Multispecific binding molecules and methods of use thereof

Abstract

Multispecific binding molecules having a first binding domain that targets a blood-brain barrier target and a second binding domain that targets a neuronal, astrocytic, and / or glial cell target, and uses thereof, in aiding in the treatment of central nervous system diseases, including neurodegenerative diseases such as Alzheimer's disease, Huntington's disease, Parkinson's disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), autism, catalepsy, encephalitis, migraine, and Tourette's.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This patent application claims the benefit of U.S. Provisional Patent Application No. 63 / 338,480, filed May 5, 2022, which is incorporated by reference in its entirety herein.

[0002] (Reference to sequence listing) This application contains a Sequence Listing that has been submitted electronically in ST.26XML format and is incorporated herein by reference in its entirety. The ST.26XML Sequence Listing was created on April 4, 2023, is named 22892.xml, and is 31,460 bytes in size. No new matter has been added.

[0003] FIELD OF THEINVENTION The present invention is in the field of medicine, specifically in the field of treating central nervous system (CNS) diseases. More specifically, the present disclosure relates to compositions comprising multispecific binding molecules having a first binding domain that targets a blood-brain barrier target and a second binding domain that targets a neuronal target, an astrocyte target, and / or a glial cell target (collectively, CNS targets), and their use in treating CNS diseases. The multispecific binding molecules of the present disclosure aid in the treatment of CNS diseases by enhancing passage of a therapeutic agent across the blood-brain barrier and its delivery to its intended CNS target. [Background technology]

[0004] The CNS is perhaps the most complex and highly organized organ system of the human body. It consists of two major organs, the brain and the spinal cord, and is composed of both glial cells and neurons. In vertebrates, such as humans, neurons are classified into three types: sensory neurons and motor neurons, some of which may span both the CNS and the peripheral nervous system, and interneurons, which are thought to be located almost exclusively within the CNS.

[0005] CNS diseases are disorders that affect the structure and / or function of the brain and / or spinal cord.CNS diseases or disorders can include neuromyelitis optica, multiple sclerosis, and anti-myelin oligodendrocyte glycoprotein; oncological disorders including astrocytoma, oligodendroglioma, glioblastoma, and other CNS tumors; neurodegenerative disorders including Alzheimer's disease, Huntington's disease, Parkinson's disease, Progressive Supranuclear Palsy (PSP), Amyotrophic Lateral Sclerosis (ALS), and Frontal Temporal Dementia (FTD); and other CNS disorders such as autism, catalepsy, encephalitis, migraine, and Tourette's.

[0006] CNS diseases have traditionally proven very difficult to treat. One reason is that therapeutic agents must cross the blood-brain barrier to enter the CNS. The blood-brain barrier is a specialized semi-permeable boundary of endothelial cells that selectively prevents the passage of solutes, including pathogens, into the CNS. The blood-brain barrier allows the passage of some molecules via passive diffusion and is involved in the selective active transport of some molecules important for neurological function. In addition to the challenges posed by the blood-brain barrier, once a therapeutic agent crosses the blood-brain barrier, the therapeutic agent must still act on its intended target within the CNS. Some studies estimate that less than 1% of some therapeutic agents cross the blood-brain barrier.

[0007] Blood-brain barrier shuttles or chaperones to improve passage of therapeutic agents across the blood-brain barrier into the CNS have been described for over 20 years. For example, WO 2003 / 009815 (U.S. Patent Application Publication No. 2003 / 0129186) describes the use of antibodies directed against the transferrin receptor ("TFR") to modulate blood-brain barrier transport. However, attempts to shuttle therapeutics across the blood-brain barrier using anti-TFR antibodies as conjugates have proven difficult and inadequate in significantly improving the treatment of CNS diseases (see, e.g., Syvanen S, Hultqvist G, Gustavsson T, et al. Efficient clearance of Abeta protofibrils in AbetaPP-transgenic mice treated with a brain-penetrating bifunctional antibody. Alzheimer's Res Ther. 2018;10:49; Couch JA, Yu YJ, Zhang Y, et al. Addressing safety liabilities of TfR bispecific antibodies that cross the blood-brain barrier. Sci Transl Med. 2013;5:183ra57, which discuss the pharmacokinetics, toxicity, and off-target issues associated with targeting TfR as a blood-brain barrier shuttle).

[0008] Thus, there remains a need for enhancing the treatment of CNS diseases that overcomes the problems described above. More specifically, there exists a need for improving the delivery of therapeutic agents across the blood-brain barrier and providing enhanced selective targeting of CNS targets within the CNS. Such enhancements must be able to deliver therapeutic agents across the blood-brain barrier with increased efficiency and improve delivery to specific CNS targets within the CNS so that the shuttled therapeutic agent is delivered to its intended therapeutic target within the CNS. Such enhancements must not be more invasive than the therapeutic molecule, allow peripheral systemic delivery of therapeutic agents to overcome the need to invasively deliver therapeutic agents directly to the CNS, should not entail unacceptable immunogenicity and off-target problems, and should demonstrate acceptable stability and developability. Such enhancements must also not interfere with the properties of the therapeutic agent required to treat CNS diseases.

[0009] The present disclosure provides multispecific binding molecules for enhancing the treatment of CNS diseases. More specifically, the present disclosure provides multispecific binding molecules having a first binding domain that specifically binds to a blood-brain barrier target ("BBB target") and a second binding domain that specifically binds to a neuronal target, an astrocyte target, and / or a glial cell target ("CNS target"), thereby enhancing the delivery of a therapeutic composition to its intended therapeutic target. Such multispecific binding molecules can be linked to therapeutic compositions to aid in the treatment of CNS diseases by enhancing the passage of a therapeutic agent across the blood-brain barrier and delivery to a specific CNS target within the CNS, such as a neuronal target, an astrocyte target, and / or a glial cell target, whereby the therapeutic composition modulates its intended therapeutic target. Summary of the Invention

[0010]

[0013] Embodiments of the present disclosure include multispecific binding molecules comprising a first binding domain and a second binding domain, where the first binding domain specifically binds to a blood-brain barrier target and the second binding domain specifically binds to at least one of a neuronal target, an astrocytic target, and / or a glial cell target, or another parenchymal target in the CNS. According to some embodiments, the blood-brain barrier target is one of TFR, lipoprotein receptor-related protein 1 (LRP1), LRP2 or megalin / GP330, LRP3, LRP5, LRP6, LRP8, insulin-like growth factor (IGF), transferrin receptor-1, CD98 heavy chain (SLC3A2), large amino acid transporter small subunit 1 (LAT-1), insulin receptor, low-density lipoprotein (LDL) receptor, CDC50A. According to some embodiments, the neuronal target is one of L1CAM, THY1, KIT, GRIA1, GRM2, TMEM130, NSG1, BASP1, NSTR1, SLC22A17, NCAM1, NCAM2, SLC6A17, SCN3B, and P2Y12R. In particular, according to some embodiments, the blood-brain barrier target is TFR and the neuronal target is L1CAM.

[0011] In some embodiments, the multispecific binding molecule of the present disclosure comprises a bispecific antibody, a heterodimeric immunoglobulin antibody, or a fragment thereof. Additionally, in some embodiments of the multispecific binding molecule of the present disclosure, the first binding domain is a Fab, scFv, Fv, or scFab. According to some embodiments, the second binding domain is a Fab, scFv, Fv, or scFab. Additionally, in some embodiments, the first binding domain and the second binding domain are linked by one of a thiol-based reactive group, an amine-based reactive group, an aldehyde-reactive group, or a carbohydrate-based group. In additional embodiments, the first binding domain and the second binding domain are linked by one of a PEG-linker, an amino acid linker, and a glycan linker.

[0012] In further embodiments of the multispecific binding molecules of the present disclosure, a therapeutic composition is linked to the multispecific binding molecule. According to some embodiments, the therapeutic composition is one of a peptide, an antibody or fragment thereof, or an oligonucleotide. In some such embodiments, the therapeutic composition target includes, but is not limited to, MAPT, SNCA, APP, BAC1, ATXN2, ATXN3, SARM1, APOE, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B.

[0013] In addition, in some embodiments, the multispecific binding molecules of the disclosure are linked to a carrier molecule.

[0014] Further embodiments of the present disclosure include pharmaceutical compositions comprising a multispecific binding molecule of the present disclosure and one or more pharma- ceutically acceptable carriers, diluents, or excipients.

[0015] Embodiments of the present disclosure also include DNA molecules comprising a polynucleotide sequence encoding a polypeptide chain comprising one of the HC, LC, HCVR, or LCVR of a multispecific binding molecule of the present disclosure.

[0016] According to some embodiments, a method of treating a central nervous system disease is provided, comprising administering to a patient in need thereof a therapeutically effective amount of a multispecific binding molecule of the present disclosure. Embodiments of the present disclosure also include a method of treating a central nervous system disease, comprising administering to a patient in need thereof a therapeutic composition, wherein the therapeutic composition is linked to a multispecific binding molecule of the present disclosure, and wherein the therapeutic composition accumulation in peripheral tissues is reduced compared to the therapeutic composition when not linked to the multispecific binding molecule. According to some embodiments, at least 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, or 144 hours after the administering step, the therapeutic composition accumulation in peripheral tissues is reduced compared to the therapeutic composition when not linked to the multispecific binding molecule.

[0017] According to some embodiments, the present disclosure provides a method of delivering a therapeutic composition to a CNS target in the CNS, comprising administering the therapeutic composition to a patient in need thereof, wherein the therapeutic composition is linked to a multispecific binding protein of the present disclosure, and the therapeutic composition demonstrates reduced accumulation in peripheral tissues of the patient. In some such embodiments, the therapeutic composition demonstrates reduced accumulation in bone marrow of the patient. According to some embodiments, the CNS target is one of a neuronal target, an astrocyte target, and a glial cell target. According to some embodiments, the central nervous system disease is a neurodegenerative disorder. According to some embodiments, the central nervous system disease is Alzheimer's disease, Huntington's disease, Parkinson's disease, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), autism, catalepsy, encephalitis, migraine, and Tourette's.

[0018] The embodiments of the present disclosure include the multispecific binding molecules of the present disclosure for use in therapy. The embodiments also include the multispecific binding molecules of the present disclosure for use in treating central nervous system diseases. According to some such embodiments, the CNS disease is Alzheimer's disease, Huntington's disease, Parkinson's disease, progressive supranuclear palsy (PSP), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), autism, catalepsy, encephalitis, migraine, and Tourette's. Furthermore, the present disclosure provides, in some embodiments, the multispecific binding molecules of the present disclosure for use in the manufacture of a medicament for use in treating a CNS disease. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a schematic diagram of a multispecific binding molecule in the form of a heterodimeric IgG antibody of the present disclosure. [Diagram 2] FIG. 1 is a schematic diagram of a multispecific binding molecule of the present disclosure in the form of a heterodimeric IgG antibody having a first binding domain that binds to the blood-brain barrier target, TFR, and a second binding domain that binds to a neuronal target, L1CAM. [Diagram 3] 1 shows high content imaging data demonstrating the cellular activity (binding, internalization, and degradation properties) of exemplary multispecific binding molecules of the disclosure. [Figure 4A] 1 shows brain pharmacokinetic data for exemplary multispecific binding molecules of the disclosure. [Figure 4B] 1 shows brain pharmacokinetic data for exemplary multispecific binding molecules of the disclosure. [Figure 5A] 1 shows in vivo pharmacodynamic data for exemplary multispecific binding molecules of the disclosure. [Figure 5B] 1 shows in vivo pharmacodynamic data for exemplary multispecific binding molecules of the disclosure. [Figure 5C] 1 shows in vivo pharmacodynamic data for exemplary multispecific binding molecules of the disclosure. [Figure 5D] 1 shows in vivo pharmacodynamic data for exemplary multispecific binding molecules of the disclosure. [Figure 5E] 1 shows in vivo pharmacodynamic data for exemplary multispecific binding molecules of the disclosure. [Figure 6A] 1 shows in vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure. [Figure 6B] 1 shows in vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure. [Figure 6C] 1 shows in vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure. [Figure 6D] 1 shows in vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure. [Figure 6E] 1 shows in vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure. [Figure 6F] 1 shows in vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure. [Figure 6G] 1 shows in vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure. [Figure 6H] 1 shows in vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure. [Figure 6I] 1 shows in vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure. [Figure 6J] 1 shows in vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The term "multispecific" as used herein refers to a molecule or compound that comprises two or more different binding domains. A multispecific binding molecule can bind to two or more different targets, a blood-brain barrier target and a CNS target, as described herein. An embodiment of a multispecific binding molecule of the present disclosure can include a bispecific antibody and / or a fragment of an antibody, such as a Fab, scFv, Fv, or scFab, where the antibody or fragment thereof portion forms the binding domain of the molecule such that the multispecific binding molecule can bind to a blood-brain barrier target and a CNS target, as described herein. An embodiment of a multispecific binding molecule includes, for example, two scFvs linked together via a peptide linker (e.g., a GS linker), where one scFv forms a binding domain for a blood-brain barrier target and the other scFv forms a binding domain for a CNS target. A multispecific binding molecule of the present disclosure also includes a VHH linked to one or more fragments of an antibody. Other multispecific binding molecules of the disclosure include heterodimeric immunoglobulin G (IgG) antibodies as illustrated in FIG. 1, in which the amino-terminus (N-terminus) of each "arm" comprising an HCVR / LCVR pair forms a binding domain that exhibits selective monovalent binding to its cognate antigen (e.g., the binding domain of one "arm" specifically binds to a blood-brain barrier target and the binding domain of the other "arm" specifically binds to a CNS target).

[0021] The term "binding domain" refers to a portion of a multispecific binding molecule of the present disclosure that binds to a target (e.g., a BBB target or a CNS target as illustrated in FIG. 1). For example, a binding domain of a multispecific binding molecule may bind to a blood-brain barrier target such as TFR. Similarly, a different binding domain of a multispecific binding molecule of the present disclosure may bind to a CNS target such as L1CAM. The terms "bind" and "binds", as used herein, unless otherwise indicated, are intended to mean the ability of a binding domain of a multispecific binding molecule to form a chemical bond or an attractive interaction with another protein or molecule, resulting in the proximity of the two proteins or molecules as determined by common methods known in the art.

[0022] Blood-brain barrier targets, as used herein, refer to membrane receptors that are presented on the apical side of the endothelial blood-brain barrier and actively assist, facilitate, and / or promote the internalization of molecules outside the CNS (e.g., in serum) into the CNS. Blood-brain barrier targets, according to the present disclosure, include, but are not limited to, TFR, low-density lipoprotein receptor-related protein 1 (LRP1), LRP2 or megalin / GP330, LRP3, LRP5, LRP6, LRP8, insulin-like growth factor receptor 1 (IGF1R), transferrin receptor-1 (or TFR / CD71), CD98 heavy chain (SLC3A2), large amino acid transporter small subunit 1 (LAT-1), insulin receptor, low-density lipoprotein (LDL) receptor, CDC50A, and the like.

[0023] As used herein, a CNS target refers to an antigen present on the outer membrane of one of neurons, astrocytes, and / or glial cells, as well as other parenchymal antigens in the CNS. The multispecific binding molecules of the present disclosure include a binding domain that targets a CNS target, facilitating the proximity of a therapeutic composition linked to the multispecific binding molecule to a desired CNS target, whereby the therapeutic composition can act on its intended therapeutic target. Antigens present on the outer membrane of neurons, including single-pass transmembrane receptors, multi-pass transmembrane protein receptors, and cell surface proteoglycans, include, but are not limited to, L1CAM, THY1, KIT, GRIA1, GRM1, GRM2, TMEM130, NSG1, BASP1, NSTR1, SLC22A17, NCAM1, NCAM2, SLC6A17, SCN3B, P2Y12R, CADM1, PARM1, NSG1, and KIDINS220. Antigens present on the outer membrane of microglial cells, including single-pass transmembrane receptors, multi-pass transmembrane protein receptors, and cell surface proteoglycans, include, but are not limited to, TREM2, TMEM119, CD11b, CD14, CD16, CD32, CD33, CD40, CD45, CD64, CD68, CD80, CD115, CD172a, CX3CR1, FCER1G, F4 / 80, FCRLS, Siglec, Glut5, and P2Y12. Antigens present on the outer membrane of astrocytes, including single-pass transmembrane receptors, multi-pass transmembrane protein receptors, and cell surface proteoglycans, include, but are not limited to, CD49f, AQP4, GLT1, GLAST, A2AR, TGFbR, S1P1, IL1R<IL6R, IL17R, and IFNgR.

[0024] The region of the target bound by the binding domain of the multispecific binding molecule can be a linear or conformational region of the target. The region of the target bound by the binding domain of the multispecific binding molecule can be determined according to different experimental techniques. It is understood that the determination of the region of the target bound by the multispecific binding molecule can vary based on the different mapping techniques used, and can also vary depending on the different experimental conditions used, for example, due to conformational changes or cleavage of the target induced by the specific experimental conditions. Binding mapping techniques are known in the art, including, but not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species swap mutagenesis, alanine scanning mutagenesis, hydrogen-deuterium exchange (HDX), and cross-blocking assays (e.g., Rockberg & Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd ed. 2018).

[0025] The term "specific for," as used herein with respect to a binding domain, refers to the ability of a binding domain of a multispecific binding molecule of the disclosure to bind, associate with, and / or modulate a particular target molecule above background levels of non-target levels. In some embodiments, "specific for" may be demonstrated by an affinity (Kd) measure for the target over a non-target.

[0026] Terms such as "modulate" or "modulates", as used herein, refer to altering or changing a measurable value, and include altering or changing such a measurable value upward (i.e., upmodulating or upmodulating) or altering or changing downward (i.e., downmodulating or downmodulating).

[0027] The multispecific binding molecules of the present disclosure may also be linked to one or more therapeutic compositions, such as therapeutic antibodies or fragments thereof, peptides, oligonucleotides, small molecules, nanoparticles, lipid nanoparticles, exosomes, or combinations thereof (it is also understood that the multispecific binding molecules or therapeutic agents may be fully or partially encapsulated by lipid nanoparticles or exosomes). Exemplary therapeutic oligonucleotides include antisense oligonucleotides, RNA interference, RNA activation, and guide RNA-directed targeted RNA editing using systems such as the CRISPR-Cas9 and ARCUS nuclease systems. Exemplary therapeutic oligonucleotides, antibodies or fragments thereof, and / or peptides directed to therapeutic target genes or proteins in the CNS (i.e., "therapeutic targets" distinct from blood-brain barrier targets and CNS targets) known in the art and associated with proteins involved in diseases of the CNS, such as beta-amyloid, BACE1, MAPT, SNCA, ATXN2, ATXN3, SARM1, APOE, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, CACNA1B, etc., whose modulation is intended to provide a therapeutic effect, may be linked to the multispecific binding molecules of the present disclosure.

[0028] Furthermore, therapeutic compositions can be linked to the multispecific binding molecules of the present disclosure in a variety of ways and at various positions or portions of the multispecific binding molecule, such that such linkages do not interfere with binding of the multispecific binding molecule to the blood-brain barrier and CNS targets, and do not interfere with the therapeutic properties of the therapeutic composition when linked.

[0029] The term linker, as used herein, refers to an atom, group of atoms, molecule, or compound (such as an amino acid or group of amino acids) that contains at least one bond that bonds or connects a molecule or compound to another molecule or compound. Linkers can be composed of a variety of compositions, including polyethylene glycol (PEG) units, linear, branched, and aromatic carbon-based units, amino acids, and glycans. Linkers, in accordance with the present disclosure, can also include hydrolyzable and / or enzymatically degradable linkers. Linkers can also attach multiple therapeutic compositions to the multispecific binding molecules of the present disclosure.

[0030] The terms "linked" and "conjugated", as used interchangeably herein, refer to a first molecule or compound, e.g., a polypeptide, being associated, bound, connected, or otherwise joined to a second molecule or compound, e.g., a nucleotide (or polynucleotide) or a polypeptide. For example, a polypeptide may be linked to a polynucleotide sequence. Similarly, a peptide sequence may be linked to a second peptide sequence via covalent or non-covalent interactions to form a multimeric peptide. In some embodiments of the present disclosure, an antibody or fragment thereof is linked to an oligonucleotide. The term "conjugated peptide" or "peptide conjugate", as used herein, refers to a peptide or protein that is covalently (including reversible covalently) or non-covalently linked to another molecule, moiety, compound, or group.

[0031] Certain embodiments of the present disclosure include heterodimeric IgG multispecific binding molecules, one arm of which specifically binds to a blood-brain barrier target, e.g., TFR-1, and the other arm of which specifically binds to a CNS target, e.g., a neuronal target such as L1CAM, where the IgG multispecific binding molecule is linked, e.g., via a chemically functionalized linker, to a siRNA therapeutic composition that provides a covalent bond to the multispecific binding molecule scaffold. Examples of chemically functionalized linkers include thiol-based reactive groups (e.g., maleimide, sulfhydryl, haloacetyl, thiosulfonate, disulfide), amine-based groups (e.g., N-hydroxysuccinimide ester, imidoester, etc.), and aldehyde-reactive groups (e.g., hydrazide and alkoxyamine), as well as carbohydrate-based groups (e.g., natural Fc glycans, engineered glycans, etc.).

[0032] The term "amino acid having a functional group available for conjugation" as used herein refers to any natural or unnatural amino acid or derivative thereof having a functional group that can be conjugated to a conjugate moiety, for example, directly or via a conjugate linker. Examples of such functional groups include, but are not limited to, alkynyl, alkenyl, amino, azido, bromo, carboxyl, chloro, iodo, and thiol groups. Similarly, examples of natural amino acids containing such functional groups include C (thiol), D (carboxyl), E (carboxyl), K (amino), and Q (amide).

[0033] The multispecific binding molecules of the present disclosure may be used to aid in the treatment of patients, particularly CNS diseases, including neurodegenerative, oncological, and autoimmune disorders of the CNS. When used with respect to the multispecific binding molecules of the present disclosure, "treatment" and / or "treating" and / or "treating" refers to the use of a multispecific binding molecule of the present disclosure in conjunction with a therapeutic composition (e.g., a therapeutic composition linked or conjugated to a multispecific binding molecule) whereby the multispecific binding molecule assists in the delivery of the therapeutic composition across the blood-brain barrier to a CNS target, whereby the therapeutic composition can modulate its intended therapeutic target, thereby completely or partially slowing, interrupting, arresting, controlling, halting, or reversing the progression of a CNS disorder, but not necessarily showing complete elimination of all disorder symptoms. Treatment includes administration of a multispecific binding molecule of the disclosure, including a therapeutic composition linked or conjugated to a multispecific binding molecule, for the treatment of a CNS disease or condition in a human that would benefit from delivery of the therapeutic composition across the blood-brain barrier to a CNS target to help the therapeutic composition produce a desired therapeutic effect.

[0034] As used interchangeably herein, the terms "patient," "subject," and "individual" refer to a human. In certain embodiments, the patient is further characterized as having a CNS disease, disorder, or condition (e.g., a CNS neurodegenerative disorder). In some embodiments, the patient may be further characterized as being at risk for developing a CNS disorder, disease, or condition.

[0035] An embodiment of the multispecific binding molecule of the present disclosure includes an antibody or a fragment thereof. As used herein, an "antibody" includes an immunoglobulin or immunoglobulin-like molecule that binds to an antigen and comprises four polypeptide chains: two heavy chains (HC) and two light chains (LC) cross-linked via interchain disulfide bonds (illustrated above). Each HC is composed of a heavy chain variable region (VH) and a heavy chain constant region. Each LC is composed of a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions, termed complementarity determining regions (CDRs), interspersed with more conserved regions, termed framework regions (FRs). The CDRs are exposed on the surface of the protein and are the regions of the antibody that are important for antigen-binding specificity. Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Herein, the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3," and the three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3." The CDRs contain most of the residues that form specific interactions with the antigen.The assignment of amino acid residues to CDRs can be done using the standard methods published by Kabat (Kabat et al., "Sequences of Proteins of Immunological Interest," National Institutes of Health, Bethesda, Md. (1991)), Chothia (Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 196, 901-917 (1987); Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins," Journal of Molecular Biology, 273, 927-948 (1997)), North (North et al., "A New Clustering of Antibody CDR Loop Conformations," Journal of Molecular Biology, 406, 228-256 (2011)), or IMGT (the international standard available at www.imgt.org). ImMunoGeneTics database; see Lefranc et al., Nucleic Acids Res. 1999;27:209-212). Exemplary embodiments of an antibody fragment or antigen-binding fragment, as used herein, include at least a portion of an antibody that retains the ability to specifically interact with an antigen or an epitope of an antigen, such as a Fab, a Fab', a F(ab')2, an Fv fragment, an scFv antibody fragment, a scFab, a disulfide-linked Fv (sdFv), an Fd fragment, etc. EXAMPLES

[0036] Exemplary Multispecific Binding Molecules Exemplary multispecific binding molecules of the present disclosure include a first binding domain specific for a blood-brain barrier target and a second binding domain specific for a CNS target. According to certain exemplary multispecific binding molecules of the present disclosure, the first binding domain binds to TFR and the second binding domain binds to L1CAM. Exemplary binding domains of the multispecific binding molecules of the present disclosure include a heavy chain variable region (HCVR) and a light chain variable region (LCVR), each of which includes three complementarity determining regions (HCDR1-3 and LCDR1-3, respectively). Exemplary binding domains are shown in Table 1.

[0037] [Table 1]

[0038] Expression of Exemplary Multispecific Binding Molecules Exemplary multispecific binding molecules can be expressed and purified essentially as follows: A glutamine synthetase (GS) expression vector containing DNA encoding a polypeptide chain comprising HCVR and LCVR (or, e.g., HC and LC) or fragments thereof, comprising the respective binding domains of the multispecific binding molecules of the present disclosure, is used to transfect a Chinese hamster cell line, CHO (GS knockout, clone 1D3), by electroporation. The expression vector encodes the SV Early (Simian Virus 40E) promoter and the gene for GS. Expression of GS allows the biochemical synthesis of glutamine, an amino acid required by CHO cells. After transfection, cells are subjected to bulk selection with 50 μM L-methionine sulfoximine (MSX). To increase the stringency of selection, inhibition of GS by MSX is utilized. Cells in which the expression vector cDNA has been integrated into a transcriptionally active region of the host cell genome can be selected against CHO wild-type cells expressing endogenous levels of GS. The transfected pools are plated at low density to allow for near-clonal growth of stable expressing cells. Master wells are screened for polyspecific binding molecule expression and then scaled up in serum-free suspension cultures used for production. The clarified medium into which the exemplary polyspecific binding molecules are secreted is applied to a Protein A affinity column that is equilibrated with a compatible buffer, such as phosphate-buffered saline (pH 7.4). The column is washed to remove non-specifically bound components. The bound polyspecific binding molecules are eluted, for example, by a pH gradient (such as 0.1 M sodium phosphate buffer pH 6.8 to 0.1 M sodium citrate buffer pH 2.5) and neutralized with Tris, pH 8 buffer. The polyspecific binding molecule fractions are detected, such as by SDS-PAGE or analytical size exclusion, and then pooled. Soluble aggregates and multimers can be effectively removed by common techniques including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The polyspecific binding molecules are concentrated and / or sterile filtered using common techniques.The purity of the multispecific binding molecules after these chromatographic steps is >98.6% (monomeric). The multispecific binding molecules can be immediately frozen at -70°C or stored at 4°C for several months.

[0039] Exemplary TFR and L1CAM Multispecific Binding Molecules Exemplary multispecific binding molecules of the present disclosure are provided, including heterodimeric IgG antibodies. Thus, the exemplary multispecific binding molecules include a first heavy chain (HC1) and a light chain (LC1), the N-terminus of the heavy chain and the light chain comprising a blood-brain barrier target (TFR) binding domain, and further include a second heavy chain (HC2) and a light chain (LC2), the N-terminus of the second heavy chain and the light chain comprising a neuronal target (L1CAM) binding domain. Each heavy chain and light chain each comprises a variable region, a heavy chain variable region (HCVR) having three complementarity determining regions (HCDR1-3), and a light chain variable region (LCVR) having three LCDRs (LCDR1-3). The amino acid sequences of each polypeptide chain and its regions of the exemplary multispecific binding molecules are shown in Table 2.

[0040] [Table 2]

[0041] Exemplary multispecific binding molecules can be linked at the CH1 of one or both of the heavy or light chain constant regions, e.g., HC1 and HC2, or at the constant regions, e.g., LC1 and LC2 (e.g., via a thiol-reactive maleimide-functionalized linker, at an engineered cysteine, either natural or non-natural, as known in the art), to a therapeutic composition, such as a siRNA therapeutic composition (represented by an asterisk in the schematic diagram below) that targets a therapeutic target described above. An illustration of an exemplary multispecific binding molecule is provided in FIG. 2.

[0042] Binding Affinities of Exemplary Multispecific Binding Molecules The binding affinity and binding stoichiometry of exemplary multispecific binding molecules to mouse TFR, and human and mouse L1CAM are determined using surface plasmon resonance assays on a Biacore T200 instrument primed with HBS-EP+(10 mM Hepes pH 7.4+150 mM NaCl+3 mM EDTA+0.05% (w / v) surfactant P20) running buffer and the analysis temperature set at 25°C. Human Fab capture kit (Cytiva P / N 28958325) is immobilized on a CM5 chip (Cytiva P / N 29104988) using standard NHS-EDC amine coupling on all four flow cells (Fc). Multispecific binding molecules ("MBM1" in Table 2, conjugated siRNA ( * Targets (e.g., mouse TFR-mIgG1-Fc, mouse L1CAM-hIgG1-Fc, and human L1CAM-hIgG1-Fc, respectively) are prepared at final concentrations of 100.0, 25.0, 6.25, 1.56, 0.39, 0.097, 0.024, and 0 (blank) nM by dilution into running buffer.

[0043] Each analytical cycle consisted of (1) capturing the multispecific binding molecule sample onto separate flow cells (Fc2, Fc3, and Fc4); (2) injecting each concentration of the blood-brain barrier target (e.g., TFR) over the entire Fc at 10 μL / min for 60 seconds, followed by a return to buffer flow for 1800 seconds to monitor the dissociation phase; (3) injecting each human L1CAM concentration over the entire Fc at 100 μL / min for 120 seconds, followed by a return to buffer flow for 1200 seconds to monitor the dissociation phase; (4) regenerating the chip surface by injecting 10 mM glycine, pH 1.5, over the entire cell at 10 μL / min for 30 seconds; and (5) equilibrating the chip surface with a 10 μL (60 seconds) injection of HBS-EP+. Data were processed using standard double referencing and fitted to a 1:1 binding model using Biacore T200 Evaluation software, version 2.0.3, to determine the association rate (kon , M -1 s -1 units), dissociation rate (k off , s -1 units), and R max Determine the number of RUs (units). D =k off / k on From the relationship, the equilibrium dissociation constant (K D ) is calculated and presented in moles. The results are provided in Table 3.

[0044] [Table 3]

[0045] These results demonstrate that exemplary multispecific binding molecules of the invention bind with high affinity to the blood-brain barrier target TFR and the CNS target (neuronal target) L1CAM at 25°C.

[0046] Simultaneous binding of TFR and L1CAM A BIAcore T200 instrument is used to determine whether TFR and L1CAM can simultaneously bind to an exemplary multispecific binding molecule (MBM1) described in detail above. Unless otherwise stated, all reagents and materials are from Cytiva (Upsala, Sweden). All measurements are performed at 25° C. HBS-EP+ buffer (150 mM sodium chloride, 3 mM EDTA, 0.05% (w / v) surfactant P-20, and 10 mM HEPES, pH 7.4) is used as running buffer and sample buffer. A human Fab capture kit (Cytiva P / N 28958325) is immobilized on a CM5 chip (Cytiva P / N 29104988) using standard NHS-EDC amine coupling on all four flow cells (Fc). An exemplary polyspecific binding molecule (diluted to 50 μg / ml) is first captured on flow cell 2 (injected at 30 μl / min for 30 seconds, resulting in 100 response units (RU) of polyspecific binding molecule capture), followed by injection of human or mouse L1CAM at 500 nM for 5 minutes to saturate the L1CAM binding sites (a binding signal of 15 ΔRU is observed). Flow cell 1 is a blank-only control. After binding of L1CAM (flow cell 2), 500 nM of mouse TFR is then injected for 5 minutes, and an additional binding signal (8 ΔRU) is observed. The chip surface is then regenerated using 10 mM glycine pH 1.5. The same process is repeated, but the order is reversed: mouse TFR first, followed by human or mouse L1CAM. The results show an increase in response units from the two ligands binding to the multispecific binding molecule (first 10 RU from TFR, then an additional 13 RU from L1CAM), demonstrating that an exemplary multispecific binding molecule of the invention can bind to human or mouse L1CAM and mouse TFR simultaneously.

[0047] Assessment of in vitro binding, internalization, and degradation in mouse cortical neurons The fluorescent signals corresponding to the total levels and internalization of antibody and binding molecule-siRNA conjugates are measured by performing a high content live cell imaging assay in primary mouse cortical neurons. Briefly, mouse primary cortical neurons are isolated from wild type C57BL6 mouse embryos at E18. Cells are plated at a density of 40k cells / well in poly-D-lysine coated 96-well plates and cultured in NbActiv1 (BrainBits, LLC) containing 1% antibiotic / antimycotic (Corning) at 37°C for 7 days in a tissue culture incubator in a humidified chamber with 5% CO2. On day 7, media was removed from each well and 5ug / ml (33nM) of (i) hIgG4 isotype control antibody (Iso mAb), (ii) monovalent TFR antibody (with a single TFR-targeting arm and a non-specific arm (mvTFR)), (iii) MBM1 (Table 2), or the above molecule with siRNA linked at HC constant region 1 ( * 1. Incubate overnight with live cells grown at 37°C in a 96 well plate.

[0048] The next day, cells are washed and incubated with NucBlue Hoechst dye (Thermo Fisher number R37605) for 20 min, washed again, and then imaged on a Cytation5 high content imager (Biotek). The DyLight650 signal measures total antibody levels, the DyLight650+BHQ3 signal measures the degradation signal that increases DyLight 650 fluorescence when the BHQ3 dye is released and FRET quenching is lost, while the pHAb pH sensor dye signal measures only internalized fluorescence. An excess of goat gamma globulin is added to reduce non-specific binding and uptake of antibodies into the cells. The intensity of the signal in each well is divided by the number of Hoechst stained nuclei to determine the signal intensity per cell. Wells are analyzed in duplicate, and for each well, approximately 20k cells are analyzed from images taken with a 4x objective. Background signal is determined from a human IgG isotype control and subtracted from the final value. Results are provided in Table 3.

[0049] High content imaging data demonstrates that the cellular activity (binding, internalization, and degradation properties) of the exemplary multispecific binding molecule is greater than that of the monovalent TFR antibody and isotype control in neurons. In addition, when conjugated to siRNA, the activity of the exemplary multispecific binding molecule is substantially unchanged. The isotype control and the isotype control conjugated to siRNA are devoid of activity, indicating that the activity shown for the exemplary multispecific binding molecule is target receptor specific.

[0050] In vitro potency assessment in mouse cortical neurons Mouse primary cortical neurons are isolated from wild-type C57BL6 mouse embryos at E18. Cells are plated at a density of 40k cells / well in poly-D-lysine coated 96-well plates and cultured in NbActiv1 (BrainBits, LLC) containing 1% antibiotic / antimycotic (Corning) in a tissue culture incubator in a humidified chamber with 5% CO2 for 7 days at 37°C. On day 7, half of the medium is removed from each well and incubated with (i) siRNA (targeting the desired therapeutic target in the CNS); (ii) control siRNA (naked siRNA not targeting a therapeutic target in the CNS); (iii) hIgG4 isotype control antibody with siRNA linked to HC constant region 1 (Iso mAb * ), (iv) monovalent TfR with cholesterol-conjugated siRNA in HC constant region 1 (mvTFR * ), or (v) MBM1 with cholesterol-conjugated siRNA in HC constant region 1 (Table 2) (MBM1 * ) in culture medium with 2% FBS is added for treatment at a range of siRNA concentrations and incubated with cells for another 7 days. At the end of siRNA treatment, RT-qPCR is performed to quantify targeted mRNA levels using a TaqMan Fast Advanced Cell-to-CT kit. Specifically, cells are lysed, cDNA is generated on a Mastercycler X50a (Eppendorf), and qPCR is performed on a QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems). Gene expression levels of therapeutic targets are normalized by β-actin (ThermoFisher Mm02619580_g1) using the respective probes. The results are provided in Table 4.

[0051] [Table 4]

[0052] The results provided in Table 4 demonstrate that the exemplary multispecific binding molecules provide potency for linked siRNAs targeting therapeutic targets that is several orders of magnitude greater than unconjugated (e.g., naked siRNAs) and an order of magnitude greater than siRNAs targeting therapeutic targets linked only to mvTFR.

[0053] Brain pharmacokinetic evaluation Brain pharmacokinetics of exemplary multispecific binding molecules of the present disclosure may be evaluated as follows: Briefly, 8-week-old male FVB mice are dosed with (i) Iso mAb, (ii) mvTFR, (iii) L1CAM antibody (L1CAM mAb), and (iv) MBM1 at 5 mg / kg and sacrificed and perfused at 0.5, 2, 6, 24, 48, 72, 168, 336, and 504 hours (n=3 per time point). Levels of each molecule in the brain are determined by ELISA (assessed by area under the curve (AUC)). Following the procedures described herein, Iso mAb and L1CAM mAb demonstrate low levels in the brain. mvTFR demonstrates brain accumulation that achieves a peak Cmax of approximately 1000 ng / g between 24 and 48 hours and is cleared by 168 hours. MBM1 demonstrates a higher Cmax than mvTFR between 24 and 48 hours and demonstrates delayed elimination from the brain with 500 ng / ml detected at 168 hours and over 100 ug detected at 336 hours. Results are provided in Tables 4A and 4B.

[0054] The results suggest that the combination of a vascular barrier target binding domain and a CNS target (neuronal) binding domain demonstrates enhanced molecular brain PK over either the mvTFR or L1CAM targeted molecules alone.

[0055] In vivo pharmacodynamic evaluation The pharmacodynamic properties of exemplary multispecific binding molecules of the present disclosure may be assessed according to the following. Briefly, Iso mAb * , mvTFR * , and MBM1 *is administered intravenously to 8-week-old FVB mice at 10 mg / kg effective siRNA concentration once a week for 4 weeks. In addition, a mouse anti-CD4 antibody (GK1.5) is administered at 10 mg / kg to deplete CD4 positive T cells and mitigate undesirable pharmacokinetic consequences resulting from spurious anti-drug antibody responses to the injected compound. Mice are sacrificed 168 hours after the last dose and perfused to harvest brains and spinal cords to assess target mRNA levels by RT-qPCR and target protein levels by ELISA in tissue homogenates. Results are provided in Figure 5A-5E.

[0056] Results demonstrate significant improvement in mRNA and protein levels over PBS and Iso mAb (Note: MBM1 * The reduction would likely be further enhanced in higher L1CAM populations).

[0057] In vivo assessment of brain accumulation, peripheral tissue accumulation, and CSF and plasma accumulation Brain and peripheral accumulation of exemplary multispecific binding molecules of the present disclosure can be assessed as follows. Briefly, following the pharmacodynamic studies described above, brain tissue, CSF, plasma, and peripheral tissues are collected and levels of each administered molecule are determined by ELISA. Results are provided in Figures 6A-6J.

[0058] The results showed that MBM1 in the brain * The level of Iso mAb * Approximately 200% higher than mvTFR * The results also demonstrate that the MBM1 * The results demonstrate that only MBM1 levels are undetectable in plasma, CSF, and all peripheral organs (bone marrow, stomach muscle, liver, kidney, heart, spleen, and testis). * Iso mAb * and mvTFR less than 10% * The results demonstrated that MBM1 * achieves increased brain presence but not peripheral accumulation, including bone marrow accumulation.

[0059] array SEQ ID NO:1 - Exemplary anti-L1CAM HC EVQLVESGGGVVQPGRSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCARGRAYGSGSLFDPW GQGTLVTVSSASTKGPCVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO:2 - Exemplary anti-L1CAM LC DIQMTQSPSSLSASVGDRVTITCKASQGISRFLSWFQQKPGKAPKSLIYAVSSLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQYNSYPYGFGGGTKVEIK RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:3 - Exemplary anti-TFR HC QSLEESGGDLVKPEGSLTLTCTASGFSFSGSYWICWVRQAPGKGLEWIGCIYSTSGGRTYYASWVKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGDDSISDAYFDLW GPGTLVTVSSASTKGPCVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKY GPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLG SEQ ID NO:4 - Exemplary anti-TFR LC ALDMTQTASPVSAAVGGTVTINCQSSQSVYNNNRLAWYQQKPGQPPKLLIYDASTLASGVPSRFKGSGSGTQFTLTISGVQSDDSATYYCQGTYFSSGWSWAFGGGTEV VVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO:5 - Exemplary anti-TFR HCVR QSVEESGGRLVTPGTPLTLTCTVSGFSLSTYAMIWVRQAPGKGLEYIGFIDNDYKAFYATWTNGRFTVSRTSTTVDLKMTSLTTEDTATYFCARYGSSSASDLWGQGTLVTVSS SEQ ID NO:6 - Exemplary anti-TFR LCVR ALVMTQTPASVSAAVGDTVTIKCQASESIITWLAWYQQRPGQPPKLLIYRASTLASGVPSRFKGSGSGTQFTLTISDLEAADAAIYYCQNNAGDSSYGFAFGGGTEVVVK SEQ ID NO:7 - Exemplary anti-TFR HCVR QSVEESGGRLVTPGTPLTLTCTVSGIDLSGSAMSWVRQAPGKGLEWIGIIYARGGTYYATWAQGRFTISKTSTTVDLKITSPTIEDTATYFCSRGYTDGFDLWGQGTLVTVSS SEQ ID NO:8 - Exemplary anti-TFR LCVR ALVMTQTPSPVSAAVGGTVSINCQSTKSIYNNKYLSWYQQKPGQPPKLLIYDASDLASGVPSRFKGSGSGTQFTLTISGVQADDAATYYCLGGYSSDSENAFGGGTEVVVK SEQ ID NO:9 - Exemplary anti-TFR HCVR ALVMTQTPASVSEPVGGTVTIKCQASQSISSWLAWYQQKPGQPPKLLIYRASTLASGVSSRFKGSGSGTDFTLTISDLEAADAATYYCQTSGAMGTYGGAFGGGTEVVVK SEQ ID NO:10 - Exemplary anti-TFR LCVR QSVEESGGRLVTPGTPLTLTCTASGFSLSSYYMSWVRQAPGKGLEWIGFIYTDGSTYYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCARYSGSGLDLWGLGTLVTVSS SEQ ID NO:11 - Exemplary anti-CD98 HCVR QSLEESGGDLVKPGASMTLTCTASGFSFSSGYWICWVRQAPGKGLEWIACIHSVRSHMTYYASWAKGRFTISKTSSTTVTLQMTSLTAAATATYFCARDASGVWNYFTLWGPGTLVTVSS SEQ ID NO:12 - Exemplary anti-CD98 LCVR AEVVMTQTPSSVSAAVGGTVTIKCQASQNINSWLSWYQQKPGQRPKLLIYSASTLASGVPSRFEGSGSGTEYTLTISDLECDDAATYYCQSSYGSSYDFGGGTEVVVK SEQ ID NO:13 - Exemplary anti-CD98 HCVR QEQLVESGGGLVQPEGALTLTCTASGLDFSSSYWICWVRQAPGKGLEWIACVHAGSGGYNYYATWAKGRFTISRTSSTTVTLQMTSLTAADTATYFCARGVFPDYVDATLFNLWGPGTLVTVSS SEQ ID NO:14 - Exemplary anti-CD98 LCVR AAVMTQTASPVSAAVGGTVTINCQASQSVSSAYLSWYQQKPGQPPKLLIYKASTLASGVSSRFKGSGSGTEYTLTISGVQCDDAATYYCLYGDYSGRSNAFGGGTEVVVK SEQ ID NO:15 - Exemplary anti-CD98 HCVR QEHMEESGGDLVKPEGSLTLTCTASGFSFSRMYWICWVRQAPGKGLEWIACIYTGDGNTYYASWAKGRFTISKTSSSTVTLQMTSLTAADTATYFCARDPDGYSIYYFNLWGPGTLVTVSS SEQ ID NO:16 - Exemplary anti-CD98 LCVR ALVMTQTPASVSTAVGGTVTISCQASQSINSWLAWYQQKPGQRPNLLIYGASKLPSGVPSRFKGSGSGTEFTLTISDLECSDAATYYCAGYKTYSNDDNAFGGGTEVVVK SEQ ID NO:17 - Exemplary anti-CD98 HCVR QQLVESGGGLVQPGASLTLTCTASGFSFSSSYWICWVRQAPGKGLEWSACIYGGIPYYASWAKGRFTISKTSSTTVTLQMTSLTVADTATYFCARDDYYKSAWGGYNLWGPGTLVTVSS SEQ ID NO:18 - Exemplary anti-CD98 LCVR AAVMTQTPSPVSAAVGGTVTISCQASQSVYGNNYFAWFQQKPGQPPKLLIYKASTLASGVPSRFKGSGSGTQFTLTISGVECDDAATYYCAGYKSYSNDGYAFGGGTEVVVK SEQ ID NO:19 - Exemplary Anti-L1CAM HCVR EVQLVESGGGVVQPGRSLRLSCAASGFTFSRFGMHWVRQAPGKGLEWVAFISNDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCARGRAYGSGSLFDPWGQGTLVTVSS SEQ ID NO:20 - Exemplary anti-L1CAM LCVR DIQMTQSPSSLSASVGDRVTITCKASQGISRFLSWFQQKPGKAPKSLIYAVSSLVDGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCVQYNSYPYGFGGGTKVEIK SEQ ID NO:21 - Exemplary anti-TFR HCVR QSLEESGGDLVKPEGSLTLTCTASGFSFSGSYWICWVRQAPGKGLEWIGCIYSTSGGRTYYASWVKGRFTISKTSSTTVTLQMTSLTAADTATYFCARGDDSISDAYFDLWGPGTLVTVSS SEQ ID NO:22 - Exemplary anti-TFR LCVR ALDMTQTASPVSAAVGGTVTINCQSSQSVYNNNRLAWYQQKPGQPPKLLIYDASTLASGVPSRFKGSGSGTQFTLTISGVQSDDSATYYCQGTYFSSGWSWAFGGGTEVVVK

Claims

1. 1. A multispecific binding molecule comprising: a first binding domain that specifically binds to a blood-brain barrier target; and a second binding domain that specifically binds to at least one of a neuronal target, an astrocytic target, and a glial cell target.

2. 2. The polyspecific binding molecule of claim 1, wherein the blood-brain barrier target is one of TFR, low density lipoprotein receptor-related protein 1 (LRP1), LRP2 or megalin / GP330, LRP3, LRP5, LRP6, LRP8, insulin-like growth factor (IGF), transferrin receptor-1, CD98 heavy chain (SLC3A2), large amino acid transporter small subunit 1 (LAT-1), insulin receptor, low density lipoprotein (LDL) receptor, CDC50A.

3. 3. The multispecific binding molecule of claim 1 or 2, wherein the second binding domain specifically binds to a neuronal target, the neuronal target being one of L1CAM, THY1, KIT, GRIA1, GRM2, TMEM130, NSG1, BASP1, NSTR1, SLC22A17, NCAM1, NCAM2, SLC6A17, SCN3B, and P2Y12R.

4. 3. The multispecific binding molecule of claim 1 or 2, wherein the second binding domain specifically binds to an astrocytic target, and the astrocytic target is one of CD49f, AQP4, GLT1, GLAST, A2AR, TGFbR, S1P1, IL1R<IL6R, IL17R, and IFNgR.

5. 3. The multispecific binding molecule of claim 1 or 2, wherein the second binding domain specifically binds to a microglial target, the microglial target being one of TREM2, TMEM119, CD11b, CD14, CD16, CD32, CD33, CD40, CD45, CD64, CD68, CD80, CD115, CD172a, CX3CR1, FCER1G, F4 / 80, FCRLS, Siglec, Glut5, and P2Y12.

6. The multispecific binding molecule of any one of claims 1 to 5, wherein the multispecific binding molecule is a heterodimeric immunoglobulin G antibody.

7. The multispecific binding molecule of any one of claims 1 to 5, wherein the first binding domain is a Fab, scFv, Fv, or scFab.

8. The multispecific binding molecule of any one of claims 1 to 5, wherein the second binding domain is a Fab, scFv, Fv, or scFab.

9. 9. The multispecific binding molecule of claim 7 or 8, wherein the first binding domain and the second binding domain are linked by one of a thiol-based reactive group, an amine-based reactive group, an aldehyde-reactive group, or a carbohydrate-based group.

10. 9. The multispecific binding molecule of claim 7 or 8, wherein the first binding domain and the second binding domain are linked by one of a PEG linker, an amino acid linker, and a glycan linker.

11. The multispecific binding molecule of any one of claims 1 to 10, wherein the blood-brain barrier target is TFR and the neuronal target is L1CAM.

12. The multispecific binding molecule of any one of claims 1 to 11, wherein a therapeutic composition is linked to the multispecific binding molecule.

13. The multispecific binding molecule of claim 12, wherein the therapeutic composition is one of a peptide, an antibody or fragment thereof, or an oligonucleotide.

14. 14. The multispecific binding molecule of claim 13, wherein the therapeutic composition is an oligonucleotide targeting one of MAPT, SNCA, APP, BACl, ATXN2, ATXN3, SARM1, APOE, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B.

15. 14. The multispecific binding molecule of claim 13, wherein the therapeutic composition is an antibody or fragment thereof that binds to one of amyloid beta, BACE1, MAPT, SNCA, APP, BACl, ATXN2, ATXN3, SARM1, APOE, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B.

16. 1. A method for delivering a therapeutic composition to the central nervous system of a mammal, comprising administering said therapeutic composition to said mammal; The therapeutic composition is linked to a multispecific binding molecule according to any one of claims 1 to 15, The method, wherein accumulation of the therapeutic composition in peripheral tissues of a mammal is reduced compared to the therapeutic composition when not linked to said multispecific binding molecule.

17. 17. The method of claim 16, wherein accumulation of the therapeutic composition in peripheral tissues is assessed at least 24 hours, 48 ​​hours, 72 hours, 96 hours, 120 hours, or 144 hours after the administering step.

18. 18. The method of claim 16 or 17, wherein the peripheral tissue is selected from one of bone marrow, liver, plasma, testis, muscle, or kidney.

19. 19. The method of any one of claims 16 to 18, wherein the administering step comprises intravenous, subcutaneous, intramuscular, or parental administration of the multispecific binding molecule.

20. 16. A method of delivering a therapeutic composition to a therapeutic target within the central nervous system of a mammal, comprising administering said therapeutic composition to a mammal, said therapeutic composition being linked to a multispecific binding protein of any one of claims 1 to 15, and said administering step comprising intravenous, subcutaneous, intramuscular or parenteral administration of said multispecific binding molecule.

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