U.S. patent application number 10/081108 was filed with the patent office on 2003-07-24 for method for determining bage expression.
Invention is credited to Boel, Pascale, Boon-Falleur, Thierry, Bruggen, Pierre van der, Coulie, Pierre, Renauld, Jean-Christophe, Wildmann, Claude.
Application Number | 20030138854 10/081108 |
Document ID | / |
Family ID | 22148499 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030138854 |
Kind Code |
A1 |
Boel, Pascale ; et
al. |
July 24, 2003 |
Method for determining BAGE expression
Abstract
A family of tumor rejection antigen precursors, and the nucleic
acid molecules which code for them, are disclosed. These tumor
rejection antigen precursors are referred to as BAGE tumor
rejection antigen precursors, and the nucleic acid molecules which
code for them are referred to as BAGE coding molecules. Various
diagnostic and therapeutic uses of the coding sequences and the
tumor rejection antigen precursor molecules are described.
Inventors: |
Boel, Pascale; (Brussels,
BE) ; Wildmann, Claude; (Brussels, BE) ;
Boon-Falleur, Thierry; (Brussels, BE) ; Bruggen,
Pierre van der; (Brussels, BE) ; Coulie, Pierre;
(Brussels, BE) ; Renauld, Jean-Christophe;
(Brussels, BE) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
666 FIFTH AVE
NEW YORK
NY
10103-3198
US
|
Family ID: |
22148499 |
Appl. No.: |
10/081108 |
Filed: |
February 20, 2002 |
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10081108 |
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09435524 |
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6465184 |
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09435524 |
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09038328 |
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6110694 |
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09038328 |
Mar 11, 1998 |
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08573186 |
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6093540 |
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08573186 |
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08389360 |
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5877017 |
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08389360 |
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08196630 |
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5683886 |
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08196630 |
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08079110 |
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5571711 |
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Current U.S.
Class: |
435/7.2 ;
435/320.1; 435/325; 435/69.1; 530/350; 536/23.5 |
Current CPC
Class: |
C07K 14/70539 20130101;
C07K 14/4748 20130101; A61K 38/00 20130101; A61P 35/00
20180101 |
Class at
Publication: |
435/7.2 ;
435/69.1; 435/320.1; 435/325; 530/350; 536/23.5 |
International
Class: |
G01N 033/53; G01N
033/567; C07H 021/04; C07K 014/74; C12P 021/02; C12N 005/06 |
Claims
We claim:
1. An isolated nucleic acid molecule consisting of the nucleotide
sequence set forth in SEQ ID NO: 1.
2. An isolated nucleic acid molecule which hybridizes, under
stringent conditions, to the nucleic acid molecule set forth in SEQ
ID NO: 1, and which codes for a tumor rejection antigen precursor,
wherein said isolated nucleic acid molecule does not code for a
MAGE tumor rejection antigen precursor.
3. An isolated molecule which is complementary to the nucleic acid
molecule of claim 1, wherein said molecule is mRNA or DNA.
4. A host cell transfected or transformed with the nucleic acid
molecule of claim 1.
5. A host cell transfected or transformed with the nucleic acid
molecule of claim 2.
6. An expression vector comprising the isolated nucleic acid
molecule of claim 1 operably linked to a promoter.
7. An expression vector comprising the isolated nucleic acid
molecule of claim 2 operably linked to a promoter.
8. The host cell of claim 4, wherein said host cell is a mammalian
cell which expresses HLk-Cw*1601.
9. The host cell of claim-5, wherein said host cell is a mammalian
cell which expresses HLA-Cw*1601.
10. The expression vector of claim 6, further comprising a nucleic
acid molecule which codes for HLA-Cw*1601.
11. The expression vector of claim 7, further comprising a nucleic
acid molecule which codes for HLA-Cw*1601.
12. An expression kit comprising a separate portion of each of: (i)
the isolated nucleic acid molecule of claim 1, and (ii) a nucleic
acid molecule which codes for HLA-Cw*1601.
13. An expression kit comprising a separate portion of each of: (i)
the isolated nucleic acid molecule of claim 2, and (ii) a nucleic
acid molecule which codes for HLA-Cw*1601.
14. A method for treating a subject with a disorder characterized
by the presence of complexes of HLA molecules and the peptide of
SEQ ID NO: 3 on cell surfaces comprising the sequence of SEQ ID NO:
1, comprising administering to said subject an amount of cytolytic
T cells specific to complexes of HLA molecules and said peptide,
sufficient to alleviate said disorder.
15. A method for treating a subject with a disorder characterized
by the presence of complexes of HLA molecules and the peptide of
SEQ ID NO: 3 on cell surfaces, comprising administering to said
subject an amount of an agent which provokes an immune response to
complexes of HLA and said peptide, sufficient to provoke said
immune response against cells presenting said complexes.
16. A method for diagnosing a disorder characterized by the
presence of complexes of HER molecules and the peptide of SEQ ID
NO: 3 on cell surfaces, comprising contacting a sample from a
subject with an agent specific for a tumor rejection antigen
consisting of the amino acid sequence of SEQ ID NO: 3, and
determining interaction between said agent and said sequence or
said expression product as a determination of said disorder.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of copending
application Ser. No. 08/196,630 filed Feb. 15, 1994, which is a
continuation-in-part of application Ser. No. 08/079,110, filed Jun.
17, 1993.
FIELD ON THE INTENTION
[0002] This invention relates to nucleic acid molecules, proteins,
and peptides which are useful in connection with the diagnosis and
treatment of pathological conditions. This invention further
relates to said proteins and peptides, which are processed to a
peptide presented by the MHC molecule HLA-Cw*1601, and the
presented peptide itself. These peptides are useful in diagnosis
and therapeutic contexts.
BACKGROUND AND PRIOR ART
[0003] The process by which the mammalian immune system recognizes
and reacts to foreign or alien materials is a complex ones An
important facet of the system is the 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 Male et al.,
Advanced Immunology (J. P. Lipincott Company, 1987), especially
chapters 6-10. The interaction of T cell and complexes of
HLA/peptide 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 HLk binding peptides. See Barinaga,
Science, 257: 880 (1992); Fremont et al., Science, 257: 919 (1992);
Matsumura et al., Science, 257: 927 (1992); and Latron et al.,
Science, 257: 964 (1992).
[0004] The mechanism by which T cells recognize cellular
abnormalities has also been implicated in cancer. For example, in
PCT application PCT/US92/04354, filed May 22, 1992, published on
Nov. 26, 1992, and incorporated by reference, a family of genes is
disclosed, 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. 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". 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. Pat. No.
5,342,774.
[0005] In U.S. patent application Ser. No. 938,334, the disclosure
of which is incorporated by reference, nonapeptides are taught
which are presented by the HLk-A1 molecule. The reference teaches
that given the known specificity of particular peptides for
particular HLA molecules, a particular peptide is expected to bind
one HLA molecule, but not others. This is important, because
different individuals possess different HLA phenotypes. As a
result, while identification of a particular peptide as being a
partner for a specific IER 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 HLk phenotype, and targeted therapy requires some
knowledge of the phenotype of the abnormal cells at issue.
[0006] In U.S. patent application Ser. No. 008,446, filed Jan. 22,
1093 and incorporated herein by reference, it is disclosed that the
MAGE-1 expression product is processed to a second TRA. This second
TRA is presented by HLA-Cw*1601 molecule. The disclosure shows that
a given TRAP can yield a plurality of TRAS.
[0007] In U.S. patent application Ser. No. 994,928, filed Dec. 22,
1992, and incorporated by reference herein, tyrosinase is described
as a tumor rejection antigen precursor. This reference discloses
that a molecule which is produced by some normal cells (e.g.,
melanocytes), is processed in tumor cells to yield a tumor
rejection antigen that is presented by HLA-A2 molecules.
[0008] In U.S. patent application Ser. No. 08/032,978, filed Mar.
18, 1993, and incorporated herein by reference, 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 by a
non MAGE gene. This disclosure shows that a particular HLA molecule
may present TRAs derived from different sources.
[0009] In U.S. patent application Ser. No. 08/079,110 filed June
17, 1993, which is incorporated herein by reference, a new family
of genes, referred to therein as the BAGE family, is disclosed. It
was observed that these genes also code for tumor rejection antigen
precursors. It is disclosed in the application that the MHC
molecule known as HLA-Cw*1601 presents a tumor rejection antigen
derived from a BAGE tumor rejection antigen precursor; however, the
tumor rejection antigen was not disclosed. The tumor rejection
antigen is disclosed in U.S. patent application Ser. No. 08/196,630
filed Feb. 15, 1994, which is incorporated herein by reference. The
application also discloses ramifications stemming from the tumor
rejection antigen, as well as therapeutic and diagnostic methods
utilizing the antigen.
[0010] The present application is directed to isolated nucleic acid
molecules which encode BAGE tumor rejection antigen precursors
described in patent application Ser. No. 08/196,630. The present
application is further directed to therapeutic and diagnostic
methods utilizing the isolated BAGE nucleic acid molecule.
[0011] The invention is elaborated upon further in the disclosure
which follows.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] The above brief description, as well as further objects and
features of the present invention, will be more fully understood by
reference to the following detailed description of the presently
preferred, albeit illustrative, embodiments of the present
invention when taken in conjunction with the accompanying drawings
wherein:
[0013] FIG. 1 is comprised of FIG. 1A and FIG. 1B. FIG. 1A shows
lytic activity of CTL clone 82/82 on MZ2-MEL sublines MZ2-MEL.3.0,
MZ2-MEL.3.1 and MZ2-MEL.B.TC.4. FIG. 1B shows lytic activity of CTL
clone 82/82 on MZ2-MEL subline MZ2-MEL.43 and on melanoma cell
lines MI4024/1-MEL and LB17-MEL, which were derived from
HLA-Cw*1601 positive patients;
[0014] FIG. 2 shows TNF release by CTL 82/82 when put into contact
with COS-7 cells transfected with HLA-Cw*1601 alone, in combination
with cDNA-AD5, or transfected with AD5 alone. CTL 82/82 was also
put into contact with MZ2-MEL.43 and MZ2-MEL.2.2.5, as
controls;
[0015] FIG. 3 is comprised of FIG. 3A and FIG. 3B. FIG. 3A shows
lysis by CTL clone 82/82 of P1.HTR mouse cells cotransfected with
expression vectors carrying HLA-Cw*1601 and cDNA-ADS, as well as
untransfected P1.HTR, and P1.HTR transfected with HLA-Cw*1601
alone. FIG. 3B shows lysis by CTL clone 82/82 of P1.HTR transfected
with HLA-Cw*1601 and BAGE-derived nonapeptide AARAVFLAL; (SEQ ID
NO: 3).
[0016] FIG. 4 sets forth nucleotide and amino acid sequences of a
BAGE tumor rejection antigen precursor. The boxed segment is a
tumor rejection antigen derived from the precursor;
[0017] FIG. 5 represents Southern blots of DNA extracted from
melanoma cell line MZ2-MEL.3.0, blood lymphocytes from patient MZ2
and mouse cell line P1.HTR;
[0018] FIG. 6 represents Northern blot analysis of the expression
of BAGE in MZ2-MEL.43 cells;
[0019] FIG. 7 represents PCR amplification of cDNAs from melanoma
lines, tumor and normal samples, and of genomic DNA from subline
MZ2-MEL.43; and
[0020] FIG. 8 represents lysis by CTL 82/82 of lymphoblastoid cell
line MZ2-EBV incubated with BAGE-encoded peptide AARAVFLAL (SEQ ID
NO: 3) or with nonapeptides ARAVFLALF (SEQ ID NO: 4) or MAARAVFLA
(SEQ ID NO: 5).
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLE 1
[0021] Melanoma cell line MZ2-MEL was derived from patient MZ2
using standard methodologies. This cell line is described in PCT
Application PCT/US92/04354, filed May 22, 1992, published Nov. 26,
1992, which is incorporated herein by reference. Once the cell line
was established, a sample of it was irradiated, so as to render it
non-proliferative. A number of subclones were obtained from
MZ2-MEL. Specifically, clonal line MZ2-MEL.3.0 was obtained from
MZ2-MEL by limiting dilution. The MZ2-MEL.3.0 culture was then
cultured further. After more than 150 generations in culture, a new
subline, denoted MZ2-MEL.3.1, was obtained. MZ2-MEL.3.1 was found
to be resistant to a large fraction of autologous CTL clones that
had strong lytic activity on MZ2-MEL.3.0. It was determined that
MZ2-MEL.3.1 had lost the genes coding for HLA-A29, B44, and Cw*1601
(see van der Bruggen et al., Eur. J. Immunol., 24:2134-2140 (1994),
which is incorporated herein by reference).
[0022] Subline MZ2-MEL.43 was derived by limiting dilution from
MZ2-MEL.3.0 cells that has survived mutagen treatment (Van den
Eynde et al., Int. J. Cancer, 44:634-640 (1989)). Clonal subline
MZ2-MEL.2.2, which does not express antigen MZ2-E, was selected
from subclone MZ2-MEL.3.1 with an autologous anti-MZ2-E CTL clone
(Van den Eynde et al., supra). Subline MZ2-MEL.2.2.5 was selected
from subline MZ2-MEL.2.2 with an anti-MZ2-F CTL clone.
MZ2-MEL.B.TC.4 was obtained by transfecting HLA-Cw*1601 gene into
subline MZ2-MEL.2.2.5 (van der Bruggen et al., supra). Melanoma
cell lines were grown as previously described by Van den Eynde et
al., supra and Traversari et al., Immunogenetics, 35:145-152
(1992).
[0023] Cytolytic T cell clones ("CTLs") specific to cell line
MZ2-MEL were obtained utilizing irradiated MZ2-MEL cells.
Specifically, a sample of peripheral blood mononuclear cells
("PBMCs") was taken from patient MZ2, and contacted to the
irradiated melanoma cells. The mixture was observed for lysis of
the melanoma cells, which indicated that CTLs specific for a
complex of peptide and HLA molecule presented by the melanoma cells
were present in the sample.
[0024] The lysis assay employed was a chromium release assay
following Herin et al., Int. J. Cancer, 39:390-396 (1987), which is
incorporated herein by reference. The assay, however, is described
e herein. The target melanoma cells were grown in vitro, and then
resuspended at 4.times.10.sup.7 cells/ml in DMEM, supplemented with
DMEM with 10 mM HEPES and 50% FCS, and incubated for 60 minutes at
37.degree. C. with 200 .mu.Ci/ml of Na(.sup.51Cr)O.sub.4. Labelled
cells were washed three times with DMEM, supplemented with 10 mM
HEPES. These were then resuspended in DMEM supplemented with 10 my
HEPES and 10% FCS, after which 100 .mu.l aliquots containing
10.sup.3 cells, were distributed into 96 well microplates. Samples
of PBLs were added in 100 .mu.l of the same medium, and assays were
carried out in duplicate. Plates were centrifuged for 4 minutes at
100 g, and incubated for four hours at 37.degree. C. in a 8%
CO.sub.2 atmosphere.
[0025] Plates were centrifuged again, and 100 .mu.l aliquots of
supernatant were collected and counted. Percentage of .sup.51Cr
release was calculated as follows: 1 % 51 Cr release = ( ER - SR )
( MR - SR ) .times. 100
[0026] where ER is observed, experimental .sup.51Cr release, SR is
spontaneous release measured by incubating 10.sup.3 labeled cells
in 200 .mu.l of medium alone, and MR is maximum release, obtained
by adding 100 .mu.l 0.3% Triton X-100 to target cells.
[0027] Those mononuclear blood samples which showed high CTL
activity were expanded and cloned via limiting dilution, and were
screened again, using the same methodology. The CTL clone MZ2-CTL
82/82 was thus isolated. The clone is referred to as "82/82"
hereafter.
[0028] MZ2-MEL sublines and other melanoma lines were put into
contact with CTL clone 82/82 and lytic activity was determined by
measuring chromium release. Chromium release was measured after 4
hours. FIG. 1A shows lysis of MZ2-MEL.3.0 and MZ2-MEL.B.TC.4.
Subline MZ2-MEL.3.1 was not lysed by CTL 82/82. FIG. 1B shows that
there is lysis of clonal line MZ2-MEL.43 by CTL 82/82. In addition,
melanoma cell lines MI4024/1-MEL and LB17-MEL, which carry the
HLA-Cw*1601 allele, were lysed by CTL 82/82.
EXAMPLE 2
[0029] The gene which codes for the antigen recognized by CTL 82/82
was identified. As described herein, the gene was identified by
cotransfecting HLA-Cw*1601 cDNA with a cDNA library. Due to the
specificity of CTL 82/82 for MZ2-MEL.43, the cDNA of this cell line
was used to construct the cDNA library. In order to construct the
cDNA library from MZ2-MEL.43, poly-A+RNA was extracted from
MZ2-MEL.43 cells using an MONA extraction kit. The mRNA was
converted to cDNA using random primers, ligated to adaptors
following standard techniques, and inserted into the EcoRI site of
expression vector pcD-SR.alpha., which contains the replication
origin of SV40. Recombinant plasmids were electrodorated into E.
coli JM101 and selected with ampicillin (50 .mu.g/ml). The library
contained 66,000 inserts and was divided into 87 pools of 400
bacteria and 297 pools of 200 bacteria. Each of these pools
comprised approximately 280 or 140 different cDNAs respectively, as
about 70% of the plasmids carried an insert. Each pool of bacteria
was amplified to saturation and plasmid DNA was extracted by the
well known alkaline lysis method.
[0030] Plasmid pcD-SR.alpha. was transfected with HLA-Cw*1601 cDNA.
cDNA pools were then cotransfected with the pcD-SR.alpha. plasmid
containing HLA-Cw*1601 cDNA into duplicate microcultures of COS-7
cells. Transfection was performed by the DEAE-dextran-chloroquine
method (Seed et al., Proc. Natl. Acad. Sci. USA, 84:3365-3369
(1987); Brichard et al., Annal. Biochem., 162:156-159 (1993);
Coulie et al., J. Exp. Med., 180:35-42 (1994)). Briefly,
1.5.times.10.sup.4 COS-7 cells were transfected with 100 ng of
plasmid pcD-SR.alpha. containing cDNA of HLA-Cw*1601, and 100 ng of
a pool of the cDNA library or 100 ng of a cDNA clone. The
HLA-Cw*1601 cDNA was isolated from a cDNA library prepared with RNA
extracted from subline MZ2-MEL.43 (van der Bruggen et al.,
supra).
[0031] Cotransfectants were tested after 24 or 48 hours for their
ability to stimulate the production of tumor necrosis factor (TNF)
by CTLs (Traversari et al., Immunogenetics, 235:145-152 (1992)).
1500 CTLs were added in 100 .mu.l of Iscove's medium (Gibco BRL)
containing 10% human serum and 20 U/ml r-hu-IL-2 to microwells
containing target cells. After 24 hours, the supernatant was
collected and its TNF content was determined by testing its
cytotoxic effect on cells of WEHI-164 clone 13 (Espevik et al., J.
Immunol. Methods, 95:99-105 (1986)) in an MTT colorimetric assay
(Hansen et al., J. Cancer, 39:390-396 (1989) and Traversari et al.,
supra).
[0032] Among the 384 pools of cDNAs (297 of 200 bacteria and 87 of
400) that were transfected, two produced positive supernatants
containing more than 40 .mu.g/ml of TNF, whereas TNF concentration
in all of the microcultures transfected with the other pools was
lower than 5 pg/ml. From one of these cDNA pools which contained
400 independent bacteria, 800 bacteria were subcloned. Plasmid DNA
was extracted from each of them and transfected into COS-7 cells
together with the HLA-Cw*1601 construct described supra. Twelve
clones conferred recognition by CTL 82/82. The result obtained with
one of them, denoted cDNA-AD5, is represented in FIG. 2.
[0033] FIG. 2 shows stimulation of CTL 82/82 by COS-7 cells
cotransfected with cDNA-AD5 and with an HLA-Cw*1601 cDNA, or
transfected with one of these cDNAs. The cDNAs were inserted in
expression vector pcD-SR.alpha. as described supra. Samples of CTL
82/82 were added one day after the transfection and the TNF content
of the supernatant was estimated one day later by testing its
toxicity on cells of WEHI-164 clone 13. Positive and negative
controls were developed with MZ2-MEL.43 and MZ2-MEL.2.2.5
cells.
[0034] To confirm the results obtained with cDNA-PD5 in transient
transfections, stable transfectants were also prepared. P1.HTR, a
highly transfectable variant derived from mouse tumor cell line
P815 (Van Pel et al., Som. Cell Genet., 11:467-475 (1985)), was
transfected with both HLA-Cw*1601 and cDNA-ADS, using the calcium
phosphate precipitate method with plasmid pSVtkneo.beta. conferring
resistance to geneticin (Nicolas et al., CSH Conferences Cell
Prolif., 10:469-485 (1983)) and HLA-Cw*1601 alone, or both
HLA-Cw*1601 and cDNA-AD5. The relevant cDNAs were inserted in
expression vector pcD-SR.alpha. as described supra. Clonal sublines
were isolated from a geneticin-resistant transfected
population.
[0035] Transfected cells, when put into contact with CTL 82/82,
were lysed by CTL 82/82, indicating that the antigen can also be
processed in these mouse cells. FIG. 3A shows lysis by CTL clone
82/82 of P1.HTR mouse cells cotransfected with expression vectors
carrying HLA-Cw*1601 and cDNA-AD5. Untransfected P1.HTR and P1.HTR
transfected with HLA-Cw*1601 alone were also tested.
EXAMPLE 3
[0036] DNA sequence analysis of cDNA-AD5 was performed by specific
priming with synthetic oligonucleotides. SEQ ID NO: 1 presents cDNA
nucleotide information for the identified gene, referred to herein
as "BAGE". The sequencing reactions were performed by the
dideoxy-chain termination method (T7 Sequencing Kit, Pharmacia
Uppsala Sweden, .DELTA.TAq.TM. Cycle-Sequencing Kit, USB,
Cleveland, Ohio). The computer search for the sequence homology was
done with programs FASTA@EMBL-Heidelberg and
blast@ncbi.nlm.nih.gov. The sequence bears no significant
similarity to any other sequence presently recorded in databanks,
except for an Alu repeat (nucleotides 385 to 484) located outside
of the coding region.
EXAMPLE 4
[0037] The region of BAGE which codes for the antigen presented by
HLA-Cw*1601 was determined. In order to identify this region, a
large number of truncated BAGE cDNA clones were produced. By
digesting BAGE with exonuclease III for various incubation times,
progressive deletions starting from the 3' end were generated. The
truncated variants were religated into pcDNAI/Amp, electroporated
into E. coli strain DH5.alpha.F'IQ, and selected via ampicillin (50
.mu.g/ml). 438 clones were obtained in this way.
[0038] The plasmid DNA was obtained from these 438 cones, and
transfected into COS-7 cells together with HLA-Cw*1601 cDNA to test
for their ability to code for the antigen. The transfectants were
tested in a TNF release assay, as described supra. Positive clones
were those which stimulated TNF release by CTL 82/82.
[0039] Once cells were divided into positive and negative
transfectants, the sequences of plasmid DNA from 5 positives and 5
negatives were determined. Clone 19C2, a positive clone, contained
part of the open reading frame for the BAGE gene described supra,
from nucleotide 201 to nucleotide 267. In contrast, clone 17G12, a
negative transfectant, contained nucleotides 201-206. This
indicated that the antigenic peptide was encoded by the first 67
nucleotides of the open reading frame.
[0040] FIG. 4, which shows the sequence of BAGE, also shows a
putative protein of 43 amino acids encoded by the largest open
reading frame. This protein was identified as containing the
sequence of the presented peptide. The 43 amino acid protein,
referred to herein as SEQ ID NO: 2, is as follows:
1 Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln
Ala Arg Leu Met Lys Glu Glu Ser Pro Val Val Ser Trp Arg Leu Glu Pro
Glu Asp Gly Thr Ala Leu Cys Phe Ile Phe
[0041] The sequence corresponding to the peptide recognized in
association with HLA-Cw*1601 by MZ2-CTL 82/82 is indicated in a
box. The sequence is referred to herein as SEQ ID NO: 3: Ala Ala
Arg Ala Val Phe Leu Ala Leu. The sequence of primers VDB85 (SEQ ID
NO: 6) (sense) and VDB86 (SEQ ID NO: 7) (anti-sense) used for PCR
amplification, as discussed in Example 4, are underlined with
arrows.
[0042] Several synthetic peptides were prepared on this basis.
Peptides were synthesized on solid phase using F-moc for transient
NH2-terminal protection as described by Atherton et al., J. Chem.
Soc. Lond. Perkin Trans., 1:538-546 (1981) and characterized by
mass spectrometry. All peptides were >90% pure as indicated by
analytical HPLC. Lyophilized peptides were dissolved at 20 mg/ml in
DMSO, diluted at 2 mg/ml in 10 mM acetic acid and stored at
80.degree. C. Peptides were tested in a CTL stimulation assay with
COS-7 cells transfected by HLA-Cw*1601 and incubated with the
peptides. They were also tested by chromium release assay as
previously described (Boon et al., J. Exp. Med., 152:1184-1193
(1980)). In this peptide sensitization assay, target cells were
.sup.51Cr-labelled for one hour at 37.degree. C. and washed
extensively. 1000 target cells were then incubated in 96-well
microplates in the presence of various concentrations of peptide
for 30 minutes at 37.degree. C. before CTL 82/82 cells were added.
Chromium release was measured after 4 hours at 37.degree. C.
[0043] FIG. 8 shows lysis by CTL 82/82 of lymphoblastoid cell line
MZ2-EBV incubated with BAGE encoded peptide AARAVFLAL (SEQ ID NO:
3). The final concentration of peptides during the incubation of
the target cells with the CTL is indicated. The arrow indicates the
percentage of lysis of MZ2-MEL.43 cells. Sensitization of a
lymphoblastoid cell line from patient MZ2 to lysis by CTL 82/82 was
observed with nonapeptide AARAVFLAL (SEQ ID NO: 3) (amino acids
2-10, FIG. 4). Half-maximal lysis was obtained at a peptide
concentration of 30 nM (FIG. 8). Nonapeptides that did not include
the N-terminal Ala, or ARAVFLALF (SEQ ID NO: 4) or the C-terminal
Leu, MAARAVFLA (SEQ ID NO: 5) were not able to sensitize target
cells to lysis. P1.HTR cells were transfected with HLA-Cw*1601 and
were incubated with nonapeptide AARAVFLAL (SEQ ID NO: 3). The
transfected cells were lysed by CTL 82/82. FIG. 3B shows lysis by
CTL clone 82/82 of P1.HTR transfected with HLA-Cw*1601 and
incubated with 1 IM of the BAGE-encoded nonapeptide AARAVFLAL (SEQ
ID NO: 3). Lysis of chromium-labelled cells was tested after 4
hours.
[0044] From two MLTCs set up with different blood samples of
patient MZ2, six CTL clones that recognized the BAGE/HLA-Cw*1601
antigen were derived. They produced TNF in the presence of COS-7
cells cotransfected with HLA-Cw*1601 and BAGE cDNA-AD5. They also
responded to cells transfected with HLA-Cw*1601 and incubated with
nonapeptide AARAVFLAL (SEQ ID NO: 3). It appears that at least 3
different CTL precursors can recognize this BAGE antigen. CTL clone
82/1 expressed V.alpha.2, V.alpha.3 and V.beta.13 while CTL clone
25/244 expressed V.alpha.8 and V.beta.8, whereas CTL clone 82/82
expressed V.alpha.3, V.alpha.4 and V.beta.13. V.alpha. and V.beta.
expression were determined as follows: Total RNA from the different
CTL clones was prepared by using RNAzol*B (Cinna/Biotecx,
Friendswood, Tex.). Single-stranded cDNA synthesis was carried with
oligo(dT) and lioloney murine leukemia virus-derived reverse
transcriptase without RNAse H activity. PCR was carried out by
amplification of TCR-.alpha. and -.beta. cDNA with the
oligonucleotide primers complementary to TCR variable
(V.alpha.1-W29, V.beta.1-W24) and constant (C.alpha., C.beta.)
region sequences described by Geneve et al., Eur. J. Immunol.,
22:1261-1269 (1992). Specificity of TCR V.alpha. and V.beta. PCR
amplification was assessed by Southern blotting and hybridization
with .sup.32P-labelled C.alpha. or c.beta. oligonucleotides
internal to the ones used for amplification.
EXAMPLE 5
[0045] The expression of BAGE in tissues was tested by reverse
transcription and nested PCR (van der Bruggen et al., supra). cDNAs
from melanoma lines, tumor and normal tissue samples, and of
genomic DNA from subline MZ2-MEL.43, were amplified by PCR. Total
RNA was extracted by the guanidine-isothiocyanate procedure as
described by Davis et al., Basic Methods in Molecular Biology, pp.
130-13S (New York, Elsevier, 1986). Reverse transcription was
performed on 2 .mu.g of total RNA in a reaction volume of 20 .mu.l
with 4 .mu.l of 5.times.reverse transcriptase buffer, 2 .mu.l of a
20 mM solution of oligo (dT) 15 primer, 20 U of RNasin, 2 .mu.l of
0.1 M dithilothreitol and 200 U of MoMLV reverse transcriptase plus
1 .mu.l of each of 10 m solution of dNTP. The reactants were
incubated at 42.degree. C. for 60 minutes. One twentieth of the
cDNA product was then supplemented with 5 .mu.l of
10.times.thermostable DNA polymerase buffer, 1 .mu.l each of 10 mM,
solution of dNTP, 1 .mu.l each of 25 .mu.M solution of primers, 1 U
of Dynazyme.TM. and water to a final volume of 50 .mu.l. The PCR
primers were 5'-TGGCTCGTCTCACTCTGG-3' (SEQ ID NO: 6) (VDB85, sense,
nucleotide 100 to 117) and 5'-CCTCCTATTGCTCCTGTTG-3- ' (SEQ ID NO:
7) (VDB86, anti-sense, nucleotide 367 to 385). PCR was performed
for 30 cycles (1 minute at 94.degree. C., 2 minutes at 62.degree.
C. and 2 minutes at 73.degree. C.). 10 .mu.l of the PCR product was
size-fractionated on a 1.5 agarose gel. The quality of RNA
preparations was tested by PCR amplification of human .beta.-actin
cDNA with primers 5'GGCATCGTGATGGACTCCG-3' (SEQ ID NO: 8) (exon 4,
sense) and 5'-GCTGGAAGGTGGACAGCGA-3' (SEQ ID NO: 9) (exon 6,
anti-sense) for 21 cycles of 1 minute at 94.degree. C, 2 minutes at
G8.degree. C. and 2 minutes at 72.degree. C. by AmpliTaq DNA
polymerase.
[0046] PCR products were visualized on a 1.5% agarose gel stained
with ethidium bromide. No expression of gene BAGE was found in
normal adult tissues except in testis (see FIG. 7 and Table 1,
below). The gene was also silent in placenta and umbilical cord and
in several tissue samples from fetuses older than 20 weeks. No
expression of BAGE was found in the twelve EBV-transformed
lymphoblastoid cell lines tested nor in blood lymphocytes
stimulated with phytohemagglutinin.
2TABLE 1 Expression of Gene BAGE by Normal Adult and Fetal Tissue
expression Adult tissues Adrenal gland - Bone marrow - Brain -
Breast - Cerebellum - Colon - Heart - Kidney - Liver - Lung -
Melanocytes - Muscle - Ovary - Prostate - Skin - Sperm -
Splenocytes - Stomach - Testis + Thymocytes - Urinary bladder -
Uterus - Placenta - Umbilical cord - Benign naevus - Fetal tissues
Fibroblasts - Brain - Liver - Spleen - Thymus - Testis -
[0047] BAGE appears to be silent in normal adult tissues including
melanocytes, except for testis. Because its expression was tested
by reverse-transcription and PCR, the absence of a detectable
product in normal tissues indicates a level of expression lower
than 0.1% of that observed in tumor MZ2-MEL.
EXAMPLE 6
[0048] Expression of BAGE gene in tumor samples and cell lines was
also determined. Six hundred samples of tumors of various
histological origins were analyzed for BAGE expression. As shown in
Table 2, below, BAGE gene is expressed mainly in melanomas (22%),
bladder carcinomas (15%), mammary carcinomas (10%) and head and
neck squamous cell carcinomas (89). A smaller proportion of
positive samples was found in sarcomas (6%) and in non-small cell
lung carcinomas (6%). No expression of BAGE was found in renal,
colorectal and prostatic carcinomas, leukemias, or lymphomas. With
very few exceptions, tumor samples that expressed BAGE also
expressed one of the MAGE genes discussed generally, supra.
3TABLE 2 Expression of gene BAGE by tumor samples Number of BAGE
Histological type positive tumors* Melanomas 40/178 primary lesions
3/38 metastatic lesions 37/140 Bladder carcinomas 9/62 superficial
tumors 0/32 infiltrating tumors 9/30 Mammary carcinomas 8/79 Head
and Neck squamous 4/53 cell carcinomas Lung carcinomas
NSCLC.degree. 4/64 Sarcomas 1/18 Renal sarcomas 0/50 Colorectal
carcinomas 0/42 Prostatic carcinomas 0/22 Leukemias and lymphomas
0/22 *Expression of gene BAGE was tested by RT-PCR amplification of
total RNA with the primers shown on FIG. 4. .degree.NSCLC = non
small cell lung carcinomas
[0049] BAGE was more frequently expressed in metastatic lesions of
melanomas (26%) than in primary lesions (8%). In transitional-cell
carcinomas of the urinary bladder, 30% of invasive tumors expressed
BAGE, while no expression was observed in superficial tumors. BAGE
was expressed in a higher proportion of tumor cell lines than of
tumor samples: 32/60 melanoma (53%) and 3/15 colorectal carcinoma
cell lines (20%) were positive. This has also been observed with
MAGE genes, and may be due to the fact that tumor cell lines are
more readily derived from metastatic tumors.
EXAMPLE 7
[0050] HLA-Cw*1601, the presenting molecule of BAGE antigen, cannot
be identified in serological assays, as useful antibodies are not
available. However, its expression can be tested by reverse
transcription and nested PCR. Approximately 7% (7/99) of Caucasian
individuals were found to express this HLA allele (van der Bruggen
et al., supra). The concentration of HLA-C molecules on the cell
surface has been reported to be about tenfold lower than that of
HLA-A and B, possibly because of less efficient binding to
62-microglobulin (Neefjes et al., Eur. J. Immunol., 18:801-810
(1988)). Nevertheless, it has ben determined that BAGE codes for a
peptide recognized on a HLA-C molecule, suggesting that HLA-C
molecules also play a significant role in the presentation of a
antigens to CTL.
EXAMPLE 8
[0051] A Southern blot with DNA extracted from blood lymphocytes of
patient MZ2 and from the melanoma cell line MZ2-MEL.3.0 was
prepared. In order to perform Southern blot analysis, DNA from
melanoma cell line MZ2-MEL.3.0, PBLs of patient MZ2 and mouse cell
line P1.HTR were digested with EcoRI or HindIII. DNA capillary
transfer was done by alkaline blotting on a Zeta-Probe.RTM.
membrane (Bio-Rad). Following transfer, the membrane was rinsed in
2.times.SSC, baked for 1 hour at 80.degree. C. and pretreated for
30 minutes at 60.degree. C. in 6.times.SSC, 10.times.Denhardt's
solution. The membrane was then hybridized for 18 hours at
65.degree. C. in 3.5.times.SSC, 1.times. Denhardt's solution, 25 mM
NaH.sub.2PO.sub.4 pH 7.0, 0.5% SDS, 2 nM EDTA, 100 .mu.g/ml of
herring sperm DNA, and 2.times.10.sup.6 cpm/ml of a 121 bp
.sup.32P-labelled probe (nucleotides 211 to 331 of SEQ ID NO: 1)
produced by PCR. The membrane was then washed at 65.degree. C. for
2.times.15 minutes in 2.times.SSC, 0.5% SDS, then for 15 minutes in
0.2.times.SSC, 0.1% SDS, and autoradiographed for 10 days.
[0052] When this blot was hybridized with the 121 bp probe
described supra, four bands were observed in lanes containing DNA
digested with EcoRI and 6 bands after HindIII digestion (FIG. 5).
Considering the small size of the probe and considering the absence
of EcoRI and HindIII restriction sites in the coding sequence,
these results indicate that BAGE belongs to a family of several
related genes.
EXAMPLE 9
[0053] A Northern blot prepared with poly-A+ RNA of subline
MZ2-MEL.43 was hybridized with a 286 bp BAGE probe including
nucleotides 100 to 385 of SEQ ID NO:-11. To perform Northern blot
analysis, poly-A+ RNA from MZ2-MEL.43 was prepared using mRNA
extraction kit. Total RNA from mouse kidney tissue was extracted by
the guanidine-isothiocyanate procedure as described by Davis et
al., supra. Poly-A+RNA was purified from total RNA on an oligo-dT
column. For the Northern blot analysis, 5 .mu.g of poly-A+ RNA from
subline MZ2-MEL.43 and 5 .mu.g of poly-A+ RNA from mouse kidney
cells were fractionated on a it agarose gel containing 0.66 M
formaldehyde and transferred on a membrane in 10.times.SSC.
[0054] The membrane was pre-hybridized for 15 minutes at 60.degree.
C. in 10% dextran sulfate, 1% SDS and 1 M NaCl and hybridized
overnight at 60.degree. C. in the same solution with
2.times.10.sup.6 cpm/ml of the 286 bp 32p-labelled probe. The
membrane was washed at room temperature in 0.2.times.SSC for 10
minutes and then 2.times.20 minutes at 60.degree. C. in
0.2.times.SSC supplemented with 0.1% SDS, and autoradiographed for
15 hours. Control hybridization was performed on the same membrane
A with a mouse .beta.-actin probe.
[0055] FIG. 6 shows the results of this work. Each lane contained 5
.mu.g of poly-A+RNA from MZ2-MEL.43 cells. Control hybridization
was performed on the same membrane with a .beta.-actin probe. Two
bands of approximately 1 and 2.4 kb were observed.
[0056] Thus far, two main classes of antigens recognized by
autologous CTL have been found on human melanoma. The antigens of
the first class are encoded by genes that are expressed very
specifically in tumors. An antigen encoded by gene MAGE-1 was the
first example (van der Bruggen et al., Science, 254:1643-1647
(1991)), followed by other antigens encoded by genes MAGE-1 and
MAGE-3 (Gaugler et al., J. Exp. Med., 179:921-930 (1994); van der
Bruggen et al., supra). A tumor rejection antigen observed on mouse
mastocytoma P815 also resulted from the activation of a gene which
is silent in all normal adult tissues with the exception of testis
(Van den Eynde et al., J. Exp. Med., 173:1373-1384 (1991)). The
second class of antigens represents differentiation antigens
encoded by genes that are expressed only in melanocytes and
melanomas. Antigens encoded by tyrosinase were the first examples
of this class (Brichard et al., Annal. Biochem., 162:156-159
(1993); Robbins et al., Cancer Res., 54:3124-3126 (1994); Wolfel et
al., Eur. J. Immunol., 24:759-764 (1994)), which also comprises
antigens encoded by Melan-A/MART-1 (Coulie et al., J. Exp. Med.,
180:35-42 (1994); Kawakami et al., Proc. Natl. Acad. Sci. USA,
91:3515-3519 (1994)) and gp 100/pmel17 (Bakker et al., J. Exp.
Med., 179:1005-1009 (1994); Cox et al., Science, 264:716-719
(1994)).
[0057] The foregoing examples show the isolation of a nucleic acid
molecule which codes for a tumor rejection antigen precursor. This
"TRAP" coding molecule, however, is not homologous with any of the
previously disclosed MAGE coding sequences described in the
references set forth supra. Hence, one aspect of the invention is
an isolated nucleic acid molecule which comprises the nucleotide
sequence set forth in SEQ ID NO: 1. This sequence is not a MAGE
coding sequence, as will be seen by comparing it to the sequence of
any of the MAGE genes described in the references. Also a part of
the invention are those nucleic acid sequences which also code for
a non-MAGE tumor rejection antigen precursor but which hybridize to
a nucleic acid molecule containing the described nucleotide
sequence, under stringent conditions. The term "stringent
conditions" as used herein refers to parameters with which the art
is familiar. More specifically, stringent conditions, as used
herein, refers to hybridization in 3.5.times.SSC, 1.times.
Denhardt's solution, 25 mM, sodium phosphate buffer (pH 7.0), 0.5%
SDS, and 2 mM EDTA for 18 hours at 65.degree. C. This is followed
by four washes of the filter at 2.times.15 minutes in 2.times.SSC,
0.5% SDS and 1.times.15 minutes in 0.2.times.SSC, 0.1% SDS at
65.degree. C. There are other conditions, reagents, and so forth
which can be used, which result in the same degree of stringency.
The skilled artisan will be familiar with such conditions, and thus
they are not provided herein.
[0058] It will also be seen from the examples that the invention
includes the use of the sequences in expression vectors, as well as
in the transformation or transfection of host cells and cell lines,
including prokaryotic cell strains (e.g., E. coli), and eukaryotic
cells (e.g., CHO or COS cells). The expression vectors require that
the sequence be operably linked to a promoter. The expression
vector may also include a nucleic acid sequence coding for
HLA-Cw*1601. Where the vector contains both coding sequences, it
can be used to transfect a cell which does not normally express
either one. The tumor rejection antigen precursor coding sequence
may be used alone, when, for example, the host cell already
expresses HLA-Cw*1601 of course, there is no limit on the
particular host cell which can be used. As the vectors which
contain the two coding sequence may be used in HLA-Cw*1601
presenting cells if desired, and the gene for tumor rejection
antigen precursor can be used in host cells which do not express
HLA-Cw*1601
[0059] The invention also includes expression kits, which allow the
artisan to prepare a desired expression vector or vectors. Such
expression kits include at least separate portions of each of the
previously discussed coding sequences. Other components may be
added, as desired, as long as the previously mentioned sequences,
which are required, are included.
[0060] To distinguish the nucleic acid molecules and the TRAPs of
the invention from the previously described MAGE family, the
invention shall be referred to as the BAGE family of genes and
TRAPs. "BAGE" refers to the tumor rejection antigen precursors
coded for by the previously described sequence. "BAGE coding
molecule" and similar terms, are used to describe the nucleic acid
molecules themselves.
[0061] Also a part of the invention are peptides, for example, the
peptide of SEQ ID NO: 3, which can be used to identify those cells
which present MHC molecule HLA-Cw*1601. Administration of the
peptides, carrying a detectable signal, e.g., followed by the
identification of cells to which the peptide has bound, is one way
to accomplish this. Another way to accomplish this is the use of
solid phase bound peptides, to which HLA-Cw*1601 presenting cells
bind, thus removing them from the sample being assayed.
[0062] Additionally, the invention permits the artisan to diagnose
a disorder characterized by expression of the TPAP. These methods
involve determining expression of the TRAP gene, and/or TRAs
derived therefrom, such as the TRA presented by HL-Cw*1601. In the
former situation, such determinations can be carried out via any
standard nucleic acid determination assay, including the polymerase
chain reaction, or assaying with labelled hybridization probes. In
the latter situation, assaying with binding partners for complexes
of TRA and HLA, such as antibodies, is especially preferred. An
alternate method for determination is a TNF release assay, of the
type described supra.
[0063] The isolation of the TRAP gene also makes it possible to
isolate the TRAP molecule itself, especially TRAP molecules
containing the amino acid sequence coded for by SEQ ID NO: 1. These
isolated molecules when presented as the TRA, or as complexes of
TRA and HLA, such as HLk-Cw*1601, may be combined with materials
such as adjuvants to produce vaccines useful in treating disorders
characterized by expression of the TRAP molecule. In addition,
vaccines can be prepared from cells which present the TRA/HLA,
complexes on their surface, such as non-proliferative cancer cells
and non-proliferative transfectants. Immunization against both BAGE
and MAGE antigens can be undertaken. In all cases where cells are
used as a vaccine, these can be cells transfected with coding
sequences for one or both of the components necessary to prove a
CTL response, or can be cells which express both molecules without
transfection. Further, the TRAP molecule, its associated TRAs, as
well as complexes of TRA and HLA, may be used to produce
antibodies, using standard techniques well known to those skilled
in the art.
[0064] When "disorder" is used herein, it refers to any
pathological condition where the tumor rejection antigen precursor
is expressed. An example of such a disorder is cancer melanoma in
particular.
[0065] Therapeutic approaches based upon the disclosure are
premised on a response by a subject's immune system, leading to
lysis of TRA presenting cells, such as HLA-Cw*1601. One such
approach is the administration of CTLs specific to the complex to a
subject with abnormal cells of the phenotype at issue. It is within
the skill of the artisan to develop such CTLs in vitro.
Specifically, a sample of cells, such as blood cells, are contacted
to a cell presenting the complex and capable of provoking a
specific CTL to proliferate. The target cell can be a transfectant,
such as a COS cell of the type described supra. These transfectants
present the desired complex on their surface and, when combined
with a CTL of interest, stimulate its proliferation. COS cells,
such as those used herein, are widely available, as are other
suitable host cells.
[0066] To detail the therapeutic methodology, referred to as
adoptive transfer (Greenberg, J. Immunol., 136(5): 1917 (1986);
Reddel et al., Science, 257: 238 (7-10-92); Lynch et al., Eur. J.
Immunol., 21: 1403-1410 (1991); Kast et al., Cell, 59: 603-614
(11-17-89)), cells presenting the desired complex are combined with
CTLs leading to proliferation of the CTLs specific thereto. The
proliferated CTLs are then administered to a subject with a
cellular abnormality which is characterized by certain of the
abnormal cells presenting the particular complex. The CTLs then
lyse the abnormal cells, thereby achieving the desired therapeutic
goal.
[0067] The foregoing therapy assumes that at least some of the
subject's abnormal cells present the relevant HLA/TRA complex. This
can be determined very easily, as the art is very familiar with
methods for identifying cells which present a particular HLA
molecule, as well as how to identify cells expressing DNA of the
pertinent sequences, in this case a BAGE sequence. Once cells
presenting the relevant complex are identified via the foregoing
screening methodology, they can be combined with a sample from a
patient, where the sample contains CTLs. If the complex presenting
cells is lysed by the mixed CTL sample, then it can be assumed that
a BAGE derived, tumor rejection antigen is being presented, and the
subject is an appropriate candidate for the therapeutic approaches
set forth supra.
[0068] Adoptive transfer is not the only form of therapy that is
available in accordance with the invention. CTLs can also be
provoked in vivo, using a number of approaches. One approach, i.e.,
the use of non-proliferative cells expressing the complex, has been
elaborated upon supra. The cells used in this approach may be those
that normally express the complex, such as irradiated melanoma
cells or cells transfected with one or both of the genes necessary
for presentation of the complex. Chen et al., Proc. Natl. Acad.
Sci. USA, 88: 110-114 (1991) exemplifies this approach, showing the
use of transfected cells expressing HPVE7 peptides in a therapeutic
regime. Various cell types may be used. Similarly, vectors carrying
one or both of the genes of interest may be used. Viral or
bacterial vectors are especially preferred. In these systems, the
gene of interest is carried by, for example, a Vaccinia virus or
the bacteria BCG, and the materials de facto "infect" host cells.
The cells which result present the complex of 7:5 interest, and are
recognized by autologous CTLs, which then proliferate. A similar
effect can be achieved by combining the tumor rejection antigen or
the precursor itself with an adjuvant to facilitate incorporation
into HLA-Cw*1601 presenting cells which present the HLA molecule of
interest. The TRAP is processed to yield the peptide partner of the
HLA molecule while the TRA is presented without the need for
further processing.
[0069] Although the invention herein has been described with
reference to particular embodiments, it is to be understood that
these embodiments are merely illustrative of various aspects of the
invention. Thus, it is to be understood that numerous modifications
may be made in the illustrative embodiments and other arrangements
may be devised without departing from the spirit and scope of the
invention.
Sequence CWU 1
1
* * * * *