Fusion protein for treatment

Abstract

To provide a fusion protein to be used in treatment or prevention of acute inflammatory organ injury (AOI).SOLUTION: Provided is a fusion protein suitable for use as a medicine or a research tool. Therapeutic use of fusion protein may include prevention or treatment of an acute or chronic inflammatory disorder and organ and microangiopathy disorders caused by an immune system, for example, an acute renal disorder, acute respiratory distress syndrome, sepsis, acute myocardial infarction, tissue fibrosis, and other organ damages caused by tissue trauma. Provided is a fusion protein for treatment for enhancing efferocytosis which includes an integrin binding domain, a phosphatidyl serine (PS) binding domain, and a solubilization domain, wherein the solubilization domain is inserted between the integrin binding domain and the PS binding domain, and the integrin binding domain binds to integrin.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing This application contains a Sequence Listing which has been submitted electronically in ASCII format, the entirety of which is as follows: The ASCII text created on August 31, 2020 is hereby incorporated by reference. The copy is named PAT058332_SL.txt and is 653,193 bytes in size. It is thread.

[0002] The present invention provides a fusion protein that contains both integrin-binding and phosphatidylserine-binding capabilities. The fusion proteins are particularly useful in treating acute or chronic inflammatory disorders, as well as the immune system or As a therapeutic agent for the prevention or treatment of organ and microvascular disorders caused by coagulation It can be used. [Background technology]

[0003] Acute inflammatory organ injury (AOI) is associated with high morbidity, mortality, and significant unmet medical needs. It is a historically challenging disease with significant medical needs. A typical AOI affects 3,200 people worldwide each year. Includes myocardial infarction (MI) and stroke, which occur in 400,000 patients. Previous MI and stroke Patients with diabetes are being treated by the World Health Organization. n), further coronary artery disease, ranked as one of the leading causes of morbidity in developed countries. Another AOI is acute kidney injury (AKI). AKI), which occurs in approximately 13.3 million people annually. In high-income countries, the incidence of AKI is The incidence is 3-5 / 1000 and is associated with a high mortality rate (14-46%) (Metha et al., (2015) Lancet, 385(9987):2616-43). M As with I and stroke, survivors of AKI often do not fully recover and are at increased risk of developing chronic kidney disease or There is a high risk of developing end-stage renal disease. To date, no drugs have been available to prevent or treat AKI. There are currently no FDA-approved drugs that can treat AKI. Developing new treatments for AKI has proven difficult. This is due to the fact that there have been no successful clinical trials to date. and / or inflammatory, microvascular dysfunction, and nephrotoxic pathology induced by nephrotoxic injury. This may be due to the multifactorial and multifaceted pathophysiology of AKI, including the biological mechanisms. These drivers act simultaneously or sequentially to induce inflammatory responses in mostly tubular cells but also in glomerular cells. This can lead to alveolar damage, loss of renal reserve, and ultimately renal failure.

[0004] One common factor in AOI is increased cell death due to tissue damage, leading to the generation of cell fragments. These are prothrombotic / proinflammatory microparticles that can increase blood flow and enter the circulation and injured tissue. After neutrophils infiltrate the tissue to prevent infection, they undergo apoptosis in the affected tissue. or other forms of cell death. Neutrophils contain proteolytic enzymes and danger-associated molecular patterns. They contain harmful substances such as DAMPs, which promote host tissue damage and propagate inflammation. Efficient uptake of dead cells may promote a non-inflammatory, pro-resolving phenotype. Reprogramming of affected macrophages (MΦ) and degradation and repair of affected tissues triggering signaling events that lead to the release of key mediators for successful This reprogramming has recently been shown to activate the phagocytic anti-inflammatory response in macrophages. This has been attributed to metabolic signaling that activates the immune system (Zhang et al., 20 19) Cell Metabolism, 29(2):443-56) Non-inflammatory methods The removal of this debris, or senescent or dead cells, in the retina is called "efferocytosis." do.

[0005] However, if efferocytosis is delayed, necrotic cells accumulate, e.g., macrophage. Triggering of pro-inflammatory cytokines (TNF-α) or immunosuppressive IL-10 by phage This can trigger an inflammatory response (Greenlee-Wacker (2016) Imm In addition, cell debris and particles If not efficiently removed, neutrophil-platelet fragment clusters, microthrombi, and other cell clumps and It can cause aggregation and / or damage dangerous molecules such as ATP, DNA, histones, or HMGB1. These molecules may release disruptive molecular patterns (DAMPS). Consequences include microvascular obstruction, dysfunction, and failure, and progression of tissue damage, primary and secondary organ failure, or significant reparative maladaptation. Sterile inflammation may be involved.

[0006] During the acute phase of AOI, the efferocytosis pathway was significantly downregulated. Inflammation or acute response to injury (organic factors, hypoxia, oxidative stress) Resistance, irradiation, inflammation, and infection) cross-link proteins and cell surface efferocytosis / Downregulation of dedicated phosphatidylserine (PS)-binding proteins, including clearance receptors By upregulating efferocytosis or phagocytosis, Examples of dysfunctional urocytosis receptors include Mer tyrosine kinase (MerTK) and Proteolytic shedding of TAM family receptors. MerTK is a phagocyte It is an integral membrane protein preferentially expressed in the as well as efferocytosis (through proteins such as Gas6 or Protein S) The soluble ectodomain of MerTK promotes inflammatory signaling and inhibits inflammatory signaling. Proteolytic cleavage and release is induced by the metalloproteinase ADAM17. The shedding process involves shedding of surface MerTK, which then triggers efferocytosis of phagocytes. Furthermore, the released ectodomain can reduce the effect of It can also inhibit eosinophilic ... Mol Cell Cardiol.,87:171-9;Miller et al. ,(2017)Clin Cancer Res.,23(3):623-629). blood Increased serum / plasma soluble Mer levels are typically associated with diabetic nephropathy or systemic lupus erythematosus. It is observed in inflammatory, malignant, or autoimmune diseases such as systemic lupus erythematosus (SLE) and indicates the severity of the disease. Obtain (Ochodnicky P (2017) Am J Pathol.,187(9) :1971-1983;Wu et al.,(2011)Arthritis Res Ther.13:R88). In addition, milk fat globule-EGF factor 8 protein (MFG- Cross-linking proteins such as E8 are also down-regulated during most acute and chronic inflammatory diseases. Similar to soluble Mer, the decrease in serum / plasma concentrations of MFG-E8 is associated with MI. It can be seen in patients with or stable angina (Dai et al., 2016 )World J Cardiol.,8(1):1-23) Regarding chronic obstructive pulmonary disease may indicate the severity of the disease, as described in

[1994] (COPD; Zhang et al. .,(2015) cited above).

[0007] Phosphatidylserine (PS) exposure to dying cells is an evolutionarily conserved pathway for immune cells. A vast number of major mammalian pathogens induce pathogenic cells utilizes PS-mediated uptake as part of the vesicle infection (Birge et al., 2016)Cell Death Diff.,23(6):962-78). For example, Viruses can bind to PS-binding receptors directly or via proteins such as Gas6 (Morizono&Chen(2014)J Virol.,88(8):4275- 90) Inactivation of the endogenous clearance pathway in response to injury prevents the release of cytotoxic agents that invade cells after injury. Reduces the efficiency of infectious pathogens in hijacking the cells, thereby circumventing the host immune response and defenses. As a result, the clearance pathways may represent evolutionarily evolved responses to prevent the Down-modulation reduces the effectiveness of innate and adaptive immune effectors to fight infection. It will improve the effectiveness of the F-12 system during acute organ damage as a result of "friendly fire." It is possible that exocytosis may be transiently affected, leading to the above complications during AOI. Accumulation of dead cells, debris, and pro-inflammatory and pro-thrombotic MPs is a hallmark of AOI. and represent the main triggers of inflammation and microvascular injury. Such accumulation is common in serious diseases with high medical needs, and It is noteworthy that steroids can contribute to the morbidity of certain conditions. Examples of such indications are sepsis and cancer. (Yang et al.,(2016)Tumour Biol.,37(6):7 881-91;Zhao et al.,(2016)J Exp Clin Canc er Res.,35:54;Muhsin-Sharafaldine et al. ,(2017) Biochim Biophys Acta Gen Subj.,18 61(2):286-295;Ma et al.,(2017)Sci Rep.,7 (1):4978;Souza et al.,(2015)Kidney Int.8 7(6):1100-8). Previous drug discovery efforts in this field have focused on PS-binding proteins. The focus is on proteins, which is explained by (Li et al., (2013) Exp O pin Ther Targets,17(11):1275-1285) As discussed above, these can serve as the basis for drug candidate design.

[0008] A subset of PS-binding proteins, αvβ3, is expressed in many cell types, including phagocytes. These proteins also recognize and bind to integrins such as αvβ5 and αvβ6. They act by exposing dead / dead cells to integrins, cross-linking PS, and inducing macrophages and Efferocytosis (also called phagocytosis) by non-professional phagocytes Several cross-linked proteins also contribute to the damage caused by inflammation during most acute and chronic inflammatory diseases. Therapeutic use of such cross-linked proteins or truncated versions thereof is also important. Therapeutic use has been previously suggested (WO 2006 / 122327 (Final Report) Ischemia), International Publication No. 2009064448 (Organ damage after ischemia / reperfusion) , International Publication No. 2012149254 Pamphlet (Cerebral Ischemia) The Feinstei n Institute for Medical Research; International Publication No. 20 No. 15025959 Brochure (Myocardial Infarction) Kyushu University and Tokyo Medical University; International Publication No. 2 No. 0150175512 Brochure (Bone Resorption) University of Pennsylvania; International Publication No. 201 No. 7018698 Brochure (Tissue Fibrosis) Korea University Re Search and Business Foundation and U.S. Patent Application Publications No. 20180334486 (tissue fibrosis) Nexel Co., Ltd.); However, the use of wild-type or naturally occurring proteins is limited by a number of problems. For example, when wild-type MFG-E8 (wtMFG-E8) is cultured in a cell expression system, It is thought to be poorly viable, poorly soluble, and expressed in very low yields. According to the study by Illanos et al. (2016), Fc MFG-E8 expressed as a -IgG fusion was fully aggregated and was not soluble in Triton X- It can only be efficiently purified by adding detergents such as 100 or CHAPS. It has been shown that (Castellanos et al., (2016) Protein in Exp. Pur., 124:10-22).

[0009] Cross-linking proteins, e.g., MFG-E8, EDIL3, Gas6, inhibiting cell death and destruction Removal of debris and particulates eliminates a major cause of sterile inflammation and microvascular dysfunction, thus reducing the This may prevent the progression of tissue damage and allow the resolution of inflammation. Therapeutic approaches that promote clearance of AOI may reduce or at least alleviate the pathology of AOI. may be used to prevent dead cells or PS-exposed particles from being sufficiently cleared. It may have implications in other disease settings, so to reduce tissue damage and inflammation and have desirable manufacturing characteristics to address the unmet medical needs of AOI. Therefore, a therapeutic agent is needed. Summary of the Invention [Means for solving the problem]

[0010] In the present disclosure, applicants address the aforementioned undesirable properties and production of wild-type crosslinked proteins. Based on the structure of naturally occurring proteins (e.g., MFG-E8) without the problem of genomic DNA fragmentation. The fusion proteins of the present disclosure are directed to integrin The fusion protein contains a binding domain, a PS-binding domain, and a solubility domain. The main biological function of type MFG-E8 protein is to inhibit, for example, PS-exposed dead cells, debris, and function to bridge microparticles to phagocytes, thus triggering efferocytosis. Furthermore, the therapeutic fusion proteins of the present disclosure function to maintain wild-type MF. Compared to the G-E8 protein (SEQ ID NO: 1), it has improved developability, especially in the adhesion Furthermore, these therapeutic fusion proteins have reduced affinity and improved solubility. Compared with the MFG-E8 protein, plasma exposure was longer and it was expressed in a cell expression system. In this case, the yield will be higher.

[0011] Integrin-binding domain, phosphatidylserine (PS)-binding domain, and solubilization Therapeutic fusion proteins for enhancing efferocytosis containing a domain are described herein. Available at.

[0012] In some embodiments, the solubility domain of the fusion protein is an integrin-binding domain. In some embodiments, the solubility domain is linked to a PS-binding domain. In some embodiments, the solubility domain is linked to an integrin domain. It is linked to both the phospho-binding domain and the PS-binding domain, i.e., integrin. In some embodiments, the ATP-binding domain is located between the ATP-binding domain and the PS-binding domain. The soluble domain may be inserted within the integrin-binding domain or within the PS-binding domain. In one embodiment, the therapeutic fusion protein is inserted into the N-terminus to C-terminus It has a structure of integrin-binding domain-solubilizing domain-PS-binding domain.

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

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

[0015] In some embodiments, the therapeutic fusion protein is linked to the N-terminus of the solubility domain. The C-terminus of the integrin-binding domain is linked to the solubilizing protein, and the PS-binding domain is linked to the C-terminus of the integrin-binding domain. In some embodiments, the therapeutic fusion protein comprises the C-terminus of the domain. The structure includes EGF-HSA-C1-C2, where EGF is MFG-E8, EDIL3 or other proteins containing an integrin-binding domain listed in Table 1. C1-C2 represent the ATP-binding EGF-like domains of MFG-E8, EDIL3, or the ATP-binding EGF-like domains listed in Table 2. Represents the PS-binding domain found in other proteins containing the listed PS-binding domain Examples of proteins containing both integrin-binding and PS-binding domains include, for example, MFG-E8 (SEQ ID NO: 1) and EDIL3 (SEQ ID NO: 11) are listed in Table 3.

[0016] In some embodiments, the integrin binding domain is an EGF-like domain. , for example, the amino acid sequence set forth in SEQ ID NO: 2, or at least 90%, 95%, or %, 96%, 97%, 98% or 99% sequence identity, or truncated forms thereof In one embodiment, the EGF-like domain has a mutation of human MFG-E8 EGF. -like domain or its functional variants containing 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 functional variants thereof containing 1, 2, 3, 4, 5, or up to 10 amino acid modifications. .

[0017] In some embodiments, the PS-binding domain comprises two discoidins C1-C2 subdomains, for example, the amino acid sequence set forth in SEQ ID NO: 3, or at least Amino acids with at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity The PS-binding domain of human MFG-E8 is a nucleotide sequence of the ... In the embodiment, the PS-binding domain is the PS-binding domain of human MFG-E8, or In one embodiment, the amino acid sequence of the present invention includes a functional variant thereof comprising at least one amino acid modification, 3, 4, 5, or up to 10 amino acid modifications. In the present invention, the PS-binding domain is the PS-binding domain of human EDIL3, or 1, 2, 3, 4 , 5, including functional variants thereof containing up to 10 amino acid modifications.

[0018] In some embodiments, the solubility domain is HSA or a functional variant thereof. , for example, the amino acid sequence set forth in SEQ ID NO: 4, or at least 90%, 95%, or %, 96%, 97%, 98% or 99% sequence identity, or truncated forms thereof In one embodiment, the HSA has a mutation that reduces the tendency of the protein to aggregate. and the amino acid sequence as set forth in SEQ ID NO:5, including the amino acid substitution C34S, which functions as In some embodiments, the solubilizing domain comprises human serum albumin (HSA). A) or functional variants thereof containing 1, 2, 3, 4, 5, up to 10 amino acid modifications, e.g. For example, HSA C34S, or truncated mutants of HSA, such as domain 3 (H In a preferred embodiment, the solubility domain comprises a soluble domain (SA D3) or a functional variant thereof. is the HSA C34S.

[0019] In an alternative embodiment, the solubility domain is the Fc region of IgG (Fc-IgG), For example, human IgG1, IgG2, IgG3, or IgG4, or functional variants thereof. In one embodiment, the solubility domain comprises an Fc region of the amino acid sequence set forth in SEQ ID NO: 7. or at least 90%, 95%, 96%, 97%, 98% or 99% of the % sequence identity to human Fc-IgG1, or truncated variants thereof. In one embodiment, the Fc-IgG1 comprises an Fc region having reduced Fc effector function. and the amino acid substitutions D265A and P329A to reduce the In another embodiment, the Fc-IgG1 has an amino acid sequence such that it does not create a "knob." or the amino acid substitution T366W to create a "hole" The Fc-IgG1 knob may contain the following acid substitutions: T366S, L368A, Y407V. The Fc-IgG1 hole may contain the amino acid substitution S354C, and the Fc-IgG1 hole may contain the amino acid substitution Y349C. This results in the formation of a cysteine ​​bridge upon pairing. Moreover, Fc-IgG1 also contains D265A and P to reduce Fc effector function. In one embodiment, the Fc-IgG1 may comprise a 329A substitution. It has the amino acid sequence as set forth in

[0020] In a preferred embodiment, the therapeutic fusion protein is a milk fat globule-EGF factor VIII protein. The protein (MFG-E8) and the solubilization domain are included, where MFG-E8 is an integrin. Phosphorus-binding EGF-like domain (SEQ ID NO: 2) and phosphatidylserine-binding C1-C2 domain MFG-E8 contains the naturally occurring or wild-type human M MFGE-E8 (SEQ ID NO: 1), or MFGE-8 having SEQ ID NO: 75 or a functional variant thereof In one embodiment, the solubility domain may comprise a soluble domain at the N- or C-terminus of MFG-E8. In one embodiment, the soluble domain is linked to an EGF-like domain and a C1 domain. The C1 domain is inserted between the C1 and C2 domains or between the C1 and C2 domains. In the present invention, the soluble domain is linked to the C-terminus of the EGF-like domain and the N-terminus of the C1 domain. The soluble domain is linked directly or indirectly to the C-terminus of the EGF-like domain. The C1 domain may be linked directly or indirectly to the N-terminus of the C1 domain. In some embodiments, the indirect linkage may be an external linker, such as a glycine-serine based linker. by.

[0021] In one embodiment, the therapeutic fusion protein is set forth in SEQ ID NO: 42 (FP330). In one embodiment, the therapeutic fusion protein comprises an amino acid sequence as described in Histidine may be used to aid in detection and / or purification in titer assays and protein expression. In one embodiment, the therapeutic fusion protein may comprise a His tag (SEQ ID NO: 67). The protein has a C-terminal His tag and the amino acid sequence set forth in SEQ ID NO: 44 (FP278). The therapeutic fusion proteins FP278 and FP330 contain a His tag attached to FP278. They share the same amino acid sequence except for an additional amino acid.

[0022] In some embodiments, the therapeutic fusion protein is SEQ ID NO: 42 (FP330) or at least 90%, 95%, 96%, 97%, or Contains amino acids with 98% or 99% sequence identity, or truncated variants thereof. For example, the therapeutic fusion protein FP776 comprises the amino acid sequence set forth in SEQ ID NO: 48: It has 97.7% sequence identity to FP330 (SEQ ID NO: 42). The fusion protein FP068 comprises the amino acid sequence set forth in SEQ ID NO: 46 and is identical to FP330( It has 98.3% sequence identity to the nucleotide sequence of SEQ ID NO: 42. For example, it is used in therapeutic fusion proteins. Protein FP816 comprises the amino acid sequence set forth in SEQ ID NO: 58, and FP330 (SEQ ID NO: 42 ) has 98.5% sequence identity to the therapeutic fusion protein FP811 comprises the amino acid sequence set forth in SEQ ID NO: 54, and has a 9:1 ratio to FP330 (SEQ ID NO: 42). For example, the therapeutic fusion protein FP010 has 9.0% sequence identity with SEQ ID NO: 56, and has a sequence identity of 99.5% with respect to FP330 (SEQ ID NO: 42). For example, the therapeutic fusion protein FP138 has sequence identity with the sequence set forth in SEQ ID NO: 52. The amino acid sequence has 99.8% sequence identity to FP330 (SEQ ID NO: 42). For example, the therapeutic fusion protein FP284 has the amino acid sequence set forth in SEQ ID NO: 50. and has 99.9% sequence identity to FP330 (SEQ ID NO: 42).

[0023] In some embodiments, and as described in the Examples section, Therapeutic fusion proteins inhibit the human endothelial cell-Jurkat cell efferocytosis assay Promotes endothelial cell efferocytosis in rats and human macrophages and neutrophils In the efferocytosis assay, basal efferocytosis by macrophages was observed. The fusion protein functions to restore damaged endothelial cells and enhance basal efferocytosis. Proteins were assessed for clearance in the human endothelial microparticle efferocytosis assay. and / or the fusion protein functions to reduce the number of plasma microparticles in a model of acute renal ischemia. provides protection against multi-organ injury in mice.

[0024] Methods, uses, diagnostic reagents, and medicaments that utilize or include these therapeutic fusion proteins Compositions and kits are also disclosed herein. Nucleic acids encoding the disclosed fusion proteins , cloning and expression vectors containing such nucleic acids, and host cells containing such nucleic acids. and producing the disclosed fusion proteins by culturing such host cells. Also provided herein are processes for: [Brief explanation of the drawings]

[0025] [Figure 1] 1 shows a schematic diagram of an example of a therapeutic fusion protein of the present disclosure. A solubility domain (labeled "SD") was linked either at the C-terminus, N-terminus, or between the EGF, C1, or C2 domains of MFG-E8. [Figure 2-1]Figure 2: 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 (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 11F shows the efferocytosis-promoting function of recombinant fusion proteins constructed as chimeric proteins fusing domains from EDIL3 and MFG-E8 to an HSA insert. The data show that FP145 (SEQ ID NO: 80) and FP146 (SEQ ID NO: 82) induced efferocytosis of killed neutrophils by human macrophages in a concentration-dependent manner. Figure 11G shows the efferocytosis-promoting function of recombinant fusion proteins constructed as chimeric proteins fusing domains from EDIL3 and MFG-E8 to an HSA insert. The data show that FP145 (SEQ ID NO: 80) and FP146 (SEQ ID NO: 82) induced efferocytosis of killed Jurkat cells by human endothelial cells (HUVEC) in a concentration-dependent manner. [Figure 11-2] (As mentioned above.) [Figure 11-3] (As mentioned above.) [Figure 11-4] (As mentioned above.) [Figure 11-5] (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 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]Figure 17 shows that 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 induced by ischemia-reperfusion-induced AKI, based on the 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 MFGE8-derived fusion protein FP278 (SEQ ID NO: 44) at 0.16 mg / kg or 0.5 mg / kg / ip. [Figure 18] Figure 1 shows the uptake of superparamagnetic iron oxide (SPIO) contrast agent (Endorem®) by the liver over time. Endorem® was injected intravenously as a 1.2-second bolus 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. DETAILED DESCRIPTION OF THE INVENTION

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

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

[0028] "comprises" and "contains" in reference to sequences (e.g., amino acid sequences) Terms such as "includes" and "comprising" are used. In all cases, the sequence may also be "consist," "consist of," " The term "consists" and "consisting" are used to describe As used herein, "essentially from" refers to a compound that is essentially a compound of a formula that is a formula that is a suffix. The phrase "targets" refers to the genus or species of active pharmaceutical agent included in the method or composition, as well as the type of active pharmaceutical agent that is included in the method or composition. refers to any excipient that is inert for the intended purpose of the composition. Thus, the phrase "consisting essentially of" refers to one or more other substances other than the multispecific binding molecules of the disclosure. In some embodiments, the term "consisting essentially of" expressly excludes the inclusion of an active agent consisting of " refers to one or more co-administered agents other than the multispecific binding molecules of the disclosure and the second co-administered agent. The inclusion of further active agents is expressly excluded.

[0029] As used herein, the term "efferocytosis" refers to the process by which cells in cell biology It refers to the process of apoptosis or necrosis or senescent cells or highly activated Dead or dying cells (collectively referred to as dead cells) such as dead cells, extracellular vesicles (microparticles), or cell debris Prey (called "prey") is removed by phagocytosis, i.e., it is engulfed by phagocytes and digested. During efferocytosis, phagocytes actively capture and engulf prey, transporting it to the endothelium. They produce large intracellular fluid-filled vesicles called ferrosomes that contain prey. During apoptosis, efferocytic cells produce lysosomal compartments where prey degradation begins. Cholesterosis occurs when the integrity of their membranes is compromised and dead cells are killed before their contents leak into the surrounding tissue. The proteins are removed, and toxic enzymes, oxidants, and other cellular enzymes such as DNA, histones, and proteases are removed. Ensure that the surrounding tissue is not exposed to intracellular DAMPs. Cells include bone marrow-derived cells such as macrophages and dendritic cells, but also other cells For example, stromal cells include epithelial and endothelial cells, as well as efferocytes such as fibroblasts. Impaired efferocytosis is associated with autoimmune diseases and tissue damage. It is used to treat cystic fibrosis, bronchiectasis, COPD, asthma, idiopathic pulmonary fibrosis, and arthritis. In diseases such as uterine arthritis, systemic lupus erythematosus, glomerulonephritis and atherosclerosis It has been proven (Vandivier RW et al (2006) Chest, 12 9(6):1673-82). Currently, there are no treatments that specifically promote efferocytosis. The method has not entered clinical trials.

[0030] The term "efferocytosis assay" as used herein and described in the Examples The term refers to a fusion process that utilizes human macrophages or human endothelial cells (HUVECs) as phagocytes. This paper describes an assay system developed for profiling of macromolecules. Phage-neutrophil efferocytosis assay, endothelial cell-transferase-induced Jurkat cell efferocytosis An endothelial cell microparticle efferocytosis assay, or an endothelial cell microparticle efferocytosis assay, is used herein. These assays are exemplified by the fusion proteins of the present disclosure, as described in more detail in the Examples. Biotherapeutics derived from MFG-E8, such as fusion proteins, are expressed by macrophages or endothelial cells. To demonstrate that IFN-γ effectively promotes efferocytosis of dead cells and microparticles, Furthermore, the described macrophage-neutrophil assay can be used in the present invention. Such compounds inhibit the production of LPS or S. aureus-infected cells. This is suitable for demonstrating that even vasopressin-induced efferocytosis can be rescued.

[0031] The terms "polypeptide" and "protein" refer to a polymer of amino acid residues. The terms are used interchangeably herein to refer to a group in which one or more amino acid residues correspond to each other. Amino acid polymers, which are artificial chemical mimics of natural amino acids, and natural amino acid polymers It also applies to polymers and unnatural amino acid polymers. A peptide sequence also implicitly encompasses conservatively modified variants thereof.

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

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

[0034] As used herein, the term "fusion protein" refers to a protein that combines several domains. This refers to a protein that contains, but does not necessarily constitute, the entire native or wild-type protein. However, it is limited to the active domain of the whole protein that is involved in binding to the corresponding receptor on the cell surface. Fusion proteins can be produced using recombinant protein engineering, The term "recombinant protein" refers to a protein prepared, expressed, produced, or otherwise processed by recombinant DNA techniques. For example, a tandem fusion refers to a protein or proteins of interest. The protein domains are simply connected at both ends via N- or C-terminal fusions between the proteins. This refers to a technique that provides a flexible bridge structure, allowing sufficient space between the fusion partners. This ensures proper folding, but the N-terminus or The C-terminus is important for obtaining the desired folding pattern of recombinant proteins The components are often complex, and simple joining of the two ends of the domain may be ineffective. The process of domain insertion allows the desired structure to be encoded in a single polypeptide chain. Fusion of consecutive protein domains and sometimes insertion of a domain within another domain In both of these aforementioned processes, domains are "directly linked" "directly linked" or "linked directly" Domain insertion is difficult because it is difficult to find a suitable nucleic acid linking site in the target gene. Therefore, it is often more difficult to perform than tandem fusion.

[0035] In addition to the direct fusion technique described above, external linkers can be used to bind proteins to the fusion protein. Such linkers allow the protein domains to be separated so that their functionality can be maintained. It refers to a series of amino acids that connects a protein domain, and is referred to herein as an "indirect linker." Therefore, the domain is called "indirectly linked" )" or "linked indirectly." For example, Those skilled in the art will recognize that a polypeptide whose structure comprises two or more functional or organizational domains is It is understood that such domains often contain a stretch of amino acids between them that connect them together. The linker allows domain interaction, enhances stability, and reduces steric hindrance. This allows for the design of engineered proteins, even when N- and C-terminal fusions are possible. In some embodiments, the linker is tend not to adopt a rigid three-dimensional structure, but rather provide flexibility to the polypeptide. Various types of naturally occurring linkers can be linked to modified proteins, e.g. Many recombinant therapeutic proteins, especially engineered antibodies, have immunoglobulins that function as linkers in their construction. It has been used for immunoglobulin hinge regions (Pack P et al., (1995) J. Mol. Biol., 246:28-34). In addition to natural linkers, many artificial linkers have been developed. Linkers have been devised, which include flexible, rigid, and in vivo cleavable linkers. These can be subdivided into three categories: (Yu K et al., (2015) Biotech.Advances,33(1):155-64;Chen X et al.,(2013)Ad.Drug Delivery Reviews,65(10 The most widely used flexible linker sequence is (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 can be adjusted by the copy number "n". In this embodiment, the polypeptide comprising the linker element has the general form D1-linker -D2, where D1 and D2 may be the same or different, In some embodiments, the linker represents two domains that are associated with each other. The peptide linker may be 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, 7 It may be 5, 80, 85, 90, 95, 100 or more amino acids in length.

[0036] As used herein, a "modification" or "mutation" of an amino acid residue / position refers to a modification of the starting amino acid. refers to a change in the primary amino acid sequence compared to the amino acid sequence of the For example, typical modifications include: ) with another amino acid (e.g., conservative or non-conservative substitution), adjacent to said residue / position This includes the insertion of one or more adjacent amino acids, and the deletion of said residues / positions. A "substitution" or modification thereof refers to the replacement of an existing amino acid residue in a given (starting) amino acid sequence with a different amino acid. Generally and preferably, the modification refers to the replacement of the starting (or "field") amino acid residue with a At least one amino acid sequence of the mutant polypeptide is at least It brings about a change in one's physical and biochemical activity.

[0037] The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants are those that contain identical or essentially identical amino acids. Those nucleic acids that encode the amino acid sequence, or if the nucleic acid does not encode the amino acid sequence, Refers to qualitatively identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids can be produced at any one time. For example, the codons GCA, GCC, GCG, and GCU all encode the protein It encodes the amino acid alanine. Therefore, alanine is specified by the codon At all positions, the codon may be substituted as described without altering the encoded polypeptide. Such nucleic acid variations are referred to as "synaptic variations." It is a "conservatively modified variation" and is a type of conservatively modified variation. All nucleic acid sequences herein that encode polypeptides also represent all possible silencers of nucleic acids. Those skilled in the art will understand the variations of each codon in a nucleic acid (usually the only codon for methionine). (except for AUG, which is the only codon for tryptophan, and TGG, which is usually the only codon for tryptophan) It will be recognized that functionally identical molecules can be obtained by modifying the polypeptides. Each silent variation of a nucleic acid encoding a peptide is implicit in each described sequence. do.

[0038] For polypeptide sequences, "conservatively modified variants" include those in which amino acids are chemically modified. Individual substitutions, deletions, or deletions in a polypeptide sequence that result in substitutions with similar amino acids Conservative substitution tables providing functionally similar amino acids are publicly known in the art. Such conservatively modified variants include polymorphic variants, interspecies homologs, and alleles. The following eight groups contain sequences that are conservatively substituted for each other: Amino acids: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamine phosphate (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine 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, for example, Creighton, Proteins (1984)). In embodiments, the phrase "conservative sequence modifications" refers to modifications of the binding domain of a variant protein of the disclosure. It is used to refer to amino acid modifications that do not significantly affect or alter the binding properties of the polypeptide. can be.

[0039] As referred to herein, a "protein variant" or "protein variant" refers to a Variants in which 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are modified As referred to herein, the term "functionality" refers to a protein containing a specific amino acid sequence. "Variants" refer to those which result in changes in the amino acid sequence but do not affect the overall properties of the protein or its function. As referred to herein, a protein variant refers to a protein containing a modification that does not alter the protein structure. "Truncated variants" of proteins are related to shortened versions of the protein, but The truncated versions retain the function of the parent protein. Functional or truncated variants These mutant proteins were analyzed to determine whether they had any changes in overall properties or function. The quality of the compounds was assessed by a comprehensive analysis of their effects in several assays as described in this disclosure. The modified or unmodified parent protein can be tested against the parent protein. For example, human endothelial cell-J Endothelial cell efferocytosis in the Urkat cell efferocytosis assay Promotion of macrophage efferocytosis in a human macrophage-neutrophil efferocytosis assay Phage-mediated recovery of impaired efferocytosis, human endothelial-microparticle efferocytosis Reduction in plasma microparticle counts due to clearance in the MRI assay and / or acute renal ischemia model The aim of this study was to provide protection against multi-organ damage in the dermatological setting.

[0040] The term "percentage identity" or "percentage sequence identity" refers to the percentage of identity between two or more In reference to a nucleic acid or polypeptide sequence, refers to two or more sequences or subsequences that are the same Compared and aligned for maximum match over a comparison window or specified region For example, using one of the following sequence comparison algorithms or by manual alignment and Identification of amino acid residues or nucleotides that are the same in two sequences, as determined by visual inspection percentage (i.e., over a specific area or unspecified) If not present, at least 60% identity across the entire sequence, optionally at least 6 5%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity), the two sequences are "substantially identical" and exhibit "sequence identity." By preference, identity is maintained over a region of at least about 50 nucleotides (or 10 amino acids) in length. Over 100 to 500, 1000, 2000, or 3000 or or more nucleotides in length, or 30 to 200, or 300, or 5 00, or 700, or 800, or 900, or 1000 or more It exists over a region of approximately 100 amino acids in length.

[0041] For sequence comparison, typically one sequence acts as a reference sequence, When using a sequence comparison algorithm, test and reference sequences are compared. The coordinates of the subarray are specified and the array algorithm program is executed as needed. Program parameters are specified. Default program parameters can be used, or Alternative parameters can be designated. The sequence comparison algorithm then runs according to the program parameters. Based on this data, the percent sequence identity of the test sequence relative to the reference sequence is calculated.

[0042] The term "comparison window" as used herein refers to a window of comparison between 20 and 600, typically about 50 from about 100 to about 200, more usually from about 100 to about 150, It includes reference to any one segment of nucleic acid or amino acid positions where two sequences After the sequence is optimally aligned, the sequence is compared to a reference sequence at the same number of consecutive positions. Methods for alignment of sequences for comparison are known in the art. Optimal alignment of sequences for comparison can be achieved using, for example, the method of Smith and Waterman an(1970)Adv.Appl.Math.2:482c local homology algorithm According to Needleman & Wunsch (1970) J. Mol. Biol. 48 :443 homology alignment algorithm, Pearson & Lipman (1 988) PNAS USA, 85:2444 similarity search method, these algorithms Rhythm (in Wisconsin Genetics Software Package) GAP, BESTFIT, FASTA and TFASTA (Genetics Comp of the Uter Group, 575 Science Dr., Madison, WI By computer implementation or by manual alignment and visual inspection (e.g., Br ent et al.,(2003)Current Protocols in Mo This can be done by conventional methods (see Leukocyte Biology).

[0043] Two examples of suitable algorithms for determining percent sequence identity and sequence similarity are , BLAST, and BLAST 2.0 algorithms, which are Alts chul et al.,(1977)Nuc.Acids Res,.25:3389 -3402; and Altschul et al., (1990) J. Mol. Biol 215:403-410. Software for performing BLAST analysis is a member of the National Center for Biotechnology Information (NCBI). This information is publicly available from the National Institute of Technology, San Jose, CA.

[0044] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., , Karlin & Altschul (1993) PNAS.USA, 90:5873-5 787). One measure of similarity provided by the BLAST algorithm is the maximum The small sum probability (P(N)) of a match between two nucleotide or amino acid sequences. provides an indication of the probability that a given sequence will occur by chance. For example, the probability that a given sequence will occur by chance in a comparison of a test nucleic acid with a reference nucleic acid. The subtotal probability is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.00 If it is less than 1, then the nucleic acid is considered to be similar to the reference sequence.

[0045] The percent identity between two amino acid sequences is calculated using the method of E. Meyers and W. Mil ler (Comput. Appl. Biosci. 4:11-17 (1988)) The residue weights can also be determined using a PAM120 residue weight table, The ALIGN program was used with a gap length penalty of 12 and a gap penalty of 4. Furthermore, the alignment between two amino acid sequences is The percent uniformity is determined using the Needleman & Wunsch (supra) algorithm. This algorithm uses the Blossom 62 matrix or PAM. Any of 250 matrices and 16, 14, 12, 10, 8, 6 or 4 gaps weights and length weights of 1, 2, 3, 4, 5, or 6 using the GCG software package ( The GAP program is incorporated into the GCG Global Action Programme (available at www.gcg.com).

[0046] Polypeptides are typically sequences of amino acids, e.g., where two peptides differ only by conservative substitutions. If the two nucleic acid sequences are substantially identical, the first polypeptide is substantially identical to the second polypeptide. Another indication that two molecules or their complements bind to each other under stringent conditions is that The goal is to hybridize.

[0047] The term "nucleic acid" is used herein synonymously with the term "polynucleotide." and deoxyribonucleotides or ribonucleic acids in either single-stranded or double-stranded form. The term refers to synthetic, natural and non-natural polymers. Known nucleotides have similar binding properties to nucleic acids and are metabolized similarly to the reference nucleotides. These include nucleic acids containing modified analogs or modified backbone residues or linkages. Examples of such analogs are: Examples include, but are not limited to, phosphorothioates, phosphoramidates, methylphosphonates, nates, chiral-methylphosphonates, 2-O-methylribonucleotides, peptide-nucleic acids Examples include polynucleotides such as poly(amino acids) (PNA).

[0048] Unless otherwise indicated, a particular nucleic acid sequence refers to the nucleic acid sequence, not just the sequence explicitly indicated. Conservatively modified variants of a sequence (e.g., degenerate codon substitutions) and complementary sequences are also implicitly encompassed. Specifically, degenerate codon substitutions are made by substituting the third codon of one or more selected (or all) codons. by generating sequences in which positions are substituted with mixed base and / or deoxyinosine residues. (Batzer et al., (1991) Nucleic Acid Res.,19:5081;Ohtsuka et al.,(1985)J Bio l Chem., 260:2605-2608; and Rossolini et al. (1994) Mol Cell Probes, 8:91-98). When a nucleotide sequence is used, the term "optimized nucleotide sequence" refers to a nucleotide sequence that is optimized for production. Uses preferred codons in cells, such as Chinese hamster ovary cells (CHO) This means that the nucleic acid has been modified to encode an amino acid sequence based on the optimized nucleic acid. The nucleotide sequence is initially coded by a starting nucleotide sequence, also known as the "parent" sequence. In certain embodiments, the amino acid sequence of ... The optimized sequences herein have codons that are preferred in CHO mammalian cells. It has been modified as follows.

[0049] Therapeutic Fusion Proteins Integrin-binding domain Integrins are transmembrane receptors that promote cell-extracellular matrix (ECM) adhesion. Upon ligand binding, integrins mediate cell cycle regulation, intracellular cellular organization, and signaling mediates cell signals, such as the organization of the nucleus and the migration of new receptors to the cell membrane. Activates the signal transduction pathway (Giancotti & Ruoslahti (1999) Sci ence,285(5430):1028-32). The presence of integrins is There are several types of integrins, which allow rapid and flexible responses to various events. Integrins are two types of integrins, and one cell can have several different types on its surface. It has subunits: α (alpha) and β (beta), each of which spans the plasma membrane. It has several cytoplasmic domains (Nermut MV et al. (1988). E MBO J.,7(13):4093-9). Acidic amino acids are essential for many ECM proteins. The integrin interaction site (e.g., the amino acid sequence arginine-glycine-asparagine) of It is characterized by a RGD moiety (as part of the single-letter amino acid code "RGD"). The chief are fibronectin, fibrinogen, vitronectin, and osteopontin. It is found in many matrix proteins and aids in cell adhesion. A conserved protein domain known as the GF-like domain is found in many proteins. , which is named after the epidermal growth factor that was first described. The EGF-like domain is It is one of the most common domains found in extracellular proteins (Hidai C (20 18)Open Access J Trans Med Res.,2(2):67- 71), some examples of EGF-like domains containing the RGD motif are listed in Table 1 below. .

[0050] [Table 1]

[0051] As used herein, the term "integrin binding domain" refers to an integrin-binding domain. It refers to a sequence of amino acids or a protein domain that functions to bind to a polypeptide. In embodiments, as used herein, an "integrin binding domain" refers to an integrin A series of amino acids or protein domains that bind to erythrin and contain the RGD motif In one embodiment of the present disclosure, the integrin binding domain is set forth in SEQ ID NO:2. is an EGF-like domain derived from human MFG-E8 having the amino acid sequence as described In an alternative embodiment of the present disclosure, the integrin binding domain is human EDIL3 The EGF-like domains are derived from the following sequences: SEQ ID NO: 11, SEQ ID NO: 77, SEQ ID NO: 9 6, 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 may be a sequence of amino acids 1 to 132 of SEQ ID NO: 11. can be found within.

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

[0053] Phosphatidylserine-binding domain As used herein, "phosphatidylserine" (PS) refers to a phospholipid that is a component of cell membranes. Regarding lipids, PS is mainly confined to the inner leaflet of the cell membrane, Phosphatidylcholine and sphingomyelin are primarily located in the outer leaflet. The asymmetric distribution of proteins is due to the role of P4-A flippases (ATP11A and ATP11C) in the plasma membrane. It is maintained by the action of ATPase, which actively transports PS from the outer leaflet to the inner leaflet. Exposure of PS to the cell surface not only inhibits apoptotic cells but also activates lymphocytes and activated blood cells. It is also observed in platelets, senescent red blood cells, and some cancer cells and their respective microparticles (Saku ragi et al.,(2019)PNAS USA,116(8):2907-1 2) PS exposure may be a biomarker for prothrombotic, inflammatory, or ischemic conditions. Obtain (Pasalic et al.,(2018)J Thromb Haemost .,16(6):1198-2010;Ma et al.,(2017) supra;Zha o et al., (2016) supra. PS functions in numerous cell signaling pathways, It functions as an essential phospholipid in coagulation and acts as a phospholipid receptor for the enzymes IXa, VIIIa, and X) and prothrombinase (factors Xa, Va, and prothrombin) complexes It can function as an enhancer (Spronk et al., 201 4) Thromb Res. 133(Suppl 1):S54-6). Externalized P The best understood function of S remains the secretion of apoptotic cells, cell debris, or PS exposure. It may be an "eat-me" marker for phagocytes such as macrophages that engulf activated cells. As used herein, "phosphatidylserine binding domain" or "P The term "PS-binding domain" refers to a sequence of amino acids or a protein domain that has the function of binding to PS. Examples of endogenous proteins with PS-binding domains are listed in Table 2 below. can be found.

[0054] [Table 2]

[0055] In one embodiment of the present disclosure, the PS domain is set forth in SEQ ID NO: 3 or SEQ ID NO: 76. The present invention is derived from human MFG-E8 having an amino acid sequence as shown in Figure 1. Alternative Embodiments of the Disclosure In one embodiment, the integrin-binding domain is a PS polypeptide from human EDIL3 (SEQ ID NO: 11). a PS-binding domain, wherein the PS-binding domain is amino acids 135-4 of SEQ ID NO: 11. Including 53.

[0056] PS binding activity can be determined by methods well known in the art. The S binding assay is described in the Examples, Section 3.1, where microtiter plates are used. Binding of the fusion proteins of the present disclosure to PS coated on a plate was confirmed by biotin. According to the present disclosure, P Preferably, the S-binding domains are tested in the same manner to determine their respective activities. When tested using the assay described in section 3.1 of the Examples, it was found to be SEQ ID NO:1 The PS-binding activity observed for the human MFG-E8 protein shown in %, for example, at least 25%, at least 50%, at least 75%, at least 80 %, preferably at least 90%, at least 95%, at least 96%, at least A protein is considered to have PS-binding activity if it has at least 97% or at least 98% PS-binding activity. can be.

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

[0058] [Table 3]

[0059] To be of therapeutic value, cross-linked proteins must be capable of cross-linking TAM family members or other P As observed for S-linked receptors, the proteolytic cleavage or shedding is typical. It contains an integrin-binding domain that recognizes integrins on typically insensitive phagocytes. It is useful when proteins with PS-binding domains and integrin-binding domains are used. Proteins such as MFG-E8 or its paralog EDIL3 / DEL1 induce effector Efferocytosis induction in AOI because it has been shown to induce efferocytosis. In contrast, the GAS6 protein, for example, As mentioned above, the receptor on phagocytes (MerTK) is proteolytically cleaved during inflammation and infection. may not be particularly effective in promoting efferocytosis in the AOI because .

[0060] An example of a cross-linked protein listed in Table 3 above is MFG-E8, which is a cross-linked protein derived from milk fat. MFG-E8 is one of the major proteins found in the membrane of the globule (MFGM). Different types of cells (e.g., mammary epithelial cells, vascular cells, epididymal epithelial cells, aortic flat cells) smooth muscle cells, activated macrophages, stimulated endometrial 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 secreted from lactadherin, BP47, and component 15 / 16, MFGM, MGP57 / 53, PAS-6 / PAS-7 glycoproteins, cell wall proteins Protein SED1, sperm surface protein SP47, breast epithelial antigen BA46, and O-acetyl It is also known by several different names, such as GD3 ganglioside synthase (AGS). The MFG-E8 gene is located on chromosome 1 in rats, chromosome 7 in mice, and chromosome 1 in humans. is located on chromosome 15. Alternative splicing of the MFG-E8 pre-mRNA is , resulting in three isoforms of the human protein and two forms of mRNA, The human MFG-E8 gene (UniProt) is expressed in the mouse mammary gland as a variant and a short variant. KB-Q08431) is a 387-residue protein that is processed to form multiple protein products. It encodes a protein of 100 kJ / s. It contains a signal peptide (residues 1-23; underlined), an EGF-like domain, and main (residues 24–67; italics), C1 domain (residues 70–225; bold), and C2 The amino acid sequence of human MFG-E8, including the domain (residues 230–387; bold and underlined), is shown in As shown below. [ka]

[0061] MFG-E8 lacks the transmembrane function of MFGM and is therefore a peripheral membrane protein. Human MFG-E8 functions as a phagocyte-expressing αvβ3 and αvβ5 integrin. It contains one N-terminal EGF-like domain (SEQ ID NO: 2) that binds to phospholipids and anionic phospholipids. Two F5 / 8-discoidin subdomains (C1 and C2) bind with high affinity. The integrin binding domain is composed of a PS-binding domain (SEQ ID NO: 3) containing human MFG-E This is the result of the RGD motif located at residues 46-48 of SEQ ID NO: 1. The cellular debris, hyperactivated cells, and most microparticles (MPs) expose PS and It is the target of FG-E8 and acts as a bridging molecule to opsonize these cells and microparticles. This cross-linking action binds to the αvβ3 and αvβ5 integrins on phagocytes. , triggering an efficient phagocytic program leading to the internalization of debris and particulates. The protein found in MFG-E8 is highly conserved across species. The structure varies depending on the species, and all currently known species contain two C domains, but E The number of GF-like domains varies. For example, the human MFG-E8 protein contains one EGF-like domain. While bovine MFG-E8 and mouse MFG-E8 (SEQ ID NO: 68) contain two Chicken, frog, and zebrafish have three EGF-like domains. The MFG-E8 domain has been previously proposed as a component of therapeutic drugs. In particular, the PS-binding domain (Kooijmans et al., (2018) Nan oscale,10(5):2413-2426) and fragments of MFG-E8 are involved in the pathogenesis of fibrosis. It has been shown to work in models (see U.S. Patent Application Publication No. 2018 / 0334486). (details).

[0062] The phagocytes, both professional and non-professional, phagocytes, kill dead cells, debris, and microorganisms. Non-inflammatory uptake of particles plays an important role in homeostasis after tissue injury (Gree (Nlee-Wacker (2016) supra). The importance of proper clearance is E8 knockout mice show, for example, an increased number of dead (uncleared) cells in tissues. Inflammation in disease models such as neonatal sepsis, autoimmunity, impaired angiogenesis, and impaired wound healing This was further clarified in a genetic model that showed an exacerbated response to the disease (Hanayama et al. al.,(2004)Science,204(5474):1147-50;Das et al.,(2016)J Immunol.,196(12):5089-510 0;Hansen et al.,(2017)J Pediatr Surg.,52 (9):1520-7).

[0063] Furthermore, MFG-E8 inhibits T while increasing the regulatory T cell subset (Treg). suppression of cell activation and proliferation, and inhibition of Th1, Th2, and Th17 subpopulations, thereby enhancing immune function. It has been shown that Tregs generate a tolerogenic environment. Inducing phage-mediated efferocytosis contributes to the resolution of inflammation ((P roto et al.,(2018)Immunity,49(4):666-77) MFG-E8 promotes allogeneic engraftment of embryonic stem cell-derived tissues across MHC barriers It has been reported that (Tan et al., (2015) Stem Cell Re ports, 5(5):741-752). MFG-E8 also has multiple nutritional uses. It promotes tissue development and helps protect against infectious agents. Proteins are potential health-promoting nutraceuticals for food and pharmaceutical applications. MFG -E8 is a nutrient-rich supplement that can be used in combination with other nutrients (e.g., probiotics, whey protein micelles, alpha-hydroxybutyric acid, may also be combined with hydroxyisocaproic acid, citrulline, and branched chain fatty acids .

[0064] Solubilization Domain As described herein, the therapeutic fusion proteins of the present disclosure comprise integrin-binding domains. Furthermore, the fusion protein also contains a nucleotide sequence that is ... It contains additional domains that confer some desirable properties. For the purposes of this application, This additional domain, called the "solubilization domain," increases solubility, reduces aggregation, and promotes bioactivity. As a result, the fusion protein can have improved biological properties, such as increased activity. Furthermore, the presence of a solubilizing domain may be beneficial for therapeutic fusion proteins. Improved protein stability and increased yield after purification in cell expression systems. This improves expression of the fusion protein compared to the wild-type protein.

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

[0066] In some embodiments, the solubilization domain is human serum albumin (HSA; sequence and albumin proteins such as albumin 4) or variants thereof, e.g., those with reduced aggregation tendency. HSA (SEQ ID NO: 5) containing the amino acid substitution C34S to achieve the desired effect, or HSA D3 (SEQ ID NO: 6) HSA has relatively large saccharin domains that reduce renal filtration. This is highly dependent on several factors, including the size and neonatal Fc receptor (FcRn) binding properties. It has a long serum half-life and avoids intracellular degradation. HSA for fusion to polypeptides The use of N-terminal fragments of the Therefore, molecules can be genetically or chemically fused or conjugated to albumin. This can stabilize or extend shelf life and / or improve the stability of the compound in solution, in vitro and in vivo. and / or in vivo, the activity of the molecule can be maintained for a prolonged period of time. Further methods are described, for example, in WO 2001 / 077137 and WO 2001 / 077137. This can be found in brochure no. 2003 / 060071.

[0067] In some embodiments, the solubility domain is human Fc immunoglobulin G1 (Fc - IgG1; SEQ ID NO: 7). The Fc domain also includes an antibody Fc domain, e.g. , using knob-into-hole (KiH)-based modification to make complementary amino acid substitutions. Modification by improving Fc heterodimerization by introducing it into the CH3 domain of Fc For example, the substitution T366W to create a "knob" in one CH3 domain can be , and substitutions T366S, L36 to create a "hole" in the other CH3 domain 8A and Y407V (Merchant et al. (1998) Nat. Biol. Technol., 16(7):677-81; EU number IgG1). In combination with modifications to improve dimerization, the Fc domain may also contain Further modifications include, for example, amino groups on cysteines to create additional cysteine ​​bonds. amino acid substitutions, e.g., S354C and / or Y349C, and Fcγ receptor and complement protein Antibodies to reduce or eliminate binding to protein C1q to "silence" immune effector function The so-called "LALA" double mutation (L234A and L235A, E U numbering) reduces effector function (Lund et al., (1992) Mol Immunol., 29:53-9). Alternatively, the "DAPA" double mutation (D26 5A and P329A (EU numbering) reduce effector function. In this embodiment, the Fc domain contains the amino acid substitution D265A, P for Fc silencing. 329A, and / or KiH amino acid substitutions T366W (knob) or T366S, L368 In one embodiment, the Fc domain may comprise human I and Y407V (whole). It is derived from gG1 and contains the amino acid substitutions D265A, P329A (SEQ ID NO: 8). In this embodiment, the Fc domain is derived from human IgG1 and contains the amino acid substitutions D265A, P3 29A, S354C and amino acid substitution T366W (Fc-IgG1-knob; SEQ ID NO: 9) In another embodiment, the Fc domain is derived from human IgG1 and comprises the amino acid substitution D265A, P329A, Y349C and amino acid substitutions T366S, L368A and Y 407V (Fc-IgG1-hole; SEQ ID NO: 10).

[0068] In some embodiments, the soluble domain is human IgA, IgD, IgE, or IgF. It contains an antibody Fc domain derived from gM.

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

[0070] Linking and Orienting Domains of Therapeutic Fusion Proteins The integrin-binding domain, PS-binding domain, and solubilizing domain of the fusion protein of the present disclosure As used herein, "coupled" or "not coupled" The term "fused to" refers to a fusion protein that is directly or indirectly linked to another domain of the fusion protein. A direct bond is a form of linkage, and is referred to herein as "a domain of a fusion protein." As an example, a molecule with the form ABC is called a "fused" or "fused" molecule. Domain A is directly linked to Domain B, which is directly linked to Domain C. In this case, domain A can be described as being fused to domain B, which is fused to domain C. In another example, domain A can be directly linked to domain B and indirectly linked to domain C. In this case, domain A is indirectly linked to domain C by an internal linker. It can also be described as being fused to domain B, which is directly linked to domain A.

[0071] In some embodiments, the linkage is a direct linkage, such that the domains are not directly linked to each other. In some embodiments, the integrin binding domain is fused to a solubilizing domain. Specifically, the PS binding domain ( For example, the C1-C2 discoidin subdomain) binds to integrin-binding domains (e.g., The C-terminus of a soluble domain (e.g., HSA) is fused to the C-terminus of a soluble domain (e.g., EGF-like domain) and the N-terminus of a soluble domain (e.g., HSA). In some embodiments, the solubility domain is fused to the PS-binding domain. fused to an integrin-binding domain. The domain (e.g., EGF-like domain) is C-terminal to the solubilizing domain (e.g., HSA). and a PS-binding domain (e.g., C1-C2 discoidin subdomain) In some embodiments, the integrin binding domain is fused to the C1- The solubilization domain is fused to a PS-binding domain containing the C2 discoidin subdomain. , inserted between the C1-C2 discoidin subdomains. The C-terminus of the binding domain (e.g., EGF-like domain) is the C1 discoidin subdomain. The C-terminus of the C1 discoidin subdomain is fused to the N-terminus of the discoidin domain, and the C-terminus of the C1 discoidin subdomain is fused to the solubilization domain ( The C-terminus of the solubilizing domain is fused to the N-terminus of a C2 discoidin. In another embodiment, the integrin-binding domain is fused to the N-terminus of the subdomain. is fused to a solubility domain which is fused to a PS binding domain. The integrin binding domain (e.g., HSA) is a cytosine-3-phosphate dehydrogenase (EGF)-like domain. the C-terminus of the discoidin and the PS-binding domain (e.g., the C1-C2 discoidin subdomain) In one embodiment, HSA is fused to the N-terminus of the EGF-like domain. The C1 discoidin domain is then fused to the N-terminus of the C1 discoidin domain.

[0072] In some embodiments, the soluble domain (e.g., HSA) binds to integrins. In some embodiments, the binding domain is fused to the PS binding domain. The tegrin-binding domain is located at the N-terminus of the fusion protein, and the PS-binding domain is located at the nucleus of the fusion protein. It is located at the C-terminus of the protein.

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

[0074] In some embodiments, the soluble domain (e.g., HSA) binds to integrins. In some embodiments, the binding domain is fused to the PS binding domain. The tegrin-binding domain is located at the N-terminus of the fusion protein, and the PS-binding domain is located at the nucleus of the fusion protein. It is located at the C-terminus of the protein.

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

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

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

[0078] In some embodiments, the solubility domain (e.g., HSA) is set forth in SEQ ID NO:5. The HSA comprises the amino acid sequence described above or a functional variant thereof.

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

[0080] In an alternative embodiment, an HSA comprising the amino acid sequence as set forth in SEQ ID NO:5. is fused to the C-terminus of the EGF-like domain of EDIL3, and the PS-binding domain of EDIL3 In one embodiment, the fusion protein is fused to the N-terminus of SEQ ID NO: 70 (FP1 068). In one embodiment, the fusion protein comprises an amino acid sequence as set forth in comprises the amino acid sequence as set forth in SEQ ID NO: 69 (FP1776).

[0081] In some embodiments, the linkage is via a polypeptide linker, e.g., For example, a polynucleotide that links a solubility domain to a PS-binding domain in the fusion protein of the present disclosure may be used. The peptide linkers are referred to as "exolinkers." These exolinkers are typically contains glycine (G) and / or serine (S), and contains glycine and leucine (GL) or In some embodiments, the linker may comprise a polynucleotide having a glycine and a valine (GL). A number of G and S residues, for example, G2S and its derivatives, such as (G2S)4 as shown in SEQ ID NO:62. a plurality of (GS)4 as set forth in SEQ ID NO: 63, a G4S as set forth in SEQ ID NO: 64, or Contains (G4S)2 as set forth in SEQ ID NO:65.

[0082] In some embodiments, the exolinker is a linker that is C-terminal to the integrin binding domain. Specifically, the external linker is fused between the N-terminus of the EGF-like domain and the N-terminus of the soluble domain. In some embodiments, the external linker is fused to the C-terminus of the polypeptide and to the N-terminus of HSA. The anchor is fused between the C-terminus of the solubility 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, the exolinker is a linker between the C-terminus of the integrin binding domain and A further external linker is fused between the N-terminus of the solubility domain and the N-terminus of the solubility domain. The exolinker is fused between the C-terminus and the N-terminus of the PS-binding domain. The EGF-like domain is fused to the C-terminus of the HSA and the N-terminus of the HSA. It is fused to the C-terminus of SA and the N-terminus of the PS-binding domain.

[0083] In some embodiments, the exolinker comprising a GS is an integrin binding domain. and the N-terminus of the solubility domain. An external linker comprising the following is fused to the C-terminus of the soluble domain and the N-terminus of the PS-binding domain: In some embodiments, the exolinker comprises (G2S)4 (SEQ ID NO: 62). The soluble domain is fused to the C-terminus of the soluble domain and the N-terminus of the PS-binding domain. In one embodiment, an exolinker comprising G4S (SEQ ID NO: 64) is attached to the C-terminus of the solubilization domain. In some embodiments, the G4S An external linker comprising 2 (SEQ ID NO: 65) connects the C-terminus of the solubility domain and the PS-binding domain. It is fused to the N-terminus of the gene.

[0084] In one embodiment, the external linker comprising GS is C-terminal to the EGF-like domain and HS A fusion protein of the present disclosure containing this structure is shown in SEQ ID NO: 42 (F It has the amino acid sequence described in (p. 330).

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

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

[0087] In one embodiment, the external linker comprising GS is C-terminal to the EGF-like domain and HS The C-terminus of HSA is fused directly to the N-terminus of the PS-binding domain. do.

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

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

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

[0091] Functional properties of therapeutic fusion proteins The present disclosure provides a method for the production of MFG-E8 derived from human MFG-E8, which is effective in promoting efferocytosis. and therefore active in eliminating the major drivers of systemic inflammation and microvascular pathology As described in the Examples, a fusion protein having the general structure EGF-H Fusion proteins carrying SA-C1-C2 were used in several efferocytosis assays For example, the fusion protein has been shown to be effective in the treatment of macrophages. Lipopolysaccharide (LPS) or Staphylococcus aureus (S. aureus)-damaging efferocytosis restores the normal function of the endothelium and promotes efferocytosis of microparticles and dead cells by endothelial cells. The fusion protein also preserved kidney function in a mouse model of acute kidney injury. It is effective in protecting against weight loss.

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

[0093] Throughout the body of this application, discrepancies between the body of the specification (e.g., Table 4) and the Sequence Listing are noted. In cases where there are any differences, the text of this specification shall take precedence.

[0094] [Table 4]

[0095] [Table 5]

[0096] [Table 6]

[0097] [Table 7]

[0098] [Table 8]

[0099] [Table 9]

[0100] [Table 10]

[0101]

Table 11

[0102]

Table 12

[0103]

Table 13

[0104]

Table 14

[0105]

Table 15

[0106] Table 16

[0107] Table 17

[0108] Table 18

[0109] Table 19

[0110] Table 20

[0111] Table 21

[0112] Table 22

[0113] Table 23

[0114] Table 24

[0115] Table 25

[0116] Table 26

[0117] Table 27

[0118] Table 28

[0119] Table 29

[0120] Table 30

[0121] Table 31

[0122] Table 32

[0123] Table 33

[0124] Table 34

[0125] Table 35

[0126] Table 36

[0127] Table 37

[0128] Table 38

[0129] Table 39

[0130] Table 40

[0131] Table 41

[0132] Table 42

[0133] Table 43

[0134] Table 44

[0135] Table 45

[0136] Table 46

[0137] Table 47

[0138] Table 48

[0139] Table 49

[0140]

Table 50

[0141] Table 51

[0142] [Table 52]

[0143] [Table 53]

[0144] [Table 54]

[0145] [Table 55]

[0146] [Table 56]

[0147] The present application also includes variants of each of SEQ ID NOs: 69, 70 and 72, The EGF-like domain of the EDIL3 sequence is represented by the following sequences: SEQ ID NO: 96, SEQ ID NO: 97, SEQ ID NO: 98, SEQ ID NO: 99, SEQ ID NO: 100, SEQ ID NO: 101, SEQ ID NO: 102, SEQ ID NO: 103, SEQ ID NO: 104, SEQ ID NO: 105, SEQ ID NO: 106, SEQ ID NO: 107, SEQ ID NO: 108, SEQ ID NO: 109, SEQ ID NO: 110, SEQ ID NO: 111, SEQ ID NO: 112, SEQ ID NO: 113, SEQ ID NO: 114, SEQ ID NO: 115, SEQ ID NO: 116, SEQ ID NO: 117, SEQ ID NO: 118, SEQ ID NO: 119 Corresponding to either sequence number 98, sequence number 99, sequence number 100, or sequence number 101 do.

[0148] The present application also relates to a method for detecting the integrin-binding domain of MFGE8 or EDIL3, and a method for detecting the integrin-binding domain of TIM4. PS such as the IgSF V domain or the GLA domain of the bridging protein GAS6 mutant Therapeutic fusion proteins include those containing the binding domain.

[0149] Modification of the Proteins of the Present Disclosure The present application provides a method for the preparation of proteins having various modifications to the variants and / or domains of the proteins described herein. and fragments thereof, as well as fusions and conjugates of the disclosed molecules. The domain of the fusion protein may have conservative modifications of amino acid residues, where the modified The resulting proteins retain or exhibit enhanced properties compared to the fusion proteins containing the parent domains. Alternatively, the domain of the therapeutic fusion protein may have a deletion of amino acid residues, wherein the modified fusion protein has enhanced activity compared to the protein containing the parent domain. Alternatively, the therapeutic fusion protein may contain an insertion of amino acid residues. wherein the modified protein has an increased activity compared to the unmodified protein. In one embodiment, such amino acid insertions are made to the parent target amino acid sequence, thereby retaining or having enhanced properties. Glycine is used in several combinations to act as a linker between protein domains. It contains a guanine or serine residue.

[0150] Site-directed mutagenesis or PCR-mediated mutagenesis can be performed to introduce mutations; The effect on integrin and / or PS binding, or other functional properties of interest, can be determined in vitro. or in vivo assays. and / or the mutations may be amino acid substitutions, additions or deletions. Typically, no more than one, two, three, four, or five residues within the binding domain are changed. do.

[0151] Amino acid sequence variants of therapeutic fusion proteins with essentially similar properties to the unmodified variants Variants can be generated by introducing appropriate nucleotide changes into the coding DNA or by modifying the desired mutation. Such variants can be prepared by synthesis of variants. This includes deletions from, or insertions or substitutions of, residues within the amino acid sequence of the gene. In some embodiments, the variants may include additional linker sequences, reduced linker sequences or The amino acid sequence may be removed, and / or the amino acid mutation or substitution and deletion of one or more amino acids may be included. Any combination of deletions, insertions, and substitutions may be made, provided that the final construct possesses the desired characteristics. Combinations are made to arrive at the final construct. Amino acid changes are also made to the possible glycosylation sites. The post-translational processing of the molecule may be altered, such as by changing the number or location of transcription sites.

[0152] Methods for producing recombinant molecules Nucleic acids and expression systems In one embodiment, the present application provides a method for treating one or more polypeptide chains of a therapeutic fusion protein. A method for recombinantly producing a multispecific binding molecule is provided, which comprises: 1) synthesizing the polypeptide chains of the multispecific binding molecule; 2) producing one or more DNA constructs containing a nucleic acid molecule encoding said DNA constructs; 3) introducing the compound into one or more expression vectors; and 3) expressing the compound in one or more host cells. and 4) expressing the molecule in a host cell or in solution. This includes assembly and assembly.

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

[0154] An "isolated nucleic acid" refers to a nucleic acid that has been isolated from a naturally occurring source. It is a nucleic acid that is isolated from the contiguous genetic sequences present in the genome of a given organism. For example, P Enzymatic or other methods of producing a nucleic acid from a template, such as a PCR product, a cDNA molecule, or an oligonucleotide. In the case of chemically synthesized nucleic acids, the nucleic acids resulting from such a process are isolated nucleic acids. It is understood that an isolated nucleic acid molecule is a nucleic acid molecule in the form of a discrete fragment or a larger fragment. In one preferred embodiment, the nucleic acid is a nucleic acid molecule that is a component of a nucleic acid construct. , substantially free from contaminating endogenous materials. The nucleic acid molecule is preferably in a substantially pure form. allowing the identification, manipulation, and recovery of its constituent nucleotide sequences by standard biochemical methods. DNA or RNA isolated at least once in an amount or concentration that allows the brook et al.,Molecular Cloning: A Laborat ory Manual,2nd ed.,Cold Spring Harbor La Laboratory, Cold Spring Harbor, NY (1989). Such sequences are preferably those typically present in eukaryotic genes. of an open reading frame uninterrupted by internal untranslated sequences or introns The sequence of the non-translated DNA is provided and / or constructed in the form of an open reading frame. It may be present 5' or 3' from the coding region and does not interfere with manipulation or expression of the coding region.

[0155] The present invention also relates to expression systems in the form of plasmids, expression vectors, transcription or expression cassettes, and Constructs are provided which comprise at least one polynucleotide as described above. Additionally, the present invention provides a host cell comprising such an expression system or construct.

[0156] In one embodiment, the present disclosure provides (a) a host cell comprising a nucleic acid encoding a fusion protein. and culturing the host cells, wherein the cultured host cells express the fusion protein. and (b) recovering the fusion protein from the host cell culture. A method for preparing a protein is provided.

[0157] Expression vectors and host cells for producing the above therapeutic fusion proteins are also disclosed in the present invention. The term "vector" refers to a vector used to transform or transfer a host cell. suitable for expression of one or more heterologous coding regions operably linked thereto Any molecule or entity containing a nucleic acid sequence that directs and / or controls (in relation to a host cell) (e.g., nucleic acid, plasmid, bacteriophage, or virus). The present vectors are used to express polynucleotides encoding the chains or binding domains of the molecules. Both viral and non-viral expression vectors can be used to express mammalian cells. Therapeutic fusion proteins can be produced in mammalian host cells. Vectors and systems typically include expression vectors for expressing proteins or RNA. Plasmids, episomal vectors, and human artificial chromosomes (e.g., Har See Rington et al., (1997) Nat Genet 15:345 For example, polynucleotides and Non-viral vectors useful for expressing polypeptides include pThioHis A and B. and C, pcDNA3.1 / His, pEBVHis A, B, and C (Invitrogen en, San Diego, CA), MPSV vectors, as well as other proteins expressing Useful vectors include many other vectors known in the art for use in the production of recombinant human ovarian tumors. Viral vectors include retroviruses, adenoviruses, adeno-associated viruses, and hepatocellular carcinoma (HCV). Rupesvirus-based vectors, SV40, papillomavirus, HBP Epstein-Barr virus - Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV)-based Examples include vectors based on Brent et al. (1995) supra; Smith , Annu. Rev. Microbiol. 49:807; and Rosenfeld e. See, e.g., et al., (1992) Cell 68:143.

[0158] The choice of expression vector will depend on the intended host cell in which the vector is to be expressed. Typically, an expression vector is engineered to encode a polynucleotide encoding a therapeutic fusion protein. Contains an operably linked promoter and other control sequences (e.g., enhancers) In some embodiments, an inducible promoter is used to allow expression of the inserted gene under conditions other than inducing conditions. Inducible promoters include, for example, arabinose, lac Z, metallothionein promoter, or heat shock promoter. Cultures of the selected organisms are then cultured in a manner that allows the expression product to be better tolerated by the host cell by cleaving the coding sequence. In addition to the promoter, other Regulatory elements may also be required or required for efficient expression of the therapeutic fusion protein. These elements typically include the ATG initiation codon and adjacent In addition, expression efficiency may vary depending on the cell line used. This can be enhanced by including an enhancer suitable for al.,(1994)Results Probl.Cell Differ.20: 125; and Bittner et al., (1987) Meth. Enzymol. , 153:516). For example, the SV40 enhancer or the CMV enhancer can be used. It may also be used to increase expression in mammalian host cells.

[0159] The expression vector may also contain the sequences of the binding domain and / or solubility domain described above. a secretion signal for forming a fusion protein with a polypeptide encoded by More often, the inserted sequence will be contained in a vector. The binding domain and the solubility domain are linked to a signal sequence before the fusion protein. Vectors that allow expression of the gene, thereby also allow production of an intact modified protein. When cultured under appropriate conditions, the host cells can then be recovered from the culture medium ( if the host cell secretes it into the medium) or is recovered directly from the host cell that produces it. The modified protein may be secreted (if not secreted) and used to express the modified protein. The choice of cells may be based on the desired expression level, activity (such as glycosylation or phosphorylation), or other factors. The desired polypeptide modifications and ease of folding into biologically active molecules are important. The host cell can be eukaryotic or prokaryotic, depending on a variety of factors.

[0160] Mammalian cell lines available as hosts for expression are known in the art and include those described in the American American Type Culture Collection Immortalized cell lines available from the American College of Cancer (ATCC) include, but are not limited to, immortalized cell lines available from the American College of Cancer (ATCC) Any cell line used in expression systems known in the art can be used to produce the recombinant fusions of the present invention. Generally, a host cell is capable of encoding the desired fusion protein. The host cell can be transformed with a recombinant expression vector containing the DNA encoding the gene. Prokaryotes include gram-negative or gram-negative organisms, yeast, or higher eukaryotic cells. Higher eukaryotic cells include bacteria, such as E. coli or bacilli. These 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 (C HO) cells, or their derivatives and related cell lines that grow in serum-free medium, HeLa cells, BHK cell line, CV-1 EBNA cell line, 293, 293EBNA or MSR293 Which human embryonic kidney (HEK) cells, human epidermal A431 cells, human Colo205 cells, For in vitro culture of other transformed primate cell lines, normal diploid cells, primary tissues, and primary explants Derived cell lines include HL-60, U937, HaK, or Jurkat cells. By choice, mammalian cells such as HepG2 / 3B, KB, NIH 3T3 or S49 may be used. The cell lines can be used to express the polypeptides in various signal transduction or reporter assays. Alternatively, lower organisms such as yeast can be used to express the polypeptide if desired. Polypeptides can be produced in eukaryotes or prokaryotes, such as bacteria. The mother is P. pastoris, S. cerevisiae siae, S. pombe, Kluyveromyces Candida, or other strains capable of expressing heterologous polypeptides. Suitable bacterial strains include Escherichia coli (E. coli), Bacillus subtilis (B. subtilis, S. typhimurium, or heterologous poly Any bacterial strain capable of expressing the peptide is included. When produced in bacteria, appropriate sites may be phosphorylated or glycosylated to obtain a functional product. It may be desirable to modify the product produced therein by sylation. Such covalent attachment can be achieved using known chemical or enzymatic methods.

[0161] Methods for introducing an expression vector containing a polynucleotide sequence of interest into a cell host include: For example, calcium chloride transfection is commonly used. It is primarily utilized by prokaryotic cells, while calcium phosphate is utilized by other cellular hosts. Treatment with ethanol or electroporation can be used. Other methods include, for example, electroporation. troporation, calcium phosphate treatment, liposome-mediated transformation, injection Microinjection, gene gun method, virosomes, immunoliposomes, polymerase chain reaction On: Nucleic acid conjugates, naked DNA, artificial virions, herpes virus structural proteins These include fusion with the protein VP22, drug-facilitated uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression is desirable. For example, cell lines that stably express modified proteins often contain viral origins of replication or The expression vectors of the present disclosure containing endogenous expression elements and a selectable marker gene are used. After the vector is introduced, the cells are grown in rich medium for 1-2 days, and then They can then be switched to selective media. The purpose of the selectable marker is to confer resistance to selection. The presence of which inhibits the growth of cells that successfully express the introduced sequences in selective medium. Resistant stably transfected cells can be cultured using tissue culture techniques appropriate for the cell type. can be propagated using

[0162] The fusion protein is typically recovered from the culture medium as a secreted polypeptide. If produced directly without a secretory signal, it may be recovered from host cell lysates. If the polypeptide is membrane-bound, use an appropriate detergent solution (e.g., Trito nX 100) can be used to release it from the membrane.

[0163] If the fusion protein is produced in a recombinant cell other than one of human origin, it is completely free of recombinant cell proteins or polypeptides, except that recombinant cell proteins or polypeptides As a first step, the fusion protein must be purified from the culture medium or The lysate is typically centrifuged to remove particulate cell debris. Luriapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography It can be easily purified by chromatography, and affinity chromatography is preferred. This is a novel purification technique. Fractionation on an ion exchange column, ethanol precipitation, reversed-phase HPLC, and silica gel chromatography are used. chromatography on heparin sepharose, anionic or Chromatography on cation exchange resins (such as polyaspartic acid columns), Protein purification methods such as microfocusing, SDS-PAGE, and ammonium sulfate precipitation Other techniques for manufacturing are available.

[0164] In certain embodiments, a polynucleotide encoding a therapeutic fusion protein of the invention is Provided herein are viral vectors comprising: The 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 The compounds can be used for the treatment and / or prevention of diseases such as those described above.

[0165] Pharmaceutical Composition In another aspect, the present disclosure provides a method for treating a cancer cell comprising administering to a patient a composition comprising one or more pharmaceutically acceptable excipients, diluents, or carriers. Compositions, e.g., pharmaceutical compositions, containing the therapeutic fusion proteins of the invention in combination with Such compositions may comprise (e.g., two or more different) therapeutic fusion proteins of the present disclosure. The protein may comprise one or a combination of:

[0166] The pharmaceutical compositions described herein may also be used in combination therapy, i.e., in combination with other drugs. For example, the combination therapy can include, for example, at least one anti-inflammatory drug. The fusion protein of the present disclosure may be combined with an antiviral, anti-infective, or immunosuppressant agent. Examples of therapeutic agents that can be used in combination therapy include those that utilize the therapeutic fusion proteins of the present disclosure. This is described in more detail below in the section on usage.

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

[0168] The phrase "pharmaceutically acceptable" means approved by a federal or state regulatory agency. or according to the United States Pharmacopoeia or other generally recognized standards for use in animals, more particularly in humans. This means that the product is listed in the pharmacopoeia in which it was produced.

[0169] The term "pharmaceutical composition" refers to a pharmaceutical composition containing at least one active ingredient (e.g., a modified protein). and at least one pharmaceutically acceptable excipient, diluent or carrier.

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

[0171] As used herein, a "pharmaceutically acceptable carrier" includes a physiologically compatible All solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. The carrier may be administered intravenously, intramuscularly, subcutaneously, parenterally, spinally, or epidermally (e.g., by injection). In one embodiment, the carrier is suitable for subcutaneous administration. Depending on the route of administration, the active compound, i.e., the fusion protein, should be administered in a coated with a material that protects the compound from the action of acids and other natural conditions that can inactivate it. It is possible.

[0172] The pharmaceutical compositions described herein may include one or more pharmaceutically acceptable salts. The pharmaceutical compositions described herein may also include a pharmaceutically acceptable antioxidant. Examples of antioxidants that may be used include ascorbic acid, cysteine ​​hydrochloride, sodium bisulfite, and sodium metabisulfite. Water-soluble antioxidants such as sodium sulfite, sodium sulphite; ascorbyl palmitate , butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), Oil-soluble antioxidants such as lecithin, propyl gallate, and alpha-tocopherol; citric acid , ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, etc. Contains additives.

[0173] Examples of suitable aqueous and non-aqueous carriers that may be used in the pharmaceutical compositions described herein include water, Ethanol, polyols (glycerol, propylene glycol, polyethylene glycol) oils such as olive oil, and oleic acid; Suitable fluidity agents include injectable organic esters such as ethyl acetate, lecithin, etc. Which coating material to use, maintaining the required particle size in case of dispersion, and the surfactant It can be maintained through use.

[0174] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms can be achieved by sterilization procedures and the use of various antibacterial and antifungal agents, e.g., parabens, By including chlorobutanol, phenol sorbic acid, etc. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like, in the compositions. Additionally, prolonged absorption of the injectable pharmaceutical form can be attributed to the use of aluminum monostearate and gelatin. This can be brought about by the inclusion of an agent that delays absorption such as benzodiazepine.

[0175] Pharmaceutically acceptable carriers include sterile water for the extemporaneous preparation of sterile injectable solutions or dispersions. Such vehicles for pharmaceutically active substances include solutions or dispersions and sterile powders. The use of such media and agents is well known in the art. Except where not otherwise specified, their use in the pharmaceutical compositions of the present invention is contemplated. Compounds can also be incorporated into compositions.

[0176] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition may be in the form of a solution, microemulsion, liposome, or other formulation suitable for high drug concentration. The carrier may be, for example, water, ethanol, polyol ( For example, glycerol, propylene glycol, and liquid polyethylene glycol. and suitable mixtures thereof. For example, the use of a coating such as lecithin, the maintenance of the required particle size in the case of dispersion, and This can be maintained by the use of isotonic agents, for example, sugars, surfactants, etc. The composition may contain polyalcohols such as mannitol, sorbitol, or sodium chloride. It is possible.

[0177] A review of the development of stable protein formulations is provided by Cleland et al., 1993)Crit Reviews Ther Drug Carrier Syst ems,10(4):307-377 and Wei W(1999) Int J Phar Maceutics, 185:129-88.

[0178] Solutions or suspensions used for intradermal or subcutaneous administration typically contain one or more of the following ingredients: Contains: water for injection, saline solution, fixed oil, polyethylene glycol, glycerin, pro Sterile diluents such as pyrene glycol or other synthetic solvents, benzyl alcohol or methyl paraben Antibacterial agents such as Raben, antioxidants such as ascorbic acid or sodium bisulfite, ethylene chelating agents such as dibenzodiaminetetraacetic acid, buffers such as acetates, citrates or phosphates, and and agents to adjust tonicity such as sodium chloride or dextrose. pH is controlled by the use of hydrochloric acid or The formulation can be prepared with an acid or base, such as sodium hydroxide. The composition may be enclosed in a plastic stick, an ampoule, a disposable syringe, or a multiple dose vial.

[0179] Sterile injectable solutions may be prepared by combining the active compound in the required amount with one or more of the ingredients enumerated above, as required. The compound can be prepared by incorporating it into a suitable solvent containing the compound and then sterile filtering it. Generally, the dispersion liquid is prepared by dissolving the fusion protein of the present invention in a basic dispersion medium and a layer of the fusion protein. by incorporating the required other ingredients from those listed above into a sterile vehicle. In the case of sterile powders for preparing sterile injections, the preparation methods are vacuum drying and freeze-drying. Lyophilized (freeze-dried), which allows the active ingredient to be extracted from a previously sterile-filtered solution. Powders of any further desired ingredients are obtained.

[0180] The amount of active ingredient which can be combined with a carrier material to produce a single dosage form varies depending on the dosage. The dosage form will vary depending on the intended subject and the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a therapeutically effective composition will generally be determined by the amount of active ingredient that can be combined with a carrier material to produce a therapeutically effective composition. Generally, out of 100 percent, this amount is between about 0.01 percent and about 99% active ingredient, about 0.1% to about 70%, or about 1% 10 to about 30 percent of the active ingredient in combination with a pharmaceutically acceptable carrier. .

[0181] The choice of dosing regimen for a therapeutic engineered protein depends on the serum or tissue turnover rate of the entity, the severity of the disease, and the severity of the disease. Several factors, including the level of morphology, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix, are important. In certain embodiments, the dosing regimen will depend on several factors: This maximizes the amount of therapeutic agent delivered to the patient. The efficacy of the antibody will depend, in part, on the specific entity and the severity of the condition being treated. Guidance for selecting appropriate doses of molecules is available (e.g., Bach (ed.) (1993) Monoclonal Antibodies and Peptides Therapy in Autoimmune Diseases,Marcel De kker,New York,NY;Baert,et al.(2003)New Engl.J.Med.348:601-608;Milgrom,et al.(1 999)New Engl.J.Med.341:1966-1973;Slamon, et al.(2001)New Engl.J.Med.344:783-792;B eniaminovitz,et al.(2000)New Engl.J.Med. 342:613-619;Ghosh,et al.(2003)New Engl.J .Med.348:24-32;Lipsky,et al.(2000)New En gl. J. Med. 343:1594-1602).

[0182] Determining the appropriate dosage is known in the art to affect treatment, e.g. parameters or factors known, suspected or predicted to affect treatment Generally, the dose is somewhat less than the optimal dose. Start with 0.05 and then gradually increase until the desired or optimal effect is achieved relative to negative side effects. Important diagnostic measures are, for example, measures of the symptoms of inflammation or the amount of inflammatory cells produced. Including Cain's level.

[0183] The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present disclosure may be determined without causing toxicity to the patient. It is not a compound that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration. The dosage level selected may be varied to obtain the desired amount of active ingredient. the activity of the particular composition, the route of administration, time of administration, rate of excretion of the particular compound used, Therapies, other drugs, compounds and / or materials used in combination with the particular composition the duration of the treatment, the age, sex, weight, condition, general health and past medical history of the patient to be treated, and various pharmacokinetic factors, including factors well known in the medical arts. This will be the case.

[0184] Dosage regimens are adjusted to provide the optimum desired response. For example, a single bolus The dose may be administered in several divided doses over time, or may be administered in a single dose over the course of a treatment. The dosage may be proportionally reduced or increased as indicated by the exigencies of the situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease and uniformity of dosage. As used herein, a dosage unit form refers to a unit dose for a subject to be treated. Each unit refers to a physically discrete unit suitable for administering the desired therapeutic agent in association with the required pharmaceutical carrier. The dosage unit forms of the present invention contain a predetermined amount of active compound calculated to produce a therapeutic effect. The nature of the method will depend on the inherent properties of the active compound and the particular therapeutic effect to be achieved, as well as the individual's sensitivity. The limitations inherent in the technology of formulating such active compounds for the treatment of directly dependent.

[0185] For administration of a therapeutic fusion protein, the dosage is about 0.0001 to 150% of the host's body weight. mg / kg, e.g., 5, 15, and 50 mg / kg subcutaneously, more usually 0.01 ~5mg / kg. Exemplary treatment regimens include once a week, once every two weeks, once every three weeks, It involves administration once, once every four weeks, once a month, once every three months, or once every three to six months.

[0186] The therapeutic fusion proteins of the present invention can be administered multiple times. The interval between single administrations is: For example, it can be weekly, monthly, quarterly, or yearly. Intervals may also be irregular, as indicated by measuring blood levels of In this case, the dose is adjusted to achieve a plasma protein concentration of approximately 1-1000 μg / ml. and some methods achieve plasma protein concentrations of approximately 25-300 μg / ml. It is adjusted as follows.

[0187] Alternatively, the therapeutic fusion protein can be administered as a sustained release formulation, in which case: Less frequent administration is required. The dosage and frequency will depend on the half-life of the protein in the patient. The dose may vary depending on whether the treatment is prophylactic or therapeutic. Doses are given at relatively infrequent intervals over an extended period of time. Some patients will need to receive treatment for the rest of their lives. For therapeutic use, the drug may be administered until the progression of the condition or disease is reduced or halted, or or at relatively short intervals until the patient shows partial or complete improvement of the symptoms of the condition or disease. Higher doses may be required. Patients may then receive a prophylactic regimen. can.

[0188] The actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be adjusted to avoid causing toxicity to the patient. It is not a compound that is effective in achieving the desired therapeutic response for a particular patient, composition, and mode of administration. The dosage level selected may be varied to obtain the desired amount of active ingredient. the activity of the particular composition, the route of administration, time of administration, rate of excretion of the particular compound used, Therapies, other drugs, compounds and / or materials used in combination with the particular composition the duration of the treatment, the age, sex, weight, condition, general health and past medical history of the patient to be treated, as well as various pharmacokinetic factors, including factors well known in the medical arts. This will be the case.

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

[0190] The compositions of the present disclosure can be prepared by one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the administration route The route and / or method of administration will vary depending on the desired results. Routes include, for example, intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal, and intravenous administration by injection or infusion. or other common routes of administration. As used herein, "parenteral administration" refers to The term "administration" generally refers to, but is not limited to, methods of administration other than enteral and topical administration by injection. However, intravenous, intramuscular, intraarterial, intrathecal, intravesical, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal , subcutaneous, subcuticular, intra-articular, subcapsular, subarachnoid, intrathecal, epidural and intrasternal injection and infusion Examples include:

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

[0192] The therapeutic fusion proteins of the present disclosure can be delivered via implants, transdermal patches, and microencapsulated delivery. with a carrier that protects the protein from rapid release, such as a controlled release formulation, including a system It can be prepared using ethylene vinyl acetate, polyanhydride, polyglycolic acid, collagen, etc. Use biodegradable, biocompatible polymers such as polypropylene, polyorthoesters, and polylactic acid. Methods for the preparation of such formulations are patented or otherwise available. These methods are generally known to those skilled in the art. For example, Release Drug Delivery Systems,JRRobin son, ed., Marcel Dekker, Inc., New York, 1978 Please refer to.

[0193] In certain embodiments, the therapeutic fusion proteins of the invention have suitable distribution in vivo. For example, the blood-brain barrier (BBB) ​​is made up of many highly hydrophilic To ensure that the therapeutic compounds of the present invention cross the BBB, If desired, they may be formulated, for example, in liposomes. For the method of producing the same, see, for example, U.S. Pat. Nos. 4,522,811 and 5,377,812. See US Pat. Nos. 4,548; and 5,399,331. Liposomes are selectively transported into specific cells or organs, thereby facilitating targeted drug delivery. It may contain one or more moieties that enhance the (See n.Pharmacol.,29:685).

[0194] 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., The method can be administered, for example in vivo, to treat, prevent, or diagnose a variety of disorders. Treatment, prevention, or diagnosis of acute or chronic inflammatory and immune system-related organ and microvascular disorders It is particularly suitable for cutting

[0195] The therapeutic fusion proteins of the present invention may be used to treat, but are not limited to, dead cells, cell fragments, and thrombi. Endogenous homeostatic clearance mechanisms or enzymes for the removal of proinflammatory / inflammatory microparticles Acute and chronic conditions where the ferrocytosis pathway is significantly downregulated The present invention is useful for the treatment, prevention, or amelioration of acute inflammatory organ damage, particularly inflammatory damage. Examples of organ damage include myocardial infarction, acute kidney injury (AKI), acute stroke and inflammation, and gastrointestinal ischemia / reperfusion of the ducts, liver, spleen, lungs, kidneys, pancreas, heart, brain, spinal cord and / or crushed Organ damage due to ischemia / reperfusion, such as in a broken limb, may occur.

[0196] The therapeutic fusion proteins of the present disclosure may also be used to inhibit or slow blood clotting, Treatment, inflammatory bowel disease (IBD), decreased fatty acid uptake and / or gastric motility, microvascular Thrombotic disorders, atherosclerosis, cardiac remodeling, tissue fibrosis, acute liver injury, Chronic liver disease, non-alcoholic steatohepatitis (NASH), vascular disease, age-related vascular disorders, intestinal disease 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 Useful for the diagnosis, treatment, prevention, or improvement of pediatric respiratory distress syndrome, wound healing, tendon repair, and neurological disorders It can be useful.

[0197] In one embodiment, the neurological disorder is a symptom of sickness syndrome, nausea, passive avoidance, behavioral agility, Neuropsychiatric, neuroinflammatory and / or neuropsychiatric symptoms, including symptoms such as depression, memory impairment and memory dysfunction or a condition having a neurodegenerative component. Examples of neurological disorders include Alzheimer's disease These include amyloid beta-related neurological disorders such as Parkinson's disease and depression.

[0198] In one embodiment, bone disorders include osteoporosis, osteomalacia, osteosclerosis, and osteopetrosis. More specifically, the administration of the fusion proteins of the present disclosure can be selected from the group consisting of NFATc 1. At least one osteoclast marker, such as cathepsin K and αvβ3 integrin In one embodiment, the administration inhibits osteoclast formation. In yet another embodiment, the administration inhibits RANKL-induced osteoclastogenesis. In yet another embodiment, the administration inhibits bone resorption. 6, IL-17A, MMP-9, Ptgs2, RANKL, Tnfsf11, CXCL1 Bone resorption stimulators, including CXCL1, CXCL2, CXCL3, CXCL5, and combinations thereof. In another embodiment, the administration inhibits the expression of at least one bone resorption stimulating agent. At least one inflammatory inducer selected from the group consisting of IL-8 and CCL2 / MCP-1 Inhibits the expression of sex cytokines.

[0199] In one embodiment, tissue fibrosis is caused by a fusion protein of the invention that reduces collagen expression. In one embodiment, the fibrosis may be in the liver, lungs, diaphragm, kidneys, brain, or heart. In one embodiment, the pulmonary fibrosis is interstitial pulmonary fibrosis (IPF). In one embodiment, the liver fibrosis is Cirrhosis of the liver, which may or may not be due to NASH.

[0200] Several respiratory diseases are characterized by the accumulation of apoptotic cells. Efferocytosis and phagocytosis by macrophages in COPD Therapeutic fusion proteins of the present disclosure also have been associated with exacerbations and severity of acute respiratory distress syndrome (ARS). and are useful in the diagnosis, treatment, prevention, or amelioration of respiratory diseases such as COPD or COPD. The therapeutic fusion proteins of the present disclosure may also be used to treat acute lung injury (ALI), e.g., toxic external inflammatory diseases. Lung injury induced by inhalation or aspiration of exogenous or endogenous compounds or drugs; pulmonary edema, shock, pancreatitis, burns, chest trauma or multiple trauma, radiation, sepsis, pathogens (bacteria, viruses) lung damage caused by parasites such as malaria parasites; These compounds may be useful in the diagnosis, treatment, prevention, or amelioration of chronic respiratory failure.

[0201] Therapeutic fusion proteins of the present disclosure may also be used to treat Corona-type viruses, e.g., ARS- of lung injury caused by CoV, SARS-CoV-2, or MERS-CoV In one embodiment, the method of the present disclosure may be useful in diagnosing, treating, preventing, or ameliorating the severity of a disease. Therapeutic fusion protein for the treatment of SARS-CoV-2 infection in COVID-19 patients Provided for use in

[0202] The therapeutic fusion proteins of the present disclosure may also be used to diagnose the severity of transfusion-associated pulmonary injury (TRALI). These compounds may be useful in the treatment, prevention, or amelioration of a condition.

[0203] The therapeutic fusion proteins of the present disclosure also reduce the severity of chronic respiratory failure leading to hypoxemia. It may be useful for diagnosis, treatment, prevention, or amelioration.

[0204] Therapeutic fusion proteins of the disclosure, e.g., therapeutic fusion proteins comprising a domain of EDIL3 of the disclosure, The fusion proteins are also useful for diagnosing, treating, preventing, or ameliorating the severity of postoperative peritoneal adhesions. It is possible.

[0205] Therapeutic fusion proteins of the present disclosure may also be used to diagnose, treat, prevent, or ameliorate the severity of heart failure. It may be useful to

[0206] Therapeutic fusion proteins of the present disclosure may also be used to diagnose, treat, prevent, or ameliorate the severity of hemodialysis. It can be useful for good.

[0207] Therapeutic fusion proteins of the present disclosure may also be used to delay graft function or reduce the severity of graft-versus-host disease. These compounds may be useful in the diagnosis, treatment, prevention, or amelioration of

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

[0209] Therapeutic fusion proteins of the present disclosure may also be used in therapeutic applications involving the administration of therapeutic agents induced by bacteria, fungi, viruses, or parasites. Possible medical conditions (e.g., sepsis or necrotizing soft tissue infections (NSTIs) such as necrotizing fasciitis) ), osteomyelitis, malaria, and other conditions directly caused by pathogens, It may be useful for treatment, prevention, or amelioration.

[0210] The therapeutic fusion proteins of the present disclosure also have utility in preventing injuries from industrial accidents, falls, traffic accidents, ballistic and combat injuries. injuries caused by accidents or other injury mechanisms, such as These may be useful in diagnosing, treating, preventing, or ameliorating the severity of trauma / multiple trauma.

[0211] The therapeutic fusion proteins of the present disclosure also can be used to diagnose, treat, or predict the severity of osteoclast-mediated pathologies. It may be useful in preventing or ameliorating

[0212] Therapeutic fusion proteins of the present disclosure may be used as the sole active ingredient or in combination, e.g. , other drugs (e.g., immunosuppressants or immunomodulators or other anti-inflammatory drugs or drugs such as cytotoxic or or anti-cancer drugs) or in combination therewith, for example, in the treatment or prevention of the above-mentioned diseases. It can be administered for the following reasons.

[0213] With respect to additional therapeutic agents, "administered in combination" means two (or more) different therapeutic agents. The drug is delivered to the subject during the course of the subject's disease, e.g., two or more treatments. The drug is administered after a subject has been diagnosed with a disease and before the disease is cured or eliminated, or before treatment is initiated by another In some embodiments, the administration of The delivery of one therapeutic agent may still be ongoing when the delivery of the second therapeutic agent begins, so that there is an overlap. This is referred to herein as "concurrent" or "concurrent delivery." In other embodiments, delivery of one therapeutic agent may occur before delivery of another therapeutic agent begins. In either embodiment, the therapeutic agents are administered in combination. For example, the second therapeutic agent may be more effective, e.g., it may be more effective than the equivalent The second therapeutic agent is less effective than the first therapeutic agent, or the second therapeutic agent is more effective than the first therapeutic agent. relieves symptoms or similar conditions to a greater extent than would be observed if administered without In some embodiments, delivery is achieved by alleviating symptoms or reducing the severity of a disease associated with the first therapeutic agent. Other parameters associated with the therapeutic agent are significantly greater than those observed with one therapeutic agent delivered without the other. The effects of the two treatments may be partially additive or completely additive. The delivery may be additive or more than additive. The effect of the first therapeutic agent delivered may be greater than the effect of the second therapeutic agent. , such that it is still detectable when the second therapeutic agent is delivered.

[0214] The term "concurrently" refers to the administration of therapies (e.g., prophylactic or therapeutic agents) at exactly the same time. Without limitation, pharmaceutical compositions comprising the therapeutic fusion proteins of the present disclosure include those in which the fusion protein Working together with additional therapeutic agents to provide increased benefit over when they are otherwise administered It means that the drugs are administered to a subject in a sequence and at time intervals that provide a Therapeutic agents may be administered to a subject simultaneously or sequentially in any order at different times; If not administered, they should be administered sufficiently close in time to provide the desired therapeutic or prophylactic effect. Each therapeutic agent should be administered separately, in any suitable form and in any suitable manner. It can be administered to a subject by any route.

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

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

[0217] When administered in combination, the therapeutic fusion proteins described herein and additional Therapeutic agents (e.g., second or third agents) may each be used individually, e.g., as monotherapy. The amount or dosage of the agent may be greater than, less than, or the same as the amount or dosage of the agent. In some embodiments, the therapeutic fusion proteins described herein may be administered in combination with an additional agent (e.g., a second or a third agent), or all of which may be used individually, e.g., as monotherapy, with the amount or dosage of each agent being lower (e.g., at least 20%, at least 30%, at least 40%, or at least 50%). In other embodiments, the desired effect (e.g., inflammatory disease or a therapeutic fusion protein as described herein resulting in the treatment of a condition, an additional agent (e.g., The amounts or dosages of the other agents (e.g., second or third agents) or all of the other agents may vary, e.g., depending on the amount of the other agent required to achieve the same therapeutic effect. The amount or dosage of each drug used individually as monotherapy is lower (e.g., less at least 20%, at least 30%, at least 40%, or at least 50% lower).

[0218] For example, the therapeutic fusion proteins of the present disclosure may be used in combination with DMARDs, e.g., gold salts, sulfasalanine, and the like. Antimalarials, methotrexate, D-penicillamine, azathioprine, mycofecoxib benzodiazepine, tacrolimus, sirolimus, minocycline, leflunomide, glucocorticoid calcineurin inhibitors, such as cyclosporin A or FK506; lymphocyte regeneration inhibitors Circulatory modulators, e.g., FTY720 and FTY720 analogs; mTOR inhibitors , e.g., rapamycin, 40-O-(2-hydroxyethyl)-rapamycin, CCI 779, ABT578, AP23573 or TAFA-93; comycins, such as ABT-281, ASM981, etc.; corticosteroids; cyclosporin Phosphamide; Azathioprine; Leflunomide; Mizoribine; Mycophenolate mofetil 15-deoxyspergualin or its immunosuppressive homologues, analogues or derivatives; immunosuppressive Inhibitory monoclonal antibodies, such as monoclonal antibodies against leukocyte receptors, e.g. MHC, CD2, CD3, CD4, CD7, CD8, CD25, CD28, CD40, C D45, CD58, CD80, CD86 or their ligands; other immunomodulatory compounds, e.g. For example, a recombinant antibody having at least a portion of the extracellular domain of CTLA4 or a variant thereof. molecules, e.g., CTLA4 or variants thereof linked to non-CTLA4 protein sequences. At least the extracellular portion, e.g., CTLA4Ig (e.g., designated ATCC68629) or a mutant thereof, e.g., LEA29Y; adhesion molecule inhibitors, e.g., LFA-1 antagomir; antagonist, ICAM-1 or -3 antagonist, VCAM-4 antagonist or VLA -4 antagonists; or chemotherapeutic agents, such as paclitaxel, gemcitabine, cisplatin, anti-TNF agents, e.g., ... monoclonal antibodies, such as infliximab, adalimumab, CDP870, or Receptor constructs for TNF-RI or TNF-RII, e.g., etanercept PEG-TNF-RI; inflammatory cytokine blockers, IL-1 blockers, e.g., anaphylactoids Kinra or IL-1 trap, canakinumab, IL-13 blockers, IL-4 blockers, IL -6 blockers; chemokine blockers, e.g., inhibitors or activators of proteases, e.g., metronidazole; Taroproteinase, anti-IL-15 antibody, anti-IL-6 antibody, anti-IL-4 antibody, anti-IL-13 antibodies, anti-CD20 antibodies, NSAIDs such as aspirin or anti-infectives; damage-associated molecular patterns and pathogen-associated molecular pattern (DAMP) or pathogen-associated molecular pattern (PAMP) antagonists, e.g., Converters, antidotes, removers, e.g., ATP converters, HMGB-1 modulators inhibitors of superantigen-induced immune responses; complement inhibitors and extracorporeal plasma exchange detoxifiers It can be used in combination with a vise.

[0219] kit Compositions, e.g., kits comprising a therapeutic fusion protein of the disclosure and instructions for use, are also provided herein. Such kits, according to the present disclosure, comprise a therapeutically effective amount of a fusion protein. Additionally, such kits may include a means for administering the therapeutic fusion protein (e.g., For example, auto-injectors, syringes and vials, pre-filled syringes, pre-filled pens ) and instructions for use. These kits can be used to treat autoimmune or inflammatory disorders or AO. Therapeutic agents for treating patients with I may include additional therapeutic agents (described below). The kit may also include instructions for administration of the therapeutic fusion protein to treat the patient. Such instructions may include dosages, routes of administration, and dosage information for use with the encapsulated fusion protein. The kit may provide a list of the intended contents of the kit. The term "label" includes any label on or associated with the kit that indicates the intended use of the kit. This includes any documentation or records supplied or otherwise accompanying the kit. A fusion protein is a compound that is administered to a patient in a therapeutically effective amount to a patient, as defined above, who responds to treatment with a therapeutic fusion protein of the invention. The device may further include a tool for diagnosing whether a patient belongs to a group that has the disease.

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

[0221] 1. Integrin-binding domain, phosphatidylserine (PS)-binding domain, and Therapeutic fusion proteins for enhancing efferocytosis containing a lytic domain.

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

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

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

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

[0226] 6. The soluble domain is an integrin-binding domain, a PS-binding domain, or both domains. 10. The fusion protein of any one of the preceding embodiments, wherein the fusion protein is directly linked to the domain.

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

[0228] 8. The soluble domain is human serum albumin (HSA), domain 3 of HSA (HSA D3), ​​Fc-IgG, or a functional variant thereof. The fusion protein according to any one of claims 1 to 4.

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

[0230] 7. The integrin binding domain has the amino acid sequence of SEQ ID NO: 2, or at least 10. The fusion protein of any one of the preceding embodiments, having at least 90% sequence identity. quality.

[0231] 8. The PS binding domain has the amino acid sequence of SEQ ID NO: 3, or at least a sequence similar thereto. or the PS-binding domain has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 76. or any of the preceding embodiments having at least 90% sequence identity thereto. The fusion protein according to any one of claims 1 to 4.

[0232] 9. The solubility domain is HSA and has the amino acid sequence of SEQ ID NO: 4, or a variant thereof 10. The multidomain peptide of any one of the preceding embodiments, having at least 90% sequence identity. In-fusion protein.

[0233] 10. The integrin binding domain has the amino acid sequence of SEQ ID NO: 2, or a variant thereof and a PS-binding domain having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 78. or having at least 90% sequence identity thereto. The fusion protein according to any one of claims 1 to 4.

[0234] 11. The integrin binding domain has the amino acid sequence of SEQ ID NO: 77, or have at least 90% sequence identity, and the PS-binding domain is SEQ ID NO: 3 or SEQ ID NO: No. 76, or any of the preceding amino acid sequences having at least 90% sequence identity thereto. 10. The fusion protein of any one of the embodiments.

[0235] 12. The fusion protein a. Human endothelial cell-Jurkat cell efferocytosis assay promotes efferocytosis by b. Macrophages in a human macrophage-neutrophil efferocytosis assay Recovering impaired efferocytosis; c. Plasma microparticle clearance in human endothelial microparticle efferocytosis assay Reduce the number of particles; and / or d. Protects against multi-organ injury in a model of acute kidney injury; 10. The fusion protein of any one of the preceding embodiments.

[0236] 13. A method for producing a hybridoma comprising an integrin-binding domain, an HSA-PS-binding domain, and the like, as previously described 2. The fusion protein of any one of the aspects.

[0237] 14. MFG-E8 is composed of, from the N-terminus to the C-terminus, an EGF-like domain, a C1 domain, and Contains the C2 domain and has wild-type human MFG-E8 (SEQ ID NO: 1) or SEQ ID NO: 75 MFG-E8 and solubility domains, including sequences from MFG-E8 or functional variants thereof 1. A therapeutic fusion protein comprising:

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

[0239] 16. The soluble domain is HSA, HSA D3, or Fc-IgG, or a combination thereof. 16. The fusion protein of embodiment 14 or embodiment 15, which is a functional variant.

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

[0241] 18. The fusion protein has the amino acid sequence of SEQ ID NO: 44, or at least 90% sequence identity to SEQ ID NO: 47, or at least 90% sequence identity thereto; sequence identity; or SEQ ID NO: 48, or at least 90% sequence identity thereto 10. The fusion protein of any one of the preceding embodiments, comprising:

[0242] 19. The fusion protein comprises the amino acid sequence of SEQ ID NO: 80, or at least 10. The fusion protein of any one of the preceding embodiments, having 90% sequence identity.

[0243] 20. The fusion protein comprises the amino acid sequence of SEQ ID NO: 82, or at least 10. The fusion protein of any one of the preceding embodiments, having 90% sequence identity.

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

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

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

[0247] 24. Embodiments in which the vector is administered to a subject in need thereof, e.g., a human subject 24. A viral vector according to 23.

[0248] 25. For use in the treatment and / or prevention of the diseases listed herein, embodiments 24. The viral vector according to embodiment 23.

[0249] 26. One or more cloning or expression vectors according to embodiment 22, and optionally Recombinant host cells suitable for the production of therapeutic fusion proteins containing secretion signals.

[0250] 27. An embodiment in which the host cell is, for example, a prokaryotic, yeast, insect, or mammalian cell. 27. A recombinant host cell according to claim 26.

[0251] 28. Expression of the protein in the host cell results in a yield of at least 10 mg / L. 21. A fusion protein according to any one of embodiments 1 to 20.

[0252] 29. Protein expression in mammalian cells is determined using wild-type MFG-E8 (SEQ ID NO: 1). 21. The method of claim 1, wherein the method results in an increase in yield of at least 100-fold. Fusion proteins.

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

[0254] 31. A therapeutically effective amount of the fusion protein according to any one of embodiments 1 to 20 is administered to an individual. 2. Treatment of an inflammatory disorder or inflammatory organ damage in an individual in need thereof, comprising administering Methods of placement or prevention.

[0255] 32. For the treatment or prevention of inflammatory disorders or inflammatory organ damage in individuals in need thereof. 21. The fusion protein according to any one of embodiments 1 to 20, for use in

[0256] 33. Inflammatory disorders or inflammatory organ damage include acute kidney injury, sepsis, myocardial infarction, and acute stroke. , burns, traumatic injury, and inflammatory and organ damage resulting from ischemia / reperfusion. 33. The method according to embodiment 31 or the use according to embodiment 32.

[0257] 34. The method of embodiment 31, wherein the inflammatory disorder or inflammatory organ injury is acute kidney injury. 33. The method or use according to embodiment 32.

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

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

[0260] 37. Inflammatory disorders or inflammatory organ damage include acute lung injury (e.g., acute respiratory distress syndrome). or liver injury or acute intestinal injury. Use of.

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

[0262] 39. Another therapeutic agent is an immunosuppressant, immunomodulator, anti-inflammatory, antioxidant, anti-infective, or cytotoxic agent. 39. The method or use of embodiment 38, wherein the agent is a toxic agent or an anti-cancer agent.

[0263] 40. A therapeutic fusion protein comprising MFG-E8 and a soluble domain, wherein the M FGF-E8 consists of an EGF-like domain, a C1 domain, and a C2 domain from the N-terminus to the C-terminus. and a sequence from wild-type human MFG-E8 (SEQ ID NO: 1) or the sequence of SEQ ID NO: 75. A therapeutic fusion protein comprising a sequence or a functional variant thereof.

[0264] 41. The solubility domain is at the N-terminus of MFG-E8 (SEQ ID NO: 1 or SEQ ID NO: 75) or 41. The fusion protein of embodiment 40, wherein the fusion protein is linked to the C-terminus.

[0265] 42. A soluble domain is inserted between the EGF-like domain and the C1 domain. 41. The fusion protein of claim 40.

[0266] 43. An embodiment in which the solubility domain is inserted between the C1 and C2 domains 41. A fusion protein according to claim 41.

[0267] 44. The soluble domain is HSA, HSA D3, or Fc-IgG, or a combination thereof. 44. The fusion protein of any one of embodiments 40 to 43, which is a functional variant.

[0268] 38. An isolated antibody encoding a fusion protein according to any one of embodiments 33 to 37. Nucleic acids.

[0269] 39. A cloning or expression vector comprising the nucleic acid of embodiment 38.

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

[0271] 41. Embodiments in which the vector is administered to a subject in need thereof, e.g., a human subject 40. A viral vector according to claim 40.

[0272] 42. For use in the treatment and / or prevention of the diseases listed herein, embodiments 41. The viral vector according to embodiment 40.

[0273] 43. One or more cloning or expression vectors according to embodiment 39, and optionally Recombinant host cells suitable for the production of therapeutic fusion proteins containing secretion signals.

[0274] 44. An embodiment in which the host cell is, for example, a prokaryotic, yeast, insect, or mammalian cell. 44. A recombinant host cell according to claim 43.

[0275] 45. Expression of protein in host cells results in a yield of at least 10 mg / L 38. A fusion protein according to any one of embodiments 33 to 37.

[0276] 46. ​​Protein expression in mammalian cells is at least 100% identical to wild-type MFG-E8. 38. The fusion protein of any one of embodiments 33 to 37, which results in a 00-fold increase in yield. quality.

[0277] To the extent such combination is consistent with the description of the embodiment, each embodiment may be combined with one or more of the other embodiments. It should be understood that the embodiments provided above may be combined with other embodiments. All embodiments are included, including those resulting from combinations of embodiments. It is further understood that:

[0278] All references cited herein, including patents, patent applications, articles, publications, textbooks, etc. The document and the references cited therein (to the extent that they are not already incorporated) are hereby incorporated by reference. No. 6,239,999, which is incorporated herein by reference in its entirety. [Example]

[0279] The following examples are provided to further illustrate the disclosure but are not intended to limit its scope. Other variations of the present disclosure will be readily apparent to those skilled in the art and are intended to fall within the scope of the appended claims. Included.

[0280] Example 1: Production of fusion proteins MFG-E8 contains an N-terminal epidermal growth factor (EGF-like) domain and two C-terminal lectins. It is a multidomain protein consisting of type C domains (C1 and C2). As previously reported, attempts to produce recombinant full-length human proteins have been hampered by protein aggregation and expression This indicates a very low velocity (Castellanos et al., (2016) P rotein Expression Purification 1124:10-2 2) Therefore, we attempted to solubilize the protein and enhance its expression. The inventors investigated the effect of fusing several proteins to MFG-E8.

[0281] Solubilization domain (SD) derived from human Fc-IgG1, human serum albumin (HSA) ), and domain 3 of HSA (HSA D3) are located in different positions, i.e., as shown schematically in FIG. As indicated, at the N-terminus or C-terminus, or between the EGF and C1 or C1 and C2 domains Furthermore, fusion to Fc-IgG1 or HSA was The protein binds to FcRn, potentially extending the half-life of the molecule in vivo. Fusion of MFG-E8 to Fc-IgG1 or HSA can be performed using the fusion It can also enhance protein production and solubility (Castellanos et al. l., (2016) cited above).

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

[0283] [Table 57]

[0284] Example 2: Generation of wild-type MFG-E8 and MFG-E8 HSA fusions; expression and purification The method for producing the fusion protein is described below. Briefly, MFG-E8 and MF G-E8 fusions and EDIL fusions, particularly to HSA, were generated according to the following method. Ta.

[0285] DNA was synthesized at GeneArt (Regensburg, Germany) and digested with restriction enzymes. Cloned into a mammalian expression vector using gating-based cloning techniques. The resulting plasmid was transfected into HEK293T cells. For transient expression, wild-type or modified strand vectors were transfected with polyethyleneimine (PEI; Suspension adaptation using (catalog number 24765 Polysciences, Inc.) HEK293T cells were transfected. Typically, 1-2 Mitochondrial cells per ml were used. To 100 ml of cells in suspension at 100-microliter density, add 100 μg of expression vector encoding the modified strand. The recombinant expression vector was then introduced into a host cell, and the host cell was transfected with DNA containing the vector. 0.1% pluronic acid, 4 mM glutamine, and 0.25 μg / ml antibiotics Culture the cells for an additional 7 days to allow secretion into supplemented medium (HEK, serum-free medium). The construct was produced by culturing the cells.

[0286] The generated constructs were then purified by immobilized metal ion affinity chromatography (I MAC), or Protein A capture, or anti-HSA capture chromatography. Purified from cell supernatant.

[0287] When His-tagged proteins were captured by IMAC, the filtered conditioned medium , 1% Triton and 20 mM NaPO4, 0.5 mM NaCl, 20 mM imidazoline The gel was mixed with IMAC resin (GE Healthcare) equilibrated at pH 7.0. The protein was eluted with 10 column volumes of elution buffer (20 mM NaPO, 0.5 M The resin was allowed to stand for 15 columns before elution with NaCl, 500 mM imidazole, pH 7.0. 100 ml of 20 mM NaPO4, 0.5 mM NaCl, 20 mM imidazole, pH 7 Washed three times with .0.

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

[0289] Finally, the eluted fractions were subjected to size exclusion chromatography (HiPrep Superdex 200, 16 / 60, GE Healthcare Life Sciences) Polished using Precision Plus Protein Unstained S for Standards marker (Biorad, ref#161-0363) The product was analyzed by DS-PAGE.

[0290] A representative expression gel of the fusion protein is shown in Figure 2. Figure 2A: EGF-HSA-C1-C 2 protein (FP330; SEQ ID NO: 42); Fig. 2B: EGF- HSA-C1-C2 (FP050; SEQ ID NO: 12); Figure 2C: Non-reduced and reduced EGF-F c(KiH)C1-C2 protein. This protein is a FP071 (EGF-Fc( Fc-IgG1 hole (SEQ ID NO: 10); Figure 2D: It is a heterodimer of the EGF-HSA-C1 protein (FP260; SEQ ID NO: 34). The proteins under reducing and non-reducing conditions are shown in Figure 2C. The heterodimer collapses under reducing conditions. Both conditions were tested because both tend to be soluble in water. The expression and yield results are shown in Table 6. As can be seen from the expression data, the HS of MFG-E8 The A fusions were at least 1% more potent than wild-type MFG-E8, even when the HSA was at a different position. As shown in the right column of Table 6, the HSA of MFG-E8 The fusion also shows at least a 100-fold increase in yield over wild-type MFG-E8.

[0291] [Table 58]

[0292] Other examples of therapeutic fusion proteins of the present disclosure are produced according to the methods described above and are SDS-P Further analysis by AGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) The proteins were separated based on their molecular weight. Before loading onto iorad, 4-20% Mini-PROTEAN TGX (stain-free) Each protein was then mixed with Laemmli buffer. After electrophoresis in running buffer at 200V for 30 minutes, the proteins contained in the gel were This was revealed using a stain-free imager (Biorad, Gel Doc EZ). As described in Figure 2E, SDS-PAGE was performed to analyze the produced and purified recombinant protein. show. Columns 1 and 12: Molecular weight markers (Biorad, Precision plus protein ein) Column 2: His6_EGF[MFG-E8]_C1[MFG-E8] 23.87k Da Column 3: EGF[MFG-E8]_C1[MFG-E8]_His6 SEQ ID NO: 115 23.87kDa Column 4: EGF[MFG-E8]_HSA_C1[MFG-E8] SEQ ID NO: 117 9 0.38kDa Column 5: EGF[MFG-E8]_HSA_C1[MFG-E8] SEQ ID NO: 74 89 .27kDa Column 6: EGF[MFG-E8]_HSA_C1[MFG-E8] SEQ ID NO: 73 88 .72kDa Column 7: EGF[EDIL3]_HSA_C1[EDIL3] SEQ ID NO: 71 98.2 2kDa Column 8: EGF[EDIL3]_HSA_C2[EDIL3] SEQ ID NO: 135 98. 20kDa Column 9: EGF[MFG-E8]_HSA_C2[MFG-E8] SEQ ID NO: 137 8 8.45kDa Column 10: EGF[EDIL3]_HSA_C1_C2[MFG-E8] SEQ ID NO: 80 115.67kDa Column 11: EGF[MFG-E8]_HSA_C1_C2[EDIL3] SEQ ID NO: 82 107.32kDa

[0293] Example 3: Characterization of MFG-E8-HSA modified proteins 3.1 Phosphatidylserine binding (biochemistry) L-α-Phosphatidylserine (brain, porcine, Avanti 840032, Alaba (ma,US) was dissolved in chloroform, diluted with methanol, and placed in a 384-well microtiter plate. on a tar plate (Corning™ 3653, Kennebunk ME, US) After overnight incubation at 4°C, the SpeedVa The solvent was analyzed using a Thermo Scientific™ system. The plates were then resuspended in a buffer containing 3% fatty acid-free bovine serum albumin (BSA). The cells were treated with phosphate-buffered saline (PBS) at room temperature for 1.5 hours.

[0294] Binding of the fusion protein to L-α-phosphatidylserine was confirmed by biotinylated mouse MFG -E8 / lactadherin (produced in-house, mMFG-E8: biotin) binding. Proteins were analyzed by PBS containing 3% fatty acid-free BSA, pH 7.4. Diluted in S and plated on L-α-phosphatidylserine-coated microtiter plates. The cells were incubated for 30 min in mMF in PBS containing 3% fatty acid-free BSA. G-E8: Biotin, pH 7.4, was added at 1 nM and incubated for an additional 30 minutes. Unbound mMFG-E8:biotin was detected by dissociation-enhanced lanthanide fluoroimmunoassay (D ELFIA™ Wash Buffer (Perkin Elmer 1244-114 The europium-labeled streptavidin was removed by three washing steps using 1000 mg of ... Visin (Perkin Elmer 1244-360, Wallac Oy, Finland ) in DELFIA™ Assay Buffer (Perkin Elmer 124 The mixture was added to a 4-111 MA, US tube at room temperature for 20 minutes. The plate was washed three times with assay buffer. Europium was purchased from the manufacturer (Perkin Elmer). Revealed in accordance with the instructions of the University of California, San Diego, CA 94440-1055, Boston, MA, USA. The time-resolved fluorescence of rB was measured using the Envision™ 2103 multilabel platelet. Quantification was performed using a chromatograph (Perkin Elmer, CT, US). Data analysis was performed using MS This was done using Excel and GraphPad Prism software.

[0295] Polypropylene plates are low protein binding microtiter plates and are typically Typically, these plates are used for serial dilutions in laboratories. has the advantage of reducing protein loss during dilution and is typically referred to as a "low protein" Dilutions of wild-type MFG-E8 were placed on polypropylene plates. When performed in a non-binding plate, wild-type MFG-E8 showed a significantly higher L These data are shown in Figure 3. Use polypropylene plates that are already optimized for low protein binding, as shown When using MFG-E8, the wild-type MFG-E8 is partially lost during the liquid handling and dilution steps. These results demonstrate that the inherent adhesiveness of wild-type MFG-E8 is consistent with that of the lab-grown MFG-E8. presents challenges for handling during the manufacturing and production of pharmaceuticals. where the steps of capture and polishing are performed to produce high yield and very high purity drug substance. In contrast, the modified protein FP278 (EGF-HSA-C1-C2 -His tag; SEQ ID NO: 44) significantly reduced adhesion compared to wild-type MFG-E8. No substantial differences were observed between dilutions performed in non-binding plates and polypropylene plates. These data support the idea that the insertion of a solubility domain into the proteins of this disclosure did not result in a significant increase in the solubility of the proteins (Figure 3B). By introducing these compounds, their technical handling can be improved, leading to improved step yields and therefore manufacturing processes. This suggests that the overall yield during the process can be improved.

[0296] The binding of the fusion protein to L-α-phosphatidylserine is shown in Figure 4. The FG-E8-derived protein FP278 (EGF-HSA-C1-C2-His tag; sequence No. 44) binds to immobilized PS and to a lesser extent to the phospholipid cardiolipin. FP278 bound to immobilized L-α-phosphatidylinositol in a concentration-dependent manner (Figure 4A). Binding to lucerin or cardiolipin (1,3-bis(sn-3'-phosphatidyl)- sn-glycerol) was found to bind to the EGF-L domain of wild-type MFG-E8. Several combinations of phosphatidylserine and immobilized L-α-phosphatidylserine were detected. The binding affinities of the recombinant fusion proteins are shown in Figure 4B. Protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) and FP 260 (EGF-HSA-C1; SEQ ID NO: 34) was prepared by 1 nM biotinylated mouse MFG-E It efficiently competed with the concentration-dependent binding of 8 to immobilized L-α-phosphatidylserine. IC obtained for protein 50 The values ​​are for the modified protein FP278 (EGF-HSA -C1-C2-His tag; SEQ ID NO: 44) of the L-α-phosphatase The binding strength of MFG-E8 is very similar to that of wild-type human MFG-E8. Surprisingly, these data support the finding that FP270 (EGF-HSA-C2; SEQ ID NO: 3) As shown in 6), the human C2 domain interacts with L-α-phosphatidylserine. This also suggests that they do not interact or only slightly interact with each other, which is consistent with the FP250( EGF-HSA (SEQ ID NO: 32) did not compete in this assay format. The F-C2-C2 protein, FP100 (SEQ ID NO: 26), was tested in this assay. The C1 domain of human MFG-E8 does not compete with the PS-binding domain of human MFG-E8 in the same manner as in the previous study (not shown). The main literature indicates that the C2 domain of MFG-E8 is the main domain involved in PS binding. This finding was surprising because it suggests that en et al.,(2000)Biochemistry,39(20):6200 -6;Shi&Gilbert(2003)Blood,101:2628-2636; Shao et al.,(2008)J Biol Chem.,283(11):7 230-41) In conclusion, these findings suggest that the C1 domain is involved in the MFG-E8 modified protein. It is the major integral PS-binding domain of proteins and is important for PS-binding-dependent functions. Therefore, the C1 domain is suitable for substitution with heterologous proteins to confer PS binding. However, the C1-C2 or C1-C1 tandem domain (the latter not shown) The fusion protein containing the ATP showed the highest PS binding.

[0297] 3.2 αv integrin adhesion assay The fusion protein was diluted in phosphate-buffered saline (PBS) pH 7.4 and diluted in 50 μL of 2 A 4 nM solution was immobilized overnight by adsorption (96-well plate, Nunc Maxis orb) (1.2 nM / well). The plates were then incubated at room temperature for 1.5 hours in 3% fatty acid The cells were treated with PBS containing free bovine serum albumin (BSA). Lymphoma cells expressing ATCC-TIB-48 BW5147.G.1.4 (ATCC, US) contains GlutaMax, 25mM HEPES, 10% FBS, Pen / Stre p, RP supplemented with 1 mM sodium pyruvate, 50 μM β-mercaptoethanol The cells were cultured in MI1640. The day before the adhesion experiment, the cells were split. The cells were treated with 3 μg / mL of 2' ,7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein Acetoxymethyl ester (BCECF AM) (Thermo Fisher Scientific The cells were labeled with BW5147.G.1.4 (BW5147.G.1.4, Scientific Inc., USA) for 30 minutes. The cells were resuspended in adhesion buffer (TBS, 0.5% BSA, 1 mM MnCl2, pH 7.4). The cells were suspended and allowed to adhere at 50,000 cells / well for 40 minutes at room temperature. The fluorescence of adherent cells was measured by Envision A 2103 Multilabel Plate Reader (Perkin Elmer, US) was used. Data analysis was performed using MS Excel and GraphPad Prism. This was carried out using m software.

[0298] Immobilized fusion protein FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42) Cell adhesion was inhibited by the αv integrin inhibitor cilengitide or 10 mM EDTA. completely blocked, demonstrating integrin-dependent cell adhesion to the immobilized modified protein (Figure 5 A). The integrin-binding motif RG of the EGF-like domain (FP280; SEQ ID NO: 38) A single point mutation in D(RGD>RGE) resulted in complete inhibition of cell adhesion, and the fusion protein A functional and accessible RGD-binding motif in αv integrin-dependent adhesion is essential for The immobilized EGF-HSA protein lacking the C1-C2 domain was shown to be deficient (Fig. 5B). The protein FP250 (SEQ ID NO: 32) is a BW5 protein despite its EGF-like domain. 147.G.1.4 Did not support or only weakly supported cell adhesion This finding supports the conclusion that EGF-like domains fused to HSA were highly potent under the experimental conditions tested (Fig. 5C). The main RGD loop is not sufficiently attached to cell surface integrins, presumably for steric reasons. This suggests that C1, C2, or C1-C2 may not be accessible. When fused to EGF-HSA at the terminal position, this defect was not apparent. Proteins such as FP330, when expressed in CHO or HEK cells, Similar to live MFG-E8, it promotes αv-integrin-dependent cell adhesion (Fig. 5D).

[0299] Taken together, these data demonstrate that the fusion proteins of the present disclosure bind to cellular integrins. , a protein that supports integrin-dependent cell adhesion and has an HSA domain insert In this study, the C-terminal EGF-like domain was integrated into the C-terminal fusion domain to support integrin binding. We show that functional benefits may be obtained from the synprotein domain.

[0300] 3.3 Human macrophage-neutrophil efferocytosis assay Human peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll gradient centrifugation (Ficoll (trademark)-Paque PLUS, GE Healthcare, Sweden), followed by Stemcell isolation kit (Stemcell 19059, Vancouver, CA) Monocytes were isolated from buffy coats by negative selection using nadine. Monocytes were cultured in 25 mM HEPES, 10% FBS, Pen / Stre for 5 days. The mixture was prepared in RPMI 1640 containing 1 mM NaPyr and 50 μM β-Merck. Recombinant human M-CSF 40ng / mL (Macrophage Colony Stimulator) "M0" macro using the rating factor (R&D Systems, US) One day before efferocytosis, the cells were incubated with Red Fluorescence Immunosorbent Assay (RIMA) for 14 days. The nt Dye Linker kit (Sigma MINI26, US) was used to Phages were labeled with PKH26. Cells were cultured in 25 mM HEPES, 10% FBS, and Pe RPMI16 containing n / Strep, 1 mM NaPyr, and 50 μM β-Merck Resuspend cells at 40,000 cells / well in a black 96-well plate (Corning ,US) and allowed to attach for 20 hours.

[0301] Neutrophils: Human neutrophils were deconvoluted with a Ficoll™ density gradient as follows: The diluted buffy coat was separated by centrifuging. The buffy coat plasma was removed by separating the cells. Diluted (Leuconostoc spp., MW 450. 000-650.000; Sigma, US) and allowed to settle on ice for 20-30 minutes.

[0302] Leukocytes were collected from the supernatant and placed on a Ficoll™-Paque layer (GE Health After centrifugation, the pellet was collected and red blood cells (RBCs) were counted. Residual red blood cells were lysed using lysis buffer (BioConcept, Switzerland). Neutrophils were cultured in culture medium (25mM HEPES, 10%FBS, Pen / Strep, 0.1mM RPMI1640 + GlutaMax containing NaPyr and 50uM b-Merc ) and stored overnight at 15°C. Apoptosis / cell death was assessed by incubating neutrophils at 1 μg / m Superfas Ligand (Enzo Life Sciences,Lau Neutrophils were induced by treatment with erythrocytes (H. sanne, Switzerland) for 3 hours at 37°C. Stained with Electron Microscopy 33342 (Life Technologies, US) for 25 minutes. The staining was performed with DRAQ5 (eBioscience, UK, 1:2000 dilution) at 37°C in the dark. Then, staining was carried out for 5 minutes.

[0303] Efferocytosis assay M0 macrophages were incubated with the fusion protein for 30 minutes. The cis-labeled neutrophils were added at a ratio of 1:4 M0 / neutrophils. Efferocytosis of apoptotic neutrophils is mediated by low pH lysosomal transport in M0 macrophages. The increase in DRAQ5 fluorescence intensity when neutrophils localize in the murine compartment was used to visualize the It has been transformed.

[0304] Efferocytosis was measured using ImageXpress Micro XLS Wide Field High-quality content analysis system (Molecular Devices, CA, US) Macrophages were identified via PKH26 fluorescence. The macrocytosis index (EI, expressed as a %) is the percentage of at least 100 macrophages in the total number of macrophages. Macros containing one ingested apoptotic neutrophil (DRAQ5high) event The data were calculated as the ratio of phage abundance to total phage abundance. Data analysis was performed using MS Excel and GraphPanel. d Performed using Prism software.

[0305] Fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44 ) on promoting efferocytosis of killed neutrophils by human macrophages. Figure 6 shows the fusion protein expressed in M0 macrophages at basal levels. Beyond its already high efferocytosis capacity, pHrodo-labeled killed human neutrophils Figure 7 shows that the recombinant fusion protein FP278 increases the internalization of human phage. Endotoxin (lipopolysaccharide)-induced damage to efferocytic cells from neutrophils killed by macrophages. Figure 7A shows that the 100p IgG antibody in three human donors can rescue the disease. Macrophage efflux of human neutrophils killed by lipopolysaccharide (LPS) at 1000 mg / ml The left panel shows the response of an individual donor, and the right panel shows the response of three donors. Figure 7B shows the mean impairment (%) of efferocytosis in donors. This endotoxin (LPS) of killed neutrophils by human macrophages using 278 1 shows rescue of impaired efferocytosis.

[0306] Killing of neutrophils by human macrophages using the fusion protein FP330 in grapevine aureus Rescue of S. aureus particle-induced efferocytosis is shown in Figure 8. Figure 8A The effect of 100 nM of fusion protein on promoting efferocytosis compared to basal levels The effect of protein concentration (dotted line; left side of the figure) and the addition of Staphylococcus aureus (S. aureus) 100 nM fusion rescues the impairment of efferocytosis caused by fusion The effect of the protein (right side of the figure) is shown in Figure 8B. Rescue of efferocytosis impairment caused by the addition of Fusion protein F for promoting efferocytosis after reaching a basal level of cytokinin P278(EC 50 The effect of increasing concentrations of ATP (8 nM) is shown.

[0307] 3.4 Human endothelial-Jurkat efferocytosis assay cell culture Human umbilical vein endothelial cells (HUVECs) were obtained from Lonza (Basel, Switzerland). The cells were cultured in flasks coated with gelatin (derived from bovine skin, P (Dilution of 2% stock solution, Sigma, Germany, final concentration of 0.2% in BS). , 10% FBS (GE Healthcare, UK), 1% Pen / Strep ( Thermo Fischer Scientific, US), 1% Glutamax (Thermo Fischer Scientific, US) and 1ng / mL recombinant Medium 199 supplemented with fibroblast growth factor-basic (Peprotech, UK) (Thermo Fischer Scientific, US). using tase (trademark) (Thermo Fischer Scientific, US). The cells were isolated for harvesting or passaging.

[0308] Jurkat E6-1 cells were purchased from ATCC (American Type Culture Collection). The medium was obtained from the National Agricultural Collection (USA) and contained 10% FBS (GE Healthcare are,UK), 1% Pen / Strep(Thermo Fischer Scie ntific, US), 10 mM sodium pyruvate (Thermo Fischer r Scientific, US) and 10 mM HEPES (4-(2-hydroxybenzoates) Ethyl)-1-piperazineethanesulfonic acid, Thermo Fisher Scientific RPMI 1640 medium (Thermo Fischer, Pharmacia, USA) supplemented with 100% ribosomal methyltransferase (RITP) The cells were grown in a medium containing 100% ethanol (Scientific, US).

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

[0310] Efferocytosis assay On day 1, HUVECs (70-90% confluence) were cultured with Accutase™. The cells were separated for 5 minutes, collected, washed with PBS, and resuspended in cell culture medium. , Guava EasyCyte flow cytometer (Merck, Germany) and Gu ava ViaCount reagent (Merck, Germany) according to the manufacturer's instructions. The required amount of cells was centrifuged at 300 x g for 5 minutes at room temperature and resuspended in medium. , 6.6×10 4 The cell number was determined as cells / mL. 150 μL / well of this cell suspension was added to HUVECs were added to a 96-well tissue culture plate (Corning™, US). Incubate the ink for another 16-20 hours in a 37°C / 5% CO2 / 95% humidity incubator. was invited.

[0311] Jurkat E6-1 cell number and viability / cell death status were measured using Guava EasyC yte flow cytometer (Merck, Germany) and Guava ViaCount The required amount of cells was evaluated using the reagent (Merck, Germany) according to the manufacturer's instructions. Centrifuge at 300xg for 5 minutes at room temperature and add 50ng / mL of recombinant human TRAIL-1 to the suspension. 1 x 10 in medium supplemented with IL 6 The cells were resuspended at a density of 1000 cells / mL. Cell death was monitored by incubation at 37°C for 5 min. % CO2 / 95% humidity overnight.

[0312] On day 2, remove the medium from the HUVECs by aspiration and add 25 μL of fresh pre-warmed (37 °C) After adding the medium, 25 μL of fusion protein or control diluted in pre-warmed (37°C) medium was added. The dilutions were performed in a non-binding surface (NBS) treated 96-well plate (Corning (trademark), US) was used. The fusion protein was incubated for 1 hour at 4°C for 1 hour before adding killed Jurkat cells. The cells were allowed to interact with HUVECs for 30 minutes at 37°C / 5% CO2 / 95% humidity.

[0313] Apoptotic / dead Jurkat E6-1 cell counts were determined using Guava EasyCyte Flow cytometer (Merck, Germany) and Guava ViaCount reagent ( The required amount of apoptotic cells was counted using a 400-well platelet counting machine (Merck, Germany). Centrifuge at 37°C for 5 min at 47°C, room temperature, and 5 x 1 6 At a density of 100 cells / mL, the final concentration was 5 μg / mL (Staining medium) supplemented with pHrodo™ Green STP Ester dye The cells were resuspended in PMI1640 medium (without FBS). After staining for 10 minutes at 37°C, the remaining cells were of reactive pHrodo™ Green STP Ester supplemented with 10% FBS The staining medium was then inactivated for an additional 5 minutes at 37°C. The cells were washed once and the number of cells was increased to 3 × 10 in HUVEC medium. 6 Adjusted to 1.5x cells / mL 10 6 pHrodo™ Green-labeled Jurkat cells per well were added to HUVECs. The mixture was then incubated at 37°C / 5% CO2 / 95% humidity for 5 hours. Wash HUVECs once with PBS and then rinse with 40 μL / well of Accutase™ solution. The cells were collected by adding 80 μL of ice-cold flow cytometry buffer and then Transfer to a 0.5 mL polypropylene 96-well block and add excess ice-cold flow cytometry data. The mixture was washed with a buffer and centrifuged at 400 x g (4°C) for 5 minutes. The supernatant was removed by aspiration. Resuspend the pellet in 80 µL of ice-cold flow cytometry buffer and plate in a 96-well plate. The mixture was then transferred to a microtiter plate (BD Biosciences, US). Samples were analyzed using a BD LSRFortessa™ flow cytometer (BD Bio Lysosomal localization of phagocytosed Jurkat cells was measured using a lysosomal immunoassay (LISA) using a lysosomal immunoassay (LAI ...). pHrodo™ Green fluorescence intensity was recorded as an indicator of flow cytometry. Data analysis was performed using FlowJo™ software. Fluorescence intensity (MFI) values ​​of pHrodo™ Green signal from rat HUVECs The median value of EC was used as the reading. 50 MS Excel for calculations The analysis was performed using GraphPad Prism software.

[0314] Fusion protein FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44 ) and FP270 (EGF-HSA-C2; SEQ ID NO: 36) by HUVEC endothelial cells The effect of HU on promoting efferocytosis of dead Jurkat cells is shown in Figure 9. Internalization of pHrodo-labeled killed human Jurkat T cells by VEC fusion proteins The results showed that endothelial cells were armed with the fusion protein. Surprisingly, in this assay, The efficacy of the fusion protein in this study was clearly dependent on the presence of the C1-C2 or C1-C1 tandem domain. For example, the fusion protein consisting of EGF-HSA-C2 (FP270) The protein is inactive in this experimental setting, as shown in Figure 9. Figure 10 shows the effect of the engineered protein The position of the HSA domain, i.e., the N-terminal or C-terminal position (HSA-EGF- C1-C2 (FP220; SEQ ID NO: 30) or EGF-C1- C2-HSA (FP11 0; SEQ ID NO: 28)) exhibited efferocytosis in macrophage efferocytosis assays. This invention confers the ability to block eosinophilic cytosis to the MFG-E8 HSA modified protein. These data demonstrate the highly surprising findings of the present inventors. For efficient promotion of efferocytosis, the integrin-binding domain and the PS-binding domain were This clearly demonstrates the importance of placing the HSA domain between the nucleotides.

[0315] Figure 11 shows the combination of the EGF domain, the C1-C2 domain, the HSA domain, and the Fc domain. We compared the promotion of endothelial efferocytosis by various forms of fusion proteins containing Figure 11A shows that HSA has a C-terminal, N-terminal, or EGF-like domain and a C1-C2 domain. between EGF-C1-C2-HSA (FP110; SEQ ID NO: 28), HS A-EGF-C1-C2 (FP220; SEQ ID NO: 30) and EGF-HSA-C1-C2 - a fusion protein containing HSA located in a His tag (FP278; SEQ ID NO: 44) Figure 11B shows a comparison of fusion proteins containing an Fc domain and those containing either an EGTA domain or an Fc domain at the C-terminus. The figure shows a comparison with the Fc region located between the F-like domain and the C1 domain. Formats are shown: FP070 (EGF-Fc-C1-C2; SEQ ID NO: 17) and and the wild-type Fc (sequence number 22) found in FP080 (EGF-C1-C2-Fc; SEQ ID NO: 22). No. 7) and Fc with KiH modifications S354C and T366W in one arm of Fc The fragment (FP060; EGF-C1-C2-Fc [S354C, T366W]; SEQ ID NO: 1 4) EU numbering (Merchant et al. (1998) supra). Figure 11C shows three At different concentrations (0.72, 7.2 and 72 nM), a fusion protein containing an N-terminally positioned Fc portion was Three FP09 fragments of the FP090 protein (Fc-EGF-C1-C2; SEQ ID NO: 24) Comparison of the 0.0 batch to the wild type MFG-E8 control is shown. Efferocytosis of rkat cells is mediated by the EGF-like domain followed by either HSA or Fc. Figure 11D shows that the insertion of the soluble domain enhanced the expression of the modified protein. However, a novel biological activity based on the endogenous cross-linking protein EDIL3, a paralog of MFG-E8, has been reported. As shown in Figure 11D, HSA can be used to generate a fusion protein with a specific activity. , EGF-like domain and C1-C2 domain of EDIL3, a paralog of MFG-E8 This EDIL3 construct (FP050 (EDIL3-based EGF-H SA-C1-C2 (SEQ ID NO: 12) contains the three EGF-like domains found in wild-type EDIL3. Surprisingly, the present inventors have found that the RGD loop is the only one of the amino acids that has a RGD loop. This construct allows the HSA domain to be isolated for the expression of novel recombinant modified proteins with very high purity. We found similar tolerance of EDIL3-mediated insertions (Fig. 2B). The recombinantly engineered protein FP050 inhibits the death of Jurkat cells (HUVECS) It promotes efferocytosis of AT cells, indicating the core function of cross-linking proteins. We demonstrate that protein domains are useful for designing novel functional recombinant proteins. It was found that:

[0316] Example 4: Efferocytosis of prothrombotic plasma microparticles 4.1 Human endothelial microparticle efferocytosis assay cell culture HUVEC cells were obtained from Lonza (Basel, Switzerland). The cells were cultured in flasks coated with 0.2% bovine skin-derived erythrocytes in PBS. degree, dilution of 2% stock solution, Sigma Aldrich / Merck, Germany). The cells were cultured in 10% FBS (GE Healthcare, UK), 1% Pen / Str ep (Thermo Fischer Scientific, US), 1% Gluta max (Thermo Fischer Scientific, US) and 1 ng / m Supplemented with recombinant fibroblast growth factor-basic (Peprotech, UK) Cultured in Medium 199 (Thermo Fischer Scientific, US). . Accutase(TM)(Thermo Fischer Scientific, US) to separate the cells for harvesting or passaging.

[0317] Platelet-derived microparticles were prepared according to the following procedure: After the consent was obtained, citrated venous blood was collected from healthy adult volunteers (Coa gulation 9NC Citrate Monovette,Sarstedt, Platelet-rich plasma (PRP) was prepared by centrifugation (200 x g, 15 min, no brake, Platelet-derived particles / debris were prepared by immersing PRP in liquid nitrogen for 3 min at room temperature. Platelets were prepared by subjecting them to 1 flash freeze / freeze cycle and thawing at 37°C. Fragments / particulates were pelleted by centrifugation at 20,000 x g for 15 minutes at room temperature. The pellet was resuspended in PBS, aliquoted, and stored at -80°C. The preparation consisted of AlexaFluor™ 488-labeled mouse MFG-E8 / lactoad When measured by flow cytometry using phosphorus (in-house at Novartis), 100% were PS positive. The number of microparticles was counted using dedicated counting beads (BioCytex / The flow cytometry buffer was 1 % FBS (GE Healthcare, UK), 0.05% w / v sodium azide (Merck, Germany), and 0.5 mM EDTA (ethylenediaminetetraacetic acid, Th PBS (Thermo Fischer Scientific, US) supplemented The samples were prepared by Fischer Scientific, USA.

[0318] 4.2 Efferocytosis assay On day 1, HUVEC cells (70-90% confluence) were incubated with Accutase ( ) for 5 minutes, collected, washed with PBS, and resuspended in cell culture medium. The rates were measured using a Guava EasyCyte flow cytometer (Merck, Germany) and Guava ViaCount reagent (Merck, Germany) was used according to the manufacturer's instructions. The required amount of cells was centrifuged at 300 x g for 5 minutes at room temperature and resuspended in medium. So, 6.6 x 10 4 The cell count was determined as cells / mL. 150 μL / well of this cell suspension was added to a 96-well tissue culture plate (Corning™, US). The EC cells were incubated in a 37°C / 5% CO2 / 95% humidity incubator for an additional 4-8 hours. The mixture was incubated for 1 hour.

[0319] On day 2, remove the medium from the HUVEC cells by aspiration and add 25 µL of fresh pre-warmed (37 After adding pre-warmed (37°C) medium, 0.3 nM, 3 nM, or 3 nM diluted in pre-warmed (37°C) medium was added. 0 nM and three different concentrations of the fusion protein FP278 (EGF-HSA-C1-C2- 25 μL of His-tag (SEQ ID NO: 44) or control was added. S) treated 96-well plates (Corning™, US) were used. The protein was incubated at 37°C / 5% CO2 / 95% humidity for 30 minutes before adding the platelet-derived microparticles. The cells were allowed to interact with HUVEC cells.

[0320] Centrifuge the required amount of microparticles at 20,000 x g for 15 minutes at 4°C to remove 2 x 10 8 particle / pHrodo™ Green at a final concentration of 5 μg / mL (staining medium) Resuspended in RPMI 1640 medium (without FBS) supplemented with STP Ester dye. After staining for 10 minutes at 37°C, the remaining reactive pHrodo™ Green STP The esters were inactivated in staining medium supplemented with 10% FBS for an additional 5 minutes at 37°C. Hrodo™ Green-labeled microparticles were incubated at 20,000 x g for 15 minutes at 4°C. The cells were washed once by centrifugation and then cultured in 1 x 10 HUVEC medium. 8 Adjusted to particles / mL 5×10 6 particles / well of pHrodo™ Green-labeled microparticles The medium was added to the C cells and incubated at 37°C / 5% CO2 / 95% humidity for 5 hours. Remove and wash HUVEC cells once with PBS and add 40 μL / well of Accutase ( The cells were separated with 80 μL of ice-cold flow cytometry buffer. The excess was collected and transferred to a 1.5 mL polypropylene 96-well block and placed in an ice-cold flow cytometer. Wash with centrifugation buffer and centrifuge at 400 x g (4°C) for 5 minutes. Aspirate the supernatant. and resuspend the pellet in 80 μL of ice-cold flow cytometry buffer. Transferred to a 96-well V-bottom microtiter plate (BD Biosciences, US). Samples were analyzed using a BD LSRFortessa™ flow cytometer (BD The lysosomal localization of phagocytosed microparticles was measured using a lysosomal immunoassay (LISA) performed by the phagocytosed microparticles phagocytosed by the lysosomal immunoassay (LISA). pHrodo™ Green fluorescence intensity was recorded as an index. Flow cytometry Data analysis was performed using FlowJo™ software. The fluorescence intensity values ​​(MFI) of the pHrodo™ Green signal from HUVEC cells were The median value of EC 50 MS Exc for calculations The analysis was performed using el and GraphPad Prism software. As shown in Figure 12, the protein FP278 inhibits the effervescence of platelet-derived microparticles by endothelial cells. The uptake was concentration-dependent and was not as potent as other types of It was also observed in endothelial cells (data not shown).

[0321] Example 5: Technical characteristics of the MFG-E8-HSA fusion protein 5.1 Surface plasmon resonance (SPR) binding of fusion protein FP330 to FcRn analysis FcRn of fusion protein FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42) To characterize the binding to α-glucan, a direct binding assay was performed. The kinetic binding affinity constant ( KD) was measured on captured proteins using recombinant human FcRn as the analyte. Measurements were performed using a BIAcore (registered trademark) T200 (GE Healthcare, G The experiments were carried out in a centrifuge (Lattbrugg, Switzerland) at room temperature and pH 5.8 and 7.4, respectively. For affinity measurements, proteins were buffered in 10 mM NaP, 150 mM NaCl, 0.05% T Ween 20, pH 5.8, diluted according to the manufacturer's recommendations (GE Healthcare) CM5 research grade sensor chips (GE Healthcare) were fabricated using standard procedures according to The antibody was immobilized on a flow cell of a fluororesin (Hcare, ref. BR-1000-14). One flow cell was immobilized with a blank for use. Binding data were then analyzed using a reference The data were obtained by injecting a series of analyte dilutions into the flow cell and the measurement flow cell. A zero concentration sample (running buffer only) was included to allow for double referencing during titration. Data evaluation involved double-referenced sensorgrams and analysis of dissociation constants (KD). .

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

[0323] 5.2 Differential scanning calorimetry (DSC) of MFG-E8 and its mutants Differential scanning calorimetry was used to characterize the modified MFG-E8 protein variant FP278 (EGF The thermal stability of the α-HSA-C1-C2-His tag (SEQ ID NO: 44) was measured. The measurements were carried out using a scanning microcalorimeter (Nano DSC, TA instruments). The tube volume was 0.5 ml and the heating rate was 1°C / min. 4) was used at a concentration of 1 mg / ml. The molar heat capacity of a protein is was estimated by comparison with a replicate sample containing the same buffer omitted The partial molar heat capacities and melting curves were analyzed using standard procedures. The baseline was corrected and the concentrations were normalized. Two melting events were observed, the first T The Tm was 50°C and the second Tm was 64°C.

[0324] 5.3 Measurement of aggregation tendency and solubility of MFG-E8 mutants First, the MFG-E8 mutant protein FP278 (EGF-HSA-C1-C2-His The aggregation tendency of the ATP-tag (SEQ ID NO: 44) was measured by dynamic light scattering (DLS, Wyatt). Dynamic light scattering was applied to quantify the dynamic fluctuations of scattered light to determine the activity of FP278 in solution. Translational diffusion coefficients were measured. Protein variant size distribution without fractionation, polydispersity estimates, The hydrodynamic radius of the fusion protein FP278 was measured at a concentration of 1 mg / ml. The dynamic radius was measured using a DynaPro™ plate reader (Wyatt Technology ogy Europe GmbH, Dernbach, Germany) and software DYN Measurements were performed using AMICS (version 7.1.0.25, Wyatt). 1 μL of undiluted filtered (0.22 μm PVDF-Filter (Millex Syringe-Driven Filter Unit, Millipore, Billerica, U S) Protein solution was placed in a 384-well plate (384 round well plate, polystyrene Thermo Scientific, Langenselbold, Germany) High molecular weight aggregates of the protein samples were not identified. The mechanical radius is approximately 5-6 nm, indicative of a monomeric protein in solution.

[0325] Next, concentration-dependent hydrodynamic radius measurements of the fusion protein FP278 were performed to determine the Protein solubility was estimated. Protein concentrations up to 22 mg / ml were applied. Hydrodynamics The effective radius was determined as described above. Increasing the concentration of the fusion protein FP278 increased the radius ( No increase in the number of nuclei (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 a concentration of about 0.2 mg / ml.

[0326] Example 6: Optimization of MFG-E8 fusion protein Panel of mutant MFG-E8-based fusion proteins optimized for improved expression and yield To generate the FP330 fusion protein, mass spectrometry (MS) was used to identify the FP330 fusion protein (EGF) The panel of mutant proteins was designed to contain a variety of proteins of different sizes and structures. linkers of this structure, for example, a linker containing GS between the EGF domain and the HSA domain, and and / or a linker (sequence) containing multiple GS or G4S between the HSA domain and the C1 domain. In addition, some mutants contain amino acids containing deletions or substitutions. The mutant fusion protein contained an acid modification (shown as HSA* in Table 7). The panels are summarized in Table 7 below.

[0327] [Table 59]

[0328] Example 7: Mutant MFG-E8 fusion proteins; expression and purification The method for producing fusion proteins in HEK cell lines is described in Example 2. Nucleic acids encoding MFG-E8 mutants were transfected into Geneart (Li The DNA was synthesized by restriction enzyme ligation-based cloning at Eppendorf Technologies. The resulting plasmid was cloned into a mammalian expression vector using the cloning technique. The fusion protein was transfected into OS cells (Thermo). For transient expression of GFP, use Expifectamine CHO transfection agent ( The expression vector was transfected into suspension-adapted CHO-S cells using a 500-kDa ELISA kit (Thermo Scientific). Typically, 400 ml of cells in suspension at a density of 6 Mio cells per ml were Transfect DNA containing 400 μg expression vector encoding the engineered protein Next, the recombinant expression vector was introduced into the host cells and the culture medium (ExpiCHO feed) was and ExpiCHO expression medium supplemented with enhancer reagent (Thermo) for an additional 7 days. The cells were secreted for 1 day.

[0329] As can be seen from the expression data shown in Table 8, the mutant fusion protein FP068 (sequence FP776 (SEQ ID NO: 46) and FP776 (SEQ ID NO: 48) are fused to the fusion protein FP330 (SEQ ID NO: 4 2) showed approximately two-fold improvement in expression.

[0330] [Table 60]

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

[0332] In the first assay, a human macrophage-neutrophil efferocytosis assay was performed. The effect of the mutant fusion proteins on the expression of β-glucan was determined according to the method described in section 3.3 above. M0 macrophages were treated with the fusion protein FP333 (EGF-HSA-C1-C2 ; SEQ ID NO: 42) or mutant FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 43) 44) or FP776 (EGF-HSA-C1-C2; SEQ ID NO: 48) for 30 min. As shown in Figure 13, the fusion proteins FP330, FP278, and FP776 inhibit the endotoxin (lipopolysaccharide ( The fusion protein FP3 can rescue LPS-induced impaired efferocytosis. 30(EC 50 =1.6 nM; Figure 13A), FP278 (EC 50 =1.78 nM; Figure 13B) and FP776 (EC 50= 0.5 nM; Figure 13C), L This led to the rescue of the impaired efferocytosis caused by the addition of PS, and It even promoted efferocytosis when it reached the bell.

[0333] Fusion proteins FP330, FP278 and FP776 were synthesized as described in section 3.4 above. Human endothelial (HUVEC) cell-Jurkat cell efferocytes were cultured according to the described method. The endothelial cell-mediated killing of Jurkat cells was further characterized by apoptosis assay. Fusion proteins FP330, FP278 and F for promoting vesicle efferocytosis The effect of P776 is shown in Figure 14. pHrodo-labeled killing of human Jurkat cells by HUVECs. Internalization of tT cells was confirmed by FP330 (EC 50 = 3.4 nM; Figure 14A), FP278 (E C 50 =2.4 nM; Figure 14B) and FP776 (EC 50 = 3 nM; Figure 14C) These results suggest that endothelial cells are sensitive to the fusion protein. Thus, they are armed to become efficient phagocytes of dead cells.

[0334] Example 9: Protection of mice from AKI and acute organ reactions caused by AKI 9.1 Acute kidney injury model Female C57BL / 6 mice (18–22 g) were purchased from Charles River (France). ) and maintained at room temperature in cages protected by filter tops on a 12-hour light / dark cycle. The animals were housed in a controlled laboratory facility. We strictly adhere to the principles of Laboratory Animal Care. The therapeutic fusion protein under investigation was administered intraperitoneally (ip) 2 hours before surgery. ) or intravenously (iv). r Schweiz AG) was administered subcutaneously at a dose of 0.1 mg / kg 60–30 minutes before surgery. Inhalation anesthesia with isoflurane was administered in the anesthesia chamber for 5 minutes before surgery. During surgery, the animals were induced with a 3.5-5% oxygen (carrier gas). Anesthesia was maintained via a face mask with 100% isoflurane / oxygen at a gas flow rate of 100 vol%. The blood flow rate was 0.8-1.2 L / min. The abdominal skin was shaved and Betaseptic (Mundi The animals were disinfected using a thermoregulator (PhysiTem Pharma, France). p,US-Physitemp Instruments LLC,US) The animals were placed on a blanket (Rothacher, Switzerland) and covered with sterile gauze. by a rectal probe (Physitemp Instruments LLC, US) Patients were monitored throughout the procedure and their body temperature was controlled to 36.5–37.5°C. All animals, including sham controls, underwent unilateral right kidney nephrectomy. After midline incision / laparotomy, the abdominal cavity The contents were retracted to the left to expose the right kidney. The right ureter and renal vessels were separated and ligated. The right kidney was removed. For animals that experienced AKI, the abdominal contents were removed from the right side of the kidney using sterile gauze. The left renal artery and vein were dissected to allow clamping for induction of ischemia. Microaneurysm clamping (artery and vein together using one clamp) Renal ischemia was induced by blocking the blood flow to the kidney using a pump (B Braun, Switzerland). 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. The abdominal contents were washed. Use warm sterile saline (approximately 2 ml, 37°C) to cleanse the wound before closing. After washing, 1 ml of sterile saline was added to the abdominal cavity as a replacement fluid. When reperfusion was initiated, the wound was closed in two layers (muscle and skin separately). The animals were kept under a red warm lamp until they were fully recovered. Buprenorphine was administered at the end of the surgery. One and four hours later, the subjects were again administered 0.1 mg / kg of ethanol in drinking water (9.09 After 24 hours, the animals were euthanized for analysis.

[0335] 9.2 Administration of Therapeutic Fusion Proteins Therapeutic fusion protein FP330 (EGF-HSA-C1-C2; SEQ ID NO: 42), F P278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) and FP776 ( EGF-HSA-C1-C2; SEQ ID NO: 48) was administered at the doses shown in Table 9 below. This was tested in an AKI model to detect the expression of serum markers and qPCR markers. In a study to determine whether ischemia-reperfusion injury was prevented, the fusion protein FP278 was administered 2 hours before surgery. FP330 and FP776 were administered intravenously 30 minutes before the onset of injury. In a study to measure the uptake of contrast agents by AKI-induced fibroblasts, the fusion protein FP776 was administered to the affected area. Administered prophylactically at 1.26 mg / kg 30 minutes before the onset of ischemia-reperfusion injury or Five hours later, 2 mg / kg iv was administered therapeutically.

[0336] [Table 61]

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

[0338] qPCR marker expression in organs: Organs (kidneys, liver, lungs, and heart) were harvested 24 hours after AKI induction and cut into 1 cm pieces. and RNA Later Buffer (Thermo Fisher Scientific The organ pieces were stored overnight at 4°C in Lysing Matrix (Cellulose, Inc., US). 134 mM beta-mercaptoethanol in a D tube (MP Biomedicals FR) RLT buffer (RNeasy Mini Kit) containing ethanol (Merck, DE) , Qiagen, DE) and then loaded onto a FastPrep-24 Instrument (MP The cardiac fibrous tissue was then homogenized using a Protease Biomedicals (Protease Biomedicals). Lysates of kidney, liver, and lung were digested with enzyme K (RNeasy Mini Kit). was 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 using RNeasy MiniMixture containing DNase digestion. The procedure was carried out according to the kit manual. The RNA concentration was determined by Nano Drop 10 00 Device (Thermo Fisher Scientific Inc.) Measurement was performed using a SimpliAmp Thermocycler (Applied Biosystems High-Capacity cDNA Reverse PCR System (USA) se Transcription Kit Manual(Thermo Fishe 2 μg of RNA per sample was reverse transcribed according to the protocol of r Scientific Inc. The cDNA was transferred to a 384-well microplate (MicroAmp Optical 384-Well Reaction Plate,Thermo Fisher S Scientific Inc., and nuclease-free water (Thermo Fish er Scientific Inc), TaqMan probes (TaqMan Gen e Expression Assay(FAM),Thermo Fisher Sc ientific Inc), and TaqMan Gene Expression M aster Mix (Thermo Fisher Scientific Inc) qPCR was performed using a ViiA 7 real-time PCR system (Applied The incubation was carried out in a 50°C (Biosystems, US) with the following settings: 1: 2 min, 50°C; 2: 10 min. 1: 95°C; 2: 15 seconds, 95°C; 3: 15 seconds, 95°C; 4: 1 minute, 60°C. Steps 3 and 4 were repeated for 45 seconds. Data analysis was performed using ViiA7 software and qPCR Data analysis software was MS Excel and GraphPad Prism software. This was carried out using software.

[0339] Uptake of contrast agents by the liver measured by magnetic resonance imaging (MRI) The method for performing MRI is described by Egger et al. ,(2015)J Magn Reson Imaging,41:829-840) The experiments were performed on a 7-T Bruker Biospec MRI system. The experiments were carried out in a lab at 1000 sq m (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 pad. After a short induction period, anesthesia was administered via a nose cone. Approximately 1.4% isoflurane in a mixture of O2 / N2O (1:2) administered via All measurements were performed in spontaneously breathing animals and were not triggered by cardiac or respiratory triggers. was not applied.

[0340] After placing the mouse in the scanner, scout high-speed images were acquired for localization purposes. The deposition was performed using superparamagnetic iron oxide (SPIO) nanoparticles (Endorem®, Guerbet). The study was conducted using intravascular agents including Endorem® (France). are animals with AKI (24 hours after disease induction) or after sham surgery (24 hours after nephrectomy) ) was injected intravenously as a bolus for 1.2 seconds. The first bolus was 400 ms / image. The CT scan was administered for 1.2 seconds in conjunction with sequential acquisition of echoplanar images at a resolution of 25. After acquiring two baseline images, inject the second bolus for 1.2 seconds. An additional 575 images were acquired in 4 minutes, for a total of 600 images. Paramagnetic contrast agents induce local changes in susceptibility, resulting in a signal proportional to renal perfusion. For the series of images, the signal intensity was located in the cortex / outer zone of the outer medulla. The location, shape, and size of the ROI were determined by the breathing pattern. Care was taken to ensure that the ROI covered approximately the same area despite the induced kidney movement. The mean signal intensity of the pre-injection images was calculated by dividing the baseline intensity (S(0)) by the mean signal intensity of the pre-injection images. The perfusion index was determined from the mean of the following ratios (Rosen et al. ,(1990)Magn Reson Med.,14:249-265): -ln[S(t) / S(0)]~TE.V.cT(t) where TE is the echo time, V is the blood volume, and cT is the concentration of the contrast agent.

[0341] The average diameter of the SPIO nanoparticles used in this study was approximately 150 nm, and Therefore, in addition to perfusion of the kidney, MRI is placed on the liver. By detecting contrast changes assessed in the selected ROI, nanoparticle uptake in the liver was assessed. It was also possible to monitor the traffic.

[0342] 9.4 Results As shown in Figure 15, the fusion protein FP330 (EGF-HSA-C1-C2; sequence No. 42), FP278 (EGF-HSA-C1-C2-His tag; SEQ ID NO: 44) and and FP776 (EGF-HSA-C1-C2; SEQ ID NO: 48) were compared with ip (FP278 ) or i.v. (FP330 and FP776), acute kidney injury ( Renal function was protected in this model of AKI. This protection was due to elevated serum creatinine (sCr) ) is reflected in the blockade of the fusion protein FP27 at both doses tested. 8 reduced blood pressure compared to vehicle-treated animals and as effectively as murine MFG-E8. The results show that serum creatinine levels were significantly reduced (p<0.0001). As shown, the fusion protein FP330 protected kidney function in a dose-dependent manner. The same was true for substance FP776 (Figure 15C), where serum creatinine levels were also used. was blocked in a dose-dependent manner.

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

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

[0345] Furthermore, as shown in Figure 17, a single administration of the fusion protein FP278 significantly reduced AKI. AKI protects distant organs from the acute phase response induced by inflammatory cytokines. AKI affects the spleen, lungs, liver, and heart. , excess measurable by qPCR in lysates of remote, highly perfused organs, such as the brain. Typical mRNAs are those expressed in response to selected lesions (NGAL, KIM- 1), induction of chemokines (not shown), or acute responses such as serum amyloid A (SAA). Figures 17A and 17B show the results of a single injection of the fusion protein. Such AKI in the mouse heart and lungs was subsequently potently blocked and returned to sham levels. An inducible response (serum amyloid A (SAA)) is illustrated.

[0346] The time course of liver uptake of the SPIO contrast agent Endorem® is shown in Figure 18. Animals with AKI had significantly more liver damage (target = Kupffer cells) compared to sham-operated animals. The results showed that the uptake of contrast agent by FP776 treatment (1.26 m g / kg administered prophylactically approximately 30 minutes before AKI induction, or 2 mg / kg administered therapeutically. 5 hours after ischemia-reperfusion injury was induced) due to the loss of contrast agent accumulation in the liver of AKI mice. These results suggest that AKI is caused by the activation of endogenous Kupffer cells in this mouse model. This leads to a significant impairment of iron particle clearance via the liver, and AKI is associated with increased iron particle production in the liver. These findings suggest that fusion proteins may cause microvascular damage that affects contrast agent accumulation. Treatment with FP776 protected against loss of clearance and microvascular damage, and was significantly superior to sham animals. When compared, both doses tested promoted contrast agent uptake.

[0347] Therapeutic fusion proteins, such as those of embodiment 19 (e.g., SEQ ID NO: 80) or embodiment 20 (e.g., SEQ ID NO: 82) when tested in the above experiments, Integrin promotes cell adhesion and, like FPJ776, promotes efferocytosis. They are therefore suitable for the therapeutic uses disclosed herein.

[0348] Taken together, these data support the use of fusion proteins of the present disclosure (e.g., HSA domain inserts). The steroids (with accompanying ingredients) are functional and effective and can be used as therapeutic agents.

[0349] The examples and embodiments described herein are for illustrative purposes only. Various modifications or changes will be suggested by those skilled in the art in view of the present application and It will be understood that all such modifications and variations are to be included within the spirit and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes. and is incorporated herein by reference.

Claims

1. Integrin-binding domain, phosphatidylserine (PS)-binding domain, and solubilization 1. A therapeutic fusion protein for enhancing efferocytosis comprising a domain, The soluble domain is inserted between the integrin-binding domain and the PS-binding domain. wherein the therapeutic fusion protein is inserted into a target cell and the integrin binding domain binds to an integrin. Plagiarism.

2. The integrin-binding domain is αvβ3 and / or αvβ5 and / or α8β1 The fusion protein of claim 1 that binds to an integrin.

3. The integrin binding domain is arginine-glycine-aspartic acid (RGD). The fusion protein of claim 1 or 2, comprising the motif.

4. the solubility domain is the integrin-binding domain, the PS-binding domain, or The fusion protein according to any one of claims 1 to 3, wherein both domains are directly linked. quality.

5. The soluble domain is linked to the integrin-binding domain and / or the precursor domain by a linker. The fusion of any one of claims 1 to 4, which is indirectly linked to the PS binding domain. protein.

6. The solubilizing domain is selected from the group consisting of human serum albumin (HSA), domain 3 of HSA (HSA D3), ​​Fc-IgG, or a functional variant thereof. The fusion protein according to paragraph 1.

7. the integrin binding domain has the amino acid sequence of SEQ ID NO: 2, or and wherein the PS binding domain has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

3. or has at least 90% sequence identity thereto, or The S-binding domain has the amino acid sequence of SEQ ID NO: 76, or at least 9 amino acids thereto. The fusion protein of any one of claims 1 to 6, having 0% sequence identity.

8. The integrin binding domain has the amino acid sequence of SEQ ID NO: 2, or a variant thereof. and wherein the PS binding domain has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

78. or having at least 90% sequence identity thereto. The fusion protein according to any one of claims 1 to 4.

9. The integrin binding domain has the amino acid sequence of SEQ ID NO: 77 or a sequence corresponding thereto. and wherein the PS-binding domain has at least 90% sequence identity with the amino acid sequence of SEQ ID NO:

3. or has at least 90% sequence identity thereto, or The PS binding domain has the amino acid sequence of SEQ ID NO: 76, or at least The fusion protein of any one of claims 1 to 8, which has 90% sequence identity.

10. The solubility domain is HSA and has the amino acid sequence of SEQ ID NO: 4 or The fusion protein according to any one of claims 1 to 9, having at least 90% sequence identity. Plagiarism.

11. the fusion protein comprises the amino acid sequence of SEQ ID NO: 42, or at least The fusion protein of any one of claims 1 to 10, having 90% sequence identity.

12. The fusion protein comprises the amino acid sequence of SEQ ID NO: 44, or at least or SEQ ID NO:47, or at least 90% sequence identity thereto; sequence identity; or SEQ ID NO: 48, or at least 90% sequence identity thereto The fusion protein according to any one of claims 1 to 11, comprising:

13. the fusion protein comprises the amino acid sequence of SEQ ID NO: 80, or at least The fusion protein of any one of claims 1 to 12, having 90% sequence identity.

14. the fusion protein comprises the amino acid sequence of SEQ ID NO: 82, or at least The fusion protein of any one of claims 1 to 13, having 90% sequence identity.

15. An isolated nucleic acid encoding an amino acid sequence according to any one of claims 11 to 14. 。

16. A cloning or expression vector comprising the nucleic acid of claim 15.

17. One or more cloning or expression vectors according to claim 16, and optionally a Recombinant host cells suitable for the production of therapeutic fusion proteins containing secretory signals.

18. A fusion protein according to any one of claims 1 to 14 and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising:

19. In the treatment or prevention of inflammatory disorders or inflammatory organ damage in individuals in need thereof. A fusion protein according to any one of claims 1 to 14 for use, Inflammatory organ damage or inflammatory organ damage includes acute kidney injury, acute respiratory distress syndrome, acute liver injury, sepsis, inflammatory and organ damage resulting from myocardial infarction, stroke, burns, traumatic injury, and ischemia / reperfusion The fusion protein is a damage.

20. 20. A fusion protein for use according to claim 19, wherein the fusion protein is a fusion protein comprising a fusion protein of another It is administered in combination with a therapeutic agent, and the therapeutic agent is an immunosuppressant, an immunomodulator, an anti-inflammatory agent ... The fusion protein is an oxidative agent, an anti-infective agent, a cytotoxic agent, or an anti-cancer agent.

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