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US-20260125665-A1 - TARGETING AND LYSING MICROTHROMBI ASSOCIATED WITH ABERRANT BLOOD CLOTTING

US20260125665A1US 20260125665 A1US20260125665 A1US 20260125665A1US-20260125665-A1

Abstract

The present invention provides means and methods for treating MVT and associated and/or similar conditions. The invention therefore provides hybrid proteins for targeted delivery of plasminogen activators to platelet-VWF complexes, or alternatively to the site where these are located, in a fibrin-independent manner. The invention further provides therapeutic methods for conditions that can be prevented or treated by local delivery/stimulation of plasminogen activation to sites of microvascular occlusion.

Inventors

  • Steven de Maat
  • Coen Maas

Assignees

  • TARGED BIOPHARMACEUTICALS B.V.

Dates

Publication Date
20260507
Application Date
20251028
Priority Date
20231219

Claims (20)

  1. 1 . A polypeptide comprising a urokinase catalytic domain, wherein the arginine residue corresponding to position 153 in SEQ ID No. 1 is substituted with a glutamine residue.
  2. 2 . The polypeptide according to claim 1 , wherein the urokinase catalytic domain further comprises a substitution of the asparagine residue corresponding to position 299 in SEQ ID No. 1.
  3. 3 . The polypeptide according to claim 2 , wherein the asparagine residue corresponding to position 299 in SEQ ID No. 1 is substituted with a glutamine residue.
  4. 4 . The polypeptide according to claim 1 , wherein the urokinase catalytic domain further comprises a lysine residue or a histidine residue at position 297 in SEQ ID No. 1.
  5. 5 - 6 . (canceled)
  6. 7 . The urokinase catalytic domain of claim 1 , that is capable of facilitating the dissolvement of microthrombi.
  7. 8 . A hybrid protein comprising a polypeptide comprising a urokinase catalytic domain, wherein an arginine residue corresponding to position 153 in SEQ ID No. 1 is substituted with a glutamine residue; wherein position 299 in SEQ ID No. 1 is an asparagine residue or a histidine residue; and wherein the hybrid protein comprises an VHH .
  8. 9 . (canceled)
  9. 10 . The hybrid protein according to claim 8 , wherein the VHH comprises complementarity determining regions (CDRs) CDR1, CDR2, and CDR3, wherein CDR3 comprises at least one of the amino acid sequences SEQ ID NO: 6-28; CDR 1 comprises at least one of the amino acid sequences SEQ ID NO: 29-51; and/or CDR2 comprises at least one of the amino acid sequences SEQ ID NO: 52-74.
  10. 11 - 13 . (canceled)
  11. 14 . The hybrid protein according to claim 8 , wherein the variable domain comprises or consists of a sequence selected from SEQ ID NO: 75-101.
  12. 15 . A hybrid protein according to claim 8 , wherein the variable domain has binding specificity for von Willebrand factor.
  13. 16 . The hybrid protein according to claim 8 , wherein at least part of the coding sequence is codon optimized.
  14. 17 - 18 . (canceled)
  15. 19 . A hybrid protein according to claim 8 , which is a chemically linked protein or is a fusion protein.
  16. 20 . (canceled)
  17. 21 . A hybrid protein according to claim 8 , further comprising a linker sequence linking the polypeptide comprising the urokinase catalytic domain to the VHH.
  18. 22 - 23 . (canceled)
  19. 24 . A hybrid protein according to claim 8 , which comprises the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.
  20. 25 . A hybrid protein according to claim 8 , for use in the prevention or treatment of microvascular thrombosis.

Description

CROSS-REFERENCE TO ELECTRONIC SEQUENCE LISTING The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on Dec. 17, 2024, is named “AMS-202305EPPC Sequence listing.xml” and is 93,282 bytes in size. The sequence listing contained in this. XML file is part of the specification and is hereby incorporated by reference herein in its entirety. FIELD OF THE INVENTION The present invention relates to the field of diseases or conditions associated with undesired blood clotting, in particular microvascular thrombosis, such as thrombotic thrombocytopenia purpura. In particular, the invention relates to a hybrid protein comprising a (urokinase) catalytic domain linked to an antibody-like molecule, in particular a VHH, capable of facilitating the lysis of microthrombi. In a particular embodiment the VHH binds to von Willebrand factor (VWF), enabling the linked urokinase catalytic domain to drive the conversion of plasminogen to plasmin, which then degrades fibrin and/or VWF into smaller fragments. The invention thus provides means and methods for treating microvascular thrombosis and associated conditions. BACKGROUND ART Primary hemostasis is a procoagulation clot forming process associated with the initiation and formation of the platelet plug, in which the binding of platelets to vWF is of importance. Secondary hemostasis is also a procoagulation clot forming process and it is associated with the formation of fibrin via the intrinsic and extrinsic coagulation cascades. In healthy people, homeostatic balance exists between procoagulant forces and anticoagulant and fibrinolytic forces. Numerous genetic, acquired, and environmental factors can tip the balance in favor of coagulation, leading to the pathologic formation of thrombi in veins (eg, deep venous thrombosis [DVT]), arteries (eg, myocardial infarction, ischemic stroke), or cardiac chambers. Thrombi can obstruct blood flow at the site of formation or detach and embolize to block a distant blood vessel (eg, pulmonary embolism, embolic stroke). In here we will focus on microvascular thrombosis. Microvascular thrombosis (MVT) is characterized by the formation of microvascular platelet aggregates. They are minimally composed of platelets and VWF. This becomes clear in thrombotic thrombocytopeniaurpura (TTP), where platelet- and VWF-rich, but fibrin-poor microthrombi obstruct microvasculature with life-threatening consequences. Thus, fibrin, which is seen in macrovascular thrombosis, is not per se required for these microvascular obstructions. MVT is a shared feature between several disease states including thrombotic thrombocytopenia purpura, hemolytic uremic syndrome, antiphospholipid antibody syndrome and complement-mediated thrombotic microangiopathy (George et al., 2014, N Engl J Med. 371 (7): 654-66). In severe cases of MVT, multi-organ failure can occur with lethal consequences. Even in less severe cases, organ damage may occur, reducing both the quality of life, as well as the life expectancy of the patient. Recent studies suggest that microvascular disease underlies cardiovascular disease/events in patients in the more generalized population with cardiovascular disease that do not show overt signs of macrovascular obstruction on radiological examination. This is ultimately thought to cause heart failure, in particular in females. Patients with TTP experience attacks of microvascular thrombosis, when platelets form complexes with ultra-large multimers of von Willebrand Factor (VWF). This is the result of severely decreased activity of the enzyme ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type I motif, member 13). ADAMTS13 normally regulates the thrombogenicity of VWF by enzymatically reducing its multimer size. Hereto, VWF needs to unfold from its globular form into an unrolled conformation, thereby exposing its A2 domain for proteolysis. The majority of TTP patients suffer from neutralizing autoantibodies against AD AMTS 13. For a small subgroup, mutations in ADAMTS13 have been described that lead to deficiency (Upshaw-Shulman syndrome). Current TTP therapy involves extensive plasma exchange to deplete inhibitory antibodies and restore ADAMTS13 activity at the same time. However, persistent autoantibodies impede elimination of microthrombi. This makes therapy time-consuming and very costly (Fijnheer et al., Ned Tijdsch Hematol 2016; 13 (1): 18-24). Besides AD AMTS 13, VWF can be cleaved by the enzyme plasmin (Berkowitz et al., J Clin Invest 1987 February; 79 (2): 524-31). We previously identified that systemic plasminogen activation (with streptokinase) was therapeutic in a mouse model for TTP, suggesting that plasmin can act as a functional alternative to ADAMTS13 (Tersteeg et al., 2014, Circulation, 129 (12): 1320-31). Although plasmin (ogen) can directly bind to unrolled (unfolded) VWF, natural plasminogen activ