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WO1998037191A1 - Plnh (peptide lymphocytaire neutrophile humain) circulant - Google Patents

Plnh (peptide lymphocytaire neutrophile humain) circulant Download PDF

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Publication number
WO1998037191A1
WO1998037191A1 PCT/EP1998/000950 EP9800950W WO9837191A1 WO 1998037191 A1 WO1998037191 A1 WO 1998037191A1 EP 9800950 W EP9800950 W EP 9800950W WO 9837191 A1 WO9837191 A1 WO 9837191A1
Authority
WO
WIPO (PCT)
Prior art keywords
hnlp
seq
polypeptide
amino acids
oligo
Prior art date
Application number
PCT/EP1998/000950
Other languages
German (de)
English (en)
Inventor
Wolf-Georg Forssmann
Burkhard Kleemeier
Peter Schulz-Knappe
Michael Nehls
Original Assignee
Forssmann Wolf Georg
Burkhard Kleemeier
Schulz Knappe Peter
Michael Nehls
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
Priority claimed from DE1997130786 external-priority patent/DE19730786C2/de
Application filed by Forssmann Wolf Georg, Burkhard Kleemeier, Schulz Knappe Peter, Michael Nehls filed Critical Forssmann Wolf Georg
Publication of WO1998037191A1 publication Critical patent/WO1998037191A1/fr

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Classifications

    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Human circulating liNLP human neutrophil ⁇ n lymphocyte peptide
  • the present invention relates to a peptide with the formula given in claim 1, polynucleotides coding for peptides with the formula given in claim 1, vectors containing a DNA from the polynucleotides according to the invention, genetically engineered host cells containing the vector according to the invention, DNA hybridizing with a polynucleotide according to the invention , Antibodies directed against the polypeptides according to the invention, antagonists / inhibitors directed against the polypeptides according to the invention, antisense oligonucleotides for inhibiting the expression of the peptides according to the invention, medicaments containing at least one polypeptide according to the invention or a polynucleotide according to the invention, method for producing the peptides according to the invention, a diagnostic agent for Detection of the polypeptides according to the invention and uses of the polypeptides according to the invention.
  • R a is NH 2 or a derivative of a primary amino group or an oligo- or polypeptide, the oligo- or polypeptide being composed of naturally occurring amino acids, their derivatives modified in the side chain and / or their stereoisomeric amino acids,
  • n is an integer between 3 and 7,
  • X is a naturally occurring amino acid, the derivative of which is modified in the side chain and / or the stereoisomeric amino acid thereof,
  • R d is COOH, a carboxylic acid derivative or an oligo- or polypeptide, the oligo- or polypeptide being composed of naturally occurring amino acids, their derivatives modified in the side chain and / or their stereoisomeric amino acids.
  • the peptide according to the invention preferably has the following structure (formula II):
  • R a , R d , n and X have the meanings given above and R b and R c are each an oligo- or polypeptide, the oligo- or polypeptide consisting of naturally occurring amino acids, their derivatives modified in the side chain and / or their stereoisomers Amino acids is built up.
  • X n is RDDSE.
  • the peptide according to the invention has in particular four molecular forms, of which three in human blood filtrate, one in urine, with the following amino acid sequences:
  • peptides are referred to as human circulating neutrophil lymphocyte peptides.
  • the peptide according to the invention can be obtained by a purification process starting from human hemofiltrate.
  • the human hemofiltrate is optionally diluted with water and acidified.
  • the pH is preferably 1.5 to 3.5, in particular 2.5 to 3.0.
  • the hemofiltrate is then passed over a cation exchanger, for example a carrier material modified with sulfonic acid groups (Fractogel SP-650 (M), Merck, Darmstadt).
  • the peptides bound to the cation exchanger are eluted with a relatively highly concentrated salt solution.
  • the ionic strength of the elution solution corresponds approximately to a 0.5 to 1 molar ammonium acetate solution.
  • the eluate collected is subjected to a further cation exchange chromatography.
  • This chromatography is preferably a step elution with buffers of increasing pH values.
  • fractions containing the peptide according to the invention are e.g. further purified by means of preparative reverse phase chromatography and subsequent semi-preparative reverse phase chromatography, for example on support materials modified with C4.
  • the degree of purification is preferably checked by means of analytical reverse phase chromatography, for example on carrier materials modified with C18 and by capillary zone electrophoresis.
  • the substances obtained by the chromatographic purification were used for structure elucidation.
  • the molecular masses of the native peptides as well as the proteolytic fragments and chemically modified derivatives were determined using a Sciex API III mass spectrometer.
  • the amino acid analysis was carried out after a total acid hydrolysis.
  • the sequence analysis was carried out using Edman degradation of the peptides, their proteolytic cleavage products and chemically modified derivatives using an ABI 473 A sequencer.
  • the polynucleotides according to the invention code for the peptides according to the invention.
  • the polynucleotides can be used Methods known to those skilled in the art derive primers for analysis and sequencing of the associated mRNA and the gene.
  • the polynucleotide is in particular a cDNA which can serve both as a starting point for the genetic engineering of the hNLP and as an analytical tool for detecting the occurrence of DNA or mRNA coding for the peptide.
  • the cDNA of the peptide according to the invention has the novel sequence below (Seq. ID No. 6):
  • GGC AGA TGT ACG CTT TAA ATT GGT CTC CAT TTC TTA GAA 605 614 623 632 641 650
  • the gene of the peptide according to the invention was cloned from human genomic leukocyte DNA and has the following sequence:
  • CTCCCCCTGT CAAGATGTGG CACCTCAAAC TTTGTGCAGT CCTCATGATC TTCCTGTTGC 300
  • mice The representation of the cDNA of mouse (Mus musculus) and production of knock-out mice were possible according to the usual methods.
  • mice cDNA sequence (Seq. ID No. 10) and the peptide sequence derived therefrom (Seq. ID No. 14) are:
  • TGA TGT CCA TAC TGG GGA AGA GTC ACC TCC AAC AGT GAT CTG TTC TGG GGA ATA TTG TTA 305 314 323 332 341 350
  • the mouse gene was isolated from a murine cosmid library and sequenced by genomic PCR. The sequence is shown below (Seq. ID No. 15):
  • GAGATCAGAA AACAGAGACC CAAGGGTAAA GCTGAAATTA GTGGTGTGGG GTGGGGGTGG 540
  • the peptide according to the invention and its cDNA, its gene and analogs, fragments and derivatives of the peptide, the cDNA and the gene and its antagonists or inhibitors can be used as medicaments.
  • Its biological activity corresponds to that of an anti-cell proliferative substance similar to the well-known interleukins. It is therefore suitable as a drug for the treatment of disorders in inflammatory processes, disturbed inflammatory reactions, tumor diseases, proliferation and maturation disorders of the hematopoietic system, infectious diseases, inflammatory and neoplastic diseases and proliferation disorders such.
  • the peptide according to the invention or its antagonists / inhibitors can be administered parenterally, intravenously, intramuscularly, intranasally, locally topically or bucally in a manner customary for peptides.
  • the amount of peptide to be administered is 1 ⁇ g to 1 g per administration unit per day.
  • the action of the peptide according to the invention can be inhibited by administration of suitable inhibitors and antagonists.
  • the diagnostic agent according to the invention contains poly- or monoclonal antibodies against the peptide according to the invention, optionally in fluorescence or radioactive labeling, in order to be used, for example, in ELISA or RIA known per se.
  • the diagnostic agent according to the invention contains DNA, RNA and / or PNA, optionally in modified and / or labeled form for use in test systems known to those skilled in the art, such as PCR or fingerprinting. The invention is described in more detail by the following examples.
  • Buffer A Hemofiltrate pH 2.7, conductivity
  • ammonium acetate eluates of the batch extraction are combined in amounts of 5,000 to 10,000 liters of hemofiltrate peptide.
  • the peptide extract is admixed with Demineralized water with a conductivity of 5.5 mS / cm applied to the preparative cation exchanger.
  • the column is rinsed with 0.01 M HCl until the conductivity is below 1 mS / cm.
  • the elution is carried out in several stages using the buffers specified below
  • Eluates 1-7 are referred to as pH pool I-VII. They are collected separately. Elution takes place until a new baseline is reached, elution volumes of 10 to 25 L being achieved for the individual pH pools I to VII.
  • Second preparative separation The individual pH pools are separated for fractionation and simultaneous desalination using reversed phase chromatography.
  • the column is rinsed with buffer A. Fractions of 200 ml are collected during the elution. The fractions are freeze-dried and stored at -20 ° C.
  • Fraction 15 from pH pool VI was separated successively in several identical chromatographies on a semi-preparative reversed-phase column.
  • Buffer B 0.1% TFA, 80% acetonitrile
  • capillary fused silica effective length 50 cm total length: 57 cm buffer: 100 mM NaH 2 P0 4 pH 2, 5,
  • amino acids After gas phase hydrolysis with 6 N HCl for 1 hour at 160 ° C, the amino acids are analyzed with an automatic amino acid analyzer (AminoQuant, Hewlett-Packard) after double labeling with OPA / Fmoc using the standard program.
  • AminoQuant Hewlett-Packard
  • the peptides contain the amino acids detectable by this method in accordance with the four molecular forms mentioned.
  • cysteines can be detected in peptide sequencing.
  • desalting is preferably followed by analytical reverse phase chromatography with a Vydac RP-C18 column (4.6 mm x 25 cm).
  • hNLP-22-39 (Seq. ID No. 4), MW 1877 as urine peptide:
  • GVSLRPIGASCRDDSECI The linkage of the cysteines to one another in a 1-3 and 2-4 arrangement is determined by analyzing the fragments of the endoproteinase cleavages and is indicated by the brackets.
  • NLP-N and NLP-C form pairs of so-called 'degenerate ' PCR primers which contain all coding possibilities for the amino and carboxy-terminal amino acid sequences of the hNLP determined in Example 2 and an additional linker sequence with restriction sites for later cloning.
  • the first strand synthesis of the cDNA is carried out with UNIP.
  • RNA was prepared from human intestinal tissue using an automatic nucleic acid extractor (ABI, 340) according to the manufacturer's instructions. From 5 ⁇ g of this RNA, the mRNA was transcribed into cDNA first strand using the primers and MMLV-RTase (Gibco-BRL).
  • Amplification of the cDNA coding for hNLP The specific amplification of the coding cDNA was carried out by PCR on a thermal cycler (Perkin-Elmer-Cetus, 7000). With about 1 ⁇ g of first cDNA strands from different human tissues, in particular of the intestinal tract, the coding cDNAs are obtained with the primers listed above according to standard methods.
  • GGC AGA TGT ACG CTT TAA ATT GGT CTC CAT TTC TTA GAA 605 614 623 632 641 650
  • the human gene sequence was amplified, cloned and sequenced by genomic PCR from human DNA from leukocytes by methods known to the person skilled in the art.
  • the gene sequence is:
  • Length of gene NLP 1255 bp, +1 at: 1; Listed from: 1 to: 1255;
  • CTCCCCCTGT CAAGATGTGG CACCTCAAAC TTTGTGCAGT CCTCATGATC TTCCTGTTGC 300
  • mice cDNA sequence (Seq. ID No. 10) and the peptide sequence derived therefrom are:
  • Length of mouse cDNA 429 bp, +1 at: 1; Listed from: 3 to: 429;
  • TGA TGT CCA TAC TGG GGA AGA GTC ACC TCC AAC AGT GAT CTG TTC TGG GGA ATA TTG TTA 305 314 323 332 341 350
  • the knock-out construct has the following structure:
  • Synthetic peptide forms of hNLP were obtained using conventional chemical synthesis methods and by means of recombinant expression in E. coli and by expression in Pichia pastoris. The following products were achieved through multiple synthesis:
  • CTCCCCCTGT CAAGATGTGG CACCTCAAAC TTTGTGCAGT CCTCATGATC TTCCTGTTGC 300
  • HYPOTHETICAL NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATGTGGCACC TCAAACTTTG TGCAGTCCTC ATGATCTTCC TGTTGCTGTT GGGCCAGATA 60
  • AATAGTTCCC CAGTACCAGA AGTGAGTTCA GCAAAGAGAT CCCGGAGAAT GACCCCATTT 120
  • GAGATCAGAA AACAGAGACC CAAGGGTAAA GCTGAAATTA GTGGTGTGGG GTGGGGGTGG 540

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biochemistry (AREA)
  • Toxicology (AREA)
  • Zoology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

L'invention a trait à un peptide de la formule (I): Ra-C-Xn-C-Rd, où Ra est NH2 ou un dérivé d'un groupe amine primaire, ou un oligopeptide ou polypeptide composé dans un cas comme dans l'autre d'acides aminés existant dans la nature, de leurs dérivés modifiés dans la chaîne latérale et/ou de leurs stéréo-isomères aminoacides; n est un entier entre 3 et 7; X est un acide aminé existant dans la nature, son dérivé modifié dans la chaîne latérale et/ou son stéréo-isomère aminoacide; et Rd est COOH, un dérivé d'acide carboxylique, ou un oligopeptide ou polypeptide composé dans un cas comme dans l'autre d'acides aminés existant dans la nature, de leurs dérivés modifiés dans la chaîne latérale et/ou de leurs stéréo-isomères.
PCT/EP1998/000950 1997-02-20 1998-02-19 Plnh (peptide lymphocytaire neutrophile humain) circulant WO1998037191A1 (fr)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
DE19706937 1997-02-20
DE19706937.1 1997-02-20
DE19712487 1997-03-25
DE19712487.9 1997-03-25
DE1997130786 DE19730786C2 (de) 1997-07-18 1997-07-18 Humanes zirkulierendes hNLP (humanes Neutrophilen-Lymphocyten-Peptid)
DE19730786.8 1997-07-18

Publications (1)

Publication Number Publication Date
WO1998037191A1 true WO1998037191A1 (fr) 1998-08-27

Family

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1998/000950 WO1998037191A1 (fr) 1997-02-20 1998-02-19 Plnh (peptide lymphocytaire neutrophile humain) circulant

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WO (1) WO1998037191A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000029621A3 (fr) * 1998-11-16 2000-11-09 Genelabs Tech Inc Methode pour mesurer des polynucleotides cibles et des biomolecules de l'asthme

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995001436A1 (fr) * 1993-06-29 1995-01-12 The Salk Institute For Biological Studies Toxines de cone presentant des proprietes de liaison au recepteur d'acetylcholine
US5585478A (en) * 1992-12-10 1996-12-17 Beth Israel Hospital Association D4 gene and methods of use thereof

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5585478A (en) * 1992-12-10 1996-12-17 Beth Israel Hospital Association D4 gene and methods of use thereof
WO1995001436A1 (fr) * 1993-06-29 1995-01-12 The Salk Institute For Biological Studies Toxines de cone presentant des proprietes de liaison au recepteur d'acetylcholine

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
EMBL Datenbank Entry HS26447; Zugriffsnummer T82264; 28 März 1995; HILLIER L. ET AL.:"The WashU-Merck EST project" *
EMBL Datenbank entry HS46277; Zugriffsnummer R11462; 21 April 1995; HILLIER L. ET AL.:"The WashU-Merck EST project" *
EMBL Datenbank Entry HS52831; Zugriffsnummer T60528; 5 März 1995; HILLIER L. ET AL.:"The WashU-Merck EST project" *
EMBL Datenbank Entry HS551134; Zugriffsnummer T83551; 31 Mai 1995 HILLIER L. ET AL.:"The WashU-Merck EST project" *
EMBL Datenbank Entry MM16134; Zugriffsnummer W82161; 27 Juni 1996; MARRA M. ET AL.:"The WashU-HHMI Mouse EST project" *
EMBL Datenbank Entry MM82512; Zugriffsnummer W29825; 10 Mai 1996; MARRA M. ET AL.:"The WashU-HHMI Mouse EST project" *
ROBERT I. LEHRER ET AL.: "Defensins: Endogenous antibiotic peptides of animal cells", CELL, vol. 64, no. 2, 25 January 1991 (1991-01-25), NA US, pages 229 - 230, XP000170612 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000029621A3 (fr) * 1998-11-16 2000-11-09 Genelabs Tech Inc Methode pour mesurer des polynucleotides cibles et des biomolecules de l'asthme

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