WO2022114838A1 - Novel brain-targeting, long-acting protein conjugate and preparation method therefor - Google Patents

Abstract

The present invention relates to a novel brain-targeting, long-acting protein conjugate and a preparation method therefor.

Description

Novel brain-targeting long-acting protein conjugate and method for preparing the same

The present invention relates to a novel brain-targetable long-acting protein conjugate and a method for preparing the same.

Although the need for effective therapeutic drug delivery to the brain is emerging, the development of new drugs for the brain is progressing at a much slower rate compared to the rest of the body. This slow progression is largely due to a situation in which most drugs cannot penetrate into the brain through the brain capillary wall forming the blood-brain barrier (BBB). The blood-brain barrier (BBB) is a structure composed of a tight junction between endothelial cells of the brain blood vessels and astrocytes that further strengthen it. It functions to prevent it from coming out easily into the substance. For this reason, many of the drugs developed for the treatment of brain diseases have a problem in that they do not pass through the blood-brain barrier well. Nearly 100% of macro-molecular drugs and more than 98% of small-molecule drugs do not cross the BBB. Very few drugs, high lipid solubility and small molecules with a molecular weight of 400-500 daltons or less actually cross the BBB. In addition, of the small molecules that cross the BBB, only a small fraction of them cross the BBB in a pharmaceutically significant amount.

Only a few brain disorders, such as depression, affective disorders, chronic pain, and epilepsy, respond to small molecule drugs that cross the BBB. much more brain diseases, such as Alzheimer's disease, stroke/neuroprotection, brain and spinal cord injuries, brain cancer, HIV infection of the brain, various ataxia-producing disorders, amyotrophic lateral sclerosis, ALS), Huntington disease, childhood inborn genetic error affecting the brain, Parkinson's disease, multiple sclerosis, lysosomal storage disease, etc. are common lipid-soluble small Does not respond to molecular weight agents.

Currently, the development of a carrier that can pass through the blood-brain barrier to deliver drugs, etc. is actively in progress. On the other hand, peptides having a specific amino acid sequence are known to have a function of passing drugs or siRNA through the blood-brain barrier, but research on whether there is a possibility of delivering a physiologically active substance with a large size of a specific structure is still insufficient. (Kumar et al ., Nature. 2007, 448:39-43). In addition, there is a need for further development and research on new and improved drugs for very few brain diseases for which effective small molecule drugs exist.

It is also required to develop new and improved drugs for brain diseases.

One object of the present invention is to provide a brain-targeting long-acting protein conjugate.

Another object of the present invention is to provide a polynucleotide encoding the brain-targeting long-acting protein conjugate, an expression vector comprising the polynucleotide, and a transformant comprising the expression vector.

Another object of the present invention is to provide a method for producing the brain-targeting long-acting protein conjugate.

Another object of the present invention is to provide a composition comprising the brain-targeting long-acting protein conjugate.

Another object of the present invention is to provide the use of the brain-targeting long-acting protein conjugate in the manufacture of a medicament.

The brain-targeting long-acting conjugate of the present invention comprising a brain-targeting peptide and a physiologically active substance can pass the blood-brain barrier in vivo, maintain physiological activity, and have the effect of significantly increasing the blood half-life. The novel long-acting brain-targeting conjugate may have improved stability and may be used as a therapeutic agent for brain-related diseases.

One aspect embodying the present invention is a brain-targeting long-acting protein conjugate.

In one embodiment, the present invention relates to a brain-targeting long-acting conjugate represented by the following formula (1):

[Formula 1]

XL ₁ -F1 : YL ₂ -F2

From here,

X and Y are each independently a physiologically active substance or a brain-targeting peptide, and if one of X and Y is a physiologically active substance, the other is a brain-targeting peptide;

L ₁ and L ₂ are each independently a peptidic linker or a non-peptidyl linker,

when either or both of L ₁ and L ₂ are peptidic linkers, the peptidic linker comprises 0 to 1000 amino acids;

: is a chemical bond between XL ₁ -F1 and YL ₂ -F2;

F1 and F2 each independently include an immunoglobulin constant region, and is a substance comprising an FcRn binding site.

In the brain-targeting long-acting conjugate according to the preceding embodiment, the immunoglobulin constant regions of F1 and F2 include 1 to 4 domains selected from the group consisting of immunoglobulin CH1, CH2, CH3, and CH4 domains. do.

In the brain-targeted long-acting conjugate according to any one of the preceding embodiments, the immunoglobulin constant region comprises a hinge region.

In the brain-targeted long-acting conjugate according to any one of the preceding embodiments, each of the immunoglobulin constant region of F1 and the immunoglobulin constant region of F2 comprises a hinge region, a CH2 domain and a CH3 domain.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the immunoglobulin constant region is an immunoglobulin Fc region.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the immunoglobulin constant region is characterized in that it is derived from IgG.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the immunoglobulin constant region is characterized in that it is derived from IgG1 or IgG4.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the immunoglobulin constant region is non-glycosylated and has an amino acid sequence derived from a human IgG1 or IgG4 Fc region.

The brain target long-acting conjugate according to any one of the preceding embodiments, wherein the F1 comprises a hinge region, a CH2 domain, and a CH3 domain of IgG1, wherein the CH3 domain is amino acid 26 in the amino acid sequence of the CH3 domain of wild-type IgG1 substitution of threonine (Thr) with leucine (Leu) and substitution of amino acid 59 aspartic acid (Asp) with arginine (Arg);

The F2 includes a hinge region, a CH2 domain and a CH3 domain of IgG1, wherein the CH3 domain is amino acid 11 in the amino acid sequence of the CH3 domain of wild-type IgG1, leucine (Leu) is substituted with glutamic acid (Glu), amino acid 67 It is characterized in that it includes a substitution of tyrosine (Tyr) with leucine (Leu), and substitution of lysine (Lys) at amino acid position 69 with valine (Val).

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the long-acting conjugate is characterized in that it passes the blood-brain barrier and delivers a physiologically active substance into the brain tissue.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the brain-targeting peptide is characterized in that it is in the form of a peptide, protein, or antibody comprising an amino acid sequence that can cross the blood-brain barrier.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the brain-targeting peptide passes through the blood-brain barrier through a pathway by passive transport or a pathway by receptor-mediated transport. characterized in that

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, an insulin receptor, a transferrin receptor, a low density lipoprotein receptor, a low density lipoprotein receptor-related protein (Low density lipoprotein) receptor-related protein, leptin receptor, nicotinic acetylcholine receptor, glutathione transporter, calcium-activated potassium channel, and RAGE (receptor for advanced) glycation endproducts), and it is characterized in that it crosses the blood-brain barrier by a receptor-mediated transduction pathway through any one selected from the group consisting of a ligand of the receptors and an antibody binding to the receptor or ligand.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the physiologically active substance is a toxin; or a GLP-1 (Glucagon like peptide-1) receptor agonist; glucagon receptor agonists; Gastric inhibitory polypeptide (GIP) receptor agonists; Fibroblast growth factor (FGF) receptor agonists; Cholecystokinin receptor agonist; gastrin receptor agonists; melanocortin receptor agonists; human growth hormone; growth hormone releasing hormone; growth hormone releasing peptide; interferon; interferon receptor; colony stimulator (granulocyte colony stimulator); interleukin; interleukin receptor; enzyme; interleukin binding protein; cytokine binding protein; macrophage activator; macrophage peptides; B cell factor; T cell factor; protein A; allergy suppressors; cell necrosis glycoprotein; immunotoxins; lymphotoxin; tumor necrosis factor; tumor suppressors; metastatic growth factor; alpha-1 antitrypsin; albumin; alpha-lactalbumin; apolipoprotein-E; erythropoietin; hyperglycosylated erythropoietin; angiopoietin; hemoglobin; thrombin; thrombin receptor activating peptide; thrombomodulin; blood factor VII; blood factor VIIa; blood factor VIII; blood factor IX; blood factor XIII; plasminogen activator; fibrin-binding peptide; urokinase; streptokinase; hirudin; protein C; C-reactive protein; renin inhibitors; collagenase inhibitors; superoxide dismutase; leptin; platelet-derived growth factor; epidermal growth factor; epidermal growth factor; angiostatin; angiotensin; osteogenic growth factor; bone-promoting protein; calcitonin; insulin; atriopeptin; cartilage inducer; elcatonin; connective tissue activator; tissue factor pathway inhibitors; follicle stimulating hormone; luteinizing hormone; luteinizing hormone releasing hormone; nerve growth factors; axogenesis factor-1; brain-natriuretic peptide; glial derived neurotrophic factor; netrin; neutrophil inhibitor factor; neurotrophic factor; nutrin; parathyroid hormone; relaxin; secretin; somatomedin; insulin-like growth factor; adrenocortical hormone; glucagon; cholecystokinin; pancreatic polypeptide; gastrin releasing peptide; corticotropin releasing factor; thyroid stimulating hormone; autotaxin; lactoferrin; myostatin; ADNP (activity-dependent neuroprotective protein), BACE1 (beta-secretase1), APP (Amyloid Precursor Protein), NCAM (Neural cell adhesion molecule), Amyloid beta, Tau, RAGE (receptor for advanced glycation endproducts) ), alpha-synuclein, or an agonist or antagonist thereof; receptor class, receptor agonist; cell surface antigen; monoclonal antibody; polyclonal antibodies; antibody fragments; virus-derived vaccine antigens; hybrid polypeptides or chimeric polypeptides that activate one or more receptor agonists; And it is characterized in that it is a bioactive substance selected from the group consisting of analogs thereof.

In the brain target long-acting conjugate according to any one of the preceding embodiments,

The toxin is selected from the group consisting of maytansine or a derivative thereof, auristatin or a derivative thereof, duocarmycin or a derivative thereof, and Pyrrolobenzodiazepine (PBD) or a derivative thereof,

GLP-1 (Glucagon like peptide-1) receptor agonist is selected from the group consisting of native exendin-3 or native exendin-4, and analogs thereof,

The FGF receptor agonist is selected from the group consisting of FGF1 or an analog thereof, FGF19 or an analog thereof, FGF21 or an analog thereof, and FGF23 or an analog thereof,

the interferon is selected from the group consisting of interferon-alpha, interferon-beta and interferon-gamma;

the interferon receptor is selected from the group consisting of an interferon-alpha receptor, an interferon-beta receptor, an interferon-gamma receptor, and a soluble type I interferon receptor,

Interleukin is interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, Interleukin-13, Interleukin-14, Interleukin-15, Interleukin-16, Interleukin-17, Interleukin-18, Interleukin-19, Interleukin-20, Interleukin-21, Interleukin-22, Interleukin-23, Interleukin-24, Interleukin- 25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, and interleukin-30;

the interleukin receptor is an interleukin-1 receptor or an interleukin-4 receptor,

Enzymes are beta-glucosidase, alpha-galactosidase, beta-galactosidase, iduronidase, iduronate 2-sulfatase ( iduronate-2-sulfatase, galactose-6-sulfatase, acid alpha-glucosidase, acid ceramidase, acid sphingomyelinase sphingomyelinsase), galactocerebrosidsase, arylsulfatase A, arylsulfatase B, beta-hexosaminidase A, beta-hexosaminidase B, heparin-N -sulfatase (heparin N-sulfatase), alpha-D-mannosidase (alpha-D-mannosidase), beta-glucuronidase (beta-glucuronidase), N-acetylgalactosamine-6-sulfatase ( N-acetylgalactosamine-6 sulfatase, lysosomal acid lipase, alpha-N-acetyl-glucosaminidase, glucocerebrosidase, butyrylcholine Butyrylcholinesterase, Chitinase, glutamate decarboxylase, imiglucerase, lipase, Uricase, platelet activator acetylhydrolase (Platelet-) Activating Factor Acetylhydrolase, neutral endopeptidase, myeloperoxidase, alpha-galactosidase-A, agalsidase alpha, agalsidase beta, alpha-L-iduronidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, and imiglucerase is selected from the group consisting of

the interleukin binding protein is IL-18bp,

The cytokine binding protein is a TNF (Tumor necrosis factor) binding protein,

Nerve growth factors include nerve growth factor, cilliary neurotrophic factor, axogenesis factor-1, brain-natriuretic peptide, glial derived neurotrophic factor factor), netrin (netrin), neutrophil inhibitor factor (neurophil inhibitor factor), neurotrophic factor, and selected from the group consisting of nutrin (neuturin),

Myostatin receptor is selected from the group consisting of TNFR (P75), TNFR (P55), IL-1 receptor, VEGF receptor, and B cell activator receptor,

The myostatin receptor antagonist is IL1-Ra,

the cell surface antigen is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD45 and CD69,

Antibody fragments are characterized in that they are selected from the group consisting of scFv, Fab, Fab', F(ab') ₂ and Fd.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the GLP-1 receptor agonist is native exendin-4, exendin-4 from which the N-terminal amine group of native exendin-4 is removed, Exendin-4 in which the N-terminal amine group of native exendin-4 is substituted with a hydroxyl group, exendin-4 in which the N-terminal amine group of native exendin-4 is modified with a dimethyl group, and native exendin exendin-4 in which the N-terminal amine group of -4 is substituted with a carboxyl group, exendin-4 in which the alpha carbon of the first amino acid (histidine) of native exendin-4 is removed; and exendin-4 in which the twelfth amino acid (lysine) of exendin-4 is substituted with serine and exendin-4 in which the twelfth amino acid (lysine) of exendin-4 is substituted with arginine. characterized by being

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, when one or both of L ₁ and L ₂ is a non-peptide linker, the non-peptide linker is polyethylene glycol, polypropylene glycol, ethylene glycol- Propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer, chitin, hyaluronic acid, fatty acid, high molecular weight polymer, low molecular weight compound, nucleotide and combinations thereof It is characterized in that it is selected from the group consisting of.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, when any one or both of L ₁ and L ₂ is a peptidic linker, the X and F1 and Y and F2 are each independently a covalent chemical bond, a non-covalent bond They are bonded to each other by L ₁ and L ₂ by a chemical bond or a combination thereof, and L ₁ and L ₂ are each independently 0 to 1000 amino acids.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, when any one or both of L ₁ and L ₂ is a peptidic linker, L ₁ and L ₂ consist of 0 amino acids, and the conjugate is (i ) a peptide bond between X and F1, (ii) a peptide bond between Y and F2; or (iii) a peptide bond between X and F1 and a peptide bond between Y and F2.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, any one of L ₁ and L ₂ is a peptidic linker, and the other is a non-peptidyl linker, wherein the peptidic linker is 0 to 1000 It is a linker comprising an amino acid, and the non-peptidyl linker is characterized in that it is polyethylene glycol.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the non-peptidyl linker is characterized in that it has a molecular weight of 1 kDa to 100 kDa.

In the brain target long-acting conjugate according to any one of the preceding embodiments, the C-terminal region of X and the N-terminal region of F1 are connected by L ₁ , and the C-terminal region of Y and N of F2 are connected by L ₂ . - Characterized in that the end regions are connected.

In the brain target long-acting conjugate according to any one of the preceding embodiments, one end of L ₁ is linked to a lysine residue or cysteine residue of X, and the other end of L ₁ is linked to the N-terminal region of F1, and one of L ₂ It is characterized in that one end is connected to a lysine residue or cysteine residue of Y, and the other end of L ₂ is connected to the N-terminal region of F2.

In the brain-targeting long-acting conjugate according to any one of the preceding embodiments, the chemical bond between XL ₁ -F1 and YL ₂ -F2 is a disulfide formed between the hinge region of the immunoglobulin constant region of F1 and the hinge region of the immunoglobulin constant region of F2. It is characterized by bonding.

Another aspect embodying the present invention is a polynucleotide encoding the brain target long-acting protein conjugate, an expression vector comprising the polynucleotide, and a transformant comprising the expression vector.

In one embodiment, the present invention includes an expression cassette comprising a polynucleotide encoding XL ₁ -F1 and an expression cassette comprising a polynucleotide encoding YL ₂ -F2, wherein X and Y are each independently physiological an active substance or brain-targeting peptide, wherein if one of X and Y is a physiologically active substance, the other is a brain-targeting peptide; L ₁ and L ₂ are each a peptidic linker, and when either or both of L ₁ and L ₂ are peptidic linkers, the peptidic linker contains 0 to 1000 amino acids, and F1 and F2 are each It relates to an expression vector expressing a brain-targeting sustained-type conjugate, characterized in that it is a material that independently includes an immunoglobulin constant region and includes an FcRn binding site.

In one embodiment, the present invention relates to a non-human transformant comprising the expression vector.

Another aspect embodying the present invention is a method for producing the brain-targeting long-acting protein conjugate.

In one embodiment, the present invention relates to a method for preparing the brain-targeting long-acting conjugate comprising the steps of:

(a) providing XL ₁ -F1 and L ₂ -Y-F2, respectively; and

(b) contacting XL ₁ -F1 and L ₂ -Y-F2 with each other to form a chemical bond therebetween;

here,

X and Y are each independently a physiologically active substance or a brain-targeting peptide, and if one of X and Y is a physiologically active substance, the other is a brain-targeting peptide;

L ₁ and L ₂ are each independently a peptidic linker or a non-peptidyl linker,

when either or both of L ₁ and L ₂ are peptidic linkers, the peptidic linker comprises 0 to 1000 amino acids;

F1 and F2 each independently include an immunoglobulin constant region, and is a substance comprising an FcRn binding site

In the preparation method according to the preceding embodiment, L ₁ and L ₂ are each independently a peptidic linker or a non-peptidyl linker.

In the preparation method according to any one of the preceding embodiments, L ₁ and L ₂ are both peptidic linkers.

In the preparation method according to any one of the preceding embodiments, each of L ₁ and L ₂ is a peptide bond, which is a peptidic linker having 0 amino acids.

In the preparation method according to any one of the preceding embodiments, L ₁ and L ₂ are both non-peptidyl linkers.

In the preparation method according to any one of the preceding embodiments, L ₁ or L ₂ is a non-peptide linker, and the non-peptide linker is an aldehyde group, a maleimide group and a succinimide derivative. It is characterized in that it forms a covalent bond with a functional group of X or Y through a functional group selected from the group consisting of.

As another embodiment, the present invention comprises the steps of (a) culturing the transformant to express a brain target long-acting conjugate; and (b) recovering the brain-targeting long-acting conjugate expressed in step (a).

Another embodiment embodying the present invention is a composition comprising the brain-targeting long-acting protein conjugate.

Another aspect embodying the present invention is the use of the brain-targeting long-acting protein conjugate in the manufacture of a medicament.

Hereinafter, the present invention will be described in detail.

On the other hand, each description and embodiment disclosed herein may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein are within the scope of the present invention. In addition, it cannot be said that the scope of the present invention is limited by the specific descriptions described below.

In addition, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Also, such equivalents are intended to be encompassed by the present invention.

Throughout this specification, conventional one-letter and three-letter codes for amino acids are used. Also, commonly accepted three-letter codes for other amino acids such as Dap (diaminopropionic acid) are used. In addition, amino acids referred to by abbreviation herein are described according to the IUPAC-IUB nomenclature.

One aspect embodying the present invention provides a brain-targeting long-acting protein conjugate.

As used herein, the term "brain-targeting long-acting protein conjugate" includes a brain-targeting peptide and a physiologically active substance, and includes an immunoglobulin constant region and a brain-targeting peptide and a physiologically active substance, respectively, directly or indirectly in each substance having an FcRn binding site. It refers to a compound having a structurally linked structure. The long-acting conjugate may be used interchangeably with “long-acting conjugate” in the present invention, and the term “persistent” refers to a bioactive substance linked to a substance including an immunoglobulin constant region and having an FcRn binding site has increased activity compared to the bioactive substance itself. indicates the duration.

The brain-targeting long-acting conjugate may pass through the blood-brain barrier and deliver a physiologically active substance into the brain tissue, but is not particularly limited thereto.

More specifically, the brain-targeting long-acting conjugate of the present invention comprises a first conjugate, wherein the brain-targeting peptide includes an immunoglobulin constant region and is linked to a substance including an FcRn binding site via a peptidic linker or a non-peptidyl linker; and a second conjugate, wherein the physiologically active substance is linked to a substance including an immunoglobulin constant region and an FcRn binding site through a peptide linker or a non-peptide linker, wherein the chemical between the first conjugate and the second conjugate The first binder and the second binder may have a structure connected to each other through bonding.

Specifically, the brain-targeting long-acting conjugate may be represented by the following structure, that is, the structure of Formula 1.

[Formula 1]

XL ₁ -F1 : YL ₂ -F2

From here,

X and Y are each independently a physiologically active substance or a brain-targeting peptide, and if one of X and Y is a physiologically active substance, the other is a brain-targeting peptide;

L ₁ and L ₂ are each independently a peptidic linker or a non-peptidyl linker,

when either or both of L ₁ and L ₂ are peptidic linkers, the peptidic linker comprises 0 to 1000 amino acids;

: is a chemical bond between XL ₁ -F1 and YL ₂ -F2;

F1 and F2 each independently include an immunoglobulin constant region, and is a substance comprising an FcRn binding site.

On the other hand, in the brain target long-acting conjugate,

(1) the C-terminal region of X and the N-terminal region of F1 are connected by L ₁ , the N-terminal region of X and the C-terminal region of F1 are connected by L ₁ , or X by L ₁ the N-terminal region of and the N-terminal region of F1 are connected, or the C-terminal region of X and the C-terminal region of F1 are connected by L ₁ ,

(2) the C-terminal region of Y and the N-terminal region of F2 are connected by L ₂ , or the N-terminal region of Y and the C-terminal region of F2 are connected by L ₂ , or Y by L ₂ The N-terminal region of and the N-terminal region of F2 may be connected, or the C-terminal region of Y and the C-terminal region of F2 may be connected by L ₂ , but the present invention is not particularly limited thereto.

More specifically, the C-terminal region of X and the N-terminal region of F1 are connected by L ₁ , and the C-terminal region of Y and the N-terminal region of F2 are connected by L ₂ , but in particular It is not limited thereto.

In addition, (3) L ₁ and L ₂ each independently has both ends or three ends, one end of L ₁ is a lysine residue or a cysteine residue of X, and the other end of L ₁ is N- of F1 It may be connected to a terminal region and/or one end of L ₂ may be connected to a lysine residue or a cysteine residue of Y, and the other end of L ₂ may be connected to an N-terminal region of F2. However, the present invention is not limited thereto.

The above-described connection direction of XL ₁ -F1 and YL ₂ -F2 passes through the blood-brain barrier in consideration of the type of brain target peptide and/or physiologically active substance, and/or delivers the physiologically active substance into the brain tissue and/or the It can be appropriately implemented so as to be in a form that can exhibit the physiological activity possessed by the material, and is not limited to the above-described examples.

In addition, in the novel brain-targeted long-acting conjugate, "a chemical bond between XL ₁ -F1 and YL ₂ -F2; or a chemical bond between the first conjugate or the second conjugate" means XL ₁ -F1 and YL ₂ -F2 to each other If it is a kind of chemical bond that connects to deliver a physiologically active substance into the brain tissue, the type is not particularly limited.

For example, the chemical bond may be a covalent bond, more specifically a disulfide bond, even more specifically a disulfide bond formed between F1 and F2, and still more specifically, the immunoglobulin constant region of F1. It may be a disulfide bond formed between the hinge region and the hinge region of the immunoglobulin constant region of F2, but is not particularly limited thereto.

As used herein, the term “N-terminal region” refers to an amino-terminal region of a peptide or protein. For example, but not limited thereto, the "N-terminal region" may include not only the most terminal amino acid residue of the N-terminal region but also all amino acid residues surrounding the N-terminal amino acid residue, specifically from the most It may include the first to twentieth amino acid residues. However, it is not particularly limited thereto.

As used herein, the term “C-terminal region” refers to a carboxyl-terminal region of a peptide or protein. For example, but not limited thereto, the "C-terminal region" may include not only the most terminal amino acid residue of the C-terminal region but also all amino acid residues surrounding the C-terminal amino acid residue, specifically from the most It may include the first to twentieth amino acid residues. However, it is not particularly limited thereto.

The elements constituting the brain target long-acting conjugate will be described in more detail as follows.

In the present invention, "brain targeting peptide" includes a peptide, protein, or antibody comprising an amino acid sequence capable of passing through the blood-brain barrier. The brain-targeting peptide corresponds to one moiety constituting the brain-targeting long-acting conjugate of the present invention.

In addition, the brain-targeting peptide is a partial sequence separated from a known peptide, protein, or antibody, and may have an activity capable of passing through the BBB and the blood-brain barrier.

As used herein, the term "amino acid" refers to an amino acid moiety comprising any naturally-occurring or non-naturally occurring or synthetic amino acid residue, ie, 1, 2, 3 or more carbon atoms, typically , means any moiety comprising at least one carboxyl moiety and at least one amino moiety directly linked by one (α) carbon atom.

The brain-targeting peptide of the present invention may be characterized in that it crosses the blood-brain barrier through a pathway by passive transport or a pathway by receptor-mediated transport, but is not limited thereto. For example, a peptide, protein or antibody that can cross the blood-brain barrier is not particularly limited in its type and size.

Meanwhile, the brain-targeting peptide may pass through the blood-brain barrier through a pathway by receptor-mediated delivery.

Specifically, the brain target peptide is an insulin receptor (insulin receptor); transferrin receptor; low density lipoprotein receptor; low density lipoprotein receptor-related protein; leptin receptor; nicotinic acetylcholine receptor; Glutathione transporter; calcium-activated potassium channel; and RAGE (receptor for advanced glycation endproducts); And through any one selected from the group consisting of a ligand of the receptors, and an antibody binding to the receptor or ligand, it may pass through the blood-brain barrier by a receptor-mediated transduction pathway, but is not particularly limited thereto.

The brain-targeting peptide is

(1) Angiopep-2: TFFYGGSRGKRNNFKTEEY- OH (SEQ ID NO: 29),

(2) ApoB (3371-3409): SVIDALQYKLEGTTRLTRKRGLKLATALSLSNKFVEGS (SEQ ID NO: 30),

(3) ApoE(159-167) ₂ : (LRKLRKRLL) ₂ (SEQ ID NO: 51),

(4) peptide-22: Ac-C(&)MPRLRGC(&)- NH2 (SEQ ID NO: 31),

(5) THR: THRPPMWSPVWP- NH2 (SEQ ID NO: 32),

(6) THR retro-enantio : pwvpswmpprht- NH2 (SEQ ID NO: 33),

(7) CRT: C (&) RTIGPSVC (&) (SEQ ID NO: 34),

(8) Leptin 30: YQQILTSMPSRNVIQISNDLENLRDLLHVL (SEQ ID NO: 35),

(9) RVG29: YTIWMPENPRPGTPCDIFTNSRGKRASNG- OH (SEQ ID NO: 36)

(10) ^D CDX: GreirtGraerwsekf-OH (SEQ ID NO: 37)

(11) Apamin: C(& ₁ )NC(& ₂ )KAPETALC(& ₁ )-ARRC(& ₂ )QQH- NH2 (SEQ ID NO: 38),

(12) MiniAp-4: [Dap] (&) KAPETALD (&) (SEQ ID NO: 39),

(13) GSH: γ-L-glutamyl-CG-OH

(14) G23: HLNILSTLWKYRC (SEQ ID NO: 40),

(15) g7: GFtGFLS(O-β-Glc) -NH2 (SEQ ID NO: 41),

(16) TGN: TGNYKALHPHNG (SEQ ID NO: 42),

(17) TAT (47-57): YGRKKRRQRRR- NH2 (SEQ ID NO: 43),

(18) SynB1: RGGRLSYSRRRFSTSTGR (SEQ ID NO: 44),

(19) Diketopiperazines: &(N-MePhe)-(N-MePhe)Diketopiperazines;

(20) PhPro: (Phenylproline) ₄ -NH2 (SEQ ID NO: 45), or

(21) HAIYPRH (SEQ ID NO: 46), but is not limited thereto.

In the above sequence & is J. Pept. Res., 2005, 65, 550-555 (J. Spengler et al.) as a symbol according to the nomenclature for a cyclic peptide described in, '&' refers to a connecting point. For example, in a single chain, the first '&' indicates the position of one end of the chemical bond, and the second '&' indicates 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. When two or more chemical bonds exist, symbols such as &1 and &2 may be used to indicate a position where the corresponding chemical bond is formed. For example, when expressed as &1Asp(&2)-Trp-Phe-Dpr(&2)-Leu-Met&1, a chemical bond is formed between Asp and Met, and a chemical bond is formed between Asp and Dpr. On the other hand, [Dap] represents diaminopropionic acid.

In the present invention, "biologically active substance" refers to a substance having any physiological action in a living body. The physiologically active substance corresponds to one moiety constituting the brain-targeting long-acting conjugate of the present invention.

Such physiologically active substances may be toxins or physiologically active polypeptides, and cytokines, interleukins, interleukin-binding proteins, enzymes, antibodies, growth factors, transcriptional regulators, blood factors used for the purpose of treating or preventing human diseases, Vaccines, structural proteins, ligand proteins or receptors, cell surface antigens, receptor antagonists, bioactive peptides secreted from the small intestine and pancreas that have therapeutic effects on diabetes and obesity, GPCR (G protein-coupled receptors) agonists or antagonists, etc. Bioactive polypeptides and analogs thereof may be exemplified, but are not limited thereto.

As used herein, the term "analog of X" is a substance capable of exhibiting the same kind of activity as X, and is an agonist of X, derivatives of X, fragments of X, and All variants and the like are included.

Specifically, the toxin may include maytansine and / or its derivatives, auristatin and / or its derivatives, duocarmycin and / or its derivatives, PBD (pyrrolobenzodiazepine) and / or its derivatives effective in killing cancer cells Although it may be selected from derivatives, toxins that are effective in killing cancer cells are included without limitation.

Specifically, the physiologically active polypeptide includes a GLP-1 receptor agonist, a glucagon receptor agonist, a Gastric inhibitory polypeptide (GIP) receptor agonist, a Fibroblast growth factor (FGF) receptor agonist (FGF1, FGF19, FGF21, FGF23, etc.), Cholecystokinin receptor agonist, gastrin receptor agonist, melanocortin receptor agonist, human growth hormone, growth hormone-releasing hormone, growth hormone-releasing peptide, interferons and interferon receptors (e.g. interferon) -alpha, -beta and -gamma, soluble type I interferon receptors, etc.), colony stimulating factors, interleukins (eg, interleukin-1, -2, -3, -4, -5, -6, -7, -8 , -9, -10, -11, -12, -13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, - 25, -26, -27, -28, -29, -30, etc.) and interleukin receptors (eg, IL-1 receptor, IL-4 receptor, etc.), enzymes (eg, beta-glucosidase ), alpha-galactosidase, beta-galactosidase, iduronidase, iduronate-2-sulfatase, galactose-6 -Sulfatase (Galactose-6-sulfatase), acid alpha-glucosidase (acid alpha-glucosidase), acid ceramidase (acid ceramidase), acid sphingomyelinsase (acid sphingomyelinsase), galactocerebrosidase ( galactocerebrosidsase), arylsulfatase A, B, beta-hexosaminidase A, B, heparin-N-sulfatase (hepari) n N-sulfatase), alpha-D-mannosidase, beta-glucuronidase, N-acetylgalactosamine-6-sulfatase (N-acetylgalactosamine- 6 sulfatase), lysosomal acid lipase, alpha-N-acetyl-glucosaminidase, glucocerebrosidase, butyrylcholinesterase ), chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, platelet-activating factor acetylhydrolase , neutral endopeptidase, myeloperoxidase, alpha-galactosidase-A, agalsidase alpha, beta, alpha-L-iduronidase (alpha-) L-iduronidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, imiglucerase, lipase, uricase, platelet-activating factor acetylhydrolase, neutral endopeptidase, and myeloperoxidase, etc.), interleukins and cytokine binding proteins (e.g. IL- 18bp, TNF-binding protein, etc.), macrophage activator, macrophage peptide, B cell factor, T cell factor, protein A, allergy suppressor, cell Necrotizing glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor suppressor, metastatic growth factor, alpha-1 antitrypsin, albumin, alpha-lactalbumin, apolipoprotein-E, erythropoietin, High glycosylated erythropoietin, angiopoietin, hemoglobin, thrombin, thrombin receptor activating peptide, thrombomodulin, blood factor VII, blood factor VIIa, blood factor VIII, blood factor IX , blood factor XIII, plasminogen activator, fibrin-binding peptide, urokinase, streptokinase, hirudin, protein C, C-reactive protein, renin inhibitor, collagenase inhibitor, superoxide dismutase, leptin , platelet-derived growth factor, epidermal growth factor, epidermal growth factor, angiostatin, angiotensin, bone growth factor, bone growth promoting protein, calcitonin, insulin, attriopeptin, cartilage inducer, elkato nin, connective tissue activator, tissue factor pathway inhibitor, follicle stimulating hormone, luteinizing hormone, luteinizing hormone releasing hormone, nerve growth factors (e.g., nerve growth factor, ciliary neurotrophic factor ( cilliary neurotrophic factor, axogenesis factor-1, brain-natriuretic peptide, glial derived neurotrophic factor, netrin, neutrophil inhibitor ( neurophil inhibitor factor), neurotrophic factor, neuturin, parathyroid hormone, relaxin, secretin, somatomedin, insulin-like growth factor, corticosteroid, glucagon, cholecystokinin, pancreatic polypeptide, gastrin-releasing peptide, corticotropin pin-releasing factor, thyroid-stimulating hormone , autotaxin, lactoferrin, myostatin, ADNP (activity-dependent neuroprotective protein), BACE1 (beta-secretase1), APP (Amyloid Precursor Protein), NCAM (Neural cell adhesion molecule), amyloid Beta (Amyloid beta), Tau (Tau), RAGE (receptor for advanced glycation endproducts), alpha-synuclein (alpha-synuclein), or their agonist or antagonist, their receptors (eg, TNFR (P75); TNFR (P55), IL-1 receptor, VEGF receptor, B cell activator receptor, etc.), receptor antagonist (eg IL1-Ra, etc.), cell surface antigen (eg CD 2, 3, 4, 5, 7, 11a, 11b, 18, 19, 20, 23, 25, 33, 38, 40, 45, 69, etc.), monoclonal antibodies, polyclonal antibodies, antibody fragments (eg scFv, Fab, Fab', F(ab) ')2 and Fd), virus-derived vaccine antigens, hybrid polypeptides or chimeric polypeptides that activate one or more receptor agonists, but are not limited thereto.

The physiologically active polypeptide applicable in the present invention may be a native type or one produced by genetic recombination in prokaryotic cells such as E. coli or eukaryotic cells such as yeast cells, insect cells or animal cells, and also have the same activity or It may be an analog having homologous activity, for example, a derivative in which one or more amino acid positions are mutated, but is not limited thereto.

On the other hand, the GLP-1 receptor agonist is a native exendin-4, an exendin-4 derivative in which the N-terminal amine group of exendin-4 is removed, and the N-terminal amine group of exendin-4 is a hydroxyl group. Exendin-4 derivatives substituted with , exendin-4 derivatives in which the N-terminal amine group of exendin-4 is modified with a dimethyl group, alpha carbon of the first amino acid (histidine) of exendin-4 is removed (deletion) exendin-4 derivatives, exendin-4 derivatives in which the twelfth amino acid (lysine) of exendin-4 is substituted with serine, and exendin-4 in which the twelfth amino acid (lysine) of exendin-4 is substituted with arginine It may be selected from the group consisting of derivatives, but is not particularly limited thereto.

In the present invention, the term "L ₁ and/or L ₂ "is a component constituting the moiety of the brain-targeting long-acting conjugate, and refers to a linker that binds a brain-targeting peptide or a physiologically active substance to F1 or F2, for example, an immunoglobulin Fc region. The linker basically means a linker that can connect two fusion partners using a covalent bond, etc. In addition to the role of connecting the fusion partners, the linker provides a gap of a certain size between the fusion partners. It may perform a role, or a role that provides flexibility or rigidity, but is not particularly limited thereto.

L ₁ and L ₂ may each independently be a peptidic linker or a non-peptidyl linker. Specifically, one of L ₁ and L ₂ may be a peptidic linker, the other may be a non-peptidyl linker, and both L ₁ and L ₂ may be a peptidic linker, and on the other hand, the L All of ₁ and L ₂ may be a non-peptidyl linker, but is not particularly limited thereto.

In one specific embodiment, in the brain-targeting long-acting conjugate, when any one or both of L ₁ and L ₂ is a peptidic linker, X and F1 and Y and F2 are each independently a covalent chemical bond, a non-covalent chemical bond A bond or a combination thereof is coupled to each other by L ₁ and L ₂ , and L ₁ and L ₂ may each independently include 0 to 1000 amino acids. When the peptidic linker is 0 amino acids, it may be bound by a peptide bond, which is a covalent chemical bond.

Specifically, in the brain-targeting long-acting conjugate, any one of L ₁ and L ₂ is a peptidic linker, and the other is a non-peptidyl linker, wherein the peptidic linker contains 0 to 1000 amino acids. a linker, and the non-peptidyl linker is polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, biodegradable polymer, lipid polymer , chitin, hyaluronic acid, fatty acids, high molecular weight polymers, low molecular weight compounds, may be a non-peptidyl linker selected from the group consisting of nucleotides and combinations thereof. However, it is not limited thereto.

For example, when any one of L ₁ and L ₂ in Formula 1 is a peptidic linker, and the other one is a non-peptidyl linker, the peptidic linker is a linker containing 0 to 1000 amino acids, and The peptidic linker may be polyethylene glycol. However, it is not limited thereto.

In addition, specifically, in the brain-targeting long-acting conjugate, both L ₁ and L ₂ are peptidic linkers, wherein L ₁ and L ₂ may each independently be a linker comprising 0 to 1000 amino acids. However, it is not limited thereto.

In addition, when any one or both of L ₁ and L ₂ is a peptidic linker, L ₁ and L ₂ consist of 0 amino acids, and the conjugate is (i) a peptide bond between X and F1, (ii) Y and peptide bond between F2; or (iii) a peptide bond between X and F1 and a peptide bond between Y and F2.

i.e. X and F1; and Y and F2 may be directly fused by a peptide bond.

In addition, specifically, in the brain target long-acting conjugate, both L ₁ and L ₂ are non-peptidyl linkers, wherein L ₁ and L ₂ are each independently polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, Polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, fatty acids, high molecular weight polymers, low molecular weight compounds, selected from the group consisting of nucleotides and combinations thereof It may be a linker that becomes However, it is not limited thereto.

In the present invention, "peptidyl linker" includes a peptide bond or a polymer of amino acids connecting two fusion partners. Specifically, the peptidic linker may include a sequence of 0 to 1000 amino acids, more specifically 0 to 900, 0 to 800, 0 to 700, 0 to 600, 0 to 500, 0 to 400, 0 to 300, 0 to 250, 0 to 200, 0 to 150, 0 to 100, 0 to 90, 0 to 80 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 25, 0 to 20, 0 to 15, Or it may include a 0 to 10 amino acid sequence, but is not particularly limited thereto.

In addition, examples of the peptidic linker include a GGGGS (SEQ ID NO: 47) motif, a GS motif, a GGGS (SEQ ID NO: 48) motif, or an amino acid sequence in which the GGSG (SEQ ID NO: 49) motif is repeated. and peptides, and the motif may be repeated 1 to 10 times, but is not particularly limited thereto.

In the present invention, the "non-peptidyl linker" refers to a biocompatible linker in which two or more repeating units are bonded, and the repeating units are linked to each other through any covalent bond rather than a peptide bond.

The non-peptide linker is polyethylene glycol, polypropylene glycol, a copolymer of ethylene glycol and propylene glycol, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, PLA (polylactic acid, polylactic acid) And it may be selected from the group consisting of biodegradable polymers such as PLGA (polylactic-glycolic acid), lipid polymers, chitin, hyaluronic acid, oligonucleotides, and combinations thereof, for example, polyethylene glycol, but , but not limited thereto. Derivatives thereof already known in the art and derivatives that can be easily prepared at the level of skill in the art are also included in the scope of the present invention.

The non-peptidyl linker that can be used in the present invention may be used without limitation as long as it is a polymer resistant to proteolytic enzymes in vivo. The molecular weight of the non-peptidyl polymer is, but is not limited to, greater than 0 in the range of about 100 kDa, specifically in the range of about 1 to about 100 kDa, and even more specifically in the range of about 1 to about 20 kDa.

In the present invention, the term “about” includes all values within a range including ±0.5, ±0.4, ±0.3, ±0.2, ±0.1, etc., and includes all values in a range equal to or similar to the value following the term about, but not limited

In addition, as the non-peptidyl linker, not only one type of polymer, but also a combination of different types of polymers may be used.

On the other hand, F1 or F2; And, before forming a conjugate, the non-peptidyl linker is F1 or F2; And it may have a functional group capable of bonding to the functional group of X or Y.

Specifically, the non-peptidyl linker may have at least two terminal functional groups, specifically, may have two or three terminal functional groups, and more specifically, may have two terminal functional groups.

The functional group may be selected from the group consisting of an aldehyde group, a maleimide group and a succinimide derivative, but is not limited thereto.

In the above, the aldehyde group may be exemplified by a propionaldehyde group or a butyl aldehyde group, but is not limited thereto.

In the above, as the succinimide derivative, succinimidyl valerate, succinimidyl methylbutanoate, succinimidyl methylpropionate, succinimidyl butanoate, succinimidyl propionate, N-hydroxysuccini Mead, hydroxy succinimidyl, succinimidyl carboxymethyl or succinimidyl carbonate may be used, but are not limited thereto.

The functional groups at both ends of the non-peptidyl linker may be the same as or different from each other.

For example, it may have a maleimide group at one end and an aldehyde group at the other end, such as a propion aldehyde group, or a butyl aldehyde group. When polyethylene glycol having a hydroxyl functional group at both ends is used as a non-peptidyl polymer, the hydroxyl group can be activated into the various reactive groups by a known chemical reaction, or by using a commercially available polyethylene glycol having a modified reactive group. The conjugates of the invention can be prepared.

In addition, the non-peptidyl linker may be a homogeneous functional non-peptidyl polymer in which both or all three ends of the non-peptidyl linker are aldehyde groups.

For example, the non-peptidyl linker may be a non-peptidyl polymer having propion aldehyde groups at both ends, specifically, PEG having propion aldehyde groups at both ends, but is not particularly limited thereto.

When the non-peptidyl linker has functional groups of reactive aldehyde groups at both ends, it is effective to minimize non-specific reactions and bind to bioactive polypeptides and immunoglobulins at both ends of the non-peptidyl polymer, respectively. The final product from reductive amination by aldehyde bonds is much more stable than those linked by amide bonds. The aldehyde functional group selectively reacts with the N-terminus at a low pH, and can form a covalent bond with a lysine residue at a high pH, for example, pH9.0.

In the present invention, "F1 and F2" refers to a substance including an immunoglobulin constant region and an FcRn binding site. Specifically, it corresponds to one moiety constituting the brain-targeting long-acting conjugate of the present invention. For example, the F1 and F2 may be an immunoglobulin Fc region containing an FcRn binding site.

The “immunoglobulin constant region” refers to the heavy and light chain variable regions of immunoglobulin, heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) except for heavy chain constant region 1 (CH1) and light chain constant region (CL1) includes The immunoglobulin constant region may be an immunoglobulin Fc region. In addition, the immunoglobulin constant region may include a hinge portion to the heavy chain constant region.

The immunoglobulin constant regions of F1 and F2 may include one to four domains selected from the group consisting of immunoglobulin CH1, CH2, CH3, and CH4 domains.

Specifically, 1) CH1 domain, CH2 domain, CH3 domain and CH4 domain, 2) CH1 domain and CH2 domain, 3) CH1 domain and CH3 domain, 4) CH2 domain and CH3 domain, 5) CH1 domain, CH2 domain, CH3 a combination of one or two or more domains of a domain and a CH4 domain with an immunoglobulin hinge region (or a part of a hinge region), 6) a heavy chain constant region, and a dimer of each domain and a light chain constant region.

Specifically, each of the immunoglobulin constant region of F1 and the immunoglobulin constant region of F2 may include a hinge region, a CH2 domain, and a CH3 domain, but is not particularly limited thereto.

More specifically, in order to effectively form a bond between XL ₁ -F1 and YL ₂ -F2, specifically a chemical bond (eg, a disulfide bond), the F1 comprises a hinge region, a CH2 domain, and a CH3 domain of IgG1, Wherein the CH3 domain is the amino acid sequence of the CH3 domain of wild-type IgG1, amino acid 26 threonine (Thr) to leucine (Leu) and / or 59 amino acid aspartic acid (Asp) to arginine (Arg) includes; wherein F2 includes a hinge region, a CH2 domain and a CH3 domain of IgG1, wherein the CH3 domain is amino acid 11 in the amino acid sequence of the CH3 domain of wild-type IgG1, leucine (Leu) is substituted with glutamic acid (Glu) and/or 67 It may include substitution of amino acid tyrosine (Tyr) with leucine (Leu) and/or substitution of lysine (Lys) with valine (Val) as amino acid 69, but is not particularly limited thereto.

The immunoglobulin Fc region having such a mutation promotes the formation of a heterodimer between XL ₁ -F1 and YL ₂ -F2, thereby producing the brain-targeting long-acting protein conjugate of the present invention.

In addition to the above-described mutations, other amino acid mutations capable of promoting heterodimer formation are included without limitation, and various combinations of these mutations are included in the scope of the present invention. In the embodiment of the present invention, as a representative example, IgG1 was produced as an Fc region, but IgG2, IgG3, IgG4, etc. are included without limitation.

The amino acid sequence of native IgG1 can be obtained through a known database such as NCBI or Unitprot. The amino acid sequence of the CH3 domain of the aforementioned wild-type IgG1 is shown in SEQ ID NO: 50, but is not particularly limited thereto.

In addition, the immunoglobulin constant region of the present invention includes a native amino acid sequence as well as a sequence derivative thereof. An amino acid sequence derivative means that one or more amino acid residues in a natural amino acid sequence have a different sequence by deletion, insertion, non-conservative or conservative substitution, or a combination thereof.

For example, in the case of IgG Fc, amino acid residues 214 to 238, 297 to 299, 318 to 322, or 327 to 331 known to be important for binding may be used as suitable sites for modification.

In addition, various types of derivatives are possible, such as a site capable of forming a disulfide bond is removed, some amino acids at the N-terminus of native Fc are removed, or a methionine residue may be added to the N-terminus of native Fc do. In addition, in order to eliminate the effector function, the complement binding site, eg, the C1q binding site, may be removed, or the ADCC (antibody dependent cell mediated cytotoxicity) site may be removed. Techniques for preparing such an immunoglobulin constant region sequence derivative are disclosed in International Patent Publication Nos. WO 97/34631 and International Patent Publication No. 96/32478.

Amino acid exchanges in proteins and peptides that do not entirely alter the activity of the molecule are known in the art (H.Neurath, R.L.Hill, The Proteins, Academic Press, New York, 1979). The most common exchanges are amino acid residues Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/ It is an exchange between Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu, Asp/Gly. In some cases, the formula (phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, etc.) may be modified).

The above-described immunoglobulin constant region derivative may exhibit biological activity equivalent to that of the immunoglobulin constant region of the present invention and increase the structural stability of the immunoglobulin constant region against heat, pH, and the like.

In addition, the immunoglobulin constant region may be obtained from a native type isolated in vivo from animals such as humans, cattle, goats, pigs, mice, rabbits, hamsters, rats, or guinea pigs, transformed animal cells or microorganisms It may be a recombinant obtained from or a derivative thereof. Here, the method of obtaining from the native type may be a method of obtaining whole immunoglobulin by isolating it from a living body of a human or animal and then treating it with a proteolytic enzyme. When treated with papain, it is cleaved into Fab and Fc, and when treated with pepsin, it is cleaved into pF'c and F(ab) ₂ . In this case, Fc or pF'c may be separated using size-exclusion chromatography or the like. In a more specific embodiment, it is a recombinant immunoglobulin Fc region obtained from a human-derived Fc region from a microorganism.

In addition, the immunoglobulin constant region may have a native sugar chain, an increased sugar chain compared to the native type, a decreased sugar chain compared to the native type, or a form in which the sugar chain is removed. Conventional methods such as chemical methods, enzymatic methods, and genetic engineering methods using microorganisms may be used for the increase or decrease or removal of such immunoglobulin Fc sugar chains. Here, the immunoglobulin Fc region from which the sugar chains are removed from the Fc has significantly reduced binding to complement (c1q) and reduced or eliminated antibody-dependent cytotoxicity or complement-dependent cytotoxicity, so that unnecessary immune responses in vivo are not induced. does not In this respect, a form more suitable for the original purpose as a drug carrier will be an immunoglobulin constant region in which sugar chains are removed or non-glycosylated.

In the present invention, "deglycosylation" refers to an Fc region from which sugars are removed with an enzyme, and aglycosylation refers to an immunoglobulin constant region that is not glycosylated produced in prokaryotes, and in a more specific embodiment, E. coli. it means.

Meanwhile, the immunoglobulin constant region may be of human or animal origin, such as cattle, goats, pigs, mice, rabbits, hamsters, rats, and guinea pigs, and in a more specific embodiment, it is of human origin.

In addition, the immunoglobulin constant region may be an immunoglobulin constant region derived from IgG, IgA, IgD, IgE, or IgM, or a combination or hybrid thereof. In a more specific embodiment, it is derived from IgG or IgM, which is most abundant in human blood, and in a more specific embodiment it is derived from IgG, which is known to enhance the half-life of ligand binding proteins. In an even more specific embodiment, the immunoglobulin Fc region is an IgG1 Fc region or an IgG4 Fc region, but is not limited thereto.

Meanwhile, in the present invention, "combination" means that when forming a dimer or a multimer, a polypeptide encoding a single-chain immunoglobulin constant region of the same origin forms a bond with a single-chain polypeptide of a different origin. That is, it is possible to prepare a dimer or multimer from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc fragment.

In the present invention, "hybrid" is a term meaning that sequences corresponding to immunoglobulin constant regions of two or more different origins exist in a single-chain immunoglobulin constant region. In the case of the present invention, various types of hybrids are possible. That is, a hybrid of domains consisting of 1 to 4 domains from the group consisting of CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc is possible, and may include a hinge.

Another aspect embodying the present invention provides a polynucleotide encoding the brain target long-acting protein conjugate, an expression vector comprising the polynucleotide, and a transformant comprising the expression vector.

The brain-targeting long-acting protein conjugate is the same as described above.

The isolated polynucleotide encoding the conjugate comprises a polynucleotide sequence having 75% or more, specifically 85% or more, more specifically 90% or more, and more specifically 95% or more sequence identity to the corresponding sequence. included in the category.

In one embodiment,

The present invention includes an expression cassette comprising a polynucleotide encoding XL ₁ -F1 and an expression cassette comprising a polynucleotide encoding YL ₂ -F2,

X and Y are each independently a physiologically active substance or a brain-targeting peptide, and if one of X and Y is a physiologically active substance, the other is a brain-targeting peptide; L ₁ and L ₂ are each a peptidic linker, and when either or both of L ₁ and L ₂ are peptidic linkers, the peptidic linker contains 0 to 1000 amino acids, and F1 and F2 are each It provides an expression vector expressing a brain-targeting sustained-type conjugate, characterized in that it is a material that independently includes an immunoglobulin constant region and includes an FcRn binding site.

The expression cassette may include elements necessary for expression of a desired protein, specifically XL ₁ -F1 or YL ₂ -F2, such as a promoter.

The recombinant vector according to the present invention may be typically constructed as a vector for cloning or as a vector for expression, and may be constructed using a prokaryotic or eukaryotic cell as a host cell.

As used herein, the term "vector" refers to a recombinant vector capable of expressing a target protein in a suitable host cell, and refers to a nucleic acid construct including essential regulatory elements operably linked to express a nucleic acid insert.

By transforming or transfecting the recombinant vector into a host cell, the brain-targeting sustained-type conjugate of the present invention can be obtained.

In the present invention, the polynucleotide encoding XL ₁ -F1 and the polynucleotide encoding YL ₂ -F2 may be operably linked to a promoter.

The method for transforming the recombinant vector including the polynucleotide according to the present invention is not limited to the above examples, and transformation or transfection methods commonly used in the art may be used without limitation.

The transformant of the present invention can be obtained by introducing a recombinant vector containing the polynucleotide according to the present invention into a host cell.

A host suitable for the present invention is not particularly limited as long as it allows expression of the polynucleotide according to the present invention. Specific examples of the host that can be used in the present invention include Escherichia bacteria such as E. coli ; Bacillus subtilis ( Bacillus subtilis ), such as Bacillus ( Bacillus ) genus bacteria; Bacteria of the genus Pseudomonas , such as Pseudomonas putida ; Yeasts such as Pichia pastoris , Saccharomyces cerevisiae , Schizosaccharomyces pombe ; insect cells such as Spodoptera frugiperda (SF9); and animal cells such as CHO, COS, and BSC.

Another aspect embodying the present invention provides a method for producing the brain target long-acting protein conjugate.

The brain-targeting long-acting protein conjugate is the same as described above.

Specifically, the method of the present invention may include the following steps:

(a) providing XL ₁ -F1 and L ₂ -Y-F2, respectively; and

(b) contacting XL ₁ -F1 and L ₂ -Y-F2 with each other to form a chemical bond therebetween;

here,

X and Y are each independently a physiologically active substance or a brain-targeting peptide, and if one of X and Y is a physiologically active substance, the other is a brain-targeting peptide;

L ₁ and L ₂ are each independently a peptidic linker or a non-peptidyl linker,

when either or both of L ₁ and L ₂ are peptidic linkers, the peptidic linker comprises 0 to 1000 amino acids;

F1 and F2 each independently include an immunoglobulin constant region, and is a substance comprising an FcRn binding site

In addition, the manufacturing method comprises the steps of (a) culturing the transformant to express a brain-targeting long-acting conjugate; and (b) recovering the brain target long-acting conjugate expressed in step (a), but is not limited thereto.

Specifically, when each of XL ₁ -F1 and L ₂ -Y-F2 is a fusion protein, the brain-targeting long-acting protein conjugate of the present invention can be prepared through the same method as described above.

When F1 and F2 are immunoglobulin constant regions including a hinge region, chemical bonds, specifically disulfide bonds, between the Fc regions during folding after expression of XL ₁ -F1 and L ₂ -Y-F2 produced through an expression host, respectively By forming a brain target long-acting protein conjugate according to the present invention can be prepared.

Another aspect embodying the present invention provides a composition comprising the brain target long-acting protein conjugate.

The brain-targeting long-acting protein conjugate is the same as described above.

Another aspect embodying the present invention provides the use of the brain-targeting long-acting protein conjugate in the manufacture of a medicament.

The brain-targeting long-acting protein conjugate is the same as described above.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Preparation of a novel brain-targeting long-acting protein conjugate in which the immunoglobulin Fc region is mutated through an expression host cell

The immunoglobulin Fc region was fused to each of iduronate-2-sulfatase (IDS) and brain-targeting peptide 5 (BTP5) at the gene level and inserted into expression vectors, respectively.

Specifically, a conjugate containing each of IDS and BTP5 as shown below was synthesized (Table 1). In the immunoglobulin Fc region binding to IDS, specifically, immunoglobulin in which threonine (Thr), amino acid 26 of the CH3 region, is substituted with leucine (Leu), and amino acid aspartic acid (Asp), amino acid number 59, is substituted with arginine (Arg). The Fc region was used, and specifically, as an immunoglobulin Fc region binding to BTP5, leucine (Leu), amino acid 11 of the CH3 region, was used as glutamic acid (Glu), and amino acid tyrosine (Tyr), amino acid number 67, was changed to leucine (Leu). , was synthesized using an immunoglobulin region in which lysine (Lys), amino acid 69, was substituted with valine (Val).

Example 2: Preparation of brain-targeted IDS long-acting conjugate

Polynucleotide sequences encoding the IDS-IgG1 Fc conjugate and the BTP5-IgG1 Fc conjugate synthesized in Example 1 were respectively inserted into an expression vector, X0GC, using a restriction enzyme.

In order to insert both sequences encoding the two conjugates into one vector, first, in an expression vector containing a polynucleotide sequence encoding the BTP5-IgG1 Fc conjugate, from the promoter to the poly A part was amplified by PCR. At this time, XhoI was inserted at the end of the forward primer and XbaI restriction enzyme was inserted at the end of the reverse primer. The XhoI and XbaI are restriction enzymes that do not cut both the polynucleotide sequence encoding the IDS-IgG1 Fc conjugate and the polynucleotide sequence encoding the BTP5-IgG1 Fc conjugate. The expression vector into which the polynucleotide encoding the IDS-IgG1 Fc conjugate is inserted and the promoter-BTP5-IgG1 Fc-Poly A PCR product are treated with XhoI and XbaI, respectively, and cleaved, followed by ligation to perform ligation to the IDS-IgG1 Fc conjugate. and one expression vector expressing each of the BTP5-IgG1 Fc conjugates was completed.

When the bicistronic expression vector is expressed in CHO cells, which are host cells, two mRNAs and two proteins for the IDS-IgG1 Fc conjugate and the BTP5-IgG1 Fc conjugate are expressed in one vector. At this time, the expressed IDS-IgG1 Fc conjugate and BTP5-IgG1 Fc conjugate form disulfide bonds with each other, and the mutated portion of IgG1 CH3 serves to promote binding between the conjugates, so that the IDS-IgG1 Fc conjugate and the BTP5-IgG1 Fc conjugate Brain-targeted IDS long-acting protein conjugates that form heterodimers with each other can be prepared. In addition, as described above, it is possible to increase the production yield of a novel brain target persistent protein by promoting the formation of heterodimers through Fc mutation.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the following claims and their equivalents.

Claims (34)

A brain-targeting long-acting conjugate represented by Formula 1 below: [Formula 1] XL ₁ -F1 : YL ₂ -F2 From here, X and Y are each independently a physiologically active substance or a brain-targeting peptide, and if one of X and Y is a physiologically active substance, the other is a brain-targeting peptide; L ₁ and L ₂ are each independently a peptidic linker or a non-peptidyl linker, when either or both of L ₁ and L ₂ are peptidic linkers, the peptidic linker comprises 0 to 1000 amino acids; : is a chemical bond between XL ₁ -F1 and YL ₂ -F2; F1 and F2 each independently include an immunoglobulin constant region, and is a substance comprising an FcRn binding site. According to claim 1, wherein the immunoglobulin constant region of F1 and F2 comprises 1 to 4 domains selected from the group consisting of immunoglobulin CH1, CH2, CH3, and CH4 domains, brain target long-acting conjugate. The brain-targeted long-acting conjugate of claim 2 , wherein the immunoglobulin constant region comprises a hinge region. The brain-targeting long-acting conjugate of claim 1, wherein each of the immunoglobulin constant region of F1 and the immunoglobulin constant region of F2 comprises a hinge region, a CH2 domain and a CH3 domain. The brain-targeting long-acting conjugate of claim 1, wherein the immunoglobulin constant region is an immunoglobulin Fc region. The brain-targeting long-acting conjugate of claim 1, wherein the immunoglobulin constant region is derived from IgG. The brain-targeting long-acting conjugate of claim 6, wherein the IgG is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4. The brain-targeting long-acting conjugate according to claim 6, wherein the immunoglobulin constant region is derived from IgG1 or IgG4. The brain-targeting long-acting conjugate according to claim 6, wherein the immunoglobulin constant region is non-glycosylated and has an amino acid sequence derived from a human IgG1 or IgG4 Fc region. According to claim 1, wherein F1 includes a hinge region, a CH2 domain, and a CH3 domain of IgG1, wherein the CH3 domain is amino acid 26 in the amino acid sequence of the CH3 domain of wild-type IgG1, threonine (Thr) is substituted with leucine (Leu), and substitution of the amino acid aspartic acid (Asp) with arginine (Arg); The F2 includes a hinge region, a CH2 domain and a CH3 domain of IgG1, wherein the CH3 domain is amino acid 11 in the amino acid sequence of the CH3 domain of wild-type IgG1, leucine (Leu) is substituted with glutamic acid (Glu), amino acid 67 A brain-targeting long-acting conjugate comprising a substitution of tyrosine (Tyr) with leucine (Leu) and a substitution of valine (Val) for lysine (Lys) at amino acid 69. The brain-targeting long-acting conjugate according to claim 1, wherein the long-acting conjugate passes the blood-brain barrier and delivers a physiologically active substance into brain tissue. The brain-targeting long-acting conjugate according to claim 1, wherein the brain-targeting peptide is in the form of a peptide, protein, or antibody comprising an amino acid sequence capable of crossing the blood-brain barrier. The brain-targeting long-acting conjugate of claim 12 , wherein the brain-targeting peptide crosses the blood-brain barrier via a pathway by passive transport or a pathway by receptor-mediated transport. 14. The method of claim 13, Insulin receptor, transferrin receptor, low density lipoprotein receptor, low density lipoprotein receptor-related protein, leptin receptor, nicotinic nicotinic acetylcholine receptor, glutathione transporter, calcium-activated potassium channel, and receptor for advanced glycation endproducts (RAGE), and ligands of the receptors and the receptor or ligand A brain-targeting long-acting conjugate that crosses the blood-brain barrier by a receptor-mediated transduction pathway through any one selected from the group consisting of a binding antibody. According to claim 1, The physiologically active substance is a toxin; or a GLP-1 (Glucagon like peptide-1) receptor agonist; glucagon receptor agonists; Gastric inhibitory polypeptide (GIP) receptor agonists; Fibroblast growth factor (FGF) receptor agonists; Cholecystokinin receptor agonist; gastrin receptor agonists; melanocortin receptor agonists; human growth hormone; growth hormone releasing hormone; growth hormone releasing peptide; interferon; interferon receptor; colony stimulating factor; interleukin; interleukin receptor; enzyme; interleukin binding protein; cytokine binding protein; macrophage activator; macrophage peptides; B cell factor; T cell factor; protein A; allergy suppressors; cell necrosis glycoprotein; immunotoxins; lymphotoxin; tumor necrosis factor; tumor suppressors; metastatic growth factor; alpha-1 antitrypsin; albumin; alpha-lactalbumin; apolipoprotein-E; erythropoietin; high glycosylated erythropoietin; angiopoietin; hemoglobin; thrombin; thrombin receptor activating peptide; thrombomodulin; blood factor VII; blood factor VIIa; blood factor VIII; blood factor IX; blood factor XIII; plasminogen activator; fibrin-binding peptide; urokinase; streptokinase; hirudin; protein C; C-reactive protein; renin inhibitors; collagenase inhibitors; superoxide dismutase; leptin; platelet-derived growth factor; epidermal growth factor; epidermal growth factor; angiostatin; angiotensin; osteogenic growth factor; bone-promoting protein; calcitonin; insulin; atriopeptin; cartilage inducer; elcatonin; connective tissue activator; tissue factor pathway inhibitors; follicle stimulating hormone; luteinizing hormone; luteinizing hormone releasing hormone; nerve growth factors; axogenesis factor-1; brain-natriuretic peptide; glial derived neurotrophic factor; netrin; neutrophil inhibitor factor; neurotrophic factor; nutrin; parathyroid hormone; relaxin; secretin; somatomedin; insulin-like growth factor; adrenocortical hormone; glucagon; cholecystokinin; pancreatic polypeptide; gastrin releasing peptide; corticotropin releasing factor; thyroid stimulating hormone; autotaxin; lactoferrin; myostatin; ADNP (activity-dependent neuroprotective protein), BACE1 (beta-secretase1), APP (Amyloid Precursor Protein), NCAM (Neural cell adhesion molecule), Amyloid beta, Tau, RAGE (receptor for advanced glycation endproducts) ), alpha-synuclein, or an agonist or antagonist thereof; receptor class, receptor agonist; cell surface antigen; monoclonal antibody; polyclonal antibodies; antibody fragments; virus-derived vaccine antigens; hybrid polypeptides or chimeric polypeptides that activate one or more receptor agonists; and a physiologically active substance selected from the group consisting of analogs thereof, a brain-targeting long-acting conjugate. 16. The method of claim 15, The toxin is selected from the group consisting of maytansine or a derivative thereof, auristatin or a derivative thereof, duocarmycin or a derivative thereof, and Pyrrolobenzodiazepine (PBD) or a derivative thereof, GLP-1 ( Glucagon like peptide-1) receptor agonist is selected from the group consisting of native exendin-3 or native exendin-4, and analogs thereof, and the FGF receptor agonist is FGF1 or an analog thereof, FGF19 or analog thereof; selected from the group consisting of FGF21 or an analog thereof, and FGF23 or an analog thereof, the interferon is selected from the group consisting of interferon-alpha, interferon-beta and interferon-gamma; the interferon receptor is selected from the group consisting of an interferon-alpha receptor, an interferon-beta receptor, an interferon-gamma receptor, and a soluble type I interferon receptor, Interleukin is interleukin-1, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, Interleukin-13, Interleukin-14, Interleukin-15, Interleukin-16, Interleukin-17, Interleukin-18, Interleukin-19, Interleukin-20, Interleukin-21, Interleukin-22, Interleukin-23, Interleukin-24, Interleukin- 25, interleukin-26, interleukin-27, interleukin-28, interleukin-29, and interleukin-30; the interleukin receptor is an interleukin-1 receptor or an interleukin-4 receptor, Enzymes are beta-glucosidase, alpha-galactosidase, beta-galactosidase, iduronidase, iduronate 2-sulfatase ( iduronate-2-sulfatase, galactose-6-sulfatase, acid alpha-glucosidase, acid ceramidase, acid sphingomyelinase sphingomyelinsase), galactocerebrosidsase, arylsulfatase A, arylsulfatase B, beta-hexosaminidase A, beta-hexosaminidase B, heparin-N -sulfatase (heparin N-sulfatase), alpha-D-mannosidase (alpha-D-mannosidase), beta-glucuronidase (beta-glucuronidase), N-acetylgalactosamine-6-sulfatase ( N-acetylgalactosamine-6 sulfatase, lysosomal acid lipase, alpha-N-acetyl-glucosaminidase, glucocerebrosidase, butyrylcholine Butyrylcholinesterase, Chitinase, glutamate decarboxylase, imiglucerase, lipase, Uricase, platelet activator acetylhydrolase (Platelet-) Activating Factor Acetylhydrolase, neutral endopeptidase, myeloperoxidase, alpha-galactosidase-A, agalsidase alpha, agalsidase beta, alpha-L-iduronidase, butyrylcholinesterase, chitinase, glutamate decarboxylase, and imiglucerase is selected from the group consisting of the interleukin binding protein is IL-18bp, The cytokine binding protein is a TNF (Tumor necrosis factor) binding protein, Nerve growth factors include nerve growth factor, cilliary neurotrophic factor, axogenesis factor-1, brain-natriuretic peptide, glial derived neurotrophic factor factor), netrin (netrin), neutrophil inhibitor factor (neurophil inhibitor factor), neurotrophic factor, and selected from the group consisting of nutrin (neuturin), Myostatin receptor is selected from the group consisting of TNFR (P75), TNFR (P55), IL-1 receptor, VEGF receptor, and B cell activator receptor, The myostatin receptor antagonist is IL1-Ra, the cell surface antigen is selected from the group consisting of CD2, CD3, CD4, CD5, CD7, CD11a, CD11b, CD18, CD19, CD20, CD23, CD25, CD33, CD38, CD40, CD45 and CD69, The antibody fragment is selected from the group consisting of scFv, Fab, Fab', F(ab') ₂ and Fd, a brain-targeting long-acting conjugate. 16. The method of claim 15, The GLP-1 receptor agonist includes native exendin-4, exendin-4 from which the N-terminal amine group of native exendin-4 has been removed, and the N-terminal amine group of native exendin-4 with hydroxyl. exendin-4 substituted with a group, exendin-4 in which the N-terminal amine group of native exendin-4 is modified with a dimethyl group, exendin- in which the N-terminal amine group of natural exendin-4 is substituted with a carboxyl group 4, exendin-4 from which the alpha carbon of the first amino acid (histidine) of native exendin-4 has been removed; and exendin-4 in which the twelfth amino acid (lysine) of exendin-4 is substituted with serine and exendin-4 in which the twelfth amino acid (lysine) of exendin-4 is substituted with arginine. A brain-targeted long-acting conjugate. According to claim 1, When any one or both of L ₁ and L ₂ is a non-peptide linker, the non-peptide linker is polyethylene glycol, polypropylene glycol, ethylene glycol-propylene glycol copolymer, polyoxyethylated polyol, polyvinyl alcohol, Polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitin, hyaluronic acid, fatty acids, high molecular weight polymers, low molecular weight compounds, selected from the group consisting of nucleotides and combinations thereof, brain target long-acting conjugate. According to claim 1, When any one or both of L ₁ and L ₂ is a peptidic linker, X and F1 and Y and F2 are each independently a covalent chemical bond, a non-covalent chemical bond, or a combination thereof by L ₁ and L ₂ bound, and L ₁ and L ₂ are each independently 0 to 1000 amino acids. According to claim 1, when either or both of L ₁ and L ₂ are peptidic linkers, L ₁ and L ₂ consist of 0 amino acids, The combination is (i) a peptide bond between X and F1, (ii) a peptide bond between Y and F2; or (iii) a peptide bond between X and F1 and a peptide bond between Y and F2; Brain-targeted long-acting conjugate. According to claim 1, Any one of L ₁ and L ₂ is a peptidic linker, and the other is a non-peptidyl linker, In this case, the peptidic linker is a linker containing 0 to 1000 amino acids, The non-peptidyl linker is polyethylene glycol, the brain-targeting long-acting conjugate. According to claim 1, wherein the non-peptidyl linker has a molecular weight of 1 kDa to 100 kDa, brain-targeting long-acting conjugate. According to claim 1, A brain-targeting long-acting conjugate, wherein the C-terminal region of X and the N-terminal region of F1 are connected by L ₁ , and the C-terminal region of Y and the N-terminal region of F2 are connected by L ₂ . According to claim 1, One end of L ₁ is linked to a lysine residue or cysteine residue of X, and the other end of L ₁ is linked to the N-terminal region of F1, One end of L ₂ is connected to a lysine residue or cysteine residue of Y, and the other end of L ₂ is connected to the N-terminal region of F2, Brain-targeted long-acting conjugate. 4. The method of claim 3, The chemical bond between XL ₁ -F1 and YL ₂ -F2 is a disulfide bond formed between the hinge region of the immunoglobulin constant region of F1 and the hinge region of the immunoglobulin constant region of F2, Brain-targeted long-acting conjugate. an expression cassette comprising a polynucleotide encoding XL ₁ -F1 and an expression cassette comprising a polynucleotide encoding YL ₂ -F2, X and Y are each independently a physiologically active substance or a brain-targeting peptide, and if one of X and Y is a physiologically active substance, the other is a brain-targeting peptide; L ₁ and L ₂ are each a peptidic linker, When any one or both of L ₁ and L ₂ is a peptidic linker, the peptidic linker comprises 0 to 1000 amino acids, F1 and F2 are each independently comprising an immunoglobulin constant region, a substance comprising an FcRn binding site, An expression vector expressing the brain-targeting long-acting conjugate of claim 1. A transformant other than a human comprising the expression vector of claim 26 . A method for preparing the brain-targeting long-acting conjugate of any one of claims 1 to 25, comprising the steps of: (a) providing XL ₁ -F1 and L ₂ -Y-F2, respectively; and (b) contacting XL ₁ -F1 and L ₂ -Y-F2 with each other to form a chemical bond therebetween; here, X and Y are each independently a physiologically active substance or a brain-targeting peptide, and if one of X and Y is a physiologically active substance, the other is a brain-targeting peptide; L ₁ and L ₂ are each independently a peptidic linker or a non-peptidyl linker, when either or both of L ₁ and L ₂ are peptidic linkers, the peptidic linker comprises 0 to 1000 amino acids; F1 and F2 each independently include an immunoglobulin constant region, and is a substance comprising an FcRn binding site. 29. The method of claim 28, The L ₁ and L ₂ are each independently a peptide linker or a non-peptidyl linker. 29. The method of claim 28, The L ₁ and L ₂ are all peptidic linkers, the production method. The method of claim 28, wherein each of L ₁ and L ₂ is a peptide bond that is a peptidic linker having 0 amino acids. The method of claim 28, wherein L ₁ and L ₂ are both non-peptidyl linkers. 29. The method of claim 28, wherein L ₁ or L ₂ is a non-peptide linker, wherein the non-peptide linker is a functional group selected from the group consisting of an aldehyde group, a maleimide group, and a succinimide derivative. A method for forming a covalent bond with a functional group of X or Y through (a) culturing the transformant of claim 27 to express the brain target long-acting conjugate; and (b) a method for preparing the brain-targeting long-acting conjugate of claim 1, comprising the step of recovering the brain-targeting long-acting conjugate expressed in step (a).