Note: Descriptions are shown in the official language in which they were submitted.
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Tumor-specific antigens, methods for their production and their use for
immunization and
diagnosis
The invention relates to new tumor-specific antigens, methods for their
production, and
their use for immunization, in particular, for the activation of cytotoxic,
tumor-specific T-
lymphocytes, and for specific diagnosis of tumor cells presenting said tumor-
specific
antigen in MHC class I-complex.
The immune system plays an important role in immunosurveillance against cancer
and in
tumor regression. The anti-tumor immune responses can be mediated through B
and T cells
which recognize tumor antigens expressed on tumor cells. The generation of
cvtotoxic T
lymphocytes (CTLs) from either peripheral blood lymphocytes or tumor-
infiltrating
lymphocytes (TILs) derived from patients with cancer in the last few years has
allowed one
to evaluate the role of T cells in the process of tumor regression in humans.
The adoptive transfer of tumor-infiltrating lymphocytes along with
interleultin 2 (II,-2) into
autologous patients with cancer can mediate the regression of tumor in humans
(Rosenberg .
et al., New Engl. J. Med. 319 (1988) 1676-1680; Rosenberg et al., J. Natl.
Cancer Inst. 86
(1994) 1159-1166), suggesting that T cells play an important role in tumor
rejection in
vivo. The ability to mediate tumor regression in vivo was associated with the
ability of
TILs to mediate specific lysis and cytoltine release when co-cultivated with
syngenic tumor
cells in vitro (Barth et al., J. Exp. Med. 173 (1991)647-658).
To understand the molecular basis of T cell-mediated anti-tumor responses, a
variety of
genes recognized by T cells encoding tumor antigens have been identified (Boon
et al.,
Annu. Rev. Immunol. 12 (1994) 337-365; Houghton, J. Exp. Med. 180 ( 1994) 1-4;
Tsomides and Eisen, Proc. Natl. Acad Sci. USA 91 (1994) 3487-3489; Pardoll,
Nature 369
(1994) 357-358; Rosenberg, Cancer J. Sci. Am. 1 (1995) 90-100). Based on their
expression pattern these antigens can be divided into several classes:
The first class of tumor antigens includes antigens (e.g. MAGE, BAGE and GAGE)
that
are shared between melanomas and other tumors of various histological types,
but not by
normal tissues other than testis and placenta {Van der Bruggen et al., Science
254 (1991)
1643-1647; Boel et al., Immunity 2 (1990 I67-175; Van den Eynde et al., J.
Exp. Med 182
(1995) 689-698). Clinical trials based on the use of these antigens recognized
by CTL-
restricted by different HLA-class I alleles are in progress (Marchand et al.,
Int. J. Cancer
Sc/So 25.9.98
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63 (1995) 883-885; Rosenberg, Immunology Today (1997) 175-182) in patients
affected by
melanoma and other neoplastic diseases. Taking into account the frequency of
expression
of each of these antigens and that of the HLA class I alleles, more than 60%
of Caucasian
melanoma patients, 40% of the head and neck, and 28% of bladder cancer
patients could be
eligible for immunization with at least one antigen of this group. No side
effects have been
detected in the treated patients. Indeed, MAGE, BAGS and GAGE gene expression
r
normally occurs in cells of testis like spermatogonia and spermatocytes (II),
which do not
express the classical MHC class I molecules (Haas et al., Am. J. Reprod.
Immunol.
Microbiol. 18 ( 1988) 47-57) needed for antigen presentation and thus will not
be targeted
by T cells.
The second class of tumor antigens contains tissue-specific antigens expressed
in normal
and neoplastic cells of the melanocyte lineage. CTL recognizing epitopes from
tyrosinase
{Brichard et al., J. Exp. Med. 178 (1993) 489-49513), MelanAMa"' (Coulie et
al., J. Exp.
Med. 180 (1994) 35-42; Castelli et al., J. Exp. Med 181 {1995) 363-368;
Kawakami et al.,
Proc. Natl. Acad. Sci. USA 91(1991) 3515-3519), gp100P""" (Bakker et al., J.
Exp. Med.
179 (1994) 1005-1009; Kawakami et al., Proc. Natl. Acad Sci. USA 91 (1994)
6458-6462),
gp75 ~P' (Wang et al., J. Exp. Med. 181 (1995) 799-804) and TRP-2 (Wang et
al., J. Exp.
Med. 184 (1996) 2207-2216) on melanoma and normal cultured melanocytes can be
expanded in vitro from many melanoma patients. Therefore, a large fraction of
melanoma
patients could potentially benefit of immunotherapy trials aimed at inducing a
T cell
response against these antigens. Indeed, differentiation antigens are
expressed in almost all
melanomas and the majority of them are presented to the immune effectors by
the HLA-A2
allele that has a high frequency in various ethnic groups. However, the
potential side
effects of these treatments due to the development of cross-reacting responses
against
normal tissues (i.e., skin melanocytes and pigmented retinal cells) must be
carefully
considered.
The third class of tumor antigens includes antigens expressed only by the
tumor cells from
which they have been isolated. Such antigens are not expressed in other normal
or
neoplastic tissues of different origin and the antigenic epitope is usually
generated by a
point-mutation occurring in an otherwise ubiquitously expressed protein. Tumor
antigens
belonging to this group have been described in the murine system (Boon et al.,
Annu. Rev.
Immunol. 12 (1994) 337-365) and in some human tumors (Wolfel et al., Science
269
(1995) 1281-1284; Coulie et al., Proc. Natl. Acad Sci. USA 92 (1995) 7976-
7980; Robbins
et al., J. Exp. Med. 183 (1996) 1185-1192). The lack of natural tolerance
against these
antigens may allow the induction of a strong immune response, while avoiding
the
development of potential auto-immune reactions. However, until a panel of
broad hot-spot
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mutations will be discovered, the clinical application of such antigens should
be limited to
the treatment of individual patients or at least to very few individuals whose
tumor carries
the given mutation (Wolfel et al., Science 269 (1995)1281-1284).
A fourth class of tumor antigens results from alternatively processed
transcripts. Robbins et
al. describe in J. Immunol. 159 (1997) 303-308 a gp100 transcript
corresponding to a part
T"
of the fourth intron and coding for additional 35 amino acids not found in the
normal
gp100 glycoprotein. However, this epitope is expressed only in low levels in
melanomas.
Robbins et al., in J. Immunol. 154 (1995) 5944-5950, describe the cloning of
gene
encoding an antigen recognized by melanoma-specific HLA-A24 restricted tumor-
infiltrating lymphocytes. This antigen is a fragment of a full-length clone
not encoded by
an intron or a part thereof.
Fujii et al., in J. Immunol. 153 (1994) 5516-5524, describe a soluble form of
the non-
tumoricidal HLA-G antigen, which is coded by mRNA containing intron 4.
However, no
specific function for the soluble HLA-G protein is known at this time.
The existence of a fifth set of tumor antigens has been recently suggested
based on the
pattern of reactivity of a panel of CTL clones able to recognize the
autologous and HLA-
matched allogeneic melanomas, but not melanocytes or other targets of
different
histological origin (Anichini et al., J. Immunol. 156 (1996) 208-217).
It is an object of the invention to provide new tumor-specific antigens which
are not
expressed in normal cells and are capable of specifically distinguishing tumor
cells from
normal cells.
Summary of the invention
The invention comprises a tumor-specific polypeptide having an antigenic
effect,
characterized in that it is partly coded by intron sequences from the gene of
a polypeptide
which is presented by the MHC class I complex on tumor cells (an exon-coded
tumor
antigen) and is obtainable by reverse transcriptase PCR from rnRNA isolated
from the
soluble cytoplasmic fraction of a tumor cell, whereby nucleic acid fragments
which
hybridize under stringent conditions with intron sequences of an exon-coded
tumor antigen
are used as a prymer; and, if a PCR product is obtained, by isolation of the
PCR product,
expression in a host cell, and isolation of the tumor-specific antigen coded
by the PCR
product.
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Said antigen can be presented as a fragment by an antigen-presenting cell
(APC) in order to
induce specific CTL response.
Another object of the invention is a method for the identification of such a
tumor-specific
polypeptide having an antigenic effect, whereby the following is earned out:
- reverse transcriptase PCR from mRNA of a tumor cell, whereby nucleic acid
fragments which hybridize under stringent conditions with intron sequences of
an
exon-coded tumor antigen are used as a primer;
if a PCR product is obtained: isolation of the PCR product, expression in a
host cell,
and isolation of the tumor-specific antigen coded by the PCR product which
hybridizes also with exon sequences of said antigen.
After isolation, the hybridization product, or a fragment thereof, is inserted
into an
expression vector, the vector is transferred into an appropriate host cell and
expressed in
said host cell. Subsequently, the resultant recombinant polypeptide is
isolated.
In a preferred embodiment of the invention, a fragment of 8 to 12 codons of
the
hybridization product is used for the expression of the antigen.
The subject-matter of the invention therefore is a tumor-specific polypeptidic
antigen
which is coded partially by an intron of an exon-coded tumor antigen and which
is obtained
by
a) reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic
fraction
of a tumor cell, whereby nucleic acid fragments which hybridize under
stringent
conditions with intron sequences of an exon-coded tumor antigen are used as a
primer,
b) isolation of the product of said PCR, expression of said PCR product or of
a fragment
thereof in a host cell, and isolation of said tumor-specific antigen which is
coded by
said PCR product or a fragment thereof and which hybridizes also with exon
sequences of said antigen.
Another subject-matter of the invention is a tumor-specific polypeptidic
antigen according
to the invention, wherein a fragment of 8 to 1? codons of said PCR product is
used for the
expression.
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Yet another subject-matter of the invention is a tumor-specific antigen
according to the
invention, wherein said exon-coded tumor antigen is a CTL recognizing antigen
like
MAGE, BAGE and GAGE, CTL recognizing epitopes from tyrosinase, MelanAM"'~,
gp100P"'e~7, °oP75TRP~ or TRP-2.
The present invention further relates to a tumor-specific polypeptidic antigen
which is
l
coded by SEQ ID NO:1.
A further subject-matter of the invention is a method for the isolation of
mRNA of a tumor-
specific antigen coded by an intron of an exon-coded tumor antigen, whereby
there is
carried out
a) reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic
fraction
of a tumor cell, whereby nucleic acid fragments which hybridize under
stringent
conditions with intron sequences of an exon-coded tumor antigens are used as a
primer, and
b) the product of said PCR is isolated which hybridizes also with exon
sequences of
said antigen.
The invention further relates to a method for measurement of proliferation of
tumor-
specific cytotoxic T-cells, wherein a tumor-specific antigen according to the
invention is
added to a sample of a body fluid of a patient, which contains antigen-
presenting cells and
cytotoxic T cells, and the proliferation of the cytotoxic T cells is measured,
preferably via
cytokine release (measurement of cytokines like TNF, IFNy, GM-CSF).
Another subject-matter of the present invention is the use of a nucleic acid
coding for a
tumor-specific antigen according to the invention for the manufacture of a
therapeutic
agent for the treatment of a tumor disease.
The invention in addition relates to the use of a tumor-specific antigen
according to the
invention for the activation of cytotoxic T cells from T precursor cells in
vivo or in vitro.
A further subject-matter of the invention is a method for the production of a
tumor-specific
polypeptidic antigen, wherein said tumor-specific antigen is obtained by
a) reverse transcriptase PCR from mRNA isolated from the soluble cytoplasmic
fraction
of a tumor cell, whereby nucleic acid fragments which hybridize under
stringent
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conditions with intron sequences of an exon-coded tumor antigen are used as a
primer,
b) isolation of the product of said PCR, expression of said PCR product or of
a fragment
thereof in a host cell, and isolation of said tumor-specific antigen which is
coded by
said PCR product or a fragment thereof which hybridizes also with exon
sequences
of said antigen.
Yet another subject-matter of the invention is a combination of two nucleic
acids which
hybridize under stringent conditions with intron sequences of an exon-coded
tumor antigen
and which can be used as a primer pair for reverse transcriptase PCR from
mRNA.
Nucleic acids which are coded by SEQ )D N0:3 to SEQ )D N0:9 are a further
subject-
matter of this invention.
The tumor antigen according to the invention is not found on the surface of
normal cells
such as melanocytes. However, it is found on more than 80% of, e.g., melanoma
cells and
hence is specific for tumor cells.
Thus, this peptide is particularly suitable for the tumor cell-specific
immunization of
patients, and also for the diagnostic differentiation of melanoma cells and
normal
melanocytes.
A cytolytic T lymphocyte clone (CTL 128), derived from peripheral blood
lymphocytes of
a patient with metastatic melanoma, was able to lyse the autologous tumor and
several
allogenic melanomas in an HLA A*6801 restricted fashion. The gene coding for
the
antigen recognized by CTL 128 was identified by transfection of a cDNA
library,
constructed from autologous melanoma mRNA, into Cos-7 cells expressing the
HLA-A*6801 allele. It has surprisingly been found that in contrast to
melanocytes splicing-
errors occur in mRNA maturation. This results in the translation of a peptide
which was
composed by a partially spliced form of the melanocyte differentiation antigen
(TRP)-2
containing exon 1-4 with retention of intron 2 and part of intron 4 (TRP-2-
INT2). TRP-2-
INT2 codes for a putative protein of 238 amino acids which runs, using the
same reading
frame of TRP-2, from the start codon in position 400 to the terminator site
(nt 1113)
located in intron 2, just 18 nt downstream the peptide coding region.
The differentiation antigen TRP-2 is described by R.F. Wang, J. Experimental
Medicine
184 (1996) 2207-2216. TRP-2 is one of the most highly expressed glycoproteins
in human
pigmented melanocytic cells and melanoma (Wang et al., J. Exp. Med. 184
(1996), 2207-
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2216). It is located on the human chromosome 13 and has been shown to be a
member of
the tyrosinase-related gene family and shares a 40-45% amino acid sequence
identity with
tyrosinase and gp75/TRP-1 {Yokoyama et al., Biochim. Biophys. Acta. 1217
(1994), 317-
321; Robbins et al., J. Immunol. 154 (1995), 5944-5950). TRP-2 encodes a
protein with
519 amino acids and has been demonstrated to have DOPAchrome tautomerase
activity
which is involved in melanin.synthesis (Bouchard et al., Eur. J. Biochem. 219
(1994), 127-
134).
In contrast to the fully spliced TRP-2 mRNA which is described by Wang et al.
to be found
in melanomas, normal skin melanocytes and in retina, the TRP-2-INT2 mRNA was
detected exclusively in melanomas. These results indicate that melanoma
antigens
recognized by CTL may derive from known lineage-related proteins by
differences in
splicing occurring in tumor but not in normal cells. This mechanism results in
a new group
of antigens that can be considered as true tumor specific and shared only by
tumors of the
same histotype while absent in normal tissues of the same lineage. This new
group of
antigens is therefore characterized by being derived from known lineage-
related proteins by
a tumor-specific and alternative splicing which does not occur in normal
cells. Normal
melanocytes, skin samples and retina proved negative in a reverse
transcriptase (RT) PCR
analysis. These features define an antigen that may allow the development of
safer and
more efficacious immunotherapy trials.
The present invention therefore relates to partially intron-coded tumor-
specific antigens
that are obtained by reverse transcriptase PCR from mRNA isolated from the
soluble
cytoplasmic fraction of a tumor cell. These antigens are recognized by
specific T
lymphocytes which then lyse the tumor cells presenting, in the MHC class I
complex, the
antigen according to the invention. It was surprisingly found that the mRNA
coding for the
antigens of the invention enriched in the cytoplasm of such tumor cells is
particularly
tumor-specific.
By "intron-coded tumor-specific antigen" is meant a tumor antigen which is
coded not only
by an exon sequence but partly (preferably about or more than 30%, more
preferably about
or more than 50%, most preferably about or more than 80%), by an intron
sequence and
which specifically recognizes tumor antigens that are presented via MHC class
I. The
expression of the intron is related to mechanisms) of altered splicing in
lineage related
proteins. "Partly" means preferably 30 to 50% or 30 to 80% intron coded. The
exon coded
and intron coded sequences of the antigens according to the invention are
directly linked in
the related genomic sequence because said antigens are caused by alternative
splicing.
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By "exon-coded tumor antigens" are meant the antigens described in the
introductory part,
such as, e.g., MAGE, BAGS and GAGE (Van der Bruggen et al., Science 254 (1991)
1643-1647; Boel et al., Immunity 2 (1995) 167-175; Van den Eynde et al., J.
Exp. Med 182
(1995) 689-698) or such as e.g. CTL recognizing epitopes from tyrosinase
(Brichard et al.,
J. Exp. Med. I78 (1993) 489-49513), MelanAMa"' (Coulie et al., J. Exp. Med.
180 (1994)
35-42; Castelli et al., J. Exp. Med 181 (1995) 363-368; Kawakami et al., Proc.
Natl. Acad.
Sci. USA 91(1991) 3515-3519), gplOOpm''7 (Bakker et al., J. Exp. Med. 179
(1994) 1005-
1009; Kawakami et al., Proc. Natl. Acad Sci. USA 91 (1994) 6458-6462), gp75~p'
(Wang
et al., J. Exp. Med. 181 (1995) 799-804) and TRP-2 (Wang et al., J. Exp. Med.
184 (1996)
2207-2216).
By "peptide or polypeptide according to the invention" is meant a polypeptide
which
preferably consists of 8 to 12 amino acids, and most preferably at least 10
amino acids, but
may also comprise the size of a protein. The peptide may be also a part of a
protein, such as
a fusion protein.
By "polypeptide having an antigenic effect" is meant a polypeptide that
elicits, in vivo and
in vitro, a specific immune response.
The term "hybridize under stringent conditions" means that two nucleic acid
fragments are
capable of hybridization to one another under standard hybridization
conditions described
in Sambrook et al., "Expression of cloned genes in E.coli" in Molecular
cloning: A
laboratory manual (1989), Cold Spring Harbor Press, New York, USA, 9.47 - 9.62
and
11.45 - 11.61.
More specifically, "stringent conditions" as used herein refer to
hybridization in 6.0 x SSC
at about 45°C, followed by a wash of 2.0 x SSC at SO°C. For
selection of the stringency the
salt concentration in the wash step can be selected, for example, from about
2.0 x SSC at
50°C, for low stringency, to about 0.2 x SSC at 50°C, for high
stringency. In addition, the
temperature in the wash step can be increased from low stringency conditions
at room
temperatures, about 22°C, to high stringency conditions at about
65°C.
For expression in a prokaryotic or eukaryotic organism, such as prokaryotic
host cells or
eukaryotic host cells, the nucleic acid sequence is integrated into suitable
expression
vectors, according to methods familiar to a person skilled in the art. Such an
expression
vector preferably contains a regulatable / inducible promoter. These
recombinant vectors
are then introduced for the expression into suitable host cells, such as,
e.?., E.coli as a
prokaryotic host cell, or Saccharomyces cerevisiae. CHO or COS cells as
eukaryotic host
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cells, and the transformed or transduced host cells are cultured under
conditions which
allow the expression of a heterologous gene. The isolation of the peptide can
be carried out
according to known methods from the host cell or from the culture supernatant
of the host
cell. Such methods are described, for example, by Ausubel L, Frederick M.,
Current
S Protocols in Mol. Bioi. (1992) John Wiley and Sons, New York.
By "host cells" are meant prokaryotic and eukaryotic cells, preferably COS or
CHO cells.
Production in prokaryotic cells will be preferred if glycosylation proves to
be of minor
importance with respect to the action of the protein.
The gene coding for the tumor-specific antigen recognized by a specific
cytotoxic
T lymphocyte (CTL) can be identified by transfection of a cDNA library,
constructed from
autologous tumor mRNA, into eukaryotic cells, preferably expressing a suitable
HLA allele
of the patient.
In order to determine the HLA antigen the entire RNA is isolated from the
patient's tumor
cells or tumor tissue, and the cDNA is obtained by reverse transcriptase PCR
with primers
1S that specifically code for the HLA antigens, and is cloned in eukaryotic
expression vectors,
such as, e.g., pcDNA3 (Invitrogen Corporation, Oxon, U.K.). In order to
determine the
intron-coded tumor-specific antigen according to the invention the poly A+ RNA
is
isolated from mRNA isolated from the soluble cytoplasmic fraction of a tumor
cell, and a
cDNA library is established by reverse transcriptase PCR with primers that
specifically
code for an exon-coded tumor antigen, such as, e.g., MAGE, BAGE and GAGE, CTL
recognizing epitopes from tyrosinase, MelanA Martt, gp100 gp7S ~P~ and TRP-2.
The
cDNA fragments from the cDNA library are cloned into eukaryotic expression
vectors,
such as, e.g., pcDNA3.1 (Invitrogen Corporation, Oxon, U.K.). After co-
transfection of the
expression vectors into eukaryotic cells, such as, e.g., Cos-7 cells
(expressing relevant
2S MHC genes which are able to present the peptide fragment of the antigen
which is capable
of activating specific tumor antigen T cells), one may then determine those
clones that
cause stimulation of the tumor-specific cytotoxic T lymphocytes. The
stimulatory effect
upon the tumor-specific cytotoxic T lymphocytes can be determined by a CTL
stimulation
assay (determination of TNF-a, IFN-y, GM-CSF) as described by Traversari et
al.,
Immunogenetics 3S (1992), 145-152.
In this manner it is possible to obtain a cell clone which specifically
activates those
cytotoxic T cells that cause lysis of the respective tumor cells.
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This cell clone expresses a polypeptide which, in isolated and purified form,
may be
administered to the patient for immunization / vaccination, either directly as
a full length
polypeptide since the antigenic polypeptide according to the invention is
processed in vivo,
or in the form of shortened polypeptide fragments.
To this end, the nucleic acid coding for the polypeptide according to the
invention is
r
isolated from this cell clone according to established methods (Sambrook et
al., Molecular
cloning (1989), Cold Spring Harbor Laboratory Press) and is shortened by
restriction
digestion. These shortened fragments, after cloning into eukaryotic expression
vectors, are
then transfected into eukaryotic cells, such as, e.g., COS-7 cells (supra),
and the stimulation
of the cytotoxic T cells is determined in a CTL stimulation assay as described
by Traversari
et al. in Immunogenetics 35 (1992) 145-152. In this manner the coding nucleic
acid
sequence can be restricted to the peptide epitope essential for the
stimulation of the
cytotoxic T cells.
In a preferred embodiment, the nucleic acid sequence coding for the protein is
coupled with
a nucleic acid sequence capable of enhancing the processing of the protein in
the host cell.
Meanwhile a number of sequences, such as, e.g., ubiquitin, are known which
enhance the
transport of the protein, the degradation of the protein and the introduction
into the MHC
class I complex (Bachmair et al., Science 234 (1986) 179-186; Gonda et al., J.
Biol. Chem.
264 (1989) 16700-16712; Bachmair et al., Cell 56 (1989) 1019-1032). A faster
degradation
of the protein and a more effective introduction into the MHC class I complex
can be
achieved by coupling a protein containing the antigenic polypeptide according
to the
invention with ubiquitin. As a result of this, the presentation of the peptide
on the cell
surface of the tumor cells is particularly efficient. In this connection, the
amino acid
between the protein containing the antigenic peptide and ubiquitin is of
special importance
regarding the coupling of a protein containing the antigenic peptide with
ubiquitin. By
selecting the appropriate amino acid one can additionally achieve a
considerable
improvement in the recognition of the melanoma cell by the specific cytotoxic
T cell.
As a preferred embodiment a fragment of 8 to 12 codons of the PCR products is
used.
In another preferred embodiment there is used a partly intron-coded tumor-
specific antigen
TRP-2-INT2 (hereafter referred to as TRP-2-INT2 fragment or TRP-2-INT2
peptide)
which is recognized by specific T lymphocytes which then lyse those melanoma
cells that
present in the MHC class I complex the polypeptide EVISCKLIKR and whose amino
acid
sequence is coded by the DNA sequence shown in SEQ ID NO:?.
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To obtain the coding sequences of the tumor-specific antigen of the TRP-2-INT2-
peptide
the cDNA coding for the HLA-A*6801 allele and the TRP-2-INT2 antigen were
isolated
from the cytoplasm of a melanoma cell line which was established from a
metastatic lesion
obtained from a surgical specimen of a patient, who was admitted to surgery to
the Istituto
~ Nazionale Tumori (Milan, Italy). After transfection of both cDNAs in COS-7
cells a clone
was isolated (TRP-2-INT2, HLA-A*6801), which was able to stimulate the
cytotoxic effect
of a cytolytic T lymphocyte (CTL128). The DNA sequencing analysis presented a
cDNA
fragment of the exon-coded TRP-2 antigen which contains exon 1-4 with
retention of
intron 2 and part of intron 4 (TRP-2-INT2). Subfragments of the TRP-2-INT2
fragment
were obtained by digestion of the TRP-2-INT2 DNA with restriction enzymes
according to
methods familiar to a person skilled in the art or according to methods which
are described
by Sambrook et al., "Expression of cloned genes in E-coli" in Molecular
cloning: A
laboratory manual (1989), Cold Spring Harbor Press, New York, USA, 5.3 - 5.10
or by
PCR amplification with fragment-specific primers (SEQ m NOS:6, 7, 8). After
transfection into COS-7 cells the CTL-stimulating effect is determined by a
CTL
Stimulation Assay as described in Traversari et al., Immunogenetics 35 (1992)
145-152.
The coding nucleic acid sequence was determined by DNA sequence analysis.
Another subject-matter of the invention is the use of tumor cells presenting
the antigen
according to the invention via MHC class I.
Another subject-matter of the invention is a method for the detection of the
expression of
the antigen according to the invention from the patient's body fluid,
preferably a blood or
tissue specimen. To this end, an intron-coded tumor-specific cDNA coding for
the antigen
according to the invention is used prior to, during, and after therapy to
diagnose tumor cells
which express the antigen according to the invention.
Prior to therapy, it is examined whether the tumor antigen according to the
invention is
expressed in the patient's tumor cells, because only if the tumor does indeed
express the
antigen according to the invention can the therapy be carried out. During the
course of
therapy, one can monitor whether the expression of the antigen according to
the invention,
being a marker for tumor cells present, can be reduced, and after therapy,
measurement of
the expression of the antigen according to the invention allows controlling of
whether the
tumor cells have been eliminated to the fullest possible extent.
In a preferred embodiment, a nucleic acid coding for the tumor-specific TRP-?-
INT2
peptide is used for the diagnosis of melanoma cells presenting on their
surface the TRP-2-
INT2 peptide.
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The sequence for the TRP-2-INT2 peptide is specifically expressed in
melanomas. Hence,
TRP-2-INT2 sequences may be used as specimens for identifying tumor cells.
Identification is by means of PCR or labelled hybridization samples or by any
of the
various nucleic acid probe based assays known in the art.
The determination of the TRP-2-INT2 expression is carried out at the mRNA
level by
reverse transcription PCR (RT-PCR) and by hybridization with specific labelled
TRP-2-INT2 probes.
TRP-2-INT2 mRNA can be specifically determined, for distinction against TRP-2
mRNA,
by
1) transcription to cDNA;
2) the use of intron-specific primers;
3) comparing the length of the amplified cDNA fragment (determining the length
of the
amplified cDNA exon-intron-exon fragment with cDNA exon to exon);
4) hybridizing with intron-specific probes.
Another subject-matter of the invention is a method for the determination of
proliferation
of tumor-specific cytotoxic T lymphocytes. This method is used for the
detection of
cytotoxic T cells which can be activated by the antigen according to the
invention, prior or
during the course of therapy. Prior to therapy, it is possible to select
patients who already
have cytotoxic T lymphocytes that can be activated by the antigen according to
the
invention.
To check whether there are tumor-specific activatable T cells in the patient,
T cells and
antigen-presenting cells are isolated from the patient's blood, and the
antigen according to
the invention is brought into contact ("pulsed"), ex vivo, with the antigen-
presenting cells.
If T cells capable of being activated by the antigen according to the
invention are present in
the patient's blood, there will be a proliferation of the specific cytotoxic L
lymphocytes
which can be detected then by a CTL stimulation assay, for instance. Such
activatable T
cells, after being stimulated with the antigen according to the invention, can
be used for
therapy then. This diagnostic procedure must be carned out prior to therapy.
Another diagnostic procedure, during therapy, serves as a control of the
cytotoxic L
lymphocytes formed, making it possible to quantitatively determine the
induction of tumor-
specific T cell activation.
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The proliferation of the cytotoxic T lymphocytes can be determined by a CTL
stimulation
assay, for instance. The stimulating cell lines are tested for their ability
to induce the
production of TNF by the CTLs as described in Traversari et al.,
Immunogenetics 35
(1992) 145-152. The TNF content is determined by testing its cytotoxic effect
on WEHI-
164.13 cells (Espevic and Nissen-Meyer, J. Immunol. Methods 95 (1986) 99-105)
in an
MTT colorimetric assay (CTL Stimulation Assay).
In a preferred embodiment, the TRP-2-1NT2 peptide is used for the
determination of
proliferation of TRP-2-INT2-specific cytotoxic T lymphocytes.
Another subject-matter of the invention is the use of a nucleic acid, which
codes for the
intron-specific antigen according to the invention, for the manufacture of a
therapeutic
agent for the treatment of tumor diseases.
In this connection, nucleic acids according to the invention can be used for
gene therapy.
The nucleic acid is introduced into the patient's body with the help of viral
or non-viral
vectors, whereby the coding sequence should be specifically expressed and the
peptide
according to the invention should, by virtue of the binding to antigen-
presenting cells, elicit
a specific cytotoxic T cell response. The immune response elicited should then
be directed
against all tumor cells that express on their cell surface the peptide
according to the
invention. The DNA sequences coded on vectors can be applied in the form of
nude DNA,
in combination with liposomes, or together with a suitable adjuvant (as well-
known in the
art), either subcutaneously, intramuscularly, or intratumorally.
In a preferred embodiment, the TRP-2-INT2 peptide is used for gene therapy.
In another embodiment, the antigen according to the invention can be used for
the
immunization and/or vaccination of tumor patients. The immunization is based
on the
activation of specific cytotoxic T cells by presenting the antigen according
to the invention
via antigen-presenting cells. Immunization can be carned out both ex vivo and
in vivo.
In doing so, antigen-presenting cells (macrophages, dendritic cells, or B
cells) and T
lymphocytes are taken from the patient's blood and brought into contact
("pulsed), ex vivo,
with the peptide according to the invention. These antigen-presenting cells
equipped with
the peptide according to the invention, which in this manner cause an
activation of specific
cytotoxic T cells, are subsequently returned to the patient's blood.
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Immunization can be carried out in vivo by subcutaneously administering to the
patient the
polypeptide according to the invention, whereby the activation of specific
cytotoxic T cells
directly in the patient is achieved. The binding of the peptide according to
the invention to
the corresponding HLA molecule on the surface of antigen-presenting cells
leads to
proliferation of specific cytotoxic T lymphocytes.
The immunogenic effect of the peptide according to the invention during
application can be
enhanced by the following measures:
1) coupling with bacterial toxins (superantigens);
2) administration in combination with Freud's adjuvant;
3) mixing the peptide according to the invention with liposomes.
In a preferred embodiment, the TRP-2-INT2 peptide is used for the immunization
and
vaccination of melanoma patients.
Another subject-matter of the invention is a primer for the detection of the
expression of
the specific tumor antigen according to the invention by RT-PCR. In this
connection, the
primer can be selected by the following measures:
1) The sense and anti-sense primer hybridizes with two difference exon
sequences of
the tumor antigen.
2) The sense primer hybridizes with an exon sequence and the anti-sense primer
(oligo-
dT primer) hybridizes with the poly-A tail of the mRNA sequence of the tumor
antigen.
In a preferred embodiment, a specific primer is used for the detection of the
expression of
the TRP-2-INT fragment whose amino acid sequence is coded by the DNA sequence
shown in SEQ ID N0:2 (PRIT-3) and SEQ >D N0:4 (INT2-1260).
The following examples, references, sequence listing and the drawings are
provided for
further illustrating various aspects and embodiments of the present invention
and are in no
way intended to be limiting in scope.
Description of the drawings:
Figure 1: CTL 128 recognized the autologous melanoma (Me18732) in an HLA-
A*6801 restricted fashion. Target cells were incubated with the
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indicated anti-HLA mAb for lh at room temperature before addition
of effectors at fixed E:T ratio {A.: Me 18732 + none; B: Me18732 +
HLA Class I; C: Me18732 + HI,A-A2, -A69; D: ME18732 + HLA-
A2, -A28; Me18732 + HLA-B, -C).
Figure 2: Recognition by CTL 128 of autologous melanoma Me18732 and
'r
HLA-A*6801+ melanoma cell lines. 1,00 CTL were added to 25,000
stimulator cells and the TNF content of the supernatant was tested 24
h later on WEHI-164.13 cells (Melanomas: A: Me18732; B: Me
20842; C: Me17697; D: Me 2559/1; E: Me12657; F: Me17088; G: Me
4023; H: LB-33; I: Lung carcinoma Calu 3; K: Breast carcinoma
SKBR3; L: Ovarian carcinoma SKOV3).
Figure 3: Stimulation of CTL clone 128 by Cos-7 cells transfected with cDNA
131 and HLA-A*6801 cDNA. Cos-7 cells were transfected with HLA-
A*6801 and with pool A255 or cDNA 131. Pool A255 is a (group of
1S 100 cDNA clones of the Me18732 cDNA library which was amplified
to saturation and from which plasmid DNA was extracted. cDNA 131
was a single clone subcloned from pool A255. The production of TNF
by CTL 128 was measured after 20 h of co-culture with the transfected
cells, using the TNF sensitive cell line WEHI-164.13. As control Cos-
7 cells were transfected with cDNA 13I or HLA-A*6801 alone (A:
Me18732; B: COS; C: COS + A*6801; D: COS + cDNA 131; E: COS
+ A*6801 + cDNA 131).
Figure 4: cDNA 131 codes for the antigen recognized by CTL 128. (A)
Recognition and (B) lysis by CTL 128 of a geneticin resistant
population of the HLA-A*6801 melanoma cell line LB33, following
transfection with the pcDNA3.1/cDNA 131 construct. TNF secretion
by CTL 128 was measured after 24 h of co-culture of 1,500 responder
cells with 20,000 stimulating cells. Lytic activity of CTL I28 was
measured on j'Cr labeled target cells after 4 h of co-incubation with
the CTL at different effector-to-target (E/T) ratios.
Figure ~: Identification of the sequence coding for the antigenic peptide
recognized by CTL 128. Exon/intron organization of cDNA 131 is
shown in the upper part of the panel (A). Exon and introns are
indicated as solid and open boxes respectively, the horizontal line at
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the extremities represents pcDNA3.1 vector, while the numbering of
the sequence is relative to the 5'end of cDNA 131. Subfra~ments
derived from cDNA 131 and PCR products, shown below cDNA 131
as open boxes, were cloned in expression vectors and transfected into
Cos-7 cells with HLA-A*6801 cDNA. Spliced full-length form of
TRP-2 cDNA was obtained by screening the i 8732 cDNA library with
an exon 8 specific oligonucleotide probe. TNF release by CTL 128
was evaluated on WEHI164.13 cells (B). The peptide encoding
sequence present in the PCR fragments JNT-2-I66 and INT-?-107 are
pointed out.
Figure 6: Lysis by CTL 128 of HLA-A*6801 cells pulsed with the synthetic
antigenic peptide. 5'Cr-labeled HLA-A*6801 EBV-LCL (LB-EBV)
were incubated with CTL 128 at an E/T ratio of 20:1, in the presence
of the synthetic peptides shown on the left, at the concentration
indicated.''Cr-release was measured after 4 h. As a negative control, a
MAGE-3 derived peptide (M3A1) able to bind HL,A-A1 was used.
Figure 7: Recognition by CTL 128 of TRP-2-INT2~2i-23, peptide when
presented by HI.A alleles of the A3-like supertype. 5'Cr-labeled EBV-
LCLs were incubated with CTL 128 at an E/T ratio of 20:1, in the
presence of peptide TRP-2-INT2,21_~;, at different concentrations.
Chromium release was measured after 4 h. c: negative control without
pepti de.
Description of the seauences:
SEQ ID NO:1 : coding sequence (nucleic acid) of the antigenic peptide
recognized by
CTL 128.
SEQ ID N0:2 : coding sequence (amino acid) of the antigenic peptide recognized
by
CTL 128.
SEQ ID N0:3 : (PRTT-1) sense primer used for the verification of unspliced
intron
TRP2-INT; located in the 5'UTR of the TRP2-gene.
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SEQ ID N0:4 : (INT2-1260) anti-sense primer used for the verification of
unspliced
intron TRP2-INT; located in the 5'IJTR and intron 2 of the TRP2-
gene.
SEQ ID NO:~ : (KS-INT2) sense primer used for the production of the
subfragments
of cDNA 131 and for cloning of TRP-2-INT2 from genomic DNA;
located in exon 2 of the TRP2-gene.
SEQ ID N0:6 : (INT2-asl) anti-sense primer used for the production of
subfragment
INT2-107 of cDNA 131; located in intron 2 of the TRP2-gene.
SEQ ID N0:7 : (INT2-as?) anti-sense primer used for the production of
subfragment
INT2-166 of cDNA 131; located in intron 2 of the TRP2-gene.
SEQ ID N0:8 : (Sp6) anti-sense primer used for the production of subfragment
INT2-434 of cDNA 131; located in the pCDNAI-plasmid.
SEQ ID N0:9 : (PR2) anti-sense primer used for cloning of TRP-2-INT2 from
genomic DNA; located in exon 3.
SEQ ID NO:10 : (PR3) sense primer used for amplification of TRP-2 DNA; located
in
exon 2.
SEQ ID NO:11 : (TRP-2L) anti-sense primer used for amplification of TRP-2 DNA;
located in exon 8.
SEQ ID N0:12 : Nucleic acid sequence of the 5'end-1500 fragment including the
coding region for the antigenic peptide. The first 45 by before the start
of cDNA 131 and belonging to pcDNA3.1 vector are omitted.
Examale 1
Identification of HLA-Ax6801 as a restriction element for CTL 128
Melanoma cell line Me18732 was established from a metastatic lesion of a
patient, typed as
LA-A2 and HLA-A28 by serological methods and then as HL.A-A*0201 and HLA
*68011
(further referred to as HLA-A*6801) by sequence-specific oligonucleotide probe
(SSOP)
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subtyping (Oh et al., Genomics 29 (1995) 24-34). Anti-tumor CTL clones were
obtained as
described by Anichini et al., J. Immunol. 156 (1996) 208-217.
CTL clone 128 recognized the autologous melanoma in the context of an allele
of the
HLA-A locus, since its cytolytic activity was reduced by the anti-HLA Ciass I
mAB W6/32, but not by the anti-HLA-B, -C mAb 4E (Fig. 1 ). HLA-A*0201 could be
excluded as presenting molecule for the antigen recognized by CTL 128 since
inhibition of
lysis was observed only with the anti-HLA-A2, -A28 mAb CRl 1.351, but not with
the
anti-HLA-A2, -A69 mAb BB7.2 (Fig. 1). The inhibitory activity of CR1 1.351,
therefore,
indicated that A28 (A*6801) was the HLA presenting molecule for the CTL 128.
Cultivation of cell lines
The melanoma cell line Me18732 was established from a metastatic lesion
obtained from a
surgical specimen of a patient who was admitted for surgery to the Istituto
Nazionale
Tumori (Milan, Italy). PBLs of this patient were serologically typed as: HLA-
A2, -A28, -
B44, -BSI, -C2, -C5. Human metastatic (Me17697, Me2559/1, Me12657, Me17088/1,
Me4023) and primary (Me20842) melanoma cell lines were established and
cultured in
10% FCS/RPMI 1640. The melanoma line LB-33, LB-40 and the Cos-7 (ATCC: CRL
1651) cell line were maintained in 10% FCS/DMEM. The carcinoma lines CALU3,
SKBR3 and SKOV3, purchased from the American Type Culture Collection (ATCC,
Rockville, MD), were kept in culture in 10% FCS/RPMI- 1640. C1RA*03301
transfectant,
the homozygous EBV-transformed LCL, the cell lines SCHU is characterized as
HLA-
A*0301, B*0702, -C7, AMA-1 is characterized as HLA-A*6802, B*5301, -C4, and WT-
100-bis is characterized as HLA-Al l, -B3~, -C4. The EBV-LCL JHAF (HLA-
A*31011, -
B51, -C8) and LB (HLA-A*68011, B*40011, -C2, -C3) were obtained from ATCC. EBV-
LCL were maintained in 10% FCS/RPMI-1640.
Evaluation of the antigenic specificity of CTL 128
To evaluate the frequency of expression of the antigen recognized by CTL 128
on other
tumors, a panel of HLA-A*6801 melanoma lines was tested in a CTL stimulation
assay.
Five out of eight melanoma cell lines induced TNF release by CTL i28 (Fig. 2).
No
reactivity was instead observed with three HLA-A*6801 carcinoma lines of
different
histological origin (Fig. 2). HLA-A*680i negative melanomas, melanocytes and
tumor
lines of other histological type failed to stimulate cytokine release by CTL
128. The pattern
of reactivity displayed by CTL 128 towards the melanoma cell lines tested did
not correlate
with expression of already described melanoma antigens in these cell lines, as
assessed by
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RT-PCR. This was confirmed by lack of TNF release by CTL 128 in the presence
of Cos-7
cells cotransfected with plasmid pcDNA3/A*6801, containing the HLA-A*6801 gene
of
patient 18732, together with each of the genes known to encode shared melanoma
antigens:
Melan-A/MART- 1, tyrosinase, gp 100, TRP-1, -2, MAGE- l, 2, -3, -4, -12, BAGS-
l, -2,
GAGE-1, -2, -3, -4, -5, -6. These results were consistent with the hypothesis
that HLA
A*6801 restricted CTL 128 recognized a new antigen shared by a number of
melanomas.
The CTL clone 128 was derived by limiting dilution of 4 week-old mixed
lymphocyte-
tumor cultures (MLTC) and grown in conditions similar to those previously
described
(Anichini et al., J. Immunol. 156 (1996) 208-217). CTL 128 expressed a CD3+,
CD4-,
CD8+, TCR-+ phenotype, as assessed by flow cytometry with specific mAbs.
Assay for cytolytic activity:
The lytic activity of CTL 128 was tested in a chromium release assay as
previously
described (Anichini et al., J. Immunol. 156 ( 1996) 208-217). Results were
expressed as:
(experimental release - spontaneous release)
% lysis =
(maximum release - spontaneous release)
where spontaneous release was assessed by incubating target cells in the
absence of
effectors, and the maximum release was determined in the presence of 1 % NP-40
detergent (BDH Biochemicals, Poole, U.K.). Inhibition of lysis against the
autologous
melanoma was performed with the following mAbs as reported (Anichini et al.,
J.
Immunol. 156 (1996) 208-217): the anti-HLA-A, - B, -C W6/32 (Parham et al., J.
Immunol. 123 (1979) 342-349), the anti-HLA-A2, -A69 BB7.2 (Parham and Brodsky,
Hum. Immunol. 3 (1981) 277-299), the anti-HLA-A2, -A28 CR11.351(Russo et al.,
Immunogenetics 18 (1983) 23-35), and the anti-HLA-B, -C 4E (Yang et al.,
Immunogenetics 19 (1984) 217-231).
Subcloning of the HLA -A*6801 allele
Total RNA was prepared from Me18732 cells by the Quanidine-isothiocyanate
method
using RNAzoI.B (Cinna/Biotecx, South Loop East, TX). Single-stranded cDNA
synthesis
was carried out on 2 pg of total RNA using oligo-dT primer and Moloney murine
leukemia
virus-derived reverse transcriptase without RNase-H activity (MMLV-RT RNase-H-
Superscript; GIBCO BRL, Gaithersburg, MD) according to the manufacturer's
instructions.
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cDNA corresponding to 300 ng of total RNA was amplified by PCR using 1 U of
DynaZymeTM (Finnzymes OY, Espoo, Finland) and a primer pair suitable for
specific
amplification and directional cloning of the full length coding region of HLA-
A alleles.
Following BamHI and Hindlll digestion, the 1.1 Kb PCR-product was subcloned
into the
BamHI/EcoRV site of the eukaryotic expression vector pcDNA3 (Invitrogen
Corporation,
Oxon, UK).
Plasmid clones encoding the HLA-A*68011 or the A*0201 (the HLA-A28 and -A2
alleles
of patient 18732) were identified using diagnostic restriction enzymes. The
HLA-A*68011
gene was then sequenced to verify the correspondence to the published DNA
sequence.
This plasmid was called pcDNA3/HI,A-A*6801.
Example 2
Cloning of a cDNA encoding the melanoma antigen recognized by CTL 128
A cDNA library was constructed in a suitable expression vector (pcDNA3.l) with
poly(A)+
RNA extracted from Me18732 cells. Poly(A)+ RNA was isolated from Me18732 cells
using the Fast Track mRNA extraction kit (Invitrogen). The library was
constructed
converting 5 pg of poly(A)+ RNA to cDNA with the Superscript Choice System kit
(GIBCO BRL, Gaithersburg, MD) using an oligo-dT primer containing a NotI site
at its 5'-
end. cDNAs were then ligated to BstXI adapters (Invitrogen) and digested with
NotI. After
size fractionation, cDNAs were unidirectionally cloned into the BstXI/NotI
site of the
mammalian expression vector pcDNA3.1 (Invitrogen). Recombinant plasmids were
electroporated into DHS- Escherichia coli and selected with ampicillin (100
mg/ml).
The library was divided into 1,300 pools of about 100 cDNA clones. Each pool
of bacteria
was amplified to saturation, plasmid DNA was extracted and transfected (100
ng) together
with pcDNA3/A*6801 (100 ng) into 1.2x10' Cos-7 cells by the DEAE-dextran-
chloroquine method (Coulie et al., J. Exp. Med. 180 ( 1994) 35-42; Seed and
Aruffo, Proc.
Natl. Acad. Sci. USA 84 (1987) 3365-3369). Using the same technique, in other
experiments Cos-7 cells were cotransfected with 100 ng of pcDNA3/A*6801 vector
and
100 ng of pcDNAI or pcD SR plasmids containing the cDNA of one of the
following
melanoma antigens: Melan A/MART-I, tyrosinase, gp100, MAGE-l, -2, -3, -4, -12,
BAGS-1, -2, GAGE-1, -2, -3, -4,-~, -6, TRP-1. FuII length TRP-2 cDNA was
amplified by
RT PCR using specific primers located in the 5'-untranslated region (UTR) and
at the end
of exon 8, cloned into pcDNA3 and sequenced to verify the correspondence to
the
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published cDNA sequence (Yokohama et al., Bioch. Bioph. Acta 1271 (1994) 317-
321).
Transfected Cos-7 cells were tested in a CTL stimulation assay after 48 h.
CTL stimulation assay
Transfectants or stimulating cell lines were tested for their ability to
induce the production
of TNF by CTL 128 as previously described {Traversari et al., Immunogenetics
35 (199?)
145-152). Briefly, 1,500 CTL were added to microwells containing target cells,
in 100 1 of
1MDM (BioWhittaker, Walkersville, MD) with 10% pooled human serum (PHS) and
25 U/ml r-hu-IL.2 (EuroCetus, Amsterdam, The Netherlands). After 24 h, the
supernatant
was collected and its TNF content was determined by testing its cytotoxic
effect on WEHI
cells, such as WEHI-164.13 (Espevic and Nissen-Meyer, J. Immunol. Methods 95
(1986)
99-105) in an MTT colorimetric assay. The WEHI-164.13 cells were kept in
culture in 10%
FCS/RPMI- 1640.
Duplicate microcultures were transfected and screened two days later for their
ability to
stimulate TNF release by CTL 128. The DNA of one of the 1,300 pools (pool
A255)
induced the production of a high level of TNF (Fig. 3), a finding confirmed in
a second
transfection experiment. Bacteria of the positive pool were cloned and their
plasmid DNA
was cotransfected with the HL,A-A*6801 construct as before. 25 out of 159
clones
stimulated TNF release by CTL 128. The results obtained with one of these,
namely cDNA
131, are shown in Fig. 3.
Transfection of melanoma cell tines
The melanoma cell line LB-33 was transfected by the calcium phosphate
precipitation
technique with cDNA 131 cloned in plasmid pcDNA3.l (Invitrogen), which
contains the
neomycin resistance gene. A clonal subline was isolated from a 6418-resistant
transfected
population. Using the same method. the melanoma cell lines LB-40, SK23-MEL and
MZ2-
MEL and maintained in 10% FCS/DMEM) were transfected with HLA-A*6801 cDNA and
selected in 6418. Expression of the transfected HLA A*6801 allele in stable
transfectants
was verified by flow cytometry with specific mAbs.
The HL,A-A*6801+ melanoma cell line LB-33, which was not recognized by CTL 128
(see
Fig. 2), when transfected with cDNA 131 acquired the property capable of
inducing TNF
release (Fig. 4A) and became sensitive to lysis by CTL 128 (Fig: 4B),
indicating that
recognition of the antigen may occur in a tumor cell and was independent of
the high,
artificial expression level achieved in Cos-7 cells.
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The sequence of the cDNA 131 proved to be 3548 by long. By searching Genbank,
it was
found that nucleotides (nt) 1-994 and nt 3081-3347 were identical to two non-
contiguous
regions of the cDNA coding TRP-2 (Yokohama et al., Bioch. Bioph. Acta 1217
(1994)
317-321), which has been recently identified as a melanoma antigen of the
melanocyte
lineage (Wang et al., J. Exp. Med. 184 (1996) 2207-2216). The first region
contained the
5'-UTR, exon 1 and exon 2, whereas the second region exon 3 and exon 4 of the
TRP-2
gene, respectively. The sequence between nt 995-3080 and that downstream nt
3347
showed no significant homology with any sequence recorded in databanks. The
two regions
which were present in cDNA 131 but absent in the TRP-2 cDNA were retained
intron
sequences. A stretch of ten amino acids flanking the exonic portions perfectly
matched the
described sequences at exon-intron junctions of the TRP-2 gene (Sturm et al.,
Genomics 29
(1995) 24-34). Moreover, the length of sequence 995-3080 (2086 nt) was
compatible with
that of the intron 2 of TRP-2, as deduced from the published genomic map
{Sturm et al.,
Genomics 29 (1995) 24-34). The identity of nt 995-3080 was therefore
consistent with that
of the intron 2 of TRP-2. The sequence downstream nt 3,347 of cDNA 131
presented a 5'
donor splice site sequence identical to that of the intron 4 of TRP-2 (Sturm
et al.,
Genomics 29 (1995) 24-34). Since it lacks the 3' acceptor splice site sequence
and its
length is considerably shorter than that estimated from the published genomic
map, this is
the sequence of intron 4 of TRP-2, truncated at nt 200. Thus, cDNA 131 is
composed of a
partially spliced form of the melanocyte differentiation antigen TRP-2
containing exon 1-4
with retention of intron 2 and of the initial portion of intron 4 (Fig. S).
DNA sequencing and homology search
DNA sequencing analysis was performed by specific priming with synthetic
oligonucleotides. The sequencing reactions were performed by the dideoxy-chain
termination method using dye-labeled dideoxynucleotides and the ABI PRISMTM
Dye
Terminator Cycle Sequencing Ready Reaction kit (Perkin Elmer, Foster City,
CA). DNA
sequence of the plasmid clone cDNA 131 was determined with an automated DNA
sequences (ABI Prism 377 DNA Sequences; Perkin Elmer). The computer search for
the
sequence homology was done with the program FASTA EMBL-Heidelberg.
Identification of the sequence encoding the antigenic peptide recognized by
CTL 128
To localize the sequence coding for the antigenic peptide recognized by CTL
128, cDNA
131 was digested with HindIll and three subfragments of 1500, 200 and 2000 bp,
respectively (5'-end-1500, 200 by and 3-end in Fig. 5) were obtained.
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Production of subfragments of cDNA 131
Subfragments of cDNA 131 (5'-end-1500, 5'-end-800, 200 by and 3'-end) were
obtained
by digestion of the plasmid with HindllI and BstUI. After purification on
agarose gel, the
fragments were cloned into the pcDNAI plasmid. From the 5'-end-1500, the
smaller
fragments INT-2-434, IN'T-2-166 and INT-2-107 were generated by PCR
amplification.
The sense primer KS-INT2 (SEQ )D NO:S) was used for amplification of all the
fragments.
This primer generates an ATG start codon (underlined), with an appropriated
Kozak
consensus sequence, in the same frame as TRP-2. The anti-sense primer used for
amplification of the INT-2-434, -166 and -107 fragments respectively were SP6
(SEQ ID NO: 8) located in the pcDNAI plasmid, INT2-as 1 (SEQ >D N0:6) and INT2-
as2
(SEQ ID N0:7), located in intron 2 of TRP-2. The anti-sense primers INT2-asl
and -as2
contained a XhoI restriction site for directional cloning. To facilitate
legations, we took
advantage of the presence of a single 3' A-overhang, due to the terminal
transferase activity
of the DNA polymerase, at the 5'-end of the DynazymeTM-amplified fragments.
The
pcDNAI plasmid was digested with EcoRV and a single thymidine was then added
at the
3' end of each fragment by incubation with DynazymeTM in the presence of 2 mM
dTTP,
as described by Marchuk et al., Nucl. Acids Res.l9 {1991) 1154. The T-vector
as well as
the PCR products were digested with XhoI, purified on agarose gel and legated.
After
legation the plasmids were electroporated into DH-5 E. coli and selected with
ampicillin
(50 mg/mI). Clones were isolated, plasmid DNA was extracted and transfected
into Cos-7
cells along with the HLA-A*6801 gene.
At this step the presence in the two 5'-end fragments of start codons
regulating their
Translation was not investigated. The level of TNF released by CTL 128 in the
presence of
Cos-7 cells transfected with the 5'-end-1500 fragment (SEQ >D N0:12) was
comparable to
that stimulated by cDNA 131 (Fig. 5), indicating that the antigenic peptide
was encoded
within this region. The nucleotide sequence of this subfragment, which
encompassed exon
1, exon 2 and the first 410 by of intron 2, is shown in SEQ ID N0:12. CTL 128
was not
stimulated by Cos-7 cells cotransfected with both the fully spliced TRP-2 cDNA
and HLA-
A*6801 (Fig. 5). This shows that the sequence coding for the antigen
recognized by CTL
128 could be entirely or partially located in the intronic portion of the TRP-
2 gene present
in cDNA 131. This notion received support by lack of recognition of Cos-7
cells
transfected with the 5'-end-800 cDNA fragment (Fig. 5), derived by truncation
of the ~'-
end-1500 fragment at a BstLJI site and comprising exon 1 and half of exon 2.
The intronic
localization of the sequence encoding the antigenic peptide was confirmed by
the ability of
a PCR amplified fragment, encompassing the first 434 by of intron 2 (INT- 2-
434), to
convey the expression of the antigen (Fig. ~). In the same reading frame of
the previous
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exons of this region, there was observed the presence of a sequence that coded
for a
decapeptide (EVISCKLIKR) (SEQ ID N0:2) which possessed anchor residues
(position 2,
9 and 10) corresponding to the HI.A-A*6801 peptide binding motif (Rammensee et
al.,
Immunogenetics 41 { 1995) 178-228).
To further define the region containing the epitope, PCR fragments were
amplified using
the sense primer KS-1NT2 {SEQ B7 NO:S), which generates an ATG that has the
appropriate Kozak consensus sequence and the reverse primers 1NT2-asl (SEQ )D
N0:6)
or INT2-as2 (SEQ 1D N0:7). The first fragment (INT-2-166) includes the entire
putative
peptide encoding sequence that is partially deleted in the second one (INT-2-
107}. Only
fragment 1NT-2-166, containing the intact putative peptide sequence was able
to stimulate
TNF release by CTL 128 (Fig. ~). Both 10-mer and 9-mer peptides, EVISCKLIKR
(SEQ ID N0:2) and EVISCKL1K (the 9-mer peptide differs from the 10-mer peptide
(SEQ 1D N0:2) by the deletion of the last peptide (R)), were then synthesized
and
incubated with the HLA-A*6801 homozygous LCL line LB. Decapeptide EVISCKLIKR
was able to sensitize LB cells to lysis by CTL 128, with half-maximum lysis
obtained at a
concentration of about 100 pM, whereas the nonapeptide had a very low
efficiency and a
control peptide was negative (Fig. 6).
To exclude that the epitope recognized by CTL 128 may be generated by a
mutation
occurred in the tumor, a 2152 by fragment, spanning the entire intron 2 (nt
995-3080 in
cDNA 131), was amplified by PCR from genomic DNA of CTL 128 and from a
different
melanoma, MZ2-mel.
Example 3
Cloning of TRP-2-INT2 from genomic DNA
Genomic DNA was purified from MZ2 melanoma cells and CTL 128, by use of the
Ql:Aamp blood kit (QIAGEN, Hilden, Germany). Intron 2 of TRP-2 gene was
amplified by
PCR from 100 ng of genomic DNA with KS-INT? (SEQ >D NO:S), located in exon 2
as
sense primer and PR2 (SEQ )D N0:9), located in exon 3, as anti-sense primer.
TRP-2-
INT2 fragments were cloned into the pcDNAI vector as described above,
sequenced and
transfected into Cos-7 cells together with the HLA-A*6801 gene.
The PCR products were cloned, sequenced and transfected into Cos-7 cells. All
clones had
the expected sequences and were able to transfer the expression of the
antigen.
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An RT-PCR analysis, performed with the sense primer PRIT-I (SEQ ID N0:3) and
the
anti-sense primer 1NT2-1260 (SEQ )D N0:4), located in exon 1 and intron 2,.
amplifies a
977 by fragment only from TRP 2 transcripts that retain intron 2.
Example 4
PCR analysis for the expression of TRP-2-INT2 and TRP2
Total RNA was prepared by the guanidine-isothiocyanate method, using RNAzoI B,
from
cultured cell lines, fresh skin, retina and tumor samples. cDNA corresponding
to 300 ng of
total RNA was amplified by PCR using 1 U of DynazymeTM (Finnzymes) and 1 mM of
each primer, in a final vclume of 50 ml. Reaction mixtures were subjected to
30
amplification cycles. For amplification of TRP-2 cDNA, PR3 (SEQ m NO:10)
located in
exon 2 and TRP-2L (SEQ )D NO:11), located in exon 8, were used as sense and
anti-sense
primers respectively. PCR were performed for 30 cycles (1 min at 94°C,
1 min at 58°C and
1 min at 72°C). To verify the expression of the unspliced intron 2,
there were used the
sense primer PRTT-1 (SEQ m N0:3) and the anti-sense primer INT2-1260
(SEQ 1D N0:4), located in the 5'-UTR and in intron 3, respectively. PCR were
performed
for 30 cycles (1 min at 94°C, 1 min at 55°C and I min at
72°C). Amplification from
contaminating genomic DNA was avoided by localization of the primers in
distant exons
for detection of TRP-2 and by the presence of a 10 kb long intron 1 (Sturm et
al.,
Genomics 29 ( 1995) 24-34) between exon 1 and exon 2, for the amplification of
TRP-2-
INT2. The quality of RNA preparations was checked by PCR amplification of -
actin cDNA
with specific primers.
Expression of the completely spliced TRP-2 messenger was detected with the
sense primer
PR3 (SEQ )D NO:IO), and the anti-sense primer TRP-2L (SEQ 1D NO:11), located
in exon
2 and 8, respectively.
Among the tumors tested only melanomas proved positive for TRP-2 and TRP-2-
INT2
antigens (Table 1). Expression of TRP-2-INT2 in the absence of TRP-2 was never
observed. This result is in agreement with the notion that TRP-2 is a
melanocytic
differentiation antigen (Wang et al., J. Exp. Med. 184 (1996) 2207-2216) and
that the same
promoter drives the synthesis of a common messenger from which both the
antigens arise.
69% of fresh melanoma samples analyzed expressed TRP-2 with 78% of the TRP-2+
melanomas also expressing TRP-2-INT2 (Table I). This was also observed in
melanoma
cell lines, where the normal form of TRP-2 and the one retaining intron 2 are
expressed in
84% and in 68% of the analyzed samples, respectively. Normal tissues in which
TRP 2 is
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known to be present were analyzed for TRP-2-1NT2 expression. Three melanocytes
cell
lines, four skin samples and one retina were negative in the RT-PCR assay
(Table 1). The
four skin samples were analyzed in comparison with bioptic primary lesions
derived from
the same patient and whereas TRP-2 was detected in all samples, TRP-2-INT2 was
exclusively present in tumor samples.
Table 1
Expression of TR.P-2-INT2 and TRP-2 in tumors and normal tissues
Number of samples
with antigen
expression
/
number of samples
tested
TRP-2-INT2 TRP-2
Melanoma cell lines 13 /19 (68%)* 16 /19 (84%)
~
Melanocyte cell lines0 /3 3/3
Fresh melanoma samples7 /13 (54%)* 9 /13 (69%)
Skins 0/4 4/4
Retina 0 / 1 1 /1
Tumors (non melanomas)0 / 3 0 /3
*TRP-2-INT2 expression was detected only in samples positive for TRP-2.
A strong correlation was found between expression of TRP-2-1NT2 mRNA in
melanoma
lines and their ability to stimulate TNF release by CTL 128 (Fig. 7).
Presentation of the antigenic peptide by alleles of the HLA-A3-like Supertype
HLA-A*6801, along with A3, Ail; A31 and A*3301, belongs to an A3-like
supertype of
I-B.,A-A alleles with similar peptide-binding characteristics (Sidney J. M. et
al., Immunol.
154 (1995) 247-259). To investigate whether the peptide EVISCKLIR (seq ID NO:
2) can
be presented to CTL 128 by HLA-A*6802 alleles of the A3-like supertype, EBV
LCL
expressing such alleles were used as targets for CTL128 after pulsing with the
peptide (Fig.
7).
The analysis also included the HLA-A*6802 allele, a subtype of HLA-A28
belonging to
the A2-like supertype. Among the A3-like supertype alleles only A*3301 was
able to
present the TRP-2-INT22z-z2s peptide with the same efficiency as A*6801. A low
level of
recognition, at the higher concentration, was observed when the peptide was
presented by
A*6802.
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Exameple 5
Antigenic peptide and CTL assay
Peptides were synthesized on solid phase using Fmoc for transient NH2-terminal
protection
and characterized by mass spectrometry (Primm, Milan, Italy). All peptides
were >90%
:~
pure as indicated by analytical HPLC. Lyophilized peptides were dissolved at
10 mM
concentration in 10% DMSO and stored at -80°C. Peptides were tested in
an assay, where
5'Cr-labeled target cells were incubated for 1 h at room temperature in 96-
wells
microplates with various concentration of the peptide before addition of CTL
128 at an
effector/target ratio of 20:1. Lysis was measured 4 h later. Presentation of
the antigenic
peptides was also tested in a TNF-release assay. Briefly, stimulator cells
were incubated for
1 h at room temperature with a fixed concentration of peptides; following
extensive
washing, CTL 128 was added and the TNF release was evaluated 18-20 h later on
WEHI-
164.13 cells.
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SEQUENCE LISTING
(1) GENERAL
INFORMATION:
(i) APPLICANT:
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(ii) TITLE OF INVENTION: Tumor-specific antigens, methods
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their production and their use for immunization and
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(ix) FEATURE:
4O (A) NAME/KEY: CDS
(B) LOCATION:1..30
(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 1:
GAA GTA ATT TCA TGC AAG TTA ATT AAG AGA 30
Glu Val Ile Ser Cys Lys Leu Ile Lys Arg
1 5 10
(2) INFORMATION FOR SEQ ID NO: 2:
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Glu Val Ile Ser Cys Lys Leu Ile Lys Arg
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
ACCTCACCAA CTCACATCTT 20
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GCCGCCATGT ATTCTGTTAG AGATACA 27
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(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
ATCCGCTCGA GCATGAAATT ACTTCCC 27
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ATTTAGGTGA CACTATAG 18
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(xi) SEQUENCE DESCRIPTION: SEQ ID N0: 9:
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30 (xi) SEQUENCE DESCRIPTION: SEQ ID N0: 10:
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SO
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AGCTAAGGAG GGAGGGAGAG GGTTTAGAAA TACCAGCATA ATAAGTAGTA TGACTGGGTG 60
CTCTGT.AAAT TAACTCAATT AGACAAAGCC TGACTTAACG GGGGAAGATG GTGAGAAGCG 120
CTACCCTCAT TAAATTTGGT TGTTAGAGGC GCTTCTAAGG AAATTAAGTC TGTTAGTTGT 180
TTGAATCACA TAAAATTGTG TGTGCACGTT CATGTACACA TGTGCACACA TGTAACCTCT 240
4S GTGATTCTTG TGGGTATTTT TTTAAGAAGA AAGGAATAGA AAGCAAAGAA AAATAAAAAA 300
TACTGAAAAG AAAAGACTGA AAGAGTAGAA GATAAGGAGA AAAGTACGAC AGAGACAAGG 360
S0
AAAGTAAGAG AGAGAGAGAG CTCTCCCAAT TATAAAGCCA TGAGCCCCCT TTGGTGGGGG 420
TTTCTGCTCA GTTGCTTGGG CTGCAAAATC CTGCCAGGAG CCCAGGGTCA GTTCCCCCGA 480
GTCTGCATGA CGGTGGACAG CCTAGTGAAC AAGGAGTGCT GCCCACGCCT GGGTGCAGAG 540
SS TCGGCCAATG TCTGTGGCTC TCAGCAAGGC CGGGGGCAGT GCACAGAGGT GCGAGCCGAC 600
ACAAGGCCCT GGAGTGGTCC CTACATCCTA CGAAACCAGG ATGACCGTGA GCTGTGGCCA 660
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AGAAAATTCT TCCACCGGAC CTGCAAGTGC ACAGGAAACT TTGCCGGCTA TAATTGTGGA 720
GACTGCAAGT TTGGCTGGAC CGGTCCCAAC TGCGAGCGGA AGAAACCACC AGTGATTCGG 780
CAGAACATCC ATTCCTTGAG TCCTCAGGAA AGAGAGCAGT TCTTGGGCGC CTTAGATCTC 840
GCGAAGAAGA GAGTACACCC CGACTACGTG ATCACCACAC AACACTGGCT GGGCCTGCTT 900
IO GGGCCCAATG.GAACCCAGCC GCAGTTTGCC AACTGCAGTG TTTATGATTT TTTTGTGTGG 960
CTCCATTATT ATTCTGTTAG AGATACATTA TTAGGTGGGT TTTTTCCTTG GCTGAAGGTA 1020
TATTATTACA GGTTTGTGAT TGGGTTGAGG GTATGGCAGT GGGAAGTAAT TTCATGCAAG 1080
1$
TTAATTAAGA GAGCAACCAC AAGGCAGCCT TAGGTTTATG AAAGTCGTAG AATTATCAAA 1140
TACCGCCTGG AGTTAGAAGG AAGCAGTTTC TTCCTGTGCA TTGGATGCAG ACACTTTAAA 1200
2O TGTTCTCTCC TCTACCGTAT GTTCTTGGTT CAAAGTGTAA ACTTTTCTCT GTGAAGCTGT 1260
TAATCATCAA AGATGTGAGT TGGTGAGGTG GAGGCGAATT CCTTTTGATT TCAGAAGAAA 1320
ATATTTGCGA ATCTGGCCAT GGAAGCCCTC TCTGACCTTT TCTCCAAATT AGAGGAATTA 1380
2$
ACTGAACATG TGCTAAGGCA CATGAAGCT 1409
