WO2009046742A1

WO2009046742A1 - Staphylokinase variant

Info

Publication number: WO2009046742A1
Application number: PCT/EP2007/008871
Authority: WO
Inventors: Désiré COLLEN
Original assignee: Thrombogenics N.V.
Priority date: 2007-10-09
Filing date: 2007-10-09
Publication date: 2009-04-16
Also published as: CN101848928A; TR201001630T1; IL204171A; CN101848928B

Abstract

The present invention relates to an optimized thrombolytically or fibrinolytically active staphylokinase variant exhibiting low T-cell immunogenicity and reduced clearance by circulating antibodies that can be expressed at high levels.

Description

STAPHYLOKINASE VARIANT

Field of the invention

Introduction

Administration of fibrinolytic agents in the early hours following onset of symptoms suggestive of myocardial ischemia preserves myocardial tissue, prevents secondary complications that result from myocardial infarction (congestive heart failure, arrhythmias, etc.), and most importantly - saves lives. At present, the two most commonly used fibrinolytic agents are streptokinase (SK) and recombinant tissue plasminogen activator (rtPA). Whereas SK has the advantage of substantially lower cost, it is less effective than rtPA (or mutants of rt-PA) in terms of arterial patency and death at 30 days (and this difference in mortality persists for at least two years) and is associated with hypotension, allergic reactions and neutralizing antibodies. Conversely, rtPA is not currently the drug of choice for all patients because of its considerable cost and the perceived "marginal" benefit in mortality (an absolute difference of 1% at 30 days).

Therefore, an alternative fibrinolytic agent, with efficacy superior to SK and equivalent to rtPA, would be of substantial importance. Initial data suggest that the novel fibrinolytic agent staphylokinase (Sak) may be such an agent. Its efficacy in inducing early coronary artery patency appears at least similar to that of rtPA, and its safety profile may be equal or better. Like other thrombolytic agents, Sak dissolves blood clots in a plasma milieu. However, unlike SK or rtPA, Sak administration does not result in fibrinogen degradation. Sak acts at the clot surface, and is more fibrin-specific than SK or rt-PA. Unlike rt-PA, Sak has been found not to induce procoagulant activity.

As Sak is an enzyme of bacterial origin, much effort has been invested in reducing the antigenicity of Sak by site-directed mutagenesis, without reducing the fibrinolytic potency. Despite these efforts, there is still an unmet need for a Sak variant combining the properties of reduced (T-cell) immunogenicity and reduced clearance (i.e., reduced antibody-binding) that moreover can be produced at levels sufficient to enable broad and wide use. Obviously, besides these characteristics the desired fibrinolytic potency should be maintained.

Summary of the Invention In a first aspect, the invention relates to an isolated fibrinolytically or thrombolytically active staphylokinase variant characterized in that the amino acid variations introduced in the variant are relative to a staphylokinase, more particularly relative to a wild-type staphylokinase selected from the group consisting of Sak42D, SakCΦc* and SakSTAR, and wherein said amino acid variations comprise one or more of the list of variations selected from E65D, K74Q, R77E, E80S, D82S, V1 12T, K130Y, and E134R. In a particular embodiment the variations comprise V112T and E134R. Most particularly, said amino acid variations consist of E65D, K74Q, R77E, E80S, D82S, V112T, K130Y, and E134R. In one embodiment, the staphylokinase variant of the invention consists of the amino acid sequence of SEQ ID NO:2. In a further embodiment, the additional amino acid variation S3C can be introduced, e.g., to enable modification of the staphylokinase molecule. Said modification can be, e.g., pegylation. Furthermore an additional N-terminal Methionine residue can be present in the staphylokinases of the invention introduced for, and/or due to, recombinant expression.

A next aspect of the invention relates to fibrinolytically or thrombolytically active proteins comprising a staphylokinase variant according to the invention.

A further aspect covers compositions comprising a staphylokinase variant according to the invention and/or comprising a fibrinolytically or thrombolytically active protein comprising a staphylokinsase variant according to the invention, and comprising at least one of a solvent, diluents or carrier.

Furthermore, any of the staphylokinase variants and/or proteins comprising them according to the invention can be used for the manufacture of a medicament for lysing blood clots in a subject. In an embodiment thereto, said blood clot can be associated with any thrombotic condition or with catheter occlusion. The invention additionally covers isolated nucleic acids and recombinant vectors comprising a nucleotide sequence encoding a staphylokinase variant according to the invention, optionally operably linked to regulatory nucleotide sequences required for expression of said staphylokinase variant.

Finally, the invention relates to methods for recombinant production of a staphylokinase variant according to the invention comprising the steps of:

(i) expressing a staphylokinase variant according to the invention in a suitable host; and

(ii) isolating the staphylokinase variant expressed in (i) from the host cells and/or from the host cell culture medium.

In order to produce a modified staphylokinase variant, the above production method can be extended with the steps of:

(iii) modifying the staphylokinase variant isolated in (ii).

In one embodiment thereto, said step of modifying said staphylokinase variant consists of pegylation of the staphylokinase molecule.

Legends to the Figures

FIGURE 1. Absorption of staphylokinase variants by human antisera.

Wild-type SakSTAR and several staphylokinase variants were analyzed using plasmon resonance (Biacore analysis) with two antisera-pools from 40 and 18 SakSTAR-immunized patients respectively. The Figure shows that the staphylokinase of the invention ("SEQ ID NO:1") as well as the further variant comprising the S3C amino acid change ("SEQ ID NO:1 S3C") are less well recognized by the antibody-pools compared to wild-type SakSTAR, SakSTAR in which the S3C variation was introduced ("SakSTAR S3C") and pegylated SakSTAR ("SakSTAR MP5"). Although not all of the alterations introduced in the staphylokinase of the invention are facing the surface of the molecule, they nevertheless result in the beneficial effect of lowering the recognition by anti-staphylokinase antibodies circulating in a subject.

FIGURE 2. In vivo pulmonary embolism lysis after staphylokinase treatment. Lysis of pulmonary embolism in hamsters was measured after treatment by staphylokinase infusion. The proteins were produced and purified concomitantly and the results were obtained in the same series of experiments. Panel A: comparison of staphylokinase variant of the invention (SEQ ID NO:1 ; triangles) and wild-type Sak42D staphylokinase (diamonds). Panel B: comparison of staphylokinase variant of the invention with the additional pegylated S3C variation (squares) and wild-type SakSTAR staphylokinase (circles). The results demonstrate that the staphylokinase variants of the invention are as efficient as wild-type staphylokinases in this in vivo model.

Detailed description of the invention

The invention is encompassed by the enabling description following hereafter, including experimental work and claims. The description and examples are not intended to limit the invention and it will be clear that common and general knowledge in the field of the invention but not included hereafter are likewise part of the description. Furthermore, any common and generally accepted terminology or methodology not explained hereafter is to be given its ordinary meaning/interpretation or explanation.

The work leading to the present invention is based in part on staphylokinase amino acid variations as previously disclosed in WO 01/40281 and as published by Warmerdam et al. (J. Immunol. 168, 155-161 (2002); Thromb. Haemost. 87, 666-673 (2002)). These documents highlight the importance of a number of staphylokinase domains (and of individual amino acids within each of these domains) in the elicitation of a T-cell response. Furthermore a number of amino acid variations and combinations of amino acid variations are suggested in order to eliminate much of staphylokinase' s T-cell reactivity. Additional work has now resulted in the identification of a thrombolytically/fibrinolytically active staphylokinase variant (see Examples describing in vitro and in vivo thrombolytic/fibrinolytic activity) not only displaying reduced T-cell antigenicity but further exhibiting the much desired advantages of

(i) reduced binding to antibodies circulating in a subject; and (ii) capability of high-level expression. This staphylokinase variant is given in SEQ ID NO: 1 and has the following amino acid variation relative to the wild-type SakSTAR staphylokinase (SEQ ID NO:2), itself initially disclosed by Collen et al. (Fibrinolysis 6, 203-213 (1992)): E65D, K74Q, R77E, E80S, D82S, Vl 12T, K130Y, and E134R. Other wild-type staphylokinases in which the same amino acid variations can be introduced are Sak42D (see DD245444) and SakCΦc* (see EP0077664). Optionally, for purposes of modification, such as pegylation, of the staphylokinase molecule of the invention an additional amino acid change (S3C) can be introduced. Pegylation can occur with any suitable type of polythelene glycol (PEG), e.g., PEG-5000 or PEG-20000, and can be performed as described by, e.g., Collen et al. (Circulation 102, 1766-1772 (2000)). Furthermore an additional N-terminal Methionine residue can be present in the staphylokinases of the invention introduced for, and due to, recombinant expression.

The reduced binding to (pre-existing) antibodies circulating in a subject is important in preventing rapid clearance and/or inactivation/neutralization of staphylokinase when administered to said subject. Or, in other words, this property of the staphylokinase variant subject of this invention can limit the amount of active compound that needs to be administered to a subject in need. Inherently this adds to the tolerability and safety of the product, e.g. by preventing an anaphylactic reaction (White, Br.Med.J. 302, 429-430 (1991)). This aspect of the staphylokinase variant of the invention is illustrated in Figure 1. The amino acid variation E65D cannot be derived from any of WO 01/40281 or Warmerdam et al. (J. Immunol. 168, 155-161 (2002); Thromb. Haemost. 87, 666-673 (2002)). In an initial staphylokinase variant (K74Q, R77E, E80S, D82S, V112T, K130Y, and E134R) this variation was not present but production of such variant was greatly hampered due to accumulation of the expression product as insoluble material as well as proteolytic degradation/truncation during fermentation and/or subsequent purification. Besides reducing yield and rendering the purification process much more difficult, said degradation included truncation of the amino-terminal serine (or, optionally, cysteine) at position 3, hence precluding pegylation of the staphylokinase variant of the invention additionally bearing the S3C mutation. This problem was solved by the change of a single amino acid, i.e., the change of the wild-type glutamate at position 65 in an aspartate (E65D).

The staphylokinase variants of the invention may be incorporated in larger molecules or can be modified (e.g., pegylated) as long as the original immunoreactivity and fibrinolytic or thrombolytic activity of the incorporated staphylokinase variant are conserved. Such larger molecules include, e.g., tagged versions of the staphylokinase variants. The purpose of any of the above changes can, e.g., be to slow down the turnover of the staphylokinase variant once administered to a subject in need. The staphylokinase variant (or modified version thereof) according to the invention will typically be administered to a subject in need in the form of a composition, e.g., a solution. Thereto, the drug of the invention can be formulated with at least one of a solvent, diluent or carrier. In particular, said solvents, diluents or carriers are pharmaceutically acceptable (as can be derived from, e.g., a pharmacopeia). The solvent, diluent or carrier can include any substance enabling efficient administration of the drug to the subject in need, e.g., salts, buffers, (poly)saccharides, (poly)alcohols, DMSO, etc.

The staphylokinase variant (or modified version thereof) according to the invention can further be used in the manufacture of a medicament for lysing blood clots in a subject.

A subject in need of thrombolytic/fibrinolytic therapy or treatment can suffer from any thrombotic condition such as (acute) myocardial infarction, coronary thrombosis, occlusive stroke, ischemic stroke, deep venous thrombosis, peripheral arterial disease, hepatic vein thrombosis, marasmic thrombosis, sinus thrombosis, venous thrombosis, arterial thrombosis and occlusion of an arterio-venal shunt. Alternatively the thrombotic condition is occurring during, e.g., infusion of any substance to a patient during which the catheter used for delivering the therapeutic agent(s) is occluded by a blood clot.

The staphylokinase variants of the invention can be administered to a subject in need as single or multiple dose(s) or bolus dose(s), by normal infusion, by accelerated infusion, or can be administered proximal to or directly within a thrombus. Combination of any of these administration routes is also an option.

Typically, in order to solubilize intravenous, intra-arterial or coronary blood clots and to restore blood vessel patency, staphylokinase variants of the invention can be administered in amounts of 1 μg to 1 mg/kg body weight, or an amount of 0,1 to 100 mg. This amount can be administered either in a single dose or divided over multiple, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 subsequent doses each interspersed by a suitable amount of time, e.g., 15, 30, or 45 seconds, or from 1 to 10 minutes. Alternatively, the drug is delivered by intravenous or intra-arterial therapy or infusion, or delivered via a catheter to a vascular or coronary thrombus, the time of delivery being dependent on the concentration of the drug in the fluid to be introduced in the vascular system. Typical duration of infusion with a staphylokinase variant of the invention ranges from 5 to 60 minutes. Alternatively an initial accelerated infusion of 1 to 10 minutes can be followed by normal infusion up to 60 minutes total infusion time. Independent of the route of administering the drug, the total dose administered can be in the range of 1 to 100 mg, of 1 to 90 mg, of 1 to 80 mg, of 1 to 70 mg, of 1 to 60 mg, of 1 to 50 mg, of 1 to 40 mg, of 1 to 30 mg, of 1 to 20 mg or of 1 to 10 mg of a staphylokinase variant of the invention. Typically, total doses can amount to 0.1, 0.5, 1, 5, 7.5 , 10, 12.5, 15, 17.5, 20, 22.5, 25, 27.5, 30, 32.5, 35, 37.5, 40, 42.5, 45, 47.5, 50, 52.5, 55, 57.5, 60, 62.5, 65, 67.5, 70, 72.5, 75, 77.5, 80, 82.5, 85, 87.5, 90, 92.5, 95, 97.5 or 100 mg. In case of catheter occlusion, such as central venous catheter occlusion (CVC), the total dose of an administered staphylokinase variant of the invention can amount to 0.1, 0.15, 0.3, 0.45, 0.5, 0.6, 0.75, 0.8, 0.9, 1, 1.25, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 4, 5, 6, 7, 7.5, 8, 9 or 10 mg and this total dose can be administered as a single dose or divided over 2, 3, 4 or 5 doses separated by, e.g., 15, 30, or 45 seconds, or by 1 to 10 minutes, e.g., by 2, 3, 4, 5, 6, 7, 8, 9 or 10 minutes. In the case of CVC, the staphylokinase of the invention is typically administered locally into the occluded catheter.

The invention further relates to any nucleic acid or recombinant vector (e.g. expression vector) comprising or consisting of a nucleotide sequence encoding a staphylokinase according to the invention. Optionally, said nucleotide sequence can be operably linked to regulatory nucleotide sequences, e.g., promoter and/or terminator sequences, enabling expression of the encoded staphylokinase. Any suitable combination of regulatory nucleotide sequence(s) and the nucleotide sequence encoding the staphylokinase of the invention is envisaged. Included in the regulatory nucleotide sequences is the methionine-coding ATG codon that can be placed at the N-terminus of the protein to be expressed. Finally, the invention relates to methods for recombinant production of a staphylokinase variant according to the invention comprising the steps of:

(i) expressing a staphylokinase variant according to the invention in a suitable host; and (ii) isolating the staphylokinase variant expressed in (i) from the host cells and/or from the host cell culture medium.

(iii) modifying the staphylokinase variant isolated in (ii).

Any suitable host or host cell and accompanying host cell culture medium is envisaged. Non- limiting exemplary host cells include: Escherichia coli, Saccharomyces species (e.g., cerevisiae), Schizosaccharamyces species (e.g., pombe), Hansenula species (e.g., polymorphά), insect cells (e.g. Spodoptera frugiperda) and mammalian cells (e.g., COS cells, CHO cells).

Examples

Staphylokinase variant production Molecular cloning, expression, (recombinant) production and purification of staphylokinase and its variants was performed essentially as extensively described in any of EP0525252 Al, WO 93/13209, WO 96/21016 and WO 99/40198. In vitro fibrinolytic/thrombolytic activity The specific activity of the staphylokinase of SEQ ID NO: 1 in a chromogenic thrombolytic assay was determined as 55 ± 15 kU/mg, this is significantly lower than the activity of wild- type SakSTAR (153 ± 33 kU/mg). The chromogenic assays was performed essentially as described in Example 2.5 of WO 99/40198 using S2403 as chromogenic substrate. In contrast thereto, the concentration of the staphylokinase of SEQ ID NO:1 required to induce 50% clot lysis in human plasma was similar to the required SakSTAR concentration (360 ± 30 ng/niL for SEQ ID NO:1 versus 343 ± 35 ng/mL for SakSTAR). The assay was performed basically as outlined in Amery et al. (Thromb. Diath. Haemorrh. 9, 175-188

(1963)), and as described in Experiment 3 of EP0525252 Al .

In vivo fibrinolytic/thrombolytic activity: hamster pulmonary embolism model

Experimental Animals: Outbred hamsters (245, Pfd, Gold, University of Leuven, Belgium) with body weights of 80 to 12O g were premedicated with 1.25 mg/kg atropine IP and anesthetized by an intraperitoneal injection of 50 mg/kg of sodium pentobarbital (Mebumal Vet, ACO Lakemedel). Body temperatures were maintained at 37°C. Catheters (Portex blue, Hythe) were inserted in the jugular vein for drug administration and in the left femoral vein for blood sampling during pharmacokinetic studies. The experiments were performed according to the guidelines of the International Society on Thrombosis and Haemostasis. (Giles, Thromb. Haemost. 58, 1078-1084 (1987)).

Thrombolytic Potency: The dosages that induced 50% clot lysis were determined from the dose-response curves obtained with TX 174 and SakSTAR in a hamster pulmonary embolism model as described by Stassen et al. (Fibrinolysis 4 (Suppl. 2), 15-21 (1990); Circulation 83 (suppl.IV), IV65-IV72 (1991)). Briefly, a 50-μL 1251-fibrin-labeled human plasma clot was produced in vitro and injected into the jugular vein of heparinized hamsters. Thrombolytic agents or saline were infused intravenously over 60 minutes, and 30 minutes after the end of the infusion the extent of clot lysis was determined as the difference between the radioactivity initially incorporated in the clot and the residual radioactivity in the lungs and the heart at the end of the experiment. At the end of the infusion and at the end of the experiment, blood samples were collected as described above.

Results: The staphylokinase of SEQ ID NO:1 was as potent as SakSTAR in the in- vivo hamster pulmonary embolism animal model (IC50 of 52 μg/kg for SEQ ID NO:1 versus 55 μg/kg for SakSTAR). This is further illustrated in Figure 2 where Sak42D (see patent DD245444) was compared with the staphylokinase of SEQ ID NO: 1.

Claims

12Claims

1. An isolated fibrinolytically active staphylokinase variant characterized in that the amino acid variations introduced in the variant are relative to a wild-type staphylokinase selected from the group consisting of Sak42D, SakCΦc* and SakSTAR, and wherein said amino acid variations consist of E65D, K74Q, R77E, E80S, D82S, V1 12T, K130Y, and E134R.

2. The staphylokinase variant according to claim 1 further characterized in that the amino acid variation S3C is introduced.

3. The staphylokinase variant according to claim 1 consisting of the amino acid sequence of SEQ ID NO : 1.

4. The staphylokinase variant according to any of claims 1 to 3 carrying an additional amino-terminal methionine residue.

5. The staphylokinase variant according to any of claims 1 to 4 which is modified.

6. The staphylokinase variant according to claim 5 which is modified by pegylation.

7. A fibrinolytically active protein comprising a staphylokinase variant according to any one of claims 1 to 6.

8. A composition comprising the staphylokinase variant according to any one of claims 1 to 6 and/or comprising the protein according to claim 7, and at least one of a solvent, diluent or carrier.

9. Use of a staphylokinase variant according to any one of claims 1 to 6 and/or a protein according to claim 7 for the manufacture of a medicament for lysing blood clots in a subject. 13

10. The use according to claim 9 wherein said blood clot is associated with a thrombotic condition or with cathether occlusion.

1 1. An isolated nucleic acid comprising a nucleotide sequence encoding a staphylokinase variant according to any of claims 1 to 4, optionally operably linked to regulatory nucleotide sequences required for expression of said staphylokinase variant.

12. A recombinant vector comprising a nucleotide sequence encoding a staphylokinase variant according to any of claims 1 to 4.

13. A method for recombinant production of a staphylokinase variant according to any of claims 1 to 4 comprising the steps of:

(i) expressing a staphylokinase variant according to any of claims 1 to 4 in a suitable host; and (ii) isolating the staphylokinase variant expressed in (i) from the host cells and/or from the host cell culture medium.

14. A method for producing a staphylokinase variant according to claim 5 comprising the steps of: (i) expressing a staphylokinase variant according to any of claims 1 to 4 in a suitable host; (ii) isolating the staphylokinase variant expressed in (i) from the host cells and/or from the host cell culture medium; and (iii) modifying the staphylokinase variant isolated in (ii).

15. The method according to claim 14 wherein the step of modifying said staphylokinase variant consists of pegylation.