J., et al., J. Immunol. 38, 3877-3884
(1987)). GPI linked proteins may interact with tyrosine kinases
(Stefanova, I., et al., Science 254, 1016-1019 (1991); Pandey, A.,
et al., Science 268, 567-569 (1995)). Antibodies to mesothelin
would be useful in inhibiting the spread or implantation of ovarian
cancer cells into the peritoneal wall that sometimes occurs, for
example, during ovarian cancer surgery. Without intending to be
bound by theory, it is our belief that mesothelin is likely
responsible for the adhesion and implantation of ovarian carcinoma
cells that frequently occurs throughout the peritoneal cavity or
the adhesion of tumor cells in the thoracic cavity. Mesothelin
plays a role in adhesion since mesothelin transfectants are more
slowly removed from culture dishes than non-transfected cells.
Mesothelial cells are extremely flat and regulate the traffic of
molecules and cells in and out of the peritoneal or thoracic
cavity.
[0032] Mesothelin is very abundant in normal mesothelial cells from
which malignant mesotheliomas and ovarian cystadenocarcinomas are
derived. These two types of tumors share a unique biological
characteristic that distinguishes them from other solid tumors. In
the early stages, both types of tumors spread aggressively
throughout the peritoneal (or thoracic) cavity and invade locally
but do not metastasize distally through lymphatics or the blood
stream. In fact, many patients succumb to their cancer before
distant metastases develop. Mesothelin likely has a role in this
process, since cells overexpressing mesothelin have altered
adhesive properties and mesothelin expression is diminished in
poorly differentiated ovarian cancers (Chang, K., et al., Int. J.
Cancer 51, 548-554 (1992); Chang, K., et al., Am. J. Surg. Pathol.
16, 259-268 (1992)). Implantation of ovarian cancer cells through a
strong adhesion mechanism may be the first step towards local
invasion and distal metastasis. Thus, blocking ovarian cancer
implantation will prevent invasion and metastasis as well as
proliferation of the cancer cells and lead cancer cells to
apoptosis and the like.
[0033] I. Detection for Mesothelin
[0034] The detection of mesothelin is useful as an indicator of the
presence of tumor cells, particularly ovarian tumor cells or
mesotheliomas. If found in serum it can be a factor indicating the
presence of residual cancer cells. Tumor tissues contain various
proteases which may be responsible for the cleavage of mesothelin.
The amount of N-terminal fragment of mesothelin present in blood or
ascitic fluid can reflect the number of residual tumor cells
present. The serological detection of mesothelin may serve as a
novel indicator for monitoring the process of disease. The basic
principle for detection of the mesothelin proteins is to detect the
protein using specific ligands that bind to mesothelin but not to
other proteins or nucleic acids in a normal human cell or its
environs. The ligands can be either nucleic acid or antibodies. The
ligands can be naturally occurring or genetically or physically
modified such as non-natural or antibody derivatives, i.e. FAB, or
chimeric antibodies.
[0035] A. Sample Collection and Processing
[0036] Mesothelin is preferably quantified in a biological sample,
such as a serum, cell, or a tissue sample derived from a patient.
In a preferred embodiment, mesothelin is quantified in samples of
serum, mesothelial cells, cervical tissue or ovarian tissue with
reference to a standard prepared from recombinant mesothelin.
[0037] The sample may be pretreated as necessary by dilution in an
appropriate buffer solution or concentrated, if desired depending
upon the assay being used. Any of a number of standard aqueous
buffer solutions, employing one of a variety of buffers, such as
phosphate, Tris, or the like, at physiological pH can be used.
[0038] B. Quantification of Mesothelin Peptides.
[0039] Mesothelin peptides may be detected and quantified by any of
a number of means well known to those of skill in the art. These
include analytic biochemical methods such as electrophoresis,
capillary electrophoresis, high performance liquid chromatography
(HPLC), thin layer chromatography (TLC), hyperdiffusion
chromatography, and the like, and various immunological methods
such as fluid or gel precipitin reactions, immunodiffusion (single
or double), immunoelectrophoresis, radioimmunoassays (RIAs),
enzyme-linked immunosorbent assays (ELISAs), immunofluorescent
assays, and the like.
[0040] C. General Techniques--Nucleic Acid Detection
[0041] Accepted means for conducting hybridization assays for
detection are known and general overviews of the technology can be
had from a review of: Nucleic Acid Hybridization: A Practical
Approach, Ed. Hames, B. D. and Higgins, S. J., IRL Press, 1985;
Hybridization of Nucleic Acids Immobilized on Solid Supports,
Meinkoth, J. and Wahl, G.; Analytical Biochemistry, Vol 238,
267-284, 1984 and Innis et al., PCR Protocols, supra, all of which
are incorporated by reference herein.
[0042] If PCR is used, for example, primers are designed to target
a specific portion of the nucleic acid of the targeted agent.
Preferably the primers are about 14 to about 24 nucleotides in
length. From the sequence information provided herein, those of
skill in the art will be able to select appropriate specific
primers.
[0043] Target specific probes may be used in the nucleic acid
hybridization diagnostic assays for mesothelin. The probes are
specific for or complementary to the target of interest. For
example, probes to one of the nucleic acid sequences in the open
reading frame for mesothelin would be effective. For precise
allelic differentiation, the probes should be about 14 nucleotides
long and preferably about 20-30 nucleotides. For more general
detection, nucleic acid probes are about 50 to about 1000
nucleotides, most preferably about 200 to about 400
nucleotides.
[0044] The detection of the mesothelin polypeptides and other
aspects of the present invention may make use of techniques such as
PCR, TAS, 3SR, QB amplification and cloning, to amplify a nucleic
acid in a biological sample which encodes a mesothelin polypeptide
for detection or for, inter alia, the production of probes and
primer tools for detection.
[0045] The presence of mesothelin nucleic acid in a biological
sample such as, for example, serum or tissue suspected to contain
tumor cells, is useful, e.g., as a probe to assess the presence of
mesothelin and subsequently provide evidence indicative of tumor
cells.
[0046] The nucleic acids of the present invention are cloned, or
amplified by in vitro methods, such as the polymerase chain
reaction (PCR), the ligase chain reaction (LCR), the
transcription-based amplification system (TAS), the self-sustained
sequence replication system (3SR) and the Q replicase amplification
system (QB). A wide variety of cloning and in vitro amplification
methodologies are well-known to persons of skill. Examples of these
techniques and instructions sufficient to direct persons of skill
through many cloning exercises are found in Berger and Kimmel,
Guide to Molecular Cloning Techniques, Methods in Enzymology 152
Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al.
(1989) Molecular Cloning--A Laboratory Manual (2nd ed.) Vol. 1-3,
Cold Spring Harbor Laboratory, Cold Spring Harbor Press, NY,
(Sambrook et al.); Current Protocols in Molecular Biology, F. M.
Ausubel et al., eds., Current Protocols, a joint venture between
Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,
(1994 Supplement) (Ausubel); Cashion et al., U.S. Pat. No.
5,017,478; and Carr, European Patent No. 0,246,864. Examples of
techniques sufficient to direct persons of skill through in vitro
amplification methods are found in Berger, Sambrook, and Ausubel,
as well as Mullis et al., (1987) U.S. Pat. No. 4,683,202; PCR
Protocols A Guide to Methods and Applications (Innis et al. eds)
Academic Press Inc. San Diego, Calif. (1990) (Innis); Arnheim &
Levinson (Oct. 1, 1990) C&EN 36-47; The Journal Of NIH Research
(1991) 3, 81-94; (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86,
1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874;
Lomell et al. (1989) J. Clin. Chem. 35, 1826; Landegren et al.,
(1988) Science 241, 1077-1080; Van Brunt (1990) Biotechnology 8,
291-294; Wu and Wallace, (1989) Gene 4, 560; and Barringer et al.
(1990) Gene 89, 117.
[0047] It will be readily understood by those of skill in the art
and it is intended here, that when reference is made to particular
sequence listings, such as SEQ ID NOs: 1 and 2, such reference
includes sequences which substantially correspond to its
complementary sequence and those described including allowances for
minor sequencing errors, single base changes, deletions,
substitutions and the like, such that any such sequence variation
corresponds to the nucleic acid sequence to which the relevant
sequence listing relates.
[0048] D. Antibodies to Mesothelin and Antibody-Ligand Binding
Assays
[0049] Antibodies (or antisera) are raised to the polypeptides of
the present invention, including individual fragments thereof, both
in their naturally occurring (full-length) forms and in recombinant
forms. Additionally, antibodies are raised to these polypeptides in
either their native configurations or in non-native configurations.
Anti-idiotypic antibodies can also be generated. Many methods of
making antibodies are known to persons of skill. The following
discussion is presented as a general overview of the techniques
available; however, one of skill will recognize that many
variations upon the following methods are known.
[0050] 1. Antibody Production
[0051] A number of immunogens are used to produce antibodies
specifically reactive with mesothelin polypeptides. Recombinant or
synthetic polypeptides of 10 amino acids in length, or greater,
selected from sub-sequences of SEQ ID NO: 1 are the preferred
polypeptide immunogen for the production of monoclonal or
polyclonal antibodies. In one class of preferred embodiments, an
immunogenic peptide conjugate is also included as an immunogen.
Naturally occurring polypeptides are also used either in pure or
impure form. Transfected mammalian cells overexpressing recombinant
mesothelin can also be used as an immunogen, either in whole intact
cells or membrane preparations. These immunogens are useful for
polyclonal or monoclonal antibody generation.
[0052] Recombinant polypeptides are expressed in eukaryotic or
prokaryotic cells and purified using standard techniques. The
polypeptide, or a synthetic version thereof, is then injected into
an animal capable of producing antibodies. Either monoclonal or
polyclonal antibodies can be generated for subsequent use in
immunoassays to measure the presence and quantity of the
polypeptide.
[0053] Methods of producing polyclonal antibodies are known to
those of skill in the art. In brief, an immunogen, preferably a
purified polypeptide, a polypeptide coupled to an appropriate
carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a
polypeptide incorporated into an immunization vector such as a
recombinant vaccinia virus (see, U.S. Pat. No. 4,722,848) is mixed
with an adjuvant and animals are immunized with the mixture. The
animal's immune response to the immunogen preparation is monitored
by taking test bleeds and determining the titer of reactivity to
the polypeptide of interest. When appropriately high titers of
antibody to the immunogen are obtained, blood is collected from the
animal and antisera are prepared. Further fractionation of the
antisera to enrich for antibodies reactive to the polypeptide is
performed where desired. See, e.g., Coligan (1991) Current
Protocols in Immunology Wiley/Greene, N.Y.; and Harlow and Lane
(1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press,
NY, which are incorporated herein by reference, and the examples
below.
[0054] Antibodies, including binding fragments and single chain
recombinant versions thereof, against predetermined fragments of
mesothelin polypeptides are raised by immunizing animals, e.g.,
with conjugates of the fragments with carrier proteins as described
above. Typically, the immunogen of interest is a peptide of at
least about 3 amino acids, more typically the peptide is 5 amino
acids in length, preferably, the fragment is 10 amino acids in
length and more preferably the fragment is 15 amino acids in length
or greater. The peptides are typically coupled to a carrier protein
(e.g., as a fusion protein), or are recombinantly expressed in an
immunization or expression vector. Antigenic determinants on
peptides to which antibodies bind are typically 3 to 10 amino acids
in length.
[0055] Monoclonal antibodies are prepared from cells secreting the
desired antibody. These antibodies are screened for binding to
normal or modified polypeptides. Specific monoclonal and polyclonal
antibodies will usually bind with a KD of at least about 0.1 mM,
more usually at least about 50 .mu.M, and most preferably at least
about 1 .mu.M or better.
[0056] In some instances, it is desirable to prepare monoclonal
antibodies from various mammalian hosts, such as mice, rodents,
primates, humans, etc. Description of techniques for preparing such
monoclonal antibodies are well known and are found in, e.g., Asai,
ed. Antibodies in Cell Biology, Academic Press, Inc., San Diego,
Calif.; Stites et al. (eds.) Basic and Clinical Immunology (4th
ed.) Lange Medical Publications, Los Altos, Calif., and references
cited therein; Harlow and Lane, Supra; Goding (1986) Monoclonal
Antibodies: Principles and Practice (2d ed.) Academic Press, New
York, N.Y.; and Kohler and Milstein (1975) Nature 256: 495-497. The
polypeptides and antibodies of the present invention are used with
or without modification, and include chimeric antibodies such as
humanized murine antibodies.
[0057] Other suitable techniques involve selection of libraries of
recombinant antibodies in phage or similar vectors. See, Huse et
al. (1989) Science 246: 1275-1281; and Ward, et al. (1989) Nature
341: 544-546.
[0058] Frequently, the polypeptides and antibodies will be labeled
by joining, either covalently or non-covalently, a substance which
provides for a detectable signal. A wide variety of labels and
conjugation techniques are known and are reported extensively in
both the scientific and patent literature. Suitable labels include
radionucleotides, enzymes, substrates, cofactors, inhibitors,
fluorescent moieties, chemiluminescent moieties, magnetic
particles, and the like. Patents teaching the use of such labels
include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;
4,277,437; 4,275,149; and 4,366,241. Also, recombinant
immunoglobulins may be produced. See, for example, Cabilly, U.S.
Pat. No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci.
USA 86: 10029-10033.
[0059] The antibodies of this invention are also used for affinity
chromatography in isolating mesothelin polypeptides. Columns are
prepared, e.g., with the antibodies linked to a solid support,
e.g., particles, such as agarose, Sephadex, or the like, where a
cell lysate is passed through the column, washed, and treated with
increasing concentrations of a mild denaturant, whereby purified
mesothelin polypeptides are released.
[0060] The antibodies can be used to screen expression libraries
for particular expression products such as mammalian mesothelin.
Usually the antibodies in such a procedure are labeled with a
moiety allowing easy detection of presence of antigen by antibody
binding.
[0061] Antibodies raised against mesothelin polypeptides can also
be used to raise anti-idiotypic antibodies. These are useful for
detecting or diagnosing various pathological conditions related to
the presence of the respective antigens.
[0062] 2. Immunoassays
[0063] A particular protein can be quantified by a variety of
immunoassay methods. For a review of immunological and immunoassay
procedures in general, see Stites and Terr (eds.) 1991 Basic and
Clinical Immunology (7th ed.). Moreover, the immunoassays of the
present invention can be performed in any of several
configurations, e.g., those reviewed in Maggio (ed.) (1980) Enzyme
Immunoassay CRC Press, Boca Raton, Fla.; Tijan (1985) "Practice and
Theory of Enzyme Immunoassays," Laboratory Techniques in
Biochemistry and Molecular Biology, Elsevier Science Publishers
B.V., Amsterdam; Harlow and Lane, supra; Chan (ed.) (1987)
Immunoassay: A Practical Guide Academic Press, Orlando, Fla.; Price
and Newman (eds.) (1991) Principles and Practice of Immunoassays
Stockton Press, NY; and Ngo (ed.) (1988) Non-isotopic Immunoassays
Plenum Press, NY.
[0064] Immunoassays also often utilize a labeling agent to
specifically bind to and label the binding complex formed by the
capture agent and the analyte. The labeling agent may itself be one
of the moieties comprising the antibody/analyte complex. Thus, the
labeling agent may be a labeled mesothelin peptide or a labeled
anti-mesothelin antibody. Alternatively, the labeling agent may be
a third moiety, such as another antibody, that specifically binds
to the antibody/mesothelin complex, or to a modified capture group
(e.g., biotin) which is covalently linked to the mesothelin peptide
or anti-mesothelin antibody.
[0065] In a preferred embodiment, the labeling agent is an antibody
that specifically binds to the capture agent (anti-mesothelin).
Such agents are well known to those of skill in the art, and most
typically comprise labeled antibodies that specifically bind
antibodies of the particular animal species from which the capture
agent is derived (e.g., an anti-idiotypic antibody). Thus, for
example, where the capture agent is a mouse derived anti-human
mesothelin antibody, the label agent may be a goat anti-mouse IgG,
i.e., an antibody specific to the constant region of the mouse
antibody.
[0066] Other proteins capable of specifically binding
immunoglobulin constant regions, such as streptococcal protein A or
protein G are also used as the labeling agent. These proteins are
normal constituents of the cell walls of streptococcal bacteria.
They exhibit a strong non-immunogenic reactivity with
immunoglobulin constant regions from a variety of species. See,
generally Kronval, et al., (1973) J. Immunol., 111: 1401-1406, and
Akerstrom, et al., (1985) J. Immunol., 135:2589-2542.
[0067] Throughout the assays, incubation and/or washing steps may
be required after each combination of reagents. Incubation steps
can vary from about 5 seconds to several hours, preferably from
about 5 minutes to about 24 hours. However, the incubation time
will depend upon the assay format, analyte, volume of solution,
concentrations, and the like. Usually, the assays are carried out
at ambient temperature, although they can be conducted over a range
of temperatures, such as 5.degree. C. to 45.degree. C.
[0068] (a) Non-Competitive Assay Formats
[0069] Immunoassays for detecting mesothelin may be either
competitive or noncompetitive. Noncompetitive immunoassays are
assays in which the amount of captured analyte (in this case
mesothelin) is directly measured. In one preferred "sandwich"
assay, for example, the capture agent (e.g., anti-mesothelin
antibodies) are bound directly to a solid substrate where they are
immobilized. These immobilized antibodies then capture mesothelin
present in the test sample. The mesothelin thus immobilized is then
bound by a labeling agent, such as a second human mesothelin
antibody bearing a label. Alternatively, the second mesothelin
antibody may lack a label, but it may, in turn, be bound by a
labeled third antibody specific to antibodies of the species from
which the second antibody is derived.
[0070] Sandwich assays for mesothelin may be constructed. As
described above, the immobilized anti-mesothelin specifically binds
to mesothelin present in the sample. The labeled anti-mesothelin
then binds to the already bound mesothelin. Free labeled
anti-mesothelin is washed away and the remaining bound labeled
anti-mesothelin is detected (e.g., using a gamma detector where the
label is radioactive).
[0071] (b) Competitive Assay Formats
[0072] In competitive assays, the amount of analyte (e.g.,
mesothelin) present in the sample is measured indirectly by
measuring the amount of an added (exogenous) analyte displaced (or
competed away) from a capture agent (e.g., anti-mesothelin
antibody) by the analyte present in the sample. In one competitive
assay, a known amount of analyte is added to the sample and the
sample is contacted with a capture agent, in this case an antibody
that specifically binds the analyte. The amount of analyte bound to
the antibody is inversely proportional to the concentration of
analyte present in the sample.
[0073] In a particularly preferred embodiment, the capture agent is
immobilized on a solid substrate. The amount of mesothelin bound to
the capture agent is determined either by measuring the amount of
mesothelin present in an mesothelin/antibody complex, or
alternatively by measuring the amount of remaining uncomplexed
mesothelin. The amount of mesothelin may be detected by providing a
labeled mesothelin.
[0074] A hapten inhibition assay is another preferred competitive
assay. In this assay, a known analyte, in this case mesothelin, is
immobilized on a solid substrate. A known amount of anti-mesothelin
antibody is added to the sample, and the sample is then contacted
with the immobilized mesothelin. In this case, the amount of
anti-mesothelin antibody bound to the immobilized mesothelin is
proportional to the amount of mesothelin present in the sample.
Again the amount of immobilized antibody is detected by detecting
either the immobilized fraction of antibody or the fraction of the
antibody that remains in solution. Detection may be direct where
the antibody is labeled, or indirect by the subsequent addition of
a labeled moiety that specifically binds to the antibody as
described above.
[0075] (c) Generation of Pooled Antisera for Use in
Immunoassays.
[0076] A mesothelin protein that specifically binds to or that is
specifically immunoreactive with an antibody generated against a
defined immunogen, such as an immunogen consisting of the amino
acid sequence of SEQ ID NO:2, is determined in an immunoassay. The
immunoassay uses a polyclonal antiserum which was raised to the
protein of SEQ ID NO:2 (the immunogenic polypeptide).
[0077] In order to produce antisera for use in an immunoassay, the
polypeptide of SEQ ID NO:2 is isolated as described herein. For
example, recombinant protein can be produced in a mammalian or
other eukaryotic cell line. An inbred strain of mice is immunized
with the protein of SEQ ID NO:2 using a standard adjuvant, such as
Freund's adjuvant, and a standard mouse immunization protocol (see
Harlow and Lane, supra). Alternatively, a synthetic polypeptide
derived from the sequences disclosed herein and conjugated to a
carrier protein is used as an immunogen. Polyclonal sera are
collected and titered against the immunogenic polypeptide in an
immunoassay, for example, a solid phase immunoassay with the
immunogen immobilized on a solid support. Polyclonal antisera with
a titer of 104 or greater are selected and tested for their cross
reactivity against proteins of interest, using a competitive
binding immunoassay such as the one described in Harlow and Lane,
supra, at pages 570-573.
[0078] Immunoassays in the competitive binding format are used for
crossreactivity determinations. For example, the immunogenic
polypeptide is immobilized to a solid support. Proteins added to
the assay compete with the binding of the antisera to the
immobilized antigen. The ability of the above proteins to compete
with the binding of the antisera to the immobilized protein is
compared to the immunogenic polypeptide. The percent
crossreactivity for the above proteins is calculated, using
standard calculations. Those antisera with less than 10%
crossreactivity with the protein of interest are combined and
pooled. The cross-reacting antibodies are then removed from the
pooled antisera by immunoadsorbtion. The immunoadsorbed and pooled
antisera are then used in a competitive binding immunoassay as
described herein to compare a second "target" polypeptide to the
immunogenic polypeptide. In order to make this comparison, the two
polypeptides are each assayed at a wide range of concentrations and
the amount of each polypeptide required to inhibit 50% of the
binding of the antisera to the immobilized protein is determined
using standard techniques. If the amount of the target polypeptide
required is less than twice the amount of the immunogenic
polypeptide that is required, then the target polypeptide is said
to specifically bind to an antibody generated to the immunogenic
protein. As a final determination of specificity, the pooled
antisera is fully immunoadsorbed with the immunogenic polypeptide
until no binding to the polypeptide used in the immunoadsorbtion is
detectable. The fully immunoadsorbed antisera is then tested for
reactivity with the test polypeptide. If no reactivity is observed,
then the test polypeptide is specifically bound by the antisera
elicited by the immunogenic protein.
[0079] D. Other Assay Formats
[0080] Western blot analysis can also be used to detect and
quantify the presence of mesothelin in the sample. The technique
generally comprises separating sample proteins by gel
electrophoresis on the basis of molecular weight, transferring the
separated proteins to a suitable solid support, (such as a
nitrocellulose filter, a nylon filter, or derivatized nylon
filter), and incubating the sample with the antibodies that
specifically bind mesothelin. The anti-mesothelin antibodies
specifically bind to mesothelin on the solid support. These
antibodies may be directly labeled or alternatively may be
subsequently detected using labeled antibodies (e.g., labeled sheep
anti-mouse antibodies where the antibody to mesothelin is a murine
antibody) that specifically bind to the anti-mesothelin.
[0081] Other assay formats include liposome immunoassays (LIAs),
which use liposomes designed to bind specific molecules (e.g.,
antibodies) and release encapsulated reagents or markers. The
released chemicals are then detected according to standard
techniques (see, Monroe et al., (1986) Amer. Clin. Prod. Rev.
5:34-41), which is incorporated herein by reference.
[0082] E. Labels
[0083] The labeling agent for the applications described herein can
be, e.g., a monoclonal antibody, a polyclonal antibody, a
mesothelin binding protein or complex such as those described
herein, or a polymer such as an affinity matrix, carbohydrate or
lipid. Detection may proceed by any known method, such as
immunoblotting, western analysis, gel-mobility shift assays,
fluorescent in situ hybridization analysis (FISH), tracking of
radioactive or bioluminescent markers, nuclear magnetic resonance,
electron paramagnetic resonance, stopped-flow spectroscopy, column
chromatography, capillary electrophoresis, or other methods which
track a molecule based upon an alteration in size and/or charge.
The particular label or detectable group used in the assay is not a
critical aspect of the invention. The detectable group can be any
material having a detectable physical or chemical property. Such
detectable labels have been well-developed in the field of
immunoassays and, in general, any label useful in such methods can
be applied to the present invention. Thus, a label is any
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means.
Useful labels in the present invention include magnetic beads (e.g.
Dynabeads.TM.), fluorescent dyes (e.g., fluorescein isothiocyanate,
texas red, rhodamine, and the like), radiolabels (e.g., 3H, 125I,
35S, 14C, or 32P), enzymes (e.g., horse radish peroxidase, alkaline
phosphatase and others commonly used in an ELISA), and colorimetric
labels such as colloidal gold or colored glass or plastic (e.g.
polystyrene, polypropylene, latex, etc.) beads.
[0084] The label may be coupled directly or indirectly to the
desired component of the assay according to methods well known in
the art. As indicated above, a wide variety of labels may be used,
with the choice of label depending on the sensitivity required,
ease of conjugation of the compound, stability requirements,
available instrumentation, and disposal provisions.
[0085] Non-radioactive labels are often attached by indirect means.
Generally, a ligand molecule (e.g., biotin) is covalently bound to
the molecule. The ligand then binds to an anti-ligand (e.g.,
streptavidin) molecule which is either inherently detectable or
covalently bound to a signal system, such as a detectable enzyme, a
fluorescent compound, or a chemiluminescent compound. A number of
ligands and anti-ligands can be used. Where a ligand has a natural
anti-ligand, for example, biotin, thyroxine, and cortisol, it can
be used in conjunction with the labeled, naturally occurring
anti-ligands. Alternatively, any haptenic or antigenic compound can
be used in combination with an antibody.
[0086] The molecules can also be conjugated directly to signal
generating compounds, e.g., by conjugation with an enzyme or
fluorophore. Enzymes of interest as labels will primarily be
hydrolases, particularly phosphatases, esterases and glycosidases,
or oxidoreductases, particularly peroxidases. Fluorescent compounds
include fluorescein and its derivatives, rhodamine and its
derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds
include luciferin, and 2,3-dihydrophthalazinediones, e.g., luminol.
For a review of various labelling or signal producing systems which
may be used, see, U.S. Pat. No. 4,391,904, which is incorporated
herein by reference.
[0087] Means of detecting labels are well known to those of skill
in the art. Thus, for example, where the label is a radioactive
label, means for detection include a scintillation counter or
photographic film as in autoradiography. Where the label is a
fluorescent label, it may be detected by exciting the fluorochrome
with the appropriate wavelength of light and detecting the
resulting fluorescence, e.g., by microscopy, visual inspection, via
photographic film, by the use of electronic detectors such as
charge coupled devices (CCDs) or photomultipliers and the like.
Similarly, enzymatic labels may be detected by providing
appropriate substrates for the enzyme and detecting the resulting
reaction product. Finally, simple colorimetric labels may be
detected simply by observing the color associated with the label.
Thus, in various dipstick assays, conjugated gold often appears
pink, while various conjugated beads appear the color of the
bead.
[0088] Some assay formats do not require the use of labeled
components. For instance, agglutination assays can be used to
detect the presence of the target antibodies. In this case,
antigen-coated particles are agglutinated by samples comprising the
target antibodies. In this format, none of the components need be
labeled and the presence of the target antibody is detected by
simple visual inspection.
[0089] F. Substrates
[0090] As mentioned above, depending upon the assay, various
components, including the antigen, target antibody, or anti-human
antibody, may be bound to a solid surface. Many methods for
immobilizing biomolecules to a variety of solid surfaces are known
in the art. For instance, the solid surface may be a membrane
(e.g., nitrocellulose), a microtiter dish (e.g., PVC,
polypropylene, or polystyrene), a test tube (glass or plastic), a
dipstick (e.g. glass, PVC, polypropylene, polystyrene, latex, and
the like), a microcentrifuge tube, or a glass, silica, plastic,
metallic or polymer bead. The desired component may be covalently
bound, or noncovalently attached through nonspecific bonding.
[0091] A wide variety of organic and inorganic polymers, both
natural and synthetic may be employed as the material for the solid
surface. Illustrative polymers include polyethylene, polypropylene,
poly(4-methylbutene), polystyrene, polymethacrylate, poly(ethylene
terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene
difluoride (PVDF), silicones, polyformaldehyde, cellulose,
cellulose acetate, nitrocellulose, and the like. Other materials
which may be employed, include paper, glasses, ceramics, metals,
metalloids, semiconductive materials, cements or the like. In
addition, substances that form gels, such as proteins (e.g.,
gelatins), lipopolysaccharides, silicates, agarose and
polyacrylamides can be used. Polymers which form several aqueous
phases, such as dextrans, polyalkylene glycols or surfactants, such
as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium
salts and the like are also suitable. Where the solid surface is
porous, various pore sizes may be employed depending upon the
nature of the system.
[0092] In preparing the surface, a plurality of different materials
may be employed, e.g., as laminates, to obtain various properties.
For example, protein coatings, such as gelatin can be used to avoid
non-specific binding, simplify covalent conjugation, enhance signal
detection or the like.
[0093] If covalent bonding between a compound and the surface is
desired, the surface will usually be polyfunctional or be capable
of being polyfunctionalized. Functional groups which may be present
on the surface and used for linking can include carboxylic acids,
aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl
groups, mercapto groups and the like. The manner of linking a wide
variety of compounds to various surfaces is well known and is amply
illustrated in the literature. See, for example, Immobilized
Enzymes, Ichiro Chibata, Halsted Press, New York, 1978, and
Cuatrecasas, J. Biol. Chem. 245 3059 (1970) which are incorporated
herein by reference.
[0094] In addition to covalent bonding, various methods for
noncovalently binding an assay component can be used. Noncovalent
binding is typically nonspecific absorption of a compound to the
surface. Typically, the surface is blocked with a second compound
to prevent nonspecific binding of labeled assay components.
Alternatively, the surface is designed such that it nonspecifically
binds one component but does not significantly bind another. For
example, a surface bearing a lectin such as Concanavalin A will
bind a carbohydrate containing compound but not a labeled protein
that lacks glycosylation. Various solid surfaces for use in
noncovalent attachment of assay components are reviewed in U.S.
Pat. Nos. 4,447,576 and 4,254,082, which are incorporated herein by
reference.
[0095] II. Targeting Effector Molecules to Mesothelin
[0096] This invention also provides for compositions and methods
for detecting the presence or absence of tumor cells bearing
mesothelin. These methods involve providing a chimeric molecule
comprising an effector molecule, that is a detectable label
attached to a targeting molecule that specifically binds
mesothelin. The mesothelin targeting moiety specifically binds the
chimeric molecule to tumor cells which are then marked by their
association with the detectable label. Subsequent detection of the
cell-associated label indicates the presence of a tumor cell.
[0097] In yet another embodiment, the effector molecule may be
another specific binding moiety such as an antibody, a growth
factor, or a ligand. The chimeric molecule will then act as a
highly specific bifunctional linker. This linker may act to bind
and enhance the interaction between cells or cellular components to
which the fusion protein binds. Thus, for example, where the
"targeting" component of the chimeric molecule comprises a
polypeptide that specifically binds to mesothelin and the
"effector" component is an antibody or antibody fragment (e.g. an
Fv fragment of an antibody), the targeting component specifically
binds cancer cells, while the effector component inhibits cell
growth or may act to enhance and direct an immune response toward
target cancer cells.
[0098] In still yet another embodiment the effector molecule may be
a pharmacological agent (e.g. a drug) or a vehicle containing a
pharmacological agent. Thus, the moiety that specifically binds to
mesothelin may be conjugated to a drug such as vinblastine,
doxirubicin, genistein (a tyrosine kinase inhibitor), an antisense
molecule, and other pharmacological agents known to those of skill
in the art, thereby specifically targeting the pharmacological
agent to tumor cells.
[0099] Alternatively, the targeting molecule may be bound to a
vehicle containing the therapeutic composition. Such vehicles
include, but are not limited to liposomes, micelles, various
synthetic beads, and the like.
[0100] One of skill in the art will appreciate that the chimeric
molecules of the present invention may include multiple targeting
moieties bound to a single effector or conversely, multiple
effector molecules bound to a single targeting moiety. In still
other embodiments, the chimeric molecules may include both multiple
targeting moieties and multiple effector molecules. Thus, for
example, this invention provides for "dual targeted" cytotoxic
chimeric molecules in which targeting molecule that specifically
binds to mesothelin is attached to a cytotoxic molecule and another
molecule (e.g. an antibody, or another ligand) is attached to the
other terminus of the toxin. Such a dual-targeted cytotoxin might
comprise a growth factor substituted for domain Ia, for example, at
the amino terminus of a PE and anti-TAC(Fv) inserted in domain III,
between amino acid 604 and 609. Other antibodies may also be
suitable.
[0101] A. The Targeting Molecule
[0102] In a preferred embodiment, the targeting molecule is a
molecule that specifically binds to mesothelin. A variety of
immunoassay formats may be used to select appropriate antibodies
and are discussed above.
[0103] B. The Effector Molecule
[0104] As described above, the effector molecule component of the
chimeric molecules of this invention may be any molecule whose
activity it is desired to deliver to cells that express mesothelin.
Particularly preferred effector molecules include cytotoxins such
as PE or DT, radionuclides, ligands such as growth factors,
antibodies, detectable labels such as fluorescent or radioactive
labels, and therapeutic compositions such as liposomes and various
drugs.
[0105] 1. Cytotoxins
[0106] Particularly preferred cytotoxins include Pseudomonas
exotoxins, Diphtheria toxins, ricin, and abrin. Pseudomonas
exotoxin and Dipthteria toxin are most preferred.
[0107] (a) Pseudomonas exotoxin (PE)
[0108] Pseudomonas exotoxin A (PE) is an extremely active monomeric
protein (molecular weight 66 kD), secreted by Pseudomonas
aeruginosa, which inhibits protein synthesis in eukaryotic cells
through the inactivation of elongation factor 2 (EF-2) by
catalyzing its ADP-ribosylation (catalyzing the transfer of the ADP
ribosyl moiety of oxidized NAD onto EF-2).
[0109] The toxin contains three structural domains that act in
concert to cause cytotoxicity. Domain Ia (amino acids 1-252)
mediates cell binding. Domain II (amino acids 253-364) is
responsible for translocation into the cytosol and domain III
(amino acids 400-613) mediates ADP ribosylation of elongation
factor 2, which inactivates the protein and causes cell death. The
function of domain Ib (amino acids 365-399) remains undefined,
although a large part of it, amino acids 365-380, can be deleted
without loss of cytotoxicity. See Siegall et al., J. Biol. Chem.
264: 14256-14261 (1989), incorporated by reference herein.
[0110] Where the targeting molecule is fused to PE, a preferred PE
molecule is one in which domain Ia (amino acids 1 through 252) is
deleted and amino acids 365 to 380 have been deleted from domain
Ib. However all of domain Ib and a portion of domain II (amino
acids 350 to 394) can be deleted, particularly if the deleted
sequences are replaced with a linking peptide such as GGGGS (SEQ ID
NO:3).
[0111] In addition, the PE molecules can be further modified using
site-directed mutagenesis or other techniques known in the art, to
alter the molecule for a particular desired application. Means to
alter the PE molecule in a manner that does not substantially
affect the functional advantages provided by the PE molecules
described here can also be used and such resulting molecules are
intended to be covered herein.
[0112] For maximum cytotoxic properties of a preferred PE molecule,
several modifications to the molecule are recommended. An
appropriate carboxyl terminal sequence to the recombinant molecule
is preferred to translocate the molecule into the cytosol of target
cells. Amino acid sequences which have been found to be effective
include, REDLK (SEQ ID NO:4) (as in native PE), REDL (SEQ ID NO:5),
RDEL (SEQ ID NO:6), or KDEL (SEQ ID NO:7), repeats of those, or
other sequences that function to maintain or recycle proteins into
the endoplasmic reticulum, referred to here as "endoplasmic
retention sequences". See, for example, Chaudhary et al, Proc.
Natl. Acad. Sci. USA 87:308-312 and Seetharam et al, J. Biol. Chem.
266: 17376-17381 (1991) and commonly assigned, U.S. Ser. No.
07/459,635 filed Jan. 2, 1990, all of which are incorporated by
reference herein.
[0113] Deletions of amino acids 365-380 of domain Ib can be made
without loss of activity. Further, amino acids 1-279 may be deleted
so that the toxin begins with a methionine followed by glycine at
position 280. A serine may be placed at position 289 to prevent
formation of improper disulfide bonds is beneficial. The targeting
molecule may be inserted in replacement for domain Ia.
[0114] Preferred forms of PE contain amino acids 253-364 and
381-608, and are followed by the native sequences REDLK (SEQ ID
NO:4) or the mutant sequences KDEL (SEQ ID NO:7) or RDEL (SEQ ID
NO:6). Lysines at positions 590 and 606 may or may not be mutated
to glutamine.
[0115] The targeting molecule may also be inserted at a point
within domain III of the PE molecule. Most preferably the targeting
molecule is fused between about amino acid positions 607 and 609 of
the PE molecule. This means that the targeting molecule is inserted
after about amino acid 607 of the molecule and an appropriate
carboxyl end of PE is recreated by placing amino acids about
604-613 of PE after the targeting molecule. Thus, the targeting
molecule is inserted within the recombinant PE molecule after about
amino acid 607 and is followed by amino acids 604-613 of domain
III. The targeting molecule may also be inserted into domain Ib to
replace sequences not necessary for toxicity. Debinski, et al. Mol.
Cell. Biol., 11: 1751-1753 (1991).
[0116] Methods of cloning genes encoding PE fused to various
ligands are well known to those of skill in the art. See, for
example, Siegall et al., FASEB J., 3: 2647-2652 (1989); Chaudhary
et al. Proc. Natl. Acad. Sci. USA, 84: 4538-4542 (1987), which are
incorporated herein by reference.
[0117] Those skilled in the art will realize that additional
modifications, deletions, insertions and the like may be made to
the chimeric molecules of the present invention or to the nucleic
acid sequences encoding mesothelin-directed chimeric molecules. All
such constructions may be made by methods of genetic engineering
well known to those skilled in the art (see, generally, Sambrook et
al., supra) and may produce proteins that have differing properties
of affinity, specificity, stability and toxicity that make them
particularly suitable for various clinical or biological
applications.
[0118] (b) Diphtheria Toxin (DT)
[0119] Like PE, diphtheria toxin (DT) kills cells by
ADP-ribosylating elongation factor 2 thereby inhibiting protein
synthesis. Diphtheria toxin, however, is divided into two chains, A
and B, linked by a disulfide bridge. In contrast to PE, chain B of
DT, which is on the carboxyl end, is responsible for receptor
binding and chain A, which is present on the amino end, contains
the enzymatic activity (Uchida et al., Science, 175: 901-903
(1972); Uchida et al. J. Biol. Chem., 248: 3838-3844 (1973)).
[0120] In a preferred embodiment, the targeting molecule-Diphtheria
toxin fusion proteins of this invention have the native
receptor-binding domain removed by truncation of the Diphtheria
toxin B chain. Particularly preferred is DT388, a DT in which the
carboxyl terminal sequence beginning at residue 389 is removed.
Chaudhary, et al., Bioch. Biophys. Res. Comm., 180: 545-551
(1991).
[0121] Like the PE chimeric cytotoxins, the DT molecules may be
chemically conjugated to a mesothelin targeting molecule, but, in a
preferred embodiment, the targeting molecule will be fused to the
Diphtheria toxin by recombinant means. The genes encoding protein
chains may be cloned in cDNA or in genomic form by any cloning
procedure known to those skilled in the art. Methods of cloning
genes encoding DT fused to various ligands are also well known to
those of skill in the art. See, for example, Williams et al. J.
Biol. Chem. 265: 11885-11889 (1990) and copending patent
application U.S. Ser. No. 07/620,939 (now U.S. Pat. No. 6,099,842)
which describe the expression of a number of growth-factor-DT
fusion proteins.
[0122] The term "Diphtheria toxin" (DT) as used herein refers to
full length native DT or to a DT that has been modified.
Modifications typically include removal of the targeting domain in
the B chain and, more specifically, involve truncations of the
carboxyl region of the B chain.
[0123] Detectable labels suitable for use as the effector molecule
component of the chimeric molecules of this invention include any
composition detectable by spectroscopic, photochemical,
biochemical, immunochemical, electrical, optical or chemical means
all as described above.
[0124] C. Attachment of the Targeting Molecule to the Effector
Molecule
[0125] One of skill will appreciate that the targeting molecule and
effector molecules may be joined together in any order. Thus, where
the targeting molecule is a polypeptide, the effector molecule may
be joined to either the amino or carboxy termini of the targeting
molecule. The targeting molecule may also be joined to an internal
region of the effector molecule, or conversely, the effector
molecule may be joined to an internal location of the targeting
molecule, as long as the attachment does not interfere with the
respective activities of the molecules.
[0126] The targeting molecule and the effector molecule may be
attached by any of a number of means well known to those of skill
in the art. Typically the effector molecule is conjugated, either
directly or through a linker (spacer), to the targeting molecule.
However, where both the effector molecule and the targeting
molecule are polypeptides it is preferable to recombinantly express
the chimeric molecule as a single-chain fusion protein.
[0127] D. Conjugation of the Effector Molecule to the Targeting
Molecule
[0128] In one embodiment, the targeting molecule is chemically
conjugated to the effector molecule (e.g. a cytotoxin, a label, a
ligand, or a drug or liposome). Means of chemically conjugating
molecules are well known to those of skill.
[0129] The procedure for attaching an agent to an antibody or other
polypeptide targeting molecule will vary according to the chemical
structure of the agent. Polypeptides typically contain variety of
functional groups; e.g., carboxylic acid (COOH) or free amine
(--NH2) groups, which are available for reaction with a suitable
functional group on an effector molecule to bind the effector
thereto.
[0130] Alternatively, the targeting molecule and/or effector
molecule may be derivatized to expose or attach additional reactive
functional groups. The derivatization may involve attachment of any
of a number of linker molecules such as those available from Pierce
Chemical Company, Rockford Ill.
[0131] A "linker", as used herein, is a molecule that is used to
join the targeting molecule to the effector molecule. The linker is
capable of forming covalent bonds to both the targeting molecule
and to the effector molecule. Suitable linkers are well known to
those of skill in the art and include, but are not limited to,
straight or branched-chain carbon linkers, heterocyclic carbon
linkers, or peptide linkers. Where the targeting molecule and the
effector molecule are polypeptides, the linkers may be joined to
the constituent amino acids through their side groups (e.g.,
through a disulfide linkage to cysteine). However, in a preferred
embodiment, the linkers will be joined to the alpha carbon amino
and carboxyl groups of the terminal amino acids.
[0132] A bifunctional linker having one functional group reactive
with a group on a particular agent, and another group reactive with
an antibody, may be used to form the desired immunoconjugate.
Alternatively, derivatization may involve chemical treatment of the
targeting molecule, e.g., glycol cleavage of the sugar moiety of a
the glycoprotein antibody with periodate to generate free aldehyde
groups. The free aldehyde groups on the antibody may be reacted
with free amine or hydrazine groups on an agent to bind the agent
thereto. (See U.S. Pat. No. 4,671,958). Procedures for generation
of free sulfhydryl groups on polypeptide, such as antibodies or
antibody fragments, are also known (See U.S. Pat. No.
4,659,839).
[0133] Many procedures and linker molecules for attachment of
various compounds including radionuclide metal chelates, toxins and
drugs to proteins such as antibodies are known. See, for example,
European Patent Application No. 188,256; U.S. Pat. Nos. 4,671,958,
4,659,839, 4,414,148, 4,699,784; 4,680,338; 4,569,789; and
4,589,071; and Borlinghaus et al. Cancer Res. 47: 4071-4075 (1987)
which are incorporated herein by reference. In particular,
production of various immunotoxins is well-known within the art and
can be found, for example in "Monoclonal Antibody-Toxin Conjugates:
Aiming the Magic Bullet," Thorpe et al., Monoclonal Antibodies in
Clinical Medicine, Academic Press, pp. 168-190 (1982), Waldmann,
Science, 252: 1657 (1991), U.S. Pat. Nos. 4,545,985 and 4,894,443
which are incorporated herein by reference.
[0134] In some circumstances, it is desirable to free the effector
molecule from the targeting molecule when the chimeric molecule has
reached its target site. Therefore, chimeric conjugates comprising
linkages which are cleavable in the vicinity of the target site may
be used when the effector is to be released at the target site.
Cleaving of the linkage to release the agent from the antibody may
be prompted by enzymatic activity or conditions to which the
immunoconjugate is subjected either inside the target cell or in
the vicinity of the target site. When the target site is a tumor, a
linker which is cleavable under conditions present at the tumor
site (e.g. when exposed to tumor-associated enzymes or acidic pH)
may be used.
[0135] A number of different cleavable linkers are known to those
of skill in the art. See U.S. Pat. Nos. 4,618,492; 4,542,225, and
4,625,014. The mechanisms for release of an agent from these linker
groups include, for example, irradiation of a photolabile bond and
acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958, for example,
includes a description of immunoconjugates comprising linkers which
are cleaved at the target site in vivo by the proteolytic enzymes
of the patient s complement system. In view of the large number of
methods that have been reported for attaching a variety of
radiodiagnostic compounds, radiotherapeutic compounds, drugs,
toxins, and other agents to antibodies one skilled in the art will
be able to determine a suitable method for attaching a given agent
to an antibody or other polypeptide.
[0136] E. Production of Fusion Proteins
[0137] Where the targeting molecule and/or the effector molecule is
relatively short (i.e., less than about 50 amino acids) they may be
synthesized using standard chemical peptide synthesis techniques.
Where both molecules are relatively short the chimeric molecule may
be synthesized as a single contiguous polypeptide. Alternatively
the targeting molecule and the effector molecule may be synthesized
separately and then fused by condensation of the amino terminus of
one molecule with the carboxyl terminus of the other molecule
thereby forming a peptide bond. Alternatively, the targeting and
effector molecules may each be condensed with one end of a peptide
spacer molecule thereby forming a contiguous fusion protein.
[0138] Solid phase synthesis in which the C-terminal amino acid of
the sequence is attached to an insoluble support followed by
sequential addition of the remaining amino acids in the sequence is
the preferred method for the chemical synthesis of the polypeptides
of this invention. Techniques for solid phase synthesis are
described by Barany and Merrifield, Solid-Phase Peptide Synthesis;
pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2:
Special Methods in Peptide Synthesis, Part A., Merrifield, et al.
J. Am. Chem. Soc., 85: 2149-2156 (1963), and Stewart et al., Solid
Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, Ill.
(1984) which are incorporated herein by reference.
[0139] In a preferred embodiment, the chimeric fusion proteins of
the present invention are synthesized using recombinant DNA
methodology. Generally this involves creating a DNA sequence that
encodes the fusion protein, placing the DNA in an expression
cassette under the control of a particular promoter, expressing the
protein in a host, isolating the expressed protein and, if
required, renaturing the protein.
[0140] DNA encoding the fusion proteins of this invention may be
prepared by any suitable method, including, for example, cloning
and restriction of appropriate sequences or direct chemical
synthesis by methods such as the phosphotriester method of Narang
et al. Meth. Enzymol. 68: 90-99 (1979); the phosphodiester method
of Brown et al., Meth. Enzymol. 68: 109-151 (1979); the
diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22:
1859-1862 (1981); and the solid support method of U.S. Pat. No.
4,458,066, all incorporated by reference herein.
[0141] Chemical synthesis produces a single stranded
oligonucleotide. This may be converted into double stranded DNA by
hybridization with a complementary sequence, or by polymerization
with a DNA polymerase using the single strand as a template. One of
skill would recognize that while chemical synthesis of DNA is
limited to sequences of about 100 bases, longer sequences may be
obtained by the ligation of shorter sequences.
[0142] Alternatively, subsequences may be cloned and the
appropriate subsequences cleaved using appropriate restriction
enzymes. The fragments may then be ligated to produce the desired
DNA sequence.
[0143] While the two molecules are preferably essentially directly
joined together, one of skill will appreciate that the molecules
may be separated by a peptide spacer consisting of one or more
amino acids. Generally the spacer will have no specific biological
activity other than to join the proteins or to preserve some
minimum distance or other spatial relationship between them.
However, the constituent amino acids of the spacer may be selected
to influence some property of the molecule such as the folding, net
charge, or hydrophobicity.
[0144] The nucleic acid sequences encoding the fusion proteins may
be expressed in a variety of host cells, including E. coli, other
bacterial hosts, yeast, and various higher eukaryotic cells such as
the COS, CHO and HeLa cells lines and myeloma cell lines. The
recombinant protein gene will be operably linked to appropriate
expression control sequences for each host. For E. coli this
includes a promoter such as the T7, trp, or lambda promoters, a
ribosome binding site and preferably a transcription termination
signal. For eukaryotic cells, the control sequences will include a
promoter and preferably an enhancer derived from immunoglobulin
genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence,
and may include splice donor and acceptor sequences.
[0145] The plasmids and vectors of the invention can be transferred
into the chosen host cell by well-known methods such as calcium
chloride transformation for E. coli and calcium phosphate treatment
or electroporation for mammalian cells. Cells transformed by the
plasmids can be selected by resistance to antibiotics conferred by
genes contained on the plasmids, such as the amp, gpt, neo and hyg
genes.
[0146] Once expressed, the recombinant fusion proteins can be
purified according to standard procedures of the art, including
ammonium sulfate precipitation, affinity columns, column
chromatography, gel electrophoresis and the like (see, generally,
R. Scopes, Protein Purification, Springer--Verlag, N.Y. (1982),
Deutscher, Methods in Enzymology Vol. 182: Guide to Protein
Purification., Academic Press, Inc. N.Y. (1990)). Substantially
pure compositions of at least about 90 to 95% homogeneity are
preferred, and 98 to 99% or more homogeneity are most preferred for
pharmaceutical uses. Once purified, partially or to homogeneity as
desired, the polypeptides may then be used therapeutically.
[0147] III. Administration to Patients of Targeting Agents to
Mesothelin
[0148] Therapeutic agents of the present invention, such as
antibodies to mesothelin or such as antibodies or other targeting
molecules attached to an effector molecule are administered in any
suitable manner, preferably with pharmaceutically acceptable
carriers. One skilled in the art will appreciate that suitable
methods of administering such compounds in the context of the
present invention to a patient are available, and, although more
than one route can be used to administer a particular compound, a
particular route can often provide a more immediate and more
effective reaction than another route. It should be recognized that
the administration of peptides are well-known for a variety of
diseases, and one of skill is able to extrapolate the information
available for use of peptides to treat these other diseases to
mesothelin peptides.
[0149] Pharmaceutically acceptable carriers are also well known to
those who are skilled in the art. The optimal choice of carrier
will be determined in part by the particular compound, as well as
by the particular method used to administer the composition.
Accordingly, there is a wide variety of suitable formulations of
the pharmaceutical compositions of the present invention.
[0150] Antibodies may be formulated into an injectable preparation.
Parenteral formulations are known and are suitable for use in the
invention, preferably for i.m. or i.v. administration. The
formulations containing therapeutically effective amounts of
antibodies or immuno-toxins are either sterile liquid solutions,
liquid suspensions or lyophilized versions and optionally contain
stabilizers or excipients. Lyophilized compositions are
reconstituted with suitable diluents, e.g., water for injection,
saline, 0.3% glycine and the like, at a level of about from 0.01
mg/kg of host body weight to 10 mg/kg where appropriate. Typically,
the pharmaceutical compositions containing the antibodies or
immunotoxins will be administered in a therapeutically effective
dose in a range of from about 0.01 mg/kg to about 5 mg/kg of the
treated mammal. A preferred therapeutically effective dose of the
pharmaceutical composition containing antibody or immunotoxin will
be in a range of from about 0.01 mg/kg to about 0.5 mg/kg body
weight of the treated mammal administered over several days to two
weeks by daily intravenous infusion, each given over a one hour
period, in a sequential patient dose-escalation regimen.
[0151] Antibody may be administered systemically by injection i.m.,
subcutaneously, intrathecally or intraperitoneally or into vascular
spaces, particularly into the peritoneal cavity or thoracic cavity,
e.g., injection at a dosage of greater than about 1 .mu.g/cc
fluid/day. A permanent intrathecal catheter would be a convenient
means to administer therapeutic antibodies. The dose will be
dependent upon the properties of the antibody or immunotoxin
employed, e.g., its activity and biological half-life, the
concentration of antibody in the formulation, the site and rate of
dosage, the clinical tolerance of the patient involved, the disease
afflicting the patient and the like as is well within the skill of
the physician.
[0152] The antibody of the present invention may be administered in
solution. The pH of the solution should be in the range of pH 5 to
9.5, preferably pH 6.5 to 7.5. The antibody or derivatives thereof
should be in a solution having a suitable pharmaceutically
acceptable buffer such as phosphate, tris
(hydroxymethyl)aminomethane-HCl or citrate and the like. Buffer
concentrations should be in the range of 1 to 100 mM. The solution
of antibody may also contain a salt, such as sodium chloride or
potassium chloride in a concentration of 50 to 150 mM. An effective
amount of a stabilizing agent such as an albumin, a globulin, a
gelatin, a protamine or a salt of protamine may also be included
and may be added to a solution containing antibody or immunotoxin
or to the composition from which the solution is prepared. Antibody
or immunotoxin may also be administered via microspheres, liposomes
or other microparticulate delivery systems placed in certain
tissues including blood.
[0153] Dosages
[0154] In therapeutic applications, the dosages of compounds used
in accordance with the invention vary depending on the class of
compound and the condition being treated. The age, weight, and
clinical condition of the recipient patient; and the experience and
judgment of the clinician or practitioner administering the therapy
are among the factors affecting the selected dosage. For example,
the dosage of an immunoglobulin can range from about 0.1 milligram
per kilogram of body weight per day to about 10 mg/kg per day for
polyclonal antibodies and about 5% to about 20% of that amount for
monoclonal antibodies. In such a case, the immunoglobulin can be
administered once daily as an intravenous infusion. Preferably, the
dosage is repeated daily until either a therapeutic result is
achieved or until side effects warrant discontinuation of therapy.
Generally, the dose should be sufficient to treat or ameliorate
symptoms or signs of the disease without producing unacceptable
toxicity to the patient.
[0155] A therapeutically effective amount of the compound is that
which provides either subjective relief of a symptom(s) or an
objectively identifiable improvement, such as inhibition of tumor
cell growth, as noted by the clinician or other qualified observer.
The dosing range varies with the compound used, the route of
administration and the potency of the particular compound.
[0156] IV. Gene Therapy and Inhibitory Nucleic Acid
Therapeutics
[0157] Using the nucleotide sequence information of this invention,
one skilled in the art can formulate strategies and methods to
isolate the mesothelin gene, describe the gene structure for
function, and may also discover specific promoters for known or
unknown transcriptional factors which may be of further value in
the genetic intervention of mesothelioma and ovarian cancers.
Analytical DNA sequencing of normal mesothelin in mesothelial cells
may lead to a discovery of mutation(s) of the gene in mesothelioma
and ovarian cancers.
[0158] Mesothelin manifests an adhesive property which can be
attributed to implantation of the mesothelioma and ovarian cancers.
By introducing antisense DNA or blocking the transcription of
mesothelin gene, novel gene therapy regimens can be set up
according to current strategies of gene therapy.
[0159] Inhibitory nucleic acid therapeutics which can block the
expression or activity of the mesothelin gene will be useful in
slowing or inhibiting the growth of mesotheliomas or ovarian tumors
or other abnormal cells which are associated with mesothelin.
Inhibitory nucleic acids may be single-stranded nucleic acids,
which can specifically bind to a complementary nucleic acid
sequence. By binding to the appropriate target sequence, an
RNA-RNA, a DNA-DNA, or RNA-DNA duplex or triplex is formed. These
nucleic acids are often termed "antisense" because they are usually
complementary to the sense or coding strand of the gene, although
recently approaches for use of "sense" nucleic acids have also been
developed. The term "inhibitory nucleic acids" as used herein,
refers to both "sense" and "antisense" nucleic acids.
[0160] By binding to the target nucleic acid, the inhibitory
nucleic acid can inhibit the function of the target nucleic acid.
This could, for example, be a result of blocking DNA transcription,
processing or poly(A) addition to mRNA, DNA replication,
translation, or promoting inhibitory mechanisms of the cells, such
as promoting RNA degradation. Inhibitory nucleic acid methods
therefore encompass a number of different approaches to altering
expression of, for example, a mesothelin gene. These different
types of inhibitory nucleic acid technology are described in
Helene, C. and Toulme, J., 1990, Biochim. Biophys. Acta.
1049:99-125, which is hereby incorporated by reference and is
referred to hereinafter as "Helene and Toulme."
[0161] In brief, inhibitory nucleic acid therapy approaches can be
classified into those that target DNA sequences, those that target
RNA sequences (including pre-mRNA and mRNA), those that target
proteins (sense strand approaches), and those that cause cleavage
or chemical modification of the target nucleic acids.
[0162] Approaches targeting DNA fall into several categories.
Nucleic acids can be designed to bind to the duplex DNA to form a
triple helical or "triplex" structure. Alternatively, inhibitory
nucleic acids are designed to bind to regions of single stranded
DNA resulting from the opening of the duplex DNA during replication
or transcription. See Helene and Toulme.
[0163] More commonly, inhibitory nucleic acids are designed to bind
to mRNA or mRNA precursors. Inhibitory nucleic acids are used to
prevent maturation of pre-mRNA. Inhibitory nucleic acids may be
designed to interfere with RNA processing, splicing or
translation.
[0164] The inhibitory nucleic acids can be targeted to mRNA. In
this approach, the inhibitory nucleic acids are designed to
specifically block translation of the encoded protein. Using this
approach, the inhibitory nucleic acid can be used to selectively
suppress certain cellular functions by inhibition of translation of
mRNA encoding critical proteins. For example, an inhibitory nucleic
acid complementary to regions of c-myc mRNA inhibits c-myc protein
expression in a human promyelocytic leukemia cell line, HL60, which
overexpresses the c-myc proto-oncogene. See Wickstrom E. L., et
al., 1988, PNAS (USA) 85:1028-1032 and Harel-Bellan, A., et al.,
1988, Exp. Med. 168:2309-2318. As described in Helene and Toulme,
inhibitory nucleic acids targeting mRNA have been shown to work by
several different mechanisms to inhibit translation of the encoded
protein(s).
[0165] The inhibitory nucleic acids introduced into the cell can
also encompass the "sense" strand of the gene or mRNA to trap or
compete for the enzymes or binding proteins involved in mRNA
translation. See Helene and Toulme.
[0166] Lastly, the inhibitory nucleic acids can be used to induce
chemical inactivation or cleavage of the target genes or mRNA.
Chemical inactivation can occur by the induction of crosslinks
between the inhibitory nucleic acid and the target nucleic acid
within the cell. Other chemical modifications of the target nucleic
acids induced by appropriately derivatized inhibitory nucleic acids
may also be used.
[0167] Cleavage, and therefore inactivation, of the target nucleic
acids may be accomplished by attaching a substituent to the
inhibitory nucleic acid which can be activated to induce cleavage
reactions. The substituent can be one that affects either chemical,
or enzymatic cleavage. Alternatively, cleavage can be induced by
the use of ribozymes or catalytic RNA. In this approach, the
inhibitory nucleic acids would comprise either naturally occurring
RNA (ribozymes) or synthetic nucleic acids with catalytic
activity.
[0168] The targeting of inhibitory nucleic acids to specific cells
of the immune system by conjugation with targeting moieties binding
receptors on the surface of these cells can be used for all of the
above forms of inhibitory nucleic acid therapy. This invention
encompasses all of the forms of inhibitory nucleic acid therapy as
described above and as described in Helene and Toulme.
[0169] This invention relates to the targeting of inhibitory
nucleic acids to sequences of mesothelin for use in inhibiting or
slowing the growth of tumors associated with mesothelin. A problem
associated with inhibitory nucleic acid therapy is the effective
delivery of the inhibitory nucleic acid to the target cell in vivo
and the subsequent internalization of the inhibitory nucleic acid
by that cell. Delivery, however, can be accomplished by linking the
inhibitory nucleic acid to a targeting moiety to form a conjugate
that binds to a specific receptor on the surface of the target
infected cell, and which is internalized after binding. Preferably,
the inhibitory nucleic acid will be delivered to the peritoneal
cavity, the thoracic cavity, as well as any other location where
cells bearing mesothelin are of interest.
[0170] Gene therapy can also correct genetic defects by insertion
of exogenous cellular genes that encode a desired function into
cells that lack that function, such that the expression of the
exogenous gene a) corrects a genetic defect or b) causes the
destruction of cells that are genetically defective. Methods of
gene therapy are well known in the art, see, for example, Lu, M.,
et al. (1994), Human Gene Therapy 5:203; Smith, C. (1992), J.
Hematotherapy 1:155; Cassel, A., et al. (1993), Exp. Hematol.
21-:585 (1993); Larrick, J. W. and Burck, K. L., Gene Therapy:
Application of Molecular Biology, Elsevier Science Publishing Co.,
Inc., New York, N.Y. (1991) and Kreigler, M. Gene Transfer and
Expression: A Laboratory Manual, W.H. Freeman and Company, New York
(1990), each incorporated herein by reference. One modality of gene
therapy involves (a) obtaining from a patient a viable sample of
primary cells of a particular cell type; (b) inserting into these
primary cells a nucleic acid segment encoding a desired gene
product; (c) identifying and isolating cells and cell lines that
express the gene product; (d) re-introducing cells that express the
gene product; (e) removing from the patient an aliquot of tissue
including cells resulting from step c and their progeny; and (f)
determining the quantity of the cells resulting from step c and
their progeny, in said aliquot. The introduction into cells in step
(b) of a vector that encodes a sequence (for a "desired gene
product") which will block mesothelin expression or activity can be
useful in inhibiting or slowing the growth of tumor cells
associated with mesothelin.
[0171] V. Vaccine Development
[0172] Vaccine development using mesothelin amino acid
sequence.
[0173] Substances suitable for use as vaccines for the prevention
of and inhibition of the growth of tumors bearing mesothelin and
methods for administering them may be employed. The vaccines are
directed against mesothelin. Preferably, the vaccines comprise
mesothelin derived antigen.
[0174] Vaccines can be made recombinantly. Typically, a vaccine
will include from about 1 to about 50 micrograms of antigen or
antigenic protein or peptide. More preferably, the amount of
protein is from about 15 to about 45 micrograms. Typically, the
vaccine is formulated so that a dose includes about 0.5
milliliters. The vaccine may be administered by any route known in
the art. Preferably, the route is intraperitoneally or
parenteral.
[0175] There are a number of strategies for amplifying an antigen's
effectiveness, particularly as related to the art of vaccines. For
example, cyclization or circularization of a peptide can increase
the peptide's antigenic and immunogenic potency. See U.S. Pat. No.
5,001,049 which is incorporated by reference herein. More
conventionally, an antigen can be conjugated to a suitable carrier,
usually a protein molecule. This procedure has several facets. It
can allow multiple copies of an antigen, such as a peptide, to be
conjugated to a single larger carrier molecule. Additionally, the
carrier may possess properties which facilitate transport, binding,
absorption or transfer of the antigen.
[0176] For parenteral administration, examples of suitable carriers
are the tetanus toxoid, the diphtheria toxoid, serum albumin and
lamprey, or keyhole limpet, hemocyanin because they provide the
resultant conjugate with minimum genetic restriction. Conjugates
including these universal carriers can function as T cell clone
activators in individuals having very different gene sets.
[0177] The conjugation between a peptide and a carrier can be
accomplished using one of the methods known in the art.
Specifically, the conjugation can use bifunctional cross-linkers as
binding agents as detailed, for example, by Means and Feeney, "A
recent review of protein modification techniques," Bioconjugate
Chem. 1:2-12 (1990).
[0178] The antigen may be combined or mixed with various solutions
and other compounds as is known in the art. For example, it may be
administered in water, saline or buffered vehicles with or without
various adjuvants or immunodiluting agents. Examples of such
adjuvants or agents include aluminum hydroxide, aluminum phosphate,
aluminum potassium sulfate (alum), beryllium sulfate, silica,
kaolin, carbon, water-in-oil emulsions, oil-in-water emulsions,
muramyl dipeptide, bacterial endotoxin, lipid X, Corynebacterium
parvum (Propionibacterium acnes), Bordetella pertussis,
polyribonucleotides, sodium alginate, lanolin, lysolecithin,
vitamin A, saponin, liposomes, levamisole, DEAE-dextran, blocked
copolymers or other synthetic adjuvants. Such adjuvants are
available commercially from various sources, for example, Merck
Adjuvant 65 (Merck and Company, Inc., Rahway, N.J.) or Freund's
Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories,
Detroit, Mich.). Other suitable adjuvants are Amphigen
(oil-in-water), Alhydrogel (aluminum hydroxide), or a mixture of
Amphigen and Alhydrogel. Only aluminum is approved for human
use.
[0179] The proportion of antigen and adjuvant can be varied over a
broad range so long as both are present in effective amounts. For
example, aluminum hydroxide can be present in an amount of about
0.5% of the vaccine mixture (A12O3 basis). On a per-dose basis, the
amount of the antigen can range from about 0.1 .mu.g to about 100
.mu.g protein per patient. A preferable range is from about 1 .mu.g
to about 50 .mu.g per dose. A more preferred range is about 15
.mu.g to about 45 .mu.g. A suitable dose size is about 0.5 ml.
After formulation, the vaccine may be incorporated into a sterile
container which is then sealed and stored at a low temperature, for
example 4.degree. C., or it may be freeze-dried. Lyophilization
permits long-term storage in a stabilized form.
[0180] The treatment may consist of a single dose of vaccine or a
plurality of doses over a period of time. It is preferred that the
doses be given to a patient suspected of having mesothelin bearing
tumor cells. The antigen of the invention can be combined with
appropriate doses of compounds including influenza antigens, such
as influenza type A antigens. Also, the antigen could be a
component of a recombinant vaccine which could be adaptable for
oral administration.
[0181] Vaccines of the invention may be combined with other
vaccines for other diseases to produce multivalent vaccines. A
pharmaceutically effective amount of the antigen can be employed
with a pharmaceutically acceptable carrier such as a protein or
diluent useful for the vaccination of mammals, particularly humans.
Other vaccines may be prepared according to methods well-known to
those skilled in the art.
[0182] Those of skill will readily recognize that it is only
necessary to expose a mammal to appropriate epitopes in order to
elicit effective immunoprotection. The epitopes are typically
segments of amino acids which are a small portion of the whole
protein. Using recombinant genetics, it is routine to alter a
natural protein's primary structure to create derivatives embracing
epitopes that are identical to or substantially the same as
(immunologically equivalent to) the naturally occurring epitopes.
Such derivatives may include peptide fragments, amino acid
substitutions, amino acid deletions and amino acid additions within
the amino acid sequence for mesothelin. For example, it is known in
the protein art that certain amino acid residues can be substituted
with amino acids of similar size and polarity without an undue
effect upon the biological activity of the protein.
[0183] Using the mesothelin amino acid sequence information, one of
skill in the art can perform epitope mapping against sera isolated
from patients with ovarian cancers or mesotheliomas. Relatively
strong epitopes may be identified and common epitope(s) may also be
recognized. The epitope mapping against human sera can also be
extended to a screening of epitope-peptides against activated human
lymphocytes in order to identify potential T-cell epitopes.
Theoretically, it is not likely that T-cell epitopes of mesothelin
will be found in human T-cells, but mutations induced in mesothelin
may create new epitopes which may be recognized by T-cells. Mutant
mesothelin can easily be generated randomly using a phage display
method. The resultant library is screened by human sera from
patients suffering from malignant mesothelioma and ovarian cancer.
Thus, suitable antigenic peptides may be identified for
mesothelin-derived vaccines.
[0184] VI. Kits.
[0185] This invention further embraces diagnostic kits for
detecting for the presence of mesothelin in tissue samples or in
serum, comprising a container having a nucleic acid or an antibody
or other targeting agent specific for mesothelin and instructional
material for the detection of mesothelin.
[0186] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
of understanding, it will be readily apparent to those of ordinary
skill in the art in light of the teachings of this invention that
certain changes and modifications may be made thereto without
departing from the spirit or scope of the appended claims.
[0187] All publications and patent applications cited in this
specification are herein incorporated by reference in their
entirety for all purposes as if each individual publication or
patent application were specifically and individually indicated to
be incorporated by reference.
[0188] VI. Models for Evaluation of Therapies Directed to
Mesothelin.
[0189] The mesothelin cDNA can be transfected into established
tumor cell lines where it will express the protein. The transfected
cell lines can be used to grow tumors in mice or other mammals to
provide a model for testing therapies directed to controlling,
suppressing or regulating mesothelin expression. Transfected tumor
cell lines can be transplanted into the test mammal. The mammal can
then be subjected to a drug of interest and subsequent tumor cell
activity can be monitored to determine whether the drug of interest
has anti-tumor effects. Tumor cell lines that have been found to be
particularly good candidates for this procedure include, mouse NIH
3T3 cells (tumorigenic cell lines), A431 human ovarian tumor cells
and MCF-7 breast tumor cells, A2780 human ovarian tumor cells and
OVCAR-3 human epidermoid carcinoma cells.
EXAMPLES
A. Materials and Methods
[0190] 1. Cells and antibodies. Human ovarian tumor cell line,
OVCAR-3, and cell lines A431, KB, MCF-7, COS-1, WI-38 and NIH 3T3
were obtained from the American Type Culture Collections (ATCC,
Rockville, Md.). Cells were cultured either in RPMI 1640 or DMEM
media (GIBCO Laboratories, Grand Island, N.Y.), supplemented with
L-glutamine (2 mM), penicillin (50 .mu.g/ml), streptomycin (50
units/ml) and 5-10% fetal bovine serum (GIBCO). NIH 3T3
transfectants were grown in DMEM with 0.8 mg/ml of G418 (GIBCO).
Cells were used when they reached 80-90% confluency after washing
three times with ice-cold PBS (GIBCO). MAb K1 and antibody MOPC-21
have been described (Chang, K., et al., Int. J. Cancer 50, 373-381
(1992)) and were used in a concentration of 5-10 .mu.g/ml.
[0191] 2. Isolation of the cDNA clones. The HeLa S3 cDNA library
(ClonTech, Palo Alto, Calif.) was screened at approximately 50,000
pfu/150 mm filter as described previously (Chang, K., and Pastan,
I., Int. J. Cancer 57, 90-97 (1994)) using protein A-purified MAb
K1 (5 .mu.g/ml) and peroxidase-conjugated goat anti-mouse IgG (H+L)
(10 .mu.g/ml, Jackson ImmunoResearch Lab, Inc., West Grove, Pa.).
Two positive plaques (6-1, 6-2) were isolated and the phages were
purified to homogeneity by three or more rounds of screening. After
verification of their specificity with MAb K1 by showing they did
not react with a control MOPC-21 antibody, single-plaque isolates
of 6-1 and 6-2 were used to prepare 5 to 10 phage-plates, followed
by extraction and purification of phage DNA with a lambda phage DNA
kit (Qiagen, Inc., Chatsworth, Calif.). Phage DNA was then digested
with EcoRI and the insert subcloned into the EcoRI site of a
pcDNAI/Amp (Invitrogen Corporation, San Diego, Calif.) vector using
a rapid ligation protocol (Chang, K., and Pastan, I., Int. J.
Cancer 57, 90-97 (1994)). Plasmid DNAs were isolated using
Qiagene's plasmid DNA isolation kit (Chang, K., and Pastan, I.,
Int. J. Cancer 57, 90-97 (1994)). Restriction mapping using XhoI,
EcoRI, SalI, BamHI, NcoI, and DNA sequencing revealed that the two
plasmid clones (p6-1 and p6-2) had identical 1500 base-pair
inserts.
[0192] To isolate a longer clone, the insert of p6-1 was purified
to make a cDNA probe (specific activity=8.5.times.105 cpm/ml) by
random priming. The HeLa S3 cDNA library was re-screened using the
filter hybridization method described previously (Chang, K., and
Pastan, I., Int. J. Cancer 57, 90-97 (1994)). 14 lambda clones were
isolated and purified, and their insert sizes were assessed by
digestion with EcoRI. Four large inserts were subcloned into a
pcDNAI/Amp plasmid vector (p9, p13-1, p16 and p18-1). p9 contained
the largest insert with a long open reading frame.
[0193] 3. DNA sequencing analysis. Using T3 and T7 promoter primers
and twenty 17 bp synthetic primers, the entire cDNA insert of p9
was sequenced using the method described by Sanger (Sanger, F., et
al., Proc. Natl. Acad. Sci. USA 74, 5463-5467 (1977)) and an
automatic cycle sequencing method.
[0194] 4. Northern blot analysis. Total RNAs (20 .mu.g) from
OVCAR-3, KB, MCF-7, A431 and WI38 were electrophoresed on a 1%
agarose gel in MOPS buffer with 16.6% formaldehyde, and then
transferred to a Nylon paper. Northern hybridization was done with
a method described before (Chang, K., and Pastan, I., Int. J.
Cancer 57, 90-97 (1994)). The blot washed and reprobed with a
32P-labeled human-actin cDNA as an internal control to assess the
integrity and quantity of the RNA samples loaded.
[0195] 5. In Vitro transcription and translation. TNT Coupled
Reticulocyte Lysate System, canine pancreatic microsomal membrane,
2 .mu.g of plasmid DNAs of p9(pcDICAK)-9), pAPK1 (Chang, K., and
Pastan, I., Int. J. Cancer 57, 90-97 (1994)), to eliminate and 3H
leucine were used in an in vitro transcription/translation and
translocation/processing experiment according to the protocol of
the manufacturer (Promega, Madison, Wis., USA). Translation
products were resolved on a 10% SDS-PAGE reducing gel. The proteins
were fixed and the unincorporated label was removed by soaking the
gel three times in 200 ml of buffer, 40% methanol and 10% acetic
acid in deionized water for 30 min. The gels were then soaked for
30 min in 200 ml of INTENSIFY Part A and Part B (NEN Research
Product, Boston, Mass.). After drying, the translated products were
visualized by autoradiography.
[0196] 6. Expression of the cloned cDNAs in mammalian cells.
Transient transfections of COS cells were performed using
pcDICAK1-9 (p9) and LipofectAMINE (GIBCO) following the
manufacturer's protocol (GIBCO). COS1 cells were plated a day
before the experiment at 2.5.times.105 cells/60 mm dish. 24 .mu.l
of LipofectAMINE and 76 g of OptiMEMI medium were mixed with 10
.mu.g of pcDNAI/Amp vector, or pcDICAK1-9 in 100 .mu.l of OptiMEMI
medium at room temperature for 30 min. After washing the COS-1
cells with OptiMEMI twice, 2.4 ml OptiMEMI were added to the
transfection mixtures and overlaid onto COS1 cells, followed by
incubation at 37.degree. C. for 5 hours. 2.6 ml of DMEM with 20%
FBS were then added into each dish. 48 hours after transfection,
the dishes were subjected to immunofluorescence labeling as
described (Chang, K., et al., Int. J. Cancer 50, 373-381 (1992);
Chang, K., et al., Cancer Res. 52, 181-186 (1992)) or other
treatments. The insert from plasmid p9 (in pcDNA1/Amp) was also
subcloned into a pcDNA3 (Invitrogen) vector for stable
transfection. Plasmid minipreps were made using Qiagen's Miniprep
Plasmid DNA Kit and orientation of the insert in individual clone
was determined by restriction mapping. The resulting plasmid,
pcD3CAK1-9, was then used to transfect NIH 3T3, MCF-7, A431 and
OVCAR-3 cells by DNA-calcium phosphate precipitation as described
(Chen, C. and Okayama, H., Mol. Cell. Biol. 7, 2745-2752 (1987)).
After overnight exposure to the precipitate, the cells were washed
with PBS three times and fed with fresh DMEM/10% FBS medium for 2-3
days. Geneticin G418 sulfate (0.8 mg/ml) was added and the cultures
were maintained until colonies of 2-3 mm in diameter were formed.
Colonies were then transferred into wells of a 96 well plate and
then into a 35 mm dish when they were 80% confluent. Transfected
cells were screened by immunofluorescence (Chang, K., et al., Int.
J. Cancer 50, 373-381 (1992); Chang, K., et al., Cancer Res. 52,
181-186 (1992)) and positive cells were further subcloned by
limited dilution as described (Chang, K., et al., Int. J. Cancer
50, 373-381 (1992)). One of the NIH 3T3 transfectant clones, NIH
3T3 K20, was chosen for further study. To localize the expression
of CAK1, both cell surface and intracellular immunofluorescence
labeling was also performed according to methods described before
(Chang, K., et al., Cancer Res. 52, 181-186 (1992)).
[0197] 7. Treatment of the transfected cells with PI-PLC. CAK1 cDNA
transfected NIH 3T3 cells (NIH 3T3 K20) were grown in 175 mm2
flasks, and when they reached 90% confluency, the cells were washed
in PBS for three times. The cells were incubated with either 5 ml
of 1.25 U/ml PI-PLC (from Bacillus cereus; Boehringer Mannheim
Biochemicals) or 0.05% trypsin/0.052 mM EDTA for 30 min at
37.degree. C. and 30 min at room temperature with shaking. The
supernatants were collected and after centrifugation at
1000.times.g and concentrated about 10 fold using Centricon 30
(Amicon, Inc., Beverly, Mass.). The concentrated supernatants were
used in SDS-PAGE and immunoblot analysis. The enzyme-treated cells
can be recultured and the recovery of CAK1 expression can be seen
after overnight culture. Treatment with PI-PLC was done in a
similar manner using 35 mm diameter dishes followed by
immunofluorescence labeling of the treated cells (Chang, K., et
al., Cancer Res. 52, 181-186 (1992)).
[0198] 8. Immunoblotting Analysis of the Transfected NIH 3T3
Cells.
[0199] Membrane and cytosolic fractions from transfected NIH 3T3
K20 cells (Chang, K., and Pastan, I., Int. J. Cancer 57, 90-97
(1994)) were subjected to 12.5% SDS-PAGE and the resolved proteins
were transferred to nitrocellulose. Immunoblotting was performed as
previously described (Chang, K., et al., Int. J. Cancer 51, 548-554
(1992); Chang, K., and Pastan, I., Int. J. Cancer 57, 90-97
(1994)).
B. Results
[0200] Expression cloning was used to isolate the CAK1 cDNA. We
previously observed that MAb K1 reacts with OVCAR-3 and HeLa cells.
Because we were unable to isolate the cDNA from an OVCAR-3 library
(Chang, K., and Pastan, I., Int. J. Cancer 57, 90-97 (1994)), we
screened a HeLa cDNA library expressed in gt11 as described above.
A total of 1.times.106 phages were screened and two phage clones
(6-1 and 6-2) were identified. DNA sequencing showed both phages
contained the same 1.5 kb insert. The insert hybridized to mRNA
from OVCAR-3 and KB cells (a HeLa subclone which also reacts with
MAb K1) but not to RNA from many other cell lines indicating that
the cDNA is specific for cells reacting with MAb K1. 20 .mu.g of
total RNA from OVCAR-3 cells (lane 1), MCF-7 cells (lane 2), KB
cells (a HeLa subclone, lane 3), A431 cells (lane 4) and W138 cells
(lane 5) were resolved by electrophoresis transferred to nylon
paper and blotted with a 32P-labeled CAK1 probe. Hybridization with
an actin probe showed that the lanes were equally loaded. The mRNA
detected is 2.2 kb in size indicating that the insert isolated was
not full length. The insert contained an open reading frame, a stop
codon and a poly A tail but the 5' end appeared to be missing.
Therefore, the phage library was rescreened with one of the inserts
and 14 new phages with cDNA inserts of various sizes isolated. The
largest insert (#9) was 2138 bp long and when sequenced contained
an open reading frame of 1884 bp (FIG. 1). It contains a typical
Kozak sequence (Kozak, M., Nucleic Acids Res. 5, 8125-8148 (1987))
(AXXATGG) followed by an open reading frame that encodes a 69 kD
protein. The sequence was not present in various data bases
examined (EMBL-GenBank). Because the CAK1 antigen was originally
found to be about 40 kD in size, several experiments were carried
out to determine if clone 9 encoded CAK1.
[0201] 1. In vitro translation. Insert 9 was cloned into a
pcDNAI/Amp vector to make pcDICAK1-9 and used in the TNT
reticulocyte system. pcDICAK1-9 plasmid DNA (lanes 1 and 2), and
pcDIAPK1 (lanes 3 and 4) were used in a TNT coupled reticulocyte
lysate system in the presence (+) or absence (-) of pancreatic
microsomal membrane (m). The products were resolved on a 10%
reducing SDS-PAGE and autoradiographed. A 69 kD protein was
produced. In the presence of pancreatic microsomes (lane 2), a
slightly larger protein was observed indicating the protein had
been inserted into microsomes and glycosylated. As a control, a
cDNA encoding a 30 kD cytosolic protein that also reacts with MAb
K1 (Chang, K., and Pastan, I., Int. J. Cancer 57, 90-97 (1994)) was
subjected to the same analysis. The size of the protein was
unaffected by the presence of microsomes.
[0202] 2. Expression in cultured cells. pcDICAK1-9 was transfected
into COS cells for transient expression. pcDNAI/Amp vectors with
insert 9 or without insert were transfected into COS cells. Two
days later, the cells were immunocytochemically labeled with MAb K1
at 4.degree. C. (for surface labeling) or at 23.degree. C. (for
intracellular labeling) and photographed (Magnification
.times.250). The specific labeling pattern of COS cells transfected
with insert 9 using MAb K1 was observed. In nonpermeabilized cells,
a typical cell surface fluorescent pattern is detected. In
permeabilized cells, strong staining of the Golgi region is
evident. No cytosolic staining was detected. Also, no
immunoreactivity was detected in cells transfected with vector
without insert or control inserts. Thus, insert 9 encodes a cell
surface protein that is also present in the Golgi.
[0203] 3. Size and processing of CAK1 antigen. To determine the
size of the antigen produced by cells transfected with insert 9,
NIH 3T3 cells were transfected with pcD3CAK1-9 to make stable cell
lines. Stably transfected clones were produced as described above
and the presence of antigen on the surface was confirmed by
immunofluorescence. Then membrane and cytosolic fractions were
prepared from NIH 3T3 K20 cells and from OVCAR-3 cells, subjected
to SDS-PAGE and analyzed by immunoblotting with MAb K1.
Approximately 100 .mu.g of membrane fraction (lanes 1 and 3) or
cytosolic fraction (lanes 2 and 4) of the transfected NIH 3T3
(pcD3CAK1) and mock control (pcD3) and membrane (lane 5) or
cytosolic fraction (lane 6) of OVCAR-3 cells were electrophoresed
and immunoblotted with MAb K1. As previously reported, the major
reactivity in OVCAR-3 cells is with a doublet of about 40 and 43 kD
that is present in membranes but not in the cytosol. In the
transfectants, two bands of equal intensity were detected in the
membrane fraction; one of about 40 kD and a second of about 71 kD.
No signal was detected in the cytosol. These data suggest that CAK1
is made as a large molecular weight precursor that is processed by
proteolysis to an approximately 40 kD form.
[0204] 4. Nature of cell surface attachment. To determine if CAK1
was attached to the transfectants via a PI linkage as it is in
OVCAR-3 cells (Chang, K., et al., Cancer Res. 52, 181-186 (1992)),
the NIH 3T3 transfectant cell line k20 was treated with PI-PLC for
60 min. The transfected NIH 3T3 k20 cells were treated with PI-PLC
and labeled with MAb K1 as described above. The CAK1 signal was
completely abolished after PI-PLC treatment. A strong cell surface
labeling pattern was observed in untreated cells. Fluorescence was
absent after treatment with PI-PLC. In phase contrast images before
(B) and after (D) treatment, the treated cells are still attached
to the dish but are slightly altered in shape. The medium from
PI-PLC treated cells was concentrated, subjected to SDS-PAGE and
analyzed with MAb K1. A band of about 70 kD was detected, but no
lower molecular weight bands were detected.
C. Summary of Results
[0205] Thus, the above describes the molecular cloning of the CAK1
antigen which is found on mesothelium, mesotheliomas, ovarian
cancers and some squamous cell carcinomas. We have designated this
antigen mesothelin to reflect its presence on mesothelial cells.
One unexpected feature of mesothelin is that its cDNA encodes a 69
kD protein, whereas the antigen present on OVCAR-3 cells, used to
isolate MAb K1, has a molecular weight of 40,000 Daltons. The DNA
sequence and the deduced amino acid sequence of CAK1 is shown in
FIG. 1. The cDNA is 2138 bp in length and contains an open reading
frame of 1884 bp. The protein it encodes contains 628 amino acids
with a calculated molecular weight of 69001 daltons. A homology
analysis was performed with nucleotide or amino acid sequences and
none was detected using EMBL-GenBank accessed by the GCG program.
The protein contains four potential N-linked glycosylation sites
N--X--S or N--X-T that are shown in bold letters. A typical signal
sequence is not present at the amino terminus. However, a short
hydrophobic segment is located 15 amino acids from the first
methionine (FIG. 1). This sequence might function as a signal
sequence for membrane insertion, because the protein is found on
the cell surface and is inserted into microsomes during cell free
translation. Also present is a putative proteolytic processing
site, RPRFRR (SEQ ID NO:8), beginning at amino acid 293 (FIG. 1).
This site is recognized by furin, a protease important in the
processing of several membrane proteins as well as in the
activation of Pseudomonas and diphtheria toxins (Chiron, M. F., et
al., J.B.C. 269(27):18169-18176 (1994)). The 40 kD form appears to
be derived from a 69 kD precursor by several processing steps.
These are summarized in FIG. 2. Initially, mesothelin is made as a
69 kD polypeptide with a hydrophobic tail which is probably removed
and replaced by phosphatidylinositol (Chang, K., et al., Cancer
Res. 52, 181-186 (1992)). After glycosylation at one or more of its
four putative N-linked glycosylation sites, it is cleaved by a
protease to yield the 40 kD fragment (or doublet) found on the
surface of OVCAR-3 cells and a smaller (31 kD) fragment. The latter
could be released into the medium and/or further degraded. The
amino terminal fragment has recently been detected in the medium of
OVCAR-3 cells (our data). In transfected NIH 3T3 and MCF-7 cells,
we find approximately equal amounts of 70 kD and 40 kD proteins. We
originally detected the 40 kD form in OVCAR-3 and HeLa cells and
did not notice a larger form. Reexamination of the OVCAR-3 and HeLa
cell gels reveals a trace amount of the 70 kD precursor.
Sequence CWU 1
1
* * * * *