JP2003523759A - TLP peptide and DNA sequence encoding the same - Google Patents

Abstract

  (57) [Summary] The present invention relates generally to DNA molecules that encode peptides derived from the proteins of the TLP complex. More specifically, the present invention is directed to cDNA sequences encoding proteins of the 100 kDa TLP complex.

Description

Detailed Description of the Invention

The present invention provides a D encoding a peptide derived from a protein of the TLP complex.
Regarding the NA molecule. More specifically, the present invention is directed to the cDNA sequence encoding the peptide of the 100 kDa TLP complex.

The abbreviation TLP (“Tumor Liberated Parti”)
cles ") indicates protein complexes present in human tumor cells, especially in lung cancer. TLP was first isolated from tumor tissue according to the procedure disclosed in EP283433. Subsequently, several proteins forming the TLP complex were identified. The 214 kDa TLP protein, which is mainly expressed in lung cancer, is disclosed in Oncology, 1983, 40: 248-253.

Epitopes specific to such proteins, and antibodies against them, are described in WO9
8/01462. Further, EP649433 discloses a 100 kDa protein isolated from lung cancer, as well as a plurality of immunogenic peptides derived therefrom. Among them, the peptide RTNKEASI has 100 kD T
It was used to generate polyclonal antibodies that recognize the LP complex. Such an antibody is
It was used to identify the expression pattern of the TLP in tissues of different origin and to validate the protein as a tumor marker (
WO98 / 15282). Studies of TLP expression in different tumor cell lines showed that
Similar expression levels have been found in cell lines derived from breast cancer (MCF7) and colorectal cancer (HT29).

Furthermore, in vitro treatment with chemotherapeutic agents showed enhanced expression of the TLP antigen in the tumor cell population (NSCLC), which was 100 kD.
a TLP protein, as well as peptides derived therefrom, showed an increase in lymphocytes with a specific antitumor effect (WO98 / 15282).

To provide a wider variety of antigenic and immunogenic peptides derived from oncoprotein TLP complexes, as well as effective means for the large-scale production of such TLP proteins, for diagnostic or therapeutic applications. The importance of is obvious. To that end, the inventor has obtained a cDNA sequence encoding the peptide of the 100 kDa TLP complex, as described below.

Tumor cell lysates expressing TLPs were prepared and total RNA was extracted therefrom, followed by a degenerate sequence corresponding to the TNKEASI peptide (forward primer) and a random hexa nucleotide sequence (reverse primer). Primer) and RT-PCR using oligonucleotide primer pairs each having The amplification product was cloned into an appropriate vector and then sequenced. An ORF of 300 bases was identified therein and the sequence is published in SEQ ID NO: 1. The amino acid sequence encoded by the ORF is published in SEQ ID NO: 2.

According to a first aspect, the invention relates to a nucleic acid molecule comprising the sequence of SEQ ID NO: 1, variants thereof due to the degeneracy of the genetic code, and under stringent conditions the complementary strand of SEQ ID NO: 1. A nucleic acid molecule that hybridizes to is provided.

The invention further includes expression vectors containing the nucleic acids disclosed herein. The vector is a plasmid, cosmid, virus, bacteriophage,
Alternatively, it could be any other vector commonly used in genetic engineering, which regulates transcription, in addition to coding sequences, including regulatory elements such as initiation and termination codons, enhancers, promoters, signal sequences and the like. It may include an array or the like. Furthermore, the present invention also includes eukaryotic or prokaryotic cells that have been transfected or transformed with the above vector.

The complete sequence of the TLP protein has been genetically engineered in vitro.
Enables large-scale preparation in. The availability of such antigen preparations allows for in-depth research into the role of TLPs in human malignancies. It also allows the creation of assays for the early diagnosis of corresponding tumors and the creation of specific anti-cancer vaccines.

A further aspect of the invention relates to a peptide encoded by the DNA sequence of SEQ ID NO: 1, as shown in SEQ ID NO: 2, or a fragment thereof. Such peptides can be prepared by known methods (eg, “Merrifield, (1986) Science”).
232: 341-347 ", or" Barany and Merri. "
field, 1979, The peptides, Gross and
Meienhofer, eds NY Academic Press, 1
~ 284 "), using the above-described cDNA, recombinant DNA
Or by chemical synthesis. The synthesis can be carried out in solution or in solid phase using an automated synthesizer. One or more amino acid residues can be replaced by different L or D residues, as long as the immunogenic effect is maintained, or immunogenicity, selectivity of induction of immune response, biological properties after administration, etc. In order to improve properties such as availability, they are chemically modified, for example by amidation of the carboxy terminus, or by attachment of a lipophilic group (eg myristyl group),
Alternatively, chemical modifications can be made by glycosylation or conjugation with other peptides. The peptides may be chemically derivatized by forming salts, esters, hydrazides, to their side chains, eg, free carboxy groups. Furthermore, the peptides can be conjugated to known epitopes in order to induce a higher cytotoxic or helper cell-mediated immune response against tumors.

The immunogenic activity of the peptide is tested in vitro, for example, in T lymphocyte induction or proliferation test (Proti MP et al., 1990, J. Immunol. 144: 1711-1720), cytotoxicity test. (Prot
ti M. P. et al. , 1996, Cancer Res. 56
: 1210-1213), or a binding test with an MHC molecule.

According to a preferred embodiment, the peptide derived from SEQ ID NO: 2 is TLP
It is used to create specific antibodies. It can be monoclonal or polyclonal and can recognize TLP epitopes specific for the peptides disclosed herein. Such antibodies are preferably used in immunoassays to detect TLP expression in tumors.

According to another aspect, the invention provides the use of a nucleic acid molecule or peptide as described above in the preparation of a pharmaceutical composition for the preventive or therapeutic treatment of tumors, in particular lung tumors.
A typical application involving the use of said cDNA is the preparation of DNA vaccines, which can be carried out as taught in US5593972 or US55808859. The peptide may be used in combination with another chemotherapeutic agent known to increase its effect (WO98 / 15282).

The composition according to the present invention comprises an effective amount of DNA or peptide together with a pharmaceutically acceptable excipient. By "effective amount" is meant an amount sufficient to activate lymphocytes and elicit a cellular or humoral response to the tumor. According to a preferred embodiment, the pharmaceutical composition is used in prophylactic vaccination of a subject having susceptibility to a neoplasm or therapeutic vaccination of a neoplastic patient. In this context, the term "vaccination" refers to active vaccination, i.e. in vvv of the peptide or cDNA for the direct activation of the patient's immune response.
By administration iv or by passive vaccination, i.e. the use of the peptide for activating cytotoxic T lymphocytes in vitro and subsequently re-vaccination of the patient. Techniques for vaccine preparation and use are known to those of skill in the art, and a comprehensive description can be found, for example, in Paul, Fundamental.
Immunology, Raven Press, New York (
1989) or Cryz, S .; J. , Immunotherapy
d Vaccines, VCH Verlagsgesselshaft
(1991). Vaccines are usually prepared in the form of solutions, injectable forms, suspensions, but may also be in the form of solid or liposome-based formulations. The immunogenic ingredients can be mixed with pharmaceutically acceptable excipients such as emulsifiers, buffers and the like in order to increase the effectiveness of the vaccine.

[0015]   The following examples explain the invention in more detail.

Example 1-Isolation and cloning of TLP cDNA Total RNA was prepared using RNAzol B reagent (TEL-TEST, INC).
Several cell lines (HT-29, SAOS-2, A549, MCF-7, H2
3, H157, H1819, W138) and reverse transcribed using the reverse transcription system (PROMEGA). Upstream degenerate oligonucleotides corresponding to the amino acid sequence RTNKEASI: ACN AAY AAR GAR G
Polymerase chain reaction (PCR) was performed for 35 cycles (1 min at 95 ° C, 2 min at 40 ° C, 1 min at 72 ° C) using CN TCN ATH TC and random hexamers as downstream primers. PCR products were electrophoresed on a 1% agarose gel containing ethidium bromide. The PCR product is p (as shown in the figure)
It was cloned into GEM-T easy vector (PROMEGA). The resulting plasmid clones were sequenced by the chain termination method using an Applied Biosystems model 373A DNA sequencer. One open reading frame was predicted (SEQ ID NO: 1).

Example 2-Production of antibodies and their identification Rabbits with 0.5 ml of Phosphate Buffered Saline (PBS) solution of 0.5 mg of the peptide of SEQ ID NO: 2 mixed with 0.5 ml of Freund's complete adjuvant. Rabbit anti-TLP serum was obtained by subcutaneously immunizing 4 birds. Booster injections with incomplete adjuvant were given at 2 week intervals. Serum was collected during the week during which blood was collected from the ear vein. Antiserum titers were assessed by radioimmunoassay on 96-well microtiter plates. Wells were coated with a fixed concentration of peptide antigen, washed and incubated with various dilutions of serum. Bound immunoglobulin was detected with ¹²⁵ I-labeled protein A and quantified by radioimmunoassay. At a dilution of 1: 1000, all sera showed a positive staining reaction with the peptide antigen. The corresponding pre-immune sera from rats were also tested and no specific reactivity was detected.

Western Blot 200 μl lysis buffer (50 mM Tris, 5 mM EDTA,
250 mM NaCl, 50 mM NaF, 0.1% Triton, 0
． 1 mM Na ₃ VO _4, protease inhibitor added) in, by resuspending the pelleted cells were prepared lysate of tumor cells. 50 μg of protein extract was run on an 8% polyacrylamide gel. The protein in the polyacrylamide gel was loaded with CAPS buffer (10 mM CAPS, 20 mM
% Methanol, pH 11) and transferred to a PVDF membrane (Millipore). The membrane was treated with TBS-T buffer (2 mM Tris, 13.7 mM NaC).
1, 5% milk in 0.1% Tween-20, pH 7.6)
Blocked and washed in TBS-T. Affinity purified anti-T
LP antibody was incubated with the membrane in 3% milk and washed in TBS-T. The membranes were then incubated with a rabbit anti-rabbit antibody conjugated 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). Specific staining was detected, corresponding to tumor cell lysates. Preimmune sera as well as control lysates showed no significant reactivity.

[Brief description of drawings]

FIG. 1 is a diagram showing the location of a TLP gene fragment cloned in pGEM-T easy vector (PROMEGA).

[Sequence list]

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Claims (9)

[Claims] 1. A nucleic acid molecule encoding a TLP peptide having the sequence of SEQ ID NO: 2. 2. The nucleic acid molecule of claim 1 having the sequence of SEQ ID NO: 1. 3. A peptide of SEQ ID NO: 2. 4. A monoclonal or polyclonal antibody against the peptide of claim 3. 5. A vector comprising the nucleic acid molecule according to claim 1 or 2. 6. A pharmaceutical composition comprising the nucleic acid molecule according to claim 1 or 2 or the peptide according to claim 3. 7. The composition according to claim 6, which is in the form of a vaccine. 8. Use according to claim 6 or 7 for use in the prevention or treatment of tumors.
The composition according to. 9. A composition according to claim 8 for use in the prevention or treatment of lung tumors.