U.S. patent application number 10/754820 was filed with the patent office on 2004-07-15 for melanoma antigens and methods of use.
This patent application is currently assigned to UAB Research Foundation. Invention is credited to Conry, Robert M., LoBuglio, Albert F., Strong, Theresa.
Application Number | 20040136999 10/754820 |
Document ID | / |
Family ID | 22575263 |
Filed Date | 2004-07-15 |
United States Patent
Application |
20040136999 |
Kind Code |
A1 |
Strong, Theresa ; et
al. |
July 15, 2004 |
Melanoma antigens and methods of use
Abstract
The present invention provides for isolated DNA and protein
corresponding to novel melanoma tumor-associated antigens,
antibodies directed towards the novel antigens of the present
invention as well as methods of using the antigens for inhibiting
the growth of a melanoma tumor and methods of screening compounds
that inhibit the novel antigens of the present invention.
Inventors: |
Strong, Theresa;
(Birmingham, AL) ; Conry, Robert M.; (Birmingham,
AL) ; LoBuglio, Albert F.; (Birmingham, AL) |
Correspondence
Address: |
Benjamin Aaron Adler, Ph.D., J.D.
Adler & Associates
8011 Candle Lane
Houston
TX
77071
US
|
Assignee: |
UAB Research Foundation
|
Family ID: |
22575263 |
Appl. No.: |
10/754820 |
Filed: |
January 9, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10754820 |
Jan 9, 2004 |
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09691538 |
Oct 18, 2000 |
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6677444 |
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60160042 |
Oct 18, 1999 |
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Current U.S.
Class: |
424/155.1 |
Current CPC
Class: |
C12Q 1/6886 20130101;
C12Q 2600/136 20130101; C07K 14/4748 20130101 |
Class at
Publication: |
424/155.1 |
International
Class: |
A61K 039/395 |
Goverment Interests
[0002] This invention was produced in part using funds obtained
through grant 5R21CA78489 from the National Institute of Health.
Consequently, the federal government has certain rights in this
invention.
Claims
What is claimed is:
1. An isolated DNA encoding a melanoma tumor-associated antigen
selected from the group consisting of: (a) isolated DNA comprises
of sequence selected from the group consisting of SEQ ID Nos. 1-12;
(b) isolated DNA which is complementary to the isolated DNA of (a)
above; and (c) isolated DNA differing from the isolated DNAs of (a)
and (b) above in codon sequence due to the degeneracy of the
genetic code.
2. A vector comprising the DNA of claim 1 and regulatory elements
necessary for expression of said DNA in a cell.
3. The vector of claim 2, wherein said DNA encodes a melanoma
tumor-associated antigen.
4. The vector of claim 2, wherein said DNA is positioned in reverse
orientation relative to said regulatory elements such that a
melanoma tumor-associated antigen antisense mRNA is produced.
5. A host cell transfected with the vector of claim 2, said vector
expressing a melanoma tumor-associated antigen.
6. The host cell of claim 5, wherein said cell is selected from
group consisting of bacterial cells, mammalian cells, plant cells
and insect cells.
7. The host cell of claim 6, wherein said bacterial cell is E.
coli.
8. Isolated and purified melanoma tumor-associated antigen encoded
for by the DNA of claim 1.
9. A method for detecting mRNA coding for a melanoma
tumor-associated antigen in a sample, comprising the steps of: (a)
contacting a sample with an oligonucleotide probe specific for a
melanoma tumor-associated antigen, wherein said probe comprises of
sequence selected from the group consisting of SEQ ID Nos. 1-12;
and (b) detecting binding of said probe to said mRNA coding for
said melanoma tumor-associated antigen in said sample.
10. A kit for detecting mRNA coding for a melanoma tumor-associated
antigen, comprising: an oligonucleotide probe specific for a
melanoma tumor-associated antigen, wherein said probe comprises of
sequence selected from the group consisting of SEQ ID Nos.
1-12.
11. The kit of claim 10, further comprising: a label with which to
label said probe; and means for detecting said label.
12. A method of detecting a melanoma tumor-associated antigen in a
sample, comprising the steps of: (a) contacting a sample with an
antibody, wherein said antibody is specific for a melanoma
tumor-associated antigen or a fragment thereof encoded by the DNA
of claim 1; and (b) detecting binding of said antibody to said
melanoma tumor-associated antigen in said sample.
13. A kit for detecting a melanoma tumor-associated antigen,
comprising: an antibody, wherein said antibody is specific for a
melanoma tumor-associated antigen or a fragment thereof encoded by
the DNA of claim 1.
14. The kit of claim 13, further comprising: means to detect said
antibody.
15. An antibody, wherein said antibody is specific for a melanoma
tumor-associated antigen or a fragment thereof encoded by the DNA
of claim 1.
16. A method of screening for compounds that inhibit the activity
of a melanoma tumor-associated antigen, comprising the steps of:
(a) contacting a sample with a compound, wherein said sample
comprises a melanoma tumor-associated antigen encoded by the DNA of
claim 1; and (b) assaying for activity of said melanoma
tumor-associated antigen, wherein a decrease in said melanoma
tumor-associated antigen activity in the presence of said compound
relative to said melanoma tumor-associated antigen activity in the
absence of said compound is indicative of a compound that inhibits
the activity of said melanoma tumor-associated antigen.
17. A method of inhibiting the growth of a melanoma tumor in an
individual, comprising the steps of: (a) administering to an
individual a therapeutic compound, wherein said therapeutic
compound comprises a thereapeutic moiety and a targeting moiety,
wherein said targeting moiety recognizes a melanoma
tumor-associated antigen encoded by the DNA of claim 1, wherein
said therapeutic compound inhibits the growth of said melanoma
tumor in said individual.
18. The method of claim 17, wherein said targeting moiety is
selected from the group consisting of an antibody or fragment
thereof and a ligand.
19. The method of claim 17, wherein said therapeutic moiety is
selected from the group consisting of a therapeutic gene or
protein, a toxin, a radiolabel and a virus.
20. A cancer vaccine composition, comprising a vector capable of
expressing a DNA molecule selected from the group consisting of SEQ
ID Nos. 1-12, and an appropriate adjuvant.
21. A method of vaccinating an individual against cancer,
comprising the steps of: administering to said individual a vector
capable of expressing a DNA molecule selected from the group
consisting of SEQ ID Nos. 1-12, wherein said expression elicits an
immune response specific towards a melonoma-specific antigen,
thereby inducing immune-mediated destruction of melanoma cells.
22. The method of claim 21, wherein said individual is at risk of
getting cancer, suspected of having cancer or has cancer.
23. A method of inhibiting the growth of a melanoma tumor,
comprising the steps of: administering the cancer vaccine of claim
20 to an individual, wherein administration of said vaccine induces
an immune response, thereby inhibiting the growth of a melanoma
tumor.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This non-provisional patent application claims benefit of
provisional patent application U.S. Serial No. 60/160,042 filed
Oct. 18, 1999, now abandoned.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates generally to the field of
cancer. More specifically, the present invention relates to
antigens specific for melanoma carcinomas.
[0005] 2. Description of the Related Art
[0006] Several methods have been employed to isolate and clone
tumor-associated antigens, and in general, these methods have
relied upon the ability of the antigens to stimulate cytolytic T
cells (1-4). These methods involve demanding techniques, including
extensive manipulation and expansion of cytolytic T cells.
[0007] Furthermore, it is becoming increasingly apparent that
tumor-bearing individuals also develop serological immune responses
to tumor antigens. Antibodies directed towards mutated cellular
genes have been described, including those reactive with mutant p53
(5, 6) and ras (7). In addition, humoral immune responses to
non-mutated, aberrantly expressed tumor antigens, such as erbB-2
(8) and cathepsin D (9), have been reported.
[0008] The presence of humoral immunity to many known
tumor-associated antigens suggests its use for identification of
novel tumor-associated antigens. The feasibility of this strategy
was demonstrated in a study by Pfreundschuh and coworkers (10, 11)
who screened tumor-derived cDNA libraries with autologous patient
sera and identified two known tumor antigens as well as several
novel, putative tumor antigens. This technology, termed SEREX, for
serological identification of antigens by recombinant expression
cloning, has since been applied by many groups and has led to the
substantial expansion of known tumor antigens (12).
[0009] Thus, the prior art is deficient in additional novel
antigens specific to melanomas. The present invention fulfills this
long-standing need and desire in the art.
SUMMARY OF THE INVENTION
[0010] Herein, the SEREX approach was used to identify melanoma
antigens in patients undergoing active immunotherapy. The primary
goal of identifying novel melanoma antigens is to expand the
potential targets for immunotherapy. In addition, characterization
of these proteins has the potential to impact on diverse areas of
melanoma research including detection, diagnosis and staging,
characterization of the genetic changes associated with
tumorigenesis, and the principles of immune activation and tumor
cell rejection.
[0011] Novel melanoma tumor-associated antigens may be useful for
detection, diagnosis, and staging of melanomas. Novel melanoma
tumor-associated antigens may also be useful for monitoring to
detect recurrence and metastatic disease and to monitor disease
burden (e.g., proteins expressed on the cell surface may provide
targets for monitoring, i.e., via detection and imaging of tumors).
Novel tumor-associated antigens may additionally be useful as
targets for immunotherapy and intervention strategies.
[0012] One object of the present invention is to provide melanoma
tumor-associated antigens and methods of using the melanoma
tumor-associated antigens.
[0013] In one embodiment of the present invention, there is
provided DNA encoding a melanoma tumor-associated antigen selected
from the group consisting of: (a) isolated DNA as shown in SEQ ID
Nos. 1-12; (b) isolated DNA which is complementary to isolated DNA
of (a) above; and (c) isolated DNA differing from the isolated DNAs
of (a) and (b) above in codon sequence due to the degeneracy of the
genetic code.
[0014] In another embodiment of the present invention, there is
provided an isolated and purified melanoma tumor-associated antigen
coded for by the DNA disclosed herein.
[0015] In another embodiment of the present invention, there is
provided a method for detecting mRNA coding for a melanoma
tumor-associated antigen in a sample, comprising the steps of: (a)
contacting a sample with an oligonucleotide probe having a sequence
such as SEQ ID Nos. 1-12; and (b) detecting binding of the probe to
the mRNA coding for a melanoma tumor-associated antigen in the
sample.
[0016] In yet another embodiment of the present invention, there is
provided a kit for detecting mRNA coding for a melanoma
tumor-associated antigen, comprising: an oligonucleotide probe
having a nucleotide sequence shown in SEQ ID Nos. 1-12. The kit may
further comprises: a label with which to label the probe; and means
for detecting the label.
[0017] In still yet another embodiment of the present invention,
there is provided a method of detecting a melanoma tumor-associated
antigen in a sample, comprising the steps of: (a) contacting a
sample with an antibody specific for a melanoma tumor-associated
antigen or a fragment thereof encoded by the DNA disclosed herein;
and (b) detecting binding of the antibody to the melanoma
tumor-associated antigen in the sample.
[0018] In another embodiment of the present invention, there is
provided a kit for detecting a melanoma tumor-associated antigen,
comprising: an antibody specific for a melanoma tumor-associated
antigen or a fragment thereof encoded by the DNA disclosed herein.
The kit may further comprise means to detect the antibody.
[0019] In another embodiment of the present invention, there is
provided an antibody specific for a melanoma tumor-associated
antigen or a fragment thereof encoded by the DNA disclosed
herein.
[0020] In still yet another embodiment of the present invention,
there is provided a method of screening for compounds that inhibit
the activity of a melanoma tumor-associated antigen, comprising the
steps of: (a) contacting a sample with a compound, wherein the
sample comprises a melanoma tumor-associated antigen encoded by the
DNA disclosed herein; and (b) assaying for activity of the melanoma
tumor-associated antigen. Generally, a decrease in the melanoma
tumor-associated antigen activity in the presence of the compound
relative to the melanoma tumor-associated antigen activity in the
absence of the compound is indicative of a compound that inhibits
the activity of the melanoma tumor-associated antigen.
[0021] In another embodiment of the present invention, there is
provided a method of inhibiting the growth of a melanoma tumor in
an individual, comprising the steps of: (a) treating an individual
with a therapeutic compound, wherein the therapeutic compound
comprises a thereapeutic moiety and a targeting moiety, wherein the
targeting moiety recognizes a melanoma tumor-associated antigen
encoded by the DNA disclosed herein; wherein the therapeutic
compound inhibits the growth of the melanoma tumor in the
individual.
[0022] In another embodiment of the present invention, there is
provided a cancer vaccine composition, comprising a vector capable
of expressing a DNA molecule such as SEQ ID Nos. 1-12, and an
appropriate adjuvant.
[0023] In another embodiment of the present invention, there is
provided a method of vaccinating an individual against cancer,
comprising the step of: (a) administering to the individual a
vector capable of expressing a DNA molecule such as SEQ ID Nos.
1-12, wherein said expression elicits an immune response specific
towards a melonoma-specific antigen, thereby inducing
immune-mediated destruction of melanoma cells.
[0024] In another embodiment of the present invention, there is
provided a method of inhibiting the growth of a melanoma tumor,
comprising the steps of: (a) administering to an individual a
cancer vaccine comprising a vector expressing a DNA such as SEQ ID
Nos. 1-12, wherein administration of said vaccine induces an immune
response, thereby inhibiting the growth of a melanoma tumor.
[0025] Other and further aspects, features, and advantages of the
present invention will be apparent from the following description
of the presently preferred embodiments of the invention. These
embodiments are given for the purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
[0026] The appended drawings have been included herein so that the
above-recited features, advantages and objects of the invention
will become clear and can be understood in detail. These drawings
form a part of the specification. It is to be noted, however, that
the appended drawings illustrate preferred embodiments of the
invention and should not be considered to limit the scope of the
invention.
[0027] FIG. 1 shows an example of a colony lift assay with a
purified positive clone. Clone 3.1 was plated in a 50:50 mix with a
negative control phage. Filters were lifted from the plates,
incubated with serum from patient 1, and reactive plaques were
detected with a labeled secondary antibody. Dark circles represent
the positive plaques for clone 3.1, while negative plaques are not
detected.
[0028] FIG. 2 shows the 5' and 3' sequences of clone 3.1 (FIG. 2A);
clone 3.14 (FIG. 2B); clone 3.3T (FIG. 2C); clone 5.17 (FIG. 2D);
clone 5.31 (FIG. 2G); and complete sequence of clone 5.23 (FIG.
2E); clone 5.28 (FIG. 2F).
DETAILED DESCRIPTION OF THE INVENTION
[0029] The primary goal in identifying novel melanoma antigens is
to expand the potential targets for immunotherapy. In addition,
characterization of tumor antigens may impact diverse areas of
melanoma research, including detection, diagnosis and staging,
characterization of the genetic changes associated with
tumorigenesis, and the principles of immune activation and tumor
cell rejection.
[0030] The tumor-associated antigens of the present invention may
be useful for detection, diagnosis, and staging of melanoma.
Detection and diagnosis of melanoma is currently based on visual
identification of melanoma lesions, while staging is based on depth
of the lesion at the time of diagnosis. While these visual
guidelines have proven useful, the use of additional marker
proteins and molecular characterization of melanoma lesions may
provide information useful in more accurately defining the clinical
course of the disease.
[0031] The tumor-associated antigens of the present invention may
also be useful for disease monitoring. Metastatic melanoma can
spread to a variety of sites. The identification of the
tumor-associated antigens of the present invention may allow
recurrence and metastatic disease to be detected and disease burden
monitored (e.g., by imaging an antigen-targeted melanoma cell).
[0032] The tumor-associated antigens of the present invention may
further be useful as targets in immunotherapy. Several
immunotherapy approaches directed towards melanoma cancers are
currently under development. The tumor-associated antigens of the
present invention will provide additional and specific therapeutic
targets for intervention.
[0033] The SEREX approach has been used herein to identify novel
melanoma antigens in patients undergoing active immunotherapy.
[0034] The present invention is directed towards a DNA encoding a
melanoma tumor-associated antigen selected from the group
consisting of: (a) isolated DNA having a sequence shown in SEQ ID
Nos. 1-12; (b) isolated DNA which is complementary to the isolated
DNA of (a) above; and (c) isolated DNA differing from the isolated
DNAs of (a) and (b) above in codon sequence due to the degeneracy
of the genetic code.
[0035] The present invention is also directed towards a vector
comprising the DNA disclosed herein and regulatory elements
necessary for expression of the DNA in a cell, wherein the DNA
encodes a melanoma tumor-associated antigen. Also included in the
present invention is a vector in which the DNA is positioned in
reverse orientation relative to the regulatory elements such that a
melanoma tumor-associated antigen antisense mRNA is produced.
Further provided are host cells transfected with the
above-described vector expressing a melanoma tumor-associated
antigen. Representative host cells are bacterial cells, mammalian
cells, plant cells and insect cells, more preferably, the bacterial
cell is E. coli.
[0036] The present invention is additionally directed towards an
isolated and purified melanoma tumor-associated antigen coded for
by DNA selected from the group consisting of: (a) isolated DNA
selected from the group consisting of SEQ ID Nos. 1-12; (b)
isolated DNA which is complementary to the isolated DNA of (a)
above; and (c) isolated DNA differing from the isolated DNAs of (a)
and (b) above in codon sequence due to the degeneracy of the
genetic code.
[0037] The present invention is further directed towards a method
for detecting mRNA coding for a melanoma tumor-associated antigen
in a sample, comprising the steps of: (a) contacting a sample with
an oligonucleotide probe having a nucleotide sequence shown in SEQ
ID Nos. 1-12; and (b) detecting binding of the probe to the mRNA
coding for a melanoma tumor-associated antigen in the sample.
[0038] The present invention is also directed towards a kit for
detecting mRNA coding for a melanoma tumor-associated antigen,
comprising: an oligonucleotide probe having a nucleotide sequence
such as SEQ ID Nos. 1-12. The above-described kit may further
comprise a label with which to label the probe; and means for
detecting the label.
[0039] The present invention is still further directed towards a
method of detecting a melanoma tumor-associated antigen in a
sample, comprising the steps of: (a) contacting a sample with an
antibody specific for a melanoma tumor-associated antigen or a
fragment thereof encoded by the DNA disclosed herein; and (b)
detecting binding of the antibody to the melanoma tumor-associated
antigen in the sample.
[0040] The present invention is additionally directed towards a kit
for detecting a melanoma tumor-associated antigen, comprising: an
antibody specific for a melanoma tumor-associated antigen or a
fragment thereof encoded by the DNA disclosed herein. The
above-described kit may further comprise means to detect the
antibody.
[0041] The present invention is further directed towards an
antibody specific for a melanoma tumor-associated antigen or a
fragment thereof encoded by the DNA disclosed herein.
[0042] The present invention is also directed towards a method of
screening for compounds that inhibit the activity of a melanoma
tumor-associated antigen, comprising the steps of: (a) contacting a
sample with a compound, wherein the sample comprises a melanoma
tumor-associated antigen encoded by the DNA disclosed herein; and
(b) assaying for activity of the melanoma tumor-associated antigen.
Typically, a decrease in the melanoma tumor-associated antigen
activity in the presence of the compound relative to the melanoma
tumor-associated antigen activity in the absence of the compound is
indicative of a compound that inhibits the activity of the melanoma
tumor-associated antigen.
[0043] The antigens reported herein may play a role in signaling
growth, activating the cell cycle, down-regulating inhibitors of
growth and/or promoting metastatic spread. Alternatively, the
antigens reported herein may normally be expressed in melanocytes
and may or may not have a direct role in tumorigenesis.
[0044] The present invention is also directed towards a method of
inhibiting the growth of a melanoma tumor in an individual,
comprising the steps of: (a) treating an individual with a
therapeutic compound, wherein the therapeutic compound comprises a
thereapeutic moiety and a targeting moiety, wherein the targeting
moiety recognizes a melanoma tumor-associated antigen encoded by
the DNA disclosed herein; wherein the therapeutic compound inhibits
the growth of the melanoma tumor in the individual. Preferred
targeting moieties are an antibody or fragment thereof, or a
ligand, while preferred therapeutic moieties are a therapeutic gene
or protein, a toxin, a radiolabel or a virus.
[0045] The present invention is also directed toward a cancer
vaccine composition, comprising a vector capable of expressing a
DNA molecule having a sequence shown in SEQ ID Nos. 1-12, and an
appropriate adjuvant.
[0046] The present invention is further directed toward a method of
vaccinating an individual against cancer, comprising the steps of:
(a) administering to the individual a vector capable of expressing
a DNA molecule shown in SEQ ID Nos. 1-12, wherein expression
elicits an immune response which is specific towards a
melonoma-specific antigen, thereby inducing immune-mediated
destruction of melanoma cells. Typically, the individual is at risk
of getting cancer, suspected of having cancer or has cancer.
[0047] The present invention is also directed toward a method of
inhibiting the growth of a melanoma tumor, comprising the steps of:
(a) administering a cancer vaccine to an individual comprising a
vector expressing a DNA such as SEQ ID Nos. 1-12, wherein
administration of the vaccine induces an immune response, thereby
inhibiting the growth of a melanoma tumor.
[0048] It will be apparent to one skilled in the art that various
substitutions and modifications may be made to the invention
disclosed herein without departing from the scope and spirit of the
invention.
[0049] In accordance with the present invention, there may be
employed conventional molecular biology, microbiology, and
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g.,
Sambrook, Fritsch & Maniatis, "Molecular Cloning: A Laboratory
Manual (2nd Ed.)", (1989); "DNA Cloning: A Practical Approach,"
Volumes I and II (D. N. Glover ed. 1985); "Oligonucleotide
Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B.
D. Hames & S. J. Higgins eds. (1985)]; "Transcription and
Translation" [B. D. Hames & S. J. Higgins eds. (1984)]; "Animal
Cell Culture" [R. I. Freshney, ed. (1986)]; "Immobilized Cells And
Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To
Molecular Cloning" (1984). Therefore, if appearing herein, the
following terms shall have the definitions set out below.
[0050] A "DNA molecule" refers to the polymeric form of
deoxyribonucleotides (adenine,. guanine, thymine, or cytosine) in
its either single stranded form or a double-stranded helix. This
term refers only to the primary and secondary structure of the
molecule, and does not limit it to any particular tertiary forms.
Thus, this term includes double-stranded DNA found, inter alia, in
linear DNA molecules (e.g., restriction fragments), viruses,
plasmids, and chromosomes. DNA structure is discussed herein
according to the normal convention of showing only the sequence in
the 5' to 3' direction along the nontranscribed strand of DNA
(i.e., the strand having a sequence homologous to the mRNA).
[0051] A "vector" is a replicon, such as plasmid, phage viral
genome or cosmid, to which another DNA segment may be attached so
as to bring about the replication of the attached segment. A
"replicon" is any genetic element (e.g., plasmid, chromosome,
virus) that functions as an autonomous unit of DNA replication in
vivo; i.e., capable of replication under its own control. An
"origin of replication" refers to those DNA sequences that
participate in and/or regulate DNA synthesis. An "expression
control sequence" or "regulatory elements necessary for expression"
are DNA sequence(s) that control and regulate the transcription and
translation of another DNA sequence. A coding sequence is "operably
linked" and "under the control" of transcriptional and
translational control sequences in a cell when RNA polymerase
transcribes the coding sequence into mRNA, which is then translated
into the protein encoded by the coding sequence.
[0052] In general, expression vectors containing promoter sequences
which facilitate the efficient transcription and translation of the
inserted DNA fragment are used in connection with the host. The
expression vector typically contains an origin of replication,
promoter(s), terminator(s), as well as specific genes which are
capable of providing phenotypic selection in transformed cells. The
transformed hosts can be fermented and cultured according to means
known in the art to achieve optimal cell growth.
[0053] A DNA "coding sequence" is a double-stranded DNA sequence
which is transcribed and translated into a polypeptide when placed
under the control of appropriate regulatory sequences. The
boundaries of the coding sequence are determined by a start codon
at the 5' (amino) terminus and a translation stop codon at the 3'
(carboxyl) terminus. A coding sequence can include, but is not
limited to, prokaryotic sequences, cDNAs from eukaryotic mRNA,
genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and
even synthetic DNA sequences. A coding sequence may alternatively
be transcribed in the opposite orientation (i.e., the
nontranscribed strand is used as the template) to produce an
antisense RNA molecule. An antisense RNA is complementary to an
mRNA molecule produced from the transcribed strand.
[0054] A polyadenylation signal and transcription termination
sequence will usually be located 3' to the coding sequence. A
"cDNA" is defined as copy-DNA or complementary-DNA, and is a
product of a reverse transcription reaction from an mRNA
transcript. An "exon" is an expressed sequence transcribed from the
gene locus, whereas an "intron" is a non-expressed, usually
spliced-out, sequence.
[0055] Transcriptional and translational regulatory sequences, such
as promoters, enhancers, polyadenylation signals, terminators, and
the like, provide for expression of a coding sequence in a host
cell. A "cis-element" is a nucleotide sequence, also sometimes
termed a "consensus sequence" or "motif", that interacts with
proteins that regulate expression of a specific gene locus. A
"signal sequence" can also be included with the coding sequence.
This sequence encodes a signal peptide, N-terminal to the
polypeptide, that communicates with the host cell and directs the
polypeptide to the appropriate cellular location. Signal sequences
can be found associated with a variety of proteins native to
prokaryotes and eukaryotes.
[0056] A "promoter sequence" is a DNA regulatory region capable of
binding RNA polymerase in a cell and initiating transcription of a
downstream (3' direction) coding sequence. For purposes of defining
the present invention, the promoter sequence is bounded at its 3'
terminus by the transcription initiation site and extends upstream
(5' direction) to include the minimum number of bases or elements
necessary to initiate transcription at levels detectable above
background. Within the promoter sequence will be found a
transcription initiation site, as well as consensus sequences
responsible for binding of RNA polymerase. Eukaryotic promoters
often, but not always, contain "TATA" boxes and "CAT" boxes.
Prokaryotic promoters typically contain the -10 and -35 consensus
sequences, as well as Shine-Dalgarno sequences for ribosome
binding.
[0057] The term "oligonucleotide" is defined as a molecule
comprised of two or more deoxyribonucleotides, preferably more than
three. Its exact size will depend upon many factors which, in turn,
depend upon the ultimate function and use of the oligonucleotide.
The term "primer" as used herein refers to an oligonucleotide,
whether occurring naturally (as in a purified restriction digest)
or produced synthetically, which is capable of acting as a point of
initiation when placed under conditions in which synthesis of a
primer extension product, which is complementary to a nucleic acid
strand, is induced, i.e., in the presence of nucleotides and a
polymerizing agent such as a DNA polymerase, and at a suitable
temperature and pH. The primer may be either single-stranded or
double-stranded and must be sufficiently long to prime the
synthesis of the desired extension product in the presence of the
inducing agent. The exact length of the primer will depend upon
many factors, including temperature, source of primer and the
method used. For example, for diagnostic applications, depending
upon the complexity of the target sequence, the oligonucleotide
primer typically contains 15-25 or more nucleotides, although it
may contain fewer nucleotides.
[0058] Primers are selected to be "substantially" complementary to
a strand of a particular target DNA sequence. This means that the
primers must be sufficiently complementary to hybridize with their
respective strands under the appropriate conditions. Therefore, the
primer sequence need not reflect the exact sequence of the
template. For example, a non-complementary nucleotide fragment may
be attached to the 5' end of the primer, with the remainder of the
primer sequence being complementary to the strand. Alternatively,
non-complementary bases or longer sequences can be interspersed
into the primer, provided that the primer sequence has sufficient
complementarity with the sequence to hybridize therewith and
thereby initiate synthesis of the extension product.
[0059] As used herein, the terms "restriction endonucleases" and
"restriction enzymes" refer to enzymes which cut double-stranded
DNA at or near a specific nucleotide sequence.
[0060] "Recombinant DNA technology" refers to techniques for
uniting two heterologous DNA molecules, usually as a result of in
vitro ligation of DNAs from different organisms. Recombinant DNA
molecules are commonly produced by experiments in genetic
engineering. Synonymous terms include "gene splicing", "molecular
cloning", "cloning" and "genetic engineering". The product of these
manipulations results in a "recombinant" or "recombinant
molecule".
[0061] A cell has been "transformed" or "transfected" with
exogenous or heterologous DNA when such DNA has been introduced
into the cell. The transforming DNA may or may not be integrated
(covalently linked) into the genome of the cell. In prokaryotes,
yeast, and mammalian cells for example, the transforming DNA may be
maintained on an replicative episomal element such as a vector or
plasmid. With respect to eukaryotic cells, a stably transformed
cell is one in which the transforming DNA has become integrated
into a chromosome so that it is inherited by daughter cells through
chromosome replication. This stability is demonstrated by the
ability of the eukaryotic cell to establish cell lines or clones
comprised of a population of daughter cells containing the
transforming DNA. A "clone" is a population of cells derived (by
mitosis) from a single cell or ancestor. A "cell line" is a clone
of a primary cell that is capable of stable growth in vitro for
many generations. An organism, such as a plant or animal, that has
been transformed with exogenous DNA is termed "transgenic".
[0062] As used herein, the term "host" is meant to include not only
prokaryotes, but also eukaryotes, such as yeast cells, plant cells
and animal cells. A recombinant DNA molecule or gene can be used to
transform a host using any of the techniques commonly known to
those of ordinary skill in the art. Prokaryotic hosts may include
E. coli, S. tymphimurium, Serratia marcescens and Bacillus
subtilis. Eukaryotic hosts include yeasts such as Pichia pastoris,
mammalian cells and insect cells, and plant cells, such as
Arabidopsis thaliana and Tobaccum nicotiana.
[0063] As used herein, "fragment," as applied to a polypeptide,
will ordinarily be at least 10 residues, more typically at least 20
residues, and preferably at least 30 (e.g., 50) residues in length,
but less than the entire, intact sequence. Fragments can be
generated by methods known to those skilled in the art, e.g., by
enzymatic digestion/cleavage of naturally occurring or recombinant
protein, by recombinant DNA techniques using an expression vector
that encodes a defined fragment, or by chemical synthesis. The
ability of a candidate fragment to exhibit an enzyme characteristic
(e.g., binding to a specific antibody, or exhibiting enzymatic or
catalytic activity) can be assessed by methods described herein.
Purified fragments or antigenic fragments can be used to generate
new regulatory enzymes using multiple functional fragments from
different enzymes, as well as to generate antibodies, by employing
standard protocols known to those skilled in the art.
[0064] Generally speaking, antibodies for use in these aspects of
the present invention will preferably recognize antigens that are
preferentially, or specifically, expressed by melanoma tumor cells.
Such antibodies will also preferably exhibit properties of high
affinity, such as exhibiting a K.sub.d of <200 nM, and
preferably, of <100 nM, and will not show significant reactivity
with normal tissues, such as tissues from heart, kidney, brain,
liver, bone marrow, colon, breast, prostate, thyroid, gall bladder,
lung, adrenals, muscle, nerve fibers, pancreas, skin, or other
life-sustaining organ or tissue in the human body. These tissues
are important for the purposes of the present invention from the
standpoint of low reactivity with the antibody. The term
"reactivity," as used herein, refers to an antibody or antibody
fragment that, when applied to the particular tissue under
conditions suitable for immunohistochemistry, will elicit staining
only in positive cells and not negative cells. Particularly
promising antibodies contemplated for use in the present invention
are those having high reactivity specific to the melanoma
tumor.
[0065] A standard Northern blot assay can be used to ascertain the
relative amounts of mRNA in a cell or tissue obtained from tissue
in accordance with conventional Northern blot hybridization
techniques known to those persons of ordinary skill in the art.
Alternatively, a standard Southern blot assay may be used to
confirm the presence and the copy number of a gene in accordance
with conventional Southern blot hybridization techniques known to
those of ordinary skill in the art. Both the Northern blot and
Southern blot use a hybridization probe, e.g., radiolabelled cDNA,
either containing the full-length, single stranded DNA or a
fragment of the DNA sequence at least 20 (preferably at least 30,
more preferably at least 50, and most preferably at least 100
consecutive nucleotides in length). The oligonucleotide
hybridization probe can be labelled by any of the many different
methods known to those skilled in this art. Conditions for Northern
and Southern hybridizations, i.e., stringency, can be determined
for that particular system empirically and/or experimentally, and
defining appropriate hybridization conditions is well within the
skill of the art. See, e.g., Maniatis et al., supra; DNA Cloning,
Vols. I & II, supra; Nucleic Acid Hybridization, supra.
[0066] By "high stringency" is meant DNA hybridization and wash
conditions characterized by high temperature and low salt
concentration, e.g., wash conditions of 65.degree. C. at a salt
concentration of approximately 0.1.times.SSC, or the functional
equivalent thereof. For example, high stringency conditions may
include hybridization at about 42.degree. C. in the presence of
about 50% formamide; a first wash at about 65.degree. C. with about
2.times.SSC containing 1% SDS; followed by a second wash at about
65.degree. C. with about 0.1.times.SSC.
[0067] By "substantially pure DNA" is meant DNA that is not part of
a milieu in which the DNA naturally occurs, by virtue of separation
(partial or total purification) of some or all of the molecules of
that milieu, or by virtue of alteration of sequences that flank the
claimed DNA. The term therefore includes, for example, a
recombinant DNA which is incorporated into a vector, into an
autonomously replicating plasmid or virus, or into the genomic DNA
of a prokaryote or eukaryote; or which exists as a separate
molecule (e.g., a cDNA or a genomic or cDNA fragment produced by
polymerase chain reaction (PCR) or restriction endonuclease
digestion) independent of other sequences. It also includes a
recombinant DNA which is part of a hybrid gene encoding additional
polypeptide sequence, e.g., a fusion protein. Also included is a
recombinant DNA which includes a portion of the nucleotides listed
in SEQ ID Nos. 1-12 or which encodes an alternative splice variant
of SEQ ID Nos. 1-12.
[0068] The labels most commonly employed for these studies are
radioactive elements, enzymes, chemicals which fluoresce when
exposed to ultraviolet light, and others. A number of fluorescent
materials are known and can be utilized as labels. These include,
for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue
and Lucifer Yellow. A particular detecting material is anti-rabbit
antibody prepared in goats and conjugated with fluorescein through
an isothiocyanate. Proteins can also be labeled with a radioactive
element or with an enzyme. The radioactive label can be detected by
any of the currently available counting procedures. The preferred
isotope may be selected from .sup.3H, .sup.14C, .sup.32P, .sup.35S,
.sup.36Cl, .sup.51Cr, .sup.57Co, .sup.58Co, .sup.59Fe, .sup.90Y,
.sup.125I, .sup.131I, and .sup.186Re.
[0069] Enzyme labels are likewise useful, and can be detected by
any of the presently utilized colorimetric, spectrophotometric,
fluorospectrophotometric, amperometric or gasometric techniques.
The enzyme is conjugated to the selected particle by reaction with
bridging molecules such as carbodiimides, diisocyanates,
glutaraldehyde and the like. Many enzymes which can be used in
these procedures are known and routinely utilized. The preferred
enzymes are peroxidase, .beta.-glucuronidase, .beta.-D-glucosidase,
.beta.-D-galactosidase, urease, glucose oxidase plus peroxidase and
alkaline phosphatase, etc. U.S. Pat. Nos. 3,654,090, 3,850,752, and
4,016,043 are referred to by way of example for their disclosure of
alternate labeling material and methods.
[0070] By "degeneracy of the genetic code" is meant that some amino
acids are specified by more than one codon. Accordingly, a single
protein sequence could be coded for by multiple DNA sequences due
to the degeneracy of the genetic code.
[0071] As used herein, "cancer vaccine" refers to a therapeutic
vaccine consisting of a vector encoding an antigenic protein or a
peptide fragment thereof. Immunization of an individual with such a
vaccine is meant to induce an immune response to the protein or
peptide, and is directed towards a method of inhibiting growth or
promoting destruction of the melanoma tumor in the individual.
[0072] As used herein, "immunotherapy", as used in the context of
cancer therapy, refers to a therapeutic method achieved by
manipulation of an individual's immune system to inhibit growth or
promote destruction of a tumor.
[0073] As used herein, "antigen" generally refers to a protein or
polypeptide which can, in certain formulations or settings, be
recognized by the immune system and elicit an immune response.
Although carbohydrate moieties may also act as antigens, as used
herein, antigens are defined as proteins or polypeptides which may
or may not be modified post-translationally.
[0074] As used herein, "tumor-associated antigen" refers to an
antigen which is associated with tumor cells. Such proteins need
not be expressed exclusively in or on tumor cells. Generally,
tumor-associated antigens are fetal proteins aberrantly expressed
in tumor cells, mutated cellular proteins which are antigenic due
to the mutation, viral proteins expressed in tumor cells, normal
cellular proteins highly expressed in the tumor compared to normal
tissue, or normal cellular proteins which are mislocalized.
[0075] As used herein, "melanoma antigen" refers to an antigen
which is expressed in melanoma cells, however, expressed need not
be limited to only melanoma cells.
[0076] As used herein, "polymerase chain reaction" or "PCR" refers
to an enzymatic reaction using primers specific for a DNA or cDNA
sequence which results in amplification of the specified
sequence.
[0077] The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant
to limit the present invention in any fashion:
EXAMPLE 1
Patient Population and Clinical Trial
[0078] Nine patients with advanced stage melanoma were enrolled in
a Phase Ib clinical trial (NCI Protocol T97-0005) of intratumoral
injection of a recombinant canarypox virus encoding the human
interleukin-12 (IL-12) gene. The recombinant virus was provided by
Pasteur Merieux Connaught through an agreement with the National
Cancer Institute. Patients were injected with the recombinant virus
encoding IL-12 on days 1, 4, 8, and 11 and serum was collected on
days 0, 18 and 43.
EXAMPLE 2
cDNA Library Construction
[0079] A cDNA expression library derived from the melanoma cell
lines MEL888 and MEL624 (kindly provided by S. Rosenberg, National
Cancer Institute) was synthesized in the 1-ZAP Express vector
(Stratagene). Briefly, total RNA was isolated from the cells using
the RNA-Stat60 reagent (TelTestB), according to the manufacturer's
directions. mRNA was isolated using a 5'.fwdarw.3' mRNA isolation
kit. Five to seven micrograms of mRNA was reverse transcribed using
an oligo dT primer with an internal XhoI site. After second strand
synthesis, EcoRI adapters were added by ligation. The cDNA was
passed through a size exclusion column (Pharmacia) which eliminates
cDNAs smaller than 400 bp in size and the cDNA fragments cloned
into the .lambda.ZapExpress vector, packaged according to the
manufacturer's instructions, and used to infect E. coli cells. As a
preliminary characterization of the library, inserts from
twenty-five randomly selected recombinant plaques were PCR
amplified using T3 and T7 primers to determine insert size ranges
(0.5 to 3.0 kb). The library contained 6.8.times.10.sup.6 primary
recombinants.
EXAMPLE 3
Immunoscreening
[0080] Table 1 shows isolated clones screened for reactivity with
all patients from the study. A cDNA expression library was
generated from the melanoma cell lines MEL888 and MEL624, and serum
from two individuals (patients one and two) was diluted to 1:250,
mixed in equal volume and used to screen the library. Recombinant
plaques were plated at a density of approximately 25,000 plaques
per 150 mm plate, and a total of approximately 200,000 plaques were
screened. After a four hour incubation of the plates at 37.degree.
C., protein expression was induced by incubation of the plates with
nitrocellulose filters saturated with isopropyl
.beta.-D-thiogalactoside (IPTG) overnight. Filters were blocked
with 1% BSA in Tris-buffered saline (TBS; 20 mM Tris (pH 7.5), 150
mM NaCl) and screened with patient sera. Primary sera is
preabsorped with E. coli phage lysate (Stratagene) and diluted
1:250 for screening. After incubating filters with diluted sera,
the filters were washed with TBST (TBS with 0.05% Tween 20 [Sigma])
and incubated with alkaline phosphate-conjugated goat anti-human
IgG (H+L) antibodies (Jackson Labs) at a dilution of 1:5,000 for 1
hour at room temperature. After washing, an NBT/BCIP colorimetric
assay was used to identify positive clones. Positive plaques were
purified to clonality for further study.
[0081] Isolated clones (FIG. 1) were then screened for reactivity
against sera from nine patients with advanced stage melanoma who
were intratumorally injected with a recombinant canarypox virus
encoding the human interleukin-12 (IL-12) gene. Patients were
injected with the recombinant virus encoding IL-12 on days 1, 4, 8,
and 11 and serum was collected on days 0, 18 and 43. Confirmed
immunoreactive plaques were evaluated for reactivity with serum
from ten normal individuals. Only those plaques which were not
reactive with ten normal sera were processed further.
1 TABLE 1 Patients Day 43 Patient 1 1 2 3 4 5 6 7 8 9 Day 18 0 3.1
+ - - - - - - - - + - 3.14 + - - - - - - - - + - 5.16 + - - - - - -
- - + - 5.17 + + - - - - - - - + - 5.23 + - - - - - - - - + - 5.28
+ - - - - - - - - + - 5.31 + - - - - - - - - + - 3.3T - - - - + - -
- + n/a n/a
[0082] Isolated clones were screened for reactivity with all
patients from the study.
EXAMPLE 4
Isotype Analysis
[0083] Upon plaque purification, the isotype of the reactive
antibodies were determined using human isotype specific antibodies
(Southern Biotech), according to the recommended procedure. Isotype
analysis demonstrated the presence of predominantly IgG antibodies,
consistent with a mature, T.sub.n-dependent immune response.
EXAMPLE 5
Isolation of Plasmid DNA and DNA Sequence Analysis
[0084] Plasmid DNA containing cDNA inserts of interest were
isolated from purified plaques by in vivo excision using a helper
phage system (ExAssist, Stratagene). For single clone excision,
approximately 10.sup.5 phage particles were used to infect
XL-1-Blue MRF cells in the presence of the helper phage and the
cells were incubated for 3 hours at 37.degree. C. To isolate the
excised phagemid which are packaged as filamentous phage particles,
the culture was heated to 70.degree. C. for 20 min, spun at
1000.times.g for 15 min and the supernatant collected. These phage
were used to infect XLOR cells (Stratagene) and the cells were
plated on selective media. These cells do not permit growth of the
helper phage and only allow propagation of the phagemid. Single
colonies were grown in liquid culture and DNA isolated by standard
miniprep procedures.
[0085] Partial DNA sequence of each insert was determined using an
automated DNA sequencer and vector specific primers. Partial DNA
sequences were used in BLAST searches through the National Center
for Biotechnology Information database to identify sequences which
matched previously described genes or expressed sequence tags
(ESTs) (Table 2).
2TABLE 2 Melanoma tumor-associated antigen homology Clone &
Identifier Homology with: Reference 3.1 (SEQ ID No. 1 & 2)
KIAA0663 13 (=3.8 = 3.16) (GenBank Accession No. AB014563) 3.14
(SEQ ID No. 3 & 4) Drosophila disc large protein 14 (GenBank
Accession No. U13896) 3.3T (SEQ ID No. 5 & 6) Ubiquilin; DA41
15 (GenBank Accession No. AF176069, HRIHFB2157) 5.17 (SEQ ID No. 7
& 8) KIAA0555 16 (GenBank Accession No. AB011127) 5.23 (SEQ ID
No. 9) EST: qu76c08.x1; 17 NCI-CGAP-ES02 (GenBank Accession No.
AI354862) 5.28 (SEQ ID No. 10) Various ESTs; Similarity with TR:
G581223 5.31 (SEQ ID No. 11 & 12) RING3* 18 (GenBank Accession
No. X96670) *RING3 has been described by Matthew Scanlan at the
Ludwig Institute for Cancer Research as a potential breast tumor
antigen based on SEREX.
[0086] The positive clones, derived from plaques that bound sera
from patients intratumorally injected with a recombinant virus
expressing IL-12, represent putative antigens specific to melanoma
tumors.
EXAMPLE 6
Uses
[0087] Novel tumor-associated antigens may be useful for detection,
diagnosis, and staging of melanoma. Detection and diagnosis of
melanoma is currently based on visual identification of melanoma
lesions, while staging is based on depth of the lesion at the time
of diagnosis. While these visual guidelines have proven quite
useful, the use of additional marker proteins and the molecular
characterization of melanoma lesions may prove more accurate in
defining the clinical course of the disease.
[0088] Novel tumor-associated antigens may also be useful for
disease monitoring. Metastatic melanoma can spread to a variety of
sites. The identification of novel tumor antigens may allow
recurrence and metastatic disease to be detected and disease burden
monitored. Antigenic proteins expressed on the cell surface may
provide targets for detection and imaging of metastatic
disease.
[0089] Tumor-associated antigens may additionally be useful as
novel targets for immunotherapy. Several immunotherapeutical
approaches to melanoma are currently under development, and
tumor-associated antigens may provide additional therapeutic
targets for intervention.
[0090] The following references were cited herein:
[0091] 1. van der Bruggen, P et al. Science 254:1643-1647,
1991.
[0092] 2. Kawakami, Y et al. Proc. Natl. Acad. Sci. USA
91:3515-3519, 1994
[0093] 3. Toso, J F et al. Cancer Res. 56:16-20, 1996.
[0094] 4. Robbins, P F et al. Cancer Res. 54:3124-3126, 1994.
[0095] 5. Schlichtholz, B et al. Cancer Res. 52: 6380-6384,
1992.
[0096] 6. Lubin, R et al. Cancer Res. 53:5872-5876, 1993.
[0097] 7. Stauss, H J J Natl Cancer Inst. 87:820-821, 1995.
[0098] 8. Disis, M L et al. Cancer Res. 54:16-20, 1994.
[0099] 9. Chinni S et al. Clin Cancer Res. 3:1557-1564, 1997.
[0100] 10. Sahin, et al. Proc. Natl Acad Sci. USA 92:11810-11813,
1995.
[0101] 11. Tureci, O et al. Cancer Res 56:4766-4772, 1996.
[0102] 12. Old, L O & Chen, Y T. J. Exp. Med. 187:1163-1167,
1998.
[0103] 13. Ishikawa, K et al. DNA Res. 5:169-176, 1998.
[0104] 14. Lu, R A et al. Proc. Natl. Acad. Sci. USA 91:9818-22,
1994.
[0105] 15. Hanaoka, E et al. J. Human Genet. 45:188-191, 2000.
[0106] 16. Ishikawa, K et al. DNA Res. 5:31-19, 1998.
[0107] 17. Natl. Cancer Institute; Cancer Genome Anatomy
Project.
[0108] 18. Thorpe, K L et al. Immunogenetics 44:391-6, 1996.
[0109] Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which
the invention pertains. Further, these patents and publications are
incorporated by reference herein to the same extent as if each
individual publication was specifically and individually indicated
to be incorporated by reference.
[0110] One skilled in the art will appreciate readily that the
present invention is well adapted to carry out the objects and
obtain the ends and advantages mentioned, as well as those objects,
ends and advantages inherent herein. The present examples, along
with the methods, procedures, treatments, molecules, and specific
compounds described herein are presently representative of
preferred embodiments, are exemplary, and are not intended as
limitations on the scope of the invention. Changes therein and
other uses will occur to those skilled in the art which are
encompassed within the spirit of the invention as defined by the
scope of the claims.
Sequence CWU 1
1
12 1 586 DNA Unknown mat_peptide
5,8,17,38,45,68,95,465,470,472,476,506,50- 7, 514,540,552,564, 5'
end of clone 3.1 encoding a melanoma tumor-associated antigen; n =
unknown 1 atggnatntg tttgaancct attttgaaac cttgccanct cgaantaacc 50
ctcataaggg caacaaanct ggagctcgcg cgcctgcagg tcganactag 100
tggttccaaa gaattcggca cgagcctaat caaggagaag actgctattt 150
ttttttctat tccacatgta ccaaaggcga cagctgccca ttccgtcact 200
gtgaagctgc aataggaaat gaaactgttt gcacattatg gcaagaaggg 250
cgctgttttc gacaggtgtg caggtttcgg cacatggaga ttgataaaaa 300
acgcagtgaa attccttgtt attgggaaaa tcagccaaca ggatgtcaaa 350
aattaaactg cgctttccat cacaatagag gaccgatatg ttgatggcct 400
tttcctacct ccgagcaaaa ctgtgttgcc cactgtgcct gagtcaccag 450
aagaggaaag tgaangctan cncaantttc agttcaagct ggaacaaaat 500
tggctnntcc aatnccaaat cccttcccct taaacctggn ggaaaccgtt 550
antgaaaagt tagnaaattt tcccgaaaat tgttct 586 2 615 DNA Unknown 3'
end of clone 3.1 encoding a melanoma tumor-associated antigen 2
cgcctggaaa agggtaagta acccagggac ggagccttgg gtaaagtgtg 50
tcatccccca attggcccaa cgtaagcagt gagatgccgc tgtgtcattg 100
ccgctgtgaa cctcagtcca caggtcctac aggaccccca gccaaaaagg 150
cagctgtggc tgttgtcccg cttgtcttga ggcaaatcag tcctgtgcct 200
gaagcagaaa atcctagagc agtcttgtgc tgcctccaac ccagtccttt 250
cagattcctc acccccagag gtgtctggcc ctcctcatcc caatgagcat 300
gaaaactgcc gactcagctt tgcctcaaca ggaaagcccc cactcttgtg 350
gaggatgatt ttagaaacta atatgggaga tttcaggagg caaattggaa 400
gctgagattg acctggatct gggaaaatga aatgaccttt gcttgagcta 450
tcaaaatgat tatagctgaa ggtggtagtg aggacccttt aaaaaaaaaa 500
tcgccaaaaa ctggcttagt ttcattattg aactttacct gagatgatct 550
tttttagtta gaatttgccc caatcaaaga accttgaatt atccaaaaaa 600
aaaaaaaaaa aaaaa 615 3 574 DNA Unknown mat_peptide
8,9,21,29,52,473,515,545,573 5' end of clone 3.14 encoding a
melanoma tumor-associated antigen; n = unknown 3 ggggtatnnt
ttgaaccttc nttctccant taaccctcat aagggaacaa 50 anctggactc
gcgcgcctgc aggtcgacac tagtggatcc aaagaattcg 100 gcacgagttt
atcttagccg gaggacctgc tgatctaagt ggagagctca 150 gaaaaggaga
tcgtattata tcggtaaaca gtgttgacct cagagctgct 200 agtcatgagc
aggcagcagc tgcattgaaa aatgctggcc aggctgtcac 250 aattgttgca
caatatcgac ctgaagaata cagtcgtttt gaagctaaaa 300 tacatgattt
acgggagcag atgatgaata gtagtattag ttcagggtca 350 ggttttcaaa
tggttcctga ggttttttgt tgttgtccgt gttgttactg 400 ttgttcttgt
catcaggttt gattttggtc cttgcccttt ccttctagtt 450 ctccttttat
taataggaaa ggnaggcaaa agcccccatt tatgtggggg 500 ggttttcccc
ttaanacagc ttttcattcc acctggttct gcacntaaaa 550 ttggccccaa
aatcttcatt ggng 574 4 528 DNA Unknown 3' end of clone 3.14 encoding
a melanoma tumor-associated antigen 4 accgtgttga actaaaactt
ttcgggccca tttttaaatg gggttttcag 50 ggcccgtttt caaaaggttc
ctaaggtttt tgttgtgccc gggttgtaac 100 tggttgttct gtcatcaggt
ttgattttgg gcccttgccc tttccttcta 150 gttctccttt tattaatagg
aaggcaggca aaagccccat ttatgtggtg 200 ttttcccctc agacagcttt
catccactgc tctgcactag aattgcacaa 250 atcttcatgg tgagcaattt
taagaaatgt tagtaaaagg tagaaattat 300 ttcacaaatc agtttctctg
gtccttcata ttaataataa tatttggctt 350 cccattgctc tttggagttg
tttattaaat atgtgttttt gacaacctcc 400 tcattagttt cttaaatgag
tactggtttg taaagaatta tcaacattat 450 ccattccatt tatgaagaag
aggagaacag ctaataaact gtattgaaat 500 ccaaaaaaaa aaaaaaaaaa aaaaaaaa
528 5 547 DNA Unknown mat_peptide
4,5,26,183,216,223,231,250,252,271,315,342,351,
356,357,358,360,367,370,3- 72,374,386,394,395,402,
409,416,420,421,435,444,461,467,482,493,495,499, 500,517,527 5' end
of clone 3.3T encoding a melanoma tumor-associated antigen; n =
unknown 5 aggnnggagc actcagctcg aaattnaccc tcactaaagg gaacaaaagc 50
tggagctcgc gcgcctgcag gtcgacacta gtggatccaa agaattcggc 100
accaggaaat ccagaaatta gtcatatgtt gaataatcca gatataatga 150
gacaaacgtt ggaacttgcc aggaatccag cantgatgca ggagatgatg 200
aggaaccagg accgancttt gancaaccta naaagcatcc cagggggatn 250
tnatgcttta aggcgcatgt ncacagatat tcatgaacca atgctgagtg 300
ctgcacaaaa acaanttggg gggaaaccat ttgcttcctt gngaacaaat 350
natccnnngn ggaaggnagn cnanccttcc cgtccngaaa tttnnattcc 400
cntcccatnc cttggncccn naactcccaa atttntaaat ttcnacggcc 450
tgcacctggg ngggcantcg gttcctgcca gnggcctttt ggnanatann 500
ctgccaaatt ggcccgngag agaactnttt tttcaacaca caaaatg 547 6 413 DNA
Unknown 3' end of clone 3.3T encoding a melanoma tumor-associated
antigen 6 ttttccgtga gaaacattca gttaaacaca gggggggatc accagctaat 50
aaagggtatg ggtcccctca tacagcattt tgtttttaaa aaatggattt 100
atttttgtaa cgggtttaaa ctttaaaaac ccgctttatt tcatttgctt 150
tgggaattgg cgttaaacca accccaatta gccttttaag ggggctaaag 200
gggggtttcg gaattttttt ttcggaggga ataagggaag gagatcttgc 250
attaatggat ttttaaaacc cccctttaaa gtgggggacc agattttgtc 300
ctgcatctgt ccagttattt gctttttaaa catagcctat ggtagtaatt 350
tatgtagaat aaaagcatta aaaagaagca aatcatttgc tctctaaaaa 400
aaaaaaaaaa aaa 413 7 616 DNA Unknown mat_peptide
4,5,11,453,481,498,501,531,552,568,569,571,572,
586,588,591,595,602,614 5' end of clone 5.17 encoding a melanoma
tumor-associated antigen; n = unknown 7 aggnntaggt naccctacta
aagggaacaa aagctggagc tcgcgcgcct 50 gcaggtcgac actagtggat
ccaaagaatt cggcacgagc cgactcggtc 100 acaaggaaaa tggattcagt
ttgcatctct ccctccttta aacagcttct 150 ccgggtctca gcatggtatc
aaagcttgaa agagagaaga ctcaagaagc 200 gaagaggatt cgtgagctgg
agcagcgcaa gcacacggtg ctggtgacag 250 aactcaaagc caagctccat
gaggagaaga tgaaggagct gcaggctgtg 300 agggagaacc ttatcaagca
gcacgagcag gaaatgtcaa ggacggtgaa 350 ggtacgtgat ggaagaagat
ccagaggctc aagtctgctc tctgtgctct 400 ccgcgacggc agcagtgacc
aaagtaagga cagcgctacc attgaggccc 450 ggnaaggagg cccgaaacct
gtttgaccca nacgccttaa gctttacngg 500 naaattgcgg acctgaaacg
gccaaaagcc ngggggccaa aggttttgcc 550 antttgatcc caaggccnna
nnttttatag tgggcntnga nggcnttatc 600 cncaaacctt taanat 616 8 510
DNA Unknown 3' end of clone 5.17 encoding a melanoma
tumor-associated antigen 8 gtacccccga aaagggttta cccttaaggg
caattgttcc ccccccccct 50 aagggttcca aagttaagat tccccctgaa
cggctaaggg ttttaaagcc 100 ttattcaagg tttcttactt gccagttcct
accaaaccct gtaaaatctc 150 caataatgct gcatttaatg aaacatggta
tatgtcaaat cagaagagaa 200 gaactataaa catatattgt gtaaagaaaa
agttcagcaa tggaactagt 250 tttgcagatc aagcaaagat gtgtcttggg
catggaacca aagttacaat 300 gaaatattca acccctgctg tgcagggggg
tcattttaat gtaacaccac 350 accccatgga aacactagtc ctgataataa
acatcatttt aaaagatcaa 400 aacaaacaaa caaaaaaaac aagggtgggt
ggggagtgaa gcacgaggaa 450 tacctatgaa gagctattta caataaaatg
tttcatttga aaaaaaaaaa 500 aaaaaaaaaa 510 9 3512 DNA Unknown
prim_transcript complete sequence of clone 5.23 encoding a melanoma
tumor-associated antigen 9 ggacaacagc tggagctcgc gcgcctgcag
gtcgacacta gtggatccaa 50 agaattcggc acgagagaaa gtaaggaaaa
gttcagggta tagaaatagc 100 tattcagtga ctttgtattt ttacttgtgc
tcttaagaac ctttattcat 150 gtaatgcaaa gtaatttgtg ttgaagttga
acttgtgaga aaatatatag 200 tacctaatgc attctcattt ggaatatgtg
atctgtagaa atggaaatat 250 ttttatttat tttactgttt ttataggagg
ttcgtaaagt gaatgaaagc 300 atcaagataa tcacccattg agaaaatgtg
ttgatacaat acttaaaaag 350 tgccctacag agtatcagga aaaaatgggt
aggaacatgg atgattatga 400 agattttgat gaaaagcata gtatctatcc
agtgaaaaaa gtctggtaaa 450 actgccataa acaggggact ttgctaatta
taagtatttt actaatgatg 500 atttttaatt agacttctaa tcattgctca
taaaaaaagg aatttttagt 550 gaatgtgtat ttaaaacttc ctttaatccc
gtccttatca ttctttgaaa 600 tattttatct ctgtgtatac cagcaggggt
attattggcg tttggggagg 650 gagaattctt cactgagcat aactgttaca
ttatataaaa ctgttacatc 700 attttggaac attaatattc tcagcctgac
ccagtaaatg ccctagcact 750 ttcccattgt tatgacaatc caaacatgct
ccctagtgga gagttgaacc 800 actgttggat cagaacactg ccaggtctac
ccccattctc ttttttaggt 850 gatttattca gttcagagac accgtcgaac
tcaagtacaa tggcagattc 900 ttttggaaca agcattttat ctagaagatg
tagcaaaaaa tgaaactagt 950 gctactcatc agtttgttca cacctttcaa
tcgccagagc cagaaaatcg 1000 atttatccaa tatttttata atcctacatt
tggtatgtaa tttgatataa 1050 atttcaaact ttaatgatga aaagttttct
gtagaaagaa gttatgtatt 1100 ttcaccaatg caaagttgaa ttttatttgt
attatttgat ttataccatg 1150 tgatattaag tatctggtaa catttcccca
aaataactgt tttacttatc 1200 atataacata taatccatca gtttccactg
ttacttcaca aataataaaa 1250 attctattaa aaaacatgta tacatcaagc
atatttttta taatgcataa 1300 tatatacaat tatgcattgc ttaatgactg
ggattactct gagaaatgta 1350 ttgttaggca atttcatcac tgcatgagca
tcatagggta tgtactaaac 1400 ctagatggta tagtacaggt aggcaaatat
gggtattggc ttattactcc 1450 taaggctaca aaacctatac agcatggtta
ctgtacctga aagtggtagg 1500 cagttgtaca ccagggtttt tggtttttaa
acttgaaaaa tatttttaaa 1550 agccgttgta atttttgggg atcacccttt
ttttgcaccc tctttggccg 1600 ggaggtgtat tgacccctat gtcctttaaa
aatagaaatt tagtattttt 1650 cttccagctt tggttttttt ttatttgaac
tatattttgg ttaattcctc 1700 ttgatattaa cctttatagt ttttcaggaa
attagttaaa atccgttgta 1750 ttttatggtc cccatttagc gtccttcatg
ggtggaagtt tttatgtgac 1800 acaaggctga taaaaaggtt aaatttttaa
gttattttct caccaggctg 1850 gggttttttc ttcagtcttg aacaaacaac
tgaaatttgg cttaagtaag 1900 tcctccttga tattaaccat ttattagtct
taattataaa accctatact 1950 ttgtaggtta tcattttttc tccttttttg
ctaaatttat gggcaatccc 2000 ttccaagtat ttgtcaaatt tagtgtgaag
aaacttaaaa gcaaggtacc 2050 aaaagtgtca tagtattaaa acttctattt
accttattta ttttaaaaaa 2100 attgttatat tcacttgatt tctccctttg
catgtttggt tttgagtatg 2150 aagacttaat ggctataaca aatatctcag
aaaactcctt taacaaaaat 2200 ccttcctaat taaatgaagg aatgatgtgt
tatctgtttt cattcattca 2250 acaaatattt gggtacatta gtgctatgta
ttattgggtg ctgggtagct 2300 tggtatatat cagtttaaaa agacagaaat
tcctgccctt gtggagtgag 2350 aaaaacagac aataaacata taaaggcata
aagattctga ataggcagtt 2400 gattatagaa attgaaattc aagggaggag
tctgaattgc agatatgaat 2450 tagggtacca tcaatgtgta gggaaccatg
gggtcaggat aaaatcaata 2500 aagaagtaat tgagatagag aaaagagaaa
agtctgagga ccaagcctga 2550 ggcactccag aatttagaga ttaggtggat
gagaagtaac tagcagaaaa 2600 gactagaaaa ggaggggcca gtgagatagg
aaaattagga caatgaagtg 2650 ttttgaggaa aagagtatat aaagtacctt
ttcaaatgtt gcacatagat 2700 taaggatcat atatattaag acctgaccat
tggattttag agaagtgagg 2750 ggagaggata aaaaagtctg actgtaattt
aaaagaaata agaagaggag 2800 caattggaga cagactagaa aactctaaaa
atgttttcct tataaaaggg 2850 aacagagaaa aggggtagta gctgaaagag
gattgggggc atagtcaaga 2900 gaaattatca catgtaatta gtaaatgata
taatagaatt tgaggccagg 2950 cgcggtggct cacacctgta atcccagcac
tttgggaggc cgaggcggca 3000 gatcacaagg tcaagagatt gagaccatcc
tggccaacat ggtgaaaccc 3050 cgtctctact aaaaatacaa aaattagctg
ggcgtggtgg tgcgtgcctg 3100 tagtcccagc tactcgggag gctgaggcag
gagaatcctt gaacaggagg 3150 cggaggttgc agtgagccga gattgtgcca
tgcactccag cctacctgta 3200 gtcccagcta ctcgggaggc tgaggcagga
gaatcacttg aacccaggag 3250 gtggaggttg cagtgagccg agattgcgcc
actgcactcc agcctacctg 3300 tagtcccagc tacttgggag atgaggcagg
agaatcgctt gaacccggga 3350 ggcagaggtt gcagtgagcc aagattgcac
cactacactc cagcctgggg 3400 acagaatgag actccgtcaa aaaaaaaaaa
aaaaactcga gagtacttct 3450 agagcggccg cgggcccatc gattttccac
ccgggtgggg taccaggtaa 3500 gtgtacccgt cg 3512 10 2634 DNA Unknown
prim_transcript complete sequence of clone 5.28 encoding a melanoma
tumor-associated antigen 10 ggacgccgct ggagctccgc gcctgcaggt
cgacactagt ggatccaaag 50 aattcggcac cagcctgcag gtactgctgc
tcgtgcctcc ggctccggcc 100 cctgagcgat ggtcctttcc ttctgccacg
gcgggatcgg gcactcaccc 150 agttgcaagt gcgagcacta tggagtagcg
cagggtctcg agctgtggcc 200 gtggacttag gcaacaggaa attagaaata
tcttctggaa agctggccag 250 atttgcagat ggctctgctg tagtacagtc
aggtgacact gcagtaatgg 300 tcacagcggt cataaaacaa aaccttcccc
ttcccagttt atgcctttgg 350 tggttgacta cagacaaaaa gctgctgcag
caggtagaat tcccacaaac 400 tatctgagaa gagaggttgg tacttctgat
aaagaaattc taacaagtcg 450 aataatagat cgttcaatta ggaccgctct
ttccagctgg ctacttctat 500 gatacacagg ttctgtgtaa tctgttagca
gtagatggtg taaatgagcc 550 tgatgtccta gcaattaatg gcgcttcgta
gccctctcat tatcagatat 600 tccttggaat ggacctgttg gggcagtacg
aataggaata attgatggag 650 aatatgttgt taacccaaca agaaaagaaa
tgtcttctag tactttaaat 700 ttagtggttg ctggagcacc taaaagtcag
attgtcatgt tggaagcctc 750 tgcagagaac attttacagc aggacttttg
ccatgctatc aaagtgggag 800 tgaaatatac ccaacaaata attcagggca
ttcagcagtt ggtaaaagaa 850 actggtgtta ccaagaggac acctcagaag
ttatttaccc cttcgccaga 900 gattgtgaaa tatactcata aacttgctat
ggagagactc tatgcagttt 950 ttacagatta cgagcatgac aaagtttcca
gagatgaagc tgttaacaaa 1000 ataagattag atacggagga acaactaaaa
gaaaaatttc cagaagcccg 1050 atccatatga aataatagaa tccttcaatg
ttgttgcaaa ggaagttttt 1100 agaagtattg ttttgaatga atacaaaagg
tgcgatggtc gggatttgac 1150 ttcacttagg aatgtaagtt gtgaggtaga
tatgtttaaa acccttcatg 1200 gatcagcatt atttcaaaga ggacaaacac
aggtgctttg taccgttaca 1250 tttgattcat tagaatctgg tattaagtca
gatcaagtta taacagctat 1300 aaatgggata aaagataaaa atttcatgct
gcactacgag tttcctcctt 1350 atgcaactaa tgaaattggc aaagtcactg
gtttaaatag aagagaactt 1400 gggcatggtg ctcttgctga gaaagctttg
tatcctgtta ttcccagaga 1450 ttttcctttc accataagag ttacatctga
agtcctagag tcaaatgggt 1500 catcttctat ggcatctgca tgtggcggaa
gtttagcatt aatggattca 1550 ggggttccaa tttcatctgc tgttgcaggc
gtagcaatag gattggtcac 1600 caaaaccgat cctgagaagg gtgaaataga
agattatcgt ttgctgacag 1650 atattttggg aattgaagat tacaatgtga
catggacttc aaaatagctg 1700 gcacttaata aaggaataac tgcattacag
gctgatatta aattacctgg 1750 aataccaata aaaattgtga tggaggctat
tcaacaagct tcagtggcaa 1800 aaaaggagat attacagatc atgaacaaaa
ctatttcaaa acctcgagca 1850 tctagaaaag aaaatggacc tgttgtagaa
actgttcagg ttccattatc 1900 aaaacgagca aaatttgttg gacctggtgg
ctataactta aaaaaacttc 1950 aggctgaaac aggtgtaact attagtcagg
tggatgaaga aacgttttct 2000 gtatttgcac caacacccag tgctatgcat
gaggcaagag acttcattac 2050 tgaaatctgc aaggatgatc aggagcagca
attagaattt ggagcagtat 2100 ataccgccac aataactgaa atcagagata
ctggtgtaat ggtaaaatta 2150 tatccaaata tggctgcggt actgcttcat
aacacacaac ttgatcaacg 2200 aaagattaaa catcctactg ccctaggatt
agaagttggc caagaaattc 2250 aggtgaaata ctttggacgt gacccagccg
atggaagaat gaggctttct 2300 cgaaaagtgc ttcagtcgcc agctacaacc
gtggtcagaa ctttgaatgc 2350 agaagtagta ttgtaatggg agaacctatt
tccagtcatc atctaattct 2400 cagtgatttt ttttttttaa agagaattct
agaattctat tttgtctagg 2450 gtgatgtgct gtagagcaac attttagtag
tatcttccat tgtgtagatt 2500 tctatataat ataaatacat tttaattatt
tgtactaaaa aaaaaaaaaa 2550 aaaactcgag agtacttcta gagcgggccg
cgggcccatc gattttccac 2600 ccgggggggt accaggtaag tgtcccggct cacc
2634 11 673 DNA Unknown mat_peptide 3,4,5,6,16,21,580,590,654,668
5' end of clone 5.31 encoding a melanoma tumor-associated antigen;
n = unknown 11 ggnnnntttg tttatnacac nccagctcga aattaaccct
cactaaaggg 50 aacaaaagct ggagctcgcg cgcctgcagg tcgacactag
tggatccaaa 100 gaattcggca cgaggtgtta ccagtgccca tcaggtgcct
gccgtctctt 150 ctgtgtcaca cacagccctg tatactcctc cacctgagat
acctaccact 200 gtcctcaaca ttccccaccc atcagtcatt tcctctccac
ttctcaagtc 250 cttgcactct gctggacccc cgctccttgc tgttactgca
gctcctccag 300 cccagcccct tgccaaggta tgatctgtgg atttcctctg
ggcagcaggg 350 aggcaagggt cttaagtaaa gtgggcttgg agtgacaggt
tccctatctt 400 gtttctttct gcagaaaaaa ggcgtaaagc ggaaagcaga
tactaccacc 450 cctacaccta cagccatctt ggctcctggt tctccagcta
gccctcctgg 500 gagtcttgag cctaaggcag cacggcttcc cctatgcgta
gagagagtgg 550 tcgcccatca agcccccacg caaagacttn ctgactctan
caacaacacc 600 agactctaag aaaggaaagc tttagaacag ttaaacattg
caatggattt 650 tgangagtac tctctaanaa cat 673 12 593 DNA Unknown 3'
end of clone 5.31 encoding a melanoma tumor-associated antigen 12
ttgaaaataa tgatgggagt tttttgtcat gtgtgtgcaa ctcaacgagg 50
tctcctgtct gacagtgtaa attggagcta tatcacttgg gggctgggag 100
tagggcctgt ttatcagcat agttttgagt ttggcctctt tctaggatga 150
tttgagttcc gttatgccaa gatgccagat gaaccactag aaccagggcc 200
tttaccagtc tctactgcca tgccccctgg cttggccaaa tcgtcttcag 250
agtcctccag tgaggaaagt agcagtgaga gctcctctga ggaagaggag 300
gaggaagatg aggaggacga ggaggaagaa gagagtgaac ctcagactca 350
gaggaagaaa gggctcatcg cttagcagaa ctacaggaac aggtattttg 400
tcactcttga aagtttttat tgggtaagag gttcatgccc tttgtcctca 450
ttttttcttc ttgttatttt atctttattt actttttcca cttcatgttt 500
tttttccttt agcttcgggc agtacatgaa caactggctg ctctgtccca 550
gggtccaata tccaagccca agaggaaaaa aaaaaaaaaa aaa 593
* * * * *