Complement c5-binding protein

Abstract

The present invention relates to: a novel anti-C5 antibody that completely inhibits the activity of complement C5 by significantly reducing free C5 (unbound C5); and use thereof. The anti-C5 antibody according to the present invention can significantly reduce the concentration of free C5 in serum and simultaneously inhibit the classical and alternative pathways of a complement system. In addition, the anti-C5 antibody according to the present invention stably inhibited complement activity of both pathways even when the serum ratio was increased in in vitro hemolytic assays, and exhibits an excellent inhibitory effect compared to previously known antibodies, and such an inhibitory effect was consistently observed even under clinical conditions with high serum concentrations. Therefore, the anti-C5 antibody of the present invention can be effectively used for the prevention or treatment of various autoimmune diseases, including myasthenia gravis, age-related macular degeneration, and paroxysmal nocturnal hemoglobinuria.

Description

complement C5 binding protein

[0001] The present invention relates to a novel anti-C5 antibody that completely inhibits complement C5 activity by significantly inhibiting free C5 (unbound C5) and its use.

[0002] The complement system is a part of the innate immunity that plays a key role in host defense. It is activated by foreign pathogens such as microorganisms, viruses, cancer cells, and antigens, and induces an antibody immune response through opsonization.

[0003] The complement system is divided into the classical pathway, which is induced by antibodies, and the alternative pathway, which is activated independent of antibodies. The signaling pathway of the complement system starts from the C1 complex, which is composed of C1q / r / s (C1qrs), and is activated through C1 complex → C2, C4 activation → C3 activation → C5 activation. Proteins such as C3b and C5b generated during the complement activation process bind to cells to form a membrane attack complex (MAC), which creates holes in the cell membrane and eliminates pathogens or cells.

[0004] When complement activation is not properly regulated, the complement system attacks the body's own cells, leading to various autoimmune and inflammatory diseases, including paroxysmal nocturnal hemoglobinuria (PNH), hemolytic uremic syndrome (HUS), myasthenia gravis (gMG), and neuromyelitis optica. The pathogenesis of these diseases is believed to involve either or both the classical and alternative pathways.

[0005] Among them, complement C5 is a protein of the terminal pathway of complement activation, involved in both the classical and alternative pathways, and is a precursor protein of C5b, which forms the inflammatory mediator C5a and the cytolytic membrane attack complex (MAC) during C5 activation. C5a and MAC are known to mediate various inflammatory and autoimmune diseases.

[0006] Based on these mechanistic research results, there have been previous attempts to use antibodies that bind to complement C5 protein and block the C5 activation pathway as therapeutic drugs. However, existing drugs have limitations in that they do not completely inhibit C5 activity, requiring additional treatment or causing difficulties in daily life, such as increasing LDH (Lactate Dehydrogenase) levels above the normal range.

[0007] Therefore, in order to solve the above problems, there is a need to develop an improved antibody that can effectively inhibit C5 activity.

[0008] (Patent Document 1) US 2020-0254092 A1

[0009] One aspect is a pharmaceutical composition for preventing or treating a complement-related disease, comprising an effective amount of an anti-C5 antibody (hereinafter also referred to as antibody A) or an antigen-binding fragment thereof, which is a full-length antibody consisting of a heavy chain sequence of sequence number 1 and a light chain sequence of sequence number 2, wherein the anti-C5 antibody or antigen-binding fragment thereof provides a composition that suppresses free C5 to 130 ng / mL or less.

[0010] Another aspect is a pharmaceutical composition for preventing or treating a complement-related disease, comprising an effective amount of an anti-C5 antibody or an antigen-binding fragment thereof that specifically binds to the MG 4 domain of the complement C5 protein beta chain consisting of the amino acid sequence of SEQ ID NO: 10, wherein the anti-C5 antibody or antigen-binding fragment thereof suppresses free C5 to 130 ng / mL or less.

[0011] In one embodiment, it was confirmed that the anti-C5 antibody or its antigen binding protein inhibits the production of free C5 in serum, thereby inhibiting complement activity, thereby inhibiting not only classical pathway-mediated hemolysis but also alternative pathway-mediated hemolysis in patients with myasthenia gravis.

[0012] One aspect is a pharmaceutical composition for preventing or treating a complement-related disease, comprising an effective amount of an anti-C5 antibody or an antigen-binding fragment thereof, which is a full-length antibody consisting of a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO: 2, wherein the anti-C5 antibody or an antigen-binding fragment thereof suppresses free C5 in human serum to 130 ng / mL or less, preferably 50 ng / mL or less, more preferably 10 ng / mL or less, and most preferably 1 ng / mL, as measured by an electrochemiluminescence (ECL) immunoassay.

[0013] According to one embodiment of the present invention, the free C5 measurement method comprises an electrochemiluminescence (ECL) immunoassay, in which an anti-C5 antibody is coated on a Meso Scale Discovery (MSD) standard plate (ECL capable) to act as a capture agent. Thereafter, C5 contained in the standard (STD), quality control (QC) material, and sample is captured by the anti-C5 antibody as a capture agent, and the plate wells are thoroughly washed, and a biotinylated anti-human C5 antibody (e.g., eculizumab) is added to the plate wells. By doing so, the biotinylated anti-human C5 antibody binds to the free C5 captured by the anti-C5 antibody coated on the plate. Excess unbound conjugate is removed by additional washing of the wells, and then SULFO-TAG Streptavidin is added to the plate wells. The SULFO-TAG Streptavidin binds to the biotinylated anti-human C5 antibody bound to the C5 / anti-C5 antibody complex. After incubation with SULFO-TAG Streptavidin, the plate is washed, MSD read buffer is added, and the assay plate is then read using an MSD plate reader, where the electrochemiluminescence signal generated is proportional to the amount of C5 present in the tested standard (STD), quality control (QC) material, and sample. The concentration of C5 is then recalculated from a five-parameter 1 / y2 nonlinear regression of the measured standard concentrations.

[0014] According to one aspect, the anti-C5 antibody or antigen-binding fragment thereof of the present invention inhibits serum complement hemolytic activity, wherein the serum complement hemolytic activity is determined through an in vitro serum hemolytic test, and stably inhibits serum hemolysis even when the ratio of serum within the test conditions increases. The anti-C5 antibody or antigen-binding fragment thereof of the present invention exhibits superior serum hemolysis inhibition ability compared to competitive antibodies as the ratio of serum increases. The ratio of serum within the test conditions at this time may be 25% or more, preferably 50% or more, and more preferably 75% or more.

[0015] In the above in vitro serum hemolytic test, IC according to the ratio of serum 50 Although values ​​may be derived differently, IC values ​​for classical pathway mediated activity are in the range of 5 to 75 ug / ml, preferably 10 to 65 ug / ml, as calculated using GraphPad Prism Software. 50 may have an IC of 0.2 to 75 ug / ml, preferably 0.2 to 70 ug / ml, more preferably 0.25 to 65 ug / ml for alternative pathway mediated activity. 50 It can have value.

[0016] According to one aspect, the anti-C5 antibody or antigen-binding fragment thereof of the present invention is characterized by binding to complement C5 protein with high binding affinity and inhibiting complement activity. The binding affinity can be measured using Surface Plasmon Resonance (SPR), Bio-Layer Interferometry (BLI), Enzyme-linked immunosorbent assay (ELISA)-based competitive binding, etc. As an example, the anti-C5 antibody or antigen-binding fragment thereof of the present invention has a binding affinity of 1.0 X 10 when measured through surface plasmon resonance. -10 2.0 X 10 -10 Equilibrium dissociation constant at M level (equilibrium K D ) is characterized by having a value.

[0017] In addition, the anti-C5 antibody or antigen-binding fragment thereof of the present invention has a dissociation rate constant (K d ) is characterized by a low dissociation rate constant value, which means that the dissociation after binding between the antibody and the antigen occurs slowly and the binding between the antibody and the antigen is stably maintained. The anti-C5 antibody or antigen-binding fragment thereof of the present invention has a dissociation rate constant of 1 X 10 -5 8 X 10 -5 / s, preferably 2 X 10 -5 6 X 10 -5 / s, more preferably 3 X 10 -5 5 X 10 -5 / s level K d It can have value.

[0018] According to one aspect, the anti-C5 antibody of the present invention is a monoclonal antibody that specifically binds to the C5 protein. In addition, the anti-C5 antibody of the present invention may be an IgG isotype. Preferably, the IgG isotype of the anti-C5 antibody of the present invention may be IgG2 / 4. The IgG2 / 4 hybrid constant region may be a fusion form of the CH1 and hinge region of IgG2, the hybrid CH2 region of IgG2 and IgG4, and the CH3 region of IgG4. The anti-C5 antibody of the present invention may have excellent production yield and structural stability by having the IgG2 / 4 hybrid form.

[0019] Another aspect is a pharmaceutical composition for preventing or treating a complement-related disease, comprising an effective amount of an anti-C5 antibody or an antigen-binding fragment thereof that specifically binds to the MG 4 domain of the complement C5 protein beta chain consisting of the amino acid sequence of SEQ ID NO: 10, wherein the anti-C5 antibody or antigen-binding fragment thereof suppresses free C5 to 130 ng / mL or less.

[0020] As used herein, the term "anti-C5 antibody" refers to a humanized monoclonal antibody that binds to complement component 5 (C5) and acts at the final step in the complement cascade reaction. The anti-C5 antibody or antigen-binding fragment thereof specifically binds to the beta chain of C5, and specifically binds to the MG4 domain of the C5 beta chain. For example, as the bindable C5 protein, the amino acid sequence of the human C5 protein may be SEQ ID NO: 8, the amino acid sequence of the beta chain of the human C5 protein may be SEQ ID NO: 9, and the amino acid sequence of the MG4 domain of the beta chain of the human C5 protein may be SEQ ID NO: 10.

[0021] In one specific example, the antibody or antigen-binding fragment thereof may have cross-reactivity between species of the subject. The subject may be a vertebrate, such as a mammal, amphibian, reptile, bird, etc., and the species may be, for example, a human (Homo sapiens), a monkey, a rat, a mouse, etc.

[0022] In another specific example, the heavy chain sequence of SEQ ID NO: 1 may comprise a heavy chain variable region comprising HCDR1 of SEQ ID NO: 3, HCDR2 of SEQ ID NO: 4, and HCDR3 of SEQ ID NO: 5; and the light chain sequence of SEQ ID NO: 2 may comprise a light chain variable region comprising LCDR1 of SEQ ID NO: 6, LCDR2 of amino acid sequence YAS, and LCDR3 of SEQ ID NO: 7.

[0023] Additionally, the heavy chain constant region and light chain constant region of the antibody may be an antibody constant region of IgG, IgM, IgE, IgA, IgD, or a combination thereof. The constant region may be derived, for example, from an IgG2 / 4 (γ2 / 4-chain) antibody constant region.

[0024] As used herein, the term "epitope" refers to a protein determinant to which an antibody can specifically bind. Epitopes typically consist of chemically active surface molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural features as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished by the fact that binding to the former is lost in the presence of denaturing solvents, but not to the latter.

[0025] In one specific example, the anti-C5 antibody or antigen-binding fragment thereof that specifically binds to the MG4 domain of the beta chain of complement C5 protein comprising the amino acid sequence of SEQ ID NO: 10 can bind to an epitope within the beta chain of the C5 protein.

[0026] The term "antibody" as used herein refers to a glycoprotein that inactivates antigens such as viruses and bacteria and induces extracellular stimulation against microorganisms that have invaded the body, and particularly refers to immunoglobulins. Since the present invention relates to anti-C5 antibodies, the term "antibody" used without modification unless otherwise specifically designated may refer to an anti-C5 antibody that specifically binds to an epitope of C5. The scope of the present invention includes not only a complete antibody form that specifically binds to C5, but also an antigen-binding fragment of the antibody molecule. A complete antibody has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types, and has subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light chain constant region has kappa (κ) and lambda (λ) types.

[0027] Antigen-binding fragments of antibodies, or antibody fragments, refer to fragments that possess antigen-binding function, and include Fab, F(ab'), F(ab')2, and Fv. Among antibody fragments, Fab has a structure with the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv is the minimum antibody fragment that has only a heavy chain variable region and a light chain variable region, and recombinant technology for producing Fv fragments is disclosed in PCT International Patent Publication Nos. WO88 / 10649, WO88 / 106630, WO88 / 07085, WO88 / 07086, and WO88 / 09344. A two-chain Fv has a heavy chain variable region and a light chain variable region linked by a non-covalent bond, and a single-chain Fv (single-chain Fv, scFv) generally has a heavy chain variable region and a light chain variable region covalently linked by a peptide linker or directly linked at the C-terminus, so that it can form a dimer-like structure like a two-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, restriction digestion of whole antibodies with papain yields Fab fragments, and digestion with pepsin yields F(ab')2 fragments), or they can be produced using genetic recombination techniques.

[0028] In one embodiment, the antibody is in the Fv form (e.g., scFv) or in the full antibody form (IgG). In addition, the heavy chain constant region can be selected from any one of the gamma (γ), mu (μ), alpha (α), delta (δ), or epsilon (ε) isotypes. For example, the constant region is gamma 1 (IgG1), gamma 3 (IgG3), or gamma 4 (IgG4). The light chain constant region can be kappa or lambda type.

[0029] As used herein, the term "heavy chain (HC or CH)" refers to a full-length heavy chain and fragments thereof, including a variable region domain VH and three constant region domains CH1, CH2 and CH3, which comprise an amino acid sequence having sufficient variable region (VR) sequence to confer specificity to an antigen. In addition, "light chain (LC or CL)" refers to a full-length light chain and fragments thereof, including a variable region domain VL and a constant region domain CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.

[0030] The antibody may include a monoclonal antibody, a multispecific antibody, a human antibody, a humanized antibody, a chimeric antibody, a single-chain Fvs (scFv), a single-chain antibody, a Fab fragment, an F(ab') fragment, a disulfide-linked Fvs (sdFv), and an anti-idiotypic (anti-Id) antibody, or an epitope-binding fragment of the above antibodies. Specifically, the antibody may be a monoclonal antibody and a fully human antibody. That is, compared to a chimeric antibody, etc., it may have a further reduced antigenicity when administered to a human subject. The monoclonal antibody refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., identical except for possible naturally occurring mutations that may be present in trace amounts among the individual antibodies comprising the population. A monoclonal antibody is highly specific, being directed against a single antigenic site.

[0031] The above "humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region of the recipient antibody are replaced with residues from the hypervariable region of a non-human species (donor antibody) that retains the desired specificity, affinity, and ability, such as mouse, rat, rabbit, or non-human primate.

[0032] The above "human antibody" means a molecule derived from human immunoglobulin, and all amino acid sequences constituting the antibody, including the complementarity determining region and structural region, are composed of human immunoglobulin.

[0033] The above "chimeric antibodies" include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies that exhibit the desired biological activity.

[0034] The above "antibody variable domain" refers to the light chain and heavy chain portions of an antibody molecule comprising the amino acid sequences of the complementary determining regions (CDRs, i.e., CDR1, CDR2, and CDR3) and the framework regions (FR). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain. Each CDR is defined according to the Kabat definition, the Chothia definition, a combination of the Kabat definition and the Chothia definition, the IMGT numbering system, the AbM definition, or the contact definition of a CDR, and more preferably according to the IMGT definition.

[0035] "Complementarity Determining Region" (CDR, i.e., CDR1, CDR2, and CDR3) may refer to the amino acid residues of an antibody variable domain that are necessary for antigen binding. Each variable domain typically has three CDR regions, identified as CDR1, CDR2, and CDR3.

[0036] The term "framework region" (FR) as used herein refers to variable domain residues other than CDR residues. Each variable domain typically has four FRs, identified as FR1, FR2, FR3, and FR4.

[0037] As used herein, "antigen-binding fragment" refers to a fragment of an antibody having antigen-binding ability. An "Fv" fragment is an antibody fragment that contains a complete antibody recognition and binding site. This region consists of one heavy chain variable domain and one light chain variable domain. A "Fab" fragment contains the variable and constant domains of a light chain, and the variable and first constant domains (CH1) of a heavy chain. An F(ab')2 antibody fragment typically comprises a pair of Fab fragments covalently linked near their carboxy termini by hinge cysteines between them. A "single-chain Fv" or "scFv" antibody fragment comprises the VH and VL domains of an antibody, where these domains are present within a single polypeptide chain. The Fv polypeptide may additionally comprise a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.

[0038] The antibody or antibody fragment of the present invention may include not only the sequence of the anti-C5 antibody described herein, but also biological equivalents thereof, as long as it can specifically recognize C5. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid mutations are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan, and tyrosine are biologically functional equivalents.

[0039] When introducing the above mutations, the hydrophobicity index of the amino acid may be considered. Each amino acid is assigned a hydrophobicity index according to its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cysteine ​​(+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The hydrophobic amino acid index is crucial for imparting interactive biological functions to proteins. It is well known that amino acids with similar hydrophobic indices must be substituted to retain similar biological activity. When introducing mutations based on hydrophobic indices, substitutions are preferably made between amino acids with a difference in hydrophobicity index of ±2, more preferably ±1, or even ±0.5.

[0040] Meanwhile, it is also known that substitutions between amino acids having similar hydrophilicity values ​​result in proteins with equivalent biological activity. As disclosed in U.S. Patent No. 4,554,101, the following hydrophilicity values ​​are assigned to each amino acid residue: arginine (+3.0); lysine (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5±1); alanine (-0.5); histidine (-0.5); cysteine ​​(-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); Phenylalanine (-2.5); tryptophan (-3.4). Amino acid exchanges in proteins that do not alter the overall activity of the molecule are well known in the art (H. Neurath, RLHill, The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0041] Considering the mutations having the above-described biological equivalent activity, the antibody of the present invention or the nucleic acid molecule encoding the same can be interpreted as also including a sequence showing substantial identity with the sequence described in the sequence number. The substantial identity means a sequence showing at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology when the sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. NCBI Basic Local Alignment Search Tool (BLAST) is accessible from NBCI, etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn, and tblastx on the Internet.

[0042] Another aspect provides a nucleic acid encoding the antibody or an antigen-binding fragment thereof, an expression vector comprising the nucleic acid, and a cell transformed with the expression vector. Specifically, a nucleic acid encoding a heavy chain of SEQ ID NO: 1 and a light chain of SEQ ID NO: 2 is provided. For example, the nucleic acid encoding the heavy chain of SEQ ID NO: 1 may be represented by SEQ ID NO: 11, and the nucleic acid encoding the light chain of SEQ ID NO: 2 may be represented by SEQ ID NO: 12.

[0043] The nucleic acid encoding the antibody or antigen-binding fragment thereof of the present invention can be isolated and recombinantly produced. The nucleic acid can be further cloned (DNA amplification) or expressed by inserting it into a replicable vector. Based on this, the present invention relates to a vector comprising the nucleic acid from another perspective.

[0044] The term "nucleic acid" as used herein encompasses DNA (gDNA and cDNA) and RNA molecules. Nucleotides, the basic structural units of nucleic acids, include not only natural nucleotides but also analogues with modified sugar or base moieties. The sequences of nucleic acids encoding the heavy and light chain variable regions of the present invention may be modified. Such modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0045] The nucleic acid is also interpreted to include a nucleotide sequence that exhibits substantial identity to the nucleotide sequence. Substantial identity means a nucleotide sequence that exhibits at least 80% homology, more preferably at least 90% homology, and most preferably at least 95% homology, when the nucleotide sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0046] DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to DNA encoding the heavy and light chains of the antibody). Many vectors are available. Vector components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence.

[0047] The term "vector" as used herein refers to a means for expressing a target gene in a host cell, including plasmid vectors; cosmid vectors; viral vectors such as bacteriophage vectors, adenovirus vectors, retrovirus vectors, and adeno-associated virus vectors. In the vector, a nucleic acid encoding an antibody is operably linked to a promoter.

[0048] "Operably linked" means a functional association between a nucleic acid expression regulatory sequence (e.g., a promoter, a signal sequence, or an array of transcription factor binding sites) and another nucleic acid sequence, whereby the regulatory sequence regulates transcription and / or translation of the other nucleic acid sequence.

[0049] In the case of a prokaryotic cell as a host, it is common to include a strong promoter that can drive transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for translation initiation, and a transcription / translation termination sequence. Also, for example, when a eukaryotic cell is used as a host, promoters derived from the genome of mammalian cells (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or promoters derived from mammalian viruses (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, Moloney virus promoter, Epstein-Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter) can be used, and generally have a polyadenylation sequence as a transcription termination sequence. In some cases, the vector may be fused with other sequences to facilitate purification of the antibody expressed therefrom. The sequences to be fused include, for example, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6xHis (hexahistidine; Quiagen, USA). The vector includes antibiotic resistance genes commonly used in the art as selectable markers, for example, resistance genes for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0050] In another aspect, the present invention relates to a cell transformed with the above-mentioned vector. The cell used to produce the antibody of the present invention may be, but is not limited to, a prokaryotic, yeast, or higher eukaryotic cell. Prokaryotic host cells such as strains of the genus Bacillus, such as Escherichia coli, Bacillus subtilis, and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis, and Staphylococcus (e.g., Staphylococcus carnosus) can be used. However, animal cells are of greatest interest, and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO / -DHFR, CV1, HEK293, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2 / 0, NS-0, U20S, or HT1080.

[0051] The present invention relates to a method for producing an antibody or an antigen-binding fragment thereof, comprising the steps of (a) culturing the cells; and (b) recovering the antibody or an antigen-binding fragment thereof from the cultured cells. The cells can be cultured in various media. Any commercially available media can be used as the culture medium without limitation. Any other necessary supplements known to those skilled in the art may also be included at appropriate concentrations. Culture conditions, such as temperature, pH, etc., are already used with host cells selected for expression and will be apparent to those skilled in the art. The antibody or antigen-binding fragment thereof can be recovered by removing impurities, for example, by centrifugation or ultrafiltration, and the resulting product can be purified, for example, by affinity chromatography. Additional purification techniques, such as anion or cation exchange chromatography, hydrophobic interaction chromatography, and hydroxylapatite chromatography, can also be used.

[0052] In addition, another aspect provides a method for preventing or treating a complement-related disease, comprising administering to a patient an effective amount of an anti-C5 antibody or antigen-binding fragment thereof according to the present invention.

[0053] The specific details of the anti-C5 antibody or antigen-binding fragment thereof are as described above. As demonstrated in the examples below, the antibody or antigen-binding fragment thereof of the present invention can effectively inhibit not only classical pathway-mediated hemolysis but also alternative pathway-mediated hemolysis, and thus can be used for the prevention and / or treatment of complement-related diseases.

[0054] Complement-related diseases to which the composition is applied are inflammatory or autoimmune-related diseases, and may be, for example, generalized myasthenia gravis (gMG), age-related macular degeneration (AMD), paroxysmal nocturnal hemoglobinuria (PNH), etc.

[0055] The above “prevention” means any act of inhibiting or delaying the progression of a complement-related disease by administering the above composition, and “treatment” means inhibiting, alleviating or eliminating the development of a complement-related disease.

[0056] In one specific example, the composition may include impurities derived from the host organism, such as host cell proteins (HCPs), host cell DNA, serum, enzymes, and viruses. In addition, the composition may include additives that may remain in the culture medium or bioreactor (e.g., antibiotics, insulin, IPTG, DMSO, endoplasmic reticulum, serum-derived antibodies, etc.), enzymes that may be used for degradation purposes, agents used in purification processes and columns (e.g., protein A, elution buffer, etc.), unbound linkers, drugs, or PEG components, binding reactants, and substances extracted and leached from the contact surface during the production and purification processes.

[0057] The above impurities are major immunogenic risk factors that can cause unpredictable immune responses when introduced into the body, and thus can potentially affect the stability and efficacy of the drug. Therefore, it is necessary to remove the above impurities to an acceptable level.

[0058] Specifically, the composition according to one aspect may comprise preferably 100 ng or less, more preferably 40 ng or less of host cell protein (HCP) per 1 mg of the anti-C5 antibody or antigen-binding fragment thereof.

[0059] That is, the composition according to the aspect has the characteristics of excellent quality and improved drug stability due to the removal of impurities such as host cell proteins and / or host cell DNA.

[0060] In another embodiment, the composition may comprise an acidic variant. The "acidic variant" is a variant of a primary species antibody that is more acidic than the primary species antibody, characterized by gaining a negative charge or losing a positive charge compared to the primary species antibody. Such acidic variants can be separated using a separation method, such as ion exchange chromatography, which separates proteins based on charge. The acidic variant of the primary species antibody elutes earlier than the primary peak when separated by cation exchange chromatography.

[0061] The acidic variants may comprise one, two, three, four, or five of a glycosylated variant, a deamidated variant, a disulfide-reduced variant, a sialylated variant, and a non-reducing variant. In addition, the total amount of all acidic variants in the composition may preferably be 25% or less, more preferably 20% or less.

[0062] Therefore, the composition according to the aspect can prevent adverse reactions that may occur in monoclonal antibody drugs by including acid variants that may affect the efficacy and stability of the drug in amounts less than a certain amount.

[0063] Pharmaceutically acceptable carriers included in the composition of the present invention are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. In addition to the above components, the composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like.

[0064] The pharmaceutical composition of the present invention can be administered orally or parenterally. Parenteral administration is preferred, and parenteral administration can be administered via intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, intradermal administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. In one embodiment, the composition can be administered via intravenous injection. Since proteins or peptides are digested during oral administration, oral compositions must be formulated to coat the active agent or protect it from degradation in the stomach. Furthermore, the pharmaceutical composition can be administered via any device capable of transporting the active agent to target cells.

[0065] The appropriate dosage of the composition according to the present invention varies depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and response sensitivity, and a physician skilled in the art can easily determine and prescribe a dosage effective for the desired treatment or prevention. The term "pharmaceutically effective amount" as used herein means an amount sufficient to prevent or treat myasthenia gravis (gMG), age-related macular degeneration (AMD), paroxysmal nocturnal hemoglobinuria (PNH), etc.

[0066] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0067] The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.

[0068] The anti-C5 antibody according to the present invention can significantly reduce the level of free C5 and stably inhibit the activity of complement even when the amount of complement increases due to an increase in the ratio of serum under test conditions, and can completely inhibit complement C5 activity by effectively inhibiting both the classical pathway (CP) and the alternative pathway (AP) of the complement system, and can be used for the prevention or treatment of various autoimmune diseases including myasthenia gravis, age-related macular degeneration, and paroxysmal nocturnal hemoglobinuria.

[0069] Figure 1 shows the structure of a vector used in the production of anti-C5 antibodies according to one aspect.

[0070] Figure 2 shows the results of measuring the purity of anti-C5 antibodies according to the daily aspect using size exclusion chromatography.

[0071] Figure 3 shows the results of measuring the purity of anti-C5 antibodies according to the daily aspect using an imaging capillary isoelectric focusing method.

[0072] Figure 4 shows the results of measuring the content of HCP, an impurity of anti-C5 antibody, according to the daily aspect.

[0073] Figure 5 shows the results of confirming the complement-mediated hemolysis inhibition activity of anti-C5 antibodies according to the daily aspect, and shows the results of experiments performed under different types of serum (Figure 5a: serum from patients with anti-acetylcholine receptor antibody positive myasthenia gravis; Figure 5b: serum from patients with anti-acetylcholine receptor antibody negative myasthenia gravis; Figure 5c: serum from patients with ocular myasthenia gravis) within the experimental conditions.

[0074] Figure 6 shows the results of free C5 measurement according to the dose and time of anti-C5 antibody according to the daily aspect.

[0075] Figures 7 to 10 show the results of measuring free C5 in human serum of anti-C5 antibodies according to one aspect, depending on the dose of anti-C5 antibodies (Figure 7: 1600 mg, Figure 8: 3200 mg, Figure 9: 6400 mg, Figure 10: 9200 mg).

[0076] Figure 11 shows the free C5 levels in human serum of eculizumab, an FDA-approved anti-C5 therapeutic.

[0077] Figure 12 shows the free C5 levels in human serum of ravulizumab, an FDA-approved anti-C5 therapeutic.

[0078] Figures 13 to 16 show the results of confirming complement-mediated hemolysis inhibition activity according to the classical pathway using anti-C5 antibody (antibody A) and a competitor anti-C5 antibody according to one aspect, and are the results of experiments performed by varying the serum ratio within the experimental conditions (Figure 13: 10%; Figure 14: 25%; Figure 15: 50%; Figure 16: 75%).

[0079] Figures 17 to 20 show the results of confirming the complement-mediated hemolysis inhibition activity according to an alternative pathway using an anti-C5 antibody (antibody A) and a competitor anti-C5 antibody according to one aspect, and are the results of experiments performed by varying the serum ratio within the experimental conditions (Figure 17: 10%; Figure 18: 25%; Figure 19: 50%; Figure 20: 75%).

[0080] Hereinafter, the present invention will be described in detail.

[0081] Hereinafter, preferred examples are presented to aid in understanding the present invention. However, the following examples are provided solely to facilitate a better understanding of the present invention, and the scope of the present invention is not limited by the following examples.

[0082] [Example]

[0083] Anti-C5 Antibody Information

[0084] The anti-C5 antibody of the present invention (hereinafter also referred to as antibody A) was prepared and used according to the following Example 1.

[0085] Eculizumab (Soliris, Alexion), pozelimab (Veopoz, Regeneron), ravulizumab (Ultomiris, Alexion), tesidolumab (MorphoSys / Novartis), and crovalimab (Piasky, Roche / Genentech), used as comparators, were purchased or produced in-house.

[0086] In case of in-house production, eculizumab was manufactured with reference to the information disclosed in https: / drugs.ncats.io / substance / A3ULP0F556, pozelimab with reference to WHO drug information, vol 33, no.3, 2019, ravulizumab with reference to the sequence disclosed in https: / drugs.ncats.io / substance / C3VX249T6L, tesidolumab with reference to the sequence disclosed in https: / drugs.ncats.io / drug / 3FO6689MSD, and crovalimab with reference to the sequence disclosed in https: / gsrs.ncats.nih.gov / ginas / app / ui / substances / H9KH1GP3UU.

[0087] Example 1. Production and screening of recombinant anti-C5 antibodies

[0088] 1-1. Production of anti-C5 antibody cell lines

[0089] A recombinant anti-C5 antibody with improved C5 inhibitory efficacy and safety was produced. Specifically, the respective DNAs for the heavy and light chains of the anti-C5 antibody were provided by the Antibody Engineering Laboratory of Professor Jeong Jun-ho at Seoul National University and used, and were inserted into vector A and vector C, respectively, by digestion with NheI (NEB, R0131S) and PmeI (NEB, R0560S) enzymes. Vector A, into which the anti-C5 antibody heavy chain DNA was inserted, and vector C, into which the anti-C5 antibody light chain DNA was inserted, were designated vector B and vector D, respectively (see Figure 1). The DNA sequences of the expression vectors were confirmed through DNA sequencing analysis.

[0090] CHO-K1 cells (purchased from ATCC, CCL-61) were seeded at 3 × 10 per well in MEM-α medium (Gibco, 12561) supplemented with 10% dFBS (Gibco, 30067-334) for transfection. 5After inoculation with the cells, they were cultured, and a total of 2.5 μg of DNA was transfected into the cells using Lipofectamine™ LTX Reagent (Invitrogen, 15338) according to the manufacturer's instructions.

[0091] Three days after transformation, the transformed cells were screened by seeding them at a density of 1,000 cells / well (200 μL) in SFM4CHO medium (HyClone, SH30549.02) without hypoxanthine and thymidine and supplemented with 500 nM MTX (Yuhan, A193722). The cells were cultured in an incubator at 37.0±1°C and 5.0±1% CO2. After approximately 3 weeks, when the cells covered more than 70% of the bottom of the well, the titer was measured by ELISA. Based on the above results, cells with high titers were sequentially cultured in 24-well plates, 12-well plates, 6-well plates, and 125 mL shake flasks.

[0092] Afterwards, stability tests were performed for 30 passages on cell lines with high titers. Specifically, 0.3×10 6 The above cell lines were inoculated into 125 mL shake flasks containing 25 mL of SFM4CHO medium at a concentration of cells / mL and subcultured every 3 days. Cells were cultured in an incubator at 37.0±1°C and 5.0±1% CO2 with shaking (120 rpm). The final RCB (Research Cell Bank) of the anti-C5 antibody was selected based on stability test and batch culture results (production stability, maximum titer, and quality consistency).

[0093] The sequences of the selected anti-C5 antibodies are shown in Table 1 below.

[0094] Anti-C5 antibody (Antibody A) Sequence number Sequence HCDR13GFSFSGRYHCDR24SGWPGATGDTHCDR35AREPVAWGGGLDLLCDR16QSINNQLCDR2-YASLCDR37QGSYYSGGWDYGHeavy chain 1EVQLVESGGG LVQPGGSLRL SCAASGFSFS GRYWIQWVRQ APGKGLEWVA SGWPGATGDT NYANWAKGRF TISRDDSKNT LYLQMNSLRA EDTAVYYCAR EPVAWGGGLD LWGQGTLVTV SSASTKGPSV FPLAPCSRST SESTAALGCL VKDYFPEPVT VSWNSGALTS GVHTFPAVLQ SSGLYSLSSV VTVPSSNFGT QTYTCNVDHK PSNTKVDKTV ERKCCVECPP CPAPPVAGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSQE DPEVQFNWYV DGVEVHNAKT KPREEQFNST YRVVSVLTVL HQDWLNGKEY KCKVSNKGLP SSIEKTISKA KGQPREPQVY TLPPSQEEMT KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSR LTVDKSRWQE GNVFSCSVMH EALHNHYTQK SLSLSLGKLight Chain2DIQMTQSPSS LSASVGDRVT ITCQASQSIN NQLSWYQQKP GKAPKLLIYY ASTLASGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQG SYYSGGWDYG FGQGTKVEIK RTVAAPSVFI FPPSDEQLKS GTASVVCLLN NFYPREAKVQ WKVDNALQSG NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

[0095] 1-2. Anti-C5 antibody culture

[0096] The final cell line selected in Example 1-1 was cultured in large quantities in a seed bioreactor to produce anti-C5 antibodies. Specifically, the vial of the anti-C5 antibody cell line was quickly thawed by placing it in a 37.0°C water bath, and then inoculated into a 125 mL shake flask containing SFM4CHO medium and cultured in a 37.0±1°C, 5.0±1% CO2 incubator. The shake flask culture was expanded through several passages for inoculation into an N-3 Seed Bioreactor. During the culture process of the anti-C5 antibody, the inoculated cell concentration during passage was approximately 0.3 x 10 6 The concentration was maintained at 100 cells / mL, and the medium used was SFM4CHO.

[0097] After inoculation into the N-3 Seed Bioreactor, the dissolved oxygen content was maintained at 10–30%, the culture temperature was 37±1℃, and the pH was 6.9±0.2. The culture medium was periodically collected and the cell status was observed under a microscope. In addition, the cell number, cell activity, pH, and osmotic pressure were analyzed, and passage was performed when sufficient cell growth was achieved to match the initial inoculation concentration of the N-2 Seed Bioreactor.

[0098] Afterwards, when inoculation was completed in the N-2 Seed Bioreactor, the reactor was maintained under the same conditions as above, the cells were analyzed using the same method as above, and when sufficient cell growth was achieved as the initial inoculation concentration of the N-1 Seed Bioreactor, passage was performed.

[0099] Afterwards, when inoculation was completed in the N-1 Seed Bioreactor, the reactor was maintained under the same conditions as above, and cells were analyzed in the same manner as above, except that metabolites were additionally analyzed. When sufficient cell growth was achieved to match the initial inoculation concentration of the 1000L Main Bioreactor, passage was performed.

[0100] Afterwards, when the inoculation in the 1000L Main Bioreactor was completed, the reactor was maintained under the same conditions as above, except that a cell booster (Hyclone) was periodically added to increase the cell number during culture and the culture temperature was maintained at 35±1℃ from 6 days after culture to maintain cell activity. In addition, the cells were analyzed using the same method as above, and the glucose and glutamine supply amounts were adjusted according to the analysis results to ensure cell growth.

[0101] After inoculating a 1000L culture vessel, when cell activity rapidly decreased, cell culture was stopped and cells were separated from the culture medium using a Millipore POD filter system. The culture supernatant from which cells were separated was filtered using a 0.1 µm membrane filter, collected in a container, and stored at ambient temperature.

[0102] 1-3. Anti-C5 antibody purification

[0103] To purify the anti-C5 antibody from the culture solution obtained in the above Examples 1-2, a two-step chromatography, concentration, and buffer exchange were performed. Specifically, affinity chromatography was performed using MabSelect PrismA (Cytiva) resin to obtain the anti-C5 antibody from the culture recovery solution. The column was equilibrated with an equilibration buffer solution (pH 5.8-6.2) containing 50 mM sodium phosphate, and then the culture recovery solution was loaded. After loading, the column was washed once with the equilibration buffer solution, then twice with a washing buffer solution containing sodium chloride, and then a third wash with the equilibration buffer solution. After the third wash, the target protein was eluted using an elution buffer solution (pH 3.4-3.6) containing 50 mM sodium citrate.

[0104] Viruses that could originate from host cells or auxiliary materials used in the process were inactivated using low pH conditions. Specifically, 1 M citric acid was added to the eluate obtained from the MabSelect PrismA chromatography step to adjust the pH to 3.5–3.7. After approximately 1 hour, 1 M Tris was added to adjust the pH to 5.6–6.0.

[0105] After virus inactivation was completed, mixed mode chromatography was performed using Capto Adhere (Cytiva) resin to remove impurities. The column was equilibrated with equilibration buffer (pH 5.6–6.0) containing 20 mM histidine and a small amount of sodium chloride, and then the virus inactivation solution was loaded. After loading was completed, the equilibration buffer was additionally loaded to recover the target protein with high purity (SE-HPLC, 99.0% or higher) and low impurity content (HCP, 100 ng or less).

[0106] To remove viruses that could be derived from host cells or auxiliary materials used in the process, ultrafiltration was performed using a virus filter (Virosart HF, Sartorius). After equilibration with equilibration buffer (pH 5.6–6.0) containing 20 mM histidine and a small amount of sodium chloride, the eluate obtained from the mixed-mode chromatography step was loaded. After loading was completed, the target protein was recovered by additional loading of the equilibration buffer.

[0107] An ultrafiltration device (Tangential Flow Filtration Membrane System) was used to adjust the protein concentration of the anti-C5 antibody and to exchange the purified protein with a formulation buffer. The membrane (Merck, Cutoff: 30 kDa) in the ultrafiltration device was washed with water for injection and equilibrated with an equilibration buffer (pH 5.6–6.0) containing 20 mM histidine and a small amount of sodium chloride. After concentrating the target protein to a concentration of approximately 60 mg / mL or more, buffer exchange was performed with a buffer solution (pH 5.3–5.7) containing 20 mM succinate and 60 mM Arg-HCl. Additional concentration was performed after buffer exchange was completed. After further concentration was completed, sucrose and polysorbate 80 were added to adjust the target protein concentration and formulation of the anti-C5 antibody (anti-C5 antibody protein concentration: 108-132 mg / mL. Formulation: 20 mM Succinate, 50 g / L sucrose, 60 mM Arg-HCl, 0.045 % (w / v) PS80).

[0108] Example 2. Measurement of purity and impurity content of anti-C5 antibody

[0109] 2-1. Purity measurement of anti-C5 antibody (1)

[0110] The purity of the anti-C5 antibody purified in Example 1 was measured using size exclusion chromatography. Specifically, the purity of the substance was measured using an Alliance 2695 HPLC system (UV / Vis detector) and analyzed using Empower 3 software. The column used was a TSKgel G3000SWXL (Tosoh Biosecience), and analysis was performed at a flow rate of 1.0 mL / min.

[0111] As a result, as shown in Fig. 2, the anti-C5 monomer was detected after about 7.7 minutes, and it was confirmed that it maintained a purity of 99.0% or more.

[0112] 2-2. Purity measurement of anti-C5 antibody (2)

[0113] To analyze the purity of the anti-C5 antibody purified in Example 1, imaged capillary isoelectric focusing was used. The imaged capillary isoelectric focusing is an analytical method mainly used to analyze the purity by confirming the charge variant of a substance. To measure the charge variant of anti-C5, imaged capillary electrophoresis (Protein Simple, iCE3) was used, and analysis was performed using Empower 3 software. The cartridge used was an Fc-coated capillary cartridge (Protein Simple), and the analysis was performed under electrophoretic conditions of 1500 V (1 min) and 3000 V (5 min).

[0114] As a result, as shown in Table 2 and Figure 3 below, it was confirmed that the acidic, basic, and main peaks of the anti-C5 antibody were observed within two pI markers (pI 4.22 / pI 9.50).

[0115] Batch scale1000L1000L1000LTest Item Acceptance StandardBatch 1Batch 2Batch 3icIEFMain47 - 89 %67 %59 %64 %Acidic0 - 43 %18 %18 %20 %Basic0 - 38 %14 %23 %17 %

[0116] 2-3. Measurement of impurity content in anti-C5 antibody

[0117] The impurity content of the anti-C5 antibody purified in Example 1 was measured. First, the content of host cell protein (HCP), one of the impurities, was measured using the CHO kit F550-1 (Cygnus technologies) and analyzed according to the manufacturer's instructions. Specifically, 100 μL of anti-CHO-HRP was added to each well of the plate provided in the kit. 50 μL each of the antibody of Example 1, the control, and the diluted sample were added to the plate wells treated with anti-CHO-HRP. The plate was covered with a sealer and stirred at 400–600 rpm for approximately 2 hours at room temperature. Upon completion of the reaction, the plate was washed four times with the wash solution provided in the kit. Afterwards, 100 μL of TMB reagent was added and the reaction was allowed to proceed at room temperature for approximately 30 minutes. Upon completion of the reaction, 100 μL of stop solution was added, and the absorbance was measured at 450 / 650 after approximately 5 minutes. The content of HCP, an impurity during the process of anti-C5, was calculated by considering the dilution factor.

[0118] Next, host cell DNA (HCD) was measured using the resDNASEQ Quantitative CHO DNA kit (including Nucleic Acid Extraction kit and Residual DNA Sample Prep kit, Applied Biosystems), and analyzed according to the manufacturer's instructions provided with the kit. In addition, residual protein A (RCA) was measured using Protein A ELISA for detection of MabSelect SuRe™ (Repligen), and analyzed according to the manufacturer's instructions provided with the kit.

[0119] As a result, as shown in Table 3 and Figure 4 below, it was confirmed that the HCP content was very low, at 100 ng / mg or less.

[0120] Batch scale1000L1000L1000LTest item acceptance criteriaBatch 1Batch 2Batch 3HCP≤ 100 ng / mg23 ng / mg35 ng / mg33 ng / mg

[0121] Example 3. Confirmation of binding affinity between anti-C5 antibody and complement C5.

[0122] In order to confirm the binding affinity of the anti-C5 antibody purified in Example 1 above to human complement C5 protein, the binding affinity between the antibody and the protein was measured using the surface plasmon resonance (SPR) method.

[0123] Specifically, anti-human IgG (Fc) antibody was immobilized onto the surface of a sensor chip (CM5, GE Healthcare, BR-1005-30) using a human antibody capture kit (GE Healthcare, BR-1008-39), and then anti-C5 antibody (0.5 μg / mL) was injected to immobilize the anti-C5 antibody onto the surface of the sensor chip. Anti-C5 antibodies were prepared using antibody A of the present invention and competitive anti-C5 antibodies such as eculizumab, ravulizumab, crobalimab, tesidolumab, and pozellimab.

[0124] Human complement C5 protein (Acrobiosystems, CO5-H52Ha) was diluted two-fold from a concentration of 20 nM to 80 nM to a concentration of 0.0625 nM or 2.5 nM, and then injected onto the sensor chip immobilized with anti-C5 antibody to determine the association rate constant (K) between anti-C5 antibody and complement C5. a , association rate constant) and dissociation rate constant (K d , dissociation rate constant) and calculate the equilibrium dissociation constant (K D , equilibrium dissociation constant) was analyzed.

[0125] As a result, as shown in Table 4 below, antibody A, the anti-C5 antibody of the present invention, had an average of 1.757 X 10 -10 Equilibrium dissociation constant at M level (K D ) and confirmed that it has excellent binding affinity.

[0126] Additionally, for antibody A, the dissociation rate constant (K) was higher than that of competing anti-C5 antibodies Eculizumab, Ravulizumab, Crovalimab, Tesidolumab, and Pozelimab. d ) was shown to be low, confirming that the binding was maintained stably for a long time after binding to the C5 protein.

[0127] Sample K a (1 / Ms)K d (1 / s)K D (M)Rmax(RU)Chi 2 (RU 2 )U-valueSE(ka)SE(kd)Antibody A- 1st test2.174X10 5 4.139X10 -5 1.903X10 -10 382.76.815.1761.2x 10 -6 Antibody A-2nd test 2.270X10 5 3.656X10 -5 1.611X10 -10 287.64.919.7881.3x 10 -6 Average 2.222X10 5 3.898X10 -5 1.757X10 -10 -----SD6.748X10 3 3.410X10 -6 2.068X10 -11 -----Eculizumab- 1st test 1.172X10 6 1.168X10 -4 9.965X10 -11 359.42.24.12.3X 10 2 6.8X10 -7Eculizumab- 2nd trial 1.291X10 6 1.119X10 -4 8.667X10 -11 270.42.24.13.3X 10 2 8.9X10 -7 Average 1.231X10 6 1.143X10 -4 9.316X10 -11 -----SD8.394X10 4 3.484X10 -6 9.181X10 -12 -----Ravulizumab - 1st trial 3.406X10 5 1.499X10 -3 4.402X10 -9 35.20.21.12.8X 10 2 2.8X10 -6 Ravulizumab - 2nd trial 3.409X10 5 1.471X10 -3 4.315X10 -9 34.20.21.12.9X 10 2 3.0X10 -6 Average 3.407X10 5 1.485X10 -3 4.359X10 -9 -----SD1.639X10 2 2.020X10 -5 6.138X10 -11 -----Crovalimab - 1st trial 5.011X10 5 2.212X10 -4 4.415X10 -10 253.917.16.93.6X 10 2 2.4X10 -6 Crovalimab-2nd trial 5.058X10 5 2.002X10 -4 3.959X10 -10 235.09.15.32.9X 10 2 1.9X10 -6 Average 5.034X10 5 2.107X10 -4 4.187X10 -10-----SD3.262X10 3 1.484X10 -5 3.218X10 -11 -----Tesidolumab - 1st test 2.184X10 6 4.098X10 -4 1.876X10 -10 342.421.23.11.3X 10 3 2.0X10 -6 Tesidolumab - 2nd trial 2.456X10 6 4.186X10 -4 1.704X10 -10 254.817.74.11.8X 10 3 2.5X10 -6 Average 2.320X10 6 4.142X10 -4 1.790X10 -10 -----SD1.924X10 5 6.215X10 -6 1.217X10 -11 -----Pozelimab - 1st trial 1.211X10 6 2.024X10 -4 1.672X10 -10 368.53.62.43.0X 10 2 8.6X10 -7 Pozelimab - 2nd trial 1.281X10 6 2.143X10 -4 1.673X10 -10 261.45.64.13.2X 10 2 7.8X10 -8 Average 1.246X10 6 2.084X10 -4 1.673X10 -10 -----SD4.949X10 4 8.415X10 -6 1.095X10 -13 -----

[0128] Example 4. Confirmation of the complement activation inhibitory effect of anti-C5 antibodies using serum from patients with myasthenia gravis.

[0129] To confirm the classical pathway-mediated hemolytic inhibition activity of anti-C5 antibodies, a hemolysis assay was performed with sera from patients with myasthenia gravis and compared with the activity of FDA-approved anti-C5 agents, eculizumab and ravulizumab.

[0130] First, sheep RBCs (Sheep RBC, Innovative Research, ISHRBC100P15ML) were prepared. Specifically, 5X Veronal buffer was diluted in DW to make 1X Veronal buffer. Sheep RBCs were gently resuspended in 10 ml of 1X Veronal buffer and centrifuged at 4°C, 500 RCF for 5 minutes. Washing was repeated twice in ice-cold 1X Veronal buffer. The sheep RBCs were then resuspended in cold 1X Veronal buffer at a final concentration of 6X10 8 The cells were resuspended at 10 cells / ml and stored on ice.

[0131] Next, an anti-sheep erythrocyte antibody solution was prepared. Specifically, an antibody stock was prepared by reconstituting anti-sheep erythrocyte antibody (Rockland, RK213-4139) with the recommended amount of DW (e.g., adding 5 mL DW to a 50 mg / mL antibody vial to make a 10 mg / mL solution), aliquoting 210 μl into a tube and storing at -80°C. Then, 200 μl of the anti-sheep erythrocyte antibody solution was mixed with 10 ml of sheep erythrocytes, and sensitization was performed by incubating in a 37°C shaking incubator at 100 rpm for 1 hour. After an additional 1 hour of incubation, the cells were centrifuged at 500 RCF at room temperature, the supernatant was discarded, and the pellet was resuspended in 1X Veronal buffer to obtain a 6X10 8 3X10 cells / ml (50 ㎕ in 7 doggy After preparing the sensitized red blood cell solution (C5 cells), it was stored on ice. The neutralizing anti-C5 antibody sample was diluted in 1X Veronal buffer and stored on ice, and a 1,125 μg / mL sample was prepared for the 25% serum status test. Then, the serum of a patient with generalized myasthenia gravis (gMG) was diluted in 1X Veronal buffer. For the 25% serum status test, 75% serum / 1X Veronal buffer was prepared and the prepared solution was stored on ice. The prepared sample was transferred to a 96-well round-bottom plate, and 50 μL of the neutralizing anti-C5 antibody sample was added per well.

[0132] For the positive control, 50 μL of 1X Veronal buffer was added per well (no antibody sample added), and for the negative control, 100 μL of 1X Veronal buffer was added per well (no antibody sample or serum sample added). 50 μL / well of the diluted gMG patient serum solution was transferred to the wells treated with antibody samples and the positive control wells. The sensitized red blood cell solution was added per well at 50 μL (3X10 7 (cells) were transferred to the plate. Afterwards, each well was mixed by gently pipetting three times with a multichannel pipette and incubated in a 37°C shaking incubator at 100 rpm for 45 minutes. Afterwards, each well was mixed by gently pipetting three times with a multichannel pipette again, the plate was centrifuged at 4°C and 4000 rpm for 10 minutes, and 80 μl of the supernatant was transferred to a new Immunoplate Strip. The absorbance of the supernatant was measured at 414 nm using SpectraMax ABS and Microplate Reader (Molecular Devices), and the lysis percentage was calculated according to the following mathematical equation 1.

[0133]

[0134] Background OD: Absorbance of negative control

[0135] Maximal OD: absorbance of positive control

[0136] As a result, as shown in Fig. 5, anti-C5 antibody (anti-C5) according to one aspect showed anti-AChR compared to eculizumab and ravulizumab. + We were able to confirm that classical pathway-mediated hemolysis was suppressed to an undetectable level in the serum of gMG patients (Fig. 5a). In addition, anti-C5 was anti-AChR - It was confirmed that complement-induced hemolysis was significantly inhibited in the serum of gMG patients (Fig. 5b) and the ocular myasthenia gravis (ocular MG) group (Fig. 5c).

[0137] Therefore, anti-C5 antibodies according to one aspect have excellent complement-mediated hemolysis inhibition activity and can be used as C5 inhibitory therapeutic agents for the prevention or treatment of autoimmune diseases such as myasthenia gravis.

[0138] Example 5. Confirmation of free C5 inhibitory activity of anti-C5 antibody (clinical phase 1)

[0139] 5-1. Phase 1 clinical trial of anti-C5 antibody

[0140] The free C5 inhibitory activity of the anti-C5 antibody of the present invention was measured in a phase 1 clinical trial (SAD: Single Ascending Dose Study) for myasthenia gravis.

[0141] The anti-C5 antibody of the present invention was administered to normal subjects via IV infusion at doses of 200 mg, 400 mg, 800 mg, 1600 mg, 3200 mg, 6400 mg, and 9200 mg, and the free C5 level was measured at different times after administration, including 15 minutes after administration, upon completion of infusion, and 1 hour after administration.

[0142] Specifically, to confirm the free C5 inhibitory effect of anti-C5 antibody, free C5 in human serum was measured in the analytical range of 1 to 2500 ng / mL using electrochemiluminescence immunoassay. First, 50 μL of 1 μg / mL anti-C5 antibody solution in 1X PBS was added per well of the plate, coated on the plate overnight at 4°C, and then stored at room temperature. The plate was washed three times with 300 μL PBST using an automatic plate washer. Then, 150 μL of PBS casein was added per well, and the solution was blocked by incubating for at least 60 minutes in a microtiter plate shaker with a 3 mm path at 600 RPM at 22°C. The CS (calibration standard), QC (quality control) material, blank control, and samples were diluted 10-fold to prepare a minimum dilution factor. For the calibration standards, quality control materials, and serum samples, pre-diluted as much as necessary before the minimum dilution factor dilution was prepared. The blocked plate was washed three times using an automated plate washing solution containing 300 μL PBST. Then, 50 μL of the calibration standards, quality control materials, and serum samples, previously prepared at a minimum dilution factor of 10, were transferred to each well of the blocked plate and incubated for 60 minutes in a microtiter plate shaker at 22°C, 600 RPM, and 3 mm path. The plate was then washed three times using an automated plate washing solution containing 300 μL PBST. 50 μL per well of 250 ng / mL anti-C5 biotin antibody solution in PBS casein was added, and the plate was incubated for 60 minutes in a microtiter plate shaker with a 3 mm path at 600 RPM at 22°C, and the plate was washed three times with 300 μL PBST.After washing, the same incubation and washing procedure was repeated except that 200 ng / mL SULFO-TAG Streptavidin solution was used instead of the anti-C5 biotin antibody solution. Then, 150 μL of MSD Read Buffer 1X was added per well, and the plate was analyzed using an MSD plate reader.

[0143] Specific measurement results are summarized in Tables 5 to 21 below.

[0144] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 1, Pre-dose (Baseline)n13566666654Mean86105.59969875.51270815.72083625.60177185.29068247.98994438.94599092.64682021.991SD19821.879126976.282322699.092012771.185728549.99946635.568718111.490412890.663521038.9471CV%23.02038.60632.05415.27236.9899.72319.17813.00925.650Geometric Mean83748.10765584.68167811.36282706.41473194.26967950.77692962.02098410.86379175.343Min46820.3135283.8146117.5360227.2849353.8355130.0268289.8786601.9535283.81Median89627.89167954.29470463.25387093.81068031.32570126.19391443.01997080.41583162.376Max113844.87110880.72105927.3894774.36124056.0373896.93120281.29118506.73124056.03Day 1, 15 Minutes Post-dosen13566666654Mean90772.7288658.629291.540112.985118.014150.24380.179113.79622750.725SD23153.525217924.777468.925349.168062.412237.403425.432719.222240576.0079CV%25.507207.01623.64243.51752.88624.89531.72016.892178.350Geometric Mean87680.3221375.367285.353104.431104.056146.42377.264112.307758.664Min45879.92311.93222.3070.8060.76109.0159.4980.6359.49Median94221.713872.100268.04297.368105.969147.94968.552114.268179.207Max122305.0040720.41410.81181.48198.07206.52124.62137.06122305.00Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0145] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 1, 15 Minutes Post-dose - CFBn13566666654Mean4667.129-61216.883-70524.180-83512.616-77067.276-68097.747-94358.766-98978.851-59271.266SD13004.681714878.090822709.736012749.797628495.66796622.606818089.333712886.667541143.2770CV%278.644-24.304-32.201-15.267-36.975-9.725-19.171-13.020-69.415Geometric Mean7867.107-------7867.107Min-21264.79-70160.31-105648.42-94652.39-123879.47-73787.92-120156.68-118390.62-123879.47Median3681.691-67065.416-70210.254-86923.250-67929.272-69961.965-91380.312-96969.524-69558.504Max34239.59-34971.89-45706.71-60156.49-49285.24-55016.92-68226.48-86464.8934239.59Day 1, End of Infusionn13566666654Mean85337.154335.800112.35446.6060.5930.0000.0000.00020592.950SD15544.5693206.448821.369222.40310.98980.00000.00000.000037535.4578CV%18.21661.48019.02048.069166.865---182.273Geometric Mean83925.176291.945110.79043.309-----Min55233.55146.1488.2829.850.000.000.000.000.00Median86073.520262.140110.46539.9100.0000.0000.0000.00039.910Max108693.84669.24151.1090.982.360.000.000.00108693.84Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0146] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 1, End of Infusion - CFBn13566666654Mean-768.445-69539.712-70703.365-83578.995-77184.697-68247.989-94438.945-99092.646-61429.040SD13513.941926781.049922706.784312760.905228549.26896635.568718111.490412890.663539730.6029CV%-1758.609-38.512-32.116-15.268-36.988-9.723-19.178-13.009-64.677Geometric Mean6777.416-------6777.416Min-27916.64-110211.48-105816.34-94729.59-124054.83-73896.93-120281.29-118506.73-124054.83Median-494.087-67692.155-70369.774-87031.212-68031.325-70126.193-91443.019-97080.415-69888.715Max20732.19-35137.67-45966.43-60197.43-49353.83-55130.02-68289.87-86601.9520732.19Day 1, 1 Hour Post-dosen13566666654Mean85359.544299.846103.48239.9735.0685.9880.0000.17520594.471SD15380.9355156.238813.379326.071211.084212.27260.00000.428937531.7820CV%18.01952.10612.92965.223218.710204.954-244.949182.242Geometric Mean84104.398265.149102.75425.950-----Min55880.10117.1487.071.700.000.000.000.000.00Median83740.253240.258104.36340.1130.6491.2120.0000.00040.113Max123170.03500.54120.9882.0527.6531.020.001.05123170.03Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0147] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 1, 1 Hour Post-dose - CFBn13566666654Mean-746.056-69575.666-70712.237-83585.628-77180.222-68242.001-94438.945-99092.471-61427.520SD13314.286426835.672922692.279812762.079828545.65746632.826118111.490412890.814939726.4899CV%-1784.623-38.571-32.091-15.268-36.986-9.720-19.178-13.009-64.672Geometric Mean5475.878-------5475.878Min-19689.81-110380.18-105806.40-94728.98-124054.73-73895.87-120281.29-118506.73-124054.73Median1347.512-67714.037-70366.422-87051.936-68030.595-70109.971-91443.019-97079.889-69872.531Max16912.57-35166.67-46002.94-60196.80-49353.83-55130.02-68289.87-86601.9516912.57Day 1, 2 Hours Post-dosen13566666654Mean86681.354307.533107.21339.3790.5060.0000.0000.00020912.553SD32976.0193107.227313.88898.73560.80100.00000.00000.000040541.4005CV%38.04334.86712.95522.184158.149---193.862Geometric Mean82904.074292.883106.50938.568-----Min61243.31189.9394.6629.440.000.000.000.000.00Median78800.584284.044104.52239.6980.0000.0000.0000.00039.698Max191265.26457.28132.0750.951.770.000.000.00191265.26Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0148] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 1, 2 Hours Post-dose - CFBn13566666654Mean575.755-69567.979-70708.507-83586.222-77184.783-68247.989-94438.945-99092.646-61109.438SD27979.287726909.510822690.120212766.758528549.33086635.568718111.490412890.663541896.4492CV%4859.583-38.681-32.090-15.274-36.988-9.723-19.178-13.009-68.560Geometric Mean15885.902-------15885.902Min-24935.89-110509.89-105795.31-94729.39-124054.76-73896.93-120281.29-118506.73-124054.76Median-8212.615-67670.250-70364.277-87053.616-68031.325-70126.193-91443.019-97080.415-69854.383Max81524.31-35093.88-46005.29-60195.76-49353.83-55130.02-68289.87-86601.9581524.31Day 1, 6 Hours Post-dosen13566666654Mean86310.011334.365121.79850.3629.9920.0000.0000.00020829.535SD20472.3385132.662819.347520.731414.55640.00000.00000.000038471.6556CV%23.72039.67615.88541.165145.674---184.698Geometric Mean83815.124310.117120.50347.472-----Min45298.50159.6795.1734.840.000.000.000.000.00Median88258.924350.980117.92144.2421.2780.0000.0000.00044.242Max118669.48460.51144.0189.3630.680.000.000.00118669.48Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0149] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 1, 6 Hours Post-dose - CFBn13566666654Mean204.412-69541.147-70693.922-83575.239-77175.297-68247.989-94438.945-99092.646-61192.456SD11993.092626868.185622698.233212761.532028536.35486635.568718111.490412890.663539988.0134CV%5867.125-38.636-32.108-15.270-36.976-9.723-19.178-13.009-65.348Geometric Mean8471.767-------8471.767Min-20337.94-110422.73-105783.66-94720.12-124025.34-73896.93-120281.29-118506.73-124025.34Median-1374.883-67603.315-70359.517-87031.505-68030.595-70126.193-91443.019-97080.415-69852.767Max19167.00-35124.14-45973.51-60192.44-49352.73-55130.02-68289.87-86601.9519167.00Day 1, 12 Hours Post-dosen13566666654Mean92817.901407.083150.20839.67911.0520.0000.0000.00022405.070SD22482.1413174.210626.315435.433316.15000.00000.00000.000041426.5150CV%24.22242.79517.51989.301146.129---184.898Geometric Mean90442.745373.253148.33917.912-----Min54425.27180.32122.511.640.000.000.000.000.00Median85851.280412.581143.59643.1121.3110.0000.0000.00043.112Max149383.76624.09184.3696.6633.470.000.000.00149383.76Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0150] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 1, 12 Hours Post-dose - CFBn13566666654Mean6712.301-69468.429-70665.511-83585.922-77174.238-68247.989-94438.945-99092.646-59616.921SD18691.255026815.270622692.304012757.576028534.85156635.568718111.490412890.663543000.7634CV%278.463-38.601-32.112-15.263-36.975-9.723-19.178-13.009-72.129Geometric Mean10819.228-------10819.228Min-18963.03-110256.63-105743.01-94772.17-124022.55-73896.93-120281.29-118506.73-124022.55Median1905.099-67541.714-70338.159-87022.377-68030.014-70126.193-91443.019-97080.415-69824.981Max42507.56-35103.49-45938.02-60225.64-49353.83-55130.02-68289.87-86601.9542507.56Day 2, 24 Hours Post-dosen13566666654Mean94263.053549.458192.42573.97518.7860.1670.0000.00022775.539SD19574.2079239.517543.095825.540720.10860.41020.00000.000041686.2602CV%20.76643.59222.39634.526107.043244.949--183.031Geometric Mean91928.697501.592188.55871.1346.763----Min46522.42243.66147.6955.061.280.000.000.000.00Median94819.354598.139182.59664.69613.5430.0000.0000.00064.696Max120299.69838.10255.37124.9944.741.000.000.00120299.69Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0151] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 2, 24 Hours Post-dose - CFBn13566666654Mean8157.453-69326.054-70623.295-83551.626-77166.504-68247.822-94438.945-99092.646-59246.452SD15130.140826759.936022685.909112760.927528530.96696635.532318111.490412890.663543224.3292CV%185.476-38.600-32.122-15.273-36.973-9.723-19.178-13.009-72.957Geometric Mean13178.480-------13178.480Min-15379.52-110042.62-105672.00-94711.41-124011.29-73896.93-120281.29-118506.73-124011.29Median9172.161-67356.155-70312.479-86995.586-68017.782-70125.690-91443.019-97080.415-69736.788Max33601.60-35040.16-45885.09-60172.22-49352.55-55130.02-68289.87-86601.9533601.60Day 3, 48 Hours Post-dosen13566666654Mean95008.3584019.538286.476127.23651.7840.7380.0000.00023296.366SD21090.67596954.326379.876352.644619.44830.57960.00000.000042035.7928CV%22.199173.01327.88241.37637.55778.572--180.439Geometric Mean92496.4031419.833278.140117.98848.836----Min48394.49332.54212.9666.6431.380.000.000.000.00Median92086.5421006.105256.634117.81548.7881.0210.0000.000117.815Max121320.9716424.39422.64197.5881.261.290.000.00121320.97Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0152] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 3, 48 Hours Post-dose - CFBn13566666654Mean8902.759-65855.974-70529.243-83498.365-77133.506-68247.252-94438.945-99092.646-58725.625SD14213.460121164.783722659.416112743.262928531.70686635.232618111.490412890.663543130.4356CV%159.652-32.138-32.128-15.262-36.990-9.722-19.178-13.009-73.444Geometric Mean13516.624-------13516.624Min-13487.56-94456.33-105504.74-94684.10-123974.76-73895.92-120281.29-118506.73-123974.76Median11580.021-66227.861-70234.814-86908.214-67986.465-70125.550-91443.019-97080.415-69579.326Max29987.31-34951.27-45778.46-60160.64-49314.59-55130.02-68289.87-86601.9529987.31Day 5, 96 Hours Post-dosen12566666653Mean92818.63420406.223540.569205.30078.54918.3520.0000.00023036.063SD23633.428616474.0748206.618574.745534.447726.31610.00000.000040328.5712CV%25.46280.73138.22236.40843.855143.395--175.067Geometric Mean89681.75010129.150510.240193.94173.1024.942---Min45658.97499.17312.82119.1649.971.410.000.000.00Median97303.01921777.451461.580189.69763.3151.6590.0000.000161.124Max129933.8140240.12875.78298.00135.5457.880.000.00129933.81Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0153] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 5, 96 Hours Post-dose - CFBn12566666653Mean5272.883-49469.289-70275.151-83420.301-77106.741-68229.637-94438.945-99092.646-59234.951SD14614.797914985.279022619.711412735.488128518.27866629.442418111.490412890.663540996.3882CV%277.169-30.292-32.187-15.267-36.986-9.716-19.178-13.009-69.210Geometric Mean14385.544-------14385.544Min-14531.01-70640.60-105051.60-94625.66-123920.48-73895.42-120281.29-118506.73-123920.48Median8936.756-48941.008-70090.017-86801.542-67977.239-70102.437-91443.019-97080.415-69401.497Max27891.36-34784.65-45419.52-60108.12-49285.40-55128.61-68289.87-86601.9527891.36Day 7, 144 Hours Post-dosen13566666654Mean93793.93332481.7351001.359318.176105.57331.0800.6000.55325749.516SD21666.818621064.6135567.1692108.298649.199333.04390.66180.608741494.0042CV%23.10064.85156.64034.03746.602106.320110.265110.061161.145Geometric Mean91271.70616444.964907.627301.30597.46010.744---Min53820.21551.37602.81165.1865.971.830.000.000.00Median96656.59245238.137816.857314.13884.20023.9160.5310.501314.138Max121949.2547755.392129.91445.33189.1172.631.281.18121949.25Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0154] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 7, 144 Hours Post-dose - CFBn13566666654Mean7688.333-37393.777-69814.361-83307.425-77079.717-68216.910-94438.345-99092.093-56272.475SD17784.644816924.234122291.062212701.303628504.18206626.359518111.900112890.641543138.7387CV%231.320-45.260-31.929-15.246-36.980-9.714-19.179-13.009-76.661Geometric Mean16371.793-------16371.793Min-17300.91-63187.85-103797.46-94474.77-123866.92-73895.10-120281.29-118505.55-123866.92Median6999.898-34732.440-69806.449-86656.096-67958.890-70093.900-91442.388-97079.847-68771.559Max47668.86-20198.90-45186.61-60062.11-49264.33-55128.19-68288.81-86601.9547668.86Day 10, 216 Hours Post-dosen13566666654Mean88279.16256414.96117749.584495.170148.16250.3631.3911.08828525.527SD23128.310428132.446612497.6529162.779586.080638.94110.26720.561340174.7758CV%26.19949.86770.41132.87458.09977.32219.20351.593140.838Geometric Mean85023.77245518.33812116.155468.324130.42623.7161.370--Min40096.439546.491480.12277.1476.602.341.040.000.00Median87080.52671352.84819225.566589.330105.76362.6871.3471.219589.330Max131476.4976567.6236314.39615.32283.1488.641.701.53131476.49Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0155] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 10, 216 Hours Post-dose - CFBn13566666654Mean2173.562-13460.551-53066.136-83130.431-77037.127-68197.627-94437.554-99091.558-53496.464SD17867.660416940.990118635.037812639.566228467.85426643.949118111.502812890.764642333.7287CV%822.045-125.857-35.117-15.205-36.953-9.742-19.178-13.009-79.134Geometric Mean9358.2803305.094------7425.896Min-25205.32-34313.10-71164.96-94192.88-123772.88-73894.59-120279.85-118505.24-123772.88Median2159.228-13865.091-57091.783-86483.258-67940.842-70083.281-91441.562-97079.066-65059.280Max35050.433398.55-23007.06-59950.14-49265.26-55076.25-68288.62-86600.8835050.43Day 14, 312 Hours Post-dosen13566666654Mean89367.11367756.03851956.0681560.502256.18691.5691.8531.33733773.292SD20042.633730136.186312447.84091077.4278160.192956.97450.24180.687641352.1332CV%22.42744.47823.95869.04462.53062.22013.05351.439122.440Geometric Mean87139.89460553.99650667.4841333.610221.41557.4301.840--Min53185.1822726.1437775.94743.37124.493.061.590.000.00Median89811.31172580.20953654.3311360.806189.34592.7361.7671.4881360.806Max117066.59105201.1465836.843655.38528.74170.842.241.90117066.59Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0156] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 14, 312 Hours Post-dose - CFBn13566666654Mean3261.514-2119.474-18859.652-82065.099-76929.103-68156.420-94437.092-99091.309-48248.699SD15705.828927717.787819384.905612148.942828396.07406631.631918111.470212890.776745177.2139CV%481.550-1307.767-102.785-14.804-36.912-9.730-19.178-13.009-93.634Geometric Mean9139.99917038.98714699.580-----10856.293Min-17116.63-38300.51-40090.54-91118.98-123527.29-73893.87-120279.49-118504.94-123527.29Median1730.712-4780.702-24844.842-85633.390-67877.653-70014.937-91441.145-97078.692-57674.281Max27698.9537246.8414699.58-59475.36-49218.78-55067.18-68288.28-86600.6037246.84Day 28, 648 Hours Post-dosen13566666654Mean88441.03571950.08382434.62674021.11921649.588338.59151.71355.98047792.474SD24528.621125253.811519119.562313772.575731023.053088.894553.94508.372843010.5215CV%27.73435.09923.19418.606143.29626.254104.31614.95789.994Geometric Mean84636.46768041.22880634.17272930.6284882.137329.64918.28955.4225531.589Min43854.1538564.7963664.1656140.66526.81250.142.9442.472.94Median94286.82780531.40079642.18374063.1866603.475326.14343.35557.59156068.888Max117597.30104112.89110169.9290784.6277780.19493.22115.7464.26117597.30Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0157] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 28, 648 Hours Post-dose - CFBn13566666654Mean2335.4362074.57111618.906-9604.482-55535.702-67909.398-94387.232-99036.666-34229.517SD21824.751824430.013819606.539612550.627111045.78566607.238518075.439912885.850945716.4977CV%934.5041177.594168.747-130.675-19.890-9.730-19.150-13.011-133.559Geometric Mean6205.98510524.30926431.2902351.961----7964.208Min-39625.30-30349.32-8844.55-24247.93-72818.18-73636.49-120165.55-118442.47-120165.55Median1896.0743280.9737926.872-9437.150-54270.224-69726.215-91439.350-97032.295-25011.356Max50773.9636158.6040762.544865.56-44200.02-54879.88-68286.93-86540.5450773.96Day 42, 984 Hours Post-dosen13556666653Mean86948.67176364.16276770.54986720.19072678.6567387.083216.952168.39754698.754SD22361.123222976.647817839.599513872.499931097.695714179.1050112.946028.689841985.4714CV%25.71830.08823.23815.99742.788191.94552.06017.03776.758Geometric Mean84001.47873723.47275068.37885872.63266506.5542525.761193.820166.41213400.383Min42691.8757331.5852757.1575174.6033896.75831.71117.59135.21117.59Median83546.43764516.63374473.73682913.06377041.1201744.031177.232160.27269662.264Max122001.43103861.14101715.46111972.03115782.7236301.96368.03206.03122001.43Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0158] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 42, 984 Hours Post-dose - CFBn13556666653Mean843.0726488.65012977.1613094.589-4506.633-60860.906-94221.993-98924.250-26872.192SD15962.269133597.321119122.624813320.730923954.185714977.464018053.743712881.305146729.2928CV%1893.347517.786147.356430.452-531.532-24.609-19.161-13.021-173.895Geometric Mean12644.74728198.24720529.82614006.67622171.854---17427.602Min-20389.78-46364.09-19887.93-12437.35-32625.58-72818.93-119940.25-118306.32-119940.25Median-512.74322047.77117362.1912057.245-5804.438-67861.389-91314.257-96928.201-12437.353Max25934.4133142.6828356.2119796.2826991.92-33145.62-68172.28-86395.9233142.68Day 56, 1320 Hours Post-dosen13556666653Mean84902.30781706.78176070.57083214.51677651.88771666.7874777.760398.66962620.273SD24561.918822198.224016048.445411462.069525258.863411425.867410290.4210306.562537248.1030CV%28.93027.16821.09713.77432.52815.943215.38276.89759.483Geometric Mean81314.30678922.34674788.10782557.67074606.48470858.542922.667334.67925409.653Min43597.1148038.2958030.5467153.7253949.8754620.00263.89210.40210.40Median86353.15179740.16676380.97583968.87475390.42175116.518513.918270.95571899.519Max122001.43103128.79101375.88101231.52122975.8682491.2225767.151005.66122975.86Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0159] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)Day 56, 1320 Hours Post-dose - CFBn13556666653Mean-1203.29211831.26912277.182-411.085466.5973418.797-89661.185-98693.977-18950.673SD20766.297126055.32387204.666416037.608924385.21968067.676222313.581612974.325444979.1882CV%-1725.790220.22458.683-3901.2925226.184235.980-24.887-13.146-237.349Geometric Mean8101.40720247.74210407.4459503.14316037.4066145.338--10337.989Min-39882.34-31140.563857.67-24188.60-42679.20-9435.28-118952.93-118089.66-118952.93Median809.44612754.48110460.903-2254.8232989.1853518.096-91116.336-96842.096-510.022Max36579.6935174.5021945.1818386.2225405.6713043.63-61112.44-85596.2936579.69End of Study, 1656 Hours Post-dosen13556666653Mean86977.97976214.30071183.51191340.72979757.06671766.81839685.48210932.58668464.130SD25369.399621332.884213723.839514934.979125735.66934718.721046125.962325068.161734587.6090CV%29.16827.99119.28016.35132.2686.575116.229229.29850.519Geometric Mean83956.31073677.01970123.54890304.20876288.28371634.2339781.6761356.85139254.371Min51449.1847896.7056517.9169967.6050238.0365465.56988.20404.96404.96Median83843.68472368.05473653.39389084.75776819.26374043.86122564.116666.94675209.327Max154815.68100042.6589335.48110112.13114843.4275597.8796902.8862098.15154815.68Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0160] Anti-C5 antibodyBiomarker (unit)Visit, TimepointStatisticAll Placebo(N=13)200 mg(N=5)400 mg(N=6)800 mg(N=6)1600 mg(N=6)3200 mg(N=6)6400 mg(N=6)9200 mg(N=6)Overall(N=54)Complement C5, Free (ng / mL)End of Study, 1656 Hours Post-dose - CFBn13556666653Mean872.3806338.7887390.1237715.1282571.7773518.829-54753.463-88160.060-13106.816SD21382.124917750.035220096.749218444.497217128.15705831.806039390.289433092.194139566.0315CV%2451.011280.023271.941239.069666.005165.731-71.941-37.537-301.874Geometric Mean10231.7445786.41819952.66712685.05211231.6616536.32810023.281-9878.019Min-31091.16-16336.09-16127.17-21374.72-19735.55-5443.13-92571.46-117204.87-117204.87Median4628.8721679.19012471.1497316.088-1402.1985318.028-66272.078-96649.6081649.595Max47939.4832088.3530252.4627266.8327101.9711029.1710023.28-24503.8047939.48 Abbreviations: PD=Pharmacodynamic(s); N=Number of subjects in the population; n=Number of observations; SD=Standard deviation; Min=Minimum; Max=Maximum; CFB=Change from baseline; CH50=Terminal complement activity, classical pathway; AP50=Terminal complement activity, alternative pathway; Complement C5=Total complement component 5; Complement C5, Free=Free complement component 5; N / A=Not Applicable; '-'=Not calculated.Note: Baseline is defined as the last result (scheduled or unscheduled) obtained prior to study drug administration.Note: The overall column includes subjects from all treatment groups, including placebo.

[0161] As a result, as shown in Figures 6 to 10, it was confirmed that the amount of free C5 significantly decreased within 1 hour after administration of the anti-C5 antibody, and although there was a difference depending on the administered dose, the decreased amount of free C5 was maintained unchanged until the 14th day. In addition, it was confirmed that as the dose of anti-C5 antibody administration increased, the amount of free C5 decreased and the amount of free C5 recovered over time decreased.

[0162] Specifically, when the anti-C5 antibody of the present invention was administered at 400 mg, the amount of free C5 that was significantly reduced after the completion of the injection was maintained at 130 ng / mL or less for 6 hours or more, when the anti-C5 antibody of the present invention was administered at 800 mg, the amount of free C5 that was significantly reduced after the completion of the injection was maintained at 50 ng / mL or less for 12 hours or more, when the anti-C5 antibody of the present invention was administered at 1600 mg, the amount of free C5 that was significantly reduced to 1 ng / mL or less after the completion of the injection was maintained at 10 ng / mL or less for 6 hours or more, and when the anti-C5 antibody of the present invention was administered at 3200 mg or more, the amount of free C5 that was rapidly reduced to 1 ng / mL or less, almost 0 ng / mL or less after the completion of the injection was maintained at a similar level for 14 days or more.

[0163] 5-2. Comparison of free C5 inhibitory activity with existing anti-C5 antibody drugs

[0164] To determine the free C5 inhibitory activity of anti-C5 antibodies, the free C5 inhibitory activity of eculizumab and ravulizumab, FDA-approved anti-C5 antibody therapeutics disclosed in U.S. Patent Publication No. US 2020-0254092 A1, was compared.

[0165] Eculizumab (900mg biweekly)DaysFree C5(ng / mL)log Free C51101836003.5563025152802.44715822501.6989729501.6989743501.69897572602.4149733717602.8808136851802.2552 725997002.8450981132202.34242271272902.4623981411502.17609131555302.72427591699402.973127918330003.4771213

[0166] Ravulizumab (3.3g bimonthly)daysFree C5(ng / mL)log Free C511018401.6020615501.6989722301.477121329301.477121343401.6020657501.6989771601.778151385301. 477121399401.60206113501.69897127601.7781513141401.60206155501.69897169601.7781513183701.845098

[0167] Additionally, as shown in Table 22 and Figure 11, eculizumab significantly increased the amount of free C5 in serum on day 8, but decreased rapidly after day 15. Thereafter, the increase and decrease were repeated, and on day 183, it was confirmed that the amount increased to a similar amount as on day 8.

[0168] In addition, as shown in Table 23 and Figure 12, rabulizumab showed an increasing pattern over time with repeated increases and decreases in free C5 in serum.

[0169] When comparing the above-described results with the free C5 inhibitory activity of the anti-C5 antibody of the present invention in Example 5-1, it can be confirmed that the anti-C5 antibody of the present invention can suppress free C5 in serum to a significantly low level, thereby effectively inhibiting the activity of complement C5.

[0170] Example 6. Confirmation of inhibition of classical pathway-mediated complement activation by anti-C5 antibodies.

[0171] The complement system activation pathways are divided into the antibody-dependent classical pathway and the antibody-independent alternative pathway. Complement C5 acts at the terminal end of the complement activation pathway, contributing to both pathways. To confirm the inhibition of complement activation mediated by the classical pathway, a hemolysis assay was performed.

[0172] In addition, considering that blood in a living body is composed of plasma and blood cells, and most of the plasma is composed of serum, we attempted to confirm the complement activation inhibitory effect of anti-C5 antibody by varying the ratio of serum in the experimental conditions.

[0173] Specifically, as described in Example 4, sheep red blood cells (Innovative research, Cat# ISHRBC100P15ML) sensitized with anti-sheep red blood cell antibody (Rockland, RK213-4139) were suspended in Veronal buffer to prepare, and human serum with live complement activity (Innovative research, Cat# ICSER100ML) and anti-C5 antibodies (antibody A of the present invention, and antibodies of competitors eculizumab, ravulizumab, crobalimab, and pozelimab) were prepared. The final concentrations of antibodies were prepared to be included as summarized in Table 24 below according to the serum ratio (serum %).

[0174] Serum%Ab conc(㎍ / ㎖) : Final treatment concentration751125.00562.50281.25140.6370.3135.1617.588.7950750.00375.00187.5093.7546.8823.4411.725.8625375.00187.5093.7546.8823.4411.725.862.9310150.0075.0037.5018.759.384.692.341.17

[0175] The degree of hemolysis of blood cells was confirmed using the sample prepared above using the following method.

[0176] For experiments under 10% serum conditions, 50 μl of sensitized sheep red blood cell solution and VBS++ (with Ca, Mg) (veronal buffer saline with Ca 2+ and Mg 2+) were mixed with 50 ㎕ of a 30% serum solution and 50 ㎕ of a VBS++ (with Ca, Mg) solution containing drugs. For the experiment under 25% serum conditions, 50 ㎕ of a sensitized sheep red blood cell solution and 50 ㎕ of a 75% serum solution containing VBS++ (with Ca, Mg) and 50 ㎕ of a VBS++ (with Ca, Mg) solution containing drugs were mixed. For the experiment under 50% serum conditions, 50 ㎕ of a sensitized sheep red blood cell solution and 100 ㎕ of a serum solution and 50 ㎕ of a VBS++ (with Ca, Mg) solution containing drugs were mixed. For the experiment under 75% serum conditions, 25 ㎕ of a sensitized sheep red blood cell solution and 150 ㎕ of a serum solution and 25 ㎕ of a VBS++ (with Ca, Mg) solution containing drugs were mixed. The positive control group used VBS++ (with Ca, Mg) instead of the drug, and the negative control group used human serum whose complement activity had been removed through heat treatment. Each mixture was incubated at 37°C and 100 rpm for 45 minutes, then gently pipetted three times with a multichannel pipette to mix each well, and the plate was centrifuged at 4°C and 4000 rpm for 10 minutes. 80 μl of the supernatant was transferred to a new Immunoplate Strip. The absorbance of the supernatant was measured at 414 nm using a SpectraMax ABS and Microplate Reader (Molecular Devices), and the dissolution ratio was calculated according to the following mathematical equation (1).

[0177] [Mathematical Formula 1]

[0178]

[0179] Background OD: Absorbance of negative control

[0180] Maximal OD: absorbance of positive control

[0181] The measurement results are summarized in Tables 25 to 44 below (10% serum condition: Tables 25 to 29, 25% serum condition: Tables 30 to 34, 50% serum condition: Tables 35 to 39, 75% serum condition: Tables 40 to 44), and the values ​​in Tables 25 to 44 are shown as graphs in Figures 13 to 16, which are analyzed using GraphPad Prism software (Nonlinear regression (curve fit) - Log (concentration) vs response - variable slope (four parameters) condition) and IC in Table 45 50 The values ​​(ug / ml) were organized.

[0182] Serum 10% Antibody A(+) control No Ab(-) control 1HI serum(-) control 2No serum concentration (㎍ / ㎖) 1507537.518.759.384.692.341st test 1.8822.00432.1022.61533.5643443.4911.94991.91732nd test 1.94272.01452.10352.51963.56143.997143.58011.97981.8876Average 1.912352.00942.102752.567453.562853.9985543.535551.964851.90245% lysis(1st)-5.272.518.7341.41101.83129.57129.57 % lysis(2nd)-1.413.168.8335.32101.65129.38129.57 % lysis average-3.342.848.7838.37101.74129.48129.57 STDEV2.730.460.074.310.130.130.00 CV-81.7616.190.7711.230.130.100.00

[0183] Serum 10% Eculizumab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 150 75 37.5 18.75 9.38 4.69 2.34 1st test 2.6 36 22.79 59 2.97 96 3.17 26 3.35 15 3.95 22 4 3.47 4 1.87 0 8 1.72 4 2nd test 2.5 36 6 2.78 76 2.95 4 6 3.15 38 3.35 84 4 3.47 6 1.84 38 1.68 5 4 Average 2.5 8 6 4 2.79 17 5 2.96 7 13.16 32 3.35 49 5 3.97 6 14 3.47 5 1.85 7 31.70 4 7% lysis(1st)42.7452.9164.6076.8988.28126.53129.57 % lysis(2nd)36.4052.3863.0175.7088.72129.57129.57 % lysis average39.5752.6563.8176.2988.50128.05129.57 STDEV4.480.371.130.850.312.150.00 CV11.330.711.761.110.351.680.00

[0184] Serum 10%Ravulizumab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)1507537.518.759.384.692.341st test3.16453.3113.5863.69613.76083.95413.8023.4741.87081.7242nd test Test 3.22443.46633.633.74663.822543.84793.4761.84381.6854Average 3.194453.388653.6083.721353.791653.977053.824953.4751.85731.7047% lysis(1st) 76.3885.70103.21110.22114.34126.65116.96 % lysis (2nd) 80.1995.59106.01113.44118.27129.57119.89 % lysis Average 78.2890.65104.61111.83116.30128.11118.42 STDEV 2.706.991.982.272.782.072.07 CV 3.447.711.892.032.391.611.74

[0185] Serum 10%Crovalimab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)1507537.518.759.384.692.341st test3.08923.19123.33223.36933.53.86253.93313.4741.87081.7242nd test3.143.12773.38423.35673.523.91753.74773.4761.84381.6854Average3.11463.159453.35823.3633.513.893.84043.4751.85731.7047% lysis(1st)71.5878.0887.0589.4297.74120.82125.31 % lysis(2nd)74.8274.0390.3688.6199.01124.32113.51 % lysis average73.2076.0688.7189.0198.37122.57119.41 STDEV2.292.862.340.570.902.488.35 CV3.123.762.640.640.922.026.99

[0186] Serum 10% Pozelimab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)1507537.518.759.384.692.341st test2.4142.66552.83763.03653.38223.81943.73913.4741.87081.7242nd test Test 2.52862.65352.84793.09253.41693.80493.37053.4761.84381.6854Average 2.47132.65952.842753.06453.399553.812153.55483.4751.85731.7047% lysis(1st) 28.6044.6155.5668.2390.24118.07112.96 % lysis (2nd) 35.8943.8456.2271.7992.45117.1589.49 % lysis Average 32.2444.2355.8970.0191.34117.61101.23 STDEV 5.160.540.462.521.560.6516.59 CV 16.001.220.833.601.710.5616.39

[0187] Serum 25% antibody A(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 375 187.59 3.75 46.88 23.44 11.72 5.86 1st test 1.85 0 5 1.95 6 72.01 26 2.32 19 3.50 0 8 3.77 9 73.85 54 3.47 4 1.87 0 8 1.72 4 2nd test Test 1.83331.94582.0222.23713.53233.79683.82623.4761.84381.6854Average 1.84191.951252.01732.27953.516553.788253.84083.4751.85731.7047% lysis(1st)-0.426.149.6028.72101.59118.84123.51 % lysis(2nd)-1.485.4710.1823.48103.54119.89121.71 % lysis Average-0.955.819.8926.10102.57119.36122.61 STDEV0.750.480.413.711.380.751.28 CV-78.988.204.1514.201.340.631.04

[0188] Serum 25% Eculizumab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 375 187.59 3.75 46.88 23.44 11.725.86 1st test 2.9 2333.19 353.27 533.36 823.534 73.726 63.85 713.474 1.870 81.724 2nd test Test 2.89 223.16 353.25 493.35 253.49 13.72 163.79 323.47 61.84 381.68 54 Average 2.90 77 53.17 85 3.26 513.36 035 3.51 28 53.72 413.82 515 3.47 51.85 73 1.70 47% lysis (1st) 65.90 82.60 87.66 93.40 103.69 115.55 123.62 % lysis (2nd) 63.97 80.74 86.39 92.43 100.99 115.24 119.67 % lysis Average64.9381.6787.0292.91102.34115.40121.64 STDEV1.361.310.890.691.910.222.79 CV2.091.611.020.741.870.192.30

[0189] Serum 25%Ravulizumab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)375187.593.7546.8823.4411.725.861st test3.36143.49143.49173.52663.5743.783.7123.4741.87081.7242nd test Test 3.35583.44753.49823.53693.57663.77413.71543.4761.84381.6854Average 3.35863.469453.494953.531753.57533.777053.71373.4751.85731.7047% lysis (1st) 92.98101.01101.03103.19106.12118.85114.65 % lysis (2nd) 92.6398.30101.43103.83106.28118.49114.86 % lysis Average92.8099.66101.23103.51106.20118.67114.76 STDEV0.241.920.280.450.110.260.15 CV0.261.930.280.430.110.220.13

[0190] Serum 25%Crovalimab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)375187.593.7546.8823.4411.725.861st test3.4213.40973.38523.45423.4833.65093.76813.4741.87081.7242nd test Test 3.38963.36733.35353.41893.54323.62813.69093.4761.84381.6854Average 3.40533.38853.369353.436553.51313.63953.72953.4751.85731.7047% lysis(1st) 96.6695.9694.4598.71100.49110.87118.12% lysis(2nd) 94.7293.3492.4996.53104.22109.46113.35% lysis Average95.6994.6593.4797.62102.36110.17115.73 STDEV1.371.851.391.542.631.003.37 CV1.431.961.481.582.570.902.92

[0191] Serum 25% Pozelimab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 375 187.59 3.75 46.88 23.44 11.72 5.86 1st test 2.73 42 2.86 512.95 54 3.13 93.46 793.55 75 3.70 393.47 41.87 081.72 4 2nd test Test 2.76 19 2.84 96 2.98 48 3.14 47 3.44 84 3.63 07 3.74 85 3.47 61.84 38 1.68 54 Average 2.74 80 52.85 7 35 2.97 0 13.14 18 5 3.45 8 15 3.59 4 13.72 6 23.47 5 1.85 7 31.70 4 7 % lysis (1st) 54.2 16 2.30 67.88 7 9.23 9 9.56 105.10 11 4.15 % lysis (2nd) 55.9 26 1.34 69.70 79.58 9 8.36 109.62 116.91 % lysis Average 55.0661.8268.7979.4198.96107.36115.53 STDEV 1.210.681.290.250.853.201.95 CV 2.201.101.870.310.862.981.69

[0192] Serum 50% antibody A(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 750 375 187.59 3.75 46.88 23.44 11.72 1st test 1.89 311.88 232.04 262.21 952.95 433.59 543.63 163.29 982.16 792.13 69 2nd test Test 1.86471.91992.03152.14412.70443.56253.57813.3462.06842.1521Average 1.87891.90112.037052.18182.829353.578953.604853.32292.118152.1445% lysis(1st)-18.68-19.58-6.278.4169.40122.62125.62 % lysis(2nd)-21.04-16.46-7.192.1548.66119.89121.18 % lysis Average-19.86-18.02-6.735.2859.03121.25123.40 STDEV1.672.210.654.4314.671.933.14 CV-8.39-12.25-9.6883.7624.851.592.54

[0193] Serum 50% Eculizumab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 750 375 187.59 3.75 46.88 23.44 11.72 1st test 3.19 0 53.26 163.35 23.41 183.44 923.58 533.67 733.29 982.16 792.13 69 2nd test Test 3.14143.26883.33773.41273.43953.4643.5693.3462.06842.1521Average 3.165953.26523.344853.412253.444353.524653.623153.32292.118152.1445% lysis (1st) 89.0194.91102.42107.38110.48121.78129.42 % lysis (2nd) 84.9395.51101.23107.45109.68111.71120.43 % lysis Average 86.9795.21101.82107.42110.08116.75124.92 STDEV 2.880.420.840.050.577.126.36 CV 3.310.440.820.050.526.105.09

[0194] Serum 50%Ravulizumab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)750375187.593.7546.8823.4411.721st test3.35753.2823.44593.42723.51013.66553.57153.29982.16792.13692nd test Test 3.37113.24633.39693.46393.513.66863.65373.3462.06842.1521Average 3.36433.264153.42143.445553.510053.667053.61263.32292.118152.1445% lysis(1st) 102.8796.61110.21108.66115.54128.44120.63 % lysis (2nd) 104.0093.64106.14111.70115.53128.69127.46 % lysis Average 103.4495.12108.18110.18115.53128.57124.05 STDEV 0.802.102.882.150.010.184.82 CV 0.772.202.661.960.010.143.89

[0195] Serum 50%Crovalimab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)750375187.593.7546.8823.4411.721st test3.38163.29433.39783.44063.45363.54833.70523.29982.16792.13692nd test Test 3.38143.29013.36193.33533.45213.61893.67693.3462.06842.1521Average 3.38153.29223.379853.387953.452853.58363.691053.32292.118152.1445% lysis(1st) 104.8797.63106.22109.77110.85118.71131.73 % lysis (2nd) 104.8697.28103.24101.03110.72124.57129.38 % lysis Average 104.86 97.45 104.73 105.40 110.79 121.64 130.56 STDEV 0.010.25 2.116.18 0.09 4.14 1.66 CV 0.010.25 2.015.86 0.08 3.4 11.27

[0196] Serum 50% Pozelimab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 750 375 187.59 3.75 46.88 23.44 11.72 1st test 2.99 53.08 683.16 493.29 773.46 853.57 943.66 363.29 982.16 792.13 69 2nd test Test 3.03823.07493.16653.26433.45753.58663.61533.3462.06842.1521Average 3.01663.080853.16573.2813.4633.5833.639453.32292.118152.1445% lysis(1st) 72.7880.4086.8997.91112.09121.29128.28 % lysis(2nd) 76.3779.4187.0295.14111.17121.89124.27 % lysis Average74.5879.9186.9596.52111.63121.59126.28 STDEV2.540.700.091.960.650.422.83 CV3.400.870.112.030.580.352.25

[0197] Serum 75% antibody A(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 1 1 2 5 5 6 2.5 2 8 1.25 14 0.6 3 7 0.3 1 3 5.16 17.58 1st test 1.8 1 2 1 1.8 8 5 3 1.9 5 6 2.10 2 5 2.0 7 8 9 3.0 9 2 8 2.8 1 2 7 3.0 1 1.9 9 6 1.6 5 2 7 2nd test Test 1.81361.83091.9212.09532.04622.99233.04063.01341.96931.7453Average 1.812851.85811.93852.09892.062553.042552.926653.01171.984451.699% lysis(1st)-16.78-9.65-2.7711.499.19107.8980.63% lysis(2nd)-16.63-14.95-6.1810.796.0198.11102.81% lysis Average-16.70-12.30-4.4711.147.60103.0091.72STDEV0.103.742.410.502.256.9215.69CV-0.62-30.44-53.864.4529.616.7217.10

[0198] Serum 75% Eculizumab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 1 1 2 5 5 6 2.5 2 8 1.2 5 14 0.6 3 7 0.3 1 3 5.1 6 17 5 8 1st test 2.4 2 6 4 7 9 2.7 5 7 7 2.8 7 6 3.0 2 1 6 3.0 19 13.0 2 8 23.0 1 1.9 9 6 1.6 5 2 7 2nd test Test 2.44332.66612.78182.88572.98763.14193.05373.01341.96931.7453Average 2.434852.6572.769752.880853.00463.08053.040953.01171.984451.699% lysis(1st) 43.0264.5975.2786.79100.96100.72101.61% lysis (2nd) 44.6766.3677.6287.7397.65112.67104.09% lysis Average 43.85 65.47 76.45 87.26 99.31 106.70 102.85 STDEV 1.16 1.25 1.66 0.67 2.34 8.45 1.76 CV 2.65 1.91 2.17 0.77 2.36 7.92 1.71

[0199] Serum 75%Ravulizumab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)1125562.5281.25140.6370.3135.1617.581st test2.75722.87782.93372.96723.08833.15573.03723.011.99961.652722nd test Test 2.8062.922.91923.01993.12243.22053.03133.01341.96931.7453Average 2.78162.89892.926452.993553.105353.18813.034253.01171.984451.699% lysis(1st) 75.2386.9792.4195.67107.46114.02102.48% lysis(2nd) 79.9891.0791.00100.80110.78120.33101.91% lysis Average77.6089.0291.7098.23109.12117.17102.20STDEV3.362.901.003.632.354.460.41CV4.333.261.093.692.153.810.40

[0200] Serum 75%Crovalimab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)1125562.5281.25140.6370.3135.1617.581st test2.77932.91712.95032.92913.42033.3542.96733.011.99961.65272nd test Test 2.80692.89332.9623.05332.98723.1192.98183.01341.96931.7453Average 2.79312.90522.956152.99123.203753.23652.974553.01171.984451.699% lysis(1st) 77.3890.7994.0291.96139.78133.3295.68% lysis(2nd) 80.0688.4795.16104.0597.61110.4597.09% lysis Average78.7289.6394.5998.00118.70121.8896.38STDEV1.901.640.818.5529.8116.181.00CV2.411.830.858.7225.1213.271.04

[0201] Serum 75% Pozelimab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 1 1 2 5 5 6 2.5 2 8 1.2 5 14 0.6 3 7 0.3 1 3 5 16 17 5 8 1st test 2.2 3 6 4 2.4 6 4 8 2.5 7 0 8 2.7 8 5 22 60 12 2.7 2 4 6 3.0 1 1 3 0 1 1.9 9 6 1.6 5 27 2nd test Test 2.16 13 2.42 122.6 18 32.73 39 2.86 46 2.98 34 2.94 27 3.01 34 1.96 9 31.74 53 Average 2.19 8 8 52.44 32.59 455 2.75 9 55 2.73 29 2.85 42.97 193.01 17 1.98 44 5 1.69 9% lysis (1st) 24.5 34 6.76 5 7.08 7 7.95 6 0.04 7 2.05 9 8.97% lysis (2nd) 17.2 24 2.5 26 1.70 7 2.96 85 68 97 25 9 3.28% lysis Average20.8744.6459.3975.4572.8684.6596.13STDEV5.173.003.273.5318.1317.814.02CV24.776.725.514.6824.8921.054.18

[0202] Serum antibody ratio AEculizumabRavulizumabCrovalimabPozelimab75%64.711507290.9272.62.59X10 9 50%49.323.104X10 19 70.1526.3375.9525%33.7729.4954.6216.5536.6810%14.059.53871.5910.4819.54

[0203] As can be seen from this, it was confirmed that the anti-C5 antibody of the present invention (antibody A) exhibited superior complement activity inhibition ability compared to competitive antibodies under all conditions, and the anti-C5 antibody of the present invention was not significantly affected by the ratio of serum and IC 50 The values ​​are stable, confirming that the suppression ability is excellent.

[0204] In particular, under the conditions of 50% serum (Fig. 15) and 75% serum (Fig. 16), the antibody of the present invention exhibits excellent inhibitory ability, while the effect of inhibiting complement activity does not increase even when the treatment concentration increases, so it can be expected to exhibit relatively better effects in an actual living body.

[0205] Example 7. Confirmation of inhibition of alternative pathway-mediated complement activation by anti-C5 antibodies.

[0206] The complement system activation pathways are divided into the antibody-dependent classical pathway and the antibody-independent alternative pathway. Complement C5 acts at the terminal end of the complement activation pathway, contributing to both pathways. To confirm the ability of complement activation inhibition mediated by the alternative pathway, a hemolysis assay was performed.

[0207] In addition, in order to confirm the change in inhibitory ability according to the serum ratio within the experimental conditions, as in Example 6, rabbit erythrocytes (Innovative research, Cat# IRBRBC10ML) were suspended in GVBMG (gelatin veronal buffer with Mg, EGTA, Novatein biosciences, Cat# NIBB-320X) solution, and human serum with live complement activity (Innovative research, Cat# ICSER100ML) was prepared. The final concentration of anti-human complement C5 antibody in the GVBMG solution was prepared as shown in Table 46 below.

[0208] Serum%Ab conc(㎍ / ㎖) : Final treatment concentration751125.00562.50281.25140.6370.3135.1617.588.7950750.00375.00187.5093.7546.8823.4411.725.8625375.00187.5093.7546.8823.4411.725.862.9310150.0075.0037.5018.759.384.692.341.17

[0209] The prepared rabbit erythrocyte samples, serum, and antibody samples were mixed in a 96-well plate and incubated with shaking at 37°C and 100 rpm for 2 hours. The positive control group was prepared by mixing only rabbit erythrocytes and serum without antibody samples, and the negative control group was prepared by mixing rabbit erythrocyte samples and human serum whose complement activity had been removed through heat treatment and incubated with shaking at 37°C and 100 rpm for 2 hours in the same conditions as the test group. After the reaction was completed, centrifugation was performed at 4°C and 4000 rpm for 10 minutes, and the supernatant was separated and transferred to a new 96-well plate, and the absorbance was measured at 414 nm. The degree of blood cell hemolysis (%) was derived according to mathematical formula 1 in the same manner as in Example 6.

[0210] The measurement results are summarized in Tables 47 to 66 below (10% serum condition: Tables 47 to 51, 25% serum condition: Tables 52 to 56, 50% serum condition: Tables 57 to 61, 75% serum condition: Tables 62 to 66), and the values ​​in Tables 47 to 66 are shown as graphs in Figures 17 to 20, which are analyzed using GraphPad Prism software (Nonlinear regression (curve fit) - Log (concentration) vs response - variable slope (four parameters) condition) and IC in Table 67. 50 The values ​​(ug / ml) were organized.

[0211] Serum 10% antibody A(+) control No Ab(-) control 1HI serum(-) control 2No serum concentration (㎍ / ㎖) 1507537.518.759.384.692.341st test 0.42510.40250.43480.66810.59941.01322.03562.29930.25660.06262nd test Test 0.42170.45380.40820.67890.58070.81651.96182.28320.29470.0647Average 0.42340.428150.42150.67350.590050.914851.99872.291250.275650.06365% lysis (1st) 7.416.297.9019.4716.0636.5987.32% lysis (2nd) 7.258.846.5820.0115.1326.8383.65% lysis Average 7.337.577.2419.7415.6031.7185.49STDEV0.121.800.930.380.666.902.59CV1.6323.7912.901.924.2121.763.03

[0212] Serum 10% Eculizumab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 150 75 37.5 18.75 9.38 4.69 2.34 1st test 0.72 89 1.16 28 1.11 23 1.20 98 0.93 43 1.58 14 1.99 7 32.29 9 30.25 66 0.06 26 2nd test Test 0.83 12 0.87 5 1.06 21 0.90 8 20.92 16 1.41 6 9 1.54 46 2.28 3 20.29 47 0.06 47 Average 0.78 00 5 1.01 8 9 1.08 7 21 05 9 0.92 7 9 5 1.49 9 15 1.77 09 5 2.29 12 5 0.27 5 6 5 0.06 36 5 % lysis (1st) 22.49 4 4.01 4 1.5 1 4 6.35 32.68 6 4.78 8 5.42 % lysis (2nd) 27.56 29.74 39.02 31.38 32.05 5 6.62 6 2.96 % lysis Average25.0236.8740.2638.8632.3660.7074.19STDEV3.5910.101.7610.580.455.7715.88CV14.3427.384.3727.221.389.5121.41

[0213] Serum 10%Ravulizumab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)1507537.518.759.384.692.341st test1.1491.28851.3441.581.18882.14851.58762.29930.25660.06262nd test Test 1.25421.21531.63781.62111.1852.2552.0282.28320.29470.0647Average 1.20161.25191.49091.600551.18692.201751.80782.291250.275650.06365% lysis(1st)43.3350.2553.0064.7145.3092.9265.09% lysis(2nd)48.5546.6267.5866.7545.1298.2086.94% lysis Average45.9448.4360.2965.7345.2195.5676.01STDEV3.692.5710.311.440.133.7415.45CV8.035.3017.102.190.293.9120.33

[0214] Serum 10%Crovalimab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)1507537.518.759.384.692.341st test1.13720.82090.87830.94911.01361.49871.99242.29930.25660.06262nd test Test 0.79960.84211.09380.93550.89031.35581.72342.28320.29470.0647Average 0.96840.83150.986050.94230.951951.427251.85792.291250.275650.06365% lysis (1st) 42.7427.0529.9033.4136.6160.6885.17% lysis (2nd) 25.9928.1040.5932.7430.4953.5971.83% lysis Average34.3727.5835.2533.0733.5557.1378.50STDEV11.840.747.560.484.335.019.44CV34.462.7021.451.4412.898.7712.02

[0215] Serum 10% Pozelimab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)1507537.518.759.384.692.341st test0.64160.93771.12460.90241.07671.671.79722.29930.25660.06262nd test Test 0.67270.70341.09291.21090.98351.61151.62522.28320.29470.0647Average 0.657150.820551.108751.056651.03011.640751.71122.291250.275650.06365% lysis (1st) 18.1632.8542.1231.0939.7469.1875.49% lysis (2nd) 19.7021.2240.5546.4035.1266.2866.96% lysis Average18.9327.0341.3338.7537.4367.7371.22STDEV1.098.221.1110.823.272.056.03CV5.7630.402.6927.938.743.038.47

[0216] Serum 25% antibody A(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 375 187.59 3.75 46.88 23.44 11.72 5.86 1st test 0.63 49 0.86 141.10 89 1.53 68 1.91 84 2.24 12.8 142.63 020.26 110.05 47 2nd test Test 0.64420.66551.13881.49981.77852.34152.74972.67130.27230.0571Average 0.639550.763451.123851.51831.848452.291252.781852.650750.26670.0559% lysis (1st) 15.4424.9435.3353.2769.2882.81106.85% lysis (2nd) 15.8316.7336.5851.7263.4187.03104.15% lysis Average 15.64 20.84 35.95 52.50 66.35 84.92 105.50 STDEV 0.28 5.81 0.89 1.104.15 2.98 1.91 CV 1.76 27.89 2.47 2.09 6.25 3.51 1.81

[0217] Serum 25% Eculizumab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 375 187.59 3.75 46.88 23.44 11.7 25.86 1st test 2.23 2.08 85 2.27 16 2.45 62.44 97 2.63 9 22.57 22.63 0 20.26 110.05 47 2nd test Test 2.18182.19122.27942.44892.4472.57262.49132.67130.27230.0571Average 2.20592.139852.27552.452452.448352.60592.531652.650750.26670.0559% lysis(1st) 82.3576.4284.1091.8391.5799.5296.70% lysis (2nd) 80.3380.7284.4291.5391.4596.7293.31% lysis Average 81.3478.5784.2691.6891.5198.1295.00 STDEV 1.433.050.230.210.081.982.39 CV 1.763.880.270.230.092.012.52

[0218] Serum 25%Ravulizumab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)375187.593.7546.8823.4411.725.861st test2.47042.5552.6082.70222.70952.75052.72612.63020.26110.05472nd test Test 2.4492.51572.49462.62042.69142.73882.80442.67130.27230.0571Average 2.45972.535352.55132.66132.700452.744652.765252.650750.26670.0559% lysis (1st) 92.4495.9898.21102.16102.46104.18103.16% lysis (2nd) 91.5494.3493.4598.73101.71103.69106.44% lysis Average91.9995.1695.83100.44102.08103.94104.80STDEV0.631.173.362.430.540.352.32CV0.691.223.512.420.530.332.22

[0219] Serum 25%Crovalimab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)375187.593.7546.8823.4411.725.861st test2.22721.94252.11342.15252.25922.31392.39692.63020.26110.05472nd test Test 2.1451.99471.94392.12312.07512.33012.54022.67130.27230.0571Average 2.18611.96862.028652.13782.167152.3222.468552.650750.26670.0559% lysis (1st) 82.2370.2977.4679.1083.5885.8789.35% lysis (2nd) 78.7972.4870.3577.8775.8586.5595.36% lysis Average 80.5 171.3 973.9 178.4 879.7 286.2 192.36 STDEV 2.44 1.55 5.0 30.87 5.46 0.48 4.25 CV 3.0 32.176.80 1.116.85 0.56 4.60

[0220] Serum 25% Pozelimab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 375 187.59 3.75 46.88 23.44 11.72 5.86 1st test 1.48 0 41.6 17 7 1.6 3 9 9 1.95 48 2.03 16 2.40 65 2.49 9 2.63 0 20.26 110.05 47 2nd test Test 1.45751.55441.58611.95882.02652.33252.52182.67130.27230.0571Average 1.468951.586051.6131.95682.029052.36952.51042.650750.26670.0559% lysis(1st)50.9156.6757.6070.8174.0389.7593.63% lysis(2nd)49.9554.0155.3470.9873.8286.6594.59% lysis Average 50.43 55.34 56.47 70.89 73.92 88.20 94.11 STDEV 0.68 1.88 1.60 0.12 0.15 2.19 0.68 CV ​​1.35 3.39 2.83 0.17 0.20 2.49 0.72

[0221] Serum 50% antibody A(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 750 375 187.59 3.75 46.88 23.44 11.72 1st test 2.13 44 1.37 59 1.68 46 2.15 0 72.22 78 2.89 0 63.27 133.01 170.59 270.07 86 2nd test Test 2.06081.92591.59151.92762.06422.85833.24863.01790.63880.0759Average 2.09761.65091.638052.039152.1462.874453.259953.01480.615750.07725% lysis(1st) 63.3031.6944.5563.9867.2094.82110.69% lysis (2nd) 60.2354.6140.6754.6860.3893.48109.75% lysis Average61.7743.1542.6159.3363.7994.15110.22STDEV2.1716.212.746.584.820.950.67CV3.5137.576.4411.087.561.010.61

[0222] Serum 50% Eculizumab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 750 3 75 18 7.59 3.75 46.88 23.44 11.72 1st test 3.15 9 ​​22.92 122.64 223.09 492.95 543.15 113.23 413.01 170.59 270.078 6 2nd test Test 3.14752.89282.48363.07372.81443.19863.24583.01790.63880.0759Average 3.153352.9072.56293.08432.88493.174853.239953.01480.615750.07725% lysis(1st) 106.0296.1084.47103.3497.52105.68109.14% lysis (2nd) 105.5394.9177.86102.4691.65107.66109.63% lysis Average 105.7895.5181.16102.9094.59106.67109.38STDEV 0.340.844.670.624.161.400.34CV 0.330.885.760.614.391.310.32

[0223] Serum 50%Ravulizumab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)750375187.593.7546.8823.4411.721st test3.24863.20253.03573.32243.27543.33173.33113.01170.59270.07862nd test Test 3.24253.19023.22313.18913.24093.31753.32693.01790.63880.0759Average 3.245553.196353.12943.255753.258153.32463.3293.01480.615750.07725% lysis (1st) 109.75107.82100.87112.82110.86113.21113.18% lysis (2nd) 109.49107.31108.68107.27109.42112.62113.01% lysis Average109.62107.57104.78110.04110.14112.91113.10STDEV0.180.365.523.931.020.420.12CV0.160.345.273.570.920.370.11

[0224] Serum 50%Crovalimab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)750375187.593.7546.8823.4411.721st test3.25832.95132.99013.06922.92023.18453.18813.01170.59270.07862nd test Test 3.27782.86672.933.0523.06113.1673.15353.01790.63880.0759Average 3.268052.9092.960053.06062.990653.175753.17083.01480.615750.07725% lysis(1st) 110.1597.3598.97102.2796.06107.07107.22% lysis (2nd) 110.9693.8396.47101.55101.93106.34105.78% lysis Average 110.56 95.59 97.72 101.9 198.99 106.7 1106.50 STDEV 0.57 2.49 1.77 0.514.15 0.52 1.02 CV 0.52 2.6 11.8 10.50 4.20 0.48 0.96

[0225] Serum 50% Pozelimab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 750 3 75 18 7.59 3.75 46.88 23.44 11.72 1st test 2.82 2.43 132.6 27 22.94 29 2.82 4 33.15 133.18 373.01 170.59 270.07 8 6 2nd test Test 2.87222.27132.72442.82892.883.1863.23963.01790.63880.0759Average 2.84712.35132.67582.88592.852153.168653.211653.01480.615750.07725% lysis (1st) 91.9675.6883.8497.0092.06105.69107.04% lysis (2nd) 94.0669.0187.9092.2594.38107.14109.37% lysis Average93.0172.3485.8794.6393.22106.41108.21STDEV1.484.722.863.361.641.021.65CV1.596.523.343.551.760.961.52

[0226] Serum 75% antibody A(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 1 1 2 5 5 6 2.5 2 8 1.25 14 0.6 3 7 0.3 1 3 5.16 17.58 1st test 1.4 0 7 3 1.5 1.6 4 7 6 1.9 9 8 12.7 0 6 3.6 9 8 34 3.5 3 0 6 1.25 7 8 0.0 9 7 7 2nd test Test 1.40 141.46 41.69 071.90 89 2.45 66 3.64 33.93 42 3.58 0 31.31 22 0.08 82 Average 1.40 43 51.48 21.66 9 151.95 352.58 133.67 065 3.96 7 13.55 54 51.28 5 0.09 29 5% lysis (1st) 5.39 9.47 15.97 31.41 62.59 106.29 119.58% lysis (2nd) 5.13 7.88 17.87 27.48 51.60 103.86 116.68% lysis Average 5.268.6816.9229.4457.09105.07118.13STDEV0.181.121.342.787.771.722.05CV3.5012.927.939.4413.601.641.73

[0227] Serum 75% Eculizumab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 1 1 2 5 5 6 2.5 2 8 1.2 5 14 0.6 3 7 0.3 1 3 5 16 17 58 1st test 3.3 4 2 5 3.5 8 9 4 3.6 0 4 2 3.6 8 16 3.9 4 7 4 3.9 0 7 5 3.9 5 3.5 3 0 6 1.2 5 7 8 0.0 9 7 7 2nd test Test 3.47753.54083.6513.69163.83353.82163.84993.58031.31220.0882Average 3.413.56513.62763.68663.890453.864553.899953.555451.2850.09295% lysis (1st) 90.62101.50102.15105.56117.26115.51117.38% lysis (2nd) 96.5799.35104.21106.00112.25111.72112.97% lysis Average93.59100.43103.18105.78114.75113.61115.17STDEV4.201.511.460.313.552.683.12CV4.491.511.410.293.092.352.71

[0228] Serum 75%Ravulizumab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)1125562.5281.25140.6370.3135.1617.581st test3.75153.75283.73393.68693.86943.941843.53061.25780.09772nd test Test 3.83383.89323.74223.7593.90273.977143.58031.31220.0882Average 3.792653.8233.738053.722953.886053.9594543.555451.2850.09295% lysis(1st) 108.63108.69107.86105.79113.83117.02119.58% lysis (2nd) 112.26114.88108.23108.97115.29118.57119.58% lysis Average110.45111.78108.04107.38114.56117.79119.58STDEV2.564.370.262.251.041.100.00CV2.323.910.242.090.910.930.00

[0229] Serum 75%Crovalimab(+) controlNo Ab(-) control 1HI serum(-) control 2No serum concentration(㎍ / ㎖)1125562.5281.25140.6370.3135.1617.581st test3.72923.83743.6953.61833.83573.88073.86773.53061.25780.09772nd test Test 3.76953.79333.72383.66743.9673443.58031.31220.0882Average 3.749353.815353.70943.642853.90153.940353.933853.555451.2850.09295% lysis(1st) 107.65112.42106.15102.77112.34114.33113.75 % lysis (2nd) 109.43110.48107.41104.93118.14119.58119.58 % lysis Average 108.54 111.45 106.78 103.85 115.24 116.95 116.67 STDEV 1.26 1.37 0.90 1.53 4.103.72 4.12 CV 1.16 1.23 0.84 1.47 3.56 3.18 3.53

[0230] Serum 75% Pozelimab(+) control No Ab(-) control 1 HI serum(-) control 2 No serum concentration (㎍ / ㎖) 1 1 2 5 5 6 2.5 2 8 1.2 5 14 0.6 3 7 0.3 1 3 5.1 6 17 5 8 1st test 2.6 7 1 3 3 1 0 4 3 7 6 5 3.6 1 1 9 4 4 3 5 3 0 6 1 2 5 7 8 1.6 5 2 7 2nd test 2.6 9 4 8 3.1 3 5 3.4 1 8 7 3.5 5 19 3.8 7 0 5 4 3 5 8 0 3 1 3 1 2 2 1.7 4 5 3 average 2.6 8 3 0 5 3.1 2 2 7 3.3 9 7 6 3.5 8 1 9 3.9 5 2 5 4 4 3 5 5 4 5 1.2 8 5 1.6 9 9 % lysis(1st)61.0680.4092.12102.49119.58119.58119.58 % lysis (2nd)62.0981.4893.9899.84113.88119.58119.58 % lysis average61.5880.9493.05101.16116.73119.58119.58 STDEV0.730.771.311.874.030.000.00 CV1.190.951.411.853.460.000.00

[0231] Serum Antibody Ratio AEculizumabRavulizumabCrovalimabPozelimab75%63.08419.270.9970.85114250%30.2124.650.15Unstable45.5225%7.75364.4974.9812.1123.4910%0.02814.858unstable4.7761.72X10 -7

[0232] As shown in Figures 17 to 20, as in the experiments for classical pathway-mediated complement activation, the antibody also exhibited superior complement activation inhibition ability compared to the competitor antibody under all serum ratio conditions for alternative pathway-mediated complement activation.

[0233] In particular, when the serum ratio is low, such as in the 10% serum condition, the difference in efficacy between the competitor antibody and the antibody of the present invention is not significant, but as the serum ratio increases, such as in the 25%, 50%, and 75% serum conditions, the competitor antibody shows a decrease in the level of blood cell lysis inhibition, and the level of blood cell lysis does not decrease significantly even when the concentration of the antibody being treated increases, but the antibody of the present invention shows a significant decrease in the level of blood cell lysis as the concentration of the antibody being treated increases even when the serum ratio is high.

[0234] Through this, it can be inferred that the antibody of the present invention will exhibit a better effect in an actual body with a high serum ratio compared to competitive antibodies.

[0235] In addition, through Examples 6 and 7, it was confirmed that the anti-C5 antibody of the present invention can effectively inhibit both the classical pathway and the alternative pathway complement activation pathway, and that the level of inhibition was superior to that of competitive antibodies.

[0236] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0237] The anti-C5 antibody according to the present invention can significantly reduce the level of free C5 and stably inhibit the activity of complement even when the amount of complement increases due to an increase in the ratio of serum under test conditions, and can completely inhibit complement C5 activity by effectively inhibiting both the classical pathway (CP) and the alternative pathway (AP) of the complement system, and thus can be used for the prevention or treatment of various autoimmune diseases including myasthenia gravis, age-related macular degeneration, and paroxysmal nocturnal hemoglobinuria, and thus has industrial applicability.

Claims

1. A pharmaceutical composition for preventing or treating complement-related diseases, comprising an effective amount of an anti-C5 antibody or an antigen-binding fragment thereof, which is a full-length antibody consisting of a heavy chain sequence of sequence number 1 and a light chain sequence of sequence number 2, A composition wherein the anti-C5 antibody or antigen-binding fragment thereof inhibits free C5 to 130 ng / mL or less.

2. In the first paragraph, the serum complement hemolytic activity of the anti-C5 antibody or its antigen-binding fragment is determined through an in vitro serum hemolytic test, and the composition inhibits complement activity when the proportion of serum in the test conditions is 50% or more.

3. A composition according to claim 1, wherein the anti-C5 antibody or antigen-binding fragment thereof inhibits both the classical pathway and the alternative pathway of complement activation.

4. In the first paragraph, the anti-C5 antibody or antigen-binding fragment thereof is IC based on an in vitro serum hemolytic test. 50 A composition that inhibits the classical pathway of C5 complement activation at a concentration of 5 to 75 ug / ml.

5. In the first paragraph, the anti-C5 antibody or antigen-binding fragment thereof is 1 X 10 -5 / s to 8 X 10 -5 / s dissociation rate constant (K d ) having a value.

6. A composition according to claim 1, wherein the host cell protein (HCP) is contained in an amount of 100 ng or less per 1 mg of the anti-C5 antibody or antigen-binding fragment thereof.

7. A composition comprising 25% or less of an acidic variant in the composition according to claim 1.

8. A composition according to claim 1, wherein the complement-related disease is any one selected from the group consisting of generalized myasthenia gravis (gMG), age-related macular degeneration (AMD), and paroxysmal nocturnal hemoglobinuria (PNH).

9. A pharmaceutical composition for preventing or treating a complement-related disease, comprising an effective amount of an anti-C5 antibody or an antigen-binding fragment thereof that specifically binds to the MG 4 domain of the complement C5 protein beta chain consisting of the amino acid sequence of SEQ ID NO: 10, A composition wherein the anti-C5 antibody or antigen-binding fragment thereof inhibits free C5 to 130 ng / mL or less.

10. In the 9th paragraph, the serum complement hemolytic activity of the anti-C5 antibody or antigen-binding fragment thereof is determined through an in vitro serum hemolytic test, and the composition inhibits complement activity when the proportion of serum in the test conditions is 50% or more.

11. A composition according to claim 9, wherein the anti-C5 antibody or antigen-binding fragment thereof inhibits both the classical pathway and the alternative pathway of complement activation.

12. In the 9th paragraph, the anti-C5 antibody or antigen-binding fragment thereof is IC based on an in vitro serum hemolytic test. 50 A composition that inhibits the classical pathway of C5 complement activation at a concentration of 5 to 75 ug / ml.

13. In the 9th paragraph, the anti-C5 antibody or antigen-binding fragment thereof is 1 X 10 -5 / s to 8 X 10 -5 / s dissociation rate constant (K d ) having a value.

14. A composition according to claim 9, wherein the host cell protein (HCP) is contained in an amount of 100 ng or less per 1 mg of the anti-C5 antibody or antigen-binding fragment thereof.

15. A composition comprising 25% or less of an acidic variant in the composition according to claim 9.

16. A composition according to claim 9, wherein the complement-related disease is any one selected from the group consisting of myasthenia gravis (gMG), age-related macular degeneration (AMD), and paroxysmal nocturnal hemoglobinuria (PNH).

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