DE4244565A1 - Lymphocytoma proliferation activating peptide structure and peptide - Google Patents

Abstract

Claimed is the lymphocytoma proliferation activating peptide (LPAP) of the prim. structure shown and the homologous-dimeric antiparallel peptide derived therefrom and its derivs.Also claimed is (i) the prodn. of LPAP using prokaryotic and eukaryotic expression and chromatographic purificn., (ii) a medicament contg. human, circulating LPAP or LPAP produced by (i).

Description

The invention relates to a method for obtaining a peptide (protein) in pure or partially purified form from human and animal Body fluids that have the ability to control the growth of cells. This substance is characterized in that it consists in particular of hemofiltrate or Hemodialysate, which is filtered off from human and animal blood, can be won. The substance is called "Lymphocytoma poliferation activating peptides "(LPAP) and may be used for the purpose of (1) analysis of diseases, (2) for medical and professional use as Drug as well as (3) can be used as a growth factor for ex vivo preparations.

The substance LPAP was first extracted from the hemofiltrate kidney disease or from Subjects after utrafiltration on Hemofiltrationsapparat won and over a proliferation test of lymphocytoma cells in cell culture functional characterized. A patented process (Forssmann, 1988; Laid-open publication DE 36 33 707 A1) for the production of protein from Refined hemofiltrate and modified for surprisingly detectable functions. With this procedure, the protein molecules are won, the one Possess molecular weight below 20 kDalton. They are used in veno-venous or arterio venous shunt compound filtered off. In the chromatographic separation This crude peptide material can be fractions containing the LPAP won become. The previously known method was therefore used to the To win crude peptide extracts, which surprisingly in the Application to lymphocytoma cells in cell culture a strong growth effect was noticed. It was further found that in special further Purification method this biological activity could be concentrated until finally a uniform protein identified and in its structure was clarified. The value of this invention is characterized in that this substance has been purified from the previously considered worthless hemofiltrate can be used as an adjunct to cell culture media. The named Fabric LPAP is further characterized by being produced by chemical synthesis and can be obtained through genetic engineering production and  Surprisingly, it can be used for numerous other concerns, u. a. For the analysis in human blood as a pathognomic diagnostic feature, as Growth factor, for example, in wound healing and as a starting point for the study of cancerous tumors, as this factor surprisingly just stimulating cell types with cancerous growth.

The present invention thus relates to a novel peptide, the LPAP (Lymphocytoma Proliferation Activating Peptide), its production, this containing medicaments and preparations containing this Preservation and breeding of ex vivo preparations and its use for this.

More recently, more and more has been shown that growth factors therapeutically of great interest and that probably still a large one Number of unknown factors exists that are of clinically useful significance. This could include factors that are important in the treatment of both Tumors as well as in hormone replacement in many diseases regular administration over a long period, possibly for life, could be administered to treat illnesses in a positive sense control.

In several papers (Tanaka et al., J. Clin. Endocrinol., Metah. 51, 1058-1063, 1980) has now been shown that certain cell lines of the white Blood cell line (Lymphocytoma cells, Nb 2 Node cell line) by already known pituitary peptide hormones (pituitary gland) in their growth be particularly specifically influenced.

The search for the existence of further, in this way characterizable Active ingredients as biologically active molecules in human plasma healthy and sick people surprisingly ended up with the result concluded that with the LPAP a molecule was found whose Structure was previously unknown and its educational site is still in the body  is largely unexplained. The therapeutic and economical use was tested and the LPAP was surprisingly important Recognized circulating peptide of human blood.

The present invention thus relates to:
A blood peptide LPAP with the amino acid sequence

which is constructed as homolog-dimeric, antiparallel peptide by two chains each, the two Cys residues in position 3 are each connected to the Cys residues in position 69 via a disulfide bridge. This results in the opposite (antiparallel) attachment of two (dimer), identical (homologous) amino acid chains, ie the homologous dimeric, antiparal peptide:

Furthermore, the invention relates to the natural and pharmacologically acceptable derivatives of LPAP, in particular amidated, acetylated, phosphorylated and glycosylated LPAP derivatives and further fragments of the peptide. By mass spectrometry, a molecular weight of 15,826.2 daltons (adjusted for 32, since there is a double oxidation of two Met) can be detected ( Figure 10), and confirmed by genomic analysis of the further portion of the amino acid sequence belonging to the cDNA, which were also recognized in several sequencing of sections after endoprotease degradation and after cyanogen bromide cleavage ( Figures 7 and 8).

Thus, another object of the present invention, a new peptide, the LPAP, which is characterized as having a good Accessible drug with biological and therapeutic activity natural substance can be used as blood-borne substance.

Another object of the present invention is to provide a manufacturing method for this LPAP and the use of the LPAP as a drug for various therapeutic and diagnostic indications. For this purpose, the LPAP can be used as a highly pure substance or - if sufficient for the particular use - within a partially purified peptide mixture (see active fractions in Fig. 2 to 4).

The present invention further relates to a process for the preparation of this LPAP or its derivatives, characterized in that it has a produced prokaryotic or eukaryotic expression and is purified by chromatography. Another method of making the LPAP-related molecules or their derivatives is characterized in that one knows it from human blood over chromatography procedure in well-known Way isolated. Finally, a method for producing the LPAP or its derivatives, characterized in that the LPAP molecules by the conventional methods of solid-phase and liquid-phase synthesis of the protected amino acids contained in the indicated sequence, deblocked and purified it by the usual chromatography method (Example 2).

The LPAP molecules were chemically synthesized (Example 2) and as Medicines prepared. Also the genetic engineering production over usual Vectors have been developed: the LPAP peptide is genetically engineered both (1) in pro- and (2) produced in eukaryotic organisms. Therefor we use u. a. Expression vectors for secretory expression (eg pSP6, pRit derivatives, Pharmacia), for direct cytoplasmic expression (eg pKK- Derivatives, Pharmacia) or expression as a fusion protein (pMC 1871, Pharmacia).  

For eukaryotic expression are various organisms and vectors available, for. Insect cells (Summers and Smith, Tex.Agric.Exp.Stn. (bull) 1555, 1987), yeasts (Hitzeman et al., Nature, 293, 717-722, 1981), filamentous fungi (Yelton et al., Proc. Natl. Acad Sci., USA 81, 1470-1474, 1984)) and mammalian cells (Zettlmeissl et al., Biotechnology, 5, 720- 725, 1987), of which we prefer to use the insect cells. The expressed LPAP peptides are purified by chromatography methods, preferably as indicated in Example 1.

The pharmaceutical preparation contains LPAP or a physiologically acceptable Salt of the LPAP. The form and composition of the drug containing the LPAP contains depends on the mode of administration. The humane LPAP can be administered parenterally, intranasally, orally and by inhalation. Preferably, LPAP becomes an injection preparation, either as a solution or as lyophilisate for dissolution immediately before use, ready-made. Also as epicutane application is the LPAP for wound healing with special made-up dressings, plasters or sprays used. The Pharmaceutical preparation may also contain adjuvants, the bottling technology are contributing to the solubility, stability or sterility of the Drug or the efficiency of absorption in the body increase.

The daily dose to be administered for LPAP depends on the indication and the Application of certain derivatives. In the therapy of wound healing by epicutane administration, the dose is 100 to 10,000 units (μg), at i.v./i.m. Injection is in the range of 100 to 1200 units (μg) / day, daily subcutaneous injection preferably at 300-2400 units (μg) / day.

The determination of biological activity for the LPAP is based on measurements against international and in-house reference preparations for human LPAP or its analogs in a common biological assay procedure for the same. For this purpose, a standardized proliferation test with lymphocytoma cells is preferably used (Example 3, FIG. 1).

The peptide according to the invention LPAP is characterized in that it is especially suitable for long-term therapy in wound healing, as it has a has excellent biological effectiveness and on the other hand Continuous treatment does not trigger an immune reaction.

The inventive preparation is further as an agent for the therapy of Kidney disease, heart disease, in intensive care and at To use lung diseases.

The preparation according to the invention is further included as an agent for therapy and diagnosis Use tumor diseases, as it is for the production of human-compatible Antibodies can be related to the tumor-specific growth inhibit. Further it is as a means to a special therapy Use tumor diseases, after preparation of human-compatible Antibodies this can be used to inhibit the growth of tumors.

The inventive preparation is further a substance for the treatment of explanted to use asthenic killer lymphocytes from tumor metastases, to use these as immune-fortified cells that the tumor also inhibit specific growth or break down the tumors.

The preparation of the invention is further as an agent for the therapy of To use diseases in the accident medicine, since it to the improvement especially the skin growth can be used, preferably also at Burn wounds and in the ex vivo cultivation of the body's own cells Patients where they are reimplanted for dermal replacement.  

The invention will be described below by way of examples and the following Illustrations referred to in the examples are explained. It shows

Fig. 1: Proliferation test to determine LPAP activity. The Nb 2 Node cells are grown in Fischer medium supplemented with 10% horse serum (HS) and 10% fetal calf serum (FCS) and counted at 24, 48 and 96 hours by means of a counter (Casy, sharpness), cell size and Cell count analyzed. The cells can be seen in a peak between 8-16 μm. To test the FCS is replaced by the Hemofiltratfraktion, or by the LPAP from the individual purification steps. The controls are bred in Fischer Medium (a) with Horse Serum (HS) alone, (b) with Horse Serum and Fetal Calf Serum (FCS). These controls show against LPAP-containing fractions (c and d) (for example, fraction 33 or fraction 35 of the chromatography in the 5th purification stage as in Fig. 6, designated there as fraction 23 and 25 , since fractionated after 10 min a significant increase in cell growth, which is dose-dependent (see also Fig. 14).

Fig. 2: Preparative large-scale RP chromatography of the alginic acid extract for coarse separation according to molecular properties and for desalting the crude peptide extract (purification step 1 ). 3.8 g of alginic acid extract, corresponding to 50 l of hemofiltrate per chromatography run (12 in total, without preliminary tests) were dissolved in 100 ml of buffer A and applied to the column. The hatched area contains the LPAP fractions determined by the proliferation test, which are used for further purification.

Pillar: Steel jacket 50 × 200 mm Material: Parcosil RP C4, 250 Å, 20-45 μm Eluent A: Water with 0.01 N HCl, Eluent B: 50% isopropanol, 30% methanol, 20% water (V: V: V) in 0.01 N HCl   Gradient: 0-0% eluent B 30 min 0-70% eluent B 100 min 70-100% eluent B 1 min 100-100% eluent B 9 min 100-0% eluent B 1 min 0-0% eluent B 20 min Flow rate: 50 ml / min fractions: 100 ml / 2 min from min. 30 Absorption: 230 nm and 280 nm

Fig. 3: Preparative large-scale RP chromatography II for the coarse separation of the biologically active fractions obtained as pool IX and X from the separation in Fig. 2 (purification step 2 ). A total of 5 runs were carried out, in each case 50 mg of the dried material Pool IX / X dissolved in 10 ml of buffer A, filtered through 0.2 μm filter, were applied. In the hatched area of 75 to 95 min retention time, the LPAP-containing fractions determined by the proliferation test are again.

Pillar: Steel jacket 30 × 125 mm Material: Parcosil RP C4, 250 Å, 20-45 μm Eluent A: Water with 0.01 N hydrochloric acid, Eluent B: 50% isopropanol, 30% methanol, 20% water (V: V: V) in 0.01 N HCl Gradient: 0-0% eluent B 10 min 0-40% eluent B 10 min 40-100% eluent B 120 min 100-100% eluent B 5 min 100-0% eluent B 5 min 0-0% eluent B 10 min Flow rate: 5 ml / min fractions: were collected as pool fractions, Pool I: 0-44 min Pool II: 45-60 min Pool III: 60-75 min   Pool IV: 76-90 min Pool V: 91 ä120 min Absorption: 230 nm and 280 nm

Fig. 4: Semipreparative RP chromatography of the separation of the poliferative fractions from preparative RP chromatography according to Fig. 3 (purification step 3 ). A total of 4 runs were carried out, wherein the active fractions from the chromatographies of Fig. 3 were treated only for evaporation of the organic solvent in a vacuum centrifuge and then used in liquid form. In the hatched area of the larger peak at retention time of 51 to 63 minutes or above 50% of acetonitrile of solvent B are the further purified LPAP fractions determined by the proliferation test.

Pillar: Steel jacket 20 × 100 mm Material: Parcosil Pro-RP C18, 100 Å, 7 μm Eluent A: Water with 0.01 N HCl, Eluent B: 80% acetonitrile, 20% water (v: v) in 0.01 N HCl Gradient: 0-0% eluent B 10 min 0-25% eluent B 5 min 25-100% eluent B 75 min 100-100% eluent B 5 min 100-0% eluent B 2 min 0-0% eluent B 10 min Flow rate: 5 ml / min fractions: 10 ml / 2 min from min. 40 to min. 70 Absorption: 214 nm and 230 nm

Fig. 5: Analytical HPLC gel chromatography of the separation of the poliferatively acting fractions from the semi-preparative RP chromatography according to Fig. 4 (purification step 4 ). All the material from these chromatographies was lyophilized and taken up in 5 ml of running buffer: 5 runs, each with 1 ml of application, were carried out. In the hatched area of the larger peak with a retention time of 38 to 50 min, the further purified LPAP fractions determined by the proliferation test are located.

Pillar: Steel column 7.2 × 600 mm Material: TSK G 3000 SW Guard column: BK G 2000 SW 7.2 × 50 mm eluent: 50 mM NaH ₂ PO ₄ + 100 mM NaCl, pH: 6.8 Gradient: isocratic Running time: 90 min Flow rate: 0.5 ml / min fractions: 0.5 ml / 1 min from min. 30, flow in pools Absorption: 214 nm and 230 nm

Fig. 6: Analytical RP-chromatography I of the separation of the poliferatively acting fractions from gel chromatography according to Fig. 5 (purification step 5 ). The non-desalted, non-lyophilized fractions were pooled and spread evenly over 4 runs (4.5 ml per run). In the shaded area of the peak at about 60% eluent B, in fraction 32 and 33 are the purified LPAP fractions determined by the proliferation test with the highest biological activity.

Pillar: Vydac, steel casing 2.1 × 100 mm Material: Vydac RP C18 Column temperature: 35 ° C Eluent A: Water with 0.06% TFA Eluent B: 20% water (V: V) with 0.05% TFA with 80% acetonitrile Gradient: 0-0% eluent B 5 min 0-25% eluent B 5 min 25-100% eluent B 150 min 100-100% eluent B 5 min 100-0% eluent B 5 min 0-0% eluent B 10 min   Flow rate: 0.2 ml / min fractions: 0.2 ml / 1 min from min. 70 to min. 100 Advance / after-run in pools Absorption: 220 nm and 280 nm

Fig. 7: Analytical RP-chromatography II of the separation of the poliferative fractions from RP-chromatography I according to Fig. 6 in a rechromatography (final cleaning, purification step 6 ). The fractions of a chromatography run according to FIG. 6 were applied after these fractions had been briefly concentrated on a vacuum centrifuge to evaporate off the organic solvent. In the region of the peak at about 80 min retention time or 50% acetonitrile from solvent B is the highly purified LPAP determined by the proliferation test, from which the remaining structural data were also obtained. This peak contained about 1 nM hochgreinigten LPAPs, which corresponds to the yield of about 100-125 l hemofiltrate (1/4 of the approach).

Pillar: Vydac, steel casing 2.1 × 100 mm Material: Vydac RP C18 Column temperature: 35 ° C Eluent A: Water with 0.06% TFA Eluent B: 20% water (V: V) with 0.05% TFA with 80% acetonitrile Gradient: 0-0% eluent B 5 min 0-25% eluent B 5 min 25-100% eluent B 150 min 100-100% eluent B 5 min 100-0% eluent B 5 min 0-0% eluent B 10 min Flow rate: 0.2 ml / min fractions: collected manually Absorption: 214 nm

Fig. 8: Capillary zone electrophoresis of the native, purified LPAP from the preparation of Fig. 6. Five μl of fraction 32 were lyophilized in a microvial, then dissolved in water with 5 μl of 0.1% TFA and used directly for CE measurement , This result shows that in purification stage 5 , a high-purity LPAP was already present in this fraction.

Device: Beckmann P / ACE System 2000 Kappilare: Uncoated fused silica 500 mm × 75 μm ID) Buffer: 100 mM sodium phosphate pH 2.5 0.02% hydroxypropylmethylcellulose Temperature: 25 ° C Injection: 20 sec corresponds to 120 nl Running time: 60 minutes Absorption: 214 nm

Fig. 9: Amino acids analysis of the purified LPAP fraction from the final purification after chromatography in Fig. 7: 5 μl of the LPAP fraction were lyophilized directly in a hydrolysis vessel and hydrolyzed for 60 minutes at 150 ° C. with 6N HCl in gas phase under vacuum. The sample was then lyophilized and taken up in 15 μl of 0.4 M sodium borate, pH 10.5. The analysis was done by OPA labeling for the primary amino acids and FMOC for the secondary amino acids. The separation and fluorescence detection was performed with a Hewlett Packard HP1090II AminoQuant according to the manufacturer's instructions. The data for the individual amino acids gave the theoretically expected values in terms of measurement accuracy, which correspond to the structure derived from MS and sequence analysis.

Fig. 10: Sequence analysis: After applying 1/4 of the material from the final purified LPAP after chromatography in Fig. 7, approximately 250 pM, the residues from position 1 to 52 could be clearly detected without detectable secondary sequence.

The type of sequencing is described in detail in the text. The others Sequence shares were analyzed by degradation and fragment sequencing as well Mass spectrometry and amino acid analysis were obtained (see following Illustrations):

Fig. 11: The enzymatic degradation and sequence analysis after application of Lys-C endoprotease digestion was carried out using 20 μl of the final purified fraction according to Fig. 7. To the dissolved aliquot was added 20 μl of H ₂ O, which was concentrated in vacuo for 20 minutes to evaporate off the acetonitrile and TFA. The residual volume of about 5 .mu.l was taken up in 100 .mu.l 0.1 M Tris-HCl buffer of pH 8.5 and treated for 3 hours at 37 ° C with 1 ul Lys-C endoprotease. The entire sample was directly chromatographed by microbore HPLC. Essential sequence information was obtained by analysis of peaks I to III.

Pillar: Vydac, steel casing 2.1 × 100 mm Material: Vydac RP C18 Column temperature: 35 ° C Eluent A: Water with 0.06% TFA Eluent B: 20% water (V: V) with 0.05% TFA with 80% acetonitrile Gradient: 0-0% eluent B 5 min 0-100% eluent B 100 min 100-100% eluent B 5 min 100-0% eluent B 5 min Flow rate: 0.2 ml / min fractions: collected manually Absorption: 206 nm

Fig. 12: In the cyanogen bromide cleavage, a chemical reaction takes place in which the methionine is converted into homoserine lactone and the peptide bond following the methionine opens. This results in relatively large partial fragments of the peptide, which can be individually sequenced after HPLC separation. For cleavage 50 pmol of LPAP were dissolved in 100 ul of 70% formic acid with 1% phenol, covered with protective gas and treated with 100 ug BrCN. This approach incubates for 24 hours at room temperature in the dark. Thereafter, the entire batch is lyophilized and separated by means of microbore HPLC.

Pillar: Vydac, steel casing 2.1 × 100 mm Material: Vydac RP C18 Column temperature: 35 ° C Eluent A: Water with 0.06% TFA   Eluent B: 20% water (V: V) with 0.05% TFA with 80% acetonitrile Gradient: 0-0% eluent B 5 min 0-100% eluent B 100 min 100-100% eluent B 5 min 100-0% eluent B 5 min Flow rate: 0.2 ml / min fractions: collected manually Absorption: 206 nm

Fig. 13: The mass spectrum of the native, purified LPAP from the highly purified preparation of Fig. 7 was measured on the SCIEX API III with variable ion source in positive ion mode (single MS mode, measuring range 1500- 2400 m / z) of 2 μl sample created. The injection was carried out at a flow rate of 1 μl / min via a microliter syringe and Harvard syringe pump. The found molecular weight of MW 15859 +/- 2.2 MU in the error range <0.1% agrees with the theoretically calculated mass of MW 15826.2 of the sequenced molecule after the mass values for two oxidized methionines have been reduced. Secondary substances are not visible. This mass determination is clearly confirmed by sequencing, namely that the peak III after Lys-C degradation contained a double sequence (see Fig. 12), which results in the detection of the dimeric attachment of two LPAP chains together with mass spectrometry.

Fig. 14: Time course of the proliferation rate of Nb 2 Node lymphocyte cells after stimulation with LPAP. One recognizes the increase of the growth compared to the control and depending on the addition of the LPAP or the LPAP-containing preparation from a final purified product according to FIG. 6, in which 1 μl corresponds to 6 pmol of LPAP.

example 1 Direct detection of the amino acid sequence of the circulating, biological active LPAP after isolation from hemofiltrate

The starting material used was hemofiltrate, which was available in large quantities in the treatment of renal failure patients and all Contains plasma components up to a molecular size of about 20,000 daltons.

I. Recovery of the crude peptide material

The hemofiltrate was analyzed by means of a hemofiltration system from Sartorius using polysulfone filters with an exclusion size of 20,000 Dalton (type SM 40042, Sartorius, Göttingen, FRG) won. The filtrate came from kidney-insulin-dependent patients undergoing long-term hemofiltration in a stable metabolism. For the present investigation Initially, several approaches were used to pre-assay hemofiltrate up to 2000 l performed to optimize the individual isolation steps and on the To adapt the question of LPAP cleaning. With the stated, optimized Technique sufficed 720 l Hemofiltrat, which immediately after the extraction using Acidification and cooling to 4 ° C protected against proteolytic degradation were. Subsequently, by alginic acid extraction according to the method of Forssmann DE 36 33 797 A1 a Rohpeptidfraktion (about 46.5 g) won.

II. Isolation of the fractions in purification step 1 with proliferative action on NB2 cells 1. Desalting and Fractionation of the Raw Peptide Material Via RP-C4 Solid Phase (Purification Stage 1 )

The crude peptide fraction from the alginic acid extraction of the hemofiltrate was separated by HPLC chromatography on a steel mantle column filled with RP-C4 material (Biotek ProRP, Heidelberg, Germany), which had been equilibrated with 0.01 M hydrochloric acid ( FIG. 2). For this purpose, a preparative HPLC system from Cedi (Lannemezan, France) equipped with a Jasco photometer (Chemodata / for Japan) and a Shimadzu integrator (Shimadzu, Kyoto, Japan) was used. Overall, the optimized cleaning was carried out with material of 12 runs of 3.8 g of applied Hemofiltratrohextrakt (corresponding to 600 l hemofiltrate).

The proliferative activity in pools IX and X was measured on Nb 2 Node cells grown in Fischer medium (Gibco, Life Technologies Ltd Paisley, Scotland) by increasing the cell by means of an automatic cell counter (Casy 1 Analyzer System, Schärfe, Reutlingen , FRG) after 24, 48 and 96 hours and compared with controls in which instead of the hemofiltrate peptides, human prolactin (NIH, USA), fetal calf serum (FCS, Gibco, Life Technologies Ltd. Palsley, Scotland) was added to the basal medium. was given or no addition in the base medium was present (see Fig. 1). Pool IX and X of this 1st purification step contained the total bioactivity measurable in the proliferation assay, while no activity was seen in the other fractions.

Pool IX and X (hatched in Fig. 2) were used for the further isolation, since the highest concentration of the sought substance occurred here due to these measurements.

2. Preparative Large-Scale RP-Chromatography II of Pool IX and X (Purification Stage 2 )

A further preparative purification of the LPAP-containing peptide fractions was carried out at room temperature via a steel jacket column filled with Parcosil RP-C4 (Biotek ProRP, Heidelberg, Germany) by means of an HPLC system (the system consists of two Kontron pumps 420, a Kontron detector 430, a Kontron Daten System 450 and a Pharmacia / LKB SuperFrac fraction collector), whereby the proliferatively effective peptide fractions could be concentrated again according to the method shown in FIG . The material of pools IX and X was applied as a spun liquid and then lyophilized material after resuspension in eluent A (see FIG. 3), in each case 50 mg per run. A total of 5 runs of this type were carried out, identical chromatograms were obtained. The fractions shown in Fig. 3 (hatched) contained the highest bioactivity with respect to the proliferation assay on Nb 2 Node cells and were pooled for further separation.

3. Semipreparative Reversed-Phase Chromatography of the LPAP-containing Pool from Preparative RP-C18 Chromatography II (Purification Step 3 )

The bioactive LPAP-containing pool (about 260 ml) was concentrated by evaporation in a vacuum to about 50% of the initial volume to remove especially the organic solvent components. Semipreparative reverse-phase (RP) chromatography was further purified as detailed in FIG. 4. The biological activity appears in the range of a larger peak at a retention time of 51 and 63 min and over 62% of the eluent B. These fractions from 4 identical runs were pooled and lyophilized and prepared for the next purification steps.

4. Analytical HPLC gel chromatography of the LPAP-containing pool from the semi-preparative RP-C18 chromatography (purification step 4 )

The work-up of the main peak fraction, which showed the proliferating, bioactive effect, was separated by means of analytical HPLC gel chromatography according to molecular weight into further subcomponents, whereby an LPAP activity under the conditions according to Fig. 5 after a retention time of 38-50 min after the injection was detected within a new, clearly visible peak (hatched in Fig. 5). These highly active fractions from four chromatographic runs were pooled again and further processed directly.

5. Analytical RP-chromatography I of the LPAP-bioactive pool from analytical HPLC gel chromatography (purification step 5 )

The entire substance of the four runs from purification step 4 was left in solution and chromatographed on a Vydac RP C 18 microbore column (MZ-Analysentechnik, Mainz, FRG) on the HPLC system (Kontron, see above) in 4 similar runs, as shown in Fig. 6 in detail. The lymphocytoma cells (Nb 2 Node) activating peptide was detected in a peak eluting substantially within 4 minutes, namely, in fractions 31 to 33 chromatography shown in Fig. 6. Depending in the ascending and descending limb of this peak, the As a result, the amino acid sequence of the LPAP could be determined properly with a small amount of the material applied to the Pulse-Liquid sequencer (473 A, Applied Biosystems, Weiterstadt, FRG). All other bioactive fraction material was finally rechromatographed on the same column (see purification step 6 ) and was available for further analysis.

6. Analytical RP-chromatography II of the LPAP bioactive fractions Pools from the analytical RP-chromatography I (purification step 6 , final cleaning)

The entire substance of each of four runs from purification step 5 was left in solution, concentrated by a brief, 5 minute vacuum evaporation to remove predominantly the organic solvents. Then, in each case one run of the RP chromatography from purification stage 5 was rechromatographed on a Vydac RP C 18 microbore column (see purification step 5 ) ( FIG. 7). The Lymphocytoma cell (Nb 2 Node) activating peptide (LPAP) was now detected in a peak collected in 4 sub-fractions by hand. The degree of purity of the isolated substance could already be determined to be <98% using a diode array system (Kontron, FRG) during the final isolation chromatogram. Each fraction was tested in the bioassay (see Fig. 1), and partially sequenced (see Fig. 10), tested for amino acid content (see Fig. 9) in the mass spectrometer analyzed (see Fig. 13), and further by means of capillary zone electrophoresis (CE) for purity tested (see Fig. 8). Furthermore, the overall structure of the amino acid sequence was elucidated by the sequencing of single fragments after Lys-C endoprotease degradation and cyanogen bromide cleavage ( Figures 10, 11, and 12).

7. Sequence analytical determination of the primary structure of the LPAP

From the peak of the first run of purification step 6 , 40 μl out of the total of 200 microl was used for amino acid sequence analysis. After isolation from hemofiltrate as described above, it was sequenced using the Pulse-Liquid Sequenator Type 473 A (Applied Biosystems, Weiterstadt, FRG). EDMAN degradation (Edman and Begg, Eur. J. Biochem 1, 80-91, 1967) was carried out using the standard program NORM. Analysis of the derivatized amino acids was performed on-line using an integrated chromatograph using the standard ABI protocol.

For this highly pure substance, a uniform sequence of 52 amino acids could be determined; further 3 amino acid residues could only be determined with reservations ( Fig. 10). Further elucidation was achieved by fragment analysis ( Figures 11 and 12) and mass spectrometry ( Figure 13). The sequence found is known to have homology with human 10 kD protein and human uteroglobin (Singh et al., Biochem Biophys Acta 950, 329-337, 1988). This first substance is said to originate from Clara cell airway secretions and was determined by molecular biology via the cDNA method; no function is known for this postulated peptide. The significance of the low sequence homology of 60% with human uteroglobin (Lipman and Pearson, Science 227 1435-1441, 1985) is completely unclear. It has not been determined in previous work that the presently surprisingly found substance is derived from human blood and also surprisingly represents a homologous dimeric antiparallel peptide.

Furthermore, from the end-stage purification product of step 6, all sub-fractions of the hand-collected peak were then sequenced to prove the identity of the LPAP in these sub-fractions. Because of the lower amount of these subfractions used for sequencing only 10 to 20 positions could be determined by sequence analysis. However, it was clear from this determination that the sub-fractions clearly gave only one main sequence, the LPAP.

The LPAP could thus be identified several times flawlessly in each sub-fraction and from the direct sequence analysis showed that the peptide had to have at least a sequence length of 50 amino acids.

8. Amino acid analysis for the determination of the peptide content

From the end-stage purification product of step 6 , an amount of 5 μl of an amino acid analysis was added to all sub-fractions of the hand-collected peak. This was intended to further prove the identity of the LPAP in these sub-fractions ( Figure 9).

The amino acid analysis (ASA) was carried out with the high-purity LPAP fraction from the final purification after chromatography in FIG. 6 by lyophilizing 5 μl of this LPAP fraction directly in a hydrolysis vessel. The hydrolysis was carried out for 60 minutes at 150 ° C with 6N HCl in gas phase under vacuum. The sample was then lyophilized and taken up in 15 μl of 0.4 M sodium borate, pH 10.5. The analysis of the amino acids was made after OPA labeling for the primary amino acids and FMOC for the secondary amino acids. The separation and fluorescence detection was performed with a Hewlett Packard HP 1090II AminoQuant according to the manufacturer's instructions. The data for the individual amino acids gave the values within the accuracy of measurement, the data obtained with the values from mass spectrometry and sequence analysis, theoretically calculated, match. Histidine could not be detected in the sample. The amino acid analysis ( Figure 9) shows the following amino acid residues:

is further proven that the subfractions comparable amounts of LPAP contained, which were to be expected according to Peakaufentrennung. Thus, due to the later proved overall structure of the LPAP on the sequencing of the Endoprotease fragments as well as the mass determination by means of Mass spectrometry subsequently the degree of contamination by not yet separated peptides  be calculated. The extremely low level of contamination of the native LPAP could thus be found in each sub-fraction as <1%, from which showed that the peptide was present in highly pure form and the biological Effect according to the current state of knowledge not on an impurity was to refer.

9. Detection of the molecular weight of the native LPAP by Mass spectrometry

From the end product of Step 6 purification, all sub-fractions of the hand collected peaks were further analyzed in mass spectrometers. The mass spectra of these native, purified LPAP fractions from the highly purified preparation of Figure 7 were recovered on the SCIEX API III variable ion source positive ion mode instrument (single MS mode, range of 1500-2400 m / z) of 2 μl samples each , The samples were injected at 1 μl / min flow rate via a microliter syringe and Harvard syringe pump. The found molecular weight of MW 15859 + - 2.2 MU was consistent with the theoretically calculated mass of MW 15826.2 of the sequenced molecule for all samples in the error range <0.1% after the mass values for two oxidized methionines were reduced. Secondary substances were not found in any fraction. It was thus determined by means of this determination that all subfractions contain LPAP with a uniform molecular weight ( FIG. 13).

10. Detection of the purity of the isolated, native LPAP capillary

Furthermore, also from the end product of the purification according to step 7, all subfractions of the peak collected by hand were tested for their purity by means of capillary zone electrophoresis. This method is suitable for detecting impurities down to the lower picogram range and also results in high-resolution reproducibility in the representation of a particular peptide. By capillary zone electrophoresis, it is confirmed that the final fraction subfractions contained only one peak relating to LPAP, which eluted under the given conditions at 15.1 min ( Figure 8). No further by-products could be identified.

11. Detection of the C-terminal amino acid sequence of the isolated, native LPAP by endoprotease degradation (endoprotease-Lys-C) and cyanogen bromide Cleavage and sequencing of subfragments Endoprotease Lys-C degradation

The enzymatic degradation and subsequent sequence analysis of the partial fragments after application of Lys-C endoprotease digestion was carried out using 20 μl of the final purified fraction according to FIG . The dissolved aliquot of the highly purified fraction was taken up in 20 μl H ₂ O, then concentrated in a vacuum centrifuge for 20 min to remove the acetonitrile and trifluoroacetic acid (TFA). The residual volume of approximately 5 .mu.l was taken up in 100 .mu.l 0.1 M Tris-HCl buffer at a pH of 8.5 and treated for 3 hours at 37 ° C with 1 ug Lys-C endoprotease (Boehringer, Mannheim) , The entire sample was directly chromatographed by microbore HPLC. This sequence information gave us the entire primary structure of the LPAP. Since a molecular weight of 15826.2 daltons was found due to the mass spectrometry, it had to be concluded about 130 to 150 amino acids. In the elucidation of the C-terminal sequence via production of partial fragments, the antiparallel, dimeric structure in peak III was clearly recognized.

The final protease degradation by Lys-C-end protease thus gave after separation several distinct peaks, as shown in Fig. 11 chromatography (for details see Figure legend to Fig. 11) can be seen. The peaks listed below were analyzed as described above in FIG. 11 and the following partial sequences were obtained:

Cyanogen bromide cleavage

Cyanogen bromide cleavage involves a chemical reaction in which the methionine is converted to homoserine lactone and the peptide bond following the methionine opens. This results in relatively large partial fragments of the peptide, which can be individually sequenced after HPLC separation ( Figure 12). For cleavage 50 pmol of LPAP were dissolved in 100 ul of 70% formic acid with 1% phenol, covered with protective gas and treated with 100 ug BrCN. This approach incubates for 24 hours at room temperature in the dark. Thereafter, the entire batch is lyophilized and separated by means of microbore HPLC. Essential sequence information was obtained by the analysis as shown in Fig. 12 chromatography (for details see Figure legend to Fig. 12). All peaks were analyzed as described above for total sequencing and the following partial sequences were obtained:

12. Summary of Structural Data on the Isolated, Native LPAP: Derivation of the structure as a homologous dimeric, antiparallel peptide

From the sequencing and the partial sequences of the cleavage products, the primary structure of the LPAP, which belongs to a molecular weight of 7913.1 daltons, could be perfectly derived in overlapping and the primary structure of the LPAP thus appeared to be elucidated. With the retention time in analytical HPLC gel chromatography, however, surprisingly, there was already an indication that LPAP had to have a molecular weight of 15,000 to 20,000 daltons (reference substances for chromatography in FIG. 5). This was confirmed by mass spectrometry that a dimeric peptide had to be deduced. The sequencing of the degradation products by the endoprotease-Lys-C cleavage also surprisingly revealed a partial fragment with a double sequence from the one hand position 1 to position 43 and on the other hand from position 63 to 70 with the same molarity. This could only conclude that it is a dimer in which each of the Cys residues in position 3 and 69 are connected via a disulfide bridge. Furthermore, the studies of LPAP by denaturing gel electrophoresis showed the same molecular weight. Even in highly reducing conditions half of the molecular weight was difficult to detect in mass spectrometry because the molecule has exceptionally high stability. The structure of LPAP as a homologous dimeric, antiparallel peptide of a total of 140 amino acids so surprisingly as follows:

and the associated, derived homolog-dimeric antiparallel peptide as a scheme:

Example 2 Chemical Synthesis of Human Lymphocytoma Cell Proliferation Activating Peptides (LPAP) 1. Synthesis of the Kieselguhr Reinforced Fmoc-Leu Resin

The Fmoc amino acid anhydride (5 equivalents) was in a minimal volume Dissolved N, N-dimethylformamide (DMF) and in a round bottomed flask to the 4- Added hydroxymethylphenoxyacetyl-norleucine resin. 4 Dimethylaminopyridine (1 equivalent) was also in a minimum volume DMF dissolved and added to the round bottom flask. After 1 hour of reaction time was The excess of the reagents is removed by filtration and the resin on a Filter thoroughly with DMF, 2-propanol and dichloromethane.  

2. Strategy of Synthesis of Human LPAP

For the synthesis of the peptide having the formula:

the flow-through method (Atherton and Sheppard, Solid phase peptide synthesis. IRL Press, Oxford, 1989). The named peptide sequence was using an automatic peptide synthesis apparatus (PepSynthesizer 9050, Millipore) using the Fmoc amino acids. The following Fmoc amino acids were used in excess (4 equivalents) (in each case the derivatives of the L-amino acids were used):

Fmoc-Ala
Fmoc-Asp (OtBu)
Fmoc-Met
Fmoc-Glu (OtBu)
Fmoc-His (Trt)
Fmoc-Leu
Fmoc-Arg (Pmc)
Fmoc-Thr (tBu)
Fmoc-Lys (Boc)
Fmoc-Ile
Fmoc-Phe
Fmoc-Gly
Fmoc-Asn (Trt)
Fmoc-Gln (Trt)
Fmoc-Val
Fmoc-Ser (t Bu)
Fmoc-Cys (Trt) at position 3, Fmoc-Cys (Acm) at position 69
Fmoc-Tyr (tBu)
Fmoc-Pro

The synthesis was performed by in situ activation with TBTU [O- (1H-benzotriazole-1) yl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate].

The peptide was removed from the carrier resin by adding a mixture of Trifluoroacetic acid-ethanedithiol-water 95: 2, 5: 2, 5 (v / v / v /) and cleaved with Ether like. The HPLC-purified peptide was analyzed with the aid of Amino acid analysis, analytical HPLC, amino acid sequence analysis and Characterized mass spectrometry.

3. Practical implementation of the synthesis of the human LPAP

The synthesis of

was performed with the automatic 9050 PepSynthesizer (version 1.5), Millipore, using the continuous-flow method. Fmoc amino acids were L-configured and used in 0.56 mmol portions. All reagents necessary for the synthesis were obtained from Millipore: N, N-dimethylformamide, 20% piperidine in N, N-dimethylformamide and 1-hydroxybenzotriazole. The L-amino acid derivatives were used in quadruplicate. The terminal amino groups were Fmoc-protected. Asparagine and glutamic acid were used as N ^W -tert-butyl ester, tyrosine, serine and threonine as tert-butyl ether, glutamine and asparagine as N ^W -Trt compounds, lysine as N ^e -tert-butyloxycarbonyl (Boc) derivative and arginine as N ^G -2,2,5,7,8-pentamethylchroman-6-sulfonyl (Pmc) - derivative used. The cysteine residues of a chain were introduced as trityl (Trt) and acetarnidomethyl (Acm) derivative. The synthesis was carried out starting from the kieselguhr resin Fmoc-Leu-PepSyn KA (Millipore), ie with a supported C-terminal amino acid (0.091 meq Leu / g, 1.60 g). All couplings were made by activation with 1 equivalent of O- (1H-benzotriazol-1-yl) -N, N, N ', N'-tetramethyluronium tetrafluoroborate (TBTU), 1-hydroxybenzotriazole (based on the amino acids). Performed using diisopropylethylamine as the auxiliary base. The following synthetic cycle was used: Fmoc cleavage with 20% piperidine in DMF (15 min), washing with DMF (12 min), acylation (30 min), and washing with DMF (10 min). The progressive synthesis was followed by continuous UV detection. The synthesis was completed with the cleavage of the N-terminal Fmoc residue. The resin-bound peptide was washed three times with 50 ml of 2-propanol, glacial acetic acid, 2-propanol and diethyl ether and dried under high vacuum.

The cleavage from the resin was carried out with trifluoroacetic acid / ethanedithiol / water 95: 2.5: 2.5 (v / v / v, 30 ml) for 90 minutes. The solution is in vacuo on a Concentrated volume of 5 ml and the product by adding cold Diethyl ether like. The resulting crude peptide, which is an α-acetamidomethyl contains protected cysteine (CysAcm), several times with diethyl ether washed and dried. From 1000 mg of peptide-resin so 250 mg Obtained crude peptide.

The formation of the antiparallel dimer was by oxidation with iron (III) initially linked two monomeric chains via a disulide bridge. For this The crude peptide was dissolved in a concentration of 1-10 mg in 1 ml of water and oxidized with potassium hexacyanoferrate (III) for 1-10 hours. To separate the  Salts, the solution was purified by reversed-phase chromatography and the product-containing fractions lyophilized. The material thus obtained was washed with a Concentration of 0.2-1.0 mg / ml dissolved in water at pH 8.0. The cleavage of the acetamidomethyl (Acm) groups and cyclization to antiparallel-oriented LPAP were made by oxidation with iodine. For this was titrated with a solution of iodine in glacial acetic acid until light yellowing. The Excess iodine was destroyed by the addition of sodium thiosulfate solution. The The resulting solution was diluted with water and directly by chromatography cleaned.

Purification was performed using an independently synthesized standard of LPAP whose correct sequence was confirmed by MS (mass spectrometry) and sequence analysis. In the first step, the deblocked crude was purified by cation exchange HPLC (Parcosil PepKat 2 x 10 cm, 300 Å, 7 μ, flow rate 9 ml / min, 230 nm, eluant A = 5 mM NaH ₂ PO ₄ , pH = 2.5, B = 5 mM NaH ₂ PO ₄ + 1 M NaCl, pH = 2.5, gradient: 5% → 100% in 60 min), with the main peak corresponding to the reference substance appearing at 28.17 min. When using capillary zone electrophoresis and Vydac Microbore Column RP-HPLC, the peaks appear according to the references (see Figures 7 and 8). In the second purification step with simultaneous RP-HPLC for desalination (Parcosil ProRP C4 2 × 10 cm, 300 Å, 7 μ, flow rate 7 ml / min, 230 nm, eluent A = 0.1% trifluoroacetic acid in water, B = 0.1 % Trifluoroacetic acid in acetonitrile / water 4: 1, gradient: 0% → 100% B in 60 min). Yield: 6.6 mg (1.5 mmol, 14.8%) gave a peak at 30.80 min which gave a sharp uniform peak in analytical RP-HPLC with C18 material.

The identity of the synthesized material with the given primary structure of LPAP was determined by MS (plasma desorption method, Bio-Ion 20, Applied Biosystems) and countersequencing in a gas phase sequenator (Model 470 A, Applied Biosystems). The biological activity of the Synthetic LPAP was in the functional test by the proliferation-stimulating Effect on Nb 2 Node Lymphocytoma cells detected: this showed that the synthetic material has a correct biological activity.  

Example 3 Proof of the proliferating effect of the biologically active, circulating human LPAP on Nb 2 node lymphocytoma cells

As described in Example 1, hemofiltrate was worked up by chromatography, with LPAP-containing fractions found in all purification steps. The active fractions found in Example 2 according to Example 1 in purification stage 2 and 6 (final cleaning) were used as substance for the test ( FIG. 14). In this way, a fraction could be detected from the hemofiltrate in both purification stages, which showed the biological, growth-promoting effect of LPAP.

The Nb 2 Node cells were seeded and used in 24-well plates after a loose cell suspension had formed (about 4-8 days). The incubation was carried out with 1 ml of cell medium at room temperature. The LPAP peptide-containing preparations were diluted with cell medium immediately before the start of the experiment. In the first approach, 1.5 x 10 ⁵ cells were preincubated for 4 days for conditioning and then treated with 6 x 10 ^-8 to 6 x 10 ^-9 M LPAP, and after 24, 48 and 96 hrs the proliferation rate was measured against controls , For control, cells were incubated with Fischer medium (1.) only with 10% horse serum and (2.) with equine serum plus 100 μl fetal calf serum per well (1 ml). The LPAP addition was carried out with 1, 5 and 10 .mu.l, corresponding to about <6, 30 and 60 pM / ml from the final purified product according to Fig. 7. The obtained values of the proliferation of Nb 2 Node cells are highly significant as averages of multiple approaches, that they can not be entered into the growth curve ( Fig. 14). The addition of LPAP or the composition of the medium was carried out at the beginning of the incubation and during the test period the samples remained in the incubator at 37 ° C.

The results show that there are significant differences in the efficacy of the LPAP compared to controls with the Nb 2 Node cell system under study. This result suggests differences in the mode of action of these peptide mixtures, which relate to the activation mechanism of the LPAP. In particular, it should be emphasized that in this case a long-acting mechanism must exist by LPAP, since the cell growth is stimulated up to and significantly after 96 hours, preferably. Compared to the other media, the proliferation by administering LPAP does not stand still after this time and continues evenly ( Fig. 14). This demonstrates that LPAP must affect the proliferation of Nb 2 Node cells via a mechanism that is probably unknown.

Similarly, it was found that other cell types on LPAP respond that kept in culture long term using the LPAP can be without the use of fetal calf serum necessary would.

Claims (15)

1. LDAP with the primary structure as well as the homolog-dimeric, antiparallel peptide derived therefrom: and its natural, synthetic and pharmacologically acceptable derivatives and analogs, in particular amidated, acetylated, phosphorylated and glycosylated LPAP derivatives. 2. A process for the preparation of the LPAP or its derivatives according to claim 1 characterized in that this via a prokaryotic or a eukaryotic expression produced and purified by chromatography becomes. 3. Process for the preparation of the LPAP or its derivatives according to claim 1, characterized in that it is made of human blood via usual Chromatography method isolated in a known manner.   4. A process for the preparation of the LPAP or its derivatives according to claim 1, characterized in that the LPAP or its derivatives by the conventional methods of solid-phase and liquid-phase synthesis of the protected amino acids contained in the indicated sequence, produces, deblocks and it with the usual chromatographic procedures cleans. 5. A pharmaceutical composition containing the human, circulating LPAP according to claim 1 or prepared according to claims 2 to 4, the LPAP as an active ingredient in addition to customary auxiliaries and additives. 6. Use of the medicament according to claim 5 for the treatment of Diseases that are more normal in the disturbance of growth pathological cells (tumors, hypoplasias, etc.). 7. Use of the medicament according to claim 5 for the treatment of degenerative diseases of the human organism, in particular the lymphocytes (asthenic lymphocytes in the absence of defense) and other mesenchymal tissue (eg bone marrow). 8. Use of the medicament according to claim 5 for the treatment of Skin diseases in the healing phase, in particular of injuries of the Epidermis. 9. Use of the medicament according to claim 5 for the treatment of Circulatory diseases (cardiovascular system), lung diseases (respiratory system), and renal diseases and diseases at Reproductive system (urogenital system). 10. Use of the medicament according to claim 5 for the treatment of Diseases of the nervous system, especially those with degeneration or Growth disorders of nerve and glial cells are associated.   11. Use of the medicament according to claim 5 for the treatment of Dysfunction of the immune system, in particular at z. B. by Immunosuppressants caused inhibitions in the immune system involved cells. 12. Use of the medicament according to claim 5 for the treatment of Cancers, especially at z. B. by application against this formed antibodies or for use in adoptive cancer therapy. 13. Use of the medicament according to claim 5 for the treatment of acute diseases, according to claims 6 to 12, by being suitable Form used for treatment in the intensive care of these diseases becomes. 14. Use of the medicament according to claim 5 for the diagnosis of Diseases, in particular according to any one of claims 6 to 13, by producing specific antibodies against the synthetic peptide and the blood concentration of LPAP via immunoassays (RIA, ELISA) is measured. 15. Use of the medicament according to claim 5 in various galenic administration forms, in particular the lyophilized, with Mannitol ingested form in sterile vials for dissolution in physiological saline and / or infusion solutions for repeated single injection and / or continuous infusion in amounts of 300 Microgram to 30 milligrams of pure LPAP per therapy unit.