Fusion proteins comprising tissue factor for controlled haemostasis

Abstract

The present invention relates to fusion proteins comprising (i) at least one blood coagulation factor or any variants thereof, and (ii) one or more Cellulose Binding Module (CBM), wherein the CBM is bound to the at least one blood coagulation factor, preferably Tissue Factor, optionally bound by a linker. Furthermore, dressing materials, uses and manufacturing methods in relation to the fusion proteins are disclosed.

Description

[0001] Fusion proteins comprising Tissue Factor for controlled Haemostasis

[0002] ABSTRACT

[0003] The present invention provides a safe and efficient system for enhancing haemostatic control by binding fusion protein comprising at least one blood coagulation factor, such as the extracellular domain of Tissue Factor (TF) also known as soluble TF (sTF), or any variants thereof to a dressing material suitable for use in a wound dressing. This configuration ensures direct contact between the blood coagulation factor and the wound site, facilitating localized haemostasis. The invention is designed to generate a localized "thrombin burst", a process that rapidly releases thrombin, the key enzyme in the coagulation cascade, thereby accelerating clot formation. Notably, the system prevents systemic release of the blood coagulation factor into the vascular bed, reducing potential adverse effects. The fusion protein comprising a blood coagulation factor may be used alone or in combination with other human proteins or components to optimize haemostatic efficacy. The fusion protein further comprises a cellulose binding mod-ule / protein domain which enhances compatibility with various types of dressing materials without compromising the biological activity of the blood coagulation factor.

[0004] FIELD OF THE INVENTION

[0005] The present invention relates to a fusion protein comprising at least one blood coagulation factor such as the extracellular domain of Tissue Factor or soluble Tissue Factor (sTF), or any variants thereof and a cellulose-binding module (CBM) immobilized on a dressing material. This is a fast-acting and safe system for promoting haemostasis without inducing systemic coagulation such as disseminated intravascular coagulation. The invention also relates to a system for promoting rapid haemostasis comprising one or more haemostatic agents, including sTF or any variant thereof, immobilized on a dressing material via a peptide containing a cellulose-binding module. This system facilitates localized clot formation at the site of injury while ensuring safety inclinical use. The invention utilizes immobilized blood coagulation factor to induce a localized "thrombin burst," a process that is rapid activation of vast amount thrombin, a key enzyme in the coagulation cascade, thereby enhancing haemostatic efficiency.

[0006] BACKGROUND

[0007] Controlling hemorrhage is critical in military settings, trauma care, and surgical procedures due to its direct impact on survival rates and patient outcomes. Uncontrolled bleeding is a leading cause of preventable death, making effective hemorrhage management a top priority. According to the American Association for the Surgery of Trauma, trauma is the leading cause of death for individuals between the ages of 1 and 45 years, with injuries accounting for over 5 million deaths annually worldwide.

[0008] In military combat situations, rapid hemorrhage control is essential. A study analyzing U.S. military casualties from October 2001 to June 2011 found that 87% of deaths occurred before reaching a medical treatment facility, with 24.3% of these potentially survivable. Notably, 90.9% of potentially survivable deaths were due to hemorrhage, highlighting the critical need for immediate bleeding control on the battlefield (Eastridge et al., The Journal of Trauma and Acute Care Surgery, 2012).

[0009] Similarly, in civilian settings, hemorrhage is the primary cause of preventable deaths following traumatic events, affecting both military personnel and the general population (Bellamy R.F ., Mil Med, 1984).

[0010] During surgical procedures, effective hemorrhage control is necessary to prevent complications such as hypovolemic shock, coagulopathy, and organ failure. Failure to manage hemorrhage leads to increased mortality, extended hospital stays, higher healthcare costs, legal consequences, and a reduced quality of life for patients. Despite advancements in medical technology, hemorrhage control remains a fundamental challenge in trauma care, surgery, and military medicine. Various products have been developed to control bleeding, yet there is an ongoing need for more effective and safer solutions. (Henry T. Peng, Haemostatic Agents for Prehospital Hemorrhage Control: A Narrative Review).The present invention addresses this need by providing a safe and efficient system for promoting rapid haemostasis without induction of systemic blood coagulation. The present invention relates to a fusion protein comprising at least one blood coagulation factor, such as the extracellular domain of Tissue Factor also known as soluble Tissue Factor (sTF), or a variant thereof, referred to as the Tissue Factor Component, and one or more cellulose-binding module (CBM) in combination with a dressing material. Said fusion protein is associated with the dressing in such a manner that it remains immobilized when exposed to a physiological environment where blood coagulation occurs. This provides for an efficient, and safe rapid haemostasis which enhances localized clot formation while minimizing the risks of systemic coagulation. The cellulose-binding module component of the fusion protein can be easily incorporated into various types of dressing material owing to its broad compatibility without comprising the biological activity of the blood coagulation factor. In particular, it has been shown that TF maintains its full biological activity. This is contrary to previous reports that immobilization of fused TF proteins on cellulose results in a loss of the biological activity of TF.

[0011] CBMs have been extensively employed in the engineering of enzymes and the modification of cellulose-based materials. Their utility spans a broad range of applications where specific binding is desirable. There is a precedence for the use of CBM-containing fusion proteins in applications such as in vitro diagnostics, affinity chromatography, and antimicrobial applications. However, CBM fused with blood coagulation factors for the purpose of providing a wound dressing has not previously been investigated.

[0012] Therefore, the present invention provides a versatile haemostasis-promoting system, in the form of a fusion protein that can be incorporated into various forms and types of dressing materials. Thus, the present invention provides a versatile, efficient, and safe haemostatic solution that enhances localized clot formation while minimizing systemic coagulation risks.SUMMARY OF THE INVENTION

[0013] The invention relates to a fusion protein comprising at least one blood coagulation factor or a variant thereof and one or more Cellulose Binding Module (CBM), wherein the CBM is linked to the C terminal of the blood coagulation factor.

[0014] In another aspect of the invention relates to a dressing material comprising the fusion protein, wherein the fusion protein is bound to the dressing material.

[0015] In a further aspect of the invention relates to a system for promoting rapid haemostasis while reducing the risk of undesirable blood coagulation, said system comprising the fusion protein bound to a dressing material.

[0016] In another aspect of the invention relates to the use of a fusion protein or a dressing material comprising the fusion protein to promote rapid haemostasis.

[0017] In another aspect of the invention relates to a method for preparing dressing material according to the present invention comprising the following steps:

[0018] (a) obtaining a fusion protein comprising sTF and at least one CBM,

[0019] (b) soaking the fusion protein from step (a) on to a dressing material capable of binding CBM, and

[0020] (c) optionally washing the dressing and drying with preservatives.

[0021] FIGURES

[0022] Figure 1 - Schematic representation of the basic principles of the invention. A fusion protein comprising a carbohydrate-binding module (CBM) linked to a blood coagulation factor (e.g., Tissue Factor) is shown bound to a dressing. The fusion protein promotes haemostasis by interacting with the coagulation cascade and initiating or enhancing fibrin clot formation. The dressing may further comprise additives such as calcium ions, preservatives, and stabilizers.

[0023] Figure 2 - Schematic illustration of various configurations of fusion proteins comprising CBM and blood coagulation factor domains. The CBM domain is indicated with a dottedline:

[0024] (A) Fusion protein with CBM directly linked to a blood factor without a linker; (B) Fusion protein with CBM and blood factor connected via a short linker; (C) Fusion protein with CBM and blood factor connected via a long linker; (D) Fusion protein comprising multiple CBM domains and a blood factor connected via a short linker;

[0025] (E) Fusion protein comprising multiple distinct CBM domains linked to a blood factor via a short linker;

[0026] (F) Fusion protein with CBM domains connected to multiple blood factors via linkers;

[0027] (G) Fusion protein with CBM domains and different blood factors at the N- and C-ter-mini, connected via linkers;

[0028] (H) Fusion protein with different CBM domains fused via short linkers and connected to different blood factors via long linkers;

[0029] (I) Fusion protein with several CBM domains arranged non-linearly and linked to blood factors via linkers;

[0030] (J) Fusion protein with several distinct CBM domains arranged non-linearly and connected to blood factors via linkers;

[0031] (K) Fusion protein with several CBM domains arranged non-linearly and connected to different blood factors via linkers.

[0032] Figure 3 - Schematic illustration of various combinations of fusion proteins comprising CBM and blood coagulation factor domains, and their association with dressings:

[0033] (A) Fusion protein with CBM and blood factor domains bound to a dressing to enhance coagulation;

[0034] (B) Fusion protein with multiple CBM domains and a blood factor connected via a short linker and bound to an absorbable or non-absorbable dressing containing storage additives;

[0035] (C) Fusion protein with CBM and blood factor domains connected via a long linker and bound to a dressing comprising enhancers and additives;

[0036] (D) Multiple fusion proteins with different CBM and blood factor domains, connected via short or long linkers and bound to a dressing comprising enhancers and additives.

[0037] Figure 4 - Schematic representation of various dressing formats bound with fusion proteins comprising CBM and blood coagulation factor domains (e.g., Tissue Factor), intended to promote haemostasis and stop bleeding:(A) Simple non-woven sheets or swabs incorporating detectable markers;

[0038] (B) Elongated sheets or rods;

[0039] (C) Threads or fiber bundles;

[0040] (D) Composite dressings comprising materials for heamostasis, liquid absorbance, and optional liners;

[0041] (E) Pellets or microspheres;

[0042] (F) Small expandable rods with detectable markers;

[0043] (G) Spear-shaped dressings designed for access to small bleeding sites;

[0044] (H) Elongated, semi-rigid dressings for laparoscopic use, optionally with detectable markers;

[0045] (I) Expandable balloon dressings for use in confined or soft tissue areas, such as during endoscopic procedures.

[0046] Figure 5 - Tissue Factor (TF) activity measurement of TF-CBM fusion proteins in solution. The TF fusion proteins were incubated with Factor VII, Factor X, and a Factor X chromogenic substrate in buffer at 37 °C. The UV absorbance at 405 nm was recorded over time (in seconds). The samples analyzed included the fusion proteins ADU112, ADU113, and ADU114; reference TF proteins P2 (3.1 pg / mL), P4 (0.75 pg / mL), and P6 (0.19 pg / mL); and assay blanks as negative controls.

[0047] Figure 6 - Tissue Factor activity measurement of TF-CBM fusion proteins in solution following buffer exchange. UV absorbance at 405 nm was recorded over time (in seconds). Samples included fusion proteins ADU112A, ADU113A, and ADU114A; reference TF proteins P2 (3.1 pg / mL), P4 (0.75 pg / mL), and P6 (0.19 pg / mL); and assay blank as control.

[0048] Figure 7 - Tissue Factor activity measurement of TF-CBM fusion proteins bound on various dressing materials (cut 5 mm diameter pieces). The bound TF was incubated with Factor VII, Factor X, and a Factor X chromogenic substrate in buffer at 37 °C. UV absorbance at 405 nm was monitored over time (in seconds). All TF-CBM fusion proteins were partially purified via ion exchange chromatography and equilibrated in low-salt buffer. Test samples included fusion proteins ADU112A (TF-CBM-cel6A with (GGGGSf linker), ADU113 A (TF-CBM-cel6A with (GGGGS)e linker), and ADU114A (TF-CBM-CBM3A with (GGGGS)e linker). Reference samples included assay blank, untreated dressing, and TF standards P2, P4, and P6.Figure 7A - Fusion proteins bound to SUGI® sponge made of cotton and regenerated cellulose.

[0049] Figure 7B - Fusion proteins bound to Dentist surgical dressing composed of non-woven fabric made of 70% rayon and 30% polyester.

[0050] Figure 7C - Fusion proteins bound to UPM-FibDex® dressing comprising nano-cellu-lose fibers on a polyethylene backbone.

[0051] Figure 8 - Summary of Tissue Factor activity for ADU112A after 60-minute incubation with UPM nano-cellulose dressing. Samples were subjected to three or six washing steps. Results were compared to assay blank, untreated dressing, and TF reference proteins P2, P4, and P6.

[0052] Figure 9A - Tissue Factor (TF) activity measurement of purified TF-CBM fusion proteins coupled to SUGI150 dressing pellets and compared to unbound proteins.

[0053] The samples were incubated with Factor VII, Factor X, and a Factor X chromogenic substrate in buffer at 37 °C. The UV absorbance at 405 nm was recorded over time (in seconds). The samples analyzed included the fusion proteins construct No.3 fraction A bound to SUGI150 pellets after 5, 15 or 30 minutes, untreated SUGI® pellets (blanks), and unbound fusion proteins construct No.3 fraction A in known concentrations.

[0054] Figure 9B - Tissue Factor (TF) activity measurement of purified TF-CBM fusion proteins coupled to SUGI150 dressing pellets and compared to unbound proteins.

[0055] The samples were incubated with Factor VII, Factor X, and a Factor X chromogenic substrate in buffer at 37 °C. The UV absorbance at 405 nm was recorded over time (in seconds). The samples analyzed included the fusion proteins construct No.3 fraction B bound to SUGI150 pellets after 5, 15 or 30 minutes, untreated SUGI® pellets (blanks), and unbound fusion proteins construct No.3 fraction B in known concentrations.

[0056] Figure 10 - Tissue Factor (TF) activity of TF-CBM-functionalized dressings, measured as time to initiation of solidification of human plasma. The figure compares construct No.3 fraction A and fraction B at different coupling times.

[0057] TF-CBM-SUGI pellets prepared with coupling times of 30 minutes (Figure 10A), 15 minutes (Figure 10B), and 5 minutes (Figure 10C) were evaluated. For each condition, the following samples were tested: (i) a native 5 mm SUGI® pellet, (ii) a TF-CBM-SUGI pellet, and (iii) an approximately 8 mm QuikClot® dressing piece. All measurements were performed in duplicateFigure 11 - Tissue Factor (TF) activity of TF-CBM-functionalized dressings, measured as time to initiation of solidification of human plasma. The figure shows TF-CBM fusion protein construct No.3 fraction A coupled to SUGI® dressing at varying protein loading levels of 100, 200, 400, 800, and 1600 ng per pellet. TF-CBM-SUGI pellets were prepared using a coupling time of 30 minutes. The following samples were evaluated: (i) a native 5 mm SUGI® pellet, (ii) a TF-CBM-SUGI pellet, and (iii) an approximately 8 mm QuikClot® dressing piece. All measurements were performed in duplicate.

[0058] Figure 12 - Tissue Factor (TF) activity measurement of four different purified TF-CBM fusion proteins (No.l-No.4) coupled to SUGI150 dressing pellets in doublets (A and B) and compared to TF references (P3, P4, P5) and untreated SUGI® pellet. The samples were incubated with Factor VII, Factor X, and a Factor X chromogenic substrate in buffer at 37 °C. The UV absorbance at 405 nm was recorded over time (in seconds).

[0059] DEFINITIONS

[0060] The term “fusion protein” as used herein, refers to a blood coagulation factor such as fibrinogen (Factor I), prothrombin (Factor II), Tissue Factor (TF - Factor III), the extracellular domain of Tissue Factor (sTF), Factor V, Provoncertin (Factor VII), Factor VIII, Christmas factor (Factor IX), Stuart-Prower protein (Factor X), Fator Xia, Hageman factor (Factor XII), or any variants thereof, including their activated counterpart and at least one cellulose binding module (CBM), linked to one another. The blood coagulation factor may be linked via either its N-terminus or C-terminus. Linked via the C -terminus is preferred. In particular, for extracellular TF domains, linkage via the C-terminus is preferred, as this configuration more closely mimics the native structure of the protein.

[0061] The term “Tissue Factor (TF)” as used herein, refers to Tissue Factor also known as Factor III or CD142, is a transmembrane glycoprotein with an extracellular domain functioning as a high-affinity receptor for coagulation Factor VII and activated Factor VII (Factor Vila). In the context of the present invention, the term “Tissue Factor” or “TF” refers to any mammalian Tissue Factor comprising at least the extracellular domain, either endogenous or recombinant, including mouse, rat, guinea pig, dog, cat,porcine, cow, and human Tissue Factor. The human Tissue Factor consists of 263 amino acids, constituting the extracellular, transmembrane, and cytoplasmic domains. The soluble Tissue Factor consists of 219 amino acids, corresponding to the extracellular domain only. The extracellular domain of human Tissue Factor also known as soluble Tissue Factor (sTF) has the following DNA and amino acid sequence (also shown in SEQ ID NO: 1 & 2, respectively).

[0062] In the context of the present invention, “a variant of Tissue Factor” is to be understood as any variant capable of binding Factor VII and / or Factor Vila, provided that the activity of Tissue Factor is maintained when bound to a dressing material for interaction with mammalian whole blood. Thus, the variant may be the extracellular domain of human Tissue Factor described above or any of the other Tissue Factor molecules or parts as described elsewhere herein. The ability of a Tissue Factor variant to bind Factor VII may be evaluated by any suitable interaction assay, such as standard pulldown assays using tagged versions of the respective proteins.

[0063] Accordingly, an variant of Tissue Factor may be defined as having at least an extracellular domain and sharing at least 59%, such as at least 69%, such as least 70%, such as at least 80%, such as at least 90%, or such as at least 95% sequence identity to SEQ ID NO: 2 and is capable of binding Factor VII and / or Factor Vila is to be understood as being part of the invention. Thus, the fusion protein according to the present invention may comprise a Tissue Factor protein having at least 59%, such as 60%, such as 70%, such as 80%, such as 90% or such as 95% sequence identity to SEQ ID NO: 2.

[0064] The term “Factor VII”, also referred to as blood-coagulation Factor Vila, activated blood coagulation Factor VII, or proconvertin (EC 3.4.21.21), in the context of the present invention is a 50-kDa, vitamin K-dependent zymogen synthesized in the liver. It plays a critical role in the initiation of tissue factor-induced coagulation, also known as the extrinsic pathway.

[0065] In the context of the present invention, the term "Factor VII" encompasses both the zymogen form and the activated form, the latter often referred to as Factor Vila. This includes recombinant human Factor Vila (Factor 7 alfa), commercially available as NovoSeven® (Novo Nordisk, Denmark), which has been developed for the treatment ofuncontrolled bleeding in hemophilia patients, as well as biosimilar forms of recombinant activated Factor VII, such as AryoSeven® (AryoGen, Iran).

[0066] The term “Fibrinogen” (Factor I) as used herein refers to the blood coagulation factor which is converted into fibrin to form the clot mesh.

[0067] The term “Prothrombin” (Factor II) as used herein refers to the blood coagulation factor, which is a precursor to thrombin (Factor Ila), which converts fibrinogen to fibrin.

[0068] The term “Factor V” as used herein refers to the blood coagulation factor, which is a cofactor helping in thrombin formation.

[0069] The term “Factor VII”, also known as “Proconvertin” or serum prothrombin conversion accelerator, as used herein, refers to the blood coagulation factor which is a key protein in the extrinsic pathway of the blood coagulation cascade. It binds to tissue factor (Factor III) to become Factor Vila, which converts Factor X to Xa. It also activates Factor IX. Factor VII is also to refer to recombinant versions, e.g., NovoSeven RT (Novo Nordisk, Denmark).

[0070] The term “Factor VIII” as used herein refers to the blood coagulation factor that is often bound to von Willebrand factor (vWF) and thereby stabilizes vWF. It is activated by thrombin to become Factor Villa, which is as a cofactor for Factor IXa. Together, they form the tenase complex activating Factor X to Factor Xa, which leads to thrombin generation and eventually fibrin clot formation. Factor VII is also to refer to recombinant versions, e.g., Efanesoctocog alfa (Altuviiio, Sanofi) and Turoctocog alfa (Novo8, Novo Nordisk, Denmark).

[0071] The term “Christmas factor” (Factor IX) as used herein refers to the blood coagulation factor which is part of the intrinsic clotting pathway. When activated by Factor Xia, it becomes Factor IXa, which amplifies clot formation. Factor IXa, with calcium and Factor Villa as a cofactor, forms the tenase complex. As described above, this complex activates Factor X to Xa, which leads to the thrombin burst, which creates a fibrin clot. Variants have improved properties, e.g. The Factor IX Fc fusion protein also known as Alprolix® (Swedish Orphan Biovitrium) extends the half-life of Factor IX, making it more efficient.The term “Stuart-Prower protein” (Factor X) as used herein refers to the blood coagulation factor which is an important enzyme that activates prothrombin. It is the key meeting point of the intrinsic and extrinsic pathways.

[0072] Modified Factor Xa proteins relevant to the present invention include An-dexanet alfa (Portola Pharmaceuticals, Astra Zeneca AB), a recombinant, catalytically inactive form of human Factor Xa. Andexanet alfa is engineered to act as a decoy receptor for Factor Xa inhibitors, including but not limited to apixaban and rivaroxaban. By binding to such inhibitors with high affinity, Andexanet alfa neutralizes their anticoagulant activity, thereby restoring or enhancing the physiological clotting function.

[0073] The term “Factor Xia” as used herein refers to the blood coagulation factor, which activates Factor IX to Factor IXa. Together with calcium and Factor Villa as a cofactor forms the tenase complex. This complex activates Factor X to Factor Xa that leads to the thrombin burst, creating a fibrin clot.

[0074] The term “Hageman factor” (Factor XII) as used herein refers to the blood coagulation factor, which is one of the proteins involved in the intrinsic pathway of the blood coagulation cascade. It gets activated into Factor Xlla when it comes in contact with e.g. negatively charged surfaces or other surfaces.

[0075] As used herein, the term “Fibrin-stabilizing factor” (Factor XIII) refers to a blood coagulation factor that functions to stabilize the fibrin clot by catalyzing the cross-linking of fibrin polymers. This enzymatic cross-linking results in the formation of a fibrin mesh with enhanced mechanical strength and increased resistance to fibrinolysis, thereby contributing to the durability and stability of the clot.

[0076] Factor XIII is a protransglutaminase zymogen, which is converted into its active form, Factor Xllla, a transglutaminase enzyme, upon activation by thrombin in the presence of calcium ions. The term also encompasses recombinant forms of Factor XIII, such as recombinant Factor XIII-A subunit (e.g., Tretten® / NovoThirteen®, Novo Nordisk, Denmark), which are functionally equivalent to plasma-derived Factor XIII and may be used interchangeably in the context of the invention.The term “Cellulose Binding Module (CBM)” as used herein, refers to non-catalytic protein domains that bind exhibit specific affinity for polymeric carbohydrate structures like cellulose or chitin and gelatines. They are often part of larger enzymes (e.g., cellulases) and help anchor the enzyme to its substrate.

[0077] The term “linker” as used herein refers to a chemical entity that connects two components, such as two molecules, proteins or protein domains by means of stable covalent bonding. A linker may for example be linear such as a PEG linker or branched linker. An example of a suitable linker is short peptide sequence which may be repeated more than one time to provide a longer more flexible linker. The linker may be designed to be flexible or rigid, by incorporating sterically hindered groups.

[0078] The term “dressing material” as used herein, refers to any material capable of binding the fusion protein and that does not enter systemic circulation via a bleeding wound, such as a traumatic injury. The dressing material may take various forms, for example it may be in a mechanically stable form or a mechanically unstable form. Examples of dressing materials which are mechanically stable include polymers, beads, matrices, solid carriers, patches, bandages, gauzes, membranes, barriers, compresses, scaffolds. Examples of dressing materials which are in a mechanically unstable form are gels, pastes, or suture materials. The dressing material may be in the form of a mechanically stable solid substrate, which may be fabricated in numerous physical configurations such as sheets, nets, woven or non-woven sheets, sutures, threads, tampons, foams, microspheres, balls, inflatable balloons, or spatulas. The dressing material may serve as a topical dressing for the local management of bleeding wounds, including lacerations, cuts, and abrasions, or be integrated into such a dressing. Preferably, the dressing possesses sufficient physical integrity to be inserted into or wrapped around a wound. The dressing is intended for direct contact with the wound, blood, or other fluids and is often secured in place using a bandage or compression dressing. The dressing may also be engineered to have a large surface area to enhance fluid absorption from the wound and optimize clot formation.

[0079] A dressing material comprising the fusion protein disclosed herein may also be in a flexible or mechanically unstable form, such as a glue, powder, spray, gel, granulate, mechanically compressed sponge or granulates, swellable sponge, gelatin, paste, fluid, or cream, intended for direct application to the wound. The fusion protein ispreferably bound to the dressing material, which may be a component of the dressing or fully integrated within it. This binding prevents the systemic administration of Tissue Factor, thereby enhancing the safety of the haemostatic dressing.

[0080] Suitable dressing materials must be capable of binding to a CBM sequence and are composed of carbohydrate polymers or blends thereof. Examples of carbohydrate polymers include but are not limited to cotton, cellulose, cellulose ethers, regenerated cellulose, oxidized cellulose, oxidized regenerated cellulose, carboxymethylcellulose, paper-based membranes, agarose, silk, catgut, chitosan and its derivatives, calcium alginate, and combinations or blends thereof.

[0081] The dressing material according to the invention may also comprise materials containing natural or synthetic polymers known to catalyze and enhance haemostasis, including carbohydrates such as chitosan and its derivatives, microfibrillar collagen, thrombin, poly-N-acetyl glucosamine, polyprolate acetate, D-glucosamine, and calcium alginate, are particularly suitable. Additionally, the dressing may incorporate inorganic or other salts that promote haemostasis, including calcium chloride or other haemostatic agents.

[0082] Dressing materials may be further classified as absorbable or non-absorba-ble, depending on whether they undergo natural degradation and absorption in vivo. In the context of the present invention, an absorbable material is one that retains structural and mechanical integrity when in contact with mammalian plasma for at least 15 minutes, preferably for at least 20 minutes, and up to several days, but not exceeding 200 days.

[0083] Absorbable dressing materials include cellulose, oxidized cellulose, silk, catgut, and synthetic polymers such as polyglycolic acid, polylactic acid, polydioxanone, and caprolactone, as well as polymer and co-block-polymer compositions derived from one or more of the following cyclic monomers: glycolide, lactide, p-dioxanone, trimethylene carbonate, or s-caprolactone. Absorbable dressing materials are particularly preferred for use in sutures. The suture thread may be monofilament or multifilament and optionally pre-coated with additional material, such as collagen, to facilitate binding of a CBM-Tissue Factor fusion protein.The person skilled in the art will appreciate that no single heamostatic dressing design, format, or material is universally optimal for controlling all types of bleeding, with the nature and location of the bleeding site significantly influencing the choice of the optimal dressing. The selection of appropriate material, design, shape, and format depends on the intended application, such as civilian first aid, emergency trauma, military use, open surgery, laparoscopic surgery, or endoscopic procedures.

[0084] For example, in military field conditions, ideal dressing should be designed for rapid packing into the wound cavity and application of compression before evacuation. Conversely, in controlled surgical environments, such as open liver resections, surgeons may prefer a flat dressing sheet to be wrapped around the bleeding site before compression. In laparoscopic procedures, dressings must be appropriately sized and flexible to fit within confined spaces and to allow for transport through a trocar. Similarly, endoscopic surgical procedures impose further requirements regarding the size and format of the dressing.

[0085] The term "immobilized", “linked” and "bound" may be used interchangeably and is in the context of the present invention to be understood as a binding of sTF via the at least one CBM to a dressing material having a strength sufficiently high to prevent sTF from being released from the dressing material under physiological conditions, e.g. when the system is exposed to mammalian whole blood. This may be achieved by providing a binding linker between sTF and a dressing material having a binding strength sufficiently high to prevent significant measurable leakage of the sTF-CBM fusion protein when measured by e.g. TF activity assays, radioactive or fluorescent label techniques. Preferably, the molecular binding applied to achieve immobilization of the Tissue Factor fusion protein to a dressing material preferably has a dissociation constant (Kd) of at least 10-5 mol / L, more preferably at least 10-8 mol / L at the relevant physiological conditions at body temperature.

[0086] The term “suitable additives” as used herein is to be understood as additives that promote haemostasis, enhance the stability of the dressing material, improve the consistency / formulation of the dressing material. Suitable additives also encompass other active agents such as painkillers and relievers and antimicrobial agents. Examples of suitable additives include coagulation factors or components thereof, such as Factor VII or Factor Vila, as well as other agents capable of inducing, promoting, or modulating thecoagulation pathway. Such additives may include blood coagulation proteins, fibrin, thrombin, stabilizing agents, detectable markers, and combinations thereof.

[0087] System (for promoting rapid haemostasis)

[0088] A system according to the present invention comprises (i) a fusion protein of at least one blood coagulation factor, such as Tissue Factor, preferably the extracellular domain, or any variant thereof; and (ii) one or more cellulose-binding module (CBM) bound to a dressing material.

[0089] The term “haemostasis” and “rapid haemostasis” as used herein refers to a fundamental physiological process that prevents excessive blood loss following vascular injury. A critical event in this process is the thrombin burst, which plays a key role in stabilizing blood clots by ensuring a rapid and amplified generation of thrombin during the coagulation cascade. The coagulation process is typically divided into three main phases: initiation, amplification, and propagation, culminating in the formation of a stable fibrin clot.

[0090] Initiation Phase: Upon vascular injury, Tissue Factor (TF) is exposed to plasma proteins, triggering the coagulation cascade. TF binds to Factor Vila, leading to the activation of Factor X into Factor Xa. Factor Xa catalyzes the conversion of prothrombin (Factor II) into small amounts of thrombin (Factor Ila).

[0091] Amplification Phase (Thrombin Burst): The initial generation of thrombin serves as a catalyst for an amplification loop, activating multiple coagulation factors: Factor V to Factor Va (cofactor for Factor Xa), Factor VIII to Factor Villa (cofactor for Factor IXa), Factor XI to Factor Xia (which further activates Factor IX) and platelets, making them procoagulant surfaces that enhance clot formation.

[0092] Propagation Phase (Massive Thrombin Generation): Activated Factor IXa, in combination with Factor Villa, forms the tenase complex, further boosting Factor Xa production. Factor Xa, along with Factor Va, forms the prothrombinase complex, catalyzing the rapid conversion of prothrombin into thrombin. The resulting thrombin surge converts fibrinogen into fibrin, leading to clot stabilization. Additionally, thrombin activates Factor XIII, which cross-links fibrin strands, reinforcing the clots mechanical strength and integrity.The thrombin burst is a pivotal mechanism ensuring the rapid and effective formation of stable clots. It plays several essential roles including:

[0093] (1) accelerated clot formation at the injury site, crucial for minimizing blood loss. (2) sufficient thrombin production to efficiently convert fibrinogen into fibrin, ensuring proper clot stabilization,

[0094] (3) enhanced platelet activation, reinforcing clot integrity and mechanical strength, and

[0095] (4) prevention of excessive bleeding by creating a robust fibrin network.

[0096] Assessment of Haemostatic Dressing Efficacy

[0097] Under normal physiological conditions, whole blood in a static environment coagulates spontaneously within an average timeframe of 10 to 30 minutes, though this duration exhibits substantial variability among individuals. Factors influencing coagulation time include, but are not limited to, gender, age, ethnicity, general physical condition, underlying diseases, and medication use, particularly anticoagulants.

[0098] The effectiveness of an active haemostatic dressing is best quantified by its ability to reduce the time to haemostasis when compared to a passive dressing or the absence of any dressing. A key metric in evaluating such dressings is the duration for which the dressing must be held against the bleeding site to achieve haemostasis.

[0099] A reduction in time to haemostasis, measured in minutes, is considered indicative of enhanced haemostatic efficacy. Even a one-minute reduction provides practical clinical benefits, while a reduction of two minutes or more constitutes a significant improvement. In certain cases, the effectiveness of the dressing may determine whether bleeding cessation is achieved upon application of pressure to the dressing at the bleeding site.

[0100] In cases of arterial bleeding, where blood pressure is high, the formation of a blood clot is not sufficient on its own; the mechanical strength of the clot is also crucial to ensure continued haemostatic control after removal of the dressing.Uncontrolled and Undesirable Haemostasis or Blood Coagulation

[0101] Any haemostasis or blood coagulation initiated by the haemostatic dressing within the vascular system or organs remote from the bleeding site is considered uncontrolled and undesirable. A haemostatic dressing that induces or initiates haemostasis away from the intended application site may contribute to the formation of thrombi, potentially resulting in vascular occlusion in critical anatomical regions.

[0102] Obstruction of blood vessels in the brain due to thrombi formation can lead to ischemic strokes, while occlusion of coronary arteries may cause myocardial infarctions. Additionally, thromboembolic events originating from deep veins in the lower extremities, pelvis, or abdomen may travel through the circulatory system and obstruct pulmonary arteries, leading to pulmonary embolism. Therefore, the prevention of undesirable blood coagulation is an essential requirement for ensuring the safety of a haemostatic dressing.

[0103] The ability of a dressing to induce efficient haemostasis can be measured in human experiments and certain animal models. The ability of a dressing comprising a fusion protein comprising Tissue Factor can be, for example, measured in a standard Thromboelastographic (TEG) viscoelastic haemostatic assay or a simplified version of the Thromboelastographic (TEG) viscoelastic haemostatic assay as described in the examples below herein. A standard Thromboelastographic (TEG) viscoelastic haemostatic assay allows for the measure of the total viscoelastic response of clot formation initiated by Tissue Factor, whether in free solution or bound to a dressing. The standard Thromboelastographic (TEG) viscoelastic haemostatic assay can be performed using whole blood, a mixture of thrombocytes and plasma proteins, or pure plasma.

[0104] Sequences referred to in the present invention are presented below in Table 1. Any part of the sequence that is underlined is referring to the signal peptide of the protein.

[0105] Table 1: Sequence list

[0106] SEQ Type Sequence

[0107] ID NO:

[0108] 1 DNA

[0109]

[0110] tcaggcactacaaatactgtggcagcatataatttaacttggaaatcaactaatttcaagacaattttggagtgggaacccaaacccgtcaatcaagtctacactgttcaaataagcact aagtcaggagattggaaaagcaaatgcttttacacaacagacacagagtgtgacctcacc gacgagattgtgaaggatgtgaagcagacgtacttggcacgggtcttctcctacccggca gggaatgtggagagcaccggttctgctggggagcctctgtatgagaactccccagagttc acaccttacctggagacaaacctcggacagccaacaattcagagttttgaacaggtggga acaaaagtgaatgtgaccgtagaagatgaacggactttagtcagaaggaacaacactttc ctaagcctccgggatgtttttggcaaggacttaatttatacactttattattggaaatct tcaagttcaggaaagaaaacagccaaaacaaacactaatgagtttttgattgatgtggat aaaggagaaaactactgtttcagtgttcaagcagtgattccctcccgaacagttaaccgg aagagtacagacagcccggtagagtgtatgggccaggagaaaggggaattcagagaatga - taa

[0111] ProSGTTNTVAAY NLTWKSTNFK TILEWEPKPV NQVYTVQIST tein KSGDWKSKCF YTTDTECDLT DEIVKDVKQT YLARVFSYPA (aa) GNVESTGSAG EPLYENSPEF TPYLETNLGQ PTIQSFEQVG TKVNVTVEDE RTLVRRNNTF LSLRDVFGKD LIYTLYYWKS SSSGKKTAKT NTNEFLIDVD KGENYCFSVQ AVIPSRTVNR KSTDSPVECM GQEKGEFRE

[0112] Protein METPAWPRVP RPETAVARTL LLGWVFAQVA GASGTT- (aa) NTVA AYNLTWKSTN FKTILEWEPK PVNQVYTVQI STKSGDWKSK CFYTTDTECD LTDEIVKDVK QTYLARVFSY PAGNVESTGS AGEPLYENSP EFTPYLETNL GQPTIQSFEQ VGTKVNVTVE DERTLVRRNN TFLSLRDVFG KDLIYTLYYW KSSSSGKKTA KTNTNEFLID VDKGENYCFS VQAVIPSRTV NRKSTDSPVE CMGQEKGEFR EGGGGSGGGG SGGGGSCSSV WGQCGGQNWS GPTCCASGST CVYSNDYYSQ CL

[0113] Protein METPAWPRVP RPETAVARTL LLGWVFAQVA GASGTT- (aa) NTVA AYNLTWKSTN FKTILEWEPK PVNQVYTVQI STKSGDWKSK CFYTTDTECD LTDEIVKDVK QTYLARVFSY PAGNVESTGS AGEPLYENSP EFTPYLETNL GQPTIQSFEQ VGTKVNVTVE DERTLVRRNN TFLSLRDVFG KDLIYTLYYW KSSSSGKKTA KTNTNEFLID VDKGENYCFS VQAVIPSRTV NRKSTDSPVE CMGQEKGEFR EGGGGSGGGG SGGGGSGGGG SGGGGSGGGG SCSSVWGQCG GQNWS- GPTCC ASGSTCVYSN DYYSQCL

[0114] Protein METPAWPRVP RPETAVARTL LLGWVFAQVA GASGTT-

[0115]

[0116] (aa) NTVA AYNLTWKSTN FKTILEWEPK PVNQVYTVQISTKSGDWKSK CFYTTDTECD LTDEIVKDVK QTYLARVFSY PAGNVESTGS AGEPLYENSP EFTPYLETNL GQPTIQSFEQ VGTKVNVTVE DERTLVRRNN TFLSLRDVFG KDLIYTLYYW KSSSSGKKTA KTNTNEFLID VDKGENYCFS VQAVIPSRTV NRKSTDSPVE CMGQEKGEFR EGGGGSGGGG SGGGGSPVSG NLKVEFYNSN PSDTTNSINP QFKVTNTGSS AIDLSKLTLR YYYTVDGQKD QTFWCDHAAI IGSNGSYNGI TSNVKGTFVK MSSSTNNADT YLEISFTGGT LEPGAHVQIQ GRFAKNDWSN YTQSNDYSFK SASQFVEWDQ VTAYLNGVLV WGKEP

[0117] 6 Protein METPAWPRVP RPETAVARTL LLGWVFAQVA GASGTT- (aa) NTVA AYNLTWKSTN FKTILEWEPK PVNQVYTVQI STKSGDWKSK CFYTTDTECD LTDEIVKDVK QTYLARVFSY PAGNVESTGS AGEPLYENSP EFTPYLETNL GQPTIQSFEQ VGTKVNVTVE DERTLVRRNN TFLSLRDVFG KDLIYTLYYW KSSSSGKKTA KTNTNEFLID VDKGENYCFS VQAVIPSRTV NRKSTDSPVE CMGQEKGEFR EGGGGSGGGG SGGGGSGGGG SGGGGSGGGG SPVSGNLKVE FYNSNPSDTT NSINPQFKVT NTGSSAIDLS KLTLRYYYTV DGQKDQTFWC DHAAIIGSNG SYNGITSNVK GTFVKMSSST NNADTYLEIS FTGGTLEPGA HVQIQGRFAK NDWSNYTQSN DYSFKSASQF VEWDQVTAYL NGVLVWGKEP

[0118] 7 Protein METPAWPRVP RPETAVARTL LLGWVFAQVA GA

[0119] (aa)

[0120] 8 Protein GGGGS

[0121] (aa)

[0122] 9 ProMETPAWPRVP RPETAVARTL LLGWVFAQVA GASGTT- tein NTVA AYNLTWKSTN FKTILEWEPK PVNQVYTVQI (aa) STKSGDWKSK CFYTTDTECD LTDEIVKDVK QTYLARVFSY PAGNVESTGS AGEPLYENS PEFTPYLETN LGQPTIQSFE QVGTKVNVTV EDERTLVRRN NTFLSLRDVF GKDLIYTLYY WKSSSSGKKT AKTNTNEFLI DVDKGENYCF SVQAVIPSRT VNRKSTDSPV ECMGQEKGEF REIFYIIGAV VFVVIILVII LAISLHKCRK AGVGQSWKEN SPLNVS

[0123] 10 ProCSSV WGQCGGQNWS GPTCCASGST CVYSNDYYSQ CL tein

[0124] (aa)

[0125] 11 ProPVSG NLKVEFYNSN PSDTTNSINP QFKVTNTGSS AIDtein LSKLTLR YYYTVDGQKD QTFWCDHAAI IGSNGSYNGI (aa) TSNVKGTFVK MSSSTNNADT YLEISFTGGT LEPGAHVQIQ GRFAKNDWSN YTQSNDYSFK SASQFVEWDQ

[0126]

[0127] VTAYLNGVLV WGKEP

[0128] DETAILED DESCRIPTION OF THE INVENTION

[0129] The present invention relates to a fusion protein comprising (i) at least one blood coagulation factor, such as Tissue Factor (TF), or any variant thereof; and (ii) oneor more Cellulose Binding Module (CBM), wherein the one or more CBM is linked to the at least one blood coagulation factor, optionally through a linker. The present invention also relates to a dressing material comprising said fusion protein, wherein the fusion protein is bound to the dressing material. The present invention further relates to a system comprising the dressing material binding the fusion protein wherein the system is capable of providing rapid haemostasis while reducing the risk of undesirable blood coagulation.

[0130] The system promotes rapid and sustained haemostasis. More specifically, the system promotes rapid haemostasis initiated by a blood coagulation factor bound on a dressing material, so that the compound is prevented from being released into the blood stream. Thereby, the compound is capable of initiating coagulation starting directly at the site of application of the blood coagulation factor bound to the dressing material placed directly onto the vascular injury. This creates efficient haemostasis that, at the same time, is safe to use.

[0131] The invention thus allows local application of an active extrinsic agent with or without co-agents to the site where they exert their pharmacological effect by direct interaction with Factor Vll / VIIa or any other component of the haemostasis cascade components. Therefore, the extrinsic agent is prevented from entering the vascular bed of the organism and thereby hindering intra-vascular systemic exposure.

[0132] In one aspect, the invention relates to a fusion protein comprising (i) at least one blood coagulation factor or any variants thereof, and (ii) one or more Cellulose Binding Module (CBM), wherein the CBM is linked to the at least one blood coagulation factor, optionally bound by a linker.

[0133] In one or more aspects, the invention relates to a fusion protein comprising (i) at least one blood coagulation factor or any variants thereof, and (ii) one or more Cellulose Binding Module (CBM), wherein the CBM is bound to the at least one blood coagulation factor, optionally bound by a linker, and wherein said at least one blood coagulation factor is Tissue Factor (TF) as set forth in SEQ ID NO: 9 or as set forth in SEQ ID NO: 2, or any variant thereof.

[0134] Despite the well-characterized role of the thrombin burst in coagulation, existing haemostatic technologies face significant challenges in achieving efficientthrombin generation, particularly in wound dressings and topical haemostatic applications.

[0135] In one or more embodiments, the at least one blood coagulation factor is selected from the group consisting of Fibrinogen (Factor I), Prothrombin (Factor II), Tissue Factor (Factor II), Proconvertin (Factor VII), Factor XIII, Christmas factor (Factor IX), Stuart-Prower protein (Factor IX), Factor Xia, and Hageman factor (Factor XII).

[0136] In a particular embodiment, the at least one blood coagulation factor is Tissue Factor (TF) as set forth in SEQ ID NO: 9, preferably the extracellular domain of TF as set forth in SEQ ID NO: 2, or any variant thereof.

[0137] In another particular embodiment, the at least one blood coagulation factor is Factor VII. In another particular embodiment, the at least one blood coagulation factor is Factor XII. In another particular embodiment, the at least one blood coagulation factor is Factor VIII.

[0138] It is advantageous to simultaneously initiate both the intrinsic and extrinsic pathways of the blood coagulation cascade. In one embodiment, this is achieved by combining Factor III (Tissue Factor), Factor XII (Hageman factor, a serine protease zymogen), and optionally Factor IV (calcium ions). The presence of these components promotes the activation of both coagulation pathways, thereby enhancing the efficiency and speed of clot formation.

[0139] In a further preferred embodiment, Tissue Factor is used in combination with Factor VII or a Factor VIII mimetic. Most preferably, the Factor VIII mimetic is selected from Emicizumab (Chugai Pharmaceutical Co., Ltd / Roche) or Mim8 (Novo Nordisk), both of which are bispecific antibodies capable of bridging activated Factor IX and Factor X, thereby functionally mimicking the activity of Factor VIII.

[0140] Emicizumab and Mim8 are recombinant proteins expressed in mammalian cell systems, with known amino acid sequences and corresponding gene sequences. These proteins may be incorporated into fusion constructs, for example by linkage to CBMs, to facilitate their immobilization onto dressing materials. Also, both proteins may be combined unbound with blood factor proteins bound to the dressing.This combination is particularly relevant for the treatment of bleeding in individuals suffering from hemophilia A, a disorder characterized by deficient or defective Factor VIII activity.

[0141] It is to be understood that the purpose of immobilizing selected coagulation factors onto wound dressings is primarily to limit systemic exposure and dilution by a blood flow. Certain coagulation factors may be more safely bound on a dressing, as they may pose a thrombosis risk if administered systemically or at high local concentrations.

[0142] It has been found that a localized concentration of Tissue Factor triggers haemostasis rapidly due to sustained thrombin generation at the site of injury. It is further believed that any localized blood coagulation factors as described herein will have the similar effect. In particular, the finding of TF has led to the development of an efficient system comprising Tissue Factor which enables a controlled and effective method for rapid and localized haemostasis in profuse bleeding scenarios while preventing systemic dissemination of Tissue Factor into the vascular bed.

[0143] The inventors of the present invention have surprisingly found that linking at least the extracellular domain of TF (sTF) to at least one Cellulose Binding Module results in an improved system for rapid haemostasis. The fusion protein comprising sTF and at least one CBM according to the invention demonstrates an unexpected and advantageous combination of properties. Specifically, the fusion protein retains its biological activity while being immobilized onto cellulose-based substrates, thereby offering superior heamostatic potential in wound dressings. The use of a fusion protein comprising tissue factor (TF) and at least one cellulose-binding module (CBM) addresses both technical and safety challenges associated with TF-based hemostatic dressings.

[0144] In particular, unbound TF present on or released from a dressing may diffuse into the systemic circulation, thereby increasing the risk of systemic exposure and undesired thrombotic events. By providing TF as part of a fusion protein comprising at least one CBM, the TF is immobilized through binding to a cellulose-containing dressing material. This immobilization significantly reduces the risk of TF release and subsequent systemic exposure, thereby improving the safety profile of the dressing.

[0145] Furthermore, the fusion protein according to the present invention addresses challenges related to the manufacturing of TF-based dressings. Moreconventional approaches rely on chemical activation of the dressing material, washing procedures and subsequent covalent coupling of TF, typically involving reactive crosslinking agents, organic solvents and multiple processing steps, followed by quenching excess reactive sites and extensive washing. In contrast, the fusion protein comprising TF and at least one CBM is capable of binding directly, rapidly, and with high affinity to the dressing material in an aqueous environment, without the need for prior chemical modification of either the dressing or the protein.

[0146] As a result, the manufacturing process according to the present invention requires fewer process steps and avoids the use of reactive chemical coupling agents. This leads to reduced material consumption and waste generation. Additionally, the simplified process is readily scalable, facilitating large-scale production of hemostatic dressings, including in situations requiring rapid and high-volume manufacturing, such as emergency or military settings.

[0147] It is known in the art that fusion proteins incorporating CBMs exhibit strong binding to cellulose substrates through well-characterized interactions. Similarly, Tissue Factor is an essential cofactor for Factor Vll / VIIa, playing a pivotal role in the coagulation cascade. However, prior attempts to immobilize TF onto substrates have resulted in an alteration of its biological activity. For example, Tissue Factor immobilized on dressing materials using the chemical conjugation method disclosed in WO2022058452A1, exhibited a slower reaction rate and a delayed signal development over time, as measured in a Tissue Factor activity assay. Furthermore, due to the inherent properties of CBMs, which typically contain aromatic residues forming hydrophobic planar surfaces crucial for cellulose binding, it would be expected that an attached highly positively charged TF segment (with 14 lysines) potentially could interfere with the CBM function through electrostatic or in particular pi-cation interactions. Such interactions could induce conformational changes of lead to the structural destabilization of TF, thereby reducing its ability to bind Factor VII and prevent initiation of initiate the clotting cascade. Likewise, internal interactions between the CBM and TF - particularly ly-sins - will impair binding of the CBMs to the cellulose material. Given these biochemical considerations, a person skilled in the art would have reasonably assumed that a fusion protein combining CBMs with TF would suffer from impaired functional performance.Contrary to these expectations, the inventors have surprisingly discovered that the sTF-CBM fusion protein binds to cellulose and, when immobilized onto cellulose-based dressing materials, retains its full biological activity. The TF activity, as measured by the activation of Factor VII, Factor X, and the cleavage of a chromogenic peptide substrate, remains substantially unchanged when compared to free, unbound TF. This finding is highly unexpected, as conventional immobilization techniques typically compromise TF function. The ability of the fusion protein to retain its full biological activity allows for efficient clotting initiation upon Factor Vll / VIIa binding.

[0148] Furthermore, various commercially available dressing materials, when treated with the fusion protein in a single-step process followed by washing, demonstrated remarkably high heamostatic potential without requiring additional processing steps. This result is highly surprising, as the prior art suggests that efficient TF immobilization generally necessitates complex chemical modifications and finetuning of reaction conditions.

[0149] Additionally, it was observed that the TF density on the dressing materials reached unexpectedly high levels, further supporting the efficiency of the immobilization method. This is particularly surprising given that no specific cellulose substrate optimization was required, nor were hazardous reagents necessary to achieve stable TF immobilization.

[0150] Binding a blood coagulation factor, such as sTF, to a Cellulose Binding Module facilitates the downstream immobilization onto different cellulose-containing dressings without the need for prior material-specific optimization. Furthermore, the fusion protein comprising a blood coagulation factor and a cellulose binding module can be bound to cellulose substrates in an aqueous buffer without the need for reactive chemicals or additional reactive steps. This significantly improves manufacturability, scalability, and commercial feasibility.

[0151] The fusion protein therefore provides for versatility and compatibility with a broad range of cellulose materials as well as other haemostatic technologies. The fusion protein is compatible with a range of existing dressing formulations, including those containing zeolite, kaolin, chitosan, oxidized regenerated cellulose (ORC), fibrinogen, orthrombin. Therefore, the present invention provides a to the long-standing challenge of immobilizing active Tissue Factor onto cellulose-based wound dressings.

[0152] A blood coagulation factor or a variant thereof may be directly linked to at least one CBM. It is preferred that the CBM is linked to the C terminal of the blood coagulation factor or any variant thereof. Alternatively, the CBM may be bound by a linker to the blood coagulation factor or any variant thereof.

[0153] In one or more embodiments, the fusion protein comprises a linker having a sequence of (GGGGS)x, wherein x is any number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one or more embodiments, X is any number selected from 1, 2, 3, 4, 5, 6, 7, or 8. In one or more embodiments, a blood coagulation factor or any variant thereof is directly linked to the CBM(s). In one or more embodiments the CBM(s) is / are linked to the C terminal of a blood coagulation factor or any variant thereof. In one or more embodiments, the CBM(s) may be bound by a linker to a blood coagulation factor or any variant thereof. In one or more embodiments the linker has a sequence of (GGGGS)x, wherein x is any number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In one or more embodiments, X is any number selected from 1, 2, 3, 4, 5, 6, 7, or 8.

[0154] To enhance flexibility and reduce potential steric hindrance or unwanted surface interactions, it is advantageous to incorporate a linker sequence between the CBM and a blood coagulation factor component. Such linker sequences may be designed to optimize spatial separation and preserve the functionality of both protein domains.

[0155] The fusion protein construct may be further modified to include multiple CBM or blood coagulation factors in order to enhance overall activity or improve binding affinity to a substrate, such as a wound dressing. These multivalent constructs may be arranged in linear, dendritic, or comb-like configurations. Furthermore, the CBM and / or blood coagulation factors within such constructs may be identical in amino acid sequence or may comprise different sequences designed to achieve complementary or synergistic functions. Similarly, linker sequences may be varied to optimize the combined performance in terms of blood coagulation activity and substrate binding.

[0156] The individual monomeric units within a multimeric fusion protein may also be identical or non-identical in amino acid sequence, as required for the intendedapplication. In general, the fusion protein constructs are preferably of a size that permits efficient expression in suitable host systems, facilitates purification, and ensures sufficient stability for storage and application.

[0157] For practical reasons, one or more purification tags — such as biotin, thiobiotin, polyhistidine (His-tag), or other affinity tags — may be included in the construct. These tags may be optionally removed prior to application, such as prior to binding to a wound dressing or other substrate.

[0158] It is preferred that the linker used between functional domains, such as a CBM and a blood coagulation factor, is selected to minimize any interference with the structure or function of the CBM, the dressing substrate, or the tissue factor domain. Preferably, the linker does not form hairpin structures, is semi-rigid and extended at physiological pH, is resistant to enzymatic cleavage, exhibits thermostability, and is compatible with expression in the selected recombinant production system.

[0159] In practice, preferred linkers are non-aromatic and may be repeated in tandem to fine-tune both the overall linker length and the spatial separation between the CBM and blood coagulation factors. Furthermore, such linker systems may be applied in both linear constructs and more complex architectures, including dendritic, comb-like, or star-shaped fusion protein configurations.

[0160] One example of a suitable linker fulfilling the above criteria is the GGGGS linker, a flexible peptide sequence comprising Gly-Gly-Gly-Gly-Ser (GtS) (SEQ ID NO: 8). This linker is commonly used in recombinant fusion proteins and can be repeated (e.g., (GGGGS)n) to adjust inter-domain spacing. The GGGGS linker is known for its resistance to proteolytic degradation and has been successfully applied in fusion proteins intended for use in challenging environments.

[0161] In one or more embodiments, the fusion protein comprises Tissue Factor (TF) as set forth in SEQ ID NO: 9 or as set forth in SEQ ID NO:2 or any variant thereof and at least one CBM, wherein the CBM is bound to the TF via a linker having a sequence of (GGGGS)x, wherein X is any number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In some preferred embodiments, X is 3. In one or more preferred embodiments, X is 6.In one or more embodiments, the fusion protein comprises a sTF having a sequence according to SEQ ID NO:3. In one embodiment, the fusion protein comprises sTF having a sequence according to SEQ ID NO:4. In one or more embodiments, the fusion protein comprises sTF having a sequence according to SEQ ID NO:5. In one or more embodiments, the fusion protein comprises sTF having a sequence ID according to SEQ IDNO:6.

[0162] In one or more embodiments, the fusion protein comprises TF which has at least 59%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, or such as at least 95% sequence identity to SEQ ID NO: 2. In one or more embodiments, TF has at least 59% sequence identity to SEQ ID NO:2. In one or more embodiments, TF has at least 60% sequence identity to SEQ ID NO:2. In one or more embodiments, TF has at least 70% sequence identity to SEQ ID NO:2. In one or more embodiments, TF has at least 80% sequence identity to SEQ ID NO:2. In one or more embodiments, TF has at least 90% sequence identity to SEQ ID NO:2. In one or more embodiments, TF has at least 95% sequence identity to SEQ ID NO:2.

[0163] The mammalian coagulation pathway is a highly conserved and robust mechanism. Applying well-known alignment software, such as Standard Protein BLAST from the National Center for Biotechnology Information (NCBI), it can be appreciated that the amino acid sequence (SEQ ID NO: 2) of the extracellular domain of human Tissue Factor shares: 59% identity with the extracellular domain of mouse Tissue Factor, 59% identity with the Norwegian rat, 69% identity with the guinea pig, 79% identity with the cat, 76% identity with the dog, 75% identity with cattle, and 74% identity with the Pig-

[0164] In addition to the conservation of the Tissue Factor amino acid sequence across species, recombinant soluble human Tissue Factor has been demonstrated to initiate coagulation when exposed to rat whole blood and similarly when exposed to porcine whole blood.

[0165] Motifs involved in interactions with FVIIa include amino acid numbers 16 to 24 (aal6-aa24), amino acid numbers 37 to 51 (aa37-aa51), amino acid numbers 56 to 61 (aa56-aa61), amino acid numbers 74 to 76 (aa74-aa76), amino acid numbers 91 to 96 (aa91-aa96), amino acid numbers 109 to 110 (aal09-aal 10), amino acid numbers 128 to135 (aal28-aal35), amino acid number 140 (aal40), amino acid numbers 158 to 164 (aal58-aal64), amino acid numbers 186 to 188 (aal86-aal88), and amino acid numbers 203 to 209 (aa203-aa209) of the sTF as set forth in SEQ ID NO: 2. Conserved sequences across species include amino acids 8 to 79 (aa8-aa79) of SEQ ID NO: and amino acid numbers 91 and 214 (aa91-aa214) of SEQ ID NO: 2.

[0166] Particularly conserved sequences include amino acid numbers 9 to 70 (aa9-aa70) and amino acid numbers 108 to 210 (aal08-aa210), with even higher conservation in amino acid numbers 17 to 19 (aal7-aal9), amino acid numbers 34 to 39 (aa34-aa39), amino acid numbers 52 to 68 (aa52-aa68), amino acid numbers 125 to 129 (aal25-aal29), amino acid numbers 132 to 137 (aal32-aal37), amino acid numbers 159 to 167 (aal59-aal67), amino acid numbers 184 to 187 (aal84-aal87), and amino acid number 209 (aa209) of sTF as set forth in SEQ ID NO: 2. The high conservation of these sequences reflects the overall strong homology and identity of the extracellular domains of Tissue Factor across species.

[0167] A variant of Tissue Factor, as used herein, can be a protein comprising one or more of the above-described conserved sequences or motifs.

[0168] Furthermore, Andrews et al. provide evidence that the functional activity of TF is maintained across species. Based on this knowledge, a skilled person can determine which amino acid variations — down to at least 59% sequence identity — can be introduced while preserving the functional integrity of TF in accordance with the present invention. As described in US 2018 / 0111977, the extracellular domain of TF alone is capable of initiating clot formation (e.g., see Example 10, page 22), demonstrating that the soluble expressed protein is sufficient to trigger the coagulation cascade.

[0169] These findings further suggest that glycosylation may influence the stability, folding, and immunogenicity of the protein. However, TF produced in E. coh. which lacks glycosylation, is known to retains specific biological activity relevant to the present invention.

[0170] In one or more embodiments, the fusion protein comprises TF or the variant thereof is of human, porcine, equine, canine or bovine origin. In one embodiment, the fusion protein comprises TF or a variant thereof of human origin. In one embodiment,the fusion protein comprises TF or a variant thereof of porcine origin. In one embodiment, the fusion protein comprises TF or a variant thereof of equine origin. In one embodiment, the fusion protein comprises TF or a variant thereof of canine origin. In one embodiment, the fusion protein comprises TF or a variant thereof bovine origin.

[0171] In one or more embodiments, TF or a variant thereof is the extracellular domain of human TF. The extracellular domain of human TF is also referred to as sTF.

[0172] The fusion protein of the invention may be a fusion of any blood coagulation factors, such as anyone of Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XII, and Factor XIII or any combination thereof. Thus, in one embodiment, the fusion protein comprises at least one blood coagulation factor selected form the group consisting of: Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XII, and Factor XIII, or any combination thereof.

[0173] In one or more embodiments, the fusion protein comprises a CBM selected from the group consisting of: CBM1, CBM2, CBM3, CBM4, CBM5, CBM17, CBM28, CBM12, CBM18, CBM33, CBM20, CBM25, CBM48, CBM32, CBM35, CBM6a andCBM6b.

[0174] Selection and Classification of CBMs

[0175] The selection or design of CBMs may be achieved by reviewing the scientific literature for efficient carbohydrate binders, often found in natural enzymes such as plant cell wall-degrading enzymes (e.g., cellulases, amylases) or industrially optimized enzymes used for carbohydrate hydrolysis or processing.

[0176] Numerous CBM families are cataloged in bioinformatics databases, including the CAZy (Carbohydrate-Active enZYmes) database (www.cazy.org). This database classifies CBMs into families, assigns CBM numbering, and provides detailed structural and ligand specificity information.

[0177] Currently, more than 80 CBM families have been identified and characterized based on sequence similarity, structural motifs, and carbohydrate-binding specificity. CBMs with >30% sequence identity are grouped within the same family, whilestructurally conserved CBMs with low sequence similarity are classified together. Each family is assigned a unique numerical designation (e.g., CBM1, CBM2, etc.).

[0178] It is noted that CBMs from the same family may exhibit binding affinity for different carbohydrates. Moreover, carbohydrate-based materials contain structural variations, leading to diverse binding interactions with CBMs. The binding specificity and efficiency are influenced by the carbohydrate’s physical structure and accessibility, as well as environmental conditions such as temperature, pH, and ionic strength. Ionic strength, particularly salt concentration, can modulate CBM binding. Typical binding conditions include low salt concentrations (<100 mM) and pH ranges between 5 and 9, although significant variation exists among CBM families.

[0179] Thermophilic CBMs

[0180] Thermophilic CBMs (e.g., from Clostridium thermocellum) often have high stability and functionality under elevated temperature conditions. Thermophilic CBMs are therefore preferred for heamostatic devices due to their enhanced stability under extreme conditions. Relevant thermophilic CBMs include: CBM3 and CBM4 (binding to cellulose), CBM5 (binding to chitin), CBM9 (binding to xylan and hemicellulose), and CBM22 (binding to xylan and arabinoxylan).

[0181] Preferred CBMs for Specific Substrates

[0182] The following CBM-substrate combinations are particularly relevant to the present invention for the formation of safe topical heamostats:

[0183] Cellulose Binding CBMs

[0184] The efficiency of CBM binding to cellulose depends on the type of cellulose (e.g., crystalline vs. amorphous). Relevant CBM families include: CBM1 (high affinity for crystalline cellulose, utilizing a planar aromatic surface for hydrophobic stacking with cellulose fibers), CBM2 (binding both amorphous and crystalline cellulose with broad substrate specificity), CBM3 (strong affinity for crystalline cellulose with a larger binding interface compared to CBM1 and CBM2), CBM4 (binding amorphous cellulose and other P-glucans, making it suitable for less-ordered cellulose or mixed substrates),CBM17 and CBM28 (binding both crystalline and amorphous cellulose but exhibit higher specificity for less-ordered regions).

[0185] The most preferred CBMs for binding to crystalline cellulose are CBM1, CBM3, and CBM4, whereas CBM17 and CBM28 are preferred for amorphous cellulose. CBM3 and CBM4 are particularly advantageous due to their thermophilic properties, maintaining stability under extreme conditions.

[0186] Chitosan and Chitin Binding CBMs

[0187] Chitosan, derived from chitin, is a positively charged linear polysaccharide at physiological pH, composed of P-(l^4)-linked D-glucosamine and N-acetyl-D-glu-cosamine. Chitosan is widely used as a heamostatic agent, making it a relevant substrate for the present invention.

[0188] Relevant CBMs for chitosan binding include: CBM5 (binding to the -(1 — >4)-l inked glucosamine backbone and partially deacetylated chitosan, as well as crystalline chitin, CBM12 (prefers chitosan substrates with higher degrees of deacetylation), CBM14 (binding to highly deacetylated chitosan analogs), CBM18 (targeting both crystalline chitin and partially deacetylated chitosan), CBM33 (interacting with crystalline and partially deacetylated chitosan substrates), and CBM50 (binding peptidoglycan, which structurally resembles chitosan). Preferred CBMs for chitosan binding include CBM5 and CBM33 for crystalline chitosan, and CBM12 and CBM18 for amorphous or solubilized chitosan.

[0189] Starch Binding CBMs

[0190] Starch, including potato starch, is available in various qualities and may be crosslinked (e.g., with epichlorohydrin) for use in gel formulations. Crosslinked starch is particularly suited for gel and paste applications and are absorbable. Preferred CBMs for starch binding include: CBM20 (binding starch, covering varying amylose-amylopectin ratios), CBM25 (binding raw starch), CBM26 (binding amylose-rich starches), CBM41 (interacting with amylopectin-rich regions), and CBM48 (binding raw starch granules). CBM20, CBM25, and CBM48 are the most preferred CBMs for starch due to their broad specificity.Pectin Binding CBMs

[0191] Pectins are non-animal sourced carbohydrates commonly used as heamostats. Relevant CBMs include: CBM28 (binding pectin and other galacturonic acid-containing polysaccharides), CBM35 (binding homogalacturonan (HG) regions of pectin), CBM67 (targeting rhamnogal acturonan I (RG-I) regions), and CBM4 and CBM6 (binding to various pectin-associated structures). The most preferred CBMs for pectin binding are CBM4 and CBM6, as they interact with multiple carbohydrate structures.

[0192] Alginate Binding CBMs

[0193] Alginates are widely used in heamostatic and wound treatment applications. Relevant CBMs include: CBM32 (recognizing uronic acid residues in alginate chains), CBM35 (binding to uronic acid-containing polysaccharides) and CBM 13 (binding sulfated and uronic acid-rich polysaccharides). Preferred CBMs for alginate binding are CBM32 and CBM35.

[0194] General binding CBMs

[0195] Some CBMs exhibit a broad and versatile binding profile, interacting with various polysaccharides. For example, Carbohydrate-Binding Module family 6 (CBM6) domains possess two distinct ligand-binding sites, referred to as cleft A and cleft B, each with unique substrate specificities.

[0196] . CBM6 - Cleft A:

[0197] o Binds cello-oligosaccharides (P-l,4-linked glucose polymers). o Interacts with xylo-oligosaccharides (P-l,4-linked xylose polymers). o Recognizes P-1, 3 -glucans, such as laminarin.

[0198] . CBM6 - Cleft B:

[0199] o Binds cello-oligosaccharides.

[0200] o Interacts with mixed-linkage P-l,4 / p-l,3-glucans, including lichenin and barley P-glucan.

[0201] The dual-binding site architecture of CBM6 modules enables interaction with a diverse range of carbohydrate structures, as described in [J. L. Henshaw, et al, 2004],This structural versatility makes CBM6 modules particularly advantageous for binding to various carbohydrate-based dressings.

[0202] Recent scientific advances have identified CBM s capable of binding to plastics, including polyethylene terephthalate, making the CBMs a very useful tool in modifying many types of dressing according to the invention (Rennison AP, Westh P, Moller MS (2023) Protein-plastic interactions: the driving forces behind the high affinity of a carbohydrate-binding module for polyethylene terephthalate. Sci Total Environ 870:161948).

[0203] Among the carbohydrate-binding modules (CBMs), CBM6 and CBM3 are the most preferred for use in combination with Tissue Factor (TF) for binding to cellu-lose-containing dressings, thereby forming a heamostatic dressing with enhanced binding affinity and functional efficacy.

[0204] Non-conventional CBMs

[0205] In the context of the present invention, the term cellulose-binding module (CBM) is understood to encompass all proteins that exhibit strong binding affinity to cellulose or other polymeric carbohydrates utilized in dressing substrates for the control of bleeding. Such proteins demonstrate cellulose affinity due to their inherent structural properties, electrostatic interactions, or hydrophobic characteristics.

[0206] While conventional CBMs are well known, certain proteins exhibit cellulose-binding properties even in the absence of a traditional CBM domain. For example, some cellulases and hemicellulases possess peptide sequences that confer strong cellulose affinity. Notable examples include lytic polysaccharide monooxygenases (LPMOs) and various glycoside hydrolases (GHs).

[0207] Additionally, various structural or adhesive proteins demonstrate a strong affinity for cellulose. Hydrophobins, which are small fungal proteins capable of selfassembly at hydrophobic-hydrophilic interfaces, can adsorb onto cellulose fibers. Similarly, silk fibroins, particularly in regenerated cellulose materials, interact with cellulose through non-covalent interactions, making them relevant for the present invention.Moreover, chitinase-like proteins from plants or insects, which typically bind chitin, a polysaccharide structurally similar to cellulose, have been observed to exhibit cross-binding with cellulose. Likewise, certain bacterial surface proteins associated with biofilms, such as Curb proteins (CsgA, CsgB) or bacterial amyloid proteins from Escherichia coli. have been reported to bind cellulose. Other examples include the TasA protein from Bacillus suhlilis. which binds with cellulose fibers.

[0208] Engineered proteins containing histidine-rich sequences can also exhibit moderate cellulose-binding properties, primarily through metal-mediated interactions. Polyhistidine-tagged proteins (His-tags), commonly employed for down-stream protein purification, are relevant to the present invention due to their extra potential to bind to suitable dressing materials. Furthermore, engineered peptides with hydrophobic domains or aromatic residues, such as tryptophan, tyrosine, and phenylalanine-rich sequences, have demonstrated the ability to adhere to cellulose.

[0209] Collagen and Gelatin-binding proteins are relevant for the present innovation, as gelatine is often used in dressing systems to promote heamostasis. Gelatin-binding proteins are typically those that recognize collagen or gelatin-like sequences. Some well-known proteins that bind to gelatin include: Fibronectin, which contains a gelatinbinding domain that facilitates interactions with collagen and other extracellular matrix components. Other gelatine binding proteins include peptide sequences from Gelatinase A (MMP-2) - A matrix metalloproteinase (MMP) that degrades gelatin and collagen. Yet other are Gelatinase B (MMP-9), Collagen-binding integrins, such as aipi and a2pi, C-reactive protein (CRP) or Heat shock proteins (Hsp47). Also, the glycoprotein, fibronectin, is known to bind to collagen and gelatine.

[0210] Microbinders, are a class of proteins, of relevance for the present invention. In one embodiment, the system comprises novel microbinders designed to mimic the binding functionality of cellulose-binding modules. By replicating the structural and functional properties of CBMs, these microbinders can facilitate stable, non-covalent attachment of bioactive agents, like tissue factor, to polysaccharide-containing materials, including dressings. The CBM-mimetic microbinders may be engineered as peptide mimetics, synthetic constructs, or recombinant fusion proteins combining the binding domain with functional entities such as tissue factor, coagulation factors, antimicrobial agents, or fibrinolysis inhibitors. This modular design allows for targeted and durablesurface attachment while preserving the bioactivity of the therapeutic components. Furthermore, the use of CBM-like microbinders enables site-specific immobilization without the need for harsh chemical coupling, enhancing both biocompatibility and manufacturing scalability.

[0211] All of these proteins are known, and part of or all of the peptide sequence can be part of a fusion protein with Tissue Factor according to the present invention.

[0212] From the above, it is evident that a person skilled in the art of designing fusion proteins and recombinant proteins can develop multiple novel fusion proteins that exhibit strong binding to dressing materials and can be effectively utilized for hemorrhage control.

[0213] This design ensures that Tissue Factor remains bound to the dressing and does not dissociate when applied to a wound. By securing Tissue Factor in place, the system triggers a localized thrombin burst — a rapid activation of thrombin that accelerates clot formation at the injury site — while preventing systemic dissemination of Tissue Factor into the vascular bed.

[0214] Another aspect of the invention relates to a dressing material comprising the fusion protein of the invention. In one or more embodiments, the dressing material comprises any one or more of the blood coagulation factors, such as the extracellular domain of human Tissue Factor or a recombinant variant thereof and one or more CBM. In one embodiment the recombinant variant of human TF is a recombinant soluble human Tissue Factor.

[0215] By incorporating the fusion protein into a dressing material, the fusion protein can be easily applied to a patient to promote the body's natural haemostatic response. This then leads to an optimized blood clot formation at the wound site while minimizing the risk of systemic thrombotic complications. This system is particularly advantageous for treating severe bleeding trauma patients, including victims of combat injuries, terrorist attacks, violence, work and traffic accidents, and other trauma-related events.

[0216] In one or more embodiments, the dressing material is selected from the group consisting of wound dressing, patches, gauzes, extracellular matrix components, gels, creams, ointments, particles, such as nanoparticles, and any combinations thereof.The dressing material can be in a mechanically stable or a flexible or a mechanically unstable form.

[0217] In one or more embodiments, the dressing material is in a mechanically stable form such as a patch, gauze, extracellular matrix components, a solid substrate. This allows for fast application to an accessible wound injury initiating a rapid onset of haemostasis.

[0218] In one or more embodiments, the dressing material is in a flexible mechanically unstable form such as a suture, powder, granules, a gel, a foam, a spray, or a sponge, particles such as nanoparticles. This allows access to hard-to-reach bleedings may be enhanced. The dressing material according to the invention can e.g. be in the form of a gel to be applied to the wound or in the wound cavity, either by hand or by a device. The advantage is the flexibility in getting to the bleeding site and the possibility to combine with any type of dressings or devices, to further put a pressure to the wound. The gel can be distributed directly to the wound or indirectly to the nearest available dressing. This is important in situations, where one may not have the correct size or type of dressing for the situation, for example during military operations at night or at civilian accidents outside the hospital.

[0219] In some embodiments, the dressing material may be a heamostatic agent such as spatulas and balloons, or other equipment used in surgeries to control bleeding rapidly and effectively. Such heamostatic agents may be understood as mechanical agents, which create a barrier to blood flow.

[0220] In one or more embodiments, the invention relates to the dressing material wherein the material is selected from the group consisting of cotton, cellulose, cellulose ethers, regenerated cellulose, such as rayon and viscose, oxidized cellulose, regenerated cellulose, carboxymethylcellulose, chitosan, agarose or paper-based membranes, polyamides, poly sulfone ether, polyvinyl alcohols, nitrocellulose, nitrocellulose mixed esters, polyethylene terephthalate (PET), blends and combinations thereof. In one or more embodiments, the dressing material comprises cotton and / or regenerated cellulose. In one or more embodiments, the dressing material is SUGI® (QuestAlpha-Ger-many / BVI Medical-Denmark). In one or more embodiments, the dressing material is QuikClot® (Teleflex Incorporated).In one or more embodiments, the dressing material comprises the fusion protein comprising Tissue Factor (TF) as set forth in SEQ ID NO: 9 or as set forth in SEQ ID NO:2, or any variant thereof and the dressing material further comprises cotton and / or regenerated cellulose.

[0221] In one or more embodiments, the dressing material comprises the fusion protein comprising Tissue Factor (TF) as set forth in SEQ ID NO: 3, or any variant thereof and the dressing material further comprises cotton and / or regenerated cellulose.

[0222] In one or more embodiments, the dressing material comprises the fusion protein comprising Tissue Factor (TF) as set forth in SEQ ID NO: 4, or any variant thereof and the dressing material further comprises cotton and / or regenerated cellulose.

[0223] In one or more embodiments, the dressing material comprises the fusion protein comprising Tissue Factor (TF) as set forth in SEQ ID NO: 5, or any variant thereof and the dressing material further comprises cotton and / or regenerated cellulose.

[0224] In one or more embodiments, the dressing material comprises the fusion protein comprising Tissue Factor (TF) as set forth in SEQ ID NO: 6, or any variant thereof and the dressing material further comprises cotton and / or regenerated cellulose.

[0225] In one or more embodiments, the dressing material comprises the fusion protein according to the present invention in an amount in the range of about 50 ng / cm2area of dressing material to about 20000 ng / cm2area of dressing material, more preferably, in the range of about 500 ng / cm2area of dressing material to about 10000 ng / cm2area of dressing material.

[0226] It has been shown that protein loadings as low as 100 ng are able to initiate effective coagulation. Thus, this indicates that the coagulation effects of the fusion protein only show a weak dependence on the amount of fusion protein loaded on the dressing material. Thus, the dressing material comprising the fusion protein according to the present invention is a highly efficient and robust system for initiating haemostasis. This in strong contrast to conventional systems that rely on high loading orextensive surface interaction with the dressing materials. Thus, the dressing material comprising the fusion protein according to the present invention provides robustness as well as a simplified manufacturing with reduced protein consumption.

[0227] The fusion proteins comprising Tissue and CBM are engineered to bind to carbohydrate substrates suitable for safe haemostatic dressings.

[0228] Polymeric carbohydrates, also known as polysaccharides, include but are not limited to cellulose, hemicellulose, starch, flax, cotton, glycogen, chitin and chitosan, peptidoglycan, agar, carrageenan, hyaluronic acid, heparin, chondroitin sulfate, pectin, alginate, dextran, xanthan gum, inulin, pullulan, levan, and their derivatives.

[0229] Materials of particular relevance as dressing materials for safe haemostatic devices include cellulose, regenerated cellulose, oxidized cellulose, oxidized regenerated cellulose, cellulose derivatives, chitosan, starch, alginates, pectins, gelatins, car-bamylated gelatins, and combinations thereof. The dressing may be designed as either non-resorbable or resorbable under physiological conditions. Several carbohydrate-based materials are already recognized for their heamostatic properties.

[0230] Although cellulose is biodegradable and resorbable, most cellulose fibers exhibit stability over prolonged periods under physiological conditions. Nano- or microcellulose variants demonstrate higher resorbability due to their increased surface area and altered crystallinity ratio.

[0231] Examples of suitable substrates include cellulose or regenerated cellulose (e.g., rayon, viscose, lyocell) and cellulose derivatives, such as carboxymethyl cellulose and hydroxypropyl cellulose. Cellulose sources may also include natural fibers, such as cotton, hemp, jute, and other materials with high cellulose content. Preferred substrates include cellulose, nano-cellulose, and regenerated cellulose due to their widespread application in medical dressing systems, either alone or in combination with other polymers to modify mechanical properties. Additionally preferred substrates include resorbable carbohydrates under physiological conditions, specifically oxidized cellulose, cellulose acetate, chitosan, starch, alginates, pectins, gelatins, and their derivatives.

[0232] Other examples of suitable dressing materials include cotton, agarose or paper-based membranes, polyamines, poly sulfone ether, polyvinyl alcohols,nitrocellulose, nitrocellulose mixed esters, and polyethylene ther ephthalate. Further examples of suitable dressing materials include small and flexible sheets designed for laparoscopic procedures or endoscopic applications, where the sheets are sufficiently small to be inserted and retracted through the narrow tubes or trocars, an “umbrella-style” dressing, which can be unfolded, applied to the bleeding site, compressed, then refolded and removed after achieving haemostasis, and haemostatic systems in the form of haemostatic fingers, spatulas, or inflatable balloons, which enable targeted compression in confined spaces while also applying pressure against soft tissue, making them particularly suitable for controlling postpartum bleeding. It should be understood, that such ballon or devices can be coated with e.g. crosslinked agarose, which can be bound to heamostatic fusions proteins of the invention.

[0233] The above-mentioned dressing materials herein can either be used alone or in blends and combinations.

[0234] In one or more embodiments, the dressing material is made from a material, which is absorbable and / or non-absorbable in mammalian plasma / blood.

[0235] In one or more embodiments, the dressing material is made from a material which is absorbable in mammalian plasma or blood such as cellulose, oxidized cellulose, ORC, silk, catgut, chitosan, and synthetic polymers such as polyglycolic acid, polylactic acid, polydioxanone, and caprolactone, as well as polymer and co-block-polymer compositions derived from one or more of the following cyclic monomers: glycolide, lactide, p-dioxanone, trimethylene carbonate, or 8-caprolactone.

[0236] In one or more embodiments, the dressing material is made from a material which is non-absorbable in mammalian plasma or blood, meaning they do not degrade or dissolve when exposed to bodily fluids and remain intact over time. Such as crystalline cellulose, acetylated cellulose, Polyurethane (PU), which are often used in foam and film dressings, polypropylene (PP) widely used in surgical meshes and dressings, polyethylene (PE) used to mechanically stabilize dressings and as a barrier layer in composite dressings, polytetrafluoroethylene (PTFE) and expanded PTFE, nylon (polyamide), commonly used in surgical sutures and wound contact layers, polyester (e.g., PET) used in woven and nonwoven fabrics for dressings, and silicone (e.g., polydimethylsiloxane) or silver-coated nylon used for antimicrobial properties or mechanical strength.The dressing material, absorbable or non-absorbable, is capable of promoting rapid haemostasis without resulting in undesirable systemic blood coagulation.

[0237] In one or more embodiments, the dressing material further comprises suitable additives. Specifically, in one embodiment, the dressing material further comprises a stabilizing agent or an antimicrobial agent. In another embodiment, the further suitable additives may be selected from: (i) one or more additional co-active agents such as Factor VII or its components and other inducers of the coagulation paths, (ii) chitosan, salts, zeolites, kaolin, thrombin, fibrin, and fibrinogen to enhance clotting efficiency, (iii) stabilizing agents, including carbohydrates, coating proteins, pH buffers, detergents, moisture control agents, radical quenchers, UV absorbers, and antimicrobial compounds for extended storage life, (iv) antimicrobial agents to prevent infections, (v) markers, (vi) topical pain relieve agents, (vii) inorganic or other salts that promote haemostasis, including calcium chloride or other haemostatic agents, and (viii) combinations thereof.

[0238] The dressing material according to the present invention can include components for further enhancement of the heamostasis, in addition to components increasing storage stability, wettability or dressing flexibility or other active agents and combinations thereof.

[0239] The dressing material may further comprise Factor VII. Preferably, Factor VII is incorporated into the dressing or applied to the dressing immediately prior to use. Localized application of Factor VII at the bleeding site minimizes systemic exposure, as Factor VII can only access the vascular system through the lesion itself. This approach allows for the use of a significantly lower quantity — preferably less than one-tenth — of the amount typically required for systemic administration, thereby substantially reducing safety concerns. The preferred embodiment includes a combination of immobilized a fusion protein comprising sTF and a CBM together with locally applied Factor VII, enabling effective haemostasis with minimal systemic risk. The bound TF fusion protein enhances clotting efficiency in the presence of unbound Factor VII, resulting in faster and more effective heamostasis.

[0240] In one embodiment, the dressing material according to the present invention comprises Factor Vll / VIIa. In one embodiment, the dressing material comprisesFactor VII which is provided in a freeze-dried and stabilized form. This allows for application of Factor VII directly onto the dressing immediately prior to use.

[0241] The dressing material according to the present invention may further comprise one or several co-active agents. Examples of co-active agent include various agents, which further enhance haemostasis and other agents, which can benefit a patient having a bleeding wound, such as agents that can cool down the wound or reduce the apparent pain.

[0242] Preferably, such co-active agent is a haemostasis enhancing agent such as an inorganic salt, gelatine or gelatine derivatives, biopolymers like cyclodextrin or chitosan or combinations hereof. Collagen or thrombin, poly-N-acetyl glucosamine, poly-prolate acetate, D-glucosamine, calcium alginate, kaolin, salts known to catalyse and enhance haemostasis, including calcium chloride, or other salts. A co-active agent may be immobilized on the dressing material together with sTF.

[0243] In another embodiment, the dressing material comprises a Prothrombin Complex Concentrate (PCC), or one or more coagulation factors selected from Factor II, Factor VII, Factor IX, and Factor X, or combinations thereof.

[0244] In one or more embodiments, the dressing material comprises Recombinant platelet Factor 4 (rPF4) and Factor XII. Recombinant platelet Factor 4 (rPF4) are considered preferred components for enhancing haemostatic performance. In one or more embodiments, the dressing material comprises agents that inhibit or delay fibrinolysis. These agents that can inhibit fibrinolysis can stabilize the formed blood clot.

[0245] In further preferred embodiments, the dressing material comprises agents that inhibit or delay fibrinolysis, thereby stabilizing the formed blood clot. Suitable examples of such inhibitors include Plasminogen Activator Inhibitor- 1 (PAI-1), a2- Antiplasmin, which directly inhibits plasmin activity, and Thrombin-Activatable Fibrinolysis Inhibitor (TAFI), which modifies fibrin to render it more resistant to plasmin-mediated degradation. Preferred fibrinolytic inhibitors according to the present invention include aprotinin (e.g., Trasylol®) and aminocaproic acid (e.g., Amicar®).

[0246] In one or more embodiments, the dressing material according to the present invention comprises agents capable of neutralizing the activity of heparins or Vitamin Kantagonists (VKAs). The neutralization of VKAs is known to promote rapid coagulation. Preferred neutralizing agents according to the invention include protamine sulfate.

[0247] Many patients are generally treated with anticoagulant agents such as Vitamin K antagonists (VKAs), for example warfarin. Other commonly used anticoagulants include unfractionated heparin (UFH), low molecular weight heparins (LMWHs) such as enoxaparin, dalteparin, and tinzaparin, as well as synthetic pentasaccharides such as fondaparinux.

[0248] The inventors have surprisingly found that dressing with bound Tissue Factor preserved its functionality for initiating haemostasis upon prolonged storage, even without being coated ("glazing") with a mixture consisting of carbohydrates, protein, alcohols and preservatives.

[0249] The dressing material according to the present invention may further comprise a stabilizing agent to improve storage stability. The preferred stabilizing agent will passively fill in the surface space of the dressing material around the active compound. Examples of stabilizing agents include serum albumin, fish gelatine, casein or protein mixtures or proteolytic degraded gelatine or other proteins. The stabilizing agent may be immobilized on the dressing material together with the fusion protein, whereby release to the vascular system and systemic exposure is minimized.

[0250] The dressing material according to the invention may also be coated with carbohydrates for further storage stabilization. Preferred carbohydrates for further storage stabilization include trehalose, lactitol, inositol, glucose, sucrose, mannose, saccharose, dextran, diethylaminoethyl (DEAE)-dextran and / or agarose.

[0251] The dressing material according to the invention may also be coated with proteins. Preferred proteins include human serum albumin, bovine serum albumin (BSA), recombinant serum albumins and derivatives, Non-fat dry milk, Casein or caseinate, Fish Gelatine. Preferred alcohols include glycerol, and preservatives include 2-chloro acetamide or other antimicrobial agents suited for wound dressings or topical use.

[0252] The dressing material according to the present invention may be combined with agents exhibiting antibacterial properties, such as nanosilver, conventional antibiotics, or antibacterial proteins, including lysozymes. These agents may be incorporated intothe dressing either by direct impregnation or by binding, for example, using a carbohydrate-binding module (CBM) binding strategy as described previously.

[0253] The dressings relating to the present invention can incorporate identification markers to facilitate detection and identification, particularly by medical professionals such as surgeons. The dressing may include markers composed of filaments or materials that are detectable via X-ray imaging. Preferably, the spacing between the X-ray-detectable markers is sufficiently small to ensure detectability, even in fragmented or small portions of the dressing.

[0254] In addition to X-ray markers, the dressing may be color-coded to enhance visibility in a surgical or wound care environment. Preferably, the selected color exhibits high contrast against fresh blood to improve detection. Suitable colors include, but are not limited to, Bright Green (wavelength range: 495-570 nm), Cyan or Turquoise (480-510 nm), Bright Yellow (570-590 nm), or White.

[0255] The markers, whether X-ray-detectable, visually distinguishable, or fluorescent, are designed to be both chemically stable and securely affixed to the dressing material. The attachment method ensures that the markers remain permanently integrated within the dressing structure, thereby preventing any risk of detachment, leakage, or degradation during use.

[0256] The dressing material according to the present invention may be covered or swelled with chemicals and buffers, which will further stabilize the functionality upon storage and transport. This includes antimicrobial agents or buffers to stabilize the pH or agents, which protect against damage from freezing or UV irradiation.

[0257] The dressing material according to the present invention may additionally be combined with topical pain-relieving agents. These may include local anesthetics, such as lidocaine or pram oxine; non-steroidal anti-inflammatory drugs (NSAIDs), such as diclofenac or ibuprofen; counterirritants, including menthol, camphor, or methyl salicylate; capsaicin; or combinations thereof.

[0258] In one or more embodiments, the dressing material according to the present invention comprises calcium donors, preferably CaCh. Calcium (also known as FactorIV of the haemostasis) is important at several steps in the extrinsic and intrinsic cascade involved in haemostasis.

[0259] One or more embodiments involve the use of gelatin- and / or agarose-based dressings as the substrate for the fusion protein of the present invention. In this embodiment, the fusion protein is specifically tailored to bind to gelatin or agarose, ensuring efficient immobilization while preserving its biological activity.

[0260] In one or more embodiments, a composite dressing is formed by combining gelatin and agarose with a fusion protein of the present invention designed to bind to agarose. This configuration offers synergistic heamostatic benefits, as it combines the clot-promoting properties of gelatin with the coagulation-triggering function of blood coagulation factor. The advantages of this embodiment, include the combination of gelatin-induced platelet aggregation and blood coagulation factor, in particular TF, thrombin generation leads to a more rapid and robust clot formation. Also, the dressing can be formulated as a solid matrix, hydrogel, or sponge, making it adaptable to various wound types and clinical needs. Further, the fusion protein remains functionally active when immobilized on the biocompatible and moisture-retaining gelatin / agarose matrix. The herein described additives herein may be added to the dressing alone or in combination.

[0261] Another aspect of the invention relates to a system for promoting rapid haemostasis while reducing the risk of undesirable blood coagulation, said system comprising the dressing material according to the invention.

[0262] Binding the fusion protein to the dressing material ensures that the blood coagulation factor acts only at the site of wound injury. The fusion protein stays bound to the dressing material via the CBM, thereby reducing the risk of permeation of blood coagulation factors into the vascular system. This results in a safe method or promoting haemostasis with reduced risk of systemic blood coagulation.

[0263] In one or more embodiments, the system according to the present invention is provided in the form of sheets, threads, fibers, or bundles of threads, which can be woven, glued, or otherwise integrated into various types of wound dressings. This configuration enables centralized and standardized manufacturing, facilitating seamless integration into existing wound dressing production processes. The density and distributionof the active material, whether in sheet, thread, or fiber form, may be optimized based on the specific clinical application to maximize haemostatic efficacy.

[0264] In one or more embodiments, the system is provided in the form of a foam, which represents a variation of a gel that can be expandable due to its gas content. An alternative variation includes a sprayed foam formulation, comprising absorbable polymers with covalently attached Tissue Factor. When applied to a wound or dressing, the neutral solvent or gas component evaporates, leaving a highly concentrated gel layer positioned close to the bleeding site. This variation enhances the flexibility and adaptability of the system, allowing it to be applied directly to a bleeding site or incorporated into wound dressings.

[0265] A further aspect of the invention relates to a system for promoting rapid haemostasis while reducing the risk of undesirable blood coagulation comprising a dressing material according to the invention.

[0266] The fusion protein comprising at least one blood coagulation factor and one or more CBM allows for the easy and effective immobilization of the blood coagulation factor on the dressing, preventing leakage into the bloodstream, which could otherwise lead to uncontrolled coagulation elsewhere in the vascular system. Unlike conventional wound dressings that may allow unbound clotting agents to disperse, this system ensures local activation of haemostasis without systemic clotting risks.

[0267] This immobilization aspect is particularly important in severe venous and arterial hemorrhage scenarios, where continuous blood flow at high pressure may wash away unbound clotting agents. The permanent binding of Tissue Factor to the dressing ensures both efficacy and safety, meeting stringent regulatory requirements set by organizations such as the EMA and FDA.

[0268] Additionally, the invention provides advantages in clinical practice. Unlike conventional haemostatic agents that become incorporated into the fibrin clot, the Tissue Factor dressing remains separate from the formed clot, allowing for easy removal or repositioning after bleeding has been controlled. This feature improves clinical handling and enhances patient safety.In one or more embodiments, the system includes sponges, such as compressible sponges. In a further embodiment, the sponges may be provided in the form of small beads, which can be delivered into a wound cavity either manually or via a dispensing device. Following mechanical expansion or swelling upon fluid absorption, these beads exert additional pressure on the bleeding sites, thereby promoting haemostasis. This configuration is particularly suitable for emergency medical services (EMS) in packing deep wound cavities and effectively managing irregular wound structures where traditional dressings may be less effective.

[0269] In one or more embodiments, the system involves a sticky glue formulation, haemostatic adhesives, which is typically absorbable (e.g. based on potato starch) and can be applied manually or via a spray or tube-based dispensing device. This adhesive serves a dual function: (1) facilitating tissue adhesion for wound closure, and (2) initiating haemostasis by delivering the blood coagulation factor directly to the bleeding site.

[0270] This formulation provides versatile application flexibility, allowing for efficient and localized haemostasis in a wide range of traumatic and surgical bleeding scenarios.

[0271] Another aspect of the invention relates to the use of the fusion protein and / or a dressing material according to the invention, comprising the fusion protein and / or a system comprising the fusion protein according to the invention for promoting rapid haemostasis.

[0272] The dressing material comprising the fusion protein according to the invention is applied directly to the bleeding site to achieve bleeding control and to induce controlled haemostasis. Optionally, the dressing may be compressed against the bleeding site to further promote cessation of blood flow. The dressing may also be removed following haemostasis, depending on clinical requirements or treatment protocols.

[0273] TF bound to a dressing material via at least one CBM, ensures that clinically significant amounts of Tissue Factor are not released into the bloodstream at the site of vascular injury. This configuration prevents systemic exposure of Tissue Factor,thereby avoiding the risk of thrombosis or embolic events caused by excessive clotting in the circulatory system.

[0274] Upon contact with a bleeding trauma, the system facilitates the delivery of Tissue Factor to the profusely bleeding area, where it interacts with Factor Vll / VIIa. The quantity of Tissue Factor provided by the system exceeds the levels of endogenous, cellbound Tissue Factor, such as that found in endothelial tissue or leukocytes, which typically initiate the coagulation cascade in smaller bleeding events that can be controlled by short-term compression.

[0275] However, in cases where profuse bleeding results in the loss of approximately 100 cc or more of blood, particularly in instances involving larger vessels or arterial bleeding, the application of a large amount of Tissue Factor in a controlled manner provides a superior and effective means of achieving haemostasis, preventing further excessive blood loss as long as the Tissue Factor or parts thereof remain applied.

[0276] In one aspect, the present invention relates to a method for preparing the dressing material according to the invention comprising the following steps:

[0277] (a) obtaining a fusion protein comprising at least one blood coagulation factor and one or more CBM,

[0278] (b) soaking or contacting the fusion protein from step (a) with a dressing material capable of binding CBM, and

[0279] (c) optionally washing the dressing and optionally drying with preservatives.

[0280] In one or more embodiments, preservatives are added when washing the dressing in step (c). In one or more embodiments, preservatives are added in the last washing step or washing cycle in step (c). In one or more embodiments, a solution of preservatives is directly added to the dressing following the washing step (c) and prior to drying. In one or more embodiments, step (c) is followed by step (d) cutting, packing and labelling, and step (e) sterilization.

[0281] In one or more embodiments, the method for preparing the dressing material according to the invention comprises the following steps:(a) obtaining a fusion protein comprising at least one blood coagulation factor and one or more CBM,

[0282] (b) soaking or contacting the fusion protein from step (a) with a dressing material capable of binding CBM, and

[0283] (c) optionally washing the dressing,

[0284] (d) adding a solution of preservatives

[0285] (e) optionally drying

[0286] (f) Cutting, packing and labelling

[0287] (g) Sterilization

[0288] Step (a) obtaining a fusion protein comprising sTF and a CBM

[0289] Fusion proteins suitable for use in the context of the present invention can be produced using standard recombinant technology.

[0290] Recombinant proteins and fusion proteins, including soluble Tissue Factor and cellulose-binding modules (CBMs), can be produced using various expression systems. Standard industrial recombinant technology includes the use of human embryonic kidney (HEK) cells, Human 293 / T, HEK-F, baby hamster kidney (BHK-21) cells, Chinese Hamster Ovary (CHO) cells, SF9 insect cells, mouse-derived cell lines, Escherichia coli. or yeast (Saccharomyces cerevisiae) cell systems. These systems facilitate the controlled and efficient production of recombinant proteins with specific structural and functional characteristics.

[0291] The production process typically involves cloning the gene of interest into an expression vector, followed by transfection into the host cell line using methods such as electroporation or lipid-based transfection. Once inside the cells, the gene is transcribed and translated into the desired protein under the control of a strong promoter for efficient expression. Fusion proteins, which combine functional domains from different proteins, can be engineered by linking the corresponding DNA sequences in-frame within the vector. Signal peptides may be included to facilitate secretion, and purification tags such as His-tags or Fc-fusions allow for efficient downstream processing.

[0292] Mammalian cell cultures, including CHO and HEK cells, are preferred due to their ability to perform post-translational modifications that influence protein functionand stability. Such modifications include glycosylation, phosphorylation, carboxylation, palmitoylation, and neuraminoglycosylation, which are particularly critical for proteins involved in coagulation. The glycosylation of recombinant proteins, especially those intended for human administration, significantly impacts biological activity, function, clearance from circulation, and antigenicity.

[0293] CHO cells, originally derived from the ovary of the Chinese hamster, are widely used in biopharmaceutical production due to their adaptability to suspension cultures in serum-free and chemically defined media. They facilitate large-scale protein production in bioreactors and reduce the risk of contamination. The CHO expression system has been instrumental in producing recombinant biotherapeutic proteins and remains a standard in industrial protein manufacturing. Optimized high-density cell culture systems, such as high-cell-density perfusion processes in single-use bioreactor systems with disposable cell culture bags, allow expansion to cell concentrations exceeding 10 million cells / ml under controlled physiological conditions (pH 6.8-7.4) with optimal oxygenation, gas exchange, and pressure regulation in bioreactors of up to 100 liters.

[0294] Following expression, recombinant proteins are harvested from the culture medium and purified using standard chromatography techniques, including affinity, ionexchange, and size-exclusion chromatography. Advances in genetic engineering, such as codon optimization, CRISPR-based genome editing, and the use of stable cell lines, further enhance the yield and quality of recombinant and fusion proteins, making them highly suitable for therapeutic, diagnostic, and industrial applications.

[0295] This method presents a simplified and efficient approach to producing fusion proteins with heamostatic properties, reducing processing steps while ensuring high purity and functional integrity of the final product.

[0296] Given the critical role of glycosylation in determining protein function, the use of human or CHO cell-based expression systems is preferred. The ability to control and optimize these post-translational modifications is essential for the development of functional recombinant proteins suitable for clinical and industrial use.

[0297] It is noted, however, that tissue factor derived from Escherichia coli (E. coli) is capable of initiating the extrinsic coagulation cascade. This recombinant E. coli-derived tissue factor, which lacks glycosylation, is commonly employed as a calibrator and initiator in tissue factor activity assays.

[0298] Additionally, Tissue Factor, CBMs, or fusion proteins described herein may also be synthesized using chemical methods. Synthetic peptide synthesis techniques, such as stepwise elongation via solid-phase peptide synthesis (SPPS) or solution-phase synthesis, are well-established. The use of chemo-selective and orthogonal protection strategies, including Boc, Fmoc, benzyl, or t-butyl protecting groups, allows precise control of synthesis. Amide bond formation can be achieved using carbodiimides, active esters, HATU / HOAt, or other coupling reagents.

[0299] Accordingly, the fusion proteins according to the present invention encompass recombinant proteins, as well as proteins assembled enzymatically or chemically from recombinant proteins or synthetic peptides.

[0300] The use of recombinant tissue factor, rather than tissue factor derived from human plasma or other biological sources, eliminates the risk of transmission of pathogens or other contaminants associated with human-to-human or cross-species biological material.

[0301] Step (b) Soaking

[0302] The inventors have realized that the number of wet process steps in combination with the various dressing types, designs and sizes can make the medical device manufacturing complicated. This in combination with the need for good manufacturing practice (GMP), which involves validation and strict monitoring of all the individual process steps makes the conjugation methods potentially laborious and costly.

[0303] The inventors have realized that a much simpler method can be designed, which omits the use of conjugations chemicals and procedures and only use protein interactions to bind the sTF to the dressing. The fusion protein can be fused to a suitable dressing material in a predominantly aqueous solution. Further, by using an oriented binding, the sTF is located in a similar fashion as in the nature without reduced risk of crosslinking to the dressing.One or more embodiments involve the application of the fusion protein according to the invention applying said fusion protein onto cellulose-based dressings via spray deposition. This approach allows for flexible and scalable production, which can be implemented in (i) Centralized manufacturing facilities, where dressings are pretreated with the fusion protein according to the invention before packaging and distribution or (ii) decentralized or point-of-care settings, where the fusion protein is applied to dressings just before use, ensuring freshness and adaptability across different surgical routines.

[0304] Such embodiments provide scalability and mass production of heamostatic dressings while maintaining cost-effectiveness. Also, it allows multiple dressing formats (e.g., gauze, sponges, bandages) to be rapidly functionalized for different clinical applications. Further, such embodiments improve user adaptability: Surgeons and healthcare providers can apply the fusion protein of the invention on-demand, ensuring compatibility with existing procedural workflows, including a highly flexible and efficient method for creating heamostatic dressings while accommodating industrial manufacturing practices and clinical usability requirements.

[0305] Alternatives to soaking or spray deposition include dip coating, roll coating (blade or gravure coating), electrospinning or electrostatic deposition, layer-by-layer (LbL) assembly using alternating layers of positively and negatively charged biomolecules, inkjet or microdroplet printing or freeze-drying (lyophilization) with rehydration activation. Preferred embodiments are spray deposition and roll coating as they are readily suited for flat sheet in a continuous process.

[0306] In one or more embodiments, the fusion protein according to the present invention is assembled on the dressing. For example, a streptavidin-CBM (carbohydrate-binding module) fusion protein may be used to bind to cellulose present in the dressing material. Subsequently, a biotinylated tissue factor protein can be bound to the streptavidin domain of the fusion protein. In this manner, the tissue factor is indirectly immobilized on the cellulose surface via a biotin-streptavidin interaction and the CBM-cellulose affinity.

[0307] This approach enables an alternative implementation of the present invention, particularly useful when, for example, the expression of a specific CBM fusionprotein is inefficient, technically challenging, or cost-prohibitive. Moreover, this method allows for flexible post-assembly modification of the dressing with additional biotinylated compounds, such as antimicrobial peptides or other biologically active moieties.

[0308] In one or more embodiments of the present invention, the fusion protein is immobilized onto a cellulose substrate using a click chemistry or other covalent conjugation approach. This method enables stable and efficient attachment of the blood coagulation factor to the substrate while preserving its biological activity.

[0309] The immobilization process can be carried out in two steps (1) Initial Binding via CBM Interaction, the CBM domain facilitates the adsorption of the fusion protein onto the cellulose substrate through non-covalent interactions (2) Covalent Attachment via Click Chemistry or Other Selective Reactions. After the initial binding, the blood coagulation factor is further secured onto the substrate using a chemoselective click reaction (e.g., azide-aryl coupling, Meldal coupling) or another chemo- or regio- selective conjugation method. The reaction is performed under mild conditions to prevent unwanted crosslinking or loss of the blood coagulation factor activity.

[0310] CBM binding

[0311] Carbohydrate-binding modules (CBMs) are known to interact with carbo-hydrate-based substrates, with their binding efficiency influenced by several factors, including temperature, pH, and ionic strength. Generally, CBM binding is most efficient at higher temperatures, low salt concentrations, and within a pH range of 4 to 9. However, the optimal conditions vary significantly depending on the specific CBM and the nature of the substrate.

[0312] The modulation of CBM binding, i.e., the reduction or enhancement of CBM binding can be achieved by adjusting water activity and ionic strength. Reagents capable of reducing water activity include sucrose, glucose, fructose, glycerol, sorbitol, mannitol, xylitol, urea, ethylene glycol, propylene glycol, polyethylene glycol (PEG), polyethyleneimine (PEI), and non-toxic salts such as sodium chloride, potassium chloride, calcium chloride, magnesium sulfate, and potassium carbonate. Ideally, these reagents should be non-toxic, non-interactive with the CBM, and capable of remaining in a haemostatic dressing after a drying step, thereby minimizing manufacturing complexity.Among the available options, calcium chloride and trehalose are particularly preferred for modulating water activity. Calcium chloride is known to enhance haemostasis, whereas trehalose stabilizes proteins and macromolecules. Crystallinity of cellulose — a major component of many dressing substrates — varies depending on its source, such as wood, cotton, or bacterial cellulose. Thus, selecting an appropriate CBM for a specific binding pattern is essential. In addition to engineering CBM fusion proteins tailored for particular dressings and materials, modifications to the dressing itself can enhance CBM binding efficiency.

[0313] The degree of crystallinity in cellulose and other dressing materials can be altered through chemical, physical, and enzymatic treatments. These modifications affect the balance between crystalline (ordered) and amorphous (disordered) regions, impacting properties such as solubility, biodegradability, mechanical strength, and CBM binding preferences.

[0314] Well-established chemical methods include alkaline treatment (so-called "mercerization"), which converts native cellulose I into the less crystalline cellulose II polymorph, as well as acid hydrolysis, which increases crystallinity by selectively removing amorphous regions. The resulting nanocrystalline cellulose exhibits distinct binding characteristics compared to native cellulose.

[0315] Other chemical modifications include esterification, such as acetylation with acetic anhydride, or etherification, both of which disrupt hydrogen bonding in crystalline regions, reducing crystallinity and enhancing solubility. Similarly, ionic liquids can be employed to dissolve cellulose and subsequently regenerate it, yielding a more amorphous structure.

[0316] Physical modifications include mechanical shearing (e.g., milling), which disrupts crystalline regions and increases amorphous content, as well as heat treatment, which can either increase or decrease crystallinity depending on the conditions. High-pressure steam treatment followed by rapid decompression is another method that can reduce crystallinity.

[0317] Enzymatic treatments, such as selective hydrolysis with cellulase enzymes, degrade amorphous regions preferentially, thereby increasing overall crystallinity.Likewise, selective oxidation via chemical or enzymatic means can weaken crystalline structures, further influencing CBM binding.

[0318] Of particular relevance to the present invention is the production of regenerated cellulose fibers using dissolution and regeneration techniques. Swelling agents or solvents such as dimethyl sulfoxide (DMSO) and ethylenediamine can expand crystalline regions, reducing overall crystallinity. Additionally, dissolution in solvents such as NaOH / urea or ionic liquids followed by reprecipitation results in a less crystalline or more amorphous cellulose state. Several established methods exist for producing regenerated cellulose fibers tailored for specific industrial and research applications.

[0319] By selecting appropriate modification techniques, cellulose crystallinity can be precisely controlled to optimize CBM binding in various applications, particularly in the development of heamostatic dressings and related materials.

[0320] Manufacturing and safety benefits

[0321] No existing haemostatic dressing design, format, or material is universally effective for controlling all types of bleeding. The present invention addresses this manufacturing challenge by providing a general method for preparing haemostatic dressings and devices through a limited number of steps, without the use of harsh chemicals or labor-intensive coupling procedures. The binding of the fusion protein to the dressing material is done in a reduced number of steps by effectively binding the fusion protein to the dressing material in an aqueous buffered solution. The reduction in manufacturing steps enables treatment of dressings regardless of their design, including but not limited to Extrusion, inkjet / drop-on, or stereolithography, 3D-printed, bulky, rigid, soft, or irregularly shaped formats. Furthermore, scalability is significantly improved due to the decreased number of unit operations involved.

[0322] EXAMPLES

[0323] Example 1 - Preparation of fusions proteins from CHO-3E7

[0324] Four different genetic constructions of CBM-TF fusion proteins were designed with two different lengths of the repeated GGGGS sequence linker between the CBM and sTFsequences. The sTF sequence (SEQ ID NO: 2) being the same and the CBM being CBM6a and CBM3a with expected different cellulose binding patterns:

[0325] Construct No. 1 - TF-CBM-cel6A with (GGGGS)3 linkers (SEQ ID NO: 3) Construct No. 2 - TF-CBM-cel6A with (GGGGS)e linkers (SEQ ID NO: 4) Construct No. 3 - TF-CBM-CBM3A, with (GGGGS)3linkers (SEQ ID NO: 5) Construct No. 4 - TF-CBM-CBM3A, with (GGGGS)6linkers (SEQ ID NO: 6) The signal peptide is indicated with underlining. The signal peptide cleavage site is suggested to be after G34, by SignalP 6.O., software, which predicts the presence of signal peptides and the location of their cleavage.

[0326] Expression:

[0327] The expressing in transient CHO-3E7 cells (Thermo Fisher) was according to the general instructions.

[0328] The amino acid sequence was checked to get the codon-optimized sequence (for CHO cell system), and the genes delivered by GeneArt (Invitrogen / ThermoFisher, Germany). In short, cloning the gene into pTT5 expression vector was followed by large-scale plasmid preparation for transfection. Plasmid constructs was confirmed by DNA sequencing of cloning junctions. After transfection of CHO-3E7 cells and production in Shake-Flasks (200 mL culture volume), the supernatant samples were collected at day 4 and day 8 and analyzed by SDS-PAGE and Western Blot.

[0329] Crude supernatant was analyzed on with coomassie stained SDS-PAGE / W estern blot. (15 microliter reduced DTT, 4-20% Tris-glycine gel) and compared to protein Mw ladder reference (Sea Blue, Plus2, Invitrogen). Western blot staining was utilized antiTF (AbCam). A non-relevant protein supernatant from same CHO-3E7 system harvested after 4 and 8 days, were included as reference. The result indicated moderately low expression, and some smeared bands due to different levels of glycosylation. Bands with the expected Mw 30-44 kDa was stained with the anti TF.

[0330] Collection and crude purification:

[0331] The fusion proteins were collected in a single IEX step. Cell supernatant harvested after 8 days were diluted 1+3 with buffer Al (20 mM Tris pH 8.0) was loaded on IEX chromatography column (HiTrap Q FF 5 mL, Cytiva, equilibrated with BufferAl, 5 ml / min flow rate) and eluted with gradient buffer Al to 50% buffer Bl (20 mM Tris, 1 M NaCl, pH 8.0). 0.50 mL fractions collected, and column stripped with 100% buffer Bl. Column clean in place with IM NaOH before re-equilibrating.

[0332] For all four fusion proteins, the run through was significant. Each fraction was analyzed with Coomassie stained SDS-PAGE / Western blot and showed weak stain-ing. Staining with anti TF (AbCam) combined with a general Ponceau stain indicated highest TF presence in two of the collected fractions, but also the presence of impurities. The pooled fraction D for fusion protein 1, 2 and 4 was used in the following experiments. Samples from Protein 3 was lost. Table 2 below summarizes the sample identities.

[0333] Table 2: Overview of sample identities

[0334] Construct FT-CBM construct After IEX, After buffer No. pool D exchange

[0335] 1 TF-CBM-cel6A with (GGGGS)3 linkers ADU112 ADU112A (SEQ ID NO: 3)

[0336] 2 TF-CBM-cel6A with (GGGGS)e linkers ADU113 ADU113A (SEQ ID NO: 4)

[0337] 3 TF-CBM-CBM3A, with (GGGGS)3linkLost Lost

[0338] ers (SEQ ID NO: 5)

[0339] 4 TF-CBM-CBM3A, with (GGGGS)6linkADU114 ADU114A ers (SEQ ID NO: 6)

[0340]

[0341] Example 2 - Tissue Factor activity in solutionThe TF activity assay was modified from a commercial assay (AbCam Abl08906, Tissue Factor Activity Assay, human, Colorimetric) with extra phospholipids (Rossix AB, PL052)

[0342] All assay components (assay buffer, substrate buffer, Factor VII, Factor X, Factor X substrate chromogen, Tissue Factor references (P0-P8), phospholipids) were prepared, store and treated carefully according to the assay instructions.

[0343] The three samples of TF-CBM from Example 1 were measured for Tissue Factor activity, together with references and an assay buffer blank.

[0344] The assay set-up followed the general AbCam assay instructions, with some minor improvements. One water ice bath labelled 1.5 mL vials was prepared with reagents in the order: 20 microliter sample or sample buffer, 40 microliter assay buffer, 60 microliter phospholipid and vortex mixed before being added 20 microliter Factor VII and Factor X solution. The closed vials was vortex mixed and incubated for 30 minutes at 37 C on a vortex (620 RPM) heating mixing block, before being added 20 microliter Fact or X substrate, vortex mixed and incubated at 37 C. 2.3 microliter reaction samples were taken out for analysis on a Nano Drop (ThermoFisher, water as blank) and UV absorbance at 405 nm recorded together with the time. The UV signal and time was plotted in a graph, (see Figure 5). The samples compared were the fusion proteins, ADU112, ADU113, ADU114, the TF references P2 (3.1 microgram / ml), P4 (0.75 microgram / ml) and P6 (0.19 microgram / ml) and two assay blanks with no samples.

[0345] Conclusion: The samples from pool D after IEX had a specific TF activity, with ADU 112 and 113 with higher activity than ADU114, irrespective of the concentration (as can be seen in Figure 5).

[0346] Example 3 - Tissue Factor activity after buffer exchange

[0347] The samples from Example 1 were measured by UV (Nano Drop) and concentrated and buffer exchanged. The samples were reduced in volume (VivoSpin6, 5 kDa MwCo, PIS membrane, 4 C, 3000 G, several hours, Sartorius-Germany) over several hours and added 25 mM acetate pH 5 buffer twice, before being collected and UV measured. Table 3 provides an overview of the results.Table 3: Overview of the UV measurements of the samples

[0348] Sample ID UV, 210 nm, UV, 280 nm, UV, 210 nm, UV, 280 nm, before before after after

[0349] ADU112 15.9 2.28 0.6 8.64

[0350] ADU113 16.55 1.46 8.66 3.17

[0351] ADU114 16.84 1.41 14.05 1.53

[0352]

[0353] The change in UV signal at 205 nm before and after buffer exchange indicates impurities in the samples. The samples, called AUDI 12a, 113a, and 114a were measured for TF activity as described in Example 2. The TF activity was estimated according to the assay described in Example 2. Samples compared were the fusion proteins, ADU112A, ADU113A, ADU114A, the TF references as in the previous example and assay blanks.

[0354] The samples had a specific TF activity, with ADU 113 A with higher activity than ADU 114A and ADU 112A, which had the approximately the same activity (see Figure 6).

[0355] Conclusion: The result indicates the fusion proteins were functional and stable at lower salt concentration and after centrifugal membrane filtration at prolonged period. This indicates good stability and ease of use at pH 5 with no or insignificant loss of TF activity.

[0356] Example 4 - Tissue Factor activity after binding to dressings

[0357] The three TF-CBM fusion protein samples from Example 2 in low salt was bound to various dressing samples, representing different cellulose properties.

[0358] Three different dressings utilized:

[0359] A - Dentist surgical dressing, Conzept super gauze, all-purpose sponges, nonwoven. (No. 45394, Zenith Dental Aps, Denmark). Traditional surgical swaps, for dentists and surgeons, 70% rayon and 30% polyester mixture, approx. 0,039 g / m2.B - SUGI® sponge, (QuestAlpha-Germany / B VI Medical-Denmark), lint-free sheet / sponge with a high tear resistance, made of cotton and regenerated cellulose, uncompressed, 150 g / m2)

[0360] C - UPM-FibDex (UPM medical / Steripolar Oy., thin paper like sheets, nano cellulose fibers on PE backbone, approx. 0,57 g / m2)

[0361] The dressings were cut in 5 mm in diameter round pieces using a metal arch puncher. Different numbers of dressings were used in the experiment, due to the very different densities and swelling properties.

[0362] 5 mm dressings were incubated with FT-CBM samples in low salt from example 3: 1 Sugi, 4 dentists or 8 UPM dressing pieces mixed with 100 microliter TF-CBM and 200 microliter Milliq water, vortexed and incubated overnight at 4 C.

[0363] After 18 hours at 4 C, the dressings were washed with 1000 microliters 1 : 1 PBS: water for 2 minutes while vortex mixing. The dressings were squeezed with a tweezer to remove remaining liquid. The supernatant discarded. This was repeated three times, before they were measured for Tissue Factor activity as described in Example 2 compared with Tissue Factor P4 and P6 reference, unmodified dressings and assay blank.

[0364] Figure 7a, Figure 7b and Figure 7c summarizes the results for the three different dressings, Dentist, SUGI® and UPM, and three different TF-CBM fusion proteins.

[0365] Conclusion: The fusion protein treated dressings had TF activity, as observed by the increase in UV signal over time. There seems to be differences in the combination of CBM and dressing type. For example, ADU114A combined with the dentist dressing gave a near linear curve, resembling the curve for free TF, as in the P4 reference.

[0366] Example 5 - Tissue Factor activity after binding to dressings with longer incubation and altered washing procedureIn a similar procedure as Example 4, TF-CBM fusion proteins were incubated with the dressings. 5 mm dressings were incubated with FT-CBM samples in low salt from example 3: 1 SUGI®, 4 dentist or 8 UPM dressing pieces were mixed with 100 microliter TF-CBM and 200 microliter Milliq water, vortexed and incubated for days at 4 C. Washing was done by first adding 300 microliter 25 mM citrate pH 5, followed by three times washing with PBS:water as Example 4.

[0367] In parallel, native dressing and TF-CBM fusion samples without the dressings were treated as references. The native dentist dressing was almost disintegrated after four days.

[0368] TF activity was measured as described in Example 2 comparing the three modified dressings, TF-CBM samples, Tissue Factor references P4 and P6 reference, unmodified dressings and assay blank.

[0369] The TF activity was estimated according to the assay described in Example 2. Samples compared were the fusion proteins, ADU112A, ADU113A, ADU114A, the TF references as in the previous example and assay blanks.

[0370] Conclusion: The fusion protein treated dressings had TF activity, as observed by the increase in UV signal at 405 nm over time. There seems to be differences in the combination of CBM and dressing type. The native dentist dressing gave a high background.

[0371] Example 6 - TF activity after binding to dressings with short incubation and repeated washing procedure

[0372] In a procedure similar to Example 5, the TF-CBM fusion protein, ADU112A in low salt, was incubated with the S dressing. In parallel, two sets of S dressings (5 mm) were mixed with 200 microliters of TF-CBM, ADU112, and 200 microliters of 50 mM acetate buffer (pH 5). The samples were vortexed and incubated for 60 minutes at room temperature (20-22°C).

[0373] Washing was performed by first adding 300 microliters of 25 mM citrate buffer (pH 5), followed by three washes with PBS:water, as described in Example 4.

[0374] Additionally, one set of S dressings underwent six washes with PBS: water.TF activity was measured as described in Example 2, comparing the two modified dressings, Tissue Factor references P4 and P6, unmodified dressings, and the assay blank.

[0375] Figure 8 summarizes the results of the TF activity measurement for ADU112A incubated for 60 minutes and washed either three or six times.

[0376] Conclusion: The activity remained preserved despite the rigorous washing procedure, indicating low leakage of fusion protein from dressing.

[0377] Example 7: Purification of TF-CBM Constructs by Cellulose Affinity Pulldown In order to optimize purification, reduce manufacturing costs, and minimize process complexity, the downstream purification of four TF-CBM fusion proteins expressed in transient CHO cells, as described in Example 1, was improved by means of an affinity-based pulldown method employing insoluble cellulose. The following fusion proteins or constructs was purified:

[0378] Construct No. 1 - TF-CBM-cel6A with (GGGGS)3 linkers (SEQ ID NO: 3) Construct No. 2 - TF-CBM-cel6A with (GGGGS)e linkers (SEQ ID NO: 4) Construct No. 3 - TF-CBM-CBM3A, with (GGGGS)3linkers (SEQ ID NO: 5) Construct No. 4 - TF-CBM-CBM3A, with (GGGGS)6linkers (SEQ ID NO: 6)

[0379] Preparation of Cellulose Matrix: Microcrystalline cellulose (Avicel; Fluka Analytical, REF 11363-250G, CAS 9004-34-6) was suspended in Type 1 water to a concentration of approximately 15 mg / mL. The suspension was washed three times under continuous stirring, twice for about 2 h and once overnight. Between washes, the cellulose was pelleted by centrifugation and the supernatant discarded. Following the final wash, the pellet was centrifuged to remove residual liquid.

[0380] Binding of TF-CBM Constructs: Culture supernatant comprising TF-CBM constructs was combined with washed cellulose to obtain a final cellulose concentration of about 10 mg / mL. The mixture was incubated for approximately 30 min at about 4°C with gentle mixing to allow binding.

[0381] Separation of Unbound Fraction: Samples were centrifuged at about 20,000 x g for approximately 10 min at about 4°C. The supernatant comprising unbound proteins was collected for analysis.Washing of Cellulose-Bound Protein: The cellulose pellet was washed by addition of approximately 0.5 mL water per 10 mg cellulose, followed by gentle mixing and centrifugation at about 20,000 x g for approximately 10 min at about 4°C. Wash fractions were collected as desired.

[0382] Elution of Bound Protein: Bound TF-CBM constructs were eluted by addition of an aqueous glycerol solution (approximately 50% v / v) at about 100 pL per 20 mg cellulose. The mixture was incubated for approximately 5 h at about 4°C with gentle mixing, followed by centrifugation at about 20,000 x g for approximately 10 min at about 4°C. A second elution step was optionally performed under similar conditions with a reduced incubation time of about 1 h. Elution fractions (A and B) were collected and optionally combined. Eluted fractions were stored at about -80°C. Cellulose pellets were retained for analytical evaluation.

[0383] Protein Quantification: Protein concentration was determined by absorbance at 280 nm using calculated extinction coefficients derived from sequence-based analysis (e.g., ProtParam, ExPASy).

[0384] SDS-PAGE Analysis: Samples were mixed with denaturing buffer comprising LDS sample buffer and reducing agent, and heated to about 95°C for approximately 15 min. Proteins were separated by electrophoresis on 4-12% Tris-glycine gels using standard running buffer at approximately 200 V for about 55 min. Molecular weight markers in the range of about 10-250 kDa were used. Gels were stained with Coomassie-based stain according to the manufacturer’s instructions.

[0385] Also, analytical samples were incubated with PNGase F enzyme for complete deglycosylation. All gels were photographed for documentation and analysis.

[0386] Results and Conclusion: The developed affinity-based pulldown method provides a highly efficient and scalable purification strategy for TF-CBM fusion proteins. In contrast to conventional chromatographic approaches, such as ion exchange or hydrophobic interaction chromatography, the present method enables direct capture of the target protein using an insoluble cellulose matrix in a simple batch format. Surprisingly, the method achieves high purity without the need for column-based separation, despite the complexity of the starting material. This demonstrates that the CBM-medi-ated affinity interaction provides sufficient selectivity to replace multi-step chromatographic purification schemes.Furthermore, the elimination of chromatography columns and associated cleaning-in-place (CIP) procedures significantly reduces process complexity, equipment requirements, and operational costs. The process is therefore inherently robust and readily scalable to industrial production.

[0387] The combination of (i) high purity, (ii) simplified processing, and (iii) elimination of chromatography represents a significant technical advantage over conventional downstream processing methods.

[0388] Example 8 - Thromboelastography (TEG) measurements demonstrating accelerated coagulation

[0389] The four TF-CBM fusion proteins purified as described in Example 7 were evaluated for their ability to initiate and enhance coagulation in whole blood using thromboelastography (TEG).

[0390] Measurements were performed using a TEG 5000 Hemostasis Analyzer System (Haemonetics, TEG500), which monitors the development of viscoelastic properties of blood during clot formation, thereby providing real-time assessment of coagulation initiation, propagation, and clot strength.

[0391] The following parameters were recorded:

[0392] • R time (reaction time): time to initial fibrin formation, reflecting coagulation initiation;

[0393] • K time: rate of clot development following initiation;

[0394] • MA (maximum amplitude): maximum clot strength, reflecting fibrin and platelet contributions.

[0395] A reduction in R time and / or K time, and / or an increase in MA, is indicative of enhanced hemostatic activity.

[0396] For each measurement, pooled human whole blood (340 pL) was combined with CaCE (20 pL, 0.2 M) and TF-CBM fusion protein (20 pL; constructs No.l-No.4, pooled fractions A and B, see Example 7), adjusted to 0.50 mg / mL with type 1 water. A kaolin-activated sample (Haemonetics) was used as a reference standard representing a clinically established coagulation activator. The results are summarized in Table 4.

[0397] Table 4: Overview of TEG-5000 measurements for constructs No.l to No.4 and the reference (Kaolin)TEG-5000 Reference Construct Construct Construct Construct (Kaolin) No. 1 No. 2 No.3 No. 4

[0398] R (Minutes) 6.9 5.5 4.9 8.2 6.3 Angel (Rate

[0399] of clot formation in degrees) 60.3 63.5 68.2 53.6 55.6 MA (mm) 60.5 61.9 64.2 59.1 62.0

[0400]

[0401] Results and Conclusion: The TF-CBM fusion proteins according to the invention demonstrate a pronounced and unexpected ability to accelerate coagulation in whole blood. In particular, Constructs No. 1 and No. 2 show a substantial reduction in R time relative to the kaolin reference, indicating significantly faster initiation of fibrin formation than achieved with a standard clinical activator.

[0402] It is noted, that despite the long CBM protein tail, all four fusion proteins performed well as initiator of coagulation in solution.

[0403] Notably, this acceleration of coagulation initiation is achieved without any reduction in clot strength. On the contrary, MA values are maintained or increased, demonstrating that rapid clot formation is accompanied by the generation of stable and mechanically robust clots.

[0404] This combination of (i) faster-than-kaolin initiation of coagulation and (ii) preserved or enhanced clot strength is unexpected, as conventional activators typically involve a trade-off between speed and clot quality.

[0405] Example 9 - Rapid coupling of TF-CBM fusion protein to cellulose dressing with preserved activity

[0406] In the following, TF-CBM fusion protein Construct No.3 (fraction A or B), as purified in Example 7, was coupled to a cellulose-based dressing, and the resulting Tissue Factor (TF) activity was evaluated.

[0407] Dressing pellets (5 mm diameter) were punched from SUGI® sheets (cot-ton / regenerated cellulose; 150 g / m2; QuestAlpha). For coupling, five pellets wereincubated with TF-CBM fusion protein at a concentration of 0.02 mg / mL in 50 mM acetate buffer (pH 5.0), using a total volume of 1.0 mL per five pellets.

[0408] Incubation was performed at room temperature for 5, 15, or 30 minutes, with vortexing every 1-2 minutes to ensure uniform exposure of the substrate. Following incubation, the pellets were washed six times with water (Type 1 water, 2 ml) under vigorous vortexing to remove unbound protein.

[0409] The pellets were compressed to remove excess liquid and subjected to a TF activity assay as described in Example 2: 20 microliter pellet, sample or sample buffer, 40 microliter assay buffer, 60 microliter phospholipid (Rossix) and vortex mixed before being added 20 microliter Factor VII and Factor X solution. The closed vials were vortex mixed and incubated for 30 minutes at 37 C on a vortex (620 RPM) heating mixing block, before being added 20 microliter Factor X substrate, vortex mixed and incubated at 37 °C. 2.3 microliter reaction samples were taken out for analysis on a Nano Drop UV spectrophotometer.

[0410] Measurements were performed in duplicate (A, B). For comparison, TF-CBM fusion protein Construct No.3 in solution was analyzed in parallel by substituting the dressing pellets with 20 pL of diluted protein sample of known concentration. Untreated SUGI® was added as reference.

[0411] The results are summarized in Figure 9A and Figure 9B for TF-CBM fusion protein Construct No.3 fractions A and B, respectively.

[0412] Results and conclusion: The slopes of the activity curves obtained for TF-CBM fusion protein in solution and for TF-CBM immobilized on the dressing pellets were essentially identical across all incubation times. This demonstrates that immobilization of the fusion protein onto the cellulose substrate does not impair Tissue Factor activity. This result is highly unexpected, as immobilization of coagulation factors on solid supports would typically be expected to reduce accessibility, alter conformation, or otherwise impair biological function resulting in an altered activity and curve. In contrast, the CBM-mediated immobilization according to the invention preserves full functional activity of the TF domain. Furthermore, by comparison of signal intensities between bound and unbound samples of known concentration, coupling yields were estimated to be approximately 20% for Construct No.3 fraction A and approximately 50% for Construct No.3 fraction B, consistent with the relative binding strengths observed during purification (Example 7). Importantly, effective coupling and full functional activity wereachieved after as little as 5 minutes of incubation, with no significant increase in activity observed at longer incubation times. This demonstrates a rapid and efficient binding process, which is particularly advantageous for scalable manufacturing.

[0413] The combination of (i) rapid coupling kinetics, (ii) substantial binding yield, and (iii) complete retention of biological activity upon immobilization represents a significant and unexpected technical advantage over conventional approaches for immobilizing bioactive proteins on dressing materials. These results confirm that TF-CBM fusion proteins can be directly and efficiently immobilized onto cellulose-based dressings to produce highly active hemostatic materials without the need for chemical coupling or complex processing steps.

[0414] Example 10 - Vial assay demonstrating rapid plasma coagulation

[0415] The 5 mm TF-CBM-SUGI pellets obtained in Example 9 were evaluated for their ability to initiate coagulation in human plasma under controlled conditions designed to distinguish intrinsic procoagulant activity from surface-mediated or adsorptionbased effects.

[0416] Citrated human plasma (single donor) was stored at -80 °C and thawed by incubation at approximately 0 °C for 30 minutes, followed by storage on ice prior to use.

[0417] For the assay, one of the following was placed in a labeled 1.5 mL polypropylene vial: (i) a native 5 mm SUGI® pellet, (ii) a TF-CBM-SUGI pellet, (iii) an approximately 8 mm QuikClot® dressing piece (simple kaolin-based reference) or (iv) no sample.

[0418] Subsequently, 340 pL of human plasma was added, followed immediately by 20 pL CaCh (0.20 M, Sigma-Aldrich). The vial was closed and vortexed for 1-2 seconds.

[0419] A camera with time recording was initiated, and all vials were placed on a static heating block at 37 °C. At one-minute intervals, each vial was assessed by inversion. Samples were classified as negative if the plasma remained fluid and positive if a stable clot had formed (no flow upon inversion). The clotting time was recorded accordingly.

[0420] Human plasma alone did not coagulate or solidify within at least 30 minutes under these conditions. Native SUGI pellets likewise did not induce clot formation within the same timeframe.All samples and controls were tested in duplicate (1, 2). The experiment was repeated using TF-CBM-SUGI pellets prepared with coupling times of 5, 15, and 30 minutes, using the same plasma sample within one hour. Figure 10A, Figure B and Figure C, summarize the results.

[0421] Results and conclusion: TF-CBM-functionalized SUGI® pellets according to the invention induced rapid and reproducible coagulation of human plasma, compared to native SUGI® dressing pellets. Plasma alone showed no coagulation within the experimental timeframe. Furthermore, full hemostatic activity was observed for pellets prepared with coupling times as short as 5 minutes, indicating that the functional performance is independent of extended incubation or high surface loading. This indicates that even partial surface modification with TF-CBM fusion protein is sufficient to trigger rapid coagulation. The results demonstrate that the TF-CBM-functionalized dressing provides (i) a direct and potent trigger of the coagulation cascade, (ii) functionality in plasma in the absence of cellular components, and (iii) robust performance independent of precise loading conditions.

[0422] Example 11 - Dose-response of TF-CBM-functionalized dressing in human plasma coagulation

[0423] TF-CBM-functionalized SUGI® pellets with varying protein loading densities were evaluated for their ability to initiate coagulation in human plasma under controlled conditions designed to distinguish intrinsic procoagulant activity from surface-mediated or adsorption-based effects.

[0424] Using a plasma vial assay as described in Example 10 and coupling conditions as in Example 9, TF-CBM fusion protein Construct No.3 fraction A was coupled to 5 mm SUGI150 pellets for 15 minutes at room temperature in 50 mM acetate buffer (pH 5.0). Protein loading levels of 100, 200, 400, 800, and 1600 ng per pellet were applied. Native SUGI® pellets and approximately 8 mm QuikClot® dressing pieces were included as references. All samples and controls were tested in duplicate. The same human plasma donor was used as in Example 10. The results are summarized in Figures 10A-C

[0425] Results and conclusion: TF-CBM-functionalized SUGI® pellets according to the invention consistently induced rapid and reproducible coagulation of humanplasma, whereas native SUGI® and plasma alone did not result in clot formation within the experimental timeframe. Importantly, only a weak dependence on TF-CBM loading density was observed across the tested range. Even at low protein loadings (e.g., 100 ng per pellet), effective coagulation was initiated. This demonstrates that only limited surface coverage is required to trigger the coagulation cascade. This weak dose-response relationship is unexpected and indicates a highly efficient and robust system for initiating hemostasis.

[0426] In contrast to conventional systems that rely on high loading or extensive surface interaction, the present invention provides reliable coagulation initiation across a broad range of protein densities.

[0427] The results demonstrate that the TF-CBM-functionalized dressing exhibits (i) high functional efficiency at low protein loadings, (ii) robustness with respect to variations in coating density, and (iii) reproducible performance under plasma conditions.

[0428] These properties are of significant industrial and clinical relevance, as they enable simplified manufacturing with reduced protein consumption while maintaining consistent hemostatic performance. The weak dose-response relationship shows that only minimal TF-CBM surface coverage is required to trigger coagulation, contrary to conventional expectations.

[0429] Example 12 - preparation of TF-CBM dressing based on SUGI150

[0430] TF-CBM fusion protein Construct No.3, fraction B, was coupled to 3 x 3 cm sheets of SUGI 150.

[0431] Coupling was performed at a loading of 0.002 mg / cm2(corresponding to 400 ng per 5 mm pellet or approximately 0.0001358 mg TF-CBM per mg SUGI) in 50 mM acetate buffer (pH 5.0) for 30 minutes at room temperature. Following incubation, the dressing was washed six times with Type 1 water to remove unbound protein.

[0432] Subsequently, the dressing was incubated in a solution of 2% (w / v) trehalose in 50 mM Tris buffer (pH 7.0) for 5 minutes, compressed to remove excess liquid, and dried overnight (approximately 18 hours) at 12-14 °C and 50% relative humidity under a gentle airflow.

[0433] The dried dressing was vacuum-packaged and stored at 5 °C until use. The resulting TF-CBM-functionalized dressing was used in subsequent in vivo experiments (see Example 13).Example 13 - Mini-pig carotid arterial bleeding model demonstrating robust hemostasis and repositioning tolerance

[0434] The present example demonstrates the hemostatic efficacy and robustness of a Tissue Factor (TF)-CBM-functionalized dressing according to the invention in a severe arterial bleeding model. In particular, the experiment evidences the ability of the dressing to achieve rapid and stable hemostasis under conditions mimicking uncontrolled trauma, including obscured bleeding sites and repeated misplacement.

[0435] Dressings according to the invention were evaluated in vivo using a male mini-pig (Gottingen minipigs, Ellegaard Gottingen Minipigs A / S, Denmark), aged 5-7 months and weighing 14-19 kg.

[0436] Anesthesia was induced by i.m. injection of a zoletil mix (Zoletil 50 (50 mg / ml) + 6.25 ml xylazine (20 mg / ml) + 1.25 ml Ketamine (100 mg / ml) + 2.5 ml butorphanol (10 mg / m)). Dose 1 ml / 10 kg i.m.. After induction, anesthesia was sustained by isoflurane (0-4%).

[0437] Intravenous (i.v.) catheter (femoral vein) was placed in addition to intraarterial blood pressure probe (in femoral arteria). The pigs were given 25 i.u. heparin / kg i.v. to ensure that the coagulation reached human Character. Basic volume-therapy was sustained by infusion of ringer-lactate (approximately 5-50 ml / h) in the i.v. catheter. Adjustment of the infusion velocity aiming to keep a mean blood pressure above 65 mmHg during the study. During the experiment, the minipig were continuously monitored for blood pressure, oxygen saturation and pulsey.

[0438] The carotid artery was surgically exposed and prepared with proximal and distal control. A arterial injury was created using a 14G cannula. The vessel was then allowed to bleed freely for approximately 30 seconds, resulting in rapid blood loss and complete obscuration of the bleeding site, thereby reproducing a clinically relevant scenario of poorly localized severe hemorrhage.

[0439] A TF-CBM-SUGI dressing (3 x 3 cm, prepared in example 10) according to the present invention was applied to an estimated bleeding location and manual compression was initiated. Under these conditions, initial placement did not immediately achieve hemostasis, and continued bleeding was observed. The dressing was subsequently repositioned following partial removal of blood using surgical swaps.Despite initial misplacement and repeated repositioning, effective hemostasis was achieved within approximately 5 minutes of compression at bleeding location. Upon gradual release of pressure, the arterial puncture remained sealed, and physiological pulsation of the artery was preserved. The hemostatic seal remained stable upon observation for an additional 5 minutes. The experiment was ended by injection of pentobarbital i.v.. All the action documented on video for post-surgery analysis.

[0440] The results demonstrate that the TF-CBM-functionalized dressing according to the invention provides rapid and reliable hemostasis in a high-pressure arterial bleeding model, even under anticoagulated conditions and in the presence of poor visual localization of the bleeding source.

[0441] Notably, and unexpectedly, the dressing retains full hemostatic functionality following misapplication and subsequent repositioning, without loss of efficacy. This robustness contrasts with conventional hemostatic materials, which typically require precise placement and may lose effectiveness upon displacement or saturation with blood.

[0442] The observed combination of (i) efficacy under anti coagulated conditions, (ii) tolerance to incorrect initial placement, and (iii) maintained function after repositioning represents a significant technical advantage and supports the suitability of the invention for use in uncontrolled bleeding scenarios, including trauma and emergency surgery.

[0443] Example 14 - TF-CBM-functionalized dressing with high binding efficiency and retained activity

[0444] Four TF-CBM fusion proteins prepared as described in Example 7 (pooled fractions A and B) were evaluated for their ability to bind to cellulose-based dressing material and retain tissue factor activity.

[0445] Coupling

[0446] Coupling was performed as described in Example 9. Briefly, each TF-CBM construct was incubated with SUGI® dressing material (150 g / m2) in the form of 5 mm pellets (n = 80) in 50 mM acetate buffer (pH 5.0) for 30 minutes at room temperature. A loading of approximately 400 ng protein per pellet was applied.

[0447] Following incubation, pellets were washed four times with purified water (30 mL) with vortex mixing (2 minutes per wash) to remove unbound protein.

[0448] Activity assessmentTissue factor activity was determined using the assay described in Example 2, analyzing both unbound and pellet-bound fusion protein. Activity was monitored by UV absorbance over time.

[0449] Results

[0450] A clear difference in performance between the different protein constructs was observed, with activity ranking in free solution:

[0451] Construct No.2 > Construct No.1 » Construct No.3 ~ Construct No.4 In free solution, the activity observed for Construct No.2 was higher than the activity for Construct No.1. The activity of Construct No.1 in free solution was much higher than that of Construct No.3 in free solution. The activities of Constructs No.3 and No.4 in free solution were approximately the same.

[0452] Pellets functionalized with TF-CBM constructs exhibited substantial procoagulant activity, whereas blank SUGI pellets showed no detectable activity. Reference TF preparations (P3, P4, P5) confirmed assay performance (see Figure 12 for summarized results).

[0453] Importantly, only minimal activity was detected in wash fractions, indicating efficient immobilization of the fusion proteins.

[0454] Conclusion: The results demonstrate that selected TF-CBM fusion proteins enable rapid and efficient immobilization to cellulose under mild aqueous conditions while retaining biological activity.

[0455] The combination of:

[0456] • high binding efficiency (low loss during extensive washing), • rapid coupling without chemical activation, and

[0457] • retention of functional tissue factor activity upon immobilization was not expected from conventional protein immobilization approaches and represents a significant technical advantage for the preparation of functionalized hemostatic dressings.Embodiments

[0458] 1. A fusion protein comprising at least one blood coagulation factor or any variants thereof, and one or more Cellulose Binding Module (CBM), wherein the CBM is bound, optionally by a linker, to the at least one blood coagulation factor or any variants thereof.

[0459] 2. The fusion protein according to embodiment 1, wherein said at least one blood coagulation factor is Tissue Factor (TF) as set forth in SEQ ID NO: 9 or as set forth in SEQ ID NO: 2, or any variant thereof.

[0460] 3. The fusion protein according to embodiments 1 or 2, wherein said fusion protein comprises a linker having a sequence of (GGGGS)x, wherein X is any number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0461] 4. The fusion protein according to any one of embodiments 2 or 3, wherein the TF or any variant thereof has at least 59%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, or such as at least 95% sequence identity to SEQ ID NO: 2.

[0462] 5. The fusion protein according to any one of embodiments 2 - 4, wherein the TF or any variant thereof is of human, porcine, equine, canine or bovine origin.

[0463] 6. The fusion protein according to any one of embodiments 2 - 5, wherein the TF or any variant thereof is the extracellular domain of human TF.

[0464] 7. The fusion protein according to any one of embodiments 1 or 3, wherein the at least one blood coagulation factor is selected from the group consisting of: Factor I, Factor II, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XII and Factor XIII, or combinations thereof.

[0465] 8. The fusion protein according to any one of the preceding embodiments, wherein the CBM is selected from the group consisting of: CBM1, CBM2, CBM3, CBM4, CBM5, CBM17, CBM28, CBM12, CBM18, CBM33, CBM20, CBM25, CBM48, CBM32, CBM35, CBM6a and CBM6b.9. A dressing material comprising the fusion protein according to any one of embodiments 1 to 8.

[0466] 10. The dressing material according to embodiment 9, wherein the material is selected from the group consisting of wound dressing, patches, gauzes, extracellular matrix components, gels, creams, ointments, particles, such as nanoparticles, and any combinations thereof.

[0467] 11. The dressing material according to any one of embodiments 9 or 10, wherein the material is selected from the group consisting of cotton, cellulose, cellulose ethers, such as rayon and viscose, oxidized cellulose, regenerated cellulose, carboxymethylcellulose, chitosan, agarose or paper-based membranes, polyamides, poly sulfone ether, polyvinyl alcohols, nitrocellulose, nitrocellulose mixed esters, polyethylene terephthalate, blends and combinations thereof.

[0468] 12. A system for promoting rapid haemostasis while reducing the risk of undesirable blood coagulation, said system comprising dressing material according to any one of embodiments 9 to 11.

[0469] 13. Use of a fusion protein according to any one of embodiments 1 to 8, a dressing material according to any one of embodiments 9 to 11, or a system according to embodiment 12 in promoting rapid haemostasis.

[0470] 14. The fusion protein according to any one of embodiments 1 to 8 or the dressing material according to any one of embodiments 9 to 11, or the system according to embodiment 12 for use in promoting rapid haemostasis.

[0471] 15. A method for preparing the dressing material according to any one of embodiments 9 to 11 comprising the following steps:

[0472] (a) obtaining a fusion protein comprising at least one blood coagulation factor and one or more CBM,

[0473] (b) soaking or contacting the fusion protein from step (a) with a dressing material capable of binding CBM, and

[0474] (c) optionally washing the dressing and optionally drying with preservatives.

Claims

CLAIMS1. A fusion protein comprising (i) at least one blood coagulation factor or any variants thereof; and (ii) one or more Cellulose Binding Module (CBM), wherein the CBM is bound, optionally by a linker, to the at least one blood coagulation factor or any variant thereof, and wherein said at least one blood coagulation factor is Tissue Factor (TF) as set forth in SEQ ID NO: 9 or as set forth in SEQ ID NO: 2, or any variant thereof.

2. The fusion protein according to claim 1, wherein said fusion protein comprises a linker having a sequence of (GGGGS)x, wherein X is any number selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

3. The fusion protein according to any one of claims 1 or 2, wherein the TF or any variant thereof has at least 59%, such as at least 60%, such as at least 70%, such as at least 80%, such as at least 90%, or such as at least 95% sequence identity to SEQ ID NO: 2.

4. The fusion protein according to any of the preceding claims, wherein the TF or any variant thereof is of human, porcine, equine, canine or bovine origin.

5. The fusion protein according to any of the preceding claims, wherein the TF or any variant thereof is the extracellular domain of human TF.

6. The fusion protein according to any of the preceding claims, wherein the CBM is selected from the group consisting of: CBM1, CBM2, CBM3, CBM4, CBM5, CBM17, CBM28, CBM12, CBM18, CBM33, CBM20, CBM25, CBM48, CBM32, CBM35, CBM6a and CBM6b.

7. A dressing material comprising the fusion protein according to any one of claims 1 to 6.

8. The dressing material according to claim 7, wherein the material is selected from the group consisting of wound dressing, patches, gauzes, extracellular matrix components, gels, creams, ointments, particles, such as nanoparticles, and any combinations thereof.

749. The dressing material according to any one of claims 7 or 8, wherein the material is selected from the group consisting of cotton, cellulose, cellulose ethers, regenerated cellulose, such as rayon and viscose, oxidized cellulose, carboxymethylcellulose, chitosan, agarose or paper-based membranes, polyamides, polysulfone ether, polyvinyl alcohols, nitrocellulose, nitrocellulose mixed esters, polyethylene terephthalate, blends and combinations thereof.

10. The dressing material according to any one of claims 7 to 9 wherein the fusion protein comprises TF as set forth in SEQ ID NO:3 or SEQ ID NO:4 or SEQ ID NO:5 or SEQ ID NO:6 and wherein the dressing material comprises cotton and / or regenerated cellulose.

11. The dressing material according to any one of claims 7 to 10, wherein the amount of fusion protein is in the range of about 50 ng / cm2area of dressing material to about 20000 ng / cm2area of dressing material, more preferably, in the range of about 500 ng / cm2area of dressing material to about 10000 ng / cm2area of dressing material.

12. A system for promoting rapid haemostasis while reducing the risk of undesirable blood coagulation, said system comprising adressing material according to any one of claims 7 to 11.

13. The fusion protein according to any one of claims 1 to 6 or the dressing material according to any one of claims 7 to 11 or the system according claim 12 for use in promoting rapid haemostasis.

14. A method for preparing the dressing material according to any one of claims 7 to 11 comprising the following steps:(a) obtaining a fusion protein comprising at least one blood coagulation factor and one or more CBM,(b) soaking or contacting the fusion protein from step (a) with a dressing material capable of binding CBM, and(c) optionally washing the dressing and optionally drying with preservatives.75

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