WO2023195834A1 - Pharmaceutical composition for treating neuropathic pain, comprising mincle inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating neuropathic pain, the composition comprising, as an active ingredient, an epitope of a macrophage-inducible C-type lectin (Mincle) protein, wherein the epitope comprises at least one polypeptide selected from the group consisting of polypeptides consisting of amino acids represented by SEQ ID NOs: 1 to 4. The present invention significantly inhibits and ameliorates the level of pain, in particular neuropathic pain which is an intractable disease that is difficult to treat and requires an improved treatment, and therefore can be effectively used as a treatment composition for neuropathic pain.

Description

Pharmaceutical composition for treating neuropathic pain comprising a MINCLE inhibitor

The present invention relates to a pharmaceutical composition for treating neuropathic pain containing a Mincle inhibitor.

Neuropathic pain is pain caused by damage or functional abnormality of the nervous system. It is incurable and chronically long-lasting, so patients suffering from this pain have a significantly reduced quality of life, not only due to the pain itself, but also to sleep disorders, It is a pain syndrome that causes social problems such as emotional disorders such as depression and decreased productivity due to poor social adaptability.

It is estimated that approximately 7 to 10% of the total population suffers from neuropathic pain, which is due to various factors (e.g. aging, increasing rates of obesity, and cancer patients being treated with interventions that can cause neuropathic pain). Due to increased survival rates, the prevalence of neuropathic pain is predicted to increase further in the future. Because the underlying cause of neuropathic pain is not known, the management and treatment of neuropathic pain is often complex and still has limitations. Current approaches include tricyclic antidepressants (e.g., amitriptyline, serotonin-norepinephrine reuptake inhibitors (SNRIs), the calcium channel alpha-2-delta ligands gabapentin and pregabrain), and opioids. It only focuses on treating the symptoms of neuropathic pain with the same pharmacological agents. These drugs often have serious side effects in many people and/or have limited efficacy. Therefore, there is a need for a more effective treatment for neuropathic pain.

Therefore, the present invention was researched and designed for an improved treatment for neuropathic pain, and relates to a pharmaceutical composition for the treatment of neuropathic pain containing a Mincle (macrophage inducible C-type lectin) inhibitor as an active ingredient. The pharmaceutical composition of the present invention is expected to be widely used in the field of treating neuropathic pain induced by nerve damage, etc., as it has been confirmed to significantly effectively improve the degree of pain in the in vivo system.

The present inventors have made extensive research efforts to develop an effective treatment for neuropathic pain, an intractable disease that is difficult to treat and requires improved treatments. As a result, the present invention was completed by confirming that the Mincle inhibitor as an active ingredient significantly suppresses and improves the degree of pain in the in vivo system.

Therefore, the purpose of the present invention is to provide a pharmaceutical composition for treating neuropathic pain containing a Mincle inhibitor as an active ingredient.

Other objects and advantages of the present invention will become clearer from the following detailed description, claims, and drawings.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

DETAILED DESCRIPTION OF THE INVENTION Various embodiments described herein are described below with reference to the drawings. In the following description, various specific details, such as specific forms, compositions, and processes, are set forth in order to provide a thorough understanding of the invention. However, certain embodiments may be practiced without one or more of these specific details or in conjunction with other known methods and forms. In other instances, well-known processes and manufacturing techniques are not described in specific detail so as not to unnecessarily obscure the invention. Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, form, composition or characteristic described in connection with the embodiment is included in one or more embodiments of the invention. Accordingly, the phrases “in one embodiment” or “an embodiment” expressed in various places throughout this specification do not necessarily refer to the same embodiment of the invention. Additionally, particular features, shapes, compositions, or properties may be combined in any suitable way in one or more embodiments.

Unless there is a special definition in the specification, all scientific and technical terms used in the specification have the same meaning as commonly understood by a person skilled in the art in the technical field to which the present invention pertains.

Throughout the specification, when a part is said to “include” a certain element, this means that it may further include other elements rather than excluding other elements, unless specifically stated to the contrary.

According to one aspect of the present invention, the present invention is an epitope of Mincle (Macrophage-inducible C-type lectin; Mincle) protein,

An epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of amino acid sequences represented by SEQ ID NO: 1 to SEQ ID NO: 4 is provided.

The present inventors have made extensive research efforts to develop an effective treatment for neuropathic pain, an intractable disease that is difficult to treat and requires improved treatments. As a result, the present invention was completed by confirming that the Mincle inhibitor as an active ingredient significantly suppresses and improves the degree of pain in the in vivo system.

As used herein, the term "Mincle (Macrophage-inducible C-type lectin)" protein is also known as CLEC-4E (C-type lectin domain family 4, member E), and is a member of the C-type lectin superfamily encoded by the CLEC4E gene. means. As a pattern recognition receptor capable of recognizing glycolipids, including mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM), Mincle receptors bind to various carbohydrate structures, mainly containing glucose or mannose, and are recognized by the immune system. It plays an important role in recognizing bacterial glycolipids.

As used herein, the term “epitope” is also called an antigenic determinant and refers to a specific part of an antigen that allows the immune system, such as antibodies, B cells, and T cells, to identify the antigen. The specific part of an antibody that identifies an epitope is called an antigen-binding site (paratope) or an antigenic determinant. In general, it is easy to think of an antigen or its epitope as a substance that comes from outside rather than itself, but cancer cells and Likewise, there are cases where it acts as an antigen even though it is its own, and in this case, it is also classified as an antigen binding site.

As used herein, the term “peptide” refers to a polymer of amino acids. Usually, a form in which a few amino acids are linked is called a peptide, and a form in which many amino acids are linked is called a protein. In these peptide and protein structures, the linkage between amino acids is made up of amide bonds or peptide bonds. A peptide bond is a bond between a carboxyl group (-COOH) and an amino group (NH2-) where water escapes and forms -CO-NH-. A variety of ligands promote signaling through Mincle, including proteins, sterols, modified or damaged glycolipids of their own, and a variety of glycolipids from pathogenic and commensal organisms.

According to one embodiment of the present invention, as an epitope of the Mincle (Macrophage-inducible C-type lectin) protein, some amino acids of the Mincle protein represented by SEQ ID NOs: 1 to 4, for example, 2 to 50; 6 to 45; Alternatively, it may consist of 10 to 44 amino acids, but is not limited thereto. According to another embodiment of the present invention, as an epitope of the Mincle protein, an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of amino acid sequences represented by SEQ ID NOs: 1 to 4 is provided.

As an example of the present invention, the epitope of the Mincle protein may be an epitope containing a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 4, but is not limited thereto.

The epitope of the present invention may be a conformational epitope.

The “three-dimensional epitope” of the present invention is composed of a discontinuous amino acid sequence, unlike a one-dimensional linear epitope composed of a continuous sequence. These three-dimensional epitopes react with the three-dimensional structure of the antibody antigen binding site.

According to another aspect of the invention, the invention provides a nucleic acid molecule encoding an epitope.

The nucleic acid molecule of the present invention includes all nucleic acid molecules in which the amino acid sequence of the polypeptide provided by the present invention has been translated into a polynucleotide sequence as known to those skilled in the art. 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.

According to another aspect of the present invention, the present invention provides an expression vector into which a nucleic acid molecule is inserted.

In the present invention, the “vector” is a nucleic acid molecule capable of transporting another nucleic acid to which a nucleic acid molecule is linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors are episomal mammalian vectors that have a bacterial origin of replication). Other vectors (e.g., non-episomal mammalian vectors) can be introduced into the host cell and integrated into the host cell's genome, thereby replicating along with the host genome. In addition, some vectors can direct the expression of genes to which they are operationally linked. Such vectors are referred to herein as “recombinant expression vectors” or simply “expression vectors”. In general, expression vectors useful in recombinant DNA techniques often exist in the form of plasmids. In this specification, “plasmid” and “vector” can be used interchangeably because plasmid is the most commonly used form of vector.

Specific examples of the expression vector in the present invention include widely used commercially available pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, and Ti vectors; cosmid; Phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμμ, T-even, T2, T3, T7; It may be selected from the group consisting of plant viruses, but is not limited thereto, and all expression vectors known to those skilled in the art as expression vectors can be used in the present invention, and when selecting an expression vector, it depends on the properties of the target host cell. Introduction of vectors into host cells may be performed by calcium phosphate transfection, viral infection, DEAE-dextran control transfection, lipofectamine transfection, or electroporation, but is not limited thereto, and those skilled in the art will use the method. An introduction method suitable for the expression vector and host cell can be selected and used. Preferably, the vector contains one or more selection markers, but is not limited to this, and selection can be made depending on whether or not the product is produced using a vector that does not contain a selection marker. The selection marker is selected based on the desired host cell, and since this method is already known to those skilled in the art, the present invention is not limited thereto.

To facilitate purification of the protein encoded by the nucleic acid molecule of the present invention, a tag sequence can be inserted into the expression vector and fused. The tag includes, but is not limited to, a hexa-histidine tag, a hemagglutinin tag, a myc tag, or a flag tag, and any tag that facilitates purification known to those skilled in the art can be used in the present invention.

According to another aspect of the present invention, the present invention provides a transformant transfected with an expression vector.

The transformation method of the present invention is any method of injecting a desired vector into a host cell, and may include any known method capable of injecting a vector into a host cell, for example, a method using CaCl ₂ , Electroporation, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, and transformation using viruses can be used, but are limited thereto. no.

According to a specific embodiment of the present invention, the transformant may be a cell or an individual transformant.

According to the present specification, transformation is when a DNA chain fragment or plasmid containing a different type of gene from that of the original cell infiltrates between cells and combines with the DNA existing in the original cell, thereby changing the genetic characteristics of the cell. It refers to a molecular biological phenomenon that changes. Transformation is commonly observed in bacteria and can be achieved through artificial genetic manipulation. Cells that undergo transformation by accepting DNA that is not their own are called transformation recipient cells. Transformed recipient cells can spread new genetic traits through conjugation and transduction.

According to another aspect of the present invention, the present invention provides an antibody that specifically binds to an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4, or Antigen-binding fragments are provided.

According to another embodiment of the present invention, an antibody or antigen-binding fragment that specifically binds to the epitope of the present invention is provided.

In the present invention, the antibody is a full-length antibody or a portion of an antibody, which has the ability to bind to the CLEC-4E protein and includes all antibody fragments that bind to the CLEC-4E antigenic determining site competitively with the binding molecule of the present invention. Includes.

In the present invention, the “antibody” refers to a protein molecule that serves as a receptor that specifically recognizes an antigen, including immunoglobulin molecules that are immunologically reactive with a specific antigen.

In the present invention, the “immunoglobulin” has a heavy chain and a light chain, and each heavy chain and light chain includes a constant region and a variable region. The variable regions of the light and heavy chains include three variable regions called complementarity determining regions (hereinafter referred to as “CDRs”) and four framework regions. The CDR mainly functions to bind to the epitope of the antigen. The CDRs of each chain are typically referred to sequentially, starting from the N-terminus, as CDR1, CDR2, and CDR3, and are also identified by the chain on which a particular CDR is located.

In the present invention, the "full-length antibody" has a structure having two full-length light chains and two full-length heavy chains, and each light chain is connected to the heavy chain by a disulfide bond, and includes IgA, IgD, IgE, Includes IgM, and IgG. The IgG subtypes include IgG1, IgG2, IgG3, and IgG4.

In addition, in the present invention, the "antigen-binding fragment" refers to a fragment that possesses an antigen-binding function, and examples of the antigen-binding fragment include (i) the variable region (VL) of the light chain and the variable region (VH) of the heavy chain and the light chain. A Fab fragment consisting of the constant region (CL) and the first constant region (CH1) of the heavy chain; (ii) Fd fragment consisting of VH and CH1 domains; (iii) an Fv fragment consisting of the VL and VH domains of a single antibody; (iv) dAb fragment consisting of VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragment, a bivalent fragment containing two linked Fab fragments; (vii) a single chain Fv molecule (scFv) linked by a peptide linker that joins the VH domain and the VL domain to form an antigen binding site; (viii) bispecific single-chain Fv dimers and (ix) diabodies, which are multivalent or multispecific fragments produced by gene fusion. The antigen-binding fragment can be obtained as a Fab or F(ab')2 fragment using a proteolytic enzyme, for example, papain or pepsin, and can be produced through genetic recombination technology.

In addition, in the present invention, the antibody may be monoclonal antibody, polyclonal antibody, chimeric antibody, humanized antibody, human antibody, bivalent, Bispecific molecule, minibody, domain antibody, bispecific antibody, antibody mimetic, unibody, diabody, triabody, tetrabody or It may be a fragment thereof, but is not limited thereto.

In addition, in the present invention, the “monoclonal antibody” refers to an antibody molecule with a single molecule composition obtained from a substantially identical antibody population, and exhibits single binding specificity and affinity for a specific epitope.

In the present invention, 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 the immune response is greatly improved compared to the mouse antibody.

Additionally, in the present invention, the “humanized antibody” refers to an antibody in which the protein sequence of an antibody derived from a non-human species has been modified to be similar to an antibody variant naturally produced in humans. For example, the humanized antibody can be manufactured by recombining mouse-derived CDRs with FRs derived from a human antibody to prepare a humanized variable region, and then recombining this with the constant region of a desired human antibody.

In the present invention, “binding” or “specific binding” refers to the affinity of the antibody or antibody composition for the antigen. In antigen-antibody binding, “specific binding” can typically be distinguished from non-specific background binding if the dissociation constant (Kd) is less than 1x10 ^-5 M or less than 1x10 ^-6 M or less than 1x10 ^-7 M. Specific binding can be detected by methods known in the art, such as ELISA, SPR (Surface plasmon resonance), immunoprecipitation, coprecipitation, etc., and non-specific binding and specific binding can be distinguished. Include an appropriate control group for differentiation.

The antibody or antigen-binding fragment of the present invention may exist as a multimer such as a dimer, trimer, tetramer, or pentamer containing at least a portion of the antigen-binding ability of the monomer. These multimers also include homomultimers and heteromultimers. Antibody multimers have superior antigen-binding ability compared to monomers because they contain multiple antigen-binding sites. Antibody multimers also facilitate the production of multifunctional (bifunctional, trifunctional, tetrafunctional) antibodies.

In the present invention, the term “multifunctionality” refers to an antibody or antigen-binding fragment having two or more activities or functions (e.g., antigen-binding ability, enzyme activity, ligand or receptor-binding ability). For example, the antibody of the present invention is It can be combined with polypeptides having enzymatic activity, such as luciferase, acetyltransferase, galactosidase, etc. Multifunctional antibodies also include antibodies in multivalent or multispecific (bispecific, trispecific, etc.) forms.

According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating neuropathic pain comprising an antibody or antigen-binding fragment as an active ingredient.

As used herein, the term “Neuropathic pain” is used with the same meaning as “neuropathic pain” and refers to pain caused by damage or disease affecting the somatosensory nervous system. The mechanism that sustains pain is caused by abnormalities in the somatosensory processes of the central or peripheral nervous system and may be related to abnormal sensations called paresthesia or pain caused by normally non-painful stimuli (allodynia). The International Association for the Study of Pain (IASP) defines neuropathic pain as pain initiated or caused by a primary lesion or dysfunction in the nervous system. It was defined as .

In the present invention, “treatment” may include, without limitation, any action that improves the symptoms of a disease or provides benefits using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may be in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be intended for human subjects.

In the present invention, the pharmaceutical composition is not limited to these, but can 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 injection solutions according to conventional methods. You can. The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. for oral administration, and buffers, preservatives, and analgesics for injections. Topics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used. The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above. For example, 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 dosage ampoules or multiple dosage forms. there is. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.

Meanwhile, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose. , methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil can be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

The route of administration of the pharmaceutical composition in the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, Includes sublingual or rectal areas. Oral or parenteral administration is preferred.

In the present invention, the term “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or injection techniques. The pharmaceutical composition of the present invention can also be administered in the form of a suppository for rectal administration.

The pharmaceutical composition of the present invention may vary depending on several factors, including the activity of the specific compound used, age, body weight, general health, gender, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. It may vary, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, route and period of administration, but may be appropriately selected by a person skilled in the art, and may range from 0.0001 to 50 mg/kg per day. Alternatively, it can be administered at 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention can be formulated into pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

According to a specific embodiment of the present invention, the composition may be a pharmaceutical composition that has an inhibitory effect on C-type lectin receptors (CLRs).

As used herein, the term “C-type lectin receptors (CLRs)” refers to important pattern recognition receptors involved in the recognition and induction of adaptive immunity against pathogens. Certain CLRs play an important role in viral infections because they interact efficiently with viruses.

According to a specific embodiment of the present invention, the C-type lectin receptor is Macrophage-inducible C-type lectin (Mincle), Dectin-1, Dentin-2, and macrophage C-type lectin. (macrophage C-type lectin; MCL), macrophage mannose receptor (MMR, CD206), or CD209 (dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin; DC-SIGN). It may be a pharmaceutical composition, which is selected.

As used herein, the term “Dectin-1” refers to C-type lectin domain family 7 member A, a protein encoded by the CLEC7A gene in humans. CLEC7A is a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily, an encoded glycoprotein of small type with an extracellular C-type lectin-like domain fold and a cytoplasmic domain with a partial immunoreceptor tyrosine-based activation motif. II It is a membrane receptor. It plays an important role in the innate immune response by acting as a pattern recognition receptor for various β-1,3-linked and β-1,6-linked glucans derived from fungi and plants. Expression is found in myeloid dendritic cells, monocytes, macrophages and B cells.

As used herein, the term “macrophage mannose receptor (MMR, CD206)” refers to a C-type lectin primarily expressed by most tissue macrophages, dendritic cells, and certain lymphatic or endothelial cells. It plays an important role in immune homeostasis by functioning in endocytosis and phagocytosis and by scavenging unwanted mannoglycoproteins.

As used herein, the term "DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin), also known as CD209 (Cluster of Differentiation 209), refers to the protein encoded by the CD209 gene in humans. DC- SIGN is a C-type lectin receptor present on the surface of macrophages and dendritic cells. DC-SIGN on macrophages binds high-mannose-type N-glycans, a class of pathogen-associated molecular patterns (PAMPs) commonly found in viruses, bacteria, and fungi. Recognizes and combines with high affinity.

As used herein, the term "macrophage-inducible C-type lectin (Mincle)" is part of the innate immune system and is a pattern recognition receptor for pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPs). It works.

According to a specific embodiment of the present invention, the C-type lectin receptor may be Mincle (Macrophage-inducible C-type lectin; Mincle), a pharmaceutical composition.

According to a specific embodiment of the present invention, the neuropathic pain is at least selected from the group consisting of Spontaneous pain, Paresthesias, Dysesthesias, Hyperalgesia, and Allodynia. It may be any one pharmaceutical composition.

As used herein, the term “spontaneous pain” refers to pain caused by chronic inflammatory conditions or neurological problems in our body, rather than temporary pain caused by stimulation. Spontaneous pain can often be stimulus-evoked pain, where the stimulus is not perceived because it is produced by activities of daily living (both external and internal stimuli generated by normal physiological processes).

As used herein, the term “Paresthesias” refers to abnormal sensations of the skin (numbness, tingling, chills, burning, tingling) without an obvious physical cause. Paresthesia can be temporary or chronic and can have dozens of potential causes. Paresthesias are generally painless and can occur anywhere on the body, but most commonly occur in the arms and legs.

As used herein, the term “Dysesthesias” refers to abnormal sensations. Its etymology comes from the Greek words "dys" meaning "bad" and "aesthesis" meaning "sensation" (strange sensation), and is defined as an unpleasant and abnormal sensation of touch. It often presents as pain, but it can also present as an inappropriate but not uncomfortable sensation. It is caused by lesions of the peripheral or central nervous system and includes spontaneous or evoked sensations such as burning, wetness, itching, electric shock, and pins and needles. Paresthesias may include sensation in any body tissue, including the mouth, scalp, skin, or legs in most cases.

As used herein, the term “Hyperalgesia” means caused by an abnormal increase in sensitivity to pain. Damage to nociceptors or peripheral nerves can cause hypersensitivity to stimuli, and prostaglandins E and F play a role in sensitizing nociceptors. Temporarily increased sensitivity to pain occurs as part of disease behavior and is an evolved response to infection.

As used herein, the term “allodynia” refers to a condition in which pain is caused by a stimulus that does not generally cause pain.

According to another aspect of the present invention, the present invention provides an antibody or antigen-binding fragment; and an antibody-drug conjugate (ADC) containing a drug.

In the present invention, the term “Antibody-Drug Conjugate (ADC)” refers to a form in which a drug and an antibody are chemically linked without reducing the biological activities of the antibody and drug. In the present invention, the antibody-drug conjugate is a form in which the drug is bound to the amino acid residue at the N-terminus of the heavy and/or light chain of the antibody, specifically, the drug is bound to the α-amine group at the N-terminus of the heavy and/or light chain of the antibody. This is the combined form.

In the present invention, the “drug” may mean any substance that has specific biological activity in cells, and this concept includes DNA, RNA, or peptides. The drug may be in a form containing a reactive group capable of crosslinking by reacting with an α-amine group, and may also include a form in which a linker containing a reactive group capable of crosslinking by reacting with an α-amine group is connected.

In the present invention, as an example of a reactive group capable of crosslinking by reacting with the α-amine group, the type is not particularly limited as long as it can crosslink by reacting with the α-amine group of the N-terminus of the heavy or light chain of the antibody, and is known in the art. Includes all types that react with known amine groups. Examples include Isothiocyanate, Isocyanates, Acyl azide, NHS ester, Sulfonyl chloride, Aldehyde, Glyoxal, Epoxide, Oxirane, Carbonate, Aryl halide, Imidoester, Carbodiimide, Anhydride and Fluorophenyl ester), but is not limited thereto.

In the present invention, the antibody-drug conjugate is the antibody or antigen-binding fragment containing the epitope of the present invention, that is, Mincle protein; or an epitope containing a polypeptide consisting of a partial amino acid sequence of the extracellular domain of the Mincle protein; or an antibody or antigen-binding fragment that specifically binds to an epitope containing a polypeptide represented by any one of the amino acid sequences of SEQ ID NOs: 1 to 4, wherein the drug is capable of treating the disease targeted by the Mincle antibody. Any drug may be included, for example, it may be a drug that can treat pain disease or neuropathic pain disease, but is not limited thereto.

Antibodies or antigen-binding fragments of the invention; And the antibody-drug conjugate specifically binds to an epitope containing a polypeptide represented by any one of the amino acid sequences of SEQ ID NOs: 1 to 4, inhibits the function of the regulatory T cells, and maintains or increases the activity of the effector T cells. It can treat various diseases very effectively.

In the present invention, the "subject" is an individual suspected of developing neuropathic pain, and the individual suspected of developing neuropathic pain is a mammal, including rats, livestock, etc., including humans, that has or may develop the disease. However, subjects that can be treated with the antibody or antibody-drug conjugate of the present invention are included without limitation.

The method of the present invention may include administering an antibody or antibody-drug conjugate in a pharmaceutically effective amount. The appropriate total daily usage amount can be determined by the treating physician within the scope of sound medical judgment, and can be administered once or in several divided doses. However, for the purposes of the present invention, the specific therapeutically effective amount for a specific patient depends on the type and degree of response to be achieved, the specific composition, including whether other agents are used as the case may be, the patient's age, weight, and general health status, It is desirable to apply it differently depending on various factors including gender and diet, administration time, administration route and secretion rate of the composition, treatment period, drugs used together or simultaneously with the specific composition, and similar factors well known in the medical field.

Meanwhile, although not limited thereto, the method for preventing or treating neuropathic pain may be a combination therapy further comprising administering a compound or substance having therapeutic activity against one or more neuropathic pain diseases.

In the present invention, the “combined use” should be understood to indicate simultaneous, separate or sequential administration. If the administration is sequential or separate, the interval between the administrations of the second components should be such that the beneficial effects of the combination are not lost.

In the present invention, the administered dose of the antibody or antibody-drug conjugate may be about 0.0001 μg to 500 mg per kg of patient body weight, but is not limited thereto.

According to another aspect of the present invention, the present invention provides an antibody that specifically binds to an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4, or Provided is a method for preventing or treating neuropathic pain, comprising administering a pharmaceutical composition containing an antigen-binding fragment as an active ingredient.

According to another aspect of the present invention, the present invention provides a treatment for neuropathic pain of an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4. Provides preventive or therapeutic use.

According to another aspect of the present invention, the present invention provides an antibody that specifically binds to an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4, or Provided is an antigen-binding fragment for use in preventing or treating neuropathic pain.

The features and advantages of the present invention are summarized as follows:

(a) The present invention is an epitope of Mincle (Macrophage-inducible C-type lectin) protein, comprising at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4. Provided is a pharmaceutical composition for treating neuropathic pain containing an epitope as an active ingredient.

(b) The present invention significantly suppresses and improves the degree of pain, especially neuropathic pain, which is an incurable disease that is difficult to treat and requires improved therapeutic agents, and can therefore be usefully used as a composition for the treatment of neuropathic pain.

Figure 1 shows an experimental schedule for animal behavioral experiments according to an embodiment of the present invention.

Figure 2 shows the results of confirming the pain reduction effect after administration of antibodies for each manufacturer, according to an experimental example of the present invention.

Figure 3 shows the results confirming the pain reduction effect after administration of Mincle antibody, the first antibody prepared according to an experimental example of the present invention.

Figure 4 shows the results confirming the pain reduction effect after administration of Mincle antibody, a second antibody prepared according to an experimental example of the present invention.

Figure 5 shows the results confirming the pain reduction effect after administration of Mincle antibody, a third antibody prepared according to an experimental example of the present invention.

In the present invention, antibodies were prepared to recognize SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4 as epitopes, and the effect on mechanical allodynia was confirmed. As a result, the Mincle antibody, the third antibody produced with SEQ ID NO: 4, significantly showed the highest pain reduction effect.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

[Example 1] Intrathecal catheterization model

After the rats arrived in the breeding room and adapted for 48 hours, a catheter (polyethylene, PE-10) was placed in the intrathecal space for administration of the experimental drug. In rats under anesthesia induced by sevoflurane, an incision was made 2 cm below both ears along the midline above the skull, and the atlantooccipital membrane was found by pulling the muscles and fascia. After incising the dura with the tip of a 24-gauge needle, the catheter was inserted 8.5 cm downward so that the tip of the catheter was located in the lumbar extension. The incision site was sutured with 3-0 silk and the rats were awakened from anesthesia. After surgery, all rats were placed in individual cages and used in this experiment after a 5-day recovery period. Rats showing signs of neurological damage after catheter insertion were immediately euthanized with an overdose of inhalation anesthetic.

[Example 2] Neuropathic pain model caused by spinal nerve ligation

A catheter was inserted into the spinal canal, and after a 5-day recovery period, spinal nerve ligation surgery for a pain-producing model was performed. Under sevoflurane anesthesia, a skin incision was made slightly to the left after identifying the spinal processes in the midline, and a longitudinal incision was made starting from the fourth lumbar region, passing through the iliac crest to the sacral region. After removing some of the back muscles and identifying the transverse process of the 6th lumbar vertebra, the posterior joint and part of the transverse process were removed to isolate the 5th lumbar nerve and the distal part of the ganglion was tightly ligated with a 6-0 silk suture. Recovery from the surgery was confirmed 5 days after the spinal nerve ligation surgery, and rats showing mechanical allodynia on the left footpad, which was on the side of the nerve ligation, were used in the experiment.

[Example 3]

1. Method of drug administration and behavioral evaluation of pain

Three different types of mincle inhibitors were administered to the neuropathic pain animal model prepared in Example 2. The mincle inhibitor is an antibody, and the definitions of the three types of antibodies are listed in Table 1 below.

first antibody	An antibody prepared to recognize the mouse mincle protein represented by SEQ ID NO: 1 as an epitope
second antibody	An antibody prepared to recognize amino acids 41-219 of the human mincle protein represented by SEQ ID NO: 2 as an epitope.
third antibody	An antibody prepared to recognize amino acids 44-89 of the human mincle protein represented by SEQ ID NO: 2 as an epitope.

Behavioral experiments were conducted using three prepared antibodies. The three Mincle antibodies were prepared to recognize SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4 as epitopes, and each antibody was injected intrathecally. The first antibody, mincle antibody, was 2 μg, 1 μg, and 0.5 μg/10 μg, the second antibody, mincle antibody, was 300 μg, 150 μg, and 75 μg/10 μg, and the third antibody, mincle antibody, was 200 μg. 100 μg and 50 μg were administered once each, and mechanical allodynia was measured at 15, 30, 60, 90, 120, 150, and 180 minutes.

To assess mechanical allodynia, von Frey filaments were applied to the hind paws and the paw withdrawal threshold (PWT) was measured. PWT was measured by applying von Frey filaments to the hind paws of rats. Using von Frey filaments of various sizes capable of applying a pressure of 0.4 g to 15 g, pressure was applied to the soles of the rat's feet to the point where the filaments were slightly bent and maintained for about 5 seconds. A positive avoidance response was considered if the animal removed or licked the paw pad during stimulation with the filament. If an avoidance response was shown, the next stimulus was stimulated with a smaller pressure filament. If an avoidance response was shown, the next stimulus was stimulated with a larger pressure filament. The avoidance response threshold was calculated using an up-down method. If the rat did not show withdrawal or licking response, a cutoff value of 15 g PWT was applied. All behavioral experiments were performed blindly. The schedule of the behavioral experiment is shown in Figure 1.

2. Statistical processing

Differences in PWT between groups using SPSS software were compared using one-way analysis of variance, and analyzed using Bonferroni as a post hoc test. A P value of 0.05 or less was considered statistically significant.

[Example 4] Results and Analysis

4-1. Comparison and confirmation of pain reduction effects by antibody

The effect of reducing mechanical allodynia was measured at 15, 30, 60, 90, 120, 150, and 180 minutes after administering 2 μg, 300 μg, and 200 μg of the three Mincle antibodies listed in Table 1, respectively. did. As a result of the experiment, the Mincle antibody, the third antibody produced with SEQ ID NO: 4, significantly showed the highest pain reduction effect (FIG. 2).

4-2. Confirmation of pain reduction effect by antibody

1. Primary antibody

The pain suppressing effect was confirmed by administering 0.5 μg, 1 μg, and 2 μg of Mincle antibody, the primary antibody intrathecally for mechanical allodynia induced by spinal nerve ligation. Compared to the Vehicle group, intrathecal administration of the first antibody, Mincle antibody, significantly reduced mechanical allodynia induced by spinal nerve ligation in a dose-dependent manner (Figure 3).

2. Secondary antibody

The pain suppressing effect was confirmed by administering 75 μg, 150 μg, and 300 μg of Mincle antibody, an intrathecal second antibody for mechanical allodynia induced by spinal nerve ligation. Compared to the Vehicle group, intrathecal administration of the second antibody, Mincle antibody, significantly reduced mechanical allodynia induced by spinal nerve ligation in a dose-dependent manner (Figure 4).

3. Third antibody

The pain suppressing effect was confirmed by administering 50 μg, 100 μg, and 200 μg of intrathecal Mincle antibody to mechanical allodynia induced by spinal nerve ligation. Compared to the Vehicle group, one-time intrathecal administration of the third antibody, Mincle antibody, significantly reduced mechanical allodynia induced by spinal nerve ligation in a dose-dependent manner (Figure 5).

As the specific parts of the present invention have been described in detail above, it is clear to those skilled in the art that these specific techniques are merely preferred embodiments and do not limit the scope of the present invention. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

The present invention relates to a pharmaceutical composition for treating neuropathic pain containing a Mincle inhibitor. The pharmaceutical composition of the present invention is expected to be widely used in the field of treating neuropathic pain induced by nerve damage, etc., as it has been confirmed to significantly effectively improve the degree of pain in the in vivo system.

SEQ ID NO: 1: Macrophage-inducible C-type lectin (Mincle) protein of mouse

MNSTKSPASH HTERGCFKNS QVLSWTIAGA SILFLSGCFI TRCVVTYRSS QISGQNLQPH RNIKELSCYS EASGSVKNCC PLNWKHYQSS CYFFSTTTLT WSSSLKNCSD MGAHLVVIDT QEEQEFLFRT KPKRKEFYIG LTDQVVEGQW QWVDDTPFTE SLSFWDAGEP NNIVLVEDCA TIRDSSNSRK NWND IPCFYS MPWICEMPEI SPLD

SEQ ID NO: 2: Human Macrophage-inducible C-type lectin (Mincle) protein

MNSSKSSETQ CTERGCFSSQ MFLWTVAGIP ILFLSACFIT RCVVTFRIFQ TCDEKKFQLP ENFTELSCYN YGSGSVKNCC PLNWEYFQSS CYFFSTDTIS WALSLKNCSA MGAHLVVINS QEEQEFLSYK KPKMREFFIG LSDQVVEGQW QWVDGTPLTK SLSFWDVGEP NNIATLEDCA TMRDSSNPRQ NWNDVT CFLN YFRICEMVGI NPLNKGKSL

SEQ ID NO: 3: Amino acids 41-219 of human Macrophage-inducible C-type lectin (Mincle) protein

RCVVTFRIFQ TCDEKKFQLP ENFTELSCYN YGSGSVKNCC PLNWEYFQSS CYFFSTDTIS WALSLKNCSA MGAHLVVINS QEEQEFLSYK KPKMREFFIG LSDQVVEGQW QWVDGTPLTK SLSFWDVGEP NNIATLEDCA TMRDSSNPRQ NWNDVTCFLN YFRICEMVGI NPLNKGKS

SEQ ID NO: 4: Amino acids 44-89 of human Macrophage-inducible C-type lectin (Mincle) protein

VTFRIFQTCD EKKFQLPENF TELSCYNYGS GSVKNCCPLN WEYFQS

Claims (14)

1. As an epitope of the Macrophage-inducible C-type lectin (Mincle) protein,

   An epitope comprising at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4.
2. A nucleic acid molecule encoding the epitope of claim 1.
3. An expression vector into which the nucleic acid molecule of claim 2 is inserted.
4. A transformant transfected with the expression vector of claim 3.
5. According to clause 4,

   The transformant is a cell or individual.
6. An antibody or antigen-binding fragment that specifically binds to an epitope containing at least one polypeptide selected from the group consisting of polypeptides consisting of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 4.
7. A pharmaceutical composition for preventing or treating neuropathic pain, comprising the antibody or antigen-binding fragment of claim 6 as an active ingredient.
8. According to claim 7,

   The composition is a pharmaceutical composition that has an inhibitory effect on C-type lectin receptors (CLRs).
9. According to claim 8,

   The C-type lectin receptors include Macrophage-inducible C-type lectin (Mincle), Dectin-1, Dectin-2, and macrophage C-type lectin (MCL). A pharmaceutical composition selected from the group consisting of macrophage mannose receptor (MMR, CD206), or CD209 (dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin; DC-SIGN).
10. According to clause 9,

    A pharmaceutical composition, wherein the C-type lectin receptor is Mincle (Macrophage-inducible C-type lectin; Mincle).
11. According to claim 8,

    The pharmaceutical composition, wherein the neuropathic pain is at least one selected from the group consisting of Spontaneous pain, Paresthesias, Dysesthesias, Hyperalgesia, and Allodynia.
12. The antibody or antigen-binding fragment of claim 6; and an antibody-drug conjugate (ADC) containing a drug.
13. A method of preventing or treating neuropathic pain comprising the step of administering a pharmaceutical composition containing the antibody or antigen-binding fragment of claim 6 as an active ingredient.
14. Use of the antibody or antigen-binding fragment of claim 6 to prevent or treat neuropathic pain.

Images