EP1265917A2 - Tlp peptides and dna sequences coding the same - Google Patents

Tlp peptides and dna sequences coding the same

Info

Publication number
EP1265917A2
EP1265917A2 EP01911660A EP01911660A EP1265917A2 EP 1265917 A2 EP1265917 A2 EP 1265917A2 EP 01911660 A EP01911660 A EP 01911660A EP 01911660 A EP01911660 A EP 01911660A EP 1265917 A2 EP1265917 A2 EP 1265917A2
Authority
EP
European Patent Office
Prior art keywords
tlp
peptides
peptide
nucleic acid
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP01911660A
Other languages
German (de)
English (en)
French (fr)
Inventor
Giulio Tarro
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Unihart Corp
Original Assignee
Unihart Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Unihart Corp filed Critical Unihart Corp
Publication of EP1265917A2 publication Critical patent/EP1265917A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • 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.
  • 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. Epitopes specific for that protein and antibodies against it are disclosed in
  • EP649433 discloses a lOOkDa protein isolated from lung carcinoma, as well as immunogenic peptides derived therefrom.
  • the peptide RTNKEASI was used to produce polyclonal antibodies recognizing the lOOkDa TLP complex.
  • Such antibodies were used for the 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).
  • chemotherapeutic agents were shown to increase the expression of TLP antigen in a tumor cell population (NSCLC), whereas the lOOkDa TLP protein, as well as the peptides derived therefrom, lymphocytes with specific antitumor effects (W098/15282).
  • the inventors have obtained the cDNA sequence encoding a peptide of the lOOkDa 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 TNKEASI 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 ORF is reported in SEQ ID N. 2.
  • the invention provides a nucleic acid molecule comprising the sequence SEQ ID N. 1 , 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, -> j enhancers, promoters, signal sequences and the like.
  • the invention comprises eukaryotic or prokaryotic cells transfected or transformed with said vector.
  • TLP protein The complete sequence of a TLP protein enables m 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 m human malignancy It also enables the preparation of an assay for early diagnosis of the corresponding tumors and the generation of a specific a i cancer vaccine.
  • a further aspect of the invention relates to a peptide encoded by the DNA sequence of SEQ ID N. 1 , as reported in SEQ ID N. 2, or a fragment thereof.
  • Such peptides can be prepared with recombmant DNA procedures, using the desc ⁇ bed cDNA, or with chemical synthesis, following known procedures (e.g. see “Mer ⁇ field, ( 1986) Science 232:341-347", or “Barany and Mer ⁇ field, 1979, The peptides, Gross and Meienhofer, eds NY Academic Press, 1-284”). The synthesis can be earned out in solution or m solid phase w th an automatic synthesizer.
  • One or more ammo 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 hpophilic groups (e.g. mynstyl), or by glycosylation or conjugation with other peptides, in order to improve properties like immunogenicity, selectivity of induction of the immune response or bioavailabihty after administration.
  • the peptides can also be chemically denvatized on their lateral chains, for instance on free carboxy groups, to form salts, esters, hydrazides.
  • said peptides can be conjugated to known epitopes m order to induce a higher cytotoxic or helper 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
  • the peptides derived from SEQ ID N are identical to SEQ ID N.
  • TLP-specific antibodies 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.
  • 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 US5593972 or
  • the peptides can also be used in combination with different chemotherapeutic agents, which are known to increase the effects thereof
  • compositions in accordance with the invention contain an effective amount of DNA or peptides together with pharmaceutically acceptable excipients.
  • effective amount is meant the amount sufficient to activate lymphocytes and to trigger a cellular or humoral response against tumors.
  • the pharmaceutical compositions are used 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 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
  • 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.
  • pharmaceutically acceptable excipients such as emulsifiers, buffering agents, adjuvants, so as to increase the vaccine effectiveness.
  • RNAs were extracted from several cell lines (HT-29, SAOS-2, A 549, MCF-7, H23, H157, H1819, WI38) with RNAzol B reagent (TEL-TEST, INC) and reverse transcribed using the Reverse Transcription System (PROMEGA). Polymerase chain reaction (PCR) was carried out for 35 cycles (1 min at 95°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 products were electrophoresed on a 1% agarose gel containing ethidium bromide.
  • PCT products were cloned in the pGEM-T easy vector (PROMEGA)
  • 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.
  • PBS phosphate -buffered saline
  • the bound immunoglobuline was detected with 125 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 (SOmMTris, 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, pH 11). The membrane was blocked with 5% milk in TBS-T buffer (2mM Tris,
  • 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.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Plant Pathology (AREA)
  • Public Health (AREA)
  • Microbiology (AREA)
  • Toxicology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
EP01911660A 2000-02-25 2001-02-20 Tlp peptides and dna sequences coding the same Withdrawn EP1265917A2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
ITMI000344 2000-02-25
IT2000MI000344A IT1317853B1 (it) 2000-02-25 2000-02-25 Peptidi tlp e sequenze di dna che li codificano.
PCT/EP2001/001857 WO2001062786A2 (en) 2000-02-25 2001-02-20 Tlp peptides and dna sequences coding the same

Publications (1)

Publication Number Publication Date
EP1265917A2 true EP1265917A2 (en) 2002-12-18

Family

ID=11444175

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01911660A Withdrawn EP1265917A2 (en) 2000-02-25 2001-02-20 Tlp peptides and dna sequences coding the same

Country Status (9)

Country Link
EP (1) EP1265917A2 (ko)
JP (1) JP2003523759A (ko)
KR (1) KR20020086564A (ko)
AU (1) AU4063401A (ko)
BR (1) BR0108557A (ko)
CA (1) CA2401031A1 (ko)
IT (1) IT1317853B1 (ko)
RU (1) RU2002122722A (ko)
WO (1) WO2001062786A2 (ko)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1836217B1 (en) 2004-12-30 2012-09-12 Vito Michele Fazio Anti-tumoral immunogenic peptides and vaccine thereof
ITRM20100256A1 (it) * 2010-05-19 2011-11-20 Farmafin Spa Un nuovo biomarcatore per la diagnosi precoce e l'immunoterapia del cancro

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1262954B (it) * 1992-07-03 1996-07-23 Ist Farmacoterapico It Spa Regioni antigeniche dei complessi tpl e anticorpi diretti contro di essi.
IT1294967B1 (it) * 1996-10-09 1999-04-23 Ist Farmacoterapico It Spa Composizione immunogenica da tlp

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0162786A2 *

Also Published As

Publication number Publication date
ITMI20000344A1 (it) 2001-08-25
ITMI20000344A0 (it) 2000-02-25
AU4063401A (en) 2001-09-03
RU2002122722A (ru) 2004-03-27
BR0108557A (pt) 2003-04-29
KR20020086564A (ko) 2002-11-18
WO2001062786A3 (en) 2002-01-17
WO2001062786A2 (en) 2001-08-30
JP2003523759A (ja) 2003-08-12
IT1317853B1 (it) 2003-07-15
CA2401031A1 (en) 2001-08-30

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