JPWO2018070521A1 - Fusion polypeptides that inhibit complement activation pathways - Google Patents

Abstract

In one embodiment, an object of the present invention is to provide an agent that inhibits the lectin pathway and the second pathway. In one embodiment, the present invention includes a first region containing a CUB1 domain and an EGF domain of the MASP-2 protein or sMAP protein on the N-terminal side, and a second region containing the SCR domain of the factor H protein as C. The present invention relates to a fusion polypeptide contained on the terminal side, a polynucleotide encoding the fusion polypeptide, a vector containing the polynucleotide, and the like.

Description

  The present invention relates to a fusion polypeptide comprising a first region comprising a CUB1 domain and an EGF domain of a MASP-2 protein or sMAP protein, and a second region comprising an SCR domain of a factor H protein, and the fusion polypeptide It relates to a polynucleotide to be encoded.

  Complement refers to a body fluid component that assists antibodies and phagocytic cells, and constitutes a reaction system (complement system) that eliminates foreign substances such as pathogens by activation. The complement system is initiated through three pathways, the classical pathway, the lectin pathway, and the second pathway, and plays a pivotal role in the host immune system. On the other hand, excessive activation of the complement system is known to result in complement-related diseases such as systemic lupus erythematosus and ischemia-reperfusion injury.

  So far, the possibility of treating systemic lupus erythematosus and ischemia-reperfusion injury by inhibiting the second pathway of the complement activation pathway has been shown (Non-Patent Document 1 and Non-Patent Document 2, respectively). Furthermore, Patent Document 1 shows that several diseases can be treated by inhibitors of MASP-2 protein involved in the lectin pathway. However, since the complement system has three activation pathways as described above, it is necessary to inhibit not only a single pathway but also multiple complement activation pathways in order to effectively treat complement-related diseases. It is believed that there is. On the other hand, when all three of the complement activation pathways are inhibited, the side effects are concerned.

  Therefore, a drug capable of selectively inhibiting a plurality of complement activation pathways has been desired.

Special Table 2014-515922

Sekine H et al., Arthritis & Rheumatism, 63: 1076-1085, 2011 Huang Y et al., The Journal of Immunology, 181: 8068-8076, 2008

  An object of the present invention is to provide an agent that inhibits the lectin pathway and the second pathway.

  The present inventor includes the first region containing the CUB1 domain and the EGF domain of the MASP-2 protein or sMAP protein on the N-terminal side, and the second region containing the SCR domain of the factor H protein on the C-terminal side. The present inventors have found that the fusion polypeptide can inhibit the lectin pathway and the second pathway, and completed the present invention.

Accordingly, the present invention includes the following aspects.
(1) The MASP-2 protein or sMAP protein includes a first region containing the CUB1 domain and the EGF domain in order from the N-terminal side on the N-terminal side,
a second region containing 3 or more SCR domains selected from the group consisting of SCR1 to 4 domains of factor H protein, on the C-terminal side;
Fusion polypeptide.
(2) The CUB1 domain is represented by SEQ ID NO: 1 or 3, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or 3, or represented by SEQ ID NO: 1 or 3 Including an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence;
In the amino acid sequence in which the EGF domain is represented by SEQ ID NO: 5 or 7, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 5 or 7, or the amino acid sequence represented by SEQ ID NO: 5 or 7 The fusion polypeptide according to (1), comprising an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted.
(3) The first region is SEQ ID NO: 49, or an amino acid sequence in which one amino acid is added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 49 on the C-terminal side of the EFG domain. The fusion polypeptide according to (1) or (2).
(4) The fusion polypeptide according to any one of (1) to (3), wherein the second region comprises SCR1 to 4 domains.
(5) The fusion polypeptide according to any one of (1) to (4), wherein the second region contains an SCR5 domain.
(6) The fusion polypeptide according to (5), wherein the second region comprises SCR1-5 domains in order from the N-terminal side.
(7) The SCR1 domain is represented by SEQ ID NO: 9 or 11, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 9 or 11, or represented by SEQ ID NO: 9 or 11 Including an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence;
In the amino acid sequence in which the SCR2 domain is represented by SEQ ID NO: 13 or 15, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 13 or 15, or the amino acid sequence represented by SEQ ID NO: 13 or 15 An amino acid sequence in which one or more amino acids are added, deleted, and / or substituted,
In the amino acid sequence in which the SCR3 domain is represented by SEQ ID NO: 17 or 19, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 17 or 19, or the amino acid sequence represented by SEQ ID NO: 17 or 19 An amino acid sequence in which one or more amino acids are added, deleted, and / or substituted,
In the amino acid sequence in which the SCR4 domain is represented by SEQ ID NO: 21 or 23, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 21 or 23, or the amino acid sequence represented by SEQ ID NO: 21 or 23 An amino acid sequence in which one or more amino acids are added, deleted, and / or substituted,
In the amino acid sequence in which the SCR5 domain is represented by SEQ ID NO: 25 or 27, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 25 or 27, or the amino acid sequence represented by SEQ ID NO: 25 or 27 Comprising an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted,
The fusion polypeptide according to (5) or (6).
(8) The first region is an amino acid sequence represented by SEQ ID NO: 29 or 31, an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 29 or 31, or SEQ ID NO: 29 or 31 An amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence shown,
The second region is the amino acid sequence shown by SEQ ID NO: 33 or 35, the amino acid sequence having 90% or more identity to the amino acid sequence shown by SEQ ID NO: 33 or 35, or the amino acid shown by SEQ ID NO: 33 or 35 Comprising an amino acid sequence in which one or more amino acids have been added, deleted and / or substituted in the sequence;
The fusion polypeptide according to (1).
(9) The fusion polypeptide according to any one of (1) to (8), wherein the first region is linked to the second region via a linker.
(10) The fusion polypeptide according to (9), wherein the linker is a peptide linker comprising 1 to 8 amino acid sequences represented by (GGGGS).
(11) A polynucleotide encoding the fusion polypeptide according to any one of (1) to (10).
(12) A vector comprising the polynucleotide according to (11).
(13) A cell comprising the polynucleotide according to (11) or the vector according to (12).
(14) An inhibitor of the lectin pathway and the second pathway in the complement activation pathway, comprising the fusion polypeptide according to any one of (1) to (10).
(15) A pharmaceutical composition comprising the fusion polypeptide according to any one of (1) to (10), the polynucleotide according to (11), or the vector according to (12).
(16) The pharmaceutical composition according to (15), which inhibits the lectin pathway and the second pathway in the complement activation pathway.
(17) The pharmaceutical composition according to (16) for treating and / or preventing a complement-related disease.
(18) Complement-related diseases are ischemia-reperfusion injury, age-related macular degeneration, systemic lupus erythematosus, antiphospholipid antibody syndrome (APS), IgA nephropathy, C3 nephropathy, membranoproliferative glomerulonephritis, joint Rheumatoid arthritis, malignant rheumatoid arthritis, IgG4-related disease, mixed cryoglobulinemia, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), myasthenia gravis The pharmaceutical composition according to (17), selected from the group consisting of symptom, Guillain-Barre syndrome, optic neuromyelitis, Alzheimer's disease, thrombotic microangiopathy (TMA), ulcerative colitis, Crohn's disease, and sepsis object.

  This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2016-203024, which is the basis for the priority of the present application.

  According to the present invention, a fusion polypeptide capable of inhibiting the lectin pathway and the second pathway, a polynucleotide encoding the fusion polypeptide, a vector comprising the polynucleotide, and an inhibitor or medicament for the lectin pathway and the second pathway comprising them Compositions and the like are provided.

1 shows a schematic diagram of the fusion polypeptide sMAP-fH prepared in Example 1. FIG. A refers to the sMAP protein portion containing the CUB1 domain and EGF domain, B refers to the linker portion consisting of (GGGGS) ₄ , and C refers to the fH protein portion including the SCR1-5 domain. FIG. 2 shows the results of CBB staining of SDS-PAGE of sMAP-fH (A) and Western blotting using anti-mouse sMAP polyclonal antibody or anti-mouse factor H antibody (B: anti-mouse sMAP polyclonal antibody, C: anti-mouse Mouse factor H antibody) results are shown. FIG. 3 shows the results of sMAP-fH C3 deposition assay. The C3 deposition level at the time of adding each concentration of sMAP-fH to wild type C57BL / 6J mouse serum is shown. The horizontal axis represents the concentration of sMAP-fH added to serum, and the vertical axis represents the absorbance at OD ₄₅₀ nm. FIG. 4 shows the measurement results of blood concentration (μg / mL) of sMAP-fH 1, 2, 4, 8, 24, and 48 hours after intraperitoneal administration of 500 μg of sMAP-fH into mice. FIG. 5 shows the results of the C4 deposition assay for serum at 0, 1, 2, 4, 8, 24, and 48 hours after intraperitoneal administration of 500 μg sMAP-fH into mice. The results are shown as relative values (% PBS) when the PBS administration group is taken as 100. Fig. 6 shows the serum-free sample (a), serum-added sample of MASP-2 deficient mice (b), serum-added sample before sMAP-fH administration (c), and intraperitoneal administration of sMAP-fH to mice. The results of zymosan assay by flow cytometry for serum-added samples after 1, 2, 4, 8, 24, and 48 hours (d to i, respectively) are shown. The left axis shows FITC fluorescence intensity, and the vertical axis shows the number of particles (count). FIG. 7 shows the evaluation results of the lectin pathway and the second pathway using the Complement System Screen kit of three types of human sMAP-fH fusion polypeptides. Polypeptides 2, 3, and 4 refer to the polypeptides prepared in Examples 4, 5, and 6, respectively.

1. Definitions Definitions of terms used in the present specification are described below.

As used herein, “complement” refers to a body fluid component that assists antibodies and phagocytic cells when a living body excludes foreign substances such as pathogens. Complement includes 9 components (complement components) from 1st component C1 to 9th component C9 (C1 further includes C1q, C1r, C1s), and this includes a series of various reaction factors, etc. This constitutes the reaction system (complement system). As used herein, “complement activation pathway” refers to a pathway by which complement or the complement system is activated, and includes the classical pathway (Classical Pathway), the lectin pathway (Lectin Pathway), and the second pathway (Alternative Pathway, 3 paths) (also called alternative paths or sub-paths). The classical pathway is initiated by the binding of complement component C1q to antigen-antibody complexes, etc., and the lectin pathway is initiated by the binding of serum proteins such as mannan-binding lectin or ficollin and bacterial or viral mannose-containing sugar chains. The two pathways are initiated by the binding of spontaneously activated complement components to the pathogen surface.
2. Fusion polypeptide In one aspect, the present invention relates to a fusion polypeptide comprising a first region on the N-terminal side and a second region on the C-terminal side.

  The first region of the fusion polypeptide includes the CUB1 domain and the EGF domain of the MASP-2 protein or sMAP protein in order from the N-terminal side.

  In this specification, “MASP-2” refers to Mannose-Binding Protein-Associated Serine Protease 2: Mannose-binding protein-related serine protease 2 and is also referred to as Mannan-binding lectin serine protease 2: Mannan-binding lectin serine protease 2. MASP-2 is known to be activated by MASP-1 and activate a pathway downstream of the complement activation pathway. MASP-2 has 6 domains, namely 2 CUB (C1r / C1s / embryonic sea urchin protein / bone morphogenetic protein: C1r / C1s / embryonic sea urchin protein / bone morphogenetic protein) domains, EGF (epidermal growth factor like: epithelium) It contains a growth factor-like) domain, two CCP (complement control protein) domains, and an SP (serine protease) domain.

  In this specification, “sMAP” refers to small mannose binding lectin (MBL) -associated protein: small mannose binding lectin (MBL) -related protein, and MAp19 (mannose-binding lectin associated protein 19: mannose binding lectin associated protein 19) It is also called MASP2-isoform (MASP2 isoform). sMAP is a splicing variant of MASP-2 and contains only the N-terminal CUB domain (CUB1 domain) and EGF domain of the MASP-2 protein. The sMAP protein and the base sequence encoding the sMAP protein are known to those skilled in the art. For example, the human sMAP protein includes the amino acid sequence represented by SEQ ID NO: 37, and the polynucleotide encoding the human sMAP protein is represented by SEQ ID NO: 38. Contains the base sequence.

  The sMAP protein is known to competitively inhibit the activity of the MASP-2 protein. Since the sMAP protein is encoded by the same MASP-2 gene as the MASP-2 protein, the CUB1 domain and EGF domain of the MASP-2 protein and the CUB1 domain and EGF domain of the sMAP protein usually contain the same amino acid sequence.

  The biological species from which the CUB1 domain and EGF domain included in the first region of the fusion polypeptide are not limited, but experiments with mammals such as primates such as humans and chimpanzees, rats and mice, etc. Examples include animals, domestic animals such as pigs, cows, horses, sheep and goats, and pets such as dogs and cats, preferably humans or mice, most preferably humans. The amino acid sequences of the CUB1 and EGF domains of the MASP-2 protein or sMAP protein in each organism or the base sequences encoding them can be obtained by any method known in the art, for example, a public database (eg NCBI (USA) , DDBJ (Japan), EMBL (Europe)), and the domain contained in the amino acid sequence can be specified by public programs such as SMART and Pfam.

  The CUB1 domain and EGF domain contained in the first region are chimeras of an amino acid sequence derived from one species (eg, human) or an equivalent sequence thereof and an amino acid sequence derived from another species (eg, mouse) or an equivalent sequence thereof. Although it may be a polypeptide, it preferably contains only an amino acid sequence derived from any biological species, preferably a human, or a sequence equivalent thereto.

  In the present specification, an amino acid sequence and an “equivalent sequence” have, for example, 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to a certain amino acid sequence. The amino acid sequence may include an amino acid sequence, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in a certain amino acid sequence. Similarly, a certain nucleotide sequence and an “equivalent sequence” are, for example, a nucleotide sequence having 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to a certain nucleotide sequence. Alternatively, it may include a base sequence in which one or more bases are added, deleted, and / or substituted in a certain base acid sequence. In the present specification, the identity value relating to the amino acid sequence and the base sequence indicates a value calculated with default settings using software (for example, FASTA, DANASYS, and BLAST) that calculates identity between a plurality of sequences. . For details of how identity is determined, see, for example, Altschul et al, Nuc. Acids. Res. 25, 3389-3402, 1977 and Altschul et al, J. Mol. Biol. 215, 403-410, 1990. . In the present specification, the range of “one or more” regarding the amino acid sequence and the base sequence is 1 to 10, preferably 1 to 7, more preferably 1 to 5, particularly preferably several. One to four or one to three, or one or two.

  The CUB1 domain included in the first region is the amino acid sequence of the CUB1 domain of human MASP-2 protein or sMAP protein (SEQ ID NO: 1), or the amino acid sequence of the CUB1 domain of mouse MASP-2 protein or sMAP protein (sequence) No. 3), or an amino acid sequence equivalent to these amino acid sequences.

  Therefore, the CUB1 domain contained in the first region is 90% or more, 95% or more, 97% or more, 98% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 or 3, or the amino acid sequence represented by SEQ ID NO: 1 or 3. An amino acid sequence having% or more, or 99% or more identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 1 or 3 Good.

  Similarly, the EGF domain contained in the first region is the amino acid sequence of the EGF domain of human MASP-2 protein or sMAP protein (SEQ ID NO: 5), or the amino acid of the EGF domain of mouse MASP-2 protein or sMAP protein. The sequence (SEQ ID NO: 7) or a sequence equivalent to these amino acid sequences may be included.

  Therefore, the EGF domain contained in the first region is an amino acid sequence represented by SEQ ID NO: 5 or 7, 90% or more, 95% or more, 97% or more, 98% or more with respect to the amino acid sequence represented by SEQ ID NO: 5 or 7. An amino acid sequence having% or more, or 99% or more identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 5 or 7 Good.

  As described above, it is preferable that the CUB1 domain and the EGF domain included in the first region include only an amino acid sequence derived from any biological species, preferably human, or a sequence equivalent thereto. Thus, for example, in the first region, the CUB1 domain has an amino acid sequence represented by SEQ ID NO: 1, 90% or more, 95% or more, 97% or more, 98% or more, or 99% of the amino acid sequence represented by SEQ ID NO: 1. An amino acid sequence having% or more identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 1; the EGF domain is SEQ ID NO: 5 The amino acid sequence shown, the amino acid sequence having 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence shown by SEQ ID NO: 5, or SEQ ID NO: 5 The amino acid sequence may include an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted.

  In addition to the above CUB1 domain and EGF domain, the first region is one amino acid sequence unique to the sMAP protein that is not present in the MASP-2 protein, that is, EQSL (SEQ ID NO: 49), or one amino acid sequence represented by SEQ ID NO: 49. An amino acid sequence in which is added, deleted, and / or substituted may preferably be included on the C-terminal side of the EFG domain. That is, the first region can include the CUB1 domain, the EGF domain, and the amino acid sequence specific to the sMAP protein in order from the N-terminal side. In addition, the amino acid sequence peculiar to sMAP protein is the same in human and mouse.

  The first region may be composed only of the CUB1 domain and EGF domain, or may be composed only of the CUB1 domain, EGF domain, and sMAP protein-specific amino acid sequence.

  The amino acid length of the first region is not limited, but may be, for example, 150 residues or more, 160 residues or more, 170 residues or more, or 180 residues or more, and 350 residues or less, 300 residues or less, It may be 250 residues or less, or 200 residues or less.

  Each domain included in the first region may be appropriately connected via a linker, but is preferably directly connected without a linker.

  The fusion polypeptide may include only one first region, or may include a plurality, for example 2-8, 2-6, 2-5, 2-4, 2-3, or 2. When a plurality of first regions are included, each region may be directly connected or may be connected via a linker.

  The fusion polypeptide includes a second region in addition to the first region. The second region of the fusion polypeptide comprises 3 or more, 4 or more, or 5 or more, 20 or less, 15 or less, 10 or less, or 5 or more SCR domains selected from the group consisting of SCR 1-4 domains of factor H protein. Hereinafter, for example, 3, 4 or 5 is included. The second region may include two or more identical SCR domains, but the SCR domains included in the second region are preferably different from each other.

  In the present specification, “factor H: factor H (fH)” is also referred to as complement factor H: complement factor H (CfH), and is a major regulator of the second pathway of the complement activation pathway. The fH protein has 20 domains that are homologous to each other, that is, SCR (Short Consensus Repeat) 1 domain to SCR20 domain (in this specification, SCR1 domain to SCR20 domain are collectively referred to as SCR1 to 20 domain. ) In order from the N-terminal side, among which the N-terminal SCR1-4 domains are known to have fH protein complement control activity. The fH protein and the base sequence encoding the same are known to those skilled in the art. For example, the human fH protein includes the amino acid sequence represented by SEQ ID NO: 39, and the polynucleotide encoding the human fH protein is represented by SEQ ID NO: 40. Contains the base sequence.

  The second region of the fusion polypeptide preferably comprises 3 or more different SCR domains selected from the group consisting of SCR1-4 domains of the factor H protein, for example, SCR1, SCR2, and SCR3 domains; SCR1, SCR2, And SCR4 domains; SCR1, SCR3, and SCR4 domains; SCR2, SCR3, and SCR4 domains; or SCR1-4 domains, preferably in order from the N-terminus.

  The second region of the fusion polypeptide preferably comprises an SCR5 domain in addition to the SCR domain selected from the group consisting of SCR1-4 domains. In this case, the second region of the fusion polypeptide may contain, for example, SCR1-5 domains in order from the N-terminus.

  Further, the second region of the fusion polypeptide comprises an SCR domain selected from SCR 6-20 domains, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 15 or less, 14 or less, 13 or less, 12 Hereinafter, 11 or less, or 10 or less may be included. In this case, it is preferable that the second region includes the SCR domain with the smallest number in order from the N-terminal. The SCR domains selected from the SCR 6 to 20 domains may include two or more identical SCR domains, but are preferably different from each other.

  Species derived from the SCR1-20 domain that can be included in the second region, such as SCR1-5 domain or SCR1-4 domain, are not limited, but mammals such as humans and primates such as chimpanzees And laboratory animals such as rats and mice, domestic animals such as pigs, cows, horses, sheep and goats, and pets such as dogs and cats, preferably humans or mice, most preferably humans. The amino acid sequence of the fH protein in each organism can be obtained by any method known in the art, for example, from public databases (eg, NCBI (US), DDBJ (Japan), EMBL (Europe)). The domain contained in the amino acid sequence can be specified by a public program such as SMART and Pfam.

  The SCR1-20 domain that can be included in the second region, such as the SCR1-5 domain or the SCR1-4 domain, is an amino acid sequence derived from one species (eg, human) or equivalent sequence, and another species (eg, mouse) Although it may be a chimeric polypeptide having an amino acid sequence derived from it or an equivalent sequence, it preferably contains only an amino acid sequence derived from any biological species, preferably human, or an equivalent sequence.

  The SCR1 domain that can be included in the second region is the amino acid sequence of the SCR1 domain of human fH protein (SEQ ID NO: 9), the amino acid sequence of the SCR1 domain of mouse fH protein (SEQ ID NO: 11), or these amino acid sequences May be an array equivalent to

  Therefore, the SCR1 domain that can be included in the second region is an amino acid sequence represented by SEQ ID NO: 9 or 11, 90% or more, 95% or more, 97% or more with respect to the amino acid sequence represented by SEQ ID NO: 9 or 11. An amino acid sequence having 98% or more, or 99% or more identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 9 or 11 It's okay.

  Similarly, the SCR2 domain that can be included in the second region is the amino acid sequence of the SCR2 domain of human fH protein (SEQ ID NO: 13), the amino acid sequence of the SCR2 domain of mouse fH protein (SEQ ID NO: 15), or these It may be a sequence equivalent to the amino acid sequence of

  Therefore, the SCR2 domain that can be included in the second region is an amino acid sequence represented by SEQ ID NO: 13 or 15, 90% or more, 95% or more, 97% or more with respect to the amino acid sequence represented by SEQ ID NO: 13 or 15. An amino acid sequence having 98% or more, or 99% or more identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 13 or 15 It's okay.

  Similarly, the SCR3 domain that can be included in the second region is the amino acid sequence of the SCR3 domain of human fH protein (SEQ ID NO: 17), the amino acid sequence of the SCR2 domain of mouse fH protein (SEQ ID NO: 19), or these It may be a sequence equivalent to the amino acid sequence of

  Therefore, the SCR3 domain that can be included in the second region is an amino acid sequence represented by SEQ ID NO: 17 or 19, 90% or more, 95% or more, 97% or more with respect to the amino acid sequence represented by SEQ ID NO: 17 or 19, An amino acid sequence having 98% or more, or 99% or more identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 17 or 19 It's okay.

  Similarly, the SCR4 domain that can be included in the second region is the amino acid sequence of the SCR4 domain of human fH protein (SEQ ID NO: 21), the amino acid sequence of the SCR2 domain of mouse fH protein (SEQ ID NO: 23), or these It may be a sequence equivalent to the amino acid sequence of

  Therefore, the SCR4 domain that can be included in the second region is an amino acid sequence represented by SEQ ID NO: 21 or 23, 90% or more, 95% or more, 97% or more with respect to the amino acid sequence represented by SEQ ID NO: 21 or 23, An amino acid sequence having 98% or more, or 99% or more identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 21 or 23 It's okay.

  Similarly, the SCR5 domain that may be included in the second region is the amino acid sequence of the SCR5 domain of human fH protein (SEQ ID NO: 25), the amino acid sequence of the SCR5 domain of mouse fH protein (SEQ ID NO: 27), or these It may be a sequence equivalent to the amino acid sequence of

  Therefore, the SCR5 domain that can be included in the second region is an amino acid sequence represented by SEQ ID NO: 25 or 27, 90% or more, 95% or more, 97% or more with respect to the amino acid sequence represented by SEQ ID NO: 25 or 27, An amino acid sequence having 98% or more, or 99% or more identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 25 or 27 It's okay.

  As described above, the SCR1-20 domain that can be included in the second region, for example, the SCR1-5 domain or the SCR1-4 domain, preferably includes only an amino acid sequence derived from any species or a sequence equivalent thereto. . Thus, for example, in the second region, the SCR1 domain has an amino acid sequence represented by SEQ ID NO: 9, 90% or more, 95% or more, 97% or more, 98% or more, or 99% of the amino acid sequence represented by SEQ ID NO: 9. An amino acid sequence having at least% identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 9; the SCR2 domain is SEQ ID NO: 13 The amino acid sequence shown, the amino acid sequence having 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence shown by SEQ ID NO: 13, or shown by SEQ ID NO: 13 An amino acid sequence in which one or more amino acids are added, deleted, and / or substituted; the SCR3 domain is represented by SEQ ID NO: 17, An amino acid sequence having 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence, or one or more amino acids in the amino acid sequence represented by SEQ ID NO: 17 Including the amino acid sequence added, deleted, and / or substituted; the SCR4 domain is 90% or more, 95% or more, 97% or more with respect to the amino acid sequence represented by SEQ ID NO: 21, the amino acid sequence represented by SEQ ID NO: 21 An amino acid sequence having 98% or more, or 99% or more identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 21; The SCR5 domain is an amino acid sequence represented by SEQ ID NO: 25, an amino acid sequence having 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence represented by SEQ ID NO: 25 ,or 1 or more amino acids in the amino acid sequence shown in SEQ ID NO: 25 is added, may comprise deletions, and / or substituted amino acid sequence.

  The second region may include the SCR domain, or may include only these domains. The amino acid length of the second region is not limited, but may be, for example, 150 residues or more, 200 residues or more, or 250 residues or more, and 450 residues or less, 400 residues or less, or 350 residues. It may be below the group.

  Each domain included in the second region may be appropriately connected via a linker, but is preferably directly connected without a linker.

  The first region comprises in particular the amino acid sequence comprising the CUB1 domain and EGF domain of human MASP-2 protein or sMAP protein (SEQ ID NO: 29) or the CUB1 domain and EGF domain of mouse MASP-2 protein or sMAP protein An amino acid sequence (SEQ ID NO: 31) or a sequence equivalent to these amino acid sequences may be included. Similarly, the second region is an amino acid sequence comprising the SCR1-5 domain of human fH protein (SEQ ID NO: 33), or an amino acid sequence comprising the SCR1-5 domain of mouse fH protein (SEQ ID NO: 35), or A sequence equivalent to these amino acid sequences may be included.

  Therefore, in the fusion polypeptide of the present invention, the first region is 90% or more, 95% or more, 97% or more with respect to the amino acid sequence represented by SEQ ID NO: 29 or 31, and the amino acid sequence represented by SEQ ID NO: 29 or 31. An amino acid sequence having 98% or more, or 99% or more identity, or an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 29 or 31 The second region is an amino acid sequence represented by SEQ ID NO: 33 or 35, 90% or more, 95% or more, 97% or more, 98% or more, or 99% with respect to the amino acid sequence represented by SEQ ID NO: 33 or 35 The amino acid sequence having the above identity or the amino acid sequence represented by SEQ ID NO: 33 or 35 may include an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted. In this embodiment, the first region may include SEQ ID NO: 49 at the C-terminus thereof, or an amino acid sequence in which one amino acid is added, deleted, and / or substituted in the amino acid sequence shown by SEQ ID NO: 49. .

  The first region and the second region are chimeric polypeptides having an amino acid sequence derived from one species (eg, human) or an equivalent sequence thereof, and an amino acid sequence derived from another species (eg, mouse) or an equivalent sequence thereof. It may be present, but preferably contains only an amino acid sequence derived from any biological species, preferably a human, or a sequence equivalent thereto. Therefore, in the fusion polypeptide of the present invention, the first region is the amino acid sequence represented by SEQ ID NO: 29, 90% or more, 95% or more, 97% or more, 98% or more with respect to the amino acid sequence represented by SEQ ID NO: 29. Or an amino acid sequence having 99% or more identity, or an amino acid sequence represented by SEQ ID NO: 29, wherein one or more amino acids are added, deleted, and / or substituted, and the second region Is an amino acid sequence represented by SEQ ID NO: 33, an amino acid sequence having 90% or more, 95% or more, 97% or more, 98% or more, or 99% or more identity to the amino acid sequence represented by SEQ ID NO: 33, or The amino acid sequence represented by SEQ ID NO: 33 may include an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted.

  In the above fusion polypeptide, the first region and the second region may be directly linked, but are preferably linked via a linker as appropriate in order to maintain the function and structure of each region. The linker in the present specification may be a chemical linker (for example, disulfide linker and maleimide linker) or a peptide linker, but is preferably a peptide linker. The peptide linker may be, for example, a peptide consisting of an amino acid sequence of 1-50, 5-40, or 10-30, and the length of the linker takes into account the function and structure of the first region and the second region. Thus, those skilled in the art can appropriately determine it. Examples of peptide linkers include linkers composed of glycine and serine (GGS linker and GS linker). The GGS linker contains 1 to a plurality of GGS, for example 2 to 6 or 2 to 4. On the other hand, the GS linker contains 1 to several amino acid sequences represented by GGGGS, for example, 1 to 8, 3 to 6, preferably 4. The linker may contain amino acid residues other than glycine and serine, but is preferably composed only of glycine and serine. This is because glycine and serine are small in size and do not easily form a higher order structure in the linker, so that the functions and structures of the first region and the second region are unlikely to be negatively affected.

  Similarly, the fusion polypeptide may comprise only one second region or may comprise a plurality, for example 2-8, 2-6, 2-5, 2-4, 2-3, or 2. . When two or more 2nd area | regions are included, each area | region may be connected directly and may be connected through a linker.

  The fusion polypeptide can be produced by a method known to those skilled in the art, for example, chemical synthesis, but is preferably produced by a genetic recombination method. For example, the fusion polypeptide can be prepared by culturing in a host cell into which a polynucleotide encoding the fusion polypeptide, for example, the vector described in “3. Polynucleotide and vector” below is introduced.

  The prepared peptide can be prepared by conventional methods, for example, gel filtration chromatography, ion exchange column chromatography, affinity chromatography, reverse phase column chromatography, HPLC chromatography, ammonium sulfate fractionation, ultrafiltration, and immunoadsorption. It can be recovered or purified by using any one of the methods or a combination of two or more thereof.

The first region of the fusion polypeptide can inhibit the lectin pathway by directly inhibiting the activity of MASP-2, an important component of the lectin pathway. MASP-2 is activated by MASP-1, but since it can be activated without MASP-1, the first region has an excellent effect in that it can directly inhibit MASP-2. In addition, the second region of the fusion polypeptide can effectively inhibit the second pathway by inhibiting the activity of C3b, C3bBb, and C3bBbP, which are important components of the second pathway. Therefore, since the first region can inhibit the lectin pathway and the second region can inhibit the second pathway, the fusion polypeptide inhibits the lectin pathway and the second pathway in one molecule. Can effectively inhibit the complement activation pathway.
3. Polynucleotides and Vectors In one aspect, the present invention relates to a polynucleotide encoding the fusion polypeptide.

  A polynucleotide encoding the amino acid sequence contained in the fusion polypeptide can be easily obtained by those skilled in the art based on the amino acid sequence. For example, polys containing the amino acid sequences shown in SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, and 49 The peptide-encoding polynucleotides are shown in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, and 50, respectively. And a polynucleotide containing the nucleotide sequence. A person skilled in the art can obtain a polynucleotide encoding the fusion polypeptide based on these correspondences.

  For example, the CUB1 domain contained in the first region is an amino acid sequence represented by SEQ ID NO: 1 or 3, 90% or more, 95% or more, 97% or more, 98% or more with respect to the amino acid sequence represented by SEQ ID NO: 1 or 3. When the amino acid sequence having% or more, or 99% or more identity, or the amino acid sequence represented by SEQ ID NO: 1 or 3 includes an amino acid sequence in which one or more amino acids are added, deleted, and / or substituted , The polynucleotide encoding the CUB1 domain is (a) a nucleotide sequence represented by SEQ ID NO: 2 or 4, (b) 90% or more, 95% or more, 97% with respect to a nucleotide sequence represented by SEQ ID NO: 2 or 4 As described above, a nucleotide sequence having 98% or more, or 99% or more identity, (c) one or more bases are added, deleted, and / or substituted in the basic acid sequence represented by SEQ ID NO: 2 or 4 Base sequence, or (d) the sequences (a) to (c) above However, it may contain a base sequence encoding the same protein due to degeneracy.

  Similarly, the polynucleotide encoding the polypeptide of the first region is (e) a base sequence represented by SEQ ID NO: 30 or 32, (f) 90% or more with respect to the base sequence represented by SEQ ID NO: 30 or 32 , 95% or more, 97% or more, 98% or more, or 99% or more of the base sequence having identity, or (g) one or more bases added in the base acid sequence represented by SEQ ID NO: 30 or 32, A base sequence that is deleted and / or substituted, or (h) a base sequence that differs from the sequences (e) to (g) above but encodes the same protein due to degeneracy. The polynucleotide encoding the polypeptide of the region of (i) the nucleotide sequence represented by SEQ ID NO: 34 or 36, (j) 90% or more, 95% or more with respect to the nucleotide sequence represented by SEQ ID NO: 34 or 36, Nucleotide sequences having 97% or more, 98% or more, or 99% or more identity, (k A base sequence in which one or more bases are added, deleted, and / or substituted in the base acid sequence represented by SEQ ID NO: 34 or 36, or (l) a base different from the sequences (i) to (k) above. A base sequence having a sequence but encoding the same protein due to degeneracy may be included.

  The polynucleotide can be prepared by methods known to those skilled in the art. For example, the polynucleotide can be prepared by genetic recombination techniques such as PCR and infusion using cDNA obtained by reverse transcription of mRNA isolated from mammals, for example, human or mouse tissues or cells. The gene recombination technique can be performed according to a known method (for example, Sambrook et al., “Molecular Cloning, A Laboratory Manual fourth edition”, Cold Spring Harber Laboratory Press, 2012).

  In one aspect, the present invention relates to a vector comprising a polynucleotide encoding the above fusion polypeptide.

  In the present invention, the type of vector is not particularly limited. Examples of vectors include vectors such as plasmids, phages, cosmids, phagemids, and viruses. Examples of plasmid vectors include, but are not limited to, plasmids for E. coli (eg, pET22b (+), pBR322, pBR325, and pUC118), plasmids for Bacillus subtilis, plasmids for yeast, and plasmids for mammals. Is mentioned. Phage vectors include, but are not limited to, T7 phage display vectors and lambda phage vectors. Examples of viral vectors include, but are not limited to, animal viruses such as retroviruses, adenoviruses, adeno-associated viruses, vaccinia viruses, and Sendai viruses, and insect viruses such as baculoviruses. Examples of cosmid vectors include, but are not limited to, Lorist 6, Charomid 9-20, and Charomid 9-42. Examples of the phagemid vector include, but are not limited to, pSKAN, pBluescript, pBK, and pComb3H.

  The vector may include a regulatory sequence so that the target DNA can be expressed, a selection marker for selecting a vector containing the target DNA, a multicloning site for inserting the target DNA, and the like. Such regulatory sequences include promoters, enhancers, terminators, Shine-Dalgarno sequences or ribosome binding sites, replication origins, poly A sites, and the like. In addition, for example, an ampicillin resistance gene, a neomycin resistance gene, a kanamycin resistance gene, or the like can be used as a selection marker.

  Among the vectors, those intended to express the target DNA are called expression vectors. In the present invention, the vector is preferably a vector suitable for expression in mammalian cells, or a shuttle vector suitable for expression in a plurality of organisms (for example, pCAG-Bsd PA tag-C).

  In order to facilitate recovery of the fusion polypeptide, a DNA encoding a signal peptide sequence that enables the secretion of the peptide is linked to the 5 ′ end of the DNA encoding the target peptide, thereby connecting the generated peptide to the cell. It may be secreted outside. In this case, the fusion peptide moves to the cell membrane, the signal peptide is cleaved by the signal peptidase, and the target peptide is secreted and released into the medium. Alternatively, the target peptide accumulated in the cells can be recovered without adding a signal peptide. In this case, the cell can be physically or chemically destroyed, and the target peptide can be purified or recovered by a conventional method.

  In one aspect, the present invention relates to a cell comprising the above polynucleotide or vector. Host cells for introducing the vector include bacteria such as Escherichia coli and Bacillus subtilis, yeast cells, animal cells (eg, insect cells or mammalian cells), and plant cells, especially post-translational modifications such as glycosylation. Therefore, animal cells, particularly mammalian cells are preferred. These cells can be transformed or transfected by, for example, the calcium phosphate method, electroporation method, lipofection method, particle gun method, PEG method and the like. The transformed cells are cultured according to a usual method used for culturing host organisms. In order to select cells into which the gene of interest has been introduced, it is preferable to select cells using a selection marker.

In the present specification, the “cell containing the polynucleotide or vector” may be a cell in which the polynucleotide or vector is transformed or transfected, or a progeny cell thereof.
4). Inhibitor of Lectin Pathway and Alternative Pathway and Pharmaceutical Composition In one aspect, the present invention relates to an inhibitor of the lectin pathway and alternative pathway in the complement activation pathway, comprising the fusion polypeptide.

  In one aspect, the present invention relates to a composition comprising the fusion polypeptide, a polynucleotide encoding the fusion polypeptide, the vector, or the inhibitor, such as a food, a supplement, and a pharmaceutical composition.

  A polynucleotide encoding the above fusion polypeptide or a pharmaceutical composition comprising the above vector can act as a DNA vaccine in which the polypeptide encoded by the polynucleotide is expressed in vivo and exerts its function.

  The amount of the fusion polypeptide, the polynucleotide encoding the fusion polypeptide, and the vector contained in the composition of the present invention can be determined by those skilled in the art based on the subject's sex, weight, age, and the course of disease, etc. It can be determined as appropriate in consideration of various factors such as symptoms. For example, the amount of fusion polypeptide, polynucleotide or vector included in the composition of the present invention is not limited, but is about 0.001 to 100.0 mg, 0.010 to 10 mg, preferably 0.010 to 1.0 mg or 0.010 to 0.1. May be mg.

  In addition to the fusion polypeptide, the polynucleotide encoding the fusion polypeptide, or the vector, the composition of the present invention includes other components such as excipients, binders, disintegrants, surfactants, lubricants. An agent, a fluidity promoter, a flavoring agent, a coloring agent, and a fragrance may be included.

  The composition of the present invention can be formulated by a conventional method. For formulation, for example, the method described in Remington's Pharmaceutical Sciences (Merck Publishing Co., Easton, Pa.) Can be referred to.

  The dosage form is not particularly limited and is appropriately selected as necessary. Generally, oral forms such as tablets, capsules, granules, fine granules, powders, solutions, syrups, suspensions, emulsions, and elixirs are used. Or parenteral preparations such as injections, drops, suppositories, inhalants, transdermal absorbents, transmucosal absorbents, patches, ointments and the like.

  Examples of the excipient include starch, lactose, sucrose, mannitol, carboxymethylcellulose, corn starch, and inorganic salts.

  Binders include, but are not limited to, crystalline cellulose, crystalline cellulose / carmellose sodium, methylcellulose, hydroxypropylcellulose, low substituted hydroxypropylcellulose, hydroxypropylmethylcellulose, carboxymethylethylcellulose, starch, polyvinylpyrrolidone, and tragacanth Is mentioned.

  Examples of the disintegrant include, but are not limited to, crystalline cellulose, methylcellulose, low-substituted hydroxypropylcellulose, carmellose, carmellose calcium, carmellose sodium, croscarmellose sodium, starch, and tragacanth.

  Surfactants include, but are not limited to, soy lecithin, sucrose fatty acid ester, polyoxyl stearate, polyoxyethylene hydrogenated castor oil, sorbitan sesquioleate, sorbitan trioleate, polysorbate, glyceryl monostearate, and A sodium lauryl sulfate is mentioned.

  Lubricants include, but are not limited to, starch, stearic acid, stearate, hydrous silicon dioxide, light silicic anhydride, talc, waxes, hydrogenated vegetable oil, and polyethylene glycol.

  Examples of flow promoters include, but are not limited to, hydrous silicon dioxide, light anhydrous silicic acid, dry aluminum hydroxide gel, synthetic aluminum silicate, and magnesium silicate.

  Since the inhibitor or composition of the present invention can effectively inhibit the complement activation pathway by inhibiting the two pathways of the lectin pathway and the second pathway, it can treat the complement-related diseases and / or Can be used for prevention. As used herein, “complement-related disease” refers to a disease caused by excessive activation of the complement system. Examples of complement-related diseases include ischemia-reperfusion injury, age-related macular degeneration, systemic lupus erythematosus, antiphospholipid antibody syndrome (APS), IgA nephropathy, C3 nephropathy, membranoproliferative glomerulonephritis, joints Rheumatoid arthritis, malignant rheumatoid arthritis, IgG4-related disease, mixed cryoglobulinemia, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), myasthenia gravis Can be selected from the group consisting of symptom, Guillain-Barre syndrome, optic neuromyelitis, Alzheimer's disease, thrombotic microangiopathy (TMA), ulcerative colitis, Crohn's disease, and sepsis. The complement-related disease is preferably selected from the group consisting of ischemia-reperfusion injury, age-related macular degeneration, systemic lupus erythematosus, and IgA nephropathy, more preferably ischemia-reperfusion injury, age-related macular degeneration, or IgA nephropathy.

  Subjects to which the inhibitor or composition of the present invention can be administered include, but are not limited to, mammals, primates such as humans and chimpanzees, laboratory animals such as rats and mice, pigs, cows, horses , Domestic animals such as sheep and goats, and pets such as dogs and cats, preferably humans or mice, most preferably humans.

  In one aspect, the invention relates to a method for treating and / or preventing a complement-related disease comprising administering to the subject the inhibitor or composition. Description of the subject and complement-related diseases is omitted because they are as described for the inhibitor and composition.

  The dosage, administration interval, and administration period of the inhibitor or composition in this method should be considered by those skilled in the art in consideration of various factors such as the subject's sex, weight, age, and the course and symptoms of the disease. Can be determined as appropriate. For example, the dosage of the inhibitor or composition in this method is about 0.001 to 100.0 mg / kg body weight, 0.010 to 10 mg / kg body weight, preferably 0.010 to 1.0 mg / kg body weight, or 0.010 to 0.1 mg / kg body weight. It may be. The number of doses is not limited, but 3 times a day, 2 times a day, 1 time a day, 1 time every 2 days, 1 time every 3 days, once a week, 1 time every 2 weeks Once a month, etc. Further, the administration period is not limited, but may be 1 day, 2 days, 3 days, 1 week, 2 weeks, 1 month, 6 months, 1 year, or more.

  In one aspect, the invention relates to the use of the inhibitor or composition described above for treating and / or preventing a complement-related disease in a subject. Description of the subject and complement-related diseases is omitted because they are as described for the inhibitor and composition.

<Example 1: Production of fusion polypeptide of sMAP and factor H (polypeptide 1)>
(Materials and methods)
1) Cloning of mouse sMAP and factor H SCR1-5 domain cDNA RNA was extracted from mouse liver using TRIzol solution (Thermo Fisher Scientific), converted to cDNA using Advantage RT-for-PCR Kit (Clontech), and then used as a template. The cDNA of mouse sMAP and mouse factor H SCR1-5 domain was cloned by PCR using the primers shown in Table 1 below. PCR for amplification of sMAP was carried out in 35 cycles of 98 ° C. for 10 seconds, 70 ° C. for 15 seconds, and 72 ° C. for 1 minute 30 seconds. PCR for amplification of factor H SCR1-5 domain was performed in 35 cycles of 98 ° C for 10 seconds, 58 ° C for 15 seconds, and 72 ° C for 1 minute.

The obtained cDNA encoding mouse sMAP and cDNA encoding mouse factor H SCR1-5 domain were each incorporated into a pCR4 Blunt-TOPO vector using Zero Blunt TOPO PCR Cloning Kit for Sequencing (Thermo Fisher Scientific). The vector was transformed into Escherichia coli DH5α strain by a heat shock method at 42 ° C. for 1 minute, sprayed on LB agar medium supplemented with ampicillin, and cultured at 37 ° C. Twelve E. coli colonies were picked up and cultured in ampicillin-added LB liquid medium. The vector was purified using QIAGEN Plasmid Midi Kit (QIAGEN), the base sequence was decoded, and the vector carrying the target base sequence (base sequence encoding SCR1-5 domain of sMAP or fH) was selected.
2) Construction of sMAP-fH expression vector Linker sequence connecting sMAP and fH SCR1-5 domains using the template containing the nucleotide sequence encoding SCR1-5 domains of sMAP or fH obtained in 1) above as a template Mouse sMAP and mouse factor H SCR1-5 domain cDNA were amplified by PCR using additional primers. PCR conditions were 98 ° C. for 10 seconds, 75 ° C. for 15 seconds, and 72 ° C. for 1 minute 30 seconds in 35 cycles. The PCR primer sequences used for amplification are shown in the following table.

  Using the In-Fusion HD Cloning Kit (Clontech), the cDNA encoding the mouse sMAP and the cDNA encoding the mouse factor H SCR1-5 domain, to which the resulting linker sequence was added, were added to the PA-tagged expression vector pCAG-Bsd PA. It was incorporated into the multi-cloning site of tag-C (Wako Pure Chemical Industries).

The above expression vector was transformed into Escherichia coli DH5α strain by a heat shock method at 42 ° C. for 1 minute, sprayed onto LB agar medium supplemented with blasticidin and cultured at 37 ° C. Twelve E. coli colonies were picked up and cultured in LB liquid medium supplemented with blasticidin. The vector is purified using the QIAGEN Plasmid Midi Kit (QIAGEN), the base sequence is decoded and the target base sequence (sMAP (SEQ ID NO: 31), linker ((GGGGS) ₄ )), and fH SCR1-5 domains ( An expression vector carrying a fusion polypeptide (hereinafter simply referred to as sMAP-fH) containing SEQ ID NO: 35) in order from the N-terminus was selected.
3) Preparation of sMAP-fH The expression vector incorporating the gene encoding sMAP-fH obtained in 2) above was transfected into CHO cells using Fugene6 transfection reagent (Promega), and serum-free medium (SIGMA ALDRICH, Ex cell 325) was used to produce sMAP-fH in a 37 ° C 5% carbon dioxide environment. The culture supernatant was collected, and sMAP-fH was purified with an anti-PA antibody-binding affinity column (Wako Pure Chemical Industries) according to the attached instructions.
4) Western blotting of sMAP-fH The sMAP-fH obtained in 3) above was developed by SDS-PAGE on a 10% acrylamide gel, transferred to a PVDF membrane, and then blocked with Blocking one (Nacalai) for 30 minutes. Anti-mouse sMAP polyclonal antibody and HRP-labeled anti-rabbit antibody (Dako) or anti-human factor H antibody (abcom) and HRP-labeled (produced by immunizing rabbits with recombinant MASP-2 and purifying from the serum) Anti-sheep antibody (chemicon) was reacted at room temperature for 30 minutes each, and sMAP-fH was detected using ECL prime kit (GE health) according to the attached instructions.
5) Mass spectrometry analysis of sMAP-fH The sMAP-fH obtained in 3) above was developed on 10% acrylamide gel by SDS-PAGE and stained with CBB. A band corresponding to sMAP-fH was cut out, destained with 50% acetonitrile (Thermo Fisher Scientific), and centrifuged. A trypsin / lysyl endopeptidase solution (Wako Pure Chemical Industries, Ltd.) was added thereto to swell the gel and digested at 37 ° C. To this solution, add 40 μL of 0.1% TFA / 50% acetonitrile solution (Thermo Fisher Scientific), sonicate for 15 minutes, centrifuge to approximately 2 μL, add 50 μL of 0.1% TFA (Thermo Fisher Scientific), and add C18 Desalting and insoluble matter removal were performed using a column. 20 μL of 0.1% formic acid / 80% acetonitrile (Thermo Fisher Scientific) was added to this to elute the peptide fragment of sMAP-fH twice. After concentration to about 2 μL, 20 μL of 0.1% formic acid / 2% acetonitrile solution (Thermo Fisher Scientific), liquid chromatography mass spectrometry with QSTAR XL Hybrid LC / MS / MS System (Thermo Fisher Scientific) using C18 column (75μm x 15cm, 200nL / min) and PEAKS Studio (Bioinformatics Solutions) Data base search was performed to identify the target protein.
(result)
In order to obtain a fusion polypeptide (sMAP-fH) containing the SCR1-5 domains of sMAP and fH, an expression vector was prepared.

The base sequence encoding sMAP-fH is incorporated into the vector using PCR and In-Fusion method, and the base sequence of the obtained expression vector is decoded to obtain the target base sequence (sMAP, linker, and fH SCR1 An expression vector having a fusion polypeptide sMAP-fH (base sequence encoding a schematic diagram shown in FIG. 1) containing ˜5 domains in order from the N-terminal was selected. When this expression vector was transfected into CHO cells and the expressed polypeptide was subjected to CBB staining, a band was observed near the molecular weight corresponding to the target peptide (FIG. 2A). When Western blotting was performed on the expressed polypeptide, it reacted with both the anti-mouse sMAP polyclonal antibody and the anti-human factor H antibody, so that a fusion polypeptide containing the sMAP and factor H SCR1-5 domains was expressed. (Figs. 2B and C). In mass spectrometry analysis, as a result of data base search of the obtained data, the prepared sMAP-fH is a peptide of a domain corresponding to the mouse sMAP and a peptide of the domain corresponding to the mouse factor H SCR1-5 domain. (Data not shown).
<Example 2: In vitro complement inhibitory action by sMAP-fH>
(Materials and methods)
100 μL of 100 μg / mL Mannan (Sigma Aldrich M3640) solution diluted with 0.1 M Carbonate Buffer (pH 9.6) was added to each well on a Maxisorp plate (Nunc) and allowed to stand overnight at 4 ° C. in a humid environment. On the next day, PBS containing 1% BSA (SIGMA A7882) was used as a blocking solution, 200 μL each was added, and allowed to stand in a humid environment at room temperature for 2 to 3 hours to obtain a mannan-coated microtiter plate. The plate blocking solution was removed, and 8-12 week old Wild type C57BL / 6J mouse serum diluted to 2% with 5 mM CaCl ₂ -containing TBS solution and 125 sMAP-fH (polypeptide 1) prepared in Example 1 were used. After serially diluting from nM to 1000 nM, the mixture was reacted at 37 ° C. for 1 hour, added to a mannan-coated microtiter plate, and incubated at 37 ° C. for 20 minutes. After washing the plate 3 times with TBS containing 0.05% Tween and 5 mM CaCl ₂ , HRP-labeled goat anti-mouse C3 antibody (Kamiya biomedical company, Complement C3 ELISA Kit) was diluted 8000 times with TBS containing 5 mM CaCl ₂ and 100 μL per well. This was added at a time and allowed to react for 30 minutes at room temperature. 100 μL of TMB reagent (KPL) was added to each well, mixed with a micro mixer, and color development was observed. After 10 minutes, 100 μL of 1M phosphoric acid was added to each well to stop the reaction.

Absorbance (OD ₄₅₀ nm) was measured with Varioscan Lux (Thermo Fisher Scientific), and the degree of complement C3 deposition on the mannan plate (the ability to activate the complement lectin pathway and the second pathway) was measured.
(result)
In order to evaluate the inhibitory effect of sMAP-fH on the complement activation pathway in vitro, a C3 deposition assay reflecting the inhibitory effect of the complement activation pathway, which activates the second pathway via the lectin pathway, was performed. It was. As a result, mouse serum supplemented with sMAP-fH showed an inhibitory effect on C3 deposition in a dose-dependent manner with sMAP-fH (Fig. 3). From the above results, it was shown that sMAP-fH has the ability to inhibit the complement activation pathway in vitro.
<Example 3: Examination of in vivo effect of sMAP-fH>
(Materials and methods)
1) Change in blood concentration after intraperitoneal administration of sMAP-fH Mice (CLEA Japan, C57BL / 6J, female, 12-14 weeks old) were administered sMAP-fH 500 μg intraperitoneally. Blood was collected from the orbital venous plexus after 8, 24, and 48 hours. As a control, blood was also collected from mice administered PBS intraperitoneally. Add 50 μL of anti-PA antibody (Wako Pure Chemical Industries 016-25861) diluted to 10 ng / mL with carbonate buffer p9.6 to Maxisorp plate (Nunc) and leave it in a humidified environment at 4 ℃ overnight. Blocking was performed for 2 to 3 hours at room temperature using PBS containing 1% BSA. To this plate, the centrifuged serum was diluted with a PBS solution containing 0.1% BSA, and sMAP-fH having a known concentration prepared in Example 1 was added as a standard solution. After incubation for 1 hour at room temperature, react with HRP-labeled anti-PA antibody (Wako Pure Chemical Industries 015-25951) for 30 minutes at room temperature, add 100 μL of TMB reagent (KPL) to each well, mix with a micro mixer, and observe color development Ten minutes later, 100 μL of 1 M phosphoric acid was added to each well to stop the reaction. Absorbance (OD ₄₅₀ nm) was measured with Varioscan Lux (Thermo Fisher Scientific), and sMAP-fH concentration in blood was measured.
2) Inhibitory effect of lectin pathway in vivo after intraperitoneal administration of sMAP-fH
100 μL of 100 μg / mL mannan solution (SIGMA M3640) diluted with 0.1 M carbonate buffer pH 9.6 was added to each well on a Maxisorp plate (Nunc) and reacted at 4 ° C. in a wet state overnight. On the next day, after blocking with PBS containing 1% BSA, 100 μL of a sample obtained by diluting the mouse serum of 1) above to 2% with TBS containing 1% BSA and 5 mM CaCl ₂ was added and reacted at 37 ° C. in a humid environment for 30 minutes. Add 100 μL of human C4 (Hycult) diluted 1000-fold with 1% BSA and 5 mM CaCl _{2 in} TBS, react on ice for 30 minutes, then dilute with 1% BSA and 5 mM CaCl _{2 in} TBS goat anti-human C4 antibody (Capple) And allowed to react for 30 minutes at room temperature. Next, biotin-labeled anti-goat antibody (Dako) diluted with TBS containing 1% BSA and 5 mM CaCl ₂ was reacted for 30 minutes at room temperature, and then VECTASTAIN ABC Kit (Vector laboratories) was reacted according to the attached instructions, onto the mannan plate. The degree of deposition of complement C4 (the ability to activate the complement lectin pathway) was measured with an absorptiometer (OD450nm), and the relative value of C4 to the value of mice administered with PBS alone was determined.
3) Inhibitory effect of the second pathway in vivo after intraperitoneal administration of sMAP-fH 5 μL of zymosan (SIGMA Z4250) prepared to 20 mg / mL was added to 5 μL of the serum collected in 1) above, and 10 mM CaCl ₂ and The total volume was diluted to 50 μL with 10 mM MgCl ₂ -containing TBS solution, and reacted at 37 ° C. for 10 minutes. FITC-labeled anti-mouse C3 antibody diluted with TBS solution containing 10 mM CaCl ₂ and 10 mM MgCl ₂ after adding 10 mM EDTA (Dojindo Laboratories) and TBS containing 0.05% Tween to the reaction solution to stop complement activation and washing (Capple) is allowed to react on ice for 30 minutes, and the degree of binding of complement C3 to zymosan (the ability to activate the alternative pathway of the complement) is measured by MASP-2-deficient mouse serum (m2KO), before sMAP-fH administration (pre) Compared with the case where no serum was added (zymosan only), the C3 deposition was measured from the FITC value by flow cytometry.
(result)
In order to examine the kinetics of sMAP-fH in vivo, sMAP-fH was administered intraperitoneally and blood concentrations were measured after 1, 2, 4, 8, 24, and 48 hours. As a result, it was shown that the blood concentration of sMAP-fH gradually decreased until 48 hours, peaking at 1 hour after intraperitoneal administration (FIG. 4).

  Subsequently, in order to evaluate the inhibitory effect of sMAP-fH intraperitoneally on the lectin pathway in vivo, a C4 deposition assay reflecting the inhibitory effect of the lectin pathway was performed on the serum of mice administered sMAP-fH intraperitoneally. Used. As a result, compared to the PBS-administered group, the sMAP-fH-administered group showed an inhibitory effect on the C4 deposition level on the mannan-coated plate, and the effect was greatest in the serum 8 hours after intraperitoneal administration of sMAP-fH. (Figure 5).

  Furthermore, in order to evaluate the inhibitory effect of sMAP-fH intraperitoneally on the second pathway in vivo, a zymosan assay reflecting the inhibitory effect of the second pathway was performed on the serum of mice administered sMAP-fH intraperitoneally. Used. As a result, compared with the serum before sMAP-fH administration, the sMAP-fH administration group showed an inhibitory effect on the level of C3 deposition on zymosan, and the effect was greatest in the serum 4 hours after intraperitoneal administration of sMAP-fH. (Fig. 6).

From the above results, it was shown that sMAP-fH has the ability to inhibit the complement lectin pathway and the second pathway in vivo.
<Example 4: Production of fusion polypeptide of human sMAP and human factor H (linker (GGGGS) × 1) (polypeptide 2)>
(Materials and methods)
1) Cloning of cDNA of human sMAP and human factor H SCR1-5 domain The human factor H SCR1-5 domain cDNA with human sMAP and N-terminal linker sequence added by nested PCR using human liver cDNA as a template Cloning was performed using the primers in Table 3. In the first PCR, a cycle of 98 ° C. for 10 seconds, 63 ° C. for 15 seconds and 72 ° C. for 1 minute 30 seconds was performed in 35 cycles. The second PCR was carried out in 35 cycles of 98 ° C. for 10 seconds, 94.5 ° C. to 75 ° C. for 15 seconds, and 72 ° C. for 1 minute and 30 seconds. Table 3 shows the outer primer and inner primer in the nested PCR method.

2) Construction of human sMAP-fH expression vector The obtained cDNA encoding human sMAP and the cDNA encoding human factor H SCR1-5 domain with a linker sequence added to the N-terminus were converted into In-Fusion HD Cloning Kit (Clontech). Was incorporated into the multiple cloning site of the PA tag-added expression vector pCAG-Bsd PA tag-C (Wako Pure Chemical Industries). The vector was transformed into Escherichia coli DH5α strain by a heat shock method at 42 ° C. for 1 minute, sprayed on LB agar medium supplemented with ampicillin, and cultured at 37 ° C. Thirty E. coli colonies were picked up and cultured in ampicillin-added LB liquid medium. NucleoBond (registered trademark) Purify the vector using Xtra Midi Olus EF (MACHEREY-NAGEL), decode the nucleotide sequence, and include the desired nucleotide sequence (human sMAP, linker sequence, human fH SCR1-5 domain in this order from the N-terminal) A vector having the nucleotide sequence encoding the polypeptide was selected.
3) Preparation of human sMAP-fH The expression vector incorporating the gene encoding sMAP-fH obtained in 2) above was transfected into CHO cells using Fugene6 transfection reagent (Promega), and serum-free medium ( Using SIGMA ALDRICH, Ex cell 325), sMAP-fH was produced in a 37 ° C. 5% carbon dioxide environment. The culture supernatant was collected, and sMAP-fH was purified with an anti-PA antibody-binding affinity column (Wako Pure Chemical Industries) according to the attached instructions.
<Example 5: Production of fusion polypeptide of human sMAP and human factor H (linker (GGGGS) × 4) (polypeptide 3)>
In the same manner as in Example 4, a vector having a base sequence encoding a polypeptide containing human sMAP, linker (GGGGS) × 4, human fH SCR1-5 domain in this order from the N-terminus was constructed, and CHO Cells were transfected to express the polypeptide of interest, and the culture supernatant was collected and purified. The outer primer described in Table 3 was used. Table 4 shows the inner primers in the nested PCR method.

<Example 6: Production of fusion polypeptide of human sMAP and human factor H (linker (GGGGS) × 7) (polypeptide 4)>
An expression vector having a base sequence encoding a polypeptide containing human sMAP, linker (GGGGS) x 7, SCR1-5 domain of human fH in this order from the N-terminus in the same manner as in Example 4 was constructed, and CHO Cells were transfected, polypeptides were expressed and purified. The outer primer described in Table 3 was used. Table 5 shows the inner primers in the nested PCR method.

<Example 7: Confirmation of fusion polypeptide of human sMAP and human factor H (polypeptides 2, 3, 4)>
1) Western blotting Each polypeptide (polypeptides 2, 3 and 4) obtained in Examples 4, 5, and 6 was developed by SDS-PAGE on a 10% acrylamide gel, transferred to a PVDF membrane, and blocked with Blocking one ( Blocking was performed for 30 minutes. PVDF membrane was immunized with anti-mouse sMAP polyclonal antibody (prepared by immunizing rabbits with recombinant MASP-2 and purified from the serum) and HRP-labeled anti-rabbit antibody (Dako), or anti-human factor H antibody (abcam) and HRP-labeled Anti-sheep antibody (chemicon) was reacted at room temperature for 30 minutes each, and sMAP-fH was detected using ECL prime kit (GE health) according to the attached instructions.
2) Mass spectrometry analysis Each polypeptide obtained in Examples 4, 5, and 6 was developed by SDS-PAGE on a 10% acrylamide gel and stained with CBB. A band corresponding to sMAP-fH was cut out, destained with 50% acetonitrile (Thermo Fisher Scientific), and centrifuged. A trypsin / lysyl endopeptidase solution (Wako Pure Chemical Industries, Ltd.) was added thereto to swell the gel and digested at 37 ° C. To this solution, add 40 μL of 0.1% TFA / 50% acetonitrile solution (Thermo Fisher Scientific), sonicate for 15 minutes, centrifuge to approximately 2 μL, add 50 μL of 0.1% TFA (Thermo Fisher Scientific), and add C18 Desalting and insoluble matter removal were performed using a column. 20 μL of 0.1% formic acid / 80% acetonitrile (Thermo Fisher Scientific) was added to this to elute the peptide fragment of sMAP-fH twice. After concentration to about 2 μL, 20 μL of 0.1% formic acid / 2% acetonitrile solution (Thermo Fisher Scientific) was added, and liquid chromatography mass spectrometry was performed with QSTAR XL Hybrid LC / MS / MS System (Thermo Fisher Scientific) using a C18 column (75 μm x 15 cm, 200 nL / min), with PEAKS Studio (Bioinformatics Solutions) Data base search was performed to identify the target protein.
(result)
Western blotting was performed on each of the polypeptides obtained in Examples 4, 5, and 6, and both polypeptides reacted with both the anti-mouse sMAP polyclonal antibody and the anti-human factor H antibody. It was confirmed that a fusion polypeptide containing the sMAP and factor H SCR1-5 domains was expressed (data not shown). Moreover, in the mass spectrometry analysis performed about polypeptide 3 prepared in Example 5, as a result of data base search of the obtained data, the peptide of the domain corresponding to mouse sMAP and the mouse factor H SCR1-5 domain And a peptide corresponding to a domain corresponding to (data not shown).
<Example 8: Fusion polypeptide of human sMAP and human factor H (complement inhibitory action of polypeptides 2, 3, and 4)
(Materials and methods)
The inhibitory action of the three polypeptides (polypeptides 2, 3, and 4) prepared in Examples 4, 5, and 6 on the lectin pathway and the second pathway was determined using the Wieslab® Complement System Screen kit (Euro Diagnostica). Was used to evaluate. Normal human serum (N = 3) and each polypeptide were mixed, reacted at 37 ° C. for 1 hour, then added to a plate for evaluating each route, and incubated at 37 ° C. for 60 minutes. After washing 3 times with Wash buffer, 100 μL of each conjugate was added and allowed to react for 30 minutes at room temperature. After washing 3 times with wash buffer, 100 μL of substrate solution was added to each well and reacted at room temperature for 30 minutes, and the reaction was stopped with 5 mM EDTA. Absorbance (OD ₄₀₅ nm) was measured with Varioscan Lux (Thermo Fisher Scientific), and the degree of deposition of the complement activation final product MAC (Membrane Attack Complex) on the plate of each pathway was measured.

In the evaluation of the inhibitory action on the lectin pathway, fH, which is assumed to have no inhibitory action on the lectin pathway, was also evaluated. Further, in the evaluation of the inhibitory action of the second pathway, sMAP, which is assumed not to have the inhibitory action of the second pathway, was also evaluated.
(result)
The evaluation results of the inhibitory action of the lectin pathway and the second pathway are shown in FIG. The vertical axis represents the absorbance (%) when the absorbance in the absence of the polypeptide is 100% and the background absorbance is 0%. As shown in FIG. 7, in both the lectin pathway and the second pathway, a concentration-dependent decrease in absorbance was observed in the presence of polypeptides 2, 3, and 4, and both polypeptides 2, 3, and 4 It was found to inhibit both the lectin pathway and the second pathway.

  According to the present invention, a fusion polypeptide capable of inhibiting the lectin pathway and the second pathway, a polynucleotide encoding the fusion polypeptide, a vector comprising the polynucleotide, and an inhibitor or medicament for the lectin pathway and the second pathway comprising them Compositions and the like are provided. The present invention may make it possible to effectively treat and / or prevent complement related diseases.

  All publications, patents and patent applications cited herein are hereby incorporated by reference in their entirety.

Claims (18)

The MASP-2 protein or sMAP protein includes a first region containing the CUB1 domain and the EGF domain in order from the N-terminal side on the N-terminal side,
a second region containing 3 or more SCR domains selected from the group consisting of SCR1 to 4 domains of factor H protein, on the C-terminal side;
Fusion polypeptide. In the amino acid sequence in which the CUB1 domain is represented by SEQ ID NO: 1 or 3, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 1 or 3, or the amino acid sequence represented by SEQ ID NO: 1 or 3 An amino acid sequence in which one or more amino acids are added, deleted, and / or substituted,
In the amino acid sequence in which the EGF domain is represented by SEQ ID NO: 5 or 7, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 5 or 7, or the amino acid sequence represented by SEQ ID NO: 5 or 7 The fusion polypeptide according to claim 1, comprising an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted.   The first region includes SEQ ID NO: 49, or an amino acid sequence in which one amino acid is added, deleted, and / or substituted in the amino acid sequence represented by SEQ ID NO: 49 on the C-terminal side of the EFG domain. Item 3. The fusion polypeptide according to Item 1 or 2.   4. The fusion polypeptide according to any one of claims 1 to 3, wherein the second region comprises SCR1-4 domains.   The fusion polypeptide according to any one of claims 1 to 4, wherein the second region comprises an SCR5 domain.   The fusion polypeptide according to claim 5, wherein the second region comprises SCR1-5 domains in order from the N-terminal side. In the amino acid sequence in which the SCR1 domain is represented by SEQ ID NO: 9 or 11, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 9 or 11, or the amino acid sequence represented by SEQ ID NO: 9 or 11 An amino acid sequence in which one or more amino acids are added, deleted, and / or substituted,
In the amino acid sequence in which the SCR2 domain is represented by SEQ ID NO: 13 or 15, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 13 or 15, or the amino acid sequence represented by SEQ ID NO: 13 or 15 An amino acid sequence in which one or more amino acids are added, deleted, and / or substituted,
In the amino acid sequence in which the SCR3 domain is represented by SEQ ID NO: 17 or 19, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 17 or 19, or the amino acid sequence represented by SEQ ID NO: 17 or 19 An amino acid sequence in which one or more amino acids are added, deleted, and / or substituted,
In the amino acid sequence in which the SCR4 domain is represented by SEQ ID NO: 21 or 23, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 21 or 23, or the amino acid sequence represented by SEQ ID NO: 21 or 23 An amino acid sequence in which one or more amino acids are added, deleted, and / or substituted,
In the amino acid sequence in which the SCR5 domain is represented by SEQ ID NO: 25 or 27, the amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 25 or 27, or the amino acid sequence represented by SEQ ID NO: 25 or 27 Comprising an amino acid sequence in which one or more amino acids have been added, deleted, and / or substituted,
The fusion polypeptide according to claim 5 or 6. The first region is an amino acid sequence represented by SEQ ID NO: 29 or 31, an amino acid sequence having 90% or more identity to the amino acid sequence represented by SEQ ID NO: 29 or 31, or the amino acid represented by SEQ ID NO: 29 or 31 An amino acid sequence in which one or more amino acids are added, deleted and / or substituted in the sequence;
The second region is the amino acid sequence shown by SEQ ID NO: 33 or 35, the amino acid sequence having 90% or more identity to the amino acid sequence shown by SEQ ID NO: 33 or 35, or the amino acid shown by SEQ ID NO: 33 or 35 Comprising an amino acid sequence in which one or more amino acids have been added, deleted and / or substituted in the sequence;
The fusion polypeptide of claim 1.   The fusion polypeptide according to any one of claims 1 to 8, wherein the first region is linked to the second region via a linker.   The fusion polypeptide according to claim 9, wherein the linker is a peptide linker comprising 1 to 8 amino acid sequences represented by (GGGGS).   A polynucleotide encoding the fusion polypeptide according to any one of claims 1-10.   A vector comprising the polynucleotide of claim 11.   A cell comprising the polynucleotide according to claim 11 or the vector according to claim 12.   An inhibitor of the lectin pathway and the second pathway in the complement activation pathway, comprising the fusion polypeptide according to any one of claims 1 to 10.   A pharmaceutical composition comprising the fusion polypeptide according to any one of claims 1 to 10, the polynucleotide according to claim 11, or the vector according to claim 12.   16. The pharmaceutical composition according to claim 15, for inhibiting the lectin pathway and the second pathway in the complement activation pathway.   The pharmaceutical composition according to claim 16, for treating and / or preventing a complement-related disease.   Complement-related diseases are ischemia reperfusion injury, age-related macular degeneration, systemic lupus erythematosus, antiphospholipid antibody syndrome (APS), IgA nephropathy, C3 nephropathy, membranoproliferative glomerulonephritis, rheumatoid arthritis, malignant Rheumatoid arthritis, IgG4-related disease, mixed cryoglobulinemia, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), myasthenia gravis, Guillain The pharmaceutical composition according to claim 17, which is selected from the group consisting of Valley syndrome, optic neuromyelitis, Alzheimer's disease, thrombotic microangiopathy (TMA), ulcerative colitis, Crohn's disease, and sepsis.

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