Antigen-binding molecules with increased penetration and retention in brain and methods of use thereof

Abstract

The present invention relates to antigen binding molecules with increased penetration and / or retention in the brain; methods of use thereof; and a production or screening method thereof.

Description

Technical Field

[0001] The present invention relates to antigen-binding molecules with increased penetration and / or retention in the brain; methods of using the same; and methods of producing or screening the same. The present disclosure relates to antigen-binding molecules with increased concentration, exposure and / or retention in the brain; methods for increasing the concentration, exposure and / or retention of antigen-binding molecules in the brain; and methods for producing and screening antigen-binding molecules with increased concentration, exposure and / or retention in the brain.

[0002] On the one hand, 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, the first target promotes the transfer of the antigen binding molecule to the mammalian brain (through the blood-brain barrier), and the second antigen binding domain specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is 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 antigen-binding molecules, nucleic acids or two or more nucleic acids encoding antigen-binding molecules, vectors or two or more vectors comprising the nucleic acids or two or more nucleic acids, host cells comprising (i) the nucleic acids or two or more nucleic acids or (ii) the vectors or two or more vectors, methods for producing antigen-binding molecules and screening methods, methods for increasing the concentration of antigen-binding molecules in the brain of a subject in need thereof, methods for increasing the exposure of antigen-binding molecules in the brain of a subject in need thereof and methods for retaining antigen-binding molecules in the brain of a subject in need thereof, as well as methods for treating subjects and medical uses. Background Art

[0003] Biological products such as antibodies offer the advantages of high specificity, efficacy and low off-target toxicity, however, delivery of these drugs to the brain remains the biggest challenge in developing therapeutic agents for the treatment of central nervous system diseases. The main obstacle is the blood-brain barrier (BBB), which hinders the entry of most molecules present in the systemic circulation, especially macromolecular drugs. Due to the presence of the BBB, it is difficult to obtain effective concentrations when administering drugs in the central nervous system, which brings difficulties to drug development.

[0004] Various delivery technologies are being studied to increase the concentration of macromolecular substances such as biologics in the brain. The technology reported mostly uses receptor-mediated transcytosis (RMT), and is expressed in the cerebral vascular endothelium to serve as a receptor of a target, including, for example, transferrin receptor, insulin receptor, insulin-like growth factor receptor (IGFR), low-density lipoprotein receptor family (LDLRf) etc. For example, it has been reported that the fusion protein by producing anti-transferrin receptor antibodies and nerve growth factor, via the technology of passing through BBB via anti-transferrin receptor. 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 have been reported (WO 2016 / 081640 (PTL 1), WO 2015 / 191934 (PTL 2), WO 2016 / 081643 (PTL 3), and fusion antibodies obtained by fusing a monovalent anti-transferrin receptor antibody to the carboxyl terminal side of an anti-β-amyloid antibody (WO 2014 / 033074 (PTL 4)). However, transferrin receptor and insulin receptor are expressed not only in cerebrovascular endothelial cells but also widely throughout the body and non-brain tissues, and therefore, drugs are also delivered to non-brain tissues. Since the antigen is expressed throughout the body, the half-life of the antibody in the blood is short. Therefore, BBB transfer techniques such as fusion of anti-transferrin receptor binding antibodies or anti-insulin receptor antibodies have the disadvantage of rapid clearance from systemic circulation (Sci Transl Med.2013;5:183ra57.pmid:23636093(NPL 1)).

[0005] Reference List

[0006] Patent Literature

[0007] [PTL 1] WO 2016 / 081640

[0008] [PTL 2] WO 2015 / 191934

[0009] [PTL 3] WO 2016 / 081643

[0010] [PTL 4] WO 2014 / 033074

[0011] Non-patent literature

[0012] [NPL 1]Sci Transl Med.2013;5:183ra57.pmid:23636093 SUMMARY OF THE INVENTION

[0014] Technical issues

[0015] There is a continuing need to provide compounds, compositions, methods and uses that are useful for brain diseases or brain disorders. The object of the present disclosure is, but is not limited to, providing compounds, compositions, methods and uses that are useful for brain diseases or brain disorders.

[0016] Solution to the problem

[0017] As a means to solve the above-mentioned problem, one aspect of the present invention provides an antigen-binding molecule, which comprises a first antigen-binding domain and a second antigen-binding domain, wherein the first antigen-binding domain specifically binds to a first target, the first target promotes the transfer of the antigen-binding molecule into the brain of a mammal, and the second antigen-binding domain specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain extracellular matrix (ECM) protein or a brain ECM polysaccharide, and a method of using the antigen-binding molecule.

[0018] Throughout this disclosure, various aspects of the present invention are provided. However, it should be understood that each aspect disclosed is merely an exemplary embodiment of the present invention, and they are not intended to rigidly limit the present invention to these embodiments. On the contrary, the present invention is intended to cover substitutions, modifications and equivalents that may be included in the spirit and scope of the present disclosure.

[0019] [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, the first target promotes the transfer of the antigen binding molecule into the brain of a mammal, and the second antigen binding domain specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain extracellular matrix (ECM) protein or a brain ECM polysaccharide.

[0020] In addition, the following embodiments are provided:

[0021] [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).

[0022] [2A] The antigen-binding molecule according to [1] or [2], wherein the first target promotes the transfer of the antigen-binding molecule across the BBB into the brain of a mammal.

[0023] [3] An antigen-binding molecule according to [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 (HSPG).

[0024] [4] The antigen-binding molecule according to any one of [1] to [3], wherein the second target is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), neurocan 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 (γ-enolase), CD68 (macrophage sialic acid protein), allogeneic transplant inflammatory factor 1 (IBA1 or AIF1), purinergic receptor (P2RY12), interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), glutamate ion channel receptor NMDA type subunit 2B (GRIN2B), voltage-gated calcium channel auxiliary subunit γ8 (CACNG8), CD11b (integrin subunit αM), SLC6A2 (sodium-dependent norepinephrine 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 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), phosphoprotein associated with glycosphingolipid-rich microdomains 1 (Csk-binding protein), plasma membrane calcium transporting ATPase 1, prominin-1, somatostatin receptor type 1, carnitine O-palmitoyltransferase 1 brain isoform (CPT1-B), epidermal growth factor receptor, protein MAL2, syntaxin-1A, sodium / calcium exchanger 1, lysophosphatidylcholine acyltransferase 1 (LPC acyltransferase 1), calcium homeostasis protein-3 (Alcadein-β), pituitary adenylate cyclase activating polypeptide type I receptor (PACAPI receptor), neutral cholesterol ester hydrolase 1 (NCEH), CD166 antigen (activated leukocyte adhesion molecule), inactivated 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), tropoelastin (elastin), collagen alpha-2 (IV) chain ( The proteins involved in the expression of the proteins are angiostatin, angiostatin, aggrecan nexin 1 (hyaluronan and aggrecan nexin 1), tenascin-R (TN-R), aggrecan nexin 2 (hyaluronan and aggrecan nexin 2), collagen alpha-1 (I) chain, neurofilament protein-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).

[0025] [5] The antigen-binding molecule according to any one of [1] to [4], wherein the brain cells comprise one or more cells selected from the group consisting of oligodendrocytes, astrocytes, neurons, and microglia.

[0026] [6] The antigen-binding molecule according to any one of [1] to [5], wherein the second target is a molecule mainly expressed on brain-specific cells.

[0027] [6A] The antigen-binding molecule according to any one of [1] to [6], wherein the second target is a molecule widely expressed in brain tissue.

[0028] [6B] The antigen-binding molecule according to [6A], wherein the second target widely expressed in brain tissue is a molecule expressed in more than one, more than two or more than three brain tissues selected from the group consisting of: cerebral cortex, hippocampal formation, amygdala, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, medulla oblongata and spinal cord.

[0029] [6C] The antigen-binding molecule according to [6B], wherein the second target is a molecule selected from the group consisting of interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), glutamate ion channel receptor NMDA-type subunit 2B (GRIN2B) and voltage-gated calcium channel auxiliary subunit γ8 (CACNG8).

[0030] [6D] The antigen-binding molecule according to any one of [1] to [4], wherein the second target is a brain ECM protein.

[0031] [6E] An antigen-binding molecule according to [6D], wherein the second target is selected from the group consisting of: versican core protein (chondroitin sulfate proteoglycan 2 or CSPG2), tropoelastin (elastin), collagen α-2 (IV) chain (angiostatin), aggrecan link protein 1 (hyaluronic acid and aggrecan link protein 1), tenascin-R (TN-R), aggrecan link protein 2 (hyaluronic acid and aggrecan link protein 2), collagen α-1 (I) chain, neurofilament protein-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).

[0032] [6F] The antigen-binding molecule according to any one of [1] to [5], wherein the second target is expressed only on the cell membrane of brain-specific cells.

[0033] [6G] The antigen-binding molecule according to [1] to [5], wherein the second target is expressed only in brain tissue.

[0034] [6H] The antigen-binding molecule according to [1] to [3], wherein the second target is brain ECM polysaccharide.

[0035] [7] An antigen binding molecule according to 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.

[0036] [7A] An antigen binding molecule according to any one of [1] to [7], wherein the first target is transferrin receptor (TfR), wherein 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 (VH) having the amino acid sequence shown in SEQ ID NO:30 or a variant thereof having one or more conservative amino acid substitutions.

[0037] [7B] The antigen-binding molecule according to any one of [1] to [7], wherein the first target is Basigin (CD147), wherein optionally, the antigen-binding domain that binds 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.

[0038] [7C] The antigen-binding molecule according to any one of [1] to [7], wherein the first target is the insulin receptor.

[0039] [7D] The antigen-binding molecule according to any one of [1] to [7], wherein the first target is a low-density lipoprotein receptor (LDLR).

[0040] [7E] The antigen-binding molecule according to any one of [1] to [7], wherein the first target is low-density lipoprotein receptor-related protein (LRP), preferably LRP1.

[0041] [7F] The antigen-binding molecule according to any one of [1] to [7], wherein the first target is a diphtheria toxin receptor.

[0042] [7G] The antigen-binding molecule according to any one of [1] to [7], wherein the first target is a glucose receptor, preferably Glut1.

[0043] [7H] The antigen-binding molecule according to any one of [1] to [7], wherein the first target is CD98hc.

[0044] [7I] The antigen-binding molecule according to any one of [1] to [7], wherein the first target is insulin-like growth factor 1 receptor (IGF1R).

[0045] [8] An antigen-binding molecule according to any one of [1] to [7I], wherein the second target is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5) and cell adhesion molecule 3 (CADM3).

[0046] [8A] The antigen-binding molecule according to any one of [1] to [8], wherein the second target is MOG, wherein optionally, the antigen-binding domain that binds MOG comprises a heavy chain variable region (VH) having an amino acid sequence of SEQ ID NO: 15 or 34 and / or a light chain variable region (VL) having an amino acid sequence shown in SEQ ID NO: 17 or 35, or variants thereof having one or more conservative amino acid substitutions.

[0047] [8B] The antigen-binding molecule according to [8A], wherein the retention of the antigen-binding molecule in central nervous tissues such as the optic nerve, spinal cord, olfactory bulb, and medulla oblongata is improved.

[0048] [8C] An antigen binding molecule according to any one of [1] to [8], wherein the second target is CSPG5, wherein optionally, the antigen binding domain that binds to CSPG5 comprises a heavy chain variable region (VH) having an amino acid sequence of SEQ ID NO: 36 or 56 and / or a light chain variable region (VL) having an amino acid sequence shown in SEQ ID NO: 37 or 57 or a variant thereof having one or more conservative amino acid substitutions.

[0049] [8D] An antigen-binding molecule according to any one of [1] to [8], wherein the second target is CADM3, wherein optionally, the antigen-binding domain that binds to CADM3 comprises a heavy chain variable region (VH) having an amino acid sequence of SEQ ID NO: 38 and / or a light chain variable region (VL) having an amino acid sequence shown in SEQ ID NO: 39 or a variant thereof having one or more conservative amino acid substitutions.

[0050] [9] The antigen-binding molecule according to any one of [1] to [8C], wherein:

[0051] (i) the first target is transferrin receptor (TfR), and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0052] (ii) the first target is transferrin receptor (TfR), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0053] (iii) the first target is transferrin receptor (TfR), and the second target is cell adhesion molecule 3 (CADM3); or

[0054] (iv) the first target is Basigin (CD147), and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0055] (v) the first target is Basigin (CD147), and the second target is Neurocan C / Chondroitin Sulfate Proteoglycan 5 (CSPG5); or

[0056] (vi) the first target is Basigin (CD147), and the second target is cell adhesion molecule 3 (CADM3); or

[0057] (vii) the first target is insulin receptor, and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0058] (viii) the first target is insulin receptor, and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0059] (ix) the first target is insulin receptor, and the second target is cell adhesion molecule 3 (CADM3); or

[0060] (x) the first target is a glucose receptor, and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0061] (xi) the first target is a glucose receptor and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0062] (xii) the first target is glucose receptor, and the second target is cell adhesion molecule 3 (CADM3); or

[0063] (xiii) the first target is low-density lipoprotein receptor (LDLR), and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0064] (xiv) the first target is low-density lipoprotein receptor (LDLR), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0065] (xv) the first target is low-density lipoprotein receptor (LDLR), and the second target is cell adhesion molecule 3 (CADM3); or

[0066] (xvi) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0067] (xvii) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0068] (xviii) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is cell adhesion molecule 3 (CADM3); or

[0069] (xix) the first target is CD98hc, and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0070] (xx) the first target is CD98hc, and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0071] (xxi) The first target is CD98hc, and the second target is cell adhesion molecule 3 (CADM3).

[0072]

[10] The antigen-binding molecule according to any one of [1] to [4], wherein the second target is not a target selected from the group consisting of: β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, TRKA, 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 receptors, and EpCAM.

[0073]

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

[10] , further comprising

[0074] (i) at least one functional portion, wherein the at least one functional portion comprises an enzyme, a therapeutic protein, an antibody or an antigen-binding fragment thereof, a peptide, a DNA, a shRNA, a siRNA, a small molecule drug or a cytotoxic agent and / or

[0075] (ii) at least one in vivo half-life extending moiety, preferably wherein said at least one in vivo half-life extending moiety 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.

[0076] [11A] The antigen-binding molecule according to [1], wherein the at least one functional part is selected from the group consisting of enkephalinase, an anti-sortilin 1 antigen-binding domain, an anti-BACE1 antigen-binding domain and acid α-glucosidase (GAA).

[0077]

[12] In a second aspect, a method for increasing the concentration of an antigen-binding molecule in the brain of a subject in need thereof is provided, comprising:

[0078] (a) providing a first antigen-binding molecule, the first antigen-binding molecule comprising:

[0079] (a1) a first antigen-binding domain that specifically binds to a first target that facilitates transfer of the antigen-binding molecule into the brain of a mammal; or

[0080] (a2) a second antigen-binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide;

[0081] (b) in the case of (a1), introducing into the first antigen-binding molecule at least one second antigen-binding domain that specifically binds to the second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide, thereby obtaining a second antigen-binding molecule; or

[0082] In the case of (a2), at least one first antigen-binding domain that specifically binds to the first target is introduced into the first antigen-binding molecule, and the first target promotes the transfer of the antigen-binding molecule into the brain of a mammal, thereby obtaining a second antigen-binding molecule;

[0083] Such that, after administration to the subject, the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule.

[0084] In addition, the following embodiments are provided:

[0085]

[13] The method according to

[12] , further comprising step (c):

[0086] (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 differs from the second antigen-binding molecule according to (b) only in that the control antigen-binding molecule does not comprise:

[0087] In the case of (a1), the at least one second antigen binding domain specifically binds to the second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide; or

[0088] In the case of (a2), the at least one first antigen-binding domain specifically binds to the first target, which promotes the transfer of the antigen-binding molecule into the brain of a mammal.

[0089]

[14] The method according to

[12] or

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

[0090]

[15] In a third aspect, a method for increasing exposure of an antigen binding molecule in the brain of a subject in need thereof is provided, comprising:

[0091] (a) providing a first antigen-binding molecule, the first antigen-binding molecule comprising:

[0092] (a1) a first antigen-binding domain that specifically binds to a first target that facilitates transfer of the antigen-binding molecule into the brain of a mammal; or

[0093] (a2) a second antigen binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain extracellular matrix (ECM) protein or brain ECM polysaccharide;

[0094] (b) In the case of (a1), introducing into the first antigen-binding molecule

[0095] at least one second antigen-binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of a brain cell, or (ii) is a brain ECM protein or brain ECM polysaccharide, thereby obtaining a second antigen-binding molecule; or

[0096] In the case of (a2), at least one first antigen-binding domain that specifically binds to a first target is introduced into the first antigen-binding molecule, and the first target promotes the transfer of the antigen-binding molecule into the brain of a mammal, thereby obtaining a second antigen-binding molecule;

[0097] Such that, after administration to the subject, exposure of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule.

[0098] In addition, the following embodiments are provided:

[0099]

[16] The method according to

[15] , further comprising step (c):

[0100] (c) determining that 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 differs from the second antigen-binding molecule according to (b) only in that the control antigen-binding molecule does not comprise:

[0101] In the case of (a1), the at least one second antigen binding domain specifically binds to the second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide; or

[0102] In the case of (a2), the at least one first antigen-binding domain specifically binds to the first target, which promotes the transfer of the antigen-binding molecule into the brain of a mammal.

[0103]

[17] The method according to

[15] or

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

[0104]

[18] In a fourth aspect, a method for retaining an antigen-binding molecule in the brain of a subject in need thereof is provided, comprising:

[0105] (a) providing a first antigen-binding molecule, the first antigen-binding molecule comprising a first antigen-binding domain that specifically binds to a first target, the first target facilitating transfer of the antigen-binding molecule into the brain of a mammal;

[0106] (b) introducing into the first antigen-binding molecule at least one second antigen-binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide, thereby obtaining a second antigen-binding molecule;

[0107] Such that, after administration to the subject, the retention of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule.

[0108] In addition, the following embodiments are provided:

[0109]

[19] The method according to

[18] , further comprising step (c):

[0110] (c) determining that the retention 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 according to (b) only in that the control antigen-binding molecule does not comprise the at least one antigen-binding domain that specifically binds to the second target.

[0111]

[20] The method according to

[18] or

[19] , wherein the retention of the second antigen-binding molecule in the brain of the subject is the in vivo half-life in the brain.

[0112] [20A] The method according to

[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.

[0113] [20B] The method according to [20A], wherein the in vivo half-life of the second antigen-binding molecule in the brain is at least 10 days.

[0114] [20C] The method according to [20A], wherein the in vivo half-life of the second antigen-binding molecule in the brain is at least 30 days.

[0115] [20D] The method according to [20A], wherein the in vivo half-life of the second antigen-binding molecule in the brain is at least 60 days.

[0116]

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

[11] or the method according to any one of

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

[0117]

[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].

[0118]

[23] The method according to 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] .

[0119]

[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] .

[0120]

[25] The method according to any one of

[12] to

[24] , wherein:

[0121] (i) the first target is transferrin receptor (TfR), and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0122] (ii) the first target is transferrin receptor (TfR), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0123] (iii) the first target is transferrin receptor (TfR), and the second target is cell adhesion molecule 3 (CADM3);

[0124] (iv) the first target is Basigin (CD147), and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0125] (v) the first target is Basigin (CD147), and the second target is Neurocan C / Chondroitin Sulfate Proteoglycan 5 (CSPG5); or

[0126] (vi) the first target is Basigin (CD147), and the second target is cell adhesion molecule 3 (CADM3);

[0127] (vii) the first target is the insulin receptor, and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0128] (viii) the first target is the insulin receptor, and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0129] (ix) the first target is insulin receptor, and the second target is cell adhesion molecule 3 (CADM3);

[0130] (x) the first target is a glucose receptor, and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0131] (xi) the first target is a glucose receptor, and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0132] (xii) the first target is a glucose receptor, and the second target is cell adhesion molecule 3 (CADM3);

[0133] (xiii) the first target is low-density lipoprotein receptor (LDLR), and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0134] (xiv) the first target is low-density lipoprotein receptor (LDLR), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0135] (xv) the first target is low-density lipoprotein receptor (LDLR), and the second target is cell adhesion molecule 3 (CADM3);

[0136] (xvi) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0137] (xvii) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0138] (xviii) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is cell adhesion molecule 3 (CADM3); or

[0139] (xix) the first target is CD98hc, and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0140] (xx) the first target is CD98hc, and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0141] (xxi) The first target is CD98hc, and the second target is cell adhesion molecule 3 (CADM3).

[0142]

[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] .

[0143] [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 retention / 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.

[0144]

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

[11] and one or more pharmaceutically acceptable carriers or excipients.

[0145] In addition, the following embodiments are provided:

[0146]

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

[11] or a pharmaceutical composition according to

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

[0147]

[29] In a sixth aspect, a nucleic acid or two or more nucleic acids encoding an antigen-binding molecule according to any one of [1] to

[11] is provided, optionally wherein the nucleic acid or two or more nucleic acids are operably linked to a promoter.

[0148]

[30] In the seventh aspect, a vector or two or more vectors comprising the nucleic acid or two or more nucleic acids according to

[29] .

[0149]

[31] In an eighth aspect, a host cell is provided, which comprises (i) a nucleic acid or two or more nucleic acids according to

[29] ; (ii) a vector or two or more vectors according to

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

[11] .

[0150]

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

[11] is provided, which comprises culturing the host cell according to

[31] to produce the antigen-binding molecule; optionally further comprising recovering the antigen-binding molecule from the host cell.

[0151] In addition, the following embodiments are provided:

[0152]

[33] The antigen-binding molecule according to 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).

[0153] [33A] An 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, DARPins, FN3, aptamer, anticalins).

[0154]

[34] An 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).

[0155] [34A] An 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 single domain antibody (VHH), or a non-antibody binder (e.g., affibody, DARPins, FN3, aptamer, anticalins).

[0156]

[35] An 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: an antibody variable region comprising 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 a single domain antibody (VHH).

[0157]

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

[11] ,

[21] and

[33] to

[35] , wherein the antigen-binding molecule contains 1, 2, 3 or 4 first antigen-binding domains and 1, 2, 3 or 4 second antigen-binding domains.

[0158]

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

[11] ,

[21] and

[33] to

[36] , wherein the antigen-binding molecule contains 1, 2, 3 or 4 first antigen-binding domains and 1, 2, 3 or 4 second antigen-binding domains and at least one Fc region.

[0159]

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

[11] ,

[21] and

[33] to

[37] , wherein the one or more first antigen-binding domains and the one or more second antigen-binding domains are independently selected from the group consisting of: an antibody variable region comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), Fab, Fab', F(ab')2, a diabody, a triabody, a scFab, a Fv, a scFv or a single domain antibody (VHH) or a non-antibody binder (e.g., affibody, DARPins, FN3, aptamer, anticalins).

[0160]

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

[11] ,

[21] , and

[33] to

[37] , which is a bispecific antibody.

[0161]

[39] The antigen-binding molecule according to

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

[0162]

[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.

[0163] [40A] The antigen-binding molecule according to

[36] or

[40] , wherein the Fc region has an ability to bind to activated Fcγ receptors that is lower than the ability of the Fc region of native human IgG to bind to activated Fcγ receptors.

[0164] [40B] The antigen-binding molecule according to [40A], wherein the activating Fcγ receptor is human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V) or human FcγRIIIa(F).

[0165] [40C] The antigen-binding molecule of [40A] or [40B], wherein the Fc region comprises one or more of the following amino acid substitutions (all positions are numbered according to EU):

[0166] Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr or Trp at position 234;

[0167] Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val or Arg at position 235;

[0168] Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro or Tyr at position 236;

[0169] Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr or Arg at position 237;

[0170] Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp or Arg at position 238;

[0171] Gln, His, Lys, Phe, Pro, Trp, Tyr or Arg at position 239;

[0172] Ala, Arg, Asn, Gln, Gly, His, He, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val at position 265;

[0173] Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp or Tyr at position 266;

[0174] Arg, His, Lys, Phe, Pro, Trp or Tyr at position 267;

[0175] Ala, Arg, Asn, Gln, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val at position 269;

[0176] Ala, Arg, Asn, Gln, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val at position 270;

[0177] Arg, His, Phe, Ser, Thr, Trp or Tyr at position 271;

[0178] Arg, Asn, Asp, Gly, His, Phe, Ser, Trp or Tyr at position 295;

[0179] Arg, Gly, Lys or Pro at position 296;

[0180] Ala at position 297;

[0181] Arg, Gly, Lys, Pro, Trp or Tyr at position 298;

[0182] Arg, Lys, or Pro at position 300;

[0183] Lys or Pro at position 324;

[0184] Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr or Val at position 325;

[0185] Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 327;

[0186] Arg, Asn, Gly, His, Lys or Pro at position 328;

[0187] Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, or Arg at position 329;

[0188] Pro or Ser at position 330;

[0189] Arg, Gly or Lys at position 331;

[0190] Arg, Lys or Pro at position 332.

[0191]

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

[11] ,

[21] and

[33] to

[40] , wherein the at least one functional part is an antibody or an antigen-binding fragment thereof, which specifically binds to the following membrane proteins i) immune cells, in particular wherein the immune cells are selected from the group consisting of: T cells, killer cells, helper T cells, regulatory T cells, B cells, memory B cells, NK cells, NKT cells, dendritic cells, macrophages, eosinophils, neutrophils and basophils, ii) tumor cells, or iii) autoreactive cells.

[0192]

[42] The antigen-binding molecule according to

[41] , wherein the antibody or antigen-binding fragment thereof specifically binds to a group of membrane proteins selected from the group consisting of T cell receptors, CD3, CD137, CD40, CTLA4, co-stimulatory molecules and co-inhibitory molecules.

[0193]

[43] In a tenth aspect, a method for producing an antigen-binding molecule is provided, comprising the following steps:

[0194] (a) selecting a first antigen binding domain that specifically binds to a first target, which promotes the transfer of the antigen binding molecule into the brain of a mammal; (b) selecting a second antigen binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells or (ii) is a brain ECM protein or brain ECM polysaccharide;

[0195] (c) obtaining one or more nucleic acids encoding antigen-binding molecules in which an antigen-binding domain is linked to the antigen-binding domains prepared in (a) and (b); and

[0196] (d) using the one or more nucleic acids prepared in (c) to produce an antigen-binding molecule.

[0197]

[44] In the eleventh aspect, a method for screening antigen-binding molecules is provided, comprising the following steps:

[0198] (a) selecting a first antigen binding domain that specifically binds to a first target, which promotes the transfer of the antigen binding molecule into the brain of a mammal; (b) selecting a second antigen binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells or (ii) is a brain ECM protein or brain ECM polysaccharide;

[0199] (c) obtaining one or more nucleic acids encoding antigen-binding molecules in which an antigen-binding domain is linked to the antigen-binding domains prepared in (a) and (b); and

[0200] (d) using the one or more nucleic acids prepared in (c) to produce an antigen-binding molecule.

[0201] In addition, the following embodiments are provided:

[0202]

[45] The method according to

[43] or

[44] , further comprising step (e):

[0203] (e) determining an increase in (i) retention, (ii) concentration or (iii) exposure of an antigen-binding molecule according to (d) in the brain of a subject compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule according to (d) only in that the control antigen-binding molecule comprises one but not both of the antigen-binding domains defined in (a) and (b).

[0204] [45A] The method according to any one of

[43] to

[45] , wherein when the second target is MOG, the increase in (i) retention (ii) concentration or (iii) exposure of the second antigen-binding molecule in the brain refers to an increase in (i) retention (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.

[0205]

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

[11] ,

[21] and

[33] to

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

[29] , (iii) a vector or two or more vectors according to

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

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

[0206]

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

[11] ,

[21] and

[33] to

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

[29] , (iii) a vector or two 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 drug.

[0207]

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

[11] ,

[21] and

[33] to

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

[29] , (iii) a vector or two 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.

[0208]

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

[11] ,

[21] and

[33] to

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

[29] , (iii) a vector or two or more vectors according to

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

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

[27] or

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

[0209]

[50] In the sixteenth aspect, the present invention provides 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) a molecular antigen binder according to any one of [1] to

[11] ,

[21] and

[33] to

[42] , (ii) a nucleic acid or two or more nucleic acids encoding the antigen binding molecule of

[29] , (iii) a vector or two or more vectors according to

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

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

[27] or

[46] .

[0210]

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

[11] ,

[21] and

[33] to

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

[29] , (iii) a vector or two or more vectors according to

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

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

[27] or

[46] , which is used for the preparation of a medicament for treating and / or preventing brain disorders or diseases.

[0211]

[52] In the eighteenth aspect, the present invention provides (i) an antigen-binding molecule according to any one of [1] to

[11] ,

[21] and

[33] to

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

[29] , (iii) a vector or two or more vectors according to

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

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

[27] or

[46] for the preparation of a medicament for treating and / or preventing brain disorders or diseases.

[0212]

[53] A pharmaceutical composition / antigen-binding molecule / nucleic acid / vector for the use of

[49] or

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

[50] , or the use according to

[52] , wherein 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-Draeger 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 syndrome, chronic wasting disease and fatal familial insomnia), bulbar palsy, motor neurone disease and degenerative diseases of the nervous system (including but not limited to Canavan disease, Huntington disease, neuronal ceroid lipofuscinosis, Alexander disease, Tourette syndrome, Menkes kinky hair syndrome, Cockayne syndrome, Halervorden-Spatz syndrome, Lafora disease, Rett syndrome, hepatolenticular degeneration, Leigh-Ny syndrome and Unverricht-Lundborg syndrome), dementia (including but not limited to Pick's disease and spinocerebellar ataxia), mental disorders, cancer (e.g., cancer of the CNS, including brain metastases from cancers elsewhere in the body).

[0213]

[54] A pharmaceutical composition / antigen-binding molecule / nucleic acid / vector for the use of

[49] or

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

[50] , or the use according to

[52] , wherein 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).

[0214] Unless otherwise expressly stated or unless there is an inconsistency in the context, any term expressed in the singular herein may be intended to include the plural form as well, and any term expressed in the plural form herein may be intended to include the singular form as well. In addition, reference to an element by the indefinite article "a / an" does not exclude the possibility that more than one element is present, unless the context clearly requires that there is one and only one element. Therefore, the indefinite article "a / an" generally means "at least one". BRIEF DESCRIPTION OF THE DRAWINGS

[0215] [ Figure 1A ] shows a schematic diagram of a molecule having a brain transfer portion (a first antigen binding domain that specifically binds to a first target that promotes transfer of the antigen binding molecule to the brain of a mammal; for example, an anti-transferrin receptor antibody).

[0216] [ Figure 1B ] shows a schematic diagram of a molecule with a brain retention / target portion (a second antigen binding domain specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide; for example, an anti-MOG antibody).

[0217] [ Figure 1C ] shows a schematic diagram of a molecule having a brain transfer portion (a first antigen binding domain that specifically binds to a first target that promotes transfer of the antigen binding molecule to the brain of a mammal; e.g., an anti-transferrin receptor domain) and a brain retention portion (a second antigen binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide; e.g., an anti-MOG domain).

[0218] [ Figure 2a ]The schematic diagram provided in (a) shows the concept of an embodiment of an antigen-binding molecule of the present disclosure, which comprises (1) a brain transfer portion, (2) a brain retention portion, (3) a functional portion and optionally an additional half-life extending portion.

[0219] [ Figure 2b] Schematic diagram showing the exemplary molecular form of the antigen binding molecules of this aspect and embodiment. In one example, the antigen binding molecule comprises the first Fab region and the second Fab region, the first Fab region combines to promote the penetration of the antigen binding molecule or transfer to the molecule (i.e., brain transfer part) in the brain, and the second Fab region combines to express on brain-specific cells, or is a molecule (i.e., brain retention part) of brain ECM protein or brain ECM polysaccharide, and optionally further comprises a functional part. The structure of the brain retention part and the brain transfer part is not limited to the Fab region, but can also be in the form of an antibody fragment, such as scFab (single-chain Fab), Fv, Fab, Fab', F(ab')2, double antibody, three antibodies, scFv, VHH, double antibody or F(ab')2 fragment. In other exemplary embodiments, the variable domain is connected to the Fc region so that there is no F(ab')2 fragment. A plurality of such embodiments are depicted in Fig. 2 (b) and Fig. 2 (c).

[0220] [ Figure 2c ] Schematic diagram showing exemplary molecular forms of antigen-binding molecules of the present aspects and embodiments. See also Figure 2b A brief description of .

[0221] [ Figure 3a ] Figure showing the pharmacokinetic profiles in the brain and the brain concentration-time profiles of KLH, MOG303, KLH / / TfR and MOG303 / / TfR. KLH, MOG303, KLH / / TfR and MOG303 / / TfR were administered intravenously at a dose of 2 mg / kg and the brain was collected after perfusion. Antibody concentrations in the brain were measured by ECL. Figure 3a Antibody concentrations in the brain are shown. Data show mean + / - SD (n=6).

[0222] [ Figure 3b ] Figure showing the pharmacokinetic profiles in the brain and the brain concentration-time profiles of KLH, MOG303, KLH / / TfR and MOG303 / / TfR. KLH, MOG303, KLH / / TfR and MOG303 / / TfR were administered intravenously at a dose of 2 mg / kg and the brain was collected after perfusion. Antibody concentrations in the brain were measured by ECL. Figure 3b The ratio of antibody concentration in brain to that in plasma is shown. Data show mean + / - SD (n=6).

[0223] [ Figure 3c] Figure showing the pharmacokinetic profiles in the brain and the brain concentration-time profiles of KLH, MOG303, KLH / / TfR and MOG303 / / TfR. KLH, MOG303, KLH / / TfR and MOG303 / / TfR were administered intravenously at a dose of 2 mg / kg and the brain was collected after perfusion. Antibody concentrations in the brain were measured by ECL. Figure 3c The percentage of injected dose per brain tissue weight (%ID / g brain) is shown. Data show mean + / - SD (n=6).

[0224] [ Figure 3d ] Figure showing the pharmacokinetic profiles in the brain and the brain concentration-time profiles of KLH, MOG303, KLH / / TfR and MOG303 / / TfR. KLH, MOG303, KLH / / TfR and MOG303 / / TfR were administered intravenously at a dose of 2 mg / kg and the brain was collected after perfusion. Antibody concentrations in the brain were measured by ECL. Figure 3d Cumulative brain AUC is shown up to day 28. Data show mean + / - SD (n=6).

[0225] [ Figure 4 ] Graph showing the plasma concentration-time curve of antibodies. KLH, MOG303, KLH / / TfR and MOG303 / / TfR were intravenously administered to mice at a dose of 2 mg / kg, and blood was collected. Antibody concentrations in plasma were measured by ECL. Data show mean + / - SD (n = 6).

[0226] [ Figure 5 ] Graph showing plasma concentration-time curves 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. Antibody concentrations in plasma were measured by ECL. Data show mean + / - SD (n = 3-4).

[0227] [ Figure 6] Graphs showing pharmacokinetic profiles in the brain 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. Antibodies were administered intravenously at a dose of 2 mg / kg, and the brain was collected 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 antibody concentration in plasma. (c) Percentage of injected dose per weight of brain tissue (%ID / g brain). Data show mean + / - SD (n = 3-4).

[0228] [ Figure 7 ] Graph showing plasma concentration-time curves of antibodies KLH, mBsg / / KLH, mBsg / / MOG303, mBsg / / CSPG5.2, mBsg / / CADM3. Antibodies were administered intravenously at a dose of 2 mg / kg, and blood was collected. Antibody concentrations in plasma were measured by ECL. Data show mean + / - SD (n=4).

[0229] [ Figure 8 ] Graphs showing pharmacokinetic profiles in the brain and brain 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 brains were collected 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 antibody concentration in plasma. (c) Percentage of injected dose per brain tissue weight (%ID / g brain). Data show mean + / - SD (n = 4).

[0230] [ Fig. 9 ] Graph showing the plasma concentration-time curves of antibodies KLH, CSPG5120 and CSPG5120-BS. Antibodies were administered intravenously at a dose of 2 mg / kg, and blood was collected. Antibody concentrations in plasma were measured by ECL. Data show mean + / - SD (n=3).

[0231] [ Fig.10] Graphs showing the pharmacokinetic profiles in the brain and the brain concentration-time profiles of antibodies KLH, CSPG5120, and CSPG5120-BS. Antibodies were administered intravenously at a dose of 2 mg / kg, and brains were collected 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 antibody concentration in plasma. (c) Percentage of injected dose per brain tissue weight (%ID / g brain). Data show mean + / - SD (n = 3).

[0232] [ Fig.11 ] Schematic diagram showing additional exemplary molecular forms of the antigen-binding molecules of the present aspects and embodiments. Fig.11 A 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 transfer of antigen-binding molecules into the brain (i.e., a brain transfer portion, such as a TfR binding domain or a basigin binding domain), and each binds to a molecule specifically expressed on brain-specific cells, or is a brain ECM protein or brain ECM polysaccharide (i.e., a brain retention portion, such as a MOG binding domain or a CSPG5 binding domain) The second and third Fab regions, and optionally further comprise a functional portion (not shown in the figure). Fig.11 B shows the schematic structure of the bispecific antibodies (f) to (m) exemplified in Example 4A, which comprise a first Fab region that binds to a molecule that promotes penetration or transfer of antigen-binding molecules into the brain (i.e., a brain transfer portion, such as a TfR binding domain or a basigin binding domain), and a second Fab region that binds to a molecule specifically expressed on brain-specific cells, or a brain ECM protein or brain ECM polysaccharide (i.e., a brain retention portion, such as a MOG binding domain, a CSPG5 binding domain, a CADM3 binding domain), and optionally further comprises a functional portion (not shown in the figure). Fig.11C to 11F show other exemplary molecular forms of antigen binding molecules that additionally include one or more functional parts. The structure of the brain retention part and the brain transfer part is not limited to the Fab region, but can also be in the form of an antibody fragment, such as scFab (single-chain Fab), Fv, Fab, Fab', F(ab')2, double antibody, three antibodies, scFv, VHH, double antibody, F(ab')2 fragment or non-antibody conjugate (e.g., affibody, DARPins, FN3, aptamer, anticalins). The functional part can be any molecule with therapeutic function, such as agonists, antagonists, enzymes, regulators, stabilizers, cell death inducers and any molecular form, such as nucleic acids, small molecules, cyclic peptides, peptides, ligands, cytokines, chemokines, growth factors, enzymes and antigen binding domains. In some instances, the functional part is selected from the group consisting of: enkephalinase, anti-sortilin1 antigen binding domain, anti-BACE1 antigen binding domain and acid alpha-glucosidase (GAA).

[0233] [ Fig.12 ] Schematic diagram showing additional exemplary molecular forms of the antigen-binding molecules of the present aspects and embodiments.

[0234] Fig.12 A shows the schematic structure of the antibodies IGF1R / / MOG303 and TfRVNAR.CloneC / / MOG303 exemplified in Example 8, each of which comprises a VHH that binds to a molecule that promotes penetration or transfer of the antigen-binding molecule into the brain (i.e., a brain transfer portion, such as a TfR binding domain or an IGF1R binding domain), and a Fab region that binds to a molecule specifically expressed on brain-specific cells, or is a brain ECM protein or brain ECM polysaccharide (i.e., a brain retention portion, such as a MOG binding domain), and optionally further comprises a functional portion (not shown in the figure).

[0235] Fig.12 B shows the schematic structure of IL6R / / MOG303-TfR-GAA or IL6R / / MOG303-TfR-shortGAA exemplified in Example 9A, which comprises a scFab that binds to a molecule that promotes penetration or transfer of antigen-binding molecules into the brain (i.e., a brain transfer portion, such as a TfR binding domain), a Fab that binds to a molecule specifically expressed on brain-specific cells, or is a brain ECM protein or brain ECM polysaccharide (i.e., a brain retention portion, such as a MOG binding domain), and one or more functional portions (e.g., GAA or NEP), and optionally a VHH that binds to another antigen.

[0236] Fig.12C shows the schematic structure of MOG303-TfR-2GAA or MOG303-TfR-2shortGAA exemplified in Example 9A, which comprises two Fabs (i.e., brain retention portions, such as MOG binding domains) each binding to a molecule specifically expressed on brain-specific cells, or a brain ECM protein or brain ECM polysaccharide, a scFab (i.e., brain transfer portion, such as TfR binding domain) that binds to a molecule that promotes penetration or transfer of antigen-binding molecules into the brain, and one or more (e.g., two) functional portions (e.g., GAA) at the C-terminus of each of the two light chains of the Fab connected to the brain retention portion.

[0237] Fig.12 D shows the schematic structure of BACE1 / / MOG303-TfR exemplified in Example 9C, which comprises a scFab that binds to a molecule that promotes penetration or transfer of antigen-binding molecules into the brain (i.e., a brain transfer portion, such as a TfR binding domain), a Fab that binds to a molecule specifically expressed on brain-specific cells, or a brain ECM protein or brain ECM polysaccharide (i.e., a brain retention portion, such as a MOG binding domain), and a functional portion of the Fab that binds to another biological target (e.g., a BACE1 binding domain).

[0238] The structures of the brain retention part and the brain transfer part are not limited to Fab, VHH or scFab, but can also be in the form of antibody fragments, such as scFab (single-chain Fab), Fv, Fab, Fab', F(ab')2, double antibody, three antibody, scFv, VHH, double antibody, F(ab')2 fragment or non-antibody conjugate (e.g., affibody, DARPins, FN3, aptamer, anticalins). The functional part can be any molecule with therapeutic function, such as agonist, antagonist, enzyme, regulator, stabilizer, cell death inducer and any molecular form, such as nucleic acid, small molecule, cyclic peptide, peptide, ligand, cytokine, chemokine, growth factor, enzyme and antigen binding domain. In some examples, the functional part is selected from the group consisting of: enkephalinase, anti-sortilin1 antigen binding domain, anti-BACE1 antigen binding domain and acid α-glucosidase (GAA).

[0239] [ Fig.13 ] A schematic diagram showing another exemplary molecular form of the antigen-binding molecule of the present aspects and embodiments illustrated in Example 9D. Fig.13 A shows the schematic structure of the bivalent anti-Sort1 antibody (Sort1). Fig.13 B shows the schematic structure of Sort1-TfR, which comprises two Fabs each binding to Sort1 and a scFab binding to TfR. Fig.13C is a schematic structure of Sort1-TfR-MOG303 (L), which comprises two Fabs (each Fab binds to a biological target, such as Sort1 (functional portion)), one scFab (binds to a molecule that promotes penetration or transfer of antigen-binding molecules into the brain (i.e., a brain transfer portion, such as a TfR binding domain)), and two scFvs (each scFv binds to a molecule expressed on brain-specific cells, or is a brain ECM protein or brain ECM polysaccharide (i.e., a brain retention portion, such as an anti-MOG scFv)), wherein each of the two brain retention portions (anti-MOG scFv) is linked to the C-terminus of the L chain of the anti-SORT1 Fab. Fig.13 D is a schematic structure of Sort1-TfR-MOG303 (H), which comprises two Fabs (each Fab binds to a biological target, such as Sort1 (functional portion)), one scFab (binds to a molecule that promotes penetration or transfer of antigen-binding molecules into the brain (i.e., a brain transfer portion, such as a TfR binding domain)), and two scFvs (each scFv binds to a molecule expressed on brain-specific cells, or is a brain ECM protein or brain ECM polysaccharide (i.e., a brain retention portion, such as an anti-MOG scFv)), wherein each of the two brain retention portions (anti-MOG scFv) is linked to the C-terminus of the H chain of the anti-SORT1 antibody.

[0240] [ Fig.14 ] shows a graph of tissue concentrations of 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 show mean + / - SD (n = 3-4).

[0241] [ Fig.15 ] shows a graph of tissue concentrations of 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 collected. Antibody concentrations in these tissues were measured by ECL. Data show mean + / - SD (n = 3-4).

[0242] [ Fig.16] Graph showing antibody concentrations in plasma 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 blood was collected. Antibody concentrations in plasma were measured by ECL. Data show mean + / - SD (n=3).

[0243] [ Fig.17 ] Graph showing antibody concentrations in the brain 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 brains were collected after perfusion. Antibody concentrations in the brain were measured by ECL. Data show mean + / - SD (n=3).

[0244] [ Fig.18 ] Fig.18 (a) shows the plasma concentrations of anti-Sortilin1 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 show mean + / - SD (n = 5). Fig.18 b shows the brain concentration of anti-Sortilin1 antibodies (Sort1, Sort1-TfR, Sort1-TfR-MOG303(L), ​​and Sort1-TfR-MOG303(H)) and 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 show mean + / - SD (n = 4-5).

[0245] [ Fig.19 ] Fig.19 (a) shows the plasma concentration-time curves of antibodies KLH, IL6R / / KLH-NEP, IL6R / / KLH-TfR-NEP and IL6R / / MOG303-TfR-NEP (administered intravenously to mice at a dose of 50 nmol / kg and blood collected). Antibody concentrations in plasma were measured by ECL. Data show mean + / - SD (n = 6).

[0246] Fig.19(b) shows the brain concentration-time curves of antibodies KLH, IL6R / / KLH-NEP, IL6R / / KLH-TfR-NEP and IL6R / / MOG303-TfR-NEP (administered intravenously to mice at a dose of 50 nmol / kg and brains collected after perfusion). Antibody concentrations in the brain were measured by ECL. Data show mean + / - SD (n = 5-6).

[0247] [ Fig. 20 ] Fig. 20 Comparison of the concentration of amyloid β 1-40 peptide in the brain of mice showing KLH, IL6R / / KLH-NEP, IL6R / / KLH-TfR-NEP and IL6R / / MOG303-TfR-NEP (administered intravenously at a dose of 50 nmol / kg and brains collected after perfusion). The concentration of amyloid β 1-40 peptide in the brain was measured by ECL. Data show mean + / - SD (n = 5-6).

[0248] [ Fig.21 ] Fig.21 (a) shows the plasma concentration-time curves of antibodies KLH, BACE1 / / KLH-TfR and BACE1 / / MOG303-TfR (administered intravenously at a dose of 25 mg / kg and blood collected). Antibody concentrations in plasma were measured by ECL. Data show mean + / - SD (n=6). Fig.21 (b) shows the brain concentration-time curves of antibodies KLH, BACE1 / / KLH-TfR and BACE1 / / MOG303-TfR (administered intravenously at a dose of 25 mg / kg and brains collected after perfusion). Antibody concentrations in the brain were measured by ELISA. Data show mean + / - SD (n=6). DETAILED DESCRIPTION

[0249] The following definitions and detailed description are provided to facilitate understanding of the invention presented herein. In particular, these definitions and detailed description are used to explain various aspects and embodiments of the present disclosure.

[0250] definition

[0251] "Affinity" refers to the strength of the sum of non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise indicated, as used herein, "binding affinity" refers to intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be represented by a dissociation constant (Kd). Affinity can be measured by conventional methods known in the art.

[0252] The terms "anti-[X] antibody," "antibody that binds [X]," and "antibody that binds to [X]" refer to an antibody that is capable of binding to a target [X] with sufficient affinity such that the antibody can be used as a diagnostic, prophylactic, and / or therapeutic agent that targets the target X, wherein the target [X] is a given target of interest. In one embodiment, the extent of binding of the anti-[X] antibody to an unrelated, non-[X] protein is less than about 10% of the extent of binding of the antibody to [X], as measured, for example, by a radioimmunoassay (RIA). In certain embodiments, the antibody that binds [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 smaller, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 In certain embodiments, an anti-[X] antibody binds to an epitope of [X] that is conserved in [X] from different species.

[0253] The term "antibody" herein is used in the broadest sense and encompasses various 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.

[0254] "Antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody and binds to the antigen to which the intact antibody binds. 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 (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0255] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species.

[0256] The "class" of an antibody refers to the type of constant domain or constant region possessed by the heavy chain of the antibody. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these antibodies can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0257] As used herein, the term "cytotoxic agent" refers to a substance that inhibits or prevents cellular function and / or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g., 211 At 131 I. 125 I. 90 Y. 186 Re, 188 Re, 153 Sm, 212 Bi, 32 P. 212 radioisotopes of Pb and Lu); chemotherapeutic agents or drugs (e.g., methotrexate, doxorubicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitors; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and / or variants thereof; and various anti-tumor or anti-cancer drugs disclosed below.

[0258] "Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with 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; downregulation of cell surface receptors (e.g., B cell receptor); and B cell activation.

[0259] 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 the desired therapeutic or prophylactic result.

[0260] The term "Fc region" herein is used to define the C-terminal region of an immunoglobulin heavy chain, which comprises at least a portion of a constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, the human IgG heavy chain Fc region extends from Cys226 or from Pro230 to the carboxyl terminus of the heavy chain. However, the C-terminal lysine (Lys447) or glycine-lysine (residues 446-447) in the Fc region may be present or absent. 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 referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Edition Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

[0261] 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 heavy chains that contain an Fc region as defined herein.

[0262] 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 transformed cell, without regard to the number of passages. Progeny may not be identical to the nucleic acid content of the parent cell, but may contain mutations. Mutant progeny having the same function or biological activity as screened or selected in the original transformed cell are included herein.

[0263] A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human or human cell, or derived from a non-human antibody using a human antibody library or other human antibody encoding sequences. This definition of a human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues.

[0264] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will substantially comprise at least one, usually two, variable domains, wherein all or substantially all HVRs (e.g., CDRs) correspond to HVRs of non-human antibodies, and all or substantially all FRs correspond to FRs of human antibodies. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. An antibody in "humanized form," such as a non-human antibody, refers to an antibody that has undergone humanization.

[0265] An "immunoconjugate" is an antibody conjugated to one or more heterologous molecules, including but not limited to cytotoxic agents.

[0266] An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cattle, 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.

[0267] An "isolated" antibody is one that has been separated from a component of its natural environment. In some embodiments, the antibody is purified to a purity greater than 95% or 99% by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reversed-phase HPLC). For a review of methods for assessing antibody purity, see, e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

[0268] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. An isolated nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0269] "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 such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present in one or more locations in a host cell.

[0270] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, except for possible variant antibodies (e.g., containing naturally occurring mutations or produced during the production of monoclonal antibody preparations, such variants are usually present in small amounts), the individual antibodies comprising the population have identity and / or bind to the same epitope. Contrary to the polyclonal antibody preparations that typically include different antibodies for different determinants (epitopes), each monoclonal antibody in the monoclonal antibody preparation is directed to a single determinant on the antigen. Therefore, the modifier "monoclonal" represents that the feature of the antibody is obtained from a substantially homogeneous antibody population, and should not be interpreted as requiring antibody to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared 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 human immunoglobulin loci, such methods and other exemplary methods for preparing monoclonal antibodies are described herein.

[0271] A "naked antibody" refers to an antibody that is not conjugated to a heterologous moiety (eg, a cytotoxic moiety) or a radiolabel. Naked antibodies can be present in pharmaceutical preparations.

[0272] "Natural antibodies" refer to naturally occurring immunoglobulin molecules with different structures. For example, natural IgG antibodies are heterotetrameric glycoproteins of about 150,000 daltons, consisting of two identical light chains and two identical heavy chains bonded by disulfide bonds. From the N-terminus to the C-terminus, each heavy chain has a variable region (VH), also referred to as a variable heavy chain domain or a heavy chain variable domain, followed by three constant domains (CH1, CH2, and CH3). Similarly, from the N-terminus to the C-terminus, each light chain has a variable region (VL), also referred to as a variable light chain domain or a light chain variable domain, followed by a constant light chain (CL) domain. The light chain of an antibody can be classified into one of two types based on the amino acid sequence of its constant domain, which are called κ and λ.

[0273] The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and / or warnings concerning the use of such therapeutic products.

[0274] " Amino acid sequence identity percentage (%) " relative to a reference polypeptide sequence is defined as after comparing a candidate sequence with a reference polypeptide sequence and introducing a space (if necessary) to achieve maximum sequence identity percentage, and without considering any conservative substitution as a component of sequence identity, the percentage of the amino acid residue in the candidate sequence that is identical to the amino acid residue in the reference polypeptide sequence. The comparison for determining the amino acid sequence identity percentage can be achieved in various ways within the technical scope of the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR) software or GENETYX (registered trademark) (Genetyx Co., Ltd.). Those skilled in the art can determine the appropriate parameters for comparing sequences, including any algorithm required for achieving maximum comparison over the full length of the sequence compared.

[0275] The ALIGN-2 sequence comparison computer program is written by Genentech, Inc., and the source code has been submitted to the US Copyright Office, Washington DC, 20559, with user documentation, where it is registered with U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and remain unchanged. In the case of using ALIGN-2 for amino acid sequence comparison, the amino acid sequence identity % of a given amino acid sequence A and a given amino acid sequence B (which can be alternatively expressed as a given amino acid sequence A having or containing a certain amino acid sequence identity % with a given amino acid sequence B) is calculated as follows:

[0276] Multiply 100 by the fraction X / Y

[0277] Where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be understood that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the preceding paragraph using the ALIGN-2 computer program.

[0278] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that permits the biological activity of the active ingredient contained therein to be effective, and that contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered.

[0279] "Pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier or excipient" refers to a component of a pharmaceutical preparation 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.

[0280] Unless otherwise indicated, the term "[X]" used herein to designate a particular target refers to any native target [X] from any vertebrate source, including mammals such as primates (e.g., humans) and rodents (such as mice and rats). The term encompasses "full-length," unprocessed [X], as well as any form of [X] produced by processing in a cell. The term also encompasses naturally occurring variants of [X], such as splice variants or allelic variants.

[0281] As used herein, "treatment" (and grammatical variations thereof, such as "treat" or "treating") refers to clinical intervention that attempts to alter the natural course of the individual being treated, and can be performed for prevention or during the course of clinical pathology. Desired effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, the antibodies of the invention are used to delay the development of a disease or slow the progression of a disease.

[0282] The term "variable region" or "variable domain" refers to the domain of an antibody heavy chain or light chain that participates in the binding of an antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies (VH and VL, respectively) generally have similar structures, wherein each domain comprises four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th edition, WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, antibodies that bind to a specific antigen can be separated using VH or VL domains from antibodies that bind to the antigen, respectively, to screen libraries of complementary VL or VH domains. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0283] The term "vector" as used herein refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. The term includes vectors that are self-replicating nucleic acid structures, as well as vectors that are incorporated into the genome of a host cell into which they have been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors."

[0284] be able to combine / combine with / combine with

[0285] In the context of the present invention, the terms "bind", "bind to", "capable of binding to" and "bind to" are used interchangeably and refer to the ability of a moiety to bind to an antigen, specifically under physiological conditions, i.e., under conditions found in animals, particularly humans. Specific exemplary methods for determining the ability are described below. As used herein, "ability to bind to an antigen" and the like may also be designated as "binding affinity for an antigen" and the like. Furthermore, in the case of an antibody, the ability may also be described by using the term "antibody against" or by using the term "anti-antigen X antibody".

[0286] Specific binding / specific binding

[0287] The terms "specific binding", "specifically binds", etc. refer to the ability of an antigen binding domain to preferentially bind to a corresponding target relative to other molecules or moieties. In certain embodiments, an antigen binding domain binds to a target with a higher affinity, avidity, greater ease, and / or greater duration than it binds to another target. Generally, but not necessarily, reference to specific binding means preferential binding, wherein the affinity of the antigen binding domain for a target is at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times, or at least 1000 times greater than the affinity of the antigen binding domain for a different target.

[0288] Compositions and methods

[0289] On the one hand, the present invention is based in part on providing an antigen binding molecule with improved blood-brain barrier permeability and retention in the brain.Antigen binding molecules comprising a first antigen binding domain and a second antigen binding domain are provided, wherein the first antigen binding domain specifically binds to promote the transfer of antigen binding molecules to a first target in the mammalian brain, and the second target antigen binding domain specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide, and pharmaceutical compositions and related methods, purposes, nucleic acids, carriers, host cells and kits.The antigen binding molecules of the present invention show improved pharmacokinetic properties.The antigen binding molecules of the present invention show increased concentration, exposure and / or retention in the brain.The antigen binding molecules of the present invention can be used for diagnosis or treatment of, for example, brain disorders or diseases. 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-Draeger 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 syndrome, chronic wasting disease and fatal familial insomnia), bulbar palsy, motor neuron disease and degenerative diseases of the nervous system (including but not limited to Canavan disease, Huntington disease, neuronal ceroid lipidosis). 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).

[0290] A first antigen binding domain that specifically binds to a first target

[0291] 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 the “first target”, which means that the first target “promotes the transfer of the antigen-binding molecule into the brain of a mammal”.

[0292] The target of "promoting antigen binding molecules to be transferred to the mammalian brain" is understood as a target in the broadest sense, which results in an increase in the transfer of molecules comprising antigen binding domains specifically bound to the target relative to a compound that does not comprise an antigen binding domain that specifically binds to a target in the mammalian brain. In certain embodiments, the increase is determined by determining a higher amount or concentration of a compound comprising an antigen binding domain specifically bound to the target. In general, but not necessarily, the increase referred to as transfer means that the amount or concentration of the compound comprising antigen binding domains in the mammalian brain in vivo is at least at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times, at least 100 times or at least 1000 times higher than the amount or concentration of the compound that does not comprise an antigen binding domain specifically bound to the target. Transfer to the mammalian brain can be from any tissue, organ or cell type different from 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 substructures, etc. In an embodiment, transfer is from circulating blood to the brain." first target " can have any structure, as long as it is conducive to transfer to the mammalian brain. For example, "first target" can be protein, carbohydrate, proteoglycan, lipid, nucleic acid, etc. "first target" can be present in different cell types and organs of mammals and is not limited to any specific situation. In an embodiment, "first 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, "first target" includes targets located outside the cell, such as proteins, proteoglycans or carbohydrates of the extracellular matrix, and targets present on the cell surface of the cell, such as cell surface proteins.

[0293] 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, which are combined by tight junction proteins to form a cerebellar vascular cavity. The endothelial cells of the blood-brain barrier are referred to herein as "vascular endothelial cells of the blood-brain barrier" or "microvascular endothelial cells of the blood-brain barrier" or "BMEC". In addition, astrocytes, particularly the protrusions and pericytes of those cells referred to as astrocyte feet contribute to the structure and function of the blood-brain barrier, and these cells and their substructures are also included as parts of the blood-brain barrier. The blood-brain barrier controls the entry of all peripheral circulating factors, such as water diffusion, some gases and fat-soluble molecules, and the selective transport of other substances, such as glucose, amino acids and micronutrients that are vital to neuronal function. On the contrary, the blood-brain barrier can protect the brain from the passage of toxic substances that may endanger the central nervous system (CNS).

[0294] The term "brain" is understood to mean the organ that serves as the center of the nervous system in all vertebrates and most invertebrates.

[0295] The term "mammalian brain" is understood to be the brain of a mammal and includes, for example, the human brain, but is not limited thereto.

[0296] In the embodiments herein, the mammalian brain is a human brain.

[0297] The term "brain cell" is to be understood in the broadest sense to encompass any cell present in the mammalian brain. The term "brain cell" includes, but is not limited to, oligodendrocytes, astrocytes, neurons, and microglia.

[0298] The term "brain-specific cells" is understood in the broadest sense to encompass any cells that are present in the brain of a mammal but are absent or substantially absent in other organs or tissues of a mammal. The term "brain-specific cells" includes, but is not limited to, oligodendrocytes, astrocytes, neurons, and microglia. Cells that are substantially absent in other organs or tissues of a mammal refer to cells that are found in the brain at least 5 times, 10 times, 100 times, or more than in any other organ or tissue of a mammal.

[0299] The term "mammal" includes any mammal, including, but not limited to, humans, monkeys, horses, cows, sheep, dogs, cats, cattle, rats and mice. In an embodiment, the mammal is a human.

[0300] In an embodiment, 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).

[0301] The terms "transferrin receptor" and "TfR" are used interchangeably herein and refer to the carrier protein of transferrin. The term includes "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 an embodiment, TfR is TfR1, preferably human TfR1.

[0302] The terms "Basigin" or "CD147" are used interchangeably herein and refer to transmembrane proteins. The UNIPROT reference number of human Basigin is P35613. To date, 4 isoforms have been described, designated as isoforms 1 to 4, all of which are encompassed by the term "Basigin". In an embodiment, Basigin is human Basigin (UNIPROT accession number is P35613).

[0303] The terms "Ldlrad3" and "class A domain-containing low-density lipoprotein receptor 3" are used interchangeably herein. The protein belongs to the scavenger receptor superfamily and is described as a receptor for Venezuelan equine encephalitis virus. In an embodiment, Ldlrad3 is human Ldlrad3 (UNIPROT accession number Q86YD5).

[0304] The term "CD320" refers to the transcobalamin receptor. In an embodiment, CD320 is human CD320 (UNIPROT accession number is Q9NPF0).

[0305] The terms "insulin receptor", "InsR" and "IR" are used interchangeably herein and refer to a transmembrane receptor activated by insulin, IGF-I and IGF-II. In an embodiment, the insulin receptor is human insulin receptor (UNIPROT accession number P06213).

[0306] The terms "low-density lipoprotein receptor", "LDL-R" and "LDLR" are used interchangeably herein and refer to the low-density lipoprotein receptor, which is a chimeric protein of 839 amino acids (after removal of a 21 amino acid signal peptide) that mediates 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) found in chylomicron remnants and very low-density lipoprotein (VLDL) remnants. In an embodiment, the LDLR is human LDLR (UNIPROT accession number P01130).

[0307] 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 term includes LRP-1, LRP-1b, LRP-2, LRP-5, and LRP-6. In an embodiment, the LRP is LRP1. In an embodiment, the LRP is human LRP1 (UNIPROT accession number is Q07954).

[0308] The terms "diphtheria toxin receptor" and "proheparin binding EGF-like growth factor" are used interchangeably herein and refer to the growth factor of the human diphtheria toxin receptor protein having UNIPROT accession number Q99075. In embodiments, the diphtheria toxin receptor is human diphtheria toxin receptor (UNIPROT accession number Q99075).

[0309] The term "glucose receptor" refers to the family of glucose transporters, including but not limited to Glut1, Glut2 and Glut3. In embodiments, the glucose receptor is Glut1. In embodiments, the glucose receptor is human Glut1 (UNIPROT accession number is P11166).

[0310] 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 an embodiment, CD98hc is human CD98hc (UNIPROT accession number P08195).

[0311] The terms "TMEM30A" and "cell cycle control protein 50A" are used interchangeably and refer to the human TMEM30A protein having UNIPROT accession number Q9NV96. In an embodiment, the TMEM30A is human TMEM30A (UNIPROT accession number Q9NV96).

[0312] The terms "leptin receptor", "Lep-R" and "LepR" are used interchangeably herein and refer to a type I cytokine receptor, a protein encoded by the LEPR gene in humans. Lep-R acts as a receptor for the adipocyte-specific hormone leptin. In an embodiment, the leptin receptor is a human leptin receptor (UNIPROT accession number P48357).

[0313] The term "heparan sulfate chain (HSPG) branched from proteoglycans" refers to a heparan sulfate chain covalently attached to a glycoprotein. The heparan sulfate chain is a glycosaminoglycan (GAG). The term includes membrane HSPGs, such as polysaccharides and proteoglycans (phosphatidylinositol) anchored by glycosylphosphatidylinositols, secreted extracellular matrix HSPGs (aggregin, perlecan, type XVIII collagen), and secreted vesicle proteoglycans, serine. In an embodiment, HSPG is human HSPG1 (UNIPROT accession number is P34741).

[0314] The "first antigen binding domain" of the molecule in the present invention may have any structure as long as it specifically binds to the "first antigen" as described above. The structure of the "first antigen binding domain" may include, but is not limited to, a polypeptide or a portion thereof, or a small or medium chemical 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 or portions thereof (e.g., extracellular domains, any unique domains thereof) expressed on cells (e.g., immune cells, such as dendritic cells); antibodies (including, but not limited to, human antibodies, chimeric antibodies, humanized antibodies, and VHH antibodies) or antigen binding domains (also referred to as a portion, part, or fragment of an antibody). The antigen binding domain of an antibody includes, but is not limited to, an antibody heavy chain variable (VH) region, an antibody light chain variable (VL) region (preferably a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region), a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, Fv, a single chain Fv2 (scFv2), Fab, and F(ab')2.

[0315] The polypeptide or a portion thereof may also be an antigen-binding polypeptide, such as a module of the A domain called Avimer (which has about 35 amino acids contained in an in vivo cell membrane protein (WO2004 / 044011 and WO2005 / 040229)), an adnectin having a 10Fn3 domain as a protein binding domain (which is derived from glycoprotein fibronectin expressed on the cell membrane (WO2002 / 032925)), an Affibody having an IgG binding domain scaffold composed of a three-helix bundle of 58 amino acids of protein A (WO1995 / 001937), DARPins (designed ankyrin repeat proteins) (which are molecular surface exposed regions of ankyrin repeat sequences (AR), Each region has a 33 amino acid residue structure folded into a turn subunit, two antiparallel helices and a loop (WO2002 / 020565)), anticalins (having four loop regions connected to eight antiparallel chains bent toward a central axis at one end of a highly conserved barrel structure in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO2003 / 029462)), and inhibitory regions in the inner parallel sheet structure of the horseshoe fold composed of repeated leucine-rich repeat (LRR) modules of the immunoglobulin unstructured variable lymphocyte receptor (VLR), as seen in the acquired immune system of jawless vertebrates such as lampreys or hagfishes (WO2008 / 016854).

[0316] An example of the polypeptide or part thereof belonging to the "first antigen binding domain" described above in the present invention includes, but is not limited to, a cell membrane protein (eg, a receptor) expressed on a cell or a part thereof (eg, an extracellular domain, any unique domain thereof).

[0317] 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 a portion, part, or fragment of an antibody). Antigen binding domains of antibodies 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 (sdAb), single chain Fv (scFv), single chain antibodies, Fv, single chain Fv2 (scFv2), Fab, and F(ab')2.

[0318] In certain embodiments herein, the mammalian brain is a human brain. Therefore, in certain embodiments herein, "promoting the transfer of antigen-binding molecules to a first target in the mammalian brain" is promoting the transfer of antigen-binding molecules to a first target in the human brain.

[0319] A second antigen binding domain that specifically binds to a second target

[0320] 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 the "second target", which means that the second target is "expressed on the cell membrane of brain cells, or is a brain ECM protein or brain ECM polysaccharide."

[0321] In embodiments, the second target is different from the first target, as described above.

[0322] A target "expressed on the cell membrane of a brain cell" is understood in the broadest sense as a target expressed on the cell membrane of at least one cell type of a brain cell. For example, a target may be expressed on the cell membrane of 2, 3, 4, 5 or more different brain cells, such as, for example, on the cell membrane of neurons and oligodendrocytes. The term "expressed on the cell membrane of a brain cell" includes molecules, such as proteins, comprising at least one transmembrane protein and at least one extracellular protein associated with a transmembrane protein, as well as secreted polypeptides and proteoglycans and polypeptides, which are inserted into the cell membrane (such as by a lipid anchor) or otherwise associated with the cell membrane of a brain cell (such as by non-covalent binding, such as by binding of a ligand to a receptor).

[0323] The "second target" may have any structure as long as it is expressed on the cell membrane of brain cells. For example, the "second target" may be a protein, carbohydrate, proteoglycan, lipid, nucleic acid, etc. The "second target" may exist in different cell types and organs of mammals and is not limited to any specific case.For example, in an embodiment, the "second target" is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), neurocan 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 (γ-enolase), CD68 (macrophage sialic acid protein), allogeneic transplant inflammatory factor 1 (IBA1 or AIF1), purinergic receptor (P2RY12), interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), glutamate ion channel receptor NMDA type subunit 2B (GRIN2B), voltage-gated calcium channel auxiliary subunit γ8 (CACNG8), CD11b (integrin subunit αM), SLC6A2 (sodium-dependent norepinephrine 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 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 LRP-4, phosphoprotein associated with glycosphingolipid-rich microdomains 1 (Csk-binding protein), plasma membrane calcium transporting ATPase 1, prominin-1, somatostatin receptor type 1, carnitine O-palmitoyltransferase 1 brain isoform (CPT1-B), epidermal growth factor receptor, protein MAL2, syntaxin-1A, sodium / calcium exchanger 1, lysophosphatidylcholine acyltransferase 1 (LPC acyltransferase 1), calcineurin-3 (alcadein-β), pituitary adenylate cyclase-activating polypeptide type I receptor (PACAP type I receptor), neutral cholesterol ester hydrolase 1 (NCEH), CD166 antigen (activated leukocyte adhesion molecule), inactivated tyrosine-protein kinase 7, claudin-11, ectonucleotide phosphatase (ENPP6), tetraspanin-2 (Tspan-2), myelin proteolipid protein (PLP), glycolipid transfer protein (GLTP).

[0324] 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, and is a macromolecular network composed primarily of polysaccharide glycosaminoglycans (e.g., hyaluronic acid), 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 alpha-2 (IV) chain (angiostatin), aggrecan nexin 1 (hyaluronic acid and aggrecan nexin 1), tenascin-R (TN-R), aggrecan nexin 2 (hyaluronic acid and aggrecan nexin 2), collagen alpha-1 (I) chain, neurofilament protein-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). Examples of brain ECM polysaccharides include, but are not limited to, hyaluronic acid.

[0325] These targets and their sequences and / or structures are well known in the art.

[0326] In some embodiments, the "second target" is primarily expressed on brain-specific cells.

[0327] "Targets expressed primarily on brain-specific cells" are understood to be targets 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 on non-brain-specific cells. "Targets expressed primarily on brain-specific cells" also include targets expressed on the cell membranes of less than 10, less than 5, less than 4, less than 3 or less than 2 other tissues or organs different from the brain. In one embodiment, the target is expressed only on the cell membrane of brain-specific cells.

[0328] In a further embodiment, the second target is a molecule widely expressed in brain tissue.

[0329] The target of "a molecule widely expressed in brain tissue" is understood to be a target expressed in more than one brain tissue selected from the group consisting of cerebral cortex, hippocampal formation, amygdala, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, medulla oblongata and spinal cord. In an embodiment, the target of "a molecule widely expressed in brain tissue" is a target expressed in more than two or more than three brain tissues selected from the group consisting of cerebral cortex, hippocampal formation, amygdala, basal ganglia, thalamus, hypothalamus, midbrain, cerebellum, pons, medulla oblongata and spinal cord. For example, the second target of "widely expressed in brain tissue" can be a molecule selected from the group consisting of the following items: interleukin 1 receptor auxiliary protein-like 1 (IL1RAPL1), glutamate ion channel receptor NMDA-type subunit 2B (GRIN2B) and voltage-gated calcium channel auxiliary subunit γ8 (CACNG8). In one embodiment, the second target is expressed only in brain tissue.

[0330] The terms "myelin oligodendrocyte glycoprotein" and "MOG" are used interchangeably herein and refer to a member of the immunoglobulin (Ig) superfamily, which is a myelin protein expressed only on the outermost surface of myelin and oligodendrocyte membranes. In an embodiment, MOG is human MOG (UNIPROT accession number is Q16653).

[0331] The terms "neurocan C", "chondroitin sulfate proteoglycan 5" and "CSPG5" are used interchangeably herein and refer to brain-specific chondroitin sulfate proteoglycans. Neurocan C is a 120-150 kDa type I transmembrane glycoprotein, a member of the neuregulin family. Depending on its expression, neurocan C can be a 120 kDa glycoprotein, or a 150 kDa chondroitin sulfate (CS) proteoglycan, and both variants are included herein. In an embodiment, neurocan C is human neurocan C (UNIPROT accession number is O95196).

[0332] The terms "IGSF4B", "cell adhesion molecule 3" and "SynCAM3 (CADM3)" are used interchangeably herein and refer to a protein involved in cell-cell adhesion. In an embodiment, IGSF4B is human IGSF4B (UNIPROT accession number Q8N126).

[0333] The terms "CNPase" and "2',3'-cyclic nucleotide 3'-phosphodiesterase" are used interchangeably herein and refer to an enzyme encoded by the CNP gene in humans. CNPase is a myelin-related enzyme that accounts for 4% of total CNS myelin proteins. In an embodiment, the CNPase is human CNPase (UNIPROT accession number is P09543).

[0334] The terms "myelin-associated glycoprotein" and "MAG" are used interchangeably herein and refer to a type 1 transmembrane protein glycoprotein located in the membranes of Schwann cells and oligodendrocytes around axons, which plays a role in glial-axon interactions. MAG is a member of the SIGLEC protein family and is a functional ligand of the NOGO-66 receptor NgR. In an embodiment, MAG is human MAG (UNIPROT accession number is P20916).

[0335] The terms "myelin basic protein" and "MBP" are used interchangeably herein and refer to myelin basic protein, which is the major component of the myelin sheath of oligodendrocytes and Schwann cells. The UNIPROT accession number for the human protein is P02686. In an embodiment, the MBP is human MBP (UNIPROT accession number is P02686).

[0336] 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 known as glutamate aspartate transporter 1 (GLAST-1). In an embodiment, EAAT1 is human EAAT1 (UNIPROT accession number P43003).

[0337] 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 known as glutamate transporter 1 (GLT-1). In an embodiment, EAAT2 is human EAAT2 (UNIPROT accession number P43004).

[0338] 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 an embodiment, MAP2 is human MAP2 (UNIPROT accession number P11137).

[0339] The terms "NEFL" and "neurofilament light polypeptide" are used interchangeably herein and refer to neurofilament light chain. NEFL is a human neurofilament protein encoded by the NEFL gene. Neurofilament light chain is a biomarker that can be measured by immunoassays in cerebrospinal fluid and plasma and reflects axonal damage in a variety of nervous system disorders. In an embodiment, NEFL is human NEFL (UNIPROT accession number is P07196).

[0340] The terms "NEFM" and "neurofilament medium chain polypeptide" are used interchangeably herein and refer to neurofilament medium chain polypeptides, which are proteins encoded by the NEFM gene in humans. Neurofilament protein is a type IV intermediate filament heteropolymer, consisting of a light chain (NEFL), a medium chain (the protein) and a heavy chain (NEFH). In an embodiment, NEFM is human NEFM (UNIPROT accession number is P07197).

[0341] The terms "NSE" and "γ-enolase" are used interchangeably herein and refer to enolase 2 (ENO2) or neuron-specific enolase (NSE), which is an enzyme encoded by the ENO2 gene in humans. γ-enolase is a phosphopyruvate hydratase. γ-enolase is one of the three enolase isozymes found in mammals. The isozyme γ-enolase is a homodimer present in mature neurons and cells of neuronal origin. In an embodiment, NSE is human NSE (UNIPROT accession number is P09104).

[0342] The terms "CD68" and "macrophage sialoprotein" are used interchangeably herein and refer to an antigen called "cluster of differentiation 68". The UNIPROT accession number for the human protein is P34810. Human CD68 is a transmembrane glycoprotein whose extracellular domain is highly glycosylated. In an embodiment, CD68 is human CD68 (UNIPROT accession number is P34810).

[0343] The terms "allogeneic inflammatory factor 1" and "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 embodiments, IBA1 is human IBA1 (UNIPROT accession number P55008).

[0344] The terms "purinergic receptor" and "P2RY12" are used interchangeably herein and refer to a chemical receptor for adenosine diphosphate (ADP) belonging to the Gi class of the G protein-coupled (GPCR) purinergic receptor group. The UNIPROT accession number for the human protein is Q9H244. In an embodiment, P2RY12 is human P2RY12 (UNIPROT accession number is Q9H244).

[0345] 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. This is a protein encoded by the IL1RAPL1 gene in humans. In an embodiment, IL1RAPL1 is human IL1RAPL1 (UNIPROT accession number Q9NZN1).

[0346] The terms "glutamate ion channel 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 a protein encoded by the GRIN2B gene in humans. The UNIPROT accession number for the human protein is Q13224. In an embodiment, GRIN2B is human GRIN2B (UNIPROT accession number is Q13224).

[0347] The terms "voltage-gated calcium channel auxiliary subunit gamma 8" and "CACNG8" are used interchangeably herein and refer to human CACGN8 protein with UNIPROT accession number Q8WXS5. In an embodiment, CACNG8 is human CACNG8 (UNIPROT accession number Q8WXS5).

[0348] The terms "CD11b" and "integrin subunit αM" are used interchangeably herein and refer to human CD11b protein having UNIPROT Accession No. P11215. In embodiments, CD11b is human CD11b (UNIPROT Accession No. P11215).

[0349] The terms "SLC6A2" and "sodium-dependent norepinephrine transporter" are used interchangeably herein and refer to the human SLC6A2 protein having UNIPROT Accession No. P23975. In an embodiment, SLC6A2 is human SLC6A2 (UNIPROT Accession No. P23975).

[0350] The terms "DPP6" and "dipeptidyl peptidase-like 6" are used interchangeably herein and refer to the human DPP6 protein having UNIPROT Accession No. P42658. In an embodiment, the DPP6 is human DPP6 (UNIPROT Accession No. P42658).

[0351] The terms "SLC18A3" and "vesicular acetylcholine transporter" are used interchangeably herein and refer to the human SLC18A3 protein having UNIPROT Accession No. Q16572. In an embodiment, SLC18A3 is human SLC18A3 (UNIPROT Accession No. Q16572).

[0352] The term "sodium / potassium transporting ATPase subunit alpha-2" refers to the human sodium / potassium transporting ATPase subunit alpha-2 protein with UNIPROT Accession No. P50993. In an embodiment, the sodium / potassium transporting ATPase subunit alpha-2 is human sodium / potassium transporting ATPase subunit alpha-2 (UNIPROT Accession No. P50993).

[0353] The term "broad substrate specific ATP binding cassette transporter ABCG2" refers to a human broad substrate specific ATP binding cassette transporter ABCG2 protein with UNIPROT accession number Q9UNQ0. In an embodiment, the broad substrate specific ATP binding cassette transporter ABCG2 is a human broad substrate specific ATP binding cassette transporter ABCG2 (UNIPROT accession number is Q9UNQ0).

[0354] The term "solute carrier family 12 member 9" refers to a human solute carrier family 12 member 9 protein having UNIPROT accession number Q9BXP2. In an embodiment, the solute carrier family 12 member 9 is a human solute carrier family 12 member 9 (UNIPROT accession number Q9BXP2).

[0355] The term "electrogenic sodium bicarbonate cotransporter 1" refers to a human electrogenic sodium bicarbonate cotransporter 1 protein having UNIPROT Accession No. Q9Y6R1. In an embodiment, the electrogenic sodium bicarbonate cotransporter 1 is human electrogenic sodium bicarbonate cotransporter 1 (UNIPROT Accession No. Q9Y6R1).

[0356] The term "excitatory amino acid transporter 2" refers to the human excitatory amino acid transporter 2 protein having UNIPROT accession number P43004. In an embodiment, the excitatory amino acid transporter 2 is human excitatory amino acid transporter 2 (UNIPROT accession number is P43004).

[0357] The term "chondroitin sulfate proteoglycan 4" refers to human chondroitin sulfate proteoglycan 4 protein having UNIPROT accession number Q6UVK1. In an embodiment, chondroitin sulfate proteoglycan 4 is human chondroitin sulfate proteoglycan 4 (UNIPROT accession number is Q6UVK1).

[0358] The term "excitatory amino acid transporter 2" refers to the human excitatory amino acid transporter 2 protein having UNIPROT accession number P43004. In an embodiment, the excitatory amino acid transporter 2 is human excitatory amino acid transporter 2 (UNIPROT accession number is P43004).

[0359] The term "immunoglobulin superfamily DCC subclass member 4" refers to a human immunoglobulin superfamily DCC subclass member 4 protein having UNIPROT accession number Q8TDY8. In an embodiment, the immunoglobulin superfamily DCC subclass member 4 is a human immunoglobulin superfamily DCC subclass member 4 (UNIPROT accession number is Q8TDY8).

[0360] The term "Vang-like protein 2" refers to human Vang-like protein 2 protein having UNIPROT accession number Q9ULK5. In an embodiment, Vang-like protein 2 is human Vang-like protein 2 (UNIPROT accession number Q9ULK5).

[0361] The terms "neural cell adhesion molecule 1" and "N-CAM-1" are used interchangeably herein and refer to human N-CAM-1 protein having UNIPROT Accession No. P13591. In embodiments, N-CAM-1 is human N-CAM-1 (UNIPROT Accession No. P13591).

[0362] The terms "low-density lipoprotein receptor-related protein 4" and "LRP-4" are used interchangeably herein and refer to the human LRP-4 protein having UNIPROT Accession No. 075096. In an embodiment, LRP-4 is human LRP-4 (UNIPROT Accession No. 075096).

[0363] The terms "phosphoprotein 1 associated with glycosphingolipid-rich microdomains" and "Csk-binding protein" are used interchangeably herein and refer to human phosphoprotein 1 associated with glycosphingolipid-rich microdomains having UNIPROT accession number Q9NWQ8. In an embodiment, the phosphoprotein 1 associated with glycosphingolipid-rich microdomains is human phosphoprotein 1 associated with glycosphingolipid-rich microdomains (UNIPROT accession number is Q9NWQ8).

[0364] The term "plasma membrane calcium transporting ATPase 1" refers to the human plasma membrane calcium transporting ATPase 1 protein with UNIPROT accession number P20020. In an embodiment, the plasma membrane calcium transporting ATPase 1 is human plasma membrane calcium transporting ATPase 1 (UNIPROT accession number is P20020).

[0365] The term "Prominin-1" refers to the human Prominin-1 protein having UNIPROT accession number 043490. In an embodiment, Prominin-1 is human Prominin-1 (UNIPROT accession number 043490).

[0366] The term "somatostatin receptor type 1" refers to a human somatostatin receptor type 1 protein having UNIPROT accession number P30872. In an embodiment, the somatostatin receptor type 1 is human somatostatin receptor type 1 (UNIPROT accession number P30872).

[0367] The terms "carnitine O-palmitoyltransferase 1, brain isoform" and "CPT1-B" are used interchangeably herein and refer to the human carnitine O-palmitoyltransferase 1, brain isoform protein having UNIPROT Accession No. Q8TCG5. In embodiments, the carnitine O-palmitoyltransferase 1, brain isoform is human carnitine O-palmitoyltransferase 1, brain isoform (UNIPROT Accession No. Q8TCG5).

[0368] The term "epidermal growth factor receptor" refers to the human epidermal growth factor receptor protein having UNIPROT accession number P00533. In an embodiment, the epidermal growth factor receptor is human epidermal growth factor receptor (UNIPROT accession number P00533).

[0369] The term "protein MAL2" refers to the human protein MAL2 protein having UNIPROT accession number Q969L2. In an embodiment, the protein MAL2 is the human protein MAL2 (UNIPROT accession number Q969L2).

[0370] The term "syntaxin-1A" refers to the human syntaxin-1A protein having UNIPROT Accession No. Q16623. In an embodiment, the syntaxin-1A is human syntaxin-1A (UNIPROT Accession No. Q16623).

[0371] The term "sodium / calcium exchanger 1" refers to human sodium / calcium exchanger 1 protein having UNIPROT accession number P32418. In an embodiment, the sodium / calcium exchanger 1 is human sodium / calcium exchanger 1 (UNIPROT accession number P32418).

[0372] The terms "lysophosphatidylcholine acyltransferase 1" and "LPC acyltransferase 1" are used interchangeably herein and refer to the human LPC acyltransferase 1 protein having UNIPROT Accession No. Q8NF37. In an embodiment, LPC acyltransferase 1 is human LPC acyltransferase 1 (UNIPROT Accession No. Q8NF37).

[0373] The terms "calcadein-3" and "Alcadein-β" are used interchangeably herein and refer to human calcadein-3 protein with UNIPROT accession number Q9BQT9. In an embodiment, calcadein-3 is human calcadein-3 (UNIPROT accession number Q9BQT9).

[0374] The terms "pituitary adenylate cyclase activating polypeptide type I receptor" and "PACAP type I receptor" are used interchangeably herein and refer to the human PACAP type I receptor protein having UNIPROT accession number P41586. In embodiments, the PACAP type I receptor is human PACAP type I receptor (UNIPROT accession number P41586).

[0375] The terms "neutral cholesterol ester hydrolase 1" and "NCEH" are used interchangeably herein and refer to the human NCEH protein having UNIPROT Accession No. Q6PIU2. In embodiments, the NCEH is human NCEH (UNIPROT Accession No. Q6PIU2).

[0376] The terms "CD166" and "activated leukocyte adhesion molecule"

[0377] Used interchangeably herein, and refers to human CD166 protein with UNIPROT accession number Q13740. In an embodiment, CD166 is human CD166 (UNIPROT accession number Q13740).

[0378] The term "inactive tyrosine protein kinase 7" refers to human inactive tyrosine protein kinase 7 protein having UNIPROT accession number Q13308. In an embodiment, inactive tyrosine protein kinase 7 is human inactive tyrosine protein kinase 7 (UNIPROT accession number is Q13308).

[0379] The term "clauin-11" refers to the human claudin-11 protein having UNIPROT accession number 075508. In an embodiment, claudin-11 is human claudin-11 (UNIPROT accession number 075508).

[0380] The terms "ectonucleotide phosphatase" and "ENPP6" are used interchangeably herein and refer to the human ENPP6 protein having UNIPROT Accession No. Q6UWR7. In an embodiment, the ENPP6 is human ENPP6 (UNIPROT Accession No. Q6UWR7).

[0381] The terms "tetraspanin-2" and "Tspan-2" are used interchangeably herein and refer to human tetraspanin-2 protein having UNIPROT Accession No. 060636. In embodiments, tetraspanin-2 is human tetraspanin-2 (UNIPROT Accession No. 060636).

[0382] The terms "myelin proteolipid protein" and "PLP" are used interchangeably herein and refer to human myelin proteolipid protein with UNIPROT Accession No. P60201. In embodiments, the myelin proteolipid protein is human myelin proteolipid protein (UNIPROT Accession No. P60201).

[0383] The terms "glycolipid transfer protein" and "GLTP" are used interchangeably herein and refer to the human GLTP protein having UNIPROT Accession No. Q9NZD2. In embodiments, the GLTP is human GLTP (UNIPROT Accession No. Q9NZD2).

[0384] The terms "versican core protein", "chondroitin sulfate proteoglycan 2" and "CSPG2" are used interchangeably herein and refer to human CSPG2 protein with UNIPROT accession number P13611. In an embodiment, CSPG2 is human CSPG2 (UNIPROT accession number P13611).

[0385] The terms "tropoelastin" and "elastin" are used interchangeably herein and refer to human tropoelastin protein having UNIPROT Accession No. P15502. In embodiments, the tropoelastin is human tropoelastin (UNIPROT Accession No. P15502).

[0386] The terms "collagen α-2 (IV) chain" and "angiostatin" are used interchangeably herein and refer to human collagen α-2 (IV) chain protein with UNIPROT accession number P08572. In an embodiment, collagen α-2 (IV) chain is human collagen α-2 (IV) chain (UNIPROT accession number is P08572).

[0387] The terms "aggrecan connexin 1" and "hyaluronic acid and aggrecan connexin 1" are used interchangeably herein and refer to human aggrecan connexin 1 protein having UNIPROT accession number P10915. In an embodiment, aggrecan connexin 1 is human aggrecan connexin 1 (UNIPROT accession number P10915).

[0388] The terms "tenascin-R" and "TN-R" are used interchangeably herein and refer to human tenascin-R protein having UNIPROT Accession No. Q92752. In embodiments, tenascin-R is human tenascin-R (UNIPROT Accession No. Q92752).

[0389] The terms "aggrecan connexin 2" and "hyaluronic acid and aggrecan connexin 2" are used interchangeably herein and refer to human aggrecan connexin 2 protein with UNIPROT accession number Q9GZV7. In an embodiment, aggrecan connexin 2 is human aggrecan connexin 2 (UNIPROT accession number Q9GZV7).

[0390] The term "collagen alpha-1 (I) chain" refers to human collagen alpha-1 (I) chain protein having UNIPROT accession number P02452. In an embodiment, collagen alpha-1 (I) chain is human collagen alpha-1 (I) chain (UNIPROT accession number is P02452).

[0391] The terms "neurofilament-3" and "NEF3" are used interchangeably herein and refer to human neurofilament-3 protein having UNIPROT Accession No. Q9UK51. In an embodiment, neurofilament-3 is human neurofilament-3 (UNIPROT Accession No. Q9UK51).

[0392] The terms "immunoglobulin superfamily member 8" and "IgSF8" are used interchangeably herein and refer to human IgSF8 protein having UNIPROT Accession No. Q969P0. In an embodiment, IgSF8 is human IgSF8 (UNIPROT Accession No. Q969P0).

[0393] The terms "laminin subunit gamma-1" and "LAMC1" are used interchangeably herein and refer to human laminin subunit gamma-1 protein having UNIPROT accession number P11047. In an embodiment, the laminin subunit gamma-1 is human laminin subunit gamma-1 (UNIPROT accession number P11047).

[0394] The terms "collagen alpha-1 (VI) chain" and "Col6a1" are used interchangeably herein and refer to human collagen alpha-1 (VI) chain protein having UNIPROT accession number P12109. In an embodiment, collagen alpha-1 (VI) chain is human collagen alpha-1 (VI) chain (UNIPROT accession number is P12109).

[0395] The terms "collagen α-3 (VI) chain" and "Col6a3" are used interchangeably herein and refer to human collagen α-3 (VI) chain protein having UNIPROT accession number P12111. In an embodiment, collagen α-3 (VI) chain is human collagen α-3 (VI) chain (UNIPROT accession number is P12111).

[0396] 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 neurotrophic factor receptor (p75NTR), caspase 6, TRK A, TRK B, TRK 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 receptors, and EpCAM.

[0397] The terms "β-secretase 1" and "BACE1" are used interchangeably herein and refer to a protein also known as "β-site amyloid precursor protein cleaving enzyme 1." The UNIPROT accession number for the human protein is P56817.

[0398] The terms "Aβ" and "amyloid β" are used interchangeably herein and refer to a peptide of 36 to 43 amino acids that is the major component of amyloid plaques found in the brains of subjects with Alzheimer's disease.

[0399] The terms "epidermal growth factor receptor" and "EGFR" are used interchangeably herein. The UNIPROT accession number for the human protein is P00533.

[0400] 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 UNIPROT accession number for the human protein is P04626.

[0401] The terms "tau" and "microtubule-associated protein tau" are used interchangeably herein. The UNIPROT accession number for the human protein is P10636.

[0402] The terms "apolipoprotein E" and "ApoE" are used interchangeably herein. Various variants and isoforms of ApoE have been described and are included in the term "ApoE", such as variant E4.

[0403] The terms "α-synuclein" and "alpha synuclein" are used interchangeably herein and refer to the protein that is encoded by the SNCA gene in humans. α-Synuclein is a neuronal protein that regulates synaptic vesicle trafficking and subsequent neurotransmitter release.

[0404] The term "CD20" refers to a protein also known as "B-lymphocyte antigen CD20." In humans, CD20 is encoded by the MS4A1 gene.

[0405] The terms "huntingtin" and "Htt" are used interchangeably herein and refer to the protein encoded by the human HTT gene, which is also known as the IT15 ("transcript of interest 15") gene.

[0406] 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 subtypes of the protein included in the term "PrP", such as the PrPC form, a protease-resistant form called PrPRes, such as the disease-causing PrPSc (scrapie) and a subtype located in the mitochondria.

[0407] 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. The protein is also known as dardarin or PARK8.

[0408] The term parkin refers to a protein that in humans is encoded by the PSEN-1 gene. Parkin is a 465 amino acid residue E3 ubiquitin ligase.

[0409] The term "presenilin-1" refers to a protein encoded by the PSEN-1 gene in humans. Presenilin-1 is one of the four core proteins in the γ-secretase complex and is believed to play an important role in the generation of β-amyloid (Aβ) from amyloid precursor protein (APP).

[0410] The term "presenilin-2" refers to the protein encoded by the PSEN2 gene in humans. It is described as a possible catalytic subunit of the γ-secretase complex.

[0411] The term "γ-secretase" refers to a multi-subunit protease complex composed of four individual proteins, PSEN1 (presenilin-1), sedrin, APH-1 (anterior pharyngeal defect 1), and PEN-2 (presenilin enhancer 2). The complex itself is an integral membrane protein that cleaves single-pass transmembrane proteins at residues within the transmembrane domain. The best-known substrate of γ-secretase is the amyloid precursor protein.

[0412] 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".

[0413] The terms "amyloid precursor protein" and "APP" are used interchangeably herein and refer to the precursor protein of A[beta].

[0414] The terms "p75 neurotrophin receptor" and "p75NTR" are used interchangeably herein and refer to the protein encoded by the NGFR gene in humans.

[0415] The terms "caspase 6" and "caspase-6" are used interchangeably herein and refer to the protein encoded by the human CASP6 gene.

[0416] The term "Trk A" refers to tropomyosin receptor kinase A. TrkA is a protein encoded by the NTRK1 gene.

[0417] The term "Trk B" refers to tropomyosin receptor kinase B. TrkB is a protein encoded by the NTRK2 gene.

[0418] The term "Trk C" refers to tropomyosin receptor kinase C. TrkC is a protein encoded by the NTRK3 gene.

[0419] 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.

[0420] 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.

[0421] The terms "vascular endothelial growth factor" and "VEGF" are used interchangeably herein and refer to the protein having UNIPROT Accession No. Q9UNS8 in humans.

[0422] The term "neuropilin" refers to a protein receptor and describes two forms thereof, NRP-1 and NRP-2, both of which are included in the term "neuropilin". In humans, the UNIPROT accession number for NRP-1 is O14786. In humans, the UNIPROT accession number for NRP-2 is O60462.

[0423] The term "Semaphorin" is 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 present in both vertebrates and invertebrates. In humans, the genes encoding Semaphorins are SEMA3A, SEMA3B, SEMA3C, SEMA3D, SEMA3E, SEMA3F, SEMA3GSEMA4A, SEMA4B, SEMA4C ("SEMAF"), SEMA4D, SEMA4F, SEMA4G, SEMA5A, SEMA5B, SEMA6A, SEMA6B, SEMA6C, SEMA6D, and SEMA7A.

[0424] The term "Semaphorin 3A" refers to the Semaphorin encoded by the SEMA3A gene in humans.

[0425] The term "Semaphorin 4A" refers to the Semaphorin encoded by the SEMA4A gene in humans.

[0426] The term "Semaphorin 6A" refers to the Semaphorin encoded by the SEMA6A gene in humans.

[0427] The term "PLP" means "myelin proteolipid protein".

[0428] The term "aquaporin-4" refers to the aquaporin encoded by the AQP4 gene in humans. This protein is also referred to as "AQP-4".

[0429] The term "glutamate receptor" refers to glutamate receptors and includes glutamate receptor 1 and glutamate receptor 2, but is not limited thereto.

[0430] The term "EpCAM" refers to a protein also known as "epithelial cell adhesion molecule".

[0431] The "second antigen binding domain" of the molecule in the present invention may have any structure as long as it specifically binds to the "second antigen" as described above. The structure of the "second antigen binding domain" may include, but is not limited to, a polypeptide or a portion thereof, or a small or medium chemical 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 or portions thereof (e.g., extracellular domains, any unique domains thereof) expressed on cells (e.g., immune cells, such as dendritic cells); antibodies (including, but not limited to, human antibodies, chimeric antibodies, humanized antibodies, and VHH antibodies) or antigen binding domains (also referred to as a portion, part, or fragment of an antibody). The antigen binding domain of an antibody includes, but is not limited to, an antibody heavy chain variable (VH) region, an antibody light chain variable (VL) region (preferably a combination of an antibody heavy chain variable (VH) region and an antibody light chain variable (VL) region), a single domain antibody (sdAb), a single chain Fv (scFv), a single chain antibody, Fv, a single chain Fv2 (scFv2), Fab, and F(ab')2.

[0432] The polypeptide or a portion thereof may also be an antigen-binding polypeptide, such as a module of the A domain called Avimer (which has about 35 amino acids contained in an in vivo cell membrane protein (WO2004 / 044011 and WO2005 / 040229)), an adnectin having a 10Fn3 domain as a protein binding domain (which is derived from glycoprotein fibronectin expressed on the cell membrane (WO2002 / 032925)), an Affibody having an IgG binding domain scaffold composed of a three-helix bundle of 58 amino acids of protein A (WO1995 / 001937), DARPins (designed ankyrin repeat proteins) (which are molecular surface exposed regions of ankyrin repeat sequences (AR), Each region has a 33 amino acid residue structure folded into a turn subunit, two antiparallel helices and a loop (WO2002 / 020565)), anticalins (having four loop regions connected to eight antiparallel chains bent toward a central axis at one end of a highly conserved barrel structure in lipocalin molecules such as neutrophil gelatinase-associated lipocalin (NGAL) (WO2003 / 029462)), and inhibitory regions in the inner parallel sheet structure of the horseshoe fold composed of repeated leucine-rich repeat (LRR) modules of the immunoglobulin unstructured variable lymphocyte receptor (VLR), as seen in the acquired immune system of jawless vertebrates such as lampreys or hagfishes (WO2008 / 016854).

[0433] An example of the polypeptide or part thereof belonging to the "second antigen binding domain" in the present invention includes, but is not limited to, a cell membrane protein (eg, a receptor) expressed on a cell or a part thereof (eg, an extracellular domain, any unique domain thereof).

[0434] 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 a portion, part, or fragment of an antibody). Antigen binding domains of antibodies 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 (sdAb), VHH, single chain Fv (scFv), single chain antibodies, Fv, single chain Fv2 (scFv2), Fab, single chain Fab (scFab), and F(ab')2.

[0435] Antigen binding molecule comprising a first antigen binding domain and a second antigen binding domain

[0436] 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, wherein these domains may be covalently or non-covalently linked. The term includes, but is not limited to, molecules having the following exemplary features.

[0437] 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 the "second antigen". Alternatively, instead of or in addition to conjugation to the Fc region, the "first antigen binding domain" can be conjugated to one of the Fab arms. In addition, more than two "first antigen binding domains" can be conjugated to the Fc region. The two Fab arms can be conjugated to the Fc region in various forms, as shown in Figures 2(b) and 2(c). As an alternative to the Fab arms, the antigen binding domains of one or both Fab arms can have different forms, such as VHH, scFv or scFab.

[0438] 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 bispecific antigen binding domain, wherein one Fab arm binds to a first target that promotes transfer of the antigen binding molecule to the mammalian brain as described above, and the other Fab arm binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells or (ii) is a brain ECM protein or a brain ECM polysaccharide. The antibody or antibody-like molecule in this example can have various forms as shown in Figure 2 (b).

[0439] 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 with or without a linker. An example of this embodiment is a fusion protein comprising a VHH or scFv that binds to TfR or a VHH or scFv that binds to MOG, optionally fused via a peptide linker. Some examples in Figures 2(b) and 2(c) illustrate such embodiments.

[0440] iv. A chemical compound comprising a "first antigen binding domain" and a "second antigen binding domain". An example of this embodiment may be a chemical compound comprising at least two parts, wherein one part binds to a first target that promotes the transfer of antigen binding molecules to the mammalian brain as described above, and the other part binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells or (ii) is a brain ECM protein or a brain ECM polysaccharide. Such chemical compounds may be carried out using bioconjugate techniques (Bioconjugate Techniques, 3rd edition, 2013, Greg T. Hermanson). The linker or compound may, for example, further comprise a photoreactive group (e.g., aryl azide, diazirine, psoralen).

[0441] The antigen binding molecule further comprises at least one functional moiety and / or at least one in vivo half-life extending moiety

[0442] In embodiments, the antigen binding molecule of any aspect and embodiments herein further comprises at least one functional moiety and / or at least one in vivo half-life extending moiety.

[0443] In an embodiment, the antigen binding molecule of any aspect and embodiment of the present invention further comprises

[0444] (i) at least one functional portion comprising an enzyme, a therapeutic protein, an antibody or an antigen-binding fragment thereof, a peptide, a DNA, a shRNA, a siRNA, a small molecule drug or a cytotoxic agent, and / or

[0445] (ii) at least one in vivo half-life extending moiety, preferably wherein said at least one in vivo half-life extending moiety 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.

[0446] In some embodiments, the antigen binding molecules of any aspect and embodiment of this article further comprise at least one functional part.In an embodiment, the functional part is selected from the group consisting of enzymes, therapeutic proteins, antibodies or antigen binding fragments thereof, peptides, DNA, shRNA, siRNA, small molecule drugs and cytotoxic agents.

[0447] In certain embodiments, the functional moiety is not an Fc region.

[0448] Exemplary embodiments of formats of antigen binding molecules further comprising at least one functional moiety are depicted in Figures 2(a) and 2(c).

[0449] In embodiments, the antigen binding molecule of any aspect and embodiment herein further comprises at least one in vivo half-life extending moiety. In embodiments, the in vivo half-life extending moiety 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.

[0450] Exemplary embodiments of the format of an antigen binding molecule further comprising an Fc region as an exemplary in vivo half-life extending moiety are depicted in Figures 2(a) and 2(c).

[0451] In an embodiment, the antigen binding molecules of any aspect and embodiment of this article further comprise at least one functional portion and at least one in vivo half-life extending portion. An exemplary embodiment of the form of an antigen binding molecule further comprising at least one functional portion and an Fc region as an exemplary in vivo half-life extending portion is depicted in Figures 2 (a) and 2 (c).

[0452] The term "in vivo half-life" of a molecule is understood to be the time required for the concentration of the molecule in a given tissue to be halved (half-life) to its steady state when the molecule is present in the corresponding tissue of the subject. In an embodiment, the subject is a mammal. In an embodiment, 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.

[0453] The term "in vivo half-life extending moiety" is understood as a moiety that, when attached to an active agent, extends the in vivo half-life in the blood compared to an active agent without such a moiety. In embodiments, an "in vivo half-life extending moiety" is understood as a moiety that, when attached to an active agent, extends the in vivo half-life by at least 10%, 20%, 30%, 40%, 50%, 100%, 200%, 500%, or 1000%.

[0454] Suitable in vivo half-life extending moieties are known in the art and can be used. In an embodiment, 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 is capable of binding to one or more Fc receptors. In particular, in the case of an Fc region, the Fc region is capable of binding to FcRn. Such Fc regions are suitable for extending the in vivo half-life.

[0455] In an embodiment, the antigen binding molecule of any aspect or embodiment is an Fc region-containing antibody.

[0456] In other embodiments, the in vivo half-life extension moiety is an FcRn binding protein or FcRn binding peptide. Such moieties are known in the art and are disclosed, for example, in US20170275373 and Datta-Mannan A et al. (Biotechnol J. 2019 March; 14(3): e1800007.doi: 10.1002 / biot.201800007).

[0457] A "PEG" moiety is understood to be a moiety comprising two or more polyethylene glycol moieties covalently linked to each other. For example, a PEG moiety with a molecular weight between 500 Da and 100 kDa, such as between 1000 Da and 50000 Da, may be used.

[0458] An "albumin binding domain" is understood to be a portion that is capable of binding to mammalian albumin. In embodiments, the mammalian albumin is human albumin. The structure of the albumin binding domain is not limited and includes, for example, proteins, peptides, and small molecules. In embodiments, the albumin binding domain is a peptide or protein. Suitable portions are known in the art and are described, for example, in Tan H et al. (Eur J Pharmacol. 2021 Jan 5; 890: 173650).

[0459] "Functional moiety" is understood to be a moiety that can exert a therapeutic, preventive and / or diagnostic effect. The skilled person is aware of such "functional moieties" and can select such "functional moieties" according to the disease or disorder to be treated, prevented and / or diagnosed. In an embodiment, the functional moiety comprises an enzyme, a therapeutic protein, an antibody or an antigen-binding fragment thereof, a peptide, a DNA, a shRNA, a 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.

[0460] Fc receptors

[0461] 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 natural human FcR. In some embodiments, the FcR is an FcR that binds to an IgG antibody (a gamma receptor), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants of these receptors and alternative splicing forms. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), both of which have similar amino acid sequences, the main difference being 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 inhibition motif (ITIM) in its cytoplasmic domain. (See, e.g., Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcRs are reviewed, for example, by 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). The term "FcR" herein encompasses other FcRs, including those to be identified in the future.

[0462] The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and regulating the homeostasis of immunoglobulins. 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.).

[0463] In vivo binding to human FcRn and the plasma half-life of human FcRn high affinity binding polypeptides can be determined, for example, in transgenic mice or transfected human cell lines expressing human FcRn or in primates administered polypeptides with variant Fc regions. WO 2000 / 42072 (Presta) describes antibody variants with increased or decreased binding to FcR. See also, for example, Shields et al. J. Biol. Chem. 9(2): 6591-6604 (2001).

[0464] Antibodies containing Fc region

[0465] The term "Fc region-containing antibody" refers to an antibody comprising 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 can be removed, for example, during the purification of the antibody or by recombinantly designing the nucleic acid encoding the antibody. Therefore, a composition comprising an antibody having an Fc region according to the present invention may comprise an antibody having G446 to K447, an antibody having G446 but not having K447, an antibody with all G446 to K447 removed, or a mixture of all three types of antibodies.

[0466] Methods for increasing the concentration of antigen-binding molecules in the brain, and methods for increasing the exposure of antigen-binding molecules in the brain Method and method for retaining antigen-binding molecules in the brain

[0467] 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:

[0468] (a) providing a first antigen-binding molecule, the first antigen-binding molecule comprising:

[0469] (a1) a first antigen-binding domain that specifically binds to a first target that facilitates transfer of the antigen-binding molecule into the brain of a mammal; or

[0470] (a2) a second antigen-binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide;

[0471] (b) in the case of (a1), introducing into the first antigen-binding molecule at least one second antigen-binding domain that specifically binds to the second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide, thereby obtaining a second antigen-binding molecule; or

[0472] In the case of (a2), at least one first antigen-binding domain that specifically binds to the first target is introduced into the first antigen-binding molecule, and the first target promotes the transfer of the antigen-binding molecule into the brain of a mammal, thereby obtaining a second antigen-binding molecule;

[0473] Such that, after administration to the subject, the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule.

[0474] In some embodiments, "the concentration of the antigen binding molecule in the brain of a subject" is understood as the concentration of the antigen binding molecule in the brain per weight of brain tissue, and is generally provided in units of micrograms / g brain. The concentration can be determined as follows ("antibodies" are provided herein as exemplary antigen binding molecules:

[0475]

[0476] In some additional embodiments, the term "increasing the concentration of the antigen binding molecule in the brain of a subject" is understood to mean increasing the concentration (micrograms per gram of 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.

[0477] In some embodiments, the "concentration of the antigen binding molecule in the brain of a subject" is understood as a percentage value of the injected dose per brain tissue weight and is usually provided as %ID (injected dose) / g brain, which can be determined as follows ("antibody" is provided herein as an exemplary antigen binding molecule:

[0478]

[0479] In some additional embodiments, the term "increasing the concentration of the antigen binding molecule in the brain of a subject" is understood to mean 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.

[0480] In an embodiment, a method for increasing the concentration of an antigen binding molecule in the brain of a subject in need thereof is provided, comprising:

[0481] (a) providing a first antigen-binding molecule, the first antigen-binding molecule comprising:

[0482] a first antigen binding domain that specifically binds to a first target that promotes transfer of the antigen binding molecule into the brain of a mammal;

[0483] (b) To

[0484] The first antigen-binding molecule introduces at least one second antigen-binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide, thereby obtaining a second antigen-binding molecule;

[0485] Such that, after administration to the subject, the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule.

[0486] In other embodiments, a method for increasing the concentration of an antigen binding molecule in the brain of a subject in need thereof is provided, comprising:

[0487] (a) providing a first antigen-binding molecule, the first antigen-binding molecule comprising:

[0488] a second antigen binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide;

[0489] (b) To

[0490] The first antigen-binding molecule introduces at least one first antigen-binding domain, which specifically binds to a first target that promotes the transfer of the antigen-binding molecule to the brain of a mammal, thereby obtaining a second antigen-binding molecule;

[0491] Such that, after administration to the subject, the concentration of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule.

[0492] For the “first antigen-binding domain”, “first target”, “second antigen-binding domain” and “second target”, the same embodiments as for the “antigen-binding molecule” of any of the above aspects and embodiments apply.

[0493] The "introducing" step in (b) can be achieved by methods known to those skilled in the art, such as by covalently linking the "second antigen-binding domain" or the "first antigen-binding domain" to the "first antigen-binding molecule", optionally using a linker. For example, the "introducing" step can include generating one or more nucleic acids encoding the second antigen-binding molecule and obtaining the second antigen-binding molecule by recombinant expression technology.

[0494] In an embodiment, the second antigen binding molecule is as defined in any aspect and embodiment of the antigen binding molecules of the invention.

[0495] In certain embodiments, the method further comprises step (c):

[0496] (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 differs from the second antigen-binding molecule according to (b) only in that the control antigen-binding molecule does not comprise:

[0497] In the case of (a1), the at least one second antigen binding domain specifically binds to the second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide; or

[0498] In the case of (a2), the at least one first antigen-binding domain specifically binds to the first target, which promotes the transfer of the antigen-binding molecule into the brain of a mammal.

[0499] The concentration of the control can be determined at the same time point or at different time points. In one embodiment, the concentration of the control can be determined at the same time point.

[0500] Therefore, in the case of (a1), the "control antigen binding molecule" does not contain the at least one second antigen binding domain that specifically binds to the second target; or in the case of (a2), the "control antigen binding molecule" does not contain the at least one first antigen binding domain that specifically binds to the first target. The rest remains unchanged.

[0501] In Examples 3, 5, 6 and 7 and Figures 3 (c), 6 (c), 8 (c) and 10 (c), it was found that the concentration of the bispecific antibody comprising the brain metastasis portion and the brain retention portion in the brain was increased (determined as % ID / g brain) compared to the control antibody comprising only the brain metastasis portion or only the brain retention portion. For example, anti-Basigin / / anti-MOG bispecific antibodies, anti-TfR / / anti-MOG bispecific antibodies and anti-TfR / / anti-CADM3 bispecific antibodies are provided in Examples 4 and 5. Surprisingly, it was found that the combination of the brain metastasis portion and the brain retention portion in these bispecific antibodies showed much higher concentrations on the 7th day compared to the antibodies having the brain metastasis portion alone or the brain retention portion alone. These further data also show that the combination of the brain metastasis portion and the brain retention portion has a synergistic effect on the retention efficacy of the antibody in the brain. In addition, anti-TfR / / anti-CSPG5 bispecific antibodies are provided in Examples 4 and 7. The results in Example 7 indicate that the combination of a brain-transferring portion that binds to TfR and a brain-retention portion that binds to CSPG5 has superior efficacy in transfer and retention into the brain compared to an antibody comprising only a portion that binds to CSPG5.

[0502] In an embodiment, the concentration of the second antigen binding molecule in the brain of the subject is Cmax."Cmax" is also called maximum concentration, and refers to the highest antibody concentration in the brain within a period of time after administration.

[0503] In the embodiments herein, the mammalian brain is a human brain. Thus, in one embodiment, the first target promotes the transfer of the antigen binding molecule into the human brain. Methods for determining Cmax are known in the art. Suitable methods for determining Cmax are described in the Examples.

[0504] For example, Example 3 shows that the cmax in the brain of a bispecific antibody comprising a brain transfer portion and a brain retention portion (anti-TfR / / anti-MOG bispecific antibody) is higher than the cmax in the brain of a bispecific antibody comprising only a brain transfer portion (anti-TfR / / anti-KLH).

[0505] In a further embodiment, the first target is as specified in any one of the embodiments of the antigen binding molecules 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 molecules 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 molecules of the first aspect herein.

[0506] Specifically, in a further embodiment, the first target and the second target are as follows:

[0507] (i) the first target is transferrin receptor (TfR), and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0508] (ii) the first target is transferrin receptor (TfR), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0509] (iii) the first target is transferrin receptor (TfR), and the second target is cell adhesion molecule 3 (CADM3);

[0510] (iv) the primary target is Basigin (CD147), and the secondary target is myelin oligodendrocyte glycoprotein (MOG);

[0511] (v) the first target is Basigin (CD147), and the second target is Neurocan C / Chondroitin Sulfate Proteoglycan 5 (CSPG5); or

[0512] (vi) the first target is Basigin (CD147), and the second target is cell adhesion molecule 3 (CADM3);

[0513] (vii) the first target is the insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0514] (viii) the first target is the insulin receptor and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0515] (ix) the first target is the insulin receptor, and the second target is cell adhesion molecule 3 (CADM3);

[0516] (x) the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0517] (xi) the first target is the glucose receptor and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0518] (xii) the first target is glucose receptor and the second target is cell adhesion molecule 3 (CADM3);

[0519] (xiii) the primary target is low-density lipoprotein receptor (LDLR), and the secondary target is myelin oligodendrocyte glycoprotein (MOG);

[0520] (xiv) the first target is low density lipoprotein receptor (LDLR) and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0521] (xv) the first target is low-density lipoprotein receptor (LDLR), and the second target is cell adhesion molecule 3 (CADM3);

[0522] (xvi) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0523] (xvii) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0524] (xviii) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is cell adhesion molecule 3 (CADM3); or

[0525] (xix) the first target is CD98hc and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0526] (xx) the first target is CD98hc and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0527] (xxi) The first target is CD98hc, and the second target is cell adhesion molecule 3 (CADM3).

[0528] Specifically, the second antigen binding molecule is an antigen binding molecule according to any one of the embodiments of the antigen binding molecule of the first aspect herein.

[0529] In another aspect, a method for increasing exposure of an antigen binding molecule in the brain of a subject in need thereof is provided, comprising:

[0530] (a) providing a first antigen-binding molecule, the first antigen-binding molecule comprising:

[0531] (a1) a first antigen-binding domain that specifically binds to a first target that facilitates transfer of the antigen-binding molecule into the brain of a mammal; or

[0532] (a2) a second antigen-binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide;

[0533] (b) In the case of (a1), introducing into the first antigen-binding molecule

[0534] at least one second antigen-binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of a brain cell, or (ii) is a brain (ECM) protein or brain ECM polysaccharide, thereby obtaining a second antigen-binding molecule; or

[0535] In the case of (a2), at least one first antigen-binding domain that specifically binds to a first target is introduced into the first antigen-binding molecule, and the first target promotes the transfer of the antigen-binding molecule into the brain of a mammal, thereby obtaining a second antigen-binding molecule;

[0536] Such that, after administration to the subject, exposure of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule.

[0537] The term "exposure of the second antigen binding molecule in the subject's brain" is understood as 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 curve of the antigen binding molecule" are used interchangeably herein and correspond to the cumulative exposure of the antibody in the brain during the study. AUC can be determined by pharmacokinetic methods known in the art, such as by measuring the concentration of the target antigen binding molecule at multiple discrete time points and using the trapezoidal rule to estimate and thus determine AUC. Suitable methods for determining AUC are described in the examples.

[0538] The term "increasing exposure of an antigen binding molecule in the brain of a subject" is understood to mean increasing 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.

[0539] For the “first antigen-binding domain”, “first target”, “second antigen-binding domain” and “second target”, the same embodiments as for the “antigen-binding molecule” of any of the above aspects and embodiments apply.

[0540] The "introducing" step in (b) can be achieved by methods known to those skilled in the art, such as by covalently linking the "second antigen-binding domain" or the "first antigen-binding domain" to the "first antigen-binding molecule", optionally using a linker. For example, the "introducing" step can include generating one or more nucleic acids encoding the second antigen-binding molecule and obtaining the second antigen-binding molecule by recombinant expression technology.

[0541] In an embodiment, the second antigen binding molecule is as defined in any aspect and embodiment of the antigen binding molecules of the invention.

[0542] In an embodiment, a method for increasing exposure of an antigen binding molecule in the brain of a subject in need thereof is provided, comprising:

[0543] (a) providing a first antigen-binding molecule, the first antigen-binding molecule comprising:

[0544] a first antigen binding domain that specifically binds to a first target that promotes transfer of the antigen binding molecule into the brain of a mammal;

[0545] (b) To

[0546] The first antigen-binding molecule introduces at least one second antigen-binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide, thereby obtaining a second antigen-binding molecule;

[0547] Such that, after administration to the subject, exposure of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule.

[0548] In an embodiment, a method for increasing exposure of an antigen binding molecule in the brain of a subject in need thereof is provided, comprising:

[0549] (a) providing a first antigen-binding molecule, the first antigen-binding molecule comprising:

[0550] a second antigen binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide;

[0551] (b) To

[0552] The first antigen-binding molecule introduces at least one first antigen-binding domain, which specifically binds to a first target that promotes the transfer of the antigen-binding molecule to the brain of a mammal, thereby obtaining a second antigen-binding molecule;

[0553] Such that, after administration to the subject, exposure of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule.

[0554] In an embodiment, the method further comprises step (c):

[0555] (c) determining that 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 differs from the second antigen-binding molecule according to (b) only in that the control antigen-binding molecule does not comprise:

[0556] In the case of (a1), the at least one second antigen binding domain specifically binds to the second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide; or

[0557] In the case of (a2), the at least one first antigen binding domain specifically binds to the first target, which promotes the transfer of the antigen binding molecule into the brain of a mammal.

[0558] In embodiments, the exposure of the second antigen binding molecule in the brain of the subject is the AUC (area under the curve) of the brain concentration-time curve of the antigen binding molecule.

[0559] In the embodiments herein, the mammalian brain is a human brain. Thus, in one embodiment, the first target promotes the transfer of the antigen binding molecule into the human brain.

[0560] In a further embodiment, the first target is as specified in any one of the embodiments of the antigen binding molecules 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 molecules 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 molecules of the first aspect herein.

[0561] Specifically, in a further embodiment, the first target and the second target are as follows:

[0562] (i) the first target is transferrin receptor (TfR), and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0563] (ii) the first target is transferrin receptor (TfR), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0564] (iii) the first target is transferrin receptor (TfR), and the second target is cell adhesion molecule 3 (CADM3);

[0565] (iv) the primary target is Basigin (CD147), and the secondary target is myelin oligodendrocyte glycoprotein (MOG);

[0566] (v) the first target is Basigin (CD147), and the second target is Neurocan C / Chondroitin Sulfate Proteoglycan 5 (CSPG5); or

[0567] (vi) the first target is Basigin (CD147), and the second target is cell adhesion molecule 3 (CADM3);

[0568] (vii) the first target is the insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0569] (viii) the first target is the insulin receptor and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0570] (ix) the first target is the insulin receptor, and the second target is cell adhesion molecule 3 (CADM3);

[0571] (x) the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0572] (xi) the first target is the glucose receptor and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0573] (xii) the first target is glucose receptor and the second target is cell adhesion molecule 3 (CADM3);

[0574] (xiii) the primary target is low-density lipoprotein receptor (LDLR), and the secondary target is myelin oligodendrocyte glycoprotein (MOG);

[0575] (xiv) the first target is low density lipoprotein receptor (LDLR) and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0576] (xv) the first target is low-density lipoprotein receptor (LDLR), and the second target is cell adhesion molecule 3 (CADM3);

[0577] (xii) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0578] (xii) the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0579] (xviii) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is cell adhesion molecule 3 (CADM3); or

[0580] (xix) the first target is CD98hc and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0581] (xx) the first target is CD98hc and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0582] (xxi) The first target is CD98hc, and the second target is cell adhesion molecule 3 (CADM3).

[0583] Specifically, the second antigen binding molecule is an antigen binding molecule according to any one of the embodiments of the antigen binding molecule of the first aspect herein.

[0584] In Example 3 of Figure 3 (d), it was found that the exposure of the bispecific antibody comprising the brain metastasis portion and the brain retention portion in the brain was increased (determined by AUC) compared to the control antibody comprising only the brain metastasis portion or only the brain retention portion. In particular, unexpectedly, MOG303 / / TfR showed a synergistic effect in terms of cumulative brain AUC (area under the curve), which represents the total antibody exposure in the brain over a period of time, wherein the cumulative brain AUC of MOG303 / / TfR was 108 times, 15.2 times, and 24.7 times higher than the brain AUC of KLH, MOG303, and KLH / / TfR, respectively (Figure 3 (d)). It is worth noting that the cumulative brain AUC of MOG303 / / TfR (73.0 micrograms / g*day) is greater than the sum of the cumulative brain AUCs of MOG303 (4.80 micrograms / g*day) and KLH / / TfR (2.95 micrograms / g*day). The results suggest that the combination of the transfer part (anti-TfR) and the retention part (anti-MOG) in the bispecific antibody provides a synergistic effect of more sustained and high antibody concentrations in the brain.

[0585] In one aspect, a method for retaining an antigen binding molecule in the brain of a subject in need thereof is provided, comprising:

[0586] (a) providing a first antigen-binding molecule, the first antigen-binding molecule comprising a first antigen-binding domain that specifically binds to a first target, the first target facilitating transfer of the antigen-binding molecule into the brain of a mammal;

[0587] (b) introducing into the first antigen-binding molecule at least one second antigen-binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide, thereby obtaining a second antigen-binding molecule;

[0588] Such that, after administration to the subject, the retention of the second antigen-binding molecule in the brain of the subject is increased compared to the first antigen-binding molecule.

[0589] Specifically, the data for the 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 in Examples 3, 5, 6, and 7 indicate that the combination of the brain transfer portion and the brain retention portion has a synergistic effect on the retention efficacy of the antibody in the brain.

[0590] The term "retention" or "retention of the antigen binding molecule in the brain of a subject" is to be understood as the in vivo half-life in the brain of a subject.

[0591] For example, Example 3 and Figure 3 show that a bispecific antibody comprising a brain transfer portion and a brain retention portion (anti-TfR / / anti-MOG bispecific antibody) has a higher in vivo half-life in the brain of a subject than a bispecific antibody comprising only a brain transfer portion (anti-TfR / / anti-KLH).

[0592] The term "half-life in vivo in the brain" of a molecule is understood to mean the time required for the concentration of the molecule in the brain to be halved (half-life) to its steady state when the molecule is present in the brain of a subject. In an embodiment, the subject is a mammal. In an embodiment, the subject is a human. In a further embodiment, "retention of the second antigen-binding molecule in the brain of the subject" is the half-life in vivo in the brain.

[0593] The term "increasing the retention of an antigen binding molecule in the brain of a subject" is understood to mean increasing the retention 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.

[0594] In an embodiment, 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. For example, 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 or at most 100 days, 150 days or 200 days, or any sub-range thereof.

[0595] Therefore, in an embodiment, compared with the first antigen binding molecule, the in vivo half-life of the second antigen binding molecule in the brain increases 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, and 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. For example, 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 or at most 100 days, 150 days or 200 days, or any sub-range thereof.

[0596] For the “first antigen-binding domain”, “first target”, “second antigen-binding domain” and “second target”, the same embodiments as for the “antigen-binding molecule” of any of the above aspects and embodiments apply.

[0597] The "introducing" step in (b) can be achieved by methods known to those skilled in the art, such as by covalently linking the "second antigen-binding domain" to the "first antigen-binding molecule", optionally using a linker. For example, the "introducing" step can encompass the generation of one or more nucleic acids encoding the second antigen-binding molecule and obtaining the second antigen-binding molecule by recombinant expression technology.

[0598] In an embodiment, the second antigen binding molecule is as defined in any aspect and embodiment of the antigen binding molecules of the invention.

[0599] In an embodiment, the method further comprises step (c):

[0600] (c) determining that the retention 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 according to (b) only in that the control antigen binding molecule does not comprise the at least one antigen binding domain that specifically binds to the second target.

[0601] In the embodiments herein, the mammalian brain is a human brain. Thus, in one embodiment, the first target promotes the transfer of the antigen binding molecule into the human brain.

[0602] In a further embodiment, the first target is as specified in any one of the embodiments of the antigen binding molecules 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 molecules 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 molecules of the first aspect herein.

[0603] Specifically, in a further embodiment, the first target and the second target are as follows:

[0604] (i) the first target is transferrin receptor (TfR), and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0605] (ii) the first target is transferrin receptor (TfR), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0606] (iii) the first target is transferrin receptor (TfR), and the second target is cell adhesion molecule 3 (CADM3);

[0607] (iv) the primary target is Basigin (CD147), and the secondary target is myelin oligodendrocyte glycoprotein (MOG);

[0608] (v) the first target is Basigin (CD147), and the second target is Neurocan C / Chondroitin Sulfate Proteoglycan 5 (CSPG5); or

[0609] (vi) the first target is Basigin (CD147), and the second target is cell adhesion molecule 3 (CADM3);

[0610] (vii) the first target is the insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0611] (viii) the first target is the insulin receptor and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0612] (ix) the first target is the insulin receptor, and the second target is cell adhesion molecule 3 (CADM3);

[0613] (x) the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0614] (xi) the first target is the glucose receptor and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5);

[0615] (xii) the first target is glucose receptor and the second target is cell adhesion molecule 3 (CADM3);

[0616] (xiii) the primary target is low-density lipoprotein receptor (LDLR), and the secondary target is myelin oligodendrocyte glycoprotein (MOG);

[0617] (xiv) the first target is low density lipoprotein receptor (LDLR) and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0618] (xv) the first target is low-density lipoprotein receptor (LDLR), and the second target is cell adhesion molecule 3 (CADM3);

[0619] (xvi) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is myelin oligodendrocyte glycoprotein (MOG);

[0620] (xvii) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0621] (xviii) the first target is low-density lipoprotein receptor-related protein (LRP), and the second target is cell adhesion molecule 3 (CADM3); or

[0622] (xix) the first target is CD98hc and the second target is myelin oligodendrocyte glycoprotein (MOG); or

[0623] (xx) the first target is CD98hc and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or

[0624] (xxi) The first target is CD98hc, and the second target is cell adhesion molecule 3 (CADM3).

[0625] Specifically, the second antigen binding molecule is an antigen binding molecule according to any one of the embodiments of the antigen binding molecule of the first aspect herein.

[0626] The "brain-to-plasma ratio" is understood to be the ratio of the concentration of the antigen-binding molecule in the brain to the concentration of the antigen-binding molecule in the plasma. The "brain-to-plasma ratio" can be calculated as follows:

[0627]

[0628] "Transfer efficiency" is understood as the percentage value of the injected dose per brain tissue weight (% ID (injected dose) / g brain). The transfer efficiency can be calculated as follows:

[0629]

[0630] Examples of the first target and the second target

[0631] As described above, for any aspect herein, the first antigen binding domain specifically binds to a first target that facilitates transfer of the antigen binding molecule into the brain of a mammal, and

[0632] The second antigen binding domain specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide.

[0633] Embodiments of the first target are described above, and embodiments of the second target are described above.Further embodiments of any aspect herein are disclosed below.

[0634] According to certain embodiments, the second target is expressed on the cell membrane of a brain cell, wherein the brain cell comprises one or more of the 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 more than one brain cell. Such second targets include, for example, CSPG5 and SynCAM3 as shown in Table 2.

[0635] According to certain embodiments, the second target is a brain ECM protein. Such second targets include, for example, versican core protein (chondroitin sulfate proteoglycan 2 or CSPG2), tropoelastin (elastin), collagen alpha-2 (IV) chain (angiostatin), aggrecan connexin 1 (hyaluronic acid and aggrecan connexin 1), tenascin-R (TN-R), aggrecan connexin 2 (hyaluronic acid and aggrecan connexin 2), collagen alpha-1 (I) chain, neurofilament protein-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.

[0636] According to another embodiment, the second target is a brain ECM polysaccharide. Such second targets include, but are not limited to, for example, hyaluronic acid (HA) or hyaluronic acid.

[0637] In one embodiment, the first target is transferrin receptor (TfR).Antigen binding molecules comprising an antigen binding domain combined with TfR are provided in the example.Specifically, anti-TfR / / anti-MOG bispecific antibodies are provided in Examples 2 and 3, which show favorable pharmacokinetic properties.The results show that the combination of brain metastasis part (anti-TfR) and brain retention part (anti-MOG) provides a synergistic effect of more lasting and high antibody concentration in the brain, resulting in increased retention in the brain and increased concentration in the brain.In addition, anti-TfR / / anti-MOG bispecific antibodies and anti-TfR / / anti-CADM3 bispecific antibodies are provided in Examples 4 and 5.It is surprisingly found that compared with the antibody with brain metastasis part alone, the combination of brain metastasis part and brain retention part in these bispecific antibodies shows the same or higher concentration on the 1st day, and shows a much higher concentration than the antibody with a separate brain metastasis part or a separate brain retention part on the 7th day.These further data also show that the combination of brain metastasis part and brain retention part is effective for the retention of antibodies in the brain have Synergistic effect. In addition, 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 transfer portion that binds to TfR and a brain retention portion that binds to CSPG5 has excellent efficacy in transfer and retention to the brain.

[0638] In another embodiment herein, the first target is Basigin (CD147).

[0639] Examples 4 and 5 provide bispecific antibodies comprising a brain transfer portion that binds to Basigin and a brain retention portion that binds to MOG (anti-Basigin / / anti-MOG bispecific antibodies). It was surprisingly found that the combination of the brain transfer portion and the brain retention portion in these bispecific antibodies showed the same or higher concentrations on day 1 compared to the antibodies with the brain transfer portion alone, and showed much higher concentrations on day 7 than the antibodies with the brain transfer portion alone or the brain retention portion alone. These further data also show that the combination of the brain transfer portion and the brain retention portion has a synergistic effect on the retention efficacy of the antibodies in the brain.

[0640] In another embodiment herein, the first target is the insulin receptor.

[0641] In another embodiment herein, the first target is insulin-like growth factor 1 receptor (IGF1R).

[0642] In another embodiment herein, the first target is the low-density lipoprotein receptor (LDLR).

[0643] In another embodiment herein, the first target is low-density lipoprotein receptor-related protein (LRP). In a preferred embodiment, the LRP is LRP1.

[0644] In another embodiment herein, the first target is a diphtheria toxin receptor.

[0645] In another embodiment herein, the first target is a glucose receptor. In a preferred embodiment, the glucose receptor is Glut1.

[0646] In another embodiment herein, the first target is CD98hc.

[0647] In yet further embodiments, the second target is selected from the group consisting of myelin oligodendrocyte glycoprotein (MOG), neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5), and cell adhesion molecule 3 (CADM3).

[0648] In one embodiment, the second target is myelin oligodendrocyte glycoprotein (MOG).Antigen binding molecules comprising an antigen binding domain combined with MOG are provided in the example.Specifically, anti-TfR / / anti-MOG bispecific antibodies are provided in Examples 2 and 3, which show favorable pharmacokinetic properties.The results show that the combination of brain metastasis part (anti-TfR) and brain retention part (anti-MOG) provides a synergistic effect of more lasting and high antibody concentration in the brain, resulting in increased retention in the brain and increased concentration in the brain.In addition, anti-TfR / / anti-MOG bispecific antibodies and bispecific antibodies (anti-Basigin / / anti-MOG bispecific antibodies) comprising a brain metastasis part combined with Basigin and a brain retention part combined with MOG are provided in Examples 4, 5 and 7.It is surprisingly found that compared with the antibody with a brain metastasis part alone, the combination of the brain metastasis part and the brain retention part in these bispecific antibodies shows the same or higher concentration on the 1st day, and shows a much higher concentration than the antibody with a single brain metastasis part or a single brain retention part on the 7th day. These further data also show that the combination of the brain transfer portion and the brain retention portion has a synergistic effect on the retention efficacy of the antibody in the brain. Examples of other anti-MOG antigen binding domains that can be used are described in, for example, WO2018123979. Examples of other anti-TfR antigen binding domains that can be used are described in, for example, WO2012075037, WO2014033074 or Johnsen, Kasper Bendix et al. "Targeting the transferrin receptor for brain drug delivery." Progress in neurobiology 181 (2019): 101665.

[0649] In one embodiment, the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5).

[0650] Anti-TfR / / anti-CSPG5 bispecific antibodies are provided in Examples 4 and 7. The results in Example 7 show that the combination of the brain transfer portion bound to TfR and the brain retention portion bound to CSPG5 has excellent efficacy in terms of transfer and retention in the brain. Examples of other anti-CSPG5 antigen binding domains that can be used are described in, for example, WO2020004490. Examples of other anti-TfR antigen binding domains that can be used are described in, for example, WO2012075037, WO2014033074 or Johnsen, Kasper Bendix et al. "Targeting the transferrin receptor for brain drug delivery." Progress in neurobiology 181 (2019): 101665.

[0651] In one embodiment, the second target is cell adhesion molecule 3 (CADM3).

[0652] In the example, anti-TfR / / anti-CADM3 bispecific antibodies are provided in Examples 4 and 5. Surprisingly, the combination of the brain metastasis part and the brain retention part in these bispecific antibodies showed the same or higher concentration on the first day compared with the antibody with the brain metastasis part alone, and showed a much higher concentration than the antibody with a separate brain metastasis part or a separate brain retention part on the 7th day. These further data also show that the combination of the brain metastasis part and the brain retention part has a synergistic effect on the retention effect of the antibody in the brain. Examples of other anti-CADM3 antigen binding domains that can be used are described in, for example, WO2020004492. Examples of other anti-TfR antigen binding domains that can be used are described in, for example, WO2012075037, WO2014033074 or Johnsen, Kasper Bendix et al. "Targeting the transferrin receptor for brain drug delivery." Progress in neurobiology 181 (2019): 101665.

[0653] In a further embodiment, the first target is transferrin receptor (TfR) and the second target is myelin oligodendrocyte glycoprotein (MOG).

[0654] Specifically, examples 2 and 3 provide anti-TfR / / anti-MOG bispecific antibodies that exhibit favorable pharmacokinetic properties. The results show that the combination of the brain metastasis portion (anti-TfR) and the brain retention portion (anti-MOG) provides a synergistic effect of more persistent and high antibody concentrations in the brain, resulting in increased retention in the brain and increased concentrations in the brain. In addition, examples 4 and 5 provide anti-TfR / / anti-MOG bispecific antibodies. It was surprisingly found that the combination of the brain metastasis portion and the brain retention portion in these bispecific antibodies showed the same or higher concentrations on the first day compared to the antibody with the brain metastasis portion alone, and showed a much higher concentration than the antibody with a separate brain metastasis portion or a separate brain retention portion on the 7th day. These further data also show that the combination of the brain metastasis portion and the brain retention portion has a synergistic effect on the retention efficacy of the antibody in the brain.

[0655] In further embodiments, the first target is transferrin receptor (TfR) and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5).

[0656] 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-transferring portion that binds to TfR and a brain-retaining portion that binds to CSPG5 has excellent efficacy in transfer and retention into the brain.

[0657] In a further embodiment, the first target is transferrin receptor (TfR) and the second target is cell adhesion molecule 3 (CADM3).

[0658] In the examples, anti-TfR / / anti-CADM3 bispecific antibodies are provided in Examples 4 and 5. Surprisingly, the combination of the brain transfer portion and the brain retention portion in these bispecific antibodies showed the same or higher concentrations on day 1 compared to the antibody with the brain transfer portion alone, and showed much higher concentrations on day 7 than the antibody with the brain transfer portion alone or the brain retention portion alone. These further data also show that the combination of the brain transfer portion and the brain retention portion has a synergistic effect on the retention efficacy of the antibody in the brain.

[0659] In a further embodiment, the first target is Basigin (CD147) and the second target is myelin oligodendrocyte glycoprotein (MOG).

[0660] Examples 4 and 5 provide bispecific antibodies comprising a brain transfer portion that binds to Basigin and a brain retention portion that binds to MOG (anti-Basigin / / anti-MOG bispecific antibodies). It was surprisingly found that the combination of the brain transfer portion and the brain retention portion in these bispecific antibodies showed the same or higher concentrations on day 1 compared to the antibodies with the brain transfer portion alone, and showed much higher concentrations on day 7 than the antibodies with the brain transfer portion alone or the brain retention portion alone. These further data also show that the combination of the brain transfer portion and the brain retention portion has a synergistic effect on the retention efficacy of the antibodies in the brain.

[0661] In further embodiments, the first target is Basigin (CD147) and the second target is Neurocan C / Chondroitin Sulfate Proteoglycan 5 (CSPG5).

[0662] In a further embodiment, the first target is Basigin (CD147) and the second target is cell adhesion molecule 3 (CADM3).

[0663] In further embodiments, the first target is the insulin receptor and the second target is myelin oligodendrocyte glycoprotein (MOG).

[0664] In further embodiments, the first target is the insulin receptor and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5).

[0665] In a further embodiment, the first target is the insulin receptor and the second target is cell adhesion molecule 3 (CADM3).

[0666] In further embodiments, the first target is a glucose receptor and the second target is myelin oligodendrocyte glycoprotein (MOG).

[0667] In further embodiments, the first target is a glucose receptor and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5).

[0668] In further embodiments, the first target is a glucose receptor and the second target is cell adhesion molecule 3 (CADM3).

[0669] In further embodiments, the first target is low density lipoprotein receptor (LDLR) and the second target is myelin oligodendrocyte glycoprotein (MOG).

[0670] In further embodiments, the first target is low density lipoprotein receptor (LDLR) and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5).

[0671] In further embodiments, the first target is low density lipoprotein receptor (LDLR) and the second target is cell adhesion molecule 3 (CADM3).

[0672] In further embodiments, the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is myelin oligodendrocyte glycoprotein (MOG).

[0673] In further embodiments, the first target is low-density lipoprotein receptor-related protein (LRP) and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5).

[0674] 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).

[0675] As described above, in aspects herein, the structure of the "second target" is not limited, as long as the second target is expressed on the cell membrane of brain cells, or is a brain ECM protein or brain ECM polysaccharide.

[0676] In embodiments, the "first target" is different from the "second target". Thus, the target specifically bound by the "first antigen binding domain" is different from the target specifically bound by the "second antigen binding domain".

[0677] Thus, in certain embodiments, the antigen binding molecules are bispecific and / or multispecific.

[0678] In addition, 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 neurotrophic factor receptor (p75NTR), caspase 6, TRK A, TRK B, TRK 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 receptors, and EpCAM.

[0679] 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 neurotrophic factor 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. 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.

[0680] Embodiments of the antigen binding molecule further comprise at least one functional portion and / or at least one in vivo half-life Extension

[0681] In an embodiment, the antigen binding molecules in any aspect and embodiment of this article further include at least one functional part and / or at least one half-life extension part in vivo. The antigen binding molecules further including at least one functional part and / or at least one half-life extension part in vivo may include at least one functional part and / or at least one half-life extension part in vivo connected in different ways. The antigen binding molecules of the present invention further including functional parts and half-life extension parts in vivo or the exemplary embodiment of the antigen binding molecules of the present invention further including functional parts (but not including half-life extension parts in vivo) are shown in Figure 2 (a). In Figure 2 (a), a part is shown for each type of part (i.e., the first antigen binding domains, the second antigen binding domains, the functional part and / or the half-life extension part in vivo). However, for each type of part, two, three, four or more parts can also exist independently. In addition, "the first antigen binding domain" and "the second antigen binding moiety" may not be directly connected, but connected via half-life extension parts in vivo, such as Fc regions or functional parts. Embodiments in which the "first antigen binding domain" and the "second antigen binding moiety" are not directly linked but are linked to the Fc region are, for example, shown in some examples in Figures 2(b) and 2(c). Figures 2(b) and 2(c) show embodiments comprising an Fc region as an exemplary in vivo half-life extending moiety.

[0682] One, two or more functional parts can be connected to antigen binding molecules.For example, one or two functional parts can be connected to one or two chains in Fc region.For example, such one or two functional parts can be Fab, scFv, scFab or VHH.Such embodiments are shown in Figure 2 (b).For example, two functional parts can form a divalent F (ab ') 2 region connected to Fc region.For example, "the first antigen binding domain" and "the second antigen binding moiety" can be connected to Fc region. "The first antigen binding domain" can be connected to the first chain in Fc region, and "the second antigen binding moiety" can be connected to the second chain in Fc region.Alternatively, "the first antigen binding domain" and "the second antigen binding moiety" can be connected to each other, for example, as a fusion protein, and then can be connected to one or two chains in Fc region.In another alternative exemplary embodiment, "the first antigen binding domain" can be connected to the first light chain, and "the second antigen binding moiety" can be connected to the second light chain. In another exemplary embodiment, the "first antigen binding domain" and the "second antigen binding moiety" can be connected to each other, for example as a fusion protein, and can then be connected to one or two functional domains. In another example, the "first antigen binding domain" and / or the "second antigen binding moiety" are connected to the functional portion. For example, a functional portion can be Fab, scFv, scFab or VHH, and the "first antigen binding domain" (or "second antigen binding moiety") can be Fab, scFv, scFab or VHH. For example, such two monovalent Fabs together form F(ab)2, which can be connected to the Fc region, and the "second antigen binding domain" (or "first antigen binding moiety") can be connected to the "first antigen binding domain" (or "second antigen binding moiety"). In a further exemplary embodiment, a functional portion can be Fab, scFv, scFab or VHH, and the "first antigen binding domain" (or "second antigen binding moiety") can be Fab, scFv, scFab or VHH. For example, such two monovalent Fabs together form F(ab)2, which can be linked to the Fc region, and the "second antigen binding domain" (or "first antigen binding moiety") can be linked to the C-terminus of the Fc region.

[0683] Examples of Domain Arrangements

[0684] In certain embodiments of any aspect herein, the antigen binding molecule comprises one or more antibody-type domains. For example, in this example, a bispecific anti-TfR anti-MOG antibody was prepared. For example, an antigen binding molecule comprising one or more variable regions, or an antibody or antibody fragment comprising such one or more variable regions can be provided.

[0685] Thus, in 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).

[0686] In specific embodiments, the first antigen binding domain is Fab, Fab', F(ab')2, diabody, triabody, scFab, Fv, scFv or single domain antibody (VHH).

[0687] Furthermore, in 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).

[0688] In specific embodiments, the second antigen binding domain is Fab, Fab', F(ab')2, diabody, triabody, scFab, Fv, scFv or single domain antibody (VHH).

[0689] In 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).

[0690] In yet further embodiments, the first antigen binding domain and the second antigen binding domain are independently selected from the group consisting of: an antibody variable region comprising 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 a single domain antibody (VHH).

[0691] In addition, there may be one first antigen binding domain or there may be two or more first antigen binding domains. In addition, there may be one second antigen binding domain or there may be two or more first antigen binding domains.

[0692] In one embodiment, the antigen binding molecule contains 1, 2, 3 or 4 first antigen binding domains and 1, 2, 3 or 4 second antigen binding domains.

[0693] In addition, in one embodiment, the antigen binding molecule further comprises at least one Fc region, such as one Fc region or two or more Fc regions. Exemplary embodiments of molecules comprising one Fc region are, for example, depicted in Figure 2(b) and Figure 2(c).

[0694] In yet further embodiments, the antigen binding molecule contains 1, 2, 3 or 4 first antigen binding domains and 1, 2, 3 or 4 second antigen binding domains and at least one Fc region.

[0695] In yet another embodiment, the one or more first antigen binding domains and the one or more second antigen binding domains are independently selected from the group consisting of: an antibody variable region comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), Fab, Fab', F(ab')2, a diabody, a triabody, scFab, Fv, scFv or a single domain antibody (VHH).

[0696] In yet another embodiment, the antigen binding molecule herein is a bispecific antibody. Various embodiments of bispecific antibodies are shown in FIG2( b ).

[0697] In one embodiment, the antigen binding molecule comprises one first antigen binding domain and one second antigen binding domain.

[0698] For example, the Fab arm exchange technology described in WO2016 / 159213 can be used to generate bispecific antibodies.

[0699] Further embodiments of the functional part

[0700] In certain embodiments, the antigen binding molecule of any aspect and embodiments herein further comprises at least one functional moiety.

[0701] In embodiments herein, at least one functional portion is an antibody or an antigen-binding fragment thereof that specifically binds to a membrane protein of i) an immune cell, particularly wherein the immune cell is selected from the group consisting of T cells, killer cells, helper T cells, regulatory T cells, B cells, memory B cells, NK cells, NKT cells, dendritic cells, macrophages, eosinophils, neutrophils and basophils, ii) a tumor cell, or iii) an autoreactive cell.

[0702] Specifically, in the embodiments, the antibody or antigen-binding fragment thereof specifically binds to a membrane protein selected from the group consisting of: T cell receptor, CD3, CD137, CD40, CTLA4, co-stimulatory molecules and co-inhibitory molecules.

[0703] For example, the antibody or its antigen-binding fragment can be selected from the group consisting of: an antibody variable region comprising an antibody heavy chain variable region (VH) and an antibody light chain variable region (VL), Fab, Fab', F(ab')2, a diabody, a triabody, scFab, Fv, scFv and a single domain antibody (VHH). For example, the example discloses a bispecific antibody comprising a Fab or a single-chain Fab (scFab).

[0704] Antibody fragments

[0705] In certain embodiments, the antigen binding molecules provided herein are antibodies, which are antibody fragments. Antibody fragments include, but are not limited to, single-chain Fab (scFab), Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments, as well as other fragments described below. For a review of certain antibody fragments, see Hudson et al. Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, for example, Pluckthun, in The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed., (Springer-Verlag, New York), pp. 269 to 315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For a discussion of Fab fragments and F(ab')2 fragments comprising salvage receptor binding epitope residues and having an extended in vivo half-life, see U.S. Pat. No. 5,869,046.

[0706] Diabodies are antibody fragments with two antigen binding sites, which can be bivalent or bispecific. See, e.g., EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993). For a description of triabodies and tetrabodies, see also Hudson et al., Nat. Med. 9:129-134 (2003).

[0707] A "single domain antibody" is an antibody fragment that contains all or a portion of the heavy chain variable domain of an antibody or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516B1).

[0708] Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells (eg, E. coli or phage), as described herein.

[0709] Chimeric and humanized antibodies

[0710] In certain embodiments, the antigen binding molecules provided herein are chimeric antibodies. Certain chimeric antibodies are described, for example, in the following references: 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 mouse, rat, hamster, rabbit or non-human primate (such as a monkey)) and a human constant region. In another example, a chimeric antibody is a "class switching" antibody in which the class or subclass has been changed from the class or subclass of the parent antibody. Chimeric antibodies include antigen binding fragments thereof.

[0711] In certain embodiments, chimeric antibodies are humanized antibodies. Typically, non-human antibodies are humanized to reduce immunogenicity to people while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies include one or more variable domains, wherein HVR, such as CDR (or a portion thereof) are derived from non-human antibodies, and FR (or a portion thereof) are derived from human antibody sequences. Humanized antibodies will optionally also include at least a portion of a human constant region. In certain embodiments, some FR residues in humanized antibodies are replaced by corresponding residues from non-human antibodies (e.g., antibodies from which HVR residues are derived), for example, to restore or improve antibody specificity or affinity.

[0712] Humanized antibodies and methods for making them are reviewed, for example, in the following references: Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and are further described, for example, in the following references: 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) transplantation); 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 the "guided selection" method of FR shuffling).

[0713] 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 the consensus sequence of human antibodies of a particular subgroup 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 (199 3)); 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 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)).

[0714] Human Antibodies

[0715] In certain embodiments, the antigen binding molecules provided herein are human antibodies or comprise human antibodies. 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).

[0716] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce complete human antibodies or complete antibodies with human variable regions in response to antigenic stimulation. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus, or exists outside the chromosome of the animal or is randomly integrated into the chromosome of the animal. In such transgenic mice, the endogenous immunoglobulin locus is typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, for example, description of XENOMOUSE TM U.S. Patent Nos. 6,075,181 and 6,150,584 describing HUMAB (registered trademark) technology; U.S. Patent No. 5,770,429 describing KM MOUSE (registered trademark) technology; and U.S. Patent No. 7,041,870 describing V ELOCI MOUSE (Registered Trademark) technology is described in US Patent Application Publication No. US2007 / 0061900. Human variable regions from intact antibodies produced by such animals can be further modified, for example by combining with different human constant regions.

[0717] Human antibodies can also be prepared by hybridoma-based methods. Human myeloma and mouse-human hybrid myeloma 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). Additional methods include, for example, those described in U.S. Pat. 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).

[0718] Human antibodies can also be produced by isolating the Fv clone variable domain sequence selected from the human phage display library. Such variable domain sequences can then be combined with the expected human constant domains. The technology of selecting human antibodies from the antibody library is described below.

[0719] Library-derived antibodies

[0720] The antigen binding domains of the antigen binding molecules provided herein, such as antibodies, can be separated by screening combinatorial libraries for antibodies with desired activity. For example, various methods are known in the art for producing phage display libraries and screening such libraries to obtain antibodies with desired binding characteristics. Such methods are reviewed in, for example, Hoogenboom et al., included in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described in, for example, 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, included in 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).

[0721] In certain phage display methods, the repertoire of VH and VL genes is cloned individually by polymerase chain reaction (PCR) and randomly recombined in a phage library, from which antigen-binding phage can then be screened, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). Phages typically display antibody fragments as single-chain Fv (scFv) fragments or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, the initial repertoire (e.g., from humans) can be cloned to provide a single source of antibodies to a wide range of non-self antigens and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12: 725-734 (1993). Finally, the initial library can also be synthesized by cloning unrearranged V gene segments from stem cells; and using PCR primers containing random sequences to encode highly variable CDR3 regions and complete in vitro rearrangement, as described in the following literature: Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example: U.S. Patent No. 5,750,373, and U.S. Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0722] Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0723] Multispecific Antibodies

[0724] In certain embodiments, the antigen binding molecules provided herein are multispecific antibodies, such as bispecific antibodies. Multispecific antibodies are monoclonal antibodies with binding specificity to at least two different sites. In certain embodiments, one of the binding specificities is directed to promoting the transfer of the antigen binding domain to the first target in the mammalian brain, and the other is directed to the second target expressed on the cell membrane of brain cells. In certain other embodiments, one of the binding specificities is directed to promoting the transfer of the antigen binding domain to the first target in the mammalian brain, and the other is directed to the second target of brain ECM protein or brain ECM polysaccharide. In addition, there may be one or more additional binding specificities for other targets. Multispecific antibodies comprising additional specificities can also be used to localize cytotoxic agents to brain cells expressing the second target. Bispecific and multispecific antibodies can be prepared as full-length antibodies or antibody fragments.

[0725] Techniques for preparing 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 prepared by the following methods: engineering electrostatic steering effects for the preparation of antibody Fc-heterodimer molecules (WO 2009 / 089004A1); 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 produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)); using "diabody" technology to prepare bispecific antibody fragments segment (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 preparing trispecific antibodies according to the method described, e.g., Tutt et al., J. Immunol. 147:60 (1991).

[0726] In order to effectively obtain the desired bispecific antibody comprising two heavy chains and two light chains, known amino acid substitutions and combinations that promote the desired H chain-L chain association (such as described in WO2019065795) can be used in the embodiments.

[0727] Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US 2006 / 0025576 A1).

[0728] The antibodies or fragments herein also include "dual-acting Fabs" or "DAFs," which include an antigen binding site that binds to a first target as well as a second target (see, e.g., US 2008 / 0069820).

[0729] Antigen binding molecule variants and specifically antibody variants

[0730] In certain embodiments, it is contemplated that the amino acid sequence variants of antigen binding molecules and particularly antibodies provided herein.For example, it may be necessary to improve the binding affinity and / or other biological properties of antigen binding molecules and particularly antibodies.Antigen binding molecules and particularly amino acid sequence variants of antibodies can be prepared by introducing suitable modifications into the nucleotide sequence encoding the antibody or by peptide synthesis.Such modifications include, for example, the disappearance and / or insertion and / or substitution of residues in the amino acid sequence of antigen binding molecules and particularly antibodies.Any combination of deletion, insertion and substitution can be performed to realize the final construct, provided that the final construct has the desired features, such as antigen binding.

[0731] Glycosylation variants

[0732] In certain embodiments, the antigen binding molecules and specific antibodies provided herein are altered to increase or decrease the degree of antibody glycosylation. The addition or deletion of glycosylation sites to an antibody can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0733] When antigen binding molecules and specific antibodies include Fc district, the carbohydrate attached thereto can be changed.Native antibodies produced by mammalian cells typically include the biantennary oligosaccharide of side chain, which is usually connected to the Asn297 of the CH2 domain in Fc district by N key.See, for example, Wright et al. TIBTECH 15:26-32 (1997).Oligosaccharides can include various carbohydrates, for example, mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and the fucose of the GlcNAc in the "trunk" of biantennary oligosaccharide structure.In certain embodiments, the oligosaccharide in the antibody of the present invention can be modified to produce antigen binding molecules and specifically antibody variants with certain improved characteristics.

[0734] In one embodiment, antigen binding molecules and specifically antibody variants are provided, which have a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the content 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 at Asn297 in the sugar chain relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) connected to Asn297 measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at about 297 in the Fc region (EU numbering of Fc region residues); however, due to minor sequence changes in antibodies, Asn297 can also be located about + / -3 amino acids upstream or downstream of 297, i.e., between 294 and 300. Such fucosylated variants may have improved ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Related "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US2003 / 0115614; US2002 / 0164328; US2004 / 0093621; US2004 / 0132140; US2004 / 0110704; US2004 / 0110282; US2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 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 protein fucosylation-deficient Lec13 CHO cells (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially in Example 11), and knockout cell lines, such as CHO cells in which the α-1,6-fucosyltransferase gene FUT8 is knocked out (see, 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).

[0735] Further provided are antigen binding molecules with bisected oligosaccharides and specifically antibody variants, for example, wherein the bisected oligosaccharide connected to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described in, for example, WO 2003 / 011878 (Jean-Mairet et al.), U.S. Patent No. 6,602,684 (Umana et al.) and US2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide connected to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described in, for example, WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0736] In a preferred embodiment of the antigen binding molecules provided herein, at least one first antigen binding domain and at least one second antigen binding domain are linked to an Fc region.

[0737] Fc region variants

[0738] In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of the antigen binding molecules and specific antibodies provided herein, thereby generating Fc region variants. Fc region variants may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0739] In a preferred embodiment of the antigen binding molecules provided herein, one first antigen binding domain and one second antigen binding domain are linked to an Fc region.

[0740] In a preferred embodiment of the antigen binding molecules provided herein, the Fc region is an Fc region whose ability to bind to an activated Fcγ receptor is reduced compared to the ability of the Fc region of native human IgG to bind to an activated Fcγ receptor.

[0741] In a preferred embodiment, the activating Fcγ receptor is human FcγRIa, human FcγRIIa(R), human FcγRIIa(H), human FcγRIIIa(V) or human FcγRIIIa(F).

[0742] In a preferred embodiment, the activated Fcγ receptor is human FcγRIa. In a preferred embodiment, the activated Fcγ receptor is human FcγRIIa (R). In a preferred embodiment, the activated Fcγ receptor is human FcγRIIa (H). In a preferred embodiment, the activated Fcγ receptor is human FcγRIIIa (V). In a preferred embodiment, the activated Fcγ receptor is human FcγRIIIa (F).

[0743] In a preferred embodiment, the Fc region comprises one or more of the following amino acid substitutions (all positions are numbered according to EU):

[0744] Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Met, Phe, Pro, Ser, Thr or Trp at position 234;

[0745] Ala, Asn, Asp, Gln, Glu, Gly, His, Ile, Lys, Met, Pro, Ser, Thr, Val or Arg at position 235;

[0746] Arg, Asn, Gln, His, Leu, Lys, Met, Phe, Pro or Tyr at position 236;

[0747] Ala, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Pro, Ser, Thr, Val, Tyr or Arg at position 237;

[0748] Ala, Asn, Gln, Glu, Gly, His, Ile, Lys, Thr, Trp or Arg at position 238;

[0749] Gln, His, Lys, Phe, Pro, Trp, Tyr or Arg at position 239;

[0750] Ala, Arg, Asn, Gln, Gly, His, He, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr or Val at position 265;

[0751] Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Lys, Phe, Pro, Ser, Thr, Trp or Tyr at position 266;

[0752] Arg, His, Lys, Phe, Pro, Trp or Tyr at position 267;

[0753] Ala, Arg, Asn, Gln, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val at position 269;

[0754] Ala, Arg, Asn, Gln, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val at position 270;

[0755] Arg, His, Phe, Ser, Thr, Trp or Tyr at position 271;

[0756] Arg, Asn, Asp, Gly, His, Phe, Ser, Trp or Tyr at position 295;

[0757] Arg, Gly, Lys or Pro at position 296;

[0758] Ala at position 297;

[0759] Arg, Gly, Lys, Pro, Trp or Tyr at position 298;

[0760] Arg, Lys, or Pro at position 300;

[0761] Lys or Pro at position 324;

[0762] Ala, Arg, Gly, His, Ile, Lys, Phe, Pro, Thr, Trp, Tyr or Val at position 325;

[0763] Arg, Gln, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val at position 327;

[0764] Arg, Asn, Gly, His, Lys or Pro at position 328;

[0765] Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, or Arg at position 329;

[0766] Pro or Ser at position 330;

[0767] Arg, Gly or Lys at position 331;

[0768] Arg, Lys or Pro at position 332.

[0769] 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.

[0770] In certain other embodiments, the present invention contemplates antigen binding molecules and specifically antibody variants with some but not all effector functions, which make them ideal candidates for applications where the half-life of the antibody in vivo is important, but certain effector functions (such as complement and ADCC) are unnecessary or harmful. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / consumption of CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcγR binding (and therefore may lack ADCC activity), but retains FcRn binding ability. The main cell NK cells that mediate ADCC express only FcγRIII, while monocytes express FcγRI, FcγRII and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu.Rev.Immunol.9:457-492 (1991) page 464 Table 3. Non-limiting examples of in vitro assays for evaluating ADCC activity of a molecule of interest are described in U.S. Pat. Nos. 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 can be used (see, e.g., ACT1 for flow cytometry). TMNon-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA); and CytoTox 96 (registered trademark) 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 additionally, ADCC activity of the molecule of interest can be assessed 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, C1q and C3c binding ELISAs in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay can 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)). FcRn binding and in vivo clearance / half-life assays 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)).

[0771] Antigen binding molecules and specifically antibodies comprising an Fc region with reduced effector function include antigen binding molecules surrounding antibodies in which one or more Fc region residues 238, 265, 269, 270, 297, 327, and 329 are substituted (U.S. Patent No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc mutant, in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0772] Certain antigen binding molecules, particularly antibody variants with increased or decreased binding to FcRs, are 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).)

[0773] In certain embodiments, antigen binding molecules and in particular antibody variants comprise an Fc region with one or more amino acid substitutions that improve ADCC, for example, substitutions at positions 298, 333 and / or 334 (EU numbering of residues) of the Fc region.

[0774] In some embodiments, alterations are made in the Fc region that result in altered (ie, increased or decreased) CIq 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).

[0775] Antigen binding molecules and specifically antibodies with increased half-life and increased binding to the neonatal Fc receptor (FcRn), responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)) are described in US 2005 / 0014934A1 (Hinton et al.). Those antibodies comprise an Fc region having one or more substitutions therein that increase the binding of the Fc region to FcRn. Such Fc variants include those having a substitution at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434, such as a substitution at Fc region residue 434 (U.S. Pat. No. 7,371,826).

[0776] See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No. 5,648,260; U.S. Pat. No. 5,624,821; and WO 94 / 29351 for other examples of Fc region variants.

[0777] Cysteine ​​engineered antibody variants

[0778] In certain embodiments, it is desirable to produce antibodies specific to cysteine-engineered antigen-binding molecules, such as "thioMAbs", in which one or more residues of the antibody are replaced by cysteine ​​residues. In a particular embodiment, the substituted residues are present in accessible sites of the antibody. As further described herein, by replacing those residues with cysteine, reactive thiol groups are thus located at accessible sites of the antibody, and can be used to conjugate the antibody to other moieties (such as drug moieties or linker-drug moieties) to produce immunoconjugates. 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) in the heavy chain Fc region. Cysteine-engineered antibodies may be generated as described in, for example, U.S. Patent No. 7,521,541.

[0779] Antibody derivatives

[0780] In certain embodiments, antigen binding molecules provided herein and specifically antibodies can be further modified to contain other non-protein parts known in the art and easily available. Suitable parts for antibody derivatization 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), copolymers of ethylene glycol / propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acid (homopolymer or random copolymer) and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol (e.g., glycerol), polyvinyl alcohol and mixtures thereof. Due to its stability in water, polyethylene glycol propionaldehyde can have advantages in manufacturing. Polymer can have any molecular weight, and can have side chains or not. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the antigen binding molecule and the particular property or function to be improved in the particular antibody, whether the antibody derivative will be used for therapy under defined conditions, etc.

[0781] In another embodiment, a conjugate of an antigen binding molecule and a specific antibody and non-proteinaceous moiety that can be selectively heated by exposure to radiation is provided. In one embodiment, the non-proteinaceous moiety is a carbon nanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can have any wavelength, and includes but is not limited to a wavelength that is not harmful to ordinary cells, but heats the non-proteinaceous moiety to a temperature at which cells near the antibody-non-proteinaceous moiety are killed.

[0782] Recombinant methods and compositions

[0783] In one aspect of this article, there is provided a nucleic acid or two or more nucleic acids encoding the antigen binding molecules according to any embodiment of this article. Optionally, the nucleic acid or two or more nucleic acids are operably connected to a promoter. Also provided are vectors or two or more vectors comprising nucleic acids or two or more nucleic acids provided herein. Further provided herein is a host cell comprising (i) nucleic acids or two or more nucleic acids provided herein; (ii) vectors or two or more vectors provided herein; and / or (iii) capable of expressing the antigen binding molecules according to any aspect and embodiment of this article.

[0784] Further provided are methods of producing an antigen binding molecule provided herein, comprising culturing a host cell provided herein to produce an antibody; optionally further comprising recovering the antibody from the host cell.

[0785] Recombinant methods and compositions can be used to produce antigen binding molecules and specific antibodies, for example, as described in U.S. Patent No. 4816567. In one embodiment, there is provided a nucleic acid encoding the separation of the first antigen-binding domains comprising the variable region described herein. Such nucleic acid can encode the amino acid sequence of the VL of the first antigen-binding domains containing the variable region (for example, the light chain and / or the heavy chain of the antibody) and / or the amino acid sequence of the VH comprising the above-mentioned first antigen-binding domains, and optionally further encodes the amino acid sequence of the VL of the second antigen-binding domains containing the variable region (for example, the light chain and / or the heavy chain of the antibody) and / or the amino acid sequence of the VH comprising the above-mentioned second antigen-binding domains. Alternatively, the sequence can be provided as two or more separate nucleic acids. In a further embodiment, one or more vectors (for example, 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): (1) a vector comprising a nucleic acid encoding an amino acid sequence of a VL comprising a first antigen binding domain and an amino acid sequence of a VH comprising the first antigen binding domain, and a nucleic acid encoding an amino acid sequence of a VL comprising a second antigen binding domain and an amino acid sequence of a VH comprising the second antigen binding domain, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence of a VL comprising a first antigen binding domain and a nucleic acid encoding an amino acid sequence of a VH comprising the first antigen binding domain; and a third vector comprising a nucleic acid encoding an amino acid sequence of a VL comprising a second antigen binding domain; and a fourth vector comprising a nucleic acid encoding an amino acid sequence of a VH encoding a second antigen binding domain. In one embodiment, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell or a lymphocyte (e.g., a Y0, NS0, Sp2 / 0 cell). In one embodiment, a method for preparing an antigen-binding molecule, and in particular an antibody, is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding an antigen-binding molecule, and in particular an antibody, as provided above under conditions suitable for expressing the antibody, and optionally recovering the antigen-binding molecule from the host cell (or host cell culture medium).

[0786] For the recombinant production of antigen binding molecules (particularly antibodies), nucleic acids encoding antigen binding molecules (such as antibodies, for example as described above) are separated and inserted into one or more vectors for further cloning and / or expression in host cells. Conventional procedures can be used to easily separate and sequence-check such nucleic acids (for example, by using oligonucleotide probes that can be bound to the gene specificity of the heavy chain and light chain of the encoding antibody).

[0787] Suitable host cells for cloning or expressing vectors encoding antibodies include prokaryotic or eukaryotic cells as described herein. For example, antibodies can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. Regarding 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 (BKCLo ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, describing the expression of antibody fragments in Escherichia coli.). Antibodies can be separated from bacterial cell paste in a soluble fraction after expression, and can be further purified.

[0788] In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are also suitable cloning or expression hosts for vectors encoding antibodies, including fungi and yeast strains whose glycosylation pathways have been "humanized" so that antibodies with partially or fully human glycosylation patterns are produced. See Gerngross, Nat. Biotech. 22: 1409-1414 (2004); and Li et al., Nat. Biotech. 24: 210-215 (2006).

[0789] Suitable host cells for expressing glycosylated antibodies also come from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant cells and insect cells. Many baculovirus strains have been identified that can be used in conjunction with insect cells, particularly for transfecting Spodoptera frugiperda cells.

[0790] Plant cell cultures can also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES for producing antibodies in transgenic plants). TM technology).

[0791] Vertebrate cells can also be used as hosts. For example, mammalian cell lines adapted to growth in suspension may be useful. Other examples of useful mammalian host cell lines are: monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (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 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 liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, such as those described in 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, including DHFR - 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 certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0792] Production and screening methods

[0793] The physical / chemical properties and / or biological activities of the antigen binding molecules provided herein can be identified, screened or characterized by various assays known in the art.

[0794] The present invention also provides a method for producing an antigen binding molecule, comprising the following steps:

[0795] (a) selecting a first antigen binding domain that specifically binds to a first target, which promotes the transfer of the antigen binding molecule into the brain of a mammal; (b) selecting a second antigen binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells or (ii) is a brain ECM protein or brain ECM polysaccharide;

[0796] (c) obtaining one or more nucleic acids encoding antigen-binding molecules in which an antigen-binding domain is linked to the antigen-binding domains prepared in (a) and (b); and

[0797] (d) using the one or more nucleic acids prepared in (c) to produce an antigen-binding molecule.

[0798] In a preferred embodiment of the present invention, the method further comprises step (e):

[0799] (e) determining an increase in (i) retention, (ii) concentration or (iii) exposure of an antigen-binding molecule according to (d) in the brain of a subject compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule according to (d) only in that the control antigen-binding molecule comprises one but not both of the antigen-binding domains defined in (a) and (b).

[0800] Embodiments of any other aspects and embodiments herein also apply to these methods.

[0801] The present invention also provides a method for screening antigen-binding molecules, comprising the following steps:

[0802] (a) selecting a first antigen binding domain that specifically binds to a first target, which promotes the transfer of the antigen binding molecule into the brain of a mammal; (b) selecting a second antigen binding domain that specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells or (ii) is a brain ECM protein or brain ECM polysaccharide;

[0803] (c) obtaining one or more nucleic acids encoding antigen-binding molecules in which an antigen-binding domain is linked to the antigen-binding domains prepared in (a) and (b); and

[0804] (d) using the one or more nucleic acids prepared in (c) to produce an antigen-binding molecule.

[0805] In a preferred embodiment of the present invention, the method further comprises step (e):

[0806] (e) determining an increase in (i) retention, (ii) concentration or (iii) exposure of an antigen-binding molecule according to (d) in the brain of a subject compared to a control antigen-binding molecule, wherein the control antigen-binding molecule differs from the antigen-binding molecule according to (d) only in that the control antigen-binding molecule comprises one but not both of the antigen-binding domains defined in (a) and (b).

[0807] Embodiments of any other aspects and embodiments herein also apply to these methods.

[0808] In one embodiment, an antigen binding molecule (such as an antibody of the invention) is tested for antigen binding activity, for example, by known methods (such as ELISA, Western blot, etc.).

[0809] Methods and compositions for diagnosis and detection.

[0810] In certain embodiments, any antigen binding molecule provided herein can be used to detect the presence of a first target and / or a second target in a biological sample. As used herein, the term "detection" encompasses quantitative or qualitative detection. In certain embodiments, the biological sample includes cells or tissues, such as brain tissue and brain cells.

[0811] In certain embodiments, labeled antigen-binding molecules, particularly antibodies, are provided. Labels include, but are not limited to, directly detected labels or moieties (such as fluorescent labels, chromogenic labels, electron-dense labels, chemiluminescent labels, and radioactive labels), and indirectly (e.g., by enzymatic reactions or molecular interactions) detected moieties (such as enzymes or ligands). 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; luciferase, such as firefly luciferase and bacterial luciferase (U.S. Pat. No. 4,737,456); luciferin; 2,3-dihydrophthalazinedione; 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 urate oxidase and xanthine oxidase; coupled to an enzyme that uses hydrogen peroxide to oxidize a dye precursor (such as HRP, lactoperoxidase, or microperoxidase); biotin / avidin; spinning labels; phage labels; stable free radicals, etc.

[0812] Pharmaceutical preparations

[0813] Provided herein is a pharmaceutical composition comprising an antigen binding molecule according to any aspect and embodiment of this invention and one or more pharmaceutically acceptable carriers or excipients. In addition, a pharmaceutical composition is provided comprising (i) an antigen binding molecule provided herein, (ii) a nucleic acid or two or more nucleic acids encoding an antigen binding molecule provided herein, (iii) a vector or two or more vectors provided herein, or (iv) a host cell provided herein, and one or more pharmaceutically acceptable carriers or excipients.

[0814] Pharmaceutical preparations of antigen binding molecules as described herein are prepared by mixing such antibodies having the desired purity with one or more optional pharmaceutical carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980)) in the form of lyophilized preparations or aqueous solutions. Acceptable carriers are nontoxic to recipients at the doses and concentrations employed; and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl alcohol or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) polyols Peptide; protein, such as serum albumin, gelatin or immunoglobulin; hydrophilic polymer, such as polyvinylpyrrolidone; amino acid, such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharide, disaccharide and other carbohydrate, including glucose, mannose or dextrin; chelating agent, such as EDTA; sugar, such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterion, such as sodium; metal complex (e.g. zinc-protein complex); and / or nonionic surfactant, such as polyethylene glycol (PEG). Exemplary pharmaceutical carriers herein further include interstitial drug dispersants such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (HYLENEX (registered trademark), Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0815] Exemplary lyophilized antibody formulations are described in US Pat. No. 6,267,958. Aqueous antibody formulations include those described in US Pat. No. 6,171,586 and WO 2006 / 044908, the latter of which contains a histidine-acetate buffer.

[0816] The formulations herein may also contain more than one active ingredient essential for the particular indication being treated, preferably active ingredients with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0817] The active ingredient can be embedded in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively); embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or embedded in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980).

[0818] Sustained-release preparations can 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, eg, films or microcapsules.

[0819] Preparations to be used for in vivo administration are generally sterile. For example, sterility can be easily achieved by filtration through sterile filtration membranes.

[0820] Methods of treatment and compositions

[0821] Any of the antigen binding molecules and specific antibodies provided herein, as well as nucleic acids, vectors or host cells and pharmaceutical compositions can be used in therapeutic methods.

[0822] In one embodiment, an antigen binding molecule antibody for use as a medicament is provided.

[0823] In embodiments, (i) an antigen binding molecule provided herein, (ii) a nucleic acid or two or more nucleic acids encoding an antigen binding molecule provided herein, (iii) a vector or two or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein, is used as a medicament.

[0824] In embodiments, (i) an antigen binding molecule provided herein, (ii) a nucleic acid or two or more nucleic acids encoding an antigen binding molecule provided herein, (iii) a vector or two or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein, is used as a diagnostic agent.

[0825] In an embodiment, provided herein is (i) an antigen binding molecule provided herein, (ii) a nucleic acid or two or more nucleic acids encoding an antigen binding molecule provided herein, (iii) a vector or two or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein for treating and / or preventing a brain disorder or disease in a subject.

[0826] In an 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) a nucleic acid or two or more nucleic acids encoding the antigen binding molecule provided herein, (iii) a vector or two or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein.

[0827] In the embodiments, provided herein are (i) an antigen binding molecule provided herein, (ii) a nucleic acid or two or more nucleic acids encoding an antigen binding molecule provided herein, (iii) a vector or two or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein for the preparation of a medicament for treating and / or preventing brain disorders or diseases.

[0828] In the embodiments, provided herein are (i) an antigen binding molecule provided herein, (ii) a nucleic acid or two or more nucleic acids encoding an antigen binding molecule provided herein, (iii) a vector or two or more vectors provided herein, or (iv) a host cell provided herein, or (iv) a pharmaceutical composition provided herein for the preparation of a medicament for treating and / or preventing brain disorders or diseases.

[0829] 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-Draeger 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 syndrome, chronic wasting disease and fatal familial insomnia), bulbar palsy, motor neuron disease and neurodegenerative diseases of the nervous system (including but not limited to Canavan disease, Huntington disease, neuronal ceroid 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).

[0830] In an embodiment, an antigen binding molecule of any aspect or embodiment of the present invention is provided for use in (i) a method of increasing the concentration of an antigen binding molecule in the brain of a subject in need thereof; (ii) a method of increasing the exposure of an antigen binding molecule in the brain of a subject in need thereof; and / or (iii) for retaining an antigen binding molecule in the brain of a subject in need thereof; optionally, wherein the subject is a human.

[0831] In one embodiment, a "subject" is a human.

[0832] In one embodiment, a "subject in need thereof" is a subject suffering from or suspected of suffering from a brain disease or disorder.

[0833] The antigen binding molecules and antibodies of the present invention can be used alone or in combination with other agents for 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 will depend on the brain disease or disorder to be treated.

[0834] Such combination therapies described above encompass combined administration (wherein two or more therapeutic agents are included in the same or separate preparations) and separate administration, in which case the administration of the antibody of the present invention can be performed before, simultaneously and / or after the administration of another therapeutic agent or medicament. In one embodiment, the administration of the antigen binding molecules and the administration of another therapeutic agent are performed within about 1 month from each other, or within about 1 week, 2 weeks or 3 weeks, or within about 1 day, 2 days, 3 days, 4 days, 5 days or 6 days. The antigen binding molecules of the present invention can also be used in conjunction with radiotherapy.

[0835] Antigen binding molecules (and any other therapeutic agents) can be administered in any suitable manner, including parenteral, intrapulmonary and intranasal, and if necessary for local treatment, intralesional administration. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Administration can be carried out by any suitable route, for example, by injection, such as intravenous or subcutaneous injection, depending in part on whether the administration is short-lived or long-term. Various administration time arrangements are contemplated herein, including but not limited to single or multiple administrations, push injection administration, and pulse infusions at various time points.

[0836] Antigen binding molecules of the present invention can be prepared, administered and used in a manner consistent with good medical practice. Factors to be considered in this case include the specific illness treated, the specific mammal treated, the clinical condition of individual patients, the cause of the illness, the delivery site of the medicament, the method of administration, the time arrangement of administration, and other factors known to practitioners. Antigen binding molecules are not necessary, but are optionally co-prepared with one or more medicaments currently used to prevent or treat the illness in question. The effective amount of such other medicaments depends on the amount of the antigen binding molecules present in the preparation, the type of illness or treatment and other factors discussed above. These are usually used with the same dosage and route of administration as described herein, or with about 1% to 99% of the dosage described herein, or with any dosage and by experience / clinically determined to be suitable any approach use.

[0837] In order to prevent or treat a disease, the appropriate dose of the antigen binding molecules of the present invention (when used alone or in combination with one or more other therapeutic agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the molecule is used for prevention or treatment purposes, the patient's medical history and the response to the antibody, and the discretion of the attending physician. The antibody is appropriately administered to the patient once or in a series of treatments. Depending on the type and severity of the disease, an antibody of about 1 μg / kg to 15 mg / kg (e.g., 0.1 mg / kg to 10 mg / kg) can be an initial candidate dose for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. Depending on the factors mentioned above, a typical daily dose can be in the range of from about 1 μg / kg to 100 mg / kg or more. For repeated administration for several days or longer, depending on the condition, treatment will generally continue until desired disease symptoms are suppressed. An exemplary dosage of the antigen binding molecules ranges from about 0.05 mg / kg to about 10 mg / kg. Therefore, 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 the patient. Such doses can be administered intermittently, for example, weekly or every three weeks (for example, so that the patient receives about twice to about twenty times, or for example, about six doses of the antibody). An initial higher loading dose can be administered, followed by one or more lower doses. The progress of the therapy can be easily monitored by conventional techniques and determinations.

[0838] Brain disorders or diseases

[0839] As used herein, "neurological disorder" refers to a disease or disorder that affects the brain / CNS and / or has 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, epileptic seizure, behavioral disorder, and lysosomal storage disease. For purposes of this application, the CNS will be understood to include the eye, which is usually isolated 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-Draeger 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 syndrome, chronic wasting disease, and fatal familial insomnia), bulbar palsy, motor neuron disease, and degenerative diseases of the nervous system (including, but not limited to, Canavan disease, Huntington disease, neuronal ceroid lipofuscinosis, 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).

[0840] In some embodiments, the corresponding functional parts 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 small molecules and any of the following active fragments: the aforementioned substances themselves or specifically recognize and / or 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, β-amyloid protein, β-secretase, γ-secretase, tau, α-synuclein, Parkin, Huntington protein, DR6, presenilin, ApoE, glioma or other CNS cancer markers and neurotrophic factors. Non-limiting examples of brain disorders or diseases described herein that can be used for treatment and corresponding functional parts contained in the antigen binding molecules of the present invention are provided in Table A below:

[0841] (Table A)

[0842] Non-limiting examples of functional moieties and corresponding brain disorders or diseases that they can be used to treat.

[0843]

[0844]

[0845] Products

[0846] In another aspect of the present invention, a product is provided, which contains a product that can be used to treat, prevent and / or diagnose the above-mentioned brain disorders or diseases. The product includes a container and a label on the container or a package insert associated with the container. Suitable containers include, for example, bottles, vials, syringes, intravenous (IV) solution bags, etc. The container can be formed of a variety of materials such as glass or plastic. The container holds a composition, which can be effectively used to treat, prevent and / or diagnose a condition by itself or in combination with another composition, and the container can have a sterile access port (for example, the container can be an intravenous solution bag or 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. In addition, the product 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 cytotoxic agent or other therapeutic agent composition. The product in this embodiment of the present invention may further include a package insert indicating that these compositions can be used to treat a specific condition. Alternatively or additionally, the article 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. The article may further include other substances desired from a commercial and user perspective, including other buffers, diluents, filters, needles and syringes.

[0847] Examples

[0848] The following are examples of methods and compositions of the invention. It is understood that various other embodiments may be practiced given the general description provided above.

[0849] Example 1. Improving antigen binding molecules by designing molecules with a brain transfer portion and a brain retention / targeting portion The concept of concentration and exposure of (such as antibodies) in the brain

[0850] One of the main limitations of known CNS field treatment is that due to low transfer efficiency, the exposure of therapeutic agent is low. In order to solve this problem, a blood-brain barrier (BBB) ​​using a receptor-mediated transfer system (RMT) has been developed to pass through technology and for therapeutic agent. One of the examples of RMT is to utilize transferrin receptor binding moieties, such as antibodies combined with transferrin receptor (TfR), which are expressed in endothelial cells (including endothelial cells of BBB), and it is allowed to be transported through BBB by RMT [Lajoie, Jason M., and Eric V.Shusta." Targeting receptor-mediated transport fordelivery of biologics across the blood-brain barrier." Annual review ofpharmacology and toxicology 55 (2015): 613].

[0851] In addition, other receptors, such as insulin receptor, low-density lipoprotein receptor (LDLR) are also reported to be used as RMT-based BBB transfer parts to deliver biologics to the RMT system of the brain [Lajoie, Jason M., and Eric V. Shusta, the same]. It is known that RMT systems such as anti-TfR antibodies and anti-insulin receptor antibodies have a higher brain metastasis rate compared with IgG in plasma. However, it is well known that the half-life of anti-transferrin receptor antibodies in plasma is shorter, because transferrin receptor is not only expressed in the vascular endothelial cells of the brain, but also expressed systemically (i.e., widely expressed throughout the body, such as in many other non-brain cell types). Therefore, anti-TfR antibodies also deliver drugs to tissues other than the brain, resulting in a shorter half-life in the blood. Similarly, other antibodies with BBB transfer ability show rapid clearance and short half-life (anti-CD98hc antibodies, Neuron Vol. 90, No. 1, 2016, 70-82). In addition, due to the regular flow turnover of the interstitial fluid (ISF) in the brain, the antibodies transferred to the brain will be quickly washed away from the brain and enter the blood circulation. Therefore, after the antibodies flow into the blood circulation, they need to be transferred back to the brain again through the BBB. Therefore, the therapeutic molecules fused to the anti-transferrin receptor antibody show that the improvement of the antibody concentration in the brain only maintains a shorter or limited duration, which limits the effectiveness of the therapeutic agent molecule in the brain (Figure 1 (a)).

[0852] A different approach has been proposed and achieved high antibody concentrations and retention in the brain by using antibodies that bind to myelin oligodendrocyte glycoprotein (MOG), a brain-specific antigen (PLoS One 2019 Apr 12;14(4)e0214404), which slows down the export of antibodies from the brain due to binding to MOG.

[0853] However, without being bound by theory, the inventors of the present invention envision that the mechanism of this "brain retention" approach, while capable of overcoming the short exposure of molecules with transferrin receptor binding domains, requires a relatively long half-life of anti-MOG antibodies in plasma to gradually accumulate and reach high concentrations in the brain, because it requires a continuous supply of antibodies from the blood circulation to the brain ( FIG. 1( b) ). Therefore, anti-MOG antibodies require a sustained high concentration in plasma (or a long half-life in plasma) to reach and maintain high concentrations in the brain, and therefore, when used in combination with therapeutic molecules with an inherently short half-life in plasma, their brain retention effect will be reduced or limited. Another limitation of this approach is that it takes a considerable amount of time for anti-MOG antibodies to gradually accumulate and reach the desired antibody concentration and exposure in the brain after administration, which is not ideal in terms of PK profiles.

[0854] Considering this situation, new technologies are needed to enable molecules with high antibody concentrations and rapid exposure in the brain for a longer duration.

[0855] As described above, given that BBB-crossing technologies such as anti-TfR antibodies are known to have a short half-life in plasma, while brain-retention technologies such as anti-MOG antibodies are expected to require anti-MOG antibodies to have a long plasma half-life so as to gradually accumulate and reach high concentrations in the brain, attempting to combine these two technologies does not seem to be a promising approach, and therefore molecules having both anti-TfR domains and anti-MOG domains have not yet been reported or tested.

[0856] In the present application, the inventors surprisingly found that molecules comprising both a brain transfer portion and a brain retention portion, despite a short half-life in plasma, can quickly achieve high antibody concentrations and exposure in the brain and high retention in the brain for a long time (Figure 1 (c)). Figure 2 (a) is a schematic diagram showing the concept of an exemplary embodiment of an antigen-binding molecule of the present invention, which comprises (1) a brain transfer portion, (2) a brain retention portion, optionally (3) a functional portion, and further optionally an additional half-life extending portion.

[0857] Brain metastasis part is the antigen binding domains that specifically binds to promote antigen binding molecules to penetrate or transfer to the molecules in the brain.For example, the molecule that promotes penetration transfer to the brain is a receptor expressed on the vascular endothelial cells of vascular endothelial cells, preferably blood-brain barrier (BBB), which allows the transfer of biological agents to the brain via the RMT approach.Fully studied and reported that biological agents are transferred to the brain (for example, using anti-TfR antibodies, anti-insulin receptor antibodies or anti-LDLR antibodies) [Lajoie, Jason M., and Eric V.Shusta, the same] via the receptor based on RMT. In a non-limiting example, the molecule is a molecule specifically expressed on the vascular endothelial cells of the blood-brain barrier (BBB).Exemplary molecules are one or more of those molecules listed in Table 1.In one example, brain metastasis part is the antigen binding domains that specifically bind to transferrin receptor (TfR).In another example, brain metastasis part is the antigen binding domains that specifically bind to insulin receptor.

[0858] In one embodiment, the brain retention part is an antigen binding domain of a molecule expressed on the cell membrane of a brain cell, or a brain ECM protein or a brain ECM polysaccharide. It is reported that the combination of brain antigens can slow down the outflow of molecules from the brain, thereby achieving higher retention 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.

[0859] In some specific embodiments, by selecting brain-specific antigens, the antibody concentration is specifically increased only in the brain and maintained for a long time in the brain. In an 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 cells combined with the brain retention portion are shown in Table 2. In addition, the target molecule can be any molecule specifically expressed on brain-specific cells by characterizing the following expression pattern; 1. Select the target molecule, 2. Check the expression level (RNA / protein / actual data) 3. Compare the expression levels of the brain and other regions (organs / tissues), 4. Extract the target molecule with higher expression levels in the brain from other regions (organs / tissues). Cell markers commonly used for FACS analysis or other analysis to identify cell types can also be used for brain retention portions.

[0860] The functional part can be any molecule with therapeutic function, such as agonists, antagonists, enzymes, regulators, stabilizers, cell death inducers and any molecular form, such as nucleic acids, small molecules, cyclic peptides, peptides, ligands, cytokines, chemokines, growth factors, enzymes and antigen binding domains, etc. Optionally, one or more half-life extension parts can also be fused to the molecule. Examples of half-life extension parts are the Fc region of immunoglobulins, albumin binding domains, FcRn binding proteins, FcRn binding peptides and PEG.

[0861] Fig. 2 (b) and (c) are schematic diagrams showing exemplary molecular forms of antigen binding molecules of the present disclosure. In one example, the antigen binding molecule includes the first Fab region and the second Fab region, the first Fab region combines to promote the penetration of antigen binding molecules or transfer to the molecule (i.e., brain transfer part) in the brain, and the second Fab region combines to the molecule (i.e., brain retention part) expressed on brain-specific cells, and optionally further includes a functional part. The structure of the brain retention part and the brain transfer part is not limited to the Fab region, but can also be in the form of an antibody fragment, such as single-chain Fab (scFab), Fv, Fab, Fab', F (ab') 2, double antibody, three antibodies, scFv, VHH, double antibody or F (ab') 2 fragment, or non-antibody conjugates (e.g., affibody, DARPins, FN3, fit, anticalins). In an example, a bispecific antibody comprising single-chain Fab (scFab) or Fab is prepared.

[0862] [Table 1]

[0863] Target of the brain transfer part (the first target that promotes the transfer of antigen-binding molecules into the mammalian brain)

[0864]

[0865]

[0866] [Table 2]

[0867] Target molecule (secondary target expressed on the cell membrane of brain cells, or brain ECM protein or brain ECM polysaccharide)

[0868]

[0869]

[0870]

[0871] Example 2. Preparation of molecules with brain transfer and brain retention moieties

[0872] An antibody comprising a transferrin receptor (TfR) binding domain (brain transfer portion) and two myelin oligodendrocyte glycoprotein (MOG) binding domains (brain retention portion), designated MOG303 / / TfR, was generated as follows. The molecular format of MOG303 / / TfR is as follows Fig.11 (A) shown.

[0873] MOG303, a bivalent antibody comprising two myelin oligodendrocyte glycoprotein (MOG) binding domains; KLH, a bivalent anti-KLH (keyhole limpet hemocyanin) antibody; and KLH / / TfR, an antibody comprising a KLH binding domain and a TfR binding domain; are also prepared by methods known in the art. Both MOG303 and KLH comprise a modified heavy chain constant region of hIgG1 (SEQ ID NO: 2).

[0874] Expression vectors encoding the heavy and light chains of MOG303 / / TfR, MOG303, KLH-TfR and KLH as shown in Table 3 were constructed by methods known in the art.

[0875] [Table 3]

[0876] Expression vectors, heavy and light chains of MOG303 / / TfR, MOG303, KLH-TfR and KLH

[0877]

[0878]

[0879] An expression vector encoding MOG303VH-SG181v11k.newmBBB (heavy chain 1: SEQ ID NO: 9) was prepared by methods known in the art, which contained the VH region of an anti-MOG antibody (SEQ ID NO: 1), a modified human IgG1 constant region (CH1-hinge-CH2-CH3, reducing binding to activating Fγ receptors (human FcγRIa, human FcγRIIa (R), human FcγRIIa (H), human FcγRIIIa (V) and human FcγRIIIa (F)), and containing mutations for heterodimeric Fc production) and a single-chain Fab domain of an anti-mouse transferrin receptor antibody, followed by a Gly-Ser linker (constant region of scFab: SEQ ID NO: 5).

[0880] An expression vector encoding MOG303VH-SG181v11h (heavy chain 2: SEQ ID NO: 10) was prepared by a method known in the art, which contains the VH region of an anti-MOG antibody (SEQ ID NO: 1), a modified human IgG1 constant region (CH1-hinge-CH2-CH3, reduced binding to activating Fγ receptors (human FcγRIa, human FcγRIIa (R), human FcγRIIa (H), human FcγRIIIa (V) and human FcγRIIIa (F)), and contains mutations for heterodimeric Fc production; SEQ ID NO: 6).

[0881] An expression vector encoding MOG303VL-SK1 (light chain: SEQ ID NO: 8), which contains the VL region of 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.

[0882] 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) (having Fig.11 The expression of the molecular format shown in (A) was completed by using the 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 several days of culture, the culture medium was collected and treated with an affinity column. The antibody protein was purified by an affinity column (protein A) and size exclusion chromatography was performed to obtain MOG303 / / TfR. Expression and purification methods were performed by methods known in the art (Nat Protoc. 2018 January; 13(1): 99-117).

[0883] Example 3. Pharmacokinetic characteristics of antibodies in mouse brain and plasma

[0884] The pharmacokinetics of anti-KLH and anti-MOG antibodies with or without anti-TfR Fab domains were evaluated in C57BL / 6J mice (male, 6 to 8 weeks) in brain and plasma.

[0885] IgG antibody, KLH-IgG (KLH) and MOG303-IgG (MOG303) and IgG antibody with anti-TfR domain, KLH / / TfR and MOG303 / / TfR were intravenously administered to mice at a dose of 2 mg / kg. Blood was collected from the 1st to the 28th day after administration, and plasma was then obtained by centrifugation (12000rpm, 4°C, 5min). After blood sampling, phosphate buffered saline (PBS) was perfused from the heart by inserting a catheter to remove blood, and then the brain was collected. 50mg brain fragments were dissolved with PBS containing NP-40 alternative detergent (Millipore) and Complete Mini protease inhibitor cocktail (Roche) and 5mm stainless steel beads, and homogenized with a homogenizer (Qiagen). After rotating at 4°C for 60min, the sample was centrifuged (15000rpm, 4°C, 20min), and the supernatant was collected.

[0886] The concentration of antibodies in plasma and brain was determined by electrochemiluminescence immunoassay (ECL). As capture antibody, anti-human IgG Fab'2 antibody (LifeSpan BioSciences) was applied to the plate. After adding PBS with Tween20 (PBST, Sigma-Aldrich) containing 1% bovine serum albumin (Sigma-Aldrich) as a blocking solution, diluted plasma and brain homogenate samples were applied. As detection antibody, biotin-labeled anti-human IgG antibody (BethyLaboratories) was added. Finally, streptavidin labeled with SULFO-label (Meso Scale Discovery) was added, and the signal was detected by ECL (Meso Scale Discovery). Phoenix WinNonlin (version 8.3) was used to analyze the concentration-time curve of antibodies in plasma and brain by non-compartmental model to reveal the area under the curve (AUC), clearance, distribution volume (Vd) and half-life. Brain / plasma ratio was calculated by dividing the concentration in brain by the concentration in plasma. The percentage of injected dose per brain tissue weight was calculated by dividing the brain concentration by the dose amount.

[0887] The pharmacokinetic curves of KLH, MOG303, KLH / / TfR and MOG303 / / TfR in the brain are shown in Figure 3. On the first day after administration, the control antibody KLH showed an antibody concentration in the brain of about 0.046 micrograms / g, a brain-to-plasma concentration ratio of 0.003, and 0.10%ID / g brain (i.e., the percentage of injected dose per brain tissue weight). The concentration on the first day for KLH was the maximum concentration (Cmax) during the study. MOG303 showed antibody concentration and brain distribution characteristics similar to KLH on the first day. On the other hand, KLH / / TfR showed an antibody concentration in the brain of 1.4 micrograms / g, a brain-to-plasma concentration ratio of 0.37, and 3.1%ID / g brain, which were higher than KLH and MOG303. The KLH / / TfR concentration on the first day was the Cmax during the study. Surprisingly, the concentration, brain to plasma concentration ratio, and %ID / g brain of MOG303 / / TfR in the brain showed approximately 2-fold higher than that of KLH / / TfR (2.8 μg / g brain, 0.55 brain to plasma concentration ratio, 6.1%ID / g brain) on day 1. These results suggest that the combination of the transfer moiety (anti-TfR) and the retention moiety (anti-MOG) contributes to a significant increase in antibody concentration and brain distribution within a short time (1 day) after antibody injection.

[0888] From the 7th day to the 28th day, the brain concentration and dose percentage of KLH gradually decreased (Figure 3 (a)), and the brain to plasma ratio was almost constant (Figure 3 (b)). The brain concentration, brain plasma ratio and dose pe...

Claims

1. 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, the first target facilitates transfer of the antigen binding molecule into the brain of a mammal, and The second antigen binding domain specifically binds to a second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain extracellular matrix (ECM) protein or brain ECM polysaccharide. 2 . The antigen-binding molecule according to 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 according to 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 (HSPG) branched from proteoglycans.

4. according to the antigen binding molecule described in any one of claims 1 to 3, wherein the second target is selected from the group consisting of the following items: 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 carrier family 1 member 3), EAAT2 (solute carrier family 1 member 2), M AP2 (microtubule-associated protein 2), NEFL (neurofilament light polypeptide), NEFM (neurofilament medium polypeptide), NSE (γ-enolase), CD68 (macrophage sialic acid protein), allogeneic transplant inflammatory factor 1 (IBA1 or AIF1), purinergic receptor (P2RY12), interleukin 1 receptor accessory protein-like 1 (IL1RAPL1), glutamate ion channel receptor NMDA type subunit 2B (GRIN2B), voltage-gated calcium channel auxiliary subunit γ8 (CACNG8), CD11b (integrin subunit αM), S LC6A2 (sodium-dependent norepinephrine 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), phosphoprotein associated with glycosphingolipid-rich microdomains 1 (Csk binding protein), plasma membrane calcium transporting ATPase 1, prominin-1, somatostatin receptor type 1, carnitine O-palmitoyltransferase 1 brain isoform (CPT1-B), epidermal growth factor receptor, protein MAL2, syntaxin-1A, sodium / calcium exchanger 1, lysophosphatidylcholine acyltransferase 1 (LPC acyltransferase 1), calcium homeostasis protein-3 (Alcadein-β), pituitary adenylate cyclase activating polypeptide type I receptor (PACAPI receptor), neutral cholesterol ester hydrolase 1 (NCEH), CD166 antigen (activated leukocyte adhesion molecule), inactivated 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), tropoelastin (elastin), collagen alpha-2 (IV) chain ( The proteins involved in the expression of the proteins are angiostatin, angiostatin, aggrecan nexin 1 (hyaluronan and aggrecan nexin 1), tenascin-R (TN-R), aggrecan nexin 2 (hyaluronan and aggrecan nexin 2), collagen alpha-1 (I) chain, neurofilament protein-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 according to any one of claims 1 to 4 , wherein the brain cells comprise one or more of cells selected from the group consisting of oligodendrocytes, astrocytes, neurons, and microglia. The antigen binding molecule according to any one of claims 1 to 5, wherein the second target is a molecule mainly expressed on brain-specific cells.

7. according to the antigen binding molecules described in 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. according to the antigen binding molecule described in any one of claims 1 to 7, wherein the second target is selected from the group consisting of: myelin oligodendrocyte glycoprotein (MOG), neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5) and cell adhesion molecule 3 (CADM3).

9. The antigen-binding molecule according to any one of claims 1 to 8, wherein: (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 neurocan 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 Neurocan 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 insulin receptor, and the second target is myelin oligodendrocyte glycoprotein (MOG); or (viii) the first target is insulin receptor, and the second target is neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (ix) the first target is 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 neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xii) the first target is 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 neurocan 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 neurocan 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 neurocan C / chondroitin sulfate proteoglycan 5 (CSPG5); or (xxi) The first target is CD98hc, and the second target is cell adhesion molecule 3 (CADM3).

10. The antigen binding molecule according to any one of claims 1 to 4, wherein the second target is not a target selected from the group consisting of: β-secretase 1 (BACE1), Aβ, epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), tau, apolipoprotein E (ApoE), α-synuclein, CD20, huntingtin, prion protein (PrP), leucine-rich repeat kinase 2 (LRRK2), parkin, presenilin 1, presenilin 2, γ-secretase, death receptor 6 (DR6), amyloid precursor protein (APP), p75 neurotrophic factor receptor (p75NTR), caspase 6, TRKA, 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 receptors, and EpCAM.

11. The antigen-binding molecule according to any one of claims 1 to 10, further comprising (i) at least one functional portion comprising an enzyme, a therapeutic protein, an antibody or an antigen-binding fragment thereof, a peptide, a DNA, a shRNA, a siRNA, a small molecule drug or a cytotoxic agent and / or (ii) at least one in vivo half-life extending moiety, preferably wherein said at least one in vivo half-life extending moiety 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.

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

13. The method according to claim 12, further comprising 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 differs from the second antigen-binding molecule according to (b) only in that the control antigen-binding molecule does not comprise: In the case of (a1), the at least one second antigen binding domain specifically binds to the second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide; or In the case of (a2), the at least one first antigen-binding domain specifically binds to the first target, which promotes the transfer of the antigen-binding molecule into the brain of a mammal.

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

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

16. The method according to claim 15, further comprising step (c): (c) determining that 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 differs from the second antigen-binding molecule according to (b) only in that the control antigen-binding molecule does not comprise: In the case of (a1), the at least one second antigen binding domain specifically binds to the second target, wherein the second target (i) is expressed on the cell membrane of brain cells, or (ii) is a brain ECM protein or brain ECM polysaccharide; or In the case of (a2), the at least one first antigen-binding domain specifically binds to the first target, which promotes the transfer of the antigen-binding molecule into the brain of a mammal.

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

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

19. The method according to claim 18, further comprising step (c): (c) determining that the retention 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 according to (b) only in that the control antigen-binding molecule does not comprise the at least one antigen-binding domain that specifically binds to the second target.

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

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

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