U.S. patent application number 11/656200 was filed with the patent office on 2007-06-07 for breast, gastric and prostate cancer associated antigens and uses therefor.
This patent application is currently assigned to Ludwig Institute for Cancer Research. Invention is credited to Yuichi Obata.
Application Number | 20070128655 11/656200 |
Document ID | / |
Family ID | 37663606 |
Filed Date | 2007-06-07 |
United States Patent
Application |
20070128655 |
Kind Code |
A1 |
Obata; Yuichi |
June 7, 2007 |
Breast, gastric and prostate cancer associated antigens and uses
therefor
Abstract
Cancer associated antigens have been identified by autologous
antibody screening of libraries of nucleic acids expressed in
breast, gastric and prostate cancer cells using antisera from
cancer patients. The invention relates to nucleic acids and encoded
polypeptides which are cancer associated antigens expressed in
patients afflicted with cancer. The invention provides, inter alia,
isolated nucleic acid molecules, expression vectors containing
those molecules and host cells transfected with those molecules.
The invention also provides isolated proteins and peptides,
antibodies to those proteins and peptides and cytotoxic T
lymphocytes which recognize the proteins and peptides. Fragments of
the foregoing including functional fragments and variants also are
provided. Kits containing the foregoing molecules additionally are
provided. The molecules provided by the invention can be used in
the diagnosis, monitoring, research, or treatment of conditions
characterized by the expression of one or more cancer associated
antigens.
Inventors: |
Obata; Yuichi; (Nagoya,
JP) |
Correspondence
Address: |
WOLF GREENFIELD & SACKS, P.C.
600 ATLANTIC AVENUE
BOSTON
MA
02210-2206
US
|
Assignee: |
Ludwig Institute for Cancer
Research
New York
NY
|
Family ID: |
37663606 |
Appl. No.: |
11/656200 |
Filed: |
January 22, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09979932 |
May 24, 2002 |
7166573 |
|
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PCT/US00/14749 |
May 26, 2000 |
|
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11656200 |
Jan 22, 2007 |
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60136526 |
May 28, 1999 |
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60153454 |
Sep 10, 1999 |
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Current U.S.
Class: |
435/6.14 ;
435/7.23 |
Current CPC
Class: |
A61P 35/00 20180101;
G01N 33/574 20130101 |
Class at
Publication: |
435/006 ;
435/007.23 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; G01N 33/574 20060101 G01N033/574 |
Claims
1. A method of diagnosing prostate cancer, comprising: contacting a
biological sample isolated from a subject with a nucleic acid probe
or primer that specifically binds to the nucleic acid molecule
which is (a) a nucleic acid molecule that encodes a polypeptide
having at least 99% sequence identity to the amino acid sequence of
SEQ ID NO: 1218, (b) a nucleic acid molecule that differs from SEQ
ID NO: 504 in codon sequence due to the degeneracy of the genetic
code, or (c) a full length complement of (a) or (b), and
determining the binding of the nucleic acid probe or primer to the
nucleic acid molecule, wherein a determination that the nucleic
acid probe or primer interacts with the nucleic acid molecule
indicates that the nucleic acid molecule is present in the sample
and that the patient has prostate cancer.
2-67. (canceled)
68. A kit for detecting the presence of the expression of a human
cancer associated antigen precursor comprising a pair of isolated
nucleic acid molecules each of which consists essentially of a
molecule selected from the group consisting of (a) a 12-32
nucleotide contiguous segment of the nucleotide sequence of (a) a
nucleic acid molecule that encodes a polypeptide having at least
99% sequence identity to the amino acid sequence of SEQ ID NO:
1218, (b) a nucleic acid molecule that differs from SEQ ID NO: 504
in codon sequence due to the degeneracy of the genetic code, or (c)
a full length complement of (a) or (b), wherein the contiguous
segments are nonoverlapping.
69. The kit of claim 68, wherein the pair of isolated nucleic acid
molecules is constructed and arranged to selectively amplify at
least a portion of an isolated nucleic acid molecule that is (a) a
nucleic acid molecule that encodes a polypeptide having at least
99% sequence identity to the amino acid sequence of SEQ ID NO:
1218, (b) a nucleic acid molecule that differs from SEQ ID NO: 504
in codon sequence due to the degeneracy of the genetic code, or (c)
a full length complement of (a) or (b).
70-104. (canceled)
105. The method of claim 1, wherein the nucleic acid probe or
primer is a nucleic acid probe.
106. The method of claim 105, wherein the nucleic acid probe is
labeled.
107. The method of claim 1, wherein the nucleic acid probe or
primer is a primer or a primer pair.
108. The method of claim 107, wherein the primer or a primer of the
primer pair is a 12-32 nucleotide contiguous segment of the
nucleotide sequence of (a) a nucleic acid molecule that encodes a
polypeptide having at least 99% sequence identity to the amino acid
sequence of SEQ ID NO: 1218, (b) a nucleic acid molecule that
differs from SEQ ID NO: 504 in codon sequence due to the degeneracy
of the genetic code, or (c) a full length complement of (a) or
(b).
109. The method of claim 1, wherein the method is carried out as an
amplification assay.
110. The method of claim 109, wherein the amplification assay is a
polymerase chain reaction (PCR).
111. The method of claim 110, wherein the PCR is RT-PCR.
112. The method of claim 1, wherein the method is carried out as a
hybridization assay.
113. The method of claim 112, wherein the hybridization assay is a
Northern blot.
114. The method of claim 1, wherein the sample is a tissue
sample.
115. The method of claim 114, wherein the tissue sample is a tissue
biopsy.
116. The method of claim 1, wherein the sample is a cell
sample.
117. The method of claim 116, wherein the cell sample is a cell
scraping.
118. The method of claim 1, wherein the sample is blood or other
bodily fluid.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of application Ser. No.
09/979,932, filed May 24, 2002, now pending, which is a national
stage filing under 35 U.S.C. .sctn. 371 of PCT International
application PCT/US00/14749, filed May 26, 2000, which was published
under PCT Article 21(2) in English, and claims the benefit under 35
U.S.C. .sctn.119 (e) of United States provisional application
60/136,526, filed May 28, 1999, and of United States provisional
application 60/153,454, filed Sep. 10, 1999.
SEQUENCE LISTING SUBMITTED ON COMPACT DISC
[0002] A Sequence Listing has been submitted on compact disc. Three
discs have been submitted; the material recorded on the compact
discs is identical. Two discs have been labeled Copy 1 and Copy 2,
in compliance with the requirements for submitting a Sequence
Listing on compact disc. The third disc is the computer readable
form of the Sequence Listing. Each disc contains one file:
L0461.70122US01-SEQUENCE LISTING.txt, 11.4 MB in size, created on
Jan. 22, 2007. The Sequence Listing is incorporated by
reference.
FIELD OF THE INVENTION
[0003] The invention relates to nucleic acids and encoded
polypeptides which are cancer associated antigens expressed in
patients afflicted with breast, gastric or prostate cancer. The
invention also relates to agents which bind the nucleic acids or
polypeptides. The nucleic acid molecules, polypeptides coded for by
such molecules and peptides derived therefrom, as well as related
antibodies and cytolytic T lymphocytes, are useful, inter alia, in
diagnostic and therapeutic contexts.
BACKGROUND OF THE INVENTION
[0004] The mechanism by which T cells recognize foreign materials
has been implicated in cancer. A number of cytolytic T lymphocyte
(CTL) clones directed against autologous melanoma antigens,
testicular antigens, and melanocyte differentiation antigens have
been described. In many instances, the antigens recognized by these
clones have been characterized.
[0005] The use of autologous CTLs for identifying tumor antigens
requires that the target cells which express the antigens can be
cultured in vitro and that stable lines of autologous CTL clones
which recognize the antigen-expressing cells can be isolated and
propagated. While this approach has worked well for melanoma
antigens, other tumor types, such as epithelial cancers including
breast and colon cancer, have proved refractory to the
approach.
[0006] More recently another approach to the problem has been
described by Sahin et al. (Proc. Natl. Acad Sci. USA
92:11810-11813, 1995). According to this approach, autologous
antisera are used to identify immunogenic protein antigens
expressed in cancer cells by screening expression libraries
constructed from tumor cell cDNA. Antigen-encoding clones so
identified have been found to elicit a high-titer humoral immune
response in the patients from which the antisera were obtained.
Such a high-titer IgG response implies helper T cell recognition of
the detected antigen. These tumor antigens can then be screened for
the presence of MHC/HLA class I and class II motifs and reactivity
with CTLs.
[0007] Since the individual tumor antigens presently known may be
expressed only in a fraction of tumors, the availability of
additional tumor antigens would significantly enlarge the
proportion of patients who are potentially eligible for therapeutic
interventions. Thus there presently is a need for additional tumor
antigens for development of therapeutics and diagnostics applicable
to a greater number of cancer patients having various cancers.
[0008] The invention is elaborated upon further in the disclosure
which follows.
SUMMARY OF THE INVENTION
[0009] Autologous antibody screening has now been applied to
breast, gastric and prostate cancer using antisera from cancer
patients. Numerous cancer associated antigens have been identified.
The invention provides, inter alia, isolated nucleic acid
molecules, expression vectors containing those molecules and host
cells transfected with those molecules. The invention also provides
isolated proteins and peptides, antibodies to those proteins and
peptides and CTLs which recognize the proteins and peptides.
Fragments including functional fragments and variants of the
foregoing also are provided. Kits containing the foregoing
molecules additionally are provided. The foregoing can be used in
the diagnosis, monitoring, research, or treatment of conditions
characterized by the expression of one or more cancer associated
antigens.
[0010] Prior to the present invention, only a handful of cancer
associated genes had been identified in the past 20 years. The
invention involves the surprising discovery of several genes, some
previously known and some previously unknown, which are expressed
in individuals who have cancer. These individuals all have denim
antibodies against the proteins (or fragments thereof) encoded by
these genes. Thus, abnormally expressed genes are recognized by the
host's immune system and therefore can form a basis for diagnosis,
monitoring and therapy.
[0011] The invention involves the use of a single material, a
plurality of different materials and even large panels and
combinations of materials. For example, a single gene, a single
protein encoded by a gene, a single functional fragment thereof, a
single antibody thereto, etc. can be used in methods and products
of the invention. Likewise, pairs, groups and even panels of these
materials and optionally other cancer associated antigen genes
and/or gene products can be used for diagnosis, monitoring and
therapy. The pairs, groups or panels can involve 2, 3, 4, 5 or more
genes, gene products, fragments thereof or agents that recognize
such materials. A plurality of such materials are not only useful
in monitoring, typing, characterizing and diagnosing cells
abnormally expressing such genes, but a plurality of such materials
can be used therapeutically. An example of the use of a plurality
of such materials for the prevention, delay of onset, amelioration,
etc. of cancer cells, which express or will express such genes
prophylactically or acutely. Any and all combinations of the genes,
gene products, and materials which recognize the genes and gene
products can be tested and identified for use according to the
invention. It would be far too lengthy to recite all such
combinations; those skilled in the art, particularly in view of the
teaching contained herein, will readily be able to determine which
combinations are most appropriate for which circumstances.
[0012] As will be clear from the following discussion, the
invention has in vivo and in vitro uses, including for therapeutic,
diagnostic, monitoring and research purposes. One aspect of the
invention is the ability to fingerprint a cell expressing a number
of the genes identified according to the invention by, for example,
quantifying the expression of such gene products. Such fingerprints
will be characteristic, for example, of the stage of the cancer,
the type of the cancer, or even the effect in animal models of a
therapy on a cancer. Cells also can be screened to determine
whether such cells abnormally express the genes identified
according to the invention.
[0013] The invention, in one aspect, is a method of diagnosing a
disorder characterized by expression of a cancer associated antigen
precursor coded for by a nucleic acid molecule. The method involves
the steps of contacting a biological sample isolated from a subject
with an agent that specifically binds to the nucleic acid molecule,
an expression product thereof, or a fragment of an expression
product thereof complexed with an MHC, preferably an HLA, molecule,
wherein the nucleic acid molecule is a NA Group 1 nucleic acid
molecule, and determining the interaction between the agent and the
nucleic acid molecule, the expression product or fragment of the
expression product as a determination of the disorder.
[0014] In one embodiment the agent is selected from the group
consisting of (a) a nucleic acid molecule comprising NA Group 1
nucleic acid molecules or a fragment thereof, (b) a nucleic acid
molecule comprising NA Group 3 nucleic acid molecules or a fragment
thereof, (c) a nucleic acid molecule comprising NA Group 5 nucleic
acid molecules or a fragment thereof, (d) an antibody that binds to
an expression product, or a fragment thereof, of NA group 1 nucleic
acids, (e) an antibody that binds to an expression product, or a
fragment thereof, of NA group 3 nucleic acids, (f) an antibody that
binds to an expression product, or a fragment thereof, of NA group
5 nucleic acids, (g) and agent that binds to a complex of an MHC,
preferably HLA, molecule and a fragment of an expression product of
a NA Group 1 nucleic acid, (h) an agent that binds to a complex of
an MHC, preferably HLA, molecule and a fragment of an expression
product of a NA group 3 nucleic acid, and (i) an agent that binds
to a complex of an MHC, preferably HLA, molecule and a fragment of
an expression product of a NA Group 5 nucleic acid.
[0015] The disorder may be characterized by expression of a
plurality of cancer associated antigen precursors. Thus the methods
of diagnosis may include use of a plurality of agents, each of
which is specific for a different human cancer associated antigen
precursor (including at least one of the cancer associated antigen
precursors disclosed herein), and wherein said plurality of agents
is at least 2, at least 3, at least 4, at least 5, at least 6, at
least 7, at least 8, at least 9 or at least 10 such agents. Any of
the diagnostic methods disclosed herein can be applied sequentially
over time to permit determination of the prognosis or progression
(or regression) of the disorder.
[0016] In each of the above embodiments the agent may be specific
for a human cancer associated antigen precursor, including the
breast, gastric and prostate cancer associated antigen precursors
disclosed herein.
[0017] In another aspect the invention is a method for determining
regression, progression or onset of a condition characterized by
expression of abnormal levels of a protein encoded by a nucleic
acid molecule that is a NA Group 1 molecule. The method involves
the steps of monitoring a sample, from a subject who has or is
suspected of having the condition, for a parameter selected from
the group consisting of (i) the protein, (ii) a peptide derived
from the protein, (iii) an antibody which selectively binds the
protein or peptide, and (iv) cytolytic T cells specific for a
complex of the peptide derived from the protein and an MHC
molecule, as a determination of regression, progression or onset of
said condition. In one embodiment the sample is a body fluid, a
body effusion or a tissue.
[0018] In another embodiment the step of monitoring comprises
contacting the sample with a detectable agent selected from the
group consisting of (a) an antibody which selectively binds the
protein of (i), or the peptide of (ii), (b) a protein or peptide
which binds the antibody of (iii), and (c) a cell which presents
the complex of the peptide and MHC molecule of (iv). In a preferred
embodiment the antibody, the protein, the peptide or the cell is
labeled with a radioactive label or an enzyme. The sample in a
preferred embodiment is assayed for the peptide. Preferably samples
are isolated from tissue or bodily fluids of the subject at
sequential time points, and the samples are assayed as a
determination of the regression, progression or onset of the
condition from a first sequential time point to a second sequential
time point.
[0019] According to another embodiment the nucleic acid molecule is
one of the following: a NA Group 3 molecule or a NA Group 5
molecule. In yet another embodiment the protein is a plurality of
proteins, the parameter is a plurality of parameters, each of the
plurality of parameters being specific for a different one of the
plurality of proteins.
[0020] The invention in another aspect is a pharmaceutical
preparation for a human subject. The pharmaceutical preparation
includes an agent which when administered to the subject enriches
selectively the presence of complexes of an HLA molecule and a
human cancer associated antigen, and a pharmaceutically acceptable
carrier, wherein the human cancer associated antigen is a fragment
of a human cancer associated antigen precursor encoded by a nucleic
acid molecule which comprises a NA Group 1 molecule. In one
embodiment the nucleic acid molecule is a NA Group 3 nucleic acid
molecule.
[0021] The agent in one embodiment comprises a plurality of agents,
each of which enriches selectively in the subject complexes of an
HLA molecule and a different human cancer associated antigen.
Preferably the plurality is at least two, at least three, at least
four or at least 5 different such agents.
[0022] In another embodiment the agent is selected from the group
consisting of (1) an isolated polypeptide comprising the human
cancer associated antigen, or a functional variant thereof, (2) an
isolated nucleic acid operably linked to a promoter for expressing
the isolated polypeptide, or functional variant thereof, (3) a host
cell expressing the isolated polypeptide, or functional variant
thereof, and (4) isolated complexes of the polypeptide, or
functional variants thereof, and an HLA molecule.
[0023] The agent may be a cell expressing an isolated polypeptide.
In one embodiment the agent is a cell expressing an isolated
polypeptide comprising the human cancer associated antigen or a
functional variant thereof. In another embodiment the agent is a
cell expressing an isolated polypeptide comprising the human cancer
associated antigen or a functional variant thereof, and wherein the
cell expresses an HLA molecule that binds the polypeptide. The cell
can express one or both of the polypeptide and HLA molecule
recombinantly. In preferred embodiments the cell is
nonproliferative. In yet another embodiment the agent is at least
two, at least three, at least four or at least five different
polypeptides, each representing a different human cancer associated
antigen or functional variant thereof.
[0024] The agent in one embodiment is a PP Group 2 polypeptide. In
other embodiments the agent is a PP Group 3 polypeptide or a PP
Group 4 polypeptide.
[0025] In an embodiment each of the pharmaceutical preparations
described herein also includes an adjuvant.
[0026] According to another aspect the invention, a composition is
provided which includes an isolated agent that binds selectively a
PP Group 1 polypeptide. In separate embodiments the agent binds
selectively to a polypeptide selected from the following: a PP
Group 2 polypeptide, a PP Group 3 polypeptide, a PP Group 4
polypeptide, and a PP Group 5 polypeptide. In other embodiments,
the agent is a plurality of different agents that bind selectively
at least two, at least three, at least four, or at least five
different such polypeptides. In each of the above described
embodiments the agent may be an antibody.
[0027] In another aspect the invention is a composition of matter
composed of a conjugate of the agent of the above-described
compositions of the invention and a therapeutic or diagnostic
agent. Preferably the conjugate is of the agent and a therapeutic
or diagnostic that is an antineoplastic.
[0028] The invention in another aspect is a pharmaceutical
composition which includes an isolated nucleic acid molecule
selected from the group consisting of: (1) NA Group 1 molecules,
and (2) NA Group 2 molecules, and a pharmaceutically acceptable
carrier. In one embodiment the isolated nucleic acid molecule
comprises a NA Group 3 or NA Group 4 molecule. In another
embodiment the isolated nucleic acid molecule comprises at least
two isolated nucleic acid molecules coding for two different
polypeptides, each polypeptide comprising a different cancer
associated antigen.
[0029] Preferably the pharmaceutical composition also includes an
expression vector with a promoter operably linked to the isolated
nucleic acid molecule. In another embodiment the pharmaceutical
composition also includes a host cell recombinantly expressing the
isolated nucleic acid molecule.
[0030] According to another aspect of the invention a
pharmaceutical composition is provided. The pharmaceutical
composition includes an isolated polypeptide comprising a PP Group
1 or a PP Group 2 polypeptide, and a pharmaceutically acceptable
carrier. In one embodiment the isolated polypeptide comprises a PP
Group 3 or a PP Group 4 polypeptide.
[0031] In another embodiment the isolated polypeptide comprises at
least two different polypeptides, each comprising a different
cancer associated antigen at least one of which is encoded by a NA
group 1 molecule as disclosed herein. In separate embodiments the
isolated polypeptides are selected from the following: breast
cancer polypeptides or HLA binding fragments thereof and gastric
cancer polypeptides or HLA binding fragments thereof.
[0032] In an embodiment each of the pharmaceutical compositions
described herein also includes an adjuvant.
[0033] Another aspect the invention is an isolated nucleic acid
molecule comprising a NA Group 3 molecule. Another aspect the
invention is an isolated nucleic acid molecule comprising a NA
Group 4 molecule.
[0034] The invention in another aspect is an isolated nucleic acid
molecule selected from the group consisting of (a) a fragment of a
nucleic acid selected from the group of nucleic acid molecules
consisting of SEQ ID Nos: 1-593, of sufficient length to represent
a sequence unique within the human genome, and identifying a
nucleic acid encoding a human cancer associated antigen precursor,
(b) complements of (a), provided that the fragment includes a
sequence of contiguous nucleotides which is not identical to any
sequence selected from the sequence group consisting of (1)
sequences having the GenBank accession numbers of Table 1 and other
sequences publicly available as of the filing date of this
application, (2) complements of (1), and (3) fragments of (1) and
(2). Preferably the unique fragments are fragments of a nucleic
acid selected from the group of nucleic acid molecules consisting
of SEQ ID NOs:12, 15, 34-59, 61, 62, 83-95, 186, 190-205, 297,
327-332, and 335-352.
[0035] In one embodiment the sequence of contiguous nucleotides is
selected from the group consisting of: (1) at least two contiguous
nucleotides nonidentical to the sequences in Table 1, (2) at least
three contiguous nucleotides nonidentical to the sequences in Table
1, (3) at least four contiguous nucleotides nonidentical to the
sequences in Table 1, (4) at least five contiguous nucleotides
nonidentical to the sequences in Table 1, (5) at least six
contiguous nucleotides nonidentical to the sequences in Table 1, or
(6) at least seven contiguous nucleotides nonidentical to the
sequences in Table 1.
[0036] In another embodiment the fragment has a size selected from
the group consisting of at least: 8 nucleotides, 10 nucleotides, 12
nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20,
nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28
nucleotides, 30 nucleotides, 50 nucleotides, 75 nucleotides, 100
nucleotides, 200 nucleotides, 1000 nucleotides and every integer
length therebetween.
[0037] In yet another embodiment the molecule encodes a polypeptide
which, or a fragment of which, binds a human HLA receptor (e.g.,
class I or class II) or a human antibody.
[0038] Another aspect of the invention is an expression vector
comprising an isolated nucleic acid molecule of the invention
described above operably linked to a promoter.
[0039] According to one aspect the invention is an expression
vector comprising a nucleic acid operably linked to a promoter,
wherein the nucleic acid is a NA Group 1 or Group 2 molecule. In
another aspect the invention is an expression vector comprising a
NA Group 1 or Group 2 molecule and a nucleic acid encoding an MHC,
preferably HLA, molecule.
[0040] In yet another aspect the invention is a host cell
transformed or transfected with an expression vector of the
invention described above.
[0041] In another aspect the invention is a host cell transformed
or transfected with an expression vector comprising an isolated
nucleic acid molecule of the invention described above operably
linked to a promoter, or an expression vector comprising a nucleic
acid operably linked to a promoter, wherein the nucleic acid is a
NA Group 1 or 2 molecule and further comprising a nucleic acid
encoding HLA.
[0042] According to another aspect of the invention an isolated
polypeptide encoded by the isolated nucleic acid molecules of the
invention, described above, is provided. These include PP Group 1-5
polypeptides. The invention also includes a fragment of the
polypeptide which is immunogenic. In one embodiment the fragment,
or a portion of the fragment, binds HLA or a human antibody.
[0043] The invention includes in another aspect an isolated
fragment of a human cancer associated antigen precursor which, or a
portion of which, binds HLA or a human antibody, wherein the
precursor is encoded by a nucleic acid molecule that is a NA Group
1 molecule. In one embodiment the fragment is part of a complex
with HLA. In another embodiment the fragment is between 8 and 12
amino acids in length. In another embodiment the invention includes
an isolated polypeptide comprising a fragment of the polypeptide of
sufficient length to represent a sequence unique within the human
genome and identifying a polypeptide that is a human cancer
associated antigen precursor.
[0044] According to another aspect of the invention a kit for
detecting the presence of the expression of a cancer associated
antigen precursor is provided. The kit includes a pair of isolated
nucleic acid molecules each of which consists essentially of a
molecule selected from the group consisting of (a) a 12-32
nucleotide contiguous segment of the nucleotide sequence of any of
the NA Group 1 molecules and (b) complements of (a), wherein the
contiguous segments are nonoverlapping. In one embodiment the pair
of isolated nucleic acid molecules is constructed and arranged to
selectively amplify an isolated nucleic acid molecule that is a NA
Group 3 molecule. Preferably, the pair amplifies a human NA Group 3
molecule.
[0045] According to another aspect of the invention a method for
treating a subject with a disorder characterized by expression of a
human cancer associated antigen precursor is provided. The method
includes the step of administering to the subject an amount of an
agent, which enriches selectively in the subject the presence of
complexes of an HLA molecule and a human cancer associated antigen,
effective to ameliorate the disorder, wherein the human cancer
associated antigen is a fragment of a human cancer associated
antigen precursor encoded by a nucleic acid molecule selected from
the group consisting of (a) a nucleic acid molecule comprising NA
group 1 nucleic acid molecules, (b) a nucleic acid molecule
comprising NA group 3 nucleic acid molecules, (c) a nucleic acid
molecule comprising NA group 5 nucleic acid molecules.
[0046] In one embodiment the disorder is characterized by
expression of a plurality of human cancer associated antigen
precursors and wherein the agent is a plurality of agents, each of
which enriches selectively in the subject the presence of complexes
of an HLA molecule and a different human cancer associated antigen.
Preferably the plurality is at least 2, at least 3, at least 4, or
at least 5 such agents.
[0047] In another embodiment the agent is an isolated polypeptide
selected from the group consisting of PP Group 1, PP Group 2, PP
Group 3, PP Group 4, and PP group 5 polypeptides.
[0048] In yet another embodiment the disorder is cancer.
[0049] According to another aspect the invention is a method for
treating a subject having a condition characterized by expression
of a cancer associated antigen precursor in cells of the subject.
The method includes the steps of (i) removing an immunoreactive
cell containing sample from the subject, (ii) contacting the
immunoreactive cell containing sample to the host cell under
conditions favoring production of cytolytic T cells against a human
cancer associated antigen which is a fragment of the precursor,
(iii) introducing the cytolytic T cells to the subject in an amount
effective to lyse cells which express the human cancer associated
antigen, wherein the host cell is transformed or transfected with
an expression vector comprising an isolated nucleic acid molecule
operably linked to a promoter, the isolated nucleic acid molecule
being selected from the group of nucleic acid molecules consisting
of NA Group 1, NA Group 2, NA Group 3, NA Group 4, NA Group 5.
[0050] In one embodiment the host cell recombinantly expresses an
HLA molecule which binds the human cancer associated antigen. In
another embodiment the host cell endogenously expresses an HLA
molecule which binds the human cancer associated antigen.
[0051] The invention includes in another aspect a method for
treating a subject having a condition characterized by expression
of a cancer associated antigen precursor in cells of the subject.
The method includes the steps of (i) identifying a nucleic acid
molecule expressed by the cells associated with said condition,
wherein said nucleic acid molecule is a NA Group 1 molecule (ii)
transfecting a host cell with a nucleic acid molecule selected from
the group consisting of (a) the nucleic acid molecule identified,
(b) a fragment of the nucleic acid molecule identified which
includes a segment coding for a cancer associated antigen, (c)
deletions, substitutions or additions to (a) or (b), and (d)
degenerates of (a), (b), or (c); (iii) culturing said transfected
host cells to express the transfected nucleic acid molecule, and;
(iv) introducing an amount of said host cells or an extract thereof
to the subject effective to increase an immune response against the
cells of the subject associated with the condition. Preferably, the
antigen is a human antigen and the subject is a human.
[0052] In one embodiment the method also includes the step of (a)
identifying an MHC molecule which presents a portion of an
expression product of the nucleic acid molecule, wherein the host
cell expresses the same MHC molecule as identified in (a) and
wherein the host cell presents an MHC binding portion of the
expression product of the nucleic acid molecule.
[0053] In another embodiment the method also includes the step of
treating the host cells to render them non-proliferative.
[0054] In yet another embodiment the immune response comprises a
B-cell response or a T cell response. Preferably the response is a
T-cell response which comprises generation of cytolytic T-cells
specific for the host cells presenting the portion of the
expression product of the nucleic acid molecule or cells of the
subject expressing the human cancer associated antigen.
[0055] In another embodiment the nucleic acid molecule is a NA
Group 3 molecule.
[0056] Another aspect of the invention is a method for treating or
diagnosing or monitoring a subject having a condition characterized
by expression of an abnormal amount of a protein encoded by a
nucleic acid molecule that is a NA Group 1 molecule. The method
includes the step of administering to the subject an antibody which
specifically binds to the protein or a peptide derived therefrom,
the antibody being coupled to a therapeutically useful agent, in an
amount effective to treat the condition.
[0057] In one embodiment the antibody is a monoclonal antibody.
Preferably the monoclonal antibody is a chimeric antibody or a
humanized antibody.
[0058] In another aspect the invention is a method for treating a
condition characterized by expression in a subject of abnormal
amounts of a protein encoded by a nucleic acid molecule that is a
NA Group 1 nucleic acid molecule. The method involves the step of
administering to a subject at least one of the pharmaceutical
compositions of the invention described above in an amount
effective to prevent, delay the onset of, or inhibit the condition
in the subject. In one embodiment the condition is cancer. In
another embodiment the method includes the step of first
identifying that the subject expresses in a tissue abnormal amounts
of the protein.
[0059] The invention in another aspect is a method for treating a
subject having a condition characterized by expression of abnormal
amounts of a protein encoded by a nucleic acid molecule that is a
NA Group 1 nucleic acid molecule. The method includes the steps of
(i) identifying cells from the subject which express abnormal
amounts of the protein; (ii) isolating a sample of the cells; (iii)
cultivating the cells, and (iv) introducing the cells to the
subject in an amount effective to provoke an immune response
against the cells.
[0060] In one embodiment the method includes the step of rendering
the cells non-proliferative, prior to introducing them to the
subject.
[0061] In another aspect the invention is a method for treating a
pathological cell condition characterized by abnormal expression of
a protein encoded by a nucleic acid molecule that is a NA Group 1
nucleic acid molecule. The method includes the step of
administering to a subject in need thereof an effective amount of
an agent which inhibits the expression or activity of the
protein.
[0062] In one embodiment the agent is an inhibiting antibody which
selectively binds to the protein and wherein the antibody is a
monoclonal antibody, a chimeric antibody, a humanized antibody or a
fragment thereof. In another embodiment the agent is an antisense
nucleic acid molecule which selectively binds to the nucleic acid
molecule which encodes the protein. In yet another important
embodiment the nucleic acid molecule is a NA Group 3 nucleic acid
molecule.
[0063] The invention includes in another aspect a composition of
matter useful in stimulating an immune response to a plurality of
proteins encoded by nucleic acid molecules that are NA Group 1
molecules. The composition is a plurality of peptides derived from
the amino acid sequences of the proteins, wherein the peptides bind
to one or more MHC molecules presented on the surface of the cells
which express an abnormal amount of the protein.
[0064] In one embodiment at least a portion of the plurality of
peptides bind to MHC molecules and elicit a cytolytic response
thereto. In another embodiment the composition of matter includes
an adjuvant. In another embodiment the adjuvant is a saponin,
GM-CSF, or an interleukin. In still another embodiment, the
compositions also includes at least one peptide useful in
stimulating an immune response to at least one protein which is not
encoded by nucleic acid molecules that are NA Group 1 molecules,
wherein the at least one peptide binds to one or more MHC
molecules.
[0065] According to another aspect the invention is an isolated
antibody which selectively binds to a complex of: (i) a peptide
derived from a protein encoded by a nucleic acid molecule that is a
NA Group 1 molecule and (ii) and an MHC molecule to which binds the
peptide to form the complex, wherein the isolated antibody does not
bind to (i) or (ii) alone.
[0066] In one embodiment the antibody is a monoclonal antibody, a
chimeric antibody, a humanized antibody or a fragment thereof.
[0067] The invention also involves the use of the genes, gene
products, fragments thereof, agents which bind thereto, and so on
in the preparation of medicaments. A particular medicament is for
treating cancers including, e.g., one or more of cancers of the
breast, cervix, ovary, prostate, testis, lung, colon, pancreas,
stomach, liver, skin (e.g., melanoma), bladder, head and neck,
thyroid, blood cells, bone and kidney. Diagnostics for specific
cancers and groups of cancers also are envisioned.
[0068] In certain preferred embodiments, the nucleic acid molecules
are selected from the group consisting of SEQ ID NOs:1-18, and the
polypeptides are encoded by these preferred nucleic acid
molecules.
[0069] Still other embodiments and aspects of the invention will
become apparent in connection with the description of the invention
which follows.
DETAILED DESCRIPTION OF THE INVENTION
[0070] In the above summary and in the ensuing description, lists
of sequences are provided. The lists are meant to embrace each
single sequence separately, two or more sequences together where
they form a part of the same gene, any combination of two or more
sequences which relate to different genes, including and up to the
total number on the list, as if each and every combination were
separately and specifically enumerated. Likewise, when mentioning
fragment size, it is intended that a range embrace the smallest
fragment mentioned to the full-length of the sequence (less one
nucleotide or amino acid so that it is a fragment), each and every
fragment length intended as if specifically enumerated. Thus, if a
fragment could be between 10 and 15 in length, it is explicitly
meant to mean 10, 11, 12, 13, 14, or 15 in length.
[0071] The summary and the claims mention antigen precursors and
antigens. As used in the summary and in the claims, a precursor is
substantially the full-length protein encoded by the coding region
of the isolated DNA and the antigen is a peptide which complexes
with MHC, preferably HLA, and which participates in the immune
response as part of that complex. Such antigens are typically 9
amino acids long, although this may vary slightly.
[0072] As used herein, a subject is a human, non-human primate,
cow, horse, pig, sheep, goat, dog, cat or rodent. In all
embodiments human cancer antigens and human subjects are
preferred.
[0073] The present invention in one aspect involves the cloning of
cDNAs encoding human cancer associated antigen precursors using
autologous antisera of subjects having breast, gastric or prostate
cancer. The sequences of the clones representing genes identified
according to the methods described herein are presented in the
attached Sequence Listing. Of the foregoing, it can be seen that
some of the clones are considered completely novel as no coding
regions were found in the databases searched. Other clones are
novel but have some nucleotide or amino acid homologies to
sequences deposited in databases (mainly EST sequences).
Nevertheless, the entire gene sequence was not previously known. In
some cases no function was suspected and in other cases, even if a
function was suspected, it was not known that the gene was
associated with cancer, or with a particular cancer. In all cases,
it was not known or suspected that the gene encoded a cancer
antigen which reacted with an antibody from autologous sera.
Analysis of the clone sequences by comparison to nucleic acid and
protein databases determined that still other of the clones
surprisingly are closely related to other previously-cloned genes.
The sequences of these related genes is also presented in the
Sequence Listing. The nature of the foregoing genes as encoding
antigens recognized by the immune systems of cancer patients is, of
course, unexpected.
[0074] The invention thus involves in one aspect cancer associated
antigen polypeptides, genes encoding those polypeptides, functional
modifications and variants of the foregoing, useful fragments of
the foregoing, as well as diagnostics and therapeutics relating
thereto.
[0075] Homologs and alleles of the cancer associated antigen
nucleic acids of the invention can be identified by conventional
techniques. Thus, an aspect of the invention is those nucleic acid
sequences which code for cancer associated antigen precursors.
Because this application contains so many sequences, the following
chart is provided to identify the various groups of sequences
discussed in the claims and in the summary:
Nucleic Acid Sequences
[0076] NA Group 1. (a) nucleic acid molecules which hybridize under
stringent conditions to a molecule consisting of a nucleic acid
sequence selected from the group consisting of nucleic acid
sequences among SEQ ID NOs: 1-593, and which code for a cancer
associated antigen precursor, [0077] (b) deletions, additions and
substitutions which code for a respective cancer associated antigen
precursor, [0078] (c) nucleic acid molecules that differ from the
nucleic acid molecules of (a) or (b) in codon sequence due to the
degeneracy of the genetic code, and [0079] (d) complements of (a),
(b) or (c). [0080] NA Group 2. Fragments of NA Group 1, which code
for a polypeptide which, or a portion of which, binds an MHC
molecule to form a complex recognized by an autologous antibody or
lymphocyte. [0081] NA Group 3. The subset of NA Group 1 where the
nucleotide sequence is selected from the group consisting of:
[0082] (a) previously unknown human nucleic acids coding for a
human cancer associated antigen precursor, e.g., SEQ ID NOs:12, 15,
34-59, 61, 62, 83-95, 186, 190-205, 297, 327-332, and 335-352,
[0083] (b) deletions, additions and substitutions which code for a
respective human cancer associated antigen precursor, [0084] (c)
nucleic acid molecules that differ from the nucleic acid molecules
of (a) or (b) in codon sequence due to the degeneracy of the
genetic code, and [0085] (d) complements of (a), (b) or (c). [0086]
NA Group 4. Fragments of NA Group 3, which code for a polypeptide
which, or a portion of which, binds to an MHC molecule to form a
complex recognized by an autologous antibody or lymphocyte. [0087]
NA Group 5. A subset of NA Group 1, comprising human cancer
associated antigens that react with allogeneic cancer antisera.
Polypeptide Sequences
[0087] [0088] PP Group 1. Polypeptides encoded by NA Group 1.
[0089] PP Group 2. Polypeptides encoded by NA Group 2. [0090] PP
Group 3. Polypeptides encoded by NA Group 3. [0091] PP Group 4.
Polypeptides encoded by NA Group 4. [0092] PP Group 5. Polypeptides
encoded by NA Group 5.
[0093] Particularly preferred polypeptides are those recognized by
allogeneic sera of cancer patients, but not by non-cancer patient
control sera. For example, as shown in the Examples below,
polypeptides encoded by SEQ ID NOs:1-18 are recognized only by
antibodies in cancer patients antisera.
[0094] The term "stringent conditions" as used herein refers to
parameters with which the art is familiar. Nucleic acid
hybridization parameters may be found in references which compile
such methods, e.g. Molecular Cloning: A Laboratory Manual, J.
Sambrook, et al., eds., Second Edition, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, New York, 1989, or Current
Protocols in Molecular Biology, F. M. Ausubel, et al., eds., John
Wiley & Sons, Inc., New York. More specifically, stringent
conditions, as used herein, refers, for example, to hybridization
at 65.degree. C. in hybridization buffer (3.5.times.SSC, 0.02%
Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin,
2.5 mM NaH.sub.2PO.sub.4(pH7), 0.5% SDS, 2 mM EDTA). SSC is 0.15 M
sodium chloride/0.15 M sodium citrate, pH7; SDS is sodium dodecyl
sulphate; and EDTA is ethylenediaminetetracetic acid. After
hybridization, the membrane upon which the DNA is transferred is
washed, for example, in 2 x SSC at room temperature and then at
0.1-0.5.times.SSC/0.1.times.SDS at temperatures up to 68.degree.
C.
[0095] There are other conditions, reagents, and so forth which can
be used, which result in a similar degree of stringency. The
skilled artisan will be familiar with such conditions, and thus
they are not given here. It will be understood, however, that the
skilled artisan will be able to manipulate the conditions in a
manner to permit the clear identification of homologs and alleles
of cancer associated antigen nucleic acids of the invention (e.g.,
by using lower stringency conditions). The skilled artisan also is
familiar with the methodology for screening cells and libraries for
expression of such molecules which then are routinely isolated,
followed by isolation of the pertinent nucleic acid molecule and
sequencing.
[0096] In general homologs and alleles typically will share at
least 80% nucleotide identity and/or at least 90% amino acid
identity to the sequences of cancer associated antigen nucleic acid
and polypeptides, respectively, in some instances will share at
least 90% nucleotide identity and/or at least 95% amino acid
identity and in still other instances will share at least 95%
nucleotide identity and/or at least 99% amino acid identity. The
homology can be calculated using various, publicly available
software tools developed by NCBI (Bethesda, Maryland) that can be
obtained through the Internet (ncbi.nlm.nih.gov/pub/). Exemplary
tools include the BLAST system available at ncbi.nlm.nih.gov,
preferably using default settings. Pairwise and ClustalW alignments
(BLOSUM30 matrix setting) as well as Kyle-Doolittle hydropathic
analysis can be obtained using the MacVector sequence analysis
software (Oxford Molecular Group). Watson-Crick complements of the
foregoing nucleic acids also are embraced by the invention.
[0097] In screening for cancer associated antigen genes, a Southern
blot may be performed using the foregoing conditions, together with
a radioactive probe. After washing the membrane to which the DNA is
finally transferred, the membrane can be placed against X-ray film
to detect the radioactive signal. In screening for the expression
of cancer associated antigen nucleic acids, Northern blot
hybridizations using the foregoing conditions can be performed on
samples taken from breast, gastric or prostate cancer patients or
subjects suspected of having a condition characterized by
expression of the cancer associated antigen genes disclosed herein.
Amplification protocols such as polymerase chain reaction using
primers which hybridize to the sequences presented also can be used
for detection of the cancer associated antigen genes or expression
thereof.
[0098] The breast, gastric and prostate cancer associated genes
correspond to SEQ ID Nos:1-593. These sequences represent genes
previously known in humans and genes previously unknown in humans
(e.g., SEQ ID NOs:12, 15, 34-59, 61, 62, 83-95, 186, 190-205, 297,
327-332, and 335-352). Preferred breast, gastric and prostate
cancer associated antigens for the methods of diagnosis disclosed
herein are those which encode polypeptides that react with
allogeneic cancer antisera (i.e. NA Group 5). Encoded polypeptides
(e.g., proteins), peptides and antisera thereto are also preferred
for diagnosis.
[0099] As used herein with respect to nucleic acids, the term
"isolated" means: (i) amplified in vitro by, for example,
polymerase chain reaction (PCR); (ii) recombinantly produced by
cloning; (iii) purified, as by cleavage and gel separation; or (iv)
synthesized by, for example, chemical synthesis. An isolated
nucleic acid is one which is readily manipulable by recombinant DNA
techniques well known in the art. Thus, a nucleotide sequence
contained in a vector in which 5' and 3' restriction sites are
known or for which polymerase chain reaction (PCR) primer sequences
have been disclosed is considered isolated but a nucleic acid
sequence existing in its native state in its natural host is not.
An isolated nucleic acid may be substantially purified, but need
not be. For example, a nucleic acid that is isolated within a
cloning or expression vector is not pure in that it may comprise
only a tiny percentage of the material in the cell in which it
resides. Such a nucleic acid is isolated, however, as the term is
used herein because it is readily manipulable by standard
techniques known to those of ordinary skill in the art. An isolated
nucleic acid as used herein is not a naturally occurring
chromosome.
[0100] As used herein with respect to polypeptides, "isolated"
means separated from its native environment and present in
sufficient quantity to permit its identification or use. Isolated,
when referring to a protein or polypeptide, means, for example: (i)
selectively produced by expression cloning or (ii) purified as by
chromatography or electrophoresis. Isolated proteins or
polypeptides may be, but need not be, substantially pure. The term
"substantially pure" means that the proteins or polypeptides are
essentially free of other substances with which they may be found
in nature or in vivo systems to an extent practical and appropriate
for their intended use. Substantially pure polypeptides may be
produced by techniques well known in the art. Because an isolated
protein may be admixed with a pharmaceutically acceptable carrier
in a pharmaceutical preparation, the protein may comprise only a
small percentage by weight of the preparation. The protein is
nonetheless isolated in that it has been separated from the
substances with which it may be associated in living systems, i.e.
isolated from other proteins.
[0101] The invention also includes degenerate nucleic acids which
include alternative codons to those present in the native
materials. For example, serine residues are encoded by the codons
TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is
equivalent for the purposes of encoding a serine residue. Thus, it
will be apparent to one of ordinary skill in the art that any of
the serine-encoding nucleotide triplets may be employed to direct
the protein synthesis apparatus, in vitro or in vivo, to
incorporate a serine residue into an elongating cancer associated
antigen polypeptide. Similarly, nucleotide sequence triplets which
encode other amino acid residues include, but are not limited to:
CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and
AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons);
AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine
codons). Other amino acid residues may be encoded similarly by
multiple nucleotide sequences. Thus, the invention embraces
degenerate nucleic acids that differ from the biologically isolated
nucleic acids in codon sequence due to the degeneracy of the
genetic code.
[0102] The invention also provides modified nucleic acid molecules
which include additions, substitutions and deletions of one or more
nucleotides. In preferred embodiments, these modified nucleic acid
molecules and/or the polypeptides they encode retain at least one
activity or function of the unmodified nucleic acid molecule and/or
the polypeptides, such as antigenicity, enzymatic activity,
receptor binding, formation of complexes by binding of peptides by
MHC class I and class II molecules, etc. In certain embodiments,
the modified nucleic acid molecules encode modified polypeptides,
preferably polypeptides having conservative amino acid
substitutions as are described elsewhere herein. The modified
nucleic acid molecules are structurally related to the unmodified
nucleic acid molecules and in preferred embodiments are
sufficiently structurally related to the unmodified nucleic acid
molecules so that the modified and unmodified nucleic acid
molecules hybridize under stringent conditions known to one of
skill in the art.
[0103] For example, modified nucleic acid molecules which encode
polypeptides having single amino acid changes can be prepared. Each
of these nucleic acid molecules can have one, two or three
nucleotide substitutions exclusive of nucleotide changes
corresponding to the degeneracy of the genetic code as described
herein. Likewise, modified nucleic acid molecules which encode
polypeptides having two amino acid changes can be prepared which
have, e.g., 2-6 nucleotide changes. Numerous modified nucleic acid
molecules like these will be readily envisioned by one of skill in
the art, including for example, substitutions of nucleotides in
codons encoding amino acids 2 and 3, 2 and 4, 2 and 5, 2 and 6, and
so on. In the foregoing example, each combination of two amino
acids is included in the set of modified nucleic acid molecules, as
well as all nucleotide substitutions which code for the amino acid
substitutions. Additional nucleic acid molecules that encode
polypeptides having additional substitutions (i.e., 3 or more),
additions or deletions (e.g., by introduction of a stop codon or a
splice site(s)) also can be prepared and are embraced by the
invention as readily envisioned by one of ordinary skill in the
art. Any of the foregoing nucleic acids or polypeptides can be
tested by routine experimentation for retention of structural
relation or activity to the nucleic acids and/or polypeptides
disclosed herein.
[0104] The invention also provides isolated unique fragments of
cancer associated antigen nucleic acid sequences or complements
thereof. A unique fragment is one that is a `signature` for the
larger nucleic acid. It, for example, is long enough to assure that
its precise sequence is not found in molecules within the human
genome outside of the cancer associated antigen nucleic acids
defined above (and human alleles). Those of ordinary skill in the
art may apply no more than routine procedures to determine if a
fragment is unique within the human genome. Unique fragments,
however, exclude fragments completely composed of the nucleotide
sequences of any of the GenBank accession numbers listed in Table 1
or other previously published sequences as of the filing date of
the priority documents for sequences listed in a respective
priority document or the filing date of this application for
sequences listed for the first time in this application which
overlap the sequences of the invention.
[0105] A fragment which is completely composed of the sequence
described in the foregoing GenBank deposits is one which does not
include any of the nucleotides unique to the sequences of the
invention. Thus, a unique fragment must contain a nucleotide
sequence other than the exact sequence of those in GenBank or
fragments thereof. The difference may be an addition, deletion or
substitution with respect to the GenBank sequence or it may be a
sequence wholly separate from the GenBank sequence.
[0106] Unique fragments can be used as probes in Southern and
Northern blot assays to identify such nucleic acids, or can be used
in amplification assays such as those employing PCR. As known to
those skilled in the art, large probes such as 200, 250, 300 or
more nucleotides are preferred for certain uses such as Southern
and Northern blots, while smaller fragments will be preferred for
uses such as PCR. Unique fragments also can be used to produce
fusion proteins for generating antibodies or determining binding of
the polypeptide fragments, or for generating immunoassay
components. Likewise, unique fragments can be employed to produce
nonfused fragments of the cancer associated antigen polypeptides,
useful, for example, in the preparation of antibodies, and in
immunoassays. Unique fragments further can be used as antisense
molecules to inhibit the expression of cancer associated antigen
nucleic acids and polypeptides, particularly for therapeutic
purposes as described in greater detail below. Unique fragments
also can be used to create chimeric nucleic acid molecule or
polypeptide molecules by, for example, joining all or part of the
unique fragment to another nucleic acid or polypeptide molecule
(homologous or not). For example, the unique fragment may be
similar or identical in large part to a known molecule but may have
a portion which is nonidentical to the known molecule; the known
molecule and the unique fragment can be used to construct a
molecule containing in large part the known molecule with the
portion unique to the unique fragment added. Other chimeric
molecules will be known to one of ordinary skill in the art and can
be prepared using standard molecular biology techniques.
[0107] As will be recognized by those skilled in the art, the size
of the unique fragment will depend upon its conservancy in the
genetic code. Thus, some regions of cancer associated antigen
sequences and complements thereof will require longer segments to
be unique while others will require only short segments, typically
between 12 and 32 nucleotides (e.g. 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32 or more bases
long), up to the entire length of the disclosed sequence. As
mentioned above, this disclosure intends to embrace each and every
fragment of each sequence, beginning at the first nucleotide, the
second nucleotide and so on, up to 8 nucleotides short of the end,
and ending anywhere from nucleotide number 8, 9, 10 and so on for
each sequence, up to the very last nucleotide (provided the
sequence is unique as described above).
[0108] Virtually any segment of the polypeptide coding region of
novel cancer associated antigen nucleic acids, or complements
thereof, that is 25 or more nucleotides in length will be unique.
Those skilled in the art are well versed in methods for selecting
such sequences, typically on the basis of the ability of the unique
fragment to selectively distinguish the sequence of interest from
other sequences in the human genome of the fragment to those on
known databases typically is all that is necessary, although in
vitro confirmatory hybridization and sequencing analysis may be
performed.
[0109] Especially preferred include nucleic acids encoding a series
of epitopes, known as "polytopes". The epitopes can be arranged in
sequential or overlapping fashion (see, e.g., Thomson et al., Proc.
Natl. Acad Sci. USA 92:5845-5849, 1995; Gilbert et al., Nature
Biotechnol. 15:1280-1284, 1997), with or without the natural
flanking sequences, and can be separated by unrelated linker
sequences if desired. The polytope is processed to generate
individual epitopes which are recognized by the immune system for
generation of immune responses.
[0110] Thus, for example, peptides derived from a polypeptide
having an amino acid sequence encoded by one of the nucleic acid
disclosed herein, and which are presented by MHC molecules and
recognized by CTL or T helper lymphocytes, can be combined with
peptides from one or more other cancer associated antigens (e.g. by
preparation of hybrid nucleic acids or polypeptides) to form
"polytopes". The two or more peptides (or nucleic acids encoding
the peptides) can be selected from those described herein, or they
can include one or more peptides of previously known cancer
associated antigens. Exemplary cancer associated peptide antigens
that can be administered to induce or enhance an immune response
are derived from tumor associated genes and encoded proteins
including MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6,
MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A12, GAGE-1,
GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8, GAGE-9,
BAGE-1, RAGE-1, LB33/MUM-1, PRAME, NAG, MAGE-B2, MAGE-B3, MAGE-B4,
tyrosinase, brain glycogen phosphorylase, Melan-A, MAGE-C1,
MAGE-C2, MAGE-C3, MAGE-C4, MAGE-C5, NY-ESO-1, LAGE-1, SSX-1, SSX-2
(HOM-MEL-40), SSX-4, SSX-5, SCP-1 and CT-7. See, for example, PCT
application publication no. WO96/10577. Other examples will be
known to one of ordinary skill in the art (for example, see Coulie,
Stem Cells 13:393-403, 1995), and can be used in the invention in a
like manner as those disclosed herein. One of ordinary skill in the
art can prepare polypeptides comprising one or more peptides and
one or more of the foregoing cancer associated peptides, or nucleic
acids encoding such polypeptides, according to standard procedures
of molecular biology.
[0111] Thus polytopes are groups of two or more potentially
immunogenic or immune response stimulating peptides which can be
joined together in various arrangements (e.g. concatenated,
overlapping). The polytope (or nucleic acid encoding the polytope)
can be administered in a standard immunization protocol, e.g. to
animals, to test the effectiveness of the polytope in stimulating,
enhancing and/or provoking an immune response.
[0112] The peptides can be joined together directly or via the use
of flanking sequences to form polytopes, and the use of polytopes
as vaccines is well known in the art (see, e.g., Thomson et al.,
Proc. Acad. Natl. Acad. Sci USA 92(13):5845-5849, 1995; Gilbert et
al., Nature Biotechnol. 15(12):1280-1284, 1997; Thomson et al., J.
Immunol. 157(2):822-826, 1996; Tam et al., J. Exp. Med.
171(1):299-306, 1990). For example, Tam showed that polytopes
consisting of both MHC class I and class II binding epitopes
successfully generated antibody and protective immunity in a mouse
model. Tam also demonstrated that polytopes comprising "strings" of
epitopes are processed to yield individual epitopes which are
presented by MHC molecules and recognized by CTLs. Thus polytopes
containing various numbers and combinations of epitopes can be
prepared and tested for recognition by CTLs and for efficacy in
increasing an immune response.
[0113] It is known that tumors express a set of tumor antigens, of
which only certain subsets may be expressed in the tumor of any
given patient. Polytopes can be prepared which correspond to the
different combination of epitopes representing the subset of tumor
rejection antigens expressed in a particular patient. Polytopes
also can be prepared to reflect a broader spectrum of tumor
rejection antigens known to be expressed by a tumor type. Polytopes
can be introduced to a patient in need of such treatment as
polypeptide structures, or via the use of nucleic acid delivery
systems known in the art (see, e.g., Allsopp et al., Eur. J.
Immunol. 26(8):1951-1959, 1996). Adenovirus, pox virus, Ty-virus
like particles, adeno-associated virus, plasmids, bacteria, etc.
can be used in such delivery. One can test the polytope delivery
systems in mouse models to determine efficacy of the delivery
system. The systems also can be tested in human clinical
trials.
[0114] In instances in which a human HLA class I molecule presents
tumor rejection antigens derived from cancer associated nucleic
acids, the expression vector may also include a nucleic acid
sequence coding for the HLA molecule that presents any particular
tumor rejection antigen derived from these nucleic acids and
polypeptides. Alternatively, the nucleic acid sequence coding for
such a HLA molecule can be contained within a separate expression
vector. In a situation where the vector contains both coding
sequences, the single vector can be used to transfect a cell which
does not normally express either one. Where the coding sequences
for a cancer associated antigen precursor and the HLA molecule
which presents it are contained on separate expression vectors, the
expression vectors can be cotransfected. The cancer associated
antigen precursor coding sequence may be used alone, when, e.g. the
host cell already expresses a HLA molecule which presents a cancer
associated antigen derived from precursor molecules. Of course,
there is no limit on the particular host cell which can be used. As
the vectors which contain the two coding sequences may be used in
any antigen-presenting cells if desired, and the gene for cancer
associated antigen precursor can be used in host cells which do not
express a HLA molecule which presents a cancer associated antigen.
Further, cell-free transcription systems may be used in lieu of
cells.
[0115] As mentioned above, the invention embraces antisense
oligonucleotides that selectively bind to a nucleic acid molecule
encoding a cancer associated antigen polypeptide, to reduce the
expression of cancer associated antigens. This is desirable in
virtually any medical condition wherein a reduction of expression
of cancer associated antigens is desirable, e.g., in the treatment
of cancer. This is also useful for in vitro or in vivo testing of
the effects of a reduction of expression of one or more cancer
associated antigens.
[0116] As used herein, the term "antisense oligonucleotide" or
"antisense" describes an oligonucleotide that is an
oligoribonucleotide, oligodeoxyribonucleotide, modified
oligoribonucleotide, or modified oligodeoxyribonucleotide which
hybridizes under physiological conditions to DNA comprising a
particular gene or to an mRNA transcript of that gene and, thereby,
inhibits the transcription of that gene and/or the translation of
that mRNA. The antisense molecules are designed so as to interfere
with transcription or translation of a target gene upon
hybridization with the target gene or transcript. Those skilled in
the art will recognize that the exact length of the antisense
oligonucleotide and its degree of complementarity with its target
will depend upon the specific target selected, including the
sequence of the target and the particular bases which comprise that
sequence. It is preferred that the antisense oligonucleotide be
constructed and arranged so as to bind selectively with the target
under physiological conditions, i.e., to hybridize substantially
more to the target sequence than to any other sequence in the
target cell under physiological conditions. Based upon the
sequences of nucleic acids encoding breast, gastric or prostate
cancer associated antigens, or upon allelic or homologous genomic
and/or cDNA sequences, one of skill in the art can easily choose
and synthesize any of a number of appropriate antisense molecules
for use in accordance with the present invention. For example, a
"gene walk" comprising a series of oligonucleotides of 15-30
nucleotides spanning the length of a cancer associated antigen can
be prepared, followed by testing for inhibition of cancer
associated antigen expression. Optionally, gaps of 5-10 nucleotides
can be left between the oligonucleotides to reduce the number of
oligonucleotides synthesized and tested.
[0117] In order to be sufficiently selective and potent for
inhibition, such antisense oligonucleotides should comprise at
least 10 and, more preferably, at least 15 consecutive bases which
are complementary to the target, although in certain cases modified
oligonucleotides as short as 7 bases in length have been used
successfully as antisense oligonucleotides (Wagner et al., Nature
Biotechnol. 14:840-844, 1996). Most preferably, the antisense
oligonucleotides comprise a complementary sequence of 20-30 bases.
Although oligonucleotides may be chosen which are antisense to any
region of the gene or mRNA transcripts, in preferred embodiments
the antisense oligonucleotides correspond to N-terminal or 5'
upstream sites such as translation initiation, transcription
initiation or promoter sites. In addition, 3'-untranslated regions
may be targeted. Targeting to mRNA splicing sites has also been
used in the art but may be less preferred if alternative mRNA
splicing occurs. In addition, the antisense is targeted,
preferably, to sites in which mRNA secondary structure is not
expected (see, e.g., Sainio et al., Cell Mol. Neurobiol.
14(5):439-457, 1994) and at which proteins are not expected to
bind. Finally, although the listed sequences are cDNA sequences,
one of ordinary skill in the art may easily derive the genomic DNA
corresponding to the cDNA of a cancer associated antigen. Thus, the
present invention also provides for antisense oligonucleotides
which are complementary to the genomic DNA corresponding to nucleic
acids encoding cancer associated antigens. Similarly, antisense to
allelic or homologous cDNAs and genomic DNAs are enabled without
undue experimentation.
[0118] In one set of embodiments, the antisense oligonucleotides of
the invention may be composed of "natural" deoxyribonucleotides,
ribonucleotides, or any combination thereof. That is, the 5' end of
one native nucleotide and the 3' end of another native nucleotide
may be covalently linked, as in natural systems, via a
phosphodiester intemucleoside linkage. These oligonucleotides may
be prepared by art recognized methods which may be carried out
manually or by an automated synthesizer. They also may be produced
recombinantly by vectors.
[0119] In preferred embodiments, however, the antisense
oligonucleotides of the invention also may include "modified"
oligonucleotides. That is, the oligonucleotides may be modified in
a number of ways which do not prevent them from hybridizing to
their target but which enhance their stability or targeting or
which otherwise enhance their therapeutic effectiveness.
[0120] The term "modified oligonucleotide" as used herein describes
an oligonucleotide in which (1) at least two of its nucleotides are
covalently linked via a synthetic internucleoside linkage (i.e., a
linkage other than a phosphodiester linkage between the 5' end of
one nucleotide and the 3' end of another nucleotide) and/or (2) a
chemical group not normally associated with nucleic acids has been
covalently attached to the oligonucleotide. Preferred synthetic
intemucleoside linkages are phosphorothioates, alkylphosphonates,
phosphorodithioates, phosphate esters, alkylphosphonothioates,
phosphoramidates, carbamates, carbonates, phosphate triesters,
acetamidates, carboxymethyl esters and peptides.
[0121] The term "modified oligonucleotide" also encompasses
oligonucleotides with a covalently modified base and/or sugar. For
example, modified oligonucleotides include oligonucleotides having
backbone sugars which are covalently attached to low molecular
weight organic groups other than a hydroxyl group at the 3'
position and other than a phosphate group at the 5' position. Thus
modified oligonucleotides may include a 2'-O-alkylated ribose
group. In addition, modified oligonucleotides may include sugars
such as arabinose instead of ribose. The present invention, thus,
contemplates pharmaceutical preparations containing modified
antisense molecules that are complementary to and hybridizable
with, under physiological conditions, nucleic acids encoding
breast, gastric or prostate cancer associated antigen polypeptides,
together with pharmaceutically acceptable carriers.
[0122] Antisense oligonucleotides may be administered as part of a
pharmaceutical composition. Such a pharmaceutical composition may
include the antisense oligonucleotides in combination with any
standard physiologically and/or pharmaceutically acceptable
carriers which are known in the art. The compositions should be
sterile and contain a therapeutically effective amount of the
antisense oligonucleotides in a unit of weight or volume suitable
for administration to a patient. The term "pharmaceutically
acceptable" means a non-toxic material that does not interfere with
the effectiveness of the biological activity of the active
ingredients. The term "physiologically acceptable" refers to a
non-toxic material that is compatible with a biological system such
as a cell, cell culture, tissue, or organism. The characteristics
of the carrier will depend on the route of administration.
Physiologically and pharmaceutically acceptable carriers include
diluents, fillers, salts, buffers, stabilizers, solubilizers, and
other materials which are well known in the art, as further
described below.
[0123] As used herein, a "vector" may be any of a number of nucleic
acids into which a desired sequence may be inserted by restriction
and ligation for transport between different genetic environments
or for expression in a host cell. Vectors are typically composed of
DNA although RNA vectors are also available. Vectors include, but
are not limited to, plasmids, phagemids and virus genomes. A
cloning vector is one which is able to replicate autonomously or
integrated in the genome in a host cell, and which is further
characterized by one or more endonuclease restriction sites at
which the vector may be cut in a determinable fashion and into
which a desired DNA sequence may be ligated such that the new
recombinant vector retains its ability to replicate in the host
cell. In the case of plasmids, replication of the desired sequence
may occur many times as the plasmid increases in copy number within
the host bacterium or just a single time per host before the host
reproduces by mitosis. In the case of phage, replication may occur
actively during a lytic phase or passively during a lysogenic
phase. An expression vector is one into which a desired DNA
sequence may be inserted by restriction and ligation such that it
is operably joined to regulatory sequences and may be expressed as
an RNA transcript. Vectors may further contain one or more marker
sequences suitable for use in the identification of cells which
have or have not been transformed or transfected with the vector.
Markers include, for example, genes encoding proteins which
increase or decrease either resistance or sensitivity to
antibiotics or other compounds, genes which encode enzymes whose
activities are detectable by standard assays known in the art
(e.g., .beta.-galactosidase, luciferase or alkaline phosphatase),
and genes which visibly affect the phenotype of transformed or
transfected cells, hosts, colonies or plaques (e.g., green
fluorescent protein). Preferred vectors are those capable of
autonomous replication and expression of the structural gene
products present in the DNA segments to which they are operably
joined.
[0124] As used herein, a coding sequence and regulatory sequences
are said to be "operably" joined when they are covalently linked in
such a way as to place the expression or transcription of the
coding sequence under the influence or control of the regulatory
sequences. If it is desired that the coding sequences be translated
into a functional protein, two DNA sequences are said to be
operably joined if induction of a promoter in the 5' regulatory
sequences results in the transcription of the coding sequence and
if the nature of the linkage between the two DNA sequences does not
(1) result in the introduction of a frame-shift mutation, (2)
interfere with the ability of the promoter region to direct the
transcription of the coding sequences, or (3) interfere with the
ability of the corresponding RNA transcript to be translated into a
protein. Thus, a promoter region would be operably joined to a
coding sequence if the promoter region were capable of effecting
transcription of that DNA sequence such that the resulting
transcript might be translated into the desired protein or
polypeptide.
[0125] The precise nature of the regulatory sequences needed for
gene expression may vary between species or cell types, but shall
in general include, as necessary, 5' non-transcribed and 5'
non-translated sequences involved with the initiation of
transcription and translation respectively, such as a TATA box,
capping sequence, CAAT sequence, and the like. Especially, such 5'
non-transcribed regulatory sequences will include a promoter region
which includes a promoter sequence for transcriptional control of
the operably joined gene. Regulatory sequences may also include
enhancer sequences or upstream activator sequences as desired. The
vectors of the invention may optionally include 5' leader or signal
sequences. The choice and design of an appropriate vector is within
the ability and discretion of one of ordinary skill in the art.
[0126] Expression vectors containing all the necessary elements for
expression are commercially available and known to those skilled in
the art. See, e.g., Sambrook et al., Molecular Cloning: A
Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory
Press, 1989. Cells are genetically engineered by the introduction
into the cells of heterologous DNA (RNA) encoding a cancer
associated antigen polypeptide or fragment or variant thereof. That
heterologous DNA (RNA) is placed under operable control of
transcriptional elements to permit the expression of the
heterologous DNA in the host cell.
[0127] Preferred systems for mRNA expression in mammalian cells are
those such as pRc/CMV (available from Invitrogen, Carlsbad, CA)
that contain a selectable marker such as a gene that confers G418
resistance (which facilitates the selection of stably transfected
cell lines) and the human cytomegalovirus (CMV) enhancer-promoter
sequences. Additionally, suitable for expression in primate or
canine cell lines is the pCEP4 vector (Invitrogen), which contains
an Epstein Barr Virus (EBV) origin of replication, facilitating the
maintenance of plasmid as a multicopy extrachromosomal element.
Another expression vector is the pEF-BOS plasmid containing the
promoter of polypeptide Elongation Factor 1.alpha., which
stimulates efficiently transcription in vitro. The plasmid is
described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990),
and its use in transfection experiments is disclosed by, for
example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still
another preferred expression vector is an adenovirus, described by
Stratford-Perricaudet, which is defective for E1 and E3 proteins
(J. Clin. Invest. 90:626-630, 1992). The use of the adenovirus as
an Adeno.P1A recombinant for the expression of an antigen is
disclosed by Warnier et al., in intradermal injection in mice for
immunization against P1A (Int. J. Cancer, 67:303-310, 1996).
Additional vectors for delivery of nucleic acid are provided
below.
[0128] The invention also embraces so-called expression kits, which
allow the artisan to prepare a desired expression vector or
vectors. Such expression kits include at least separate portions of
a vector and one or more of the previously discussed cancer
associated antigen nucleic acid molecules. Other components may be
added, as desired, as long as the previously mentioned nucleic acid
molecules, which are required, are included. The invention also
includes kits for amplification of a cancer associated antigen
nucleic acid, including at least one pair of amplification primers
which hybridize to a cancer associated antigen nucleic acid. The
primers preferably are 12-32 nucleotides in length and are
non-overlapping to prevent formation of "primer-dimers". One of the
primers will hybridize to one strand of the cancer associated
antigen nucleic acid and the second primer will hybridize to the
complementary strand of the cancer associated antigen nucleic acid,
in an arrangement which permits amplification of the cancer
associated antigen nucleic acid. Selection of appropriate primer
pairs is standard in the art. For example, the selection can be
made with assistance of a computer program designed for such a
purpose, optionally followed by testing the primers for
amplification specificity and efficiency.
[0129] The invention also permits the construction of cancer
associated antigen gene "knock-outs" and transgenic overexpression
in cells and in animals, providing materials for studying certain
aspects of cancer and immune system responses to cancer.
[0130] The invention also provides isolated polypeptides (including
whole proteins and partial proteins) encoded by the foregoing
cancer associated antigen nucleic acids. Such polypeptides are
useful, for example, alone or as fusion proteins to generate
antibodies, as components of an immunoassay or diagnostic assay or
as therapeutics. Cancer associated antigen polypeptides can be
isolated from biological samples including tissue or cell
homogenates, and can also be expressed recombinantly in a variety
of prokaryotic and eukaryotic expression systems by constructing an
expression vector appropriate to the expression system, introducing
the expression vector into the expression system, and isolating the
recombinantly expressed protein. Short polypeptides, including
antigenic peptides (such as are presented by MHC molecules on the
surface of a cell for immune recognition) also can be synthesized
chemically using well-established methods of peptide synthesis.
[0131] A unique fragment of a cancer associated antigen
polypeptide, in general, has the features and characteristics of
unique fragments as discussed above in connection with nucleic
acids. As will be recognized by those skilled in the art, the size
of the unique fragment will depend upon factors such as whether the
fragment constitutes a portion of a conserved protein domain. Thus,
some regions of cancer associated antigens will require longer
segments to be unique while others will require only short
segments, typically between 5 and 12 amino acids (e.g. 5, 6, 7, 8,
9, 10, 11 or 12 or more amino acids including each integer up to
the full length).
[0132] Unique fragments of a polypeptide preferably are those
fragments which retain a distinct functional capability of the
polypeptide. Functional capabilities which can be retained in a
unique fragment of a polypeptide include interaction with
antibodies, interaction with other polypeptides or fragments
thereof, selective binding of nucleic acids or proteins, and
enzymatic activity. One important activity is the ability to act as
a signature for identifying the polypeptide. Another is the ability
to complex with HLA and to provoke in a human an immune response.
Those skilled in the art are well versed in methods for selecting
unique amino acid sequences, typically on the basis of the ability
of the unique fragment to selectively distinguish the sequence of
interest from non-family members. A comparison of the sequence of
the fragment to those on known databases typically is all that is
necessary.
[0133] The invention embraces variants of the cancer associated
antigen polypeptides described above. As used herein, a "variant"
of a cancer associated antigen polypeptide is a polypeptide which
contains one or more modifications to the primary amino acid
sequence of a cancer associated antigen polypeptide. Modifications
which create a cancer associated antigen variant can be made to a
cancer associated antigen polypeptide 1) to reduce or eliminate an
activity of a cancer associated antigen polypeptide; 2) to enhance
a property of a cancer associated antigen polypeptide, such as
protein stability in an expression system or the stability of
protein-protein binding; 3) to provide a novel activity or property
to a cancer associated antigen polypeptide, such as addition of an
antigenic epitope or addition of a detectable moiety; or 4) to
provide equivalent or better binding to an HLA molecule.
Modifications to a cancer associated antigen polypeptide are
typically made to the nucleic acid which encodes the cancer
associated antigen polypeptide, and can include deletions, point
mutations, truncations, amino acid substitutions and additions of
amino acids or non-amino acid moieties. Alternatively,
modifications can be made directly to the polypeptide, such as by
cleavage, addition of a linker molecule, addition of a detectable
moiety, such as biotin, addition of a fatty acid, substitution of
L-amino acids with D-amino acids, and the like. Modifications also
embrace fusion proteins comprising all or part of the cancer
associated antigen amino acid sequence. One of skill in the art
will be familiar with methods for predicting the effect on protein
conformation of a change in protein sequence, and can thus "design"
a variant cancer associated antigen polypeptide according to known
methods. One example of such a method is described by Dahiyat and
Mayo in Science 278:82-87, 1997, whereby proteins can be designed
de novo. The method can be applied to a known protein to vary a
only a portion of the polypeptide sequence. By applying the
computational methods of Dahiyat and Mayo, specific variants of a
cancer associated antigen polypeptide can be proposed and tested to
determine whether the variant retains a desired conformation. Other
computational and computer modeling methods for designing
polypeptide mimetics which retain activity of the polypeptides
described herein, as well as selection methods such as phage
display of peptide libraries are known in the art.
[0134] In general, variants include cancer associated antigen
polypeptides which are modified specifically to alter a feature of
the polypeptide unrelated to its desired physiological activity.
For example, cysteine residues can be substituted or deleted to
prevent unwanted disulfide linkages. Similarly, certain amino acids
can be changed to enhance expression of a cancer associated antigen
polypeptide by eliminating proteolysis by proteases in an
expression system (e.g., dibasic amino acid residues in yeast
expression systems in which KEX2 protease activity is present).
[0135] Mutations of a nucleic acid which encode a cancer associated
antigen polypeptide preferably preserve the amino acid reading
frame of the coding sequence, and preferably do not create regions
in the nucleic acid which are likely to hybridize to form secondary
structures, such a hairpins or loops, which can be deleterious to
expression of the variant polypeptide.
[0136] Mutations can be made by selecting an amino acid
substitution, or by random mutagenesis of a selected site in a
nucleic acid which encodes the polypeptide. Variant polypeptides
are then expressed and tested for one or more activities to
determine which mutation provides a variant polypeptide with the
desired properties. Further mutations can be made to variants (or
to non-variant cancer associated antigen polypeptides) which are
silent as to the amino acid sequence of the polypeptide, but which
provide preferred codons for translation in a particular host. The
preferred codons for translation of a nucleic acid in, e.g., E.
coli, are well known to those of ordinary skill in the art. Still
other mutations can be made to the noncoding sequences of a cancer
associated antigen gene or cDNA clone to enhance expression of the
polypeptide. The activity of variants of cancer associated antigen
polypeptides can be tested by cloning the gene encoding the variant
cancer associated antigen polypeptide into a bacterial or mammalian
expression vector, introducing the vector into an appropriate host
cell, expressing the variant cancer associated antigen polypeptide,
and testing for a functional capability of the cancer associated
antigen polypeptides as disclosed herein. For example, the variant
cancer associated antigen polypeptide can be tested for reaction
with autologous or allogeneic sera as disclosed in the Examples.
Preparation of other variant polypeptides may favor testing of
other activities, as will be known to one of ordinary skill in the
art.
[0137] The skilled artisan will also realize that conservative
amino acid substitutions may be made in cancer associated antigen
polypeptides to provide functionally equivalent variants of the
foregoing polypeptides, i.e, the variants retain the functional
capabilities of the cancer associated antigen polypeptides. As used
herein, a "conservative amino acid substitution" refers to an amino
acid substitution which does not alter the relative charge or size
characteristics of the protein in which the amino acid substitution
is made. Variants can be prepared according to methods for altering
polypeptide sequence known to one of ordinary skill in the art such
as are found in references which compile such methods, e.g.
Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds.,
Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, New York, 1989, or Current Protocols in Molecular Biology,
F. M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
Exemplary functionally equivalent variants of the cancer associated
antigen polypeptides include conservative amino acid substitutions
of in the amino acid sequences of proteins disclosed herein.
Conservative substitutions of amino acids include substitutions
made amongst amino acids within the following groups: (a) M, I, L,
V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g)
E, D.
[0138] For example, upon determining that a peptide derived from a
cancer associated antigen polypeptide is presented by an MHC
molecule and recognized by CTLs, one can make conservative amino
acid substitutions to the amino acid sequence of the peptide,
particularly at residues which are thought not to be direct contact
points with the MHC molecule. For example, methods for identifying
functional variants of HLA class II binding peptides are provided
in a published PCT application of Strominger and Wucherpfennig
(PCT/US96/03182). Peptides bearing one or more amino acid
substitutions also can be tested for concordance with known HLA/MHC
motifs prior to synthesis using, e.g. the computer program
described by D'Amaro and Drijfhout (D'Amaro et al., Human Immunol.
43:13-18, 1995; Drijfhout et al., Human Immunol. 43:1-12, 1995).
The substituted peptides can then be tested for binding to the MHC
molecule and recognition by CTLs when bound to MHC. These variants
can be tested for improved stability and are useful, inter alia, in
vaccine compositions.
[0139] Conservative amino-acid substitutions in the amino acid
sequence of cancer associated antigen polypeptides to produce
functionally equivalent variants of cancer associated antigen
polypeptides typically are made by alteration of a nucleic acid
encoding a cancer associated antigen polypeptide. Such
substitutions can be made by a variety of methods known to one of
ordinary skill in the art. For example, amino acid substitutions
may be made by PCR-directed mutation, site-directed mutagenesis
according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci.
U.S.A. 82: 488-492, 1985), or by chemical synthesis of a gene
encoding a cancer associated antigen polypeptide. Where amino acid
substitutions are made to a small unique fragment of a cancer
associated antigen polypeptide, such as an antigenic epitope
recognized by autologous or allogeneic sera or cytolytic T
lymphocytes, the substitutions can be made by directly synthesizing
the peptide. The activity of functionally equivalent fragments of
cancer associated antigen polypeptides can be tested by cloning the
gene encoding the altered cancer associated antigen polypeptide
into a bacterial or mammalian expression vector, introducing the
vector into an appropriate host cell, expressing the altered cancer
associated antigen polypeptide, and testing for a functional
capability of the cancer associated antigen polypeptides as
disclosed herein. Peptides which are chemically synthesized can be
tested directly for function, e.g., for binding to antisera
recognizing associated antigens.
[0140] The invention as described herein has a number of uses, some
of which are described elsewhere herein. First, the invention
permits production and/or isolation of the cancer associated
antigen protein molecules. A variety of methodologies well-known to
the skilled practitioner can be utilized to obtain isolated cancer
associated antigen molecules. The polypeptide may be purified from
cells which naturally produce the polypeptide by chromatographic
means or immunological recognition. Alternatively, an expression
vector may be introduced into cells to cause production of the
polypeptide. In another method, mRNA transcripts may be
microinjected or otherwise introduced into cells to cause
production of the encoded polypeptide. Translation of mRNA in
cell-free extracts such as the reticulocyte lysate system also may
be used to produce polypeptide. Those skilled in the art also can
readily follow known methods for isolating cancer associated
antigen polypeptides. These include, but are not limited to,
immunochromatography, HPLC, size-exclusion chromatography,
ion-exchange chromatography and immune-affinity chromatography.
[0141] The isolation and identification of cancer associated
antigen genes also makes it possible for the artisan to diagnose a
disorder characterized by expression of cancer associated antigens.
These methods involve determining expression of one or more cancer
associated antigen nucleic acids, and/or encoded cancer associated
antigen polypeptides and/or peptides derived therefrom. In the
former situation, such determinations can be carried out via any
standard nucleic acid determination assay, including the polymerase
chain reaction, or assaying with labeled hybridization probes. In
the latter situation, such determinations can be carried out by
screening patient antisera for recognition of the polypeptide.
[0142] The invention also makes it possible isolate proteins which
bind to cancer associated antigens as disclosed herein, including
antibodies and cellular binding partners of the cancer associated
antigens. Additional uses are described further herein.
[0143] The invention also provides, in certain embodiments,
"dominant negative" polypeptides derived from cancer associated
antigen polypeptides. A dominant negative polypeptide is an
inactive variant of a protein, which, by interacting with the
cellular machinery, displaces an active protein from its
interaction with the cellular machinery or competes with the active
protein, thereby reducing the effect of the active protein. For
example, a dominant negative receptor which binds a ligand but does
not transmit a signal in response to binding of the ligand can
reduce the biological effect of expression of the ligand. Likewise,
a dominant negative catalytically-inactive kinase which interacts
normally with target proteins but does not phosphorylate the target
proteins can reduce phosphorylation of the target proteins in
response to a cellular signal. Similarly, a dominant negative
transcription factor which binds to a promoter site in the control
region of a gene but does not increase gene transcription can
reduce the effect of a normal transcription factor by occupying
promoter binding sites without increasing transcription.
[0144] The end result of the expression of a dominant negative
polypeptide in a cell is a reduction in function of active
proteins. One of ordinary skill in the art can assess the potential
for a dominant negative variant of a protein, and using standard
mutagenesis techniques to create one or more dominant negative
variant polypeptides. For example, given the teachings contained
herein of breast, gastric and prostate cancer associated antigens,
especially those which are similar to known proteins which have
known activities, one of ordinary skill in the art can modify the
sequence of the cancer associated antigens by site-specific
mutagenesis, scanning mutagenesis, partial gene deletion or
truncation, and the like. See, e.g., U.S. Pat. No. 5,580,723 and
Sambrook et al., Molecular Cloning: A Laboratory Manual, Second
Edition, Cold Spring Harbor Laboratory Press, 1989. The skilled
artisan then can test the population of mutagenized polypeptides
for diminution in a selected and/or for retention of such an
activity. Other similar methods for creating and testing dominant
negative variants of a protein will be apparent to one of ordinary
skill in the art.
[0145] The invention also involves agents such as polypeptides
which bind to cancer associated antigen polypeptides. Such binding
agents can be used, for example, in screening assays to detect the
presence or absence of cancer associated antigen polypeptides and
complexes of cancer associated antigen polypeptides and their
binding partners and in purification protocols to isolated cancer
associated antigen polypeptides and complexes of cancer associated
antigen polypeptides and their binding partners. Such agents also
can be used to inhibit the native activity of the cancer associated
antigen polypeptides, for example, by binding to such
polypeptides.
[0146] The invention, therefore, embraces peptide binding agents
which, for example, can be antibodies or fragments of antibodies
having the ability to selectively bind to cancer associated antigen
polypeptides. Antibodies include polyclonal and monoclonal
antibodies, prepared according to conventional methodology.
[0147] Significantly, as is well-known in the art, only a small
portion of an antibody molecule, the paratope, is involved in the
binding of the antibody to its epitope (see, in general, Clark, W.
R. (1986) The Experimental Foundations of Modem Immunology Wiley
& Sons, Inc., New York; Roitt, I. (1991) Essential Immunology,
7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and
Fc regions, for example, are effectors of the complement cascade
but are not involved in antigen binding. An antibody from which the
pFc' region has been enzymatically cleaved, or which has been
produced without the pFc' region, designated an F(ab').sub.2
fragment, retains both of the antigen binding sites of an intact
antibody. Similarly, an antibody from which the Fc region has been
enzymatically cleaved, or which has been produced without the Fc
region, designated an Fab fragment, retains one of the antigen
binding sites of an intact antibody molecule. Proceeding further,
Fab fragments consist of a covalently bound antibody light chain
and a portion of the antibody heavy chain denoted Fd. The Fd
fragments are the major determinant of antibody specificity (a
single Fd fragment may be associated with up to ten different light
chains without altering antibody specificity) and Fd fragments
retain epitope-binding ability in isolation.
[0148] Within the antigen-binding portion of an antibody, as is
well-known in the art, there are complementarity determining
regions (CDRs), which directly interact with the epitope of the
antigen, and framework regions (FRs), which maintain the tertiary
structure of the paratope (see, in general, Clark, 1986; Roitt,
1991). In both the heavy chain Fd fragment and the light chain of
IgG immunoglobulins, there are four framework regions (FR1 through
FR4) separated respectively by three complementarity determining
regions (CDR1 through CDR3).
[0149] The CDRs, and in particular the CDR3 regions, and more
particularly the heavy chain CDR3, are largely responsible for
antibody specificity.
[0150] It is now well-established in the art that the non-CDR
regions of a mammalian antibody may be replaced with similar
regions of conspecific or heterospecific antibodies while retaining
the epitopic specificity of the original antibody. This is most
clearly manifested in the development and use of "humanized"
antibodies in which non-human CDRs are covalently joined to human
FR and/or Fc/pFc' regions to produce a functional antibody. See,
e.g., U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,762 and
5,859,205.
[0151] Thus, for example, PCT International Publication Number WO
92/04381 teaches the production and use of humanized murine RSV
antibodies in which at least a portion of the murine FR regions
have been replaced by FR regions of human origin. Such antibodies,
including fragments of intact antibodies with antigen-binding
ability, are often referred to as "chimeric" antibodies.
[0152] Thus, as will be apparent to one of ordinary skill in the
art, the present invention also provides for F(ab').sub.2, Fab, Fv
and Fd fragments; chimeric antibodies in which the Fc and/or FR
and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been
replaced by homologous human or non-human sequences; chimeric
F(ab').sub.2 fragment antibodies in which the FR and/or CDR1 and/or
CDR2 and/or light chain CDR3 regions have been replaced by
homologous human or non-human sequences; chimeric Fab fragment
antibodies in which the FR and/or CDR1 and/or CDR2 and/or light
chain CDR3 regions have been replaced by homologous human or
non-human sequences; and chimeric Fd fragment antibodies in which
the FR and/or CDR1 and/or CDR2 regions have been replaced by
homologous human or non-human sequences. The present invention also
includes so-called single chain antibodies.
[0153] Thus, the invention involves polypeptides of numerous size
and type that bind specifically to cancer associated antigen
polypeptides, and complexes of both cancer associated antigen
polypeptides and their binding partners. These polypeptides may be
derived also from sources other than antibody technology. For
example, such polypeptide binding agents can be provided by
degenerate peptide libraries which can be readily prepared in
solution, in immobilized form or as phage display libraries.
Combinatorial libraries also can be synthesized of peptides
containing one or more amino acids. Libraries further can be
synthesized of peptoids and non-peptide synthetic moieties.
[0154] Phage display can be particularly effective in identifying
binding peptides useful according to the invention. Briefly, one
prepares a phage library (using e.g. m13, fd, or lambda phage),
displaying inserts from 4 to about 80 amino acid residues using
conventional procedures. The inserts may represent, for example, a
completely degenerate or biased array. One then can select
phage-bearing inserts which bind to the cancer associated antigen
polypeptide. This process can be repeated through several cycles of
reselection of phage that bind to the cancer associated antigen
polypeptide. Repeated rounds lead to enrichment of phage bearing
particular sequences. DNA sequence analysis can be conducted to
identify the sequences of the expressed polypeptides. The minimal
linear portion of the sequence that binds to the cancer associated
antigen polypeptide can be determined. One can repeat the procedure
using a biased library containing inserts containing part or all of
the minimal linear portion plus one or more additional degenerate
residues upstream or downstream thereof. Yeast two-hybrid screening
methods also may be used to identify polypeptides that bind to the
cancer associated antigen polypeptides. Thus, the cancer associated
antigen polypeptides of the invention, or a fragment thereof, can
be used to screen peptide libraries, including phage display
libraries, to identify and select peptide binding partners of the
cancer associated antigen polypeptides of the invention. Such
molecules can be used, as described, for screening assays, for
purification protocols, for interfering directly with the
functioning of cancer associated antigen and for other purposes
that will be apparent to those of ordinary skill in the art.
[0155] As detailed herein, the foregoing antibodies and other
binding molecules may be used for example to identify tissues
expressing protein or to purify protein. Antibodies also may be
coupled to specific diagnostic labeling agents for imaging of cells
and tissues that express cancer associated antigens or to
therapeutically useful agents according to standard coupling
procedures. Diagnostic agents include, but are not limited to,
barium sulfate, iocetamic acid, iopanoic acid, ipodate calcium,
diatrizoate sodium, diatrizoate meglumine, metrizamide, tyropanoate
sodium and radiodiagnostics including positron emitters such as
fluorine-18 and carbon-11, gamma emitters such as iodine-123,
technetium-99m, iodine-131 and indium-111, and nuclides for nuclear
magnetic resonance such as fluorine and gadolinium. Other
diagnostic agents useful in the invention will be apparent to one
of ordinary skill in the art. As used herein, "therapeutically
useful agents" include any therapeutic molecule which desirably is
targeted selectively to a cell expressing one of the cancer
antigens disclosed herein, including antineoplastic agents,
radioiodinated compounds, toxins, other cytostatic or cytolytic
drugs, and so forth. Antineoplastic therapeutics are well known and
include: aminoglutethimide, azathioprine, bleomycin sulfate,
busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide,
cyclosporine, cytarabidine, dacarbazine, dactinomycin,
daunorubicin, doxorubicin, taxol, etoposide, fluorouracil,
interferon-.alpha., lomustine, mercaptopurine, methotrexate,
mitotane, procarbazine HCl, thioguanine, vinblastine sulfate and
vincristine sulfate. Additional antineoplastic agents include those
disclosed in Chapter 52, Antineoplastic Agents (Paul Calabresi and
Bruce A. Chabner), and the introduction thereto, 1202-1263, of
Goodman and Gilman's "The Pharmacological Basis of Therapeutics",
Eighth Edition, 1990, McGraw-Hill, Inc. (Health Professions
Division). Toxins can be proteins such as, for example, pokeweed
anti-viral protein, cholera toxin, pertussis toxin, ricin, gelonin,
abrin, diphtheria exotoxin, or Pseudomonas exotoxin. Toxin moieties
can also be high energy-emitting radionuclides such as
cobalt-60.
[0156] In the foregoing methods and compositions, antibodies
prepared according to the invention also preferably are specific
for the cancer associated antigen/MHC complexes described
herein.
[0157] When "disorder" is used herein, it refers to any
pathological condition where the cancer associated antigens are
expressed. An example of such a disorder is cancer, including
breast, gastric and prostate cancer as particular examples.
[0158] Samples of tissue and/or cells for use in the various
methods described herein can be obtained through standard methods
such as tissue biopsy, including punch biopsy and cell scraping,
and collection of blood or other bodily fluids by aspiration or
other methods.
[0159] In certain embodiments of the invention, an immunoreactive
cell sample is removed from a subject. By "immunoreactive cell" is
meant a cell which can mature into an immune cell (such as a B
cell, a helper T cell, or a cytolytic T cell) upon appropriate
stimulation. Thus immunoreactive cells include CD34.sup.+
hematopoietic stem cells, immature T cells and immature B cells.
When it is desired to produce cytolytic T cells which recognize a
cancer associated antigen, the immunoreactive cell is contacted
with a cell which a expresses a cancer associated antigen under
conditions favoring production, differentiation and/or selection of
cytolytic T cells; the differentiation of the T cell precursor into
a cytolytic T cell upon exposure to antigen is similar to clonal
selection of the immune system.
[0160] Some therapeutic approaches based upon the disclosure are
premised on a response by a subject's immune system, leading to
lysis of antigen presenting cells, such as breast, gastric or
prostate cancer cells which present one or more cancer associated
antigens. One such approach is the administration of autologous
CTLs specific to a cancer associated antigen/MHC complex to a
subject with abnormal cells of the phenotype at issue. It is within
the ability of one of ordinary skill in the art to develop such
CTLs in vitro. An example of a method for T cell differentiation is
presented in International Application number PCT/US96/05607.
Generally, a sample of cells taken from a subject, such as blood
cells, are contacted with a cell presenting the complex and capable
of provoking CTLs to proliferate. The target cell can be a
transfectant, such as a COS cell. These transfectants present the
desired complex at their surface and, when combined with a CTL of
interest, stimulate its proliferation. COS cells are widely
available, as are other suitable host cells. Specific production of
CTL clones is well known in the art. The clonally expanded
autologous CTLs then are administered to the subject.
[0161] CTL proliferation can be increased by increasing the level
of tryptophan in T cell cultures, by inhibiting enzymes which
catabolize tryptophan, such as indoleamine 2,3-dioxygenase (IDO),
or by adding tryptophan to the culture. Proliferation of T cells is
enhanced by increasing the rate of proliferation and/or extending
the number of divisions of the T cells in culture. In addition,
increasing tryptophan in T cell cultures also enhances the lytic
activity of the T cells grown in culture.
[0162] Another method for selecting antigen-specific CTL clones has
recently been described (Altman et al., Science 274:94-96, 1996;
Dunbar et al., Curr. Biol. 8:413-416, 1998), in which fluorogenic
tetramers of MHC class I molecule/peptide complexes are used to
detect specific CTL clones. Briefly, soluble MHC class I molecules
are folded in vitro in the presence of .beta..sub.2-microglobulin
and a peptide antigen which binds the class I molecule. After
purification, the MHC/peptide complex is purified and labeled with
biotin. Tetramers are formed by mixing the biotinylated peptide-MHC
complex with labeled avidin (e.g. phycoerythrin) at a molar ratio
or 4:1. Tetramers are then contacted with a source of CTLs such as
peripheral blood or lymph node. The tetramers bind CTLs which
recognize the peptide antigen/MHC class I complex. Cells bound by
the tetramers can be sorted by fluorescence activated cell sorting
to isolate the reactive CTLs. The isolated CTLs then can be
expanded in vitro for use as described herein.
[0163] To detail a therapeutic methodology, referred to as adoptive
transfer (Greenberg, J. Immunol. 136(5): 1917, 1986; Riddel et al.,
Science 257: 238, 1992; Lynch et al, Eur. J Immunol. 21:
1403-1410,1991; Kast et al., Cell 59: 603-614, 1989), cells
presenting the desired complex (e.g., dendritic cells) are combined
with CTLs leading to proliferation of the CTLs specific thereto.
The proliferated CTLs are then administered to a subject with a
cellular abnormality which is characterized by certain of the
abnormal cells presenting the particular complex. The CTLs then
lyse the abnormal cells, thereby achieving the desired therapeutic
goal.
[0164] The foregoing therapy assumes that at least some of the
subject's abnormal cells present the relevant HLA/cancer associated
antigen complex. This can be determined very easily, as the art is
very familiar with methods for identifying cells which present a
particular HLA molecule, as well as how to identify cells
expressing DNA of the pertinent sequences, in this case a cancer
associated antigen sequence. Once cells presenting the relevant
complex are identified via the foregoing screening methodology,
they can be combined with a sample from a patient, where the sample
contains CTLs. If the complex presenting cells are lysed by the
mixed CTL sample, then it can be assumed that a cancer associated
antigen is being presented, and the subject is an appropriate
candidate for the therapeutic approaches set forth supra.
[0165] Adoptive transfer is not the only form of therapy that is
available in accordance with the invention. CTLs can also be
provoked in vivo, using a number of approaches. One approach is the
use of non-proliferative cells expressing the complex. The cells
used in this approach may be those that normally express the
complex, such as irradiated tumor cells or cells transfected with
one or both of the genes necessary for presentation of the complex
(i.e. the antigenic peptide and the presenting HLA molecule). Chen
et al. (Proc. Natl. Acad Sci. USA 88: 110-114,1991) exemplifies
this approach, showing the use of transfected cells expressing
HPV-E7 peptides in a therapeutic regime. Various cell types may be
used. Similarly, vectors carrying one or both of the genes of
interest may be used. Viral or bacterial vectors are especially
preferred. For example, nucleic acids which encode a cancer
associated antigen polypeptide or peptide may be operably linked to
promoter and enhancer sequences which direct expression of the
cancer associated antigen polypeptide or peptide in certain tissues
or cell types. The nucleic acid may be incorporated into an
expression vector. Expression vectors may be unmodified
extrachromosomal nucleic acids, plasmids or viral genomes
constructed or modified to enable insertion of exogenous nucleic
acids, such as those encoding cancer associated antigens, as
described elsewhere herein. Nucleic acids encoding one or more
cancer associated antigens also may be inserted into a retroviral
genome, thereby facilitating integration of the nucleic acid into
the genome of the target tissue or cell type. In these systems, the
gene of interest is carried by a microorganism, e.g., a Vaccinia
virus, pox virus, herpes simplex virus, retrovirus or adenovirus,
and the materials de facto "infect" host cells. The cells which
result present the complex of interest, and are recognized by
autologous CTLs, which then proliferate.
[0166] A similar effect can be achieved by combining the cancer
associated antigen or a stimulatory fragment thereof with an
adjuvant to facilitate incorporation into antigen presenting cells
in vivo. The cancer associated antigen polypeptide is processed to
yield the peptide partner of the HLA molecule while a cancer
associated antigen peptide may be presented without the need for
further processing. Generally, subjects can receive an intradermal
injection of an effective amount of the cancer associated antigen.
Initial doses can be followed by booster doses, following
immunization protocols standard in the art. Preferred cancer
associated antigens include those found to react with allogeneic
cancer antisera, shown in the examples below.
[0167] The invention involves the use of various materials
disclosed herein to "immunize" subjects or as "vaccines". As used
herein, "immunization" or "vaccination" means increasing or
activating an immune response against an antigen. It does not
require elimination or eradication of a condition but rather
contemplates the clinically favorable enhancement of an immune
response toward an antigen. Generally accepted animal models can be
used for testing of immunization against cancer using a cancer
associated antigen nucleic acid. For example, human cancer cells
can be introduced into a mouse to create a tumor, and one or more
cancer associated antigen nucleic acids can be delivered by the
methods described herein. The effect on the cancer cells (e.g.,
reduction of tumor size) can be assessed as a measure of the
effectiveness of the cancer associated antigen nucleic acid
immunization. Of course, testing of the foregoing animal model
using more conventional methods for immunization can include the
administration of one or more cancer associated antigen
polypeptides or peptides derived therefrom, optionally combined
with one or more adjuvants and/or cytokines to boost the immune
response. Methods for immunization, including formulation of a
vaccine composition and selection of doses, route of administration
and the schedule of administration (e.g. primary and one or more
booster doses), are well known in the art. The tests also can be
performed in humans, where the end point is to test for the
presence of enhanced levels of circulating CTLs against cells
bearing the antigen, to test for levels of circulating antibodies
against the antigen, to test for the presence of cells expressing
the antigen and so forth.
[0168] As part of the immunization compositions, one or more cancer
associated antigens or stimulatory fragments thereof are
administered with one or more adjuvants to induce an immune
response or to increase an immune response. An adjuvant is a
substance incorporated into or administered with antigen which
potentiates the immune response. Adjuvants may enhance the
immunological response by providing a reservoir of antigen
(extracellularly or within macrophages), activating macrophages and
stimulating specific sets of lymphocytes. Adjuvants of many kinds
are well known in the art. Specific examples of adjuvants include
monophosphoryl lipid A (MPL, SmithKline Beecham), a congener
obtained after purification and acid hydrolysis of Salmonella
minnesota Re 595 lipopolysaccharide; saponins including QS21
(SmithKline Beecham), a pure QA-21 saponin purified from Quillja
saponaria extract; DQS21, described in PCT application WO96/33739
(SmithKline Beecham); QS-7, QS-17, QS-18, and QS-L1 (So et al.,
Mol. Ce{acute over (ll)}s 7:178-186, 1997); incomplete Freund's
adjuvant; complete Freund's adjuvant; montanide; alum; CpG
oligonucleotides (see e.g. Kreig et al., Nature 374:546-9, 1995);
and various water-in-oil emulsions prepared from biodegradable oils
such as squalene and/or tocopherol. Preferably, the peptides are
administered mixed with a combination of DQS21/MPL. The ratio of
DQS21 to MPL typically will be about 1:10 to 10:1, preferably about
1:5 to 5:1 and more preferably about 1:1. Typically for human
administration, DQS21 and MPL will be present in a vaccine
formulation in the range of about 1 pg to about 100 .mu.g. Other
adjuvants are known in the art and can be used in the invention,
(see, e.g. Goding, Monoclonal Antibodies: Principles and Practice,
2nd Ed., 1986). Methods for the preparation of mixtures or
emulsions of peptide and adjuvant are well known to those of skill
in the art of vaccination.
[0169] Other agents which stimulate the immune response of the
subject can also be administered to the subject. For example, other
cytokines are also useful in vaccination protocols as a result of
their lymphocyte regulatory properties. Many other cytokines useful
for such purposes will be known to one of ordinary skill in the
art, including interleukin-12 (IL-12) which has been shown to
enhance the protective effects of vaccines (see, e.g., Science 268:
1432-1434, 1995), GM-CSF and IL-18. Thus cytokines can be
administered in conjunction with antigens and adjuvants to increase
the immune response to the antigens.
[0170] There are a number of immune response potentiating compounds
that can be used in vaccination protocols. These include
costimulatory molecules provided in either protein or nucleic acid
form. Such costimulatory molecules include the B7-1 and B7-2 (CD80
and CD86 respectively) molecules which are expressed on dendritic
cells (DC) and interact with the CD28 molecule expressed on the T
cell. This interaction provides costimulation (signal 2) to an
antigen/MHC/TCR stimulated (signal 1) T cell, increasing T cell
proliferation and effector function. B7 also interacts with CTLA4
(CD152) on T cells and studies involving CTLA4 and B7 ligands
indicate that the B7-CTLA4 interaction can enhance antitumor
immunity and CTL proliferation (Zheng P., et al. Proc. Natl. Acad.
Sci. USA 95 (11):6284-6289 (1998)).
[0171] B7 typically is not expressed on tumor cells so they are not
efficient antigen presenting cells (APCs) for T cells. Induction of
B7 expression would enable the tumor cells to stimulate more
efficiently CTL proliferation and effector function. A combination
of B7/IL-6/IL-12 costimulation has been shown to induce IFN-gamma
and a Th1 cytokine profile in the T cell population leading to
further enhanced T cell activity (Gajewski et al., J. Immunol,
154:5637-5648 (1995)). Tumor cell transfection with B7 has ben
discussed in relation to in vitro CTL expansion for adoptive
transfer immunotherapy by Wang et al., (J. Immunol., 19:1-8
(1986)). Other delivery mechanisms for the B7 molecule would
include nucleic acid (naked DNA) immunization (Kim J., et al. Nat
Biotechnol., 15:7:641-646 (1997)) and recombinant viruses such as
adeno and pox (Wendtner et al., Gene Ther., 4:7:726-735 (1997)).
These systems are all amenable to the construction and use of
expression cassettes for the coexpression of B7 with other
molecules of choice such as the antigens or fragment(s) of antigens
discussed herein (including polytopes) or cytokines. These delivery
systems can be used for induction of the appropriate molecules in
vitro and for in vivo vaccination situations. The use of anti-CD28
antibodies to directly stimulate T cells in vitro and in vivo could
also be considered. Similarly, the inducible co-stimulatory
molecule ICOS which induces T cell responses to foreign antigen
could be modulated, for example, by use of anti-ICOS antibodies
(Hutloff et al., Nature 397:263-266, 1999).
[0172] Lymphocyte function associated antigen-3 (LFA-3) is
expressed on APCs and some tumor cells and interacts with CD2
expressed on T cells. This interaction induces T cell IL-2 and
IFN-gamma production and can thus complement but not substitute,
the B7/CD28 costimulatory interaction (Parra et al., J. Immunol.,
158:637-642 (1997), Fenton et al., J. Immunother., 21:2:95-108
(1998)).
[0173] Lymphocyte function associated antigen-1 (LFA-1) is
expressed on leukocytes and interacts with ICAM-1 expressed on APCs
and some tumor cells. This interaction induces T cell IL-2 and
IFN-gamma production and can thus complement but not substitute,
the B7/CD28 costimulatory interaction (Fenton et al., J.
Immunother., 21:2:95-108 (1998)). LFA-1 is thus a further example
of a costimulatory molecule that could be provided in a vaccination
protocol in the various ways discussed above for B7.
[0174] Complete CTL activation and effector function requires Th
cell help through the interaction between the Th cell CD40L (CD40
ligand) molecule and the CD40 molecule expressed by DCs (Ridge et
al., Nature, 393:474 (1998), Bennett et al., Nature, 393:478
(1998), Schoenberger et al., Nature, 393:480 (1998)). This
mechanism of this costimulatory signal is likely to involve
upregulation of B7 and associated IL-6/IL-12 production by the DC
(APC). The CD40-CD40L interaction thus complements the signal 1
(antigen/MHC-TCR) and signal 2 (B7-CD28) interactions.
[0175] The use of anti-CD40 antibodies to stimulate DC cells
directly, would be expected to enhance a response to tumor antigens
which are normally encountered outside of a inflammatory context or
are presented by non-professional APCs (tumor cells). In these
situations Th help and B7 costimulation signals are not provided.
This mechanism might be used in the context of antigen pulsed DC
based therapies or in situations where Th epitopes have not been
defined within known cancer antigen precursors.
[0176] A cancer associated antigen polypeptide, or a fragment
thereof, also can be used to isolate their native binding partners.
Isolation of such binding partners may be performed according to
well-known methods. For example, isolated cancer associated antigen
polypeptides can be attached to a substrate (e.g., chromatographic
media, such as polystyrene beads, or a filter), and then a solution
suspected of containing the binding partner may be applied to the
substrate. If a binding partner which can interact with cancer
associated antigen polypeptides is present in the solution, then it
will bind to the substrate-bound cancer associated antigen
polypeptide. The binding partner then may be isolated.
[0177] It will also be recognized that the invention embraces the
use of the cancer associated antigen cDNA sequences in expression
vectors, as well as to transfect host cells and cell lines, be
these prokaryotic (e.g., E. coli), or eukaryotic (e.g., dendritic
cells, B cells, CHO cells, COS cells, yeast expression systems and
recombinant baculovirus expression in insect cells). Especially
useful are mammalian cells such as human, mouse, hamster, pig,
goat, primate, etc. They may be of a wide variety of tissue types,
and include primary cells and cell lines. Specific examples include
keratinocytes, peripheral blood leukocytes, bone marrow stem cells
and embryonic stem cells. The expression vectors require that the
pertinent sequence, i.e., those nucleic acids described supra, be
operably linked to a promoter.
[0178] The invention also contemplates delivery of nucleic acids,
polypeptides or peptides for vaccination. Delivery of polypeptides
and peptides can be accomplished according to standard vaccination
protocols which are well known in the art. In another embodiment,
the delivery of nucleic acid is accomplished by ex vivo methods,
i.e. by removing a cell from a subject, genetically engineering the
cell to include a cancer associated antigen, and reintroducing the
engineered cell into the subject. One example of such a procedure
is outlined in U.S. Pat. No. 5,399,346 and in exhibits submitted in
the file history of that patent, all of which are publicly
available documents. In general, it involves introduction in vitro
of a functional copy of a gene into a cell(s) of a subject, and
returning the genetically engineered cell(s) to the subject. The
functional copy of the gene is under operable control of regulatory
elements which permit expression of the gene in the genetically
engineered cell(s). Numerous transfection and transduction
techniques as well as appropriate expression vectors are well known
to those of ordinary skill in the art, some of which are described
in PCT application WO95/00654. In vivo nucleic acid delivery using
vectors such as viruses and targeted liposomes also is contemplated
according to the invention.
[0179] In preferred embodiments, a virus vector for delivering a
nucleic acid encoding a cancer associated antigen is selected from
the group consisting of adenoviruses, adeno-associated viruses,
poxviruses including vaccinia viruses and attenuated poxviruses,
Semliki Forest virus, Venezuelan equine encephalitis virus,
retroviruses, Sindbis virus, and Ty virus-like particle. Examples
of viruses and virus-like particles which have been used to deliver
exogenous nucleic acids include: replication-defective adenoviruses
(e.g., Xiang et al., Virology 219:220-227, 1996; Eloit et al., J.
Virol. 7:5375-5381, 1997; Chengalvala et al., Vaccine 15:335-339,
1997), a modified retrovirus (Townsend et al., J. Virol.
71:3365-3374, 1997), a nonreplicating retrovirus (Irwin et al., J.
Virol. 68:5036-5044, 1994), a replication defective Semliki Forest
virus (Zhao et al., Proc. Natl. Acad. Sci. USA 92:3009-3013, 1995),
canarypox virus and highly attenuated vaccinia virus derivative
(Paoletti, Proc. Natl. Acad. Sci. USA 93:11349-11353, 1996),
non-replicative vaccinia virus (Moss, Proc. Natl. Acad Sci. USA
93:11341-11348, 1996), replicative vaccinia virus (Moss, Dev. Biol.
Stand 82:55-63, 1994), Venzuelan equine encephalitis virus (Davis
et al., J. Virol. 70:3781-3787, 1996), Sindbis virus (Pugachev et
al., Virology 212:587-594, 1995), and Ty virus-like particle
(Allsopp et al., Eur. J. Immunol 26:1951-1959, 1996). In preferred
embodiments, the virus vector is an adenovirus.
[0180] Another preferred virus for certain applications is the
adeno-associated virus, a double-stranded DNA virus. The
adeno-associated virus is capable of infecting a wide range of cell
types and species and can be engineered to be
replication-deficient. It further has advantages, such as heat and
lipid solvent stability, high transduction frequencies in cells of
diverse lineages, including hematopoietic cells, and lack of
superinfection inhibition thus allowing multiple series of
transductions. The adeno-associated virus can integrate into human
cellular DNA in a site-specific manner, thereby minimizing the
possibility of insertional mutagenesis and variability of inserted
gene expression. In addition, wild-type adeno-associated virus
infections have been followed in tissue culture for greater than
100 passages in the absence of selective pressure, implying that
the adeno-associated virus genomic integration is a relatively
stable event. The adeno-associated virus can also function in an
extrachromosomal fashion.
[0181] In general, other preferred viral vectors are based on
non-cytopathic eukaryotic viruses in which non-essential genes have
been replaced with the gene of interest. Non-cytopathic viruses
include retroviruses, the life cycle of which involves reverse
transcription of genomic viral RNA into DNA with subsequent
proviral integration into host cellular DNA. Adenoviruses and
retroviruses have been approved for human gene therapy trials. In
general, the retroviruses are replication-deficient (i.e., capable
of directing synthesis of the desired proteins, but incapable of
manufacturing an infectious particle). Such genetically altered
retroviral expression vectors have general utility for the
high-efficiency transduction of genes in vivo. Standard protocols
for producing replication-deficient retroviruses (including the
steps of incorporation of exogenous genetic material into a
plasmid, transfection of a packaging cell lined with plasmid,
production of recombinant retroviruses by the packaging cell line,
collection of viral particles from tissue culture media, and
infection of the target cells with viral particles) are provided in
Kriegler, M., "Gene Transfer and Expression, A Laboratory Manual,"
W.H. Freeman Co., New York (1990) and Murry, E. J. Ed. "Methods in
Molecular Biology," vol. 7, Humana Press, Inc., Clifton, New Jersey
(1991).
[0182] Preferably the foregoing nucleic acid delivery vectors: (1)
contain exogenous genetic material that can be transcribed and
translated in a mammalian cell and that can induce an immune
response in a host, and (2) contain on a surface a ligand that
selectively binds to a receptor on the surface of a target cell,
such as a mammalian cell, and thereby gains entry to the target
cell.
[0183] Various techniques may be employed for introducing nucleic
acids of the invention into cells, depending on whether the nucleic
acids are introduced in vitro or in vivo in a host. Such techniques
include transfection of nucleic acid-CaPO.sub.4 precipitates,
transfection of nucleic acids associated with DEAE, transfection or
infection with the foregoing viruses including the nucleic acid of
interest, liposome mediated transfection, and the like. For certain
uses, it is preferred to target the nucleic acid to particular
cells. In such instances, a vehicle used for delivering a nucleic
acid of the invention into a cell (e.g., a retrovirus, or other
virus; a liposome) can have a targeting molecule attached thereto.
For example, a molecule such as an antibody specific for a surface
membrane protein on the target cell or a ligand for a receptor on
the target cell can be bound to or incorporated within the nucleic
acid delivery vehicle. Preferred antibodies include antibodies
which selectively bind a cancer associated antigen, alone or as a
complex with a MHC molecule. Especially preferred are monoclonal
antibodies. Where liposomes are employed to deliver the nucleic
acids of the invention, proteins which bind to a surface membrane
protein associated with endocytosis may be incorporated into the
liposome formulation for targeting and/or to facilitate uptake.
Such proteins include capsid proteins or fragments thereof tropic
for a particular cell type, antibodies for proteins which undergo
internalization in cycling, proteins that target intracellular
localization and enhance intracellular half life, and the like.
Polymeric delivery systems also have been used successfully to
deliver nucleic acids into cells, as is known by those skilled in
the art. Such systems even permit oral delivery of nucleic
acids.
[0184] The therapeutics of the invention can be administered by any
conventional route, including injection or by gradual infusion over
time. The administration may, for example, be oral, intravenous,
intraperitoneal, intramuscular, intracavity, subcutaneous, or
transdermal. When cancer associated antigen peptides are used for
vaccination, modes of administration which effectively deliver the
cancer associated antigen and adjuvant, such that an immune
response to the antigen is increased, can be used. For
administration of a cancer associated antigen peptide in adjuvant,
preferred methods include intradermal, intravenous, intramuscular
and subcutaneous administration. Although these are preferred
embodiments, the invention is not limited by the particular modes
of administration disclosed herein. Standard references in the art
(e.g., Remington's Pharmaceutical Sciences, 18th edition, 1990)
provide modes of administration and formulations for delivery of
immunogens with adjuvant or in a non-adjuvant carrier. When
antibodies are used therapeutically, a preferred route of
administration is by pulmonary aerosol. Techniques for preparing
aerosol delivery systems containing antibodies are well known to
those of skill in the art. Generally, such systems should utilize
components which will not significantly impair the biological
properties of the antibodies, such as the paratope binding capacity
(see, for example, Sciarra and Cutie, "Aerosols," in Remington's
Pharmaceutical Sciences, 18th edition, 1990, pp 1694-1712;
incorporated by reference). Those of skill in the art can readily
determine the various parameters and conditions for producing
antibody aerosols without resort to undue experimentation. When
using antisense preparations of the invention, slow intravenous
administration is preferred.
[0185] The compositions of the invention are administered in
effective amounts. An "effective amount" is that amount of a cancer
associated antigen composition that alone, or together with further
doses, produces the desired response, e.g. increases an immune
response to the cancer associated antigen. In the case of treating
a particular disease or condition characterized by expression of
one or more cancer associated antigens, such as breast, gastric or
prostate cancers, the desired response is inhibiting the
progression of the disease. This may involve only slowing the
progression of the disease temporarily, although more preferably,
it involves halting the progression of the disease permanently.
This can be monitored by routine methods or can be monitored
according to diagnostic methods of the invention discussed herein.
The desired response to treatment of the disease or condition also
can be delaying the onset or even preventing the onset of the
disease or condition.
[0186] Such amounts will depend, of course, on the particular
condition being treated, the severity of the condition, the
individual patient parameters including age, physical condition,
size and weight, the duration of the treatment, the nature of
concurrent therapy (if any), the specific route of administration
and like factors within the knowledge and expertise of the health
practitioner. These factors are well known to those of ordinary
skill in the art and can be addressed with no more than routine
experimentation. It is generally preferred that a maximum dose of
the individual components or combinations thereof be used, that is,
the highest safe dose according to sound medical judgment. It will
be understood by those of ordinary skill in the art, however, that
a patient may insist upon a lower dose or tolerable dose for
medical reasons, psychological reasons or for virtually any other
reasons.
[0187] The pharmaceutical compositions used in the foregoing
methods preferably are sterile and contain an effective amount of
cancer associated antigen or nucleic acid encoding cancer
associated antigen for producing the desired response in a unit of
weight or volume suitable for administration to a patient. The
response can, for example, be measured by determining the immune
response following administration of the cancer associated antigen
composition via a reporter system by measuring downstream effects
such as gene expression, or by measuring the physiological effects
of the cancer associated antigen composition, such as regression of
a tumor or decrease of disease symptoms. Other assays will be known
to one of ordinary skill in the art and can be employed for
measuring the level of the response.
[0188] The doses of cancer associated antigen compositions (e.g.,
polypeptide, peptide, antibody, cell or nucleic acid) administered
to a subject can be chosen in accordance with different parameters,
in particular in accordance with the mode of administration used
and the state of the subject. Other factors include the desired
period of treatment. In the event that a response in a subject is
insufficient at the initial doses applied, higher doses (or
effectively higher doses by a different, more localized delivery
route) may be employed to the extent that patient tolerance
permits.
[0189] In general, for treatments for eliciting or increasing an
immune response, doses of cancer associated antigen are formulated
and administered in doses between 1 ng and 1 mg, and preferably
between 10 ng and 100 .mu.g, according to any standard procedure in
the art. Where nucleic acids encoding cancer associated antigen of
variants thereof are employed, doses of between 1 ng and 0.1 mg
generally will be formulated and administered according to standard
procedures. Other protocols for the administration of cancer
associated antigen compositions will be known to one of ordinary
skill in the art, in which the dose amount, schedule of injections,
sites of injections, mode of administration (e.g., intra-tumoral)
and the like vary from the foregoing. Administration of cancer
associated antigen compositions to mammals other than humans, e.g.
for testing purposes or veterinary therapeutic purposes, is carried
out under substantially the same conditions as described above.
[0190] When administered, the pharmaceutical compositions of the
invention are applied in pharmaceutically-acceptable amounts and in
pharmaceutically-acceptable preparations. The term
"pharmaceutically acceptable" means a non-toxic material that does
not interfere with the effectiveness of the biological activity of
the active ingredients. Such preparations may routinely contain
salts, buffering agents, preservatives, compatible carriers, and
optionally other therapeutic agents. When used in medicine, the
salts should be pharmaceutically acceptable, but
non-pharmaceutically acceptable salts may conveniently be used to
prepare pharmaceutically-acceptable salts thereof and are not
excluded from the scope of the invention. Such pharmacologically
and pharmaceutically-acceptable salts include, but are not limited
to, those prepared from the following acids: hydrochloric,
hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic,
salicylic, citric, formic, malonic, succinic, and the like. Also,
pharmaceutically-acceptable salts can be prepared as alkaline metal
or alkaline earth salts, such as sodium, potassium or calcium
salts.
[0191] A cancer associated antigen composition may be combined, if
desired, with a pharmaceutically-acceptable carrier. The term
"pharmaceutically-acceptable carrier" as used herein means one or
more compatible solid or liquid fillers, diluents or encapsulating
substances which are suitable for administration into a human. The
term "carrier" denotes an organic or inorganic ingredient, natural
or synthetic, with which the active ingredient is combined to
facilitate the application. The components of the pharmaceutical
compositions also are capable of being co-mingled with the
molecules of the present invention, and with each other, in a
manner such that there is no interaction which would substantially
impair the desired pharmaceutical efficacy.
[0192] The pharmaceutical compositions may contain suitable
buffering agents, including: acetic acid in a salt; citric acid in
a salt; boric acid in a salt; and phosphoric acid in a salt.
[0193] The pharmaceutical compositions also may contain,
optionally, suitable preservatives, such as: benzalkonium chloride;
chlorobutanol; parabens and thimerosal.
[0194] The pharmaceutical compositions may conveniently be
presented in unit dosage form and may be prepared by any of the
methods well-known in the art of pharmacy. All methods include the
step of bringing the active agent into association with a carrier
which constitutes one or more accessory ingredients. In general,
the compositions are prepared by uniformly and intimately bringing
the active compound into association with a liquid carrier, a
finely divided solid carrier, or both, and then, if necessary,
shaping the product.
[0195] Compositions suitable for oral administration may be
presented as discrete units, such as capsules, tablets, lozenges,
each containing a predetermined amount of the active compound.
Other compositions include suspensions in aqueous liquids or
non-aqueous liquids such as a syrup, elixir or an emulsion.
[0196] Compositions suitable for parenteral administration
conveniently comprise a sterile aqueous or non-aqueous preparation
of cancer associated antigen polypeptides or nucleic acids, which
is preferably isotonic with the blood of the recipient. This
preparation may be formulated according to known methods using
suitable dispersing or wetting agents and suspending agents. The
sterile injectable preparation also may be a sterile injectable
solution or suspension in a non-toxic parenterally-acceptable
diluent or solvent, for example, as a solution in 1,3-butane diol.
Among the acceptable vehicles and solvents that may be employed are
water, Ringer's solution, and isotonic sodium chloride solution. In
addition, sterile, fixed oils are conventionally employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono-or di-glycerides. In
addition, fatty acids such as oleic acid may be used in the
preparation of injectables. Carrier formulation suitable for oral,
subcutaneous, intravenous, intramuscular, etc. administrations can
be found in Remington's Pharmaceutical Sciences, Mack Publishing
Co., Easton, Pa.
EXAMPLES
Example 1
SEREX Screening of Breast, Gastric and Prostate Cancer Cells
[0197] Breast, gastric and prostate cancer cDNA libraries were
established, using standard techniques, and the libraries were
screened, using the SEREX methodology described by Sahin et al.,
Proc. Natl. Acad. Sci. USA 92: 11810 (1995), and by Chen et al.,
Proc. Natl. Acad. Sci. USA 94: 1914 (1997), each of which is
incorporated by reference in its entirety.
[0198] To be specific, total RNA was isolated by homogenizing tumor
samples in 4M guanidinium thiocyanate/0.5% sodium N-lauryl
sarcosine/25 mM EDTA followed by centrifugation in 5.7 M CsCI/25 mM
sodium acetate/10 .mu.M EDTA at 32,000 rpm. Total mRNA was removed
by passing the sample over an oligo-dT cellulose column. The cDNA
libraries were then constructed by taking 5 .mu.g of mRNA, using
standard methodologies to reverse transcribe the material. Breast
cancer libraries were prepared from two different breast cancer
patients, referred to as "MT" and "MK". Gastric cancer libraries
were prepared from a gastric cancer patient, referred to as
"YS".
[0199] The cDNA was used to construct a lambda phage library, and
500 phages were plated onto XL1-Blue MRF E. coli, and incubated for
eight hours at 37.degree. C. A nitrocellulose membrane was then
placed on the plate, followed by overnight incubation. The membrane
was then washed, four times, with Tris buffered saline (TBS) which
contained 0.05% Tween, and was then immersed in TBS containing 5%
non-fat dried milk. After one hour, the membrane was incubated with
conjugates of peroxidase-goat anti human IgG specific for Fc
portions of human antibodies (1:2000, diluted in TBS with 1% BSA).
The incubation was carried out for one hour, at room temperature,
and the membrane was then washed three times with TBS. Those clones
which produced antibodies were visualized with 0.06%
3,3'diaminobenzidine tetrachloride and 0.015% H.sub.2O.sub.2, in 50
mM Tris (pH 7.5). Any clones which produced immunoglobulin were
marked, and then the membrane was washed, two further times, with
TBS that contained 0.05% Tween, and then twice with "neat" TBS.
[0200] The membranes were then incubated in 1:100 diluted patient
serum, overnight, at 4.degree. C. The patient serum had been
pretreated. Specifically, 5 ml samples were diluted to 10 ml with
TBS containing 1% bovine serum albumin, and 0.02% Na.sub.3N. The
serum had been treated to remove antibodies to bacteriophage, by
passing it through a 5 ml Sepharose column, to which a lysate of E.
coli Y1090 had been attached, followed by passage over a second
column which had E. coli lysate and lysate of E. coli infected with
lambda bacteriophage. The screening was carried out five times. The
samples were then diluted to 50 ml, and kept at -80.degree. C.,
until used as described herein.
[0201] Following the overnight incubation with the membrane, the
membrane was washed twice with TBS/0.05% Tween 20, and then once
with TBS. A further incubation was carried out, using the protocols
discussed supra, for the peroxidase labeled antibodies.
[0202] The positive clones were then sequenced, using standard
techniques. Following comparison of the sequences to information
available in data banks, clones were resolved into known and
unknown genes. Some clones corresponded to previously identified
human proteins and nucleotide sequences, and other clones have not
been identified in humans previously, although there were related
molecules found in other species. Still other clones represent
molecules for which no related sequences were found (most clones
contained very short sections (e.g. 25 or fewer nucleotides) that
corresponded to portions of unrelated sequences). Some GenBank
accession numbers representative of sequences having homology to
the cancer associated antigen nucleotide sequences of the invention
are presented in Table 1. All of the homologous sequences are
accessible in publicly-available databases by reference to the
sequences' accession numbers provided in Table 1.
[0203] Breast Cancer Clones:
[0204] The nucleotide sequences of clones derived from breast
cancer patients "MT" and "MK" are presented as SEQ ID NOs: 1-205.
Polypeptides encoded by open reading frames of the nucleic acid
clones are presented as SEQ ID Nos: 594-829; the correspondence
between nuicleic acid molecules and encoded polypeptides is shown
in Table 2.
[0205] Gastric Cancer Clones:
[0206] The nucleotide sequences of clones derived from gastric
cancer patient "YS" are presented as SEQ ID NOs:206-352 (clones
beginning with "YS"). Polypeptides encoded by open reading frames
of the YS nucleic acid clones are presented as SEQ ID Nos:830-1083;
the correspondence between nucleic acid molecules and encoded
polypeptides is shown in Table 2.
[0207] Prostate Cancer Clones
[0208] The nucleotide sequences of clones derived from prostate
cancer patient "ZH" are presented as SEQ ID NOs:353-593(clones
beginning with "ZH"). Polypeptides encoded by open reading frames
of the ZH nucleic acid clones are presented as SEQ ID Nos:
1084-1332; the correspondence between nucleic acid molecules and
encoded polypeptides is shown in Table 2. TABLE-US-00001 LENGTHY
TABLE REFERENCED HERE US20070128655A1-20070607-T00001 Please refer
to the end of the specification for access instructions.
TABLE-US-00002 LENGTHY TABLE REFERENCED HERE
US20070128655A1-20070607-T00002 Please refer to the end of the
specification for access instructions.
Example 2
Preparation of Recombinant Cancer Associated Antigens
[0209] To facilitate screening of patients' sera for antibodies
reactive with cancer associated antigens, for example by ELISA,
recombinant proteins are prepared according to standard procedures.
Where gaps exist in the gene sequences represented by the clones
disclosed herein, or where flanking sequences are desired, such
nucleic acid sequences can be isolated according to standard
procedures. For example, where 5' and 3' clones of a gene sequence
are known, PCR primers can be designed for amplification of the
nucleotide sequence between the clones. Flanking sequences can be
isolated using procedures such as RACE PCR. Such sequences also can
be isolated by standard hybridization cloning protocols.
[0210] In one method of preparing recombinant cancer associated
antigens, the clones encoding cancer associated antigens are
subcloned into a baculovirus expression vector, and the recombinant
expression vectors are introduced into appropriate insect cells.
Baculovirus/insect cloning systems are preferred because
post-translational modifications are carried out in the insect
cells. Another preferred eukaryotic system is the Drosophila
Expression System from Invitrogen. Clones which express high
amounts of the recombinant protein are selected and used to produce
the recombinant proteins. The recombinant proteins are tested for
antibody recognition using serum from the patient which was used to
isolated the particular clone, or in the case of cancer associated
antigens recognized by allogeneic sera, by the sera from any of the
patients used to isolate the clones or sera which recognize the
clones' gene products.
[0211] Alternatively, the cancer associated antigen clones are
inserted into a prokaryotic expression vector for production of
recombinant proteins in bacteria. Other systems, including yeast
expression systems and mammalian cell culture systems also can be
used.
Example 3
Preparation of Antibodies to Cancer Associated Antigens
[0212] The recombinant cancer associated antigens produced as in
Example 2 above are used to generate polyclonal antisera and
monoclonal antibodies according to standard procedures. The
antisera and antibodies so produced are tested for correct
recognition of the cancer associated antigens by using the
antisera/antibodies in assays of cell extracts of patients known to
express the particular cancer associated antigen (e.g. an ELISA
assay). These antibodies can be used for experimental purposes
(e.g. localization of the cancer associated antigens,
immunoprecipitations, Western blots, etc.) as well as diagnostic
purposes (e.g., testing extracts of tissue biopsies, testing for
the presence of cancer associated antigens).
Example 4
Expression of Breast, Gastric and Prostate Cancer Associated
Antigens in Cancers of Similar and Different Origin
[0213] The expression of one or more of the breast, gastric and/or
prostate cancer associated antigens is tested in a range of tumor
samples to determine which, if any, other malignancies should be
diagnosed and/or treated by the methods described herein. Tumor
cell lines and tumor samples are tested for cancer associated
antigen expression, preferably by RT-PCR according to standard
procedures. Northern blots also are used to test the expression of
the cancer associated antigens. Antibody based assays, such as
ELISA and western blot, also can be used to determine protein
expression. A preferred method of testing expression of cancer
associated antigens (in other cancers and in additional same type
cancer patients) is allogeneic serotyping using a modified SEREX
protocol (as described above).
[0214] In all of the foregoing, extracts from the tumors of
patients who provided sera for the initial isolation of the cancer
associated antigens are used as positive controls. The cells
containing recombinant expression vectors described in the Examples
above also can be used as positive controls.
[0215] The results generated from the foregoing experiments provide
panels of multiple cancer associated nucleic acids and/or
polypeptides for use in diagnostic (e.g. determining the existence
of cancer, determining the prognosis of a patient undergoing
therapy, etc.) and therapeutic methods (e.g., vaccine composition,
etc.).
Example 5
HLA Typing of Patients Positive for Cancer Associated Antigen
[0216] To determine which HLA molecules present peptides derived
from the cancer associated antigens, cells of the patients which
express the breast and/or gastric cancer associated antigens are
HLA typed. Peripheral blood lymphocytes are taken from the patient
and typed for HLA class I or class II, as well as for the
particular subtype of class I or class II. Tumor biopsy samples
also can be used for typing. HLA typing can be carried out by any
of the standard methods in the art of clinical immunology, such as
by recognition by specific monoclonal antibodies, or by HLA
allele-specific PCR (e.g. as described in WO97/31126).
Example 6
Characterization of Cancer Associated Antigen Peptides Presented by
MHC Class I and Class II Molecules
[0217] Antigens which provoke an antibody response in a subject may
also provoke a cell-mediated immune response. Cells process
proteins into peptides for presentation on MHC class I or class II
molecules on the cell surface for immune surveillance. Peptides
presented by certain MHC/HLA molecules generally conform to motifs.
These motifs are known in some cases, and can be used to screen the
breast and/or gastric cancer associated antigens for the presence
of potential class I and/or class II peptides. Summaries of class I
and class II motifs have been published (e.g., Rammensee et al.,
Immunogenetics 41:178-228, 1995). Based on the results of
experiments such as those described above, the HLA types which
present the individual breast cancer associated antigens are known.
Motifs of peptides presented by these HLA molecules thus are
preferentially searched.
[0218] One also can search for class I and class II motifs using
computer algorithms. For example, computer programs for predicting
potential CTL epitopes based on known class I motifs has been
described (see, e.g., Parker et al, J. Immunol. 152:163, 1994;
D'Amaro et al., Human Immunol. 43:13-18, 1995; Drijfhout et al.,
Human Immunol. 43:1-12, 1995). HLA binding predictions can
conveniently be made using an algorithm available via the Internet
on the National Institutes of Health World Wide Web site at URL
bimas.dcrt.nih.gov. Methods for determining HLA class II peptides
and making substitutions thereto are also known (see, e.g.
International applications PCT/US96/03182 and PCT/US98/01373).
Computer software for selecting HLA class II binding peptides is
also available (TEPITOPE; Sturniolo et al., Nature Biotechnol.
17:555-561, 1999; Manici et al., J. Exp. Med. 189:871-876, 1999).
Peptides which are thus selected can be for inducing specific
CD4.sup.+ lymphocytes and identification of peptides. Additional
methods of selecting and testing peptides for HLA class II binding
are well known in the art.
Example 7
Identification of the Portion of a Cancer Associated Polypeptide
Encoding an Antigen
[0219] To determine if the cancer associated antigens isolated as
described above can provoke a cytolytic T lymphocyte response, the
following method is performed. CTL clones are generated by
stimulating the peripheral blood lymphocytes (PBLs) of a patient
with autologous normal cells transfected with one of the clones
encoding a cancer associated antigen polypeptide or with irradiated
PBLs loaded with synthetic peptides corresponding to the putative
protein and matching the consensus for the appropriate HLA class I
molecule (as described above) to localize an antigenic peptide
within the cancer associated antigen clone (see, e.g., Knuth et
al., Proc. Natl. Acad. Sci. USA 81:3511-3515, 1984; van der Bruggen
et al., Eur. J. Immunol.24:3038-3043, 1994). These CTL clones are
screened for specificity against COS cells transfected with the
cancer associated antigen clone and autologous HLA alleles as
described by Brichard et al. (Eur. J. Immunol. 26:224-230, 1996).
CTL recognition of a cancer associated antigen is determined by
measuring release of TNF from the cytolytic T lymphocyte or by
.sup.51Cr release assay (Herin et al., Int. J. Cancer 39:390-396,
1987). If a CTL clone specifically recognizes a transfected COS
cell, then shorter fragments of the cancer associated antigen clone
transfected in that COS cell are tested to identify the region of
the gene that encodes the peptide. Fragments of the cancer
associated antigen clone are prepared by exonuclease III digestion
or other standard molecular biology methods. Synthetic peptides are
prepared to confirm the exact sequence of the antigen.
[0220] Optionally, shorter fragments of cancer associated antigen
cDNAs are generated by PCR. Shorter fragments are used to provoke
TNF release or .sup.51Cr release as above.
[0221] Synthetic peptides corresponding to portions of the shortest
fragment of the cancer associated antigen clone which provokes TNF
release are prepared. Progressively shorter peptides are
synthesized to determine the optimal cancer associated antigen
tumor rejection antigen peptides for a given HLA molecule.
[0222] A similar method is performed to determine if the cancer
associated antigen contains one or more HLA class II peptides
recognized by T cells. One can search the sequence of the cancer
associated antigen polypeptides for HLA class II motifs as
described above. In contrast to class I peptides, class II peptides
are presented by a limited number of cell types. Thus for these
experiments, dendritic cells or B cell clones which express HLA
class II molecules preferably are used.
Example 8
Recognition of Cancer Antigens by Cancer Patient Sera
[0223] Several of the cancer antigen identified herein were tested
for reactivity with sera from normal and breast cancer patients
according to standard procedures (e.g., the SEREX procedure
outlined above). TABLE-US-00003 TABLE 3 Serology of antigens SEQ
Breast Cancer Normal ID NO Gene/Clone Patient Sera Sera 1
Br-38/HSP105 (MK) 6/31 0/30 2, 3 Br-39/HSP105 (MK) 3/31 0/30 4, 5
RGS-GAIP interacting protein GIPC (MK) 3/31 0/30 6, 7 NS1-binding
protein/KIAA0850 (MK) 3/31 0/30 8 Opa-interacting protein OIP2 (MK)
3/31 0/30 9, 10 Kinesin family protein 3B (KIF3B) (MT) 2/31 0/30 11
Endothelial-monocyte activating protein (EMAP2) (MT) 2/31 0/30 12
Unknown TOM1 protein (MT311) 2/31 0/30 13 Outer mitochodrial
membrane protein 34 kDa (MT) 1/31 0/30 14, 15 IPL (MK) 1/31 0/30
16, 17 Mus ACF7 neural isoform (MK) 1/31 0/30 18 Cyclin D3 (MT)
1/31 0/30
[0224] The data show that proteins encoded by SEQ ID NO:1-12 were
recognized by multiple breast cancer patients' sera, but not by
control individuals' sera. Proteins encoded by SEQ ID NO:13-18 were
recognized by only a single breast cancer patient's sera, but not
by control individuals' sera. The
Equivalents
[0225] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
[0226] All references disclosed herein are incorporated by
reference in their entirety. TABLE-US-00004 LENGTHY TABLE The
patent application contains a lengthy table section. A copy of the
table is available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070128655A1).
An electronic copy of the table will also be available from the
USPTO upon request and payment of the fee set forth in 37 CFR
1.19(b)(3).
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070128655A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20070128655A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References