Multispecific binding molecules and methods of use thereof

Abstract

Multi-specific binding molecules having a first binding domain targeting a blood-brain barrier target and a second binding domain targeting a neuronal, astrocyte and / or glial cell target, and their use in aiding the treatment of central nervous system diseases, including neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative diseases, neurodegenerative The present invention relates to compounds of formula (I), such as Alzheimer's disease, Huntington's disease, Parkinson's disease, progressive supranuclear paralysis (PSP), amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), autism, stiff, encephalitis, migraine, and Tourette's syndrome.

Description

[0001] Cross-reference to related applications This patent application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 338,480, filed on May 5, 2022, which is hereby incorporated by reference in its entirety.

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

[0003] The present invention belongs to the field of medicine, in particular to the field of treatment of central nervous system (CNS) diseases. More specifically, the present disclosure relates to compositions comprising multispecific binding molecules and their uses in the treatment of CNS diseases, wherein the multispecific binding molecules have 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, a CNS target). The multispecific binding molecules of the present disclosure help treat CNS diseases by enhancing the passage of therapeutics across the blood-brain barrier and delivering them to the intended CNS target. Background Art

[0004] The CNS is arguably the most complex and highly organized organ system in the human body. The CNS consists of two major organs, the brain and the spinal cord, and is composed of glial cells and neurons. In vertebrates such as humans, neurons are divided into three categories: sensory neurons and motor neurons, parts of which may span both the CNS and the peripheral nervous system, as well as interneurons that are considered to be almost entirely within the CNS.

[0005] Diseases of the CNS are disorders that affect the structure and / or function of the brain and / or spinal cord. CNS diseases or disorders may include autoimmune disorders, including neuromyelitis optica, multiple sclerosis, and anti-myelin oligodendrocyte glycoprotein; oncological disorders, including astrocytomas, oligodendrogliomas, glioblastomas, 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 (Frontal Temporal Dementia) (FTD); and other CNS disorders, such as autism, catalepsy, encephalitis, migraine, and Tourette's syndrome.

[0006] Traditionally, CNS diseases have proven to be 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 special semi-permeable boundary of endothelial cells that selectively prevents solutes (including pathogens) from entering the CNS. The blood-brain barrier allows some molecules to pass through via passive diffusion and participates in the selective, active transport of some molecules that are critical to neural function. In addition to the challenges caused 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 have estimated that less than 1% of some therapeutic agents cross the blood-brain barrier.

[0007] Blood-brain barrier shuttling mechanisms or molecular chaperones for improving the passage of therapeutic agents across the blood-brain barrier and into the CNS have been described for more than two decades. For example, WO2003 / 009815 (US2003 / 0129186) describes the use of antibodies against the transferrin receptor ("TFR") for modulating blood-brain barrier transport. However, attempts to use anti-TFR antibodies as conjugates to shuttle therapeutics across the blood-brain barrier have proven both challenging and insufficient to significantly improve the treatment of CNS diseases (see, e.g., Syvanen S, Hultqvist G, Gustavsson T, et al., Efficient clearance of Abetaprotofibrils 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 pharmacokinetic, toxicity, and off-target issues associated with targeting TfR as a blood-brain barrier shuttling mechanism).

[0008] Therefore, there is still a need for enhancing the treatment of CNS diseases that overcome the above challenges. More particularly, there is a need for improving the delivery of therapeutic agents across the blood-brain barrier and providing enhanced selective targeting of CNS targets in the CNS. This enhancement must be able to deliver therapeutic agents across the blood-brain barrier with increased efficiency, and improve the delivery of specific CNS targets in the CNS, so that the therapeutic agent shuttled is delivered to its expected therapeutic target in the CNS. This enhancement should not be more invasive than therapeutic molecules, so that the peripheral systemic delivery of therapeutic agents can be achieved to overcome the need for the direct invasive delivery of therapeutic agents to the CNS, should not be accompanied by unacceptable immunogenicity and off-target problems, and should show acceptable stability and developability. This enhancement should not interfere with the properties of the therapeutic agents necessary for the treatment of CNS diseases.

[0009] The present disclosure provides multispecific binding molecules for enhancing the treatment of CNS diseases. More particularly, 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 connected to a therapeutic composition for assisting the treatment of CNS diseases by enhancing the passage of therapeutic agents through the blood-brain barrier and delivery to specific CNS targets (e.g., neuronal targets, astrocyte targets and / or glial cell targets) within the CNS, thereby regulating its intended therapeutic target with the therapeutic composition. Summary of the invention

[0010] Embodiments of the present disclosure include multispecific binding molecules comprising a first binding domain and a second binding domain, wherein 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 astrocyte 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, 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. 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 specific embodiments, the blood-brain barrier target is TFR, and the neuronal target is L1CAM.

[0011] In some embodiments, the multispecific binding molecules of the present disclosure include bispecific antibodies, heterodimeric immunoglobulin antibodies or fragments thereof. In addition, in some embodiments of the multispecific binding molecules of the present disclosure, the first binding domain is Fab, scFv, Fv or scFab. According to some embodiments, the second binding domain is Fab, scFv, Fv or scFab. In addition, in some embodiments, the first binding domain and the second binding domain are connected by one of a thiol-based reactive group, an amine-based reactive group, an aldehyde-based reactive group or a carbohydrate-based group. In other embodiments, the first binding domain and the second binding domain are connected by one of a PEG-linker, an amino acid linker and a polysaccharide linker.

[0012] In further embodiments of the multispecific binding molecules of the present disclosure, the 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] Furthermore, in some embodiments, the multispecific binding molecules of the present 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 pharmaceutically 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 the multispecific binding molecules of the present disclosure.

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

[0017] According to some embodiments, the present disclosure provides a method for delivering a therapeutic composition to a CNS target within 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, wherein the therapeutic composition exhibits reduced accumulation in the patient's peripheral tissues. In some such embodiments, the therapeutic composition exhibits reduced accumulation in the patient's bone marrow. According to some embodiments, the CNS target is one of a neuronal target, an astrocyte target, and a glial target. According to some embodiments, the CNS disease is a neurodegenerative disorder. According to some embodiments, the CNS disease is Alzheimer's disease, Huntington's disease, Parkinson's disease, progressive supranuclear palsy (PSP), frontotemporal dementia (FTD), autism, catalepsy, encephalitis, migraine, and Tourette syndrome.

[0018] Embodiments of the present disclosure include multispecific binding molecules of the present disclosure for use in therapy. Embodiments also include multispecific binding molecules of the present disclosure for use in the treatment of 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 syndrome. Further, the present disclosure provides, in some embodiments, multispecific binding molecules of the present disclosure for use in the preparation of medicaments for the treatment of CNS diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a simplified diagram of a multispecific binding molecule of the present disclosure in the form of a heterodimeric IgG antibody.

[0020] Figure 2 is a simplified 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 the neuronal target L1CAM.

[0021] Figure 3 High content imaging data showing the cellular activities (binding, internalization and degradation properties) of exemplary multispecific binding molecules of the present disclosure are shown.

[0022] Figure 4A and 4B Brain pharmacokinetic data for exemplary multispecific binding molecules of the present disclosure are shown.

[0023] Figures 5A to 5E In vivo pharmacokinetic data for exemplary multispecific binding molecules of the present disclosure are shown.

[0024] Figures 6A to 6J In vivo brain and peripheral accumulation of exemplary multispecific binding molecules of the disclosure are shown. DETAILED DESCRIPTION

[0025] The term "multispecific" as used herein refers to a molecule or compound that comprises two or more different binding domains. As described herein, a multispecific binding molecule can bind to two or more different targets; a blood-brain barrier target and a CNS target. Embodiments of the multispecific binding molecules of the present disclosure may include bispecific antibodies and / or fragments of antibodies, such as Fab, scFv, Fv or scFab, wherein portions of the antibody or fragment thereof form 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. Embodiments of the multispecific binding molecules include two scFvs linked together, for example via a peptide linker (e.g., a GS linker), wherein one scFv forms a binding domain for a blood-brain barrier target and the other scFv forms a binding domain for a CNS target. The multispecific binding molecules of the present disclosure also include VHHs linked to one or more fragments of an antibody. Other multispecific binding molecules of the present disclosure include heterodimeric immunoglobulin G (IgG) antibodies, such as Figure 1 As exemplified in , the amino terminus (N-terminus) of each "arm" comprising the 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 a blood-brain barrier target, while the binding domain of the other "arm" specifically binds a CNS target).

[0026] The term "binding domain" refers to a binding target of a multispecific binding molecule of the present disclosure (e.g., a BBB target or a CNS target, such as Figure 1 For example, a binding domain of a multispecific binding molecule can bind to a blood-brain barrier target, such as TFR. Likewise, different binding domains of a multispecific binding molecule of the present disclosure can bind to a CNS target, such as L1CAM. Unless otherwise indicated, the terms "bind" and "binds" as used herein are intended to mean the ability of a binding domain of a multispecific binding molecule to form a chemical bond or attractive interaction with another protein or molecule that results in the proximity of the two proteins or molecules, as determined by common methods known in the art.

[0027] As used herein, a blood-brain barrier target refers to a membrane receptor that is present on the apical surface of the endothelial blood-brain barrier and that actively aids, facilitates, and / or promotes the internalization into the CNS of molecules outside the CNS (e.g., in serum). According to the present disclosure, blood-brain barrier targets 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 neutral amino acid transporter subunit 1 (LAT-1), insulin receptor, low density lipoprotein (LDL) receptor, CDC50A, and the like.

[0028] As used herein, a CNS target refers to an antigen present on the outer membrane of one of neurons, astrocytes, and / or glial cells and other parenchymal antigens in the CNS. The multispecific binding molecules of the present disclosure comprise binding domains that target CNS targets, thereby 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 include single transmembrane receptors, multipass membrane protein receptors, and cell surface proteoglycans, including but 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 microglia include single transmembrane receptors, multipass membrane protein receptors, and cell surface proteoglycans, including but 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 include single transmembrane receptors, multipass membrane protein receptors, and cell surface proteoglycans, including but not limited to CD49f, AQP4, GLT1, GLAST, A2AR, TGFbR, S1P1, IL1R<IL6R, IL17R, and IFNgR.

[0029] The region of the target that the binding domain of the multispecific binding molecule binds to can be a linear region or a conformational region of the target. The region of the target that the binding domain of the multispecific binding molecule binds to can be determined according to different experimental techniques. It is understood that the determination of the target region bound by the multispecific binding molecule may vary based on the different mapping techniques used, and may also vary with the different experimental conditions used, for example, due to conformational changes or cracking of the target induced by specific experimental conditions. Binding location techniques are known in the art (e.g., Rockberg & Nilvebrant, Epitope Mapping Protocols: Methods in Molecular Biology, Humana Press, 3rd edition. 2018), including but not limited to, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, site-directed mutagenesis, species swap mutagenesis, alanine partitioning mutagenesis, hydrogen-deuterium exchange (HDX) and cross-blocking assays.

[0030] The term "specific for" as used herein in relation to a binding domain refers to the ability of a binding domain of a multispecific binding molecule of the present disclosure to bind to, associate with, and / or modulate a specific target molecule above background levels of non-target levels. In some embodiments, "specific for" can be demonstrated by measuring affinity (Kd) for a target compared to a non-target.

[0031] As used herein, the terms "regulate" or "modulate" and the like refer to altering or changing a measurable value and include altering or changing such a measurable value upward (ie, upregulating or upmodulating) or downward (ie, downregulating or downmodulating).

[0032] The multispecific binding molecules of the present disclosure can 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 will also be appreciated that the multispecific binding molecules or therapeutic agents can be fully or partially encapsulated by lipid nanoparticles or exosomes). Exemplary therapeutic oligonucleotides include antisense oligonucleotides, RNA interference, RNA activation, and guide RNA-directed target RNA editing using systems such as CRISPR-Cas9 and ARCUS nuclease systems. Exemplary therapeutic oligonucleotides, antibodies or fragments thereof and / or peptides that are known in the art and directed against therapeutic target genes or proteins within the CNS associated with proteins involved in diseases of the CNS (i.e., "therapeutic targets" other than blood-brain barrier targets and CNS targets), such as β-amyloid, BACE1, MAPT, SNCA, ATXN2, ATXN3, SARM1, APOE, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, CACNA1B, etc., whose modulation is intended to cause a therapeutic effect, can be linked to the multispecific binding molecules of the present disclosure.

[0033] Furthermore, therapeutic compositions can be linked to the multispecific binding molecules of the disclosure in a variety of ways and at various locations or portions of the multispecific binding molecules such that such linkage does not interfere with the binding of the multispecific binding molecules to the blood-brain barrier and CNS targets and does not interfere with the therapeutic properties of the therapeutic composition when linked.

[0034] As used herein, the term linker refers to an atom, a group of atoms, a molecule or a compound (e.g., an amino acid or amino acid group) that contains at least one bond that attaches or links one molecule or compound to another molecule or compound. The linker can contain various compositions, including polyethylene glycol (PEG) units, straight, branched and aromatic carbonyl units, amino acids and polysaccharides. According to the present disclosure, the linker can also include a hydrolyzable and / or enzymatically degradable linker. The linker can also attach multiple therapeutic compositions to the multispecific binding molecules of the present disclosure.

[0035] The terms "linked" and "conjugated" as used interchangeably herein refer to a first molecule or compound, such as a polypeptide, that is associated, attached, connected or otherwise bound to a second molecule or compound, such as a nucleotide (or polynucleotide) or a polypeptide. For example, a polypeptide can be linked to a polynucleotide sequence. Similarly, a peptide sequence can 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 terms "conjugated peptide" or "peptide conjugate" as used herein refer to a peptide or protein that is covalently (including reversibly covalently) or non-covalently linked to another molecule, moiety, compound or group.

[0036] Specific embodiments of the present disclosure include heterodimeric IgG multispecific binding molecules, wherein one arm specifically binds to a blood-brain barrier target, such as TFR-1, and the other arm specifically binds to a CNS target, such as a neuronal target, such as L1CAM, and the IgG multispecific binding molecule is connected to a siRNA therapeutic composition, for example, via a chemically functionalized linker that provides covalent attachment to the multispecific binding molecule scaffold. Examples of chemically functionalized linkers include thiol-based reactive groups (e.g., maleimide, sulfhydryl, haloacetyls, thiosulfonates, disulfides), amine-based groups (e.g., N-hydroxysuccinimide esters, imidoesters, etc.), and aldehyde-based reactive groups (e.g., hydrazides and alkoxyamines), as well as carbohydrate-based groups (e.g., natural Fc glycans, artificially engineered glycans, etc.).

[0037] As used herein, the term "amino acid having a functional group available for conjugation" refers to any natural or non-natural amino acid or derivative thereof having a functional group that can be conjugated to a conjugated moiety directly or through, for example, a conjugation linker. Examples of such functional groups include, but are not limited to, alkynyl, alkenyl, amino, azido, bromo, carboxyl, chloro, iodo, and thiol. Likewise, examples of natural amino acids containing such functional groups include C (thiol), D (carboxyl), E (carboxyl), K (amino), and Q (amide).

[0038] The multispecific binding molecules of the present disclosure can be used to aid in the treatment of patients, in particular to aid in the treatment of CNS diseases, including neurodegeneration, oncology and autoimmune disorders of the CNS. "Treatment" and / or "treating" and / or "treat" when used in relation to the multispecific binding molecules of the present disclosure refers to the use of the multispecific binding molecules of the present disclosure with a therapeutic composition (e.g., a therapeutic composition linked or conjugated to the multispecific binding molecule), whereby the multispecific binding molecule aids in the delivery of the therapeutic composition across the blood-brain barrier and to the CNS target, whereby the therapeutic composition is able to modulate its intended therapeutic target, thereby completely or partially: slowing, interrupting, preventing, controlling, stopping or reversing the progression of the CNS disorder, but does not necessarily indicate complete elimination of all disease symptoms. Treatment includes the administration of a multispecific binding molecule of the present disclosure, comprising a therapeutic composition linked or conjugated thereto, for the treatment of a CNS disease or condition in a human, which disease or condition would benefit from the delivery of the therapeutic composition across the blood-brain barrier and to the CNS target to aid the therapeutic composition in causing the desired therapeutic effect.

[0039] As used interchangeably herein, the terms "patient," "subject," and "individual" refer to a human. In certain embodiments, the patient is further characterized as suffering from 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 of developing a CNS disorder, disease, or condition.

[0040] Embodiments of multispecific binding molecules of the present disclosure include antibodies or fragments thereof. As used herein, "antibodies" include immunoglobulins or immunoglobulin-like molecules that bind to an antigen and comprise four polypeptide chains: two heavy chains (HC) and two light chains (LC), which are cross-linked via interchain disulfide bonds (as exemplified above). Each HC comprises a heavy chain variable region (VH) and a heavy chain constant region. Each LC comprises a light chain variable region (VL) and a light chain constant region. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions, called framework regions (FRs). CDRs are exposed on the surface of the protein and are important regions of the antigen binding specificity of the antibody. Each VH and VL consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: 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 antigens.The assignment of amino acid residues to CDRs can be performed according to well-known schemes, including 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, 274, 937-951 (1998)), and Chothia et al., "Canonical structures for the hypervariable regions of immunoglobulins," Journal of Molecular Biology, 276, 937-951 (1998)). Biology, 406, 228-256 (2011) or those described in IMGT (International ImMunoGeneTics database available at www.imgt.org; see Lefranc et al., Nucleic Acids Res. 1999; 27: 209-212). As used herein, exemplary embodiments of antibody fragments or antigen-binding fragments 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 Fab, Fab', F(ab')2, Fv fragments, scFv antibody fragments, scFab, disulfide-linked Fvs (sdFv), Fd fragments.

[0041] Example Exemplary multispecific binding molecules The exemplified multispecific binding molecules of the present disclosure comprise a first binding domain specific for a blood-brain barrier target and a second binding domain specific for a CNS target. According to the specific exemplified multispecific binding molecules of the present disclosure, the first binding domain binds to TFR and the second binding domain binds to L1CAM. The exemplified binding domains of the multispecific binding molecules of the present disclosure comprise a heavy chain variable region (HCVR) and a light chain variable region (LCVR), each of which comprises three complementarity determining regions (HCDRs 1-3 and LCDRs 1-3, respectively). The exemplified binding domains are set forth in Table 1.

[0042] Table 1: Exemplary binding domain regions Expression of Exemplary Multispecific Binding Molecules Exemplary multispecific binding molecules can be expressed and purified essentially as follows. A glutamine synthetase (GS) expression vector (or vectors) containing DNA encoding a polypeptide chain comprising HCVR and LCVR (or, for example, HC and LC) or fragments thereof comprising each binding domain of a multispecific binding molecule of the present disclosure is used to transfect the 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 for biochemical synthesis of glutamine, a required amino acid for CHO cells. Following transfection, the cells are subjected to bulk selection using 50 μM L-methionine sulfoximine (MSX). Inhibition of GS by MSX is used to increase the stringency of the selection. Cells in which the expression vector cDNA is integrated into a transcriptionally active region of the host cell genome can be selected against CHO wild-type cells that express endogenous levels of GS. The transfected library is plated at low density to allow close-to-clonal outgrowth of stably expressing cells. The masterwells are screened for expression of the multispecific binding molecules and then scaled up in serum-free suspension cultures to be used for production. The clarified culture medium into which the exemplified multispecific binding molecules have been secreted is applied to a protein A affinity column that has been equilibrated with a compatible buffer such as phosphate buffered saline (pH 7.4). The column is washed to remove nonspecific binding components. The bound multispecific binding molecules are eluted, for example, by a pH gradient (e.g., 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 multispecific binding molecule fractions are detected, for example, by SDS-PAGE or analytical size exclusion, and then combined. Soluble aggregates and polymers can be effectively removed by common techniques, including size exclusion, hydrophobic interactions, ion exchange, or hydroxyapatite chromatography. The multispecific binding molecules are concentrated and / or sterile filtered using conventional techniques. The purity of the multispecific binding molecules after these chromatography steps is greater than 98.6% (monomeric). The multispecific binding molecules can be frozen immediately at -70°C or stored at 4°C for several months.

[0043] Exemplary TFR and L1CAM multispecific binding molecules Provided are exemplary multispecific binding molecules of the present disclosure, including heterodimeric IgG antibodies. Thus, the exemplary multispecific binding molecules comprise a first heavy chain (HC1) and a light chain (LC1), wherein the N-termini of the heavy chain and the light chain comprise a blood-brain barrier target TFR binding domain, and further comprise a second heavy chain (HC2) and a light chain (LC2), wherein the N-termini of the second heavy chain and the light chain comprise a neuronal target L1CAM binding domain. Each heavy chain and light chain comprises a variable region, which is a heavy chain variable region (HCVR) having three complementarity determining regions (HCDRs 1-3) and a light chain variable region (LCVR) having three LCDRs (LCDRs 1-3). Table 2 sets forth the amino acid sequences of each polypeptide chain and its region of the exemplary multispecific binding molecules.

[0044] Table 2: Amino acid sequences constituting an exemplary multispecific binding molecule ("MBM1"). area SEQ ID NO Heavy chain 1 (HC1) SEQ ID NO: 3 Heavy chain variable region (HCVR1) SEQ ID NO: 21 Light chain 1 (LC1) SEQ ID NO: 4 Light chain variable region (LCVR1) SEQ ID NO: 22 Heavy chain 2 (HC2) SEQ ID NO: 1 Heavy chain variable region (HCVR2) SEQ ID NO: 19 Light chain 2 (LC2) SEQ ID NO: 2 Light chain variable region (LCVR2) SEQ ID NO: 20 Exemplary multispecific binding molecules can be linked at the heavy or light chain constant region, e.g., CH1 of one or both of HC1 and HC2, or e.g., the constant region of one or both of LC1 and LC2 (e.g., at a cysteine, whether natural or non-naturally engineered as known in the art, via a thiol-reactive maleimide-functionalized linker) to a therapeutic composition, e.g., an siRNA therapeutic composition targeted to a therapeutic target as described above (indicated by an asterisk in the schematic below). Figure 2 Illustrations of exemplary multispecific binding molecules are provided in .

[0045] Binding Affinity of Exemplary Multispecific Binding Molecules Surface plasmon resonance measurements were used to determine the binding affinity and binding stoichiometry of the exemplified multispecific binding molecules to mouse TFR and human and mouse L1CAM on a Biacore T200 instrument prepared 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 assay temperature was set to 25°C. Human Fab capture kits (Cytiva P / N 28958325) were immobilized on CM5 chips (Cytiva P / N 29104988) using standard NHS-EDC amine coupling on all four flow cells (Fc). Multispecific binding molecules ("MBM1" in Table 2, with or without conjugated siRNA as described above (indicated by *)) were prepared at 10 μg / mL by diluting into running buffer. Targets (e.g., mouse TFR-mIgG1-Fc, mouse L1CAM-hlgG1-Fc, and human L1CAM-hlgG1-Fc, respectively) were prepared by dilution into running buffer at final concentrations of 100.0, 25.0, 6.25, 1.56, 0.39, 0.097, 0.024, and 0 (blank) nM.

[0046] Each analysis cycle consisted of the following: (1) capture of multispecific binding molecule samples on separate flow cells (Fc2, Fc3, and Fc4); (2) injection of each concentration of a blood-brain barrier target (e.g., TFR) at 10 μL / min for 60 seconds on all Fcs, followed by return buffer flow for 1800 seconds to monitor the dissociation phase; (3) injection of each human L1CAM concentration at 100 μL / min for 120 seconds on all Fcs, followed by return buffer flow for 1200 seconds to monitor the dissociation phase; (4) regeneration of the chip surface using injection of 10 mM glycine, pH 1.5, at 10 μL / min on all cells for 30 seconds; and (5) equilibration of the chip surface with a 10 μL (60 second) injection of HBS-EP+. Data were processed using Biacore T200 Evaluation Software Version 2.0.3 using standard double-referencing and fitted to a 1:1 binding model to determine association rates (k on , M -1 s -1 Unit), dissociation rate (k off ,s -1 Unit) and R max (RU units). Equilibrium dissociation constant (K D ) According to the relationship K D =k off / k onThe calculations are shown in molar units. The results are provided in Table 3.

[0047] Table 3: Binding affinities of the exemplified binding domains to the respective targets at 25°C.

[0048] These results demonstrate that exemplified multispecific binding molecules of the invention bind with high affinity to both the blood-brain barrier target TFR and the CNS target (neuronal target) L1CAM at 25°C.

[0049] Simultaneous binding of TFR and L1CAM The BIAcore T200 instrument was used to determine whether TFR and L1CAM can be simultaneously bound to the multispecific binding molecules (MBM1) described in detail above. Except as noted, all reagents and materials were from Cytiva (Upsala, Sweden). All measurements were performed at 25°C. HBS-EP+ buffer (150mM sodium chloride, 3mM EDTA, 0.05% (w / v) surfactant P-20 and 10mM HEPES, pH 7.4) was used as running buffer and sample buffer. On all four flow cells (Fc), human Fab capture kit (Cytiva P / N 28958325) was fixed on a CM5 chip (Cytiva P / N 29104988) using standard NHS-EDC amine coupling. The exemplified multispecific binding molecules (diluted to 50 μg / ml) were first captured on flow cell 2 (injected for 30 seconds at 30 μl / min, resulting in 100 response units (RU) of multispecific binding molecule capture), followed by an injection of 500 nM human or mouse L1CAM for 5 minutes to saturate the L1CAM binding sites (15ΔRU of binding signal was observed). Flow cell 1 was a blank control only. After binding to L1CAM (flow cell 2), 500 nM mouse TFR was then injected for 5 minutes, and an additional binding signal (8ΔRU) was observed. The chip surface was then regenerated using 10 mM glycine pH 1.5. The same process was repeated, except with the reverse order of first mouse TFR followed by human or mouse L1CAM. The results showed an increase in response units from both ligands bound to the multispecific binding molecules (initially 10 RU from TFR, and then an additional 13 RU from L1CAM), confirming that the exemplified multispecific binding molecules of the present invention can bind to human or mouse L1CAM and mouse TFR simultaneously.

[0050] In vitro Binding, Internalization, and Degradation Assessment in Rat Cortical Neurons Fluorescence signals corresponding to the total level and internalization of antibody and binding molecule-siRNA conjugates were measured by high-content live cell imaging assays in primary mouse cortical neurons. Briefly, primary mouse cortical neurons were isolated from wild-type C57BL6 mouse embryos at E18. Cells were plated at a density of 40k cells / well in poly-D-lysine-coated 96-well plates and cultured at 37°C for 7 days in NbActiv1 (BrainBits, LLC) containing 1% antibiotic / antimycotic (Corning) in a tissue culture incubator in a humidified chamber with 5% CO2. On day 7, the medium was removed from each well and replaced with medium containing 5ug / ml (33nM) of any of the following: (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 molecules with siRNA linked at the HC constant region 1 (indicated by *), together with 10ug / ml (0.2uM) anti-human IgG labeled with DyLight650 (Thermo Fisher #62266), DL650 with BHQ3 dye (BioSearch Tech BHQ-3000S-5) or pHAb dye (Promega #G9845) in medium with 6.7uM (1mg / ml) goat gamma globulin (Jackson Immuno #005-000-002). Fcγ fragment specific Fab fragment (Jackson Immuno #109-007-008) and incubated with live cells grown in 96-well plates at 37°C overnight.

[0051] The next day, cells were washed, incubated with NucBlue Hoechst dye (Thermo Fisher #R37605) for 20 minutes, washed again, and then imaged using a Cytation 5 High Content Imager (Biotek). The DyLight 650 signal measures total antibody levels, the DyLight 650 plus BHQ3 signal measures the degradation signal, which increases DyLight 650 fluorescence when the BHQ3 dye is released and FRET quenching is lost, and the pHAb pH sensor dye signal only measures internalized fluorescence. Excess goat gamma globulin was added to reduce nonspecific binding and antibody uptake into cells. The signal intensity in each well was divided by the number of Hoechst-stained nuclei to determine the signal intensity per cell. The wells were analyzed in duplicate, and for each well, approximately 20k cells were analyzed from images taken using a 4x objective. Background signal was determined by a human IgG isotype control and subtracted from the final value. Results are provided in Figure 3middle.

[0052] High content imaging data confirmed that the cellular activities (binding, internalization and degradation properties) of the exemplified multispecific binding molecules were higher than those of the monovalent TFR antibodies and isotype controls in neurons. In addition, the activities of the exemplified multispecific binding molecules were not substantially altered when conjugated to siRNA. The isotype control and the isotype control conjugated to siRNA lacked activity, indicating that the activities exhibited by the exemplified multispecific binding molecules were target receptor specific.

[0053] In vitro potency assessment in mouse cortical neurons Murine primary cortical neurons were isolated from wild-type C57BL6 mouse embryos at E18. Cells were 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, half of the medium was removed from each well and one of the following was added at 2x concentration in medium with 2% FBS: (i) siRNA (targeting the desired therapeutic target within the CNS); (ii) control siRNA (naked siRNA that does not target the therapeutic target within the CNS); (iii) hIgG4 isotype control antibody with siRNA attached at HC constant region 1 (Iso mAb*), (iv) monovalent TfR antibody with cholesterol-conjugated siRNA attached at HC constant region 1 (mvTFR*), or (v) MBM1 (MBM1*) with cholesterol-conjugated siRNA attached at HC constant region 1 (Table 2) for treatment at a range of siRNA concentrations and incubated with cells for another 7 days. At the end of siRNA treatment, RT-qPCR was performed using the TaqMan Fast Advanced Cell-to-CT kit to quantify the targeted mRNA levels. Specifically, cells were lysed, cDNA was generated on MastercyclerX50a (Eppendorf), and qPCR was performed on QuantStudio 7Flex Real-Time PCR System (Applied Biosystems). Gene expression levels of therapeutic targets were normalized by β-actin (ThermoFisher Mm02619580_g1) using the corresponding probe. The results are provided in Table 4: Table 4: In vitro potency in mouse cortical neurons The results provided in Table 4 demonstrate that the exemplified multispecific binding molecules provide linked siRNAs targeting therapeutic targets with potency that is multiple orders of magnitude higher than unconjugated (e.g., naked siRNA) and one order of magnitude higher than siRNAs targeting therapeutic targets linked only to mvTFR.

[0054] Brain Pharmacokinetic Assessment The brain pharmacokinetics of the exemplified multispecific binding molecules of the present disclosure can be evaluated as follows. Briefly, 8-week-old male FVB mice were dosed with (i) Iso mAb, (ii) mvTFR, (iii) L1CAM antibody (L1CAM mAb), and (iv) MBM1 at 5 mg / kg, sacrificed and perfused at the following time points: 0.5, 2, 6, 24, 48, 72, 168, 336, and 504 hours (n=3 per time point). The levels of the corresponding molecules in the brain were determined by ELISA (as assessed by the area under the curve (AUC)). Following the procedures described herein, Iso mAb and L1CAM mAb exhibited low levels in the brain. mvTFR reached a peak Cmax of approximately 1000 ng / g between 24 and 48 hours and exhibited brain accumulation, which was cleared within 168 hours. MBM1 exhibited a higher Cmax than mvTFR between 24 and 48 hours and showed delayed clearance from the brain, with 500 ng / ml detectable at 168 hours and more than 100 ug detectable at 336 hours. Results are provided in Figure 4A and Figure 4B middle.

[0055] The results suggest that the combination of the blood-brain barrier target binding domain and the CNS target (neuron) binding domain exhibits enhanced brain PK of the molecule compared to either the mvTFR or L1CAM targeting molecules alone.

[0056] In vivo pharmacokinetic evaluation The pharmacokinetic properties of the exemplified multispecific binding molecules of the present disclosure can be evaluated as follows. Briefly, Iso mAb*, mvTFR*, and MBM1* were administered intravenously once a week for 4 weeks in 8-week-old FVB mice at an effective siRNA concentration of 10 mg / kg. In addition, mouse anti-CD4 antibody (GK1.5) was administered at 10 mg / kg to deplete CD4-positive T cells to mitigate undesirable pharmacokinetic consequences caused by spurious anti-drug antibody responses to the injected compounds. Mice were sacrificed 168 hours after the last dose and perfused to collect brain and spinal cord to evaluate target mRNA levels by RT-qPCR in tissue homogenates and target protein levels by ELISA. The results are provided in Figures 5A-5E middle.

[0057] Results show significant improvement in mRNA and protein levels compared to PBS and Iso mAb (note: it is believed that reduction of MBM1* would be further enhanced in the higher L1CAM population).

[0058] 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 pharmacokinetic studies as described above, brain tissue, CSF, plasma, and peripheral tissues were collected and the levels of the respective administered molecules were determined by ELISA. The results are provided in Figures 6A to 6J middle.

[0059] The results confirmed that the level of MBM1* in the brain was about 200% higher than that of Iso mAb* and about 100% higher than that of mvTFR*. The results also confirmed that only the level of MBM1* was undetectable in plasma, CSF and all peripheral organs (bone marrow, gastrocnemius muscles, liver, kidney, heart, spleen and testis). In addition, the level of MBM1* in the bone marrow was comparable to that of Iso mAb* and lower than 10% of mvTFR*. The results confirmed that MBM1* achieved increased brain presence but did not achieve peripheral accumulation including bone marrow accumulation.

[0060] sequence SEQ ID NO. 1 - Exemplary anti-L1CAM HC SEQ ID NO. 2 - Exemplary anti-L1CAM LC SEQ ID NO. 3 - Exemplary anti-TFR HC SEQ ID NO.4 - Exemplary anti-FR LC SEQ ID NO.5 - Exemplary anti-TFR HCVR SEQ ID NO. 6 - Exemplary anti-TFR LCVR SEQ ID NO.7 - Exemplary anti-TFR HCVR SEQ ID NO. 8 - Exemplary anti-TFR LCVR SEQ ID NO.9 - Exemplary anti-TFR HCVR SEQ ID NO. 10 - Exemplary anti-TFR LCVR SEQ ID NO. 11 - Exemplary anti-CD98 HCVR SEQ ID NO. 12 - Exemplary anti-CD98 LCVR SEQ ID NO. 13 - Exemplary anti-CD98 HCVR SEQ ID NO. 14 - Exemplary anti-CD98 LCVR SEQ ID NO. 15 - Exemplary anti-CD98 HCVR SEQ ID NO. 16 - Exemplary anti-CD98 LCVR SEQ ID NO. 17 - Exemplary anti-CD98 HCVR SEQ ID NO. 18 - Exemplary anti-CD98 LCVR SEQ ID NO. 19 - Exemplary anti-L1CAM HCVR SEQ ID NO. 20 - Exemplary anti-L1CAM LCVR SEQ ID NO.21 - Exemplary Anti-TFR HCVR SEQ ID NO.22-Exemplary Anti-TFR LCVR

Claims

1. A multispecific binding molecule, the 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 astrocyte target, and a microglial target.

2. The multispecific binding molecule according to 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 neutral amino acid transporter subunit 1 (LAT-1), insulin receptor, low density lipoprotein (LDL) receptor, and CDC50A.

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

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

5. The multispecific binding molecule according to claim 1 or claim 2, wherein the second binding domain specifically binds to a microglial target, and the microglial target is 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 according to any one of claims 1-5, wherein the multispecific binding molecule is a heterodimeric immunoglobulin G antibody.

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

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

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

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

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

12. The multispecific binding molecule of any one of claims 1-11, 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. The multispecific binding molecule of claim 13, wherein the therapeutic composition is an oligonucleotide that targets one of MAPT, SNCA, APP, BAC1, ATXN2, ATXN3, SARM1, APOE, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B.

15. The multispecific binding molecule of claim 13, wherein the therapeutic composition is an antibody or fragment thereof that binds one of amyloid beta, BACE1, MAPT, SNCA, APP, BAC1, ATXN2, ATXN3, SARM1, APOE, FMR1, LRRK2, HTT, SOD1, SCN10A, SCN9A, or CACNA1B.

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

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

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

20. A method for delivering a therapeutic composition to a therapeutic target within the central nervous system of a mammal, comprising administering the therapeutic composition to the mammal, wherein the therapeutic composition is linked to a multispecific binding protein according to any one of claims 1-15, wherein the administering step comprises intravenous, subcutaneous, intramuscular or intraperitoneal administration of the multispecific binding molecule.

Images