Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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T.~OT.ATED NUCLEIC ACID MnT.~ul.~ WHICH ENCODES MURINE
TUMOR REJECTION ANTIGEN PRE~ SMAGE-3
FIELD OF THE lNv~NllON
This invention relates to a nucleic acid molecule which
codes for a murine, tumor rejection antigen precursor. The
tumor rejection antigen precursor encoding sequence is a
murine sequence, isolated from a murine autosome in contrast
to all previously identified MAGE and MAGE related tumor
rejection antigen encoding sequences.
0 RA~R~.R~UND AND PRIOR ART
The process by which the m~mm~l ian immune system
recognizes and reacts to foreign or alien materials is a
complex one. An important facet of the system is the T
lymphocyte, or "T cell" response. This response requires that
T cells recognize and interact with complexes of cell surface
molecules, referred to as human leukocyte antigens ("HLA"), or
major histocompatibility complexes ("MHCs"), and peptides.
The peptides are derived from larger molecules which are
processed by the cells which also present the HLA/MHC
molecule. See in this regard Male et al., Advanced Immunoloqv
(J.P. Lipincott Company, 1987), especially chapters 6-10. The
interaction of T cells and HLA/peptide complexes is
restricted, requiring a T cell specific for a particular
combination of an HLA molecule and a peptide. If a specific
T cell is not present, there is no T cell response even if its
partner complex is present. Similarly, there is no response
if the specific complex is absent, but the T cell is present.
This mechanism is involved in the immune system's response to
foreign materials, in autoimmune pathologies, and in responses
to cellular abnormalities. Much work has focused on the
mechanisms by which proteins are processed into the HLA
binding peptides. See, in this regard, Barinaga, Science 257:
880 (1992); Fremont et al., Science 257: 919 (1992); Matsumura
et al., Science 257: 927 (1992); Latron et al., Science 257:
964 (1992).
The mechanism by which T cells recognize cellular
: :~
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abnormalities has also been implicated in cancer. For
example, in PCT application PCT/US92/04354, filed May 22,
1992, published on November 26, 1992, and in U.S. Serial No.
08/142,368 filed May 2, 1994, both incorporated by reference,
a family of genes is disclosed, and which are processed into
peptides which, in turn, are expressed on cell surfaces, which
can lead to lysis of the tumor cells by specific CTLs
cytolytic T lymphocytes, or "CTLs" hereafter. The genes are
said to code for "tumor rejection antigen precursors" or
"TRAP" molecules, and the peptides derived therefrom are
referred to as "tumor rejection antigens" or "TRAs". The
applications disclose, inter alia, Smage I and Smage II, two
murine tumor rejection antigen precursor encoding sequences.
See Traversari et al., Immunogenetics 35: 145 (1992); van der
Bruggen et al., Science 254: 1643 (1991), for further
information on this family of genes. Also, see U.S. patent
application Serial Number 807,043, filed December 12, 1991,
now U.S. Patent No. 5,342,774, incorporated by reference in
its entirety. The "MAGE" family of tumor rejection antigen
precursors is disclosed in this patent.
In U.S. patent application Serial Number 938,334, now
U.S. Patent No. 5,405,940, April 15, 1995, the disclosure of
which is incorporated by reference, it is explained that the
MAGE-1 gene codes for a tumor rejection antigen precursor
which is processed to nonapeptides which are presented by the
HLA-Al molecule. The nonapeptides which bind to HLA-Al follow
a "rule" for binding in that a motif is satisfied. In this
regard, see e.g. PCT/US93/07421; Falk et al., Nature 351: 290-
296 (1991); Engelhard, Ann Rev. Immunol. 12: 181-207 (1994);
Ruppert et al., Cell 74: 929-937 (1993); Rotzschke et al.,
Nature 348: 252-254 (1990); Bjorkman et al., Nature 329: 512-
518 (1987); Traversari et al., J. Exp. Med. 176: 1453-1457
(1992). The reference teaches that given the known
specificity of particular peptides for particular H~A
molecules, one should expect a particular peptide to bind to
one HLA molecule, but not to others. This is important,
because different individuals possess different HLA
.
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phenotypes. As a result, while identification of a particular
peptide as being a partner for a specific HLA molecule has
diagnostic and therapeutic ramifications, these are only
relevant for individuals with that particular HLA phenotype.
There is a need for further work in the area, because cellular
abnormalities are not restricted to one particular HLA
phenotype, and targeted therapy requires some knowledge of the
phenotype of the abnormal cells at issue.
In U.S. Patent Application Serial Number 008,446, filed
January 22, 1993 and incorporated by reference, the fact that
the MAGE-1 expression product is processed to a second TRA is
disclosed. This second TRA is presented by HLA-Cw~1601
molecules. The disclosure shows that a given TRAP can yield
a plurality of TRAs, each of which will satisfy a motif rule
for binding to an MHC molecule.
In U.S. Patent Application Serial Number 994,928, filed
December 22, 1992, and incorporated by reference herein
teaches that tyrosinase, a molecule which is produced by some
normal cells (e.g., melanocytes), is processed in tumor cells
to yield peptides presented by HLA-A2 molecules.
In U.S. patent application Serial No. 08/032,978, filed
March 18, 1993, and incorporated by reference in its entirety,
a second TRA, not derived from tyrosinase is taught to be
presented by HLA-A2 molecules. The TRA is derived from a
TRAP, but is coded for ~y a non-MAGE gene. This disclosure
shows that a particular HLA molecule may present TRAs derived
from different sources.
In U.S. patent application Serial No.08/079,110, filed
June 17, 1993 and incorporated by reference herein, an
unrelated tumor rejection antigen precursor, the so-called
"BAGE" precursor is described. The BAGE precursor is not
related to the MAGE family.
In U.S. patent applications Serial No. 08/096,039 and
Serial No. 08/250,162, both of which are incorporated by
reference, non-related TRAP precursor GAGE is also disclosed.
The work which is presented by the papers, patent, and
patent applications cited supra deal, in large part, with the
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MAGE family of genes, and the unrelated BAGE, GAGE and DAGE
genes, showing that there are different, additional tumor
rejection antigen precursors expressed by cells.
As was pointed out, supra, PCT Application PCT/US92/04354
and U.S. Serial No. 08/142,368 filed May 2, 1994 disclose
murine sequences Smage-I and Smage-II. These sequences are
isolated from the murine X chromosome, which is typical of the
MAGE family of tumor rejection antigen precursors.
A third murine tumor rejection antigen precursor coding
10. sequence has now been isolated and cloned. This sequence,
referred to hereafter as Smage-3, was isolated from a murine
autosome. It hybridizes to human MAGE encoding sequences, and
thus is useful, e.g., as a diagnostic reagent useful in
determining expression of human MAGE tumor rejection antigen
precursors, and hence the presence of tumors in a sample or
patient.
The invention is explained in further detail in the
disclosure which follows.
BRIEF D~rRTPTION OF THE FIGURES
Figure 1 shows the result of Southern blotting
experiments which establish that Smage-3 is autosomal in
origin .
n~TATr.~n D~TPTION OF PREFERRED EMBOD
Example 1
In a first experiment, a probe based upon the human MAGE-
1 tumor re~ection antigen precursor gene was used to determine
if comparable sequences were found in the murine genome.
A PCR fragment of 1111 base pairs was used as the probe.
This probe was prepared by carrying out standard PCR with
MAGE-1 as a template, using sense primer:
5'-AGT CCT CAG GGA GCC TCC-3' (SEQ ID NO: 1)
and antisense primer:
5'-TAT CCC AAT TCA CAA AA-3 (SEQ ID NO: 2)
Two successive PCR amplifications were performed (30 cycles:
1 minute at 94~C, 2 minutes at 45~C, 3 minutes at 72~C). One
ng of plasmid DNA (contalning the 2.4 kb BamHI fragment of
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MAGE-1 cloned into pTZ19R~ was used as substrate for the first
round of PCR. For the second round, 1 ul out of 100 ul of
product of the first round was used. The fragment was
~ purified from a low melting point agarose gel, and then was
labeled by incorporation of ~32_p dCTP (3000 Ci/mole), using
random primer extension methodologies.
The probe was then used in genomic Southern blotting
experiments, using the protocol taught by Lurquin et al., Cell
58: 293-303 (1989), incorporated by reference. The probe was
hybridized to genomic DNA obtained from murine DBA/2 kidney
cells. The washing conditions were 2xSSC, 0.1% SDS, at 65 C.
The experiment revealed several faint bands, which
suggested that there were in fact, sequences in the murine
genome related to human MAGE sequences.
~xample 2
In this example, more specific information about the
murine sequence was sought, and found. Genomic DNA o~ DBA/2
kidney cells was partially digested with endonuclease Sau3A,
and cloned into cosmid vector c2RB, in accordance with DePlaen
et al., Proc. Natl. Acad. Sci. USA 85: 2274-2278 (1988),
~incorporated by reference. The library was then screened
using the same probe as in the Southern blotting of example 1.
A 1.7 kb, EcoRI fragment was found to hybridize to the human
probe. Two EcoRI fragments of 3.4 kb and 4.5 kb were also
found.
In a set of follow up experiments, the 1.7 kb fragment
was itself used as a probe, this time on a murine library from
NIH/3T3 cells, cloned in vector AFIXII (purchased from
Stratagene). The hybridization conditions differed in that
the wash was more rigorous, i.e., 0.2xSSC, 0.1% SDS at 65~C.
Three clones were found, each of which contained a 4.5 kb
EcoRI fragment.
These fragments were then sequenced, using standard
methodologies. A portion of the sequence from NIH/3T3, now
= referred to as Smage-3, is set forth in SEQ ID N0: 3.
Comparison of the sequence of Smage-3 with exon-3 of MAGE-1
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reveals 57% identity of the homologous region of the murine
se~uence with MAGE-1 exon 3. The portion set forth in SEQ
ID N0: 3 corrresponds to a sequence beginning at position 472
of the 4.5 kb fragment discussed supra.
Analysis of the se~uence suggests that Smage-3 is a
processed pseudogene. It seems to be intronless, has a 3'
poly(A) tail, and is flanked by inverted repeats of 10
nucleotides each.
Example 3
It had been ascertained, previously, that all MAGE genes
map to the X chromosome. It was therefore of interest to
determine if Smage-3 also maps to the X chromosome.
Somatic cell hybrid VI-6 is a mouse/chinese hamster
hybrid cell, which contains mouse X chromosome and mouse
chromosome 16. See Cox et al., Ann. N.Y. Acad. Sci. 450: 169-
177 (1985). Using Southern blotting, as described supra, the
1.7 kb EcoRI fragment described supra was used to probe VI-6
chromosomal DNA, as well as chromosomal DNA from "clone 8",
which is a hybrid cont~; ni ng, as its only murine chromosome,
the murine X chromosome. See Herman et al., Genomics 10: 961-
970 (1991). Blotting was also carried out on clone "Ell",
which contains only the proximal half of murine X chromosome,
and BALB/c chromosomal DNA.
Figure 1, which follows, shows that the Smage-3 se~uence
is autosomal in nature, i.e., it does not appear in the sex
chromosomes. This is different from Smage-l and Smage-2, two
previously identified murine genes, as described in Boon et
al., PCT Application PCT/US92/04354, which are located on the
X chromosome. Smage-3 is thus the first example of an
autosomal tumor rejection antigen precursor.
Example 4
Northern blot analysis was then carried out on a panel of
tissues to determine expression of the Smage genes.
Total RNA was extracted using the st~n~A~d, guanidine
isothiocyanate procedure of Davis et al., Basic Methods In
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Molecular Biology (Elsevier, N.Y., pp. 130-146 (1986). The
poly(A)~ RNA was purified on an oligo dT cellulose column, and
then Northern analysis was carried out, in accordance with
Davis, supra. No expression of Smage-3 was found; however,
S more sensitive RT-PCR proved to the contrary.
In the RT-PCR, 2 ug of total RNA of each cell type tested
was incubated at 42 C for 40 minutes in a 20 ul volume
con~;n;ng 50 mM Tris-HCl tpH 8.3), 40 mM KCl, 6 mM MgCl2, 1
mM dithiothreitol, 0.1 mg/ml BSA, 0.5 mM of each dNTP, 2 ~M
oligo(dT)12-18, 20 units of RNAsin, and 200 units of MMLV
reverse transcriptase. One tenth of the cDNA obtained was
amplified, for 32 cycles (94 C, 1 minute; 58 C, 2 minutes;
72 C, 3 minutes), using various primers. Primers were chosen
for specificity to known Smage sequences:
5'-GAGCTTGATCCACGAGTTC-3' (SEQ ID NO: 4);
5'-AGGAGACCTGTCCTAGGC-3' (SEQ ID NO: 5);
corresponding to sense and anti-sense sequences for Smage-I
and Smage-II. PCR was carried out in a total volume of 100 ul
containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2,
0.2 mM each dNTP, 50 pmoles of each primer, and 2.5 units Ta~
DNA. In order to distinguish the amplification products,
labelled probes speci~ic for Smage-I and Smage-II, i.e.:
5'-GTCTGCCAGCTCTTTT-3' (SEQ ID NO: 6)
or for Smage-3
5'GCCTGTCAGCTCTTCT-3' (SEQ ID NO: 7),
were used, following gel electrophoresis and blotting in
nitrocellulose paper. The only normal tissue ~ound to express
Smage-3 was testis, with all of brain, heart, kidney, ovary,
sperm lung spleen, thymus blood mononuclear cells, bone
marrow, colon, stomach, liver and pancreas ~eing negative ~or
Smage-3 expression. Leydig cell line TM3, and Sertoli cell
line 7M4 were found negative. In further experiments,
embryonic stem cells and whole mouse embryos 10-18 days old
were tested by RT-PCR. Smage 3 expression was found in the
embryonic stem cell line E14 ES, and in 11-15 day old embryos.
Of the three Smage genes, only Smage-3 message was found in
11-15 day old embryos.
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The foregoing disclosure sets forth an isolated nucleic
acid molecule which encodes a murine tumor rejection antigen
precursor, referred to hereafter as Smage-3. The sequence
shows about 50% homology to previously published human MAGE
sequences, as well as the sequences described in Serial No.
08/403,388 filed March 14, 1995 incorporated by reference.
Thus, Smage-3 can be used in a hybridization assay to
determine expression of MAGE TRAPS. Smage-3 will hybridize to
these sequences under the conditions described, e.g., by
Lurquin, et al., Cell 58: 293-303 (1989), cited supra and
incorporated by reference. For example, an assay carried out
in 50 ul/cm2 of 3.5xSSC, lxDenhardt's solution, 25 mM sodium
phosphate buffer (pH7), 0.5% SDS, 2 mM EDTA and 3X106 cpm/ml
of radiolabelled probe ( ~_32p dCTP, 2-3000 Ci/mmol), using an
Amersham Multiprime labelling kit). This assay is carried out
for 18 hours, at 65 C, followed by a low stringency wash,
e.g., 65 C, 2xSSC, 1% SDS, as indicated in example 1, supra.
The Smage-3 sequence is most closely homologous to MAGE-4 and
MAGE-10, both of which have been found to be expressed in
tumors. See, e.g., U.S. patent application Serial No.
08/346,774 filed November 30, 1994, incorporated by reference,
and DePlaen, et al., Immunogenetics 40: 360-369 (1994), also
incorporated by reference, Table 2 in particular for MAGE-4,
and page 367, first column for MAGE-10.
"Nucleic acid molecule" as used herein refers to all
species of DNA and RNA which possess the properties discussed
supra. Genomic and complementary DNA, or "cDNA" both code for
particular proteins, and as the examples directed to isolation
of Smage-3 coding sequences show, this disclosure teaches the
artisan how to secure both of these.
All isolated nucleic acid molecules which encode Smage-3
proteins, are encompassed by this invention. As used herein,
this refers to conditions such as hybridization with 5X106
cpm/ml for 18 hours at 65 C, followed by 4, 20 minute washes
at 65 C, with each wash using 2xSSC, 0.5% SDS and lxDenhardt's
solution, followed by two washes at 0.2xSSC, 1~ SDS (20
minutes, each wash), and, finally, two washes at 68 C, 1~ SDS,
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a varying concentration of SSC, each of these washes being for
20 minutes. The final concentration of SSC should be no
greater than 0.5xSSC, more preferably it is 0.2xSSC, and most
preferably it is O.lxSSC.
Similarly, RNA molecules, such as mRNA can be secured.
Again, with reference to the skilled artisan, once one has a
coding sequence in hand, mRNA can be isolated or synthesized.
Complementary sequences which do not code for Smage-3,
such as "antisense DNA" or mRNA are useful, e.g., in probing
for the coding sequence as well as in methodologies for
blocking its expression.
It will also be clear that one may manufacture
biologically pure cultures of cell lines which have been
transfected with nucleic acid sequences which code for or
express the Smage-3 molecules. Such cultures can be used as
a source for tumor rejection antigens, e.g., or as
therapeutics. This aspect of the invention is discussed
infra.
Cells transfected with the Smage-3 coding sequences may
also be transfected with other coding sequences. Examples of
other coding sequences include cytokine genes, such as
interleukins (e.g., IL-2 or IL-4), or major histocompatibility
complex (MHC) or human leukocyte antigen (HLA) molecule.
Cytokine gene transfection is of value because expression of
these is expected to enhance the therapeutic efficacy of the
biologically pure culture of the cells in vivo. The art is
well aware of therapies where interleukin transfectants have
been administered to subjects for treating cancerous
conditions. In a particularly preferred embodiment, cells are
transfected with sequence coding for each of (i) Smage-3
molecule, (ii) an HLA/MHC molecule, and (iii) a cytokine.
Such systems are useful in, e.g., the screening of potentially
valuable therapeutic agents.
Transfection with an MHC/HLA coding sequence is desirable
because certain of TRAs derived from Smage-3 may be
preferentially or especially presented only by particular
MHC/HLA molecules. Thus, where a recipient cell already
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expresses the MHC/HLA molecule associated with presentation of
a TRA, additional transfection may not be necessary although
further transformation could be used to cause overexpression
of the antigen. On the other hand, it may be desirable to
transfect with a second sequence when the recipient cell does
not normally express the relevant MHC/HLA molecule. It is
to be understood, of course, that transfection with one
additional se~uence does not preclude further transfection
with other sequences.
The term "biologically pure" as used in connection with
the cell line described herein simply means that these are
essentially free of other cells. Strictly speaking, a "cell
line" by definition is "biologically pure", but the recitation
will establish this fully.
Transfection of cells requires that an appropriate vector
be used. Thus, the invention encompasses expression vectors
where a coding sequence for the Smage-3 TRAP of interest is
operably linked to a promoter. The promoter may be a strong
promoter, such as those well known to the art, or a
differential promoter, i.e., one which is operative only in
specific cell types. The expression vectors may also contain
all or a part of a viral or bacterial genome, such as vaccinia
virus or BCG. Such vectors are especially useful in preparing
vaccines.
The expression vectors may incorporate several coding
sequences, as long as the Smage-3 sequence is contained
therein. The cytokine and/or HLA genes discussed supra may be
included in a single vector with the TRAP sequence. Where
this is not desired, then an expression system may be
provided, where two or more separate vectors are used where
each coding sequence is operably linked to a promoter. Again,
the promoter may be a strong or differential promoter. Co-
transfection is a well known technique, and the artisan in
this field is expected to have this technology available for
utilization. The vectors may be constructed so that they code
for the TRA molecule directly, rather than the MAGE-Xp TRAP.
This eliminates the need for post-translational processing.
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As the foregoing discussion makes clear, the sequences
code for "tumor rejection antigen precursors" ("TRAPs") which,
in turn, are processed into tumor rejection antigens ("TRAs").
Perhaps their most noteworthy aspect is as vaccines for
treating various cancerous conditions. The evidence points to
presentation of TRAs on tumor cells, followed by the
development of an immune response and deletion of the cells.
The evidence in the art shows that when various TRAs are
administered to cells, a CTL response is mounted and
presenting cells are deleted. This is behavior characteristic
of vaccines, and hence TRAPs, which are processed into TRAs,
and the TRAs themselves may be used, either alone or in
pharmaceutically appropriate compositions, as vaccines.
Similarly, presenting cells may be used in the same manner,
either alone or as combined with ingredients or yield
pharmaceutical compositions. Additional materials which may
be used as vaccines include isolated cells which present the
TRA molecule on their surface, as well as TRAP fragments,
mutated viruses, especially etiolated forms, and transformed
bacteria. "Fragments" as used herein refers to peptides which
are smaller than the TRA, but which possess the properties
required of a vaccine, as discussed supra. Another vaccine
comprises or consists of complexes of TRA and HLA molecule.
Vaccines of the type described herein may be used
preventively, i.e., via administration to a subject in an
amount sufficient to prevent onset of a cancerous condition.
The generation of an immune response, be it T-cell or B-
cell related, is characteristic of the effect of the presented
tumor rejection antigen. With respect to the B-cell response,
this involves, inter alia, the generation of antibodies to the
TRA, i.e., which speci~ically bind thereto. In addition, the
TRAP molecules are of sufficient size to render them
immunogenic, and antibodies which specifically bind thereto
are a part of this invention. These antibodies may be
polyclonal or monoclonal, the latter being prepared by any of
the well recognized methodologies for their preparation which
need not be repeated here. For example, mAbs may be prepared
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12
using an ~ni~-~l model, e.g., a Balb/C mouse or in a test tube,
using, e.g., EBV transformants. In addition, antiserum may be
isolated from a subject afflicted with a cancerous condition
where certain cells present a TRA. Such antibodies may also
be generated to epitope defined by the inter-action of TRA and
HLA/MHC molecules. --
Review of the foregoing disclosure will show that there
are a number of facets to the system which may be referred to
as "tumor rejection antigen presentation and recognition".
Recognition of these phenomena has diagnostic consequences.
For example, the existence of specific CTL clones, or
antibodies to the TRA makes it possible to diagnose or monitor
cancerous conditions (explained infra), by monitoring the CTLs
in a sample from a subject, binding of antibodies to TRAs, or
the activity of anti-TRA CTLs in connection with subject
samples. Similarly, the expression of nucleic acid molecules
for TRAPs can be monitored via amplification (e.g.,
"polymerase chain reactionl'), anti-sense hybridization, probe
technologies, and so forth. Various subject samples,
including body fluids (blood, serum, and other exudates,
e.g.), tissues and tumors may be so assayed.
A particular manner of diagnosis is to use an adaptation
of the standard "tuberculin test" c7~rrently used for diagnosis
of tuberculosis. This standard skin test ~m;n; sters a stable
form of "purified protein derivative" or "PPD" as a diagnostic
aid. In a parallel fashion, TRAs in accordance with this
invention may be used in such a skin test as a diagnostic aid
or monitoring method.
The term "cancerous condition" is used herein to embrace
all physiological events that commence with the initiation of
the cancer and result in final clinical manifestation. Tumors
do not spring up "ab initio" as visible tumors; rather there
are various events associated with the transformation of a
normal cell to malignancy, followed by development of a growth
of biomass, such as a tumor, metastasis, etc. In addition,
remission may be conceived of as part of "a cancerous
condition" as tumors seldom spontaneously disappear. The
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13
diagnostic aspects of this invention include all events
involved in carcinogenesis, from the first transformation to
malignancy of a single cell, through tumor development and
~ metastasis, as well as remission. All are embraced herein.
Where "subject" is used, the term embraces any species
which can be afflicted with a cancerous condition. This
includes humans and non-humans, such as domesticated animals,
breeding stock, and so forth.
There are therapeutic aspects of this invention as well.
The efficacy of administration of effective amounts of TRAPs
and TRAs as vaccines have already been discussed supra.
Similarly, one may develop the specific CTLs L~ vitro and then
administer these to the subject. Antibodies may be
administered, either polyclonal or monoclonal, which
specifically bind to cells presenting the TRA of interest.
These antibodies may be coupled to specific antitumor agents,
including, but not being limited to, methotrexate radio-
iodinated compounds, toxins such as ricin, other cytostatic or
cytolytic drugs, and so forth. Thus, "targeted" antibody
therapy is included herein, as is the application of deletion
of the cancerous cells by the use of CTLs.
The terms and expression which have been employed are
used as terms of description and not of limitation, and there
is no intention in the use of such terms and expression of
excluding any equivalents of the features shown and described
or portions thereof, it being recognized that various
modifications are possible within the scope of the invention.
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(1) GENERAL INFORMATION:
(i) APPLICANTS: DeBacker, Olivier
DePlaen, Etienne
Boon-Falleur, Thiery
(ii) TITLE OF I~V~llON: ISOLATED NUCLEICACID MOLECULE
WHICH ENCODES MURINE TUMOR
REJECTION ANTIGEN PRECURSOR
SMAGE-3
(iii) NUMBER OF SEQUENCES: 3
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Felfe & Lynch
(B) STREET: 805 Third Avenue
(C) CITY: New York City
(D) STATE: New York
(E) COUNTRY: USA
(F) ZIP: 10022
( V ) COM~U'1'~K READABLE FORM:
(A) MEDIUM TYPE: Diskette, 5.25 inch, 360 kb
storage
(B) COM~U'1'~K: IBM PS/2
(C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: Wordperfect
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/496,517
(B) FILING DATE: 29-JUNE-1995
(C) CLASSIFICATION: 435
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hanson, Norman D.
(B) REGISTRATION NUMBER: 30,946
(C) REFERENCE/DOCKET NUMBER: LUD 5407
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 688-9200
(B) TELEFAX: (212) 838-3884
CA 02224662 1997-12-11
WO 97/01574 PCTrUS96/10518
INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 18 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
AGTCCTCAGG GAGCCTCC
INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 17 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
TATCCCAATT CACAAAA
INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1609 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
TGCTGATTGA GACAGGAGAG GCTTGGACGC CAGTCTGAAG AGAACAATCC 50
TAGGGTTTGC AGAAAAGAGC TTGATCCACG AGTTCGGAAG TCCTGATTCC 100
TGCCTGTCAG CTCTTCTACT TCAGCCCTGA GCACAGTCAA CATGCCTAGG 150
GGTCA~AA~.~ GTAAGAGCCG CTCCCGTGCT AAACGACAGC AGTCACGCAG 200
GGAGGTTCAA GTAGTTCAGC CCACTGCAGA GGAAGCAGGG TCTTCTCCTG 250
CA 02224662 l997-l2-ll
W O 97/01574 PCT~US96/10518
16
TTGACCTGAG TGCTGGGTCC AACTTCCCTG GTGGTTCTGC TCCTCAGGGT 300
GTGAAAAACC CTGAATCTTT TGGTGCAGGT GTATCCTGCA CAGGCTCTGG 350
TATAGGTGGT AGAAATGCTA CTGTCCTGCC TGATACAAAA AGTTCAGATG 400
GCACCCAGGC AGGGACTTCC ATTCAGCACA CACT~-AAA~A TCCTATCATG 450
AGGAAGGCTA GTGTGCTGAT AGA~TTCCTG CTAGATAAGT TTAAGATGAA 500
AGAAGCAGTT ACAAGGAGTG AAATGCTGGC AGTAGTTAAC AAGAAGTATA 550
AGGAGCAATT CCCTGAGATC CTCAGGAGAA CTTCTGCACG CCTAGAATTG 600
GTCTTTGGTC TTGAGTTGAA GGAAATTGAT CCCAGCACTC ATTCCTATTT 650
GCTGGTGGGC AAACTGGGTC TTTCCACTGA GGGAAGTTTG AGTAGTAACT 700
GGGGGTTCCC TAGGACAGGT CTCCTAATGT CTGTCCTAGG TGTGATCTTC 750
ATGAAGGGTA ACCGTGCCAC TGAG~AA~.AG GTCTGGCAAT TTCTGCATGG 800
AGTGGGGGTA TATGCTGGGA AGAAGCACTT GATCTTTGGC GAGCCTGAGG 850
AGTTTATAAG AGATGTAGTG CAGÇAAAATT ACCTGGAGTA CCGCCAGGTA 900
CCTGGCAGTG ATCCCCCAAG CTATGAGTTC CTGTGGGGAC CCAGAGCCCA 950
TGCTGAAACA ACCAAGATGA AAGTCCTGGA AGTTTTAGCT AAAGTCAATG 1000
GCACAGTCCC TAGTGCCTTC CCTAATCTCT ACCAGTTGGC TCTTAGAGAT 1050
CAGGCAGGAG GGGTGCCAAG AAGTAGAGTT CAAGGCAAGG GTGTTCATTC 1100
CAAGGCCCCA TCC~AAAAGT CCTCTAATGT GTAGTTGAGT CTGTTCTGTT 1150
GT~l~ GAAA AACAGTCAGG GTCCTAATCA GTA~A~AGTT CATAGCCTAC 1200
CAGAACCAGC ATGCATCCAT TCTTGGCCTG TTATACATTA ATA~AATGGA 1250
GGCTATTTTT GTTACTTTTC TAATGTTTGT TTAACTAAAC AGTG~ 1300
GCCATGCTTT TTGTTAACTG CATACAGAAG TAACTGTCAC TTGTCAGGTT 1350
AGGACTTGTT TTGTTATTTG CAACAAACTG GAAAACATTA ~ ACTA 1400
AAACATTGTG TAACATTGCA TTGGAGAACG GATTGTCATG GCAATGTGAT 1450
ATCATACAGT GGTGAAACAA CAGTGAAGTG GGAAAGTTTA TATTGTTAGT 1500
TTTGAAAATT TTATGAGTGT GATTGCTGTA TA~ C ~ GTAT 1550
AATGCTAAGT GAAATAAAGT TGGATTTGAT GACTTTACTC AAAAAAAAAT 1600
GCTGATTGA 1609
