Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an organic...
Reexamination Certificate
1999-05-06
2001-04-10
Weber, Jon P. (Department: 1651)
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an organic...
C435S178000, C435S179000, C435S180000, C435S212000, C435S188000
Reexamination Certificate
active
06214594
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates generally to the field of biochemistry and in particular to size modification of a fibrinolytic enzyme.
BACKGROUND OF THE INVENTION
Fibrolase is a metalloproteinase isolated from the venom of the southern copperhead (
Agkistrodon contortrix contortrix
). It is typical of the small venom metalloproteinases of approximately 23 kDa. The enzyme possesses proteolytic activity directed to the cleavage of the &agr;- and &bgr;-chains of fibrin and fibrinogen. In that fibrin is a major component of occlusive thrombi, the degradative action of fibrolase leads to thrombus lysis and elimination. Fibrinolytic activity of fibrolase has been examined in both test tube and animal models. The enzyme has been shown to effectively lyse fibrin clots in-vitro [Guan, A. L., et al.,
Arch. Biochem. Biophys.,
289:197-207 (1991)] and in-vivo [Markland, F. S., et al.,
Circulation,
90:2448-2456 (1994); Markland, F. S., in
Natural Toxins II
(Singh, B. R. and Tu, A. T., eds.), pp 427-438, Plenum Press, New York (1996)]. While fibrolase can degrade mature thrombi it has no effect on the formation of these structures.
Although fibrolase degrades fibrin(ogen) in the test tube, in the circulation the enzyme is efficiently inactivated by alpha-2 macroglobulin (&agr;2M). To enable complete thrombus dissolution in vivo a modification to fibrolase must be made to block its rapid inactivation by &agr;2M. &agr;2M is a general protease inhibitor present in the circulatory system at fairly high concentrations (≅3 &mgr;M). This inhibitor has the ability to bind to and sequester small proteases and remove them from the circulation via the formation of a covalent bond between the proteinase and the very large, 720 kDa, tetrameric inhibitor molecule. Interactions between proteinases and &agr;2M are sterically influenced and appear to be directly related to the size of the proteinase [Werb, Z., et al.,
Biochemical Journal,
139:359-368 (1974)]. A 68 kDa hemorrhagic metalloproteinase from Crotalus atrox is not inhibited by &agr;2M, while another closely related but smaller, 23 kDa, metalloproteinase is rapidly and effectively bound and inhibited by &agr;2M [Baramova, E. N., et al.,
Biochemistry,
29:1069-1074 (1990)]. Once bound to &agr;2M the proteinase is essentially removed from circulation, unable to act on the target molecule, in the case of fibrolase, a thrombus.
SUMMARY OF THE INVENTION
In accordance with this invention, a fibrinolytic enzyme, fibrolase, is modified by covalent attachment with a water soluble large organic molecule which alters the size of fibrolase while not changing the effectiveness of its thrombolytic activity. The size modified fibrolase, due to its increased bulk and rotational size, can interact with &agr;2M with much slower kinetics and thus have a longer active circulatory half-life. Slowing the interaction of fibrolase with &agr;2M allows for more rapid and effective thrombolysis by the fibrinolytic enzyme and at lower administered dosage.
Size modification of the fibrinolytic enzyme (including, but not limited to, fibrolase or similar fibrinolytic enzymes from the Crotalus or Agkistridon species, such as the fibrinolytic enzyme from
A. piscovirus conanti
[Retzios, A. D., et al.,
Protein Expressing Purification,
1(1):33-39 (1990)] or from
C. basiliscus basiliscus
[Retzios, A. D., et al.,
Protein Expressing Purification,
1(1):33-39 (1990); Retzios, A. D. et al.,
Biochemistry,
31:4547-4557 (1992)] or enzymes isolated from other living organisms) can be made via a number of different methods. The adducted groups can be any chemically inert large water soluble non-charged organic molecule, having a molecular weight from about 5,000 Da to about 50,000 Da, containing a moiety for adduction to the enzyme. Polyalkylene glycols from 2 to 5 carbon atoms, exemplified by polyethylene glycol, are suitable molecules for adduction to the enzyme. Other suitable large organic molecules include polymers of a single amino acid such as: poly-alanine; poly-glycine; and poly-lysine. Alternatively, large organic polymers such as polyethylenes and poly-vinyls can be used. Furthermore, natural human proteins such as Fab fragments from antibodies can be suitable large organic molecules.
Each adducted large organic molecule imparts a large increase in rotational size of the enzyme while not adding any reactive moieties. One method of large organic molecule adduction to the fibrinolytic enzyme is exemplified by covalent attachment through an NHS ester included in the modifying large organic molecule. An NHS ester reacts with the &egr;-amino groups of surface lysine residues of fibrolase or any other venom fibrinolytic enzyme to yield a covalent amide linkage.
The method for the construction of one type of size modified fibrinolytic enzyme is detailed in the following sections. Also included are the details pertaining to the specific assays we have performed to determine the extent of size modifying agent adduction on retention of fibrinolytic enzyme activity.
REFERENCES:
patent: 4378435 (1983-03-01), Tagagi et al.
patent: 4378803 (1983-04-01), Tagagi et al.
patent: 4446316 (1984-05-01), Chazov et al.
patent: 4610879 (1986-09-01), Markland, Jr. et al.
patent: 4640835 (1987-02-01), Shimizu et al.
patent: 4935465 (1990-06-01), Garman
patent: 5234903 (1993-08-01), Nho et al.
patent: 5514572 (1996-05-01), Veronese et al.
patent: 0183503 (1986-06-01), None
Baramova, E.N., et al., Biochemistry, 29:1069-1074 (1990).
Clark, R., et al., Journal of Biological Chemistry, 271(36):21969-21977 (1996).
Markland Francis S.
Swenson Stephen D.
Fulbright & Jaworski L.L.P.
University of California
Weber Jon P.
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