Peptide modified with an uncharged hydrophilic polymer modified with a GLUT1 ligand so as to be cleavable under a reducing or low pH environment, and antibody containing said peptide

Abstract

The present invention relates to a peptide which is modified with a non-charged hydrophilic polymer segment modified with a GLUT1 ligand, in which the peptide is modified in a manner such that the peptide can be cleaved from the segment under a reductive environment or a low-pH environment. The present invention also provides an antibody comprising the peptide moiety.

Description

[Technical Field]

[0001] The present invention relates to a peptide modified with an uncharged hydrophilic polymer modified with a GLUT1 ligand so as to be cleavable in a reducing or low pH environment. The present invention also relates to an antibody comprising the peptide. [Background technology]

[0002] The development of drug delivery systems aims to efficiently and selectively deliver drugs to target tissues or organs, and drug delivery systems can also play a role in increasing the retention of drugs in the blood.

[0003] As a drug delivery system for the brain, Patent Document 1 discloses micelles whose surfaces are modified with glucose. Patent Document 1 reveals that administration of an antibody conjugated with a GLUT1 ligand while controlling blood glucose levels significantly increases the accumulation of the conjugated antibody in the brain. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] WO2015 / 075942 Brief description of the invention

[0005] The present invention provides a peptide modified with an uncharged hydrophilic polymer modified with a GLUT1 ligand so as to be cleavable in a reducing environment or a low pH environment, and an antibody comprising the peptide.

[0006] The present inventors have invented peptides and antibodies modified with an uncharged hydrophilic polymer modified with a GLUT1 ligand so as to be cleavable under a reducing or low pH environment. The present inventors have found that peptides modified with an uncharged hydrophilic polymer modified with a GLUT1 ligand, for example, via a linker, where the modification is covalently linked to the side chain amino group of a lysine residue of the peptide, and the linker can be cleaved from the modified moiety under a reducing environment such as the pH in the endosomes of vascular endothelial cells, can improve their physiological activity in the brain (particularly, in the case of antibodies, their binding properties, which have been reduced by the modification). The examples clearly show that the same applies when the peptide is an antibody and when an antibody contains the peptide.

[0007] According to the present invention, for example, the following industrially applicable inventions are provided. [1] A peptide modified with an uncharged hydrophilic polymer segment (e.g., polyethylene glycol or polyoxazoline) modified with a GLUT1 ligand so that the segment can be cleaved under a reducing environment or an environment of pH 6.5 or less. [2] The peptide according to [1] above, wherein the uncharged hydrophilic polymer segment modified with a GLUT1 ligand and the peptide are linked via a linker that is cleavable in a reducing environment or a low pH environment of pH 6.5 or less, thereby modifying the segment so that it can be cleaved in a reducing environment or a low pH environment of pH 6.5 or less. [3] The peptide according to [2] above, wherein the linker has a disulfide bond therein and is modified so as to be cleavable from the segment under a reducing environment. [4] A peptide according to any one of [1] to [3] above, wherein the linker is -CO-O-L1-SS-L2- {wherein L1 is a substituted or unsubstituted alkylene or a substituted or unsubstituted heteroalkylene, and L2 is a linker stable in the bloodstream}, which is linked to the side chain amino group of a lysine residue of the peptide via a carbamate bond (hereinafter, this may be referred to as an "amide bond," including an amide bond), and L2 is linked to an uncharged hydrophilic polymer segment. [5] The peptide according to any one of the above [1] to [4], wherein the GLUT1 ligand is glucose. [6] An antibody comprising the peptide moiety described in any one of [1] to [5] above. [7] The antibody according to [6] above, which binds to a cell surface antigen or an extracellular antigen in the brain parenchyma. [8] Extracellular antigens are Aβ 1-40 The antibody according to [7] above, [9] The antibody according to any one of [1] to [7] above, further linked to a cytotoxic agent.

[10] The antibody according to any one of [1] to [8] above, further linked to an imaging agent selected from the group consisting of a fluorescent dye, a radioisotope, and a contrast agent.

[11] A pharmaceutical composition comprising the peptide or antibody according to any one of [1] to [9] above.

[12] A composition for use in brain imaging, comprising the antibody according to

[10] above.

[13] The composition according to

[11] or

[12] above, which is administered according to the following dosage regimen: lowering the blood glucose of a subject, and then Inducing an increase in blood glucose level in the subject so that more antibodies are transferred to the brain parenchyma than when the increase in blood glucose level is not induced, and administering the composition according to

[11] or

[12] above. composition.

[14] A peptide modified with an uncharged hydrophilic polymer segment (e.g., polyethylene glycol or polyoxazoline) so that the segment can be cleaved under a reducing environment or an environment of pH 6.5 or less.

[15] The peptide according to

[14] above, wherein the uncharged hydrophilic polymer segment and the peptide are linked via a linker that is cleavable under a reducing environment or an environment of pH 6.5 or less, thereby modifying the segment so that it can be cleaved under a reducing environment or an environment of pH 6.5 or less.

[16] The peptide according to

[15] above, wherein the linker, -CO-O-L1-SS-L2- {wherein L1 is a substituted or unsubstituted alkylene or a substituted or unsubstituted heteroalkylene, and L2 is a linker stable in the bloodstream}, is linked to the side chain amino group of a lysine residue of the peptide via an amide bond, and L2 is linked to an uncharged hydrophilic polymer segment.

[0008]

[17] An antibody (modified antibody) modified with an uncharged hydrophilic polymer segment (e.g., polyethylene glycol or polyoxazoline) modified with a GLUT1 ligand so that the segment can be cleaved under a reducing environment or an environment with a pH of 6.5 or less, and which has an antigen-binding affinity of 10% or less compared to an unmodified antibody.

[18] An antibody (modified antibody) modified with an uncharged hydrophilic polymer segment (e.g., polyethylene glycol or polyoxazoline) modified with a GLUT1 ligand so that the segment can be cleaved under a reducing environment or an environment with a pH of 6.5 or less, and which has an antigen-binding affinity of 5% or less compared to an unmodified antibody.

[19] An antibody (modified antibody) modified with an uncharged hydrophilic polymer segment (e.g., polyethylene glycol or polyoxazoline) modified with a GLUT1 ligand so that the segment can be cleaved under a reducing environment or an environment of pH 6.5 or below, and which has 1% or less of or no antigen-binding ability compared to an unmodified antibody.

[20] The antibody according to

[17] above, wherein the antibody binds to a cell surface antigen or an extracellular antigen in the brain parenchyma.

[21] The antibody according to

[18] above, wherein the antibody binds to a cell surface antigen or an extracellular antigen in the brain parenchyma.

[22] The antibody according to

[19] above, wherein the antibody binds to a cell surface antigen or an extracellular antigen in the brain parenchyma.

[23] The antibody according to

[17] above, which is an antibody that binds to Aβ.

[24] The antibody according to

[18] above, which is an antibody that binds to Aβ.

[25] The antibody according to

[19] above, which is an antibody that binds to Aβ.

[26] A composition comprising the antibody according to any one of

[17] to

[25] above.

[27] A pharmaceutical composition comprising the antibody according to any one of

[17] to

[25] above.

[0009] [1A] An antibody modified with polyethylene glycol modified with a GLUT1 ligand so as to be cleavable from the polyethylene glycol under a reducing environment or a low pH environment of pH 6.5 or lower. [2A] An antibody described in [1A] above, in which the uncharged hydrophilic polymer segment modified with a GLUT1 ligand and the peptide are linked via a linker that is cleavable in a reducing environment or an environment with a pH of 6.5 or less, thereby modifying the antibody so that it can be cleaved in a reducing environment or an environment with a pH of 6.5 or less. [2A] The antibody described in [1] above, in which the uncharged hydrophilic polymer segment modified with a GLUT1 ligand and the peptide are linked via a linker that is cleavable in a reducing environment or an environment with a pH of 6.5 or less, thereby modifying the segment so that it can be cleaved in a reducing environment or an environment with a pH of 6.5 or less. [4A] The antibody according to any one of [1A] to [3A] above, wherein the linker has the structure -CO-O-L1-SS-L2- {wherein L1 is a substituted or unsubstituted alkylene or a substituted or unsubstituted heteroalkylene, and L2 is a bond or a linker stable in the bloodstream}, and is linked to the side chain amino group of a lysine residue of the antibody via an amide bond, and L2 is linked to the uncharged hydrophilic polymer segment. [5A] The antibody according to any one of [1A] to [4A] above, wherein the GLUT1 ligand is glucose. [6A] The antibody according to any one of [1A] to [5A] above, which binds to a cell surface antigen or an extracellular antigen in the brain parenchyma. [7A] Extracellular antigen is Aβ 1-40 The antibody according to [6A] above, [8A] The antibody according to any one of [1A] to [6A] above, further linked to a cytotoxic agent. [9A] The antibody according to any one of [1A] to [7A] above, further linked to an imaging agent selected from the group consisting of a fluorescent dye, a radioisotope, and a contrast agent. [10A] A pharmaceutical composition comprising the antibody according to any one of [1A] to [9A] above. [11A] A composition for use in brain imaging, comprising the antibody according to [9A] above. [12A] The composition according to [10A] or [11A] above, which is administered according to the following dosage regimen: lowering the blood glucose of a subject, and then Inducing an increase in blood glucose level in the subject so that more antibodies are transferred to the brain parenchyma than when the increase in blood glucose level is not induced, and administering the composition described in [10A] or [11A] above to the subject. composition. [13A] An antibody modified with polyethylene glycol so that it can be cleaved from the polyethylene glycol under a reducing environment or an environment of pH 6.5 or lower.

[0010] [1B] An antibody modified with an uncharged hydrophilic polymer segment, wherein the antibody is capable of binding to an antigen, and in the blood, the antibody is in the form of the antibody modified with the uncharged hydrophilic polymer segment, and in the brain parenchyma, the antibody is in a form dissociated from the uncharged hydrophilic polymer segment. [2B] The antibody according to [1B] above, wherein dissociation from the uncharged hydrophilic polymer segment is induced in an environment of pH 6.5 or less or in the reducing environment of brain parenchyma. [3B] The antibody according to [1B] or [2B] above, wherein an antibody modified with an uncharged hydrophilic polymer segment has a weaker binding affinity to an antigen than an unmodified antibody, and an antibody dissociated from the uncharged hydrophilic polymer segment has a stronger binding affinity to an antigen than an antibody modified with an uncharged hydrophilic polymer segment. [4B] The antibody according to any one of [1B] to [3B] above, wherein the uncharged hydrophilic polymer segment and the antibody are linked via a disulfide bond, and the disulfide bond is cleaved under the reducing environment in the brain, thereby dissociating the uncharged hydrophilic polymer segment and the antibody. [5B] The antibody according to any one of [1B] to [4B] above, wherein the uncharged hydrophilic polymer segment is polyethylene glycol. [6B] The antibody according to any one of [1B] to [5B] above, wherein the uncharged hydrophilic polymer segment is polyethylene glycol modified with glucose. [7B] An antibody described in any of [1B] to [6B] above, in which the uncharged hydrophilic polymer segment modified with a GLUT1 ligand and the peptide are linked via a linker that is cleavable in a reducing environment or an environment of pH 6.5 or less, thereby modifying the antibody so that it can be cleaved in a reducing environment or an environment of pH 6.5 or less. [8B] An antibody described in [7B] above, in which the linker has the structure -CO-O-L1-SS-L2- {wherein L1 is a substituted or unsubstituted alkylene or a substituted or unsubstituted heteroalkylene, and L2 is a bond or a linker stable in the bloodstream}, and is linked to the side chain amino group of a lysine residue of the antibody via an amide bond, and L2 is linked to an uncharged hydrophilic polymer segment. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows an example of an antibody to which a glucose-modified PEG is bound via a linker that is reduction-sensitive under reducing conditions, and a scheme showing how the glucose-modified PEG dissociates under reducing conditions in this example. [Figure 2]Panel (a) of Figure 2 shows the percentage of amino groups in PEG-modified anti-BACE1 antibodies as a function of reaction time and various PEG ratios (the PEG:amino group ratio was 5:1). Panel (b) of Figure 2 shows the percentage of PEG-modified amino groups in antibodies obtained after 6 hours of reaction at various PEG:amino group ratios. [Figure 3] Figure 3 shows the results of characterization of PEGylated anti-BACE1. Panel (a) shows the efflux spectra of native anti-BACE1, PEGylated anti-BACE1, and anti-BACE1 measured by gel permeation chromatography (GPC) after incubating these antibodies with 2 mM GSH for 4 hours at 37 °C. Panel (b) shows the hydrodynamic diameters of native anti-BACE1 and PEGylated anti-BACE1 measured by dynamic light scattering (DLS). Panel (c) shows the results of measuring Aβ1-40 aggregation by thioflavin T assay after incubation (6 hours at room temperature) of various ratios of Aβ1-40 with native anti-Aβ1-40, PEGylated anti-Aβ1-40, or anti-Aβ1-40 induced by PEGylated anti-Aβ1-40 release. Panel (d) shows Aβ1-40 aggregation measured after preincubation of solutions of native anti-Aβ1-40 and various PEGylated anti-Aβ1-40 antibodies (PEG12.5-anti-Aβ1-40, PEG25.5-anti-Aβ1-40, PEG52.1-anti-Aβ1-40, and PEG73.2-anti-Aβ1-40) for 4 hours at 37°C. In the assay in panel (d), the ratio of anti-Aβ1-40 antibody to Aβ1-40 was 1:2.5. [Figure 4] Figure 4 shows antibody staining of nuclear pore complex protein (NPCP) in human breast cancer (MCF-7) cells. Panel (a) shows native anti-NPCP, panel (b) shows PEG25.5-anti-NPCP, and panel (c) shows anti-NPCP whose linker was cleaved under reducing conditions. Anti-NPCP was contacted with cells after fixation with 4% PFA. Cell nuclei were stained with Hoechst 33258 (blue), and anti-NPCP was labeled with Alexa Fluor 488 (green). The scale bar indicates 20 μm. [Figure 5]Figure 5 shows the brain accumulation and biological activity of anti-BACE1. Panel (a) shows the amount of anti-BACE1 antibody in the serum of C57BL / 6J mice at various times after administration of native, G0-PEGylated, G25-PEGylated, G50-PEGylated, G75-PEGylated, and G100-PEGylated anti-BACE1, expressed as the relative fluorescence intensity of labeled Alexa 647. Panel (b) shows the accumulation of anti-BACE1 in the brain at 12, 24, and 48 hours after sample injection, as determined by quantifying Alexa 647 fluorescence in perfused brain homogenates. C57BL / 6J mice were intravenously administered native anti-BACE1 and Gluc-PEGylated anti-BACE1 (all Alexa647-labeled Fabs, containing anti-BACE1 at a concentration of 0.15 mg / mL) under glycemic control (24-hour fasting, intraperitoneal glucose injection 30 minutes before treatment injection). Panel (c) shows the effect of glucose-modified PEG-conjugated anti-BACE1 on Aβ production in an Alzheimer's disease mouse model. APP / PS1 mice (6 weeks old) were treated with PBS, free native anti-BACE1, or glucose-modified PEG-modified anti-BACE1 (50% glucose-modified, G50-PEGylated anti-BACE1) (both at a dose of 20 mg / kg). Aβ levels were measured in whole brain homogenates by ELISA (n = 5) at 0, 12, 24, and 48 hours post-injection. Specific Description of the Invention

[0012] As used herein, "lowering blood glucose" refers to lowering blood glucose levels in a subject below the blood glucose level that would be present if the treatment were not performed. Methods for lowering blood glucose include dietary restriction or administration of diabetes medication. For example, when lowering blood glucose, it is acceptable to take other medications or drink beverages such as water, as long as the goal of lowering blood glucose is achieved. Lowering blood glucose may involve other treatments that do not substantially affect blood glucose.

[0013] As used herein, "fasting" means causing a subject to fast, e.g., for 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours or more, 24 hours or more, 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 hours or more, 34 hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more, 46 hours or more, 47 hours or more, or 48 hours or more. A subject's blood glucose level can be lowered by fasting for a sufficient period of time. The fasting period is determined by a physician or other professional taking into account the subject's health condition, and is preferably set to a period of time equal to or longer than the time required for the subject to reach fasting blood glucose levels. The fasting period may be set to a period of time equal to or longer than the time required for GLUT1 expression on the intravascular surface of cerebrovascular endothelial cells to increase or plateau. The fasting period may be, for example, 12 hours or longer, 24 hours or longer, or 36 hours or longer. Fasting may also be accompanied by other treatments that do not substantially affect blood glucose levels or GLUT1 expression on the intravascular surface.

[0014] As used herein, "inducing an increase in blood glucose level" refers to increasing blood glucose level in a subject. In particular, an increase in blood glucose level can be induced in a subject whose blood glucose level has been reduced or whose reduced blood glucose level has been maintained. Blood glucose level can be increased by various methods known to those skilled in the art, for example, by administering a substance that induces an increase in blood glucose level, for example, by administering a monosaccharide that induces an increase in blood glucose level, such as glucose, fructose, or galactose, or by administering a polysaccharide that induces an increase in blood glucose level, such as maltose, or by ingesting a carbohydrate that induces an increase in blood glucose level, such as starch, or by eating.

[0015] As used herein, "glycemic manipulation" refers to lowering a subject's blood glucose level and then increasing the blood glucose level. After lowering a subject's blood glucose level, the lowered blood glucose level can be maintained. The time period for lowering a subject's blood glucose level can be, for example, 0 hours or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 11 hours or more, 12 hours or more, 13 hours or more, 14 hours or more, 15 hours or more, 16 hours or more, 17 hours or more, 18 hours or more, 19 hours or more, 20 hours or more, 21 hours or more, 22 hours or more, 23 hours or more, 24 hours or more, or the like. The period may be 25 hours or more, 26 hours or more, 27 hours or more, 28 hours or more, 29 hours or more, 30 hours or more, 31 hours or more, 32 hours or more, 33 hours or more, 34 hours or more, 35 hours or more, 36 hours or more, 37 hours or more, 38 hours or more, 39 hours or more, 40 hours or more, 41 hours or more, 42 hours or more, 43 hours or more, 44 hours or more, 45 hours or more, 46 hours or more, 47 hours or more, or 48 hours or more. Thereafter, blood glucose levels can be increased. As used herein, "maintaining blood glucose" refers to, for example, taking other medications or drinking beverages such as water, as long as the purpose of maintaining the lowered blood glucose level in the subject is achieved. Lowering blood glucose may involve other treatments that do not substantially affect blood glucose.

[0016] As used herein, a "subject" refers to a mammal, including a human. The subject may be healthy or may be suffering from a disease. Examples of diseases include neurological disorders, such as psychotic disorders, depression, mood disorders, anxiety, sleep disorders, dementia, and substance-related disorders. Dementia includes, but is not limited to, Alzheimer's disease and Creutzfeldt-Jakob disease.

[0017] As used herein, "antibody" refers to an intact immunoglobulin of any isotype (i.e., a protein consisting of two heavy chains (H chains) and two light chains (L chains) stabilized by a pair of disulfide bonds) associated with one another, or a fragment thereof capable of competing with an intact antibody for binding to an antigen. Antibodies include antibodies derived from a single non-human vertebrate (e.g., antibodies derived from a non-human mammal), chimeric antibodies, humanized antibodies, human antibodies, and bispecific antibodies. Antibodies may be monoclonal or polyclonal, and when administered to humans, monoclonal antibodies are preferred. Antibody isotypes include IgG, IgM, IgA, IgD, and IgE. "Humanized antibodies" refer to antibodies in which the corresponding positions in a human antibody have been substituted with amino acid sequences characteristic of antibodies of non-human origin, and include, for example, antibodies that have heavy chain CDRs 1 to 3 and light chain CDRs 1 to 3 of an antibody produced by immunizing a mouse or rat, with all other regions, including the four framework regions (FR) of each of the heavy and light chains, derived from a human antibody. Such antibodies are sometimes called CDR-grafted antibodies. A "human chimeric antibody" is an antibody of non-human origin in which the constant regions of the non-human antibody have been replaced with the constant regions of a human antibody. Antigen-binding antibody fragments include, but are not limited to, Fab, which consists of VL, VH, CL, and CH1 regions; F(ab')2, in which two Fabs are linked by a disulfide bond at the hinge region; Fv, which consists of VL and VH; scFv, which is a single-chain antibody in which VL and VH are linked by an artificial polypeptide linker; and bispecific antibodies such as diabody type, scDb type, tandem scFv type, and leucine zipper type. An antibody that binds to substance A may be referred to herein as "anti-substance A" or "anti-substance A."

[0018] As used herein, the term "blood-brain barrier" refers to a functional barrier that exists between the blood circulation and the brain and has selectivity for the permeation of substances. The blood-brain barrier is thought to consist of cerebral vascular endothelial cells and the like. Although much is unknown about the substance permeability of the blood-brain barrier, it is known that glucose, alcohol, and oxygen easily pass through the blood-brain barrier, and it is thought that lipid-soluble substances and small molecules (e.g., molecular weight less than 500) tend to pass through the blood-brain barrier more easily than water-soluble molecules and polymers (e.g., molecular weight greater than 500). Many brain disease therapeutic agents and brain diagnostic agents do not pass through the blood-brain barrier, which is a major obstacle to the treatment of brain diseases and brain analysis.

[0019] As used herein, the term "GLUT1 ligand" refers to a substance that specifically binds to GLUT1. Various GLUT1 ligands are known, including, but not limited to, molecules such as glucose and hexose. Any of these GLUT1 ligands can be used in place of glucose to prepare carriers or conjugates in the present invention. The GLUT1 ligand is glucose, or preferably a substance with an affinity for GLUT1 equal to or greater than that of glucose. 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2-propylamine (ATB-BMPA), 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (6-NBDG), 4,6-O-ethylidene-α-D-glucose, 2-deoxy-D-glucose, and 3-O-methylglucose are also known to bind to GLUT1, and these molecules can also be used as GLUT1 ligands in the present invention.

[0020] As used herein, "modified with a GLUT1 ligand" means modified so that the GLUT1 ligand can be recognized by GLUT1.

[0021] As used herein, "alkyl" means straight-chain (i.e., unbranched) or branched-chain or combinations thereof, is fully saturated, and has a specified number of carbon atoms (i.e., C1 to C6). 10 has 1 to 10 carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, (cyclohexyl)methyl, homologs, isomers, etc., of n-pentyl, n-hexyl, n-heptyl, n-octyl, etc. Thus, the term "alkyl group" refers to any group having C1 to C6 16 Straight chain saturated aliphatic hydrocarbons, C1-C 16 Branched-chain saturated aliphatic hydrocarbons, C3-C8 cyclic saturated aliphatic hydrocarbons, and C1-C cyclic saturated aliphatic hydrocarbons substituted with C3-C8 cyclic saturated aliphatic hydrocarbon groups having a specific number of carbon atoms. 16 It may also refer to a straight or branched chain saturated aliphatic hydrocarbon group. For example, this definition includes, but is not limited to, methyl (Me), ethyl (Et), propyl (Pr), butyl (Bu), pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, isopropyl (i-Pr), isobutyl (i-Bu), tert-butyl (t-Bu), sec-butyl (s-Bu), isopentyl, neopentyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclopropylmethyl, and the like.

[0022] As used herein, "alkylene" means a divalent radical derived from alkyl, by way of example, but not limitation, -(CH) n -{where n is any natural number from 1 to 24}. Typically, alkyl (or alkylene) groups have 1 to 24 carbon atoms, and these groups preferably have 10 or fewer carbon atoms. A "lower alkyl" or "lower alkylene" is a short-chain alkyl group or short-chain alkylene group, generally having 8 or fewer, 6 or fewer, or 4 or fewer (e.g., 2 or 3) carbon atoms.

[0023] As used herein, "heteroalkyl" means a stable linear or branched chain, or combinations thereof, consisting of at least one carbon atom and at least one heteroatom selected from the group consisting of O, N, P, Si, and S, wherein N and S are optionally oxidized, and the N heteroatom is optionally quaternized. The heteroatom(s) O, N, P, S, and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. For example, heteroalkyl groups include, but are not limited to, -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-CH3, -CH2S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH2-CH2-S(O)-CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, -CH=CH-N(CH3)-CH3, -O-CH3, -O-CH2-CH3, and -CN. Up to two heteroatoms may be consecutive in the chain, such as, for example, -CH2-NH-OCH3. Heteroalkyl groups can contain, for example, up to two heteroatoms. Heteroalkyl groups can contain, for example, one heteroatom. Heteroalkyl groups can be lower heteroalkyl.

[0024] As used herein, "heteroalkylene" refers to a straight- or branched-chain divalent radical derived from heteroalkyl, including, but not limited to, -CH-CH-S-CH-CH- and -CH-S-CH-CH-NH-CH-. For alkylene heterogroups or alkylenediheterogroups, heteroatoms are at one or both chain ends, particularly terminal (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, etc.). Furthermore, for alkylene and heteroalkylene linking groups, the direction of the written formula of the linking group does not imply orientation of the linking group. For example, the formula -C(O)R'- represents both -C(O)OR'- and -R'C(O)O-. As discussed above, heteroalkyl groups, as used herein, include those groups that are attached to the remainder of the molecule through a heteroatom, such as -C(O)R', -C(O)NR', ​​-NR'R'', -OR', -SR', and / or -SOR'.

[0025] The terms "cycloalkyl" and "heterocycloalkyl" refer to cyclic versions of "alkyl" and "heterocycloalkyl," respectively. Additionally, for heterocycloalkyl, a heteroatom can be located at the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. The terms "cycloalkylene" and "heterocycloalkylene," alone or as part of another substituent, mean divalent radical derivatives derived from cycloalkyl and heterocycloalkyl, respectively.

[0026] A linker that is stable in the bloodstream refers to a linker that is stable enough to remain stable in the blood for the time required for translocation from administration to the brain parenchyma. A bond selected from the group consisting of a carbon-carbon bond, an amide bond, a phosphodiester bond, an ester bond, an ether bond, a thioester bond, a thioether bond, and a disulfide bond may be a linker that is stable in the bloodstream. In the present invention, a linker that is stable in the bloodstream is pharmaceutically acceptable. Therefore, the uncharged hydrophilic polymer segment and the antibody can be linked by such a linker. Examples of linkers that are stable in the bloodstream include, but are not limited to, substituted or unsubstituted alkylenes or substituted or unsubstituted heteroalkylenes. Whether a linker is stable in the bloodstream can be determined, for example, by evaluating the stability of the linker in isolated blood or physiological saline containing serum. The time required for translocation from administration to the brain parenchyma can be determined appropriately by those skilled in the art. The time required for transfer from administration to the brain parenchyma can be, for example, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, 7 hours or more, 8 hours or more, 9 hours or more, 10 hours or more, 12 hours or more, 15 hours or more, 18 hours or more, 21 hours or more, 24 hours or more, 2 days or more, 3 days or more, 4 days or more, 5 days or more, 6 days or more, or 7 days or more. The time required for transfer from administration to the brain parenchyma can be, for example, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less. The time required for transfer from administration to the brain parenchyma can be, for example, 1 hour or more to 1 day or less.

[0027] As used herein, a "substituent" can be selected from the group consisting of -OH, -NH2, -SH, -CN, -CF3, -NO2, oxo, halogen, -COOH, unsubstituted alkyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl, unsubstituted heterocycloalkyl, unsubstituted aryl, and unsubstituted aryl. As used herein, a "substituent" can be a "lower substituent," and a substituted or unsubstituted alkyl is a substituted or unsubstituted C1-C8 alkyl, each substituted or unsubstituted heteroalkyl is a substituted or unsubstituted 2- to 8-membered heteroalkyl, each substituted or unsubstituted cycloalkyl is a substituted or unsubstituted C5-C7 cycloalkyl, and each substituted or unsubstituted heterocycloalkyl is a substituted or unsubstituted 5- to 7-membered heterocycloalkyl.

[0028] According to the present invention, there is provided an antibody modified with an uncharged hydrophilic polymer segment, wherein the antibody is capable of binding to an antigen, and is in the form of the antibody modified with the uncharged hydrophilic polymer segment in the blood, and in the brain parenchyma after migration from the intravascular space to the brain parenchyma, the antibody is in a form dissociated from the uncharged hydrophilic polymer segment. According to one embodiment of the present invention, when the antibody enters a low pH environment, such as the pH in the endosomes of cerebrovascular endothelial cells from a blood vessel, it dissociates from the uncharged hydrophilic polymer segment in response to a decreased pH. In this embodiment, the antibody is linked to the uncharged hydrophilic polymer segment by a pH-responsive bond, which is cleaved, for example, in an environment of pH less than 7, pH 6.5 or less, pH 6.4 or less, pH 6.3 or less, pH 6.2 or less, pH 6.1 or less, or pH 6 or less (e.g., the pH in the endosome). When the linker has a pH-sensitive moiety selected from the group consisting of ester, sulfonate ester, boronate ester, phosphate ester, amide (including carbamate), acetal, ketal, hydrazone, imine, imide, enamine, and thiosuccinimidyl, the linker can be cleaved in response to a decreased pH. In one embodiment, the linker can have the structure -NH-CO-CH=CH-L3-CO-NH-, where L3 is a linker moiety that is stable in blood. The linker is cleaved in a low pH environment to release the antibody according to the following scheme (where L3 is represented as R):

[0029] [ka]

[0030] The effectiveness of the above linker is evident from the fact that after linker cleavage, not only is the uncharged hydrophilic segment (e.g., PEG as shown in the figure above) removed, but also the intact antibody (the amino group of the lysine side chain used for PEG modification returns to its original amino group).

[0031] According to one embodiment of the present invention, the antibody dissociates from the uncharged hydrophilic polymer segment during transcytosis of the BBB or in the brain parenchyma after passing through vascular endothelial cells and entering the brain parenchyma. In this embodiment, the antibody is linked to the uncharged hydrophilic polymer segment via a linker that is responsive to a reducing environment, and the bond is cleaved in the reducing environment in the brain. In this embodiment, the linkage that is responsive to a reducing environment may be a disulfide bond. According to one embodiment of the present invention, an antibody dissociated from an uncharged hydrophilic polymer segment has stronger binding affinity to an antigen than an antibody modified with an uncharged hydrophilic polymer segment. Under a reducing environment, the antibody can partially or completely recover (or reactivate) its binding affinity to the antigen. The greater the recovery or reactivation, the more preferable. For example, the binding affinity of the recovered or reactivated antibody may be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the binding affinity of the unmodified antibody. According to one embodiment of the present invention, an antibody modified with an uncharged hydrophilic polymer segment has weaker binding affinity to an antigen than the antibody before modification (unmodified antibody). The greater the reduction in binding affinity, the more preferable. For example, the binding affinity of the modified antibody may be 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.1% or less of that of the unmodified antibody. Binding affinity can be measured, for example, in serum or physiological saline. Thus, the antibody of the present invention may be inactivated by modification. In particular, the antibody of the present invention may have reduced or eliminated binding affinity to its antigen by modification. That is, the antibody of the present invention may be an inactivated antibody. The reduction or elimination of the binding affinity of an antibody to an antigen can be caused by steric hindrance by the uncharged hydrophilic polymer block. Therefore, by increasing the bulk of the uncharged hydrophilic polymer block and / or by increasing the modification rate, the binding affinity of an antibody to an antigen can be further reduced or eliminated. According to one embodiment of the present invention, an antibody modified with an uncharged hydrophilic polymer segment has weaker binding affinity to an antigen than the antibody before modification (unmodified antibody), and an antibody dissociated from the uncharged hydrophilic polymer segment has stronger binding affinity to an antigen than the antibody modified with an uncharged hydrophilic polymer segment. According to the present invention, an antibody modified with an uncharged hydrophilic polymer segment is cleaved from the segment in the brain parenchyma, thereby restoring the antibody's binding properties, which had been impaired by the modification. Such an antibody can reach the brain parenchyma without special blood glucose control or glucose modification, and can restore its binding properties in the brain parenchyma, making it useful, for example, in patients with diseases in which the blood-brain barrier is weakened. In this embodiment, according to the present invention, the uncharged hydrophilic polymer segment may be modified with a GLUT1 ligand. Modification with a GLUT1 ligand is useful in delivering the antibody into the endosomes of cerebrovascular endothelial cells or into the brain parenchyma in subjects with a normal blood-brain barrier.

[0032] In one embodiment of the present invention, the antibody and the uncharged hydrophilic polymer segment are linked by -CO-O-L1-SS-L2- (wherein L1 is a substituted or unsubstituted alkylene or substituted or unsubstituted heteroalkylene, and L2 is a linker stable in the bloodstream), which is linked to the side chain amino group of a lysine residue of the peptide via an amide bond, and L2 is linked to the uncharged hydrophilic polymer segment. The above -CO-O-L1-SS-L2- is cleaved at the SS bond in the reducing environment of the brain parenchyma. This causes the S-L2-uncharged hydrophilic polymer segment to dissociate from the antibody. The antibody remains linked to -CO-O-L1-S, or S attacks the C=O to form a ring, which allows it to be released from the antibody. When S attacks the C=O to form a ring and is released from the antibody, the antibody can return to its original state (the antibody before modification or the unmodified antibody).

[0033] The present invention provides an antibody modified with an uncharged hydrophilic polymer via a linker, wherein the uncharged hydrophilic polymer is modified with a GLUT1 ligand. The antibody can bind to GLUT1 expressed on the luminal surface of cerebrovascular endothelial cells via the GLUT1 ligand. When glucose (or a GLUT1 ligand) is administered to a subject whose blood glucose level has been reduced, substances bound to the luminal surface of the cerebrovascular endothelial cells of the subject are taken up into the vascular endothelial cells by endocytosis, and at least a portion of the substances are delivered to the brain parenchyma by transcytosis. GLUT1 is expressed on the luminal surface of cerebrovascular endothelial cells of a subject whose blood glucose level has been reduced. The antibody of the present invention binds to GLUT1 via the GLUT1 ligand, and when glucose (or a GLUT1 ligand) is administered, the antibody is taken up into the vascular endothelial cells by endocytosis, and at least a portion of the substances are delivered to the brain parenchyma by transcytosis. The binding of the antibody to GLUT1 can be confirmed in vitro by an assay evaluating the binding of the antibody to isolated GLUT1. Examples of uncharged hydrophilic polymers include uncharged hydrophilic polymers such as polyethylene glycol (PEG) and polyoxazoline. Here, "uncharged" means that the charge is neutral throughout the polymer segment. Uncharged hydrophilic polymers are biocompatible. In one embodiment, the antibodies of the present invention may or may not have an uncharged hydrophilic polymer segment modified with a ligand (second ligand) for a receptor in the brain other than the GLUT1 ligand.

[0034] According to the present invention, in the above-described antibodies of the present invention, the linker can be linked to the side chain amino group of a lysine residue of the antibody. The linkage can preferably be a covalent bond. In one embodiment, the lysine residue linked to the linker can be present in the heavy and / or light chain variable regions of the antibody. In one embodiment, the lysine residue linked to the linker can be present in the CDR region of the heavy and / or light chain variable regions of the antibody. Modification with a GLUT1 ligand is used for transport from the bloodstream to the brain parenchyma and may become unnecessary after transport. Therefore, after the antibody has transported to the brain parenchyma, the PEG modified with the GLUT1 ligand that modifies the antibody may be cleaved from the antibody. Also, according to the present invention, modification of the side chain amino group of the lysine residue of the antibody may reduce the binding properties of the antibody depending on the strength of the modification. Therefore, after the antibody has transported to the brain parenchyma, the PEG modified with the GLUT1 ligand that modifies the antibody may be cleaved from the antibody. The GLUT1 ligand may modify the PEG. The GLUT1 ligand may modify the terminal carbon atom or oxygen atom of the PEG. For example, the brain parenchyma has a reducing environment. The reducing environment of the brain parenchyma is a reducing environment with a strength equivalent to that of a 2 mM aqueous solution of glutathione (GSH). By including a cleavage site that can be cleaved in a reducing environment in the linker, the linker can be configured so that the linker is cleaved after the antibody is delivered to the brain parenchyma. Such a linker is called a linker that can be cleaved in a reducing environment. The present invention provides an antibody modified with an uncharged hydrophilic polymer (e.g., PEG) modified with a GLUT1 ligand via a linker that can be cleaved in a reducing environment. The antibody of the present invention can preferably be cleaved in the brain parenchyma, which provides a reducing environment. The linker that can be cleaved in a reducing environment can have, for example, a disulfide bond as the cleavage site. By linking an antibody to a PEG modified with a GLUT1 ligand using a linker that can be cleaved in a reducing environment, the linker can be cleaved after the antibody is delivered to the brain parenchyma, releasing the antibody within the brain parenchyma.

[0035] A linker that is cleavable under a reducing environment can be configured to leave the side chain amino group of a lysine residue of an antibody unsubstituted upon cleavage. For example, when the linker is (antibody-NH)-CO-O-C2H4-SS-L2-(uncharged hydrophilic polymer) (where L2 is a bond or a linker stable in the bloodstream) and the disulfide bond in the linker is cleaved, the side chain amino acid of the lysine residue of the antibody is restored by the following reaction: The following describes the mechanism by which an antibody of the present invention, in which the GLUT1 ligand is glucose (Gluc), the uncharged hydrophilic polymer is polyethylene glycol (PEG), and L2 is C2H4-O-CO-, dissociates from the linker under a reducing environment and returns to the original antibody. This mechanism is also applicable when the GLUT1 ligand is a GLUT1 ligand other than glucose, the uncharged hydrophilic polymer is an uncharged hydrophilic polymer other than PEG, and the antibody is a peptide other than an antibody (e.g., a protein or a fragment thereof). The modified peptide may have 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or 0.1% or less of the physiological activity of the unmodified peptide. Furthermore, the physiological activity of the modified peptide can be partially or completely restored (or reactivated) under, for example, a reducing environment. The greater the restoration or reactivation, the more preferable. For example, the binding affinity of the restored or reactivated peptide may be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more of the binding affinity of the unmodified peptide.

[0036] [ka]

[0037] By being configured so that cleavage dissociates the linker moiety from the side chain amino group of the lysine residue of the antibody, returning the amino group to an unsubstituted amino group, a linker that is cleavable in a reducing environment can return the antibody to its unsubstituted state in brain parenchyma. It has been pointed out that the binding properties of an antibody can be reduced by modifying the side chain amino group of a lysine residue. Even if the binding properties of an antibody are reduced by modifying the side chain amino group of a lysine residue, when the antibody is delivered to the reducing environment of brain parenchyma, the modification of the side chain amino group of the lysine residue of the antibody is removed, returning the antibody to its unsubstituted state, thereby allowing the antibody to regain its binding properties. Such linker design can be performed appropriately by those skilled in the art.

[0038] Thus, the antibodies of the present invention may be antibodies whose binding properties, which have been reduced by modification, can be restored by cleavage of the linker. Restoration of binding properties can be achieved by various techniques, for example, by restoring the side chain amino group of the lysine residue to an unsubstituted state, as described above. In this case, the linker can be (antibody-NH)-CO-O-L1-SS-L2-(uncharged hydrophilic polymer segment) {where L1 is a substituted or unsubstituted alkylene or substituted or unsubstituted heteroalkylene, and L2 is a linker that is stable in a bond or in the bloodstream} (the structure in parentheses is not included in the linker; the structure in parentheses is shown to illustrate the connection between the linker and the structure). Thus, preferred antibodies of the present invention include an antibody in which the linker has the structure -NH-CO-O-C2H4-SS-C2H4-O-CO- and is linked to the side chain amino group of the lysine residue of the antibody via an amide bond. The present invention provides the following: (antibody)-CO-O-L1-SS-L2-(uncharged hydrophilic polymer segment) {wherein L1 is substituted or unsubstituted alkylene or substituted or unsubstituted heteroalkylene, and L2 is a linker stable in the bloodstream} {the structure in parentheses is not included in the linker. The structure in parentheses is shown to explain the connection between the linker and the structure}. The present invention also provides a PEG-linked linker having a structure represented by: (antibody)-CO-O-L1-SS-L2-PEG(uncharged hydrophilic polymer segment) {wherein L1 is substituted or unsubstituted alkylene or substituted or unsubstituted heteroalkylene, and L2 is a bond or a linker stable in the bloodstream}. The PEG-linked linker may be modified with a GLUT1 ligand.

[0039] In some preferred embodiments of the present invention, L1 can be ethylene and L2 can be -C2H4-OC(=O)-.

[0040] In a preferred embodiment of the antibody of the present invention, the GLUT1 ligand may be glucose. In this embodiment, glucose may be linked to PEG, for example, via its carbon atom at position 2, 3, or 6, and may favorably interact with GLUT1.

[0041] In the antibody of the present invention, the number average degree of polymerization of PEG can be 2,000 to 12,000, for example, 5,000.

[0042] In the antibodies of the present invention, 10 to 90%, 20 to 80%, 30 to 70%, or 40 to 60% of the side chain amino groups (primary amines) of lysine residues may be modified with polyethylene glycol (PEG) via a linker. Herein, the percentage of PEG-modified amino groups among the side chain amino groups of lysine residues may be expressed by adding the percentage (%) after "PEG."

[0043] The antibodies of the present invention may be modified both with PEG modified with a GLUT1 ligand and with PEG not modified with a GLUT1 ligand. In the antibodies of the present invention, for example, 10 to 100%, 30 to 80%, or 40 to 60% of the PEG modifying the antibody may be modified with GLUT1. Herein, the percentage of PEG modified with a GLUT1 ligand may be expressed by adding the percentage (%) after the letter "G."

[0044] The antibody of the present invention can be, for example, an antibody that binds to an antigen in the brain parenchyma (e.g., an intracellular antigen, a cell surface antigen, or an extracellular antigen). In this embodiment, the antibody of the present invention can be an antibody that does not show significant binding to components in the blood. Antigens in the brain parenchyma include, for example, amyloid β (Aβ, e.g., Aβ 1-40 and Aβ 1-42), its soluble oligomers, its extracellular deposits, β-secretase 1 (BACE1), abnormal prion protein (misfolded prion protein), superoxide dismutase 1 (SOD1), and α-synuclein. Aβ may preferably be an extracellular deposit.

[0045] When the antigen of the antibody is a surface antigen of a tumor in the brain parenchyma, the antibody may be an antibody having antibody-dependent cellular cytotoxicity (ADCC activity) and / or complement-dependent cytotoxicity (CDC activity). ADCC activity can be enhanced, for example, by making the subclass of the antibody IgG1. When the antigen of the antibody is a surface antigen of a tumor in the brain parenchyma, the antibody may be in the form of an antibody-drug conjugate (ADC) with a cytotoxic agent. The antibody may also be in the form of, for example, a non-ADC.

[0046] The antibody of the present invention may be linked to an imaging agent. Examples of imaging agents include biocompatible fluorescent dyes (e.g., fluorescent dyes that emit fluorescence in the visible light region or near-infrared region) and luminescent dyes (e.g., luciferase), as well as radioisotopes, ultrasound probes, MRI contrast agents, and CT contrast agents. Conjugates of imaging agents and antibodies can be appropriately prepared by those skilled in the art.

[0047] The antibody of the present invention may be linked to a physiologically active substance (e.g., an enzyme or a nucleic acid). This allows the physiologically active substance to be delivered to the brain parenchyma. Conjugates of physiologically active substances and antibodies can be appropriately prepared by those skilled in the art.

[0048] The present invention provides pharmaceutical compositions for treating or preventing brain diseases, comprising the peptides or antibodies of the present invention. Brain diseases include, for example, anxiety, depression, sleep disorders, Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Antibodies also include those that bind to causative factors of these diseases and neutralize their activity. For example, antibodies of the present invention that bind to Aβ, its soluble oligomers, and its extracellular deposits (e.g., antibodies that bind to Aβ and / or its soluble oligomers and inhibit the formation or buildup of extracellular deposits), or antibodies of the present invention that bind to cellular deposits of Aβ and suppress their buildup or reduce their accumulation, can be administered to, for example, patients with Alzheimer's disease or patients at risk of developing the disease, thereby being used to treat Alzheimer's disease. Antibodies of the present invention that bind to abnormal prion protein (e.g., antibodies that bind to abnormal prion protein and suppress or reduce the accumulation of abnormal prion protein) can be administered to patients with Creutzfeldt-Jakob disease or patients at risk of developing it, thereby treating Creutzfeldt-Jakob disease. Antibodies of the present invention that bind to SOD1 (e.g., antibodies that bind to and activate SOD1 mutants found in amyotrophic lateral sclerosis (ALS), e.g., anti-SOD1 antibodies or anti-Derlin-1 antibodies that block the binding of SOD1 to Derlin-1) can be administered to patients with ALS or patients at risk of developing it, thereby treating ALS. Antibodies of the present invention that bind to α-synuclein (e.g., antibodies that bind to α-synuclein and suppress or reduce the accumulation of α-synuclein) can be administered to patients with synucleinopathies such as dementia with Lewy bodies and Parkinson's disease or patients at risk of developing them, thereby treating synucleinopathies. The present invention also provides antibodies for use in treating diseases. As used herein, "treatment of a disease" refers to prevention of a disease and treatment of a disease. Prevention of a disease is used to mean preventing the onset of a disease, delaying the onset, and reducing the incidence of a disease.Treating a disease is used to include slowing the rate of progression of the disease, slowing progression, preventing progression, alleviating the symptoms of the disease, curing the disease, and causing remission of the disease.

[0049] The peptide or antibody or pharmaceutical composition of the present invention may be administered according to a dosing regimen, wherein the dosing regimen comprises: lowering the blood glucose of a subject, and then The method may include inducing an increase in blood glucose levels in the subject, and administering the peptide or antibody or pharmaceutical composition of the present invention to the subject so that more antibody migrates to the brain parenchyma than when the increase in blood glucose levels is not induced.

[0050] In the administration regimen according to the present invention, the composition can be administered to the subject simultaneously, consecutively, or sequentially with the induction of an increase in blood glucose level in the subject. The administration regimen may or may not have an interval between the administration of the composition to the subject and the induction of an increase in blood glucose level in the subject. When the composition is administered simultaneously with the induction of an increase in blood glucose level in the subject, the composition may be administered to the subject in a form mixed with the agent that causes the induction of an increase in blood glucose level, or may be administered in a form separate from the agent that causes the induction of an increase in blood glucose level in the subject. Furthermore, when the composition is administered to the subject consecutively or sequentially with the induction of an increase in blood glucose level in the subject, the composition may be administered to the subject before or after the induction of an increase in blood glucose level in the subject, but preferably, the composition can be administered to the subject before the induction of an increase in blood glucose level in the subject. When the blood glucose level is increased in the subject prior to administration of the composition to the subject, it is preferable to administer the composition to the subject within 1 hour, 45 minutes, 30 minutes, 15 minutes, or 10 minutes after inducing an increase in blood glucose level in the subject.When the blood glucose level is increased in the subject after administration of the composition to the subject, it is preferable to induce an increase in blood glucose level in the subject within 6 hours, 4 hours, 2 hours, 1 hour, 45 minutes, 30 minutes, 15 minutes, or 10 minutes after administration of the composition to the subject.The above-mentioned cycle of administration schedule may be performed two or more times.The order of glucose administration and sample administration can be determined by the timing of passing through the blood-brain barrier.

[0051] The pharmaceutical composition of the present invention may further contain a pharmaceutically acceptable excipient in addition to the antibody of the present invention. The pharmaceutical composition of the present invention can be in various forms, such as a liquid (e.g., an injection), a dispersion, a suspension, a tablet, a pill, a powder, a suppository, etc. In a preferred embodiment, the pharmaceutical composition of the present invention is an injection and can be administered parenterally (e.g., intravenously, transdermally, intraperitoneally, and intramuscularly).

[0052] The present invention provides a method for administering an antibody to a subject, the method comprising administering to the subject an antibody of the present invention. The present invention also provides a method for delivering an antibody to the brain parenchyma of a subject, the method comprising administering to the subject an antibody of the present invention or a pharmaceutical composition comprising the antibody. Administration may be intravenous. In these methods, the antibody of the present invention may be administered according to the dosing regimen provided by the present invention.

[0053] The present invention provides a method for producing an antibody, comprising linking glucose-modified polyethylene glycol to the side chain amino acid of a lysine residue of the antibody via a linker cleavable under a reducing environment. In the present invention, the linker can be (GLUT1 ligand-PEG)-L2'-C2H4-SS-C2H4-O-CO-(NH-antibody) (the structure in parentheses is not included in the linker. The structure in parentheses is shown to explain the connection relationship between the linker and the structure. L2' is a bond or a linker that is stable in the bloodstream, preferably a carbamate bond NH-CO-O).

[0054] In all aspects of the present invention, a peptide can be used instead of an antibody. In some aspects of the present invention, the peptide has multiple lysine residues. In some aspects of the present invention, the peptide has multiple lysine residues on its surface. In some aspects of the present invention, the peptide has multiple lysine residues on its surface, and multiple of the lysine residues are modified via a linker with an uncharged hydrophilic polymer modified with a GLUT1 ligand. In these aspects, the GLUT1 ligand can be glucose. In these aspects, the uncharged hydrophilic polymer can be polyethylene glycol or polyoxazoline. In these aspects, the linker can be cleavable in a reducing environment. In these aspects, the GLUT1 ligand can be glucose, the uncharged hydrophilic polymer can be polyethylene glycol or polyoxazoline, and the linker can be a linker cleavable in a reducing environment. The linker is preferably cleaved from the peptide so as to return the modified amino group to a primary amine. After the linker is cleaved, the peptide can retain its inherent biological activity.

[0055] According to the present invention, there is provided a peptide having a lysine residue modified with GLUT1 ligand-modified PEG via -N-L2'-C2H4-SS-C2H4-O-CO- {L2' is defined as above}. The peptide may further have a lysine residue modified with PEG via -NH-L2'-C2H4-SS-C2H4-O-CO- {L2' is defined as above}. According to the present invention, the modified peptide may have a plurality of lysine residues modified with GLUT1 ligand-modified PEG via -L2-C2H4-SS-C2H4-O-CO- {L2' is defined as above}.

[0056] The present invention provides a method for producing a peptide, comprising linking glucose-modified polyethylene glycol to the side chain amino acid of a lysine residue of the peptide via a linker that can be cleaved under a reducing environment. In the present invention, the linker can be (GLUT1 ligand-PEG)-L2'-C2H4-SS-C2H4-O-CO-(peptide) {the structure in parentheses is not included in the linker. The structure in parentheses is shown to explain the connection relationship between the linker and the structure. L2' is defined as above.}

[0057] The present invention provides a composition comprising a modified peptide of the present invention. The modified peptide can be administered according to the dosage regimen of the present invention, thereby delivering it to the brain and allowing the linker to be cleaved in the brain. The composition can be a pharmaceutical composition. The peptide can be a peptide used in peptide replacement therapy. The peptide can comprise the full-length amino acid sequence of a protein. In some embodiments, the peptide can be a part of an antibody. In some embodiments, the peptide can be an antibody. In some embodiments, the antibody of the present invention can comprise the modified peptide.

[0058] The present invention provides use of the peptide or antibody of the present invention in the manufacture of the pharmaceutical composition of the present invention. [Example]

[0059] Example 1: Materials and Methods Materials and animals. 1,2:3,4-Di-O-isopropylidene-α-D-glucofuranoside (DIG) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Ethylene oxide (EO) was obtained from Nippon Ekitan Co., Ltd. (Tokyo, Japan) and purified with CaH2 by the trap-trap method. The solvents used in the polymerization (THF, CHCl, and DMF) were purified by passing through two columns packed with neutral alumina purchased from Nikko Hansen Co., Ltd. (Osaka, Japan). α-Methoxy-ω-aminopoly(ethylene glycol) (MeO-PEG-NH) (PEG Mw 2,200, Mw / Mn = 1.05) was purchased from NOF Co., Ltd. (Tokyo). Dulbecco's phosphate-buffered saline (D-PBS(-)), D-(+)-glucose in phosphate buffer, and paraformaldehyde were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Cell lysis buffer was purchased from Promega Corporation (Madison, WI). Glutathione (GSH) was purchased from Thermo Fisher Scientific. Dimethyl sulfoxide (DMSO) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Bis(2-hydroxyethyl) disulfide and 4-nitrophenyl chloroformate were purchased from Sigma. Anti-nuclear pore complex protein (anti-NPCP, product number: ab60080), anti-Aβ 1-40 , and anti-BACE1 (product number: ab2077) were purchased from Abcam Inc. All animal experiments were conducted in compliance with the guidelines for the care and use of laboratory animals established by iCONM (Innovation Center of Nanomedicine, Kawasaki, Japan).

[0060] Ring-opening polymerization of ethylene oxide (EO) from 1,2:5,6-di-O-isopropylidene-α-D-glucofuranoside (DIG). EO polymerization was carried out according to our previous work. 26Briefly, 520 mg (2.0 mmol) of DIG dissolved in THF was sublimed in a reaction tube under vacuum at 70 °C. Next, a 0.3 M solution of potassium naphthalene (6.6 mL, 2.0 mmol) in THF (6.6 mL, 2.0 mmol) was added dropwise to 4.6 mL of EO(92)-Alzheimer's HF-dissolved DIG solution while stirring under an Ar atmosphere. After stirring at 25 °C for 48 h, 2 mL of MeOH was added to the solution. The mixture was then washed with ice-cold diethyl ether with stirring to obtain a white precipitate of DIG-PEG-OH, where PEG had a number-average molecular weight of 5,000.

[0061] Terminal amination of DIG-PEG-OH. The ω-hydroxyl group of DIG-PEG-OH was 28 The ω-amino group was converted to the ω-amino group by the standard method reported in

[2012] : 2.0 g of DIG-PEG-OH (1 mmol, in THF) and 830 μL of TEA (6.0 mmol) were added to a THF solution of methanesulfonyl chloride (387 μL, 5.0 mmol) in ice. The solution was stirred at 25 °C for 6 h, and then added dropwise to ice-cold diethyl ether to obtain a white precipitate of DIG-PEG-OMs. After drying in vacuo, the white solid of DIG-PEG-OMs was dissolved in 200 mL of 25% aqueous ammonia and stirred at 25 °C for 48 h. The mixture was evaporated, dialyzed, and lyophilized, and then subjected to ion-exchange chromatography (Sephadex C-25, GE Healthcare) to remove the nonaminated PEG fraction. After evaporation and lyophilization, a white powder of pure DIG-PEG-NH2 was obtained.

[0062] Preparation and characterization of PEGylated antibodies Preparation of bis(NP-ethyl) disulfide (NP-CC-SS-CC-NP). First, bis(2-hydroxyethyl) disulfide (0.5 mmol) and 4-nitrophenyl chloroformate (NPC, 1.5 mmol) were dissolved in anhydrous DCM (8 mL). The solution was stirred at 0°C for 10 min, and then trimethylamine (1.5 mmol) was added. The reaction was carried out at room temperature for 5 h. After purification by silica gel flash column chromatography, bis(NP-ethyl) disulfide was recovered. 1It was analyzed by H NMR (fig. S1). Preparation of MeO-PEG-CC-SS-CC-NP and glucose (Gluc)-PEG-CC-SS-CC-NP The synthesized bis(NP-ethyl) disulfide was added to DIG-PEG-NH2 or MeO-PEG-NH2 dissolved in DMF at a molar ratio of bis(NP-ethyl) disulfide:DIG-PEG-NH2 or MeO-PEG-NH2 = 20:1, respectively, and the reaction was carried out for 12 hours to obtain DIG-PEG-CC-SS-CC-NP and MeO-PEG-CC-SS-CC-NP, respectively. 1 Deprotection of DIG using 80% TFA gave Gluc-PEG-CC-SS-CC-NPs, as identified by H NMR (Figure S2).

[0063] PEGylated antibodies were prepared as described below. The effect of glucose modification on PEGylated antibody formation was evaluated by varying the ratio of Gluc- and MeO-PEG-CC-SS-CC-NP (10 mg / ml, DMF) complexed with 2 mg / ml antibody in phosphate buffer (200 mM, pH 8.5). The mixture was then stirred at 4°C for various reaction times. The solution was then purified with Vivaspin 6 (three times, MW cutoff 30,000 Da, 10 mM pH 7.4 phosphate-buffered saline) to obtain Gluc-PEGylated antibodies with 0, 25, 50, 75, and 100% glucose modification (determined by percentage polymer composition). Gluc-PEGylated antibodies with 0, 25, 50, 75, and 100% glucose modifications are referred to as G0-PEGylated antibody, G25-PEGylated antibody, G50-PEGylated antibody, G75-PEGylated antibody, and G100-PEGylated antibody, respectively.

[0064] The PEGylated antibodies were analyzed using size exclusion chromatography (Superose 6 Elight 10 / 300 column, GE). The sample volume was set to 100 μl and eluted with phosphate-buffered saline (10 mM, pH 7.4, 150 mM NaCl) at a flow rate of 0.5 mL / min. Fluorescence signals were detected at Ex and Em, 652 nm and 668 nm, respectively.

[0065] DLS measurements of the size distribution of PEGylated antibodies were performed using a Zetasizer Nano ZS90 (Malvern Instruments Ltd., Worcestershire, UK) in phosphate-buffered saline (10 mM, pH 7.4) at 25 °C.

[0066] Fluorescent labeling of antibodies. Fluorescently labeled antibodies were prepared for antibody recovery measurements from PEGylated antibodies (i.e., recovery to free antibody after PEG removal; see Figure 1). The antibodies were dissolved in a pH 8.0, 0.1 M NaHCO3 buffer solution. Alexa-Fluor 647 or Alexa-Fluor 488 succinimidyl ester (Invitrogen, USA) (10 mg / mL in DMSO; 10 antibody equivalents) was then added to the antibody solution and stirred at 4 °C for 4 hours. The samples were purified using a Vivaspin 6 (MWCO = 10,000) (x4, 0.01 M, pH = 7.4 PBS). The concentrations of labeled Alexa-Fluor 647 or Alexa-Fluor 488 were determined based on a standard calibration curve of serially diluted free Alexa-Fluor 647. The molar ratios between labeled Alexa-Fluor 647 or Alexa-Fluor 488 and antibody were calculated to be 1.31 and 1.27, respectively, by comparing the dye and antibody concentrations.

[0067] In vitro recovery of PEGylated antibodies. The PEGylated antibody was incubated with 2 mM glutathione (GSH), which mimics the reducing conditions in the brain, at 37°C for 4 hours. As shown in Figure 1, the PEGylated moiety is released from the PEGylated antibody in the reducing environment, and the antibody is thought to return to its pre-modification state (antibody recovery step). The solution was then purified with Vivaspin 6 (three times, cutoff MW 30,000 Da, 10 mM, pH 7.4 phosphate-buffered saline buffer). The recovered antibody was confirmed by GPC using the same method as above. Anti-Aβ 1-40The biological activity of the recovered antibodies was evaluated using anti-NPCP. MCF-7 cells were seeded onto eight-chamber cover glasses and incubated in 500 μL of Dulbecco's modified Eagle's medium (DMEM) (10% FBS) for 18 hours. The MCF-7 cells were then fixed with 4% paraformaldehyde for 15 minutes and then treated with 0.2% Triton X-100 for 5 minutes to disrupt the membrane. The cells were then treated with 1% BSA solution for 10 minutes to prevent nonspecific recognition, and then incubated with Alexa-488-labeled native anti-NPCP, PEGylated anti-NPCP, and recovered anti-NPCP, each with the same concentration of anti-NPCP (2 μg / mL; 15 nM) in 1% BSA solution for 1 hour. All samples were observed, and images were taken using a confocal laser scanning microscope (Zeiss, Germany) after nuclear staining with DAPI. Anti-Aβ antibodies were then detected. 1-40 The biological activity of Aβ was assessed using the ThT assay. 1-40 was evaluated by its effect on the aggregation of 29 .

[0068] Thioflavin T (ThT) assay. Aβ 1-40 Anti-Aβ effects on aggregation 1-40 The effect of was analyzed by ThT assay. 29 Briefly, Aβ dissolved in hexafluoroisopropanol 1-40 The mixture was air-dried and then dissolved in 50 mM Tris-HCl buffer (pH 7.4) at a constant concentration of 20 μM. Next, various molar ratios of anti-Aβ 1-40 :Aβ 1-40 Free native anti-Aβ (1:50, 1:10, 1:2.5, 1:1 and 0:1) 1-40 , PEGylated anti-Aβ 1-40 and reducing agent (GSH)-treated anti-Aβ 1-40 Aβ 1-40 Then, 10 μM ThT was added to the solution, and Aβ was measured at room temperature using an Infinite M1000 PRO spectrophotometer at an excitation wavelength of 440 nm and an emission wavelength of 480 nm. 1-40 Aggregation was monitored.

[0069] Antibody circulation.Mice were intravenously administered 200 μl of 0.15 mg / ml Alexa647-labeled PEGylated antibodies (G0-, G25-, G50-, G75-, and G100-PEGylated anti-BACE1 and free anti-BACE1 in 10 mM phosphate-buffered saline (PBS) at the same concentration). Thirty μl of blood was collected from the mouse vein at various times after injection and concentrated at 15,000 rpm to obtain serum. Serum anti-BACE1 was then quantified by fluorescence measurement using an Infinite M1000 PRO spectrophotometer (Tecan Group Ltd., Mennedorf, Switzerland) after dilution with 10 mM phosphate-buffered saline (pH 7.4).

[0070] Antibody accumulation in in vivo studies. To analyze the distribution of delivered antibodies in vivo, we administered antibodies to fasted mice, following the method described in WO 2015 / 075942A, followed by glucose administration to elevate blood glucose levels and promote antibody translocation to the brain. Specifically, BALB / c mice (6 weeks, n = 5) were fasted for 24 hours. Then, 200 μl of 0.15 mg / ml Alexa647-labeled PEGylated antibodies (G0-, G25-, G50-, G75-, and G100-PEGylated antibodies and free antibodies in 10 mM phosphate-buffered saline) was intravenously administered to the mice. Thirty minutes later, 200 μl of D-PBS(-) containing 20 wt% D-(+)-glucose was intraperitoneally administered. Mice were sacrificed, and excess blood was flushed out by perfusion with D-PBS(-) at various times (12, 24, and 48 h) after antibody injection. Brains were excised, washed with D-PBS(-), weighed, and after removing excess wash solution, homogenized in 600 μL of cell lysis buffer. Blood was collected from the inferior vena cava, heparinized, and centrifuged to obtain plasma. Accumulation of Alexa 647-labeled antibodies was quantified by fluorescence measurement using an Infinite M1000 PRO spectrophotometer (Tecan Group Ltd., Mennedorf, Switzerland).

[0071] Suppression of amyloid-β production in APP / PS1 mice. Brain Aβ 1-40 was collected in the same manner as previously reported 30Six-week-old mice were intravenously injected with antibody (20 mg / kg) and PBS. Mice were then perfused at various time points after injection. 1-40 For Aβ measurement, collected brain samples were homogenized in 5 M guanidine hydrochloride buffer. These samples were rotated at 25°C for 3 hours and then diluted (1:10) with PBS containing freshly added aprotinin (20 mg / ml) and leupeptin (10 mg / ml), 0.25% casein, and 5 mM EDTA (pH 8.0). The diluted homogenate was centrifuged at 14K rpm for 20 minutes to detect Aβ levels. 1-40 The supernatant containing Aβ was isolated and analyzed by sandwich ELISA (Wako, Japan). 1-40 The concentration of was measured.

[0072] Results and Discussion Preparation and characterization of PEGylated antibodies First, bis(NPC-ethyl) disulfide was synthesized from bis(2-hydroxyethyl) disulfide and 4-nitrophenyl chloroformate dissolved in anhydrous DCM. Then, the synthesized bis(NPC-ethyl) disulfide was added to DMF-dissolved DIG-PEG-NH2 and MeO-PEG-NH2 solutions for 12 hours to obtain DIG-PEG-SS-NPC and MeO-PEG-SS-NPC, respectively. DIG is 1 The resulting product was deprotected to give Gluc-PEG-SS-NPC, as identified by H NMR ( Figure S2 ). All of the reaction procedures were described in detail in the Materials and Methods section.

[0073] The degree of modified PEG was quantified by the number of unmodified amines available for coupling with fluorescamine. PEGylated antibody (4 μL, 0.5 mg / mL; 10 mM phosphate buffer) was incubated with fluorescamine (2 μL, 3 mg / mL; DMF) at room temperature for 15 minutes, and the fluorescence signal was measured using an ND-3300 fluorescence spectrometer (Nanodrop, Wilmington, DE, USA). The resulting fluorescence signal was used to estimate the concentration of primary amines based on a standard calibration curve of serially diluted BSA with 30 known primary amines. For example, for anti-BACE1, a total of 80 primary amines in the native antibody was calculated and defined as 100%. First, MeO-PEG-SS-NPC was used to evaluate the effective PEGylation and PEGylated antibody. As shown in Figure 2a, when the PEG:amino molar ratio was set to 5:1, conjugation to amines increased with increasing reaction time, reaching a plateau of 73.2% at 15 hours. By controlling the conjugation time, the degree of primary amine modification in the antibody was determined to be 12.5%, 25.5%, 52.1%, and 73.2%, respectively. These antibodies were named PEG12.5, PEG25.5, PEG52.1, and PEG73.2, respectively. Meanwhile, the degree of amine conjugation increased with increasing amounts of activated PEG molecules (Figure 2b). For example, at a 10:1 PEG / amino molar ratio, the amine modification level reached 76.7% at 6 hours of conjugation, approximately four times higher than at a 1:1 PEG / amino molar ratio (Figure 2b).

[0074] The PEG-conjugated antibodies were then further analyzed by gel permeation chromatography (GPC). Here, PEG25.5-anti-BACE1 was taken as an example. The prepared PEGylated anti-BACE1 had a much earlier elution peak time than the native antibody when measured by GPC, indicating that PEG was successfully conjugated to the antibody (Figure 3a). The size of the antibody also increased for the PEGylated antibody compared to the native antibody. For example, the size increased from 8.57 nm for the native anti-BACE1 to 11.51 nm for the PEGylated anti-BACE1 (PEG25.5-anti-BACE1), in which 25.5% of the amine groups were conjugated with PEG, and it had a narrow particle size distribution (PDI < 0.15) as characterized by dynamic light scattering (DLS) (Figure 3b).

[0075] Restoration of biological activity of PEGylated antibodies treated with reducing agents PEG modification is a widely studied technique for protein modification. However, PEG erosion can significantly affect the biological activity of the modified protein. Here, we demonstrate that PEG, a reversible linker, can restore the conjugated amino group under reducing conditions, allowing the conjugated PEG to be irretrievably removed from the antibody. To perform antibody erosion, PEGylated anti-BACE1 was treated with 2 mM GSH, which creates a reducing environment in solution similar to that found in the brain. As shown in Figure 3a, characterization of anti-BACE1 by GPC revealed that the 2 mM GSH-treated anti-BACE1 exhibited nearly identical flux peak times to the native antibody, indicating that the treated anti-BACE1 successfully removed the conjugated PEG from the antibody and restored its molecular weight to that of the native antibody.

[0076] To confirm the antibody was subjected to appropriate disruption from the conjugated PEG and thus bioactivity was recovered under reducing conditions, and the reductively released anti-Aβ 1-40 (collected and quantified as described above) Aβ was then purified. 1-40 The ability to inhibit Aβ aggregation was assessed (Fig. 3c). 1-40 Aβ 1-40Inhibition of aggregation was first confirmed in vitro by the thioflavin T (ThT) assay. 1-40 and soluble Aβ 1-40 Co-incubation with (6 h in PBS, pH 7.4) resulted in a clear dose-dependent inhibition of aggregation. 1-40 :Aβ 1-40 At a molar ratio of 1:1, anti-Aβ 1-40 Aβ incubated in the absence of 1-40 Even at a low molar ratio (1:50), the anti-Aβ antibody inhibited aggregation by 92.2% (Fig. 3c). 1-40 The PEGylated anti-Aβ antibody still showed a strong inhibitory effect (63.9% inhibition). 1-40 and released anti-Aβ 1-40 The inhibitory activity of PEGylated anti-Aβ 1-40 is the natural anti-Aβ antibody at all concentrations tested. 1-40 The inhibitory effect on Aβ aggregation was found to be much weaker compared to that of the released anti-Aβ (Fig. 3c). 1-40 The inhibitory activity of natural anti-Aβ 1-40 The inhibitory activity of the released anti-Aβ was comparable to that of the 1-40 demonstrates efficient destruction of conjugated PEG and restoration of bioactivity.

[0077] Then, various percentages of PEG-conjugated anti-Aβ were added to the PBS solution while incubating it under reducing conditions (2 mM GSH, 37°C) for 4 hours. 1-40 As shown in Figure 3d, the recovery of the anti-Aβ bioactivity was evaluated. 1-40 :Aβ 1-40 The ratio was set at 1:2.5, and the reducing agent-treated PEG12.5%-anti-Aβ 1-40 and PEG25.5%-anti-Aβ 1-40 For the solution of Aβ 1-40 The aggregation inhibition efficiency was almost the same. However, the PEG52.1-anti-Aβ treated with a reducing agent was significantly higher than that of the PEG52.1-anti-Aβ treated with a reducing agent. 1-40 and PEG73.2-anti-Aβ 1-40 In the solution, Aβ 1-40 The efficiency of inhibiting aggregation was clearly reduced under these reducing conditions, and anti-Aβ 1-40The biological activity of the antibody was not fully restored under reducing conditions, and the PEGylation effect was high. On the other hand, considering that a sufficient number of glucose units on the nanoparticles is necessary for BBB crossing, the following BBB crossing experiments and therapeutic experiments were carried out using PEG 25.5% anti-Aβ. 1-40 The antibody was selected.

[0078] The biological activity of anti-nuclear pore complex protein (anti-NPCP) microrelease antibodies was also evaluated using confocal imaging. Anti-NPCP is an antibody that targets nuclear pore complex proteins, which regulate the transport of biomolecules across the nuclear membrane. 31 While native anti-NPCP IgG can selectively recognize NPCP on fixed MCF-7 cells (Fig. 4a), PEG-modified anti-NPCP IgG (PEG25.5 anti-NPCP IgG) loses this selectivity (Fig. 4b). However, the binding activity of anti-NPCP IgG to NPCP was largely restored after 4 hours of incubation with 2 mM GSH (Fig. 4c).

[0079] Antibody circulation time Next, Gluc-PEG-SS-NPC and MeO-PEG-SS-NPC were mixed in various molar ratios to obtain various glucose modification ratios (ratio of glucose-modified polymer to total polymer): 0%, 25%, 50%, 75%, and 100%. These mixed polymers were then conjugated to anti-BACE1 to obtain PEGylated antibodies with various glucose modification ratios. These were named G0-PEG-anti-BACE1, G25-PEG-anti-BACE1, G50-PEG-anti-BACE1, G75-PEG-anti-BACE1, and G100-PEG-anti-BACE1, respectively, according to their glucose modification ratios. The pharmacokinetic profiles of these Alexa 647-labeled PEGylated antibodies were measured after intravenous administration to mice. We investigated the blood residence time of serum collected from mouse tail veins using an Infinite M1000 PRO spectrophotometer (Tecan Group Ltd., Mennedorf, Switzerland) based on the fluorescence signal. As shown in Figure 5a, all PEGylated anti-BACE1 antibodies exhibited prolonged blood residence times regardless of their glucose modification ratio (i.e., surface glucose density). The half-lives for G0-PEGylated anti-BACE1, G25-PEGylated anti-BACE1, G50-PEGylated anti-BACE1, G75-PEGylated anti-BACE1, and G100-PEGylated anti-BACE1 were 2.34 days, 3.01 days, 3.59 days, and 3.30 days, respectively. In contrast, the native antibody labeled with Alexa-647 exhibited a shorter circulation time, with a half-life of 1.73 days. These results suggest that PEGylated antibody systems significantly improve the blood residence time of antibodies, potentially enhancing their therapeutic efficacy. Furthermore, the blood retention time of the antibody improved as the glucose modification ratio at the end of the PEG increased, but no adverse effects such as a decrease in the blood retention time were observed.

[0080] Accumulation of antibodies in the brain We then assayed antibody accumulation in the brain to verify whether this Gluc-PEG-antibody could sufficiently deliver functional antibody across the BBB to the CNS via the GLUT1 recycling mechanism. Based on our previous report on the glucose-induced GLUT1 recycling mechanism, we fasted mice for 24 hours to localize more GLUT1 on the luminal cell membrane of BCECs. Then, we intraperitoneally injected 20% free glucose to achieve peak blood glucose concentrations within 30 minutes. Alexa647-labeled PEGylated and Alexa647-labeled free antibodies were administered via tail vein injection. At sacrifice 90 minutes after antibody injection, brain tissue was collected, washed, and homogenized. The fluorescence intensity of these samples was then determined using an Infinite M1000 Pro microplate reader (TECAN Ltd.). As shown in Figure 5b, only trace amounts of free antibody accumulated in the brain, at only 0.07% dose / g brain. The non-glucose-modified PEGylated antibody also showed very limited brain accumulation, at only 0.14% dose / g brain, despite its long blood circulation time. However, significant brain accumulation was observed with glucose-modified PEGylated antibodies, with the G50-PEGylated antibody showing the highest accumulation rate of 1.38% dose / g brain, approximately 20-fold higher than that of free antibody and 10-fold higher than that of G0-PEGylated antibodies. These results demonstrate that enhanced antibody accumulation can be achieved through this glucose-modified PEG delivery system and support our previous theory that antibody brain accumulation can be controlled by adjusting the glucose moiety density.

[0081] Reduction of amyloid-β accumulation in Alzheimer's disease (AD) model mice After achieving highly efficient brain accumulation of the delivered anti-BACE1, we attempted to evaluate the effect of administering this reduction-sensitive glucose-modified PEGylated anti-BACE1 on the accumulation of Aβ in an AD mouse model. Generally, Aβ accumulation in the brain of APP / PS1 model mice (8 weeks old) was significantly reduced. 1-40The Aβ expression was quantitatively analyzed by anti-Aβ ELISA (Wako) at various time points: 0, 12, 24, and 48 hours after administration of saline, free native anti-BACE1, and G50PEGylated anti-BACE1 (anti-BACE1 20 mg / kg). Brains were harvested at various times after administration, and anti-Aβ 1-40 Aβ using ELISA 1-40 As shown in Figure 6, saline-treated mice showed significantly higher Aβ production at these time points. 1-40 There was little change in Aβ levels. Mice treated with G50-PEGylated anti-BACE1 and native anti-BACE1 showed a decrease at 12 hours after administration. Mice treated with G50-PEGylated anti-BACE1 showed a 43.2% decrease in Aβ levels at 12 hours after administration. 1-40 The levels of Aβ were significantly lower than those of both saline-treated and free native anti-BACE1-treated animals at all 12-hour measurement points (Fig. 5c). Even 48 hours after administration of G50-PEGylated anti-BACE1, the Aβ levels were still significantly higher. 1-40 A high decrease in values ​​(39.4%) was observed.

[0082] conclusion In this study, we developed a novel reduction-sensitive glucose-modified PEG linker approach for the delivery of therapeutic antibodies across the BBB, taking advantage of GLUT1 recycling induced by glucose administration after hypoglycemia. We achieved significantly higher accumulation of anti-BACE1 antibodies in the brain and subsequently restored their function in the brain. More importantly, the delivered anti-BACE1 antibodies, which subsequently restored their function under the reducing environment in the brain, suppressed Aβ production in Alzheimer's disease model mice. 1-40 Thus, our approach represents a viable strategy for delivering antibodies across the BBB for the treatment of neurodegenerative diseases.

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Claims

1. A peptide modified via a linker with an uncharged hydrophilic polymer segment modified with a GLUT1 ligand so as to be cleavable under a reducing environment or an environment of pH 6.5 or less, wherein the peptide is in a form dissociated from the polymer segment after cleavage, A peptide in which the linker has the structure -CO-OL 1 -S-SL 2 - (wherein L 1 is a substituted or unsubstituted alkylene or a substituted or unsubstituted heteroalkylene, and L 2 is a linker that is stable in the bloodstream), which is connected to the side chain amino group of a lysine residue of the peptide via an amide bond, and L 2 is connected to an uncharged hydrophilic polymer segment, or the linker has the structure -CO-CH=CH-L 3 -CO-NH- (wherein L 3 is a linker moiety that is stable in the blood), which is connected to the side chain amino group of a lysine residue of the peptide via an amide bond, and -NH- is connected to an uncharged hydrophilic polymer segment.

2. The peptide of claim 1 , wherein the GLUT1 ligand is glucose.

3. An antibody comprising a peptide portion described in claim 1 or 2.

4. The antibody of claim 3, which binds to a cell surface antigen or an extracellular antigen in the brain parenchyma.

5. The extracellular antigen is Aβ １－４０ The antibody of claim 4,

6. The antibody of any one of claims 3 to 5, further linked to a cytotoxic agent.

7. The peptide of claim 1 or 2, or the antibody of any one of claims 3 to 5, further linked to an imaging agent selected from the group consisting of a fluorescent dye, a radioisotope, and a contrast agent.

8. A pharmaceutical composition comprising a peptide described in any one of claims 1 or 2, or an antibody described in any one of claims 3 to 6.

9. A composition for use in brain imaging, comprising the antibody described in claim 7.

10. 10. The composition of claim 8 or 9, administered according to the following dosing regimen: The dosing regimen is lowering the blood glucose of a subject, and then Inducing an increase in blood glucose levels in the subject so that more peptides are translocated to the brain parenchyma than when the increase in blood glucose levels is not induced, and administering the composition of claim 11 or 12 to the subject. composition.

11. an antibody (modified antibody) modified via a linker with an uncharged hydrophilic polymer segment modified with a GLUT1 ligand so as to be cleavable from the segment in a reducing environment or an environment of pH 6.5 or less, the antibody having an antigen-binding ability of 10% or less compared to an unmodified antibody; An antibody having a linker of -CO-O-L 1 -S-S-L 2 - (wherein L 1 is a substituted or unsubstituted alkylene or substituted or unsubstituted heteroalkylene, and L 2 is a linker that is stable in the blood stream), which is linked to the side chain amino group of a lysine residue of the antibody via an amide bond, and L 2 is linked to the uncharged hydrophilic polymer segment; or a linker of -CO-CH=CH-L 3 -CO-NH- (wherein L 3 is a linker moiety that is stable in the blood), which is linked to the side chain amino group of a lysine residue of the antibody via an amide bond, and -NH- is linked to the uncharged hydrophilic polymer segment.

12. The antibody of claim 11, which has antigen-binding ability of 1% or less or is eliminated compared to an unmodified antibody.

13. The antibody according to claim 11, wherein the binding activity of the modified antibody is restored to 50% or more of that of the unmodified antibody under a reducing environment or an environment of pH 6.5 or less.

14. The antibody according to claim 12, wherein the binding activity of the modified antibody is restored to 50% or more of that of the unmodified antibody under a reducing environment or an environment of pH 6.5 or less.

15. The antibody according to claim 11, wherein the binding activity of the modified antibody is restored to 80% or more of that of the unmodified antibody under a reducing environment or an environment of pH 6.5 or less.

16. The antibody according to claim 12, wherein the binding activity of the modified antibody is restored to 80% or more of that of the unmodified antibody under a reducing environment or an environment of pH 6.5 or less.

17. A composition comprising an antibody of any one of claims 11 to 16.

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