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 myocardial infarction, acute respiratory distress or chronic obstructive pulmonary disease fibrosis, and other organ damage resulting from tissue trauma and acute and chronic injury.

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 containing both integrin-binding and phosphatidylserine-binding capabilities, which can be used as therapeutic agents, particularly 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 associated with high morbidity, mortality, and significant unmet medical needs. Typical AOIs include myocardial infarction (MI) and stroke, which occur in 32.4 million patients worldwide annually. Patients with a prior MI or stroke are considered by the World Health Organization to be at highest risk for further coronary and cerebral events, ranking them among the leading causes of morbidity in developed countries. Another AOI is acute kidney injury (AKI), which affects approximately 13.3 million people annually. In high-income countries, the incidence of AKI is 3–5 / 1000 and is associated with a high mortality rate (14–46%) (Metha et al., (2015) Lancet, 385(9987):2616–43). Similar to MI and stroke, survivors of AKI often do not fully recover and are at high risk for developing chronic kidney disease 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 challenging, with no successful clinical trials to date. 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 damage to glomerular cells, mostly tubular cells, loss of renal functional reserve, and ultimately renal failure.

[0004] One common factor underlying AOI is increased cell death due to tissue injury, increased generation of cell fragments, and prothrombotic / proinflammatory microparticles that can enter the circulation and damaged tissue. After neutrophils infiltrate tissues 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 the reprogramming of macrophages (MΦs) toward a non-inflammatory, pro-resolving phenotype and the release of key mediators for successful degradation and repair of affected tissues. 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). This non-inflammatory removal of debris, or senescent or dead cells, is called "efferocytosis."

[0005] However, if efferocytosis is delayed, necrotic cells can accumulate, triggering an inflammatory response that, for example, triggers the production of proinflammatory cytokines (TNF-α) or immunosuppressant IL-10 by macrophages (Greenlee-Wacker (2016) Immunol. Reviews, 273:357-370). Furthermore, if cellular debris and particles are not efficiently removed, they can 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 can include microvascular obstruction, dysfunction, and significant sterile inflammation leading to the progression of tissue damage, primary and secondary organ failure, or maladaptive repair.

[0006] During the acute phase of AOI, the efferocytic pathway appears to be significantly downregulated. Inflammation or acute responses to injury (organic factors, hypoxia, oxidative stress, irradiation, inflammation, and infection) inhibit effective efferocytosis or phagocytosis by downregulating cross-linking proteins and dedicated phosphatidylserine (PS)-binding proteins, including cell surface efferocytosis / clearance receptors. An example of dysfunction of efferocytic receptors is the proteolytic shedding of TAM family receptors, such as Mer tyrosine kinase (MerTK). MerTK is an integral membrane protein preferentially expressed in phagocytes. It not only functions as a signaling protein but also promotes efferocytosis (via 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 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 can 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 down-regulated 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 endogenous 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. Consequently, downmodulation of clearance pathways may improve the effectiveness of innate and adaptive immune effectors to fight infection. As a result of "friendly fire," efferocytosis may be transiently affected during acute organ injury, leading to the above-mentioned complications in AOI. The accumulation of dead cells, debris, and 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 illnesses with high medical needs and may contribute to their morbidity. Examples of such indications are sepsis and cancer (Yang et al., (2016) Tumor 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 recognizes and binds to integrins, such as αvβ3 and αvβ5, which are expressed on many cell types, including phagocytes. These proteins act to crosslink PS on apoptotic / dying cells, exposing them to integrins and leading to efferocytosis (also called phagocytosis) by macrophages and nonprofessional phagocytes. Several crosslinking proteins are also downregulated during most acute and chronic inflammatory diseases. Therapeutic uses of such cross-linked proteins or truncated versions thereof have been suggested previously (WO 2006122327 (Sepsis), WO 2009064448 (Organ Injury After Ischemia / Reperfusion), WO 2012149254 (Cerebral Ischemia) The Feinstein Institute for Medical Research; WO 2015025959 (Myocardial Infarction) Kyushu University and Tokyo Medical University; WO 20150175512 (Bone Resorption) University of Pennsylvania; WO 2017018698 (Tissue Fibrosis) Korea University Research and Business Foundation and U.S. Patent Application Publication No. 20180334486 (Tissue Fibrosis) Nexel Co., Ltd.); WO 2020084344; however, the use of wild-type or naturally occurring proteins is limited by a number of issues. 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 cell expression systems. Castellanos et al. (2016) demonstrated 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] The main reported functions of MFG-E8 are enhancing efferocytosis (Hanayama 2004 Science) and regulating lipid uptake / processing (Nat Med. 2014). rMFG-E8 regulates enterocyte-specific lipid storage by promoting enterocyte triglyceride hydrolase (TG) activity (JCI 2016). Intracellular MFG-E8 has been shown to suppress hepatic lipid accumulation and inflammation, acting through inhibition of the ASK1-JNK / p38 signaling cascade (Zhang et al. 2020). Furthermore, anti-inflammatory properties, promotion of angiogenesis, atherosclerosis, tissue remodeling, and hemostasis regulation have been described for MFG-E8. Furthermore, MFG-E8 has been reported to remove excess collagen from lung tissue by binding collagen via its C1 domain. Interestingly, MFG-E8− / − macrophages exhibited defective collagen uptake that could be rescued by recombinant MFG-E8, which contains at least one discoidin domain (Atabai et al., 2009).

[0010] In preclinical studies, recombinant MFG-E8 has demonstrated robust protection in a variety of, mostly rodent, models of acute inflammatory and organ disease, including models of disease involving abnormal healing. Recombinant MFG-E8 has been shown to promote wound healing in diabetic and I / R-induced wounds / ulcers (Uchiyama et al., 2015 / 2017), promote intestinal epithelial repair after colitis (Bu et al., 2007), and promote tendon repair after injury (Shi et al., 2019). Recombinant MFG-E8 reduced renal injury and fibrosis in a ureteral obstruction (UUO) model (Brisette et al., 2016). Furthermore, efficacy has been demonstrated in classic models of fibrosis, where recombinant MFG-E8 promoted the resolution of TAA- and CCl4-induced liver fibrosis (SY, Gastroenterology, 2016) and protected in a bleomycin-induced pulmonary fibrosis model (Atabai et al., 2009). Recently, a C2-depleted truncated version has been published to show similar or even superior efficacy in several preclinical fibrosis models, including the TAA liver fibrosis model (WO 2020084344).

[0011] EDIL3 (EGF-like repeat and discoidin I-like domain-containing protein 3) was recently reviewed by Hajishengallis and Chavakis (2019). EDIL3 (also known as DEL-1) has been shown to mediate efferocytosis, regulate neutrophil recruitment and inflammation, trigger emergency myelopoiesis in the hematopoietic stem cell niche (αvb3-integrin dependent), suppress osteoclast formation in rodents and non-human primates, and inhibit inflammatory bone loss. EDIL3 has been found to be an essential component of immune privilege in the central nervous system. The potential of EDIL3 as a therapeutic protein was tested as a fusion protein with the Fc fragment of human IgG (DEL-1-Fc). DEL-1-Fc administration inhibited neutrophil infiltration and blocked IL-17-induced inflammatory bone loss in a mouse model of periodontitis (Eskan et al., 2012 doi:10.1038 / ni.2260). Furthermore, DEL-1-Fc improved periodontitis, tissue destruction, and bone loss in a non-human primate model of periodontitis (Shin et al., 2015 doi:10.1126 / scitranslmed.aac5380). Furthermore, DEL-1-Fc improved relapsing-remitting experimental autoimmune encephalomyelitis (EAE), a translational multiple sclerosis model (Choi et al., 2014 doi:10.1038 / mp.2014.146). DEL-1-Fc further reduced the incidence and severity of postoperative peritoneal adhesions in a mouse model (Fu et al., 2018).

[0012] Removal of dead cells, debris, and microparticles by cross-linking proteins such as 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. Therefore, therapeutic approaches that promote the clearance of dead cells during AOI may be used to alleviate or at least mitigate the pathology of AOI and may have implications in other disease settings where dead cells or PS-exposed microparticles are not adequately cleared. Therefore, therapeutic agents that can be used to reduce tissue damage and inflammation and have desirable manufacturing properties to address the unmet medical needs of AOI are needed. Summary of the Invention [Means for solving the problem]

[0013] In the present disclosure, applicants have generated recombinant therapeutic fusion proteins based on the structure of naturally occurring binding proteins (e.g., MFG-E8) without the aforementioned undesirable properties and production problems of wild-type proteins. The fusion proteins of the present disclosure comprise an integrin-binding domain (e.g., an EGF-like domain), a solubilizing domain, and a phosphatidylserine-binding domain (e.g., the C1 domain from MFG-E-8 or its paralog, EDIL3). The proteins of the present invention are suitable for the prevention or treatment of acute or chronic inflammatory organ damage, organ damage caused by the immune system, or fibrosis. The proteins of the present invention may also find use in enabling, accelerating, and promoting repair and regeneration.

[0014] The fusion proteins maintain the primary biological functions of wild-type MFG-E8 or EDIL3 proteins, e.g., by crosslinking PS-exposed dead cells, debris, and microparticles to phagocytes, thereby triggering efferocytosis. Furthermore, the therapeutic fusion proteins of the present disclosure have improved developability, particularly reduced adhesion and improved solubility, compared to wild-type MFG-E8 protein (SEQ ID NO: 1), or recombinant MFG-E8 and C2-truncated MFG-E8 (EGF_C1). Furthermore, these therapeutic fusion proteins exhibit longer plasma exposure and higher yields when expressed in cell expression systems compared to wild-type MFG-E8 protein. Therapeutic fusion proteins of the present invention have increased macrophage-selective activity (enhanced efferocytosis). Furthermore, therapeutic fusion proteins of the present invention have improved safety compared to full-length, wild-type MFG-E8, or other full-length functional variants.

[0015] Provided herein are therapeutic fusion proteins for enhancing efferocytosis comprising an integrin-binding domain, a phosphatidylserine (PS)-binding domain, and a solubilization domain, wherein the PS-binding domain is a truncated mutant of at least one PS-binding domain listed in Table 2.

[0016] 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-SC, where EGF represents the integrin-binding domain, e.g., of MFG-E8, EDIL3, or any other protein containing an integrin-binding domain listed in Table 1; S represents the solubility domain; and C represents a truncated PS-binding domain, e.g., a truncated mutant of the PS-binding domain found in MFG-E8, EDIL3, or any other protein containing either C1 and / or C2 of the PS-binding domain listed in Table 2. Examples of proteins containing both an integrin-binding domain and a PS-binding domain, e.g., MFG-E8 (SEQ ID NO: 1) and EDIL3 (SEQ ID NO: 11), are listed in Table 3.

[0017] In some embodiments, the PS-binding domain comprises one of the two discoidin C1-C2 subdomains or a functional variant thereof. For example, the PS-binding domain of human MFG-E8 having the 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 truncated PS-binding domain comprises the truncated PS-binding 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 PS-binding domain comprises the truncated PS-binding domain of human EDIL3, or a functional variant thereof comprising 1, 2, 3, 4, 5, or up to 10 amino acid modifications.

[0018] In certain aspects, provided herein are fusion proteins comprising an epidermal growth factor (EGF)-like domain, a solubility domain, a C1 domain, but lacking a functional C2 domain. In some embodiments, the fusion protein comprises an epidermal growth factor (EGF)-like domain, a solubility domain, a C1 domain, but lacks a medin polypeptide or fragment thereof.

[0019] In some embodiments, the solubility domain of the fusion protein is linked to the integrin-binding domain. In some embodiments, the solubility domain is linked to the PS-binding domain. In some embodiments, the solubility 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 solubility 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- to C-terminus: integrin-binding domain-solubility domain-PS-binding domain.

[0020] 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.

[0021] In some embodiments, the soluble domain of the therapeutic fusion protein is linked directly to the integrin-binding domain and / or to the PS-binding domain, i.e., inserted between said domains. In alternative embodiments, the soluble 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 soluble domain comprises human serum albumin (HSA), domain 3 of HSA (HSA D3), or the Fc region of IgG (Fc-IgG), or functional variants thereof.

[0022] In some embodiments, the integrin-binding domain is an EGF-like domain, e.g., having the amino acid sequence 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 truncated 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.

[0023] In some embodiments, the solubility domain is HSA or a functional variant thereof, e.g., 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 truncation 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 solubility domain comprises human serum albumin (HSA) or a functional variant thereof comprising 1, 2, 3, 4, 5, or up to 10 amino acid modifications, e.g., HSA C34S, or a truncation variant of HSA, e.g., domain 3 of HSA (HSA D3), or a functional variant thereof. In a preferred embodiment, the solubility domain is HSA C34S.

[0024] In an alternative embodiment, the soluble domain comprises the Fc region of an IgG (Fc-IgG), for example, the Fc region of human IgG1, IgG2, IgG3, or IgG4, or a functional variant thereof. In one embodiment, the soluble domain comprises the 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 mutant 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 can contain the amino acid substitution S354C, and the Fc-IgG1 hole can 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 can also contain D265A and P329A substitutions to reduce Fc effector function. In one embodiment, the Fc-IgG1 has the amino acid sequence set forth in SEQ ID NO: 9 or 10.

[0025] In a preferred embodiment, the therapeutic fusion protein comprises a milk fat globule-EGF factor 8 protein (MFG-E8) and a solubilization domain, wherein MFG-E8 comprises an integrin-binding EGF-like domain (SEQ ID NO: 2) and a functional variant of the 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 solubilization domain is linked to the N- or C-terminus of MFG-E8. In one embodiment, the solubilization domain is inserted between the EGF-like domain and the C1 domain or between the EGF-like domain and the C2 domain. In a preferred embodiment, the solubilization domain is linked to the C-terminus of the EGF-like domain and to the N-terminus of the C1 domain. The solubilization domain may be linked directly or indirectly to the C-terminus of the EGF-like domain and to the N-terminus of the C1 domain. In some embodiments, the indirect linkage is by an external linker, eg, a glycine-serine based linker.

[0026] In some embodiments, and as described in the Examples section, therapeutic fusion proteins of the present disclosure function to promote efferocytosis by endothelial cells in a human endothelial cell-Jurkat cell efferocytosis assay, restore impaired basal efferocytosis 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.

[0027] Also disclosed herein are methods, uses, diagnostic reagents, pharmaceutical compositions, and kits that utilize or include 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 explanation of the drawings]

[0028] [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: SDS-PAGE protein gels of fusion proteins expressed in HEK cells. 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 mentioned above.) [Figure 3]Figure 3 illustrates the effect of loss of fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) protein relative to wild-type (wt) MFG-E8 during practical handling. Figure 3A shows the loss of efficacy of wild-type MFG-E8 in an 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, Figure 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 a PS competition assay when protein dilutions are made on polypropylene plates (symbol: □) versus non-binding plates (symbol: ●). [Figure 4] 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) in 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 BW5147.G.1.4 cell adhesion, 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 similarly to wild-type MFG-E8 when expressed in CHO or HEK cells. [Figure 5-2] (As mentioned above.) [Figure 6] Figure 1 shows the effect of the 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 the 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) can rescue endotoxin (lipopolysaccharide)-injured 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 an individual donor, and the right panel shows the average impairment (%) of efferocytosis for the three donors. Figure 7B shows the rescue of this endotoxin (LPS)-injured efferocytosis of killed neutrophils by human macrophages in the presence of the therapeutic fusion protein FP278. The efferocytosis index for three different human macrophage donors was normalized and plotted as efferocytosis (%). [Figure 7-2](As mentioned above.) [Figure 8-1] Figure 8: Rescue of Staphylococcus aureus (S. aureus) particle-induced impairment of efferocytosis of killed neutrophils by human macrophages using the 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 rescuing the impairment of efferocytosis caused by administration of S. aureus and on promoting efferocytosis after the basal level of efferocytosis was reached. [Figure 8-2] (As mentioned above.) [Figure 9] Figure 9 shows the effect of the therapeutic fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) on promoting efferocytosis of dying Jurkat cells by human endothelial cells (HUVEC). As shown in Figure 9, the efficiency of fusion proteins in the endothelial cell efferocytosis assay depends on the presence of the C1-C2 or C1-C1 tandem domains, as a fusion protein of the structure EGF-HSA-C2 (FP270; SEQ ID NO: 36) is ineffective in this assay. [Figure 10] Figure 1 shows that the position of the HSA domain in the therapeutic fusion protein, i.e., at the N- or C-terminus (FP220 (HSA-EGF-C1-C2; SEQ ID NO: 30) or FP110 (EGF-C1-C2-HSA; SEQ ID NO: 28), respectively, confers 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 the promotion of efferocytosis by various formats of therapeutic fusion proteins containing HSA or an Fc portion. 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, 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. Figure 11B shows a comparison of fusion proteins containing an Fc portion, in which 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 the fusion protein FP090 (Fc-EGF-C1-C2; SEQ ID NO: 24) containing an N-terminally positioned 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 present disclosure, such as chimeric variants (FP114 or FP260; SEQ ID NO: 34, FP147 or FP1777; SEQ ID NO: 71, FP1149, FP1150, FP145; SEQ ID NO: 80, FP1145; SEQ ID NO: 103, FP146; SEQ ID NO: 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 11-2] (As mentioned above.) [Figure 11-3] (As mentioned above.) [Figure 11-4] (As mentioned above.) [Figure 11-5] (As mentioned above.) [Figure 11-6] (As mentioned 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 efferocytosis increasing 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) can rescue endotoxin (lipopolysaccharide)-induced efferocytosis of killed neutrophils by human macrophages. Fusion protein concentration is shown on the x-axis, and efferocytosis [%] is shown on the y-axis. [Figure 13-2] (As mentioned above.) [Figure 13-3] (As mentioned 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). Fusion protein concentration is shown on the x-axis, and efferocytosis [%] is shown on the y-axis. [Figure 14-2] (As mentioned above.) [Figure 15-1]Figure 15: Single administration of the 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 of FP278 (SEQ ID NO: 44) (x-axis) reduced 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 mentioned above.) [Figure 16] FIG. 1 shows that a single administration 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 17-1] Figure 17: A single administration of the therapeutic fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) protects remote organs from the acute phase response elicited 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 the mouse heart, and Figure 17B illustrates such an AKI-induced response (SAA) in the murine lung, both of which were potently blocked after a single i.p. injection of the MFG-E8-derived fusion protein FP278 (SEQ ID NO: 44) at 0.16 mg / kg or 0.5 mg / kg / ip. [Figure 17-2] (As mentioned above.) [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 into 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. [Figure 19] The therapeutic fusion protein FP114 (EGF-HSA-C1 SEQ ID NO: 34), also referred to herein as FP260, was tested at 1.5 mg / kg / iv in an AKI model as described in the Examples. In this study, FP114 was administered 30 minutes before the onset of ischemia-reperfusion injury. Serum markers and kidney weights were assessed 24 hours after induction of disease. The reduction in serum creatinine and BUN, as well as normal kidney weights, suggest protection from AKI in this model. [Figure 20] The therapeutic fusion protein FP135 (EGF-HSA-C1 SEQ ID NO: 73), also referred to herein as FP261, was tested at 0.8 mg / kg / ip in the CCL4 fibrosis model. Treatment began 4 weeks after fibrosis induction (with CCL4) (11 doses total) or 5 weeks after fibrosis induction with CCL4 (8 doses total), with three doses per week. A third group of animals was treated 6 weeks after cessation of disease induction with CCL4 (4 doses total). In all groups, FP135 was administered once daily for the final 3 days. Liver stiffness was assessed at baseline (the start of the experiment), upon cessation of CCL4, and 3 days after cessation of CCL4. The data suggest that animals treated with FP135 (starting after weeks 4 and 5 of CCL4) achieved significantly accelerated resolution of CCL4-induced liver stiffness. [Figure 21-1]Figure 21: Figure 21A. The therapeutic fusion protein FP135 (EGF-HSA-C1 SEQ ID NO: 73) was tested at 0.8 mg / kg / ip in the CCL4 fibrosis model. Treatment was initiated 4 weeks after fibrosis induction (with CCL4) (11 doses total), or 5 weeks after fibrosis induction with CCL4 (8 doses total), with 3 doses per week, or 6 weeks after cessation of disease induction with CCL4 (4 doses total). In all groups, FP135 was administered once daily for the final 3 days. The reduction in serum ALT suggests that treatment with FP135 helped accelerate the resolution of CCL4-induced liver damage in groups that began treatment at weeks 4 and 5 of CCL4. Figure 21B: Therapeutic fusion protein FP135 (EGF-HSA-C1 SEQ ID NO: 73) was tested at 0.8 mg / kg / ip in the CCL4 fibrosis model as described in Figure 21A. Collagen content in the livers of sacrificed animals was quantified by hydroxyproline assay. The reduction observed in animals dosed 8 and 11 times suggests that treatment with FP135 helped accelerate the resolution of CCL4-induced liver fibrosis. Figure 21C: Therapeutic fusion protein FP135 (EGF-HSA-C1 SEQ ID NO: 73) was tested at 0.8 mg / kg / ip in the CCL4 fibrosis model as described in Figure 21A. Collagen expression in the livers of sacrificed animals was quantified by hydroxyproline assay. The reduction observed in animals dosed 8 and 11 times suggests that treatment with FP135 helped accelerate the resolution of CCL4-induced liver fibrosis. [Figure 21-2] (As mentioned above.) [Figure 22] Integrin adhesion data for sections of truncated proteins FP137, FP135 and FP147 are shown. [Figure 23] Dynamic light scattering (DLS) of C2 truncated MFG-E8 (EGF-C1; SEQ ID NO: 115) and HSA fusion (EGF-HSA-C1; SEQ ID NO: 73) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0029] Disclosed herein are therapeutic fusion proteins comprising an integrin-binding domain, a PS-binding domain, and a solubility domain. Also disclosed herein are methods of treatment using the fusion proteins of the disclosure, as well as assays, such as efferocytosis assays, useful for characterizing the fusion proteins.

[0030] definition In order that this disclosure may be more readily understood, certain terms are specifically defined throughout the detailed description. Unless defined otherwise, 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.

[0031] In all instances 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" explicitly 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" explicitly excludes the inclusion of one or more additional active agents other than the multispecific binding molecules of the present disclosure and a second co-administered agent.

[0032] As used herein, the term "efferocytosis" refers to a process in cell biology in which dead or dying cells, such as apoptotic, necrotic, senescent, highly activated, 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 prey, generating large intracellular fluid-filled vesicles containing prey called efferosomes, and lysosomal compartments where prey degradation begins. During apoptosis, efferocytosis ensures that dead cells are removed before their membrane integrity is compromised and their contents leak into surrounding tissues, preventing exposure of surrounding tissues 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, epithelial and endothelial cells, and fibroblasts, can also perform efferocytosis. 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). Currently, no therapies that specifically promote efferocytosis are in clinical trials.

[0033] The term "efferocytosis assay" as used herein and described in the Examples refers to assay systems developed for profiling fusion proteins that utilize human macrophages or human endothelial cells (HUVECs) as phagocytes. Examples of macrophage-neutrophil efferocytosis assays, endothelial cell-Jurkat cell efferocytosis assays, and endothelial cell microparticle efferocytosis assays are exemplified herein. These assays can be used to demonstrate that MFG-E8-derived biotherapeutics, 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 for demonstrating that such compounds of the present invention can rescue even LPS- or S. aureus-injured efferocytosis of dead cells.

[0034] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. This phrase applies to amino acid polymers in which one or more amino acid residues are artificial chemical mimetics of corresponding naturally occurring amino acids, 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.

[0035] 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.

[0036] 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 (-NH).

[0037] As used herein, the term "fusion protein" refers to a protein containing 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 involved in binding to the corresponding receptor on the cell surface. Fusion proteins can be generated using recombinant protein engineering, and the term "recombinant protein" refers to proteins 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, ensuring sufficient space between the fusion partners and ensuring 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 domain ends may be ineffective. Alternatively, the process of domain insertion involves fusing consecutive protein domains by encoding the desired structure into a single polypeptide chain, and sometimes inserting 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 due to the difficulty of finding suitable nucleic acid ligation sites in the gene of interest.

[0038] In addition to the direct fusion techniques described above, external linkers can be used to maintain the function of protein domains in fusion proteins. Such linkers, which refer to a series of amino acids connecting one protein domain to another, 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 structures comprise two or more functional or organizational domains often contain a series of amino acids between such domains that connect them to one another. Linkers can allow domain interaction, enhance stability, and reduce steric hindrance, making them often preferred for use in engineered protein design, even when N- and C-terminal fusions are possible. In some embodiments, linkers are characterized by their tendency to not adopt a rigid three-dimensional structure but rather provide flexibility to the polypeptide. Various types of naturally occurring linkers have been used in engineered proteins, such as many recombinant therapeutic proteins, especially immunoglobulin hinge regions, which function as linkers 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, which may be the same or different, 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.

[0039] 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 alteration involving said amino acid residue / position. For example, typical modifications include substitution of a residue (or said position) with another amino acid (e.g., conservative or non-conservative substitution), insertion of one or more amino acids adjacent to said residue / position, and deletion of said residue / position. An amino acid "substitution" or variation thereof refers to replacing 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 variant polypeptide compared to a polypeptide comprising the starting (or "wild-type") amino acid sequence.

[0040] 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 encode identical or essentially identical amino acid sequences, or, if the nucleic acid does not encode an amino acid sequence, essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, the codons GCA, GCC, GCG, and GCU all encode the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be altered 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 encodes a polypeptide also describes every possible silent variation of the nucleic acid. Those of skill in the art will recognize that each codon in 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 encodes a polypeptide is implicit in each described sequence.

[0041] With respect to polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions to a polypeptide sequence that result in the substitution 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 an engineered protein of the disclosure.

[0042] As referred to herein, a "protein variant" or "protein variant" refers to a protein containing a variation in which one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, amino acids are modified. As referred to herein, a "functional variant" of a protein refers to a protein variant containing 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 "truncation variant" of a protein or protein domain refers to a shortened version of the protein or protein domain, where the shortened version of the protein retains the function of the parent protein. To determine whether functional variants or truncation variants have changes in overall properties or function, these variant proteins can be tested against the full-length or unmodified parent protein for their effect in several assays, such as those described in this disclosure. For example, promoting endothelial cell efferocytosis in a human endothelial cell-Jurkat cell efferocytosis assay, reversing impaired macrophage efferocytosis in a human macrophage-neutrophil efferocytosis assay, reducing plasma microparticle numbers by clearance in a human endothelial-microparticle efferocytosis assay, and / or providing protection against multi-organ injury in an acute renal ischemia model.

[0043] 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. Two sequences are "substantially identical" and exhibit "sequence identity" when compared and aligned for maximum correspondence over a comparison window, or designated region, if two sequences 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), as measured, for example, using one of the sequence comparison algorithms below or by manual alignment and visual inspection. Optionally, 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 to 500, or 1000, or 2000, or 3000 or more nucleotides in length, or over a region that is 30 to 200, or 300, or 500, or 700, or 800, or 900, or 1000 or more amino acids in length.

[0044] 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 needed.Default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the sequence identity percentage of test sequence compared with reference sequence based on program parameters.

[0045] As used herein, the term "comparison window" 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 aligning 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, e.g., Brent et al., (2003) Current Protocols in Molecular Biology).

[0046] 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.

[0047] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul (1993) PNAS.USA, 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest 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 smallest sum probability in the comparison of the test nucleic acid with 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.

[0048] 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. Furthermore, the percent identity between two amino acid sequences can be determined using the Needleman & Wunsch (supra) algorithm, 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.

[0049] 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.

[0050] The term "nucleic acid" is used interchangeably herein with the term "polynucleotide" and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- 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 similarly 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).

[0051] Unless otherwise indicated, a particular nucleic acid sequence implicitly encompasses not only the explicitly indicated sequence, 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 substituted 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 altered to encode an amino acid sequence using codons preferred in a production cell, such as a Chinese hamster ovary cell (CHO). The optimized nucleotide sequences are 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.

[0052] 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 recruiting 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 a single cell may have multiple different types on its surface. Integrins have two subunits: α (alpha) and β (beta), each of which spans 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-interacting sites of many extracellular matrix proteins (e.g., as part of the amino acid sequence arginine-glycine-aspartic acid (RGD in the single-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, named after the epidermal growth factor, which was first described in the EGF-like domain. 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). Some examples of EGF-like domains containing the RGD-binding motif are listed in Table 1 below.

[0053] [Table 1]

[0054] As used herein, the term "integrin-binding domain" refers to a sequence of amino acids or a protein domain that functions to bind to integrins. In one embodiment of the present disclosure, as used herein, "integrin-binding domain" refers to a sequence of amino acids or a protein domain that functions to bind to integrins and that includes an RGD motif. In one embodiment of the present disclosure, the integrin-binding domain is an EGF-like domain derived from human MFG-E8 having the amino acid sequence set forth in SEQ ID NO:2. In an alternative embodiment of the present disclosure, the integrin-binding domain is an EGF-like domain derived 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 sequence of amino acids 1 to 132 of SEQ ID NO:11.

[0055] As used herein, the term "binds to an integrin" 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 adhesion of fluorescently labeled αβ integrin-expressing lymphoma cells to a therapeutic fusion protein of the present disclosure was 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%, or 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 for determining the respective activity, preferably using the assay described in the Examples, Section 3.2.

[0056] Phosphatidylserine-binding domain As used herein, "phosphatidylserine" (PS) refers to a phospholipid, a component of the cell membrane. PS is primarily confined to the inner leaflet of the cell membrane, whereas phosphatidylcholine and sphingomyelin are primarily 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 translocate 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 conditions (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, serves as an essential phospholipid in coagulation, and can function 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 engulf 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.

[0057] [Table 2]

[0058] In one embodiment of the present disclosure, the PS domain is derived from human MFG-E8 having the amino acid sequence as set forth in SEQ ID NO: 3. 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.

[0059] 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 binding of a fusion protein of the present disclosure to PS coated on a microtiter plate was assessed 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, when tested in the same manner as for determining its respective activity, preferably using the assay described in the Examples, Section 3.1, 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%, or at least 98% of the PS-binding activity observed for the human MFG-E8 protein set forth in SEQ ID NO: 1.

[0060] Cross-linked 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.

[0061] [Table 3]

[0062] To be of therapeutic value, cross-linked proteins would be useful if they contained 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 inducers of AOI efferocytosis. In contrast, for example, GAS6 proteins may not be particularly effective at promoting AOI efferocytosis because their receptor on phagocytes (MerTK) is proteolytically cleaved during inflammation and infection, as described above.

[0063] One 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 cell types (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 mRNA, the long and short variants of which are expressed in the mouse mammary gland. The human MFG-E8 gene (UniProtKB-Q08431) encodes a 387-residue protein that is processed to form multiple protein products. The amino acid sequence of human MFG-E8, including the signal peptide (residues 1–23; underlined), EGF-like domain (residues 24–67; italicized), C1 domain (residues 70–225; bold), and C2 domain (residues 230–387; bold and underlined), is shown below. [ka]

[0064] MFG-E8 lacks the transmembrane function of MFGM and therefore functions as a peripheral membrane protein. Human MFG-E8 consists of an 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) containing two F5 / 8-discoidin subdomains (C1 and C2) that bind anionic phospholipids with high affinity. Integrin binding is the result of an RGD motif located at residues 46-48 of human MFG-E8 (SEQ ID NO: 1). Apoptotic cells, cellular debris, hyperactivated cells, and most microparticles (MPs) expose PS, which are targets for MFG-E8, which acts as a bridging molecule, opsonizing these cells and microparticles and linking them to αvβ3 and αvβ5 integrins on phagocytes. This crosslinking 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 by 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. MFG-E8 domains have previously been proposed as components of therapeutic drugs, and the PS-binding domain in particular (Kooijmans et al., (2018) Nanoscale, 10(5):2413-2426) and fragments of MFG-E8 have been described to act in fibrosis models (U.S. Patent Application Publication No. 2018 / 0334486).

[0065] Non-inflammatory uptake of dead cells, debris, and particulates by professional and non-professional phagocytes plays an important role in homeostasis after tissue injury (Greenlee-Wacker (2016) supra). The importance of proper clearance was further demonstrated in genetic models in which MFG-E8 knockout mice showed 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).

[0066] 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 may 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).

[0067] Solubilization Domain As described herein, the therapeutic fusion proteins of the present disclosure comprise an integrin-binding domain and a PS-binding domain. Additionally, the fusion proteins also comprise an additional domain that confers several desirable properties to the fusion protein. For purposes of this application, this additional domain, referred to as a "solubilization domain," provides improved biological properties, such as increased solubility, reduced aggregation, and increased biological activity. As a result, the fusion proteins exhibit desirable pharmacokinetic profiles. Furthermore, the presence of the solubilization domain improves the stability of the therapeutic fusion protein and improves expression of the fusion protein compared to the wild-type protein in cellular expression systems, as evidenced by increased yields after purification.

[0068] The presence of a solubilizing domain can 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 their plasma half-life and enhance their 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).

[0069] 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) containing the 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 its neonatal Fc receptor (FcRn) binding property. The use of N-terminal fragments of HSA for fusion to polypeptides has also been proposed (e.g., European Patent Application No. 399666). Thus, genetic or chemical fusion or conjugation of molecules to albumin can stabilize or extend their shelf life and / or retain the activity of the molecules for extended periods 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.

[0070] 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 can also be modified to improve Fc heterodimerization by introducing complementary amino acid substitutions into the CH3 domain of the Fc, e.g., using knob-into-hole (KiH)-based modifications. For example, 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 the 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 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).

[0071] In some embodiments, the soluble domain comprises an antibody Fc domain derived from human IgA, IgD, IgE, or IgM.

[0072] In some embodiments, the solubilization domain comprises SUMO (small ubiquitin-like modifier), ubiquitin, GST (glutathione S-transferase), or a variant thereof.

[0073] Linking and Orienting Domains of Therapeutic Fusion Proteins The integrin-binding domain, PS-binding domain, and solubility domain of the fusion proteins of the present disclosure are linked. As used herein, the term "linked" or "linked" refers to one domain of a 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." Using a molecule with the format ABC as an example: 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.

[0074] In some embodiments, the linkage is direct, such that the domains are fused to one another. 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 fused to the N-terminus of the solubility domain (e.g., 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., HSA) and 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 comprising 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., an 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 solubility domain (e.g., HSA), and the C-terminus of the solubility domain is fused to the N-terminus of the C2 discoidin subdomain. In another embodiment, the integrin-binding domain is fused to a solubility domain that is fused to a PS-binding domain. Specifically, the solubility domain (e.g., HSA) is fused to the C-terminus of the integrin-binding domain (e.g., an EGF-like domain) and the N-terminus of the PS-binding domain (e.g., a C1-C2 discoidin subdomain). In one embodiment, HSA is fused to the C-terminus of the EGF-like domain and to the N-terminus of the C1 discoidin domain.

[0075] 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.

[0076] 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 or Fc), and a third region containing a PS-binding domain, e.g., a C1 and / or C2 discoidin domain. 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.

[0077] In some embodiments, the solubility domain (eg, HSA or Fc) is HSA.

[0078] In some embodiments, the solubility domain is HSA, or a functional variant thereof.

[0079] In some embodiments, the solubility domain is the antibody Fc-immunoglobulin G1 (Fc-IgG1; SEQ ID NO: 7).

[0080] In a preferred embodiment, HSA comprising the amino acid sequence 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 set forth in SEQ ID NO:46 (FP068). In one embodiment, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:48 (FP776).

[0081] In an alternative embodiment, HSA comprising the amino acid sequence 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 set forth in SEQ ID NO: 70 (FP1068). In one embodiment, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 69 (FP1776).

[0082] In some embodiments, the linkage is via a polypeptide linker; for example, the polypeptide linker that connects the solubility domain to the PS-binding domain in the fusion proteins of the present disclosure is referred to as an "external linker." These external linkers typically contain glycine (G) and / or serine (S), and may contain glycine and leucine (GL) or glycine and valine (GL). In some embodiments, the linker contains multiple G and S residues, e.g., G2S and multiples thereof, such as (G2S)4 set forth in SEQ ID NO:62, (GS)4 set forth in SEQ ID NO:63, G4S set forth in SEQ ID NO:64, or (G4S)2 set forth in SEQ ID NO:65.

[0083] In some embodiments, an exolinker is fused between the C-terminus of the integrin-binding domain and the N-terminus of the soluble domain. Specifically, the exolinker is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA. In some embodiments, the exolinker is fused between the C-terminus of the soluble domain and the N-terminus of the PS-binding domain. Specifically, the exolinker is fused to the C-terminus of HSA and the N-terminus of the PS-binding domain. In some embodiments, an exolinker is fused between the C-terminus of the integrin-binding domain and the N-terminus of the soluble domain, and an additional exolinker is fused between the C-terminus of the soluble domain and the N-terminus of the PS-binding domain. Specifically, the exolinker is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA, and an additional exolinker is fused to the C-terminus of HSA and the N-terminus of the PS-binding domain.

[0084] In some embodiments, an exolinker 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 exolinker 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 exolinker 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 exolinker 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 exolinker 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.

[0085] In one embodiment, an external linker containing 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 containing this structure has the amino acid sequence set forth in SEQ ID NO: 42 (FP330).

[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 an additional 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.

[0087] In one embodiment, an external linker containing GS is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA, and an additional external linker containing (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).

[0088] In one embodiment, an exolinker containing GS is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA, and the C-terminus of HSA is fused directly to the N-terminus of the PS-binding domain.

[0089] In one embodiment, an external linker containing GS is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA, and an additional external linker containing 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).

[0090] In one embodiment, an external linker containing GS is fused to the C-terminus of the EGF-like domain and the N-terminus of HSA, and an additional external linker containing (GS) (SEQ ID NO: 65) 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: 56 (FP010).

[0091] 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).

[0092] Functional properties 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-injured 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 preventing weight loss in a mouse model of acute kidney injury.

[0093] Exemplary Protein Sequences The amino acid sequences in Table 4 include examples of therapeutic fusion proteins and portions thereof of the present disclosure.

[0094] Throughout the body of this application, in the event of any discrepancy between the body of the specification (e.g., Table 4) and the Sequence Listing, the body of the specification shall control.

[0095] [Table 4]

[0096] [Table 5]

[0097] [Table 6]

[0098] [Table 7]

[0099] [Table 8]

[0100] [Table 9]

[0101] [Table 10]

[0102] [Table 11]

[0103] [Table 12]

[0104] Table 13

[0105] Table 14

[0106] Table 15

[0107] Table 16

[0108] Table 17

[0109] Table 18

[0110] Table 19

[0111] Table 20

[0112] Table 21

[0113] Table 22

[0114] Table 23

[0115] Table 24

[0116] Table 25

[0117] Table 26

[0118] Table 27

[0119] Table 28

[0120] Table 29

[0121] Table 30

[0122] Table 31

[0123] Table 32

[0124] Table 33

[0125] Table 34

[0126] Table 35

[0127] Table 36

[0128] Table 37

[0129] Table 38

[0130] Table 39

[0131] Table 40

[0132] Table 41

[0133] Table 42

[0134] Table 43

[0135] Table 44

[0136] Table 45

[0137] Table 46

[0138] Table 47

[0139] Table 48

[0140] Table 49

[0141] Table 50

[0142] Table 51

[0143] Table 52

[0144] Table 53

[0145] [Table 54]

[0146] [Table 55]

[0147] [Table 56]

[0148] [Table 57]

[0149] 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.

[0150] The present application also includes therapeutic fusion proteins comprising the integrin-binding domain of MFGE8 or EDIL3 and a truncated PS-binding domain, such as a truncated mutant of the IgSF V domain of TIM4 or a truncated mutant of the GLA domain of the bridging protein GAS6 mutant.

[0151] Modification 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 can have conservative modifications of amino acid residues, 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 can have deletions of amino acid residues, where the modified fusion protein retains or has enhanced properties compared to a protein comprising the parent domain. Alternatively, a therapeutic fusion protein can have insertions of amino acid residues, 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 proteins.

[0152] 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 assessed in in vitro or in vivo assays. Conservative modifications (as described above) can be introduced, and / or 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.

[0153] 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 encoding DNA or by synthesizing 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, reduced, or removed linker sequences, and / or amino acid mutations or substitutions and deletions of one or more amino acids. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties. Amino acid changes may also alter post-translational processes of the molecule, such as changing the number or location of potential glycosylation sites.

[0154] Methods for producing recombinant molecules Nucleic acids and expression systems In one embodiment, the present 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.

[0155] 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 corresponding complementary sequences. Nucleic acid molecules of the present invention include full-length genes or cDNA molecules, as well as combinations of fragments thereof. While the nucleic acids of the present invention are derived from human sources, the present invention also encompasses those derived from non-human species.

[0156] An "isolated nucleic acid" is a nucleic acid isolated from a naturally occurring source, separated from adjacent gene sequences present in the genome of the organism from which the nucleic acid is isolated. In the case of nucleic acids enzymatically or chemically synthesized from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide, the nucleic acid resulting from such a process is understood to be 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 a 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 permits 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.

[0157] 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. Additionally, the present invention provides host cells comprising such expression systems or constructs.

[0158] In one embodiment, the present disclosure provides a method for preparing a therapeutic fusion protein, comprising: (a) culturing host cells comprising a nucleic acid encoding the fusion protein, wherein the cultured host cells express the fusion protein; and (b) recovering the fusion protein from the host cell culture.

[0159] Expression vectors and host cells for producing the above-described therapeutic fusion proteins 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 contains a nucleic acid sequence (in relation to the host cell) that directs and / or controls the expression of one or more heterologous coding regions operably linked thereto. Various expression vectors can be used to express polynucleotides encoding molecular chains or binding domains. 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 carrying expression cassettes for protein or RNA expression, episomal vectors, and human artificial chromosomes (see, e.g., Harrington et al., (1997) Nat Genet 15:345). 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, CA), MPSV vectors, and 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, papillomaviruses, 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.

[0160] 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 toward coding sequences whose expression products are better tolerated by the host cell. In addition to a promoter, other control elements may also be necessary or required for efficient expression of a 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.

[0161] The expression vector may also provide a secretion signal sequence site for forming a fusion protein with the encoded polypeptide by inserting the above-described binding domain and / or solubility domain sequences. More often, the inserted sequences are linked to a signal sequence before being incorporated into the vector. Vectors that allow expression of the binding domain and solubility domain as a fusion protein thereby result 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 culture medium (if the host cells secrete it into the culture medium) or directly recovered from the host cells that produce it (if not secreted). The selection of an appropriate host cell depends on various factors, such as the desired expression level, polypeptide modifications 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.

[0162] Mammalian cell lines available as hosts for expression are known in the art, 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, including, but not limited to, immortalized cell lines available from the American Type Culture Collection (ATCC). Generally, host cells are transformed with a recombinant expression vector containing 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 grown 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 to express polypeptides if it is desired to use the polypeptides in various signal transduction or reporter assays. Alternatively, polypeptides can be produced in lower eukaryotes such as yeast or 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 produced 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 achieved using known chemical or enzymatic methods.

[0163] 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 herpesvirus 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 desirable. For example, cell lines stably expressing engineered proteins can be generated using expression vectors of the present disclosure containing viral origins of replication or endogenous expression elements and a selectable marker gene. After vector introduction, cells can be grown in an enriched medium for 1-2 days before switching to a selective medium. The purpose of the selectable marker is to confer resistance to selection, and its presence allows cells that successfully express the introduced sequences to grow in a selective medium. Resistant, stably transfected cells can be grown using tissue culture techniques appropriate to the cell type.

[0164] Fusion proteins are typically recovered from the culture medium as secreted polypeptides, but may also be recovered from host cell lysates if produced directly 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).

[0165] When a fusion protein is produced in a recombinant cell other than one 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, including fractionation on ion-exchange columns, ethanol precipitation, reverse-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.

[0166] In certain aspects, provided herein is 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 certain embodiments, the viral vector is administered to a subject, e.g., 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.

[0167] Pharmaceutical Composition 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 pharmaceutically acceptable excipients, diluents, or carriers. Such compositions can include one or a combination of (e.g., two or more different) therapeutic fusion proteins of the present disclosure.

[0168] 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 a fusion protein of the present disclosure combined with, for example, 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 regarding uses of the therapeutic fusion proteins of the present disclosure.

[0169] To prepare a pharmaceutical or sterile composition comprising a fusion protein of the present disclosure, the fusion protein is mixed with a pharmaceutically acceptable carrier or excipient.

[0170] The phrase "pharmaceutically acceptable" means approved by a regulatory agency of a federal or state government or listed in the United States Pharmacopoeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans.

[0171] The term "pharmaceutical composition" refers to a mixture of at least one active ingredient (e.g., a variant protein) with at least one pharmaceutically acceptable excipient, diluent or carrier.

[0172] "Drug" refers to a substance used in medical treatment.

[0173] As used herein, "pharmaceutically acceptable carrier" includes 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 administration. 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.

[0174] The pharmaceutical compositions described herein may contain one or more pharmaceutically acceptable salts.The pharmaceutical compositions described herein may also contain a pharmaceutically acceptable antioxidant.Examples of pharmaceutically acceptable antioxidants include water-soluble antioxidants such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, sodium metabisulfite, sodium sulfite, etc.; oil-soluble antioxidants such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, etc.; metal chelators such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc.

[0175] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions described herein include water, ethanol, polyols (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 using coating materials such as lecithin, by maintaining the required particle size in the case of dispersion, and by using surfactants.

[0176] 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 the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. Furthermore, prolonged absorption of the injectable dosage form can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0177] 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 conventional media or agents are incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated.A supplementary active compound can also be incorporated into the composition.

[0178] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., 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.

[0179] 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.

[0180] Solutions or suspensions used for intradermal or subcutaneous administration typically contain one or more of the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerin, propylene glycol, or other synthetic solvents; an antibacterial agent such as benzyl alcohol or methylparaben; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as ethylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and an agent 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.

[0181] Sterile injectable solutions can be prepared by incorporating the required amount of active compound into a suitable solvent containing one or a combination of the ingredients listed above, as needed, followed by sterile filtration. Generally, dispersions are prepared by incorporating the fusion protein of the present invention into a sterile vehicle containing a basic dispersion medium and the necessary other ingredients listed above. For sterile powders to prepare sterile injectable solutions, the preparation method is vacuum drying and freeze-drying (lyophilization), which yields a powder of the active ingredient plus any additional desired ingredients from the previously sterile-filtered solution.

[0182] The amount of active ingredient that can be combined with a carrier material 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 a carrier material to produce a single dosage form will generally be the amount of the composition that produces a therapeutic effect. Generally, out of 100 percent, this amount will range from about 0.01 percent to about 99 percent active ingredient, from about 0.1 percent to about 70 percent, or from about 1 percent to about 30 percent active ingredient combined with a pharmaceutically acceptable carrier.

[0183] The choice of dosing regimen for a therapeutic engineered 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 will depend, in part, on the particular entity and the severity of the condition being treated. Guidance for selecting appropriate doses of biological molecules 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 al. (2000) New Engl. J. Med. 343:1594-1602).

[0184] 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 begins at an amount somewhat less than the optimal dose and is then increased by small increments until a desired or optimal effect is achieved relative to negative side effects. Important diagnostic measures include, for example, measures of inflammatory symptoms or levels of inflammatory cytokines produced.

[0185] The actual dosage level of the active ingredient in the pharmaceutical compositions 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 employed, the route of administration of the particular compound employed, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and past medical history of the patient being treated, and factors such as are known in the medical arts.

[0186] 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 a physically discrete unit suitable as a unitary dosage for the subject 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 characteristics 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 to treat individual sensitivities.

[0187] For administration of therapeutic fusion proteins, dosages range from about 0.0001 to 150 mg / kg, e.g., 5, 15, and 50 mg / kg, subcutaneously, and more usually 0.01 to 5 mg / kg, of the host body weight. 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.

[0188] Therapeutic fusion proteins 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 concentration 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.

[0189] Alternatively, therapeutic fusion proteins can be administered as sustained-release formulations, in which case less frequent administration is required. The 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 dosages 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 in the symptoms of the condition or disease. The patient can then receive a prophylactic administration regimen.

[0190] The actual dosage level of the active ingredient in the pharmaceutical compositions 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 employed, the route of administration of the particular compound employed, the time of administration, the rate of excretion, the duration of treatment, other drugs, compounds, and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, and past medical history of the patient being treated, and factors well known in the medical arts.

[0191] 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.

[0192] 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 results. Routes of administration of the engineered 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" generally refers to methods of administration other than enteral and topical administration by injection, and includes, but is 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.

[0193] Alternatively, the therapeutic fusion proteins of the invention can be administered by parenteral routes, such as topical, epidermal, or mucosal routes of administration.

[0194] The therapeutic fusion proteins of the present disclosure can be prepared with carriers that protect the protein from rapid release, such as controlled-release formulations, 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*, *J.R. Robinson*, ed., *Marcel Dekker*, Inc., New York, 1978.

[0195] In certain embodiments, 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 therapeutic compounds of the present invention cross the BBB (if necessary), they can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, e.g., 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, e.g., Ranade VV (1989) J. Clin. Pharmacol., 29:685).

[0196] Therapeutic Uses and Methods of the Invention The therapeutic fusion proteins of the present 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 for treating, preventing, or diagnosing acute or chronic inflammatory and immune system-driven organ and microvascular disorders.

[0197] The therapeutic fusion proteins of the present invention are useful for treating, preventing, or ameliorating acute and chronic inflammatory organ damage, particularly inflammatory damage when endogenous homeostatic clearance mechanisms or efferocytosis pathways for removing dead cells, cell fragments, and prothrombotic / inflammatory microparticles are significantly downregulated, including, but not limited to, myocardial infarction, acute kidney injury (AKI), acute stroke and inflammation, and organ damage resulting from ischemia / reperfusion of the gastrointestinal tract, liver, spleen, lungs, kidneys, pancreas, heart, brain, spinal cord, and / or crushed limbs.

[0198] 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 respiratory distress syndrome, wound healing, tendon repair, and neurological disorders.

[0199] In one embodiment, the neurological disorder may be selected from conditions with 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 disorders include amyloid beta-related neurological disorders such as Alzheimer's disease, Parkinson's disease, and depression.

[0200] 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 the expression of at least one osteoclast marker, such as NFATc1, cathepsin K, and αvβ3 integrin. In one embodiment, the administration inhibits osteoclastogenesis. In another embodiment, the administration inhibits RANKL-induced osteoclastogenesis. In yet another embodiment, the administration inhibits bone resorption. In yet another embodiment, the administration inhibits the expression of at least one bone resorption stimulator, such as TNF, IL-6, IL-17A, MMP-9, Ptgs2, RANKL, Tnfsf11, CXCL1, CXCL2, CXCL3, CXCL5, and combinations thereof. In another embodiment, the administration inhibits the expression of at least one pro-inflammatory cytokine selected from the group consisting of IL-8 and CCL2 / MCP-1.

[0201] In one embodiment, tissue fibrosis can be the fibrosis in liver, lung, diaphragm, kidney, brain, heart, and the 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 cirrhosis, which may or may not be caused by NASH.

[0202] 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 the worsening and severity of the disease. 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 (ARDS), 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), for example, 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); and chronic respiratory failure leading to hypoxemia.

[0203] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of lung injury caused by Coronaviruses, such as ARS-CoV, SARS-CoV-2, or MERS-CoV. In one embodiment, a therapeutic fusion protein of the present disclosure is provided for use in treating SARS-CoV-2 infection in COVID19 patients.

[0204] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of transfusion-associated pulmonary injury (TRALI).

[0205] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of chronic respiratory failure that leads to hypoxemia.

[0206] Therapeutic fusion proteins of the present disclosure, for example, therapeutic fusion proteins comprising a domain of EDIL3 of the present disclosure, may also be useful in diagnosing, treating, preventing, or ameliorating the severity of post-surgical peritoneal adhesions.

[0207] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of heart failure.

[0208] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of hemodialysis.

[0209] 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.

[0210] 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.

[0211] 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.).

[0212] 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 that result in injury, such as work-related accidents, falls, traffic accidents, ballistic and combat injuries, etc.

[0213] Therapeutic fusion proteins of the present disclosure may also be useful in diagnosing, treating, preventing, or ameliorating the severity of osteoclast-mediated pathologies.

[0214] Therapeutic fusion proteins of the present disclosure can 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), for the treatment or prevention of, e.g., the diseases listed above.

[0215] With respect to additional therapeutic agents, "administered 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, there is overlap in administration, such that delivery of one therapeutic agent is still occurring when delivery of a second therapeutic agent begins. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, delivery of one therapeutic agent ends before 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., an equivalent effect is seen with the second therapeutic agent to a lesser extent, or the second therapeutic agent alleviates symptoms to a greater extent than would be seen if the second therapeutic agent were administered without the first therapeutic agent, or a similar condition 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 that 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 remains detectable when the second therapeutic agent is delivered.

[0216] The term "concurrently" is not limited to administration of therapies (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 over when they are otherwise administered. For example, each therapeutic agent can be administered to a subject at the same time or at different times, sequentially in any order; if not administered simultaneously, they should be administered sufficiently close in time to provide the desired therapeutic or prophylactic effect. Each therapeutic agent can be administered to a subject separately, in any appropriate form and by any suitable route.

[0217] The therapeutic fusion protein described herein and the additional therapeutic agent can be administered simultaneously, in the same or separate pharmaceutical compositions 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 a different route of administration as the fusion protein.

[0218] 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.

[0219] When administered in combination, a therapeutic fusion protein described herein and an additional therapeutic agent (e.g., a second or third agent) can be administered in an amount or dosage that is 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, a therapeutic fusion protein described herein, an additional agent (e.g., a second or third agent), or all, is administered at a dosage that is 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 a therapeutic fusion protein described herein, an additional agent (e.g., a second or third agent), 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 seeking to achieve the same therapeutic effect.

[0220] 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 cyclosporin 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, for example, recombinant binding molecules having at least a portion of the extracellular domain of CTLA4 or a variant thereof, for example, at least the extracellular portion of CTLA4 or a variant thereof linked to a non-CTLA4 protein sequence, for example, CTLA4Ig (e.g., designated ATCC68629) or a variant thereof, for example, LEA29Y; adhesion molecule inhibitors, for example, 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, for example, infliximab, adalimumab, CDP870, or receptor constructs against TNF-RI or TNF-RII, for example, etanercept, PEG-TNF-RI; blockers of inflammatory cytokines, IL-1 blockers, for example, anakinra or IL-1 trap, canakinumab, IL-13 blockers, IL-4 blockers, IL-6 blockers;Chemokine blocking agents, such as inhibitors or activators of proteases, e.g., 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, e.g., converters, detoxifiers, removers, e.g., ATP converters, HMGB-1 modulators, histone detoxifiers; inhibitors of superantigen-induced immune responses; complement inhibitors and extracorporeal plasma exchange devices;

[0221] kit Kits comprising a composition, e.g., a therapeutic fusion protein of the present disclosure, and instructions for use are also within the scope of the present invention. Such kits comprise a therapeutically effective amount of the fusion protein according to the present disclosure. Furthermore, 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 also include additional therapeutic agents (described below) for treating patients with autoimmune or inflammatory disorders or AOI. Such kits may also include instructions for administering the therapeutic fusion protein to treat patients. Such instructions may provide the dosage, route of administration, regimen, and total treatment duration for use with the enclosed fusion protein. The kit typically includes a label indicating the intended use of the contents of the kit. The term label includes any written or recorded material supplied on or with the kit, or otherwise associated with the kit. The kit 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.

[0222] Embodiment The present disclosure provides the following embodiments.

[0223] 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 PS-binding domain is a truncation mutant.

[0224] 2. The fusion protein of embodiment 1, wherein the PS-binding domain is a truncated mutant of at least one PS-binding domain listed in Table 2.

[0225] 3. A fusion protein according to embodiment 1 or embodiment 2, wherein the PS-binding domain is a truncated mutant of the PS-binding motif of MFG-E8 or EDIL3.

[0226] 4. The fusion protein of embodiment 3, wherein the PS-binding domain is a truncated mutant of the PS-binding motif of MFG-E8.

[0227] 5. The fusion protein of embodiment 4, wherein the PS-binding domain is a discoidin domain.

[0228] 6. A fusion protein according to any one of embodiments 1 to 5, wherein the PS binding domain is a C1 domain.

[0229] 7. A fusion protein according to any one of embodiments 1 to 6, wherein the PS binding domain does not comprise a C2 domain.

[0230] 8. A 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 PS-binding domain is a C1 domain.

[0231] 9. The fusion protein of any one of embodiments 1 to 8, wherein the integrin binding domain binds to one or more integrins.

[0232] 10. The fusion protein of embodiment 9, wherein the integrin-binding domain binds to αvβ3 and / or αvβ5 and / or α8β1 integrin.

[0233] 11. The fusion protein of any one of embodiments 9 to 11, wherein the integrin binding domain comprises an arginine-glycine-aspartic acid (RGD) motif.

[0234] 12. Therapeutic fusion proteins of the formula EGF-SC (Formula I), wherein: (i) EGF is an integrin-binding domain, and the integrin-binding domain binds to one or more integrins; (ii) S is a solubilization domain; (iii) C is a truncated PS-binding domain; Therapeutic fusion proteins.

[0235] 13. The therapeutic fusion protein of embodiment 12, wherein the integrin binding domain binds to αvβ3 and / or αvβ5 and / or α8β1 integrin.

[0236] 14. The therapeutic fusion protein of embodiment 12 or embodiment 13, wherein the integrin binding domain comprises an arginine-glycine-aspartic acid (RGD) motif.

[0237] 15. The therapeutic fusion protein of any one of embodiments 12-14, wherein the integrin-binding domain is an EGF-like domain of MFG-E8, EDIL3, or a protein containing an integrin-binding domain listed in Table 1.

[0238] 16. The therapeutic fusion protein of any one of embodiments 12-15, wherein the truncated PS-binding domain is a truncated mutant of a PS-binding domain listed in Table 2.

[0239] 17. The therapeutic fusion protein according to any one of embodiments 12 to 16, wherein the PS-binding domain is a truncated mutant of the PS-binding motif of MFG-E8 or EDIL3.

[0240] 18. A fusion protein according to any one of embodiments 12 to 16, wherein the PS-binding domain is a truncated mutant of the PS-binding motif of MFG-E8.

[0241] 19. A fusion protein according to any one of embodiments 12 to 18, wherein the PS-binding domain is a discoidin domain.

[0242] 20. The therapeutic fusion protein according to any one of embodiments 11 to 16, wherein the truncated PS-binding domain comprises any of the C1 and / or C2 domains of the PS-binding domains listed in Table 2.

[0243] 21. The therapeutic fusion protein according to any one of embodiments 11 to 18, wherein the truncated PS-binding domain is the C1 domain.

[0244] 22. The therapeutic fusion protein of any one of embodiments 11-21, wherein the truncated PS-binding domain does not comprise the C2 domain.

[0245] 23. The fusion protein of any one of embodiments 12 to 22, wherein the solubility domain is directly linked to the integrin-binding domain, the PS-binding domain, or both domains.

[0246] 24. The fusion protein according to any one of embodiments 12 to 23, wherein the solubility domain is indirectly linked to the integrin-binding domain and / or the PS-binding domain by a linker.

[0247] 25. The fusion protein of any one of embodiments 12 to 24, wherein the integrin binding domain has the amino acid sequence of SEQ ID NO: 2, or at least 90% sequence identity thereto.

[0248] 26. The fusion protein of any one of embodiments 12 to 25, wherein the integrin binding domain has the amino acid sequence of SEQ ID NO: 77, or at least 90% sequence identity thereto.

[0249] 27. The fusion protein of any one of embodiments 12 to 26, wherein the integrin binding domain has an amino acid sequence selected from 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, or at least 90% sequence identity thereto.

[0250] 28. The fusion protein of any one of embodiments 12 to 27, wherein the PS-binding domain has the amino acid sequence of SEQ ID NO: 141 or SEQ ID NO: 142, or at least 90% sequence identity thereto.

[0251] 29. The fusion protein of any one of embodiments 12 to 28, wherein the PS-binding domain has the amino acid sequence of SEQ ID NO: 144, or at least 90% sequence identity thereto.

[0252] 30. The fusion protein of any one of embodiments 12 to 29, wherein the solubility domain is HSA and has the amino acid sequence of SEQ ID NO: 4, or at least 90% sequence identity thereto.

[0253] 31. A fusion protein according to any one of embodiments 12 to 30, comprising in this order an integrin-binding domain-HSA-PS-binding domain.

[0254] 32. 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, and a C1 domain and / or a C2 domain; and the fusion protein comprises a sequence from wild-type human MFG-E8 (sequence number 1) or a functional variant thereof.

[0255] 33. The fusion protein of embodiment 32, wherein the solubility domain is inserted between the EGF-like domain and the C1 or C2 domain.

[0256] 34. The fusion protein of embodiment 32 or 33, wherein the solubility domain is HSA, HSA D3 or Fc-IgG, or a functional variant thereof.

[0257] 35. The fusion protein of any one of embodiments 32 to 34, wherein the solubility domain comprises human serum albumin (HSA), or a functional variant thereof.

[0258] 36. A fusion protein, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 34, or at least 90% sequence identity thereto.

[0259] 37. A fusion protein, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 36, or at least 90% sequence identity thereto.

[0260] 38. A fusion protein, wherein the fusion protein has an amino acid sequence selected from SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:125, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, or SEQ ID NO:147, or at least 90% sequence identity thereto.

[0261] 39. A fusion protein, wherein the fusion protein has the amino acid sequence of SEQ ID NO: 147, or at least 90% sequence identity thereto.

[0262] 40. The fusion protein is a. Recovering impaired macrophage efferocytosis in a human macrophage-neutrophil efferocytosis assay; b. Reduces the number of plasma microparticles by clearance in a human endothelial microparticle efferocytosis assay; and / or c. Protects against multi-organ injury in a model of acute renal ischemia; d. Improve disease burden in models of liver fibrosis; 40. The fusion protein of any one of embodiments 1 to 39.

[0263] 41. An isolated nucleic acid encoding the amino acid sequence of any one of embodiments 36-39.

[0264] 42. A cloning or expression vector comprising a nucleic acid according to embodiment 41.

[0265] 43. A viral vector comprising the isolated nucleic acid of embodiment 41, preferably wherein the viral vector comprising the isolated nucleic acid of embodiment 41 is derived from AAV.

[0266] 44. The viral vector of embodiment 43, wherein the vector is administered to a subject in need thereof, for example a human subject.

[0267] 45. The viral vector of embodiment 43, for use in the treatment and / or prevention of diseases listed herein.

[0268] 46. ​​A recombinant host cell suitable for producing a therapeutic fusion protein, comprising one or more cloning or expression vectors according to embodiment 42, and optionally a secretion signal.

[0269] 47. The recombinant host cell of embodiment 46, wherein the host cell is, for example, a prokaryotic, yeast, insect or mammalian cell.

[0270] 48. A fusion protein according to any one of embodiments 1 to 40, wherein expression of the protein in a host cell results in a yield of at least 10 mg / L.

[0271] 49. A fusion protein according to any one of embodiments 1 to 40 or 48, wherein expression of the protein in mammalian cells results in at least a 100-fold increase in yield over wild-type MFG-E8 (SEQ ID NO: 1).

[0272] 50. A pharmaceutical composition comprising a fusion protein according to any one of embodiments 1 to 40, and at least one pharmaceutically acceptable carrier.

[0273] 51. 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 of any one of embodiments 1-40.

[0274] 52. A fusion protein according to any one of embodiments 1 to 40, for use in the treatment or prevention of an inflammatory disorder or inflammatory organ damage in an individual in need thereof.

[0275] 53. The method according to embodiment 51 or the use according to embodiment 52, wherein the inflammatory disorder or inflammatory organ damage is acute kidney injury, sepsis, myocardial infarction, acute stroke, burns, traumatic injury, and inflammation and organ damage due to ischemia / reperfusion.

[0276] 54. The method according to embodiment 51 or the use according to embodiment 52, wherein the fusion protein is administered in combination with another therapeutic agent.

[0277] 55. The method or use according to embodiment 54, wherein the other therapeutic agent is an immunosuppressant, immunomodulatory agent, anti-inflammatory agent, antioxidant, anti-infective agent, cytotoxic agent or anti-cancer agent.

[0278] 56. 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 or a C2 domain, and comprises a functional variant of a sequence from wild-type human MFG-E8 (sequence number 1).

[0279] 57. The fusion protein of embodiment 56, wherein the solubility domain is inserted between the EGF-like domain and the C1 domain.

[0280] 58. The fusion protein of embodiment 56, wherein the solubility domain is inserted between the EGF-like domain and the C2 domain.

[0281] 59. The fusion protein according to any one of embodiments 56 to 58, wherein the solubility domain is HSA, HSA D3 or Fc-IgG, or a functional variant thereof.

[0282] 60. The fusion protein of any one of embodiments 56-59, wherein the fusion protein has an amino acid sequence selected from the SEQ ID NOs listed in Table 4.

[0283] 61. An isolated nucleic acid encoding a fusion protein according to any one of embodiments 56 to 60.

[0284] 62. A viral vector comprising the isolated nucleic acid of embodiment 61, preferably wherein the viral vector comprising the isolated nucleic acid of embodiment 61 is derived from AAV.

[0285] 63. The viral vector of embodiment 62, wherein the vector is administered to a subject in need thereof, for example a human subject.

[0286] 64. The viral vector of embodiment 62, for use in the treatment and / or prevention of diseases listed herein.

[0287] 65. A cloning or expression vector comprising a nucleic acid according to embodiment 61.

[0288] 66. A recombinant host cell suitable for producing a therapeutic fusion protein, comprising one or more cloning or expression vectors according to embodiment 65, and optionally a secretion signal.

[0289] 67. The recombinant host cell of embodiment 66, wherein the host cell is, for example, a prokaryotic, yeast, insect or mammalian cell.

[0290] 68. A fusion protein according to any one of embodiments 56 to 60, wherein expression of the protein in a host cell results in a yield of at least 10 mg / L.

[0291] 69. A fusion protein according to any one of embodiments 56 to 60, wherein expression of the protein in mammalian cells results in at least a 100-fold increase in yield over wild-type MFG-E8.

[0292] It is to be understood that each embodiment may be combined with one or more other embodiments to the extent that such combination is consistent with the description of the embodiment. It is further to be understood that the embodiments provided above are understood to include all embodiments, including such embodiments as a result of combinations of embodiments.

[0293] 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. [Example]

[0294] 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 skilled in the art and are encompassed by the appended claims.

[0295] Example 1: Production of fusion proteins MFG-E8 is a multidomain 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 have shown very low protein aggregation and expression rates (Castellanos et al., (2016) Protein Expression Purification 1124:10-22). Therefore, to attempt to solubilize the protein and enhance its expression, we investigated the effect of fusing several proteins to MFG-E8.

[0296] The solubilizing domain (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 Figure 1. Furthermore, fusion to Fc-IgG1 or HSA may extend the half-life of the molecule in vivo due to the binding of these proteins to FcRn. Fusion of MFG-E8 to Fc-IgG1 or HSA can also enhance the production and solubility of the fusion protein, as shown in the following example (Castellanos et al., (2016) supra).

[0297] 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).

[0298] [Table 58]

[0299] Example 2: Generation of wild-type MFG-E8 and MFG-E8 HSA fusions; expression and purification The methods for producing fusion proteins are described below. Briefly, MFG-E8 and MFG-E8 fusions and EDIL fusions, particularly fusions to HSA, were produced according to the following methods.

[0300] 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 protein expression, the wild-type or modified strand vector was 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. The recombinant expression vector was then introduced into the host cells, and the construct was produced by culturing the cells for an additional 7 days to allow secretion into medium (HEK, serum-free medium) supplemented with 0.1% pluronic acid, 4 mM glutamine, and 0.25 μg / ml antibiotic.

[0301] 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.

[0302] 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 mM NaCl, 20 mM imidazole, pH 7.0. The resin was washed three times with 15 column volumes of 20 mM NaPO4, 0.5 mM NaCl, 20 mM imidazole, pH 7.0 before the protein was eluted with 10 column volumes of elution buffer (20 mM NaPO4, 0.5 mM NaCl, 500 mM imidazole, pH 7.0).

[0303] When proteins were captured by Protein A or anti-HSA chromatography, the filtered conditioned medium 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 the proteins were eluted with 10 column volumes of elution buffer (50 mM citrate, 90 mM NaCl, pH 2.5), the pH of which was neutralized using 1 M TRIS pH 10.0.

[0304] 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).

[0305] Representative expression gels of fusion proteins are shown in Figure 2. 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). Proteins under reducing and non-reducing conditions are shown in Figure 2C. Because the heterodimer tends to collapse under reducing conditions, both conditions were tested. Expression and yield results after purification of an additional 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 at a different location. 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.

[0306] [Table 59]

[0307] 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), in which 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 200 V for 30 minutes, the proteins contained in the gel were visualized using a stain-free imager (Biorad, Gel Doc EZ). As illustrated in Figure 2E, SDS-PAGE shows the produced and purified recombinant proteins. Columns 1 and 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 Lane 7: EGF[EDIL3]_HSA_C1[EDIL3] SEQ ID NO: 71 98.22 kDa Lane 8: EGF[EDIL3]_HSA_C2[EDIL3] SEQ ID NO: 135 98.20 kDa Lane 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

[0308] 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 a 384-well microtiter plate (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 plate was treated with phosphate-buffered saline (PBS) containing 3% fatty acid-free bovine serum albumin (BSA) for 1.5 hours at room temperature.

[0309] Binding of the fusion protein to L-α-phosphatidylserine was assessed by competing with the binding of biotinylated mouse MFG-E8 / lactadherin (produced in-house, mMFG-E8:biotin). The protein was diluted in PBS containing 3% fatty acid-free BSA, pH 7.4, and incubated on L-α-phosphatidylserine-coated microtiter plates for 30 minutes. 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 minutes. Unbound mMFG-E8:biotin was removed by three wash 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 to DELFIA™ assay buffer (Perkin Elmer 1244-111, MA, US) for 20 minutes at room temperature, followed by three washes with DELFIA™ assay buffer. Europium was revealed according to the manufacturer's instructions (Perkin Elmer 1244-105, Boston MA, US). Time-resolved fluorescence of europium was quantified using an Envision™ 2103 multilabel plate reader (Perkin Elmer, CT, US). Data analysis was performed using MS Excel and GraphPad Prism software.

[0310] Polypropylene plates are low-protein-binding microtiter plates 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 on polypropylene plates, wild-type MFG-E8 lost potency in the L-α-phosphatidylserine competition assay compared to dilutions performed on non-binding plates. These data, shown in Figure 3, indicate that when polypropylene plates, already optimized for low protein binding, are used, wild-type MFG-E8 is partially lost during the liquid handling and dilution steps (Figure 3A). These results indicate that the inherent stickiness of wild-type MFG-E8 poses challenges for handling in the laboratory and possibly during pharmaceutical manufacturing and production, where capture and polishing steps are required to produce high-yield and very pure drug substance. In contrast, the stickiness of the engineered protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) was significantly reduced compared to wild-type MFG-E8, and no substantial difference was observed between dilutions performed on non-binding and polypropylene plates (Figure 3B). These data suggest that inserting solubilization domains into the disclosed proteins can improve their technical handling, leading to improved step yields and therefore overall yields during the manufacturing process.

[0311] 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). Binding of FP278 to immobilized L-α-phosphatidylserine or 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. The IC values ​​obtained for the fusion proteins were 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 for FP270 (EGF-HSA-C2; SEQ ID NO: 36), which, along with FP250 (EGF-HSA; SEQ ID NO: 32), did not compete in this assay format. 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 primary PS-binding moiety of human MFG-E8. This finding was surprising, because the literature suggests that the C2 domain of MFG-E8 is the primary 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 primary integral PS-binding domain of the MFG-E8 engineered protein and is crucial 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 observed with fusion proteins containing the C1-C2 or C1-C1 tandem domains (the latter not shown).

[0312] 3.2 αv integrin adhesion assay The fusion protein was diluted in phosphate-buffered saline (PBS) pH 7.4, and 50 μL of a 24 nM solution was immobilized overnight by adsorption (96-well plate, Nunc Maxisorb) (1.2 nM / well). The plate was then treated with PBS containing 3% fatty acid-free bovine serum albumin (BSA) for 1.5 hours at room temperature. αvβ3 integrin-expressing lymphoma cells (ATCC-TIB-48 BW5147.G.1.4, ATCC, USA) were cultured in RPMI 1640 medium supplemented with GlutaMax, 25 mM HEPES, 10% FBS, Pen / Strep, 1 mM sodium pyruvate, and 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 minutes. BW5147.G.1.4 cells were resuspended in adhesion buffer (TBS, 0.5% BSA, 1 mM MnCl2, pH 7.4) and allowed to adhere at 50,000 cells / well for 40 minutes at room temperature. Nonadherent 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.

[0313] 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, demonstrating integrin-dependent cell adhesion to the immobilized engineered protein (Figure 5A). A single point mutation in the integrin-binding motif RGD (RGD>RGE) of the EGF-like domain (FP280; SEQ ID NO: 38) resulted in complete inhibition of cell adhesion, demonstrating that a functional and accessible RGD-binding motif in the fusion protein is essential for αv integrin-dependent adhesion (Figure 5B). An immobilized EGF-HSA protein lacking the C1-C2 domain, FP250 (SEQ ID NO: 32), did not or only weakly support the adhesion of BW5147.G.1.4 cells, despite the presence of 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, likely due to steric reasons. This defect 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, promoted αv-integrin-dependent cell adhesion similarly to wild-type MFG-E8 when expressed in CHO or HEK cells (Figure 5D).

[0314] 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.

[0315] 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 40 ng / mL recombinant human M-CSF (Macrophage Colony Stimulating Factor, R&D Systems, US) in RPMI 1640 containing 25 mM HEPES, 10% FBS, Pen / Strep, 1 mM NaPyr, and 50 μM β-Merck for 5 days. One day before efferocytosis, macrophages were labeled with PKH26 using a Red Fluorescent Dye Linker kit (Sigma MINI26, US). Cells were resuspended in RPMI1640 containing 25 mM HEPES, 10% FBS, Pen / Strep, 1 mM NaPyr, and 50 μM β-Merck, seeded at 40,000 cells / well into black 96-well plates (Corning, US), and allowed to adhere for 20 hours.

[0316] Neutrophils: Human neutrophils were isolated from buffy coats by dextran sedimentation combined with a Ficoll™ density gradient as follows: Plasma was removed from the buffy coat by centrifugation of the diluted buffy coat. The cell harvest was diluted with 1% dextran (from Leuconostoc spp., MW 450,000–650,000; Sigma, USA) and allowed to settle on ice for 20–30 min.

[0317] 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 medium (RPMI 1640 + GlutaMax containing 25 mM HEPES, 10% FBS, Pen / Strep, 0.1 mM NaPyr, 50 μM b-Merck) 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.

[0318] Efferocytosis assay M0 macrophages were incubated with the fusion protein for 30 minutes. Apoptotic labeled neutrophils were added at a ratio of 1:4 M0 / neutrophils. Efferocytosis of apoptotic neutrophils by macrophages was visualized by the increase in DRAQ5 fluorescence intensity upon localization of neutrophils in the low-pH lysosomal compartment of M0 macrophages.

[0319] Efferocytosis was quantified using an ImageXpress Micro XLS Widefield High Content Analysis System (Molecular Devices, CA, USA). 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.

[0320] Figure 6 shows 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. The fusion protein increases the internalization of pHrodo-labeled killed human neutrophils into macrophages, exceeding the already high efferocytic capacity of M0 macrophages, shown as a basal level. Figure 7 shows that the recombinant fusion protein FP278 can rescue endotoxin (lipopolysaccharide)-induced 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 an individual donor, and the right panel shows the average impairment (%) of efferocytosis across the three donors. FIG. 7B shows the rescue of this endotoxin (LPS)-injured efferocytosis of killed neutrophils by human macrophages using the fusion protein FP278.

[0321] Rescue of Staphylococcus aureus (S. aureus) particle-injured efferocytosis of killed neutrophils by human macrophages using 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 the 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 the figure). Figure 8B shows the effect of 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 was reached. 50 The effect of increasing concentrations of ATP (8 nM) is shown.

[0322] 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 (derived 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). Cells were dissociated for harvesting or passage using Accutase™ (Thermo Fischer Scientific, US).

[0323] Jurkat E6-1 cells were obtained from ATCC (American Type Culture Collection, US) and grown in RPMI 1640 medium (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).

[0324] 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 with 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).

[0325] 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 and resuspended in culture medium to obtain a total of 6.6 x 10 cells. 4 The cell count was determined as cells / mL. 150 μL of this cell suspension was added per well to a 96-well tissue culture plate (Corning™, US). HUVECs were incubated for an additional 16–20 hours in a 37°C / 5% CO2 / 95% humidity incubator.

[0326] 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 × g for 5 min at room temperature and then cultured at 1 × 10 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.

[0327] On day 2, the medium was removed from the HUVECs by aspiration, and 25 μL of fresh prewarmed (37°C) medium was added, followed by 25 μL of fusion protein or control diluted in prewarmed (37°C) medium. Dilutions were performed in a non-binding surface (NBS)-treated 96-well plate (Corning™, US). The fusion protein was allowed to interact with the HUVECs for 30 minutes at 37°C / 5% CO2 / 95% humidity before adding killed Jurkat cells.

[0328] 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 × g for 5 min at room temperature, and 5 × 10 6 The cells were resuspended at a density of 1000 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 minutes at 37°C, the remaining reactive pHrodo™ Green STP Ester was inactivated with staining medium supplemented with 10% FBS for an additional 5 minutes at 37°C. pHrodo™ Green-labeled cells were washed once and counted at 3 x 10 cells in HUVEC medium. 6 The concentration was adjusted to 1.5 x 10 cells / mL. 6pHrodo™ Green-labeled Jurkat cells were added to HUVECs at 1000p / well and incubated at 37°C / 5% CO2 / 95% humidity for 5 hours. The medium was removed, and HUVECs 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 x g (4°C) for 5 minutes. 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 then measured using a BD LSRFortessa™ flow cytometer (BD Biosciences, US). pHrodo™ Green fluorescence intensity was recorded as an indicator of lysosomal localization of engulfed Jurkat cells. 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.

[0329] The effects 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 are shown in Figure 9. Internalization of pHrodo-labeled dead human Jurkat T cells by HUVEC is strongly promoted by the fusion protein FP278. The results indicate that endothelial cells armed with the fusion protein become efficient phagocytes of dead cells. Surprisingly, the effectiveness of the fusion proteins in this assay clearly depends on the presence of the C1-C2 or C1-C1 tandem domains. For example, a fusion protein consisting of EGF-HSA-C2 (FP270) is inactive in this experimental setting, as shown in Figure 9. Figure 10 shows our highly surprising finding that the location of the HSA domain in the modified protein, i.e., at either 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.

[0330] Figure 11 shows a comparison of the promotion of endothelial efferocytosis by various fusion protein formats 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, 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 Fc moiety formats are shown: the 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 positioned Fc moiety, at three different concentrations (0.72, 7.2, and 72 nM), to the wild-type MFG-E8 control. Efferocytosis of dead Jurkat cells by HUVEC was promoted only by engineered proteins in which the HSA or Fc portion was inserted after the EGF-like domain. Figure 11D shows that insertion of a solubility 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)) contains 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, resulting in the expression of novel recombinant engineered proteins of very high purity (Figure 2B). Furthermore, we surprisingly found that the recombinant engineered protein FP050 derived from EDIL3 promoted efferocytosis of dying Jurkat cells by endothelial cells (HUVECS), demonstrating the core function of cross-linking proteins and demonstrating that cross-linking protein domains are useful for the design of functional novel recombinant engineered proteins.

[0331] 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 (derived 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). Cells were dissociated for harvesting or passage using Accutase™ (Thermo Fischer Scientific, US).

[0332] 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 (200 x g, 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 20,000 x g for 15 min at room temperature. The pellet was resuspended in PBS, aliquots were prepared, and stored at -80 °C. The microparticle preparation was 85–100% PS-positive as measured by flow cytometry using AlexaFluor™ 488-labeled mouse MFG-E8 / lactadherin (in-house, Novartis). The number of microparticles was determined using dedicated counting beads (BioCytex / Stago, France). Flow cytometry buffer was prepared with 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).

[0333] 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 x 10 cells. 4The cell count was determined as cells / mL. 150 μL of this cell suspension was added per well to a 96-well tissue culture plate (Corning™, US). HUVEC cells were incubated for an additional 16–20 hours in a 37°C / 5% CO2 / 95% humidity incubator.

[0334] On day 2, the medium was removed from the HUVEC cells by aspiration, and 25 μL of fresh prewarmed (37°C) medium was added. Then, 25 μL of the fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) or control diluted in prewarmed (37°C) medium at three different concentrations (0.3 nM, 3 nM, or 30 nM) was added. For the dilutions, a non-binding surface (NBS)-treated 96-well plate (Corning™, US) was used. The test protein was allowed to interact with the HUVEC cells for 30 minutes at 37°C / 5% CO2 / 95% humidity before the addition of platelet-derived microparticles.

[0335] Centrifuge the required amount of microparticles at 20,000 x g for 15 minutes at 4°C to remove 2 x 10 8 The microparticles were resuspended at a density of 10 ... 8 Adjusted to 5 x 10 particles / mL 6pHrodo™ Green-labeled microparticles (particles / well) were added to HUVEC cells and incubated for 5 hours at 37°C, 5% CO2, and 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 x g (4°C) for 5 minutes. 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 using a BD LSRFortessa™ flow cytometer (BD Biosciences, US). pHrodo™ Green fluorescence intensity was recorded as an indicator of lysosomal localization of endocytosed 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. The 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).

[0336] 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 A direct binding assay was performed to characterize the binding of the fusion protein FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42) to FcRn. The kinetic binding affinity constant (KD) was measured on the captured protein using recombinant human FcRn as the 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, the protein was diluted in 10 mM NaP, 150 mM NaCl, 0.05% Tween 20, pH 5.8, and immobilized on the 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 for use 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 for double referencing during data evaluation. Double-referenced sensorgrams were used for data evaluation and the dissociation constants (KD) were analyzed.

[0337] The fusion protein FP330 binds to FcRn at pH 5.8 with an affinity of 1380 nM, but no binding was observed at pH 7.4 (see Table 5 above). These results are in good agreement with those of wild-type HSA (1000-2000 nM, pH 5.8, data not shown).

[0338] 5.2 Differential scanning calorimetry (DSC) of MFG-E8 and its mutants The thermal stability of the modified MFG-E8 protein variant FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) was measured using differential scanning calorimetry. 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 but omitting the protein. Partial molar heat capacities 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.

[0339] 5.3 Measurement 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 fluctuations of scattered light. The size distribution of the protein variants without fractionation, the estimated 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 were identified in the protein sample. The hydrodynamic radius of the protein was approximately 5-6 nm, indicating a monomeric protein in solution.

[0340] Next, concentration-dependent hydrodynamic radius measurements of the fusion protein FP278 were performed to estimate the protein solubility. 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) was 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.

[0341] 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, we investigated the fusion protein FP330 (EGF-HSA-C1-C2) using mass spectrometry (MS). The panel of mutant proteins was generated with linkers of various sizes and structures, e.g., a linker containing a GS between the EGF and HSA domains and / or a linker containing multiple GS or G4S between the HSA and C1 domains. Additionally, some mutants contained amino acid modifications, including deletions or substitutions (denoted as HSA* in Table 7). The panel of mutant fusion proteins is summarized in Table 7 below.

[0342] [Table 60]

[0343] 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 a proprietary CHO cell line, nucleic acids encoding MFG-E8 variants were synthesized by Geneart (Life Technologies) and cloned into a mammalian expression vector 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 Expifectamine CHO transfection reagent (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 variant 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)).

[0344] As can be seen from the expression data shown in Table 8, the mutant fusion proteins FP068 (SEQ ID NO: 46) and FP776 (SEQ ID NO: 48) showed approximately a 2-fold improvement in expression over the fusion protein FP330 (SEQ ID NO: 42).

[0345] [Table 61]

[0346] Further therapeutic fusion proteins were obtained following the method described in Example 1. For example, the expression levels (mg / l) obtained after the complete purification process (capture and polishing) are 4.3 for SEQ ID NO: 80 and 8.4 for SEQ ID NO: 82.

[0347] 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.

[0348] 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 the 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 minutes. As shown in Figure 13, the fusion proteins FP330, FP278, and FP776 can rescue endotoxin (lipopolysaccharide (LPS))-induced efferocytosis of killed neutrophils by human macrophages. The 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 impaired efferocytosis caused by the addition of LPS and even promoted efferocytosis once basal levels were reached.

[0349] Fusion proteins FP330, FP278, and FP776 were further characterized in a human endothelial (HUVEC) cell-Jurkat cell efferocytosis assay, according to 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 Jurkat T cells by HUVEC was significantly enhanced by FP330 (EC 50 = 3.4 nM; Figure 14A), FP278 (EC 50 =2.4 nM; Figure 14B) and FP776 (EC 50 This was strongly enhanced by increasing the concentration of fusion protein (=3 nM; Figure 14C). These results indicate that endothelial cells are armed with the fusion protein to become efficient phagocytes of dead cells.

[0350] 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 adherence to Swiss Federal Law and the NIH Principles of Laboratory Animal Care. The therapeutic fusion protein under study was administered either intraperitoneally (i.p.) or intravenously (i.v.) 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% by volume, carrier gas: oxygen) for 5 min before surgery. During surgery, animals were maintained under anesthesia via a face mask with 1–2% isoflurane / oxygen at a gas flow rate of 0.8–1.2 L / min. The abdominal skin was shaved and disinfected with Betaseptic (Mundipharma, France). Animals were placed on a homeothermic blanket (Rothacher, Switzerland) equipped with a homeothermic monitoring system (PhysiTemp, US - Physitemp Instruments LLC, US) and covered with sterile gauze. Body temperature was monitored throughout the procedure using a rectal probe (Physitemp Instruments LLC, US) and controlled to a temperature of 36.5–37.5°C. All animals, including sham controls, underwent unilateral right nephrectomy. 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 dissected and ligated, and the right kidney was removed. For animals experiencing 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 (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 the kidney changing 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 irrigate the abdominal cavity and rehydrate the tissue before wound closure. After irrigation, an additional 1 ml of sterile saline was added to the abdominal cavity as replacement fluid. When reperfusion began, the wound was closed in two layers (muscle and skin separately). The animals were then maintained under a red warm lamp until 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.

[0351] 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 serum and qPCR marker expression, 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 contrast agent uptake by magnetic resonance imaging, the fusion protein FP776 was administered prophylactically at 1.26 mg / kg 30 minutes before AKI induction or therapeutically at 2 mg / kg iv 5 hours after the induction of ischemia-reperfusion injury.

[0352] [Table 62]

[0353] 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 clinic analyzer according to the manufacturer's instructions (Axonlab, Switzerland).

[0354] 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 Lysing Matrix D tubes (MP Biomedicals, FR) in RLT buffer (RNeasy Mini Kit, Qiagen, DE) containing 134 mM beta-mercaptoethanol (Merck, DE) 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 minutes in a microcentrifuge (Eppendorf, DE). The supernatant was transferred to a QIAshredder spin column (Qiagen, DE) and centrifuged for 2 minutes. RNA extraction from the flow-through was performed according to the RNeasy Mini Kit manual, including DNase digestion. RNA concentration was measured using a 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 probes (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 using 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 s, 95°C; 4: 1 min, 60°C. Steps 3 and 4 were repeated 45 cycles. Data analysis was performed using ViiA7 software, and qPCR data analysis software was performed using MS Excel and GraphPad Prism software.

[0355] Uptake of contrast agents by the liver measured by magnetic resonance imaging (MRI) The method for performing MRI was adapted from a 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 a supine position in a Plexiglas cradle. Body temperature was maintained at 37 ± 1 °C using a heating pad. After a short induction period, 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.

[0356] 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 intravenously injected as a 1.2-second bolus in animals with AKI (24 hours after disease induction) or after sham surgery (24 hours after nephrectomy). The first bolus was administered for 1.2 seconds in conjunction with sequential acquisition of echoplanar images at a resolution of 400 ms per image. After acquiring 25 baseline images, a second bolus was injected over 1.2 seconds, and an additional 575 images were acquired after the bolus, resulting in a total of 600 images acquired over 4 minutes. The superparamagnetic contrast agent induced regional changes in susceptibility, resulting in signal attenuation proportional to renal perfusion. For each series of images, signal intensity was assessed in a region of interest (ROI) located in the cortex / external zone of the outer medulla. The position, shape, and size of the ROI were carefully selected so that the ROI covered approximately the same area despite respiration-induced kidney movement. The mean signal intensity of the pre-injection images provided the baseline intensity (S(0)). The perfusion index was determined from the mean value 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.

[0357] The SPIO nanoparticles used in this study had an average diameter of approximately 150 nm and were taken up by Kupffer cells in the liver. Therefore, in addition to renal perfusion, MRI could also monitor hepatic nanoparticle uptake by detecting contrast changes assessed in ROIs placed in the liver.

[0358] 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 i.p. (FP278) or i.v. (FP330 and FP776). This protection is reflected in a blockade of serum creatinine elevation (sCr). Figure 15A shows that the fusion protein FP278 at both tested doses significantly (p<0.0001) reduced serum creatinine levels compared with vehicle-treated animals and as effectively as murine MFG-E8. As shown in Figure 15B, the fusion protein FP330 protected kidney function in a dose-dependent manner, as did the fusion protein FP776 (Figure 15C), where serum creatinine levels were also blocked in a dose-dependent manner.

[0359] 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.

[0360] In summary, as shown in Figures 15 and 16, the fusion proteins FP278, FP330, and FP776 potently protected against elevation of these markers used in the clinical diagnosis of renal failure, and the observed efficacy was confirmed by histology (not shown).

[0361] Furthermore, 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 the spleen, lung, liver, heart, and brain. Representative mRNAs induced by selected injury-associated proteins (NGAL, KIM-1), induction of chemokines (not shown), or induction of acute phase response proteins such as serum amyloid A (SAA) are shown. Figures 17A and 17B illustrate such AKI-induced responses (serum amyloid A (SAA)) in the heart and lungs of mice, which were potently blocked and returned to sham levels after a single injection of the fusion protein.

[0362] Figure 18 shows the time course of liver uptake of the SPIO contrast agent Endorem®. Animals with AKI demonstrated 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 ischemia-reperfusion injury induction) protected against loss of contrast agent accumulation in the liver of AKI mice. These results suggest that in this mouse model, AKI causes significant impairment of particle clearance via endogenous Kupffer cells and that AKI causes microvascular damage that affects the accumulation of iron particle contrast agent in the liver. Treatment with the fusion protein FP776 protected against loss of clearance and microvascular damage and promoted contrast agent uptake at both doses tested when compared to sham animals.

[0363] Example 10: Characterization of MFG-E8-HSA modified proteins 10.2 αv integrin adhesion assay Fusion proteins were diluted in phosphate-buffered saline (PBS) pH 7.4, and 50 μL of the indicated concentrations were immobilized overnight by adsorption (96-well plates, Nunc Maxisorb). The plates were then treated with PBS containing 3% fatty acid-free bovine serum albumin (BSA) for 1.5 hours at room temperature. αvβ3 integrin-expressing lymphoma cells (ATCC-TIB-48 BW5147.G.1.4, ATCC, USA) were cultured in RPMI 1640 medium supplemented with GlutaMax, 25 mM HEPES, 10% FBS, Pen / Strep, 1 mM sodium pyruvate, and 50 μM β-mercaptoethanol. Cells were labeled with 3 μg / mL 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein, acetoxymethyl ester (BCECF AM) (Thermo Fisher Scientific Inc., USA) for 30 minutes. BW5147.G.1.4 cells were resuspended in adhesion buffer (TBS, 0.5% BSA, 1 mM MnCl, pH 7.4) and allowed to adhere at 50,000 cells / well for 40 minutes at room temperature. Non-adherent cells were removed by manual 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.

[0364] Adhesion of BW5147.G.1.4 cells to immobilized EGF-like domain containing fusion proteins. This finding suggests that under the experimental conditions tested, the RGD loop of the EGF-like domain fused to HSA in MFG-E8 or EDIL3 / DEL-1-based fusion proteins is accessible, allowing interaction with cellular av integrins.

[0365] Taken together, these data demonstrate that the fusion proteins of the present disclosure bind to cellular integrins, support integrin-dependent cell adhesion, and retain functionality in proteins with HSA domain inserts.

[0366] 10.3 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 40 ng / mL recombinant human M-CSF (Macrophage Colony Stimulating Factor, R&D Systems, US) in RPMI 1640 containing 25 mM HEPES, 10% FBS, Pen / Strep, 1 mM NaPyr, and 50 μM β-Merck for 5 days. One day before efferocytosis, macrophages were labeled with PKH26 using a Red Fluorescent Dye Linker kit (Sigma MINI26, US). Cells were resuspended in RPMI1640 containing 25 mM HEPES, 10% FBS, Pen / Strep, 1 mM NaPyr, and 50 μM β-Merck, seeded at 40,000 cells / well into black 96-well plates (Corning, US), and allowed to adhere for 20 hours.

[0367] Neutrophils: Human neutrophils were isolated from buffy coats by dextran sedimentation combined with a Ficoll™ density gradient as follows: Plasma was removed from the buffy coat by centrifugation of the diluted buffy coat. The cell harvest was diluted with 1% dextran (from Leuconostoc spp., MW 450,000–650,000; Sigma, USA) and allowed to settle on ice for 20–30 min.

[0368] 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 medium (RPMI 1640 + GlutaMax containing 25 mM HEPES, 10% FBS, Pen / Strep, 0.1 mM NaPyr, 50 μM b-Merck) 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.

[0369] Efferocytosis assay M0 macrophages were incubated with the fusion protein for 30 minutes. Apoptotic labeled neutrophils were added at a ratio of 1:4 M0 / neutrophils. Efferocytosis of apoptotic neutrophils by macrophages was visualized by the increase in DRAQ5 fluorescence intensity upon localization of neutrophils in the low-pH lysosomal compartment of M0 macrophages.

[0370] Efferocytosis was quantified using an ImageXpress Micro XLS Widefield High Content Analysis System (Molecular Devices, CA, USA). 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.

[0371] The effect of fusion proteins FP114 and FP133 (MFG-E8-derived EGF-HSA-C1, SEQ ID NO: xxx) on rescuing and promoting efferocytosis of killed neutrophils by LPS-treated human macrophages is shown in Figure 13D. The fusion proteins increase the internalization of pHrodo-labeled killed human neutrophils into macrophages, exceeding the already high efferocytic capacity of M0 macrophages. Figure 13E shows that recombinant fusion protein FP147 (EDIL / DEL-1-derived EGF_EGF_EGF_HSA_C1) can rescue endotoxin (lipopolysaccharide)-injured efferocytosis of killed neutrophils by human macrophages. Overall, the data demonstrate the surprising finding that C2-truncated MFG-E8- or EDIL3 / DEL-1-derived fusion proteins promote efferocytosis with low nM potency in vitro.

[0372] Example 11: Protection of mice from AKI 11.1 Acute Kidney Injury Models 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 adherence to Swiss Federal Law and the NIH Principles of Laboratory Animal Care. The therapeutic fusion protein under study was administered either intraperitoneally (i.p.) or intravenously (i.v.) 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% by volume, carrier gas: oxygen) for 5 min before surgery. During surgery, animals were maintained under anesthesia via a face mask with 1–2% isoflurane / oxygen at a gas flow rate of 0.8–1.2 L / min. The abdominal skin was shaved and disinfected with Betaseptic (Mundipharma, France). Animals were placed on a homeothermic blanket (Rothacher, Switzerland) equipped with a homeothermic monitoring system (PhysiTemp, US - Physitemp Instruments LLC, US) and covered with sterile gauze. Body temperature was monitored throughout the procedure using a rectal probe (Physitemp Instruments LLC, US) and controlled to a temperature of 36.5–37.5°C. All animals, including sham controls, underwent unilateral right nephrectomy. 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 dissected and ligated, and the right kidney was removed. For animals experiencing 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 (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 the kidney changing 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 irrigate the peritoneal cavity and rehydrate the tissue before wound closure. After irrigation, an additional 1 ml of sterile saline was added to the peritoneal cavity as replacement fluid. When reperfusion was initiated, the wound was closed in two layers (muscle and skin separately). The animals were then maintained under a red warm lamp until 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. The therapeutic fusion protein FP135 (EGF-HSA-C1; SEQ ID NO: x) was tested in the AKI model and administered at 1.5 mg / kg i.v. 30 minutes before the onset of ischemia-reperfusion injury. 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 clinic analyzer according to the manufacturer's instructions (Axonlab, Switzerland).

[0373] Example 12: EGF_HSA_C1 protects in a liver fibrosis model (CCL4 model) Liver fibrosis is a wound-healing response to various types of injury. Its progression can lead to cirrhosis and subsequently to hepatocellular carcinoma (HCC). Common causes of liver fibrosis in developed countries are alcohol abuse, viral hepatitis infection, and metabolic syndrome due to obesity, insulin resistance, and diabetes.

[0374] Long-term injury results in inflammation and deposition of extracellular matrix (ECM) proteins by myofibroblast-like cells, which are essentially activated hepatic stellate cells (HSCs). These cells produce alpha-smooth muscle actin (αSMA), deposit type I and III collagen, and produce matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs). As the disease becomes chronic, the composition of the ECM changes from type IV and VI collagens, glycoproteins, and proteoglycans to type I and III collagens and fibronectin.

[0375] If the injury is not severe, the liver can regenerate, whereby adjacent adult hepatocytes can replace apoptotic or necrotic cells. Resolution of fibrosis occurs when activated HSCs undergo apoptosis or revert to a more quiescent phenotype.

[0376] There are several in vivo models available that attempt to mimic various aspects of disease. Liver fibrosis models must be able to reflect various pathological and molecular features of human disease, be simple to set up, and have good reproducibility. Chemical-induced fibrosis models most closely meet these ideal characteristics, one of which is the rodent carbon tetrachloride (CCl4) liver fibrosis model. Repeated intraperitoneal injections of this hepatotoxin induce liver fibrosis, which has been shown to closely resemble human liver fibrosis. Furthermore, cessation of the injurious agent results in resolution of fibrosis, and thus the model is reversible.

[0377] In the first phase, CYP2E1 enzymes metabolize CCl4 to generate trichloromethyl free radicals, which contribute to an acute-phase response characterized by damage to lipid membranes and internal organelles of hepatocytes, ultimately leading to necrosis. Acute CCl4-mediated liver fibrosis is then characterized by activation of Kupffer cells and induction of an inflammatory response, resulting in the secretion of cytokines, chemokines, and other proinflammatory factors. This attracts and activates monocytes, neutrophils, and lymphocytes, contributing to liver necrosis and a subsequent robust regenerative response, resulting in substantial proliferation of hepatocytes and nonparenchymal hepatocytes approximately 48 hours after the initial CCl4 application. Histological fibrosis and scarring appear in the second phase of the disease, 2–3 weeks later. The third phase, with extensive fibrosis and massive hepatic fat accumulation, as well as increased serum levels of triglycerides and AST, can be observed 4–6 weeks after CCl4 injury. Complete resolution of CCl4-induced liver fibrosis in mice is typically observed within a few weeks after removal of the CCl4 toxin. Drugs with properties that promote fibrosis resolution may be particularly relevant for patients with established disease. For example, in patients with NASH (nonalcoholic steatohepatitis), chronic kidney disease, or scleroderma who have established fibrosis, demonstration of fibrosis resolution may be a primary clinical endpoint, potentially not only halting the disease but also restoring organ function. (Yanguaset al. 2016. Experimental models of liver fibrosis. Arch Toxicol. 2016;90:1025-1048. doi:10.1007 / s00204-015-1543-4)

[0378] CCL4 liver fibrosis model: Induction of the disease: CCl4 was freshly diluted in olive oil and injected intraperitoneally at a dose of 500 μl / kg three times a week for 6 weeks into 8-12 week-old male BALB / c mice (Netherlands). To induce liver fibrosis, CCl4 was administered for a total of 6 weeks. Treatment with EGF_HSA_C1 (FP135) began 4, 5, or 6 weeks after CCL4 treatment. EGF_HSA_C1 (FP135) was administered intraperitoneally at 0.8 mg / kg three times a week until the end of the experiment (3 days after CCL4 withdrawal).

[0379] reading: Liver enzymes such as ALT (alanine transaminase) and AST (aspartate transaminase) were measured as an assessment of liver damage in serum samples obtained at the time of CCL4 cessation (day 0) and 3 days after the end of the experiment. ALT and AST were analyzed using a Hitachi M40 clinical analyzer according to the manufacturer's instructions (Axonlab, Switzerland).

[0380] To quantify the collagen content in the animal livers, a hydroxyproline assay was performed using a total collagen assay (QuickZyme Biosciences, The Netherlands) according to the manufacturer's instructions. Expression of the collagen genes COL1A1 and COL1A2 by qPCR was performed as described in section 9.3.

[0381] Sonoelastography is a reliable and reproducible noninvasive method for assessing liver elasticity (stiffness) and has been shown to positively correlate with liver fibrosis (Li, R., Ren, X., Yan, F. et al. Liver fibrosis detection and staging: a comparative study of T1ρ MR imaging and 2D real-time shear-wave elastography. Abdom Radiol 43, 1713-1722 (2018). https: / / doi.org / 10.1007 / s00261-017-1381-3). Furthermore, this technique has been used in the clinic and will help translate preclinical data more appropriately to human liver disease with fibrosis. Liver stiffness was determined using ultrasound-based shear wave elastography (SWE) assessment. SWE was performed using an Aixplorer® device (Supersonic Imagine, Aix-en-Provence, France). For acquisition, mice were anesthetized with isoflurane (approximately 1.5%) and placed on a heating pad. An ultrasound probe (model SL25-15, SuperSonic Imagine, 25 MHz bandwidth, 256 elements) was attached to a support and brought close to the liver for evaluation. The probe allowed sufficient wave penetration for both B-mode and SWE acquisition.

[0382] Elastograms were acquired during exhalation to minimize respiratory motion artifacts. Three elastograms were acquired per mouse and time point. The average stiffness was then extracted from the three elastograms. Ultrasound examinations lasted approximately 5 minutes.

[0383] Example 13. Generation of C2-truncated MFG-E8 (EGF-C1) and HSA fusion (EGF-HSA-C1); expression and purification. Methods for producing the proteins disclosed herein are described below.

[0384] DNA was synthesized at GeneArt (Regensburg, Germany) and cloned into a mammalian expression vector using a restriction enzyme ligation-based cloning technique. For transient protein expression, the resulting plasmid was transfected into HEK293T cells. Briefly, the vector was 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 an expression vector encoding the protein of interest. The recombinant expression vector was then introduced into the host cells, and the construct was produced by culturing the cells for an additional 7 days to allow secretion into medium (HEK, serum-free medium) supplemented with 0.1% pluronic acid, 4 mM glutamine, and 0.25 μg / ml of antibiotic.

[0385] The produced constructs were then purified from the cell-free supernatant using immobilized metal ion affinity chromatography (IMAC) or anti-HSA capture chromatography.

[0386] When his-tagged proteins were captured by IMAC, the filtered conditioned medium was mixed with IMAC resin (GE Healthcare) equilibrated with 20 mM NaPO4, 0.5 mM NaCl, 20 mM imidazole, pH 7.0. The resin was washed three times with 15 column volumes of 20 mM NaPO4, 0.5 mM NaCl, 20 mM imidazole, pH 7.0 before the proteins were eluted with 10 column volumes of elution buffer (20 mM NaPO4, 0.5 mM NaCl, 500 mM imidazole, pH 7.0).

[0387] When proteins were captured by anti-HSA chromatography, the filtered conditioned medium was mixed with 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, and the pH was neutralized using 1 M TRIS pH 10.0 before the proteins were eluted with 10 column volumes of elution buffer (50 mM citrate, 90 mM NaCl, pH 2.5).

[0388] Finally, the eluted fractions were polished using size exclusion chromatography (HiPrep Superdex 200, 16 / 60, GE Healthcare Life Sciences).

[0389] The aggregate contents were followed during the purification process by analytical size exclusion chromatography (Superdex 200 Increase 3.2 / 300 GL, GE Healthcare Life Sciences).

[0390] The aggregation levels after the capture step and expression yields after purification for C2-truncated MFG-E8 and HSA fusion are shown in Table 10. The HSA fusion of C2-truncated MFG-E8 shows at least 40-fold improved expression over C2-truncated MFG-E8. Furthermore, the HSA fusion of C2-truncated MFG-E8 shows at least 4-fold less aggregation compared to C2-truncated MFG-E8. These data suggest that the HSA fusion of C2-truncated MFG-E8 exhibits superior production characteristics compared to C2-truncated MFG-E8. As a result, the HSA fusion appears to have better development potential for use as a drug.

[0391] [Table 63]

[0392] Example 14: Dynamic Light Scattering (DLS) of C2-Truncated MFG-E8 (EGF-C1) and HSA Fusion (EGF-HSA-C1) The aggregation tendency of C2-truncated MFG-E8 and HSA fusion proteins was measured by dynamic light scattering (DLS, Wyatt). Dynamic light scattering was applied to measure the translational diffusion coefficient of proteins in solution by quantifying the dynamic fluctuations of scattered light. As an indicator of aggregate formation, the hydrodynamic radius upon thermal stress was measured at a concentration of 3 mg / ml using a DynaPro™ plate reader (Wyatt Technology Europe GmbH, Dernbach, Germany) in combination with the software DYNAMICS (version 7.1.0.25, Wyatt). Protein solutions were measured in 384-well plates (384 round well plates, polystyrene, Thermo Scientific, Langenselbold, Germany).

[0393] As shown in Figure 23, C2-truncated MFG-E8 exhibits an overall higher hydrodynamic radius compared to the HSA fusion (5 nm vs. 80 nm at 25°C). Furthermore, C2-truncated MFG-E8 exhibits a strong increase in hydrodynamic radius starting at 45°C, indicating strong aggregation, while the HSA fusion retains the same hydrodynamic radius up to at least 55°C. These data suggest that HSA fusions of C2-truncated MFG-E8 are more stable and exhibit superior biophysical properties compared to C2-truncated MFG-E8. As a result, the HSA fusion appears to have better exploitation potential for use as a drug.

[0394] Taken together, these data indicate that the fusion proteins of the present disclosure (eg, with an HSA domain insert) are functional and effective and may be used as therapeutic agents.

[0395] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes in light thereof 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 PS-binding domain is a truncated mutant. [Invention 2] The fusion protein according to invention 1, wherein the PS-binding domain is a truncated mutant of at least one PS-binding domain listed in Table 2. [Invention 3] A fusion protein according to Invention 1 or Invention 2, wherein the PS-binding domain is a truncated mutant of the PS-binding motif of MFG-E8 or EDIL3. [Invention 4] A fusion protein according to invention 3, wherein the PS-binding domain is a truncated mutant of the PS-binding motif of MFG-E8. [Invention 5] 5. The fusion protein according to claim 4, wherein the PS-binding domain is a discoidin domain. [Invention 6] 6. The fusion protein according to any one of Inventions 1 to 5, wherein the PS-binding domain is a C1 domain. [Invention 7] 7. The fusion protein according to any one of Inventions 1 to 6, wherein the PS-binding domain does not contain a C2 domain. [Invention 8] 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 PS-binding domain is a C1 domain. [Invention 9] 9. The fusion protein according to any one of Inventions 1 to 8, wherein the integrin-binding domain binds to one or more integrins. [Invention 10] 10. The fusion protein according to claim 9, wherein the integrin-binding domain binds to αvβ3 and / or αvβ5 and / or α8β1 integrin. [Invention 11] 11. The fusion protein according to claim 9 or 10, wherein the integrin-binding domain comprises an arginine-glycine-aspartic acid (RGD) motif. [Invention 12] 12. The fusion protein according to any one of Inventions 8 to 11, wherein the soluble domain is directly linked to the integrin-binding domain, the PS-binding domain, or both domains. [Invention 13] 13. The fusion protein according to any one of Inventions 8 to 12, wherein the soluble domain is indirectly linked to the integrin-binding domain and / or the PS-binding domain via a linker. [Invention 14] 14. The fusion protein according to any one of inventions 8 to 13, wherein the integrin-binding domain has the amino acid sequence of SEQ ID NO: 2 or at least 90% sequence identity thereto. [Invention 15] 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 or a C2 domain, and comprises a sequence from wild-type human MFG-E8 (sequence number 1) or a functional variant thereof. [Invention 16] 16. The fusion protein according to claim 15, wherein the soluble domain is inserted between the EGF-like domain and the C1 or C2 domain. [Invention 17] 17. The fusion protein according to invention 15 or 16, wherein the soluble domain is HSA, HSA D3, or Fc-IgG, or a functional variant thereof. [Invention 18] 18. The fusion protein according to any one of inventions 15 to 17, wherein the soluble domain comprises human serum albumin (HSA) or a functional variant thereof. [Invention 19] 19. The fusion protein according to any one of inventions 15 to 18, 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 inflammation and organ damage due to ischemia / reperfusion. [Invention 20] 20. The fusion protein according to any one of inventions 15 to 19 for use in the treatment or 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, graft 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. [Invention 21] 21. A fusion protein for use according to invention 19 or invention 20, wherein the fusion protein is administered in combination with another therapeutic agent, the therapeutic agent being an immunosuppressant, immunomodulator, anti-inflammatory agent, antioxidant, anti-infective agent, cytotoxic agent or anti-cancer agent.

Claims

1. A therapeutic fusion protein for enhancing efferocytosis, said fusion protein having the amino acid sequence of SEQ ID NO:

73.

2. 10. The fusion protein of claim 1 for use in the treatment or prevention of an inflammatory disorder or inflammatory organ damage in an individual in need thereof, wherein the inflammatory disorder or inflammatory organ damage is at least one selected from the group consisting of acute kidney injury, acute respiratory distress syndrome, acute liver injury, sepsis, myocardial infarction, stroke, burns, traumatic injury, and inflammation and organ damage due to ischemia / reperfusion.

3. 10. The fusion protein of claim 1 for use in the treatment or prevention or amelioration of at least one selected from the group consisting of inhibiting or slowing blood clotting, microbiome treatment, 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.

4. 4. The fusion protein for use according to claim 2 or 3, wherein the fusion protein is administered in combination with another therapeutic agent, the therapeutic agent being an immunosuppressant, immunomodulator, anti-inflammatory agent, antioxidant, anti-infective agent, cytotoxic agent or anti-cancer agent.

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