Biopolymer target-specific complement inhibitors, their production methods and applications

Abstract

The present invention provides a biopolymer target-specific complement inhibitor that not only has a significant target complement inhibitory effect but also can promote increased drug formation and / or production for the treatment and prevention of various human diseases associated with abnormal complement activation. The present application relates to a fusion protein of a biopolymer target-specific complement inhibitor, comprising: (i) the CRIg extracellular domain, (ii) a complement regulatory domain, and (iii) an enhancer domain, as well as methods for producing and using the fusion protein and pharmaceutical compositions containing the fusion protein.

Description

[Technical Field]

[0001] The present application relates to the field of biopharmaceuticals, and more particularly to the design, preparation and clinical application of biopolymer target-specific complement inhibitors. [Background technology]

[0002] The complement system is an important component of innate immunity, as well as a key regulator of adaptive immunity, and acts as a bridge between the two. The complement system is a highly complex self-regulating cascade consisting of more than 30 activators, inhibitors, and complement receptors. Its main physiological function is to remove invading pathogenic microorganisms and host cell debris, and to regulate the entire immune and inflammatory process, making it an important system for immune surveillance and self-stabilization (Ricklin et al., 2011). et al., 2010 ).

[0003] The activation of the complement system is generally classified into three pathways: the classical pathway, which is mediated by IgG and IgM antibodies; the lectin pathway, which is mediated by mannose on the bacterial surface; and the alternative pathway, which is mediated by various components of the cell wall / cytosol of pathogens and complement C3b analogues such as serotonin. Furthermore, C3 is automatically activated by hydration. It can be done.

[0004] Complement activation exerts its physiological effects in three main ways (Dunkelberger and First, C3a, especially C5a, produced by complement components C3 and C5, respectively, binds to receptors C3aR or C5aR1 and C5L2 expressed on immune cells, thereby inducing various immune cells. These cells recruit inflammatory cytokines (TNF-α, IL-1, IL-6, etc.) and chemokines (MCP-1, MIP-2, KC, CINC, etc.) (Riedemann et al., 2003), which then activate complement. It produces a strong pro-inflammatory effect locally at the site, i.e. it produces a pro-inflammatory effect.

[0005] Second, another product of C3 activation, C3b, and its further degradation product, iC3b, They are inserted onto the surface of target cells under complement attack, "tagging" these foreign components and binding to multiple receptors expressed on immune cells, such as CR1 / CR2 / CR3 / CR4 / CRIg, which ultimately allow them to be phagocytosed or lysed by immune cells. These foreign components are then engulfed by immune cells, i.e., phagocytosis is mediated.

[0006] Third, another product of C5 activation, C5b, also acts as a surface receptor for target cells attacked by complement. It is inserted into the surface of the ribosomal membrane and binds complement C6 and C7 to form the stable complex C5b-7, which then binds to C8 and C9. Finally, C9 multimerizes to form the C5b-9n complex, also known as the membrane attack complex (MAC), This allows the complex to form in the inner membrane of the target cell. This creates a hole in the target cell membrane with an inner diameter of 5 nm, an outer diameter of 20 nm, and a height of 15 nm, which changes the osmotic pressure inside and outside the cell and causes the cell to It exerts a direct lytic effect on the vesicles, i.e., cell lysis effect (Tegla et al., 2011).

[0007] To avoid the physiological effects of complement activation on normal somatic cells, the body has evolved more than 10 types of complement regulatory proteins, which are expressed on cell membranes or circulate in the blood system, thereby suppressing complement activation at each stage of complement activation. For example, complement inhibitory proteins CR1, CD46, CD55, and CD59 are expressed on cell membranes, and C1-INH, C4BP, FH, vitronectin, and S protein circulate freely in the blood. Somatic cells respond to complement-mediated activation during complement activation. It is this complement regulatory protein that allows them to avoid killing.

[0008] The complement system is a self-defense immune mechanism under normal conditions. However, certain abnormal conditions in the body, such as an increase in the concentration of complement activation products or a decrease or deficiency of complement regulatory proteins, can cause the complement to become overly activated and attack the body's own tissue cells, ultimately leading to seizures. Nocturnal hemoglobinuria (PNH), hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), neuromyelitis optica spectrum disorder (NMOSSD), age-related macular degeneration (AMD), autoimmune hemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, ankylosing rheumatoid arthritis, ankylosing spondylitis, arteriosclerosis, Parkinson's disease, This can lead to diseases such as Alzheimer's disease (dementia), asthma, allergies, psoriasis, multiple sclerosis, and Crohn's disease (Ricklin and Lambris, 2007). Complement system inhibitors are particularly strongly associated with the onset of autoimmune diseases in the early stages of their development. Therefore, inhibitors of the complement system could be of great clinical benefit and demand, as they could intervene early in the progression of these diseases.

[0009] Based on the importance of the complement system in the pathogenesis of numerous autoimmune diseases and acute and chronic infectious diseases, nearly 50 complement inhibitors from nearly 30 pharmaceutical companies are in various stages of development, indirectly indicating that the development of more optimal complement inhibitors is still necessary. Summary of the Invention

[0010] The present application provides a fusion protein, comprising: (i) a CRIg extracellular domain; (ii) a complement control (iii) a complement regulatory domain, wherein the complement regulatory domain comprises a protein or functional fragment thereof selected from the group consisting of Factor H (FH), CD55, CD46, CD59, and CR1; and (iv) an enhancement domain, wherein the enhancement The domain comprises a protein or functional fragment thereof selected from the group consisting of an IgG Fc domain and human serum albumin.

[0011] The present inventors have unexpectedly found that the IL-14-14 fusion protein binds to other complement regulatory proteins FH, CD55, CD46, CD59 or CR1. CRIg conjugated to IgG Fc fragment or HSA has a more pronounced complement inhibitory effect Furthermore, we found that this method can contribute to improving drug formulation properties such as purification and improved pharmacokinetic (PK) / pharmacodynamic (PD) effects.

[0012] In some embodiments, the present invention provides a method for targeting complement component C3 by binding to CRIg, which exerts its targeting effect by binding to the fragment C3b and / or iC3b generated after activation of complement component C3, and another component having a complement inhibitory effect, such as FH, CD55, CD46, CD59, or CR1 (all components except CD59). All CDs are located at different positions in C3b and / or iC3b compared to single CRIg, FH, CD55, and CR1. and (at the same time) stably bind to C3b / iC3b, thereby more effectively inhibiting its action), which can be further linked to an IgG Fc fragment or HSA to produce a fusion protein (e.g., by genetic engineering). This method allows recombinant conjugated complement regulatory proteins to be delivered to local sites of complement activation, ultimately targeting and inhibiting complement activation. This class of drugs can be used to treat and prevent a variety of human diseases associated with abnormal complement activation.

[0013] On the other hand, the present application provides a fusion protein, comprising: (i) a CRIg extracellular domain; (ii) a complement; (iii) a complement regulatory domain, wherein the complement regulatory domain comprises a protein or functional fragment thereof selected from the group consisting of Factor H (FH), CD55, CD46, CD59, and CR1; and The enhancement domain comprises a protein or functional fragment thereof selected from the group consisting of an IgG Fc domain and human serum albumin.

[0014] In some embodiments, the CRIg extracellular domain comprises the amino acid sequence set forth in SEQ ID NO:6.

[0015] In some embodiments, the C-terminus of the CRIg extracellular domain is is directly or indirectly connected to the N-terminus of

[0016] In some embodiments, the C-terminus of the complement control domain is N-terminus of the enhancement domain. are directly or indirectly connected to

[0017] In some embodiments, the N-terminus of the CRIg extracellular domain is is directly or indirectly connected to the C-terminus of

[0018] In some embodiments, the C-terminus of the CRIg extracellular domain is linked directly or indirectly to the N-terminus of the potentiation domain.

[0019] In some embodiments, the indirect link is via a linker.

[0020] In some embodiments, the linker may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 44, 46, 48, and 50.

[0021] In some embodiments, the complement regulatory domain may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 8, 18, 20, 22, 62, and 64.

[0022] In some embodiments, the enhancement domain is human serum albumin.

[0023] In some embodiments, the human serum albumin comprises the amino acid sequence set forth in SEQ ID NO:12.

[0024] In some embodiments, the fusion protein is a single chain structure.

[0025] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the CRIg extracellular domain, the complement control domain, and the potentiation domain.

[0026] In some embodiments, the fusion protein comprises, from N-terminus to C-terminus, the complement regulatory domain, the CRIg extracellular domain, and the potentiation domain.

[0027] In some embodiments, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO:14.

[0028] In some embodiments, the enhancing protein comprises an IgG Fc domain.

[0029] In some embodiments, the IgG is selected from the group consisting of human IgG1 and human IgG4. Contains proteins.

[0030] In some embodiments, the IgG domain is any of SEQ ID NOs: 10, 30, 32, and 34. It may comprise the amino acid sequence set forth in paragraph 1.

[0031] In some embodiments, the fusion protein comprises a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises the CRIg extracellular domain, the first complement control domain, and the first IgG Fc domain, and the second polypeptide chain comprises the CRIg extracellular domain, the second complement control domain, and the second IgG Fc domain.

[0032] Here, the first IgG Fc domain and the second IgG Fc domain can interact to form a dimer.

[0033] In some embodiments, each of the first complement regulatory domain and the second complement regulatory domain independently encodes a protein selected from the group consisting of factor H (FH), CD55, CD46, CD59, and CR1. It includes proteins or functional fragments thereof.

[0034] In some embodiments, the first complement control domain is identical to the second complement control domain.

[0035] In some embodiments, the first IgG Fc domain is identical to the second IgG Fc domain.

[0036] In some embodiments, the first IgG Fc domain and the second IgG Fc domain may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 10 and 30.

[0037] In some embodiments, the first polypeptide chain is identical to the second polypeptide chain.

[0038] In some embodiments, the first polypeptide chain and / or the second polypeptide chain , may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 14, 24, 26, 28, 58, 60, 66 and 68.

[0039] In some embodiments, the first complement control domain is different from the second complement control domain.

[0040] In some embodiments, the first complement regulatory domain and the second complement regulatory domain each independently comprise a protein or functional fragment thereof selected from the group consisting of CD59 and CD55.

[0041] In some embodiments, the first complement control domain and the second complement control domain each independently comprise a protein or functional fragment thereof selected from the group consisting of FH and CD55.

[0042] In some embodiments, the first complement regulatory domain and the second complement regulatory domain each independently comprise a protein or functional fragment thereof selected from the group consisting of CD46 and CD59.

[0043] In some embodiments, the first IgG Fc domain is identical to the second IgG Fc domain.

[0044] In some embodiments, the first IgG Fc domain is different from the second IgG Fc domain.

[0045] In some embodiments, the first IgG Fc domain is any one of SEQ ID NOs: 32 and 34. The amino acid sequence may include the amino acid sequence shown in the paragraph.

[0046] In some embodiments, the second IgG Fc domain is any one of SEQ ID NOs: 32 and 34. The amino acid sequence may include the amino acid sequence shown in the paragraph.

[0047] In some embodiments, the first polypeptide chain is selected from the group consisting of SEQ ID NOs: 36, 38, 40, and 42. It may comprise any one of the amino acid sequences shown above.

[0048] In some embodiments, the second polypeptide chain is selected from the group consisting of SEQ ID NOs: 36, 38, 40, and 42. It may comprise any one of the amino acid sequences shown above.

[0049] In some embodiments, the fusion protein comprises: the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:38 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:40; or the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:36 and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:42; The first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:38, and the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:42.

[0050] On the other hand, the present application provides one or more isolated nucleic acid molecules encoding said fusion protein or fragments thereof. to provide.

[0051] On the other hand, the present application provides a vector comprising the nucleic acid molecule.

[0052] On the other hand, the present application provides a cell containing the vector or a cell expressing the fusion protein.

[0053] On the other hand, the present application may provide a method for preparing the fusion protein, which may include a step of synthesizing the fusion protein or a fragment thereof, and / or a step of culturing the cell under conditions for expressing the fusion protein or a fragment thereof.

[0054] On the other hand, the present application provides a pharmaceutical composition comprising the fusion protein and, optionally, a pharmaceutically acceptable vector.

[0055] Meanwhile, the present application provides the use of said fusion protein or said pharmaceutical composition in the preparation of a medicament, said medicament being used for treating a disease associated with targeted inhibition of complement activation.

[0056] In some embodiments, the disease comprises an autoimmune disease.

[0057] In some embodiments, the disease comprises autoimmune myasthenia gravis.

[0058] Those skilled in the art can readily ascertain other aspects and advantages of the present application from the following detailed description. The following detailed description shows and describes only exemplary embodiments of the present application. As those skilled in the art will recognize, the present application will enable those skilled in the art to make modifications to the particular embodiments disclosed without departing from the spirit and scope of the invention of the present application. Accordingly, the accompanying drawings and description of the present application are illustrative only and not restrictive.

[0059] Specific features of the present invention are set forth in the appended claims. A better understanding of the features and advantages of the present invention can be obtained by reference to the exemplary embodiments described in detail herein and the accompanying drawings, the brief description of which is as follows: [Brief explanation of the drawings]

[0060] [Figure 1] Figure 1 shows the schematic design of (CRIg-FH-IgG4Fc) × 2 and CRIg-FH-HSA. [Figure 2] FIG. 2 shows the identification of the (CRIg-FH-IgG4Fc)×2 and CRIg-FH-HSA recombinant proteins. [Figure 3] FIG. 3 shows the inhibitory effect of (CRIg-FH-IgG4Fc)×2 on the classical pathway of human serum complement. [Figure 4] FIG. 4 shows the inhibitory effect of (CRIg-FH-IgG4Fc)×2 on the alternative pathway of human serum complement. [Figure 5] FIG. 5 shows the inhibitory effect of CRIg-FH-HSA on the classical pathway of human serum complement. [Figure 6] FIG. 6 shows the inhibitory effect of CRIg-FH-HSA on the alternative pathway of human serum complement. [Figure 7] Figure 7 shows the results of a pharmacokinetic study of (CRIg-FH-IgG4Fc) x 2 in rats. [Figure 8]FIG. 8 shows the results of a pharmacokinetic study of CRIg-FH-HSA in rats. [Figure 9] Figure 9 shows the schematic designs of (CRIg-FH-IgG4Fc) x 2, (CRIg-CD55-IgG4Fc) x 2, (CRIg-CD46-IgG4Fc) x 2, and (CRIg-CD59-IgG4Fc) x 2. [Figure 10] Figure 10 shows the identification of four recombinant proteins: (CRIg-FH-IgG4Fc) x 2, (CRIg-CD55-IgG4Fc) x 2, (CRIg-CD46-IgG4Fc) x 2, and (CRIg-CD59-IgG4Fc) x 2. [Figure 11] FIG. 11 shows the inhibitory effect of (CRIg-CD55-IgG4Fc)×2 on the classical pathway of human serum complement. [Figure 12] FIG. 12 shows the inhibitory effect of (CRIg-CD55-IgG4Fc)×2 on the alternative pathway of human serum complement. [Figure 13] FIG. 13 shows the inhibitory effect of (CRIg-CD46-IgG4Fc)×2 on the classical pathway of human serum complement. [Figure 14] FIG. 14 shows the inhibitory effect of (CRIg-CD46-IgG4Fc)×2 on the alternative pathway of human serum complement. [Figure 15] FIG. 15 shows the inhibitory effect of (CRIg-CD59-IgG4Fc)×2 on the classical pathway of human serum complement. [Figure 16] Figure 16 shows the inhibitory effect of (CRIg-CD59-IgG4Fc) x 2 on the alternative pathway of human serum complement. [Figure 17] Figure 17 shows the schematic designs of the three recombinant proteins: (CRIg-CD55-IgG1 Fc), (CRIg-FH-IgG1 Fc), (CRIg-CD46-IgG1 Fc), (CRIg-CD59-IgG1 Fc), and (CRIg-CD55-IgG1 Fc) (CRIg-CD59-IgG1 Fc). [Figure 18]Figure 18 shows the identification of three recombinant proteins: (CRIg-CD55-IgG1 Fc), (CRIg-FH-IgG1 Fc), (CRIg-CD46-IgG1 Fc), (CRIg-CD59-IgG1 Fc), and (CRIg-CD55-IgG1 Fc) (CRIg-CD59-IgG1 Fc). [Figure 19] FIG. 19 shows the inhibitory effect of (CRIg-CD55-IgG1 Fc) (CRIg-FH-IgG1 Fc) on the classical pathway of human serum complement. [Figure 20] FIG. 20 shows the inhibitory effect of (CRIg-CD55-IgG1 Fc) (CRIg-FH-IgG1 Fc) on the alternative pathway of human serum complement. [Figure 21] FIG. 21 shows the inhibitory effect of (CRIg-CD46-IgG1 Fc) (CRIg-CD59-IgG1 Fc) on the classical pathway of human serum complement. [Figure 22] Figure 22 shows the inhibitory effect of (CRIg-CD46-IgG1 Fc) (CRIg-CD59-IgG1 Fc) on the alternative pathway of human serum complement. [Figure 23] FIG. 23 shows the inhibitory effect of (CRIg-CD55-IgG1 Fc) (CRIg-CD59-IgG1 Fc) on the classical pathway of human serum complement. [Figure 24] Figure 24 shows the inhibitory effect of (CRIg-CD55-IgG1 Fc) (CRIg-CD59-IgG1 Fc) on the alternative pathway of human serum complement. [Figure 25] Figure 25 shows the changes in body weight of EAMG rats administered with (CRIg-CD59-IgG4Fc) x 2. [Figure 26] Figure 26 shows changes in clinical scores in EAMG rats administered with (CRIg-CD59-IgG4Fc) x 2. [Figure 27] Figure 27 shows the change in mortality rate of EAMG rats administered with (CRIg-CD59-IgG4Fc) x 2. [Figure 28] FIG. 28 shows the results of a pharmacokinetic study of CRIg-FH in rats. [Figure 29] Figure 29 shows the schematic designs of the five recombinant proteins: (CRIg-IgG4 Fc)x2, (FH-IgG4 Fc)x2, (FH-CRIg-IgG4 Fc)x2, (CRIg-FH-IgG4 Fc)x2, and (CRIg-L-FH-IgG4 Fc)x2. [Figure 30] Figure 30 shows the identification of five recombinant proteins: (CRIg-IgG4 Fc)x2, (FH-IgG4 Fc)x2, (FH-CRIg-IgG4 Fc)x2, CRIg-FH-IgG4 Fc)x2, and (CRIg-L-FH-IgG4 Fc)x2. [Figure 31] Figure 31 shows the inhibitory effects of five recombinant proteins, (CRIg-IgG4 Fc)x2, (FH-IgG4 Fc)x2, (FH-CRIg-IgG4 Fc)x2, (CRIg-FH-IgG4 Fc)x2, and (CRIg-L-FH-IgG4 Fc)x2, on the alternative pathway of human serum complement. [Figure 32] Figure 32 shows the schematic design of (CRIg-CR1-IgG4Fc)x2. [Figure 33] FIG. 33 shows the identification of the (CRIg-CR1-IgG4Fc)×2 recombinant protein. [Figure 34] Figure 34 shows the inhibitory effect of (CRIg-CR1-IgG4Fc) x 2 on the classical pathway of human serum complement. [Figure 35] Figure 35 shows the inhibitory effect of (CRIg-CR1-IgG4Fc) x 2 on the alternative pathway of human serum complement. DETAILED DESCRIPTION OF THE INVENTION

[0061] In the following, embodiments of the present invention will be described using certain specific embodiments, but those familiar with the art will be able to easily understand other advantages and effects of the present invention from the disclosure herein.

[0062] Term definition In this application, the term "complement regulatory domain" generally refers to a substance capable of inhibiting activation of the complement system, which is generally activated through the classical pathway by IgG and IgM antibodies, the lectin pathway by mannose on the surface of bacteria, various components of the cell wall / cytosol of pathogens, and the like. There are three alternative pathways, one mediated by complement C3b analogues such as serotonin, and the other by C3b hydration, which can be activated automatically. The complement regulatory domain can inhibit different stages of activation. The complement regulatory domain described herein may be derived from a complement regulatory protein or a functional fragment thereof, and includes complement inhibitors present in the blood circulation and complement membrane regulatory proteins associated with cell membrane surfaces, including, but not limited to, C1-INH (C1 inhibitor, C1 inhibitory protein), C4BP (C4 binding protein), factor I (F1), factor H (FH), S-protein, clusterin, CD35 (also known as CR1), CD46 (also known as MCP), CD55 (also known as DAF), and / or CD59. , and even the full-length or partial sequence of CRIg itself.

[0063] In this application, the term "CD55" generally refers to a complement component that also becomes a complement decay-accelerating factor or DAF. CD55 is a phospholipase C1 (C1)-dependent phospholipase C1 (C2)-dependent phospholipase C2 (C3)-dependent phospholipase C3 (C4b)-dependent phospholipase C4 (C4b)-dependent phospholipase C3 (C3 ... It is used to block the conversion of Bb by factor B, thereby generating the C3bBc C3 convertase of the alternative pathway. The CD55 described herein can include the full-length CD55 protein or fragments thereof, as well as various variants (e.g., mutants, isoforms) thereof. For example, an exemplary nucleic acid molecule encoding CD55 can include the nucleotide sequence set forth in SEQ ID NO: 17, and an exemplary CD55 protein can include the protein sequence set forth in SEQ ID NO: 18.

[0064] As used herein, the term "CD59" generally refers to a complement regulatory protein and is a "membrane attack complex" Also known as "MAC inhibitory protein" (MAC-IP), "membrane inhibitor of reactive lysis" (MIRL), human erythrocyte membrane MAC formation inhibitory factor (MACIF) or protectin, and a member of the LY6 / uPAR / α-neurotoxin family CD59 is a glycophosphatidylinositol (GPI)-anchored protein. CD59 can bind to host cells via the C5b678 complex. Upon deposition of the C5b678 complex on host cells upon complement activation, CD59 can prevent polymerization of C9 and formation of the complement membrane attack complex. CD59 as used herein can include full-length CD59 proteins or fragments thereof, as well as various variants (e.g., mutants, isoforms) thereof. For example, an exemplary nucleic acid molecule encoding CD59 can include the nucleotide sequence set forth in SEQ ID NO:21, and an exemplary CD59 protein can include the protein sequence set forth in SEQ ID NO:22.

[0065] As used herein, the term "CD46" generally refers to a complement regulatory protein, also known as a membrane cofactor protein (MCP). Generally, CD46 has cofactor activity and protects host cells from complement damage by inactivating (thermolyzing) complement components C3b and C4b via serum factor I. CD46, as used herein, can include the full-length CD46 protein or fragments thereof, as well as various variants (e.g., mutants, isoforms) thereof. For example, an exemplary nucleic acid molecule encoding CD46 can include the nucleotide sequence set forth in SEQ ID NO: 19, and an exemplary CD59 protein can include the protein sequence set forth in SEQ ID NO: 20.

[0066] In this application, the term "factor H (FH)" generally refers to a member of the family of complement activation regulators. FH stands for complement regulatory protein, a member of the FH family. Its main function is to regulate the alternative pathway of the complement system, allowing it to function against pathogens and other dangerous substances without damaging host tissues. The sequence of FH is usually called a short consensus repeat (SCR). FHs have many highly conserved motifs that are recognized by their specificity. Each SCR consists of approximately 60 amino acids, two disulfide bonds, a highly substituted loop, and a short SCR linker of 3-8 residues. FHs are typically composed of 20 SCRs, and the functional properties of FHs are typically localized in the SCRs. The FHs described herein can include full-length FH proteins or fragments thereof (e.g., one or more SCRs), as well as various variants (e.g., mutants, isoforms) thereof. For example, an exemplary nucleic acid molecule encoding the FH may comprise the nucleotide sequence set forth in SEQ ID NO:3, and an exemplary FH protein may comprise the protein sequence set forth in SEQ ID NO:4. For example, the FHs described herein may comprise the SCR1-5 domain, and an exemplary nucleic acid molecule encoding the FH SCR1-5 domain may comprise the nucleotide sequence set forth in SEQ ID NO:7, and an exemplary FH SCR1-5 domain may comprise the protein sequence set forth in SEQ ID NO:8. In some embodiments, the FHs may also comprise other SCR fragments.

[0067] In this application, the term "CR1" generally refers to a complement regulatory protein, also known as the C3b / C4b receptor or CD35. CR1 has a total of 30 SCR or CCP sequences, of which CCP1-3 CCP8-11 and CCP15-18 exert their complement inhibitory effects by binding to C4b, and CCP8-11 and CCP15-18 exert their complement inhibitory effects by binding to C3b. The CR1 described herein may include CCP8-11 or CCP15-18, as well as various variants (e.g., mutants, isoforms) thereof. For example, the CR1 described herein may include a CCP8-11 domain, and an exemplary nucleic acid molecule encoding the CR1 protein CCP8-11 domain may include the nucleotide sequence set forth in SEQ ID NO: 61, and the exemplary CR1 protein CCP8-11 domain may include the protein sequence set forth in SEQ ID NO: 62. For example, the CR1 described herein may include a CCP15-18 domain, and an exemplary nucleic acid molecule encoding the CR1 protein CCP15-18 domain may include the nucleotide sequence set forth in SEQ ID NO: 63, and the exemplary CR1 protein CCP15-18 domain may include the protein sequence set forth in SEQ ID NO: 64. In some embodiments, the CR1 may contain other CCP fragments. The domains encoding the CR1 proteins CCP8-11 and CCP15-18 differ by only three amino acids and are functionally identical. Therefore, in this application, we decided to use the CCP8-11 domain of the CR1 protein as an example.

[0068] In this application, the term "single-chain structure" generally refers to a single-chain structure that is bound by a covalent bond (e.g., a peptide bond). The term "single-chain structure" refers to a chain of amino acids linked by a single chain. The single-chain structure may be produced by linking polypeptide fragments, or may be produced by linking nucleic acids encoding the polypeptide fragments prior to expression. For example, multiple polypeptide chains of the same or different origin may form a single-chain fusion protein.

[0069] As used herein, the term "human serum albumin (HSA)" generally refers to the globular protein encoded by the human gene ALB, which is normally present in plasma. is a single polypeptide with three domains: domain I, domain II, and domain III. Each domain consists of two subregions, A and B, with opposing grooves. The structure of HSA is relatively flexible, as it can form a cylindrical structure. As used herein, the term refers to naturally occurring (e.g., plasma-derived) HSA, artificially synthesized HSA, and the like. The term may include HSA (e.g., synthesized by recombinant techniques). The term encompasses full-length HSA or functional fragments thereof. An exemplary nucleic acid molecule encoding an HSA protein is An exemplary HSA protein may comprise the nucleotide sequence shown in SEQ ID NO: It may comprise the amino acid sequence shown in SEQ ID NO:12.

[0070] As used herein, the term "enhancement domain" generally refers to a polypeptide domain that enhances a complement inhibitory effect. The complement inhibitory effect may include increasing the strength of the complement inhibitory activity of a substance having complement inhibitory activity (e.g., a complement control domain), lengthening the complement inhibition period of a substance having strong complement activity inhibition (e.g., a complement control domain), shortening the degradation period of a substance having complement activity inhibition (e.g., a complement control domain), and / or lengthening the blood half-life of a substance having complement activity inhibition (e.g., a complement control domain). The enhancement domain may include a domain consisting of HSA, IgG1, IgG2, IgG3, and / or IgG4 Fc. As used herein, the enhancement domain and complement control domain may be located on the same polypeptide chain or on different polypeptide chains.

[0071] As used herein, the term "IgG Fc domain" generally refers to an immunoglobulin Fc region or domain thereof, and may include the Fc domains of IgG1, IgG2, IgG3 and / or IgG4.

[0072] In this application, the term "CRIg" generally refers to a complement membrane regulatory protein belonging to the immunoglobulin superfamily. CRIg specifically recognizes iC3b (non-activated C3b) and inhibits the activation of C3 convertase, thereby exerting an inhibitory effect at the early stage of the complement cascade reaction. The CRIg may include CRIg from a different species, for example, human or mouse CRIg. In some embodiments, the CRIg may be derived from human CRIg. Human CRIg may include a long form of CRIg consisting of V-terminal and C2-terminal Ig domains. Human CRIg also includes a short form of CRI consisting of a V-terminal Ig domain. An exemplary nucleic acid sequence encoding CRIg may be as shown in SEQ ID NO: 1, and an exemplary CRIg protein may be as shown in SEQ ID NO: 2. Generally, the functional region of CRIg is present in its extracellular domain. The term "CRIg extracellular domain" also generally includes the CRIg extracellular functional region, and an exemplary nucleic acid sequence encoding the CRIg extracellular domain is shown in SEQ ID NO:5. An exemplary extracellular domain of CRIg may be as shown in SEQ ID NO:6. It may also contain an acid sequence.

[0073] In this application, the term "vector" generally refers to a nucleic acid molecule that can self-replicate in a suitable host cell and transfers an inserted nucleic acid molecule into and / or between host cells. The vector may include a vector that is primarily used to insert DNA or RNA into a cell, a vector that is primarily used to replicate DNA or RNA, and a vector that is primarily used for transcriptional and / or translational expression of DNA or RNA. The vector also includes vectors having the various functions described above. The vector can be introduced into a suitable host cell and used to express the inserted nucleic acid molecule in a suitable manner. Alternatively, the vector may be a polynucleotide that is transcribed or translated into a polypeptide when introduced into a host cell. Typically, the vector can produce a desired expression product by culturing an appropriate host cell containing the vector.

[0074] As used herein, the term "cell" generally refers to an individual cell, cell line, or cell culture that can contain, or has contained, a plasmid or vector comprising a nucleic acid molecule described herein, or that is capable of expressing an antigen-binding fragment described herein. The cell may be the progeny of a single host cell. Due to natural, accidental, or deliberate mutations, the daughter cells may not necessarily be completely morphologically or genomically identical to the original parent cell, but may still express the antibodies or antigen-binding fragments thereof described herein. The cell may be obtained by in vitro transfection of the cell with a vector described herein. The cell may be a prokaryotic cell (e.g., Escherichia coli) or a eukaryotic cell (e.g., yeast cell, e.g., COS cell, Chinese hamster ovary (CHO) cell, HeLa cell, HEK293 cell, COS-1 cell, NS0 cell, or myeloma cell). In some embodiments, the cell may be a mammalian cell. For example, the mammalian cell may be a CHO-K1 cell.

[0075] As used herein, the term "recombinant cell" generally refers to a cell into which a recombinant expression vector has been introduced. The recombinant host cell includes not only the particular cell but also the progeny of such a cell.

[0076] In this application, the term "pharmaceutically acceptable vector" generally refers to a vector that is suitable for use in a particular dosage regimen. The physiologically acceptable vector includes a pharmaceutically acceptable adjuvant, excipient, or stabilizer that is nontoxic to cells or mammals exposed thereto at the appropriate concentration. Generally, a physiologically acceptable vector is a pH-buffered solution. Examples of physiologically acceptable vectors include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid; proteins such as low molecular weight (less than about 10 residues) polypeptides, serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

[0077] As used herein, the term "disease associated with targeted inhibition of complement activation" generally refers to the inhibition of disorders resulting from excessive complement activation, which may be caused by abnormal complement activation, elevated levels and / or activity of complement activation products, or decreased levels and / or activity of complement regulatory proteins (e.g., CD55, CD59, and / or FH). For example, unregulated complement activation can occur due to decreased expression of complement regulatory proteins, decreased replication and / or transcription levels of complement regulatory protein genes, or abnormalities in the translation process of complement regulatory proteins, compared to normal conditions.

[0078] Excessive activation of complement can result in aberrant (e.g., excessive) inflammatory responses, opsonization, and / or cytolysis. In some embodiments, the body produces autoantibodies against normal autoantigens, which, upon binding to the antigen, activate the complement pathway, resulting in immune damage to the body's own tissues, organs, and cells due to reduced levels and / or activity of substances associated with complement inhibitory activation (e.g., complement regulatory proteins), ultimately leading to autoimmune disease. In some embodiments, diseases associated with targeted inhibition of complement activation include autoimmune diseases. In some embodiments, the diseases associated with targeted inhibition of complement activation can be caused by acute or chronic infections.

[0079] In some embodiments, the disease associated with targeted inhibition of complement activation may be caused by a genetic mutation. The disease associated with targeted inhibition of complement activation may be selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), generalized myasthenia gravis (gMG), neuromyelitis optica spectrum disorder (NMOSSD), age-related macular degeneration (AMD), autoimmune hemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, ankylosing rheumatoid arthritis, ankylosing spondylitis, and vascular endothelial cell carcinoma (VEc). atherosclerosis, Parkinson's disease, Alzheimer's disease (dementia), asthma, allergies, psoriasis, multiple sclerosis, Crohn's disease. For example, the disease is autoimmune myasthenia gravis.

[0080] In this application, the term "autoimmune myasthenia gravis" generally refers to a condition characterized by neuromuscular signaling. It is a chronic autoimmune disease that blocks the supply of acetylcholine to the nervous system, thereby affecting skeletal muscle strength. It develops when complement becomes overactive, triggering an immune response that attacks acetylcholine receptors and receptor-associated proteins, proteins in the postsynaptic membrane of the neuromuscular junction. Over time, symptoms of autoimmune myasthenia gravis gradually spread from the eye muscles to the face and neck muscles, causing weakness, slurred speech, difficulty chewing and swallowing, and / or difficulty breathing. The symptoms gradually spread from the head and neck to the rest of the body, eventually resulting in systemic myasthenia gravis.

[0081] fusion proteins On the other hand, the present application provides a fusion protein, which comprises: (i) a CRIg extracellular domain; The CRIg extracellular domain described herein may comprise the amino acid sequence set forth in SEQ ID NO: 6. In some embodiments, the CRIg extracellular domain may comprise the amino acid sequence set forth in SEQ ID NO: 6. The amino acid sequence may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence of the target polypeptide, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0082] In the present application, the fusion protein may comprise (ii) a complement regulatory domain.

[0083] In some embodiments, the protein may comprise a protein derived from FH or a functional fragment thereof, and the FH may comprise the amino acid sequence shown in SEQ ID NO:4.

[0084] In some embodiments, the FH has an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO:4, e.g., at least 95%, at least 96%, at least 97% , at least 98%, or at least 99% homologous to the amino acid sequence of the present invention.

[0085] In some embodiments, the complement regulatory domain may comprise one or more SCR domains of FH. For example, the complement regulatory domain may comprise the SCR1-5 domain of FH, which may comprise the amino acid sequence set forth in SEQ ID NO:8. .

[0086] In some embodiments, the FH has an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO:8, e.g., at least 95%, at least 96%, at least 97%. , at least 98%, or at least 99% homologous to the amino acid sequence of the present invention.

[0087] In some embodiments, the complement regulatory domain may comprise a protein derived from CD55 or a functional fragment thereof, and the CD55 may comprise the amino acid sequence set forth in SEQ ID NO:18.

[0088] In some embodiments, the CD55 may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 18, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0089] In some embodiments, the complement regulatory domain may comprise a protein derived from CD59 or a functional fragment thereof, and the CD59 may comprise the amino acid sequence set forth in SEQ ID NO:22.

[0090] In some embodiments, the CD59 may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 22, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0091] In some embodiments, the complement regulatory domain may comprise a protein derived from CD46 or a functional fragment thereof, and the CD46 may comprise the amino acid sequence set forth in SEQ ID NO:20.

[0092] In some embodiments, the CD46 may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 20, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0093] In some embodiments, the complement regulatory domain is derived from CR1 or a functional fragment thereof. The CR1 may comprise a protein having the amino acid sequence shown in SEQ ID NO: 62. It's fine.

[0094] In some embodiments, the CR1 has at least the amino acid sequence set forth in SEQ ID NO:62. The CR1 may comprise an amino acid sequence that is at least 90% identical to the CR1, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the CR1. Alternatively, the CR1 may comprise the amino acid sequence set forth in SEQ ID NO:64.

[0095] In some embodiments, the CR1 has at least the amino acid sequence set forth in SEQ ID NO:64. The amino acid sequence may comprise an amino acid sequence that is at least 90% homologous, for example, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0096] The complement regulatory domain described herein may comprise a protein or functional fragment thereof from the group selected from Factor H (FH), CD55, CD46, CD59 and CR1.

[0097] In some embodiments, the complement regulatory domain may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 8, 18, 20, 22, 62, and 64.

[0098] In some embodiments, the complement control domain comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in any one of SEQ ID NOs: 8, 18, 20, 22, 62, and 64, e.g., For example, it may comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to any one of the amino acid sequences.

[0099] The complement regulatory domain described herein may comprise a protein or functional fragment thereof from the group selected from Factor H (FH), CD55, CD59 and CR1.

[0100] In some embodiments, the complement regulatory domain may comprise an amino acid sequence set forth in any one of SEQ ID NOs: 8, 18, 20, 22, 62, and 64.

[0101] In some embodiments, the complement control domain comprises an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in any one of SEQ ID NOs: 8, 18, 20, 22, 62, and 64, e.g., For example, it may comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to any one of the amino acid sequences.

[0102] In the present application, the fusion protein may comprise (iii) an enhancement domain.

[0103] In some embodiments, the enhancement domain may comprise an IgG Fc domain or a functional fragment thereof.

[0104] In some embodiments, the IgG Fc domain may be an IgG Fc4 domain; The IgG Fc4 domain may comprise the amino acid sequence shown in SEQ ID NO:10.

[0105] In some embodiments, the enhancement domain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 10, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0106] In some embodiments, the IgG Fc domain may be an IgG Fc1 domain; The IgG Fc1 domain may include a variant thereof. For example, the IgG Fc domain may include: The IgG Fc1 may be mutated to obtain a desired spatial structure, for example, to form a knob-hole structure to improve dimer formation. For example, the IgG Fc1 may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 30, 32, and 34.

[0107] In some embodiments, the enhancement domain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in any one of SEQ ID NOs: 30, 32, and 34, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to any one of SEQ ID NOs: 30, 32, and 34.

[0108] In the present application, the enhancement domain may comprise human serum albumin or a functional fragment thereof, and the human serum albumin may comprise the amino acid sequence shown in SEQ ID NO:12.

[0109] In some embodiments, the enhancement domain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 12, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0110] In the present application, the fusion protein comprises (i) a CRIg extracellular domain, (ii) a complement regulatory domain, and and (iii) an enhancement domain. In the fusion protein, the C-terminus of the CRIg extracellular domain may be directly or indirectly linked to the N-terminus of the complement regulatory domain, or Alternatively, the C-terminus of the complement control domain may be linked directly or indirectly to the N-terminus of the potentiation domain. For example, the fusion protein may comprise, from N-terminus to C-terminus, the CRIg extracellular domain, the complement control domain, and the potentiation domain, in that order. For example, the fusion protein may comprise, from N-terminus to C-terminus, the complement control domain, the CRIg extracellular domain, and the potentiation domain, in that order.

[0111] In the present application, the indirect linkage may include a linkage via a linker. For example, the linker may include a connecting peptide.

[0112] In some embodiments, the C-terminus of the CRIg extracellular domain is connected to the The linker may be linked to the N-terminus of the complement regulatory domain, and the linker may be selected from SEQ ID NOs: 44, 46, 48 and and 50.

[0113] In some embodiments, the nucleotide sequence encoding the linker may be as set forth in any one of SEQ ID NOs: 43, 45, 47, and 49. For example, the linker may be (Gly4Ser)3.

[0114] In some embodiments, the C-terminus of the complement inhibitory domain is connected to the augmented domain by a linker. The linker may be any of SEQ ID NOs: 44, 46, 48 and 50. It may comprise any one of the amino acid sequences set forth in the above.

[0115] In some embodiments, the nucleotide sequence encoding the linker may be as set forth in any one of SEQ ID NOs: 43, 45, 47, and 49. For example, the linker may be (Gly4Ser)3.

[0116] In the present application, the fusion protein may comprise, from the N-terminus to the C-terminus, the CRIg extracellular domain, the linker, the complement control domain, and the potentiation domain, in that order.

[0117] In the present application, the fusion protein may comprise, from the N-terminus to the C-terminus, the complement regulatory domain, the linker, the CRIg extracellular domain, and the potentiation domain, in that order.

[0118] In the present application, the fusion protein may comprise, from the N-terminus to the C-terminus, the CRIg extracellular domain, the FH, and the IgG Fc domain (e.g., IgG1 Fc or IgG4 Fc). For example, the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO: 14 or 40.

[0119] In the present application, the fusion protein may comprise, from the N-terminus to the C-terminus, the CRIg extracellular domain, the linker, the FH, and the IgG Fc domain (e.g., IgG1 Fc or IgG4 Fc). For example, the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO: 60.

[0120] In the present application, the fusion protein may comprise, from the N-terminus to the C-terminus, the CRIg extracellular domain and the IgG Fc domain (e.g., IgG1 Fc or IgG4 Fc). For example, the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:58.

[0121] In the present application, the fusion protein may comprise, from the N-terminus to the C-terminus, the CRIg extracellular domain, the FH, and the HSA. For example, the fusion protein may comprise a CRIg extracellular domain, a FH, and a HSA, as set forth in SEQ ID NO: 16. It may comprise the amino acid sequence shown.

[0122] In the present application, the fusion protein may comprise, from the N-terminus to the C-terminus, the CRIg extracellular domain, the CD59, and the IgG Fc domain (e.g., IgG1 Fc or IgG4 Fc). For example, the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO: 28 or 42.

[0123] In the present application, the fusion protein is composed of the CRIg extracellular domain in order from the N-terminus to the C-terminus. The fusion protein may comprise the amino acid sequence set forth in SEQ ID NO: 24 or 38.

[0124] In the present application, the fusion protein may comprise, from the N-terminus to the C-terminus, the CRIg extracellular domain, the CR1, and the IgG Fc domain (e.g., IgG1 Fc or IgG4 Fc). For example, the fusion protein may comprise the amino acid sequence shown in SEQ ID NO: 66 or 68.

[0125] In the present application, the fusion protein may be a single-chain structure.

[0126] In the present application, the fusion protein may comprise, from the N-terminus to the C-terminus, the CRIg extracellular domain, the FH SCR1-5, and the human serum albumin. The fusion protein may comprise the amino acid sequence set forth in SEQ ID NO: 16. For example, the fusion protein may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in SEQ ID NO: 16, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous.

[0127] In the present application, the fusion protein can be a first polypeptide chain or a second polypeptide chain.

[0128] In some embodiments, the first polypeptide chain comprises the CRIg extracellular domain, the In some embodiments, the first complement regulatory domain comprises factor H (FH), CD55, CD46, CD59, and CR1. The protein may comprise a protein or functional fragment thereof selected from the group:

[0129] In some embodiments, the first complement regulatory domain may comprise a protein or functional fragment thereof selected from the group consisting of factor H (FH), CD55, CD46, CD59, and CR1.

[0130] In some embodiments, the first IgG Fc domain is selected from the group consisting of IgG1 Fc and IgG4 Fc. The protein may comprise a protein or a functional fragment thereof selected from:

[0131] In some embodiments, the second polypeptide chain comprises the CRIg extracellular domain, the It may comprise a second complement control domain, and said second IgG Fc domain.

[0132] In some embodiments, the second complement regulatory domain may comprise a protein or functional fragment thereof selected from the group consisting of factor H (FH), CD55, CD46, CD59, and CR1.

[0133] In some embodiments, the second complement regulatory domain may comprise a protein or functional fragment thereof selected from the group consisting of factor H (FH), CD55, CD46, CD59, and CR1.

[0134] In some embodiments, the second IgG Fc domain is selected from the group consisting of IgG1 Fc and IgG4 Fc. The protein may comprise a protein or a functional fragment thereof selected from:

[0135] In some embodiments, the first polypeptide chain can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 14, 24, 26, 28, 38, 40, 42, 58, 60, 66, and 68. For example, the first polypeptide chain may comprise an amino acid sequence that is at least 90% homologous to any one of the amino acid sequences set forth in SEQ ID NOs: 14, 24, 26, 28, 38, 40, 42, 58, 60, 66, and 68. For example, it may comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the amino acid sequence.

[0136] In some embodiments, the first polypeptide chain can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 14, 24, 28, 38, 40, 42, 58, 60, 66, and 68. For example, the first polypeptide chain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in any one of SEQ ID NOs: 14, 24, 28, 38, 40, 42, 58, 60, 66 and 68, e.g., an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to any one of SEQ ID NOs: 14, 24, 28, 38, 40, 42, 58, 60, 66 and 68.

[0137] In some embodiments, the second polypeptide chain can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 14, 24, 26, 28, 38, 40, 42, 58, 60, 66, and 68. For example, the second polypeptide chain may comprise an amino acid sequence that is at least 90% homologous to any one of the amino acid sequences set forth in SEQ ID NOs: 14, 24, 26, 28, 38, 40, 42, 58, 60, 66, and 68. For example, it may comprise an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to the amino acid sequence.

[0138] In some embodiments, the second polypeptide chain can comprise the amino acid sequence set forth in any one of SEQ ID NOs: 14, 24, 28, 38, 40, 42, 58, 60, 66, and 68. For example, the second polypeptide chain may be any of SEQ ID NOs: 14, 24, 28, 38, 40, 42, 58, 60, and 68. It may comprise an amino acid sequence that is at least 90% homologous to any one of the amino acid sequences set forth in any one of the above, for example, an amino acid sequence that is at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to any one of the above.

[0139] In the present application, the fusion protein may comprise a first polypeptide chain and a second polypeptide chain, wherein the first IgG Fc domain of the first polypeptide chain and the second polypeptide chain can interact with the second IgG Fc domain to form a dimer.

[0140] In the present application, the first complement control domain of the first polypeptide chain of the fusion protein may be the same as the second complement control domain of the second polypeptide chain. For example, the first complement control domain and the second complement control domain may comprise a protein or functional fragment thereof selected from the group consisting of factor H (FH), CD46, CD55, CD59, and CR1. For example, the first complement control domain and the second complement control domain may comprise a protein or functional fragment thereof selected from the group consisting of factor H (FH), CD55, and CD59. The protein may comprise a protein or a functional fragment thereof selected from the group consisting of:

[0141] In some embodiments, the first IgG Fc domain of the first polypeptide chain of the fusion protein may be the same as the second IgG Fc domain of the second polypeptide chain, for example, the first IgG Fc domain and / or the second IgG Fc domain may be the same as any one of SEQ ID NOs: 10 and 30. The amino acid sequence may include the amino acid sequence shown in the paragraph.

[0142] For example, the first IgG Fc domain and / or the second IgG Fc domain may comprise an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in any one of SEQ ID NOs: 10 and 30, e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous to any one of SEQ ID NOs: 10 and 30. For example, the fusion protein may comprise a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain The first polypeptide chain and the second polypeptide chain may be identical. For example, the first polypeptide chain and / or the second polypeptide chain may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 14, 24, 26, 28, 58, and 60. For example, the first polypeptide chain and / or the second polypeptide chain may comprise the amino acid sequence set forth in any one of SEQ ID NOs: 14, 24, 28, and 60. For example, the first polypeptide chain and / or the second polypeptide chain may comprise is an amino acid sequence that is at least 90% homologous to the amino acid sequence set forth in any one of SEQ ID NOs: 14, 24, 26, 28, 58, and 60, for example, at least 95%, at least 96%, or at least The amino acid sequence may be at least 97%, at least 98%, or at least 99% homologous to the amino acid sequence of the target gene.

[0143] In the present application, the first complement control domain of the first polypeptide chain of the fusion protein may be different from the second complement control domain of the second polypeptide chain. For example, the first complement control domain and the second complement control domain each independently comprise a protein or functional fragment thereof selected from the group consisting of CD59 and CD55. For example, the first complement control domain may be different from the second complement control domain of the second polypeptide chain. each of said domain and said second complement regulatory domain independently selected from the group consisting of FH and CD55 The selected protein or functional fragment thereof includes, for example, the first complement regulatory domain and and the second complement regulatory domain is independently selected from the group consisting of CD46 and CD59. This includes proteins or functional fragments thereof.

[0144] In some embodiments, the first IgG Fc domain of the first polypeptide chain of the fusion protein may be the same as the second IgG Fc domain of the second polypeptide chain.

[0145] For example, the first IgG Fc domain and / or the second IgG Fc domain may comprise the amino acid sequence shown in any one of SEQ ID NOs: 10 and 30. In some embodiments, the first IgG Fc domain of the first polypeptide chain and the second IgG Fc domain of the second polypeptide chain of the fusion protein may be different.

[0146] For example, the first IgG Fc domain may comprise the amino acid sequence shown in SEQ ID NO: 32, The second IgG Fc domain may comprise the amino acid sequence shown in SEQ ID NO:34.

[0147] For example, the first polypeptide chain or the second polypeptide chain of the fusion protein comprises, from the N-terminus to the C-terminus, the CRIg extracellular domain, CD46, and IgG1 Fc, in that order. That's fine.

[0148] For example, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 36. Good too.

[0149] For example, the first polypeptide chain or the second polypeptide chain of the fusion protein comprises, from the N-terminus to the C-terminus, the CRIg extracellular domain, CD55, and IgG1 Fc, in that order. That's fine.

[0150] For example, the first polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 38. Good too.

[0151] For example, the second polypeptide chain of the fusion protein or the second polypeptide chain comprises, from the N-terminus to the C-terminus, the CRIg extracellular domain, FH, and IgG1 Fc, in this order. Good too.

[0152] For example, the second polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO: 40. Good too.

[0153] For example, the second polypeptide chain of the fusion protein or the second polypeptide chain comprises, from the N-terminus to the C-terminus, the CRIg extracellular domain, CD59, and IgG1 Fc, in that order. The second polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:42.

[0154] For example, the first polypeptide chain may be any one of SEQ ID NOs: 36, 38, 40, and 42. It may also contain an amino acid sequence.

[0155] For example, the first polypeptide chain may be an amino acid sequence set forth in any one of SEQ ID NOs: 38, 40, and 42. It may also contain an amino acid sequence.

[0156] For example, the second polypeptide chain may be any one of SEQ ID NOs: 36, 38, 40, and 42. It may also contain an amino acid sequence.

[0157] For example, the first polypeptide chain may be an amino acid sequence set forth in any one of SEQ ID NOs: 38, 40, and 42. It may also contain an amino acid sequence.

[0158] For example, the first polypeptide chain of the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:38. and said second polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:42. It's okay to do that.

[0159] For example, the first polypeptide chain of the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:38. and said second polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:40. It's okay to do that.

[0160] For example, the first polypeptide chain of the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:38. and said second polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:42. It's okay to do that.

[0161] For example, the first polypeptide chain of the fusion protein may comprise the amino acid sequence set forth in SEQ ID NO:36. and said second polypeptide chain may comprise the amino acid sequence set forth in SEQ ID NO:42. It's okay to do that.

[0162] In the present application, the first polypeptide chain, the second polypeptide chain, the CRIg extracellular domain, the complement regulatory domain (e.g., factor H (FH), CD46, CD55, and CD59), the enhancer domain, The host (for example, the IgG Fc domain and / or human serum albumin and / or the fusion protein) may not only contain the above-mentioned respective amino acid sequences, but may also contain variants of the respective amino acid sequences.

[0163] In the present application, a variant of the amino acid sequence is defined as having 1) a sequence that is at least 90% identical to the corresponding amino acid sequence; an amino acid sequence having sequence identity (e.g., at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%); and / or 2) It may include amino acid sequences obtained by substituting, deleting or adding one or more amino acids (e.g., 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-11, 1-12 or more) in the corresponding amino acid sequence.

[0164] As used herein, the term "homology" generally refers to sequence similarity or interchangeability between two or more polynucleotide sequences or two or more polypeptide sequences. When using a computer program or software (e.g., Emboss Needle or BestFit) to determine sequence identity, similarity, or homology between different amino acid sequences, default parameter settings can be used. To optimize the identity, similarity, or homology score, an appropriate scoring matrix, such as blosum45 or blosum80, can be selected. In some embodiments, a homologous polynucleotide can hybridize to a reference polynucleotide sequence under stringent conditions and achieve at least 60%, at least 65%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% sequence similarity compared to the reference polynucleotide sequence. A homologous polypeptide may be a polypeptide that has at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or at least 100% sequence identity to a reference polypeptide sequence when sequence comparison is performed under optimized conditions.

[0165] To determine sequence identity, sequence comparison can be performed using various means known to those skilled in the art, such as BLAST, BLAST-2, ALIGN, NEEDLE, or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters to use for comparison, including algorithms necessary to achieve optimal comparison across the full length sequences to be compared.

[0166] In the present application, the amino acid substitution may be a conservative amino acid substitution or a non-conservative amino acid substitution. The first polypeptide chain, the second polypeptide chain, the CRIg extracellular domain, and the complement regulatory domain (e.g., the factor H (FH), CD55, and CD59) after the substitution are and / or the enhancement domain (e.g., the IgG Fc domain and / or human serum albumin) is still the same as the first polypeptide chain, the second polypeptide chain, the CRIg extracellular domain, the complement control domain (e.g., the factor H (FH), CD55, and CD59) and / or the enhancement domain (e.g., the IgG Fc domain and / or human serum albumin) before substitution. or has a similar functional activity.

[0167] For example, the amino acid substitution may be a non-conservative substitution, which may involve non-conservatively changing an amino acid residue in a target protein or polypeptide, for example, changing an amino acid residue having a certain side chain size or certain properties (e.g., hydrophilicity) to an amino acid residue having a different side chain size or different properties (e.g., hydrophobicity).

[0168] Alternatively, the amino acid substitution may be a conservative substitution. The non-conservative substitution may involve conservatively changing an amino acid residue in a target protein or polypeptide, for example, changing an amino acid residue having a certain side chain size or certain properties (e.g., hydrophilicity) to an amino acid residue having the same or similar side chain size or the same or similar properties (e.g., still hydrophilicity). Such conservative substitutions usually do not significantly affect the structure or function of the resulting protein. In the present application, the first polypeptide chain, the second polypeptide chain, the CRIg extracellular domain, the complement control domain (e.g., factor H (FH), CD46, CD55, CD59, and CR1), and / or the enhancement domain (e.g., amino acid sequence variants of an IgG Fc domain and / or human serum albumin) are selected from the group consisting of amino acid sequences that are consistent with the structure or function of a protein. Conservative amino acid substitutions that do not significantly alter the activity of the polypeptide may be included.

[0169] By way of example, substitutions between amino acids within each of the following groups for one another can be considered conservative substitutions herein: groups of amino acids having non-polar side chains: alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, and methionine.

[0170] A group of amino acids with uncharged polar side chains: glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine.

[0171] A group of amino acids with a negative charge in the polar side chain: aspartic acid, glutamic acid. Positively charged basic amino acids: lysine, arginine, histidine. Amino acids with phenyl groups: phenylalanine, tryptophan, and tyrosine.

[0172] Nucleic acid molecules, vectors and cells On the other hand, the present application also provides one or more isolated nucleic acid molecules. The nucleic acid molecules may encode the fusion proteins described herein or fragments thereof. For example, each of the one or more nucleic acid molecules may encode the entire fusion protein (e.g., For example, if the fusion protein is a single chain), or a portion of the fusion protein, such as the first polypeptide chain, the second polypeptide chain, the CRIg extracellular domain, the complement regulatory domain (e.g., the Factor H (FH), CD46 CD55, CD59, and CR1), and / or the enhancement domain (e.g., the IgG Fc domain), and / or human serum albumin, or one or more of these.

[0173] In the present application, the nucleic acid molecule may comprise the base sequence set forth in any one of SEQ ID NOs: 13, 15, 23, 25, 27, 35, 37, 39, 41, 57, 59, 65 and 67.

[0174] The nucleic acid molecules described herein may be isolated, for example, (i) in vitro, e.g. The isolated nucleic acid may be produced or synthesized by, for example, polymerase chain reaction (PCR) amplification, (ii) by clonal recombination, (iii) by purification, for example, enzymatic digestion and stepwise separation by gel electrophoresis, or (iv) synthetically, for example, chemically. In some embodiments, the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA techniques.

[0175] In this application, nucleic acids encoding fusion proteins or fragments thereof can be synthesized by any method known in the art, including, but not limited to, restriction fragment manipulation or overlap extension PCR using synthetic oligonucleotides. For details, see Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al., Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York, NY, 1993.

[0176] On the other hand, the present application provides one or more vectors comprising one or more of the nucleic acid molecules described herein. Each vector may comprise one or more of the nucleic acid molecules. The vector may contain other genes, such as a marker gene, that allows the vector to be selected under appropriate conditions in a suitable host cell. The vector may also contain expression control elements that enable the coding region to be properly expressed in a suitable host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation. In some embodiments, the expression control sequence is a regulatable element. The specific structure of the expression control sequence may vary depending on the species or cell type, but typically includes 5' non-transcribed sequences involved in transcription and translation initiation, such as TATA, capping sequences, CAAT sequences, and 5' and 3' non-translated sequences. For example, the 5' non-transcribed expression control sequence may include a promoter region, The region may include a promoter sequence operably linked to the nucleic acid for transcriptional control. The expression control sequence may also include an enhancer sequence or an upstream activator sequence. Suitable promoters herein include, for example, promoters for SP6, T3, and T7 polymerases. The present application provides a host cell that can express one or more nucleic acid molecules described herein. The vectors can be, for example, a plasmid, a slime mold, a virus, a phage, or other vectors commonly used in genetic engineering. For example, the vector can be an expression vector. On the other hand, the present application provides a host cell that can express one or more nucleic acid molecules described herein. and / or one or more of the vectors described herein. Each cell may contain one or more nucleic acid molecules or vectors described herein. In some embodiments, each cell contains multiple (e.g., two or more) or multiple types of the vectors described herein. The nucleic acid molecule or vector may comprise a plurality of (e.g., two or more) nucleic acid molecules or vectors. The vectors described herein may be introduced into the host cells, e.g., eukaryotic cells such as plant, fungal or yeast cells. The carriers described herein may be introduced into the host cells by electroporation, transfection with Lipofectamine, or other suitable methods. The vector can be introduced into the host cell by methods known in the art, such as transfection.

[0177] Pharmaceutical Composition The present application provides a pharmaceutical composition comprising the fusion protein and, optionally, a pharmaceutically acceptable vector. A pharmaceutically acceptable vector generally refers to a vector that can be used to prepare a pharmaceutical composition or formulation and is generally safe, non-toxic, and not biologically or otherwise undesirable. The vector used is generally suitable for administration to mammals, such as humans. In preparing the composition, the active ingredient is generally mixed with the vector and diluted or encapsulated by the vector. When a carrier is used as a diluent, it may be solid, semi-solid, or liquid, as long as it functions as a vehicle, vector, or medium for the antibody active ingredient. The pharmaceutically acceptable vector may also contain a buffer, antioxidant, preservative, small peptide, protein, hydrophilic polymer, amino acid, sugar, chelating agent, counterion, metal complex, and / or non-ionic surfactant.

[0178] In this application, the pharmaceutical composition is formulated for oral administration, intravenous administration, intramuscular administration, in situ administration at a tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration via a subcutaneous reservoir. Solutions or suspensions for transdermal administration or administration via a subcutaneous reservoir may contain the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol and methylparabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and tonicity adjusters such as sodium chloride and glucose. pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide.

[0179] Methods and Uses Meanwhile, the present application provides a method for preparing the fusion protein or a fragment thereof. The method may include a step of synthesizing the fusion protein or a fragment thereof and / or a step of culturing the cells under conditions for expressing the fusion protein or a fragment thereof. For example, the method can be performed using a method known to those skilled in the art, such as using an appropriate medium, an appropriate temperature, and an appropriate incubation time.

[0180] The present application provides a method for producing a fusion protein or a fragment thereof described herein using genetic engineering techniques, such as the first polypeptide chain, the second polypeptide chain, the CRIg extracellular domain, the complement control domain (e.g., factor H (FH), CD46, CD55, CD59, and CR1), and / or the enhancement domain (e.g., the IgG Fc domain and / or human serum albumin), or the amino acid residues may be linked in order according to the protein sequence.

[0181] The present application also provides a method for using genetic engineering techniques to generate the coding sequence of the fusion protein or fragment thereof described herein, for example, the coding sequence of the first polypeptide chain, the second polypeptide chain, the CRIg extracellular domain, the complement control domain (e.g., the factor H (FH), CD46, CD55, CD59, and CR1), and / or the enhancement domain (e.g., the IgG Fc domain and / or human serum albumin); or the bases may be linked in order according to a nucleic acid sequence.

[0182] Meanwhile, the present application provides use of the fusion protein or the pharmaceutical composition in the preparation of a medicament for treating a disease associated with targeted inhibition of complement activation, which may be selected from the group consisting of paroxysmal nocturnal hemoglobinuria (PNH), hemoglobinuria (PNH), atypical hemolytic uremic syndrome (aHUS), Generalized myasthenia gravis (gMG), neuromyelitis optica spectrum disorder (NMOSSD), age-related macular degeneration (AMD), autolytic hemolytic anemia, autoimmune thrombocytopenia, aplastic anemia, systemic lupus erythematosus, ankylosing rheumatoid arthritis, ankylosing spondylitis, arteriosclerosis, Parkinson's disease , Alzheimer's disease (dementia), asthma, allergies, psoriasis, multiple sclerosis, Crohn's disease. The agents described herein can inhibit complement activation and / or protect cells from complement attack. In some embodiments, the disease can include an autoimmune disease. For example, the disease can include autoimmune myasthenia gravis.

[0183] The following examples are not intended to be limited by theory and are only used to explain the fusion protein, preparation method, use, etc. of the present application, and are not used to limit the scope of the present application. ns: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Example]

[0184] Example 1: Prolongation of the blood half-life of a complement inhibitor by addition of an enhancement domain fragment Objective: To develop two recombinant proteins by introducing human IgG Fc or HSA into the CRIg-FH complement inhibitor. Expression vectors for protein (CRIg-FH-IgG4 Fc) × 2 and His × 6-CRIg-FH-HSA were constructed and used for the eukaryotic expression of the protein. To screen and identify proteins that can extend the half-life of CRIg-FH without affecting its complement inhibitory effect through expression and purification, and measurement of complement inhibitory activity and in vivo half-life.

[0185] 1. Equipment and materials: Electric thermostat (DK-8D, Shanghai Jinghong Laboratory Equipment Co., Ltd.), PCR machine (Mastercycler pro-Eppendorf, Eppendorf, Germany), RNA / DNA concentration / purity meter (NANODROP 2000c, Thermo Scientific, USA), gel imager (Tanon 1600, Shanghai Tanen Technology Co., Ltd.) , CO2 incubator (240i, Thermo Scientific, USA), BioRAD Mini protein Tera system (BioRAD, USA), low-temperature horizontal centrifuge (Allegra X-15R Centrifuge, Beckman Coulter, USA).

[0186] 2. Method: 2.1 Gene cloning and vector construction Total RNA was extracted from the human hepatocyte cell line Hep3B using NucleoZOL (Macherey-Nagel, Germany). The RNA was extracted from the sarcoma and reverse transcribed into cDNA using reverse transcriptase (PrimeScript™ RT Master Mix, Takara, Japan). The cDNA was amplified by PCR using primers appropriate for the SCR1-5 domain (E19-K323) encoding the functional fragment of FH complement inhibitory activity and the human serum albumin (HSA) gene sequence. Total RNA was also extracted from lymphoma U937 cells in the same manner and reverse transcribed into cDNA. Then, the gene sequence encoding the extracellular structural domain (G19-K137) of the CRIg gene was amplified by PCR. did.

[0187] The nucleic acid sequence encoding the FH protein is shown in SEQ ID NO: 3, and the amino acid sequence of the FH protein is shown in SEQ ID NO: 4. In column number 4, the nucleic acid sequence encoding the SCR1-5 domain of FH is shown as SEQ ID NO: 7, and the amino acid sequence of the SCR1-5 domain of FH is shown as SEQ ID NO: 8.

[0188] The nucleic acid sequence encoding the HSA protein is shown in SEQ ID NO: 11, and the amino acid sequence of the HSA protein is shown in SEQ ID NO: 12.

[0189] The nucleic acid sequence encoding the CRIg protein is shown in SEQ ID NO: 1, and the amino acid sequence of the CRIg protein is shown in SEQ ID NO: 2. are shown in SEQ ID NO: 2. The nucleic acid sequence encoding the CRIg extracellular domain is shown in SEQ ID NO: 5, and the amino acid sequence of the CRIg extracellular domain is shown in SEQ ID NO: 6.

[0190] Primers containing both the CRIg extracellular domain and the FH SCR1-5 domain were designed and used. Overlapping PCR was used to insert the CRIg extracellular domain gene and the FH SCR1-5 gene into the pFUSE-hIgG4-Fc1 eukaryotic expression vector (InvivoGen). The nucleic acid sequence encoding hIgG4-Fc is shown in SEQ ID NO: 9, and the amino acid sequence of the hIgG4-Fc protein is shown in SEQ ID NO: are shown in SEQ ID NO: 10. Furthermore, the CRIg extracellular domain gene was cloned into FH SCR1-5 The resulting fragment was ligated to the HSA gene and inserted into the pcDNA3.1 / His A (Invitrogen) expression vector by overlap PCR. The vector was then checked for correct insertion sequence before further testing. The nucleotide sequences of both sides were determined to confirm the identity of the fragments, and the fragments were named pFUSE-CRIg-FH-hIgG4Fc1 and pCDNA / His-CRIg-FH-HSA, respectively.

[0191] 2.2 Protein expression 2.0 × 10 293FT cells were placed in a 15 cm diameter dish. 7 The cells were spread uniformly at a density of 1 cell / dish, grown to the logarithmic phase, and then transfected with Lipofectamine 2000 (Invitrogen, USA) reagent as described above. The constructed PFUSE-CRIg-FH-hIgG4-Fc1 or pCDNA / His-CRIg-FH-HSA extracted plasmid was transfected. After 6 hours of incubation at 37°C in a 5% CO2 incubator, 293 protein expression was observed. The medium was replaced with serum-free medium (Gibco, USA) and cultured for 3 days, after which the supernatant was collected, centrifuged to remove cells and cell debris, concentrated using an ultrafiltration tube, and then purified.

[0192] 2.3 Protein affinity purification, buffer exchange, and concentration Protein A antibody purification magnetic beads kit (Beaver Nano, Suzhou, China) was used to purify the cell supernatant. The (CRIg-FH-IgG4Fc) × 2 recombinant protein was purified and dissolved in elution buffer and subjected to ultrafiltration. The solution was transferred to a tube (Millipore, USA), and then centrifugation was performed at low temperature to replace the solution with PBS. Alternatively, the Hisx6-CRIg-FH-HSA fusion protein expressed according to the manufacturing method was purified using a His-Bind affinity Purification Kit (Novagen / Merck Millipore), and the buffer was replaced with PBS in the same manner. The resulting recombinant proteins were designated (CRIg-FH-IgG4Fc) × 2 and CRIg-FH-HSA, respectively (Figure 1).

[0193] 2.4 Measurement of complement inhibitory activity The commercially available kits WIESLAB™ Complement System Classical Pathway (COMPLCP310) and WIESLAB™ Complement Alternative Pathway (COMPLAP330) were used, respectively. The inhibition of the classical and alternative complement pathways by the two recombinant proteins was investigated. Detected.

[0194] 2.5 Measurement of half-life of recombinant proteins in vivo SD rats (half male and half female) were intravenously injected with (CRIg-FH-IgG4Fc) × 2. Before administration (first day), At different times, 5 minutes after the start of administration (first day) (i.e., 2 minutes after the end of administration), 30 minutes, 4 hours, 24 hours, and on the 2nd, 3rd, 5th, 7th, 10th, 14th, 21st, and 28th days, respectively. Blood samples were collected and serum was separated. The recombinant protein concentration in each sample was determined by ELISA. Or CRIg-FH-HSA, 1 mg / kg, was intravenously injected, and the results were analyzed before administration, 5 minutes, 30 minutes after administration, and 1 Serum samples were collected at 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, and on days 2, 3, 5, 7, 10, 14, 21, and 28. CRIg-FH (20 mg / kg) was administered intravenously. (The preparation method is as disclosed by the inventors, Qiao Q., et al., A novel CRIg-targeted complement inhibitor protects cells from complement damage, FASEB J.2014 Nov;28 (11) :4986-99.) Before administration, 5 minutes, 30 minutes, 1 hour, 3 hours, 6 hours, 12 hours, and 18 hours after administration. Serum was isolated after 24, 36, 48, 72, and 120 hours.

[0195] ELISA was performed using the following procedure: Encapsulated rabbit anti-human CRIg monoclonal antibody The antibody (clone 202, sino biological, catalog 12163-H08H) is a standard product that binds to recombinant proteins or recombinant proteins in serum, and is a mouse anti-human IgG 4 fragment secondary antibody. ry (5 c 7) [HRP] (NOVUS, Cat. NB 110-7081 H), enzyme-conjugated secondary antibody (CRIg-FH-IgG 4 Fc) × 2 was used to detect CRIg-FH-HSA with human serum albumin polyclonal antibody, HRP (ThermoFisher, Cat. #PA 1-72058), enzyme-conjugated secondary antibody, or HRP-targeted CRIg-FH-HSA. CRIg-FH was detected using the rabbit anti-human FH antibody EPR 6225 (catalog #ab133536) that was specifically labeled with CRIg-FH. The blood concentration of the recombinant protein in serum was calculated from a standard curve, and a time-drug serum concentration curve was plotted. The data were plotted and the half-life was calculated.

[0196] 3.Results: 3.1 The two recombinant proteins obtained by the above-mentioned eukaryotic expression and purification, (CRIg-FH-IgG4Fc)×2 and His×6-CRIg-FH-HSA, were detected with a purity of more than 95% by electrophoresis (PAGE). The theoretical molecular weights were 146 kDa and 114 kDa, respectively, and the actual sizes were consistent with the theoretical values ​​(Figure 2). The nucleic acid sequence of the protein encoding CRIg-FH-IgG4 Fc is shown in SEQ ID NO: 13, and the amino acid sequence of the CRIg-FH-IgG4 Fc protein is shown in SEQ ID NO: 14. The nucleic acid sequence of the protein is shown in SEQ ID NO: 15, and the amino acid sequence of the CRIg-FH-HSA protein is shown in SEQ ID NO: The nucleic acid sequence encoding CRIg-FH is shown in SEQ ID NO: 51, and the amino acid sequence of the CRIg-FH protein is shown in SEQ ID NO: 52, respectively.

[0197] 3.1 (CRIg-FH-IgG4 Fc)×2 and CRIg-FH-HSA inhibit the classical and alternative pathways of human serum complement The inhibitory activity of both (CRIg-FH-IgG4 Fc) × 2 and CRIg-FH-HSA was investigated. The IC50 of the former to inhibit the classical complement pathway was 91.38 nM (Figure 3), The IC50 for inhibiting the alternative complement pathway was 1.04 nM (Fig. 4), the IC50 for inhibiting the classical complement pathway was 1355 nM (Fig. 5), and the IC50 for inhibiting the alternative complement pathway was 10.39 nM (Fig. 6).

[0198] 3.2 (CRIg-FH-IgG4 Fc) × 2 and CRIg-FH-HSA The concentrations of (CRIg-FH-IgG4 Fc) × 2, CRIg-FH-HSA, or CRIg-FH in serum collected at different time points were measured, and the time-concentration pharmacokinetic curves were plotted. The curve for (CRIg-FH-IgG4 Fc) × 2 was The curve for CRIg-FH-HSA is shown in Figure 7, with a half-life of 142.2 hours; the curve for CRIg-FH-HSA is shown in Figure 8, with a half-life of 32.8 hours; and the curve for CRIg-FH is shown in Figure 28, with a half-life of 0.56 hours. The results showed that adding an enhancement domain (e.g., antibody Fc fragment or single-chain HSA) enhanced the CRIg-FH It was shown that the half-lives of the compounds were significantly extended by more than 250 and 58 times, respectively.

[0199] Example 2: Excellent complement inhibitory effect by linking CRIg with a complement regulatory domain Objective: To construct expression vectors for nine recombinant proteins, (CRIg-FH-IgG4Fc) x 2, (CRIg-CD55-IgG4Fc) x 2, (CRIg-CD46-IgG4Fc) x 2, (CRIg-CD59-IgG4Fc) x 2, (CRIg-CR1-IgG4Fc) x 2, (CRIg-IgG4Fc) x 2, (FH-IgG4Fc) x 2, (FH-CRIg-IgG4Fc) x 2, and (CRIg-L-FH-IgG4Fc) x 2, and to elucidate which of the five important complement inhibitory proteins, FH, CD55, CD46, CD59, and CR1, exhibits superior complement inhibitory effects when linked to CRIg, and to determine the effects of linking complement activation regulatory and enhancing domains on complement inhibitory activity.

[0200] 1. Equipment and materials: Electric thermostat (DK-8D, Shanghai Jinghong Laboratory Equipment Co., Ltd.), PCR machine (Mastercycler pro-Eppendorf, Eppendorf, Germany), RNA / DNA concentration / purity meter (NANODROP 2000c, Thermo Scientific, USA), gel imager (Tanon 1600, Shanghai Tanen Technology Co., Ltd.) , CO2 incubator (240i, Thermo Scientific, USA), BioRAD Mini protein Tera system (BioRAD, USA), low-temperature horizontal centrifuge (Allegra X-15R Centrifuge, Beckman Coulter, USA)

[0201] 2. Method: 2.1 Gene cloning and vector construction Total RNA was extracted from human normal pancreatic duct epithelial cells HPDE6-C7 using NucleoZOL (Macherey-Nagel, Germany) and reverse transcribed into cDNA using reverse transcriptase (PrimeScript™ RT Master Mix, Takara, Japan). The cDNA was then cloned into DNA sequences encoding CD55, CD46, and CD59 ( primers suitable for amplifying the DNA sequence encoding the signal peptide The DNA was amplified by PCR using the same primers. Total RNA was extracted from lymphoma U937 cells using a PCR kit, reverse transcribed into cDNA, and the SCR1-5 domain (E19-K323) encoding the FH complement inhibitory fragment was amplified by PCR. The DNA was then reverse transcribed into cDNA, and the gene sequences encoding the extracellular domain (G19-K137) of the CRIg gene and the CR1 CCP8-11 and CCP15-18 domains were amplified by PCR. The nucleic acid sequence encoding CD55 is shown in SEQ ID NO: 17, and the amino acid sequence of the CD55 protein is shown in SEQ ID NO: 18. The nucleic acid sequence encoding CD46 is shown in SEQ ID NO: 19, and the amino acid sequence of the CD46 protein is shown in SEQ ID NO: 20. The nucleic acid sequence encoding CD59 is shown in SEQ ID NO: 21, and the amino acid sequence of the CD59 protein is shown in SEQ ID NO: 22. The DNA sequences encoding CR1 CCP8-11 and CCP15-18 were amplified by PCR. The nucleic acid sequences of the CR1 CCP8-11 and CCP15-18 domains are shown in SEQ ID NOs: 61 and 63, respectively. The protein sequences are shown in SEQ ID NOs: 62 and 64, respectively.

[0202] Primers containing the coding sequence for the CRIg extracellular domain, the flexible linker peptide (Gly4Ser)3, the CRIg extracellular domain and / or the FH SCR1-5 domain (specifically, It is expressed in four forms: the CRIg extracellular domain alone, FH SCR1-5 alone, and the N-terminal CRIg domain. The extracellular domain was designed with the C-terminus of FH SCR1-5, the N-terminus of FH SCR1-5 and the CRIg extracellular domain, CD55, CD46, CD59, or CR1 (CCP15-18 domain) simultaneously. FH SCR1-5 alone, CRIg extracellular domain gene and FH SCR1-5 with or without a flexible linker peptide, CD46, CD55, CD59, or CR1 CCP15-18 gene The resulting fragments were ligated by overlap PCR and then synthesized into the pFUSE-hIgG4-Fc1 eukaryotic expression vector (InvivoGen ) was inserted into the vector. The vectors were sequenced in both directions to confirm that the correct insert sequence was identified for subsequent experiments, and these vectors were named pFUSE-CRIg-L-FH-IgG4Fc, pFUSE-CRIg-IgG4Fc, pFUSE-FH-IgG4Fc, pFUSE-FH-CRIg-IgG4Fc, pFUSE-CRIg-FH-, pFUSE-FH-IgG4Fc, pFUSE-CRIg-CD55-IgG4Fc, pFUSE-CRIg-CD46-IgG4Fc, pFUSE-CRIg-CD59-IgG4Fc, and pFUSE-CRIg-CR1-IgG4Fc, respectively.

[0203] 2.2 Protein expression 2.0 × 10 293FT cells were placed in a 15 cm diameter dish. 7 The cells were spread uniformly at a density of 1 cell / dish, grown to the logarithmic phase, and then transfected with Lipofectamine 2000 (Invitrogen, USA) reagent as described above. Constructed pFUSE-CRIg-L-FH-IgG4Fc, pFUSE-CRIg-IgG4Fc, pFUSE-FH-IgG4Fc, pFUSE-FH-CRIg-IgG4Fc, pFUSE-CRIg-FH-IgG4Fc, pFUSE-CRIg-CD55-IgG4Fc, pFUSE-CRIg-CD46-IgG4Fc, The cells were transfected with the pFUSE-CRIg-CD59-IgG4Fc or pFUSE-CRIg-CR1-IgG4Fc extracted plasmid. After culturing for 6 hours at 37°C in a 5% CO2 incubator, the cells were incubated in serum-free medium for 293 protein expression. The medium was replaced with new medium (Gibco, USA), and after culturing for 3 days, the supernatant was collected, centrifuged to remove cells and cell debris, and concentrated using an ultrafiltration tube before purification.

[0204] 2.3 Protein affinity purification, buffer exchange, and concentration Protein A antibody purification magnetic beads kit (Beaver Nano, Suzhou, China) was used to purify the cell supernatant. Inside (CRIg-L-FH-IgG4Fc) x 2, (CRIg-IgG4Fc) x 2, (FH-IgG4Fc) x 2, (FH-CRIg-IgG4Fc) x 2 , (CRIg-FH-IgG4Fc) × 2, (CRIg-CD55-IgG4Fc) × 2, (CRIg-CD46-IgG4Fc) × 2, (CRIg-CD59-IgG4Fc) × 2, or (CRIg-CR1-IgG4Fc) × 2, the purified recombinant proteins were dissolved in elution buffer, transferred to ultrafiltration tubes (Millipore, USA), and replaced with PBS by low-temperature centrifugation to concentrate the proteins for storage.

[0205] 2.4 Measurement of complement inhibitory activity The above constructs were analyzed using the commercially available kits WIESLAB™ Complement System Classical Pathway (COMPLCP310) and WIESLAB™ Complement Alternative Pathway (COMPLAP330). The inhibition of the classical and alternative complement pathways by the recombinant proteins was detected, respectively.

[0206] 3.Results: 3.1 Nine recombinant proteins obtained by applying the above-mentioned eukaryotic induction and purification (See Figures 9, 29, and 32 for the structures), (CRIg-IgG4Fc) x 2, (FH-IgG4Fc) x 2, (FH-CRIg-IgG4Fc) x 2, (CRIg-L-FH-IgG4Fc) x 2, (CRIg-FH-IgG4Fc) x 2, (CRIg-CD55-IgG4Fc) x 2, (CRIg-CD46-IgG4Fc) x 2, (CRIg-CD59-IgG4Fc) x 2, and (CRIg-CR1-IgG4Fc) x 2 are , detected by PAGE with a purity of more than 95% and theoretical molecular weights of 77, 119, and 146, respectively. , 146 and 148 kDa (Fig. 30), 146, 153, 157 and 101 kDa (Fig. 10), and 134 kDa (Fig. 33). The size of the particles agreed with the theoretical value.

[0207] The nucleic acid sequence of the protein encoding CRIg-IgG4 Fc is shown in SEQ ID NO: 53, and the amino acid sequence of the CRIg-IgG4 Fc protein is shown in SEQ ID NO: 54. The amino acid sequence of the FH-hIgG4 Fc protein is shown in SEQ ID NO: 55, and the amino acid sequence of the FH-hIgG4 Fc protein is shown in SEQ ID NO: 56. The nucleic acid sequence of the protein encoding FH-CRIg-IgG4 Fc is shown in SEQ ID NO: 57, and the amino acid sequence of the FH-CRIg-IgG4 Fc protein is shown in SEQ ID NO: 58. The nucleic acid sequence of the encoding protein is shown in SEQ ID NO: 59, and the amino acid sequence of the CRIg-L-FH-IgG4 Fc protein is shown in SEQ ID NO: 60. The amino acid sequences are shown in SEQ ID NO: 60. Protein encoding CRIg-CD55-IgG4 Fc The nucleic acid sequence of CRIg-CD55-IgG4 Fc protein is shown in SEQ ID NO: 23, and the amino acid sequence of CRIg-CD55-IgG4 Fc protein is shown in SEQ ID NO: 24. The nucleic acid sequence of the protein encoding CRIg-CD46-IgG4 Fc is shown in SEQ ID NO: 25, and the amino acid sequence of the CRIg-CD46-IgG4 Fc protein is shown in SEQ ID NO: 26. The nucleic acid sequence of the protein encoding CRIg-CD59-IgG4 Fc is shown in SEQ ID NO: 27, and the amino acid sequence of the CRIg-CD59-IgG4 Fc protein is shown in SEQ ID NO: 28. The nucleic acid sequence of the protein encoding CRIg-CR1(CCP15-18)-IgG4 Fc is shown in SEQ ID NO: 67, and the amino acid sequence of the CRIg-CR1(CCP15-18)-IgG4 Fc protein is shown in SEQ ID NO: 68.

[0208] 3.2 The authors investigated the inhibitory activity of five recombinant proteins, (CRIg-IgG4 Fc) × 2, (FH-IgG4 Fc) × 2, (FH-CRIg-IgG4Fc) × 2, (CRIg-FH-IgG4Fc) × 2, and (CRIg-L-FH-IgG4Fc) × 2, against the human serum alternative complement pathway, and five recombinant proteins, (CRIg-FH-IgG4Fc) × 2, (CRIg-CD55-IgG4Fc) × 2, (CRIg-CD46-IgG4Fc) × 2, (CRIg-CD59-IgG4Fc) × 2, and (CRIg-CR1-IgG4Fc) × 2. The inhibitory activity of these proteins against the classical and alternative complement pathways in human serum was measured. The inhibitory effects of five recombinant proteins, (CRIg-IgG4 Fc) × 2, (FH-IgG4 Fc) × 2, (FH-CRIg-IgG4 Fc) × 2, (CRIg-FH-IgG4 Fc) × 2, and (CRIg-L-FH-IgG4 Fc) × 2, on the alternative complement pathway in human serum are shown in Figure 31. The detailed IC50 values ​​were 248.4 nM, 138.8 nM, 1.48 nM, 0.75 nM, and 0.69 nM, respectively. The results showed that (1) the inhibitory effect of CRIg or FH alone on complement was lower than that of the two molecules after they were bound together, and that the inhibitory effect of CRIg on complement could be enhanced when CRIg was bound to the N-terminus or C-terminus of FH. Next, when the two molecules were bound together, CRIg was located at the N-terminus of FH, and the inhibitory effect of complement was enhanced. It was shown that the inhibitory effect on complement is slightly better than when it is located at the C-terminus of FH. Furthermore, it was also found that the inhibitory effect on complement can be enhanced by linking CRIg and FH directly or via a linker such as the flexible peptide (Gly4Ser)3.

[0209] In addition, the inhibitory effects of (CRIg-FH-IgG4Fc) × 2 on the classical and alternative complement pathways were investigated. Figures 3 and 4 show the inhibitory effects of (CRIg-CD55-IgG4Fc) × 2 on the classical and alternative complement pathways. The results are shown in Figures 11 and 12, respectively, and the classical and alternative pathways of complement activation in (CRIg-CD46-IgG4Fc) × 2. The inhibitory effects of (CRIg-CD59-IgG4Fc) × 2 on the classical and alternative pathways of complement are shown in Figures 13 and 14. The inhibitory effect of (CRIg-CR1-IgG4Fc) × 2 on the classical and alternative pathways of complement is shown in Figures 15 and 16, respectively, and the inhibitory effect of (CRIg-CR1-IgG4Fc) × 2 on the classical and alternative pathways of complement is shown in Figures 34 and 35, respectively. The details of the IC50 for inhibiting the classical pathway of complement are shown in Table 2. Specifically, the inhibitory effect of (CRIg-FH-IgG4 Fc) × 2 on the classical pathway of complement was shown in Figure 34. The IC50 values ​​for the classical and alternative pathways were 91.38 nM and 1.04 nM, respectively (there was a discrepancy between the two assays of inhibitory activity against the alternative pathway, i.e., 0.75 nM and 1.04 nM, which may be due to the differences in the test lots); the IC50 values ​​for (CRIg-CD55-IgG4Fc) x 2 for classical and alternative pathway complement inhibition were 23.25 nM and 19.66 nM, respectively; the IC50 value for (CRIg-CD46-IgG4Fc) x 2 for classical pathway complement inhibition was 44.06 nM; the IC50 value for (CRIg-CD59-IgG4Fc) x 2 for classical pathway complement inhibition was 44.84 nM; and the IC50 values ​​for (CRIg-CR1-IgG4Fc) x 2 for classical and alternative pathway complement inhibition were 35.75 nM and 30.26 nM, respectively. All five fusion proteins were able to inhibit both the classical and alternative pathways. Understood.

[0210] [Table 1]

[0211] Example 3 Development of bispecific targeted complement inhibitors Objective: To construct three bispecific targeting recombinant protein mp expression vectors: (CRIg-CD55-IgG1 Fc) (CRIg-FH-IgG1 Fc), (CRIg-CD46-IgG1 Fc) (CRIg-CD59-IgG1 Fc), and (CRIg-CD55-IgG1 Fc) (CRIg-CD59-IgG1 Fc), and to enable the eukaryotic expression and purification of these fusion proteins, as well as the assay of their complement inhibitory activity, to screen for more potent complement inhibitors.

[0212] 1. Equipment and materials: Electric thermostat (DK-8D, Shanghai Jinghong Laboratory Equipment Co., Ltd.), PCR device (Mastercycler pro-Eppendorf, Eppendorf, Germany), RNA / DNA concentration / purity meter (NANODROP 2000c, Thermo Scientific, USA), gel imager (Tanon 1600, Shanghai Tanon Technology Co., Ltd.), CO2 incubator (240i, Thermo Scientific, USA), BioRAD Mini protein Tera system (BioRAD, USA), low-temperature horizontal centrifuge (Allegra X-15R Centrifuge, Beckman Coulter, USA).

[0213] 2. Method: 2.1 Gene cloning and vector construction Mutagenic primers were designed and a point mutagenesis kit (Toyobo, Japan) was used to mutate the primer sites of the pFUSE-hIgG1-Fc1 eukaryotic expression vector (Alegre et al., 1992; Carter, 2001; Merchant et al., 1998; Ridgway et al., 1996; Xu et al., 2000). Two mutant vectors, pFUSE-hIgG1-Fc1 knob mutant and pFUSE-hIgG1-Fc1 hole mutant, were then constructed. Transfected.

[0214] Here, the nucleic acid sequence encoding IgG1 Fc is shown in SEQ ID NO: 29, and the amino acid sequence of the IgG1 Fc protein is shown in SEQ ID NO: 30. The nucleic acid sequence encoding the Knob mutant is shown in SEQ ID NO: 31. The amino acid sequence of the Knob mutant protein is shown in SEQ ID NO: 32. The nucleic acid sequence encoding the Hole mutant is shown in SEQ ID NO: 33, and the amino acid sequence of the Hole mutant protein is shown in SEQ ID NO: 34. Number 34 shows each.

[0215] The CRIg-FH, CRIg-CD46, CRIg-CD55, and CRIg-CD59 gene sequences constructed in Examples 1 and 2 were inserted into the pFUSE-hIgG1-Fc knob and hIgG1-Fc mutant vectors using a One-Step Rapid Cloning Kit (Shanghai Meisheng Biotechnology Co., Ltd.), and the resulting plasmids were designated pFUSE-CRIg-CD46-hIgG1 Fc knob and pFUSE-CRIg-CD55-hIgG1 Fc knob, respectively. Furthermore, the fusion fragments CRIg-FH and CRIg-CD59 were inserted into pFUSE-IgG1-FC hole using the kit, and the resulting plasmids were designated pFUSE-CRIg-FH-IgG1 Fc hole and pFUSE-CRIg-CD59-IgG1 Fc hole, respectively.

[0216] Here, the nucleic acid sequence encoding CRIg-CD46-IgG1 Fc knob is shown in SEQ ID NO: 35, and the amino acid sequence of CRIg-CD46-IgG1 Fc knob protein is shown in SEQ ID NO: 36. CRIg-CD55-IgG1 The nucleic acid sequence encoding the Fc knob is shown in SEQ ID NO: 37, and the amino acid sequence of the CRIg-CD55-IgG1 Fc knob protein is shown in SEQ ID NO: 38. The nucleic acid sequence encoding the CRIg-FH-IgG1 Fc hole is shown in SEQ ID NO: 39, and the amino acid sequence of the CRIg-FH-IgG1 Fc hole protein is shown in SEQ ID NO: 40. The nucleic acid sequence encoding the CRIg-CD59-IgG1 Fc hole is shown in SEQ ID NO: 41, and the amino acid sequence of the CRIg-CD59-IgG1 Fc hole protein is shown in SEQ ID NO: 42.

[0217] 2.2 Protein expression 2.0 × 10 293FT cells were placed in a 15 cm diameter dish. 7 The cells were uniformly spread at a density of 1 cell per dish, grown to the logarithmic phase, and transfected with pFUSE-CRIg-CD46-hIgG1 Fc knob, pFUSE-CRIg-CD59-hIgG1 Fc hole plasmid, pFUSE-CRIg-CD55-hIgG1 Fc knob, pFUSE-CRIg-CD59-hIgG1 Fc hole, pFUSE-CRIg-CD55-hIgG1 Fc knob, and pFUSE-CRIg-FH-hIgG1 Fc hole using Lipofectamine 2000 (Invitrogen, USA). After culturing for 6 hours at 37°C in a 5% CO2 incubator, the medium was replaced with serum-free 293 protein expression medium (Gibco, USA) and incubated for 3 days. After culturing for 1 h, the supernatant was collected, centrifuged to remove cells and cell debris, and concentrated using an ultrafiltration tube before purification.

[0218] 2.3 Protein affinity purification, buffer exchange, and protein concentration Protein A antibody purification magnetic beads kit (Beaver Nano, Suzhou, China) was used to purify the cell supernatant. The purified recombinant proteins (CRIg-CD46-IgG1 Fc), (CRIg-CD59-IgG1 Fc), (CRIg-CD55-IgG1 Fc), (CRIg-CD59-IgG1 Fc), and (CRIg-CD55-IgG1 Fc) (CRIg-FH-IgG1 Fc) were dissolved in elution buffer, transferred to ultrafiltration tubes (Millipore, USA), and centrifugally replaced with PBS at low temperature to concentrate and store the proteins.

[0219] 2.4 Measurement of complement inhibitory activity The inhibitory effects of these two recombinant proteins on the classical and alternative complement pathways were detected using commercially available kits, WIESLAB™ Complement System Classical Pathway (COMPLCP310) and WIESLAB™ Complement Alternative Pathway (COMPLAP330), respectively, and the data were analyzed using Graphpad Prism 7.0 software.

[0220] 3.Results: 3.1 Three recombinant proteins obtained by applying the above-mentioned eukaryotic induction and purification , (CRIg-CD55-IgG1 Fc) (CRIg-FH-IgG1 Fc), (CRIg-CD46-IgG1 Fc) (CRIg-CD59-IgG1 Fc), and (CRIg-CD55-IgG1 Fc) (CRIg-CD59-IgG1 Fc) showed over 95% chromatin resorption by PAGE (Figure 17). The theoretical molecular weights were 151, 130, and 128 kDa, respectively, and the actual sizes were smaller than the theoretical values. This coincided with the results shown in Figure 18.

[0221] 3.2 IC50 inhibition of three bispecific targeting complement inhibitors against the classical and alternative complement pathways The inhibitory activity of CRIg-CD55-IgG1 Fc and CRIg-FH-IgG1 Fc is shown in Table 2. The inhibitory effects of the alternative pathway are shown in Figures 19 and 20. (CRIg-CD46-IgG1 Fc) (CRIg-CD59-Ig The inhibitory effects of (CRIg-CD55-IgG1 Fc) and (CRIg-CD59-IgG1 Fc) on the classical and alternative complement pathways are shown in Figures 21 and 22, respectively. The inhibitory effects of (CRIg-CD55-IgG1 Fc) and (CRIg-CD59-IgG1 Fc) on the classical and alternative complement pathways are shown in Figures 23 and 24, respectively. All three bispecific targeted complement inhibitors inhibited either the classical or alternative complement pathway. The pathway can be inhibited.

[0222] [Table 2]

[0223] Example 4 Therapeutic effect of (CRIg-FH-IgG4Fc) × 2 on a rat myasthenia gravis model Objective: To select the complement inhibitor (CRIg-FH-IgG4Fc) × 2 from the above fusion proteins and verify its efficacy against diseases with hyperactivation (excessive activation) of complement in vivo.

[0224] Autoantibodies against the acetylcholine receptor (AChR) bind to AChR at the neuromuscular junction, either directly reducing AChR expression or indirectly through the classical pathway of complement activation, resulting in impaired neuromuscular conduction and ultimately the clinical symptoms of MG, such as muscle weakness (Gomez et al., 2010; Phillips and Vincent, 2016). The authors investigated the therapeutic effects of (CRIg-FH-IgG4Fc)×2 complement inhibitors in rat models of myasthenia gravis and experimental autoimmune diseases. This was verified in epidemic myasthenia gravis (EAMG), laying the foundation for its potential subsequent clinical application.

[0225] 1. Equipment and materials: Animal scale (Shanghai Precision Instruments Co., Ltd., YP2001N), Lewis rats (Beijing Vital River Laboratory Animal Technology Co., Ltd.), InVivo MAb anti-human / rat / fish AChR (Bio X Cell, West Lebanon, NH).

[0226] 2. Method: 2.1 EAMG induction Intraperitoneal administration of anti-AChR antibody mAb35 (1–3 mg / kg) to female rats for approximately 4 weeks is an internationally accepted method for inducing a generalized myasthenia gravis-like experimental autoimmune myasthenia (EAMG) model (Hepburn et al., 2007; Liu et al., 2007; Papanastasiou et al., 2000; Poulas et al., 2000). Rats were divided into seven groups: a control group without anti-AChR antibody injection; a group injected with anti-AChR antibody (1.5 mg / kg) intraperitoneally and PBS; and a group injected with anti-AChR antibody intraperitoneally and five doses of (CRIg-FH-IgG4Fc) × 2 drug treatment (0.5, 1, 2, 5, and 10 mg / kg). Two experiments were completed. 40 rats were used in the first experiment, and 50 rats in the second. Into groups This is shown in Table 3.

[0227] [Table 3]

[0228] 2.2 Monitoring the EAMG phenotype The rats were tested after an adaptation period of at least 5-7 days. Before and 24 hours after each experiment, they were weighed and clinically scored. The specific scoring criteria were: 0 if they could grasp and lift the cage lid; 1 if they could grasp but could not lift the lid; 2 if they could not grasp; 0 if they could not grasp. The results were as follows: 1) the PBS control group of the model group had almost no hind limb paralysis within 48 hours; and 2) the death or dead state was scored as 4 (Piddlesden et al., 1996; Soltys et al., 2009). Because the rats died or were facing death, observation of this group was terminated at 48 hours in accordance with ethical standards. On the other hand, the clinical scores of the (CRIg-FH-IgG4Fc) × 2 drug-administered group were almost completely recovered by 96 hours, so this group was discontinued. Groups were terminated at 96 hours and animals were counted for 24 and 48 hour mortality.

[0229] 2.3 Statistical processing The experimental data were expressed as mean ± SEM. Body weight was analyzed by two-tailed t-test, clinical scores by two-way ANOVA, and mortality rates by Chi-Square and Fisher's Exact Test. ns: no significance, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0230] 3.Results: Normal rats without anti-AChR antibody and (CRIg-FH-IgG4Fc) × 2 drugs gradually gained weight. However, rats injected with anti-AChR antibodies but not administered drugs lost 11.2% of their body weight at 24 hours and 15.5% at 48 hours. 82% (14 / 17) had a clinical score of 3-4 at 24 hours, and 88% (15 / 17) had a clinical score of 3-4 at 48 hours. Of the 17 rats, 2 were moribund or dead at 24 hours, and 9 died at 48 hours, resulting in a mortality rate of 52.9% (9 / 17). Of the remaining 8 surviving rats, 6 were moribund. It was shown that the model was successfully constructed (see Figures 25 to 27).

[0231] Pretreatment with (CRIg-FH-IgG4Fc) × 2 significantly improved EAMG symptoms, with the minimum dose of 0.5 mg / kg At 24 hours after administration, weight loss was only 1.6%, clinical score was 1.1, and at 48 hours, weight loss was only 2.3%. The clinical score was 1.3, and the animal mortality rate was 14.3% (1 / 7), demonstrating significant efficacy. 24 hours after administration of the high-dose drug, there was no significant decrease in body weight, and the clinical score gradually decreased in a dose-dependent manner. After 48 hours, body weight gradually recovered and the clinical score gradually decreased, demonstrating a dose-dependent relationship, i.e., the higher the dose, the greater the weight gain and the decrease in clinical score (Figures 25-27). Furthermore, when all animals in the drug-treated group were observed for up to 96 hours, it was confirmed that after 48 hours, the body weight of surviving rats had gradually increased, the clinical score had gradually decreased to normal values, and EAMG symptoms had gradually returned to normal.

[0232] 4. Consideration: The EAMG phenotype was induced in female rats by intraperitoneal injection of anti-AChR antibody mAb35 (1.5 mg / kg) within approximately 4 weeks, and was characterized by significant weight loss, rapid decrease in locomotion, decreased forelimb grip strength, and a 48-hour period. Body weight monitoring, clinical scoring, and animal mortality monitoring showed that LM007 was effective in treating EAMG at a dose of 0.5 mg / kg, with a dose-dependent effect as the dose increased, and doses of 5 mg / kg and above significantly inhibited the disease process. Although excessive complement activation was the primary effect in the anti-AChR antibody-induced EAMG rat model, a small proportion of rats may exhibit pathogenicity independent of complement activation.

Claims

1. A fusion protein comprising a first polypeptide chain and a second polypeptide chain, The first polypeptide chain and the second polypeptide chain are, (i) CRIg extracellular domain, (ii) A complement regulatory domain comprising a protein selected from the group consisting of CD55, CD59, and CR1, and (iii) Enhanced domains including the IgG Fc domain Includes, The enhancing domain of the first polypeptide chain and the enhancing domain of the second polypeptide chain can interact with each other to form a dimer. The aforementioned fusion protein.

2. The fusion protein according to claim 1, wherein the CRIg extracellular domain comprises the amino acid sequence shown in SEQ ID NO:

6.

3. The C-terminus of the CRIg extracellular domain is directly or indirectly ligated to the N-terminus of the complement regulatory domain, or The N-terminus of the CRIg extracellular domain is directly or indirectly ligated to the C-terminus of the complement regulatory domain. The fusion protein according to claim 1 or 2.

4. The C-terminus of the complement control domain is directly or indirectly connected to the N-terminus of the enhancement domain, or The C-terminus of the CRIg extracellular domain is directly or indirectly ligated to the N-terminus of the enhancement domain. A fusion protein according to any one of claims 1 to 3.

5. The complement regulatory domain includes the amino acid sequence shown in any one of SEQ ID NOs: 18, 22, 62, and 64. The fusion protein according to any one of claims 1 to 4.

6. The CRIg extracellular domain, the complement regulatory domain, and the enhancement domain are included in order from the N-terminus to the C-terminus, or The complement regulatory domain, the CRIg extracellular domain, and the enhancement domain are included in order from the N-terminus to the C-terminus. A fusion protein according to any one of claims 1 to 5.

7. The fusion protein according to any one of claims 1 to 6, wherein the IgG Fc domain comprises a protein selected from the group consisting of human IgG1 and human IgG4.

8. The fusion protein according to any one of claims 1 to 7, wherein the IgG Fc domain comprises the amino acid sequence shown in any one of SEQ ID NOs: 10, 30, 32, and 34.

9. The first polypeptide chain comprises the CRIg extracellular domain, the first complement regulatory domain, and the first IgG Fc domain, The second polypeptide chain comprises the CRIg extracellular domain, the second complement regulatory domain, and the second IgG Fc domain, The first IgG Fc domain and the second IgG Fc domain can interact with each other to form a dimer. The fusion protein according to any one of claims 1 to 8.

10. The fusion protein according to claim 9, wherein the first polypeptide chain is identical to the second polypeptide chain.

11. The fusion protein according to claim 10, wherein the first polypeptide chain and / or the second polypeptide chain comprises the amino acid sequence shown in any one of SEQ ID NOs: 24, 28, 66, and 68.

12. The fusion protein according to claim 9, wherein the first complement regulatory domain is different from the second complement regulatory domain.

13. The first complement regulatory domain comprises a protein selected from the group consisting of factor H (FH), CD55, CD59, and CR1. The second complement regulatory domain comprises a protein selected from the group consisting of CD55, CD59, and CR1. The fusion protein according to claim 12.

14. The first polypeptide chain and / or the second polypeptide chain contains the amino acid sequence shown in any one of SEQ ID NOs: 14, 24, 28, 60, 66, 68, 36, 38, 40, and 42. The fusion protein according to claim 12 or 13.

15. The first complement control domain and the second complement control domain each operate independently, (1) CD59 and CD55, (2) FH and CD55 A protein or functional fragment thereof selected from the group consisting of the following: The fusion protein according to any one of claims 12 to 14.

16. The fusion protein according to claim 15, which is any of the following: (1) The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 38, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 40, or (2) The first polypeptide chain comprises the amino acid sequence shown in SEQ ID NO: 38, and the second polypeptide chain comprises the amino acid sequence shown in SEQ ID NO:

42.

17. An isolated nucleic acid or isolated nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 16.

18. A pharmaceutical composition comprising a fusion protein according to any one of claims 1 to 16 and a pharmaceutically acceptable carrier.

19. A vector comprising the isolated nucleic acid molecule described in claim 17.

20. A cell that contains the vector described in claim 19, or expresses the fusion protein described in any one of claims 1 to 16.

21. In the manufacture of pharmaceuticals for treating diseases related to targeted inhibition of complement activation, Use of the fusion protein described in any one of claims 1 to 16 or the pharmaceutical composition described in claim 18.

22. The use according to claim 21, wherein the disease includes autoimmune diseases.

23. The use according to claim 21 or 22, wherein the disease includes autoimmune myasthenia gravis.

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