KR20240015800A - The bispecific antibody and the conjugate that can cross the BBB to treat brain nervous disease - Google Patents

Abstract

The present invention relates to a bispecific antibody or a conjugate of an antibody and a peptide that can effectively pass through the blood-brain barrier (BBB) and prevent or treat cranial nervous system diseases.
A binding molecule that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells) provided by the present invention and a blood-brain barrier receptor antibody ( Bispecific antibodies (blood-brain barrier receptor antibodies) can effectively penetrate the blood-brain barrier (BBB) to effectively prevent or treat brain and nervous system diseases.
Additionally, a binding molecule or binding fragment thereof that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells); and a peptide, through a conjugate of a binding molecule that specifically binds to the Lrig-1 protein and a peptide, it is possible to effectively prevent or treat brain and nervous system diseases by effectively passing through the blood brain barrier (BBB).
The bispecific antibody and conjugate of the present invention have the effect of passing the blood-brain barrier in vivo, maintaining physiological activity, and significantly increasing blood half-life. The stability of the bispecific antibody and conjugate is also improved, so it can be used as a treatment for brain-related diseases.

Description

Bispecific antibody and the conjugate that can cross the BBB to treat brain nervous disease}

The present invention relates to a bispecific antibody or a conjugate of an antibody and a peptide that can effectively pass through the blood-brain barrier (BBB) and prevent or treat cranial nervous system diseases.

Deterioration in memory, attention, cognitive ability, and emotional control is observed in degenerative cranial nerve diseases such as stroke, dementia, and Alzheimer's disease. This is the result of death of nerve cells and atrophy of nerve branches. The elongation of nerve branches of nerve cells plays an important role in the memory and learning functions of neural circuits by increasing nerve plasticity. Accordingly, it is predicted that active ingredients that promote nerve branch elongation and nerve regeneration have the potential to be developed as new treatments for brain and nervous system diseases.

However, in the treatment of such brain and nervous system diseases, the blood-brain barrier (hereinafter referred to as BBB) effectively protects the brain from circulating toxins by acting as a physical and functional barrier that separates the brain parenchyma from systemic circulation. It represents a major obstacle in the pharmacological treatment of brain diseases such as Alzheimer's disease, Parkinson's disease, and brain cancer.

Since most of the components of the BBB are phospholipids, fat-soluble substances pass through it, but most water-soluble substances do not.

Therefore, most charged molecules and molecules exceeding 700 daltons in size cannot cross the blood-brain barrier. Many therapeutic agents for treating diseases and disorders of the brain and CNS are so hydrophilic that they do not pass directly through the BBB. Additionally, these drugs and preparations are prone to decomposition in the blood and peripheral tissues, increasing the amount required to achieve effective serum concentrations.

Since the BBB blocks the delivery of drugs for the treatment of brain disease patients, how to deliver drugs through the BBB to the brain has been a problem for scientists for a long time.

An object of the present invention is to provide a binding molecule or binding fragment thereof that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells); and a binding molecule/blood-brain barrier receptor antibody that specifically binds to the Lrig-1 protein, including a blood-brain barrier receptor antibody or an antigen-binding fragment thereof. ) to provide a dual specific antibody.

Another object of the present invention is to provide a binding molecule or binding fragment thereof that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells); And to provide a conjugate of a peptide and a binding molecule that specifically binds to the Lrig-1 protein, including a peptide.

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.

The bispecific antibody of the binding molecule/blood-brain barrier receptor antibody that specifically binds to the Lrig-1 protein according to the present invention is an anti-insulin-like growth factor receptor (Insulin-like Growth Factor Receptor). Factor 1 Receptor (hereinafter referred to as IGF1R)) allows binding molecules or antigen-binding fragments thereof, including antibodies or antigen-binding fragments thereof, that specifically bind to the Lrig-1 protein to pass through the blood-brain barrier, thereby allowing them to enter the brain. It allows the drug to exert its action and extends its half-life to maintain its effectiveness for a long period of time.

Moreover, the dual specific antibody of the binding molecule/blood-brain barrier receptor antibody that specifically binds to the Lrig-1 protein according to the present invention is the insulin-like growth factor receptor on the cell surface. Like Growth Factor 1 Receptor, IGF1R), but does not interfere with the binding of IGF1R's ligands, IGF-1, IGF-2, and/or insulin, to IGF1R. Specifically, the anti-IGF1R antibody or antigen-binding fragment does not prevent the IGF1R ligand from binding to the IGF1R located on the cell membrane in cells expressing IGF1R and does not inhibit signaling through IGF1R, and also does not inhibit IGF1R signaling on the cell surface. It has the advantage of not affecting expression. Accordingly, the anti-IGF1R antibody or antigen-binding fragment according to the present invention can be effectively used to pass through the blood brain barrier through transcytosis. Compared to other transcytosis targets known to be expressed in endothelial cells of the brain, which are mainly used to improve BBB passage ability, IGF1R was found to have a relatively high expression level in the brain. In one embodiment, IGF1R is a target of peripheral tissue compared to other targets currently being developed for improving the BBB crossing ability of therapeutic antibodies, such as transferrin receptor, insulin receptor, etc. ), for example, it was found to have a relatively low expression level in the liver, lung, and colon.

IGF1R is a target of RMT (Receptor Mediated Transcytosis), which can deliver useful substances into the brain by passing through the blood-brain barrier (BBB). However, in order to be used as a drug delivery target for crossing the blood brain barrier, it is desirable to have the properties of binding to IGF1R on the cell surface but not affecting the binding of the ligand and not affecting the signaling pathway through IGF1R. Therefore, the anti-IGF1R antibody and antigen-binding fragment thereof according to the present invention do not inhibit IGF1R and its ligand binding and signaling through IGF1R, and therefore can be used as a shuttle means to pass through the blood brain barrier.

The anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention is capable of transcytosis and can pass through brain endothelial cells. Additionally, when the antibody according to the present invention is intravascularly injected into a mouse, it is located at the same location as the mouse's brain blood vessel. These results indicate that the antibody or antigen-binding fragment according to the present invention can be effectively used as a drug carrier that passes through the blood brain barrier.

Therefore, the anti-IGF1R antibody or antigen-binding fragment thereof according to the present invention allows bioactive substances acting in the brain to pass through the blood-brain barrier. As used herein, a biological barrier refers to a cell, membrane, or structure that prevents the effective passage, diffusion, or transfer of cells, tissues, membranes, or biological molecules. These biological barriers include nerve cells/tissue, connective tissue, muscle, membrane or epithelial (e.g. mucosal or vascular) cells. A representative example is the blood brain barrier.

In particular, the anti-IGF1R antibody or antigen-binding fragment according to the present invention specifically recognizes IGF1R (Insulin-like Growth Factor 1 Receptor) and recognizes IGF1R, especially human IGF1R, mouse IGF1R, rat IGF1R, and monkey IGF1R. and binds to it, does not interfere with the binding of IGF1R's ligands, IGF-1, IGF-2, and/or insulin to IGF1R, does not inhibit signaling through IGF1R, and can be used for transcytosis to form a barrier in the blood. It has the ability to pass through and does not have ADCC (Antibody-dependent cell-mediated cytotoxicity), so it does not reduce the level of IGF1R in the brain even when administered repeatedly to animals, so it is not toxic.

In particular, the anti-IGF1R antibody according to the present invention binds to IGF1R present on the surface of brain endothelial cells constituting the BBB and is internalized into the cells.

For example, the anti-IGF1R antibody according to the present invention may have an scFv form and may be produced by binding to a therapeutic antibody in various ways. For example, the scFv of the anti-IGF1R antibody is a bispecific antibody in which two are bound to the C-terminus of a therapeutic antibody, e.g., an α-syn antibody, or one is It can be produced as a combined bispecific antibody, that is, a monovalent form of bispecific antibody, and all of the bispecific antibodies are internalized into cells expressing IGF1R. The high binding affinity of IGF1R antibodies to antigens on the cell surface increases the internalization effect, which may lead to the ability to pass the BBB. If the antibody has the ability to pass through the BBB and interferes with IGF1R signaling, it may cause side effects. The anti-IGF1R antibody according to the present invention has the binding ability to function as a BBB shuttle and at the same time interferes with IGF1R signaling. It is an antibody that does not do this.

The anti-IGF1R antibody or antigen-binding fragment has excellent ease of development. In this regard, an attempt was made to remove post-translational modifications, such as deamidation, that occur in the CDR region of the anti-IGF1R antibody and reduce the stability and efficacy of the antibody. Alternatively, at least one of the amino acids located on both sides of the deamidation site of the antibody may be substituted, preferably the amino acid immediately next to the C-terminus of the deamidation site of the antibody. For example, by substituting G next to Asn located at the deamidation site of the antibody with A or S next to Asn with V, it has a binding affinity similar to that of the parental anti-IGF1R antibody and at the same time has excellent stability and BBB passage ability. Eggplant can produce deamidated antibodies.

In one embodiment of the present invention, by eliminating deamidation of amino acids in the anti-IGF1R antibody, there is no change in the binding ability of the antigen, IGF1R, to ECD, and the risk of antibody degradation, which is disadvantageous for process development, storage, etc., is reduced. Amino acid positions where deamidation is eliminated in anti-IGF1R antibodies include, for example, N51D, N51Q, S52V of light chain LCDR2 at 1564 (IgG), 1564 (scFv), or 1564-3, N95aK, N95aH, N95aR, N95aD, G95bA of LCDR3. or N54D, N54Q or G55A in HCDR2 of the heavy chain. Deamidation removal is not limited to the clones mentioned above.

CDR sequences of heavy and light chain variable regions of major anti-IGF1R antibodies Clone ID Hv-CDR1 Hv-CDR2 Hv-CDR3 Lv-CDR1 Lv-CDR2 Lv-CDR3 GTC-01 SEQ ID NO: 53 sequence number
54 sequence number
57 sequence number
58 sequence number
59 sequence number
61 GTC-02 SEQ ID NO: 53 sequence number
55 sequence number
57 sequence number
58 sequence number
60 sequence number
62 GTC-03 SEQ ID NO: 53 sequence number
56 sequence number
57 sequence number
58 sequence number
60 sequence number
62 GTC-04 SEQ ID NO: 53 sequence number
55 sequence number
57 sequence number
58 sequence number
60 sequence number
62

Additionally, when the anti-IGF1R antibody according to the present invention is linked to a bioactive substance that acts in the brain, it can induce improved BBB passage ability and efficacy compared to the bioactive substance alone. An anti-IGF1R antibody or antigen-binding fragment according to the present invention is said to “specifically bind” to its target, such as an antigen, when it binds with an affinity with a dissociation constant (K _D) of ≤ 1x10 ^-6 M. . Antibodies specifically bind to the target with high affinity when K _D is ≤ 1x10 ^-6 M or EC50 (effective concentration 50) is 2 nM or less. In one embodiment, the antibody or antigen-binding fragment thereof is capable of binding IGF1R or human IGF1R with a K _D of ≤ 1x10 ^-8 M.

In the present invention, the term “epitope” is interpreted as an antigenic determinant, meaning a portion of an antigen recognized by an antibody. According to one embodiment, the binding site of the anti-IGF1R antibody according to the present invention may be the extracellular domain of IGF1R protein, for example, human IGF1R protein. More specifically, the binding site of the anti-IGF1R antibody according to the present invention, such as the 1564 clone antibody, to the human IGF1R protein is: In the human IGF1R protein, binding site 1 is Y775, P776, F778, R650, S791, L798 and Contains Glu779, binding site 2 includes L641, H808, E809 and L813, and binding site 3 includes V397, D435, W434, Y460 and C488.

Accordingly, the epitope of the IGF1R antibody according to the present invention is a conformational epitope and may include all or part of the three binding sites above.

The "Blood Brain Barrier (BBB)" of the present invention is based on basement membrane, microglia, pericytes, astrocytes, and endothelial cells. They form tight junctions, and the basement membrane surrounds and protects endothelial cells. Microglia play a major role in inducing immune responses in the central nervous system, and periepithelial cells account for more than 20% of blood capillaries. It produces components of the basement membrane, which is a barrier that regulates ionic balance within the brain and protects the brain from extracellular toxicity.

In the bispecific antibody or conjugate according to the present invention, the bispecific antibody or peptide passes through the blood-brain barrier through a passive transport route or a receptor-mediated transport route. It may be characterized by, but is not limited to, this.

In the present invention, the pathway by receptor-mediated transport includes insulin receptor, transferrin receptor, low density lipoprotein receptor, and low density lipoprotein receptor-related protein (low density lipoprotein receptor). density lipoprotein receptor-related protein, leptin receptor, nicotinic acetylcholine receptor, glutathione transporter, calcium-activated potassium channel, and RAGE (receptor for advanced glycation endproducts), and a ligand of the receptor and an antibody that binds to the receptor or ligand, and passes through the blood-brain barrier by a receptor-mediated transport pathway, but is not limited thereto. .

In the present invention, the peptide may be selected from the group consisting of peptides, proteins, and antibodies that pass through the blood-brain barrier through this route, or may be a partial peptide sequence separated from the peptide, protein, or antibody. Not limited. For example, peptides such as HAITYPRH peptide of SEQ ID NO: 64, THR peptide of SEQ ID NO: 65, Angiopep-2 peptide of SEQ ID NO: 66, ApoB peptide of SEQ ID NO: 68, and ApoE peptide of SEQ ID NO: 67 may be used, but the present invention Peptides that are included in the conjugate and can deliver an antibody that specifically binds to the bound Lrig-1 protein through the BBB and into the brain are included without limitation.

As another embodiment, the peptide is

(1) Angiopep-2: TFFYGGSRGKRNNFKTEEYOH (SEQ ID NO: 66),

(2) ApoB (3371-3409): SVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO: 68),

(3) ApoE(159-167) ₂ : (LRKLRKRLL) ₂ (SEQ ID NO: 67),

(4) Peptide-22: Ac-C(&)MPLRRGC(&)- NH ₂ (SEQ ID NO: 69),

(5) THR: THRPPMWSPVWP- NH ₂ (SEQ ID NO: 65),

(6) THR retro-enantio : pwvpswmpprht- NH ₂ (SEQ ID NO: 70),

(7) CRT: C(&)RTIGPSVC(&) (SEQ ID NO: 71),

(8) Leptin 30: YQQILTSMPSRRNVIQISNDLENLRDLLHVL (SEQ ID NO: 72),

(9) RVG29: YTIWMPENRPGTPCDIFTNSRGKRASNGOH (SEQ ID NO: 73)

(10) ^D CDX: GreirtGraerwsekf-OH (SEQ ID NO: 74)

(11) Apamin: C(& ₁ )NC(& ₂ )KAPETALC(& ₁ )-ARRC(& ₂ )QQH- NH ₂ (SEQ ID NO: 75),

(12) MiniAp-4: [Dap](&)KAPETALD(&) (SEQ ID NO: 76),

(13) GSH: γ-L-glutamyl-CG-OH

(14) G23: HLNILSTLWKYRC (SEQ ID NO: 77),

(15) g7: GFtGFLS(O-β-Glc) -NH ₂ (SEQ ID NO: 78),

(16) TGN: TGNYKALHPHNG (SEQ ID NO: 79),

(17) TAT(47-57): YGRKKRRQRRR- NH ₂ (SEQ ID NO: 80),

(18) SynB1: RGGRLSYSRRRFSTSTGR (SEQ ID NO: 81),

(19) Diketopiperazines: &(N-MePhe)-(N-MePhe)Diketopiperazines, or

(20) PhPro: It may be (Phenylproline) ₄ -NH ₂ (SEQ ID NO: 82), but is not limited thereto.

In the above sequence, & refers to J. Pept. As a symbol according to the nomenclature for cyclic peptides described in Res., 2005, 65, 550-555 (J. Spengler et al.), '&' refers to the connecting point. For example, in a single chain, '&' written first refers to the position of one end of a chemical bond, and '&' written second refers to the site to which the chemical bond is attached. For example, “&Ala-Ala-Phe-Leu-Pro&” indicates that a chemical bond is formed between alanine and proline to form a cyclic peptide. If two or more chemical bonds exist, symbols such as &1, &2 can be used to indicate the position where the chemical bond is formed. For example, if it is expressed as &1Asp(&2)-Trp-Phe-Dpr(&2)-Leu-Met&1, it means that a chemical bond is formed between Asp and Met, and a chemical bond is formed between Asp and Dpr. Meanwhile, [Dap] represents diaminopropionic acid.

One embodiment of the present invention is a bispecific device that specifically recognizes IGF1R (Insulin-like Growth Factor 1 Receptor), but has no effect on the binding of IGF1R ligand, does not inhibit signaling through the IGF1R receptor, and is capable of transcytosis. An antibody or antigen-binding fragment thereof is provided.

One embodiment of the present invention is an antibody comprising an antigen binding site for Lrig-1 protein and an antigen binding site for IGF1R, specifically an antibody specifically binding to Lrig-1 protein and a bispecific antibody for IGF1R. It's about. Accordingly, the bispecific antibody according to the present invention can recognize and bind to both Lrig-1 protein and IGF1R as antigens.

The antibody specifically binding to the Lrig-1 protein and the bispecific antibody against IGF1R according to the present invention include an antibody or antigen-binding fragment thereof that specifically binds to the Lrig-1 protein. It can specifically recognize and bind, and can be used for the prevention, treatment and/or diagnosis of brain and nervous system diseases.

As used herein, a “bivalent antigen binding protein” or “bivalent antibody” contains two antigen binding sites. The two antigen binding sites included in such a bivalent antibody may have the same antigen specificity, or may be bispecific antibodies that each bind to different antigens. As used herein, a “multispecific antigen binding protein” or “multispecific antibody” is one that targets two or more antigens or epitopes.

In one embodiment of the present invention, the active ingredient contains a binding molecule that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells). , and provides a pharmaceutical composition for combined administration for the prevention or treatment of cranial nervous system diseases. For the purposes of the present invention, the regulatory T cells may be CD4+ T cells, but are not limited thereto.

In the present invention, the Lrig-1 protein may include the amino acid sequence represented by SEQ ID NO: 19 or 21, but is not limited thereto.

Additionally, in the present invention, the Lrig-1 protein may be encoded by the gene represented by SEQ ID NO: 20 or 22, but is not limited thereto.

In the present invention, the Lrig-1 protein is a transmembrane protein consisting of 1091 amino acids present on the surface of immune cells, especially regulatory T cells, and contains a leucine-rich repeat (LRR) sequence on the extracellular or lumen side. It consists of three immunoglobulin-like domains, a transmembrane sequence, and a cytoplasmic tail. The LRIG gene family includes LRIG1, LRIG2, and LRIG3, and the amino acids among them are highly conserved. The LRIG1 gene is highly expressed in normal skin, and can regulate the proliferation of epithelial stem cells by expressing it in basal and hair follicle cells. Therefore, it plays an important role in maintaining epidermal homeostasis, and its absence can lead to psoriasis or skin cancer. It has been reported that if the chromosome 3p14.3 part where LRIG1 is located is cut, there is a high possibility of developing into cancer cells. In fact, the expression of LRIG1 is greatly reduced in renal cell carcinoma and cutaneous squamous cell carcinoma. It was confirmed that Currently, LRIG1 degrades proteins through ubiquitination of EGFR (Epidermarl growth factor receptor) through c-Cbl, thereby blocking signal transduction by phosphorylation of MAPK and AKT, which exist downstream and are involved in cell proliferation, and caspase ( It has the potential to be a cancer inhibitor by increasing the secretion of Caspase-8, causing apoptosis. For the purpose of the present invention, the Lrig-1 protein may be a protein existing in humans or mice, but is not limited thereto.

“Binding molecule” as used in the present invention refers to an intact immunoglobulin, including a monoclonal antibody such as a chimeric, humanized or human monoclonal antibody, or an immunoglobulin that binds to an antigen, e.g. It refers to a variable domain containing an immunoglobulin fragment that competes with intact immunoglobulin for binding to the monomeric HA or trimeric HA of the influenza A virus. Regardless of structure, the antigen-binding fragment binds the same antigen recognized by the intact immunoglobulin. An antigen-binding fragment is a sequence of two or more consecutive amino acid residues, at least 20 consecutive amino acid residues, at least 25 consecutive amino acid residues, at least 30 consecutive amino acid residues, at least 35 consecutive amino acid residues, or at least 40 consecutive amino acid residues of the amino acid sequence of the binding molecule. , 50 or more consecutive amino acid residues, 60 or more consecutive amino acid residues, 70 or more consecutive amino acid residues, 80 or more consecutive amino acid residues, 90 or more consecutive amino acid residues, 100 or more consecutive amino acid residues, 125 or more consecutive amino acid residues, It may comprise a peptide or polypeptide comprising an amino acid sequence of at least 150 consecutive amino acid residues, at least 175 consecutive amino acid residues, at least 200 consecutive amino acid residues, or at least 250 consecutive amino acid residues. “Antigen-binding fragment” refers specifically to Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragment, single-chain antibody (scFv), bivalent Single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, polypeptides containing one or more fragments of an immunoglobulin sufficient to bind to a specific antigen as a polypeptide, and the like. The fragments may be produced synthetically or by enzymatic or chemical digestion of intact immunoglobulins, or may be genetically engineered by recombinant DNA techniques. Methods of production are well known in the art.

In the present invention, the binding molecule may further include an Fc region (Fragment crystallization region) or a constant region. At this time, the Fc region may be the Fc region of an IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 antibody, may be derived therefrom, or may be a hybrid Fc (hybrid Fc) region.

In the present invention, the Fc region may be the Fc region of a mammalian IgA, IgD, IgE, IgM, IgG1, IgG2, IgG3 or IgG4 antibody, preferably a human-derived IgA, IgD, IgE, IgM, IgG1, IgG2, It may be the Fc region of an IgG3 or IgG4 antibody, but is not limited thereto.

As an example of the present invention, the Fc region may be a human-derived immunoglobulin lambda constant region, but is not limited thereto.

In the present invention, the “hybrid Fc” may be derived from a combination of human IgG subclasses or a combination of human IgD and IgG. When the hybrid Fc binds to a biologically active molecule, polypeptide, etc., it not only increases the serum half-life of the biologically active molecule, but also has the effect of increasing the expression level of the polypeptide when the nucleotide encoding the Fc-polypeptide fusion protein is expressed.

In the binding molecule of the present invention, the Fc or constant region may be connected to the variable region with a linker. At this time, a linker is connected to the C-terminus of the Fc or constant region, and the N-terminus of the binding molecule of the present invention may be connected to the linker, but is not limited thereto.

In the present invention, the “linker” may include a sequence that can be cleaved by an enzyme overexpressed in the tissue or cell of the desired disease. If it can be cleaved by an overexpressed enzyme as described above, it can effectively prevent the activity of the polypeptide from being reduced due to the Fc or constant region. In the present invention, a preferred example of a linker is a peptide linker consisting of 33 amino acids located at positions 282 to 314 of human albumin, which is most abundant in the blood, more preferably a peptide linker consisting of 13 amino acids located at positions 292 to 304. It may be a peptide linker, and this part is mostly exposed to the outside due to its three-dimensional structure and has a minimal possibility of inducing an immune response in the body. However, it is not limited to this.

The binding molecule of the present invention is characterized as being an antibody or a fragment thereof, but is not limited thereto. The antibody has the ability to bind to the Lrig-1 protein as a monoclonal antibody, full-length antibody, or part of an antibody, and binds to the Lrig-1 antigenic determining site competitively with the binding molecule of the present invention. Includes all possible antibody fragments.

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. For the purposes of the present invention, the antigen may be the Lrig-1 protein present on the surface of regulatory T cells. Preferably, it may specifically recognize the leucine rich region or immunoglobulin-like domain of the Lrig-1 protein, but is not limited thereto.

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 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 "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.

Additionally, in the present invention, the “antibody fragment” refers to a fragment that possesses an antigen-binding function and includes Fab, Fab', F(ab')2, and Fv. The Fab has a structure that includes the variable regions of the light and heavy chains, the constant region of the light chain, and the first constant region (CH1 domain) of the heavy chain, and has one antigen binding site. Additionally, Fab' differs from Fab in that it has a hinge region containing one or more cysteine residues at the C terminus of the heavy chain CH1 domain. F(ab')2 antibody is produced when the cysteine residue in the hinge region of Fab' forms a disulfide bond. Fv (Variable fragment) refers to the minimum antibody fragment containing only the heavy chain variable region and light chain variable region. In double-chain Fv (dsFv), the heavy chain variable region and light chain variable region are connected by a disulfide bond, and in single-chain Fv (scFv), the heavy chain variable region and light chain variable region are generally covalently connected through a peptide linker. . The antibody 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 is a chimeric antibody, humanized antibody, bivalent, bispecific molecule, minibody, domain antibody, bispecific antibody, antibody mimetic, and unispecific antibody. The body (unibody) may be a diabody, triabody, tetrabody, or a fragment thereof, but is not limited thereto.

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, the binding molecule can bind to the Lrig-1 protein and can also be provided as a bispecific antibody or bispecific antigen-binding fragment that can bind to other proteins.

In the present invention, the bispecific antibody and bispecific antigen binding fragment may include a binding molecule according to the present invention. As an example in the present invention, the bispecific antibody and the bispecific antigen binding fragment include an antigen binding domain capable of binding to the Lrig-1 protein, wherein the antigen binding domain capable of binding to the Lrig-1 protein is It may comprise or consist of a binding molecule according to the present invention and may comprise an antigen binding domain capable of binding to a blood-brain barrier receptor.

The bispecific antibodies and bispecific antigen-binding fragments provided by the present invention include an antigen-binding domain, which is a binding molecule capable of binding to the Lrig-1 protein according to the present invention, and a blood-brain barrier receptor. Contains an antigen binding domain capable of binding to.

Bispecific antibodies and bispecific antigen binding fragments according to the invention may be provided in any suitable format, for example, those described in the literature incorporated herein by reference in their entirety. For example, a bispecific antibody or bispecific antigen binding fragment may be a bispecific antibody conjugate (e.g. an IgG2, F(ab')2 or CovX-body), a bispecific IgG or an IgG-type molecule (e.g. IgG, scFv4-Ig, IgG-scFv, scFv-IgG, DVD-Ig, IgG-sVD, sVD-IgG, or 2 in 1-IgG, mAb2, or Tandemab common LC), asymmetric bispecific IgG or IgG-type molecules (e.g. kih IgG, kih IgG common LC, CrossMab, kih IgG-scFab, mAb-Fv, charge pair or SEED-body), small bispecific antibody molecules (e.g. diabody (Db), dsDb, DART, scDb, tandAbs, tandem scFv (taFv), tandem dAb/VHH, triple body, triple head, Fab-scFv, or F(ab')2-scFv2), bispecific Fc and CH3 fusion proteins (e.g. : taFv-Fc, di-diabody, scDb-CH3, scFv-Fc-scFv, HCAb-VHH, scFv-kih-Fc, or scFv-kih-CH3), or bispecific fusion protein (e.g. scFv2-albumin , scDb-albumin, taFv-toxin, DNL-Fab3, DNL-Fab4-IgG, DNL-Fab4-IgG-cytokine2). Those skilled in the art can design and prepare bispecific antibodies and bispecific antigen binding fragments according to the present invention.

The method for producing the bispecific antibody in the present invention includes chemical cross-linking of an antibody or antibody fragment with a reducing disulfide or non-reducing thioether bond. For example, N-succinimidyl-3-(-2-pyridyldithio)-propionate (SPDP) can be used to generate disulfide-linked bispecific F(ab)2 heterodimers, e.g. It can be used to chemically cross-link Fab fragments through the hinge region SH- group.

In addition, another method for producing the bispecific antibody in the present invention is to fuse the antibody-producing hybridoma with, for example, polyethylene glycol to generate quadroma cells capable of secreting the bispecific antibody. Includes.

Bispecific antibodies and bispecific antigen-binding fragments according to the invention can be produced recombinantly, for example, by expression from a nucleic acid construct encoding a polypeptide for the antigen-binding molecule.

For example, two antigen binding domains (i.e., light and heavy chain variable domains for an antigen binding domain capable of binding PD-1, etc., and light and heavy chain variable domains for an antigen binding domain capable of binding other target proteins). A DNA construct encoding light and heavy chain variable domains for ) and comprising sequences encoding a suitable linker or dimerization domain between the antigen binding domains can be prepared by molecular cloning techniques. The recombinant bispecific antibody can then be produced by expression (e.g., in vitro) of the construct in a suitable host cell (e.g., a mammalian host cell), and the expressed recombinant bispecific antibody can then optionally be purified.

Antibodies may be produced by an affinity maturation process in which modified antibodies are produced with improved affinity of the antibody for the antigen compared to the unmodified parent antibody. Affinity mature antibodies can be produced by procedures known in the art.

In addition, the binding molecule provided by the present invention may include a variant of the above amino acid sequence as long as it can specifically bind to the Lrig-1 protein. For example, changes can be made to the amino acid sequence of an antibody to improve its binding affinity and/or other biological properties. Such modifications include, for example, deletions, insertions and/or substitutions of amino acid sequence residues of the antibody.

These amino acid mutations are made based on the relative similarity of amino acid side chain substitutions, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape and type of amino acid side chain substitutions shows that arginine, lysine and histidine are all positively charged residues; Alanine, glycine and serine have similar sizes; It can be seen that phenylalanine, tryptophan and tyrosine have similar shapes. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine, and serine; And phenylalanine, tryptophan, and tyrosine can be said to be biologically equivalent in function.

In introducing mutations, the hydrophobic index of the amino acid may be considered. Each amino acid is assigned a hydrophobicity index based on its hydrophobicity and charge: isoleucine (+4.5); Valine (+4.2); leucine (+3.8); phenylalanine (+2.8); Cysteine/Cysteine (+2.5); Methionine (+1.9); Alanine (+1.8); Glycine (-0.4); Threonine (-0.7); Serine (-0.8); tryptophan (-0.9); Tyrosine (-1.3); Proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); Aspartate (-3.5); Asparagine (-3.5); Lysine (-3.9); and arginine (-4.5). The hydrophobic amino acid index is very important in imparting interactive biological functions to proteins. It is a known fact that similar biological activity can be maintained only when substituted with an amino acid having a similar hydrophobic index. When introducing a mutation with reference to the hydrophobicity index, substitution is made between amino acids showing a difference in the hydrophobicity index, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

Meanwhile, it is also well known that substitution between amino acids with similar hydrophilicity values results in proteins with equal biological activity. As already known, the following hydrophilicity values are assigned to each amino acid residue: arginine (+3.0); Lysine (+3.0); Asphaltate (+3.0± 1); glutamate (+3.0±1); serine (+0.3); Asparagine (+0.2); Glutamine (+0.2); Glycine (0); Threonine (-0.4); Proline (-0.5±1); Alanine (-0.5); histidine (-0.5); Cysteine (-1.0); Methionine (-1.3); Valine (-1.5); leucine (-1.8); isoleucine (-1.8); Tyrosine (-2.3); Phenylalanine (-2.5); Tryptophan (-3.4). When introducing a mutation with reference to the hydrophilicity value, substitution can be made between amino acids showing a difference in hydrophilicity value, preferably within ±2, more preferably within ±1, and even more preferably within ±0.5.

Amino acid exchanges in proteins that do not overall alter the activity of the molecule are known in the art. The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, and Gln/Glu.

Considering the mutations with bioequivalent activity described above, the binding molecule of the present invention is interpreted to also include sequences showing substantial identity with the sequences listed in the sequence listing.

In the present invention, the term "substantial identity" means that the sequence of the present invention and any other sequence are paralleled to the maximum extent possible, and when the parallel sequences are analyzed using an algorithm commonly used in the art, the identity of the sequence is at least 61%. It means a sequence showing homology, more preferably 70% homology, even more preferably 80% homology, and most preferably 90% homology. Alignment methods for sequence comparison are known in the art. Various methods and algorithms for alignment are accessible from NCBI Basic Local Alignment Search Tool (BLAST), NBCI (National Center for Biological Information), etc., and are linked to sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. Available. BLSAT can be accessed at this address (www.ncbi.nlm.nih.gov/BLAST/). The sequence homology comparison method using this program can be checked online (www.ncbi.nlm.nih.gov/BLAST/blast_help.html).

In the present invention, the binding molecule, preferably the antibody, may be produced by a conventional method for producing antibodies, but may also be produced by affinity maturation.

In the present invention, “affinity maturation” refers to the process by which activated B cells produce antibodies with increased affinity for an antigen during an immune response. For the purposes of the present invention, the affinity maturation can produce antibodies or antibody fragments produced by affinity maturation based on the principles of mutation and selection, identical to the process that occurs in nature.

In the pharmaceutical composition of the present invention, the active ingredient of the present invention promotes endocytosys of beta amyloid and decomposition of endocytosed beta amyloid in microglial cells of the brain, thereby causing beta amyloid accumulation. Diseases can be treated and improved.

In the pharmaceutical composition, the disease caused by beta amyloid accumulation is Alzheimer's disease, Lewy body dementia, myositis, or cerebral amyloid angiopathy. You can.

Additionally, the composition provided by the present invention can be used as a pharmaceutical composition or a food composition, but is not limited thereto.

The "prevention" of the present invention may include, without limitation, any action that can block, suppress or delay symptoms caused by a brain nervous system disease using the composition of the present invention.

The “treatment” and “improvement” of the present invention may include, without limitation, any action that improves symptoms caused by a cranial nervous system disease or provides benefits by using the 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.

The pharmaceutical composition of the present invention 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. there is. 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, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may 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 according to 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.

As used herein, “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion 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 varies 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. The dosage of the pharmaceutical composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by a person skilled in the art, and may range from 0.0001 to 50 mg/kg per day. It can be administered at 0.001 to 50 mg/kg. The above administration may be administered once a day or in divided doses. The above 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 pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

Food compositions containing the composition of the present invention as an active ingredient can be manufactured in the form of various foods, such as beverages, gum, tea, vitamin complexes, powders, granules, tablets, capsules, snacks, rice cakes, bread, etc. . The food composition of the present invention is composed of plant extracts with little toxicity and side effects, so it can be used safely even when taken for long periods of time for preventive purposes.

When the composition of the present invention is included in a food composition, it can be added in an amount of 0.1 to 50% of the total weight.

Here, when the food composition is manufactured in the form of a beverage, there are no particular limitations other than containing the food composition in the indicated ratio, and various flavoring agents or natural carbohydrates can be contained as additional ingredients like ordinary beverages. That is, natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, polysaccharides such as sucrose, dextrin, cyclodextrin, etc., and sugar alcohols such as xylitol, sorbitol, and erythritol. can do. Examples of the flavoring agent include natural flavoring agents (thaumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.).

In addition, the food composition of the present invention contains various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, and protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc.

These ingredients can be used independently or in combination. The proportion of these additives is not critical, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.

In the present invention, the “individual” refers to an individual suspected of having a brain nervous system disease, and the subject suspected of having the disease refers to a mammal, including humans, rats, livestock, etc., that has or may develop the disease. , subjects that can be treated with the effective substance provided by the present invention are included without limitation.

The brain nervous system disease treated through the method of the present invention may be a neurodegenerative disease or a neuroinflammatory disease, and specific examples include stroke, dementia, Alzheimer's disease, Parkinson's disease, and Huntington's disease. ), Niemann-Pick disease, multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis , Paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy disease, progressive supranuclear palsy, nervous system autoimmune disease, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury ( It may be selected from the group consisting of spinal cord injury and tauopathy, but is not limited thereto.

In one embodiment of the present invention, a binding molecule or binding fragment thereof that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells); and a binding molecule/blood-brain barrier receptor antibody that specifically binds to the Lrig-1 protein, including a blood-brain barrier receptor antibody or an antigen-binding fragment thereof. ) provides a dual specific antibody.

In another embodiment of the present invention, the blood-brain barrier receptor is transferrin receptor, insulin receptor, insulin-like growth factor receptor, low-density lipoprotein receptor-related protein 8. (low density lipoprotein receptor-related protein 8), low density lipoprotein receptor-related protein 1 (low density lipoprotein receptor-related protein 1), heparin-binding epidermal growth factor-like growth factor ), leptin receptor, nicotinic acetylcholine receptor, glutathione transporter, calcium-activated potassium channel, and RAGE (receptor for advanced glycation endproducts). A bispecific antibody of choice is provided.

In another embodiment of the present invention, the insulin-like growth factor receptor (insulin-like growth factor receptor) specifically binds to the Lrig-1 protein, which is the insulin-like growth factor 1 receptor (IGF1R). Bispecific antibodies of binding molecules/blood-brain barrier receptor antibodies are provided.

In another embodiment of the present invention, the antigen-binding fragment of the insulin-like growth factor 1 receptor (IGF1R) antibody includes scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab) ') It provides a bispecific antibody of a binding molecule/blood-brain barrier receptor antibody that specifically binds to the Lrig-1 protein.

In another embodiment of the present invention, the binding molecule that specifically binds to the Lrig-1 protein is a complete binding molecule, and the insulin-like growth factor 1 receptor (IGF1R) antibody is an Fv-fragment of the antibody. phosphorus, provides a bispecific antibody.

In another embodiment of the present invention, a bispecific antibody is provided in which the complete binding molecule that specifically binds to the Lrig-1 protein is in the form of IgG1, IgG2, IgG3, or IgG4.

In another embodiment of the present invention, the unit price of one molecule of the insulin-like growth factor 1 receptor (IGF1R) antibody bound to one heavy chain CH3 of a binding molecule that specifically binds to the Lrig-1 protein Provides a bispecific antibody, which is a (monovalent) bispecific antibody.

In another embodiment of the present invention, the binding molecule that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in the regulatory T cells (Treg cells) is SEQ ID NO: A heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 33, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 35; And a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 36, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 37, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 38. , provides bispecific antibodies.

In another embodiment of the present invention, the insulin-like growth factor 1 receptor (IGF1R) antibody includes (a) a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 53, and a heavy chain CDR1 shown in SEQ ID NO: 54. A heavy chain variable region comprising a heavy chain CDR2 consisting of an amino acid sequence, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 57; And a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 58, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 59, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 61. , insulin-like growth factor 1 receptor (IGF1R) antibody; (b) a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 53, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 55, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 57; And a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 58, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 60, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 62. , insulin-like growth factor 1 receptor (IGF1R) antibody; (c) a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 53, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 56, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 57; And a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 58, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 60, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 63. , insulin-like growth factor 1 receptor (IGF1R) antibody; (d) a heavy chain variable region comprising heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 53, heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 555, and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 57; And a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 58, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 60, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 63. It provides a bispecific antibody selected from the group consisting of an insulin-like growth factor 1 receptor (IGF1R) antibody.

In another embodiment of the present invention, a pharmaceutical composition is provided wherein the brain nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

In another embodiment of the present invention, the neurodegenerative disease or neuroinflammatory disease includes stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and Niemann-Pick disease. ), multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal Degeneration, multiple system atrophy disease, progressive supranuclear paralysis, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury and tauopathy. Provided is a pharmaceutical composition selected from the group consisting of:

In another embodiment of the present invention, a binding molecule or binding fragment thereof that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells). ; and a BBB shuttle peptide, and a conjugate of a binding molecule that specifically binds to the Lrig-1 protein and a BBB shuttle peptide.

In another embodiment of the present invention, the binding molecule that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in the regulatory T cells (Treg cells) is SEQ ID NO: A heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 33, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 25; And a light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 36, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 37, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 38. , provides a conjugate of a binding molecule that specifically binds to the Lrig-1 protein and a BBB shuttle peptide.

In another embodiment of the present invention, the BBB shuttle peptide is Angiopep-2, ApoB (3371-3409), ApoE (159-167) _2, Peptide-22, THR, THR retro-enatio , CRT, Leptin 30, RVG29, ^D A binding molecule that specifically binds to the Lrig-1 protein and the BBB, selected from the group consisting of CDX, Apamin, MiniAp-4, GSH, G23, g7, TGN, TAT (47-57), SynB1, Diketopiperazines, and PhPro. A conjugate of shuttle peptide is provided.

In another embodiment of the present invention, the BBB shuttle peptide provides a conjugate of a BBB shuttle peptide and a binding molecule that specifically binds to the Lrig-1 protein, including an amino acid sequence selected from the group consisting of SEQ ID NOs: 64 to 81. do.

In another embodiment of the present invention, a pharmaceutical composition is provided wherein the brain nervous system disease is a neurodegenerative disease or a neuroinflammatory disease.

In another embodiment of the present invention, the neurodegenerative disease or neuroinflammatory disease includes stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, and Niemann-Pick disease. ), multiple sclerosis, prion disease, Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, amyotrophic lateral sclerosis, paraneoplastic syndrome, corticobasal Degeneration, multiple system atrophy disease, progressive supranuclear paralysis, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular disease, spinal cord injury and tauopathy. Provided is a pharmaceutical composition selected from the group consisting of:

A binding molecule that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells) provided by the present invention and a blood-brain barrier receptor antibody ( Bispecific antibodies (blood-brain barrier receptor antibodies) can effectively penetrate the blood-brain barrier (BBB) to effectively prevent or treat brain and nervous system diseases.

Additionally, a binding molecule or binding fragment thereof that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells); and a peptide, through a conjugate of a binding molecule that specifically binds to the Lrig-1 protein and a peptide, it is possible to effectively prevent or treat brain and nervous system diseases by effectively passing through the blood brain barrier (BBB).

Figure 1 shows the structure of Lrig-1 protein according to an embodiment of the present invention.
Figure 2 shows the structure of Lrig-1 protein according to an embodiment of the present invention.
Figure 3 shows the expression level of Lrig-1 mRNA according to an embodiment of the present invention.
Figure 4 shows the expression level of Lrig-1 mRNA according to an embodiment of the present invention.
Figure 5 shows the expression level of Lrig-1 mRNA according to an embodiment of the present invention.
Figure 6 shows the expression levels of Lrig-1, Lrig-2, and Lrig-3 mRNA according to an embodiment of the present invention.
Figure 7 shows the results of comparing the expression level of Lrig-1 protein in regulatory T cells and non-regulatory T cells according to an embodiment of the present invention.
Figure 8 shows the expression of Lrig-1 protein on the surface of regulatory T cells according to an embodiment of the present invention.
Figure 9 shows an experimental design of 5xFAD mice and 6xTg mice, which are Alzheimer's induction mice, to confirm the Alzheimer's treatment effect of a monoclonal antibody according to an embodiment of the present invention.
Figure 10 is a graph showing the change in spontaneous alteration (%) values as a result of the Y-maze experiment after treatment of 5xFAD mice and 6xTg mice, which are Alzheimer's-induced mice, with a monoclonal antibody according to an embodiment of the present invention. will be.
Figure 11 is a graph showing the change in preference values as a result of a novel object recognition experiment after treatment of 6xTg mice, which are Alzheimer's-induced mice, with a monoclonal antibody according to an embodiment of the present invention.
Figure 12 is a graph showing the change in retention time as a result of a passive avoidance test in 5xFAD mice and 6xTg mice, which are Alzheimer's-induced mice, after treatment with a monoclonal antibody according to an embodiment of the present invention.

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

[Preparation Example 1] T cell subtype cell culture

To confirm that Lrig-1 protein is expressed only in regulatory T cells (Treg), T cell subsets Th0, Th1, Th2, Th17, and iTreg were prepared. The iTreg, unlike naturally isolated nTreg, refers to cells whose differentiation is artificially induced in a medium containing the following composition.

T cell subtypes were first isolated from naïve T cells obtained from the spleen of mice, and then RPMI1640 (Invitrogen Gibco, Grand Island, NY) containing 10% fetal bovine serum (FBS; hyclone, logan, UT). The nutrient medium was further included with the ingredients listed in Table 1 below, and differentiation into each cell was induced through culturing for 72 hours in an incubator at 37°C and 5% CO ₂ .

differentiated cells Furtherance Th0 anti-CD3, anti-CD28 Th1 IL-12, anti-IL-4 antibody Th2 IL-4, anti-IFNβ Th17 IL-6, TGFβ, anti-IFNβ, anti-IL-4 iTreg IL-2, TGFβ

[Example 1] Lrig-1 structural analysis

In order to produce a specific antibody against the Lrig-1 protein, a surface protein of regulatory T cells, the three-dimensional structure of the extracellular domain of the Lrig-1 protein was predicted.

First, in order to predict the epitope sequence, Uniprot (www.uniprot.org) and RCSB Protein Data Bank (www.rcsb. org/pdb) tool was used to predict the three-dimensional structure, and the results are shown in Figures 1 and 2.

As shown in Figure 1, a total of 15 leucine rich regions from LRR1 to LRR15 were present within the Lrig-LRR domain (amino acid sequence numbers 41 to 494) of the extracellular domain of the Lrig-1 protein. Each of the LRR domains consisted of 23 to 27 amino acids and contained 3 to 5 leucines.

Additionally, as shown in Figure 2, among the extracellular domains of the Lrig-1 protein, three immunoglobulin-like domains were present at amino acid sequence numbers 494 to 781 of the Lrig-1 protein.

[Example 2] Lrig-1 epitope amino acid sequence prediction

The base sequence was predicted using the Ellipro server (tools.iedb.org/ellipro/), an epitope prediction software based on the structure of the Lrig-1 protein. The Ellipro search engine was used because it is known to be the most reliable among existing antigenic determinant prediction algorithms.

After inputting the extracellular domain analyzed in Example 1 into the antigenic determinant prediction software, the predicted continuous or discontinuous amino acid sequence of the predicted antigenic determinant was predicted.

A total of 22 consecutive epitope amino acid sequences were predicted, and a total of 8 discontinuous epitope amino acid sequences were predicted.

[Preparation Examples 1 to 2] Preparation of Lrig-1 antibody

An antibody specific to the Lrig-1 protein according to the present invention was produced. This antibody was not produced with a specific antigenic determinant, but was produced as an antibody that can bind to any site on the Lrig-1 protein.

To produce the antibody, cells expressing Lrig-1 protein were produced. The DNA fragment corresponding to SEQ ID NO: 20 and pcDNA (hygro) were cut with a cutting enzyme, then cultured at 37 degrees Celsius for ligation to produce pcDNA in which the DNA sequence of the Lrig-1 protein was inserted. did. The prepared pcDNA inserted with SEQ ID NO: 20 was introduced into L cells through transfection, allowing Lrig-1 protein to be expressed on the surface of L cells.

The light chain and heavy chain amino acid sequences capable of binding to Lrig-1 expressed on the cell surface were selected from the Human scFv library, and a total of eight heavy and light chains were selected.

A monoclonal antibody was produced by fusing the selected heavy and light chain amino acid sequences with the mlgG2a Fc region. The sequence numbers of the monoclonal antibodies are shown in Table 2 below.

division clone location Sequence information Manufacturing Example 1 GTC310-01 mouse antibody heavy chain SEQ ID NO: 49 light chain SEQ ID NO: 50 Production example 2 GTC310-01 humanized antibody heavy chain SEQ ID NO: 51 light chain SEQ ID NO: 52

[Example 3] Confirmation of specific expression of Lrig-1 mRNA in regulatory T cells

We verified whether Lrig-1 protein can act as a specific biomarker for regulatory T cells.

For the above verification, CD4 ⁺ T cells were isolated from the spleen of mice using magnet-activated cell sorting (MACS) using CD4 beads. Afterwards, regulatory T (CD4 ⁺ CD25 ⁺ T) cells and non-regulatory T (CD4 ⁺ CD25 ^- T) cells were separated using fluorescence activated cell sorter (FACS) using CD25 antibody. After extracting mRNA from each cell and the cells differentiated in Preparation Example 1 using Trizol, gDNA was removed from genomic RNA using a gDNA extraction kit (Qiagen) according to the protocol provided by the company. . The mRNA from which gDNA was removed was synthesized into cDNA using the BDsprint cDNA synthesis kit (Clonetech).

To quantitatively confirm the expression level of Lrig-1 mRNA in the cDRNA, real-time polymerase chain reaction (real time PCR) was performed.

The real-time polymerase chain reaction was conducted using SYBR Green (Molecular Probes), using the primers in Table 3 below under the conditions of 40 cycles of 3 minutes at 95, 15 seconds at 61, and 30 seconds at 72 according to the protocol provided by the company. The relative gene expression level was calculated using the ΔCT method, normalized using HPRT, and the results are shown in Figures 5 to 8.

primer order Sequence information Rat Lrig-1 Forward primer SEQ ID NO: 23 Reverse primer SEQ ID NO: 24 Rat Lrig-2 forward primer SEQ ID NO: 25 reverse primer SEQ ID NO: 26 Rat Lrig-3 forward primer SEQ ID NO: 27 reverse primer SEQ ID NO: 28 Rat FOXP3 forward primer SEQ ID NO: 29 reverse primer SEQ ID NO: 30 ACTG1 forward primer SEQ ID NO: 31 reverse primer SEQ ID NO: 32

As shown in Figure 5, it can be seen that the expression of Lrig-1 is 18.1 times higher in regulatory T ((CD4 ⁺ CD25 ⁺ T) cells than in non-regulatory T (CD4 ⁺ CD25 ^- T) cells. Compared to Lag3 and Ikzf4, known regulatory T cell markers, the expression level was approximately 10 times higher.

Additionally, as shown in Figure 6, the expression of Lrig-1 was the highest among Lrig-1, Lrig-2, and Lrig-3 corresponding to the Lrig family.

From the above results, it can be seen that the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells, especially naturally occurring regulatory T cells.

[Example 4] Confirmation of specific expression of Lrig-1 protein in regulatory T cells

It was confirmed whether Lrig-1 protein expressed from Lrig-1 mRNA was specifically expressed only in regulatory T cells.

Using FOXP3-RFP-injected (knock-in) mice in which RFP (Red fluorescent protein) was bound to the FOXP3 promoter, a transcription factor specific for regulatory T cells, a magnet-activated cell sorter (magnet-activated cell sorter) was performed using CD4 beads from the spleens of the mice. CD4 ⁺ T cells were isolated using cell sorting (MACS). Afterwards, using RFP protein, regulatory T (CD4 ⁺ RFP ⁺ T) cells and non-regulatory T (CD4 ⁺ RFP ^- T) cells were separated and obtained through fluorescence-activated cell sorter (FACS). Each of the above cells was stained with the purchased Lrig-1 antibody and the negative control was isotype, and the expression level of Lrig-1 was measured using a fluorescence-activated cell sorter, and the results are shown in Figure 7.

As shown in Figure 7, non-regulatory T cells indicated by a dotted line showed an expression level of Lrig-1 that was almost the same as that of the negative control, but in the case of regulatory T cells, there were many cells with a high expression level of Lrig-1.

The above results show that the Lrig-1 protein according to the present invention is specifically expressed in regulatory T cells.

[Example 5] Confirmation of specific expression of Lrig-1 protein on the surface of regulatory T cells

In order for the Lrig-1 protein to be a target for cell therapy, it must be expressed on the surface of regulatory T cells for more effective target treatment. Therefore, we confirmed whether the Lrig-1 protein was expressed on the surface.

Each differentiated T cell subtype in Preparation Example 1 was stained with anti-CD4-APC and anti-Lrig-1-PE antibodies, and the surface of each cell was sorted using a fluorescence-activated cell sorter (FACS). The expression level of Lrig-1 was measured, and the results are shown in Figure 8.

As shown in Figure 8, the expression of Lrig-1 is expressed in an amount of 0.77 to 15.3 in activated T cells, Th1 cells, Th2 cells, Th17 cells, and naive T cells, while differentiation was highly expressed at 83.9 in induced T cells (iTreg).

The above results show that the Lrig-1 protein according to the present invention is not only specifically expressed in regulatory T cells (Treg) cells, but is especially highly expressed on the surface of Treg cells.

[Example 6] Evaluation of the Alzheimer's treatment ability of the antibody according to the present invention - 6xTg mice

In order to evaluate the Alzheimer's treatment ability of the antibody according to the present invention, groups were designed and experiments were conducted as shown in Figure 9. Specifically, the GTC310-01 antibody of Preparation Example 1 was injected intravenously every other week for 2 months in an amount of 10 mpk into female Alzheimer's-induced 5xFAD mice and female 6xTg mice that had undergone an adaptation period for 4.5 months. However, for comparison, glatiramer acetate (GA, Copaxone®) was injected intravenously every other week for 2 months in an amount of 100 ug as a positive control. Two months later, a Y-maze experiment, a new object recognition experiment, and a passive avoidance experiment were conducted.

1. Characteristics of 6xTg mice

The 5xFAD mouse is widely used as an Alzheimer's induction model, but the 6xTg mouse used in the experiment below is a mouse model in which not only amyloid-β (Aβ42) but also tau protein (MAPT) is mutated and accumulated, and is a model closer to the human disease model than the 5xFAD mouse. Therefore, it is an ideal animal model as a model for brain and nervous system diseases, including Alzheimer's.

2. Y-maze test

The Y-maze experiment used a maze frame made by arranging three identical arms of 40 cm in length (wall height 15 cm) at an angle of 120 degrees. This experiment was a behavioral experiment that utilized the instinctive exploration habits of rodents, and was based on the high possibility of exploring new areas. By remembering the arm searched just before and not entering the same arm, the higher the memory value. An exploration time of 8 minutes is provided per object, and the final results are expressed as spontaneous alteration (%) values in Figure 10. The spontaneous alteration (%) value was calculated using Equation 1 below. Here, analysis of behavioral patterns was conducted using SMART VIDEO TRACKING Software (Panlab, USA).

[Equation 1]

Spontaneous alternation (%) = The number of triplet / (total arm entry-2)

As shown in Figure 10, in this experiment in which the relative frequency of experimental animals identifying surrounding clues and sequentially entering the maze was measured, when the GTC310-01 antibody according to the present invention was administered to Alzheimer's-induced mice, the normal control group It was confirmed that the spontaneous shift value was at a similar level to . Furthermore, it was confirmed that the 6xTg model mouse, which is closer to the human disease model, had spontaneous alternation values at a level more similar to the normal control group than the 5xFAD mouse.

3. Novel Object Recognition

The novel object recognition experiment evaluated memory using two different objects in an acrylic cage of 40 cm x 40 cm. After becoming familiar with the acrylic cage, two objects were positioned at a certain location and freely recognized, and the time spent exploring each object was measured. A delay time of 24 hours was given to each object, and then only one object was replaced with another object in the same location. At this time, the changed object is recognized as a new object, and the longer the search time, the better the memory. If you cannot remember an object from 24 hours ago, it is possible that you will not be able to distinguish between a new object and an old object and will search for both objects equally. You are allowed to explore freely for a total of 10 minutes, and the results are shown in Figure 19 as a preference index (preference index = Novel object exploring time/total exploring time). At this time, the analysis was conducted using SMART VIDEO TRACKING Software (Panlab, USA).

As shown in Figure 11, the preference index for new objects was observed to be lower in the Alzheimer's induced group compared to the normal control group. However, when the GTC310-01 antibody according to the present invention was administered, it was confirmed that the affinity value increased above the normal control group in the 6xTg model, which is closer to the human disease model.

4. Passive Avoidance Test

The passive avoidance test experimental equipment is divided into two areas, a bright chamber with lighting and a dark chamber, and the floor is made of wire mesh. First, on the first day, the mouse was allowed to move freely and get used to it. The next day, each 5xFAD mouse and 6xTg mouse were acclimatized in a lighted chamber for 1 min without the light on, then turned on the light, acclimatized for 2 min, and then received an electric shock at 0.5 mA, 1 as soon as the mouse was moved to the dark chamber. A learning test was conducted by applying pressure for seconds. The day after the learning test, a memory test (teat trial) was conducted for each mouse. 5xFAD mice and 6xTg mice were placed in a lighted chamber, and the time taken for all four paws of the 5xFAD mouse and 6xTg mouse to enter (latency time: retention) time) was set to within 300 seconds and executed. As a result, as shown in Figure 12, it was found that not only 5xFAD mice but also 6xTg mice recovered to a level close to the normal control group when the GTC310-01 antibody was administered.

Although the present invention has been described in detail above, the scope of the present invention is not limited thereto, and it is known in the art that various modifications and variations are possible without departing from the technical spirit of the present invention as set forth in the claims. It will be self-evident to those with knowledge.

Claims (17)

A binding molecule or binding fragment thereof that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells); and a binding molecule/blood-brain barrier receptor antibody that specifically binds to the Lrig-1 protein, including a blood-brain barrier receptor antibody or an antigen-binding fragment thereof. ) of bispecific antibodies. According to clause 1,
The blood-brain barrier receptors include transferrin receptor, insulin receptor, insulin-like growth factor receptor, and low density lipoprotein receptor-related protein. 8), low density lipoprotein receptor-related protein 1, heparin-binding epidermal growth factor-like growth factor, leptin receptor , a bispecific antibody selected from nicotinic acetylcholine receptor, glutathione transporter, calcium-activated potassium channel, and receptor for advanced glycation endproducts (RAGE). According to clause 2,
The insulin-like growth factor receptor is a binding molecule/blood-brain barrier receptor that specifically binds to the Lrig-1 protein, which is the insulin-like growth factor 1 receptor (IGF1R). Bispecific antibody (blood-brain barrier receptor antibody). According to clause 3,
The antigen-binding fragment of the insulin-like growth factor 1 receptor (IGF1R) antibody consists of scFv, (scFv)2, scFv-Fc, Fab, Fab', and F(ab')2, Bispecific antibody of a binding molecule/blood-brain barrier receptor antibody that specifically binds to the Lrig-1 protein. According to clause 3,
The binding molecule that specifically binds to the Lrig-1 protein is a complete binding molecule, and the insulin-like growth factor 1 receptor (IGF1R) antibody is a bispecific antibody that is an Fv-fragment of the antibody. According to clause 5,
The complete binding molecule that specifically binds to the Lrig-1 protein is a bispecific antibody in the form of IgG1, IgG2, IgG3, or IgG4. According to clause 5,
A monovalent bispecific antibody in which one molecule of the insulin-like growth factor 1 receptor (IGF1R) antibody binds to one heavy chain CH3 of a binding molecule that specifically binds to the Lrig-1 protein. Bispecific antibodies. According to clause 1,
The binding molecule that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in the regulatory T cells (Treg cells) is,
A heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 33, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 35; and
A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 36, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 37, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 38, Bispecific antibodies. According to clause 3,
The insulin-like growth factor 1 receptor (IGF1R) antibody,
(a) a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 53, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 54, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 57; and
A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 58, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 59, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 61, Insulin-like growth factor 1 receptor (IGF1R) antibody;
(b) a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 53, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 55, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 57; and
A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 58, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 60, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 62, Insulin-like growth factor 1 receptor (IGF1R) antibody;
(c) a heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 53, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 56, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 57; and
A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 58, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 60, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 63, Insulin-like growth factor 1 receptor (IGF1R) antibody;
(d) a heavy chain variable region comprising heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 53, heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 555, and heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 57; and
A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 58, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 60, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 63, A bispecific antibody selected from the group consisting of an insulin-like growth factor 1 receptor (IGF1R) antibody. According to any one of claims 1 to 9,
A pharmaceutical composition, wherein the brain nervous system disease is a neurodegenerative disease or a neuroinflammatory disease. According to clause 10,
The neurodegenerative disease or neuroinflammatory disease includes stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease ( prion disease), Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, Amyotrophic lateral sclerosis, Paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy disease, A pharmaceutical composition selected from the group consisting of progressive supranuclear paralysis, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular diseases, spinal cord injury and tauopathy. . A binding molecule or binding fragment thereof that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in regulatory T cells (Treg cells); and a conjugate of a binding molecule that specifically binds to the Lrig-1 protein and a BBB shuttle peptide, including a BBB shuttle peptide. According to clause 12,
The binding molecule that specifically binds to the Lrig-1 (leucine-rich and immunoglobulin-like domains 1) protein present in the regulatory T cells (Treg cells) is,
A heavy chain variable region comprising a heavy chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 33, a heavy chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 34, and a heavy chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 25; and
A light chain variable region comprising a light chain CDR1 consisting of the amino acid sequence shown in SEQ ID NO: 36, a light chain CDR2 consisting of the amino acid sequence shown in SEQ ID NO: 37, and a light chain CDR3 consisting of the amino acid sequence shown in SEQ ID NO: 38, A conjugate of a binding molecule that specifically binds to the Lrig-1 protein and a BBB shuttle peptide. According to clause 13,
The BBB shuttle peptides are Angiopep-2, ApoB (3371-3409), ApoE (159-167) _2, Peptide-22, THR, THR retro-enatio , CRT, Leptin 30, RVG29, ^D CDX, Apamin, MiniAp-4 , GSH, G23, g7, TGN, TAT (47-57), SynB1, Diketopiperazines, and PhPro, a conjugate of a binding molecule that specifically binds to the Lrig-1 protein and a BBB shuttle peptide. According to clause 13,
The BBB shuttle peptide is a conjugate of a BBB shuttle peptide and a binding molecule that specifically binds to the Lrig-1 protein, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 64 to 81. According to any one of claims 13 to 15,
A pharmaceutical composition, wherein the brain nervous system disease is a neurodegenerative disease or a neuroinflammatory disease. According to clause 16,
The neurodegenerative disease or neuroinflammatory disease includes stroke, dementia, Alzheimer's disease, Parkinson's disease, Huntington's disease, Niemann-Pick disease, multiple sclerosis, prion disease ( prion disease), Creutzfeldt-Jakob disease, frontotemporal dementia, Lewy dementia, Amyotrophic lateral sclerosis, Paraneoplastic syndrome, corticobasal degeneration, multiple system atrophy disease, A pharmaceutical composition selected from the group consisting of progressive supranuclear paralysis, autoimmune diseases of the nervous system, spinocerebellar ataxia, inflammatory and neuropathic pain, cerebrovascular diseases, spinal cord injury and tauopathy. .