Therapeutic Fusion Proteins

Abstract

The present invention relates to fusion proteins suitable for use as pharmaceuticals or research tools. Therapeutic uses of the fusion proteins may include the prevention or treatment of acute or chronic inflammatory and immune system-induced organ and microvascular disorders, such as acute kidney injury, acute respiratory distress syndrome, sepsis, acute myocardial infarction, tissue fibrosis, and other organ damage resulting from tissue trauma.

Description

[Technical field]

[0001] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy, created on August 31, 2020, is named PAT058332_SL.txt and is 653,193 bytes in size.

[0002] The present invention relates to fusion proteins that contain both integrin-binding and phosphatidylserine-binding capabilities, which can be used as therapeutic agents, especially for the prevention or treatment of acute or chronic inflammatory disorders, as well as organ and microvascular disorders caused by the immune system or coagulation. [Background technology]

[0003] Acute inflammatory organ injury (AOI) is a historically challenging disease with high morbidity, mortality, and significant unmet medical needs. Typical AOI include myocardial infarction (MI) and stroke, which occur in 32.4 million patients worldwide each year. Patients with previous MI and stroke are considered the highest risk group for further coronary and cerebral events, ranked by the World Health Organization as one of the top causes of morbidity in developed countries. Another AOI is acute kidney injury (AKI), which occurs in approximately 13.3 million people annually. In high-income countries, the incidence of AKI is 3-5 / 1000 and is associated with high mortality (14-46%) (Metha et al., (2015) Lancet, 385(9987):2616-43). As with MI and stroke, survivors of AKI often do not fully recover and are at high risk of developing chronic or end-stage renal disease. To date, there are no FDA-approved drugs available to prevent or treat AKI. The development of novel treatments for AKI has proven difficult, with no successful results in clinical trials so far. This may be due to the multifactorial and multifaceted pathophysiology of AKI, including inflammatory, microvascular dysfunction, and nephrotoxic pathomechanisms induced by sepsis, ischemia / reperfusion, and / or nephrotoxic injury. These drivers may act simultaneously or sequentially to cause injury to mostly tubular but also glomerular cells, loss of renal functional reserve, and ultimately renal failure.

[0004] One common common factor in AOI is increased cell death due to tissue injury, increased generation of cell fragments, and prothrombotic / proinflammatory microparticles that can enter the circulation and the injured tissue. After neutrophils infiltrate tissue to prevent infection, they undergo apoptosis or other forms of cell death in the affected tissue. Neutrophils contain harmful substances such as proteolytic enzymes and danger-associated molecular patterns (DAMPs), which can promote host tissue damage and propagate inflammation. Efficient uptake of dead cells triggers signaling events that lead to reprogramming of macrophages (MΦ) toward a non-inflammatory pro-resolving phenotype and release of key mediators for successful degradation and repair of affected tissue. This reprogramming has recently been attributed to metabolic signaling that activates a phagocytic anti-inflammatory response in macrophages (Zhang et al., (2019) Cell Metabolism, 29(2): 443-56). The removal of this debris, or senescent or dead cells, in a non-inflammatory manner is called "efferocytosis."

[0005] However, if efferocytosis is delayed, necrotic cells may accumulate and cause an inflammatory response, triggering, for example, proinflammatory cytokines (TNF-α) or immunosuppressive IL-10 by macrophages (Greenlee-Wacker (2016) Immunol. Reviews, 273:357-370). Furthermore, if cellular debris and particles are not efficiently removed, they may cause cell clumps and aggregates such as neutrophil-platelet fragment clusters, microthrombi, and / or release danger-associated molecular patterns (DAMPS) such as ATP, DNA, histones, or HMGB1. Consequences may include microvascular obstruction, dysfunction, and prominent sterile inflammation leading to progression of tissue damage, primary and secondary organ failure, or repair maladaptation.

[0006] In the acute phase of AOI, the efferocytosis pathway appears to be significantly downregulated. Inflammation or acute responses to injury (organic factors, hypoxia, oxidative stress, irradiation, inflammation, and infection) suppress effective efferocytosis or phagocytosis by downregulation of cross-linking proteins and dedicated phosphatidylserine (PS)-binding proteins, including cell surface efferocytosis / clearance receptors. An example of a dysfunctional efferocytosis receptor is the proteolytic shedding of TAM family receptors, such as Mer tyrosine kinase (MerTK). MerTK is an integral membrane protein preferentially expressed in phagocytes that not only functions as a signaling protein but also promotes efferocytosis (through proteins such as Gas6 or Protein S) and inhibits inflammatory signaling. Proteolytic cleavage and release of the soluble ectodomain of MerTK is induced by the metalloproteinase ADAM17. The shedding process can reduce phagocyte efferocytosis by shedding of surface MerTK. Furthermore, the released ectodomain can also inhibit efferocytosis in vitro (Zhang et al., (2015) J Mol Cell Cardiol., 87: 171-9; Miller et al., (2017) Clin Cancer Res., 23(3): 623-629). Increased serum / plasma soluble Mer levels are typically observed in inflammatory, malignant, or autoimmune diseases such as diabetic nephropathy or systemic lupus erythematosus (SLE) and may indicate disease severity (Ochodnicky P (2017) Am J Pathol., 187(9): 1971-1983; Wu et al., (2011) Arthritis Res Ther. 13: R88). Furthermore, cross-linking proteins such as milk fat globule-EGF factor 8 protein (MFG-E8) are also downregulated during most acute and chronic inflammatory diseases.Similar to soluble Mer, decreased serum / plasma concentrations of MFG-E8 can be seen in patients with MI or stable angina (Dai et al., (2016) World J Cardiol., 8(1):1-23) and may indicate disease severity, as described for chronic obstructive pulmonary disease (COPD; Zhang et al., (2015) supra).

[0007] Phosphatidylserine (PS) exposure to dying cells is an evolutionarily conserved anti-inflammatory and immunosuppressive signal for immune cells. A vast number of major mammalian pathogens utilize PS-mediated uptake as part of their pathogenic cell infection (Birge et al., (2016) Cell Death Diff., 23(6): 962-78). For example, viruses can bind to PS-binding receptors directly or through proteins such as Gas6 (Morizono & Chen (2014) J Virol., 88(8): 4275-90). Inactivation of intrinsic clearance pathways in response to injury may reduce the efficiency of infectious pathogens to invade and hijack cells after injury, thereby representing an evolutionarily evolved response to evade host immune responses and defenses. As a result, downmodulation of clearance pathways would improve the effectiveness of innate and adaptive immune effectors to fight infection. As a consequence of "friendly fire", efferocytosis may be transiently affected during acute organ injury, leading to the above-mentioned complications in AOI. Accumulation of dead cells, debris, proinflammatory and prothrombotic MPs is a hallmark of AOI and represents a major trigger of inflammation and microvascular injury. It is noteworthy that such accumulation of inflammatory and prothrombotic microparticles is common in critical diseases with high medical need and may contribute to their morbidity. Examples of such indications are sepsis and cancer (Yang et al., (2016) Tumour Biol., 37(6):7881-91; Zhao et al., (2016) J Exp Clin Cancer Res., 35:54; Muhsin-Sharafaldine et al., (2017) Biochim Biophys Acta Gen Subj., 1861(2):286-295; Ma et al., (2017) Sci Rep., 7(1):4978; Souza et al., (2015) Kidney Int. 87(6):1100-8).Previous drug discovery efforts in this field have focused on PS-binding proteins, which can serve as the basis for drug candidate design, as reviewed by (Li et al., (2013) Exp Opin Ther Targets, 17(11):1275-1285).

[0008] A subset of PS-binding proteins also recognize and bind to integrins, such as αvβ3 and αvβ5, which are expressed in many cell types, including phagocytes. These proteins act to crosslink PS exposing apoptotic / dying cells to integrins, leading to efferocytosis (also called phagocytosis) by macrophages and non-professional phagocytes. Several crosslinking proteins are also downregulated during most acute and chronic inflammatory diseases. Therapeutic use of such cross-linked proteins or truncated versions thereof has been suggested previously (WO2006122327 (sepsis), WO2009064448 (organ injury after ischemia / reperfusion), WO2012149254 (cerebral ischemia) The Feinstein Institute for Medical Research; WO2015025959 (myocardial infarction) Kyushu University and Tokyo Medical University; WO20150175512 (bone resorption) University of Pennsylvania; WO2017018698 (tissue fibrosis) Korea University Research and Business Foundation and US20180334486 (tissue fibrosis) Nexel Co., Ltd.); however, the use of wild-type or naturally occurring proteins is limited by a number of problems. For example, wild-type MFG-E8 (wtMFG-E8) is thought to be poorly viable, poorly soluble, and expressed in very low yields when cultured in a cell expression system. A study by Castellanos et al. (2016) showed that MFG-E8 expressed as an Fc-IgG fusion in insect or CHO cells was completely aggregated and could only be efficiently purified by adding detergents such as Triton X-100 or CHAPS (Castellanos et al., (2016) Protein Exp. Pur., 124: 10-22).

[0009] Clearance of dead cells, debris, and particulates by cross-linking proteins, e.g., MFG-E8, EDIL3, and Gas6, may eliminate a major cause of sterile inflammation and microvascular dysfunction, thus preventing the progression of tissue damage and allowing inflammation to resolve. Thus, therapeutic approaches that promote the clearance of dead cells during the course of AOI may be used to reduce or at least alleviate the pathology of AOI and may have implications in other disease settings where dead cells or PS-exposed particulates are not adequately cleared. Therefore, there is a need for therapeutic agents that can be used to reduce tissue damage and inflammation and that have desirable manufacturing properties to address the unmet medical needs of AOI. Summary of the Invention [Means for solving the problem]

[0010] In the present disclosure, applicants have generated recombinant therapeutic fusion proteins based on the structure of naturally occurring proteins (e.g., MFG-E8) without the aforementioned undesirable properties and production problems of wild-type cross-linked proteins. The fusion proteins of the present disclosure include an integrin binding domain, a PS binding domain, and a solubilization domain. The fusion proteins maintain the main biological functions of the wild-type MFG-E8 protein, for example, by functioning to cross-link PS-exposed dead cells, debris, and particulates to phagocytes, thus triggering efferocytosis. Furthermore, the therapeutic fusion proteins of the present disclosure have improved developability, particularly reduced adhesion and improved solubility, compared to the wild-type MFG-E8 protein (SEQ ID NO: 1). Furthermore, these therapeutic fusion proteins have longer plasma exposure and higher yields when expressed in a cell expression system when compared to the wild-type MFG-E8 protein.

[0011] Provided herein is a therapeutic fusion protein for enhancing efferocytosis that comprises an integrin-binding domain, a phosphatidylserine (PS)-binding domain, and a solubility domain.

[0012] In some embodiments, the soluble domain of the fusion protein is linked to the integrin binding domain. In some embodiments, the soluble domain is linked to the PS binding domain. In some embodiments, the soluble domain is linked to both the integrin binding domain and the PS binding domain, i.e., is located between the integrin binding domain and the PS binding domain. In some embodiments, the soluble domain is inserted within the integrin binding domain or inserted within the PS binding domain. In one embodiment, the therapeutic fusion protein has the following structure from N-terminus to C-terminus: integrin binding domain-soluble domain-PS binding domain.

[0013] In some embodiments, the integrin binding domain of the therapeutic fusion protein comprises an arginine-glycine-aspartic acid (RGD) binding motif and binds to αvβ3 and / or αvβ5 or α8β1 integrin.

[0014] In some embodiments, the solubility domain of the therapeutic fusion protein is directly linked to the integrin binding domain and / or linked to the PS binding domain, i.e., inserted between said domains. In alternative embodiments, the solubility domain is indirectly linked to the integrin binding domain and / or the PS binding domain by a linker, such as an external linker. In some embodiments, the solubility domain comprises human serum albumin (HSA), domain 3 of HSA (HSA D3) or the Fc region of IgG (Fc-IgG), or functional variants thereof.

[0015] In some embodiments, a therapeutic fusion protein comprises the C-terminus of an integrin binding domain linked to the N-terminus of a solubility domain, and the C-terminus of the solubility domain linked to a PS binding domain. In some embodiments, a therapeutic fusion protein comprises the general structure EGF-HSA-C1-C2, where EGF represents the integrin-binding EGF-like domain of MFG-E8, EDIL3, or other proteins containing an integrin binding domain listed in Table 1, and C1-C2 represents the PS binding domain found in MFG-E8, EDIL3, or other proteins containing a PS binding domain listed in Table 2. Examples of proteins containing both an integrin binding domain and a PS binding domain, such as MFG-E8 (SEQ ID NO: 1) and EDIL3 (SEQ ID NO: 11), are listed in Table 3.

[0016] In some embodiments, the integrin binding domain is an EGF-like domain, e.g., having an amino acid sequence as set forth in SEQ ID NO:2, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a truncation variant thereof. In one embodiment, the EGF-like domain comprises the EGF-like domain of human MFG-E8, or a functional variant thereof comprising 1, 2, 3, 4, 5, or up to 10 amino acid modifications. In one embodiment, the EGF-like domain comprises the EGF-like domain of human EDIL3, or a functional variant thereof comprising 1, 2, 3, 4, 5, or up to 10 amino acid modifications.

[0017] In some embodiments, the PS binding domain comprises two discoidin C1-C2 subdomains, e.g., the PS binding domain of human MFG-E8 having an amino acid sequence set forth in SEQ ID NO:3, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a truncated variant thereof. In one embodiment, the PS binding domain comprises the PS binding domain of human MFG-E8, or a functional variant thereof comprising 1, 2, 3, 4, 5, up to 10 amino acid modifications. In one embodiment, the PS binding domain comprises the PS binding domain of human EDIL3, or a functional variant thereof comprising 1, 2, 3, 4, 5, up to 10 amino acid modifications.

[0018] In some embodiments, the solubilization domain is HSA or a functional variant thereof, for example having the amino acid sequence set forth in SEQ ID NO: 4, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a truncated variant thereof. In one embodiment, HSA comprises the amino acid substitution C34S, which functions to reduce the aggregation tendency of the protein, and has the amino acid sequence as set forth in SEQ ID NO: 5. In some embodiments, the solubilization domain comprises human serum albumin (HSA) or a functional variant thereof comprising 1, 2, 3, 4, 5, up to 10 amino acid modifications, for example HSA C34S, or a truncated variant of HSA, for example domain 3 of HSA (HSA D3), or a functional variant thereof. In a preferred embodiment, the solubilization domain is HSA C34S.

[0019] In an alternative embodiment, the solubility domain comprises an Fc region of an IgG (Fc-IgG), such as the Fc region of human IgG1, IgG2, IgG3 or IgG4 or a functional variant thereof. In one embodiment, the solubility domain comprises an Fc region of human Fc-IgG1 having the amino acid sequence set forth in SEQ ID NO: 7, or amino acids having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a truncation variant thereof. In one embodiment, the Fc-IgG1 comprises the amino acid substitutions D265A and P329A to reduce Fc effector function and has the amino acid sequence as set forth in SEQ ID NO: 8. In another embodiment, the Fc-IgG1 may comprise the amino acid substitution T366W to create a "knob" or the amino acid substitutions T366S, L368A, Y407V to create a "hole". Furthermore, the Fc-IgG1 knob may contain the amino acid substitution S354C and the Fc-IgG1 hole may contain the amino acid substitution Y349C, resulting in the formation of a cysteine ​​bridge upon pairing. In addition to the knob-in-hole modification, the Fc-IgG1 may also contain D265A and P329A substitutions to reduce Fc effector function. In one embodiment, the Fc-IgG1 has an amino acid sequence as set forth in SEQ ID NO: 9 or 10.

[0020] In a preferred embodiment, the therapeutic fusion protein comprises a milk fat globule-EGF factor 8 protein (MFG-E8) and a solubility domain, where MFG-E8 comprises an integrin-binding EGF-like domain (SEQ ID NO:2) and a phosphatidylserine-binding C1-C2 domain (SEQ ID NO:3 or SEQ ID NO:76). MFG-E8 may comprise native or wild-type human MFG-E8 (SEQ ID NO:1), or MFG-E8 having SEQ ID NO:75 or a functional variant thereof. In one embodiment, the solubility domain is linked to the N- or C-terminus of MFG-E8. In one embodiment, the solubility domain is inserted between the EGF-like domain and the C1 domain, or between the C1 domain and the C2 domain. In a preferred embodiment, the solubility domain is linked to the C-terminus of the EGF-like domain and to the N-terminus of the C1 domain. The solubility domain may be linked directly or indirectly to the C-terminus of the EGF-like domain and may be linked directly or indirectly to the N-terminus of the C1 domain. In some embodiments, the indirect linkage is by way of an external linker, for example a glycine-serine based linker.

[0021] In one embodiment, the therapeutic fusion protein comprises the amino acid sequence as set forth in SEQ ID NO: 42 (FP330). In one embodiment, the therapeutic fusion protein may comprise a histidine tag (His tag; SEQ ID NO: 67) to aid in detection and / or purification in characterization assays and protein expression. In one embodiment, the therapeutic fusion protein has a C-terminal His tag and comprises the amino acid sequence set forth in SEQ ID NO: 44 (FP278). The therapeutic fusion proteins FP278 and FP330 share the same amino acid sequence, except for the addition of a His tag to FP278.

[0022] In some embodiments, the therapeutic fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 42 (FP330), or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity thereto, or a truncation variant thereof. For example, the therapeutic fusion protein FP776 comprises the amino acid sequence set forth in SEQ ID NO: 48 and has 97.7% sequence identity to FP330 (SEQ ID NO: 42). For example, the therapeutic fusion protein FP068 comprises the amino acid sequence set forth in SEQ ID NO: 46 and has 98.3% sequence identity to FP330 (SEQ ID NO: 42). For example, the therapeutic fusion protein FP816 comprises the amino acid sequence set forth in SEQ ID NO: 58 and has 98.5% sequence identity to FP330 (SEQ ID NO: 42). For example, the therapeutic fusion protein FP811 comprises the amino acid sequence set forth in SEQ ID NO: 54 and has 99.0% sequence identity to FP330 (SEQ ID NO: 42). For example, therapeutic fusion protein FP010 comprises the amino acid sequence set forth in SEQ ID NO:56 and has 99.5% sequence identity to FP330 (SEQ ID NO:42). For example, therapeutic fusion protein FP138 comprises the amino acid sequence set forth in SEQ ID NO:52 and has 99.8% sequence identity to FP330 (SEQ ID NO:42). For example, therapeutic fusion protein FP284 comprises the amino acid sequence set forth in SEQ ID NO:50 and has 99.9% sequence identity to FP330 (SEQ ID NO:42).

[0023] In some embodiments, and as described in the Examples section, the therapeutic fusion proteins of the disclosure function to promote efferocytosis by endothelial cells in a human endothelial cell-Jurkat cell efferocytosis assay, restore impaired and enhance basal efferocytosis by macrophages in a human macrophage-neutrophil efferocytosis assay; the fusion proteins function to reduce the number of plasma microparticles by clearance in a human endothelial-microparticle efferocytosis assay; and / or the fusion proteins provide protection against multi-organ injury in an acute renal ischemia model.

[0024] Also disclosed herein are methods, uses, diagnostic reagents, pharmaceutical compositions and kits that utilize or comprise these therapeutic fusion proteins. Also provided herein are nucleic acids encoding the disclosed fusion proteins, cloning and expression vectors containing such nucleic acids, host cells containing such nucleic acids, and processes for producing the disclosed fusion proteins by culturing such host cells. [Brief description of the drawings]

[0025] [Figure 1] 1 shows a schematic diagram of an example of a therapeutic fusion protein of the present disclosure. A solubility domain (labeled "SD") was linked either at the C-terminus, N-terminus, or between the EGF, C1 or C2 domains of MFG-E8. [Figure 2-1] Figure 2: A number of SDS-PAGE protein gels of fusion proteins expressed in HEK cells are shown. Figure 2A: EGF-HSA-C1-C2 protein (FP330; SEQ ID NO: 42); Figure 2B: EDIL3 protein EGF-HSA-C1-C2 (FP050; SEQ ID NO: 12); Figure 2C: Non-reduced and reduced EGF-Fc(KiH)C1-C2 protein (this protein is a heterodimer of FP071 (EGF-Fc(knob)-C1-C2; SEQ ID NO: 18) and Fc-IgG1 hole (SEQ ID NO: 10); Figure 2D: EGF-HSA-C1 protein (FP260; SEQ ID NO: 34). For each of Figures 2A, 2C, and 2D, the first column shows the Precision Plus Protein unstained standard marker and the second column shows the respective fusion protein. For Figure 2B, the first column shows the fusion protein and the second column shows the Precision Plus Protein unstained standard marker. FIG. 2E shows additional recombinant proteins that were produced and purified. [Figure 2-2] (As stated above.) [Diagram 3]FIG. 3 illustrates the effect of loss of fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) protein on wild-type (wt) MFG-E8 during practical handling. FIG. 3A shows the loss of efficacy of wild-type MFG-E8 in the L-α-phosphatidylserine competition assay when protein dilutions are made on polypropylene plates (symbol: □) compared to dilutions made on non-binding plates (symbol: ●). In contrast, FIG. 3B shows that there is virtually no loss of efficacy of fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) in the PS competition assay when protein dilutions are made on polypropylene plates (symbol: □) versus non-binding plates (symbol: ●). [Figure 4] Binding of fusion proteins to L-α-phosphatidylserine. Figure 4A shows the concentration-dependent binding of FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) to immobilized L-α-phosphatidylserine and to a lesser extent to the phospholipid cardiolipin. Figure 4B shows the concentration-dependent binding of human wild-type MFG-E8 and several therapeutic fusion proteins: FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44), FP250 (EGF-HSA; SEQ ID NO: 32), FP260 (EGF-HSA-C1; SEQ ID NO: 34), and FP270 (EGF-HSA-C2; SEQ ID NO: 36) to immobilized L-α-phosphatidylserine in a competitive assay format (competition for binding of biotinylated mouse wild-type MFG-E8 to L-α-phosphatidylserine). [Figure 5-1]Figure 5: αv-integrin-dependent cell adhesion to fusion proteins. Figure 5A shows that cell adhesion to FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42) is completely blocked by the αv-integrin inhibitor Cilengitide or 10 mM EDTA. Single point mutation of the integrin binding motif RGD (RGD>RGE) of the EGF-like domain (FP280; SEQ ID NO: 38) results in complete inhibition of cell adhesion as shown in Figure 5B. Figure 5C shows that immobilized EGF-HSA protein (FP250; SEQ ID NO: 32) does not or only moderately supports adhesion of BW5147.G.1.4 cells despite the EGF-like domain. As shown in Figure 5D, the fusion protein of the present disclosure (FP330; SEQ ID NO: 42) promotes αv-integrin-dependent cell adhesion similar to wild-type MFG-E8 when expressed in CHO or HEK cells. [Figure 5-2] (As stated above.) [Figure 6] Figure 4 shows the effect of therapeutic fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) on promoting efferocytosis of killed neutrophils by human macrophages. The concentration of fusion protein is shown on the x-axis and efferocytosis [%] is shown on the y-axis. [Figure 7-1] Figure 7: The therapeutic fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) is able to rescue endotoxin (lipopolysaccharide)-impaired efferocytosis of killed neutrophils by human macrophages. Figure 7A shows the impairment of macrophage efferocytosis of killed human neutrophils by 100 pg / ml lipopolysaccharide (LPS) in three human donors. The left panel shows the response of individual donors, and the right panel shows the average impairment (%) of efferocytosis for the three donors. Figure 7B shows the rescue of this endotoxin (LPS)-impaired efferocytosis of killed neutrophils by human macrophages in the presence of the therapeutic fusion protein FP278. The efferocytosis index of three different human macrophage donors was normalized and plotted as efferocytosis (%). [Figure 7-2](As stated above.) [Figure 8-1] Figure 8: Rescue of S. aureus particle-induced impairment of efferocytosis of killed neutrophils by human macrophages with therapeutic fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44). Figure 8A shows the effect of a concentration of 100 nM FP278 on promoting efferocytosis compared to basal levels (dotted line; left side of figure) and the effect of 100 nM FP278 in rescuing the impairment of efferocytosis caused by administration of S. aureus (right side of figure). Figure 8B shows the effect of increasing concentrations of the fusion protein FP278 (EC50 8 nM) on the rescue of the impairment of efferocytosis caused by administration of S. aureus and on promoting efferocytosis after the basal level of efferocytosis has been reached. [Figure 8-2] (As stated above.) [Figure 9] 9 shows the effect of therapeutic fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) on promoting efferocytosis of dead Jurkat cells by human endothelial cells (HUVEC). As shown in FIG. 9, the efficiency of fusion proteins in the endothelial cell efferocytosis assay depends on the presence of C1-C2 or C1-C1 tandem domains, since a fusion protein of the structure EGF-HSA-C2 (FP270; SEQ ID NO: 36) is ineffective in this assay. [Figure 10] The position of the HSA domain of the therapeutic fusion protein, i.e., N- or C-terminal position (FP220 (HSA-EGF-C1-C2; SEQ ID NO: 30) or FP110 (EGF-C1-C2-HSA; SEQ ID NO: 28), respectively, is shown to confer efferocytosis blocking function to the MFG-E8 HSA fusion protein in a macrophage efferocytosis assay. Fusion protein concentration is shown on the x-axis and efferocytosis [%] is shown on the y-axis. [Figure 11-1]Figure 11: Comparison of promotion of efferocytosis by various formats of therapeutic fusion proteins containing HSA or Fc moieties. Fusion protein concentration is shown on the x-axis (nM) and efferocytosis [MFI] is shown on the y-axis. Figure 11A shows a comparison of fusion proteins containing HSA, where HSA is located at the C-terminus, or the N-terminus, or between the EGF-like domain and the C1 domain; FP110 (EGF-C1-C2-HSA; SEQ ID NO: 28), FP220 (HSA-EGF-C1-C2; SEQ ID NO: 30) and FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44), respectively. FIG. 11B shows a comparison of fusion proteins containing an Fc portion, where the Fc is located at the C-terminus (FP060 (EGF-C1-C2-Fc [S354C, T366W]; SEQ ID NO: 14) and FP080 (EGF-C1-C2-Fc; SEQ ID NO: 22)) or between the EGF-like domain and the C1 domain (FP070 (EGF-Fc-C1-C2; SEQ ID NO: 16)), to wild-type MFG-EG (SEQ ID NO: 1). Two formats of the Fc portion are shown: wild-type Fc (FP080; SEQ ID NO: 22) and an Fc portion with modifications S354C and T366W (EU numbering; FP060; SEQ ID NO: 14). Figure 11C shows a comparison of three batches of fusion protein FP090 (Fc-EGF-C1-C2; SEQ ID NO: 24) containing an N-terminally placed Fc portion at three different concentrations (0.72, 7.2 and 72 nM) to the wild-type MFG-E8 control. Figure 11D shows the promotion of efferocytosis by the fusion protein construct FP050 (EDIL3-based EGF-HSA-C1-C2; SEQ ID NO: 12) containing HSA inserted between the EGF-like and C1-C2 domains of EDIL3. Figure 11E shows further examples of fusion proteins of the disclosure, such as chimeric variants (FP145; sequence number 80, FP1145; sequence number 103, FP146; sequence number 82, FP1146) and combinations of the integrin binding domain of MFGE8 or EDIL3 with a PS binding domain such as the IgSFV domain of TIM4 or the GLA domain of the bridging protein GAS6 (FP1147 and FP1148).Figure 11F shows the efferocytosis-promoting function of recombinant fusion proteins constructed as chimeric proteins fusing domains from EDIL3 and MFG-E8 to HSA inserts. The data show that FP145 (SEQ ID NO: 80) and FP146 (SEQ ID NO: 82) induced efferocytosis of killed neutrophils by human macrophages in a concentration-dependent manner. Figure 11G shows the efferocytosis-promoting function of recombinant fusion proteins constructed as chimeric proteins fusing domains from EDIL3 and MFG-E8 to HSA inserts. The data show that FP145 (SEQ ID NO: 80) and FP146 (SEQ ID NO: 82) induced efferocytosis of killed Jurkat cells by human endothelial cells (HUVEC) in a concentration-dependent manner. [Figure 11-2] (As stated above.) [Figure 11-3] (As stated above.) [Figure 11-4] (As stated above.) [Figure 11-5] (As stated above.) [Figure 12] 1 shows the promotion of efferocytosis by HUVEC cells of the therapeutic fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) tested at three different concentrations up to 30 nM. The promotion of efferocytosis was concentration-dependent, with increased efferocytosis as the concentration of the fusion protein FP278 increased. [Figure 13-1] Figure 13: Therapeutic fusion proteins FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42; Figure 13A), FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44; Figure 13B) and FP776 (EGF-HSA-C1-C2; SEQ ID NO: 48; Figure 13C) are able to rescue endotoxin (lipopolysaccharide)-induced efferocytosis of killed neutrophils by human macrophages. Fusion protein concentrations are shown on the x-axis and efferocytosis [%] on the y-axis. [Figure 13-2] (As stated above.) [Figure 14-1]Figure 14: Effect of fusion proteins FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42; Figure 14A), FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44; Figure 14B) and FP776 (EGF-HSA-C1-C2; SEQ ID NO: 48; Figure 14C) on promoting efferocytosis of killed Jurkat cells by human endothelial cells (HUVEC). The concentration of fusion proteins is shown on the x-axis and efferocytosis [%] is shown on the y-axis. [Figure 14-2] (As stated above.) [Figure 15-1] Figure 15: Single administration of therapeutic fusion proteins FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44), FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42) or FP776 (EGF-HSA-C1-C2; SEQ ID NO: 48) protects renal function in a model of ischemia-reperfusion injury-induced acute kidney injury (AKI). Figure 15A shows that intraperitoneal (ip) administration of 0.16 mg / kg or 0.5 mg / kg FP278 (SEQ ID NO: 44) (x-axis) reduces the rise in serum creatinine (sCr) (mg / dL; y-axis). As shown in Figure 15B, intravenous (iv) administration of 0.5 mg / kg or 1.5 mg / kg of the fusion protein FP330 (SEQ ID NO: 42) significantly reduced serum creatinine levels. FIG. 15C shows that intravenous administration of the fusion protein FP776 (SEQ ID NO: 48) reduced serum creatinine in a dose-dependent manner. [Figure 15-2] (As stated above.) [Figure 16] FIG. 1 shows that a single dose of either 0.16 mg / kg or 0.5 mg / kg of the therapeutic fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) reduced blood urea nitrogen (BUN) levels in a mouse model of acute kidney injury. [Figure 17]Figure 17 shows that a single administration of therapeutic fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) protects remote organs from acute phase responses induced by ischemia-reperfusion-induced AKI based on gene expression of injury markers. Figure 17A illustrates such an AKI-induced response (SAA) of serum amyloid protein in mouse heart, and Figure 17B illustrates such an AKI-induced response (SAA) in murine lung, both of which were potently blocked after a single ip injection of MFGE8-derived fusion protein FP278 (SEQ ID NO: 44) at 0.16 mg / kg or 0.5 mg / kg / ip. [Figure 18] Figure 1 shows the uptake of superparamagnetic iron oxide (SPIO) contrast agent (Endorem®) by the liver over time. Endorem® was injected intravenously as a 1.2 second bolus in animals with AKI (24 hours after disease induction) or after sham surgery (animals 24 hours after nephrectomy). Animals with AKI showed significantly reduced uptake of contrast agent by the liver (target = Kupffer cells) compared to sham-operated animals. Treatment with the fusion protein FP776 (EGF-HSA-C1-C2; SEQ ID NO: 48), administered prophylactically approximately 30 minutes before AKI induction or therapeutically 5 hours after induction of ischemia-reperfusion injury, protected against loss of contrast agent accumulation in the liver of AKI mice. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] Therapeutic fusion proteins comprising an integrin binding domain, a PS binding domain and a solubility domain are disclosed herein. Treatment methods using the fusion proteins of the present disclosure, as well as assays such as efferocytosis assays useful for characterizing the fusion proteins are also disclosed herein.

[0027] definition In order that this disclosure may be more readily understood, certain terms are specifically defined throughout the detailed description. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0028] In all cases where the terms "comprise", "comprises", "comprising", etc. are used with respect to a sequence (e.g., an amino acid sequence), it is understood that the sequence may also be limited by terms such as "consist", "consists", "consisting of", etc. As used herein, the phrase "consisting essentially of" refers to the genus or species of active pharmaceutical agent included in a method or composition, as well as any excipients that are inert for the intended purpose of the method or composition. In some embodiments, the phrase "consisting essentially of" expressly excludes the inclusion of one or more additional active agents other than the multispecific binding molecules of the present disclosure. In some embodiments, the phrase "consisting essentially of" expressly excludes the inclusion of one or more additional active agents other than the multispecific binding molecules of the present disclosure and the second co-administered agent.

[0029] As used herein, the term "efferocytosis" refers to a process in cell biology in which dead or dying cells, such as apoptotic or necrotic or senescent cells or highly activated cells or extracellular vesicles (microparticles) or cell debris (collectively referred to as "prey"), are removed by phagocytosis, i.e., engulfed and digested by phagocytes. During efferocytosis, phagocytes actively tether and engulf the prey, generating large intracellular fluid-filled vesicles containing the prey called efferosomes, giving rise to lysosomal compartments where degradation of the prey begins. During apoptosis, efferocytosis ensures that dead cells are removed before the integrity of their membranes is compromised and their contents leak into the surrounding tissue, preventing exposure of the surrounding tissue to DAMPs such as toxic enzymes, oxidants, and other intracellular components, such as DNA, histones, and proteases. Professional phagocytes include bone marrow-derived cells such as macrophages and dendritic cells, but other cells, such as stromal cells, can also perform efferocytosis, including epithelial and endothelial cells, and fibroblasts. Impaired efferocytosis has been linked to autoimmune diseases and tissue damage, and has been demonstrated in diseases such as cystic fibrosis, bronchiectasis, COPD, asthma, idiopathic pulmonary fibrosis, rheumatoid arthritis, systemic lupus erythematosus, glomerulonephritis, and atherosclerosis (Vandivier RW et al (2006) Chest, 129 (6): 1673-82). At present, no therapeutic approaches that specifically promote efferocytosis have entered clinical trials.

[0030] The term "efferocytosis assay" as used herein and described in the Examples refers to assay systems developed for profiling of fusion proteins that utilize human macrophages or human endothelial cells (HUVEC) as phagocytes. Exemplary herein are macrophage-neutrophil efferocytosis assay, endothelial cell-di-Jurkat cell efferocytosis assay, or endothelial cell microparticle efferocytosis assay. These assays can be used to demonstrate that biotherapeutics derived from MFG-E8, such as the fusion proteins of the present disclosure, effectively promote efferocytosis of dead cells and microparticles by macrophages or endothelial cells, as described in more detail in the Examples. Furthermore, the described macrophage-neutrophil assay is suitable to demonstrate that such compounds of the present invention can rescue even LPS- or S. aureus-impaired efferocytosis of dead cells.

[0031] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The phrase also applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of a corresponding naturally occurring amino acid, as well as to naturally occurring and non-naturally occurring amino acid polymers. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.

[0032] The term "stickiness" as used herein with respect to proteins of the present disclosure refers to the result of protein misfolding that promotes protein clumping or aggregation. These undesirable non-functional effects are the result of surface hydrophobic interactions.

[0033] As used herein, "C-terminus" refers to the carboxyl-terminal amino acid of a polypeptide chain having a free carboxyl group (-COOH). As used herein, "N-terminus" refers to the amino-terminal amino acid of a polypeptide chain having a free amine group (-NH2).

[0034] As used herein, the term "fusion protein" refers to a protein that includes several domains, which may not necessarily constitute the entire native or wild-type protein, but may be limited to the active domains of the entire protein that are involved in binding to the corresponding receptor on the cell surface. Fusion proteins can be generated using recombinant protein design, and the term "recombinant protein" refers to a protein that is prepared, expressed, produced, or isolated by recombinant DNA techniques. For example, tandem fusion refers to a technique in which proteins or protein domains of interest are simply connected at both ends via N- or C-terminal fusion between proteins. This provides a flexible bridge structure, allowing sufficient space between the fusion partners to ensure proper folding. However, the N- or C-terminus of a peptide is often a critical component in obtaining the desired folding pattern of a recombinant protein, and simple joining of both ends of a domain may be ineffective. Alternatively, the process of domain insertion involves the fusion of consecutive protein domains by encoding the desired structure into a single polypeptide chain, and sometimes the insertion of a domain within another domain. In both of these aforementioned processes, the domains are "directly linked" or "linked directly". Domain insertions are often more difficult to perform than tandem fusions because of the difficulty in finding suitable nucleic acid ligation sites in the gene of interest.

[0035] In addition to the above-mentioned direct linkage fusion techniques, external linkers may be used to maintain the function of the protein domains of the fusion protein. Such linkers refer to a series of amino acids that connect a protein domain to another protein domain and are referred to herein as "indirect linkers". Thus, the domains are "indirectly linked" or "linked indirectly". For example, those skilled in the art will appreciate that polypeptides whose structure includes two or more functional or organizational domains often include a series of amino acids between such domains that connect them to each other. Linkers can allow domain interaction, enhance stability, and reduce steric hindrance, which often makes them preferable for use in engineered protein design, even when N- and C-termini can be fused. In some embodiments, the linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility to the polypeptide. Various types of naturally occurring linkers have been used to engineer proteins, such as many recombinant therapeutic proteins, especially the immunoglobulin hinge region, which functions as a linker in engineered antibody constructs (Pack P et al., (1995) J. Mol. Biol., 246: 28-34). In addition to natural linkers, numerous artificial linkers have been devised, which can be subdivided into three categories: flexible, rigid, and in vivo cleavable linkers. (Yu K et al., (2015) Biotech. Advances, 33(1): 155-64; Chen X et al., (2013) Ad. Drug Delivery Reviews, 65(10): 1357-69). The most widely used flexible linker sequences are (Gly)n (Sabourin et al., (2007) Yeast, 24:39-45) and (Gly4Ser)n (SEQ ID NO:64) (Huston et al., 1988, 85:5879-83), where the linker length is adjustable with the copy number "n".In some embodiments, a polypeptide comprising a linker element has an overall structure of the general form D1-linker-D2, where D1 and D2 may be the same or different and represent two domains associated with each other by the linker. In some embodiments, the polypeptide linker is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more amino acids in length.

[0036] As used herein, a "modification" or "mutation" of an amino acid residue / position refers to a change in the primary amino acid sequence compared to the starting amino acid sequence, where the change results from a sequence change involving said amino acid residue / position. For example, typical modifications include the substitution of a residue (or said position) with another amino acid (e.g., conservative or non-conservative substitution), the insertion of one or more amino acids adjacent to said residue / position, and the deletion of said residue / position. An amino acid "substitution" or modification thereof refers to the replacement of an existing amino acid residue in a given (starting) amino acid sequence with a different amino acid residue. Generally and preferably, the modification results in a change in at least one physico-biochemical activity of the mutant polypeptide compared to the polypeptide comprising the starting (or "wild-type") amino acid sequence.

[0037] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, conservatively modified variants refer to those nucleic acids that code for the same or essentially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations," a species of conservatively modified variation. Every nucleic acid sequence herein that codes for a polypeptide also describes every possible silent variation of the nucleic acid. One of skill in the art will recognize that each codon of a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Thus, every silent variation of a nucleic acid that codes for a polypeptide is implicit in each sequence described.

[0038] For polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions to a polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles. The following eight groups contain amino acids that are conservatively substituted for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M) (see, e.g., Creighton, Proteins (1984)). In some embodiments, the phrase "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or alter the binding properties of the binding domain of the engineered proteins of the disclosure.

[0039] As referred to herein, a "protein variant" or "variant of a protein" refers to a protein that includes a variation in which one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acids are modified. As referred to herein, a "functional variant" of a protein refers to a variant of a protein that includes a modification that results in a change in the amino acid sequence, but does not change the overall properties of the protein or its function. As referred to herein, a "truncated variant" of a protein refers to a shortened version of a protein, which retains the function of the parent protein. To determine whether a functional variant or a truncated variant has a change in the overall properties or function, these variant proteins can be tested against the full-length or unmodified parent protein for their effect in several assays as described in this disclosure. For example, promoting endothelial cell efferocytosis in a human endothelial cell-Jurkat cell efferocytosis assay, restoring impaired macrophage efferocytosis in a human macrophage-neutrophil efferocytosis assay, reducing the number of plasma microparticles by clearance in a human endothelial-microparticle efferocytosis assay, and / or providing protection against multi-organ injury in an acute renal ischemia model.

[0040] The term "percentage identity" or "percentage sequence identity," in the context of two or more nucleic acid or polypeptide sequences, refers to two or more sequences or subsequences that are the same. When compared and aligned for maximum correspondence over a comparison window or designated region, for example, as measured using one of the sequence comparison algorithms below or by manual alignment and visual inspection, two sequences are "substantially identical" and exhibit "sequence identity" if they have a specified percentage of the same amino acid residues or nucleotides (i.e., at least 60% identity, optionally at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity, over a specified region, or, if not specified, over the entire sequence). Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or over a region that is 100-500, or 1000, or 2000, or 3000 or more nucleotides in length, or over a region that is 30-200, or 300, or 500, or 700, or 800, or 900, or 1000 or more amino acids in length.

[0041] For sequence comparison, typically, one sequence serves as a reference sequence, and test sequence is compared with it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, subsequence coordinates are designated, and sequence algorithm program parameters are designated as necessary.Default program parameters can be used, or alternative parameters can be designated.The sequence comparison algorithm then calculates the percent sequence identity of test sequence compared to reference sequence based on program parameters.

[0042] The term "comparison window" as used herein includes reference to any one segment of several contiguous nucleic acid or amino acid positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more usually about 100 to about 150, where the two sequences are optimally aligned and then the sequence can be compared to a reference sequence of the same number of contiguous positions. Methods for alignment of sequences for comparison are known in the art. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman & Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson & Lipman (1988) PNAS USA, 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package (Genetics Computer Group, 575 Science Dr., Madison, WI)), or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

[0043] Two examples of suitable algorithms for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25:3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.

[0044] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul (1993) PNAS. USA, 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered to be similar to a reference sequence if the minimum sum probability in the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.

[0045] The percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci. 4:11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 residue weight table, a gap length penalty of 12, and a gap penalty of 4. Additionally, the percent identity between two amino acid sequences can be determined using the algorithm of Needleman & Wunsch (supra), which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0046] A polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions.

[0047] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single-stranded or double-stranded form. The term encompasses nucleic acids that are synthetic, natural and unnatural, and contain known nucleotide analogs or modified backbone residues or linkages that have similar binding properties as the reference nucleic acid and are metabolized in a similar manner to the reference nucleotide. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs).

[0048] Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the sequence explicitly indicated, but also conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences of that nucleic acid sequence. Specifically, degenerate codon substitutions can be achieved by generating sequences in which the third position of one or more selected (or all) codons is replaced with mixed base and / or deoxyinosine residues (Batzer et al., (1991) Nucleic Acid Res., 19:5081; Ohtsuka et al., (1985) J Biol Chem., 260:2605-2608; and Rossolini et al., (1994) Mol Cell Probes, 8:91-98). As used herein, the term "optimized nucleotide sequence" means that the nucleotide sequence has been modified to code for an amino acid sequence using codons that are preferred in a production cell, such as Chinese hamster ovary cells (CHO). The optimized nucleotide sequence is modified to completely retain the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence. In certain embodiments, the optimized sequences herein are modified to have codons that are preferred in CHO mammalian cells.

[0049] Therapeutic Fusion Proteins Integrin Binding Domain Integrins are transmembrane receptors that promote cell-extracellular matrix (ECM) adhesion. Upon ligand binding, integrins activate signal transduction pathways that mediate cell signals, such as regulating the cell cycle, organizing the intracellular cytoskeleton, and mobilizing new receptors to the cell membrane (Giancotti & Ruoslahti (1999) Science, 285 (5430): 1028-32). The presence of integrins allows for rapid and flexible responses to events at the cell surface. There are several types of integrins, and one cell may have several different types on its surface. Integrins have two subunits: α (alpha) and β (beta), each of which penetrates the plasma membrane and has several cytoplasmic domains (Nermut MV et al (1988). EMBO J., 7 (13): 4093-9). Acidic amino acids characterize the integrin interaction sites of many ECM proteins (e.g., as part of the amino acid sequence arginine-glycine-aspartic acid ("RGD" in the one-letter amino acid code). The RGD motif is found in many matrix proteins, such as fibronectin, fibrinogen, vitronectin, and osteopontin, and aids in cell adhesion. The RGD motif is found in many proteins in a conserved protein domain known as the EGF-like domain, a name derived from the epidermal growth factor, which was first described. The EGF-like domain is one of the most common domains found in extracellular proteins (Hidai C (2018) Open Access J Trans Med Res., 2(2):67-71), and some examples of EGF-like domains containing the RGD motif are listed in Table 1 below.

[0050] [Table 1]

[0051] As used herein, the term "integrin binding domain" refers to a series of amino acids or a protein domain having the function of binding to an integrin. In one embodiment of the present disclosure, as used herein, "integrin binding domain" refers to a series of amino acids or a protein domain having the function of binding to an integrin and comprising an RGD motif. In one embodiment of the present disclosure, the integrin binding domain is an EGF-like domain from human MFG-E8 having an amino acid sequence as set forth in SEQ ID NO:2. In an alternative embodiment of the present disclosure, the integrin binding domain is an EGF-like domain from human EDIL3 (any of the following sequences: SEQ ID NO:11, SEQ ID NO:77, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, or SEQ ID NO:101); for example, the EGF-like domain can be found within the series of amino acids 1-132 of SEQ ID NO:11.

[0052] The term "binds to integrin" as used herein refers to integrin binding activity. Integrin binding activity can be determined by methods well known in the art. For example, an integrin adhesion assay is described in the Examples, Section 3.2, where the adhesion of fluorescently labeled αvβ3 integrin-expressing lymphoma cells to therapeutic fusion proteins of the present disclosure is determined. An integrin binding domain is considered to have integrin binding activity if it has at least 10%, e.g., at least 25%, at least 50%, at least 75%, more preferably at least 80%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of the integrin binding activity observed for human MFG-E8 protein (SEQ ID NO:1) when tested in the same manner as determining the respective activity, preferably using the assay described in the Examples, Section 3.2.

[0053] Phosphatidylserine-binding domain "Phosphatidylserine" (PS) as used herein refers to phospholipids that are components of cell membranes. PS is mainly restricted to the inner leaflet of the cell membrane, whereas phosphatidylcholine and sphingomyelin are mainly localized in the outer leaflet. The asymmetric distribution of phospholipids is maintained by the action of flippases (P4-ATPases such as ATP11A and 11C) in the plasma membrane, which actively move PS from the outer leaflet to the inner leaflet. Cell surface exposure of PS is observed not only in apoptotic cells, but also in activated lymphocytes, activated platelets, senescent erythrocytes, and some cancer cells and their respective microparticles (Sakuragi et al., (2019) PNAS USA, 116(8):2907-12). PS exposure can be a biomarker for prothrombotic, inflammatory, or ischemic pathologies (Pasalic et al., (2018) J Thromb Haemost., 16(6):1198-2010; Ma et al., (2017) supra; Zhao et al., (2016) supra). PS functions in numerous cell signaling pathways, as an essential phospholipid in coagulation, and as an enhancer of tenase (factors IXa, VIIIa, and X) and prothrombinase (factors Xa, Va, and prothrombin) complex formation (Spronk et al., (2014) Thromb Res. 133(Suppl 1):S54-6). The best understood function of externalized PS may still be as an "eat-me" marker for phagocytes such as macrophages that phagocytose apoptotic cells, cellular debris, or PS-exposed activated cells. As used herein, the term "phosphatidylserine-binding domain" or "PS-binding domain" refers to a set of amino acids or a protein domain that functions to bind to PS. Examples of endogenous proteins with PS-binding domains can be found in Table 2 below.

[0054] [Table 2]

[0055] In one embodiment of the present disclosure, the PS domain is derived from human MFG-E8 having an amino acid sequence as set forth in SEQ ID NO: 3 or SEQ ID NO: 76. In an alternative embodiment of the present disclosure, the integrin binding domain is a PS binding domain from human EDIL3 (SEQ ID NO: 11), wherein the PS binding domain comprises amino acids 135-453 of SEQ ID NO: 11.

[0056] PS binding activity can be determined by methods well known in the art. For example, a PS binding assay is described in the Examples, Section 3.1, in which the binding of the fusion proteins of the present disclosure to PS coated on a microtiter plate was evaluated by competing with the binding of biotinylated mouse MFG-E8. According to the present disclosure, a PS binding domain is considered to have PS binding activity if it has at least 10%, e.g., at least 25%, at least 50%, at least 75%, at least 80%, preferably at least 90%, at least 95%, at least 96%, at least 97%, at least 98% of the PS binding activity observed for the human MFG-E8 protein shown in SEQ ID NO:1, when tested in the same manner as determining the respective activity, preferably using the assay described in the Examples, Section 3.1.

[0057] Cross-linking proteins There are several endogenous proteins that contain both integrin-binding and PS-binding domains. Examples of such "cross-linking proteins" are shown in Table 3 below.

[0058] [Table 3]

[0059] To be of therapeutic value, it is useful if the cross-linked protein contains an integrin-binding domain that recognizes an integrin on phagocytes that is typically not susceptible to proteolytic cleavage or shedding, as observed with TAM family members or other PS-binding receptors. Proteins with PS-binding and integrin-binding domains, such as MFG-E8 or its paralog EDIL3 / DEL1, have been shown to induce efferocytosis in vitro and may therefore be of therapeutic value as efferocytosis inducers of AOI. In contrast, for example, GAS6 protein may not be particularly effective at promoting efferocytosis of AOI because its receptor on phagocytes (MerTK) is proteolytically cleaved during inflammation and infection as described above.

[0060] An example of a cross-linked protein listed in Table 3 above is MFG-E8, which is one of the major proteins found in milk fat globule membranes (MFGMs). MFG-E8 is expressed and secreted by several different types of cells (e.g., mammary epithelial cells, vascular cells, epididymal epithelial cells, aortic smooth muscle cells, activated macrophages, stimulated endometrium, and immature dendritic cells) and tissues (e.g., heart, lung, mammary gland, spleen, intestine, liver, kidney, brain, blood, and endothelium). The MFG-E8 protein is also known by several different names, including lactadherin, BP47, component 15 / 16, MFGM, MGP57 / 53, PAS-6 / PAS-7 glycoprotein, cell wall protein SED1, sperm surface protein SP47, mammary epithelial antigen BA46, and O-acetyl GD3 ganglioside synthase (AGS). The MFG-E8 gene is located on chromosome 1 in rats, chromosome 7 in mice, and chromosome 15 in humans. Alternative splicing of the MFG-E8 pre-mRNA results in three isoforms of the human protein and two forms of the mRNA, with the long and short variants expressed in the mouse mammary gland. The human MFG-E8 gene (UniProtKB-Q08431) encodes a 387-residue long protein that is processed to form multiple protein products. The amino acid sequence of human MFG-E8 is shown below, including the signal peptide (residues 1-23; underlined), EGF-like domain (residues 24-67; italics), C1 domain (residues 70-225; bold), and C2 domain (residues 230-387; bold underlined). [ka]

[0061] MFG-E8 lacks the transmembrane function possessed by MFGM and therefore functions as a peripheral membrane protein. Human MFG-E8 consists of one N-terminal EGF-like domain (SEQ ID NO:2) that binds to αvβ3 and αvβ5 integrins expressed on phagocytes, and a PS-binding domain (SEQ ID NO:3) that contains two F5 / 8-discoidin subdomains (C1 and C2) that bind with high affinity to anionic phospholipids. Integrin binding is the result of an RGD motif located at residues 46-48 of human MFG-E8 (SEQ ID NO:1). Apoptotic cells, cell debris, overactivated cells, and most microparticles (MPs) expose PS and are targets for MFG-E8, which acts as a bridging molecule, opsonizing and linking these cells and microparticles to αvβ3 and αvβ5 integrins on phagocytes. This bridging action triggers an efficient phagocytic program that leads to the internalization of cells, debris, and microparticles. The proteins found in MFGM are highly conserved across species. The structure of the MFG-E8 protein varies from species to species, with all currently known species containing two C domains, but differing in the number of EGF-like domains. For example, human MFG-E8 protein contains one EGF-like domain, while bovine MFG-E8 and mouse MFG-E8 (SEQ ID NO: 68) have two EGF-like domains, and chicken, frog, and zebrafish have three EGF-like domains. Domains of MFG-E8 have previously been proposed as components of therapeutic drugs, and in particular the PS-binding domain (Kooijmans et al., (2018) Nanoscale, 10(5): 2413-2426) and fragments of MFG-E8 have been described to act in models of fibrosis (US Patent Application Publication No. 2018 / 0334486).

[0062] Non-inflammatory uptake of dead cells, debris, and particulates by professional and non-professional phagocytes plays a key role in homeostasis after tissue injury (Greenlee-Wacker (2016) supra). The importance of proper clearance was further revealed in genetic models in which MFG-E8 knockout mice showed, for example, increased numbers of (uncleared) dead cells in tissues and exacerbated inflammatory responses in disease models such as neonatal sepsis, autoimmunity, poor angiogenesis, and impaired wound healing (Hanayama et al., (2004) Science, 204(5474):1147-50; Das et al., (2016) J Immunol., 196(12):5089-5100; Hansen et al., (2017) J Pediatr Surg., 52(9):1520-7).

[0063] Furthermore, MFG-E8 has been shown to generate a tolerogenic environment by suppressing T cell activation and proliferation, inhibiting Th1, Th2, and Th17 subpopulations, while increasing regulatory T cell subsets (Tregs). Interestingly, Tregs contribute to the resolution of inflammation by inducing efferocytosis by macrophages (Proto et al., (2018) Immunity, 49(4): 666-77). MFG-E8 has been reported to promote allogeneic engraftment of embryonic stem cell-derived tissues across MHC barriers (Tan et al., (2015) Stem Cell Reports, 5(5): 741-752). MFG-E8 also has multiple nutritional uses, which can help promote tissue development and protect against infectious pathogens. Glycoproteins such as MFG-E8 are potential health-promoting nutraceuticals for food and pharmaceutical applications. MFG-E8 can also be combined with other nutrients (e.g., probiotics, whey protein micelles, α-hydroxyisocaproic acid, citrulline, and branched chain fatty acids).

[0064] Solubilization Domain As described herein, the therapeutic fusion protein of the present disclosure comprises an integrin binding domain and a PS binding domain. In addition, the fusion protein also comprises an additional domain that confers several desirable properties to the fusion protein. For the purposes of this application, this additional domain, referred to as a "solubilization domain," results in improved biological properties, such as increased solubility, reduced aggregation, and increased biological activity. As a result, the fusion protein exhibits a desirable pharmacokinetic profile. Furthermore, the presence of the solubility domain improves the stability of the therapeutic fusion protein and improves the expression of the fusion protein compared to the wild-type protein in a cellular expression system, as shown by increased yields after purification.

[0065] The presence of a solubilization domain may also confer an extended half-life to a therapeutic fusion protein. For example, many protein drugs have been linked to polyethylene glycol (PEG), reCODE PEG, antibody scaffolds, polysialic acid (PSA), hydroxyethyl starch (HES), and serum proteins such as albumin, IgG, and FcRn to extend plasma half-life and enhance therapeutic efficacy (Kim et al., (2010) J Pharmacol Exp Ther., 334:682-92; Weimer et al., (2008) Thromb Haemost. 99:659-67; Dumont et al., (2006) BioDrugs, 20:151-60; Schellenberger et al., (2009) Nat Biotechnol., 27:1186-90).

[0066] In some embodiments, the solubilization domain is an albumin protein, such as human serum albumin (HSA; SEQ ID NO: 4) or a variant thereof. For example, HSA (SEQ ID NO: 5) with amino acid substitution C34S to reduce aggregation tendency, or a domain of HSA, such as HSA D3 (SEQ ID NO: 6). HSA has a very long serum half-life and avoids intracellular degradation due to several factors, including its relatively large size, which reduces renal filtration, and neonatal Fc receptor (FcRn) binding properties. The use of N-terminal fragments of HSA for fusion to polypeptides has also been proposed (e.g., European Patent Application No. 399666). Thus, by genetically or chemically fusing or conjugating molecules to albumin, it is possible to stabilize or extend the shelf life and / or retain the activity of the molecules for a long period of time in solution, in vitro and / or in vivo. Further methods related to HSA fusion can be found, for example, in WO 2001 / 077137 and WO 2003 / 060071.

[0067] In some embodiments, the solubility domain comprises an antibody Fc domain, such as human Fc immunoglobulin G1 (Fc-IgG1; SEQ ID NO: 7). The Fc domain may also be modified to improve Fc heterodimerization by introducing complementary amino acid substitutions into the CH3 domain of the Fc, for example, using knob-into-hole (KiH) based modifications, such as substitution T366W to create a "knob" in one CH3 domain, and substitutions T366S, L368A, and Y407V to create a "hole" in the other CH3 domain (Merchant et al (1998) Nat. Biotechnol., 16(7):677-81; EU numbering IgG1). Additional modifications that can be included in the Fc domain, alone or in combination with modifications to improve heterodimerization, can include, for example, amino acid substitutions to cysteine ​​to create additional cysteine ​​bonds, e.g., S354C and / or Y349C, and amino acid substitutions to reduce or eliminate binding to Fcγ receptors and complement protein C1q to "silence" immune effector function. The so-called "LALA" double mutation (L234A and L235A, EU numbering) reduces effector function (Lund et al., (1992) Mol Immunol., 29:53-9). Alternatively, the "DAPA" double mutation (D265A and P329A, EU numbering) reduces effector function. In one embodiment of the present disclosure, the Fc domain may contain amino acid substitutions D265A, P329A, and / or KiH amino acid substitutions T366W (knob) or T366S, L368A, and Y407V (hole) for Fc silencing. In one embodiment, the Fc domain is derived from human IgG1 and contains amino acid substitutions D265A, P329A (SEQ ID NO: 8). In another embodiment, the Fc domain is derived from human IgG1 and contains amino acid substitutions D265A, P329A, S354C, and amino acid substitution T366W (Fc-IgG1-knob; SEQ ID NO: 9). In another embodiment, the Fc domain is derived from human IgG1 and contains amino acid substitutions D265A, P329A, Y349C, and amino acid substitutions T366S, L368A, and Y407V (Fc-IgG1-hole; SEQ ID NO: 10).

[0068] In some embodiments, the solubility domain comprises an antibody Fc domain derived from human IgA, IgD, IgE, or IgM.

[0069] In some embodiments, the solubilization domain comprises SUMO (small ubiquitin-like modifier), ubiquitin, GST (glutathione S-transferase), or a variant thereof.

[0070] Linking and Orienting Domains of Therapeutic Fusion Proteins The integrin-binding domain, PS-binding domain, and solubility domain of the fusion protein of the present disclosure are linked. As used herein, the term "linked" or "linked" refers to one domain of the fusion protein that is directly or indirectly linked to another domain of the fusion protein. Direct linkage is a form of linkage and is referred to herein as "fused" or "fusion". As an example, use a molecule with the format ABC: domain A is directly linked to domain B, which is directly linked to domain C. In that case, domain A can also be described as fused to domain B, which is fused to domain C. As another example, domain A is directly linked to domain B, which is indirectly linked to domain C. In that case, domain A can also be described as fused to domain B, which is indirectly linked to domain C by an internal linker.

[0071] In some embodiments, the linkage is a direct linkage, and thus the domains are fused to each other. In some embodiments, the integrin binding domain is fused to a PS binding domain that is fused to a solubility domain. Specifically, the PS binding domain (e.g., the C1-C2 discoidin subdomain) is fused to the C-terminus of the integrin binding domain (e.g., the EGF-like domain) and is fused to the N-terminus of the solubility domain (e.g., the HSA). In some embodiments, the solubility domain is fused to an integrin binding domain that is fused to a PS binding domain. Specifically, the integrin binding domain (e.g., the EGF-like domain) is fused to the C-terminus of the solubility domain (e.g., the HSA) and is fused to the N-terminus of the PS binding domain (e.g., the C1-C2 discoidin subdomain). In some embodiments, the integrin binding domain is fused to a PS binding domain that includes the C1-C2 discoidin subdomain, and the solubility domain is inserted between the C1-C2 discoidin subdomains. Specifically, the C-terminus of the integrin binding domain (e.g., EGF-like domain) is fused to the N-terminus of the C1 discoidin subdomain, the C-terminus of the C1 discoidin subdomain is fused to the N-terminus of the soluble domain (e.g., HSA), and the C-terminus of the soluble domain is fused to the N-terminus of the C2 discoidin subdomain. In another embodiment, the integrin binding domain is fused to a soluble domain that is fused to a PS binding domain. Specifically, the soluble domain (e.g., HSA) is fused to the C-terminus of the integrin binding domain (e.g., EGF-like domain) and the N-terminus of the PS binding domain (e.g., C1-C2 discoidin subdomain). In one embodiment, HSA is fused to the C-terminus of the EGF-like domain and fused to the N-terminus of the C1 discoidin domain.

[0072] In some embodiments, a solubility domain (e.g., HSA) is fused between the integrin binding domain and the PS binding domain, hi some embodiments, the integrin binding domain is located at the N-terminus of the fusion protein and the PS binding domain is located at the C-terminus of the fusion protein.

[0073] In some embodiments, the fusion protein comprises a first region containing an integrin binding domain, e.g., an EGF-like domain, a second region containing a solubility domain (e.g., HSA), and a third region containing a PS binding domain, e.g., the C1 and / or C2 discoidin domains. In some embodiments, the integrin binding domain is located at the N-terminus of the fusion protein and the PS binding domain is located at the C-terminus of the fusion protein.

[0074] In some embodiments, a solubility domain (e.g., HSA) is fused between the integrin binding domain and the PS binding domain, hi some embodiments, the integrin binding domain is located at the N-terminus of the fusion protein and the PS binding domain is located at the C-terminus of the fusion protein.

[0075] In some embodiments, the fusion protein comprises a first region containing an integrin binding domain, e.g., an EGF-like domain, a second region containing a solubilization domain (e.g., HSA or Fc), and a third region containing a PS binding domain, e.g., the C1 and / or C2 discoidin domains. In some embodiments, the integrin binding domain is located at the N-terminus of the fusion protein and the PS binding domain is located at the C-terminus of the fusion protein.

[0076] In some embodiments, the solubilization domain is HSA.

[0077] In some embodiments, the solubilization domain is the antibody Fc-immunoglobulin G1 (Fc-IgG1; SEQ ID NO:7).

[0078] In some embodiments, the solubility domain (eg, HSA) is HSA comprising the amino acid sequence set forth in SEQ ID NO:5, or a functional variant thereof.

[0079] In a preferred embodiment, HSA comprising the amino acid sequence as set forth in SEQ ID NO:5 is fused to the C-terminus of the EGF-like domain of MFG-E8 and to the N-terminus of the PS-binding domain of MFG-E8. In one embodiment, the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO:46 (FP068). In one embodiment, the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO:48 (FP776).

[0080] In an alternative embodiment, HSA comprising the amino acid sequence as set forth in SEQ ID NO:5 is fused to the C-terminus of the EGF-like domain of EDIL3 and to the N-terminus of the PS-binding domain of EDIL3. In one embodiment, the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO:70 (FP1068). In one embodiment, the fusion protein comprises the amino acid sequence as set forth in SEQ ID NO:69 (FP1776).

[0081] In some embodiments, the linkage is via a polypeptide linker, e.g., the polypeptide linker that connects the solubility domain to the PS binding domain in the fusion proteins of the disclosure is referred to as an "external linker." These external linkers typically include glycine (G) and / or serine (S), and may include glycine and leucine (GL) or glycine and valine (GL). In some embodiments, the linker includes multiple G and S residues, e.g., G2S and multiples thereof, such as (G2S)4 as set forth in SEQ ID NO:62, (GS)4 as set forth in SEQ ID NO:63, G4S as set forth in SEQ ID NO:64, or (G4S)2 as set forth in SEQ ID NO:65.

[0082] In some embodiments, the external linker is fused between the C-terminus of the integrin binding domain and the N-terminus of the soluble domain. Specifically, the external linker is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA. In some embodiments, the external linker is fused between the C-terminus of the soluble domain and the N-terminus of the PS binding domain. Specifically, the external linker is fused to the C-terminus of HSA and the N-terminus of the PS binding domain. In some embodiments, the external linker is fused between the C-terminus of the integrin binding domain and the N-terminus of the soluble domain, and a further external linker is fused between the C-terminus of the soluble domain and the N-terminus of the PS binding domain. Specifically, the external linker is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA, and a further external linker is fused to the C-terminus of HSA and the N-terminus of the PS binding domain.

[0083] In some embodiments, an external linker comprising GS is fused to the C-terminus of the integrin binding domain and the N-terminus of the soluble domain. In some embodiments, an external linker comprising GL is fused to the C-terminus of the soluble domain and the N-terminus of the PS binding domain. In some embodiments, an external linker comprising (G2S)4 (SEQ ID NO: 62) is fused to the C-terminus of the soluble domain and the N-terminus of the PS binding domain. In some embodiments, an external linker comprising G4S (SEQ ID NO: 64) is fused to the C-terminus of the soluble domain and the N-terminus of the PS binding domain. In some embodiments, an external linker comprising (G4S)2 (SEQ ID NO: 65) is fused to the C-terminus of the soluble domain and the N-terminus of the PS binding domain.

[0084] In one embodiment, an external linker comprising GS is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA. A fusion protein of the present disclosure comprising this structure has the amino acid sequence set forth in SEQ ID NO: 42 (FP330).

[0085] In one embodiment, an external linker comprising GS is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA, and a further external linker comprising (GS)4 (SEQ ID NO: 63) is fused to the C-terminus of HSA and the N-terminus of the PS-binding domain.

[0086] In one embodiment, an external linker comprising GS is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA, and a further external linker comprising (G2S)4 (SEQ ID NO:62) is fused to the C-terminus of HSA and the N-terminus of the PS-binding domain. A fusion protein of the present disclosure comprising this structure has the amino acid sequence set forth in SEQ ID NO:42 (FP330).

[0087] In one embodiment, a GS-containing exolinker is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA. The C-terminus of HSA is fused directly to the N-terminus of the PS-binding domain.

[0088] In one embodiment, an external linker comprising GS is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA, and a further external linker comprising G4S (SEQ ID NO: 64) is fused to the C-terminus of HSA and the N-terminus of the PS-binding domain. A fusion protein of the present disclosure comprising this structure has the amino acid sequence set forth in SEQ ID NO: 54 (FP811).

[0089] In one embodiment, an external linker comprising GS is fused to the C-terminus of the EGF-like domain and to the N-terminus of HSA, and a further external linker comprising (G4S)2 (SEQ ID NO:65) is fused to the C-terminus of HSA and to the N-terminus of the PS-binding domain. A fusion protein of the present disclosure comprising this structure has the amino acid sequence set forth in SEQ ID NO:56 (FP010).

[0090] In some embodiments, the His tag is fused to an external linker comprising GS (GS-6xHis; SEQ ID NO: 66) fused to the C-terminus of the PS binding domain. In one embodiment, a fusion protein of the present disclosure comprising a His tag has the amino acid sequence set forth in SEQ ID NO: 44 (FP278) or SEQ ID NO: 60 (FP114 or FP260).

[0091] Functional characterization of therapeutic fusion proteins The present disclosure provides a fusion protein derived from human MFG-E8 that is effective in promoting efferocytosis and is therefore active in eliminating key drivers of systemic inflammation and microvascular pathology. As described in the Examples, a fusion protein with the general structure EGF-HSA-C1-C2 has been shown to be effective in several efferocytosis assays. For example, the fusion protein is effective in restoring lipopolysaccharide (LPS) or Staphylococcus aureus (S. aureus)-impaired efferocytosis in macrophages and promoting efferocytosis of microparticles and dead cells by endothelial cells. The fusion protein is also effective in protecting kidney function and protecting against weight loss in a mouse model of acute kidney injury.

[0092] Exemplary Protein Sequences The amino acid sequences in Table 4 include examples of therapeutic fusion proteins and portions thereof of the present disclosure.

[0093] Throughout the text of this application, in the event of any discrepancies between the text of the specification (eg, Table 4) and the Sequence Listing, the text of the specification shall control.

[0094] [Table 4]

[0095] [Table 5]

[0096] [Table 6]

[0097] [Table 7]

[0098]

Table 8

[0099]

Table 9

[0100]

Table 10

[0101]

Table 11

[0102]

Table 12

[0103]

Table 13

[0104]

Table 14

[0105]

Table 15

[0106]

Table 16

[0107]

Table 17

[0108]

Table 18

[0109]

Table 19

[0110]

Table 20

[0111]

Table 21

[0112]

Table 22

[0113]

Table 23

[0114]

Table 24

[0115]

Table 25

[0116]

Table 26

[0117]

Table 27

[0118]

Table 28

[0119]

Table 29

[0120]

Table 30

[0121]

Table 31

[0122]

Table 32

[0123]

Table 33

[0124]

Table 34

[0125]

Table 35

[0126]

Table 36

[0127]

Table 37

[0128]

Table 38

[0129]

Table 39

[0130]

Table 40

[0131]

Table 41

[0132]

Table 42

[0133]

Table 43

[0134]

Table 44

[0135]

Table 45

[0136]

Table 46

[0137]

Table 47

[0138]

Table 48

[0139]

Table 49

[0140] [Table 50]

[0141] [Table 51]

[0142] [Table 52]

[0143] [Table 53]

[0144] [Table 54]

[0145] [Table 55]

[0146] [Table 56]

[0147] The present application also includes variants of each of SEQ ID NOs: 69, 70 and 72, wherein the EGF-like domain of the EDIL3 sequence contained therein corresponds to any one of the following sequences: SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, or SEQ ID NO: 101.

[0148] The application also includes therapeutic fusion proteins comprising an integrin-binding domain of MFGE8 or EDIL3 and a PS-binding domain, such as the IgSF V domain of TIM4 or the GLA domain of the bridging protein GAS6 mutant.

[0149] Modifications of the Proteins of the Present Disclosure The present application includes variants of the proteins described herein and / or fragments thereof with various modifications to the domains, as well as fusions and conjugates of the disclosed molecules. For example, a domain of a therapeutic fusion protein may have a conservative modification of an amino acid residue, where the modified protein retains or has enhanced properties compared to a fusion protein comprising the parent domain. Alternatively, a domain of a therapeutic fusion protein may have a deletion of an amino acid residue, where the modified fusion protein retains or has enhanced properties compared to a protein comprising the parent domain. Alternatively, a therapeutic fusion protein may have an insertion of an amino acid residue, where the modified protein retains or has enhanced properties compared to the unmodified protein. In one embodiment, such amino acid insertions include glycine or serine residues in some combination to function as linkers between the domains of the parent protein.

[0150] Site-directed or PCR-mediated mutagenesis can be performed to introduce mutations, and the effect on integrin and / or PS binding, or other functional properties of interest, can be evaluated in in vitro or in vivo assays. Conservative modifications (as described above) can be introduced, and / or the mutations can be amino acid substitutions, additions, or deletions. Furthermore, typically no more than one, two, three, four, or five residues within the binding domain are altered.

[0151] Amino acid sequence variants of therapeutic fusion proteins with essentially similar properties to the unmodified variants can be prepared by introducing appropriate nucleotide changes into the coding DNA or by synthesis of the desired variant. Such variants include, for example, deletions from, or insertions or substitutions of, residues within the amino acid sequence of the current molecule. In some embodiments, variants may include additional linker sequences, reduced linker sequences or removal of linker sequences, and / or amino acid mutations or substitutions and deletions of one or more amino acids. Any combination of deletions, insertions and substitutions may be made to arrive at the final construct, provided that the final construct has the desired properties. Amino acid changes may also alter post-translational processes of the molecule, such as changing the number or location of possible glycosylation sites.

[0152] Methods for Producing Recombinant Molecules Nucleic Acids and Expression Systems In one embodiment, the application provides a method for recombinantly producing one or more polypeptide chains of a therapeutic fusion protein, comprising: 1) producing one or more DNA constructs comprising nucleic acid molecules encoding the polypeptide chains of a multispecific binding molecule; 2) introducing the DNA constructs into one or more expression vectors; 3) co-transfecting the expression vectors in one or more host cells; and 4) expressing and assembling the molecules in the host cells or in solution.

[0153] In this regard, the present disclosure provides isolated nucleic acids, e.g., one or more polynucleotides, encoding the therapeutic fusion proteins described herein. Nucleic acid molecules include DNA and RNA in both single-stranded and double-stranded forms, as well as the corresponding complementary sequences. The nucleic acid molecules of the present invention include full-length genes or cDNA molecules, as well as combinations of fragments thereof. Although the nucleic acids of the present invention are derived from human sources, the present invention includes those derived from non-human species.

[0154] An "isolated nucleic acid" is a nucleic acid that is separated from adjacent gene sequences present in the genome of the organism from which the nucleic acid is isolated, in the case of a nucleic acid isolated from a naturally occurring source. In the case of a nucleic acid that is enzymatically or chemically synthesized from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, it is understood that the nucleic acid resulting from such a process is an isolated nucleic acid. An isolated nucleic acid molecule refers to a nucleic acid molecule in the form of a separate fragment or as a component of a larger nucleic acid construct. In one preferred embodiment, the nucleic acid is substantially free of contaminating endogenous material. The nucleic acid molecule is derived from DNA or RNA that has been isolated at least once, preferably in substantially pure form and in an amount or concentration that allows the identification, manipulation, and recovery of its constituent nucleotide sequences by standard biochemical methods (such as those reviewed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)). Such sequences are preferably provided and / or constructed in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, that are typically present in eukaryotic genes. Sequences of non-translated DNA may be present 5' or 3' from the open reading frame and do not interfere with manipulation or expression of the coding regions.

[0155] The present invention also provides expression systems and constructs in the form of plasmids, expression vectors, transcription or expression cassettes, which comprise at least one polynucleotide as described above. Furthermore, the present invention provides host cells comprising such expression systems or constructs.

[0156] In one embodiment, the disclosure provides a method for preparing a therapeutic fusion protein comprising: (a) culturing a host cell comprising a nucleic acid encoding the fusion protein, wherein the cultured host cell expresses the fusion protein; and (b) recovering the fusion protein from the host cell culture.

[0157] Expression vectors and host cells for producing the therapeutic fusion proteins described above are also provided in the present disclosure. The term "vector" refers to any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage, or virus) that is suitable for transformation or transfection of a host cell and includes a nucleic acid sequence (in relation to a host cell) that directs and / or controls the expression of one or more heterologous coding regions operably linked thereto. A variety of expression vectors can be used to express the polynucleotides that code for the chains or binding domains of the molecule. Both viral and non-viral expression vectors can be used to produce therapeutic fusion proteins in mammalian host cells. Non-viral vectors and systems typically include plasmids, episomal vectors, and human artificial chromosomes (see, e.g., Harrington et al., (1997) Nat Genet 15:345) that carry expression cassettes for expressing proteins or RNA. For example, non-viral vectors useful for expressing polynucleotides and polypeptides in mammalian (e.g., human) cells include pThioHis A, B and C, pcDNA3.1 / His, pEBVHis A, B and C (Invitrogen, San Diego, Calif.), MPSV vectors, as well as numerous other vectors known in the art for expressing other proteins. Useful viral vectors include vectors based on retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, SV40, papilloma viruses, HBP Epstein-Barr virus, vaccinia virus vectors, and Semliki Forest virus (SFV) vectors. See Brent et al., (1995) supra; Smith, Annu. Rev. Microbiol. 49:807; and Rosenfeld et al., (1992) Cell 68:143.

[0158] The choice of expression vector depends on the intended host cell in which the vector will be expressed. Typically, expression vectors contain a promoter and other control sequences (e.g., enhancers) operably linked to the polynucleotide encoding the therapeutic fusion protein. In some embodiments, an inducible promoter is used to prevent expression of the inserted sequence except under inducing conditions. Inducible promoters include, for example, arabinose, lacZ, metallothionein promoters, or heat shock promoters. Cultures of transformed organisms can be grown under non-inducing conditions that do not bias the population in favor of coding sequences whose expression products are better tolerated by the host cell. In addition to promoters, other control elements may also be necessary or required for efficient expression of the therapeutic fusion protein. These elements typically include an ATG initiation codon and adjacent ribosome binding sites or other sequences. In addition, expression efficiency can be increased by including enhancers appropriate for the cell system used (see, e.g., Scharf et al., (1994) Results Probl. Cell Differ. 20:125; and Bittner et al., (1987) Meth. Enzymol., 153:516). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.

[0159] The expression vector may also provide a secretion signal sequence position for forming a fusion protein with the encoded polypeptide by inserting the above-mentioned binding domain and / or solubility domain sequences. More often, the inserted sequences are linked to a signal sequence before being included in the vector. A vector that allows the expression of the binding domain and solubility domain as a fusion protein thereby results in the production of an intact modified protein. When cultured under appropriate conditions, the host cells can be used to express a modified protein that can then be recovered from the medium (if the host cell secretes it into the medium) or directly from the host cell that produces it (if not secreted). The selection of an appropriate host cell depends on various factors, such as the desired expression level, the polypeptide modifications that are desired or necessary for activity (such as glycosylation or phosphorylation), and the ease of folding into a biologically active molecule. The host cell may be eukaryotic or prokaryotic.

[0160] Mammalian cell lines available as hosts for expression are known in the art, including, but not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC), and any cell line used in an expression system known in the art can be used to produce the recombinant fusion proteins of the invention. Generally, a host cell is transformed with a recombinant expression vector that contains DNA encoding the desired fusion protein. Among the host cells that can be used are prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive organisms, such as E. coli or bacilli. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include COS-7 cells, L cells, Cl27 cells, 3T3 cells, Chinese hamster ovary (CHO) cells, or their derivatives and related cell lines growing in serum-free medium, HeLa cells, BHK cell lines, CV-1 EBNA cell lines, human embryonic kidney (HEK) cells such as 293, 293EBNA or MSR293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, primary tissues, cell lines derived from in vitro culture of primary explants, HL-60, U937, HaK or Jurkat cells. Optionally, mammalian cell lines such as HepG2 / 3B, KB, NIH 3T3 or S49 can be used for the expression of the polypeptides, if it is desired to use the polypeptides in various signal transduction or reporter assays. Alternatively, it is possible to produce the polypeptides in lower eukaryotes such as yeast or in prokaryotes such as bacteria. Suitable yeasts include P. pastoris, S. cerevisiae, S. pombe, Kluyveromyces strains, Candida, or any yeast strain capable of expressing a heterologous polypeptide. Suitable bacterial strains include E. coli, B. subtilis, S. typhimurium, or any bacterial strain capable of expressing a heterologous polypeptide.If the fusion protein is made in yeast or bacteria, it may be desirable to modify the product produced therein, for example by phosphorylation or glycosylation of appropriate sites, to obtain a functional product. Such covalent attachments can be accomplished using known chemical or enzymatic methods.

[0161] Methods for introducing expression vectors containing a polynucleotide sequence of interest vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, while calcium phosphate treatment or electroporation may be used for other cellular hosts. Other methods include, for example, electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, gene gun techniques, virosomes, immunoliposomes, polycation:nucleic acid conjugates, naked DNA, artificial virions, fusion with herpes virus structural protein VP22, drug-facilitated uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression is often desired. For example, cell lines stably expressing modified proteins can be generated using the expression vectors of the present disclosure that contain a viral origin of replication or endogenous expression elements and a selectable marker gene. After introduction of the vector, the cells can be grown in rich medium for 1-2 days before switching them to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells that successfully express the introduced sequences in a selective medium. Resistant, stably transfected cells can be grown using tissue culture techniques appropriate to the cell type.

[0162] Fusion proteins are typically recovered from the culture medium as secreted polypeptides, but may also be recovered from host cell lysates if directly produced without a secretory signal. If the polypeptide is membrane-bound, it can be released from the membrane using an appropriate detergent solution (e.g., Triton-X 100).

[0163] When the fusion protein is produced in a recombinant cell other than that of human origin, it is completely free of proteins or polypeptides of human origin. However, it is necessary to purify the fusion protein from the recombinant cell proteins or polypeptides. As a first step, the culture medium or lysate is usually centrifuged to remove particulate cell debris. The produced molecule can be conveniently purified by hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography, with affinity chromatography being the preferred purification technique. Other techniques for protein purification are also available, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

[0164] In certain aspects, the present specification provides a viral vector comprising a polynucleotide encoding the therapeutic fusion protein of the present invention.In some embodiments, the viral vector is derived from AAV.In some embodiments, the viral vector is administered to a subject, such as a human, in which the therapeutic fusion protein is expressed, and can be used for the treatment and / or prevention of diseases such as those listed herein.

[0165] Pharmaceutical Compositions In another aspect, the present disclosure provides compositions, e.g., pharmaceutical compositions, containing a therapeutic fusion protein of the present disclosure in combination with one or more pharma- ceutically acceptable excipients, diluents, or carriers. Such compositions may include one or a combination of (e.g., two or more different) therapeutic fusion proteins of the present disclosure.

[0166] The pharmaceutical compositions described herein can also be administered in combination therapy, i.e., in combination with other agents. For example, the combination therapy can include the fusion protein of the present disclosure, for example, in combination with at least one anti-inflammatory, anti-infective, or immunosuppressant agent. Examples of therapeutic agents that can be used in combination therapy are described in more detail below in the section on the use of therapeutic fusion proteins of the present disclosure.

[0167] To prepare a pharmaceutical or sterile composition comprising a fusion protein of the present disclosure, the fusion protein is mixed with a pharma- ceutically acceptable carrier or excipient.

[0168] The phrase "pharmaceutical acceptable" means approved by a regulatory agency of the Federal or state government or listed in the United States Pharmacopeia or other generally recognized pharmacopoeias for use in animals, or more specifically, in humans.

[0169] The term "pharmaceutical composition" refers to a mixture of at least one active ingredient (eg, a variant protein) and at least one pharma- ceutically acceptable excipient, diluent or carrier.

[0170] "Drug" refers to a substance used in medical treatment.

[0171] As used herein, "pharmaceutically acceptable carriers" include all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The carrier should be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion). In one embodiment, the carrier should be suitable for subcutaneous routes. Depending on the route of administration, the active compound, i.e., the fusion protein, may be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0172] The pharmaceutical compositions described herein may include one or more pharma- ceutically acceptable salts.The pharmaceutical compositions described herein may also include pharma- ceutically acceptable antioxidants.Examples of pharma-ceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, and sodium sulfite; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, and alpha tocopherol; and metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, and phosphoric acid.

[0173] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.) and suitable mixtures thereof, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants.

[0174] These compositions may also contain auxiliary agents such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be ensured both by sterilization procedures and by including various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents in the compositions, such as sugars, sodium chloride, etc. In addition, prolonged absorption of the injectable dosage form can be brought about by including agents that delay absorption, such as aluminum monostearate and gelatin.

[0175] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.The use of such media and agents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present invention is contemplated.Supplementary active compounds can also be incorporated into the compositions.

[0176] Therapeutic compositions must typically be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by maintaining the required particle size in the case of dispersion, and by the use of surfactants. In many cases, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, can be included in the composition.

[0177] Reviews on the development of stable protein formulations can be found in Cleland et al., (1993) Crit Reviews Ther Drug Carrier Systems, 10(4):307-377 and Wei W (1999) Int J Pharmaceutics, 185:129-88.

[0178] Solutions or suspensions used for intradermal or subcutaneous administration typically contain one or more of the following components: sterile diluents such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methylparabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates, and agents for adjusting tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Such formulations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic.

[0179] Sterile injectable solution can be prepared by incorporating the required amount of active compound into a suitable solvent containing one or a combination of the above-listed ingredients as necessary, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating the fusion protein of the present invention into a sterile vehicle containing a basic dispersion medium and other necessary ingredients from the above-listed ones.For the preparation of sterile powder for preparing sterile injectable solution, the preparation method is vacuum drying and freeze-drying (lyophilization), which obtains a powder of active ingredient plus any additional desired ingredients from the solution previously sterilized and filtered.

[0180] The amount of active ingredient that can be combined with carrier materials to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of active ingredient that can be combined with carrier materials to produce a single dosage form is generally the amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount ranges from about 0.01 percent to about 99 percent of active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent of active ingredient combined with a pharma- ceutically acceptable carrier.

[0181] The choice of dosing regimen for a therapeutic modified protein depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix. In certain embodiments, the dosing regimen maximizes the amount of therapeutic agent delivered to the patient consistent with an acceptable level of side effects. Thus, the amount of protein delivered depends, in part, on the particular entity and the severity of the condition being treated. Guidance for selecting appropriate doses of biological and small molecules is available (e.g., Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom, et al. (1999) New Engl. J. Med. 341:1966-1973; Slamon, et al. (2001) New Engl. J. Med. 344:783-792; Beniaminovitz, et al. (2000) New Engl. J. Med. 342:613-619; Ghosh, et al. (2003) New Engl. J. Med. 348:24-32; Lipsky, et al. (see, e.g., et al. (2000) New Engl. J. Med. 343:1594-1602).

[0182] The determination of the appropriate dose is made by the clinician, for example, using parameters or factors known or suspected in the art to affect treatment, or predicted to affect treatment. Generally, administration is started at an amount somewhat less than the optimal dose, and then increased in small increments until a desired or optimal effect is obtained relative to negative side effects. Important diagnostic measures include, for example, measures of symptoms of inflammation or levels of inflammatory cytokines produced.

[0183] The actual dosage level of the active ingredient in the pharmaceutical composition of the present disclosure may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present disclosure used, the route of administration of the particular compound used, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and past medical history of the patient being treated, and factors known in the medical arts.

[0184] Dosage regimens are adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subjects to be treated. Each unit contains a predetermined amount of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are determined and directly depend on the unique properties of the active compound and the particular therapeutic effect to be achieved, as well as the limitations inherent in the technology of compounding such active compounds for the treatment of individual sensitivities.

[0185] For administration of therapeutic fusion proteins, dosages range from about 0.0001 to 150 mg / kg of the host body weight subcutaneously, e.g., 5, 15, and 50 mg / kg, more usually 0.01 to 5 mg / kg. Exemplary treatment regimens involve administration once per week, once every two weeks, once every three weeks, once every four weeks, once per month, once every three months, or once every three to six months.

[0186] The therapeutic fusion protein of the invention can be administered multiple times. The interval between single doses can be, for example, weekly, monthly, every three months, or yearly. The intervals can also be irregular, as indicated by measuring the patient's blood levels of the modified protein. In some methods, the dosage is adjusted to achieve a plasma protein concentration of about 1-1000 μg / ml, and in some methods, to achieve a plasma protein concentration of about 25-300 μg / ml.

[0187] Alternatively, the therapeutic fusion protein can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency will vary depending on the half-life of the protein in the patient and may vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, relatively low doses are administered at relatively infrequent intervals over an extended period of time. Some patients may continue to receive treatment for the rest of their lives. In therapeutic applications, relatively high doses at relatively short intervals may be required until the progression of the condition or disease is alleviated or terminated, or until the patient shows partial or complete improvement of the symptoms of the condition or disease. The patient can then receive a prophylactic regimen.

[0188] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without causing toxicity to the patient. The selected dosage level will depend on various pharmacokinetic factors, including the activity of the particular composition of the present disclosure used, the route of administration of the particular compound used, the time of administration, the rate of excretion, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and past medical history of the patient being treated, and factors well known in the medical arts.

[0189] A "therapeutically effective dose" of a fusion protein of the invention may result in a reduction in the severity of a condition or symptom or disease and / or prevention of impairment or disability due to a condition.

[0190] The compositions of the present disclosure can be administered by one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or method of administration will vary depending on the desired outcome. Routes of administration of the modified proteins of the present invention include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other common routes of administration by injection or infusion. As used herein, the phrase "parenteral administration" refers to methods of administration other than enteral and topical administration, usually by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intrathecal, epidural and intrasternal injection and infusion.

[0191] Alternatively, the therapeutic fusion proteins of the invention can be administered by parenteral routes, such as topical, epidermal, or mucosal routes of administration.

[0192] The therapeutic fusion protein of the present disclosure can be prepared with a carrier that protects the protein from rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for preparing such formulations are patented or generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0193] In certain embodiments, the therapeutic fusion proteins of the present invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compounds of the present invention cross the BBB (if necessary), they can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Pat. Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more moieties that selectively transport into specific cells or organs, thereby facilitating targeted drug delivery (see, for example, Ranade VV (1989) J. Clin. Pharmacol., 29:685).

[0194] Therapeutic Uses and Methods of the Invention The therapeutic fusion proteins of the invention have in vitro and in vivo diagnostic and therapeutic uses. For example, these molecules can be administered to cells in culture, e.g., in vitro, or to a subject, e.g., in vivo, to treat, prevent, or diagnose a variety of disorders. The methods are particularly suited to treat, prevent, or diagnose acute or chronic inflammatory and immune system-driven organ and microvascular disorders.

[0195] The therapeutic fusion proteins of the present invention are useful for treating, preventing, or ameliorating acute and chronic inflammatory organ damage, particularly inflammatory damage, including, but not limited to, when endogenous homeostatic clearance mechanisms or efferocytosis pathways for removing dead cells, cell fragments, and prothrombotic / inflammatory particulates are significantly downregulated. Examples of acute inflammatory organ damage include myocardial infarction, acute kidney injury (AKI), acute stroke and inflammation, and organ damage resulting from ischemia / reperfusion, such as the gastrointestinal tract, liver, spleen, lungs, kidneys, pancreas, heart, brain, spinal cord, and / or crushed limbs.

[0196] Therapeutic fusion proteins of the present disclosure may also be useful in the diagnosis, treatment, prevention, or amelioration of inhibiting or slowing blood clotting, microbiome manipulation, inflammatory bowel disease (IBD), reduced fatty acid uptake and / or gastric motility, microthrombus dependent disorders, atherosclerosis, cardiac remodeling, tissue fibrosis, acute liver injury, chronic liver disease, non-alcoholic steatohepatitis (NASH), vascular disease, age-related vascular disorders, intestinal disease, sepsis, bone disorders, cancer, thalassemia, pancreatitis, hepatitis, endocarditis, pneumonia, acute lung injury, osteoarthritis, periodontitis, tissue trauma induced inflammation, colitis, diabetes, hemorrhagic shock, transplant rejection, radiation induced injury, splenomegaly, sepsis induced AKI or multiple organ failure, acute burns, adult and pediatric respiratory distress syndrome, wound healing, tendon repair, and neurological disorders.

[0197] In one embodiment, the neurological disease may be selected from conditions having neuropsychiatric, neuroinflammatory and / or neurodegenerative components, including symptoms such as sickness syndrome, nausea, passive avoidance, suppressed behavioral alertness, memory impairment and memory dysfunction, etc. Examples of neurological diseases include amyloid beta-related neurological diseases such as Alzheimer's disease, Parkinson's disease, and depression.

[0198] In one embodiment, the bone disorder may be selected from conditions including osteoporosis, osteomalacia, osteosclerosis, and osteopetrosis. More specifically, administration of the fusion protein of the present disclosure may inhibit expression of at least one osteoclast marker, such as NFATc1, cathepsin K, and αvβ3 integrin. In one embodiment, the administration inhibits osteoclast formation. In another embodiment, the administration inhibits RANKL-induced osteoclast formation. In yet another embodiment, the administration inhibits bone resorption. In yet another embodiment, the administration inhibits expression of at least one bone resorption stimulator, such as bone resorption stimulators including TNF, IL-6, IL-17A, MMP-9, Ptgs2, RANKL, Tnfsf11, CXCL1, CXCL2, CXCL3, CXCL5, and combinations thereof. In another embodiment, the administration inhibits expression of at least one proinflammatory cytokine selected from the group consisting of IL-8 and CCL2 / MCP-1.

[0199] In one embodiment, tissue fibrosis can be fibrosis in liver, lung, diaphragm, kidney, brain, heart, where fusion protein of the present invention reduces collagen expression.In one embodiment, pulmonary fibrosis is interstitial pulmonary fibrosis (IPF).In one embodiment, liver fibrosis is liver cirrhosis, which may or may not be caused by NASH.

[0200] Several respiratory diseases are characterized by the accumulation of apoptotic cells. Furthermore, defective efferocytosis and phagocytosis by macrophages in chronic obstructive pulmonary disease (COPD) are associated with exacerbation and severity. The therapeutic fusion proteins of the present disclosure may also be useful for the diagnosis, treatment, prevention, or amelioration of respiratory diseases such as acute respiratory distress syndrome, or COPD. The therapeutic fusion proteins of the present disclosure may also be useful for the diagnosis, treatment, prevention, or amelioration of acute lung injury (ALI), such as lung injury induced by inhalation or aspiration of toxic exogenous or endogenous compounds or drugs; lung injury caused by pulmonary edema, shock, pancreatitis, burns, chest trauma or multiple trauma, radiation, sepsis, pathogens (bacteria, viruses, or parasites such as malaria parasites); chronic respiratory failure leading to hypoxemia.

[0201] The therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of lung injury caused by Corona-type viruses, such as ARS-CoV, SARS-CoV-2, or MERS-CoV. In one embodiment, the therapeutic fusion proteins of the present disclosure are provided for use in treating SARS-CoV-2 infection in COVID19 patients.

[0202] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of transfusion-associated pulmonary insufficiency (TRALI).

[0203] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of chronic respiratory failure leading to hypoxemia.

[0204] Therapeutic fusion proteins of the disclosure, for example therapeutic fusion proteins comprising a domain of EDIL3 of the disclosure, may also be useful in diagnosing, treating, preventing, or ameliorating the severity of post-surgical peritoneal adhesions.

[0205] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of heart failure.

[0206] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of hemodialysis.

[0207] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating delayed graft function or the severity of graft-versus-host disease.

[0208] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of severe frostbite, trench foot, pyoderma gangrenosum / gangrene.

[0209] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of bacterial, fungal, viral, or parasitic induced conditions (e.g., sepsis, or other conditions directly induced by pathogens, such as necrotizing soft tissue infections (NSTIs such as necrotizing fasciitis), osteomyelitis, malaria, etc.).

[0210] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of trauma / polytrauma caused by accidents or other injury mechanisms resulting in injury, such as work-related accidents, falls, traffic accidents, ballistic and combat injuries, etc.

[0211] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of osteoclast-mediated pathologies.

[0212] The therapeutic fusion proteins of the present disclosure may be administered as the sole active ingredient or in combination, e.g., as an adjunct to or in combination with other drugs (e.g., immunosuppressants or immunomodulators or other anti-inflammatory agents or e.g., cytotoxic or anti-cancer agents), e.g., for the treatment or prevention of the diseases listed above.

[0213] With respect to additional therapeutic agents, administering "in combination" means that two (or more) different therapeutic agents are delivered to a subject during the course of the subject's disease, e.g., two or more therapeutic agents are delivered after the subject is diagnosed with a disease and before the disease is cured or eliminated or before treatment is stopped for other reasons. In some embodiments, the delivery of one therapeutic agent is still occurring when the delivery of the second therapeutic agent begins, so that there is an overlap in administration. This may be referred to herein as "concurrent" or "concurrent delivery." In other embodiments, the delivery of one therapeutic agent ends before the delivery of the other therapeutic agent begins. In either embodiment, the therapeutic agents are more effective when administered in combination. For example, the second therapeutic agent is more effective, e.g., the same effect is seen with the second therapeutic agent but to a lesser extent, or the second therapeutic agent relieves symptoms to a greater extent than would be seen if the second therapeutic agent were administered without the first therapeutic agent, or a similar situation is seen with the first therapeutic agent. In certain embodiments, delivery is such that relief of symptoms or other parameters associated with the disease is greater than observed with one therapeutic agent delivered without the other. The effects of the two therapeutic agents may be partially additive, fully additive, or greater than additive. Delivery may be such that the effect of the first therapeutic agent delivered is still detectable when the second therapeutic agent is delivered.

[0214] The term "concurrently" is not limited to administration of therapeutic agents (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather means that a pharmaceutical composition comprising the therapeutic fusion protein of the present disclosure is administered to a subject in an order and within a time interval such that the fusion protein may act together with the additional therapeutic agents to provide an increased benefit than if they were otherwise administered. For example, each therapeutic agent may be administered to a subject at the same time or at different times, sequentially in any order; however, if not administered simultaneously, they should be administered sufficiently close in time to provide the desired therapeutic or prophylactic effect. Each therapeutic agent may be administered to a subject separately, in any appropriate form and by any suitable route.

[0215] The therapeutic fusion protein described herein and the additional therapeutic agent can be administered simultaneously, in the same or separate pharmaceutical composition as the fusion protein of the present disclosure, or sequentially. In the case of sequential administration, the fusion protein as described herein can be administered first, and the additional agent can be administered second, or the order of administration can be reversed. The additional therapeutic agent can be administered to the subject by the same or different administration route as the fusion protein.

[0216] The therapeutic fusion proteins described herein and / or additional therapeutic agents, procedures or modalities can be administered during periods of active disorder or during periods of remission or less active disease. Therapeutic fusion proteins as described herein can be administered prior to, concurrently with, or after other treatments, or during remission of the disorder.

[0217] When administered in combination, the therapeutic fusion proteins described herein and the additional therapeutic agents (e.g., second or third agents) may be administered in amounts or dosages that are greater than, less than, or the same as the amount or dosage of each agent used individually, e.g., as a monotherapy. In certain embodiments, the therapeutic fusion proteins described herein, the additional agents (e.g., second or third agents), or all are lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) than the amount or dosage of each agent used individually, e.g., as a monotherapy. In other embodiments, the amount or dosage of the therapeutic fusion proteins described herein, the additional agents (e.g., second or third agents), or all that produces a desired effect (e.g., treatment of an inflammatory disease or condition) is lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50% lower) than the amount or dosage of each agent used individually, e.g., as a monotherapy to achieve the same therapeutic effect.

[0218] For example, therapeutic fusion proteins of the disclosure may be used in combination with DMARDs, such as gold salts, sulfasalazine, antimalarials, methotrexate, D-penicillamine, azathioprine, mycophenolic acid, tacrolimus, sirolimus, minocycline, leflunomide, glucocorticoids; calcineurin inhibitors, such as cyclosporine A or FK506; modulators of lymphocyte recirculation, such as FTY720 and FTY720 analogs; mTOR inhibitors, such as rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI77, 9, ABT578, AP23573 or TAFA-93; ascomycins with immunosuppressive properties, such as ABT-281, ASM981, etc.; corticosteroids; cyclophosphamide; azathioprine; leflunomide; mizoribine; mycophenolate mofetil; 15-deoxyspergualin or its immunosuppressive homologues, analogues or derivatives; immunosuppressive monoclonal antibodies, such as monoclonal antibodies against leukocyte receptors, such as MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, CD45 , CD58, CD80, CD86 or their ligands; other immunomodulatory compounds, such as recombinant binding molecules having at least a portion of the extracellular domain of CTLA4 or a variant thereof, such as at least the extracellular portion of CTLA4 or a variant thereof linked to a non-CTLA4 protein sequence, such as CTLA4Ig (e.g., referred to as ATCC68629) or a variant thereof, such as LEA29Y; adhesion molecule inhibitors, such as LFA-1 antagonists, ICAM-1 or -3 antagonists, VCAM-4 antagonists or VLA-4 antagonists. or chemotherapeutic agents such as paclitaxel, gemcitabine, cisplatin, doxorubicin or 5-fluorouracil; anti-TNF agents such as monoclonal antibodies against TNF, such as infliximab, adalimumab, CDP870, or receptor constructs against TNF-RI or TNF-RII, such as etanercept, PEG-TNF-RI; blockers of inflammatory cytokines, IL-1 blockers such as anakinra or IL-1 trap, canakinumab, IL-13 blockers, IL-4 blockers, IL-6 blockers;Chemokine blockers, such as inhibitors or activators of proteases, such as metalloproteases, anti-IL-15 antibodies, anti-IL-6 antibodies, anti-IL-4 antibodies, anti-IL-13 antibodies, anti-CD20 antibodies, NSAIDs such as aspirin or anti-infectives; Damage-associated molecular pattern (DAMP) or pathogen-associated molecular pattern (PAMP) antagonists, such as converters, detoxifiers, removers, such as ATP converters, HMGB-1 modulators, histone detoxifiers; inhibitors of superantigen-induced immune responses; complement inhibitors and extracorporeal plasma exchange devices;

[0219] kit Also within the scope of the present invention are kits consisting of compositions, such as therapeutic fusion proteins of the present disclosure, and instructions for use. Such kits include a therapeutically effective amount of the fusion protein according to the present disclosure. In addition, such kits may include a means for administering the therapeutic fusion protein (e.g., an autoinjector, syringe and vial, prefilled syringe, prefilled pen) and instructions for use. These kits may include additional therapeutic agents (described below) for treating patients with autoimmune or inflammatory disorders or AOI. Such kits may also include instructions for administration of the therapeutic fusion protein to treat patients. Such instructions may provide dosages, routes of administration, regimens, and total treatment duration for use with the enclosed fusion protein. The kits typically include a label indicating the intended use of the contents of the kit. The term label includes any written or recorded matter provided on or with the kit, or otherwise associated with the kit. The kits may further include a tool for diagnosing whether a patient belongs to a group that will respond to treatment with the therapeutic fusion protein of the present invention, as defined above.

[0220] Embodiment The present disclosure provides the following embodiments.

[0221] 1. A therapeutic fusion protein for enhancing efferocytosis comprising an integrin-binding domain, a phosphatidylserine (PS)-binding domain, and a solubility domain.

[0222] 2. The solubility domain is (i) linked to an integrin-binding domain; (ii) linked to a PS-binding domain; (iii) inserted between the integrin-binding domain and the PS-binding domain; (iv) is inserted into an integrin-binding domain; or (v) inserted into the PS-binding domain; 2. The fusion protein of embodiment 1.

[0223] 3. The fusion protein of embodiment 1 or embodiment 2, wherein the integrin binding domain binds to one or more integrins.

[0224] 4. The fusion protein of embodiment 3, wherein the integrin binding domain binds to αvβ3 and / or αvβ5 and / or α8β1 integrin.

[0225] 5. The fusion protein of embodiment 3 or embodiment 4, wherein the integrin binding domain comprises an arginine-glycine-aspartic acid (RGD) motif.

[0226] 6. A fusion protein according to any one of the previous embodiments, wherein the solubility domain is directly linked to the integrin binding domain, the PS binding domain, or both domains.

[0227] 7. The fusion protein according to any one of embodiments 1 to 6, wherein the solubility domain is indirectly linked to the integrin-binding domain and / or the PS-binding domain by a linker.

[0228] 8. The fusion protein according to any one of the previous embodiments, wherein the solubilization domain comprises human serum albumin (HSA), domain 3 of HSA (HSA D3), Fc-IgG, or a functional variant thereof.

[0229] 9. The fusion protein of any one of the previous embodiments, wherein the solubility domain comprises human serum albumin (HSA), or a functional variant thereof.

[0230] 7. The fusion protein of any one of the previous embodiments, wherein the integrin binding domain has the amino acid sequence of SEQ ID NO:2, or at least 90% sequence identity thereto.

[0231] 8. A fusion protein according to any one of the previous embodiments, wherein the PS binding domain has the amino acid sequence of SEQ ID NO: 3, or at least 90% sequence identity thereto; or the PS binding domain has the amino acid sequence of SEQ ID NO: 76, or at least 90% sequence identity thereto.

[0232] 9. The multidomain fusion protein of any one of the previous embodiments, wherein the solubility domain is HSA and has the amino acid sequence of SEQ ID NO: 4, or at least 90% sequence identity thereto.

[0233] 10. The fusion protein of any one of the previous embodiments, wherein the integrin binding domain has the amino acid sequence of SEQ ID NO: 2, or at least 90% sequence identity thereto, and the PS binding domain has the amino acid sequence of SEQ ID NO: 78, or at least 90% sequence identity thereto.

[0234] 11. The fusion protein of any one of the previous embodiments, wherein the integrin binding domain has the amino acid sequence of SEQ ID NO: 77, or at least 90% sequence identity thereto, and the PS binding domain has the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 76, or at least 90% sequence identity thereto.

[0235] 12. The fusion protein, a. Promotes efferocytosis by endothelial cells in a human endothelial cell-Jurkat cell efferocytosis assay; b. Recovering impaired macrophage efferocytosis in a human macrophage-neutrophil efferocytosis assay; c. reducing the number of plasma microparticles by clearance in a human endothelial microparticle efferocytosis assay; and / or d. Protects against multi-organ injury in models of acute kidney injury; 2. A fusion protein according to any one of the preceding embodiments.

[0236] 13. A fusion protein according to any one of the preceding embodiments, comprising in that order an integrin binding domain-HSA-PS binding domain.

[0237] 14. A therapeutic fusion protein comprising MFG-E8 and a solubilization domain, wherein MFG-E8 comprises, from the N-terminus to the C-terminus, an EGF-like domain, a C1 domain and a C2 domain, and comprises wild-type human MFG-E8 (sequence number 1) or a sequence from MFG-E8 having sequence number 75 or a functional variant thereof.

[0238] 15. The fusion protein of embodiment 14, wherein the solubility domain is inserted between the EGF-like domain and the C1 or C2 domain.

[0239] 16. The fusion protein according to embodiment 14 or embodiment 15, wherein the solubility domain is HSA, HSA D3 or Fc-IgG, or a functional variant thereof.

[0240] 17. The fusion protein of any one of embodiments 1 to 16, wherein the modified protein has the amino acid sequence of SEQ ID NO: 42, or at least 90% sequence identity thereto.

[0241] 18. The fusion protein of any one of the previous embodiments, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 44, or at least 90% sequence identity thereto; or SEQ ID NO: 47, or at least 90% sequence identity thereto; or SEQ ID NO: 48, or at least 90% sequence identity thereto.

[0242] 19. The fusion protein of any one of the previous embodiments, wherein the fusion protein has an amino acid sequence of SEQ ID NO: 80, or at least 90% sequence identity thereto.

[0243] 20. The fusion protein of any one of the previous embodiments, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 82, or at least 90% sequence identity thereto.

[0244] 21. An isolated nucleic acid encoding the amino acid sequence of any one of embodiments 17 to 20.

[0245] 22. A cloning or expression vector comprising a nucleic acid according to embodiment 21.

[0246] 23. A viral vector comprising the isolated nucleic acid of embodiment 21, preferably wherein the viral vector comprising the isolated nucleic acid of embodiment 21 is derived from AAV.

[0247] 24. The viral vector of embodiment 23, wherein the vector is administered to a subject in need thereof, such as a human subject.

[0248] 25. The viral vector according to embodiment 23, for use in the treatment and / or prevention of diseases listed herein.

[0249] 26. A recombinant host cell suitable for producing a therapeutic fusion protein, comprising one or more cloning or expression vectors according to embodiment 22 and optionally a secretion signal.

[0250] 27. The recombinant host cell according to embodiment 26, wherein the host cell is, for example, a prokaryotic, yeast, insect or mammalian cell.

[0251] 28. A fusion protein according to any one of embodiments 1 to 20, wherein expression of the protein in a host cell results in a yield of at least 10 mg / L.

[0252] 29. A fusion protein according to any one of embodiments 1 to 20, wherein expression of the protein in a mammalian cell results in at least a 100-fold increase in yield over wild-type MFG-E8 (sequence number 1).

[0253] 30. A pharmaceutical composition comprising a fusion protein according to any one of embodiments 1 to 20, and at least one pharma- ceutically acceptable carrier.

[0254] 31. A method for treating or preventing an inflammatory disorder or inflammatory organ damage in an individual in need thereof, comprising administering to the individual a therapeutically effective amount of the fusion protein according to any one of embodiments 1 to 20.

[0255] 32. A fusion protein according to any one of embodiments 1 to 20 for use in the treatment or prevention of an inflammatory disorder or inflammatory organ damage in an individual in need thereof.

[0256] 33. The method according to embodiment 31 or the use according to embodiment 32, wherein the inflammatory disorder or inflammatory organ damage is acute kidney injury, sepsis, myocardial infarction, acute stroke, burns, traumatic injury, and inflammatory and organ damage due to ischemia / reperfusion.

[0257] 34. The method according to embodiment 31 or the use according to embodiment 32, wherein the inflammatory disorder or inflammatory organ damage is acute kidney injury.

[0258] 35. The method according to embodiment 31 or the use according to embodiment 32, wherein the inflammatory disorder or inflammatory organ damage is myocardial infarction.

[0259] 36. The method according to embodiment 31 or the use according to embodiment 32, wherein the inflammatory disorder or inflammatory organ damage is stroke.

[0260] 37. The method according to embodiment 31 or the use according to embodiment 32, wherein the inflammatory disorder or inflammatory organ injury is acute lung injury (e.g. acute respiratory distress syndrome) or liver injury or acute intestinal injury.

[0261] 38. The method according to embodiment 31 or the use according to embodiment 32, wherein the fusion protein is administered in combination with another therapeutic agent.

[0262] 39. The method or use according to embodiment 38, wherein the further therapeutic agent is an immunosuppressant, an immunomodulatory agent, an anti-inflammatory agent, an antioxidant, an anti-infective agent, a cytotoxic agent or an anti-cancer agent.

[0263] 40. A therapeutic fusion protein comprising MFG-E8 and a solubilization domain, wherein the MFG-E8 comprises, from the N-terminus to the C-terminus, an EGF-like domain, a C1 domain and a C2 domain, and comprises a sequence from wild-type human MFG-E8 (sequence number 1) or the sequence of sequence number 75 or a functional variant thereof.

[0264] 41. The fusion protein of embodiment 40, wherein the solubility domain is linked to the N-terminus or C-terminus of MFG-E8 (sequence number 1 or sequence number 75).

[0265] 42. The fusion protein of embodiment 40, wherein the solubility domain is inserted between the EGF-like domain and the C1 domain.

[0266] 43. The fusion protein of embodiment 41, wherein the solubility domain is inserted between the C1 and C2 domains.

[0267] 44. A fusion protein according to any one of embodiments 40 to 43, wherein the solubility domain is HSA, HSA D3 or Fc-IgG, or a functional variant thereof.

[0268] 38. An isolated nucleic acid encoding a fusion protein according to any one of embodiments 33 to 37.

[0269] 39. A cloning or expression vector comprising a nucleic acid according to embodiment 38.

[0270] 40. A viral vector comprising the isolated nucleic acid of embodiment 38, preferably wherein the viral vector comprising the isolated nucleic acid of embodiment 38 is derived from AAV.

[0271] 41. The viral vector of embodiment 40, wherein the vector is administered to a subject in need thereof, such as a human subject.

[0272] 42. The viral vector according to embodiment 40, for use in the treatment and / or prevention of diseases listed herein.

[0273] 43. A recombinant host cell suitable for producing a therapeutic fusion protein, comprising one or more cloning or expression vectors according to embodiment 39 and optionally a secretion signal.

[0274] 44. The recombinant host cell according to embodiment 43, wherein the host cell is, for example, a prokaryotic, yeast, insect or mammalian cell.

[0275] 45. A fusion protein according to any one of embodiments 33 to 37, wherein expression of the protein in a host cell results in a yield of at least 10 mg / L.

[0276] 46. ​​A fusion protein according to any one of embodiments 33 to 37, wherein expression of the protein in a mammalian cell results in at least a 100-fold increase in yield over wild-type MFG-E8.

[0277] It is to be understood that each embodiment may be combined with one or more other embodiments to the extent such combination is consistent with the description of the embodiment. It is to be further understood that the embodiments provided above are understood to include all embodiments, including such embodiments as a result of combinations of embodiments.

[0278] All references cited herein, including patents, patent applications, articles, publications, textbooks, etc., and references cited therein (to the extent they are not already incorporated), are hereby incorporated by reference in their entirety. EXAMPLES

[0279] The following examples are provided to further illustrate the disclosure, but not to limit its scope. Other variations of the present disclosure will be readily apparent to those of ordinary skill in the art and are encompassed by the scope of the appended claims.

[0280] Example 1: Production of fusion proteins MFG-E8 is a multi-domain protein consisting of an N-terminal epidermal growth factor (EGF-like) domain and two C-terminal lectin-type C domains (C1 and C2). As described in the literature, attempts to produce recombinant full-length human protein show very low protein aggregation and expression rates (Castellanos et al., (2016) Protein Expression Purification 1124:10-22). Therefore, to try to solubilize the protein and enhance its expression, we investigated the effect of fusing several proteins to MFG-E8.

[0281] Solubilization domains (SD) from human Fc-IgG1, human serum albumin (HSA), and domain 3 of HSA (HSA D3) were fused to MFG-E8 at different positions, i.e., at the N-terminus or C-terminus, or between the EGF and C1 or C1 and C2 domains, as shown diagrammatically in FIG. 1. Furthermore, fusion to Fc-IgG1 or HSA may increase the half-life of the molecule in vivo, since these proteins bind to FcRn. Fusion of MFG-E8 to Fc-IgG1 or HSA can also increase the production and solubility of the fusion protein, as shown in the following example (Castellanos et al., (2016) supra).

[0282] Table 5 shows the binding of the fusion protein FP330 containing an HSA insert (EGF-HSA-C1-C2; SEQ ID NO: 42) to the human neonatal Fc receptor (see also Example 5.1).

[0283] [Table 57]

[0284] Example 2: Generation of wild-type MFG-E8 and MFG-E8 HSA fusions; expression and purification Methods for producing fusion proteins are described below: Briefly, MFG-E8 and MFG-E8 fusions and EDIL fusions, particularly to HSA, were produced according to the following methods.

[0285] DNA was synthesized at GeneArt (Regensburg, Germany) and cloned into a mammalian expression vector using a restriction enzyme ligation-based cloning technique. The resulting plasmid was transfected into HEK293T cells. For transient expression of proteins, vectors of wild-type or modified strands were transfected into suspension-adapted HEK293T cells using polyethyleneimine (PEI; catalog number 24765 Polysciences, Inc.). Typically, 100 ml of cells in suspension at a density of 1-2 Mio cells per ml were transfected with DNA containing 100 μg of expression vector encoding the modified strand. Constructs were then produced by introducing the recombinant expression vector into the host cells and culturing the cells for an additional 7 days to allow secretion into medium supplemented with 0.1% pluronic acid, 4 mM glutamine, and 0.25 μg / ml antibiotics (HEK, serum-free medium).

[0286] The produced constructs were then purified from the cell-free supernatant using immobilized metal ion affinity chromatography (IMAC), or protein A capture, or anti-HSA capture chromatography.

[0287] When his-tagged proteins were captured by IMAC, the filtered conditioned medium was mixed with IMAC resin (GE Healthcare) equilibrated with 1% Triton and 20 mM NaPO4, 0.5 Mn NaCl, 20 mM imidazole, pH 7.0. The resin was washed three times with 15 column volumes of 20 mM NaPO4, 0.5 Mn NaCl, 20 mM imidazole, pH 7.0 before the proteins were eluted with 10 column volumes of elution buffer (20 mM NaPO4, 0.5 Mn NaCl, 500 mM imidazole, pH 7.0).

[0288] If proteins were captured by Protein A or anti-HSA chromatography, the filtered conditioned media was mixed with Protein A resin (CaptivA PriMab™, Repligen) or anti-HSA resin (Capture Select Human Albumin affinity matrix, Thermo) and equilibrated with PBS, pH 7.4. The resin was washed three times with 15 column volumes of PBS, pH 7.4, before proteins were eluted with 10 column volumes of elution buffer (50 mM citrate, 90 mM NaCl, pH 2.5) and the pH was neutralized using 1 M TRIS pH 10.0.

[0289] Finally, the eluted fractions were polished using size exclusion chromatography (HiPrep Superdex 200, 16 / 60, GE Healthcare Life Sciences) and analyzed by SDS-PAGE against Precision Plus Protein Unstained Standards markers (Biorad, ref#161-0363).

[0290] Representative expression gels of the fusion proteins are shown in Figure 2. Figure 2A: EGF-HSA-C1-C2 protein (FP330; SEQ ID NO: 42); Figure 2B: EGF-HSA-C1-C2 of EDIL3 protein (FP050; SEQ ID NO: 12); Figure 2C: Non-reduced and reduced EGF-Fc(KiH)C1-C2 protein. The protein is a heterodimer of FP071 (EGF-Fc(knob)-C1-C2; SEQ ID NO: 18) and Fc-IgG1 hole (SEQ ID NO: 10); Figure 2D: EGF-HSA-C1 protein (FP260; SEQ ID NO: 34). The protein under reducing and non-reducing conditions is shown in Figure 2C. Both conditions were tested, since the heterodimer tends to collapse under reducing conditions. Expression and yield results after purification of a further set of fusion proteins are shown in Table 6. As can be seen from the expression data, the HSA fusions of MFG-E8 show at least a 100-fold improvement in expression over wild-type MFG-E8, even when the HSA is in a different position. As shown in the right column of Table 6, the HSA fusions of MFG-E8 also show at least a 100-fold increase in yield over wild-type MFG-E8.

[0291] [Table 58]

[0292] Other examples of therapeutic fusion proteins of the present disclosure were produced according to the above method and further analyzed by SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis), where the proteins were separated based on their molecular weight. Each protein was mixed with Laemmli buffer before loading onto a polyacrylamide gel (Biorad, 4-20% Mini-PROTEAN TGX stain-free). After running in Tris-glycine-SDS running buffer at 200V for 30 minutes, the proteins contained in the gel were revealed with a stain-free imager (Biorad, Gel Doc EZ). As illustrated in Figure 2E, SDS-PAGE shows the produced and purified recombinant proteins. Rows 1, 12: Molecular weight markers (Biorad, Precision plus protein) Column 2: His6_EGF[MFG-E8]_C1[MFG-E8] 23.87kDa Column 3: EGF[MFG-E8]_C1[MFG-E8]_His6 SEQ ID NO: 115 23.87 kDa Column 4: EGF[MFG-E8]_HSA_C1[MFG-E8] SEQ ID NO: 117 90.38 kDa Column 5: EGF[MFG-E8]_HSA_C1[MFG-E8] SEQ ID NO: 74 89.27 kDa Column 6: EGF[MFG-E8]_HSA_C1[MFG-E8] SEQ ID NO: 73 88.72 kDa Column 7: EGF[EDIL3]_HSA_C1[EDIL3] SEQ ID NO: 71 98.22 kDa Column 8: EGF[EDIL3]_HSA_C2[EDIL3] SEQ ID NO: 135 98.20 kDa Column 9: EGF[MFG-E8]_HSA_C2[MFG-E8] SEQ ID NO: 137 88.45 kDa Column 10: EGF[EDIL3]_HSA_C1_C2[MFG-E8] SEQ ID NO: 80 115.67 kDa Column 11: EGF[MFG-E8]_HSA_C1_C2[EDIL3] SEQ ID NO:82 107.32 kDa

[0293] Example 3: Characterization of MFG-E8-HSA modified proteins 3.1 Phosphatidylserine binding (biochemistry) L-α-phosphatidylserine (brain, porcine, Avanti 840032, Alabama, US) was dissolved in chloroform, diluted with methanol, and coated onto 384-well microtiter plates (Corning™ 3653, Kennebunk ME, US) at 1 μg / mL. After overnight incubation at 4° C., the solvent was evaporated using a SpeedVac™ system (Thermo Scientific™). The plates were treated with phosphate-buffered saline (PBS) containing 3% fatty acid-free bovine serum albumin (BSA) for 1.5 hours at room temperature.

[0294] Binding of the fusion proteins to L-α-phosphatidylserine was assessed by competing with the binding of biotinylated mouse MFG-E8 / lactadherin (produced in-house, mMFG-E8:biotin). Proteins were diluted in PBS containing 3% fatty acid-free BSA, pH 7.4, and incubated for 30 min on L-α-phosphatidylserine-coated microtiter plates. mMFG-E8:biotin in PBS containing 3% fatty acid-free BSA, pH 7.4, was added at 1 nM and incubated for an additional 30 min. Unbound mMFG-E8:biotin was removed by three washing steps using Dissociation Enhanced Lanthanide Fluorescence Immunoassay (DELFIA™) Wash Buffer (Perkin Elmer 1244-114 MA, US). Europium-labeled streptavidin (Perkin Elmer 1244-360, Wallac Oy, Finland) was added in DELFIA™ assay buffer (Perkin Elmer 1244-111 MA, US) for 20 min at room temperature. This was followed by three washes with DELFIA™ assay buffer. Europium was revealed according to the manufacturer's instructions (Perkin Elmer 1244-105, Boston MA, US). Europium time-resolved fluorescence was quantified with an Envision™ 2103 multilabel plate reader, Perkin Elmer, CT, US). Data analysis was performed using MS Excel and GraphPad Prism software.

[0295] Polypropylene plates are low protein binding microtiter plates and are typically used in laboratories for serial dilutions. Compared to polystyrene, these plates have the advantage of reducing protein loss during dilution and are typically classified as "low protein binding" plates. When dilutions of wild-type MFG-E8 were performed in polypropylene plates, wild-type MFG-E8 lost potency in the L-α-phosphatidylserine competition assay compared to dilutions performed in non-binding plates. These data show that wild-type MFG-E8 is partially lost during liquid handling and dilution steps when using polypropylene plates already optimized for low protein binding, as shown in Figure 3 (Figure 3A). These results indicate that the inherent stickiness of wild-type MFG-E8 poses challenges for handling in the laboratory and possibly during drug manufacturing and production, where capture and polishing steps are required to produce high yield and very high purity drug substance. In contrast, the adhesion of the modified protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) was significantly reduced compared to wild-type MFG-E8, and no substantial differences were observed between dilutions performed in non-binding and polypropylene plates (Figure 3B). These data suggest that the insertion of solubilization domains into the proteins of the present disclosure can improve their technical handling, improving step yields and therefore the overall yield during the manufacturing process.

[0296] The evaluation of the binding of fusion proteins to L-α-phosphatidylserine is shown in Figure 4. The modified MFG-E8-derived protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) bound to immobilized PS and to a lesser extent to the phospholipid cardiolipin in a concentration-dependent manner (Figure 4A). The binding of FP278 to immobilized L-α-phosphatidylserine or to cardiolipin (1,3-bis(sn-3'-phosphatidyl)-sn-glycerol) was detected using an antibody against the EGF-L domain of wild-type MFG-E8. The binding intensity of several recombinant fusion proteins to immobilized L-α-phosphatidylserine is shown in Figure 4B. Human wild-type MFG-E8 and the fusion proteins FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) and FP260 (EGF-HSA-C1; SEQ ID NO: 34) efficiently competed with the concentration-dependent binding of 1 nM biotinylated mouse MFG-E8 to immobilized L-α-phosphatidylserine. ICs obtained for the fusion proteins 50The values ​​show that the L-α-phosphatidylserine binding strength of the C1-C2 domain of the modified protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) is very similar compared to human wild type MFG-E8. Surprisingly, these data also suggest that the human C2 domain does not interact or only weakly interacts with L-α-phosphatidylserine, as shown by the results of FP270 (EGF-HSA-C2; SEQ ID NO: 36), which did not compete in this assay format, together with FP250 (EGF-HSA; SEQ ID NO: 32). The EGF-C2-C2 protein, FP100 (SEQ ID NO: 26), was tested and did not compete in this assay format (not shown), leaving the C1 domain as the major PS-binding moiety of human MFG-E8. This finding was surprising, since the primary literature suggests that the C2 domain of MFG-E8 is the major domain involved in PS binding (Andersen et al., (2000) Biochemistry, 39(20):6200-6; Shi & Gilbert (2003) Blood, 101:2628-2636; Shao et al., (2008) J Biol Chem., 283(11):7230-41). In conclusion, these findings indicate that the C1 domain is the major integral PS-binding domain of the MFG-E8 engineered protein and is important for PS-binding-dependent functions. Therefore, the C1 domain is useful for substitution into heterologous proteins to confer PS binding. However, the highest PS binding was shown for fusion proteins containing C1-C2 or C1-C1 tandem domains (the latter not shown).

[0297] 3.2 αv integrin adhesion assay Fusion proteins were diluted in phosphate-buffered saline (PBS) pH 7.4 and 50 μL of a 24 nM solution was immobilized overnight by adsorption (96-well plates, Nunc Maxisorb) (1.2 nM / well). Plates were subsequently treated with PBS containing 3% fatty acid-free bovine serum albumin (BSA) for 1.5 h at room temperature. αvβ3 integrin-expressing lymphoma cells (ATCC-TIB-48 BW5147.G.1.4, ATCC, US) were cultured in RPMI1640 supplemented with GlutaMax, 25 mM HEPES, 10% FBS, Pen / Strep, 1 mM sodium pyruvate, 50 μM β-mercaptoethanol. Cells were split the day before adhesion experiments. Cells were labeled with 3 μg / mL 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF AM) (Thermo Fisher Scientific Inc, US) for 30 min. BW5147.G.1.4 cells were resuspended in adhesion buffer (TBS, 0.5% BSA, 1 mM MnCl2, pH 7.4) and 50000 cells / well were allowed to adhere for 40 min at room temperature. Non-adherent cells were removed by repeated washing with adhesion buffer. Fluorescence of adherent cells was quantified using an Envision™ 2103 multilabel plate reader, Perkin Elmer, US. Data analysis was performed using MS Excel and GraphPad Prism software.

[0298] Cell adhesion to the immobilized fusion protein FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42) was completely blocked by the αv integrin inhibitor Cilengitide or 10 mM EDTA, indicating integrin-dependent cell adhesion to the immobilized engineered protein (Figure 5A). Single point mutation of the integrin-binding motif RGD (RGD>RGE) of the EGF-like domain (FP280; SEQ ID NO: 38) resulted in complete inhibition of cell adhesion, indicating that a functional and accessible RGD-binding motif of the fusion protein is essential for αv integrin-dependent adhesion (Figure 5B). FP250 (SEQ ID NO: 32), an immobilized EGF-HSA protein lacking the C1-C2 domain, did not or only weakly supported adhesion of BW5147.G.1.4 cells, despite the EGF-like domain (Figure 5C). This finding suggests that under the experimental conditions tested, the RGD loop of the EGF-like domain fused to HSA may not be sufficiently accessible to cell surface integrins, possibly due to steric reasons. This impairment was not evident when C1, C2, or C1-C2 were fused to EGF-HSA at the C-terminus.Recombinant proteins of the present disclosure, such as FP330, promote αv-integrin-dependent cell adhesion similar to wild-type MFG-E8 when expressed in CHO or HEK cells (FIG. 5D).

[0299] Taken together, these data indicate that the fusion proteins of the present disclosure bind to cellular integrins and support integrin-dependent cell adhesion, and that in proteins with HSA domain inserts, the C-terminal EGF-like domain may derive functional benefit from the C-terminal fusion protein domain to support integrin binding.

[0300] 3.3 Human macrophage-neutrophil efferocytosis assay Human peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats by Ficoll gradient centrifugation (Ficoll®-Paque PLUS, GE Healthcare, Sweden) followed by negative selection of monocytes using a Stemcell isolation kit (Stemcell 19059, Vancouver, Canada). Monocytes were differentiated into "M0" macrophages using recombinant human M-CSF 40ng / mL (Macrophage Colony Stimulating Factor, R&D Systems, US) in RPMI1640 containing 25mM HEPES, 10% FBS, Pen / Strep, 1mM NaPyr, 50μM β-Merc for 5 days. One day prior to efferocytosis, macrophages were labeled with PKH26 using the Red Fluorescent Dye Linker kit (Sigma MINI26, US). Cells were resuspended in RPMI1640 containing 25 mM HEPES, 10% FBS, Pen / Strep, 1 mM NaPyr, 50 μM β-Merc, plated at 40,000 cells / well in black 96-well plates (Corning, US) and allowed to adhere for 20 hours.

[0301] Neutrophils: Human neutrophils were isolated from the buffy coat by dextran sedimentation combined with a Ficoll™ density gradient as follows: Plasma in the buffy coat was removed by centrifugation of the diluted buffy coat. The cell harvest was diluted with 1% dextran (from Leuconostoc spp., MW 450.000-650.000; Sigma, US) and allowed to sediment for 20-30 min on ice.

[0302] Leukocytes were collected from the supernatant and placed on a Ficoll™-Paque layer (GE Healthcare Sweden). After centrifugation, the pellet was collected and the remaining red blood cells were lysed using red blood cell (RBC) lysis buffer (BioConcept, Switzerland). Neutrophils were washed once with culture medium (RPMI1640+GlutaMax containing 25 mM HEPES, 10% FBS, Pen / Strep, 0.1 mM NaPyr, 50 uM b-Merc) and stored overnight at 15°C. Apoptosis / cell death was induced by treating neutrophils with 1 μg / mL Superfas Ligand (Enzo Life Sciences, Lausanne, Switzerland) for 3 hours at 37°C. Neutrophils were stained with Hoechst 33342 (Life Technologies, US) for 25 minutes and then with DRAQ5 (eBioscience, UK, 1:2000 dilution) for 5 minutes at 37°C in the dark.

[0303] Efferocytosis assay M0 macrophages were incubated with the fusion proteins for 30 min. Apoptotic labeled neutrophils were added at a ratio of 1:4 M0 / neutrophils. Efferocytosis of apoptotic neutrophils by macrophages was visualized using the increase in DRAQ5 fluorescence intensity upon localization of neutrophils to the low pH lysosomal compartment of M0 macrophages.

[0304] Efferocytosis was quantified using an ImageXpress Micro XLS widefield high content analysis system (Molecular DEVICES, CA, US). Macrophages were identified via PKH26 fluorescence. The efferocytosis index (EI, expressed as %) was calculated as the ratio of macrophages containing at least one ingested apoptotic neutrophil (DRAQ5high) event to the total number of macrophages. Data analysis was performed using MS Excel and GraphPad Prism software.

[0305] The effect of the fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) on promoting efferocytosis of killed neutrophils by human macrophages is shown in FIG. 6. The fusion protein increases the internalization of pHrodo-labeled killed human neutrophils into macrophages, above the already high efferocytosis capacity of M0 macrophages, shown as basal levels. In FIG. 7, it is shown that the recombinant fusion protein FP278 can rescue endotoxin (lipopolysaccharide)-impaired efferocytosis of killed neutrophils by human macrophages. FIG. 7A shows the impairment of macrophage efferocytosis of killed human neutrophils by 100 pg / ml lipopolysaccharide (LPS) in three human donors. The left panel shows the response of individual donors, and the right panel shows the average impairment (%) of efferocytosis of the three donors. FIG. 7B shows the rescue of this endotoxin (LPS)-induced efferocytosis of killed neutrophils by human macrophages using the fusion protein FP278.

[0306] Rescue of S. aureus particle-impaired efferocytosis of killed neutrophils by human macrophages with fusion protein FP330 is shown in Figure 8. Figure 8A shows the effect of a concentration of 100 nM fusion protein on promoting efferocytosis compared to basal levels (dotted line; left side of figure) and the effect of 100 nM fusion protein in rescuing the impairment of efferocytosis caused by the addition of S. aureus (right side of figure). Figure 8B shows the effect of the fusion protein FP278 (EC278) on rescuing the impairment of efferocytosis caused by the addition of S. aureus and promoting efferocytosis after the basal level of efferocytosis has been reached. 50 The effect of increasing concentrations of β-lactam (from 0.01 to 0.8 nM) is shown.

[0307] 3.4 Human endothelial-Jurkat efferocytosis assay cell culture Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Basel, Switzerland). Cells were cultured in gelatin-coated flasks (from bovine skin, 0.2% final concentration in PBS, dilution of 2% stock solution, Sigma, Germany). Cells were cultured in medium 199 (Thermo Fischer Scientific, US) supplemented with 10% FBS (GE Healthcare, UK), 1% Pen / Strep (Thermo Fischer Scientific, US), 1% Glutamax (Thermo Fischer Scientific, US) and 1 ng / mL recombinant fibroblast growth factor-basic (Peprotech, UK). Accutase™ (Thermo Fischer Scientific, US) was used to dissociate cells for harvesting or passage.

[0308] Jurkat E6-1 cells were obtained from ATCC (American Type Culture Collection, US) and grown in medium RPMI1640 (Thermo Fischer Scientific, US) supplemented with 10% FBS (GE Healthcare, UK), 1% Pen / Strep (Thermo Fischer Scientific, US), 10 mM sodium pyruvate (Thermo Fischer Scientific, US) and 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, Thermo Fischer Scientific, US).

[0309] Apoptosis of Jurkat E6-1 cells was induced using recombinant human TRAIL (R&D Systems, US). Apoptotic cells were labeled with pHrodo™ Green STP ester dye (Thermo Fischer Scientific, US). Flow cytometry buffer was prepared in PBS (Thermo Fischer Scientific, US) supplemented with 1% FBS (GE Healthcare, UK), 0.05% w / v sodium azide (Merck, Germany), and 0.5 mM EDTA (ethylenediaminetetraacetic acid, Thermo Fischer Scientific, US).

[0310] Efferocytosis assay On day 1, HUVECs (70-90% confluence) were harvested by detachment with Accutase™ for 5 min, washed with PBS, and resuspended in cell culture medium. Cell number and viability were assessed using a Guava EasyCyte flow cytometer (Merck, Germany) and Guava ViaCount reagent (Merck, Germany) according to the manufacturer's instructions. The required amount of cells was centrifuged at 300 x g for 5 min at room temperature, resuspended in culture medium, and harvested at 6.6 × 10 4 The cell count was given as cells / mL. 150 μL / well of this cell suspension was added to a 96-well tissue culture plate (Corning™, US). HUVECs were incubated for an additional 16–20 h in a 37°C / 5% CO2 / 95% humidity incubator.

[0311] Jurkat E6-1 cell number and viability / cell death status were assessed using a Guava EasyCyte flow cytometer (Merck, Germany) and Guava ViaCount reagent (Merck, Germany) according to the manufacturer's instructions. The required amount of cells was centrifuged at 300 x g for 5 min at room temperature and then diluted with 1 × 10 cells in medium supplemented with recombinant human TRAIL at a final concentration of 50 ng / mL. 6 The cells were resuspended at a density of 1000 cells / mL. Cell death was induced overnight at 37°C / 5% CO2 / 95% humidity.

[0312] On day 2, the medium was removed from the HUVECs by aspiration and 25 μL of fresh pre-warmed (37° C.) medium was added, followed by 25 μL of fusion protein or control diluted in pre-warmed (37° C.) medium. Non-binding surface (NBS) treated 96-well plates (Corning™, US) were used for dilutions. Fusion proteins were allowed to interact with HUVECs for 30 min at 37° C. / 5% CO2 / 95% humidity before adding killed Jurkat cells.

[0313] The number of apoptotic / dead Jurkat E6-1 cells was counted using a Guava EasyCyte flow cytometer (Merck, Germany) and Guava ViaCount reagent (Merck, Germany). The required amount of apoptotic cells was centrifuged at 400 x g for 5 min at room temperature and 5 × 10 6 Cells were resuspended at a density of 1000000 cells / mL in RPMI 1640 medium (without FBS) supplemented with pHrodo™ Green STP Ester dye at a final concentration of 5 μg / mL (staining medium). After staining for 10 min at 37°C, remaining reactive pHrodo™ Green STP Ester was inactivated with staining medium supplemented with 10% FBS for an additional 5 min at 37°C. pHrodo™ Green-labeled cells were washed once and re-quantified to 3 x 10 cells in HUVEC medium. 6 Adjusted to 1.5 x 10 cells / mL. 61000 / well pHrodo™ Green-labeled Jurkat cells were added to HUVECs and incubated for 5 h at 37°C / 5% CO2 / 95% humidity. The medium was removed and HUVECs were washed once with PBS and detached with 40 μL / well Accutase™ solution. Cells were harvested by adding 80 μL ice-cold flow cytometry buffer, transferred to a 1.5 mL polypropylene 96-well block, washed with excess ice-cold flow cytometry buffer, and centrifuged at 400xg (4°C) for 5 min. The supernatant was removed by aspiration and the pellet was resuspended in 80 μL ice-cold flow cytometry buffer and transferred to a 96-well V-bottom microtiter plate (BD Biosciences, US). Samples were then measured on a BD LSRFortessa™ flow cytometer (BD Biosciences, US). pHrodo™ Green fluorescence intensity as an indicator of lysosomal localization of phagocytosed Jurkat cells was recorded. Flow cytometry data analysis was performed using FlowJo™ software. The median fluorescence intensity (MFI) values ​​of the pHrodo™ Green signal from singlet-gated HUVECs were used as the readout. Data analysis was performed using EC 50 Calculations were performed using MS Excel and GraphPad Prism software.

[0314] The effect of the fusion proteins FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) and FP270 (EGF-HSA-C2; SEQ ID NO: 36) on promoting efferocytosis of dead Jurkat cells by HUVEC endothelial cells is shown in FIG. 9. The internalization of pHrodo-labeled dead human Jurkat T cells by HUVEC is strongly promoted by the fusion protein FP278. The results show that endothelial cells are armed with the fusion protein to become efficient phagocytes of dead cells. Surprisingly, the effectiveness of the fusion proteins in this assay is clearly dependent on the presence of the C1-C2 or C1-C1 tandem domains. For example, the fusion protein consisting of EGF-HSA-C2 (FP270) is inactive in this experimental setting, as shown in FIG. 9. 10 shows our highly surprising finding that the location of the HSA domain in the modified protein, i.e., at the N- or C-terminus (HSA-EGF-C1-C2 (FP220; SEQ ID NO: 30) or EGF-C1-C2-HSA (FP110; SEQ ID NO: 28), respectively, confers efferocytosis-blocking ability to the MFG-E8 HSA modified protein in a macrophage efferocytosis assay. These data clearly demonstrate the importance of placing the HSA domain between the integrin-binding domain and the PS-binding domain for efficient promotion of efferocytosis by the fusion proteins of the present disclosure.

[0315] Figure 11 shows a comparison of the promotion of endothelial efferocytosis by various formats of fusion proteins containing combinations of EGF domain, C1-C2 domain, HSA or Fc domain. Figure 11A shows a comparison of fusion proteins containing HSA, where HSA is located at the C-terminus, or N-terminus, or between the EGF-like domain and the C1-C2 domain; EGF-C1-C2-HSA (FP110; SEQ ID NO:28), HSA-EGF-C1-C2 (FP220; SEQ ID NO:30) and EGF-HSA-C1-C2-His tag (FP278; SEQ ID NO:44), respectively. Figure 11B shows a comparison of fusion proteins containing the Fc domain with Fc located at the C-terminus or between the EGF-like domain and the C1 domain. Two formats of Fc moieties are shown: wild-type Fc (SEQ ID NO: 7) found in FP070 (EGF-Fc-C1-C2; SEQ ID NO: 17) and FP080 (EGF-C1-C2-Fc; SEQ ID NO: 22) and an Fc moiety with KiH modifications S354C and T366W in one arm of the Fc (FP060; EGF-C1-C2-Fc [S354C, T366W]; SEQ ID NO: 14) EU numbering (Merchant et al (1998) supra). Figure 11C shows a comparison of three batches of FP090 (Fc-EGF-C1-C2; SEQ ID NO: 24), a fusion protein containing an N-terminally placed Fc moiety, at three different concentrations (0.72, 7.2 and 72 nM), against the wild-type MFG-E8 control. Efferocytosis of killed Jurkat cells by HUVEC was promoted only by engineered proteins in which the EGF-like domain was followed by HSA or Fc moieties. Figure 11D shows that insertion of a solubilization domain can result in a novel bioactive fusion protein based on the endogenous cross-linking protein EDIL3, a paralog of MFG-E8. As shown in Figure 11D, HSA was inserted between the EGF-like domain and the C1-C2 domain of EDIL3, a paralog of MFG-E8. This EDIL3 construct (FP050 (EDIL3-based EGF-HSA-C1-C2; SEQ ID NO: 12)) has only one of the three EGF-like domains (including the RGD loop) found in wild-type EDIL3.Surprisingly, we found a similar tolerance of HSA domain insertions in this construct for expression of novel recombinant engineered proteins of very high purity (Figure 2B).Moreover, surprisingly, we found that the recombinant engineered protein FP050 derived from EDIL3 promoted efferocytosis of dead Jurkat cells by endothelial cells (HUVECS), indicating the core function of the cross-linking protein and demonstrating that the cross-linking protein domain is useful for designing functional novel recombinant engineered proteins.

[0316] Example 4: Efferocytosis of prothrombotic plasma microparticles 4.1 Human endothelial microparticle efferocytosis assay cell culture HUVEC cells were obtained from Lonza (Basel, Switzerland). Cells were cultured in gelatin-coated flasks (from bovine skin, 0.2% final concentration in PBS, dilution of 2% stock solution, Sigma Aldrich / Merck, Germany). Cells were cultured in medium 199 (Thermo Fischer Scientific, US) supplemented with 10% FBS (GE Healthcare, UK), 1% Pen / Strep (Thermo Fischer Scientific, US), 1% Glutamax (Thermo Fischer Scientific, US) and 1 ng / mL recombinant fibroblast growth factor-basic (Peprotech, UK). Accutase™ (Thermo Fischer Scientific, US) was used to dissociate cells for harvesting or passaging.

[0317] Platelet-derived microparticles were prepared according to the following procedure: After written informed consent was granted, citrated venous blood was collected from healthy adult volunteers (Coagulation 9NC Citrate Monovette, Sarstedt, Germany). Platelet-rich plasma (PRP) was prepared by centrifugation (200xg, 15 min, no brake, room temperature). Platelet-derived microparticles / debris were generated by subjecting PRP to three flash-freeze / freeze cycles using liquid nitrogen and thawing at 37°C. Platelet fragments / microparticles were pelleted by centrifugation at 20,000xg, room temperature, for 15 min. The pellet was resuspended in PBS and aliquots were prepared and stored at -80°C. Microparticle preparations were 85-100% PS positive as measured by flow cytometry using AlexaFluor™ 488-labeled mouse MFG-E8 / lactadherin (in-house at Novartis). The number of microparticles was determined using dedicated counting beads (BioCytex / Stago, France). Flow cytometry buffer was prepared in PBS (Thermo Fischer Scientific, US) supplemented with 1% FBS (GE Healthcare, UK), 0.05% w / v sodium azide (Merck, Germany), and 0.5 mM EDTA (ethylenediaminetetraacetic acid, Thermo Fischer Scientific, US).

[0318] 4.2 Efferocytosis assay On day 1, HUVEC cells (70-90% confluence) were harvested by detachment with Accutase™ for 5 min, washed with PBS, and resuspended in cell culture medium. Cell number and viability were assessed using a Guava EasyCyte flow cytometer (Merck, Germany) and Guava ViaCount reagent (Merck, Germany) according to the manufacturer's instructions. The required amount of cells was centrifuged at 300 x g for 5 min at room temperature and resuspended in culture medium to obtain a total of 6.6 × 10 4The cell count was given as cells / mL. 150 μL / well of this cell suspension was added to a 96-well tissue culture plate (Corning™, US). HUVEC cells were incubated for an additional 16–20 h in a 37°C / 5% CO2 / 95% humidity incubator.

[0319] On day 2, the medium was removed from the HUVEC cells by aspiration and 25 μL of fresh pre-warmed (37° C.) medium was added, followed by the addition of 25 μL of fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) or control at three different concentrations of 0.3 nM, 3 nM, or 30 nM diluted in pre-warmed (37° C.) medium. Non-binding surface (NBS) treated 96-well plates (Corning™, US) were used for the dilutions. The test proteins were allowed to interact with the HUVEC cells for 30 min at 37° C. / 5% CO2 / 95% humidity before the addition of platelet-derived microparticles.

[0320] Centrifuge the required amount of microparticles at 20,000 x g for 15 min at 4 °C to remove 2 × 10 8 The particles were resuspended in RPMI 1640 medium (without FBS) supplemented with pHrodo™ Green STP Ester dye at a final concentration of 5 μg / mL (staining medium) at a density of 1000 particles / mL. After staining for 10 min at 37°C, remaining reactive pHrodo™ Green STP Ester was inactivated with staining medium supplemented with 10% FBS for an additional 5 min at 37°C. The pHrodo™ Green-labeled microparticles were washed once by centrifugation at 20,000 x g for 15 min at 4°C and diluted to 1 x 10 in HUVEC cell medium. 8 Adjusted to 5×10 particles / mL 6particles / well of pHrodo™ Green-labeled microparticles were added to HUVEC cells and incubated for 5 h at 37° C. / 5% CO2 / 95% humidity. The medium was removed and HUVEC cells were washed once with PBS and detached with 40 μL / well of Accutase™ solution. Cells were harvested by adding 80 μL of ice-cold flow cytometry buffer, transferred to a 1.5 mL polypropylene 96-well block, washed with excess ice-cold flow cytometry buffer, and centrifuged at 400×g (4° C.) for 5 min. The supernatant was removed by aspiration and the pellet was resuspended in 80 μL of ice-cold flow cytometry buffer and transferred to a 96-well V-bottom microtiter plate (BD Biosciences, US). Samples were measured on a BD LSRFortessa™ flow cytometer (BD Biosciences, US). pHrodo™ Green fluorescence intensity was recorded as an indicator of lysosomal localization of phagocytosed microparticles. Flow cytometry data analysis was performed using FlowJo™ software. The median fluorescence intensity value (MFI) of the pHrodo™ Green signal from singlet-gated HUVEC cells was used as the readout. Data analysis was performed using EC 50 Calculations were performed using MS Excel and GraphPad Prism software. Fusion protein FP278 promoted efferocytosis of platelet-derived microparticles by endothelial cells in a concentration-dependent manner, as shown in Figure 12. The promotion of uptake was concentration-dependent and was also observed in other types of endothelial cells (data not shown).

[0321] Example 5: Technical characteristics of the MFG-E8-HSA fusion protein 5.1 Surface Plasmon Resonance (SPR) Binding Analysis of Fusion Protein FP330 to FcRn To characterize the binding of the fusion protein FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42) to FcRn, a direct binding assay was performed. Kinetic binding affinity constants (KD) were measured on captured proteins using recombinant human FcRn as analyte. Measurements were performed on a BIAcore® T200 (GE Healthcare, Glattbrugg, Switzerland) at room temperature and pH 5.8 and 7.4, respectively. For affinity measurements, proteins were diluted in 10 mM NaP, 150 mM NaCl, 0.05% Tween 20, pH 5.8 and immobilized on flow cells of a CM5 research grade sensor chip (GE Healthcare, ref BR-1000-14) using standard procedures according to the manufacturer's recommendations (GE Healthcare). One flow cell was immobilized with a blank to be used as a reference. Binding data were then acquired by injecting a series of analyte dilutions into the reference and measurement flow cells. A zero concentration sample (running buffer only) was included to allow double referencing during data evaluation. Double-referenced sensorgrams were used for data evaluation and dissociation constants (KD) were analyzed.

[0322] The fusion protein FP330 binds to FcRn at pH 5.8 with an affinity of 1380 nM, whereas no binding was observed at pH 7.4 (see Table 5 above). These results are in good agreement with wild-type HSA (1000-2000 nM, pH 5.8, data not shown).

[0323] 5.2 Differential scanning calorimetry (DSC) of MFG-E8 and its mutants Differential scanning calorimetry was used to measure the thermal stability of the modified MFG-E8 protein variant FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44). Measurements were performed in a differential scanning microcalorimeter (Nano DSC, TA instruments). The cell volume was 0.5 ml and the heating rate was 1°C / min. The protein was used at a concentration of 1 mg / ml in PBS (pH 7.4). The molar heat capacity of the protein was estimated by comparison with a replicate sample containing the same buffer in which the protein was omitted. The partial molar heat capacity and melting curves were analyzed using standard procedures. Thermograms were baseline corrected and concentration normalized. Two melting events were observed, the first with a Tm of 50°C and the second with a Tm of 64°C.

[0324] 5.3 Determination of aggregation tendency and solubility of MFG-E8 mutants First, the aggregation tendency of the MFG-E8 mutant protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) was measured by dynamic light scattering (DLS, Wyatt). Dynamic light scattering was applied to measure the translational diffusion coefficient of FP278 in solution by quantifying the dynamic fluctuation of scattered light. The protein variant size distribution without fractionation, the estimate of polydispersity, and the hydrodynamic radius were measured at a concentration of 1 mg / ml. The hydrodynamic radius of the fusion protein FP278 was measured using a DynaPro™ plate reader (Wyatt Technology Europe GmbH, Dernbach, Germany) in combination with the software DYNAMICS (version 7.1.0.25, Wyatt). 50 μL of undiluted filtered (0.22 μm PVDF-Filter (Millex® Syringe-Driven Filter Unit, Millipore, Billerica, US)) protein solution was measured in a 384-well plate (384 round well plate, polystyrene, Thermo Scientific, Langenselbold, Germany). No high molecular weight aggregates of the protein samples could be identified. The hydrodynamic radius of the protein was approximately 5-6 nm, indicating a monomeric protein in solution.

[0325] Next, concentration-dependent hydrodynamic radius measurements of the fusion protein FP278 were performed to estimate the solubility of the protein. Protein concentrations up to 22 mg / ml were applied. The hydrodynamic radius was determined as described above. With increasing concentrations of the fusion protein FP278, no increase in radius (5-7 nm) could be observed, and dynamic light scattering measurements of wild-type MFG-E8 (SEQ ID NO: 1) failed due to high aggregation at concentrations of approximately 0.2 mg / ml.

[0326] Example 6: Optimization of MFG-E8 fusion protein To generate a panel of mutant MFG-E8-based fusion proteins optimized for improved expression and yield, the fusion protein FP330 (EGF-HSA-C1-C2) was investigated using mass spectrometry (MS). The panel of mutant proteins was generated with linkers of various sizes and structures, for example, a linker containing GS between the EGF and HSA domains, and / or a linker containing multiple GS or G4S between the HSA and C1 domains (SEQ ID NO: 64). In addition, some mutants contained amino acid modifications including deletions or substitutions (shown as HSA* in Table 7). The panel of mutant fusion proteins is summarized in Table 7 below.

[0327] [Table 59]

[0328] Example 7: Mutant MFG-E8 fusion proteins; expression and purification The method for producing fusion proteins in HEK cell lines is described in Example 2. For expression in CHO cell lines, nucleic acids encoding MFG-E8 variants were synthesized at Geneart (LifeTechnologies) and cloned into mammalian expression vectors using a restriction enzyme ligation-based cloning technique. The resulting plasmids were transfected into CHO-S cells (Thermo). Briefly, for transient expression of fusion proteins, the expression vectors were transfected into suspension-adapted CHO-S cells using ExpifectamineCHO transfection agent (Thermo). Typically, 400 ml of cells in suspension at a density of 6 Mio cells per ml were transfected with DNA containing 400 μg of expression vector encoding the modified protein. The recombinant expression vectors were then introduced into the host cells and secreted for an additional 7 days in culture medium (ExpiCHO expression medium supplemented with ExpiCHO feed and enhancer reagent (Thermo)).

[0329] As can be seen from the expression data shown in Table 8, mutant fusion proteins FP068 (SEQ ID NO: 46) and FP776 (SEQ ID NO: 48) showed approximately a 2-fold improvement in expression over fusion protein FP330 (SEQ ID NO: 42).

[0330] [Table 60]

[0331] Example 8: Properties of mutant fusion proteins The effect of the mutant fusion proteins on efferocytosis was determined by performing an efferocytosis assay as described in Example 3.

[0332] In the first assay, the effect of mutant fusion proteins in a human macrophage-neutrophil efferocytosis assay was determined according to the method described in section 3.3 above. M0 macrophages were incubated with fusion protein FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42) or mutant FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) or FP776 (EGF- HSA-C1-C2; SEQ ID NO: 48) for 30 min. As shown in Figure 13, fusion proteins FP330, FP278, and FP776 are able to rescue endotoxin (lipopolysaccharide (LPS))-induced efferocytosis of killed neutrophils by human macrophages. Fusion protein FP330 (EC 50 = 1.6 nM; Figure 13A), FP278 (EC 50 = 1.78 nM; Figure 13B) and FP776 (EC 50 = 0.5 nM; Figure 13C ), led to rescue of the efferocytosis impairment caused by LPS addition and even promoted efferocytosis once basal levels were reached.

[0333] Fusion proteins FP330, FP278 and FP776 were further characterized in a human endothelial (HUVEC) cell-Jurkat cell efferocytosis assay, following the method described in section 3.4 above. The effect of fusion proteins FP330, FP278 and FP776 on promoting efferocytosis of killed Jurkat cells by HUVEC endothelial cells is shown in Figure 14. Internalization of pHrodo-labeled killed human JurkatT cells by HUVEC was enhanced by FP330 (EC 50 = 3.4 nM; Figure 14A), FP278 (EC 50 = 2.4 nM; Figure 14B) and FP776 (EC 50 The effect of endothelial cells on the expression of the fusion protein was strongly promoted by increasing concentrations of β-lactamase (β-lactamase = 3 nM; Fig. 14C). These results indicate that endothelial cells are armed with the fusion protein to become efficient phagocytes of dead cells.

[0334] Example 9: Protection of mice from AKI and acute organ reactions caused by AKI 9.1 Acute kidney injury model Female C57BL / 6 mice (18–22 g) were purchased from Charles River (France) and housed in a temperature-controlled facility in cages protected by filter tops with a 12-h light / dark cycle. Animals were handled in strict compliance with Swiss Federal Law and the NIH Principles of Laboratory Animal Care. The therapeutic fusion protein under study was administered either intraperitoneally (ip) or intravenously (iv) 2 h before surgery. Buprenorphine (Indivior Schweiz AG) was applied subcutaneously (sc) at a dose of 0.1 mg / kg 60–30 min before surgery. Inhalation anesthesia with isoflurane was induced in an anesthesia chamber (3.5–5 vol.%, carrier gas: oxygen) for 5 min before surgery. During surgery, animals were maintained under anesthesia via face mask with 1–2 Vol.% isoflurane / oxygen, with a gas flow rate of 0.8–1.2 l / min. The abdominal skin was shaved and disinfected with Betaseptic (Mundipharma, France). The animals were placed on a homeothermic blanket (Rothacher-Switzerland) with a homeothermic monitoring system (PhysiTemp, US-Physitemp Instruments LLC, US) and covered with sterile gauze. Body temperature was monitored throughout the surgery by a rectal probe (Physitemp Instruments LLC, US) and controlled to maintain a temperature of 36.5-37.5 °C. All animals, including sham controls, underwent unilateral nephrectomy of the right kidney. After a midline incision / laparotomy, the abdominal contents were retracted to the left to expose the right kidney. The right ureter and renal vessels were separated and ligated, after which the right kidney was removed. For animals that experienced AKI, the abdominal contents were placed on the right side of the sterile gauze and the left renal artery and vein were incised to allow clamping for ischemia induction. A microaneurysm clamp (B Braun, Switzerland) was used to clamp the renal pedicle (artery and vein together using one clamp) to block blood flow to the kidney and induce renal ischemia. Successful ischemia was confirmed by a change in kidney color from red to deep purple, which occurred within seconds. After ischemia induction (35–38 min), the microaneurysm clamp was removed.Warm sterile saline (approximately 2 ml, 37°C) was used to wash the abdominal cavity contents to rehydrate the tissue before wound closure. After washing, an additional 1 ml of sterile saline was added into the abdominal cavity as replacement fluid. When reperfusion was initiated, the wound was closed in two layers (muscle and skin separately). The animals were then kept under a red warm lamp until they were fully recovered. Buprenorphine was administered again at a dose of 0.1 mg / kg 1 and 4 hours after surgery and was also included in the drinking water (9.091 μg / mL). After 24 hours, the animals were euthanized for analysis.

[0335] 9.2 Administration of Therapeutic Fusion Proteins The therapeutic fusion proteins FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42), FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) and FP776 (EGF-HSA-C1-C2; SEQ ID NO: 48) were tested in the AKI model as described above at the doses shown in Table 9 below. In studies to detect the expression of serum and qPCR markers, the fusion protein FP278 was administered 2 hours before surgery. FP330 and FP776 were administered intravenously 30 minutes before the onset of ischemia-reperfusion injury. In studies to measure the uptake of contrast agents by magnetic resonance imaging, the fusion protein FP776 was administered prophylactically at 1.26 mg / kg 30 minutes before the induction of AKI or therapeutically at 2 mg / kg iv 5 hours after the induction of ischemia-reperfusion injury.

[0336] [Table 61]

[0337] 9.3 Readout / Analysis for AKI Protection: Serum markers: Serum samples were collected 24 h after induction of ischemia-reperfusion and analyzed for serum creatinine and blood urea nitrogen (BUN) content using a Hitachi M40 clinical analyzer according to the manufacturer's instructions (Axonlab, Switzerland).

[0338] qPCR marker expression in organs: Organs (kidney, liver, lung and heart) were harvested 24 hours after AKI induction, cut into 1 cm pieces and stored overnight at 4°C in RNA Later Buffer (Thermo Fisher Scientific Inc, US). Organ pieces were transferred to RLT buffer (RNeasy Mini Kit, Qiagen, DE) containing 134 mM beta-mercaptoethanol (Merck, DE) in Lysing Matrix D tubes (MP Biomedicals FR) and homogenized using a FastPrep-24 Instrument (MP Biomedicals). Cardiac fibrous tissue was then digested with proteinase K (RNeasy Mini Kit) and kidney, liver and lung lysates were directly centrifuged at full speed for 3 min in a microcentrifuge (Eppendorf, DE). The supernatant was transferred to a QIAshredder spin column (Qiagen, DE) and centrifuged for 2 min. RNA extraction of the flow-through was performed according to the RNeasy Mini Kit Manual including DNase digestion. RNA concentration was measured by Nano Drop 1000 device (Thermo Fisher Scientific Inc). 2 μg of RNA per sample was reverse transcribed using a SimpliAmp Thermocycler (Applied Biosystems, US) according to the High-Capacity cDNA Reverse Transcription Kit Manual (Thermo Fisher Scientific Inc). cDNA was combined with Nuclease-free water (Thermo Fisher Scientific Inc), TaqMan probe (TaqMan Gene Expression Assay (FAM), Thermo Fisher Scientific Inc), and TaqMan Gene Expression Master Mix (Thermo Fisher Scientific Inc) in a 384-well microplate (MicroAmp Optical 384-Well Reaction Plate, Thermo Fisher Scientific Inc).qPCR was performed on a ViiA 7 real-time PCR system (Applied Biosystems, US). The settings were: 1: 2 min, 50°C; 2: 10 min, 95°C; 3: 15 sec, 95°C; 4: 1 min, 60°C. Steps 3 and 4 were repeated for 45 cycles. Data analysis was performed using ViiA7 software, and qPCR data analysis software was performed using MS Excel and GraphPad Prism software.

[0339] Uptake of contrast agents by the liver measured by magnetic resonance imaging (MRI) The method for performing MRI was adapted from the publication by Egger et al. (Egger et al., (2015) J Magn Reson Imaging, 41:829-840). Experiments were performed on a 7-T Bruker Biospec MRI system (Bruker Biospin, Ettlingen, Germany). During MRI signal acquisition, mice were placed in supine position in a Plexiglas cradle. Body temperature was maintained at 37 ± 1 °C using a heating pad. After a short induction, anesthesia was maintained with approximately 1.4% isoflurane in a mixture of O2 / N2O (1:2), administered via a nose cone. All measurements were performed in spontaneously breathing animals and no cardiac or respiratory triggers were applied.

[0340] After placing the mouse in the scanner, scout high-speed images were acquired for localization purposes. Perfusion analysis was performed using an intravascular agent containing superparamagnetic iron oxide (SPIO) nanoparticles (Endorem®, Guerbet, France). Endorem® was injected intravenously as a bolus for 1.2 seconds in animals with AKI (24 hours after disease induction) or after sham surgery (animals 24 hours after nephrectomy). The first bolus was administered for 1.2 seconds in conjunction with sequential acquisition of echo-planar images at a resolution of 400 ms / image. After acquisition of 25 baseline images, a second bolus was injected for 1.2 seconds and an additional 575 images were acquired after the bolus, resulting in a total of 600 images acquired in 4 minutes. The superparamagnetic contrast agent induced a regional change in susceptibility, resulting in signal attenuation proportional to renal perfusion. For the series of images, signal intensity was evaluated in regions of interest (ROIs) located in the cortex / external zone of the outer medulla. The location, shape, and size of the ROI were carefully selected to ensure that the ROI covered approximately the same area despite respiration-induced kidney movement. The average signal intensity of the pre-injection images provided the baseline intensity (S(0)). The perfusion index was determined from the average of the following ratios (Rosen et al., (1990) Magn Reson Med., 14:249-265): -ln[S(t) / S(0)]~TE.V.cT(t) where TE is the echo time, V is the blood volume, and cT is the concentration of the contrast agent.

[0341] The SPIO nanoparticles used in this study have an average diameter of approximately 150 nm and are taken up by Kupffer cells in the liver. Therefore, in addition to renal perfusion, MRI could also monitor the uptake of nanoparticles in the liver by detecting contrast changes assessed in ROIs placed in the liver.

[0342] 9.4 Results As shown in Figure 15, the fusion proteins FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42), FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) and FP776 (EGF-HSA-C1-C2; SEQ ID NO: 48) protected renal function in this model of acute kidney injury (AKI) when administered either ip (FP278) or iv (FP330 and FP776). This protection is reflected in the blockade of serum creatinine rise (sCr). Figure 15A shows that the fusion protein FP278 at both doses tested significantly (p<0.0001) reduced serum creatinine levels compared to vehicle-treated animals and as effectively as mouse MFG-E8. As shown in FIG. 15B, the fusion protein FP330 protected renal function in a dose-dependent manner, as did the fusion protein FP776 (FIG. 15C), where serum creatinine levels were also blocked in a dose-dependent manner.

[0343] Renal dysfunction was also reflected in the blood urea nitrogen (BUN) levels of the mice tested, and the effect of the fusion protein FP278 on BUN levels is shown in FIG.

[0344] In summary, the fusion proteins FP278, FP330 and FP776 strongly protected against the elevation of these markers used in the clinical diagnosis of renal failure, as shown in Figures 15 and 16. The observed efficacy was confirmed by histology (not shown).

[0345] Moreover, as shown in Figure 17, a single administration of the fusion protein FP278 protects distant organs from the acute phase response induced by AKI. AKI induces a plethora of mRNA responses measurable by qPCR in lysates of distant highly perfused organs such as spleen, lung, liver, heart, and brain. Exemplary mRNAs induced selected injuries (NGAL, KIM-1), induction of chemokines (not shown), or induction of acute phase response protein introduction such as serum amyloid A (SAA). Figures 17A and 17B illustrate such AKI-induced responses (serum amyloid A (SAA)) in the heart and lungs of mice that were potently blocked and returned to sham levels after a single injection of the fusion protein.

[0346] The uptake of the SPIO contrast agent Endorem® by the liver over time is shown in Figure 18. Animals with AKI showed significantly reduced uptake of the contrast agent by the liver (target = Kupffer cells) compared to sham-operated animals. FP776 treatment (administered prophylactically at 1.26 mg / kg, approximately 30 minutes before AKI induction, or therapeutically at 2 mg / kg, 5 hours after induction of ischemia-reperfusion injury) protected against loss of contrast agent accumulation in the liver of AKI mice. These results suggest that in this mouse model, AKI causes a significant impairment of clearance of particles via endogenous Kupffer cells, and that AKI causes microvascular dysfunction that affects accumulation of iron particle contrast agent in the liver. Treatment with the fusion protein FP776 protected against loss of clearance and microvascular dysfunction and promoted uptake of the contrast agent at both doses tested when compared to sham animals.

[0347] Therapeutic fusion proteins, such as those according to embodiment 19 (e.g., SEQ ID NO: 80) or embodiment 20 (e.g., SEQ ID NO: 82), promote av integrin cell adhesion and efferocytosis similar to FPJ776 when tested in the above experiments, and are therefore suitable for the therapeutic applications disclosed herein.

[0348] Taken together, these data indicate that the fusion proteins of the present disclosure (eg, with an HSA domain insert) are functional and effective and can be used as therapeutic agents.

[0349] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light of this will be suggested by those skilled in the art and are to be included within the spirit and scope of this application and the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes. The inventions described in the original claims of this application are listed below. [Invention 1] A therapeutic fusion protein for enhancing efferocytosis comprising an integrin binding domain, a phosphatidylserine (PS) binding domain, and a solubility domain, wherein the solubility domain is inserted between the integrin binding domain and the PS binding domain, and the integrin binding domain binds to an integrin. [Invention 2] The fusion protein of invention 1, wherein the integrin binding domain binds to αvβ3 and / or αvβ5 and / or α8β1 integrin. [Invention 3] 3. The fusion protein according to claim 1 or 2, wherein the integrin binding domain comprises an arginine-glycine-aspartic acid (RGD) motif. [Invention 4] 4. The fusion protein according to any one of Inventions 1 to 3, wherein the soluble domain is directly linked to the integrin-binding domain, the PS-binding domain, or both domains. [Invention 5] 5. The fusion protein according to any one of Inventions 1 to 4, wherein the soluble domain is indirectly linked to the integrin-binding domain and / or the PS-binding domain via a linker. [Invention 6] 6. The fusion protein according to any one of claims 1 to 5, wherein the soluble domain comprises human serum albumin (HSA), domain 3 of HSA (HSA D3), Fc-IgG, or a functional mutant thereof. [Invention 7] A fusion protein according to any one of Inventions 1 to 6, wherein the integrin-binding domain has the amino acid sequence of SEQ ID NO: 2 or has at least 90% sequence identity thereto, and the PS-binding domain has the amino acid sequence of SEQ ID NO: 3 or has at least 90% sequence identity thereto, or the PS-binding domain has the amino acid sequence of SEQ ID NO: 76 or has at least 90% sequence identity thereto. [Invention 8] The fusion protein according to any one of Inventions 1 to 7, wherein the integrin-binding domain has the amino acid sequence of SEQ ID NO: 2 or has at least 90% sequence identity thereto, and the PS-binding domain has the amino acid sequence of SEQ ID NO: 78 or has at least 90% sequence identity thereto. [Invention 9] A fusion protein according to any one of Inventions 1 to 8, wherein the integrin-binding domain has the amino acid sequence of SEQ ID NO: 77 or has at least 90% sequence identity thereto, and the PS-binding domain has the amino acid sequence of SEQ ID NO: 3 or has at least 90% sequence identity thereto, or the PS-binding domain has the amino acid sequence of SEQ ID NO: 76 or has at least 90% sequence identity thereto. [Invention 10] 10. The fusion protein according to any one of claims 1 to 9, wherein the solubility domain is HSA and has the amino acid sequence of SEQ ID NO: 4 or at least 90% sequence identity thereto. [Invention 11] 11. The fusion protein according to any one of claims 1 to 10, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 42 or at least 90% sequence identity thereto. [Invention 12] 12. The fusion protein according to any one of claims 1 to 11, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 44, or at least 90% sequence identity thereto; or SEQ ID NO: 47, or at least 90% sequence identity thereto; or SEQ ID NO: 48, or at least 90% sequence identity thereto. [Invention 13] 13. The fusion protein according to any one of claims 1 to 12, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 80 or at least 90% sequence identity thereto. [Invention 14] 14. The fusion protein according to any one of claims 1 to 13, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 82 or at least 90% sequence identity thereto. [Invention 15] An isolated nucleic acid encoding an amino acid sequence according to any one of inventions 11 to 14. [Invention 16] A cloning or expression vector comprising the nucleic acid according to invention 15. [Invention 17] 17. A recombinant host cell suitable for the production of a therapeutic fusion protein, comprising one or more cloning or expression vectors according to invention 16 and optionally a secretion signal. [Invention 18] A pharmaceutical composition comprising the fusion protein according to any one of inventions 1 to 14 and a pharma- ceutically acceptable carrier. [Invention 19] 15. The fusion protein according to any one of claims 1 to 14 for use in the treatment or prevention of an inflammatory disorder or inflammatory organ damage in an individual in need thereof, wherein said inflammatory disorder or inflammatory organ damage is acute kidney injury, acute respiratory distress syndrome, acute liver injury, sepsis, myocardial infarction, stroke, burns, traumatic injury, and inflammatory and organ damage due to ischemia / reperfusion. [Invention 20] 20. A fusion protein for use according to invention 19, wherein the fusion protein is administered in combination with another therapeutic agent, said therapeutic agent being an immunosuppressant, an immunomodulatory agent, an anti-inflammatory agent, an antioxidant, an anti-infective agent, a cytotoxic agent or an anti-cancer agent.

Claims

1. A therapeutic fusion protein for enhancing efferocytosis comprising an integrin binding domain, a phosphatidylserine (PS) binding domain, and a solubilization domain, wherein the fusion protein has an amino acid sequence of SEQ ID NO:42 or has an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

42.

2. A therapeutic fusion protein for enhancing efferocytosis comprising an integrin binding domain, a phosphatidylserine (PS) binding domain, and a solubilization domain, wherein the fusion protein has an amino acid sequence of SEQ ID NO:44 or has an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

44.

3. A therapeutic fusion protein for enhancing efferocytosis comprising an integrin binding domain, a phosphatidylserine (PS) binding domain, and a solubilization domain, wherein the fusion protein has an amino acid sequence of SEQ ID NO:46 or has an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

46.

4. A therapeutic fusion protein for enhancing efferocytosis comprising an integrin binding domain, a phosphatidylserine (PS) binding domain, and a solubilization domain, wherein the fusion protein has an amino acid sequence of SEQ ID NO:48 or has an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

48.

5. A therapeutic fusion protein for enhancing efferocytosis comprising an integrin binding domain, a phosphatidylserine (PS) binding domain, and a solubilization domain, wherein the fusion protein has an amino acid sequence of SEQ ID NO: 80 or has an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

80.

6. A therapeutic fusion protein for enhancing efferocytosis comprising an integrin binding domain, a phosphatidylserine (PS) binding domain, and a solubilization domain, wherein the fusion protein has an amino acid sequence of SEQ ID NO:82 or has an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

82.

7. An isolated nucleic acid encoding the amino acid sequence of the fusion protein according to any one of claims 1 to 6.

8. A cloning or expression vector comprising the nucleic acid of claim 7.

9. A recombinant host cell suitable for producing a therapeutic fusion protein comprising one or more cloning or expression vectors according to claim 8.

10. The recombinant host cell of claim 9, comprising a secretion signal.

11. A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 6 and a pharma- ceutically acceptable carrier.

12. 7. The fusion protein of any one of claims 1 to 6 for use in the treatment or prevention of inflammatory disorders or inflammatory organ damage in an individual in need thereof, wherein said inflammatory disorder or inflammatory organ damage is acute kidney injury, acute respiratory distress syndrome, acute liver injury, sepsis, myocardial infarction, stroke, burns, traumatic injury, and inflammatory and organ damage due to ischemia / reperfusion.

13. 13. The fusion protein for use according to claim 12, wherein the fusion protein is administered in combination with another therapeutic agent, the therapeutic agent being an immunosuppressant, an immunomodulatory agent, an anti-inflammatory agent, an antioxidant, an anti-infective agent, a cytotoxic agent or an anti-cancer agent.

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