Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Patent
1988-10-19
1991-12-10
Schwartz, Richard A.
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
435226, C12N 948, C12N 964, C07H 1700
Patent
active
050719726
DESCRIPTION:
BRIEF SUMMARY
This invention relates to substances having tissue plasminogen activator-type (t-PA) activity. More specifically, this invention relates to "recombinant" thrombolytic proteins, a process for obtaining the proteins from genetically engineered cells, and the therapeutic use of the substances as thrombolytic agents.
These proteins are active thrombolytic agents which, it is contemplated, possess improved fibrinolytic profiles relative to native human t-PA. This may be manifested as increased affinity to fibrin, decreased reactivity with inhibitors of t-PA, faster rate of thrombolysis, increased fibrinolytic activity and/or prolonged biological half-life. It is also contemplated that proteins of this invention can be more conveniently prepared in more homogeneous form than can native human t-PA. An improved overall pharmacokinetic profile is contemplated for these proteins.
The structure of native human t-PA can be viewed as comprising an amino (N-) terminus of about 91 amino acid residues, two so-called "kringle" regions, and at the carboxy terminus a serine protease-type domain. We have found that the N-terminus contains several sub-domains which play functional roles, inter alia, in fibrin binding and in the in vivo clearance of the protein. Recently the recovery of another form of t-PA which lacks the native N-terminus and first kringle region has been reported, see European Published Patent Application No. 0 196 920 (published 08 Oct. 1986). According to that report the truncated form of t-PA, which begins with Ala-160 of native human t-PA, is fibrinolytically active.
As described in greater detail hereinafter, this invention provides novel protein analogs of human t-PA which retain both kringle regions of native human t-PA, but contain modifications within the N-terminus. While in certain embodiments the modifications involve deletions in the N-terminus, the first kringle region is left intact, and the N-terminal deletion is never greater than 94 amino acids. Most embodiments involve significantly smaller deletion(s) and/or amino acid substitution(s). By retaining more of the structure of native human t-PA, it is contemplated that the proteins of this invention selectively retain more of the desirable biological activities of native human t-PA and may be less immunogenic than more drastically modified analogs of t-PA. It is therefore contemplated that the proteins of this invention possess improved fibrinolytic and pharmacokinetic profiles relative to both native human t-PA and the truncated Ala-160 t-PA, as well as other modified forms of t-PA.
The polypeptide backbone of natural human t-PA also includes four consensus Asn-linked glycosylation sites. It has been shown that two of these sites are typically glycosylated in t-PA from melanoma-derived mammalian cells, i.e. at Asn.sub.117 and Asn.sub.448. Asn.sub.184 is glycosylated sometimes and Asn.sub.218 is typically not glycosylated. t-PA from melanoma-derived mammalian cells, e.g. Bowes cells, is also referred to herein as "native" or "natural" human t-PA.
This invention, as mentioned above, involves novel protein analogs of human t-PA which possess t-PA-type thrombolytic activity. The proteins of this invention differ in structure from human t-PA in that they contain modifications in peptide sequence (i) at up to three of the Asn-linked glycosylation sites present in native t-PA; (ii) within the N-terminus of the proteins corresponding to the 94 amino acid mature N-terminus of native t-PA; and/or (iii) at the proteolytic cleavage site spanning Arg-.sub.275 and Ile-.sub.276. These features of the proteins of this invention are described in greater detail below. Notwithstanding the various modifications, the numbering of amino acids as shown in the one-letter code sequence of Table 1 is retained.
A. Modifications at the N-terminus
In one aspect of this invention the proteins are characterized by deletion of 1-94 amino acids within the peptide region spanning Gly-(-3) or Ser-1 through Thr-91, relative to native human t-PA. In one embodiment, for examp
REFERENCES:
patent: 4766075 (1988-08-01), Goeddel et al.
Pennica, D. et al., Nature, vol. 301, pp. 214-221, 1983.
Ny, T. et al., Proc. Natl. Acad. Sci., vol. 81, pp. 5355-5359, 1984.
van Zonneveld, A. et al., International Congress for the Society on Thrombosis and Haemostasis, Abstract 022 as evidence of presentation, Jul. 1985.
van Zonneveld, A. et al., Proc. Natl. Acad. Sci., vol. 83, pp. 4670-4674, Jul. 1986.
Zoller, M. et al., Nuc. Acid Res, vol. 10, pp. 6487-6500, 1982.
Pohl, G. et al., Biochemistry, vol. 23, pp. 3701-3707, 1984.
Cserr Luann
Eisen Bruce
Genetics Institute Inc.
Porta Marianne
Schwartz Richard A.
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