Tissue type plasminogen activator (t-PA) variants:...

Details Tissue type plasminogen activator (t-PA) variants:... Tissue type plasminogen activator (t-PA) variants:...

2000-11-13

2004-03-16

Carlson, Karen Cochrane (Department: 1653)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Reexamination Certificate

active

06706504

ABSTRACT:

The invention comprises protein single chain variants of tissue type plasminogen activator, also referred to as t-PA as well as nucleic acids encoding such protein single chain variants of tissue type plasminogen activator. The t-PA protein variants have higher zymogenicity than the wild-type single chain t-PA form. Methods of making and using the t-PA variant compositions are also described.
BACKGROUND
Tissue-type plasminogen activator (t-PA) is a serine protease that plays a critical role in the process of fibrinolysis, the dissolution of clots, by activating plasminogen to the protease plasmin. t-PA has been fully identified and characterized by underlying DNA sequence and deduced amino acid sequence. See Pennica et al.,
Nature
, 301: 214 (1983) and U.S. Pat. No. 4,853,330, issued Aug. 1, 1989, the teachings of both of which are incorporated by reference. The nucleotide sequence and deduced primary amino acid sequence of human t-PA is depicted in
FIG. 1A
, FIG.
1
B and FIG.
1
C.
The group of amino acid residues from −35 to −1 preceding the sequence of the mature t-PA is the “pro” sequence. The mature t-PA molecule (amino acid residues 1-527) contains five domains that have been defined with reference to homologous or otherwise similar structures identified in various other proteins such as trypsin, chymotrypsin, plasminogen, prothrombin, fibronectin, and epidermal growth factor (EGF). These domains have been designated, starting at the N-terminus of the amino acid sequence of mature t-PA, as 1) the finger region (F) that has variously been defined as including amino acid residues 1 to about 44, 2) the growth factor region (G) that has been variously defined as stretching from about amino acid residues 45 to 91 (based upon its homology with EGF), 3) kringle one (K1) that has been defined as stretching from about amino acid residue 92 to about amino acid residue 173, 4) kringle two (K2) that has been defined as stretching from about amino acid residue 180 to about amino acid residue 261, and 5) the so-called serine protease domain (P) that generally has been defined as stretching from about amino acid residue 264 to the C-terminal end of the molecule at amino acid residue 527. These domains, which are situated generally adjacent to one another, or are separated by short “linker” regions, account for the entire amino acid sequence of from 1 to 527 amino acid residues of the mature form of t-PA.
Each domain has been described variously as contributing certain specific biologically significant properties. The finger domain has been characterized as containing a sequence of at least major importance for high binding affinity to fibrin. (This activity is thought important for the high specificity that t-PA displays with respect to clot lysis at the locus of a fibrin-rich thrombus.) The growth factor-like region likewise has been associated with cell surface binding activity. The kringle 2 region also has been strongly associated with fibrin binding and with the ability of fibrin to stimulate the activity of t-PA. The serine protease domain is responsible for the enzymatic cleavage of plasminogen to produce plasmin.
t-PA is unusual among proteases in the level of the enzymatic activity of its precursor. In general, proteases are synthesized as zymogens, inactive precursors that must either be proteolytically processed or bind to a specific co-factor to develop substantial catalytic activity. The increase in catalytic efficiency after zymogen activation, or zymogenicity, is dramatic in almost all cases, although varying widely among individual members of the chymotrypsin family. For example, strong zymogens, i.e., those having high zymogenicity, such as trypsinogen, chymotrypsinogen, or plasminogen are almost completely inactive, with measured zymogenicities of 10
4
to 10
6
(Robinson, N. C., Neurath, H., and Walsh, K. A. (1973)
Biochemistry
12, 420-426; Gertler, A., Walsh, K. A., and Neurath, H. (1974)
Biochemistry
13, 1302-1310). Other serine proteases exhibit intermediate zymogenicity. For example, the enzymatic activity of Factor XIIa is 4000-fold greater than that of its corresponding zymogen, Factor XII (Silverberg, M., and Kaplan, A. P. (1982) Blood 60, 64), and the catalytic efficiency of urokinase is 250-fold greater than that of pro-urokinase (Lijnen, H. R., Van Hoef, B., Nelles, L., and Collen, D. (1990)
J. Biol. Chem
. 265, 5232-5236). By contrast, the catalytic activities of single and two chain t-PA vary by a factor of only 5-10.
The zymogenicity, expressed as the ratio of the activity of the mature cleaved two-chain enzyme to that of the single chain precursor form, is only 5-10 for wild-type t-PA, in contrast to other precursors of other proteases that have little or no catalytic activity. Thus, the single chain form of wild-type t-PA is not a true zymogen.
There have been many attempts to improve the usefulness of t-PA by genetic engineering. These efforts have been only partially successful. The state of the art has been reviewed by Krause, J., & Tanswell, P.
Arzneim.-Forsch
. 39: 632-637 (1989) and in U.S. Pat. No. 5,616,486, the teachings of both of which are incorporated by reference.
Despite the profound advantages associated with natural t-PA as a clot-dissolving agent, it is not believed that the natural protein necessarily represents the optimal t-PA agent under all circumstances. Therefore, several variants have been proposed or devised to enhance specific properties of t-PA. Certain of those variants address disadvantages associated with the use of natural t-PA in situations where an agent with a longer half-life or slower clearance rate would be preferred, e.g., in the treatment of deep-vein thrombosis and following reperfusion of an infarct victim, or where a single-chain agent is preferred.
For example, removal of a substantial portion or all of the finger domain results in a molecule with substantially diminished fibrin binding characteristics, albeit in return there is a decrease in the overall rate of clearance of the resultant entity—See WO 89/00197 published Jan. 12, 1989.
Variants are described in EPO Pat. Publ. No. 199,574 that have amino acid substitutions at the proteolytic cleavage sites at positions 275, 276, and 277. These variants, characterized preferentially as t-PA variants having an amino acid other than arginine at position 275, are referred to as protease-resistant one-chain t-PA variants in that, unlike natural t-PA, which can exist in either a one-chain or two-chain form, they are resistant to protease cleavage at position 275 and are therefore not converted metabolically in vivo into a two-chain form. This form is thought to have certain advantages biologically and commercially, in that it is more stable while the fibrin binding and fibrin stimulation are increased relative to two-chain t-PA. Furthermore, plasminogen activators are described that comprise one domain capable of interacting with fibrin and the protease domain of urokinase, with one embodiment being urokinase altered to make it less susceptible to forming two-chain urokinase. See WO 88/05081 published Jul. 14, 1988.
For further patent literature regarding modification of the protease cleavage site of t-PA, see, for example, EPO Pat. Nos. 241,209; EP 201,153 published Nov. 12, 1986; EP 233,013 published Aug. 19, 1987; EP 292,009 published Nov. 23, 1988, EP 293,936 published Dec. 7, 1988; and EP 293,934 published Dec. 7, 1988; and WO 88/10119.
Glycosylation mutants at positions 117-119, 184-186, and 448-450 exhibited higher specific activity as the mole percent carbohydrate was reduced. See EPO Pub. No. 227,462 published Jul. 1, 1987. This patent application additionally discloses using an assay of fibrin/fibrin degradation products and teaches that one may also modify the t-PA molecule at positions 272-280 or delete up to 25 amino acids from the C-terminus. Further, the t-PA mutants with Asn 119, Ala 186 and Asn 450, which have the N-glycosylation sites selectively removed by DNA modification but contain residual O-linked carbohydrate, were foun

Affiliated with

Also associated with

Rating

Tissue type plasminogen activator (t-PA) variants:... does not yet have a rating. At this time, there are no reviews or comments for this patent.

If you have personal experience with Tissue type plasminogen activator (t-PA) variants:..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Tissue type plasminogen activator (t-PA) variants:... will most certainly appreciate the feedback.

Rate now

Comments { 0 }

Profile ID: LFUS-PAI-O-3280470