WO2024058590A1 - Novel human interleukin-4 receptor binding nanobody and use thereof - Google Patents
Novel human interleukin-4 receptor binding nanobody and use thereof Download PDFInfo
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- WO2024058590A1 WO2024058590A1 PCT/KR2023/013874 KR2023013874W WO2024058590A1 WO 2024058590 A1 WO2024058590 A1 WO 2024058590A1 KR 2023013874 W KR2023013874 W KR 2023013874W WO 2024058590 A1 WO2024058590 A1 WO 2024058590A1
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- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
- C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
Definitions
- the present invention relates to a nanobody that specifically binds to the IL-4 receptor and a method of preventing or treating various IL-4-mediated inflammatory diseases using the nanobody as a pharmacological ingredient.
- Chronic rhinosinusitis is one of the most common chronic inflammatory diseases that occurs in the nasal mucosa. It has a high infection rate worldwide and causes a socioeconomic burden by reducing the quality of life of patients. am. Chronic rhinosinusitis (CRS) is generally divided into two special forms: CRS without nasal polyps (CRSsNP) and CRS with nasal polyps (CRSwNP). Although CRSsNP is the predominant form, CRSwNP also accounts for approximately 20 of all CRS cases. It constitutes an important form of the disease, accounting for % to 33%. CRSwNP is often accompanied by asthma, fungal rhinosinusitis, and aspirin hypersensitivity respiratory diseases, with poor treatment outcomes after standard medical therapy and endoscopic sinus surgery.
- Dupilumab a monoclonal antibody (mAb) against interleukin (IL)-4R- ⁇ , has previously shown therapeutic efficacy and safety in moderate to severe eosinophilic asthma, but recent clinical trials have shown chronic rhinosinusitis. The treatment effect was also confirmed in (CRSwNP).
- dupilumab is currently administered in the form of an injection, which has the disadvantage of causing systemic side effects such as blepharitis, keratitis, and oral herpes.
- nanobodies which have recently been actively developed in the therapeutic and diagnostic fields, are antigen recognition variable regions of heavy chain-specific antibodies found in camelid animals (camels, llamas, alpacas, etc.), and are small in size and Based on its high stability, it is attracting attention as an alternative solution that can solve the problems of existing antibody treatments.
- Most antibodies have flat or slightly grooved CDRs (Complementarity Determining Regions) regions that interact with antigens, so their binding efficiency is low for proteins such as enzymes whose active sites have a concave shape.
- CDRs Complementarity Determining Regions
- the CDR-H3 region consists of an average of 19 amino acids, and compared to the average human CDR-H3 of 12 amino acids, it has a longer CDR region, enabling the formation of a convex loop and compared to a typical antibody. It has a simple structure and small size, which has the advantage of high structural stability.
- the present inventors sought to develop a new type of antibody for IL-4R inhibition that exhibits an equivalent or greater IL-4 receptor inhibitory effect compared to the existing dupilumab, has high stability, and can be applied to various administration routes due to its small molecular weight. did.
- the present inventors have made extensive research efforts to develop an excellent antibody-based therapeutic composition that can efficiently inhibit the IL-4 receptor, which is being actively studied as a mediator and treatment target for various inflammatory diseases.
- the CDR sequences of SEQ ID NOs. 1 to 4 are used as the antigen recognition site, it has a higher binding affinity to the IL-4 receptor than the commercialized antibody treatment dupilumab (DUPIXENT ® ), while having a simple structure and low molecular weight.
- the present invention was completed by discovering that an antibody fragment with significantly improved structural stability, productivity and permeability could be used as a nanobody, specifically a nanobody.
- the purpose of the present invention is to provide an antibody or antigen-binding fragment thereof against the IL-4 receptor (IL-4R) and a nucleic acid molecule encoding the same.
- IL-4R IL-4 receptor
- Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases.
- the present invention provides an anti-IL-4R antibody or antigen-binding fragment thereof comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4.
- the present inventors have made extensive research efforts to develop an excellent antibody-based therapeutic composition that can efficiently inhibit the IL-4 receptor, which is being actively studied as a mediator and treatment target for various inflammatory diseases.
- the CDR sequences of SEQ ID NOs. 1 to 4 are used as the antigen recognition site, it has a higher binding affinity to the IL-4 receptor than the commercialized antibody treatment dupilumab (DUPIXENT ® ), while having a simple structure and low molecular weight. It was discovered that it could be used as an antibody fragment with significantly improved structural stability, productivity, and permeability.
- the antibody fragment of the present invention is applied in the form of a nanobody that is delivered directly to the lesion area through, for example, the nasal mucosa, making it useful as an efficient local treatment for various inflammatory diseases that occur in the nasal cavity without systemic immunosuppression. It can be used.
- antibody refers to an antibody against IL-4-R, a peptide that specifically recognizes and binds to a specific epitope thereof, and includes not only the complete antibody form but also an antigen-binding fragment of the antibody molecule (antibody). includes fragments).
- a complete antibody has a structure of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond.
- the heavy chain constant region has gamma ( ⁇ ), mu ( ⁇ ), alpha ( ⁇ ), delta ( ⁇ ), and epsilon ( ⁇ ) types and is subclassed as gamma1 ( ⁇ 1), gamma2 ( ⁇ 2), and gamma3 ( ⁇ 3). ), gamma 4 ( ⁇ 4), alpha 1 ( ⁇ 1), and alpha 2 ( ⁇ 2).
- the constant region of the light chain has kappa ( ⁇ ) and lambda ( ⁇ ) types.
- antigen-binding fragment of an antibody refers to a fragment that has a significant antigen-antibody binding function within the entire antibody molecule, including Fab, F(ab'), F(ab')2, Fv, and nanobody. (nanobody or sybody) etc.
- Fab has a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen binding site.
- Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
- F(ab')2 antibody is produced when cysteine residues in the hinge region of Fab' form a disulfide bond.
- Fv is a minimal antibody fragment containing only the heavy chain variable region and the light chain variable region.
- Double-chain Fv is a non-covalent bond between the heavy chain variable region and light chain variable region
- single-chain Fv single-chain Fv
- They can be connected by a bond or directly connected at the C-terminus to form a dimer-like structure, such as double-chain Fv.
- antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), and gene It can also be produced through recombinant technology.
- proteolytic enzymes for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin
- gene It can also be produced through recombinant technology.
- heavy chain refers to a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2, and CH3, including an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a full-length heavy chain thereof. It means all fragments.
- light chain refers to both a full-length light chain and fragments thereof including a variable region domain VL and a constant region domain CL containing an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.
- CDR complementarity determining region
- antibodies or antibody fragments of the present invention includes variants with conservative amino acid substitutions in the CDR regions.
- the antibody or antibody fragment of the present invention may include variants of the amino acid sequence described in the attached sequence list within the range that can specifically recognize the IL-4 receptor.
- additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody.
- Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. These amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc.
- arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that 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 said to be biologically equivalent in function.
- Amino acid exchanges in proteins that do not overall alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979).
- the most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/ Exchanges between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.
- the amino acid sequence constituting the antibody of the present invention is interpreted to also include a sequence showing substantial identity with the sequence listed in the sequence listing.
- the above substantial identity is at least 61% when aligning the sequence of the present invention and any other sequence to correspond as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art.
- Homology refers to a sequence showing 70% homology, according to another specific example, 80% homology, and according to another specific example, 90% homology.
- Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 and Pearson et al. Meth. Mol. Biol. (1994) 24:307-31, etc.
- IL-4 refers to a cytokine that induces differentiation from unactivated helper T cells (na ⁇ ve T cells, Th0 cells) to Th2 cells.
- IL-4 has various biological functions, such as stimulating activated B cells and T cells or differentiating B cells into plasma cells, and is an important regulator of humoral and adaptive immunity.
- IL-4R refers to the receptor for IL-4, and induces signaling related to the production of IgE antibodies by binding to its ligand, IL-4.
- the receptor for IL-4 is known as IL-4R ⁇ , and it exists in two types of complexes in the body.
- Type 1 receptors generally exist as a complex of ⁇ chain ( ⁇ c) and IL-4R ⁇ and are specific for IL-4.
- Type 2 receptors generally exist as a complex of IL-4R ⁇ and IL-13R ⁇ 1 and are specific for both IL-4 and IL13.
- dupilumab refers to a monoclonal antibody against IL-4R ⁇ , and is an antibody treatment product commercialized under the product name DUPIXENT ® .
- the mechanism of action of dupilumab is to inhibit signal transduction by blocking the binding of IL-4 and IL-4R ⁇ at the type 1 IL-4 receptor or by blocking the dimerization reaction of IL-4 ⁇ and ⁇ chain, and type 2 IL-4 receptor.
- the -4 receptor is known to inhibit signal transduction by blocking the dimerization reaction of IL-4R ⁇ and IL-13R ⁇ 1.
- the antigen-binding fragment of the invention is selected from the group consisting of F(ab')2, Fab', Fab, Fv, scFv and nanobodies.
- Antibodies of the present invention include monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and anti-idio Type (anti-Id) antibodies, and epitope-binding fragments and nanobodies (Nanobodies or sybodies) of the above antibodies, but are not limited thereto.
- the term “monoclonal antibody” refers to an antibody molecule with a single molecular composition obtained from a substantially identical antibody population, and a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.
- the antigen-binding fragment is a nanobody.
- the anti-IL-4R antibody or antigen-binding fragment thereof of the present invention includes one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8.
- SEQ ID NOs: 5, 6, 7 and 8 are the amino acid sequences of a full-length nanobody comprising the CDR sequences of SEQ ID NOs: 1, 2, 3 and 4, respectively.
- Nanobody refers to a single domain antibody analog composed of only one heavy chain variable region, and has the same meaning as “single domain antibody (VHH antibody)” or “synthetic nanoantibody (sybody),” and has the same meaning as “synthetic nanobody (sybody).”
- VHH antibody single domain antibody
- sibody single domain antibody
- Sybody single domain antibody
- CH1 light and heavy chain constant region 1
- Constructed single domain antibodies Variable Domain of Heavy-chain Antibodies, VHH can be constructed.
- Nanobodies can be manufactured by artificially processing antibody fragments extracted from camelid animals such as camels, llamas, and alpacas, and are one-tenth the size of regular antibodies, making them resistant to external environments such as temperature changes and easy to administer. It has the advantage of high production yield.
- the nanobody has a convex scaffold structure.
- nanobody CDR-H3 consists of an average of 19 amino acids, which is longer than human CDR-H3, which consists of an average of 12 amino acids. It forms a convex loop and can effectively bind to the concave-shaped active site of enzymes or proteases.
- scFv one of the antigen-binding fragments
- nanobodies can also bind well to concave active sites.
- they can bind with high specificity even to flat active sites, and this high affinity is known to be due to the flexible scaffold of nanobodies.
- nanobodies also have the advantage of having a lower degree of nonspecific backgound binding compared to scFvs.
- the nanobody has an average molecular weight of 10 to 18 kDa. More specifically, it is 11 to 17 kDa, even more specifically it is 12 to 17 kDa, and most specifically it is 13 to 15 kDa.
- the invention provides a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention.
- nucleic acid molecule is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogs with modified sugar or base sites. (analogue) is also included ( Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman et al., Chemical Reviews (1990)90:543-584).
- sequences of the nucleic acid molecules encoding the heavy chain, light chain variable regions and nanobodies of the invention may be modified. The modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.
- the nucleic acid molecule of the present invention is interpreted to also include a nucleotide sequence showing substantial identity to the above-mentioned nucleotide sequence.
- the above substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art.
- Homology in one specific example, means a nucleotide sequence showing at least 90% homology, and in another specific example, at least 95% homology.
- the antibody or antigen-binding fragment thereof of the present invention can be obtained recombinantly by expressing the nucleic acid molecule encoding the same in a host cell.
- the term “express” means causing a target cell to express an exogenous gene or artificially introducing it using a gene delivery system to increase the natural expression level of an endogenous gene, thereby allowing the gene to enter the target cell. This means that replication becomes possible as an extrachromosomal factor or through completion of chromosomal integration. Accordingly, the term “expression” has the same meaning as “transformation,” “transfection,” or “transduction.” More specifically, in the present invention, “express” means causing a target cell to artificially express a foreign gene.
- gene carrier or “gene delivery system” refers to any means for transporting a gene into a cell, and gene delivery has the same meaning as transduction of a gene into a cell.
- gene transfer is synonymous with gene spread. Accordingly, the gene delivery system of the present invention can be described as a gene penetration system and a gene diffusion system.
- the nucleotide sequence of the present invention is present in a suitable expression construct, wherein the nucleotide sequence of the present invention can be operably linked to an expression control sequence.
- operably linked refers to a functional linkage between a nucleic acid expression control sequence, such as a promoter, a signal sequence, or an array of transcriptional regulator binding sites, and another nucleic acid sequence, thereby regulating said expression. The sequence will regulate transcription and/or translation of the other nucleic acid sequences.
- the gene delivery system of the present invention can be manufactured in various forms, including (1) a naked recombinant DNA molecule, (2) a plasmid, (3) a viral vector, and (4) the naked recombinant DNA molecule or It can be produced in the form of a liposome or niosome containing a plasmid.
- the present invention provides an antibody or antigen-binding fragment thereof of the present invention.
- a pharmaceutical composition for preventing or treating inflammatory or autoimmune diseases comprising a nucleic acid molecule encoding the same as an active ingredient is provided.
- prevention refers to suppressing the occurrence of a disease or disease in a subject who has not been diagnosed as having the disease or disease but is likely to develop the disease or disease.
- treatment refers to (a) inhibiting the development of a disease, condition or symptom; (b) alleviation of a disease, condition or symptom; or (c) means eliminating a disease, condition or symptom.
- the composition of the present invention is administered to a subject, IL-4-mediated signaling is blocked, thereby suppressing, eliminating, or alleviating the development of symptoms caused by inflammatory or autoimmune diseases.
- the composition of the present invention may itself be a composition for treating these diseases, or may be administered together with other pharmacological ingredients and applied as a treatment adjuvant for these diseases.
- treatment or “therapeutic agent” includes the meaning of “therapeutic aid” or “therapeutic aid.”
- inflammatory disease refers to a general term for diseases whose main etiology is inflammatory reactions.
- autoimmune disease refers to all diseases whose etiology is excessive or unwanted immune response. Specifically, the induction or continuous maintenance of self-tolerance is not normally achieved, resulting in an immune response to self-antigens. This refers to a disease that is caused by a process in which one's own tissues are damaged.
- inflammatory or autoimmune diseases that can be prevented or treated with the composition of the present invention include rhinitis, conjunctivitis, periodontitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, and hemorrhoids.
- gout ankylosing spondylitis, rheumatic fever, rheumatoid arthritis, polymyalgia rheumatica, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, periarthritis, tendonitis, tenosynovitis, peritendinitis, myositis, polymyositis, dermatomyositis, hepatitis, Cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, inflammatory bowel disease, asthma, type 1 diabetes, psoriasis, eczema, scleroderma, vitiligo, peripheral neuritis, uveitis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis.
- the rhinitis is nasal polyps or sinusitis.
- nasal polyp generally refers to a pathological condition in which benign edematous mucosa in the shape of a bunch of grapes, originating from the middle meatus (middle nasal passage), protrudes into the nasal cavity.
- the term “sinusitis” refers to a disease in which the natural pores called paranasal sinuses in the facial bones around the nose are blocked and the paranasal sinuses are not properly ventilated and excreted, causing secondary inflammation and worsening inflammation as purulent secretions accumulate. do.
- the sinusitis of the present invention is chronic sinusitis (CRS).
- CRS chronic sinusitis
- chronic sinusitis generally refers to cases where symptoms of sinusitis persist for a long period of time, approximately 3 months or more, but is not limited thereto and includes all cases that persist beyond the normal recovery period of the disease.
- the chronic sinusitis of the present invention is chronic sinusitis with nasal polyp (CRSwNP).
- chronic sinusitis with nasal polyp refers to a disease in which nasal polyps (nasal polyps) appear along with chronic sinusitis, compared to cases where nasal polyps are not accompanied. It is referred to as “chronic sinusitis without nasal polyp (CRSsNP).”
- the pharmaceutical composition of the present invention is for nasal administration.
- nasal administration refers to a method of administering a drug through the nasal cavity or nasal mucosa, and the above-described administration method may be a non-invasive route.
- the present invention provides an antibody or antigen-binding fragment thereof of the present invention.
- a method for preventing or treating inflammatory or autoimmune diseases comprising administering to a subject a pharmaceutical composition containing a nucleic acid molecule encoding the same as an active ingredient.
- the present invention relates to an antibody or antigen-binding fragment thereof of the present invention; Since the route of administration of a pharmaceutical composition containing a nucleic acid molecule encoding the same as an active ingredient and the inflammatory or autoimmune diseases that can be prevented or treated using the pharmaceutical composition have already been described in detail, this is omitted to avoid excessive duplication. .
- the present invention relates to an antibody or antigen-binding fragment thereof against the IL-4 receptor, specifically a nanobody; and a pharmaceutical composition for preventing or treating inflammatory or autoimmune diseases containing the same as an active ingredient.
- the nanobody of the present invention exhibits significantly better IL-4 receptor binding affinity than commercialized antibody therapeutics, but also has excellent structural stability, productivity, and permeability due to its simple structure and low molecular weight, and can be used in various ways, such as nasal spray. It can be formulated without limitation in form.
- the nanobody of the present invention not only effectively blocks IL-4 signaling, which causes inflammatory diseases such as sinusitis, but also concentrates the pharmacological effect specifically on the lesion without lowering systemic immunity due to intravenous administration, thereby causing chronic inflammatory diseases. It can be used more safely for long-term administration.
- Figure 1 shows the final process of anti-IL-4R nanobody screening, where the Y axis represents the fold-change of OD values for IL-4R and MBP, which represents the IL-4R of each nanobody clone. Specificity is indicated, and clones boxed in red represent the finally selected nanobodies with a fold change value higher than the threshold (1.7).
- Figure 2 shows the results of purifying each nanobody of the four final nanobody candidates to confirm their binding affinity to the IL-4R molecule, and examining nanobodies at the same concentration through ELISA, H5 It was found that the nanobody had the highest binding affinity to the IL-4R molecule among the four candidates at the same concentration.
- Figure 3 shows the results of selecting three nanobody candidates according to ELISA results and investigating whether the binding affinity is related to the IL-4 signal blocking function.
- the degree of signal blocking of each nanobody was measured at three different concentrations. was compared with the degree of signal blocking of dupilumab. Reporter cells were incubated with 83pM of IL-4 and each nanobody (or antibody) for 24 hours, and the results showed that dupilumab and H5 nanobody had the most effective IL-4 signal blocking effect among the candidates.
- Figure 4 shows the results of comparing the signal blocking effect of the H5 nanobody selected as the final IL-4R blocking nanobody with that of dupilumab at three different concentrations.
- Figure 5 is a diagram showing the results of evaluating the binding affinity of dupilumab and H5 nanobody to HEK293 cells using a plate reader.
- Figures 6a to 6d show the results of immunofluorescence staining and image analysis on the regulation of FOXJ1 expression by IL-4, dupilumab, and nanobodies.
- HEK293T and HEK293 cell lines were purchased from the Korean Cell Line Bank. These cells were cultured in DMEM high glucose medium (Gibco, 11995-065) supplemented with 10% FBS (Gibco, 26140-079) and penicillin/streptomycin (Gibco, 15140-122) at 37°C with 5% CO 2 .
- DMEM high glucose medium Gibco, 11995-065
- FBS Gibco, 26140-079
- penicillin/streptomycin Gabco, 15140-122
- Primer information was referenced from a paper on the production of synthetic nanobodies (Zimmermann et al., 2018) 1 .
- FW_c_for CAA GTC CAG CTG GTG GAA TCG (SEQ ID NO: 9)
- FW_c_rev GCC GCT AGC CGC ACA G (SEQ ID NO: 10)
- Link2_c_for ATA TAT GAA GAC CTC TGC GCG GC (SEQ ID NO: 11)
- Link2_c_rev ATG CAT GGT CTC AGC AGT AAT ACA AAG CAG TAT CTT CCG G (SEQ ID NO: 12)
- CDR3_c GAA GAC CTC TGC GCG GCA GCC 111 111 GGC 111 111 CCG CTG 111 111 111 TAT 222 TAC TGG GGT CAG GGC ACC CAA GTT ACC GTT TCT (SEQ ID NO: 13)
- tolAK_rev CCG CAC ACC AGT AAG GTG TGC GGT TTC AGT TGC CGC TTT CTT TCT (SEQ ID NO: 17)
- RT primer CTT CAG TTG CCG CTT TCT TTC TTG (SEQ ID NO: 18)
- pRDV pRDV (Addgene, 132696) containing Sb-convex was selected as the template for PCR.
- Primers FW_c_for and Link2_c_rev were used to amplify the former region of the convex scaffold containing the CDR1 and CDR2 regions.
- Link2_c_for, FW_c_rev and CDR3_c primers were used to amplify the latter region of the convex scaffold with randomization of the CDR3 region.
- Random primer CDR3_c contains 10 different trinucleotide residues, 9 (111) of which are enriched in A, S, T, N, Y (10.6% each), D, E, Q, R, K , H, and W (5% each), and is characterized by almost no nonpolar amino acids F, M, V, I, L, and G (2% each) 1 .
- Another trinucleotide residue (222) is missing the amino acids D and A because the two aforementioned amino acids are underrepresented at the ends of the ⁇ -sheet 2 .
- BbsI restriction sites are present at both ends of the former (scaffold) and latter (randomized) convex fragments, which are restricted and ligated to form a convex fragment with a diversity of 3.17 ⁇ 10 12
- a library (convex library) was formed.
- Both 5' and 3' regions of the CDR3-randomized convex library were flanked before transcription.
- the 5' flanking region and the 3' flanking region are primers 5'_flank_for and 5'_flank_rev, respectively; and 3'_flank_for and tolAK_rev primers were used to amplify from plasmid pRDV5 containing Sb-convex (Addgene, 132696). Flanking regions were restricted with BspQI and then linked to each end of the library. Libraries with flanking regions were transcribed using the T7 RibomaxTM RNA Production System (Promega, P1320) according to the manual.
- Ribosome display can offer the advantage of displaying approximately 10 to 12 different library members with minimal effort1 .
- this method has not been widely applied due to several reasons regarding reagents and unfavorable RNase activity 3 .
- the in vitro translation kit PUREfrex2.1 (GeneFrontier, PF213-0.25-EX) was adopted 1 .
- the kit lacks oxidized glutathione (GSSG) and disulfide bridge isomerase (DsbC), which are additionally supported to form disulfide bond folding. 70ng of RNA library (approximately 1.7 ⁇ 10 12 molecules) was used as input, and the experimental procedure followed the manual.
- the in vitro translation reaction was performed at 37°C for 1 hour.
- 12ul of DynabeadsTMMyOneTMStreptavidinT1 beads (Invitrogen, 65601) were washed twice at 0.5% with WTB-BSA buffer (50mM Tris/acetate pH7.4, 150mM NaCl, 50mM MgAc 2 , supplemented with BSA). Then, the magnetic beads were blocked with WTB-BSA buffer for more than 20 minutes.
- the ribosomal complex was added to 100ul of panning solution (WTB-D-BSA (WTB-BSA with 0.1% tween20) supplemented with 500ug of heparin and 1ul of RNaseIn (Promega N2611)), and then the mixture was Centrifuged at 20,000xg for 5 minutes. The supernatant was mixed with biotinylated IL-4R (Acro Biosystems, ILR-H82E9) and incubated on ice for 2 hours. Magnetic beads were washed three times with WTB-B-BSA, and the panning-IL-4R mixture was added to the beads and incubated on ice for 1 hour.
- WTB-D-BSA WTB-BSA with 0.1% tween20
- RNaseIn Promega N2611
- the entire mixture was then washed three times with WTB-D (50mM Tris/acetate pH7.4, 150mM NaCl, 50mM MgAc 2 , supplemented with 0.1% Tween20).
- WTB-D 50mM Tris/acetate pH7.4, 150mM NaCl, 50mM MgAc 2 , supplemented with 0.1% Tween20.
- the supernatant was purified using the RNeasy kit (Qiagen, 74004) with an elution volume of 15ul.
- RNA eluted from the ribosome display was reverse transcribed using SuperiorScript III reverse transcriptase (Enzynomics, RT006M) in a total volume of 30ul according to the manual.
- the generated cDNA was purified using a PCR purification mini kit (Favorgen, FAGCK 001-1) with an elution volume of 30ul.
- the generated cDNA was purified using a PCR purification mini kit (Favorgen, FAGCK 001-1) with an elution volume of 30ul. 1ul of the eluate was used as a template for qPCR analysis, and 29ul of the elution was used for amplification.
- Purified DNA in the form of cDNA was amplified by PCR using Long_FX_for and Long_FX_rev primers. The total reaction volume was 100ul, and after the reaction was completed, it was divided into two tubes.
- qPCR analysis was used to monitor the enrichment of the library during the selection step by assessing the quality of cDNA resulting from ribosome display and phage display selection4 .
- QuantStudio 3 Real-time PCR instrument (Applied Biosystems) was used with AccuPower® 2X Greenstar qPCR Master Mix (Bioneer, K-6251). PCR program conditions were as follows: 95°C, 2 minutes (initial denaturation) / 95°C, 10 seconds; 63°C, 30 s (denaturation, annealing, extension, measurement)/melting curve steps followed the default settings in the machine manual.
- the enriched nanobody (sybody) library was introduced into the phagemid vector pDXinit (Addgene, 110101) using FX Cloning 5 .
- the cell mixture was pulsed with a Biorad Gene Pulser II electroporation system using 2.4 kV, 25 uF, and 300 ⁇ .
- the electroporated cells were immediately transferred to 25 ml of SOC medium and incubated at 37°C and 160 rpm for 30 minutes.
- the recovered culture was then transferred to 225 ml of 2TY medium supplemented with 200 ug/ml ampicillin and 2% glucose and incubated overnight at 37°C and 160 rpm.
- 2YT medium supplied with 200 ug/ml ampicillin and 2% glucose
- E. coli SS320 was cultured in 50 ml of 2YT medium (supplemented with 10 ⁇ g/ml tetracycline), and half of a 96-well plate was coated with 100 ⁇ l of 67 nM neutravidin at 4°C one day before phage display.
- Neutravidin coated plates were washed with 250ul of TBS for each well and blocked with 250ul of TBS-BSA (TBS supplemented with 0.5% BSA). Biotinylated IL-4R was added to 4.9 ml of purified phage (1012 phages/ml) to bring the protein concentration to 50 nM, and then incubated on ice for 2 hours. Neutravidin coated plates were washed with 250ul TBS-BSA-D (TBS supplemented with 0.5% BSA and 0.1% Tween 20). 100ul of phage-IL-4R mixture was added to each well of the plate and incubated on ice for 1 hour.
- TBS-D TBS with 0.1% Tween 20
- 100ul of PD elution buffer TBS with 0.25ml/ml trypsin added as powder
- AEBSF solution AEBSF solution
- Phages generated in the first phage display were harvested and purified in TBS-BSA-D (5 ⁇ 10 12 phages/ml). Biotinylated IL-4R was added to 100ul of 50nM phage solution and incubated on ice for 2 hours. 12ul of DynabeadsTMMyOneTMStreptavidinC1 beads (Invitrogen, 65001) were washed twice with 500ul of TBS-WTB-BSA and then blocked with 500ul of WTB-BSA for more than 20 minutes on ice. The magnetic beads were washed three times with 500 ⁇ l of TBS-BSA-D, then resuspended and incubated with the phage-IL-4R complex solution for 1 hour on ice.
- TBS-BSA-D TBS-BSA-D supplemented with 5uM non-biotinylated IL-4R (Sino Biological, 10402-H08H). It was resuspended with 100ul of competition buffer and incubated on ice for 3 minutes.
- Competitive non-biotinylated IL-4R was washed twice with 500ul of TBS-D. The beads were resuspended in 100ul of PD elution buffer and incubated for 10 minutes at room temperature. 0.8ul of ABESF was added to the resulting solution and mixed by pipetting.
- E. coli SS320 was cultured overnight at 37°C and 160 rpm in 2YT medium supplemented with 200 ug/ml ampicillin and 2% glucose.
- Plasmid pBXNH3CA_MBP (addgene, 132700) was transformed into E. coli BL21 for expression. It was cultured in TB medium supplemented with 100ug/ml ampicillin at 37°C and 160rpm for 4 hours. For expression and biotinylation, arabinose and biotin were added at concentrations of 0.02% and 100 uM, respectively. Afterwards, culture was performed overnight at 22°C and 160 rpm for expression. The culture was sonicated overnight and purified using MBP MiniExcellose® (Takara, AEx-MC-M03).
- the enriched library was cloned twice into the pSBinit vector (addgene, 110100) through phage display 5 .
- the cloned plasmid was transformed into E. coli SS320 through electroporation, cultured overnight at 37°C and 160 rpm, and then the plasmid was extracted.
- the extracted plasmid was transformed into E. coli BL21 (Enzynomics, CP110), plated on LB-agar plates supplemented with 25ug/ml chloramphenicol, and incubated at 37°C overnight.
- 1.2 ml of TB medium supplemented with 25 ug/ml chloramphenicol was prepared in each well of a 96-well deep well plate labeled ‘preculture’.
- Ninety-five colonies were selected from the incubated plate, each colony was inoculated into each well, and the first well was filled with the positive control pSb_init containing the MBP nanobody Sb_MBP#1 (addgene, 132699).
- Deep well plates were sealed with gas permeability and grown at 37°C and 300 rpm for 4 hours.
- a new 96-well deepwell plate filled with 1 ml of pre-warmed TB medium supplemented with 25 ug/ml chloramphenicol was labeled ‘expression culture’.
- the palette of 'preculture' was stored at -20°C for DNA purification, and the palette of 'expression culture' was stored in 100ul of periplasmic extraction buffer (20% sucrose, 50mM Tris pH8.0, 0.5mM EDTA, and 0.5ug/ml). of lysozyme (indicated as DW) was resuspended by intensive vortexing. After incubation on ice for 30 minutes, 900ul of TBS containing 1mM MgCl 2 was added to each well. The plate was centrifuged at 5,000xg, 4°C for 15 minutes, and the supernatant was used as periplasmic extraction for ELISA.
- Two 96-well immune plates were coated with 100ul of 5ug/ml protein A solution and incubated overnight at 4°C with adhesive seals. Plates were washed with 250ul of TBS per well and then blocked with 150ul of TBS-BSA per well. Next, 100ul of anti-c-Myc antibody (Biolegend, 626802) diluted 1:2,000 in TBS-BSA-D was added per well and incubated for 20 minutes. After washing three times with 250ul of TBS-D, 80ul of TBS-BSA-D was added to each well to compare nanobody binding between IL-4R and the degree of binding of nanobody to IL-4R and MBP. , 20ul of the same periplasmic extract was added side by side and incubated for 20 minutes.
- the plate was washed with 250ul of TBS-D per well, 100ul of 50nM MBP was added to the first two wells, 100ul of 50nM biotinylated IL-4R was added to each well, and the plate was incubated for 20 minutes. After washing three times with 250ul TBS-D, 100ul of streptavidin-peroxidase (Invitrogen, 434323) diluted 1:5,000 in TBS-BSA-D was added to each well and incubated for 20 minutes. The plate was washed again three times with TBS-D, and 100ul of TMB substrate (biolegend, 421101) was added to each well. The reaction took approximately 15 minutes until individual wells turned blue. Absorbance was measured at 650 nm with a plate reader.
- TALON® SuperflowTM (Cytiva, 28957502) slurry was pre-equilibrated with TBS pH 8.0. After binding, the beads were washed three times with washing buffer (50mM Tris pH 8.0, 300mM NaCl, 5mM imidazole), and then the nanobodies were eluted with elution buffer (50nM Tris pH 8.0, 300mM NaCl, 200mM imidazole). Eluted nanobodies were buffer exchanged overnight using Slide-A-Lyzer® Dialysis Cassette (Thermo, 66330).
- the HEK293 cell line stably expressed human STAT6 (addgene, 81950) by a puromycin-resistant lentivirus system and luciferase (addgene, 35554) induced by pSTAT6 by a blasticidin-resistant system, thereby producing IL- 4 luciferase was allowed to be stably expressed under the control of the STAT6 response 6,7 .
- HNE cells in the transwell were washed with PBS and fixed with 500ul of 4% paraformaldehyde (Biosesang, P2031) for 30 minutes at room temperature. HNE cells were then incubated with 100 ul (1:200) of FOXJ1 primary antibody (GeneTex, GTX114408) for 1 h, followed by 100 ul (1:1000) mouse anti-rabbit FITC for 30 min. Washed three times with PBS. Cells were photographed on a Zeiss LSM 700 using parameter settings for the 405 and 488 nm lasers and applying the Z-stack multidimensional acquisition function. Images of Z-stack slices were obtained with the spacing set to 8 ⁇ m.
- H5 CDR sequence (SEQ ID NO: 1): DKGREYTLARAKYWYW
- E9 CDR sequence (SEQ ID NO: 2): AYGIWEPLRYRNYSYW
- G5 CDR sequence (SEQ ID NO: 4): RTGIWEPLAHRNYNYW
- H5 full-length sequence (SEQ ID NO: 5):
- E9 full-length sequence (SEQ ID NO: 6):
- G5 full sequence (SEQ ID NO: 8):
- nanobody clones were screened.
- ELISA was performed for IL-4R and MBP.
- the y-axis of the graph represents the fold-change of OD values for IL-4R and MBP, which indicates the specificity of each nanobody clone for IL-4R.
- Clones marked with red boxes are the finally selected nanobodies with fold change values higher than the threshold (1.7). Excluding overlapping sequences, the inventors finally obtained four different nanobody sequences: H5, G5, E9, and B3 ( Figure 1).
- H5 nanobody was selected as the final IL-4R blocking nanobody.
- the signal blocking activity of each nanobody was compared with dupilumab at three different concentrations. Reporter cells were incubated with 667 pM of IL-4 and nanobody (or antibody) for 24 hours. It was found that H5 nanobody exhibited similar blocking activity as dupilumab at about twice the concentration (Figure 4).
- the binding affinity of dupilumab and H5 nanobody to HEK293 cells was evaluated using a plate reader.
- Anti-human IgG-FITC was used as a secondary antibody for dupilumab
- anti-Myc-FITC was used as a secondary antibody for H5 nanobody.
- the fluorescence units of each antibody (nanobody) were normalized to the fluorescence units of each secondary antibody. The results in Figures 4 and 5 suggest that the signal blocking activity depends on the binding of the antibody (nanobody) to the cell.
- FOXJ1 is one of the forkhead box family transcription factors, and its expression is suppressed by IL-4/IL-13 signaling and STAT6 is phosphorylated.
- Figure 6A is an ortho image of human nasal epithelial cells (HNE) treated with IL-4, dupilumab and H5 nanobody. Administration of dupilumab and H5 was shown to block IL-4 signaling and subsequently induce the expression of FOXJ1.
- Figure 6c shows that FOXJ1 expression is induced when dupilumab and H5 are treated without IL-4, which means that the antibody and nanobody do not have any agonistic activity against IL-4R.
- FIGS. 6b and 6c are 3D images of the corresponding HNE samples.
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Abstract
The present invention relates to an antibody against an IL-4 receptor or antigen-binding fragment thereof, specifically a nanobody; and a pharmaceutical composition for preventing or treating inflammatory or autoimmune diseases containing same as an active ingredient. The nanobody of the present invention exhibits a significantly higher IL-4 receptor binding affinity than commercially available antibody therapeutics, while the simple structure and low molecular weight thereof result in excellent structural stability, productivity, and permeability, and can be formulated in a variety of forms, including a nasal spray, without limitation. Accordingly, the nanobody of the present invention not only effectively blocks IL-4 signaling, which causes inflammatory diseases such as sinusitis, but also concentrates pharmacological effects specifically on a lesion without systemic immunosuppression by means of intravenous administration, such that the nanobody of the present invention can be used more safely for long-term administration for chronic inflammatory diseases.
Description
본 발명은 IL-4 수용체에 특이적으로 결합하는 나노바디 및 이를 약리성분으로 이용하여 다양한 IL-4 매개-염증성 질환을 예방 또는 치료하는 방법에 관한 것이다.The present invention relates to a nanobody that specifically binds to the IL-4 receptor and a method of preventing or treating various IL-4-mediated inflammatory diseases using the nanobody as a pharmacological ingredient.
만성 부비동염(Chronic Rhinosinusitis, CRS)은 비강(sinonasal) 점막에 발생하는 가장 흔한 만성 염증성 질환 중 하나로서, 세계적으로 높은 감염률을 나타내고, 환자의 삶의 질 저하를 유발함으로서 사회경제적인 부담을 초래하는 질병이다. 만성 비부비동염(CRS)은 일반적으로 두 가지 특수 형태인 비강 용종이 없는 CRS(CRSsNP)와 비강 용종이 있는 CRS(CRSwNP)로 분류되는데, 비록 CRSsNP가 우세한 형태이나, CRSwNP 또한 모든 CRS 사례의 약 20% 내지 33%를 차지하여 중요한 질환 형태에 해당한다. CRSwNP는 종종 천식, 진균성 비부비동염 및 아스피린 과민성 호흡기 질환을 동반하며 표준 의료 요법 및 내시경 부비동 수술 후에 치료 결과가 좋지 않다.Chronic rhinosinusitis (CRS) is one of the most common chronic inflammatory diseases that occurs in the nasal mucosa. It has a high infection rate worldwide and causes a socioeconomic burden by reducing the quality of life of patients. am. Chronic rhinosinusitis (CRS) is generally divided into two special forms: CRS without nasal polyps (CRSsNP) and CRS with nasal polyps (CRSwNP). Although CRSsNP is the predominant form, CRSwNP also accounts for approximately 20 of all CRS cases. It constitutes an important form of the disease, accounting for % to 33%. CRSwNP is often accompanied by asthma, fungal rhinosinusitis, and aspirin hypersensitivity respiratory diseases, with poor treatment outcomes after standard medical therapy and endoscopic sinus surgery.
인터류킨(IL)-4R-α에 대한 단일클론항체(mAb)인 두필루맙(Dupilumab)의 경우, 기존에 중등도 내지 중증의 호산구성 천식에서 치료 효과와 안정성을 보였는데, 최근 임상시험에서 만성 비부비동염(CRSwNP)에서 치료 효과 또한 확인되었다. 다만, 두필루맙은 현재 주사제의 형태로 투여되어 안검염(conjunctivitis), 각막염(keratitis) 및 구강 헤르페스(oral herpes) 등 전신적인 부작용을 수반하는 단점을 가진다.Dupilumab, a monoclonal antibody (mAb) against interleukin (IL)-4R-α, has previously shown therapeutic efficacy and safety in moderate to severe eosinophilic asthma, but recent clinical trials have shown chronic rhinosinusitis. The treatment effect was also confirmed in (CRSwNP). However, dupilumab is currently administered in the form of an injection, which has the disadvantage of causing systemic side effects such as blepharitis, keratitis, and oral herpes.
한편, 특정 표적 항원과의 결합이 가능한 항체만을 대량생산할 수 있는 혼성세포기술(hybridoma technology) 및 이를 이용한 단일클론항체(monoclonal antibody) 제조 방법이 1970년대에 개발된 이후 항체 의약품 분야는 급성장하였으나, 일반적인 전장(full length) 항체는 큰 분자량으로 인해 막 투과가 어려워 세포 표면 항원 또는 수용성 항원들을 표적으로 한 치료제로 활용이 제한되기 쉽고, 보다 진보된 형태의 항체 제형인 항체 단편(single-chain variable fragment, scFv 등) 역시 여전히 낮은 안정성으로 상용화에 난점이 있다. Meanwhile, the field of antibody pharmaceuticals has grown rapidly since hybridoma technology, which can mass-produce only antibodies capable of binding to specific target antigens, and the monoclonal antibody manufacturing method using it were developed in the 1970s, but general Full-length antibodies have difficulty penetrating membranes due to their large molecular weight, which limits their use as therapeutic agents targeting cell surface antigens or water-soluble antigens, and single-chain variable fragments, which are a more advanced form of antibody formulation, scFv, etc.) are still difficult to commercialize due to low stability.
이러한 상황에서, 최근 치료제 및 진단영역에서 활발히 개발되고 있는 나노바디(Nanobody)는 낙타과 동물(낙타, 라마, 알파카 등)에서 발견되는 중쇄 전용 항체의 항원인식 가변부(variable region)로서, 작은 크기와 높은 안정성에 기반하여 기존 항체 치료제들의 문제점을 해결할 수 있는 대안책으로 주목받고 있다. 대부분의 항체는 항원과 상호작용하는 CDRs(Complementarity Determining Regions) 영역이 편평하거나 약간의 홈이 파져 있는 형태를 가지고 있어서, 활성부위(active site)가 오목한 형태를 가지는 효소 등의 단백질에 대하여 결합 효율이 낮은 반면, CDR 영역이 볼록한 형태를 가지는 나노바디(Nanobody)는 결합 효율이 보다 높다. 아울러 나노바디의 경우 CDR-H3 부위가 평균 19개의 아미노산으로 이루어져, 인간의 CDR-H3이 평균 12개의 아미노산으로 이루어진 것과 비교하여 더 긴 CDR 부위를 가짐으로서 볼록한 루프의 형성이 가능하고 일반적인 항체에 비해 구조가 단순하며 크기가 작아 구조적 안정성이 높다는 장점이 있다. In this situation, nanobodies, which have recently been actively developed in the therapeutic and diagnostic fields, are antigen recognition variable regions of heavy chain-specific antibodies found in camelid animals (camels, llamas, alpacas, etc.), and are small in size and Based on its high stability, it is attracting attention as an alternative solution that can solve the problems of existing antibody treatments. Most antibodies have flat or slightly grooved CDRs (Complementarity Determining Regions) regions that interact with antigens, so their binding efficiency is low for proteins such as enzymes whose active sites have a concave shape. On the other hand, nanobodies with a convex CDR region have higher binding efficiency. In addition, in the case of nanobodies, the CDR-H3 region consists of an average of 19 amino acids, and compared to the average human CDR-H3 of 12 amino acids, it has a longer CDR region, enabling the formation of a convex loop and compared to a typical antibody. It has a simple structure and small size, which has the advantage of high structural stability.
이에, 본 발명자들은 기존의 두필루맙과 비교하여 동등 이상의 IL-4 수용체 억제 효과를 보이면서도 높은 안정성을 가지고 작은 분자량으로 인해 다양한 투여 루트에 적용될 수 있는 새로운 형태의 IL-4R 억제용 항체를 개발하고자 하였다.Accordingly, the present inventors sought to develop a new type of antibody for IL-4R inhibition that exhibits an equivalent or greater IL-4 receptor inhibitory effect compared to the existing dupilumab, has high stability, and can be applied to various administration routes due to its small molecular weight. did.
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.Numerous papers and patent documents are referenced and citations are indicated throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to more clearly explain the content of the present invention and the level of technical field to which the present invention pertains.
본 발명자들은 다양한 염증성 질환의 매개자이자 치료 타겟으로 활발하게 연구 중인 IL-4 수용체를 효율적으로 억제할 수 있는 우수한 항체 기반 치료 조성물을 개발하기 위하여 예의 연구 노력하였다. 그 결과, 서열번호 1 내지 4의 CDR 서열을 항원 인식부위로 이용할 경우 상용화된 항체 치료제인 두필루맙(DUPIXENT®) 보다도 IL-4 수용체에 대한 높은 결합 친화도를 가지면서 구조가 단순하고 분자량이 낮아 구조적 안정성, 생산성 및 투과성이 현저히 개선된 항체 단편, 구체적으로는 나노바디(Nanobody)로 이용될 수 있음을 발견함으로써, 본 발명을 완성하게 되었다.The present inventors have made extensive research efforts to develop an excellent antibody-based therapeutic composition that can efficiently inhibit the IL-4 receptor, which is being actively studied as a mediator and treatment target for various inflammatory diseases. As a result, when the CDR sequences of SEQ ID NOs. 1 to 4 are used as the antigen recognition site, it has a higher binding affinity to the IL-4 receptor than the commercialized antibody treatment dupilumab (DUPIXENT ® ), while having a simple structure and low molecular weight. The present invention was completed by discovering that an antibody fragment with significantly improved structural stability, productivity and permeability could be used as a nanobody, specifically a nanobody.
따라서 본 발명의 목적은 IL-4 수용체(IL-4R)에 대한 항체 또는 이의 항원 결합단편과 이를 인코딩하는 핵산 분자를 제공하는 데 있다.Therefore, the purpose of the present invention is to provide an antibody or antigen-binding fragment thereof against the IL-4 receptor (IL-4R) and a nucleic acid molecule encoding the same.
본 발명의 다른 목적은 염증 질환 또는 자가 면역 질환의 예방 또는 치료용 약학적 조성물을 제공하는 데 있다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory diseases or autoimmune diseases.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.
본 발명의 일 양태에 따르면, 본 발명은 서열번호 1, 2, 3 및 4로 구성된 군으로부터 선택되는 하나 이상의 아미노산 서열을 포함하는 항-IL-4R 항체 또는 이의 항원 결합단편을 제공한다.According to one aspect of the present invention, the present invention provides an anti-IL-4R antibody or antigen-binding fragment thereof comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4.
본 발명자들은 다양한 염증성 질환의 매개자이자 치료 타겟으로 활발하게 연구 중인 IL-4 수용체를 효율적으로 억제할 수 있는 우수한 항체 기반 치료 조성물을 개발하기 위하여 예의 연구 노력하였다. 그 결과, 서열번호 1 내지 4의 CDR 서열을 항원 인식부위로 이용할 경우 상용화된 항체 치료제인 두필루맙(DUPIXENT®) 보다도 IL-4 수용체에 대한 높은 결합 친화도를 가지면서 구조가 단순하고 분자량이 낮아 구조적 안정성, 생산성 및 투과성이 현저히 개선된 항체 단편으로 이용될 수 있음을 발견하였다. 특히 본 발명의 항체 단편은 예를 들어 비강 점막을 통하여 병변부에 직접적으로 전달되는 나노바디(Nanobody) 형태로 적용됨으로써 전신적인 면역 억제 없이도 비강에서 발병하는 다양한 염증성 질환에 대한 효율적인 국소 치료제로 유용하게 이용될 수 있다. The present inventors have made extensive research efforts to develop an excellent antibody-based therapeutic composition that can efficiently inhibit the IL-4 receptor, which is being actively studied as a mediator and treatment target for various inflammatory diseases. As a result, when the CDR sequences of SEQ ID NOs. 1 to 4 are used as the antigen recognition site, it has a higher binding affinity to the IL-4 receptor than the commercialized antibody treatment dupilumab (DUPIXENT ® ), while having a simple structure and low molecular weight. It was discovered that it could be used as an antibody fragment with significantly improved structural stability, productivity, and permeability. In particular, the antibody fragment of the present invention is applied in the form of a nanobody that is delivered directly to the lesion area through, for example, the nasal mucosa, making it useful as an efficient local treatment for various inflammatory diseases that occur in the nasal cavity without systemic immunosuppression. It can be used.
본 명세서에서 용어“항체(antibody)”는 IL-4-R에 대한 항체로서, 이의 특정 에피토프를 특이적으로 인식하여 결합하는 펩타이드를 의미하며, 완전한 항체 형태뿐만 아니라 항체 분자의 항원 결합 단편(항체 단편)을 포함한다.As used herein, the term “antibody” refers to an antibody against IL-4-R, a peptide that specifically recognizes and binds to a specific epitope thereof, and includes not only the complete antibody form but also an antigen-binding fragment of the antibody molecule (antibody). includes fragments).
완전한 항체는 2개의 전체 길이의 경쇄 및 2개의 전체 길이의 중쇄를 가지는 구조이며 각각의 경쇄는 중쇄와 다이설파이드 결합으로 연결되어 있다. 중쇄 불변영역은 감마(γ), 뮤(μ), 알파(α), 델타(δ) 및 엡실론(ε) 타입을 가지고 서브클래스로 감마1(γ1), 감마2(γ2), 감마3(γ3), 감마4(γ4), 알파1(α1) 및 알파2(α2)를 가진다. 경쇄의 불변영역은 카파(κ) 및 람다(λ) 타입을 가진다.A complete antibody has a structure of two full-length light chains and two full-length heavy chains, with each light chain connected to the heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and is subclassed as gamma1 (γ1), gamma2 (γ2), and gamma3 (γ3). ), gamma 4 (γ4), alpha 1 (α1), and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.
본 명세서에서 용어“항체의 항원 결합 단편”은 전체 항체 분자 내에서 유의한 항원-항체 결합 기능을 가지는 단편을 의미하며, Fab, F(ab'), F(ab')2, Fv 및 나노바디(nanobody 또는 sybody) 등을 포함한다. As used herein, the term “antigen-binding fragment of an antibody” refers to a fragment that has a significant antigen-antibody binding function within the entire antibody molecule, including Fab, F(ab'), F(ab')2, Fv, and nanobody. (nanobody or sybody) etc.
항체 단편 중 Fab는 경쇄 및 중쇄의 가변영역과 경쇄의 불변영역 및 중쇄의 첫 번째 불변영역(CH1)을 가지는 구조로 1개의 항원 결합 부위를 가진다. Fab'는 중쇄 CH1 도메인의 C-말단에 하나 이상의 시스테인 잔기를 포함하는 힌지 영역(hinge region)을 가진다는 점에서 Fab와 차이가 있다. F(ab')2 항체는 Fab'의 힌지 영역의 시스테인 잔기가 디설파이드 결합을 이루면서 생성된다. Fv는 중쇄 가변영역 및 경쇄 가변영역만을 가지고 있는 최소의 항체조각으로 Fv 단편을 생성하는 재조합 기술은 WO88/10649, WO88/106630, WO88/07085, WO88/07086 및 WO88/09344에 개시되어 있다. 이중쇄 Fv(two-chain Fv)는 비공유 결합으로 중쇄 가변영역과 경쇄 가변영역이 연결되어 있고 단쇄 Fv(single-chain Fv)는 일반적으로 펩타이드 링커를 통하여 중쇄의 가변영역과 단쇄의 가변영역이 공유결합으로 연결되거나 또는 C-말단에서 바로 연결되어 있어서 이중쇄 Fv와 같이 다이머와 같은 구조를 이룰 수 있다. 이러한 항체 단편은 단백질 가수분해 효소를 이용해서 얻을 수 있고(예를 들어, 전체 항체를 파파인으로 제한 절단하면 Fab를 얻을 수 있고 펩신으로 절단하면 F(ab')2 단편을 얻을 수 있다), 유전자 재조합 기술을 통하여 제작할 수도 있다.Among antibody fragments, Fab has a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen binding site. Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibody is produced when cysteine residues in the hinge region of Fab' form a disulfide bond. Fv is a minimal antibody fragment containing only the heavy chain variable region and the light chain variable region. Recombinant techniques for generating Fv fragments are disclosed in WO88/10649, WO88/106630, WO88/07085, WO88/07086, and WO88/09344. Double-chain Fv (two-chain Fv) is a non-covalent bond between the heavy chain variable region and light chain variable region, and single-chain Fv (single-chain Fv) is generally shared between the heavy chain variable region and the short chain variable region through a peptide linker. They can be connected by a bond or directly connected at the C-terminus to form a dimer-like structure, such as double-chain Fv. These antibody fragments can be obtained using proteolytic enzymes (for example, Fab can be obtained by restriction digestion of the entire antibody with papain, and F(ab')2 fragment can be obtained by digestion with pepsin), and gene It can also be produced through recombinant technology.
본 명세서에서 용어“중쇄”는 항원에 대한 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VH 및 3개의 불변영역 도메인 CH1, CH2 및 CH3을 포함하는 전체길이 중쇄 및 이의 단편을 모두 의미한다. As used herein, the term “heavy chain” refers to a full-length heavy chain comprising a variable region domain VH and three constant region domains CH1, CH2, and CH3, including an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen, and a full-length heavy chain thereof. It means all fragments.
본 명세서에서 용어“경쇄”는 항원에 대한 특이성을 부여하기 위한 충분한 가변영역 서열을 갖는 아미노산 서열을 포함하는 가변영역 도메인 VL 및 불변영역 도메인 CL을 포함하는 전체길이 경쇄 및 이의 단편을 모두 의미한다.As used herein, the term “light chain” refers to both a full-length light chain and fragments thereof including a variable region domain VL and a constant region domain CL containing an amino acid sequence having sufficient variable region sequence to confer specificity to an antigen.
본 명세서에서, 용어“CDR(complementarity determining region)”은 면역글로블린 중쇄 및 경쇄의 고가변 영역(hypervariable region)의 아미노산 서열을 의미한다(Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)). 중쇄(HCDR1, HCDR2 및 HCDR3) 및 경쇄(LCDR1, LCDR2 및 LCDR3)에는 각각 3개의 CDR이 포함되어 있으며, 이들 CDR은 항체가 항원 또는 에피토프에 결합하는 데 있어서 주요한 접촉 잔기를 제공한다.As used herein, the term “complementarity determining region (CDR)” refers to the amino acid sequence of the hypervariable region of immunoglobulin heavy and light chains (Kabat et al. Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)). The heavy chain (HCDR1, HCDR2, and HCDR3) and light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs, which provide key contact residues for the antibody to bind to the antigen or epitope.
본 발명의 항체 또는 항체 단편의 범위에는 CDR 영역에 보존적 아미노산 치환을 갖는 변이체가 포함된다. 또한, 본 발명의 항체 또는 항체 단편은, IL-4 수용체를 특이적으로 인식할 수 있는 범위 내에서 첨부한 서열목록에 기재된 아미노산 서열의 변이체를 포함할 수 있다. 예를 들면, 항체의 결합 친화도 및/또는 기타 생물학적 특성을 보다 더 개선시키기 위하여 항체의 아미노산 서열에 추가적인 변화를 줄 수 있다. 이러한 변형은, 예를 들어 항체의 아미노산 서열 잔기의 결실, 삽입 및/또는 치환을 포함한다. 이러한 아미노산 변이는 아미노산 곁사슬 치환체의 상대적 유사성, 예컨대, 소수성, 친수성, 전하, 크기 등에 기초하여 이루어진다. 아미노산 곁사슬 치환체의 크기, 모양 및 종류에 대한 분석에 의하여, 아르기닌, 라이신과 히스티딘은 모두 양전하를 띤 잔기이고; 알라닌, 글라이신과 세린은 유사한 크기를 갖으며; 페닐알라닌, 트립토판과 타이로신은 유사한 모양을 갖는다는 것을 알 수 있다. 따라서, 이러한 고려 사항에 기초하여, 아르기닌, 라이신과 히스티딘; 알라닌, 글라이신과 세린; 그리고 페닐알라닌, 트립토판과 타이로신은 생물학적으로 기능 균등물이라 할 수 있다.The scope of antibodies or antibody fragments of the present invention includes variants with conservative amino acid substitutions in the CDR regions. Additionally, the antibody or antibody fragment of the present invention may include variants of the amino acid sequence described in the attached sequence list within the range that can specifically recognize the IL-4 receptor. For example, additional changes can be made to the amino acid sequence of the antibody to further improve the binding affinity and/or other biological properties of the antibody. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody. These amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutions shows that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that 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 said to be biologically equivalent in function.
분자의 활성을 전체적으로 변경시키지 않는 단백질에서의 아미노산 교환은 당해 분야에 공지되어 있다(H. Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). 가장 통상적으로 일어나는 교환은 아미노산 잔기 Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu 및 Asp/Gly 간의 교환이다.Amino acid exchanges in proteins that do not overall alter the activity of the molecule are known in the art (H. Neurath, R.L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thr/Phe, Ala/ Exchanges between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly.
상술한 생물학적 균등 활성을 갖는 변이를 고려한다면, 본 발명의 항체를 구성하는 아미노산 서열은 서열목록에 기재된 서열과 실질적인 동일성(substantial identity)을 나타내는 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인 하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인 된 서열을 분석한 경우에, 최소 61%의 상동성, 일특정예에 따르면 70%의 상동성, 다른 특정예에 따르면 80%의 상동성, 또 다른 특정예에 따르면 90%의 상동성을 나타내는 서열을 의미한다. 서열비교를 위한 얼라인먼트 방법은 당업계에 공지되어 있다. 얼라인먼트에 대한 다양한 방법 및 알고리즘은 Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 및 Pearson et al. Meth. Mol. Biol. (1994) 24:307-31 등에 개시되어 있다. Considering the mutations having the above-mentioned biological equivalent activity, the amino acid sequence constituting the antibody of the present invention is interpreted to also include a sequence showing substantial identity with the sequence listed in the sequence listing. The above substantial identity is at least 61% when aligning the sequence of the present invention and any other sequence to correspond as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Homology, according to one specific example, refers to a sequence showing 70% homology, according to another specific example, 80% homology, and according to another specific example, 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are described in Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 and Pearson et al. Meth. Mol. Biol. (1994) 24:307-31, etc.
본 명세서에서 용어 “IL-4”는 활성화 되지 않은 상태의 헬퍼 T 세포(naive T cell, Th0 cell)에서 Th2 세포로의 분화를 유도하는 사이토카인(cytokine)을 지칭한다. IL-4는 활성화된 B 세포와 T 세포를 자극하거나 B 세포를 형질세포로 분화시키는 등의 다양한 생물학적 기능을 가지며, 체액성 면역과 후천성 면역의 중요한 조절자이다.As used herein, the term “IL-4” refers to a cytokine that induces differentiation from unactivated helper T cells (naïve T cells, Th0 cells) to Th2 cells. IL-4 has various biological functions, such as stimulating activated B cells and T cells or differentiating B cells into plasma cells, and is an important regulator of humoral and adaptive immunity.
본 명세서에서 용어 “IL-4R”은 IL-4의 수용체를 의미하며, 리간드인 IL-4와 결합함으로서 IgE 항체 생성과 관련된 신호전달을 유도한다. 일반적으로 IL-4의 수용체는 IL-4Rα로 알려져 있으며, 신체에서 2가지 형태의 복합체로 존재한다. 1형(Type 1) 수용체는 일반적으로 γ사슬(γc) 및 IL-4Rα의 복합체로 존재하며, IL-4에 특이적이다. 2형(Type2) 수용체는 일반적으로 IL-4Rα 및 IL-13Rα1의 복합체로 존재하며, IL-4 및 IL13 모두에 특이적이다.As used herein, the term “IL-4R” refers to the receptor for IL-4, and induces signaling related to the production of IgE antibodies by binding to its ligand, IL-4. Generally, the receptor for IL-4 is known as IL-4Rα, and it exists in two types of complexes in the body. Type 1 receptors generally exist as a complex of γ chain (γc) and IL-4Rα and are specific for IL-4. Type 2 receptors generally exist as a complex of IL-4Rα and IL-13Rα1 and are specific for both IL-4 and IL13.
본 명세서에서 용어“두필루맙”은 IL-4Rα에 대한 단일클론항체로서, 제품명 DUPIXENT®으로 상용화된 항체 치료제이다. 두필루맙의 작용기전은 1형 IL-4 수용체에서 IL-4와 IL-4Rα의 결합을 차단하거나 IL-4α과 γ사슬의 이합체화 반응(dimerization)을 차단함으로써 신호전달을 억제하고, 2형 IL-4 수용체에서는 IL-4Rα 및 IL-13Rα1의 이합체화 반응을 차단함으로써 신호전달을 억제하는 것으로 알려져 있다.As used herein, the term “dupilumab” refers to a monoclonal antibody against IL-4Rα, and is an antibody treatment product commercialized under the product name DUPIXENT ® . The mechanism of action of dupilumab is to inhibit signal transduction by blocking the binding of IL-4 and IL-4Rα at the type 1 IL-4 receptor or by blocking the dimerization reaction of IL-4α and γ chain, and type 2 IL-4 receptor. The -4 receptor is known to inhibit signal transduction by blocking the dimerization reaction of IL-4Rα and IL-13Rα1.
본 발명의 구체적인 구현예에 따르면, 본 발명의 항원 결합 단편은 F(ab')2, Fab', Fab, Fv, scFv 및 나노바디로 구성된 군으로부터 선택된다.According to a specific embodiment of the invention, the antigen-binding fragment of the invention is selected from the group consisting of F(ab')2, Fab', Fab, Fv, scFv and nanobodies.
본 발명의 항체는 단일클론 항체, 인간 항체, 인간화 항체, 키메라 항체, 단쇄 Fvs(scFV), 단쇄항체, Fab 단편, F(ab')단편, 다이설파이드-결합 Fvs(sdFV) 및 항-이디오타입(항-Id) 항체, 그리고 상기 항체들의 에피토프-결합 단편 및 나노바디(Nanobody 또는 sybody) 등을 포함하나, 이에 한정되는 것은 아니다.Antibodies of the present invention include monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, single chain Fvs (scFV), single chain antibodies, Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFV) and anti-idio Type (anti-Id) antibodies, and epitope-binding fragments and nanobodies (Nanobodies or sybodies) of the above antibodies, but are not limited thereto.
본 명세서에서 용어“단일클론 항체”는, 실질적으로 동일한 항체 집단에서 수득한 단일 분자 조성의 항체분자를 의미하며, 단일클론 항체는 특정 에피토프에 대해 단일 결합 특이성 및 친화도를 나타낸다.As used herein, the term “monoclonal antibody” refers to an antibody molecule with a single molecular composition obtained from a substantially identical antibody population, and a monoclonal antibody exhibits a single binding specificity and affinity for a specific epitope.
본 발명의 구체적인 구현예에 따르면, 상기 항원 결합 단편은 나노바디(Nanobody)이다.According to a specific embodiment of the present invention, the antigen-binding fragment is a nanobody.
본 발명의 구체적인 구현예에 따르면, 본 발명의 항-IL-4R 항체 또는 이의 항원 결합단편은 서열번호 5, 6, 7 및 8로 구성된 군으로부터 선택되는 하나의 아미노산 서열을 포함한다.According to a specific embodiment of the present invention, the anti-IL-4R antibody or antigen-binding fragment thereof of the present invention includes one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8.
본 발명에 따르면, 서열번호 5, 6, 7 및 8은 각각 서열번호 1, 2, 3 및 4의 CDR 서열을 포함하는 전장(full-length) 나노바디의 아미노산 서열이다.According to the present invention, SEQ ID NOs: 5, 6, 7 and 8 are the amino acid sequences of a full-length nanobody comprising the CDR sequences of SEQ ID NOs: 1, 2, 3 and 4, respectively.
본 명세서에서 용어 “나노바디”는 하나의 중쇄 가변영역만으로 구성된 단일 도메인의 항체 유사체를 의미하며,“단일 도메인 항체(VHH antibody)”또는“합성나노항체(sybody)”와 동일한 의미를 가지고, 상호 교환적으로 사용될 수 있다. 나노바디는 완전한 기능을 가진 가장 작은 항원 결합 단편이며, 통상적으로 경쇄 및 중쇄 불변영역1(CH1)이 자연적으로 결여된 항체를 먼저 수득한 후 항체 중쇄의 가변영역을 클로닝하여 하나의 중쇄 가변영역만으로 구성된 단일 도메인 항체(Variable Domain of Heavy-chain Antibodies, VHH)를 구축할 수 있다. 나노바디는 낙타, 라마, 알파카 등의 낙타과 동물에서 추출한 항체 조각을 인공적으로 가공하여 제조할 수 있으며, 일반 항체 크기의 10분의 1수준의 크기를 가져 온도 변화 등 외부 환경에 강하고, 투여가 용이하며, 생산수율이 높은 장점이 있다.As used herein, the term “nanobody” refers to a single domain antibody analog composed of only one heavy chain variable region, and has the same meaning as “single domain antibody (VHH antibody)” or “synthetic nanoantibody (sybody),” and has the same meaning as “synthetic nanobody (sybody).” Can be used interchangeably. Nanobodies are the smallest fully functional antigen-binding fragments, and typically antibodies that naturally lack the light and heavy chain constant region 1 (CH1) are first obtained, and then the variable region of the antibody heavy chain is cloned to produce only one heavy chain variable region. Constructed single domain antibodies (Variable Domain of Heavy-chain Antibodies, VHH) can be constructed. Nanobodies can be manufactured by artificially processing antibody fragments extracted from camelid animals such as camels, llamas, and alpacas, and are one-tenth the size of regular antibodies, making them resistant to external environments such as temperature changes and easy to administer. It has the advantage of high production yield.
본 발명의 구체적인 구현예에 따르면, 상기 나노바디는 볼록 스캐폴드(convex scaffold)구조를 가진다.According to a specific embodiment of the present invention, the nanobody has a convex scaffold structure.
본 명세서에서 용어 “볼록 스캐폴드(convex scaffold)”란, 나노바디가 가지는 볼록한 모양의 형태를 의미한다. 나노바디의 CDR-H3는 평균 19개의 아미노산으로 이루어져 평균 12개의 아미노산으로 이루어진 인간의 CDR-H3보다 길어, 볼록한 모양의 루프를 형성하여 오목한 형태의 효소나 프로테아제 등의 활성부위에 효과적으로 결합할 수 있다. 예를 들어, 항원 결합 단편 중 하나인 scFv의 경우, 평평한 선형 에피토프와 잘 결합하지만 이에 반해 나노바디의 경우 오목한 활성 부위에도 잘 결합할 수 있다. 다만, 나노바디의 경우 평평한 활성 부위에도 높은 특이도로 결합할 수 있으며, 이러한 높은 친화도는 나노바디의 유연한 스캐폴드에 기인하는 것으로 알려져 있다. 나아가, 나노바디의 경우 scFv와 비교하여 비특이적 배경 결합(nonspecific backgound binding) 정도가 낮은 장점도 가진다.As used herein, the term “convex scaffold” refers to the convex shape of a nanobody. Nanobody CDR-H3 consists of an average of 19 amino acids, which is longer than human CDR-H3, which consists of an average of 12 amino acids. It forms a convex loop and can effectively bind to the concave-shaped active site of enzymes or proteases. . For example, scFv, one of the antigen-binding fragments, binds well to flat linear epitopes, whereas nanobodies can also bind well to concave active sites. However, in the case of nanobodies, they can bind with high specificity even to flat active sites, and this high affinity is known to be due to the flexible scaffold of nanobodies. Furthermore, nanobodies also have the advantage of having a lower degree of nonspecific backgound binding compared to scFvs.
본 발명의 구체적인 구현예에 따르면, 상기 나노바디는 10 내지 18kDa의 평균 분자량을 가진다. 보다 구체적으로는 11 내지 17kDa이고, 보다 더 구체적으로는 12 내지 17kDa이며, 가장 구체적으로는 13 내지 15kDa이다. According to a specific embodiment of the present invention, the nanobody has an average molecular weight of 10 to 18 kDa. More specifically, it is 11 to 17 kDa, even more specifically it is 12 to 17 kDa, and most specifically it is 13 to 15 kDa.
본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 항체 또는 이의 항원 결합 단편을 인코딩하는 핵산 분자를 제공한다.According to another aspect of the invention, the invention provides a nucleic acid molecule encoding an antibody or antigen-binding fragment thereof of the invention.
본 명세서에서 용어“핵산 분자”는 DNA(gDNA 및 cDNA) 그리고 RNA 분자를 포괄적으로 포함하는 의미를 가지며, 핵산 분자에서 기본 구성단위인 뉴클레오타이드는 자연의 뉴클레오타이드뿐만 아니라, 당 또는 염기 부위가 변형된 유사체(analogue)도 포함한다(Scheit, Nucleotide Analogs, John Wiley, New York(1980); Uhlman er al., Chemical Reviews (1990)90:543-584). 본 발명의 중쇄, 경쇄 가변영역 및 나노바디를 코딩하는 핵산 분자의 서열은 변형될 수 있다. 상기 변형은 뉴클레오타이드의 추가, 결실, 또는 비보존적 치환 또는 보존적 치환을 포함한다.As used herein, the term “nucleic acid molecule” is meant to comprehensively include DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units in nucleic acid molecules, include not only natural nucleotides but also analogs with modified sugar or base sites. (analogue) is also included ( Scheit, Nucleotide Analogs , John Wiley, New York (1980); Uhlman et al., Chemical Reviews (1990)90:543-584). The sequences of the nucleic acid molecules encoding the heavy chain, light chain variable regions and nanobodies of the invention may be modified. The modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.
본 발명의 핵산 분자는 상기한 뉴클레오타이드 서열에 대하여 실질적인 동일성을 나타내는 뉴클레오타이드 서열도 포함하는 것으로 해석된다. 상기의 실질적인 동일성은, 상기한 본 발명의 뉴클레오타이드 서열과 임의의 다른 서열을 최대한 대응되도록 얼라인하고, 당업계에서 통상적으로 이용되는 알고리즘을 이용하여 얼라인 된 서열을 분석한 경우에, 최소 80%의 상동성, 일 특정예에서는 최소 90%의 상동성, 다른 특정예에서는 최소 95%의 상동성을 나타내는 뉴클레오타이드 서열을 의미한다.The nucleic acid molecule of the present invention is interpreted to also include a nucleotide sequence showing substantial identity to the above-mentioned nucleotide sequence. The above substantial identity is at least 80% when the nucleotide sequence of the present invention and any other sequence are aligned to correspond as much as possible, and the aligned sequence is analyzed using an algorithm commonly used in the art. Homology, in one specific example, means a nucleotide sequence showing at least 90% homology, and in another specific example, at least 95% homology.
본 발명에 따르면, 본 발명의 항체 또는 이의 항원 결합 단편은 이를 인코딩하는 핵산 분자를 숙주 세포에서 발현시킴으로서 재조합적으로 수득될 수 있다. According to the present invention, the antibody or antigen-binding fragment thereof of the present invention can be obtained recombinantly by expressing the nucleic acid molecule encoding the same in a host cell.
본 명세서에서 용어“발현시키다”는 대상 세포가 외래(exogenous) 유전자를 발현하게 하거나 또는 내인성(endogenous) 유전자의 자연적 발현량을 증가시키기 위해 유전자 전달체를 이용하여 인위적으로 이를 도입함으로써 유전자가 대상 세포 내에서 염색체외 인자로서 또는 염색체 통합 완성에 의해 복제 가능하게 되는 것을 의미한다. 따라서, 용어“발현”은“형질전환(transformation)”, "형질감염(transfection)" 또는 "형질도입(transduction)"과 동일한 의미이다. 보다 구체적으로는, 본 발명에서“발현시키다”는 대상 세포가 외래 유전자를 인위적으로 발현하도록 하는 것을 의미한다. As used herein, the term “express” means causing a target cell to express an exogenous gene or artificially introducing it using a gene delivery system to increase the natural expression level of an endogenous gene, thereby allowing the gene to enter the target cell. This means that replication becomes possible as an extrachromosomal factor or through completion of chromosomal integration. Accordingly, the term “expression” has the same meaning as “transformation,” “transfection,” or “transduction.” More specifically, in the present invention, “express” means causing a target cell to artificially express a foreign gene.
본 명세서에서, 용어“유전자 전달체”또는“유전자 전달 시스템(gene delivery system)”은 유전자를 세포 내로 운반하는 모든 수단을 의미하며, 유전자 전달은 유전자의 세포내 침투(transduction)와 동일한 의미를 가진다. 조직 또는 세포 수준에서, 유전자 전달은 유전자의 확산(spread)과 동일한 의미를 가진다. 따라서, 본 발명의 유전자 전달 시스템은 유전자 침투 시스템 및 유전자 확산 시스템으로 기재될 수 있다.As used herein, the term “gene carrier” or “gene delivery system” refers to any means for transporting a gene into a cell, and gene delivery has the same meaning as transduction of a gene into a cell. At the tissue or cellular level, gene transfer is synonymous with gene spread. Accordingly, the gene delivery system of the present invention can be described as a gene penetration system and a gene diffusion system.
본 발명의 유전자 전달체을 제조하기 위해, 본 발명의 뉴클레오타이드 서열은 적합한 발현 컨스트럭트(expression construct) 내에 존재하며, 상기 발현 컨스트럭트 내에서 본 발명의 뉴클레오타이드 서열은 발현조절서열에 작동적으로 연결될 수 있다. 본 명세서에서, 용어“작동적으로 결합된”은 프로모터, 시그널 서열, 또는 전사조절인자 결합 위치의 어레이와 같은 핵산 발현 조절서열과 다른 핵산 서열사이의 기능적인 결합을 의미하며, 이에 의해 상기 발현조절서열은 상기 다른 핵산 서열의 전사 및/또는 해독을 조절하게 된다. To prepare the gene carrier of the present invention, the nucleotide sequence of the present invention is present in a suitable expression construct, wherein the nucleotide sequence of the present invention can be operably linked to an expression control sequence. there is. As used herein, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence, such as a promoter, a signal sequence, or an array of transcriptional regulator binding sites, and another nucleic acid sequence, thereby regulating said expression. The sequence will regulate transcription and/or translation of the other nucleic acid sequences.
본 발명의 유전자 전달체는 다양한 형태로 제작할 수 있는 데, 이는 (1) 내이키드(naked) 재조합 DNA 분자, (2) 플라스미드, (3) 바이러스 벡터, 그리고, (4) 상기 내이키드 재조합 DNA 분자 또는 플라스미드를 내포하는 리포좀 또는 니오좀의 형태로 제작할 수 있다.The gene delivery system of the present invention can be manufactured in various forms, including (1) a naked recombinant DNA molecule, (2) a plasmid, (3) a viral vector, and (4) the naked recombinant DNA molecule or It can be produced in the form of a liposome or niosome containing a plasmid.
본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 항체 또는 이의 항원 결합 단편; 또는 이를 인코딩하는 핵산분자를 유효성분으로 포함하는 염증 또는 자가 면역 질환의 예방 또는 치료용 약학적 조성물을 제공한다.According to another aspect of the present invention, the present invention provides an antibody or antigen-binding fragment thereof of the present invention; Alternatively, a pharmaceutical composition for preventing or treating inflammatory or autoimmune diseases comprising a nucleic acid molecule encoding the same as an active ingredient is provided.
본 명세서에서 용어“예방”은 질환 또는 질병을 보유하고 있다고 진단된 적은 없으나, 이러한 질환 또는 질병에 걸릴 가능성이 있는 대상체에서 질환 또는 질병의 발생을 억제하는 것을 의미한다.As used herein, the term “prevention” refers to suppressing the occurrence of a disease or disease in a subject who has not been diagnosed as having the disease or disease but is likely to develop the disease or disease.
본 명세서에서 용어“치료”는 (a) 질환, 질병 또는 증상의 발전의 억제; (b) 질환, 질병 또는 증상의 경감; 또는 (c) 질환, 질병 또는 증상을 제거하는 것을 의미한다. 본 발명의 조성물을 대상체에 투여하면 IL-4 매개 신호전달이 차단되면서 염증 질환 또는 자가 면역 질환에 의한 증상의 발전을 억제하거나, 이를 제거하거나 또는 경감시키는 역할을 한다. 따라서, 본 발명의 조성물은 그 자체로 이들 질환 치료의 조성물이 될 수도 있고, 혹은 다른 약리성분과 함께 투여되어 상기 질환에 대한 치료 보조제로 적용될 수도 있다. 이에, 본 명세서에서 용어“치료”또는“치료제”는“치료 보조”또는“치료 보조제”의 의미를 포함한다.As used herein, the term “treatment” refers to (a) inhibiting the development of a disease, condition or symptom; (b) alleviation of a disease, condition or symptom; or (c) means eliminating a disease, condition or symptom. When the composition of the present invention is administered to a subject, IL-4-mediated signaling is blocked, thereby suppressing, eliminating, or alleviating the development of symptoms caused by inflammatory or autoimmune diseases. Accordingly, the composition of the present invention may itself be a composition for treating these diseases, or may be administered together with other pharmacological ingredients and applied as a treatment adjuvant for these diseases. Accordingly, in this specification, the term “treatment” or “therapeutic agent” includes the meaning of “therapeutic aid” or “therapeutic aid.”
본 명세서에서 용어“염증성 질환”은 염증 반응을 주 병인으로 하는 질병을 총칭하는 의미이다.As used herein, the term “inflammatory disease” refers to a general term for diseases whose main etiology is inflammatory reactions.
본 명세서에서 용어“자가면역질환”이란, 과도하거나 원치 않는 면역반응을 병인으로 하는 모든 질환을 총칭하는 의미로서, 구체적으로는 자기관용의 유도 또는 계속적 유지가 정상적으로 이루어지지 않아 자기항원에 대한 면역반응이 일어나고, 이로 인해 자신의 조직이 손상받는 과정에 의해 발생되는 질환을 의미한다.As used herein, the term “autoimmune disease” refers to all diseases whose etiology is excessive or unwanted immune response. Specifically, the induction or continuous maintenance of self-tolerance is not normally achieved, resulting in an immune response to self-antigens. This refers to a disease that is caused by a process in which one's own tissues are damaged.
본 발명의 구체적인 구현예에 따르면, 본 발명의 조성물로 예방 또는 치료될 수 있는 염증 또는 자가 면역 질환은 비염, 결막염, 치주염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 치질, 통풍, 강직성 척추염, 류마티스 열, 류마티스 관절염, 류마티스성 다발성 근육통, 루프스, 섬유근통 (fibromyalgia), 건선성 관절염, 골관절염, 견관절 주위염, 건염, 건초염, 건주위염, 근육염, 다발성 근육염, 피부 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증, 염증성 장질환, 천식, 제1형 당뇨병, 건선, 습진, 피부경화증, 백반증, 말초성 신경염, 포도막염, 자가면역 혈구감소증, 자가면역 심근염, 아토피피부염, 일차성간경변, 안구건조증, 굿파이처 증후군, 자가면역 뇌수막염, 애디슨병, 자가면역성 이하선염, 이영양성 수포성 표피박리증, 부고환염, 사구체 신염, 그레이브스병, 셀리악병, 길랑바레 증후군, 하시모토병, 용혈성 빈혈, 중증 근무력증, 근위축성측색경화증, 심상천포창, 유육종증, 척추관절증, 갑상선염, 혈관염, 점액수종, 악성빈혈, 항인지질증후군, 고형장기 이식 후기 및 만성 거부증, 및 이식편대숙주질환으로 구성된 군으로부터 선택된다.According to a specific embodiment of the present invention, inflammatory or autoimmune diseases that can be prevented or treated with the composition of the present invention include rhinitis, conjunctivitis, periodontitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, and hemorrhoids. , gout, ankylosing spondylitis, rheumatic fever, rheumatoid arthritis, polymyalgia rheumatica, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, periarthritis, tendonitis, tenosynovitis, peritendinitis, myositis, polymyositis, dermatomyositis, hepatitis, Cystitis, nephritis, Sjogren's syndrome, multiple sclerosis, inflammatory bowel disease, asthma, type 1 diabetes, psoriasis, eczema, scleroderma, vitiligo, peripheral neuritis, uveitis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis. , primary cirrhosis, dry eye, Goodfeitzer syndrome, autoimmune meningitis, Addison's disease, autoimmune parotitis, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic Selected from the group consisting of anemia, myasthenia gravis, amyotrophic lateral sclerosis, pemphigus vulgaris, sarcoidosis, spondyloarthrosis, thyroiditis, vasculitis, myxedema, pernicious anemia, antiphospholipid syndrome, late and chronic rejection of solid organ transplantation, and graft-versus-host disease. do.
본 발명의 구체적인 구현예에 따르면, 상기 비염은 비용종(nasal polyps) 또는 부비동염(Sinusitis)이다.According to a specific embodiment of the present invention, the rhinitis is nasal polyps or sinusitis.
본 명세서에서 용어“비용종”이란, 일반적으로 중비도(중간 콧길)에서 유래된 포도송이 모양의 양성 부종성 점막이 비강 내로 돌출된 병적 상태를 의미한다.As used herein, the term “nasal polyp” generally refers to a pathological condition in which benign edematous mucosa in the shape of a bunch of grapes, originating from the middle meatus (middle nasal passage), protrudes into the nasal cavity.
본 명세서에서 용어“부비동염”이란, 코 주위의 얼굴 뼈 속에 있는 부비동이라는 자연공이 막혀서 부비동이 제대로 환기 및 배설되지 않아 이차적으로 부비동에 염증이 발생하고, 농성 분비물이 고이면서 염증이 심해지는 질환을 의미한다.As used herein, the term “sinusitis” refers to a disease in which the natural pores called paranasal sinuses in the facial bones around the nose are blocked and the paranasal sinuses are not properly ventilated and excreted, causing secondary inflammation and worsening inflammation as purulent secretions accumulate. do.
본 발명의 구체적인 구현예에 따르면, 본 발명의 부비동염은 만성 부비동염(Chronic Sinusitis, CRS)이다.According to a specific embodiment of the present invention, the sinusitis of the present invention is chronic sinusitis (CRS).
본 명세서에서 용어“만성 부비동염”이란, 일반적으로 부비동염의 증상이 약 3개월 이상 장기적으로 지속되는 경우를 의미하나, 이에 국한되지 않고 해당 질환의 보통의 회복기간을 넘어 지속되는 모든 경우를 포함한다.As used herein, the term “chronic sinusitis” generally refers to cases where symptoms of sinusitis persist for a long period of time, approximately 3 months or more, but is not limited thereto and includes all cases that persist beyond the normal recovery period of the disease.
본 발명의 구체적인 구현예에 따르면, 본 발명의 만성 부비동염은 비용종을 동반한 만성 부비동염(CRS with nasal polyp, CRSwNP)이다.According to a specific embodiment of the present invention, the chronic sinusitis of the present invention is chronic sinusitis with nasal polyp (CRSwNP).
본 명세서에서 용어“비용종을 동반한 만성 부비동염(CRS with nasal polyp, CRSwNP)”란, 만성적인 부비동염과 함께 비용종(비강용종)이 나타나는 질환을 의미하며, 이와 비교하여 비용종이 동반되지 않는 경우는 “비용종을 동반하지 않는 만성 부비동염(CRS without nasal polyp, CRSsNP)”라고 지칭된다.In this specification, the term “chronic sinusitis with nasal polyp (CRSwNP)” refers to a disease in which nasal polyps (nasal polyps) appear along with chronic sinusitis, compared to cases where nasal polyps are not accompanied. It is referred to as “chronic sinusitis without nasal polyp (CRSsNP).”
본 발명의 구체적인 구현예에 따르면, 본 발명의 약학적 조성물은 비강 투여용이다.According to a specific embodiment of the present invention, the pharmaceutical composition of the present invention is for nasal administration.
본 명세서에서 용어“비강 투여”란, 비강 또는 비강의 점막을 통하여 약물을 투여하는 방식을 의미하며, 전술한 투여 방식은 비침습적(non-invasive)인 경로에 의한 것일 수 있다.As used herein, the term “nasal administration” refers to a method of administering a drug through the nasal cavity or nasal mucosa, and the above-described administration method may be a non-invasive route.
본 발명의 또 다른 양태에 따르면, 본 발명은 본 발명의 항체 또는 이의 항원 결합 단편; 또는 이를 인코딩하는 핵산분자를 유효성분으로 포함하는 약학적 조성물을 대상체에 투여하는 단계를 포함하는 염증 또는 자가 면역 질환의 예방 또는 치료 방법을 제공한다.According to another aspect of the present invention, the present invention provides an antibody or antigen-binding fragment thereof of the present invention; Alternatively, a method for preventing or treating inflammatory or autoimmune diseases is provided, comprising administering to a subject a pharmaceutical composition containing a nucleic acid molecule encoding the same as an active ingredient.
본 발명은 본 발명의 항체 또는 이의 항원 결합 단편; 또는 이를 인코딩하는 핵산분자를 유효성분으로 포함하는 약학적 조성물의 투여 경로 및 상기 약학적 조성물을 이용하여 예방 또는 치료할 수 있는 염증 또는 자가 면역 질환에 대해서는 이미 상술하였으므로, 과도한 중복을 피하기 위하여 이를 생략한다.The present invention relates to an antibody or antigen-binding fragment thereof of the present invention; Since the route of administration of a pharmaceutical composition containing a nucleic acid molecule encoding the same as an active ingredient and the inflammatory or autoimmune diseases that can be prevented or treated using the pharmaceutical composition have already been described in detail, this is omitted to avoid excessive duplication. .
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 IL-4 수용체에 대한 항체 또는 이의 항원 결합단편, 구체적으로는 나노바디; 및 이를 유효성분으로 포함하는 염증 또는 자가 면역 질환의 예방 또는 치료용 약학적 조성물을 제공한다.(a) The present invention relates to an antibody or antigen-binding fragment thereof against the IL-4 receptor, specifically a nanobody; and a pharmaceutical composition for preventing or treating inflammatory or autoimmune diseases containing the same as an active ingredient.
(b) 본 발명의 나노바디는 상용화된 항체 치료제보다 현저히 우수한 IL-4 수용체 결합 친화도를 보이면서도 단순한 구조와 낮은 분자량으로 인해 구조적 안정성, 생산성 및 투과성이 우수할 뿐 아니라, 비강 분무 등의 다양한 형태로 제한 없이 제형화될 수 있다.(b) The nanobody of the present invention exhibits significantly better IL-4 receptor binding affinity than commercialized antibody therapeutics, but also has excellent structural stability, productivity, and permeability due to its simple structure and low molecular weight, and can be used in various ways, such as nasal spray. It can be formulated without limitation in form.
(c) 본 발명의 나노바디는 부비동염 등의 염증 질환의 원인이 되는 IL-4 신호전달을 효과적으로 차단할 뿐 아니라, 정맥 투여에 의한 전신적인 면역 저하 없이 병변 특이적으로 약리효과를 집중시킴으로서 만성 염증 질환에 대한 장기 투여에 보다 안전하게 이용될 수 있다.(c) The nanobody of the present invention not only effectively blocks IL-4 signaling, which causes inflammatory diseases such as sinusitis, but also concentrates the pharmacological effect specifically on the lesion without lowering systemic immunity due to intravenous administration, thereby causing chronic inflammatory diseases. It can be used more safely for long-term administration.
도 1은 항-IL-4R 나노바디 스크리닝의 최종적인 프로세스에 관한 것으로, Y축은 IL-4R과 MBP에 대한 OD값의 배수 변화(fold-change)를 나타내며, 이는 각 나노바디 클론의 IL-4R 특이성을 의미하고, 붉은색 상자로 표시된 클론은 임계값(1.7)보다 폴드 변경 값이 더 높은 최종적으로 선택된 나노바디를 나타낸다.Figure 1 shows the final process of anti-IL-4R nanobody screening, where the Y axis represents the fold-change of OD values for IL-4R and MBP, which represents the IL-4R of each nanobody clone. Specificity is indicated, and clones boxed in red represent the finally selected nanobodies with a fold change value higher than the threshold (1.7).
도 2는 4개의 최종 나노바디 후보에 대하여, IL-4R 분자에 대한 결합 친화도를 확인하기 위해 각각의 나노바디를 정제하고, 동일한 농도의 나노바디를 ELISA를 통해 조사한 결과를 도시한 것으로, H5 나노바디가 동일한 농도의 4가지 후보 중 IL-4R 분자에 대한 결합 친화도가 가장 높은 것으로 조사되었다.Figure 2 shows the results of purifying each nanobody of the four final nanobody candidates to confirm their binding affinity to the IL-4R molecule, and examining nanobodies at the same concentration through ELISA, H5 It was found that the nanobody had the highest binding affinity to the IL-4R molecule among the four candidates at the same concentration.
도 3은 3개의 나노바디 후보를 ELISA 결과에 따라 선택하여, 결합 친화도가 IL-4 신호 차단 기능과 관련이 있는지 조사한 결과를 도시한 그림으로, 각 나노바디의 신호 차단 정도를 3가지 다른 농도에서 두필루맙의 신호 차단 정도와 비교하였다. 리포터 세포를 83pM의 IL-4 및 각 나노바디(또는 항체)와 함께 24시간 동안 인큐베이션하였으며, 그 결과 두필루맙과 H5 나노바디가 후보 중 가장 효과적인 IL-4 신호 차단 효과를 가짐을 알 수 있었다.Figure 3 shows the results of selecting three nanobody candidates according to ELISA results and investigating whether the binding affinity is related to the IL-4 signal blocking function. The degree of signal blocking of each nanobody was measured at three different concentrations. was compared with the degree of signal blocking of dupilumab. Reporter cells were incubated with 83pM of IL-4 and each nanobody (or antibody) for 24 hours, and the results showed that dupilumab and H5 nanobody had the most effective IL-4 signal blocking effect among the candidates.
도 4는 최종 IL-4R 차단 나노바디로 선택된 H5 나노바디의 신호 차단 효과를 3가지 다른 농도에서 두필루맙의 신호 차단과 비교한 결과를 도시한 그림이다.Figure 4 shows the results of comparing the signal blocking effect of the H5 nanobody selected as the final IL-4R blocking nanobody with that of dupilumab at three different concentrations.
도 5는 두필루맙과 H5 나노바디의 HEK293 세포에 대한 결합 친화도를 플레이트 판독기를 통해 평가한 결과를 도시한 그림이다.Figure 5 is a diagram showing the results of evaluating the binding affinity of dupilumab and H5 nanobody to HEK293 cells using a plate reader.
도 6a 내지 도6d는 IL-4, 두필루맙 및 나노바디에 의한 FOXJ1 발현의 조절을 면역형광 염색 및 이미지 분석을 수행한 결과를 도시한 그림이다.Figures 6a to 6d show the results of immunofluorescence staining and image analysis on the regulation of FOXJ1 expression by IL-4, dupilumab, and nanobodies.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .
실시예Example
실험방법 및 분석방법Experimental methods and analysis methods
세포cell
HEK293T 및 HEK293 세포주는 한국 세포주 은행에서 구입하였다. 이러한 세포들을 10% FBS(Gibco, 26140-079) 및 페니실린/스트렙토마이신(Gibco, 15140-122)이 보충된 DMEM 고 포도당 배지(Gibco, 11995-065)에서 37℃, 5% CO2으로 배양하였다.HEK293T and HEK293 cell lines were purchased from the Korean Cell Line Bank. These cells were cultured in DMEM high glucose medium (Gibco, 11995-065) supplemented with 10% FBS (Gibco, 26140-079) and penicillin/streptomycin (Gibco, 15140-122) at 37°C with 5% CO 2 .
사용된 프라이머의 서열Sequence of primers used
프라이머 정보는 합성 나노바디 생성에 관한 논문에서 참고하였다(Zimmermann et al., 2018)1.Primer information was referenced from a paper on the production of synthetic nanobodies (Zimmermann et al., 2018) 1 .
FW_c_for : CAA GTC CAG CTG GTG GAA TCG (서열번호 9)FW_c_for: CAA GTC CAG CTG GTG GAA TCG (SEQ ID NO: 9)
FW_c_rev : GCC GCT AGC CGC ACA G (서열번호 10)FW_c_rev: GCC GCT AGC CGC ACA G (SEQ ID NO: 10)
Link2_c_for : ATA TAT GAA GAC CTC TGC GCG GC (서열번호 11)Link2_c_for: ATA TAT GAA GAC CTC TGC GCG GC (SEQ ID NO: 11)
Link2_c_rev : ATG CAT GGT CTC AGC AGT AAT ACA AAG CAG TAT CTT CCG G (서열번호 12)Link2_c_rev: ATG CAT GGT CTC AGC AGT AAT ACA AAG CAG TAT CTT CCG G (SEQ ID NO: 12)
CDR3_c : GAA GAC CTC TGC GCG GCA GCC 111 111 GGC 111 111 111 CCG CTG 111 111 111 111 TAT 222 TAC TGG GGT CAG GGC ACC CAA GTT ACC GTT TCT (서열번호 13)CDR3_c: GAA GAC CTC TGC GCG GCA GCC 111 111 GGC 111 111 111 CCG CTG 111 111 111 111 TAT 222 TAC TGG GGT CAG GGC ACC CAA GTT ACC GTT TCT (SEQ ID NO: 13)
5’_flank_for : CGA AAT TAA TAC GAC TCA CTA TAG GGA GAC (서열번호 14)5’_flank_for: CGA AAT TAA TAC GAC TCA CTA TAG GGA GAC (SEQ ID NO: 14)
5’_flank_rev : TAT AGC TCT TCA ACT ACC CAT GGA TAT ATC TCC (서열번호 15)5’_flank_rev: TAT AGC TCT TCA ACT ACC CAT GGA TAT ATC TCC (SEQ ID NO: 15)
3’_flank_for : TAT AGC TCT TCT GCA AAG CTT TAT ATG GCC TC (서열번호 16)3’_flank_for: TAT AGC TCT TCT GCA AAG CTT TAT ATG GCC TC (SEQ ID NO: 16)
tolAK_rev : CCG CAC ACC AGT AAG GTG TGC GGT TTC AGT TGC CGC TTT CTT TCT (서열번호 17)tolAK_rev: CCG CAC ACC AGT AAG GTG TGC GGT TTC AGT TGC CGC TTT CTT TCT (SEQ ID NO: 17)
RT primer : CTT CAG TTG CCG CTT TCT TTC TTG (서열번호 18)RT primer: CTT CAG TTG CCG CTT TCT TTC TTG (SEQ ID NO: 18)
Long_FX_for : ATA TGC TCT TCT AGT CAA GTC CAG CTG GTG GAA TCG (서열번호 19)Long_FX_for: ATA TGC TCT TCT AGT CAA GTC CAG CTG GTG GAA TCG (SEQ ID NO: 19)
Long_FX_rev : TAT AGC TCT TCA TGC AGA AAC GGT AAC TTG GGT GCC C (서열번호 20)Long_FX_rev: TAT AGC TCT TCA TGC AGA AAC GGT AAC TTG GGT GCC C (SEQ ID NO: 20)
qPCR_RD_5’_for : GGG AGA CCA CAA CGG TTT CCC (서열번호 21)qPCR_RD_5’_for: GGG AGA CCA CAA CGG TTT CCC (SEQ ID NO: 21)
qPCR_ RD_L_5_rev : GCC GCT AGC CGC ACA GCT C (서열번호 22)qPCR_ RD_L_5_rev: GCC GCT AGC CGC ACA GCT C (SEQ ID NO: 22)
무작위 볼록 스캐폴드(Convex Scaffold) DNA 라이브러리 구축Construction of Random Convex Scaffold DNA Library
Sb-convex를 포함하는 pRDV(Addgene, 132696)가 PCR을 위한 주형으로 선택하였다. FW_c_for 및 Link2_c_rev 프라이머는 CDR1 및 CDR2 영역을 포함하는 볼록 스캐폴드의 전자 영역(former region)을 증폭하는 데 사용하였다. Link2_c_for, FW_c_rev 및 CDR3_c 프라이머는 CDR3 영역의 무작위화로 볼록 스캐폴드의 후자 영역(latter region)을 증폭하는 데 사용하였다. 무작위 프라이머 CDR3_c는 10개의 서로 다른 트리뉴클레오티드 잔기를 포함하며, 그 중 9개(111)는 A, S, T, N, Y(각각 10.6%)가 풍부하고, D, E, Q, R, K, H, W(각각 5%)를 포함하며, 무극성 아미노산 F, M, V, I, L, G가 거의 없는 것을 특징으로 한다(각각 2%)1. 또 다른 트리뉴클레오타이드 잔기(222)에는 아미노산 D와 A가 없는데, 그 이유는 전술한 두 가지의 아미노산이 β-시트의 말단에서 과소 대표되기 때문이다2. BbsI 제한 부위는 전자(뼈대 부위, scaffold) 및 후자(무작위화 부위, randomized) 볼록 단편의 양 말단에 존재하는데, 이들은 제한되었고(restricted), 연결(ligated)되어 3.17×1012의 다양성을 갖는 볼록 라이브러리(convex library)를 형성하였다.pRDV (Addgene, 132696) containing Sb-convex was selected as the template for PCR. Primers FW_c_for and Link2_c_rev were used to amplify the former region of the convex scaffold containing the CDR1 and CDR2 regions. Link2_c_for, FW_c_rev and CDR3_c primers were used to amplify the latter region of the convex scaffold with randomization of the CDR3 region. Random primer CDR3_c contains 10 different trinucleotide residues, 9 (111) of which are enriched in A, S, T, N, Y (10.6% each), D, E, Q, R, K , H, and W (5% each), and is characterized by almost no nonpolar amino acids F, M, V, I, L, and G (2% each) 1 . Another trinucleotide residue (222) is missing the amino acids D and A because the two aforementioned amino acids are underrepresented at the ends of the β-sheet 2 . BbsI restriction sites are present at both ends of the former (scaffold) and latter (randomized) convex fragments, which are restricted and ligated to form a convex fragment with a diversity of 3.17 × 10 12 A library (convex library) was formed.
인-비트로 전사In-vitro transcription
CDR3 무작위 볼록 라이브러리(CD3-randomized convex library)의 5' 및 3' 영역은 모두 전사되기 전에 플랭킹되게(flanked) 하였다. 5' 측면 영역 및 3' 측면 영역은 각각 5'_flank_for와 5'_flank_rev 프라이머; 및 3'_flank_for 와 tolAK_rev 프라이머;를 사용하여 Sb-convex(Addgene, 132696)를 포함하는 플라스미드 pRDV5로부터 증폭시켰다. 측면 영역(flanking regions)은 BspQI로 제한한 다음 라이브러리의 각 끝에 연결시켰다. 측면 영역이 있는 라이브러리는 매뉴얼에 따라 T7 Ribomax™ RNA 생산 시스템(Promega, P1320)을 사용하여 전사하였다.Both 5' and 3' regions of the CDR3-randomized convex library were flanked before transcription. The 5' flanking region and the 3' flanking region are primers 5'_flank_for and 5'_flank_rev, respectively; and 3'_flank_for and tolAK_rev primers were used to amplify from plasmid pRDV5 containing Sb-convex (Addgene, 132696). Flanking regions were restricted with BspQI and then linked to each end of the library. Libraries with flanking regions were transcribed using the T7 Ribomax™ RNA Production System (Promega, P1320) according to the manual.
리보솜 디스플레이ribosome display
리보솜 디스플레이는 최소한의 노력으로 약 1012개의 서로 다른 라이브러리 구성원을 표시할 수 있는 이점을 제공할 수 있다1. 그러나 이 방법은 시약 및 불리한 RNase 활성에 관한 몇 가지 이유로 인해 널리 적용되지 않았다3. 기술적인 장애물을 극복하기 위해 체외 번역 키트(in vitro translation kit) PUREfrex2.1(GeneFrontier, PF213-0.25-EX)을 채택하였다1. 키트는 산화 글루타티온(GSSG)과 이황화 다리 이성질화 효소(DsbC)가 결여되어 있으며, 이들은 이황화 결합 폴딩(disulfide bond folding)을 형성하기 위해 추가로 지지(supported)되었다. 70ng의 RNA 라이브러리(약 1.7×1012 분자)를 입력(input)으로 사용하였으며, 실험 과정은 매뉴얼을 따랐다. 리보솜 복합체 형성을 위해 시험관 내 번역 반응을 37℃에서 1시간 동안 진행하였다. 리보솜 디스플레이 전에 Dynabeads™MyOne™StreptavidinT1 비드(Invitrogen, 65601) 12ul를 WTB-BSA 완충액(50mM Tris/acetate pH7.4, 150mM NaCl, 50mM MgAc2, BSA 보충)으로 0.5%로 두 번 세척하였다. 그런 다음 자기 비드(magnetic bead)를 WTB-BSA 버퍼로 20분 이상 차단하였다. 리보솜 복합체(ribosomal complex)를 100ul의 패닝 용액(500ug의 heparin과 1ul의 RNaseIn(Promega N2611)이 첨가된 WTB-D-BSA(0.1% tween20이 첨가된 WTB-BSA))에 첨가한 다음, 혼합물을 20,000xg에서 5분간 원심분리하였다. 상층액을 비오틴화된 IL-4R(Acro Biosystems, ILR-H82E9)과 혼합하고 얼음에서 2시간 동안 인큐베이션하였다. 자기 비드(magnetic bead)를 WTB-B-BSA로 3회 세척하고 패닝-IL-4R 혼합물을 비드에 첨가한 다음 얼음에서 1시간 동안 인큐베이션하였다. 그런 다음 전체 혼합물을 WTB-D(50mM Tris/아세테이트 pH7.4, 150mM NaCl, 50mM MgAc2, 0.1% Tween20이 보충됨)로 3회 세척하였다. 상층액은 15ul 용출량의 RNeasy 키트(Qiagen, 74004)를 사용하여 정제하였다.Ribosome display can offer the advantage of displaying approximately 10 to 12 different library members with minimal effort1 . However, this method has not been widely applied due to several reasons regarding reagents and unfavorable RNase activity 3 . To overcome technical obstacles, the in vitro translation kit PUREfrex2.1 (GeneFrontier, PF213-0.25-EX) was adopted 1 . The kit lacks oxidized glutathione (GSSG) and disulfide bridge isomerase (DsbC), which are additionally supported to form disulfide bond folding. 70ng of RNA library (approximately 1.7×10 12 molecules) was used as input, and the experimental procedure followed the manual. To form the ribosome complex, the in vitro translation reaction was performed at 37°C for 1 hour. Before ribosome display, 12ul of Dynabeads™MyOne™StreptavidinT1 beads (Invitrogen, 65601) were washed twice at 0.5% with WTB-BSA buffer (50mM Tris/acetate pH7.4, 150mM NaCl, 50mM MgAc 2 , supplemented with BSA). Then, the magnetic beads were blocked with WTB-BSA buffer for more than 20 minutes. The ribosomal complex was added to 100ul of panning solution (WTB-D-BSA (WTB-BSA with 0.1% tween20) supplemented with 500ug of heparin and 1ul of RNaseIn (Promega N2611)), and then the mixture was Centrifuged at 20,000xg for 5 minutes. The supernatant was mixed with biotinylated IL-4R (Acro Biosystems, ILR-H82E9) and incubated on ice for 2 hours. Magnetic beads were washed three times with WTB-B-BSA, and the panning-IL-4R mixture was added to the beads and incubated on ice for 1 hour. The entire mixture was then washed three times with WTB-D (50mM Tris/acetate pH7.4, 150mM NaCl, 50mM MgAc 2 , supplemented with 0.1% Tween20). The supernatant was purified using the RNeasy kit (Qiagen, 74004) with an elution volume of 15ul.
역전사reverse transcription
리보솜 디스플레이에서 용출된 RNA는 매뉴얼에 따라 총 부피 30ul의 SuperiorScript III 역전사효소(Enzynomics, RT006M)를 사용하여 역전사하였다. 생성된 cDNA를 30ul 용출량의 PCR 정제 미니 키트(Favorgen, FAGCK 001-1)를 사용하여 정제하였다. 생성된 cDNA를 30ul 용출량의 PCR 정제 미니 키트(Favorgen, FAGCK 001-1)를 사용하여 정제하였다. 1ul의 용출액을 qPCR 분석의 주형로 사용하고 29ul의 elution을 증폭에 사용하였다. 생성된 cDNA 형태의 DNA 정제는 Long_FX_for 및 Long_FX_rev 프라이머를 사용한 PCR에 의해 증폭하였다. 총 반응 부피는 100ul이었고 반응이 끝나면 두 개의 튜브로 나누었다.RNA eluted from the ribosome display was reverse transcribed using SuperiorScript III reverse transcriptase (Enzynomics, RT006M) in a total volume of 30ul according to the manual. The generated cDNA was purified using a PCR purification mini kit (Favorgen, FAGCK 001-1) with an elution volume of 30ul. The generated cDNA was purified using a PCR purification mini kit (Favorgen, FAGCK 001-1) with an elution volume of 30ul. 1ul of the eluate was used as a template for qPCR analysis, and 29ul of the elution was used for amplification. Purified DNA in the form of cDNA was amplified by PCR using Long_FX_for and Long_FX_rev primers. The total reaction volume was 100ul, and after the reaction was completed, it was divided into two tubes.
qPCRqPCR
qPCR 분석은 리보솜 디스플레이 및 파지 디스플레이 선택으로 인한 cDNA의 품질을 평가하여 선택 단계 동안 라이브러리의 농축을 모니터링하는 데 사용하였다4. 실험을 위해 AccuPower® 2X Greenstar qPCR 마스터 믹스(Bioneer, K-6251)와 함께 QuantStudio 3 Real-time PCR 기기(Applied Biosystems)를 사용하였다. PCR 프로그램 조건은 다음과 같다: 95℃, 2분 (초기 변성) / 95℃, 10초 ; 63℃, 30초(변성, 어닐링, 연장, 측정) / 용융 곡선 단계는 기계 설명서의 기본 설정을 따랐다.qPCR analysis was used to monitor the enrichment of the library during the selection step by assessing the quality of cDNA resulting from ribosome display and phage display selection4 . For the experiment, QuantStudio 3 Real-time PCR instrument (Applied Biosystems) was used with AccuPower® 2X Greenstar qPCR Master Mix (Bioneer, K-6251). PCR program conditions were as follows: 95°C, 2 minutes (initial denaturation) / 95°C, 10 seconds; 63°C, 30 s (denaturation, annealing, extension, measurement)/melting curve steps followed the default settings in the machine manual.
전기천공electroporation
FX 클로닝5을 사용하여 농축된(enriched) 나노바디(sybody) 라이브러리를 파지미드 벡터 pDXinit(Addgene, 110101)에 도입하였다. E. coli SS320(Lucigen, 60512-1) 350ul를 얼음 위에서 해동하고, 농축 라이브러리(enriched library)를 포함하는 파지미드(phagemid)의 ligation mix 50ul를 SS320이 있는 전기천공 큐벳(Biorad, 1652086)에 넣고 부드럽게 피펫팅하여 혼합하였다. 세포 혼합물은 2.4kV, 25uF 및 300Ω을 사용하는 Biorad Gene Pulser II 전기천공 시스템으로 펄스화(pulsed)하였다. 전기천공된 세포를 즉시 25ml의 SOC 배지로 옮기고 37℃, 160rpm에서 30분 동안 배양하였다. 이어서, 회수 배양물을 200ug/ml 암피실린 및 2% 글루코스가 보충된 2TY 배지 225ml로 옮기고, 37℃, 160rpm에서 밤새 인큐베이션하였다.The enriched nanobody (sybody) library was introduced into the phagemid vector pDXinit (Addgene, 110101) using FX Cloning 5 . Thaw 350ul of E. coli SS320 (Lucigen, 60512-1) on ice, and add 50ul of ligation mix of phagemid containing the enriched library to an electroporation cuvette containing SS320 (Biorad, 1652086). Mix by gently pipetting. The cell mixture was pulsed with a Biorad Gene Pulser II electroporation system using 2.4 kV, 25 uF, and 300 Ω. The electroporated cells were immediately transferred to 25 ml of SOC medium and incubated at 37°C and 160 rpm for 30 minutes. The recovered culture was then transferred to 225 ml of 2TY medium supplemented with 200 ug/ml ampicillin and 2% glucose and incubated overnight at 37°C and 160 rpm.
파지의 생산 및 정제Production and purification of phages
1ml의 전기천공 전배양물을 50ml의 2YT 배지(200ug/ml 암피실린 및 2% 포도당 보충)에 첨가한 다음 OD600 = 0.6이 될 때까지 37℃, 160rpm에서 배양하였다. 그런 다음 배양액 10ml를 M13KO7 헬퍼 파지(NEB, N0315S) 30ul와 혼합하고 37℃에서 1시간 동안 진탕하지 않고 배양하였다. 인큐베이션된 배양물을 5,000xg에서 10분간 원심분리한 후 상층액을 제거하였다. 펠렛을 50ml의 2YT 배지(200ug/ml 암피실린 및 25ug/ml 카나마이신 보충)로 재현탁하고 37℃, 160rpm에서 밤새 인큐베이션하였다. 1 ml of electroporation preculture was added to 50 ml of 2YT medium (supplemented with 200 ug/ml ampicillin and 2% glucose) and then cultured at 37°C and 160 rpm until OD 600 = 0.6. Then, 10 ml of the culture medium was mixed with 30 ul of M13KO7 helper phage (NEB, N0315S) and cultured at 37°C for 1 hour without shaking. The incubated culture was centrifuged at 5,000xg for 10 minutes and the supernatant was removed. The pellet was resuspended in 50 ml of 2YT medium (supplemented with 200 ug/ml ampicillin and 25 ug/ml kanamycin) and incubated overnight at 37°C and 160 rpm.
밤새 인큐베이션된 배양물을 6,000xg, 4℃에서 30분 동안 원심분리하였다. 40ml의 상등액을 새로운 50ml 튜브에 옮기고, 10ml의 PEG6000/NaCl을 첨가하고 튜브를 약 5회 뒤집었다. 혼합물을 얼음에서 밤새 인큐베이션한 다음, 10,000xg, 4℃에서 1시간 동안 원심분리하였다. 상층액을 제거한 후, 튜브를 PBS 40ml로 부드럽게 세척하고, 이어서 PBS 1ml로 재현탁시켰다. 재현탁된 파지를 20,000xg, 4℃에서 5분간 원심분리하고 상층액을 새 튜브에 수집하였다. 수집된 파지의 역가는 269nm 및 320nm에서 UV 가시광선 분광기를 이용하여 측정한 후 다음 식에 의해 계산하였다: Cultures incubated overnight were centrifuged at 6,000xg, 4°C for 30 minutes. 40ml of supernatant was transferred to a new 50ml tube, 10ml of PEG6000/NaCl was added, and the tube was inverted approximately 5 times. The mixture was incubated on ice overnight and then centrifuged at 10,000xg, 4°C for 1 hour. After removing the supernatant, the tube was gently washed with 40 ml of PBS and then resuspended in 1 ml of PBS. The resuspended phage was centrifuged at 20,000xg, 4°C for 5 minutes, and the supernatant was collected in a new tube. The titer of the collected phages was measured using UV-visible spectroscopy at 269 nm and 320 nm and then calculated by the following equation:
파지 디스플레이Phage display
E. coli SS320을 50ml의 2YT 배지(10ug/ml 테트라사이클린 보충)에서 배양하고 파지 디스플레이 하루 전에 4℃에서 96웰 플레이트의 절반을 100ul의 67nM 뉴트라비딘으로 코팅하였다.E. coli SS320 was cultured in 50 ml of 2YT medium (supplemented with 10 μg/ml tetracycline), and half of a 96-well plate was coated with 100 μl of 67 nM neutravidin at 4°C one day before phage display.
- 파지 디스플레이의 첫 번째 라운드- First round of phage display
뉴트라비딘 코팅된 플레이트를 각 웰에 대해 250ul의 TBS로 세척하고, 250ul의 TBS-BSA(0.5% BSA가 보충된 TBS)로 차단하였다. Biotinylated IL-4R을 4.9ml의 정제된 파지(1012 phages/ml)에 첨가하여 단백질 농도가 50nM이 되도록 한 후 얼음에서 2시간 동안 배양하였다. 뉴트라비딘 코팅된 플레이트를 250ul TBS-BSA-D(0.5% BSA 및 0.1% 트윈 20으로 보충된 TBS)로 세척하였다. 100ul의 파지-IL-4R 혼합물을 플레이트의 각 웰에 첨가하고, 얼음에서 1시간 동안 인큐베이션하였다. 플레이트의 각 웰을 150ul의 TBS-D(0.1% 트윈 20이 첨가된 TBS)로 3회 세척하고, 플레이트를 각 세척 단계 후 2분 동안 페이퍼 타월 상에서 건조시켰다. 100ul의 PD 용출 완충액(분말로 첨가된 0.25ml/ml 트립신이 첨가된 TBS)을 각 웰에 첨가하고, 플레이트를 실온에서 10분 동안 인큐베이션하였다. 용출된 용액을 새로운 튜브에 옮기고, 튜브에 AEBSF 용액(Merck, A8456) 40ul를 첨가하였다. 1ul의 샘플을 qPCR을 통해 분석하고, 나머지 샘플을 50ml의 배양된 SS320에 첨가하였다. 혼합물을 37℃에서 1.5시간 동안 진탕하지 않고 배양한 후, 혼합물 50ml를 2YT 배지(200ug/ml 암피실린 및 2% 포도당 보충) 200ml에 옮기고 37℃, 160rpm에서 밤새 배양하였다. Neutravidin coated plates were washed with 250ul of TBS for each well and blocked with 250ul of TBS-BSA (TBS supplemented with 0.5% BSA). Biotinylated IL-4R was added to 4.9 ml of purified phage (1012 phages/ml) to bring the protein concentration to 50 nM, and then incubated on ice for 2 hours. Neutravidin coated plates were washed with 250ul TBS-BSA-D (TBS supplemented with 0.5% BSA and 0.1% Tween 20). 100ul of phage-IL-4R mixture was added to each well of the plate and incubated on ice for 1 hour. Each well of the plate was washed three times with 150ul of TBS-D (TBS with 0.1% Tween 20), and the plate was dried on paper towels for 2 minutes after each wash step. 100ul of PD elution buffer (TBS with 0.25ml/ml trypsin added as powder) was added to each well, and the plate was incubated for 10 minutes at room temperature. The eluted solution was transferred to a new tube, and 40ul of AEBSF solution (Merck, A8456) was added to the tube. 1ul of sample was analyzed through qPCR, and the remaining sample was added to 50ml of cultured SS320. After the mixture was incubated at 37°C for 1.5 h without shaking, 50 ml of the mixture was transferred to 200 ml of 2YT medium (supplemented with 200 ug/ml ampicillin and 2% glucose) and incubated overnight at 37°C and 160 rpm.
- 파지 디스플레이의 두 번째 라운드- Second round of phage display
1차 파지 디스플레이에서 생성된 파지를 TBS-BSA-D(5×1012 phages/ml)에서 수확하고 정제하였다. 50nM 농도의 파지 용액 100ul에 Biotinylated IL-4R을 첨가하고 얼음에서 2시간 동안 배양하였다. Dynabeads™MyOne™StreptavidinC1 비드(Invitrogen, 65001) 12ul를 TBS-WTB-BSA 500ul로 두 번 세척한 다음, 얼음에서 WTB-BSA 500ul로 20분 이상 동안 차단하였다. 마그네틱 비드를 500ul의 TBS-BSA-D로 3회 세척한 다음, 재현탁하고 얼음에서 파지-IL-4R 복합체 용액과 함께 1시간 동안 인큐베이션하였다. 인큐베이션 후, 혼합물을 500ul의 TBS-BSA-D로 세척한 다음, 비-바이오티닐화(non-biotinylated) IL-4R(Sino Biological, 10402-H08H) 5uM로 보충한 TBS-BSA-D인 ‘경쟁 완충제(competition buffer)’100ul로 재현탁하였고, 얼음에서 3분 동안 인큐베이션하였다. 경쟁적인 비-바이오티닐화(non-biotinylated) IL-4R은 TBS-D 500ul로 2회 세척하여 세척하였다. 비드를 PD 용출 완충액 100ul로 재현탁하고, 실온에서 10분 동안 인큐베이션하였다. 생성된 용액에 0.8ul의 ABESF를 첨가하여 피펫팅하여 혼합하고, 1ul의 용액을 qPCR 분석에 사용하고, 나머지 용액은 SS320의 감염에 사용하였다. E. coli SS320을 200ug/ml 암피실린 및 2% 글루코스가 보충된 2YT 배지에서 37℃, 160rpm에서 밤새 배양하였다.Phages generated in the first phage display were harvested and purified in TBS-BSA-D (5×10 12 phages/ml). Biotinylated IL-4R was added to 100ul of 50nM phage solution and incubated on ice for 2 hours. 12ul of Dynabeads™MyOne™StreptavidinC1 beads (Invitrogen, 65001) were washed twice with 500ul of TBS-WTB-BSA and then blocked with 500ul of WTB-BSA for more than 20 minutes on ice. The magnetic beads were washed three times with 500 μl of TBS-BSA-D, then resuspended and incubated with the phage-IL-4R complex solution for 1 hour on ice. After incubation, the mixture was washed with 500ul of TBS-BSA-D and then incubated with 'competition', TBS-BSA-D supplemented with 5uM non-biotinylated IL-4R (Sino Biological, 10402-H08H). It was resuspended with 100ul of competition buffer and incubated on ice for 3 minutes. Competitive non-biotinylated IL-4R was washed twice with 500ul of TBS-D. The beads were resuspended in 100ul of PD elution buffer and incubated for 10 minutes at room temperature. 0.8ul of ABESF was added to the resulting solution and mixed by pipetting. 1ul of the solution was used for qPCR analysis, and the remaining solution was used for infection of SS320. E. coli SS320 was cultured overnight at 37°C and 160 rpm in 2YT medium supplemented with 200 ug/ml ampicillin and 2% glucose.
비오틴화된 MBP의 생산 및 정제Production and purification of biotinylated MBP
플라스미드 pBXNH3CA_MBP(addgene, 132700)를 발현을 위해 E.coli BL21로 형질전환시켰다. 이를 37℃, 160rpm에서 4시간 동안 100ug/ml 암피실린이 보충된 TB 배지에서 배양하였다. 발현 및 비오틴화(biotinylation)를 위해 아라비노스(arabinose) 및 비오틴(biotin)을 각각 0.02% 및 100uM 농도로 첨가하였다. 그 후 발현을 위해 22℃, 160 rpm에서 밤새 배양을 진행하였다. 밤새 배양물을 초음파 처리한 후 MBP MiniExcellose® (Takara, AEx-MC-M03)를 사용하여 정제하였다.Plasmid pBXNH3CA_MBP (addgene, 132700) was transformed into E. coli BL21 for expression. It was cultured in TB medium supplemented with 100ug/ml ampicillin at 37°C and 160rpm for 4 hours. For expression and biotinylation, arabinose and biotin were added at concentrations of 0.02% and 100 uM, respectively. Afterwards, culture was performed overnight at 22°C and 160 rpm for expression. The culture was sonicated overnight and purified using MBP MiniExcellose® (Takara, AEx-MC-M03).
효소 결합 면역 흡착 분석법 (ELISA)Enzyme-linked immunosorbent assay (ELISA)
FX 클로닝 방법을 사용하여, 파지 디스플레이를 통하여 2회에 걸쳐 농축된 라이브러리(enriched library)를 pSBinit 벡터(addgene, 110100)에 클로닝하였다5. 클로닝된 플라스미드를 전기천공법을 통해 E. coli SS320에 형질전환시킨 후, 37℃, 160rpm에서 밤새 배양한 다음, 플라스미드를 추출하였다. 추출된 플라스미드를 E. coli BL21(Enzynomics, CP110)로 형질전환하고 25ug/ml 클로람페니콜이 보충된 LB-한천 플레이트에 플레이팅한 다음 37℃에서 밤새 인큐베이션하였다.Using the FX cloning method, the enriched library was cloned twice into the pSBinit vector (addgene, 110100) through phage display 5 . The cloned plasmid was transformed into E. coli SS320 through electroporation, cultured overnight at 37°C and 160 rpm, and then the plasmid was extracted. The extracted plasmid was transformed into E. coli BL21 (Enzynomics, CP110), plated on LB-agar plates supplemented with 25ug/ml chloramphenicol, and incubated at 37°C overnight.
25ug/ml 클로람페니콜이 보충된 1.2ml의 TB 배지를 '전배양'이라고 표시된 96-웰 딥웰 플레이트의 각 웰에 준비하였다. 인큐베이션된 플레이트에서 95개의 콜로니를 선택하고 각 콜로니를 각 웰에 접종하고 첫 번째 웰을 MBP 나노바디 Sb_MBP#1(addgene, 132699)을 포함하는 양성 대조군 pSb_init로 채웠다. 딥 웰 플레이트는 기체 투과성으로 밀봉되었고 37℃, 300rpm에서 4시간 동안 성장시켰다. 25ug/ml 클로람페니콜이 보충된 미리 데운 TB 배지 1ml로 채워진 새로운 96웰 딥웰 플레이트는 '발현 배양'으로 표시되었다. 50ul의 '전배양'을 발현 배양물의 해당 웰에 첨가하고 플레이트를 OD600 = 0.4~0.8이 될 때까지 37℃, 300rpm에서 2시간, 22℃, 300rpm에서 인큐베이션하였다. 충분히 성장시킨 후, 0.02%(wt/vol)의 L-(+)-아라비노스를 첨가하여 발현을 유도하고, 22℃, 150rpm에서 밤새 배양하였다. 다음날, 세포를 5,000xg, 4℃에서 15분 동안 원심분리하여 수집하였다. '전배양'의 팔레트는 DNA 정제를 위해 -20℃에 보관되었고, '발현 배양'의 팔레트는 100ul의 주변세포질 추출 완충액(20% sucrose, 50mM Tris pH8.0, 0.5mM EDTA 및 0.5ug/ml의 리소자임(DW로 표시))으로 강한 소용돌이(intensive vortex)로 재현탁하였다. 얼음에서 30분간 인큐베이션한 후, 1mM MgCl2가 첨가된 TBS 900ul를 각 웰에 첨가하였다. 플레이트를 5,000xg, 4℃에서 15분 동안 원심분리하고 상층액을 ELISA를 위한 주변 세포질 추출액(periplasmic extraction)으로 사용하였다. 2개의 96-웰 면역 플레이트를 100ul의 5ug/ml 단백질 A(protein A) 용액으로 코팅하고 접착제 밀봉과 함께 4℃에서 밤새 인큐베이션하였다. 플레이트를 웰당 250ul의 TBS로 세척한 다음, 웰당 150ul의 TBS-BSA로 차단하였다. 그 다음, TBS-BSA-D에 1:2,000으로 희석된 항-c-Myc 항체(Biolegend, 626802) 100ul를 웰당 첨가하고, 20분 동안 배양하였다. 250ul의 TBS-D로 3회 세척한 후, IL-4R 간의 나노바디 결합을 비교하기 위해 각 웰에 TBS-BSA-D 80ul를 첨가하고 나노바디의 IL-4R 및 MBP와의 결합 정도를 비교하기 위하여, 동일한 주변 세포질 추출액 20ul를 나란히 첨가하였으며, 20분 동안 인큐베이션하였다. 플레이트를 웰당 250ul의 TBS-D로 세척하고, 처음 두 개의 웰에 100ul의 50nM MBP를 첨가하고, 각각의 웰에 100ul의 50nM 비오틴화된 IL-4R을 첨가하고, 플레이트를 20분 동안 인큐베이션하였다. 250ul TBS-D로 3회 세척한 후, TBS-BSA-D에 희석된 1:5,000 streptavidin-peroxidase(Invitrogen, 434323) 100ul를 각 웰에 첨가하고 20분 동안 배양하였다. 플레이트를 다시 TBS-D로 3회 세척하고, 100ul의 TMB 기질(biolegend, 421101)을 각 웰에 첨가하였다. 반응은 개별 웰이 파란색으로 변할 때까지 약 15분이 소요되었다. 흡광도는 플레이트 판독기로 650nm에서 측정하였다.1.2 ml of TB medium supplemented with 25 ug/ml chloramphenicol was prepared in each well of a 96-well deep well plate labeled ‘preculture’. Ninety-five colonies were selected from the incubated plate, each colony was inoculated into each well, and the first well was filled with the positive control pSb_init containing the MBP nanobody Sb_MBP#1 (addgene, 132699). Deep well plates were sealed with gas permeability and grown at 37°C and 300 rpm for 4 hours. A new 96-well deepwell plate filled with 1 ml of pre-warmed TB medium supplemented with 25 ug/ml chloramphenicol was labeled ‘expression culture’. 50ul of 'preculture' was added to the corresponding well of the expression culture and the plate was incubated at 37°C, 300rpm for 2 hours, 22°C, 300rpm until OD 600 = 0.4-0.8. After sufficient growth, expression was induced by adding 0.02% (wt/vol) L-(+)-arabinose, and cultured overnight at 22°C and 150 rpm. The next day, cells were collected by centrifugation at 5,000xg, 4°C for 15 minutes. The palette of 'preculture' was stored at -20°C for DNA purification, and the palette of 'expression culture' was stored in 100ul of periplasmic extraction buffer (20% sucrose, 50mM Tris pH8.0, 0.5mM EDTA, and 0.5ug/ml). of lysozyme (indicated as DW) was resuspended by intensive vortexing. After incubation on ice for 30 minutes, 900ul of TBS containing 1mM MgCl 2 was added to each well. The plate was centrifuged at 5,000xg, 4°C for 15 minutes, and the supernatant was used as periplasmic extraction for ELISA. Two 96-well immune plates were coated with 100ul of 5ug/ml protein A solution and incubated overnight at 4°C with adhesive seals. Plates were washed with 250ul of TBS per well and then blocked with 150ul of TBS-BSA per well. Next, 100ul of anti-c-Myc antibody (Biolegend, 626802) diluted 1:2,000 in TBS-BSA-D was added per well and incubated for 20 minutes. After washing three times with 250ul of TBS-D, 80ul of TBS-BSA-D was added to each well to compare nanobody binding between IL-4R and the degree of binding of nanobody to IL-4R and MBP. , 20ul of the same periplasmic extract was added side by side and incubated for 20 minutes. The plate was washed with 250ul of TBS-D per well, 100ul of 50nM MBP was added to the first two wells, 100ul of 50nM biotinylated IL-4R was added to each well, and the plate was incubated for 20 minutes. After washing three times with 250ul TBS-D, 100ul of streptavidin-peroxidase (Invitrogen, 434323) diluted 1:5,000 in TBS-BSA-D was added to each well and incubated for 20 minutes. The plate was washed again three times with TBS-D, and 100ul of TMB substrate (biolegend, 421101) was added to each well. The reaction took approximately 15 minutes until individual wells turned blue. Absorbance was measured at 650 nm with a plate reader.
나노바디의 생산 및 정제Production and purification of nanobodies
발현 벡터(pSBinit) 내의 나노바디 클론을 E. coli BL21에 형질전환시키고, 37℃, 160 rpm에서 밤새 배양하였다. 밤새 배양한 배양액 2ml를 클로람페니콜 25ug/ml가 첨가된 TB 배지 200ml에 접종하고 OD600 = 0.6이 될 때까지 37℃, 200rpm에서 배양한 후, 배양액을 22℃, 150rpm으로 1시간 동안 이동시켰다. 발현 유도를 위해 아라비노스 0.02%를 첨가하고 37℃, 150rpm에서 밤새 배양하였다. 밤새 배양한 배양물을 5,000xg, 4℃에서 15분 동안 원심분리하고, 팔레트를 얼음에서 20ml의 주변 세포질 추출 완충액으로 30분 동안 재현탁하였다. 인큐베이션 후, 1mM MgCl2가 첨가된 TBS 180ml를 첨가하고, 5,000xg, 4℃에서 15분 동안 원심분리하였다. The nanobody clone in the expression vector (pSBinit) was transformed into E. coli BL21 and cultured at 37°C and 160 rpm overnight. 2ml of the overnight culture was inoculated into 200ml of TB medium supplemented with 25ug/ml of chloramphenicol and cultured at 37°C and 200rpm until OD 600 = 0.6, then the culture was moved to 22°C and 150rpm for 1 hour. To induce expression, 0.02% arabinose was added and cultured overnight at 37°C and 150 rpm. Overnight cultures were centrifuged at 5,000×g at 4°C for 15 min, and pallets were resuspended in 20 ml of periplasmic extraction buffer for 30 min on ice. After incubation, 180ml of TBS containing 1mM MgCl 2 was added and centrifuged at 5,000xg and 4°C for 15 minutes.
1ml의 TALON®Superflow™(Cytiva, 28957502) 슬러리를 TBS pH 8.0으로 미리 평형화(pre-equilibrated)하였다. 결합 후 비드를 세척 완충액(50mM Tris pH 8.0, 300mM NaCl, 5mM 이미다졸)으로 3회 세척한 후, 나노바디를 용출 완충액(50nM Tris pH 8.0, 300mM NaCl, 200mM 이미다졸)으로 용출시켰다. 용출된 나노바디는 Slide-A-Lyzer® Dialysis Cassette(Thermo, 66330)을 사용하여 밤새 완충액을 교환하였다.1 ml of TALON® Superflow™ (Cytiva, 28957502) slurry was pre-equilibrated with TBS pH 8.0. After binding, the beads were washed three times with washing buffer (50mM Tris pH 8.0, 300mM NaCl, 5mM imidazole), and then the nanobodies were eluted with elution buffer (50nM Tris pH 8.0, 300mM NaCl, 200mM imidazole). Eluted nanobodies were buffer exchanged overnight using Slide-A-Lyzer® Dialysis Cassette (Thermo, 66330).
IL-4R 억제 측정을 위한 STAT6-루시페라제 리포터 시스템STAT6-luciferase reporter system for measuring IL-4R inhibition
HEK293 세포주는 퓨로마이신(puromycin) 내성 렌티바이러스 시스템(lentivirus system)에 의해 human STAT6(addgene, 81950)을, blasticidin 내성 system에 의해 pSTAT6에 의해 유도된 luciferase(addgene, 35554)를 안정적으로 발현시켜 IL-4 신호의 STAT6 응답에 의한 조절하에 luciferase가 안정적으로 발현될 수 있도록 하였다6,7. 5 x 104개의 리포터 세포를 96-웰 세포 배양 플레이트의 각 웰에 시딩하고 hIL-4 및 무혈청 RMPI 배지에서 두필루맙 또는 나노바디 중 하나와 함께 37℃ 및 5% CO2 환경으로 24시간 동안 배양하였다. 세포 용해(cell lysis)를 백색 96-웰 플레이트로 옮기고, 50ul의 루시퍼라제 기질(Promega, E1501)을 첨가하였다. 랜덤 발광 단위(Random luminescence unit)는 SpectraMax®M5 발광계(Molecular Devices)로 측정하였다.The HEK293 cell line stably expressed human STAT6 (addgene, 81950) by a puromycin-resistant lentivirus system and luciferase (addgene, 35554) induced by pSTAT6 by a blasticidin-resistant system, thereby producing IL- 4 luciferase was allowed to be stably expressed under the control of the STAT6 response 6,7 . 5 Cultured. Cell lysis was transferred to a white 96-well plate, and 50ul of luciferase substrate (Promega, E1501) was added. Random luminescence units were measured with a SpectraMax®M5 luminometer (Molecular Devices).
면역형광 염색 및 이미지 분석Immunofluorescence staining and image analysis
트랜스웰의 HNE 세포를 PBS로 세척하고 500ul의 4% 파라포름알데히드(Biosesang, P2031)로 실온에서 30분 동안 고정하였다. 그런 다음 HNE 세포를 100ul(1:200)의 FOXJ1 1차 항체(GeneTex, GTX114408)와 함께 1시간 동안 인큐베이션하였고, 그 다음 100ul(1:1000)의 마우스 항-토끼 FITC와 30분 동안 인큐베이션한 후 PBS로 세 번 세척하였다. 세포들은 405, 488nm 레이저의 매개변수(parameter) 설정을 사용하여 Zeiss LSM 700에서 사진을 촬영했으며 Z-stack 다차원 획득 기능을 적용하였다. Z-stack 슬라이스의 이미지는 간격을 8um로 설정하여 얻었다.HNE cells in the transwell were washed with PBS and fixed with 500ul of 4% paraformaldehyde (Biosesang, P2031) for 30 minutes at room temperature. HNE cells were then incubated with 100 ul (1:200) of FOXJ1 primary antibody (GeneTex, GTX114408) for 1 h, followed by 100 ul (1:1000) mouse anti-rabbit FITC for 30 min. Washed three times with PBS. Cells were photographed on a Zeiss LSM 700 using parameter settings for the 405 and 488 nm lasers and applying the Z-stack multidimensional acquisition function. Images of Z-stack slices were obtained with the spacing set to 8 μm.
나노바디 후보들의 서열Sequence of nanobody candidates
H5 CDR 서열 (서열번호 1) : DKGREYTLARAKYWYWH5 CDR sequence (SEQ ID NO: 1): DKGREYTLARAKYWYW
E9 CDR 서열 (서열번호 2) : AYGIWEPLRYRNYSYWE9 CDR sequence (SEQ ID NO: 2): AYGIWEPLRYRNYSYW
B3 CDR 서열 (서열번호 3) : AQRGKYRPLYAAKYSYWB3 CDR sequence (SEQ ID NO: 3): AQRGKYRPLYAAKYSYW
G5 CDR 서열 (서열번호 4) : RTGIWEPLAHRNYNYWG5 CDR sequence (SEQ ID NO: 4): RTGIWEPLAHRNYNYW
H5 전장서열 (서열번호 5) :H5 full-length sequence (SEQ ID NO: 5):
QVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREGVAALSTSSGTTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAADKGREYTLARAKYWYWGQGTQVTVSAGRAGEQKLISEEDLNSAVDHHHHHHQVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREGVAALSTSSGTTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAADKGREYTLARAKYWYWGQGTQVTVSAGRAGEQKLISEEDLNSAVDHHHHHH
E9 전장서열 (서열번호 6) : E9 full-length sequence (SEQ ID NO: 6):
QVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREGVAALSTSSGTTYYADSVKGRFTVSLDNAKNTVYLQLNSLKPEDTALYYCAAAAYGIWEPLRYRNYSYWGQGTQVTVSAGRAGEQKLISEEDLNSAVDHHHHHHQVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREGVAALSTSSGTTYYADSVKGRFTVSLDNAKNTVYLQLNSLKPEDTALYYCAAAAYGIWEPLRYRNYSYWGQGTQVTVSAGRAGEQKLISEEDLNSAVDHHHHHH
B3 전체서열 (서열번호 7) : B3 full sequence (SEQ ID NO: 7):
QVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREGVAALSTSSGTTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAAQRGKYRPLYAAKYSYWGQGTQVTVSAGRAGEQKLISEEDLNSAVDHHHHHHQVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREGVAALSTSSGTTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAAQRGKYRPLYAAKYSYWGQGTQVTVSAGRAGEQKLISEEDLNSAVDHHHHHH
G5 전체서열 (서열번호 8) : G5 full sequence (SEQ ID NO: 8):
QVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREGVAALSTSSGTTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAARTGIWEPLAHRNYNYWGQGTQVTVSAGRAGEQKLISEEDLNSAVDHHHHHHQVQLVESGGGSVQAGGSLRLSCAASGSISSITYLGWFRQAPGKEREGVAALSTSSGTTYYADSVKGRFTVSLDNAKNTVYLQMNSLKPEDTALYYCAAARTGIWEPLAHRNYNYWGQGTQVTVSAGRAGEQKLISEEDLNSAVDHHHHHH
실험결과Experiment result
항IL-4R 나노바디 스크리닝의 최종 과정Final steps in anti-IL-4R nanobody screening
리보솜 디스플레이 및 2회의 파지 디스플레이 후, 약 102개의 나노바디 클론을 스크리닝하였다. 이 나노바디들의 풀을 풍부(enrich)하게 하기 위해 IL-4R 및 MBP에 대해 ELISA를 수행하였다. 그래프의 y축은 IL-4R과 MBP에 대한 OD 값의 배수변화도(fold-change) 나타내며, 이는 각 나노바디 클론의 IL-4R에 대한 특이성을 의미한다. 붉은색 박스로 표시된 클론은 임계값(1.7)보다 폴드 변경 값이 더 높은, 최종적으로 선택된 나노바디들이다. 겹치는 시퀀스를 제외하고 발명자들은 최종적으로 4개의 다른 나노바디 시퀀스인 H5, G5, E9 및 B3을 얻었다(도 1).After ribosome display and two rounds of phage display, approximately 102 nanobody clones were screened. To enrich the pool of these nanobodies, ELISA was performed for IL-4R and MBP. The y-axis of the graph represents the fold-change of OD values for IL-4R and MBP, which indicates the specificity of each nanobody clone for IL-4R. Clones marked with red boxes are the finally selected nanobodies with fold change values higher than the threshold (1.7). Excluding overlapping sequences, the inventors finally obtained four different nanobody sequences: H5, G5, E9, and B3 (Figure 1).
IL-4R에 대한 나노바디 후보들의 결합 친화도Binding affinity of nanobody candidates to IL-4R
IL-4R에 대한 4개의 최종 나노바디 후보를 리보솜 디스플레이, 파지 디스플레이 및 ELISA를 통해 선별하였다. 각각의 나노바디를 정제하고, 동일한 농도의 나노바디의 IL-4R 분자에 대한 결합 친화도를 확인하기 위해 ELISA를 통해 조사하였다. 그 결과, H5 나노바디가 동일한 농도의 4가지 후보 중에서 IL-4R 분자에 대한 결합 친화도가 가장 높다는 것을 알 수 있었다(도 2).Four final nanobody candidates against IL-4R were screened through ribosome display, phage display and ELISA. Each nanobody was purified and examined through ELISA to confirm the binding affinity of the nanobody at the same concentration to the IL-4R molecule. As a result, it was found that H5 nanobody had the highest binding affinity for the IL-4R molecule among the four candidates at the same concentration (Figure 2).
나노바디 후보의 IL-4 신호 차단 활성IL-4 signaling blocking activity of nanobody candidates
3개의 나노바디 후보를 이전 ELISA 결과에 따라 선택하여 결합 친화도가 IL-4 신호 차단 기능과 관련이 있는지 조사하였다. 3가지 다른 농도의 각 나노바디의 신호 차단 기능을 두필루맙(Dupilumab)의 신호 차단 기능과 비교하였다. 리포터 세포를 83pM의 IL-4 및 각 나노바디(또는 항체)와 함께 24시간 동안 인큐베이션하였다. 그 결과 두필루맙과 H5 나노바디가 후보 중 가장 효과적인 IL-4 신호 차단제임을 확인할 수 있었다(도 3).Three nanobody candidates were selected based on previous ELISA results to investigate whether their binding affinity was related to their ability to block IL-4 signaling. The signal blocking function of each nanobody at three different concentrations was compared with that of Dupilumab. Reporter cells were incubated with 83 pM of IL-4 and each nanobody (or antibody) for 24 hours. As a result, it was confirmed that dupilumab and H5 nanobody were the most effective IL-4 signal blockers among the candidates (Figure 3).
두필루맙(Dupilumab)과 H5의 IL-4 신호 차단 활성의 비교Comparison of IL-4 signaling blocking activities of Dupilumab and H5
이전 결과에 따라, H5 나노바디를 최종 IL-4R 차단 나노바디로 선택하였다. 3가지 다른 농도에서 각 나노바디의 신호 차단 활성을 두필루맙과 비교하였다. 리포터 세포를 667pM의 IL-4 및 나노바디(또는 항체)와 함께 24시간 동안 인큐베이션하였다. H5 나노바디는 약 2배 농도에서 두필루맙과 유사한 차단 활성을 나타냄을 알 수 있었다(도 4).According to previous results, H5 nanobody was selected as the final IL-4R blocking nanobody. The signal blocking activity of each nanobody was compared with dupilumab at three different concentrations. Reporter cells were incubated with 667 pM of IL-4 and nanobody (or antibody) for 24 hours. It was found that H5 nanobody exhibited similar blocking activity as dupilumab at about twice the concentration (Figure 4).
두필루맙(Dupilumab)과 H5의 세포 결합 친화도의 비교Comparison of cell binding affinities of Dupilumab and H5
HEK293 세포에 대한 두필루맙과 H5 나노바디의 결합 친화도를 플레이트 판독기를 통해 평가하였다. 항-인간 IgG-FITC를 두필루맙의 2차 항체로 사용하였고, 항-Myc-FITC를 H5 나노바디의 2차 항체로 사용하였다. 각 항체(나노바디)의 형광 단위를 각 2차 항체의 형광 단위로 표준화하였다. 도 4 및 도 5의 결과는 신호 차단 활성이 항체(나노바디)와 세포의 결합에 의존함을 시사한다.The binding affinity of dupilumab and H5 nanobody to HEK293 cells was evaluated using a plate reader. Anti-human IgG-FITC was used as a secondary antibody for dupilumab, and anti-Myc-FITC was used as a secondary antibody for H5 nanobody. The fluorescence units of each antibody (nanobody) were normalized to the fluorescence units of each secondary antibody. The results in Figures 4 and 5 suggest that the signal blocking activity depends on the binding of the antibody (nanobody) to the cell.
IL-4, 두필루맙(Dupilumab) 및 나노바디에 의한 FOXJ1 발현 조절 (도 6)Regulation of FOXJ1 expression by IL-4, Dupilumab and nanobodies (Figure 6)
FOXJ1은 forkhead box family 전사인자 중 하나로 IL-4/IL-13 신호전달에 의해 발현이 억제되고 STAT6이 인산화된다. 도 6a는 IL-4, 두필루맙 및 H5 나노바디로 처리된 인간 비강 상피 세포(HNE)의 정사 이미지(ortho image)이다. 두필루맙 및 H5의 투여는 IL-4 신호전달을 차단하고 후속적으로 FOXJ1의 발현을 유도하는 것으로 나타났다. 도 6c는 두필루맙과 H5를 IL-4 없이 처리했을 때 FOXJ1 발현이 유도됨을 보여주고 있으며, 이는 항체와 나노바디가 IL-4R에 대한 어떤 작용제(agonistic) 활성을 가지지 않음을 의미한다. 그러나 FOXJ1의 발현은 IL-4 및 항-MBP 나노바디를 모두 처리했을 때 억제되는데, 이는 나노바디 스캐폴드 자체가 IL-4R에 대한 길항 효과가 없음을 시사한다. 도 6b 및 6c는 해당 HNE 샘플의 3D 이미지이다.FOXJ1 is one of the forkhead box family transcription factors, and its expression is suppressed by IL-4/IL-13 signaling and STAT6 is phosphorylated. Figure 6A is an ortho image of human nasal epithelial cells (HNE) treated with IL-4, dupilumab and H5 nanobody. Administration of dupilumab and H5 was shown to block IL-4 signaling and subsequently induce the expression of FOXJ1. Figure 6c shows that FOXJ1 expression is induced when dupilumab and H5 are treated without IL-4, which means that the antibody and nanobody do not have any agonistic activity against IL-4R. However, FOXJ1 expression was inhibited when treated with both IL-4 and anti-MBP nanobodies, suggesting that the nanobody scaffold itself does not have an antagonistic effect on IL-4R. Figures 6b and 6c are 3D images of the corresponding HNE samples.
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
참고문헌references
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Claims (13)
- 서열번호 1, 2, 3 및 4로 구성된 군으로부터 선택되는 하나 이상의 아미노산 서열을 포함하는 항-IL-4R 항체 또는 이의 항원 결합단편.An anti-IL-4R antibody or antigen-binding fragment thereof comprising one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1, 2, 3, and 4.
- 제 1 항에 있어서, 상기 항-IL-4R 항체 또는 이의 항원 결합단편은 서열번호 5, 6, 7 및 8로 구성된 군으로부터 선택되는 어느 하나의 아미노산 서열을 포함하는 것을 특징으로 하는 항체 또는 이의 항원 결합 단편.The antibody or antigen thereof according to claim 1, wherein the anti-IL-4R antibody or antigen-binding fragment thereof comprises any one amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 6, 7, and 8. Combined fragment.
- 제 1 항에 있어서, 상기 항원 결합단편은 F(ab')2, Fab', Fab, Fv, scFv 및 나노바디(Nanobody)로 구성된 군으로부터 선택되는 것을 특징으로 하는 항체 또는 이의 항원 결합단편.The antibody or antigen-binding fragment thereof according to claim 1, wherein the antigen-binding fragment is selected from the group consisting of F(ab')2, Fab', Fab, Fv, scFv, and Nanobody.
- 제 3 항에 있어서, 상기 항원 결합단편은 나노바디인 것을 특징으로 하는 항체 또는 이의 항원 결합 단편.The antibody or antigen-binding fragment thereof according to claim 3, wherein the antigen-binding fragment is a nanobody.
- 제 4 항에 있어서, 상기 나노바디는 볼록 스캐폴드(convex scaffold)구조를 가지는 것을 특징으로 하는 항체 또는 이의 항원 결합 단편.The antibody or antigen-binding fragment thereof according to claim 4, wherein the nanobody has a convex scaffold structure.
- 제 4 항에 있어서, 상기 나노바디는 10 내지 18kDa의 평균 분자량을 가지는 것을 특징으로 하는 항체 또는 이의 항원 결합 단편.The antibody or antigen-binding fragment thereof according to claim 4, wherein the nanobody has an average molecular weight of 10 to 18 kDa.
- 제 1 항 내지 제 6 항 중 어느 한 항의 항체 또는 이의 항원 결합 단편을 인코딩하는 핵산 분자.A nucleic acid molecule encoding the antibody or antigen-binding fragment thereof of any one of claims 1 to 6.
- 제 1 항 내지 제 6 항 중 어느 한 항의 항체 또는 이의 항원 결합 단편; 또는 제 7 항의 핵산분자를 유효성분으로 포함하는 염증 또는 자가 면역 질환의 예방 또는 치료용 약학적 조성물.The antibody or antigen-binding fragment thereof of any one of claims 1 to 6; Or a pharmaceutical composition for preventing or treating inflammatory or autoimmune diseases comprising the nucleic acid molecule of claim 7 as an active ingredient.
- 제 8 항에 있어서, 상기 염증 또는 자가 면역 질환은 비염, 결막염, 치주염, 중이염, 인후염, 편도염, 폐렴, 위궤양, 위염, 크론병, 대장염, 치질, 통풍, 강직성 척추염, 류마티스 열, 류마티스 관절염, 류마티스성 다발성 근육통, 루프스, 섬유근통(fibromyalgia), 건선성 관절염, 골관절염, 견관절 주위염, 건염, 건초염, 건주위염, 근육염, 다발성 근육염, 피부 근육염, 간염, 방광염, 신장염, 쇼그렌 증후군(sjogren's syndrome), 다발성 경화증, 염증성 장질환, 천식, 제1형 당뇨병, 건선, 습진, 피부경화증, 백반증, 말초성 신경염, 포도막염, 자가면역 혈구감소증, 자가면역 심근염, 아토피피부염, 일차성간경변, 안구건조증, 굿파이처 증후군, 자가면역 뇌수막염, 애디슨병, 자가면역성 이하선염, 이영양성 수포성 표피박리증, 부고환염, 사구체 신염, 그레이브스병, 셀리악병, 길랑바레 증후군, 하시모토병, 용혈성 빈혈, 중증 근무력증, 근위축성측색경화증, 심상천포창, 유육종증, 척추관절증, 갑상선염, 혈관염, 점액수종, 악성빈혈, 항인지질증후군 및 이식편대숙주질환으로 구성된 군으로부터 선택되는 염증임을 특징으로 하는 조성물.The method of claim 8, wherein the inflammatory or autoimmune diseases include rhinitis, conjunctivitis, periodontitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, rheumatoid arthritis, and rheumatoid arthritis. Polymyalgia, lupus, fibromyalgia, psoriatic arthritis, osteoarthritis, periarthritis, tendonitis, tenosynovitis, peritendinitis, myositis, polymyositis, dermatomyositis, hepatitis, cystitis, nephritis, Sjogren's syndrome, multiple sclerosis. , inflammatory bowel disease, asthma, type 1 diabetes, psoriasis, eczema, scleroderma, vitiligo, peripheral neuritis, uveitis, autoimmune cytopenia, autoimmune myocarditis, atopic dermatitis, primary cirrhosis, dry eye, Goodficher syndrome, Autoimmune meningitis, Addison's disease, autoimmune parotitis, dystrophic epidermolysis bullosa, epididymitis, glomerulonephritis, Graves' disease, celiac disease, Guillain-Barré syndrome, Hashimoto's disease, hemolytic anemia, myasthenia gravis, amyotrophic lateral sclerosis, pemphigus vulgaris, A composition characterized in that it is an inflammatory selected from the group consisting of sarcoidosis, spondyloarthrosis, thyroiditis, vasculitis, myxedema, pernicious anemia, antiphospholipid syndrome, and graft-versus-host disease.
- 제 9 항에 있어서, 상기 비염은 비용종(nasal polyps) 또는 부비동염(Sinusitis)인 것을 특징으로 하는 조성물.The composition according to claim 9, wherein the rhinitis is nasal polyps or sinusitis.
- 제 10 항에 있어서, 상기 부비동염은 만성 부비동염(Chronic Sinusitis, CRS)인 것을 특징으로 하는 조성물.The composition according to claim 10, wherein the sinusitis is chronic sinusitis (CRS).
- 제 11 항에 있어서, 상기 만성 부비동염은 비용종을 동반한 만성 부비동염(CRS with nasal polyp, CRSwNP)인 것을 특징으로 하는 조성물.The composition according to claim 11, wherein the chronic sinusitis is chronic sinusitis with nasal polyp (CRSwNP).
- 제 8 항에 있어서, 상기 조성물은 비강 투여용 조성물인 것을 특징으로 하는 조성물.The composition according to claim 8, wherein the composition is a composition for nasal administration.
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| WO2016077675A1 (en) * | 2014-11-14 | 2016-05-19 | Sanofi Biotechnology | Methods for treating chronic sinusitis with nasal polyps by administering an il-4r antagonist |
| CN106604744A (en) * | 2014-09-03 | 2017-04-26 | 免疫医疗有限公司 | Stable anti-IL-4R-alpha antibody formulation |
| CN111690066A (en) * | 2020-06-22 | 2020-09-22 | 南京融捷康生物科技有限公司 | anti-IL-4R alpha single-domain antibody and application and medicament thereof |
| WO2021170020A1 (en) * | 2020-02-27 | 2021-09-02 | Chia Tai Tianqing Pharmaceutical Group Co., Ltd. | Antibodies binding il4r and uses thereof |
| KR20220091566A (en) * | 2019-10-31 | 2022-06-30 | 상하이 노바맙 바이오파아슈티컬 컴퍼니 리미티드 | Anti-IL-4R single domain antibodies and applications thereof |
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| US7608693B2 (en) | 2006-10-02 | 2009-10-27 | Regeneron Pharmaceuticals, Inc. | High affinity human antibodies to human IL-4 receptor |
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| CN106604744A (en) * | 2014-09-03 | 2017-04-26 | 免疫医疗有限公司 | Stable anti-IL-4R-alpha antibody formulation |
| WO2016077675A1 (en) * | 2014-11-14 | 2016-05-19 | Sanofi Biotechnology | Methods for treating chronic sinusitis with nasal polyps by administering an il-4r antagonist |
| KR20220091566A (en) * | 2019-10-31 | 2022-06-30 | 상하이 노바맙 바이오파아슈티컬 컴퍼니 리미티드 | Anti-IL-4R single domain antibodies and applications thereof |
| WO2021170020A1 (en) * | 2020-02-27 | 2021-09-02 | Chia Tai Tianqing Pharmaceutical Group Co., Ltd. | Antibodies binding il4r and uses thereof |
| CN111690066A (en) * | 2020-06-22 | 2020-09-22 | 南京融捷康生物科技有限公司 | anti-IL-4R alpha single-domain antibody and application and medicament thereof |
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| CHI LIANHUA; NA MOON-HEE; JUNG HYUN-KYUNG; VADEVOO SRI MURUGAN POONGKAVITHAI; KIM CHEONG-WUN; PADMANABAN GURUPRASATH; PARK TAE-IN;: "Enhanced delivery of liposomes to lung tumor through targeting interleukin-4 receptor on both tumor cells and tumor endothelial cells", JOURNAL OF CONTROLLED RELEASE, ELSEVIER, AMSTERDAM, NL, vol. 209, 12 May 2015 (2015-05-12), AMSTERDAM, NL , pages 327 - 336, XP029173953, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2015.05.260 * |
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