Modified urokinase-type plasminogen activator polypeptides and methods of use

Patent metadata

Field	Value
Publication number	US-12331334-B2
Application number	US-202318167534-A
Country	US
Kind code	B2
Filing date	Feb 10, 2023
Priority date	Dec 28, 2018
Publication date	Jun 17, 2025
Grant date	Jun 17, 2025

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.

First claim

What is claimed:

1. A nucleic acid molecule, comprising a sequence of nucleotides encoding a modified urokinase-type plasminogen activator (u-PA) polypeptide that comprises one or more amino acid modifications selected from among replacements corresponding to R35Q, R35W, R35Y, H37E, H37Y, V41R, Y40Q, D60aP, L97bA, L97bG, T97aI, H99Q, and conservative amino acid modifications therefor, whereby the modified u-PA polypeptide has increased activity and/or specificity for a complement protein compared to the unmodified active form of the u-PA polypeptide, wherein:
any further amino acid modifications in the encoded modified u-PA polypeptide are selected from among replacements, insertions and deletions in the primary sequence of the modified u-PA polypeptide;
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;
the complement protein is C3;
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;
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;
the residues are 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); and
the conservative modifications are selected from among R35F or N; H37R, Q, W or F; V41K; D60aS; T97aL or V; L97bS; and H99N.

2. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide cleaves within residues QHARASHLG (residues 737-745) of human C3 (SEQ ID NO: 47).

3. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide has increased activity for cleavage of C3 that is least 3-fold greater than the unmodified u-PA polypeptide comprising the protease domain of SEQ ID NO: 5, or a corresponding form of u-PA set forth in any of SEQ ID NOs: 1-4 and 6.

4. The nucleic acid molecule of claim 1 , wherein:
the encoded modified u-PA polypeptide has an ED50 for inactivation cleavage of C3 of less than 100 nM in an in vitro assay; and
the modified u-PA polypeptide has stability of greater than 50% after incubation in PBS, or a body fluid for 7 days.

5. The nucleic acid molecule of claim 1 , wherein the unmodified u-PA polypeptide consists of the sequence of amino acids set forth in any of SEQ ID NOs: 1-6.

6. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide comprises up to 20 amino acid replacements, insertions, and/or deletions, compared to the unmodified u-PA polypeptide of any of SEQ ID NOs: 1-6 or a catalytically active portion thereof.

7. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide comprises one or more amino acid modifications selected from among replacements corresponding to R35Q, H37Y, V41R, Y40Q, D60aP, L97bA, T97al, and H99Q.

8. The nucleic acid molecule of claim 7 , wherein the encoded modified u-PA polypeptide comprises V41R and one or more of the replacements L97bA, R35Q, H99Q, D60aP, and T97al.

9. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide comprises the replacement V41R, or the replacements V41R and C122S.

10. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide further comprises the replacement V38E.

11. The nucleic acid molecule of claim 7 , wherein the encoded modified u-PA polypeptide comprises the replacement H37Y.

12. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide comprises the modifications V38E/V41R.

13. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide comprises the replacements R35Y/H37S/V38E/V41R or R35Y/H37Y/V38E/V41R.

14. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide comprises the replacements H37Y/V38E, R35Y/H37K, R35Q/H37K, R35Q/H37Y, V38E/V41R, V38E/V41R/Y149R, T39Y/V41R/D60aP/L97bA/H99Q/C122S, T39Y/V41R/D60aP/L97bA/H99Q, T39Y/V41R/Y60bQ/L97bA/H99Q,or T39Y/V41R/Y60bQ/L97bA/H99Q/C122S.

15. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide comprises the replacements R35Q/H37Y/T39Y/V41R, R35Q/H37Y/T39Y/V41R/C122S, R35Q/H37Y/T39Y/V41R/L97bA/H99Q/C122S, or R35Q/H37Y/T39Y/V41R/L97bA/H99Q.

16. The nucleic acid molecule of claim 1 , wherein the encoded modified u-PA polypeptide comprises amino acid modifications selected from:
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L;
R35W/R36Q/H37S/V38P/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y151L;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K82R/T97aI/L97bA/H99Q/K110aR/C122S/Y149R/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K/K179R;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K92R/T97aI/L97bA/H99Q/C122S/Y149R/M157K;
F30Y/R35V/R36H/H37G/V38E/T39W/Y40H/V41R/Y60bW/T97aI/L97bA/H99Q/C122S/Y149E/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K92S/T97aI/L97bA/H99Q/C122S/Y149R/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K61R/K62R/T97aI/L97bA/H99Q/C122S/Y149R/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K/K179S;
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/T97aI/L97bA/H99Q/C122S;
F30Y/R35W/R36T/H37S/V38S/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y151L/M157R/Q192Y;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/T97aI/L97bA/H99Q/C122S/M157K;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K61S/K62S/T97aI/L97bA/H99Q/C122S/Y149R/M157K;
R35A/H37E/R37aG/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L;
R35W/R36Q/H37S/V38T/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y151P/M157R;
F30Y/R35W/H37Y/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R;
V38E/T39W/V41R/D60aW/Y60bP/L97bG/H99L/C122S;
R35W/R36K/H37S/V38E/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y151L/M157S/Q192H;
R35Q/H37Y/R37aP/V38E/T39Y/V41R/D60aQ/Y60bP/T97aI/L97bA/H99Q/C122S/Y149R;
I17V/F30Y/R35Q/R36H/H37W/V38E/Y40H/V41R/T97aI/L97bA/H99Q/C122S/M157K/T158A;
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S/Y151L/Q192H;
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40F/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K;
R35W/R36N/H37S/V38E/T39Y/Y40M/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/M157S;
R35Y/H37D/V38E/T39W/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R;
F30Y/R35Y/R36H/H37K/V38E/T39F/Y40F/V41R/K82S/T97aI/L97bA/H99Q/K110aS/C122S/Y149R/M157K;
R35W/H37P/R37aN/V38E/T39Y/V41R/D60aP/Y60bL/D97T/T97aE/L97bG/A98S/H99L/C122S;
R35Y/H37S/R37aP/V38E/T39Y/V41R/D60aP/Y60bD/T97aI/L97bA/H99Q/C122S;
F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aI/L97bA/H99Q/C122S/Y149R/M157K;
F30Y/R35W/H37S/V38E/T39Y/Y40H/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/M157K;
R35H/V38E/T39Y/V41R/D60aP/Y60bQ/L97bA/H99Q/C122S/T158A;
R35Q/R36H/H37Y/V38E/T39Y/Y40L/V41R/T97aI/L97bA/H99Q/C122S/Y149R/M157K;
R35W/H37P/R37aG/V38E/T39Y/V41R/D60aP/Y60bE/T97aI/L97bA/H99Q/C122S/Y149R;
V38D/V41Q/D60aH/Y60bS/T97aW/L97bR/H99E/C122S/Y151L/E175D/R217E/K224R;
F30Y/R35W/R36H/H37P/R37aQ/V38E/T39Y/Y40F/V41R/Y60bQ/T97aE/L97bA/H99Q/C122S/Y149R/M157K;
F30Y/R35W/R36Q/H37S/V38P/T39Y/Y40L/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/M157R;
F30H/R35W/R36T/H37S/V38P/T39Y/V41R/Y60bN/T97aE/L97bA/H99Q/C122S/Y149R/Y151L/M157S;
F30Y/R35W/R36H/H37D/V38E/T39Y/Y40H/V41R/Y60bD/T97aI/L97bA/H99Q/C122S/M157K;
F30Y/R3

Assignees

• Vertex Pharma

Inventors

• Madison Edwin L
• Thanos Christopher
• Soros Vanessa
• Popkov Mikhail
• Tipton Kimberly
• Traylor Matthew John
• Furfine Eric Steven
• Way Jeffrey Charles

Classifications

• C12Y304/21073

  u-Plasminogen activator (3.4.21.73), i.e. urokinase · CPC title

• C07K2319/50

  containing protease site · CPC title

• C07K2319/02

  containing a signal sequence · CPC title

• C07K14/765

  Serum albumin, e.g. HSA · CPC title

• A61K38/00

  Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00) · CPC title