Note: Descriptions are shown in the official language in which they were submitted.
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1
ISOLATED PEBTIDEB WHICH FORM COMPLE7CE8 WITH MBC
MOLECULE BLA-C-CLONE 10 AND D8EB THEREOF
j'IELD OF THE INVENTION
This invention relates to peptides which are useful in
connection with the diagnosis and treatment of pathological
conditions. More particularly, it relates to peptides which
are processed to a peptide presented by the MHC molecule HLA-
C-clone 10, and the presented peptide itself. These peptides
are useful in diagnosis and therapeutic contexts.
~~C1CGRODND 1~~!JD PRIOR ART
The process by which the mammalian immune system
recognizes and reacts to foreign or alien materials is a
complex one. An important fx~cet 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 in
this regard 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 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:
CA 02165435 2003-03-03
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964 (1992j.
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 November 26, 1992,
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
l0 precursors" or "T;RAP" 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. patent
No. 5,342,774.
In U.S. patent No. S,G05,940~
nonapeptides
are taught which are presented by the HLA-A1 molecule. The
reference teaches that given the known specificity of
particular peptides for particular HLA molecules, one should
expect a particular peptide 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 H?~A molecule has diagnostic and therapeutic
ramifications, these axe 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 No. 5,5.58,995 .-
the fact that
the MAGE-1 expression product is processed to a second TRA is
disclosed" This second TRA is presented by HLA-C clone 10
molecules. The disclosure shows that a given TRAP can yield
CA 02165435 2003-03-03
a plurality of TRAs.
In U.~~. Patent Number '_>, r87, X374, 1=yrosinase is described
as a tumor rE=jecr:ion ~:mtigen pre~;ursor. This reference discloses
that a molecule which. i.~; produ:,ed by some normal cells (e.g.,
melanocytes) ,, is processed ire tumor cells t:o yield a tumor
rejection ant=igen that i.s presert:e<1 )v~y HLA--A2 molecules.
In L1.S. Paten':. I'lurnber 5, 620, 886, a second TRA, not
derived from tyrosin<nse is taught to be presented by HLA-A2
molecules. The TRA is derived from a TRAP, but is coded f=or by a
non MAGE gene. This di~c:losur:~ shows that a particular HLA
molecule may present 'I"RAs der_iver_1 from different sources.
In U.S. patent No. _'i,571.,'711, <3 new family of genes,
referred to therein a; the BAt~F; :family, was disclosed. It was
observed that these gE.me~> also ~_°.ode for r.umo~ rejection antigen
precursors. It was of>served in that: application that the MHC
molecule known as HL~-~wi~'-clone 10 presented a tumor rejection
antigen derived from ..~ BAC~E tumor rejection antigen precursor;
however, the tumor rejection <3ntig~ru was not disclosed. The
present application dE:eals with. this peptide, referred to herein
as SEQ ID 1VO:10, as ~,aeLa. as other ramificatvons stemming from
the identifi<:ation of this peptide.
The invention i.s elaborated upon further in the
disclosure which follo~as.
BRIEF DESCRIPTIOI\1 OF THE FIGURES
Figures 1A to LL~ set forth the results of a chromium
release assay using C'1'u c;-Lone 82/8% .against M'Z2-KIEL cell line in
Fig. lA, K56<'? cell lime ~n Fig. 1B, and MZ2-MEL 2.2.5 cell line
for Figs. 1C and 1D, where f'ig. 1C is the transfected variation.
Figure 2 shows the results of <~ TNF release assay when
CTL clone 82/82 was used against a panel of different cell
lines.
CA 02165435 2003-03-03
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Figure 3 depicts the results of a TNF release assay using
transfected COS cells.
Figure 4 presents the results of a test of various
transfectants using CTL clone 82/82, and measuring TNF
release.
Figure 5 shows results obtained when studies were
undertaken to determine half maximal lysis of cells, following
stimulation of cytolytic T cell clone CTL 82/82 with the
peptide of SEQ ID NO: 10.
DETAILED pE8CRIPTION OF PREFERRED EMBODII~iENTB
example 1
A melanoma cell line, MZ2-MEL was established from
melanoma cells taken from patient MZ2, using standard
methodologies. This cell line is described, e.g., in PCT
Application PCT/US92/04354, filed May 22, 1992, published
November 26, 199..
Once the cell line was established, a sample
thereof was irradiated, so as to render it non-proliferative.
These irradiated cells were then used to isolate cytolytic T
cell clones ("CTLs") specific thereto.
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.
The lysis assay employed was a chromium release assay
following Herin et al., Int. J. Cancer 39:390-396 (1987),
The assay,
however, is described herein. The target melanoma cells were
grown ~ Y'~, and then resuspended at 10' cells/ml in DMEM,
supplemented with 10 mM NEPES and 30~ FCS, and incubated for
minutes at 37°C with 200 ~Ci/ml of Na(S~Cr)O'. Labelled
cells were washed three times with DMEM, supplemented with 10 ,
mM Iiepes. These were then resuspended in DMEM supplemented
40 with 10 mM Hepes and 10~ FCS, after which 100 ul aliquots
containing 103 cells, were distributed into 96 well
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5 microplates. Samples of PBLs were added in 100 ul of the same
medium, and assays were carried out in duplicate. Plates were
centrifuged for 4 minutes at 1008, and incubated for four
hours at 37°C in a 8% Co2 atmosphere.
Plates were centrifuged again, and 100 ul aliquots of
:10 supernatant were collected and counted. Percentage of S~Cr
release was calculated as follows:
% ~~Cr release = jER-SRS x 100
(MR-SR)
:15
where ER is observed, experimental S~Cr release, SR is
spontaneous release measured by incubating 103 labeled cells
in 200 ul of medium alone, and MR is maximum release, obtained
by adding 100 ul 0.3% Tritori~X-100 to target cells.
:20 Those mononuclear blood samples which showed high CTL
activity were expanded and clamed 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.
a5 The same method was used to test target K562 cells, as
well as a melanoma cell line. These results, presented in
Figure lA, show that this CTL clone recognizes and ,lyses the
melanoma cell line, but not K562. The CTL clone, 82/82, was
then tested against melanoma cell lines in the same manner
:30 described ,~,g,~. Figure 1 shows that, while MZ2-MEL.43 is
lysed by CTL clone 82/82, the cell line M22-M~L 2.2.5, a
variant which has lost expression of HLA-A29, HLA-B44, and
HLA-C clone 10 is not, suggesting that the TRA is presented by
one of these HLA molecules. When cell line MZ2-MEL 2.2.5 was
:35 transfected with DNA coding for HLA-C clone 10, using well
known techniques, the cells became sensitive to lysis by CTL
clone 82/82, thus demonstrating that the antigen recognized by
CTL clone 82/82 is presented by HLA-C clone 10.
Trade-mark
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WO 95100159 PCT/US94106534
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Example 2
Further studies were carried out to determine if 82/82
also produced tumor necrosis factor ("TNF") when contacted
with target cells. The method used was that described by
Traversari et al., Immunogenetics 35: 145-152 (1992)
Eriefly,
samples of the CTL line were combined with samples of a target
cell of interests in culture medium. After 24 hours,
supernatant from the cultures was removed, and then tested
on TNF sensitive WEHI cells. A panel of fourteen different
1!i cell lines were tested, as shown in Figure 2.
The results, presented i.n terms of the percentage of WEHI
cells which died upon exposure to the supernatant, are shown
in Figure 2. The results demonstrate that three melanoma cell.
lines present this antigen. As a result of the strong
response by MZ2 MEL43, it was used in the experiments which
follow.
Example 3
The results from Example 2 indicated that MZ2.MEL.43
presented the target antigen of interest. As such, it was
used as a source of total mRNA to prepare a cDNA library.
Total RNA was isolated from the cell line. The mRNA was
isolated using an align-dT binding kit, following well
recognized techniques. Once the mRNA was secured, it was
transcribed into cDNA, again using standard methodologies.
The cDNA was then ligated to EcoRI adaptors and cloned into
the EcoRI site of plasmid pcD-SRa, in accordance with the
manufacturer's instructions. The recombinant plasmids were
then electroporated into 0'M101 ~. coli (electroporation
conditions: 1 pulse at 25 farads, 2500 V).
The transfected bacteria were selected with ampicillin -
(50 ~g/ml), and then divided into 87 pools of 400 bacteria
and 297 pools of 200 bacteria. Each pool represented either
about 280 or about 140 cDNAs, as analysis showed that about
70% of plasmids contained an insert. Each pool was amplified
to saturation, and plasmid DNA was isolated via alkaline
°
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I
:5 lysis, potassium acetate precipitation without phenol
extraction, following Maniatis et al., in Molecular Cloning:
A Laboratory Manual (Cold Spring Harbor, N.Y., 1982).
sample 4
Following preparation of the library described in Example
3, the cDNA was transfected into eukaryotic cells. The
transfections, described herein, were carried out in
duplicate. Samples of COS-7 cells were seeded, at 15,000
cells/well into tissue culture flat bottom microwells, in
Dulbeco~s modified Eagles Medium ("DMEM") supplemented with
1!5 l0% fetal calf serum. The cells were incubated overnight at
37°C, medium was removed and then replaced by 50 ~cl/well of
DMEM medium containing 10% Nu~serum, 400 ~Cg/ml DEAF-dextran;
and 100 ~uM chloroquine, plus 100 ng of subject plasmids.
These plasmids were the plasmids of the various pools
described supra, and 100 ng of plasmids containing DNA coding
for HLA-C clone 10 in plasmid pcD-SRa. Following four hours
of incubation at 3T°C, the medium was removed, and replaced by
50 ~l of PBS containing 10% DMSO. This medium was removed
after two minutes and replaced by 200 ~1 of DMEM supplemented
2!5 with 10% FCS.
Following this change in medium, COS cells were incubated
for 24-48 hours at 37°C. Medium was then discarded, and 1500
cells of CTL clone 82/82 were added, in 100 ~C1 of Iscove
medium containing 10% pooled human serum supplemented with 20
3~D U/ml of IL-2. Supernatant was removed after 24 hours, and TNF
content was determined in an assay on WEHI cells, as
described by Traversari et al., Immunogenetics 35: 145-152
(1992),
3!5 Of the 384 pools tested, 99% stimulated TNF, at a
' concentration below 5 pg/ml. Two pools gave concentrations
above 40 pg/ml, with duplicate wells giving equivalent
results. Figure 3 shows this. The bacteria from one of these
pools, i.e., pool .19, were selected for further experiments.
4 ~~
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WO 95/00159 , PCT/LTS94/06534
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Examule 5
The bacteria of pool 19 were cloned, and 800 bacteria
were tested. Plasmid DNA was extracted therefrom, transfected
into a new sample of COS cells in the same manner as described
supra, and the cells were again tested for stimulation of CTL
clone 82/82. Twelve positive clones were found. One of
these, referred to as cDNA clone AD5 was tested further. In
a comparative test COS cells were transfected with cDNA clone
AD5 and the HLA-C clone 10. HLA-C clone 10 and MAGE-1, AD5
alone, or HLA-C clone 10 alone. Control cell lines MZ2-MEL
2.2.5 and MZ2-MEL.43 were also used. TNF release in CTL
supernatant was measured by testing it on WEHI cells, as
referred to supra. The optical density of the surviving WEHI
cells was measured using MTT. Figure 4 shows that only the
COS cells transfected with HLA-C clone 10 and cDNA-ADS, and
the original cell line MZ2-MEL 43 stimulated TNF release from
CTL clone 82/82.
Example 6
The cDNA AD5 was sequenced following art known
techniques. A sequence search revealed that the plasmid
insert showed no homology to known genes or proteins.
SEQUENCE ID NO: 1 presents cDNA nucleotide information for the
identified gene, referred to hereafter as "BAGE-1". A
putative open reading frame is located at bases 201-332 of the
molecule.
Example 7
Following the sequencing of the cDNA, as per example 6,
experiments were carried out to determine if cells of normal
tissues expressed the gene. To determine this, RNA isolated
from normal tissues was reverse transcribed, using oligo-dT as
primer. The resulting cDNA was then amplified, using primers:
5' CAG AAG ATG AAG CAC AGA G-3' (SEQ ID NO: 2)
and
5'-GAG CGG TTT TTC TGG CAT TG-3~ (SEQ ID NO: 3)
and standard PCR methodologies. Radioactive nucleotides were
WO 95/00159 216 5 4 3 5 ~T~S94/06534
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added so that the amount of amplification product could be
determined via phosphor imaging.
The amount of product was expressed as a percentage of
the product secured from cell line MZ2-MEL 3.0, which was
shown to express the gene. The results are as follows:
MZ2-MEL 3.0 100%
Lung < 0.5%
Breast "
Stomach "
Skin "
Brain
Prostate "
Kidney "
Testis 8%
In additional experiments not elaborated upon herein, the
DNA of cell line MZ2-MEL was digested with EcoRI, and then
hybridized with a PCR probe corresponding to the first 300
nucleotides of the cDNA described herein. Following standard
Southern blotting, four bands, corresponding to sizes of
approximately 5.8, 7.5, 8.5 and 11 kilobases were identified,
suggesting the existence of a family of Bage genes.
Example 8
Expression of the gene by tumor samples and tumor cell
lines was also determined. cDNA was secured just as in
example 4, and then nested primer methodologies were carried
out to amplify the pertinent sequences. First, twenty cycles
of amplification were carried out, using primers:
5'-CGG CTT AGA GGA CCA GGA GAA-3' (SEQ ID NO: 4)
and
5'CAG AAG ATG AAG CAC AGA G-3' (SEQ ID NO: 5)
This was followed by twenty additional cycles using primers:
5'-GGC TCC AAC CTC CAG CTC AC-3' (SEQ ID NO: 6)
AND
5'-TTA GAG GAC CAG GAG AAG G-3' (SEQ ID NO: 7)
The results are presented below. The first figure is the
WO 95/00159 PCT/US94/06534
5 number of positive samples; the second is the total number
of
samples tested:
Melanoma 12/20
Breast Carcinoma 2/5
Small Cell lung carcinoma 2/8
10 Non-small cell lung carcinoma 2/5
Sarcoma 1/4
Head and neck tumors 1/6
Colon carcinoma 0/4
Kidney tumor 0/5
Leukemia/Lymphoma 0/3
Example 9
The experiments set forth supra showed that cytolytic T
cell clone CTL 82/82 recognized an antigen encoded by a GAGE
gene and presented by HLA-C-clone 10. The work described in
this example details how the amino acid sequence of the
presented antigen was determined.
The cDNA clone identified as coding for BAGE, i.e., ADS,
was used to generate a large number of incomplete cDNA
molecules. The cDNA clone was inserted into expression vector
pcDNAI/Amp, and digested with NotI and SphI restriction
endonucleases, followed by treatment with exonuclease III, in
accordance with the manufacturer's instructions. By using
exonuclease III for varying lengths of time, progressive
deletions of AD5 at its 3' end were obtained. The truncated
variants were religated into pcDNAI/Amp, electroporated into
_E. co i strain DHSaF'IQ, and selected via ampicillin (50
~g/ml). Four hundred clones were obtained in this way.
The plasmid DNA was obtained from these 400 cones, and
transfected into COS-7 cells together with HLA-C-clone 10
coding cDNA. The transfectants were then tested in a TNF
release assay, as described supra. Positive clones were those
which stimulated TNF release by CTL 82/82.
Once cells were divided into positive and negative
transfectants, the sequences of plasmid DNA from 10 positives
and 10 negatives was determined. Clone 19C2, a positive
WO 95100159 216 5 4 3 5 ' PCT/US94/06534
11
clone, contained part of the open reading frame for the BAGE
gene described.supra,, from nucleotide 1 to nucleotide 67. In
contrast, clone 17612, a negative transfectant, contained
nucleotides 1-6 of the gene.
By comparing the inserts of positive and negative clones,
a region of 22 amino acids was identified as probably
containing the sequence of the presented peptide, i.e.:
Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln
Ala Arg Leu Met Lys Glu
(SEQ ID NO: 8)
Synthetic peptides were then prepared, based upon this
sequence, and were tested for their ability to render COS-7
cells transfected with HLA-C-clone l0 capable of stimulating
TNF release. The first positive peptide was a 16-mer:
Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln
(SEQ ID NO: 9)
Testing of smaller peptides led to the identification of
peptide
Ala Ala Arg Ala Val Phe Leu Ala Leu
(SEQ ID NO: 10)
This peptide effectively stimulated CTL 82/82, with half
maximal lysis being reached at a peptide concentration of 80
nM, which is shown in Figure 5.
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
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WO 95/00159 PCT/(1S94/06534
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with which the art is familiar. More specifically, stringent
conditions, as used herein, refers to hybridization in
3.5xSSC, ixDenhardt~s solution, 25 mM sodium phosphate buffer
(pH 7.0), 0.5% SDS, and 2 mM EDTA for 18 hours at 65°C. This
is followed by four washes of the filter, at 65°C for 20
minutes, in 2xSSC, 0. 1 % SDS, and one wash for up to 20 minutes
in 0.3xSSC, 0.1% SDS. 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 given here.
It will also be seen from the examples that the invention
embraces the use of the sequences in expression vectors, as
well as to transfect host cells and cell lines, be these
prokaryotic (e.g., E. coli), or eukaryotic (e.g., CHO or COS
cells). The expression vectors require that the pertinent
sequence, i.e., those described supra, be operably linked to
a promoter. As it has been found that human leukocyte antigen
HLA-C clone 10 presents a tumor rejection antigen derived from
these genes, the expression vector may also include a nucleic
acid sequence coding for HLA-C clone 10. In a situation 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, e.g., the host cell already expresses HLA-C
clone 10. Of course, there is no limit on the particular host
cell which can be used. As the vectors which contain the two
coding sequences may be used in HLA-C clone 10 presenting
cells if desired, and the gene for tumor rejection antigen
precursor can be used in host cells which do not express HLA-C
clone 10.
The invention also embraces so called expression kits,
which allow the artisan to prepare a desired expression vector
or vectors. Such expression kits include at least separate
portions of 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.
WO 95/00159 216 5 4 3 5 PCT/US94/06534
13
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. Hence, whenever "BAGE" is used herein, it refers
to the tumor rejection antigen precursors coded for by the
previously described sequences. "BAGE coding molecule" and
similar terms, are used to describe the nucleic acid molecules
themselves.
Also a part of the invention are the peptides of SEQ ID
NO: 8, 9 and 10 which are set forth in Example 9. These
peptides can be used, for example, to identify those cells
which present MHC molecule HLA-C-clone 10. 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, as is the use of solid phase bound
peptides, to which the HLA-C-clone 10 presenting cells bind,
thus removing them from the sample being assayed.
Additionally, the invention permits the artisan to diagnose a
disorder characterized by expression of the TRAP. These
methods involve determining expression of the TRAP gene,
and/or TRAs derived therefrom, such as the TRA presented by
HLA-C clone 10. 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.
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 HLA-C clone 10, 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
WO 95/00159 PCT/LTS94/06534
14
cells which present the TRA/HLA complexes on their surface,
such as non-proliferative cancer cells, non-proliferative
transfectants, etcetera. 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 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 the art.
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.
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-c clone l0
cells. 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.
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
WO 95/00159 2 ~ 6 5 4 ~ 5 PCT/US94/06534
characterized by certain of the abnormal cells presenting the
particular complex. The CTLs then lyse the abnormal cells,
thereby achieving;the desired therapeutic goal.
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 are 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.
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
(January, 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, e.g., a Vaccinia
virus or the bacteria BCG, and the materials de facto "infect"
host cells. The cells which result present the complex of
interest, and are recognized by autologous CTLs, which then
proliferate. A similar effect can be achieved by combining
the tumor rejection antigen or the precursor itself with an
WO 95/00159 PCT/US94/06534
16
adjuvant to facilitate incorporation into HLP.-C clone 10
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.
Other aspects of the invention will be clear to the
skilled artisan and need not be repeated here.
The terms and expressions 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 expressions 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.
WO 95/00159 216 5 4 3 5
PCTIUS94/06534
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SEQUENCE LISTIN~3
(1) GENERAL INFORMATION:
(i) APPLICANT: van der Bruggen, Pierre
Boon-Falleur, Thierry
(ii) TITLE OF INVENTION: ISOLATED PEPTIDES WHICH FORM COMPLEXES WITH
MHC MOLECULE HLA-C-CLONE 10 AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 10
(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) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette, 5.25 inch, 360 kb storage
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: PC-DOS
(D) SOFTWARE: Wordperfect
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 08/196,630
(B) FILING DATE: 15-FEB-1994
(C) CLASSIFICATION: 435
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/079,110
(B) FILING DATE: 17-JUN-1993
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Hanson, Norman D.
(B) REGISTRATION NUMBER: 30,946
(C) REFERENCE/DOCRET NUMBER: LUD 310.1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 688-9200
(B) TELEFAX: (212) 838-3884
WO 95/00159 216 5 4 3 5 PCT/US94106534
18
(2) INFORMATION FOR SEQ ID NO: 1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1032 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: l:
CGCCAATTTA GGGTCTCCGG TATCTCCCGC TGAGCTGCTC TGTTCCCGGC TTAGAGGACC 60
AGGAGAAGGG GGAGCTGGAG GCTGGAGCCT GTAACACCGT GGCTCGTCTC ACTCTGGATG 120
GTGGTGGCAA CAGAGATGGC AGCGCAGCTG GAGTGTTAGG AGGGCGGCCT GAGCGGTAGG 180
AGTGGGGCTG GAGCAGTAAG ATGGCGGCCA GAGCGGTTTT TCTGGCATTG TCTGCCCAGC 240
TGCTCCAAGC CAGGCTGATG AAGGAGGAGT CCCCTGTGGT GAGCTGGAGG TTGGAGCCTG 300
AAGACGGCAC AGCTCTGTGC TTCATCTTCT GAGGTTGTGG CAGCCACGGT GATGGAGACG 360
GCAGCTCAAC AGGAGCAATA GGAGGAGATG GAGTTTCACT GTGTCAGCCA GGATGGTCTC 420
GATCTCCTGA CCTCGTGATC CGCCCGCCTT GGCCTTCCAA AGTGCCGAGA TTACAGCGAT 480
GTGCATTTTG TAAGCACTTT GGAGCCACTA TCAAATGCTG TGAAGAGAAA TGTACCCAGA 540
TGTATCATTA TCCTTGTGCT GCAGGAGCCG GCTCCTTTCA GGATTTCAGT CACATCTTCC 600
TGCTTTGTCC AGAACACATT GACCAAGCTC CTGAAAGATG TAAGTTTACT ACGCATAGAC 660
TTTTAAACTT CAACCAATGT ATTTACTGAA AATAACAAAT GTTGTAAATT CCCTGAGTGT 720
TATTCTACTT GTATTAAAAG GTAATAATAC ATAATCATTA AAATCTGAGG GATCATTGCC 780
AGAGATTGTT GGGGAGGGAA ATGTTATCAA CGGTTTCATT GAAATTAAAT GTTATCAACG 840
GTTTCATTGA AATTAAATCC AAAAAGTTAT TTCCTCAGAA AAATCAAATA AAGTTTGCAT 900
GTTTTTTATT CTTAAAACAT TTTAAAAACC ACTGTAGAAT GATGTAAATA GGGACTGTGC 960
AGTATTTCTG ACATATACTA TAAAATTATT AAAAAGTCAA TCAGTATTCA ACATCTTTTA 1020
CACTAAAAAG CC 1032
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ZD NO: 2:
CAGAAGATGA AGCACAGAG
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
GAGCGGTTTT TCTGGCATTG 20
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
WO 95/00159 ~ PCT/US94/06534
19
CGGCTTAGAG GACCAGGAGA A 21
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(8) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
CAGAAGATGA AGCACAGAG 19
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
GGCTCCAACC TCCAGCTCAC 20
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
TTAGAGGACC AGGAGAAGG 19
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acid residues
(B) TYPE: amino acid
(D) TOPOLOGY: single
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln
10 15
Ala Arg Leu Met Lys Glu
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
WO 95/00159 2 ~ b 5 4 3 5 PCT/US94/06534
(A) LENGTH: 16 amino acid residues
(B) TYPE: amino acid
(D) TOPOLOGY: single
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
Met Ala Ala Arg Ala Val Phe Leu Ala Leu Ser Ala Gln Leu Leu Gln
5 10 15
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acid residues
(B) TYPE: amino acid
(D) TOPOLOGY: single
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
Ala Ala Arg Ala Val Phe Leu Ala Leu
5
