AU634955B2 - Peptides, a process for the preparation thereof, the use thereof for obtaining antibodies, and the use thereof for blocking pai 1 activity in human blood - Google Patents

Description

i' COMMONWEALTH OF AUSTRALIA PATENTS ACT 1952-69 COMPLETE SPECIFICATION

(ORIGINAL)

Class Application Number: Lodged: Complete Specification Lodged: Accepted: S, Published: Priority R* A SRelated Art: Form Int. Class Name of Applicant: BEHRINGWERKE AKTIENGESELLSCHAFT Adress of Applicant: D-3550 Marburg, Federal Republic of Germany Actual Inventor: Address for Service Address for Service: THOMAS STIEF and WERNER STUBER EDWD. WATERS SONS, 50 QUEEN STREET, MELBOURNE, AUSTRALIA, 3000.

Complete Specification for the invention entitled: PEPTIDES, A PROCESS FOR THE PREPARATION THEREOF, THE USE THEREOF FOR OBTAINING ANTIBODIES, AND THE USE THEREOF FOR BLOCKING PAI 1 ACTIVITY IN HUMAN BLOOD The following statement is a full description of this invention, including the best method of performing it known to US BEHRINGWERKE AKTIENGESELLSCHAFT 87/B 044 Ma 657 Dr. Ha/Sd/Bn Peptides, a process for the preparation thereof, the use thereof for obtaining antibodies, and the use thereof for blocking PAI 1 activity in human blood The invention relates to peptides which contain the amino acid sequence of the active center of the endothelial plasminogen activator inhibitor (PAI to a process for the preparation thereof, to antibodies which are raised by immunization of animals with the former peptides, and to the use of these antibodies for blocking PAI 1 in human blood.

It was recently disclosed that the activity of the two 15 human plasminogen activators of greatest physiological importance, t-PA and u-PA, is controlled via two specific inhibitors in the blood. These are, on the one hand, endothelial plasminogen activator inhibitor, called PAI 1, which is formed and released, inter alia, by endothelial S 20 cells and platelets, and, on the other hand, placental plasminogen activator inhibitor, called PAI 2, from the trophoblastic cells of the placenta. The two inhibitors g resemble one another in their high association constants S with plasminogen activators, but are not immunologically 25 related.

S These inhibitors appear to be of great clinical significance because, in particular, greatly increased concentrations of PAI 1 may lead to life-threatening complications. Associations of this type have already been described in patients with deep leg vein thrombosis, acute myocardial infarct, bacteremia, liver disease, pancreatitis or cancer, as well as late in pregnancy and in patients with toxemia of pregnancy.

A substance which is able specifically to block the activity of this inhibitor is of diagnostic and therapeutic interest.

2 It has been found, surprisingly, that PAI 1 activity can be blocked with antibodies obtained by using as immunogen, for example, a peptide of the sequence Ser-Thr-Ala-Val- Ile-Val-Ser--Aa-Arg-Met-Ala-Pro-Glu-Glu-Ile-Ile, which is optionally coupled to a carrier molecule.

Hence the invention relates to a peptide containing from 4 to 40 amino acids whose sequence coincides with the amino acid sequence of the peptide Ser-Thr-Ala-Val-Ile- Val-Ser-Ala-Arg-Met-ALa-Pro-Glu-Glu-Ile-ILe or with parts of this sequence.

The sequence -Arg-Met- appears to be of particular signi- ,i ficance, so that preferred peptides contain this sequence.

Preferred peptides have 6 to 25 amino acids with the Arg- Met sequence being flanked by at least 2 amino acids on each side.

S.

Particularly preferred peptides are Ser-Thr-Ala-Val-Ile- Val-Ser-Ala-Arg-Met-ALa-Pro-GLuuG-Ile-Ile or Cys-Ser- SThr-Ala-Val-ILe-Val-Ser-a-Aa-Arg-Met-Ala-Pro-Glu-Glu-le- 0. ILe.

25 Peptides of this type can be used to obtain antibodies suitable within the scope of the invention.

*o Antibodies of this type can be purified by immunoadsorption using the corresponding peptides. The,- antibodies specifically block the activity of the PAI 1 present in human blood.

The peptides according to the invention are prepared by processes known per se, for example by the classical technique in solution, where protected amino acids or peptide segments are condensed together and, after elimination of the protective groups, the desired peptide is obtained. However, the peptides according to the invention are particularly preferably prepared by the 3 solid-phase method, so that the invention Likewise relates to a process for the preparation of these peptides.

In the solid-phase peptide synthesis the peptides are built up on a matrix such as crosslinked polystyrene, polyacrylamide or the Like, which are optionally provided with anchor molecules, and are eliminated therefrom.

Preferably used for these syntheses was an insoluble polystyrene matrix crosslinked with divinylbenzene) Loaded with the C-terminal amino acid to the level of 0.1 1 mmol/g, preferably 0.4 0.6 mmol/g of resin. Since the peptides were built up using the h~a-tLabile Fmoc group, p-alkoxybenzyl ester compounds according to the state of the art were used as anchor molecule. Preferably used as solvents in the synthesis were dichloro- 15 methane, N-methylpyrrolidone and, very particularly preferably, dimethylformamide. Normally used in every washing or reaction step were 10 25 ml/g of resin of solution or solvent, with 15 ml being particularly preferred. Since the alpha-NH2 groups of the amino acids were protected with 20 the Fmov group, the following protective groups were chosen for the side groups of the trifunctional amino acids: serine, threonine tert.-butyl ether S 25 glutaminic acid tert.-butyL ester arginine 4-methoxy-2,3,6-trimethylphenylsulfonyl cysteine tert.-butylmercapto.

S

The amino acids were activated and coupled, for example, via active esters or iixed or symmetric anhydrides, but with in situ activation being carried out with HOBt/carbodiimide, particularly preferably N,N'-diisopropylcarbodiimide.

The alpha-NH2 protective groups were eliminated with a base, with 20% piperidine in DMF at room temperature being particularly preferably used. After each of these reaction steps, the resin was washed, preferably with DMF and 4 isopropyl alcohol.

The peptides were preferably eliminated from the support by acidolysis with simultaneous removal of the protective groups on the side groups. The sulfhydryl group of cysteine was Liberated by thiols, such as dithiothreitol, or, preferably, by butylphosphine. The peptides according to the invention were purified by methods known per se, for example by gel permeation on agarose gels, but preferably by high performance liquid chromatography.

The synthetic peptides were examined for their chemical composition and purity. The composition of the peptides was determined by amino acid analysis, For this purpose, 15 a small sample was heated with 6 N hydrochloric acid in the presence of phenol at 110 0 C for 24 hours or 72 hours.

S* It was demonstrated in this way that the amino acids had been incorporated in the expected region in the peptide chain and, likewise, it was possible in this way to determine the peptide content of the synthetic antigens to be equal to or greater than The purity of the peptides was determined by high performance liquid chromatography on C18 reverse phases. A 25 phosphate/acetonitrile gradient was used for this purpose.

It was found that the peptides had a purity of greater than To obtain antibodies, a peptide of this type is bonded, where appropriate via a cysteine residue, to a high-molecular weight carrier. A product of this type is used to immunize animals, and antibodies which can be used within the scope of the invention are obtained.

An example of a high-molecular weight carrier of this type is albumin or keyhole limpet hemocyanin, to which the peptide is coupled via a thioether bond. Other suitable coupling techniques are those in which the peptides are bonded to a carrier protein via amino groups, for example the glutaraldehyde method.

Using these products as immunization antigens, polyclonal or monoclonal antibodies can be produced.

These antibodies can be purified by affinity chromatography. Suitable affinity materials have proven to be those which either have as Ligand a peptide as described above or use protein A for this purpose.

The invention also reLates to antibodies of this type, and to the use thereof as pharmaceuticals.

Abbreviations 66 666 6 0@ 6 6 6* 6 .6 S 6 6*6f 66 6 6 66 6~ 6 6 6* 66 6 6

S.

6* 0 6 6 66 6**666 66 6 66..

666666 6 15 Cys Ser Thr ALa VaL 1L e A rg Met Pro G Lu 25 Fmoc

DMF

tBu Mtr StBu

TFA

EDTA

Nva

CHA

Lys

GMBS

The exampL L-Cys te ine L-Serime L-Th reon ine L-AI an ine L-VaLine L- IsoL euc ine L-Argimine L-Meth ionine L-ProL mne L-GLutamic acid 9-F LuorenyLmethy LoxycarbonyL DimethyLformamide tert ButyL 4-Met ho xy-2 6-t rimet hyLph eny IsuLf onyL tert.-ButyLmercapto TrifLuoroactic acid EthyLenediamine tetraacetic acid NorvaLime C y c10hex y Ia Ia nin e L ys in e y'-Maleimidobutyric acid N-hydroxysuccinimide ester es which foLLow explain the invention in detail.

Example 1 6 Example 1 a) Synthesis of Cys-Ser-Thr-ALa-Val-ILe-VaL-Ser-ALa-Arg- Met-Ala-Pro-Glu-GLu-Ile-Ile

S

The synthesis was carried out in a semiautomatic peptide synthesizer. 1 g of Fmoc-ILe-p-aLkoxybenzyL-ester-polystyrene (crosslinked with 1% divinylbenzene) was treated with 15 mg of 20% piperidine/DMF and washed with DMF and isopropanol. 1.65 mmol of Fmoc-Ile and 2.25 mmoL of HOBt were dissolved in 14 ml of DMF, and 1.1 ml of a 1 M diisopropylcarbodiimide solution were added. The mixture was shaken at room temperature for 1 hour and then washed with DMF and isopropano until all the excess reagents 15 and soluble byproducts had been removed. This process was continued until the N-terminal amino acid was reached, using the following amino acid derivatives: Fmoc- Glu(OtBu), Fmoc-Pro, Fmoc-Ala, Fmoc-ILe, Fmoc-Met, Fmoc- Arg(Mtr), Fmoc-Ser(tSu), Fmoc-Val, Fmoc-Thr(tBu), Boccys(SStBu).

.15 mL of trifluoroethanol, 200 VL of water, 30 p1 of Nmethylmorpholine and 100 IL of butyLphosphine were added to the protected peptide resin, which was shaken at room temperature for 12 hours. The resin was washed with 1% glacial acetic acid in methanol and dried under high vacuum. The resin was mixed with 18 ml of TFA, 1 mL of thioanisole, 500 p1 of dimercaptoethane and 500 mg of resorcino and stirred at 35 0 C for 3 hours. The eliminated peptide was removed by filtration and crystallized with 300 ml of diethyl ether. The crystallized product was purified by gel permeation in portions (100 mg) on a RSephadex G25 column (3 x 100 cm, 0.5% acetic acid). 328 mg of peptide were obtained after freeze drying.

b) Preparation of conjugate mg of keyhole limpet hemocyanin (KLH) were dissolved 7 in 0.05 mmol/L sodium phosphate buffer pH 8.0, and were stirred with 3 mg of GMBS for 1 hour. The protein was chromatographed on a Sephadex G50 column (2 x 30 cm) (0.1 mol/l sodium phosphate, 0.5 mmol/L EDTA, pH The activated protein was concentrated to 5 ml and incubated with 30 mg of the peptide for 1 hour. 35 mg of immunization antigen were obtained after dialysis and freeze-drying.

Example 2 Immunization of rabbits 5 rabbits were immunized with 1.5 mg of antigen per ani- 15 mal on each occasion for a period of 4 weeks. The peptide-KLH conjugate was administered subcutaneously and *intravenously. The animals were then exsanguinated, and the crude antisera were stabilized with preservative.

Yield per animal: 150 ml of antiserum.

Example 3 Preparation of immunoadsorbents S25 Crude antisera obtained as described in Example 2 were purified by affinity chromatography. For this purpose, 80 mg of the 16-peptide Ser-Thr-Ala-Val-Ile-Val-Ser-Ala- Arg-Met-Ala-Pro-Glu-Glu-Ile-Ile were coupled onto 25 nl of Sepharose, which had been activated with cyanogen bromide, by the process described by Axen et at. (Nature 214, 1302, 1967). The immunoadsorbent was then washed with phosphate-buffered saline (PBS, 150 mmol/l, pH 7.2) and acetic acid (500 mmol/l, pH 2.5) and equilibrated with 3 times the gel volume of PBS.

Exlimple 4 Obtaining antibodies 8 ml of crude antiserum were passed through the PBS-equilibrated Sepharose (from Example 3) at a flow rate of about 60 ml/h at 23 0 C. The column was then washed with three times this volume of PBS, 1 M NaCI and distilled water (pH The antibodies were eluted with water (pH The eluate was adjusted to pH 7.5 with solid Tris-HCl and was stored at -20 0 C. Yield: about mg of antibodies.

Example Testing of the antibodies obtained by immunoadsorption a) Blocking of the PAI 1 activity in a functional test 50 p1 of PAI-deficient plasma, prepared by preincubation 15 of 40 IU of urokinase/mL of plasma and subsequent immunoadsorption on anti-urokinase-antibody-Sepharose, and 50 pL of PAl-rich plasma (10 units/ml) were incubated with 50 pl of antibody solution in PBS (from Example 4) and 1:10, 1:100 and 1:1000 dilutions thereof, as well as only PBS as control, at 23 0 C for 15 min, and then a functional S@ test of the PAI activity was carried out. For this purpose, 1 IU of urokinase in Tris buffer (100 mmol/l Tris, 100 mmol/l NaCI, 1% polygeline, 0.1% Triton X 100) was added, the mixture was incubated at 23 0 C for 10 min, S 25 and 200 Al of 10 mmol/l chloramine T, 200 pl of 10 CTA-U/ ml plasminogen in Tris buffer containing 10 mmol/l tranexamic acid were added, and the mixture was incubated at 23 0 C for 10 min. 500 p1 of 0.6 pmol/l chromogenic plasmin substrate Nva-CHA-Lys-pNA (pNA para-nitroanilide) in 500 mmol/l NaCI started the detection reaction, which was stopped after 10 min at 23 0 C by 100 pl of 8.5 M acetic acid. There is a linear inverse proportional relation between the content of functional PAI in a sample and the measured change irn the extinction at 405 nm.

9 Result: PAI-deficient plasma PAI-rich plasma (mE) (mE) Added: PBS 800 350 Antibodies 1:1000 805 368 Antibodies 1:100 793 590 Antibodies 1:10 810 685 Undiluted 802 700 The antibody was able to block about 80% of the fast inhibitory capacity of the PAl-rich plasma.

b) Removal of PAI 1 activity from biological fluids by 15 immunoadsorption **pe 14 mg of antibodies were immobilized on 10 ml of Sepharose activated with cyanogen bromide. 5 ml of PAI-rich plasma

S.

S. were passed through the Sepharose at a flow rate of 10 ml/ h. A sample of this eluate was investigated for function- S. al PAI 1 activity, taking account of the dilution factor.

The plasma had lost about 70% of its PAI activity.

Example 6 a) Synthesis cf ALa-Val-Ie-Val-Ser-Ala-Arg-Met-ALa-Pro- Glu-Cys Starting from 1 g of Fmoc-Cys(SStBu)-p-alkoxybenzylester-polystyrene, the peptide was built up, eliminated and purified in analogy to ExampLe 1 Yield 245 mg.

b) Preparation of conjugate The conjugate was prepared as in Example 1 Yield 36 mg.

Rabbits were immunized, immunoadsorbent was prepared, and antibodies were obtained as in Examples 2 4. Testing as in Example 5 of the antibodies obtained by 10 immunoadsorption revealed antibodies blocking PAI activity, (about 80% of the PAl activity blocked) as far as a titer of 1:20.

0 0" 41 94

Claims (5)

1. A peptide of Formula I A-Ala-Val-Ile-Val-Ser-Ala-Arg-Met-Ala-Pro-Glu-Glu-B (I) wherein A is Ser-Thr-, Cys-Ser or and B is lie-lie, Cys or HO-.

2. The peptide of claim 1 bonded to a high molecular weight carrier.

3. A process for the preparation of the peptide of claim 1, which comprises I protected amino acid derivatives or peptide segments being coupled together in solution or on a solid phase, and the peptides of claim 1 being obtained by elimination of the protective groups, and in the case of a solid phase, by elimination from the support resin. S.

4. An antibody obtained using as antigen the peptide of claim 1 or 2.

5. The use of an antibody as claimed in claim 4 as a pharmaceutical or for diagnosis. o DATED this 5th day January 1993. BEHRINGWERKE AKTIENGESELLSCHAFT WATERMARK PATENT TRADEMARK ATTORNEYS THE ATRIUM 290 BURWOOD ROAD HAWTHORN VICTORIA 3122 AUSTRALIA Doc 27 AU269J.-88.WPC DBM/KJS/BAS