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WO2024058590A1 - Nouveau nanocorps de liaison au récepteur de l'interleukine-4 humaine et son utilisation - Google Patents

Nouveau nanocorps de liaison au récepteur de l'interleukine-4 humaine et son utilisation Download PDF

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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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antibody
nanobody
antigen
binding fragment
present
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김주영
안태영
이동혁
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Industry Academic Cooperation Foundation of Yonsei University
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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
    • 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
    • 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
    • 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]
    • 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
    • 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
    • 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
    • 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

La présente invention concerne un anticorps dirigé contre un récepteur d'IL-4 ou un fragment de liaison à l'antigène de celui-ci, en particulier un nanocorps ; et une composition pharmaceutique pour prévenir ou traiter des maladies inflammatoires ou auto-immunes le contenant en tant que principe actif. Le nanocorps selon la présente invention présente une affinité de liaison au récepteur d'IL-4 significativement plus élevée que les agents thérapeutiques d'anticorps disponibles dans le commerce, tandis que sa structure simple et son faible poids moléculaire offrent une excellente stabilité structurale, une excellente productivité et une excellente perméabilité, et peuvent être formulés sous une variété de formes, y compris une pulvérisation nasale, sans limitation. En conséquence, le nanocorps selon la présente invention bloque non seulement efficacement la signalisation d'IL-4, qui provoque des maladies inflammatoires telles que la sinusite, mais concentre également des effets pharmacologiques particulièrement sur une lésion sans immunosuppression systémique au moyen d'une administration intraveineuse, de telle sorte que le nanocorps selon la présente invention peut être utilisé de manière plus sûre pour une administration à long terme pour des maladies inflammatoires chroniques.
PCT/KR2023/013874 2022-09-16 2023-09-15 Nouveau nanocorps de liaison au récepteur de l'interleukine-4 humaine et son utilisation Ceased WO2024058590A1 (fr)

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CN106604744A (zh) * 2014-09-03 2017-04-26 免疫医疗有限公司 稳定的抗IL‑4R‑α抗体配制品
CN111690066A (zh) * 2020-06-22 2020-09-22 南京融捷康生物科技有限公司 抗IL-4Rα的单域抗体以及应用和药物
WO2021170020A1 (fr) * 2020-02-27 2021-09-02 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Anticorps se liant à il4r et leurs utilisations
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WO2021170020A1 (fr) * 2020-02-27 2021-09-02 Chia Tai Tianqing Pharmaceutical Group Co., Ltd. Anticorps se liant à il4r et leurs utilisations
CN111690066A (zh) * 2020-06-22 2020-09-22 南京融捷康生物科技有限公司 抗IL-4Rα的单域抗体以及应用和药物

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