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WO2025053658A1 - Nouvel anticorps à maturation d'affinité - Google Patents

Nouvel anticorps à maturation d'affinité Download PDF

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Publication number
WO2025053658A1
WO2025053658A1 PCT/KR2024/013460 KR2024013460W WO2025053658A1 WO 2025053658 A1 WO2025053658 A1 WO 2025053658A1 KR 2024013460 W KR2024013460 W KR 2024013460W WO 2025053658 A1 WO2025053658 A1 WO 2025053658A1
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seq
sequence represented
light chain
heavy chain
amino acid
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Korean (ko)
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김정호
김범석
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Good T Cells Inc
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Good T Cells Inc
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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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/10—Cellular immunotherapy characterised by the cell type used
    • A61K40/11—T-cells, e.g. tumour infiltrating lymphocytes [TIL] or regulatory T [Treg] cells; Lymphokine-activated killer [LAK] cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K40/00—Cellular immunotherapy
    • A61K40/30—Cellular immunotherapy characterised by the recombinant expression of specific molecules in the cells of the immune system
    • A61K40/31—Chimeric antigen receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/7051—T-cell receptor (TcR)-CD3 complex
    • 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
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to a novel binding molecule having superior binding affinity to the Lrig-1 antigen compared to existing antibodies and its use in the prevention, improvement or treatment of cancer, immune-related diseases and neurological diseases.
  • immunologic unresponsiveness or tolerance This tolerance is caused by eliminating lymphocytes that may have specific receptors for self-antigens, or by inactivating the function of responding to self-antigens after exposure to them. If there is a problem in inducing or maintaining tolerance, an immune response to self-antigens occurs, and the disease caused by this is called an autoimmune disease.
  • suppressor T cells which are T cells that can control and suppress the effector function of conventional T cells
  • Treg regulatory T cells
  • regulatory T cells play an important role in naturally preventing the occurrence of excessive inflammation and immune responses, but the function and number of regulatory T cells are significantly reduced when autoimmune diseases and chronic inflammatory diseases occur. Therefore, it is important for patients with immune diseases and inflammatory diseases to produce regulatory T cells at a normal level, and this can be one of the treatments for the above diseases.
  • One purpose of the present invention is to provide a binding molecule having superior binding ability to Lrig-1 protein present on the surface of regulatory T cells (Treg) compared to existing antibodies and having excellent effects in preventing, improving or treating immune-related diseases.
  • Another object of the present invention is to provide a composition for preventing, improving or treating a neurological disease comprising a binding molecule according to the present invention.
  • Another object of the present invention is to provide a composition for preventing, improving or treating cancer comprising a binding molecule according to the present invention.
  • Another object of the present invention is to provide a composition for diagnosing cancer, immune-related diseases or neurological diseases; a kit and a method for providing information for diagnosing them.
  • Antigen-binding fragments include, in particular, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of an immunoglobulin sufficient to bind a specific antigen to the polypeptide, and the like.
  • the fragments may be produced synthetically or by enzymatic or chemical degradation of complete immunoglobulins, or may be produced genetically by recombinant DNA techniques. Methods for their production are well known in the art.
  • the "Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein” is a transmembrane protein consisting of 1091 amino acids present on the surface of regulatory T cells, and is composed of a leucine repeat sequence (leucine-rich repeat (LRR)) on the extracellular or lumen side, three immunoglobulin-like domains, a transmembrane sequence, and a cytoplasmic tail.
  • LRR leucine-rich repeat
  • the LRIG gene family consists of LRIG1, LRIG2, and LRIG3, and the amino acids among them are very conservatively composed.
  • the LRIG1 gene is highly expressed in normal skin, and can regulate the proliferation of epithelial stem cells by being expressed in basal and hair follicle cells.
  • the Lrig-1 protein may be a protein existing in humans or mice, but is not limited thereto.
  • the binding molecule may further include an Fc region (Fragment crystallization region) or a constant region.
  • the Fc region may be an Fc region of an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 antibody, or may be derived therefrom, or may be a hybrid Fc region.
  • the Fc region may be an Fc region of a mammalian IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 antibody, and preferably, may be an Fc region of a human-derived IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 antibody, but is not limited thereto.
  • the Fc region may be, but is not limited to, a human-derived immunoglobulin lambda constant region.
  • the "hybrid Fc" can be derived from a combination of human IgG subclasses or a combination of human IgD and IgG.
  • the hybrid Fc binds to a biologically active molecule, polypeptide, etc., it not only increases the serum half-life of the biologically active molecule, but also has the effect of increasing the expression level of the polypeptide when a nucleotide encoding the Fc-polypeptide fusion protein is expressed.
  • the Fc or constant region may be connected to the variable region by a linker.
  • the linker may be connected to the C-terminus of the Fc or constant region, and the N-terminus of the binding molecule of the present invention may be connected to the linker, but is not limited thereto.
  • the "linker” may include a sequence that can be cleaved by an enzyme that is overexpressed in the tissue or cell of the target disease.
  • the linker can be cleaved by the overexpressed enzyme as described above, the activity of the polypeptide can be effectively prevented from being reduced due to the Fc or constant region.
  • a preferred example of the linker may be a peptide linker consisting of 33 amino acids located at positions 282 to 314 of human albumin, which is most abundant in blood, more preferably a peptide linker consisting of 13 amino acids located at positions 292 to 304, and such a portion is a portion that is mostly exposed to the outside in terms of its three-dimensional structure and is a portion that has a minimal possibility of inducing an immune response in the body.
  • the present invention is not limited thereto.
  • the binding molecule of the present invention is characterized by, but is not limited to, an antibody or a fragment thereof.
  • the antibody includes all antibody fragments that have the ability to bind to Lrig-1 protein as a monoclonal antibody, a full-length antibody, or a portion of an antibody and can competitively bind to the Lrig-1 antigenic determinant site with the binding molecule of the present invention.
  • the "antibody” refers to a protein molecule that acts as a receptor that specifically recognizes an antigen, including an immunoglobulin molecule that immunologically has a specific antigen reactivity.
  • the antigen may be an Lrig-1 protein present on the surface of a regulatory T cell. Preferably, it may be one that specifically recognizes the Leucine Rich Region or the immunoglobulin-like domain of the Lrig-1 protein, but is not limited thereto.
  • the "immunoglobulin” has a heavy chain and a light chain, and each of the heavy chain and the light chain includes a constant region and a variable region.
  • the variable regions of the light chain and the heavy chain include three variably variable regions called complementarity determining regions (hereinafter referred to as "CDRs") and four framework regions.
  • CDRs mainly play a role in binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 in sequence starting from the N-terminus, and are also identified by the chain on which a specific CDR is located.
  • the "monoclonal antibody” in the present invention refers to an antibody molecule of a single molecular composition obtained from a substantially identical antibody population, which exhibits a single binding specificity and affinity for a specific antigenic determinant (Epitope).
  • the "full-length antibody” has a structure having two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond, and includes IgA, IgD, IgE, IgM, and IgG.
  • the IgG includes IgG1, IgG2, IgG3, and IgG4 as its subtypes.
  • the "antibody fragment” in the present invention means a fragment having an antigen binding function, and includes Fab, Fab', F(ab')2, and Fv, etc.
  • the Fab has one antigen binding site in a structure having variable regions of the light chain and the heavy chain, a constant region of the light chain, and a first constant region (CH1 domain) of the heavy chain.
  • Fab' differs from Fab in that it has a hinge region including one or more cysteine residues at the C-terminus of the heavy chain CH1 domain.
  • F(ab')2 antibody is generated when cysteine residues in the hinge region of Fab' form a disulfide bond.
  • Fv (Variable fragment) means the minimum antibody fragment having only a heavy chain variable region and a light chain variable region.
  • Double-chain Fv (dsFv) has a heavy chain variable region and a light chain variable region linked by a disulfide bond
  • single-chain Fv (scFv) has a heavy chain variable region and a light chain variable region covalently linked, usually via a peptide linker.
  • the antibody fragments can be obtained as Fab or F(ab')2 fragments using a protein hydrolytic enzyme, such as papain or pepsin, and can be produced through genetic recombination technology.
  • the antibody may be, but is not limited to, a chimeric antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a unibody, a diabody, a triabody, a tetrabody, or a fragment thereof.
  • the "chimeric antibody” is an antibody in which the variable region of a mouse antibody and the constant region of a human antibody are recombined, and is an antibody with a greatly improved immune response compared to a mouse antibody.
  • the binding molecule can be provided as a bispecific antibody or bispecific antigen-binding fragment that can bind to Lrig-1 protein and also bind to other proteins.
  • the method for producing the bispecific antibody of the present invention comprises chemical cross-linking of antibodies or antibody fragments with a reducing disulfide or non-reducing thioether bond.
  • a reducing disulfide or non-reducing thioether bond For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to chemically cross-link Fab fragments, for example, via hinge region SH- groups, to produce disulfide-linked bispecific F(ab)2 heterodimers.
  • SPDP N-succinimidyl-3-(-2-pyridyldithio)-propionate
  • Antibodies can be produced by an affinity maturation process, which produces a modified antibody having improved affinity for the antigen compared to the unmodified parent antibody. Affinity matured antibodies can be produced by procedures known in the art.
  • amino acid mutations are made on the basis of the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc.
  • Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine, and histidine; alanine, glycine, and serine; and phenylalanine, tryptophan, and tyrosine can be said to be biologically functional equivalents.
  • hydrophobic index of amino acids can be considered.
  • Each amino acid is assigned a hydrophobic index according to its hydrophobicity and charge: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the hydrophobic amino acid index is very important in imparting interactive biological functions to proteins. It is a known fact that amino acids having similar hydrophobic indices must be substituted in order to have similar biological activities. When introducing mutations with reference to hydrophobic indices, substitutions are made between amino acids having hydrophobic indices that are preferably within ⁇ 2, more preferably within ⁇ 1, and even more preferably within ⁇ 0.5.
  • Amino acid exchanges in proteins that do not alter the overall activity of the molecule are known in the art.
  • the most common exchanges are those between amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Gln/Glu.
  • binding molecule of the present invention also includes sequences showing substantial identity with the sequences described in the sequence listing.
  • substantially identical means a sequence that exhibits at least 61% homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology when the sequence of the present invention and any other sequence are paralleled to the greatest extent possible and the parallel sequences are analyzed using an algorithm commonly used in the art. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are accessible from the NCBI Basic Local Alignment Search Tool (BLAST), NBCI (National Center for Biological Information), etc., and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet.
  • BLAST Basic Local Alignment Search Tool
  • NBCI National Center for Biological Information
  • BLAST can be accessed at this address (ncbi.nlm.nih.gov/BLAST/).
  • a method for comparing sequence homology using this program can be checked online (ncbi.nlm.nih.gov/BLAST/blast_help.html).
  • the binding molecule preferably the antibody
  • the binding molecule may be produced by a conventional method for producing antibodies, but may be produced by affinity maturation.
  • the “H6 antibody” or “GTC110-04” antibody is a monoclonal antibody identical to a known antibody that has a preventive and therapeutic effect on cancer or brain and nervous system diseases.
  • the “GTC310-01 antibody” or “1C07” antibody is a monoclonal antibody identical to a known antibody that has a preventive and therapeutic effect on brain and nervous system diseases.
  • the affinity maturation refers to a process in which activated B cells produce antibodies with increased affinity for an antigen, i.e., antibodies with enhanced binding ability to an antigen, during an immune response process.
  • the affinity maturation can produce antibodies or antibody fragments produced by affinity maturation based on the principles of mutation and selection, in the same manner as processes occurring in nature.
  • the ten affinity-matured antibody clones (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01) provided in the present invention are affinity-matured antibodies from the known H6 or GTC310-01 antibody, in which only one of the six CDRs of the GTC310-01 antibody is partially modified (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8 and MH6) or in which only three CDRs included in the light chain are modified (6F01), and have the characteristic of higher binding affinity to Lrig-1 than the H6 or GTC310-01 antibody.
  • these antibodies have improved binding affinity for binding to the antigen Lrig-1 while retaining the pharmacological effect of the existing H6 or GTC310-01 antibodies because only a portion of the entire 6CDR has been modified.
  • an antibody or antigen-binding fragment is determined by which antigen the antibody or antigen-binding fragment binds to. Therefore, it is already widely known in the relevant technical field that an antibody with a superior binding affinity for the same antigen compared to an antibody with a known existing pharmacological effect will have the same pharmacological effect, but with an effect that is the same or superior to that of an antibody with a known existing effect.
  • the 10 affinity-matured antibody clones (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01) provided by the present invention have a KD value that is 2 to 737 times lower than that of the GTC310-01 antibody or H6 antibody, which have previously been shown to have pharmacological effects, indicating a high binding affinity the lower the KD value, or a binding affinity for CD4+FoxP3+Lrig1+ regulatory T cells that is 90% or higher when 5 ug of the antibodies are administered.
  • the antibodies provided in the present invention which have a KD value that is 2 to 737 times lower than that of the GTC310-01 antibody, which has a known pharmacological effect, and thus exhibit a high binding affinity the lower the KD value, or have a binding affinity for CD4+FoxP3+Lrig1+ regulatory T cells that is 90% or higher when 5 ug of the antibodies are administered, have pharmacological effects that are the same as or superior to the existing H6 or GTC310-01 antibodies.
  • the binding molecule preferably an antibody, provided by the present invention can effectively prevent, improve or treat immune-related diseases, neurological diseases, and cancer.
  • nucleic acid molecule encoding the binding molecule provided in the present invention is provided.
  • the nucleic acid molecule of the present invention includes all nucleic acid molecules translated into polynucleotide sequences, as is known to those skilled in the art, of the amino acid sequence of the binding molecule provided in the present invention. Therefore, various polynucleotide sequences can be prepared by ORF (open reading frame), and all of these are also included in the nucleic acid molecule of the present invention.
  • ORF open reading frame
  • an expression vector into which the isolated nucleic acid molecule provided by the present invention is inserted is provided.
  • the "vector” is a nucleic acid molecule capable of transporting another nucleic acid to which a nucleic acid molecule is linked.
  • a vector which refers to a circular double-stranded DNA to which additional DNA segments can be ligated.
  • a phage vector Another type of vector is a viral vector, to which additional DNA segments can be ligated to the viral genome.
  • Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., a bacterial vector is an episomal mammalian vector having a bacterial origin of replication).
  • vectors e.g., a non-episomal mammalian vector
  • a non-episomal mammalian vector can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome.
  • some vectors can direct the expression of genes to which they are linked in an operational manner.
  • Such vectors are referred to herein as "recombinant expression vectors” or simply "expression vectors.”
  • expression vectors useful in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” may be used interchangeably, as the plasmid is the most commonly used form of vector.
  • the "antibody-drug conjugate (ADC)" refers to a form in which a drug and an antibody are chemically linked without reducing the biological activity of the antibody and the drug.
  • the antibody-drug conjugate refers to a form in which a drug is bound to an amino acid residue at the N-terminus of the heavy chain and/or light chain of an antibody, specifically, a form in which a drug is bound to an ⁇ -amine group at the N-terminus of the heavy chain and/or light chain of an antibody.
  • the drug may be included regardless of its type as long as it is a drug that can treat immune-related diseases, brain and nervous system diseases, and cancer.
  • the "autoimmune disease” of the present invention refers to a disease that can occur when an abnormality in the immune function occurs and attacks the organs or tissues of our body.
  • it can occur when the function of T cells, particularly regulatory T cells, does not function normally and an immune response occurs against self-antigens.
  • the autoimmune disease may be selected from the group consisting of, for example, rheumatoid arthritis, systemic scleroderma, systemic lupus erythematosus, atopic dermatitis, psoriasis, alopecia areata, asthma, Crohn's disease, Behcet's disease, Sjogren's syndrome, Guilla-Barre syndrome, chronic thyroiditis, multiple sclerosis, polymyositis, ankylosing spondylitis, fibromyalgia, and polyarteritis nodosa, but is not limited thereto.
  • the “neurological disease” that the composition provided in the present invention is intended to prevent, improve or treat may be a neurodegenerative disease or a neuroinflammatory disease.
  • the “neurodegenerative disease” may mean a disease caused by a decrease or loss of function of nerve cells
  • the “neuroinflammatory disease” may mean a disease caused by an excessive inflammatory response of the nervous system.
  • specific examples of the neurodegenerative disease or neuroinflammatory disease include multiple sclerosis, stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy. May be selected from the military, but is not limited thereto.
  • neuroinflammation and degenerative neurological diseases that may arise from it by reducing the expression level of proinflammatory mediators within these glial cells.
  • Activated glial cells have been reported to cause various central nervous system diseases such as multiple sclerosis, Parkinson's disease, Alzheimer's disease, and Huntington's disease by sequentially inducing the activity of nitric oxide (NO), reactive oxygen species (ROS), inflammatory enzymes such as inducible nitric oxidesynthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory mediators such as IL-1 ⁇ , IL-6, and tumor necrosis factor- ⁇ (Gao et al., 2002; Nelson et al., 2002; Eikelenboom and Van Gool, 2004).
  • NO nitric oxide
  • ROS reactive oxygen species
  • COX-2 cyclooxygenase-2
  • proinflammatory mediators such as IL-1 ⁇ , IL-6, and tumor necrosis factor- ⁇
  • composition provided by the present invention can be used as a pharmaceutical composition or a food composition, but is not limited thereto.
  • the above “prevention” of the present invention may include, without limitation, any act that can block, suppress or delay symptoms caused by a neurodegenerative disease or a neuroinflammatory disease by using the above composition of the present invention.
  • treatment and “improvement” of the present invention may include, without limitation, any act that can improve or benefit symptoms caused by a neurodegenerative disease or a neuroinflammatory disease by using the above composition of the present invention.
  • the pharmaceutical composition may be characterized as being in the form of a capsule, tablet, granule, injection, ointment, powder or beverage, and the pharmaceutical composition may be characterized as being intended for humans.
  • the pharmaceutical composition of the present invention is not limited thereto, but may be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injectable solutions, each according to a conventional method.
  • the pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, pigments, fragrances, etc. for oral administration, and may include buffers, preservatives, analgesics, solubilizers, isotonic agents, stabilizers, and the like for injections.
  • compositions of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers described above.
  • pharmaceutically acceptable carriers for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dose ampoules or multiple doses.
  • it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.
  • examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate or mineral oil.
  • fillers, anticoagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be additionally included.
  • the routes of administration of the pharmaceutical composition according to the present invention include, but are not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal. Oral or parenteral administration is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.
  • the pharmaceutical composition of the present invention may vary depending on various factors including the activity of the specific compound used, age, body weight, general health, sex, dosage, administration time, administration route, excretion rate, drug combination, and the severity of the specific disease to be prevented or treated, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by those skilled in the art, and may be administered at 0.0001 to 50 mg/kg or 0.001 to 50 mg/kg per day. The administration may be administered once a day or divided into several times. The dosage does not limit the scope of the present invention in any way.
  • the pharmaceutical composition according to the present invention may be formulated as a pill, a dragee, a capsule, a solution, a gel, a syrup, a slurry, or a suspension.
  • a food composition containing the composition of the present invention as an active ingredient can be manufactured in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, etc. Since the food composition of the present invention is composed of plant extracts with almost no toxicity or side effects, it can be used safely even when taken for a long period of time for preventive purposes.
  • the amount may be added in a ratio of 0.1 to 50% of the total weight.
  • the food composition when the food composition is manufactured in the form of a beverage, there is no special limitation other than containing the food composition in the indicated ratio, and various flavoring agents or natural carbohydrates, etc. can be contained as additional ingredients like a typical beverage. That is, as the natural carbohydrate, it can include monosaccharides such as glucose, disaccharides such as fructose, sucrose, and polysaccharides, dextrin, cyclodextrin, and typical sugars, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • flavoring agent there can be mentioned natural flavoring agents (thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.).
  • natural flavoring agents thaumatin, stevia extracts (e.g., rebaudioside A, glycyrrhizin, etc.)
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.
  • These components can be used independently or in combination.
  • the proportion of these additives is not so critical, but is typically selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.
  • a binding molecule comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26; and a light chain CDR2 represented by a selected amino acid sequence represented by SEQ ID NO: 27.
  • the binding molecule comprises a heavy chain variable region further comprising a heavy chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 25 and 43; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 26 and 79; a light chain CDR2 represented by an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 and 80; and a light chain CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 47, 51, 55, 59, 63, 67, 71, 75 and 81.
  • the binding molecule comprises a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; and a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; and a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35.
  • the binding molecule comprises a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 33 and 45; and a light chain variable region comprising a light chain CDR1 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 34 and 84; a light chain CDR2 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 35 and 85; and a light chain CDR3 encoded by a base sequence selected from the group consisting of SEQ ID NOs: 36, 49, 53, 57, 61, 65, 69, 73, 77 and 86.
  • a binding molecule comprising a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 25; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26, a light chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 27, and a light chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 28; and a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23, a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24, and a heavy chain CDR3 comprising an amino acid sequence represented by SEQ ID NO: 43; And a light chain variable region comprising a light chain CDR
  • the binding molecule comprises a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 33; and a light chain variable region comprising a light chain CDR1 encoded by a base sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a base sequence represented by SEQ ID NO: 35; and a light chain CDR3 encoded by a base sequence represented by SEQ ID NO: 36; and a binding molecule comprising a heavy chain variable region comprising a heavy chain CDR1 encoded by a base sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a base sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a base sequence represented by SEQ ID NO: 45;
  • a binding molecule comprising a light chain variable region comprising a light chain CDR2 encoded by a nucleotide sequence represented by SEQ ID NO: 35; a light chain CDR3 encoded by a nucleotide sequence represented by SEQ ID NO: 36; and a heavy chain variable region comprising a heavy chain CDR1 encoded by a nucleotide sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a nucleotide sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a nucleotide sequence represented by SEQ ID NO: 33; and a light chain CDR1 encoded by a nucleotide sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a nucleotide sequence represented by SEQ ID NO: 35; a light chain CDR3 encoded by a nucleotide sequence represented by SEQ ID NO: 49; and a heavy chain variable region comprising a heavy chain C
  • a binding molecule comprising a light chain variable region comprising a light chain CDR3; encoded by a nucleotide sequence represented by SEQ ID NO: 73; and a heavy chain variable region comprising a heavy chain CDR1 encoded by a nucleotide sequence represented by SEQ ID NO: 31; a heavy chain CDR2 encoded by a nucleotide sequence represented by SEQ ID NO: 32; a heavy chain CDR3 encoded by a nucleotide sequence represented by SEQ ID NO: 33; and a light chain CDR1 encoded by a nucleotide sequence represented by SEQ ID NO: 34; a light chain CDR2 encoded by a nucleotide sequence represented by SEQ ID NO: 35; a light chain CDR3 encoded by a nucleotide sequence represented by SEQ ID NO: 77; and a light chain variable region comprising a heavy chain CDR1 encoded by a nucleotide sequence represented by SEQ ID NO: 31; a
  • a binding molecule comprising a light chain variable region, wherein the light chain CDR2 is encoded by a nucleotide sequence represented by SEQ ID NO: 85; and a light chain CDR3 is encoded by a nucleotide sequence represented by SEQ ID NO: 86.
  • a heavy chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 29, 44 and 82;
  • a binding molecule comprising a light chain variable region comprising any one amino acid sequence selected from the group consisting of SEQ ID NOs: 30, 48, 52, 56, 60, 64, 68, 72, 76 and 83.
  • a heavy chain variable region encoded by any one of the base sequences selected from the group consisting of SEQ ID NOs: 37, 46 and 87;
  • a binding molecule comprising a light chain variable region encoded by any one nucleotide sequence selected from the group consisting of SEQ ID NOs: 38, 50, 54, 58, 62, 66, 70, 74, 78 and 88.
  • the binding molecule further comprises an Fc region or a constant region.
  • the binding molecule provides a Fc region of an IgG1, IgG2, IgG3 or IgG4 antibody, or a hybrid Fc region.
  • the binding molecule further comprises a heavy chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 3, 4, 7, 8, 9, 10 and 12.
  • the binding molecule further comprises a light chain constant region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 6 and 11.
  • a binding molecule further comprising: a heavy chain constant region encoded by a base sequence selected from the group consisting of SEQ ID NOs: 13, 15, and 17; and a light chain constant region encoded by a base sequence selected from the group consisting of SEQ ID NOs: 14, 16, and 18.
  • a binding molecule is provided, wherein the binding molecule is an antibody or a fragment thereof.
  • the antibody provides a binding molecule which is a chimeric antibody, a humanized antibody, a bivalent, a bispecific molecule, a minibody, a domain antibody, a bispecific antibody, an antibody mimetic, a diabody, a triabody, a tetrabody or a fragment thereof.
  • a nucleic acid molecule encoding the binding molecule is provided.
  • an expression vector having the nucleic acid molecule inserted therein is provided.
  • a host cell line transfected with the expression vector is provided.
  • an antibody-drug conjugate comprising the binding molecule and a drug is provided.
  • a chimeric antigen receptor comprising an antigen-specific binding domain, a linking domain and a CD3 zeta ( ⁇ ) signaling domain
  • the antigen-specific binding domain comprises a heavy chain variable region comprising a heavy chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 23; a heavy chain CDR2 comprising an amino acid sequence represented by SEQ ID NO: 24; and a light chain variable region comprising a light chain CDR1 comprising an amino acid sequence represented by SEQ ID NO: 26; and a light chain CDR2 represented by a selected amino acid sequence represented by SEQ ID NO: 27; and a chimeric antigen receptor which is a fusion protein comprising an antigen-specific binding domain; and an immunoglobulin Fc region.
  • a pharmaceutical composition for preventing or treating cancer comprising the binding molecule as an active ingredient.
  • a pharmaceutical composition wherein the cancer is a solid cancer.
  • a pharmaceutical composition wherein the cancer is gastric cancer, liver cancer, glioma, ovarian cancer, colon cancer, head and neck cancer, bladder cancer, renal cell cancer, breast cancer, metastatic cancer, prostate cancer, pancreatic cancer, melanoma, or lung cancer.
  • a pharmaceutical composition for preventing or treating a neurological disease comprising the binding molecule as an active ingredient is provided.
  • a pharmaceutical composition wherein the neurological disease is a neurodegenerative disease or a neuroinflammatory disease.
  • the neurodegenerative disease or neuroinflammatory disease is selected from the group consisting of stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy, progressive supranuclear palsy, nervous system autoimmune diseases, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury, and tauopathy.
  • a pharmaceutical composition of choice is provided.
  • a pharmaceutical composition for preventing or treating an immune-related disease comprising the binding molecule as an active ingredient.
  • a pharmaceutical composition wherein the immune-related disease is an autoimmune disease; graft-versus-host disease; organ transplant rejection; asthma; atopy; or an acute or chronic inflammatory disease.
  • the binding molecule provided by the present invention has a superior binding affinity for the Lrig-1 antigen, and thus can specifically prevent, improve, or treat various immune-related diseases, brain and nervous system diseases, and cancer.
  • Figure 2 is a line graph showing the ELISA results of 11 other antibodies that underwent affinity maturation with mLrig1-His.
  • Figure 4 is a graphical comparison of the binding affinities of 10 affinity-matured antibodies compared to the control H6 antibody and GTC310-01 antibody.
  • Figure 5 is a schematic diagram of an experimental design for the cancer treatment effect using a monoclonal antibody (H6) specific for the Lrig-1 protein according to one embodiment of the present invention.
  • H6 monoclonal antibody
  • Figure 6 shows the results of analyzing the cancer treatment effect using a monoclonal antibody (H6) specific for Lrig-1 protein according to one embodiment of the present invention.
  • FIG. 7 is a schematic diagram of an experimental design for the cancer treatment effect using an affinity-matured monoclonal antibody (ML2) specific for Lrig-1 protein according to one embodiment of the present invention.
  • ML2 affinity-matured monoclonal antibody
  • Figure 8 shows the results of analyzing the cancer treatment effect using an affinity matured monoclonal antibody (ML2) specific for Lrig-1 protein according to one embodiment of the present invention.
  • ML2 affinity matured monoclonal antibody
  • Figure 10 is a graph showing the results of calculating the ThS+ area ratio (%) compared to a normal mouse after staining the brain cortex and hippocampus tissue of the mouse obtained after each treatment including a monoclonal antibody according to one embodiment of the present invention in an Alzheimer's disease-induced mouse with thioflavin S (ThS).
  • ThS thioflavin S
  • Figure 11 is a graph showing the change in spontaneous alteration (%) value in a Y maze experiment after each treatment including a monoclonal antibody according to one embodiment of the present invention in Alzheimer's disease-induced mice.
  • Figure 12 is a graph showing the change in preference value of a novel object recognition experiment after each treatment including a monoclonal antibody according to one embodiment of the present invention in an Alzheimer's disease-induced mouse.
  • Figure 13 shows an experimental design diagram of 5xFAD mice and 6xTg mice, which are Alzheimer's disease-induced mice, to confirm the Alzheimer's treatment effect of a monoclonal antibody according to one embodiment of the present invention.
  • Figure 14 is a graph showing the change in spontaneous alteration (%) value in a Y maze experiment after each treatment including a monoclonal antibody according to one embodiment of the present invention in 5xFAD mice and 6xTg mice, which are Alzheimer's disease-induced mice.
  • Figure 15 is a graph showing the change in preference value of a novel object recognition experiment after each treatment including a monoclonal antibody according to one embodiment of the present invention in 6xTg mice, which are Alzheimer's disease-induced mice.
  • Figure 16 is a graph showing the change in retention time as a result of a passive avoidance experiment after each treatment including a monoclonal antibody according to one embodiment of the present invention in 5xFAD mice and 6xTg mice, which are Alzheimer's disease-induced mice.
  • Figure 17 illustrates an experimental design diagram for confirming the therapeutic effect of a monoclonal antibody on neurological diseases and autoimmune diseases according to one embodiment of the present invention.
  • Figure 18 shows the therapeutic effects of MH6 and 6F01 antibodies on neurological diseases and autoimmune diseases.
  • Figure 19 illustrates an experimental design diagram for confirming the therapeutic effect of a monoclonal antibody on neurological diseases and autoimmune diseases according to one embodiment of the present invention.
  • Figure 20 shows the therapeutic effect of ML4 antibody on neurological diseases and autoimmune diseases.
  • cells expressing the Lrig-1 protein were produced. More specifically, the DNA fragment corresponding to sequence number 20 and pcDNA (hygro) were cut with a cutting enzyme, and then cultured at 37°C and ligated to produce pcDNA having the DNA sequence of the Lrig-1 protein inserted. The produced pcDNA having the sequence number 20 inserted was introduced into L cells through transfection, so that the Lrig-1 protein could be expressed on the surface of the L cells.
  • sequences of light and heavy chain amino acids that can bind to Lrig-1 expressed on the cell surface were selected from a human scFv library.
  • 3% skim milk and HA-HRP secondary antibody
  • 50ul/well was loaded, and 50ul of substrate solution was added.
  • 50ul of sulfuric acid or Stop buffer was added to stop the reaction. Then, the absorbance at 450nm was measured using a plate reader.
  • the Kd value of the previously manufactured 6F01 antibody was about half that of the H6 antibody, indicating that it binds to his-tagged mouse Lrig-1 (antigen) about twice as well as the H6 antibody.
  • PBMCs Peripheral blood mononuclear cells
  • naive CD4+ T cells were dispensed per well and treated with TGF- ⁇ 5 ng/mL and IL-2 20 ng/mL at a concentration. The cells were then cultured in an incubator for 4 days. Each differentiated cell (200ul) was pipetted and transferred to a round well. After centrifugation at 2,500 rpm for 2 minutes, for staining of cell surface proteins, the medium was removed and 50 ⁇ l of PBS and antibodies were mixed and pipetted per well. The plate was wrapped in foil and incubated at 4°C for 30 minutes.
  • the plate was wrapped in foil and incubated at 4°C for 30 minutes.
  • 150 ⁇ l of 1x permeation buffer was additionally added, and centrifuged at 2,500 rpm for 2 minutes.
  • 200 ⁇ l of PBS was added, dissolved, and centrifuged at 2,500 rpm for 2 minutes to wash.
  • the KD values which indicate higher affinity for the antigen as they are lower, are at least 2-fold and at most 737-fold lower for the affinity-matured novel antibodies than for the GTC310-01 antibody. Therefore, it was confirmed that the binding affinity for the Lrig-1 antigen is at least 2-fold and at most 737-fold higher than that of the existing GTC310-01 antibody.
  • the existing Lrig-1 protein-specific monoclonal antibody can effectively prevent, improve, or treat various solid cancer cells by inhibiting their growth.
  • the ML2 antibody which binds specifically to CD4+Foxp3+Lrig1+ cells at a concentration of 5 ug approximately 9 times more, i.e., more than 90%, compared to the existing H6 antibody, was selected based on Fig. 4, and its cancer therapeutic effect was confirmed.
  • a CT26 mouse model was prepared, and CT26 cancer cells were subcutaneously injected into the back of 7-week-old female mice at a dose of 3X105 cells/200ul, and the size of the cancer cells was measured after 7 days.
  • ML2 antibody was injected intraperitoneally at concentrations of 100ug, 200ug, and 500ug at 2-day intervals, and the size of the cancer cells was measured, and the results are shown in Fig. 8.
  • the novel affinity-matured ML2 antibody provided by the present invention can treat cancer by reducing the size of cancer compared to the control group. Therefore, it will be clearly understood by those skilled in the art that other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01) that specifically bind to CD4+Foxp3+Lrig1+ cells at a concentration of 5 ug, that is, more than 90%, can treat cancer by specifically binding to the Lrig-1 protein of regulatory T cells and inhibiting the function of regulatory T cells, thereby activating effector T cells.
  • affinity-matured antibodies ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6 and 6F01
  • the H6 antibody with relatively low binding affinity for the Lrig-1 protein also has an anticancer effect, and among other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01) that bind specifically to CD4+Foxp3+Lrig1+ cells about 9 times more, i.e., more than 90%, at a concentration of 5 ug compared to the existing H6 antibody, the anticancer effect of the ML2 antibody was confirmed, so it was confirmed that other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01) that bind specifically to CD4+Foxp3+Lrig1+ cells about 9 times more, i.e., more than 90%, at a concentration of 5 ug compared to the existing H6 antibody also have an anticancer effect on regulatory T cells.
  • affinity-matured antibodies ML1, ML2,
  • a group was designed as shown in Fig. 9.
  • the GTC310-01 mouse antibody of the above-mentioned manufacturing example was intravenously injected at a dose of 10 mpk for 3 weeks into male Alzheimer's disease-induced 5xFAD mice aged 5 to 6 months.
  • glatiramer acetate glatiramer acetate, GA, Copaxone®
  • the H6 antibody GTC110-04 antibody
  • the above-mentioned manufacturing example was intravenously injected at a dose of 10 mpk for 4 weeks.
  • mice were euthanized, and the cortical and hippocampal tissues were obtained and fixed in 4% paraformaldehyde (pH 7.4) for 16 h.
  • the fixed cortical and hippocampal brain tissues were immersed in 30% sucrose for cryoprotection and sliced to 35 ⁇ m thickness using a Cryostat (Microm HM 525, Thermo Scientific, Waltham, MA, USA).
  • Cryostat Merom HM 525, Thermo Scientific, Waltham, MA, USA.
  • Thioflavin S was purchased from Sigma-Aldrich (catalog no. T-1892). 500 ⁇ M of ThS was dissolved in 50% ethanol.
  • the ThS+ area was significantly increased in Alzheimer's-induced mice, but when the existing GTC310-01 and H6 (GTC110-04) antibodies according to the above-mentioned manufacturing example were administered, the ThS+ area was significantly reduced, especially in the cortex, indicating that the effect of reducing amyloid- ⁇ plaques was very excellent.
  • the Y-maze experiment used a maze frame with three identical arms, each 40 cm long (wall height 15 cm), arranged at an angle of 120 degrees.
  • This experiment is a behavioral experiment that utilizes the instinctive exploratory behavior of rodents, and was a method that took into account the high possibility of exploring a new area. It showed a higher memory value the more it remembered the arm it explored just before and did not enter the same arm.
  • Each individual was given an exploration time of 8 minutes, and the final result was expressed as the spontaneous alteration (%) value in Fig. 13.
  • the spontaneous alteration (%) value was calculated using the following equation 1.
  • the analysis of the behavior pattern was conducted using SMART VIDEO TRACKING Software (Panlab, USA).
  • the preference index for a novel object was observed to be lower in the Alzheimer's-induced group than in the normal control group.
  • the existing GTC310-01 antibody or H6 (GTC110-04) antibody of the above-mentioned manufacturing example was administered, it was confirmed that the preference index increased more than that of the normal control group.
  • the existing GTC310-01 antibody or H6 (GTC110-04) antibody of the above-mentioned manufacturing example has an effect in preventing, improving, or treating brain nervous system diseases.
  • the GTC310-01 antibody of Manufacturing Example 1 was intravenously injected at a dose of 10 mpk every other week for 2 months into female Alzheimer's-induced 5xFAD mice and female 6xTg mice that had undergone an adaptation period of 4.5 months.
  • glatiramer acetate (GA, Copaxone®) was intravenously injected at a dose of 100 ug every other week for 2 months as a positive control. After 2 months, a Y-maze test, a novel object recognition test, and a passive avoidance test were conducted.
  • 5xFAD mice are widely used as an Alzheimer's disease model, but the 6xTg mice used in the following experiments are a mouse model in which not only amyloid- ⁇ (A ⁇ 42) but also tau protein (MAPT) is mutated and accumulates, making them a model closer to human disease models than the 5xFAD mice, making them an ideal animal model for brain and nervous system diseases including Alzheimer's disease.
  • a ⁇ 42 amyloid- ⁇
  • MTT tau protein
  • the Y-maze experiment used a maze frame with three identical arms, each 40 cm long (wall height 15 cm), arranged at an angle of 120 degrees.
  • This experiment is a behavioral experiment that utilizes the instinctive exploratory behavior of rodents, and was a method that took into account the high possibility of exploring a new area. It showed a higher memory value the more it remembered the arm it explored just before and did not enter the same arm.
  • Each individual was given an exploration time of 8 minutes, and the final result was expressed as the spontaneous alteration (%) value in Fig. 14.
  • the spontaneous alteration (%) value was calculated using the following equation 1.
  • the analysis of the behavior pattern was conducted using SMART VIDEO TRACKING Software (Panlab, USA).
  • the novel object recognition experiment evaluated memory using two different objects in a 40 cm x 40 cm acrylic cage. After familiarizing the inside of the acrylic cage, the two objects were placed in a fixed location and freely recognized, and the time spent exploring each object was measured. A delay time of 24 hours was given to each subject, and then only one object was changed to a different object in the same location. At this time, the changed object was recognized as a novel object, and the longer the exploration time, the better the memory. If the subject cannot remember the object from 24 hours ago, it is likely that the subject will not be able to distinguish between the novel object and the existing object and will explore both objects equally.
  • the preference index for a novel object was observed to be lower in the Alzheimer's-induced group than in the normal control group.
  • the GTC310-01 antibody according to the present invention was administered, it was confirmed that the preference index increased above the normal control group in the 6xTg model, which is closer to the human disease model.
  • the passive avoidance test experimental apparatus was divided into two areas, a bright chamber with lighting and a dark chamber, and the floor was made of wire mesh.
  • the mice were allowed to move freely to adapt.
  • each 5xFAD mouse and 6xTg mouse was allowed to adapt for 1 minute in the lighted chamber with the light turned off, then the light was turned on and allowed to adapt for 2 minutes.
  • an electric shock of 0.5 mA for 1 second was applied to conduct a learning test.
  • a memory test (teat trial) was conducted on each mouse.
  • 5xFAD mice and 6xTg mice were placed in the chamber with the light on, and the time it took for the 5xFAD and 6xTg mice to enter the chamber with all four paws (latency time) was set to 300 seconds and the test was performed.
  • latency time As a result, as shown in Fig. 16, it was found that when GTC310-01 antibody was administered to not only 5xFAD mice but also 6xTg mice, recovery was close to the normal control group.
  • the antibodies showing the lowest affinity and the antibodies showing the highest affinity were selected in terms of affinity to confirm the therapeutic effects on autoimmune diseases and neurological diseases. If the therapeutic effects of the above antibodies are confirmed, those skilled in the art can clearly understand the therapeutic effects of other antibodies having intermediate binding affinities compared to the above antibodies without excessive experiments or special knowledge.
  • the MH6 antibody which has a KD value about 2 times lower than that of the existing GTC310-01 antibody, and thus has the lowest Lrig-1 binding affinity among other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01), although it has a binding affinity for Lrig-1 protein about 2 times higher than that of the GTC310-01 antibody, and conversely, the ML4 antibody, which has a KD value that is 737 times lower than that of the existing GTC310-01 antibody and thus has the highest Lrig-1 binding affinity among other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01), and based on Table 2, the KD value of the existing H6 is 1.2x10 10 In comparison, the 6F01 antibody, which has a KD value of 6.1x10 11 , which is about twice as low, was confirmed to have
  • EAE experimental autoimmune encephalomyelitis
  • mice 9-week-old female C57Bl/6J mice were injected subcutaneously with MOG peptide and intraperitoneally with 260 ng of PTX.
  • 260 ng of PTX was injected once more to enhance the immune system of the mice.
  • the mice were administered 50 ug of the 6F01 and MH6 antibodies produced in the above manufacturing example, and 50 ug of anti-IL-17a antibody was administered as a positive control.
  • the ML4 antibody which has the highest binding affinity for Lrig-1 among other affinity-matured antibodies in the multiple sclerosis-induced model, can also significantly treat multiple sclerosis.
  • the KD value was about 2 times lower than that of the existing GTC310-01 antibody based on FIG. 4, so although the binding affinity for Lrig-1 protein was about 2 times higher than that of the GTC310-01 antibody, the MH6 antibody had the lowest Lrig-1 binding affinity among other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01), while the MH6 antibody had the highest Lrig-1 binding affinity among other affinity-matured antibodies (ML1, ML2, ML3, ML4, ML5, ML6, ML7, ML8, MH6, and 6F01) because the KD value was 737 times lower than that of the existing GTC310-01 antibody.

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Abstract

La présente invention concerne une molécule de liaison qui peut se lier de manière spécifique à une protéine Lrig-1, qui est une protéine présente à la surface d'un lymphocyte T régulateur. La molécule de liaison fournie par la présente invention peut prévenir, atténuer ou traiter efficacement le cancer, en particulier le cancer solide, par la suppression de la fonction des lymphocytes T régulateurs, peut cibler efficacement la protéine Lrig-1, par comparaison avec des anticorps anti-Lrig-1 disponibles dans le commerce, et a une excellente force d'affinité de liaison pour celle-ci.
PCT/KR2024/013460 2023-09-06 2024-09-06 Nouvel anticorps à maturation d'affinité Pending WO2025053658A1 (fr)

Applications Claiming Priority (2)

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KR20230118149 2023-09-06
KR10-2023-0118149 2023-09-06

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WO2025053658A1 true WO2025053658A1 (fr) 2025-03-13

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180081465A (ko) * 2017-01-06 2018-07-16 에이비엘바이오 주식회사 항 α-syn 항체 및 그 용도
KR20180117067A (ko) * 2017-04-18 2018-10-26 주식회사 굳티셀 Lrig-1 단백질에 특이적인 결합 분자 및 이의 용도
KR20190124665A (ko) * 2018-04-26 2019-11-05 주식회사 굳티셀 신규한 융합 단백질 및 이를 포함하는 암의 예방 또는 치료용 약학적 조성물
KR20200112745A (ko) * 2019-03-20 2020-10-05 주식회사 굳티셀 뇌신경계 질환의 예방 또는 치료용 조성물
KR20220108747A (ko) * 2021-01-27 2022-08-03 주식회사 굳티셀 신규한 결합 분자 및 이의 용도

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20180081465A (ko) * 2017-01-06 2018-07-16 에이비엘바이오 주식회사 항 α-syn 항체 및 그 용도
KR20180117067A (ko) * 2017-04-18 2018-10-26 주식회사 굳티셀 Lrig-1 단백질에 특이적인 결합 분자 및 이의 용도
KR20180117066A (ko) * 2017-04-18 2018-10-26 주식회사 굳티셀 Lrig-1 단백질에 특이적인 결합 분자 및 이의 용도
KR20190124665A (ko) * 2018-04-26 2019-11-05 주식회사 굳티셀 신규한 융합 단백질 및 이를 포함하는 암의 예방 또는 치료용 약학적 조성물
KR20200112745A (ko) * 2019-03-20 2020-10-05 주식회사 굳티셀 뇌신경계 질환의 예방 또는 치료용 조성물
KR20220108747A (ko) * 2021-01-27 2022-08-03 주식회사 굳티셀 신규한 결합 분자 및 이의 용도

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