Anti-ne antibody and pharmaceutical composition for preventing or treating ne hyperactivation-related diseases comprising same

Abstract

The present invention relates to an anti-neutrophil elastase (NE) antibody or antigen-binding fragment thereof that specifically binds to NE, wherein the anti-NE antibody or antigen-binding fragment thereof comprises: a heavy chain variable region (VH) including a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, 7, or 13, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, 8, or 14, and a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3, 9, or 15; and a light chain variable region (VL) including a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, 10, or 16, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, 11, or 17, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6, 12, or 18. The antibody of the present invention exhibits excellent NE neutralizing activity, and thus can be used for preventing or treating NE hyperactivation-related diseases.

Description

Anti-NE antibody and pharmaceutical composition for preventing or treating NE-hyperactivation-related diseases comprising the same

[0001] The present invention relates to an anti-NE antibody or an antigen-binding fragment thereof that specifically binds to NE (neutrophil elastase). The anti-NE antibody can specifically bind to NE and neutralize it.

[0002] Neutrophil elastase (NE) is a serine protease, responsible for approximately 80% of the total proteolytic activity of the human body. NE is primarily found in neutrophils, where it plays a crucial role in the innate immune response, and is stored in azurophil granules within neutrophils, along with other proteases involved in antimicrobial defense.

[0003] NE is synthesized in a premature form, and the terminal end containing the C-terminus and signal peptide is cleaved, and the terminal dipeptide is further cleaved by DPP1 to form the mature form. NE is activated by a trimeric catalytic structure composed of Asp, His, and Ser, but exists in an inactive state within granules compartmented at low pH (Pharmacol Rev 62:726-59).

[0004] Meanwhile, in cases of infection or other inflammatory stimuli, neutrophils rapidly infiltrate organs and undergo phagocytosis. During this process, neutrophils are activated and degranulated, releasing NE into the cytoplasm at a neutral pH, where it becomes activated. At this time, NE plays an important role in pathogen attack, along with NADPH oxidase, myeloperoxidase (MPO), and reactive oxygen species (ROS) (Clin Immunol 99:7-17).

[0005] High concentrations of NE released locally outside the cell kill pathogens directly or through neutrophil extracellular traps (NETs). Furthermore, they can degrade extracellular matrix (ECM) components such as elastin, collagen, and fibronectin, thereby contributing to the physical support and stability of tissues (Nat Rev Immunol 6:541-50).

[0006] However, NE can also induce a wide range of inflammation through various substrates. NE plays a dual role in inducing inflammatory responses by activating IL-1β and IL-18, precursors of inflammatory cytokines, and promoting the production of IL-8, or conversely, directly degrading already produced TNF-α, IL-1β, and IL-8 to modulate cytokine signaling (J Immunol 175:7594-601, J Biomed Sci 11:49-58). Furthermore, fragments generated by NE cleavage of extracellular matrix (ECM), surface receptors, and cytokines influence cell signaling (Nat Rev Immunol 6:541-50).

[0007] When NE is overactivated in this way, it actually promotes inflammation, progression of bacterial infection, and excessive mucus secretion (Biochem Pharmacol 83:695-704). Therefore, protein-tissue inhibitor proteins such as serpin and macroglobulin, which exist as essential components of body fluids, quickly neutralize NE and prevent destruction of ECM and host cells by NE. Representative examples include elafin and SLPI (secretory leucocyte protease inhibitor), which are produced and secreted by epithelial cells, neutrophils, and macrophages, contributing to local defense, and AAT (α-1 antitrypsin), which is produced in the liver and circulates throughout the body to regulate NE (Clin Sci (Lond) 110:21-35).

[0008] If they fail to properly neutralize NE, the balance between proteases and inhibitor proteins becomes disrupted, leading to NE overactivity that can cause a number of diseases (Rheumatology (Oxford) 49:1618-31, Annu Rev Immunol 30:459-89). In addition to acute diseases, prolonged increased NE activity can lead to tissue destruction and remodeling due to infection and chronic inflammation, which can induce various serious chronic diseases, including lung and vascular diseases, skin diseases, obesity, and cancer (Curr Opin Hematol 21:23-8, Int J Biol Sci 14:1343-1360, J Pharm Anal 13:355-366). In addition, the amount and activity of NE are known to reflect the disease state and severity (Biomolecules 11:1065).

[0009] To control overactive NE, several synthetic NE inhibitors have been developed to date. However, while preclinical studies have demonstrated a multifaceted reduction in NE-induced damage and inflammation, none have been approved as therapeutics in clinical trials due to lack of efficacy and concerns about toxicity. Therefore, the development of antibodies or antigen-binding fragments that can specifically bind and neutralize extracellular overactive NE and minimize side effects remains essential.

[0010] Accordingly, the inventors of the present invention invented a novel antibody that specifically binds to NE with high affinity and confirmed the efficient neutralizing ability of the antibody, thereby confirming its potential as an effective therapeutic agent and combination agent for various diseases caused by overactivated NE, and completed the present invention.

[0011] The purpose of the present invention is to provide a means for preventing or treating diseases related to NE overactivation by providing an antibody or an antigen-binding fragment thereof that specifically binds to NE or a fragment thereof and has a neutralizing ability.

[0012] To achieve the above objectives:

[0013] One aspect of the present invention provides an anti-NE antibody or antigen-binding fragment thereof that specifically binds to NE (neutrophil elastase), comprising a heavy chain variable region (VH) comprising a heavy chain CDR1 represented by an amino acid sequence of SEQ ID NO: 1, 7 or 13, a heavy chain CDR2 represented by an amino acid sequence of SEQ ID NO: 2, 8 or 14 and a heavy chain CDR3 represented by an amino acid sequence of SEQ ID NO: 3, 9 or 15; and a light chain variable region (VL) comprising a light chain CDR1 represented by an amino acid sequence of SEQ ID NO: 4, 10 or 16, a light chain CDR2 represented by an amino acid sequence of SEQ ID NO: 5, 11 or 17 and a light chain CDR3 represented by an amino acid sequence of SEQ ID NO: 6, 12 or 18.

[0014] Another aspect of the present invention provides a polynucleotide encoding the antibody or an antigen-binding fragment thereof.

[0015] Another aspect of the present invention provides an expression vector comprising the polynucleotide.

[0016] Another aspect of the present invention provides a host cell transformed with the expression vector.

[0017] Another aspect of the present invention comprises: (a) a step of culturing the host cell; and

[0018] (b) A method for producing an antibody or an antigen-binding fragment thereof that specifically binds to NE is provided, comprising the step of recovering the antibody or an antigen-binding fragment thereof from the cultured cells.

[0019] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating a disease associated with NE hyperactivation, comprising the anti-NE antibody or an antigen-binding fragment thereof as an active ingredient.

[0020] The antibody of the present invention or an antigen-binding fragment thereof has high binding affinity and affinity for NE and has an effect of reducing cell adhesion and inhibiting cell death by NE, and therefore can be effectively used for the prevention or treatment of diseases related to NE overactivation.

[0021] However, the effects of the present invention are not limited to the above-described effects, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0022] Figure 1 shows the results of selecting three types of antibodies (NI-10, NI-11, NI-50) with neutralizing ability against the antigen NE through antibody single concentration screening evaluation.

[0023] Figure 2 shows the IC for NE neutralization ability of NI-10 antibody and humanized antibodies (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45). 50 Shows the value.

[0024] Figures 3a and 3b show the EC values ​​for NE binding affinity of NI-10, NI-11, NI-50 and humanized antibodies (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45). 50 Shows the value.

[0025] Figure 4 shows the cell adhesion reduction inhibitory effect and cell death inhibition effect of NI-10 and humanized antibody NI-10_v38.

[0026] Figure 5 shows the results of a competition ELISA in which the binding affinity of NI-10 is reduced when the endogenous protein elafin and NI-10, which bind to the active site of NE, are simultaneously bound to the antigen NE.

[0027] Figure 6 shows the results of confirming the validity of the epitope by observing the competitive binding of other endogenous proteins alpha-1 antitrypsin (AAT) and NI-10_v38, which bind to the NE active site, using BLI.

[0028] Figure 7 shows the results of gel electrophoresis confirming the resistance of humanized antibodies NI-10_v38, AAT, and mutated AAT-Fc fusion protein INBRX-101 to protein cleavage by high concentrations of NE.

[0029] Figure 8 is a graph comparing the stability of humanized antibody NI-10_v38 against oxidation with AAT, and comparing the hNE residual activity over time under oxidative conditions.

[0030] Figure 9 shows the results of evaluating the lung damage inhibition effect and anti-inflammatory effect of humanized antibody NI-10_v38 in an acute lung injury mouse model.

[0031] Hereinafter, the present invention will be described in detail.

[0032] In the present invention, the term “consisting of” means that the proportion of a specific component(s) totals 100%. The components or features listed below the term “consisting of” may be essential or mandatory.

[0033] In the present invention, the term "comprising" means the presence of the features, steps, or components described below, and does not exclude the presence or addition of one or more features, steps, or components. While the components or features described below "comprising" in this specification may be essential or mandatory, some embodiments may further include other optional or non-essential components or features.

[0034] In the present invention, the term “comprising” may, in some embodiments, be modified to refer to “consisting essentially of” or “consisting of.”

[0035] As used herein, the term “binds specifically to” or “specific for” means determinative for the presence of a target in the presence of a heterogeneous population of molecules, including biological molecules, and refers to a measurable and reproducible interaction, such as binding between a target and an antibody. For example, an antibody that specifically binds to a particular target (e.g., an epitope) means an antibody that binds to that target with greater affinity, greater binding affinity, more readily, and / or with a longer duration of binding than an antibody that binds to other targets.

[0036] In the present invention, the term “K D ” refers to the binding equilibrium dissociation constant of a specific antibody-antigen interaction, K D = K dis / K a It is calculated through the calculation formula (at this time, K a is the association rate constant, and K dis is the dissociation rate constant), the constant K D has units of M. K for antibodies D The value can be measured using methods widely established in the art. The K of an antibody D Specific methods for measuring the values ​​include surface plasmon resonance (SPR), more specifically, using a biosensor system, for example, the Biacore® system, or bio-layer interferometry (BLI), for example, the Octet® system.

[0037] Anti-NE antibody or antigen-binding fragment thereof

[0038] The present invention provides an anti-neutrophil elastase (NE) antibody or an antigen-binding fragment thereof that specifically binds to NE.

[0039] The present invention provides an anti-NE antibody or antigen-binding fragment thereof that specifically binds to NE (neutrophil elastase), comprising a heavy chain variable region (VH) comprising a heavy chain CDR1 represented by an amino acid sequence of SEQ ID NO: 1, 7 or 13, a heavy chain CDR2 represented by an amino acid sequence of SEQ ID NO: 2, 8 or 14, and a heavy chain CDR3 represented by an amino acid sequence of SEQ ID NO: 3, 9 or 15; and a light chain variable region (VL) comprising a light chain CDR1 represented by an amino acid sequence of SEQ ID NO: 4, 10 or 16, a light chain CDR2 represented by an amino acid sequence of SEQ ID NO: 5, 11 or 17, and a light chain CDR3 represented by an amino acid sequence of SEQ ID NO: 6, 12 or 18.

[0040] Another aspect of the present invention provides an anti-NE antibody or antigen-binding fragment thereof, wherein the anti-NE antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising an amino acid sequence of SEQ ID NO: 37, 39, 41, 56, 58 or 60; and a light chain variable region (VL) comprising an amino acid sequence of SEQ ID NO: 38, 40, 42, 57 or 59.

[0041] Another aspect of the present invention provides an anti-NE antibody or antigen-binding fragment thereof, wherein the anti-NE antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) represented by an amino acid sequence of SEQ ID NO: 37, 39, 41, 56, 58 or 60; and a light chain variable region (VL) represented by an amino acid sequence of SEQ ID NO: 38, 40, 42, 57 or 59.

[0042] In another aspect of the present invention, the anti-NE antibody or antigen-binding fragment thereof comprises a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 9, a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 12; Or, an antibody or antigen-binding fragment thereof is provided, which comprises a heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 14, a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 15, a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 18.

[0043] In the present invention, the term "NE" may refer to a concept that collectively refers to NE itself, any variants, isotypes, and paralogs existing in animals or humans. For example, it may refer to NE derived from humans or mice, and "human NE" refers to NE derived from humans.

[0044] In the present invention, the term “antibody or antigen-binding fragment thereof” means an anti-NE antibody molecule that specifically binds to NE, or a fragment of the antibody molecule that has the function of binding to an antigen even if it is not in the form of a complete antibody.

[0045] Antibodies of the present invention include, but are not limited to, monoclonal antibodies, bispecific antibodies, polyspecific antibodies, human antibodies, mouse antibodies, humanized antibodies, chimeric antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4, IgG-delta CH2, CrossMabs, Trimab, DVD-Igs, CovX-bodies, duobodies or triomAbs antibodies.

[0046] The above monoclonal antibody refers to an antibody against a single determinant on an antigen, and refers to an antibody obtained from a substantially homogeneous antibody population.

[0047] The above humanized antibody utilizes amino acid sequences derived from human antibodies or has modified portions thereof, except for the CDR sequences. Therefore, when administered to the human body, it is less likely to induce HAMA (Human Anti-Mouse Antibody), and thus has low immunogenicity. Accordingly, the humanized antibody of the present invention has the advantage of being able to resolve the problem of immune response that may arise when using mouse-derived antibodies.

[0048] Antigen-binding fragments of the present invention include, but are not limited to, Fab, F(ab'), F(ab')2, Fv, single-chain antibodies (scFv), diabodies, triabodies, tetrabodies, minibodies, phenomers, DARTs, BiTEs, TandAbs, DAFs, SMIPs, anticalins, FN3 monobodies, DARPins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Avimers, Im7, VLR, VNAR, nanobodies, bi-nanobodies, di-sdFv, etc. Among antibody fragments, Fab has a structure having variable regions of light and heavy chains, constant region of light chain, and first constant region (CH1) of heavy chain and has one antigen-binding site. F(ab') 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 cysteine ​​residues in the hinge region of Fab' form disulfide bonds. Fv refers to the smallest antibody fragment that contains only the heavy chain variable region and the light chain variable region.

[0049] The term "epitope" as used herein refers to a protein determinant to which an antibody can specifically bind. Epitopes are typically composed of chemically active surface molecules, such as amino acids or sugar side chains, and typically have specific three-dimensional structural characteristics.

[0050] In the present invention, the term "heavy chain" means a full-length heavy chain or a fragment thereof comprising a heavy chain variable region (VH) that specifically binds to an antigen and three constant regions CH1, CH2, and CH3. In addition, the term "light chain" in the present invention means a full-length light chain or a fragment thereof comprising a light chain variable region (VL) that specifically binds to an antigen and a constant region CL.

[0051] In the present invention, the term "variable region" refers to the light chain and heavy chain portions of an antibody molecule, including complementarity determining regions (CDRs; i.e., CDR1, CDR2, and CDR3), and framework regions (FR).

[0052] In the present invention, the term "complementarity determining region" (CDR; i.e., CDR1, CDR2, and CDR3) refers to a portion of an antibody variable region necessary for antigen binding. The heavy and light chain variable regions each typically have three CDR regions, identified as CDR1, CDR2, and CDR3.

[0053] The anti-NE antibody of the present invention or an antigen-binding fragment thereof may include not only the anti-NE sequence of the present invention described herein, but also biological equivalents thereof, as long as it can specifically recognize NE. For example, additional changes may be made to the amino acid sequence of the antibody to improve biological properties such as binding affinity of the antibody. Such changes include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody. Such amino acid modifications are made based on the relative similarity of 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; Phenylalanine, tryptophan, and tyrosine are biologically functional equivalents of each other. The above classification is merely an example and is not intended to be limiting.

[0054] Based on this, considering a modification having biologically equivalent activity, the antibody or antigen-binding fragment thereof of the present invention is interpreted to also include a sequence that shows substantial identity with the specified sequence described in the specification. In terms of such substantial identity, the antibody or antigen-binding fragment thereof of the present invention means including a sequence that has 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology compared to the sequence described in the specification.

[0055] Specifically, the antibody or antigen-binding fragment thereof of the present invention may be an anti-NE antibody or antigen-binding fragment thereof comprising a CDR having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with a CDR of the anti-NE antibody or antigen-binding fragment thereof.

[0056] Polynucleotide encoding anti-NE antibody or antigen-binding fragment thereof, expression vector, host cell and production method

[0057] One aspect of the present invention provides a polynucleotide encoding an anti-NE antibody or an antigen-binding fragment thereof of the present invention.

[0058] Another aspect of the present invention provides a polynucleotide comprising a heavy chain variable region (VH) coding region comprising a heavy chain CDR1 coding sequence represented by SEQ ID NO: 19, 25 or 31, a heavy chain CDR2 coding sequence represented by SEQ ID NO: 20, 26, 32 or 49, and a heavy chain CDR3 coding sequence represented by SEQ ID NO: 21, 27, 33 or 50; and a light chain variable region (VL) coding region comprising a light chain CDR1 coding sequence represented by SEQ ID NO: 22, 28, 34, 51 or 54, a light chain CDR2 coding sequence represented by SEQ ID NO: 23, 29, 35, 52 or 55, and a light chain CDR3 coding sequence represented by SEQ ID NO: 24, 30, 36 or 53.

[0059] Another aspect of the present invention provides a polynucleotide comprising a heavy chain variable region (VH) encoding base sequence represented by SEQ ID NO: 43, 45, 47, 61, 63 or 65; and a light chain variable region (VL) encoding base sequence represented by SEQ ID NO: 44, 46, 48, 62 or 64.

[0060] In another aspect of the present invention, the polynucleotide comprises a heavy chain CDR1 coding base sequence represented by SEQ ID NO: 19, a heavy chain CDR2 coding base sequence represented by SEQ ID NO: 20, a heavy chain CDR3 coding base sequence represented by SEQ ID NO: 21, a light chain CDR1 coding base sequence represented by SEQ ID NO: 22, a light chain CDR2 coding base sequence represented by SEQ ID NO: 23, and a light chain CDR3 coding base sequence represented by SEQ ID NO: 24; a heavy chain CDR1 coding base sequence represented by SEQ ID NO: 25, a heavy chain CDR2 coding base sequence represented by SEQ ID NO: 26, a heavy chain CDR3 coding base sequence represented by SEQ ID NO: 27, a light chain CDR1 coding base sequence represented by SEQ ID NO: 28, a light chain CDR2 coding base sequence represented by SEQ ID NO: 29, and a light chain CDR3 coding base sequence represented by SEQ ID NO: 30; Or, a polynucleotide is provided, which comprises a heavy chain CDR1 coding sequence represented by SEQ ID NO: 31, a heavy chain CDR2 coding sequence represented by SEQ ID NO: 32, a heavy chain CDR3 coding sequence represented by SEQ ID NO: 33, a light chain CDR1 coding sequence represented by SEQ ID NO: 34, a light chain CDR2 coding sequence represented by SEQ ID NO: 35, and a light chain CDR3 coding sequence represented by SEQ ID NO: 36.

[0061] Another aspect of the present invention provides a polynucleotide comprising a heavy chain CDR1 coding sequence represented by SEQ ID NO: 19, a heavy chain CDR2 coding sequence represented by SEQ ID NO: 49, a heavy chain CDR3 coding sequence represented by SEQ ID NO: 50, a light chain CDR1 coding sequence represented by SEQ ID NO: 51, a light chain CDR2 coding sequence represented by SEQ ID NO: 52, and a light chain CDR3 coding sequence represented by SEQ ID NO: 53; or a heavy chain CDR1 coding sequence represented by SEQ ID NO: 19, a heavy chain CDR2 coding sequence represented by SEQ ID NO: 49, a heavy chain CDR3 coding sequence represented by SEQ ID NO: 50, a light chain CDR1 coding sequence represented by SEQ ID NO: 54, a light chain CDR2 coding sequence represented by SEQ ID NO: 55, and a light chain CDR3 coding sequence represented by SEQ ID NO: 24.

[0062] One aspect of the present invention provides an expression vector comprising the polynucleotide of the present invention.

[0063] One aspect of the present invention provides a host cell transformed with the expression vector of the present invention.

[0064] One aspect of the present invention provides a method for producing an anti-NE antibody or an antigen-binding fragment thereof that specifically binds to NE, comprising the steps of: (a) culturing a host cell of the present invention; and (b) recovering the antibody or an antigen-binding fragment thereof from the cultured cell.

[0065] The term "polynucleotide" used in the present invention has a comprehensive meaning that includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogs with modified sugar or base moieties. The sequence of the nucleic acid molecule 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. The polynucleotide may be isolated.

[0066] The term "expression vector" used in the present invention refers to a recombinant vector capable of expressing a target protein in a host cell, and refers to an expression system that includes essential regulatory elements operably linked to enable expression of the inserted gene. The polynucleotide encoding the anti-NE antibody or antigen-binding fragment thereof of the present invention may be inserted into a single vector, or may be used in a form inserted into one or more separate vectors. For example, the polynucleotides encoding the heavy chain and light chain of the anti-NE antibody of the present invention may be inserted into a single vector, or may be used in a form inserted into separate vectors.

[0067] The term "operably linked" as used herein means that a nucleic acid expression control sequence and a nucleic acid sequence encoding a desired protein are functionally linked to perform a desired function.

[0068] An expression vector suitable for the production of the above-described anti-NE antibody or antigen-binding fragment thereof may include, in addition to expression regulatory elements such as a promoter, an initiation codon, a stop codon, a polyadenylation signal, and an enhancer, a signal sequence for membrane targeting or secretion. The initiation codon and stop codon are generally considered to be part of the nucleotide sequence encoding the immunogenic target protein, and must be functional in a subject when the gene expression system is administered, and must be located in frame with the coding sequence. A common promoter may be a constitutive or inducible promoter. Promoters include, but are not limited to, the prokaryotic promoters of lac, tac, T3, and T7, simian virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV) such as the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Rous sarcoma virus (RSV) promoters, as well as eukaryotic promoters derived from the β-actin promoter, human hemoglobin, human muscle creatine, and human metallothionein.

[0069] The above expression vector may include a selectable marker for selecting host cells containing the vector. The selectable marker is used to select cells transformed with the vector, and markers that confer a selectable phenotype, such as drug resistance, nutrient requirement, cytotoxic agent resistance, or expression of a surface protein, may be used. In an environment treated with a selective agent, only cells expressing the selectable marker survive, allowing for selection of transformed cells. In addition, if the vector is a replicable expression vector, it may include a replication origin, which is a specific nucleic acid sequence where replication begins.

[0070] Various types of vectors, such as plasmids, viruses, and cosmids, can be used as recombinant expression vectors for inserting foreign genes. The type of recombinant vector is not particularly limited, as long as it functions to express the desired gene and produce the desired protein in various host cells, either prokaryotic or eukaryotic. However, a vector that exhibits a strong promoter activity and possesses strong expression capacity while being capable of mass-producing a foreign protein in a form similar to that in the natural state may be preferable.

[0071] The term "host cell" used in the present invention refers to a cell system that is transformed with an expression vector and can express a protein encoded by a polynucleotide included in the vector.

[0072] Suitable host cells for the above vector may be prokaryotic cells such as Escherichia coli, Bacillus subtilis, Streptomyces sp., Pseudomonas sp., Proteus mirabilis or Staphylococcus sp. In addition, it may be eukaryotic cells such as fungi such as Aspergillus sp., yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa, other lower eukaryotic cells, and cells of higher eukaryotes such as cells from insects. In addition, the host cell may be derived from plants or mammals. Preferably, Chinese hamster ovary (CHO) cells, W138 cells (diploid human cell culture), baby hamster kidney (BHK) cells, and HEK293 cells can be used, and CHO cells can be preferably used, but are not limited thereto.

[0073] The method for producing an anti-NE antibody or an antigen-binding fragment thereof of the present invention may specifically include the steps of: producing a recombinant vector by inserting a polynucleotide sequence encoding the anti-NE antibody or an antigen-binding fragment thereof into an expression vector; transforming the recombinant vector into a host cell and culturing the same; and isolating, purifying, and recovering the antibody or an antigen-binding fragment thereof from the cultured transformant.

[0074] Antibodies can be mass-produced by culturing transformants expressing the above recombinant vector in nutrient media. The media and culture conditions can be appropriately selected based on the host cell type. During culture, conditions such as temperature, medium pH, and culture time can be appropriately adjusted to ensure cell growth and mass production of proteins.

[0075] The anti-NE antibody or antigen-binding fragment thereof produced recombinantly as described above can be recovered from the medium or cell lysate. If it is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., Triton-X 100) or by enzymatic cleavage. The cells used to express the antibody or antigen-binding fragment thereof can be disrupted by various physical and chemical means, such as freeze-thaw purification, sonication, mechanical disruption, or cell lysing agents, and can be separated and purified by conventional biochemical separation techniques. Electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunoadsorption chromatography, size exclusion chromatography, etc.), isoelectric focusing, etc. can be used, but are not limited thereto.

[0076] Pharmaceutical composition comprising an anti-NE antibody or an antigen-binding fragment thereof

[0077] One aspect of the present invention provides a pharmaceutical composition for preventing or treating a disease associated with NE hyperactivation, comprising the anti-NE antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

[0078] In the present invention, the disease related to NE overactivation may be any one selected from the group consisting of chronic obstructive pulmonary disease (COPD), fibrosis, cystic fibrosis, alpha-1-antitrypsin deficiency (AATD), bronchiectasis, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), cyclic neutropenia, and emphysema.

[0079] The above pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. For oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, coloring agents, fragrances, etc. may be used. For injections, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used. For topical administration, bases, excipients, lubricants, preservatives, etc. may be used.

[0080] The pharmaceutical composition may be prepared in various forms by mixing it with the pharmaceutically acceptable carrier described above. For example, for oral administration, it may be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it may be prepared in the form of unit dose ampoules or multiple doses.

[0081] Additionally, the pharmaceutical composition may include a surfactant capable of improving membrane permeability. Such surfactants may be, but are not limited to, those derived from steroids, cationic lipids such as N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA), or various compounds such as cholesterol hemisuccinate and phosphatidylglycerol.

[0082] The pharmaceutical composition may be administered together with or sequentially with the aforementioned pharmacological or physiological components, or may be administered in combination with additional conventional therapeutic agents, either sequentially or simultaneously. Such administration may be single or multiple administrations. Taking all of the above factors into account, it is important to administer an amount that achieves maximum effect with the minimum amount possible without causing side effects, a determination readily made by those skilled in the art.

[0083] The term "subject" as used herein means a mammal suffering from or at risk of a condition or disease that can be alleviated, suppressed, prevented or treated by administering the pharmaceutical composition, and preferably means a human.

[0084] The term "administration" as used herein refers to introducing a given substance into a subject by any suitable method, and the pharmaceutical composition may be administered via any route as long as it can reach the target tissue. Such administration methods include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, and rectal administration. However, since proteins are digested during oral administration, it may be desirable to formulate oral compositions to coat the active agent or protect it from degradation in the stomach. Furthermore, the pharmaceutical composition may be administered by any device that allows the active agent to travel to the target cells.

[0085]

[0086] Compositions, uses, therapeutic methods and kits comprising anti-NE antibodies or antigen-binding fragments thereof

[0087] One aspect of the present invention provides a biomarker composition for diagnosing a disease associated with NE hyperactivation, comprising the anti-NE antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

[0088] One aspect of the present invention provides a composition for analyzing or detecting NE, comprising the anti-NE antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

[0089] One aspect of the present invention provides a use of the anti-NE antibody or antigen-binding fragment thereof of the present invention for diagnosing a disease associated with NE hyperactivation.

[0090] One aspect of the present invention provides a use of the anti-NE antibody or antigen-binding fragment thereof of the present invention for preventing or treating a disease associated with NE hyperactivation.

[0091] One aspect of the present invention provides the use of an anti-NE antibody or antigen-binding fragment thereof of the present invention for the manufacture of a medicament for preventing or treating a disease associated with NE hyperactivation.

[0092] One aspect of the present invention provides a method for preventing or treating a disease associated with NE hyperactivation, comprising administering to a subject an effective amount of an anti-NE antibody or antigen-binding fragment thereof of the present invention.

[0093] One aspect of the present invention provides a kit for preventing or treating a disease associated with NE overactivation, comprising the anti-NE antibody or antigen-binding fragment thereof of the present invention as an active ingredient.

[0094] Among the terms or elements mentioned in the above composition, use, prevention or treatment method and kit, the composition of the anti-NE antibody or antigen-binding fragment thereof is understood to be the same as mentioned in the description of the anti-NE antibody or antigen-binding fragment thereof and the pharmaceutical composition comprising the same.

[0095] The kit may additionally include a user manual describing optimal reaction conditions. The manual may include a pamphlet, leaflet-style guidebook, a label attached to the kit, or a description on the package containing the kit. Furthermore, the manual may include information disclosed or provided through electronic media, such as the Internet.

[0096] Hereinafter, the present invention will be described in more detail through examples. The following examples are described for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0097]

[0098] Example 1: Production and screening of anti-NE antibodies

[0099] We screened anti-NE antibodies and analyzed their amino acid sequences to determine the complementary determining region (CDR) of each antibody.

[0100] Specifically, first, a DNA sequence encoding an amino acid sequence in which serine present in the active site of human or mouse NE is substituted with alanine was inserted into a pcDNA3.4 vector that was constructed to encode a TCE sequence and histidine at the C-terminus of the protein, and gene synthesis was performed to express an inactive NE protein.

[0101] Using the above inactive NE as an antigen, 8 female BALB / c and A / J mice (Biocytogen), each 6 to 8 weeks old, were subcutaneously inoculated 6 to 7 times at 2-week intervals at a dose of 50 μg / mouse. Blood samples were collected a total of 3 times during the inoculation period, and ELISA analysis was performed, and mice expressing sufficient anti-NE antibodies were selected.

[0102] B lymphocytes were isolated from the spleen of these mice and subjected to Beacon screening. The CDR sequences of the resulting anti-NE antibodies were analyzed, and a primary selection of representative antibody candidates was performed using the neighbor-joining tree technique.

[0103] Example 2: Neutralizing ability screening of anti-NE antibodies

[0104] The final aim was to select antibodies with anti-NE antibody neutralizing ability from the first-selected antibodies.

[0105] Specifically, the Neutrophil Elastase Inhibitor Assay Kit (Abcam, #ab118971) was used for the evaluation based on the protocol. However, NE (EPC, #SE563) was diluted with the buffer provided in the kit to a concentration of 50 ng / well, and 50 μL was dispensed into each 96-well plate (Thermofisher, #7905). 25 μL of anti-NE antibody diluted in PBS was added. After incubation at 37°C for 5 minutes, 2 μL of substrate and 23 μL of buffer provided in the kit were mixed and added to each well. Signals were measured at 400 / 505 nm using a Victor Nivo (PerkinElmer).

[0106] Figure 1 illustrates the results of secondary screening of three antibodies (NI-10, NI-11, and NI-50) with neutralizing ability against the antigen NE through antibody single-concentration screening evaluation (Figure 1). The sequences of the selected antibodies are as described in Tables 1 to 4 below.

[0107]

[0108] CDR amino acid sequence of antibodies (NI-10, NI-11, NI-50) with neutralizing ability against NE Antibody name Variable region Amino acid Sequence SEQ ID NO. NI-10 Heavy chain CDR1 DYWIE1 CDR2 EILPGSFSTNYNEKFKG2 CDR3 DNY3 Light chain CDR1 KAGQNVGTNVA4 CDR2 SASYRYS5 CDR3 QQYNSYPLT6 NI-11 Heavy chain CDR1 NYGVH7 CDR2 LIWAGGSTSYNSALMS8 CDR3 EKDYYYSDYRFAY9 Light chain CDR1 KASQSVHYDGDSYMN10 CDR2 AASNLES11 CDR3 QQNNEDPWT12 NI-50 Heavy chain CDR1 SYGVH13 CDR2 VIWAGGSTNYKSALMS14 CDR3 DRAIYYSTYEFAY15 Light chain CDR1 KASQSVDYDGDSYIT16 CDR2 VASNLES17 CDR3 QQTNEDPWT18

[0109]

[0110] CDR base sequence of antibodies (NI-10, NI-11, NI-50) with neutralizing ability against NE Antibody name Variable region base sequence Sequence number NI-10 heavy chain CDR1 GACTACTGGATCGAG CDR2 GAGATCTGCCCGGCTCTTTCAGTACCAACTACAACGAGAAATTCAAGGGC CDR3 GACAACTAT CDR2 Light chain CDR1 AAGGCCGGACAGAACGTGGGAACAAACGTGGCT CDR2 CDR2 TCCGCCTCATACCGTTACAGC CDR3 CAGCAGTACAACAGCTACCCCCTGACC CDR2 NI-11 heavy chain CDR1 AACTACGGCGTCCAC CDR2 CDR2 CTCATCTGGGCTGGTGGATCTACCTCCTACAACTCCGCCCTGATGTCT CDR3 GAAAAGGACTATTACTACTCCGACTACCGCTTTGCCTAC CDR3 Light chain CDR1 AAG GCTTCCCAGTCCGTGCACTATGACGGGGATTCCTACATGAAC28CDR2GCCGCCTCCAACCTGGAGTCT29CDR3CAGCAGAACAACGAAGATCCTTGGACC30NI-50 Heavy ChainCDR1AGCTACGGCGTCCAC31CDR2GTGATTTGGGCCGGCGGATCTACCAACTACAAAAGC GCCCTGATGTCA32CDR3GACAGGGCAATCTACTACTCCACATACGAGTTCGCCTAC33Light chainCDR1AAGGCCTCCCAGTCCGTGGACTACGACGGTGATTCCTACATCACC34CDR2GTGGCTTCCAACCTGGAGTCC35CDR3CAGCAGACCAACGAAGATCCTTGGACC36

[0111]

[0112] Variable region amino acid sequence of antibodies (NI-10, NI-11, NI-50) with neutralizing ability against NE Antibody name Variable region amino acid Sequence SEQ ID NO: NI-10 Heavy chain QVQLQQSGAELMKPGSSVKLSCKATGYTFTDYWIEWVKQRPGHGLEWIGEILPGSFSTNYNEKFKGKATFTADISSNTAYMQLSSLTSEDSAVYYCASDNYWGQGTLVTVSA37 Light chain DIVMTQSQKFMSASVGDRVSVTCKAGQNVGTNVAWYQQQPGQSPKALIYSASYRYSGVPDRFTGSGSGTDFTLTISTVQSEDLAEYFCQQYNSYPLTFGAGTKLELK38 NI-11 Heavy chain QVQLKESGPGLLAPSQSLSITCTVSGFSLTNYGVHWVRQPPGKGLEWLGLIWAGGSTSYNSALMSRLSISKDNSKSQVFLKMNSLQTDDTAIYYCAREKDYYYSDYRFAYWGQGTLVTV SA39Light ChainDIVLTQSPTSLAVSLGQRATISCKASQSVHYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFTLNIHPVEED DAATYYCQQNNEDPWTFGGGTKLEIK40NI-50 Heavy ChainQVQLKESGPGLVAPSQSLSITCTVSGFSLTSYGVHWVRQPPGKGLEWLGVIWAGGS TNYKSALMSRLSISKDNSKSQVFLKMNSLQTDDTAMYYCARDRAIYYSTYEFAYWGQGTLVTVSA41Light chainDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYITWYQQKPGQPPKLLIYVASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQTNEDPWTFGGGTKLEIK42

[0113]

[0114]

[0115]

[0116] Example 3: Humanization of NI-10 antibody

[0117] Humanization was performed using NI-10 antibody, which has the highest NE neutralizing ability.

[0118] A chimeric antibody of NI-10 was constructed, in which the framework sequence excluding the CDR of the antibody was substituted with a human antibody sequence, and its activity was measured using a Neutrophil Elastase Inhibitor Assay Kit (Abcam, #ab118971). Specifically, NE (EPC, #SE563) was diluted with the kit buffer to a concentration of 50 ng / well, and 50 μL was dispensed into a 96-well plate (Thermofisher, #7905). 25 μL of the chimeric antibody of NI-10, in which the human antibody sequence was substituted and diluted in PBS, was added. After incubation at 37°C for 5 minutes, 2 μL of substrate and 23 μL of buffer (in the kit) were mixed and added to each well. The signal was measured at 400 / 505 nm using a Victor Nivo (PerkinElmer). As a result of the measurement, four types of antibodies with neutralizing ability were selected (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45). The sequences of the selected antibodies are as described in Tables 5 to 8 below.

[0119]

[0120] CDR amino acid sequences of humanized antibodies (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45) Antibody name Variable region Amino acid sequence Sequence number NI-10_v38 Heavy chain CDR1 Same as SEQ ID NO: 1 1 CDR2 Same as SEQ ID NO: 2 2 CDR3 Same as SEQ ID NO: 3 3 Light chain CDR1 Same as SEQ ID NO: 4 4 CDR2 Same as SEQ ID NO: 5 5 CDR3 Same as SEQ ID NO: 6 6 NI-10_v39 Heavy chain CDR1 Same as SEQ ID NO: 1 1 CDR2 Same as SEQ ID NO: 2 2 CDR3 Same as SEQ ID NO: 3 3 Light chain CDR1 Same as SEQ ID NO: 4 4 CDR2 Same as SEQ ID NO: 5 5 CDR3 Same as SEQ ID NO: 6 6 NI-10_v44 Heavy chain CDR1 Same as SEQ ID NO: 1 1 CDR2 Sequence number 2Same as 2CDR3Seq No. 3Same as 3Light chainCDR1Seq No. 4Same as 4CDR2Seq No. 5Same as 5CDR3Seq No. 6Same as 6NI-10_v45Heavy chainCDR1Same as 1CDR2Same as 2CDR3Seq No. 3Same as 3Light chainCDR1Same as 4CDR2Same as 5CDR3Same as 66

[0121]

[0122] CDR base sequence of humanized antibody (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45) Antibody name Variable region base sequence Sequence number NI-10_v38 Heavy chain CDR1 Same as SEQ ID NO. 19 19 CDR2 GAGATCTGCCCGGCTCCTTCTCCACCAACTACAACGAGAAGTTCAAGGGC 49 CDR3 GACAACTAC 50 Light chain CDR1 AAAGCCGGCCAGAACGTGGGAACCAACGTGGCT 51 CDR2 TCCGCCTCCTACAGATACAGC 52 CDR3 CAGCAGTACAACTCCTACCCACTGACC 53 NI-10_v39 Heavy chain CDR1 Same as SEQ ID NO. 19 19 CDR2 Same as SEQ ID NO. 49 49 CDR3 Same as SEQ ID NO. 50 Identical to 50 light chain CDR1 AAGGCTGGACAGAACGTGGGAACCAACGTGGCT 54 CDR2 TCCGCCTCCTACAGGTACTCC 55 CDR3 Identical to SEQ ID NO: 24 24 NI-10_v44 heavy chain CDR1 Identical to SEQ ID NO: 19 19 CDR2 Identical to SEQ ID NO: 49 49 CDR3 Identical to SEQ ID NO: 50 50 light chain CDR1 Identical to SEQ ID NO: 51 51 CDR2 Identical to SEQ ID NO: 52 52 CDR3 Identical to SEQ ID NO: 53 53 NI-10_v45 heavy chain CDR1 Identical to SEQ ID NO: 19 19 CDR2 Identical to SEQ ID NO: 49 49 CDR3 Identical to SEQ ID NO: 50 50 light chain CDR1 Identical to SEQ ID NO: 54 54 CDR2 Identical to SEQ ID NO: 55 55 CDR3 Identical to SEQ ID NO: 24 24

[0123]

[0124] Variable region amino acid sequence of humanized antibodies (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45) Antibody name Variable region amino acid Sequence SEQ ID NO. NI-10_v38Heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWIEWVRQAPGQGLEWMGEILPGSFSTNYNEKFKGRVTMTADTSTSTVYMELSSLRSEDTAVYYCASDNYWGQGTLVTVSS56Light chain EIVMTQSPATLSVSPGERATLSCKAGQNVGTNVAWYQQKPGQAPRALIYSASYRYSGIPARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNSYPLTFGGGTKVEIK57NI-10_v39Heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWIEWVRQAPGQGLEWMGEI LPGSFSTNYNEKFKGRVTMTADTSTSTVYMELSSLRSEDTAVYYCASDNYWGQGTLVTVSS58Light chainDIQMTQSPSTLSASVGDRVTITCKAGQNVGTNVAWYQQKPGKAPKALIYSASYRYSGVPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYPLTFGGGTKVEIK59NI-10_v44Heavy chainQVQLVQSGAEVKKPGSSVKVSCKASGYTFTDYWIEWVRQAPGQGLEWMGEILPGSFSTNYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCASDNYWGQGTLVTVSS60Light chainSame as SEQ ID NO: 5757NI-10_v45Heavy chainSame as SEQ ID NO: 6060Light chainSame as SEQ ID NO: 5959

[0125]

[0126]

[0127] The NE neutralizing ability of the selected humanized antibodies (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45) was measured using the Neutrophil Elastase Inhibitor Assay Kit (Abcam, #ab118971). NE (EPC, #SE563) was diluted to a concentration of 50 ng / well with the kit buffer, and 50 μL was dispensed into a 96-well plate (Thermofisher, #7905). Then, 25 μL of the four selected humanized antibodies, diluted to different concentrations with PBS buffer, were added. After reacting at 37°C for 5 minutes, 2 μL of substrate and 23 μL of buffer in the kit were mixed and added to each well. The signal was measured at 400 / 505 nm using Victor Nivo (PerkinElmer). The IC confirmed by the measurement results 50 The values ​​are as described in Table 9 (Fig. 2).

[0128]

[0129] Antibody name NE Neutralizing capacity IC 50 (nM)NI-10119.6NI-10_v38118.2NI-10_v39115.1NI-10_v44190NI-10_v45441.4

[0130]

[0131] Example 4: Confirmation of binding affinity and affinity of anti-NE antibodies

[0132] The binding affinity and affinity of the selected anti-NE antibodies were determined using ELISA and bio-layer interferometry (BLI).

[0133] First, to confirm the specific binding to NE by ELISA, 50 ng / well of NE (LeeBiosolutions, #342-40) was added to a 96-well plate (Corning, #3690) and incubated overnight at 4°C to coat (NI-10, NI-11, and NI-50 in Fig. 3a).

[0134] In addition, pre-mature recombinant NE produced in the form of adding a dipeptide to the N-terminus was coated with active mature NE produced by reacting DPP1 (R&D systems, #2336-CY-010) in a 50 mM MES, 50 mM NaCl, pH 5.5 buffer condition at 37°C for 2 hours (NI-10_v38, NI-10_v39, NI-10_v44, and NI-10_v45 in Fig. 3b).

[0135] The next day, the membrane was blocked by incubation with 3% BSA / PBS at 37°C for 1 hour, and then the primary antibody, anti-NE antibody, was added and reacted at 37°C for 2 hours and washed 4 times. Rabbit anti-Human IgG Fc gamma (Thermofisher, #31142) was used as the secondary antibody and reacted at 37°C for 1 hour and washed 4 times. TMB and STOP solutions were added sequentially to induce a color reaction, and the absorbance was measured at 450 nm using Victor Nivo (PerkinElmer). The EC was derived by confirming the binding affinity to NE by ELISA. 50 The values ​​are as listed in Tables 10 and 11.

[0136]

[0137] Antibody name NE binding affinity EC 50 (nM)NI-100.769NI-110.422NI-500.308

[0138]

[0139] Antibody name NE binding affinity EC 50(nM)NI-10_v380.398NI-10_v390.618NI-10_v446.571NI-10_v4523.870

[0140]

[0141] Next, BLI evaluation was performed using Octet RED96e (FORTEBIO) to analyze the NE affinity of the anti-NE antibody. The antibody candidates selected in Example 1 (NI-10, NI-10_v37, NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45) were loaded onto the AHC2 biosensor (Sartorius, #18-5142) as ligands, and the binding constant (K) was measured using the same type of NE used for immunization in Example 1 as an analyte. D ), association rate constant (K a ), dissociation rate constant (K dis ) values ​​were derived, and each value was as shown in Table 12.

[0142]

[0143] Antibody name K D (M)K a (1 / Ms)K dis (1 / s)NI-101.07E-093.23E+053.47E-04NI-10_v373.61E-103.42E+051.23E-04NI-10_v386.61E-104.92E+053.25E-0 4NI-10_v398.02E-104.87E+053.90E-04NI-10_v447.69E-105.12E+053.94E-04NI-10_v459.74E-103.00E+052.92E-04

[0144]

[0145] Example 5: Confirmation of the efficacy of anti-NE antibodies in human alveolar epithelial cells.

[0146] We sought to confirm whether the antibody of the present invention could prevent the decrease in cell adhesion and the occurrence of cell death caused by NE.

[0147] Human alveolar epithelial cells, A549, were used, and 2 x 10 A549 were seeded in a 96-well plate (Corning, #3690). 4 / well The day after seeding, serum-free medium, 20% PBS, the antibody of the present invention, and 125 nM NE were treated and reacted for 24 hours. The medium was washed with PBS, color was developed using the CCK-8 kit (Chromogen, #CH-3000), and the absorbance was measured at 450 nm using Victor Nivo (PerkinElmer).

[0148] As a result, the amount of adherent cells increased in proportion to the amount of NI-10 and humanized antibody NI-10_v38 treated, confirming the inhibitory effect of cell adhesion reduction and cell death according to the NE neutralizing effect of the antibody of the present invention. In addition, similar aspects were observed in NI-10 and humanized antibody NI-10_v38, confirming that the above effects were not inhibited during the humanization process (Fig. 4).

[0149]

[0150] Example 6: Validation of the antigenic determinant of the selected representative antibody

[0151] Based on the results of Example 5 above, NI-10 and NI-10_v38 were selected as final candidates, and competition ELISA or BLI was performed to confirm the validity of the antigen determinant.

[0152] First, when endogenous proteins elafin and NI-10, known to bind broadly to the NE active site and inhibit NE activity, were simultaneously bound to the antigen NE using ELISA, a decrease in the binding affinity of NI-10 was observed (Fig. 5). In addition, competitive binding of other endogenous proteins, alpha-1 antitrypsin (AAT) and NI-10_v38, which bind to the NE active site, was confirmed using BLI (Fig. 6).

[0153] At this time, NE bound to N-01, an antibody obtained through biopanning that was not selected as a final candidate, showed binding to AAT, whereas NE bound to NI-10_v38 showed no additional binding of AAT, confirming that the epitope of NI-10_v38 was valid (Fig. 6). Since NI-10_v38 is a humanized antibody of NI-10, the epitopes of these antibodies are identical.

[0154]

[0155] Example 7: Confirmation of NE target cleavage resistance of humanized antibody NI-10_v38

[0156] The protein stability of each substance was compared in a pathological environment simulating the presence of high concentrations of NE, such as acute lung injury (AATD) patients' lung tissues, where neutrophils infiltrate in large quantities, targeting humanized antibody NI-10_v38 and AAT, a mutated AAT-Fc fusion protein INBRX-101.

[0157] Specifically, a pseudo-inflammatory environment in which high concentrations of NE and inhibitors coexisted was created by mixing 1 μM of NE with three different inhibitors (NI-10_v38, AAT, INBRX-101) at concentrations of 0.4 μM, 0.59 μM, and 0.89 μM, respectively, and incubating at room temperature for 16 hours. Afterwards, 10 μL of each reaction mixture was analyzed by NuPAGE TM After electrophoresis on a 4-12% Bis-Tris 1.0 mm gel (Invitrogen, #NP0323BOX), the protein stability of the inhibitors in each sample was analyzed by staining with Coomassie Brilliant Blue (CBB).

[0158] As a result, it was confirmed that AAT and INBRX-101 were severely degraded, such that they were cleaved by NE at all three concentration conditions (0.4 μM, 0.59 μM, and 0.89 μM) or some of the cleaved inhibitors bound to NE and could not maintain their own protein band patterns (Fig. 7). This suggests that these inhibitors may be rapidly eliminated before exerting their effect at the site of inflammation. On the other hand, in the case of humanized antibody NI-10_v38, it was observed that the same band pattern was maintained regardless of the presence or absence of NE in all three concentration conditions.

[0159] Through this, it was confirmed that humanized antibody NI-10_v38 is a stable substance with high cleavage resistance to NE compared to AAT and INBRX-101.

[0160]

[0161] Example 8: Oxidation stability of humanized antibody NI-10_v38

[0162] To evaluate whether humanized antibody NI-10_v38 maintains NE neutralization ability under oxidative conditions, hNE residual activity was measured according to the ratio of AAT or NI-10_v38 and time.

[0163] Specifically, the oxidation condition was created by mixing NI-10_v38 and AAT (final concentration 1.5 μM) with 0.1% hydrogen peroxide (H2O2, v / v) solution at a 1:1 ratio and incubating at room temperature. Samples were collected at 0, 6, and 24 h, and the NE inhibitory activity was evaluated. The neutralizing activity was evaluated based on the protocol using the EnzChek® Elastase Assay Kit (Invitrogen, #E12056). NE was fixed at a concentration of 20 nM per 200 μL of assay well, and NI-10_v38 and AAT were used at concentrations of 20 nM and 40 nM, which correspond to a 1:1 or 2:1 ratio of NE. Signals were measured at 480 / 520 nm using a Victor Nivo (PerkinElmer). Each bar in Figure 8 represents time points of 0, 6, and 24 hours after chemical treatment under oxidizing conditions, and the experiment was repeated twice and the average was calculated (N=2).

[0164] As a result, AAT loses its inhibitory activity within 6 hours when mixed with NE at both 1:1 and 2:1 ratios. In contrast, the humanized antibody NI-10_v38 fully retains its inhibitory activity for up to 24 hours under oxidative conditions when treated with NE at 1:1 and 2:1 ratios. Therefore, the humanized antibody NI-10_v38 appears to be highly stable compared to AAT under oxidative conditions (Fig. 8).

[0165]

[0166] Example 9: Efficacy of NI-10_v38 in an acute lung injury mouse model

[0167] The aim of this study was to evaluate the lung injury inhibitory and anti-inflammatory effects of humanized antibody NI-10_v38 in an acute lung injury mouse model.

[0168] Specifically, C57BL / 6 mice were intratracheally administered the provocative human NE (10 μg / head) alone or mixed with the test substances NI-10_v38 (250, 50, and 10 μg / head), IgG-Fc (91.5 μg / head), or INBRX-101 (250 μg / head) immediately before individual dosing. IgG-Fc was used as a control as an isotype control having the same Fc as NI-10_v38, and INBRX-101 was used as a competitive drug to compare the effects. The test substance Alvelestat (10 mg / kg) was orally administered at a dose of 10 mg / kg, calculated based on body weight, 1 hour before intratracheal administration of the provocative human NE. Bronchoalveolar lavage fluid (BALF) was collected using 1 mL of PBS 4 hours after human NE administration. Blood cell counts in BALF were measured using a CBC analyzer (Mindray BC-5000 Vet), and cytokines IL-6 (Abcam, ab222503) and total MMP-9 (RnD Systems, MMPT90) in BALF were evaluated based on the protocol using an ELISA kit.

[0169] As a result, the number of red blood cells (RBC), neutrophils, lymphocytes, and hemoglobin (HGB) concentrations were confirmed to be statistically significantly increased in the hNE only administration group (G2) compared to the vehicle (G1), and a statistically significant decrease in RBC and HGB was confirmed in the NI-10_v38 treatment group (G3~G5), and a statistically significant decrease in neutrophils and lymphocytes was confirmed in the NI-10_v38 treatment group (G3, G4).

[0170] In addition, the concentrations of acute inflammatory cytokines, IL-6 and total MMP9, were statistically significantly increased in the hNE only administration group (G2), while the concentration of IL-6 in the NI-10_v38 treatment groups (G3, G4) and the concentration of total MMP9 in the NI-10_v38 treatment groups (G3 to G5) were both statistically significantly decreased. Through this, it was confirmed that NI-10_v38 effectively inhibited the leakage of RBC and HGB into BALF induced by hNE, and significantly reduced the infiltration of neutrophils and lymphocytes and the inflammatory response (Fig. 9).

Claims

An anti-NE antibody or an antigen-binding fragment thereof that specifically binds to NE (neutrophil elastase), Heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, 7 or 13; A heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, 8 or 14, and A heavy chain variable region (VH) comprising a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3, 9, or 15; and A light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, 10 or 16; A light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, 11, or 17, and A light chain variable region (VL) comprising a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6, 12 or 18, Anti-NE antibody or antigen-binding fragment thereof. In paragraph 1, The above anti-NE antibody or antigen-binding fragment thereof A heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 37, 39, 41, 56, 58, or 60; and An anti-NE antibody or antigen-binding fragment thereof comprising a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 38, 40, 42, 57 or 59. In paragraph 1, The above anti-NE antibody or antigen-binding fragment thereof A heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 1, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 2, a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 3, a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 4, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 5, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 6; A heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 7, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 8, a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 9, a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 10, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 11, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 12; or A heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO: 13, a heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO: 14, a heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO: 15, a light chain CDR1 represented by the amino acid sequence of SEQ ID NO: 16, a light chain CDR2 represented by the amino acid sequence of SEQ ID NO: 17, and a light chain CDR3 represented by the amino acid sequence of SEQ ID NO: 18 An antibody or antigen-binding fragment thereof comprising: In paragraph 1, An anti-NE antibody or antigen-binding fragment thereof, wherein the NE comprises NE derived from a human or mouse. In paragraph 1, The above anti-NE antibody is a humanized antibody, an anti-NE antibody or an antigen-binding fragment thereof. A polynucleotide encoding an antibody or an antigen-binding fragment thereof according to any one of claims 1 to 5. In paragraph 6, The above polynucleotide The heavy chain CDR1 encoding sequence represented by SEQ ID NO: 19, 25, or 31, A heavy chain CDR2 encoding base sequence represented by SEQ ID NO: 20, 26, 32, or 49, and A heavy chain variable region (VH) coding region comprising a heavy chain CDR3 coding sequence represented by SEQ ID NO: 21, 27, 33, or 50; and A light chain CDR1 encoding sequence represented by SEQ ID NO: 22, 28, 34, 51, or 54; a light chain CDR2 encoding sequence represented by SEQ ID NO: 23, 29, 35, 52 or 55, and A light chain variable region (VL) encoding region comprising a light chain CDR3 encoding sequence represented by SEQ ID NO: 24, 30, 36 or 53, Polynucleotide. In paragraph 6, The above polynucleotide A heavy chain variable region (VH) encoding sequence represented by SEQ ID NO: 43, 45, 47, 61, 63, or 65; and comprising a light chain variable region (VL) encoding base sequence represented by SEQ ID NO: 44, 46, 48, 62 or 64; Polynucleotide. In paragraph 6, The above polynucleotide A heavy chain CDR1 coding sequence represented by SEQ ID NO: 19, a heavy chain CDR2 coding sequence represented by SEQ ID NO: 20, a heavy chain CDR3 coding sequence represented by SEQ ID NO: 21, a light chain CDR1 coding sequence represented by SEQ ID NO: 22, a light chain CDR2 coding sequence represented by SEQ ID NO: 23, and a light chain CDR3 coding sequence represented by SEQ ID NO: 24; a heavy chain CDR1 coding sequence represented by SEQ ID NO: 25, a heavy chain CDR2 coding sequence represented by SEQ ID NO: 26, a heavy chain CDR3 coding sequence represented by SEQ ID NO: 27, a light chain CDR1 coding sequence represented by SEQ ID NO: 28, a light chain CDR2 coding sequence represented by SEQ ID NO: 29, and a light chain CDR3 coding sequence represented by SEQ ID NO: 30; or A heavy chain CDR1 coding sequence represented by SEQ ID NO: 31, a heavy chain CDR2 coding sequence represented by SEQ ID NO: 32, a heavy chain CDR3 coding sequence represented by SEQ ID NO: 33, a light chain CDR1 coding sequence represented by SEQ ID NO: 34, a light chain CDR2 coding sequence represented by SEQ ID NO: 35, and a light chain CDR3 coding sequence represented by SEQ ID NO: 36 A polynucleotide comprising: In paragraph 6, The above polynucleotide a heavy chain CDR1 coding sequence represented by SEQ ID NO: 19, a heavy chain CDR2 coding sequence represented by SEQ ID NO: 49, a heavy chain CDR3 coding sequence represented by SEQ ID NO: 50, a light chain CDR1 coding sequence represented by SEQ ID NO: 51, a light chain CDR2 coding sequence represented by SEQ ID NO: 52, and a light chain CDR3 coding sequence represented by SEQ ID NO: 53; or A heavy chain CDR1 coding sequence represented by SEQ ID NO: 19, a heavy chain CDR2 coding sequence represented by SEQ ID NO: 49, a heavy chain CDR3 coding sequence represented by SEQ ID NO: 50, a light chain CDR1 coding sequence represented by SEQ ID NO: 54, a light chain CDR2 coding sequence represented by SEQ ID NO: 55, and a light chain CDR3 coding sequence represented by SEQ ID NO: 24 A polynucleotide comprising: An expression vector comprising the polynucleotide of claim 6. A host cell transformed with the expression vector of Article 11. A method for producing an anti-NE antibody or an antigen-binding fragment thereof that specifically binds to NE, comprising the following steps: (a) a step of culturing the host cell of clause 12; and (b) A step of recovering an antibody or an antigen-binding fragment thereof from the cultured cells. A pharmaceutical composition for preventing or treating a disease related to NE overactivation, comprising an anti-NE antibody or an antigen-binding fragment thereof according to claim 1 as an active ingredient. In Article 14, A pharmaceutical composition, wherein the disease associated with NE overactivation is any one selected from the group consisting of chronic obstructive pulmonary disease (COPD), fibrosis, cystic fibrosis, alpha-1 antitrypsin deficiency (AATD emphysema), bronchiectasis, acute lung injury (ALI), acute respiratory distress syndrome (ARDS), cyclic neutropenia, and emphysema. Use of an anti-NE antibody or an antigen-binding fragment thereof according to Article 1 for preventing or treating a disease associated with NE hyperactivation. Use of an anti-NE antibody or an antigen-binding fragment thereof according to claim 1 for the manufacture of a medicament for preventing or treating a disease associated with NE overactivation. A method for preventing or treating a disease associated with NE overactivation, comprising administering to a subject an effective amount of an anti-NE antibody or an antigen-binding fragment thereof according to claim 1.

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