KR102784657B1 - A Pharmaceutical composition for treating neuropathic pain comprising Mincle inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating neuropathic pain, which comprises as an active ingredient an epitope of Mincle (Macrophage-inducible C-type lectin) protein, wherein the epitope comprises 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. The present invention can be usefully utilized as a composition for treating neuropathic pain, since it significantly suppresses and improves the degree of pain, particularly neuropathic pain, which is an intractable disease that is difficult to treat and requires an improved therapeutic agent.

Description

{A Pharmaceutical composition for treating neuropathic pain comprising Mincle inhibitor}

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

Neuropathic pain is pain that occurs due to damage or functional abnormality of the nervous system. It is intractable and chronic, and patients suffering from this pain experience a significant decline in quality of life due to its long-lasting nature. It is a pain syndrome that causes not only the pain itself, but also social problems such as sleep disorders, emotional disorders such as depression, and decreased productivity due to decreased social adaptability.

It is estimated that approximately 7-10% of the general population suffers from neuropathic pain, and the prevalence of neuropathic pain is expected to increase in the future due to various factors such as aging, increasing obesity rates, and increased survival of cancer patients treated with interventions that can cause neuropathic pain. Because the underlying cause of neuropathic pain is unknown, the management and treatment of neuropathic pain is often complex and still has limitations. Current approaches focus solely on treating the symptoms of neuropathic pain with pharmacological agents such as tricyclic antidepressants (e.g., amitriptyline, serotonin-norepinephrine reuptake inhibitors (SNRIs), calcium channel alpha-2-delta ligands gabapentin and pregabrane), and opioids. These agents often have serious side effects and/or have limited efficacy in many people. Therefore, more effective treatments for neuropathic pain are needed.

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

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

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

Other objects and advantages of the present invention will become more apparent from the detailed description of the invention, the claims and the drawings which follow.

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

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, various specific details, such as specific configurations, compositions, and processes, are set forth in order to provide a thorough understanding of the present invention. However, certain embodiments may be practiced without one or more of these specific details, or in conjunction with other known methods and configurations. In other instances, well-known processes and manufacturing techniques have not been described in specific detail so as not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in one or more embodiments of the present invention. Thus, the appearances of "in one embodiment" or "an embodiment" in various places throughout this specification are not necessarily referring to the same embodiment of the present invention. Additionally, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments.

Unless otherwise specifically defined in the specification, all scientific and technical terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

Throughout the specification, whenever a part is said to "include" a component, this does not mean that it excludes other components, but rather that it may include other components, unless otherwise stated.

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

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

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

The term "Mincle (Macrophage-inducible C-type lectin)" protein in this specification, also known as CLEC-4E (C-type lectin domain family 4, member E), refers to a member of the C-type lectin superfamily encoded by the CLEC4E gene. As a pattern recognition receptor that can recognize glycolipids including mycobacterial cord factor, trehalose-6,6'-dimycolate (TDM), the Mincle receptor primarily binds to various carbohydrate structures containing glucose or mannose and plays an important role in the recognition of bacterial glycolipids by the immune system.

The term "epitope" in this specification 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 the antigenic determinant is called a paratope or antigenic determinant. In general, it is easy to think of an antigen or its antigenic determinant as a substance from outside the body, but there are cases where it functions as an antigen even though it is one's own, such as cancer cells, and in this case, it is also classified as an antigen binding site.

The term "peptide" in this specification refers to a polymer of amino acids. Usually, a form in which a small number of amino acids are connected is called a peptide, and a form in which many amino acids are connected is called a protein. In these peptide and protein structures, the connection between amino acids is made of an amide bond or a peptide bond. A peptide bond is a bond in which water is lost between a carboxyl group (-COOH) and an amino group (NH2-) to form -CO-NH-. Various ligands, including proteins, sterols, modified or damaged self-glycolipids, and various glycolipids of pathogenic and commensal organisms, promote signal transduction through Mincle.

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

As an example of the present invention, the epitope of the Mincle protein may be an epitope comprising a polypeptide consisting of an amino acid sequence represented by 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 "stereoscopic epitope" of the present invention is composed of a discontinuous amino acid sequence, unlike a one-dimensional linear epitope composed of a continuous sequence. This stereoscopic epitope reacts with the three-dimensional structure of the antibody antigen-binding site.

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

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 polypeptide provided in the present invention. Therefore, various polynucleotide sequences can be produced 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 having a nucleic acid molecule inserted therein.

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 to which additional DNA segments can be ligated. Another type of vector is 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). Other vectors (e.g., a non-episomal mammalian vector) can be integrated into the genome of a host cell upon introduction into the host cell, thereby replicating together with the host genome. In addition, 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." In general, expression vectors useful in recombinant DNA techniques are often in the form of plasmids. In this specification, “plasmid” and “vector” may be used interchangeably, since plasmids are the most commonly used form of vector.

In the present invention, specific examples of the expression vector may be selected from the group consisting of commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμμ, T-even, T2, T3, T7; and plant viruses, but are 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. When introducing a vector into a host cell, it may be performed by calcium phosphate transfection, virus infection, DEAE-dextran controlled transfection, lipofectamine transfection, or electroporation, but is not limited thereto, and those skilled in the art can select and use an introduction method appropriate for the expression vector and host cell to be used. Preferably, the vector contains one or more selection markers, but is not limited thereto, and selection is possible based on whether or not the product is produced using a vector that does not contain a selection marker. The selection of the selection marker is performed by selecting the desired host cell, and since this is performed using a method already known to those skilled in the art, the present invention is not limited thereto.

In order 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 known to those skilled in the art that facilitates purification 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 for injecting a desired vector into a host cell, and any known method capable of injecting a vector into a host cell can be included, and for example, a method using CaCl ₂ , electroporation, microinjection, calcium phosphate precipitation, electroporation, liposome-mediated transfection, DEAE-dextran treatment, gene bombardment, and virus-based transformation methods can be used, but are not limited thereto.

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

According to this specification, transformation refers to a molecular biological phenomenon in which a DNA chain fragment or plasmid containing a different type of gene from that of the original cell penetrates between cells and combines with the DNA present in the original cell, thereby changing the genetic characteristics of the cell. Transformation is commonly observed in bacteria and can also be achieved through artificial genetic manipulation. A cell that has accepted DNA other than its own and has undergone transformation is called a transformed recipient cell. The transformed recipient cell can spread new genetic characteristics through conjugation and transduction.

According to another aspect of the present invention, the present invention provides an antibody or antigen-binding fragment that specifically binds to an epitope comprising 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.

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

In the present invention, the antibody includes both a full-length antibody or an antibody fragment having the ability to bind to CLEC-4E protein as a portion of the antibody and binding to the CLEC-4E antigenic determinant region competitively with the binding molecule of the present invention.

In 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 reactivity with a specific antigen.

In the present invention, 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. The 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.

In the present invention, 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.

In addition, the "antigen-binding fragment" in the present invention means a fragment having an antigen-binding function, and examples of the antigen-binding fragment include (i) a Fab fragment consisting of a variable region (VL) of a light chain and a variable region (VH) of a heavy chain and a constant region (CL) of a light chain and a first constant region (CH1) of a heavy chain; (ii) a Fd fragment consisting of a VH and a CH1 domain; (iii) a Fv fragment consisting of the VL and VH domains of a single antibody; (iv) a dAb fragment consisting of a VH domain; (v) an isolated CDR region; (vi) an F(ab')2 fragment which is a bivalent fragment including two linked Fab fragments; (vii) a single-chain Fv molecule (scFv) linked by a peptide linker which links the VH domain and the VL domain to form an antigen-binding site; (viii) a bispecific single-chain Fv dimer, and (ix) a diabody which is a multivalent or multispecific fragment produced by gene fusion. The above antigen binding fragment can be obtained as a fragment of Fab or F(ab')2 by using a protein hydrolytic enzyme, such as papain or pepsin, and can be produced through genetic recombination technology.

In addition, in the present invention, the antibody may be, but is not limited to, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody, a human 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.

In addition, 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).

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 is an antibody with a greatly improved immune response compared to a mouse antibody.

In addition, the "humanized antibody" in the present invention means an antibody in which the protein sequence of an antibody derived from a non-human species is modified to be similar to an antibody variant naturally produced in a human. For example, the humanized antibody can be produced by recombining a mouse-derived CDR with a human antibody-derived FR to produce a humanized variable region, and recombining this with a desired human antibody constant region to produce a humanized antibody.

In the present invention, the "binding" or "specific binding" refers to the affinity of the antibody or antibody composition of the present invention for the antigen. In antigen-antibody binding, "specific binding" can typically be distinguished from nonspecific background binding when the dissociation constant (Kd) is less than 1x10 ^-5 M, 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 includes an appropriate control that can distinguish between nonspecific binding and specific binding.

The antibody or antigen-binding fragment of the present invention may exist as a multimer, such as a dimer, trimer, tetramer, or pentamer, which contains at least a portion of the antigen-binding ability of the monomer. Such multimers also include homomultimers or heteromultimers. Antibody multimers contain a large number of antigen-binding sites, so they have superior antigen-binding ability compared to monomers. Antibody multimers are also easy to produce multifunctional (bifunctional, trifunctional, or tetrafunctional) antibodies.

The term "multifunctional" as used herein refers to an antibody or antigen-binding fragment having two or more activities or functions (e.g., antigen-binding ability, enzymatic activity, ligand or receptor binding ability), for example, the antibody of the present invention can bind to a polypeptide having enzymatic activity, such as luciferase, acetyltransferase, galactosidase, etc. Multifunctional antibodies also include antibodies in the form of multivalent or multispecific (bispecific, trispecific, etc.).

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.

The term "neuropathic pain" in this specification is used interchangeably with "neuropathic pain" and refers to pain caused by damage or disease affecting the somatosensory nervous system. The mechanism that sustains the pain is caused by an abnormality in the somatosensory process of the central or peripheral nervous system and may be associated with abnormal sensations called paresthesia or pain caused by stimuli that are not normally painful (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 nerve system.

In the present invention, “treatment” may include, without limitation, any act that improves symptoms of a disease or is beneficial by using the pharmaceutical composition of the present invention.

In 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.

In the present invention, the pharmaceutical composition 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 injection solutions, respectively, according to conventional methods. 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, etc. for injections, and may include bases, excipients, lubricants, preservatives, etc. for topical administration. The formulation of the pharmaceutical composition of the present invention may be prepared in various ways by mixing with the pharmaceutically acceptable carriers 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 dose ampoules or multiple doses. In addition, it can be formulated in the form of solutions, suspensions, tablets, capsules, sustained-release preparations, etc.

Meanwhile, 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. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers and preservatives may be additionally included.

The routes of administration of the pharmaceutical composition of 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.

The term "parenteral" in the present invention 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 form, 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, pharmaceutical 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.

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

The term "C-type lectin receptors (CLRs)" as used herein 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 may be a pharmaceutical composition selected from the group consisting of macrophage-inducible C-type lectin (Mincle), Dectin-1, Dectin-2, macrophage C-type lectin (MCL), macrophage mannose receptor (MMR, CD206), or CD209 (dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin; DC-SIGN).

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

The term "macrophage mannose receptor (MMR, CD206)" in this specification refers to a C-type lectin expressed primarily by most tissue macrophages, dendritic cells, and certain lymphoid or endothelial cells. It functions in endocytosis and phagocytosis and plays a critical role in immune homeostasis by clearing unwanted mannoglycoproteins.

The term "DC-SIGN (dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin), also known as CD209 (Cluster of Differentiation 209), refers to a 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, and DC-SIGN on macrophages recognizes and binds with high affinity to high-mannose type N-glycans, a class of PAMPs (pathogen-associated molecular patterns) commonly found on viruses, bacteria, and fungi.

The term "macrophage-inducible C-type lectin (Mincle)" as used herein refers to a part of the innate immune system that acts as a pattern recognition receptor for pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPs).

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

According to a specific embodiment of the present invention, the pharmaceutical composition may be one in which the neuropathic pain is at least one selected from the group consisting of spontaneous pain, paresthesias, dysesthesias, hyperalgesia, and allodynia.

The term "spontaneous pain" in this specification refers to pain caused by chronic inflammatory conditions or neurological problems in our body, rather than temporary pain caused by a stimulus. Spontaneous pain can often be stimulus-induced pain, and may be a case where the stimulus is not recognized because it is generated by daily life activities (both external and internal stimuli generated by normal physiological processes).

The term "paresthesia" in this specification refers to abnormal sensations (tingling, tingling, chills, burning, numbness) of the skin without an apparent physical cause. Paresthesia can be temporary or chronic and can have dozens of potential causes. Paresthesia is usually painless and can occur anywhere in the body, but most commonly occurs in the arms and legs.

The term "Dysesthesia" in this specification means an abnormal sensation. It is derived from the Greek "dys" meaning "bad" and "aesthesis" meaning "sense" (an abnormal sensation), and is defined as an unpleasant and abnormal sense of touch. It often manifests as pain, but can also manifest as a sensation that is inappropriate but not uncomfortable. It is caused by lesions of the peripheral or central nervous system and includes spontaneous or induced sensations such as burning, wetness, itching, electric shock, and pins and needles. Paresthesia can involve sensations in any body tissue, most often the mouth, scalp, skin, or legs.

The term "Hyperalgesia" in this specification means 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. Temporary increased sensitivity to pain occurs as part of disease behavior and is an evolved response to infection.

The term "Allodynia" as used herein refers to a condition in which pain is induced by stimuli that do not normally cause pain.

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

In the present invention, 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. In the present invention, 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 α-amine group at the N-terminus of the heavy chain and/or light chain of an antibody.

In the present invention, the "drug" may mean any substance having a specific biological activity in cells, and is a concept including DNA, RNA, or peptide. The drug may be in a form including a reactive group capable of cross-linking by reacting with an α-amine group, and also includes a form in which a linker including a reactive group capable of cross-linking by reacting with an α-amine group is connected.

In the present invention, examples of reactive groups capable of cross-linking by reacting with the α-amine group include, but are not limited to, any groups known in the art that react with amine groups as long as they can react with the α-amine group of the N-terminus of the heavy or light chain of an antibody to cross-link. Examples thereof include, but are not limited to, any one of isothiocyanate, isocyanates, acyl azide, NHS ester, sulfonyl chloride, aldehyde, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, anhydride, and fluorophenyl ester.

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

The antibody or antigen-binding fragment of the present invention; and the antibody-drug conjugate specifically bind to an epitope comprising a polypeptide represented by any one of the amino acid sequences of SEQ ID NOS: 1 to 4, thereby inhibiting the function of the regulatory T cells and maintaining or increasing the activity of effector T cells, thereby enabling the very effective treatment of various diseases.

In the present invention, the "subject" refers to a subject suspected of having developed neuropathic pain. The subject suspected of having developed neuropathic pain refers to a mammal including a human, a rat, a livestock, etc. that has developed or can develop the disease. However, a subject that can be treated with the antibody or antibody-drug conjugate of the present invention is included without limitation.

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

Meanwhile, but 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 term "combination" should be understood to mean simultaneous, separate or sequential administration. When the administration is sequential or separate, the interval between the administrations of the second component should be such that the beneficial effect of the combination is not lost.

In the present invention, the administration dose of the antibody or antibody-drug conjugate may be about 0.0001 μg to 500 mg per 1 kg of patient body weight, but is not limited thereto.

Sequence number 1: Macrophage-inducible C-type lectin (Mincle) protein of mouse

10 20 30 40 50

MNSTKSPASH HTERGCFKNS QVLSWTIAGA SILFLSGCFI TRCVVTYRSS

60 70 80 90 100

QISGQNLQPH RNIKELSCYS EASGSVKNCC PLNWKHYQSS CYFFSTTTLT

110 120 130 140 150

WSSSLKNCSD MGAHLVVIDT QEEQEFLFRT KPKRKEFYIG LTDQVVEGQW

160 170 180 190 200

QWVDDTPFTE SLSFWDAGEP NNIVLVEDCA TIRDSSNSRK NWNDIPCFYS

210

MPWICEMPEI SPLD

Sequence number 2: Human Macrophage-inducible C-type lectin (Mincle) protein

10 20 30 40 50

MNSSKSSETQ CTERGCFSSQ MFLWTVAGIP ILFLSACFIT RCVVTFRIFQ

60 70 80 90 100

TCDEKKFQLP ENFTELSCYN YGSGSVKNCC PLNWEYFQSS CYFFSTDTIS

110 120 130 140 150

WALSLKNCSA MGAHLVVINS QEEQEFLSYK KPKMREFFIG LSDQVVEGQW

160 170 180 190 200

QWVDGTPLTK SLSFWDVGEP NNIATLEDCA TMRDSSNPRQ NWNDVTCFLN

210 220

YFRICEMVGI NPLNKGKSL

Sequence number 3: Amino acids 41-219 of human Macrophage-inducible C-type lectin (Mincle) protein

10 20 30 40 50

RCVVTFRIFQ TCDEKKFQLP ENFTELSCYN YGSGSVKNCC PLNWEYFQSS

60 70 80 90 100

CYFFSTDTIS WALSLKNCSA MGAHLVVINS QEEQEFLSYK KPKMREFFIG

110 120 130 140 150

LSDQVVEGQW QWVDGTPLTK SLSFWDVGEP NNIATLEDCA TMRDSSNPRQ

160 170 180

NWNDVTCFLN YFRICEMVGI NPLNKGKS

Sequence number 4: Amino acids 44-89 of human Macrophage-inducible C-type lectin (Mincle) protein

10 20 30 40 50

VTFRIFQTCD EKKFQLPENF TELSCYNYGS GSVKNCCPLN WEYFQS

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

(a) The present invention provides a pharmaceutical composition for treating neuropathic pain, comprising as an active ingredient an epitope of Mincle (Macrophage-inducible C-type lectin) protein, wherein the epitope comprises 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.

(b) Since the present invention significantly suppresses and improves the degree of pain, particularly neuropathic pain, which is an intractable disease that is difficult to treat and requires improved treatment, it can be usefully utilized as a composition for treating neuropathic pain.

Figure 1 illustrates an experimental schedule for an animal behavior experiment according to one embodiment of the present invention.
Figure 2 shows the results of confirming the pain reduction effect after antibody administration by each manufacturer according to one experimental example of the present invention.
Figure 3 shows the results of confirming the pain reduction effect after administration of Mincle antibody, the first antibody manufactured according to one experimental example of the present invention.
Figure 4 shows the results of confirming the pain reduction effect after administration of Mincle antibody, a manufactured second antibody according to one experimental example of the present invention.
Figure 5 shows the results of confirming the pain reduction effect after administration of Mincle antibody, a manufactured third antibody according to one experimental example of the present invention.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to explain the present invention more specifically, 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 intended to explain the present invention more specifically, 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 at the breeding room and acclimated for 48 hours, a catheter (polyethylene, PE-10) was placed in the spinal space for administering the experimental drug. The rats were anesthetized with sevoflurane. An incision was made along the midline above the skull 2 cm inferior to both ears, and the muscles and fascia were tractioned to find the atlas-occipital membrane. After the dura was incised with the tip of a 24-gauge needle, the catheter was inserted 8.5 cm inferiorly so that the tip of the catheter was positioned at the lumbar enlargement. 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 the present experiment after a 5-day recovery period. Rats showing signs of neurological damage after catheter insertion were immediately euthanized with an overdose of inhaled anesthetic.

[Example 2] Neuropathic pain model 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-induced model was performed. Under sevoflurane anesthesia, a skin incision was made slightly to the left after identifying the spinal process in the midline, and a longitudinal incision was made starting from the 4th lumbar vertebra, passing the iliac crest and reaching the sacrum. After removing part of the back muscles, the transverse process of the 6th lumbar vertebra was identified, and the posterior facet joint and part of the transverse process were removed to isolate only the 5th lumbar nerve, which was then tightly ligated distally with a 6-0 silk suture. Recovery from the surgery was confirmed on the 5th day after spinal nerve ligation surgery, and rats showing mechanical allodynia in the sole of the left foot on the nerve-ligated side were used for the experiment.

[Example 3]

1. Drug administration and behavioral assessment of pain

Three different mincle inhibitors were administered to the neuropathic pain animal model prepared in Example 2 above. The mincle inhibitors are antibodies, and the definitions of the three antibodies are described in Table 1 below.

first antibody An antibody manufactured to recognize the mouse mincle protein represented by sequence number 1 as an epitope. Second antibody An antibody manufactured to recognize amino acids 41-219 of the human mincle protein represented by sequence number 2 as an epitope. Third antibody An antibody manufactured to recognize amino acids 44-89 of the human mincle protein represented by sequence number 2 as an epitope.

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

To assess mechanical allodynia, von Frey filaments were applied to the hind paws to measure paw withdrawal thresholds (PWT). PWT was measured by applying von Frey filaments to the hind paws of rats. Using von Frey filaments of various sizes that can apply pressures ranging from 0.4 g to 15 g, pressure was applied to the paws of rats to the extent that the filaments slightly bent and maintained for approximately 5 s. If the paws were removed or licked during the filament stimulation, it was considered a positive withdrawal response. If an withdrawal response was shown, the next stimulus was stimulated with a filament of lower pressure, and if an withdrawal response was shown, the next stimulus was stimulated with a filament of higher pressure. The withdrawal response threshold was obtained by the up-down method. If the rats did not show withdrawal or licking responses, the cutoff value of 15 g PWT was applied. All behavioral experiments were performed in a blind manner. The schedule of the behavioral experiments is shown in Fig. 1.

2. Statistical processing

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

[Example 4] Results and Analysis

4-1. Comparison of pain reduction effects by antibody

The effects of reducing mechanical allodynia were measured at 15, 30, 60, 90, 120, 150, and 180 minutes after administration of 2 μg, 300 μg, and 200 μg of the three Mincle antibodies described in Table 1, respectively. As a result of the experiment, the third antibody, Mincle antibody produced with sequence number 4, significantly showed the highest pain reduction effect (Fig. 2).

4-2. Confirmation of pain reduction effect by antibody

1. First antibody

The pain-suppressing effect of Mincle antibody, the first antibody intrathecally, on mechanical allodynia induced by spinal nerve ligation was confirmed by administering 0.5 μg, 1 μg, and 2 μg, respectively. Compared with the vehicle group, a single intrathecal administration of Mincle antibody, the first antibody, significantly dose-dependently reduced mechanical allodynia induced by spinal nerve ligation (Fig. 3).

2. Second antibody

The pain-suppressing effect of intrathecal second antibody, Mincle antibody, on mechanical allodynia induced by spinal nerve ligation was confirmed by administering 75 μg, 150 μg, and 300 μg, respectively. Compared with the vehicle group, a single intrathecal second antibody, Mincle antibody, significantly reduced spinal nerve ligation-induced mechanical allodynia in a dose-dependent manner (Fig. 4).

3. Third antibody

The pain-suppressing effect of intrathecal Mincle antibody on mechanical allodynia induced by spinal nerve ligation was confirmed by administering 50 μg, 100 μg, and 200 μg, respectively. Compared with the vehicle group, a single intrathecal administration of the third antibody, Mincle antibody, significantly reduced pain in a dose-dependent manner on mechanical allodynia induced by spinal nerve ligation (Fig. 5).

While the specific parts of the present invention have been described in detail above, it is obvious to those skilled in the art that such specific descriptions are merely preferred embodiments and that the scope of the present invention is not limited thereto. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

<110> Industry Foundation of Channam National University <120> A pharmaceutical composition for treating neuropathic pain comprising Mincle inhibitors <130> PDPB222212 <160> 4 <170> KoPatentIn 3.0 <210> 1 <211> 214 <212> PRT <213> Unknown <220> <223> Mouse Macrophage-inducible C-type lectin (Mincle) <400> 1 Met Asn Ser Thr Lys Ser Pro Ala Ser His His Thr Glu Arg Gly Cys 1 5 10 15 Phe Lys Asn Ser Gln Val Leu Ser Trp Thr Ile Ala Gly Ala Ser Ile 20 25 30 Leu Phe Leu Ser Gly Cys Phe Ile Thr Arg Cys Val Val Thr Tyr Arg 35 40 45 Ser Ser Gln Ile Ser Gly Gln Asn Leu Gln Pro His Arg Asn Ile Lys 50 55 60 Glu Leu Ser Cys Tyr Ser Glu Ala Ser Gly Ser Val Lys Asn Cys Cys 65 70 75 80 Pro Leu Asn Trp Lys His Tyr Gln Ser Ser Cys Tyr Phe Phe Ser Thr 85 90 95 Thr Thr Leu Thr Trp Ser Ser Ser Leu Lys Asn Cys Ser Asp Met Gly 100 105 110 Ala His Leu Val Val Ile Asp Thr Gln Glu Glu Gln Glu Phe Leu Phe 115 120 125 Arg Thr Lys Pro Lys Arg Lys Glu Phe Tyr Ile Gly Leu Thr Asp Gln 130 135 140 Val Val Glu Gly Gln Trp Gln Trp Val Asp Asp Thr Pro Phe Thr Glu 145 150 155 160 Ser Leu Ser Phe Trp Asp Ala Gly Glu Pro Asn Asn Ile Val Leu Val 165 170 175 Glu Asp Cys Ala Thr Ile Arg Asp Ser Ser Asn Ser Arg Lys Asn Trp 180 185 190 Asn Asp Ile Pro Cys Phe Tyr Ser Met Pro Trp Ile Cys Glu Met Pro 195 200 205 Glu Ile Ser Pro Leu Asp 210 <210> 2 <211> 219 <212> PRT <213> Homo sapiens <400> 2 Met Asn Ser Ser Lys Ser Ser Glu Thr Gln Cys Thr Glu Arg Gly Cys 1 5 10 15 Phe Ser Ser Gln Met Phe Leu Trp Thr Val Ala Gly Ile Pro Ile Leu 20 25 30 Phe Leu Ser Ala Cys Phe Ile Thr Arg Cys Val Val Thr Phe Arg Ile 35 40 45 Phe Gln Thr Cys Asp Glu Lys Lys Phe Gln Leu Pro Glu Asn Phe Thr 50 55 60 Glu Leu Ser Cys Tyr Asn Tyr Gly Ser Gly Ser Val Lys Asn Cys Cys 65 70 75 80 Pro Leu Asn Trp Glu Tyr Phe Gln Ser Ser Cys Tyr Phe Phe Ser Thr 85 90 95 Asp Thr Ile Ser Trp Ala Leu Ser Leu Lys Asn Cys Ser Ala Met Gly 100 105 110 Ala His Leu Val Val Ile Asn Ser Gln Glu Glu Gln Glu Phe Leu Ser 115 120 125 Tyr Lys Lys Pro Lys Met Arg Glu Phe Phe Ile Gly Leu Ser Asp Gln 130 135 140 Val Val Glu Gly Gln Trp Gln Trp Val Asp Gly Thr Pro Leu Thr Lys 145 150 155 160 Ser Leu Ser Phe Trp Asp Val Gly Glu Pro Asn Asn Ile Ala Thr Leu 165 170 175 Glu Asp Cys Ala Thr Met Arg Asp Ser Ser Asn Pro Arg Gln Asn Trp 180 185 190 Asn Asp Val Thr Cys Phe Leu Asn Tyr Phe Arg Ile Cys Glu Met Val 195 200 205 Gly Ile Asn Pro Leu Asn Lys Gly Lys Ser Leu 210 215 <210> 3 <211> 178 <212> PRT <213> Homo sapiens <400> 3 Arg Cys Val Val Thr Phe Arg Ile Phe Gln Thr Cys Asp Glu Lys Lys 1 5 10 15 Phe Gln Leu Pro Glu Asn Phe Thr Glu Leu Ser Cys Tyr Asn Tyr Gly 20 25 30 Ser Gly Ser Val Lys Asn Cys Cys Pro Leu Asn Trp Glu Tyr Phe Gln 35 40 45 Ser Ser Cys Tyr Phe Phe Ser Thr Asp Thr Ile Ser Trp Ala Leu Ser 50 55 60 Leu Lys Asn Cys Ser Ala Met Gly Ala His Leu Val Val Ile Asn Ser 65 70 75 80 Gln Glu Glu Gln Glu Phe Leu Ser Tyr Lys Lys Pro Lys Met Arg Glu 85 90 95 Phe Phe Ile Gly Leu Ser Asp Gln Val Val Glu Gly Gln Trp Gln Trp 100 105 110 Val Asp Gly Thr Pro Leu Thr Lys Ser Leu Ser Phe Trp Asp Val Gly 115 120 125 Glu Pro Asn Asn Ile Ala Thr Leu Glu Asp Cys Ala Thr Met Arg Asp 130 135 140 Ser Ser Asn Pro Arg Gln Asn Trp Asn Asp Val Thr Cys Phe Leu Asn 145 150 155 160 Tyr Phe Arg Ile Cys Glu Met Val Gly Ile Asn Pro Leu Asn Lys Gly 165 170 175 Lys Ser <210> 4 <211> 46 <212> PRT <213> Homo sapiens <400> 4 Val Thr Phe Arg Ile Phe Gln Thr Cys Asp Glu Lys Lys Phe Gln Leu 1 5 10 15 Pro Glu Asn Phe Thr Glu Leu Ser Cys Tyr Asn Tyr Gly Ser Gly Ser 20 25 30 Val Lys Asn Cys Cys Pro Leu Asn Trp Glu Tyr Phe Gln Ser 35 40 45

Claims (12)

As an epitope of the Mincle (Macrophage-inducible C-type lectin; Mincle) protein,
An epitope consisting of an amino acid sequence represented by sequence number 4.
A nucleic acid molecule encoding an epitope of claim 1.
An expression vector into which the nucleic acid molecule of claim 2 has been inserted.
delete delete An antibody or antigen-binding fragment that specifically binds to an epitope consisting of the amino acid sequence represented by SEQ ID NO: 4.
A pharmaceutical composition for preventing or treating neuropathic pain, comprising an antibody or antigen-binding fragment of claim 6 as an active ingredient.
In paragraph 7,
A pharmaceutical composition having a C-type lectin receptors (CLRs) inhibitory effect.
In Article 8,
A pharmaceutical composition, wherein the C-type lectin receptor is selected from the group consisting of macrophage-inducible C-type lectin; Mincle, Dectin-1, Dectin-2, macrophage C-type lectin (MCL), macrophage mannose receptor (MMR, CD206), or CD209 (dendritic cell-specific intercellular adhesion molecule-3 grabbing non-integrin; DC-SIGN).
In Article 9,
A pharmaceutical composition, wherein the C-type lectin receptor is Mincle (macrophage-inducible C-type lectin).
In Article 8,
A pharmaceutical composition, wherein the neuropathic pain is at least one selected from the group consisting of spontaneous pain, paresthesias, dysesthesias, hyperalgesia, and allodynia.
delete