WO1998037191A1

WO1998037191A1 - Human circulating hnlp (human neutrophil lymphocyte peptide)

Info

Publication number: WO1998037191A1
Application number: PCT/EP1998/000950
Authority: WO
Inventors: Wolf-Georg Forssmann; Burkhard Kleemeier; Peter Schulz-Knappe; Michael Nehls
Original assignee: Forssmann Wolf Georg; Burkhard Kleemeier; Schulz Knappe Peter; Michael Nehls
Priority date: 1997-02-20
Filing date: 1998-02-19
Publication date: 1998-08-27

Abstract

The invention relates to a peptide of the formula (I): Ra-C-Xn-C-Rd, where Ra is NH2 or a derivative of a primary amino group or an oligo- or polypeptide, whereby the oligo- or polypeptide consists of naturally occurring amino acids, of derivatives of said amino acids having modified side chains and/or of their stereoisomeric amino acids; n is an integer between 3 and 7; X is a naturally occurring amino acid, a derivative of said amino acid having a modified side chain and/or its stereoisomeric ammoniated; and Rd is a carboxylic acid derivative or an oligo- or polypeptide, whereby the oligo- or polypeptide consists of naturally occurring amino acids, derivatives of said amino acids having a modified side chain and/or their stereoisomeric amino acids.

Description

Human circulating liNLP (human neutrophil βn 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.

According to the invention, peptides are provided with the following structure (formula I)

^R a ^{~ C "X} n ^{" C_R} d

in which 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):

K.-, ^- C ^- Λ._ - C ^- R ~ C —R „- C, ^- RJ II

in which

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.

In a preferred embodiment, X _{n is} RDDSE.

In a further preferred embodiment, 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:

hNLP-17-55, MW 4451:

TPFWRGVSLRP1GASCRDDSECITRLCRKRRCS SVAQE

hNLP - 22-55, MW 3762

GVSLRPIGASCRDDSECITRLCRKRRCSLSVAQE

hNLP-26-55, MW 3406

RP1GASCRDDSECITRLCRKRRCSLSVAQE

hNLP-22-39, MW 1877 as urine peptide:

GVSLRPIGASCRDDSECI

These 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.

The 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.

Corresponding derivatives can be used as hybridization probes. For example, the cDNA of the peptide according to the invention has the novel sequence below (Seq. ID No. 6):

GA GGG GCG AAA GGG TCC ATG CCA GCA ACA CAA TCA CTC AAA GTC CAG ATG AGG GAT 11 20 29 38 47 56

CAG TAA ATA CAA CGT GCC TGA AAG GTG GCC CTT GAG CAC ATT CCT CCG GTA GAC ATT AAC 65 74 83 92 101 110

TTA TTA AAT TGA TTC TGA TTA CAA ATA TAA ACT TTG CCC CCA TCT CAC CCA GTA ACA ATG 125 134 143 152 161 170

CAA GAG TTG ATG TCA GTC TAT AAA AGG AAG TAG GAA CTG TCC CTG GCT TTC AGG CTC CAA 185 194 203 212 221 230

MWHLKLCAVMIF CAT CCT CCC CCT GTC AAG ATG TGG CAC CTC AAA CTT TGT GCA GTC CTC ATG ATC TTC CTG 245 254 263 272 281 290 LLGQIDGSPIPEVSSAKRR TTG CTG TTG GGC CAG ATA GAT GGC TCC CCA ATA CCAGGG 314 323 332 341 350

P R R M T P F W R G V S L R P I G A S C CCA CGG AGA ATG ACC CCA TTT TGG AGA GGG GTT TCC CTC AGG CCT ATT GGA GCC TCC TGC 365 374 383 392 401 410

R D D S E C I T R L C R K R R C S L S V CGG GAT GAT TCT GAG TGT ATC ACA AGG CTA TGC AGA AAA AGA CGC TGT TCC TTA AGT GTG 425 434 443 452 461 470

A Q E

GCC CAG GAA TGA TGT ACA TAC CAG GGA AAG AAA GGA CAG CAG TCA CCT CCG ACA ATG CTC 485 494 503 512 521 530

CGT TCT ATG GAA TAT TGA TTA ACT GCA TTT TGG CTG GAG ACA CCC AAG TGA AGC AAT CTT 545 554 563 572 581 590

GTA TTT TTA ATA TTT AAA GGC AGA TGT ACG CTT TAA ATT GGT CTC CAT TTC TTC TTA GAA 605 614 623 632 641 650

TGT TGA TAT ATG GAT AAG CAT AAC TAA ACT TGT CAA TTT AGA GTT TAT TTT TCT ATG GAT 665 674 683 692 701 710

ACT ATT AAA TGT CTC AAA TTG poly (A) tail 725 734 The gene of the peptide according to the invention was cloned from human genomic leukocyte DNA and has the following sequence:

GAGGGGCGAA AGGGTCCATG CCAGCAACAC AATCACTCAA AGTCCAGATG AGGGATCAGT 60

AAATACAACG TGCCTGAAAG GTGGCCCTTG AGCACATTCC TCCGGTAGAC ATTAACTTAT 120

TAAATTGATT CTGATTACAA ATATAAACTT TGCCCCCATC TCACCCAGTA ACAATGCAAG 180

AGTTGATGTC AGTCTATAAA AGGAAGTAGG AACTGTCCCT GGCTTTCAGG CTCCAACATC 240

CTCCCCCTGT CAAGATGTGG CACCTCAAAC TTTGTGCAGT CCTCATGATC TTCCTGTTGC 300

TGTTGGGCCA GGTAAGGAGG GAAGGATACT TATGTGTGTG TGTGGAGTGT GGAGATGATA 360

GTGGTGGTGG AACTTGAAAG CTAGATTCAG TCCTGAGGAA TGGTTCCTCT GTTCTGAGTC 420

TACAGCATCT GCGGAATGGA ATGATCACTC TTCCAAGGTG TGCAGCAGGG TGTCAACACT 480

TTCATATCTG AATGTCTTTG CCCTTACAGA TAGATGGCTC CCCAATACCA GAAGTGAGTT 540

CGGCAAAGAG AAGGCCACGG AGAATGACCC CATTTTGGAG AGGGGTTTCC CTCAGGCCTA 600

TTGGAGCCTC CTGCCGGGAT GATTCTGAGT GTATCACAAG GCTATGCAGG TACTCCCTGA 660

ACCTGGGAGC AGGGTTGGGC CAGAGAGCCC TGGGAAGCTG GGCAGAGGAG TGACAAGGGG 720

ACACATGAAC CTAAGAATAA AGCTGGGATG GAGGAGTTCT AGACTGAGAC TGGGAGCTCC 780

ATGGAAAATC CCTTAGGAGT TAGGAGCCAA GAACAGCTCT ATGTGGGTAC CATGAAAGAG 840

TGGTGCCTTT CCATTCTTCA GTCTTTCCTG AGACTGGACA GATCAAGCAA AGAGGAAGGA 900

AATGCAGCAT AGGTGGCCCT GGTCATTCCT AGACCCAGTC TTAAAAAACT ACCCTTTCTT 960

TTCCCCTAGA AAAAGACGCT GTTCCTTAAG TGTGGCCCAG GAATGATGTA CATACCAGGG 1020

AAAGAAAGGA CAGCAGTCAC CTCCGACAAT GCTCCGTTCT ATGGAATATT GATTAACTGC 1080

ATTTTGGCTG GAGACACCCA AGTGAAGCAA TCTTGTATTT TTAATATTTA AAGGCAGATG 1140

TACGCTTTAA ATTGGTCTCC ATTTCTTCTT AGAATGTTGA TATATGGATA AGCATAACTA 1200 AACTTGTCAA TTTAGAGTTT ATTTTTCTAT GGATACTATT AAATGTCTCA AATTG

Exon 1: 1-311 Intron 1: 312-509 Exon 2: 510-649 Intron 2: 650-969 Exon 3: 970-1255

The representation of the cDNA of mouse (Mus musculus) and production of knock-out mice were possible according to the usual methods.

The mouse cDNA sequence (Seq. ID No. 10) and the peptide sequence derived therefrom (Seq. ID No. 14) are:

GT CGG TCT ATA AAA GGA AAC GGG AAC TGC CCC CTG GCT TTC AGA CTC CAA CAT CAT 11 20 29 38 47 56 LQLKFAVLLTCLL TCT TTT GCC AAG ATG CTA CAG CTA AAA CTC TTT GCA GTG CTC CTG ACT TGC CTG CTG CTG 65 74 83 92 101 110

L G Q V N S S P V P E V S S A K R S R R CTG GGT CAG GTC AAT AGT TCC CCA GTA CCA GAA GTG AGT TCA GCA AAG AGA TCC CGG AGA 125 134 143 152 161 170

M T P F R G V S L R P I G A S C R D D ATG ACC CCA TTT TGG AGA GGG GTT TCC CTC AGG CCC ATT GGT GCC TCG TGC CGG GAT GAC 185 194 203 212 221 230

S .E C I T R L C R K R R C S L S V A Q E TCT GAG TGT ATC ACA AGA CTA TGC AGA AAA AGA CGC TGT TCC CTA AGT GTG GCC CAG GAG 245 254 263 272 281 290

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

ACG ACA TTT TGG TTG GAG ATA GAT AAG TGA AGT AGT CTT TGT TTT TAA TAT TTA AAG ACG 365 374 383 392 401 410

GAT ACT TGC TTT A 425

The mouse gene was isolated from a murine cosmid library and sequenced by genomic PCR. The sequence is shown below (Seq. ID No. 15):

Mouse NLP gene: 799 bp, +1 at: 1

GTCGGTCTAT AAAAGGAAAC GGGAACTGCC CCCTGGCTTT CAGACTCCAA CATCATTCTT 60

TTGCCAAGAT GCTACAGCTA AAACTCTTTG CAGTGCTCCT GACTTGCCTG CTGCTGCTGG 120

GTCAGGTAAG GAAGGAAACA GTAGTGGCGT GGAATTCCAA GTCAGACTCA GTCCTTAGAA 180

ATGCAGTTCG ATCTCCTGCA ACCTGCAAAG GGCCCTTCTT CTGAGGCTGT GCAGCAGAGG 240

GTGGGTGCTT CCTCAGCTAA AGTGAGTATG CCTTTGCTCT ACAGGTCAAT AGTTCCCCAG 300

TACCAGAAGT GAGTTCAGCA AAGAGATCCC GGAGAATGAC CCCATTTTGG AGAGGGGTTT 360

CCCTCAGGCC CATTGGTGCC TCGTGCCGGG ATGACTCTGA GTGTATCACA AGACTATGCA 420

GGTATTTGAT GATCCCAGGT ACAGGGTGGA CCAGAGAGCA CTGGGAAAGC TGGGAGAGAT 480

GAGATCAGAA AACAGAGACC CAAGGGTAAA GCTGAAATTA GTGGTGTGGG GTGGGGGTGG 540

GGGTGGGGGG AATATATGAA GCTGTGAACT CCATGGAGGG GATTCCGTGA CAGTCAAGAA 600

CCATTCAGAT TCCTAACACT GAACGACAGA TGAAACAAGG AATAATGGAG GCAGCAGAGG 660

TGTCCCAGGG CTCCTTAGGT CTACATCCAA TCTTAATCAC TTCTTTTCTT CCCCCCACTA 720

GAAAAAGACG CTGTTCCCTA AGTGTGGCCC AGGAGTGATG TCCATACTGG GGAAGAGTCA 780 CCTCCAACAG TGATCTGTT

Exon 1: 1-125

Intron 1: 126-284

Exon 2: 285-421

Intron 2: 422-721

Exon 3: 722-799 In addition to genetic engineering, total synthesis based on conventional solid phases in the sense of Merrifield synthesis or liquid phase synthesis is also possible. The synthetic strategy and the structure of the peptide and derivatives derived therefrom, for example with the correspondingly protected amino acids, are known to the person skilled in the art.

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. B. the respiratory tract, gastrointestinal tract and urogenital system, especially the blood and the hematopoietic system. 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 μg 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.

example 1

Hemofil rat batch extraction

800 to 1,000 L of hemofiltrate are adjusted to a pH of 2.7 with HCl and diluted to a conductivity of 5.5 mS / cm with water and applied to a strong cation exchanger at a flow rate of 3 L / min.

Chromatography conditions:

Column: Vantage VA 250 (Amicon, Witten)

Column material: Fractogel TSK SP 650 (M),

25 cm diameter x 5 cm length flow: 3 L / min

Detection: 280 nm, pH, conductivity

Buffer A: Hemofiltrate pH 2.7, conductivity

5.5 S / cm buffer B: 0.5 M ammonium acetate

System: autopilot chromatography system,

(PerSeptive Biosystems, Wiesbaden)

After application of the total of 4,000 L of liquid overnight, 5 mM HCl is rinsed with several column volumes. The bound peptides are eluted as a batch elution with 0.5 M ammonium acetate. Here, a complete elution of the peptides over increasing pH (2.7 - 7.2) and increasing conductivity (56 mS / cm) is achieved in about 5 L eluate.

First preparative separation

The ammonium acetate eluates of the batch extraction are combined in amounts of 5,000 to 10,000 liters of hemofiltrate peptide. After pH adjustment to 2.7, the peptide extract is admixed with Demineralized water with a conductivity of 5.5 mS / cm applied to the preparative cation exchanger.

Chromatography conditions:

Column: Vantage 250 VA column material Fractogel TSK SP 650 (M), 25 cm diameter x 5 cm length

Flow: up to 3 L / min during the application 0.5 to 1 L during the elution

Detection: 280 nm, pH, conductivity Sample: hemofiltrate pH 2.7, conductivity 5.5 mS / cm System: autopilot chromatography system, (PerSeptive Biosystems, Wiesbaden)

After applying the crude extract over 240 min, 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

Buffer ppHH-- W value buffer substances conductivity (mS / cm)

Wash buffer: 2.0 0.01 M HCl 1

Elution buffer 1 3, 6 0.1 M citric acid 1-hydrate 2, 9

Elution buffer 2 4, 5 0.1 M acetic acid +

0.1 M sodium acetate 4.0

Elution buffer 3 5, 0 0.1 M malic acid 6, 2

Elution buffer 4 5 6 0.1 M succinic acid 6, 1

Elution buffer 5 6 6 0, 1 M NaH ₂ P0 ₄ 4, 9

Elution buffer 6 7 4 0, 1 M NaH ₂ P0 ₄ 6, 7

Elution buffer 7 9 0 0, 1 M ammonium carbonate 6, 7

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.

Chromatography conditions:

Column: FineLine 100 (Pharmacia, Freiburg) Column material Source RPC, 15 μm

10 cm diameter x 12.5 cm length

(FineLine 100)

Flow: 150 mL / min

Detection: 280 nm, conductivity, pH buffer A: 10 mM HCl buffer B: 80% acetonitrile in 10 mM HCl gradient 0 - 60% buffer B in 5 column volumes

After the pH pools have been applied, 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.

Semi-preparative reverse phase Cl8 chromatography

Fraction 15 from pH pool VI was separated successively in several identical chromatographies on a semi-preparative reversed-phase column.

Chromatography conditions:

Column: 1 cm x 25 cm steel column

Filling material Vydac RP-C18 5 μm, 300 Ä

Buffer A 0.1% TFA

Buffer B 0.1% TFA, 80% acetonitrile

Gradient 5 - 50% B in 45 min, 50 - 100% B in 10 min

Flow: 2 ml / min

Detection: 220 nm

Chromatography system: Kontron fractions: 1 min from the start of the gradient

Analytical reversed-phase C18 chromatography Fraction 37 contained the substance according to the invention. It was further purified on an analytical column (chromatogram see Fig. 4).

Chromatography conditions:

Column: 0.46 cm x 25 cm steel column

Filling material Vydac RP-C18, 5 μm, 300 Ä buffer A: 0.1% TFA buffer B: 0.1% TFA, 80% acetonitrile gradient 5 - 50% B in 45 min, 50 - 100% B in 10 min

Flow: 0.7 ml / min detection: 214 nm

Chromatography system Kontron

In this rechromatography the purity of the isolated peptide, as it is used for further analysis, becomes clear. The peptides HF 4451 (hNLP-17-55) and HF 3406 (hNLP-26-55) were also purified analogously to this procedure.

Example 2

Capillary zone electrophoresis

To check the purity, a capillary zone electrophoresis was made for the different hNLPs. The fraction containing hNLPs show the substances which have been purified to over 95%.

Conditions: capillary: fused silica effective length 50 cm total length: 57 cm buffer: 100 mM NaH ₂ P0 ₄ pH 2, 5,

0.2% hydroypropylmethyl cellulose

Current: 120 μA const. Detection: 200 nm Temperature: 25 ° C

Example 3

Mass determinations

All mass determinations of the unmodified, modified and / or peptides fragmented by endoproteinase cleavage were carried out on an electrospray mass spectrometer (Sciex API III, Perkin Elmer). The molecular masses of the peptides were determined in positive ion mode in accordance with the mass numbers (MW) shown above.

Amino acid analysis

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. The peptides contain the amino acids detectable by this method in accordance with the four molecular forms mentioned.

Determination of cysteines

After prior chemical derivatization, for example after reduction with ß-mercaptoethanol and carboxamidomethylation with iodoacetamide, cysteines can be detected in peptide sequencing. After derivatization, desalting is preferably followed by analytical reverse phase chromatography with a Vydac RP-C18 column (4.6 mm x 25 cm). Some of the peptides modified in this way are sent to the sequence analysis, with the other part the mass determinations result in a molecular weight increased by 57 Da per cysteine. From the mass difference to the native peptide, it is concluded that the peptides from hemofiltrate contain four cysteines, which are also connected to one another by two disulfide bridges. Sequence determination

Both the native and the carboxamidomethylated peptides and the fragments after cleavage with the endoproteinases Glu-C and Arg-C are analyzed by Edman degradation on an ABI 473 A sequencer using the standard program. The samples are applied to a Polybrene membrane in amounts between 100 and 400 pmol. The following sequences result in accordance with the results from the amino acid analyzes and the mass determinations:

hNLP-17-55, MW 4451 (Seq. ID No. 1):

TPFWRGVSLRP1GASCRDDSECITRLCRKRRCSLSVAQE

1 I

hNLP-22-55, MW 3762 (Seq. ID No. 2)

GVSLRPIGASCRDDSECITRLCRKRRCSLSVAQE

hNLP-26-55, MW 3406 (Seq. ID No. 3)

RPIGASCRDDSECITRLCRKRRCSLSVAQE

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.

Database comparison

A database comparison was carried out using the HUSAR program package on the SwissProt, PIR, EMBL nucleic acid and GenBank databases. The peptide sequence is surprisingly new. No sequence homology to members of known peptide families was found, so that the hNLPs have a surprisingly novel sequence.

Example 4

Synthesis of oligonucleotides

Primers were synthesized on a nucleic acid synthesizer (Applied Biosystems) according to the phosphoamidite method according to the manufacturer's instructions, cleaned on OPC columns and in bidist. H ₂ 0 solved. 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.

Preparation of cDNA

Total RNA was prepared from human intestinal tissue using an automatic nucleic acid extractor (ABI, 340) according to the manufacturer's instructions. From 5 μg 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 μg 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.

Nuc oleic acid sequencing

The nucleic acid sequence after routine fluorescence sequencing resulted in the following sequence with a deduced amino acid sequence (Seq. ID No. 6):

M W H K L C A V L M I F CAT CCT CCC CCT GTC AAG ATG TGG CAC CTC AAA CTT TGT GCA GTC CTC ATG ATC TTC CTG 245 254 263 272 281 290

L L L G Q I D G S P I P E V S S A K R R TTG CTG TTG GGC CAG ATA GAT GGC TCC CCA ATA CCA GAA GTG AGT TCG GCA AAG AGA AGG 305 314 323 332 341 350

A Q E

ACT ATT AAA TGT CTC AAA TTG poly (A) taιl 725 734 The polyadenylation signal is underlined.

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;

Thu, Feb 20, 1997 12:51 PM

GAGGGGCGAA AGGGTCCATG CCAGCAACAC AATCACTCAA AGTCCAGATG AGGGATCAGT 60

AAATACAACG TGCCTGAAAG GTGGCCCTTG AGCACATTCC TCCGGTAGAC ATTAACTTAT 120

TAAATTGATT CTGATTACAA ATATAAACTT TGCCCCCATC TCACCCAGTA ACAATGCAAG 180

AGTTGATGTC AGTCTATAAA AGGAAGTAGG AACTGTCCCT GGCTTTCAGG CTCCAACATC 240

CTCCCCCTGT CAAGATGTGG CACCTCAAAC TTTGTGCAGT CCTCATGATC TTCCTGTTGC 300

TGTTGGGCCA GGTAAGGAGG GAAGGATACT TATGTGTGTG TGTGGAGTGT GGAGATGATA 360

GTGGTGGTGG AACTTGAAAG CTAGATTCAG TCCTGAGGAA TGGTTCCTCT GTTCTGAGTC 420

TACAGCATCT GCGGAATGGA ATGATCACTC TTCCAAGGTG TGCAGCAGGG TGTCAACACT 480

TTCATATCTG AATGTCTTTG CCCTTACAGA TAGATGGCTC CCCAATACCA GAAGTGAGTT 540

CGGCAAAGAG AAGGCCACGG AGAATGACCC CATTTTGGAG AGGGGTTTCC CTCAGGCCTA 600

TTGGAGCCTC CTGCCGGGAT GATTCTGAGT GTATCACAAG GCTATGCAGG TACTCCCTGA 660

ACCTGGGAGC AGGGTTGGGC CAGAGAGCCC TGGGAAGCTG GGCAGAGGAG TGACAAGGGG 720

ACACATGAAC CTAAGAATAA AGCTGGGATG GAGGAGTTCT AGACTGAGAC TGGGAGCTCC 780

ATGGAAAATC CCTTAGGAGT TAGGAGCCAA GAACAGCTCT ATGTGGGTAC CATGAAAGAG 840

TGGTGCCTTT CCATTCTTCA GTCTTTCCTG AGACTGGACA GATCAAGCAA AGAGGAAGGA 900

AATGCAGCAT AGGTGGCCCT GGTCATTCCT AGACCCAGTC TTAAAAAACT ACCCTTTCTT 960

TTCCCCTAGA AAAAGACGCT GTTCCTTAAG TGTGGCCCAG GAATGATGTA CATACCAGGG 1020

AAAGAAAGGA CAGCAGTCAC CTCCGACAAT GCTCCGTTCT ATGGAATATT GATTAACTGC 1080

ATTTTGGCTG GAGACACCCA AGTGAAGCAA TCTTGTATTT TTAATATTTA AAGGCAGATG 1140

Exon 1: 1-311

Intron 1: 312-509

Exon 2: 510-649

Intron 2: 650-969

Exon 3: 970-1255

The processing of the peptide hormone hNLP can be seen in Figure 1. The cDNA of mouse (Mus musculus) and the production of knock-out mice were possible using the usual methods:

The mouse 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;

Translated from: 69 to: 296 (ORFs); Genetic Code used: Universal; Thu, Feb 20, 1997 4:32 PM

Frame 3

GT CGG TCT ATA AAA GGA AAC GGG AAC TGC CCC CTG GCT TTC AGA CTC CAA CAT CAT 11 20 29 38 47 56

M Q L K L F A V L L T C L L L TCT TTT GCC AAG ATG CTA CAG CTA AAA CTC TTT GCA GTG CTC CTG ACT TGC CTG CTG CTG 65 74 83 92 101 110

M T P F W R G V S L R P I G A S C R D D ATG ACC CCA TTT TGG AGA GGG GTT TCC CTC AGG CCC ATT GGT GCC TCG TGC CGG GAT GAC 185 194 203 212 221 230

S E C I T R L C R K R R C S L S V A Q E TCT GAG TGT ATC ACA AGA CTA TGC AGA AAA AGA CGC TGT TCC CTA AGT GTG GCC CAG GAG 245 254 263 272 281 290

GAT ACT TGC TTT A 425 The knock-out construct has the following structure:

targeting vector

poiyA

• gcnomic

Example 5

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:

hNLP-17-55, MW 4451 (Seq. ID No. 1):

TPFWRGVSLRPIGASCRDDSECITRLCRKRRCSLSVAQE hNLP-22-55, MW 3762 (Seq. ID No. 2):

GVSLRPIGASCRDDSECITR CRKRRCSLSVAQE

hNLP-26-55, MW 3406 (Seq. ID No. 3]

RPIGASCRDDSECITRLCRKRRCSLSVAQE

hNLP- 1-55, (Seq. ID No. 5]

SPIPEVSSAKRRPRR TPFWRGVSLRPIGASCRDDSECITRLCRKRRCSLSVAQE

SEQUENCE LOG

(1. GENERAL INFORMATION:

(i) APPLICANT:

(A) NAME: Prof. Dr. Wolf-Georg Forssmann

(B) STREET: Feodor-Lynen-Str. 31

(C) LOCATION: Hanover

(E) COUNTRY: Germany

(F) POSTAL NUMBER: 30625

(ii) NAME OF THE INVENTION: Human Circulating hNLP (Human Neurophyte Lymphocyte Peptide) (iii) NUMBER OF SEQUENCES: 15 (iv) COMPUTER READABLE VERSION:

(A) DISK: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS -DOS

(D) SOFTWARE: Patentin Release # 1.0, Version # 1.30 (EPA)

(2) INFORMATION ON SEQ ID NO: 1:

(i) SEQUENCE LABEL:

(A) LENGTH: 39 amino acids

(B) TYPE: amino acid

(C) STRAND FORM:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

Thr Pro Phe Trp Arg Gly Val Ser Leu Arg Pro Ile Gly Ala Ser Cys 1 5 10 15

Arg Asp Asp Ser Glu Cys Ile Thr Arg Leu Cys Arg Lys Arg Arg Cys 20 25 30

Ser Leu Ser Val Ala Gin Glu 35

(2) INFORMATION ABOUT SEQ ID NO: 2: (i) SEQUENCE LABEL:

(A) LENGTH: 34 amino acids

(B) TYPE: amino acid

(C) STRAND FORM: (ii) MOLECULE TYPE: Peptide (iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:

Gly Val Ser Leu Arg Pro Ile Gly Ala Ser Cys Arg Asp Asp Ser Glu

1 5 10 15

Cys Ile Thr Arg Leu Cys Arg Lys Arg Arg Cys Ser Leu Ser Val Ala 20 25 30

Gin glu

(2) INFORMATION ABOUT SEQ ID NO: 3: (i) SEQUENCE LABEL:

(A) LENGTH: 30 amino acids

(B) TYPE: amino acid

(C) STRAND FORM:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

Arg Pro Ile Gly Ala Ser Cys Arg Asp Asp Ser Glu Cys Ile Thr Arg 1 5 10 15

Leu Cys Arg Lys Arg Arg Cys Ser Leu Ser Val Ala Gin Glu 20 25 30

(2) INFORMATION ABOUT SEQ ID NO: 4: (i) SEQUENCE LABEL:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) STRAND FORM:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

Gly Val Ser Leu Arg Pro Ile Gly Ala Ser Cys Arg Asp Asp Ser Glu 1 5 10 15

Cys Ile (2) INFORMATION ABOUT SEQ ID NO: 5: (i) SEQUENCE LABEL:

(A) LENGTH: 55 amino acids

(B) TYPE: amino acid

(C) STRAND FORM:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide

(iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Ser Pro Ile Pro Glu Val Ser Ser Ala Lys Arg Arg Pro Arg Arg Met 1 5 10 15

Thr Pro Phe Trp Arg Gly Val Ser Leu Arg Pro Ile Gly Ala Ser Cys 20 25 30

Arg Asp Asp Ser Glu Cys Ile Thr Arg Leu Cys Arg Lys Arg Arg Cys 35 40 45

Ser Leu Ser Val Ala Gin Glu 50 55

(2) INFORMATION ABOUT SEQ ID NO: 6: (i) SEQUENCE LABEL:

(A) LENGTH: 737 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:

GAGGGGCGAA AGGGTCCATG CCAGCAACAC AATCACTCAA AGTCCAGATG AGGGATCAGT 60

AAATACAACG TGCCTGAAAG GTGGCCCTTG AGCACATTCC TCCGGTAGAC ATTAACTTAT 120

TAAATTGATT CTGATTACAA ATATAAACTT TGCCCCCATC TCACCCAGTA ACAATGCAAG 180

AGTTGATGTC AGTCTATAAA AGGAAGTAGG AACTGTCCCT GGCTTTCAGG CTCCAACATC 240

CTCCCCCTGT CAAGATGTGG CACCTCAAAC TTTGTGCAGT CCTCATGATC TTCCTGTTGC 300

TGTTGGGCCA GATAGATGGC TCCCCAATAC CAGAAGTGAG TTCGGCAAAG AGAAGGCCAC 360 GGAGAATGAC CCCATTTTGG AGAGGGGTTT CCCTCAGGCC TATTGGAGCC TCCTGCCGGG 420

ATGATTCTGA GTGTATCACA AGGCTATGCA GAAAAAGACG CTGTTCCTTA AGTGTGGCCC 480

AGGAATGATG TACATACCAG GGAAAGAAAG GACAGCAGTC ACCTCCGACA ATGCTCCGTT 540

CTATGGAATA TTGATTAACT GCATTTTGGC TGGAGACACC CAAGTGAAGC AATCTTGTAT 600

TTTTAATATT TAAAGGCAGA TGTACGCTTT AAATTGGTCT CCATTTCTTC TTAGAATGTT 660

GATATATGGA TAAGCATAAC TAAACTTGTC AATTTAGAGT TTATTTTTCT ATGGATACTA 720

TTAAATGTCT CAAATTG 737

(2) INFORMATION ABOUT SEQ ID NO: 7: (i) SEQUENCE LABEL:

(A) LENGTH: 254 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:

GAGGGGCGAA AGGGTCCATG CCAGCAACAC AATCACTCAA AGTCCAGATG AGGGATCAGT 60

AAATACAACG TGCCTGAAAG GTGGCCCTTG AGCACATTCC TCCGGTAGAC ATTAACTTAT 120

TAAATTGATT CTGATTACAA ATATAAACTT TGCCCCCATC TCACCCAGTA ACAATGCAAG 180

AGTTGATGTC AGTCTATAAA AGGAAGTAGG AACTGTCCCT GGCTTTCAGG CTCCAACATC 240

CTCCCCCTGT CAAG 254

(2) INFORMATION ON SEQ ID NO: 8: (i) SEQUENCE LABEL:

(A) LENGTH: 231 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: ATGTGGCACC TCAAACTTTG TGCAGTCCTC ATGATCTTCC TGTTGCTGTT GGGCCAGATA 60

GATGGCTCCC CAATACCAGA AGTGAGTTCG GCAAAGAGAA GGCCACGGAG AATGACCCCA 120

TTTTGGAGAG GGGTTTCCCT CAGGCCTATT GGAGCCTCCT GCCGGGATGA TTCTGAGTGT 180

ATCACAAGGC TATGCAGAAA AAGACGCTGT TCCTTAAGTG TGGCCCAGGA A 231

(2) INFORMATION ABOUT SEQ ID NO: 9: (i) SEQUENCE LABEL:

(A) LENGTH: 252 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

TGATGTACAT ACCAGGGAAA GAAAGGACAG CAGTCACCTC CGACAATGCT CCGTTCTATG 60

GAATATTGAT TAACTGCATT TTGGCTGGAG ACACCCAAGT GAAGCAATCT TGTATTTTTA 120

ATATTTAAAG GCAGATGTAC GCTTTAAATT GGTCTCCATT TCTTCTTAGA ATGTTGATAT 180

ATGGATAAGC ATAACTAAAC TTGTCAATTT AGAGTTTATT TTTCTATGGA TACTATTAAA 240

TGTCTCAAAT TG 252

(2) INFORMATION ON SEQ ID NO: 10: (i) SEQUENCE LABEL:

(A) LENGTH: 429 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

GTCGGTCTAT AAAAGGAAAC GGGAACTGCC CCCTGGCTTT CAGACTCCAA CATCATTCTT 60

TTGCCAAGAT GCTACAGCTA AAACTCTTTG CAGTGCTCCT GACTTGCCTG CTGCTGCTGG 120

GTCAGGTCAA TAGTTCCCCA GTACCAGAAG TGAGTTCAGC AAAGAGATCC CGGAGAATGA 180 CCCCATTTTG GAGAGGGGTT TCCCTCAGGC CCATTGGTGC CTCGTGCCGG GATGACTCTG 240

AGTGTATCAC AAGACTATGC AGAAAAAGAC GCTGTTCCCT AAGTGTGGCC CAGGAGTGAT 300

GTCCATACTG GGGAAGAGTC ACCTCCAACA GTGATCTGTT CTGGGGAATA TTGTTAACGA 360

CATTTTGGTT GGAGATAGAT AAGTGAAGTA GTCTTTGTTT TTAATATTTA AAGACGGATA 420

CTTGCTTTA 429

(2) INFORMATION ABOUT SEQ ID NO: 11: (i) SEQUENCE LABEL:

(A) LENGTH: 68 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

GTCGGTCTAT AAAAGGAAAC GGGAACTGCC CCCTGGCTTT CAGACTCCAA CATCATTCTT 60

TTGCCAAG 68

(2) INFORMATION ABOUT SEQ ID NO: 12: (i) SEQUENCE LABEL:

(A) LENGTH: 228 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

ATGCTACAGC TAAAACTCTT TGCAGTGCTC CTGACTTGCC TGCTGCTGCT GGGTCAGGTC 60

AATAGTTCCC CAGTACCAGA AGTGAGTTCA GCAAAGAGAT CCCGGAGAAT GACCCCATTT 120

TGGAGAGGGG TTTCCCTCAG GCCCATTGGT GCCTCGTGCC GGGATGACTC TGAGTGTATC 180

ACAAGACTAT GCAGAAAAAG ACGCTGTTCC CTAAGTGTGG CCCAGGAG 228 (2) INFORMATION ABOUT SEQ ID NO: 13: (i) SEQUENCE LABEL:

(A) LENGTH: 133 base pairs

(B) TYPE: nucleotide

(C) STRAND FORM: Single strand

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

TGATGTCCAT ACTGGGGAAG AGTCACCTCC AACAGTGATC TGTTCTGGGG AATATTGTTA 60

ACGACATTTT GGTTGGAGAT AGATAAGTGA AGTAGTCTTT GTTTTTAATA TTTAAAGACG 120

GATACTTGCT TTA 133

(2) INFORMATION ABOUT SEQ ID NO: 14: (i) SEQUENCE LABEL:

(A) LENGTH: 76 amino acids

(B) TYPE: amino acid

(C) STRAND FORM:

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein

(iii) HYPOTHETICAL: YES (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:

Met Leu Gin Leu Lys Leu Phe Ala Val Leu Leu Thr Cys Leu Leu Leu 1 5 10 15

Leu Gly Gin Val Asn Ser Ser Pro Val Pro Glu Val Ser Ser Ala Lys 20 25 30

Arg Ser Arg Arg Met Thr Pro Phe Trp Arg Gly Val Ser Leu Arg Pro 35 40 45

Ile Gly Ala Ser Cys Arg Asp Asp Ser Glu Cys Ile Thr Arg Leu Cys 50 55 60

Arg Lys Arg Arg Cys Ser Leu Ser Val Ala Gin Glu 65 70 75

(2) INFORMATION ON SEQ ID NO: 15: (i) SEQUENCE LABEL: (B) TYPE: nucleotide

(C) STRANDFORM: not known

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(iii) HYPOTHETICAL: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

GTCGGTCTAT AAAAGGAAAC GGGAACTGCC CCCTGGCTTT CAGACTCCAA CATCATTCTT 60

TTGCCAAGAT GCTACAGCTA AAACTCTTTG CAGTGCTCCT GACTTGCCTG CTGCTGCTGG 120

GTCAGGTAAG GAAGGAAACA GTAGTGGCGT GGAATTCCAA GTCAGACTCA GTCCTTAGAA 180

ATGCAGTTCG ATCTCCTGCA ACCTGCAAAG GGCCCTTCTT CTGAGGCTGT GCAGCAGAGG 240

GTGGGTGCTT CCTCAGCTAA AGTGAGTATG CCTTTGCTCT ACAGGTCAAT AGTTCCCCAG 300

TACCAGAAGT GAGTTCAGCA AAGAGATCCC GGAGAATGAC CCCATTTTGG AGAGGGGTTT 360

CCCTCAGGCC CATTGGTGCC TCGTGCCGGG ATGACTCTGA GTGTATCACA AGACTATGCA 420

GGTATTTGAT GATCCCAGGT ACAGGGTGGA CCAGAGAGCA CTGGGAAAGC TGGGAGAGAT 480

GAGATCAGAA AACAGAGACC CAAGGGTAAA GCTGAAATTA GTGGTGTGGG GTGGGGGTGG 540

GGGTGGGGGG AATATATGAA GCTGTGAACT CCATGGAGGG GATTCCGTGA CAGTCAAGAA 600

CCATTCAGAT TCCTAACACT GAACGACAGA TGAAACAAGG AATAATGGAG GCAGCAGAGG 660

TGTCCCAGGG CTCCTTAGGT CTACATCCAA TCTTAATCAC TTCTTTTCTT CCCCCCACTA 720

GAAAAAGACG CTGTTCCCTA AGTGTGGCCC AGGAGTGATG TCCATACTGG GGAAGAGTCA 780

CCTCCAACAG TGATCTGTT 799

Claims

claims

Peptide of formula I.

R _a -CX _n -CR _d

in which

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,

2. Peptide according to claim 1 with the formula II

R _a -CX _n -CR _b -CR _c -CR _d 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.

3. Polypeptide according to claim 1 or 2 with the designation native hNLP (human neutrophil lymphocyte peptide) with the following amino acid sequences:

hNLP-17-55, MW 4451 (Seq. ID No. 1):

TPFWRGVSLRPIGASCRDDSECITRLCRKRRCSLSVAQE

hNLP-22-55, MW 3762 (Seq. ID No. 2)

GVSLRPIGASCRDDSECITRLCRKRRCSLSVAQE

I I

hNLP-26-55, MW 3406 (Seq. ID No. 3)

RPIGASCRDDSECITRLCRKRRCSLSVAQE

hNLP-22-39, MW 1877 (Seq. ID No. 4): GVSLRPIGASCRDDSECI hNLP - 1 - 55, (Seq. ID No. 5)

SPIPEVSSAKRRPRRMTPFWRGVSLRPIGASCRDDSECITRLCRKRRCSLSVAQE l J

their bio-active fragments and / or derivatives, in particular amidated, acetylated, sulfated, phosphorylated and / or glycosylated derivatives.

4. Polynucleotides coding for peptides according to at least one of claims 1 to 3 and / or its fragments, derivatives and analogs.

5. Polynucleotides according to claim 4 with the Seq. ID # 6 to 9 and 11 to 14.

6. Polynucleotides according to claim 4 and / or 5, characterized in that the polynucleotide is composed of DNA, RNA or PNA.

7. Vector containing a DNA according to claim 6.

8. Genetically manipulated host cell containing the vector according to claim 7.

9. DNA hybridizing with a polynucleotide according to claim 4, which codes for a polypeptide with hNLP activity.

10. Antibody directed against the polypeptides according to at least one of claims 1 to 3.

11. Antagonist / inhibitor directed against the polypeptides from claim 1.

12. Antisense oligonucleotides to inhibit hNLP expression.

13. Medicament containing at least one polypeptide according to one of claims 1 to 3 or at least one polynucleotide according to one of claims 4 to 6 or polynucleotides with the Seq. ID. # 10 and, if applicable, compatible carriers and auxiliary substances.

14. Medicament containing at least one of the substances mentioned in claims 10 to 12.

15. Medicament according to one of claims 13 or 14 for oral, parenteral, intravenous, intramuscular, intracutaneous, intrathecal, intranasal and topical application, and as an aerosol for transpulmonary application.

16. A method for producing an hNLP according to any one of claims 1 to 3 by extraction of hemofiltrate by cation exchanger extraction with subsequent elution of the adsorbed substances, a renewed cation exchange chromatography of the extract containing the peptides and multi-stage reverse phase chromatography.

17. A method for producing an hNLP according to any one of claims 1 to 3 by Merrifield solid phase synthesis or liquid phase synthesis according to methods known to those skilled in the art with protected amino acids.

18. A method for producing an hNLP according to one of claims 1 to 3 by methods known to the person skilled in the art of heterologous expression using common biotechnological vectors.

19. Diagnostic agent for detecting the polypeptides according to one of claims 1 to 3 or the polynucleotides according to one of claims 4 to 6 or polynucleotides with the Seq. ID # 10, containing poly- or monoclonal antibodies against hNLP or with the nucleic acid coding for the hNLP or mRNA hybridizing DNA, RNA or PNA.

20. Diagnostic agent according to claim 19, containing polypeptides according to one of claims 1 to 3 or antibodies against it for test systems for controlling tissue, plasma, urine and cerebrospinal fluid levels of these polypeptides.

21. Diagnostics for the detection of functional disorders of the bone marrow, the lymph organs, the gastrointestinal tract, the immune system and inflammatory and neoplastic processes containing the polypeptides according to one of claims 1 to 3.

22. Use of the polypeptides or polynucleotides according to at least one of claims 1 to 6 for the manufacture of a medicament for the treatment of disorders in inflammatory processes, disturbed inflammatory reactions, tumor diseases, proliferation and maturation disorders of the hematopoietic system.

23. Use of DNA according to claim 9, antibodies according to claim 10, antagonists / inhibitors according to claim 11, antisense oligonucleotides according to claim 12 for the manufacture of a medicament for the treatment of infectious diseases, inflammatory and neoplastic diseases and proliferation disorders e.g. of the respiratory tract, gastrointestinal tract and urogenital system, especially the blood and blood-forming systems.

24. Vectors for generating knock-out mutants in mice for establishing chronic animal models with maturation disorders of the hematopoietic systems.