Production of thrombin inhibitors

Details Production of thrombin inhibitors Production of thrombin inhibitors

1990-02-27

2002-06-25

Yucel, Remy (Department: 1636)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Using a micro-organism to make a protein or polypeptide

C435S069100, C435S483000, C435S212000, C435S226000, C435S254800, C435S254210, C435S320100, C536S023100, C536S023500, C536S024100

Reexamination Certificate

active

06410272

ABSTRACT:

The invention pertains to the field of recombinant DNA technology and concerns a method for the preparation of the thrombin inhibitor desulphatohirudin with the aid of genetically engineered eukaryotic cells, said genetically engineered eukaryotic cells, hybrid vectors carrying a gene for desulphatohirudin and methods for the preparation of said eukaryotic cells and said hybrid vectors.
Hirudin is an anticoagulant agent that occurs naturally in leeches (
Hirudo medicinalis
). Hirudin is not a single protein species but consists of at least three components designated hirudin variant 1 (HV1), hirudin variant 2 (HV2) (cf. European Patent Application No. 158,564) and “des-(Val)
2
-hirudin” (cf. European Patent Application No. 158,986). The variants differ in structure from each other by a number of amino acids (especially, the N-terminal sequence of HV1 is Val-Val-Tyr, that of HV2 is Ile-Thr-Tyr and that of “des-(Val)
2
-hirudin” is Thr-Tyr) but have an accumulation of hydrophobic amino acids at the N-terminus and of polar amino acids at the C-terminus, a tyrosine residue (Tyr
63
) present as sulphate monoester, three disulphide bridges and the anticoagulant activity in common.
Hirudin, with a K
i
-value (complex dissociation constant) of 6×10
−11
M, is the strongest thrombin inhibitor known and is characterised by a specific affinity to thrombin. Other enzymes of the blood coagulation cascade are not inhibited by hirudin. In contrast to heparin which is the preferred anticoagulant in conventional anticoagulation therapy, hirudin exerts its inhibiting action directly on thrombin and, unlike the former, does not act through antithrombin III. The only pharmacologically detectable effect of purified hirudin is the inhibition of blood coagulation and the prophylaxis of thrombosis. No effect on heart rate, respiration, blood pressure, thrombocyte count, fibrinogen and haemoglobin could be observed when administered intravenously to dogs, even in high doses. In tests on rats, pigs and dogs, hirudin has proved effective in experimental thrombosis (induced either by stasis or by the injection of thrombin), in endotoxin shock, and also in DIC (disseminated intravascular coagulation). Whenever direct comparison tests have been carried out, hirudin has proved to be superior to heparin. Furthermore, hirudin has an extremely low toxicity, is non antigenic and shows an almost complete clearance via the kidneys in a biologically active form.
Although long known, hirudin has not as yet achieved the broad therapeutic use that might be expected on the basis of its excellent biological properties. Its extremely limited availability is a serious drawback which stands in the way of its widespread use in medicine. Up to now, hirudin preparations have been obtainable essentially from natural material(leech extracts), which is expensive and difficult to obtain, and employing time-consuming and costly isolation and purification processes [cf. P. Walsmann et al., Pharmazie 36, 653 (1981); European Patent Application No. 158,986]. In view of the relatively long sequence of 65 amino acids also the conventional peptide synthesis offers little hope of success for economic reasons. New methods must therefore be applied for the manufacture of adequate amounts of hirudin that render possible detailed clinical tests of its therapeutic potential and its broad therapeutic use in anticoagulation therapy.
Such methods are offered especially by recombinant DNA technology. By means of this technology it is possible to manufacture the most varied physiologically active polypeptides by culturing correspondingly genetically modified host organisms. In this context, it has to be mentioned that hirudin is presumably produced by leeches via a desulphatohirudin precursor which is post-translationally sulphated. It is expected that hosts other than leech lack the specific sulphate-transferring enzyme system and will therefore produce desulphatohirudins rather than hirudins. However, the biological activity is not injured by the absence of the sulphate group as is evidenced by the desulphatohirudin protein obtained by enzymatic removal of the sulphate group from the phenolic hydroxy group of the Tyr 63 residue of the corresponding hirudin protein (cf. European Patent Application No. 142,860).
In the recently published European Patent Application No. 158,564 the production of desulphatohirudin variants 1 and 2 and of analogues thereof by means of
E. coli
cells transformed with plasmids containing the structural genes for the respective desulphatohirudirsis disclosed. The anticoagulant activity measured in cell extracts of cultured
E. coli
cells and given in terms of anti-thrombin units (“ATU”) amounts to 3,000-4,000 ATU/D
600
/l culture which corresponds to a concentration of 0.2 mg hirudin/OD/l (based upon an estimated specific activity of 15,000-20,000 ATU/mg of pure hirudin).
Presumably the poor yield in hirudin activity obtained from transformed
E. coli
cells is attributable to an accumulation of most of the hirudin protein in an inactive form in the cytoplasm due to incorrect folding of the molecule. The correct folding depends on the formation of three disulphide bridges within the hirudin molecule which are essential for enzyme activity(cf. P. Walsmann et al., supra). Other naturally secreted mammalian proteins, such as bovine growth hormone, human tissue plasminogen activator and human &ggr;-interferon are likewise essentially inactive when produced and accumulated in the cytoplasm of microorganisms [cf. R. A. Smith et al., Science 229, 1219 (1985)]. It is apparent that the secretion pathway may favor disulphide bond formation since most proteins containing disulphide bridges are extracellular. There are other features that make secretion most desirable:
Secreted proteins are generally easier to detect and to purify than intracellularly accumulated products;
secretion of desired products into the medium avoids the necessity of breaking up the host organisms in order to recover the product;
some heterologous proteins may have a toxic effect on the host organism. When secreted they are less likely to interfere with normal cellular functions;
secreted proteins are less likely to be digested by proteolytic enzymes than intracellularly accumulated proteins which are attacked by these enzymes upon disintegration of the cells.
Most secreted proteins are encoded on the DNA as preproteins with a signal peptide as an appended amino terminal extension of the mature amino acid sequence. The signal peptide plays an essential role during cotranslational insertion of the protein into the membranes of the endoplasmatic reticulum (ER). A signal peptidase cleaves the signal peptide in an early event on the luminal side of the ER. Further transport to the outer cell membrane involves Golgi and secretory vesicles. The protein is either secreted into the periplasmic space (e.g. acid phosphatase, invertase) or into the culture medium (e.g. &agr; factor, killer toxin).
Since all eukaryotes seem to share the mechanisms for the expression of genetic information and for sorting the expressed proteins, the expression of eukaryotic genes proceeds with greater efficiency in an eukaryotic host than in
E. coli
. Among the eukaryotic organisms yeast is the easiest to handle and to cultivate. A number of heterologous proteins have been expressed successfully in yeast. However, it has not been possible so far to define essential features of a protein—except for an appended signal peptide—that would allow efficient secretion into the medium. Therefore, it is not possible to make reliable predictions whether or not a protein will be secreted. Thus, while 90% of total glucoamylase produced by transformed yeast (containing the genetic information for pre-glucoamylase) is secreted into the medium (PCT-Patent Application No. 84/2921) and high titers of epidermal growth factor (EGF) are found in the medium of cultured yeast containing the EGF gene with the appended &agr; factor signal peptide (European Patent Application

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