Antigen-binding molecules with increased brain penetration and persistence in the brain, and methods for using same

Abstract

The present invention relates to antigen-binding molecules with increased brain permeability and / or persistence in the brain; methods for using the same; and methods for producing or screening for the same.

Description

[Technical Field]

[0001] The present invention relates to antigen-binding molecules with increased brain penetration and / or persistence in the brain, methods for using the same, and methods for producing or screening for the same. The present disclosure relates to antigen-binding molecules with increased brain concentration, exposure, and / or persistence, methods for improving the concentration, exposure, and / or persistence of antigen-binding molecules in the brain, and methods for producing and screening antigen-binding molecules with increased brain concentration, exposure, and / or persistence. In one aspect, the present disclosure relates to an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain (crossing the blood-brain barrier), and the second antigen-binding domain specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain extracellular matrix (ECM) protein or brain ECM polysaccharide. The present disclosure further relates to an antigen-binding molecule wherein the first target is a molecule expressed on vascular endothelial cells of the blood-brain barrier (BBB). The present disclosure further relates to methods for producing antigen-binding molecules, pharmaceutical compositions comprising the antigen-binding molecules, one or more nucleic acids encoding the antigen-binding molecules, one or more vectors comprising the one or more nucleic acids, host cells (i) comprising the one or more nucleic acids or (ii) comprising the one or more vectors, methods for producing and screening for antigen-binding molecules, methods for increasing the concentration of an antigen-binding molecule in the brain of a subject in need thereof, methods for increasing the exposure of an antigen-binding molecule in the brain of a subject in need thereof, and methods for the persistence of an antigen-binding molecule in the brain of a subject in need thereof, as well as methods and medical uses for treating a subject. [Background technology]

[0002] Although biologics such as antibodies offer the advantages of high specificity, efficacy, and reduced off-target toxicity, delivering these drugs to the brain remains the greatest challenge in the development of therapeutics for treating diseases of the central nervous system. The primary barrier is the blood-brain barrier (BBB), which prevents the entry of most molecules present in the systemic circulation, especially large drug molecules. The BBB makes it difficult to achieve effective concentrations when drugs are administered to the central nervous system, complicating drug development.

[0003] Various delivery technologies have been investigated to increase the brain concentration of macromolecular substances, such as biologics. Most of the reported technologies use receptor-mediated transcytosis (RMT), and the target receptors expressed in the vascular endothelium of the brain include the transferrin receptor, insulin receptor, insulin-like growth factor receptor (IGFR), and low-density lipoprotein receptor family (LDLRf). For example, a technology for crossing the BBB via the transferrin receptor has been reported, which involves creating a fusion protein of an anti-transferrin receptor antibody and nerve growth factor (NGF). Other techniques using anti-transferrin receptor antibodies include bispecific antibodies of anti-transferrin receptor antibodies and anti-β-secretase (BACE1) antibodies, and other bispecific antibodies (WO 2016 / 081640 (Patent Document 1), WO 2015 / 191934 (Patent Document 2), WO 2016 / 081643 (Patent Document 3)), and fusion antibodies obtained by fusing a monovalent anti-transferrin receptor antibody to the carboxyl terminal of an anti-amyloid β antibody (WO 2014 / 0330 (Patent Document 4)). No. 74 (Patent Document 4) has also been reported. However, transferrin receptors and insulin receptors are not only expressed in vascular endothelial cells in the brain, but are also widely expressed throughout the body and in tissues other than the brain, and therefore drugs are delivered to tissues other than the brain. Because the antigens are expressed throughout the body, the half-life of the antibodies in the blood is short. As a result, BBB-crossing techniques, such as fusion of anti-transferrin receptor-binding antibodies or anti-insulin receptor antibodies, have the disadvantage of rapid clearance from the systemic circulation (Sci Transl Med. 2013;5:183ra57. pmid:23636093 (Non-Patent Document 1)). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2016 / 081640 [Patent Document 2] International Publication No. 2015 / 191934 [Patent Document 3] International Publication No. 2016 / 081643 [Patent Document 4] International Publication No. 2014 / 033074 [Non-patent literature]

[0005] [Non-Patent Document 1] Sci Transl Med. 2013;5: 183ra57. pmid:23636093 Summary of the Invention [Problem to be solved by the invention]

[0006] There is a continuing need to provide compounds, compositions, methods and uses that are useful in the context of brain disease or brain disorders.One of the objectives of the present disclosure is to provide, but is not limited to, compounds, compositions, methods and uses that are useful in the context of brain disease or brain disorders. [Means for solving the problem]

[0007] As a means for solving the problems, one aspect of the present disclosure provides an antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, and a method of using the antigen-binding molecule, wherein the first antigen-binding domain specifically binds to a first target that promotes translocation of the antigen-binding molecule into a mammalian brain, and the second antigen-binding domain specifically binds to a second target, which is (i) expressed on the cell membrane of a brain cell, or (ii) a brain extracellular matrix (ECM) protein or brain ECM polysaccharide.

[0008] Throughout this disclosure, various aspects of the invention are provided. However, it should be understood that each disclosed aspect is merely an exemplary embodiment of the invention and is not intended to strictly limit the invention to that embodiment. Rather, the present invention is intended to cover alternatives, modifications, and equivalents that may be included within the spirit and scope of the present disclosure.

[0009] [1] In a first aspect, an antigen-binding molecule is provided comprising a first antigen-binding domain and a second antigen-binding domain, wherein: The first antigen-binding domain specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; and The second antigen-binding domain specifically binds to a second target, which is (i) expressed on the cell membrane of a brain cell or (ii) a brain extracellular matrix (ECM) protein or brain ECM polysaccharide. Additionally, the following aspects are provided: [2] The antigen-binding molecule according to [1], wherein the first target is a molecule expressed on vascular endothelial cells of the blood-brain barrier (BBB). [2A] The antigen-binding molecule of [1] or [2], wherein the first target promotes migration of the antigen-binding molecule across the BBB into the mammalian brain. [3] The antigen-binding molecule of [1], [2], or [2A], wherein the first target is selected from the group consisting of transferrin receptor (TfR), basigin (CD147), Glut1, Ldlrad3, CD320, insulin receptor, insulin-like growth factor 1 receptor (IGF1R), low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor-related protein (LRP), preferably LRP1, diphtheria toxin receptor, glucose receptor, CD98hc, TMEM30A, leptin receptor (LepR), and heparan sulfate chains branched from proteoglycans (HSPGs). [4] The second target is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5), IGSF4B / SynCAM3 / cell adhesion molecule 3 (CADM3), CNPase (2',3'-cyclic nucleotide 3'-phosphodiesterase), myelin-associated glycoprotein (MAG), myelin basic protein (MBP), EAAT1 (solute transporter family 1 member 3), EAAT2 (solute transporter family 1 member 2), MAP2 (microtubule-associated protein 2), and NEFL. (neurofilament light polypeptide), NEFM (neurofilament medium polypeptide), NSE (gamma-enolase), CD68 (macrosialin), allograft inflammatory factor 1 (IBA1 or AIF1), purinergic receptor (P2RY12), interleukin-1 receptor accessory protein-like 1 (IL1RAPL1), glutamate ionotropic receptor NMDA type subunit 2B (GRIN2B), voltage-gated calcium channel auxiliary subunit gamma 8 (CACNG8), CD11b (integrin subunit alpha M), SLC6A2 (sodium-dependent noradrenaline transporter), DPP6 (dipeptidyl peptidase-like 6), SLC18A3 (vesicular acetylcholine transporter), sodium / potassium transporting ATPase subunit α-2, broad substrate specificity ATP-binding cassette transporter ABCG2, solute transporter family 12 member 9, electrogenic sodium bicarbonate cotransporter 1, excitatory amino acid transporter 2, chondroitin sulfate proteoglycan 4, immunoglobulin superfamily DCC subunit class member 4, Vang-like protein 2, neural cell adhesion molecule 1 (N-CAM-1), low-density lipoprotein receptor-related protein 4 (LRP-4), glycosphingolipid-enriched microdomain-associated phosphoprotein 1 (Csk-binding protein), plasma membrane calcium-transporting ATPase 1, prominin-1, somatostatin receptor type 1, brain isoform of carnitine O-palmitoyltransferase 1 (CPT1-B), epidermal growth factor receptor, protein MAL2, syntaxin-1A, sodium / calcium exchanger 1,Lysophosphatidylcholine acyltransferase 1 (LPC acyltransferase 1), calsyntenin-3 (alcadein-β), type I receptor for pituitary adenylate cyclase-activating polypeptide (type I receptor for PACAP), neutral cholesterol ester hydrolase 1 (NCEH), CD166 antigen (activated leukocyte cell adhesion molecule), inactive tyrosine protein kinase 7, claudin-11, ectonucleotide phosphatase (ENPP6), tetraspanin-2 (Tspan-2), myelin proteolipid protein (PLP), glycolipid transfer protein (GLTP), versican core protein (chondroitin sulfate proteoglycan 2 or CSPG2), troponin The antigen-binding molecule according to any one of [1] to [3], wherein the antigen-binding molecule is selected from the group consisting of elastin (elastin), collagen α-2(IV) chain (canstatin), proteoglycan link protein 1 (hyaluronan and proteoglycan link protein 1), tenascin-R (TN-R), proteoglycan link protein 2 (hyaluronan and proteoglycan link protein 2), collagen α-1(I) chain, neurofilament-3 (NEF3), immunoglobulin superfamily member 8 (IgSF8), laminin subunit γ-1 (LAMC1), collagen α-1(VI) chain (Col6a1), and collagen α-3(VI) chain (Col6a3). [5] The antigen-binding molecule according to any one of [1] to [4], wherein the brain cells comprise one or more types of cells selected from the group consisting of oligodendrocytes, astrocytes, neurons, and microglia. [6] The antigen-binding molecule according to any one of [1] to [5], wherein the second target is a molecule that is primarily expressed on brain-specific cells. [6A] The antigen-binding molecule of any one of [1] to [6], wherein the second target is a molecule that is widely expressed in brain tissue. [6B] The antigen-binding molecule of [6A], wherein the second target that is widely expressed in brain tissue is a molecule expressed in more than one, more than two, or more than three types of brain tissue selected from the group consisting of the cerebral cortex, hippocampus, amygdala, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, medulla oblongata, and spinal cord. [6C] The antigen-binding molecule of [6B], wherein the second target is a molecule selected from the group consisting of interleukin-1 receptor accessory protein-like 1 (IL1RAPL1), glutamate ionotropic receptor NMDA-type subunit 2B (GRIN2B), and voltage-dependent calcium channel auxiliary subunit gamma 8 (CACNG8). [6D] The antigen-binding molecule of any one of [1] to [4], wherein the second target is a brain ECM protein. [6E] The antigen-binding molecule of [6D], wherein the second target is selected from the group consisting of versican core protein (chondroitin sulfate proteoglycan 2 or CSPG2), tropoelastin (elastin), collagen alpha-2(IV) chain (canstatin), proteoglycan link protein 1 (hyaluronan and proteoglycan link protein 1), tenascin-R (TN-R), proteoglycan link protein 2 (hyaluronan and proteoglycan link protein 2), collagen alpha-1(I) chain, neurofilament-3 (NEF3), immunoglobulin superfamily member 8 (IgSF8), laminin subunit gamma-1 (LAMC1), collagen alpha-1(VI) chain (Col6a1), and collagen alpha-3(VI) chain (Col6a3). [6F] The antigen-binding molecule of any one of [1] to [5], wherein the second target is expressed only in the cell membrane of brain-specific cells. [6G] The antigen-binding molecule of any one of [1] to [5], wherein the second target is expressed only in brain tissue. [6H] The antigen-binding molecule according to any one of [1] to [3], wherein the second target is a brain ECM polysaccharide. [7] The antigen-binding molecule of any one of [1] to [6H], wherein the first target is selected from the group consisting of transferrin receptor (TfR), basigin (CD147), insulin receptor, insulin-like growth factor 1 receptor (IGF1R), low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor-related protein (LRP), preferably LRP1, diphtheria toxin receptor, glucose receptor, preferably Glut1, and CD98hc. [7A] The antigen-binding molecule of any one of [1] to [7], wherein the first target is transferrin receptor (TfR), and optionally the antigen-binding domain that binds to TfR comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 29 and / or a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 30, or a variant thereof having one or more conservative amino acid substitutions. [7B] The antigen-binding molecule of any one of [1] to [7], wherein the first target is basigin (CD147), and optionally the antigen-binding domain that binds to basigin comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 31 and / or a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 32, or a variant thereof having one or more conservative amino acid substitutions. [7C] The antigen-binding molecule of any one of [1] to [7], wherein the first target is an insulin receptor. [7D] The antigen-binding molecule of any one of [1] to [7], wherein the first target is low-density lipoprotein receptor (LDLR). [7E] The antigen-binding molecule of any one of [1] to [7], wherein the first target is low-density lipoprotein receptor-related protein (LRP), preferably LRP1. [7F] The antigen-binding molecule of any one of [1] to [7], wherein the first target is a diphtheria toxin receptor. [7G] The antigen-binding molecule of any one of [1] to [7], wherein the first target is a glucose receptor, preferably Glut1. [7H] The antigen-binding molecule of any one of [1] to [7], wherein the first target is CD98hc. [7I] The antigen-binding molecule of any one of [1] to [7], wherein the first target is insulin-like growth factor 1 receptor (IGF1R). [8] The antigen-binding molecule of any one of [1] to [7I], wherein the second target is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5), and cell adhesion molecule 3 (CADM3). [8A] The antigen-binding molecule of any one of [1] to [8], wherein the second target is MOG, and optionally the antigen-binding domain that binds to MOG comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 15 or 34 and / or a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 17 or 35, or a variant thereof having one or more conservative amino acid substitutions. [8B] The antigen-binding molecule according to [8A], which has increased persistence in central nervous tissues, such as the optic nerve, spinal cord, olfactory bulb, and medulla oblongata. [8C] The antigen-binding molecule of any one of [1] to [8], wherein the second target is CSPG5, and optionally the antigen-binding domain that binds to CSPG5 comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 36 or 56 and / or a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 37 or 57, or a variant thereof having one or more conservative amino acid substitutions. [8D] The antigen-binding molecule of any one of [1] to [8], wherein the second target is CADM3, and optionally the antigen-binding domain that binds to CADM3 comprises a heavy chain variable region (VH) having the amino acid sequence shown in SEQ ID NO: 38 and / or a light chain variable region (VL) having the amino acid sequence shown in SEQ ID NO: 39, or a variant thereof having one or more conservative amino acid substitutions. [9] (i) the first target is transferrin receptor (TfR) and the second target is myelin oligodendrocyte glycoprotein (MOG); or (ii) the first target is transferrin receptor (TfR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (iii) the first target is transferrin receptor (TfR) and the second target is cell adhesion molecule 3 (CADM3); or (iv) the first target is basigin (CD147) and the second target is myelin oligodendrocyte glycoprotein (MOG); or (v) the first target is basigin (CD147) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (vi) the first target is basigin (CD147) and the second target is cell adhesion molecule 3 (CADM3); or (vii) the first target is an insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); or (viii) the first target is the insulin receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (ix) the first target is an insulin receptor and the second target is cell adhesion molecule 3 (CADM3); or (x) the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xi) the first target is a glucose receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xii) the first target is a glucose receptor and the second target is cell adhesion molecule 3 (CADM3); or (xiii) the first target is low-density lipoprotein receptor (LDLR) and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xiv) the first target is low-density lipoprotein receptor (LDLR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xv) the first target is low density lipoprotein receptor (LDLR) and the second target is cell adhesion molecule 3 (CADM3); or (xvi) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xvii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xviii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is cell adhesion molecule 3 (CADM3); or (xix) the first target is CD98hc and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xx) the first target is CD98hc and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xxi) the first target is CD98hc and the second target is cell adhesion molecule 3 (CADM3); The antigen-binding molecule according to any one of [1] to [8C].

[10] The second target is selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, TRK A, TRK B, and TRK The antigen-binding molecule of any one of [1] to [4], which is not a target selected from the group consisting of C, α-synuclein, β-synuclein, γ-synuclein, vascular endothelial growth factor (VEGF), neuropilin, semaphorin, semaphorin 3A, semaphorin 4A, semaphorin 6A, myelin basic protein (MBP), MOG, PLP, MAG, aquaporin 4, glutamate receptor, and EpCAM.

[11] (i) at least one functional moiety, including an enzyme, a therapeutic protein, an antibody or antigen-binding fragment thereof, a peptide, DNA, shRNA, siRNA, a small molecule drug, or a cytotoxic agent; and / or (ii) at least one in vivo half-life extending moiety, preferably selected from the group consisting of an Fc region, an albumin binding domain, an FcRn binding protein, an FcRn binding peptide, and a PEG moiety; The antigen-binding molecule according to any one of [1] to

[10] , further comprising: [11A] The antigen-binding molecule of [1], wherein at least one functional moiety is selected from the group consisting of neprilysin, an anti-sortilin 1 antigen-binding domain, an anti-BACE1 antigen-binding domain, and acid α-glucosidase (GAA).

[12] In a second aspect, there is provided a method for increasing the concentration of an antigen-binding molecule in the brain of a subject in need thereof, the method comprising: so that the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) (a1) a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; or (a2) A second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide. providing a first antigen-binding molecule; (b) In the case of (a1), introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule; or In the case of (a2), introducing at least one first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. Includes. Additionally, the following aspects are provided:

[13] Step (c): (c) determining that the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to a control antigen-binding molecule, wherein the control antigen-binding molecule is: In the case of (a1), at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide; or In the case of (a2), at least one first antigen-binding domain that specifically binds to a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain. (b) a second antigen-binding molecule, which differs from the second antigen-binding molecule described in (b) only in that it does not contain The method according to

[12] , further comprising:

[14] The method of

[12] or

[13] , wherein the concentration of the second antigen-binding molecule in the subject's brain is Cmax.

[15] In a third aspect, there is provided a method for increasing exposure of an antigen-binding molecule in the brain of a subject in need thereof, the method comprising: so that the exposure amount of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) (a1) a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; or (a2) a second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain extracellular matrix (ECM) protein or brain ECM polysaccharide. providing a first antigen-binding molecule; (b) In the case of (a1), introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule; or In the case of (a2), introducing at least one first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. Includes. Additionally, the following aspects are provided:

[16] Step (c): (c) determining that the exposure of the second antigen-binding molecule in the brain of the subject is increased compared to a control antigen-binding molecule, wherein the control antigen-binding molecule is In the case of (a1), at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide; or In the case of (a2), at least one first antigen-binding domain that specifically binds to a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain. (b) a second antigen-binding molecule, which differs from the second antigen-binding molecule described in (b) only in that it does not contain The method according to

[15] , further comprising:

[17] The method according to

[15] or

[16] , wherein the exposure amount of the second antigen-binding molecule in the subject's brain is the AUC (area under the curve) of the brain concentration-time profile of the antigen-binding molecule.

[18] In a fourth aspect, there is provided a method for persistence of an antigen-binding molecule in the brain of a subject in need thereof, the method comprising: such that the persistence of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) providing a first antigen-binding molecule comprising a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the brain of a mammal; (b) introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. Includes. Additionally, the following aspects are provided:

[19] Step (c): (c) determining that the persistence of the second antigen-binding molecule in the brain of the subject is increased compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the second antigen-binding molecule of (b) only in that it does not contain at least one antigen-binding domain that specifically binds to a second target. The method according to

[18] , further comprising:

[20] The method according to

[18] or

[19] , wherein the persistence of the second antigen-binding molecule in the subject's brain is its in vivo half-life in the brain. [20A] The method of

[20] , wherein the in vivo half-life of the second antigen-binding molecule in the brain is at least 10 days, 12 days, 15 days, 20 days, 30 days, 45 days, 60 days, or 90 days. [20B] The method of [20A], wherein the second antigen-binding molecule has an in vivo half-life in the brain of at least 10 days. [20C] The method of [20A], wherein the in vivo half-life of the second antigen-binding molecule in the brain is at least 30 days. [20D] The method of [20A], wherein the in vivo half-life of the second antigen-binding molecule in the brain is at least 60 days.

[21] The antigen-binding molecule of any one of [1] to

[11] or the method of any one of

[12] to [20D], wherein the mammalian brain is a human brain.

[22] The method according to any one of

[12] to

[21] , wherein the first target is as specified in any one of [2] to [3] and [7] to [7I].

[23] The method of any one of

[12] to

[22] , wherein the second target is as specified in any one of [4], [5], [6] to [6H], [8] to [8C], and

[10] .

[24] The method according to any one of

[12] to

[23] , wherein the first target and the second target are as specified in any one of [2] to

[11] .

[25] (i) the first target is the transferrin receptor (TfR) and the second target is myelin oligodendrocyte glycoprotein (MOG); (ii) the first target is transferrin receptor (TfR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (iii) the first target is transferrin receptor (TfR) and the second target is cell adhesion molecule 3 (CADM3); (iv) the first target is basigin (CD147) and the second target is myelin oligodendrocyte glycoprotein (MOG); (v) the first target is basigin (CD147) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (vi) the first target is basigin (CD147) and the second target is cell adhesion molecule 3 (CADM3); (vii) the first target is the insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); (viii) the first target is the insulin receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (ix) the first target is the insulin receptor and the second target is cell adhesion molecule 3 (CADM3); (x) the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); (xi) the first target is a glucose receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (xii) the first target is a glucose receptor and the second target is cell adhesion molecule 3 (CADM3); (xiii) the first target is low-density lipoprotein receptor (LDLR) and the second target is myelin oligodendrocyte glycoprotein (MOG); (xiv) the first target is low-density lipoprotein receptor (LDLR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xv) the first target is low-density lipoprotein receptor (LDLR) and the second target is cell adhesion molecule 3 (CADM3); (xvi) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is myelin oligodendrocyte glycoprotein (MOG); (xvii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xviii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is cell adhesion molecule 3 (CADM3); or (xix) the first target is CD98hc and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xx) the first target is CD98hc and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xxi) The first target is CD98hc and the second target is cell adhesion molecule 3 (CADM3); The method according to any one of

[12] to

[24] .

[26] The method according to any one of

[12] to

[25] , wherein the second antigen-binding molecule is the antigen-binding molecule according to any one of [1] to

[11] . [26A] The method according to any one of

[15] to

[25] , wherein, when the second target is MOG, the second antigen-binding molecule has increased persistence / concentration / exposure in central nervous tissues, such as the optic nerve, spinal cord, olfactory bulb, and medulla oblongata, compared to a control antigen-binding molecule.

[27] In a fifth aspect, there is provided a pharmaceutical composition comprising the antigen-binding molecule according to any one of [1] to

[11] and one or more pharmaceutically acceptable carriers or excipients. Additionally, the following aspects are provided:

[28] An antigen-binding molecule or pharmaceutical composition according to any one of [1] to

[11] , or

[27] , for (i) use in a method for increasing the concentration of an antigen-binding molecule in the brain of a subject in need thereof; (ii) use in a method for increasing the exposure of an antigen-binding molecule in the brain of a subject in need thereof; and / or (iii) use for maintaining the antigen-binding molecule in the brain of a subject in need thereof, wherein the subject is optionally a human.

[29] In a sixth aspect, there is provided one or more nucleic acids encoding the antigen-binding molecule of any one of [1] to

[11] , wherein the one or more nucleic acids are optionally operably linked to a promoter.

[30] In a seventh aspect, one or more vectors comprising one or more nucleic acids according to

[29] .

[31] In an eighth aspect, there is provided a host cell that (i) comprises one or more types of nucleic acids according to

[29] ; (ii) comprises one or more types of vectors according to

[30] ; and / or (iii) is capable of expressing an antigen-binding molecule according to any one of [1] to

[11] .

[32] In a ninth aspect, there is provided a method for producing the antigen-binding molecule according to any one of [1] to

[11] , the method comprising the step of culturing the host cell according to

[31] so as to produce the antigen-binding molecule; and optionally, further comprising the step of recovering the antigen-binding molecule from the host cell. Additionally, the following aspects are provided:

[33] The antigen-binding molecule of any one of [1] to

[11] and

[21] , wherein the first antigen-binding domain is an antibody variable region comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL). [33A] The antigen-binding molecule according to any one of [1] to

[11] and

[21] , wherein the first antigen-binding domain is Fab, Fab', F(ab')2, diabody, triabody, scFab, Fv, scFv, or a single-domain antibody (VHH), or a non-antibody binder (e.g., affibody, DARPin, FN3, aptamer, anticalin, etc.).

[34] The antigen-binding molecule according to any one of [1] to

[11] ,

[21] ,

[33] , and [33A], wherein the second antigen-binding domain is an antibody variable region comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL). [34A] The antigen-binding molecule according to any one of [1] to

[11] ,

[21] ,

[33] , and [33A], wherein the second antigen-binding domain is Fab, Fab', F(ab')2, diabody, triabody, scFab, Fv, scFv, or a single-domain antibody (VHH), or a non-antibody binder (e.g., affibody, DARPin, FN3, aptamer, anticalin, etc.).

[35] The antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to [34A], wherein the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of antibody variable regions including an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), and Fab, Fab', F(ab')2, diabody, triabody, scFab, Fv, scFv, or single-domain antibody (VHH).

[36] The antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[35] , which comprises one, two, three, or four first antigen-binding domains and one, two, three, or four second antigen-binding domains.

[37] The antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[36] , comprising one, two, three, or four first antigen-binding domains, one, two, three, or four second antigen-binding domains, and at least one Fc region.

[38] The antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[37] , wherein the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of antibody variable regions including an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), Fab, Fab', F(ab')2, diabodies, triabodies, scFab, Fv, scFv, or single-domain antibodies (VHH), or non-antibody binders (e.g., affibodies, DARPins, FN3, aptamers, anticalins, etc.).

[38] The antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[37] , which is a bispecific antibody.

[39] The antigen-binding molecule of

[38] , comprising one first antigen-binding domain and one second antigen-binding domain.

[40] The antigen-binding molecule according to

[38] or

[39] , wherein one first antigen-binding domain and one second antigen-binding domain are linked to an Fc region. [40A] The antigen-binding molecule according to

[36] or

[40] , wherein the Fc region has a reduced ability to bind to an activating Fcγ receptor compared to the ability of the Fc region of native human IgG to bind to an activating Fcγ receptor. [40B] The antigen-binding molecule of [40A], wherein the activating Fcγ receptor is human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), or human FcγRIIIa(F). [40C] The Fc region contains the following amino acid substitutions (all positions are according to EU numbering): Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, or Trp at position 234; Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, or Arg at position 235; Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, or Tyr at position 236; Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, or Arg at position 237; Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, or Arg at position 238; Gln, His, Lys, Phe, Pro, Trp, Tyr, or Arg at position 239; Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val at position 265; Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, or Tyr at position 266; Arg, His, Lys, Phe, Pro, Trp, or Tyr at position 267; Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 269; Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 270; Arg, His, Phe, Ser, Thr, Trp, or Tyr at position 271; Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, or Tyr at position 295; Arg, Gly, Lys, or Pro at position 296; Ala at 297th position; Arg, Gly, Lys, Pro, Trp, or Tyr at position 298; Arg, Lys, or Pro at position 300; Lys or Pro at position 324; Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, or Val at position 325; Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 327; Arg, Asn, Gly, His, Lys, or Pro at position 328; Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, or Arg at position 329; Pro or Ser at position 330; Arg, Gly, or Lys at position 331; Arg, Lys, or Pro at position 332 The antigen-binding molecule according to [40A] or [40B], comprising one or more of:

[41] The antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[40] , wherein at least one functional moiety is an antibody or an antigen-binding fragment thereof that specifically binds to a membrane protein of (i) an immune cell, particularly an immune cell selected from the group consisting of a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, a NK cell, a NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil cell, and a basophil, (ii) a tumor cell, or (iii) an autoreactive cell.

[42] The antigen-binding molecule according to

[41] , wherein the antibody or antigen-binding fragment thereof specifically binds to a membrane protein selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a costimulatory molecule, and a costimulatory molecule.

[43] In a tenth aspect, there is provided a method for producing an antigen-binding molecule, the method comprising the steps of: (a) selecting a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; (b) selecting a second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide; (c) obtaining one or more nucleic acids encoding an antigen-binding molecule to which the antigen-binding domain prepared in (a) and the antigen-binding domain prepared in (b) are linked; and (d) Producing an antigen-binding molecule using one or more nucleic acids prepared in (c).

[44] In an eleventh aspect, there is provided a method for screening an antigen-binding molecule, the method comprising the steps of: (a) selecting a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; (b) selecting a second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide; (c) obtaining one or more nucleic acids encoding an antigen-binding molecule to which the antigen-binding domain prepared in (a) and the antigen-binding domain prepared in (b) are linked; and (d) Producing an antigen-binding molecule using one or more nucleic acids prepared in (c). Additionally, the following aspects are provided:

[45] Step (e): (e) determining whether the antigen-binding molecule of (d) has increased (i) persistence, (ii) concentration, or (iii) exposure in the subject's brain compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule of (d) only in that it contains either one of the antigen-binding domains defined in (a) and (b), but not both. The method according to

[43] or

[44] , further comprising: [45A] The method of any one of

[43] to

[45] , wherein when the second target is MOG, the increase in (i) persistence, (ii) concentration, or (iii) exposure of the second antigen-binding molecule in the brain is an increase in (i) persistence, (ii) concentration, or (iii) exposure in central nervous tissues, such as the optic nerve, spinal cord, olfactory bulb, and medulla oblongata, compared to a control antigen-binding molecule.

[46] In a twelfth aspect, there is provided a pharmaceutical composition comprising: (i) the antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[42] ; (ii) one or more nucleic acids encoding the antigen-binding molecule according to

[29] ; (iii) one or more vectors according to

[30] ; or (iv) the host cell according to

[31] , together with one or more pharmaceutically acceptable carriers or excipients.

[47] In a thirteenth aspect, the present specification provides (i) an antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[42] , (ii) one or more nucleic acids encoding the antigen-binding molecule according to

[29] , (iii) one or more vectors according to

[30] , (iv) a host cell according to

[31] , or (iv) a pharmaceutical composition according to

[27] or

[46] , for use as a pharmaceutical.

[48] In a fourteenth aspect, the present specification provides (i) an antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[42] , (ii) one or more nucleic acids encoding the antigen-binding molecule according to

[29] , (iii) one or more vectors according to

[30] , (iv) a host cell according to

[31] , or (iv) a pharmaceutical composition according to

[27] or

[46] , for use as a diagnostic agent.

[49] In a fifteenth aspect, provided herein is (i) an antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[42] , (ii) one or more nucleic acids encoding the antigen-binding molecule according to

[29] , (iii) one or more vectors according to

[30] , (iv) a host cell according to

[31] , or (iv) a pharmaceutical composition according to

[27] or

[46] , for use in treating and / or preventing a brain disorder or brain disease in a subject.

[50] In a sixteenth aspect, provided herein is a method for treating and / or preventing a brain disorder or brain disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of: (i) the antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[42] ; (ii) one or more nucleic acids encoding the antigen-binding molecule according to

[29] ; (iii) one or more vectors according to

[30] ; (iv) the host cell according to

[31] ; or (iv) the pharmaceutical composition according to

[27] or

[46] .

[51] In a seventeenth aspect, the present specification provides (i) an antigen-binding molecule according to any one of [1] to

[11] ,

[21] , and

[33] to

[42] , (ii) one or more nucleic acids encoding the antigen-binding molecule according to

[29] , (iii) one or more vectors according to

[30] , (iv) a host cell according to

[31] , or (iv) a pharmaceutical composition according to

[27] or

[46] , for use in preparing a medicament for treating and / or preventing a brain disorder or brain disease.

[52] In an eighteenth aspect, provided herein is use of (i) the antigen-binding molecule of any one of [1] to

[11] ,

[21] , and

[33] to

[42] , (ii) one or more nucleic acids encoding the antigen-binding molecule of

[29] , (iii) one or more vectors of

[30] , (iv) the host cell of

[31] , or (iv) the pharmaceutical composition of

[27] or

[46] , for the preparation of a medicament for the treatment and / or prevention of a brain disorder or brain disease.

[53] The brain disorder or disease is a neurodegenerative disorder (including, but not limited to, Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and nervous system heterogeneous degenerative disorders (Cavana disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexandria syndrome, a pharmaceutical composition / antigen-binding molecule / nucleic acid / vector for use as described in

[49] or

[51] , a method for treatment and / or prevention as described in

[50] , or a use as described in

[52] , wherein the pharmaceutical composition / antigen-binding molecule / nucleic acid / vector for use as described in

[49] or

[51] is selected from the group consisting of: dementia (including but not limited to Pick's disease and spinocerebellar ataxia), psychiatric disorders, cancer (e.g., cancer of the CNS, including metastases to the brain arising from cancer elsewhere in the body), including but not limited to Darr syndrome, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including but not limited to Pick's disease and spinocerebellar ataxia), psychiatric disorders, cancer (e.g., cancer of the CNS, including metastases to the brain arising from cancer elsewhere in the body),

[54] The pharmaceutical composition / antigen-binding molecule / nucleic acid / vector for use according to

[49] or

[51] , the method for treatment and / or prevention according to

[50] , or the use according to

[52] , wherein the brain disorder or brain disease is selected from the group consisting of Alzheimer's disease, Pompe disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS).

[0010] Unless expressly specified otherwise or contradicted by context, any term expressed in the singular herein may be intended to include the plural as well, and any term expressed in the plural herein may be intended to include the singular as well. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of that element is present, unless the context clearly requires that one and only one such element is present. Thus, the indefinite article "a" or "an" typically means "at least one." [Brief explanation of the drawings]

[0011] [Figure 1A] FIG. 1 is a schematic diagram showing a molecule having a brain-localizing moiety (a first antigen-binding domain that specifically binds to a first target that promotes the transport of the antigen-binding molecule into the mammalian brain; for example, an anti-transferrin receptor antibody). [Figure 1B] Schematic diagram showing a molecule having a brain retention / targeting moiety (a second antigen-binding domain that specifically binds to a second target that is (i) expressed on the cell membrane of a brain cell or (ii) a brain ECM protein or brain ECM polysaccharide; e.g., an anti-MOG antibody). [Figure 1C] Schematic diagram showing a molecule having a brain-localizing moiety (a first antigen-binding domain that specifically binds to a first target that promotes the transport of the antigen-binding molecule into the mammalian brain; e.g., an anti-transferrin receptor domain) and a brain-persisting moiety (a second antigen-binding domain that specifically binds to a second target that is (i) expressed on the cell membrane of a brain cell or (ii) a brain ECM protein or brain ECM polysaccharide; e.g., an anti-MOG domain). [Figure 2a] A schematic diagram illustrating the concept of an embodiment of an antigen-binding molecule of the present disclosure is provided in (a), which comprises (1) a brain-translocating moiety, (2) a brain-persistent moiety, and (3) a functional moiety, and optionally further comprises a half-life extending moiety. [Figure 2b] Schematic diagram showing exemplary molecular configurations of antigen-binding molecules of this aspect and embodiment. In one example, the antigen-binding molecule comprises a first Fab region (i.e., brain-translocating moiety) that binds to a molecule that promotes penetration or migration of the antigen-binding molecule into the brain, and a second Fab region (i.e., brain-persisting moiety) that binds to a molecule that is specifically expressed on brain-specific cells or is a brain ECM protein or brain ECM polysaccharide, and optionally further comprises a functional moiety. The structures of the brain-persisting moiety and brain-translocating moiety are not limited to the Fab region and may be in the form of an antibody fragment such as scFab (single-chain Fab), Fv, Fab, Fab', F(ab')2, diabody, triabody, scFv, VHH, diabody, or F(ab')2 fragment. In other exemplary embodiments, the variable domain is linked to the Fc region such that there is no F(ab')2 fragment. Several such embodiments are shown in Figures 2(b) and 2(c). [Figure 2c] Schematic diagram showing exemplary molecular architectures of antigen-binding molecules according to this aspect and embodiment. See also brief description of Figure 2b. [Figure 3a] Figure 3a shows the pharmacokinetic and brain concentration-time profiles of KLH, MOG303, KLH / / TfR, and MOG303 / / TfR in the brain. KLH, MOG303, KLH / / TfR, and MOG303 / / TfR were administered intravenously at a dose of 2 mg / kg, and brains were harvested after perfusion. Antibody concentrations in the brain were measured by ECL. Figure 3a shows antibody concentrations in the brain. Data represent the mean + / - SD (n = 6). [Figure 3b] Figure 3b shows the pharmacokinetic and concentration-time profiles of KLH, MOG303, KLH / / TfR, and MOG303 / / TfR in the brain. KLH, MOG303, KLH / / TfR, and MOG303 / / TfR were administered intravenously at a dose of 2 mg / kg, and brains were harvested after perfusion. Antibody concentrations in the brain were measured by ECL. Figure 3b shows the ratio of antibody concentrations in the brain to those in plasma. Data represent the mean + / - SD (n = 6). [Figure 3c] Figure 3c shows the pharmacokinetic and brain concentration-time profiles of KLH, MOG303, KLH / / TfR, and MOG303 / / TfR in the brain. KLH, MOG303, KLH / / TfR, and MOG303 / / TfR were administered intravenously at a dose of 2 mg / kg, and brains were harvested after perfusion. Antibody concentrations in the brain were measured by ECL. Figure 3c shows the percent injected dose per weight of brain tissue (%ID / g brain). Data represent the mean + / - SD (n = 6). [Figure 3d] Figure 3d shows the pharmacokinetic and brain concentration-time profiles of KLH, MOG303, KLH / / TfR, and MOG303 / / TfR in the brain. KLH, MOG303, KLH / / TfR, and MOG303 / / TfR were administered intravenously at a dose of 2 mg / kg, and brains were harvested after perfusion. Antibody concentrations in the brain were measured by ECL. Figure 3d shows the cumulative AUC in the brain up to day 28. Data represent the mean + / - SD (n = 6). [Figure 4] Figure 1 shows the plasma concentration-time profiles of antibodies. KLH, MOG303, KLH / / TfR, and MOG303 / / TfR were administered intravenously to mice at a dose of 2 mg / kg, and blood was collected. Plasma antibody concentrations were measured by ECL. Data represent the mean + / - SD (n = 6). [Figure 5] Figure 1 shows the plasma concentration-time profiles of antibodies KLH, mTfR / / KLH, mBsg / / KLH, KLH / / MOG303, KLH / / MOG307, KLH / / CADM3, mTfR / / MOG303, mTfR / / MOG307, mTfR / / CADM3, mBsg / / MOG303, and mBsg / / MOG307. Antibodies were administered intravenously at a dose of 2 mg / kg, and blood was collected. Plasma antibody concentrations were measured by ECL. Data represent the mean + / - SD (n = 3-4). [Figure 6]Figure 1 shows the pharmacokinetic and brain concentration-time profiles of antibodies KLH, mTfR / / KLH, mBsg / / KLH, KLH / / MOG303, KLH / / MOG307, KLH / / CADM3, mTfR / / MOG303, mTfR / / MOG307, mTfR / / CADM3, mBsg / / MOG303, and mBsg / / MOG307 in the brain. Antibodies were administered intravenously at a dose of 2 mg / kg, and brains were harvested after perfusion. Antibody concentrations in the brain were measured by ECL. (a) Antibody concentrations in the brain. (b) Ratio of antibody concentration in the brain to that in plasma. (c) Percentage of injected dose per weight of brain tissue (%ID / g brain). Data represent the mean ± SD (n = 3–4). [Figure 7] Figure 1 shows plasma concentration-time profiles of antibodies KLH, mBsg / / KLH, mBsg / / MOG303, mBsg / / CSPG5.2, and mBsg / / CADM3. Antibodies were administered intravenously at a dose of 2 mg / kg, and blood was collected. Plasma antibody concentrations were measured by ECL. Data represent the mean + / - SD (n = 4). [Figure 8] Figure 1 shows the pharmacokinetic and brain concentration-time profiles of antibodies KLH, mBsg / / KLH, mBsg / / MOG303, mBsg / / CSPG5.2, and mBsg / / CADM3 in the brain. Antibodies were administered intravenously at a dose of 2 mg / kg, and brains were harvested after perfusion. Antibody concentrations in the brain were measured by ECL. (a) Antibody concentration in the brain. (b) Ratio of antibody concentration in the brain to that in plasma. (c) Percentage of injected dose per weight of brain tissue (%ID / g brain). Data represent the mean + / - SD (n = 4). [Figure 9] Figure 1 shows the plasma concentration-time profiles of antibodies KLH, CSPG5120, and CSPG5120-BS. Antibodies were administered intravenously at a dose of 2 mg / kg, and blood was collected. Plasma antibody concentrations were measured by ECL. Data represent the mean + / - SD (n = 3). [Figure 10]Figure 1 shows the pharmacokinetic and brain concentration-time profiles of antibodies KLH, CSPG5120, and CSPG5120-BS in the brain. Antibodies were administered intravenously at a dose of 2 mg / kg, and brains were harvested after perfusion. Antibody concentrations in the brain were measured by ECL. (a) Antibody concentration in the brain. (b) Ratio of antibody concentration in the brain to that in plasma. (c) Percentage of injected dose per weight of brain tissue (%ID / g brain). Data represent the mean + / - SD (n = 3). [Figure 11]11A shows the schematic structure of the antibodies MOG303 / / TfR and CSPG5120-BS exemplified in Example 2 and Example 4B, respectively, which comprise a first Fab region that binds to a molecule that promotes penetration or migration of the antigen-binding molecule into the brain (i.e., a brain-translocating moiety, such as a TfR-binding domain or a basigin-binding domain), and a second and third Fab region that bind to a molecule that is specifically expressed on brain-specific cells or is a brain ECM protein or brain ECM polysaccharide (i.e., a brain-persisting moiety, such as an MOG-binding domain or a CSPG5-binding domain), and optionally further comprise a functional moiety (not shown). Figure 11B shows the schematic structures of bispecific antibodies (f) to (m) exemplified in Example 4A, which comprise a first Fab region that binds to a molecule that promotes penetration or migration of the antigen-binding molecule into the brain (i.e., a brain-localizing moiety, such as a TfR-binding domain or a basigin-binding domain), and a second Fab region that binds to a molecule that is specifically expressed on brain-specific cells or is a brain ECM protein or brain ECM polysaccharide (i.e., a brain-persisting moiety, such as an MOG-binding domain, a CSPG5-binding domain, a CADM3-binding domain, etc.), and optionally further comprises a functional moiety (not shown). Figures 11C to 11F show additional exemplary molecular configurations of antigen-binding molecules that further comprise one or more functional moieties. The structure of the brain-persistent moiety and the brain-translocating moiety is not limited to the Fab region, but can also be in the form of an antibody fragment, such as, for example, scFab (single-chain Fab), Fv, Fab, Fab', F(ab')2, diabody, triabody, scFv, VHH, diabody, F(ab')2 fragment, or a non-antibody binder (e.g., affibody, DARPin, FN3, aptamer, anticalin).The functional moiety can be any molecule with a therapeutic function, such as, for example, an agonist, antagonist, enzyme, modulator, stabilizer, cell death inducer, etc., and can be any molecular construct, such as, for example, a nucleic acid, a small molecule, a cyclic peptide, a peptide, a ligand, a cytokine, a chemokine, a growth factor, an enzyme, and an antigen-binding domain, etc. In some examples, the functional moiety is selected from the group consisting of neprilysin, an anti-sortilin 1 antigen-binding domain, an anti-BACE1 antigen-binding domain, and acid alpha-glucosidase (GAA). [Figure 12]12A shows the schematic structure of IGF1R / / MOG303 and TfRVNAR.CloneC / / MOG303, which are antibodies exemplified in Example 8, each of which comprises a VHH (i.e., a brain-transmigrating moiety, such as a TfR-binding domain or an IGF1R-binding domain) that binds to a molecule that promotes penetration or migration of the antigen-binding molecule into the brain, and a Fab region (i.e., a brain-persisting moiety, such as an MOG-binding domain) that binds to a molecule that is specifically expressed on brain-specific cells or is a brain ECM protein or brain ECM polysaccharide, and optionally further comprises a functional moiety (not shown in the figure). Figure 12B shows the schematic structure of IL6R / / MOG303-TfR-GAA or IL6R / / MOG303-TfR-shortGAA exemplified in Example 9A, which comprises an scFab (i.e., a brain-translocating moiety, such as a TfR-binding domain) that binds to a molecule that promotes penetration or migration of the antigen-binding molecule into the brain, a Fab (i.e., a brain-persisting moiety, such as an MOG-binding domain) that binds to a molecule that is specifically expressed on brain-specific cells or is a brain ECM protein or brain ECM polysaccharide, and one or more functional moieties (e.g., GAA or NEP), and optionally a VHH that binds to another antigen. Figure 12C shows the schematic structure of MOG303-TfR-2GAA or MOG303-TfR-2shortGAA exemplified in Example 9A, which comprises two Fabs (i.e., brain persistence moieties, such as MOG-binding domains) that each bind to a molecule that is specifically expressed on brain-specific cells or is a brain ECM protein or brain ECM polysaccharide, one scFab (i.e., a brain-localizing moiety, such as a TfR-binding domain) that binds to a molecule that promotes penetration or migration of the antigen-binding molecule into the brain, and one or more (e.g., two) functional moieties (e.g., GAA) linked to the C-terminus of each of the two light chains of the Fabs that are the brain persistence moieties.Figure 12D shows the schematic structure of BACE1 / / MOG303-TfR exemplified in Example 9C, which comprises an scFab (i.e., a brain-translocating moiety, such as a TfR-binding domain) that binds to a molecule that promotes penetration or migration of the antigen-binding molecule into the brain, a Fab (i.e., a brain-persisting moiety, such as a MOG-binding domain) that binds to a molecule that is specifically expressed on brain-specific cells or is a brain ECM protein or brain ECM polysaccharide, and a functional moiety (e.g., a BACE1-binding domain), which is a Fab that binds to another biological target. The structures of the brain-persistent moiety and the brain-translocating moiety are not limited to Fab, VHH, or scFab, but can also be in the form of an antibody fragment, such as scFab (single-chain Fab), Fv, Fab, Fab', F(ab')2, diabody, triabody, scFv, VHH, diabody, F(ab')2 fragment, or non-antibody binder (e.g., affibody, DARPin, FN3, aptamer, anticalin). The functional moiety can be any molecule with therapeutic function, such as, for example, an agonist, antagonist, enzyme, modulator, stabilizer, cell death inducer, and can be any molecular configuration, such as, for example, a nucleic acid, a small molecule, a cyclic peptide, a peptide, a ligand, a cytokine, a chemokine, a growth factor, an enzyme, and an antigen-binding domain. In some instances, the functional moiety is selected from the group consisting of neprilysin, an anti-sortilin 1 antigen binding domain, an anti-BACE1 antigen binding domain, and acid alpha-glucosidase (GAA). [Figure 13]Schematic diagrams showing further exemplary molecular configurations of antigen-binding molecules of this aspect and embodiment, as exemplified in Example 9D. Figure 13A shows the schematic structure of a bivalent anti-Sort1 antibody (Sort1). Figure 13B shows the schematic structure of Sort1-TfR, which contains two Fabs that each bind to Sort1 and one scFab that binds to TfR. Figure 13C shows the schematic structure of Sort1-TfR-MOG303(L), which comprises two Fabs (functional moieties), each of which binds to a biological target, such as Sort1; one scFab (i.e., a brain-localizing moiety, such as a TfR-binding domain) that binds to a molecule that promotes penetration or translocation of the antigen-binding molecule into the brain; and two scFvs (i.e., brain-persisting moieties, such as anti-MOG scFvs) that each bind to a molecule that is specifically expressed on brain-specific cells or is a brain ECM protein or brain ECM polysaccharide, wherein each of the two brain-persisting moieties (anti-MOG scFvs) is linked to the C-terminus of the L chain of the anti-SORT1 Fab. Figure 13D shows the schematic structure of Sort1-TfR-MOG303(H), which comprises two Fabs (functional moieties), each of which binds to a biological target, such as Sort1; one scFab (i.e., a brain-localizing moiety, such as a TfR-binding domain) that binds to a molecule that promotes penetration or translocation of the antigen-binding molecule into the brain; and two scFvs (i.e., brain-persisting moieties, such as anti-MOG scFvs) that each bind to a molecule that is specifically expressed on brain-specific cells or is a brain ECM protein or brain ECM polysaccharide, wherein each of the two brain-persisting moieties (anti-MOG scFvs) is linked to the C-terminus of the heavy chain of the anti-SORT1 antibody. [Figure 14] The figure shows tissue concentrations of the antibodies KLH, MOG303, KLH / / TfR, and MOG303 / / TfR tested in Example 10. Antibodies were administered intravenously at a dose of 10 mg / kg, and plasma, brain, liver, muscle, spleen, and lung were collected. Antibody concentrations in these tissues were measured by ECL. Data represent the mean + / - SD (n = 3-4). [Figure 15] The figure shows the tissue concentrations of the antibodies KLH, MOG303, KLH / / TfR, and MOG303 / / TfR tested in Example 10. Antibodies were administered intravenously at a dose of 10 mg / kg, and the optic nerve, spinal cord, olfactory bulb, retina, and medulla oblongata were harvested. Antibody concentrations in these tissues were measured by ECL. Data represent the mean + / - SD (n = 3-4). [Figure 16] The figure shows plasma antibody concentrations of (a) IGF1R / / KLH, IGF1R / / MOG303, (b) TfRVNAR.CloneC / / KLH, TfRVNAR.CloneC / / MOG303, and control antibodies. Antibodies were administered intravenously at a dose of 2 mg / kg, and blood was collected. Plasma antibody concentrations were measured by ECL. Data represent the mean + / - SD (n = 3). [Figure 17] The figure shows antibody concentrations in the brain for (a) IGF1R / / KLH, IGF1R / / MOG303, (b) TfRVNAR.CloneC / / KLH, TfRVNAR.CloneC / / MOG303, and control antibodies. Antibodies were administered intravenously at a dose of 2 mg / kg, and brains were harvested after perfusion. Antibody concentrations in the brain were measured by ECL. Data represent the mean + / - SD (n = 3). [Figure 18]Figure 18(a) shows the plasma concentrations of anti-Sortilin 1 antibodies (Sort1, Sort1-TfR, Sort1-TfR-MOG303(L), and Sort1-TfR-MOG303(H)) and a negative control antibody (KLH). Antibodies were administered intravenously at a dose of 200 nmol / kg, and blood was collected. Antibody concentrations in plasma were measured by ECL. Data represent the mean + / - SD (n = 5). Figure 18b shows the brain concentrations of anti-Sortilin 1 antibodies (Sort1, Sort1-TfR, Sort1-TfR-MOG303(L), and Sort1-TfR-MOG303(H)) and a control antibody (KLH). Antibodies were administered intravenously at a dose of 200 nmol / kg, and brains were collected after perfusion. Antibody concentrations in the brain were measured by ECL. Data represent mean + / - SD (n = 4–5). [Figure 19] Figure 19(a) shows the plasma concentration-time profiles of antibodies KLH, IL6R / / KLH-NEP, IL6R / / KLH-TfR-NEP, and IL6R / / MOG303-TfR-NEP, which were administered intravenously to mice at a dose of 50 nmol / kg and blood samples were collected. Plasma antibody concentrations were measured by electron chromatin spectroscopy (ECL). Data represent the mean ± SD (n = 6). Figure 19(b) shows the brain concentration-time profiles of antibodies KLH, IL6R / / KLH-NEP, IL6R / / KLH-TfR-NEP, and IL6R / / MOG303-TfR-NEP, which were administered intravenously to mice at a dose of 50 nmol / kg and brains were collected after perfusion. Brain antibody concentrations were measured by ECL. Data represent the mean ± SD (n = 5-6). [Figure 20]Figure 20 shows a comparison of amyloid-β1-40 peptide concentrations in the brains of mice that were intravenously administered KLH, IL6R / / KLH-NEP, IL6R / / KLH-TfR-NEP, and IL6R / / MOG303-TfR-NEP at a dose of 50 nmol / kg and then harvested after perfusion. Amyloid-β1-40 peptide concentrations in the brain were measured by ECL. Data represent the mean ± SD (n = 5-6). [Figure 21] Figure 21(a) shows the plasma concentration-time profiles of antibodies KLH, BACE1 / / KLH-TfR, and BACE1 / / MOG303-TfR, which were administered intravenously at a dose of 25 mg / kg and blood samples were collected. Plasma antibody concentrations were measured by ECL. Data represent the mean + / - SD (n = 6). Figure 21(b) shows the brain concentration-time profiles of antibodies KLH, BACE1 / / KLH-TfR, and BACE1 / / MOG303-TfR, which were administered intravenously at a dose of 25 mg / kg and brains were collected after perfusion. Brain antibody concentrations were measured by ELISA. Data represent the mean + / - SD (n = 6). DETAILED DESCRIPTION OF THE INVENTION

[0012] To facilitate understanding of the invention exemplified herein, the following definitions and detailed descriptions are provided, which are particularly helpful in interpreting aspects and embodiments of the present disclosure.

[0013] definition "Affinity" refers to the overall strength of non-covalent interactions between a binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., between an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd). Affinity can be measured by common methods known in the art.

[0014] The terms "anti-[X] antibody," "an antibody that binds [X]," and "an antibody that binds to [X]" refer to an antibody that is capable of binding to target [X] with sufficient affinity such that the antibody is useful as a diagnostic, prophylactic, and / or therapeutic agent in targeting target [X], a given target of interest. In one embodiment, the extent to which an anti-[X] antibody binds to unrelated proteins other than [X] is less than about 10% of the antibody's binding to [X], as measured, for example, by radioimmunoassay (RIA). In certain embodiments, an antibody that binds to [X] has an affinity of 1 μM or less, 100 nM or less, 10 nM or less, 1 nM or less, 0.1 nM or less, 0.01 nM or less, or 0.001 nM or less (e.g., 10 -8 M or less, for example, 10 -8 M~10 -13 M, for example 10 -9 M~10 -13 In certain embodiments, the anti-[X] antibody binds to an epitope of [X] that is conserved among [X] from different species.

[0015] The term "antibody" as used herein is used in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity.

[0016] "Antibody fragment" refers to a molecule that is not an intact antibody and that contains a portion of an intact antibody and binds to the same antigen as the intact antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (such as scFv); and multispecific antibodies formed from antibody fragments.

[0017] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from one particular source or species, while the remainder of the heavy and / or light chain is derived from another source or species.

[0018] The "class" of an antibody refers to the type of constant domain or constant region possessed by its heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy-chain constant domains that correspond to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0019] The term "cytotoxic agent," as used herein, refers to a substance that inhibits or prevents the function of cells and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioisotopes (e.g., 211 At, 131 I, 125 I, 90 Y, 186 Re,188 Re, 153 Sm, 212 Bi, 32 P, 212 Pb, and radioactive isotopes of Lu, etc.); chemotherapeutic agents or drugs (e.g., methotrexate, adriamycin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin, or other intercalating agents, etc.); growth inhibitory agents; enzymes and fragments thereof, such as nucleolytic enzymes; antibiotics; toxins, such as small molecule toxins or enzymatically active toxins, including fragments and / or variants, of bacterial, fungal, plant, or animal origin; and various anti-tumor or anti-cancer agents disclosed below.

[0020] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (such as B cell receptors); and B cell activation.

[0021] An "effective amount" of an agent, eg, a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic or prophylactic result.

[0022] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. The term includes native-sequence Fc regions and variant Fc regions. In one embodiment, the Fc region of a human IgG heavy chain extends from Cys226 or Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat et al., "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0023] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein to refer to an antibody having a structure substantially similar to a native antibody structure or having a heavy chain that includes an Fc region as defined herein.

[0024] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived from the primary cell, regardless of the number of transfers. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are also included herein.

[0025] A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human or human cell, or derived from a non-human source that utilizes the human antibody repertoire or other sequences encoding human antibodies. This definition of a human antibody specifically excludes humanized antibodies, which contain non-human antigen-binding residues.

[0026] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and human FRs. In certain embodiments, a humanized antibody comprises substantially all of at least one, typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has been humanized.

[0027] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents.

[0028] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domestic animals (e.g., cows, sheep, cats, dogs, and horses), primates (e.g., humans and non-human primates, such as monkeys), rabbits, and rodents (e.g., mice and rats). In certain embodiments, the individual or subject is a human.

[0029] An "isolated" antibody is an antibody that has been separated from components of its natural environment. In some embodiments, the antibody is purified to greater than 95% or 99% purity, as determined, for example, by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, etc.) or chromatography (e.g., ion-exchange HPLC or reverse-phase HPLC, etc.). For a review of methods for assessing antibody purity, see, for example, Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0030] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. Isolated nucleic acid includes a nucleic acid molecule that is contained in a cell that normally contains the nucleic acid molecule, but in which the nucleic acid molecule is present extrachromosomally or in a chromosomal location that is different from its natural chromosomal location.

[0031] An "isolated nucleic acid encoding an anti-[X] antibody" refers to one or more nucleic acid molecules encoding the antibody heavy and light chains (or fragments thereof), including nucleic acid molecules in a single vector or separate vectors, and nucleic acid molecules present in one or more locations in a host cell.

[0032] The term "monoclonal antibody," as used herein, refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., a population of antibodies in which the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variant antibodies, which may, for example, include natural mutations or arise during the preparation of monoclonal antibodies, and such variants are generally present in small amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be made by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci; such methods, or other exemplary methods for making monoclonal antibodies, are described herein.

[0033] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (such as a cytotoxic moiety) or a radiolabel. The naked antibody may be present in a pharmaceutical formulation.

[0034] "Native antibodies" refer to naturally occurring immunoglobulin molecules with various structures. For example, native IgG antibodies are heterotetrameric glycoproteins of approximately 150,000 daltons, composed of two identical light chains and two identical heavy chains that are disulfide-bonded. Each heavy chain has, from N- to C-terminus, a variable region (VH), also called a variable heavy domain or heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, each light chain has, from N- to C-terminus, a variable region (VL), also called a variable light domain or light chain variable domain, followed by a light chain constant (CL) domain. The light chain of an antibody can be classified into one of two types, called kappa and lambda, based on the amino acid sequence of its constant domain.

[0035] The term "package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products, which contain information about the indications, uses, dosages, administration, concomitant therapies, contraindications, and / or warnings associated with the use of such therapeutic products.

[0036] "Percent (%) amino acid sequence identity" to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and without considering any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be achieved in a variety of ways within the skill of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software, or GENETYX® (Genetyx Co., Ltd.). Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences to be compared.

[0037] ALIGN-2, a computer program for sequence comparison, was written by Genentech, Inc., and its source code, along with user documentation, has been filed with the U.S. Copyright Office, Washington, DC 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or it may be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not change. In situations where ALIGN-2 is used to compare amino acid sequences, the percent amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (which can alternatively be expressed as "given amino acid sequence A having or containing a certain percent amino acid sequence identity to given amino acid sequence B") is calculated as follows: Fraction X / Y multiplied by 100 where X is the number of amino acid residues in an alignment of A and B by the sequence alignment program ALIGN-2 that are scored as identical matches by the program, and where Y is the total number of amino acid residues in B. It is understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, then the % amino acid sequence identity of A to B may not be equal to the % amino acid sequence identity of B to A. Unless specifically specified otherwise, all % amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program, as described in the preceding paragraph.

[0038] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient contained therein to be effective, and that does not contain additional components that have unacceptable toxicity to the subject to which the formulation may be administered.

[0039] A "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier or excipient" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0040] The term "[X]" referring to a particular target, as used herein, refers to any natural target [X] from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise specified. The term encompasses not only "full-length," unprocessed [X], but also any form of [X] resulting from processing in cells. The term also encompasses naturally occurring variants of [X], such as splice variants or allelic variants.

[0041] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, slowing the rate of disease progression, reversal or temporary palliation of the disease state, and remission or improved prognosis. In some embodiments, the antibodies of the invention are used to delay the onset of disease or slow the progression of the disease.

[0042] The term "variable region" or "variable domain" refers to the domain of an antibody's heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three hypervariable regions (HVR) (see, for example, Kindt et al. "Kuby Immunology," 6th ed., WH Freeman and Co., p. 91 (2007)). One VH domain or one VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated by screening a library of complementary VL or VH domains, respectively, using the VH or VL domain derived from an antibody that binds to the antigen. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0043] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes not only a vector as a self-replicating nucleic acid structure, but also a vector that is integrated into the genome of a host cell into which it is introduced. Certain vectors are capable of causing the expression of a nucleic acid that is operably linked thereto. Such vectors are referred to herein as "expression vectors."

[0044] It is possible to combine / to combine / to combine In the context of the present invention, the terms "bind," "bind," "capable of binding," and "binding to" are used interchangeably and refer to the ability of a moiety to bind to an antigen, particularly under physiological conditions, i.e., conditions found in the body of an animal, particularly a human. Detailed exemplary methods for determining said ability are described herein below. As used herein, "ability to bind to an antigen" and similar terms can also mean "binding affinity for an antigen" and similar terms. Furthermore, in the case of antibodies, said ability can also be expressed by using the term "antibody directed against" or by using the term "antibody against antigen X."

[0045] Specific binding / specific binding The terms "specific binding," "specifically binds," and the like refer to the ability of an antigen-binding domain to bind preferentially to its respective target compared to other molecules or other moieties. In certain embodiments, an antigen-binding domain binds to a target with higher affinity, higher avidity, more readily, and / or with longer duration than the antigen-binding domain binds to another target. Generally, but not necessarily, reference to specific binding refers to preferential binding such that the affinity of the antigen-binding domain for the target is at least 2-fold higher, at least 3-fold higher, at least 4-fold higher, at least 5-fold higher, at least 6-fold higher, at least 7-fold higher, at least 8-fold higher, at least 9-fold higher, at least 10-fold higher, at least 20-fold higher, at least 30-fold higher, at least 40-fold higher, at least 50-fold higher, at least 60-fold higher, at least 70-fold higher, at least 80-fold higher, at least 90-fold higher, at least 100-fold higher, or at least 1000-fold higher than the affinity of the antigen-binding domain for another target.

[0046] Compositions and Methods In one aspect, the present invention provides antigen-binding molecules with improved blood-brain barrier permeability and brain persistence. Provided are antigen-binding molecules comprising a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain, and the second antigen-binding domain specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain extracellular matrix protein or polysaccharide. The antigen-binding molecules of the present invention also provide pharmaceutical compositions and related methods, uses, nucleic acids, vectors, host cells, and kits. The antigen-binding molecules of the present invention exhibit improved pharmacokinetic properties. The antigen-binding molecules of the present invention exhibit increased concentration, exposure, and / or persistence in the brain. The antigen-binding molecules of the present invention are useful, for example, for the diagnosis or treatment of brain disorders or diseases. In some examples, the brain disorder or disease is selected from the group consisting of: neurodegenerative diseases (including but not limited to: Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including but not limited to Alzheimer's disease and supranuclear palsy), prion diseases (including but not limited to bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), Bulbar palsies, motor neuron diseases, and heterogeneous degenerative disorders of the nervous system (including but not limited to Canavan disease, Huntington's disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome), dementia (including but not limited to Pick's disease and spinocerebellar ataxia), psychiatric disorders, cancer (e.g., cancer of the CNS, including metastases to the brain arising from cancer elsewhere in the body).In some specific examples, the brain disorder or disease is selected from the group consisting of Alzheimer's disease, Pompe disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS).

[0047] a first antigen-binding domain that specifically binds to a first target; In the present invention, the "first antigen-binding domain" of the "antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain" binds to a "first target," which is referred to as "the first target promoting the translocation of the antigen-binding molecule into the mammalian brain."

[0048] A target that "promotes the translocation of an antigen-binding molecule into the brain of a mammal" is understood in the broadest sense as a target that causes an increase in the translocation of a compound comprising an antigen-binding domain that specifically binds to the target to the brain of a mammal in vivo, compared to a compound that does not comprise an antigen-binding domain that specifically binds to the target. In certain embodiments, the increase is determined by determining a greater amount or a higher concentration of a compound comprising an antigen-binding domain that specifically binds to the target. Generally, but not necessarily, a reference to increased translocation refers to an amount or concentration of a compound comprising an antigen-binding domain in the mammalian brain in vivo that is at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, at least 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70-fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, or at least 1000-fold greater than the amount or concentration of a compound not comprising an antigen-binding domain that specifically binds to a target. Translocation into the mammalian brain may be from any tissue, organ, or cell type other than the brain, and includes, for example, blood (particularly circulating blood), cerebrospinal fluid, skin, saliva, skin and its substructures, eyes and its substructures, and ears and its substructures. In some embodiments, translocation is from circulating blood to the brain. The "primary target" may have any structure, as long as it facilitates its entry into the mammalian brain. For example, the "primary target" may be a protein, carbohydrate, proteoglycan, lipid, nucleic acid, etc. The "primary target" may be present in various cell types and organs in a mammal and is not limited to any specific occurrence. In some embodiments, the "primary target" is present near or on the surface of one or more cells of the blood-brain barrier (BBB), but is not limited thereto.For example, a "first target" includes targets located extracellularly, such as, for example, proteins, proteoglycans, or carbohydrates of the extracellular matrix, and also includes targets present on the cell surface of a cell, such as, for example, cell surface proteins.

[0049] The terms "blood-brain barrier" and "BBB" are used interchangeably herein and refer to a selective barrier separating circulating blood from the brain. The blood-brain barrier comprises a monolayer of endothelial cells connected by tight junction proteins that form the lumen of small brain vessels. The endothelial cells of the blood-brain barrier are referred to herein as "blood-brain barrier vascular endothelial cells" or "blood-brain barrier microvascular endothelial cells" or "BMECs." In addition, astrocytes, particularly their processes called astrocyte foot processes, and pericytes contribute to the structure and function of the blood-brain barrier, and these cells and their substructures are also included as part of the blood-brain barrier. The blood-brain barrier controls the entry of all peripheral circulating factors, such as the diffusion of water, some gases, and lipid-soluble molecules, and controls the selective transport of other substances, such as glucose, amino acids, and micronutrients important for neurological function. Conversely, the blood-brain barrier protects the brain from the passage of toxic substances that could endanger the central nervous system (CNS).

[0050] The term "brain" is understood as the organ that serves as the center of the nervous system in all vertebrates and most invertebrates.

[0051] The term "mammalian brain" is understood as the brain of a mammal, and includes, but is not limited to, for example, the brain of a human. In some embodiments herein, the mammalian brain is a human brain.

[0052] The term "brain cell" is understood in its broadest sense to encompass any cell present in a mammalian brain, including, but not limited to, oligodendrocytes, astrocytes, neurons, and microglia.

[0053] The term "brain-specific cells" is understood in the broadest sense to encompass any cell that is present in the mammalian brain but is absent or essentially absent in other mammalian organs or tissues. The term "brain-specific cells" includes, but is not limited to, oligodendrocytes, astrocytes, neurons, and microglia. Cells that are essentially absent in other mammalian organs or tissues are those that are found at least 5 times, 10 times, 100 times, or more times more frequently in the brain than in any other mammalian organ or tissue.

[0054] The term "mammal" includes all mammals, including, but not limited to, humans, monkeys, horses, cows, sheep, dogs, cats, cattle, rats, and mice. In some embodiments, the mammal is a human.

[0055] In certain embodiments, the "first target" is selected from the group consisting of transferrin receptor (TfR), basigin (CD147), Glut1, Ldlrad3, CD320, insulin receptor, insulin-like growth factor 1 receptor (IGF1R), low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor-related protein (LRP), preferably LRP1, diphtheria toxin receptor, glucose receptor, CD98hc, TMEM30A, leptin receptor (LepR), and heparan sulfate chains branching from proteoglycans (HSPGs).

[0056] The terms "transferrin receptor" and "TfR" are used interchangeably herein and refer to carrier proteins for transferrin. The terms include "transferrin receptor protein 1" or "TfR1," also known as "Cluster of Differentiation 71" (CD71), and "transferrin receptor protein 2" or "TfR2." TfR1 and TfR2 are transmembrane glycoproteins. In one embodiment, the TfR is TfR1, preferably human TfR1. The terms "basigin" or "CD147" are used interchangeably herein and refer to a transmembrane protein. The UNIPROT reference number for human basigin is P35613. Four isoforms have been described so far, referred to as isoforms 1 to 4, all of which are encompassed by the term "basigin." In one embodiment, the basigin is human basigin (UNIPROT accession number is P35613). The terms "Ldlrad3" and "Low Density Lipoprotein Receptor Class A Domain Containing 3" are used interchangeably herein. The protein belongs to the scavenger receptor superfamily and has been described as a receptor for Venezuelan equine encephalitis virus. In one embodiment, Ldlrad3 is human Ldlrad3 (UNIPROT accession number is Q86YD5). The term "CD320" refers to the transcobalamin receptor. In one embodiment, the CD320 is human CD320 (UNIPROT accession number is Q9NPF0). The terms "insulin receptor," "InsR," and "IR" are used interchangeably herein and refer to a transmembrane receptor that is activated by insulin, IGF-I, and IGF-II. In one embodiment, the insulin receptor is the human insulin receptor (UNIPROT accession number is P06213). The terms "low-density lipoprotein receptor," "LDL-R," and "LDLR" are used interchangeably herein and refer to the low-density lipoprotein receptor, a mosaic protein of 839 amino acids (after removing a 21-amino acid signal peptide) that mediates the endocytosis of cholesterol-rich low-density lipoprotein (LDL). It is a cell surface receptor that recognizes apolipoprotein B100 (ApoB100), which is embedded in the outer phospholipid layer of LDL particles. The receptor also recognizes apolipoprotein E (ApoE), which is found in chylomicron remnants and very low-density lipoprotein (VLDL) remnants. In one embodiment, the LDLR is human LDLR (UNIPROT accession number is P01130). The terms "low-density lipoprotein receptor-related protein" and "LRP" are used interchangeably herein and refer to members of the low-density lipoprotein receptor-related protein family. The terms include LRP-1, LRP-1b, LRP-2, LRP-5, and LRP-6. In some embodiments, the LRP is LRP1. In one embodiment, the LRP is human LRP1 (UNIPROT accession number is Q07954). The terms "diphtheria toxin receptor" and "proheparin-binding EGF-like growth factor" are used interchangeably herein and refer to a growth factor for which the human diphtheria toxin receptor protein has UNIPROT accession number Q99075. In one embodiment, the diphtheria toxin receptor is the human diphtheria toxin receptor (UNIPROT accession number is Q99075). The term "glucose receptor" refers to the glucose transporter protein family, including, but not limited to, Glut1, Glut2, and Glut3. In some embodiments, the glucose receptor is Glut1. In one embodiment, the glucose receptor is human Glut1 (UNIPROT accession number is P11166). The terms "CD98hc" and "CD98 heavy chain" are used interchangeably herein and refer to a cell surface protein also known as "4F2 cell surface antigen heavy chain" or "SLC3A2." It is a component of several heterodimeric complexes involved in amino acid transport, including the glucose receptor. In one embodiment, the CD98hc is human CD98hc (UNIPROT accession number is P08195). The terms "TMEM30A" and "cell cycle control protein 50A" are used interchangeably and refer to the protein, where human TMEM30A has UNIPROT accession number Q9NV96. In one embodiment, TMEM30A is human TMEM30A (UNIPROT accession number is Q9NV96). The terms "leptin receptor," "Lep-R," and "LepR" are used interchangeably herein and refer to a type I cytokine receptor, which is a protein encoded by the LEPR gene in humans. Lep-R functions as a receptor for leptin, a hormone specific to adipocytes. In one embodiment, the leptin receptor is a human leptin receptor (UNIPROT accession number is P48357). The term "heparan sulfate chains branching from proteoglycans (HSPGs)" refers to heparan sulfate chains covalently attached to glycoproteins. Heparan sulfate chains are a type of glycosaminoglycan (GAG). The term includes membrane HSPGs, such as syndecans and glycosylphosphatidylinositol-anchored proteoglycans (glypicans), secreted extracellular matrix HSPGs (agrin, perlecan, type XVIII collagen), and the secretory vesicle proteoglycan serglycin. In one embodiment, the HSPG is human HSPG1 (UNIPROT accession number P34741).

[0057] The "first antigen-binding domain" of a molecule of the present invention may have any structure, as long as it specifically binds to the above-mentioned "first antigen." The structure of the "first antigen-binding domain" can include, but is not limited to, a polypeptide or a portion thereof, a small molecule or medium molecule compound or a portion thereof, or a polynucleotide or a portion thereof. Polypeptides or portions thereof include, but are not limited to, cell membrane proteins expressed on cells (e.g., immune cells such as dendritic cells) or portions thereof (e.g., their extracellular domains, any unique domains thereof, etc.); antibodies (including, but not limited to, human antibodies, chimeric antibodies, humanized antibodies, and VHH antibodies), or antigen-binding domains (also referred to as portions, parts, or fragments of antibodies). Antibody antigen-binding domains include, but are not limited to, antibody heavy chain variable (VH) regions, antibody light chain variable (VL) regions (preferably a combination of antibody heavy chain variable (VH) regions and antibody light chain variable (VL) regions), single domain antibodies (sdAbs), single-chain Fvs (scFvs), single-chain antibodies, Fvs, single-chain Fv2s (scFv2s), Fabs, and F(ab')2s. The polypeptide or a portion thereof may also be an antigen-binding polypeptide, such as: an avimer module called the A domain, which has approximately 35 amino acids contained in an in vivo cell membrane protein (WO 2004 / 044011 and WO 2005 / 040229); an adnectin (WO 2002 / 032925), which has an Fn3 domain that acts as a protein-binding domain derived from fibronectin, a glycoprotein expressed on the cell membrane; an affibody (WO 1995 / 001937), which has an IgG-binding domain scaffold that assembles into a three-helix bundle composed of 58 amino acids of protein A; and a 33 amino acid residue structure that folds into a subunit with one turn, two antiparallel helices, and one loop. DARPins (designed ankyrin repeat proteins) (WO 2002 / 020565), which contain ankyrin repeats (ARs) that are exposed on the molecular surface, an anticalin (WO 2003 / 029462), which has four loop regions connecting eight antiparallel strands that curve toward the central axis at one end of a barrel structure highly conserved in lipocalin molecules such as neutrophil gelatinase-binding lipocalin (NGAL), and variable lymphocyte receptors (VLRs), which lack immunoglobulin structure and are found in the adaptive immune systems of jawless vertebrates such as lampreys or hagfish (WO 2008 / 016854), contain a concave region in the inner parallel sheet structure of a horseshoe-folded structure composed of repeated leucine-rich repeat (LRR) modules.

[0058] In one embodiment of the present invention, the polypeptide or a portion thereof belonging to the above-mentioned "first antigen-binding domain" includes, but is not limited to, a cell membrane protein (e.g., a receptor) expressed on a cell, or a portion thereof (e.g., its extracellular domain, any unique domain thereof, etc.). Polypeptides or portions thereof include, but are not limited to, antibodies (including, but not limited to, human antibodies, chimeric antibodies, humanized antibodies, and VHH antibodies) or antigen-binding domains (also referred to as portions, parts, or fragments of antibodies). Antibody antigen-binding domains include, but are not limited to, antibody heavy chain variable (VH) regions, antibody light chain variable (VL) regions (preferably a combination of antibody heavy chain variable (VH) and antibody light chain variable (VL) regions), single-domain antibodies (sdAbs), single-chain Fvs (scFvs), single-chain antibodies, Fvs, single-chain Fv2s (scFv2s), Fabs, and F(ab')2s. In certain embodiments herein, the mammalian brain is a human brain. Thus, in certain embodiments herein, the "primary target for promoting the translocation of an antigen-binding molecule into the mammalian brain" refers to the primary target for promoting the translocation of an antigen-binding molecule into the human brain.

[0059] a second antigen-binding domain that specifically binds to a second target In the present invention, the "second antigen-binding domain" of the "antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain" binds to a "second target," which is referred to as being "expressed on the cell membrane of a brain cell, or being a brain ECM protein or brain ECM polysaccharide." In some embodiments, the second target is different from the first target described above.

[0060] A target "expressed on the plasma membrane of a brain cell" is understood in the broadest sense as a target expressed on the plasma membrane of at least one cell type of brain cell. For example, a target may be expressed on the plasma membrane of two, three, four, five, or more different types of brain cells, such as the plasma membrane of neurons and oligodendrocytes. The term "expressed on the plasma membrane of a brain cell" includes molecules such as proteins including at least one transmembrane protein and at least one extracellular protein bound to the transmembrane protein, as well as secreted polypeptides and proteoglycans, and polypeptides that are inserted into the plasma membrane, for example, by a lipid anchor, or otherwise bound to the plasma membrane of a brain cell, for example, by non-covalent binding, for example, by ligand-receptor binding.

[0061] A "secondary target" may have any structure, provided that it is expressed on the cell membrane of a brain cell. For example, a "secondary target" may be a protein, carbohydrate, proteoglycan, lipid, nucleic acid, etc. A "secondary target" may be present in a variety of cell types and organs in a mammal and is not limited to any specific occurrence. For example, in certain embodiments, the "second target" is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); IGSF4B / SynCAM3 / cell adhesion molecule 3 (CADM3); CNPase (2',3'-cyclic nucleotide 3'-phosphodiesterase); myelin-associated glycoprotein (MAG), myelin basic protein (MBP), EAAT1 (solute carrier family 1 member 3), EAAT2 (solute carrier family 1 member 2), MAP2 (microtubule-associated protein 2), NEFL (neurofilament light polypeptide), NEFM (neurofilament medium polypeptide), NSE (gamma-enolase), CD68 (macrosialin), allograft inflammatory factor 1 (IBA1 or AIF1), purinergic receptor (P2RY12), interleukin-1 receptor accessory protein-like 1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL-59, IL1RAPL1), glutamate ionotropic receptor NMDA-type subunit 2B (GRIN2B), voltage-gated calcium channel auxiliary subunit gamma 8 (CACNG8), CD11b (integrin subunit alpha M), SLC6A2 (sodium-dependent noradrenaline transporter), DPP6 (dipeptidyl peptidase-like 6), SLC18A3 (vesicular acetylcholine transporter), sodium / potassium-transporting ATPase subunit alpha-2, broad substrate specificity ATP-binding cassette transporter ABCG2, solute carrier family 12 member 9, electrogenic sodium bicarbonate cotransporter 1, excitatory amino acid transporter 2, chondroitin sulfate proteoglycan 4, immunoglobulin superfamily DCC subclass member 4, Vang-like protein 2, neural cell adhesion molecule 1 (N-CAM-1), low-density lipoprotein receptor-related protein 4 (LRP-4),Glycosphingolipid-enriched microdomain-associated phosphoprotein 1 (Csk-binding protein), plasma membrane calcium-transporting ATPase 1, prominin-1, somatostatin receptor type 1, brain isoform of carnitine O-palmitoyltransferase 1 (CPT1-B), epidermal growth factor receptor, protein MAL2, syntaxin-1A, sodium / calcium exchanger 1, lysophosphatidylcholine acyltransferase 1 (LPC acyltransferase 1), and kaolin. Lusintenin-3 (alcadein-β), type I receptor for pituitary adenylate cyclase-activating polypeptide (type I receptor for PACAP), neutral cholesterol ester hydrolase 1 (NCEH), CD166 antigen (activated leukocyte cell adhesion molecule), inactive tyrosine protein kinase 7, claudin-11, ectonucleotide phosphatase (ENPP6), tetraspanin-2 (Tspan-2), myelin proteolipid protein (PLP), glycolipid transfer protein (GLTP). A target that is "a brain ECM protein or brain polysaccharide" is understood in the broadest sense as a protein or polysaccharide that is part of the brain extracellular matrix (ECM). "Brain ECM" is understood as the "extracellular matrix" or "ECM" of the brain, which is a polymeric network primarily composed of polysaccharide glycosaminoglycans (e.g., hyaluronan), proteoglycans (e.g., neurocan, brevican, versican, and aggrecan), glycoproteins (e.g., tenascin-R), and low levels of fibrous proteins (e.g., collagen, fibronectin, and vitronectin). Examples of brain ECM proteins include, but are not limited to, versican core protein (chondroitin sulfate proteoglycan 2 or CSPG2), tropoelastin (elastin), collagen α-2(IV) chain (canstatin), proteoglycan link protein 1 (hyaluronan and proteoglycan link protein 1), tenascin-R (TN-R), proteoglycan link protein 2 (hyaluronan and proteoglycan link protein 2), collagen α-1(I) chain, neurofilament-3 (NEF3), immunoglobulin superfamily member 8 (IgSF8), laminin subunit gamma-1 (LAMC1), collagen α-1(VI) chain (Col6a1), and collagen α-3(VI) chain (Col6a3). Examples of brain ECM polysaccharides include, but are not limited to, hyaluronan. These targets, and their sequences and / or structures, are well known in the art.

[0062] In some embodiments, the "secondary target" is predominantly expressed on brain-specific cells. A "target that is predominantly expressed on brain-specific cells" is understood as a target whose expression on the cell membrane of brain-specific cells is at least 2-fold, 5-fold, 10-fold, 50-fold, or 100-fold higher than that in cells other than brain-specific cells. A "target that is predominantly expressed on brain-specific cells" also includes targets that are only expressed on the cell membrane of less than 10, less than 5, less than 4, less than 3, or less than 2 tissues or organs other than the brain. In one embodiment, the target is only expressed on the cell membrane of brain-specific cells.

[0063] In a further embodiment, the second target is a molecule that is widely expressed in brain tissue. A target that is "broadly expressed in brain tissue" is understood to be a target that is expressed in multiple brain tissues selected from the group consisting of the cerebral cortex, hippocampal formation, amygdala, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, medulla oblongata, and spinal cord. In one embodiment, a target that is "broadly expressed in brain tissue" is a target that is expressed in more than two or more than three brain tissues selected from the group consisting of the cerebral cortex, hippocampal formation, amygdala, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, medulla oblongata, and spinal cord. For example, a second target that is "broadly expressed in brain tissue" can be a molecule selected from the group consisting of interleukin-1 receptor accessory protein-like 1 (IL1RAPL1), glutamate ionotropic receptor NMDA-type subunit 2B (GRIN2B), and voltage-gated calcium channel auxiliary subunit gamma 8 (CACNG8). In one embodiment, the second target is expressed only in brain tissue.

[0064] The terms "myelin oligodendrocyte glycoprotein" and "MOG" are used interchangeably herein and refer to a member of the immunoglobulin (Ig) superfamily, a myelin protein expressed exclusively on the outermost surface of the myelin sheath and in the membranes of oligodendrocytes. In one embodiment, the MOG is human MOG (UNIPROT accession number is Q16653). The terms "neuroglycan C," "chondroitin sulfate proteoglycan 5," and "CSPG5" are used interchangeably herein and refer to a brain-specific chondroitin sulfate proteoglycan. Neuroglycan C is a 120-150 kDa type I transmembrane glycoprotein and a member of the neuregulin protein family. Depending on its expression, neuroglycan C can be either a 120 kDa glycoprotein or a 150 kDa chondroitin sulfate (CS) proteoglycan, and both variants are included herein. In one embodiment, neuroglycan C is human neuroglycan C (UNIPROT accession number is O95196). The terms "IGSF4B," "cell adhesion molecule 3," and "SynCAM3 (CADM3)" are used interchangeably herein and refer to proteins involved in cell-cell adhesion. In one embodiment, the IGSF4B is human IGSF4B (UNIPROT accession number is Q8N126). The terms "CNPase" and "2',3'-cyclic nucleotide 3'-phosphodiesterase" are used interchangeably herein and refer to the enzyme encoded by the CNP gene in humans. CNPase is a myelin-associated enzyme that constitutes 4% of all CNS myelin proteins. In one embodiment, the CNPase is human CNPase (UNIPROT accession number is P09543). The terms "myelin-associated glycoprotein" and "MAG" are used interchangeably herein and refer to a glycoprotein that is a type 1 transmembrane protein that is localized in the membranes of Schwann cells and oligodendrocytes surrounding axons, where it plays a role in glia-axon interactions. MAG is a member of the SIGLEC protein family and is the functional ligand of NgR, the NOGO-66 receptor. In one embodiment, MAG is human MAG (UNIPROT accession number is P20916). The terms "myelin basic protein" and "MBP" are used interchangeably herein and refer to myelin basic protein, a major component of the myelin sheath made from oligodendrocytes and Schwann cells. The human protein has UNIPROT accession number P02686. In one embodiment, the MBP is human MBP (UNIPROT accession number is P02686). The terms "EAAT1" and "solute carrier F" are used interchangeably herein and refer to the protein excitatory amino acid transporter 1. EEAT1 is a protein encoded by the SLC1A3 gene in humans. EAAT1 is also called glutamate-aspartate transporter 1 (GLAST-1). In one embodiment, the EAAT1 is human EAAT1 (UNIPROT accession number is P43003). The terms "EAAT2" and "solute carrier family 1 member 2" are used interchangeably herein and refer to the protein excitatory amino acid transporter 2. EEAT2 is a protein encoded by the SLC1A2 gene in humans. EAAT2 is also called glutamate transporter 1 (GLT-1). In one embodiment, EAAT2 is human EAAT2 (UNIPROT accession number is P43004). The terms "MAP2" and "microtubule-associated protein 2" are used interchangeably herein and refer to a protein belonging to the microtubule-associated protein family. In humans, the protein is encoded by the MAP2 gene. In one embodiment, the MAP2 is human MAP2 (UNIPROT accession number is P11137). The terms "NEFL" and "neurofilament light chain polypeptide" are used interchangeably herein and refer to neurofilament light chain. NEFL is a neurofilament protein encoded by the NEFL gene in humans. Neurofilament light chain is a biomarker that can be measured using immunoassays of cerebrospinal fluid and plasma, and reflects axonal damage in a wide variety of neurological disorders. In one embodiment, NEFL is human NEFL (UNIPROT accession number is P07196). The terms "NEFM" and "neurofilament medium polypeptide" are used interchangeably herein and refer to neurofilament medium polypeptide, a protein encoded by the NEFM gene in humans. Neurofilaments are heteropolymers of type IV intermediate filaments composed of a light chain (NEFL), a medium chain (the protein), and a heavy chain (NEFH). In one embodiment, the NEFM is human NEFM (UNIPROT accession number is P07197). The terms "NSE" and "γ-enolase" are used interchangeably herein and refer to enolase 2 (ENO2) or neuron-specific enolase (NSE), an enzyme encoded by the ENO2 gene in humans. γ-enolase is a phosphopyruvate hydratase. γ-enolase is one of three enolase isoenzymes found in mammals. The isoenzyme γ-enolase is a homodimer and is found in mature neurons and cells of neural origin. In one embodiment, the NSE is human NSE (UNIPROT accession number is P09104). The terms "CD68" and "macrosialin" are used interchangeably herein and refer to an antigen called "cluster of differentiation 68." The human protein has UNIPROT accession number P34810. Human CD68 is a transmembrane glycoprotein that is heavily glycosylated in its extracellular domain. In one embodiment, the CD68 is human CD68 (UNIPROT accession number is P34810). The terms "allograft inflammatory factor 1," as well as "IBA1" and "AIF1," are used interchangeably herein and refer to ionized calcium-binding adaptor molecule 1. AIF1 is a protein encoded by the AIF1 gene in humans. In one embodiment, IBA1 is human IBA1 (UNIPROT accession number is P55008). The terms "purinergic receptor" and "P2RY12" are used interchangeably herein and refer to adenosine diphosphate (ADP) chemoreceptors, belonging to the Gi class of G protein-coupled (GPCR) purinergic receptors. The human protein has UNIPROT accession number Q9H244. In one embodiment, P2RY12 is human P2RY12 (UNIPROT accession number is Q9H244). The terms "interleukin-1 receptor accessory protein-like 1" and "IL1RAPL1" are used interchangeably herein and refer to X-linked interleukin-1 receptor accessory protein-like 1, which is a protein encoded by the IL1RAPL1 gene in humans. In one embodiment, IL1RAPL1 is human IL1RAPL1 (UNIPROT accession number is Q9NZN1). The terms "glutamate ionotropic receptor NMDA type subunit 2B" and "GRIN2B" are used interchangeably herein and refer to N-methyl D-aspartate receptor subtype 2B (NMDAR2B or NR2B). This is the protein encoded by the GRIN2B gene in humans. The human protein has UNIPROT accession number Q13224. In one embodiment, GRIN2B is human GRIN2B (UNIPROT accession number is Q13224). The terms "voltage-gated calcium channel accessory subunit gamma 8" and "CACNG8" are used interchangeably herein and refer to the protein, where human CACGN8 has UNIPROT accession number Q8WXS5. In one embodiment, CACNG8 is human CACNG8 (UNIPROT accession number is Q8WXS5). The terms "CD11b" and "integrin subunit αM" are used interchangeably herein and refer to the protein, where human CD11b has UNIPROT accession number P11215. In one embodiment, CD11b is human CD11b (UNIPROT accession number is P11215). The terms "SLC6A2" and "sodium-dependent noradrenaline transporter" are used interchangeably herein and refer to the protein, where human SLC6A2 has UNIPROT accession number P23975. In one embodiment, the SLC6A2 is human SLC6A2 (UNIPROT accession number is P23975). The terms "DPP6" and "dipeptidyl peptidase-like 6" are used interchangeably herein and refer to the protein, where human DPP6 has UNIPROT accession number P42658. In one embodiment, the DPP6 is human DPP6 (UNIPROT accession number is P42658). The terms "SLC18A3" and "vesicular acetylcholine transporter" are used interchangeably herein and refer to the protein, where human SLC18A3 has UNIPROT accession number Q16572. In one embodiment, SLC18A3 is human SLC18A3 (UNIPROT accession number is Q16572). The term "sodium / potassium transport ATPase subunit alpha-2" refers to the protein human sodium / potassium transport ATPase subunit alpha-2 has UNIPROT accession number P50993. In one embodiment, the sodium / potassium transport ATPase subunit alpha-2 is human sodium / potassium transport ATPase subunit alpha-2 (UNIPROT accession number is P50993). The term "broad substrate specificity ATP-binding cassette transporter ABCG2" refers to the protein human broad substrate specificity ATP-binding cassette transporter ABCG2 has UNIPROT accession number Q9UNQ0. In one embodiment, the broad substrate specificity ATP-binding cassette transporter ABCG2 is human broad substrate specificity ATP-binding cassette transporter ABCG2 (UNIPROT accession number is Q9UNQ0). The term "solute carrier family 12 member 9" refers to the protein human solute carrier family 12 member 9 has UNIPROT accession number Q9BXP2. In one embodiment, solute carrier family 12 member 9 is human solute carrier family 12 member 9 (UNIPROT accession number is Q9BXP2). The term "electrogenic sodium bicarbonate cotransporter 1" refers to the protein human electrogenic sodium bicarbonate cotransporter 1 has UNIPROT accession number Q9Y6R1. In one embodiment, the electrogenic sodium bicarbonate cotransporter 1 is human electrogenic sodium bicarbonate cotransporter 1 (UNIPROT accession number is Q9Y6R1). The term "excitatory amino acid transporter 2" refers to the protein human excitatory amino acid transporter 2 has UNIPROT accession number P43004. In one embodiment, the excitatory amino acid transporter 2 is human excitatory amino acid transporter 2 (UNIPROT accession number is P43004). The term "chondroitin sulfate proteoglycan 4" refers to the protein human chondroitin sulfate proteoglycan 4 has the UNIPROT accession number Q6UVK1. In one embodiment, the chondroitin sulfate proteoglycan 4 is human chondroitin sulfate proteoglycan 4 (UNIPROT accession number is Q6UVK1). The term "excitatory amino acid transporter 2" refers to the protein human excitatory amino acid transporter 2 has UNIPROT accession number P43004. In one embodiment, the excitatory amino acid transporter 2 is human excitatory amino acid transporter 2 (UNIPROT accession number is P43004). The term "immunoglobulin superfamily DCC subclass member 4" refers to the protein in which human immunoglobulin superfamily DCC subclass member 4 has UNIPROT accession number Q8TDY8. In one embodiment, the immunoglobulin superfamily DCC subclass member 4 is human immunoglobulin superfamily DCC subclass member 4 (UNIPROT accession number is Q8TDY8). The term "Vang-like protein 2" refers to the protein where human Vang-like protein 2 has UNIPROT accession number Q9ULK5. In one embodiment, the Vang-like protein 2 is human Vang-like protein 2 (UNIPROT accession number is Q9ULK5. The terms "neural cell adhesion molecule 1" and "N-CAM-1" are used interchangeably herein and refer to the protein, where human N-CAM-1 has UNIPROT accession number P13591. In one embodiment, the N-CAM-1 is human N-CAM-1 (UNIPROT accession number is P13591). The terms "low density lipoprotein receptor-related protein 4" and "LRP-4" are used interchangeably herein and refer to the protein, where human LRP-4 has UNIPROT accession number O75096. In one embodiment, the LRP-4 is human LRP-4 (UNIPROT accession number is O75096). The terms "glycosphingolipid-enriched microdomain-associated phosphoprotein 1" and "Csk-binding protein" are used interchangeably herein and refer to the protein human glycosphingolipid-enriched microdomain-associated phosphoprotein 1 having UNIPROT accession number Q9NWQ8. In one embodiment, glycosphingolipid-enriched microdomain-associated phosphoprotein 1 is human glycosphingolipid-enriched microdomain-associated phosphoprotein 1 (UNIPROT accession number is Q9NWQ8). The term "plasma membrane calcium transporting ATPase 1" refers to the protein human plasma membrane calcium transporting ATPase 1 has UNIPROT accession number P20020. In one embodiment, the plasma membrane calcium transporting ATPase 1 is human plasma membrane calcium transporting ATPase 1 (UNIPROT accession number is P20020). The term "prominin-1" refers to the protein where human prominin-1 has UNIPROT accession number O43490. In one embodiment, the prominin-1 is human prominin-1 (UNIPROT accession number is O43490). The term "somatostatin receptor type 1" refers to the protein where human somatostatin receptor type 1 has UNIPROT accession number P30872. In one embodiment, the somatostatin receptor type 1 is human somatostatin receptor type 1 (UNIPROT accession number is P30872). The terms "brain isoform of carnitine O-palmitoyltransferase 1" and "CPT1-B" are used interchangeably herein and refer to the protein in which the brain isoform of human carnitine O-palmitoyltransferase 1 has UNIPROT accession number Q8TCG5. In one embodiment, the brain isoform of carnitine O-palmitoyltransferase 1 is the brain isoform of human carnitine O-palmitoyltransferase 1 (UNIPROT accession number is Q8TCG5). The term "epidermal growth factor receptor" refers to the protein where human epidermal growth factor receptor has UNIPROT accession number P00533. In one embodiment, the epidermal growth factor receptor is human epidermal growth factor receptor (UNIPROT accession number is P00533). The term "protein MAL2" refers to the protein where human protein MAL2 has UNIPROT accession number Q969L2. In one embodiment, protein MAL2 is human protein MAL2 (UNIPROT accession number is Q969L2). The term "syntaxin-1A" refers to the protein, where human syntaxin-1A has the UNIPROT accession number Q16623. In one embodiment, the syntaxin-1A is human syntaxin-1A (UNIPROT accession number is Q16623). The term "sodium / calcium exchanger 1" refers to the protein, human sodium / calcium exchanger 1 has UNIPROT accession number P32418. In one embodiment, the sodium / calcium exchanger 1 is human sodium / calcium exchanger 1 (UNIPROT accession number is P32418). The terms "lysophosphatidylcholine acyltransferase 1" and "LPC acyltransferase 1" are used interchangeably herein and refer to the protein, where human LPC acyltransferase 1 has UNIPROT accession number Q8NF37. In one embodiment, LPC acyltransferase 1 is human LPC acyltransferase 1 (UNIPROT accession number is Q8NF37). The terms "calsyntenin-3" and "alcadein-β" are used interchangeably herein and refer to the protein, where human calsyntenin-3 has UNIPROT accession number Q9BQT9. In one embodiment, the calsyntenin-3 is human calsyntenin-3 (UNIPROT accession number is Q9BQT9). The terms "type I receptor for pituitary adenylate cyclase-activating polypeptide" and "type I receptor for PACAP" are used interchangeably herein and refer to the protein, where the human type I receptor for PACAP has UNIPROT accession number P41586. In one embodiment, the type I receptor for PACAP is the human type I receptor for PACAP (UNIPROT accession number is P41586). The terms "neutral cholesterol ester hydrolase 1" and "NCEH" are used interchangeably herein and refer to the protein, where human NCEH has UNIPROT accession number Q6PIU2. In one embodiment, the NCEH is human NCEH (UNIPROT accession number is Q6PIU2). The terms "CD166" and "activated leukocyte cell adhesion molecule" are used interchangeably herein and refer to the protein, where human CD166 has UNIPROT accession number Q13740. In one embodiment, CD166 is human CD166 (UNIPROT accession number is Q13740). The term "inactive tyrosine protein kinase 7" refers to the protein human inactive tyrosine protein kinase 7 has UNIPROT accession number Q13308. In one embodiment, the inactive tyrosine protein kinase 7 is human inactive tyrosine protein kinase 7 (UNIPROT accession number is Q13308). The term "claudin-11" refers to the protein, where human claudin-11 has UNIPROT accession number O75508. In one embodiment, the claudin-11 is human claudin-11 (UNIPROT accession number is O75508). The terms "ectonucleotide phosphatase" and "ENPP6" are used interchangeably herein and refer to the protein, where human ENPP6 has UNIPROT accession number Q6UWR7. In one embodiment, ENPP6 is human ENPP6 (UNIPROT accession number is Q6UWR7). The terms "tetraspanin-2" and "Tspan-2" are used interchangeably herein and refer to the protein, where human tetraspanin-2 has UNIPROT accession number 060636. In one embodiment, the tetraspanin-2 is human tetraspanin-2 (UNIPROT accession number is 060636). The terms "myelin proteolipid protein" and "PLP" are used interchangeably herein and refer to the protein, where human myelin proteolipid protein has UNIPROT accession number P60201. In one embodiment, the myelin proteolipid protein is human myelin proteolipid protein (UNIPROT accession number is P60201). The terms "glycolipid transfer protein" and "GLTP" are used interchangeably herein and refer to the protein, where human GLTP has UNIPROT accession number Q9NZD2. In one embodiment, the GLTP is human GLTP (UNIPROT accession number is Q9NZD2). The terms "versican core protein," "chondroitin sulfate proteoglycan 2," and "CSPG2" are used interchangeably herein and refer to the protein, where human CSPG2 has UNIPROT accession number P13611. In one embodiment, the CSPG2 is human CSPG2 (UNIPROT accession number is P13611). The terms "tropoelastin" and "elastin" are used interchangeably herein and refer to the protein, where human tropoelastin has UNIPROT accession number P15502. In one embodiment, the tropoelastin is human tropoelastin (UNIPROT accession number is P15502). The terms "collagen alpha-2(IV) chain" and "Canstatin" are used interchangeably herein and refer to the protein where human collagen alpha-2(IV) chain has UNIPROT accession number P08572. In one embodiment, the collagen alpha-2(IV) chain is human collagen alpha-2(IV) chain (UNIPROT accession number is P08572). The terms "proteoglycan link protein 1" and "hyaluronan and proteoglycan link protein 1" are used interchangeably herein and refer to the protein, where human proteoglycan link protein 1 has UNIPROT accession number P10915. In one embodiment, the proteoglycan link protein 1 is human proteoglycan link protein 1 (UNIPROT accession number is P10915). The terms "tenascin-R" and "TN-R" are used interchangeably herein and refer to the protein, where human tenascin-R has UNIPROT accession number Q92752. In one embodiment, the tenascin-R is human tenascin-R (UNIPROT accession number is Q92752). The terms "proteoglycan link protein 2" and "hyaluronan and proteoglycan link protein 2" are used interchangeably herein and refer to the protein, where human proteoglycan link protein 2 has UNIPROT accession number Q9GZV7. In one embodiment, the proteoglycan link protein 2 is human proteoglycan link protein 2 (UNIPROT accession number is Q9GZV7). The term "collagen alpha-1(I) chain" refers to the protein where human collagen alpha-1(I) chain has UNIPROT accession number P02452. In one embodiment, the collagen alpha-1(I) chain is human collagen alpha-1(I) chain (UNIPROT accession number is P02452). The terms "neurofilament-3" and "NEF3" are used interchangeably herein and refer to the protein, where human neurofilament-3 has UNIPROT accession number Q9UK51. In one embodiment, the neurofilament-3 is human neurofilament-3 (UNIPROT accession number is Q9UK51). The terms "immunoglobulin superfamily 8" and "IgSF8" are used interchangeably herein and refer to the protein, where human IgSF8 has UNIPROT accession number Q969P0. In one embodiment, IgSF8 is human IgSF8 (UNIPROT accession number is Q969P0). The terms "laminin subunit gamma-1" and "LAMC1" are used interchangeably herein and refer to the protein where human laminin subunit gamma-1 has UNIPROT accession number P11047. In one embodiment, laminin subunit gamma-1 is human laminin subunit gamma-1 (UNIPROT accession number is P11047). The terms "collagen alpha-1(VI) chain" and "Col6a1" are used interchangeably herein and refer to the protein where human collagen alpha-1(VI) chain has UNIPROT accession number P12109. In one embodiment, the collagen alpha-1(VI) chain is human collagen alpha-1(VI) chain (UNIPROT accession number is P12109). The terms "collagen alpha-3(VI) chain" and "Col6a3" are used interchangeably herein and refer to the protein where human collagen alpha-3(VI) chain has UNIPROT accession number P12111. In one embodiment, the collagen alpha-3(VI) chain is human collagen alpha-3(VI) chain (UNIPROT accession number is P12111).

[0065] In certain embodiments herein, the second target is not a target selected from the group consisting of: beta-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, TRK A, TRK B, TRK C, α-synuclein, β-synuclein, γ-synuclein, vascular endothelial growth factor (VEGF), neuropilin, semaphorin, semaphorin 3A, semaphorin 4A, semaphorin 6A, myelin basic protein (MBP), MOG, PLP, MAG, aquaporin 4, glutamate receptor, and EpCAM.

[0066] The terms "β-secretase 1" and "BACE1" are used interchangeably herein and refer to the protein also known as "β-site amyloid precursor protein-cleaving enzyme 1." The human protein has UNIPROT accession number P56817. The terms "Aβ" and "amyloid beta" are used interchangeably herein and refer to a 36-43 amino acid peptide that is the major component of amyloid plaques found in the brains of subjects with Alzheimer's disease. The terms "epidermal growth factor receptor" and "EGFR" are used interchangeably herein. The human protein has UNIPROT accession number P00533. The terms "human epidermal growth factor receptor 2" and "HER2" are used interchangeably herein and refer to a protein also known as "receptor tyrosine protein kinase erbB-2." The human protein has UNIPROT accession number P04626. The terms "tau" and "microtubule-associated protein tau" are used interchangeably herein. The human protein has UNIPROT accession number P10636. The terms "apolipoprotein E" and "ApoE" are used interchangeably herein. Included within the term "ApoE" are various variants and isoforms of ApoE, such as variant E4. The terms "alpha-synuclein" and "α-synuclein" are used interchangeably herein and refer to the protein encoded by the SNCA gene in humans. Alpha-synuclein is a neuronal protein that regulates synaptic vesicle trafficking and subsequent neurotransmitter release. The term "CD20" refers to a protein also known as "B lymphocyte antigen CD20." In humans, CD20 is encoded by the MS4A1 gene. The terms "huntingtin" and "Htt" are used interchangeably herein and refer to the protein encoded by the HTT gene, which in humans is also known as the T15 ("interesting transcript 15") gene. The terms "prion protein" and "PrP" are used interchangeably herein and refer to the protein encoded by the PRNP gene in humans. There are various isoforms of the protein encompassed by the term "PrP," including PrP, protease-resistant forms called PrPRes, such as the disease-causing PrPSc (scrapie) form, and mitochondrial isoforms. The terms "leucine-rich repeat kinase 2" and "LRRK2" are used interchangeably herein and refer to the protein encoded by the LRKK2 gene in humans, which is also known as dardarin or PARK8. The term "parkin" refers to the protein encoded by the PSEN-1 gene in humans. Parkin is a 465 amino acid residue E3 ubiquitin ligase. The term "presenilin-1" refers to the protein encoded by the PSEN-1 gene in humans. Presenilin-1 is one of the four core proteins in the gamma-secretase complex, which is thought to play a key role in the production of amyloid beta (Aβ) from amyloid precursor protein (APP). The term "presenilin-2" refers to the protein encoded in humans by the PSEN2 gene, which has been described as the putative catalytic subunit of the γ-secretase complex. The term "γ-secretase" refers to a multisubunit protease complex consisting of four separate proteins: PSEN1 (presenilin-1), nicastrin, APH-1 (anterior pharynx-defective 1), and PEN-2 (presenilin enhancer 2). The complex itself is an integral membrane protein that cleaves single-pass transmembrane proteins at residues within their transmembrane domains. The best-known substrate of γ-secretase is the amyloid precursor protein. The terms "death receptor 6" and "DR6" are used interchangeably herein and refer to a protein also known as "tumor necrosis factor receptor superfamily member 21." The terms "amyloid precursor protein" and "APP" are used interchangeably herein and refer to the precursor protein of Aβ. The terms "p75 neurotrophin receptor" and "p75NTR" are used interchangeably herein and refer to the protein that in humans is encoded by the NGFR gene. The terms "caspase 6" and "caspase-6" are used interchangeably herein and refer to the protein that in humans is encoded by the CASP6 gene. The term "Trk A" refers to tropomyosin receptor kinase A. TrkA is a protein encoded by the NTRK1 gene. The term "Trk B" refers to tropomyosin receptor kinase B. TrkB is a protein encoded by the NTRK2 gene. The term "Trk C" refers to tropomyosin receptor kinase C. TrkC is a protein encoded by the NTRK3 gene. The terms "β-synuclein" and "β-synuclein" are used interchangeably herein and refer to the protein that in humans is encoded by the SNCB gene. The terms "γ-synuclein" and "gamma synuclein" are used interchangeably herein and refer to the protein that in humans is encoded by the SNCG gene. The terms "vascular endothelial growth factor" and "VEGF" are used interchangeably herein and refer to the protein having, in humans, UNIPROT accession number Q9UNS8. The term "neuropilin" refers to two described protein receptors, NRP-1 and NRP-2, both of which are included in the term "neuropilin." Human NRP-1 has UNIPROT accession number O14786. Human NRP-2 has UNIPROT accession number O60462. The term "semaphorin" refers to a class of secreted and membrane proteins that are divided into eight major classes. Classes 3, 4, 6, and 7 are found only in vertebrates, and class 5 is found in both vertebrates and invertebrates. Genes encoding semaphorins in humans are SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3G, SEMA4A, SEMA4B, SEMA4C ("SEMAF"), SEMA4D, SEMA4F, SEMA4G, SEMA5A, SEMA5B, SEMA6A, SEMA6B, SEMA6C, SEMA6D, and SEMA7A. The term "semaphorin 3A" refers to the semaphorin that in humans is encoded by the SEMA3A gene. The term "semaphorin 4A" refers to the semaphorin that in humans is encoded by the SEMA4A gene. The term "semaphorin 6A" refers to the semaphorin that in humans is encoded by the SEMA6A gene. The term "PLP" refers to "myelin proteolipid protein." The term "aquaporin-4" refers to the water channel protein encoded by the AQP4 gene in humans. The protein is also known as "AQP-4." The term "glutamate receptor" refers to a receptor for glutamate and includes, but is not limited to, glutamate receptor 1 and glutamate receptor 2. The term "EpCAM" refers to a protein also known as "epithelial cell adhesion molecule."

[0067] The "second antigen-binding domain" of a molecule of the present invention may have any structure, as long as it specifically binds to the above-mentioned "second antigen." The structure of the "second antigen-binding domain" may include, but is not limited to, a polypeptide or a portion thereof, a small molecule or medium molecule compound or a portion thereof, or a polynucleotide or a portion thereof. Polypeptides or portions thereof include, but are not limited to, cell membrane proteins expressed on cells (e.g., immune cells such as dendritic cells) or portions thereof (e.g., their extracellular domains, any unique domains thereof, etc.); antibodies (including, but not limited to, human antibodies, chimeric antibodies, humanized antibodies, and VHH antibodies), or antigen-binding domains (also referred to as portions, parts, or fragments of antibodies). Antibody antigen-binding domains include, but are not limited to, antibody heavy chain variable (VH) regions, antibody light chain variable (VL) regions (preferably a combination of antibody heavy chain variable (VH) regions and antibody light chain variable (VL) regions), single domain antibodies (sdAbs), single-chain Fvs (scFvs), single-chain antibodies, Fvs, single-chain Fv2s (scFv2s), Fabs, and F(ab')2s. The polypeptide or a portion thereof may also be an antigen-binding polypeptide, such as: an avimer module called the A domain, which has approximately 35 amino acids contained in an in vivo cell membrane protein (WO 2004 / 044011 and WO 2005 / 040229); an adnectin (WO 2002 / 032925), which has an Fn3 domain that acts as a protein-binding domain derived from fibronectin, a glycoprotein expressed on the cell membrane; an affibody (WO 1995 / 001937), which has an IgG-binding domain scaffold that assembles into a three-helix bundle composed of 58 amino acids of protein A; and a 33 amino acid residue structure that folds into a subunit with one turn, two antiparallel helices, and one loop. DARPins (designed ankyrin repeat proteins) (WO 2002 / 020565), which contain ankyrin repeats (ARs) that are exposed on the molecular surface, an anticalin (WO 2003 / 029462), which has four loop regions connecting eight antiparallel strands that curve toward the central axis at one end of a barrel structure highly conserved in lipocalin molecules such as neutrophil gelatinase-binding lipocalin (NGAL), and variable lymphocyte receptors (VLRs), which lack immunoglobulin structure and are found in the adaptive immune systems of jawless vertebrates such as lampreys or hagfish (WO 2008 / 016854), contain a concave region in the inner parallel sheet structure of a horseshoe-folded structure composed of repeated leucine-rich repeat (LRR) modules. In one embodiment of the present invention, the polypeptide or a portion thereof belonging to the above-mentioned "second antigen-binding domain" includes, but is not limited to, a cell membrane protein (e.g., a receptor) expressed on a cell, or a portion thereof (e.g., its extracellular domain, any unique domain thereof, etc.). Polypeptides or portions thereof include, but are not limited to, antibodies (including, but not limited to, human antibodies, chimeric antibodies, humanized antibodies, and VHH antibodies) or antigen-binding domains (also referred to as portions, parts, or fragments of antibodies). Antibody antigen-binding domains include, but are not limited to, antibody heavy chain variable (VH) regions, antibody light chain variable (VL) regions (preferably a combination of antibody heavy chain variable (VH) regions and antibody light chain variable (VL) regions), single domain antibodies (sdAbs), VHHs, single-chain Fvs (scFvs), single-chain antibodies, Fvs, single-chain Fv2 (scFv2), Fabs, single-chain Fabs (scFabs), and F(ab')2s.

[0068] An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain In the present invention, the term "antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain" includes molecules comprising at least one first antigen-binding domain and at least one second antigen-binding domain, where the domains may be covalently or non-covalently linked. The term includes, but is not limited to, molecules with the following exemplary characteristics: i. An antibody in which the "first antigen-binding domain" is conjugated to the Fc region and at least one of the two Fabs binds to a "second antigen." Alternatively, the "first antigen-binding domain" may be conjugated to one of the Fab arms rather than or in addition to being conjugated to the Fc region. Furthermore, more than two "first antigen-binding domains" may be conjugated to the Fc region. The two Fab arms can be conjugated to the Fc region in various configurations, as illustrated in Figures 2(b) and 2(c). Instead of Fab arms, the antigen-binding domains of one or both Fab arms may have different configurations, such as VHH, scFv, or scFab. ii. An antibody in which one Fab arm binds to a "first antigen" and the other Fab arm binds to a "second antigen." An example of this embodiment can be a bispecific antibody, or a bispecific antigen-binding domain, in which one Fab arm binds to a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain, as described above, and the other Fab arm binds to a second target that is (i) expressed on the cell membrane of brain cells or (ii) a brain ECM protein or brain ECM polysaccharide. The antibody or antibody-like molecule in this example can have a variety of configurations, as illustrated in Figure 2(b). iii. A polypeptide comprising a "first antigen-binding moiety" and a "second antigen-binding moiety," wherein the first antigen-binding moiety and the second antigen-binding moiety are fused together with or without a linker. An example of this embodiment is a fusion protein comprising a VHH or scFv that binds to TfR and a VHH or scFv that binds to MOG, optionally fused together via a peptide linker. Several examples in Figures 2(b) and 2(c) illustrate such embodiments. iv. Compounds comprising a "first antigen-binding domain" and a "second antigen-binding domain." An example of this embodiment may be a compound comprising at least two moieties, one of which binds to a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain, as described above, and another of which binds to a second target that is (i) expressed on the cell membrane of a brain cell or (ii) a brain ECM protein or brain ECM polysaccharide. Such compounds can be achieved using bioconjugate technology ("Bioconjugate Techniques," 3rd Edition, 2013, by Greg T. Hermanson). The linker or compound may further comprise, for example, a photoreactive group (e.g., aryl azide, diazirine, psoralen, etc.).

[0069] Antigen-binding molecules further comprising at least one functional moiety and / or at least one in vivo half-life extending moiety In certain embodiments, the antigen binding molecule of any of the aspects and embodiments herein further comprises at least one functional moiety and / or at least one in vivo half-life extending moiety. In some embodiments, the antigen binding molecule of any of the aspects and embodiments herein further comprises: (i) at least one functional moiety, including an enzyme, a therapeutic protein, an antibody or antigen-binding fragment thereof, a peptide, DNA, shRNA, siRNA, a small molecule drug, or a cytotoxic agent; and / or (ii) at least one in vivo half-life extending moiety, preferably selected from the group consisting of an Fc region, an albumin binding domain, an FcRn binding protein, an FcRn binding peptide, and a PEG moiety.

[0070] In some embodiments, the antigen-binding molecule of any of the aspects and embodiments herein further comprises at least one functional moiety. In certain embodiments, the functional moiety is selected from the group consisting of an enzyme, a therapeutic protein, an antibody or antigen-binding fragment thereof, a peptide, DNA, shRNA, siRNA, a small molecule drug, and a cytotoxic agent. In certain embodiments, the functional moiety is not an Fc region. Exemplary embodiments for the construction of antigen-binding molecules further comprising at least one functional moiety are shown in Figures 2(a) and 2(c). In certain embodiments, the antigen-binding molecule of any of the aspects and embodiments herein further comprises at least one in vivo half-life extending moiety, which in certain embodiments is selected from the group consisting of an Fc region, an albumin-binding domain, an FcRn-binding protein, an FcRn-binding peptide, and a PEG moiety. Exemplary embodiments for the construction of antigen-binding molecules further comprising an Fc region as an exemplary in vivo half-life extending moiety are shown in Figures 2(a) and 2(c). In some embodiments, the antigen-binding molecule of any of the aspects and embodiments herein further comprises at least one functional moiety and at least one in vivo half-life extending moiety. Exemplary embodiments of the configuration of an antigen-binding molecule further comprising at least one functional moiety and an Fc region as an exemplary in vivo half-life extending moiety are shown in Figures 2(a) and 2(c).

[0071] The term "in vivo half-life" of a molecule refers to the time (half-life) it takes for the concentration of the molecule in a given tissue of a subject to decrease by half when the molecule is present in the tissue at steady state. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. Methods for determining in vivo half-life are known in the art, and suitable methods are described in the Examples.

[0072] The term "in vivo half-life extending moiety" is understood to mean a moiety that, when linked to an active agent, extends the in vivo half-life in the blood relative to the active agent without said moiety. In some embodiments, an "in vivo half-life extending moiety" is understood to mean a moiety that, when linked to an active agent, extends the in vivo half-life by at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 500%, or 1000%.

[0073] Suitable in vivo half-life extending moieties are known and available in the art. In some embodiments, an Fc region, an albumin-binding domain, an FcRn-binding protein, an FcRn-binding peptide, or a PEG moiety can be used. For example, in the case of an Fc region, the Fc region can bind to one or more Fc receptors. Specifically, in the case of an Fc region, the Fc region can bind to FcRn. Such an Fc region is suitable for extending in vivo half-life. In some embodiments, the antigen binding molecule of this aspect or any of the present embodiments is an Fc region-containing antibody. In other embodiments, the in vivo half-life extending moiety is an FcRn-binding protein or peptide. Such moieties are known in the art and are disclosed, for example, in US Patent Application Publication No. 20170275373 and Datta-Mannan A et al (Biotechnol J. 2019 Mar;14(3):e1800007. doi: 10.1002 / biot.201800007).

[0074] A "PEG" moiety is understood to be a moiety comprising two or more polyethylene glycol moieties covalently linked to one another. For example, PEG moieties having molecular weights of 500 Da to 100 kDa, e.g., 1000 Da to 50000 Da, can be used. "Albumin binding domain" is understood to mean a moiety that can bind to mammalian albumin. In some embodiments, the mammalian albumin is human albumin. The structure of the albumin binding domain includes, but is not limited to, for example, proteins, peptides, and small molecules. In some embodiments, the albumin binding domain is a peptide or protein. Suitable moieties are known in the art and are described, for example, in Tan H et al. (Eur J Pharmacol. 2021 Jan 5;890:173650).

[0075] A "functional moiety" is understood as a moiety capable of exerting a therapeutic, preventive, and / or diagnostic effect. Those skilled in the art will recognize such "functional moieties" and will be able to select such "functional moieties" depending on the disease or disorder to be treated, prevented, and / or diagnosed. In certain embodiments, the functional moiety includes an enzyme, a therapeutic protein, an antibody or an antigen-binding fragment thereof, a peptide, DNA, shRNA, siRNA, a small molecule drug, or a cytotoxic agent. The structure of the functional moiety is not limited, as long as the functional moiety exerts the desired therapeutic, preventive, or diagnostic effect.

[0076] Fc receptors The term "Fc receptor" or "FcR" refers to a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native human FcR. In some embodiments, the FcR binds to an IgG antibody (a gamma receptor), and includes the FcγRI, FcγRII, and FcγRIII subclasses of receptors, including allelic variants and alternatively spliced forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibitory receptor"), which have similar amino acid sequences but differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, e.g., Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed, for example, in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991); Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are also encompassed by the term "FcR" herein. The term "Fc receptor" or "FcR" also includes the fetal receptor, FcRn, which is responsible for the transport of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) and for regulating immunoglobulin homeostasis. Methods for measuring binding to FcRn are known (see, e.g., Ghetie and Ward., Immunol. Today 18(12):592-598 (1997); Ghetie et al., Nature Biotechnology, 15(7):637-640 (1997); Hinton et al., J. Biol. Chem. 279(8):6213-6216 (2004); WO 2004 / 92219 (Hinton et al.)). The in vivo binding to human FcRn and plasma half-life of human FcRn high-affinity binding polypeptides can be assayed, for example, in transgenic mice or transfected human cell lines expressing human FcRn, or in primates administered with polypeptides having variant Fc regions. International Publication No. 2000 / 42072 (Presta) describes antibody variants with increased or decreased FcR binding. See also, for example, Shields et al. J. Biol. Chem. 9(2):6591-6604 (2001).

[0077] Fc region containing antibody The term "Fc region-containing antibody" refers to an antibody that contains an Fc region. The C-terminal lysine (residue 447 according to the EU numbering system) or the C-terminal glycine-lysine (residues 446-447) of the Fc region may be removed, for example, during antibody purification or by recombinant manipulation of a nucleic acid encoding the antibody. Thus, a composition containing an antibody with an Fc region according to the present invention may contain an antibody with G446 to K447, an antibody with G446 but not K447, an antibody from which all of G446 to K447 have been removed, or a mixture of the three types of antibodies.

[0078] Methods for increasing the concentration of antigen-binding molecules in the brain, methods for increasing the exposure of antigen-binding molecules in the brain, and methods for maintaining the persistence of antigen-binding molecules in the brain In one aspect, a method for increasing the concentration of an antigen-binding molecule in the brain of a subject in need thereof is provided, the method comprising: so that the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) (a1) a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; or (a2) a second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell or (ii) a brain ECM protein or brain ECM polysaccharide. providing a first antigen-binding molecule; (b) In the case of (a1), introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule; or In the case of (a2), introducing at least one first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. Includes.

[0079] In some embodiments, the "concentration of an antigen-binding molecule in the brain of a subject" refers to the concentration of the antigen-binding molecule in the brain per weight of brain tissue, and is typically given in units of μg / g brain. The concentration can be determined as follows (here, an "antibody" is described as an exemplary antigen-binding molecule): TIFF2025525307000001.tif9128 In some further embodiments, the term "increasing the concentration of an antigen-binding molecule in the brain of a subject" is understood as increasing the concentration (μg / g brain) by 1% or more, 5% or more, 10% or more, 15% or more, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% or more compared to the first antigen-binding molecule.

[0080] In some embodiments, the "concentration of an antigen-binding molecule in the brain of a subject" is understood as the percentage of the injected dose per weight of brain tissue, and is typically given as %ID(injected dose) / g brain, and can be determined as follows (here, an "antibody" is described as an exemplary antigen-binding molecule): TIFF2025525307000002.tif16128

[0081] In some further embodiments, the term "increasing the concentration of an antigen-binding molecule in the brain of a subject" is understood as increasing the concentration (%ID / g brain) by 1% or more, 5% or more, 10% or more, 15% or more, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% or more compared to the first antigen-binding molecule.

[0082] In one embodiment, a method is provided for increasing the concentration of an antigen binding molecule in the brain of a subject in need thereof, the method comprising: so that the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) providing a first antigen-binding molecule comprising a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the brain of a mammal; (b) introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. Includes.

[0083] In another aspect, a method for increasing the concentration of an antigen binding molecule in the brain of a subject in need thereof is provided, the method comprising: so that the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) providing a first antigen-binding molecule comprising a second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or a brain ECM polysaccharide; (b) introducing into the first antigen-binding molecule at least one first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain, thereby obtaining a second antigen-binding molecule. Includes.

[0084] With regard to the "first antigen-binding domain," "first target," "second antigen-binding domain," and "second target," the same aspects as for the "antigen-binding molecule" of any of the above aspects and embodiments apply. The "introducing" step in (b) can be achieved by methods known to those skilled in the art, such as by covalently linking a "second antigen-binding domain" or a "first antigen-binding domain" to a "first antigen-binding molecule," optionally using a linker. For example, the "introducing" step can include producing one or more nucleic acids encoding the second antigen-binding molecule and obtaining the second antigen-binding molecule by recombinant expression techniques.

[0085] In certain embodiments, the second antigen binding molecule is as defined in any of the aspects and embodiments of the antigen binding molecule of the present invention. In certain embodiments, the method further comprises step (c) of: (c) determining that the concentration of the second antigen-binding molecule in the subject's brain is increased compared to a control antigen-binding molecule, wherein the control antigen-binding molecule is: In the case of (a1), at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide; or In the case of (a2), at least one first antigen-binding domain that specifically binds to a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain. (b) differs from the second antigen-binding molecule described in (b) only in that it does not comprise The control concentration may be determined at the same time point or at a different time point. In one embodiment, the control concentration may be determined at the same time point.

[0086] Thus, a "control antigen-binding molecule" does not contain, in the case of (a1), at least one second antigen-binding domain that specifically binds to a second target; or, in the case of (a2), it does not contain at least one first antigen-binding domain that specifically binds to a first target, while the remaining portions remain identical. In Examples 3, 5, 6, and 7, and in Figures 3(c), 6(c), 8(c), and 10(c), it was found that the concentration of bispecific antibodies comprising a brain-localizing moiety and a brain-persisting moiety, determined as %ID / g brain, was increased in the brain compared to control antibodies comprising only a brain-localizing moiety or only a brain-persisting moiety. For example, an anti-basigin / / anti-MOG bispecific antibody, an anti-TfR / / anti-MOG bispecific antibody, and an anti-TfR / / anti-CADM3 bispecific antibody are provided in Examples 4 and 5. Surprisingly, it was found that the combination of a brain-localizing moiety and a brain-persisting moiety in these bispecific antibodies exhibited much higher concentrations at day 7 than antibodies comprising only a brain-localizing moiety or only a brain-persisting moiety. These additional data also suggest that the combination of a brain-translocating moiety and a brain-retaining moiety has a synergistic effect on the ability of an antibody to persist in the brain. Further, anti-TfR / / anti-CSPG5 bispecific antibodies are provided in Examples 4 and 7. The results in Example 7 show that the combination of a brain-translocating moiety that binds to TfR and a brain-retaining moiety that binds to CSPG5 has superior ability to penetrate and persist in the brain compared to an antibody containing only a moiety that binds to CSPG5. In some embodiments, the concentration of the second antigen-binding molecule in the subject's brain is Cmax. "Cmax" is also known as the maximum concentration, and is the highest concentration of the antibody in the brain during a certain period after administration. In one embodiment herein, the mammalian brain is human brain.Therefore, in one embodiment, the first target promotes the translocation of antigen-binding molecule into human brain.Methods for determining Cmax are known in the art.Suitable methods for determining Cmax are described in Examples. For example, Example 3 shows that the cmax in the brain of a bispecific antibody comprising a brain-translocating moiety and a brain-retaining moiety (anti-TfR / / anti-MOG bispecific antibody) is higher than the cmax in the brain of a bispecific antibody comprising only a brain-translocating moiety (anti-TfR / / anti-KLH).

[0087] In a further embodiment, the first target is as specified in any one of the embodiments of the antigen-binding molecule of the first aspect herein. In a further embodiment, the second target is as specified in any one of the embodiments of the antigen-binding molecule of the first aspect herein. In a further embodiment, the first target and the second target are as specified in any one of the embodiments of the antigen-binding molecule of the first aspect herein. In a further embodiment, specifically, the first target and second target are as follows: (i) the first target is the transferrin receptor (TfR) and the second target is myelin oligodendrocyte glycoprotein (MOG); (ii) the first target is transferrin receptor (TfR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (iii) the first target is transferrin receptor (TfR) and the second target is cell adhesion molecule 3 (CADM3); (iv) the first target is basigin (CD147) and the second target is myelin oligodendrocyte glycoprotein (MOG); (v) the first target is basigin (CD147) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (vi) the first target is basigin (CD147) and the second target is cell adhesion molecule 3 (CADM3); (vii) the first target is the insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); (viii) the first target is the insulin receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (ix) the first target is the insulin receptor and the second target is cell adhesion molecule 3 (CADM3); (x) the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); (xi) the first target is a glucose receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (xii) the first target is a glucose receptor and the second target is cell adhesion molecule 3 (CADM3); (xiii) the first target is low-density lipoprotein receptor (LDLR) and the second target is myelin oligodendrocyte glycoprotein (MOG); (xiv) the first target is low-density lipoprotein receptor (LDLR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xv) the first target is low-density lipoprotein receptor (LDLR) and the second target is cell adhesion molecule 3 (CADM3); (xii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is myelin oligodendrocyte glycoprotein (MOG); (xii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xviii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is cell adhesion molecule 3 (CADM3); or (xix) the first target is CD98hc and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xx) the first target is CD98hc and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xxi) The first target is CD98hc and the second target is cell adhesion molecule 3 (CADM3). Specifically, the second antigen-binding molecule is an antigen-binding molecule according to any of the embodiments of the antigen-binding molecule of the first aspect herein.

[0088] In a further aspect, a method for increasing the exposure of an antigen-binding molecule in the brain of a subject in need thereof is provided, the method comprising: so that the exposure amount of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) (a1) a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; or (a2) A second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide. providing a first antigen-binding molecule; (b) In the case of (a1), introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule; or In the case of (a2), introducing at least one first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. Includes.

[0089] The term "exposure of a second antigen-binding molecule in the subject's brain" refers to the area under the curve (AUC) in the brain. "Area under the curve" or "AUC" or "AUC (area under the curve) of the brain concentration-time profile of an antigen-binding molecule" are used interchangeably herein and represent the cumulative exposure of an antibody in the brain during the test period. AUC can be determined by pharmacokinetic methods known in the art, such as measuring the concentration of the antigen-binding molecule of interest at multiple separate time points and using the trapezoidal rule for approximation calculations, thereby determining the AUC. Suitable methods for determining AUC are described in the Examples.

[0090] The term "increasing the exposure of an antigen-binding molecule in the brain of a subject" is understood as increasing the exposure by 1% or more, 5% or more, 10% or more, 15% or more, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% or more compared to the first antigen-binding molecule.

[0091] With regard to the "first antigen-binding domain," "first target," "second antigen-binding domain," and "second target," the same aspects as for the "antigen-binding molecule" of any of the above aspects and embodiments apply.

[0092] The "introducing" step in (b) can be achieved by methods known to those skilled in the art, such as by covalently linking a "second antigen-binding domain" or a "first antigen-binding domain" to a "first antigen-binding molecule," optionally using a linker. For example, the "introducing" step can include producing one or more nucleic acids encoding the second antigen-binding molecule and obtaining the second antigen-binding molecule by recombinant expression techniques. In certain embodiments, the second antigen binding molecule is as defined in any of the aspects and embodiments of the antigen binding molecule of the present invention.

[0093] In one embodiment, a method for increasing exposure of an antigen-binding molecule in the brain of a subject in need thereof is provided, the method comprising: so that the exposure amount of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) providing a first antigen-binding molecule comprising a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the brain of a mammal; (b) introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. Includes. In one embodiment, a method for increasing exposure of an antigen-binding molecule in the brain of a subject in need thereof is provided, the method comprising: so that the exposure amount of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) providing a first antigen-binding molecule comprising a second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or a brain ECM polysaccharide; (b) introducing into the first antigen-binding molecule at least one first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain, thereby obtaining a second antigen-binding molecule. Includes.

[0094] In one embodiment, the method further comprises the step (c) of: (c) determining that the exposure of the second antigen-binding molecule in the subject's brain is increased compared to a control antigen-binding molecule, wherein the control antigen-binding molecule is: In the case of (a1), at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide; or In the case of (a2), at least one first antigen-binding domain that specifically binds to a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain. (b) differs from the second antigen-binding molecule described in (b) only in that it does not comprise In one embodiment, the exposure amount of the second antigen-binding molecule in the subject's brain is the AUC (area under the curve) of the brain concentration-time profile of the antigen-binding molecule.

[0095] In one embodiment herein, the mammalian brain is a human brain. Thus, in one embodiment, the first target promotes the translocation of the antigen-binding molecule into the human brain. In a further embodiment, the first target is as specified in any one of the embodiments of the antigen-binding molecule of the first aspect herein. In a further embodiment, the second target is as specified in any one of the embodiments of the antigen-binding molecule of the first aspect herein. In a further embodiment, the first target and the second target are as specified in any one of the embodiments of the antigen-binding molecule of the first aspect herein. In a further embodiment, specifically, the first target and second target are as follows: (i) the first target is the transferrin receptor (TfR) and the second target is myelin oligodendrocyte glycoprotein (MOG); (ii) the first target is transferrin receptor (TfR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (iii) the first target is transferrin receptor (TfR) and the second target is cell adhesion molecule 3 (CADM3); (iv) the first target is basigin (CD147) and the second target is myelin oligodendrocyte glycoprotein (MOG); (v) the first target is basigin (CD147) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (vi) the first target is basigin (CD147) and the second target is cell adhesion molecule 3 (CADM3); (vii) the first target is the insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); (viii) the first target is the insulin receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (ix) the first target is the insulin receptor and the second target is cell adhesion molecule 3 (CADM3); (x) the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); (xi) the first target is a glucose receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (xii) the first target is a glucose receptor and the second target is cell adhesion molecule 3 (CADM3); (xiii) the first target is low-density lipoprotein receptor (LDLR) and the second target is myelin oligodendrocyte glycoprotein (MOG); (xiv) the first target is low-density lipoprotein receptor (LDLR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xv) the first target is low-density lipoprotein receptor (LDLR) and the second target is cell adhesion molecule 3 (CADM3); (xii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is myelin oligodendrocyte glycoprotein (MOG); (xii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xviii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is cell adhesion molecule 3 (CADM3); or (xix) the first target is CD98hc and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xx) the first target is CD98hc and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xxi) The first target is CD98hc and the second target is cell adhesion molecule 3 (CADM3). Specifically, the second antigen-binding molecule is an antigen-binding molecule according to any of the embodiments of the antigen-binding molecule of the first aspect herein. In Example 3, Figure 3(d), it was found that the exposure, determined as AUC, of a bispecific antibody comprising a brain-translocating moiety and a brain-retaining moiety was increased in the brain compared to a control antibody comprising only a brain-translocating moiety or only a brain-retaining moiety. Notably, unexpectedly, MOG303 / / TfR exhibited a synergistic effect with respect to the brain cumulative AUC (area under the curve), which represents the total exposure of the antibody in the brain over a period of time, where the brain cumulative AUC of MOG303 / / TfR was approximately 108-fold, 15.2-fold, and 24.7-fold higher than those of KLH, MOG303, and KLH / / TfR, respectively (Figure 3(d)). Notably, the cumulative brain AUC of MOG303 / / TfR (73.0 μg / g*day) was greater than the sum of the cumulative brain AUCs of MOG303 (4.80 μg / g*day) and KLH / / TfR (2.95 μg / g*day), suggesting that the combination of a transferable moiety (anti-TfR) and a sustained moiety (anti-MOG) in a bispecific antibody results in a synergistic effect for longer persistence and higher antibody concentrations in the brain.

[0096] In one aspect, a method for persistence of an antigen-binding molecule in the brain of a subject in need thereof is provided, the method comprising: such that the persistence of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) providing a first antigen-binding molecule comprising a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the brain of a mammal; (b) introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. Includes. In particular, the data in Examples 3, 5, 6, and 7 for anti-TfR / / anti-CSPG5 bispecific antibody, anti-basigin / / anti-MOG bispecific antibody, anti-TfR / / anti-MOG bispecific antibody, and anti-TfR / / anti-CADM3 bispecific antibody suggest that the combination of a brain-translocating moiety and a brain-retaining moiety has a synergistic effect on the persistence of the antibody in the brain.

[0097] The term "persistence" or "persistence of an antigen-binding molecule in the brain of a subject" is understood as the in vivo half-life in the brain of a subject. For example, Example 3 and Figure 3 show that a bispecific antibody comprising a brain-translocating moiety and a brain-retaining moiety (anti-TfR / / anti-MOG bispecific antibody) has a longer in vivo half-life in the brain of a subject than a bispecific antibody comprising only a brain-translocating moiety (anti-TfR / / anti-KLH).

[0098] The term "in vivo half-life in the brain" of a molecule refers to the time (half-life) required for the steady-state concentration of the molecule in the brain of a subject to be reduced by half when the molecule is present in the brain. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In a further embodiment, the "persistence of the second antigen-binding molecule in the brain of a subject" refers to the in vivo half-life in the brain.

[0099] The term "increasing the persistence of an antigen-binding molecule in the brain of a subject" is understood as increasing the persistence in the brain or the in vivo half-life in the brain by 1% or more, 5% or more, 10% or more, 15% or more, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% or more, compared to the first antigen-binding molecule.

[0100] In some embodiments, the in vivo half-life of the second antigen-binding molecule in the brain is at least 10, 12, 15, 20, 30, 45, 60, or 90 days. For example, the in vivo half-life of the second antigen-binding molecule in the brain is at least 10, 12, 15, 20, 30, 45, 60, or 90 days, or up to 100, 150, or 200 days, or any subrange thereof.

[0101] Thus, in some embodiments, the in vivo half-life of the second antigen-binding molecule in the brain is increased by 1% or more, 5% or more, 10% or more, 15% or more, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or 200% or more compared to the first antigen-binding molecule, and the in vivo half-life of the second antigen-binding molecule in the brain is at least 10, 12, 15, 20, 30, 45, 60, or 90 days. For example, the in vivo half-life of the second antigen-binding molecule in the brain is at least 10, 12, 15, 20, 30, 45, 60, or 90 days, or up to 100, 150, or 200 days, or any subrange thereof.

[0102] With regard to the "first antigen-binding domain," "first target," "second antigen-binding domain," and "second target," the same aspects as for the "antigen-binding molecule" of any of the above aspects and embodiments apply.

[0103] The "introducing" step in (b) can be achieved by methods known to those skilled in the art, such as by covalently linking a "second antigen-binding domain" to a "first antigen-binding molecule," optionally using a linker. For example, the "introducing" step can include preparing one or more nucleic acids encoding the second antigen-binding molecule and obtaining the second antigen-binding molecule by recombinant expression techniques.

[0104] In certain embodiments, the second antigen binding molecule is as defined in any of the aspects and embodiments of the antigen binding molecule of the present invention.

[0105] In one embodiment, the method further comprises the step (c) of: (c) determining that the persistence of the second antigen-binding molecule in the subject's brain is increased compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the second antigen-binding molecule described in (b) only in that it does not contain at least one antigen-binding domain that specifically binds to the second target.

[0106] In one embodiment herein, the mammalian brain is a human brain. Thus, in one embodiment, the first target promotes the translocation of the antigen-binding molecule into the human brain. In a further embodiment, the first target is as specified in any one of the embodiments of the antigen-binding molecule of the first aspect herein. In a further embodiment, the second target is as specified in any one of the embodiments of the antigen-binding molecule of the first aspect herein. In a further embodiment, the first target and the second target are as specified in any one of the embodiments of the antigen-binding molecule of the first aspect herein. In a further embodiment, specifically, the first target and second target are as follows: (i) the first target is the transferrin receptor (TfR) and the second target is myelin oligodendrocyte glycoprotein (MOG); (ii) the first target is transferrin receptor (TfR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (iii) the first target is transferrin receptor (TfR) and the second target is cell adhesion molecule 3 (CADM3); (iv) the first target is basigin (CD147) and the second target is myelin oligodendrocyte glycoprotein (MOG); (v) the first target is basigin (CD147) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (vi) the first target is basigin (CD147) and the second target is cell adhesion molecule 3 (CADM3); (vii) the first target is the insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); (viii) the first target is the insulin receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (ix) the first target is the insulin receptor and the second target is cell adhesion molecule 3 (CADM3); (x) the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); (xi) the first target is a glucose receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); (xii) the first target is a glucose receptor and the second target is cell adhesion molecule 3 (CADM3); (xiii) the first target is low-density lipoprotein receptor (LDLR) and the second target is myelin oligodendrocyte glycoprotein (MOG); (xiv) the first target is low-density lipoprotein receptor (LDLR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xv) the first target is low-density lipoprotein receptor (LDLR) and the second target is cell adhesion molecule 3 (CADM3); (xii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is myelin oligodendrocyte glycoprotein (MOG); (xii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xviii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is cell adhesion molecule 3 (CADM3); or (xix) the first target is CD98hc and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xx) the first target is CD98hc and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xxi) The first target is CD98hc and the second target is cell adhesion molecule 3 (CADM3). Specifically, the second antigen-binding molecule is an antigen-binding molecule according to any of the embodiments of the antigen-binding molecule of the first aspect herein.

[0107] The "brain-to-plasma ratio" is understood as the ratio between the concentration of an antigen-binding molecule in the brain and the concentration of an antigen-binding molecule in the plasma. The "brain-to-plasma ratio" can be calculated as follows: TIFF2025525307000003.tif18128

[0108] "Translocation efficiency" is understood as the percentage of injected dose per weight of brain tissue (%ID(injected dose) / g brain). "Translocation efficiency" can be calculated as follows: TIFF2025525307000004.tif18128

[0109] Embodiments Relating to First and Second Targets As described above, for any of the aspects herein, the first antigen-binding domain specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; and The second antigen-binding domain specifically binds to a second target, which is (i) expressed on the cell membrane of a brain cell or (ii) a brain ECM protein or brain ECM polysaccharide.

[0110] Embodiments for the first target are described above, and the second target is also described above. Further embodiments for any of the aspects herein are disclosed below. According to certain embodiments, the second target is expressed on the cell membrane of brain cells, wherein the brain cells include one or more types of cells selected from the group consisting of oligodendrocytes, astrocytes, neurons, and microglia. For example, the second target is expressed on the cell membrane of oligodendrocytes. Such second targets include, for example, myelin oligodendrocyte glycoprotein, CNPase, and MAG, as shown in Table 2. For example, the second target is expressed on the cell membrane of astrocytes. Such second targets include, for example, EEAT1 and EEAT2, as shown in Table 2. For example, the second target is expressed on the cell membrane of microglia. Such second targets include, for example, P2RY12 and AIF1, as shown in Table 2. For example, the second target is expressed on the cell membrane of multiple types of brain cells. Such second targets include, for example, CSPG5 and SynCAM3, as shown in Table 2.

[0111] According to certain embodiments, the second target is a brain ECM protein, including, but not limited to, versican core protein (chondroitin sulfate proteoglycan 2 or CSPG2), tropoelastin (elastin), collagen alpha-2(IV) chain (canstatin), proteoglycan link protein 1 (hyaluronan and proteoglycan link protein 1), tenascin-R (TN-R), proteoglycan link protein 2 (hyaluronan and proteoglycan link protein 2), collagen alpha-1(I) chain, neurofilament-3 (NEF3), immunoglobulin superfamily member 8 (IgSF8), laminin subunit gamma-1 (LAMC1), collagen alpha-1(VI) chain (Col6a1), and collagen alpha-3(VI) chain (Col6a3), as shown in Table 2.

[0112] In another embodiment, the secondary target is a brain ECM polysaccharide, such as, but not limited to, hyaluronic acid (HA) or hyaluronan. In one embodiment, the first target is the transferrin receptor (TfR). In the examples, an antigen-binding molecule comprising an antigen-binding domain that binds to TfR is provided. Specifically, an anti-TfR / / anti-MOG bispecific antibody is provided in Examples 2 and 3, which exhibits advantageous pharmacokinetic properties. The results suggest that the combination of a brain-translocating moiety (anti-TfR) and a brain-persistent moiety (anti-MOG) has a synergistic effect on longer persistence and higher antibody concentration in the brain, resulting in increased persistence and concentration in the brain. Furthermore, an anti-TfR / / anti-MOG bispecific antibody and an anti-TfR / / anti-CADM3 bispecific antibody are provided in Examples 4 and 5. Surprisingly, it was found that the combination of a brain-localizing moiety and a brain-retaining moiety in these bispecific antibodies exhibited comparable or higher concentrations compared to antibodies having only a brain-localizing moiety on day 1, and significantly higher concentrations compared to antibodies having only a brain-localizing moiety or only a brain-retaining moiety on day 7. These further data also demonstrate that the combination of a brain-localizing moiety and a brain-retaining moiety has a synergistic effect on the persistence of the antibody in the brain. have Furthermore, anti-TfR / / anti-CSPG5 bispecific antibodies are provided in Examples 4 and 7. The results in Example 7 show that the combination of a brain-localizing moiety that binds to TfR and a brain-retaining moiety that binds to CSPG5 has superior ability to enter and persist in the brain.

[0113] In another embodiment herein, the first target is basigin (CD147). Bispecific antibodies comprising a brain-localizing moiety that binds basigin and a brain-persisting moiety that binds MOG (anti-basigin / / anti-MOG bispecific antibody) are provided in Examples 4 and 5. Surprisingly, it was found that the combination of a brain-localizing moiety and a brain-persisting moiety in these bispecific antibodies exhibited equivalent or higher concentrations compared to antibodies having only a brain-localizing moiety on day 1, and significantly higher concentrations than antibodies having only a brain-localizing moiety or only a brain-persisting moiety on day 7. These further data also suggest that the combination of a brain-localizing moiety and a brain-persisting moiety has a synergistic effect on the persistence of the antibody in the brain. In another embodiment herein, the first target is the insulin receptor. In another embodiment herein, the first target is the insulin-like growth factor 1 receptor (IGF1R). In another embodiment herein, the first target is the low density lipoprotein receptor (LDLR). In another embodiment herein, the first target is a low-density lipoprotein receptor-related protein (LRP). In a preferred embodiment, the LRP is LRP1. In another embodiment herein, the first target is a diphtheria toxin receptor. In another embodiment herein, the first target is a glucose receptor. In a preferred embodiment, the glucose receptor is Glut1. In another embodiment herein, the first target is CD98hc. In still further embodiments, the second target is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5), and cell adhesion molecule 3 (CADM3). In one embodiment, the second target is myelin oligodendrocyte glycoprotein (MOG). In the examples, an antigen-binding molecule comprising an antigen-binding domain that binds to MOG is provided. Specifically, an anti-TfR / / anti-MOG bispecific antibody is provided in Examples 2 and 3, which exhibits advantageous pharmacokinetic properties. The results suggest that the combination of a brain-localizing moiety (anti-TfR) and a brain-persistent moiety (anti-MOG) has a synergistic effect on longer persistence and higher antibody concentration in the brain, resulting in increased persistence and increased concentration in the brain. Furthermore, in addition to the anti-TfR / / anti-MOG bispecific antibody, a bispecific antibody (anti-basigin / / anti-MOG bispecific antibody) comprising a brain-localizing moiety that binds to basigin and a brain-persistent moiety that binds to MOG is provided in Examples 4, 5, and 7. Surprisingly, it was found that the combination of a brain-localizing moiety and a brain-persisting moiety in these bispecific antibodies showed comparable or higher concentrations compared to antibodies having only a brain-localizing moiety on day 1, and showed significantly higher concentrations than antibodies having only a brain-localizing moiety or only a brain-persisting moiety on day 7. These further data also suggest that the combination of a brain-localizing moiety and a brain-persisting moiety has a synergistic effect on the persistence of the antibody in the brain. Examples of other anti-MOG antigen-binding domains that can be used are described, for example, in WO2018123979. Examples of other anti-TfR antigen-binding domains that can be used are described, for example, in WO 2012075037, WO 2014033074, or Johnsen, Kasper Bendix, et al. "Targeting the transferrin receptor for brain drug delivery." Progress in neurobiology 181 (2019): 101665. In one embodiment, the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5). Anti-TfR / / anti-CSPG5 bispecific antibodies are provided in Examples 4 and 7. The results in Example 7 show that the combination of a brain-translocating moiety that binds to TfR and a brain-retaining moiety that binds to CSPG5 has excellent capabilities for translocating into and persisting in the brain. Examples of other anti-CSPG5 antigen-binding domains that can be used are described, for example, in International Publication No. 2020004490. Examples of other anti-TfR antigen-binding domains that can be used are described, for example, in International Publication No. 2012075037, International Publication No. 2014033074, or Johnsen, Kasper Bendix, et al. "Targeting the transferrin receptor for brain drug delivery." Progress in neurobiology 181 (2019): 101665. In one embodiment, the second target is cell adhesion molecule 3 (CADM3). In the examples, anti-TfR / / anti-CADM3 bispecific antibodies are provided in Examples 4 and 5. Surprisingly, it was found that the combination of a brain-localizing moiety and a brain-persisting moiety in these bispecific antibodies exhibited equivalent or higher concentrations compared to antibodies having only a brain-localizing moiety on day 1, and significantly higher concentrations on day 7 than antibodies having only a brain-localizing moiety or only a brain-persisting moiety. These additional data also suggest that the combination of a brain-localizing moiety and a brain-persisting moiety has a synergistic effect on the persistence of the antibody in the brain. Examples of other anti-CADM3 antigen-binding domains that can be used are described, for example, in WO2020004492. Examples of other anti-TfR antigen-binding domains that can be used are described, for example, in WO 2012075037, WO 2014033074, or Johnsen, Kasper Bendix, et al. "Targeting the transferrin receptor for brain drug delivery." Progress in neurobiology 181 (2019): 101665.

[0114] In a further embodiment, the first target is transferrin receptor (TfR) and the second target is myelin oligodendrocyte glycoprotein (MOG). Specifically, anti-TfR / / anti-MOG bispecific antibodies are provided in Examples 2 and 3, which exhibit beneficial pharmacokinetic properties. The results suggest that the combination of a brain-localizing moiety (anti-TfR) and a brain-persisting moiety (anti-MOG) has a synergistic effect on longer persistence and higher antibody concentrations in the brain, resulting in increased persistence and increased concentration in the brain. Furthermore, anti-TfR / / anti-MOG bispecific antibodies are provided in Examples 4 and 5. Surprisingly, it was found that the combination of a brain-localizing moiety and a brain-persisting moiety in these bispecific antibodies exhibited equivalent or higher concentrations compared to antibodies having only a brain-localizing moiety on day 1, and significantly higher concentrations than antibodies having only a brain-localizing moiety or only a brain-persisting moiety on day 7. These additional data also suggest that the combination of a brain-translocating moiety and a brain-persisting moiety has a synergistic effect on the persistence of the antibody in the brain. In a further embodiment, the first target is transferrin receptor (TfR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5). Anti-TfR / / anti-CSPG5 bispecific antibodies are provided in Examples 4 and 7. The results in Example 7 show that the combination of a brain-localizing moiety that binds to TfR and a brain-retaining moiety that binds to CSPG5 has superior ability to enter and persist in the brain. In a further embodiment, the first target is transferrin receptor (TfR) and the second target is cell adhesion molecule 3 (CADM3). In the examples, anti-TfR / / anti-CADM3 bispecific antibodies are provided in Examples 4 and 5. Surprisingly, it was found that the combination of a brain-localizing moiety and a brain-persisting moiety in these bispecific antibodies exhibited equivalent or higher concentrations compared to antibodies having only a brain-localizing moiety on day 1, and significantly higher concentrations than antibodies having only a brain-localizing moiety or only a brain-persisting moiety on day 7. These further data also suggest that the combination of a brain-localizing moiety and a brain-persisting moiety has a synergistic effect on the persistence of antibodies in the brain. In a further embodiment, the first target is basigin (CD147) and the second target is myelin oligodendrocyte glycoprotein (MOG). Bispecific antibodies comprising a brain-localizing moiety that binds basigin and a brain-persisting moiety that binds MOG (anti-basigin / / anti-MOG bispecific antibody) are provided in Examples 4 and 5. Surprisingly, it was found that the combination of a brain-localizing moiety and a brain-persisting moiety in these bispecific antibodies exhibited equivalent or higher concentrations compared to antibodies having only a brain-localizing moiety on day 1, and significantly higher concentrations than antibodies having only a brain-localizing moiety or only a brain-persisting moiety on day 7. These further data also suggest that the combination of a brain-localizing moiety and a brain-persisting moiety has a synergistic effect on the persistence of the antibody in the brain. In a further embodiment, the first target is basigin (CD147) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5). In a further embodiment, the first target is basigin (CD147) and the second target is cell adhesion molecule 3 (CADM3). In a further embodiment, the first target is the insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG). In a further embodiment, the first target is the insulin receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5). In a further embodiment, the first target is the insulin receptor and the second target is cell adhesion molecule 3 (CADM3). In a further embodiment, the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG). In a further embodiment, the first target is a glucose receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5). In a further embodiment, the first target is a glucose receptor and the second target is cell adhesion molecule 3 (CADM3). In a further embodiment, the first target is low density lipoprotein receptor (LDLR) and the second target is myelin oligodendrocyte glycoprotein (MOG). In a further embodiment, the first target is low density lipoprotein receptor (LDLR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5). In a further embodiment, the first target is low density lipoprotein receptor (LDLR) and the second target is cell adhesion molecule 3 (CADM3). In a further embodiment, the first target is low density lipoprotein receptor-related protein (LRP) and the second target is myelin oligodendrocyte glycoprotein (MOG). In a further embodiment, the first target is low density lipoprotein receptor-related protein (LRP) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5). In a further embodiment, the first target is low density lipoprotein receptor-related protein (LRP) and the second target is cell adhesion molecule 3 (CADM3).

[0115] As described above, in the aspects herein, the structure of the "second target" is not limited, provided that the second target is expressed on the cell membrane of a brain cell or is a brain ECM protein or brain ECM polysaccharide. In some embodiments, the "first target" is different from the "second target." Thus, the "first antigen-binding domain" specifically binds to a different target than the target to which the "second antigen-binding domain" specifically binds. Thus, in certain embodiments, the antigen-binding molecule is bispecific and / or multispecific.

[0116] Additionally, in some embodiments, the second target is not a target selected from the group consisting of: beta-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, TRK A, TRK B, TRK C, α-synuclein, β-synuclein, γ-synuclein, vascular endothelial growth factor (VEGF), neuropilin, semaphorin, semaphorin 3A, semaphorin 4A, semaphorin 6A, myelin basic protein (MBP), MOG, PLP, MAG, aquaporin 4, glutamate receptor, and EpCAM.

[0117] In some embodiments, the second target is not beta-secretase 1 (BACE1). In some embodiments, the second target is not Aβ. In some embodiments, the second target is not epidermal growth factor receptor (EGFR). In some embodiments, the second target is not human epidermal growth factor receptor 2 (HER2). In some embodiments, the second target is not tau. In some embodiments, the second target is not apolipoprotein E (ApoE). In some embodiments, the second target is not alpha-synuclein. In some embodiments, the second target is not CD20. In some embodiments, the second target is not huntingtin. In some embodiments, the second target is not prion protein (PrP). In some embodiments, the second target is not leucine-rich repeat kinase 2 (LRRK2). In some embodiments, the second target is not parkin, presenilin 1. In some embodiments, the second target is not presenilin 2. In some embodiments, the second target is not gamma-secretase. In some embodiments, the second target is not death receptor 6 (DR6). In some embodiments, the second target is not amyloid precursor protein (APP). In some embodiments, the second target is not p75 neurotrophin receptor (p75NTR). In some embodiments, the second target is not caspase 6. In some embodiments, the second target is not TRK A. In some embodiments, the second target is not TRK B. In some embodiments, the second target is not TRK C. In some embodiments, the second target is not alpha-synuclein. In some embodiments, the second target is not beta-synuclein. In some embodiments, the second target is not gamma-synuclein. In some embodiments, the second target is not vascular endothelial growth factor (VEGF). In some embodiments, the second target is not neuropilin. In some embodiments, the second target is not semaphorin 3A. In some embodiments, the second target is not semaphorin 4A. In some embodiments, the second target is not semaphorin 6A.In some embodiments, the second target is not myelin basic protein (MBP). In some embodiments, the second target is not MOG. In some embodiments, the second target is not PLP. In some embodiments, the second target is not MAG. In some embodiments, the second target is not aquaporin 4. In some embodiments, the second target is not a glutamate receptor. In some embodiments, the second target is not EpCAM.

[0118] Embodiments of antigen-binding molecules further comprising at least one functional moiety and / or at least one in vivo half-life extending moiety In some embodiments, the antigen-binding molecule of any of the aspects and embodiments herein further comprises at least one functional moiety and / or at least one in vivo half-life extending moiety. An antigen-binding molecule further comprising at least one functional moiety and / or at least one in vivo half-life extending moiety may comprise at least one functional moiety and / or at least one in vivo half-life extending moiety linked in different ways. An exemplary embodiment of an antigen-binding molecule of the present invention further comprising a functional moiety and an in vivo half-life extending moiety, or an exemplary embodiment of an antigen-binding molecule of the present invention further comprising a functional moiety (but not an in vivo half-life extending moiety), is shown in Figure 2(a). In Figure 2(a), one moiety is shown for each type of moiety (i.e., first antigen-binding domain, second antigen-binding domain, functional moiety, and / or in vivo half-life extending moiety). However, it is also possible for two, three, four, or more moieties to be independently present for each type of moiety. Furthermore, rather than being directly linked, the "first antigen-binding domain" and the "second antigen-binding moiety" can be linked via an in vivo half-life extending moiety, such as an Fc region, or via a functional moiety. Embodiments in which the "first antigen-binding domain" and the "second antigen-binding moiety" are linked to an Fc region rather than directly are shown, for example, in several examples in Figures 2(b) and 2(c). Figures 2(b) and 2(c) show embodiments including an Fc region as an exemplary in vivo half-life extending moiety.

[0119] One, two, or more functional moieties may be linked to an antigen-binding molecule. For example, one or two functional moieties may be linked to one chain or both chains of the Fc region. For example, such one or two functional moieties may be Fab, scFv, scFab, or VHH. Such an embodiment is shown in Figure 2(b). For example, two functional moieties may form a bivalent F(ab')2 region linked to the Fc region. For example, a "first antigen-binding domain" and a "second antigen-binding moiety" may be linked to the Fc region. The "first antigen-binding domain" may be linked to the first chain of the Fc region, and the "second antigen-binding moiety" may be linked to the second chain of the Fc region. Alternatively, the "first antigen-binding domain" and the "second antigen-binding moiety" may be linked to each other, e.g., as a fusion protein, and may further be linked to one or both chains of the Fc region. In yet another exemplary embodiment, the "first antigen-binding domain" may be linked to a first light chain, and the "second antigen-binding moiety" may be linked to a second light chain. In a further exemplary embodiment, the "first antigen-binding domain" and the "second antigen-binding moiety" may be linked to each other, e.g., as a fusion protein, and may further be linked to one or two functional domains. In a further example, the "first antigen-binding domain" and / or the "second antigen-binding moiety" are linked to functional moieties. For example, one functional moiety may be a Fab, scFv, scFab, or VHH, and the "first antigen-binding domain" (or "second antigen-binding moiety") may be a Fab, scFv, scFab, or VHH. For example, two such monovalent Fabs that together form F(ab)2 may be linked to the Fc region, and the "second antigen-binding domain" (or "first antigen-binding moiety") may be linked to the "first antigen-binding domain" (or "second antigen-binding moiety").In still further exemplary embodiments, one functional moiety may be a Fab, scFv, scFab, or VHH, and the "first antigen-binding domain" (or "second antigen-binding moiety") may be a Fab, scFv, scFab, or VHH. For example, two such monovalent Fabs that together form F(ab)2 may be linked to an Fc region, and the "second antigen-binding domain" (or "first antigen-binding moiety") may be linked to the C-terminus of the Fc region.

[0120] Aspects of Domain Arrangement In certain embodiments of any of the aspects herein, the antigen-binding molecule comprises one or more antibody-type domains. For example, in this example, an anti-TfR / anti-MOG bispecific antibody was prepared. For example, it is possible to provide an antigen-binding molecule comprising one or more variable regions, or antibodies and antibody fragments comprising such one or more variable regions. Thus, in some embodiments of the antigen-binding molecules provided herein, the first antigen-binding domain is an antibody variable region comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL). In certain specific embodiments, the first antigen-binding domain is a Fab, Fab', F(ab')2, diabody, triabody, scFab, Fv, scFv, or single-domain antibody (VHH).

[0121] Furthermore, in certain embodiments of the antigen-binding molecules provided herein, the second antigen-binding domain is an antibody variable region, comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL). In certain specific embodiments, the second antigen-binding domain is a Fab, Fab', F(ab')2, diabody, triabody, scFab, Fv, scFv, or single-domain antibody (VHH).

[0122] In certain embodiments of the antigen-binding molecules provided herein, the first antigen-binding domain is an antibody variable region comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), and the second antigen-binding domain is an antibody variable region comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL). In still further embodiments, the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of antibody variable regions, including an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), and a Fab, Fab', F(ab')2, diabody, triabody, scFab, Fv, scFv, or single domain antibody (VHH).

[0123] Furthermore, there can be one first antigen-binding domain or two or more first antigen-binding domains, and there can be one second antigen-binding domain or two or more second antigen-binding domains. In one embodiment, the antigen-binding molecule comprises one, two, three, or four first antigen-binding domains and one, two, three, or four second antigen-binding domains. Furthermore, in one embodiment, the antigen-binding molecule further comprises at least one Fc region, e.g., one Fc region, or two or more Fc regions. Exemplary embodiments of molecules comprising one Fc region are shown, for example, in Figures 2(b) and 2(c). In still further embodiments, the antigen-binding molecule comprises one, two, three, or four first antigen-binding domains and one, two, three, or four second antigen-binding domains, and at least one Fc region. In an even further preferred embodiment thereof, the first antigen-binding domain and the second antigen-binding domain are independently selected from the group consisting of antibody variable regions, including an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), Fab, Fab', F(ab')2, diabodies, triabodies, scFab, Fv, scFv, and single-domain antibodies (VHH). In yet a further embodiment, the antigen-binding molecule herein is a bispecific antibody. Several embodiments of a bispecific antibody are shown in Figure 2(b). In one embodiment, the antigen-binding molecule comprises one first antigen-binding domain and one second antigen-binding domain. To generate bispecific antibodies, Fab arm exchange technology can be used, for example as described in WO 2016 / 159213.

[0124] Further Aspects Regarding Functional Moieties In certain embodiments, the antigen-binding molecule of any of the aspects and embodiments herein further comprises at least one functional moiety. In certain embodiments herein, at least one functional moiety is an antibody or antigen-binding fragment thereof that specifically binds to a membrane protein of i) an immune cell, particularly an immune cell selected from the group consisting of a T cell, a killer cell, a helper T cell, a regulatory T cell, a B cell, a memory B cell, a NK cell, a NKT cell, a dendritic cell, a macrophage, an eosinophil, a neutrophil cell, and a basophil; ii) a tumor cell; or iii) an autoreactive cell. In certain specific embodiments, the antibody or antigen-binding fragment thereof specifically binds to a membrane protein selected from the group consisting of a T cell receptor, CD3, CD137, CD40, CTLA4, a costimulatory molecule, and a co-inhibitory molecule. For example, the antibody or antigen-binding fragment thereof may be selected from the group consisting of antibody variable regions including an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), Fab, Fab', F(ab')2, diabodies, triabodies, scFab, Fv, scFv, and single domain antibodies (VHH). For example, the Examples disclose bispecific antibodies including Fab or single-chain Fab (scFab).

[0125] antibody fragment In certain embodiments, the antigen-binding molecule provided herein is an antibody fragment. Antibody fragments include, but are not limited to, fragments such as single-chain Fab (scFab), Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv, as well as other fragments described below. For a review of several antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, "The Pharmacology of Monoclonal Antibodies" vol. 113, Rosenburg and Moore eds. (Springer-Verlag, New York), pp. 269-315 (1994); WO 93 / 16185; and U.S. Patent Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab')2 fragments that contain salvage receptors that bind epitope residues and have increased in vivo half-lives.

[0126] Diabodies are antibody fragments that have two antigen binding sites, and can be bivalent or bispecific.See, for example, European Patent No. 404,097; International Publication No. 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9:129-134 (2003).

[0127] Single-domain antibodies are antibody fragments that contain all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, single-domain antibodies are human single-domain antibodies (Domantis, Inc., Waltham, MA; see, e.g., U.S. Patent No. 6,248,516 B1).

[0128] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (such as E. coli or phage), as described herein.

[0129] Chimeric and humanized antibodies In certain embodiments, the antigen-binding molecule provided herein is a chimeric antibody.Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984).In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a non-human primate such as a mouse, rat, hamster, rabbit, or monkey) and a human constant region.In another example, a chimeric antibody is a "class-switched" antibody whose class or subclass is changed from that of the parent antibody.A chimeric antibody includes its antigen-binding fragment.

[0130] In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while maintaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains, in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody, and the FRs (or portions thereof) are derived from a human antibody sequence. Optionally, a humanized antibody also comprises at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0131] Humanized antibodies and methods for making them are reviewed, e.g., by Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25-34 (2005) (describing specificity-determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"); Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"); and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach for FR shuffling).

[0132] Human framework regions that can be used for humanization include, but are not limited to, framework regions selected using the "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); framework regions derived from consensus sequences of human antibodies of particular subgroups of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151:2623 (1993)); human mature (somatically mutated) framework regions, or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996).

[0133] Human antibodies In certain embodiments, the antigen-binding molecule provided herein is a human antibody or comprises a human antibody.Human antibodies can be produced using various techniques known in the art.Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20: 450-459 (2008).

[0134] Human antibodies can be prepared by administering an immunogen to a transgenic animal, which has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of a human immunoglobulin locus, which replaces the endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin locus is generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See, for example, U.S. Pat. Nos. 6,075,181 and 6,150,584, which describe XENOMOUSE™ technology; U.S. Pat. No. 5,770,429, which describes HUMAB® technology; U.S. Pat. No. 7,041,870, which describes KM MOUSE® technology; and U.S. Pat. ELOCI M OUSE See also U.S. Patent Application Publication No. 2007 / 0061900, which describes the .TM. technology. The human variable regions from intact antibodies produced by such animals may be further modified, for example, by combining with different human constant regions.

[0135] Human antibodies can also be produced by hybridoma-based methods. Human myeloma cell lines and mouse-human heteromyeloma cell lines for producing human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., "Monoclonal Antibody Production Techniques and Applications", pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced by human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005), and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0136] Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0137] Library-derived antibodies Antigen-binding domains of the antigen-binding molecules provided herein, such as antibodies, may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, various methods are known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are reviewed, for example, in Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are further described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

[0138] In some phage display methods, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994), VH and VL gene repertoires can be separately cloned by polymerase chain reaction (PCR) and randomly recombined in a phage library, which can then be screened for phage that bind to the antigen. Phages typically display antibody fragments as either single-chain Fv (scFv) fragments or Fab fragments. Libraries derived from immunized sources yield high-affinity antibodies against immunogens without the need for hybridoma construction. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), naive repertoires can be cloned (e.g., from humans) to provide a single source of antibodies against a wide range of non-self and self antigens without any immunization. Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode highly variable CDR3 regions and achieve rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent literature describing human antibody phage libraries includes, for example, U.S. Pat. No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0139] Antibodies or antibody fragments isolated from a human antibody library are considered herein to be human antibodies or human antibody fragments.

[0140] multispecific antibodies In certain embodiments, the antigen-binding molecules provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, one of the binding specificities is for a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain, and one is for a second target that is expressed on the cell membrane of a brain cell. In another certain embodiment, one of the binding specificities is for a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain, and one is for a second target that is a brain ECM protein or brain ECM polysaccharide. In addition, one or more additional binding specificities for other targets may be present. Multispecific antibodies containing additional specificities may be used to localize cytotoxic agents to brain cells that express the second target. Bispecific and multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0141] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, Nature 305: 537 (1983)), WO 93 / 08829, and Traunecker et al., EMBO J. 10: 3655 (1991)), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be generated by: manipulating electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004 A1); cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)); using leucine zippers to create bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to create bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (scFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)); and the preparation of trispecific antibodies, for example, as described in Tutt et al. J. Immunol. 147:60 (1991). To efficiently obtain a bispecific antibody of interest comprising two heavy chains and two light chains, there are known substitutions and combinations of amino acids at the CH1-CL domain interface that facilitate the desired association of the H and L chains (e.g., those described in WO2019065795), which can be used in one embodiment.

[0142] Engineered antibodies with three or more functional antigen-binding sites, including "octopus antibodies," are also included herein (see, e.g., U.S. Patent Application Publication No. 2006 / 0025576 A1).

[0143] The antibodies or fragments herein also include "Dual Acting Fabs" or "DAFs" that contain an antigen-binding site that binds to a first target as well as a second target (see, e.g., U.S. Patent Application Publication No. 2008 / 0069820).

[0144] Antigen-binding molecule variants and in particular antibody variants In certain embodiments, amino acid sequence variants of the antigen-binding molecules and particularly antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antigen-binding molecules and particularly antibodies. Amino acid sequence variants of antigen-binding molecules and particularly antibodies can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletion of residues from the amino acid sequence of the antigen-binding molecules and particularly antibodies, insertion of residues into the amino acid sequence, and / or substitution of residues within the amino acid sequence. Any combination of deletion, insertion, and substitution may be made to create the final construct, provided that the final construct has the desired characteristics, such as antigen binding.

[0145] Glycosylation variants In certain embodiments, the antigen-binding molecules, and particularly antibodies, provided herein are modified to increase or decrease the degree to which the antibody is glycosylated. Addition of glycosylation sites to an antibody or deletion of such sites in an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0146] When an antigen-binding molecule, and particularly an antibody, contains an Fc region, the carbohydrate attached thereto may be altered. Natural antibodies produced by mammalian cells typically contain biantennary branched oligosaccharides, which are usually N-linked to Asn297 in the CH2 domain of the Fc region. See, for example, Wright et al. TIBTECH 15:26-32 (1997). Oligosaccharides can include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, oligosaccharide modifications may be made in the antibodies of the present invention to generate antigen-binding molecules, and particularly antibody variants, with improved properties.

[0147] In one embodiment, antigen-binding molecules, and particularly antibody variants, are provided that have carbohydrate structures lacking fucose bound (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycostructures (e.g., complex structures, hybrid structures, and high-mannose structures) bound to Asn297, as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of residues in the Fc region); however, due to slight antibody sequence variations, Asn297 may also be located approximately + / - 3 amino acids upstream or downstream of position 297, i.e., at positions 294-300. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Application Publication Nos. 2003 / 0157108 (Presta, L.); 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include: U.S. Patent Application Publication No. 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; U.S. Patent Application Publication No. 2003 / 0115614; U.S. Patent Application Publication No. 2002 / 0164328; U.S. Patent Application Publication No. 2004 / 0093621; U.S. Patent Application Publication No. 2004 / 0132 140; U.S. Patent Application Publication No. 2004 / 0110704; U.S. Patent Application Publication No. 2004 / 0110282; U.S. Patent Application Publication No. 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO 2005 / 053742; WO 2002 / 031140; Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells, which are unable to fucosylate proteins (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application Publication No. 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), as well as knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene, FUT8, has been knocked out (e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688). (2006); and WO 2003 / 085107).

[0148] Antigen-binding molecule variants, and particularly antibody variants, are also provided that have bisected oligosaccharides, for example, biantennary oligosaccharides attached to the Fc region of the antibody that are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Pat. No. 6,602,684 (Umana et al.); and U.S. Patent Application Publication No. 2005 / 0123546 (Umana et al.). Antibody variants that have at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0149] In one preferred embodiment of the antigen-binding molecule provided herein, at least one first antigen-binding domain and at least one second antigen-binding domain are linked to an Fc region.

[0150] Fc region variants In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antigen-binding molecules, and particularly antibodies, provided herein, thereby creating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (such as a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0151] In one preferred embodiment of the antigen-binding molecule provided herein, one first antigen-binding domain and one second antigen-binding domain are linked to an Fc region. In one preferred embodiment of the antigen-binding molecules provided herein, the Fc region is an Fc region that has a reduced ability to bind to an activating Fcγ receptor, compared to the ability of the Fc region of native human IgG to bind to an activating Fcγ receptor. In one preferred embodiment, the activating Fcγ receptor is human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), or human FcγRIIIa(F). In one preferred embodiment, the activating Fcγ receptor is human FcγRIa. In one preferred embodiment, the activating Fcγ receptor is human FcγRIIa(R). In one preferred embodiment, the activating Fcγ receptor is human FcγRIIa(H). In one preferred embodiment, the activating Fcγ receptor is human FcγRIIIa(V). In one preferred embodiment, the activating Fcγ receptor is human FcγRIIIa(F).

[0152] In one preferred embodiment, the Fc region comprises one or more of the following amino acid substitutions (all positions according to EU numbering): Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, or Trp at position 234; Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val, or Arg at position 235; Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro, or Tyr at position 236; Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr, or Arg at position 237; Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp, or Arg at position 238; Gln, His, Lys, Phe, Pro, Trp, Tyr, or Arg at position 239; Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val at position 265; Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp, or Tyr at position 266; Arg, His, Lys, Phe, Pro, Trp, or Tyr at position 267; Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 269; Ala, Arg, Asn, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 270; Arg, His, Phe, Ser, Thr, Trp, or Tyr at position 271; Arg, Asn, Asp, Gly, His, Phe, Ser, Trp, or Tyr at position 295; Arg, Gly, Lys, or Pro at position 296; Ala at 297th position; Arg, Gly, Lys, Pro, Trp, or Tyr at position 298; Arg, Lys, or Pro at position 300; Lys or Pro at position 324; Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr, or Val at position 325; Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 327; Arg, Asn, Gly, His, Lys, or Pro at position 328; Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, or Arg at position 329; Pro or Ser at position 330; Arg, Gly, or Lys at position 331; Arg, Lys, or Pro at position 332. In a preferred embodiment, the Fc region comprises 1 to 10, 1 to 5, 1 to 4, 1 to 3, or 1, 2, 3, 4, or 5 substitutions in the Fc region.

[0153] In certain other embodiments, the present invention contemplates antigen-binding molecules, and particularly antibody variants, that possess some, but not all, effector functions that make them desirable candidates for applications in which in vivo antibody half-life is important and certain effector functions (e.g., complement and ADCC) are unnecessary or harmful. To confirm the reduction / loss of CDC and / or ADCC activity, in vitro and / or in vivo cytotoxicity assays can be performed. For example, Fc receptor (FcR) binding assays can be performed to confirm that an antibody lacks FcγR binding (and thus likely lacks ADCC activity) but retains the ability to bind FcRn. NK cells, the primary cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. Expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Pat. No. 5,500,362 (see, e.g., Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)), and Hellstrom, I. et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985); 5,821,337 (see, Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assays may be utilized (see, e.g., ACT1™, a non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc., Mountain View, CA); and CytoTox 96®, a non-radioactive cytotoxicity assay (Promega, Madison, WI)).Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or in addition, the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model such as that disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can also be performed to confirm that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the C1q and C3c binding ELISAs in WO2006 / 029879 and WO2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). Determination of FcRn binding and in vivo clearance / half-life can also be performed using methods known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0154] Antigen-binding molecules, and particularly antibodies, comprising an Fc region with reduced effector function include those with substitutions at one or more of residues 238, 265, 269, 270, 297, 327, and 329 in the Fc region (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0155] Several antigen-binding molecules, and in particular antibodies, antibody variants with increased or decreased binding to FcRs have been described (see, e.g., U.S. Pat. No. 6,737,056; WO 2004 / 056312; and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0156] In certain embodiments, the antigen binding molecules, and in particular antibody variants, comprise an Fc region with one or more amino acid substitutions that improve ADCC, such as substitutions at positions 298, 333, and / or 334 (residues according to EU numbering) of the Fc region.

[0157] In some embodiments, alterations are made in the Fc region that result in altered (i.e., increased or decreased) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99 / 51642, and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0158] Antigen-binding molecules, and in particular antibodies, with increased half-life and increased binding to fetal Fc receptors (FcRn), which mediate maternal IgG transport to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in U.S. Patent Application Publication No. 2005 / 0014934 A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions therein that increase binding of the Fc region to FcRn. Such Fc variants include those with a substitution at one or more of residues 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 of the Fc region, e.g., a substitution at residue 434 of the Fc region (U.S. Patent No. 7,371,826).

[0159] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent No. 5,648,260; U.S. Patent No. 5,624,821; and WO 94 / 29351, relating to other examples of Fc region variants.

[0160] Cysteine Engineered Antibody Variants In certain embodiments, it may be desirable to generate cysteine-engineered antigen-binding molecules, particularly antibodies, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In certain embodiments, the substituted residues are located at accessible sites of the antibody. By substituting these residues with cysteine, reactive thiol groups are located at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues may be substituted with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region. Cysteine-engineered antibodies may be generated, for example, as described in U.S. Patent No. 7,521,541.

[0161] antibody derivative In certain embodiments, the antigen-binding molecules, and particularly the antibodies, provided herein can be further modified to contain additional nonproteinaceous moieties known and readily available in the art. Moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (such as glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and when multiple polymers are attached, they may be the same or different molecules. Generally, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the specific properties or functions of the antigen-binding molecule, and particularly the antibody, that are to be improved, and whether the antibody derivative will be used for therapy under defined conditions.

[0162] In another embodiment, the conjugate of antigen-binding molecule, and in particular antibody, and non-proteinaceous moiety can be selectively heated by exposure to radiation is provided.In one embodiment, such non-proteinaceous moiety is carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)).The radiation can be of any wavelength, and includes, but is not limited to, the wavelength that heats the non-proteinaceous moiety to a temperature that kills the cells adjacent to the antibody-non-proteinaceous moiety, while not damaging normal cells.

[0163] Recombinant methods and compositions In one aspect herein, one or more nucleic acids are provided that encode the antigen-binding molecule described in any one of the embodiments herein.Optionally, the one or more nucleic acids are operably linked to a promoter.Further provided is one or more vectors that comprise one or more nucleic acids provided herein.Further provided herein is a host cell that (i) comprises one or more nucleic acids provided herein; (ii) comprises one or more vectors provided herein; and / or (iii) is capable of expressing the antigen-binding molecule described in any one of the aspects and embodiments herein.

[0164] Further provided is a method for producing the antigen-binding molecules provided herein, the method comprising culturing the host cells provided herein to produce antibodies; and optionally, further comprising recovering the antibodies from the host cells. Antigen-binding molecules, and particularly antibodies, can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. In one embodiment, an isolated nucleic acid is provided encoding a first antigen-binding domain comprising a variable region described herein. Such a nucleic acid may encode an amino acid sequence comprising a VL and / or a VH of a first antigen-binding domain comprising a variable region (e.g., an antibody light chain and / or heavy chain), and may optionally further encode an amino acid sequence comprising a VL and / or a VH of a second antigen-binding domain comprising a variable region (e.g., an antibody light chain and / or heavy chain). Alternatively, the sequences may be provided as two or more separate nucleic acids. In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acids are provided. In a further embodiment, a host cell comprising such nucleic acids is provided. In one such embodiment, the host cell comprises (e.g., has been transformed with) the following: (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of a first antigen-binding domain and an amino acid sequence comprising the VH of the first antigen-binding domain, and a nucleic acid encoding an amino acid sequence comprising the VL of a second antigen-binding domain and an amino acid sequence comprising the VH of the second antigen-binding domain, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the first antigen-binding domain, a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the first antigen-binding domain, a third vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the second antigen-binding domain, and a fourth vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the second antigen-binding domain. In one embodiment, the host cell is a eukaryotic cell, e.g., a Chinese hamster ovary (CHO) cell, or a lymphoid cell (e.g., a YO cell, an NS0 cell, or an Sp2 / 0 cell).In one embodiment, a method for producing an antigen-binding molecule, and particularly an antibody, is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antigen-binding molecule, particularly an antibody, as provided above under conditions suitable for expression of the antibody, and optionally recovering the antigen-binding molecule or antibody from the host cell (or culture medium of the host cell).

[0165] For recombinant production of antigen-binding molecules, particularly antibodies, nucleic acids encoding the antigen-binding molecules described above, such as antibodies, are isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody).

[0166] Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. In particular, when glycosylation and Fc effector function are not required, antibodies can be produced, for example, in bacteria. For the expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523 (see also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli). After expression, the antibody can be isolated as a soluble fraction from the bacterial cell paste and can be further purified.

[0167] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains whose glycosylation pathways have been "humanized" to result in production of antibodies with partial or fully human glycosylation patterns. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li et al., Nat. Biotech. 24:210-215 (2006).

[0168] Suitable host cells for expressing glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. In particular, numerous baculovirus strains have been identified that can be used in conjunction with insect cells for transfection into Spodoptera frugiperda cells.

[0169] Plant cell cultures can also be used as hosts. See, e.g., U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (which describe PLANTIBODIES™ technology for producing antibodies in transgenic plants).

[0170] Vertebrate cells can also be used as hosts, for example, mammalian cell lines adapted to growth in suspension can be useful. Other examples of useful mammalian host cell lines are the following: SV40-transformed monkey kidney CV1 line (COS-7); human embryonic kidney cell lines (such as 293 or 293 cells, as described, for example, in Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse Sertoli cells (such as TM4 cells, as described, for example, in Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK); buffalo rat hepatocytes (BRL 3A); human lung cells (W138); human hepatocytes (Hep G2); mouse mammary tumor (MMT 060562); see, for example, Mather et al., Annals NY Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, such as DHFR - Cells including CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of several mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0171] Preparation and screening methods The antigen-binding molecules provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays known in the art. Further provided herein is a method for producing an antigen-binding molecule, the method comprising the steps of: (a) selecting a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; (b) selecting a second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide; (c) obtaining one or more nucleic acids encoding an antigen-binding molecule to which the antigen-binding domain prepared in (a) and the antigen-binding domain prepared in (b) are linked; and (d) Producing an antigen-binding molecule using one or more nucleic acids prepared in (c). In one preferred embodiment herein, the method further comprises the following step (e): (e) determining whether the antigen-binding molecule of (d) has increased (i) persistence, (ii) concentration, or (iii) exposure in the brain of the subject compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule of (d) only in that it contains either one of the antigen-binding domains defined in (a) and (b), but not both. Embodiments of any of the other aspects and embodiments herein also apply to these methods.

[0172] There is further provided a method for screening an antigen-binding molecule, the method comprising the steps of: (a) selecting a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; (b) selecting a second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide; (c) obtaining one or more nucleic acids encoding an antigen-binding molecule to which the antigen-binding domain prepared in (a) and the antigen-binding domain prepared in (b) are linked; and (d) Producing an antigen-binding molecule using one or more nucleic acids prepared in (c). In one preferred embodiment herein, the method further comprises the following step (e): (e) determining whether the antigen-binding molecule of (d) has increased (i) persistence, (ii) concentration, or (iii) exposure in the brain of the subject compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule of (d) only in that it contains either one of the antigen-binding domains defined in (a) and (b), but not both. Embodiments of any of the other aspects and embodiments herein also apply to these methods.

[0173] In one embodiment, an antigen-binding molecule such as an antibody of the present invention is tested for its antigen-binding activity by known methods, such as, for example, ELISA, Western blot, and the like.

[0174] Methods and Compositions for Diagnostics and Detection In certain embodiments, any of the antigen binding molecules provided herein is useful for detecting the presence of first target and / or second target in biological sample.As used herein, the term "detection" includes quantitative detection or qualitative detection.In certain embodiments, biological sample comprises cell or tissue, for example, brain tissue and brain cell.

[0175] In certain embodiments, labeled antigen-binding molecules, particularly labeled antibodies, are provided. Labels include directly detectable labels or moieties (e.g., fluorescent labels, chromophore labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and also indirectly detectable moieties, such as enzymes or ligands, for example, via enzymatic reactions or molecular interactions. Exemplary labels include, but are not limited to, radioisotopes. 32 P, 14 C. 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates, or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luciferases such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, β-galactosidase, glucoamylase, lysozyme, sugar oxidases such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, those coupled with enzymes such as HRP, lactoperoxidase, or microperoxidase that utilize hydrogen peroxide to oxidize dye precursors, biotin / avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

[0176] Pharmaceutical preparations Provided herein is a pharmaceutical composition comprising an antigen-binding molecule according to any one of the aspects and embodiments herein, and one or more pharmaceutically acceptable carriers or excipients. Also provided is a pharmaceutical composition comprising (i) an antigen-binding molecule provided herein, (ii) one or more nucleic acids encoding the antigen-binding molecule provided herein, (iii) one or more vectors provided herein, or (iv) a host cell provided herein, and one or more pharmaceutically acceptable carriers or excipients.

[0177] Pharmaceutical formulations of the antigen-binding molecules described herein are prepared in the form of lyophilized formulations or aqueous solutions by mixing such antibodies having the desired purity with one or more pharmaceutically acceptable carriers ("Remington's Pharmaceutical Sciences," 16th ed., Osol, A., ed. (1980)). Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to, buffers such as phosphate, citric acid, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol, or benzyl alcohol; alkylparabens such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-chloroisothiazolinone). resols, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes, etc.); and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include agents that distribute the agent in the intercellular matrix, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.).Some exemplary sHASEGPs, including rHuPH20, and methods of use are described in U.S. Patent Application Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0178] Exemplary lyophilized antibody formulations are described in U.S. Patent No. 6,267,958. Aqueous antibody formulations include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter formulations comprising a histidine-acetate buffer.

[0179] The formulations herein may also contain more than one active ingredient as required for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other, and such active ingredients are suitably present in combination in amounts that are effective for the purpose intended.

[0180] The active ingredient may be encapsulated in microcapsules, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, prepared by coacervation techniques or interfacial polymerization, colloidal drug delivery systems (such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in "Remington's Pharmaceutical Sciences," 16th ed., Osol, A. (1980).

[0181] Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.

[0182] Preparations to be used for in vivo administration are generally sterile. Sterilization may be readily accomplished, for example, by filtration through sterile filtration membranes.

[0183] Therapeutic Methods and Compositions Any of the antigen-binding molecules and particularly antibodies provided herein, as well as the nuclei, vectors, or host cells and pharmaceutical compositions, may be used in therapeutic methods. In one embodiment, an antigen-binding molecule or antibody is provided for use as a pharmaceutical. In one embodiment, provided herein is (i) an antigen-binding molecule provided herein, (ii) one or more nucleic acids encoding the antigen-binding molecule provided herein, (iii) one or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein, for use as a pharmaceutical. In one embodiment, provided herein are (i) an antigen-binding molecule provided herein, (ii) one or more nucleic acids encoding the antigen-binding molecule provided herein, (iii) one or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein, for use as a diagnostic agent. In one embodiment, provided herein is (i) an antigen-binding molecule provided herein, (ii) one or more nucleic acids encoding the antigen-binding molecule provided herein, (iii) one or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein, for use in treating and / or preventing a brain disorder or brain disease in a subject. In one embodiment, provided herein is a method for treating and / or preventing a brain disorder or disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of (i) an antigen-binding molecule provided herein, (ii) one or more nucleic acids encoding the antigen-binding molecules provided herein, (iii) one or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein. In one embodiment, provided herein is (i) an antigen-binding molecule provided herein, (ii) one or more nucleic acids encoding the antigen-binding molecule provided herein, (iii) one or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein, for use in the preparation of a medicament for the treatment and / or prevention of a brain disorder or brain disease. In one embodiment, provided herein is (i) an antigen-binding molecule provided herein, (ii) one or more nucleic acids encoding the antigen-binding molecule provided herein, (iii) one or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein, for preparing a medicament for the treatment and / or prevention of a brain disorder or brain disease. In some embodiments, the brain disorder or disease is selected from the group consisting of neurodegenerative diseases (including but not limited to: Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including but not limited to Alzheimer's disease and supranuclear palsy), prion diseases (including but not limited to bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsies, motor neuron diseases, and heterogeneous degenerative disorders of the nervous system (including but not limited to Canavan disease, Huntington's disease, Brain disorders or diseases include, but are not limited to, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome, dementia (including but not limited to Pick's disease and spinocerebellar ataxia), psychiatric disorders, cancer (e.g., cancer of the CNS, including metastases to the brain arising from cancer elsewhere in the body). In some embodiments, the brain disorder or disease is selected from the group consisting of Alzheimer's disease, Pompe disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS). In one embodiment, there is provided an antigen-binding molecule of any of the aspects and embodiments herein for use (i) in a method of increasing the concentration of an antigen-binding molecule in the brain of a subject in need thereof; (ii) in a method of increasing the exposure of an antigen-binding molecule in the brain of a subject in need thereof; and / or (iii) for use in the persistence of an antigen-binding molecule in the brain of a subject in need thereof; wherein optionally the subject is a human. In one embodiment, the "subject" is a human. In one embodiment, a "subject in need thereof" is a subject having or suspected of having a brain disease or disorder.

[0184] The antigen-binding molecules of the present invention, particularly antibodies, can be used alone or in combination with other agents in therapy. For example, the antibodies of the present invention can be co-administered with at least one additional therapeutic agent. The choice of additional therapeutic agent depends on the brain disease or brain disorder to be treated.

[0185] Such combination therapy as described above includes mixed administration (two or more therapeutic agents contained in the same or different formulations) and separate administration, in which case the administration of the antibody of the present invention can be performed before, simultaneously with, and / or after the administration of one or more additional therapeutic agents. In one embodiment, the administration of the antigen-binding molecule and the administration of the additional therapeutic agent are performed within about one month, or within about one, two, or three weeks, or within about one, two, three, four, five, or six days of each other. The antigen-binding molecule of the present invention can also be used in combination with radiation therapy.

[0186] The antigen-binding molecule (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal administration, and, if local treatment is desired, intralesional administration. Parenteral administration includes intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Administration can be by any suitable route, such as injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-term or long-term. Various administration schedules are contemplated herein, including, but not limited to, a single administration or multiple administrations at various time points, bolus administration, and pulse infusion.

[0187] The antigen-binding molecules of the present invention can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the specific disorder being treated, the specific mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the delivery site of the agent, the administration method, the administration schedule, and other factors known to the physician. The antigen-binding molecules are optionally formulated with one or more agents currently used to prevent or treat the disorder in question, but this is not required. The effective amount of such other agents depends on the amount of antigen-binding molecules present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and via the administration routes described herein, or at about 1 to 99% of the dosages described herein, or at any dosage and via any route determined experimentally / clinically to be appropriate.

[0188] The appropriate dosage of the antigen-binding molecule of the present invention for disease prevention or treatment (when used alone or in combination with one or more other additional therapeutic agents) depends on the type of disease being treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, the individual's medical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient in a single treatment or over a series of treatments. For example, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) may be an initial candidate dosage for administration to a patient, depending on the type and severity of the disease, whether administered in a single administration or multiple separate administrations, or by continuous infusion. A typical daily dosage may range from about 1 μg / kg to 100 mg / kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, treatment can generally be sustained until a desired suppression of disease symptoms occurs, depending on the condition. An exemplary dosage of an antigen-binding molecule can be in the range of about 0.05 mg / kg to about 10 mg / kg. Thus, one or more doses of about 0.5 mg / kg, 2.0 mg / kg, 4.0 mg / kg, or 10 mg / kg (or any combination thereof) can be administered to a patient. Such doses can be administered intermittently, for example, weekly or every three weeks (e.g., so that the patient receives about two to about 20, or, for example, about six, doses of the antibody). A larger initial loading dose can be administered, followed by one or more smaller doses. The progress of such therapy is easily monitored by conventional techniques and assays.

[0189] Brain damage or disease As used herein, "neurological disorder" refers to a disease or disorder that affects and / or has its etiology in the brain / CNS. Exemplary brain diseases or disorders include, but are not limited to, neuropathy, amyloidosis, cancer, eye disease or disorder, viral or microbial infection, inflammation, ischemia, neurodegenerative disease, stroke, behavioral disorder, and lysosomal storage disease. For purposes of this application, the CNS is understood to include the eye, which is normally separated from the rest of the body by the blood-retinal barrier. Specific examples of brain disorders or diseases include, but are not limited to, neurodegenerative diseases (including, but not limited to, Lewy body disease, post-polio syndrome, Shy-Drager syndrome, olivopontocerebellar atrophy, Parkinson's disease, multiple system atrophy, striatonigral degeneration, tauopathies (including, but not limited to, Alzheimer's disease and supranuclear palsy), prion diseases (including, but not limited to, bovine spongiform encephalopathy, scrapie, Creutzfeldt-Jakob syndrome, kuru, Gerstmann-Straussler-Scheinker disease, chronic wasting disease, and fatal familial insomnia), bulbar palsies, motor neuron diseases, and heterogeneous degenerative disorders of the nervous system (including, but not limited to, Canavan disease, Huntington's disease, Brain disorders or diseases include, but are not limited to, neuronal ceroid lipofuscinosis, Alexander disease, Tourette's syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Hallervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Lesch-Nyhan syndrome, and Unverricht-Lundborg syndrome, dementia (including but not limited to Pick's disease and spinocerebellar ataxia), psychiatric disorders, cancer (e.g., cancer of the CNS, including metastases to the brain arising from cancer elsewhere in the body). In some specific examples, the brain disorder or disease is selected from the group consisting of Alzheimer's disease, Pompe disease, frontotemporal dementia (FTD), and amyotrophic lateral sclerosis (ALS). In some embodiments, the corresponding functional moieties contained in the antigen-binding molecules of the present invention are described herein, and include, but are not limited to, antibodies, aptamers, proteins, peptides, inhibitory nucleic acids, and inhibitory small molecules, and active fragments of any of the foregoing, which either themselves or specifically recognize and / or specifically act on (i.e., inhibit, activate, or detect) CNS antigens or target molecules, such as, but not limited to, amyloid precursor protein or a portion thereof, amyloid beta, beta-secretase, gamma-secretase, tau, alpha-synuclein, parkin, huntingtin, DR6, presenilin, ApoE, markers of glioma or other CNS cancers, and neurotrophins. Non-limiting examples of brain disorders or diseases that can be used to treat and the corresponding functional moieties contained in the antigen-binding molecules of the present invention described herein are provided in Table A below: [Table A] TIFF2025525307000006.tif56133

[0190] product In another aspect of the present invention, an article of manufacture containing a substance useful for the treatment, prevention, and / or diagnosis of the above-mentioned brain disorders or diseases is provided. The article of manufacture includes a container and a label attached to the container or a package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The container can be formed from a variety of materials, such as glass or plastic. The container holds the composition alone or in combination with another composition effective for treating, preventing, and / or diagnosing a condition and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active ingredient in the composition is an antigen-binding molecule of the present invention, such as an antibody. The label or package insert indicates that the composition is used to treat a selected condition. Furthermore, the article of manufacture may include: (a) a first container containing a composition comprising an antigen-binding molecule of the present invention; and (b) a second container containing a composition comprising a cytotoxic or otherwise therapeutic agent. The article of manufacture in this aspect of the invention may further comprise a package insert indicating that the composition can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. Other materials desirable from a commercial and user standpoint may further be included, including other buffers, diluents, filters, needles, and syringes. [Example]

[0191] The following are examples of methods and compositions of the present invention. It will be understood that various other embodiments may be practiced in light of the general description provided above.

[0192] Example 1. Concept for improving brain concentration and exposure of antigen-binding molecules such as antibodies by designing molecules with brain-translocating and brain-retentive / targeting moieties One of the major limitations of therapeutic drugs in the CNS is their low therapeutic exposure due to their low transport capacity. To address this issue, receptor-mediated transport systems (RMTs) have been developed and used to cross the blood-brain barrier (BBB). One example of an RMT utilizes a transferrin receptor-binding moiety, such as an antibody that binds to the transferrin receptor (TfR). The transferrin receptor is expressed on endothelial cells, including those of the BBB, and enables transport across the BBB via RMT [Lajoie, Jason M., and Eric V. Shusta. "Targeting receptor-mediated transport for delivery of biologics across the blood-brain barrier." Annual review of pharmacology and toxicology 55 (2015): 613].

[0193] In addition, other receptors, such as the insulin receptor and low-density lipoprotein receptor (LDLR), have also been reported to be usable as RMT-based BBB-crossing moieties for delivering biologics to the brain [Lajoie, Jason M., and Eric V. Shusta, supra]. RMT systems, such as anti-TfR antibodies and anti-insulin receptor antibodies, are known to have higher brain penetration ratios compared to IgG in plasma. However, it is well known that anti-transferrin receptor antibodies have a short half-life in plasma due to the fact that transferrin receptors are expressed not only in vascular endothelial cells in the brain but also systemically (i.e., widely throughout the body, including many other cell types other than brain cells). Therefore, anti-TfR antibodies deliver drugs to tissues other than the brain, resulting in a short blood half-life. Similarly, other antibodies with BBB-crossing ability have also shown rapid clearance and short half-lives (anti-CD98hc antibody, Neuron Vol. 90, Issue 1, 2016, 70-82). In addition, because interstitial fluid (ISF) in the brain is constantly replaced, antibodies that enter the brain quickly drain from the brain into the blood circulation. Therefore, after draining into the blood circulation, the antibodies must be transported back to the brain via the BBB. For this reason, therapeutic molecules fused to anti-transferrin receptor antibodies only show a short or limited period of sustained improvement in antibody concentration in the brain, limiting the efficacy of the therapeutic molecules in the brain (Figure 1(a)).

[0194] Another approach has been proposed by using antibodies that bind to myelin oligodendrocyte glycoprotein (MOG, a brain-specific antigen), and this approach achieves high concentrations and persistence of the antibody in the brain (PLoS One 2019, Apr 12;14(4)e0214404), which slows the exit of the antibody from the brain as a result of binding to MOG. However, without being bound by theory, the inventors of the present invention predicted that while the mechanism of this "brain-persistence" approach allows overcoming the low exposure of molecules having a transferrin receptor-binding domain, the anti-MOG antibody must have a long half-life in plasma in order to gradually accumulate and achieve a high concentration in the brain. This is because this approach requires the antibody to be continuously delivered from the blood circulation to the brain (Figure 1(b)). For this reason, the anti-MOG antibody requires a sustained high concentration in plasma (or a long half-life in plasma) to achieve and maintain a high concentration in the brain, and therefore, its brain-persistence effect will be reduced or limited when combined with a therapeutic molecule that originally has a short half-life in plasma. Another limitation of this approach is that the anti-MOG antibody requires a fairly long time after administration to gradually accumulate and reach the desired concentration and exposure of the antibody in the brain, which is not ideal from the perspective of the PK profile. Given this situation, novel technologies are needed that allow molecules to have both high antibody concentrations and exposure in the brain quickly, and that are maintained over long periods of time.

[0195] As explained above, considering that BBB-crossing technologies, such as anti-TfR antibodies, are known to have short half-lives in plasma, while brain-persistent technologies, such as anti-MOG antibodies, are expected to gradually accumulate in the brain and require long half-lives in plasma to achieve high concentrations, attempting to combine both technologies does not appear to be a likely successful approach, and therefore, for this reason, molecules with both anti-TfR and anti-MOG domains have not been reported or tested. In the present application, the present inventors surprisingly found that a molecule comprising both a brain-translocating moiety and a brain-retaining moiety can rapidly achieve high antibody concentrations and exposure in the brain and achieve high persistence in the brain over a long period of time, despite having a short half-life in plasma (Figure 1(c)). Figure 2(a) is a schematic diagram illustrating the concept of an exemplary embodiment of an antigen-binding molecule of the present invention, which comprises (1) a brain-translocating moiety, (2) a brain-retaining moiety, optionally (3) a functional moiety, and, optionally, a half-life-extending moiety in addition to the above. The brain-translocating moiety is an antigen-binding domain that specifically binds to a molecule that promotes the penetration or migration of an antigen-binding molecule into the brain. For example, the molecule that promotes penetration or migration into the brain is a receptor expressed on vascular endothelial cells, preferably on vascular endothelial cells of the blood-brain barrier (BBB), which allows biologics to be transported to the brain via the RMT pathway. For example, the RMT-based receptor-mediated brain migration of biologics using anti-TfR antibodies, anti-insulin receptor antibodies, or anti-LDLR antibodies has been extensively studied and reported [Lajoie, Jason M., and Eric V. Shusta, supra]. In a non-limiting example, the molecule is a molecule that is specifically expressed on vascular endothelial cells of the blood-brain barrier (BBB). Exemplary molecules are one or more molecules selected from those listed in Table 1. In one example, the brain-translocating moiety is an antigen-binding domain that specifically binds to transferrin receptor (TfR). In another example, the brain-localizing moiety is an antigen-binding domain that specifically binds to the insulin receptor. In one embodiment, the brain persistence moiety is an antigen binding domain that binds to a molecule that is expressed on the cell membrane of brain cells or is a brain ECM protein or brain ECM polysaccharide.It has been reported that binding to brain antigens can slow down the outflow of molecules from the brain, thereby achieving higher persistence in the brain (Nakano, Ryosuke, et al. "A new technology for increasing therapeutic protein levels in the brain over extended periods." Plos one 14.4 (2019): e0214404; WO2018123979, WO2020004490, WO2020004492).Exemplary targets are one or more targets selected from those listed in Table 2.

[0196] In some specific embodiments, by selecting a brain-specific antigen, it is possible to increase the antibody concentration specifically only in the brain and maintain it in the brain for a long period of time. In one example of the present disclosure, the brain cell can be any brain cell, such as a brain cell selected from the group consisting of oligodendrocytes, astrocytes, neurons, and microglia. Exemplary target molecules and target cells to which the brain-persistent moiety binds are shown in Table 2. In addition, the target molecule can be any molecule that has been determined to be specifically expressed on brain-specific cells by characterizing its expression pattern as follows: 1. Select a molecule of interest; 2. Confirm the expression level (either RNA / protein / actual data); 3. Compare the expression level in the brain and other regions (organs / tissues); and 4. Select the molecule of interest that has a higher expression level in the brain than in other regions (organs / tissues). Cell markers commonly used to identify cell types in FACS analysis or other analyses are also useful for the brain-persistent moiety.

[0197] The functional moiety can be any molecule with a therapeutic function, such as, for example, an agonist, antagonist, enzyme, modulator, stabilizer, cell death inducer, and can be any molecular configuration, such as, for example, a nucleic acid, a small molecule, a cyclic peptide, a peptide, a ligand, a cytokine, a chemokine, a growth factor, an enzyme, and an antigen-binding domain. Optionally, one or more half-life extending moieties can also be fused to the molecule. Examples of half-life extending moieties are the Fc region of an immunoglobulin, an albumin-binding domain, an FcRn-binding protein, an FcRn-binding peptide, and PEG. 2(b) and 2(c) are schematic diagrams showing exemplary molecular configurations of antigen-binding molecules of the present disclosure. In one example, the antigen-binding molecule comprises a first Fab region (i.e., a brain-localizing moiety) that binds to a molecule that promotes penetration or migration of the antigen-binding molecule into the brain, and a second Fab region (i.e., a brain-persisting moiety) that binds to a molecule specifically expressed on brain-specific cells, and optionally further comprises a functional moiety. The structures of the brain-persisting moiety and the brain-localizing moiety are not limited to the Fab region and can also be in the form of an antibody fragment, such as a single-chain Fab (scFab), Fv, Fab, Fab', F(ab')2, diabody, triabody, scFv, VHH, diabody, or F(ab')2 fragment, or a non-antibody binder (e.g., an affibody, DARPin, FN3, aptamer, anticalin). In the examples, bispecific antibodies comprising single-chain Fab (scFab) or Fab were prepared.

[0198] [Table 1]

[0199] [Table 2] TIFF2025525307000009.tif146163TIFF2025525307000010.tif209163TIFF2025525307000011.tif194163TIFF2025525307000012.tif225163

[0200] Example 2. Preparation of molecules having brain-translocating and brain-retaining moieties An antibody designated MOG303 / / TfR, which contains one transferrin receptor (TfR)-binding domain (brain-translocating moiety) and two myelin oligodendrocyte glycoprotein (MOG)-binding domains (brain-persisting moieties), was generated as described below. The molecular structure of MOG303 / / TfR is shown in Figure 11(A). MOG303, a bivalent antibody containing two myelin oligodendrocyte glycoprotein (MOG)-binding domains; KLH, a bivalent anti-KLH (keyhole limpet hemocyanin) antibody; and KLH / / TfR, an antibody containing a KLH-binding domain and a TfR-binding domain, were also prepared by methods known in the art. Both MOG303 and KLH contain a modified hIgG1 heavy chain constant region (SEQ ID NO: 2). Expression vectors encoding the heavy and light chains of MOG303 / / TfR, MOG303, KLH-TfR, and KLH shown in Table 3 were constructed by methods known in the art.

[0201] [Table 3]

[0202] An expression vector encoding MOG303VH-SG181v11k.newmBBB (heavy chain 1: SEQ ID NO: 9) containing the VH region of an anti-MOG antibody (SEQ ID NO: 1), a modified human IgG1 constant region (CH1-hinge-CH2-CH3, with mutations that reduce binding to activating Fγ receptors (human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), and human FcγRIIIa(F)) and generate a heterodimeric Fc), and a single-chain Fab domain of an anti-mouse transferrin receptor antibody (constant region to scFab: SEQ ID NO: 5) followed by a Gly-Ser linker was prepared by methods known in the art. An expression vector encoding MOG303VH-SG181v11h (heavy chain 2: SEQ ID NO: 10) containing the VH region of the anti-MOG antibody (SEQ ID NO: 1), a modified human IgG1 constant region (CH1-hinge-CH2-CH3, with mutations that reduce binding to activating Fγ receptors (human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V), and human FcγRIIIa(F) and generate a heterodimeric Fc; SEQ ID NO: 6) was prepared by methods known in the art. An expression vector encoding MOG303VL-SK1 (light chain: SEQ ID NO: 8), which contains the VL region of an anti-MOG antibody (SEQ ID NO: 3) fused to a human kappa constant region (SEQ ID NO: 4), was prepared by methods known in the art. The MOG303 / / TfR antibody (heavy chain 1: SEQ ID NO: 9; heavy chain 2: SEQ ID NO: 10; light chain 1 and 2 sequences: SEQ ID NO: 8) with the molecular structure shown in FIG. 11(A) was expressed using a HEK293 cell expression system. Heavy chain 1, heavy chain 2, and light chains 1 and 2 were transfected into Expi293 cells according to the manufacturer's protocol. After culturing for several days, the culture medium was collected and subjected to affinity column chromatography. The antibody protein was purified using an affinity column (protein A) and subjected to size exclusion chromatography to obtain MOG303 / / TfR. Expression and purification methods were performed according to methods known in the art (Nat Protoc. 2018 Jan;13(1):99-117).

[0203] Example 3. Pharmacokinetic profile of antibodies in mouse brain and plasma The pharmacokinetics in brain and plasma of anti-KLH and anti-MOG antibodies with or without the anti-TfR Fab domain were evaluated in C57BL / 6J mice (male, 6-8 weeks old). The IgG antibodies KLH-IgG (KLH) and MOG303-IgG (MOG303), as well as the IgG antibodies KLH / / TfR and MOG303 / / TfR containing the anti-TfR domain, were intravenously administered to mice at a dose of 2 mg / kg. Blood was collected from days 1 to 28 after administration, and plasma was obtained by centrifugation (12,000 rpm, 4°C, 5 min). After blood collection, the brains were harvested after cardiac perfusion with phosphate-buffered saline (PBS) via catheter insertion. Fifty mg sheared brain fragments were lysed in PBS containing NP-40 substitute surfactant (Millipore) and Complete Mini protease inhibitor cocktail (Roche) with 5 mm stainless steel beads, and homogenized using a homogenizer (Qiagen). After rotating for 60 min at 4°C, the samples were centrifuged (15000 rpm, 4°C, 20 min) and the supernatant was collected. Antibody concentrations in plasma and brain were determined by electrochemiluminescence immunoassay (ECL). Anti-human IgG Fab'2 antibody (LifeSpan BioSciences) was applied to the plate as the capture antibody. After adding 1% bovine serum albumin (Sigma-Aldrich) in PBS containing Tween 20 (PBST, Sigma-Aldrich) as a blocking solution, diluted plasma and brain homogenate samples were applied. Biotin-labeled anti-human IgG antibody (Bethyl Laboratories) was added as the detection antibody. Finally, SULFO-tagged streptavidin (Meso Scale Discovery) was added, and the signal was detected by ECL (Meso Scale Discovery). The concentration-time profiles of the antibodies in plasma and brain were analyzed using a noncompartmental model with Phoenix WinNonlin (ver. 8.3) to determine the area under the curve (AUC), clearance, volume of distribution (Vd), and half-life. The brain / plasma ratio was calculated by dividing the concentration in the brain by the concentration in the plasma. The percent of injected dose per brain tissue weight was calculated by dividing the concentration in the brain by the administered dose.

[0204] The pharmacokinetic profiles of KLH, MOG303, KLH / / TfR, and MOG303 / / TfR in the brain are shown in Figure 3. On day 1 after administration, the control antibody, KLH, exhibited an antibody concentration in the brain of approximately 0.046 μg / g, a brain-to-plasma concentration ratio of 0.003, and 0.10% ID / g brain (i.e., percent of injected dose per brain tissue weight). For KLH, the concentration on day 1 was the highest concentration (Cmax) during the study period. MOG303 exhibited antibody concentration and brain distribution profiles similar to those of KLH on day 1. Meanwhile, KLH / / TfR exhibited an antibody concentration in the brain of 1.4 μg / g, a brain-to-plasma concentration ratio of 0.37, and 3.1% ID / g brain, which were higher than those of KLH and MOG303. The concentration of KLH / / TfR on day 1 was the Cmax of the study period. Surprisingly, MOG303 / / TfR exhibited approximately two-fold higher brain concentrations, brain-to-plasma concentration ratios, and %ID / g brain than KLH / / TfR on day 1 (2.8 μg / g brain, brain-to-plasma concentration ratio of 0.55, 6.1%ID / g brain). These results indicate that the combination of a transitory moiety (anti-TfR) and a long-acting moiety (anti-MOG) contributes to a significant increase in antibody concentration and distribution in the brain during the short period (1 day) after antibody infusion. From days 7 to 28, the brain concentration and percent dose of KLH gradually decreased (Figure 3(a)), and the brain-to-plasma ratio remained nearly constant (Figure 3(b)). The brain concentration, brain-to-plasma ratio, and percent dose of KLH / / TfR rapidly disappeared. The concentration after day 14 was lower than that of KLH, and the concentration at day 28 was below the limit of quantification (<0.0013 μg / g). The half-life of KLH / / TfR in the brain was 8.77 days. These results suggest that the antibody that entered the brain was cleared by CSF / ISF bulk flow and reverse transcytosis to plasma. On the other hand, MOG303 accumulated in the brain in a time-dependent manner after entering the brain due to binding to MOG protein in the brain. Finally, MOG303 concentrations reached Cmax on day 28 (0.32 μg / g and 0.70%ID / g). Interestingly, MOG303 / / TfR did not show a significant decrease in antibody concentration over time in the brain, and maintained high concentrations and distribution in the brain (2.2-3.1 μg / g and 4.7-7.0%ID / g) from day 1 through at least day 28. Cmax over the study period was 3.1 μg / g and 7.0%ID / g.

[0205] Antibodies with TfR-binding domains have been reported to be rapidly cleared from the brain, which was also observed with the control antibody with an anti-TfR domain that we tested, i.e., KLH / / TfR. Therefore, the observed high initial antibody concentrations in the brain of MOG303 / / TfR at day 1 and their persistence over at leas...

Claims

1. An antigen-binding molecule comprising a first antigen-binding domain and a second antigen-binding domain, The first antigen-binding domain specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; and the second antigen-binding domain specifically binds to a second target, which is (i) expressed on the cell membrane of a brain cell, or (ii) a brain extracellular matrix (ECM) protein or brain ECM polysaccharide; The antigen-binding molecule.

2. The antigen-binding molecule of claim 1, wherein the first target is a molecule expressed on vascular endothelial cells of the blood-brain barrier (BBB).

3. The antigen-binding molecule of claim 1 or 2, wherein the first target is selected from the group consisting of transferrin receptor (TfR), basigin (CD147), Glut1, Ldlrad3, CD320, insulin receptor, insulin-like growth factor 1 receptor (IGF1R), low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor-related protein (LRP), preferably LRP1, diphtheria toxin receptor, glucose receptor, CD98hc, TMEM30A, leptin receptor (LepR), and heparan sulfate chains branched from proteoglycans (HSPG).

4. The second target is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5), IGSF4B / SynCAM3 (CADM3), CNPase (2',3'-cyclic nucleotide 3'-phosphodiesterase), myelin-associated glycoprotein (MAG), myelin basic protein (MBP), EAAT1 (solute transporter family 1 member 3), EAAT2 (solute transporter family 1 member 2), MAP2 (microtubule-associated protein 2), and NEFL (neurofibrillar protein 2). Neurofilament light polypeptide), NEFM (neurofilament medium polypeptide), NSE (gamma-enolase), CD68 (macrosialin), allograft inflammatory factor 1 (IBA1 or AIF1), purinergic receptor (P2RY12), interleukin-1 receptor accessory protein-like 1 (IL1RAPL1), glutamate ionotropic receptor NMDA-type subunit 2B (GRIN2B), voltage-gated calcium channel auxiliary subunit gamma 8 (CACNG8), CD11b (integrin subunit αM), S LC6A2 (sodium-dependent noradrenaline transporter), DPP6 (dipeptidyl peptidase-like 6), SLC18A3 (vesicular acetylcholine transporter), sodium / potassium transporting ATPase subunit α-2, broad substrate specificity ATP-binding cassette transporter ABCG2, solute transporter family 12 member 9, electrogenic sodium bicarbonate cotransporter 1, excitatory amino acid transporter 2, chondroitin sulfate proteoglycan 4, immunoglobulin superfamily DCC subclone Smember 4, Vang-like protein 2, neural cell adhesion molecule 1 (N-CAM-1), low-density lipoprotein receptor-related protein 4 (LRP-4), glycosphingolipid-enriched microdomain-associated phosphoprotein 1 (Csk-binding protein), plasma membrane calcium-transporting ATPase 1, prominin-1, somatostatin receptor type 1, brain isoform of carnitine O-palmitoyltransferase 1 (CPT1-B), epidermal growth factor receptor, protein MAL2, syntaxin-1A, sodium / calcium exchanger 1,Lysophosphatidylcholine acyltransferase 1 (LPC acyltransferase 1), calsyntenin-3 (alcadein-β), type I receptor for pituitary adenylate cyclase-activating polypeptide (type I receptor for PACAP), neutral cholesterol ester hydrolase 1 (NCEH), CD166 antigen (activated leukocyte cell adhesion molecule), inactive tyrosine protein kinase 7, claudin-11, ectonucleotide phosphatase (ENPP6), tetraspanin-2 (Tspan-2), myelin proteolipid protein (PLP), glycolipid transfer protein (GLTP), versican core protein (chondroitin sulfate proteoglycan 2 or CSPG2), troponin The antigen-binding molecule of any one of claims 1 to 3, wherein the antigen-binding molecule is selected from the group consisting of elastin (elastin), collagen alpha-2(IV) chain (canstatin), proteoglycan link protein 1 (hyaluronan and proteoglycan link protein 1), tenascin-R (TN-R), proteoglycan link protein 2 (hyaluronan and proteoglycan link protein 2), collagen alpha-1(I) chain, neurofilament-3 (NEF3), immunoglobulin superfamily member 8 (IgSF8), laminin subunit gamma-1 (LAMC1), collagen alpha-1(VI) chain (Col6a1), and collagen alpha-3(VI) chain (Col6a3).

5. The antigen-binding molecule of any one of claims 1 to 4, wherein the brain cells comprise one or more types of cells selected from the group consisting of oligodendrocytes, astrocytes, neurons, and microglia.

6. The antigen-binding molecule of any one of claims 1 to 5, wherein the second target is a molecule that is predominantly expressed on brain-specific cells.

7. The antigen-binding molecule of any one of claims 1 to 6, wherein the first target is selected from the group consisting of transferrin receptor (TfR), basigin (CD147), insulin receptor, insulin-like growth factor 1 receptor (IGF1R), low-density lipoprotein receptor (LDLR), low-density lipoprotein receptor-related protein (LRP), preferably LRP1, diphtheria toxin receptor, glucose receptor, preferably Glut1, and CD98hc.

8. The antigen-binding molecule of any one of claims 1 to 7, wherein the second target is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5), and cell adhesion molecule 3 (CADM3).

9. (i) the first target is transferrin receptor (TfR) and the second target is myelin oligodendrocyte glycoprotein (MOG); or (ii) the first target is transferrin receptor (TfR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (iii) the first target is transferrin receptor (TfR) and the second target is cell adhesion molecule 3 (CADM3); or (iv) the first target is basigin (CD147) and the second target is myelin oligodendrocyte glycoprotein (MOG); or (v) the first target is basigin (CD147) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (vi) the first target is basigin (CD147) and the second target is cell adhesion molecule 3 (CADM3); or (vii) the first target is an insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); or (viii) the first target is the insulin receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (ix) the first target is an insulin receptor and the second target is cell adhesion molecule 3 (CADM3); or (x) the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xi) the first target is a glucose receptor and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xii) the first target is a glucose receptor and the second target is cell adhesion molecule 3 (CADM3); or (xiii) the first target is low-density lipoprotein receptor (LDLR) and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xiv) the first target is low-density lipoprotein receptor (LDLR) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xv) the first target is low-density lipoprotein receptor (LDLR) and the second target is cell adhesion molecule 3 (CADM3); or (xvi) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xvii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xviii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is cell adhesion molecule 3 (CADM3); or (xix) the first target is CD98hc and the second target is myelin oligodendrocyte glycoprotein (MOG); or (xx) the first target is CD98hc and the second target is neuroglycan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xxi) the first target is CD98hc and the second target is cell adhesion molecule 3 (CADM3); The antigen-binding molecule of any one of claims 1 to 8.

10. The second target is selected from the group consisting of beta-secretase 1 (BACE1), Abeta, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), alpha-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, gamma secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophin receptor (p75NTR), caspase 6, TRK A, TRK B, TRK The antigen-binding molecule of any one of claims 1 to 4, which is not a target selected from the group consisting of C, alpha-synuclein, beta-synuclein, gamma-synuclein, vascular endothelial growth factor (VEGF), neuropilin, semaphorin, semaphorin 3A, semaphorin 4A, semaphorin 6A, myelin basic protein (MBP), MOG, PLP, MAG, aquaporin 4, glutamate receptor, and EpCAM.

11. (i) at least one functional moiety, including an enzyme, a therapeutic protein, an antibody or antigen-binding fragment thereof, a peptide, DNA, shRNA, siRNA, a small molecule drug, or a cytotoxic agent; and / or (ii) at least one in vivo half-life extending moiety, preferably selected from the group consisting of an Fc region, an albumin binding domain, an FcRn binding protein, an FcRn binding peptide, and a PEG moiety; The antigen-binding molecule of any one of claims 1 to 10, further comprising:

12. 1. A method for increasing the concentration of an antigen-binding molecule in the brain of a subject in need thereof, comprising: so that the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) (a1) a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; or (a2) A second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide. providing a first antigen-binding molecule, comprising: (b) In the case of (a1), introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule; or In the case of (a2), introducing at least one first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. The method comprising:

13. Step (c): (c) determining that the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to a control antigen-binding molecule, wherein the control antigen-binding molecule is: In the case of (a1), at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide; or In the case of (a2), at least one first antigen-binding domain that specifically binds to a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain. (b) a second antigen-binding molecule, which differs from the second antigen-binding molecule described in (b) only in that it does not contain 13. The method of claim 12, further comprising:

14. The method of claim 12 or 13, wherein the concentration of the second antigen-binding molecule in the subject's brain is Cmax.

15. A method for increasing the exposure of an antigen-binding molecule in the brain of a subject in need thereof, comprising: so that the exposure amount of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) (a1) a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain; or (a2) A second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide. providing a first antigen-binding molecule, comprising: (b) In the case of (a1), introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule; or In the case of (a2), introducing at least one first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the mammalian brain into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. The method comprising:

16. Step (c): (c) determining that the exposure of the second antigen-binding molecule in the brain of the subject is increased compared to a control antigen-binding molecule, wherein the control antigen-binding molecule is In the case of (a1), at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide; or In the case of (a2), at least one first antigen-binding domain that specifically binds to a first target that promotes the translocation of the antigen-binding molecule into the mammalian brain. (b) a second antigen-binding molecule, which differs from the second antigen-binding molecule described in (b) only in that it does not contain 16. The method of claim 15, further comprising:

17. The method of claim 15 or 16, wherein the exposure amount of the second antigen-binding molecule in the subject's brain is the AUC (area under the curve) of the brain concentration-time profile of the antigen-binding molecule.

18. 1. A method for persistence of an antigen-binding molecule in the brain of a subject in need thereof, comprising: such that the persistence of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule upon administration to the subject. (a) providing a first antigen-binding molecule comprising a first antigen-binding domain that specifically binds to a first target that promotes translocation of the antigen-binding molecule into the brain of a mammal; (b) introducing at least one second antigen-binding domain that specifically binds to a second target, wherein the second target is (i) expressed on the cell membrane of a brain cell, or (ii) a brain ECM protein or brain ECM polysaccharide, into the first antigen-binding molecule, thereby obtaining a second antigen-binding molecule. The method comprising:

19. Step (c): (c) determining that the persistence of the second antigen-binding molecule in the brain of the subject is increased compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the second antigen-binding molecule of (b) only in that it does not contain at least one antigen-binding domain that specifically binds to a second target.

20. The method of claim 18, further comprising:

20. The method of claim 18 or 19, wherein the persistence of the second antigen-binding molecule in the subject's brain is its in vivo half-life in the brain.

21. The antigen-binding molecule of any one of claims 1 to 11, or the method of any one of claims 12 to 20, wherein the mammalian brain is a human brain.

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