Modified urokinase-type plasminogen activator polypeptides and methods of use
Inventors
Madison, Edwin L. • Thanos, Christopher • Soros, Vanessa • Popkov, Mikhail • Tipton, Kimberly • Traylor, Matthew John • Furfine, Eric Steven • Way, Jeffrey Charles
Assignees
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Publication Number
US-12331334-B2
Publication Date
2025-06-17
Expiration Date
Abstract
Provided are nucleic acid molecules, including vectors and plasmids, encoding modified u-PA polypeptides and fusion proteins containing the modified u-PA polypeptides. The u-PA polypeptides are modified to have altered activity and/or specificity so that they cleave a complement protein, such as complement protein C3, to thereby inhibit complement activation. The nucleic acids and encoded modified u-PA polypeptides and fusion proteins that inhibit complement activation can be used for treatment of diseases and conditions that are mediated by complement activation, or in which complement activation plays a role. These disorders include ischemic and reperfusion disorders, including myocardial infarction and stroke, sepsis, autoimmune diseases, diabetic retinopathies, age-related macular degeneration, transplanted organ rejection, inflammatory diseases and diseases with an inflammatory component.
Core Innovation
The invention relates to nucleic acid molecules that encode modified urokinase-type plasminogen activator (u-PA) polypeptides. The modified u-PA polypeptides comprise one or more amino acid modifications selected from replacements corresponding to R35Q, R35W, R35Y, H37E, H37Y, V41R, Y40Q, D60aP, L97bA, L97bG, T97aI, and H99Q, with conservative amino acid modifications therefor. The modified u-PA polypeptides have increased activity and/or specificity for a complement protein compared to the unmodified active form of u-PA.
The modified u-PA polypeptides cleave a complement protein to inhibit or reduce complement activation compared to the unmodified u-PA polypeptide that does not contain the amino acid modifications, and the complement protein is C3. The modified u-PA polypeptides also have reduced activity or specificity for cleavage of a substrate sequence in plasminogen compared to the unmodified u-PA polypeptide.
The invention further defines sequence and numbering constraints for the modified u-PA polypeptides, including at least 90% sequence identity with polypeptides of any of SEQ ID NOs: 1-6 or a catalytically active portion thereof, with residues numbered by chymotrypsin numbering. The unmodified u-PA polypeptide comprises the sequence set forth in any of SEQ ID NOs: 1-6 or a catalytically active fragment thereof that includes the amino acid modification position(s).
Claims Coverage
The independent claim coverage centers on a nucleic acid molecule encoding a modified u-PA polypeptide. The claims recite specified amino acid modifications, C3 cleavage that inhibits or reduces complement activation, reduced plasminogen substrate cleavage activity or specificity, and sequence identity and chymotrypsin-numbering constraints.
Modified u-PA encoded in a nucleic acid with specified amino acid replacements
A nucleic acid molecule encoding a modified urokinase-type plasminogen activator polypeptide comprising amino acid modifications selected from replacements corresponding to R35Q, R35W, R35Y, H37E, H37Y, V41R, Y40Q, D60aP, L97bA, L97bG, T97aI, and H99Q, with conservative amino acid modifications therefor, wherein the modified u-PA has increased activity and/or specificity for a complement protein compared to the unmodified active form.
C3 cleavage that inhibits or reduces complement activation
The encoded modified u-PA polypeptide cleaves a complement protein to thereby inhibit or reduce complement activation compared to the unmodified u-PA polypeptide that does not contain the amino acid modifications, wherein the complement protein is C3.
Reduced plasminogen substrate cleavage activity or specificity
The encoded modified u-PA polypeptide has reduced activity or specificity for cleavage of a substrate sequence in plasminogen compared to the unmodified u-PA polypeptide.
Defined sequence identity to SEQ ID NOs: 1-6 and chymotrypsin numbering
The encoded modified u-PA polypeptide has at least 90% sequence identity with the polypeptides of any of SEQ ID NOs: 1-6 or a catalytically active portion thereof, and the residues are numbered by chymotrypsin numbering.
Overall, the claims cover nucleic acids encoding modified u-PA polypeptides that enhance activity and/or specificity for complement protein C3, cleave C3 to inhibit or reduce complement activation, and reduce plasminogen substrate cleavage, under defined sequence and numbering constraints.
Stated Advantages
Increased activity and/or specificity for a complement protein C3 compared to unmodified active u-PA.
Inhibition or reduction of complement activation by cleaving C3.
Reduced activity or specificity for cleavage of a plasminogen substrate sequence compared to unmodified u-PA.
In vitro C3 inactivation cleavage potency described in the document.
Improved stability after incubation in PBS or a body fluid described in the document.
Documented Applications
Complement-mediated diseases and conditions, including ischemia-reperfusion injury (myocardial infarction, stroke), sepsis, autoimmune/inflammatory disorders, ocular diseases such as AMD and diabetic retinopathy, renal delayed graft function (DGF), neurodegenerative disorders including Alzheimer’s, inflammatory transplant rejection, and other complement-related disorders listed in the document.
Functional characterization in plasma and vitreous humor and evaluation in an AMD-relevant assessment.
Assessment of C3 cleavage site confirmation and analysis of immunogenic hotspots using NetMHCII for HLA-DR binding.
Toxicity/tolerability assessment including NOAEL in monkeys.
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