Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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TLP PEPTIDES AND DNA SEQUENCES CODING THE SAME
The present invention relates to DNA molecules encoding peptides derived
from proteins of the TLP complex. More specifically, the invention is directed
to
a cDNA sequence coding for a peptide of the 100 kDa TLP complex.
Under the acronym TLP ("Tumor Liberated Particles"), protein complexes
present in human tumor cells, particularly in lung carcinoma, are indicated.
TLPs
were isolated for the first time from tumor tissues following the procedure
disclosed in EP283433. Afterwards, certain proteins forming the TLP complexes
were identified. A 214 kDa TLP protein prevalently expressed in lung carcinoma
is disclosed in Oncology, 1983, 40:248-253.
1 o Epitopes specific for that protein and antibodies against it are disclosed
in
W098/01462. In addition, EP649433 discloses a 100kDa protein isolated from
lung carcinoma, as well as immunogenic peptides derived therefrom. Among
those, the peptide RTNKEASI was used to produce polyclonal antibodies
recognizing the 100kDa TLP complex. Such antibodies were used for the
1 S characterisation of the TLP expression pattern in tissues of different
origin and
for the validation of the same protein as tumor marker (W098/15282). The study
of TLP expression in different tumor cell lines has revealed similar
expression
levels among cell lines derived from breast carcinoma (MCF7) and colorectal
carcinoma (HT29).
2 0 Furthermore, the in vitro treatment with chemotherapeutic agents was shown
to increase the expression of TLP antigen in a tumor cell population (N SCLC),
whereas the 100kDa TLP protein, as well as the peptides derived therefrom,
CONFIRMATION COPY
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lymphocytes with specific antitumor effects (W098/15282).
It is evident the importance to provide a larger variety of antigenic and
immunogenic peptides from the tumor protein TLP complex, as well as an
effective means for the extensive production of the same TLP proteins, for
diagnostic or therapeutic uses. To that propose, the inventors have obtained
the
cDNA sequence encoding a peptide of the 100kDa TLP complex, as described
hereafter.
Lysates of tumor cells expressing TLP were prepared, from which the total
RNA was -extracted and then subjected to RT-PCR with pairs of oligonucleotide
primers having degenerate sequences corresponding to the TN-KEASI peptide
(forward primer) and, respectively, random hexanucleotide sequences (reverse
primer). The amplification product was cloned in a suitable vector and then
sequenced. Inside it, an ORF of 300 bases was identified, whose sequence is
reported in SEQ ID N. 1. The amino acid sequence encoded by the OR)r is
1 ~ reported in SEQ ID N. 2.
According to a first aspect, the invention provides a nucleic acid molecule
comprising the sequence SEQ ID N. l, the variants thereof due to genetic code
degeneration, and the nucleic acid molecules hybridising to the complementary
strand of SEQ ID N. 1 under stringent conditions.
The invention further comprises an expression vector that contains a nucleic
acid molecule herein disclosed. The vector can be a plasmid, cosmid, virus,
bacteriophage, or any other vector commonly used in genetic techniques, which,
in addition to the coding sequence, may comprise control elements, such as
sequences regulating the transcription, including start and stop codons,
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-,
enhancers, promoters, signal sequences and the like. Furthermore, the
invention
comprises eukaryotic or prokaryotic cells transfected or transformed with said
vector.
The complete sequence of a TLP protein enables in vitro preparations of
large quantities of it by genetic engineering. The availability of such
antigen
preparations allows for deep investigations on the role of TLP in human
malignancy. It also enables the preparation of an assay for early diagnosis of
the
corresponding tumors and the generation of a specific anti cancer vaccine.
A further aspect of the invention relates to a peptide encoded by the DNA
sequence of SEQ ID N. l, as reported in SEQ ID N. 2, or a fragment thereof.
Such peptides can be prepared with recombinant DNA procedures, using the
described cDNA, or with chemical synthesis, following known procedures (e.g.
see "Merrifield, ( 1986) Science 232:341-347", or "Barany and Merrifield,
1979,
The peptides, Gross and Meienhofer, eds NY Academic Press, 1-284"). The
synthesis can be earned out in solution or in solid phase with an automatic
synthesizer. One or more amino acid residues can be replaced by different L-
or
D-residues, so as to maintain the immunogenic effects, or they can be
chemically
modified, for instance by amidation of the carboxy terminus, by linking
lipophilic
groups (e.g. myristyl), or by alycosylation or conjugation with other
peptides, in
order to improve properties like immunogenicity, selectivity of induction of
the
immune response or bioavailability after administration. The peptides can also
be
chemically derivatized on their lateral chains, for instance on free carboxy
groups, to form salts, esters, hydrazides. Furthermore, said peptides can be
conjugated to known epitopes in order to induce a higher cytotoxic or helper
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immune response against tumors.
The immunogenic activity of the peptides can be easily determined by means
of in vitro tests, for instance using t-lymphocyte propagation or
proliferation
essays (Protti M.P. et al., 1990, J. Immunol. 144:1711-1720), cytotoxicity
essays
(Protti M. P. et al., 1996, Cancer Res. 56:1210-1213), or binding essays with
MHC molecules.
According to a preferred embodiment, the peptides derived from SEQ ID N.
2 are used to generate TLP-specific antibodies. The latter can be monoclonal
or
polyclonal- and recognize the TLP epitopes specific for the peptides herein
disclosed. Such antibodies are preferably used in immunoessays to detect TLP
expression in tumors.
According to a further aspect, the invention provides the use of the above
nucleic acid molecules or peptides for the preparation of a pharmaceutical
composition for the preventive or therapeutic treatment of tumors, especially
of
lung tumors. A typical application involving the use of the cDNA is the
preparation of DNA vaccines, which can be effected as taught in US»93972 or
US5580859. The peptides can also be used in combination with different
chemotherapeutic agents, which are known to increase the effects thereof
(W098/15282).
The compositions in accordance with the invention contain an effective
amount of DNA or peptides together with pharmaceutically acceptable
excipients. By "effective amount" is meant the amount sufficient to activate
lymphocytes and to trigger a cellular or humoral response against tumors.
According to a preferred embodiment, the pharmaceutical compositions are used
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in the preventive vaccination of subjects with susceptibility to neoplasias or
in the
therapeutic vaccination of neoplastic patients. The term "vaccination" in this
context is referred either to the active vaccination, i.e. the in vivo
administration
of the peptides or cDNA to activate the immune response directly in the
patient,
or to the passive vaccination, i.e. the use of peptides for the in vitro
activation of
T cytotoxic lymphocytes and their subsequent re-inoculation in the patient.
The
procedures for the preparation and use of vaccines are known to anyone skilled
in
the art; an extensive description is given for example in Paul, Fundamental
Immunology, Raven Press, New York ( 1989) or Cryz, S.J., Immunotherapy and
Vaccines, VCH Verlagsgesselschaft (1991). Vaccines are usually prepared in
form of solutions, injectables, suspensions, but also in form of solid or
liposome-
based preparations. The immunogenic ingredients can be mixed with
pharmaceutically acceptable excipients, such as emulsifiers, buffering agents,
adjuvants, so as to increase the vaccine effectiveness.
1 S The following examples illustrate the invention in greater detail.
EXAMPLES
Example 1 - Isolation and clonin~of TLP cDNA
Total RNAs were extracted from several cell lines (HT-29, SAOS-2, A 549,
MCF-7, H23, H157, H1819, WI38) with RNAzoI B reagent (TEL-TEST, INC)
and reverse transcribed using the Reverse Transcription System (PROMEGA).
Polymerase chain reaction (PCR) was carried out for 3~ cycles (1 min at
9~°C, 2
min at 40°C and 1 min at 72°C) using an upstream degenerate
oligonucleotide:
ACN AAY AAR GAR GCN TCN ATH TC, that corresponds to the amino acid
sequence RTNKEASI and Random Hexamers as the downstream primer. PCR
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products were electrophoresed on a 1 °,% agarose gel containing
ethidium bromide.
The PCT products were cloned in the pGEM-T easy vector (PROMEGA)
(Figure). Resulting plasmid clones were sequenced with the chain termination
method using the Applied Biosystems model 373A DNA sequencer. An open
reading frame has been deducted (SEQ ID N. 1).
Example ~ - Production and characterization of antibodies
Rabbit anti-TLP serum was obtained by immunizing four rabbits
subcutaneously with 0.5 mg of the peptide of SEQ ID N. 2, in 0.5 ml of
phosphate-buffered saline (PBS), mixed with 0.5 ml of Freund's complete
adjuvant. Booster injections with incomplete adjuvant were given at 2-week
intervals. Sera were collected on alternate weeks via ear vein bleeds. Titers
of
antisera were performed by radioimmunoassay on 96-well microtiter plates.
Wells were coated with a fixed concentration of peptide antigen, washed, and
incubated with various dilutions of sera. The bound immunoglobuline was
detected with 12'I-labeled protein A and quantified by radioimmunoassay. At a
dilution of 1:1,000, all sera showed a positive staining reaction with the
peptide
antigen. Preimmune sera from the corresponding rats were also tested and non
significant reactivity was detected.
Western Blot
Tumor cell lysates were prepared by resuspending pelletted cells in 200 ~l
lysis buffer (50mMTris, 5mM EDTA, 250 mM Na Cl, 50 mM NaF, 0.1% Triton,
0.1 mM Na3V04, plus protease inhibitors). 50 ~g of protein extract was run on
a
8% polyacrylamide gel. Proteins within the polyacrylamide gel were transferred
to a PVDF membrane (Millipore) in CAPS buffer (10 mM CAPS. 20% methanol,
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pH 11). The membrane was blocked with ~°% milk in TBS-T buffer (2mM
Tris,
13.7 mM NaCI, 0.1% Tween-20, pH 7.6) and then washed in TBS-T. Affinity
purified anti-TLP antibodies were incubated with the membrane in 3% milk and
then washed in TBS-T. The membrane was then incubated with a rabbit anti-
rabbit Ab coupled with horseradish peroxidase and washed in TBS-T. The
presence of secondary antibody bound to the membrane was detected using the
ECL system (Dupont NEN). A specific staining was detected in correspondence
of the tumor cell lysates. Preimmune sera and control lysates did not show a
significant reactivity.
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SEQUENCE LISTING
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Thr Asn Lys Glu Ala Ser Ile Cys Pro Ser Val Tyr Leu Ser Thr Leu
1 5 10 15
Pro Ser Ile His Ser Phe Thr Asn Ser Ser Ile Tyr Tyr Pro Cys Ile
20 25 30
His Leu Ser Ile Ser Leu Ser Val His Pro Pro Leu Ile His Pro Ser
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Ile Tyr Pro Ser Tyr Ser Ser Ile His Ser Ser Ser His His Pro Cys
50 55 60
Thr His Leu Ser Thr His Ser Val Ile Asn Pro Val Lys Asn Phe Glu
65 70 75 80
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His Leu Leu Pro Ile Arg Pro Cys Thr Trp T::r Leu Gly
85 90
