KR20260035744A - Anti-NE antibody and pharmaceutical composition for preventing or treating NE-hyperactivation related diseases comprising the same - Google Patents

Abstract

The present invention relates to an anti-NE antibody or an 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 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) comprising 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 has excellent NE neutralizing ability and can be used for the prevention or treatment of diseases related to NE overactivation.

Description

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

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

Neutrophil elastase (NE) is one of the serine proteases and accounts for 80% of the total protease hydrolysis activity in the human body. NE is primarily found in neutrophils, which are important for the innate immune response, and is stored in azurophil granules within neutrophils along with other proteases involved in antimicrobial defense.

NE is synthesized in a pre-mature form, and the C-terminus and the terminal containing the signal peptide are cleaved, and the terminal dipeptide is further cleaved by DPP1 to become the mature form. NE is activated by a tristructural catalyst composed of Asp, His, and Ser, but exists in an inhibited state within granules compartmentalized at low pH (Pharmacol Rev 62:726-59).

Meanwhile, in the presence of infection or other inflammatory stimuli, NE is released into the cytoplasm with a neutral pH and becomes activated through the activation and degranulation of neutrophils as they rapidly infiltrate organs and perform phagocytosis. At this time, NE plays an important role in attacking pathogens along with NADPH oxidase, myeloperoxidase (MPO), and reactive oxygen species (ROS) (Clin Immunol 99:7-17).

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

However, in addition to this, NE can induce widespread inflammation through various substrates. NE performs a dual role: inducing an inflammatory response by activating IL-1β and IL-18, which are precursors of inflammatory cytokines, and promoting the production of IL-8, or conversely, regulating cytokine signaling by directly degrading already produced TNF-α, IL-1β, and IL-8 (J Immunol 175:7594-601, J Biomed Sci 11:49-58). Furthermore, fragments generated by the cleavage of the extracellular matrix (ECM), surface receptors, and cytokines by NE affect cell signaling (Nat Rev Immunol 6:541-50).

As such, the overactivation of NE actually promotes inflammation, the progression of bacterial infections, and excessive mucus secretion (Biochem Pharmacol 83:695-704). Therefore, protease inhibitor proteins such as serpins and macroglobulins, which are essential components of body fluids, rapidly neutralize NE to prevent the destruction of the ECM and host cells caused by NE. Representative examples include elafin and SLPI (secretory leukocyte protease inhibitor), which are produced and secreted by epithelial cells, neutrophils, and macrophages to contribute to local defense, while AAT (α-1 antitrypsin) is produced in the liver and circulates throughout the body to regulate NE (Clin Sci (Lond) 110:21-35).

If they fail to adequately neutralize NE, thereby disrupting the balance between protease and inhibitor proteins, NE overactivity can lead to numerous diseases (Rheumatology (Oxford) 49:1618-31, Annu Rev Immunol 30:459-89). In addition to acute diseases, prolonged elevated NE activity can cause tissue destruction and remodeling due to infection and chronic inflammation, potentially leading to various severe chronic diseases including pulmonary 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). Furthermore, the amount and activity of NE are known to reflect the disease state and severity (Biomolecules 11:1065).

To date, various synthetic NE inhibitors have been developed to regulate overactive NE. However, for example, although preclinical studies demonstrated a multifaceted reduction in NE-induced damage and inflammation, it was not approved as a therapeutic agent in actual clinical practice due to insufficient efficacy and concerns regarding toxicity. Therefore, there is still a need to develop antibodies or antigen-binding fragments that can specifically bind to and neutralize extracellular overactive NE to minimize side effects.

Accordingly, the inventors invented a novel antibody that has a high affinity for NE and specifically binds to it, confirmed the efficient neutralizing ability of the antibody, confirmed its potential as an effective therapeutic agent and combination therapy for various diseases caused by overactive NE, and completed the present invention.

The objective of the present invention is to provide a means for the prevention or treatment of 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 neutralizing ability.

To achieve the above objective:

One aspect of the present invention provides an anti-NE antibody or an 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 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) comprising 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.

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

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

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

Another aspect of the present invention is a step of (a) culturing the host cell; and

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

Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of NE overactivation-related diseases, comprising the anti-NE antibody or an antigen-binding fragment thereof as an active ingredient.

The antibody or its antigen-binding fragment of the present invention has high binding strength and affinity for NE and has the effect of reducing cell adhesion and inhibiting apoptosis caused by NE, so it can be effectively used for the prevention or treatment of diseases related to NE overactivation.

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

Figure 1 shows the results of selecting three antibodies (NI-10, NI-11, NI-50) that have neutralizing ability against antigen NE through antibody single-concentration screening evaluation.
Figure 2 shows the IC ₅₀ values for the NE neutralization ability of NI-10 antibodies and humanized antibodies (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45).
Figures 3a and 3b show the EC ₅₀ values for the 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).
Figure 4 shows the inhibitory effects of NI-10 and the humanized antibody NI-10_v38 on reducing cell adhesion and inhibiting cell death.
Figure 5 shows the results of a competition ELISA in which the binding affinity of NI-10 is reduced when the endogenous proteins elafin and NI-10, which bind to the active site of NE, are simultaneously bound to the antigen NE.
Figure 6 shows the results of confirming the efficacy of epitopes by observing the competitive binding of other endogenous proteins, alpha-1 antitrypsin (AAT) and NI-10_v38, to the NE active site using BLI.
Figure 7 shows 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, as confirmed by gel electrophoresis results.
Figure 8 is a graph comparing the hNE residual activity over time under oxidation conditions, comparing the stability of the humanized antibody NI-10_v38 against oxidation with that of AAT.
Figure 9 shows the results of evaluating the lung damage inhibitory effect and anti-inflammatory effect of the humanized antibody NI-10_v38 in an acute lung injury mouse model.

The present invention will be described in detail below.

In the present invention, the term “consisting of” means that the proportion of a specific component(s) is 100% of the total. The component or feature following the term “consisting of” may be essential or mandatory.

In the present invention, the term “comprising” means the presence of the features, steps, or components described below the above term, and does not exclude the presence or addition of one or more features, steps, or components. In this specification, the components or features described below “comprising” may be essential or mandatory, but some embodiments may further include other optional or non-essential components or features.

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

In the present invention, the terms “to bind specifically to” or “specific to” mean having determinative power regarding the presence of a target in the presence of a heterogeneous population of molecules including biological molecules, and refer to a measurable and reproducible interaction such as binding between a target and an antibody. For example, an antibody that binds specifically to a specific target (e.g., an epitope) means an antibody that binds to said target with greater affinity, greater binding strength, more easily, and/or for a longer duration than an antibody that binds to other targets.

In the present invention, the term “K _D ” refers to the binding equilibrium dissociation constant of a specific antibody-antigen interaction, and is calculated through the formula K _D = K _dis / K _a (where K _a is the binding rate constant and K _dis is the dissociation rate constant), and the constant K _D has units of M. The K _D value for an antibody can be measured using methods widely established in the art. Specific methods for measuring the K _D value of an antibody may use surface plasmon resonance (SPR), more specifically, a biosensor system, e.g., a Biacore® system, or bio-layer interferometry (BLI), e.g., an Octet® system.

anti-NE antibody or its antigen-binding fragment

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

The present invention provides an anti-NE antibody or an 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 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) comprising 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.

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

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

Another aspect of the present invention is that the anti-NE antibody or its antigen-binding fragment 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; Alternatively, an antibody or an antigen-binding fragment thereof is provided, comprising 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.

In the present invention, the term "NE" may refer to a concept collectively encompassing NE itself, any variant, isotype, and paralog that exist 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.

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 that has the function of binding to an antigen as a fragment of said antibody molecule, even if it is not in the form of a complete antibody.

The antibodies of the present invention include, but are not limited to, monoclonal antibodies, bispecific antibodies, multiple 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, CrossMab, Trimab, DVD-Igs, CovX-bodies, duobodies, or triomAbs antibodies.

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 population of antibodies.

Since the sequences of the humanized antibody described above, excluding the CDR sequence, utilize amino acid sequences derived from human antibodies or are modified versions thereof, it has the effect of having low immunogenicity and a low likelihood of inducing HAMA (Human Anti-Mouse Antibody) when administered to the human body. Accordingly, the humanized antibody of the present invention has the advantage of resolving the problems of immune responses that may arise from using mouse-derived antibodies.

The 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, triabdies, tetrabodies, minibodies, phenomers, DART, BiTEs, TandAbs, DAFs, SMIPs, anticalin, FN3 monobodies, DARPins, Affibodies, Affilins, Affimers, Affitins, Alphabodies, Avimers, Im7, VLR, VNAR, nanobodies, bi-nanobodies, D-sdFv, etc. Among the antibody fragments, Fab has a structure having a variable region of the light chain and heavy chain, a constant region of the light chain, and a first constant region (CH1) of the 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 having only a heavy chain variable region and a light chain variable region.

The term "epitope" as used in the present invention refers to a protein determinant to which an antibody can specifically bind. An epitope typically consists of a group of chemically active surface molecules, for example, amino acids or sugar side chains, and generally has specific three-dimensional structural features.

In the present invention, the term "heavy chain" refers to a heavy chain of full length 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. Additionally, in the present invention, the term "light chain" refers to a light chain of full length and a fragment thereof comprising a light chain variable region (VL) and a constant region CL that specifically binds to an antigen.

In the present invention, the term "variable region" refers to a light chain and heavy chain portion of an antibody molecule comprising a complementarity determining region (CDR; i.e., CDR1, CDR2, and CDR3) and a backbone region (FR).

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

The anti-NE antibody of the present invention or its antigen-binding fragment may include not only the anti-NE sequence of the present invention described herein but also biological equivalents thereof, within a range capable of specifically recognizing NE. For example, additional changes may be made to the amino acid sequence of the antibody to improve biological properties such as the binding affinity of the antibody. Such modifications include, for example, deletion, insertion, and/or substitution 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, e.g., 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 can be considered biologically functional equivalents of each other. The above classification is merely an example and is not limited thereto.

Based on this, considering a modification having biologically equivalent activity, the antibody of the present invention or its antigen-binding fragment is interpreted to include a sequence exhibiting substantial identity with the specified sequence described in the specification. In terms of substantial identity, this means that the antibody of the present invention or its antigen-binding fragment includes having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more homology with respect to the sequence described in the specification.

Specifically, the antibody of the present invention or its antigen-binding fragment may be an anti-NE antibody or its antigen-binding fragment 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 the CDR of the anti-NE antibody or its antigen-binding fragment.

Polynucleotide encoding an anti-NE antibody or its antigen-binding fragment, expression vector, host cell, and method of preparation

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

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.

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

Another aspect of the present invention is that the polynucleotide comprises a heavy chain CDR1 encoding sequence represented by SEQ ID NO. 19, a heavy chain CDR2 encoding sequence represented by SEQ ID NO. 20, a heavy chain CDR3 encoding sequence represented by SEQ ID NO. 21, a light chain CDR1 encoding sequence represented by SEQ ID NO. 22, a light chain CDR2 encoding sequence represented by SEQ ID NO. 23, and a light chain CDR3 encoding sequence represented by SEQ ID NO. 24; a heavy chain CDR1 encoding sequence represented by SEQ ID NO. 25, a heavy chain CDR2 encoding sequence represented by SEQ ID NO. 26, a heavy chain CDR3 encoding sequence represented by SEQ ID NO. 27, a light chain CDR1 encoding sequence represented by SEQ ID NO. 28, a light chain CDR2 encoding sequence represented by SEQ ID NO. 29, and a light chain CDR3 encoding sequence represented by SEQ ID NO. 30; Alternatively, a polynucleotide is provided comprising a heavy chain CDR1 encoding sequence represented by SEQ ID NO. 31, a heavy chain CDR2 encoding sequence represented by SEQ ID NO. 32, a heavy chain CDR3 encoding sequence represented by SEQ ID NO. 33, a light chain CDR1 encoding sequence represented by SEQ ID NO. 34, a light chain CDR2 encoding sequence represented by SEQ ID NO. 35, and a light chain CDR3 encoding sequence represented by SEQ ID NO. 36.

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

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

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

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

The term "polynucleotide" as used in the present invention has a meaning that comprehensively includes DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic building blocks of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base regions are modified. The sequence of a nucleic acid molecule encoding the heavy chain and light chain variable regions of the present invention may be modified. Such modification includes the addition, deletion, non-conservative, or conservative substitution of nucleotides. The polynucleotide may be isolated.

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

As used herein, the term "operably linked" means that a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein are functionally linked to perform a desired function.

An expression vector suitable for the production of the above anti-NE antibody or its antigen-binding fragment may include a signal sequence for membrane targeting or secretion in addition to expression regulatory elements such as a promoter, start codon, stop codon, polyadenylation signal, and enhancer. The start codon and stop codon are generally considered to be part of a nucleotide sequence encoding an immunogenic target protein, must exhibit action in the individual when the gene expression system is administered, and must be located in a position in-frame with the encoding sequence. The general promoter may be a constitutive or inducible promoter. Promoters include, but are not limited to, prokaryotic cells of lac, tac, T3, and T7, monkey virus 40 (SV40), mouse mammary tumor virus (MMTV) promoters, human immunodeficiency virus (HIV), for example, the long terminal repeat (LTR) promoter of HIV, Moloney virus, cytomegalovirus (CMV), Epstein-Barr virus (EBV), and Rhoese's sarcoma virus (RSV) promoters, as well as eukaryotic cell promoters derived from β-actin promoters, human hemoglobin, human muscle creatine, and human metallothionein.

The expression vector may include a selective marker for selecting host cells containing it. The selective marker is intended to select cells transformed by the vector, and markers conferring selectable phenotypes, such as drug resistance, nutritional requirements, resistance to cytotoxic agents, or the expression of surface proteins, may be used. Since only cells expressing the selective marker survive in an environment treated with a selective agent, the transformed cells can be selected. Additionally, if the vector is a replicable expression vector, it may include a replication origin, which is a specific nucleic acid sequence at which replication is initiated.

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 of prokaryotic and/or eukaryotic cells; however, a vector capable of producing large quantities of foreign proteins in a form similar to their natural state while possessing a promoter exhibiting potent activity and strong expression capacity may be desirable.

The term "host cell" as used in the present invention refers to a cell system that is transformed by an expression vector and is capable of expressing a protein encoded by a polynucleotide contained in the vector.

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. Additionally, they may be eukaryotic cells such as fungi like Aspergillus sp., yeasts like Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp., and Neurospora crassa, other lower eukaryotic cells, and cells of higher eukaryotes such as insects. Furthermore, the host cells 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, etc. may be used, and preferably, CHO cells may be used, but are not limited thereto.

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

Antibodies can be produced in large quantities by culturing transformants expressing the above-mentioned recombinant vector in a nutrient medium, and the medium and culture conditions can be appropriately selected to suit the host cell. During culture, conditions such as temperature, pH of the medium, and culture time can be appropriately controlled to be suitable for cell growth and large-scale protein production.

The anti-NE antibody or its antigen-binding fragment produced by recombinant as described above may be recovered from the culture medium or cell lysis products. In the case of membrane-bound types, they may be released from the membrane by using a suitable surfactant solution (e.g., Triton-X 100) or by enzymatic cleavage. Cells used for expression of the antibody or its antigen-binding fragment may be destroyed by various physical and chemical means such as freeze-thaw purification, sonication, mechanical destruction, or cell lysis agents, and may be separated and purified by conventional biochemical separation techniques. Electrophoresis, centrifugation, gel filtration, precipitation, dialysis, chromatography (ion exchange chromatography, affinity chromatography, immunosorbent chromatography, size exclusion chromatography, etc.), isoelectric point focusing, etc., may be used, but are not limited thereto.

A pharmaceutical composition comprising an anti-NE antibody or an antigen-binding fragment thereof

One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of NE overactivation-related diseases, comprising the anti-NE antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

In the present invention, the NE overactivation-related disease 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.

The above pharmaceutical composition may additionally include a pharmaceutically acceptable carrier. For oral administration, binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavors, etc. may be used; for injectables, buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. may be mixed and used; and for topical administration, bases, excipients, lubricants, preservatives, etc. may be used.

The formulations of the above pharmaceutical composition can be prepared in various ways by mixing with the pharmaceutically acceptable carrier described above. For example, for oral administration, it can be prepared in the form of tablets, troches, capsules, ellipsis, suspensions, syrups, wafers, etc., and for injectables, it can be prepared in the form of a unit dosing ampoule or a multi-dose formulation.

In addition, the above pharmaceutical composition may include a surfactant capable of improving membrane permeability. Such surfactant may be derived from a steroid, cationic lipids such as N-[1-(2,3-dioleoyl)propyl-N,N,N-trimethylammonium chloride (DOTMA), or various compounds such as cholesterol hemisuccinate, phosphatidylglycerol, but is not limited thereto.

The above pharmaceutical composition may be administered together with or sequentially with the pharmacological or physiological components described above, and may also be administered in combination with additional conventional therapeutic agents, or sequentially or simultaneously with conventional therapeutic agents. Such administration may be a single or multiple administration. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

As used herein, the term "individual" means a mammal suffering from or at risk of having a condition or disease that can be alleviated, suppressed, prevented, or treated by administering the pharmaceutical composition, and preferably means a human being.

As used herein, the term "administration" means introducing a specific substance into an individual by any appropriate method, and the pharmaceutical composition may be administered by any route as long as it can reach the target tissue. Such methods of administration include, but are not limited to, intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intranasal administration, intrapulmonary administration, or rectal administration. However, in the case of oral administration, since proteins are digested, it may be desirable to formulate the oral composition to coat the active agent or protect it from degradation in the stomach. Additionally, the pharmaceutical composition may be administered by any device so that the active substance can be transferred to the target cells.

Composition, use, treatment method, and kit comprising an anti-NE antibody or an antigen-binding fragment thereof

One aspect of the present invention provides a biomarker composition for diagnosing NE overactivation-related diseases, comprising the anti-NE antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

One aspect of the present invention provides a composition for the analysis or detection of NE, comprising an anti-NE antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

One aspect of the present invention provides the use of the anti-NE antibody of the present invention or its antigen-binding fragment for diagnosing diseases related to NE overactivation.

One aspect of the present invention provides the use of the anti-NE antibody of the present invention or its antigen-binding fragment for the prevention or treatment of NE overactivation-related diseases.

One aspect of the present invention provides the use of the anti-NE antibody of the present invention or its antigen-binding fragment for the manufacture of a drug for the prevention or treatment of diseases related to NE overactivation.

One aspect of the present invention provides a method for preventing or treating NE overactivation-related diseases, comprising administering an effective amount of the anti-NE antibody of the present invention or an antigen-binding fragment thereof to an individual.

One aspect of the present invention provides a kit for the prevention or treatment of NE overactivation-related diseases, comprising the anti-NE antibody of the present invention or an antigen-binding fragment thereof as an active ingredient.

The composition of the anti-NE antibody or its antigen-binding fragment among the terms or elements mentioned in the above composition, use, prevention or treatment method and kit is understood to be as described in the description of the anti-NE antibody or its antigen-binding fragment and the pharmaceutical composition containing it.

The above kit may additionally include a user manual describing the optimal reaction execution conditions. The manual may include a pamphlet, an instruction booklet in the form of a leaflet, a label attached to the kit, or a description on the surface of a package containing the kit. Additionally, the manual may include information disclosed or provided through electronic media, such as the Internet.

The present invention will be explained in more detail below through examples. The following examples are described for the purpose of illustrating the present invention and do not limit the scope of the present invention.

Example 1: Production and Screening of Anti-NE Antibodies

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

Specifically, 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 first inserted into a pcDNA3.4 vector constructed to encode a TCE sequence and histidine at the C-terminus of the protein, and gene synthesis was carried out to express an inactive NE protein.

Using the above-mentioned inactive NE as an antigen, 8 female BALB/c and A/J mice (Biocytogen) aged 6 to 8 weeks 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.

B lymphocytes were isolated from the spleen of the mice and Beacon screening was performed. The CDR sequences of the anti-NE antibodies obtained as a result were analyzed, and representative antibody candidates were initially selected using the neighbor-joining tree technique.

Example 2: Screening of neutralizing ability of anti-NE antibodies

We intended to finally select antibodies that have anti-NE antibody neutralizing ability from the first selected antibodies.

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

Figure 1 illustrates the results of secondary screening of three antibodies (NI-10, NI-11, NI-50) that have neutralizing ability against 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.

CDR amino acid sequences of antibodies (NI-10, NI-11, NI-50) with neutralizing ability against NE Antibody names variable area amino acid sequence Sequence number NI-10 Heavy chain CDR1 DYWIE 1 CDR2 EILPGSFSTNYNEKFKG 2 CDR3 DNY 3 Light chain CDR1 KAGQNVGTNVA 4 CDR2 SASYRYS 5 CDR3 QQYNSYPLT 6 NI-11 Heavy chain CDR1 NYGVH 7 CDR2 LIWAGGSTSYNSALMS 8 CDR3 EKDYYYSDYRFAY 9 Light chain CDR1 KASQSVHYDGDSYMN 10 CDR2 AASNLES 11 CDR3 QQNNEDPWT 12 NI-50 Heavy chain CDR1 SYGVH 13 CDR2 VIWAGGSTNYKSALMS 14 CDR3 DRAIYYSTYEFAY 15 Light chain CDR1 KASQSVDYDGDSYIT 16 CDR2 VASNLES 17 CDR3 QQTNEDPWT 18

CDR base sequences of antibodies (NI-10, NI-11, NI-50) with neutralizing ability against NE Antibody names variable area base sequence Sequence number NI-10 Heavy chain CDR1 GACTACTGGATCGAG 19 CDR2 GAGATCCTGCCCGGGCTCTTTCAGTACCAACTACAACGAGAAATTCAAGGGC 20 CDR3 GACAACTAT 21 Light chain CDR1 AAGGCCGGACAGAACGTGGGAACAAACGTGGCT 22 CDR2 TCCGCCTCATACCGTTACAGC 23 CDR3 CAGCAGTACAACAGCTACCCCCTGACC 24 NI-11 Heavy chain CDR1 AACTACGGCGTCCAC 25 CDR2 CTCATCTGGGCTGGTGGATCTACCTCCTACAACTCCGCCCTGATGTCT 26 CDR3 GAAAAGGACTATTACTACTCCGACTACCGCTTTGCCTAC 27 Light chain CDR1 AAGGCTTCCCAGTCCGTGCACTATGACGGGATTCCTACATGAAC 28 CDR2 GCCGCCTCCAACCTGGAGTCT 29 CDR3 CAGCAGAACAACGAAGATCCTTGGACC 30 NI-50 Heavy chain CDR1 AGCTACGGCGTCCAC 31 CDR2 GTGATTTGGGCCGGCGGATCTACCAACTACAAAAGCGCCCTGATGTCA 32 CDR3 GACAGGGGCAATCTACTACTCCACATACGAGTTCGCCTAC 33 Light chain CDR1 AAGGCCTCCCAGTCCGTGGACTACGACGGTGATTCCTACATCACC 34 CDR2 GTGGCTTCCAACCTGGAGTCC 35 CDR3 CAGCAGACCAACGAAGATCCTTGGACC 36

Variable region amino acid sequences of antibodies (NI-10, NI-11, NI-50) having neutralizing ability against NE Antibody names variable area amino acid sequence Sequence number NI-10 Heavy chain QVQLQQSGAELMKPGSSVKLSCKATGYTFT DYWIE WVKQRPGHGLEWIG EILPGSFSTNYNEKFKG KATFTADISSNTAYMQLSSLTSEDSAVYYCAS DNY WGQGTLVTVSA 37 Light chain DIVMTQSQKFMSASVGDRVSVTC KAGQNVGTNVA WYQQQPGQSPKALIY SASYRYS GVPDRFTGSGSGTDFTLTISTVQSEDLAEYFC QQYNSYPLT FGAGTKLELK 38 NI-11 Heavy chain QVQLKESGPGLLAPSQSLSITCTVSGFSLT NYGVH WVRQPPGKGLEWLG LIWAGGSTSYNSALMS RLSISKDNSKSQVFLKMNSLQTDDTAIYYCAR EKDYYYSDYRFAY WGQGTLVTVSA 39 Light chain DIVLTQSPTSLAVSLGQRATISC KASQSVHYDGDSYMN WYQQKPGQPPKLLIY AASNLES GIPARFSGSGSGTDFTLNIHPVEEDDAATYYC QQNNEDPWT FGGGTKLEIK 40 NI-50 Heavy chain QVQLKESGPGLVAPSQSLSITCTVSGFSLT SYGVH WVRQPPGKGLEWLG VIWAGGSTNYKSALMS RLSISKDNSKSQVFLKMNSLQTDDTAMYYCAR DRAIYYSTYEFAY WGQGTLVTVSA 41 Light chain DIVLTQSPASLAVSLGQRATISC KASQSVDYDGDSYIT WYQQKPGQPPKLLIY VASNLES GIPARFSGSGSGTDFTLNIHPVEEEDAATYYC QQTNEDPWT FGGGTKLEIK 42

Variable region nucleotide sequences of antibodies (NI-10, NI-11, NI-50) having neutralizing ability against NE Antibody names variable area base sequence Sequence number NI-10 Heavy Chain CAGGTTCAGCTCCAGCAGTCAGGAGCTGAGCTCATGAAGCCCGGAAGCTCAGTCAAACTCTCCTGCAAGGCCACAGGCTACACATTCACA GACTACTGGATCGAG TGGGTCAAACAGAGGCCTGGCCACGGATTGGAGTGGATTGGA GAGATCCTGCCCGGGCTCTTTCAGTACCAACTACAACGAGAAATTCAAGGGC AAGGCCACATTCACCGCCGACATTAGCAGTAACACCGCATACATGCAGCTGAGCAGCCTCACCAGCGAAGATTCTGCTGTGTACTACTGCGCCTCC GACAACTAT TGGGGGCAGGGAACACTGGTGACCGTGTCCGCT 43 Light chain GACATCGTGATGACCCAGTCCCAGAAATTTATGAGCGCCTCCGTGGGAGACAGAGTGAGTGTTACCTGC AAGGCCGGACAGAACGTGGGAACAAACGTGGCT TGGTACCAGCAGCAGCCCGGACAGTCCCCTAAAGCTCTGATCTAC TCCGCCTCATACCGTTACAGC GGCGTGCCTGATAGATTCACAGGAAGCGGCTCCGGGACAGACTTCACATTGACTATCTCCACCGTCCAGTCTGAAGATTTAGCTGAGTACTTCTGC CAGCAGTACAACAGCTACCCCCTGACC TTCGGCGCTGGAACCAAATTGGAGCTCAAG 44 NI-11 Heavy Chain CAGGTTCAGCTCAAAGAGAGCGGCCCAGGACTCTTGGCACCATCCCAGTCCTTGTCCATCACCTGCACCGTCTCAGGCTTCTCACTCACA AACTACGGCGTCCAC TGGGTCAGGCAGCCTCCTGGAAAAGGACTCGAATGGCTCGGA CTCATCTGGGCTGGTGGATCTACCTCCTACAACTCCGCCCTGATGTCT CGCCTGTCCATCTCTAAGGACAATTCCAAGTCCCAGGTCTTTCTGAAGATGAACTCCCTCCAGACCGATGACACCGCCATCTATTACTGCGCCCGC GAAAAGGACTATTACTACTCCGACTACCGCTTTGCCTAC TGGGGCCAGGGAACATTGGTGGCAGTGAGCGCT 45 Light chain GACATCGTGCTGACCCAGTCTCCTACCTCCCTGGCTGTTTCCTTGGGGCAGAGAGCTACCATCTCCTGC AAGGCTTCCCAGTCCGTGCACTATGACGGGATTCCTACATGAAC TGGTACCAGCAGAAACCCGCCAGCCTCCTAAGTTGCTCATCTAC GCCGCCTCCAACCTGGAGTCT GGTATCCCTGCTCGCTTCTCCGGTTCCGGTTCTGGAACAGACTTCACCCTCAACATCCACCCCGTCGAAGAGGACGACGCTGCTACCTATTACTGC CAGCAGAACAACGAAGATCCTTGGACC TTCGGGGGCGGTACAAAGTTGGAAATCAAA 46 NI-50 Heavy chain CAGGTTCAGCTCAAAGAGAGCGGCCCCGGACTCGTTGCTCCATCTCAGTCACTCTCCATCACCTGCACAGTCAGCGGCTTCAGCCTCACA AGCTACGGCGTCCAC TGGGTGCGTCAGCCTCCTGGAAAAGGCCTGGAATGGCTCGGA GTGATTTGGGCCGGCGGATCTACCAACTACAAAAGCGCCCTGATGTCA AGGTTGTCCATCTCCAAGGATAATTCCAAGTCCCAGGTTGTTCCTCAAGATGAACAGTCTGCAGACCGACGACACCGCCATGTACTACTGCGCCCGA GACAGGGCAATCTACTACTCCACATACGAGTTCGCCTAC TGGGGCCAGGGGACATTGGTTACCGTGAGCGCT 47 Light chain GATATCGTGCTGACCCAGTCCCCCGCTTCCTTGGCTGTTTCCCTGGGACAGAGGGCCACCATCTCCTGT AAGGCCTCCCAGTCCGTGGACTACGACGGTGATTCCTACATCACC TGGTACCAGCAGAAACCCGCCAGCCTCCTAAGCTGTTGATTTAC GTGGCTTCCAACCTGGAGTCC GGCATCCCAGCTCGTTTTCAGCGGAAGCGGAAGCGGTACCGACTTCACCCTGAACATCCACCCCGTGGAGGAAGAGGACGCCGCTACATACTACTACTGC CAGCAGACCAACGAAGATCCTTGGACC TTCGGAGGCGGCACCAAGTTGGAGATCAAA 48

Example 3: Humanization of NI-10 antibody

Humanization was performed using the NI-10 antibody, which has the best NE neutralizing ability.

Chimeric antibodies against NI-10 were prepared by substituting the framework sequence, excluding the CDR, with a human antibody sequence, and their activity was measured using the Neutrophil Elastase Inhibitor Assay Kit (Abcam, #ab118971). Specifically, NE (EPC, #SE563) was diluted to a concentration of 50 ng/well with the kit's buffer and dispensed into 96-well plates (Thermofisher, #7905) in 50 μL aliquots. Subsequently, 25 μL of the chimeric antibody against NI-10, diluted with PBS and substituted with the human antibody sequence, was added. After incubating at 37°C for 5 minutes, 2 μL of substrate and 23 μL of buffer from 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 measurement, four types of antibodies having 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.

CDR amino acid sequences of humanized antibodies (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45) Antibody names variable area amino acid sequence Sequence number NI-10_v38 Heavy Chain CDR1 Identical to Sequence No. 1 1 CDR2 Identical to Sequence No. 2 2 CDR3 Identical to Sequence No. 3 3 Light chain CDR1 Identical to Sequence No. 4 4 CDR2 Identical to Sequence No. 5 5 CDR3 Identical to Sequence No. 6 6 NI-10_v39 Heavy chain CDR1 Identical to Sequence No. 1 1 CDR2 Identical to Sequence No. 2 2 CDR3 Identical to Sequence No. 3 3 Light chain CDR1 Identical to Sequence No. 4 4 CDR2 Identical to Sequence No. 5 5 CDR3 Identical to Sequence No. 6 6 NI-10_v44 Heavy chain CDR1 Identical to Sequence No. 1 1 CDR2 Identical to Sequence No. 2 2 CDR3 Identical to Sequence No. 3 3 Light chain CDR1 Identical to Sequence No. 4 4 CDR2 Identical to Sequence No. 5 5 CDR3 Identical to Sequence No. 6 6 NI-10_v45 Heavy chain CDR1 Identical to Sequence No. 1 1 CDR2 Identical to Sequence No. 2 2 CDR3 Identical to Sequence No. 3 3 Light chain CDR1 Identical to Sequence No. 4 4 CDR2 Identical to Sequence No. 5 5 CDR3 Identical to Sequence No. 6 6

CDR sequences of humanized antibodies (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45) Antibody names variable area base sequence Sequence number NI-10_v38 Heavy chain CDR1 Identical to Sequence No. 19 19 CDR2 GAGATCCTGCCCGGCTCCTTCTCCACCAACTACAACGAGAAGTTCAAGGGC 49 CDR3 GACAACTAC 50 Light chain CDR1 AAAGCCGGCCAGAACGTGGGAACCAACGTGGCT 51 CDR2 TCCGCCTCCTACAGATACAGC 52 CDR3 CAGCAGTACAACTCCTACCCACTGACC 53 NI-10_v39 Heavy chain CDR1 Identical to Sequence No. 19 19 CDR2 Identical to sequence number 49 49 CDR3 Identical to sequence number 50 50 Light chain CDR1 AAGGCTGGACAGAACGTGGGAACCAACGTGGCT 54 CDR2 TCCGCCTCCTACAGGTACTCC 55 CDR3 Identical to Sequence No. 24 24 NI-10_v44 Heavy chain CDR1 Identical to Sequence No. 19 19 CDR2 Identical to sequence number 49 49 CDR3 Identical to sequence number 50 50 Light chain CDR1 Identical to sequence number 51 51 CDR2 Identical to sequence number 52 52 CDR3 Identical to sequence number 53 53 NI-10_v45 Heavy chain CDR1 Identical to Sequence No. 19 19 CDR2 Identical to sequence number 49 49 CDR3 Identical to sequence number 50 50 Light chain CDR1 Identical to Sequence No. 54 54 CDR2 Identical to sequence number 55 55 CDR3 Identical to Sequence No. 24 24

Variable region amino acid sequences of humanized antibodies (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45) Antibody names variable area amino acid sequence Sequence number NI-10_v38 Heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFT DYWIE WVRQAPGQGLEWMG EILPGSFSTNY NEKFKGRVTMTADTSTSTVYMELSSLRSEDTAVYYCAS DNY WGQGTLVTVSS 56 Light chain EIVMTQSPATLSVSPGERATLSC KAGQNVGTNVA WYQQKPGQAPRALIY SASYRYS GIPARFSGSGSGTEFTLTISSLQSEDFAVYYC QQYNSYPLT FGGGTKVEIK 57 NI-10_v39 Heavy chain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYWIEWVRQAPGQGLEWMGEILPGSFSTNYNEKFKGRVTMTADTSTSTVYMELSSLRSEDTAVYYCASDNYWGQGTLVTVSS 58 Light chain DIQMTQSPSTLSASVGDRVTITC KAGQNVGTNVA WYQQKPGKAPKALIY SASYRYS GVPSRFSGSGSGTEFTLTISSLQPDDFATYYC QQYNSYPLT FGGGTKVEIK 59 NI-10_v44 Heavy chain QVQLVQSGAEVKKPGSSVKVSCKASGYTFT DYWIE WVRQAPGQGLEWMG EILPGSFSTNY NEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCAS DNY WGQGTLVTVSS 60 Light chain Identical to sequence number 57 57 NI-10_v45 Heavy chain Identical to sequence number 60 60 Light chain Identical to sequence number 59 59

Variable region nucleotide sequences of humanized antibodies (NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45) Antibody names variable area base sequence Sequence number NI-10_v38 Heavy chain CAGGTTCAGCTCGTCCAGAGCGGAGCAGAGGTCAAAAAGCCCGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACC GACTACTGGATCGAG TGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGGTGGATGGGC GAGATCCTGCCCGGCTCCTTCTCCACCAACTACAACGAGAAGTTCAAGGGC AGGGTGACCATGACCGCCGACACCTCCACCTCCACCGTGTACATGGAGCTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTACTGCGCCTCC GACAACTAC TGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC 61 Light chain GAGATCGTGATGACACAGAGCCCCGCTACACTGAGCGTTTCCCCTGGAGAACGCGCTACACTGAGTTGT AAAGCCGGCCAGAACGTGGGAACCAACGTGGCT TGGTATCAGCAGAAGCCCCGGCCAGGCTCCTAGAGCTCTGATTTAC TCCGCCTCCTACAGATACAGC GGCATCCCAGCTAGGTTTTCCGGGAGTGGATCAGGTACAGAGTTCACCCTGACTATCTCCAGCCTGCAGAGTGAGGATTTCGCAGTGTACTACTGC CAGCAGTACAACTCCTACCCACTGACC TTCGGAGGGGGTACTAAGGTGGAGATCAAG 62 NI-10_v39 Heavy chain CAGGTTCAGCTCGTCCAGAGCGGAGCAGAGGTCAAAAAGCCCGGCGCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACC GACTACTGGATCGAG TGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGAGGTGGATGGGC GAGATCCTGCCCGGCTCCTTCTCCACCAACTACAACGAGAAGTTCAAGGGC AGGGTGACCATGACCGCCGACACCTCCACCTCCACCGTGTACATGGAGCTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTACTGCGCCTCC GACAACTAC TGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC 63 Light chain GACATCCAGATGACCCAGTCCCCATCCACCCTGTCTGCTTCTGTGGGAGACAGGGTGACCATCACCTGC AAGGCTGGACAGAACGTGGGAACCAACGTGGCT TGGTACCAGCAGAAACCAGGCAAAGCCCCAAAGGCCCTCATCTAC TCCGCCTCCTACAGGTACTCC GGCGTTCCTTCTCGGTTTTCCGGGTCTGGAAGCGGTACAGAATTTACCCTGACTATCTCCTCCCTGCAGCCCGATGACTTCGCTACATACTACTGC CAGCAGTACAACAGCTACCCCCTGACC TTCGGCGGAGGAACCAAAGTTGAAATCAAA 64 NI-10_v44 Heavy chain CAGGTTCAGCTCGTCCAGAGCGGAGCAGAGGTCAAAAAGCCCGGGCTCCTCCGTGAAGGTGTCCTGCAAGGCCTCCGGCTACACCTTCACC GACTACTGGATCGAG TGGGTGAGGCAGGCCCCCGGCCAGGGCCTGGGAGTGGATGGGC GAGATCCTGCCCGGCTCCTTCTCCACCAACTACAACGAGAAGTTCAAGGGC AGGGTGACCATCACCGCCGACGAGTCCACCTCCACCGCCTACATGGAGCTGTCCTCCCTGAGGTCCGAGGACACCGCCGTGTACTACTGCGCCTCC GACAACTAC TGGGGCCAGGGCACCCTGGTGACCGTGTCCTCC 65 Light chain Identical to sequence number 62 62 NI-10_v45 Heavy chain Identical to sequence number 65 65 Light chain Identical to Sequence No. 64 64

The NE neutralizing ability of the above-mentioned 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 using the kit's buffer, and 50 μL was dispensed into each well of a 96-well plate (Thermofisher, #7905). Subsequently, 25 μL of each of the four selected humanized antibodies, diluted to different concentrations using PBS buffer, was added. After incubating at 37°C for 5 minutes, 2 μL of substrate and 23 μL of buffer from the kit were mixed and added to each well. The signal was measured at 400/505 nm using a Victor Nivo (PerkinElmer). The IC ₅₀ value confirmed by the measurement result is as listed in Table 9 (Fig. 2).

Antibody names NE neutralization ability IC ₅₀ (nM) NI-10 119.6 NI-10_v38 118.2 NI-10_v39 115.1 NI-10_v44 190 NI-10_v45 441.4

Example 4: Confirmation of binding strength and affinity of anti-NE antibody

We intended to confirm the binding strength and affinity of the selected anti-NE antibody using ELISA and bio-layer interferometry (BLI).

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

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

The next day, blocking was achieved by incubating with 3% BSA/PBS at 37°C for 1 hour, followed by the addition of the primary anti-NE antibody, 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, reacted at 37°C for 1 hour, and washed 4 times. TMB and STOP solution were added sequentially to induce a color reaction, and absorbance was measured at 450 nm using Victor Nivo (PerkinElmer). The _EC50 values derived by confirming the binding affinity to NE via ELISA are listed in Tables 10 and 11.

Antibody names NE bonding strength EC ₅₀ (nM) NI-10 0.769 NI-11 0.422 NI-50 0.308

Antibody names NE bonding strength EC ₅₀ (nM) NI-10_v38 0.398 NI-10_v39 0.618 NI-10_v44 6.571 NI-10_v45 23.870

Next, BLI evaluation was performed using Octet RED96e (FORTEBIO) to analyze the NE affinity of the anti-NE antibody. After loading the antibody candidates selected in Example 1 (NI-10, NI-10_v37, NI-10_v38, NI-10_v39, NI-10_v44, NI-10_v45) as ligands onto an AHC2 biosensor (Sartorius, #18-5142), the binding constant ( _KD ), binding rate constant ( _Ka ), and dissociation rate constant ( _Kdis ) were derived using the same type of NE used for immunization in Example 1 as analytes, and the respective values are shown in Table 12.

Antibody names K _D (M) K _a (1/Ms) K _dis (1/s) NI-10 1.07E-09 3.23E+05 3.47E-04 NI-10_v37 3.61E-10 3.42E+05 1.23E-04 NI-10_v38 6.61E-10 4.92E+05 3.25E-04 NI-10_v39 8.02E-10 4.87E+05 3.90E-04 NI-10_v44 7.69E-10 5.12E+05 3.94E-04 NI-10_v45 9.74E-10 3.00E+05 2.92E-04

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

We intended to determine whether the antibody of the present invention could prevent the reduction of cell adhesion and the occurrence of apoptosis caused by NE.

Human alveolar epithelial cells A549 were used. A549 cells were seeded at a density of 2 x ^10⁴ /well in a 96-well plate (Corning, #3690). The following day, serum-free medium, 20% PBS, the antibody of the present invention, and 125 nM NE were added, and the mixture was incubated for 24 hours. The medium was washed with PBS, and color development was performed using a CCK-8 kit (Chromogen, #CH-3000). The absorbance was then measured at 450 nm using a Victor Nivo (PerkinElmer).

As a result, the amount of adhered cells increased in proportion to the throughput of NI-10 and the humanized antibody NI-10_v38, confirming the inhibitory effect on the reduction of cell adhesion and the inhibitory effect on cell death due to the NE neutralization effect of the antibody of the present invention. In addition, since similar patterns were observed in NI-10 and the humanized antibody NI-10_v38, it was confirmed that the aforementioned effects were not inhibited during the humanization process (Fig. 4).

Example 6: Confirmation of Epitope Validity of Selected Representative Antibody

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 efficacy of the epitopes.

First, using ELISA, it was observed that when the endogenous proteins elafin and NI-10, known to inhibit NE activity by broadly binding to the active site of NE, were simultaneously bound to the antigen NE, the binding affinity of NI-10 was reduced (Fig. 5). In addition, competitive binding of other endogenous proteins that bind to the NE active site, such as alpha-1 antitrypsin (AAT) and NI-10_v38, was confirmed using BLI (Fig. 6).

At this time, NE bound to N-01, an antibody obtained by the biopanning method that was not selected as a final candidate, showed binding to AAT, whereas NE bound to NI-10_v38 did not show additional binding to AAT, confirming that the epitope of NI-10_v38 is valid (Fig. 6). Since NI-10_v38 is a humanized antibody of NI-10, the epitopes of these antibodies are identical.

Example 7: Confirmation of NE-targeted cleavage resistance of humanized antibody NI-10_v38

The protein stability of the humanized antibody NI-10_v38, AAT, and the mutated AAT-Fc fusion protein INBRX-101 was compared under conditions simulating pathological environments in which neutrophils have infiltrated in large quantities, such as acute lung injury with high concentrations of NE and lung tissue of AATD patients.

Specifically, a pseudo-inflammatory environment in which high concentrations of NE and inhibitors coexist 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 then incubating at room temperature for 16 hours. Subsequently, 10 μL of each reaction mixture was run on a NuPAGE ^™ 4-12% Bis-Tris 1.0 mm gel (Invitrogen, #NP0323BOX) for electrophoresis, followed by CBB (Coomassie Brilliant Blue) staining to analyze the protein stability of the inhibitors in each sample.

As a result, it was confirmed that AAT and INBRX-101 were severely degraded at all three concentration conditions (0.4 μM, 0.59 μM, and 0.89 μM), either by being cleaved by NE or by binding to some inhibitors cleaved by NE, failing to maintain their own protein band patterns (Fig. 7). This suggests that these inhibitors may be rapidly lost before exerting their effects at the site of inflammation. On the other hand, in the case of the humanized antibody NI-10_v38, it was observed that the same band pattern was maintained at all three concentration conditions, regardless of whether NE was administered.

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

Example 8: Oxidation Stability of Humanized Antibody NI-10_v38

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

Specifically, oxidation conditions were established by mixing NI-10_v38 and AAT (final concentration 1.5 μM) with a 0.1% hydrogen peroxide ( _H₂O₂ , v/v) solution in a 1:1 ratio and incubating at room temperature. Subsequently, samples were collected at 0, 6, _and 24 hours to evaluate NE inhibitory activity. Neutralization ability was evaluated based on the protocol using the EnzChek® Elastase Assay Kit (Invitrogen, #E12056). The concentration of NE in the 200 μL sample in the assay well was fixed at 20 nM, while NI-10_v38 and AAT were used at concentrations of 20 nM and 40 nM, corresponding to the 1:1 or 2:1 ratio of NE. Signals were measured at 480/520 nm using a Victor Nivo (PerkinElmer). Each bar in Fig. 8 represents the time points at 0, 6, and 24 hours after chemical treatment under oxidation conditions, and the experiment was repeated twice and the average was calculated (N=2).

As a result, AAT loses its inhibitory activity within 6 hours when mixed with NE at both 1:1 and 2:1 ratios. On the other hand, the humanized antibody NI-10_v38 fully maintains its inhibitory activity 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 was found to be very stable compared to AAT under oxidative conditions (Fig. 8).

Example 9: Efficacy of NI-10_v38 in an Acute Lung Injury Mouse Model

We aimed to evaluate the lung injury inhibitory and anti-inflammatory effects of the humanized antibody NI-10_v38 in an acute lung injury mouse model.

Specifically, the inducer human NE (10 μg/head) was administered intratracheally to C57BL/6 mice, either alone or mixed with the test substance NI-10_v38 (250, 50, and 10 μg/head), IgG-Fc (91.5 μg/head), or INBRX-101 (250 μg/head) immediately before individual administration. IgG-Fc was used as a control group, an isotype control possessing the same Fc as NI-10_v38, while INBRX-101 was used as a competing drug to compare efficacy. The test substance Alvelestat (10 mg/kg) was administered orally at a dose of 10 mg/kg, calculated based on body weight, one hour prior to intratracheal administration of the inducer human NE. Four hours after human NE administration, bronchoalveolar lavage fluid (BALF) was collected using 1 mL of PBS. The blood cell count in BALF was measured using a CBC analyzer (Mindray BC-5000 Vet), and cytokine IL-6 (Abcam, ab222503) and total MMP-9 (RnD Systems, MMPT90) in BALF were evaluated based on a protocol using an ELISA kit.

As a result, the number of red blood cells (RBC), neutrophils, lymphocytes, and hemoglobin (HGB) concentrations were found to be statistically significantly higher in the hNE-alone group (G2) compared to the Vehicle (G1), while a decrease in RBCs and HGB was found in the NI-10_v38 treatment groups (G3~G5), and a statistically significant decrease in neutrophils and lymphocytes was found in the NI-10_v38 treatment groups (G3, G4).

In addition, the concentrations of acute inflammatory cytokines IL-6 and total MMP9 were statistically significantly increased in the hNE-alone administration group (G2), while IL-6 concentrations and total MMP9 concentrations were statistically significantly decreased in the NI-10_v38 treatment groups (G3, G4) and NI-10_v38 treatment groups (G3~G5). Through this, it was confirmed that NI-10_v38 effectively inhibits the leakage of RBCs and HGBs into BALF by hNE and significantly reduces neutrophil and lymphocyte infiltration and inflammatory responses (Fig. 9).

Attached electronic file of sequence list

Claims (15)

As an anti-NE antibody that specifically binds to NE (neutrophil elastase) or an antigen-binding fragment thereof,
Heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO. 1, 7, or 13,
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
Light chain CDR1 represented by the amino acid sequence of SEQ ID NO. 4, 10, or 16,
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 its antigen-binding fragment. In the first paragraph,
The above anti-NE antibody or its antigen-binding fragment is
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 its antigen-binding fragment comprising a light chain variable region (VL) containing the amino acid sequence of SEQ ID NO. 38, 40, 42, 57, or 59. In the first paragraph,
The above anti-NE antibody or its antigen-binding fragment is
Heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO. 1, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO. 2, heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO. 3, light chain CDR1 represented by the amino acid sequence of SEQ ID NO. 4, light chain CDR2 represented by the amino acid sequence of SEQ ID NO. 5, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO. 6;
Heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO. 7, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO. 8, heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO. 9, light chain CDR1 represented by the amino acid sequence of SEQ ID NO. 10, light chain CDR2 represented by the amino acid sequence of SEQ ID NO. 11, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO. 12; or
Heavy chain CDR1 represented by the amino acid sequence of SEQ ID NO. 13, heavy chain CDR2 represented by the amino acid sequence of SEQ ID NO. 14, heavy chain CDR3 represented by the amino acid sequence of SEQ ID NO. 15, light chain CDR1 represented by the amino acid sequence of SEQ ID NO. 16, light chain CDR2 represented by the amino acid sequence of SEQ ID NO. 17, and light chain CDR3 represented by the amino acid sequence of SEQ ID NO. 18
An antibody or its antigen-binding fragment comprising In the first paragraph,
The above NE is an anti-NE antibody or an antigen-binding fragment thereof comprising NE of human or mouse origin. In the first paragraph,
The above anti-NE antibody is an anti-NE antibody or an antigen-binding fragment thereof that is a humanized antibody. 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 is
Heavy chain CDR1 encoding sequence represented by SEQ ID NO. 19, 25, or 31,
Heavy chain CDR2 encoding sequences represented by SEQ ID NOs 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
Light chain CDR1 encoding sequences represented by sequence numbers 22, 28, 34, 51, or 54,
Light chain CDR2 encoding sequences represented by SEQ ID NOs 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 is
A heavy chain variable region (VH) encoding sequence represented by SEQ ID NOs 43, 45, 47, 61, 63, or 65; and
Comprising a nucleotide sequence encoding a light chain variable region (VL) represented by SEQ ID NOs 44, 46, 48, 62, or 64,
Polynucleotide. In paragraph 6,
The above polynucleotide is
A heavy chain CDR1 encoding sequence represented by SEQ ID NO. 19, a heavy chain CDR2 encoding sequence represented by SEQ ID NO. 20, a heavy chain CDR3 encoding sequence represented by SEQ ID NO. 21, a light chain CDR1 encoding sequence represented by SEQ ID NO. 22, a light chain CDR2 encoding sequence represented by SEQ ID NO. 23, and a light chain CDR3 encoding sequence represented by SEQ ID NO. 24;
A heavy chain CDR1 encoding sequence represented by SEQ ID NO. 25, a heavy chain CDR2 encoding sequence represented by SEQ ID NO. 26, a heavy chain CDR3 encoding sequence represented by SEQ ID NO. 27, a light chain CDR1 encoding sequence represented by SEQ ID NO. 28, a light chain CDR2 encoding sequence represented by SEQ ID NO. 29, and a light chain CDR3 encoding sequence represented by SEQ ID NO. 30; or
The heavy chain CDR1 encoding sequence indicated by SEQ ID NO. 31, the heavy chain CDR2 encoding sequence indicated by SEQ ID NO. 32, the heavy chain CDR3 encoding sequence indicated by SEQ ID NO. 33, the light chain CDR1 encoding sequence indicated by SEQ ID NO. 34, the light chain CDR2 encoding sequence indicated by SEQ ID NO. 35, and the light chain CDR3 encoding sequence indicated by SEQ ID NO. 36
A polynucleotide containing In paragraph 6,
The above polynucleotide is
A heavy chain CDR1 encoding sequence represented by SEQ ID NO. 19, a heavy chain CDR2 encoding sequence represented by SEQ ID NO. 49, a heavy chain CDR3 encoding sequence represented by SEQ ID NO. 50, a light chain CDR1 encoding sequence represented by SEQ ID NO. 51, a light chain CDR2 encoding sequence represented by SEQ ID NO. 52, and a light chain CDR3 encoding sequence represented by SEQ ID NO. 53; or
The encoding sequence of heavy chain CDR1 represented by SEQ ID NO. 19, the encoding sequence of heavy chain CDR2 represented by SEQ ID NO. 49, the encoding sequence of heavy chain CDR3 represented by SEQ ID NO. 50, the encoding sequence of light chain CDR1 represented by SEQ ID NO. 54, the encoding sequence of light chain CDR2 represented by SEQ ID NO. 55, and the encoding sequence of light chain CDR3 represented by SEQ ID NO. 24
A polynucleotide containing An expression vector comprising the polynucleotide of claim 6. Host cell transformed with the expression vector of claim 11. A method for preparing an anti-NE antibody that specifically binds to NE or an antigen-binding fragment thereof, comprising the following steps:
(a) a step of culturing the host cells of paragraph 12; and
(b) A step of recovering an antibody or an antigen-binding fragment thereof from the cultured cells. A pharmaceutical composition for the prevention or treatment of NE overactivation-related diseases, comprising an anti-NE antibody according to claim 1 or an antigen-binding fragment thereof as an active ingredient. In Paragraph 14,
A pharmaceutical composition wherein the above-mentioned NE overactivation-related disease 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.